Phase separation polypeptide and application thereof in bionic protocyte construction

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a phase separation polypeptide and application thereof in bionic protocyte construction. The self-assembled polypeptide provided by the invention has inherent mitochondrial targeting and oxidation resistance, the synthesis method is simple, the biocompatibility is good, and the self-assembled polypeptide can be self-assembled to form condensed liquid drops. The coagulated liquid drops have strong loading capacity, can load mitochondria, have good oxidation resistance, can provide effective protection for co-loaded exogenous mitochondria, and co-construct a bionic protocyte system with complete functions.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a phase-separated polypeptide and its application in the construction of biomimetic protocells. Background Technology

[0002] Liquid-liquid phase separation is an important physicochemical process within cells and a fundamental physical mechanism for the formation and maintenance of many membrane-free organelles. It allows specific biomolecules to spontaneously separate from a homogeneous solution within the complex and crowded cytoplasm through multivalent interactions, forming enriched, dynamic droplet-like condensates. These biomolecular condensates, such as nucleoli and stress granules, act as key biochemical reaction centers within cells, precisely regulating various core physiological activities, such as gene transcription, signal transduction, and stress response, across the spatiotemporal dimensions through their unique dynamic characteristics.

[0003] Biomimetic protocells, also known as "primitive cells" or "prototype cells," are simplified life models assembled from biomolecules or synthetic materials using a bottom-up approach. Their core goal is not to precisely replicate the full complexity of modern cells, but rather to capture the most fundamental characteristics of living systems, such as spatial separation, material exchange, energy metabolism, and information response. As a cutting-edge intersection of synthetic biology and biomaterials, research on biomimetic protocells not only helps to unravel the mysteries of the origin of life but also aims to develop next-generation cell-mimicking drug delivery systems and intelligent therapeutic platforms.

[0004] The inherent characteristics of liquid-liquid phase separation—autonomous assembly, dynamic response, and efficient enrichment—make it an ideal molecular basis for constructing biomimetic protocells. By rationally designing sequences, short peptides can be synthesized to mimic the behavior of natural phase-separating proteins, spontaneously assembling in vitro to form a microenvironment with a cytoplasmic structure. These peptide-based artificial condensates not only mimic the morphology and material enrichment capacity of protocells but also, by loading specific active substances (such as enzymes and organelles), create a programmable and functional internal space, thereby performing complex biomimetic functions.

[0005] Constructing artificial cell-like entities with hierarchical structures and biological functions is a fundamental approach to revealing the origin of life. Existing technologies have constructed a series of synthetic cell analogs with life-like behaviors as protocell models, including fatty acid vesicles, liposomes, polymer vesicles, protein vesicles, colloidal vesicles, and condensates. The core of constructing protocells based on liposomes or polymer vesicles is mimicking the phospholipid bilayer of cells. The preparation process of these systems is complex, and their closed membrane structure restricts efficient material exchange and information communication with the external environment and target cells.

[0006] Colloids have demonstrated significant advantages as primitive cell models. Compared to lipid vesicles, colloid droplets exhibit a stronger capacity for enriching chemical components and can maintain dynamic material exchange with the external aqueous phase, thereby supporting the formation of chemical gradients. These properties enable them to effectively mimic the compartmentalization function of modern cells. Constructing colloids using short peptides not only simplifies material preparation processes but also reduces experimental costs. However, current short peptide-based colloids still have relatively limited functions, and challenges remain in enriching functional units such as organelles and maintaining their biological activity. Summary of the Invention

[0007] This invention provides polypeptides with good antioxidant and organelle protection functions, which can be assembled to form aggregate droplets and can form biomimetic protocells with mitochondria.

[0008] The purpose of this invention is to provide a phase-separated polypeptide.

[0009] Another object of the present invention is to provide the application of the above-mentioned phase-separated polypeptide in the construction of biomimetic protocells and drug delivery systems.

