Self-assembled polypeptide targeting mitochondria, preparation method and application of self-assembled polypeptide, self-assembled polypeptide microneedle patch and preparation method of self-assembled polypeptide microneedle patch

Abstract

The invention belongs to the technical field of biological medicine, and provides self-assembled polypeptide targeting mitochondria, a preparation method and application of the self-assembled polypeptide, a self-assembled polypeptide microneedle patch and a preparation method of the self-assembled polypeptide microneedle patch. According to the self-assembly polypeptide of the targeted mitochondria, the amino acid sequence is GFFGKXRXKXRX, and X represents cyclohexyl alanine. A targeting peptide (Mito) sequence KXRXKXRX can recognize cell mitochondria in a targeting manner, a self-assembly sequence GFFG is introduced to an N terminal of the Mito sequence KXRXKXRX, and the targeting peptide sequence is endowed with a self-assembly capability, so that the self-assembly polypeptide can cause morphological function abnormality of mitochondria in tumor cells, the tumor cells are killed, and inhibition of the tumor cells is realized. The self-assembled polypeptide disclosed by the invention is applied to treatment of tumor cells through in-situ administration of the self-assembled polypeptide microneedle patch. Oral squamous cell carcinoma serves as a highly proliferated tumor cell, rapid proliferation of the oral squamous cell carcinoma needs a large amount of energy supply for mitochondria, and therefore attacking of the mitochondria can serve as an effective treatment target.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a mitochondrial-targeting self-assembling polypeptide, a preparation method and application thereof, a self-assembling polypeptide microneedle patch, and a preparation method thereof. Background Art

[0002] Over 90% of oral cancers are oral squamous cell carcinoma (OSCC), defined as an invasive epithelial tumor with varying degrees of differentiation. It is the most common malignant tumor of the oral and maxillofacial region. Delayed diagnosis reduces the 5-year survival rate to less than 60%. Furthermore, the annual rate of secondary tumor formation, at 3% to 7%, is unmatched by other malignancies and can lead to recurrence.

[0003] Current clinical treatment options include surgery, radiotherapy, and chemotherapy. However, surgery is generally only suitable for certain solid tumors and may cause facial tissue loss. Chemotherapeutic drugs can easily lead to multidrug resistance and off-target effects, and radiotherapy often has significant toxic side effects. Consequently, the mortality rate of OSCC remains high.

[0004] Therefore, precise and effective targeted treatment methods and technologies are the best approach for anti-tumor treatment. In recent years, targeting important subcellular compartments as a method for cancer treatment has become a hot topic in research due to its rich organelle-specific conjugates and highly precise and effective therapeutic effects. For example, the Au@Pt-Ce6-HN-1 nanozyme can target oral squamous cell carcinoma, but the specific targeting location and mechanism have not been fully analyzed. Summary of the Invention

[0005] In light of this, the present invention aims to provide a mitochondrial-targeting self-assembling polypeptide, its preparation method and application, and a self-assembling polypeptide microneedle patch and its preparation method. The self-assembling polypeptide of the present invention can accurately and efficiently target mitochondria in tumor cells and accumulate in tumor cells that require a large amount of mitochondrial energy. By causing morphological and functional abnormalities in these mitochondria, it ultimately damages or inhibits tumor cells.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A mitochondrial-targeted self-assembling polypeptide has an amino acid sequence of GFFGKXRXKXRX (SEQ ID NO. 1), wherein X represents cyclohexylalanine.

[0008] Preferably, the two Fs in the amino acid sequence are both in L configuration or both in D configuration.

[0009] Preferably, both Rs in the amino acid sequence are in D configuration.

[0010] The present invention also provides a method for preparing the mitochondrial-targeting self-assembling polypeptide described in the above technical solution, comprising the following steps:

[0011] Activating the Rink-amide resin to obtain an active resin matrix;

[0012] Assembling a first cyclohexylalanine, a first arginine, a second cyclohexylalanine, a first lysine, a third cyclohexylalanine, a second arginine, a fourth cyclohexylalanine, a second lysine, a first glycine, a first phenylalanine, a second phenylalanine, and a second glycine in sequence on the active resin matrix to obtain a peptide chain precursor;

[0013] The peptide chain precursor is cut to cut the peptide chain from the resin matrix, and the obtained cutting liquid is post-treated to obtain the mitochondria-targeted self-assembling polypeptide.

[0014] The present invention also provides the use of the mitochondria-targeted self-assembling polypeptide described in the above technical solution or the mitochondria-targeted self-assembling polypeptide prepared by the preparation method described in the above technical solution in the preparation of anti-tumor drugs, wherein the tumor cells are cells with high oxidative phosphorylation activity.

[0015] Preferably, the tumor comprises oral squamous cell carcinoma.

[0016] Preferably, the dosage form of the drug is an external dosage form.

[0017] Preferably, the external dosage form is a microneedle patch.

[0018] The present invention also provides a self-assembling polypeptide microneedle patch, comprising a support layer and a plurality of microneedles located on the support layer;

[0019] The needle tip of the microneedle contains an active ingredient;

[0020] The active ingredient is the mitochondria-targeted self-assembling polypeptide described in the above technical solution or the mitochondria-targeted self-assembling polypeptide prepared by the preparation method described in the above technical solution.

