CN118121547B

CN118121547B - Polypeptide guide molecule embedded lipid complex and preparation method and application thereof

Info

Publication number: CN118121547B
Application number: CN202410254290.6A
Authority: CN
Inventors: 李翀; 唐忠捷; 于洋
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2024-03-06
Filing date: 2024-03-06
Publication date: 2024-10-01
Anticipated expiration: 2044-03-06
Also published as: CN118121547A

Abstract

The invention belongs to the field of medicines, and particularly relates to a polypeptide guide molecule embedded lipid compound and a preparation method and application thereof. The invention takes polypeptide ligand which can naturally embed into cell membrane and specifically recognize protein transmembrane region in the membrane as guide molecule, and the polypeptide can also be embedded into phospholipid bilayer to form lipid complex embedded with polypeptide guide molecule. The polypeptide guiding molecule embedded lipid complex can avoid the competitive recognition of endogenous ligand or antibody to target receptor, and can effectively avoid the target deletion caused by extracellular region mutation or shedding. The plug-and-play embedded modification mode can remarkably improve the immune compatibility of the lipid complex in vivo.

Description

Polypeptide guide molecule embedded lipid complex and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a polypeptide guide molecule embedded lipid complex taking a membrane protein transmembrane region as a target recognition site, and a preparation method and application thereof.

Background

The membrane proteins specific to the target cells provide important recognition sites for "targeting" the drug to the target tissue, and are an important biological basis for achieving targeted drug delivery. The polypeptide has natural high affinity and specificity to protein, and has the advantages of stronger permeability, lower potential immunogenicity and the like compared with macromolecules such as antibodies and the like. Thus, polypeptide ligands for specific target (membrane) proteins have become an important class of targeting molecules that mediate targeted drug delivery. The recognition of extracellular regions of target proteins exposed on the cell surface by polypeptide-like targeting molecules modified on the surface of the carrier is one of the important strategies for the current mode of targeted drug delivery. However, some disease-associated membrane proteins may have their extracellular regions shed under certain conditions (ectodomain shedding) resulting in "no target" and the shed extracellular regions may also become "pseudo targets". In vivo natural ligands or endogenous antibodies, which also have the extracellular region of the membrane protein as the binding region, may also cause competitive interference. Direct modification of liposomes with polyethylene glycol (PEG) can be problematic, where densely distributed polyethylene glycol on the surface of the liposome can cause significant steric hindrance to recognition of target proteins by polypeptide-directed molecules embedded in the phospholipid layer; repeated injections of polyethylene glycol liposomes have also been found in recent years to accelerate blood clearance (ACCELERATED BLOOD CLEARANCE, ABC) and to lose long circulation. At the same time, exogenous ligand molecules need to be exposed on the surface of the carrier to effectively recognize the extracellular region of the target receptor, but this modification is sterically hindered and shows varying degrees of immunogenicity. For example, RGD cyclic peptides are generally considered non-immunogenic, inducing an acute immune response in mice after conjugation with PEG on the surface of liposomes and multiple administrations, and even death (Wang X,Wang H,Jiang K,et al.Liposomes with cyclic RGD peptide motif triggers acute immune response in mice[J].Journal of Controlled Release,2019,293:201-214.); CDX peptides targeting nicotinic acetylcholine receptors (nicotinic acetylcholine receptors, nAChRs) on the blood brain barrier, when modified on liposomes, tend to adsorb native IgM, resulting in a more strongly immunogenic (Guan J,Shen Q,Zhang Z,et al.Enhanced immunocompatibility of ligand-targeted liposomes by attenuating natural IgM absorption[J].Nature communications,2018,9(1):2982.). modification of the polypeptide targeting molecule into the lipid complex phospholipid bilayer, can reduce steric hindrance and immunogenicity, but at the same time, whether the polypeptide sequence concealment resulting from the embedded modification affects its targeting function, and needs further experimental investigation.

Membrane proteins account for over 50% of the currently available drug targets, and research based on membrane proteins has focused mainly on extracellular and intracellular areas. With the development of structural biology, researchers have further developed into the transmembrane region of membrane proteins, which not only supports proteins across the cell membrane, but also plays an important role in the molecular and cellular recognition processes and is involved in protein-protein interactions. Interactions between transmembrane regions are associated with cell migration, proliferation, differentiation, and specific recognition of membrane proteins can be achieved by recognizing the transmembrane regions.

Therefore, the drug delivery system constructed in the form of the embedded polypeptide targeting molecule with the transmembrane region of the membrane protein as the target receptor recognition site is helpful to avoid various challenges caused by exposure of the polypeptide targeting molecule with the extracellular region of the membrane protein as the recognition site. The embedded modification mode can avoid the direct contact of the polypeptide and protease in vivo, and the constructed polypeptide guiding molecule embedded lipid complex can realize active targeting drug delivery perhaps through oral administration.

Disclosure of Invention

In view of the above, the present invention aims to provide a polypeptide targeting molecule embedded lipid complex using a membrane protein transmembrane region as a target recognition site, and a preparation method and application thereof.

In order to achieve the above purpose, the present invention provides the following technical solutions:

The invention provides a polypeptide targeting molecule embedded lipid complex, wherein the polypeptide targeting molecule can be embedded in a phospholipid bilayer of a lipid carrier and specifically identify a membrane protein transmembrane region, and the lipid complex is obtained by embedding a polypeptide targeting molecule into the lipid carrier.

The invention selects some polypeptide guide molecules taking membrane protein transmembrane regions as recognition sites for research, but is not limited to the polypeptides, including:

Polypeptide directed against the insulin receptor transmembrane region: ELGILIFLYLFSLILGIIYWKK (ITP peptide, 22 aa) as shown in SEQ ID NO. 1;

polypeptide directed against the insulin receptor transmembrane region: LFGILIFLFLFSLIIGSIYLWKKK (P5 peptide, 24 aa) as shown in SEQ ID NO. 2;

Polypeptide directed against the integrin receptor transmembrane region: AYVFILLSFILGTLLGFLVMFWAKK (P4 peptide, 25 aa) as shown in SEQ ID NO. 3;

polypeptide directed against the integrin receptor transmembrane region: AYVFILLSFILGTLLGFLVMFWA (P0 peptide, 23 aa) as shown in SEQ ID NO. 4;

Polypeptide directed against the integrin receptor transmembrane region: FILLSFILGTLLGFL (P1 peptide, 15 aa) as shown in SEQ ID NO. 5;

Polypeptide directed against the integrin receptor transmembrane region: FILLSFILGT (P2 peptide, 10 aa) as shown in SEQ ID NO. 6;

Polypeptide directed against the integrin receptor transmembrane region: PEEFILKSFILGTLKGFLKMFYS (P3 peptide, 23 aa) as shown in SEQ ID NO. 7;

Polypeptide directed against the integrin receptor transmembrane region: DDEFILLSFILGTLKGFLKMFWS (AY peptide, 23 aa) as shown in SEQ ID NO. 8;

polypeptide directed against the epidermal growth factor receptor-2 transmembrane region: TFIIATVEGVLLFLILVVVVGILIKRR (TF peptide, 27 aa) as shown in SEQ ID NO: 9.

