Fluorine-containing modified polysarcosylated lipid, and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a fluorine-containing modified polysarcosylized lipid, a preparation method and application thereof.The fluorine-containing modified polysarcosylized lipid has a general structure as shown in formula (I) or formula (II). The fluorine-containing modified polysarcosylized lipid obtained by chemically modifying polysarcosylized lipid with different fluorine-containing groups can replace PEGylated lipid to prepare a lipid nanoparticle (LNP), is used for loading nucleic acid and other drugs, and achieves the purpose of improving drug delivery performance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a fluorine-modified polysarcosinate lipid, its preparation method, and its application. Background Technology

[0002] mRNA has emerged as a novel class of biological macromolecules for the prevention and treatment of various diseases, including pathogen infections and genetic disorders. However, due to its susceptibility to degradation by nucleases in the body, and its high molecular weight and negative charge, mRNA struggles to penetrate cell membrane barriers. Therefore, delivery vectors are needed to enhance its stability and improve its internalization and transfection capabilities. Lipid nanoparticles (LNPs) represent the most advanced delivery vector technology currently available, successfully applied in COVID-19 mRNA vaccines (Comirnaty and Spikevax). This has accelerated the development of LNPs for other mRNA therapies, such as protein replacement therapy, tumor immunotherapy, and gene editing. LNPs typically consist of four lipid components: ionized cationic lipids, accessory lipids (usually phospholipids), cholesterol, and PEGylated lipids. PEGylated lipids play a crucial role in enhancing LNP stability and prolonging blood circulation. However, studies have shown that varying levels of anti-PEG antibodies exist in the human body due to environmental exposure, cosmetics, or drug use. This leads to problems such as allergic reactions and accelerated blood clearance effects after repeated administration of LNPs containing PEGylated lipids, limiting the application of traditional LNP technology for repeated administration of mRNA drugs. Therefore, the need to explore alternative technologies for PEGylated lipids to improve LNP delivery performance remains unmet.

[0003] To address current challenges, hydrophilic polymers such as poly(N-vinylamide), peptides, poly(oxazoline), or polyglycerol can be used to modify the formulation of lactones (LNPs) to improve drug delivery performance. Polysarcosine (pSar), a polypeptide derived from the endogenous amino acid N-methylglycine (sarcosine) monomer, possesses excellent stealth properties, high biocompatibility, and low immunogenicity. Previous studies have shown that LNPs prepared from sarcosine-modified lipids exhibit better safety after systemic administration compared to LNPs prepared from traditionally PEGylated lipids. However, current LNPs prepared from polysarcosine-modified lipids still suffer from insufficient in vivo mRNA delivery efficiency, severely limiting the applicability of mRNA therapy. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a fluorine-modified polysarcosinate lipid, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a fluorine-modified polysarcosinate lipid having a structure as shown in formula (I) or formula (II):

[0007]

[0008] The n or m is the number of repeating units (i.e., degree of polymerization) of polysarcosine; n or m is 5-150;

[0009] X1 is independently selected from hydrogen or alkyl; X2 is independently selected from alkyl or alkenyl; R1 is a fluorinated modification group; X3 is independently selected from carbonyl-substituted alkyl, carbonyl-substituted alkenyl, or hydroxy-substituted alkyl; R2 is independently selected from hydrogen, alkyl, or fluorinated substituted alkyl; R3 is independently selected from fluorinated substituted alkyl, or fluorinated and amide-substituted alkyl.

[0010] In a preferred embodiment of the fluorinated modified polysarcosinate lipid of the present invention, X1 is independently selected from any group selected from hydrogen or alkyl groups having 6-22 carbon atoms; and / or X2 is independently selected from alkyl or alkenyl groups having 6-22 carbon atoms; R1 is a fluorinated modification group; and / or X3 is independently selected from any group selected from carbonyl-substituted alkyl, carbonyl-substituted alkenyl, and hydroxyl-substituted alkyl groups having 6-22 carbon atoms; and / or R2 is independently selected from any group selected from hydrogen, alkyl groups having 1-6 carbon atoms, and fluorinated substituted alkyl groups having 1-6 carbon atoms; and / or R3 is independently selected from any group selected from fluorinated substituted alkyl groups having 1-6 carbon atoms, or fluorinated and amide-substituted alkyl groups having 1-6 carbon atoms.

[0011] As a further preferred embodiment of the fluorine-modified polysarcosinate lipid of the present invention, X1 is independently selected from any group consisting of hydrogen or alkyl groups having 8-20 carbon atoms; and / or

[0012] The X2 is independently selected from any one of alkyl or alkenyl groups having 8-20 carbon atoms; and / or

[0013] R1 is independently selected from carbonyl or hydroxyl-substituted fluorine-containing groups; and / or

[0014] X3 is independently selected from any one of the following groups: carbonyl-substituted alkyl group with 8-20 carbon atoms, carbonyl-substituted alkenyl group with 8-20 carbon atoms, and hydroxyl-substituted alkyl group with 8-20 carbon atoms.

[0015] As a further preferred embodiment of the fluorine-modified polysarcosinate lipid of the present invention, X1 is independently selected from H,

[0016] and / or

[0017] The X2 is independently selected from

[0018] and / or

[0019] R1 is independently selected from

[0020] and / or

[0021] The X3 is independently selected from

[0022] and / or

[0023] R2 is independently selected from H, and / or

[0024] The R3 is independently selected from

[0025] As a further preferred embodiment of the fluorine-modified polysarcosinate lipids described in this invention, R1 is independently selected from...

[0026] R2 is independently selected from H, and / or

[0027] The R3 is independently selected from

[0028] Secondly, the present invention provides a method for preparing fluorine-modified polysarcosinate lipids, comprising the following steps:

[0029] (1) The sarcosine N-carboxylate anhydride (NCA) monomer, 18-crown ether-6 catalyst and amine initiator were dissolved in dichloromethane solvent to carry out ring-opening polymerization reaction. After precipitation, purification and drying, polysarcosine lipids with terminal secondary amine groups were obtained.

