Fluorine-containing modified poly-sarcosine acidified lipid as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses fluorine-containing modified poly-sarcosine acidified lipid as well as a preparation method and application thereof. The fluorine-containing modified poly-sarcosine acidified lipid disclosed by the invention has a structural general formula as shown in a formula (I) or a formula (II). The fluorine-containing modified poly-sarcosine acidified lipid obtained by chemically modifying the poly-sarcosine acidified lipid through different fluorine-containing groups can replace PEGylated lipid to prepare lipid nanoparticles (LNP), and is used for loading drugs such as nucleic acid and the like, so that the purpose of improving the drug delivery performance is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a fluorine-modified polysarcosinate lipid and a preparation method and application thereof. Background Art

[0002] mRNA has emerged as a new class of biomacromolecule drugs, potentially useful for preventing or treating a wide range of diseases, including pathogen infections and genetic disorders. Because mRNA is easily degraded by nucleases in the body, its high molecular weight and negative charge make it difficult to penetrate cell membranes. Therefore, delivery vehicles are needed to enhance its stability and improve its cellular internalization and transfection capabilities. Lipid nanoparticles (LNPs) are currently the most advanced delivery vehicle technology, having been successfully applied to COVID-19 mRNA vaccines (Comirnaty and Spikevax). This has accelerated the development of LNPs for other mRNA therapeutics, such as protein replacement therapy, tumor immunotherapy, and gene editing. LNPs typically consist of four lipid components: ionizable cationic lipids, helper lipids (typically phospholipids), cholesterol, and PEGylated lipids. PEGylated lipids play a crucial role in enhancing LNP stability and prolonging blood circulation. However, studies have shown that anti-PEG antibodies often exist in the human body due to various factors, such as environmental exposure, cosmetics, or pharmaceutical use. This can lead to allergic reactions and accelerated blood clearance after repeated administration of LNPs containing PEGylated lipids, limiting the application of traditional LNP technology for the repeated administration of mRNA therapeutics. Therefore, there is an unmet need to explore alternative technologies for PEGylated lipids to improve the delivery performance of LNPs.

[0003] In order to meet the current challenges, the formulation of LNPs is modified with hydrophilic polymers such as poly (N-vinylamide), polypeptides, poly (oxazoline) or polyglycerol to improve drug delivery performance. Among them, polysarcosine (pSar) is a polypolypeptide material derived from the endogenous amino acid N-methylglycine (i.e., sarcosine) monomer, which has the characteristics of excellent stealth, high biocompatibility and low immunogenicity. Previous studies have shown that compared with LNPs prepared with traditional PEGylated lipids, LNPs prepared with sarcosinated lipids exhibit better safety after systemic administration. However, the current LNPs prepared with polysarcosinated lipids still have problems such as insufficient mRNA delivery efficiency in vivo, which seriously limits the applicability of mRNA therapy. Summary of the Invention

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

[0005] To achieve the above object, 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] Said n or m is the number of repeating units of polysarcosine (i.e., degree of polymerization); n or m is 5-150;

[0009] The X1 is independently selected from hydrogen or alkyl; the X2 is independently selected from alkyl or alkenyl; the R1 is a fluorine-containing modified group; the X3 is independently selected from carbonyl-substituted alkyl, carbonyl-substituted alkenyl or hydroxyl-substituted alkyl; the R2 is independently selected from hydrogen, alkyl or fluorine-containing substituted alkyl; the R3 is independently selected from fluorine-containing substituted alkyl, or fluorine-containing and amide-substituted alkyl.

[0010] As a preferred embodiment of the fluorine-modified polysarcosinate lipid of the present invention, the X1 is independently selected from any one of hydrogen or an alkyl group with a carbon number of 6-22; and / or the X2 is independently selected from an alkyl group or an alkenyl group with a carbon number of 6-22; the R1 is a fluorine-containing modified group; and / or the X3 is independently selected from any one of a carbonyl-substituted alkyl group with a carbon number of 6-22, a carbonyl-substituted alkenyl group with a carbon number of 6-22, and a hydroxyl-substituted alkyl group with a carbon number of 6-22; and / or the R2 is independently selected from any one of hydrogen, an alkyl group with a carbon number of 1-6, and a fluorine-substituted alkyl group with a carbon number of 1-6; and / or the R3 is independently selected from any one of a fluorine-substituted alkyl group with a carbon number of 1-6, or a fluorine-containing and amide-substituted alkyl group with a carbon number of 1-6.

