Amphiphilic compounds and pharmaceutical compositions thereof

Abstract

The invention discloses an amphiphilic compound and a pharmaceutical composition thereof. The compound has a structure of formula I, wherein the cyclic disulfide can undergo dynamic covalent disulfide bond exchange with the thiol on the extracellular surface to mediate its entry into the cytoplasm; betaine has cationic and anionic charge groups, and forms a strong hydration layer through electrostatic interaction. The amphiphilic compound with the betaine structure makes the cyclic disulfide easy to be exposed to the surface of the nanocarrier and achieves the effect of anti-protein adsorption, thereby prolonging the blood circulation time. The nanocarrier modified with the functionalized amphiphilic compound can efficiently and stably deliver the drug directly to the cytoplasm through a non-endocytic pathway.

Description

Technical Field

[0001] The present invention relates to an amphiphilic compound and a pharmaceutical composition thereof, and in particular to an amphiphilic compound capable of efficiently and stably delivering drugs and a pharmaceutical composition thereof. Background Art

[0002] In recent years, nanoformulations have developed rapidly. Nanocarriers can change the physicochemical properties of their payloads, reduce off-target effects, increase cellular internalization and improve targeting. Nanocarrier delivery of bioactive drugs is a promising strategy that can improve the bioavailability of small molecule drugs and bioactive drugs, retain their biological activity to the greatest extent and reduce their immunogenicity.

[0003] Ideal nanocarriers must have good blood circulation stability and high endosomal escape ability. However, most traditional nanocarriers are internalized by cells based on the endocytosis pathway and complete drug cytoplasmic delivery through endosomal escape. Although drug design is based on the strategy of releasing drug molecules to be captured by the endosomal escape delivery pathway, these conventional strategies are often limited by low cytoplasmic release rate (<2%) and loss of biological activity of the delivered drug molecules. In addition, nanocarriers are easily taken up by the mononuclear phagocyte system (MPS) in the body and quickly cleared. To solve this problem, polyethylene glycol (PEGylation) is introduced into the carrier to increase serum stability and blood circulation time. However, injection of PEGylated NPs stimulates the production of anti-PEG antibodies, which in turn leads to complement activation and recognition by the immune system. Therefore, repeated injection of PEGylated NPs will induce the accelerated blood clearance (ABC) phenomenon, thereby reducing the blood circulation time of PEGylated NPs and its therapeutic effect. Summary of the invention

[0004] Purpose of the invention: In view of the shortcomings of existing drug delivery carriers in terms of in vivo circulation stability and endosomal escape ability, the present invention aims to provide an amphiphilic compound and a pharmaceutical composition thereof that can deliver drugs via a non-endocytic pathway mediated by dynamic covalent bond exchange and can prolong blood circulation time.

[0005] Technical solution: As the first invention of the present invention, the amphiphilic compound of the present invention has a structure of formula I,

[0006]

[0007] in:

[0008] P is

[0009] Q is -S(O) 2 O - or -C(O)O - ;

[0010] R 1 is at least one hydrogen, double bond or C 14 -C 18 Straight chain alkyl substituted C 14 -C 19 Straight chain alkoxy, C 14 -C 19 Straight chain alkylamino or C 14 -C 20 Straight chain acyl;

[0011] R 2 for

[0012] Preferably, the amphiphilic compound has a structure of Formula II or Formula III,

[0013]

[0014] Among them, Q, R 1 , R 2 , n is defined as above.

[0015] The amphiphilic compound of the present invention uses lysine and glycerol as connecting segments, connects sulfobetaine and carboxybetaine betaine head segments with lipoic acid and asparagus acid cyclic disulfide head segments through ester bonds or amide bonds, and simultaneously connects a single-chain alkyl, alkenyl or double-chain alkyl hydrophobic chain tail to the connecting segment.

[0016] The cyclic disulfide in the above amphiphilic compound can undergo dynamic covalent disulfide bond exchange with the thiol on the extracellular surface to mediate its entry into the cytoplasm; at the same time, betaine has cationic and anionic charge groups, which form a strong hydration layer through electrostatic interaction. Therefore, the amphiphilic compound with betaine structure makes the cyclic disulfide easy to be exposed to the surface of the nanocarrier and achieve the effect of anti-protein adsorption, thereby prolonging the blood circulation time. The nanocarrier modified with the functionalized amphiphilic compound can efficiently and stably deliver the drug directly to the cytoplasm through a non-endocytic pathway.

[0017] Preferably, in the above structure, R 1 Select any of the following substituents:

[0018]

[0019] Preferably, n is 1 or 3 in the above structure.

[0020] Preferably, the amphiphilic compound is selected from any one of the following compounds:

[0021]

[0022]

[0023]

[0024] In view of the problems existing in the existing biopharmaceutical nano-delivery system, the present invention provides an amphiphilic compound containing a cyclic disulfide and a betaine structure. The cell surface contains thiols to protect the cells from the influence of the oxidative environment. The covalent disulfide bond exchange between the "cargo" containing disulfides and the thiols on the extracellular surface can significantly promote cell uptake and mediate its direct entry into the cytoplasm via a non-endocytic pathway. However, it is difficult to expose the cyclic disulfide alone on the surface of the nanocarrier. In view of the characteristics of high dipole moment and high charge group, betaine has equimolar cationic and anionic groups, maintains overall electrical neutrality and high hydrophilicity, and is different from PEG that binds water molecules through hydrogen bond interactions. Betaine can form a stronger hydration shell with denser and more tightly adsorbed water through ion-dipole interactions. Therefore, the use of amphiphilic compounds with a betaine structure can ensure that the cyclic disulfide is exposed to the surface of the nanocarrier and achieves the effect of anti-protein adsorption without the ABC effect. Modifying nanocarriers with amphiphilic compounds containing cyclic disulfide and betaine structures is expected to simultaneously solve the problems of drug delivery system (ABC) phenomenon and endosomal capture, significantly improving the physiological stability of biological drug delivery systems and cytoplasmic delivery efficiency.

[0025] The preparation method of the above-mentioned amphiphilic compound comprises the following steps:

[0026] In the presence of a catalyst (EDC / HOBT, EDC / NHS or DCC / DMAP) and / or an acid-binding agent (triethylamine (TEA), N-ethyldiisopropylamine (DIPEA) or pyridine), the linker molecule is coupled with the hydrophobic tail containing an amine or hydroxyl group through an amide reaction or an esterification reaction, and then deprotected step by step under acidic or alkaline conditions, and then coupled with a cyclic disulfide and betaine through an amide / esterification reaction to obtain a functionalized amphiphilic compound. Among them, the reagents used for acidic deprotection include ethyl hydrochloride, methanol hydrochloride, and trifluoroacetic acid; the alkaline deprotection reagents include piperidine, concentrated ammonia, and ethanolamine.

[0027] The specific preparation method is as follows:

[0028] (1) Lysine as a linker molecule

[0029] A certain amount of N-α-fluorenylmethoxycarbonyl-N-ε-tert-butyloxycarbonyl-L-lysine (Fmoc-Lys(Boc)-OH) was dissolved in the reaction solvent, and then an appropriate amount of catalyst and acid binding agent were added. After activation for a period of time, the -OH or -NH 2The hydrophobic chain is added to the reaction mixture, and the reaction is stirred at room temperature overnight. After the reaction is completed, the intermediate product is separated and purified to obtain the intermediate product; the intermediate product obtained in the previous step is reacted under alkaline conditions to remove the Fmoc group, and the obtained de-Fomc product reacts with an appropriate amount of 4-(dimethylamino)butyric acid in the presence of a catalyst and an acid-binding agent overnight, and after the reaction is completed, the intermediate product is separated and purified to obtain the intermediate product; the intermediate product is dissolved and then reacted under acidic conditions to remove the Boc group, and the obtained de-Boc product reacts with a certain amount of cyclic disulfide in the presence of a catalyst and an acid-binding agent overnight, and after the reaction is completed, the intermediate product is separated and purified to obtain the intermediate product, and the intermediate product is then reacted with an excess of 1,3-propanesultone or chloroacetic acid, and the functionalized amphiphilic compound can be obtained after separation and purification.

