Design and application of a temperature-sensitive peptide-based coacervate probe for solid-liquid phase transition

Abstract

The application discloses a design and application of a temperature-sensitive solid-liquid phase change peptide-based condensate probe, and belongs to the field of biomedical technologies. The peptide-based probe can form a condensate droplet through solid-liquid phase change near body temperature, and forms a gel at room temperature, can penetrate a cell membrane and produce fluorescence when combined with RNA.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to the design and application of a thermosensitive peptide-based condensate probe capable of solid-liquid phase transition. Background Technology

[0002] Liquid-liquid phase separation (LLPS) refers to the process by which a homogeneous aqueous solution forms two immiscible liquid phases of different components through the aggregation and multi-molecular assembly of solutes. Recent studies have found that the liquid-liquid phase separation process of proteins plays a crucial role in cell biology, controlling the formation of membraneless organelles and regulating their physiological functions and activities.

[0003] Within the nucleolus and nucleosome of organisms, there exists a class of RNA-binding proteins (RNP proteins) that can bind to RNA and undergo liquid-liquid phase separation. These RNP proteins contain simple, disordered, repetitive amino acid sequences, typically comprising an RNA-binding domain, an intrinsically disordered region, and other structural and functional domains. The RNA-binding domain, upon binding to RNA, promotes condensate formation.

[0004] LARKS fragments are a class of polypeptides commonly used in biochemical and biophysical research. Their full name is low-complexity, aromatic-rich, reversible kinked segments. They are kinked structures characterized by low complexity, rich in aromatic amino acids, and capable of reversible fibrillation assembly. Low-complexity: LARKS sequences consist of only a small number of different types of amino acids, typically rich in glycine. Kinked structure: The amino acid arrangement of LARKS sequences usually results in a bent structure during folding, rather than a linear structure such as a β-sheet; Aromatic-rich: LARKS sequences are typically rich in aromatic amino acids such as tyrosine and phenylalanine, which can enhance the overall stability of the aggregate structure through weak intermolecular forces. These characteristics give LARKS fragments unique structures and functions in biology, and they can aggregate or decompose through protein-protein interactions, thus exhibiting a certain degree of temperature responsiveness. For example, solutions containing LARKS fragments can undergo fibrillation assembly to form gels upon cooling.

[0005] Fluorescent agents are often used to construct fluorescent probes. Some fluorescent agent molecules emit almost no light in solution, but their luminescence is greatly enhanced in the aggregated state or under solid film conditions. Because this enhanced luminescence is caused by aggregation, this phenomenon is defined as aggregation-induced emission (AIE). Traditional AIE units and probes modified with hydrophilic peptides generally lack temperature responsiveness, cannot bind to RNA and maintain its stability, and cannot form droplet-like aggregates. Coupled with AIE luminescent units to IDPs, RNA concentration-responsive fluorescent aggregate probes can be obtained, but these probes lack temperature responsiveness and cannot be converted into gels through a thermosensitive phase transition.

[0006] In the existing technology, the traditional thermo-responsive polymer polyacrylamide (PNIPAM) cannot form droplet-like aggregates; although thermo-sensitive polypeptide molecules such as elastin-like proteins (ELP) and arthropod elastin can form aggregates under low molecular weight conditions, their ability to cross cell membranes is poor, making them difficult to use as probes, and they do not have RNA binding ability. Summary of the Invention

[0007] To address the aforementioned problems in existing technologies, this invention provides the design and application of a thermosensitive, solid-liquid phase-change peptide-based condensate probe. This peptide-based condensate probe is a solid particle or fiber at room temperature, exhibiting good dimensional stability, self-fluidity, and low cell membrane penetration capability. When the temperature rises to body temperature or higher, it spontaneously transforms into a droplet-like condensate, thus possessing good fluidity and membrane penetration capability. This peptide-based condensate probe can simultaneously meet the storage and in vivo use requirements of bioactive macromolecular drugs such as protein drugs and nucleic acids, and is expected to be used as a carrier for macromolecular drug delivery, improving the efficacy of gene therapy, vaccine delivery, and other biomedical applications.

[0008] To achieve the above-mentioned objectives, the present invention employs the following technical solution: a thermosensitive peptide condensate probe capable of solid-liquid phase transition, wherein the peptide condensate probe comprises three parts: a photoresponsive group, a fibrillable fragment LARKS, and a repeating sequence fragment of a nucleic acid binding domain.

[0009] Furthermore, the photoresponsive groups include, but are not limited to, tetraphenylethylene, diphenylanthracene, porphyrin, or indocyanine green.

[0010] Furthermore, the fibrillable fragment LARKS is a type of short peptide fragment rich in glycine and aromatic amino acids, composed of 6-20 amino acids, which can spontaneously assemble into nanofibers and can be specifically stained by thioflavone T.

[0011] Furthermore, the "repetitive sequence fragment of the nucleic acid binding domain" is a cationic short peptide fragment or a zwitterionic short peptide fragment with a repetitive sequence.

[0012] Furthermore, the cationic or zwitterionic short peptide fragments can bind to RNA and form droplet-like aggregates.

