An activatable cell-penetrating peptide and preparation method, drug delivery method and use thereof

Abstract

The application discloses an activatable cell-penetrating peptide, a preparation method, a drug delivery method and an application thereof. The activatable cell-penetrating peptide comprises polypeptide segment 1 and polypeptide segment 2; the polypeptide segment 1 comprises a peptide chain 1, the N terminal of the peptide chain 1 is acetylated and connected with a cyclic peptide RGD, and a group with a cyano group is further connected to the peptide chain 1; the polypeptide segment 2 comprises a peptide chain 2, the N terminal of the peptide chain 2 is also connected with a cyclic peptide RGD, and the peptide chain 2 contains cysteine. The application designs a novel strategy of in-situ activatable cell-penetrating peptide based on an activation nitrile-amino thiol coupling reaction, and the cell-penetrating peptide is composed of two independent low cell-penetrating activity polyarginine polypeptides; after reaching a target site, the two are coupled in-situ through the above-mentioned click chemistry reaction to generate an octa-arginine sequence with high cell-penetrating ability. The design of the 'on-demand activation' not only restores the cell-penetrating function, but also significantly reduces the cytotoxicity caused by non-specific penetration.

Description

Technical Field

[0001] This invention relates to the field of cell-penetrating peptide technology, and in particular to an activatable cell-penetrating peptide, its preparation method, drug delivery method, and uses. Background Technology

[0002] Cell-penetrating peptides (CPPs) have shown promise as drug delivery carriers, but several issues hinder their practical application. These issues include: (1) lack of cell type specificity, leading to non-specific uptake and potential adverse reactions; (2) toxicity at high concentrations, causing hemolysis, apoptosis, or necrosis, limiting their safe administration window; and (3) rapid blood clearance, resulting in extremely short drug residence time at the target site, making it impossible to maintain effective therapeutic concentrations.

[0003] To address these issues, traditional methods primarily rely on cyclizing peptides to enhance stability; however, their targeting specificity has not met expectations. Cyclic transmembrane peptides are more prone to forming intramolecular hydrogen bonds, which facilitates their crossing of cell membranes, but this does not solve the problem of poor cell and tissue specificity. Therefore, there is an urgent need to develop novel tissue-specific delivery methods to achieve sustained and efficient drug delivery in tumor therapy. Summary of the Invention

[0004] The main objective of this invention is to provide an activatable membrane-penetrating peptide, its preparation method, drug delivery method, and uses, aiming to solve at least one of the aforementioned technical problems.

[0005] To achieve the above objectives, the present invention provides an activatable transmembrane peptide, comprising polypeptide fragment 1 and polypeptide fragment 2; The polypeptide fragment 1 includes a peptide chain 1, the N-terminus of which is acetylated and linked with a cyclic peptide RGD, and the peptide chain 1 is also linked with a cyano group. The polypeptide fragment 2 includes a peptide chain 2, the N-terminus of which is also connected to a cyclic peptide RGD, and the peptide chain 2 contains cysteine. The polypeptide fragment 1 and the polypeptide fragment 2 can be reactively coupled under activation conditions to form a membrane-penetrating peptide with cell-penetrating ability.

[0006] Furthermore, the activation condition is an alkaline reaction environment.

[0007] Furthermore, the sequence of peptide chain 1 is CWRRRRK, and the sequence of peptide chain 2 is CRRRRK.

[0008] Furthermore, the structural formula of polypeptide fragment 1 is shown in Formula 1 or Formula 2, and the structural formula of polypeptide fragment 2 is shown in Formula 3 or Formula 4:

[0009] Formula 1

[0010] Formula 2

[0011] Formula 3

[0012] Formula 4 Wherein, R1 is selected from , , , , , , , , , Any one of the following.

[0013] Furthermore, the structural formula of the polypeptide fragment 1 is shown in Formula 5 or Formula 6:

[0014] Formula 5

[0015] Formula 6.

[0016] The present invention also provides a method for synthesizing the above-mentioned membrane-penetrating peptide, wherein the method for synthesizing the polypeptide fragment 1 includes the following steps: firstly, synthesizing peptide chain 1 by solid-phase polypeptide synthesis, then attaching a cyano group to peptide chain 1 by SN2 nucleophilic substitution reaction, and attaching the cyclic peptide RGD to peptide chain 1 by disulfide bond exchange reaction. The method for synthesizing polypeptide fragment 2 includes the following steps: firstly, polypeptide chain 2 is synthesized by solid-phase polypeptide synthesis, and then the cyclic peptide RGD is linked to polypeptide chain 2 by disulfide bond exchange reaction.