[0030] (2) Glycerol as a connecting molecule

[0031] A certain amount of a hydrophobic tail chain containing a carboxyl group is dissolved in a reaction solvent, and then an appropriate amount of 1-benzylglycerol is added, and the reaction is carried out under low temperature conditions in the presence of a catalyst for 6 to 8 hours. After the reaction is completed, the intermediate product is separated and purified to obtain the intermediate product; the intermediate product is reacted with an appropriate amount of 4-(dimethylamino)butyric acid at room temperature overnight in the presence of a catalyst, and after the reaction is completed, the intermediate product is separated and purified to obtain the intermediate product; the intermediate product is exposed to dry dichloromethane containing boron trichloride at low temperature to remove the hydroxyl protecting group benzyl (Bn), and the obtained Bn-free product is reacted with a certain amount of cyclic disulfide at room temperature overnight in the presence of a catalyst, and after the reaction is completed, the intermediate product is separated and purified to obtain the intermediate product, and the intermediate product is then reacted with an excess of 1,3-propanesultone or chloroacetic acid, and the functionalized amphiphilic compound can be obtained after separation and purification.

[0032] As the second aspect of the present invention, the above-mentioned amphiphilic compound and bioactive drug lipid nanoparticles form the pharmaceutical composition of the present invention.

[0033] Preferably, the above-mentioned bioactive drugs are polypeptide drugs, protein drugs, DNA drugs, RNA drugs, CRISPR gene editing systems, small molecule drugs, etc. Among them, the polypeptide drugs are salmon calcitonin and hirudin; the protein drugs are insulin, interleukin, lymphotoxin, interferon or tumor necrosis factor; the DNA drugs are plasmid DNA; the RNA drugs are mRNA, small interfering RNA, microRNA, antisense oligonucleotides, nucleic acid aptamers, ribozymes or guide RNA; the CRISPR gene editing system is CRISPR / Cas9, CRISPR / Cas12a, CRISPR / Cas13a or CRISPR / dCas9.

[0034] Preferably, the lipids in the above-mentioned lipid nanoparticles contain phospholipids and steroidal lipids, wherein the phospholipids are soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, hydrogenated lecithin, dipalmitoylphosphatidylserine, dioleoylphosphatidylserine, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, trimethyl-2,3-dioleyloxypropylammonium bromide, trimethyl-2,3-dioleyloxypropylammonium chloride, and one or more of soybean lecithin and egg yolk lecithin; the steroidal lipids are one or more of cholesterol, ergosterol, fuccosterol, alginosterol, bile acid, glycocholic acid, deoxycholic acid and lithocholic acid, and preferably cholesterol.

[0035] Preferably, the molar ratio of the amphiphilic compound, phospholipid and steroidal lipid is (0-30):(30-90):(5-50), preferably (2-15):(40-80):(10-35).

[0036] The lipid nanoparticles can be prepared by conventional lipid nanoparticle preparation methods, such as thin film dispersion method, double emulsion method, reverse evaporation method, etc. The average particle size of the lipid nanoparticles is 10-1000nm, and the polydispersity index (PDI) is less than 0.3.

[0037] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0038] 1. The amphiphilic compound and its pharmaceutical composition significantly promote cellular uptake and mediate direct entry into cells via non-endocytic pathways through dynamic covalent disulfide bond exchange, effectively avoiding endosomal capture; at the same time, they are highly resistant to nonspecific protein adsorption through electrostatic interactions, extending the blood circulation time of the delivery system; in addition, they can effectively load drugs to achieve efficient delivery without obvious cytotoxicity.

[0039] 2. Using natural amino acids and glycerol as linker molecules, new functionalized amphiphilic compounds with betaine and cyclic disulfide are prepared through simple acylation and esterification reactions. The method is simple, the raw materials are readily available, and large-scale preparation is easy to achieve. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a graph showing the anti-protein adsorption results of Ⅰa-TA / siRNA LNP, Ⅱa-TA / siRNA LNP, Ⅲa-TA / siRNA LNP, and Ⅳa-TA / siRNA LNP prepared in Example 7;

[0041] Figure 2This is a graph showing the serum stability results of Ⅰa-TA / siRNA LNP, Ⅱa-TA / siRNA LNP, Ⅲa-TA / siRNA LNP, and Ⅳa-TA / siRNA LNP prepared in Example 7;

[0042] Figure 3 This is a graph showing the stability results of the culture medium of Ⅰa-TA / siRNA LNP, Ⅱa-TA / siRNA LNP, Ⅲa-TA / siRNA LNP, and Ⅳa-TA / siRNA LNP prepared in Example 7;

[0043] Figure 4 This is a graph showing the encapsulation efficiency test results of Ⅰa-TA / siRNALNP, Ⅱa-TA / siRNALNP, Ⅲa-TA / siRNALNP, and Ⅳa-TA / siRNALNP prepared in Example 7;

[0044] Figure 5 The cytotoxicity results of Ⅰa-TA / siRNALNP, Ⅱa-TA / siRNALNP, Ⅲa-TA / siRNALNP and Ⅳa-TA / siRNALNP prepared in Example 7 are shown in FIG.

[0045] Figure 6 This is a graph showing the cellular uptake results of Ⅰa-TA / siRNALNP, Ⅱa-TA / siRNALNP, Ⅲa-TA / siRNALNP, and Ⅳa-TA / siRNA LNP prepared in Example 7;

[0046] Figure 7 This is a diagram showing the intracellular lysosome co-localization results of Ⅰa-TA / siRNALNP, Ⅱa-TA / siRNALNP, Ⅲa-TA / siRNALNP, and Ⅳa-TA / siRNA LNP prepared in Example 7;

[0047] Figure 8 This is a graph showing the results of studying the cellular uptake mechanism of Ⅰa-TA / siRNA LNP, Ⅱa-TA / siRNA LNP, Ⅲa-TA / siRNA LNP, and Ⅳa-TA / siRNA LNP prepared in Example 7;

[0048] Fig. 9 This is a graph showing the transfection efficiency of Ⅰa-TA / siRNA LNP, Ⅱa-TA / siRNA LNP, Ⅲa-TA / siRNA LNP, and Ⅳa-TA / siRNA LNP prepared in Example 7 in 4T1-luc cells. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is further described below in conjunction with embodiments.

[0050] Example 1: Synthesis of Compounds 1a-1c

[0051]

[0052] 1. Accurately weigh compound 1 (N-α-fluorenylmethoxycarbonyl-N-ε-tert-butyloxycarbonyl-L-lysine, Fmoc-Lys(Boc)-OH) (1.0 mol) in a round-bottom flask, add tetrahydrofuran and stir to dissolve it, then add EDC (1.3 mol), HOBT (1.3 mol) and acid-binding agent triethylamine (3 mol). Stir the mixture for activation for 2 h, then add R a-c (1.2 mol) was added to the reaction mixture and stirred at room temperature for overnight reaction. After the reaction was completed, the reaction solvent was removed by rotary evaporation, the precipitate was washed by suction filtration, and the solid obtained after vacuum drying was the target product with a yield of about 80%.