[0021] The present invention also provides a method for preparing the self-assembling polypeptide patch described in the above technical solution, comprising the following steps:

[0022] mixing hyaluronic acid, mitochondria-targeted self-assembling peptides, and water to obtain an active mixture;

[0023] pouring the active mixed solution into the microneedle portion of the microneedle patch mold so that the needle tips of the microneedles are filled with the active mixed solution, and performing a first centrifugation;

[0024] Then, the microneedle portion of the microneedle patch mold is filled with a hyaluronic acid solution, and a second centrifugation and a first drying are performed in sequence. After the hyaluronic acid solution in the microneedle portion has dropped by half, the microneedle portion of the microneedle patch mold is filled with a hyaluronic acid solution again, and a third centrifugation and a second drying are performed in sequence.

[0025] After the hyaluronic acid solution in the microneedle portion is dried, the support layer portion of the microneedle patch mold is filled with a polyvinyl alcohol solution and subjected to a third drying step to obtain the self-assembling polypeptide microneedle patch.

[0026] The present invention provides a mitochondrial-targeted self-assembling polypeptide.

[0027] Mitochondria are the energy factories of cells, participating in important cellular life activities such as bioenergy synthesis and catabolism, apoptosis induction, and reactive oxygen species (ROS) production. In particular, cells are tumors of cells with high oxidative phosphorylation activity. The mitochondria in such tumor cells are different from the mitochondria in normal cells in structure and function, and actively participate in metabolic reprogramming. The mitochondria behave actively and are closely involved in their development. Therefore, mitochondria become promising targets for anticancer therapy. The amino acid sequence of the self-assembling polypeptide of the present invention is GFFGKXRXKXRX, wherein the targeting peptide (Mito) sequence KXRXKXRX is capable of targeting the mitochondria of cells with high oxidative phosphorylation activity, and the self-assembly sequence GFFG is introduced at the N-terminus of the targeting peptide sequence, giving the targeting peptide self-assembly ability, improving the targeting density and quantity of the targeting peptide, so that the self-assembling polypeptide can accurately and efficiently target the mitochondria in cells with high oxidative phosphorylation activity, and enrich in cells with high oxidative phosphorylation activity, by causing abnormal morphological and functional changes of the mitochondria therein, ultimately leading to damage or inhibition of cells with high oxidative phosphorylation activity. The self-assembling polypeptide of the present invention is made into a microneedle patch and applied to treat tumors through in situ administration. When the cells of such tumors are cells with high oxidative phosphorylation activity, especially oral squamous cell carcinoma cells, the self-assembling polypeptide can efficiently and accurately target and identify the mitochondria of tumor cells and be enriched in tumor cells, thereby having an excellent anti-tumor effect. In addition, the self-assembling polypeptide of the present invention has the advantage of strong targeting effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following detailed description, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0029] Figure 1 The molecular structure of the mitochondrial-targeting self-assembling peptide GFFGMito(L);

[0030] Figure 2 Figure 2 is the molecular structure of the mitochondrial-targeting self-assembling polypeptide GffGMito (D);

[0031] Figure 3 The MS image of the mitochondrial-targeted self-assembling peptide GFFGMito (L);

[0032] Figure 4 (D) is the MS image of the self-assembling peptide GffGMito targeting mitochondria;

[0033] Figure 5 This is the particle size distribution diagram of the mitochondrial-targeting self-assembling polypeptide in Test Example 1;

[0034] Figure 6 This is a photo of the self-assembled polypeptide microneedle patch in Test Example 2;

[0035] Figure 7 This is a photo of BALB / c nude mouse skin treated with the self-assembling peptide microneedle patch in Test Example 2;

[0036] Figure 8 This is a graph showing the inhibitory effect of GFFGMito (L) / GffGMito (D) on CAL-27 cells in Test Example 3;

[0037] Figure 9 This is a diagram showing the effect of GFFGMito(L) / GffGMito(D) on the mitochondrial morphology and structure in CAL-27 cells in Test Example 4;

[0038] Figure 10 This is a diagram showing the effect of GFFGMito(L) / GffGMito(D) on tumor size in test example 5. DETAILED DESCRIPTION

[0039] Explanation of terms

[0040] X represents cyclohexylalanine, the English name is Cyclohexylalanine;

[0041] R stands for arginine, the English name is Arginine;

[0042] K represents lysine, the English name is Lysine;

[0043] G stands for glycine, and its English name is Glycine;

[0044] F stands for phenylalanine, whose English name is phenylalanine.

[0045] The present invention provides a mitochondrial-targeted self-assembling polypeptide, the amino acid sequence of which is: GFFGKXRXKXRX.

[0046] In the present invention, the two phenylalanines (F) in the amino acid sequence are preferably both in L configuration or both in D configuration.

[0047] In the present invention, the two arginines (R) in the amino acid sequence are preferably in the D configuration.

[0048] In the present invention, since the two phenylalanines (F) in the mitochondrial-targeted self-assembling polypeptide have two configurations, the mitochondrial-targeted self-assembling polypeptide also has two configurations, namely, L configuration and D configuration. Specifically, the amino acid sequence is GF L F L GKXRXKXRX, denoted as GFFGMito(L), or GF D F D GKXRXKXRX, recorded as GffGMito(D).

