CN110078834B - Short-like peptide, auxiliary membrane penetrating agent and application thereof - Google Patents

Short-like peptide, auxiliary membrane penetrating agent and application thereof Download PDF

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CN110078834B
CN110078834B CN201910367417.4A CN201910367417A CN110078834B CN 110078834 B CN110078834 B CN 110078834B CN 201910367417 A CN201910367417 A CN 201910367417A CN 110078834 B CN110078834 B CN 110078834B
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CN110078834A (en
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张玉慧
张朦
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Huazhong University of Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • 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

Abstract

The invention discloses a short peptide-like, an auxiliary membrane penetrating agent and application thereof. The short peptide-like is a plurality of coupled electropositive short peptides, and the electropositive short peptides comprise lysine residues modified by positively charged groups and membrane-penetrating peptide segments; preferably, the short-like peptide is 2 coupled electropositive peptide fragments. The auxiliary membrane penetration agent comprises the short peptide. The auxiliary membrane penetrating agent is applied to the intracellular delivery of living cells of the mediated small-molecule organic fluorescent probe, is simple to use, only needs co-incubation operation and is low in cost; the membrane penetration effect is good, the action time is short, and the operation time can be effectively reduced.

Description

Short-like peptide, auxiliary membrane penetrating agent and application thereof
Technical Field
The invention belongs to the field of fluorescence imaging, and particularly relates to a short peptide-like auxiliary membrane penetrating agent and application thereof.
Background
There are two broad categories of current strategies for fluorescence labeling and microscopic imaging of biological macromolecules within living cells: first, fluorescent protein labeling. Specifically, a specific fluorescent protein is expressed at a target site through a plasmid transfection mode, and although the mode has good living cell compatibility, the problems of overlarge volume, low quantum yield, poor light stability and the like of the exogenous fluorescent protein are generally required to be overcome.
With the development of super-resolution microscopic imaging technology, research on basic structures in living cells is also developing towards ultrahigh resolution. In the existing super-resolution imaging strategy, the super-resolution microscopic imaging strategy based on light modulation requires that fluorescent molecules have the properties of high brightness, photobleaching resistance and the like so as to obtain better time and space resolution; based on such imaging requirements, fluorescent proteins are often not satisfactory, which requires a second labeling strategy: by small molecule organic fluorescent dyes. Compared with fluorescent protein, the organic fluorescent dye has higher brightness and better stability, and the targeting property of the marker is realized by forming an organic fluorescent probe through covalent connection with a specific recognition group. However, the application of the small-molecule organic fluorescent dye still has a problem: most dyes with excellent fluorescence performance cannot permeate cell membranes, some dyes which can permeate the membranes can lose the membrane permeability after being combined with a targeted recognition group, cannot well penetrate cell membrane barriers, and can only be used for dead cells or structural markers outside the live cell membranes but not for structural markers inside the live cells.
At present, the following approaches are mainly used for solving the problem of membrane permeability: first, chemical modification on fluorescent dye molecules or de novo design of new fluorescent dyes are performed, but this method is time and labor consuming and has low success rate. Second, electroporation is used in which a small hole or opening is temporarily formed in the cell membrane after a few microseconds to a few milliseconds by applying an electric field to the cell, and a membrane-impermeable fluorescent probe in the cell culture fluid is brought into contact with the cell membrane by the action of an electrophoretic force and is diffused into the cytoplasm. However, this method requires expensive equipment (electroporator) and the applied field strength can irreversibly damage the cell membrane to lyse a part of the cells, so the method is more harmful to the cells and the number of cells labeled at one time is small. Thirdly, the penetrating membrane of the small molecular fluorescent dye is realized by using a peptide probe which covalently combines the penetrating membrane peptide, the target recognition molecule and the fluorescent dye together. However, this method has a limitation that it is designed from the beginning only for a part of structures in living cells and when the structures are changed.
Disclosure of Invention
Aiming at the defects or improvement requirements of the prior art, the invention provides a short-like peptide, an auxiliary membrane permeation agent and application thereof, and aims to solve the technical problem that the existing molecular probe is difficult to penetrate a cell membrane or has poor fluorescence labeling effect by properly designing the molecular structure and the electrical property of the short-like peptide to enable the short-like peptide to have a mechanism of triggering endocytosis escape and assisting reagents such as a small-molecule fluorescent probe and the like to realize membrane permeation into a cell.
To achieve the above object, according to one aspect of the present invention, there is provided a short peptide-like molecule comprising a plurality of coupled electropositive short peptides, wherein the electropositive short peptides comprise a lysine residue modified with a positively charged group and a membrane-penetrating peptide segment; preferably, the short-like peptide is 2 coupled electropositive peptide fragments.
Preferably, the short-like peptide, wherein the plurality of electropositive short peptides are coupled by disulfide bonds.
Preferably, the short-like peptide has the following structure:
Figure BDA0002048656940000021
wherein K is lysine; c is cysteine; r1 is a positively charged group; CPP is cell penetrating peptide segment.
Preferably, the peptide-like short peptide has a positive charge group of rhodamine, preferably rhodamine B or rhodamine 6G.
Preferably, the cell-penetrating peptide segment of the short peptide-like is a primary structure cell-penetrating peptide, contains 4 to 40 amino acid residues and has positive charge.
Preferably, the short-like peptide, the cell-penetrating peptide segment TAT and/or (rR)3R2。
According to another aspect of the present invention, there is provided an auxiliary membrane-penetrating agent comprising said short-like peptide, preferably at a concentration of between 200 μ M and 2 mM.
Preferably, the auxiliary membrane penetration agent comprises a cell-penetrating peptide, and the mixing ratio of the cell-penetrating peptide to the short-like peptide is 10-60: 2.