[0010] According to a specific embodiment of the present invention, a phase-separated polypeptide, wherein the amino acid sequence of the polypeptide is WGDVYGGRD mt KF, its structure is as follows:

[0011] The phase-separated peptides of this invention are synthesized using the classic Fmoc solid-phase synthesis method for amino acids. During the reaction, HBTU is used as an activator for the carboxyl groups of the amino acids, DIEA is used as a catalyst, and piperidine is used to remove the Fmoc protecting groups, thereby exposing the amino group and allowing it to undergo a cross-linking condensation reaction with the carboxyl group of the next amino acid activated by HBTU, forming a peptide bond. Once the peptide chain is complete, it is cleaved from the dichloropolymer resin using 1% trifluoroacetic acid (TFA).

[0012] The phase-separated peptides were dissolved in a buffer solution with a pH of 3-4 to obtain a peptide solution with a concentration of 10-50 mmol / L. The pH of the peptide solution was gradually adjusted to neutral to obtain submicron-sized peptide aggregates, which were uniformly distributed in the solution and exhibited a spherical droplet structure, with typical morphological characteristics of liquid-liquid phase-separated aggregates.

[0013] Preferably, the buffer solution is selected from citrate-sodium citrate buffer, acetate-sodium acetate buffer, potassium dihydrogen phosphate-disodium hydrogen phosphate buffer, and formic acid-sodium formate buffer. More preferably, the buffer solution is Tris-HCl buffer.

[0014] Preferably, the pH of the buffer solution is 3.0-4.0, or 3.0-3.5, 3.5-4.0, or the pH of the buffer solution is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0.

[0015] Preferably, the concentration of the obtained polypeptide solution is 10-50 mmol / L, or 15-45 mmol / L, 20-40 mmol / L, 25-35 mmol / L, or 15-35 mmol / L, or the concentration of the polypeptide solution is 10 mmol / L, 15 mmol / L, 20 mmol / L, 21 mmol / L, 22 mmol / L, 23 mmol / L, 24 mmol / L, 25 mmol / L, 26 mmol / L, 27 mmol / L, 28 mmol / L, 29 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, or 50 mmol / L, or any value within the above range. These will not be elaborated here.

[0016] This invention provides the application of the above-mentioned phase-separated polypeptide in the preparation of biomimetic protocells.

[0017] In this invention, the biomimetic protocell is an artificially synthesized microsystem that mimics the structure and function of natural cells. Its core is the replication of fundamental cellular characteristics (segmentation, responsiveness, metabolic / signal transduction, etc.) to replace or supplement the functions of natural cells. The biomimetic protocell can carry functional modules and embed enzymes, nucleic acids, proteins, or nanomaterials to achieve biomimetic functions such as metabolic reactions, signal sensing, and targeted delivery. The size of the biomimetic protocell is close to that of natural cells (micrometer scale), enabling it to adapt to the in vivo environment or specific application scenarios.

[0018] Preferably, the phase-separated polypeptide forms the interface of the biomimetic protocell through self-assembly. Here, "interface" refers to the biomimetic interface formed by the self-assembly of the phase-separated polypeptide; preferably, the biomimetic interface is manifested as a morphologically stable spherical droplet, accompanied by significant interfacial recombination and mixing of internal substances, thus possessing fluid properties and a dynamic interface, and its internal components also exhibit good mobility and recombination energy.

[0019] The biomimetic protocell according to a specific embodiment of the present invention contains mitochondria. Preferably, the biomimetic cell contains polypeptides, proteins, or nucleic acids.

[0020] This invention also provides the application of the phase-separated polypeptide in the preparation of mitochondrial transplantation-related products, which includes... Mitochondria, and polypeptide aggregates The polypeptide condensate is assembled with the mitochondria to form a mitochondrial transplantation-related product; The polypeptide condensate is formed by the self-assembly of the phase-separated polypeptides described above.

[0021] Preferably, the mitochondria are isolated from cells or tissues.

[0022] Mitochondria can be isolated from the vast majority of mammalian cells, with cells rich in mitochondria being the preferred source. Mitochondria can also be isolated from cells obtained from fresh animal tissues, such as adipocytes, blood cells, bone cells, or liver cells. Mitochondria can also be isolated from cultured cells in the laboratory, such as NIH3T3 cells, HeLa cells, 293T cells, RAW 264.7 cells, and HepG2 cells.

