CN109553659B

CN109553659B - Cell penetrating peptide and transdermal interferon

Info

Publication number: CN109553659B
Application number: CN201811418006.5A
Authority: CN
Inventors: 李冠英; 张德宝; 李海红; 刘惠
Original assignee: SHANGHAI HUAXIN HIGH BIOTECHNOLOGY Inc
Current assignee: SHANGHAI HUAXIN HIGH BIOTECHNOLOGY Inc
Priority date: 2018-11-26
Filing date: 2018-11-26
Publication date: 2022-06-21
Anticipated expiration: 2038-11-26
Also published as: CN109553659A

Abstract

The invention belongs to the field of biological medicines, and discloses a cell penetrating peptide and a transdermal interferon. The cell penetrating peptide provided by the invention is a peptide for mediating and delivering bioactive molecules into cells, and the amino acid sequence of the cell penetrating peptide comprises a sequence shown in SEQ ID NO. 1; wherein the amino acid at position 3, 8 and/or 11 from the N-terminus may be replaced with another amino acid. The cell penetrating peptide has high transdermal activity, can be used as a carrier to carry target substances (such as protein, DNA, siRNA and the like) to permeate through the epidermis layer of the skin, and has wide application; the transdermal interferon formed by fusing the cell penetrating peptide and the interferon is easier to be absorbed through skin and can exert the effect more quickly.

Description

Cell penetrating peptide and transdermal interferon

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a cell penetrating peptide and a transdermal interferon.

Background

Transdermal drug delivery refers to a drug delivery system in which a drug, after being delivered through the skin surface, penetrates the stratum corneum of the skin and enters the capillaries to enter the blood circulation, thereby performing systemic therapeutic action. As a special administration route, compared with the traditional administration mode of oral administration and injection, the method has the unique advantages that: the first pass effect of the liver and the inactivation of the medicine in the gastrointestinal tract can be avoided, better medicine effect can be achieved, and the individual difference of medicine application is reduced; the constant and effective blood concentration is maintained, the peak-valley phenomenon of the blood concentration is avoided, and the toxic and side effects are reduced; the action time of the medicine is prolonged, the administration times are reduced, and most patients are easy to accept; has no pain, is convenient to use, and can be independently used by a patient or withdrawn at any time.

During transdermal drug absorption, the drug needs to diffuse across the skin's physical barrier into the blood circulation and ultimately into the targeted tissue. Many internal and external factors in this process affect the transdermal process of the drug. These factors include the condition of the skin itself, the physicochemical properties of the drug, the formulation of the preparation, etc. The physicochemical properties of the drug directly affect the absorption rate of the drug in transdermal administration. The dosage form and formulation composition of the formulation also have a direct effect on the transdermal delivery efficiency. The condition of the skin is also an important factor affecting the transdermal absorption of the drug when the drug is administered transdermally, and the site of the skin, the degree of damage, the thickness, the pore distribution, the temperature and humidity, the degree of cleanliness, and the like all affect the transdermal absorption efficiency of the drug.

Therefore, enhanced transdermal drug delivery has been gradually applied in clinical treatment, and most of them focus on the use of chemical transdermal enhancers and physical assistance.

The cell membrane is a semipermeable barrier between the cell and the extracellular environment, and has a selective permeation function so as to maintain the constant internal environment of the cell. While this phospholipid bilayer is essential for cell survival and function, it presents challenges to the exchange of cargo molecules inside and outside the cell. Since macromolecular substances and imaging agents of drugs such as proteins, polypeptides, nucleotides and the like must reach the inside of cells to exert corresponding effects, transmembrane transport of the substances becomes necessary.

Currently, there is a class of biological polypeptides that rapidly penetrate into mammalian cells and still retain their original structure and function, but which do not rely on endocytosis for entry into the cell, such polypeptides being referred to as cell-penetrating peptides (CPPs). Such polypeptides are rich in basic amino acids and therefore are generally positively charged. CPP can not only penetrate cells by itself, but also load other substances and promote the cell penetration of the substances, such as protein, DNA, siRNA, liposome, nano material and the like. Cell-penetrating peptides also have penetrating power to epidermal cells of animals, and thus in recent years, cell-penetrating peptides have been used as transdermal enhancers for transdermal administration of macromolecular drugs. The discovery of such agents provides a convenient route to transdermal delivery techniques.

