CN115960164A - Preparation method and application of scorpion toxin-derived cell-penetrating polypeptide - Google Patents

Abstract

The invention relates to the technical field of biomedical engineering, and provides a preparation method and application of a scorpion toxin-derived transmembrane polypeptide. The OpiCalcin1 polypeptide is optimized in a truncation mode, and two polypeptides Opi 1-33 CSKKCKRRGTNPEKRCR and Opi 1-33 RRGTNPEKRCR with excellent membrane penetration function and low cytotoxicity are provided. Experiments prove that the two cell-penetrating peptides have excellent penetrating effect and low cytotoxicity, simultaneously overcome the defects of time and labor waste in synthesizing the complete sequence of the scorpion toxin OpiCalcin1, and can be used for preparing transfer carriers of medicaments, nuclear localization sequences, probes and the like, and improve the targeting efficiency. Compared with the traditional scorpion venom as a medicine, the scorpion venom is synthesized to explore the membrane penetrating activity of the scorpion venom, and a new choice is provided for a medicine carrier.

Description

Preparation method and application of scorpion toxin-derived cell-penetrating polypeptide

Technical Field

The invention relates to the technical field of biomedical engineering, in particular to a cell-penetrating peptide and application thereof.

Background

Scorpion, also called Scorpion, is the essential herb for wind, and has the actions of extinguishing wind, relieving spasm, dredging collaterals, alleviating pain, counteracting toxic pathogen and resolving hard mass. The active components in scorpion venom mainly comprise two parts, namely a protein component and a non-protein component, wherein the non-protein component mainly comprises mucopolysaccharide, histamine, 5-hydroxytryptamine, organic acid, trace elements and the like, while the protein component playing a main functional role mainly comprises small molecular polypeptides which are usually called scorpion toxins and usually comprise 20-80 amino acids, and the amino acids forming the scorpion toxins also comprise a plurality of cysteine which can mutually react to form a plurality of disulfide bonds.

There are 300 scorpion venom polypeptides, which are ion channel inhibitors and have the biological activities of resisting epilepsy, thrombus, tumor, pain, etc. In addition, some scorpion venom polypeptides have the characteristics of cell-penetrating peptides, and the amino acid sequences of scorpion toxins which can be used as the cell-penetrating peptides often contain a large amount of arginine, lysine and other basic groups, so that the scorpion toxins with the characteristics can penetrate through cell membranes to enter cells to exert biological activity.

OpiCalcin1 is a peptide toxin which is separated from scorpions and consists of 33 amino acid residues and has a cell penetrating function, the peptide consists of three pairs of disulfide bonds of Cys3-Cys17, cys10-Cys21 and Cys16-Cys32, and the OpiCalcin1 contains an ICK motif of a cysteine knot and three beta chains through 1HNMR measurement. OpiCalcin1 is a basic polypeptide, of the 33 amino acids that make up OpiCalcin1, 11 amino acid residues carry a positive charge, 4 amino acid residues carry a negative charge, and the net charge of the entire OpiCalcin1 chain is positive; in addition, opiCalcin1 is a hydrophilic polypeptide, and the proportion of polar amino acid residues in all 33 amino acid residues is 58%.

The research mainly focuses on the synthesis, purification and transmembrane activity analysis of the scorpion toxin OpiCalcin1 series polypeptide, so as to perform further functional research on the basis of the previous research, and perform old medicine new use on the scorpion toxin OpiCalcin1 to play the new medicinal value thereof.

Disclosure of Invention

The invention is based on the research, different fragments of scorpion toxin OpiCalcin1 (Opi 1 for short) are synthesized by a chemical synthesis method, and are fluorescently labeled by fluorescein isothiocyanate, the membrane penetrating effect of the scorpion toxin Opi1 series membrane penetrating peptides is researched, after the comparison with TAT, the cell penetrating peptides Opi 1-33 and Opi 1-33 with better membrane penetrating effect and lower toxicity are found, and the cell penetrating peptides are expected to become novel cell penetrating peptides, so that a new choice is provided for a drug carrier.

The first aim of the invention is to optimize the OpiCalcin1 polypeptide in a truncated manner, providing two polypeptides Opi 1-33 CSKKCKRRGTNPEKRCR and Opi 1-33 RRGTNPEKRCR with excellent transmembrane function and low cytotoxicity.

The invention analyzes the complete sequence of scorpion toxin OpiCalcin1, and divides the whole sequence into the following parts according to the sequence length or base motif: opi 1-16, opi 1-33, opi 1-11, opi 1-22, opi 1-33, FITC-Opi 1-11, FITC-Opi 1-24, FITC-Opi 1-25-33, and then mature cell-penetrating peptide TAT as a positive control polypeptide. The amino acid sequences and molecular weights of the peptide fragments and the positive control polypeptide are shown in the following table 1:

TABLE 1 different OpiCalcin1 peptide sequences

Flow cytometry results show that cell-penetrating peptides of Opi 1-33 and Opi 1-33 have good penetrating effect, and the average fluorescence intensity in cells is far higher than that of a positive control peptide TAT. Next, cytotoxicity analysis was performed on the nine polypeptides, and the results showed that TAT, opi 1-16, opi 1-33, opi 1-11 were less cytotoxic, and in particular that the polypeptide Opi 1-33 not only was more membrane-penetrating than TAT, but also was much less cytotoxic than TAT.

