Preparation and application of self-assembled membrane active peptide

Abstract

This invention provides a method for preparing and applying a self-assembled membrane-active peptide, belonging to the field of biomedical technology. The amino acid backbone sequence of the self-assembled membrane-active peptide is Lys-Leu-Leu-Lys-Leu-Leu, with an N-terminal fluorene methoxycarbonyl group (Fmoc) modification and a C-terminal amidation, resulting in the structure Fmoc-Lys-Leu-Leu-Lys-Leu-Leu-CONH2. It can be prepared using a solid-phase synthesis method. This peptide maintains both cation membrane affinity and hydrophobic intercalation properties, thereby promoting the self-assembly of the short peptide in an aqueous medium to form ultra-small spherical nanoassemblies.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the preparation and application of an N-terminal Fmoc-modified self-assembled membrane active peptide. Background Technology

[0002] Biomembranes are fundamental structures for tumor cells to maintain survival and for bacteria to maintain their membrane integrity, participating in various processes such as substance exchange, energy metabolism, and stress adaptation. Compared to therapeutic strategies that rely on single receptors or specific signaling pathways, interventions targeting cell and bacterial membranes act at a higher biophysical level, are less affected by tumor heterogeneity or changes in drug resistance pathways, and therefore have significant potential in anti-tumor and anti-infective therapies. Existing membrane-active peptides typically require long amino acid sequences, complex structural motifs, or extensive chemical modifications to achieve sufficient membrane-disrupting activity and in vivo stability, which increases synthesis costs and subsequent transformation difficulties. How to integrate cation adsorption, hydrophobic intercalation, and ordered intermolecular stacking capabilities into short peptide sequences to obtain concise and effective membrane-active systems remains a crucial technical challenge in this field.

[0003] On the other hand, postoperative local tumor recurrence and wound repair are two common problems in clinical management. Postoperative residual lesions usually require continuous local intervention, while infected wounds are often accompanied by bacterial colonization, biofilm formation, and slow tissue repair. General local drug delivery methods have limited retention time at the lesion or wound site, making it difficult to meet the multiple needs of continuous release, anti-tumor, and antibacterial repair.

[0004] Therefore, developing a short peptide system with a simple structure, easy synthesis, self-assembly capability, and both anti-tumor immune activation and antibacterial repair functions, especially a self-assembled membrane active peptide system suitable for postoperative local administration, has important application prospects. Summary of the Invention

[0005] In view of this, the present invention provides a method for preparing and applying self-assembled membrane active peptides to solve or alleviate the technical problems existing in the prior art, and at least provides a beneficial alternative.

[0006] The technical solution of this invention is achieved as follows: This invention provides a self-assembly membrane-active peptide with an amino acid backbone sequence of Lys-Leu-Leu-Lys-Leu-Leu, an N-terminal fluorene methoxycarbonyl group (Fmoc) modification, and an amidation at the C-terminus, resulting in the structure Fmoc-Lys-Leu-Leu-Lys-Leu-Leu-CONH2. By introducing an Fmoc group at the N-terminus of the short peptide sequence while maintaining the C-terminal amidation, the peptide retains the cation-membrane-loving properties of lysine and the hydrophobic membrane-intercalation properties of leucine, further enhancing intermolecular hydrophobic interactions and aromatic stacking interactions, thereby constructing a short peptide molecule possessing both membrane activity and self-assembly capability.

[0007] Furthermore, the self-assembly membrane-active peptides self-assemble in an aqueous medium to form nanoassemblies, which are spherical or near-spherical with an average hydrated particle size of 5–100 nm. By setting the peptides to the above self-assembly form, they form nanoaggregates with small particle size and good dispersibility in an aqueous environment, which is beneficial to improving the system stability, dispersion uniformity, and subsequent local delivery adaptability.

