Preparation method and application of a scorpion toxin-derived transmembrane polypeptide

Abstract

The application relates to the technical field of biological medicine engineering, and provides a preparation method and application of a scorpion toxin-derived transmembrane polypeptide. The OpiCalcin1 polypeptide is optimized in a truncation mode, two polypeptides Opi1 17-33 CSKKCKRRGTNPEKRCR and Opi1 23-33 RRGTNPEKRCR with excellent transmembrane function and low cytotoxicity are provided. Through experiment verification, the two cell transmembrane peptides have excellent transmembrane effect and low cytotoxicity, meanwhile, the defects of time-consuming and laborious in synthesizing the complete sequence of scorpion toxin OpiCalcin1 are overcome, the cell transmembrane peptides can be used for preparing drug, nuclear localization sequence and probe transport carriers, and the targeting efficiency is improved. Compared with traditional scorpion toxin medicine, the synthesis of the scorpion toxin to explore the transmembrane activity provides a new selection for the drug carrier.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, specifically to a cell-penetrating peptide and its applications. Background Technology

[0002] Scorpions, also known as whole scorpions, are a key medicinal ingredient for treating wind-related ailments. They possess the effects of calming wind and relieving spasms, clearing the meridians and relieving pain, and attacking toxins and dispersing nodules. Ancient texts often describe the whole scorpion as the primary ingredient in medicine, but the medicinal value of scorpions lies mainly in their pharmacological toxin components. The active components of scorpion venom are mainly composed of two parts: protein and non-protein components. The non-protein components primarily contain mucopolysaccharides, histamine, serotonin, organic acids, and trace elements. The protein components, which play a major functional role, are mainly composed of small molecule polypeptides, commonly known as scorpion venom. These polypeptides typically consist of 20-80 amino acids, including several cysteine ​​residues. These cysteine ​​residues can interact to form multiple disulfide bonds, a structure that allows the scorpion venom polypeptides to exert their biological functions and enhances their stability.

[0003] Currently, over 300 scorpion venom polypeptides have been discovered. These polypeptides are ion channel inhibitors and possess biological activities such as anti-epileptic, anti-thrombotic, anti-tumor, and analgesic effects. In addition, some scorpion venom polypeptides also exhibit the characteristics of cell-penetrating peptides. The amino acid sequences of scorpion venom that can act as cell-penetrating peptides often contain a large number of basic groups such as arginine or lysine, allowing them to cross the cell membrane and enter the cell to exert their biological activities.

[0004] OpiCalcin1 is a 33-amino acid peptide toxin isolated from scorpions, possessing cell-penetrating capabilities. This peptide is composed of three disulfide bonds: Cys3-Cys17, Cys10-Cys21, and Cys16-Cys32. 1H NMR analysis revealed that OpiCalcin1 contains an ICK motif with a cysteine ​​residue and three β chains. OpiCalcin1 is a basic polypeptide; of its 33 amino acids, 11 are positively charged and 4 are negatively charged, resulting in a net positive charge. Furthermore, OpiCalcin1 is a hydrophilic polypeptide, with polar amino acid residues comprising 58% of its 33 amino acid residues.

[0005] This study focuses on the synthesis, purification, and transmembrane activity analysis of the scorpion venom OpiCalcin1 series of peptides, aiming to further explore their functions based on previous research and to repurpose the scorpion venom OpiCalcin1 to realize its new medicinal value. Summary of the Invention

[0006] Based on the above research, this invention synthesized different fragments of scorpion venom OpiCalcin1 (Opi1 for short) using a chemical synthesis method and fluorescently labeled them with fluorescein isothiocyanate. The membrane penetration effect of the Opi1 series of scorpion venom membrane-penetrating peptides was investigated. After comparison with TAT, a cell membrane-penetrating peptide Opi1 17-33 and Opi1 23-33 with better membrane penetration effect and lower toxicity was found. This cell membrane-penetrating peptide is expected to become a new type of cell membrane-penetrating peptide, providing a new option for drug carriers.

[0007] The primary objective of this invention is to optimize the OpiCalcin1 peptide in a truncated manner, providing two peptides, Opi1 17-33 CSKKCKRRGTNPEKRCR and Opi1 23-33RRGTNPEKRCR, which exhibit excellent membrane-penetrating capabilities and low cytotoxicity.

[0008] This invention analyzes the complete sequence of scorpion venom OpiCalcin1 and divides it into eight segments based on sequence length or base sequence: Opi1 1-16, Opi1 17-33, Opi1 1-11, Opi1 12-22, Opi1 23-33, FITC-Opi13-11, FITC-Opi1 16-24, and FITC-Opi1 25-33. The mature cell-penetrating peptide TAT is used as a positive control polypeptide. The amino acid sequences and molecular weights of each peptide segment and the positive control polypeptide are shown in Table 1 below.