The second objective of the invention provides a preparation method of the cell-penetrating peptide, which mainly comprises the following steps:

1) The complete sequence of scorpion toxin opiocalcin 1 was analyzed and classified according to sequence length and base motif: opi 1-16, opi 1-33, opi 1-11, opi 1-22, opi 1-33, FITC-Opi 1-11, FITC-Opi 1-16, FITC-Opi 1-24, FITC-Opi 1-25-33, and then mature cell-penetrating peptide TAT as a positive control polypeptide.

2) After the sequence is determined, the peptide fragment is synthesized by adopting Fmoc solid phase synthesis method: amino resin or wang resin is used as a solid phase carrier, then Oxyma is selected as an activating agent, DIC is used as a condensing agent, condensation reaction is carried out between amino acids at the temperature of 60 ℃, ninhydrin detection reagent is used for judging whether the condensation reaction is complete, if the reaction is complete, the resin connected with the amino acids and the ninhydrin detection reagent do not develop color under the water bath heating condition, if the reaction is incomplete, bluish purple is developed, and at the moment, the reaction needs to be carried out again.

3) After the crude polypeptide is successfully connected according to the amino acid sequence, a connecting group is required to be connected at the tail end so as to connect fluorescein with the polypeptide, and fluorenylmethyloxycarbonyl-6-aminocaproic Acid (ACP) is selected as the connecting group to connect FITC fluorescein so as to obtain a green fluorescent label for the peptide segment.

4) And (3) purifying and analyzing the polypeptide which is marked by fluorescence: analyzing and purifying in a C18 column by using a liquid chromatography, judging the position of a target peak by using LC-MS, then collecting the target peak according to the peak-appearing time, judging the purity of the target peak by using analytical LC, then freeze-drying the pure cell-penetrating peptide by using a freeze dryer, and storing the obtained fluorescent cell-penetrating peptide in a dark place to avoid fluorescence quenching.

5) Cell-penetrating peptide synthesized and purified in the early stage is subjected to penetrating activity analysis on HEK293T cells: and qualitatively and quantitatively analyzing the cells incubated by the cell-penetrating peptide by adopting a fluorescence microscope and flow cytometry, and further judging the penetrating effect of the cell-penetrating peptide.

6) Cytotoxicity analysis was performed on the cell-penetrating peptide synthesized in the previous stage by the CCK8 method.

In a third aspect, the invention provides the use of a cell-penetrating peptide, in particular in the preparation of a transfer vehicle. The active ingredients in the transfer carrier can comprise cell-penetrating peptides and other active ingredients, and the cell-penetrating peptides can also be used as the only active ingredients.

Preferably, the delivery carrier comprises a drug delivery carrier, a nuclear localization sequence delivery carrier and a probe delivery carrier.

The cell-penetrating peptide can enter cells under the condition of not causing any damage to the cells, can be successfully used as a carrier of siRNA, nucleotide, macromolecular drugs, protein and the like, and is widely applied to the field of novel drug carriers.

In addition, applications of CPPs in cellular imaging, nuclear localization, pH sensitivity, and thermal targeting have received attention:

in cellular imaging, cell-penetrating peptides are used to facilitate internalization by capturing labeled peptide probes at a target site. The high efficiency characteristics of CPPs make them a promising tool in the imaging field; in nuclear localization, in order to improve the efficiency of nuclear localization, CPPs having nuclear localization sequences (NLSS) are directly or indirectly linked to DNA or gene vectors to facilitate nuclear localization.

In addition, due to the pH gradient between the tumor environment and the physiological environment, cell-penetrating peptides allow their control in the tumor microenvironment by attachment to nanoparticles, such as acid-degradable cross-linking, removal of protective layers or enzymatic degradation. When the cell-penetrating peptide is connected with the drug-containing nanoparticles, the treatment efficiency of the nanoparticles is improved, and the drug toxicity is reduced.

The invention also provides a transport carrier, which takes the cell-penetrating peptide as an active ingredient, and transports the medicine containing siRNA, nucleotide, macromolecular medicine, protein and the like, probes, nuclear localization sequences (NLSS) and the medicine containing nano-particles into cells to realize corresponding curative effect.

The invention has the following beneficial guarantee and effects:

the invention provides a cell-penetrating peptide, a preparation method and application, wherein OpiCalcin1 polypeptide is optimized in a truncation mode, and two polypeptides Opi 1-33 CSKKCKRRGTNPEKRCR and Opi 1-33 RRGTNPEKRCR with excellent cell-penetrating function and low cytotoxicity are provided. Experiments prove that the cell-penetrating peptide has excellent penetrating effect and low cytotoxicity, and simultaneously overcomes the defects of time and labor waste in synthesizing the complete sequence of the scorpion toxin OpiCalcin 1.

