Polypeptide, polypeptide-nucleic acid nano-composite as well as preparation method and application of polypeptide-nucleic acid nano-composite

Abstract

The invention provides a polypeptide, the polypeptide is BS-PEG4-N (GCP)-MCP, the structural formula of the polypeptide is as follows: the invention also provides a preparation method of the polypeptide and an application of the polypeptide in nucleic acid drug delivery. Meanwhile, the invention also provides a polypeptide-nucleic acid nano-composite as well as a preparation method and application thereof, and the raw materials of the polypeptide-nucleic acid nano-composite comprise the polypeptide and the nucleic acid molecule. The polypeptide not only is easy to synthesize, but also has the advantages of high nucleic acid loading efficiency and high safety, and can be used as a nucleic acid delivery carrier including macromolecular nucleic acids such as pDNA and mRNA.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, and particularly to a polypeptide, a polypeptide-nucleic acid nanocomplex, its preparation method and application. Background Technology

[0002] Nucleic acid drugs include small nucleic acid molecules (such as siRNA, ASO, and aptamers) and large nucleic acid molecules (such as mRNA and pDNA). The development of nucleic acid drugs has driven the rapid advancement of gene therapy. Gene therapy, which works through multiple mechanisms such as gene inhibition, gene insertion, gene editing, and protein expression, has become a research hotspot in the biomedical field. In recent years, gene therapy has shown great potential in the treatment of hereditary diseases, cancer, and infectious diseases, especially gene editing technologies based on plasmid DNA (pDNA) and mRNA (such as CRISPR-Cas9), which can achieve precise repair, insertion, or regulation of target genes, providing new treatment strategies for many intractable diseases. However, nucleic acid drugs face many challenges in clinical application. Due to their strong negative charge, high hydrophilicity, and susceptibility to degradation by nucleases, nucleic acid drugs struggle to effectively penetrate cell membranes and reach their target sites. Therefore, developing efficient and safe nucleic acid delivery systems has become a key research direction in the field of gene therapy.

[0003] Currently, nucleic acid delivery vectors are mainly divided into two categories: viral vectors and non-viral vectors. Viral vectors (such as adenoviruses and lentiviruses) are widely used due to their high transfection efficiency, but their high immunogenicity, potential gene integration risk, and high production costs limit their clinical application. In contrast, non-viral vectors have attracted widespread attention due to their high safety, strong designability, and ease of large-scale production. In recent years, various novel non-viral vectors (such as lipid-based delivery systems, inorganic nanoparticles, cationic polymers, extracellular vesicles, hydrogels, and peptides) have shown excellent performance in improving nucleic acid stability and delivery efficiency, while also achieving significant progress in biocompatibility, toxicity, immunogenicity, and targeting.

[0004] Mesoporous silica nanoparticles, as representatives of inorganic nanocarriers, enable efficient nucleic acid loading due to their mesoporous structure and large specific surface area. Furthermore, their easily modifiable surface offers possibilities for further improving the stability and targeting of nucleic acid drugs. However, inorganic nanomaterials are difficult to degrade in vivo, potentially triggering toxic reactions and limiting their application. Polyethyleneimine (PEI), as a representative cationic polymer, can effectively induce nucleic acid aggregation, but the toxicity of high molecular weight PEI cannot be ignored. To improve the biocompatibility of PEI, researchers have introduced biocompatible molecules such as cyclodextrin and polyethylene glycol (PEG) to reduce the charge density of PEI, thereby improving its safety while maintaining its nucleic acid delivery performance. In addition, extracellular vesicles, represented by exosomes, have also been developed for nucleic acid delivery to further improve the biocompatibility of carriers and reduce their immunogenicity. Exosomes have shown good specificity in targeting nucleic acids to specific tissues and organs, but their high extraction cost limits their widespread application. As a representative of lipid delivery systems, lipid nanoparticles (LNPs) have become the preferred carrier for nucleic acid drug delivery due to their nucleic acid protection capabilities, high delivery efficiency, good biosafety, and industrialization advantages.

[0005] Despite the numerous advantages of LNP delivery systems, unmodified LNP systems still suffer from limitations such as potential cytotoxicity, lack of target selectivity, short cycle times, low endosome escape efficiency, and the need for extreme storage conditions (e.g., freezing). In contrast, while peptide carriers exhibit poor stability and insufficient endosome escape capacity, they have the potential to overcome some of the limitations of LNPs. First, peptides are derived from natural products, possessing good biocompatibility and biodegradability. Furthermore, peptides are considered an effective tool for crossing various biological barriers, including the blood-brain barrier, conjunctival barrier, nasal barrier, and skin barrier. Currently, over 40 peptide-based materials are used for nucleic acid delivery. The abundant chemical modification sites in amino acids endow peptides with extremely high design flexibility, allowing for optimization of nucleic acid delivery system performance at multiple levels, including targeting, stability, and biocompatibility. Through solid-phase synthesis techniques, peptide synthesis and purification can be standardized, reducing batch-to-batch variability. Simultaneously, peptide carrier-nucleic acid complexes can be prepared into dry powder formulations without affecting nucleic acid activity, thereby reducing storage and transportation costs.