[0030] (2) The polysarcosine lipids with terminal secondary amine groups are subjected to amidation reaction with fluorinated anhydride or to ring-opening reaction with fluorinated epoxy compound, and then fluorinated modified polysarcosine lipids as shown in formula (I) are obtained by precipitation, purification and drying.

[0031] In a preferred embodiment of the preparation method described in this invention, the amine initiator is... At least one of them; and / or

[0032] The fluoro anhydride is

[0033] At least one of them; and / or

[0034] The fluorinated epoxy compound is

[0035] At least one of them.

[0036] Thirdly, the present invention provides another method for preparing fluorine-modified polysarcosinate lipids, comprising the following steps:

[0037] (1) The N-carboxylate anhydride (NCA) monomer of sarcosine, 18-crown ether-6 catalyst and fluorinated substituted amine initiator were dissolved in dichloromethane solvent to carry out ring-opening polymerization reaction. After precipitation, purification and drying, fluorinated polysarcosine with secondary amine groups at the end was obtained.

[0038] (2) The fluorinated polysarcosine with a secondary amine group at the end is subjected to an amidation reaction with an alkyl or alkenyl substituted acid anhydride, or to a ring-opening reaction with an alkyl substituted epoxy compound, and then fluorinated modified polysarcosine lipids as shown in formula (II) are obtained by precipitation, purification and drying.

[0039] In a preferred embodiment of another preparation method described in this invention, the fluorinated substituted amine initiator is...

[0040]

[0041] At least one of them; and / or

[0042] The alkyl or alkenyl substituted acid anhydride is

[0043] At least one of them; and / or

[0044] The alkyl-substituted epoxy compound is

[0045] At least one of them.

[0046] Fourthly, the present invention provides lipid nanoparticles comprising the aforementioned fluorine-modified polysarcosinate lipids.

[0047] As a preferred embodiment of the lipid nanoparticles described in this invention, it further includes at least one of ionizable cationic lipids, sterols, and auxiliary lipids.

[0048] The ionizable cationic lipids carry a positive charge at low pH and can bind to RNA. They are nearly electroneutrally neutral at physiological pH, reducing in vivo toxicity. In addition, ionizable cationic lipids can promote endosome escape of LNPs and improve RNA transfection efficiency. Sterols stabilize the LNP structure and regulate membrane fluidity. Auxiliary lipids are structural lipids of LNPs, which stabilize and improve drug encapsulation efficiency or regulate targeting. Fluorine-modified polysarcosinate lipids are used to stabilize LNPs, prolong blood circulation time, reduce protein adsorption, improve endosome escape efficiency, and enhance RNA transfection capacity.

[0049] As a further preferred embodiment of the lipid nanoparticles of the present invention, the auxiliary lipid is at least one of 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), dioleoylphosphatidylcholine, and dipalmitoylphosphatidylcholine; the sterol is at least one of cholesterol, sitosterol, stigmasterol, and cholesterol derivatives.

[0050] As a further preferred embodiment of the lipid nanoparticles of the present invention, the molar ratio of the ionizable cationic lipid, cholesterol, auxiliary lipid, and fluorine-modified polysarcosinate lipid is (20-70):(20-50):(2-30):(0.1-20); more preferably, it is 50:38.5:10:1.5, or 40:47.5:10:2.5, or 30.7:23.7:44.7:0.9.

[0051] Fifthly, the present invention provides a drug-loaded lipid nanoparticle, comprising the lipid nanoparticle and a drug.

[0052] In a preferred embodiment of the drug-loaded lipid nanoparticles of the present invention, the drug is at least one of small molecule compounds, nucleic acid molecules, protein or polypeptide molecules, and gene editing complexes.

[0053] As a further preferred embodiment of the drug-loaded lipid nanoparticles of the present invention, the nucleic acid molecule is at least one of messenger RNA, transfer RNA, dsRNA, shRNA, DNA, plasmid DNA, siRNA, antisense oligonucleotide, circular RNA (circRNA), and miRNA; the gene editing complex is mRNA / sgRNA or Cas9 / sgRNA.

[0054] As a further preferred embodiment of the drug-loaded lipid nanoparticles of the present invention, the mass ratio of the ionizable cationic lipid to the nucleic acid molecule is ionizable cationic lipid: nucleic acid molecule = (2-50): 1.

[0055] Sixthly, the present invention provides a method for preparing the drug-loaded lipid nanoparticles, comprising the following steps:

[0056] (1) Dissolve the lipid component in an organic solution to obtain an organic phase;

[0057] (2) Dissolve the drug in a buffer solution to obtain an aqueous phase;

[0058] The volume of the aqueous phase : the volume of the organic phase = (1-6) : 1;

[0059] (3) The aqueous phase and the organic phase are rapidly mixed evenly and dialyzed to obtain the final product.

[0060] In a seventh aspect, the fluorine-modified polysarcosinate lipids, lipid nanoparticles, and drug-loaded lipid nanoparticles of the present invention are used in the preparation of delivery or transport of molecular drugs or nucleic acid vaccines.

[0061] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0062] This invention modifies polysarcosinate lipids using a fluorination strategy. A fluorinated group is chemically modified at one end of the polysarcosinate lipid to obtain fluorinated modified polysarcosinate lipids with the general chemical structure shown in formula (I) or (II). Because the fluorinated group exhibits a significant phase separation tendency in both polar and nonpolar environments, and possesses hydrophobic and lipophobic properties, its fluorine effect can efficiently promote intracellular escape of the carrier and reduce the adsorption of immune-related proteins on the carrier surface, thereby prolonging the cycle time. Compared to unmodified polysarcosinate lipids, lipid nanoparticles (LNPs) prepared using fluorinated modified polysarcosinate lipids can significantly improve the in vivo transfection performance of mRNA, can replace PEGylated lipids, avoid in vivo anti-PEG antibody problems, and achieve the goal of improving the efficacy of mRNA therapy and expanding its applications. Attached Figure Description

[0063] Figure 1 R18-pSar 70 In deuterated chloroform (CDCl3) 1 H NMR spectrum.