[0023] In this invention, mitochondrial transplantation-related products refer to products obtained based on the aforementioned polypeptide condensates and mitochondria. Mitochondrial transplantation technology is used for research in treating diseases, improving cell function, or conducting related non-disease treatment and diagnostic studies.

[0024] The interior of peptide condensates can form a highly dynamic microenvironment that supports rapid material exchange and chemical reactions, and can effectively protect mitochondria from oxidative damage.

[0025] This invention also provides the application of phase-separated peptides in the preparation of drug delivery systems.

[0026] According to a specific embodiment of the present invention, a drug delivery system includes a polypeptide condensate formed by the self-assembly of the aforementioned phase-separated polypeptides.

[0027] Preferably, the drug delivery system comprises peptides, proteins, or nucleic acids.

[0028] This invention demonstrates through experiments that polypeptide condensate droplets possess excellent and broad molecular loading and recruitment capabilities. The recruitment effect is independent of the polarity, molecular weight, or chemical structure of the loaded molecules. For example, molecules such as Dil, Rhodamine B, MB, Hoechst, Nile Red, FITC, FITC-BSA, and FAM-siRNA can be successfully recruited and encapsulated in polypeptide condensate droplets.

[0029] Based on the aforementioned molecular loading and recruitment capabilities, the drug delivery system or biomimetic progenitor cell made from the polypeptide condensate of the present invention has the following advantages: for example, it can be used for labeling liposome-based drug carriers to track the distribution of the carrier within cells or its circulation process in vivo; or for fluorescent labeling of small molecule drugs to detect the cellular uptake efficiency of the drug; or for labeling enzyme drugs to track their sites of action; or for labeling mitochondrial-targeted drugs, or as a photosensitizer for photodynamic therapy in combination with chemotherapeutic drugs; or as a co-labeling tool for nuclear-targeted drugs (such as DNA alkylating agents and topoisomerase inhibitors) to observe the effects of drugs on the cell nucleus (such as DNA damage and nuclear morphology changes); or for labeling lipid-soluble drugs (such as paclitaxel and curcumin) to track the lipid-soluble distribution of the drug within cells; or to detect the effects of lipid metabolism-related drugs; or as a fluorescent labeling group coupled with various drugs (such as antibody drugs, polypeptide drugs, and small molecule targeted drugs) for targeted drug binding detection, intracellular tracking, or in vivo imaging.

[0030] Therefore, the polypeptide condensate of the present invention, based on its carrying of cell-targeting components (membrane, nucleus, lipid, mitochondria / nucleic acid (MB), cytoplasm / protein) or universally labeled components, can form a drug delivery system or biomimetic progenitor cell that can directly target specific cell structures, or can be used as a labeling tool to conjugate drugs, enabling visual tracking.

[0031] The beneficial effects of this invention are: This invention provides a self-assembling peptide WGDVYGGRD mt KF, where RD mt KF is a mitochondrial antioxidant targeting peptide. By introducing this fragment into the molecular structure, the sequence is endowed with inherent mitochondrial targeting and antioxidant capabilities. The self-assembling polypeptide of this invention has a simple synthesis method, good biocompatibility, and can self-assemble into condensate droplets. These condensate droplets possess strong loading capacity, can load mitochondria, have excellent antioxidant capacity themselves, and can also provide effective protection for co-loaded exogenous mitochondria, jointly constructing a fully functional biomimetic protocell system. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 Display WGDVYGGRD mt Mass spectrum of KF (molecular weight 1375.50).

[0034] Figure 2 It is WGDVYGGRD mt Microscopic image of KF in Tris-HCl buffer.

[0035] Figure 3 The fluorescence spectrum of the MPC recruiting molecules is shown.

[0036] Figure 4 The image shows (A) optical microscope images of MPC at 0, 1, and 2 s, (B) photobleaching experimental images, and (C) photobleaching data analysis.

[0037] Figure 5 The free radical scavenging ability of MPC is shown in (A) in vitro antioxidant experiment, (B) intracellular ROS scavenging flow cytometry and (C) intracellular ROS scavenging fluorescence image.

[0038] Figure 6 The fluorescence spectrum of MitoSOX Red after the addition of MPC is shown.

