A polypeptide coacervate, methods of making and using the same

Abstract

The present application relates to a kind of polypeptide condensate and its preparation method and application, the polypeptide condensate is, by fusion polypeptide TAT-n × NoLS in solution containing Tris-HCl, NaCl and polyethylene glycol 8000, incubation is carried out at room temperature, self-assembly is formed in vitro phase separation condensate structure, the polypeptide condensate can be loaded nucleic acid or protein and be delivered into cell, wherein, the fusion polypeptide TAT-n × NoLS is formed n × NoLS module by NoLS polypeptide sequence with phase separation characteristics n=2-5 by n times series connection;And at least one TAT transmembrane peptide sequence is fused in N end;The sequence of the NoLS polypeptide is as shown in SEQ ID NO.1, and the sequence of the TAT transmembrane peptide is as shown in SEQ ID NO.2.The TAT-3xNoLS polypeptide condensate provided by the present application has the loading, intracellular delivery and functional output capacity of nucleic acid and protein biological macromolecule, and shows good application potential in biological macromolecule delivery, also provides experimental basis for subsequent carrying out multifunctional expansion and mechanism optimization research.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a polypeptide condensate, its preparation method, and its application. Background Technology

[0002] Lipid-based, polymer-based, and inorganic-based delivery systems have all played important roles in drug delivery and have made significant progress in the delivery of some drugs, especially small molecule drugs and some nucleic acid drugs. Lipid-based systems have good biocompatibility and a good foundation for clinical translation, polymer-based systems have strong structural designability, and inorganic-based systems have unique advantages in stimulus response and therapeutic integration. However, for biomacromolecules such as proteins, RNA, and DNA, traditional delivery systems still generally face problems such as limited loading efficiency, insufficient structural stability, low intracellular transport efficiency, weak subcellular targeting ability, and difficulty in precisely controlling functional release. In addition, different material systems are also constrained by factors such as biosafety, metabolic clearance, and batch-to-batch consistency. These limitations indicate that existing traditional delivery systems cannot fully meet the comprehensive requirements of biomacromolecule delivery for efficient loading, stable protection, precise delivery, and functional release. Therefore, it is necessary to further develop new delivery strategies and material systems.

[0003] Liquid-liquid phase separation (LLPS) refers to the process by which biomacromolecules such as proteins, peptides, and nucleic acids, under specific conditions, separate from a homogeneous solution through multivalent weak intermolecular interactions (such as electrostatic interactions, hydrogen bonds, hydrophobic interactions, π-π stacking, and cation-π interactions), forming a dense phase rich in macromolecules that coexists with a diluted phase. The aggregates formed in this process have liquid-like characteristics such as spherical morphology, droplet fusion, reversible dissociation, and dynamic exchange of internal components with the external environment. LLPS is widely regarded as an important physical basis for the formation of membraneless organelles in cells, participating in the assembly of functional compartments such as nucleoli, stress granules, and P granules, and playing an important role in key life activities such as signal transduction, autophagy, chromatin organization, and DNA damage repair.

[0004] Recent studies have shown that abnormal regulation of LLPS is closely related to a variety of diseases. For example, liquid condensates of proteins such as Tau, TDP-43, and FUS may transform into irreversible aggregates under pathological conditions, participating in the development of neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. Abnormal LLPS can also participate in the malignant transformation of tumors by affecting transcriptional regulation and signaling pathways. Therefore, LLPS is not only an important theoretical model for understanding the spatial organization and functional regulation of intracellular structures, but also provides a new perspective for disease mechanism research and the development of novel treatment strategies.

[0005] In the fields of materials science and drug delivery, LLPS-based biomolecule condensates show promising application prospects. Compared with traditional delivery systems such as liposomes, polymer nanoparticles, and inorganic nanocarriers, LLPS condensates have the following advantages: First, they do not require complex exogenous framework materials and can self-assemble under mild aqueous conditions, which is beneficial for maintaining the natural conformation and bioactivity of sensitive payloads such as proteins and nucleic acids. Second, peptide systems composed of natural amino acids have good biocompatibility and degradability, which can reduce immunogenicity and the risk of long-term accumulation. Third, LLPS condensates can efficiently encapsulate a variety of biomolecules, including short peptides, proteins, mRNA, and oncolytic viruses, and can achieve stimulus-response release by responding to environmental signals such as pH, redox state, and ionic strength. Fourth, some LLPS systems can achieve subcellular level targeted delivery by introducing functional sequences such as nuclear localization signals, and to a certain extent overcome the "endosome trap" problem, achieving direct cytoplasmic delivery.