[0053] 2. Take the intermediate product 2 (1 mol) obtained above and dissolve it in 40 mL of 20% (v / v) piperidine in DCM solution, and stir the reaction to remove the Fmoc group. After the reaction is completed, the reaction solvent is removed by vacuum rotary evaporation, and the obtained reaction product is separated and purified by column chromatography, and the product yield is about 86%.

[0054] 3. Take 4-(dimethylamino)butyric acid (1.2 mol) in a round-bottom flask, add chloroform and stir to dissolve, then add EDC (1.5 mol), HOBT (1.5 mol) and acid-binding agent triethylamine (3 mol). The above mixture is stirred and activated for 2 hours, and then the de-Fomc product 3 (1 mol) is added. The reaction mixture is stirred and reacted overnight at room temperature. After the reaction is completed, the reaction solution is transferred to a separatory funnel and extracted and washed with water. The organic phase is dried over anhydrous magnesium sulfate, filtered and evaporated under reduced pressure to remove the organic solvent. The resulting solid is the target product with a yield of about 75%.

[0055] 4. Take the intermediate product 4 (1 mol) obtained above and dissolve it in 40 mL of hydrochloric acid ethyl acetate solution, stir and react at room temperature for 4 hours to remove the Boc group. After the reaction is completed, evaporate the organic solvent under reduced pressure, and the product yield is about 90%.

[0056] 5. Take asparagus acid (1.2 mol) in a round-bottom flask, add chloroform and ultrasonically dissolve it, then add EDC (1.5 mol), HOBT (1.5 mol) and acid-binding agent triethylamine (3 mol). The above mixture is stirred for activation, and then the de-BOC product 5 (1.0 mol) is added. The reaction mixture is stirred at room temperature overnight. After the reaction is completed, the reaction solution is transferred to a separatory funnel and extracted with water. The organic phase is dried over anhydrous magnesium sulfate, filtered and evaporated under reduced pressure to remove the organic solvent. The resulting solid is the target product with a yield of about 70%.

[0057] 6. The intermediate product 6 (1 mol) obtained above was mixed with 1,3-propane sultone (3 mol) in chloroform and stirred at room temperature for 3 days. After the reaction was completed, the organic solvent was removed by rotary evaporation under reduced pressure, and the obtained solid was washed with petroleum ether to finally obtain the final product 1a-1c. The yield of this step was about 88%.

[0058] 1a: MS m / z(ESI):764.46[M-1]; 1 H NMR (300 MHz, D 2 O): δ4.24(m,1H),3.42(s,6H),3.19(m,6H),3.24(m,3H),3.02(s,2H),2.62(s,2H),2.55(s,2H), 2.16(s,2H),2.04(d,J=5.5Hz,2H)1.63(d,J=1.8Hz,2H),1.57(m,6H),1.33(m,30H),0.92(s,3H).

[0059] 1b: MS m / z(ESI):762.45[M-1]; 1 H NMR (300 MHz, D 2 O): δ5.42(s,2H),4.10(s,1H),3.42(s,6H),3.19(m,6H),3.24(m,3H),3.02(s,2H),2.62(s,2H),2.45(s ,2H),2.20(s,6H),2.09(d,J=5.5Hz,2H)1.69(d,J=1.8Hz,2H),1.53(m,6H),1.33(m,26H),0.88(s,3H).

[0060] 1c: MS m / z(ESI):1030.76[M-1]; 1 HNMR (300MHz, D 2 O): δ4.34(s,1H),3.61(m,1H),3.36(s,6H),3.18(m,4H),3.07(m,5H),2.58(m,4H),2.32(m,2H),2.07(d, J=0.9Hz,2H),1.96(d,J=5.5Hz,2H),1.69(m,2H),1.54–1.42(m,6H),1.22(d,J=1.4Hz,66H),0.83(s,6H).

[0061] Example 2: Synthesis of Compounds 2a-2c

[0062]

[0063] 1. Accurately weigh compound 1 (N-α-fluorenylmethoxycarbonyl-N-ε-tert-butyloxycarbonyl-L-lysine, Fmoc-Lys(Boc)-OH) (1.0 mol) in a round-bottom flask, add tetrahydrofuran and stir to dissolve it, then add EDC (1.3 mol), HOBT (1.3 mol) and acid-binding agent triethylamine (3 mol). Stir the mixture for activation for 2 h, then add R a-c (1.2 mol) was added to the reaction mixture and stirred at room temperature for overnight reaction. After the reaction was completed, the reaction solvent was removed by rotary evaporation, the precipitate was washed by suction filtration, and the solid obtained after vacuum drying was the target product with a yield of about 80%.

[0064] 2. Take the intermediate product 2 (1 mol) obtained above and dissolve it in 40 mL of 20% (v / v) piperidine in DCM solution, and stir the reaction to remove the Fmoc group. After the reaction is completed, the reaction solvent is removed by vacuum rotary evaporation, and the obtained reaction product is separated and purified by column chromatography, and the product yield is about 86%.

[0065] 3. Take 4-(dimethylamino)butyric acid (1.2 mol) in a round-bottom flask, add chloroform and stir to dissolve, then add EDC (1.5 mol), HOBT (1.5 mol) and acid-binding agent triethylamine (3 mol). The above mixture is stirred and activated for 2 hours, and then the de-Fomc product 3 (1 mol) is added. The reaction mixture is stirred and reacted overnight at room temperature. After the reaction is completed, the reaction solution is transferred to a separatory funnel and extracted and washed with water. The organic phase is dried over anhydrous magnesium sulfate, filtered and evaporated under reduced pressure to remove the organic solvent. The resulting solid is the target product with a yield of about 75%.

[0066] 4. Take the intermediate product 4 (1 mol) obtained above and dissolve it in 40 mL of hydrochloric acid ethyl acetate solution, stir and react at room temperature for 4 hours to remove the Boc group. After the reaction is completed, evaporate the organic solvent under reduced pressure, and the product yield is about 90%.

[0067] 5. Take asparagus acid (1.2 mol) in a round-bottom flask, add chloroform and ultrasonically dissolve it, then add EDC (1.5 mol), HOBT (1.5 mol) and acid-binding agent triethylamine (3 mol). The above mixture is stirred for activation, and then the de-BOC product 5 (1.0 mol) is added. The reaction mixture is stirred at room temperature overnight. After the reaction is completed, the reaction solution is transferred to a separatory funnel and extracted with water. The organic phase is dried over anhydrous magnesium sulfate, filtered and evaporated under reduced pressure to remove the organic solvent. The resulting solid is the target product with a yield of about 70%.

[0068] 6. Take the intermediate product 6 (1.0 mol) obtained above and chloroacetic acid (3.0 mol) and add them to an ethanol / water mixed solution, adjust the pH to 11-13, and use a condenser to maintain the reaction mixture at 60°C. After the reaction is completed, evaporate and concentrate the sample. Use ethyl acetate to precipitate the final product, filter it, wash the solid with ethyl acetate, and then vacuum dry it to finally obtain the final product 2a-2c. The yield of this step is about 80%.

[0069] 2a: MS m / z(ESI):700.46[M-1]; 1 H NMR (300 MHz, D 2 O): δ4.30(s,1H),3.79(d,J=12.3Hz,2H),3.42(m,2H),3.23(d,J=17.0Hz,8H),3.10(s,4H),2.98(s,1H),2.65(s ,2H),2.24(d,J=4.9Hz,2H),2.04(d,J=12.3Hz,2H),1.77(m,2H),1.48(d,2H),1.35–1.25(m,34H),0.89(s,3H).