The lipid carrier in the polypeptide targeting molecule embedded lipid complex with the membrane protein transmembrane region as a target point has a phospholipid bilayer, and mainly comprises the following three types, but is not limited to the three types, including:

Liposome: the liposome is a closed vesicle with a similar biological membrane structure and composed of phospholipid and the like, and the phospholipid layer is easy to modify hydrophobic polypeptide targeting molecules, has good cell affinity and tissue compatibility and is an ideal carrier;

Exosomes: exosomes are multivesicular bodies derived from the invagination of intracellular lysosomal microparticles, which are fused with the cell membrane via the outer membrane of the multivesicular body and released into the extracellular matrix, comprising proteins, RNAs, DNA, lipids, metabolites and cytoplasmic molecules. Therefore, the exosomes have rich functions of achieving intercellular communication, transferring antigens to Dendritic Cells (DCs), promoting angiogenesis, achieving homing targets, and the like;

Lipid nanoparticles: lipid nanoparticles are lipid vesicles with a homogeneous lipid core, widely used for delivery of small molecules and nucleic acid drugs. The lipid nanoparticle consists of ionizable cationic lipid (combined with nucleic acid electrostatically), neutral auxiliary phospholipid (accelerating the structural transformation of the lipid nanoparticle in cells to promote drug release), cholesterol (stabilizing the lipid nanoparticle structure), and PEG lipid (prolonging the plasma circulation time of the lipid nanoparticle).

In order to make the polypeptide targeting molecule embedded lipid complex of the invention have long circulation function, be better applied to in vivo drug delivery, the lipid complex can be modified by adopting gold standard 'PEG modification' with long circulation at present, and specifically DSPE-PEG ₂₀₀₀ is selected as a functional modification molecule.

The preparation method of the polypeptide guiding molecule embedded lipid compound comprises the step of preparing the polypeptide guiding molecule embedded lipid compound by adopting a film hydration method when the lipid carrier is lecithin. Such as: taking raw material polypeptide guide molecules and lecithin according to a mole ratio of 0.25-4%, precisely weighing, respectively dissolving raw materials by using an organic solvent, uniformly mixing, removing the organic solvent to form a lipid film, and drying the residual organic solvent. Immediately adding PBS buffer solution, oscillating until lipid membrane is fully dissolved and dispersed, controlling particle size by ultrasonic and pushing, and filtering to obtain the final product.

When the lipid carrier is exosome, the preparation method is as follows:

The RAW 264.7 cell supernatant was collected and centrifuged to remove cells and other debris. The obtained supernatant was centrifuged at 135,000g for 1h, and the precipitate was washed with PBS to obtain exosomes. The polypeptide targeting molecule is dissolved in an organic solvent and incubated with the exosomes. Unbound polypeptide-directed molecules are removed by ultracentrifugation.

When the lipid carrier is a lipid nanoparticle, the preparation method is as follows: the ionizable lipid ingredient, DSPC, steroid, DMG-PEG ₂₀₀₀, and polypeptide targeting molecule were dissolved in an organic solvent and mixed in a molar ratio (50:10:34.5:1.5:4), respectively. The aqueous phase and the organic solution were mixed at a volume ratio of 3:1 with DEPC water as the aqueous phase at a mixing rate of 12 mL/min. Dialyzing overnight with dialysis bag with molecular weight of 8-14K. The ionizable lipid ingredient is Dlin-MC3-DMA or A1 (2- (di ((9E, 12E) -octadeca-9, 12-dien-1-yl) amino) ethyl (4 ¹ R,13 aS) -13 a-ethyl-2, 3,4 ¹, 5,6,13 a-hexahydro-1H-indolo [3,2,1-de ] pyrido [3,2,1-ij ] [1,5] naphthyridine-12-carboxylate); the steroid compound is cholesterol (Chol) or ginsenoside Rg5. Wherein A1 is a vincamine derivative, and has the structural formula:

The polypeptide targeting molecule embedded lipid complex of the invention can be applied as a targeted drug delivery system. Such as brain-targeted drug delivery systems and tumor-targeted drug delivery systems.

The invention has the beneficial effects that:

(1) The invention takes polypeptide ligand which can naturally embed into cell membrane and specifically recognize protein transmembrane region in the membrane as guide molecule, and the polypeptide can also be embedded into phospholipid bilayer of lipid complex to form lipid complex with embedded polypeptide guide molecule. The polypeptide targeting molecule embedded lipid complex can avoid the competitive recognition of the target receptor by endogenous ligands or antibodies;

(2) Different from many drug delivery researches taking extracellular regions of membrane proteins as recognition sites, the polypeptide embedded lipid complex provided by the invention recognizes more conserved and stable transmembrane regions of membrane proteins, can effectively avoid target deletion caused by extracellular region mutation or shedding, and can also effectively prevent competitive interference of natural ligands or endogenous antibodies in vivo.

(3) The lipid complex embedded in the polypeptide targeting molecule does not need to expose the targeting molecule, the in vivo stability of some polypeptides with a plurality of enzyme cutting sites is obviously improved, the coupling of the carrier and the polypeptide by a chemical method is avoided, the preparation is easy, the preparation can be used in a plug-and-play way, and the constructed preparation has low immunogenicity and good stability.

(4) The polypeptide guiding molecule embedded lipid complex has universality and can be expanded to other targeted drug delivery mediated by a transmembrane receptor.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objects and other advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the specification.

Drawings

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in the following preferred detail with reference to the accompanying drawings, in which:

FIG. 1 is an analysis of the response of ITP peptides (A) and SP (B) to IR recombinant liposome interactions in example 1;

FIG. 2 is a graph showing insulin competition binding to primary brain endothelial cell uptake results in example 2;

FIG. 3 shows the anisotropy values of NBD-PE probes at 30, 35, 40℃for different liposomes in example 3, respectively;

FIG. 4 is Young's modulus values for Lip and ITP-Lip in example 3;

FIG. 5 shows uptake results of various proportions of ITP peptide modified liposomes by bEnd.3 cells in example 4;

FIG. 6 shows the uptake results of bEnd.3 cells of example 5 on different ratios of DSPE-PEG ₂₀₀₀ modified ITP-Lip;

FIG. 7 is a morphology of the polypeptide targeting molecule embedded lipid complex of example 6;

FIG. 8 is a fluorescence spectrum of Lip, ITP and ITP-Lip at a wavelength of 400-305 nm in example 7;

FIG. 9 is a fluorescence spectrum of Lip, AY and AY-Lip at a wavelength of 400 to 305nm in example 7;

FIG. 10 is a FRET fluorescence spectrum and corresponding quantitative result of FRET efficiency for DiI and DiD liposomes entrapped in example 8;

FIG. 11 shows the change in FRET efficiency in serum for different liposomes in example 8;

FIG. 12 shows the results of cellular uptake of liposomes modified with different polypeptides of example 9;

FIG. 13 is a photograph of a living small animal in example 9;

FIG. 14 is an SDS-PAGE protein adsorption analysis in example 10;

FIG. 15 shows the FRET imaging results of ITP peptide-intercalating modified liposomes in small intestine sections of example 11.

