Hairpin peptide as well as preparation and application thereof

Abstract

The invention belongs to the field of gene engineering and gene therapy, and discloses a hairpin peptide as well as preparation and application thereof. According to the invention, polypeptide molecules are amphiphilic through molecular design, so that the amphiphilic polypeptide with low toxicity is fully combined with plasmid DNA or mRNA, and the amphiphilic polypeptide has almost no toxicity to cells and has the advantages of low toxicity and high transfection efficiency.

Description

Technical Field

[0001] This invention relates to a hairpin peptide and its preparation and application, and more particularly to the application of a hairpin peptide in delivering bioactive substances such as nucleic acids. Background Technology

[0002] Gene therapy involves delivering genes (DNA, RNA, mRNA, siRNA, antisense oligonucleotides, etc.) to patients to repair the damaged genes that cause the disease, thereby achieving a therapeutic effect.

[0003] Gene delivery vectors are generally classified into viral vectors and non-viral vectors. Viral delivery systems are typically the most efficient vectors for gene delivery. They can selectively target cells and usually have high transfection efficiency. However, their separation and processing from biological sources can be very expensive. Furthermore, their safety risks, due to their carcinogenic potential and inflammatory and immunogenic effects, limit the clinical application of viral vector-based delivery strategies.

[0004] Compared to viral delivery vectors, non-viral delivery vectors offer advantages such as simplicity of use, ease of large-scale production, and lack of specific immune responses. Common non-viral delivery vectors, such as cationic lipids, polymers, dendritic polymers, and peptides, have been developed into numerous delivery technologies. Among these non-viral vector materials, peptides are increasingly popular due to their sequence and functional diversity. Various combinations of 20 natural amino acids can produce peptides with different three-dimensional conformations, charges, polarities, hydrophobicities, and hydrophilicities. These unique sequences, within a relatively small molecular weight range, can exhibit a variety of functions, including nucleic acid binding, membrane penetration, endosome disruption, and targeting—all essential for targeted gene delivery. However, existing gene vectors often cannot simultaneously achieve high transfection efficiency and low toxicity; either high transfection efficiency results in high toxicity, or low toxicity results in low transfection efficiency. Therefore, in gene therapy, there is an urgent need for a gene vector that can balance high transfection efficiency and low toxicity. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention utilizes molecular design to obtain an amphiphilic hairpin peptide that can bind to nucleic acids such as pDNA or mRNA, thereby compressing the nucleic acid to form a nanocomplex, which is then carried into cells for expression.

[0006] Therefore, a first aspect of the present invention provides a polypeptide in a hairpin-like form having the structure shown in formula (1):

[0007] [TS]K p1 H n1 X m1 GPGGX m2 H n2 K p2 (1),

[0008] in:

[0009] H stands for histidine; K for lysine; G for glycine; P for proline.

[0010] X may be the same or different, and is independently selected from tryptophan (W), leucine (L), valine (V), phenylalanine (F), or alanine (A);

[0011] m1 and m2 may be the same or different, and are independent integers from 1 to 8; preferably, m1 is 6 and m2 is 5.

[0012] n1 and n2 may be the same or different, and are independently integers from 2 to 5; preferably, n1 and n2 are independently integers from 2 to 3, and more preferably, n1 and n2 are both 3;

[0013] p1 and p2 may be the same or different, and are independently integers from 3 to 8. Preferably, p1 and p2 are independently integers from 3 to 5; more preferably, p1 is 3 and p2 is 5.

[0014] [TS] is a peptide targeting sequence; preferably, the peptide targeting sequence is selected from RGD, GPR, AEIDGIEL, LDT, CS-1peptide, CS-5peptide, and IDAPS; more preferably, the peptide targeting sequence is RGD;

[0015] In a preferred embodiment, the polypeptide has the structure shown in formula (2):

[0016] RGDKKKHHHWLVFFAGPGGLVFFAHHHKKKKK(2).

[0017] Those skilled in the art will understand that the hairpin peptide of the present invention can be prepared by methods known in the art, such as solid-phase synthesis.

[0018] Another aspect of the present invention provides a method for preparing the hairpin peptide of the present invention, which includes the following steps:

[0019] 1. Resin activation; preferably, the resin is Fmoc-Lys(Boc)-Wang resin;

[0020] 2. Removal of Fmoc protecting groups; preferably, Kjeldahl analysis is performed to determine whether Fmoc deprotection is complete;

[0021] 3. Amino acid cyclic coupling: Fmoc-protected amino acids are cyclically coupled from the C-terminus to the N-terminus of the polypeptide sequence, and the Fmoc protecting groups are removed; preferably, the coupling reagents are HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate) and DIPEA (N,N-diisopropylethylamine); preferably, Kjeldahl assays are performed after coupling the Fmoc-protected amino acids and after removing the Fmoc protecting groups to determine whether the coupling and Fmoc deprotection are complete; preferably, the amount of Fmoc-protected amino acids and HBTU is 4 equivalents, and the amount of DIPEA is 8 equivalents.

[0022] In a preferred embodiment of the present invention, the following steps are also included:

[0023] 4. Vacuum drying and cutting of the resin. Preferably, the cutting fluid is trifluoroacetic acid (TFA):water:triisopropylsilane (TIS) = 95:2.5:2.5.