The cell-penetrating peptide obtained by screening can be used for preparing a transfer carrier of medicaments, nuclear localization sequences, probes and the like, and the targeting efficiency is improved. Compared with the traditional scorpion venom as a medicine, the activity of penetrating membranes of scorpion venom is explored by synthesizing the scorpion venom, and a new choice is provided for a medicine carrier.

Drawings

FIG. 1 shows the synthesis route of Opi1 cell-penetrating peptides: (A) FITC-Opi 1-16 synthetic route; (B) FITC-Opi 1-33 synthetic route; (C) FITC-Opi 1-11 synthetic route; (D) FITC-Opi 1-22 synthetic route; (E) FITC-Opi 1-33 synthetic route.

FIG. 2 shows the synthesis route of TAT and Opi1 cell-penetrating peptides: (A) FITC-TAT synthetic route; (B) FITC-Opi 1-11 synthetic route; (C) FITC-Opi 1-24 synthetic route; (D) FITC-Opi 1-33 synthetic route.

FIG. 3 shows the results of LC and ESI-MS analysis of Opi1 different cell-penetrating peptides: (A) An LC spectrogram of the FITC-Opi 1-16 crude polypeptide has a target peak emergence time of 26.053min; (B) ESI-MS of FITC-Opi 1-16 showed the molecular weight was 2346.6580; (C) As a result of purification of FITC-Opi 1-16 polypeptide, the purity was: 97.071%; (D) An LC spectrogram of the FITC-Opi 1-33 crude polypeptide has a target peak emergence time of 13.981min; (E) ESI-MS of FITC-Opi 1-33 results, molecular weight 2552.0160; (F) The purification results of FITC-Opi 1-33 polypeptide showed the following purity: 95.872%; (G) An LC spectrogram of the FITC-Opi 1-11 crude product polypeptide has a target peak emergence time of 38.213min; (H) ESI-MS of FITC-Opi 1-11 showed a molecular weight of 1771.1080; (I) Purification results for the FITC-Opi 1-11 polypeptide, purity: 99.586%; (J) An LC spectrogram of the FITC-Opi 1-22 crude polypeptide has a target peak emergence time of 16.112min; (K) ESI-MS of FITC-Opi 1-22 showed the molecular weight was 1773.0030; (L) purification of FITC-Opi 1-22 polypeptide with a purity of: 96.668%; (M) an LC spectrogram of a FITC-Opi 1-33 crude polypeptide, wherein the peak emergence time of a target peak is 32.805min; (N) ESI-MS of FITC-Opi 1-33 with a molecular weight of 1875.1190; (O) purification of the FITC-Opi 1-33 polypeptide results in a purity of: 91.655%.

FIG. 4 shows the LC and ESI-MS analysis results of TAT and Opi1 cell-penetrating peptides: (A) An LC spectrogram of the FITC-TAT crude product polypeptide has the target peak emergence time of 29.787min; (B) ESI-MS of FITC-TAT showed that the molecular weight was 2061.0704; (C) purification of FITC-TAT polypeptide results in a purity of: 99.204 percent; (D) An LC spectrogram of the FITC-Opi 1-11 crude polypeptide has a target peak emergence time of 33.833min; (E) ESI-MS of FITC-Opi 1-11 showed a molecular weight of 1597.7356; (F) purification of FITC-Opi13-11 polypeptide with the following purity: 99.586%; (G) An LC spectrogram of the FITC-Opi 1-24 crude polypeptide has a target peak emergence time of 31.247min; (H) ESI-MS of FITC-Opi 1-24 with a molecular weight of 1612.6771; (I) The purification results of FITC-Opi 1-24 polypeptide showed the following purity: 96.370 percent; (J) An LC spectrogram of the FITC-Opi125-33 crude polypeptide has a target peak emergence time of 31.318min; (K) ESI-MS of FITC-Opi 1-33 with a molecular weight of 1561.6442; (L) purification of FITC-Opi 1-33 polypeptide with purity: 92.106 percent.

FIG. 5 is a comparison of the cellular uptake of FITC-labeled TAT and different cell-penetrating peptides of the Opi1 series in HEK293T cells at a concentration of 10. Mu.M (scale bar 100 px).

FIG. 6 is a comparison of the flow cytometric assay results of FITC-labeled TAT and Opi1 series of different transmembrane peptides in HEK293T cells at a concentration of 10. Mu.M.

Fig. 7 is a comparison of cytotoxicity of different transmembrane peptides of TAT and Opi1 series in HEK293T cells.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

The experimental procedures, in which specific conditions are not specified, in the following examples are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Percentages and parts are by volume unless otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Moreover, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are exemplary only.