[0006] Furthermore, compared to oligonucleotides (such as siRNA, ASO, and aptamers), large nucleic acid molecules like pDNA and mRNA have longer sequences, enabling transient expression of target proteins in transfected cells. Therefore, they hold broad application prospects in areas such as protein substitution, gene editing, and vaccine development. However, the strong negative charge and high hydrophilicity of pDNA and mRNA make their delivery more challenging. Moreover, gene-editing plasmids (a type of pDNA with special functions, such as pDNA carrying the CRISPR-Cas9 system) are much larger than conventional plasmids, further increasing the delivery difficulty.

[0007] Currently, peptide vectors used for the delivery of large nucleic acid molecules such as pDNA and mRNA, especially peptide vectors used for gene editing plasmids, usually require long amino acid sequences (generally more than 15 amino acids), which not only increases the complexity of synthesis but also limits their widespread application.

[0008] Therefore, developing an easy-to-synthesize, highly efficient, and safe polypeptide carrier for the efficient delivery of large nucleic acid molecules such as pDNA and mRNA remains an urgent research need. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a polypeptide, a polypeptide-nucleic acid nanocomposite, a preparation method thereof and its application. The polypeptide contains 9 natural amino acids, uses β-sitosterol (BS) as a hydrophobic group, and introduces a guanidine carbonyl pyrrole (GCP) functional group that has a strong binding ability with nucleic acids. It is not only easy to synthesize, but also has the advantages of high nucleic acid loading efficiency and high safety. It can be used as a nucleic acid delivery carrier including large nucleic acid molecules such as pDNA and mRNA.

[0010] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0011] A polypeptide, wherein the polypeptide is BS-PEG4-N(GCP)-MCP, has the following structural formula:

[0012]

[0013] The polypeptide of this invention has an overall positive charge.

[0014] A method for preparing the above-mentioned polypeptide includes the following steps:

[0015] (1) A linear polypeptide containing two methionines was prepared by solid-phase synthesis. The polypeptide sequence was BS-PEG4-K(GCP)-RRMEHRMEW.

[0016] (2) After dissolving the linear polypeptide obtained in step (1) in an organic solvent, add 1,2-o-dibromobenzyl and react at room temperature.

[0017] As one of the preferred embodiments of the present invention, in step (2), the organic solvent is a solvent containing 5% formic acid.

[0018] As one of the preferred embodiments of the present invention, in step (2), after the linear polypeptide is dissolved in an organic solvent, 10 equivalents of 1,2-o-dibromobenzyl are added, and the reaction is carried out at room temperature for 9 to 12 hours; more preferably, the reaction is carried out at room temperature for 10 hours.

[0019] In this process, the two methionines of the polypeptide react with 1,2-o-dibromobenzyl to form a cyclic closure; the resulting cyclic polypeptide has two positively charged sulfonium salt centers and enhanced stability.

[0020] An application of the aforementioned polypeptide in nucleic acid drug delivery, wherein the polypeptide serves as a nucleic acid delivery carrier.

[0021] A polypeptide-nucleic acid nanocomposite, the raw materials of which include the aforementioned polypeptide and nucleic acid molecules.

[0022] As one of the preferred embodiments of the present invention, the nucleic acid molecule is one or more of pDNA, mRNA, and sgRNA.

[0023] A method for preparing the above-mentioned polypeptide-nucleic acid nanocomposite: the polypeptide and nucleic acid molecules are incubated at room temperature to form the polypeptide-nucleic acid nanocomposite by self-assembly.

[0024] As one of the preferred embodiments of the present invention, the nitrogen-to-phosphorus ratio of the polypeptide and nucleic acid molecules is ≥6:1.

[0025] In this invention, positively charged peptides can form stable nanoparticles with negatively charged nucleic acids (such as pDNA and mRNA) through electrostatic interactions, protecting the nucleic acids from degradation and promoting their intracellular delivery. Simultaneously, the aforementioned methionine side-chain modification can be reduced in GSH-rich intracellular environments, thereby weakening the positive charge of the peptide and facilitating the release of nucleic acids from the nanoparticles, ultimately leading to intracellular expression of the target protein.

[0026] Application of the above-mentioned polypeptide-nucleic acid nanocomposite in the preparation of gene therapy drugs.

[0027] The advantages of this invention compared to the prior art are:

[0028] The polypeptide of this invention contains 9 natural amino acids, with β-sitosterol (BS) as a hydrophobic group, and introduces a guanidine carbonyl pyrrole (GCP) functional group that has a strong binding ability with nucleic acids. It is not only easy to synthesize, but also has the advantages of high nucleic acid loading efficiency and high safety.

[0029] Cytotoxicity assays demonstrated that the peptides of this invention possess high biocompatibility. Flow cytometry data further confirmed that the peptide-nucleic acid nanocomplexes of this invention can efficiently express proteins intracellularly. In editing experiments targeting the EGFP gene, it was further confirmed that the peptides of this invention can deliver large-sized gene-editing plasmids and achieve gene-editing function intracellularly.

[0030] Accordingly, the polypeptide of the present invention can serve as a novel, low-toxicity, and highly efficient nucleic acid delivery vector, especially suitable for the delivery of large-size gene editing plasmids, and can be applied in gene editing or gene therapy. Attached Figure Description

[0031] Figure 1 The synthetic route and mass spectrometry results of β-sitosterol succinate monoester of the present invention are shown in the figure (Figure a is the synthetic route diagram and Figure b is the mass spectrometry spectrum).