[0049] Figure 1 This is the molecular structure of the mitochondrial-targeting self-assembling peptide GFFGMito(L). Figure 2 The molecular structure diagram of the self-assembling polypeptide GffGMito (D) targeting mitochondria.

[0050] The present invention also provides a method for preparing the mitochondrial-targeting self-assembling polypeptide described in the above technical solution, comprising the following steps:

[0051] Activating the Rink-amide resin to obtain an active resin matrix;

[0052] Assembling a first cyclohexylalanine, a first arginine, a second cyclohexylalanine, a first lysine, a third cyclohexylalanine, a second arginine, a fourth cyclohexylalanine, a second lysine, a first glycine, a first phenylalanine, a second phenylalanine, and a second glycine in sequence on the active resin matrix to obtain a peptide chain precursor;

[0053] The peptide chain precursor is cut to cut the peptide chain from the resin matrix, and the obtained cutting liquid is post-treated to obtain the mitochondria-targeted self-assembling polypeptide.

[0054] Unless otherwise specified, the raw materials used in the present invention are preferably commercially available products.

[0055] The present invention activates Rink-amide resin to obtain an active resin matrix.

[0056] In the present invention, the activation preferably includes performing swelling and deprotection reactions in sequence. In the present invention, the swelling reagent is preferably dichloromethane; the swelling temperature is preferably 20-30°C, the time is preferably 5-15 minutes, and specifically preferably 10 minutes. After the swelling, the present invention preferably further includes extruding the reaction liquid and washing (referred to as the first washing). The present invention does not specifically limit the operation of extruding the reaction liquid, and operations familiar to those skilled in the art can be used. In the present invention, the first washing reagent is preferably N,N-dimethylformamide (DMF), the number of the first washing is preferably 3 times, and the time for each washing is preferably 5 minutes. In the present invention, the swelling can swell the Rink-amide resin, provide reaction sites for subsequent deprotection reactions, and provide active connection sites for the assembly of amino acids.

[0057] In the present invention, the reagent for the deprotection reaction is preferably a pyridine aqueous solution, the volume concentration of the pyridine aqueous solution is preferably 10-25%, specifically preferably 10%, 15%, 20% or 25%, the temperature of the deprotection reaction is preferably room temperature, and the time is preferably 20-40 minutes, specifically preferably 30 minutes. In the present invention, the deprotection reaction can remove the protecting group 9-fluorenylmethylcarbonyl (Fmoc) in the Rink-amide resin, exposing the active reaction site amino group, and realizing the subsequent assembly of amino acids.

[0058] After the deprotection reaction, the present invention preferably further comprises washing (referred to as the second washing). The reagent and method of the second washing are preferably the same as those of the first washing, and will not be described in detail here.

[0059] After obtaining an active resin matrix, the present invention sequentially assembles a first cyclohexylalanine, a first arginine, a second cyclohexylalanine, a first lysine, a third cyclohexylalanine, a second arginine, a fourth cyclohexylalanine, a second lysine, a first glycine, a first phenylalanine, a second phenylalanine, and a second glycine on the active resin matrix to obtain a peptide chain precursor.

[0060] In the present invention, the reagent for assembling the first cyclohexylalanine, assembling the first arginine, assembling the second cyclohexylalanine, assembling the first lysine, assembling the third cyclohexylalanine, assembling the second arginine, assembling the fourth cyclohexylalanine, assembling the second lysine, assembling the first glycine, assembling the first phenylalanine, assembling the second phenylalanine or assembling the second glycine preferably includes a corresponding amino acid precursor, a coupling agent, a catalyst and an organic solvent, the coupling agent is preferably benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU), the catalyst is preferably N, N-diisopropylethylamine (DIEA), and the organic solvent preferably includes dichloromethane. In the present invention, the ratio of the amount of substance of the amino acid precursor, the coupling agent and the catalyst is preferably 1: 1: 2. The present invention does not specifically limit the structure of the corresponding amino acid precursor, and those skilled in the art can arrange it according to actual needs.

[0061] In the present invention, the temperature of assembling the first cyclohexylalanine, assembling the first arginine, assembling the second cyclohexylalanine, assembling the first lysine, assembling the third cyclohexylalanine, assembling the second arginine, assembling the fourth cyclohexylalanine, assembling the second lysine, assembling the first glycine, assembling the first phenylalanine, assembling the second phenylalanine or assembling the second glycine is independently preferably 20-25° C., and the time is preferably 2-2.5 h. In the present invention, the assembling the first cyclohexylalanine, assembling the first arginine, assembling the second cyclohexylalanine, assembling the first lysine, assembling the third cyclohexylalanine, assembling the second arginine, assembling the fourth cyclohexylalanine, assembling the second lysine, assembling the first glycine, assembling the first phenylalanine, assembling the second phenylalanine or assembling the second glycine is preferably carried out in a solid phase reactor.