Detailed Description

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention. It should be noted that the illustrations provided in the following embodiments merely illustrate the basic idea of the present invention by way of illustration, and the following embodiments and features in the embodiments may be combined with each other without conflict.

1. Experimental cell strain

Mouse brain microvascular endothelial cells (bend.3), mouse mononuclear macrophages (RAW 264.7) were purchased from marsupenario life technologies limited.

The primary brain endothelial cells (BMECs) were extracted according to the following experimental procedure:

(1) Female SD rats of 4-6 weeks old were sacrificed with excess anesthesia, sterilized by 75% ethanol twice (1 st quick wash, 2 nd appropriate soak, no more than 5 min), the scalp and skull of the rats were sequentially sheared off under aseptic conditions, the brains carefully removed, and placed in petri dishes containing cold D-Hanks solution (tissue isolation was performed entirely on ice). Carefully removing meninges and large blood vessels by using sterilizing filter paper, removing olfactory bulb, thalamus, midbrain, cerebellum, pontine and the like, leaving only cerebral cortex layer, and carefully removing large blood vessels;

(2) The cortical layer was sheared, centrifuged (1200 rpm,5 min) in a sterile 15mL centrifuge tube, 25% BSA (W: V) solution was added to the pellet layer at a 1.5:1 (V: V) ratio, and repeatedly blown with a pipette to form a cortical homogenate, which was then centrifuged at 2500rpm for 5min, and the pellet vessel segments were collected. Collecting supernatant, repeatedly blowing with a pipettor, centrifuging at 2500rpm for 5min, collecting the precipitated vessel segment again, and combining the vessel lengths collected twice to extract the microvasculature to the maximum extent;

(3) The vessel sections obtained by the two centrifugation were rinsed with cold D' Hanks solution and centrifuged (2500 rpm,5 min), 0.1% type II collagenase (W: V) was added at 1:5 (V: V), and digested at 37℃for 5min. Taking out the sample after digestion, adding a complete culture medium containing 20% of serum, blowing for about 10 times by using a pipette, and centrifuging for 5min under the condition of 1200rpm to obtain the cerebral cortex microvascular endothelial cells containing the microvascular segments. Cells were suspended in a complete medium containing 20% FBS, and the cell suspension was inoculated into a flask, and after culturing in a 5% CO ₂ incubator at 37℃for 24 hours, the flask was changed (the flask was shaken thoroughly to detach the attached tissue mass). When the confluency of the cultured cells reaches 80%, the cultured cells can be subjected to passage purification and then used for experiments.

Unless otherwise specified, the culture media used in the experiments of bEnd.3 and BMECs in the examples were DMEM complete medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin and streptomycin mixed solution (P/S).

2. Experimental animal

Balb/c mice, females, 6-8 weeks old, SD rats, females, 4-6 weeks old, provided by Chongqing Chinese traditional medicine institute laboratory animal research institute. Animals were kept in SPF-class animal houses and the animals were fasted for 24 hours prior to the experiment without water deprivation. The experimental study is strictly carried out according to the guidance opinion on animals to be tested of the Chinese scientific technical section.

The construction method of the mouse breast cancer TUBO subcutaneous tumor model comprises the following steps:

After digestion of 5×10 ⁶ mouse breast cancer TUBO cells, the cells were washed 2 times with PBS, and after centrifugation to collect cell pellet, resuspended in 200 μl PBS. 100. Mu.L of the cell suspension was aspirated using a 500. Mu.L syringe and subcutaneously injected into the Balb/c mouse forelimb armpit. The TUBO breast cancer tumor-bearing mouse model is normally fed in an SPF-level feeding environment, the tumor growth state is detected on time, and the model can be used for subsequent experiments when the subcutaneous tumor volume is about 150mm ³.

3. Experimental materials

The specific preparation of compound A1 (2- (di ((9E, 12E) -octadeca-9, 12-dien-1-yl) amino) ethyl (4 ¹ R,13 aS) -13 a-ethyl-2, 3,4 ¹, 5,6,13 a-hexahydro-1H-indolo [3,2,1-de ] pyrido [3,2,1-ij ] [1,5] naphthyridine-12-carboxylate) is shown below:

(1) (6Z, 9Z) -18-bromooctadecane-6, 9-diene (C5-Br) (400 mg, 1.21 mmol), 2-aminoethanol (HO-B1-NH ₂) (35. Mu.L, 0.552 mmol), potassium carbonate (330 mg, 2.43 mmol) and potassium iodide (9 mg, 0.552 mmol) were dissolved in 5mL of acetonitrile in a molar ratio of 1.21:0.552:2.43:0.552, and the reaction mixture was heated and stirred at 65℃for 24h;

(2) Cooling to room temperature, washing the filter cake obtained by filtration with n-hexane for 3 times, extracting the filtrate obtained by filtration with n-hexane, concentrating under reduced pressure, separating by silica gel chromatography (methanol/dichloromethane=15/85 (v/v)), and obtaining an intermediate compound by rotary evaporation;

(3) A1-OH (50 mg, 0.15 mmol), intermediate compound (88 mg,1.59 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl,35mg, 0.183 mmol) and 4-dimethylaminopyridine (DMAP, 1mg,0.0081 mmol) were dissolved in 6mL pyridine/dichloromethane (1/1, v/v) and the reaction mixture was stirred at room temperature for 24h;

(4) The organic phase obtained by the separation was diluted with dichloromethane (6 mL), washed 4 times with 5mL of a 2M hydrochloric acid solution, saturated brine each, then dried over sodium sulfate, concentrated under reduced pressure, and separated by silica gel chromatography (acetone/n-hexane=1/1 (v/v); dichloromethane/methanol=1/3 (v/v)), and the final product was obtained as a vincamine derivative A1 by rotary evaporation ¹H NMR(400MHz,DMSO-d₆)δ7.43(dd,J＝6.7,2.7Hz,1H),7.05(d,J＝6.9Hz,3H),5.32(pd,J＝11.0,4.5Hz,10H),2.74(t,J＝6.3Hz,4H),2.18(s,4H),2.02(q,J＝6.8Hz,9H),1.78(tq,J＝7.0,3.6Hz,4H),1.35-1.22(m,44H),1.05(t,J＝7.1Hz,3H),0.87(q,J＝7.0,5.6Hz,9H)..