[0032] Figure 2 The synthetic route and NMR results of 2,5-pyrrolodic acid of the present invention are shown in the figure (Figure a is the synthetic route diagram and Figure b is the NMR result diagram).

[0033] Figure 3 This is the synthetic route for the polypeptide BS-PEG4-N(GCP)-MCP of this invention;

[0034] Figure 4 The mass spectrometry and HPLC results of the polypeptide BS-PEG4-N(GCP)-MCP of this invention are shown in the figure (Figure a is the mass spectrum and Figure b is the HPLC spectrum).

[0035] Figure 5 The results of agarose gel electrophoresis of the present invention's polypeptides and nucleic acids assembled into nanocomposites in different ratios are shown.

[0036] Figure 6 The diagram shows the particle size results of the polypeptide-nucleic acid nanocomplex under different ratios in this invention.

[0037] Figure 7 This is a potential result diagram of the polypeptide-nucleic acid nanocomposite of the present invention;

[0038] Figure 8 The agarose gel electrophoresis results of the release of nucleic acid by the polypeptide-nucleic acid nanocomplex in response to GSH in this invention are shown in the figure (lane 1 is plasmid pEGFP-N1, lane 2 is polypeptide-(pEGFP-N1) nanocomplex, and lane 3 is the product of polypeptide-(pEGFP-N1) nanocomplex after incubation with 10mM GSH).

[0039] Figure 9The average fluorescence intensity of EGFP and inverted fluorescence microscopy images of the polypeptide-(pEGFP-N1) nanocomposite of the present invention after administration to HEK293T cells (Figure a shows the average fluorescence intensity of EGFP after administration of the nanocomposite to HEK293T cells, and Figure b shows the inverted fluorescence microscopy image of the nanocomposite prepared by self-assembly with a nitrogen-phosphorus ratio of 10 / 1 after intracellular administration).

[0040] Figure 10 The cell proliferation results of HEK293T cells after administration of the polypeptide-(pEGFP-N1) nanocomposite of the present invention;

[0041] Figure 11 An inverted fluorescence microscope image of HEK293T cells after administration of the polypeptide-(EGFP mRNA) nanocomposite of the present invention.

[0042] Figure 12 The average fluorescence intensity of EGFP protein expression, the expression rate of EGFP-positive cells, cell proliferation, and scores of HEK293T cells after administration of the polypeptide-(pX601-GFP) nanocomposite of the present invention are shown in the figure (Figure a shows the average fluorescence intensity of EGFP protein expression after administration of the nanocomposite to HEK293T cells, Figure b shows the expression rate of EGFP-positive cells after administration of the nanocomposite to HEK293T cells, Figure c shows the cell proliferation after administration of the nanocomposite to HEK293T cells, and Figure d shows the score comparing the average fluorescence intensity and cytotoxicity).

[0043] Figure 13 EGFP protein expression in HEK293T-EGFP cells after administration of the polypeptide-(pX601-sgEGFP) nanocomposite of the present invention (in the figure, "ctrl" is the control group without administration, "Lipo 2000" is the Lipofectamine 2000-(pX601-sgEGFP) nanocomposite treatment group, "Lipo 3000" is the Lipofectamine 3000-(pX601-sgEGFP) nanocomposite treatment group, and "BS-PEG4-N(GCP)-MCP" is the polypeptide-(pX601-sgEGFP) nanocomposite treatment group of the present invention). Detailed Implementation

[0044] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents used in the following embodiments and experimental examples of the present invention are all commercially available products; the methods and equipment used are all conventional methods and equipment in this technical field, and will not be described in detail again.

[0045] Example 1: Synthesis of compound β-sitosterol succinate monoester:

[0046] The synthetic route of β-sitosterol succinate monoester is as follows: Figure 1 As shown in a, the specific steps are as follows:

[0047] (1) Dissolve 1 g of β-sitosterol (1 equivalent) in 9 mL of dichloromethane (DCM), and dissolve 0.195 g of succinic acid (1 equivalent) in 1 mL of N,N-dimethylformamide (DMF). After mixing the two, add 1.321 mL of N,N-diisopropylethylamine (DIPEA) (4 equivalents) and mix thoroughly. Set up a reflux apparatus and react in an oil bath at 50 °C for 48 h.

[0048] (2) Thin-layer chromatography (TLC) was used to monitor the reaction progress, with petroleum ether: ethyl acetate = 4:1 as the developing solvent and phosphomolybdic acid as the colorimetric agent.

[0049] (3) When the monitoring results showed that there was almost no β-sitosterol remaining, water was added for extraction, and the target product was in the organic phase.

[0050] (4) The organic reagents were removed using a rotary evaporator, and the crude product β-sitosterol succinate was obtained. The molecular weight was determined by mass spectrometry. Figure 1 b).