[0062] After each amino acid is assembled, the present invention preferably further comprises sequentially performing a first wash, removing the protecting groups on the corresponding amino acid precursors, and a second wash. In the present invention, the reagents and methods of the first wash and the second wash are preferably consistent with the first wash described in the above technical solution, and will not be repeated here. In the present invention, the reagent for removing the protecting groups on the corresponding amino acid precursors is preferably a pyridine aqueous solution, and the volume fraction of the pyridine aqueous solution is preferably 10 to 30%, specifically preferably 20%; the temperature for removing the protecting groups on the corresponding amino acid precursors is preferably 20 to 25°C, and the time is preferably 20 to 40 minutes, specifically preferably 30 minutes.

[0063] After obtaining the peptide chain precursor, the present invention cuts the peptide chain precursor to cut the peptide chain from the resin matrix, and post-treats the obtained cutting liquid to obtain the mitochondrial-targeting self-assembling polypeptide.

[0064] In the present invention, the cutting agent is preferably a trifluoroacetic acid solution, the volume fraction of the trifluoroacetic acid solution is preferably 95%, the solvent of the trifluoroacetic acid solution is preferably triisopropylsilane and water, and the volume ratio of triisopropylsilane and water is preferably 1:1.

[0065] In the present invention, the usage ratio of the peptide chain precursor and the cleavage reagent is preferably 1 g:10 mL.

[0066] In the present invention, the cutting temperature is preferably room temperature, and the cutting time is preferably 40 to 50 minutes, specifically preferably 45 minutes.

[0067] After the cutting, the present invention preferably further comprises cleaning the Rink-amide resin after the cutting (denoted as the third cleaning), collecting the eluent, and merging the eluent with the cutting liquid obtained after the cutting as the cutting liquid. In the present invention, the reagent of the third cleaning is preferably dichloromethane (DCM), and the number of the third cleanings is preferably 3 times. In the present invention, the peptide chains remaining on the Rink-amide resin after the cutting can be collected by the third cleaning.

[0068] In the present invention, the post-treatment preferably includes sequentially performing vacuum rotary evaporation, washing (denoted as the fourth washing) and ether precipitation, collecting the precipitate, and purifying the precipitate to obtain the mitochondria-targeting self-assembling polypeptide.

[0069] The present invention does not specifically limit the parameters of the vacuum rotary evaporation, as long as trifluoroacetic acid (TFA) and dichloromethane (DCM) can be removed. In the present invention, the reagent for the fourth cleaning is preferably dichloromethane (DCM), and the number of the fourth cleaning is preferably 2 to 5 times. It is preferably spin-dried after each cleaning. The present invention does not specifically limit the parameters of the spin-dried, as long as a viscous material can be obtained. In the present invention, the reagent for the ether precipitation is preferably ether, more preferably anhydrous ether; the ether precipitation method is preferably standing; after the ether precipitation is completed, the present invention preferably further includes: discarding the ether precipitation reagent, vacuum-drying the obtained suspension, and obtaining a precipitate.

[0070] The present invention also provides the use of the mitochondria-targeted self-assembling polypeptide described in the above technical solution or the mitochondria-targeted self-assembling polypeptide prepared by the preparation method described in the above technical solution in the preparation of anti-tumor drugs, wherein the tumor cells are cells with high oxidative phosphorylation activity.

[0071] In the present invention, the tumor preferably includes oral squamous cell carcinoma.

[0072] In the present invention, the dosage form of the drug is preferably an external dosage form; the external dosage form is preferably a microneedle patch.

[0073] The present invention also provides a self-assembling polypeptide microneedle patch, comprising a support layer and a plurality of microneedles located on the support layer;

[0074] The needle tip of the microneedle contains an active ingredient;

[0075] The active ingredient is the mitochondria-targeted self-assembling polypeptide described in the above technical solution or the mitochondria-targeted self-assembling polypeptide prepared by the preparation method described in the above technical solution.

[0076] The self-assembling polypeptide microneedle patch provided by the present invention comprises a support layer, wherein the material of the support layer is preferably polyvinyl alcohol, and the thickness of the support layer is preferably 0.1 mm.

[0077] The self-assembling polypeptide microneedle patch provided by the present invention comprises a plurality of microneedles positioned on a supporting layer; the tips of the microneedles contain an active ingredient; the active ingredient is the mitochondrial-targeting self-assembling polypeptide described in the above technical solution. In the present invention, the microneedles preferably comprise a tip and a shaft; the shaft is made of hyaluronic acid. In the present invention, the microneedles are preferably 1 mm in height; and the tip-to-shaft height ratio of the microneedles is preferably 1-2:2-1.

[0078] The present invention also provides a method for preparing the self-assembling polypeptide patch described in the above technical solution, comprising the following steps:

[0079] mixing hyaluronic acid, mitochondria-targeted self-assembling peptides, and water to obtain an active mixture;

[0080] pouring the active mixed solution into the microneedle portion of the microneedle patch mold so that the needle tips of the microneedles are filled with the active mixed solution, and performing a first centrifugation;

[0081] Then, the microneedle portion of the microneedle patch mold is filled with a hyaluronic acid solution, and a second centrifugation and a first drying are performed in sequence. After the hyaluronic acid solution in the microneedle portion has dropped by half, the microneedle portion of the microneedle patch mold is filled with a hyaluronic acid solution again, and a third centrifugation and a second drying are performed in sequence.

