Polypeptide compound DTTPE as well as preparation method and application thereof

Abstract

The invention provides a polypeptide compound DTTPE as well as a preparation method and application thereof. The polypeptide compound DTTPE has the following structure: # imgabs0 #. The polypeptide compound DTTPE can form a peptidyl condensation body with small interfering RNA (siRNA), the peptidyl condensation body is delivered into cells through the modified cell-penetrating peptide RRRQRRKKR, nucleic acid drugs are effectively delivered into the cells, and a promising platform is provided for gene therapy application. And the peptidyl condensation body has the characteristics of high lysosome escape efficiency and excellent serum stability, can be used for high-efficiency delivery of nucleic acid drugs in various cells, and has a huge application prospect in the field of biomedical treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a polypeptide complex DTTPE and a preparation method and application thereof. Background Art

[0002] The clinical application of nucleic acids faces numerous challenges, including their negative charge, susceptibility to nuclease degradation, and low endosomal escape efficiency (<5%). Biomolecular condensates offer a novel and promising solution to enhance cargo uptake and facilitate endosomal escape. Peptide-based condensates interact with cargo through weak non-covalent interactions, allowing for rapid recruitment of diverse cargo types in aqueous environments while maintaining the bioactivity of macromolecular therapeutics. However, existing peptide-based condensates present challenges in the delivery of nucleic acid drugs. Summary of the Invention

[0003] In view of this, the present invention provides a polypeptide complex DTTPE and a preparation method and application thereof, which can effectively deliver nucleic acid drugs into cells to achieve the effect of gene therapy.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a polypeptide complex DTTPE having the following structure: .

[0005] In a second aspect, the present invention provides a method for preparing the polypeptide complex DTTPE, comprising the following steps: Under the action of a catalyst, the polypeptide RRRQRRKKR reacts with a tetraphenylethylene derivative TPE in a solvent to obtain a polypeptide complex DTTPE.

[0006] Preferably, the molar ratio of the polypeptide RRRQRRKKR to the tetraphenylethylene derivative TPE is (2-4):1.

[0007] Preferably, the catalyst comprises cuprous bromide and sodium ascorbate.

[0008] Preferably, the solvent comprises dimethyl sulfoxide and water.

[0009] In a third aspect, the present invention provides a use of the polypeptide complex DTTPE or the polypeptide complex DTTPE prepared by the preparation method as an infrared fluorescence emission probe.

[0010] In a fourth aspect, the present invention provides an application of the polypeptide complex DTTPE or the polypeptide complex DTTPE prepared by the preparation method in delivering siRNA drugs for gene silencing.

[0011] In a fifth aspect, the present invention provides a peptide-based condensate nucleic acid delivery reagent, comprising the polypeptide complex DTTPE or the polypeptide complex DTTPE prepared by the preparation method.

[0012] Preferably, the peptide-based condensate drug delivery agent comprises a living cell nucleic acid drug delivery agent.

[0013] Preferably, the cells of the living cell nucleic acid drug delivery agent include at least one of B16, HaCaT, HeLa, SiHa, SK-OV-3 and RAW264.7 cells.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1) The polypeptide complex DTTPE provided by the present invention has infrared fluorescence emission capability and efficient coacervate formation capability.

[0015] 2) The polypeptide complex provided by the present invention can form peptide-based aggregates with siRNA (small interfering RNA) drugs and be delivered into cells through the modified cell-penetrating peptide RRRQRRKKR. The peptide-based aggregates have the characteristics of efficient lysosomal escape and excellent serum stability, and can be used for efficient delivery of nucleic acid drugs in a variety of cells, solving the problem of difficult nucleic acid drug delivery and achieving the interference effect of silencing the target gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a synthetic route for the polypeptide complex DTTPE provided in Example 1 of the present invention; Figure 2 This is a liquid chromatogram of the polypeptide complex DTTPE provided in Example 1 of the present invention; Figure 3 This is a mass spectrometry characterization diagram of the polypeptide complex DTTPE provided in Example 1 of the present invention; Figure 4 The UV-visible absorption spectrum and fluorescence spectrum characterization diagram of the polypeptide complex DTTPE provided in Example 2 of the present invention; Figure 5 This is a confocal imaging image of the peptide-based aggregates provided in Example 3 of the present invention; Figure 6 This is a confocal image of the peptide-based aggregates provided in Example 4 of the present invention delivering siRNA drugs; Figure 7 This is a confocal image of the localization of the peptide aggregates and lysosomes in living cells provided in Example 5 of the present invention; Figure 8 These are confocal images of siRNA delivered by the peptide-based aggregates provided in Example 6 of the present invention in different cells; Figure 9 This is a graph showing the stability test of the peptide-based aggregates provided in Example 7 of the present invention under different serum concentrations; Figure 10 This is a test diagram of gene silencing caused by the peptide-based aggregates provided in Example 8 of the present invention delivering siRNA drugs. DETAILED DESCRIPTION

[0017] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0018] Description of the sources of key experimental materials: The peptide RRRQRRKKR was purchased from Nanjing Source Peptide Biotechnology Co., Ltd. and has the following chemical structure: .