[0024] In a preferred embodiment of the present invention, the following steps are also included:

[0025] 5. Preparation and purification of crude peptides. Preferably, the peptides are purified by reversed-phase high-performance liquid chromatography.

[0026] Another aspect of the present invention provides a nanocomposite comprising the hairpin peptide and bioactive substance described in this invention. Preferably, the nanocomposite is composed of the hairpin peptide and bioactive substance described in this invention.

[0027] Those skilled in the art will understand that the bioactive substance is selected from one or more of therapeutic agents, preventive agents, or diagnostic agents. Various bioactive substances can be used in this invention. In a preferred embodiment, the bioactive substance is selected from nucleic acids, enzymes, and small molecule drugs; more preferably, the bioactive substance is a nucleic acid.

[0028] Those skilled in the art will understand that various nucleic acids can be used in this invention, including but not limited to any nucleic acid that can be used to treat, prevent or diagnose diseases, and types including but not limited to one or more of siRNA, mRNA, microRNA, circular mRNA, snRNA, snoRNA, tRNA, rRNA, gRNA, shRNA, piRNA, rasiRNA, hnRNA, long non-coding RNA, plasmid DNA, ceDNA, mini circle DNA, antisense oligonucleotides (ASOs).

[0029] Nucleic acids used for treatment include, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), aptamers, ribozymes, and guide RNA (gRNA).

[0030] Nucleic acids used for prevention include, but are not limited to, mRNA vaccines and DNA vaccines.

[0031] Nucleic acids used for diagnosis include, but are not limited to, nucleic acid probes and nucleic acid aptamers.

[0032] In one specific embodiment of the present invention, the nucleic acid is mRNA or plasmid DNA (pDNA); preferably, the nucleic acid is mRNA or plasmid DNA expressing EGFP protein.

[0033] In a preferred embodiment, the molar ratio (N / P) of the positively charged amino nitrogen element in the hairpin peptide to the negatively charged phosphorus element in the phosphate group of the nucleic acid is at least 0.5, preferably at least 1, more preferably 1-20, even more preferably 2-16, and most preferably 7-15.

[0034] Another aspect of the present invention provides a method for preparing the nanocomposite of the present invention, comprising the following steps:

[0035] 1. Prepare a polypeptide solution; preferably, the solvent is DMSO;

[0036] 2. Preparation of aqueous solutions of bioactive substances;

[0037] 3. Use a syringe to draw up the polypeptide solution and the aqueous solution of the bioactive substance, respectively;

[0038] 4. Place the syringe on a dual-channel microfluidic device, with one channel carrying a peptide solution and the other channel carrying an aqueous solution of a bioactive substance (e.g., mRNA solution). The two solutions flow into the chip and mix thoroughly to obtain a complex solution of the peptide and the bioactive substance. Preferably, the microfluidic flow rate is 1-8 mL / min; more preferably, the microfluidic flow rate is 4 mL / min.

[0039] 5. Dialyze the complex solution overnight; preferably, dialyze overnight in a 10k dialysis bag at 4°C.

[0040] Another aspect of the present invention provides a pharmaceutical composition comprising the nanocomposite described herein.

[0041] Those skilled in the art will understand that the pharmaceutical compositions of the present invention can be administered to humans and / or animals orally, rectally, intravenously, intramuscularly, vaginally, intranasally, intraperitoneally, buccally, or via oral or nasal spray. Those skilled in the art will also understand that the pharmaceutical compositions of the present invention may contain various pharmaceutically acceptable carriers.

[0042] Another aspect of the present invention provides a method for treating, diagnosing, or preventing a disease in a subject, comprising administering to the subject the polypeptide, nanocomposite, or pharmaceutical composition described herein.

[0043] Another aspect of the invention provides the use of the polypeptides, nanocomposites, or pharmaceutical compositions described herein in the preparation of medicaments for treating, diagnosing, or preventing diseases.

[0044] Those skilled in the art will understand that the effective amount of the polypeptide, nanocomposite, or pharmaceutical composition of the present invention can be determined based on the subject's age, weight, disease type, and / or severity.

[0045] Compared with existing gene vectors, the hairpin peptide of the present invention, its preparation and application, can fully bind the hairpin peptide to bioactive substances such as plasmid DNA or mRNA, with an encapsulation rate of over 95%, close to 100%, and is almost non-toxic to cells, while also having the advantage of high transfection efficiency. Attached Figure Description

[0046] Figure 1 This is a liquid chromatogram of the TY-10 polypeptide prepared in Example 1 of this invention;

[0047] Figure 2 This is the mass spectrum of the TY-10 polypeptide prepared in Example 1 of this invention;

[0048] Figure 3 This is an agarose gel electrophoresis image of the TY-10 polypeptide bound to mRNA in Example 2 of this invention;

[0049] Figure 4 This is a diagram showing the encapsulation efficiency of the TY-10 polypeptide on mRNA in Example 3 of this invention;

[0050] Figure 5 This is an agarose gel electrophoresis image of the TY-10 polypeptide bound to pDNA in Example 4 of this invention;