[0051] Example 2, Synthesis of compound 2,5-pyrrolic acid:

[0052] The synthetic route for 2,5-pyrrolic acid is as follows: Figure 2 As shown in Figure a, this reaction is a two-step cascade reaction. The first step is esterification, synthesizing the intermediate pyrrole dicarboxylate from methyl pyrrole carboxylate. The second step is hydrolysis to obtain 2,5-pyrrole dicarboxylic acid. In the preparation of pyrrole derivatives, it is crucial to ensure oxygen isolation at high temperatures; otherwise, oxidation easily occurs, resulting in a charred product. The specific steps are as follows:

[0053] (1) Esterification reaction to prepare intermediate product pyrrole dicarboxylate

[0054] Take methyl pyrrole (5.48 g, 438 mmol, 1.0 equivalent) and add it to a 350 mL wide-mouth sealed tube. Then add a magnetic stir bar, iron acetylacetone catalyst (Fe(acac)3) (928.4 mg, 2.6 mmol, 0.06 equivalent), carbon tetrachloride (CCl4) (12 mL, 131.4 mmol, 3 equivalent), and approximately 66 mL of methanol (MeOH). Purge with nitrogen for 5 min to remove dissolved oxygen from the solvent, then quickly seal the tube. React in an oil bath at 115 °C for 48 h. After cooling, slowly open the tube; dimethyl ether (Me2O) will be released. Once the gas has completely released, transfer the reaction mixture to a 500 mL flask and carefully rotary evaporate to remove most of the solvent.

[0055] (2) Post-processing

[0056] Take 350 mL of C-type sintered silica gel and add 200-300 mesh silica gel soaked in ethyl acetate (EA), ensuring the silica gel layer is approximately half the height of the sintered silica gel core. After dissolving the product in EA, carefully add it to the top of the silica gel layer for coarse separation. Wash with EA until the lower silica gel layer turns white, indicating that product washing is nearly complete. TLC with UV detection can be used for spotting. Rotary evaporation removes most of the solvent, yielding the crude intermediate product, pyrrole dicarboxylate, which can be used directly in the next step.

[0057] (3) Hydrolysis reaction

[0058] Dissolve 10g of NaOH in 100mL of ice water. Use this NaOH solution to transfer pyrrole dicarboxylate from the flask to a 350mL wide-mouth sealed tube. Dissolve any residue in the flask with a small amount of methanol before transferring. Purge with nitrogen for 5 minutes to remove dissolved oxygen from the solution, then quickly seal the tube. React overnight in a 95℃ oil bath for approximately 8–12 hours, finally yielding a black solution with a small amount of insoluble matter.

[0059] (4) Post-processing

[0060] After returning to room temperature, the reaction solution was transferred to a 500 mL Erlenmeyer flask, a magnetic stir bar was added, and 100 mL of ethyl acetate (EA) was added in an ice-water bath. The solution was then acidified with concentrated hydrochloric acid to bring the pH of the aqueous phase to 1–2. Diatomaceous earth was then added and stirred for 5 min to allow for complete adsorption of the Fe(OH)3 colloid. The solid was removed by filtration using a M-type sintered glass filter. The liquid phase was extracted using a separatory funnel, and the aqueous phase was extracted once with 50–100 mL of EA. The organic phases were combined and dried over Na2SO4. Careful rotary evaporation was performed to remove most of the EA. Precipitation was then carried out in a 1000 mL Erlenmeyer flask with stirring, using 3–4 times the product volume of petroleum ether (PE) to precipitate the product. Finally, the product was filtered using an M-type sintered glass filter to obtain a red solid, which was the final product, 2,5-pyrroledicarboxylic acid. The product was dried under vacuum for 30–60 min. Nuclear magnetic resonance analysis confirmed that the structure conformed to (…). Figure 2 b).

[0061] Example 3: Synthesis of polypeptide BS-PEG4-N(GCP)-MCP:

[0062] The synthetic route of BS-PEG4-N(GCP)-MCP is as follows: Figure 3 As shown, the specific steps are as follows:

[0063] (1) Synthesis, separation and purification of linear polypeptides

[0064] ① Following standard solid-phase peptide synthesis techniques using -Fmoc, peptides were synthesized on MBHA resin. Specifically, Rink amide MBHA resin (loading capacity 0.655 mmol / g) was weighed into a peptide inoculation tube, DCM was added, and N2 was applied for 20 min. A DMF solution containing 50% (v / v) morpholine was added, and N2 was applied for 30 min, followed by two washes with alternating DCM and DMF to remove the Fmoc protecting groups. The resin was washed with alternating DCM and DMF three times each, for 10 s each time. Fmoc-AA-OH (5 equivalents) and O-benzotriazole-tetramethylurea hexafluorophosphate (HATU) (5 equivalents) were dissolved in DMF, followed by the addition of DIPEA (5 equivalents), and mixed to obtain the amino acid reaction solution. The amino acid reaction solution was added to the resin, and nitrogen was applied for 1.5 h. The reaction solution was removed, and the resin was washed with alternating DCM and DMF three times each, for 10 s each time. The Fmoc protecting group is removed using the method described above, and then the reaction of the next amino acid is carried out until the synthesis of the "Fmoc-K(Mtt)RRMEHRMEW" amino acid sequence is completed.

[0065] ② Remove the Fmoc protecting group according to the method described above. Then, dissolve Fmoc-NH-PEG2-CH2COOH (5 equivalents) and HATU (5 equivalents) in DMF, followed by the addition of DIPEA (5 equivalents), and mix well to obtain the PEG2 reaction solution. Add the PEG2 reaction solution to the resin and purge with nitrogen for 1.5 hours. Remove the reaction solution and wash three times alternately with DCM and DMF, 10 seconds each time. Repeat this step once more.