Example 1 Opi1 polypeptide fragment Synthesis

OpiCalcin1 sequences are divided into five peptide segments Opi 1-16, opi 1-33, opi 1-11, opi 1-22 and Opi 1-23-33 according to the length difference, and the synthesized peptide segments are purified and prepared for the research of a transmembrane experiment.

In addition, most cell-penetrating peptides have the common characteristic that the amino acid sequences of the cell-penetrating peptides all comprise a large number of basic amino acids, and when OpiCalcin1 sequences are analyzed, three base motifs, namely Opi 1-11 KRCK, opi 1-19-24 KKCKRR and Opi 1-30 KRCR, are found in 33 amino acids of OpiCalcin1, so that new peptide segments are synthesized around the three base motifs. The OpiCalcin1 sequence is divided into three sections Opi 1-11, opi 1-24 and Opi 1-25-33 according to the distribution of the base motif of OpiCalcin 1.

The eight peptide sequences are shown in the table 1, the eight polypeptides are synthesized by adopting an Fmoc solid phase synthesis method, are labeled by FITC fluorescence and are used for a subsequent cell-penetrating activity experiment, and cell-penetrating peptide TAT is selected as a positive control polypeptide.

Example 2 Synthesis of cell-penetrating peptides and fluorescent labeling

The synthesis method of the cell-penetrating peptides with different sequences is similar, and the synthesis routes of FITC-Opi 1-16, FITC-Opi 1-17-33, FITC-Opi 1-11, FITC-Opi 1-12-22 and FITC-Opi 1-23-33 are shown in figure 1; the synthetic routes for FITC-Opi 1-11, FITC-Opi 1-24, FITC-Opi 1-33, and the positive reference peptide FITC-TAT are shown in FIG. 2. The specific synthetic process is as follows:

A. synthesis of cell-penetrating peptide Opi1

1) Resin preparation and swelling: 400mg of Rink Amide Resin (substitution =0.43 mmol/g) was weighed into a polypeptide synthesis tube, and 25ml of Dichloromethane (DCM) was added and left to stand for 30min to sufficiently swell. After swelling, the DCM was aspirated off and washed 5 times with N, N-Dimethylformamide (DMF), and the resin layer was seen to thicken after the end of the run.

2) Removing Fmoc protection: the Rink Amide Resin used has an Fmoc protecting group which we need to remove to attach the amino acid to the Resin. Removing Fmoc protection by adding 20% piperidine/7.1% Oxyma/DMF solvent for deprotection, adding 6ml of deprotection mixed reagent into a peptide connecting tube for the first time, placing the mixture into a 35-DEG gas bath constant-temperature oscillator for shaking for 5min, draining after the reaction is finished, adding 6ml of deprotection mixed reagent again, placing the mixture into a 35-DEG gas bath constant-temperature oscillator for shaking for 5min, washing the mixture for 5 times by using DCM and DMF after the deprotection is finished, taking out part of resin for ninhydrin test, and heating the mixture in a water bath for 3min, wherein the resin is changed into bluish purple, which represents that the deprotection is successful.

3) First amino acid to activate C-terminus: weighing 1mmol of first amino acid at the C end, adding an activating agent Oxyma, fully dissolving by NMP, adding DIC, uniformly mixing again, activating the amino acid for 1min, adding the activated amino acid into a peptide connecting tube containing resin, and reacting for 20min at 60 ℃ and 60R to perform condensation reaction on the amino acid and the resin. After the condensation reaction is finished, washing the resin for 5 times by using DCM and DMF in sequence, dipping a small amount of numerical values by using a capillary tube, adding ninhydrin detection reagent for detection, if the resin does not change bluish purple, indicating that the amino acid is successfully connected with the carrier resin, if the resin partially changes color, indicating that the condensation reaction of the amino acid is incomplete, re-weighing 1mmol of amino acid, adding an Oxyma activator and a DIC condensing agent, putting the amino acid into a peptide connecting tube again for reaction, reacting for 20min under the condition that the reaction temperature is still 60 ℃ and 60R, continuously detecting by using the ninhydrin reagent after the reaction is finished, and observing whether the reaction is complete.

4) Removing Fmoc protection: after the first amino acid at the C-terminus is ligated, the Fmoc protecting group of the amino group of the first amino acid is removed to allow the amino acid to be ligated to the next amino acid. Removing Fmoc protection by adding 20% piperidine/7.1% Oxyma/DMF solvent for deprotection, adding 6ml of deprotection mixed reagent into a peptide connecting tube for the first time, placing the mixture into a 35-DEG gas bath constant-temperature oscillator for shaking for 5min, draining after the reaction is finished, adding 6ml of deprotection mixed reagent again, placing the mixture into a 35-DEG gas bath constant-temperature oscillator for shaking for 5min, washing the mixture for 5 times by using DCM and DMF after the deprotection is finished, taking out part of resin for ninhydrin test, and heating the mixture in a water bath for 3min, wherein the resin is changed into bluish purple, which represents that the deprotection is successful.