[0082] After the hyaluronic acid solution in the microneedle portion is dried, the support layer portion of the microneedle patch mold is filled with a polyvinyl alcohol solution and subjected to a third drying step to obtain the self-assembling polypeptide microneedle patch.

[0083] The present invention mixes hyaluronic acid, mitochondria-targeted self-assembling polypeptide and water to obtain an active mixed solution.

[0084] In the present invention, mixing hyaluronic acid, a mitochondrial-targeting self-assembling polypeptide, and water preferably includes: mixing hyaluronic acid and water to obtain a hyaluronic acid solution; mixing the mitochondrial-targeting self-assembling polypeptide and water to obtain a polypeptide solution; and mixing the hyaluronic acid solution and the polypeptide solution. In the present invention, the mass concentration of the hyaluronic acid solution is preferably 10%, and the concentration of the polypeptide solution is preferably 1 mmol / L. In the present invention, the volume ratio of the hyaluronic acid solution to the polypeptide solution is preferably 200:7.

[0085] After obtaining the active mixed solution, the present invention pours the active mixed solution into the microneedle portion of the microneedle patch mold so that the needle tip of the microneedle is filled with the active mixed solution, and performs a first centrifugation.

[0086] In the present invention, the rotation speed of the first centrifugation is preferably 3000 rpm, and the time is preferably 40 min. In the present invention, the first centrifugation can enrich the active ingredients at the needle tip of the microneedle.

[0087] After the first centrifugation, the present invention fills the microneedle portion of the microneedle patch mold with a hyaluronic acid solution, performs a second centrifugation and a first drying in sequence, and after the hyaluronic acid solution in the microneedle portion drops by half, the microneedle portion of the microneedle patch mold is filled with a hyaluronic acid solution again, and performs a third centrifugation and a second drying in sequence.

[0088] In the present invention, the mass concentration of the hyaluronic acid solution is preferably 10%.

[0089] In the present invention, the second centrifugation speed is preferably 3000 rpm, and the time is preferably 40 minutes. In the present invention, the temperature of the first drying is preferably 32°C, and the first drying is preferably performed with a fan turned on. The present invention does not impose a specific limit on the time of the first drying, and the drying can be stopped as soon as the first hyaluronic acid solution in the mold has dropped by half.

[0090] In the present invention, the mass concentration of the hyaluronic acid solution is preferably 10%. In the present invention, the rotation speed of the third centrifugation is preferably 3000 rpm, and the duration is preferably 40 minutes. In the present invention, the temperature of the second drying is preferably 32°C, and the second drying is preferably performed with a fan turned on. The present invention does not impose any specific restrictions on the duration of the second drying, as long as the hyaluronic acid solution is dried and the resulting hyaluronic acid film does not warp.

[0091] In the present invention, after the hyaluronic acid solution in the microneedle portion is dried, the support layer portion of the microneedle patch mold is filled with a polyvinyl alcohol solution and subjected to a third drying step to obtain the self-assembling polypeptide microneedle patch.

[0092] In the present invention, the mass concentration of the polyvinyl alcohol solution is preferably 20%. In the present invention, the temperature of the third drying is preferably 32° C., and the third drying is preferably performed with a fan turned on.

[0093] The following examples describe in detail the mitochondrial-targeting self-assembling polypeptide, its preparation method and application, and the self-assembling polypeptide microneedle patch and its preparation method provided by the present invention. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0094] Example

[0095] (1) Preparation of self-assembling peptides targeting mitochondria

[0096] a. Weigh 0.5 g of Rink-amide resin and swell it in 20 mL of 20°C dichloromethane (DCM) for 10 min. Use an ear bulb to squeeze out the dichloromethane solvent in the solid phase reactor and wash it with N,N-dimethylformamide (DMF). Then, add 20% piperidine aqueous solution and react for 30 min to remove the terminal 9-fluorenylmethylcarbonyl (Fmoc) group of Rink-amide, and then wash it with DMF. Prepare cyclohexyl amino acid: weigh 1 mmol of the first amino acid precursor N-fluorenylmethylcarbonylcyclohexylalanine and add 2 mmol of N,N-diisopropylethylamine (DIEA) and 1 mmol of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) to fully dissolve them in DMF, then add them to a solid phase reactor and react at room temperature for 2 hours; wash with DMF 3 times (5 minutes / time), react with a 20% volume fraction of piperidine aqueous solution for 30 minutes to remove the amino protecting group Fmoc on the cyclohexylalanine to expose the active amino group; wash the residual piperidine with DMF.