[0017] The present invention also provides a drug delivery method utilizing the above-mentioned activatable membrane-penetrating peptide, comprising the following steps: (1) During the synthesis of the polypeptide fragment 2, drug molecules are attached to the polypeptide fragment 2; (2) The polypeptide fragment 1 and the polypeptide fragment 2 linked with the drug molecule are simultaneously delivered into the diseased tissue. The polypeptide fragment 1 and the polypeptide fragment 2 react and couple in the diseased tissue environment to form a membrane-penetrating peptide with cell-penetrating ability and carry the drug molecule into the diseased cell.

[0018] The present invention also provides the use of the above-mentioned activatable membrane-penetrating peptide in the preparation of a delivery system for delivering drugs into cells.

[0019] The present invention also provides a drug delivery system comprising the above-mentioned activatable membrane-penetrating peptide and a drug molecule, wherein the drug molecule is attached to the polypeptide fragment 2 of the activatable membrane-penetrating peptide.

[0020] Furthermore, the structural formula of the polypeptide fragment 2 linked with the drug molecule is shown in Formula 7 or Formula 8:

[0021] Formula 7

[0022] Formula 8 Wherein, R2 is the drug molecule, which is selected from any one of cyanine dye Cy5, camptothecin, paclitaxel, doxorubicin, oxaliplatin, protein degradation targeting chimera, and gemcitabine.

[0023] Furthermore, the polypeptide fragment shown in Formula 1 or Formula 2 is coupled with the polypeptide fragment 2 linked to the drug molecule shown in Formula 7 or Formula 8 under activation conditions to form a membrane-penetrating peptide with cell-penetrating efficacy, as shown in Formula 9 or Formula 10 below:

[0024] Formula 9

[0025] Formula 10.

[0026] The design principles and beneficial effects of this invention are reflected in: Cyclic peptide RGD possesses superior conformational stability and tumor-targeting ability. This invention enhances the targeting ability of peptides by introducing RGD. Specifically, RGD exhibits specific affinity for binding to the integrin αvβ3 receptor; therefore, introducing peptides into the RGD sequence can improve their tumor-targeting ability.

[0027] This invention presents a novel strategy for in-situ activation of membrane-penetrating peptides based on an activated nitrile-aminothiol coupling reaction. This reaction exhibits excellent biocompatibility and is suitable for in-situ coupling within biological systems. The designed membrane-penetrating peptide consists of two independent polyarginine polypeptides (R4) with low membrane-penetrating activity; upon reaching the target site, the two polypeptides are in-situ coupled via the aforementioned click chemistry reaction to generate an octaarginine (R8) sequence with highly efficient membrane-penetrating ability. This "on-demand activation" design not only restores membrane-penetrating function but also significantly reduces cytotoxicity caused by non-specific penetration.

[0028] To verify its feasibility and versatility, this invention further utilized the system to successfully deliver different functional molecules such as Cy5 and CPT, effectively demonstrating the feasibility and versatility of this strategy in terms of loading. This result provides new ideas and experimental evidence for the development of novel and intelligent drug delivery systems. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the synthesis and working principle of the present invention.

[0030] Figure 2 cRGD- AC Synthesis route map of R4-CPM.

[0031] Figure 3 This is the synthetic route diagram for cRGD-R4-Cy5.

[0032] Figure 4 cRGD- AC A schematic diagram of the coupling reaction between R4-CPM and cRGD-R4-Cy5.

[0033] Figure 5 cRGD- AC Mass spectrum of R4-CPM.

[0034] Figure 6 cRGD- AC Mass spectrum of r4-CPM.

[0035] Figure 7 This is the mass spectrum of cRGD-R4-Cy5.

[0036] Figure 8 This is the mass spectrum of cRGD-r4-Cy5.

[0037] Figure 9 for AC Mass spectrum of R4-R4-Cy5.

[0038] Figure 10 This is the mass spectrum of cRGD-R4-CPT.

[0039] Figure 11 This is the mass spectrum of cRGD-r4-CPT.

[0040] Figure 12 This is the mass spectrum of R4-Cy5.

[0041] Figure 13 This is the mass spectrum of r4-Cy5.

[0042] Figure 14 This is the mass spectrum of R8-Cy5.