[0013] Preferably, the short peptide fragment sequence is (XXASL)n, (XGGY)n, (XGG)n, (XGY)n, (XG)n, (XG)n, (XGDG)n or (XGEG)n, where X represents a positively charged amino acid, including arginine, lysine, and histidine; and n is 2-5.

[0014] Furthermore, the peptide-based condensate probe exhibits a solid-liquid phase transition capability with temperature changes, forming microgels at temperatures below 30°C and condensate droplets at temperatures between 30-42°C.

[0015] Furthermore, the peptide-based condensate probe is capable of crossing the cell membrane and binding to RNA within the cell nucleus to emit light.

[0016] Another objective of this invention is to provide a thermosensitive, solid-liquid phase-change-capable peptide condensate probe for use as a smart phase-change carrier to deliver exogenous RNA molecules into (tumor) cells, or for inducing solid-liquid phase change of the probe in combination with photothermal therapy, thereby improving the targeting of small molecule anticancer drugs to tumors.

[0017] The peptide-based condensate probe of the present invention can spontaneously assemble into droplet-shaped condensates at a body temperature of 35-42°C, and the condensates can undergo coalescence, fusion and growth at the liquid-liquid interface; when the temperature drops below 35°C, the condensate probe forms water-insoluble solid particles or micro / nanofibers, which can maintain size stability, and the above-mentioned solid-liquid phase transition is temperature-responsive and reversible.

[0018] The peptide-based condensate probe of this invention penetrates the cell membrane in the form of droplet-like condensates at body temperature (35-42°C). When the temperature drops below 35°C, the peptide-based condensate probe usually cannot directly enter the cell, or it penetrates the membrane as solid particles, but its membrane penetration efficiency is low and it is easily degraded by lysosomes. Overall, the membrane penetration efficiency of the peptide-based condensate probe is temperature-dependent.

[0019] The peptide-based condensate probe of the present invention can deliver biomolecules such as nucleic acids or proteins into tumor cells, either alone or in combination with photothermal agents. It can also improve the targeting of small molecule anticancer drugs to the tumor cell nucleus, thereby optimizing the process of anti-tumor radiotherapy, chemotherapy, photothermal, and photodynamic therapy for tumors and improving treatment efficacy. Attached Figure Description

[0020] Figure 1 The mass spectrum of the target peptide TPE-RRASLRRASL-EPTGLGFG.

[0021] Figure 2 The graph shows the change in turbidity of the target peptide TPE-RRASLRRASL-EPTGLGFG with temperature.

[0022] Figure 3 The images show the morphology of the target peptide TPE-RRASLRRASL-EPTGLGFG at different temperatures, where a represents 25℃, b represents 37℃, and c represents 60℃.

[0023] Figure 4 A light micrograph of cells after co-incubation with the target peptide TPE-RRASLRRASL-EPTGLGFG.

[0024] Figure 5 Fluorescence image of the target peptide TPE-RRASLRRASL-EPTGLGFG after co-incubation with cells. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0026] Example

[0027] Synthesis of the target peptide TPE-RRASLRRASL-EPTGLGFG: (TPE is a tetraphenylethylene (TPE) luminescent group, an aggregation-induced luminescent molecule. During phase separation, the peptide is enriched in the condensed phase, resulting in significantly enhanced fluorescence. RRASLRRASL is a phase-change transmembrane peptide that promotes peptide entry into cells and recognition of nucleic acids. EPTGLGFG is a LARKS peptide that incubates the peptide in a temperature-responsive manner.)

[0028] The solid-state synthesis method was used for synthesis, and the specific steps are as follows:

[0029] (1) Coupling of amino acids with resin: 2-chloro resin (1 g, 1 mmol / g) was used as a solid substrate and swelled in dichloromethane for 30 min. After swelling, amino-protected isoleucine Fmoc-Leu-OH (424 mg, 1.2 mmol), N,N-diisopropylethylamine (155 mg, 1.2 mmol), and DCC (379 mg, 3 mmol) were mixed with 20 mL of DMF and reacted for 1 h. After the coupling reaction was completed, methanol (1 mL) was added to the above mixture to stop the reaction for 10 min. The resulting leucine-labeled resin was washed twice with N,N-dimethylformamide (DMF).

[0030] (2) Removal of amino protecting groups Fmoc: The resin was then soaked in a 20% (v / v) piperidine / DMF solution for 15 minutes and then washed twice with DMF.

[0031] (3) Gradual growth of peptide chains: Fmoc-amino acids (2 mmol), 1-hydroxybenzotriazole (270 mg, 2 mmol), and DCC (757 mg, 6 mmol) were reacted with the above resin in DMF for 1 h. After the reaction, the reaction solution was filtered and washed twice with 30 mL of DMF. The binding of fmoc-amino acids to the resin was tested by ninhydrin.