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications. In the following examples, the operating temperature and pressure are generally room temperature and normal pressure unless otherwise specified. Wherein, the room temperature is 10-30 ℃; atmospheric pressure refers to a standard atmospheric pressure.

EXAMPLE 1SPR analysis of ITP peptide affinity with Insulin Receptor (IR) recombinant liposomes

10Mg of lecithin (EPC) is taken, precisely weighed, dissolved in a methanol-chloroform (1:1, V:V) mixed solution, transferred into a round bottom distillation flask, the organic solvent is removed by adopting a rotary evaporator, and after lipid films are formed at the bottom of the flask, nitrogen is introduced to blow dry the residual organic solvent. Immediately adding 1mL of IR recombinant protein into a round bottom flask, putting into a constant temperature oscillator for shaking until lipid membranes are fully dissolved and dispersed, and finally putting into an ultrasonic cell disruption instrument for ultrasonic preparation of liposome, wherein the ultrasonic treatment is carried out for 10 times, 5 s/time and 5s at intervals. Repeatedly squeezing with liposome extruder to control particle diameter, and sequentially passing through polycarbonate filter membranes of 400, 200 and 100nm to obtain IR recombinant liposome.

The SPR instrument was ligated and calibrated, the surface of an L1 chip (model SLD0930; nicoya, KITCHENER, ON, canada) was initialized by injecting 200. Mu.L of 20mM CHAPS buffer, the sample loop was washed with PBS buffer and emptied with air 3 times, the prepared IR recombinant liposomes were immobilized ON the L1 chip, and after each injection of solutions of ITP peptides (amino acid sequence ELGILIFLYLFSLILGIIYWKK) at different concentrations (125, 250, 500, 1000 and 2000nM, respectively) and the affinity of the polypeptides to the IR recombinant liposomes was evaluated by fitting the resulting data with out-of-order peptides (Scrambled peptide, SP, amino acid sequence WEILGIGLFLYILSIFILLIKK, available from Shanghai Fin Biotechnology Co., ltd.) as control polypeptide solutions at concentrations of 125, 250, 500, 1000 and 2000nM, respectively.

From FIGS. 1A and 1B, it can be seen that at the same polypeptide concentration, the response of ITP peptide to IR recombinant liposome action was significantly higher than that of the disordered peptide SP. The fit of the ITP to the IR recombinant liposome effect was 2.10x ^-7 M, significantly lower than the fit between SP and IR recombinant liposome results, with smaller KD values indicating greater affinity effects between the test samples.

Example 2 verification of ITP peptide specific binding to IR transmembrane region

Primary brain endothelial cells (BMECs) were pre-seeded in 12-well plates (2X 10 ⁵ per well) and incubated at 37℃for 24 hours. The medium was discarded, replaced with fresh medium containing 1.25. Mu.M insulin or ITP peptide at concentrations of 1.25, 2.5, 5 and 10. Mu.M, and incubated for 1 hour at 37 ℃. The medium was discarded, fresh medium was added, 20. Mu.g/mL FITC-insulin was added to the corresponding 12-well plate, and incubated at 37℃for 1 hour. Cells were then collected, washed three times with PBS and the uptake of FITC-insulin by the cells was determined using a flow cytometer.

Following insulin pre-incubation, BMECs uptake of FITC-insulin was inhibited due to competitive binding of previously pre-incubated insulin to the extracellular region of IR. Whereas preincubation with ITP peptide did not interfere with BMECs uptake of FITC-insulin (fig. 2), indicating that ITP specifically binds to the IR transmembrane region.

Example 3 Effect of ITP peptide modification on phospholipid Membrane fluidity and Liposome elastic modulus

The membrane hydration method is adopted to prepare NBD-PE fluorescence labeling liposome, and the change of the membrane fluidity is evaluated by measuring the fluorescence anisotropy of a fluorescence probe NBD-PE in the liposome.

Preparation of blank liposomes:

Similar to the preparation method of the IR recombinant liposome, the difference is that: 1mL of IR recombinant protein was replaced with 1mL of PBS buffer.

Preparation of NBD-PE fluorescence labeled liposome:

Firstly, a proper amount of NBD-PE is taken, precisely weighed, dissolved in a small amount of chloroform and diluted with methanol to a final concentration of 1mg/mL of stock solution. According to cholesterol: EPC or ITP: the EPC (0.25%, 0.5%,1%,2%,4%, mole ratio) is prepared by taking cholesterol and ITP polypeptide, precisely weighing, respectively dissolving with a methanol-chloroform (1:1, V: V) mixed solution and ethanol in proper amounts, respectively mixing with lecithin uniformly, and according to NBD-PE: lecithin (1:200, molar ratio) was added to the corresponding volume of NBD-PE stock solution. And (3) uniformly mixing, transferring to a round bottom distillation flask, removing the organic solvent by adopting a rotary evaporator, and obtaining the NBD-PE fluorescence-labeled cholesterol liposome (CHL-Lip) and the NBD-PE fluorescence-labeled ITP-modified liposome (ITP-Lip) respectively in the subsequent steps consistent with the preparation method of the blank liposome.

And (3) measuring the anisotropic change of the NBD-PE fluorescent probes in different liposomes under different temperature conditions (30, 35 and 40 ℃) by using a multifunctional enzyme-labeled instrument. The fluorescence anisotropy (r) is calculated as follows: r= (I/-I Γ)/(I/+2i), where I/, and I Γ are the fluorescence intensities measured on the polarization plane of the emitted light, parallel and perpendicular to the polarized excitation beam, respectively. The excitation and emission wavelengths of NPD-PE were 463nm and 534nm, respectively.

Changes in Young's Modulus (Young's Modulus) of the preparation after ITP modification of the liposomes were measured by Atomic Force Microscopy (AFM), and the effect of ITP modification on the elastic Modulus of the liposomes was evaluated. And (3) taking mica sheets matched with the AFM, and coating the mica sheets with polylysine solution in advance. Diluting the prepared liposome sample with ultrapure water until the final concentration of phospholipid is 2mg/mL, taking 10 mu L of diluted sample, carefully dripping the diluted sample onto the mica sheet, and standing for 10min to enable the liposome to be adsorbed on the mica sheet. And then blowing off the unadsorbed sample with nitrogen at a low speed, and obtaining the sample, wherein the sample is successfully prepared, and the Young modulus of the sample is analyzed by AFM measurement.