Interferons (IFNs) are a class of cytokines secreted by recipient cells after human and animal cells have been infected with viruses or induced by various factors such as nucleic acids, bacterial endotoxins, mitogens, and the like. Antiviral activity is one of the basic functions of the interferon family. Interferons exert their antiviral action primarily through body cells and do not themselves kill or inhibit viruses. Viral infection of cells induces production and release of IFN, followed by death and disintegration of the infected cells, and the IFN molecules are released and spread throughout the body with blood circulation. IFN molecules bind specifically to receptors on the surrounding cell membrane, enhancing the activity of cellular membrane adenylate cyclase, and promoting the formation of cyclic adenosine monophosphate. The increase of cyclic adenosine monophosphate can inhibit DNA synthesis, cell division and the like, and further inhibit virus replication. The transdermal administration of the interferon is researched, so that the compliance of patients is improved in the aspect of treating viral infections such as herpes virus, HPV and the like.

The present invention has been made in view of this situation.

Disclosure of Invention

The cell penetrating peptide has high transdermal activity, can be used as a carrier to carry target substances (such as protein, DNA, siRNA and the like) to permeate through a skin epidermal layer, and has wide application; the transdermal interferon formed by fusing the cell penetrating peptide and the interferon is easier to be absorbed transdermally, and is beneficial to more quickly exerting the effect.

In order to solve the technical problems, the invention adopts the technical scheme that:

the first object of the present invention is to provide a cell-penetrating peptide, which is a peptide mediating the delivery of a bioactive molecule into a cell, the amino acid sequence of the cell-penetrating peptide comprising the sequence shown in SEQ ID NO. 1;

alternatively, the amino acid at position 3, and/or position 8, and/or position 11 from the N-terminus is replaced with another amino acid.

The invention adopts phage in vivo display technology, and through multiple rounds of screening, the amino acid sequence of the cell penetrating peptide obtained by screening comprises 15 amino acids, such as the sequence shown in SEQ ID NO. 1, namely RKTWLRRLARRPQLK.

The applicant has found that the sequence shown in SEQ ID NO. 1 has various choices from the 3 rd position, the 8 th position and the 11 th position from the N-terminal, and when the amino acids are replaced respectively, or two of the amino acids are replaced, or other amino acids are replaced simultaneously, the obtained cell penetrating peptide can also have high penetrability. The amino acid sequence of the thus selected cell-penetrating peptide can be represented as RKX₁WLRR X₂AR X₃PQLK, wherein X₁、X₂、X₃May be as shown in SEQ ID NO. 1, or may be substituted with other amino acids, either individually or simultaneously. The applicant finds that the cell penetrating peptide obtained by screening has high transdermal activity, can be used as a carrier to carry target substances (such as protein, DNA, siRNA and the like) to permeate through the epidermal layer of the skin, and has wide application.

In a further embodiment, the sequence of the cell-penetrating peptide is substituted from the amino acid at position 3 from the N-terminus to S or A or R;

preferably, in the amino acid sequence, the amino acid at position 3 from the N-terminus is replaced with S.

In a further embodiment, the sequence of the cell-penetrating peptide is substituted by I or V from the amino acid at position 8 of the N-terminus;

preferably, in the amino acid sequence, the amino acid at position 3 from the N-terminus is replaced with I.

In a further embodiment, the sequence of the cell-penetrating peptide is substituted by K, H or N at the 11 th amino acid from the N-terminus;

preferably, in the amino acid sequence, the amino acid at position 11 from the N-terminus is replaced with K.

In the cell-penetrating peptide described above, the combination of the amino acid sequences includes a plurality of amino acids, and the amino acids at

positions

3, 8 and 11 may be replaced individually, or two of them may be replaced, or they may be replaced simultaneously.

It is a second object of the present invention to provide a nucleotide sequence encoding the cell penetrating peptide as described above.

The nucleotide sequence of the cell-penetrating peptide referred to in this scheme is any sequence that can encode any of the amino acid sequences of the cell-penetrating peptides described above.

It is a third object of the present invention to provide a vector, a recombinant bacterium or a recombinant cell of the nucleotide sequence as described above.

The vector in the scheme can be an expression vector, a shuttle vector, an integration vector and the like, the recombinant bacteria can be bacteria and fungi, and the recombinant cells can be viruses and animal cells and the like.

It is a fourth object of the present invention to provide a use of the cell-penetrating peptide as described above as an intracellular delivery vehicle;

preferably, the use of said cell penetrating peptide as a carrier for intracellular delivery of a therapeutic agent.