[0051] Figure 6 This is a particle size diagram of the TY-10 polypeptide / mRNA complex in Example 5 of the present invention;

[0052] Figure 7 This is a Zeta potential diagram of the TY-10 polypeptide / mRNA complex in Example 5 of the present invention;

[0053] Figure 8 This is a particle size diagram of the TY-10 polypeptide / pDNA complex in Example 6 of the present invention;

[0054] Figure 9 This is a Zeta potential diagram of the TY-10 polypeptide / pDNA complex in Example 6 of the present invention;

[0055] Figure 10 This is a cytotoxicity diagram of the TY-10 polypeptide / mRNA complex in Example 7 of the present invention;

[0056] Figure 11 This is a cytotoxicity diagram of the TY-10 polypeptide / pDNA complex in Example 8 of the present invention;

[0057] Figure 12 This is a graph showing the transfection efficiency of the TY-10 polypeptide / mRNA complex in Example 9 of this invention;

[0058] Figure 13 This is a transfection distribution diagram of the TY-10 polypeptide / mRNA complex in HEK-293T cells in Example 9 of the present invention;

[0059] Figure 14 This is a transfection distribution diagram of the TY-10 polypeptide / pDNA complex of Example 10 of the present invention in HEK-293T cells. Detailed Implementation

[0060] To better illustrate the purpose and advantages of this method, the specific implementation of the present invention will be further described in detail with reference to the accompanying drawings and specific embodiments.

[0061] Example 1: Preparation of amphiphilic hairpin peptide (hereinafter referred to as "TY-10 polypeptide")

[0062] This embodiment employs a solid-phase synthesis method, using Fmoc-Lys(Boc)-Wang resin as the matrix, and synthesizing the peptide sequentially from the C-terminus to the N-terminus according to the amino acid sequence. The TY-10 peptide sequence is as follows:

[0063] RGDKKKHHHWLVFFAGPGGLVFFAHHHKKKKK

[0064] Specific synthesis steps:

[0065] (1) Resin activation: Weigh 610 mg (200 μmol) of Fmoc-Lys(Boc)-Wang resin (purchased from Jier Biochemical (Shanghai) Co., Ltd.) and place it in a dry solid-phase synthesis reaction tube. Add 10 mL of anhydrous DMF (N,N-dimethylformamide) and allow the resin to swell for 2 h. Remove DMF by vacuum filtration.

[0066] (2) Removal of Fmoc protecting groups: A DMF solution containing 20% ​​piperidine was added to the solid-phase synthesis reaction tube in two separate additions, 10 mL each time. The reaction tube was placed on a rotary mixer, and the reaction was carried out for 10 min each time. After removing the piperidine solution by vacuum filtration, the resin was thoroughly washed with DMF and DCM (dichloromethane). A small amount of resin was taken for Kjeldahl analysis.

[0067] Kjeldahl assay: This involves using Kaiser's reagent to determine whether Fmoc has been completely deprotected.

[0068] Kjeldahl reagent composition: A. Detection solution: 2g ninhydrin solid completely dissolved in 50mL anhydrous ethanol; B. Detection solution: a mixed solution of 20g phenol and 5mL ethanol; C. Detection solution: a dilute pyridine solution of 2% 0.001M potassium cyanide.

[0069] Detection procedure: Place a small amount of resin in a test tube, add 2-3 drops each of the three test solutions A, B, and C to the test tube, and heat to boiling. If the resin turns blue, it indicates the presence of free amino acids; if the resin does not turn blue during heating, it indicates the absence of free amino acids.

[0070] (3) Weigh out 4 equivalents (i.e., 800 μmol) of the amino acid to be added (i.e., Fmoc-Lys(Boc)-OH), 4 equivalents of HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate), and 8 equivalents of DIPEA (N,N-diisopropylethylamine). Dissolve each in 2 mL of DMF, mix thoroughly with the resin, and react at room temperature for 1 h. Take a small amount of resin for Kjeldahl analysis.

[0071] (4) Cyclic coupling of amino acids: The coupling process uses 4 equivalents of Fmoc-AA-OH (Fmoc-protected amino acid), 4 equivalents of HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate), and 8 equivalents of DIPEA (N,N-diisopropylethylamine). Steps 2-3 are repeated until the last amino acid Arg is coupled, and then the Fmoc protecting group on Arg is removed with a 20% piperidine DMF solution.

[0072] (5) The resin is dried under vacuum.

[0073] (6) Resin cutting: After the resin dries, the polypeptide is cut off from the resin.

[0074] 5mL of cutting fluid is prepared as follows:

[0075] Trifluoroacetic acid (TFA): water: triisopropylsilane (TIS) = 95: 2.5: 2.5.

[0076] Transfer the dried resin to a fixed, dried pectoral flask, slowly add the cutting reagent, add a magnetic stir bar, and stir the mixture at room temperature for 3 hours. After the reaction is complete, transfer the reactants to a sintered glass funnel, rinse the pectoral flask with TFA, and then transfer the solution to the sintered glass funnel. After vacuum filtration, collect the filtrate in a round-bottom flask and purge the filtrate with nitrogen until it reaches a semi-viscosity.