According to another aspect of the invention, the application of the auxiliary membrane-permeable agent is provided, and the auxiliary membrane-permeable agent is characterized in that the auxiliary membrane-permeable agent is used for mediating the intracellular delivery of living cells of small-molecule organic fluorescent probes; the living cell intracellular delivery of the mediated small molecule organic fluorescent probe is the living cell intracellular delivery of one mediated small molecule organic fluorescent probe or a mixture of mediated small molecule organic fluorescent probes.
Preferably, the auxiliary membrane-permeable agent is used for incubating the auxiliary membrane-permeable agent and the small-molecule organic fluorescent probe with living cells, and preferably, the concentration of the short-like peptide is between 2 and 10 mu M.
In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects:
the oligopeptide-like material provided by the invention is used as an auxiliary membrane permeation agent, can trigger a cell endocytosis escape mechanism, so that small molecules are assisted to permeate cell membranes, is simple to use, only needs co-incubation operation, and is low in cost; the membrane penetration effect is good, the action time is short, and the operation time can be effectively reduced.
The short-like peptide provided by the invention has low toxicity, so that the toxicity to cells can be reduced to the minimum degree while the probe is delivered, and better cell activity is obtained during imaging.
When the method is applied to super-resolution imaging of a microstructure in a living cell by assisting a small-molecule fluorescent probe to penetrate a membrane, the probe is simpler in design without considering middle-link membrane-penetrating peptide, and non-target labeling of the probe is reduced to the minimum extent; meanwhile, due to the rapid and efficient probe delivery of the auxiliary membrane-permeable agent, when the auxiliary membrane-permeable agent is used for assisting the membrane-permeable marking of the fluorescent probe, the auxiliary membrane-permeable agent has higher marking efficiency, and can also realize that various non-membrane-permeable organic fluorescent probes are brought into living cells to realize multicolor marking.
According to the preferable technical scheme, the quasi-short peptide and the cell-penetrating peptide provided by the invention are mixed to serve as an auxiliary membrane-penetrating agent, so that the delivery efficiency of the non-membrane-penetrating organic fluorescent probe can be further improved.
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FIG. 1 is a MS mass spectrum of a short-peptide-like PV-1 provided in example 1 of the present invention;
FIG. 2 is a graph showing the effect of the fluorescent labeling of the short-like peptide PV-1 as an auxiliary membrane-penetrating agent provided in example 1 of the present invention;
FIG. 3 is the MS mass spectrum of the short-peptide-like PV-2 provided in example 2 of the present invention;
FIG. 4 is a graph showing the effect of the fluorescent labeling of the short-like peptide PV-2 as an auxiliary membrane-penetrating agent provided in example 2 of the present invention;
FIG. 5 is the MS mass spectrum of the short-peptide-like PV-3 provided in example 3 of the present invention;
FIG. 6 is a graph showing the effect of the fluorescent labeling of the short-like peptide PV-3 as an auxiliary membrane-penetrating agent provided in example 3 of the present invention;
FIG. 7 is a graph showing the effect of the fluorescent labeling of the short-like peptide PV-6 as an auxiliary membrane-penetrating agent provided in example 4 of the present invention;
FIG. 8 is a graph showing the effect of single-chain PV-1 provided in example 5 of the present invention as an auxiliary membrane-penetrating agent for fluorescent labeling;
FIG. 9 is a graph showing the effect of intracellular fluorescent labeling by PV-1 as an auxiliary permeant for delivering different fluorescent probes in example 9 of the present invention;
FIG. 10 is a graph showing the labeling effect of the fluorescent probe docetaxel-Alexa 647 serving as an auxiliary membrane-permeable agent on the short-like peptide PV-1 provided in example 1 of the invention;
FIG. 11 is a diagram showing the labeling effect of three-color imaging by using the short-like peptide PV-1 provided in example 1 as an auxiliary membrane-permeable agent to deliver multiple probes simultaneously;
FIG. 12 is a graph showing the effect of delivering an organic fluorescent probe docetaxel-atto 488 fluorescence labeling as an auxiliary membrane agent to a mixture of the short-like peptide PV-1 and the cell-penetrating peptide provided in example 1 of the present invention;
FIG. 13 is a graph showing the effect of delivering an organic fluorescent probe SNAP-549 fluorescent label as an auxiliary membrane agent by using the mixture of the short-like peptide PV-1 and the cell-penetrating peptide provided in example 1 of the present invention;
FIG. 14 is a graph showing the results of the cell viability assay of example 11.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The short-like peptide molecule provided by the invention is a plurality of coupled electropositive short peptides, and the short peptide comprises a lysine residue modified by a positively charged group and a membrane-penetrating peptide segment; (lysine only)
Preferably, the short peptide-like molecule is 2 coupled electropositive peptide fragments.
Preferably, the plurality of electropositive short peptides are coupled by disulfide bonds.
Preferably, the short-peptide-like molecule has the following structure:
Figure BDA0002048656940000051
wherein K is lysine; c is cysteine; r1 is a positively charged group; CPP is cell penetrating peptide segment. When 2 pieces of electropositive short peptide are coupled together through a disulfide bond formed by cysteine, the number of positive charges of the cell-penetrating peptide is increased to promote the cell-penetrating peptide to enter cells through pinocytosis, and the cell-penetrating peptide can reduce the stability of vesicle membranes after entering the cells so as to promote the release of probes in the vesicle membranes due to vesicle rupture.