[0064] Figure 2 For DR14-pSar 70 In deuterated dimethyl sulfoxide (DMSO-d6) 1 H NMR spectrum.

[0065] Figure 3 Fluorine-modified polysarcosinate lipid R18-pSar 70 -A11 1 H NMR spectrum.

[0066] Figure 4 Fluorine-modified polysarcosinate lipid R18-pSar 70 Mass spectrum of -A11.

[0067] Figure 5 DR14-pSar, a fluorine-modified polysarcosinate lipid 70 -A1 1 H NMR spectrum.

[0068] Figure 6 DR14-pSar, a fluorine-modified polysarcosinate lipid 70 -A1 mass spectrum.

[0069] Figure 7 DR14-pSar, a fluorine-modified polysarcosinate lipid 70 -A7 1 H NMR spectrum.

[0070] Figure 8 DR14-pSar, a fluorine-modified polysarcosinate lipid 70 -A7 mass spectrum.

[0071] Figure 9 For the fluorine-modified polysarcosinate lipid DR14-pSar70-BF5 in deuterated dimethyl sulfoxide 1 H NMR spectrum.

[0072] Figure 10 This is the mass spectrum of fluorine-modified polysarcosinate lipid DR14-pSar70-BF5 in deuterated dimethyl sulfoxide.

[0073] Figure 11 DR14-pSar, a fluorine-modified polysarcosinate lipid 70 -BF12 in deuterated dimethyl sulfoxide 1 H NMR spectrum.

[0074] Figure 12 DR14-pSar, a fluorine-modified polysarcosinate lipid 70 Mass spectrum of -BF12 in deuterated dimethyl sulfoxide.

[0075] Figure 13 Fluorinated polysarcosine CF6-pSar 70 of 1 H NMR spectrum.

[0076] Figure 14 For fluorine-modified polysarcosinate lipid CF6-pSar70-C18 1 H NMR spectrum.

[0077] Figure 15 This is the mass spectrum of fluorine-modified polysarcosinate lipid CF6-pSar70-C18.

[0078] Figure 16 For fluorine-modified polysarcosinate lipid CF6-pSar70-CE18 1 H NMR spectrum.

[0079] Figure 17 This is the mass spectrum of fluorine-modified polysarcosinate lipid CF6-pSar70-CE18.

[0080] Figure 18 Particle size (A) and PDI (B) of Fluc-mRNA LNP prepared based on fluorine-modified polysarlylated lipids (n=3).

[0081] Figure 19 Particle size (A), PDI (B), and average mRNA encapsulation efficiency (C) of Fluc-mRNA LNPs based on fluorine-modified polysarlylated lipids obtained by microfluidic preparation method (n=3).

[0082] Figure 20 The results show the in vitro cell transfection of Fluc-mRNA LNP. Detailed Implementation

[0083] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0084] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0085] Example 1: Synthesis of sarcosine N-carboxylic anhydride (NCA) monomer (Sar-NCA)

[0086] The synthesis method is based on the reference [J Polym Sci. 2024; 62:4908-4920], as follows:

[0087] 3.0 g of Boc-sarcosine (15.9 mmol, 1.0 eq) was dissolved in 60 mL of acetonitrile. 11.1 mL of propylene oxide (158.7 mmol, 10.0 eq) and the sarcosine solution were added sequentially to a 250 mL flask, which was then placed in an ice-water bath. 2.36 g of triphosgene (8.1 mmol, 0.5 eq) was then added, keeping the flask partially open. After reacting for 1.5 h, 10 mL of 4 °C cold water was added to quench excess triphosgene. The organic phase was then extracted with ethyl acetate, washed with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. After removing the solvent by rotary evaporation under vacuum, the collected product was further purified by crystallization at low temperature using a hexane / dichloromethane mixture (1:1, v / v). The final product was a white crystal (yield approximately 65%). The Sar-NCA monomer was stored at -20 °C for use.

[0088] Example 2: Synthesis of polysarcosinate-coated lipids

[0089] The synthesis method is based on the reference [J Polym Sci. 2024; 62:4908-4920], as follows:

[0090] The Sar-NCA monomer obtained in Example 1 was dissolved in dichloromethane (DCM) solvent. The 18-crown ether-6 (18-C-6) catalyst and different amine initiators were dissolved in DCM solvent and then added sequentially to the monomer solution. After ring-opening polymerization for 2 hours, the product was purified by ether precipitation and then dried under vacuum to obtain a polysarcinized lipid product with a terminal secondary amine group. The obtained polysarcinized lipid was labeled as "amine initiator-pSar..." n The naming convention is "n", where n represents the number of polystyrene repeating units (i.e., degree of polymerization).

[0091] The synthesis route is shown below:

[0092]

[0093] The structure of the amine initiator is shown below:

[0094]

[0095] By adjusting the molar ratio of Sar-NCA monomer and amine initiator, polysarcosinate lipids with polysarcosinate repeating unit number (degree of polymerization n) ranging from 35 to 150 were obtained. Representative products and their structures are shown in Table 1.

[0096] Table 1 Product Names and Structural Formulas

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] Using nuclear magnetic resonance hydrogen spectroscopy (NMR) 1 The structure of polysarcosinate-treated lipids was characterized by 1H NMR, and the molecular weight and dispersibility index (Mn) of polysarcosinate-treated lipids were characterized by gel permeation chromatography (SEC). w / M n ).

