Polypeptide-modified liposome for targeted drug delivery and use thereof

Abstract

A polypeptide-modified liposome for targeted drug delivery and a use thereof, relating to the technical field of biomedicine. The raw materials for the preparation of the liposome comprise a polypeptide-modified lipid material. The polypeptide-modified lipid material is obtained by coupling a polypeptide to a lipid material containing a polyethylene glycol chain; the polypeptide is Cys-(X)n, wherein X is any one or any two of Arg, Asp, His, Glu or Thr, n is the total number of X and is an integer in the range of greater than 1 and less than 11; and the polypeptide is coupled to the polyethylene glycol chain by means of a thiol group on a cysteine residue of the polypeptide. The provided polypeptide-modified liposome can achieve targeted delivery of drugs to organs, tissue and cells in the body. The provided polypeptide-modified lipid material has a certain degree of universality, and can be used in combination with multiple currently commercially available liposome excipients; and the type of an ionized lipid material and the type of a phospholipid can be adjusted arbitrarily.

Description

A polypeptide-modified liposome for targeted drug delivery and its application Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a polypeptide-modified liposome for targeted drug delivery and applications thereof.

[0002] Cross-references

[0003] This application claims the benefit of Chinese patent application CN202310977908.7 filed on August 4, 2023, the contents of which are incorporated herein in their entirety.

[0004] Sequence listing file submitted at the same time

[0005] The entire contents of the following XML file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Format (CRF) TFH01072 PCT - sequence listing (created on August 4, 2024, file size 19.8 KB). Background Art

[0006] Currently, mRNA vaccines have been widely used. The successful application of mRNA vaccines is not only a milestone in the vaccine field, but also greatly promotes the development of nucleic acid drugs in many fields, such as tumor immunotherapy, tissue regeneration and repair, cell fate regulation, gene editing, and many other life and health fields. In principle, nucleic acid drugs have modular production characteristics. They can quickly produce their mRNA or DNA sequences based on the target protein, and then deliver them to the targeted organs or tissues in the body with the help of specific nucleic acid delivery vectors. Therefore, nucleic acid drugs have the advantages of personalization, standardization, and modularization. This feature gives them great potential for rapid and widespread application.

[0007] However, due to the influence of many factors on the stability and targeting of naked nucleic acid drugs (mRNA, siRNA and DNA, etc.), it is difficult for them to be actively transported to specific target sites to exert their effects. Therefore, nucleic acid drugs must rely on specific delivery carriers. Delivery carriers can not only protect the encapsulated nucleic acid drugs from degradation by nucleases, but also deliver nucleic acid drugs to specific tissues and organs or even specific cell types with the help of the targeting effect of the carrier, thereby achieving targeted delivery of nucleic acid drugs. Therefore, safe, efficient and targeted delivery carriers are the key to the real function of nucleic acid drugs in vivo. Due to the presence of various biochemical and physical barriers in the body, once nucleic acid drugs enter the body, the blood flow shearing effect, protein corona coating effect, mononuclear macrophage system and nucleic acid degrading enzymes and other effects make nucleic acid drugs very likely to fail before entering specific targeted cells. After being internalized by cells, nucleic acid drugs need to further escape from endosomes, which further limits the delivery efficiency of nucleic acid drugs to a certain extent. Therefore, it is extremely challenging to successfully deliver nucleic acid drugs to specific sites in vivo. Currently, inorganic nanomaterials, peptides, polymers, liposomes (LNPs), etc. have all been developed into nucleic acid drug delivery vehicles. Among the many carriers, liposomes have been widely used as delivery vehicles for nucleic acids, proteins, and chemical drugs in many fields such as COVID-19 vaccines, cancer immunotherapy, tissue repair and regeneration. For example, in the field of liver disease treatment, the Onpattro product, which was approved by the FDA in August 2018, has been widely used in clinical practice. This product is an siRNA-loaded LNP dosage form drug used to treat polyneuropathy caused by the hereditary disease transthyretin-mediated amyloidosis (hATTR), and works by inhibiting the synthesis of hepatic thyroxine transporter (TTR). Currently, the main target organ of widely used liposome-based drugs is the liver. How to enable liposome-based drugs to achieve targeting of extrahepatic organs or tissues and overcome the obstructive effects of the liver is the key to the effectiveness of the delivered drugs.

[0008] To achieve targeted delivery to extrahepatic organs, there are currently three main approaches: (1) Changing the delivery method and delivering drugs to specific sites through local injection, thereby avoiding the multiple barriers that nucleic acid drugs encounter during systemic intravenous injection, and thus achieving targeted drug delivery to specific tissue sites; (2) Liposome chemical library construction and screening: Researchers currently mainly construct nanolibraries by modifying the chemical structure of ionized liposomes, and then screen out organ-targeted carriers through high-throughput screening. (3) Addition of additional components: Researchers add a fifth component to the original four components of liposomes. By regulating the charge properties of the added component, targeted delivery to extrahepatic organs can be achieved. In summary, organ-targeted carriers administered intravenously, whether through chemical library design screening or charge regulation, only optimize the chemical structure or properties of liposomes from the perspective of chemical structure and composition, thereby changing their targeted delivery behavior in vivo. However, the carriers designed by this method obviously do not have modular control characteristics. Peptide nanocarriers with encodable properties have the advantages of excellent biocompatibility, rich structure, and diverse functions. Therefore, combining peptide-modified nanoliposomes is of great significance for screening, optimizing, and designing multi-scale targeted nanocarriers for organs, tissues, cells, etc. in the body.

[0009] Summary of the Invention

[0010] The purpose of the present invention is to provide a polypeptide-modified liposome for targeted drug delivery and its application to solve the problems existing in the above-mentioned prior art. The polypeptide-modified liposome provided by the present invention can achieve targeted delivery of drugs to organs, tissues and cells in the body.

[0011] The present invention proposes a peptide-modified liposome that can achieve organ-targeted drug delivery. First, different types and numbers of amino acids are dehydrated and condensed to form a peptide, and then the prepared peptide is coupled to Mal-PEG. 2k -DSPE, dissolving the five liposome components in an alcohol phase and the nucleic acid in an aqueous phase. The two were then blended via microfluidics to form nucleic acid-encapsulated nanoliposomes. Finally, the encapsulated nucleic acid drug-encapsulated nanoliposomes were injected into mice via the tail vein. In vivo imaging allowed for the observation of the target organ of the delivered nucleic acid drug. The following procedures and studies were performed:

[0012] S1. Peptide Preparation

[0013] According to experimental requirements, specific types and numbers of amino acids or amino acid derivatives are selected, and specific encoded polypeptides are prepared using peptide synthesis (such as the Fmoc solid-phase peptide synthesis method). The type and length of the polypeptide depend on the type and number of the input amino acids or amino acid derivatives (computer-aided design). By regulating the properties of the prepared polypeptide (charge, hydrophobic interaction, hydrogen bonding, etc.), the carrier can achieve targeted drug delivery to specific organs, tissues, and cells in the body.

[0014] S2. Peptide Characterization

[0015] First, the main peak of the synthesized peptide was determined by liquid chromatography (HPLC), and then its molecular weight was determined by mass spectrometry (MS). It was compared with the molecular weight of the synthesized target peptide to confirm that the synthesized peptide met the expected target.

[0016] S3. Preparation of peptide-modified liposomes

[0017] Cysteine ​​is introduced into the end of the polypeptide (C-terminus or N-terminus) to participate in the reaction, and the thiol group of cysteine ​​is used to react with Mal-PEG 2k -DSPE reaction, the peptide prepared in step S1 can be coupled to PEG 2k -DSPE surface, and finally its chemical structure was analyzed by nuclear magnetic hydrogen spectrum to confirm the successful coupling of the peptide to PEG 2k -DSPE surface. A certain proportion of ionized lipids (such as SM102), cholesterol, phospholipids (such as DSPC), and peptide-coupled PEG are then dissolved in ethanol, and a specific amount of nucleic acid (such as mRNA) is dissolved in citrate buffer. The aqueous phase and the alcohol phase are then blended at a specific flow rate through a microfluidic device. During the microfluidic blending process, the nucleic acid and lipid components self-assemble to prepare peptide-modified liposomes.