The positively charged group is preferably a rhodamine-based group. The positive charge group has positive charge quantity which can interact with the outer side of a cell membrane on one hand, and is connected with the cell-penetrating peptide through a covalent bond on the other hand, so that the positive charge quantity carried by the cell-penetrating peptide is increased, and as a result, the positive charge quantity carried by the cell-penetrating peptide is more and is easier to be endocytosed into cells. Preferably, the plurality of electropositive short peptides carry the same positively charged groups.
The auxiliary membrane penetrating agent taking the endocytosis triggering phenomenon as the action principle has the design difficulty that the delivery efficiency and the cytotoxicity are balanced, the charge quantity of a positive charge modification group on the surface of a molecule needs to be improved as much as possible in order to improve the delivery efficiency, however, the cytotoxicity is improved along with the improvement of the charge quantity.
The cell-penetrating peptide segment contains 4 to 40 amino acid residues and has positive charges. The cell-penetrating peptide segment interacts with the cell extracellular side to trigger endocytosis, thereby mediating the membrane penetrating process. The cell-penetrating peptide segment is preferably TAT and/or (rR)3R2Has higher delivery efficiency and lower toxicity. Preferably, the plurality of electropositive short peptides carry the same membrane penetrating peptide.
The invention provides an auxiliary membrane-penetrating agent, which comprises the short-like peptide provided by the invention, and preferably, the concentration of the short-like peptide is between 200 mu M and 2 mM. In a preferable scheme, the auxiliary membrane penetration agent further comprises a membrane penetrating peptide; the molar concentration ratio of the cell-penetrating peptide to the short-like peptide is 10-60:2, wherein the concentration of the short-like peptide is between 2 mu M and 10 mu M, and preferably 100ul of auxiliary membrane penetration agent contains 30 mu M of cell-penetrating peptide and 2 mu M of PV-1. The membrane-penetrating peptide is preferably L17E (Akishiba, Misao, et al. cytotoxic antibody delivery by lipid-sensitive intracellular peptide. Nature chemistry 9,2017).
The short-like peptide can be synthesized by a solid phase peptide synthesis method.
The auxiliary membrane-penetrating agent provided by the invention is applied to the intracellular delivery of living cells of a mediated small-molecule organic fluorescent probe.
The living cell intracellular delivery of the mediated small molecule organic fluorescent probe is the living cell intracellular delivery of one mediated small molecule organic fluorescent probe or a mixture of mediated small molecule organic fluorescent probes.
The specific operation is as follows:
co-incubating the auxiliary membrane-penetrating agent provided by the invention with a small-molecule organic fluorescent probe and living cells; so that the concentration of the short-like peptide in the cell culture solution is between 2 and 10. mu.M, preferably between 3 and 5. mu.M, preferably the short-like peptide is PV-1 provided in example 1, and the concentration is preferably 4. mu.M; or
The molar concentration ratio of the cell-penetrating peptide to the short-like peptide is 10-60:2, wherein the concentration of the short-like peptide is between 2 mu M and 10 mu M, and the preferred use amount is that 30 mu M L17E and 2 mu M PV-1 are contained in 100 mu L of the incubation solution.
Preferably, serum-free medium is adopted at 37 ℃ and 5% CO2Incubating with living cells under conditions for 1 to 3 hours;
after CO-incubation, the incubation solution was removed and fresh serum-containing medium was added at 37 ℃ with 5% CO2The cells are cultured for more than 1 hour under the condition, so that the release of the probe from the vesicle is facilitated, and the labeling efficiency is improved.
The following are examples:
example 1
An auxiliary membrane-penetrating agent which is a short-like peptide PV-1, wherein the short-like peptide PV-1 has the following structure:
Figure BDA0002048656940000071
wherein RKKRRQRRRG is penetrating peptide TAT (penetrating peptide fragment CPP), K is additional lysine, and R1 is
Figure BDA0002048656940000072
C is cysteine.
The peptide is obtained by a solid phase peptide synthesis method, the MS mass spectrum detection report of the peptide is shown in figure 1, and the structure is as follows:
PV-1
Figure BDA0002048656940000073
PV-1 and docetaxel-FITC as an organic fluorescent probe provided in the embodiment adopt a serum-free culture medium with the temperature of 37 ℃ and the CO content of 5%2Incubated with live cells under conditions for 1 hour, and taken into labeled cells. Wherein the concentration of the short-like peptide is 2 muM, 4 muM, 8 muM and 10 muM respectively, and the concentration of the probe is 5 muM.
After incubation, the incubation solution was removed and fresh serum-containing medium was added at 37 ℃ in 5% CO2Cells were cultured under conditions for 1 hour and fluorescence imaging was performed. The results are shown in FIG. 2.
Compared with the Control group (Control) without the short peptide, when the PV-1 provided by the embodiment is used as an auxiliary membrane permeation agent and is used for incubating cells with the organic fluorescent probe, the probe can enter the cells and can be well combined with the microtubes.
The method comprises the following specific steps:
cell preparation: u2OS cells (1.5X 10) in good growth status4cells/well) 200uL of the solution was inoculated into sterile glass-bottom petri dishes (glass bottom. phi. 15mm, NEST Biotechnology Co., Ltd., China). 37 ℃ and 5% CO2And McCoy's 5A medium containing 10% fetal calf serum for about 40 hours until the cells re-adhere and the density is moderate.