[0008] This invention also provides a method for preparing a self-assembled membrane-active peptide, comprising the following steps: using Rink amide MBHA resin as a solid-phase support, amino acids are sequentially coupled from the C-terminus to the N-terminus using the Fmoc solid-phase synthesis method to obtain a resin peptide; after sequence assembly, the N-terminal Fmoc group is retained, and the peptide is cleaved from the resin using a cleavage system composed of trifluoroacetic acid, triisopropylsilane, and water to obtain the self-assembled membrane-active peptide. By adopting the above solid-phase synthesis route, target peptide products with well-defined sequences and specific terminal modifications can be obtained relatively stably, facilitating subsequent purification and application.

[0009] Furthermore, the volume ratio of trifluoroacetic acid, triisopropylsilane, and water was 95:2.5:2.5 (v / v / v). After lysis, the crude product was precipitated with cold diethyl ether and purified by reversed-phase high-performance liquid chromatography. By further defining the lysis system and purification steps, the yield efficiency and purification quality of the target product can be improved, making the obtained peptide more suitable for subsequent assembly characterization and formulation construction.

[0010] This invention also provides a nanoformulation comprising a self-assembled membrane-active peptide and a pharmaceutically acceptable carrier. By combining the self-assembled membrane-active peptide with a pharmaceutically acceptable carrier, its dosing compatibility and ease of local application can be further improved, thereby expanding its application in local therapeutic scenarios.

[0011] Furthermore, the carrier is a thermosensitive hydrogel containing Pluronic F127, with a mass-volume concentration of Pluronic F127 of 15%–30%. By using the above-mentioned thermosensitive hydrogel as a delivery carrier, the nano-formulation remains in a flowable state under low-temperature conditions and forms a gel state upon contact with physiological temperatures, thereby enhancing its retention ability at local lesion sites or wound sites and achieving the continuous release of self-assembled membrane active peptides.

[0012] This invention also provides the application of self-assembled membrane-active peptides, or nanoformulations, in the preparation of antitumor drugs. By utilizing the membrane activity characteristics and local delivery properties of self-assembled membrane-active peptides, they can be used to construct local antitumor drugs to achieve interventional treatment at tumor lesion sites.

[0013] Furthermore, antitumor drugs are used to prevent or treat postoperative local tumor recurrence. By applying self-assembled membrane-active peptides or nanoformulations to the postoperative residual lesion site, the retention and sustained action of local drug delivery can be improved, making them suitable for the suppression of postoperative local tumor recurrence.

[0014] This invention also provides the application of self-assembled membrane-active peptides, or nanoformulations, in the preparation of antibacterial and / or antibiofilm drugs. By utilizing the disruptive effect of self-assembled membrane-active peptides on bacterial envelope structures and their local delivery and retention properties, they can be used in the construction of antibacterial or antibiofilm drugs to meet the needs of infection-related local treatment.

[0015] Furthermore, the drug is used to inhibit or kill Gram-positive and Gram-negative bacteria and to treat infected wounds. By applying self-assembling membrane-active peptides or nanoformulations to infected wound sites, the needs for local treatment and wound repair can be met while inhibiting bacterial growth and intervening in biofilm formation.

[0016] The embodiments of the present invention have the following advantages due to the adoption of the above technical solutions: I. The self-assembled membrane active peptide provided by this invention adopts a Lys-Leu-Leu-Lys-Leu-Leu short peptide backbone, and introduces Fmoc modification at the N-terminus and performs amidation treatment at the C-terminus. This allows the peptide to maintain its cationic membrane affinity and hydrophobic intercalation properties while further enhancing intermolecular hydrophobic interactions and aromatic stacking interactions, thereby forming nano-assemblies with small particle size, good dispersibility, and high stability. At the same time, this technical solution has a simple structure and a clear preparation route, which is convenient for preparation and purification using solid-phase synthesis methods.

