CN114805594B - Dynamic protein binding peptide capable of penetrating biological barrier and aggregating perinuclear and application thereof - Google Patents

Dynamic protein binding peptide capable of penetrating biological barrier and aggregating perinuclear and application thereof Download PDF

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CN114805594B
CN114805594B CN202110068300.3A CN202110068300A CN114805594B CN 114805594 B CN114805594 B CN 114805594B CN 202110068300 A CN202110068300 A CN 202110068300A CN 114805594 B CN114805594 B CN 114805594B
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polypeptide
seq
drug
intracellular
delivery
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CN114805594A (en
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郑爱萍
刘楠
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
    • C07—ORGANIC CHEMISTRY
    • 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
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • 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
    • 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
    • A61K47/64—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
    • A—HUMAN NECESSITIES
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

The invention discloses a motor protein binding peptide capable of penetrating biological barriers and aggregating perinucleuses and application thereof. The invention provides a dynamic protein binding peptide capable of penetrating biological barriers and gathering the periphery of a nucleus, which sequentially comprises 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 of the invention can realize the intracellular pericellular delivery of drugs/vectors, and has biological barrier permeability. Compared with the prior art, the invention can improve intracellular transport capacity of macromolecules and/or nano carriers, improve effect intensity of intracellular acting drugs, and reduce or improve multi-drug resistance (tumor multi-drug resistance).

Description

Dynamic protein binding peptide capable of penetrating biological barrier and aggregating perinuclear and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a motor protein binding peptide capable of penetrating a biological barrier and aggregating the perinucleuses 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 in 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 against mitochondria; lysosomal enzymes must reach lysosomal compartments etc. to exert a pharmaceutical effect. Intracellular transport of different bioactive molecules is often one of the key problems in drug delivery, for example, intracellular administration in tumor therapy can overcome important obstacles such as multi-drug resistance caused by P-glycoprotein in anticancer chemotherapy and the like. The lipophilicity of biological membranes limits the direct delivery of many compounds within cells. The cell membrane prevents macromolecules such as peptides, proteins and deoxyribonucleic acids from spontaneously entering the cell unless there is an active transport means, such as some short peptides, into the cell. In some cases, these molecules, even small particles, can enter the cell from the extracellular space through receptor-mediated endocytosis. However, this situation suffers from the problem that the molecules/particles that enter the cell via the endocytic pathway are often trapped in the endosome, ending up in the lysosome, where the active degradation process occurs under the action of the lysosomal enzyme. As a result, only a small fraction of unaffected material is present in the cytoplasm. This results in many compounds that show promising potential in vitro being unusable in vivo due to bioavailability problems. More successful attempts have been directed to introducing various macromolecular drugs and drug-loaded drug carriers into the cytoplasm, bypassing the endocytic pathway to protect the drug and DNA from lysosomal degradation, thereby increasing the efficiency of drug or DNA incorporation into the cell genome, but the methods described above are invasive in nature in cell experiments for microinjection or electroporation of delivery membrane impermeable molecules and may damage cell membranes. More effective is a non-invasive method such as using a ph sensitive carrier, including ph sensitive liposomes, to destabilize the membrane of phagocytic vesicles at low ph inside the endosome, releasing entrapped drug into the cytoplasm, and employing cell penetrating molecules (e.g., transmembrane peptides).
The above applications circumvent phagocytic digestion of cells (drugs are delivered into the cytoplasm) and 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, where viral vectors used for gene delivery have the inherent risks of non-specificity and virus-induced complications.