[0018] S4. Peptide-modified liposomes enable organ-targeted delivery of mRNA in vivo

[0019] The liposomes synthesized in step S3 with a certain content were intravenously injected into mice. To determine the organ distribution of the peptide-modified liposomes in vivo, mRNA encoding firefly luciferase was used as the encapsulated nucleic acid. A few hours after the intravenous injection of the mouse liposomes, the various organs of the mouse were imaged by in vivo imaging under the action of the substrate to determine the target organ of the delivered mRNA. To determine whether the peptide-modified liposome method for achieving organ targeting has a certain degree of universality and mobility, the peptide-modified liposomes formed by changing the type of ionized lipids, such as the most widely commercially available MC3, SM102, ALC0315, LP01, etc., were delivered in vivo, and their enriched organs were determined by in vivo imaging. In addition, by changing the phospholipid composition, such as DSPC, DOPC, DOPE, HSPC, etc., the organs enriched in the prepared liposomes were characterized. Finally, by changing the content of the modified peptide in the liposomes, the effect of different concentrations of peptide-modified liposomes on their organ targeting in vivo was analyzed.

[0020] Based on the above research, the present invention provides the following solutions:

[0021] The present invention provides a polypeptide-modified lipid material, which is obtained by coupling a polypeptide to a lipid material containing a polyethylene glycol chain;

[0022] The polypeptide is Cys-(X) n , wherein X is any one or any two of Arg, Asp, His or Glu, and n is the total number of X and is an integer greater than 1 and less than 11;

[0023] The polypeptide is coupled to the polyethylene glycol chain via the thiol group on its cysteine ​​residue.

[0024] Furthermore, some or all of the n Xs in the polypeptide are Arg.

[0025] Furthermore, when some of the n Xs are Arg, the remaining Xs are Lys.

[0026] Furthermore, when some or all of n X's in the polypeptide are Arg, n=3, 4, 5, 6, 7 or 8.

[0027] Furthermore, when X in the polypeptide is His, n=2, 3, 4, 5, 6, 7, 8 or 10.

[0028] Furthermore, X in the polypeptide is any one of Asp, Glu or Thr.

[0029] Furthermore, the X is Asp and Glu; or the X is Asp and Thr; or the X is Asp.

[0030] Furthermore, the lipid material containing polyethylene glycol chain is Mal-PEG 2k -DSPE.

[0031] The present invention also provides use of the above-mentioned polypeptide-modified lipid material in preparing polypeptide-modified liposomes for targeted drug delivery.

[0032] Furthermore, the polypeptide-modified liposome for targeted drug delivery targets an organ in the body, and the organ in the body is the liver, spleen, lung and / or bone marrow.

[0033] The present invention also provides a polypeptide-modified liposome for targeted drug delivery, and the preparation raw materials include the above-mentioned polypeptide-modified lipid material.

[0034] Furthermore, the polypeptide-modified liposomes also include ionized lipids, phospholipids, cholesterol and phospholipid-polyethylene glycol.

[0035] Furthermore, the molar ratio of the ionized lipid, the phospholipid, the cholesterol, the polypeptide-modified lipid material and the phospholipid-polyethylene glycol is (40-60):(5-15):(30-45):(3-12):(0.5-1.5).

[0036] Furthermore, the phospholipid is distearoylphosphatidylcholine, 1,2-dioleoyl-SN-glycero-3-phosphocholine, 1,2-dioleoylglycerol 3-phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine or egg sphingomyelin.

[0037] Furthermore, the ionized lipid is MC3, SM102, ALC0315 or LP01.

[0038] The present invention also provides the use of the above-mentioned polypeptide-modified lipid material or polypeptide-modified liposome in improving drug targeting.

[0039] The present invention also provides the use of the above-mentioned polypeptide-modified lipid material or polypeptide-modified liposome in the preparation of a drug targeting an internal organ, wherein the internal organ is the liver, spleen, lung and / or bone marrow.

[0040] The present invention also provides a method for improving drug targeting, which comprises using the above-mentioned polypeptide-modified lipid material or polypeptide-modified liposome.

[0041] The present invention discloses the following technical effects:

[0042] 1. The polypeptide-modified liposomes provided by the present invention can achieve targeted delivery of drugs to organs, tissues and cells in the body.

[0043] 2. The encodable polypeptide liposomes provided by the present invention can be used to construct a modular polypeptide-modified nanoliposome delivery platform. By adjusting the type and number of amino acids or amino acid derivatives of the encoded polypeptide, a programmable nanocarrier library can be constructed.

[0044] 3. The present invention has a certain degree of universality and can be used in combination with a variety of currently commercially available liposome excipients. The types of ionized lipid materials and phospholipids can be adjusted arbitrarily, and the polypeptide-modified liposomes can be preferentially enriched in specific extrahepatic organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0046] FIG1 is a chemical structure diagram of 3R(A), 6D(B) and 6R(C) prepared and synthesized in Example 1;

[0047] FIG2 is a liquid chromatography (HPLC) test result of 3R prepared and synthesized in Example 1;

[0048] FIG3 is the mass spectrometry (MS) detection result of 3R prepared and synthesized in Example 1;

[0049] FIG4 is a liquid chromatography (HPLC) test result of 6D prepared and synthesized in Example 1;

[0050] FIG5 is the mass spectrometry (MS) detection result of 6D prepared and synthesized in Example 1;

[0051] FIG6 is a liquid chromatography (HPLC) test result of 6R prepared and synthesized in Example 1;

[0052] FIG7 is the mass spectrometry (MS) detection result of 6R prepared and synthesized in Example 1;

[0053] Figure 8 shows the characterization of DSPE-PEG 2k -3R / 6D / 6R H NMR spectrum results; AC are DSPE-PEG 2k -3R、DSPE-PEG 2k -6D and DSPE-PEG 2k -6R;

[0054] FIG9 is a schematic diagram of a microfluidic device for preparing mRNA-encapsulated polypeptide-modified liposomes;

[0055] Figure 10 shows the results of Firefly Luciferase mRNA targeted to various organs in vivo by 3R, 6R, and 6D peptide-modified liposomes (MC3, SM102, ALC0315, LP01 as ionized lipids, and DSPC as phospholipid); A is the in vivo imaging of 3R, 6R, and 6D; B is the percentage of protein expressed by the delivered mRNA in the liver, lung, and spleen; C and E are the fluorescence statistics of 3R, 6R, and 6D, respectively;

[0056] Figure 11 shows the results of Firefly Luciferase mRNA targeting in vivo by 3R, 6R, and 6D peptide-modified liposomes (MC3 as the ionized lipid, DOPC, DOPE, and HSPC as phospholipids); A is the in vivo imaging of 3R, 6R, and 6D; B is the percentage of protein expressed by the delivered mRNA in the liver, lung, and spleen; C and E are the fluorescence statistics of 3R, 6R, and 6D, respectively.

[0057] Figure 12 shows the in vivo fluorescence results of Firefly Luciferase mRNA delivered via liposomes modified with peptides encoding different numbers and combinations of arginine (R) and histidine (H); A shows 2R-10R; B shows 2H-10H; and C shows the normalized comparison of peptides of different sizes targeting three organs.