[0039] Figure 7 The construction of the biomimetic protocell is shown in the following: (A) Flow cytometry of Mito, MPC, and MPC+Mito; (B) Microscopic images of Mito, MPC, and MPC+Mito; (C) Membrane potential of the biomimetic protocell and individual mitochondria; (D) ATP content of the biomimetic protocell.

[0040] Figure 8 (A) Representative flow cytometry plot of cell uptake of biomimetic progenitor cells, (B) Quantitative analysis of cell uptake of Mito, and (C) Results of quantitative analysis of cell uptake of biomimetic progenitor cells. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0042] The reagents used in this invention are as follows: Dil: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindole carbocyanine perchlorate; Rhodamine B: Rhodamine B; MB (Methylene Blue): Methylene Blue; Hoechst: Hoechst dyes, including subtypes such as "Hoechst 33258" and "Hoechst 33342"; Nile Red; FITC (Fluorescein Isothiocyanate): Fluorescein isothiocyanate; FITC-BSA (FITC-labeled Bovine Serum Albumin): Fluorescein isothiocyanate-labeled bovine serum albumin; FAM-siRNA (FAM-labeled Small Interfering RNA, FAM being a fluorescein derivative): Carboxyfluorescein-labeled small interfering RNA.

[0043] Mitochondria: derived from NIH3T3 cells; obtained by differential centrifugation after cell disruption.

[0044] This invention combines "WGDVY" and "RD" mt Using the two KF sequences as "stickers," two glycine residues (GG) were selectively inserted between the two sequences as "spacers," resulting in the mitochondrial protective polypeptide sequence "WGDVYGGRD." mt KF (Mitochondrial protective peptide, MPP). The MPP peptide was dissolved in Tris-HCl buffer and the pH was adjusted to neutral. With non-covalent forces such as hydrogen bonding, electrostatic interaction, π-π interaction, cation-π interaction and van der Waals forces as the main driving forces for phase separation, the peptide successfully self-assembled to form submicron-sized mitochondrial protection condensate (MPC).

[0045] Example 1 The amino acids were synthesized using the classic Fmoc solid-phase synthesis method, with dichloropolymer resin as the solid support. After swelling with DCM, amino acids of a specific sequence were sequentially coupled, and then activated using the HBTU / DIEA system. The specific steps are as follows: (1) First, weigh about 0.5 g of dichloro resin into a solid synthesis tube using an electronic balance, then add 10 mL of dichloromethane to the tube to allow the resin to swell fully, and squeeze out the solvent after 5 minutes.

[0046] (2) Weigh 0.5 mmol of phenylalanine into a vial using an electronic balance, add 1 mmol of DIEA (200 μL) and 10 mL of dichloromethane to the vial to fully dissolve the amino acid, and then transfer it to a solid-phase synthesis tube to react for 2 hours.

[0047] (3) After the reaction is complete, squeeze out the reaction liquid and wash the resin with dichloromethane 5 times, each time for 1 minute (the same below). Then add 10 mL of the pre-prepared blocking solution (DCM:CH3OH:DIEA=17:2:1) to the synthesis tube to block the unreacted active chloride ions on the dichloromethane resin. The blocking time is 30 minutes.

[0048] (4) After the sealing is completed, the resin is washed with DCM 5 times, then washed with DMF 5 times. Then, 10 mL of 20% piperidine (piperidine:DMF=1:4) is used to remove the Fmoc protecting group on the amino acid and react for 30 minutes.

[0049] (5) Wash dichlororesin with DMF 5 times to remove excess piperidine. Weigh 1 mmol of lysine and 1 mmol of HBTU (379.25 mg) into a vial using an electronic balance. Add 2 mmol of DIEA (400 μL) and 10 mL of DMF to the vial and stir until the amino acid is completely dissolved. Then add it dropwise into a solid-phase synthesis tube and react for 2 hours.

[0050] (6) Wash the resin with DMF 5 times, add 10 mL of 20% piperidine to the synthesis tube, and react for 30 minutes to remove the Fmoc protecting group.

[0051] (7) Repeat the steps of DMF washing-amino acid addition reaction-removal of Fmoc protecting group-DMF washing in (5) and (6) above until the last tryptophan is used.

