Molecule with targeting function and application

Abstract

The invention relates to the technical field of medicines, in particular to a molecule with a targeting function, the molecule with the targeting function can specifically deliver a medicine to a specific cell, the specific cell can be accurately programmed in vivo, the curative effect of the medicine can be exerted, the administration dosage and side effects of the medicine can be greatly reduced, and the curative effect of the medicine can be improved. The effect of precisely treating in-vivo cells is achieved.

Description

[0001] Claim of priority

[0002] This application claims priority to Chinese patent application number CN202311627230.6, filed on November 30, 2023, which is hereby incorporated by reference in its entirety. This application claims priority to Chinese patent application number CN202410918685.1, filed on July 10, 2024, which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present invention relates to the field of medical technology, and in particular to a molecule with targeting function and its application. Background Art

[0004] Lipid nanoparticles (LNPs) are composed of four components: an ionizable lipid compound, cholesterol, phospholipids, and PEG lipids. LNPs are a well-established delivery system technology platform, suitable for delivering RNA drugs, vaccines, or gene editing tools. Research on LNP composition is currently in-depth.

[0005] The composition of LNPs significantly influences their drug delivery performance. Ionizable lipid compounds are the core components of LNPs, determining mRNA delivery and transfection efficiency. In practice, nucleic acid drugs are susceptible to nonspecific cellular uptake during delivery, leading to higher dosages and significant side effects.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a molecule with targeting function and its application, which can target the delivery of pharmacological molecules to specific cells in the body, reduce the uptake of drugs by nonspecific cells, thereby reducing the dosage and side effects of drugs, and achieve more effective in vivo precision treatment effects on diseases.

[0008] Specifically, the technical solutions provided by the present invention are as follows: In a first aspect, the present invention provides a molecule with targeting function, comprising: (I) one or more hydrophobic chains; (II) one or more linkers; (III) one or more Nanobodies.

[0009] Preferably, the number of said Nanobodies is ≤20; preferably 1-7.

[0010] Preferably, an amino acid containing a reactive site is connected between the Nanobody and the linker, and the amino acid includes but is not limited to cysteine ​​and lysine.

[0011] Preferably, the Nanobodies are connected via a linker sequence, which is located between the Nanobodies and the amino acids, and the linker sequence comprises (GS)n and / or (GGGS)m, wherein n and m are the same or different values, and the value of n or m is selected from 0, 1, 2, 3 or 4.

[0012] Preferably, the hydrophobic chain comprises one or more substituted or unsubstituted C12-C18 alkyl groups.

[0013] Preferably, the linker comprises one or more structural units selected from -C(=O)O-, -OC(=O)O-, -C(=O)NH-, -C(=O)S-, -S-, -SS-, a triazole group, a hydrazone bond, SMCC, Sulfo-SMCC, SATA, a polypeptide as a structural unit, PEG or a PEG derivative.

[0014] The PEG derivative can be maleimide-functionalized polyethylene glycol, such as PEG-MAL.

[0015] Preferably, the relative molecular mass of the PEG or PEG derivative is 500-5000, preferably 1000, 2000, 3000, 4000 or 5000.

[0016] Preferably, the nanobody comprises an antigen-binding domain that is specific for an antigen, and the antigen is a membrane protein molecule.

[0017] Preferably, the membrane protein molecule is an immune cell or immune-related cell membrane protein molecule, and the immune cell or immune-related cell membrane protein molecule includes but is not limited to one or more of CD3, CD4, CD5, CD7, CD8, CD25, CD38, CD61, CD42a, CD105, CD90, CD15, CD127, CD56, CD68, CD19, CD11c, CD138, F4 / 80, CD62P, CD49f, CD31, RANK, ALPL, PDPN, CD34, FcεR1α, CD203c, CD63, CD193, CD66b, CD41, CD117, and ASGPR; Preferably, the membrane protein molecule is a central nervous system cell membrane protein molecule, and the central nervous system cell membrane protein molecule includes but is not limited to one or more of GD2, GD3, MOG, and TMEM119; Preferably, the membrane protein molecule is a tumor-associated antigen, including but not limited to one or more of CD133, PSMA, CLDN18.2, DLL3, TROP2, EGFRVIII, CA125, MUC1, MUC16, MSLN, CA9, HER2, HER3, TGM4, PSCA, CLDN6, STEAP2, GPC3, IL-13Ra2, EGFR, STEAP1, BCMA, FRa, VEGFR2, PDGFR-β, CEA, NCAM, FAP, SLC2A2, SEZ6L2, and LRP11; Preferably, the membrane protein molecule is an immune regulatory molecule, including but not limited to one or more of B7-H1, B7-H3, and B7-H4; Preferably, the membrane protein molecules include but are not limited to the following targets: CD3, CD4, CD5, CD7, CD8, CD25, CD38, CD61, CD42a, CD105, CD90, CD15, CD127, CD56, CD68, CD19, CD11c, CD138, F4 / 80, CD62P, CD49f, CD31, RANK, ALPL, PDPN, CD34, FcεR1α, CD203c, CD63, CD193, CD66b, CD41, CD117, ASGPR, GD2, GD3, MOG, One or more of TMEM119, CD133, PSMA, CLDN18.2, DLL3, TROP2, EGFRVIII, CA125, MUC1, MUC16, MSLN, CA9, HER2, HER3, TGM4, PSCA, CLDN6, STEAP2, GPC3, IL-13Ra2, EGFR, STEAP1, BCMA, FRa, VEGFR2, PDGFR-β, CEA, NCAM, FAP, SLC2A2, SEZ6L2, LRP11, B7-H1, B7-H3, B7-H4.

[0018] Preferably, the nanobody is a nanobody targeting CD8, and the amino acid sequence of the nanobody is shown in any one of SEQ ID NO: 1-3; And / or, the Nanobody is a Nanobody targeting CD19, and the amino acid sequence of the Nanobody is shown in any one of SEQ ID NOs: 79-81; And / or, the nanobody is a nanobody targeting CD56; And / or, the Nanobody is a Nanobody targeting CD5, and the amino acid sequence of the Nanobody is shown in any one of SEQ ID NO: 4-78; And / or, the Nanobody is a PSMA-targeted Nanobody, and the amino acid sequence of the Nanobody is as shown in any one of SEQ ID NOs: 82-83; And / or, the Nanobody is a Nanobody targeting CD133, and the amino acid sequence of the Nanobody is shown in any one of SEQ ID NOs: 84-85.

