WO2022156531A1 - Dynein binding peptide capable of permeating through biological barrier, and use thereof - Google Patents

Abstract

Disclosed are a dynein binding peptide capable of permeating through a biological barrier, and the use thereof. The polypeptide provided by the present invention is a polypeptide A or a polypeptide B, wherein the polypeptide A is sequentially composed of a core region A and a cell-penetrating peptide from the N-terminus to the C-terminus, and the amino acid sequence of the core region A is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; and the polypeptide B is sequentially composed of a core region B and a cell-penetrating peptide from the N-terminus to the C-terminus, and the amino acid sequence of the core region B is SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. According to the present invention, the polypeptide A can achieve intracellular perinuclear delivery, the polypeptide B can achieve intracellular intranuclear delivery, and the two polypeptides both have biological barrier permeability.

Description

A dynein-binding peptide capable of penetrating biological barriers and its application technical field

The invention relates to the field of biotechnology, in particular to a dynein-binding peptide capable of penetrating biological barriers and applications thereof.

Background technique

Many drugs, including various macromolecules (proteins, enzymes, antibodies, DNA), as well as drug nanocarriers, require intracellular delivery to achieve intracellular delivery in the cytoplasm or nucleus or other specific organelles such as lysosomes, mitochondria or endoplasm Internet) to play its therapeutic role. Including gene and antisense therapeutic drugs, which must reach the nucleus; pro-apoptotic drugs targeting mitochondria; lysosomal enzymes, which must reach the lysosomal compartment to exert drug effects. Intracellular transport of different bioactive molecules is usually one of the key issues in drug delivery. For example, intracellular delivery in tumor therapy can overcome important obstacles such as multidrug resistance caused by P-glycoprotein in anticancer chemotherapy. . The lipophilic nature of biological membranes limits the direct intracellular delivery of many compounds. Cell membranes prevent large molecules, such as peptides, proteins, and DNA, from entering the cell spontaneously unless there is an active transport mechanism, such as the way some short peptides enter the cell. In some cases, these molecules and even small particles can enter cells from the extracellular space through receptor-mediated endocytosis. However, the problem encountered in this case is that molecules/particles entering cells through endocytosis are often trapped in endosomes and eventually end up in lysosomes, where active degradation processes occur under the action of lysosomal enzymes. As a result, only a small fraction of the unaffected material is present in the cytoplasm. This has resulted in many compounds showing promising potential in vitro that cannot be used in vivo due to bioavailability issues. The more successful attempts are to directly introduce various macromolecular drugs and drug-loaded drug carriers into the cytoplasm, bypassing the endocytic pathway, to protect drugs and DNA from lysosomal degradation, thereby improving drug efficiency or DNA incorporation into the cell genome. efficiency, but methods such as microinjection or electroporation used to deliver membrane-impermeable molecules in cell experiments are inherently invasive and can damage cell membranes. More effective are non-invasive methods such as the use of pH-sensitive carriers, including pH-sensitive liposomes, to destabilize the membranes of phagocytic vesicles at low pH inside the endosome, release the entrapped drug into the cytoplasm, and Cell penetrating molecules (eg, penetrating peptides) are employed.

The above applications circumvent phagocytosis of cells (drugs are delivered into the cytoplasm), they still have to find a way to reach specific organelles (nucleus, lysosomes, mitochondria) where they are expected to exploit their therapeutic potential. This is even more important in the context of gene drug delivery, where viral vectors for gene delivery carry an inherent risk of non-specificity and virus-induced complications.