[0006] In summary, polypeptide or protein-based LLPS condensates have significant potential for the delivery of biopharmaceutical drugs. However, related research is still in its early stages. Therefore, this invention provides a polypeptide condensate, its preparation method, and its applications. Summary of the Invention

[0007] The purpose of this invention is to provide a polypeptide condensate, its preparation method, and its application in order to solve the above-mentioned problems.

[0008] The present invention achieves the above objectives through the following technical solutions: This invention provides a polypeptide condensate, which is an in vitro phase-separated condensate structure formed by the self-assembly of a fusion polypeptide TAT-n×NoLS in a solution containing Tris-HCl, NaCl and polyethylene glycol 8000 at room temperature. The polypeptide condensate can encapsulate nucleic acids or proteins and deliver them into cells. The fusion polypeptide TAT-n×NoLS is formed by tandemly connecting a NoLS polypeptide sequence with phase separation characteristics n times to form an n×NoLS module, where n=2-5; and at least one TAT membrane-penetrating peptide sequence is fused to the N-terminus; the sequence of the NoLS polypeptide is shown in SEQ ID NO.1, and the sequence of the TAT membrane-penetrating peptide is shown in SEQ ID NO.2.

[0009] As a further optimization of the present invention, the fusion polypeptide TAT-n×NoLS is a fusion polypeptide TAT-3×NoLS, and the sequence of the fusion polypeptide TAT-3×NoLS is shown in SEQ ID NO.3.

[0010] As a further optimization of the present invention, the polypeptide condensate is an in vitro phase separation condensate structure formed by self-assembly of the fusion polypeptide TAT-n×NoLS with a final concentration of 2-10 μM in a solution containing Tris-HCl with a final concentration of 10-30 mM, NaCl with a final concentration of 120-180 mM, and polyethylene glycol 8000 with a final concentration of 10%-20% at room temperature for 5-15 min.

[0011] The present invention also provides a polypeptide condensate delivery system, the polypeptide condensate delivery system including the polypeptide condensate, and further including nucleic acids or proteins encapsulated by the polypeptide condensate.

[0012] This invention also provides an application of polypeptide condensates in intracellular delivery of biological macromolecules. The application involves adding nucleic acids or proteins to a solution containing the fusion polypeptide TAT-n×NoLS, Tris-HCl, NaCl, and polyethylene glycol 8000, and incubating them together at room temperature for 5-15 minutes to obtain polypeptide condensates encapsulating nucleic acids or proteins, i.e., the polypeptide condensate delivery system. The polypeptide condensate delivery system is then used for intracellular delivery of nucleic acids or proteins. The fusion polypeptide TAT-n×NoLS is formed by tandemly connecting a NoLS polypeptide sequence with phase separation characteristics n times to form an n×NoLS module, where n=2-5; and at least one TAT membrane-penetrating peptide sequence is fused to the N-terminus; the sequence of the NoLS polypeptide is shown in SEQ ID NO.1, and the sequence of the TAT membrane-penetrating peptide is shown in SEQ ID NO.2; The fusion polypeptide TAT-n×NoLS is a fusion polypeptide TAT-3×NoLS, and the sequence of the fusion polypeptide TAT-3×NoLS is shown in SEQ ID NO.3.

[0013] As a further optimization of the present invention, in the polypeptide condensate delivery system, the final concentration of the nucleic acid is 0.5-10 μM and the final concentration of the protein is 0.1-2 μM.

[0014] As a further optimization of the present invention, the nucleic acid includes DNA, RNA, siRNA, plasmid DNA or mRNA; the protein includes mCherry protein.

