CN114805595A - Dynein binding peptides with biological barrier permeability and nuclear aggregation properties and application thereof - Google Patents

Dynein binding peptides with biological barrier permeability and nuclear aggregation properties and application thereof Download PDF

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CN114805595A
CN114805595A CN202110069415.4A CN202110069415A CN114805595A CN 114805595 A CN114805595 A CN 114805595A CN 202110069415 A CN202110069415 A CN 202110069415A CN 114805595 A CN114805595 A CN 114805595A
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polypeptide
derivative
drug
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CN114805595B (en
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刘楠
郑爱萍
王增明
张慧
高静
高翔
杜祎萌
吕佳琦
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Academy of Military Medical Sciences AMMS of PLA
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    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
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    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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Abstract

The invention discloses an dynein binding peptide with biological barrier permeability and nuclear aggregation characteristics and application thereof. The dynein binding peptide with biological barrier permeability and nuclear aggregation characteristics sequentially consists of a core region and a membrane-penetrating peptide from an N end to a C end; the amino acid sequence of the core region is SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 4. The polypeptide can realize intracellular delivery of a medicament/carrier, and has biological barrier permeability. Compared with the prior art, the invention can improve the intracellular transport capacity of macromolecules and/or nano-carriers, improve the effect strength of intracellular acting drugs, and reduce or improve multidrug resistance (tumor multidrug resistance).

Description

Dynein binding peptides with biological barrier permeability and nuclear aggregation properties and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to dynein binding peptide with biological barrier permeability and nuclear aggregation characteristics and application thereof.
Background
Many drugs, including various macromolecules (proteins, enzymes, antibodies, DNA), as well as drug nanocarriers, need to be delivered intracellularly to achieve their therapeutic effect within the cytoplasm or on the nucleus or other specific organelles (e.g., lysosomes, mitochondria, or endoplasmic reticulum). This group includes genes and antisense therapeutics, which must reach the nucleus; pro-apoptotic drugs for mitochondria; lysosomal enzymes, must reach the lysosomal compartment etc. to exert a pharmaceutical effect. Intracellular transport of different bioactive molecules is often one of the key issues in drug delivery, for example, intracellular administration in tumor therapy can overcome important obstacles such as multidrug resistance caused by P glycoprotein in anticancer chemotherapy. The lipophilicity of biological membranes limits the direct intracellular delivery of many compounds. Cell membranes prevent macromolecules, such as peptides, proteins and deoxyribonucleic acids, from spontaneously entering cells unless there is an active transport means, such as some short peptides. In some cases, these molecules, even small particles, can enter the cell from the extracellular space through receptor-mediated endocytosis. However, a problem encountered in this case is that the molecules/particles entering the cell via the endocytic pathway are often trapped in the endosome and eventually end up in the lysosome where active degradation processes take place by the action of lysosomal enzymes. As a result, only a small fraction of unaffected material is present in the cytoplasm. This has led to the inability of many compounds that show promising potential in vitro to be used in vivo due to bioavailability problems. More successful attempts have been made to introduce various macromolecular drugs and drug-loaded drug carriers directly into the cytoplasm, bypassing the endocytic pathway, to protect the drugs and DNA from lysosomal degradation, thereby increasing the efficiency of the drug or the efficiency of DNA incorporation into the cell genome, but these methods are inherently invasive and may damage the cell membrane in cell experiments, such as microinjection or electroporation, which is used to deliver membrane-impermeable molecules. More effective non-invasive methods such as the use of ph-sensitive carriers include ph-sensitive liposomes destabilizing the membrane of the phagocytic vesicle at low ph inside the endosome, releasing the entrapped drug into the cytoplasm, and the use of cell penetrating molecules (e.g., cell penetrating peptides).
The above applications circumvent the phagocytic digestion of cells (drugs already transported into the cytoplasm), they still have to find ways to reach specific organelles (nucleus, lysosomes, mitochondria) where they are expected to exploit their therapeutic potential. This is even more important in gene drug delivery processes, and viral vectors used for gene delivery have an inherent risk of non-specificity and virus-induced complications.