[0097] b. Add the second amino acid precursor N-fluorenylmethylcarbonylarginine (L-Fmoc-Arg-OH), the third amino acid precursor N-fluorenylmethylcarbonylcyclohexylalanine, the fourth amino acid precursor N-fluorenylmethylcarbonyllysine, the fifth amino acid precursor N-fluorenylmethylcarbonylcyclohexylalanine, the sixth amino acid precursor N-fluorenylmethylcarbonylcyclohexylarginine, the seventh amino acid precursor N-fluorenylmethylcarbonylcyclohexylalanine, the eighth amino acid precursor N-fluorenylmethylcarbonylcyclohexyllysine, the ninth amino acid precursor N-fluorenylmethylcarbonylcyclohexylglycine, the tenth amino acid precursor N-fluorenylmethylcarbonylcyclohexylphenylalanine, the eleventh amino acid precursor N-fluorenylmethylcarbonylcyclohexylphenylalanine and the twelfth amino acid precursor N-fluorenylmethylcarbonylcyclohexylglycine to the system in sequence, and perform arginine assembly, cyclohexylalanine assembly, lysine assembly in sequence. Assembly, cyclohexylalanine assembly, arginine assembly, cyclohexylalanine assembly, lysine assembly, glycine assembly, phenylalanine assembly, phenylalanine assembly and glycine assembly to obtain a peptide chain precursor; during each assembly process, N,N-diisopropylethylamine (DIEA) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) were added, and the molar ratio of each amino acid precursor, HBTU and DIEA was 1:1:2; the temperature of each assembly was room temperature, and the time was 2h; after each assembly, the first washing, removal of protecting groups and the second washing were carried out in sequence, the reagents for the first washing and the second washing were both DMF, the number of times was 3, each washing lasted 5 minutes, the reagent for removing protecting groups was pyridine aqueous solution, the volume fraction of pyridine aqueous solution was 20%, the temperature for each removal of protecting groups was room temperature, and the time was 30min.

[0098] c. Then, 10 mL of 95% TFA (the volume ratio of TFA:TIS:H2O is 95:2.5:2.5) was added to 1 g of peptide chain precursor for 45 min to cut the peptide chain from the resin. The obtained cutting liquid was collected in a 100 mL eggplant flask, and dichloromethane (DCM) was added to wash the resin 3 times. The washing liquid was collected and combined as the cutting liquid. TFA and DCM were removed from the cutting liquid by vacuum rotary evaporation, and appropriate DCM was added to wash several times and continued to spin dry. Anhydrous ether was added to the viscous material to precipitate the crude polypeptide product. After standing for a period of time, the anhydrous ether was carefully discarded, and the suspension was vacuum-evaporated to obtain the crude polypeptide product. The self-assembling polypeptide targeting mitochondria was purified, and GF L F L GKFxRFxKFxRFx(GFFGMito(L)) or GF D F D GKFxRFxKFxRFx(GffGMito(D)).

[0099] (2) Preparation of self-assembled polypeptide microneedle patches

[0100] A, GFFGMito (L) or GffGMito (D) that 7 μ L concentration is 1mM are mixed on glass plate with 200 μ L hyaluronic acid solution (mass concentration is 10%) respectively, be tiled in the mould of clean dry, balancing, on centrifuge in the centrifugal 40min of 3000rpm.Fill up mould with the hyaluronic acid solution (mass concentration is 10%) without adding medicine, vacuumize 5min, on centrifuge in the centrifugal 40min of 3000rpm, mould is placed baking oven, 32 ℃ of auxiliary heatings, and open blower fan, when hyaluronic acid solution in mould descends half, again fill up hyaluronic acid solution (mass concentration is 10%), vacuumize 5min, on centrifuge in the centrifugal 40min of 3000rpm, 32 ℃ of auxiliary heatings in baking oven, and open blower fan. When the hyaluronic acid solution in the mold becomes dry but does not curl up, it is filled with polyvinyl alcohol (PVA) with a mass concentration of 20%, placed in an oven until dried, and demolded to obtain a self-assembled polypeptide microneedle patch.

[0101] Test Example 1

[0102] The mitochondrial self-assembling polypeptides GFFGMito (L) and GffGMito (D) obtained in Example 1 were subjected to MS (mass spectrometry) detection. The specific results are shown in Figure 3 and Figure 4 .

[0103] pass Figures 3 and 4 It can be seen that the self-assembling polypeptide structure targeting mitochondria prepared by the present invention is indeed as Figure 1 and Figure 2 shown.

[0104] The mitochondrial self-assembling polypeptides GFFGMito (L) and GffGMito (D) prepared in Example 1 were subjected to DLS (dynamic light scattering) detection. The GFFGMito (L) and GffGMito (D) powders were simply dissolved in DMSO and then distilled water was added to obtain 1 mM GFFGMito (L) and GffGMito (D) solutions. The hydrodynamic diameters of the two polypeptide materials were measured by DLS. The specific results are shown in FIG. Figure 5 : The average diameter of GffGMito (D) is 531±7 nm, and the average diameter of GFFGMito (L) is 459±8 nm.

[0105] Test Example 2

[0106] The self-assembling polypeptide microneedle patch prepared in Example 1 was photographed. The specific results are shown in Figure 6 .

[0107] The self-assembled polypeptide microneedle patch prepared in Example 1 was subjected to a BALB / c nude mouse skin penetration test. The specific experimental process was as follows: the self-assembled polypeptide microneedle patch was attached to the skin of the right hind limb of the BALB / c nude mouse, a 10N vertical force was applied for 1 minute, and then the microneedles were removed and photographed by a camera for research. The specific results are shown in Figure 7 .

[0108] pass Figure 6 and Figure 7 It can be seen that the self-assembling polypeptide microneedle patch prepared by the present invention can penetrate the skin, act on the tumor site in situ, construct microchannels, and enhance drug delivery.