In order to avoid such problems of phagocytic digestion, there are studies on the ability of drug nanocarriers to perform multiple functions simultaneously or sequentially, such as specific recognition of target cells and endosomal escape, aiming at individual organelles, a very desirable feature. However, the specific subcellular delivery of bioactive molecules remains a challenging problem. One possible approach is to bind a drug molecule, or more preferably a drug-loaded drug nanocarrier, to another compound that has a specific affinity for the organelle of interest. Among the organelles of most interest for specific targeting, lysosomes and mitochondria can be mentioned. Thus, the use of lysosomal targeted drug nanocarriers can significantly improve delivery of therapeutic enzymes and partners into defective lysosomes for the treatment of lysosomal storage disorders, while specific delivery of certain drugs to mitochondria may be helpful in the treatment of various diseases, including neurodegenerative and neuromuscular diseases, obesity, diabetes, ischemia reperfusion injury, and cancer. However, delivery of all intracellular drug carriers, such as nuclear delivery or macromolecular drugs or carriers that require extensive intracellular distribution, cannot be achieved by this means of targeting the organelles.
Targeting vector delivery has the problem that delivery of macromolecular drugs in cells at the action target point is still problematic while delivery in whole cells is not achieved, and some studies have employed ways of reducing the stability of phagocytic vesicles to rupture phagocytic vesicles, but if the vector is loaded with macromolecular drugs, diffusion of the delivered drugs in cells still is problematic. All living cells are internally filled with macromolecules, which are very different in thermodynamics and kinetics of biological reactions in vivo and in vitro, and studies have shown that the "excluded volume effect" in the cytoplasm is insufficient to account for the large reduction in macromolecular diffusion observed in vivo, whereas hydrodynamic interactions greatly reduce the diffusivity of monodisperse colloids, especially in dense systems. The cytoplasm is crowded, the concentration of macromolecules can reach 300g/L, and the volume occupancy reaches 30%, the environment is greatly different from the dilution and idealization conditions commonly adopted in biophysical research, for example, in the cell nucleus, all DNA fragments are almost motionless, the highly restricted diffusion of DNA fragments in the cytoplasm is due to extensive binding to immovable obstacles, and the reduced lateral mobility of DNA >250bp in the cytoplasm is due to molecular crowding. Based on the crowded state of the macromolecules, the nano-drug carrier and linear macromolecules such as DNA are difficult to realize the transportation in the cells by means of simple diffusion, so that how to develop a way capable of actively transporting the carrier or the drug to realize the efficient transportation in the cells is very important.
The new intracellular/intercellular delivery mode is derived from the knowledge of the mechanism of entering and exiting cells after the virus infects cells, the particles with the virus size cannot realize the rapid infection in the cells by means of simple diffusion, and through the intensive research in the related field, the processes of entering and exiting cells when the virus infects the cells are found to be by means of intracellular dynamic proteins and kinesins, the dynamic proteins can realize the transportation of the virus from cell membranes to microtubule tissue centers (in the direction of cell nuclei), and the kinesins transport the virus from the cell nuclei to the cell membrane sides.
The dynamic protein is highly conserved in different organisms, and is used as a potential method for drug/carrier delivery, on one hand, the dynamic protein can realize wide delivery to the cell nucleus and the coverage area of the microtubules because of being transported from the positive electrode to the negative electrode along the microtubules (in the direction of the cell nucleus), on the other hand, the dynamic protein has various movement directions as a drug/carrier transport engine, and related researches show that the dynamic protein has a certain proportion of the possibility of turning, reversing, stagnating, dissociating and the like at the crossing position of the microtubules besides passing through the crossing point, and the characteristic provides possibility for the dynamic protein to be used as a drug delivery carrier so that the drug/carrier can be widely transported in cells and even realize transcellular transport. Meanwhile, the quasi-transport efficiency of the dynamic protein is very high, the movement speed of the dynamic protein in eukaryotic cells can reach 1-3 mu m/s, and the traction force can reach pN level.