[0058] FIG13 shows the results of co-delivery of two messenger RNAs (Luciferase mRNA and EGFP green fluorescent protein) by peptide-modified LNPs;

[0059] Figure 14 shows the results of delivering EGFP-ASO to targeted organs; A is an in vivo image of targeted delivery of EGFP-ASO to the liver, lung, and spleen; B is the flow cytometry analysis of EGFP in the targeted organs, with the gray curve representing the negative group (PBS injection); C is a fluorescent section image of targeted delivery of EGFP-ASO to the liver, lung, and spleen; scale bar: 50 μm;

[0060] Figure 15 shows organ-specific editing achieved by delivering Cre mRNA into mT-mG mice; A is a schematic diagram of inducing EGFP expression by delivering Cre into mT-mG mice; B is an EGFP fluorescence image in the targeted organ after Cre mRNA delivery; C is a confocal image of the targeted organ (confirming EGFP protein expression); scale bar: 50 μm;

[0061] Figure 16 shows the delivery of Cas9 mRNA and sgSTOP into mT-mG mice to achieve organ-specific gene editing (CRISPR); A is a schematic diagram of the co-delivery of Cas9 mRNA and sgSTOP into Ai14 tool mice to induce tdTomato expression; B is the in vitro tdTomato live imaging result of the targeted organ after CRISPR-Cas gene editing; C is a confocal image of the targeted organ (confirming the expression of tdTomato protein); scale bar: 50 μm;

[0062] Figure 17 shows organ-specific PTEN gene editing in C57 / BL6 wild-type mice; A is a schematic diagram of organ-specific PTEN gene editing; B is an immunohistochemistry (IHC) image of Cas9 mRNA or Cas9 protein and sgPTEN co-delivered to the targeted organ; scale bar: 100 μm; C is the normalized statistical result of positive PTEN protein in each tissue section (n=10 tissue sections);

[0063] Figure 18 shows PCSK9 gene editing in the liver of C57 / BL6 wild-type mice; A is a Western blot (WB) analysis of PCSK9 knockout; B is an Elisa quantitative analysis of PCSK9 protein; C is a quantitative analysis of cholesterol;

[0064] Figure 19 is a chemical structure diagram of RRRRRK prepared and synthesized in Example 1;

[0065] Figure 20 is the H-NMR spectrum results of RRRRRK prepared and synthesized in Example 1;

[0066] Figure 21 shows the in vivo fluorescence results of liposome-delivered Firefly Luciferase mRNA modified with RRRRRR and RRRRRK peptides; A is the in vivo imaging of RRRRRR and RRRRRK; B is the percentage of mRNA expression in the liver, lung, and spleen delivered by RRRRRR and RRRRRK; C is the statistical results of fluorescent protein expression of RRRRRR and RRRRRK in the lungs;

[0067] FIG22 is a chemical structure diagram of DDEEDD (A), DDDDEE (B), DDTTDD (C), and DDDDTT (D) prepared and synthesized in Example 1;

[0068] FIG23 is the H-NMR spectra of DDEEDD (A), DDDDEE (B), DDTTDD (C), and DDDDTT (D) prepared and synthesized in Example 1;

[0069] Figure 24 shows the in vivo fluorescence results of liposome-delivered Firefly Luciferase mRNA modified with DDDDDD, DDEEDD, DDDDEE, DDTTDD, and DDDDTT polypeptides; A shows the in vivo imaging of DDDDDD, DDEEDD, DDDDEE, DDTTDD, and DDDDTT; B shows the protein expression ratio of DDDDDD, DDEEDD, DDDDEE, DDTTDD, and DDDDTT delivered mRNA in the liver, lung, and spleen; C shows the fluorescence statistics of DDDDDD, DDEEDD, DDDDEE, DDTTDD, and DDDDTT in the spleen;

[0070] Figure 25 shows the results of bone marrow-targeted liposome-delivered EGFP mRNA targeting in various organs in vivo; A is an in vivo imaging image; B is the fluorescence statistics of mRNA expression in the liver, lung, spleen, and bone marrow; C is the percentage of mRNA expression in the liver, lung, spleen, and bone marrow delivered by bone marrow-targeted liposomes;

[0071] Figure 26 is a chemical structure diagram of 6H prepared and synthesized in Example 1;

[0072] FIG27 is the H-NMR spectrum of 6H prepared and synthesized in Example 1;

[0073] All the above data were derived from n = 3 biologically independent mice. All data are plotted as the mean ± SD, and the P values ​​were calculated using the unpaired two-sided Student's t-test, *P < 0.05, **P < 0.001, ****P < 0.0001. DETAILED DESCRIPTION

[0074] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0075] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0076] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0077] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0078] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0079] Terminology Notes:

[0080] Fmoc-Arg(Pbf)-OH refers to Fmoc-protected arginine, CAS number 154445-77-9.

[0081] HOBT refers to 1-hydroxybenzotriazole, CAS No. 2592-95-2.

[0082] DIC refers to N,N'-diisopropylcarbodiimide, CAS No. 693-13-0.

[0083] Fmoc-Cys(Trt)-OH refers to N-Fmoc-S-trityl-L-cysteine, CAS No. 103213-32-7

[0084] Fmoc-Asp(OtBu)-OH refers to 4-tert-butyl fluorenylmethoxycarbonyl-aspartate, CAS No. 71989-14-5.

[0085] DSPC refers to distearoylphosphatidylcholine, CAS number 816-94-4.

[0086] DOPC refers to 1,2-dioleoyl-SN-glycero-3-phosphocholine, CAS No. 4235-95-4

[0087] DOPE refers to 1,2-dioleoylglycerol-3-phosphatidylethanolamine, CAS No. 4004-05-1.

[0088] HSPC refers to hydrogenated soy phosphatidylcholine, CAS number 92128-87-5.

[0089] ESM refers to egg sphingomyelin, CAS number 860061C.

[0090] Mal-PEG 2k -DSPE refers to maleimide-polyethylene glycol-phospholipid, which was purchased from Guangzhou Carbon Water Co., Ltd.

[0091] DSPE-PEG refers to phospholipid-polyethylene glycol, purchased from Guangzhou Carbon Water Co., Ltd.

[0092] The excipients used to prepare liposomes, such as ionized lipids SM102, MC3, ALC0315, and LP01, were purchased from Xiamen Sinobond Biotechnology Co., Ltd.

[0093] The corresponding relationship between the names of amino acids or amino acid residues in the polypeptides of this application is as follows:

[0094] Cys: cysteine, abbreviated as C;

[0095] Arg: Arginine, abbreviated as R;

[0096] Asp: aspartic acid, abbreviated as D;

[0097] His: histidine, abbreviated as H;

[0098] Glu: glutamate, abbreviated as E;

[0099] Thr: threonine, abbreviated as T;

[0100] Lys: lysine, abbreviated as K.

[0101] The present application provides a polypeptide-modified lipid material, wherein the polypeptide-modified lipid material is obtained by coupling a polypeptide to a lipid material containing a polyethylene glycol chain;

[0102] The polypeptide is Cys-(X) n , wherein X is any one or any two of Arg, Asp, His or Glu, and n is the total number of X and is an integer greater than 1 and less than 11;

[0103] The polypeptide is coupled to the polyethylene glycol chain via the thiol group on its cysteine ​​residue.

[0104] In a specific embodiment, X in the polypeptide described in the present application is Arg, that is, the polypeptide is Cys-(Arg) n .

[0105] In some specific embodiments, when the polypeptide is Cys-(Arg) nWhen n=3, 4, 5, 6, 7 or 8. For example, the polypeptide is Cys-(Arg)3, Cys-(Arg)4, Cys-(Arg)5, Cys-(Arg)6, Cys-(Arg)7 or Cys-(Arg)8.

[0106] In one specific embodiment, when X in the polypeptide described herein is Arg, Arg may be partially replaced by Lys. In this case, n is the total number of Arg and Lys. For example, in some specific embodiments, the polypeptide is Cys-(Arg)3(Lys)1, Cys-(Arg)4(Lys)2, or Cys-(Arg)5(Lys)1, etc.