[0019] Tetraphenylethylene derivative TPE, its chemical structure is .

[0020] For the synthesis method, please refer to "Spatial order of functional modules enabling diverse intracellular performance of fluorescent probes", DOI: doi.org / 10.1002 / anie.202106195.

[0021] Example 1 A preparation method of peptide complex DTTPE, please refer to Figure 1 , including the following steps: A 100 mL round-bottom flask was added with tetraphenylethylene derivative TPE (2.34 mg, 2.94 μmol), peptide RRRQRRKKR (10.00 mg, 6.47 μmol), 2 mL of dimethyl sulfoxide, 2 mL of water, sodium ascorbate (5.82 mg, 29.42 μmol), and cuprous bromide (4.22 mg, 29.42 μmol). Under nitrogen atmosphere, the mixture was stirred at room temperature for 24 hours to obtain a crude product, which was purified by high performance liquid chromatography and freeze-dried to obtain the product peptide complex DTTPE (5.5 mg, yield: 48%). Among them, HPLC purification uses reverse HPLC gradient elution. The purification process conditions of reverse HPLC gradient elution are as follows: the sample is dissolved in aqueous solution or acetonitrile, applied to an Ultimate XB-C18 column (10 μm, 250×10 mm), eluted at a rate of 2 mL / min, solvent A is an aqueous solution containing 0.1% TFA, solvent B is an acetonitrile solution containing 0.1% TFA, and the gradient program is: 0.01 min, 20% solvent B; 20 min, 40% solvent B; 25 min, 60% solvent B; 30 min, 90% solvent B; 50 min, 100% solvent B. The liquid chromatogram is shown in the figure below. Figure 2 shown.

[0022] The obtained polypeptide complex DTTPE was characterized by high resolution mass spectrometry to confirm the structure of the polypeptide complex DTTPE. Figure 3 shown.

[0023] MALDI-TOF / TOF m / z: [M-2PF6 - ] + Calculated value: 3882.3063, measured value: 3882.246.

[0024] Example 2 The spectral characterization test of the polypeptide complex DTTPE prepared in Example 1 includes the following steps: S1, dissolve the solid peptide complex DTTPE with water to obtain a 10 μM peptide complex DTTPE solution.

[0025] S2, use UV spectrophotometer to record the spectrum of peptide complex DTTPE solution. Figure 4 As shown in Figure (A). Figure 4 (A) It can be seen that the peptide complex DTTPE has good UV-visible light absorption.

[0026] S3, further fluorescence spectroscopy characterization was performed on 10 μM peptide complex DTTPE solution. Figure 4 As shown in (B). Figure 4 (B) It can be seen that the peptide complex DTTPE shows a broad emission spectrum from 600 nm to 800 nm, with the emission peak located near 750 nm.

[0027] Example 3 The peptide complex DTTPE prepared in Example 1 was subjected to a peptide-based aggregate characterization test, comprising the following steps: S1, dissolving the solid peptide complex DTTPE with water to obtain a 1.6 μM peptide complex DTTPE solution.

[0028] S2, dissolve the solid Cy5 fluorescently labeled siRNA in pure water to obtain a 20 μM siRNA stock solution.

[0029] S3, take 10 μL of the above siRNA stock solution and add it to 1 mL of the above peptide complex DTTPE solution, and observe it with a laser confocal microscope. Figure 5 shown.

[0030] from Figure 5 It can be seen that the fluorescence signal of siRNA highly overlaps with the fluorescence signal of the polypeptide complex DTTPE, which indicates that the polypeptide complex DTTPE has the ability to form aggregates with siRNA.

[0031] Example 4 The experiment of intracellular delivery of siRNA was conducted on the polypeptide complex DTTPE prepared in Example 1, comprising the following steps: S1, dissolve the solid peptide complex DTTPE in cell culture medium to obtain a 1.6 μM peptide complex DTTPE solution.

[0032] S2, dissolving solid Cy5 fluorescently labeled siRNA in pure water, wherein siFOXO4 is an siRNA drug for FOXO4 gene silencing, to obtain a 20 μM siRNA stock solution.