[0109] Test Example 3

[0110] The CAL-27 cell line has been used to study anticancer activity. To determine the effect of the self-assembly sequence on the efficacy of mitochondrial-targeted peptides, cytotoxicity studies were performed using the same concentrations of GFFGMito(L) / GffGMito(D).

[0111] First, plate the cells. Once the CAL-27 cells have grown to 80-90% of the dish, aspirate the culture medium from the dish and rinse once or twice with 2-3 mL of PBS to remove dead cells. Add 1 mL of trypsin to the dish and place it in a 37°C, 5% CO2 incubator for 2 minutes. Then, add 3 mL of DMEM to terminate digestion. Repeatedly pipette the dish with a pipette and transfer the cell suspension to a 15 mL centrifuge tube. Centrifuge at 1200 rpm for 3 minutes and discard the supernatant. Add 1 mL of DMEM to the collected CAL-27 cells and resuspend them with repeated pipetting. Dilute the cell suspension to a desired ratio, aspirate 10 μL, and add it to a cell counting plate for microscopic observation and counting. After counting, plate a 96-well plate with 5,000 CAL-27 cells per well in 100 μL of DMEM. Place the plate in a 37°C, 5% CO2 incubator and incubate for 24 hours. GFFGMito(L) / GffGMito(D) was prepared with serum-free DMEM medium to prepare seven concentration gradients of 0μM, 1μM, 2μM, 5μM, 10μM, 20μM, and 50μM. Take out the 96-well plate, aspirate the culture medium, add 100μL of PBS to wash once, add 100μL of culture medium containing GFFGMito(L) / GffGMito(D) at different concentrations, place in a 37°C 5% CO2 constant temperature incubator, and culture for 24 hours. Take out a certain amount of cck8 reagent and mix it with the culture medium in a weight ratio of 1:10, add 100μL of the mixture of CCK8 and culture medium to each well, place in a 37°C 5% CO2 constant temperature incubator, and culture for 4 hours. Use an enzyme marker to measure its fluorescence at 450nm. All results were confirmed by three independent reagent repetitions and triplicate tests each time. See the specific results for details. Figure 8 .

[0112] Depend on Figure 8 It can be seen that there is a significant difference in the cell inhibition rate of CAL-27 cells between the polypeptides GFFGMito (L) and GffGMito (D). The inhibition rate of GFFGMito (L) on CAL-27 cells is significantly stronger than that of GFFGMito (D), which further indicates that the mitochondrial-targeting self-assembling polypeptide provided by the present invention has excellent targeting and self-assembly effects; it is also found that the higher the drug concentration, the greater the cell inhibition rate.

[0113] Test Example 4

[0114] In order to determine the effect of mitochondria-targeted self-assembling peptide drugs on tumor cell mitochondria, the same concentrations of GFFGMito (L) and GffGMito (D) were used for research.

[0115] First, plate the cells. When the CAL-27 cells reach 80-90% cell mass in the dish, aspirate the culture medium from the dish and rinse once or twice with 2-3 mL of PBS to remove dead cells. Add 1 mL of trypsin to the dish and place it in a 37°C, 5% CO2 incubator for 2 minutes. Then, add 3 mL of DMEM to terminate digestion. Repeatedly pipette the dish with a pipette and transfer the cell suspension to a 15 mL centrifuge tube. Centrifuge at 1200 rpm for 3 minutes and discard the supernatant. Add 1 mL of DMEM to the collected CAL-27 cells and resuspend them with repeated pipetting. Dilute the cell suspension to a desired ratio, aspirate 10 μL, and apply it to a cell counting plate for microscopic observation and counting. After counting, plate the cells in a confocal microplate with 5,000 CAL-27 cells per well in 1 mL of DMEM. Place the cells in a 37°C, 5% CO2 incubator and incubate for 24 hours. GFFGMito(L) / GffGMito(D) were diluted to 6 μM in serum-free DMEM. The confocal culture dish was removed, the culture medium aspirated, and the cells were rinsed once with 1 mL of PBS. 1 mL of 6 μM GFFGMito(L) / GffGMito(D) culture medium was added and the cells were placed in a 37°C, 5% CO2 incubator. After 20 hours of incubation, the drug-containing medium was aspirated. A certain amount of Mito-TrackerRed CMXRos (a high-purity mitochondrial red fluorescent probe) was diluted to 50 nM in serum-free DMEM. 1 mL of the solution was added to the confocal culture dish and placed in a 37°C, 5% CO2 incubator for 15 minutes. The Mito-Tracker Red CMXRos working solution was removed and the cells were rinsed once with serum-free DMEM pre-incubated at 37°C. Finally, 1 mL of PBS was added. Fluorescence was measured in the 579-599 nm range using a laser confocal microscope (LSM800, Zeiss, Germany). All results were confirmed by three independent replicates of the reagents and tested in triplicate each time. Figure 9 .

[0116] Depend on Figure 9 It can be seen that the mitochondrial self-assembling polypeptides GFFGMito (L) and GffGMito (D) caused abnormal mitochondrial morphology and structure in CAL-27 cells, and GFFGMito (L) had a stronger effect than GffGMito (D), indicating that the mitochondrial self-assembling polypeptides provided by the present invention have excellent mitochondrial targeting effects, causing abnormal mitochondrial morphology and function, and thus leading to apoptosis of tumor cells.