5) And activating the second amino acid after the first amino acid is deprotected, wherein the activation step is carried out according to the step of the first amino acid, the activated amino acid is put into a peptide grafting tube, the second amino acid and the first amino acid are subjected to condensation reaction, ninhydrin detection is carried out after the reaction is finished, after the connection is successful, fmoc protection is removed, the next amino acid is continuously activated, and the operation steps are consistent with the previous amino acid until the connection of the last amino acid is finished.

7) Linking of Linker: for better attachment of FITC to the peptide chain, fluorenylmethyloxycarbonyl-6-aminocaproic Acid (ACP) was chosen as the attachment group for FITC fluorescein attachment. And (2) after the last amino acid is deprotected, adding 1mmol of ACP, adding an activating agent Oxyma, fully dissolving by NMP, adding DIC, mixing uniformly, activating the ACP for 1min, adding the activated ACP into a peptide connecting tube containing resin, reacting for 20min at 60 ℃ and 60R to connect the ACP with the peptide chain, washing the peptide chain for 5 times by DCM and DMF after connection, and detecting by ninhydrin to see whether the reaction is complete.

In the above steps, the formulation of ninhydrin detection reagent is shown in table 2:

TABLE 2 Ninhydrin test reagent preparation

The ninhydrin test method is used for qualitatively detecting whether the amino protecting group Fmoc in the amino acid used in the polypeptide synthesis process is removed and whether the amino acid condensation reaction is complete. When the amino group is exposed, the amino group can react with ninhydrin to generate color reaction, except that proline, hydroxyproline and ninhydrin react to generate yellow substance, all alpha-amino acids can react with ninhydrin to generate bluish purple substance. And (3) testing according to the reagent 1: and (2) mixing the reagents 2=2 and 1, adding the polypeptide to be detected into the ninhydrin detection reagent, heating in a water bath for 3min, and observing the color change of the polypeptide.

B. Fluorescent labeling of cell-penetrating peptide Opi1

1) Attachment of FITC: because the amino group on ACP also has an Fmoc protecting group, the Fmoc protection of the ACP needs to be removed before FITC is connected, after the deprotection of the ACP, 6 times of equivalent of FITC is weighed and dissolved by DMF, 12 times of equivalent of DIEA is added, a peptide connecting tube is added to enable the FITC and a peptide chain to carry out overnight photophobic reaction, the peptide connecting tube is taken out after the reaction is finished, DCM and DMF are used for washing the peptide connecting tube for 5 times, and the FITC which does not completely react and the used reagents are washed away.

3) Resin cutting: after all the amino acids in the sequence were linked, TFA, phenol, water and TIPS were prepared and cleaved, the cleavage solution was prepared according to 88% TFA, 5% phenol, 5% ddH2O and 2% TIPS, the prepared cleavage solution was added to a linker, and the solid phase resin carrier was separated from the desired peptide chain by vigorous shaking for 3 hours, and the protecting group of the amino acid side chain was removed.

4) Obtaining a crude polypeptide: and filtering the cut polypeptide into a centrifuge tube, blowing TFA by using nitrogen to remove TFA in the filtrate, then precipitating the peptide chain by using prepared ethyl glacial ether, centrifuging at 3000rpm for 5min, and discarding the supernatant to obtain the crude polypeptide.

C. Purification and analysis of cell-penetrating peptide Opi1 fluorescently labeled with FITC

And (3) purifying a crude polypeptide: dissolving crude polypeptide with water and acetonitrile at a certain ratio, filtering the dissolved polypeptide with 0.22 μm filter membrane, and performing LC-MS analysis on partial filtrate to determine the peak position of the target peptide segment and verify the molecular weight of the target peptide segment. After the LC-MS analysis was completed, LC analysis and purification of preparative LC were performed according to the elution gradient of table 3 below, and the purity and retention time of the polypeptide were calculated.

TABLE 3 LC elution gradient

The results of LC and ESI-MS analysis of different cell-penetrating peptides for Opi1 are shown in fig. 3 and 4, and are summarized in table 4 below:

TABLE 4 LC and ESI-MS analysis results of Opi1 different cell-penetrating peptides

Example 3 analysis of the transmembrane Activity of OpiCalcin1 series of transmembrane peptides

1. And (5) recovering the cells.

And (4) taking out the frozen HEK293T cells from a refrigerator at the temperature of-80 ℃, and quickly placing the frozen tube in water at the temperature of 37 ℃ to quickly shake and shake so as to quickly melt the frozen tube. Then wiping the mouth of the freezing tube with alcohol, sucking the cell suspension by a suction tube, transferring the cell suspension into a centrifuge tube, adding fresh culture medium, mixing uniformly, centrifuging at 800rpm for 5min. After the centrifugation is finished, the supernatant is discarded, and a proper amount of DMEM medium containing 10% fetal calf serum is added to the supernatant, and then the mixture is inoculated into a culture bottle and placed into CO ₂ Culturing in a moist heat sterile incubator. After 24 hours of culture, the cell growth morphology was observed.