[0066] ③ Removal of the K(Mtt) protecting group. Specifically, the MBHA resin linked with "Fmoc-PEG2-PEG2--K(Mtt)RRMEHRMEW" was continuously washed three times with DCM. Then, DCM containing 3% (v / v) trifluoroacetic acid (TFA) was added to the resin, and nitrogen was blown in for 10 min. The reaction solution was removed, and the resin was washed twice with DCM. The reaction step with DCM containing 3% (v / v) trifluoroacetic acid (TFA) was repeated nine times, followed by six washes with alternating DCM and DMF to end this step.

[0067] ④ Couple guanidine carbonylpyrrole (GCP) to the peptide. Specifically, tert-butylcarbonylguanidine (3 equivalents), HATU (3 equivalents), and the compound 2,5-pyrrolic acid synthesized in Example 2 (3 equivalents) were dissolved in DMF, and DIPEA (3 equivalents) was added to obtain a reaction solution. The reaction solution was stirred at room temperature for 12 h. Then, HATU (3 equivalents) was added, mixed well, and added to the resin obtained after step ③, and purged with nitrogen for 12 h. Subsequently, the mixture was washed 6 times alternately with DCM and DMF to end this step.

[0068] ⑤ Couple β-sitosterol to the polypeptide. Specifically, add a DMF solution containing 50% (v / v) morpholine, purge with N2 for 30 min, remove twice, alternating between washing with DCM and DMF twice to remove the Fmoc protecting group. Wash the resin with DCM and DMF alternately three times each, 10 s each time. Dissolve the β-sitosterol succinate monoester (3 equivalents) and HATU (3 equivalents) synthesized in Example 1 in a mixed solvent of DCM and DMF (40% DCM + 60% DMF), add DIPEA (3 equivalents) to obtain the reaction solution. Subsequently, the reaction solution is added to the corresponding resin after the above treatment, purge with nitrogen for 1 h, controlling the nitrogen flow rate to prevent DCM volatilization and precipitation of the reactants.

[0069] ⑥ Wash the resin three times with DMF and DCM alternately, 10 seconds each time, then wash it twice with methanol, 5 minutes each time. Drain the resin and wait for the next step.

[0070] ⑦ Remove the peptide from the resin. Specifically, mix the shearing buffer with the peptide resin (100 mg resin corresponds to 2 mL of shearing buffer) and react on a shaker at room temperature for 40 min. Then filter the resin with cotton and collect the liquid portion. Dry the liquid portion with nitrogen gas, and then add ice-cold ether to precipitate the peptide. The shearing buffer formulation is trifluoroacetic acid (TFA): triisopropylsilane (TIPS): water = 95%: 2.5%: 2.5% (v / v / v).

[0071] ⑧ Centrifuge at 4000 rpm for 5 min to remove the ether. Repeat this step 2-3 times to obtain the crude linear polypeptide product.

[0072] ⑨ Linear peptide purification. Specifically, a solution was prepared using acetonitrile, formic acid, and water at a volume ratio of 50%, 10%, and 40%, respectively. The crude peptide product was dissolved in the above solution, filtered through a 0.22 μm nylon membrane, and analyzed by high-performance liquid chromatography (HPLC). The molecular weight was identified by mass spectrometry to obtain a linear peptide with the sequence BS-PEG4-K(GCP)-RRMEHRMEW.

[0073] The high-performance liquid chromatography (HPLC) purification conditions mentioned were as follows: a 20x250mm column (Shimadzu, 5μm, C-18); a mobile phase consisting of water and acetonitrile containing 0.1% TFA; and a linear gradient elution program in which the acetonitrile ratio was linearly increased from 30% to 98% over a period of 25 minutes at a flow rate of 1 mL / min.

[0074] (2) Synthesis and preparation of cyclic peptides

[0075] ① Mix formic acid, acetonitrile, and water in a volume ratio of 5:50:45. Dissolve the linear polypeptide in the mixed solution, add 1,2-o-dibromobenzyl (10 equivalents), and place on a shaker to react at room temperature for about 9–12 hours.

[0076] ② The liquid fraction was filtered through a 0.22 μm nylon membrane and purified by high-performance liquid chromatography (HPLC). The molecular weight was then determined by mass spectrometry to obtain the cyclic polypeptide. Figure 4 a) HPLC was used to determine the purity of the product. Figure 4 b). The peptide was lyophilized and then dissolved in water. The concentration of the peptide was determined by Nanodrop at 280 nm absorption light, because the peptide sequence contains one tryptophan. The concentration calculation formula is:

[0077] Peptide concentration (mM) = Absorbance 280 × dilution factor ÷ 0.55.

[0078] Example 4: Peptide-nucleic acid nanocomposite:

[0079] The polypeptide-nucleic acid nanocomposite in this embodiment is composed of polypeptide BS-PEG4-N(GCP)-MCP and nucleic acid molecules (such as pDNA, mRNA, sgRNA, etc.). The preparation method is as follows:

[0080] Peptides and nucleic acid molecules are mixed at a peptide-nucleic acid nitrogen-phosphorus ratio of 6:1, and then incubated at room temperature for 30 minutes to self-assemble into corresponding peptide-nucleic acid nanocomposites. The positively charged peptides can form stable nanoparticles with the negatively charged nucleic acids through electrostatic interactions.

[0081] Example 5: Peptide-nucleic acid nanocomposite:

[0082] The polypeptide-nucleic acid nanocomposite in this embodiment is basically the same as that in Example 4, except that the nitrogen-phosphorus ratio of the polypeptide and nucleic acid molecules is 8:1.