[0070] 2b: MS m / z(ESI):698.45[M-1]; 1 H NMR (300 MHz, D 2 O): δ5.35(s,2H),3.80(m,2H),3.40(m,2H),3.23(d,J=17.0Hz,8H),3.11(s,2H),3.07(s,3H),2.69(s, 2H),2.24(m,2H),2.03(s,4H),1.95(m,2H),1.78(m,2H),1.56(m,2H),1.35–1.20(m,26H),0.87(s,3H).

[0071] 2c: MS m / z(ESI):966.75[M-1]; 1 H NMR (300 MHz, CDCl 3 ): δ4.53(m,1H),4.28(s,2H),3.41(m,3H),3.30(s,2H),3.21(s,6H),3.07(s,3H),2.78(s, 2H),2.29(m,2H),1.92(m,2H),1.74(m,2H),1.58(m,6H),1.33–1.25(m,66H),0.78(s,6H).

[0072] Example 3: Synthesis of Compounds 3a-3c

[0073]

[0074] 1. Accurately weigh compound 1 (N-α-fluorenylmethoxycarbonyl-N-ε-tert-butyloxycarbonyl-L-lysine, Fmoc-Lys(Boc)-OH) (1.0 mol) in a round-bottom flask, add tetrahydrofuran and stir to dissolve it, then add EDC (1.3 mol), HOBT (1.3 mol) and acid-binding agent triethylamine (3 mol). Stir the mixture for activation for 2 h, then add R a-c (1.2 mol) was added to the reaction mixture and stirred at room temperature for overnight reaction. After the reaction was completed, the reaction solvent was removed by rotary evaporation, the precipitate was washed by suction filtration, and the solid obtained after vacuum drying was the target product with a yield of about 80%.

[0075] 2. Take the intermediate product 2 (1 mol) obtained above and dissolve it in 40 mL of 20% (v / v) piperidine in DCM solution, and stir the reaction to remove the Fmoc group. After the reaction is completed, the reaction solvent is removed by vacuum rotary evaporation, and the obtained reaction product is separated and purified by column chromatography, and the product yield is about 86%.

[0076] 3. Take 4-(dimethylamino)butyric acid (1.2 mol) in a round-bottom flask, add chloroform and stir to dissolve, then add EDC (1.5 mol), HOBT (1.5 mol) and acid-binding agent triethylamine (3 mol). The above mixture is stirred and activated for 2 hours, and then the de-Fomc product 3 (1 mol) is added. The reaction mixture is stirred and reacted overnight at room temperature. After the reaction is completed, the reaction solution is transferred to a separatory funnel and extracted and washed with water. The organic phase is dried over anhydrous magnesium sulfate, filtered and evaporated under reduced pressure to remove the organic solvent. The resulting solid is the target product with a yield of about 75%.

[0077] 4. Take the intermediate product 4 (1 mol) obtained above and dissolve it in 40 mL of hydrochloric acid ethyl acetate solution, stir and react at room temperature for 4 hours to remove the Boc group. After the reaction is completed, evaporate the organic solvent under reduced pressure, and the product yield is about 90%.

[0078] 5. Take thioctic acid (1.2 mol) in a round-bottom flask, add chloroform and ultrasonically dissolve it, then add EDC (1.5 mol), HOBT (1.5 mol) and acid-binding agent triethylamine (3 mol). The above mixture is stirred for activation, and then the de-BOC product 5 (1.0 mol) is added. The reaction mixture is stirred at room temperature overnight. After the reaction is completed, the reaction solution is transferred to a separating funnel and extracted with water. The organic phase is dried over anhydrous magnesium sulfate, filtered and evaporated under reduced pressure to remove the organic solvent. The resulting solid is the target product with a yield of about 70%.

[0079] 6. The intermediate product 6 (1 mol) obtained above was mixed with 1,3-propane sultone (3 mol) in chloroform and stirred at room temperature for 3 days. After the reaction was completed, the organic solvent was removed by rotary evaporation under reduced pressure, and the obtained solid was washed with petroleum ether to finally obtain the final product 1a-1c. The yield of this step was about 88%.

[0080] 3a: MS m / z(ESI):820.51[M-1]; 1 H NMR (300 MHz, D 2 O): δ4.11(s,1H),3.45–3.38(m,3H),3.20(s,8H),3.10(s,2H),3.06(s,2H),2.95(d,J=17.4Hz,2H),2.65(d,J=1 2.4Hz,2H),2.26(d,J=0.7Hz,2H),2.02–1.95(m,3H),1.74(m,3H),1.53(m,6H),1.32–1.25(m,36H),0.87(s,3H).

[0081] 3b: MS m / z(ESI):818.49[M-1]; 1 H NMR (300 MHz, D 2 O): δ5.35(s,2H),4.11(s,1H),3.45–3.38(m,3H),3.30–3.22(m,3H),3.16(s,8H),2.98(d,4H),2.65(d,2H) 2.26(d,J=0.7Hz,2H),2.01–1.95(m,7H),1.78(m,3H),1.50-1.56(m,6H),1.40–1.20(m,28H),0.79(s,3H).

[0082] 3c: MS m / z(ESI):1086.83[M-1]; 1 H NMR (300 MHz, CDCl 3 ): δ4.1–3.8(m,2H),3.41–3.30(m,2H),3.30–3.22(m,2H),3.20(s,7H),2.97(s,2H),2.58(d,2H),2.27(d,J=0.9 Hz,2H),2.19(s,2H),2.01–1.95(m,4H),1.66–1.54(m,8H),1.48–1.41(m,4H),1.36–1.23(m,68H),0.92(s,6H).

[0083] Example 4: Synthesis of Compounds 4a-4c

[0084]

[0085] 1. Accurately weigh compound 1 (N-α-fluorenylmethoxycarbonyl-N-ε-tert-butyloxycarbonyl-L-lysine, Fmoc-Lys(Boc)-OH) (1.0 mol) in a round-bottom flask, add tetrahydrofuran and stir to dissolve it, then add EDC (1.3 mol), HOBT (1.3 mol) and acid-binding agent triethylamine (3 mol). Stir the mixture for activation for 2 h, then add R a-c (1.2 mol) was added to the reaction mixture and stirred at room temperature for overnight reaction. After the reaction was completed, the reaction solvent was removed by rotary evaporation, the precipitate was washed by suction filtration, and the solid obtained after vacuum drying was the target product with a yield of about 80%.

[0086] 2. Take the intermediate product 2 (1 mol) obtained above and dissolve it in 40 mL of 20% (v / v) piperidine in DCM solution, and stir the reaction to remove the Fmoc group. After the reaction is completed, the reaction solvent is removed by vacuum rotary evaporation, and the obtained reaction product is separated and purified by column chromatography, and the product yield is about 86%.

[0087] 3. Take 4-(dimethylamino)butyric acid (1.2 mol) in a round-bottom flask, add chloroform and stir to dissolve, then add EDC (1.5 mol), HOBT (1.5 mol) and acid-binding agent triethylamine (3 mol). The above mixture is stirred and activated for 2 hours, and then the de-Fomc product 3 (1 mol) is added. The reaction mixture is stirred and reacted overnight at room temperature. After the reaction is completed, the reaction solution is transferred to a separatory funnel and extracted and washed with water. The organic phase is dried over anhydrous magnesium sulfate, filtered and evaporated under reduced pressure to remove the organic solvent. The resulting solid is the target product with a yield of about 75%.

[0088] 4. Take the intermediate product 4 (1 mol) obtained above and dissolve it in 40 mL of hydrochloric acid ethyl acetate solution, stir and react at room temperature for 4 hours to remove the Boc group. After the reaction is completed, evaporate the organic solvent under reduced pressure, and the product yield is about 90%.