[0103] Representative polysarcosinate lipid R18-pSar 70 and DR14-pSar 70 of 1 The H NMR spectra are as follows: Figure 1 and Figure 2 As shown.

[0104] The molecular weight and dispersibility index of the above-mentioned polysarcosinate lipids were measured by gel permeation chromatography (SEC), and the data are shown in Table 2. The molecular weight and dispersibility index were determined by hydrogen nuclear magnetic resonance spectroscopy (1H NMR). 1 The molecular weight of polysarcosinate lipids was calculated from peaks a and c in 1H NMR and compared with the data.

[0105] Table 2. Molecular weight and polydispersity data of different polysarcosinate lipids

[0106] Sample Name <![CDATA[[M]0 / [I]0]]> <![CDATA[M n,SEC (kg / mol)]]> <![CDATA[M n,NMR (kg / mol)]]> <![CDATA[M w / M n ]]> <![CDATA[R12-pSar 50 ]]> 50 14.0 4.1 1.10 <![CDATA[R14-pSar 50 ]]> 50 14.1 3.6 1.09 <![CDATA[R16-pSar 50 ]]> 50 13.0 3.6 1.12 <![CDATA[R18-pSar 50 ]]> 50 13.2 4.2 1.12 <![CDATA[R18-1-pSar 50 ]]> 50 12.8 4.2 1.14 <![CDATA[R18-pSar 35 ]]> 35 12.5 2.8 1.06 <![CDATA[R18-pSar 70 ]]> 70 22.8 5.5 1.07 <![CDATA[R18-pSar 100 ]]> 100 25.9 7.9 1.09 <![CDATA[R18-pSar 150 ]]> 150 40.8 11.3 1.09 <![CDATA[DR8-pSar 35 ]]> 35 11.7 2.4 1.04 <![CDATA[DR8-pSar 50 ]]> 50 14.4 3.7 1.08 <![CDATA[DR8-pSar 70 ]]> 70 22.0 5.2 1.08 <![CDATA[DR8-pSar 100 ]]> 100 26.1 6.4 1.14 <![CDATA[DR10-pSar 35 ]]> 35 12.9 2.6 1.08 <![CDATA[DR10-pSar 50 ]]> 50 11.7 3.4 1.16 <![CDATA[DR10-pSar 70 ]]> 70 20.0 5.1 1.08 <![CDATA[DR10-pSar 100 ]]> 100 22.3 7.4 1.08 <![CDATA[DR12-pSar 35 ]]> 35 10.2 2.4 1.07 <![CDATA[DR12-pSar 50 ]]> 50 15.9 4.0 1.11 <![CDATA[DR12-pSar 70 ]]> 70 16.9 4.9 1.07 <![CDATA[DR12-pSar 100 ]]> 100 21.2 7.9 1.11 <![CDATA[DR14-pSar 35 ]]> 35 12.4 2.9 1.08 <![CDATA[DR14-pSar 50 ]]> 50 14.3 3.9 1.14 <![CDATA[DR14-pSar 70 ]]> 70 21.6 4.9 1.09 <![CDATA[DR14-pSar 100 ]]> 100 24.8 7.8 1.13 <![CDATA[DR16-pSar 35 ]]> 35 11.9 2.9 1.09 <![CDATA[DR16-pSar 50 ]]> 50 12.8 3.8 1.10 <![CDATA[DR16-pSar 70 ]]> 70 17.8 5.3 1.09 <![CDATA[DR16-pSar 100 ]]> 100 25.2 7.0 1.10 <![CDATA[DR18-pSar 35 ]]> 35 11.9 3.0 1.18 <![CDATA[DR18-pSar 50 ]]> 50 14.5 4.1 1.14 <![CDATA[DR18-pSar 70 ]]> 70 17.7 5.1 1.08 <![CDATA[DR18-pSar 100 ]]> 100 24.7 7.8 1.15

[0107] Note: [M]0 / [I]0 represents the molar ratio of monomer to initiator; M n,SEC (kg / mol) represents the number-average molecular weight as measured by SEC;

[0108] M n,NMR (kg / mol) represents the molecular weight calculated from the 1H NMR spectrum; M w / M n The dispersion index represents the polymer.

[0109] Example 3: Synthesis of Fluorine-Modified Polysarcosinate Lipids

[0110] Fluoroanhydrides can be obtained commercially or prepared from the corresponding fluorinated carboxylic acids through the following steps:

[0111] 50 mg of fluorinated carboxylic acid (1.0 eq) was dissolved in 1 mL of tetrahydrofuran, and 0.5 eq of dicyclohexylcarbodiimide (DCC) was added. After reacting at room temperature for 2 h, the precipitate was removed by filtration, and the solvent was removed by rotary evaporation under vacuum to obtain fluorinated anhydride.

[0112] The polysarcosine-containing lipids with terminal secondary amine groups obtained in Example 2 were subjected to amidation reactions with fluoro anhydrides, as detailed below:

[0113] 100 mg of polysarcosinate-coated lipids (1.0 eq) was dissolved in 2 mL of N,N-dimethylformamide, followed by the sequential addition of N,N-diisopropylethylamine (10.0 eq) and afluorinated anhydride (5.0 eq). The reaction was carried out at room temperature for 12 h, and the product was purified by precipitation with ice-cold diethyl ether. This process was repeated three times. The collected product was then dialyzed against pure water for 24 h for further purification. The solvent was removed by freeze-drying.

[0114] The resulting fluorinated modified polysarcosinate lipids were obtained using "amine initiator-pSar" n The name is in the form of "-fluorinated anhydride", where n represents the number of repeating units of polysarcosine (i.e., degree of polymerization).

[0115] Using the same synthetic method, polysarcosinate lipids with terminal secondary amine groups were chemically modified with acetic anhydride (control 1) or butyric anhydride (control 2) as controls for subsequent experiments.