[0077] (7) Add 20 mL of ice-cold ether to a round-bottom flask to precipitate the polypeptide. After breaking up the precipitate, transfer it to a 50 mL centrifuge tube and centrifuge at 4000 rpm for 5 min. Discard the supernatant and break up the precipitate with 20 mL of ice-cold ether again. Repeat this process 4 times. Dry the precipitate under vacuum to obtain crude TY-10 polypeptide.

[0078] (8) The crude TY-10 polypeptide was purified by reversed-phase high-performance liquid chromatography, and the results are shown in the attached figure. Figure 1 As shown.

[0079] (9) The purified target peptide was identified by mass spectrometry, and the results are shown in the appendix. Figure 2 As shown. (From the appendix) Figure 2 The molecular weight of the synthesized TY-10 polypeptide was measured to be 3804, which is consistent with its theoretical molecular weight, indicating that the target polypeptide was successfully prepared in this embodiment.

[0080] Example 2: Experiment on the binding of TY-10 peptide to mRNA

[0081] Taking the TY-10 polypeptide prepared in Example 1 as an example, a polypeptide / mRNA complex was prepared, and the binding ability between the two was detected. This example uses mRNA capable of expressing EGFP protein. The specific operating steps are as follows:

[0082] (1) Preparation of peptide / mRNA complexes: Peptide DMSO solutions and mRNA aqueous solutions were prepared according to N / P ratios (defined as the molar ratio of positively charged amino nitrogen in the peptide to phosphorus in the negatively charged phosphate group of the mRNA) of 0, 0.5, 1, 3, 5, 7, 10, and 15, respectively. The concentration of mRNA was 0.04 mg / mL; the peptide concentrations prepared according to different N / P ratios were: 0 mg / mL, 0.0276 mg / mL, 0.0551 mg / mL, 0.1654 mg / mL, 0.2757 mg / mL, 0.3860 mg / mL, 0.5514 mg / mL, and 0.8271 mg / mL. The volume of both peptide and mRNA solutions was 500 μL. The peptide and mRNA solutions were drawn up separately using a 3 mL syringe. The syringe was placed on a dual-channel microfluidic device. One channel was used for the peptide solution, and the other channel was used for the mRNA solution. The microfluidic flow rate was set to 4 mL / min. The peptide solution and mRNA solution were fed into the chip (ChipShop, FluidicDesign: 1079; Product Code: 10001480) through the channel and thoroughly mixed to obtain a peptide / mRNA complex. The complex was dialyzed overnight at 4°C into PBS to obtain the final sample.

[0083] (2) Gel preparation: Agarose gel was prepared using 1×TAE solution. The gel concentration used in the experiment was 1%. The gel was heated in a microwave oven until the agarose was completely dissolved. After the temperature was slightly cooled, the RNA dye SYBR Gold (purchased from Beijing Bairddi Biotechnology Co., Ltd.) was added and the mixture was shaken well.

[0084] (3) Pour the gel: Pour the dissolved agarose into the mold. After the gel solidifies, remove the comb and place the gel into the electrophoresis tank, with the gel well side facing the negative electrode of the electrophoresis tank. Then add 1×TAE solution to submerge the gel.

[0085] (4) Sample loading: Mix the prepared sample thoroughly with 5 μL of 5×RNA loading buffer (purchased from Beijing Bairddi Biotechnology Co., Ltd.), and bring the volume up to 50 μL with water. Then add the sample to the gel wells.

[0086] (5) Electrophoresis: Electrophoresis was performed at 120V for about 40 minutes. Electrophoresis was stopped when the front end of the loading buffer dye was observed to have run to about 2 / 3.

[0087] (6) After electrophoresis, take pictures using a gel imaging system.

[0088] As attached Figure 3 As shown, the TY-10 polypeptide of the present invention has a compressive effect on mRNA. When N / P≥1, the TY-10 polypeptide can completely bind to mRNA.

[0089] Example 3: Determination of the encapsulation efficiency of TY-10 peptide on mRNA

[0090] Taking the TY-10 polypeptide prepared in Example 1 as an example, the encapsulation efficiency of the hairpin peptide on mRNA was determined. The mRNA used in this example is the same as that in Example 2. The specific operating steps are as follows:

[0091] (1) Preparation of the peptide / mRNA complex: Refer to Example 2. Experimental group: Take 5 μL of the peptide / mRNA complex prepared in Example 2, and dilute the complex mRNA concentration to 0.002 mg / mL with TE buffer, with a volume of 100 μL. Take 1 μL of RiboGreen reagent and dilute it 100 times with TE buffer. Then, mix the two in equal volumes (total volume 200 μL). Background group: Take 1 μL of RiboGreen reagent (purchased from Thermo Fisher Scientific), and dilute it 200 times with TE buffer. Blank group: Take 5 μL of mRNA solution with a concentration of 0.04 mg / mL, and dilute it to 0.001 mg / mL with TE buffer, with a volume of 200 μL.

[0092] (2) Transfer the above sample to an enzyme-labeled plate and incubate at 37°C for 10 min.

[0093] (3) The fluorescence intensity of RiboGreen dye after binding to mRNA was detected using a multifunctional microplate reader. The excitation wavelength and emission wavelength were set to 480 nm and 520 nm, respectively.