Specifically, the cell penetrating peptide screened by the invention is used as a carrier for carrying a drug molecule, and the carried drug molecule is delivered to the cytoplasm and/or nucleus of a target cell. The drug may include antiviral infection drug, antitumor drug, cytotoxic drug, biomembrane damaging drug, gene drug, neurotrophic molecule, photosensitive drug, stem cell regulatory factor, etc.

It is a fifth object of the present invention to provide a complex comprising a cell penetrating peptide as described above and a cargo molecule;

preferably, the cell penetrating peptide and the cargo molecule are coupled to each other;

preferably, the cargo molecule is coupled to the end of the cell penetrating peptide.

In this embodiment, the cargo molecule is a macromolecule that needs to enter the cell interior by using a cell-penetrating peptide as a carrier, and the macromolecule is preferably, but not limited to, at least one of a molecule with pharmaceutical activity, a molecule with labeling effect, and a molecule with targeting effect.

The coupling may be by covalent or non-covalent linkage.

The sixth object of the present invention is to provide a transdermal interferon comprising a fusion protein of the cell-penetrating peptide as described above linked to the N-terminus of interferon and interferon.

The screened cell penetrating peptide is connected to the N end of the interferon, the cell penetrating peptide-interferon fusion protein is constructed on the carrier to express the cell penetrating peptide-interferon fusion protein, and the obtained fusion protein has the activity of the interferon, has the capability of penetrating the skin, has good patient compliance and can improve the treatment effect in the aspect of virus infection.

In a further embodiment, the interferon is selected from alpha interferon, beta interferon or gamma interferon and homologous type interferon;

preferably, the interferon is interferon alpha-2 b.

It is a seventh object of the present invention to provide a method for preparing transdermal interferon as described above, comprising the steps of:

(1) connecting the cell penetrating peptide to the N end of the interferon to construct a recombinant vector with a cell penetrating peptide-interferon fusion protein sequence;

(2) transferring the recombinant vector into a target strain, and screening to obtain a recombinant strain;

(3) and (3) fermenting and culturing the engineering bacteria, collecting bacteria, cracking and purifying to obtain the transdermal interferon.

After adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects:

1. the cell penetrating peptide is easy to prepare, has small molecular weight, is not easy to generate immunological rejection, has strong penetrating power and higher transdermal activity, can be used as a carrier to carry target substances (such as protein, DNA, siRNA and the like) to penetrate through the epidermis layer of the skin, and has wide application.

2. The transdermal interferon formed by fusing the cell penetrating peptide and the interferon has the activity of the interferon, has the capability of penetrating the skin, is easier to be absorbed through the skin, can exert the effect more quickly, has good patient compliance, and can improve the treatment effect in the aspect of virus infection.

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention, are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention without limiting the invention to the right. It is obvious that the drawings in the following description are only some embodiments, and that for a person skilled in the art, other drawings can be derived from them without inventive effort. In the drawings:

FIG. 1 is the secondary structure and physicochemical property analysis of the cell-penetrating peptide CPP of the present invention;

FIG. 2 is an in vivo transdermal assay of the CPP-IFN α -2b of the invention;

FIG. 3 is an in vivo transdermal comparison experiment of CPP-IFN alpha-2 b of the present invention with PD-1-IFN alpha-2 b and PEP-1-IFN alpha-2 b.

It should be noted that the drawings and the description are not intended to limit the scope of the inventive concept in any way, but to illustrate it by a person skilled in the art with reference to specific embodiments.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and the following embodiments are used for illustrating the present invention and are not intended to limit the scope of the present invention.

Example one

1. Screening of CPP

Screening CPP capable of penetrating skin by using phage in vivo display technology. The specific method is that,

1) the phage peptide library is smeared on the surface of the skin of a nude mouse in a transdermal drug delivery mode;

2) after a period of transdermal time, blood was drawn from the rat;

3) adding escherichia coli into blood, infecting the escherichia coli with the phage, and then culturing;

4) finally, counting plaques, and calculating the titer of the phage.

The first round of screening is performed according to the above steps, and usually many nonspecific phages or phages with weak transdermal capacity are screened in the first round of screening, and the number of the phages is large. Therefore, repeated screening is required. By repeating the above method. The phage with certain purity is enriched through 3-4 rounds of screening.

Then, the DNA sequence of the polypeptide carried by the phage is sequenced by extracting the phage, so that the polypeptide sequence is obtained.