2. Cell passage

From CO ₂ HEK293T cells are taken out from the moist-heat sterile incubator and observed under a microscope, and when the confluence degree of the cells reaches more than 85%, passage can be carried out. After aspirating the medium from the flask, the flask was washed with 3ml of sterile 1 XPBS. Adding 1ml pancreatin into the bottle for digestion, soaking the bottom surface, placing at 37 deg.C and CO ₂ And incubating in the incubator for 1-2min. After incubation, observing whether the cells become round and float under an inverted microscope, if the cells are completely digested, directly adding 2ml of complete culture medium into the culture flask, gently blowing the cells down by using a pipette, and sucking the cell mixed solution into a 15ml centrifuge tube. Centrifuging at 800rpm for 5min; centrifuging, discarding supernatant, uniformly beating cells with 1ml of complete culture medium, preparing 2T-25 culture bottles, adding 4ml of complete culture medium into the T-25 culture bottles, and then resuspendingThe cells were transferred to 2T-25 flasks. Finally, placing the culture flask after the cell seeding horizontally, shaking and mixing uniformly, placing at 37 ℃, and 5 percent of CO ₂ And (5) standing and culturing in an incubator.

3. Cell plate

From CO ₂ A bottle of HEK293T cells was removed from the incubator, aspirated of cell culture medium and washed with PBS. Then 1mL of pancreatin was added to the HEK293T cell culture flask for digestion for 1-2min. After the cells floated, 2mL of medium was added to stop the digestion, the cells were gently blown down with a pipette and transferred to a 15mL centrifuge tube. Centrifuging at 800rpm for 5min, removing supernatant after centrifuging, adding complete culture medium containing 10% fetal calf serum, gently blowing and beating cell suspension uniformly, mixing uniformly, adding cell suspension into 12-pore plate, and culturing in 37 deg.C incubator until adherent.

4. Cell uptake assay

After cell digestion counting, HEK293T cells are inoculated into a 24-well plate by using a serum-containing DMEM medium according to the number of 10000 cells per dish, cultured for 48 hours, and whether the cells are completely attached to the wall is observed under a microscope. After the cells are completely attached to the wall, the old culture medium is sucked away, and 1ml of serum-free culture medium is added into each hole for incubation for 2 hours. Then, the old medium was aspirated off, and 1ml of DMEM solution containing 10. Mu.M FITC-CPPs was added to each well, followed by incubation at 37 ℃ for 2 hours. After incubation, the cells were washed 2 times with PBS buffer. After fixing the cells by adding 200. Mu.l of cell fixing solution, the cells were washed 2 times with PBS, and then incubated with DAPI for 10min, and washed again 2 times with PBS. Fluorescence effects of FITC-CPPs and DAPI were observed using a fluorescence microscope, selecting the green channel and the blue channel.

5. Flow cytometry

After cell digestion counting, HEK293T cells were seeded in a 12-well cell plate in a number of 20 ten thousand cells per well in DMEM medium containing serum and cultured overnight. After observing the cell state under a microscope, the old medium was aspirated off, and l ml of serum-free medium was added to each well and incubated at 37 ℃ for 2 hours. Old medium was aspirated off, and to each well was added l ml of DMEM solution containing 10. Mu.M FITC-CPP, 3 duplicate wells per sample were set, and incubated at 37 ℃ for 2h. PBS buffer was added and washed 3 times. After digesting the cells with trypsin for 2min, the digestion was terminated by adding cold medium, and then gently pipetting the cells with a pipette and transferring the cells to a centrifuge tube at 1200rpm,4 ℃ for 5min. After centrifugation, the supernatant was discarded, cold 0.5ml PBS buffer was added, the cells were resuspended and transferred to a flow tube, and the FITC fluorescence intensity of each sample was measured on a flow cytometer.

Fluorescence microscopy revealed that positive controls FITC-TAT, FITC-Opi 1-33, FITC-Opi 1-22, FITC-Opi 1-33 penetrated cells better, while FITC-Opi 1-16, FITC-Opi 1-11, FITC-Opi 1-25-33 penetrated cells less effectively (FIG. 5).

The 9 peptide chains FITC-TAT, FITC-Opi 1-16, FITC-Opi 1-33, FITC-Opi 1-11, FITC-Opi 1-22, FITC-Opi 1-23-33, FITC-Opi 1-11, FITC-Opi 1-24, FITC-Opi 1-25-33 were incubated at a concentration of 10. Mu.M for flow cytometry analysis, and the intracellular mean fluorescence intensity was quantitatively analyzed by flow cytometry, and the results of the analysis showed: the intracellular mean fluorescence intensity of FITC-Opi 1-33 and FITC-Opi 1-33 is stronger than that of FITC-TAT in the positive control group, while the FITC of the unlinked polypeptide in the negative control group hardly fluoresces intracellularly, and the transmembrane effect of the rest polypeptides FITC-Opi 1-16, FITC-Opi 1-11, FITC-Opi 1-22, FITC-Opi 1-3, FITC-Opi 1-24 and FITC-Opi 1-33 is poorer (FIG. 6).