[0009] Table 1. Sequences of different OpiCalcin1 peptide segments

[0010]

[0011] Flow cytometry results showed that Opi1 17-33 and Opi1 23-33, the cell-penetrating peptides, exhibited better membrane penetration, with significantly higher average intracellular fluorescence intensity than the positive control peptide TAT. Subsequently, cytotoxicity analysis of these nine peptides revealed that TAT, Opi1 1-16, Opi1 17-33, and Opi1 1-11 showed low cytotoxicity. In particular, peptide Opi1 17-33 not only demonstrated higher membrane penetration than TAT but also exhibited significantly lower cytotoxicity.

[0012] The second objective of this invention is to provide a method for preparing the above-mentioned cell-penetrating peptides, which mainly includes the following steps:

[0013] 1) The complete sequence of scorpion venom OpiCalcin1 was analyzed and divided into eight parts according to sequence length and base sequence: Opi1 1-16, Opi1 17-33, Opi1 1-11, Opi1 12-22, Opi1 23-33, FITC-Opi1 3-11, FITC-Opi1 16-24, and FITC-Opi1 25-33. The mature cell-penetrating peptide TAT was used as a positive control peptide.

[0014] 2) After the sequence is determined, the peptide is synthesized using the Fmoc solid-phase synthesis method: using amino resin or Wang resin as the solid-phase carrier, Oxyma is selected as the activator and DIC as the condensing agent. The amino acids are condensed at 60°C. The ninhydrin test reagent is then used to determine whether the condensation reaction is complete. If the reaction is complete, the resin with amino acids and the ninhydrin test reagent will not show color under water bath heating conditions. If the reaction is incomplete, it will show blue-purple color. In this case, the reaction needs to be repeated.

[0015] 3) After the crude peptide is successfully linked according to the amino acid sequence, a linker group needs to be attached to the end to link the fluorescein to the peptide. Fluorenylmethoxycarbonyl-6-aminohexanoic acid (ACP) is selected as the linker group to link the FITC fluorescein, so that the peptide is labeled with green fluorescence.

[0016] 4) Purify and analyze the fluorescently labeled peptides: Analyze and purify them using liquid chromatography in a C18 column. Determine the position of the target peak using LC-MS, collect the target peaks according to the elution time, and determine their purity using analytical LC. Then, freeze-dry the pure transmembrane peptides using a lyophilizer. The obtained fluorescent transmembrane peptides should be stored away from light to avoid fluorescence quenching.

[0017] 5) The cell-penetrating peptides synthesized and purified in the early stage were analyzed for their cell-penetrating activity on HEK293T cells: The cells incubated with the cell-penetrating peptides were analyzed qualitatively and quantitatively using fluorescence microscopy and flow cytometry to determine the cell-penetrating effect of the cell-penetrating peptides.

[0018] 6) The CCK8 assay was used to analyze the cytotoxicity of the previously synthesized cell-penetrating peptides.

[0019] A third objective of this invention is to provide the application of cell-penetrating peptides, specifically their application in the preparation of transport carriers. The active ingredient in this transport carrier may include cell-penetrating peptides and other active ingredients, or it may use cell-penetrating peptides as the sole active ingredient.

[0020] Preferably, the transport vector includes a drug transport vector, a nuclear localization sequence transport vector, or a probe transport vector.

[0021] Because cell-penetrating peptides can enter cells without causing any damage and can be successfully used as carriers for siRNA, nucleotides, macromolecular drugs, proteins, etc., they have been widely used in the field of novel drug carriers.

[0022] In addition, CPPs have received attention for their applications in cell imaging, nuclear localization, pH sensitivity, and thermal targeted delivery.

[0023] In cell imaging, cell-penetrating peptides (CPPs) are used to promote internalization by capturing labeled peptide probes at target sites. The high efficiency of CPPs makes them a promising tool in the imaging field. In nuclear localization, to improve nuclear localization efficiency, CPPs with nuclear localization sequences (NLSSs) can be directly or indirectly linked to DNA or gene vectors to facilitate nuclear localization.

[0024] In addition, due to the pH gradient between the tumor environment and the physiological environment, cell-penetrating peptides can exert a controlling effect in the tumor microenvironment by linking with nanoparticles, such as through acid degradation cross-linking, removal of the protective layer, or enzymatic degradation. When cell-penetrating peptides are linked with drug-containing nanoparticles, the therapeutic efficiency of the nanoparticles is improved and the drug toxicity is reduced.

[0025] The fourth objective of this invention is to provide a transport carrier that uses the aforementioned cell-penetrating peptide as an active ingredient to transport drugs such as siRNA, nucleotides, macromolecular drugs, and proteins, probes, nuclear localization sequences (NLSS), and drug-containing nanoparticles into cells to achieve corresponding therapeutic effects.