There are 2 main attempts to deliver drugs using dynein, one is to construct a transcription expression system, prepare a portion of the dynein subunit, and achieve delivery by binding the subunit to the target cargo, and the study in this regard is in a stage of achieving DNA sequence transport and demonstrating higher transfection efficiency, and the other is to use a dynein binding peptide for related attempts, and further modification by a photodynamic binding peptide can achieve more functions. The fluorescent particles are modified on the surfaces of nano gold particles, fluorescent labeled polystyrene particles and PLGA nano particles, the above researches mainly comprise modification of fluorescence on the nano particles or sequences, observation of the transfer capacity of the sequences in cells after the sequences are combined with the dynein, and development of related researches, and observation of aggregation of the fluorescent particles to nuclear movement and transfer/delivery between cells (transcellular delivery). However, none of them has been advanced to the study and application of the combination of carrier and drug, and attempts have been made to develop the study of the permeation of the carrier through biological barriers.
Disclosure of Invention
Aiming at the problems that the macromolecular drugs/carriers cannot realize drug effect by means of diffusion due to the crowded state of the cytoplasm, and the drugs and the carriers need to cross a plurality of layers of cells in the process of crossing biological barriers, such as blood brain barriers, the drug/carrier permeation barriers still need to be further optimized and solved, the invention is realized by means of cell self-power proteins: (1) the macromolecular drugs/nano-carriers are rapidly transported in cells, so that the effect of the action target in the cells is realized; (2) drug/carrier permeation biological barrier issues; (3) drug core delivery.
In a first aspect, the invention claims a polypeptide.
The polypeptide of the invention comprises a core region (with the binding capacity of the dynein) and a membrane penetrating peptide from the N end to the 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.
Wherein the transmembrane peptide may consist of 6-9 consecutive arginine residues (R).
In a specific embodiment of the invention, the transmembrane peptide consists of in particular 8 consecutive arginine residues (R).
In a second aspect, the invention claims a polypeptide derivative.
The polypeptide derivatives claimed in the present invention are obtained after linking a linker to the N-terminus of the polypeptide of the first aspect, which linker can be used for linking a carrier or a drug or a fluorophore.
Further, the linker may be one or several glycine residues (G), cysteine residues (C) and/or lysine residues (K), etc.
In a specific embodiment of the invention, the linker is specifically 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 the polypeptide derivative according to the second aspect, which is obtained by linking the linker to a carrier or a drug or a fluorophore.
Wherein the carrier is a carrier for transporting medicines, such as nanoparticles, micelles, liposomes, vesicles and the like.
In a specific embodiment of the present invention, the linker (GK) is specifically carboxytetramethyl rhodamine (TAMRA). Wherein glycine residue (G) is a residue for reducing steric hindrance when linked to a carrier, and lysine residue (K) is linked to TAMRA.
In a fourth aspect, the invention claims the use of a polypeptide according to the first aspect of the invention or a polypeptide derivative according to the second aspect of the invention for the preparation of a transporter according to the third aspect of the invention.
In a fifth aspect, the invention claims the use of a polypeptide according to the first aspect of the foregoing or a polypeptide derivative according to the second aspect of the foregoing for the preparation of a drug transporter having biological barrier permeability and/or perinuclear aggregation properties.
In a sixth aspect, the invention claims the use of a polypeptide according to the first aspect of the foregoing or a polypeptide derivative according to the second aspect of the foregoing for the preparation of a formulation capable of improving the intracellular transport capacity of macromolecules and/or nanocarriers.
In a seventh aspect, the invention claims the use of a polypeptide according to the first aspect of the invention or a polypeptide derivative according to the second aspect of the invention for the preparation of a formulation capable of improving the potency of an intracellular acting drug.
In an eighth aspect, the invention claims the use of a polypeptide according to the first aspect or a polypeptide derivative according to the second aspect of the invention for the preparation of a formulation capable of reducing or ameliorating multi-drug resistance (tumor multi-drug resistance) due to efflux factors such as P-glycoprotein.
In a specific embodiment of the invention, the biological barrier is in particular the blood brain barrier.