[0017] II. The self-assembled membrane active peptides and their nano-formulations provided by this invention have both anti-tumor and antibacterial application bases. Among them, the self-assembled membrane active peptides can cause membrane damage to tumor cells and can be used to inhibit local tumor recurrence after surgery. The self-assembled membrane active peptides can also destroy the bacterial envelope structure, inhibit bacterial growth, and interfere with biofilm formation. Furthermore, when combined with Pluronic F127 thermosensitive hydrogel, their retention and continuous release capabilities at local lesion sites or infected wound sites can be improved, thereby benefiting postoperative local treatment and repair of infected wounds. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application 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 this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 For KLLKLL and Fmoc Chemical structure and self-assembly characterization diagram of KLLKLL.

[0020] Figure 2 For Fmoc Experimental diagram of KLLKLL's effect on tumor cell membrane damage.

[0021] Figure 3 For Fmoc Experimental diagram showing KLLKLL-induced mitochondrial dysfunction, mtDNA release, STING signaling pathway activation, and immunogenic cell death in tumor cells.

[0022] Figure 4 For Fmoc Key results of KLLKLL's disruption of bacterial membranes and antibacterial effects.

[0023] Figure 5 For Fmoc KLLKLL / F127 thermosensitive hydrogel sol Gel transition and release diagrams and their therapeutic effects in a postoperative tumor recurrence model.

[0024] Figure 6 For Fmoc Key results of KLLKLL / F127 thermosensitive hydrogel effectively stimulating immunity (Figure) Figure 7 For Fmoc The image shows the effect of treating an infected wound model with KLLKLL / F127 thermosensitive hydrogel.

[0025] Figure 8 For Fmoc H&E sections of the heart, liver, spleen, lungs and kidneys after local administration of KLLKLL / F127. Detailed Implementation

[0026] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0027] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0028] This invention provides a self-assembled membrane-active peptide, the amino acid backbone sequence of which is Lys-Leu-Leu-Lys-Leu-Leu; The N-terminus is modified with a fluorenemethyloxycarbonyl group (Fmoc), and the C-terminus is amidated, with the structure Fmoc-Lys-Leu-Leu-Lys-Leu-Leu-CONH2. The self-assembled membrane active peptide can form nanoassemblies in aqueous media and can be further combined with pharmaceutically acceptable carriers to form locally delivered formulations.

[0029] Example 1: Synthesis and purification of self-assembled membrane-active peptide Fmoc-KLLKLL Rink amide MBHA resin was used as a solid-phase support. The target peptide was synthesized according to the Fmoc solid-phase peptide synthesis method. During the synthesis, amino acids were coupled sequentially from the C-terminus to the N-terminus, and Leu, Leu, Lys, Leu, Leu and Lys were introduced in sequence to complete the assembly of the Lys-Leu-Leu-Lys-Leu-Leu backbone sequence. After the peptide chain assembly was completed, the N-terminal Fmoc group was retained and the C-terminus was kept in an amidated state to obtain the target resin peptide.

[0030] After peptide assembly, the target peptide was cleaved from the resin using a cleavage system composed of trifluoroacetic acid, triisopropylsilane, and water, with a volume ratio of 95:2.5:2.5 and a cleavage time of 2.5 h. After cleavage, cold diethyl ether was added to the lysis buffer for precipitation, and the crude product was collected by centrifugation. Subsequently, preparative reversed-phase high-performance liquid chromatography (RP-HPLC) was used for purification to obtain the target self-assembled membrane active peptide Fmoc-KLLKLL.

[0031] The obtained product was detected by mass spectrometry. The main peak of Fmoc-KLLKLL was located at m / z 474.9015, and an ion peak at m / z 948.8065 was also observed. This indicates that the obtained product is consistent with the target molecular weight, indicating that the target peptide was successfully synthesized.

[0032] The amino acid backbone sequence of the self-assembled membrane active peptide described in this application is SEQ ID NO:1, and its length is 6 amino acids. The short peptide sequence is Lys-Leu-Leu-Lys-Leu-Leu.