[0026] The beneficial protections and effects of this invention are as follows:

[0027] This invention provides a cell-penetrating peptide, its preparation method, and its application. By truncating the OpiCalcin1 peptide, two peptides with excellent membrane-penetrating function and low cytotoxicity, Opi1 17-33CSKKCKRRGTNPEKRCR and Opi1 23-33 RRGTNPEKRCR, are optimized. Experimental verification shows that the cell-penetrating peptides of this invention have excellent membrane-penetrating effect and low cytotoxicity, while also overcoming the time-consuming and laborious drawbacks of synthesizing the complete OpiCalcin1 scorpion venom sequence.

[0028] The cell-penetrating peptides screened in this invention can be used to prepare delivery vectors for drugs, nuclear localization sequences, and probes, thereby improving targeting efficiency. Compared with the traditional use of scorpion venom as a drug, synthesizing scorpion venom to explore its cell-penetrating activity provides a new option for drug delivery. Attached Figure Description

[0029] Figure 1Synthetic routes for Opi1 cell-penetrating peptides: (A) FITC-Opi1 1-16 synthesis route; (B) FITC-Opi1 17-33 synthesis route; (C) FITC-Opi1 1-11 synthesis route; (D) FITC-Opi1 12-22 synthesis route; (E) FITC-Opi1 23-33 synthesis route.

[0030] Figure 2 Synthetic routes for TAT and Opi1 cell-penetrating peptides: (A) FITC-TAT synthesis route; (B) FITC-Opi1 3-11 synthesis route; (C) FITC-Opi1 16-24 synthesis route; (D) FITC-Opi1 25-33 synthesis route.

[0031] Figure 3 LC and ESI-MS analysis results of different cell-penetrating peptides of Opi1: (A) LC spectrum of crude FITC-Opi1 1-16 peptide, with a target peak elution time of 26.053 min; (B) ESI-MS result of FITC-Opi1 1-16, with a molecular weight of 2346.6580; (C) Purification result of FITC-Opi1 1-16 peptide, with a purity of 97.071%; (D) LC spectrum of crude FITC-Opi1 17-33 peptide, with a target peak elution time of 13.981 min; (E) ESI-MS result of FITC-Opi1 17-33, with a molecular weight of 2552.0160; (F) FITC-Opi1 The purification results of peptide 17-33 were as follows: purity: 95.872%; (G) LC spectrum of crude FITC-Opi11-11 peptide, target peak elution time: 38.213 min; (H) ESI-MS results of FITC-Opi11-11, molecular weight: 1771.1080; (I) Purification results of FITC-Opi11-11 peptide, purity: 99.586%; (J) LC spectrum of crude FITC-Opi112-22 peptide, target peak elution time: 16.112 min; (K) ESI-MS results of FITC-Opi112-22, molecular weight: 1773.0030; (L) Purification results of FITC-Opi112-22 peptide, purity: 96.668%; (M) FITC-Opi11 LC spectrum of crude peptide 23-33, with target peak elution time of 32.805 min; ESI-MS result of (N)FITC-Opi1 23-33, molecular weight of 1875.1190; purification result of (O)FITC-Opi1 23-33 peptide, purity of 91.655%.

[0032] Figure 4LC and ESI-MS analysis results of TAT and Opi1 cell-penetrating peptides: (A) LC spectrum of crude FITC-TAT peptide, target peak elution time is 29.787 min; (B) ESI-MS result of FITC-TAT, molecular weight is 2061.0704; (C) Purification result of FITC-TAT peptide, purity is 99.204%; (D) LC spectrum of crude FITC-Opi1 3-11 peptide, target peak elution time is 33.833 min; (E) ESI-MS result of FITC-Opi1 3-11, molecular weight is 1597.7356; (F) Purification result of FITC-Opi1 3-11 peptide, purity is 99.586%; (G) FITC-Opi1 LC spectrum of crude FITC-Opi1 16-24 peptide, with a target peak elution time of 31.247 min; (H) ESI-MS result of FITC-Opi1 16-24, molecular weight 1612.6771; (I) Purification result of FITC-Opi1 16-24 peptide, purity: 96.370%; (J) LC spectrum of crude FITC-Opi1 25-33 peptide, with a target peak elution time of 31.318 min; (K) ESI-MS result of FITC-Opi1 25-33, molecular weight 1561.6442; (L) Purification result of FITC-Opi1 25-33 peptide, purity: 92.106%.

[0033] Figure 5 Comparison of cellular uptake results of FITC-labeled TAT and different transmembrane peptides of the Opi1 series in HEK293T cells at a concentration of 10 μM (scale bar is 100px).