The effect of ITP modification on liposome membrane fluidity was evaluated using cholesterol modification as a control. As shown in fig. 3, ITP was significantly better than cholesterol in reducing membrane fluidity at the same molar ratio of cholesterol or ITP modification, and there was a significant difference and positive correlation with ITP concentration change. When the temperature was lowered, both cholesterol and ITP modifications, the fluorescence probe NBD-PE anisotropy increased to some extent, indicating a decrease in liposome membrane fluidity. In conclusion, ITP modifications have better stabilizing effects on liposomes than cholesterol.

As can be seen from fig. 4, the young modulus of the ITP-Lip is about 35.19 times that of the unmodified liposome Lip, which indicates that the ITP modification can significantly improve the rigidity of the liposome, and has good supporting effect on the liposome structure, so that the liposome is stabilized, and the probability of drug leakage can be reduced.

Example 4 Effect of ITP peptide modification ratio on uptake of liposomes by cells

Coumarin 6 (C6) fluorescein-labeled ITP-Lip is prepared by a film hydration method. The method comprises the following specific steps: and (3) taking a proper amount of C6 fluorescein, precisely weighing, and dissolving in a methanol-chloroform (1:1, V:V) mixed solution until the final concentration of the fluorescein is 1mg/mL to obtain a C6 fluorescein stock solution. Lecithin 10mg was taken, precisely weighed, and dissolved in a methanol-chloroform (1:1, V: V) mixed solution. According to ITP: the ITP polypeptide and EPC are taken according to the proportion of 0.25%,0.5%,1%,2%,4% and mole ratio, and the ITP polypeptide is dissolved in proper amount of ethanol. And mixing lecithin solution and ITP polypeptide solution, adding appropriate amount of C6 stock solution to make the final concentration of the stock solution in liposome solution be 10 mug/mL, transferring to a round bottom distillation flask, removing organic solvent by adopting a rotary evaporator in the dark, forming a lipid film at the bottom of the flask, and blowing nitrogen into the flask to dry the residual organic solvent. Immediately adding 1mL of PBS buffer solution into a round-bottom flask, putting into a constant-temperature oscillator for oscillation until the lipid membrane is fully dissolved and dispersed, and carrying out subsequent steps in the same way as the preparation of blank liposome in the embodiment 3 to obtain the C6 fluorescein labeled liposome modified by different ITP proportions.

The bEnd.3 cells in the logarithmic growth phase were digested and counted using a hemocytometer, seeded into 12-well plates at an seeding concentration of 2X 10 ⁵ cells/well, and placed in a cell incubator for 24h (37 ℃,5% CO ₂). The medium in the wells is discarded, the fresh complete medium is replaced, the prepared liposomes modified by different ITP ratios of the C6 markers are added to a final concentration of 1 mug/mL, and the liposomes are placed in an incubator and incubated in a dark place for ingestion for 2 hours. After the end of the uptake, the liposome-containing medium was aspirated, the cells were digested with trypsin for about 3min, pancreatin was discarded and washed three times with PBS, the cells were resuspended with PBS, centrifuged at 1000g for 5min, the supernatant was discarded and the pelleted cells were collected, 500. Mu.L of resuspended cells were added, and the cell uptake fluorescence intensity was measured using a flow cytometer.

As shown in fig. 5, the fluorescence intensity of 2% ITP-modified liposomes was about 1.21 times that of the control group, and the fluorescence intensity of 4% ITP-modified liposomes was about 1.72 times that of the control group, each of which was significantly different. Considering the complexity of the subsequent in vivo environment, the proportion of ITP modified liposomes was chosen to be 4% in order to reduce the influence of factors such as non-target sites and endogenous substances as much as possible.

Example 5 Effect of DSPE-PEG ₂₀₀₀ modification ratio on uptake of liposomes by cells

In order to enable the ITP-Lip to have a long circulation function, the ITP-Lip is better applied to in vivo drug delivery, the ITP-Lip is modified by adopting gold standard 'PEG modification' of the current long circulation, and DSPE-PEG ₂₀₀₀ is specifically selected as a functional modification molecule. According to EPC: ITP: DSPE-PEG ₂₀₀₀ (100:4:5/100:4:10/100:4:15/100:4:20, molar ratio) was taken in proportion EPC, ITP, DSPE-PEG ₂₀₀₀, precisely weighed. Except for the ITP, the materials were dissolved in ethanol with a methanol-chloroform (1:1, V: V) mixture. The EPC, ITP and DSPE-PEG ₂₀₀₀ solutions corresponding to each formulation were mixed uniformly, transferred to a round bottom distillation flask, the organic solvent was removed using a rotary evaporator, and the subsequent steps were examined with reference to the "blank liposome preparation" in example 3 and the flow detection method in example 4.

The quantitative analysis results of flow cytometry are shown in fig. 6, in which the 5% PEG-ITP-Lip group showed a decrease in uptake compared to the ITP-Lip group, but there was still a significant difference (P < 0.001) from the Lip group. In view of the long circulation and active targeting of the balanced formulation, 5% DSPE-PEG ₂₀₀₀ was used to modify ITP-Lip to prepare PEG-ITP-Lip.

EXAMPLE 6 particle size and morphology characterization of polypeptide-directed molecular intercalating lipid complexes

The liposome is prepared by adopting a film hydration method. Precisely weighing nine polypeptides according to the optimal molar ratio of the screened polypeptides to DSPE-PEG ₂₀₀₀ (EPC: polypeptide: DSPE-PEG ₂₀₀₀ =100:4:5), wherein the nine polypeptides comprise:

polypeptide directed against the insulin receptor transmembrane region: ELGILIFLYLFSLILGIIYWKK (ITP peptide, 22 aa);

Polypeptide directed against the insulin receptor transmembrane region: LFGILIFLFLFSLIIGSIYLWKKK (P5 peptide, 24 aa);

Polypeptide directed against the integrin receptor transmembrane region: AYVFILLSFILGTLLGFLVMFWAKK (P4 peptide, 25 aa);

Polypeptide directed against the integrin receptor transmembrane region: AYVFILLSFILGTLLGFLVMFWA (P0 peptide, 23 aa);

polypeptide directed against the integrin receptor transmembrane region: FILLSFILGTLLGFL (P1 peptide, 15 aa);

Polypeptide directed against the integrin receptor transmembrane region: FILLSFILGT (P2 peptide, 10 aa);

polypeptide directed against the integrin receptor transmembrane region: PEEFILKSFILGTLKGFLKMFYS (P3 peptide, 23 aa);

polypeptide directed against the integrin receptor transmembrane region: DDEFILLSFILGTLKGFLKMFWS (AY peptide, 23 aa);

Polypeptide directed against the epidermal growth factor receptor-2 transmembrane region: TFIIATVEGVLLFLILVVVVGILIKRR (TF peptide, 27 aa).