[0083] Example 6: Peptide-nucleic acid nanocomposite:

[0084] The polypeptide-nucleic acid nanocomposite in this embodiment is basically the same as that in Example 4, except that the nitrogen-phosphorus ratio of the polypeptide and nucleic acid molecules is 10:1.

[0085] Example 7: Peptide-nucleic acid nanocomposite:

[0086] The polypeptide-nucleic acid nanocomposite in this embodiment is basically the same as that in Example 4, except that the nitrogen-phosphorus ratio of the polypeptide and nucleic acid molecules is 12:1.

[0087] Experiment Example 1: Verification of the assembly ratio of peptides and nucleic acid molecules:

[0088] This experimental example uses a gel retardation experiment to verify the appropriate assembly ratio of peptides and nucleic acids.

[0089] BS-PEG4-N(GCP)-MCP was used as the peptide, and plasmid pEGFP-N1 (a commercially available pDNA expressing enhanced green fluorescent protein) was selected as the nucleic acid molecule. Different peptide-nucleic acid nitrogen-phosphorus ratios were set: 0:1, 0.5:1, 1:1, 2:1, 4:1, 6:1, 8:1, and 10:1. The amount of nucleic acid was fixed at 10 ng / µl, and the volume was 20 µL. Different concentrations of 20 µL peptide solutions were prepared according to the set concentrations. The nucleic acid solution was added to the peptide solution, mixed well, and incubated at room temperature for 30 min for assembly. After adding 6× DNA loading buffer to the complex, 1.5% agarose gel electrophoresis was performed. The nitrogen-phosphorus ratio was defined as the molar ratio of the positive charge on the peptide to the negative charge on the nucleic acid; water was used as the solvent in this experiment.

[0090] The results showed that when the nitrogen-to-phosphorus ratio of the peptide BS-PEG4-N(GCP)-MCP to the nucleic acid pEGFP-N1 was 6:1 or higher, the peptide could completely load the nucleic acid. Figure 5 ).

[0091] Example 2: Physicochemical characterization of peptide-nucleic acid nanocomposites:

[0092] This experimental example uses a nanocomposite of peptide BS-PEG4-N(GCP)-MCP and nucleic acid pEGFP-N1 as an example. The particle size and zeta potential of the corresponding nanocomposite are characterized by a nanoparticle size potentiometry instrument.

[0093] Different peptide-nucleic acid nitrogen-phosphorus ratios were set (6:1, 8:1, 10:1, 12:1). The amount of nucleic acid was fixed at 60 ng / μL, and the volume was 150 μL. Based on the set concentration, 150 μL peptide solutions of different concentrations were prepared. The nucleic acid solution was added to the peptide solution, and the mixture was vigorously pipetted approximately 40 times briefly. The mixture was allowed to stand at room temperature for 30 minutes, and then 700 μL of water was added before testing.

[0094] Verification showed that the particle size of the prepared nanocomposite was between 66 and 77 nm. Figure 6 The prepared nanocomposite has a positive surface potential. Figure 7 ).

[0095] Experiment Example 3: Verification of the release of nucleic acid from nanoparticles in response to GSH:

[0096] This experiment uses a nanocomposite of peptide BS-PEG4-N(GCP)-MCP and nucleic acid pEGFP-N1 as an example to verify the release of nucleic acid by nanoparticles in response to GSH using a gel retardation experiment.

[0097] The polypeptide BS-PEG4-N(GCP)-MCP of this invention has two positively charged sulfonium salt centers that respond to GSH reduction. The GSH content in cells is 1-10 mM. It is envisioned that the polypeptide of this invention, co-assembled with nucleic acids into a nanocomposite, upon entering the cell, would have its positively charged sulfonium salt centers reduced by intracellular GSH, thereby weakening the positive charge of the polypeptide carrier and achieving the goal of releasing nucleic acids intracellularly.

[0098] After incubating the peptide-nucleic acid nanocomposite with 10 mM GSH at 37 °C for 12 h, the mixture was electrophoresed at 100 V for 30 min on a 1.5% (w / v) agarose gel containing GelRed at room temperature. The mixture was then observed and photographed under UV light to determine the extent to which nucleic acids were released under the action of GSH.

[0099] Experimental results show that after incubation of the polypeptide-nucleic acid nanocomposite of the present invention with GSH, pDNA is released ( Figure 8 ).

[0100] Experiment Example 4: In vitro transfection assay of peptide-pDNA nanocomplexes:

[0101] The gene transfection effect of the polypeptide-pDNA nanocomplex of the present invention was determined in HEK293T cells.

[0102] The enhanced green fluorescent protein gene from pEGFP-N1 was selected as a reporter gene. HEK293T cells were cultured in 24-well plates at a density of 1 × 10⁶ cells per well. 5 Cells were cultured at a density of 100 cells per cell for 24 h. Cells were then incubated for 24 h with peptide-(pEGFP-N1) complexes with different N / P ratios. Lipofectamine 3000 (Lipo 3000) was used as a positive control, and the complexes were formed according to the manufacturer's protocol. Each experimental group was divided into triplicate. Subsequently, cells were washed twice with PBS and treated with 200 μL of 0.05% trypsin-EDTA at room temperature for 2 min before terminating digestion with complete culture medium. The cell pellet was collected by centrifugation, and the cells were resuspended in ice-cold PBS and placed on ice. The mean fluorescence intensity of transfected cells was determined by counting and analyzing the EGFP fluorescence signal of cells using flow cytometry.