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

[0012] Said X2 is independently selected from any one of an alkyl group or an alkenyl group having 8 to 20 carbon atoms; and / or

[0013] Said R1 is independently selected from a carbonyl or hydroxy substituted fluorine-containing group; and / or

[0014] The X3 is independently selected from any one of a carbonyl-substituted alkyl group having 8 to 20 carbon atoms, a carbonyl-substituted alkenyl group having 8 to 20 carbon atoms, and a hydroxyl-substituted alkyl group having 8 to 20 carbon atoms.

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

[0016] and / or

[0017] The X2 is independently selected from

[0018] and / or

[0019] The R1 is independently selected from

[0020] and / or

[0021] The X3 is independently selected from

[0022] and / or

[0023] Said 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 lipid of the present invention, the R1 is independently selected from

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

[0027] Said R3 is independently selected from

[0028] In a second aspect, the present invention provides a method for preparing a fluorine-modified polysarcosinate lipid, comprising the following steps:

[0029] (1) dissolving sarcosine N-carboxylic anhydride (NCA) monomer, 18-crown ether-6 catalyst and amine initiator in dichloromethane solvent to carry out ring-opening polymerization reaction, and obtaining polysarcosinate lipid containing terminal secondary amino group through precipitation, purification and drying;

[0030] (2) subjecting the polysarcosinate lipid containing a secondary amine group at the end to an amidation reaction with a fluorine-containing acid anhydride, or a ring-opening reaction with a fluorine-containing epoxy compound, and then preparing a fluorine-modified polysarcosinate lipid as shown in formula (I) by precipitation, purification, and drying.

[0031] As a preferred embodiment of the preparation method of the present invention, the amine initiator is At least one of; and / or

[0032] The fluorine-containing anhydride is

[0033] At least one of; and / or

[0034] The fluorine-containing epoxy compound is

[0035] At least one of .

[0036] In a third aspect, the present invention provides another method for preparing a fluorine-modified polysarcosinate lipid, comprising the following steps:

[0037] (1) dissolving sarcosine N-carboxylic anhydride (NCA) monomer, 18-crown ether-6 catalyst and fluorinated substituted amine initiator in dichloromethane solvent to carry out ring-opening polymerization reaction, and obtaining fluorinated polysarcosine containing a secondary amino group at the end through precipitation, purification and drying;

[0038] (2) subjecting the fluorinated polysarcosine containing a secondary amine group at the terminal 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 preparing a fluorinated modified polysarcosinate lipid as shown in formula (II) by precipitation, purification, and drying.

[0039] As another preferred embodiment of the preparation method of the present invention, the fluorine-containing substituted amine initiator is

[0040]

[0041] At least one of; and / or

[0042] The alkyl or alkenyl substituted anhydride is

[0043] At least one of; and / or

[0044] The alkyl substituted epoxy compound is

[0045] At least one of .

[0046] In a fourth aspect, the present invention provides a lipid nanoparticle comprising the fluorine-modified polysarcosinate lipid.

[0047] As a preferred embodiment of the lipid nanoparticles of the present invention, at least one of ionizable cationic lipids, sterols, and auxiliary lipids is also included.

[0048] The ionizable cationic lipid carries a positive charge at low pH and can bind to RNA. It is nearly electrically neutral at physiological pH, reducing in vivo toxicity. In addition, the ionizable cationic lipid can promote the endosomal escape of LNP and improve RNA transfection efficiency; the sterol stabilizes the LNP structure and regulates membrane fluidity; the auxiliary lipid is the structural lipid of the LNP, which stabilizes and improves the drug encapsulation efficiency or regulates the targeting purpose; the fluorine-modified polysarcosine lipid is used to stabilize the LNP, prolong blood circulation time, reduce protein adsorption, improve endosomal escape efficiency and RNA transfection ability, etc.