Except for ITP and P5, the polypeptides and materials were dissolved in a methanol-chloroform (1:1, V: V) mixture. And (3) uniformly mixing the polypeptide solution, the EPC solution and the DSPE-PEG ₂₀₀₀ solution, transferring to a round bottom distillation flask, removing the organic solvent by adopting a rotary evaporator, and performing the subsequent steps on the polypeptide solution, the EPC solution and the DSPE-PEG ₂₀₀₀ solution to obtain the polypeptide-targeted molecule embedded liposome. The particle size distribution of each liposome prepared was measured using a malvern laser particle sizer.

The RAW 264.7 cell-derived exosomes were isolated by ultracentrifugation and exosomes embedded in ITP polypeptides (ITP-exosomes) were prepared. RAW 264.7 cell supernatants were collected and centrifuged at 5,000g for 30min,10,000g for 1h to remove cells and other debris. The obtained supernatant was centrifuged at 135,000g for 1h, and the precipitate was washed with PBS to obtain exosomes. ITP was dissolved in absolute ethanol and incubated with exosomes for 30min at 37 ℃. Unbound ITP was removed by ultracentrifugation at 135,000g for 1h and the pellet was the ITP-Exosome.

ITP polypeptide embedded lipid nanoparticles (PEG-ITP-LNP) were prepared using a microfluidic device (model: MPE, ai Tesen pharmaceutical Equipment Co., ltd.). Dlin-MC3-DMA, DSPC, chol, DMG-PEG ₂₀₀₀ and ITP were dissolved in ethanol and mixed in the appropriate molar ratio (50:10:34.5:1.5:4), respectively. The DEPC water is taken as an aqueous phase, and the aqueous phase and the ethanol solution are mixed according to the volume ratio of 3:1 at the mixing rate of 12 mL/min. Dialyzing overnight with dialysis bag with molecular weight of 8-14K to obtain PEG-ITP-LNP.

ITP polypeptide embedded ionizable lipid nanoparticles (PEG-ITP-VIP, PEG-ITP-Rg 5-VIP) were prepared using a microfluidic device (model: MPE, ai Tesen pharmaceutical Equipment Co., st.). The ionizable lipid fraction in the PEG-ITP-LNP preparation step was replaced by Dlin-MC3-DMA with A1 (2- (di ((9E, 12E) -octadeca-9, 12-dien-1-yl) amino) ethyl (4 ¹ R,13 aS) -13 a-ethyl-2, 3,4 ¹, 5,6,13 a-hexahydro-1H-indolo [3,2,1-de ] pyrido [3,2,1-ij ] [1,5] naphthyridine-12-carboxylate) and the other materials were unchanged, and PEG-ITP-VIP was obtained according to the preparation method described above. The PEG-ITP-Rg5-VIP is prepared by substituting ionizable lipid component Dlin-MC3-DMA with A1, substituting Chol with ginsenoside Rg5 (Xiamen Bensu pharmaceutical Co., ltd.) and other materials.

The particle size and morphology of each lipid complex prepared were characterized using a malvern laser particle sizer and a transmission electron microscope.

TABLE 1 particle size of polypeptide targeting molecule Embedded lipid complexes

Formulations	Particle size (nm)	PDI
			PEG-ITP-Lip	87.40±9.81	0.146±0.049
ITP-Exosome	174.20±5.54	0.112±0.005
			PEG-ITP-LNP	123.39±3.12	0.224±0.015
PEG-ITP-VIP	86.60±1.21	0.117±0.013
			PEG-ITP-Rg5-VIP	89.95±2.32	0.132±0.009
PEG-P5-Lip	85.88±5.54	0.157±0.021
			PEG-P1-Lip	76.33±8.92	0.208±0.022
PEG-P2-Lip	75.89±6.77	0.116±0.009
			PEG-P3-Lip	98.08±6.18	0.165±0.032
PEG-P4-Lip	95.37±8.04	0.187±0.025
			PEG-P0-Lip	92.90±6.22	0.110±0.008
PEG-AY-Lip	85.12±3.98	0.106±0.058
			PEG-TF-Lip	75.44±5.12	0.219±0.011

The results are shown in Table 1 and FIG. 7, in which the PDI value of the polypeptide targeting molecule-embedded lipid complex is small, and the particle size is uniform and the morphology is round.

EXAMPLE 7 fluorescence Spectroscopy characterization of ITP and AY peptide-embedded liposomes

Fluorescence intensity was measured on an F-7000 fluorescence spectrometer using a cuvette of 0.1cm path length. Unmodified blank liposomes (Lip) were prepared as in example 3; ITP and AY peptide-embedded liposomes (ITP-Lip, AY-Lip) were prepared by thin film hydration (EPC, ITP, AY was taken at 4% (molar ratio) of ITP/AY: EPC; except for the dissolution of ITP with ethanol, the remaining materials were dissolved with a methanol-chloroform (1:1, V: V) mixture, the EPC, ITP or AY solutions corresponding to each formulation were transferred to a round bottom distillation flask, the organic solvent was removed by rotary evaporator, and ITP, AY, lip, ITP-Lip and AY-Lip were diluted to 1mg/mL with 2, 2-trifluoroethanol for the subsequent steps with reference to "blank liposome preparation" in example 3. The excitation and emission slit widths were set to 2.5nm and 1.5nm, respectively, with an average time of 1.0 seconds. 280nm excitation is set, and emission scanning is carried out on the wavelength of 400-305 nm.

As shown in figures 8 and 9, at the wavelength of 400-305 nm, the Lip has no obvious fluorescence signal, and the maximum absorption wavelength of the fluorescence spectrum of the ITP-Lip and the AY-Lip has obvious blue shift compared with the free ITP and AY, and the fluorescence signal intensity is increased, thus proving that the polypeptide molecules are successfully embedded into the liposome phospholipid bilayer.

EXAMPLE 8 stability investigation of ITP peptide-embedded modified liposomes

The serum stability of the formulations was characterized using Fluorescence Resonance Energy Transfer (FRET) experiments, with fluorescein DiI as the fluorescent pair and di as the donor molecule (ex=549 nm, em=565 nm) and di as the acceptor molecule (ex=644 nm, em=663 nm). Proper amounts of fluorescein DiI and DiD are taken, precisely weighed, dissolved in ethanol and diluted to a final concentration of 1 mug/mL of stock solution for later use. Fluorescein DiI and DiD double-fluorescence labeled Lip were prepared according to the preparation method of coumarin 6 fluorescence labeled liposomes in example 4, wherein final concentration of fluorescein DiI in the prepared liposomes was 10 μg/mL, diD fluorescein was entrapped according to DiI: diD (1:1/1:2/1:3, mass ratio) to screen for preferred FRET fluorescence pair combinations. Separately, liposomes were prepared with fluorescein DiI and DiD, respectively, and two single fluorescently labeled liposomes were physically mixed as a control group. Scanning FRET fluorescence spectrum by using a multifunctional enzyme-labeled instrument, and calculating FRET efficiency, wherein the calculation formula is as follows: fretratio= IDiD/(IDiD + IDiI), where IDiD and IDiI are fluorescence intensity values at 663nm and 565nm, respectively.