[0103] Experimental results show that the polypeptide-pDNA nanocomposite of this invention exhibits the best transfection effect under the condition of a nitrogen-phosphorus ratio of 10:1, which is superior to the positive control Lipo 3000. Figure 9 a). The transfection effect was visualized using an inverted fluorescence microscope. Figure 9 b).

[0104] The assembly process of the polypeptide-pDNA nanocomplex was as follows: 1 μg of pEGFP-N1 plasmid (4.7 kb) expressing enhanced green fluorescent protein (EGFP) was mixed with BS-PEG4-N(GCP)-MCP in 100 μL of serum-free DMEM medium at different nitrogen-to-phosphorus ratios (6:1 to 12:1); after incubation at room temperature for 30 min, 200 μL of 5% FBSDMEM medium was added; after mixing, the mixture was added to the cells.

[0105] Experimental Example 5: Cytotoxicity Assay of Peptide-pDNA Nanocomplex:

[0106] As a delivery vector, cytotoxicity is an important indicator. The cytotoxicity of the peptide-pEGFP-N1 plasmid (4.7kb) complex (i.e., peptide-pDNA nanocomplex) of this invention in HEK293T cells was evaluated using the CCK-8 assay.

[0107] HEK293T cells were stored at 2.5 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 μL in 96-well plates. Three replicates were set up for each experimental group. After 24 h, the complete medium was replaced with 100 μL of a peptide-pEGFP-N1 complex containing a range of different N:P ratios (6:1–12:1). The plates were incubated at 37 °C for 24 h. Then, each well was replaced with serum-free medium containing 5% CCK-8 and incubated at 37 °C for 2–4 h. Absorbance at 450 nm was measured using a microplate reader. Results are expressed as a percentage of control values ​​based on absorbance from untreated cells.

[0108] The assembly process of the polypeptide-pDNA nanocomplex is as follows: the polypeptide BS-PEG4-N(GCP)-MCP and 0.3 μg pEGFP-N1 plasmid are mixed in serum-free DMEM medium with different nitrogen-phosphorus ratios (6:1 to 12:1); after standing at room temperature for 30 min, 70 μL of 5% FBSDMEM medium is added; after mixing, it is added to the cells.

[0109] Ultimately, at concentrations where the peptide-pDNA nanocomplex effectively achieves transfection, over 90% of cells survive. Figure 10 ).

[0110] Experiment Example 6: In vitro transfection assay of peptide-mRNA nanocomplexes:

[0111] The gene transfection effect of the polypeptide-mRNA nanocomplex of the present invention was determined in HEK293T cells.

[0112] EGFP mRNA was selected as the reporter gene. HEK293T cells were cultured in 24-well plates at a density of 1 × 10⁶ cells per well. 5 Cells were cultured at a density of 1000 mRNA for 24 h. Then, the cells were incubated with a polypeptide-(EGFP mRNA) complex at a nitrogen-to-phosphorus ratio of 10:1 for 24 h. The protein level of EGFP was observed using an inverted fluorescence microscope.

[0113] Experimental results show that the invented polypeptide-(EGFP mRNA) nanoparticles can increase EGFP expression in most cells. Figure 11 ).

[0114] Experiment Example 7: In vitro transfection efficiency and cytotoxicity of peptide-gene editing plasmid nanocomposites:

[0115] Gene editing plasmids are typically larger than conventional plasmids because they need to contain the Cas9 protein-coding sequence (the gene is longer than 3000 bp), the guide RNA sequence, and the necessary promoters and regulatory elements. Many nucleic acid vectors (such as viral vectors) have strict limitations on plasmid size; large plasmids may lead to reduced vector loading efficiency or ineffective encapsulation, thereby affecting cellular uptake and endosome escape efficiency.

[0116] In this experiment, the gene transfection effect of the polypeptide-gene editing plasmid nanocomplex of the present invention was determined in HEK293T cells.

[0117] pX601-GFP was selected as the reporter gene. HEK293T cells were cultured in 24-well plates at a density of 1 × 10⁶ cells per well. 5 Cells were cultured at a density of 1000 cells / h for 24 h. Then, the cells were incubated with a peptide-(pX601-GFP) complex at a nitrogen-to-phosphorus ratio of 10:1 for 48 h. Lipofectamine 2000 (Lipo 2000), Lipofectamine 3000 (Lipo 3000), PEI (25 kDa), DOTAP, and C18-R9 were used as controls. Each experimental group was divided into three replicates. Subsequently, the cells were washed twice with PBS and treated with 200 μL of 0.05% trypsin-EDTA at room temperature for 2 min before terminating digestion with complete culture medium. The cell pellet was collected by centrifugation, and the cells were resuspended in ice-cold PBS and placed on ice. The percentage of transfected cells and the mean fluorescence intensity were determined by counting and analyzing the EGFP fluorescence signal by flow cytometry.

[0118] Meanwhile, the cytotoxicity of the peptide-(pX601-GFP) plasmid complex of the present invention in HEK293T cells was evaluated using the CCK-8 assay, and the cell-killing effect of other vectors delivering the same mass of pX601-GFP was compared.