[0019] The specific sequences of the present invention are as follows: Anti-human CD8 Nb seq (corresponding sequence SEQ ID NO: 1-3) SEQ ID NO: 1: QVQLQESGGGSVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSTINWNGGSAEYAEPVKGRFTISRDNAKNTVYLQMNSLKLEDTAVYYCAKDADLVWYNLSTGQGTQVTVSSAAAYPYDVPDYGS SEQ ID NO: 2: QVQLQESGGGLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSTINWNGGSAEYAEPVKGRFTISRDNAKNTVYLQMNSLKLEDTAVYYCAKDADLVWYNLRTGQGTQVTVSSAAAYPYDVPDYGS SEQ ID NO:3: QVQLQESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADPVKGRFTISSDNAKNTVYLQMNSLKPEDAAVYYCAAGSLYTCVQSIVVPARPYYDMDYWGKGTQVTVSSAAAYPYDVPDYGS Anti-human CD5 Nb seq (corresponding sequence SEQ ID NO: 4-78) SEQ ID NO:4: DVQLQESGGGLVQPGDSLRLSCAASGRTFSSYDMGWFRQAPGKEREFVAAISRSGRDTLHADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYICAGTGFEYAGSTPYKYPYWGQGTQVTVSS SEQ ID NO:5: DVQLQESGGGLVQAGGSLRLSCTASGSIVSIISMGWYRQAPGKQRELIAKITTDGTTNYADSVRGRFTISRDIPKKIMYLQMSSLTPEDTAVYFCNRDRLLYGLVKSDWGQGTQVTVSS SEQ ID NO:6: DVQLQESGGGFVRPGGSLRLSCAASGPTVSSYTVGWFRQAPGKEREYVATISWSDASTYYADFVKGRFTISRDYAKQVVYLQMNSLKPEDTAVYHCAAVFRRVPISATLYDYWGQGTQVTVSS SEQ ID NO:7: DVQLQESGGGLVQPGGSLRLSCAASGSAFSRIGVGWYRQAPGKEREFVAVITAGGNTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCNYPGRPWGQGTQVTVSS SEQ ID NO:8: DVQLQESGGGLVQPGGSLRLSCAASGSAFSRIGVGWYRQAPGKEREFVAVITAGGNTNYADSVKGRFTISRDNAKNTVYLQMNSLKPGDTGVYYCNYPGRPWGQGTQVTVSS SEQ ID NO:9: DVQLQESGGGLVQPGGSLRLACAASGNIFEIHAMYWYRQAPGKQREWVAAITTRGSTNVADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYLCNADVFSALTGAGTEYWGQGTQVTVSS SEQ ID NO:10: DVQLQESGGGLVQPGGSLRLSCVASGSAFSRIGVGWYRQAPGKEREFVAEITAGGNTDYADSVKGRFTISRDNAKNMVYLQMNSLKPEDTGVYYCNYPGRPWGQGTQVTVSS SEQ ID NO:11: DVQLQESGGGLVQAGGSLRLSCAASGHTLSIYAMTWFRQDSGKDREFVAAITWPDYSTYYADSVKGRFTISGDRPKNTVYLEMNSVKPEDTAVYYCAADRYVNSVPTATPQEWRYWGQGTQVTVSS SEQ ID NO:12: DVQLQESGGGLVQAGGSLRLSCAVSGRTISGMSVGWFRQAPGKEREFVAADNWAGAKTSYADSVKGRFTISRDNAKAMVYLQMNGLKPEDTAVYYCAGRTSDGLSTYARIEDYYGWGQGTQVTVSS SEQ ID NO:13: DVQLQESGGGLAQAGGSLRLSCAASGGIFSIDAAAWYRQAPGKERELVAHIFHGGSTTYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYGGPPGMAIWGKGTQVTVSS SEQ ID NO:14: DVQLQESGGGLVQAGASLSLSCAASGRTVNYVFGMGWFRQALGKEREFVAAITRSGGIPLYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAIDRQYYSGRNYPQSDPAEYDYWGQGTQVTVSS SEQ ID NO:15: DVQLQESGGGLVQAGGSLRLSCAASVRTFSKYDIGWFRQAPGKEREFVATISSSGRSTYYADFAKGRFGISRDYAKNTVYLEMNSLKPEDTAVYACAVRLGYSGSETDPRQYAYWGQGTQVTVSS SEQ ID NO:16: DVQLQESGGGFVQPGGSLRLSCAASGPTVSSYTVGWFRQAPGKEREYVATISWSDASTYYADFVKGRFTISRDYAKQVVYLQMNSLKPEDTAVYHCAAVFRRVPISATLYDYWGQGTQVTVSS SEQ ID NO:17: DVQLQESGGGLVQAGDSLRLSCAPSGGIFSINAAAWYRQAPGKERELVAHINHGGSTTYVGSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYGGPPGMAIWGKGTQVTVSS SEQ ID NO:18: DVQLQESGGGLVQAGGSLRLSCTASGTIFNVMGWYRQAPGKQRELVATITFGLTTNYADSVKGRFAISRDNSEKTVYLQMNSLKPEDTAVYYCNAGSRYWGQGTQVTVSS SEQ ID NO:19: DVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTTEADSVKGRFTISRDNAKNTVYLQMYSLKPEDTAVYYCAAARDLVSTVVAETSEYDYWGQGTQVTVSS SEQ ID NO:20: DVQLQESGGGLVQAGGSLRLSCAVSGRTINDMSVGWFRQAPGKEREFVAADIWPGAKTSYADPVKGRFTITRDNAKAMVYLQMNGLKPEDTAVYYCAGRTSDGLRTYTRIEDYYGWGQGTQVTVSS SEQ ID NO:21: DVQLQESGGGLVQPGGSLRLACAASGSGFSINAMGWYRQAPGKQREWVAAINSVGMTNYRDSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCNVKPLGTADYWGQGTQVTVSS SEQ ID NO:22: DVQLQESGGGLVQAGGSLRLSCAVSGRTISGMSVGWFRQAPGKEREFVAADSWAGAKTSYADSVKGRFTISRDNAKAMVYLQMKGLKPEDTAVYYCAGRTSDGLFNYARIEDYYGWGQGTQVTVSS SEQ ID NO:23: DVQLQESGGGLVQAGDSLRLSCAVSGRTISGMSVGWFRQAPGKEREFVAADSSAGAKTSYADSVKGRFTISRDNAKAMVYLQMNGLKPEDTAVYYCAGRTSYGLSTYARMEDYYGWGQGTQVTVSS SEQ ID NO:24: DVQLQESGGGLVQPGGSLRLSCAASGSIFSSNVMGWYRQAPGKQREWVSTIISDGRTNYAGSVKGRFTISRDNAKNTVYLQMNNLKPEDTALYYCKLSTLNRDYWGQGTLVTVGQGTQVTVSS SEQ ID NO:25: DVQLQESGGGLVQAGGSLRLSCAASGRTSTIYTMGWFRQAPGKEREFVAATSWSGGNTNYAASMKGRFTISRNNAKNTVYLQMNSLKPEDTAVYYCAANRGSYYLGPSPYEYDWWGQGTQVTVSS SEQ ID NO:26: DVQLQESGGGLVQPGGSLRLSCAASGSIFSINAMGWYRQAPGKQRDVVAYIMSDGNTHYADSVKGRFTISRDENTSYLQMNSLKPDDTAVYYCNADIQPPMPEATIYVYWGQGTQVTVSS SEQ ID NO:27: DVQLQESGGGLVQTGGSLRVSCAASGRTFSSAYMGWFRQAPGKERVLVAEIWLSSGQTYYADSVKGRFTISRENAKNTVYLQMNSLKPEDTAVYYCAAQNRATGRYDYWGQGTQVTVSS SEQ ID NO:28: DVQLQESGGGLVQPGGSLRLSCAASGSIFSSNVMGWYRQAPGKQREWVSTIISDGRTNYAGSVKGRFTISRDNAKNTVYLQMNNLKPEDTALYYCKLSTLNRDYWGQGTQVTVGQGTQVTVSS SEQ ID NO:29: DVQLQESGGGLVQPGGSLRLSCAASGGIFSINAAAWYRQAPGKERELVAHIYHGGSTTYVGSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYGGPPGMAIWGKGTQVTVSS SEQ ID NO:30: DVQLQESGGGFVQPGGSLRLSCAASGPTVSSYTVGWFRQAPGKEREYVATISWSDASTYYADFVKGRFTISRDYAKKVVYLQMNSLKPEDTAVYHCAAVFRRVPISATLYDYWGQGTQVTVSS SEQ ID NO:31: DVQLQESGGGLVQAGGSLRLSCAASGGIFSINAAAWYRQAPGKERELVAHITHGGSTTYEGSVKGRFTISKDNAKNTVYLQMNSLKPEDTAVYYCYGGPPGMAIWGKGTQVTVSS SEQ ID NO:32: DVQLQESGGGLVQAGGSLRLSCAASGSIVSIISVGWYRQAPGKQRELVAKITTDGTTNYADSVRGRFTISRDIAKKMLYLQMSSLTPEDTAVYFCNRDRLVYGLVKADWGQGTQVTVSS SEQ ID NO:33: DVQLQESGGGLVQAGGSLRLSCAASGRTSTIYTMGWFRQAPGREREFVAATSWSGGNTNYAGSVKGRFTISRNDAKNTVYLQMNSLKPEDTAVYYCAANRGSYYLGPSFYEYDYWGQGTQVTVSS SEQ ID NO:34: DVQLQESGGGLVQAGGSLRLSCTASGSIVSIISMGWYRQAPGKQRELIAKITTDGTTNYADSVRGRFTTSRDIPKKIMYLQMSSLTPEDTAVYFCNRDRLLYGLVKSDWGQGTQVTVSS SEQ ID NO:35: DVQLQESGGGLVQTGGSLRVSCAASGRTFSSSYMGWFRQAPGQERELVAEIWLSSGQTYYADSVKGRFTISRENAKNTAYLQMNSLTPEDTAVYYCAAQNRATGRYDYWGQGTQVTVSS SEQ ID NO:36: DVQLQESGGGLVQAGGSLRLSCTASGSIVSIISMGWYRQAPGKQRELIAKITTDGTTSYADSVRGRFTISRDIPKKIMYLQMSSLTPEDTAVYFCNRDRLLYGLVKSDWGQGTQVTVSS SEQ ID NO:37: DVQLQESGGGLVQAGGSLRLSCAASGGIFSINAAAWYRQAPGKERELVAHIYHGGSTTYVGSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYGGPPGMAIWGKGTQVTVSS SEQ ID NO:38: DVQLQESGGGLVQAGGSLRLSCAASGRTFSSTGMGWFRQAPGKEREFVAAINWSGEDTYYADSVKGRFTISGDNAKNVVYLQMNSLKPEDTAVYYCAASPYYNPYGPRLRTPSWYDYWGQGTQVTVSS SEQ ID NO:39: DVQLQESGGGLVQAGGSLRLSCAASGGTFSSSGMGWYRQAPGKEREFVAAINWSGEDTLYADSVKGRFTISGDNAKNTVHLQMNSLKPEDTAVYYCAASPYYNPYGPRLRTPSWYDYWGQGTQVTVSS SEQ ID NO:40: DVQLQESGGGLVQAGGSLRLSCTASGTIFNVMGWYRQAPGKQRELVATITFDLSTNYADSVKGRFAISRDVTEKTVYLQMDSLKPEDTAVYYCNAGSRYWGQGTQVTVSS SEQ ID NO:41: DVQLQESGGGLVQAGGSLRLSCAASGSTSGYSFRFWTMNWYRQGSGKQRELVATITSSGITTYADSVKGRFTVSRDNTKNTLYLQMNSLKPEDTATYYCNFPGNSWGQGTQVTVSS SEQ ID NO:42: DVQLQESGGGLVQAGDSLRLSCAVSGRTISDMSVGWFRQAPGKEREFVAADSWAGAKTSYADSVKGRFTISRNNAKAMVYLQMNGLKPEDTAVYYCAGRTSDGLSTYEWIEDYYGWGQGTQVTVSS SEQ ID NO:43: DVQLQESGGGLVQAGDSLRLSCVASGGIFSINAAAWYRQAPGKERELVAHIYHGGSTTYVGSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYGGPPGMAIWGKGTQVTVSS SEQ ID NO:44: DVQLQESGGGLVQPGGSLTLSCAASGSIFSRDVMGWYRQAPGKQRELVAAITIGGYADYADSVKGRFTISRDNAKNTVYLRMNSLKPEDTAVYFCKLGYGSGSWGQGTQVTVSS SEQ ID NO:45: DVQLQESGGGLVQPGGSLRLSCAASRSTFSIDIMAWYRQAPEKKRELVALITRRGTTNYADSVKGRFTISRDNAKNTMYLQMNNLKPEDTAVYYCNSDDTETRWGLRQYWGQGTQVTVSS SEQ ID NO:46: DVQLQESGGGLVQAGGSLRLSCAASGHTLSIYAMTWFRQDSGKDREFVAAITWPDYSTYYADSVKGRFTISGDRPKNTVHLEMNSVKPEDTAVYYCAADRYVNSVPTATPQEWRYWGQGTQVTVSS SEQ ID NO:47: DVQLQESGGGLVQPGASLSLSCAASGRTVNYVFGMGWFRQALGKEREFVAAITRSGGIPLYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAIDRQYYSGRNYPQSDPAEYDYWGQGTQVTVSS SEQ ID NO:48: DVQLQESGGGLVQAGGSLRLSCSASGSIVSIISMGWYRQAPGKQRELIAKITTDGTTNYADSVRGRFTISRDIPKKIMYLQMSSLTPEDTAVYFCNRDRLLYGLVKSDWGQGTQVTVSS SEQ ID NO:49: DVQLQESGGGLVQAGGSLRLSCAASGGIFSINAAAWYRQAPGKERELVAHIFHGGSTTYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYGGPPGMAIWGKGTQVTVSS SEQ ID NO:50: DVQLQESGGGLVQPGGSLRLSCAASGSIFSINAMGWYRQAPGKQRDVVAYIMSDGNTHYADSVKGRFTISRDKNTSYLQMNSLKPDDTAVYYCNADIQPPMPEATIYVYWGQGTQVTVSS SEQ ID NO:51: DVQLQESGGGLVQAGGSLRLSCTASGTIFNVMGWYRQAPGKQRELVATITFGLATNYADSVKGRFAISRDDSEKTVYLQMNSLKPEDTAVYYCNAGSRYWGQGTQVTVSS SEQ ID NO:52: DVQLQESGGGLVQAGGSLRLSCAASGSTSGYSFGFWTMNWYRQGSGKQRELVATITSSGITTYADSVKGRFTVSRDNTKNTLYLQMNSLKPEDTATYYCNFPGNSWGQGTQVTVSS SEQ ID NO:53: DVQLQESGGGLVQAGGSLRLSCTASGSIVSIISMGWYRQAPGKQRELIAKVTTDGTTNYADSVRGRFTISRDIPKKIMYLQMSSLTPEDTAVYFCNRDRLLYGLVKSDWGQGTQVTVSS SEQ ID NO:54: DVQLQESGGGLVQPGGSLRLSCLVSGSIYSIPAMYWYRQAPGKQREFVADITNRGSTNYADSVKGRFTISRDNAKNTVNLQMNSLKPEDTAVYYCNVLGTLGPRGSLQDYWGQGTQVTVSS SEQ ID NO:55: DVQLQESGGGLVQPGGSLRLSCTASGSIVSIISMGWYRQAPGKQRELIAKITTDGTTNYADSVRGRFTISRDIPKKIMYLQMSSLTPEDTAVYFCNRDRLLYGLVKSDWGQGTQVTVSS SEQ ID NO:56: DVQLQESGGGLVQAGGSLRLSCAASVRTFSKYDIGWFRQAPGKEREFVATISSSGRSTYYADFVKGRFGISRDYAKNTVYLEMNSLKPEDTAVYACAVRLGYSGSETDPRQYAYWGQGTQVTVSS SEQ ID NO:57: DVQLQESGGGLVQAGGSLRLSCTASGSIVSIISMGWYRQAPGKQRELIAKITTDGTTNYADSVRGRFTISRGIPKKIMYLQMSSLTPEDTAVYFCNRDRLLYGLVKSDWGQGTQVTVSS SEQ ID NO:58: DVQLQESGGGLVQTGGSLRLSCAASGRTFEHYTMGMGWFRQAPGKEREFVAAISWSDGSTYYEDSVKGRFTISRGNAKTTVYLQMNSLEPEDTGVYICAAKRGIGSDYPATLLYEYDYWGQGTQVTVSS SEQ ID NO:59: DVQLQESGGGLVQAGGSLRLSCAVSGRTISDMSVGWFRQAPGKEREFVAADTWAGAKTSYADSVKGRFTISRDTAKAMVYLQMNGLKPEDTAVYYCAGRTSYGLSTYARIEDYYGWGQGTQVTVSS SEQ ID NO:60: DVQLQESGGGLVQAGGSLRLSCAASGRTFNTNAMGWFRQAPGKEREFVAAIDWSGDSTFYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADGLLYYSGSYYPDYWGQGTQVTVSS SEQ ID NO:61: DVQLQESGGGLVQAGGSLRLSCAVSGRTISDMSVGWFRQAPGKEREFVAADSWAGAKASYADSVKGRFTISRDNTKAMVHLQMNGLKPEDTAVYYCAGRTSDGLSTYARIEDYYGWGQGTQVTVSS SEQ ID NO:62: DVQLQESGGGLVQAGGSLRLSCTASGTIFNVMGWYRQAPGKQRELVATITFGLTTNYADSVKGRFAISRDSSEKTVYLQMNSLKPEDTAVYYCNAGSRYWGQGTQVTVSS SEQ ID NO:63: DVQLQESGGGLVQAGGSLRLSCAVSGRTISDMSVGWFRQAPGKEREFVAADTWAGAKTSYADSVKGRFTISRDNAKAMVYLQMNGLKPEDTAVYYCAGRTSYGLSTYARIEDYYGWGQGTQVTVSS SEQ ID NO:64: DVQLQESGGGLVQPGGSLRLSCAASGSAFSRIGVGWYRQAPGKEREFVAVITAGGNTNCADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCNYPGRPWGQGTQVTVSS SEQ ID NO:65: DVQLQESGGGLGQAGGSLRLSCAVSEIIFRFTTLNWYRQAPGKQRELVASISSGGSTNYDDSVKGRFTISRDNAKKLVYLQMNSLKPEDTAVYYCNVPGNPWGQGTQVTVSS SEQ ID NO:66: DVQLQESGGGLVQPGGSLKLSCAVSGRTISGMSVGWFRQAPGKEREFVAADSWAGAKTSYADSVKGRFTISRDNAKAMVYLQMKGLKPEDTAVYYCAGRTSDGLFNYARIEDYYGWGQGTQVTVSS SEQ ID NO:67: DVQLQESGGGFVQAGDSLRLSCAASGRAVISYTLGWFRQAPGKEREFVATISWTDGSTYYSDSVKGRFTMSRDDAKKTVYLQMNSLKPEDTAVYYCAAVFRRVPTSATLYDYWGQGTQVTVSS SEQ ID NO:68: DVQLQESGGGLVQAGDSLRLSCAVSGRTISGMAVGWFRQAPGKEREFVAADSRAGAKTSYADSVKGRFTLSRDNAKAMVYLQMNGLKPEDTAVYYCAGRTSDGLSTYARIQDYYGWGQGTQVTVSS SEQ ID NO:69: DVQLQESGGGLVQPGGSLRLSCAASGSAFSRIGVGWYRQAPGKEHEFVAEINAGGNTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCNYPGRPWGQGTQVTVSS SEQ ID NO:70: DVQLQESGGGLVQPGGSLRLSCAASGRFFSISAMGWYRQAPGKQRELVAAIINGGGTHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADAQMESGTLYFIVFWGQGTQVTVSS SEQ ID NO:71: DVQLQESGGGLVQAGDSLRLSCAVSGRTISDMSVGWFRQAPGKEREFVAADSWAGAKTSYADSVKGRFTISRDNAKAMVYLQMNGLKPEDTAVYYCAGRTSDGLSTYARIEDYYGWGQGTQVTVSS SEQ ID NO:72: DVQLQESGGGLVQPGDSLRLSCAASGRTFSSYDMGWFRQAPGKEREFVAAISRSGRDTLHADSVKGRFTISRDNAKNTVYLQMYSLKPEDTAVYICAGTGFEYAGSTPYKYPYWGQGTQVTVSS SEQ ID NO:73: DVQLQESGGGLVQPGGSLRLSCAASGSAFSRIGVGWYRQAPGKEREFVAVIAAGGNTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCNYPGRPWGQGTQVTVSS SEQ ID NO:74: DVQLQESGGGLVQAGGSLRLSCAASGGIFSRDVMGWYRQAPGEQRELVAAITIGGSASYADSVKGRFTISRDNAKNTVYLRMNSLTPEDTAVYFCKLGYGSGSWGQGTQVTVSS SEQ ID NO:75: DVQLQESGGGLVQAGGSLRLSCVVSGRTLSGMSVGWFRQAPGKEREFVAADSWAGAKASYADSVKGRFTISRDNAEAIVYLQMNGLKPEDTAVYYCAGRTSYGLSTYARIEDYYGWGQGTQVTVSS SEQ ID NO:76: DVQLQESGGGLVQAGGSLRLSCAVSGRTISGMSVGWFRQAPGKEREFVAADTWAGAKTSYADSVKGRFTISRDNTKAMVYLQMNGLKPEDTAVYYCAGRTSDGLSTYARIEDYYGWGQGTQVTVSS SEQ ID NO:77: DVQLQESGGGLVQPGGSLRLSCAASGSIFSINAMGWYRQAPGKQRELVAHITNIGTTNYADSVKGRFTISRDNGKNTVYLQMNSLKPEDTAVYICNADLPSMVETDYYLVYWGQGTQVTVSS SEQ ID NO:78: DVQLQESGGGLVQAGDSLRLSCAVSGRTISDMSVGWFRQAPGKEREFVAADSWAGAKTSYADSVKGRFTISRDNAKAMVYLQMSGLKPEDTAVYYCAGRTSSGLSTYARIEDYYGWGQGTQVTVSS SEQ ID NO:79: QVQLQESGGGSVQAGGSLRLSCAAVSGITFRRNCMGWFRQVPGKEREGVATLDGASLTRYADSVKGRFIISQDNAKNTLYLQMNALKPEDTAMYYCAASPTRYGCGGADFDSWGQGTQVTVSS SEQ ID NO:80: QVQLVESGGGLVQAGDSVTLSCTASGRTFSSYALAWFRQRPGKEREFVAGLRWGGPTNYADSVKDRFTISGDSAKHTMYLQMNSLKPEDTAVYYCGYNPGGWAVPSQYEYDSWGQGTQVTVSSAHHSEDPS SEQ ID NO:81: QLQLVESGGGAAQTGGSLRLSCAASGGDSNINIMGWNRQVPGKQRELVAAITSDGNSNYGGSAKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTVWTYSGKYEGQGTQVTVSSEPKTPKPQD SEQ ID NO:82: ESGGGSVQTGGSLRLSCTASEYTYMNNCMGWFRQTPGKEREGIAAISSDGRRIEYDDSVKGRFVLSRDNDRNALILEMNALKSEDSGRYYCAAVRWGYPYSKSSYLGPGDPGHRLL SEQ ID NO:83: ATGGCCCAGGTGCAGCTCGTGGAGTCAGGCGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCCTCTGGACGCGCCTTTCGTAGATATACCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAACAATTAACTGGAGTGGTCGTAATACAGCGTATGCCGACTCCGTGAAGGGCC GATTCACCATCTCCAGAGACAGCCGCAAAAACACCGCGTATCTCCAAATGAACAGCCTGAAACCTGAAGATACGGCCGTTTATTGTGCACAATCGCGAGCGATTACAGGTGGCACAGTTCCCGCCGGTTATAACATCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAGCGGCCGC SEQ ID NO:84: QVQLVESGGGLVHSGGSLRLSCAASGFTFSSEWMYWVRRDPLQWVSSINSPGSKTYYADSVRGRFTISRDNDKNMVYLQMNSLKPEDTGIYYCVRGVAQSVTGGEWYKVDLGDLEEWGQGTQVTVSS SEQ ID NO:85: EVQLVESGGDQVQPGGSLRLSCVGSGFSFGTYTMMWFRHSPGKRLEWVSTINSDGSNPRYLNSVEDRFIISRDNAKNTLYLQMNNLKSEDTGLYYCTNGGTRGRGTQVTVSS In a second aspect, the present invention provides a Nanobody with targeting function, whose amino acid sequence is shown in any one of SEQ ID NO: 4-78; or, the amino acid sequence of the Nanobody has an identity of ≥80% with the amino acid sequence shown in any one of SEQ ID NO: 4-78; preferably ≥90%; more preferably ≥95%.

[0020] In a third aspect, the present invention provides a lipid nanoparticle, the components of which include the molecule with targeting function, wherein the content of the molecule with targeting function in the total lipid of the lipid nanoparticle is: 0.01-1.0 mol%, preferably 0.01-0.5 mol%.

[0021] Preferably, the components of the lipid nanoparticles include polymer-bound lipids and ionizable amino lipids; the polymer-bound lipids are selected from lipids modified with PEG or its derivatives; The relative molecular mass of the lipid modified with PEG or its derivatives is 2000-5000, preferably 2000, 3000, 4000 or 5000; Preferably, the content of the polymer-bound lipid in the total lipid of the lipid nanoparticles is 0.2-5 mol%, preferably 0.5-2 mol%.

[0022] Preferably, the PEG derivative comprises maleimide-functionalized polyethylene glycol and non-maleimide-functionalized polyethylene glycol; wherein the polyethylene glycol can be selected from DMG-PEG2000; the maleimide-functionalized polyethylene glycol can be selected from PEG-MAL; the content of maleimide-functionalized polyethylene glycol in the total lipid of the lipid nanoparticles is: 0.01-1.0 mol%, preferably 0.01-0.5 mol%.

[0023] Preferably, the ionizable amino lipid accounts for 30-70 mol% of the total lipids in the lipid nanoparticles, preferably 40-60 mol%. More preferably, the ionizable amino lipid has a structure represented by general formula (I), or is an isomer, pharmaceutically acceptable salt, prodrug or solvate of the structure represented by general formula (I); .

[0024] wherein G is selected from H, OR, CN, -C(=O)OR', -OC(=O)R', -C(=O)NR'R'', -NR'C(=O)R'', NR'R'' or a cyclic alkyl structure containing at least one heteroatom; wherein the heteroatom is O or N; Wherein, R, R', R'' are the same or different from each other and are independently selected from H, C1-C 10 Alkyl, C3-C 10 Cycloalkyl, C3-C 10 Alkenyl or C3-C 10 Cycloalkenyl, C1-C 10 Alkyl, C3-C 10 Cycloalkyl, C3-C 10 an alkenyl group or a cyclic alkyl structure containing at least one heteroatom; Preferably, R, R', and R'' are the same or different from each other and are independently selected from H, C1-C8 alkyl, C3-C8 cycloalkyl, C3-C8 alkenyl, C3-C8 cycloalkenyl, C1-C8 terminally connected tertiary amine,10 Alkyl, C3-C 10 Cycloalkyl, C3-C 10 an alkenyl group or a cyclic alkyl structure containing at least one heteroatom; wherein the substitutable carbon atoms or heteroatoms in the cyclic alkyl structure are unsubstituted or substituted with one or more hydroxyl groups, C1-C4 alkyl groups, C2-C4 alkenyl groups, C3-C8 cycloalkyl groups, or C3-C8 cycloalkenyl groups; Preferably, the cyclic alkyl structure is unsubstituted or substituted with one or more C1-C4 alkyl groups, C3-C8 cycloalkyl groups or hydroxyl groups; More preferably, R' and R'' are the same or different from each other and are independently selected from H, C1-C4 alkyl, C1-C4 alkyl terminally connected to a tertiary amine, 10 Alkyl, C3-C 10 Cycloalkyl or a cyclic alkyl structure containing at least one heteroatom; the cyclic alkyl structure is unsubstituted or substituted by one or more C1-C4 alkyl or C3-C8 cycloalkyl groups.