[0052] After all coupling was completed, the target peptide was cleaved from the resin using 95% TFA cleavage buffer. The crude product was obtained by rotary evaporation and precipitation with ice-cold ether. The crude product was then concentrated under vacuum and purified by high-performance liquid chromatography (HPLC) to finally obtain the mitochondrial protection peptide sequence “WGDVYGGRD”. mt KF".

[0053] The chemical structural formula of MPP is as follows: Mass spectrometry characterization of the peptide MPP is shown in [reference needed]. Figure 1 .

[0054] Example 2 The peptide MPP was dissolved in Tris-HCl buffer (pH=3) to obtain a 20 mmol / L peptide solution. 1 M NaOH solution was added dropwise to the peptide solution to gradually adjust the pH to neutral, and the solution was observed under a microscope.

[0055] The results show that... Figure 2After adjusting to a neutral pH, numerous, uniformly distributed spherical droplet structures appeared in the field of view. These droplets exhibited typical morphological characteristics of liquid-liquid phase separation condensates, contrasting sharply with the homogeneous solution background. This result directly confirms that peptide MPP can successfully self-assemble into submicron-sized peptide condensates MPC under neutral Tris-HCl buffer conditions.

[0056] Example 3 To verify that MPC possesses the biomolecule enrichment function necessary for serving as the basis of biomimetic protocells, and to provide a theoretical basis for its subsequent specific recruitment and loading of functional mitochondria, a series of characterization experiments on molecular recruitment capabilities were conducted.

[0057] To investigate the recruitment ability of this short peptide for fluorescent molecules, proteins, and nucleic acids, after the peptide was formed into an aggregate, various small molecule dyes, proteins, and siRNA working solutions, including Dil, Rhodamine B, MB, Hoechst, Nile Red, FITC, FITC-BSA, and FAM-siRNA, were added to the aggregate solution.

[0058] (1) At room temperature (25°C), the peptide was dissolved in 100 μL of 20 mM Tris-HCl buffer (containing 0.2 M NaCl, pH = 3) to prepare a 30 mM solution; 1 M NaOH solution was added dropwise to the peptide solution. As the pH value gradually increased, the solution became turbid, and the aggregate solution was obtained. At this time, the pH value was about 7.

[0059] (2) Prepare a 10 mM FITC DMSO stock solution, then dilute the FITC DMSO stock solution 50 times with PBS solution to prepare the FITC working solution. Finally, add 2 μL of working solution to 100 μL of aggregate solution to obtain an aggregate solution encapsulated with FITC.

[0060] (3) For various small molecule dyes, proteins, and siRNAs such as Dil, Rhodamine B, MB, Hoechst, Nile Red, FITC-BSA, and FAM-siRNA, refer to the above methods for loading. Pipette 20 μL of the above mixed solution onto a glass slide and observe and photograph it using a confocal microscope.

[0061] like Figure 3 As shown, all the tested substances were successfully recruited and encapsulated within the MPC condensate, forming a stable complex. This result demonstrates that MPC possesses excellent and broad molecular loading capabilities; its recruitment effect is independent of the polarity, molecular weight, or chemical structure of the loaded molecules, highlighting its enormous potential as a universal nanomedicine delivery platform.

[0062] The droplet fusion process was observed using optical microscopy to investigate the dynamic physical properties of MPC peptide condensates. The results are as follows: Figure 4 (A) shows that when different polypeptide condensate droplets come into contact with each other, they can rapidly fuse within 2 seconds, eventually forming a stable spherical droplet, accompanied by significant interfacial remodeling and internal mixing. This phenomenon directly reflects the fluid properties and dynamic interface of polypeptide condensates, indicating that their internal components possess good mobility and remodeling capabilities.

[0063] Based on this, fluorescence recovery after bleaching (FRAP) experiments were further used to analyze the physical state and mobility of the recruited molecules within the condensate. The specific steps were as follows: High-intensity laser pulses were applied to selected condensate regions using confocal microscopy to quench the fluorescence of labeled molecules within those regions; subsequently, low-intensity lasers were used to continuously monitor the recovery process of fluorescence intensity in the quenched regions over time.