[0116] The specific synthesis route is shown below:

[0117]

[0118] The molecular structure of fluoroanhydride is shown below:

[0119]

[0120] The corresponding fluorine-containing group R1 is:

[0121]

[0122] Representative polysarcosinate lipid R18-pSar 70 and DR14-pSar 70 The names and structures of the products modified with terminal fluorine groups are shown in Table 3:

[0123] Table 3 Product Names and Structures

[0124]

[0125]

[0126]

[0127]

[0128] Using nuclear magnetic resonance hydrogen spectroscopy (NMR) 1 Characterized by ¹H NMR and mass spectrometry, the representative fluorine-modified polysarcosinate-containing lipid R18-pSar 70 -A11、DR14-pSar 70 -A1 and DR14-pSar 70 -A7 structure, such as Figure 3-8 As shown.

[0129] Based on the above nuclear magnetic resonance hydrogen spectrum ( 1 Analysis by 1H NMR or mass spectrometry results showed that fluorine-modified polysarcosinate lipids were successfully prepared.

[0130] Example 4: Synthesis of Fluorine-Modified Polysarlysinized Lipids

[0131] The polysarcosine-containing lipids with terminal secondary amine groups obtained in Example 2 were subjected to ring-opening reactions with fluorinated epoxy compounds, as detailed below:

[0132] 100 mg of polysarcosinate-coated lipids (1.0 eq) were dissolved in 2 mL of methanol, and a fluorinated epoxy compound (7.0 eq) was added. The mixture was reacted at 70 °C for 8 h. After removing the organic solvent by vacuum rotary evaporation, the collected product was dialyzed against pure water for 24 h for further purification. The product was obtained by freeze-drying to remove the solvent.

[0133] The resulting fluorinated modified polysarcosinate lipids were obtained using "amine initiator-pSar" n The name is in the form of "fluorinated epoxy compound", where n represents the number of polysarcosine repeating units (i.e., degree of polymerization).

[0134] The synthesis route is shown below:

[0135]

[0136] The molecular structure of fluorinated epoxy compounds is shown below:

[0137]

[0138] The corresponding fluorine-containing group R1 is

[0139]

[0140] Representative R18-pSar 70 and DR14-pSar 70 The names and structures of the terminal fluorine-modified products are shown in Table 4:

[0141] Table 4 Product Names and Structures

[0142]

[0143]

[0144]

[0145] Using nuclear magnetic resonance hydrogen spectroscopy (NMR) 1 Characterized by ¹H NMR and mass spectrometry, the representative fluorine-modified polysarcosinate-containing lipid DR14-pSar 70 -BF5 and DR14-pSar 70 The structure of -BF12, data as follows Figure 9-12 As shown.

[0146] Based on the above nuclear magnetic resonance hydrogen spectrum ( 1 Analysis of H NMR and mass spectrometry results showed that fluorine-modified polysarcosinate lipids were successfully prepared.

[0147] Example 5: Synthesis of Fluorinated Polysarcosine

[0148] The sarcosine NCA monomer (Sar-NCA) prepared in Example 1 was dissolved in dichloromethane (DCM) solution. Then, 18-crown ether-6 (18-C-6) catalyst and different fluorinated substituted amine initiators were dissolved in DCM solution and added sequentially to the monomer solution. After reacting at room temperature for 1 hour, the product was purified by precipitation in diethyl ether and then dried under vacuum to obtain fluorinated polysarcosine with a terminal secondary amine group. The obtained fluorinated polysarcosine was labeled as "fluorinated amine initiator-pSar". n The naming convention is "n", where n represents the number of polystyrene repeating units (i.e., degree of polymerization).

[0149] The specific synthesis route is shown below:

[0150]

[0151] The structure of the fluorinated amine initiator is as follows:

[0152]

[0153] The R2 group is H,

[0154] The R3 group is

[0155] Representative fluorinated polysarcosine CF2-pSar 70 CF3-pSar 70 and CF6-pSar 70 The structure is shown in Table 5:

[0156] Table 5 Product Names and Structures

[0157]

[0158] Using nuclear magnetic resonance hydrogen spectroscopy (NMR) 1 Representative fluorinated polysarcosine CF6-pSar was characterized by ¹H NMR. 70 Structure, data such as Figure 13 As shown above. The above 1H NMR spectrum (…) 1 Analysis of the H NMR results showed that fluorinated polysarcosine was successfully prepared.

[0159] Example 6: Synthesis of Fluorine-Modified Polysarlysinized Lipids

[0160] The fluorinated polysarcosine with a secondary amine group at the end prepared in Example 5 was subjected to an amidation reaction with an alkyl or alkenyl-substituted acid anhydride, as detailed below:

[0161] 100 mg of fluorinated polysarcosine (1.0 eq) was dissolved in 2 mL of N,N-dimethylformamide, followed by the sequential addition of N,N-diisopropylethylamine (10.0 eq) and alkyl or alkenyl substituted anhydrides (5.0 eq). The reaction was carried out at room temperature for 12 h, and the product was purified by precipitation with ice-cold diethyl ether. This process was repeated three times. The collected product was then dialyzed against pure water for 24 h for further purification. The solvent was removed by freeze-drying to obtain the final product.

[0162] The resulting fluorinated modified polysarcosinate lipids were prepared using "fluorinated amine initiator-pSar" m The name is in the form of "-anhydride", where m represents the number of repeating units of polysarcosine (degree of polymerization).