PV-1 provided in example 1 was made up to 200. mu.M in PBSSolution, docetaxel-FITC was also made into 200. mu.M stock solution in PBS. 2 μ L of the above PV-1 solution and 2.5 μ L of the above docetaxel-FITC solution were added to 95.5 μ L of McCoy's 5A medium without fetal calf serum to prepare an experimental group incubation solution. Then, 2.5. mu.L of the docetaxel-FITC solution was added to 97.5. mu.L of McCoy's 5A medium without fetal calf serum to prepare a control incubation solution. Prior to the experiment, the cells were removed, the original medium was aspirated, and the serum on the cell surface was washed with 200. mu.L of PBS solution and serum-free McCoy's 5A medium, respectively. Adding the prepared probe incubation liquid into a glass bottom culture dish respectively, and performing 5% CO treatment at 37 DEG C2Incubate in the dark for 1 hour. The probe solution was removed and the cell surface was washed 2 times with McCoy's 5A medium without serum. McCoy's 5A medium containing 10% fetal calf serum was added at 37 ℃ with 5% CO2Incubate in the dark for 1 hour. The medium was washed again with McCoy's 5A containing 10% fetal bovine serum before the microscopic examination.
Imaging conditions are as follows: observed by a rotary disk confocal microscope (Olympus IX 83). FITC was excited at 488nm and received at 510-530 nm.
The results of confocal imaging of the rotating disc after docetaxel-FITC labeling of cells are shown in figure 2. FIG. 2 shows that in the absence of the short peptide of example 1 in the incubation solution, docetaxel-FITC was unable to penetrate the microtubules labeled on the cell membrane, whereas filamentous microtubules were visible in the cells after 4. mu.M of short peptide PV-1 was added.
Example 2
An auxiliary membrane-penetrating agent which is a short-like peptide PV-2, wherein the short-like peptide PV-2 has the following structure:
Figure BDA0002048656940000091
wherein RKKRRQRRRG is cell penetrating peptide TAT (cell penetrating peptide fragment CPP), K is added lysine, R1 is rhodamine 6G, and C is cysteine.
The peptide is obtained by a solid phase peptide synthesis method, the MS mass spectrum detection report of the peptide is shown in FIG. 3, and the structure is as follows:
PV-2
Figure BDA0002048656940000092
PV-2 and organic fluorescent probe docetaxel-FITC (5 μ M) provided in the example were cultured in serum-free medium at 37 ℃ in 5% CO2Incubated with live cells under conditions for 1 hour, and taken into labeled cells. Wherein the concentrations of the short-like peptides are respectively 4 muM, 8 muM and 10 muM.
After incubation, the incubation solution was removed and fresh serum-containing medium was added at 37 ℃ in 5% CO2Cells were cultured under conditions for 1 hour and fluorescence imaging was performed. The results are shown in FIG. 4.
Compared with the Control group (Control) without the short peptide, when the PV-2 provided by the embodiment is used as an auxiliary membrane permeation agent and is used for incubating cells with the organic fluorescent probe, the probe can enter the cells and can be well combined with the microtubes.
The method comprises the following specific steps;
u2OS cells that grew well were prepared (preparation method was as in example 1).
PV-2 provided in example 2 was made up to 200. mu.M stock solution in PBS, and docetaxel-FITC was also made up to 200. mu.M stock solution in PBS. 2 μ L of the above PV-2 solution and 2.5 μ L of the above docetaxel-FITC solution were added to 95.5 μ L of McCoy's 5A medium without fetal calf serum to prepare an experimental group incubation solution. Then, 2.5. mu.L of the docetaxel-FITC solution was added to 97.5. mu.L of McCoy's 5A medium without fetal calf serum to prepare a control incubation solution. Prior to the experiment, the cells were removed, the original medium was aspirated, and the serum on the cell surface was washed with 200uL of PBS solution and serum-free McCoy's 5A medium, respectively. Adding the prepared probe incubation liquid into a glass bottom culture dish respectively, and performing 5% CO treatment at 37 DEG C2Incubate in the dark for 1 hour. The probe solution was removed and the cell surface was washed 2 times with McCoy's 5A medium without serum. McCoy's 5A medium containing 10% fetal calf serum was added at 37 ℃ with 5% CO2Incubate in the dark for 1 hour. Re-wash with McCoy's 5A medium containing 10% fetal bovine serum prior to endoscopyOne pass.
Imaging conditions are as follows: observed by a rotary disk confocal microscope (Olympus IX 83). FITC was excited at 488nm and received at 510-530 nm.
The results of confocal imaging of the rotating disc after docetaxel-FITC labeling of cells are shown in figure 4. FIG. 4 shows that in the absence of the short peptide of example 3 in the incubation solution, docetaxel-FITC was unable to penetrate the microtubules labeled on the cell membrane, whereas filamentous microtubules were visible in the cells after 4. mu.M of short peptide PV-2 was added.
Example 3
An auxiliary membrane-penetrating agent which is a short-like peptide PV-4, wherein the short-like peptide PV-4 has the following structure:
Figure BDA0002048656940000101
wherein rRrRrRRR is a cell-penetrating peptide fragment CPP, R is D-type arginine, R is L-type arginine, K is additional lysine, and R1 is
Figure BDA0002048656940000111
C is cysteine.
The peptide is obtained by a solid phase peptide synthesis method, the MS mass spectrum detection report of the peptide is shown in FIG. 5, and the structure is as follows:
PV-4
Figure BDA0002048656940000112
PV-4 and docetaxel-FITC (5 μ M) as an organic fluorescent probe provided in the example were cultured in a serum-free medium at 37 ℃ in 5% CO2Incubated with live cells under conditions for 1 hour, and taken into labeled cells. Wherein the concentrations of the short-like peptides are respectively 4 muM, 8 muM and 10 muM.
After incubation, the incubation solution was removed and fresh serum-containing medium was added at 37 ℃ in 5% CO2Cells were cultured under conditions for 1 hour and fluorescence imaging was performed.
Imaging conditions are as follows: observed by a rotary disk confocal microscope (Olympus IX 83). FITC was excited at 488nm and received at 510-530 nm. The results of confocal imaging of the rotating disc after docetaxel-FITC labeling of cells are shown in figure 6.