[0089] 5. Take thioctic acid (1.2 mol) in a round-bottom flask, add chloroform and ultrasonically dissolve it, then add EDC (1.5 mol), HOBT (1.5 mol) and acid-binding agent triethylamine (3 mol). The above mixture is stirred for activation, and then the de-BOC product 5 (1.0 mol) is added. The reaction mixture is stirred at room temperature overnight. After the reaction is completed, the reaction solution is transferred to a separating funnel and extracted with water. The organic phase is dried over anhydrous magnesium sulfate, filtered and evaporated under reduced pressure to remove the organic solvent. The resulting solid is the target product with a yield of about 70%.

[0090] 6. Take the intermediate product 6 (1.0 mol) obtained above and chloroacetic acid (3.0 mol) and add them to an ethanol / water mixed solution, adjust the pH to 11-13, and use a condenser to maintain the reaction mixture at 60°C. After the reaction is completed, evaporate and concentrate the sample. Use ethyl acetate to precipitate the final product, filter it, wash the solid with ethyl acetate, and then vacuum dry it to finally obtain the final product 2a-2c. The yield of this step is about 80%.

[0091] 4a: MS m / z(ESI):756.49[M-1]; 1 H NMR (300 MHz, D 2 O): δ4.23(s,1H),3.76–3.33(m,5H),3.21(s,6H),3.08(s,4H),2.98(d,J=17.4Hz,2H),2.24(d,J=4. 9Hz,2H),2.19(s,2H),2.07–1.98(m,4H),1.63(m,2H),1.57(m,6H),1.39–1.22(m,36H),0.83(s,3H).

[0092] 4b: MS m / z(ESI):754.53[M-1]; 1 H NMR (300 MHz, D 2 O): δ5.35(s,2H),4.34(s,1H),3.73–3.39(m,5H),3.21(s,6H),3.06(s,4H),2.98(d,J=17.4Hz,2H),2.29–2.22(m,2H),2. 19(s,2H),2.03(s,6H),1.98(t,J=6.2Hz,2H),1.67(d,J=1.8Hz,2H),1.59–1.49(m,6H),1.45–1.20(m,28H),0.85(s,3H).

[0093] 4c: MS m / z(ESI):1022.80[M-1]; 1 H NMR (300 MHz, CDCl 3 ): δ4.15(m,1H),3.89(m,1H),3.50–3.39(m,5H),3.25(s,6H),2.19(m,4H),2.02–1 .92(m,4H),1.66–1.54(m,8H),1.46–1.39(m,4H),1.37–1.24(m,68H),0.87(s,6H).

[0094] Example 5: Synthesis of Compounds 9a-9c

[0095]

[0096] 1. 1-Benzylglycerol (1.0 mol), carboxyl-containing hydrophobic chain (R a -R c ) (1.5 mol) and DMAP (0.1 mol) were dissolved in a solution of dry dichloromethane, followed by the addition of DCC (1.5 mol) and stirring at 0°C for 6-8 hours. The solid was filtered off and the filtrate was concentrated under reduced pressure. The residue was then purified by silica gel column (eluted with petroleum ether / ethyl acetate) to obtain the intermediate product 1-benzyl-3-acylglycerol, with a yield of about 75% for this step.

[0097] 2. Dissolve 1-benzyl-3-acylglycerol (1.0 mol), 4-(dimethylamino)butyric acid (0.5 mol) and DMAP (0.15 mol) in dry dichloromethane, then add DCC (0.5 mol) to the above solution, and stir the reaction at room temperature for 24 hours. Filter out the solid and evaporate the solvent under vacuum. The residue is separated and purified to obtain intermediate product 3, and the yield of this step is about 62%.

[0098] 3. Take the intermediate product 3 (1.0 mol) obtained in the previous step and dissolve it in dry dichloromethane. Under low temperature conditions, a dichloromethane solution of trichloroborane (2.0 mol) is slowly added dropwise to the above solution. Under nitrogen protection, the reaction mixture is stirred for another 30-40 minutes at the same temperature. Then the mixture is poured into ice water. Dichloromethane is separated and washed with ice water, dried over anhydrous magnesium sulfate and concentrated at room temperature. The residue is purified by silica gel column to obtain intermediate product 4, and the step yield is about 60%.

[0099] 4. The intermediate product 4 (1.0 mol), asparagus acid (1.5 mol) and DMAP (0.1 mol) obtained in the previous step were dissolved in dry dichloromethane, and then DCC (1.5 mol) was added to the above solution, and the reaction was stirred at room temperature for 24 hours. The solid was filtered out and the solvent was evaporated under vacuum. The residue was purified to obtain the intermediate product 5.

[0100] 5. The intermediate product 5 (1.0 mol) obtained above was mixed with anhydrous 1,3-propane sultone (3 mol) in chloroform and stirred at room temperature for 3 days. After the reaction was completed, the organic solvent was removed by rotary evaporation under reduced pressure, and the obtained solid was washed with petroleum ether to finally obtain the final product 9a-9c.

[0101] 9a: MS m / z(ESI):725.37[M-1]; 1 H NMR (300 MHz, D 2O):δ:4.7(m,2H),4.19(s,3H),3.45–3.24(m,5H),3.18(s,7H),2.90(m,5H),2.62(m,2H), 2.46(s,2H),2.25(s,2H),1.87(d,J=5.1Hz,2H),1.50(s,2H),1.25(m,28H),0.85(s,3H).

[0102] 9b: MS m / z(ESI):723.35[M-1]; 1 H NMR (300 MHz, D 2 O): δ5.35(s,2H),4.68(m,2H),4.19(s,3H),3.44–3.24(m,5H),3.18(s,7H),2.98(s,2H),2.89(d,J=4.4Hz,3 H),2.61(m,2H),2.41(s,2H),2.28(s,2H),2.03(s,4H),1.95(m,2H),1.50(s,2H),1.26(m,20H),0.92(s,3H).

[0103] 9c: MS m / z(ESI):1005.68[M-1]; 1 H NMR (300 MHz, CDCl 3 ): δ4.70(m,2H),4.19(d,J=1.1Hz,3H),3.48–3.34(m,3H),3.27(dd,J=12.5,1.1Hz,2H),3.18(s,7H),3.00(s,2H),2.95 -2.84(m,4H),2.58(m,2H),2.41(s,2H),1.96(d,J=1.5Hz,2H),1.63(m,4H),1.5–1.20(m,64H),0.78(s,6H).

[0104] Example 6: Synthesis of Compounds 10a-10c

[0105]

[0106] 1. 1-Benzylglycerol (1.0 mol), carboxyl-containing hydrophobic chain (R a -R c) (1.5 mol) and DMAP (0.1 mol) were dissolved in a solution of dry dichloromethane (100 ml), followed by the addition of DCC (1.5 mol) and stirring at 0°C for 6-8 hours. The solid was filtered off and the filtrate was concentrated under reduced pressure. The residue was then purified by silica gel column (eluted with petroleum ether / ethyl acetate) to obtain the intermediate product 1-benzyl-3-acylglycerol, with a yield of about 75% for this step.

[0107] 2. Dissolve 1-benzyl-3-acylglycerol (1.0 mol), 4-(dimethylamino)butyric acid (0.5 mol) and DMAP (0.15 mol) in dry dichloromethane, then add DCC (0.5 mol) to the above solution, and stir the reaction at room temperature for 24 hours. Filter out the solid and evaporate the solvent under vacuum. The residue is separated and purified to obtain intermediate product 3, and the yield of this step is about 62%.