[0032] Ninhydrin test: The ninhydrin test is used to check the completion of the coupling reaction and the removal of the Fmoc group. Simply put, add 20-30 resin beads to an Eppendorf tube (1.5 mL). Next, mix 5 wt.% ninhydrin (dissolved in ethanol), 80% phenol (dissolved in ethanol), and anhydrous pyridine in a 2:1:1 volume ratio in the centrifuge tube. Then, heat the centrifuge tube to 110°C for 2 minutes. The complete reaction of the amine terminal groups can be seen from the colorimetric analysis of the product.

[0033] a) If the product is blue, it indicates the presence of unreacted primary amines. (Positive)

[0034] b) If the product is brown or red, it indicates the presence of a secondary amine. (Positive)

[0035] c) If the product is pale yellow, it indicates the absence of terminal amines (negative).

[0036] If a positive sample is obtained after the coupling reaction, a second coupling reaction with the same amino acid is required. If the ninhydrin test is negative, the new amino acid has been successfully coupled, and peptide synthesis should then continue to remove the Fmoc group in order to proceed with the next coupling reaction.

[0037] (4) Repeat this step until the polypeptide sequence RRASLRRASL-EPTGLGFG is obtained.

[0038] (5) Coupling of tetraphenylethylene carboxylic acid (TPE-COOH): After the peptide sequence is completely synthesized, a mixture of TPE-COOH (1.5 g, 4 mmol), 1-hydroxybenzotriazole T (540 mg, 4 mmol) and DIC (1.26 g, 10 mmol) in DMF is mixed with resin and reacted for 2 hours to couple TPE-COOH to the N-terminus of the repeating peptide.

[0039] (6) Peptide cleavage from resin: The peptide-bound resin was dried by vacuum filtration and then transferred to a 50 mL centrifuge tube. Subsequently, the resin was soaked in a mixture of TFA / water / triisopropylsilane at a volume ratio of 95:2.5:2.5 for 2 hours. After vacuum impregnation, the resin peptide was precipitated in ice ether. The peptide / ether mixture was centrifuged at 5000 g for 10 minutes, and the crude peptide was collected and washed three times with ice ether. Further purification by reversed-phase high-performance liquid chromatography (RP-HPLC) yielded the target peptide sequence SEQ NO.1 TPE-RRASLRRASL-EPTGLGFG. Mass spectrometry detection MALDI-MS: m / z 1151.95 [M+2H]2+, indicating that the peptide has bound two protons and has two positively charged peaks (see...). Figure 1 ).

[0040] The application effects of this invention will be further explained below in conjunction with performance testing.

[0041] 1. Temperature response test of the target peptide TPE-RRASLRRASL-EPTGLGFG:

[0042] 0.05, 0.15, and 0.25 mM peptide solutions were prepared and sonicated to ensure uniform distribution. Turbidity was measured at 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, and 60℃. The solutions were then cooled, and turbidity was measured again at 55℃, 50℃, 45℃, 40℃, 35℃, 30℃, and 25℃. The results indicate that peptide phase separation disappears at high temperatures, and turbidity increases as temperature decreases, indicating that phase separation re-occurs (see...). Figure 2 ).

[0043] 2. Microscopic observation of the morphology of the 0.15 mM target peptide at 25℃, 37℃, and 60℃.

[0044] The results showed that at 25℃ it was a gel, at 37℃ it was a liquid condensate, and at 60℃ the phases separated and disappeared, which corresponds to the above turbidity results (see...). Figure 3 ).

[0045] 3. Cell membrane crossing assay of the target peptide TPE-RRASLRRASL-EPTGLGFG

[0046] Human gallbladder cancer cells (SGC-996) were seeded in 24-well plates at a density of 10,000 cells / well. The target peptide was added at a concentration of 0.15 mM, and the plates were incubated at 37°C for 2 hours. The culture medium was washed off, and the cells were observed. (See attached image.) Figure 4 and Figure 5 This indicates that the target polypeptide can enter the cell.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A thermosensitive peptide-based condensate probe capable of solid-liquid phase transition, characterized in that, The peptide-based condensate probe is composed of a photoresponsive group, a repeating sequence fragment of a nucleic acid binding domain, and a fibrillable fragment LARKS connected sequentially from the N-terminus to the C-terminus via amide bonds and peptide bonds. The photoresponsive group is tetraphenylethylene. The fibrillable fragment LARKS is a short peptide of 7 amino acids with the sequence EPTGLGFG. This short peptide is rich in glycine and aromatic amino acids and can spontaneously assemble into nanofibers, which can be specifically stained by thioflavin T. The repeating sequence fragment of the nucleic acid binding domain is (RRASL)2, i.e., RRASLRRASL, which is a cationic short peptide fragment that can bind to RNA and form droplet-like condensates.

2. The thermosensitive, solid-liquid phase-change-capable peptide condensate probe according to claim 1, characterized in that, The peptide-based condensate probe exhibits a solid-liquid phase transition capability with temperature changes, forming microgels at temperatures below 30°C and condensate droplets at temperatures between 30°C and 42°C.

3. The thermosensitive, solid-liquid phase-change-capable peptide condensate probe according to claim 1, characterized in that, The peptide-based condensate probe can cross the cell membrane and bind to RNA in the cell nucleus to emit light.

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