[0107] In a specific embodiment, X in the polypeptide described in the present application is His, that is, the polypeptide is Cys-(His) n .

[0108] In some specific embodiments, when the polypeptide is Cys-(His) n When n=2, 3, 4, 5, 6, 7, 8 or 10. For example, the polypeptide is Cys-(His)2, Cys-(His)3, Cys-(His)4, Cys-(His)5, Cys-(His)6, Cys-(His7, Cys-(His)8 or Cys-(His) 10 In a specific embodiment,

[0109] In some specific embodiments, X in the polypeptide described herein is Asp, that is, the polypeptide is Cys-(Asp) n For example, the polypeptide is Cys-(Asp)2, Cys-(Asp)3, Cys-(Asp)4, Cys-(Asp)5, Cys-(Asp)6, Cys-(Asp)7, Cys-(Asp)8, Cys-(Asp)9 or Cys-(Asp) 10 .

[0110] In some specific embodiments, X in the polypeptides described herein is Asp and Glu. In this case, n is the total number of Asps and Glu. For example, in some specific embodiments, the polypeptide is Cys-(Asp)4(Glu)2, Cys-(Asp)6(Glu)2, or Cys-(Asp)8(Glu)1, etc.

[0111] In some specific embodiments, X in the polypeptides described herein is Asp and Thr. In this case, n is the total number of Asp and Thr. For example, in some specific embodiments, the polypeptide is Cys-(Asp)4(Thr)2, Cys-(Asp)6(Thr)3, or Cys-(Asp)8(Thr)1, etc.

[0112] In this application, the polypeptides may be represented by abbreviations.

[0113] For example, Cys-(Arg)3 can be represented as RRR or 3R, Cys-(Arg)6 can be represented as RRRRRR or 6R, and Cys-(Asp)6 can be represented as DDDDDD or 6D.

[0114] When X is two amino acids or part of R is replaced by K, the position of the abbreviation in the polypeptide can also represent the available amino acid or residue. For example, RRRRRK represents Cys-(Arg)5(Lys)1, and Lys is located at the end of the peptide; DDEEDD represents Cys-(Asp)4(Glu)2, and the two Glu are located in the middle of the peptide; DDDDTT represents Cys-(Asp)4(Thr)2, and the two Thr are located at the end of the peptide.

[0115] In a specific embodiment, the lipid material containing polyethylene glycol chains is Mal-PEG 2k -DSPE.

[0116] The present application also provides the use of the above-mentioned polypeptide-modified lipid material in the preparation of polypeptide-modified liposomes for targeted drug delivery.

[0117] In a specific embodiment, the drug is a nucleic acid drug.

[0118] In a specific embodiment, the polypeptide-modified liposomes for targeted drug delivery are targeted to organs in vivo.

[0119] In a specific embodiment, the internal organ is liver, spleen, lung and / or bone marrow.

[0120] The present application also provides a polypeptide-modified liposome for targeted drug delivery, wherein the raw materials for preparing the polypeptide-modified liposome include the above-mentioned polypeptide-modified lipid material.

[0121] In a specific embodiment, the raw materials for preparing the polypeptide-modified liposomes further include ionized lipids, phospholipids, cholesterol and phospholipid-polyethylene glycol.

[0122] In some specific embodiments, the phospholipid can be selected from distearoylphosphatidylcholine, 1,2-dioleoyl-SN-glycero-3-phosphocholine, 1,2-dioleoylglycerol 3-phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine or egg sphingomyelin.

[0123] In some specific embodiments, the ionized lipid can be selected from MC3, SM102, ALC0315 or LP01.

[0124] In a specific embodiment, in the polypeptide-modified liposomes, the molar ratio of the ionized lipid, the phospholipid, the cholesterol, the polypeptide-modified lipid material and the phospholipid-polyethylene glycol is (40-60):(5-15):(30-45):(3-12):(0.5-1.5).

[0125] In a specific embodiment, the molar ratio of the ionized lipid, the phospholipid, the cholesterol, the polypeptide-modified lipid material and the phospholipid-polyethylene glycol is 50:10:38.5:6:0.75.

[0126] In another specific embodiment, the molar ratio of the ionized lipid, the phospholipid, the cholesterol, the polypeptide-modified lipid material and the phospholipid-polyethylene glycol is 50:10:38.5:3:0.75.

[0127] The present application also provides the use of the above-mentioned polypeptide-modified lipid material or polypeptide-modified liposome in improving drug targeting.

[0128] The present application also provides the use of the above-mentioned polypeptide-modified lipid material or polypeptide-modified liposome in the preparation of drugs targeting organs in the body.

[0129] In a specific embodiment, the internal organ is liver, spleen, lung and / or bone marrow.

[0130] The present application also provides a method for improving drug targeting, which comprises using the above-mentioned polypeptide-modified lipid material or polypeptide-modified liposome.

[0131] For ease of illustration, the following example illustrates the preparation of a peptide-modified liposome, demonstrating in vivo targeted mRNA delivery to organs such as the liver, spleen, lung, and bone marrow. This invention primarily focuses on the organ-targeted delivery technology of peptide (including other peptide derivatives, etc.)-modified liposomes. Through modular peptide-modified nanocarriers, it is expected that the screening, optimization, and design of carriers for specific organs, tissues, and cell types will be achieved, ultimately achieving targeted drug delivery. However, the actual application is not limited to this.

[0132] For modular peptides, 3R, 6D, and 6R were selected and coupled to Mal-PEG respectively.2k -DSPE, then the five lipid components, ionized lipid: phospholipid: cholesterol: polypeptide conjugate: DSPE-PEG, were dissolved in the alcohol phase at a molar ratio of 50:10:38.5:6:0.75. Firefly Luciferase mRNA was dissolved in the aqueous phase, with the N / P ratio of ionized lipid to nucleic acid being 5.67. The two were then blended at a flow rate of 1:3 using a microfluidic device to prepare nanoliposomes encapsulating Firefly Luciferase mRNA. It is worth noting that the specific polypeptide modification method and liposome preparation technology encapsulating Firefly Luciferase mRNA used here are only one example. The organ and tissue-targeted liposome modification involved in the present invention is not limited to polypeptides encoded by natural amino acids, but is also applicable to polypeptides encoded by unnatural amino acids and other amino acid derivatives.

[0133] Cas9 mRNA used in the following examples was purchased from Trilink, and Cas9 protein, modified sgSTOP, sgPTEN, sgPCSK9, and EGFP-ASO (EGFP antisense oligonucleotide) were purchased from GeneScript.

[0134] Example 1

[0135] S1. Synthesis of peptides

[0136] In this example, three peptides, 3R, 6D, and 6R, were designed. Their chemical structures are shown in Figures A to C in Figure 1, respectively. The specific synthesis methods are as follows:

[0137] (1) Synthesis of 3R (sequence shown in SEQ ID No. 1):

[0138] a) Select 1 g of Wang resin with a loading of 1 mmol / g, add 1.3 g of the first amino acid Fmoc-Arg(Pbf)-OH, 1 g of HOBT, and 1 ml of DIC, blow nitrogen protection, and react for 6 hours.

[0139] b) The reaction liquid was drawn and tested with ninhydrin. When the resin was colorless, it was washed four times alternately with DMF and DCM. 20 ml of piperidine solution was added and reacted for 15 minutes to remove the Fmoc protection.

[0140] c) Wash the resin four times alternately with DMF and DCM. Detect with ninhydrin. If the resin turns brown, it means that Fmoc has been removed and the next amino acid reaction can be carried out.

[0141] d) Add the second amino acid Fmoc-Arg(Pbf)-OH 1.3 g, HOBT 0.5 g, and DIC 0.5 ml, and react for 2 hours with ninhydrin monitoring. If the resin is colorless, remove the Fmoc protection with piperidine solution and proceed to the next step.