Compared with the Control group (Control) without the short peptide, when the PV-4 provided by the embodiment is used as an auxiliary membrane permeation agent and is used for incubating cells with the organic fluorescent probe, the probe can enter the cells and can be well combined with the microtubes.
The specific method comprises the following steps:
u2OS cells that grew well were prepared (preparation method was as in example 1).
PV-4 provided in example 2 was made up to 200. mu.M stock solution in PBS, and docetaxel-FITC was also made up to 200. mu.M stock solution in PBS. 2 μ L of the above PV-4 solution and 2.5 μ L of the above docetaxel-FITC solution were added to 95.5 μ L of McCoy's 5A medium without fetal calf serum to prepare an experimental group incubation solution. Then, 2.5. mu.L of the docetaxel-FITC solution was added to 97.5. mu.L of McCoy's 5A medium without fetal calf serum to prepare a control incubation solution. Prior to the experiment, the cells were removed, the original medium was aspirated, and the serum on the cell surface was washed with 200. mu.L of PBS solution and serum-free McCoy's 5A medium, respectively. Adding the prepared probe incubation liquid into a glass bottom culture dish respectively, and performing 5% CO treatment at 37 DEG C2Incubate in the dark for 1 hour. The probe solution was removed and the cell surface was washed 2 times with McCoy's 5A medium without serum. McCoy's 5A medium containing 10% fetal calf serum was added at 37 ℃ with 5% CO2Incubate in the dark for 1 hour. The medium was washed again with McCoy's 5A containing 10% fetal bovine serum before the microscopic examination.
Imaging conditions are as follows: observed by a rotary disk confocal microscope (Olympus IX 83). FITC was excited at 488nm and received at 510-530 nm.
The results of confocal imaging of the rotating disc after docetaxel-FITC labeling of cells are shown in figure 6. FIG. 6 shows that in the absence of the short peptide of example 2, docetaxel-FITC was unable to penetrate the microtubules labeled on the cell membrane, whereas filamentous microtubules were visible in the cells after 4. mu.M of short peptide PV-4 was added.
Example 4:
an auxiliary membrane-penetrating agent which is a short-like peptide PV-6, wherein the short-like peptide PV-6 has the following structure:
Figure BDA0002048656940000131
wherein RKKRRQRRRG is a cell-penetrating peptide TAT, K is an additional lysine, a connected cationic group is a TMR, and the middle part is connected in a non-disulfide bond mode, so that the protein is not easy to break after being endocytosed into cells, and the degradation efficiency is low.
The PV-6 and the organic fluorescent probe docetaxel-FITC provided in the embodiment adopt a serum-free culture medium with the temperature of 37 ℃ and the CO content of 5%2Incubated with live cells under conditions for 1 hour, and taken into labeled cells. Wherein the concentration of the short-like peptide is 5 mu M.
After incubation, the incubation solution was removed and fresh serum-containing medium was added at 37 ℃ in 5% CO2Cells were cultured under conditions for 1 hour and fluorescence imaging was performed.
Imaging conditions are as follows: observed by a rotary disk confocal microscope (Olympus IX 83). FITC was excited at 488nm and received at 510-530 nm; PV-6 was excited at 560nm and received at 570-590 nm. The imaging results are shown in fig. 7.
Compared with the Control group (Control) without the short peptide, when the PV-6 provided by the embodiment is used as an auxiliary membrane permeation agent and an organic fluorescent probe is used for incubating cells, the probe cannot enter the cells efficiently and can not be combined on the microtubes.
The imaging result shows that the fluorescent signal of the green channel is not sent to the cell, the fluorescent signal of the red channel is strong, the PV-6 sends the probe to the inside of the cell efficiently, and the result proves that the probe cannot be sent to the cell efficiently if the probe is not connected by the disulfide bond
Example 5 comparative example:
treating PV-1 with TCEP for half an hour to break disulfide bonds in the middle of PV-1 to obtain single-chain PV-1.
The single-chain PV-1 and the organic fluorescent probe docetaxel-FITC (5 mu M) provided in the embodiment adopt a serum-free culture medium with the temperature of 37 ℃ and the CO content of 5%2Under conditions and live cellsAfter 1 hour of incubation, the cells were taken into labeled cells. Wherein the concentration of the single-chain short-like peptide is 8 mu M.
After incubation, the incubation solution was removed and fresh serum-containing medium was added at 37 ℃ in 5% CO2Cells were cultured under conditions for 1 hour and fluorescence imaging was performed.
Imaging conditions are as follows: observed by a rotary disk confocal microscope (Olympus IX 83). FITC was excited at 488nm and received at 510-530 nm; single-stranded PV-1 was excited at 560nm and received at 570-590 nm. The results are shown in FIG. 8.
When the single-stranded PV-1 provided in this example was used as an auxiliary membrane permeation agent to incubate cells with an organic fluorescent probe, the single-stranded PV-1 could not deliver the probe to the cells efficiently, as compared to a Control group (Control) that did not use TCEP treatment.
Example 6
PV-1 provided in example 1 was mixed with organic fluorescent probes docetaxel-atto 488, docetaxel-atto 565, docetaxel-Alexa 647, Hoechst-Alexa488 and SNAP-Alexa488 respectively in a serum-free medium at 37 ℃ in the presence of 5% CO2Incubated with live cells under conditions for 1 hour, and taken into labeled cells. Wherein the concentration of the short-like peptide is 4 mu M, and the concentration of the probe is 5 mu M.
After incubation, the incubation solution was removed and fresh serum-containing medium was added at 37 ℃ in 5% CO2Cells were cultured under conditions for 1 hour and SIM super-resolution imaging was performed.