[0025] And / or, M1, M2, M3, M4 are the same as or different from each other and are each independently selected from C1-C 24 Alkylene, C3-C 24 Cycloalkylene, C2-C 24 Alkenylene or C3-C 24 Preferably, M1, M2, M3, M4 are the same as or different from each other, and M1 and M4 are each independently selected from C4-C 22 Alkylene, branched C4-C 22 Cycloalkylene, C4-C 22 Alkenylene or branched C4-C 22 Cycloalkenylene, M2 and M3 are each independently selected from C4-C 22 Alkylene, C4-C 22 Cycloalkylene, C4-C 22 Alkenylene or C4-C 22 Preferably, M2 and M3 are the same and are C4-C 22 Alkylene; M1 is the same as M4 and is a C4-C 22 alkylene; and / or, R 1 、R 2 are the same as or different from each other and are independently selected from H, C1-C 24 Alkyl, C3-C 24 Cycloalkyl, C2-C 24 Alkenyl or C3-C 24 Cycloalkenyl; preferably, R1 、R 2 are the same as or different from each other and are each independently selected from C4-C 22 Alkyl or C4-C 22 alkenyl; and / or, L1, L2, L3, L4 are identical or different from each other and are independently selected from -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=O)NR-, -NRC(=O)-, -S(=O)-, -OS(=O)2-, -S(=O)2O-, -O-, -S- or -SS-; preferably, L1, L2, L3, L4 are identical or different from each other and are independently selected from -C(=O)O-, -OC(=O)-, -C(=O)NR-, -NRC(=O)- or -SS-; when L1, L2, L3, L4 are independently selected from -C(=O)NR- or -NRC(=O)-, wherein R is independently selected from H or Cl-C 10 Alkyl; preferably, L1 and L4 are the same and are -C(=O)O- or -OC(=O)-; And / or, M5 is selected from a single bond, C1-C 16 Alkylene, C2-C 16 Preferably, M5 is selected from a single bond, a C2-C 16 Alkylene, C2-C 16 Alkenylene, C4-C8 cycloalkylene or C3-C8 cycloalkenylene; preferably, M5 is selected from a single bond, C2-C 16 Alkylene or C4-C6 cycloalkylene; Further preferably, M5 and G are connected to form , specifically one of A1~A52 can be selected: .

[0026] Among them, the Preferably one selected from A1-A18, A22-A24, A28-A52; further preferably one selected from A15, A23, A29, A33, A39-A52; and / or, R 1 -L1-M1-L2-M2- fragment is R 1 -C(=O)O-M1-OC(=O)-M2-、R 1 -C(=O)NR-M1-OC(=O)-M2-or R 1 -C(=O)O-M1-SS-M2-;R 2 -L4-M4-L3-M3- fragment is R 2-C(=O)O-M4-OC(=O)-M3-、R 2 -C(=O)NR-M4-OC(=O)-M3-or R 2 -C(=O)O-M4-SS-M3-; More preferably, the structure of general formula (I) is selected from one of the following structures: .

[0027] Preferably, the lipid nanoparticles further comprise at least one of a steroid and a neutral lipid; Preferably, the molar ratio of ionizable amino lipid, steroid, neutral lipid and polymer-bound lipid is 30-70:0-65:0-30:0.2-5; more preferably 40-60:35-60:0-20:0.5-2; Further preferably, the steroid is one or more of cholesterol and its derivatives, cholesterol esters, steroid hormones, steroid vitamins, and phytosterols, preferably cholesterol; and / or the neutral lipid is a phospholipid; and / or the polymer-bound lipid is a pegylated lipid, preferably DMG-PEG2000; More preferably, the molar ratio of ionizable amino lipid, cholesterol, phospholipid and PEGylated lipid is 40-50:40-45:0-15:0.5-2.

[0028] Preferably, the lipid nanoparticles are targeted to deliver nucleic acids to target cells of a subject, including immune cells, central nervous cells, tumor cells, etc. When the target cells are contacted with the lipid nanoparticles, the lipid nanoparticles provide at least one of the following benefits: (i) increased specificity of targeted delivery to the target cells compared to a reference LNP; (ii) the half-life of the nucleic acid or the polypeptide encoded by the nucleic acid in the cell of interest is increased compared to a reference LNP; (iii) increased transfection efficiency compared to reference LNPs; and (iv) Low levels of dye-accessible mRNA (<15%) and high RNA encapsulation efficiency, with at least 80% of the mRNA recovered in the final formulation relative to the total RNA used in the LNP bulk preparation.

[0029] In a fourth aspect, the present invention provides a method for preparing the lipid nanoparticles, which comprises the following steps: mixing polymer-bound lipids, ionizable amino lipids, steroids and auxiliary phospholipids to obtain lipid nanoparticles without targeting function, and then connecting the molecules with targeting function to the lipid nanoparticles without targeting function through membrane fusion or chemical bonds to obtain lipid nanoparticles with targeting function.

[0030] In a fifth aspect, the present invention provides another method for preparing the lipid nanoparticles, comprising the steps of: mixing a polymer-bound lipid, an ionizable amino lipid, a steroid, and an auxiliary phospholipid to obtain lipid nanoparticles without a targeting function, and then chemically linking a nanobody to the lipid nanoparticles without a targeting function to obtain lipid nanoparticles with a targeting function; Preferably, the polymer-binding lipid comprises maleimide-functionalized polyethylene glycol, and the Nanobody is linked to the maleimide-functionalized polyethylene glycol.

[0031] In a sixth aspect, the present invention provides another method for preparing the lipid nanoparticles, which comprises the following steps: mixing polymer-bound lipids, ionizable amino lipids, steroids, auxiliary phospholipids and the molecules with targeting function to obtain lipid nanoparticles with targeting function.

[0032] In a seventh aspect, the present invention provides a method for targeted delivery of a pharmacodynamic molecule to specific cells of a subject, the method comprising: contacting the specific cells with the lipid nanoparticles; preferably, the specific cells include but are not limited to immune cells, immune-related cells, central nervous cells, tumor cells, somatic cells, cells infected by viruses, bacteria, and fungi; preferably, the targeted cells are B cells, T cells, or NK cells.

[0033] In an eighth aspect, the present invention provides a method for expressing a target protein or polypeptide in a target cell of a subject, the method comprising: contacting the target cell with the lipid nanoparticle; preferably, the target cell includes but is not limited to immune cells, immune-related cells, central nervous cells, tumor cells, somatic cells, cells infected by viruses, bacteria, and fungi; preferably, the target cell is a B cell, a T cell, or a NK cell.

[0034] In a ninth aspect, the present invention provides a method for treating, ameliorating or preventing symptoms of a disease in a subject in need thereof, the method comprising: administering the lipid nanoparticles to the subject to deliver the pharmacodynamic molecule to the target cells of the subject; preferably, the target cells include but are not limited to immune cells, immune-related cells, central nervous cells, tumor cells, somatic cells, cells infected by viruses, bacteria, or fungi; preferably, the disease is an immune disease, an inflammatory disease, a central nervous system disease, a degenerative disease, a bone-related disease, a pathogen infection, a metabolic disease, an endocrine disease, aging, or cancer; preferably, the pharmacodynamic molecule includes an antigen used in a therapeutic or preventive vaccine for the treatment or prevention of an immune disease, an inflammatory disease, a central nervous system disease, a degenerative disease, a bone-related disease, a pathogen infection, a metabolic disease, an endocrine disease, aging, or cancer; preferably, the target cells are B cells, T cells, or NK cells.

[0035] In a tenth aspect, the present invention provides the use of the aforementioned lipid nanoparticles as a drug delivery carrier.

[0036] In an eleventh aspect, the present invention provides a pharmaceutical composition comprising the lipid nanoparticles, wherein the lipid nanoparticles contain pharmacodynamic molecules; Preferably, the pharmacodynamic molecule comprises one or more of mRNA, DNA, siRNA, saRNA, shRNA, miRNA, circle RNA, lnc RNA, gRNA, polypeptide or protein.

[0037] Preferably, the pharmacodynamic molecule includes mRNA, which is used to encode a CAR molecule, a TCR molecule, an immunomodulatory molecule, a functional protein molecule, or a gene editing tool such as a base editor or a Cas9 protein.

[0038] Preferably, the CAR molecule comprises at least one of a sequence that can specifically recognize a tumor-specific antigen, a transmembrane linker sequence, and / or a sequence that assists the immune function of T cells.

[0039] Preferably, the sequence encoding the TCR molecule comprises a sequence that can recognize at least one of NY-ESO-1, AFP, HBsAg, MAGEA1, Mesothelin, MART-1, CD19, CD28, PD-1, CD8, CT83, E6, E7, E8, GPC3, H3.3-K27M, HIV Gagpolyprotein, HLA-A / AFP, KRASG12 (V / D), KRASG12D, KRASG12V, LMP1, LMP2, EBNA1, MAGEA10, MAGEA3, MAGEA4, MC2R, mHag HA-1, MYO1G, PRAME, WT1, gp100, CEA, p53, HLA-A2, EGFR, DR5, RAS, LAGE-1, CMV, HCV, MCPyv, and HA-1H.

[0040] Preferably, the sequences encoding immunomodulatory molecules include CD28, PD-1, CTLA-4, RGMB, ICOS, CD28H, NKp30, HVEM, OX40, Fas, 4-1BB, CD27, CD30, APO-2, APO-3, BCMA, BAFFR, GITR, IL-2, IL-12A, IL-12B, IL-15, IL-23, IL-27, FLT3L, IL-36A, IL-36B, IL-36C, GM-CSF, CCL20, CXCL9, CXCL10, CXCL11, CXCL12, CCR7, CXCL11, CXCL12, CCR8, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL18, CXCL19, CXCL20, CXCL21, CXCL22, CXCL23, CXCL24, CXCL25, CXCL26, CXCL27, CXCL28, CXCL29, CXCL30, CXCL31, CXCL32, CXCL33, CXCL34, CXCL35, CXCL36, CXCL37, CXCL38, CXCL39, CXCL31, CXCL39, CXCL31, CXCL33, CXCL34, CXCL35, CXCL37, CXCL38, CXCL39, CXCL31, CXCL33, CXCL34, CXCL35 The sequence of at least one of CR4, CCR5, CCL4, CCL5, CCL19, CXCR3, CCR6, B7-H1, B7-DC, B7-H3, B7-H4, PD-1H, IL-10, TGF-β, HGF, B7-1, B7-2, B7-H2, B7-H3, CD40, FasL, CD70, CD30L, 4-1BBL, OX40L, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, GITRL, CD73, STAT1, IRF4, CCAR2, BCL6, iNOS, and CD103.

[0041] Preferably, the nucleic acid encoding the CAR molecule comprises a sequence that can recognize at least one molecule of CD19, BMCA, CD22, CD20, CD123, GD2, CD30, GPC3, CLDN18.2, Mesothelin, CD33, CD38, EGFRvlll, CD138, CEA, HER2, PSMA, CLL1, CD56, EGFR, MUC-1, EpCAM, CD7, NKG2D, PD-L1, LewisY, FAP, c-MET, ROR1, IL13Rα2, AFP, CD133, CD4, BTK, ROBO1, CD5, CD70, LILRB4, FLT3, Sigle-6, CD229, and SLAMF7.

[0042] In a twelfth aspect, the present invention provides a pharmaceutical preparation comprising the pharmaceutical composition and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical preparation is an aqueous injection, a lyophilized powder or a spray.

[0043] Beneficial effects: The present invention provides a molecule with a targeting function, which can specifically deliver drugs to specific cells, such as T cells, B cells, NK cells, NKT cells, γδT cells, regulatory T cells, eosinophils, basophils, mast cells, neutrophils, platelets, mesenchymal stem cells, macrophages, plasma cells, dendritic cells, microglia, neurons, astrocytes, endothelial cells, osteoclasts, osteoblasts, synovial cells, tumor cells, etc., such as specifically delivering the CAR molecule nucleic acid sequence to T cells. For example, by specifically delivering TCR molecule nucleic acid sequences into T cells, TCR-T cells targeting specific antigen sequences can be directly generated in the body to kill virus-infected cells or tumors. For example, by specifically delivering immune functional molecules or molecules that block cell function inactivation into immune cells, immune cell function can be altered and immune response can be precisely regulated. For example, by specifically delivering functional regulatory molecules to other types of cells, target cells can be precisely programmed in the body to achieve the desired effect of the drug molecules. Specific cell delivery can significantly improve drug efficacy and reduce side effects caused by drugs acting on other cells, achieving the following: 1. Pharmacological molecules can directly enter the target cells, even target cells that traditional drugs cannot enter, and 2. Extremely low dosages can achieve maximum therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0045] Figure 1 This is the quality control information of the LNP containing coupling sites in Example 20 of the present invention.

[0046] Figure 2 This is the in vitro transfection situation of LNPs with different targeting molecular structures in Example 20 of the present invention.

[0047] Figure 3 This is the in vivo transfection situation of TLNPs with different targeting molecular structures in Example 20 of the present invention.

[0048] Figure 4 These are the specific characterization data of Pre-TLNP in Example 21 of the present invention.

[0049] Figure 5 These are the specific characterization data of TLNP in Example 21 of the present invention.

[0050] Figure 6 This is the in vivo delivery efficiency of TLNP with different targeting molecule ratios in Example 21 of the present invention.

[0051] Figure 7 This is the flow cytometric analysis of the EGFP-mRNA expression efficiency of cLNP-1 and TLNP-2 in hPBMC in Example 24 of the present invention.

[0052] Figure 8 This is the in vivo transfection situation of TLNP-2 at different dosages in Example 24 of the present invention.

[0053] Figure 9 This is a statistical diagram of the in vivo transfection status of TLNP-2 with different ratios in Example 25 of the present invention.

[0054] Figure 10 This is a long-term stability study of traditional LNP and targeted TLNP in Example 27 of the present invention.

[0055] Figure 11 These are the results of the biodistribution study in Balb / c mice using Cy5 fluorescence, Luc imaging, and qPCR in Example 29 of the present invention.

[0056] Figure 12 These are the results of the Luc imaging and qPCR studies on the biodistribution of NGC in mice in Example 29 of the present invention.

[0057] Figure 13 These are the dosage safety study data of TLNP-2 at different working concentrations in hPBMC in Example 30 of the present invention.

[0058] Figure 14These are the liver toxicity index test results of the acute toxicity experiment in mice of traditional cLNP and targeted TLNP at different doses in Example 31 of the present invention.

[0059] Figure 15 This is the EGFP expression of TLNP-2 in NHP in Example 32 of the present invention.

[0060] Figure 16 These are the EGFP targeting and off-target results of TLNP-2 in NHP in Example 32 of the present invention.

[0061] Figure 17 This is the dynamic changes of EGFP expression of TLNP-2 in NHP in Example 32 of the present invention.

[0062] Figure 18 The weight change of NHP after administration of TLNP-2 in Example 33 of the present invention.

[0063] Figure 19 The temperature change of NHP after administration of TLNP-2 in Example 33 of the present invention.

[0064] Figure 20 The following are the changes in the routine blood test of NHP after administration of TLNP-2 in Example 33 of the present invention.

[0065] Figure 21 The changes in cytokines in NHP serum after administration of TLNP-2 in Example 33 of the present invention.

[0066] Figure 22 This is the blood biochemistry of NHP after administration of TLNP-2 in Example 33 of the present invention.

[0067] Figure 23 These are the in vitro transfection results of CD5-TLNPs containing different targeting molecules in Example 34 of the present invention.

[0068] Figure 24 These are the in vitro transfection results of CD5-TLNPs containing different targeting molecule ratios in Example 35 of the present invention.

[0069] Figure 25 This is the in vivo transfection situation of L2-TLNP with different ratios in Example 36 of the present invention.

[0070] Figure 26 These are the experimental results of Luc imaging and qPCR in Example 37 of the present invention to study the biodistribution of hCD5 in humanized mice. DETAILED DESCRIPTION

[0071] The present invention provides molecules and applications with targeting capabilities. Specifically, the present invention provides lipid nanoparticles with targeting capabilities. These lipid nanoparticles with targeting capabilities comprise ionizable amino lipids, polymer-bound lipids, and nanobodies; optionally, these lipid nanoparticles with targeting capabilities also include steroids and / or phospholipids.

[0072] In order to better illustrate the method of obtaining the lipid nanoparticles with targeting function described in the present invention, the following is described by way of example.

[0073] (I) Preparation of five-component cell-targeting nanoparticles (comprising ionizable amino lipids, polymer-bound lipids, steroids, phospholipids, and nanobodies) Method 1: Post-insertion chemical coupling Ionizable amino lipids E12LA6B6O3, phospholipids, cholesterol, DMG-PEG 2000 The PEG lipid derivative containing chemical coupling sites was dissolved in ethanol at a specific molar ratio, and the mRNA was dissolved in an acidic buffer solution to ensure that the mRNA concentration in the aqueous phase was 0.33 mg / mL. The molar ratio of ionizable amino lipid to mRNA base (N / P) was 6.5.

[0074] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a 1:3 ethanol to aqueous ratio. The resulting crude LNPs were terminated by the addition of 25 mM Tris buffer. Ultrafiltration was then performed once at 4°C and 1200 rcf, followed by two additional ultrafiltration cycles using 25 mM Tris buffer. The LNPs containing the chemical conjugation sites (Pre-TLNPs) were collected. After adding 0.278 times the volume of a 400 mg / mL sucrose solution, the mixture was sterile filtered using a 0.22 μm PES filter in a clean hood.

[0075] Take a certain amount of the above-mentioned Pre TLNP, mix it with the corresponding nanobody in a certain molar ratio, and o Incubate overnight at 40°C. Dilute the LNP mixture after overnight incubation with 87 mg / mL sucrose solution and purify using a 100 kD ultrafiltration tube. Sterile filtration using a PES filter with a pore size of 0.22 μm in a clean bench yields the final nanobody-modified cell-targeting nanoparticles (TLNPs).

[0076] Method 2: Targeted functional molecule post-insertion incubation method The PEG lipid derivative containing chemical coupling sites and the nanobody were mixed in a PBS buffer solution at a certain molar ratio and then oIncubate overnight at 400°C. The reaction solution is purified by column to obtain a PEG lipid-nanoantibody conjugate with targeting function (PEG-Nb).

[0077] Ionizable amino lipids, phospholipids, cholesterol and DMG-PEG 2000 Dissolve the lipids in ethanol and mRNA in an acidic buffer solution at a specific molar ratio to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio (N / P) of ionizable amino lipid to mRNA was 6.5. The LNP preparation process was similar to that described in Method 1.