[0034] Figure 6 Comparison of flow cytometry results of FITC-labeled TAT and different membrane-penetrating peptides of the Opi1 series in HEK293T cells at a concentration of 10 μM.

[0035] Figure 7 Comparison of cytotoxicity of different transmembrane peptides from the TAT and Opi1 series in HEK293T cells. Detailed Implementation

[0036] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0037] Experimental methods in the following examples, unless otherwise specified, were generally performed under standard conditions or as recommended by the manufacturer. Percentages and parts are by volume unless otherwise defined. All technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0038] Example 1: Synthesis of Opi1 polypeptide fragment

[0039] Based on the different lengths of the OpiCalcin1 sequence, it was divided into five peptide segments: Opi1 1-16, Opi1 17-33, Opi1 1-11, Opi1 12-22, and Opi1 23-33. The synthesized peptide segments were then purified and prepared for use in membrane penetration experiments.

[0040] Furthermore, a common characteristic of most cell-penetrating peptides is the presence of a large number of basic amino acids in their amino acid sequences. Analysis of the OpiCalcin1 sequence revealed three base motifs within its 33 amino acids: Opi1 8-11 KRCK, Opi1 19-24 KKCKRR, and Opi1 30-33 KRCR. Therefore, we synthesized new peptide segments based on these three base motifs. According to the distribution of the OpiCalcin1 base motifs, the OpiCalcin1 sequence was divided into three segments: Opi1 3-11, Opi1 16-24, and Opi1 25-33.

[0041] The sequences of the eight peptides mentioned above are shown in Table 1. The eight peptides were synthesized using the Fmoc solid-phase synthesis method and labeled with FITC fluorescence for subsequent membrane penetration activity experiments. The cell membrane penetration peptide TAT was selected as a positive control peptide.

[0042] Example 2: Synthesis and Fluorescent Labeling of Cell-Penetrating Peptides

[0043] The synthesis methods for cell-penetrating peptides with different sequences are similar. The synthetic routes for FITC-Opi1 1-16, FITC-Opi1 17-33, FITC-Opi1 1-11, FITC-Opi1 12-22, and FITC-Opi1 23-33 are shown below. Figure 1 The synthetic routes for FITC-Opi1 3-11, FITC-Opi1 16-24, FITC-Opi1 25-33, and the positive reference peptide FITC-TAT are described in [link to synthetic routes]. Figure 2 The specific synthesis process is as follows:

[0044] A. Synthesis of cell-penetrating peptide Opi1

[0045] 1) Resin preparation and swelling: Weigh 400 mg of Rink Amide Resin (substitution value = 0.43 mmol / g) and place it in a peptide synthesis tube. Add 25 ml of dichloromethane (DCM) and let it stand for 30 min to allow it to swell completely. After swelling is complete, remove the DCM and rinse the tube 5 times with N,N-dimethylformamide (DMF). After the operation, the resin layer will be thickened.

[0046] 2) Deprotection with Fmoc: The Rink Amide Resin used has an Fmoc protecting group, which needs to be removed to allow the amino acids to connect to the resin. For deprotection, 20% piperidine / 7.1% Oxyma / DMF solvent is added. For the first deprotection, 6 ml of the deprotection mixture is added to the peptide bond tube and shaken in a 35°C air bath for 5 minutes. After the reaction, the solution is dried, and another 6 ml of the deprotection mixture is added and shaken in a 35°C air bath for 5 minutes. After deprotection, the solution is washed five times sequentially with DCM and DMF. A portion of the resin is then tested for ninhydrin. If the resin turns blue-purple after heating in a water bath for 3 minutes, deprotection is successful.

[0047] 3) Activation of the first C-terminal amino acid: Weigh 1 mmol of the first C-terminal amino acid, add the activator Oxyma, dissolve it thoroughly with NMP, then add DIC and mix again. Activate the amino acid for 1 min. Add the activated amino acid to the peptide bond tube containing resin and react at 60℃, 60 rpm for 20 min to allow the amino acid to condense with the resin. After the condensation reaction, wash five times with DCM and DMF sequentially. Then, use a capillary tube to take a small amount of the solution and add ninhydrin reagent for detection. If the resin does not turn blue-purple, it indicates that the amino acid and the carrier resin are successfully linked. If there is partial color change, it indicates that the amino acid condensation reaction is incomplete. In this case, weigh 1 mmol of amino acid again, add Oxyma activator and DIC condenser, and re-add it to the peptide bond tube for reaction. The reaction conditions remain 60℃, 60 rpm for 20 min. After the reaction, continue to use ninhydrin reagent to detect whether the reaction is complete.