To avoid this phagocytic digestion problem, there is a study to make drug nanocarriers multifunctional, i.e., 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 desirable property. However, specific subcellular delivery of biologically active molecules remains a challenging problem. One possible approach is to combine a drug molecule, or more preferably a drug-loaded drug nanocarrier, with another compound having a specific affinity for the organelle of interest. Among the organelles of most interest for specific targeting, lysosomes and mitochondria may be mentioned. Thus, the use of lysosome-targeted drug nanocarriers can significantly improve the delivery of therapeutic enzymes and chaperones into defective lysosomes for the treatment of lysosomal storage disorders, while specific delivery of certain drugs to the mitochondria may be helpful in the treatment of a variety of diseases, including neurodegenerative and neuromuscular diseases, obesity, diabetes, ischemia-reperfusion injury, and cancer. However, the cellular organelle targeting approach does not allow the delivery of all intracellular drug carriers, such as nuclear delivery or the need for large molecule drugs or carriers that are widely distributed in the cell.
While targeting vector delivery has not been possible to achieve delivery of the entire cell, the delivery of macromolecular drugs targeted to the cell remains problematic, and some studies have been directed to disrupting phagocytes in a manner that reduces the stability of the phagocytes, but if the vector is loaded with macromolecular drugs, diffusion of the delivered drug within the cell remains problematic. The interior of all living cells is filled with macromolecules, which differ considerably in the thermodynamics and kinetics of in vivo and in vitro biological reactions, and studies have shown that the "excluded volume effect" in the cytoplasm is not sufficient to explain the large reduction in macromolecular diffusion observed in vivo, whereas the hydrodynamic interactions greatly reduce the diffusivity of monodisperse colloids, especially in dense systems. Cytoplasmic crowding, with macromolecular concentrations up to-300 g/L and volume occupancy up to 30%, differs greatly from the dilute, idealized conditions typically employed in biophysical studies, e.g., in the nucleus, all DNA fragments are nearly immobile, highly restricted diffusion of DNA fragments in the nuclear mass is due to extensive binding to immobile obstacles, and reduced lateral mobility of >250bp DNA in the cytoplasm is due to molecular crowding. Based on the crowded state of macromolecules, the intracellular transport of nano-drug carriers and linear macromolecules such as DNA is difficult to realize by simple diffusion, so how to develop a way capable of actively transporting carriers or drugs to realize the efficient intracellular transport is very important.
The novel intracellular/intercellular delivery mode is derived from the mechanism recognition that virus infects cells and then goes out of the cells, particles with the virus size cannot realize rapid infection in the cells by simple diffusion, and through the deep research in the related fields, the intracellular and extracellular processes of the virus infecting the cells are found to be based on the dynein and the kinesin in, the dynein can realize the transportation of the virus from the cell membrane side to the center of the microtubule tissue (the direction of the cell nucleus), and the kinesin can realize the transportation of the virus from the cell nucleus side to the cell membrane side.
The dynein is highly conserved in different organisms, and a potential method for delivering the dynein as a drug/carrier is that on one hand, the dynein can be widely delivered to a nucleus and a microtubule coverage area due to the fact that the dynein is transported from a positive pole to a negative pole along microtubules (in the direction of the nucleus), and on the other hand, the more important characteristic of the dynein as a drug/carrier transport engine is that the movement direction of the dynein is diverse, and related researches show that the dynein at the position where the microtubules cross passes through the cross point, the possibility of steering, reversing, arresting, dissociating and the like exist in a certain proportion, and the characteristic provides possibility for the dynein as a drug delivery carrier to enable the drug/carrier to be widely transported in a cell and even realize trans-cell transport. Meanwhile, the efficiency of the motor protein transportation is very high, the movement speed of the motor protein in eukaryotic cells can reach 1-3 mu m/s, and the traction force can reach the pN level.
The existing main attempts to carry out drug delivery by using dynein are 2 ways, one way is to adopt a transcription expression system to prepare a partial subunit of dynein, and the subunit is combined with target goods to realize delivery, the research in the aspect is in the stage of realizing the transportation of a DNA sequence and proving that the transfection efficiency is higher, the other way is to adopt dynein binding peptide to carry out related attempts, and more functions can be realized by further modifying the dynein binding peptide. The fluorescent particles are modified on the surfaces of gold nanoparticles, fluorescence-labeled polystyrene particles and PLGA nanoparticles at the present stage, the research mainly comprises the steps of modifying fluorescence on the nanoparticles or sequences, observing the transport capacity of the sequences in cells after combining with dynein, carrying out related research, and observing the phenomena of nuclear motion aggregation and intercellular transport of the fluorescent particles. However, the research and application of the combination of the carrier and the drug are not developed, and the research of the permeation of the biological barrier is also attempted.