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

[0050] As a further preferred embodiment of the lipid nanoparticles described in the present invention, the molar ratio of the ionizable cationic lipid, cholesterol, helper lipid, and fluorine-modified polysarcosine 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] In a fifth aspect, the present invention provides a drug-loaded lipid nanoparticle, comprising the lipid nanoparticle and a drug.

[0052] As a preferred embodiment of the drug-loaded lipid nanoparticles described in the present invention, the drug is at least one of a small molecule compound, a nucleic acid molecule, a protein or polypeptide molecule, and a gene editing complex.

[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; and 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] In a sixth aspect, the present invention provides a method for preparing the drug-loaded lipid nanoparticles, comprising the following steps:

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

[0057] (2) dissolving 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 quickly mixed and dialyzed to obtain the product.

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

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] The present invention modifies polysarcosinate lipids through a fluorination modification strategy, and chemically modifies one end of the polysarcosinate lipid with a fluorine-containing group to obtain a fluorine-modified polysarcosinate lipid having a chemical structure shown in formula (I) or formula (II); because the fluorine-containing group has a significant phase separation tendency in both polar and non-polar environments, and has hydrophobic and lipophobic characteristics, its fluorine effect can efficiently promote the intracellular escape of the carrier, and can also reduce the adsorption of immune-related proteins on the carrier surface, thereby prolonging the circulation time. Compared with unmodified polysarcosinate lipids, lipid nanoparticles (LNPs) prepared using fluorine-modified polysarcosinate lipids can significantly improve the in vivo transfection performance of mRNA, can replace PEGylated lipids, avoid the problem of anti-PEG antibodies in vivo, and achieve the purpose of improving the effect of mRNA therapy and expanding its application. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0065] Figure 3 Fluorinated polysarcosine lipid R18-pSar 70 -A11 1 H NMR spectrum.

[0066] Figure 4 Fluorinated polysarcosine lipid R18-pSar 70 -Mass spectrum of A11.

[0067] Figure 5 Fluorinated polysarcosine lipid DR14-pSar 70 -A1 1 H NMR spectrum.

[0068] Figure 6 Fluorinated polysarcosine lipid DR14-pSar 70 -Mass spectrum of A1.

[0069] Figure 7 Fluorinated polysarcosine lipid DR14-pSar 70 -A7 1 H NMR spectrum.

[0070] Figure 8 Fluorinated polysarcosine lipid DR14-pSar 70 -Mass spectrum of A7.

[0071] Figure 9 Fluorinated polysarcosine-modified lipid DR14-pSar70-BF5 in deuterated dimethyl sulfoxide 1 H NMR spectrum.

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

[0073] Figure 11 Fluorinated polysarcosine lipid DR14-pSar 70 -BF12 in deuterated dimethyl sulfoxide 1 H NMR spectrum.

[0074] Figure 12 Fluorinated polysarcosine lipid DR14-pSar 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 Fluorinated polysarcosine lipid CF6-pSar70-C18 1 H NMR spectrum.

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

[0078] Figure 16 Fluorinated polysarcosine lipid CF6-pSar70-CE18 1 H NMR spectrum.

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

[0080] Figure 18 Figure 3 shows the particle size (A) and PDI (B) of Fluc-mRNA LNPs prepared based on fluorine-modified polysarcosine lipids (n=3).

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

[0082] Figure 20 The results of in vitro cell transfection with Fluc-mRNA LNP are shown. DETAILED DESCRIPTION

[0083] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0084] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0085] Example 1: Synthesis of Sarcosine N-Carboxylic Anhydride (NCA) Monomer (Sar-NCA)

[0086] The synthesis method is 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 and placed in an ice-water bath. 2.36 g of triphosgene (8.1 mmol, 0.5 eq) was then added, and the flask was kept half-open for the reaction. After 1.5 h of reaction, 10 mL of cold water at 4°C was added to quench the 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 in a vacuum, the collected product was further purified by crystallization at low temperature from a mixture of n-hexane / dichloromethane (1:1, v / v). The final product was white crystals (yield approximately 65%). Sar-NCA monomer was stored at -20°C until use.