[0033] S3, 10 μL of the above siRNA stock solution was added to 1 mL of the above peptide complex DTTPE solution and allowed to stand for 5 minutes to obtain a peptide aggregate mixed solution, which was then co-cultured with lung fibroblasts for 12 hours and observed using a laser confocal microscope. Figure 6 shown.

[0034] from Figure 6 It can be seen that siRNA alone cannot enter lung fibroblasts, but when siRNA forms a peptide-based aggregate with the polypeptide complex DTTPE, siRNA can be delivered into cells.

[0035] Example 5 This example further incubates the peptide aggregates with lung fibroblasts and then performs confocal microscopy imaging, including the following steps: S1, dissolve the solid peptide complex DTTPE in cell culture medium to obtain a 1.6 μM peptide complex DTTPE solution.

[0036] S2, dissolve the solid Cy5 fluorescently labeled siRNA in pure water to obtain a 20 μM siRNA stock solution.

[0037] S3. Take 10 μL of the above siRNA stock solution and add it to 1 mL of the above peptide complex DTTPE solution and let it stand for 5 minutes to obtain a peptide-based aggregate mixed solution.

[0038] S4, culture lung fibroblasts in a 37°C culture flask in a humidified atmosphere containing 5% CO2 using a cell culture medium containing 10% FBS and 1% penicillin-streptomycin. 4 After overnight culture, the cells were washed three times with phosphate-buffered saline. The peptide aggregates were then added, and the lung fibroblasts were incubated at 37°C in a 5% CO2 atmosphere for 12 hours. The supernatant was then discarded, and the cells were gently washed twice with PBS buffer. The lysosome indicator, Lyso-tracker, was then added, and the lung fibroblasts were incubated at 37°C in a 5% CO2 atmosphere for 30 minutes. The supernatant was then discarded, and the cells were gently washed twice with PBS.

[0039] S5, the cell culture dish after the above operation was examined using a confocal microscope. Figure 7 shown.

[0040] from Figure 7 It can be seen that the peptidyl aggregates are poorly localized on the cell lysosomes, indicating that the peptidyl aggregates fail to enter the lysosomes.

[0041] Example 6 This example provides a test of the universal effect of peptide-based aggregates on siRNA delivery to various cells, including the following steps: S1, dissolve the solid peptide complex DTTPE in cell culture medium to obtain a 1.6 μM peptide complex DTTPE solution.

[0042] S2, dissolve the solid Cy5 fluorescently labeled siRNA in pure water to obtain a 20 μM siRNA stock solution.

[0043] S3. Take 10 μL of the above siRNA stock solution and add it to 1 mL of the above peptide complex DTTPE solution and let it stand for 5 minutes to obtain a peptide-based aggregate mixed solution.

[0044] S4. Culture B16, HaCaT, HeLa, SiHa, SK-OV-3, and RAW264.7 cells in culture flasks at 37°C in a humidified atmosphere containing 5% CO2 using cell culture medium containing 10% FBS and 1% penicillin-streptomycin. Seed B16, HaCaT, HeLa, SiHa, SK-OV-3, and RAW264.7 cells in the above-mentioned medium at a density of 2 × 10 4Cells were cultured in a dish containing 100 μg of culture medium. After overnight incubation, the cells were washed three times with phosphate-buffered saline. The peptide-based aggregates were then added, and the cells were incubated at 37°C in a 5% CO2 atmosphere for 12 hours. The supernatant was then discarded, and the cells were gently washed twice with PBS.

[0045] S5, the cell culture dish after the above operation was examined using a confocal microscope. Figure 8 shown from Figure 8 It can be seen that the peptide-based polymers can deliver siRNA into B16, HaCaT, HeLa, SiHa, SK-OV-3 and RAW264.7 cells, and the drug delivery ability is universal across cell types.

[0046] Example 7 This example provides a stability test of peptide-based aggregates against serum at different concentrations, comprising the following steps: S1, dissolve the solid peptide complex DTTPE in cell culture medium to obtain a 1.6 μM peptide complex DTTPE solution.

[0047] S2, dissolve the solid Cy5 fluorescently labeled siRNA in pure water to obtain a 20 μM siRNA stock solution.

[0048] S3. Take 10 μL of the above siRNA stock solution and add it to 1 mL of the above peptide complex DTTPE solution and let it stand for 5 minutes to obtain a peptide-based aggregate mixed solution.