Imaging conditions are as follows: imaging by structured light illumination super-resolution microscopy (Nikon N-SIM). The results of the super-resolution imaging are shown in fig. 9.
Probes targeting microtubules and nuclei:
PV-1 provided in example 1 was mixed with PBS to prepare 200. mu.M stock solution, and probes docetaxel-atto 488, docetaxel-atto 565, docetaxel-Alexa 647, Hoechst-Alexa647, and Hoechst-Alexa488 were mixed with PBS to prepare 200. mu.M stock solution, respectively. mu.L of the probe solution was added to 95.5. mu.L of McCoy's 5A medium containing no fetal calf serum, and 2. mu.L of the solution of dfTAT (RhB) was added to prepare an incubation solution. Prior to the experiment, cells were removed, aspirated and treated with 200. mu.L PBS and serum-free McCoy's 5A medium washed the serum from the cell surface. Adding the prepared probe incubation liquid into a glass bottom culture dish respectively, and performing 5% CO treatment at 37 DEG C2Incubate in the dark for 1 hour. The probe solution was removed and the cell surface was washed 2 times with McCoy's 5A medium without serum. McCoy's 5A medium containing 10% fetal calf serum was added at 37 ℃ with 5% CO2Incubate in the dark for 1 hour. The medium was washed again with McCoy's 5A containing 10% fetal bovine serum before the microscopic examination.
Imaging conditions are as follows: observed by SIM super-resolution microscope (Nikon, N-SIM).
Endoplasmic reticulum-targeting probe:
cell preparation: u2OS cells (1.5X 10) in good growth status4cells/well) 400. mu.L of the solution was inoculated into sterile 24-well plates. 37 ℃ and 5% CO2And culturing overnight in McCoy's 5A culture medium containing 10% fetal calf serum until the cells are attached to the wall again and the confluency rate is 80% -90%. According to the transfection reagent LipofectamineTM2000(Invitrogen, Grand Island, NY, USA), plasmid SNAP-sec61 β was transferred into cells. After 6 hours, the supernatant was removed and fresh McCoy's 5A medium containing 10% fetal calf serum was added at 37 ℃ with 5% CO2The culture was carried out overnight. The cells were digested with pancreatin containing 0.25% EDTA and treated at 1.5X 104cells/wells were seeded in sterile glass-bottom petri dishes (glass bottom. phi.15 mm, NEST Biotechnology Co., Ltd., China). 37 ℃ and 5% CO2And McCoy's 5A medium containing 10% fetal calf serum for about 40 hours until the cells re-adhere and the density is moderate.
PV-1 provided in example 1 was prepared as a 200. mu.M stock solution in PBS, and the probe SNAP-Alexa488 was prepared as a 1mM stock solution in DMSO. mu.L of the PV-1 solution and 0.5. mu.L of the SNAP-Alexa488 solution were added to 97.5. mu.L of McCoy's 5A medium without fetal calf serum to prepare a culture. Prior to the experiment, the cells were removed, the original medium was aspirated, and the serum on the cell surface was washed with 200uL of PBS solution and serum-free McCoy's 5A medium, respectively. Adding the prepared probe incubation liquid into a glass bottom culture dish respectively, and performing 5% CO treatment at 37 DEG C2Hatching in dark environmentIncubate for 1 hour. The probe solution was removed and the cell surface was washed 2 times with McCoy's 5A medium without serum. McCoy's 5A medium containing 10% fetal calf serum was added at 37 ℃ with 5% CO2Incubate in the dark for 1 hour. The medium was washed again with McCoy's 5A containing 10% fetal bovine serum before the microscopic examination.
Imaging conditions are as follows: observation was done by structured light illumination (SIM) super-resolution microscope (Nikon, N-SIM).
The results of SIM imaging after labeling the cells with each probe are shown in figure 9.
Example 7
PV-1 provided in example 1 and docetaxel-Alexa 647 as organic fluorescent probe were cultured in serum-free medium at 37 deg.C and 5% CO2Incubated with live cells under conditions for 1 hour, and taken into labeled cells. Wherein the concentration of the short-like peptide is 4 mu M, and the concentration of the docetaxel-Alexa 6475 mu M.
After incubation, the incubation solution was removed and fresh serum-containing medium was added at 37 ℃ in 5% CO2Cells were cultured under conditions for 1 hour and random optical reconstruction (STORM _ super-resolution imaging) was performed.
Imaging conditions are as follows: super-resolution images were reconstructed at 10ms/frame and 5000frames, observed by a STORM super-resolution microscope (Nikon, N-STORM). The results of the super-resolution imaging are shown in fig. 10.
The method comprises the following specific steps:
u2OS cells that grew well were prepared (preparation method was as in example 1).
PV-1 provided in example 1 was made up to 200. mu.M stock solution in PBS, and docetaxel-Alexa 647 was also made up to 200. mu.M stock solution in PBS. mu.L of the above PV-1 solution and 2.5. mu.L of the above docetaxel-Alexa 647 solution were added to 95.5. mu.L of McCoy's 5A medium without fetal calf serum to prepare an incubation solution for each experimental group. Then, 2.5. mu.L of the above docetaxel-Alexa 647 solution was added to 97.5. mu.L of McCoy's 5A medium without fetal bovine serum to prepare a control incubation solution. Prior to the experiment, the cells were removed, the original medium was aspirated, and the serum on the cell surface was washed with 200. mu.L of PBS solution and serum-free McCoy's 5A medium, respectively. Adding the prepared probe incubation liquid into the glass bottom culture medium respectivelyIn a culture dish, 5% CO at 37 ℃2Incubate in the dark for 1 hour. The probe solution was removed and the cell surface was washed 2 times with McCoy's 5A medium without serum. McCoy's 5A medium containing 10% fetal calf serum was added at 37 ℃ with 5% CO2Incubate in the dark for 1 hour. The medium was washed with McCoy's 5A containing 10% fetal bovine serum before the microscopic examination, and the imaging results are shown in FIG. 10.