[0064] The results are as follows Figure 4 (B, C) shows that after high-intensity laser bleaching, the fluorescence intensity in the preset region was rapidly quenched within 20 s. Subsequently, the fluorescence began to recover rapidly, with the fastest recovery period occurring between 20 and 60 s, during which the recovery kinetic curve was the steepest. After 60 s, the recovery rate gradually slowed down. By 80 s, the fluorescence intensity had essentially recovered to 95% of its pre-bleaching level. The rapid fluorescence recovery of the encapsulated fluorescent molecules after quenching indicates that they possess significant diffusion capacity and dynamic mobility within the condensate.

[0065] The above results collectively demonstrate that the interior of this polypeptide condensate is a highly dynamic microenvironment capable of supporting rapid material exchange and chemical reactions. Therefore, it can serve as a highly efficient microreactor, laying a solid foundation for its subsequent applications in organelle loading and biological function execution.

[0066] Example 4 The mitochondrial targeting ability and antioxidant efficacy of MPC were systematically evaluated through in vitro enzymological experiments, flow cytometry experiments and confocal microscopy to verify its biological activity.

[0067] DPPH was used for free radical scavenging experiments. Peptide samples (including monomer and aggregate forms) were mixed with DPPH ethanol solution, reacted in the dark, and the absorbance was measured at 517 nm.

[0068] First, prepare 0.1 mM DPPH anhydrous ethanol solution and 30 mM MPP and MPC solutions. Then, perform a quantitative free radical scavenging experiment using a 96-well plate, with three replicates per group. The specific sample loading protocol is as follows: Experimental group: Add 100 μL of MPP or MPC sample solution (30 mM) and 100 μL of DPPH ethanol solution to each well.

[0069] Control group: 100 μL of ultrapure water and 100 μL of DPPH ethanol solution were added to each well.

[0070] All wells were incubated at room temperature in the dark for 30 minutes. After the reaction, the absorbance (As) of the experimental group and the absorbance (Ac) of the control group at 517 nm were measured using a full-wavelength microplate reader. The DPPH radical scavenging rate was calculated using the following formula: Sweep rate (%) = (Ac-As) / Ac × 100%.

[0071] like Figure 5 As shown in (A), both forms of the peptide exhibit significant free radical scavenging ability, confirming the inherent antioxidant activity of the peptide MPP, which is unaffected by the formation of aggregates through self-assembly.

[0072] To assess the intracellular ROS scavenging capacity, cells were first treated with 6 Gy of γ-rays to induce an increase in intracellular ROS levels, followed by co-incubation with peptide aggregates.

[0073] (1) NIH3T3 cells were placed at a density of 2 × 10⁶ cells per dish. 5 Cells were seeded at a density of [insert density here] in six-well plates. After culturing for 24 h, the cells were co-incubated for 12 h with culture media containing PBS, 30 mM MPP, and 30 mM MPC solutions, respectively.

[0074] (2) Wash the cells three times with PBS to remove material that did not enter the cells. Then add 1 mL of DMEM medium containing 5 μL of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe (probe stock solution concentration is 1 mg / mL) to each dish and incubate the cells in a 37 ℃ incubator in the dark for 30 min.

[0075] (3) After washing away the dye, the cells were irradiated with 6 Gy of γ rays, then digested with trypsin and collected by centrifugation. The average fluorescence intensity of the cell population was detected by flow cytometry and photographed by confocal microscopy.

[0076] result( Figure 5As shown in B and C), after irradiation with 6 Gy γ-rays, cells in different treatment groups exhibited significant differences in fluorescence intensity. Cells in the PBS control group showed the strongest green fluorescence, indicating the highest level of reactive oxygen species (ROS) accumulation within the cells. The fluorescence intensity of cells in the MPP treatment group was slightly reduced compared to the control group, but still significant, suggesting that free MPP has some ROS scavenging ability, but the effect is limited. Cells in the MPC treatment group showed a weak fluorescence signal, with significantly lower intensity than the control and MPP groups. The images clearly showed that these cells also had more intact and healthy morphology. These results intuitively demonstrate that MPC peptide condensates can most effectively scavenge excess ROS induced by radiation, thereby greatly alleviating the oxidative stress state of cells.