Disclosure of Invention
Aiming at the problems that the macromolecular medicament/carrier can not realize the medicament effect by means of diffusion due to the macromolecular crowding state of cytoplasm, and the medicament and the carrier need to cross a plurality of layers of cells in the process of penetrating a biological barrier, such as blood brain barrier, so that the medicament/carrier penetration barrier still needs to be further optimized and solved, the invention realizes the following effects by means of the cell self-motor protein: the macromolecular drug/nano-carrier is rapidly transported in cells, and the drug effect of an action target in the cells is realized; ② the problem of drug/carrier penetration through biological barrier; ③ the delivery of drug core.
In a first aspect, the invention claims a polypeptide.
The polypeptide required to be protected by the invention sequentially consists of a core region (with binding capacity of dynein) and a cell-penetrating peptide from an N end to a C end; the amino acid sequence of the core region is SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 4.
Wherein, the cell-penetrating peptide can be composed of 6-9 continuous arginine residues (R).
In a particular embodiment of the invention, said cell-penetrating peptide consists in particular of 8 consecutive arginine residues (R).
In a second aspect, the invention claims a polypeptide derivative.
The polypeptide derivative claimed by the invention is obtained after a linker is connected to the N-terminal of the polypeptide of the first aspect, and the linker can be used for connecting a carrier or a drug or a fluorescent group.
Further, the linker may be one or several glycine residues (G), cysteine residues (C) and/or lysine residues (K), etc.
In a particular embodiment of the invention, the linker is in particular GK, i.e. consists of one glycine residue (G) and one lysine residue (K).
In a third aspect, the invention claims a transporter.
The transporter claimed in the present invention is obtained by linking the polypeptide derivative of the second aspect to a carrier or a drug or a fluorophore by means of the linker.
Wherein, the carrier refers to a carrier for transferring drugs, such as nanoparticles, micelles, liposomes, vesicles and the like.
In a specific embodiment of the invention, the linker (GK) is specifically carboxytetramethylrhodamine (TAMRA). Among them, the glycine residue (G) is a residue for reducing steric hindrance when the carrier is linked, and the lysine residue (K) is linked to TAMRA.
In a fourth aspect, the invention claims the use of a polypeptide according to the first aspect or a derivative of a polypeptide according to the second aspect for the preparation of a transporter according to the third aspect.
In a fifth aspect, the present invention claims the use of a polypeptide according to the first aspect or a derivative of a polypeptide according to the second aspect for the manufacture of a drug transporter with biological barrier permeability and/or nuclear aggregation properties.
In a sixth aspect, the invention claims the use of a polypeptide according to the first aspect or a derivative of a polypeptide according to the second aspect for the preparation of a formulation capable of improving the intracellular transport capacity of a macromolecule and/or a nanocarrier.
In a seventh aspect, the invention claims the use of a polypeptide according to the first aspect or a derivative of a polypeptide according to the second aspect for the manufacture of a formulation for improving the strength of the effect of an intracellularly acting drug.
In an eighth aspect, the present invention claims the use of a polypeptide according to the first aspect or a derivative of a polypeptide according to the second aspect for the manufacture of a formulation for reducing or improving multidrug resistance (tumor multidrug resistance) caused by exogenous factors such as P-gp.
In a particular embodiment of the invention, the biological barrier is in particular the blood brain barrier.
In order to improve the delivery efficiency of intracellular drugs/carriers, a series of polypeptides with dynein binding capacity are designed according to a core sequence of a virus combined with dynein, and experiments prove that the polypeptides can realize intracellular delivery and have biological barrier permeability. Compared with the prior art, the polypeptide disclosed by the invention can improve the intracellular transport capacity of macromolecules and/or nano-carriers, improve the effect strength of intracellular acting drugs, realize the aggregation of the drugs/carriers in cell nuclei and also has potential application (such as tumor multidrug resistance) in reducing or improving multidrug resistance caused by P glycoprotein by being compared with a control polypeptide.
Drawings
FIG. 1 shows the intracellular fluorescence behavior of each polypeptide.
FIG. 2 shows the intracellular fluorescence behavior of control polypeptides.
FIG. 3 shows the intracellular specific behavior (intranuclear aggregation) of polypeptide No. 1.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 design of dynein binding peptides with biological barrier permeability and intranuclear aggregation Properties
In order to improve the delivery efficiency of intracellular drugs/carriers, the invention designs a series of polypeptides with binding capacity of dynein by referring to the core sequence of viruses for binding with dynein. The basic structure of polypeptide design is: GK/C + + core sequence + cell-penetrating peptide, and preparing polypeptide sequence with purity of 95% by solid phase synthesis (general method).