[0142] e) Adding raw materials in sequence according to the target polypeptide sequence until the last amino acid Fmoc-Cys(Trt)-OH in the polypeptide sequence is synthesized and the Fmoc protection is removed.

[0143] f) The resin was washed, dried, and cleaved with TFA; the resin was precipitated with icy ether and centrifuged to obtain 3R solid.

[0144] (2) Synthesis of 6D (sequence shown in SEQ ID No. 2):

[0145] a) Select 1 g of Wang resin with a loading of 1 mmol / g, add 1 g of the first amino acid Fmoc-Asp(OtBu)-OH, 1 g of HOBT, and 1 ml of DIC, blow nitrogen protection, and react for 6 hours.

[0146] b) The reaction liquid was drawn and tested with ninhydrin. When the resin was colorless, it was washed four times alternately with DMF and DCM. 20 ml of piperidine solution was added and reacted for 15 minutes to remove the Fmoc protection.

[0147] c) Wash the resin four times alternately with DMF and DCM. Detect with ninhydrin. If the resin turns brown, it means that Fmoc has been removed and the next amino acid reaction can be carried out.

[0148] d) Add the second amino acid Fmoc-Asp(OtBu)-OH 1 g, HOBT 0.5 g, and DIC 0.5 ml, and react for 2 hours with ninhydrin monitoring. If the resin is colorless, remove the Fmoc protection with piperidine solution and proceed to the next step.

[0149] e) Adding raw materials in sequence according to the target polypeptide sequence until the last amino acid Fmoc-Cys(Trt)-OH in the polypeptide sequence is synthesized and the Fmoc protection is removed.

[0150] f) The resin was washed, dried, and cleaved with TFA; the resin was precipitated with icy ether and centrifuged to obtain 6D solid.

[0151] (3) Synthesis of 6R (sequence shown in SEQ ID No. 3):

[0152] a) Select 1 g of Wang resin with a loading of 1 mmol / g, add 1.3 g of the first amino acid Fmoc-Arg(Pbf)-OH, 1 g of HOBT, and 1 ml of DIC, under nitrogen protection, and react for 6 hours.

[0153] b) The reaction liquid was drawn and tested with ninhydrin. When the resin was colorless, it was washed four times alternately with DMF and DCM. 20 ml of piperidine solution was added and reacted for 15 minutes to remove the Fmoc protection.

[0154] c) Wash the resin four times alternately with DMF and DCM. Detect with ninhydrin. If the resin turns brown, it means that Fmoc has been removed and the next amino acid reaction can be carried out.

[0155] d) Add the second amino acid Fmoc-Arg(Pbf)-OH 1.3 g, HOBT 0.5 g, and DIC 0.5 ml, and react for two hours with ninhydrin monitoring. If the resin is colorless, remove the Fmoc protection with piperidine solution and proceed to the next step.

[0156] e) Adding raw materials in sequence according to the target polypeptide sequence until the last amino acid Fmoc-Cys(Trt)-OH in the polypeptide sequence is synthesized and the Fmoc protection is removed.

[0157] f) The resin was washed, dried, and cleaved with TFA; the resin was precipitated with icy ether and centrifuged to obtain 6R solid.

[0158] In this example, S1-B designed a RRRRRK polypeptide (sequence shown in SEQ ID No. 4), the chemical structure of which is shown in FIG19 , and the specific synthesis method is as follows:

[0159] a) Select 1 g of Wang resin with a loading of 1 mmol / g, add 1 g of the first amino acid Fmoc-Lys(Boc)-OH, 1 g of HOBT, and 1 ml of DIC, blow nitrogen protection, and react for 4 hours.

[0160] b) The reaction liquid was drawn and tested with ninhydrin. When the resin was colorless, it was washed four times alternately with DMF and DCM. 20 ml of piperidine solution was added and reacted for 15 minutes to remove the Fmoc protection.

[0161] c) Wash the resin four times alternately with DMF and DCM. Detect with ninhydrin. If the resin turns brown, it means that Fmoc has been removed and the next amino acid reaction can be carried out.

[0162] d) Add the second amino acid Fmoc-Arg(Pbf)-OH 1 g, HOBT 0.5 g, and DIC 0.5 ml, and react for 2 hours with ninhydrin monitoring. If the resin is colorless, remove the Fmoc protection with piperidine solution and proceed to the next step.

[0163] e) Adding raw materials in sequence according to the target polypeptide sequence until the last amino acid Fmoc-Cys(Trt)-OH in the polypeptide sequence is synthesized and the Fmoc protection is removed.

[0164] f) The resin was washed clean, dried, and cleaved with TFA; precipitated with icy ether and centrifuged to obtain RRRRRK solid.

[0165] S1-C In this embodiment, a DDEEDD polypeptide (sequence shown in SEQ ID No. 5) was designed, and its chemical structure is shown in FIG22A . The specific synthesis method is as follows:

[0166] a) Select 1 g of Wang resin with a loading of 1 mmol / g, add 1 g of the first amino acid Fmoc-Asp(OtBu)-OH, 1 g of HOBT, and 1 ml of DIC, blow nitrogen protection, and react for 4 hours.

[0167] b) The reaction liquid was drawn and tested with ninhydrin. When the resin was colorless, it was washed four times alternately with DMF and DCM. 20 ml of piperidine solution was added and reacted for 15 minutes to remove the Fmoc protection.

[0168] c) Wash the resin four times alternately with DMF and DCM. Detect with ninhydrin. If the resin turns brown, it means that Fmoc has been removed and the next amino acid reaction can be carried out.

[0169] d) Add the second amino acid Fmoc-Asp(OtBu)-OH 1 g, HOBT 0.5 g, and DIC 0.5 ml, and react for 2 hours with ninhydrin monitoring. If the resin is colorless, remove the Fmoc protection with piperidine solution and proceed to the next step.

[0170] e) Adding raw materials in sequence according to the target polypeptide sequence until the last amino acid Fmoc-Cys(Trt)-OH in the polypeptide sequence is synthesized and the Fmoc protection is removed.

[0171] f) The resin was washed, dried, and cleaved with TFA; the resin was precipitated with icy ether and centrifuged to obtain DDEEDD solid.

[0172] S1-D In this embodiment, a DDDDEE polypeptide (sequence shown in SEQ ID No. 6) was designed, and its chemical structure is shown in FIG22B . The specific synthesis method is as follows:

[0173] a) Select 1 g of Wang resin with a loading of 1 mmol / g, add 1 g of the first amino acid Fmoc-Glu(OtBu)-OH, 1 g of HOBT, and 1 ml of DIC, blow nitrogen protection, and react for 4 hours.

[0174] b) The reaction liquid was drawn and tested with ninhydrin. When the resin was colorless, it was washed four times alternately with DMF and DCM. 20 ml of piperidine solution was added and reacted for 15 minutes to remove the Fmoc protection.

[0175] c) Wash the resin four times alternately with DMF and DCM. Detect with ninhydrin. If the resin turns brown, it means that Fmoc has been removed and the next amino acid reaction can be carried out.

[0176] d) Add the second amino acid Fmoc-Glu(OtBu)-OH 1 g, HOBT 0.5 g, and DIC 0.5 ml, and react for 2 hours with ninhydrin monitoring. If the resin is colorless, remove the Fmoc protection with piperidine solution and proceed to the next step.

[0177] e) Adding raw materials in sequence according to the target polypeptide sequence until the last amino acid Fmoc-Cys(Trt)-OH in the polypeptide sequence is synthesized and the Fmoc protection is removed.

[0178] f) The resin was washed, dried, and cleaved with TFA; the resin was precipitated with glacial ether and centrifuged to obtain DDDDEE solid.

[0179] S1-E In this embodiment, a DDTTDD polypeptide (sequence shown in SEQ ID No. 7) was designed, and its chemical structure is shown in FIG22C . The specific synthesis method is as follows:

[0180] a) Select 1 g of Wang resin with a loading of 1 mmol / g, add 1 g of the first amino acid Fmoc-Asp(OtBu)-OH, 1 g of HOBT, and 1 ml of DIC, blow nitrogen protection, and react for 4 hours.