[0015] As a further optimization of the present invention, the intracellular delivery includes delivering nucleic acids and proteins into the cytoplasm and / or the cell nucleus.

[0016] As a further optimization of the present invention, the cells include HEK293T cells, PC9 cells, or A549 cells.

[0017] The beneficial effects of this invention are as follows: 1) The TAT-3×NoLS polypeptide condensate provided by the invention utilizes the synergistic effect of TAT membrane-penetrating peptide and NoLS nuclear localization signal to have both cell membrane penetration ability and cell nuclear targeting ability, which can efficiently deliver biomacromolecules such as nucleic acids and proteins to the cytoplasm and / or cell nucleus, providing an experimental basis for subsequent research on multifunctional expansion and mechanism optimization. 2) The TAT-3×NoLS polypeptide condensate provided by this invention is formed by the self-assembly of polypeptides composed of natural amino acids under mild aqueous conditions. It does not require complex exogenous backbone materials, and the preparation process is simple. It can be obtained by incubation at room temperature for 5-15 minutes. It has shown good intracellular delivery ability in various cell lines such as HEK293T, PC9, and A549, and has good application potential in the field of biomolecule delivery. Attached Figure Description

[0018] Figure 1 It is the MTT cytotoxicity of TAT-3×NoLS polypeptide aggregates ( Figure 1 A) Results of cell death experiment ( Figure 1 (B in the diagram) and the delivery of TAT-3×NoLS peptide condensates in HEK293T cells ( Figure 1 (C in the middle).

[0019] Figure 2 This is a diagram showing the encapsulation results of nucleic acids (RNA, DNA) and mCherry proteins by TAT-3×NoLS polypeptide condensates.

[0020] Figure 3 This is a diagram of TAT-3×NoLS polypeptide condensate-mediated delivery of nucleic acid (DNA) and mCherry protein in HEK293T cells.

[0021] Figure 4 This is a diagram showing the delivery of mCherry protein mediated by TAT-3×NoLS polypeptide condensates in PC9 and A549 cells.

[0022] Figure 5 TAT-3×NoLS peptide condensate-siRNA based on Western blot analysis Figure 5 A) and plasmid DNA ( Figure 5 The evaluation diagram for delivery efficiency is shown in section B). Detailed Implementation

[0023] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0024] I. Materials 1. Tris-HCl (pH=8.0), polyethylene glycol 8000 (PEG8000; Sigma-Aldrich), mCherry protein; Unless otherwise specified, the methods used in this embodiment are conventional methods known to those skilled in the art, and the reagents and materials used are commercially available products.

[0025] II. Methods 2.1 Formation of in vitro phase separation system 5 μM TAT-3×NoLS peptide was added to a solution of 20 mM Tris-HCl (pH=8.0), 150 mM NaCl and 15% (w / w) PEG8000, and after gentle mixing, it was incubated at room temperature for 10 min, and TAT-3×NoLS peptide aggregates were formed by self-assembly.

[0026] The TAT-3×NoLS polypeptide is formed by tandemly connecting three natural disordered sequences with phase separation potential (i.e., NoLS polypeptide sequences, such as SEQ ID NO.1: QERRSKKRELHCK) to form a 3×NoLS module, and then fusing a TAT membrane-penetrating peptide sequence (such as SEQ ID NO.2: YGRKKRRQRRR) at the N-terminus. The final TAT-3×NoLS polypeptide sequence is as follows: SEQ ID NO. 3: YGRKKRRQRRRQERRSKKRELHCKQERRSKKRELHCKQERRSKKRELHCK.