[0043] Figure 15This is the mass spectrum of r8-Cy5.

[0044] Figure 16 This is the mass spectrum of r8-CPT.

[0045] Figure 17 Left: Reaction process in 10 mM TPCEP solution; Right: Reaction process in 2 mM MGSH solution.

[0046] Figure 18 Left: B16F10’s ability to take up peptide fragments; Right: Quantitative analysis of fluorescence values ​​by flow cytometry.

[0047] Figure 19 This is a diagram showing the cell penetration results of the membrane-penetrating peptides that can be activated.

[0048] Figure 20 This is a graph showing the cytotoxicity results of polypeptide fragments and membrane-penetrating peptides.

[0049] Figure 21 Top: In vivo mouse imaging (scale bar: 40 μm); Bottom: Fluorescence image of ex vivo tissue. Detailed Implementation

[0050] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0051] Unless otherwise specified, the raw materials, reagents or devices used in the following embodiments can be obtained from conventional commercial sources or by existing known methods; unless otherwise specified, the methods used in the embodiments of the present invention are methods mastered by those skilled in the art.

[0052] This invention achieves barrier protection of the penetration ability of traditional membrane-penetrating peptides through structural modification, and restores their membrane-penetrating ability in situ upon activation. Specifically, as... Figure 1 As shown, the R4 peptide, which is functionalized by two RGD segments triggered by the tumor microenvironment, is coupled to form octaarginine (R8) via an activated cyano-aminothiol click chemistry reaction, thus possessing both tumor targeting (RGD) and deep penetration capabilities (R8). In tumor therapy, an activatable membrane-penetrating peptide with precise drug delivery was constructed, aiming to obtain a dual-function activatable membrane-penetrating peptide with both low toxicity and deep penetration capabilities.

[0053] Example 1 Screening cyanide-based compounds In the series of cyano-based compounds shown in Table 1, the reactivity with cysteine ​​thiol groups increases progressively as the number of nitrogen atoms on the aromatic ring increases from 0 (benzonitrile) to 2 (2-cyanopyrimidine). The fundamental reason for this is the introduction of nitrogen heteroatoms: compared to carbon atoms, nitrogen atoms have higher electronegativity and carry unshared electron pairs. This allows aromatic heterocycles such as pyrimidine and pyridine rings to effectively reduce the electron cloud density on the carbon atom connecting the cyano group through significant electron-withdrawing inductive effects and resonance effects, thereby enhancing the polarization of the cyano group and making it more susceptible to nucleophilic attack by the thiol group. Therefore, 2-cyanopyrimidine, containing two nitrogen atoms, exhibits the best reactivity (hence the use of cyanopyrimidine for the cyano group in the following examples), followed by 2-cyanopyridine, while benzonitrile shows the weakest reactivity.

[0054] Table 1 Reaction rates of various cyanide-based compounds

[0055] Example 2 Drug delivery system (cRGD- AC Synthesis and activation of R4-CPM+cRGD-R4-Cy5 The drug delivery system includes peptide fragment 1 and peptide fragment 2 linked with a drug molecule.

[0056] To achieve in vitro characterization and in vivo tracking, this embodiment introduces the drug molecule cyanine dye Cy5 to label the peptide. The structural formula of peptide fragment 1 in the drug delivery system of this embodiment is shown in Formula 1-1, denoted as cRGD- AC R4-CPM, the structure of the polypeptide fragment 2 linked to the drug molecule is shown in Formula 2-1, and is denoted as cRGD-R4-Cy5.

[0057]