TABLE 1 Polypeptides of the invention (core sequence)
Serial number Intracellular administration Extracellular administration Core sequence numbering
1 TILVSRSTQTGF TILVSRSTQTGF-Rn SEQ ID No.1
2 VKLVDAESQTL VKLVDAESQTL-Rn SEQ ID No.2
3 VQMAKSTQT VQMAKSTQT-Rn SEQ ID No.3
4 RSSEDKSTQTT RSSEDKSTQTT-Rn SEQ ID No.4
Note: rn represents n consecutive arginine residues (R), n being 6-9. Rn is the cell-penetrating peptide. The linker used for connecting the N-terminal of the core polypeptide with a drug or a carrier or a fluorescent group is GK; among them, the glycine residue (G) is a residue for reducing steric hindrance when the carrier is linked, and the lysine residue (K) is linked to TAMRA.
The polypeptide can realize the delivery of a drug carrier in 2 ways, namely, the polypeptide is connected with a drug to be delivered by a synthesis method, so that the delivery of the drug in cells or the permeation of biological barriers is realized; another way is to link the polypeptide to a drug delivery vehicle (e.g., nanoparticle, micelle, liposome, etc.) to achieve intracellular delivery of the drug delivery vehicle and a biological barrier.
Example 2 intracellular transport Capacity, cell proliferative toxicity and biological Barrier Permeability assays of the Polypeptides of the invention
This example will observe the intracellular behavior after the polypeptide N-terminal GK is linked with carboxytetramethylrhodamine in example 1, mainly relating to intracellular transport ability, cell proliferation toxicity and biological barrier permeability. Specifically, the polypeptide "administered extracellularly" in table 1 of example 1, where Rn is specifically 8R.
For each experiment, a control polypeptide (control) was set up, specifically "SLVSSDESVLHGSHESGEHV" replacing the core sequence in the polypeptide of example 1.
Synthesis and identification of polypeptide sequence
A solid phase synthesis method adopting a polypeptide synthesizer.
The specific synthesis method adopts Fmoc circulation method, and amino acids are added one by one according to designed sequence according to deprotection (removal of amino protecting group), activated crosslinking (peptide bond synthesis), elution and deprotection.
And product identification is carried out by comparing the molecular weights of the synthesized sequences by an HPLC-MS method.
The purity is determined by HPLC method, and the purity of the polypeptide is calculated by peak area integral ratio.
Second, cell staining method
1. Adopting bEnd.3 cells (mouse brain microvascular endothelial cells), inoculating the cells in a 24-well plate, wherein the initial inoculation density is 1 × 10 per well 5 A plurality of;
2、37℃，5％CO 2 allowing the cells to grow and adhere to the wall under the condition;
3. taking out the culture hole plate, removing the original culture solution by suction, and washing with 1ml PBS per hole for 2 times;
4. adding 10 mu M (system final concentration) of each polypeptide of the carboxyl tetramethyl rhodamine which is marked;
5、37℃，5％CO 2 incubating for 5-60 min;
6. discarding the incubation solution, washing with 1ml PBS per well for 2 times, and fixing with 4% paraformaldehyde for 10 min;
7. and (4) observing under a fluorescence microscope.
Cell proliferation toxicity method
1. Taking a bottle of T25 cultured bEnd.3 cells, digesting and counting, and fully dispersing the culture medium;
2. 2 pieces of 96-well plate are taken, and 1X 10 seeds are inoculated in each well 4 A plurality of;
3. after 48 hours of growth, the labeled polypeptide solution is added to make the final concentration reach 25, 50 and 100 mu M (the final concentration in the system after addition), each concentration is 3 holes, after incubation for 12h and 24h, 10 mu l of CCK-8 solution is added to each hole, after incubation for 1h in the incubator, the absorbance value at 450nm is read by an enzyme-labeling instrument.