[0027] HEK293T cells were trypsinized and seeded in 96-well cell culture dishes. Medium containing 5 μM TAT-3×NoLS (DMEM medium with 5% fetal bovine serum) was added, and the cells were incubated at 37°C in a 5% CO2 incubator for 24 or 48 hours. OD was then measured using an MTT assay kit (Beyotime Biotechnology). 490 The result is as follows Figure 1As shown in A, there were no significant differences between the experimental group and the control group after 24h or 48h. HEK293T cells were digested with trypsin and seeded in 60mm cell culture dishes. Medium containing 5μM TAT-3×NoLS (DMEM medium with 5% fetal bovine serum) was added, and the cells were incubated at 37℃ in a 5% CO2 incubator for 24h or 48h. The cell death rate was then detected using a cell death detection kit (Beyotime Biotechnology). The results are shown in Figure A. Figure 1 As shown in B, there was no significant difference between the experimental group and the control group after 24h or 48h, indicating that TAT-3×NoLS is non-toxic to cells at the working concentration of 5μM. The MTT cytotoxicity and cell death assay showed that TAT-3×NoLS peptide condensates have low cytotoxicity within the 5μM usage concentration range, which is a safe usage concentration.

[0028] FAM labeling results of TAT-3×NoLS peptide condensates, such as Figure 1 As shown in the upper figure of C, FAM-labeled TAT-3×NoLS polypeptide aggregates exhibit green fluorescence. HEK293T cells were trypsinized and seeded in 30mm cell culture dishes. When the cell confluence reached 70%, polypeptide aggregates formed by 5μM TAT-3×NoLS were added to the HEK293T cells. The confocal laser scanning microscopy image of the TAT-3×NoLS polypeptide aggregates after 2 hours is shown below. Figure 1 As shown in the figure below C, FAM-labeled TAT-3×NoLS is green fluorescent; blue fluorescent represents DAPI-labeled cell nuclei; Merge shows the relative spatial distribution and overlap of TAT-3×NoLS polypeptide condensates within the cell, with the Merge channel in the right column; the scale bar is 10 μm. The results show that TAT-3×NoLS polypeptide condensates can enter HEK293T cells and are located in the cell nucleus.

[0029] 2.2. In vitro encapsulation of nucleic acids and proteins to form polypeptide aggregates loaded with nucleic acids or proteins Nucleic acid and mCherry encapsulation experiments were performed under the basic conditions of 20 mM Tris-HCl (pH=8.0), 150 mM NaCl and 15% (mass concentration) PEG8000. First, according to the set system, 5 μM of TAT-3×NoLS peptide was added to a solution containing 20 mM Tris-HCl (pH=8.0), 150 mM NaCl and 15% (mass concentration) PEG8000. Then, nucleic acid molecules (5 μM) or mCherry protein (1 μM) were added respectively. After the sample was gently mixed, it was incubated at room temperature for 10 min to promote the entry of the loaded molecules into the condensate system.

[0030] In the nucleic acid encapsulation experiment, RNA or DNA was added to the peptide system to form a composite sample. The distribution and enrichment of nucleic acid fluorescence signals in the peptide condensates were then observed using a laser confocal microscope to evaluate the encapsulation ability of TAT-3×NoLS peptide condensates for nucleic acids. In the mCherry encapsulation experiment, purified mCherry protein was co-incubated with the peptide system. The encapsulation effect of mCherry protein in TAT-3×NoLS peptide condensates was evaluated by observing the localization of mCherry protein fluorescence in the peptide condensates.

[0031] Based on the confocal imaging results, systems with better encapsulation effects were selected and used for subsequent cell uptake and delivery experiments. Finally, ImageJ software was used to perform quantitative analysis on the captured confocal images.

[0032] The results are as follows Figure 2 As shown, Figure 2 Confocal laser scanning microscopy images of peptide condensates formed by the fusion peptide TAT-3×NoLS with nucleic acids RNA / DNA and mCherry protein. The right column shows the fluorescence colocalization analysis of the horizontally lined area within the white dashed box of the Merge channel. FAM-labeled TAT-3×NoLS is green fluorescent, ALEXA-649-labeled RNA or DNA is red fluorescent, and mCherry protein is also red fluorescent. The scale bar is 10 μm. The results showed that TAT-3×NoLS peptide condensates can effectively encapsulate nucleic acids and mCherry proteins, forming corresponding in vitro phase separation systems.