[0033] SEQ ID NO:1 can be denoted as KLLKLL.

[0034] Molecular type: AA.

[0035] Source: Artificially synthesized sequence.

[0036] Type: Artificially synthesized linear short peptide.

[0037] In a preferred embodiment, the N-terminus retains a fluorenemethyloxycarbonyl (Fmoc) protecting group, while the C-terminus undergoes amidation. The resulting Fmoc-KLLKLL exhibits stronger self-assembly capability and membrane activity.

[0038] Comparative Example 1: Synthesis of the parent peptide KLLKLL This comparative example is used to compare with the Fmoc-KLLKLL obtained in Example 1.

[0039] Using the same solid-phase synthesis route as in Example 1, with Rink amide MBHA resin as the solid-phase support, amino acids were sequentially coupled from the C-terminus to the N-terminus to construct the Lys-Leu-Leu-Lys-Leu-Leu backbone sequence. The difference from Example 1 is that the N-terminal Fmoc group was not retained after peptide assembly. The remaining cleavage, precipitation, and purification steps were consistent with Example 1, yielding the unmodified parent peptide KLLKLL.

[0040] The obtained KLLKLL was detected by mass spectrometry, and its main peak was located at m / z 363.7864, indicating that the comparative peptide was successfully synthesized.

[0041] Table 1. Synthesis and characterization results of the target peptide and control peptide.

[0042] As shown in Table 1, both Fmoc-KLLKLL and the control peptide KLLKLL of the present invention can be obtained by solid-phase synthesis. The mass spectrometry characteristic peaks of Fmoc-KLLKLL are consistent with the target structure, indicating that the self-assembled membrane active peptide of the present invention was successfully prepared.

[0043] Example 2: Preparation of Fmoc-KLLKLL / F127 thermosensitive hydrogel This embodiment is used to illustrate the nano-formulation and topical drug delivery formulation of the present invention.

[0044] Pluronic F127 was selected as the temperature-sensitive delivery carrier. A 20% (w / v) F127 hydrogel matrix was prepared. Under low-temperature conditions, the Fmoc-KLLKLL obtained in Example 1 was added to the F127 system and stirred to disperse evenly, resulting in the Fmoc-KLLKLL / F127 temperature-sensitive hydrogel formulation. The formulation remained in a fluid state at 4°C and gelled at 37°C, thus forming a localized in-situ gel.

[0045] Comparative Example 2: Blank F127 Thermosensitive Hydrogel A Pluronic F127 hydrogel system with a mass-volume concentration of 20% was prepared using the same method as in Example 2, but without adding any peptide components, resulting in a blank F127 thermosensitive hydrogel, denoted as PBS@F127.

[0046] Comparative Example 3: KLLKLL / F127 Thermosensitive Hydrogel Using the same method as in Example 2, KLLKLL obtained in Comparative Example 1 was added to a Pluronic F127 system with a mass-volume concentration of 20%, and after mixing, KLLKLL / F127 thermosensitive hydrogel was obtained, denoted as KLLKLL@F127.

[0047] Test Example 1: Self-assembly behavior and protease environment stability test like Figure 1 As shown, the KLLKLL obtained in Comparative Example 1 and the Fmoc-KLLKLL obtained in Example 1 have significant differences in structure and self-assembly behavior. The KLLKLL obtained in Comparative Example 1 and the Fmoc-KLLKLL obtained in Example 1 were dispersed in an aqueous medium, and their assembly behavior was analyzed by transmission electron microscopy, dynamic light scattering, circular dichroism spectroscopy, and Zeta potential.

[0048] The results showed that KLLKLL mainly formed large and irregular aggregates, while Fmoc-KLLKLL formed more uniform spherical nanoassemblies. Dynamic light scattering results showed that the average hydrated particle size of KLLKLL was 262.5 nm, with a PDI of 0.237; while the average hydrated particle size of Fmoc-KLLKLL was 12.56 nm, with a PDI of 0.285. These results indicate that N-terminal Fmoc modification results in short peptides that can form nanoassemblies with smaller particle size and better dispersibility.