To avoid this problem of phagocytosis, studies have multifunctionalized drug nanocarriers, that is, capable of performing multiple functions simultaneously or sequentially, such as specific recognition of target cells and endosomal escape, the ability to target individual organelles is a very ideal feature. However, specific subcellular delivery of bioactive molecules remains a challenging problem. One possible approach is to combine drug molecules, or better yet, drug-loaded drug nanocarriers, with another compound that has a specific affinity for the organelle of interest. Among the organelles of greatest interest for specific targeting are lysosomes and mitochondria. Therefore, the use of lysosome-targeted drug nanocarriers could significantly improve the delivery of therapeutic enzymes and chaperones into defective lysosomes for the treatment of lysosomal storage disorders, while the specific delivery of certain drugs to mitochondria may Helps treat a variety of diseases, including neurodegenerative and neuromuscular diseases, obesity, diabetes, ischemia-reperfusion injury, and cancer. However, the delivery of all intracellular drug carriers cannot be achieved by targeting organelles in this way, such as nuclear delivery or macromolecular drugs or carriers that require wide intracellular distribution.

Nanocarrier delivery cannot achieve rapid intracellular transport, and macromolecular drugs with intracellular targets still have the problem of delivery. Some studies use methods to reduce the stability of phagocytic vesicles to rupture phagosomes, but if the carrier Loaded with macromolecular drugs, the intracellular diffusion of the delivered drugs is still problematic. The interior of all living cells is filled with macromolecules, which differs greatly in the thermodynamics and kinetics of biological reactions in vivo and in vitro, and studies have shown that the "excluded volume effect" in the cytoplasm is not sufficient to explain the macromolecular diffusion observed in vivo The large reduction of , and the hydrodynamic interaction, greatly reduce the diffusivity of monodisperse colloids, especially in dense systems. The cytoplasm is crowded, with macromolecular concentrations up to about 300 g/L and volume occupancy reaching 30%, an environment that is quite different from the dilute, idealized conditions commonly used in biophysical research, such as in the nucleus, where all the deoxyribose Nucleic acid fragments are nearly all immobile, the highly restricted diffusion of DNA fragments in the nucleoplasm is due to extensive binding to immobile barriers, and the reduced lateral mobility of DNA >250 bp in the cytoplasm is due to molecular crowding. Based on this macromolecular crowding state, it is difficult for nano-drug carriers and linear macromolecules like DNA to rely on simple diffusion to achieve intracellular transport. So how to develop a way to actively transport carriers or drugs to achieve Methods for efficient intracellular transport are important.

This new intracellular/intercellular delivery method is derived from the understanding of the mechanism of virus infecting cells into and out of cells. Virus-sized particles cannot rely on simple diffusion to achieve rapid infection in cells. Through in-depth research in related fields, it was found that virus infection The process of entering and exiting cells relies on the dynein and kinesin in the cell. The dynein can transport the virus from the side of the cell membrane to the center of the microtubule organization (in the direction of the nucleus), while the kinesin is used to transport the virus from the nucleus. Lateral transport to the cell membrane side.

Dynein is highly conserved in different organisms, and it is a potential method for drug/vehicle delivery, on the one hand, because of its transport from positive to negative (nuclear direction) along microtubules, which can achieve extensive coverage to the nucleus and the area covered by microtubules. On the other hand, its more important feature as a drug/carrier transport engine is the diversity of its movement directions. Relevant studies have shown that the position of dynein at the intersection of microtubules not only passes through the intersection, but also has a certain proportion of steering, The possibility of reverse, stagnation, dissociation, etc., this feature makes it possible for the drug/carrier to be widely transported in cells and even transcellular as a drug delivery carrier. At the same time, the quasi-transport efficiency of dynein is very high, the movement speed of dynein in eukaryotic cells can reach 1-3 μm/s, and the traction force can reach pN level.