Example 4 cytotoxicity assay of OpiCalcin1 series of cell-penetrating peptides

Cell-penetrating peptides as a novel drug delivery vehicle need no or only low toxicity to cells, so we performed cytotoxicity analysis on 8 peptide chains of synthetic FITC-Opi 1-16, FITC-Opi 1-33, FITC-Opi 1-11, FITC-Opi 1-22, FITC-Opi 1-33, FITC-Opi 1-11, FITC-Opi 1-16, FITC-Opi 1-24, FITC-Opi 1-25-33, and a positive control polypeptide TAT to evaluate the safety of the polypeptides.

CCK8 cytotoxicity evaluation was used. After counting the cell digestions, HEK293T cells were seeded in 96-well cell culture plates at 8000 cells per well in DMEM medium containing serum and cultured overnight. After observing the cell state under a microscope, the old medium was aspirated, 100. Mu.l of a DMEM solution containing 10. Mu.M FITC-CPP was added to each well, 4 wells were set in parallel for each sample, and the samples were incubated at 37 ℃ for 2 hours, followed by addition of 10. Mu.l of WST-8 reagent and incubation in a cell incubator for 2 hours. Finally, the 96-well plate is placed on a microplate reader, and the absorption value is measured at the wavelength of 450 nm.

The cytotoxicity assay results are shown in figure 7: after incubating the cells with 10 μ M cell-penetrating peptide for two hours, the cell-penetrating peptides FITC-TAT, FITC-Opi 1-16, FITC-Opi 1-33, FITC-Opi 1-11 have almost no cytotoxicity, especially the effects of FITC-Opi 1-33, FITC-Opi 1-11 on the cells are smaller than that of the positive control peptide TAT, which is consistent with the characteristic of low toxicity of the cell-penetrating peptide; five polypeptides, FITC-Opi 1-22, FITC-Opi 1-23-33, FITC-Opi 1-11, FITC-Opi 1-24, and FITC-Opi 1-25-33, were slightly cytotoxic.

In conclusion, FITC-Opi 1-33 had better transmembrane effect than the positive control peptide TAT as analyzed by fluorescence microscopy and flow cytometry; the cytotoxicity of FITC-Opi 1-33 was found to be less than that of TAT, which is a positive control peptide, by CCK8 experiment, while the cell-penetrating effect of FITC-Opi 1-33 was also better than that of TAT, which is a positive control peptide, but the cytotoxicity of FITC-Opi 1-33 was greater than that of FITC-Opi 1-33 and TAT. In the case of low cytotoxicity requirements, the penetrating peptide may be selected because of its small sequence, ease of synthesis and human uptake, and the FITC-Opi 1-33 is preferred when the cytotoxicity requirements are high.

The present invention is not limited to the above embodiments, and those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are included in the scope of the present invention defined by the claims.

Claims (9)

1. A cell-penetrating peptide, wherein the amino acid sequence of the cell-penetrating peptide is shown as SEQ ID NO.1 or SEQ ID NO. 2.

2. The method for synthesizing a cell-penetrating peptide according to claim 1, comprising the steps of:

A. peptide fragment synthesized by Fmoc solid phase synthesis method

Amino resin or wang resin is taken as a solid phase carrier, oxyma is taken as an activating agent, DIC is taken as a condensing agent, and the condensation reaction is carried out between amino acids at the temperature of 60 +/-10 ℃; then, judging whether the condensation reaction is complete by using a ninhydrin detection reagent; after the reaction is completed, the tail end is connected with a connecting group so as to connect the fluorescein with the polypeptide,

B. fluorescent markers

After Fmoc protection of a connecting group is removed, adding excessive equivalent fluorescein and a reaction reagent in sequence to carry out fluorescein labeling; then using cutting fluid composed of TFA, phenol, water and TIPS to separate the target peptide chain from the solid phase resin carrier, completing crude polypeptide after precipitation treatment,

C. purification and analysis

Dissolving the crude polypeptide with water and acetonitrile in a certain proportion, filtering with a filter membrane, taking part of the filtrate, performing LC-MS analysis, determining the peak position of the target peptide segment, and verifying the molecular weight of the target peptide segment.