[0048] 4) Deprotection with Fmoc: After linking the first amino acid at the C-terminus, the Fmoc protecting group on the amino group of the first amino acid needs to be removed to allow the amino acid to link with the next amino acid. For deprotection, 20% piperidine / 7.1% Oxyma / DMF solvent is added. For the first deprotection, 6 ml of the deprotection mixture is added to the peptide bond tube and shaken in a 35°C air bath for 5 minutes. After the reaction, the mixture is dried, and another 6 ml of the deprotection mixture is added and shaken in a 35°C air bath for 5 minutes. After deprotection, the resin is washed five times sequentially with DCM and DMF. A portion of the resin is then tested for ninhydrin. If the resin turns blue-purple after heating in a water bath for 3 minutes, deprotection is successful.

[0049] 5) After the first amino acid is deprotected, the second amino acid is activated, following the same steps as the first amino acid. The activated amino acid is then added to the peptide bond tube, allowing the second amino acid to undergo a condensation reaction with the first amino acid. After the reaction, ninhydrin detection is performed. If the ligation is successful, the amino acid is deprotected from Fmoc and the next amino acid is activated, following the same steps as the previous amino acid, until the ligation of the last amino acid is completed.

[0050] 7) Linker Ligation: To better link FITC to the peptide chain, we selected fluorenemethoxycarbonyl-6-aminohexanoic acid (ACP) as the linker group for FITC fluorescein. After deprotecting the last amino acid, 1 mmol of ACP was added, along with the activator Oxyma. After thorough dissolution with NMP, DIC was added and mixed again. The ACP was activated for 1 min. The activated ACP was then added to a peptide linker tube containing resin and reacted at 60℃ and 60 rpm for 20 min to link the ACP to the peptide chain. After linking, the peptide chain was washed five times sequentially with DCM and DMF, and then ninhydrin was used to detect whether the reaction was complete.

[0051] The preparation of the ninhydrin detection reagent in the above steps is shown in Table 2:

[0052] Table 2 Preparation of ninhydrin test reagent

[0053]

[0054] The ninhydrin assay is used for qualitative testing to determine whether the amino protecting group (Fmoc) of amino acids used in peptide synthesis has been removed, and whether the amino acid condensation reaction has been completed. When the amino group is exposed, it reacts with ninhydrin to produce a color reaction. Except for proline and hydroxyproline, which react with ninhydrin to produce a yellow substance, all α-amino acids react with ninhydrin to produce a blue-purple substance. In the test, reagent 1 and reagent 2 are mixed in a ratio of 2:1. The peptide to be tested is added to the ninhydrin assay reagent, heated in a water bath for 3 minutes, and the color change is observed.

[0055] B. Fluorescent labeling of Opi1, a cell-penetrating peptide.

[0056] 1) FITC ligation: Since the amino group on ACP also has Fmoc protecting group, ACP needs to be deprotected before FITC ligation. After ACP is deprotected, weigh 6 equivalents of FITC, dissolve it in DMF, add 12 equivalents of DIEA, and add it to the peptide initiation tube to allow the FITC and peptide chain to react overnight in the dark. After the reaction is complete, take out the peptide initiation tube and wash it 5 times with DCM and DMF to wash away the unreacted FITC and the reagents used.

[0057] 3) Resin cleavage: After all amino acids in the sequence are linked, prepare TFA, phenol, water and TIPS for cleavage. Prepare the cleavage solution according to 88% TFA, 5% phenol, 5% ddH2O and 2% TIPS. Add the prepared cleavage solution to the peptide linker tube and shake vigorously for 3 hours to separate the solid resin support from the target peptide chain and remove the protecting groups of the amino acid side chains.

[0058] 4) Obtaining crude peptides: After the peptides have been cut, filter them into centrifuge tubes, blow TFA with nitrogen to remove TFA from the filtrate, and then precipitate the peptide chains with prepared ice-cold ether. Centrifuge at 3000 rpm for 5 min, discard the supernatant, and the precipitate is the crude peptide.

[0059] C. Purification and analysis of FITC-labeled cell-penetrating peptide Opi1.

[0060] Purification of crude peptides: The crude peptides were dissolved in a certain proportion of water and acetonitrile. The dissolved peptides were filtered through a 0.22 μm filter membrane. A portion of the filtrate was analyzed by LC-MS to determine the peak position of the target peptide and verify its molecular weight. After LC-MS analysis, LC analysis and preparative LC purification were performed according to the elution gradient in Table 3 below. The purity and retention time of the peptides were calculated.

[0061] Table 3 LC elution gradient

[0062]

[0063] LC and ESI-MS analysis results of different cell-penetrating peptides of Opi1 are shown in [reference]. Figure 3 and Figure 4 The results are summarized in Table 4 below:

[0064] Table 4. LC and ESI-MS analysis results of different cell-penetrating peptides of Opi1

[0065]

[0066]

[0067] Example 3: Membrane activity analysis of OpiCalcin1 series transmembrane peptides

[0068] 1. Cell resuscitation.