[0163] The specific synthesis route is shown below:

[0164]

[0165] The structures of alkyl or alkenyl substituted acid anhydrides are as follows:

[0166]

[0167] The X3 group is:

[0168]

[0169] The structures of representative fluorinated modified polysarcosinate lipids are shown in Table 6:

[0170] Table 6 Product Names and Structures

[0171]

[0172] Using nuclear magnetic resonance hydrogen spectroscopy (NMR) 1 The structure of CF6-pSar70-C18 was characterized by 1H NMR and mass spectrometry, and the data are as follows: Figure 14 and 15 As shown above. The above 1H NMR spectrum (…) 1 Analysis of H NMR and mass spectrometry results showed that fluorine-modified polysarcosinate lipids were successfully prepared.

[0173] Example 7: Synthesis of Fluorine-Modified Polysarlysinized Lipids

[0174] The fluorinated polysarcosine with a secondary amine group at the end, obtained in Example 5, was subjected to a ring-opening reaction with an alkyl-substituted epoxy compound, as detailed below:

[0175] 100 mg of fluorinated polysarcosine (1.0 eq) with a terminal secondary amine group was dissolved in 2 mL of methanol, and an alkyl-substituted epoxy compound (7.0 eq) was added. The reaction was carried out at 70 °C for 8 h. After removing the organic solvent by vacuum rotary evaporation, the collected product was dialyzed against pure water for 24 h for further purification. The product was obtained by freeze-drying to remove the solvent.

[0176] The resulting fluorinated modified polysarcosinate lipids were prepared using "fluorinated amine initiator-pSar" m The name is in the form of "-epoxide compound", where m represents the number of polysarcosine repeating units (i.e., degree of polymerization).

[0177] The specific synthesis route is shown below:

[0178]

[0179] The structures of alkyl-substituted epoxy compounds are as follows:

[0180]

[0181] The X3 group is:

[0182]

[0183] The structures of representative fluorinated modified polysarcosinate lipids are shown in Table 7:

[0184] Table 7 Product Names and Structures

[0185]

[0186] Using nuclear magnetic resonance hydrogen spectroscopy (NMR) 1 CF6-pSar characterization by ¹H NMR and mass spectrometry 70 The structure and data of -CE18 are as follows: Figure 16 and 17 As shown above. The above 1H NMR spectrum (…) 1Analysis of the 1H NMR results showed that fluorine-modified polysarcosinate lipids were successfully prepared.

[0187] Example 8: Preparation and Characterization of Lipid Nanoparticles (LNPs)

[0188] Taking the preparation method of drug-loaded lipid nanoparticles (Fluc-mRNA LNP) loaded with mRNA encoding luciferase (Fluc-mRNA) as an example:

[0189] Fluorinated polysarcosinate-based LNPs were prepared using a manual rapid mixing method: The organic phase was prepared by dissolving four lipid components (ionizable cationic lipid A4I18R2C18-2, see patent application 202210101998.9), cholesterol, DSPC, and fluorinated polysarcosinate-based lipids (using unmodified polysarcosinate-based lipids and commercial DMG-PEG2000 as controls) in ethanol at a molar ratio of 50:38.5:10:1.5. The aqueous phase was prepared by diluting the Fluorinated mRNA in 10 mM citrate buffer (pH=4). The organic and aqueous phases were then manually and rapidly mixed using a pipette at a volume ratio of 1:3 (the mass ratio of ionizable cationic lipids to mRNA was 11:1) to obtain the corresponding LNPs. The obtained LNPs were incubated at room temperature for 15 min and then used for in vitro cell transfection experiments. For in vivo experiments, the prepared LNPs need to be dialyzed in a PBS environment for 2 hours using a 1000MWCO dialysis bag to remove residual ethanol before use.

[0190] Using a similar method, Fluc-mRNA LNPs were also prepared with an ionizable cationic lipid (A4I18R2C18-2), cholesterol, DSPC, and fluorinated polysarcosinate lipids in a molar ratio of 40:47.5:10:2.5 and an ionizable cationic lipid to mRNA mass ratio of 11:1; and Fluc-mRNA LNPs were also prepared with an ionizable cationic lipid (A4I18R2C18-2), cholesterol, DOTAP, and fluorinated polysarcosinate lipids in a molar ratio of 30.7:23.7:44.7:0.9 and an ionizable cationic lipid to mRNA mass ratio of 11:1.

[0191] LNP was diluted to a concentration of 10 μg / mL mRNA and measured using a dynamic light scattering instrument (Malvern Zetasizer Nano ZS) to characterize its physicochemical parameters, including particle size and polydispersity index (PDI).

[0192] Taking the above-mentioned ionizable cationic lipid A4I18R2C18-2, cholesterol, DSPC, and fluorine-modified polysarcosinate lipids (with unmodified polysarcosinate lipids and commercial DMG-PEG2000 as controls) in a molar ratio of 50:38.5:10:1.5 as an example, the physicochemical characterization results of LNP are as follows: Figure 18 As shown above, the results indicate the successful preparation of Fluc-mRNA LNPs based on fluorinated polysarcosinate-modified lipids.

[0193] Example 9: Preparation and Characterization of Lipid Nanoparticles (LNPs)

[0194] Taking the preparation method of drug-loaded lipid nanoparticles (Fluc-mRNA LNP) loaded with Fluc-mRNA as an example:

[0195] Ionizable cationic lipids, cholesterol, DSPC, and fluorine-modified polysarcosinate lipids (using unmodified polysarcosinate lipids, DR14-pSar) were used. 70 -AA、DR14-pSar 70 (Using BA and commercial DMG-PEG2000 as references) were dissolved in ethanol to obtain the organic phase; the corresponding mass ratio of mRNA drug was dissolved in citrate buffer (25 mM, pH = 4.0) to obtain the aqueous phase, with the volume ratio of aqueous phase to organic phase being 3:1; the aqueous and organic phases were rapidly and uniformly mixed using a microfluidic device at a flow rate of 3 mL / min (adjustable from 1 to 10 mL / min), allowed to stand for 10 minutes, and then placed into a 1000 Da dialysis bag. Dialysis was performed at 4℃ in 1×PBS solution for 2 hours to obtain drug-loaded lipid nanoparticles that can be directly injected. The mass ratio of ionizable cationic lipid to nucleic acid drug was 11:1.