[0117] Test Example 5

[0118] All animal experiments were performed according to protocols approved by the local ethics committee and in compliance with the Chinese Regulations on the Use of Laboratory Animals. Female BALB / c mice weighing between 18 and 20 g (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were housed in a pathogen-free animal facility with unlimited access to food and water.

[0119] For CAL-27 cells: Place the frozen GelNestTM high concentration Matrigel (Cat.No.: 211252) on ice at 4°C overnight to melt and render liquid, then use a pre-cooled pipette tip to mix the Matrigel until it is uniform. Prepare cells in the logarithmic growth phase with a cell density of approximately 80%-90%, and replace the culture medium with new one the day before cell collection. Digest the cells with trypsin and add PBS to generate a cell suspension. After centrifuging and washing the cells once, resuspend them to a final concentration of 8×10 7 cells / ml. Dilute the cell suspension and GelNest™ high-concentration Matrigel at a ratio of 2:1 at 4°C.

[0120] 100 μL of cell suspension was injected subcutaneously into the right hind limb of BALB / c nude mice. The mice were returned to their cages and continued to be fed. About one week after inoculation, the average tumor volume reached 100 mm. 3 . Tumor-bearing mice were randomly divided into three different groups, each containing 5 mice. These mice received transdermal patch administration of the self-assembling peptide microneedle patch, and a blank microneedle patch without any chemical additives was used as a negative control group. The treatment was repeated every two days for a treatment period of 15 days. Tumor volume (using digital calipers), cachexia and body weight changes were recorded throughout the treatment process, and the tumors were photographed on the 15th day. The ellipse equation was used to estimate the volume of the tumor: (length × width × width) / 2.

[0121] Depend on Figure 10 It can be seen that the mitochondrial-targeted self-assembling polypeptide microneedle patch inhibited tumor growth in CAL-27 cell-bearing mice, and the effect of GFFGMito(L) was stronger than that of GffGMito(D).

[0122] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A mitochondrial-targeting self-assembling polypeptide, characterized in that: The amino acid sequence is: GFFGKXRXKXRX, wherein X represents cyclohexylalanine.

2. The mitochondrial-targeting self-assembling polypeptide according to claim 1, characterized in that In the amino acid sequence, both Fs are in L configuration or both Fs are in D configuration.

3. The mitochondrial-targeting self-assembling polypeptide according to claim 1, characterized in that In the amino acid sequence, both Rs are in D configuration.

4. The method for preparing the mitochondrial-targeting self-assembling polypeptide according to any one of claims 1 to 3, characterized in that: The following steps are involved: Activating the Rink-amide resin to obtain an active resin matrix; Assembling a first cyclohexylalanine, a first arginine, a second cyclohexylalanine, a first lysine, a third cyclohexylalanine, a second arginine, a fourth cyclohexylalanine, a second lysine, a first glycine, a first phenylalanine, a second phenylalanine, and a second glycine in sequence on the active resin matrix to obtain a peptide chain precursor; The peptide chain precursor is cut to cut the peptide chain from the resin matrix, and the obtained cutting liquid is post-treated to obtain the mitochondria-targeted self-assembling polypeptide.

5. Use of the mitochondria-targeted self-assembling polypeptide according to any one of claims 1 to 3 or the mitochondria-targeted self-assembling polypeptide prepared by the preparation method according to claim 4 in the preparation of anti-tumor drugs, wherein the tumor cells are cells with high oxidative phosphorylation activity.

6. The use according to claim 5, characterized in that Such tumors include oral squamous cell carcinoma.

7. The use according to claim 5, characterized in that The dosage form of the medicine is an external dosage form.

8. The use according to claim 7, characterized in that The external dosage form is a microneedle patch.

9. A self-assembling polypeptide microneedle patch, characterized in that: It includes a support layer and a plurality of microneedles located on the support layer; The needle tip of the microneedle contains an active ingredient; The active ingredient is the mitochondria-targeted self-assembling polypeptide according to any one of claims 1 to 3 or the mitochondria-targeted self-assembling polypeptide prepared by the preparation method according to claim 4.

10. The method for preparing the self-assembling polypeptide microneedle patch according to claim 9, characterized in that: The following steps are involved: mixing hyaluronic acid, mitochondria-targeted self-assembling peptides, and water to obtain an active mixture; The active mixed solution is poured into the microneedle portion of the microneedle patch mold so that the needle tip of the microneedle is filled with the active mixed solution, and a first centrifugation is performed; Then, the microneedle portion of the microneedle patch mold is filled with a hyaluronic acid solution, and a second centrifugation and a first drying are performed in sequence. After the hyaluronic acid solution in the microneedle portion has dropped by half, the microneedle portion of the microneedle patch mold is filled with a hyaluronic acid solution again, and a third centrifugation and a second drying are performed in sequence. After the hyaluronic acid solution in the microneedle portion is dried, the support layer portion of the microneedle patch mold is filled with a polyvinyl alcohol solution and subjected to a third drying step to obtain the self-assembling polypeptide microneedle patch.

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