[0077] Example 5 All groups of cells were loaded with MitoSOX Red, and the specific steps are as follows: (1) NIH3T3 cells were placed at a density of 2 × 10⁶ cells per dish. 5 Cells were seeded at a density of [insert density here] in six-well plates. After culturing for 24 h, the cells were co-incubated for 12 h with culture media containing PBS, 30 mM MPP, and 30 mM MPC solutions, respectively.

[0078] (2) Discard the culture medium, wash twice with PBS, add mitochondrial probe working solution (400 nM) preheated at 37 ℃ and incubate in the dark for 45 min; (3) After washing the cells with PBS three times, they were observed and photographed using a confocal microscope.

[0079] Figure 6 The confocal microscopy results for MPC-containing MitoSOX Red show that the probe itself exhibits weak fluorescence. However, upon entering the mitochondria of living cells, it is specifically oxidized by superoxide, producing strong red fluorescence. The intensity of this red fluorescence is directly proportional to the level of superoxide within the mitochondria. This result directly demonstrates that MPC not only reaches the mitochondria but also exerts its in-situ antioxidant function at the "source" of ROS production, effectively protecting mitochondria from oxidative damage. Thus, the peptide condensate MPC possesses mitochondrial protective function.

[0080] Example 6 Intact, functional mitochondria were isolated from cells using differential centrifugation. The mitochondria were then fluorescently labeled using a MitoTracker Red probe at 37°C in the dark for 30 minutes to facilitate subsequent microscopic observation.

[0081] Add 100 μg of purified mitochondria to 100 μl of MPC peptide condensate buffer (mitochondrial preservation solution containing ADP and substrate), and gently pipette several times to mix, avoiding vigorous shaking to protect mitochondrial integrity. Incubate for 30 minutes to form the MPC+Mito complex.

[0082] To confirm the successful construction of the complex at the population level, free mitochondria (Mito group), empty MPC condensates (MPC group), and their complex (MPC+Mito group) were loaded onto a flow cytometer. Scatter plots were generated using forward scattering (FSC) and side scattering (SSC).

[0083] The results are as follows Figure 7 As shown in (A), the MPC+Mito group forms a new population that is different from free mitochondria in terms of scattering light characteristics, but is highly similar to the MPC population, proving that mitochondria were successfully encapsulated and formed a stable composite structure.

[0084] Figure 7 (B) shows the results of confocal microscopy. Mitotracker Red-labeled mitochondria are effectively encapsulated within the MPC, forming a colocalization signal. This clearly demonstrates that the "organelle" is successfully encapsulated in the "bionic cytoplasm," marking the successful construction of the functionalized bionic protocell.

[0085] Two key functional indicators, mitochondrial membrane potential and ATP synthesis capacity, were measured to assess whether this biomimetic protocell could maintain the functional integrity of its core organelles. Free mitochondria and mitochondria encapsulated in MPC were incubated with the JC-1 probe at 37°C in the dark for 15-20 minutes. The ratio of red to green fluorescence intensity was detected using a fluorescence microplate reader.

[0086] The results are as follows Figure 7 (C) shows that the relative membrane potential of the mitochondrial protectant + Mito group was 1, while the relative membrane potential of the MPC + Mito group was significantly increased to 1.78. Compared with free mitochondria, the mitochondria encapsulated inside the biomimetic protocell showed a higher proportion of red fluorescent aggregates, indicating that their membrane potential was better maintained. This suggests that the polypeptide condensate matrix provides a superior microenvironment for mitochondria, maintaining the structural and functional integrity of mitochondria.

[0087] To quantitatively assess the association between the biomimetic protocell's energy synthesis function and its core component, mitochondria, we incubated samples containing equal amounts of mitochondrial proteins in the mitochondrial protectant-Mito group and the MPC-Mito group at 37°C for 30 min.

[0088] Immediately after incubation, quantification was performed using an ATP assay kit.