In order to improve the delivery efficiency of intracellular drugs/vectors, the invention designs a series of polypeptides with the binding capacity of the dynein by referring to the core sequence of the binding of the virus and the dynein, and experiments prove that the polypeptides can realize the pericellular delivery and have biological barrier permeability. Compared with the prior art, the invention can improve intracellular transport capacity of macromolecular drugs (such as polypeptides, DNA, RNA and the like) and/or nano-carriers, improve effect intensity of intracellular acting drugs and reduce or improve multi-drug resistance (tumor multi-drug resistance) through comparing with control polypeptides.
Drawings
FIG. 1 shows the intracellular fluorescence behavior of each polypeptide.
FIG. 2 is the intracellular fluorescence behavior of the control polypeptides.
FIG. 3 shows the intracellular specific behavior (perinuclear aggregation) of polypeptide No. 1.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 design of a motor protein binding peptide with biological Barrier permeance and Nuclear aggregation Properties
In order to increase the delivery efficiency of intracellular drugs/vectors, the present invention designs a series of polypeptides with the ability to bind to the kinesin protein with reference to the core sequence of the virus binding to the kinesin protein. The basic structure of the polypeptide design is as follows: GK/C+core sequence+membrane penetrating peptide, and solid phase synthesis (general method) is adopted to prepare the polypeptide sequence with the purity of 95%.
TABLE 1 Polypeptides of the invention (core sequence)
Sequence number Intracellular administration Extracellular administration Core sequence numbering
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
Note that: rn represents n consecutive arginine residues (R), n=6-9. Rn is the penetrating peptide. The linker of the N end of the core polypeptide is used for being connected with a drug or a carrier or a fluorescent group, and is specifically GK; wherein glycine residue (G) is a residue for reducing steric hindrance when linked to a carrier, and lysine residue (K) is linked to TAMRA.
The polypeptide can realize the delivery of the drug carrier in 2 modes, firstly, the polypeptide is connected with the drug to be delivered through a synthesis method, so that the drug is delivered in cells or permeates biological barriers; another approach is to achieve intracellular delivery of the drug carrier as well as a biological barrier by linking the polypeptide to the drug delivery carrier (e.g., nanoparticles, micelles, liposomes, etc.).
Example 2 identification of intracellular transport Capacity, cell proliferation toxicity and biological Barrier permeabilities of the Polypeptides of the invention
This example will observe intracellular behavior by linking the GK at the N-terminus of the polypeptide of example 1 with carboxytetramethyl rhodamine (TAMRA), mainly concerning intracellular transport capacity, cell proliferative toxicity and biological barrier permeability. Specifically, the "extracellular administration" polypeptides in Table 1 of example 1 were used, and Rn was specifically 8R.
For each experiment a control polypeptide (control) was set up, in particular the core sequence of the polypeptide of example 1 was replaced by "SLVSSDESVLHGSHESGEHV".
1. Synthesis and identification of polypeptide sequences
A solid phase synthesis method using a polypeptide synthesizer.
The specific synthesis method adopts Fmoc circulation method, and amino acids are added one by one according to the designed sequence according to deprotection (removing amino protecting groups), activation crosslinking (peptide bond synthesis), elution and deprotection.
The molecular weight of the synthetic sequence is compared by HPLC-MS method for product identification.
Purity determination the purity of the polypeptide was calculated by the peak area integral ratio using HPLC method.
2. Cell staining method
1. bEnd.3 cells (mouse brain microvascular endothelial cells) were used and seeded in 24-well plates at an initial density of 1X 10 per well 5 A plurality of;
2、37℃，5％CO 2 the cell is grown and attached under the condition;
3. taking out the culture pore plate, sucking and discarding the original culture solution, and washing 1ml PBS (phosphate buffered saline) in each pore for 2 times;
4. adding 10 mu M (final system concentration) of each polypeptide of the labeled carboxyl tetramethyl rhodamine;
5、37℃，5％CO 2 incubating for 5-60min under the condition;
6. discarding the incubation liquid, washing with 1ml PBS for 2 times per well, and fixing with 4% paraformaldehyde for 10min;
7. and observing under a fluorescence microscope.