[0078] A certain amount of the prepared LNP was mixed with the targeting functional molecule (PEG-Nb) in a certain molar ratio and then o The cells were incubated overnight at 400 °C. The subsequent LNP purification and quality control methods were carried out according to the procedures described in Method 1, and nanobody-modified cell-targeted nanoparticles (TLNPs) were finally obtained.

[0079] Method 3: One-step insertion of targeted functional molecules Ionizable amino lipids, phospholipids, cholesterol and DMG-PEG 2000 The targeting functional molecule (PEG lipid-nanobody conjugate) and mRNA were dissolved in ethanol at a specific molar ratio in an acidic buffer solution to ensure a mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio (N / P) of the ionizable amino lipid to mRNA was 6.5. Subsequent LNP preparation, purification, and quality control procedures were carried out as described in Method 1 to obtain nanobody-modified cell-targeting nanoparticles (TLNPs).

[0080] (II) Preparation of Four-Component Cell-Targeting Nanoparticles (Comprising Ionizable Amino Lipids, Polymer-Bound Lipids, Steroids, and Nanobodies) Ionizable amino lipids, cholesterol, DMG-PEG 2000 and DSPE-PEG 2000 -Mal lipids were dissolved in ethanol at a molar ratio of 40:58.5:1.45:0.05, and mRNA was dissolved in an acidic buffer solution to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio (N / P) of ionizable amino lipid to mRNA was 6.5.

[0081] The specific preparation process of LNP can be carried out according to method 1 in (I) to obtain the final four-component cell-targeted nanoparticles (TLNP).

[0082] (III) Preparation of Four-Component Cell-Targeting Nanoparticles (Comprising Ionizable Amino Lipids, Polymer-Bound Lipids, Phospholipids, and Nanobodies) Ionizable amino lipids, phospholipid DSPC, DMG-PEG 2000 and DSPE-PEG 2000 -Mal lipids were dissolved in ethanol at a molar ratio of 47.5:51.0:1.45:0.05, and mRNA was dissolved in an acidic buffer solution to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio (N / P) of ionizable amino lipid to mRNA was 6.5.

[0083] The specific preparation process of LNP can be carried out according to method 1 in (I) to obtain the final four-component cell-targeted nanoparticles (TLNP).

[0084] The following examples provide a detailed description of the technical solutions provided by the present invention, but they should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are all commercially available.

[0085] Example 1: In this example, a targeting functional molecule post-insertion incubation method was used to prepare five-component cell-targeting nanoparticles (containing ionizable amino lipids, polymer-bound lipids, steroids, phospholipids, and nanobodies).

[0086] DSPE-PEG2000-Mal lipids containing chemical coupling sites were mixed with CD8 nanobodies (with or without GS-cysteine ​​at the end) at a molar ratio of 1:1 in a PBS reaction medium and incubated for 4 o The reaction mixture was incubated overnight at 400 °C. After purification by hydrophobic interaction chromatography, the coupling product of CD8 nanobody and DSPE-PEG2000-Mal lipid was obtained, which is the CD8 targeting functional molecule.

[0087] Ionizable amino lipids E12LA6B6O3, phospholipids, cholesterol CHO and DMG-PEG 2000 Dissolve the mRNA in ethanol and the aqueous phase in an acidic buffer solution according to the molar ratios shown in Table 1, ensuring an mRNA concentration of 0.33 mg / mL. The molar ratio of ionizable amino lipid to mRNA (N / P) is 6.5.

[0088] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0089] The crude LNP product, supplemented with the stop solution, was purified by ultrafiltration once at 4°C and 1200 rcf. Subsequently, the ultrafiltrate was concentrated twice at 4°C and 1200 rcf by adding 25 mM Tris buffer. After the LNP product was collected, 0.278 times the volume of 400 mg / mL sucrose solution (prepared to volume using 25 mM Tris buffer, pH = 7.4-7.5) was added and sterile filtered using a 0.22 μm PES filter in a laminar flow hood. A 30 μL sample was aspirated for particle size, dispersion coefficient, and encapsulation efficiency determination. The remaining sample was stored in a refrigerator at 4°C.

[0090] A certain amount of the aforementioned LNPs was mixed with the CD8-targeting functional molecule according to the recipe in Table 1, and then incubated overnight at 4°C. The LNP mixture was diluted with 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH 7.4-7.5) and purified using a 100 kD ultrafiltration tube. After sterile filtration using a 0.22 μm pore size PES filter in a laminar flow hood, CD8-antibody-modified cell-targeting nanoparticles (CD8-TLNPs) were obtained.

[0091] Table 1: Proportional formula of lipids and targeting molecules for preparing CD8-TLNPs by post-insertion method

[0092] Example 2: This example uses a one-step insertion method of targeting functional molecules to prepare five-component cell-targeting nanoparticles (containing ionizable amino lipids, polymer-bound lipids, steroids, phospholipids, and targeting functional molecules). The CD8 targeting functional molecule was prepared according to the method described in Example 1.

[0093] Ionizable amino lipid E12LA6B6O3, phospholipids, cholesterol and DMG-PEG 2000Dissolve the CD8 targeting functional molecules and mRNA in ethanol according to the molar ratios listed in Table 2. Dissolve the CD8 targeting functional molecules and mRNA in an acidic buffer solution to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio (N / P) of the ionizable amino lipid to mRNA was 6.5.

[0094] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0095] The crude LNP product, supplemented with the stop solution, was purified by ultrafiltration once at 4°C and 1200 rcf. Subsequently, the ultrafiltrate was concentrated twice more at 4°C and 1200 rcf by adding 25 mM Tris buffer. After the LNP product was collected, 0.278 times the volume of 400 mg / mL sucrose solution (prepared to volume using 25 mM Tris buffer, pH = 7.4-7.5) was added. The product was sterile-filtered in a clean hood using a PES filter with a pore size of 0.22 μm to obtain CD8-antibody-modified cell-targeting nanoparticles (CD8-TLNPs).

[0096] Table 2: Proportional formula of lipids and targeting molecules for one-step preparation of CD8-TLNPs

[0097] Example 3: In this example, a post-insertion chemical coupling method was used to prepare five-component cell-targeting nanoparticles (containing ionizable amino lipids, polymer-bound lipids, steroids, phospholipids, and nanobodies). The specific steps are as follows: Ionizable amino lipids E12LA6B6O3, phospholipids, cholesterol, DMG-PEG 2000 Dissolve DSPE-PEG2000-Mal in ethanol according to the molar ratios listed in Table 3. Dissolve mRNA in an acidic buffer solution to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio (N / P) of ionizable amino lipid to mRNA was 6.5.

[0098] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0099] The crude LNPs, supplemented with the stop solution, were purified by ultrafiltration once at 4°C and 1200 rcf. Subsequently, the ultrafiltrate was concentrated twice at 4°C and 1200 rcf by adding 25 mM Tris buffer. The LNPs containing chemical coupling sites (Pre-TLNPs) were collected and then sterile-filtered using a 0.278-fold volume of 400 mg / mL sucrose solution (prepared to volume using 25 mM Tris buffer, pH 7.4-7.5) in a clean hood using a 0.22 μm PES filter. A 30 μL sample was removed for particle size, dispersion coefficient, and encapsulation efficiency determination, and the remaining sample was stored in a refrigerator at 4°C.

[0100] A certain amount of the above-mentioned Pre TLNP was taken, DSPE-PEG2000-Mal and the corresponding CD8 nanobody (with or without GS-cysteine ​​at the end) were mixed at a molar ratio of 1:1, and the mixture was stirred at 4 ℃ for 1 h. o Incubate overnight at 4°C. Dilute the LNP mixture after overnight incubation with 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH 7.4-7.5) and purify using a 100 kD ultrafiltration tube. Sterile filtration using a 0.22 μm pore size PES filter in a clean hood yields the final nanobody-modified cell-targeting nanoparticles (TLNPs). A 30 μL sample is aspirated for particle size, dispersion coefficient, and encapsulation efficiency testing, and the remaining sample is stored in a refrigerator at 4°C. After encapsulation efficiency testing, the sample is diluted to the desired concentration with 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH 7.4-7.5) and stored until further use.

[0101] Table 3: Lipid ratio formula for preparation of CD8-TLNPs by post-insertion method

[0102] Example 4: (I) Preparation of nanoparticles coupled with anti-CD19 nanoantibodies Refer to the preparation method in Example 3. Ionizable amino lipid: DSPC: CHO: DMG-PEG2000: DSPE-PEG2000-Mal were dissolved in ethanol at a molar ratio of 47.5: 10: 41: 1.49: 0.01. mRNA was dissolved in 75 mM citric acid buffer (pH 4) to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0103] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0104] The crude LNP was purified by ultrafiltration three times at 4°C and 1200 rcf, and the LNP containing chemical coupling sites (Pre-TLNP) was collected. It was then mixed with anti-CD19 nanoantibodies with cysteine ​​attached to the end at a molar ratio of 1:1 and then heated at 4 o The mixture was incubated overnight at 400 °C. Subsequently, sucrose solution with a concentration of 87 mg / mL was added for ultrafiltration and sterile filtration to obtain the final anti-CD19 nanoantibody-coupled nanoparticles (CD19-TLNP).

[0105] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD19 cells Anti-CD3 coating: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL per well, and store at 4°C overnight.

[0106] Preparation before cell plating: Aspirate the liquid in the 24-well plate, then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0107] Cell plating: Dilute human PBMC to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double antibody) supplemented with anti-CD28 antibody (concentration 1 μg / mL) and IL-2 (working concentration 400 U / mL). Add 1 million cells to each well of a 24-well plate. After plating, incubate at 37 o C, LNP was added after stabilization for 4 h under 5% CO2.

[0108] CD19-TLNPs encapsulating eGFP-mRNA were added to the plate to a working concentration of 0.1 μg / mL. A negative control group containing PBS was also added. After 24 hours, the expression of eGFP-mRNA in the CD19+ cell subset of PBMCs was determined by flow cytometry.

[0109] Table 4: CD19-TLNP in vitro transfection results

[0110] The experimental results showed that CD19-TLNP exhibited obvious targeting effect on CD19+ cells and had good transfection efficiency.

[0111] Example 5: Preparation of nanoparticles coupled with anti-CD133 nanoantibody Refer to the preparation method in Example 3. Ionizable amino lipid: DSPE: CHO: DMG-PEG4000: DSPE-PEG4000-Mal were dissolved in ethanol at a molar ratio of 50.5: 5: 43: 1.47: 0.03. mRNA was dissolved in 75 mM citric acid buffer (pH 4) to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0112] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0113] The crude LNP was purified by ultrafiltration three times at 4°C and 1200 rcf, and the LNP containing the chemical coupling site (Pre-TLNP) was collected. It was then mixed with the anti-CD133 nanobody with cysteine ​​attached to the end at a molar ratio of 1:1.2 and then o The mixture was incubated overnight at 400 °C. Subsequently, sucrose solution with a concentration of 87 mg / mL was added for ultrafiltration and sterile filtration to obtain the final anti-CD133 nanoantibody-coupled nanoparticles (CD133-TLNP).

[0114] Example 6: (I) Preparation of nanoparticles coupled with anti-PSMA nanobody Refer to the preparation method in Example 3. Ionizable amino lipid: DOPC: CHO: DMG-PEG2000: DSPE-PEG3000-Mal were dissolved in ethanol at a molar ratio of 43.5: 12: 43: 1.2: 0.3. mRNA was dissolved in 75 mM citric acid buffer (pH 4) to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0115] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0116] The crude LNP was purified by ultrafiltration three times at 4°C and 1200 rcf, and the LNP containing chemical coupling sites (Pre-TLNP) was collected. It was then mixed with anti-PSMA nanobody with cysteine ​​attached to the end at a molar ratio of 1.2:1 and then o The mixture was incubated overnight at 400 °C. Subsequently, sucrose solution with a concentration of 87 mg / mL was added for ultrafiltration and sterile filtration to obtain the final anti-PSMA nanobody-coupled nanoparticles (PSMA-TLNP).

[0117] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human PSMA cells After thawing and culturing LNCaP cells, PSMA-TLNPs encapsulating eGFP mRNA were added to achieve a working concentration of 0.1 μg / mL. A negative control group containing PBS was also established. After 24 hours, the expression of PSMA+ eGFP mRNA in LNCaP cells was determined by flow cytometry.

[0118] Table 5: PSMA-TLNP in vitro transfection results

[0119] The experimental results showed that PSMA-TLNP exhibited obvious targeting effect on PSMA+ cells and had good transfection efficiency.

[0120] Example 7: (I) Preparation of nanoparticles coupled with anti-TMEM119 nanoantibody Refer to the preparation method in Example 3. Ionizable amino lipid: DOPE: cholesterol ester: DMG-PEG2000: DSPE-PEG2000-Mal were dissolved in ethanol at a molar ratio of 55.5: 0: 43: 1.495: 0.005. mRNA was dissolved in 75 mM citric acid buffer (pH 4) to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0121] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0122] The crude LNP was purified by ultrafiltration three times at 4°C and 1200 rcf, and the LNP containing the chemical coupling site (Pre-TLNP) was collected. It was then mixed with the anti-TMEM119 nanobody with cysteine ​​attached to the end at a molar ratio of 1:1 and then heated at 4°C. o The cells were incubated overnight at 400 °C. Subsequently, 87 mg / mL sucrose solution was added for ultrafiltration and sterile filtration to obtain the final anti-TMEM119 nanoantibody-coupled nanoparticles (TMEM119-TLNP).

[0123] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human TMEM119 cells SH-SY5Y cells were revived and cultured, and TMEM119-TLNPs encapsulating eGFP mRNA were added to achieve a working concentration of 0.1 μg / mL in the well plate. A negative control group containing PBS was also established. After 24 hours, the expression of eGFP mRNA in TMEM119+ cells in SH-SY5Y cells was determined by flow cytometry.

[0124] Table 6: TMEM119-TLNP in vitro transfection results

[0125] The experimental results showed that TMEM119-TLNP exhibited obvious targeting effect on TMEM119+ cells and had good transfection efficiency.

[0126] Example 8: Preparation of nanoparticles coupled with anti-B7-H1 nanobody Refer to the preparation method in Example 3. Ionizable amino lipid: DSPC: CHO: DMG-PEG2000: DSPE-PEG2000-Mal were dissolved in ethanol at a molar ratio of 62.5: 5: 31: 1.49: 0.01. mRNA was dissolved in 75 mM citric acid buffer (pH 4) to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0127] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0128] The crude LNP was purified by ultrafiltration three times at 4°C and 1200 rcf to collect the LNP containing chemical coupling sites (Pre-TLNP). It was then mixed with anti-B7-H1 nanoantibodies with cysteine ​​attached at a molar ratio of 1:1 and then heated at 4°C. o The mixture was incubated overnight at 400 °C. Subsequently, sucrose solution with a concentration of 87 mg / mL was added for ultrafiltration and sterile filtration to obtain the final anti-B7-H1 nanobody-coupled nanoparticles (B7-H1-TLNP).

[0129] Example 9: (I) Preparation of nanoparticles coupled with anti-HER2 nanoantibodies Refer to the preparation method in Example 3. Ionizable amino lipid: DOPC: CHO: DMG-PEG2000: DSPE-PEG2000-Mal were dissolved in ethanol at a molar ratio of 58.5: 10: 30: 1.49: 0.01. mRNA was dissolved in 75 mM citric acid buffer (pH 4) to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0130] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0131] The crude LNP was purified by ultrafiltration three times at 4°C and 1200 rcf, and the LNP containing the chemical coupling site (Pre-TLNP) was collected. It was then mixed with the anti-HER2 nanobody with cysteine ​​attached to the end at a molar ratio of 1:1 and then heated at 4 o The mixture was incubated overnight at 400 °C. Subsequently, sucrose solution with a concentration of 87 mg / mL was added for ultrafiltration and sterile filtration to obtain the final anti-HER2 nanoantibody-coupled nanoparticles (HER2-TLNP).

[0132] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human HER2 cells Human breast cancer SK-BR-3 cells were revived and cultured, and cLNPs encapsulating eGFP mRNA and HER2-TLNPs were added to achieve a working LNP concentration of 0.1 μg / mL in the well plate. A negative control group containing PBS was also established. After 24 hours, flow cytometry was used to measure the expression of eGFP mRNA in the HER2+ cell subpopulation within PBMCs.

[0133] Table 7: HER2-TLNP in vitro transfection results

[0134] The experimental results showed that HER2-TLNP exhibited obvious targeting effect on HER2+ cells and had good transfection efficiency.

[0135] Example 10: Preparation of nanoparticles coupled with anti-EGFR nanoantibodies Refer to the preparation method in Example 3. Ionizable amino lipid: DSPC: CHO: DMG-PEG2000: DSPE-PEG2000-Mal were dissolved in ethanol at a molar ratio of 45.5: 15: 38: 1.49: 0.01. mRNA was dissolved in 75 mM citric acid buffer (pH 4) to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0136] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0137] The crude LNP was purified by ultrafiltration three times at 4°C and 1200 rcf, and the LNP containing the chemical coupling site (Pre-TLNP) was collected. It was then mixed with the anti-EGFR nanobody with cysteine ​​attached to the end at a molar ratio of 1:1 and then heated at 4 o The mixture was incubated overnight at 400 °C. Subsequently, 87 mg / mL sucrose solution was added for ultrafiltration washing and sterile filtration to obtain the final anti-EGFR nanobody-coupled nanoparticles (EGFR-TLNP).

[0138] Example 11: (1) Preparation of nanoparticles targeting human CD7 cells Refer to the preparation method in Example 3. Pre-TLNP was mixed with anti-CD7 nanobody with cysteine ​​linked to the end at a molar ratio of 1:1 and o The mixture was incubated overnight at 400 °C. Subsequently, sucrose solution with a concentration of 87 mg / mL was added for ultrafiltration and sterile filtration to obtain the final anti-CD7 nanobody-coupled nanoparticles (CD7-TLNP).