[0094] (4) Calculate the encapsulation efficiency of the polypeptide on mRNA using the following formula.

[0095]

[0096] Wherein, Fs is the fluorescence intensity of the experimental group, Fb is the fluorescence intensity of the background group, and Fc is the fluorescence intensity of the blank group.

[0097] As attached Figure 4 As shown, the encapsulation rate of TY-10 peptide on mRNA can reach over 95%, approaching 100%.

[0098] Example 4: TY-10 peptide binding experiment with plasmid DNA (pDNA)

[0099] Taking the TY-10 polypeptide prepared in Example 1 as an example, a polypeptide / pDNA complex was prepared, and the binding ability between the two was detected. In this example, pDNA capable of expressing EGFP protein was used. The specific operating steps are as follows:

[0100] (1) Preparation of peptide / pDNA complex: Peptide DMSO solution and pDNA aqueous solution were prepared according to N / P ratios (defined as the molar ratio of positively charged amino nitrogen in the peptide to phosphorus in the negatively charged phosphate group of the pDNA) of 0, 0.5, 1, 3, 5, 7, 10, and 15, respectively. The concentration of pDNA was 0.04 mg / mL; the peptide concentrations prepared according to different N / P ratios were: 0 mg / mL, 0.0276 mg / mL, 0.0551 mg / mL, 0.1654 mg / mL, 0.2757 mg / mL, 0.3860 mg / mL, 0.5514 mg / mL, and 0.8271 mg / mL. The volume of both peptide and pDNA solutions was 500 μL. The peptide and pDNA solutions were drawn up separately using a 3 mL syringe. The syringe was placed on a dual-channel microfluidic device. One channel was used for the peptide solution, and the other for the pDNA solution. The microfluidic flow rate was set to 4 mL / min. The peptide solution and pDNA solution flowed through the channel into the chip (ChipShop, FluidicDesign: 1079; Product Code: 10001480) and were thoroughly mixed to obtain a peptide / pDNA complex. The complex was dialyzed overnight at 4°C into PBS to obtain the final sample.

[0101] (2) Gel preparation: Agarose gel was prepared using 1×TAE solution. The gel concentration used in the experiment was 1%. The gel was heated in a microwave oven until the agarose was completely dissolved. After the temperature was slightly cooled, the DNA dye SYBR Green I (purchased from Beijing Bairddi Biotechnology Co., Ltd.) was added and the mixture was shaken well.

[0102] (3) Pour the gel: Pour the dissolved agarose into the mold. After the gel solidifies, remove the comb and place the gel into the electrophoresis tank, with the gel well side facing the negative electrode of the electrophoresis tank. Then add 1×TAE solution to submerge the gel.

[0103] (4) Sample loading: Mix the prepared sample thoroughly with 5 μL of 10× loading buffer (purchased from Beijing Bairddi Biotechnology Co., Ltd.), and bring the volume up to 50 μL with water. Then add the sample to the gel wells.

[0104] (5) Electrophoresis: Electrophoresis was performed at 120V for about 40 minutes. Electrophoresis was stopped when the front end of the loading buffer dye was observed to have run to about 2 / 3.

[0105] (6) After electrophoresis, take pictures using a gel imaging system.

[0106] As attached Figure 5 As shown, the TY-10 polypeptide of the present invention has a compressive effect on pDNA. When N / P ≥ 0.5, the TY-10 polypeptide can completely bind to pDNA.

[0107] Example 5: Particle size and potential analysis of TY-10 peptide / mRNA complex

[0108] The mRNA used in this embodiment is the same as in Embodiment 2.

[0109] (1) Referring to Example 2, prepare 500 μL each of peptide DMSO solution and 0.04 mg / mL mRNA aqueous solution with N / P ratios of 0, 1, 3, 5, 7, 10, and 15. Mix the two solutions in equal volumes using microfluidics to obtain 1 mL of peptide / mRNA complex, and dialyze to PBS overnight at 4°C to obtain the final sample.

[0110] (2) Take 100 μL of the above solution after dialysis, dilute the polypeptide / mRNA complex solution to 1 mL, and mix thoroughly.

[0111] (3) The particle size and zeta potential of the composite were measured using a Malvern Nano ZSE. A standard sample cell was used for particle size measurement; a bent capillary sample cell was used for zeta potential measurement.

[0112] (4) Measure each sample three times and take the average value.

[0113] As attached Figure 6 As shown, the particle size of the TY-10 peptide / mRNA complex of the present invention is approximately 100 nm. The potential of the complex is shown in the appendix. Figure 7 .

[0114] Example 6: Particle size and potential analysis of TY-10 peptide / pDNA complex

[0115] The pDNA used in this embodiment is the same as in Example 4.

[0116] (1) Referring to Example 4, prepare 500 μL each of polypeptide DMSO solution and 0.04 mg / mL pDNA aqueous solution with N / P ratios of 0, 1, 3, 5, 7, 10, and 15. Mix the two solutions in equal volumes using microfluidics to obtain 1 mL of polypeptide / pDNA complex, and dialyze to PBS overnight at 4°C to obtain the final sample.