Through the steps, a CPP polypeptide sequence is screened out as follows:

RKTWLRRLARRPQLK

or other embodiments, wherein amino acid T at position 3 can be amino acid S or A or R; wherein amino acid L at position 8 can be amino acid I or V; wherein the 11 th amino acid R can be the amino acid K or H or N.

Wherein, one, two or 3 amino acids can be substituted at 3 positions.

2. CPP physical and chemical property analysis

The physical and chemical property analysis of CPP sequence (RKTWLRRLARRPQLK) by using tool ProtParam shows that the molecular weight is 2.0KD, the isoelectric point PI is higher than 12.6, and the hydrophilicity is stronger (GRAVY: -1.59); the secondary structure and physicochemical properties of the CPP sequence (RKTWLRRLARRPQLK) were analyzed by Protean software, and the result is shown in FIG. 1, the sequence is in alpha-helix structure, and all amino acids are hydrophilic to different degrees.

The sequence of CPP with amino acid substitutions at position 3, and/or position 8, and/or position 11 was analyzed by the same method and the results were similar to RKTWLRRLARRPQLK.

Example two

Obtaining the fusion protein CPP-IFN alpha-2 b

(1) Construction of engineering bacteria

Splicing the selected cpp with IFN alpha-2 b gene sequence (cpp-IFN alpha-2 b) to carry out whole gene synthesis. Wherein the nucleotide sequence of Cpp encodes amino acid sequence RKTWLRRLARRPQLK linked to the N-stretch of IFN α -2 b. The nucleotide sequence of cpp-IFN alpha-2 b is shown in SEQ ID NO. 2, and the amino acid sequence is shown in SEQ ID NO. 3.

The cpp-IFN alpha-2 b gene segment is constructed on a prokaryotic soluble expression vector PHX53 of the company, competent cells BL21(DE3) are transformed, and the genetic engineering bacteria containing PHX53-cpp-IFN alpha-2 b are obtained by enzyme digestion, sequencing and screening.

(2) Engineering bacteria for fermentation culture

Inoculating the obtained genetically engineered bacteria into 2 XYT culture medium, and culturing at 37 deg.C in 15L fermentation tank;

and (3) fermentation process: regulating rotation speed to control dissolved oxygen value at 50-65%, and controlling pH at 6.0-7.5 with ammonia water; when OD600 reaches 0.8, adding IPTG (isopropyl thiogalactoside) with sterilization and filtration to a working concentration of 1mM, and continuing to perform induction culture at 25 ℃ for 6 hours; placing the mixture in a tank at room temperature, and collecting thalli by tubular centrifugation at 12000 rpm; the thalli is washed and centrifuged twice by pure water to remove impurities in the fermentation liquor.

The thalli is prepared according to the following steps of 1: 20(m/v), suspended in a lysis solution (20mMPB,5mmol/L EDTA, 0.1% Triton-x100, pH6.0), placed on a mixture of ice and water and sonicated; centrifuged at 12000rpm for 30 minutes, and the supernatant was collected.

(3) Chromatographic purification

And loading the cell lysate onto a CM column well balanced by an equilibrium solution (50mM Tris-HCL, pH8.0), eluting the CM column by 5 column volumes by the equilibrium solution (50mM Tris-HCL, pH8.0), carrying out gradient elution on an eluent (50mM Tris-HCL, pH 9.5), and collecting an elution peak.

Adjusting the pH of the protein collected by the CM column to 8.0, loading the protein onto DEAE (DEAE) balanced by a balancing solution (50mM Tris-HCL, pH8.0), collecting a breakthrough peak, continuously leaching the protein with the balancing solution (50mM Tris-HCL, pH8.0) after loading, and continuously collecting the breakthrough peak. The collected protein is stored at-20 ℃ for later use.

Test example 1

Detection of fusion protein CPP-IFN alpha-2 b activity

The biological activity of the interferon fusion was measured by the "interferon activity assay" described in pharmacopoeia of the people's republic of China 2015 edition (third). The results showed that the fusion protein CPP-IFN alpha-2 b, has 1.43X 10⁸Ratio of (A to B)And (4) activity.