[0033] 2.3 Nuclear delivery manifestations in HEK293T, PC9, and A549 cell lines mediated by the peptide condensate TAT-3×NoLS 1) After digesting HEK293T, PC9 or A549 cells purchased from ATCC with trypsin, seed them into cell culture dishes, add culture medium (DMEM medium containing 5% fetal bovine serum), and incubate in a constant temperature incubator at 37℃ and 5% CO2 for 12-24h. When the cell adhesion rate reaches 70%-80% and the growth status is good, they can be used for subsequent experiments. 2) Prepare nucleic acid-loaded polypeptide condensates (5 μM polypeptide, 5 μM nucleic acid) or mCherry protein-loaded polypeptide condensates (5 μM polypeptide, 1 μM mCherry protein) in advance according to the method in step 2.2 above. After incubating at room temperature for 10 min, add them to cell culture dishes, gently shake to distribute the sample evenly, and continue to incubate at 37℃, 5% CO2 for the set time (3-4 h for nucleic acid group, 2-3 h for mCherry protein group). After incubation, aspirate the culture medium and gently wash the cells 3 times with pre-warmed PBS to remove free sample that has not been taken up by the cells. 3) Subsequently, 4% paraformaldehyde was added to the culture dish and the cells were fixed at room temperature. After fixation, the cells were washed three times with PBS to remove residual fixative. Hoechst staining solution was added as needed for the experiment to stain the cell nuclei. After incubation in the dark, the cells were washed again. Finally, the distribution of intracellular fluorescence signals was observed using a laser confocal microscope to evaluate the cell delivery effect of peptide condensates loaded with nucleic acids or mCherry proteins. The images were processed and analyzed using ImageJ software.

[0034] The results are as follows Figure 3 , Figure 4 As shown, Figure 3 Representative confocal laser scanning microscopy images of HEK293T cells after incubation for different times with FAM-labeled TAT-3×NoL peptide (green) and ALEXA-649-labeled nucleic acid (red). Blue fluorescence represents DAPI-labeled cell nuclei. Merge shows the relative spatial distribution and overlap of peptide and nucleic acid within the cell. Scale bar is 10 μm. Figure 4 The phase-separated condensates formed by FAM-labeled TAT-3×NoL peptide (green) and mCherry protein were incubated with PC9 and A549 cells and then entered into representative intracellular laser confocal scanning microscopy images. The blue fluorescence represents the cell nucleus labeled with DAPI. Merge shows the relative spatial distribution and overlap of peptides and nucleic acids in the cell. The scale bar is 10 μm. The results showed that TAT-3×NoLS polypeptide condensates could mediate the entry of nucleic acids and mCherry proteins into cells, and a certain degree of fluorescence enrichment was observed in the nuclear region of some cells.

[0035] 2.4 Case Study on the Delivery Efficiency of TAT-3×NoLS Peptide Aggregates for siRNA and Plasmid DNA To verify the delivery efficiency of TAT-3×NoLS peptide condensates for siRNA and plasmid DNA, total protein was collected after cells were treated with different delivery systems and analyzed by Western blotting, as follows: For the siRNA delivery experiment, HEK293T cells were digested with trypsin and then seeded in 30 mm diameter culture dishes. 1 mL of culture medium (DMEM medium containing 5% fetal bovine serum) was added, and the cells were incubated in a 37°C, 5% CO2 incubator for 12-24 h. When the cell confluence reached 70%, commercially synthesized KPNA1 (siKPNA1) and control siRNA (siControl) were delivered using the TAT-3×NoLS phase separation system and PEI transfection reagent, respectively. A system containing 100 μL of TAT-3×NoLS-encapsulated siRNA was prepared using TAT-3×NoLS. The final concentration of 5 μM TAT-3×NoLS peptide was added to a solution containing 20 mM Tris-HCl (pH=8.0), 150 mM NaCl and 15% (w / w) PEG8000, and then siRNA (500 nM) was added. The sample was gently mixed and incubated at room temperature for 10 min. The PEI (Yisheng Bio) transfection system was prepared as follows: siRNA (final concentration 500 nM) and 4 uL of PEI (1 mg / mL) were added to 100 uL of DMEM medium (commercially purchased). The mixture was incubated at room temperature for 15 min. The prepared system was then added dropwise to cell culture dishes, and the cells were cultured for another 24 h. The treated cell samples were lysed on ice for 30 min with RIPA lysis buffer (Beyotime), centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was added to SDS-Loading buffer and boiled for 10 min. The expression level of KPNA1 protein was detected by Western blot to evaluate the delivery of siRNA and the gene silencing effect.