[0069] HEK 293T cells were removed from the -80℃ freezer and rapidly thawed by shaking in 37℃ water. The cryovials were then wiped with alcohol, and the cell suspension was aspirated and transferred to a centrifuge tube. Fresh culture medium was added and mixed well. The tubes were centrifuged at 800 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and an appropriate amount of DMEM medium containing 10% fetal bovine serum was added. The cells were then seeded into culture flasks and placed in a CO2-controlled aseptic incubator. Cell growth and morphology were observed after 24 hours of culture.

[0070] 2. Cell passage

[0071] HEK293T cells were removed from a CO2-heated sterile incubator and observed under a microscope. When cell confluence reached 85% or higher, passage was possible. After aspirating the culture medium from the flask, the cells were washed with 3 ml of sterile 1×PBS. Then, 1 ml of trypsin was added to the flask for digestion, and the cells were incubated at 37°C in a CO2 incubator for 1–2 minutes. After incubation, the cells were observed under an inverted microscope to see if they had become rounded and floated. If all cells had been digested, 2 ml of complete culture medium was added directly to the culture flask, and the cells were gently blown off with a pipette. The cell mixture was then transferred to a 15 ml centrifuge tube. The cells were centrifuged at 800 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and the cells were thoroughly mixed with 1 ml of complete culture medium. Two T-25 culture flasks were prepared, each containing 4 ml of complete culture medium. The resuspended cells were then transferred into both T-25 culture flasks. Finally, place the culture flasks containing the seeded cells horizontally, shake to mix, and then incubate at 37°C in a 5% CO2 incubator.

[0072] 3. Cell seeding plate

[0073] Remove a flask of HEK 293T cells from the CO2 incubator, aspirate the cell culture medium, and wash the cells with PBS. Then, add 1 mL of trypsin to the HEK 293T cell culture flask and digest for 1-2 minutes. Once the cells float, add 2 mL of culture medium to stop the digestion. Gently pipette the cells off and transfer them to a 15 mL centrifuge tube. Centrifuge at 800 rpm for 5 minutes. Discard the supernatant, add complete culture medium containing 10% fetal bovine serum, and gently pipette the cell suspension until well mixed. Add the cell suspension to a 12-well plate and incubate at 37°C until adherent.

[0074] 4. Cell uptake experiment

[0075] After cell digestion and counting, HEK293T cells were seeded at a rate of 10,000 cells per well in serum-containing DMEM medium and cultured for 48 h. Cell adhesion was observed under a microscope. After complete cell adhesion, the old medium was removed, and 1 ml of serum-free medium was added to each well for incubation for 2 h. Then, the old medium was removed, and 1 ml of DMEM solution containing 10 μM FITC-CPPs was added to each well, and the cells were incubated at 37°C for 2 h. After incubation, the cells were washed twice with PBS buffer. 200 μl of cell fixation solution was added to fix the cells, followed by washing twice with PBS, incubation with DAPI for 10 min, and washing twice more with PBS. The fluorescence effects of FITC-CPPs and DAPI were observed using a fluorescence microscope, selecting the green and blue channels.

[0076] 5. Flow cytometry experiment

[0077] After cell digestion and counting, HEK 293T cells were seeded in 12-well agar plates at a density of 200,000 cells per well using DMEM medium containing serum and cultured overnight. After observing good cell condition under a microscope, the old medium was removed, and 1 ml of serum-free medium was added to each well. The cells were incubated at 37°C for 2 hours. The old medium was then removed, and 1 ml of DMEM solution containing 10 μM FITC-CPP was added to each well. Three replicates were prepared for each sample, and the cells were incubated at 37°C for 2 hours. The cells were washed three times with PBS buffer. After trypsin digestion for 2 minutes, cold medium was added to stop the digestion. The cells were then gently pipetted and transferred to centrifuge tubes. The cells were centrifuged at 1200 rpm, 4°C, for 5 minutes. After centrifugation, the supernatant was discarded, and 0.5 ml of cold PBS buffer was added to resuspend the cells. The cells were then transferred to flow cytometry tubes, and the FITC fluorescence intensity of each sample was measured using a flow cytometer.

[0078] Fluorescence microscopy revealed that the positive control groups FITC-TAT, FITC-Opi1 17-33, FITC-Opi1 12-22, and FITC-Opi1 23-33 exhibited better cell penetration, while FITC-Opi1 1-16, FITC-Opi1 1-11, and FITC-Opi1 25-33 showed poorer cell penetration. Figure 5 ).