3. The method for synthesizing a cell-penetrating peptide according to claim 2, wherein:

wherein, step A includes the following substeps:

1) Resin preparation and swelling: weighing Rinkamide Resin with a substitution value of 0.43mmol/g and an Fmoc protecting group, adding dichloromethane according to a mass-to-volume ratio of 15-16, standing to fully swell the Resin, removing dichloromethane after swelling is finished, and washing the Resin for many times by using DMF (dimethyl formamide);

2) Removing Fmoc protection: adding 20% piperidine and 7.1% deprotection mixed reagent composed of Oxyma or DMF solvent for deprotection, adding the deprotection mixed reagent into the peptide-joining tube for the first time, placing the mixture into a 35 + -5 ℃ gas bath constant temperature oscillator for shaking, draining after the reaction is finished, adding the deprotection mixed reagent again, and oscillating under the same conditions; after the deprotection is finished, washing the resin for 3-5 times by using DCM and DMF in sequence, taking out part of the resin for ninhydrin test, and if the resin is changed into bluish purple after heating in a water bath, then the deprotection is successful;

3) C-terminal connection of the first amino acid: weighing the first amino acid at the C terminal, adding an activating agent Oxyma, fully dissolving the first amino acid by NMP, adding DIC, uniformly mixing the amino acids again, wherein the activation time of the amino acids is not more than 2min, adding the activated amino acids into a peptide connecting tube containing resin, and reacting for 20 +/-10 min at the temperature of 60 +/-10 ℃ and at the RPM of 60 +/-10 to perform condensation reaction on the amino acids and the resin; after the condensation reaction is finished, sequentially cleaning the mixture for 3-5 times by using DCM and DMF, dipping a small amount of numerical values by using a capillary tube, adding ninhydrin detection reagent for detection, and observing whether the reaction is complete;

4) Removing Fmoc protection: after the first amino acid at the C terminal is connected, the Fmoc protecting group of the amino group on the first amino acid needs to be removed so as to connect the amino acid with the next amino acid, and the reaction conditions are the same as those in step 2);

5) Activating a second amino acid after the first amino acid is deprotected, wherein the activation step is carried out according to the step of the first amino acid, the activated amino acid is put into a peptide grafting tube, the second amino acid and the first amino acid are subjected to condensation reaction, ninhydrin detection is carried out after the reaction is finished, after the connection is successful, fmoc protection is removed, the next amino acid is continuously activated, and the operation steps are consistent with the previous amino acid until the connection of the last amino acid is finished;

6) Linking with a Linker: selecting fluorenylmethyloxycarbonyl-6-aminocaproic acid as a connecting group to connect the FITC fluorescein; after the last amino acid is deprotected, sequentially adding fluorenylmethyloxycarbonyl-6-aminocaproic acid and an activating agent Oxyma, fully dissolving by NMP, adding DIC, and uniformly mixing for connecting group activation; adding activated ACP into a peptide connecting tube with resin, reacting for 20 +/-10 min at 60 +/-10 ℃ and 60 +/-10 RPM to connect a connecting group with a peptide chain, washing the peptide chain for 3-5 times by DCM and DMF after connection, and detecting by ninhydrin to see whether the reaction is complete.

4. The method for synthesizing a cell-penetrating peptide according to claim 2, wherein:

wherein, step B includes the following substeps:

1) FITC ligation: after carrying out deprotection on fluorenylmethyloxycarbonyl-6-aminocaproic acid, weighing FITC with 4-6 times of equivalent weight, dissolving the FITC with DMF, and then adding DIEA with 8-12 times of equivalent weight; performing overnight light-resistant reaction on FITC and a peptide chain, taking out a peptide connecting tube after the reaction is finished, washing the peptide connecting tube for 3-5 times by using DCM and DMF in sequence, and washing the unreacted FITC and used reagents;

2) Cutting resin: after completion of all the amino acid linkages in the sequence, according to 88% TFA, 5% phenol, 5% ₂ O, 2 percent, preparing a cutting fluid by TIPS, adding the prepared cutting fluid into a peptide connecting tube, violently shaking for 3 hours (no more than 4 hours), separating the solid-phase resin carrier from a target peptide chain, and removing a protecting group of an amino acid side chain;

3) Obtaining a crude polypeptide: and filtering the cut polypeptide into a centrifuge tube, blowing TFA by using nitrogen to remove TFA in the filtrate, then precipitating the peptide chain by using prepared ethyl glacial ether, centrifuging at 3000rpm for 5min, and discarding the supernatant to obtain the crude polypeptide.

5. The method for synthesizing a cell-penetrating peptide according to claim 2, wherein:

wherein, in the step C, the dissolved polypeptide is filtered by a filter membrane of 0.22 μm, part of the filtrate is taken for LC-MS analysis, LC analysis and purification of preparative LC are carried out according to the elution gradient shown in the following table after the LC-MS analysis is finished, the purity and retention time of the polypeptide are calculated,

6. use of a cell-penetrating peptide according to claim 1 for the preparation of a transporter.

7. Use according to claim 6, characterized in that:

wherein the active component of the transfer carrier comprises a cell-penetrating peptide or takes the cell-penetrating peptide as the only active component.

8. Use according to claim 6, characterized in that:

wherein, the transport carrier comprises a drug transport carrier, a nuclear localization sequence transport carrier, a probe transport carrier and a nanoparticle carrier.

9. A transport vector comprising the cell-penetrating peptide according to claim 1 as an active ingredient.

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