[0117] (2) Take 100 μL of the above solution after dialysis, dilute the polypeptide / pDNA complex solution to 1 mL, and mix thoroughly.

[0118] (3) The particle size and zeta potential of the composite were measured using a Malvern Nano ZSE. A standard sample cell was used for particle size measurement; a bent capillary sample cell was used for zeta potential measurement.

[0119] (4) Measure each sample three times and take the average value.

[0120] As attached Figure 8 As shown, the particle size of the TY-10 polypeptide / pDNA complex of the present invention is approximately 100 nm. The potential of the complex is shown in the appendix. Figure 9 .

[0121] Example 7: Cytotoxicity assay of the TY-10 peptide / mRNA complex

[0122] The mRNA used in this embodiment is the same as in Embodiment 2.

[0123] The cytotoxicity assay of the TY-10 peptide / mRNA complex was performed using the CCK-8 assay (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt). The specific operating steps are as follows:

[0124] Cell culture medium preparation: It was prepared by mixing 90% DMEM medium (purchased from Gibco, catalog number: GMS12052.3.1) and 10% FBS.

[0125] The cells used in the experiment were HEK-293T cells and HuH-7 cells. The experimental steps are shown below using HEK-293T cells as an example.

[0126] (1) When the HEK-293T cells in the culture flask reach more than 80%, add 1 mL of 0.25% trypsin (purchased from Sigma) to digest for 30 seconds. When the digestion of cells is observed to be complete under an inverted microscope, add 4 mL of cell culture medium to stop the process and centrifuge at 1000 rpm for 4 min. Discard the supernatant, add cell culture medium and gently pipette to disperse the cells evenly in the culture medium.

[0127] (2) Calculate cell density using a cell counter.

[0128] (3) Take the cell suspension and spread it on a plate. Add 100 μL to each well and add 5000 cells per well. Then incubate overnight in a 5% CO2, 37°C incubator to allow the cells to adhere.

[0129] (4) After incubation, remove the original culture medium and add 100 μL to each well. 1. Reduced Serum Media (Opti-DMEM, purchased from Gibco, catalog number: 31985070). Referring to Example 2, TY-10 peptide / mRNA complexes with N / P ratios of 1, 5, 10, 15, and 20 were prepared, with an mRNA concentration of 0.1 mg / mL. 10 μL of the peptide / mRNA complex was added to a 96-well plate and incubated with HEK-293T cells for 8 hours.

[0130] Among them, blank group: only cell culture medium was added; control group: HEK-293T cells were added to cell culture medium containing only mRNA (N / P=0); experimental group: HEK-293T cells were added to TY-10 peptide / mRNA complexes with different N / P ratios.

[0131] (5) After incubation for 8 hours, remove the serum-reduced culture medium and add fresh cell culture medium. Add 10 μL of LCK-8 solution to each well; then place the 96-well plate in a 5% CO2, 37°C incubator.

[0132] (6) After 4 hours of incubation, the OD value of the solution at 450 nm was measured using a microplate reader; the survival rate of HEK-293T cells in the presence of the TY-10 peptide / mRNA complex was calculated. Results are attached. Figure 10 .

[0133] As attached Figure 10 As shown, the TY-10 peptide / mRNA complex is almost non-toxic to HEK-293T and HuH-7 cells.

[0134] Example 8: Cytotoxicity assay of the TY-10 peptide / pDNA complex

[0135] The pDNA used in this embodiment is the same as in Example 4.

[0136] The cytotoxicity assay of the TY-10 peptide / pDNA complex was performed using the CCK-8 assay (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt). The specific operating steps are as follows:

[0137] Preparation of cell culture medium: Prepare DMEM medium containing 10% FBS (purchased from Gibco, catalog number: GMS12052.3.1);

[0138] The cells used in the experiment were HEK-293T cells and HuH-7 cells. The experimental steps are shown below using HEK-293T cells as an example.

[0139] (1) When the HEK-293T cells in the culture flask reach more than 80%, add 1 mL of 0.25% trypsin (purchased from Sigma) to digest for 30 seconds. When the digestion of cells is observed to be complete under an inverted microscope, add 4 mL of cell culture medium to stop the process and centrifuge at 1000 rpm for 4 min. Discard the supernatant, add cell culture medium and gently pipette to disperse the cells evenly in the culture medium.

[0140] (2) Calculate cell density using a cell counter.

[0141] (3) Take the cell suspension and spread it on a plate. Add 100 μL to each well and add 5000 cells per well. Then incubate overnight in a 5% CO2, 37°C incubator to allow the cells to adhere.

[0142] (4) After incubation, remove the original culture medium and add 100 μL to each well. 1. Reduced Serum Media (Opti-DMEM, purchased from Gibco, catalog number: 31985070). Referring to Example 4, TY-10 peptide / pDNA complexes with N / P ratios of 1, 5, 10, 15, and 20 were prepared, with a pDNA concentration of 0.1 mg / mL. 10 μL of the peptide / pDNA complex was added to a 96-well plate and incubated with HEK-293T cells for 8 hours.

[0143] Among them, blank group: only cell culture medium was added; control group: HEK-293T cells were added to cell culture medium containing only pDNA (N / P=0); experimental group: HEK-293T cells were added to TY-10 polypeptide / pDNA complexes with different N / P ratios.