[0181] b) The reaction liquid was drawn and tested with ninhydrin. When the resin was colorless, it was washed four times alternately with DMF and DCM. 20 ml of piperidine solution was added and reacted for 15 minutes to remove the Fmoc protection.

[0182] c) Wash the resin four times alternately with DMF and DCM. Detect with ninhydrin. If the resin turns brown, it means that Fmoc has been removed and the next amino acid reaction can be carried out.

[0183] d) Add the second amino acid Fmoc-Asp(OtBu)-OH 1 g, HOBT 0.5 g, and DIC 0.5 ml, and react for 2 hours with ninhydrin monitoring. If the resin is colorless, remove the Fmoc protection with piperidine solution and proceed to the next step.

[0184] e) Adding raw materials in sequence according to the target polypeptide sequence until the last amino acid Fmoc-Cys(Trt)-OH in the polypeptide sequence is synthesized and the Fmoc protection is removed.

[0185] f) The resin was washed, dried, and cleaved with TFA; the resin was precipitated with icy ether and centrifuged to obtain DDTTDD solid.

[0186] S1-F In this embodiment, a DDDDTT polypeptide (sequence shown in SEQ ID No. 8) was designed, and its chemical structure is shown in FIG22D . The specific synthesis method is as follows:

[0187] a) Select 1 g of Wang resin with a loading of 1 mmol / g, add 1 g of the first amino acid Fmoc-Thr(tBu)-OH, 1 g of HOBT, and 1 ml of DIC, blow nitrogen protection, and react for 4 hours.

[0188] b) The reaction liquid was drawn and tested with ninhydrin. When the resin was colorless, it was washed four times alternately with DMF and DCM. 20 ml of piperidine solution was added and reacted for 15 minutes to remove the Fmoc protection.

[0189] c) Wash the resin four times alternately with DMF and DCM. Detect with ninhydrin. If the resin turns brown, it means that Fmoc has been removed and the next amino acid reaction can be carried out.

[0190] d) Add the second amino acid Fmoc-Thr(tBu)-OH (1 g), HOBT (0.5 g), and DIC (0.5 ml) and react for 2 hours with ninhydrin monitoring. If the resin is colorless, remove the Fmoc protection with piperidine solution and proceed to the next step.

[0191] e) Adding raw materials in sequence according to the target polypeptide sequence until the last amino acid Fmoc-Cys(Trt)-OH in the polypeptide sequence is synthesized and the Fmoc protection is removed.

[0192] f) The resin was washed, dried, and cleaved with TFA; the resin was precipitated with icy ether and centrifuged to obtain DDDDTT solid.

[0193] In this example, a 6H polypeptide (sequence shown in SEQ ID No. 9) was designed. Its chemical structure is shown in FIG26 . The specific synthesis method is as follows:

[0194] a) Select 1 g of Wang resin with a loading of 1 mmol / g, add 1.3 g of the first amino acid Fmoc-His(Trt)-OH, 1 g of HOBT, and 1 ml of DIC, blow nitrogen protection, and react for 6 hours.

[0195] b) The reaction liquid was drawn and tested with ninhydrin. When the resin was colorless, it was washed four times alternately with DMF and DCM. 20 ml of piperidine solution was added and reacted for 15 minutes to remove the Fmoc protection.

[0196] c) Wash the resin four times alternately with DMF and DCM. Detect with ninhydrin. If the resin turns brown, it means that Fmoc has been removed and the next amino acid reaction can be carried out.

[0197] d) Add the second amino acid Fmoc-His(Trt)-OH 1.3 g, HOBT 0.5 g, and DIC 0.5 ml, and react for 2 hours with ninhydrin monitoring. If the resin is colorless, remove the Fmoc protection with piperidine solution and proceed to the next step.

[0198] e) Adding raw materials in sequence according to the target polypeptide sequence until the last amino acid Fmoc-Cys(Trt)-OH in the polypeptide sequence is synthesized and the Fmoc protection is removed.

[0199] f) The resin was washed clean, dried, and cleaved with TFA; precipitated with icy ether and centrifuged to obtain 6H solid.

[0200] S2. Characterization of peptides.

[0201] The molecular weight of each polypeptide solid prepared in step S1 was confirmed by MS, and the main peak was determined by HPLC, as shown in Figures 2-7. The product was then purified by liquid phase preparation and lyophilized.

[0202] S3. Preparation of peptide-modified liposomes (LNP)

[0203] Take 1mmol of each peptide synthesized in step S1 and mix with 1mmol Mal-PEG 2k -DSPE was added to the reaction flask, and then the following operations were performed: 10 mL of ultra-dry solvent (tetrahydrofuran) was added to dissolve under argon protection, and then 10 μL of freshly distilled acid binding agent was added. After sealing, the reaction was stirred at room temperature for 24 hours. The above reaction solutions were placed in dialysis bags, dialyzed with ultrapure water for 3 days, and freeze-dried to obtain the final product DSPE-PEG. 2k -3R / 6D / 6R / 6H / RRRRRK / DDEEDD / DDDDEE / DDTTDD / DDDDTT (peptide-modified lipid material), using nuclear magnetic resonance spectroscopy to characterize the chemical structure of the product and verify whether the peptide was successfully coupled to PEG 2k -DSPE, the results are shown in Figures 8A to C, 20, 23 and 27. Five lipid components (ionized lipid MC3, phospholipid DSPC, cholesterol, DSPE-PEG 2k-3R / 6D / 6R / RRRRRK / DDEEDD / DDDDEE / DDTTDD / DDDDTT, DSPE-PEG) were dissolved in ethanol (alcohol phase) at a molar ratio of 50:10:38.5:6:0.75, Firefly Luciferase mRNA was dissolved in citrate buffer (aqueous phase), ionized lipids and mRNA were configured at an N / P ratio of 5.67, the volume ratio of the alcohol phase to the aqueous phase was 1:3, and the alcohol phase and the aqueous phase were passed through the microfluidic channel as shown in Figure 9 at a flow rate ratio of 1:3 (alcohol phase and aqueous phase, respectively, 0.3 mL / min and 0.9 mL / min), and the polypeptide-modified liposomes were collected. Finally, the collected liquid was dialyzed against 1×PBS overnight to obtain liposomes modified with each polypeptide encapsulating Luciferase mRNA.

[0204] S4. Peptide-modified liposomes enable organ-targeted mRNA delivery in vivo

[0205] S4-A. The polypeptide-modified liposomes encapsulating Firefly Luciferase mRNA obtained in step S3 above were injected into mice via the tail vein at a concentration of 5 μg mRNA per mouse. Three hours later, each mouse was intraperitoneally injected with 200 μL of Firefly Luciferase substrate (concentration: 20 mg / mL). Three minutes after substrate injection, the five major organs (heart, liver, spleen, lung, and kidney) of the mice were removed for in vivo imaging. The fluorescence intensity of the five organs was quantitatively analyzed using in vivo imaging software.

[0206] S4-B: Effects of ionized lipid species on organ targeting

[0207] To determine whether the organ targeting of peptide-modified liposomes depends on specific ionized lipid components, the effect of changing the type of ionized lipid on organ specificity was explored. MC3, SM102, ALC0315, and LP01, the three most widely used ionized lipid components in commercial use, were selected as liposomes. DSPC, cholesterol, and DSPE-PEG were still selected as the other four liposome components. 2kThe fluorescence intensity of organs encapsulating Firefly Luciferase mRNA was statistically compared using liposomes formed with PEG-3R / 6D / 6R and DSPE-PEG, following the same steps as in S4-A. The results are shown in Figure 10. Although the ionized lipid composition of the liposomes was changed, the primary enriched organs remained the same, with only differences in the fluorescence intensity of the Firefly Luciferase protein. This is due to differences in the physicochemical properties of liposomes formed with different ionized lipids, resulting in differences in the efficiency of successful in vivo delivery of Firefly Luciferase mRNA.