[0088] Example 2: Synthesis of polysarcosinate lipids

[0089] The synthesis method is as follows:

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

[0091] The synthetic route is as follows:

[0092]

[0093] Among them, the structure of the amine initiator is as follows:

[0094]

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

[0096] Table 1 Product name and structural formula

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] Using nuclear magnetic resonance spectroscopy ( 1 H NMR was used to characterize the structure of polysarcosine lipids, and gel permeation chromatography (SEC) was used to characterize the molecular weight and dispersity index (M w / M n ).

[0103] Representative polysarcosinate lipid R18-pSar 70 and DR14-pSar 70 of 1 H NMR spectra were Figure 1 and Figure 2 shown.

[0104] The molecular weight and dispersity index of the polysarcosine lipids were measured by gel permeation chromatography (SEC), and the data are shown in Table 2. 1 The molecular weights of polysarcosinate lipids were calculated from peaks a and c in H NMR for data comparison.

[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 measured by SEC;

[0108] M n,NMR (kg / mol) represents the molecular weight calculated by H NMR spectroscopy; M w / M n Represents the dispersibility index of the polymer.

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

[0110] Fluorinated anhydrides can be obtained commercially or prepared from the corresponding fluorinated carboxylic acids by 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 in vacuo to obtain a fluorinated acid anhydride.

[0112] The polysarcosinate lipids containing secondary amine groups at the end prepared in Example 2 were subjected to amidation reaction with fluorinated anhydrides, as follows:

[0113] 100 mg of polysarcosinate lipid (1.0 eq) was dissolved in 2 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (10.0 eq) and fluorinated anhydride (5.0 eq) were added sequentially. After reacting at room temperature for 12 h, the product was purified by precipitation with glacial ether. This process was repeated three times. The collected product was further purified by dialyzing against pure water for 24 h. The solvent was removed by freeze-drying.

[0114] The obtained fluorinated modified polysarcosine lipid was reacted with "amine initiator-pSar n -fluorinated anhydride" form, where n represents the number of polysarcosine repeating units (i.e., degree of polymerization).

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

[0116] The specific synthetic route is as follows:

[0117]

[0118] The molecular structure of fluorinated anhydride is shown below:

[0119]

[0120] The corresponding fluorinated 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-containing groups are shown in Table 3:

[0123] Table 3 Product name and structure

[0124]

[0125]

[0126]

[0127]

[0128] Using nuclear magnetic resonance spectroscopy ( 1 H NMR) and mass spectrometry characterization of a representative fluorinated modified polysarcosinate lipid R18-pSar 70 -A11、DR14-pSar 70 -A1 and DR14-pSar 70 -A7 structure, such as Figure 3-8 shown.

[0129] By the above nuclear magnetic resonance spectroscopy ( 1 H NMR) or mass spectrometry analysis showed that the fluorine-modified polysarcosine lipid was successfully prepared.

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

[0131] The polysarcosinate lipids containing secondary amino groups at the end prepared in Example 2 were subjected to ring-opening reactions with fluorinated epoxy compounds, as follows:

[0132] 100 mg of polysarcosinate lipid (1.0 eq) was dissolved in 2 mL of methanol, and a fluorinated epoxy compound (7.0 eq) was added. The reaction was allowed to proceed at 70°C for 8 h. After removing the organic solvent by rotary evaporation in a vacuum, the collected product was dialyzed against pure water for 24 h for further purification. The solvent was removed by freeze drying to obtain the product.

[0133] The obtained fluorinated modified polysarcosine lipid was reacted with "amine initiator-pSar n -fluorinated epoxy compounds" in the form of "-fluorinated epoxy compounds", where n represents the number of polysarcosine repeating units (i.e., the degree of polymerization).

[0134] The synthetic route is as follows:

[0135]

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

[0137]

[0138] The corresponding fluorinated 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 name and structure

[0142]

[0143]

[0144]

[0145] Using nuclear magnetic resonance spectroscopy ( 1 H NMR) and mass spectrometry characterization of a representative fluorinated polysarcosinate lipid DR14-pSar 70 -BF5 and DR14-pSar 70 -BF12 structure, data such as Figure 9-12 shown.