[0139] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD7 cells Anti-CD3 coating: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL per well, and store at 4°C overnight.

[0140] Preparation before cell plating: Aspirate the liquid in the 24-well plate, then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0141] Cell plating: Dilute human PBMC to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double antibody) supplemented with anti-CD28 antibody (concentration 1 μg / mL) and IL-2 (working concentration 400 U / mL). Add 1 million cells to each well of a 24-well plate. After plating, incubate at 37 oC, LNP was added after stabilization for 4 h under 5% CO2.

[0142] CD7-TLNPs encapsulating eGFP-mRNA were added to the plate to a working concentration of 0.1 μg / mL. A negative control group containing PBS was also added. After 24 hours, the expression of eGFP-mRNA in the CD7+ cell subset of PBMCs was determined by flow cytometry.

[0143] Table 8: CD7-TLNP in vitro transfection results

[0144] The experimental results showed that CD7-TLNP exhibited obvious targeting effect on CD7+ cells and had good transfection efficiency.

[0145] Example 12: (1) Preparation of nanoparticles targeting human CD4 cells Reference is made to the preparation method in Example 3. Pre-TLNP was mixed with anti-CD4 nanobody with cysteine ​​linked to the terminal end at a molar ratio of 1:1 and o The mixture was incubated overnight at 400 °C. Subsequently, sucrose solution with a concentration of 87 mg / mL was added for ultrafiltration washing and sterile filtration to obtain the final anti-CD4 nanobody-coupled nanoparticles (CD4-TLNP).

[0146] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD4 cells Anti-CD3 coating: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL per well, and store at 4°C overnight.

[0147] Preparation before cell plating: Aspirate the liquid in the 24-well plate, then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0148] Cell plating: Dilute human PBMC to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double antibody) supplemented with anti-CD28 antibody (concentration 1 μg / mL) and IL-2 (working concentration 400 U / mL). Add 1 million cells to each well of a 24-well plate. After plating, incubate at 37 o C, LNP was added after stabilization for 4 h under 5% CO2.

[0149] CD4-TLNPs encapsulating eGFP-mRNA were added to the plate to a working concentration of 0.1 μg / mL. A negative control group containing PBS was also added. After 24 hours, the expression of eGFP-mRNA in the CD4+ cell subset of PBMCs was determined by flow cytometry.

[0150] Table 9: CD4-TLNP in vitro transfection results

[0151] The experimental results showed that CD4-TLNP exhibited obvious targeting effect on CD4+ cells and had good transfection efficiency.

[0152] Example 13: (1) Preparation of nanoparticles targeting human CD38 cells Refer to the preparation method in Example 3. Pre-TLNP and anti-CD38 nanobody were mixed at a molar ratio of 1:1 and o The mixture was incubated overnight at 400 °C. Subsequently, sucrose solution with a concentration of 87 mg / mL was added for ultrafiltration and sterile filtration to obtain the final anti-CD38 nanoantibody-coupled nanoparticles (CD38-TLNP).

[0153] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD38 cells Anti-CD3 coating: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL per well, and store at 4°C overnight.

[0154] Preparation before cell plating: Aspirate the liquid in the 24-well plate, then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0155] Cell plating: Dilute human PBMC to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double antibody) supplemented with anti-CD28 antibody (concentration 1 μg / mL) and IL-2 (working concentration 400 U / mL). Add 1 million cells to each well of a 24-well plate. After plating, incubate at 37 o C, LNP was added after stabilization for 4 h under 5% CO2.

[0156] CD38-TLNPs encapsulating eGFP-mRNA were added to the plate to a working concentration of 0.1 μg / mL. A negative control group containing PBS was also added. After 24 hours, the expression of eGFP-mRNA in the CD38+ cell subset of PBMCs was determined by flow cytometry.

[0157] Table 10: CD38-TLNP in vitro transfection results

[0158] The experimental results showed that CD38-TLNP exhibited obvious targeting effect on CD38+ cells and had good transfection efficiency.

[0159] Example 14: (1) Preparation of nanoparticles targeting human CD68 cells Refer to the preparation method in Example 3. Pre-TLNP and anti-CD68 nanobody were mixed at a molar ratio of 1:1 and o The mixture was incubated overnight at 400 °C. Subsequently, sucrose solution with a concentration of 87 mg / mL was added for ultrafiltration and sterile filtration to obtain the final anti-CD68 nanoantibody-coupled nanoparticles (CD68-TLNP).

[0160] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD68 cells Anti-CD3 coating: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL per well, and store at 4°C overnight.

[0161] Preparation before cell plating: Aspirate the liquid in the 24-well plate, then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0162] Cell plating: Dilute human PBMC to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double antibody) supplemented with anti-CD28 antibody (concentration 1 μg / mL) and IL-2 (working concentration 400 U / mL). Add 1 million cells to each well of a 24-well plate. After plating, incubate at 37 o C, LNP was added after stabilization for 4 h under 5% CO2.

[0163] CD68-TLNPs encapsulating eGFP-mRNA were added to the plate to a working concentration of 0.1 μg / mL. A negative control group containing PBS was also added. After 24 hours, the expression of eGFP-mRNA in the CD68+ cell subset of PBMCs was determined by flow cytometry.

[0164] Table 11: CD68-TLNP in vitro transfection results

[0165] The experimental results showed that CD68-TLNP exhibited obvious targeting effect on CD68+ cells and had good transfection efficiency.

[0166] Example 15: (1) Preparation of nanoparticles targeting human CD117 cells Refer to the preparation method in Example 3. Pre-TLNP and anti-CD117 nanobody were mixed at a molar ratio of 1:1 and o The mixture was incubated overnight at 400 °C. Subsequently, sucrose solution with a concentration of 87 mg / mL was added for ultrafiltration and sterile filtration to obtain the final anti-CD117 nanoantibody-coupled nanoparticles (CD117-TLNP).

[0167] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD117 cells HLF cells were revived and cultured, and CD117-TLNPs encapsulating eGFP mRNA were added to achieve a working concentration of 0.1 μg / mL in the plate. A negative control group containing PBS was also established. After 24 hours, the expression of CD117+ eGFP mRNA in HLF cells was determined by flow cytometry.

[0168] Table 12: CD117-TLNP in vitro transfection results

[0169] The experimental results showed that CD117-TLNP exhibited obvious targeting effect on CD117+ cells and had good transfection efficiency.

[0170] Example 16: (1) Preparation of nanoparticles targeting human B7H3 cells Refer to the preparation method in Example 3. Pre-TLNP and anti-B7H3 nanobody were mixed at a molar ratio of 1:1 and oThe mixture was incubated overnight at 400 °C. Subsequently, sucrose solution with a concentration of 87 mg / mL was added for ultrafiltration and sterile filtration to obtain the final anti-B7H3 nanobody-coupled nanoparticles (B7H3-TLNP).

[0171] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human B7H3 cells A2780 cells were revived and cultured, and B7H3-TLNPs encapsulating eGFP mRNA were added to achieve a working concentration of 0.1 μg / mL in the well plate. A negative control group containing PBS was also established. After 24 hours, the expression of B7H3+ eGFP mRNA in A2780 cells was determined by flow cytometry.

[0172] Table 13: B7H3-TLNP in vitro transfection results

[0173] The experimental results showed that B7H3-TLNP exhibited obvious targeting effect on B7H3+ cells and had good transfection efficiency.

[0174] Example 17: (1) Preparation of nanoparticles targeting human GPC3 cells Refer to the preparation method in Example 3. Pre-TLNP and anti-GPC3 nanobody with cysteine ​​terminally linked were mixed at a molar ratio of 1:1 and o The mixture was incubated overnight at 400 °C. Subsequently, sucrose solution with a concentration of 87 mg / mL was added for ultrafiltration washing and sterile filtration to obtain the final anti-GPC3 nanobody-coupled nanoparticles (GPC3-TLNP).

[0175] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human GPC3 cells HepG2 cells were revived and cultured, and GPC3-TLNPs encapsulating eGFP mRNA were added to achieve a working concentration of 0.1 μg / mL in the plate. A negative control group containing PBS was also established. After 24 hours, the expression of GPC3+ eGFP mRNA in the HepG2 cells was determined by flow cytometry.

[0176] Table 14: GPC3-TLNP in vitro transfection results

[0177] The experimental results showed that GPC3-TLNP exhibited obvious targeting effect on GPC3+ cells and had good transfection efficiency.

[0178] Example 18: DSPE-PEG 2000 -Lys-Mal2 lipid coupled to multiple nanoantibodies to form a targeting functional molecule targeting human CD8 + Preparation of T cell nanoparticles Refer to the preparation method in Example 3. Ionizable amino lipid: DSPC: CHO: DMG-PEG2000: DSPE-PEG2000-Lys-Mal2 were dissolved in ethanol at a molar ratio of 47.5: 10: 41: 1.49: 0.01. mRNA was dissolved in 75 mM citric acid buffer (pH 4) to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0179] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0180] The crude LNP was purified by ultrafiltration three times at 4°C and 1200 rcf, and 0.278 times the volume of 400 mg / mL sucrose solution was added to obtain LNP containing chemical coupling sites (Pre-TLNP). It was then mixed with anti-CD8 nanoantibodies at a molar ratio of 1:1 and o The mixture was incubated overnight at 400 °C. Subsequently, sucrose solution with a concentration of 87 mg / mL was added for ultrafiltration and sterile filtration to obtain the final anti-CD8 nanobody-coupled nanoparticles (CD8-TLNP).

[0181] Example 19: By Mal-PEG 2000 -Mal lipid coupled to multiple nanoantibodies to form a targeting functional molecule targeting human CD8 + Preparation of T cell nanoparticles Refer to the preparation method in Example 3. Ionizable amino lipid: DSPC: CHO: DMG-PEG2000: Mal-PEG2000-Mal were dissolved in ethanol at a molar ratio of 47.5: 10: 41: 1.49: 0.01. mRNA was dissolved in 75 mM citric acid buffer (pH 4) to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0182] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0183] The crude LNP was purified by ultrafiltration three times at 4°C and 1200 rcf, and 0.278 times the volume of 400 mg / mL sucrose solution was added to obtain LNP containing chemical coupling sites (Pre-TLNP). It was then mixed with anti-CD8 nanoantibodies at a molar ratio of 1:1 and o The mixture was incubated overnight at 400 °C. Subsequently, sucrose solution with a concentration of 87 mg / mL was added for ultrafiltration and sterile filtration to obtain the final anti-CD8 nanobody-coupled nanoparticles (CD8-TLNP).

[0184] Example 20: This example is a structural screening of targeting functional molecules in nanoparticles targeting human CD8+ T cells (1) Preparation of nanoparticles targeting human CD8 T cells Table 15: Preparation of LNPs with different targeting molecular structures (Lkx)

[0185] 1) Preparation of LNPs containing coupling sites Ionizable amino lipid: DSPC: CHO: DMG-PEG 2000The five lipid components, Lk:Lkx (Lk1 and Lk3 contain PEG segments, while Lk2, Lk4, and Lk5 do not), were dissolved in ethanol at a molar ratio of 47.5:10:41:1.49:0.01. eGFP-φ-mRNA was dissolved in 75 mM citric acid buffer (pH 4) to maintain an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0186] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0187] The LNPs were purified by ultrafiltration once at 4°C and 1200 rcf after adding the stop solution. Subsequently, the ultrafiltrate was concentrated twice more at 4°C and 1200 rcf by adding 25 mM Tris buffer. The prepared eGFP-φ-LNPs were collected and added with 0.278 times the volume of a 400 mg / mL sucrose solution (prepared to volume using 25 mM Tris buffer, pH = 7.4-7.5). The mixture was sterile-filtered using a 0.22 μm PES filter in a laminar flow hood. A 30 μL sample was aspirated for particle size, dispersion dilution, and encapsulation efficiency testing. The remaining sample was stored in a refrigerator at 4°C. After encapsulation efficiency testing, the sample was diluted to the desired concentration using 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH = 7.4-7.5).

[0188] 2) Preparation of Nanobody-Modified TLNPs In addition to cLNP-1, a certain amount of the other groups of eGFP-φ-Pre TLNPs containing the above-mentioned coupling sites was taken, and the molar amount of DSPE-PEG2000-Mal in the lipid nanoparticles corresponding to each group of eGFP-φ-Pre TLNPs was calculated according to the LNP formula. Then, according to the molar ratio of Lkx and anti-human-CD8-nanobody (concentration 2.3 mg / mL, stored in a mixed buffer consisting of 20 mM Tris, 150 mM NaCl and 5% glycerol at pH = 8.0) connected to the end, Pre-TLNP and nanoantibody were mixed and incubated for 4 hr. o Incubate overnight at 4°C.

[0189] After overnight incubation, the TLNPs were diluted with 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH 7.4-7.5) and purified using a 100 kD ultrafiltration tube. After sterile filtration using a 0.22 μm PES filter in a laminar flow hood, a 30 μL sample was aspirated for particle size, dispersion dilution, and encapsulation efficiency testing. The remaining sample was stored in a refrigerator at 4°C. After encapsulation efficiency testing, the sample was diluted to the desired concentration using 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH 7.4-7.5).

[0190] Table 16: Quality control information of TLNPs modified with nanoantibodies containing different targeting molecular structures

[0191] Experimental results show that CD8+ T cell-targeted lipid nanoparticles prepared using targeting molecules of different structures all exhibited good characterization data, falling within the generally accepted numerical range in the LNP field. This demonstrates that the preparation process for the targeted LNPs described in this patent is stable and generally applicable.

[0192] For detailed statistical data of LNP characterization, please see Figure 1 .

[0193] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD8+ T cells Anti-CD3 coating: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL per well, and store at 4°C overnight.

[0194] Preparation before cell plating: Aspirate the liquid in the 24-well plate, then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0195] Cell plating: Dilute human PBMC to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double antibody) supplemented with anti-CD28 antibody (concentration 1 μg / mL) and IL-2 (working concentration 400 U / mL). Add 1 million cells to each well of a 24-well plate. After plating, incubate at 37 o C, LNP was added after stabilization for 4 h under 5% CO2.

[0196] Drug addition: Each of the six LNPs prepared in Table 16 was added to each well, resulting in a working concentration of 0.1 μg / mL. PBS was also added as a negative control.

[0197] Culture: After adding LNP, PBMC cells were cultured at 37 o C, Transfection for 24 h under 5% CO2 conditions.

[0198] Detection of eGFP-mRNA expression: Cells from each well plate were collected and stained with commercially available flow cytometry antibodies. The expression rate and mean fluorescence intensity (MFI) of eGFP-mRNA in CD4+ T and CD8+ T cell subsets in PBMC were detected by flow cytometry.

[0199] Table 17: In vitro transfection results of TLNPs containing different targeting molecular structures

[0200] The experimental results showed that TLNPs prepared by Lk1, Lk2 and Lk4 exhibited obvious CD8+ T cell targeting effects at extremely low dosages and had good transfection efficiency.

[0201] Detailed statistics of LNP in vitro transfection with different targeting molecular structures are shown in Figure 2 .

[0202] (III) Evaluation of the in vivo delivery efficiency of nanoparticles targeting human CD8+ T cells Twelve 6-8-week-old NCG female mice that passed quarantine and met SPF (Specific Pathogen Free) standards were selected and divided into four groups. Each NCG mouse was injected with 20 M hPBMCs in a volume of 200 μL via the tail vein. Approximately 30 minutes later, after the venous wound healed, the TLNPs (Lk1, Lk2, and Lk4) prepared as listed in Table 16 were injected again via the tail vein. Each NCG mouse received 20 μg of mRNA-LNP in a volume of 200 μL. After 24 hours of expression, peripheral blood and spleen lysates were collected and flow cytometry was used to analyze the expression rate of eGFP mRNA in various cell subsets.

[0203] Table 18: Grouping of TLNPs with different targeting molecular structures in vivo

[0204] The experimental results showed that TLNPs prepared with Lk1 showed the best CD8+ T cell targeting effect and the lowest off-target rate in NCG mice. Subsequent experiments were conducted based on Lk1-TLNPs.

[0205] Detailed statistics of TLNP in vivo transfection with different targeting molecular structures are shown in Figure 3 .

[0206] Example 21: This example is a screening experiment for the proportion of targeting functional molecules in nanoparticles targeting human CD8+ T cells.

[0207] (1) Preparation of nanoparticles targeting human CD8 T cells Table 19: Preparation of Pre-LNPs with different targeting molecule ratios

[0208] 1) Preparation of LNPs containing coupling sites Ionizable amino lipid: DSPC: CHO: DMG-PEG 2000 DSPE-PEG2000-Mal was dissolved in ethanol according to the molar ratios described in Table 19. eGFP-φ-mRNA was dissolved in 75 mM citric acid buffer (pH 4) to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0209] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0210] The LNPs were purified by ultrafiltration once at 4°C and 1200 rcf after adding the stop solution. Subsequently, the ultrafiltrate was concentrated twice more at 4°C and 1200 rcf by adding 25 mM Tris buffer. The prepared eGFP-φ-LNPs were collected and added with 0.278 times the volume of a 400 mg / mL sucrose solution (prepared to volume using 25 mM Tris buffer, pH = 7.4-7.5). The mixture was sterile-filtered using a 0.22 μm PES filter in a laminar flow hood. A 30 μL sample was aspirated for particle size, dispersion dilution, and encapsulation efficiency testing. The remaining sample was stored in a refrigerator at 4°C. After encapsulation efficiency testing, the sample was diluted to the desired concentration using 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH = 7.4-7.5).

[0211] The experimental results show that: through the quality control data analysis of the seven groups of LNPs prepared, compared with the original four-component LNPs, when 0.0001 mol%-0.50 mol% DSPE-PEG2000-Mal lipids are introduced into the LNP formula, the particle size of the corresponding LNPs will be reduced.