[0036] For plasmid DNA delivery experiments, HEK293T cells were digested with trypsin and then plated in 30 mm diameter culture dishes. 1 mL of culture medium (DMEM medium containing 5% fetal bovine serum) was added, and the cells were incubated at 37°C and 5% CO2 for 12-24 h. When the cell confluence reached 70%, the eukaryotic expression plasmid pcDNA3.1-6×NoLS-EGFP was delivered using the TAT-3×NoLS phase separation system and PEI transfection reagent, respectively.

[0037] A system containing 100 μL of TAT-3×NoLS plasmid pcDNA3.1-6×NoLS-EGFP was prepared using TAT-3×NoLS. A final concentration of 5 μM TAT-3×NoLS peptide was added to a solution containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 15% (w / w) PEG8000. Then, 2 µg of plasmid pcDNA3.1-6×NoLS-EGFP was added. The sample was gently mixed and incubated at room temperature for 10 min. The PEI (Yisheng Biotechnology) transfection system was prepared as follows: Plasmid pcDNA3.1-6×NoLS-EGFP (2µg) and PEI (1mg / mL) were added to 100uL of DMEM medium (commercially purchased). The mixture was incubated at room temperature for 15min. The prepared system was then added dropwise to cell culture dishes, and the cells were cultured for another 24h. The treated cell samples were lysed on ice for 30min with RIPA lysis buffer (Beyotime), centrifuged at 12000rpm for 10min at 4℃, and the supernatant was added to SDS-Loading buffer and boiled for 10min. The expression level of 6×NoLS-EGFP protein was detected by Western blot to evaluate the plasmid DNA delivery and gene expression effect. The Western blot detection method is as follows: After determining the protein concentration of the collected samples using the BCA method, an equal amount of protein sample was added to the loading buffer, denatured, and then separated by SDS-PAGE electrophoresis. The separated protein was then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk powder or BSA at room temperature for 1 hour, and then anti-KPNA1, anti-GFP, and anti-GAPDH primary antibodies were added respectively. The membrane was incubated overnight at 4°C. The next day, after washing with TBST, the membrane was incubated with the corresponding HRP-labeled secondary antibody at room temperature for 1 hour. After washing, the membrane was developed with ECL chemiluminescence reagent, and images were acquired using a gel imaging system. The gray values ​​of the bands were analyzed using ImageJ software. GAPDH was used as an internal control. The delivery efficiency of the TAT-3×NoLS phase separation system for siRNA and plasmid DNA was evaluated by comparing the relative expression levels of KPNA1 or 6×NoLS-EGFP protein in different treatment groups.

[0038] The results are as follows Figure 5 As shown, Figure 5 In the table, 'A' represents the Western blot analysis of KPNA1 protein expression levels after treatment with siKPNA1 under different delivery conditions. The treatment groups were PEI+siControl, PEI+siKPNA1, and TAT-3×NoLS+siKPNA1, respectively. The top image shows representative immunoblot bands, and the bottom image shows the relative protein expression quantification results after the band gray values ​​were normalized by GAPDH. Figure 5In the figure, B represents the Western blot analysis of GFP protein expression levels after plasmid DNA (Plasmid) treatment under different delivery conditions. The model payload used was the plasmid pcDNA3.1-6×NoLS-EGFP (Plasmid) encoding 6×NoLS-EGFP. The treatment groups were free plasmid, PEI+ plasmid, and TAT-3×NoLS+ plasmid, respectively. The top shows the representative immunoblot bands (IB: GFP indicates that immunoblotting was performed using anti-GFP antibody). The bottom shows the relative protein expression quantification results after the gray values ​​of the bands were normalized by GAPDH. GAPDH is the internal reference protein.