[0049] KLLKLL and Fmoc-KLLKLL were further treated with trypsin for 30 min and 120 min respectively to simulate a protease environment. The results showed that the particle size distribution of KLLKLL changed significantly, while Fmoc-KLLKLL maintained a relatively stable small particle size distribution, indicating that the self-assembled membrane active peptide of the present invention has good structural stability in a protease environment.

[0050] To further illustrate the effect of Fmoc modification on molecular assembly state, molecular dynamics simulations were performed on KLLKLL and Fmoc-KLLKLL. The results showed that Fmoc-KLLKLL exhibited a lower radius of gyration and a smaller solvent-accessible surface area, indicating that it formed a more compact molecular packing conformation.

[0051] Table 2 Comparison of self-assembly and stability results between KLLKLL and Fmoc-KLLKLL

[0052] As shown in Table 2, under the same test conditions, Fmoc-KLLKLL can form nano-assemblies with smaller particle size and maintain better structural stability in the protease environment, indicating that N-terminal Fmoc modification is beneficial to improving the self-assembly ability and system stability of short peptides.

[0053] Test Example 2: Antitumor-related effect test The cytotoxicity of Fmoc-KLLKLL obtained in Example 1 and KLLKLL obtained in Comparative Example 1 was evaluated using 4T1, MC38, HeLa, and KPC cells. The results showed that Fmoc-KLLKLL exhibited stronger inhibitory effects on all the aforementioned tumor cell types, with 4T1 cells used for subsequent mechanism evaluation.

[0054] like Figure 2 As shown, after treating 4T1 cells with 15 μM peptide for 30 min, the intracellular reactive oxygen species level in the Fmoc-KLLKLL treatment group was significantly increased, and cell membrane damage-related indicators changed significantly. After treating 4T1 cells with 15 μM peptide for 30 min, intracellular reactive oxygen species (ROS) levels were detected using DCFH-DA. The results showed that, compared with the blank group and the KLLKLL group, the green fluorescence of the Fmoc-KLLKLL treatment group was significantly enhanced, and flow cytometry analysis revealed a significant rightward shift of the ROS peak, indicating that Fmoc-KLLKLL can induce ROS accumulation in tumor cells.

[0055] Meanwhile, bright-field microscopy revealed that cells treated with Fmoc-KLLKLL exhibited swelling and abnormal morphology; further analysis showed increased release of lactate dehydrogenase and secretion of interleukin-1β; and detection using the DiBAC4(3) membrane potential probe revealed more pronounced membrane depolarization in the Fmoc-KLLKLL-treated group. These results demonstrate that the self-assembled membrane-active peptides of this invention can significantly damage tumor cell membranes.

[0056] like Figure 3 As shown, Fmoc-KLLKLL treatment resulted in abnormal mitochondrial morphology and decreased mitochondrial membrane potential in 4T1 cells, accompanied by increased mtDNA release and activation of the STING-related signaling pathway. Further analysis using transmission electron microscopy and TMRE revealed changes in mitochondrial state and membrane potential. The results indicated that Fmoc-KLLKLL treatment caused abnormal mitochondrial morphology and decreased mitochondrial membrane potential. Further analysis of mtDNA release and STING pathway-related protein expression revealed increased expression of p-STING, p-TBK1, and p-IRF3, indicating that self-assembled membrane-active peptides can activate related immune signaling pathways.

[0057] The results showed that Fmoc-KLLKLL could induce immunogenic cell death by detecting indicators such as ATP release, calreticulin exposure, and extracellular translocation of high-mobility group box 1.