[0146] By the above nuclear magnetic resonance spectroscopy ( 1 H NMR) and mass spectrometry analysis showed that the fluorine-modified polysarcosine lipid was successfully prepared.

[0147] Example 5: Synthesis of fluorinated polysarcosine

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

[0149] The specific synthetic route is as follows:

[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 name and structure

[0157]

[0158] Using nuclear magnetic resonance spectroscopy ( 1 H NMR) characterization of representative fluorinated polysarcosine CF6-pSar 70 Structure, data such as Figure 13 As shown. Through the above nuclear magnetic resonance hydrogen spectrum ( 1 H NMR) analysis showed that fluorinated polysarcosine was successfully prepared.

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

[0160] The fluorinated polysarcosine containing a secondary amine group at the end obtained in Example 5 was subjected to an amidation reaction with an alkyl or alkenyl substituted acid anhydride as follows:

[0161] 100 mg of fluorinated polysarcosine (1.0 eq) was dissolved in 2 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (10.0 eq) and an alkyl or alkenyl substituted anhydride (5.0 eq) were added sequentially. After reacting at room temperature for 12 hours, the product was purified by precipitation with glacial ether. This process was repeated three times. The collected product was then dialyzed against pure water for 24 hours for further purification. The solvent was removed by freeze drying to obtain the product.

[0162] The obtained fluorinated modified polysarcosine lipid was reacted with "fluorinated amine initiator-pSar m -anhydride" form, where m represents the number of polysarcosine repeating units (degree of polymerization).

[0163] The specific synthetic route is as follows:

[0164]

[0165] The structure of alkyl or alkenyl substituted anhydride is as follows:

[0166]

[0167] The X3 group is:

[0168]

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

[0170] Table 6 Product name and structure

[0171]

[0172] Using nuclear magnetic resonance spectroscopy ( 1 H NMR) and mass spectrometry to characterize the structure of CF6-pSar70-C18. The data are shown in Figure 14 and 15 As shown. Through the above nuclear magnetic resonance hydrogen spectrum ( 1 H NMR) and mass spectrometry analysis showed that the fluorine-modified polysarcosine lipid was successfully prepared.

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

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

[0175] 100 mg of fluorinated polysarcosine containing a secondary amino group at the end (1.0 eq) was dissolved in 2 mL of methanol, and an alkyl-substituted epoxide (7.0 eq) was added. The reaction was allowed to proceed at 70°C for 8 h. After removing the organic solvent by rotary evaporation in a vacuum, the collected product was dialyzed against pure water for 24 h for further purification. The solvent was removed by freeze drying to obtain the product.

[0176] The obtained fluorinated modified polysarcosine lipid was reacted with "fluorinated amine initiator-pSar m -epoxide" form, where m represents the number of polysarcosine repeating units (i.e., the degree of polymerization).

[0177] The specific synthetic route is as follows:

[0178]

[0179] The structure of alkyl-substituted epoxy compounds is as follows:

[0180]

[0181] The X3 group is:

[0182]

[0183] The structures of representative fluorine-modified polysarcosine lipids are shown in Table 7:

[0184] Table 7 Product name and structure

[0185]

[0186] Using nuclear magnetic resonance spectroscopy ( 1 H NMR) and mass spectrometry characterization of CF6-pSar 70 -CE18 structure, data such as Figure 16 and 17 As shown. Through the above nuclear magnetic resonance hydrogen spectrum ( 1H NMR) analysis showed that the fluorine-modified polysarcosine lipid was successfully prepared.