[0108] 3. Take the intermediate product 3 (1.0 mol) obtained in the previous step and dissolve it in dry dichloromethane. Under low temperature conditions, a dichloromethane solution of trichloroborane (2.0 mol) is slowly added dropwise to the above solution. Under nitrogen protection, the reaction mixture is stirred for another 30-40 minutes at the same temperature. Then the mixture is poured into ice water. Dichloromethane is separated and washed with ice water, dried over anhydrous magnesium sulfate and concentrated at room temperature. The residue is purified by silica gel column to obtain intermediate product 4, and the step yield is about 60%.

[0109] 4. The intermediate product 4 (1.0 mol), asparagus acid (1.5 mol) and DMAP (0.1 mol) obtained in the previous step were dissolved in dry dichloromethane, and then DCC (1.5 mol) was added to the above solution, and the reaction was stirred at room temperature for 24 hours. The solid was filtered out and the solvent was evaporated under vacuum. The residue was purified to obtain the intermediate product 5, and the yield of this step was about 70%.

[0110] 5. The intermediate product 5 (1.0 mol) obtained above and chloroacetic acid (3 mol) were added to an ethanol / water mixed solution, the pH was adjusted to 11-13, and the reaction mixture was maintained at 60°C with a condenser. After the reaction was completed, the sample was evaporated and concentrated. The final product was precipitated with ethyl acetate, filtered, and the solid was washed with ethyl acetate and then vacuum dried to finally obtain the final product 10a-10c. The yield of this step was about 65%.

[0111] 10a: MS m / z(ESI):661.37[M-1]; 1 H NMR (300 MHz, D 2O): δ4.29(s,3H),3.78(m,2H),3.45–3.35(m,3H),3.21(s,6H),2.95(s,5H),2 .42(m,2H),2.26(m,2H),2.00(s,2H),1.51(s,2H),1.26(m,28H),0.87(s,3H).

[0112] 10b: MS m / z(ESI):659.35[M-1]; 1 H NMR (300 MHz, D 2 O): δ5.35(s,2H),4.76(m,2H),4.19(s,3H),3.45–3.35(m,4H),3.21(s,6H),2.93 (s,5H),2.42-2.26(m,4H),2.05(m,6H),1.55(s,2H),1.23(m,20H),0.89(s,3H).

[0113] 10c: MS m / z(ESI):941.49[M-1]; 1 H NMR (300 MHz, CDCl 3 ): δ4.70(m,2H),4.21(d,J=1.1Hz 3H),3.50–3.39(m,4H),3.21(s,6H),3.00(s,2H),2.90(s,3H),2.42(s,2H),1.99(s, 2H),1.63(d,J=12.4Hz,2H),1.54(d,J=12.4Hz,2H),1.38–1.20(m,64H),0.80(s,6H).

[0114] Referring to the above preparation method, functionalized amphiphilic compounds with other structures can be prepared by using different reaction raw materials, which will not be repeated here.

[0115] Example 7: Preparation of lipid nanoparticles modified with amphiphilic compounds

[0116] 1. Preparation of amphiphilic compound-modified TA / siRNA LNPs

[0117] First, tannic acid (TA) and siRNA were dissolved in RNase Free water to prepare a mother solution. The siRNA solution was mixed with an equal volume of TA dilution solution at a mass ratio of 1:10, gently vortexed for 20 seconds, and then allowed to stand at room temperature for 30 minutes. The TA / siRNA solution was used as the inner aqueous phase W1. Soybean lecithin and cholesterol were dissolved in chloroform according to the prescribed amount to form the oil phase. The inner aqueous phase was added to the oil phase, and the probe was ultrasonically formed into a W / O emulsion. The W / O emulsion was dropped into the outer aqueous phase containing the prescribed amount of soybean lecithin and functionalized amphiphilic compounds (1a, 2a, 3a, 4a), and a double emulsion was formed by water bath ultrasonication. The organic solvent was removed by reduced pressure evaporation to obtain lipid nanoparticles modified with functionalized amphiphilic compounds Ⅰa-TA / siRNA LNP, Ⅱa-TA / siRNA LNP, Ⅲa-TA / siRNA LNP, and Ⅳa-TA / siRNA LNP. The core particle size and polydispersity index (PDI) were determined using a Zetasizer 3000HS instrument (Malvern Instruments, Malvern, UK).

[0118] In this example, the specific results are as follows: the average particle size of the obtained lipid nanoparticles is about 145.0 nm, and the PDI is less than 0.3.

[0119] 2. Preparation of cationic liposomes modified with amphiphilic compounds

[0120] The prescribed amount of egg yolk lecithin, DOTAP, cholesterol and functionalized amphiphilic compounds (1a, 2a, 3a, 4a) were placed in an eggplant-shaped bottle and completely dissolved with chloroform / methanol. The organic solvent was removed by rotary evaporation at 120 rpm for 20 min at 37°C to form a uniform film, which was then vacuum dried to remove the residual organic solvent. The film was dispersed by adding an enzyme-free solution containing siRNA, and the amphiphilic compound-modified liposomes loaded with siRNA were obtained after 5-10 min of water bath ultrasound. The inner core particle size and polydispersity index (PDI) were determined using a Zetasizer 3000HS instrument particle size analyzer (Malvern Instruments, Malvern, UK).

[0121] In this example, the specific results are as follows: the average particle size of the obtained lipid nanoparticles is about 135.0 nm, and the PDI is less than 0.3.

[0122] 3. Preparation of TA / mRNA LNPs modified with amphiphilic compounds

[0123] First, TA and mRNA were dissolved in RNase Free water to prepare a mother solution. The mRNA solution was mixed with an equal volume of TA dilution solution at a mass ratio of 1:10, gently vortexed for 20 seconds, and then allowed to stand at room temperature for 30 minutes. The TA / mRNA solution was used as the inner aqueous phase W1. Soybean lecithin and cholesterol were dissolved in chloroform or in a prescribed amount as the oil phase, and the inner aqueous phase was added to the oil phase, and a W / O emulsion was formed by probe ultrasound. The W / O emulsion was added dropwise to the outer aqueous phase containing the prescribed amount of soybean lecithin and functionalized amphiphilic compounds (1a, 2a, 3a, 4a), and a double emulsion was formed by water bath ultrasound. The organic solvent was removed by reduced pressure evaporation to obtain lipid nanoparticles modified with functionalized amphiphilic compounds Ⅰa-TA / mRNA LNP, Ⅱa-TA / mRNA LNP, Ⅲa-TA / mRNA LNP, and Ⅳa-TA / mRNA LNP. The core particle size and polydispersity index (PDI) were determined using a Zetasizer 3000HS instrument (Malvern Instruments, Malvern, UK).

[0124] In this example, the specific results are as follows: the average particle size of the obtained lipid nanoparticles is about 145.0 nm, and the PDI is less than 0.3.

[0125] 4. Preparation of amphiphilic compound-modified TA / GFP LNPs

[0126] First, the green fluorescent protein (GFP) solution and the TA solution were mixed in equal volumes at a mass ratio of 1:10, gently vortexed for 20 seconds, and then allowed to stand at room temperature for 30 minutes. The TA / GFP solution was used as the inner aqueous phase W1. Soybean lecithin and cholesterol were dissolved in chloroform according to the prescribed amount to form the oil phase, and the inner aqueous phase was added to the oil phase, and the probe was ultrasonically formed into a W / O emulsion. The W / O emulsion was added dropwise to the outer aqueous phase containing the prescribed amount of soybean lecithin and functionalized amphiphilic compounds (1a, 2a, 3a, 4a), and a double emulsion was formed by water bath ultrasonication. The organic solvent was removed by reduced pressure evaporation to obtain lipid nanoparticles modified with functionalized amphiphilic compounds Ⅰa-TA / GFP LNP, Ⅱa-TA / GFP LNP, Ⅲa-TA / GFP LNP, and Ⅳa-TA / GFP LNP. The core particle size and polydispersity index (PDI) were measured using a Zetasizer 3000HS instrument particle size analyzer (Malvern Instruments, Malvern, UK).