Test example two

Fusion protein CPP-IFN alpha-2 b transdermal activity detection

In vivo experiments were performed to verify whether CPP-IFN α -2b was able to penetrate rat skin into the body. The method comprises the following steps:

1) 100ul of CPP-IFN alpha-2 b with the concentration of 0.5mg/ml is taken and smeared on the exposed skin surface of the abdomen of a rat (so that the rat is in an anesthesia state and the rat is prevented from rubbing off the medicine) to be used as an experimental group. 100ul IFN alpha-2 b with concentration of 0.5mg/ml was applied to the exposed skin surface of the abdomen of the rat as a control group. At 0min, 30min, 60min and 90min respectively, 200ul of blood is extracted from tail vein of rat, serum is obtained after centrifugation, and the content of CPP-IFN alpha-2 b in the serum is detected by ELISA technology.

The results show (as in figure 2), in 30 minutes, IFN alpha-2 b can hardly be detected; whereas CPP-IFN alpha-2 b penetrated 180pg, after 60 minutes IFN alpha-2 b only detected 5pg penetration through the skin; whereas the CPP-IFN alpha-2 b permeability has been increased to 410 pg. After 90 minutes, IFN alpha-2 b, although the transdermal volume is increased, also only at 8pg, still very low; the transdermal amount of CPP-IFN alpha-2 b reaches 420pg, at which point the drug may reach a balance of permeation-clearance.

Through in vivo experiments, we have demonstrated the transdermal capacity of CPP-IFN alpha-2 b. Further, the CPP provided by the invention has the capability of carrying protein to penetrate the skin. The results are shown in FIG. 2.

Using the same method, the sequence of CPP with amino acid substitutions at position 3, and/or position 8, and/or position 11 was tested for fusion protein with IFN α -2b, and the result was similar to RKTWLRRLARRPQLK, with the ability of the carrier protein to penetrate the skin.

Test example three

To further demonstrate the advancement of the transdermal capacity of the cell-penetrating peptide CPP of the present invention, we prepared fusion proteins PD-1-IFN α -2b and PEP-1-IFN α -2b separately for transdermal activity assays for comparison with currently used cell-penetrating peptides PD-1 and PEP-1. See test example two for experimental methods.

The results show (as in FIG. 3), that after 60 minutes of transdermal experiment, the transdermal amounts of CPP-IFN alpha-2 b (CPP sequence is RKTWLRRLARRPQLK), PD-1-IFN alpha-2 b and PEP-1-IFN alpha-2 b all reached the maximum concentration, and the transdermal capacity of CPP-IFN alpha-2 b in the experimental group was higher than that in the other two groups throughout the experimental period.

The results show that the cell penetrating peptide CPP carrying IFN alpha-2 b not only has transdermal capacity, but also has better effect than the current commonly used cell penetrating peptides PD-1 and PEP-1.

The test of the fusion protein with IFN alpha-2 b is carried out on the CPP sequence with the amino acid substitution at the 3 rd position, the 8 th position and the 11 th position by the same method, and the result is similar to RKTWLRRLARRPQLK, and the transdermal effect is superior to that of the currently common cell penetrating peptides PD-1 and PEP-1.

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A cell penetrating peptide, wherein the cell penetrating peptide is a peptide that mediates delivery of a bioactive molecule into a cell, and the amino acid sequence of the cell penetrating peptide is represented by SEQ ID NO. 1.

2. A gene encoding the cell penetrating peptide of claim 1.

3. A vector, recombinant bacterium or recombinant cell comprising the gene of claim 2.

4. Use of the cell penetrating peptide of claim 1 as an intracellular delivery vehicle.

5. Use according to claim 4, wherein said cell penetrating peptide is used as a carrier for intracellular delivery of a therapeutic agent.

6. A complex comprising the cell penetrating peptide of claim 1 and a cargo molecule.

7. The complex of claim 6, wherein the cell penetrating peptide and the cargo molecule are coupled to each other.

8. The complex of claim 7, wherein the cargo molecule is coupled to the terminus of the cell penetrating peptide.

9. A transdermal interferon comprising a fusion protein of the cell-penetrating peptide of claim 1 and an interferon, wherein the cell-penetrating peptide is linked to the N-terminus of the interferon.

10. The transdermal interferon of claim 9, wherein the interferon is selected from alpha interferon, beta interferon, or gamma interferon.

11. The transdermal interferon of claim 10, wherein the interferon is interferon alpha-2 b.

12. A method of preparing the transdermal interferon of claim 9, comprising the steps of:

(3) and (3) carrying out fermentation culture on the recombinant bacteria, collecting bacteria, cracking and purifying to obtain the transdermal interferon.