[0208] S4-C, Study on the influence of phospholipid types on organ targeting

[0209] The phospholipid components in the liposome components were adjusted to DOPC, DOPE or HSPC, and the other four liposome components were selected from MC3, cholesterol, DSPE-PEG 2k -3R / 6D / 6R, DSPE-PEG, and then the fluorescence intensity of the organs was statistically compared according to the same steps as S4-A. The statistical results are shown in Figure 11. The results show that the peptide-modified liposomes formed by three different phospholipid components can still achieve organ-specific delivery of mRNA in mice. This shows that the organ enrichment characteristics of lipids modified with 3R, 6D, and 6R peptides are independent of the ionized lipids and phospholipid types of the liposomes. The peptide-modified liposomes formed by this method have a certain degree of mobility and universality in terms of organ enrichment characteristics.

[0210] The phospholipid component in the liposome-forming component was adjusted to ESM (egg sphingomyelin), and the polypeptide-modified lipid material was adjusted to DSPE-PEG 2k -6H, the other three liposome components were selected from MC3 and cholesterol DSPE-PEG to form liposomes encapsulating EGFP mRNA, which were then injected into the mice through the tail vein at a concentration of 20 μg mRNA per mouse. Six hours later, the five major organs (heart, liver, spleen, lung, and kidney) and bone marrow of the mice were removed for in vivo imaging. The fluorescence intensity of the five major organs and bone marrow was quantitatively counted using in vivo imaging software, and the statistical results are shown in Figure 25.

[0211] The drug delivery vector designed by the present invention has modular characteristics. By adjusting the number of amino acids in the encoded polypeptide, the size of the polypeptide modified in the liposome can be changed. According to the synthesis process of step S1, for arginine, the present invention sequentially synthesized and prepared nine polypeptides of different sizes: 2R (SEQ ID No. 10, polypeptide sequence is CRR), 3R (SEQ ID No. 1), 4R (SEQ ID No. 11), 5R (SEQ ID No. 12), 6R (SEQ ID No. 3), 7R (SEQ ID No. 13), 8R (SEQ ID No. 14), 9R (SEQ ID No. 15), and 10R (SEQ ID No. 16), where nR represents Cys-(Arg) n , n=2, 3, 4, 5, 6, 7, 8, 9 or 10. For histidine, the present invention sequentially synthesized and prepared nine polypeptides of different sizes: 2H (SEQ ID No. 17, polypeptide sequence is CHH), 3H (SEQ ID No. 18), 4H (SEQ ID No. 19), 5H (SEQ ID No. 20), 6H (SEQ ID No. 9), 7H (SEQ ID No. 21), 8H (SEQ ID No. 22), 9H (SEQ ID No. 23), and 10H (SEQ ID No. 24) (method reference step S1), wherein nH represents Cys-(His) n , n = 2, 3, 4, 5, 6, 7, 8, 9, or 10. Characterization was then performed according to step S2, and peptide-modified liposomes were prepared in step S3 (the other four groups were ionized lipid MC3, phospholipid DSPC, cholesterol, and DSPE-PEG, respectively). The synthesized liposomes were then injected intravenously in vivo according to the same steps as in step S4-A, and the fluorescence intensity of the organs was statistically compared. The statistical results are shown in Figure 12. The results show that the organ targeting of liposomes is highly dependent on the size of the modified peptide. For example, the primary target organ of 3H is the liver, while that of 8H is the spleen. The target organ of the 3R-modified vector is the liver, while that of 5R becomes the liver and lungs. When the number of arginines in the coding sequence is changed to 6, the primary target organ becomes the lung. This shows that modular fine-tuning based on the coding size can change the target organ of the delivery vector. In addition, changing the amino acid sequence of the 6R peptide to lysine (K) changes the original target organ from the lung to the liver and spleen, further demonstrating the molecular sensitivity of the encoded peptide-modified liposomes to the peptide.

[0212] These results demonstrate that modular adjustments to the encoded polypeptides enable both selective organ delivery of nanocarriers and regulation of delivery efficiency, with both targeting and efficiency exhibiting single-molecule sensitivity. Furthermore, the selection of arginine and histidine numbers for encoding in this invention is merely an example; a wide variety of permutations and combinations of amino acids and amino acid derivatives are possible. This modular approach can be combined with artificial intelligence algorithms, such as machine learning, to achieve intelligent nanocarrier design.

[0213] S6. In vivo delivery of multiple messenger RNAs

[0214] In many fields such as immunotherapy, tissue repair, and cell programming, mRNA-based therapies often require multiple proteins to work together, so it is crucial to deliver multiple mRNAs to the target organs at the same time. To achieve this goal, peptide-modified LNPs encapsulated mRNAs that can encode Luc and EGFP proteins, and then C57BL / 6 mice received a tail vein injection at a total dose of 0.4 mg / kg (0.1 mg / kg of Luc mRNA and 0.3 mg / kg of EGFP). After 6 hours, the organs were separated, and Luc and EGFP fluorescence were imaged using the IVIS Lumina system. As shown in Figure 13, significant Luc luminescence and EGFP fluorescence were observed in the target organs of the LNP system. This shows that peptide-modified organ-targeted nanoparticles can co-deliver multiple nucleic acid molecules to the target organs.

[0215] S7. ASO delivery in vivo

[0216] In order to broaden the application range of LNPs, the present invention encapsulated GFP-ASO with LNPs and attempted to deliver EGFP-ASO to specific organs. ASO can degrade mRNA and has been approved by the U.S. Food and Drug Administration for clinical treatment, but organ-targeted delivery of ASOs remains challenging. For ASO encapsulation, the ratio of lipid and ASO is fixed at 15 / 1 (wt / wt). The polypeptide-modified LNPs encapsulating EGFP-ASO were intravenously injected into EGFP tool mice through the tail vein three times (day 0, day 1 and day 2), and the target organs were isolated on day 3 for fluorescence imaging. The main organs (liver, lung and spleen) sections were stained and the EGFP reduction effect was detected using a confocal microscope (Zeiss LSM710). In addition, in order to quantitatively analyze the inhibition efficiency of EGFP, the target organs were dissociated into single cell suspensions according to the protocol of the Miltenyi kit, and further quantitative analysis was performed by flow cytometry (SONY ID7000).

[0217] The in vivo imaging results of the target organs confirmed the functional targeted delivery of ASO (Figure 14 A), and were consistent with the trend of the flow analysis results (Figure 14 B). Due to the significant autofluorescence of the organs in the EGFP channel, the present invention also selected confocal imaging as a detection method to verify the functional delivery of organ-specific ASO. A significant reduction in EGFP-positive cells was found in the sections of specific target organs (Figure 14 C), which once again confirmed that the polypeptide-modified LNP delivery system can achieve organ-targeted delivery of ASO molecules.

[0218] S8. Delivery of Cre mRNA into the mT-mG mouse model

[0219] In order to verify the ability of polypeptide-modified LNP for organ-specific gene editing, the present invention first delivered Cre recombinase (Cre) mRNA into mT-mG tool mice. The genetically engineered mice are able to express tdTomato protein in all tissues. After Cre-mediated recombination, the CMV b-actin enhancer promoter (pCA) promotes the expression of enhanced green fluorescent protein, as shown in Figure 15 A. The present invention injects polypeptide-modified LNPs encapsulating Cre mRNA into mT-mG mice, and isolates organs for in vivo imaging two days later. The in vivo imaging results are shown in Figure 15 B, and significant EGFP fluorescence was observed in the target organs liver, lungs, and spleen. In addition, consistent with the in vivo imaging results, obvious EGFP green fluorescence was also detected in the tissue sections of the target organs (Figure 15 C), further providing a polypeptide-modified LNP delivery system that can achieve organ-specific gene editing.