At present, there are two main attempts to use dynein for drug delivery. One way is to construct a transcriptional expression system to prepare a partial subunit of dynein, and to combine the subunit with the target cargo to achieve delivery. At the stage of realizing the transport of DNA sequences and proved to be more efficient in transfection, another way is to use dynein-binding peptides to make related attempts, and more functions can be achieved by further modification of multi-dynein-binding peptides. At this stage, it has been modified to the surface of gold nanoparticles, fluorescently labeled polystyrene particles, and PLGA nanoparticles. The above studies are mainly to modify the fluorescence on nanoparticles or sequences to observe the intracellular transport ability of the sequences after binding to dynein. And carried out related research, observed the phenomenon of fluorescent particles moving to the nucleus, aggregation and intercellular transport. However, none of them has progressed to the research and application of the combination of carrier and drug, and it is also an attempt to carry out research on its penetration through biological barriers (such as the blood-brain barrier).

Invention Disclosure

In view of "due to the crowded state of macromolecules in the cytoplasm, macromolecular drugs/carriers cannot rely on diffusion to achieve drug effects, and drugs and carriers need to cross multiple layers of cells in the process of passing through biological barriers, such as the blood-brain barrier, so drugs/carriers permeate the barrier. It still needs to be further optimized and solved”. The present invention uses the cell’s own dynein to achieve: 1. The macromolecular drug/nanocarrier is rapidly transported in the cell to achieve the intracellular drug effect of the target; 2. The problem of the drug/carrier passing through the biological barrier ; ③ drug core delivery.

In a first aspect, the present invention claims a polypeptide.

The polypeptide claimed in the present invention is polypeptide A or polypeptide B.

The polypeptide A is composed of a core region A (having dynein binding ability) and a penetrating peptide from the N-terminus to the C-terminus in turn; the amino acid sequence of the core region A is SEQ ID No.1, SEQ ID No.2, SEQ ID No.2, and SEQ ID No. 2. ID No.3 or SEQ ID No.4.

The polypeptide B is composed of a core region B (having dynein binding ability) and a membrane-penetrating peptide from the N-terminus to the C-terminus in turn; the amino acid sequence of the core region B is SEQ ID No.5, SEQ ID No.6, SEQ ID No. ID No. 7 or SEQ ID No. 8.

Wherein, the penetrating peptide can be composed of 6-9 consecutive arginine residues (R).

In a specific embodiment of the present invention, the penetrating peptide is specifically composed of 8 consecutive arginine residues (R).

In a second aspect, the present invention claims a polypeptide derivative.

The polypeptide derivative claimed in the present invention is polypeptide derivative A or polypeptide derivative B.

The polypeptide derivative A is obtained by connecting a linker to the N-terminus of the polypeptide A in the first aspect above, and the linker can be used to connect a carrier or a drug or a fluorescent group.

The polypeptide derivative B is obtained by linking the N-terminus of the polypeptide B described in the first aspect above with a linker, and the linker can be used to link a carrier or a drug or a fluorescent group.

Further, the linker can be one or several glycine residues (G), cysteine residues (C) and/or lysine residues (K) and the like.

In a specific embodiment of the present invention, the linker is specifically GK, that is, it consists of a glycine residue (G) and a lysine residue (K).

In a third aspect, the present invention claims a transporter.

The transporter claimed in the present invention is either transporter A or transporter B.

The transporter A is obtained by linking the polypeptide derivative A described in the second aspect above with a carrier or a drug or a fluorescent group by means of the linker.

The transporter B is obtained by linking the polypeptide derivative B described in the second aspect above with a carrier or a drug or a fluorescent group by means of the linker.

Wherein, the carrier refers to a carrier for transporting drugs, such as nanoparticles, micelles, liposomes, vesicles, and the like.

In a specific embodiment of the present invention, the linker (GK) is specifically linked to carboxytetramethylrhodamine (TAMRA). Among them, the glycine residue (G) is a residue for reducing steric hindrance when the carrier is connected, and the lysine residue (K) is connected to TAMRA.

In the fourth aspect, the present invention claims the use of the polypeptide described in the first aspect above or a derivative of the polypeptide described in the second aspect above in the preparation of the transporter described in the third aspect above.