[0144] (5) After incubation for 8 hours, remove the serum-reduced culture medium and add fresh cell culture medium. Add 10 μL of LCK-8 solution to each well; then place the 96-well plate in a 5% CO2, 37°C incubator.

[0145] (6) After 4 hours of incubation, the OD value of the solution at 450 nm was measured using a microplate reader; the survival rate of HEK-293T cells in the presence of the TY-10 polypeptide / pDNA complex was calculated. Results are attached. Figure 11 .

[0146] As attached Figure 11 As shown, the TY-10 peptide / pDNA complex is virtually non-toxic to HEK-293T and HuH-7 cells.

[0147] Example 9: Assay of the transfection capability of the TY-10 peptide / mRNA complex

[0148] The mRNA used in this example is the same as in Example 2; the HEK-293T cells and culture medium are the same as in Example 7.

[0149] (1) When the HEK-293T cells in the culture flask reach more than 80%, add 1 mL of 0.25% trypsin (purchased from Sigma) to digest for 30 seconds. When the digestion of cells is observed to be complete under an inverted microscope, add 4 mL of cell culture medium to stop the digestion and centrifuge at 1000 rpm for 4 min. Discard the supernatant, add cell culture medium and gently pipette to disperse the cells evenly in the culture medium.

[0150] (2) Calculate cell density using a cell counter.

[0151] (3) Add the cell suspension to a 24-well plate, adding 1 mL to each well to make 100,000 cells per well. Then incubate overnight in a 5% CO2, 37°C incubator to allow the cells to adhere.

[0152] (4) Remove the culture medium, add 1 mL of Opti-MEM serum-depleted medium (purchased from Gibco, catalog number: 31985070), and then add 1 mL of Opti-MEM serum-depleted medium diluted with different N / P TY-10 peptide / mRNA complex solutions. The control group consisted of: blank group (only Opti-MEM serum-depleted medium added); control group (HEK-293T cells added to Opti-MEM serum-depleted medium containing only mRNA); positive control group (HEK-293T cells added to Opti-MEM serum-depleted medium containing Lipo3000); and experimental group (HEK-293T cells added to TY-10 peptide / mRNA complexes with different N / P).

[0153] (5) After co-incubating the TY-10 polypeptide / mRNA complex (N / P = 7, 10, 15) with the cells for 6 h, the culture medium was removed, 2 mL of complete culture medium was added, and then the cells were placed in a 5% CO2, 37℃ incubator for 24 h.

[0154] (6) After the culture is completed, remove the original culture medium and add 1 mL of Opti-MEM culture medium to each well. Use a fluorescence microscope to observe the fluorescence distribution in the cells.

[0155] (7) Subsequently, the cells were washed with PBS, digested with 0.25% trypsin, centrifuged to obtain cells, and then added to PBS to make a cell suspension.

[0156] (8) The percentage of successfully transfected positive cells was determined by flow cytometry; the gating signal was calibrated using untreated cells as a negative control.

[0157] The results are attached. Figure 12 and attached Figure 13 As shown, the TY-10 peptide / mRNA complex exhibited the highest green fluorescence intensity and transfection efficiency in cells at an N / P ratio of 15.

[0158] Example 10: Determination of the transfection capability of the TY-10 peptide / pDNA complex

[0159] The pDNA used in this example is the same as in Example 4; the HEK-293T cells and culture medium are the same as in Example 7.

[0160] (1) When the HEK-293T cells in the culture flask reach more than 80%, add 1 mL of 0.25% trypsin (purchased from Sigma) to digest for 30 seconds. When the digestion of cells is observed to be complete under an inverted microscope, add 4 mL of cell culture medium to stop the digestion and centrifuge at 1000 rpm for 4 min. Discard the supernatant, add cell culture medium and gently pipette to disperse the cells evenly in the culture medium.

[0161] (2) Calculate cell density using a cell counter.

[0162] (3) Add the cell suspension to a 24-well plate, adding 1 mL to each well to make 100,000 cells per well. Then incubate overnight in a 5% CO2, 37°C incubator to allow the cells to adhere.

[0163] (4) Remove the culture medium, add 1 mL of Opti-MEM serum-depleted medium (purchased from Gibco, catalog number: 31985070), and then add 1 mL of Opti-MEM serum-depleted medium diluted with different N / P TY-10 peptide / pDNA complex solutions. The control group consisted of: blank group (only Opti-MEM serum-depleted medium added); control group (HEK-293T cells added to Opti-MEM serum-depleted medium containing only pDNA); positive control group (HEK-293T cells added to Opti-MEM serum-depleted medium containing Lipo3000); and experimental group (HEK-293T cells added to TY-10 peptide / pDNA complexes with different N / P ratios).

[0164] (5) After co-incubating the TY-10 polypeptide / pDNA complex (N / P = 7, 10, 15) with the cells for 6 h, the culture medium was removed, 2 mL of complete culture medium was added, and then the cells were placed in a 5% CO2, 37℃ incubator for 24 h.

[0165] (6) After the culture is completed, remove the original culture medium and add 1 mL of Opti-MEM culture medium to each well. Use a fluorescence microscope to observe the fluorescence distribution in the cells.