[0187] Example 8: Preparation and characterization of lipid nanoparticles (LNPs)

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

[0189] Fluc-mRNA LNPs based on fluorinated polysarcosinate lipids were prepared using a manual rapid mixing method. The organic phase was prepared by dissolving four lipid components (the ionizable cationic lipid A4I18R2C18-2, see invention patent: A Cationic Lipid Analog, Composition, and Application thereof; Application No. 202210101998.9), cholesterol, DSPC, and a fluorinated polysarcosinate lipid (unmodified polysarcosinate lipid and commercial DMG-PEG2000 were used as controls) in ethanol at a molar ratio of 50:38.5:10:1.5. The aqueous phase was prepared by diluting the fluc-mRNA into 10 mM citrate buffer (pH 4). The organic and aqueous phases were then rapidly mixed manually using a pipette at a volume ratio of 1:3 (the mass ratio of ionizable cationic lipid to mRNA was 11:1) to produce the corresponding LNPs. The resulting LNPs were incubated at room temperature for 15 minutes and used for in vitro cell transfection experiments. For in vivo experiments, the prepared LNPs needed to be dialyzed in PBS for 2 h using a 1000 MWCO dialysis bag to remove residual ethanol before use.

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

[0191] LNPs were diluted to a concentration of 10 μg / mL mRNA and measured by dynamic light scattering (Malvern Zetasizer Nano ZS) to characterize their physicochemical parameters, including particle size and polydispersity index (PDI).

[0192] Taking the above-mentioned ionizable cationic lipid A4I18R2C18-2, cholesterol, DSPC and fluorine-modified polysarcosine lipid (unmodified polysarcosine lipid and commercial DMG-PEG2000 were used as controls) with a molar ratio of 50:38.5:10:1.5 as an example, the physical and chemical properties of LNP are characterized as follows Figure 18 The above results indicate that Fluc-mRNA LNP based on fluorine-modified polysarcosine lipids was successfully prepared.

[0193] Example 9: Preparation and characterization of lipid nanoparticles (LNPs)

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

[0195] Ionizable cationic lipids, cholesterol, DSPC and fluorine-modified polysarcosine lipids (using unmodified polysarcosine lipids, DR14-pSar 70 -AA、DR14-pSar 70 -BA and commercial DMG-PEG2000 as a reference) were dissolved in ethanol to prepare an organic phase. The corresponding mass ratio of the mRNA drug was dissolved in citric acid buffer (25 mM, pH 4.0) to prepare an aqueous phase, with a volume of aqueous phase:organic phase = 3:1. The aqueous and organic phases were rapidly mixed using a microfluidic device at a controlled flow rate of 3 mL / min (adjustable from 1 to 10 mL / min). After standing for 10 minutes, the mixture was placed in a 1000 Da dialysis bag and dialyzed against 1× PBS at 4°C for 2 hours to obtain drug-loaded lipid nanoparticles suitable for direct injection. The mass ratio of the ionizable cationic lipid to the nucleic acid drug was 11:1.

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

[0197] The above results show that the Fluc-mRNA LNP based on fluorine-modified polysarcosine lipids prepared by microfluidics has smaller and more uniform particle size and higher mRNA drug encapsulation efficiency.

[0198] Example 10: In vitro cell transfection assay

[0199] HepG2 cells were cultured at 5×10 3Cells were seeded at a density of 100 μg per well in a 96-well plate and incubated in an incubator for 24 h. The culture medium was then removed and fresh Opti-MEM containing LNPs was added, with a Fluc-mRNA dose of 0.1 μg / well. After 48 h of incubation, cells were treated with reporter gene cell lysate (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 the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Luciferase activity was expressed as RLU / mg protein.

[0200] In vitro cell transfection results Figure 20 The results showed that with the increase of carbon chain length in different amine initiators, the in vitro transfection ability of LNP was not much different, but the degree of polymerization of polysarcosine had a certain effect on the in vitro transfection performance. Among them, the polysarcosine lipids with better LNP transfection ability included DR12-pSar 50 、DR14-pSar 50 、DR14-pSar 70 、DR16-pSar 50 .

[0201] Example 11: In vivo animal transfection test

[0202] Different Fluc mRNA LNPs were injected intravenously with an mRNA dose of 0.125 mg / kg per C57BL / 6 mouse (6-8 weeks old). After 6 hours of administration, luciferase substrate (D-Luciferin potassium salt, Promega) was injected intraperitoneally with a dosage of 150 mg / kg per mouse. After waiting for 6 minutes, the mice were anesthetized and placed in a small animal imaging system (IVIS, PerkinElmer) for bioluminescence imaging. If necessary, mice were killed and major organs were collected for in vitro bioluminescence imaging. The luminous intensity of firefly luciferase in the organs was used to judge the LNP delivery efficiency in vivo, and the results are shown in Tables 8, 9 and 10. The LNP name is named after the polymer lipid used.