[0119] The results showed that after incubation with cells, the nanocomplex formed by the peptide of this invention and pX601-GFP mediated protein expression efficiency (indicated by the percentage of EGFP-positive cells with average fluorescence intensity) was significantly lower than that of the Lipo2000 and PEI (25kDa) control groups, but higher than that of the peptide carrier C18-R9 and lipid carrier DOTAP control groups, and comparable to that of the Lipo 3000 group. Figure 12 a, b). Furthermore, the nanocomplex formed by the peptide of this invention and pX601-GFP exhibits good cellular safety, with significantly lower cytotoxicity than the Lipo 2000, Lipo 3000, DOTAP, and PEI (25kDa) control groups. Figure 12 c). Furthermore, a direct comparison of average fluorescence intensity and cytotoxicity (with equal weights) shows that the peptide-gene editing plasmid nanocomposite of this invention scores significantly better than other vectors ( Figure 12 d).

[0120] Experiment Example 8: Detection of the efficiency of nanocomposite in editing the EGFP gene in cells:

[0121] The sgRNA targeting EGFP was constructed in the pX601-mCherry vector. This pDNA was named pX601-sgEGFP. The sequence of the EGFP-targeting sgRNA is: 5'-GCAACATCCTGGGGCACAAGC-3'.

[0122] HEK293T-EGFP cells were cultured in 24-well plates at a rate of 1 × 10⁶ cells per well. 5 Cells were cultured at a density of 1,000 cells for 24 hours. pX601-sgEGFP was transfected into HEK293T-EGFP cells. 72 hours after transfection, the expression level of EGFP in the cells was detected by flow cytometry to evaluate the EGFP gene editing efficiency.

[0123] Experimental results show that the editing efficiency of the polypeptide-(pX601-sgEGFP) nanocomplex of this invention on the EGFP gene is comparable to that of Lipo 3000. Figure 13 ).

[0124] In summary, the peptides of this invention can form stable and uniform nanoparticles after physical mixing with pDNA or mRNA expressing green fluorescent (GFP) protein and incubation at room temperature. The formation of nanoparticles was verified by gel retardation analysis, particle size analysis using dynamic optical particle size scattering (DOPS), and surface potential analysis. After incubation with cells, flow cytometry and inverted fluorescence microscopy clearly showed that the peptides delivered pDNA or mRNA into the cells and released pDNA or mRNA intracellularly, ultimately enabling the pDNA or mRNA to express green fluorescent protein. Cytotoxicity experiments demonstrated that the assembled nanoparticles have high biocompatibility. The peptides of this invention can form nanoparticles with a plasmid (pX601-GFP) expressing Cas9-T2A-EGFP protein and sgRNA. Flow cytometry verified that the prepared nanoparticles could express gene editing-related proteins and sgRNA in HEK293T cells, indicating that the peptide vector of this invention has high nucleic acid loading efficiency. Furthermore, the cytotoxicity of the nanoparticles formed by the peptide vector of this invention and pX601-GFP is significantly lower than that of Lipo 2000, Lipo 3000, and PEI, and its transfection efficiency is significantly higher than that of the peptide vector C18-R9 and the lipid vector DOTAP. This demonstrates that the peptide of this invention can serve as a novel, low-toxicity, and highly efficient nucleic acid delivery vector, especially suitable for the delivery of large-size gene editing plasmids, and can be applied in gene editing or gene therapy.

[0125] 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, and 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 polypeptide, characterized in that, The polypeptide is BS-PEG4-N(GCP)-MCP, with the following structural formula:

2. A method for preparing the polypeptide as described in claim 1, characterized in that, Includes the following steps: (1) A linear polypeptide containing two methionines was prepared by solid-phase synthesis. The polypeptide sequence was BS-PEG4-K(GCP)-RRMEHRMEW. (2) After dissolving the linear polypeptide obtained in step (1) in an organic solvent, add 1,2-o-dibromobenzyl and react at room temperature.

3. The method for preparing the polypeptide according to claim 2, characterized in that, In step (2), the organic solvent is a solvent containing 5% formic acid.

4. The method for preparing the polypeptide according to claim 2, characterized in that, In step (2), after the linear polypeptide is dissolved in an organic solvent, 10 equivalents of 1,2-o-dibromobenzyl are added, and the reaction is carried out at room temperature for 9-12 hours.

5. The application of the polypeptide as described in claim 1 in nucleic acid drug delivery, characterized in that, The polypeptide serves as a nucleic acid delivery carrier.

6. A polypeptide-nucleic acid nanocomposite, characterized in that, Its constituent raw materials include the polypeptides and nucleic acid molecules described in claim 1.

7. The polypeptide-nucleic acid nanocomposite according to claim 6, characterized in that, The nucleic acid molecule is one or more of pDNA, mRNA, and sgRNA.

8. A method for preparing a polypeptide-nucleic acid nanocomposite as described in claim 6 or 7, characterized in that, The polypeptide and nucleic acid molecules can be incubated at room temperature to form the polypeptide-nucleic acid nanocomposite.

9. The method for preparing the polypeptide-nucleic acid nanocomposite according to claim 8, characterized in that, The nitrogen-to-phosphorus ratio of the polypeptide and nucleic acid molecules is ≥6:

1.

10. The use of a polypeptide-nucleic acid nanocomposite as described in claim 6 or 7 in the preparation of gene therapy drugs.

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