In the fifth aspect, the present invention claims the use of the polypeptide A described in the first aspect above or the polypeptide derivative A described in the second aspect above in the preparation of a drug transporter with biological barrier permeability and/or perinuclear aggregation properties.

In the sixth aspect, the present invention claims the use of the polypeptide B described in the first aspect above or the polypeptide derivative B described in the second aspect above in the preparation of a drug transporter with biological barrier permeability and/or intranuclear aggregation properties.

In a seventh aspect, the present invention claims the use of the polypeptide of the first aspect above or the polypeptide derivative of the second aspect above in the preparation of a formulation capable of improving the intracellular transport capacity of macromolecules and/or nanocarriers.

In the eighth aspect, the present invention claims the use of the polypeptide according to the first aspect or the derivative of the polypeptide according to the second aspect in the preparation of a preparation capable of improving the effect strength of a drug acting in cells.

In the ninth aspect, the present invention claims that the polypeptide described in the first aspect or the polypeptide derivative described in the second aspect can be prepared to reduce or improve the multidrug resistance (tumor multidrug resistance) caused by efflux factors such as P glycoprotein. drug resistance).

In a tenth aspect, the present invention claims a method of preparing a drug transporter with biological barrier permeability and/or perinuclear aggregation properties.

The method for preparing a drug transporter with biological barrier permeability and/or perinuclear aggregation properties as claimed in the present invention may include the following steps: using the polypeptide A described in the first aspect above or the polypeptide derivative A described in the second aspect above. Prepare.

In an eleventh aspect, the present invention claims a method of preparing a drug transporter with biological barrier permeability and/or intranuclear aggregation properties.

The method for preparing a drug transporter with biological barrier permeability and/or nuclear aggregation properties as claimed in the present invention may include the following steps: using the polypeptide B described in the first aspect above or the polypeptide derivative B described in the second aspect above. Prepare.

In a twelfth aspect, the present invention claims a method of preparing a formulation capable of improving the intracellular transport capacity of macromolecules and/or nanocarriers.

The method for preparing a preparation capable of improving the intracellular transport capacity of a macromolecule and/or a nanocarrier as claimed in the present invention may comprise the steps of: preparing using the polypeptide described in the first aspect above or a derivative of the polypeptide described in the second aspect above.

In a thirteenth aspect, the present invention claims a method of preparing a formulation capable of reducing or improving multidrug resistance.

The method for preparing a preparation capable of reducing or improving multidrug resistance as claimed in the present invention may comprise the steps of: preparing using the polypeptide described in the first aspect above or the derivative of the polypeptide described in the second aspect above.

In a specific embodiment of the present invention, the biological barrier is specifically the blood-brain barrier.

Description of drawings

Figure 1 shows the intracellular fluorescence behavior of each polypeptide.

Figure 2 shows the intracellular fluorescence behavior of control polypeptides.

Figure 3 shows the intracellular specific behavior of polypeptide No. 1 and No. 5. A is the perinuclear aggregation of No. 1 polypeptide; B is the intranuclear aggregation of No. 5 polypeptide.

Best way to implement your invention

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.

Example 1. Design of dynein-binding peptides with biological barrier permeability

In order to improve the delivery efficiency of intracellular drugs/carriers, the present invention designs a series of polypeptides with dynein binding ability with reference to the core sequence of virus binding to dynein. The basic structure of polypeptide design is: GK/C + core sequence + penetrating peptide, and a polypeptide sequence with a purity of 95% is prepared by solid-phase synthesis method (general method).