[0089] The results are as follows Figure 7 (D) shows that the ATP concentration in the mitochondrial protectant-Mito group was 3.48 μM, while the ATP concentration in the MPC+Mito group was significantly higher, reaching 4.12 μM. Compared with the commercially available mitochondrial protectant-Mito group, the mitochondria encapsulated in polypeptide condensates (MPC-Mito group) produced significantly higher ATP production. The MPC condensate matrix not only does not impair mitochondrial function but also provides a superior microenvironment, effectively maintaining and even enhancing its energy synthesis activity, thus demonstrating that this biomimetic protocell possesses the functional basis as an energy factory.

[0090] Example 7 The cell uptake efficiency of the biomimetic progenitor cells was evaluated using flow cytometry to verify whether the cells could be effectively taken up by tumor cells, thus providing experimental evidence for their application in tumor treatment.

[0091] First, mitochondria used for encapsulation were pre-labeled with MitoTracker Red fluorescent dye, followed by the construction of biomimetic progenitor cells. After co-incubation with tumor cells for a specific time, untaken particles were removed by thorough washing and then analyzed by flow cytometry.

[0092] (1) 4T1 cells were placed at 2 × 10⁻⁶ cells per dish 5 Cells were seeded at a density of 100% in six-well plates. After culturing for 24 h, the cells were incubated for 0.5, 1, and 2 h in a culture medium containing Mito and 30 mM MPC+Mito complex solution, respectively.

[0093] (2) At the corresponding time points, trypsin digestion was performed, cells were collected by centrifugation, and the cell pellet was washed twice with PBS.

[0094] (3) The cell pellet was resuspended in PBS and the fluorescence intensity in different cells at different times was detected by flow cytometry.

[0095] The results are as follows Figure 8As shown, the mean fluorescence intensity of cells after taking up free mitochondria (Mito) and biomimetic proto-cells (MPC+Mito) was quantitatively compared at different time points of 0.5h, 1h, and 2h. The fluorescence intensity of the free Mito group was consistently not significantly different from the blank control group, and its mean fluorescence intensity remained at extremely low levels at all time points. This result indicates that free mitochondria are almost impossible for 4T1 cells to effectively internalize. In stark contrast, the MPC+Mito experimental group showed a significant and time-dependent enhancement in uptake. A noticeable fluorescence signal was detected after 0.5 hours of co-incubation, indicating that cells had begun rapid uptake. At the 1-hour time point, the uptake efficiency reached its peak, at which point approximately 38% of the cell population showed a significant positive fluorescence signal.

[0096] The above results clearly demonstrate that the MPC biomimetic progenitor cells constructed in this invention can be efficiently internalized by tumor cells, with an optimal uptake window of approximately one hour, a capability not found in mitochondria alone. This lays the foundation for subsequent direct intervention in tumor cell metabolism using this system.

[0097] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A phase separation polypeptide, characterized in that, The polypeptide has the following structure: 。 2. Use of the phase separating polypeptide of claim 1 in the preparation of a biomimetic protocell.

3. A biomimetic protocell, characterized by, The biomimetic protocell comprises the phase separating polypeptide of claim 1, which forms the interface of the biomimetic protocell through self-assembly.

4. The biomimetic protocell according to claim 3, wherein The biomimetic cell contains mitochondria.

5. The biomimetic protocell according to claim 3 or 4, wherein The biomimetic cell contains a polypeptide, a protein, or a nucleic acid.

6. Use of the phase separation polypeptide of claim 1 for the preparation of a mitochondria transplantation related product, characterized in that, comprises mitochondria, and a polypeptide condensate, which assembles with the mitochondria into a mitochondria transplantation related product; The polypeptide condensate is self-assembled from the phase separating polypeptide of claim 1.

7. The use according to claim 1, characterized in that, The mitochondria are isolated from a cell or a tissue.

8. Use of the phase separating polypeptide of claim 1 in the preparation of a drug delivery system.

9. A drug delivery system, characterized by The drug delivery system comprises a polypeptide condensate self-assembled from the phase separating polypeptide of claim 1, or the biomimetic protocell of claim 3, or the mitochondria transplantation related product of claim 6.

10. The drug delivery system of claim 9, wherein, The drug delivery system comprises a polypeptide, a protein, or a nucleic acid.

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