Four, Transwell permeability experimental method
1. A24-well tranwell chamber was used and each well was seeded with bEnd.3 cells at 1X 10 5 At 37 ℃ with 5% CO 2 Culturing under the condition;
2. the transmembrane resistance is periodically detected until the TEER value reaches 60 omega cm 2 On the left and right, the TEER value was calculated as TEER (culture cell transmembrane resistance-blank cell transmembrane resistance) x cell basal area cm 2 ；
3. The liquid level difference can be maintained for more than 4 hours by carrying out a leakage experiment for 4 hours;
4. adding each polypeptide solution labeled with TAMRA with final concentration of 100 μ M into the upper chamber, sampling at 5 min, 10min and 30min, and reading fluorescence intensity with enzyme-labeling instrument;
5. and (3) calculating the permeability coefficient by the following formula: p app ＝dQ/dt×1/A×1/C 0 。
Fifth, result and analysis
Cell staining experiments showed that polypeptides nos. 1-4 in example 1 all achieved a broad intracellular distribution (fig. 1), whereas the control peptide (fig. 2) only resided on the cell surface. Among them, polypeptide No.1 showed more excellent intranuclear aggregation (FIG. 3), suggesting that polypeptide No.1 has a stronger intranuclear delivery ability.
In terms of cell proliferative toxicity, tests were carried out on the bEnd.3 cells by the CCK-8 method, and it was revealed that each of the polypeptides in example 1 had no significant difference in cell number from the control polypeptide at a concentration of 100. mu.M, and did not exhibit cell proliferative toxicity (Table 2), which is a good level of safety.
In the barrier permeability study, the blood-brain barrier model was constructed by transwell using the bEnd.3 cells, and each of the polypeptides in example 1 showed more than 2X 10 in most cases -6 The apparent permeability coefficient in cm/s (Table 3) indicates a good penetration of the biological barrier.
TABLE 2 cell proliferation toxicity test results
Figure BDA0002905249510000071
Note: "-" indicates p > 0.05, with no significant difference from Ctrl.
TABLE 3 results of apparent permeability coefficient experiments
5min(cm/s) 10min(cm/s) 30min(cm/s)
1 2.129×10 -5 2.767×10 -5 3.796×10 -5
2 5.654×10 -5 3.756×10 -5 8.896×10 -5
3 2.567×10 -5 2.678×10 -5 7.768×10 -5
4 6.324×10 -5 3.542×10 -7 2.879×10 -5
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
<110> military medical research institute of military science institute of people's liberation force of China
<120> dynein-binding peptides with biological barrier permeability and nuclear aggregation properties and uses thereof
<130> GNCLN210211
<160> 4
<170> PatentIn version 3.5
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Thr Ile Leu Val Ser Arg Ser Thr Gln Thr Gly Phe
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Val Lys Leu Val Asp Ala Glu Ser Gln Thr Leu
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Val Gln Met Ala Lys Ser Thr Gln Thr
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Arg Ser Ser Glu Asp Lys Ser Thr Gln Thr Thr
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Claims (10)

1. The polypeptide consists of a core region and a membrane-penetrating peptide from an N end to a C end in sequence; the amino acid sequence of the core region is SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 4.
2. The polypeptide of claim 1, wherein: the cell-penetrating peptide consists of 6-9 continuous arginine residues.
3. A polypeptide derivative obtained by attaching a linker to the N-terminus of the polypeptide of claim 1 or 2, wherein the linker can be used for attaching a carrier or a drug or a fluorophore.
4. The polypeptide derivative according to claim 3, characterized in that: the linker is one or several glycine residues, cysteine residues and/or lysine residues.
5. A transporter obtained by linking the polypeptide derivative of claim 3 or 4 to a carrier or a drug or a fluorophore by means of said linker.
6. Use of a polypeptide according to claim 1 or 2 or a derivative of a polypeptide according to claim 3 or 4 for the preparation of a transporter according to claim 5.
7. Use of a polypeptide according to claim 1 or 2 or a derivative of a polypeptide according to claim 3 or 4 for the preparation of a drug transporter with biological barrier permeability and/or nuclear aggregation properties.
8. Use of a polypeptide according to claim 1 or 2 or a derivative of a polypeptide according to claim 3 or 4 for the preparation of a formulation capable of improving the intracellular transport capacity of a macromolecule and/or a nanocarrier.
9. Use of a polypeptide according to claim 1 or 2 or a derivative of a polypeptide according to claim 3 or 4 for the manufacture of a formulation capable of improving the strength of the effect of an intracellularly acting drug.
10. Use of a polypeptide according to claim 1 or 2 or a derivative of a polypeptide according to claim 3 or 4 in the manufacture of a medicament for reducing or ameliorating multidrug resistance.
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