[0127] In this example, the specific results are as follows: the average particle size of the obtained lipid nanoparticles is about 150.0 nm, and the PDI is less than 0.3.

[0128] 5. Preparation of PTX LNP modified with amphiphilic compounds

[0129] The prescribed amount of soybean lecithin, cholesterol, PTX and functionalized amphiphilic compounds (1a, 2a, 3a, 4a) were placed in an eggplant-shaped bottle and completely dissolved with chloroform / methanol. The organic solvent was removed by rotary evaporation at 120 rpm for 20 min at 37°C to form a uniform film, and then vacuum dried to remove the residual organic solvent. PBS was added to disperse the film, and liposomes modified with amphiphilic compounds loaded with PTX were obtained after 5-10 min of water bath ultrasound. The core particle size and polydispersity index (PDI) were measured using a Zetasizer 3000HSinstrument particle size analyzer (Malvern Instruments, Malvern, UK).

[0130] In this example, the specific results are as follows: the average particle size of the obtained lipid nanoparticles is about 150 nm, and the PDI is less than 0.3.

[0131] Example 8: Investigation of the anti-protein adsorption ability of TA / siRNA LNP modified with amphiphilic compounds

[0132] After each group of lipid nanoparticles was diluted with PBS, 1 mL of each was taken and mixed with an equal volume of bovine serum albumin (BSA) solution (3 mg / mL), placed in an air bath constant temperature oscillator for incubation for 2 h, and then centrifuged to obtain the supernatant. The ultraviolet absorbance value A at 280 nm was measured using a UV-visible spectrophotometer. The BSA concentration of each supernatant was calculated using the BSA standard curve, and the BSA adsorption value of each sample was calculated.

[0133] The results are as follows Figure 1 As shown, lipid nanoparticles modified with functionalized amphiphilic compounds have good ability to resist protein adsorption.

[0134] Example 9: Stability study of TA / siRNA LNP modified with amphiphilic compounds

[0135] The stability of lipid nanoparticles Ⅰa-LNP / siRNA, Ⅱa-LNP / siRNA, Ⅲa-LNP / siRNA, and Ⅳa-LNP / siRNA prepared according to Example 7 in phosphate buffer containing 10% serum and basal culture medium at 37°C was investigated using particle size change as an indicator. The results are as follows Figure 2 and 3 As shown, the particle size of the four preparations, Ⅰa-LNP / siRNA, Ⅱa-LNP / siRNA, Ⅲa-LNP / siRNA, and Ⅳa-LNP / siRNA, did not change significantly in the two media within 48 hours, and the stability was good, which was related to the anti-protein adsorption ability of the functionalized amphiphilic compounds.

[0136] Example 10: Investigation of encapsulation efficiency of lipid nanoparticles modified with amphiphilic compounds

[0137] The lipid nanoparticles Ⅰa-TA / Cy3-siRNA LNP, Ⅱa-TA / Cy3-siRNA LNP, Ⅲa-TACy3-siRNA LNP, and Ⅳa-TA / Cy3-siRNA LNP loaded with Cy3-siRNA were prepared according to the method in Example 7. Each group of lipid nanoparticles was dissolved in a lysis buffer (2 mM EDTA and 0.05% Triton X-100 in pH 7.8 Tris buffer), and after incubation at 65° C. for 10 min, the concentration of Cy3-siRNA released from the dissolved lipid nanoparticles was determined by measuring the fluorescence intensity, and then the encapsulation efficiency of Cy3-siRNA loaded into the LNP NPs was calculated.

[0138] The results are as follows Figure 4 As shown, the encapsulation efficiency of siRNA in lipid nanoparticles modified with functionalized amphiphilic compounds and unmodified groups was about 60%, namely 59.8%, 60.2%, 63.2%, 62.6% and 61.8% respectively.

[0139] Example 11: Evaluation of cytotoxicity of lipid nanoparticles modified with amphiphilic compounds

[0140] The MTT method was used to investigate the cytotoxicity of the lipid nanoparticles Ⅰa-LNP / siRNA, Ⅱa-LNP / siRNA, Ⅲa-LNP / siRNA, and Ⅳa-LNP / siRNA prepared in Example 7. 4T1 cells were cultured at 5×10 3 The cells were seeded at a density of 100 cells / well in a 96-well plate and the plate was placed at 37°C and 5% CO 2 Incubator for 24 hours. After 24 hours, the culture medium in the wells was aspirated and the cells were washed three times with PBS. 200nM siRNA doses of Ⅰa-LNP / siRNA, Ⅱa-LNP / siRNA, Ⅲa-LNP / siRNA, and Ⅳa-LNP / siRNA were added respectively and incubated for 48 hours under serum-free conditions. Blank culture medium was used as negative control (Control) and incubated in the same way. After the incubation was completed, 20μL of 5mg / mL MTT solution was added to each cell well and incubated at 37℃ for 4h. Then the liquid in the well was discarded, 150μL DMSO was added to each well to dissolve the formazan crystals, and the absorbance value of each cell well at 570nm was measured by microplate reader to calculate the cell survival rate.

[0141] Specific results such as Figure 5As shown in the figure, Ⅰa-LNP / siRNA, Ⅱa-LNP / siRNA, Ⅲa-LNP / siRNA, and Ⅳa-LNP / siRNA lipid nanoparticles did not show obvious cytotoxicity at the administered concentrations in the cell line, which indicates that each group of preparations has a high safety

[0142] Example 12: Investigation of cellular uptake of amphiphilic compound-modified lipid nanoparticles in 4T1 model cells

[0143] Flow cytometry analysis was used to quantitatively evaluate the cellular uptake of each group of lipid nanoparticles in the 4T1 cell line. 4T1 cells were cultured at a rate of 1×10 5 Cells / well were seeded in 6-well plates and incubated at 37°C and 5% CO 2 Cultured in an incubator for 24 hours. The original culture medium was discarded, and LNP / Cy3-siRNA, Ⅰa-LNP / Cy3-siRNA, Ⅱa-LNP / Cy3-siRNA, Ⅲa-LNP / Cy3-siRNA, and Ⅳa-LNP / Cy3-siRNA solutions diluted to the same Cy3-siRNA concentration (100nM) in serum-free RPMI-1640 medium were added and incubated for 2 hours. The blank culture medium was used as the negative control (Control), and PEI 25K / Cy3-siRNA (w / w=1) was used as the positive control. The original culture medium was discarded, the cells were washed 3 times with PBS, trypsinized and collected, and the cells were resuspended in PBS. The cells were then analyzed by flow cytometry. The mean fluorescence intensity (MFI) was used to represent the cellular uptake efficiency of lipid nanoparticles.

[0144] The results are as follows Figure 6 As shown in the figure, in 4T1 cells, the cellular uptake of lipid nanoparticles modified with amphiphilic compounds was higher than that of the control group, which was comparable to the cationic transfection reagent PEI 25K. The cellular uptake of the Ⅰa-LNP / Cy3-siRNA and Ⅱa-LNP / Cy3-siRNA groups was slightly higher than that of the Ⅲa-LNP / Cy3-siRNA and Ⅳa-LNP / Cy3-siRNA groups. This may be because the amphiphilic compounds modified on the Ⅰa-LNP / Cy3-siRNA and Ⅱa-LNP / Cy3-siRNA lipid nanoparticles have high tension on the cyclic disulfide, which further promotes the exchange of disulfide with the cell surface and enhances the cellular uptake.