[0079] Nine peptide chains, namely FITC-TAT, FITC-Opi1 1-16, FITC-Opi1 17-33, FITC-Opi1 1-11, FITC-Opi1 12-22, FITC-Opi1 23-33, FITC-Opi1 3-11, FITC-Opi1 16-24, and FITC-Opi1 25-33, were incubated with cells at a concentration of 10 μM and analyzed by flow cytometry. The intracellular mean fluorescence intensity was quantitatively analyzed by flow cytometry. The results showed that the intracellular mean fluorescence intensity of FITC-Opi1 17-33 and FITC-Opi1 23-33 was stronger than that of the positive control group FITC-TAT, while the negative control group FITC without peptides showed almost no intracellular fluorescence. The remaining peptides FITC-Opi1 1-16, FITC-Opi1 1-11, and FITC-Opi1... The film penetration effect of FITC-Opi1 12-22, FITC-Opi1 3-11, FITC-Opi1 16-24, and FITC-Opi1 25-33 is relatively poor. Figure 6 ).

[0080] Example 4: Cytotoxicity analysis of OpiCalcin1 series transmembrane peptides

[0081] Cell-penetrating peptides, as novel drug delivery carriers, need to have no or low toxicity to cells. Therefore, we performed cytotoxicity analysis on eight synthesized peptide chains (FITC-Opi1 1-16, FITC-Opi1 17-33, FITC-Opi1 1-11, FITC-Opi1 12-22, FITC-Opi1 23-33, FITC-Opi1 3-11, FITC-Opi1 16-24, and FITC-Opi1 25-33) and the positive control peptide TAT to assess the safety of the peptides.

[0082] CCK-8 cytotoxicity was evaluated. After cell digestion and counting, HEK293T cells were seeded at a density of 8000 cells per well in DMEM medium containing serum and cultured overnight. After observing good cell condition under a microscope, the old medium was aspirated, and 100 μl of DMEM solution containing 10 μM FITC-CPP was added to each well. Four replicates were set up for each sample, and the cells were incubated at 37°C for 2 h. Then, 10 μl of WST-8 reagent was added, and the cells were cultured in a cell culture incubator for 2 h. Finally, the 96-well plate was placed on a microplate reader, and the absorbance was measured at a wavelength of 450 nm.

[0083] Cytotoxicity analysis results as follows Figure 7 As shown, after incubating cells with 10 μM cell-penetrating peptides for two hours, the cell-penetrating peptides FITC-TAT, FITC-Opi1 1-16, FITC-Opi1 17-33, and FITC-Opi1 1-11 showed almost no cytotoxicity. In particular, FITC-Opi1 17-33 and FITC-Opi1 1-11 had a smaller effect on cells than the positive control peptide TAT, consistent with the low cytotoxicity of cell-penetrating peptides. However, the five peptides FITC-Opi1 12-22, FITC-Opi1 23-33, FITC-Opi1 3-11, FITC-Opi1 16-24, and FITC-Opi1 25-33 showed slight cytotoxicity.

[0084] In summary, fluorescence microscopy and flow cytometry analysis showed that FITC-Opi1 17-33 and FITC-Opi1 23-33 exhibited better membrane penetration than the positive control peptide TAT. CCK8 assays revealed that FITC-Opi1 17-33 showed lower cytotoxicity than TAT. While FITC-Opi1 23-33 also demonstrated better cell penetration than TAT, its cytotoxicity was relatively higher than both FITC-Opi1 17-33 and TAT. For applications where cytotoxicity requirements are not high, FITC-Opi1 23-33, with its small sequence size, ease of synthesis, and human absorption, is a suitable choice. For applications requiring high cytotoxicity, FITC-Opi1 17-33 is the preferred option.

[0085] The examples of the present invention have been specifically described above, but the present invention is not limited to the embodiments described. 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 all included within the scope defined by the claims of this application.

Claims

1. A cell-penetrating peptide, characterized in that, The amino acid sequence of this cell-penetrating peptide is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The method for synthesizing the conjugate of cell-penetrating peptide and fluorescein according to claim 1, characterized in that, Includes the following steps: A. Fmoc solid-phase synthesis method for peptide synthesis Using amino resin or wang resin as the solid-phase support, Oxyma as the activator, and DIC as the condensing agent, a condensation reaction was induced between amino acids at 60±10℃. The completeness of the condensation reaction was then determined using a ninhydrin assay. After complete reaction, a linker group was attached to the end of the peptide to allow the fluorescein to connect with the peptide. B. Fluorescent labeling After the linker group was deprotected from Fmoc, excess equivalents of fluorescein and reaction reagents were added sequentially for fluorescein labeling. Then, a cleavage buffer consisting of TFA, phenol, water, and TIPS was used to separate the target peptide chain from the solid-phase resin support. After precipitation, the crude peptide was obtained. C. Purification and Analysis The crude peptide was dissolved in a certain proportion of water and acetonitrile, filtered through a filter membrane, and a portion of the filtrate was analyzed by LC-MS to determine the peak position of the target peptide and verify the molecular weight of the target peptide.