Table 1. Polypeptides of the present invention (core sequence)

serial number intracellular delivery extracellular delivery core sequence number 1 PKTRNSQTQTD PKTRNSQTQTD-R _n SEQ ID No.1 2 VVSYSKETQTP VVSYSKETQTP-R _n SEQ ID No.2 3 IVTYTKETQTP IVTYTKETQTP-R _n SEQ ID No.3 4 PRMLRSTQTT PRMLRSTQTT-R _n SEQ ID No.4 5 TILVSRSTQTGF TILVSRSTQTGF-R _n SEQ ID No.5 6 VKLVDAESQTL VKLVDAESQTL-R _n SEQ ID No.6 7 VQMAKSTQT VQMAKSTQT-R _n SEQ ID No.7 8 RSSEDKSTQTT RSSEDKSTQTT-R _n SEQ ID No.8

Note: R _n represents n consecutive arginine residues (R), n=6-9. R _{n is} the penetrating peptide (it can also be other penetrating peptide CPPs). The linker used to connect the N-terminus of the core polypeptide of the present invention to a drug or a carrier or a fluorescent group is specifically GK; wherein, the glycine residue (G) is a residue used to reduce steric hindrance when the carrier is connected, and is connected to TAMRA is the lysine residue (K).

The polypeptide of the present invention can be delivered to the drug carrier in two ways. One is to connect the polypeptide with the drug to be delivered by a synthetic method, so as to realize the intracellular delivery of the drug or to pass through the biological barrier; Polypeptides are linked to drug delivery carriers (such as nanoparticles, micelles, liposomes, etc.) to achieve intracellular delivery of drug carriers and penetration of biological barriers.

Example 2. Identification of the intracellular transport ability, cell proliferation toxicity and biological barrier permeability of the polypeptide of the present invention

In this example, GK at the N-terminus of the polypeptide in Example 1 is linked with carboxytetramethylrhodamine (TAMRA) to observe the intracellular behavior, which mainly involves intracellular transport ability, cell proliferation toxicity and biological barrier permeability. Specifically, the "extracellular administration" polypeptide in Table 1 of Example 1 is used, and R _n is specifically 8 Rs.

A control polypeptide (control) was set in each experiment, specifically replacing the core sequence in the polypeptide of Example 1 with "SLVSSDESVLHGSHESGEHV". The control peptide was reported in the reference.

1. Synthesis and Identification of Polypeptide Sequences

Solid-phase synthesis method using peptide synthesizer.

The specific synthesis method adopts the Fmoc cycle method, and according to the designed sequence, amino acids are added one by one according to deprotection (removal of amino protecting group), activation of cross-linking (peptide bond synthesis), elution and deprotection.

Product Identification The molecular weights of the synthesized sequences were compared by HPLC-MS.

Purity identification was carried out by HPLC, and the peptide purity was calculated by the peak area integral ratio.

2. Cell staining method

1. Use bEnd.3 cells (mouse brain microvascular endothelial cells) to inoculate in a 24-well plate, with an initial density of 1×10 ⁵ cells per well;

2. Under the conditions of 37°C, 5% CO ₂ , the cells are allowed to grow and adhere;

3. Take out the culture plate, aspirate and discard the original culture solution, and wash twice with 1ml PBS per well;

4. Add 10 μM (final system concentration) of each polypeptide labeled with carboxytetramethylrhodamine;

5. Incubate for 5-60min at 37℃, 5% _CO2 ;

6. Discard the incubation solution, wash twice with 1 ml of PBS per well, and fix with 4% paraformaldehyde for 10 minutes;

7. Observation under a fluorescence microscope.

3. Cell proliferation toxicity method

1. Take a T25 bottle to culture bEnd.3 cells, digest and count, and the medium is fully dispersed;

2. Take 2 96-well plates and inoculate 1×10 ⁴ in each well;

3. After 48 hours of growth, add labeled polypeptide solution to make the final concentration reach 25, 50, 100 μM (the final concentration in the system after addition), 3 wells for each concentration, after incubation for 12h and 24h respectively, add 10μl to each well CCK-8 solution, continue to incubate for 1 h in the incubator, and then read the absorbance value at 450 nm with a microplate reader.