[0058] Test Example 3: Antibacterial and Anti-biofilming Activity Test like Figure 4 As shown, Fmoc-KLLKLL exhibited significantly enhanced antibacterial or bactericidal effects against both Staphylococcus aureus and Escherichia coli. Using Staphylococcus aureus and Escherichia coli as model strains, the antibacterial activity of Fmoc-KLLKLL obtained in Example 1 and KLLKLL obtained in Comparative Example 1 were compared in the concentration range of 2–32 μM. Bacterial growth was determined using the plate coating method and OD600.

[0059] The results showed that KLLKLL had a weak antibacterial effect, while Fmoc-KLLKLL showed significantly enhanced antibacterial or bactericidal effects on both types of bacteria, and the effect was concentration-dependent, with a more significant inhibitory effect on Staphylococcus aureus.

[0060] The effects of Fmoc-KLLKLL on biofilm disruption and biofilm formation inhibition were further evaluated using crystal violet staining. The results showed that Fmoc-KLLKLL could both disrupt existing biofilms and inhibit the formation of new biofilms, with better effects than the KLLKLL group.

[0061] Further investigation was conducted to detect bacterial protein leakage, changes in intracellular reactive oxygen species, live and dead staining results, and scanning electron microscopy morphology.

[0062] The results showed that after treatment with Fmoc-KLLKLL, bacterial protein leakage increased, intracellular reactive oxygen species levels rose, bacterial membrane permeability increased, and bacterial cell morphology collapsed, indicating that its antibacterial effect is closely related to membrane structure disruption.

[0063] Test Example 4: In vitro release and postoperative local recurrence inhibition test like Figure 5 As shown, the Fmoc-KLLKLL / F127 thermosensitive hydrogel exhibits sustained release characteristics in vitro; the in vitro release of the Fmoc-KLLKLL / F127 thermosensitive hydrogel obtained in Example 2 was evaluated. The results showed that the formulation could continuously release peptide molecules within 12 hours, and the release rate was faster in a serum-containing environment than in a PBS environment, indicating that it has sustained release characteristics.

[0064] A postoperative local tumor recurrence model was further established by subcutaneous seeding of 4T1 cells. When the tumor volume was approximately 200 mm3, about 90% of the tumor was removed. After surgery, the mice were given 0.4 mg of the following hydrogels: Comparative Example 2 blank F127 thermosensitive hydrogel, Comparative Example 3 KLLKLL / F127 thermosensitive hydrogel, and Example 2 Fmoc-KLLKLL / F127 thermosensitive hydrogel.

[0065] The results showed that, compared with the PBS@F127 group and the KLLKLL@F127 group, the Fmoc-KLLKLL@F127 group significantly inhibited the growth of recurrent tumors, reduced the endpoint tumor weight, and did not show significant abnormal fluctuations in mouse body weight. Further analysis of tumor tissue revealed elevated levels of IFN-γ, IL-6, CXCL10, and IFN-β. Test Example 5: Infected Wound Repair and Safety Test like Figure 6 The flow cytometry results showed an increased proportion of CD80+CD86+ mature dendritic cells and an increased proportion of CD8+ / CD4+ T cells in the tumor tissue, indicating that local hydrogel delivery can significantly enhance anti-tumor immune activation at the site of postoperative residual lesions.

[0066] like Figure 7 As shown, in the infected wound model, the Fmoc-KLLKLL@F127 group had faster wound closure and lower bacterial load; a full-thickness skin defect with a diameter of 8 mm was prepared on the back of BALB / c mice, and 50 μL of 1×10⁻⁶ mol / L methyl methacrylate (MCMA) was locally inoculated into the wound. 8A Staphylococcus aureus suspension at CFU / mL was used to establish an infected wound model. After infection was established, local treatments were performed using the blank F127 thermosensitive hydrogel (Comparative Example 2), the KLLKLL / F127 thermosensitive hydrogel (Comparative Example 3), and the Fmoc-KLLKLL / F127 thermosensitive hydrogel (Example 2), with the Fmoc-KLLKLL concentration being 200 μg / mL.