[0145] Example 13: Study on the subcellular distribution of amphiphilic lipid nanoparticles in 4T1 model cells

[0146] 4T1 cells were cultured at a rate of 1×10 5 cells / well were inoculated in a confocal culture dish and placed in an incubator at 37°C and 5% CO2 The cells were cultured in an incubator for 24 h. The original culture medium was discarded, and Ⅰa-LNP / Cy3-siRNA, Ⅱa-LNP / Cy3-siRNA, Ⅲa-LNP / Cy3-siRNA, and Ⅳa-LNP / Cy3-siRNA solutions diluted to the same Cy3-siRNA concentration (100 nM) with serum-free RPMI-1640 medium were added and incubated for 1 h. The culture medium was discarded, and the cells were washed with PBS three times. Lyso-Tracker Green (100 nM) was added to stain the lysosomes for 30 min. After washing with PBS, Hoechst 33342 (10 μg / mL) was added to stain the nuclei for 10 min. After washing with PBS, the cells were fixed with 4% paraformaldehyde for 10 min. After washing with PBS, the cells were observed and analyzed under CLSM. Colocalization analysis was performed using Image J software, and Pearson Qs correlation coefficients (PCCs) were calculated to analyze the relationship between lipid nanoparticles and lysosomes.

[0147] The results are as follows Figure 7 As shown, the PCCs of amphiphilic lipid nanoparticles ranged from 0.1 to 0.40, which is well below the threshold of >0.5 required for correlation. In contrast, the PCC of unmodified lipid nanoparticles was approximately 0.86, indicating lysosomal capture. These results suggest that amphiphilic lipid nanoparticles can directly enter the cytoplasm via a non-endocytic pathway.

[0148] Example 14: Study on the cellular uptake mechanism of amphiphilic compound-modified lipid nanoparticles

[0149] 4T1 cells were cultured at a rate of 1×10 5 Cells / well were seeded in 6-well plates and incubated at 37°C and 5% CO 2 The cells were cultured in an incubator for 24 h. Before administration, the cells were pretreated with different inhibitors (4.8 mM DTNB, 10 μg / mL chlorpromazine, 50 nM wortmannin or 50 μM methyl-β-cyclodextrin) at 37 ° C for 30 min, and then the Ⅰa-LNP / Cy3-siRNA, Ⅱa-LNP / Cy3-siRNA, Ⅲa-LNP / Cy3-siRNA, and Ⅳa-LNP / Cy3-siRNA solutions diluted to the same Cy3-siRNA concentration (100 nM) in serum-free RPMI-1640 medium were added and incubated for 2 h. The original culture medium was discarded, the cells were washed 3 times with PBS, trypsinized and collected, and the cells were resuspended in PBS. The cells were then analyzed by flow cytometry. The relative fluorescence intensity (MFI) was used to represent the effect of each treatment agent on the cellular uptake of lipid nanoparticles.

[0150] The results are as follows Figure 8As shown in the figure, the clathrin-mediated endocytosis inhibitor chlorpromazine, the caveolin-mediated endocytosis inhibitor methyl-β-cyclodextrin and the macropinocytosis inhibitor wortmannin had no significant effect on the cellular uptake efficiency of amphiphilic compound-modified lipid nanoparticles. Blocking the external surface thiol on the cell surface with 5,5'-dithio-2-nitrobenzoic acid (DTNB) significantly inhibited the internalization of amphiphilic compound-modified lipid nanoparticles, but had no significant effect on the cellular uptake of unmodified lipid nanoparticles LNP. In general, the above results indicate that amphiphilic compound-modified liposomes enter cells through a non-endocytic pathway mediated by cell surface thiol.

[0151] Example 15: Transfection efficiency of amphiphilic lipid nanoparticles in 4T1-luc model cells

[0152] 4T1-luc cells were cultured at 5×10 4 The cells were seeded at a density of 100 cells / well in a 24-well plate and the plate was placed at 37°C and 5% CO 2 The cells were cultured in an incubator for 24 hours. After 24 hours, the culture medium in the wells was aspirated and the cells were washed three times with PBS. Ia-LNP / siLuc, IIa-LNP / siLuc, IIIa-LNP / siLuc, and IVa-LNP / siLuc were added respectively and incubated in serum-containing medium for 12 hours. The medium was then replaced with complete medium and incubated for another 36 hours. Blank medium was used as negative control. 3000 / siLuc was used as the positive control. After the incubation, the liquid in the well was aspirated and the luciferase activity was measured using a luciferase detection kit.

[0153] The results are as follows Fig. 9 As shown in Figure 2, when the drug concentration was 200 nM, the silencing efficiency of the four preparations of Ⅰa-LNP / siLuc, Ⅱa-LNP / siLuc, Ⅲa-LNP / siLuc, and Ⅳa-LNP / siLuc was about 65%, which was The transfection efficiency of 3000 / siLuc was comparable. Among them, the gene silencing efficiency of Ⅰa-LNP / siLuc and Ⅱa-LNP / siLuc groups was slightly higher than that of Ⅲa-LNP / siLuc and Ⅳa-LNP / siLuc groups.

Claims

1. An amphiphilic compound, characterized in that Having the structure of Formula I, in: P is Q is -S(O)2O - or -C(O)O - ; n is 1 or 3; R1 is at least one hydrogen, double bond or C 14 -C 18 Straight chain alkyl substituted C 14 -C 19 Straight chain alkoxy, C 14 -C 19 Straight chain alkylamino or C 14 -C 20 Straight chain acyl; R2 is 2. The amphiphilic compound according to claim 1, characterized in that Having a structure of Formula II or Formula III, Wherein, the definitions of Q, R1, R2, and n are the same as those in claim 1.

3. The amphiphilic compound according to claim 1, characterized in that In the structure, R1 is selected from any of the following substituents:

4. The amphiphilic compound according to claim 1, characterized in that Select any of the following compounds:

5. A pharmaceutical composition, characterized in that The invention is composed of the amphiphilic compound according to claim 1 and bioactive drug lipid nanoparticles.

6. The pharmaceutical composition according to claim 5, characterized in that The bioactive drug is a polypeptide drug, a protein drug, a DNA drug, an RNA drug, a CRISPR gene editing system or a small molecule drug.

7. The pharmaceutical composition according to claim 6, characterized in that The polypeptide drug is salmon calcitonin or hirudin; the protein drug is insulin, interleukin, lymphotoxin, interferon or tumor necrosis factor; the DNA drug is plasmid DNA; the RNA drug is mRNA, small interfering RNA, microRNA, antisense oligonucleotide, nucleic acid aptamer, ribozyme or guide RNA; the CRISPR gene editing system is CRISPR / Cas9, CRISPR / Cas12a, CRISPR / Cas13a or CRISPR / dCas9.

8. The pharmaceutical composition according to claim 5, characterized in that The lipids in the lipid nanoparticles contain phospholipids and steroidal lipids, wherein the phospholipids are one or more of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, hydrogenated lecithin, dipalmitoylphosphatidylserine, dioleoylphosphatidylserine, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, trimethyl-2,3-dioleyloxypropylammonium bromide, and trimethyl-2,3-dioleyloxypropylammonium chloride; and the steroidal lipids are one or more of cholesterol, ergosterol, fuccosterol, alginosterol, cholic acid, glycocholic acid, deoxycholic acid, and lithocholic acid.

9. The pharmaceutical composition according to claim 8, characterized in that The molar ratio of the amphiphilic compound, phospholipid and steroidal lipid is (0-30):(30-90):(5-50).

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