[0220] Gene Editing in S9 and Ai14 Mouse Models (Cas9mRNA / sgSTOP)

[0221] Cas9 mRNA and sgTom (2 / 1, wt / wt) were encapsulated with POST LNPs and co-delivered to Ai14 mice (n=3 per group) three times (day 0, day 1, and day 2) at a total RNA dose of 2 mg / kg. Seven days after the first injection, tdTomato fluorescence in major organs was detected by IVIS Lumina system and confocal microscopy.

[0222] By co-delivering Cas9mRNA and sgRNA into selected organs, the LNP system is used for CRISPR-Cas gene editing. The present invention uses Ai14 model mice with Loxp-STOP-Loxp-tdTomato in all organs, and Cas 9mRNA and sgSTOP can delete stop and activate the expression of tdTomoto protein (Figure 16, A). Cas 9mRNA and sgSTOP are simultaneously encapsulated in polypeptide-modified LNPs at a ratio of 2: 1 and injected intravenously into Ai14 mice. As shown in Figure 16, B, after 7 days, bright tdTomato fluorescence can be detected in the specific organs of the corresponding LNP, which means effective organ-specific CRISPR-Cas gene editing of the LNP system. Similarly, confocal fluorescence images show tdTomato-positive cells (Figure 16, C).

[0223] Co-delivery of Cas9 mRNA / sgPTEN and RNPs in S10 and C57BL / 6 mice

[0224] In order to confirm the application potential of the peptide-modified LNP delivery system, the present invention attempts to achieve CRISPR-Cas gene editing of endogenous targets. Here, PTEN was chosen because it plays a key role in cell proliferation, DNA repair and tumor suppression, and has become an important target of widespread attention. The present invention focuses on co-delivering Cas9mRNA and sgPTEN into wild-type C57BL / 6 mice to delete the PTEN target. In order to verify the gene editing ability of peptide-modified LNPs in wild-type C57BL / 6 mice, the present invention selects the phosphatase and tensin homolog (PTEN) gene as the endogenous target. Cas9mRNA and sgPTEN (4 / 1, wt / wt) were encapsulated with peptide-modified LNPs and co-delivered to C57BL / 6 mice at a dose of 4 mg / kg for three consecutive days. After 10 days, the mice were sacrificed and the lungs and livers were collected for further IHC staining evaluation. For Cas9 / sgPTEN RNPs delivery, the molar ratio of Cas9 protein to sgPTEN was fixed at 1 / 3. It is worth noting that the RNP mixture was first dissolved in 1×PBS and incubated for 15 minutes, and then quickly mixed with ethanol. As shown in Figure 17A, after three consecutive injections, the IHC results of the liver and lungs showed a significant decrease in PTEN protein. Similarly, the peptide-modified LNP also achieved co-delivery of the Cas9 protein / sgPTEN complex (Figure 17B), achieving the knockout of the PTEN gene. Quantitative analysis of the normalized results of positive PTEN protein expression demonstrated the targeting of the peptide-modified LNP system (Figure 17C).

[0225] S11. In vivo delivery of Cas9 mRNA / sgPCSK9 for PCSK9 gene knockout in the liver

[0226] In order to confirm whether the peptide-modified LNP system can achieve organ-specific gene knockout of clinically relevant PCSK9 targets, Cas9mRNA and sgPCSK9 (1 / 1, wt / wt) were encapsulated with peptide-modified LNPs and co-delivered to wild-type C57BL / 6 mice at a dose of 2 mg / kg, and injected into the tail vein three times (day 0, day 2, day 4). On the seventh day, the liver was collected for further protein blotting evaluation. The blood of the mice was collected for ELISA analysis (day 10) and cholesterol detection (day 15), respectively. The results showed that the PCSK9 protein in the liver and serum was significantly reduced (A and B in Figure 18). The reduction in cholesterol in the serum also confirmed the successful knockout of the PCSK9 gene (C in Figure 18). Therefore, the peptide-modified LNP system design can provide a potential platform for the treatment of related genetic diseases.

[0227] Based on the above delivery vehicle platform design, modular peptide modification can be used to construct a programmable modular nanocarrier library, from which corresponding targeted vectors can be screened using high-throughput screening tools. Modular peptide modification is a key component of nanocarriers and an important foundation for the intelligent design and construction of targeted vectors by combining artificial intelligence and big data technologies.

[0228] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A polypeptide-modified lipid material, characterized in that: The polypeptide-modified lipid material is obtained by coupling the polypeptide to a lipid material containing a polyethylene glycol chain; The polypeptide is Cys-(X) n , wherein X is any one or any two of Arg, Asp, His, Glu or Thr, and n is the total number of X and is an integer greater than 1 and less than 11; The polypeptide is coupled to the polyethylene glycol chain via the thiol group on its cysteine ​​residue.

2. The polypeptide-modified lipid material according to claim 1, wherein Some or all of the n Xs are Arg.

3. The polypeptide-modified lipid material according to claim 2, wherein When some of the n Xs are Arg, the remaining Xs are Lys.

4. The polypeptide-modified lipid material according to claim 2, wherein n=3, 4, 5, 6, 7 or 8.

5. The polypeptide-modified lipid material according to claim 1, wherein The X is His.

6. The polypeptide-modified lipid material according to claim 5, wherein n=2, 3, 4, 5, 6, 7, 8 or 10.

7. The polypeptide-modified lipid material according to claim 1, wherein The X is any one of Asp, Glu or Thr.

8. The polypeptide-modified lipid material according to claim 7, wherein Said X is Asp and Glu; or Said X is Asp and Thr; or Said X is Asp.

9. The polypeptide-modified lipid material according to claim 1, characterized in that: The lipid material containing polyethylene glycol chain is Mal-PEG 2k -DSPE.

10. Use of the polypeptide-modified lipid material according to any one of claims 1 to 9 in preparing polypeptide-modified liposomes for targeted drug delivery.

11. The use according to claim 10, wherein: The polypeptide-modified liposome for targeted drug delivery is targeted to an in vivo organ, which is the liver, spleen, lung and / or bone marrow.

12. A polypeptide-modified liposome for targeted drug delivery, characterized in that: The preparation raw materials include the polypeptide-modified lipid material according to any one of claim 9.

13. The polypeptide-modified liposome according to claim 12, characterized in that: The polypeptide-modified liposomes also include ionized lipids, phospholipids, cholesterol and phospholipid-polyethylene glycol.

14. The polypeptide-modified liposome according to claim 13, characterized in that: The molar ratio of the ionized lipid, the phospholipid, the cholesterol, the polypeptide-modified lipid material and the phospholipid-polyethylene glycol is (40-60):(5-15):(30-45):(3-12):(0.5-1.5).

15. The polypeptide-modified liposome according to claim 13, characterized in that: The phospholipid is distearoylphosphatidylcholine, 1,2-dioleoyl-SN-glycero-3-phosphocholine, 1,2-dioleoylglycerol 3-phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine or egg sphingomyelin.

16. The polypeptide-modified liposome according to claim 13, characterized in that: The ionized lipid is MC3, SM102, ALC0315 or LP01.

17. Use of the polypeptide-modified lipid material according to any one of claims 1 to 9 or the polypeptide-modified liposome according to any one of claims 10 to 16 in improving drug targeting.

18. Use of the polypeptide-modified lipid material according to any one of claims 1 to 9 or the polypeptide-modified liposome according to any one of claims 10 to 16 in the preparation of a drug targeting an organ in vivo, wherein the organ in vivo is liver, spleen, lung and / or bone marrow.

19. A method for improving drug targeting, comprising using the polypeptide-modified lipid material according to any one of claims 1 to 9 or the polypeptide-modified liposome according to any one of claims 10 to 16.

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