[0067] Dynamic observation of wound healing revealed that, compared with the PBS@F127 group and the KLLKLL@F127 group, the Fmoc-KLLKLL@F127 group showed faster wound area reduction, higher degree of final closure, and continuous decrease in bacterial load in the wound swab plate culture.

[0068] Histological examination further showed that the Fmoc-KLLKLL@F127 group exhibited more complete epithelial regeneration, more continuous collagen deposition, and stronger expression of CD31 and α-SMA, indicating that this formulation can promote tissue repair and vascular remodeling while inhibiting infection. H&E staining of major organs showed no significant pathological abnormalities, indicating that the local treatment system has good in vivo safety. Figure 8 As shown, H&E staining of the major organs revealed no obvious pathological abnormalities, indicating that the local treatment system has good in vivo safety.

[0069] Table 3 Summary of the application effects of F127 topical formulation in different models

[0070] As shown in Table 3, the Fmoc-KLLKLL / F127 thermosensitive hydrogel of the present invention exhibits good local therapeutic effects in both the postoperative local tumor recurrence model and the infected wound model, indicating that the self-assembled membrane active peptides can achieve both local anti-tumor and antibacterial repair applications after being delivered by a thermosensitive carrier.

[0071] 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 person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in the present invention, and these should all 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 self-assembled membrane-active peptide, characterized in that, The amino acid skeleton sequence of the self-assembled membrane active peptide is Lys Leu Leu Lys Leu Leu, which is modified by a fluorenylmethyloxycarbonyl (Fmoc) at the N-terminus and is amidated at the C-terminus, and has a structure of Fmoc Lys Leu Leu Lys Leu Leu CONH2, which is denoted as Fmoc KLLKLL; wherein, unmodified KLLKLL is the parent sequence of the peptide.

2. The self-assembling membrane active peptide of claim 1, wherein, The self-assembled membrane active peptides self-assemble in an aqueous medium to form nano-assemblies, which are spherical or near-spherical with an average hydrated particle size of 5–100 nm.

3. A method of producing the self-assembling membrane active peptide according to claim 1 or 2, characterized by, Includes the following steps: Using Rink amide MBHA resin as a solid-phase support, amino acids were sequentially coupled from the C-terminus to the N-terminus using the Fmoc solid-phase synthesis method to obtain resin peptides. After the sequence assembly was completed, the N-terminal Fmoc group was retained, and the peptides were cleaved from the resin using a cleavage system composed of trifluoroacetic acid, triisopropylsilane and water to obtain the self-assembled membrane active peptides.

4. The preparation method according to claim 3, characterized in that, The volume ratio of trifluoroacetic acid, triisopropylsilane, and water is 95:2.5:2.5; after pyrolysis, the crude product is precipitated with cold diethyl ether and purified by reversed-phase high-performance liquid chromatography.

5. A nanopharmaceutical formulation characterized in that, The nanoformulation comprises the self-assembled membrane active peptide as described in claim 1 or 2, and a pharmaceutically acceptable carrier.

6. The nanoformulation of claim 5, wherein, The carrier is a thermosensitive hydrogel containing Pluronic F127, and the mass-volume concentration of Pluronic F127 is 15% to 30%.

7. The use of the self-assembled membrane active peptide according to claim 1 or 2, or the nanoformulation according to claim 5 or 6, in the preparation of antitumor drugs.

8. Use according to claim 7, characterized in that, The antitumor drug is used to prevent or treat local tumor recurrence after surgery.

9. The use of the self-assembled membrane active peptide of claim 1 or 2, or the nanoformulation of claim 5 or 6, in the preparation of antibacterial and antibiofilm drugs.

10. The application according to claim 9, characterized in that, The drug is used to inhibit or kill Gram-positive and Gram-negative bacteria, and to treat infected wounds.

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