[0212] Table 20: Quality control information of Pre TLNP containing coupling sites

[0213] For detailed characterization data statistics of Pre-TLNP, please see Figure 4 .

[0214] 2) Preparation of Nanobody-Modified TLNPs In addition to cLNP-1, a certain amount of the other groups of eGFP-φ-Pre TLNPs containing coupling sites were taken, and the molar amount of DSPE-PEG2000-Mal in the lipid nanoparticles corresponding to each group of eGFP-φ-Pre TLNPs was calculated according to the LNP formula. 2000 Pre-TLNPs were mixed with anti-human-CD8-nanobody (anti-human-CD8-nanobody) with a molar ratio of 1:1 (2.3 mg / mL, stored in a mixed buffer solution consisting of 20 mM Tris, 150 mM NaCl, and 5% glycerol at pH 8.0) and the nanobody was added for 4 h. o Incubate overnight at 4°C.

[0215] After overnight incubation, the TLNPs were diluted with 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH 7.4-7.5) and purified using a 100 kD ultrafiltration tube. After sterile filtration using a 0.22 μm PES filter in a laminar flow hood, a 30 μL sample was aspirated for particle size, dispersion dilution, and encapsulation efficiency testing. The remaining sample was stored in a refrigerator at 4°C. After encapsulation efficiency testing, the sample was diluted to the desired concentration using 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH 7.4-7.5).

[0216] Table 21: Quality control information of TLNPs containing different ratios of target molecules

[0217] The experimental results show that when the Pre TLNP containing the coupling site is connected to the nanoantibody, the corresponding TLNP- shows good characterization data, which is within the numerical range recognized in the LNP field, indicating that the preparation process of the targeted LNP in this patent is stable and feasible.

[0218] Detailed characterization data statistics of TLNP are shown in Figure 5 .

[0219] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD8+ T cells Anti-CD3 coating: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL per well, and store at 4°C overnight.

[0220] Preparation before cell plating: Aspirate the liquid in the 24-well plate, then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0221] Cell plating: Dilute human PBMC to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double antibody) supplemented with anti-CD28 antibody (concentration 1 μg / mL) and IL-2 (working concentration 400 U / mL). Add 1 million cells to each well of a 24-well plate. After plating, incubate at 37 o C, LNP was added after stabilization for 4 h under 5% CO2.

[0222] Drug addition: Add each LNP group listed in Table 21 to each well, so that the working concentration of LNP in the well plate is 1.0 μg / mL. In addition, add PBS as a negative control group.

[0223] Culture: After adding LNP, PBMC cells were cultured at 37 o C, Transfection for 24 h under 5% CO2 conditions.

[0224] Detection of eGFP-mRNA expression: Cells from each well plate were collected and stained with commercially available flow cytometry antibodies. The expression rate of eGFP-mRNA in all cells in PBMCs, CD4+ T cells, and CD8+ T cell subsets was detected by flow cytometry.

[0225] Table 22: In vitro transfection results of TLNPs containing different targeting molecule ratios

[0226] (III) Evaluation of the in vivo delivery efficiency of nanoparticles targeting human CD8+ T cells Nine 6-8-week-old NCG female mice that passed quarantine and met SPF (Specific Pathogen Free) standards were selected and divided into three groups. Each NCG mouse was injected with 20 M hPBMCs in a volume of 200 μL via the tail vein. Approximately 30 minutes later, the mice's venous wounds healed, and the TLNPs prepared in Table 20 were injected again via the tail vein. Each NCG mouse was given a dose of 20 μg mRNA-LNPs in a volume of 200 μL. After 24 hours of expression, peripheral blood and spleen lysates were collected, and flow cytometry was used to analyze the expression rate of eGFP mRNA in various cell subsets.

[0227] Table 23: In vivo experimental groups of TLNPs with different targeting molecule ratios

[0228] The experimental results showed that TLNP-2 containing 0.01% targeting molecule ratio showed better CD8+ T cell targeting delivery effect in NCG mice than TLNP-1 in the 0.0001% group.

[0229] Detailed statistics of TLNP in vivo transfection with different targeting molecule ratios are shown in Figure 6 .

[0230] Example 22: This example targets human CD8 + Study on the ratio of DMG-PEG2000 lipids in nanoparticles for T cells (1) Preparation of nanoparticles targeting human CD8 T cells Table 24: Preparation of CD8-TLNPs with different PEG lipid ratios

[0231] The lipid nanoparticles used in this example were prepared according to the method described in Example 20.

[0232] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD8+ T cells Anti-CD3 coating: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL per well, and store at 4°C overnight.

[0233] Preparation before cell plating: Aspirate the liquid in the 24-well plate, then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0234] Cell plating: Dilute human PBMC to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double antibody) supplemented with anti-CD28 antibody (concentration 1 μg / mL) and IL-2 (working concentration 400 U / mL). Add 1 million cells to each well of a 24-well plate. After plating, incubate at 37 o C, LNP was added after stabilization for 4 h under 5% CO2.

[0235] Drug addition: Add each LNP group listed in Table 24 to each well, so that the working concentration of LNP in the well plate is 1.0 μg / mL. In addition, add PBS as a negative control group.

[0236] Culture: After adding LNP, PBMC cells were cultured at 37 o C, Transfection for 24 h under 5% CO2 conditions.

[0237] Detection of eGFP-mRNA expression: Cells from each well plate were collected and stained with commercially available flow cytometry antibodies. The expression rate of eGFP-mRNA in all cells in PBMCs, CD4+ T cells, and CD8+ T cell subsets was detected by flow cytometry.

[0238] Table 25: In vitro transfection results of TLNPs containing different PEG lipid ratios

[0239] The experimental results showed that TLNPs containing 1.0% and 1.5%-DMG-PEG2000 showed better CD8+ T cell targeted delivery effect compared with PEG groups with other ratios.

[0240] Example 23: This example targets human CD8 by ionizable amino lipids of different structures. + Study on the delivery efficiency of nanoparticles to T cells (1) Preparation of nanoparticles targeting human CD8 T cells Table 26: Preparation of TLNPs composed of different ionizable amino lipids

[0241] The lipid nanoparticles used in this example were prepared according to the ratio of ionizable amino lipid: DSPC: CHO: DMG-PEG2000: DSPE-PEG2000-Mal = 47.5: 10: 41: 1.5: 0.01 and the method described in Reference Example 20.

[0242] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD8+ T cells Anti-CD3 coating: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL per well, and store at 4°C overnight.

[0243] Preparation before cell plating: Aspirate the liquid in the 24-well plate, then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0244] Cell plating: Dilute human PBMC to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double antibody) supplemented with anti-CD28 antibody (concentration 1 μg / mL) and IL-2 (working concentration 400 U / mL). Add 1 million cells to each well of a 24-well plate. After plating, incubate at 37 o C, LNP was added after stabilization for 4 h under 5% CO2.

[0245] Drug addition: Add each LNP group listed in Table 26 to each well, so that the working concentration of LNP in the well plate is 1.0 μg / mL. In addition, add PBS as a negative control group.

[0246] Culture: After adding LNP, PBMC cells were cultured at 37 o C, Transfection for 24 h under 5% CO2 conditions.

[0247] Detection of eGFP-mRNA expression: Cells from each well plate were collected and stained with commercially available flow cytometry antibodies. The expression rate of eGFP-mRNA in all cells in PBMCs, CD4+ T cells, and CD8+ T cell subsets was detected by flow cytometry.

[0248] Table 27: In vitro transfection results of CD8-TLNPs composed of different ionizable amino lipids

[0249] Experimental results showed that CD8-TLNPs composed of E12LA6B6O3, E10LA8B6O3, and the commercially available lipid SM-102 had comparable or even slightly better transfection efficiency in CD8+ T cell populations, but SM-102 exhibited a higher off-target rate in non-CD8+ T cell populations. Comprehensive analysis showed that E12LA6B6O3 and E10LA8B6O3 exhibited superior targeted delivery to CD8+ T cell subsets.

[0250] Example 24: This example is a study on the dose-dependence of nanoparticles targeting human CD8+ T cells in vitro and in vivo (1) Preparation of nanoparticles targeting human CD8+ T cells The two lipid nanoparticles used in this example were cLNP-1 and TLNP-2 (0.01% targeting molecule ratio) prepared in Example 21.

[0251] (II) Investigation of the dose-dependence of nanoparticle delivery targeting human CD8+ T cells in vitro Anti-CD3 coating: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL per well, and store at 4°C overnight.

[0252] Preparation before cell plating: Aspirate the liquid in the 24-well plate, then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0253] Cell plating: Dilute human PBMC to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double antibody) supplemented with anti-CD28 antibody (concentration 1 μg / mL) and IL-2 (working concentration 400 U / mL). Add 1 million cells to each well of a 24-well plate. After plating, incubate at 37 o C, LNP was added after stabilization for 4 h under 5% CO2.

[0254] Drug addition: Add the corresponding targeting molecule-modified TLNP-2 to each well so that the working concentration of LNP in the well plate is as shown in the table below. Add PBS as a negative control group and add LNP-1 as a positive control group.

[0255] Table 28: Information on adding LNP to the well plate

[0256] Culture: After adding LNP, PBMC cells were cultured at 37 o C, Transfection for 24 h under 5% CO2 conditions.

[0257] Detection of eGFP-mRNA expression: Cells were collected from each well plate and stained with commercially available flow cytometry antibodies. Flow cytometry was then used to detect the expression of eGFP-mRNA in all cells of hPBMCs, CD3+ T cells, CD4+ T cells, and CD8+ T cell subsets.

[0258] Table 29: Summary of the in vitro targeting effect of nanobody-modified LNPs on T cells

[0259] The experimental results show that in the in vitro transfection experiment of hPBMC, when the working concentration of TLNP-2 is as low as 0.025 μg / mL, it still maintains a high proportion of CD8 T cell targeting effect, indicating that the TLNP developed in this patent has the ability to achieve high cell targeting at low doses.

[0260] Flow cytometry analysis of EGFP-mRNA expression efficiency of cLNP-1 and TLNP-2 in hPBMCs is shown in the figure. Figure 7 .

[0261] (III) Investigation of the dose-dependence of nanoparticle delivery targeting human CD8+ T cells in vivo Twelve 6-8-week-old NCG female mice that passed quarantine and met SPF (Specific Pathogen Free) standards were selected and divided into four groups. Each NCG mouse was injected with 20 M hPBMCs in a 200 μL volume via the tail vein. Approximately 30 minutes later, after the venous wound healed, the mice were injected again with different doses of TLNP-2 in a 200 μL volume. After 24 hours of expression, peripheral blood and spleen lysates were collected and flow cytometry was used to analyze the expression rate of eGFP mRNA in various cell subsets.

[0262] Table 30: TLNP-2 in vivo experimental grouping

[0263] The experimental results showed that TLNP-2 containing 0.01% targeting molecule ratio had a good CD8+ T cell targeting delivery effect at a dose of 0.5 mpk in NCG mice.

[0264] The statistical diagram of TLNP-2 in vivo transfection at different dosages is shown in Figure 8 .

[0265] Example 25: This example is a study on the formulation of nanoparticle components targeting human CD8+ T cells (II) Preparation of Nanoparticles Targeting Human CD8 T Cells Table 31: Preparation of TLNPs with different ratios

[0266] The two lipid nanoparticles TLNP-2-F1 and TLNP-2-F2 used in this example were prepared according to the method described in Example 20.

[0267] (II) Investigation of the in vivo delivery efficiency of nanoparticles targeting human CD8+ T cells with different formulations Nine 6-8-week-old NCG female mice that passed quarantine and met SPF (Specific Pathogen Free) standards were selected and divided into three groups. Each NCG mouse was injected with 20 M hPBMCs in a volume of 200 μL via the tail vein. Approximately 30 minutes later, the venous wounds of the mice healed, and the prepared TLNP-2-F1 and TLNP-2-F2 were injected again via the tail vein. Each NCG mouse was given a dose of 10 μg mRNA-LNP in a volume of 200 μL. After 24 hours of expression, peripheral blood and spleen lysates were collected and flow cytometry was used to analyze the expression rate of eGFP mRNA in various cell subsets.

[0268] Table 32: In vivo experimental groups of TLNP with different ratios

[0269] The experimental results showed that TLNP-2-F2 prepared by formula F2 and containing 0.01% targeting molecule ratio showed better CD8+ T cell targeting delivery effect in NCG mice than the F1 group at a dosage of 0.5mpk.

[0270] The statistical diagram of TLNP-2 in vivo transfection with different ratios is shown in Figure 9 .

[0271] Example 26: This example is an in vitro freeze-thaw stability evaluation of nanoparticles targeting human CD8+ T cells (I) Preparation of Nanoparticles Targeting Human CD8+ T Cells The two lipid nanoparticles used in this example are cLNP-1 and TLNP-2 prepared in Example 21.

[0272] (II) Investigation of freeze-thaw stability of nanoparticles targeting human CD8+ T cells The prepared LNPs were frozen and stored in a -80° refrigerator for more than 24 hours, then taken out and quickly re-dissolved in a 4° refrigerator. This sample was frozen and thawed once. The reconstituted sample was frozen again in a -80° refrigerator for more than 24 hours, and then taken out and quickly reconstituted in a 4° refrigerator. This sample was frozen and thawed twice. The reconstituted sample was frozen again in a -80° refrigerator for more than 24 hours, and then taken out and quickly reconstituted in a 4° refrigerator. This sample was frozen and thawed three times. The particle size, PDI and encapsulation efficiency of LNP after each reconstitution were tested to investigate the freeze-thaw stability of LNP.

[0273] Table 33: Characterization information of LNP after freeze-thaw

[0274] Example 27: This example targets human CD8 + Evaluation of the long-term stability of nanoparticles in T cells (I) Preparation of Nanoparticles Targeting Human CD8+ T Cells Table 34: LNP preparation formula with different targeting molecule ratios

[0275] In this example, the preparation methods of LNPs containing coupling sites and LNPs modified with nanobodies refer to the process described in Example 21.

[0276] (II) Investigation of the stability of nanoparticles targeting human CD8+ T cells 1. Nanoparticles prepared on day 0: 1) Characterization of the newly prepared nanobody-modified LNPs Table 35: Quality control information of nanobody-modified LNPs (Day 0)

[0277] 2) Detection of in vitro delivery efficiency of the newly prepared nanobody-modified LNPs Anti-CD3 coating: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL per well, and store at 4°C overnight.

[0278] Preparation before cell plating: Aspirate the liquid in the 24-well plate, then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0279] Cell plating: Dilute PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double-antibody). Plate 1 million cells per well in a 24-well plate. After plating, allow cells to stabilize for 4 hours before adding LNPs.

[0280] Drug addition: Add the corresponding targeting molecule-modified LNP to each well to achieve a working concentration of 1 μg / mL. In addition, add PBS as a negative control.

[0281] Culture: After adding LNP, PBMC cells were cultured at 37 o C, Transfection for 24 h under 5% CO2 conditions.

[0282] Detection of eGFP-mRNA expression: Cells in each well plate were collected and stained with commercially available flow cytometry antibodies. The expression rate of eGFP-mRNA in all cells in PBMC, CD4 T cells, and CD8 T cells was detected by flow cytometry.

[0283] Table 36: Summary of the targeting effect of nanobody-modified LNPs on T cells in vitro (Day 0)

[0284] 2. 3 days after nanoparticle preparation: 1) Characterization of the prepared nanobody-modified LNPs (Day 3) Table 37: Quality control information of nanobody-modified LNPs (Day 3)

[0285] 3) Testing the in vitro delivery efficiency of the prepared nanobody-modified LNPs (Day 3) Same steps as above Table 38: Summary of the targeting effect of nanobody-modified LNPs on T cells in vitro (Day 3)

[0286] 3. 7 days after nanoparticle preparation: 1) Characterization of the prepared nanobody-modified LNPs (Day 7) Table 39: Quality control information of nanobody-modified LNPs (Day 7)

[0287] 3) Testing the in vitro delivery efficiency of the prepared nanobody-modified LNPs (Day 7) Same steps as above Table 40: Summary of the targeting effect of nanobody-modified LNPs on T cells in vitro (Day 7)

[0288] 4. 14 days after nanoparticle preparation: 1) Characterization of the prepared nanobody-modified LNPs (Day 14) Table 41: Quality control information of nanobody-modified LNPs (Day 14)

[0289] 3) Testing the in vitro delivery efficiency of the prepared nanobody-modified LNPs (Day 14) Same steps as above Table 42: Summary of the targeting effect of nanobody-modified LNPs on T cells in vitro (Day 14)

[0290] 5. One month after nanoparticle preparation: 1) Characterization of the prepared nanobody-modified LNPs (Month 1) Table 43: Quality control information of nanobody-modified LNPs (month 1)

[0291] 2) Testing the in vitro delivery efficiency of the prepared nanobody-modified LNPs (Month 1) Same steps as above Table 44: Summary of the in vitro targeting effect of nanobody-modified LNPs on T cells (Month 1)

[0292] The experimental results show that: the nanoantibody modified LNP in 4 o After being stored at 47°C for one month, although the changing trends and amplitudes of its physical and chemical properties were similar to those of the traditional four-component cLNP, it was still able to maintain a good CD8 T cell targeting effect, indicating that the TLNP developed in this patent has good stability and can maintain a high cell targeting ability for a long time.

[0293] For detailed statistical data of long-term stability study of traditional LNP and targeted TLNP, please see Figure 10 .

[0294] Example 28: This example targets human CD8 + Functional evaluation of T cells after nanoparticle drug delivery (I) Preparation of Nanoparticles Targeting Human CD8+ T Cells The two lipid nanoparticles used in this example are cLNP-1 and TLNP-2 prepared in Example 21.

[0295] 2. Functional evaluation of nanoparticle drug delivery targeting human CD8+ T cells Anti-CD3 coating: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL per well, and store at 4°C overnight.