4. Transwell permeability test method

1. Take a 24-well tranwell chamber, inoculate 1×10 ⁵ bEnd.3 cells in each well, and culture at 37°C, 5% CO ₂ ;

2. Regularly measure the transmembrane resistance until the TEER value reaches about 60Ω·cm ² . The formula for calculating the TEER value is TEER=(transmembrane resistance of culture chamber-transmembrane resistance of blank chamber)×cell bottom area cm ² ;

3. Carry out a 4-hour leak test, and the liquid level difference can be maintained for more than 4 hours;

4. Add each polypeptide solution labeled with TAMRA with a final concentration of 100 μM in the upper chamber, take samples at 5, 10, and 30 minutes respectively, and read the fluorescence intensity with a microplate reader;

5. Calculate the permeability coefficient, the calculation formula is: P _app =dQ/dt×1/A×1/C ₀ .

V. Results and Analysis

1. Cell staining experiment

The results of the cell staining experiment showed that the peptides No. 1-8 in Example 1 all achieved widespread intracellular distribution (Fig. 1), while the control peptide (Fig. 2) could only reside on the cell surface. Among them, peptide 1 showed better perinuclear aggregation, and peptide 5 showed better intranuclear aggregation (Fig. 3), suggesting that peptide 1 has stronger perinuclear delivery ability, and peptide 5 has better perinuclear delivery ability. Strong intranuclear delivery capability.

2. Cell proliferation toxicity test

In terms of cell proliferation and toxicity, the CCK-8 method was used to test bEnd.3 cells, and it was shown that each polypeptide in Example 1 and the control polypeptide were in contrast to the negative control (Ctrl, that is, without any growth-influencing substances added) when the concentration reached 100 μM. cell group), there was no significant difference (Table 2), no cell proliferation toxicity was shown, and the level of safety was good.

Table 2. Results of cell proliferation toxicity test

Note: "*" means p>0.05, no significant difference from Ctrl.

3. Research on the ability of permeability barrier

In the research on the permeability of the barrier, through the transwell experiment, bEnd.3 cells were used to construct the blood-brain barrier model. In most cases, each polypeptide in Example 1 showed an apparent permeability coefficient greater than 10 ^-6 cm/s (Table 3 ), indicating that it can penetrate the biological barrier well.

Table 3. Experimental results of apparent permeability coefficient

  5min(cm/s) 10min(cm/s) 30min(cm/s) 1 2.022× ^10-5 5.234× ^10-5 5.123× ^10-5 2 10.427× ^10-5 22.345× ^10-5 11.923× ^10-5 3 1.250× ^10-5 2.866× ^10-5 2.564× ^10-5 4 2.354× ^10-5 4.567× ^10-5 4.543× ^10-5 5 2.129× ^10-5 2.767× ^10-5 3.796× ^10-5 6 5.654× ^10-5 3.756× ^10-5 8.896× ^10-5 7 2.567× ^10-5 2.678× ^10-5 7.768× ^10-5 8 6.324× ^10-5 3.542× ^10-5 2.879× ^10-5

Industrial application

In order to improve the delivery efficiency of intracellular drugs/carriers, the present invention designs a series of polypeptides with dynein binding ability with reference to the core sequence of virus-dynein binding. Experiments show that polypeptide A can achieve pericellular delivery, and polypeptide B can achieve cellular Delivered within the core, both polypeptides are biologically barrier permeable. And compared with the prior art, the present invention finds that it can improve the intracellular transport ability of macromolecular drugs (such as polypeptides, DNA, RNA, etc.) and/or nanocarriers, and improve the effect strength of intracellular drugs by comparing with control polypeptides. , can reduce or improve multidrug resistance (tumor multidrug resistance).