[0058] Equation 1-1

[0059] Equation 2-1 I. Synthesis of Peptide Fragment 1 The synthetic route of peptide fragment 1 is as follows: Figure 2 As shown, the peptide chain (R4 peptide) of peptide fragment 1 has the sequence CWRRRRK. A peptide fragment modified with a functional molecule (denoted as R4-CPM) was synthesized by an SN2 nucleophilic substitution reaction between the R4 peptide and the active ester of 2-cyano-5-fluoropyrimidine. Then, a peptide fragment coupled with RGD (cRGD-) was prepared using a disulfide bond exchange reaction. AC R4-CPM). The specific synthesis method is as follows: (1) Synthesis of 2-cyano-5-fluoropyrimidine (CPM) derivative: 2-cyano-5-fluoropyrimidine (1 eq.) and 3-mercaptopropionic acid (2 eq.) were weighed and dissolved in DMF. After dissolution, cesium carbonate was added and the reaction was carried out at 60 °C for 1 h. After the reaction was completed, water and ethyl acetate were added and extracted three times to remove 3-mercaptopropionic acid. The aqueous phase was recovered and 1 mmol HCl was added to adjust to acidity and then ethyl acetate was added for extraction. The organic phases were combined and dried with anhydrous Na2SO4. The solvent was removed by vacuum distillation to obtain the intermediate. The intermediate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (2 eq.) and 371 mg N-hydroxysuccinimide (2 eq.) were dissolved in DCM and reacted at room temperature for 4 h. After the reaction was completed, water and DCM were added and extracted three times. The organic phases were combined and dried with anhydrous Na2SO4. The solvent was removed by vacuum distillation to obtain a yellow liquid crude product. The crude product was purified by silica gel column chromatography (elution buffer: ethyl acetate: petroleum ether = 1:1) to obtain the product CPM-NHS with a yield of 12.1%.

[0060] (2) Solid-phase peptide synthesis of CWR4K peptide: Rink Amide-MBHA resin was swollen in N,N-dimethylformamide (DMF) for 20 min. The Fmoc protecting group was removed using a deprotection solution (piperazine:DMF = 8:2, v / v). The peptide chain was assembled using the standard Fmoc solid-phase synthesis method. Each amino acid was coupled twice in DMF under nitrogen for 1 hour each time using HATU / DIPEA as the coupling system to obtain CWR4K resin.

[0061] (3) Acetylation of the amino terminus of the polypeptide backbone: After the polypeptide sequence is synthesized according to the above method, 200 mg of CWR4K resin is weighed and placed in the polypeptide reaction tube. 10 ml of DMF is added to swell for 5 min. After removing the filtered DMF, 2 ml of deprotection solution is added to remove the Fmoc protecting group. After completion, DMF, DCM, and DMF are added sequentially to wash the resin. Acetic anhydride (3 eq.) and pyridine (2 eq.) are weighed and dissolved in DMF. The mixed solution is added to the polypeptide reaction tube and bubbled with nitrogen for 2 h. After the reaction is completed, DMF, DCM, and DMF are added to wash the resin.

[0062] (4) Peptide cleavage from the solid support: After the peptide sequence was synthesized, methanol was added to reduce the resin volume, and nitrogen gas was introduced to dry the resin. After the resin was dried, peptide cleavage solution (95% trifluoroacetic acid / 2.5% deionized water / 2.5% triisopropylsilane, v / v / v) was added, sealed, and placed on a shaker for 2 h. After the reaction was completed, nitrogen gas was introduced to evaporate the trifluoroacetic acid, 10 mL of diethyl ether was added to precipitate the peptide, and the precipitated peptide was collected after high-speed centrifugation. The peptide was dissolved in water and acetonitrile (1:1), filtered to remove the resin, and the purity and structure of the crude peptide were analyzed by high-performance liquid chromatography (HPLC) and mass spectrometry (LCMS). AC CWR4K peptide.

[0063] (5) Targeting peptide (cRGD) coupled peptide AC CWR4K: Weigh the cut pieces AC 100 mg of CWR4K peptide was dissolved in 5 ml of DMF, and 40 mg of 2,2'-dithiopyridine (2 eq.) was added and the reaction was carried out overnight. After the reaction was completed, 52 mg of cRGD (1 eq.) was added and the reaction was continued for 4 h. Diethyl ether was added to precipitate the peptide, and the crude peptide was purified by HPLC to obtain cRGD- AC CWR4K peptide.

[0064] (6) CPM-NHS conjugated polypeptide: Weigh out cRGD- AC CWR4K peptide (10 mg, 1 eq.) and CPM-NHS (3.6 mg, 2 eq.) were dissolved in 1 ml DMSO, and DIPEA (3 mg, 4 eq.) was added. The mixture was reacted at room temperature for 3 h. After the reaction, the DMSO was removed by vacuum drying, and the crude peptide was purified by HPLC to obtain cRGD- AC R4-CPM.

[0065] cRGD- AC See the mass spectrum of R4-CPM. Figure 5 This confirmed the successful synthesis of the corresponding structural polypeptide fragment 1.