[0296] Preparation before cell plating: Aspirate the liquid in the 24-well plate, then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0297] Cell plating: Dilute PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double-antibody). Plate 1 million cells per well in a 24-well plate. After plating, allow cells to stabilize for 4 hours before adding LNPs.

[0298] Drug addition: Add the corresponding targeting molecule-modified LNP to each well to achieve a working concentration of 1 μg / mL. In addition, add PBS as a negative control.

[0299] Culture: After adding LNP, PBMC cells were cultured at 37 o C, Transfection for 24 h under 5% CO2 conditions.

[0300] Killing experiment: Collect cells from each well plate and count them, and make them into 1 million / ml. Prepare CD19+ B lymphocyte tumor cell line, count them, and make them into 1 million / ml. Resuscitated human PBMCs were used as a control for cells transfected with CD19 CAR-LNP. Different ET ratios were added according to the table below. Each numbered group was treated with triplicate wells: Incubate at 37°C for 4-8 hours; Prepare the dead cell dye 7-AAD at a stock concentration of 2 mg / ml and dilute it to 0.1 μg / 10 μl with water; Add 2.5 μL of 7-AAD to the flow cytometer and immediately test on the flow cytometer. 7-AAD is detected on the PerCP-Cy5.5 channel and Violet is detected on the BV421 channel.

[0301] The killing rate is CFSE+7-AAD+ / Total CFSE+.

[0302] Table 45: Summary of data on functional evaluation of specific killing effects of CAR delivered by nanoparticles targeting human CD8+ T cells (mean of 3 replicate wells)

[0303] Example 29: This example targets human CD8 + Study on the distribution of nanoparticles in T cells in vivo (1) Preparation of nanoparticles targeting human CD8 T cells The preparation method described in Example 21 was used. The two lipid nanoparticles used in this example were cLNP-1 and TLNP-2, which encapsulated Luciferase mRNA or 25% Cy5 dye-labeled Luciferase mRNA, respectively.

[0304] (II) Cy5-mRNA biodistribution of human CD8 T cell-targeted nanoparticles in Balb / c mice Nine 6-8-week-old female Balb / c mice (3 per group) that had passed quarantine and met SPF (Specific Pathogen Free) standards were selected. Each Balb / c mouse in the G1 group was injected via the tail vein with 200 μL of PBS. Each Balb / c mouse in the G2 and G3 groups was injected via the tail vein with 5 μg of 25%-Cy5-Luc-φ-mRNA LNPs in a 200 μL injection volume.

[0305] Two hours after drug administration, mice were anesthetized with isoflurane and dissected to remove the brain, heart, liver, spleen, lungs, kidneys, thymus, lymph nodes, and bone marrow for ex vivo organ imaging. Images were taken using a Perkin Elmer Living Imaging instrument from Shenzhen Bay Laboratory, using the system's default Cy5 imaging mode.

[0306] Table 46: Cy5 fluorescence labeling study of LNP in vivo distribution experimental group

[0307] The results showed that the biodistribution of conventional LNP and cell-targeted TLNP in normal Balb / c mice was similar, with Cy5 mRNA enrichment highest in the liver, followed by the spleen, kidney, and lung.

[0308] (III) Luc-mRNA biodistribution of human CD8 T cell-targeted nanoparticles in Balb / c mice Nine 6-8-week-old female Balb / c mice (3 per group) that had passed quarantine and met SPF (Specific Pathogen Free) standards were selected. Each Balb / c mouse in the G1 group was injected via the tail vein with 200 μL of PBS. Each Balb / c mouse in the G2 and G3 groups was injected via the tail vein with 5 μg of Luc-φ-mRNA LNPs in a 200 μL injection volume.

[0309] Six hours after drug administration, mice were anesthetized with isoflurane and intraperitoneally injected with 250 μL of luciferase substrate. Ten minutes later, the mice were dissected, and the brain, heart, liver, spleen, lungs, kidneys, thymus, lymph nodes, and bone marrow were removed for ex vivo organ imaging. Images were taken using a Perkin Elmer Living Imaging instrument from Shenzhen Bay Laboratory, set to F8 mode with a 2-second exposure time. Following imaging, mRNA was extracted from the organs and quantitative PCR was performed to determine mRNA enrichment in each organ.

[0310] Table 47: Luc imaging study LNP in vivo distribution experimental group

[0311] The results showed that the biodistribution of conventional LNPs and cell-targeted TLNPs in normal Balb / c mice was similar. Luc protein expression and Luc mRNA enrichment were highest in the spleen, with the TLNP-2 group slightly higher than the cLNP group.

[0312] See the statistical graphs for the biodistribution results of Cy5 fluorescence, Luc imaging, and qPCR studies in Balb / c mice. Figure 11 .

[0313] (IV) Study on the biodistribution of Luc-mRNA of human CD8 T cell-targeted nanoparticles in NCG mice Nine 6-8-week-old NCG female mice, which had passed quarantine and met SPF (Specific Pathogen Free) standards, were selected and divided into three groups. Each NCG mouse was injected with 20 M hPBMCs in a volume of 200 μL via the tail vein. Approximately 30 minutes later, after the venous wound had healed, the prepared cLNPs and TLNP-2 were injected via the tail vein. Each NCG mouse received 5 μg of Luc-LNP in a volume of 200 μL.

[0314] Six hours after drug administration, mice were anesthetized with isoflurane and intraperitoneally injected with 250 μL of luciferase substrate. Ten minutes later, the mice were dissected, and the brain, heart, liver, spleen, lungs, kidneys, and bone marrow were removed for ex vivo organ imaging. Images were taken using a Perkin Elmer Living Imaging instrument from Shenzhen Bay Laboratory, set to F8 mode with a 2-second exposure time. Following imaging, mRNA was extracted from the spleen, liver, and peripheral blood, and quantitative PCR was performed to measure mRNA enrichment in each organ.

[0315] Table 48: Luc imaging study LNP in vivo distribution experimental grouping

[0316] The experimental results showed that in the NCG mouse model, compared with traditional LNP, CD8+ T cell-targeted TLNP-2 can more selectively deliver Luc-mRNA to organs rich in hPBMCs, and it exhibits a higher spleen-liver fluorescence intensity ratio.

[0317] Luc imaging and qPCR analysis of NGC biodistribution in mice: Figure 12 .

[0318] Example 30: This example targets human CD8+ Evaluation of Nanoparticle Dose Toxicity in Vitro for T Cells (1) Preparation of nanoparticles targeting human CD8 T cells The two lipid nanoparticles used in this example are cLNP-1 and TLNP-2 prepared in Example 21.

[0319] (II) Investigation of the toxicity of nanoparticle delivery targeting human CD8+ T cells Anti-CD3 coating: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL per well, and store at 4°C overnight.

[0320] Preparation before cell plating: Aspirate the liquid in the 24-well plate, then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0321] Cell plating: Dilute PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double-antibody). Plate 0.05 million cells per well of a 96-well plate in duplicate. After plating, allow cells to stabilize for 4 hours before adding LNPs.

[0322] Drug addition: TLNP-2 modified with the corresponding targeting molecule was added to each well so that the working concentration of LNP in the well plate was as shown in the table below. PBS was added as a negative control group.

[0323] Table 49: Information on adding LNP to the well plate

[0324] Culture: After adding LNP, PBMC cells were cultured at 37 o C, Transfection for 24 h under 5% CO2 conditions.

[0325] Cytotoxicity assay: Add 20 μL of CCK8 reagent into a 96-well plate and incubate in a 37° incubator protected from light. Detect the OD value. The calculation formula is: Cell viability = [(As-Ab) / (Ac-Ab)] × 100% Wherein, As is the absorbance of the experimental well (containing cells, culture medium, CCK8 solution, and drug solution); Ac is the absorbance of the control well (containing cells, culture medium, and CCK8 solution, but no drug); Ab is the absorbance of blank wells (containing culture medium and CCK8 solution, but not cells or drugs).

[0326] Analyze the test data to confirm the toxicity of LNP to cells.

[0327] The experimental results showed that TLNP-2 at different working concentrations showed no cytotoxicity, indicating that the nanoantibody-modified TLNP is safe and cells have a very high dose tolerance to this LNP.

[0328] The data on the safety of TLNP-2 at different working concentrations in hPBMC are detailed in the Figure 13 .

[0329] Example 31: This example targets human CD8 + Evaluation of the safety of T cell nanoparticles in mice (I) Preparation of Nanoparticles Targeting Human CD8+ T Cells The two lipid nanoparticles used in this example are cLNP-1 and TLNP-2 prepared in Example 21.

[0330] (II) Investigation of the in vivo safety of nanoparticles targeting human CD8+ T cells The prepared LNPs were injected into mice via the tail vein at 80 μg per mouse (acute toxicity) or 20 μg per mouse for 6 consecutive injections, once a week (chronic toxicity); Twenty-four hours after the onset of acute poisoning, blood was collected from mice to separate serum, and liver and kidney function and cytokine levels were tested. The main tissues and organs of the mice were dissected and subjected to histopathological examination. One week after the last injection, blood was collected from mice in the long-term poisoning group to separate serum and test liver and kidney function and cytokine levels. The main tissues and organs of the mice were dissected and subjected to histopathological examination. Analyze the test data and confirm the toxicity of LNP.

[0331] The experimental results showed that the liver and kidney functions, cytokine levels and tissue sections of mice in both the acute poisoning group and the chronic poisoning group were basically consistent with those in the non-drug group, indicating that TLNP did not show obvious toxicity and was relatively safe.

[0332] The liver toxicity index test results of the acute toxicity experiment of traditional cLNP and targeted TLNP in mice at different doses are detailed in Figure 14 .

[0333] Example 32: This example targets human CD8 + Evaluation of the in vivo delivery efficiency of nanoparticles for T cells in NHPs (I) Preparation of Nanoparticles Targeting Human CD8+ T Cells The lipid nanoparticles used in this example are the TLNP-2 prepared in Example 21.

[0334] (II) Evaluation of the delivery efficiency of nanoparticles targeting human CD8+ T cells Two cynomolgus macaques (NHPs) that had passed quarantine and met the required weight were divided into two groups. They received intravenous injections of the prepared TLNP-EGFP at a concentration of 100 μg / mL. One group received a high-dose dose of 0.2 mpk (high dose), while the other group received a low-dose dose of 0.1 mpk (low dose). Peripheral blood was collected before administration and on days 1-7, 10, and 16 after administration, and flow cytometry was used to measure the expression of eGFP mRNA in various cell subsets.

[0335] Experimental results showed that compared with the control group, TLNP administration demonstrated significant targeted delivery in CD8 cells in NHPs, with the high-dose group demonstrating higher targeted delivery than the low-dose group. Both the high-dose and low-dose groups demonstrated high on-target activity and low off-target effects. Dynamic changes in EGFP expression after TLNP administration demonstrated that eGFP mRNA expression remained high in NHPs for one week.

[0336] The details of EGFP expression of TLNP-2 in NHP are shown in Figure 15 .

[0337] The details of EGFP targeting and off-target of TLNP-2 in NHP are as follows Figure 16 .

[0338] The dynamic changes of EGFP expression in TLNP-2 NHP are detailed in Figure 17 .

[0339] Example 33: This example targets human CD8 + In vivo safety evaluation of T cell-derived nanoparticles for NHPs (I) Preparation of Nanoparticles Targeting Human CD8+ T Cells The lipid nanoparticles used in this example are the TLNP-2 prepared in Example 21.

[0340] (II) Safety evaluation of nanoparticles targeting human CD8+ T cells The NHP dosage was the same as in Example 32. The safety evaluation of nanoparticles targeting human CD8+ T cells in NHP included: measuring the body weight of NHP before administration and on days 25, 37, 39, 43, and 46 after administration; measuring the body temperature of NHP before administration and on days 1-7, 14, 21, and 25 after administration; performing a routine blood test before administration and on day 1 after administration; collecting serum for solid phase chip cytokine detection before administration and on days 1-6 after administration; and collecting plasma for blood biochemical analysis before administration and on days 1-7, 10, and 16 after administration.

[0341] Experimental results showed that continuous monitoring of NHP weight and temperature after TLNP administration maintained pre-dose levels. Routine blood tests revealed a slight increase in granulocytes after administration, with minimal changes in other routine blood tests. Continuous cytokine analysis revealed minimal fluctuations in cytokine levels. Blood biochemistry analysis revealed only transient changes in CK and AST levels after administration, which quickly returned to pre-dose levels. These findings demonstrate the high safety of the human CD8+ T cell-targeting nanoparticles in NHPs.

[0342] The changes in NHP body weight after TLNP-2 administration are detailed in Figure 18 .

[0343] The changes in NHP body temperature after TLNP-2 administration are detailed in Figure 19 .

[0344] The changes in NHP blood routine after TLNP-2 administration are detailed in Figure 20 .

[0345] The changes in NHP serum factors after TLNP-2 administration are detailed in Figure 21 .

[0346] The details of NHP blood biochemistry after TLNP-2 administration are as follows Figure 22 .

[0347] Example 34: This example targets human CD5 + Screening of T cell targeting molecules in nanoparticles (I) Preparation of human CD5-targeted + Nanoparticles for T cells Table 50: Preparation of LNPs with different targeting molecules (Lx)

[0348] The five groups of TLNPs required in this embodiment were prepared by the method described in Example 20. The ionizable amino lipids: DSPC: CHO: DMG-PEG2000 The five-component lipid complex (DSPE-PEG2000-Mal) was dissolved in ethanol at a molar ratio of 47.5:10:41:1.49:0.01. eGFP-φ-mRNA was dissolved in 75 mM citric acid buffer (pH 4) to maintain an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio (N / P) of ionizable amino lipid to mRNA was 6.5.

[0349] Table 51: Quality control information of Lx-TLNP containing different targeting molecules modified with nanoantibodies

[0350] (II) Targeting human CD5 + Evaluation of the in vitro delivery efficiency of nanoparticles to T cells Anti-CD3 coating: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL per well, and store at 4°C overnight.

[0351] Preparation before cell plating: Aspirate the liquid in the 24-well plate, then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0352] Cell plating: Dilute human PBMC to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double antibody) supplemented with anti-CD28 antibody (concentration 1 μg / mL) and IL-2 (working concentration 400 U / mL). Add 1 million cells to each well of a 24-well plate. After plating, incubate at 37 o C, LNP was added after stabilization for 4 h under 5% CO2.

[0353] Drug addition: Each well was added with the five LNPs prepared in Table 50, so that the working concentration of LNP in the plate was 0.5 μg / mL. PBS was also added as a negative control group.

[0354] Culture: After adding LNP, PBMC cells were cultured at 37 o C, Transfection for 24 h under 5% CO2 conditions.

[0355] Detection of eGFP-mRNA expression: Cells from each well plate were collected and stained with commercially available flow cytometry antibodies. The expression rate and mean fluorescence intensity (MFI) of eGFP-mRNA in CD5+ T and CD3+ T cell subsets in PBMC were detected by flow cytometry.

[0356] Table 52: In vitro transfection results of TLNPs containing different targeting molecules

[0357] The experimental results showed that TLNPs prepared by L1, L2 and L3 showed obvious CD5+ T cell targeting effect at a lower dosage and had good transfection efficiency.

[0358] The results of in vitro transfection of CD5-TLNP containing different targeting molecules are detailed in Figure 23 .

[0359] Example 35: This example targets human CD5 + Screening of the ratio of T cell targeting molecules in nanoparticles (1) Preparation of targeted human CD5 + Nanoparticles for T cells Table 53: Preparation of TLNPs with different targeting molecule ratios

[0360] The twelve groups of TLNPs required in this embodiment were prepared by the method described in Example 20. The ionizable amino lipids: DSPC: CHO: DMG-PEG 2000 The five-component lipid structure (DSPE-PEG2000-Mal) was dissolved in ethanol at a molar ratio of 47.5:10:41:X:Y (the sum of X and Y was 1.5). eGFP-φ-mRNA was dissolved in 75 mM citric acid buffer (pH 4) to maintain an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0361] Table 54: TLNP quality control information with different targeting molecule ratios

[0362] (2) Targeting human CD5 + Evaluation of the in vitro delivery efficiency of nanoparticles to T cells Anti-CD3 coating: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL per well, and store at 4°C overnight.

[0363] Preparation before cell plating: Aspirate the liquid in the 24-well plate, then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0364] Cell plating: Dilute human PBMC to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double antibody) supplemented with anti-CD28 antibody (concentration 1 μg / mL) and IL-2 (working concentration 400 U / mL). Add 1 million cells to each well of a 24-well plate. After plating, incubate at 37 o C, LNP was added after stabilization for 4 h under 5% CO2.

[0365] Drug addition: Each well was added with 12 sets of LNPs prepared in Table 53, so that the working concentration of LNPs in the well plate was 0.5 μg / mL. PBS was also added as a negative control group.

[0366] Culture: After adding LNP, PBMC cells were cultured at 37 o C, Transfection for 24 h under 5% CO2 conditions.

[0367] Detection of eGFP-mRNA expression: Cells in each well plate were collected and stained with commercially available flow cytometry antibodies. The expression rate and mean fluorescence intensity (MFI) of eGFP-mRNA in the CD5+ T cell subsets in PBMC were detected by flow cytometry.

[0368] Table 55: In vitro transfection results of TLNPs containing different targeting molecule ratios

[0369] Experimental results showed that L2-TLNPs prepared with L2 exhibited the best CD5+ T cell targeting and delivery efficiency at lower dosages. Furthermore, when the targeting molecule ratio was higher than 0.05%, no significant differences in delivery efficacy were observed among the different TLNP ratios. Considering the physical and chemical properties of LNPs and material cost factors, a 0.05% targeting molecule ratio was used for subsequent studies.

[0370] The results of in vitro transfection of CD5-TLNPs containing different targeting molecule ratios are detailed in Figure 24 .