Claims (22)

Polypeptide, which is Polypeptide A or Polypeptide B; The polypeptide A is composed of a core region A and a membrane-penetrating peptide from the N-terminus to the C-terminus in turn; the amino acid sequence of the core region A is SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4; The polypeptide B is composed of a core region B and a membrane-penetrating peptide from the N-terminus to the C-terminus in turn; the amino acid sequence of the core region B is SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 or SEQ ID No.8. The polypeptide according to claim 1, wherein the penetrating peptide is composed of 6-9 consecutive arginine residues. The polypeptide according to claim 2, wherein the penetrating peptide consists of 8 consecutive arginine residues. A polypeptide derivative, which is a polypeptide derivative A or a polypeptide derivative B; The polypeptide derivative A is obtained by connecting a linker to the N-terminus of the polypeptide A in any one of claims 1-3, and the linker can be used to connect a carrier or a drug or a fluorescent group; The polypeptide derivative B is obtained by connecting a linker to the N-terminus of the polypeptide B in any one of claims 1-3, and the linker can be used to connect a carrier or a drug or a fluorescent group. The polypeptide derivative according to claim 4, wherein the linker is one or several glycine residues, cysteine residues and/or lysine residues. The polypeptide derivative according to claim 5, wherein the linker is GK, that is, it consists of a glycine residue and a lysine residue. transporter, which is transporter A or transporter B; The transporter A is obtained by connecting the polypeptide derivative A described in any one of claims 4-6 with a carrier or a drug or a fluorescent group by means of the linker; The transporter B is obtained by linking the polypeptide derivative B described in any one of claims 4-6 with a carrier or a drug or a fluorescent group by means of the linker. The transporter according to claim 7, wherein the carrier is a carrier for transporting drugs. The transporter according to claim 7, wherein the carrier is a nanoparticle, a micelle, a liposome or a vesicle. The transporter according to claim 7, wherein the transporter is obtained by linking the polypeptide derivative with carboxytetramethylrhodamine by means of K in GK as the linker. Use of the polypeptide according to any one of claims 1-3 or a derivative of the polypeptide according to any one of claims 4-6 in the preparation of the transporter according to any one of claims 7-10. Use of the polypeptide A described in any one of claims 1-3 or the polypeptide derivative A described in any one of claims 4-6 in the preparation of a drug transporter with biological barrier permeability and/or perinuclear aggregation properties. Use of the polypeptide B described in any one of claims 1-3 or the polypeptide derivative B described in any one of claims 4-6 in the preparation of a drug transporter with biological barrier permeability and/or intranuclear aggregation properties. The application according to claim 12 or 13, wherein the biological barrier is a blood-brain barrier. Use of the polypeptide according to any one of claims 1-3 or the derivative of the polypeptide according to any one of claims 4-6 in the preparation of a preparation capable of improving the intracellular transport capacity of macromolecules and/or nanocarriers. Use of the polypeptide according to any one of claims 1-3 or the polypeptide derivative according to any one of claims 4-6 in the preparation of a preparation capable of improving the effect strength of a drug acting in cells. Use of the polypeptide according to any one of claims 1-3 or the polypeptide derivative according to any one of claims 4-6 in the preparation of a preparation capable of reducing or improving multidrug resistance. A method for preparing a drug transporter with biological barrier permeability and/or perinuclear aggregation properties, comprising the steps of: using polypeptide A described in any one of claims 1-3 or any one of claims 4-6 Polypeptide Derivative A was prepared. A method for preparing a drug transporter with biological barrier permeability and/or aggregation characteristics in the nucleus, comprising the steps of: using the polypeptide B described in any one of claims 1-3 or any one of claims 4-6. The polypeptide derivative B was prepared. The method according to claim 18 or 19, wherein the biological barrier is a blood-brain barrier. A method for preparing a preparation capable of improving the intracellular transport ability of macromolecules and/or nanocarriers, comprising the steps of: derivatizing with any one of the polypeptides of claims 1-3 or any of the polypeptides of claims 4-6 things are prepared. A method for preparing a preparation capable of reducing or improving multidrug resistance, comprising the steps of: preparing with the polypeptide according to any one of claims 1-3 or the polypeptide derivative according to any one of claims 4-6.

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