The experimental results are shown in fig. 10, where FRET efficiency is highest when DiI: did=1:3 (mass ratio). Preparing DiI and DiD double fluorescent marks Lip in the ratio, preparing DiI and DiD double fluorescent marks ITP-Lip, PEG-Lip and PEG-ITP-Lip (DiI: diD=1:3 (mass ratio) fluorescein is properly weighed, dissolved in a mixed solution of methanol-chloroform (1:1, V: V) until the final concentration of total fluorescein is 1mg/mL, namely a fluorescein stock solution, weighing phospholipid 10mg, precisely weighing polypeptide and DSPE-PEG ₂₀₀₀ according to the ratio of EPC to DSPE-PEG ₂₀₀₀ (100:4:5), dissolving the polypeptide in proper ethanol, uniformly mixing lecithin solution and polypeptide solution, adding proper amount of fluorescein stock solution to ensure that the final concentration of the lecithin solution in liposome solution is 10 mug/mL, transferring the liposome stock solution into a round bottom distillation flask, removing an organic solvent by adopting a rotary evaporator, forming a lipid film at the bottom, introducing nitrogen residual organic solvent, immediately drying the solution into a constant temperature buffer solution of 1mL until the bottle bottom is dried, adding the constant temperature buffer solution into a PBS (100:4:5), shaking the bottle bottom until the liposome is completely dissolved in PBS, and performing the same shaking step of shaking to prepare the liposome, and fully dissolving the liposome in a blank. The prepared liposome and serum are mixed according to the proportion of (1:9, V:V), placed in a constant temperature oscillator, incubated at 37 ℃ and 120rpm, and each sample is taken at 0, 0.5, 1, 2,4,8, 12, 24 and 48 hours, and FRET fluorescence spectrum is scanned by a multifunctional enzyme-labeled instrument to calculate FRET efficiency.

Stability results as shown in fig. 11, the FRET efficiency of four groups of liposomes in serum decreased with time, but ITP-Lip and PEG-ITP-Lip groups modified with ITP were found to be significantly higher than Lip and PEG-Lip groups, indicating that ITP modification can increase serum stability of liposomes.

Example 9 investigation of targeting of ITP, AY, TF peptide-intercalating modified liposomes

The bEnd.3 cells in the logarithmic growth phase were digested and counted using a hemocytometer, seeded into 12-well plates at an seeding concentration of 2X 10 ⁵ cells/well, and placed in a cell incubator for 24h (37 ℃,5% CO ₂). Fresh complete medium was replaced by medium in wells, and prepared C6-labeled different polypeptides (ITP, AY, P0, P1, P2, P3, P4, P5 peptides) -modified liposomes (preparation method see example 4) were added to a final concentration of C6 of 1 μg/mL and incubated in an incubator protected from light for uptake for 2h. After the end of the uptake, the liposome-containing medium was aspirated, the cells were digested with trypsin for about 3min, pancreatin was discarded and washed three times with PBS, the cells were resuspended with PBS, centrifuged at 1000g for 5min, the supernatant was discarded and the pelleted cells were collected, 500. Mu.L of resuspended cells were added, and the cell uptake fluorescence intensity was measured using a flow cytometer.

As shown in FIG. 12, the fluorescence intensity of several polypeptide-embedded modified liposomes was significantly higher than that of PEG-Lip, demonstrating that the polypeptide-modified liposomes had active targeting to bEnd.3 cells.

And ITP, AY, TF polypeptides are selected for further in vivo targeting investigation. Lip, PEG-Lip (preparation method refers to example 8, and double fluorescent labels are changed into near infrared fluorescent probes DiR) and liposome modified by different polypeptides ITP, AY, TF (PEG-ITP-Lip, PEG-AY-Lip, PEG-TF-Lip; preparation method refers to example 6) for in vivo imaging of small animals. Wherein TF peptide group Balb/c mice adopts a right armpit mouse breast cancer TUBO subcutaneous tumor model, and ITP and AY peptide groups use healthy Balb/c mice.

DiR-labeled Lip, PEG-Lip, and different polypeptide (ITP, AY, TF) -modified liposome three-group formulations (100 μg DiR/kg) were injected into the tail vein, mice were anesthetized with isoflurane at 4h, and the in vivo fluorescence distribution of the mice was photographed and recorded using a VISQUE-animal fluorescence imaging system.

As shown in fig. 13, the brain fluorescence of the mice in the group of PEG-ITP-Lip and PEG-AY-Lip is stronger, which proves that the ITP and AY peptide embedded modified liposome has brain targeting; the PEG-TF-Lip group tumor tissue has stronger fluorescence, which indicates that the TF peptide embedded modified liposome shows tumor targeting. The results show that the three polypeptide guiding molecules embedded liposomes taking the membrane protein transmembrane region as the target have good in vivo targeting function.

EXAMPLE 10 immunogenicity investigation of AY peptide-embedded modified liposomes

Whole blood was collected from healthy Balb/c mice and serum was centrifuged. mu.L of liposomes (PEG-Lip, AY-Lip, PEG-AY-Lip, RGD-PEG-Lip) were mixed with an equal volume of serum and incubated for 1h at 37 ℃. The pellet was collected and washed twice with PBS and centrifuged at 14000g for 40min. The collected pellet was resuspended in 60. Mu.L PBS and 15. Mu.L 5 XSDS-PAGE sample buffer, and the mixture was boiled for 10min to denature the protein. Proteins were separated by SDS-PAGE and stained with a rapid silver staining kit. The preparation methods of AY-Lip, PEG-AY-Lip and RGD-PEG-Lip were the same as in example 6. The preparation method of the PEG-Lip comprises the following steps: 10mg of phospholipid was weighed according to EPC: DSPE-PEG ₂₀₀₀ (100:5, molar ratio) was precisely weighed DSPE-PEG ₂₀₀₀, dissolved in methanol-chloroform (1:1, V: V) mixed solution, transferred to a round bottom distillation flask, the organic solvent was removed by rotary evaporator, and after lipid film was formed at the bottom of the flask, nitrogen was introduced to blow dry the residual organic solvent. Immediately adding 1mL of PBS buffer solution into a round bottom flask, putting into a constant temperature oscillator for shaking until the lipid membrane is fully dissolved and dispersed, and carrying out the following steps in the same way as in the preparation of blank liposome in the example 3.