[0371] Example 36: This example targets human CD5 + Screening of nanoparticle formulations for T cell therapy (1) Preparation of targeted human CD5 + Nanoparticles for T cells Table 56: Preparation of CD5-TLNPs with different ratios

[0372] The two lipid nanoparticles L2-TLNP-F1 and L2-TLNP-F2 used in this example were prepared by the method described in Example 21.

[0373] (II) Investigation of the in vivo delivery efficiency of nanoparticles targeting human CD5+ T cells with different formulations Nine 6-8-week-old hCD5 humanized female mice that passed quarantine and met SPF (Specific Pathogen Free) standards were selected and divided into three groups. The mice were injected with the prepared L2-TLNP-F1 and L2-TLNP-F2 via the tail vein. Each mouse received a dose of 20 μg mRNA-LNP in a 200 μL injection volume. After 24 hours of expression, peripheral blood, spleen, lymph nodes, and tibia were collected for flow cytometry analysis to determine the expression rate of eGFP mRNA in cell subsets in each organ.

[0374] Table 57: In vivo experimental groups of L2-TLNP with different ratios

[0375] The experimental results showed that L2-TLNP-F2 prepared by formula F2 and containing 0.05% targeting molecule ratio showed better CD5 expression in various organs of hCD5 humanized mice than F1 group at a dosage of 1.0 mpk. + T cell targeted delivery effect.

[0376] The statistical diagram of L2-TLNP in vivo transfection with different ratios is shown in Figure 25 .

[0377] Example 37: This example targets human CD5 + Study on the distribution of nanoparticles in T cells in vivo (1) Preparation of nanoparticles targeting human CD5 T cells The preparation method described in Example 21 was used. The two lipid nanoparticles used in this example were cLNP-1 and L2-TLNP-F2, which encapsulated Luciferase mRNA, respectively.

[0378] (II) Luc-mRNA biodistribution of human CD5 T cell-targeted nanoparticles in hCD5 humanized mice Nine hCD5 humanized female mice (6-8 weeks old) that had passed quarantine and met SPF (Specific Pathogen Free) standards were selected (3 mice per group). Each mouse in the G1 group received a tail vein injection of 200 μL of PBS. Each mouse in the G2 and G3 groups received a tail vein injection of 2 μg of Luc-φ-mRNA LNPs in a 200 μL injection volume.

[0379] Six hours after drug administration, mice were anesthetized with isoflurane and intraperitoneally injected with 250 μL of luciferase substrate. Ten minutes later, the mice were dissected, and the brain, heart, liver, spleen, lungs, kidneys, thymus, lymph nodes, and bone marrow were removed for ex vivo organ imaging. Images were taken using a Perkin Elmer Living Imaging instrument from Shenzhen Bay Laboratory, set to F8 mode with a 2-second exposure time. Following imaging, mRNA was extracted from the organs and quantitative PCR was performed to determine mRNA enrichment in each organ.

[0380] Table 58: Luc imaging study LNP in vivo distribution experimental grouping

[0381] The experimental results showed that in the hCD5 humanized mouse model, compared with traditional LNP, CD5+ T cell-targeted L2-TLNP-F2 can more selectively deliver Luc-mRNA to T cell-rich organs, which shows a higher spleen-liver fluorescence intensity ratio.

[0382] Luc imaging and qPCR analysis of hCD5 biodistribution in humanized mice. Figure 26 .

[0383] The above-described embodiments merely illustrate several implementations of the present invention, and are provided to facilitate a specific and detailed understanding of the technical solutions of the present invention. They should not be construed as limiting the scope of protection of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, and all such modifications and improvements fall within the scope of protection of the present invention.

Claims

1. A molecule with targeting function, characterized in that: include: (I) one or more hydrophobic chains; (II) one or more linkers; (III) one or more Nanobodies; Preferably, the number of the Nanobodies is ≤20; preferably 1-7; Preferably, an amino acid containing a reactive site is connected between the Nanobody and the linker, and the amino acid includes but is not limited to cysteine ​​and lysine; Preferably, the Nanobodies are connected via a linker sequence, the linker sequence being located between the Nanobodies and the amino acids, the linker sequence comprising (GS)n and / or (GGGS)m, wherein n and m are the same or different values, and the value of n or m is selected from 0, 1, 2, 3 or 4; Preferably, the hydrophobic chain comprises one or more substituted or unsubstituted C12-C18 alkyl groups; Preferably, the linker comprises one or more structural units selected from -C(=O)O-, -OC(=O)O-, -C(=O)NH-, -C(=O)S-, -S-, -SS-, triazole, hydrazone bond, SMCC, Sulfo-SMCC, SATA, a polypeptide as a structural unit, PEG or a PEG derivative; Wherein, the PEG derivative may be maleimide-functionalized polyethylene glycol, including but not limited to PEG-MAL; Preferably, the relative molecular mass of the PEG or PEG derivative is 500-5000, preferably 1000, 2000, 3000, 4000 or 5000.

2. The molecule with targeting function according to claim 1, characterized in that The nanobody comprises an antigen-binding domain that is specific to an antigen, and the antigen is a membrane protein molecule; Preferably, the membrane protein molecule is an immune cell or immune-related cell membrane protein molecule, and the immune cell or immune-related cell membrane protein molecule includes but is not limited to one or more of CD3, CD4, CD5, CD7, CD8, CD25, CD38, CD61, CD42a, CD105, CD90, CD15, CD127, CD56, CD68, CD19, CD11c, CD138, F4 / 80, CD62P, CD49f, CD31, RANK, ALPL, PDPN, CD34, FcεR1α, CD203c, CD63, CD193, CD66b, CD41, CD117, and ASGPR; Preferably, the membrane protein molecule is a central nervous system cell membrane protein molecule, and the central nervous system cell membrane protein molecule includes but is not limited to one or more of GD2, GD3, MOG, and TMEM119; Preferably, the membrane protein molecule is a tumor-associated antigen, which includes but is not limited to one or more of CD133, PSMA, CLDN18.2, DLL3, TROP2, EGFRVIII, CA125, MUC1, MUC16, MSLN, CA9, HER2, HER3, TGM4, PSCA, CLDN6, STEAP2, GPC3, IL-13Ra2, EGFR, STEAP1, BCMA, FRa, VEGFR2, PDGFR-β, CEA, NCAM, FAP, SLC2A2, SEZ6L2, and LRP11; Preferably, the membrane protein molecule is an immune modulatory molecule, which includes but is not limited to one or more of B7-H1, B7-H3, and B7-H4; Preferably, the membrane protein molecules include but are not limited to the following targets: CD3, CD4, CD5, CD7, CD8, CD25, CD38, CD61, CD42a, CD105, CD90, CD15, CD127, CD56, CD68, CD19, CD11c, CD138, F4 / 80, CD62P, CD49f, CD31, RANK, ALPL, PDPN, CD34, FcεR1α, CD203c, CD63, CD193, CD66b, CD41, CD117, ASGPR, GD2, GD3, MOG, One or more of TMEM119, CD133, PSMA, CLDN18.2, DLL3, TROP2, EGFRVIII, CA125, MUC1, MUC16, MSLN, CA9, HER2, HER3, TGM4, PSCA, CLDN6, STEAP2, GPC3, IL-13Ra2, EGFR, STEAP1, BCMA, FRa, VEGFR2, PDGFR-β, CEA, NCAM, FAP, SLC2A2, SEZ6L2, LRP11, B7-H1, B7-H3, B7-H4; Preferably, the nanobody is a nanobody targeting CD8, and the amino acid sequence of the nanobody is shown in any one of SEQ ID NO: 1-3; And / or, the Nanobody is a Nanobody targeting CD19, and the amino acid sequence of the Nanobody is shown in any one of SEQ ID NOs: 79-81; And / or, the nanobody is a nanobody targeting CD56; And / or, the Nanobody is a Nanobody targeting CD5, and the amino acid sequence of the Nanobody is shown in any one of SEQ ID NO: 4-78; And / or, the Nanobody is a Nanobody targeting PSMA, and the amino acid sequence of the Nanobody is shown in any one of SEQ ID NO: 82-83; And / or, the nanobody is a nanobody targeting CD133, and the amino acid sequence of the nanobody is shown in any one of SEQ ID NO: 84-85.

3. A nanobody with targeting function, characterized in that: The amino acid sequence of the Nanobody is as shown in any one of SEQ ID NO: 4-78; or, the amino acid sequence of the Nanobody has a homology of ≥80% with the amino acid sequence shown in any one of SEQ ID NO: 4-78.

4. Lipid nanoparticles, characterized in that Its components include the molecule with targeting function according to claim 1 or 2, and the content of the molecule with targeting function in the total lipid of the lipid nanoparticle is: 0.0001-1.0 mol%, preferably 0.01-0.5 mol%; Preferably, the components of the lipid nanoparticles include polymer-bound lipids and ionizable amino lipids; the polymer-bound lipids are selected from lipids modified with PEG or its derivatives; The relative molecular mass of the lipid modified by PEG or its derivatives is 2000-5000, preferably 2000, 3000, 4000 or 5000; Preferably, the content of the polymer-bound lipid in the total lipid of the lipid nanoparticle is: 0.2-5 mol%, preferably 0.5-2 mol; Preferably, the PEG derivative comprises maleimide-functionalized polyethylene glycol and non-maleimide-functionalized polyethylene glycol; wherein the maleimide-functionalized polyethylene glycol can be selected from PEG-MAL; the content of maleimide-functionalized polyethylene glycol in the total lipid of the lipid nanoparticle is: 0.0001-1.0 mol%, preferably 0.01-0.5 mol%; Preferably, the ionizable amino lipid accounts for 30-70 mol% of the total lipids in the lipid nanoparticles, preferably 40-60 mol%; More preferably, the ionizable amino lipid has a structure represented by general formula (I), or is an isomer, a pharmaceutically acceptable salt, a prodrug or a solvate of the structure represented by general formula (I); wherein G is selected from H, OR, CN, -C(=O)OR', -OC(=O)R', -C(=O)NR'R'', -NR'C(=O)R'', NR'R'' or a cyclic alkyl structure containing at least one heteroatom; the substitutable carbon atoms or heteroatoms in the cyclic alkyl structure are unsubstituted or substituted with one or more hydroxyl groups, C1-C4 alkyl groups, C2-C4 alkenyl groups, C3-C8 cycloalkyl groups or C3-C8 cycloalkenyl groups; M1, M2, M3, and M4 are the same or different from each other and are each independently selected from C1-C 24 Alkylene, C3-C 24 Cycloalkylene, C2-C 24 Alkenylene or C3-C 24 Cycloalkenylene; R 1 , R 2 are the same as or different from each other and are each independently selected from H, C1-C 24 Alkyl, C3-C 24 Cycloalkyl, C2-C 24 Alkenyl or C3-C 24 Cycloalkenyl; L1, L2, L3, L4 are the same as or different from each other and are independently selected from -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=O)NR-, -NRC(=O)-, -S(=O)-, -OS(=O)2-, -S(=O)2O-, -O-, -S- or -SS-; R, R', R'' are the same or different from each other, and are each independently selected from H, Cl-C 10 Alkyl, C3-C 10 Cycloalkyl, C3-C 10 Alkenyl or C3-C 10 Cycloalkenyl, C1-C 10 Alkyl, C3-C 10 Cycloalkyl, C3-C 10 an alkenyl group or a cyclic alkyl structure containing at least one heteroatom; the cyclic alkyl structure is unsubstituted or substituted by one or more C1-C4 alkyl groups, C2-C4 alkenyl groups, C3-C8 cycloalkyl groups, or C3-C8 cycloalkenyl groups; M5 is selected from a single bond, C1-C 16 Alkylene, C2-C 16 Alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene; Preferably, the lipid nanoparticles further include at least one of a steroid and a neutral lipid; Further preferably, the steroid is one or more of cholesterol and its derivatives, cholesterol esters, steroid hormones, steroid vitamins, and phytosterols, preferably cholesterol; and / or, the neutral lipid is a phospholipid; and / or, the polymer-bound lipid is a pegylated lipid, and the pegylated lipid is preferably DMG-PEG2000; Preferably, the molar ratio of ionizable amino lipids, steroids, neutral lipids and polymer-bound lipids is 30-70:0-65:0-30:0.2-5; further preferably 40-60:35-60:0-20:0.5-2; further preferably 40-55:30-50:0-20:0.5-2; more preferably 40-50:30-45:0-15:0.5-2; more preferably 47-50:34-36:13-16:1.0-1.1; more preferably 47.5:36:15:1.

0.

5. The method for preparing lipid nanoparticles according to claim 4, characterized in that: The method comprises the following steps: mixing polymer-bound lipids, ionizable amino lipids, steroids and auxiliary phospholipids to obtain lipid nanoparticles without targeting function, and then connecting the molecules with targeting function described in claim 1 or 2 to the lipid nanoparticles without targeting function through membrane fusion or chemical bonds to obtain lipid nanoparticles with targeting function.

6. The method for preparing lipid nanoparticles according to claim 4, characterized in that: The method comprises the following steps: mixing a polymer-bound lipid, an ionizable amino lipid, a steroid and an auxiliary phospholipid to obtain lipid nanoparticles without a targeting function, and then connecting a nano antibody to the lipid nanoparticles without a targeting function through a chemical bond to obtain lipid nanoparticles with a targeting function; Preferably, the polymer-binding lipid comprises maleimide-functionalized polyethylene glycol, and the Nanobody is linked to the maleimide-functionalized polyethylene glycol.

7. The method for preparing lipid nanoparticles according to claim 4, characterized in that: The method comprises the following steps: mixing polymer-bound lipids, ionizable amino lipids, steroids, auxiliary phospholipids and the molecules with targeting function according to claim 1 or 2 to obtain lipid nanoparticles with targeting function.

8. A method for targeted delivery of a pharmacodynamic molecule to specific cells of a subject, the method comprising: The specific cells are contacted with the lipid nanoparticles according to claim 4; preferably, the specific cells include but are not limited to immune cells, immune-related cells, central nervous cells, tumor cells, somatic cells, cells infected by viruses, bacteria, and fungi; preferably, the targeted cells are B cells, T cells, or NK cells.

9. A method for expressing a protein or polypeptide of interest in a targeted cell of a subject, the method comprising: The target cells are contacted with the lipid nanoparticles according to claim 4; preferably, the target cells include but are not limited to immune cells, immune-related cells, central nervous cells, tumor cells, somatic cells, cells infected by viruses, bacteria, and fungi; preferably, the target cells are B cells, T cells, or NK cells.

10. Use of the lipid nanoparticles according to claim 4 as a drug delivery carrier.

11. A pharmaceutical composition, characterized in that The lipid nanoparticles according to claim 4, wherein the lipid nanoparticles contain pharmacodynamic molecules; Preferably, the pharmacodynamic molecule comprises one or more of mRNA, DNA, siRNA, saRNA, shRNA, miRNA, circle RNA, lncRNA, gRNA, polypeptide or protein; Preferably, the pharmacodynamic molecule includes mRNA, which is used to encode a CAR molecule, a TCR molecule, an immunomodulatory molecule, a functional protein molecule, or a gene editing tool such as a base editor or a Cas9 protein; Preferably, the CAR molecule comprises at least one of a sequence that can specifically recognize a tumor-specific antigen, a transmembrane connection sequence, and / or a sequence that assists the immune function of T cells; Preferably, the sequence encoding the TCR molecule comprises a sequence that can recognize at least one molecule among NY-ESO-1, AFP, HBsAg, MAGEA1, Mesothelin, MART-1, CD19, CD28, PD-1, CD8, CT83, E6, E7, E8, GPC3, H3.3-K27M, HIV Gagpolyprotein, HLA-A / AFP, KRASG12 (V / D), KRASG12D, KRASG12V, LMP1, LMP2, EBNA1, MAGEA10, MAGEA3, MAGEA4, MC2R, mHag HA-1, MYO1G, PRAME, WT1, gp100, CEA, p53, HLA-A2, EGFR, DR5, RAS, LAGE-1, CMV, HCV, MCPyv, and HA-1H; Preferably, the sequences encoding immunomodulatory molecules include CD28, PD-1, CTLA-4, RGMB, ICOS, CD28H, NKp30, HVEM, OX40, Fas, 4-1BB, CD27, CD30, APO-2, APO-3, BCMA, BAFFR, GITR, IL-2, IL-12A, IL-12B, IL-15, IL-23, IL-27, FLT3L, IL-36A, IL-36B, IL-36C, GM-CSF, CCL20, CXCL9, CXCL10, CXCL11, CXCL12, CCR7, CX The sequence of at least one of CR4, CCR5, CCL4, CCL5, CCL19, CXCR3, CCR6, B7-H1, B7-DC, B7-H3, B7-H4, PD-1H, IL-10, TGF-β, HGF, B7-1, B7-2, B7-H2, B7-H3, CD40, FasL, CD70, CD30L, 4-1BBL, OX40L, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, GITRL, CD73, STAT1, IRF4, CCAR2, BCL6, iNOS, and CD103; Preferably, the nucleic acid encoding the CAR molecule comprises a sequence that can recognize at least one molecule of CD19, BMCA, CD22, CD20, CD123, GD2, CD30, GPC3, CLDN18.2, Mesothelin, CD33, CD38, EGFRvlll, CD138, CEA, HER2, PSMA, CLL1, CD56, EGFR, MUC-1, EpCAM, CD7, NKG2D, PD-L1, LewisY, FAP, c-MET, ROR1, IL13Rα2, AFP, CD133, CD4, BTK, ROBO1, CD5, CD70, LILRB4, FLT3, Sigle-6, CD229, and SLAMF7.

12. A pharmaceutical preparation comprising the pharmaceutical composition according to claim 11 and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical preparation is an aqueous injection, a lyophilized powder or a spray.

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