[0049] S4, prepare cell culture medium containing different serum concentrations (0%, 10%, 50%, 100%), replace the peptide aggregate solution, and then let it stand for 12 hours, and detect it using confocal microscopy. Figure 9 shown.

[0050] from Figure 9 It can be seen that the peptide-based aggregates can remain stable in culture media with different serum concentrations.

[0051] Example 8 This example provides a test of the siRNA gene silencing efficiency of peptide-based aggregates, comprising the following steps: S1, dissolve the solid peptide complex DTTPE in cell culture medium to obtain a 1.6 μM peptide complex DTTPE solution.

[0052] S2. Dissolve solid siRNA in pure water, wherein siFOXO4 is an siRNA drug for silencing the FOXO4 gene, and siNC is a scrambled siRNA without gene silencing effect, to obtain a 20 μM siRNA stock solution.

[0053] S3. Take 10 μL of the above siRNA stock solution and add it to 1 mL of the above peptide complex DTTPE solution and let it stand for 5 minutes to obtain a peptide-based aggregate mixed solution.

[0054] S4, culture the chemotherapeutic drug-induced senescent lung fibroblasts in a culture flask at 37°C in a humidified atmosphere containing 5% CO2 using cell culture medium containing 10% FBS and 1% penicillin-streptomycin. The lung fibroblasts were seeded into the above-mentioned medium at a density of 2×10 4 After overnight culture, the cells were washed three times with phosphate-buffered saline. The peptide aggregates were then added to the mixed solution and the lung fibroblasts were incubated at 37°C in a 5% CO2 atmosphere for 72 hours. The supernatant was then discarded, and the cells were gently washed twice with PBS buffer. The cells were then collected for real-time quantitative polymerase chain reaction analysis and protein immunoblotting analysis. The results are shown in Figure 2. Figure 10 shown.

[0055] from Figure 10 It can be seen that the peptide-based polymer delivery of siRNA drugs achieved the knockdown of target genes in cells and the inhibition of downstream protein expression.

[0056] In addition, it should be noted that in the preparation method of the polypeptide complex DTTPE of the present invention, it is necessary to control the reaction molar ratio of the polypeptide RRRQRRKKR and the tetraphenylethylene derivative TPE to (2~4):1, the molar addition amounts of cuprous bromide and sodium ascorbate are preferably 4.5 times the amount of the polypeptide RRRQRRKKR, the solvent is preferably a mixed solvent of dimethyl sulfoxide and water in a volume ratio of (0.5~2):1, the stirring reaction time is not less than 24 hours, and then purified by high performance liquid chromatography to ensure the overall high yield (≥45%) and high purity (≥95%) of the polypeptide complex DTTPE.

[0057] The raw materials not specifically described in the present invention are all existing materials that can be directly purchased from the market.

[0058] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A polypeptide complex DTTPE, characterized in that Has the following structure: 。 2. The method for preparing the polypeptide complex DTTPE according to claim 1, characterized in that: The following steps are involved: Under the action of a catalyst, the polypeptide RRRQRRKKR reacts with a tetraphenylethylene derivative TPE in a solvent to obtain a polypeptide complex DTTPE.

3. The method for preparing the polypeptide complex DTTPE according to claim 2, characterized in that: The molar ratio of the polypeptide RRRQRRKKR to the tetraphenylethylene derivative TPE is (2-4):

1.

4. The method for preparing the polypeptide complex DTTPE according to claim 2, characterized in that: The catalyst includes cuprous bromide and sodium ascorbate.

5. The method for preparing the polypeptide complex DTTPE according to claim 2, characterized in that: The solvents include dimethyl sulfoxide and water.

6. Use of the polypeptide complex DTTPE according to claim 1 or the polypeptide complex DTTPE prepared by the preparation method according to any one of claims 2 to 5 as an infrared fluorescence emission probe.

7. Use of the polypeptide complex DTTPE according to claim 1 or the polypeptide complex DTTPE prepared by the preparation method according to any one of claims 2 to 5 in delivering siRNA drugs for gene silencing.

8. A peptide-based condensate drug delivery agent, characterized in that: The invention comprises the polypeptide complex DTTPE according to claim 1 or the polypeptide complex DTTPE prepared by the preparation method according to any one of claims 2 to 5.

9. The peptide-based condensate drug delivery reagent according to claim 8, characterized in that: The peptide-based condensate drug delivery reagent includes a living cell nucleic acid drug delivery agent.

10. The peptide-based condensate drug delivery agent according to claim 9, characterized in that: The cells of the living cell nucleic acid drug delivery agent include at least one of B16, HaCaT, HeLa, SiHa, SK-OV-3 and RAW264.7 cells.

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