[0203] Table 8 In vivo expression of mRNA drug-loaded nanoparticles prepared by manual method and injected intravenously

[0204]

[0205]

[0206] The results in Table 8 show that compared with the unmodified polysarcosine lipid (DR14-pSar 70), LNPs prepared by fluorine-modified polysarcosine lipids have a higher expression effect in delivering mRNA to the liver, 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 drug-loaded nanoparticles prepared by microfluidics 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] The results in Table 9 show that compared with the unmodified polysarcosine lipid DR14-pSar 70 , and polysarcosinate lipid DR14-pSar chemically modified with acetic anhydride or butyric anhydride 70 -AA and DR14-pSar 70 -BA, LNPs prepared by microfluidics using fluorinated polysarcosine lipids delivered mRNA to the liver with significantly higher expression efficacy. Furthermore, under the same conditions, the in vivo delivery performance of mRNA-loaded LNPs prepared by microfluidics was significantly superior to that of LNPs prepared by manual methods as shown in Table 8.

[0210] Table 10 In vivo expression of mRNA drug-loaded nanoparticles prepared by microfluidics and injected intravenously

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

[0212] The results in Table 10 demonstrate that Fluc mRNA LNPs prepared using a formula comprising an ionizable cationic lipid (A4I18R2C18-2), cholesterol, DOTAP, and fluorine-modified polysarcosinate lipid 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, 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 the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A fluorine-modified polysarcosinate lipid, characterized in that: It has a structure as shown in formula (I) or formula (II): The n or m is 5-150; the X1 is independently selected from hydrogen or alkyl; the X2 is independently selected from alkyl or alkenyl; the R1 is a fluorine-containing modified group; the X3 is independently selected from carbonyl-substituted alkyl, carbonyl-substituted alkenyl or hydroxyl-substituted alkyl; the R2 is independently selected from hydrogen, alkyl or fluorine-containing substituted alkyl; the R3 is independently selected from fluorine-containing substituted alkyl, or fluorine-containing and amide-substituted alkyl.

2. The fluorine-modified polysarcosinate lipid according to claim 1, characterized in that The X1 is independently selected from any one of hydrogen or an alkyl group having 6-22 carbon atoms; and / or the X2 is independently selected from an alkyl group or an alkenyl group having 6-22 carbon atoms; the R1 is a fluorine-containing modified group; and / or the X3 is independently selected from any one of a carbonyl-substituted alkyl group having 6-22 carbon atoms, a carbonyl-substituted alkenyl group having 6-22 carbon atoms, and a hydroxyl-substituted alkyl group having 6-22 carbon atoms; and / or the R2 is independently selected from any one of hydrogen, an alkyl group having 1-6 carbon atoms, and a fluorine-substituted alkyl group having 1-6 carbon atoms; and / or the R3 is independently selected from any one of a fluorine-substituted alkyl group having 1-6 carbon atoms, or a fluorine-containing and amide-substituted alkyl group having 1-6 carbon atoms.

3. The fluorine-modified polysarcosinate lipid according to claim 2, characterized in that Said X1 is independently selected from any one of hydrogen or an alkyl group having 8 to 20 carbon atoms; and / or Said X2 is independently selected from any one of an alkyl group or an alkenyl group having 8 to 20 carbon atoms; and / or Said R1 is independently selected from a carbonyl or hydroxy substituted fluorine-containing group; and / or The X3 is independently selected from any one of a carbonyl-substituted alkyl group having 8 to 20 carbon atoms, a carbonyl-substituted alkenyl group having 8 to 20 carbon atoms, and a hydroxyl-substituted alkyl group having 8 to 20 carbon atoms.