[0166] The results are attached. Figure 14 As shown, the TY-10 peptide / pDNA complex exhibited the highest green fluorescence intensity and transfection efficiency in cells at an N / P ratio of 15.

[0167] The above description is merely a preferred embodiment of the present invention, and the present invention should not be limited to the content disclosed in this embodiment and the accompanying drawings. Any equivalent or modified embodiments made without departing from the spirit of the present invention fall within the scope of protection of the present invention.

Claims

1. A hairpin peptide, characterized in that, It has the structure shown in equation (1): [TS]K p1 H n1 X m1 GPGGX m2 H n2 K p2 (1), in: H stands for histidine; K for lysine; G for glycine; P for proline; X may be the same or different, and is independently selected from tryptophan (W), leucine (L), valine (V), phenylalanine (F), or alanine (A); m1 and m2 may be the same or different, and are independent integers from 1 to 8; preferably, m1 is 6 and m2 is 5. n1 and n2 may be the same or different, and are independently integers from 2 to 5; preferably, n1 and n2 are independently integers from 2 to 3, and more preferably, n1 and n2 are both 3; p1 and p2 may be the same or different, and are independently integers from 3 to 8. Preferably, p1 and p2 are independently integers from 3 to 5; more preferably, p1 is 3 and p2 is 5. [TS] is a hairpin peptide targeting sequence; preferably, the hairpin peptide targeting sequence is selected from RGD, GPR, AEIDGIEL, LDT, CS-1peptide, CS-5peptide, and IDAPS; more preferably, the hairpin peptide targeting sequence is RGD.

2. The hairpin peptide as described in claim 1, characterized in that, It has the structure shown in equation (2): RGDKKKHHHWLVFFAGPGGLVFFAHHHKKKKK(2).

3. The method for preparing hairpin peptide as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Resin activation; preferably, the resin is Fmoc-Lys(Boc)-Wang resin; (2) Removal of Fmoc protecting groups; preferably, Kjeldahl test is performed to determine whether Fmoc deprotection is complete; (3) Cyclic coupling of amino acids: Cyclic coupling of Fmoc-protected amino acids from C to N segments according to the hairpin peptide sequence, and removal of Fmoc protecting groups; preferably, the coupling reagents are HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate) and DIPEA (N,N-diisopropylethylamine); preferably, Kjeldahl assay is performed after coupling of Fmoc-protected amino acids and removal of Fmoc protecting groups to determine whether the coupling and deprotection of Fmoc are complete; preferably, the amount of Fmoc-protected amino acids and HBTU is 4 equivalents, and the amount of DIPEA is 8 equivalents. Preferably, the following steps are also included: (4) Vacuum drying and cutting of resin. Preferably, the cutting fluid is trifluoroacetic acid (TFA): water: triisopropylsilane (TIS) = 95: 2.5: 2.

5. More preferably, it also includes the following steps: (5) Preparation and purification of crude hairpin peptide. Preferably, the hairpin peptide is purified by reversed-phase high-performance liquid chromatography.

4. A nanocomposite, characterized in that, It contains the hairpin peptide and bioactive substance as described in claim 1 or 2. Preferably, the nanocomposite is composed of hairpin peptides and bioactive substances as described in claim 1 or 2.

5. The nanocomposite according to claim 4, characterized in that, The bioactive substances are selected from nucleic acids, enzymes, and small molecule drugs. Preferably, the bioactive substance is mRNA or plasmid DNA. Preferably, the molar ratio (N / P) of the positively charged amino nitrogen element in the hairpin peptide to the negatively charged phosphorus element in the phosphate group of the nucleic acid is at least 0.5, more preferably at least 1, further preferably 1-20, even more preferably 2-16, and most preferably 7-15.

6. The method for preparing the nanocomposite as described in claim 4 or 5, characterized in that, Includes the following steps: (1) Prepare hairpin peptide solution; preferably, the solvent is DMSO; (2) Preparation of aqueous solutions of bioactive substances; (3) Use a syringe to draw up the hairpin peptide solution and the aqueous solution of the bioactive substance, respectively; (4) Place the syringe on the dual-channel microfluidic device, with one channel flowing through the hairpin peptide solution and the other channel flowing through the aqueous solution of the bioactive substance. The two flow into the chip and mix thoroughly to obtain a complex solution of hairpin peptide and bioactive substance. Preferably, the microfluidic flow rate is 1-8 mL / min; more preferably, the microfluidic flow rate is 4 mL / min. (5) Dialyze the complex solution overnight; preferably, dialyze overnight in a 10k dialysis bag at 4°C.

7. A pharmaceutical composition, characterized in that, It contains the nanocomposite as described in claim 4 or 5.

8. A method of treating, diagnosing, or preventing a disease in a subject, comprising administering to the subject a hairpin peptide as claimed in claim 1 or 2, a nanocomposite as claimed in claim 4 or 5, or a pharmaceutical composition as claimed in claim 7.

9. The use of the hairpin peptide of claim 1 or 2, the nanocomposite of claim 4 or 5, or the pharmaceutical composition of claim 7 in the preparation of a medicament for the treatment, diagnosis, or prevention of a disease.

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