Example 8
The PV-1 provided in example 1 and the combination with organic fluorescent probe or probes were performed using serum-free medium at 37 deg.C and 5% CO2Incubated with live cells under conditions for 1 hour, and taken into labeled cells. Wherein the concentration of the short-like peptide is 4 mu M, and the concentration of the organic fluorescent probe is 5 mu M.
After incubation, the incubation solution was removed and fresh serum-containing medium was added at 37 ℃ in 5% CO2Culturing the cells under the condition for 1 hour, and performing rotary table confocal microscope imaging or SIM super-resolution imaging.
Imaging conditions are as follows: the two-color images were imaged by a rotating disk confocal microscope (Olympus IX 83). The results are shown in FIG. 11. The trichromatography is imaging by a SIM super-resolution microscope (Nikon N-SIM), and the super-resolution imaging results are shown in fig. 11.
The method comprises the following specific steps:
u2OS cells that grew well were prepared (preparation method was as in example 1).
PV-1 provided in example 1 was made up to 200. mu.M stock solution in PBS. The probe and PV-1 of the indicated combination were prepared at the indicated concentrations in serum-free McCoy's 5A medium, respectively, and prior to the experiment, the cells were removed, the original medium was aspirated, and the cell surface was washed with 200. mu.L of PBS solution and serum-free McCoy's 5A medium, respectively. Adding the prepared probe incubation liquid into a glass bottom culture dish respectively, and performing 5% CO treatment at 37 DEG C2Incubate in the dark for 1 hour. The probe solution was removed and the cell surface was washed 2 times with McCoy's 5A medium without serum. McCoy's 5A medium containing 10% fetal calf serum was added at 37 ℃ with 5% CO2Incubate in the dark for 1 hour. Upper mirrorThe medium of McCoy's 5A containing 10% fetal calf serum was washed once more before examination, and the results of super-resolution imaging are shown in FIG. 11.
The results of examples 6 to 8 show that the PV-1 provided by example 1 as an auxiliary film-permeable agent has good film-permeable effect and labeling activity on different organic fluorescent probes, and has high imaging resolution.
Example 9 the auxiliary membrane permeabilizing agent consisting of PV-1 and L17E provided in example 1 and the organic fluorescent probe docetaxel-atto 488 were mixed in a serum-free medium at 37 ℃ and 5% CO2Incubated with live cells under conditions for 1 hour, and taken into labeled cells. Wherein the concentration of the short-like peptide is 2 mu M, L17E 30 mu M and docetaxel-atto 488(5 mu M).
After incubation, the incubation solution was removed and fresh serum-containing medium was added at 37 ℃ in 5% CO2Cells were cultured under conditions for 1 hour and imaged by fluorescence confocal microscopy.
Imaging conditions are as follows: observed by a rotary disk confocal microscope (Olympus IX 83). Attlo 488 was excited at 488nm and at 510-530 nm. The results are shown in FIG. 12.
The method comprises the following specific steps:
cell preparation: u2OS cells (1.5X 10) in good growth status4cells/well) 200uL of the solution was inoculated into sterile glass-bottom petri dishes (glass bottom. phi. 15mm, NEST Biotechnology Co., Ltd., China). 37 ℃ and 5% CO2And McCoy's 5A medium containing 10% fetal calf serum for about 40 hours until the cells re-adhere and the density is moderate.
PV-1 provided in example 1 was made up to 200. mu.M stock solution in PBS, L17E was made up to 1mM stock solution in PBS, and docetaxel-Atto 488 was also made up to 200. mu.M stock solution in PBS. mu.L of the above PV-1 solution, 3. mu.L of the above L17E solution and 2.5. mu.L of the above docetaxel-atto 488 solution were added to 93.5. mu.L of McCoy's 5A medium without fetal calf serum to prepare an experimental group incubation solution. Prior to the experiment, the cells were removed, the original medium was aspirated, and the serum on the cell surface was washed with 200uL of PBS solution and serum-free McCoy's 5A medium, respectively. Adding the prepared probe incubation liquid into a glass bottom culture dish respectively, and performing 5% CO treatment at 37 DEG C2Incubate in the dark for 1 hour. The probe solution was removed and the cell surface was washed 2 times with McCoy's 5A medium without serum. McCoy's 5A medium containing 10% fetal calf serum was added at 37 ℃ with 5% CO2Incubate in the dark for 1 hour. The medium was washed again with McCoy's 5A containing 10% fetal bovine serum before the microscopic examination.
Imaging conditions are as follows: observed by a rotary disk confocal microscope (Olympus IX 83).
Example 10
The auxiliary membrane permeability agent consisting of PV-1 and L17E provided in example 1 and the organic fluorescent probe SNAP-549 adopt a serum-free culture medium with the temperature of 37 ℃ and 5 percent CO2Under conditions of incubation for 1 hour with live cells expressing SNAPtag-Sec61 beta (Sec61 beta is an endoplasmic reticulum marker), the cells were taken into the labeled cells. Wherein the concentration of the short-like peptide is 2 mu M, L17E 30 mu M and SNAP-5495 mu M.
After incubation, the incubation solution was removed and fresh serum-containing medium was added at 37 ℃ in 5% CO2Cells were cultured under conditions for 1 hour and imaged by fluorescence confocal microscopy.
Imaging conditions are as follows: observed by a rotary disk confocal microscope (Olympus IX 83). SNAP-549 was excited at 560nm and received at 570-590 nm. The results are shown in FIG. 12.