[0196] LNPs were diluted to a concentration of 10 μg / mL mRNA and their physicochemical parameters, including particle size and polydispersity index (PDI), were characterized by dynamic light scattering (DLS) measurements using a Malvern Zetasizer Nano ZS. The encapsulation efficiency of mRNA in LNPs was determined using the RiboGreen RNA assay. Results are as follows: Figure 19 As shown.

[0197] The above results indicate that the Fluc-mRNALNP based on fluorinated polysarcosinate lipids prepared by microfluidic methods has smaller and more uniform particle size, as well as higher mRNA drug encapsulation efficiency.

[0198] Example 10: In vitro cell transfection assay

[0199] HepG2 cells were loaded at 5 × 10 3Cells were seeded at a density per well in 96-well plates and incubated for 24 h. The culture medium was then removed, and fresh Opti-MEM containing LNP (0.1 μg / well of Flux-mRNA) was added. After 48 h of incubation, cells were treated with reporter gene cell lysis buffer (40 μL) and firefly luciferase substrate (Beyotime, 50 μL). Relative fluorescence units (RLU) were measured using a microplate reader, and protein levels were determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific). Luciferase activity was expressed as RLU / mg protein expression.

[0200] In vitro cell transfection results as follows Figure 20 As shown in the figure. The results indicate that the in vitro transfection ability of LNP does not differ significantly with the increase of carbon chain length in different amine initiators, but the degree of polymerization of polysarcosine has a certain impact on the in vitro transfection performance. Among them, polysarcosine-modified lipids with better LNP transfection ability include DR12-pSar 50 DR14-pSar 50 DR14-pSar 70 DR16-pSar 50 .

[0201] Example 11: In vivo animal transfection experiment

[0202] Different Fluc mRNA LNPs were administered intravenously to each C57BL / 6 mouse (6-8 weeks old) at a dose of 0.125 mg / kg mRNA. Six hours after administration, each mouse was intraperitoneally injected with a luciferase substrate (D-Luciferin potassium salt, Promega). After a 6-minute wait, the mice were anesthetized and placed in a small animal imaging system (IVIS, PerkinElmer) for bioluminescence imaging. If necessary, the mice were sacrificed, and major organs were collected for in vitro bioluminescence imaging. The in vivo delivery efficiency of LNPs was determined by the luminescence intensity of firefly luciferase in the organs, and the results are shown in Tables 8, 9, and 10. The LNPs were named according to the polymer lipids used.

[0203] Table 8. In vivo expression of mRNA-loaded nanoparticles prepared manually and administered intravenously.

[0204]

[0205]

[0206] Table 8 shows that, compared to unmodified polysarcosinate lipids (DR14-pSar), 70LNPs prepared using fluorine-modified polysarcosinate lipids deliver mRNA to the liver with higher expression efficiency, and the delivery performance of some materials is comparable to or better than that of commercial DMG-PEG 2000.

[0207] Table 9. In vivo expression of mRNA-loaded nanoparticles prepared by microfluidic method after intravenous injection.

[0208] LNP name Liver fluorescence intensity DMG-PEG 2000 2.89E+09 <![CDATA[DR14-pSar 70 ]]> 1.72E+09 <![CDATA[DR14-pSar 70 -AA]]> 2.52E+09 <![CDATA[DR14-pSar 70 -NO]]> 1.37E+09 <![CDATA[DR14-pSar 70 -A1]]> 6.77E+09 <![CDATA[DR14-pSar 70 -A7]]> 1.12E+10 <![CDATA[DR14-pSar 70 -A11]]> 5.72E+09

[0209] Table 9 shows that, compared to unmodified polysarcosinate-based lipids DR14-pSar 70 And DR14-pSar, a polysarcosine-modified lipid DR14-pSar, modified with acetic anhydride or butyric anhydride. 70 -AA and DR14-pSar 70 -BA, using fluorine-modified polysarcosinate lipids prepared via microfluidic methods, resulted in significantly higher mRNA expression in the liver. Furthermore, under the same conditions, the in vivo delivery performance of the microfluidic-prepared mRNA-loaded LNPs was significantly superior to that of the manually prepared LNPs shown in Table 8.

[0210] Table 10. In vivo expression of mRNA-loaded nanoparticles prepared by microfluidic method via intravenous injection.

[0211] LNP name Lung fluorescence intensity <![CDATA[DR14-pSar 70 -A7]]> 5.15E+07

[0212] Table 10 shows that Fluc mRNA LNPs prepared using a formulation with a molar ratio of ionizable cationic lipid (A4I18R2C18-2), cholesterol, DOTAP, and fluorinated modified polysarcosinate lipid of 30.7:23.7:44.7:0.9, and a mass ratio of ionizable cationic lipid to mRNA of 11:1, can achieve lung-targeted mRNA delivery after intravenous administration.

[0213] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A fluorine-containing modified polysarcosylized lipid, characterized in that, having a structure as shown in formula (I) or formula (II): （I）； (I); and (II). n or m is 5-150; X1 is independently selected from any one of hydrogen or alkyl having carbon number of 6-22; X2 is independently selected from alkyl or alkenyl having carbon number of 6-22; X3 is independently selected from any one of carbonyl-substituted alkyl having carbon number of 6-22, carbonyl-substituted alkenyl having carbon number of 6-22, hydroxyl-substituted alkyl having carbon number of 6-22; R2 is independently selected from any one of hydrogen, alkyl having carbon number of 1-6, fluorine-substituted alkyl having carbon number of 1-6; R3 is independently selected from any one of fluorine-substituted alkyl having carbon number of 1-6, or fluorine- and amide-substituted alkyl having carbon number of 1-6; said R1is independently selected from , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

2. The fluorine-containing modified poly sarcosinylated lipid according to claim 1, characterized in that, X1 is independently selected from any one of hydrogen or alkyl having carbon number of 8-20; and / or X2 is independently selected from any one of alkyl or alkenyl having carbon number of 8-20; and / or X3 is independently selected from any one of carbonyl-substituted alkyl having carbon number of 8-20, carbonyl-substituted alkenyl having carbon number of 8-20, hydroxyl-substituted alkyl having carbon number of 8-20.