3. The method for synthesizing the conjugate of cell-penetrating peptide and luciferin according to claim 2, characterized in that: in, Step A includes the following sub-steps: 1) Resin preparation and swelling: Weigh RinkAmide Resin with a substitution value of 0.43 mmol / g and Fmoc protecting group, add dichloromethane at a mass-volume ratio of 15-16:1, let it stand to allow it to swell fully, remove the dichloromethane after swelling is complete, and rinse it several times with DMF. 2) Deprotection with Fmoc: Deprotection was performed by adding a deprotection mixture consisting of 20% piperidine and 7.1% Oxyma or DMF solvent. The first time, the deprotection mixture was added to the peptide inoculation tube and shaken in a 35±5℃ gas bath constant temperature shaker. After the reaction was completed, the tube was dried and the deprotection mixture was added again and shaken under the same conditions. After deprotection, the tube was washed with DCM and DMF 3 to 5 times in sequence. Then, a portion of the resin was taken out for ninhydrin test. If the resin turned blue-purple after heating in a water bath, it indicated that the deprotection was successful. 3) C-terminal ligation of the first amino acid: Weigh the first amino acid at the C-terminus, add the activator Oxyma, dissolve it thoroughly with NMP, then add DIC and mix again. The amino acid activation time should not exceed 2 minutes. Add the activated amino acid to the peptide initiation tube containing resin and react at 60±10℃ and 60±10 RPM for 20±10 minutes to allow the amino acid to condense with the resin. After the condensation reaction, wash with DCM and DMF 3 to 5 times in sequence. Then, use a capillary tube to take a small amount of the solution and add ninhydrin detection reagent to detect whether the reaction is complete. 4) Deprotection with Fmoc: After attaching the first amino acid at the C-terminus, the Fmoc protecting group on the amino group of the first amino acid needs to be removed so that the amino acid can be attached to the next amino acid. The reaction conditions are the same as in step 2). 5) After the first amino acid is deprotected, the second amino acid is activated. The activation steps are the same as those for the first amino acid. The activated amino acid is then put into the peptide bond tube to allow the second amino acid to undergo a condensation reaction with the first amino acid. After the reaction is complete, ninhydrin is detected. After successful ligation, Fmoc protection is removed and the next amino acid is activated. The operation steps are the same as those for the previous amino acid until the ligation of the last amino acid is completed. 6) Linker ligation: Fluorenylmethoxycarbonyl-6-aminohexanoic acid (ACA) was selected as the linker group to FITC fluorescein. After deprotection of the last amino acid, ACA and activator Oxyma were added sequentially. After thorough dissolution with NMP, DIC was added and mixed again to activate the linker group. The activated ACP was added to a peptide bonder tube containing resin and reacted at 60±10℃ and 60±10 RPM for 20±10 min to allow the linker group to connect with the peptide chain. After ligation, the peptide chain was washed 3-5 times with DCM and DMF, and then ninhydrin was used to detect whether the reaction was complete.

4. The method for synthesizing the conjugate of cell-penetrating peptide and luciferin according to claim 2, characterized in that: in, Step B includes the following sub-steps: 1) FITC ligation: After deprotection of fluorenylmethoxycarbonyl-6-aminohexanoic acid, weigh 4 to 6 equivalents of FITC, dissolve it in DMF, and then add 8 to 12 equivalents of DIEA; FITC and peptide chain are reacted overnight in the dark. After the reaction is completed, remove the peptide incorporation tube and rinse it with DCM and DMF 3 to 5 times in sequence to wash away the unreacted FITC and the reagents used. 2) Resin cleavage: After all amino acids in the sequence are ligated, the cleavage solution is prepared according to 88% TFA, 5% phenol, 5% ddH2O and 2% TIPS. The prepared cleavage solution is added to the peptide incorporation tube and shaken vigorously for 3 hours to separate the solid resin support from the target peptide chain and remove the protecting groups of the amino acid side chains. 3) Obtaining crude peptides: After the peptides have been cut, filter them into centrifuge tubes, blow TFA with nitrogen to remove TFA from the filtrate, and then precipitate the peptide chains with prepared ice-cold ether. Centrifuge at 3000 rpm for 5 min, discard the supernatant, and the precipitate is the crude peptide.

5. The method for synthesizing the conjugate of cell-penetrating peptide and luciferin according to claim 2, characterized in that: in, In step C, the dissolved peptide was filtered through a 0.22 μm filter membrane, and a portion of the filtrate was analyzed by LC-MS. After the LC-MS analysis was completed, LC analysis and preparative LC purification were performed according to the elution gradient in the table below, and the purity and retention time of the peptide were calculated.

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