The method comprises the following specific steps:
cell preparation: u2OS cells (1.5X 10) in good growth status4cells/well) 200uL of the solution was inoculated into sterile glass-bottom petri dishes (glass bottom. phi. 15mm, NEST Biotechnology Co., Ltd., China). 37 ℃ and 5% CO2After culturing in McCoy's 5A medium containing 10% fetal calf serum for about 12 hours, the plasmid SNAP-tag-Sec61 beta was transfected.
24-36 hours after transfection of plasmid PV-1 provided in example 1 was made up to 200. mu.M stock with PBS, L17E was made up to 1mM stock with PBS, and SNAP-549 was also made up to 200. mu.M stock with PBS. mu.L of the PV-1 solution, 3. mu.L of the L17E solution and 2.5. mu.L of the SNAP-549 solution were added to 93.5. mu.L of McCoy's 5A medium without fetal calf serum to prepare an incubation solution for each test group. Before the experiment, the cells were removed, the original culture medium was aspirated, and 20 cells were used separatelyThe serum on the cell surface was washed with 0. mu.L of PBS solution and serum-free McCoy's 5A medium. Adding the prepared probe incubation liquid into a glass bottom culture dish respectively, and performing 5% CO treatment at 37 DEG C2Incubate in the dark for 1 hour. The probe solution was removed and the cell surface was washed 2 times with McCoy's 5A medium without serum. McCoy's 5A medium containing 10% fetal calf serum was added at 37 ℃ with 5% CO2Incubate in the dark for 1 hour. The medium was washed again with McCoy's 5A containing 10% fetal bovine serum before the microscopic examination.
Imaging conditions are as follows: observed by a rotary disk confocal microscope (Olympus IX 83).
Example 11 toxicity testing of the polypeptides provided in example 1, example 2, example 3.
Cell preparation: well-grown U2OS cells (3000-. Cells were re-attached overnight at 37 ℃ in 5% CO2 in McCoy's 5A medium containing 10% fetal bovine serum.
PV-1, PV-2 and PV-4 were mixed with PBS to prepare 200. mu.M stock solution, and cultured with McCoy's 5A without fetal calf serum to prepare 4. mu.M, 4. mu.M and 8. mu.M incubation solutions, respectively. 25 μ L of McCoy's 5A without fetal bovine serum was then used to prepare a blank incubation in another EP tube. Prior to the experiment, the 96-well plate was removed, the original medium was aspirated, and the serum on the cell surface was washed with 100. mu.L of PBS solution and serum-free McCoy's 5A medium, respectively. The prepared incubation solutions of the experimental group and the blank control were added to a 96-well plate, and incubated at 37 ℃ for 1 hour in the dark in an environment of 5% CO 2. The incubation solution was removed and the residual incubation solution on the cell surface was washed with 100uL of PBS solution and serum-free McCoy's 5A medium, respectively.
To each well, 80. mu.L of McCoy's 5A medium containing 10% fetal bovine serum and 20. mu.L of MTS solution (Co.) were added, and alternatively, one well without cells was used as blank, and 80. mu.L of McCoy's 5A medium containing 10% fetal bovine serum and 20. mu.L of MTS solution were also added. After incubation for 3 hours at 37 ℃ in the absence of light in a 5% CO2 environment, the cells were placed on a microplate reader, and the absorbance of each well was measured at 490 nm. Finally, one maximum value was removed from 5 replicates of the experimental group, and the average was calculated after removing one minimum value, and the cell viability was calculated as follows:
Figure BDA0002048656940000201
the results are shown in FIG. 14, and it can be seen from the histogram that the activity values of the cells after the polypeptide incubation were 85%, 50% and 93% within three hours, which are all within the acceptable range, and have no major influence on the basic life activities of the cells.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (9)

1. The short-like peptide is characterized in that the short-like peptide molecule is 2 coupled electropositive short peptides, and the electropositive short peptides comprise lysine residues modified by positively charged groups and membrane-penetrating peptide segments; the plurality of electropositive short peptides are coupled by disulfide bonds; the short peptide-like molecule has the following structure:
Figure 593419DEST_PATH_IMAGE001
wherein K is lysine; c is cysteine; r1Is a positively charged group; CPP is cell penetrating peptide; the positively charged group is a rhodamine-based group; the cell-penetrating peptide segment is TAT or (rR)3R2。
2. The short peptide-like molecule of claim 1, wherein said positively charged group is rhodamine B or rhodamine 6G.
3. An auxiliary membrane-permeable agent comprising the short-like peptide according to claim 1 or 2.
4. An auxiliary membrane-permeable agent according to claim 3, wherein the concentration of the short-like peptide is between 200 μ M and 2 mM.
5. An auxiliary membrane-penetrating agent as claimed in claim 3, which comprises a membrane-penetrating peptide, wherein the mixing ratio of the membrane-penetrating peptide to the short-like peptide is 10-60: 2.
6. Use of an auxiliary membrane permeabilizing agent according to any one of claims 3 to5, for mediating the intracellular delivery of living cells of a small molecule organic fluorescent probe.
7. The use of an auxiliary membrane permeabilizing agent according to claim 6, wherein said living cell intracellular delivery of a small molecule organic fluorescent probe is mediated by one small molecule organic fluorescent probe or a mixture of small molecule organic fluorescent probes.
8. Use of an auxiliary membrane permeabilizing agent according to claim 7, wherein the auxiliary membrane permeabilizing agent according to any one of claims 3 to5 is incubated with a small molecule organic fluorescent probe and living cells.
9. An auxiliary membrane-permeable agent for use according to claim 8, wherein the concentration of the short-like peptide is between 2 μ M and 10 μ M.
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