As shown in fig. 14, three groups of liposome modified by polypeptide (AY-Lip, PEG-AY-Lip, RGD-PEG-Lip) all have more remarkable IgM (72 kDa) adsorption than PEG-Lip, wherein RGD cyclic peptide modification results in the highest IgM adsorption level, and embedded modification of AY peptide can reduce IgM adsorption to some extent.

EXAMPLE 11ITP peptide-embedded modified Liposome intestinal stability investigation

The stability of the polypeptide targeting molecule embedded liposome small intestine is characterized by adopting a Fluorescence Resonance Energy Transfer (FRET) experiment. Proper amounts of fluorescein DiI and DiD are taken, precisely weighed, dissolved in ethanol and diluted to a final concentration of 1 mug/mL of stock solution for later use. The film hydration method is adopted to prepare different liposome (Lip, ITP-Lip, PEG-ITP-Lip) with fluorescein DiI and DiD double fluorescence marks, wherein the final concentration of the fluorescein DiI and DiD in the prepared liposome is 10 mug/mL and 30 mug/mL respectively.

Healthy Balb/c mice were taken in 12 groups of three randomly. The mice were given an appropriate amount of 1.4% sodium bicarbonate solution to neutralize the gastric acid by gavage, and after 0.5h, each group of liposomes (100 μg DiI/kg) was gavaged. Mice were sacrificed at 2h with excess isoflurane anesthesia, the duodenum, jejunum, ileum were carefully dissected out, tissue was embedded with OCT embedding medium and frozen sections were performed and samples transferred to adhesive slides. The slide was placed in a wet box, 4% paraformaldehyde fixing solution was added dropwise to the sample, and the mixture was fixed at room temperature for 30min. After the fixation is finished, the fixation liquid is discarded, PBS is used for soaking and washing for 3 times, and a high content imaging system is used for fluorescent imaging shooting.

The FRET imaging result of the polypeptide guide molecule embedded liposome in the small intestine section is shown in figure 15, and the FRET DiD fluorescence signal intensity of ITP-Lip and PEG-ITP-Lip groups modified by ITP is obviously higher than that of Lip and PEG-Lip groups, which indicates that the stability of the liposome in the small intestine can be obviously improved by ITP modification.

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the present invention, which is intended to be covered by the claims of the present invention.

Claims

1. The polypeptide targeting molecule embedded lipid complex is characterized in that the polypeptide targeting molecule can be embedded in a phospholipid bilayer of a lipid carrier and specifically recognizes a membrane protein transmembrane region, and the lipid complex is obtained by embedding the polypeptide targeting molecule into the lipid carrier; the polypeptide targeting molecule comprises:

polypeptide directed against the insulin receptor transmembrane region: ELGILIFLYLFSLILGIIYWKK, ITP peptides;

Polypeptide directed against the insulin receptor transmembrane region: LFGILIFLFLFSLIIGSIYLWKKK, P5 peptide;

polypeptide directed against the integrin receptor transmembrane region: AYVFILLSFILGTLLGFLVMFWAKK, P4 peptide;

Polypeptide directed against the integrin receptor transmembrane region: AYVFILLSFILGTLLGFLVMFWA, P0 peptide;

polypeptide directed against the integrin receptor transmembrane region: FILLSFILGTLLGFL, P1 peptide;

polypeptide directed against the integrin receptor transmembrane region: FILLSFILGT, P2 peptide;

Polypeptide directed against the integrin receptor transmembrane region: PEEFILKSFILGTLKGFLKMFYS, P3 peptide;

Polypeptide directed against the integrin receptor transmembrane region: DDEFILLSFILGTLKGFLKMFWS, AY peptides;

polypeptide directed against the epidermal growth factor receptor-2 transmembrane region: TFIIATVEGVLLFLILVVVVGILIKRR, TF peptides;

the lipid carrier comprises liposome, exosome and lipid nanoparticles;

When the lipid carrier is lecithin, a thin film hydration method is adopted to prepare the polypeptide guide molecule embedded lipid complex;

When the lipid carrier is exosome, the preparation method is as follows: collecting RAW 264.7 cell supernatant, centrifuging to remove cells and fragments, centrifuging 1h at 135,000 g, and washing the precipitate with PBS to obtain exosomes; dissolving the polypeptide targeting molecule in an organic solvent, and incubating with exosomes; removing unbound polypeptide-directed molecules by ultracentrifugation;

When the lipid carrier is a lipid nanoparticle, the preparation method is as follows: respectively dissolving an ionizable lipid ingredient, DSPC, a steroid compound, DMG-PEG ₂₀₀₀ and a polypeptide targeting molecule in an organic solvent and mixing in a molar ratio of 50:10:34.5:1.5:4; taking DEPC water as a water phase, and mixing the water phase and the organic solution according to the volume ratio of 3:1 at the mixing rate of 12 mL/min; dialyzing overnight with dialysis bag with molecular weight of 8-14K; the ionizable lipid ingredient is Dlin-MC3-DMA or 2- (di ((9E, 12E) -octadeca-9, 12-dien-1-yl) amino) ethyl (4 ¹ R,13 aS) -13 a-ethyl-2, 3,4 ¹, 5,6,13 a-hexahydro-1H-indolo [3,2,1-de ] pyrido [3,2,1-ij ] [1,5] naphthyridine-12-carboxylate, and the structural formula of 2- (di ((9E, 12E) -octadeca-9, 12-dien-1-yl) amino) ethyl (4 ¹ R,13 aS) -13 a-ethyl-2, 3,4 ¹, 5,6,13 a-hexahydro-1H-indolo [3,2,1-de ] pyrido [3,2,1-ij ] [1,5] naphthyridine-12-carboxylate is aS follows:

。

2. The polypeptide targeting molecule intercalating lipid complex according to claim 1, wherein the lipid complex further comprises a modifying molecule, PEG.

3. The polypeptide targeting molecule embedded lipid complex according to claim 1, wherein the polypeptide targeting molecule: the lecithin is 0.25-4% of the mole ratio, the raw materials are taken, precisely weighed, the raw materials are respectively dissolved by using an organic solvent, the organic solvent is removed after uniform mixing, the residual organic solvent is dried after forming a lipid film, PBS buffer solution is immediately added, the mixture is vibrated until the lipid film is fully dissolved and dispersed, and the particle size is controlled by ultrasonic and pushing and filtering, thus obtaining the lecithin.

4. The polypeptide targeting molecule embedded lipid complex according to claim 1, wherein the steroid compound is cholesterol or ginsenoside.

5. Use of a polypeptide targeting molecule embedded lipid complex according to claim 1 for the preparation of a targeted drug delivery system.