4. The fluorine-modified polysarcosinate lipid according to claim 3, characterized in that Said X1 is independently selected from H, and / or The X2 is independently selected from and / or The R1 is independently selected from and / or The X3 is independently selected from and / or Said R2 is independently selected from H, and / or The R3 is independently selected from 5. The fluorine-modified polysarcosinate lipid according to claim 4, characterized in that The R1 is independently selected from Said R2 is independently selected from H, and / or Said R3 is independently selected from 6. A method for preparing a fluorine-modified polysarcosinate lipid, characterized in that: The following steps are involved: (1) dissolving sarcosine N-carboxylic anhydride (NCA) monomer, 18-crown ether-6 catalyst and amine initiator in dichloromethane solvent to carry out ring-opening polymerization reaction, and obtaining polysarcosinate lipid containing terminal secondary amino group through precipitation, purification and drying; (2) subjecting the polysarcosinate lipid containing a secondary amine group at the end to an amidation reaction with a fluorine-containing acid anhydride, or a ring-opening reaction with a fluorine-containing epoxy compound, and then preparing a fluorine-modified polysarcosinate lipid by precipitation, purification, and drying.

7. The preparation method according to claim 6, characterized in that The amine initiator is At least one of; and / or The fluorine-containing anhydride is At least one of; and / or The fluorine-containing epoxy compound is At least one of .

8. A method for preparing a fluorine-modified polysarcosinate lipid, characterized in that: The following steps are involved: (1) dissolving sarcosine N-carboxylic anhydride (NCA) monomer, 18-crown ether-6 catalyst and fluorinated substituted amine initiator in dichloromethane solvent to carry out ring-opening polymerization reaction, and obtaining fluorinated polysarcosine containing a secondary amino group at the end through precipitation, purification and drying; (2) subjecting the fluorinated polysarcosine containing a secondary amine group at the terminal 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 preparing a fluorine-modified polysarcosine lipid by precipitation, purification, and drying.

9. The preparation method according to claim 7, characterized in that 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 .

10. A lipid nanoparticle, characterized in that The invention comprises the fluorine-containing modified polysarcosinate lipid according to any one of claims 1 to 5.

11. The lipid nanoparticle according to claim 10, characterized in that It also includes at least one of ionizable cationic lipids, sterols, and helper lipids.

12. The lipid nanoparticle according to claim 11, characterized in that The auxiliary lipid is at least one of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), dioleoylphosphatidylcholine, and dipalmitoylphosphatidylcholine; the sterol is at least one of cholesterol, sitosterol, stigmasterol, and a cholesterol derivative.

13. The lipid nanoparticle according to claim 11, characterized in that The molar ratio of the ionizable cationic lipid, cholesterol, auxiliary lipid and fluorine-modified polysarcosine lipid is (20-70): (20-50): (2-30): (0.1-20).

14. A drug-loaded lipid nanoparticle, characterized in that: The invention comprises the lipid nanoparticles according to claims 10-13 and a drug.

15. The drug-loaded lipid nanoparticles according to claim 14, characterized in that The drug is at least one of a small molecule compound, a nucleic acid molecule, a protein or polypeptide molecule, and a gene editing complex.

16. The drug-loaded lipid nanoparticle according to claim 15, characterized in that 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; and the gene editing complex is mRNA / sgRNA or Cas9 / sgRNA.

17. The drug-loaded lipid nanoparticles according to claim 15, characterized in that The mass ratio of the ionizable cationic lipid to the nucleic acid molecule is ionizable cationic lipid:nucleic acid molecule=(2-50):

1.

18. A method for preparing the drug-loaded lipid nanoparticles according to any one of claims 14 to 17, characterized in that: The following steps are involved: (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 of the aqueous phase: the volume of the organic phase = (1-6): 1; (3) The aqueous phase and the organic phase are quickly mixed and dialyzed to obtain the product.

19. Use of the fluorine-modified polysarcosinate lipid according to any one of claims 1 to 5, the lipid nanoparticle according to any one of claims 10 to 13, or the drug-loaded lipid nanoparticle according to any one of claims 14 to 17 in the preparation of a delivery or transportation molecular drug or nucleic acid vaccine.

Images