3. Cell proliferation toxicity method
1. Taking a T25 bottle to culture bEnd.3 cells, performing digestion counting, and fully dispersing the culture medium;
2. taking 2 96-well plates, inoculating 1×10 each 4 A plurality of;
3. after 48 hours of growth, the labeled polypeptide solution was added so that the final concentration reached 25, 50, 100. Mu.M (to the final concentration in the system after addition), each concentration was 3 wells, after incubation for 12h and 24h, respectively, 10. Mu.l of CCK-8 solution was added to each well, and after continued incubation in the incubator for 1h the microplate reader read the absorbance at 450 nm.
4. Transwell permeability experiment method
1. 24-well tranwell cells were taken and plated with bEnd.3 cells 1X 10 per well 5 And 5% CO at 37 ℃C 2 Culturing under the condition;
2. the transmembrane resistance was periodically checked until the TEER value reached 60. Omega. Cm 2 About, TEER value calculation formula teer= (culture cell transmembrane resistance-blank cell transmembrane resistance) ×cell bottom area cm 2 ；
3. 4 hours of leakage experiments are carried out, and the liquid level difference can be maintained for more than 4 hours;
4. adding each polypeptide solution of the label TAMRA with the final concentration of 100 mu M into the upper chamber, sampling for 5, 10 and 30min respectively, and reading the fluorescence intensity by using an enzyme-labeling instrument;
5. calculating the permeability coefficient, and calculating the formula: p (P) app ＝dQ/dt×1/A×1/C 0 。
5. Results and analysis
Cell staining experiments showed that the polypeptides 1-4 of example 1 all achieved a broad intracellular distribution (FIG. 1), whereas the control peptide (FIG. 2) was only resident on the cell surface. Wherein polypeptide No.1 exhibited better perinuclear aggregation (fig. 3), suggesting that polypeptide No.1 has greater perinuclear delivery capacity.
In terms of cell proliferation toxicity, the bEnd.3 cells were tested using the CCK-8 method, which showed no significant difference in cell numbers between each of the polypeptides of example 1 and the control polypeptide at a concentration of 100. Mu.M, and no cell proliferation toxicity was shown (Table 2), which was safe at this level.
In the transwell, blood brain barrier model was constructed using bEnd.3 cells, each of the polypeptides in example 1 was shown to be greater than 2X 10 -6 The apparent permeability coefficient in cm/s (Table 3) demonstrates that the biological barrier is well permeable.
TABLE 2 results of cell proliferation toxicity experiments
Note that: ". Times." indicates that p > 0.05, no significant difference from Ctrl.
TABLE 3 apparent osmotic coefficient test results
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
The present invention is described in detail above. It will be apparent to those skilled in the art that the present 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 respect to specific embodiments, it will be appreciated that the invention may 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 application of some of the basic features may be done in accordance with the scope of the claims that follow.
<110> military medical institute of the military academy of China's civil liberation army
<120> a motor protein-binding peptide capable of penetrating biological barrier and aggregating perinuclear and use thereof
<130> GNCLN210210
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<170> PatentIn version 3.5
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Pro Lys Thr Arg Asn Ser Gln Thr Gln Thr Asp
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Claims (1)

1. Use of a polypeptide or polypeptide derivative for the preparation of a drug transporter having blood brain barrier permeability; the polypeptide sequentially comprises 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 membrane penetrating peptide consists of 6-9 continuous arginine residues; the polypeptide derivative is obtained by connecting a linker with the N-terminal of the polypeptide, wherein the linker can be used for connecting a carrier or a drug or a fluorescent group, and the linker is one or a plurality of glycine residues, cysteine residues and/or lysine residues; the transporter is obtained by connecting a polypeptide derivative with a carrier or a drug or a fluorescent group by means of the linker.
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PCT/CN2022/070230 WO2022156531A1 (en) 2021-01-19 2022-01-05 Dynein binding peptide capable of permeating through biological barrier, and use thereof

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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