3. The fluorine-containing modified poly sarcosinated lipid of claim 2, wherein, said X1is independently selected from H, , , , , , or ; and / or X2is independently selected from , , , , , , , or ; and / or X3is independently selected from , , , , , , , , or ; and / or said R2is independently selected from H, , or ; and / or said R3is independently selected from , , , , , , , .

4. The fluorine-containing modified poly sarcosinated lipid of claim 3, wherein, said R1is independently selected from , , , , , , , , , , , , or ; said R2is independently selected from H, or ; and / or R3is independently selected from the group consisting of , , , or .

5. A method of preparing the fluorine-containing modified polysarcosylized lipid of claim 1, wherein, comprising the following steps: (1) dissolving sarcosine N-carboxyanhydride monomer, 18-crown-6 catalyst and amine initiator in dichloromethane solvent to perform ring-opening polymerization reaction, and then performing precipitation, purification and drying to obtain poly-sarcosyl lipid containing secondary amine group at the end; (2) performing amide reaction of the poly-sarcosyl lipid containing secondary amine group at the end with fluorine-containing acid anhydride, or ring-opening reaction with fluorine-containing epoxy compound, and then performing precipitation, purification and drying to obtain fluorine-modified poly-sarcosyl lipid having a structure as shown in formula (I).

6. The production method according to claim 5, characterized by, The amine initiator is at least one of and / or the fluorine-containing acid anhydride is at least one of: and / or the fluorine-containing epoxy compound is at least one of R1, R2, R3, R4 7. A method of preparing the fluorine-containing modified polysarcosylized lipid of claim 1, wherein, comprising the following steps: (1) dissolving sarcosine N-carboxyanhydride monomer, 18-crown-6 catalyst and fluorine-substituted amine initiator in dichloromethane solvent to perform ring-opening polymerization reaction, and then performing precipitation, purification and drying to obtain fluorinated poly-sarcosine containing secondary amine group at the end; (2) performing amide reaction of the fluorinated poly-sarcosine containing secondary amine group at the end with alkyl or alkenyl substituted acid anhydride, or ring-opening reaction with alkyl substituted epoxy compound, and then performing precipitation, purification and drying to obtain fluorine-modified poly-sarcosyl lipid having a structure as shown in formula (II).

8. The production method according to claim 7, characterized by, The fluorine-containing substituted amine initiator is at least one of and / or The alkyl or alkenyl substituted anhydride is at least one of and / or The alkyl-substituted epoxy compound is at least one of alkyl-substituted epoxy compounds.

9. A lipid nanoparticle characterized in that, The fluorine-modified poly-sarcosyl lipid as claimed in any one of claims 1-4.

10. The lipid nanoparticle of claim 9, wherein, Further comprising at least one of ionizable cationic lipid, solid sterol, auxiliary lipid.

11. The lipid nanoparticle of claim 10, wherein, The auxiliary lipid is at least one of 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, (2,3-dioleoyl-propyl)-trimethylammonium chloride, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine; the solid sterol is at least one of cholesterol, sitosterol, stigmasterol, cholesterol derivative.

12. The lipid nanoparticle of claim 10, wherein, The molar ratio of the ionizable cationic lipid, the solid sterol, the auxiliary lipid, and the fluorine-modified polysarcosylated lipid is (20-70):(20-50):(2-30):(0.1-20).

13. Drug-loaded lipid nanoparticles, characterized in that, The drug comprises the lipid nanoparticle of claim 9.

14. Drug loaded lipid nanoparticles, characterized in that, The drug comprises the lipid nanoparticle of any one of claims 10-12.

15. The drug loaded lipid nanoparticle of claim 13, wherein, The drug is at least one of a small molecule compound, a nucleic acid molecule, a protein or polypeptide molecule, or a gene editing complex.

16. The drug loaded lipid nanoparticle of claim 14, wherein, The drug is at least one of a small molecule compound, a nucleic acid molecule, a protein or polypeptide molecule, or a gene editing complex.

17. The drug loaded lipid nanoparticle of claim 15 or 16, wherein, The nucleic acid molecule is at least one of a messenger RNA, a transfer RNA, a dsRNA, a shRNA, a DNA, a plasmid DNA, a siRNA, an antisense oligonucleotide, a circular RNA, or a miRNA; and the gene editing complex is an mRNA / sgRNA or a Cas9 / sgRNA.

18. The drug loaded lipid nanoparticle of claim 16, wherein, The mass ratio of the ionizable cationic lipid to the nucleic acid molecule is (2-50):

1.

19. A method of preparing the drug-loaded lipid nanoparticle of any one of claims 13-18, wherein, The method comprises the following steps: (1) dissolving the lipid component in an organic solution to obtain an organic phase; (2) dissolving the drug in a buffer solution to obtain an aqueous phase; The volume ratio of the aqueous phase to the organic phase is (1-6):1; (3) rapidly mixing the aqueous phase and the organic phase uniformly, and then dialyzing to obtain the lipid nanoparticle.

20. Use of the fluorine-modified polysarcosylated lipid of any one of claims 1-4, the lipid nanoparticle of any one of claims 9-12, or the drug-loaded lipid nanoparticle of any one of claims 13-18 in the preparation of a delivery or transport of a small molecule drug or a nucleic acid vaccine.

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