[0039] The results show that, by Figure 5 Results A show that the TAT-3×NoLS phase separation system significantly reduced intracellular KPNA1 protein levels in siRNA delivery experiments, with even lower levels of KPNA1 protein compared to the PEI group, indicating better siRNA delivery efficiency compared to the PEI group. Figure 5 Results B in the study show that the protein 6×NoLS-EGFP encoded by the plasmid delivered by the TAT-3×NoLS phase separation system can be expressed normally in cells, and the expression efficiency is higher than that of the PEI group. Therefore, these two experiments demonstrate the good intracellular delivery efficiency of the two TAT-3×NoLS phase separation systems.

[0040] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A polypeptide condensate, characterized in that, The polypeptide condensate is an in vitro phase-separated condensate structure formed by the self-assembly of the fusion polypeptide TAT-n×NoLS in a solution containing Tris-HCl, NaCl and polyethylene glycol 8000 at room temperature. The polypeptide condensate can encapsulate nucleic acids or proteins and deliver them into cells. The fusion polypeptide TAT-n×NoLS is formed by tandemly connecting a NoLS polypeptide sequence with phase separation characteristics n times to form an n×NoLS module, where n=2-5; and at least one TAT membrane-penetrating peptide sequence is fused to the N-terminus; the sequence of the NoLS polypeptide is shown in SEQ ID NO.1, and the sequence of the TAT membrane-penetrating peptide is shown in SEQ ID NO.

2.

2. The polypeptide condensate according to claim 1, characterized in that: The fusion polypeptide TAT-n×NoLS is a fusion polypeptide TAT-3×NoLS, and the sequence of the fusion polypeptide TAT-3×NoLS is shown in SEQ ID NO.

3.

3. A polypeptide condensate according to claim 2, characterized in that: The polypeptide condensate is an in vitro phase-separated condensate structure formed by the self-assembly of the fusion polypeptide TAT-n×NoLS at a final concentration of 2-10 μM in a solution containing Tris-HCl at a final concentration of 10-30 mM, NaCl at a final concentration of 120-180 mM, and polyethylene glycol 8000 at a final concentration of 10%-20% at room temperature for 5-15 min.

4. A polypeptide condensate delivery system, characterized in that: The polypeptide condensate delivery system includes a polypeptide condensate as described in any one of claims 1-3, and further includes nucleic acids or proteins encapsulated by the polypeptide condensate.

5. The application of a polypeptide condensate as described in any one of claims 1-3 in intracellular delivery of biomolecules, characterized in that: The application involves adding nucleic acids or proteins to a solution containing the fusion polypeptide TAT-n×NoLS, Tris-HCl, NaCl, and polyethylene glycol 8000, and incubating them together at room temperature for 5-15 minutes to obtain polypeptide condensates carrying nucleic acids or proteins, i.e., the polypeptide condensate delivery system as described in claim 4, and using the polypeptide condensate delivery system for intracellular delivery of nucleic acids or proteins. The fusion polypeptide TAT-n×NoLS is formed by tandemly connecting a NoLS polypeptide sequence with phase separation characteristics n times to form an n×NoLS module, where n=2-5; and at least one TAT membrane-penetrating peptide sequence is fused to the N-terminus; the sequence of the NoLS polypeptide is shown in SEQ ID NO.1, and the sequence of the TAT membrane-penetrating peptide is shown in SEQ ID NO.2; The fusion polypeptide TAT-n×NoLS is a fusion polypeptide TAT-3×NoLS, and the sequence of the fusion polypeptide TAT-3×NoLS is shown in SEQ ID NO.

3.

6. The application according to claim 5, characterized in that: In the polypeptide condensate delivery system, the final concentration of the nucleic acid is 0.5-10 μM, and the final concentration of the protein is 0.1-2 μM.

7. The application according to claim 6, characterized in that: The nucleic acids include DNA, RNA, siRNA, plasmid DNA, or mRNA; the proteins include mCherry proteins.

8. The application according to claim 7, characterized in that: The intracellular delivery includes delivering nucleic acids and proteins into the cytoplasm and / or nucleus.

9. The application according to claim 8, characterized in that: The cells include HEK293T cells, PC9 cells, or A549 cells.

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