[0066] II. Synthesis of polypeptide fragment 2 linked to drug molecules The synthetic route for polypeptide fragment 2 linked to a drug molecule is as follows: Figure 3 As shown, the peptide chain (R4 peptide) sequence of polypeptide fragment 2 is CRRRRK. A fluorescently labeled polypeptide fragment (denoted as R4-Cy5) was obtained through amide condensation, and then a polypeptide fragment coupled with RGD (cRGD-R4-RGD) was prepared using a disulfide bond exchange reaction. The specific synthesis method is as follows: (1) Synthesis of CR4K peptide by solid-phase peptide synthesis: Rink Amide-MBHA resin was swollen in N,N-dimethylformamide (DMF) for 20 min. The Fmoc protecting group was removed using a deprotection solution (piperazine:DMF = 8:2, v / v). The peptide chain was assembled by standard Fmoc solid-phase synthesis. Each amino acid was coupled twice in DMF under nitrogen for 1 hour each, using HATU / DIPEA as the coupling system, to obtain CR4K resin.

[0067] (2) Removal of the Mtt protecting group from the side chain (solid-phase reaction): A deprotection solution was prepared using DCM / Tis / TFA in a volume ratio of 92:5:3. CR4K resin was weighed and placed in a peptide reaction tube, and DCM was added to fully swell the resin. After swelling, the DCM was filtered off. 3 ml of the deprotection solution was added, and the reaction was carried out for 2 min. The deprotection solution was then removed by filtration. This process was repeated 10 times. After the resin turned white, the removal of the Mtt protecting group was complete. After the protecting group was removed, DMF, DCM, and DMF were added sequentially for washing three times each.

[0068] (3) Cy5-coupled peptide: 200 mg of CR4K resin with Mtt protection removed was placed in a peptide reaction tube. Cy5 (158.1 mg, 2 eq.), PyBOP (136 mg, 2 eq.), HOBT (35 mg, 2 eq.), and DIPEA (67.7 mg, 4 eq.) were dissolved in DMF. After complete dissolution, the mixture was added to the peptide reaction tube and reacted under nitrogen bubbling in the dark at room temperature for 4 h. After the reaction was completed, DMF, DCM, and DMF were added sequentially to wash and dry the resin. After Cy5 coupling was completed, the N-terminal protecting group was removed and a cysteine ​​residue (L-type) was coupled to obtain CR4K-Cy5 resin.

[0069] (4) The polypeptide is cleaved from the solid support: the method is the same as step (4) of the polypeptide fragment 1 synthesis process, to obtain the R4-Cy5 polypeptide.

[0070] (5) Target peptide (cRGD) coupled with peptide R4-Cy5: 100 mg of the cleaved R4-Cy5 peptide was weighed and dissolved in 5 ml of DMF, and 2,2'-dithiopyridine (30.1 mg, 2 eq.) was added and reacted overnight. After the reaction was completed, cRGD (39.6 mg, 1 eq.) was added and the reaction was continued for 4 h. Ether was added to precipitate the peptide, and the crude peptide was purified by HPLC to obtain cRGD-R4-Cy5.

[0071] See the mass spectrum of intermediate R4-Cy5. Figure 12 See the mass spectrum of cRGD-R4-Cy5. Figure 7This confirmed the successful synthesis of polypeptide fragment 2, which has the drug molecule Cy5 linked to the corresponding structure.

[0072] III. Activation of Drug Delivery Systems In-situ activatable RGD-R8 transmembrane peptide coupling method: Two polypeptide fragments cRGD- AC R4-CPM and cRGD-R4-Cy5 were dissolved in 10 mM TCEP Tris buffer at pH 7.5 at a molar ratio of 1:1 and reacted for 30 min to obtain the R8 transmembrane peptide, denoted as R8. AC R4-R4-Cy5.

[0073] AC The structural formula of R4-R4-Cy5 (RGD has been removed, and CPM participated in the reaction and disappeared) is shown in Equation 3-1. See mass spectrum for details. Figure 9 This confirmed that peptide fragment 1 and peptide fragment 2, which is linked to a drug molecule, reacted and coupled to form a transmembrane peptide with the corresponding structure.

[0074]

[0075] Equation 3-1 Example 3 Drug delivery system (cRGD- AC Synthesis and activation of r4-CPM+cRGD-r4-Cy5 The drug delivery system includes peptide fragment 1 and peptide fragment 2 linked with a drug molecule.

[0076] This embodiment also introduces the drug molecule cyanine dye Cy5 to label the peptide. Unlike Example 2, the amino acid configuration in this embodiment is D-type. The structural formula of peptide fragment 1 in the drug delivery system of this embodiment is shown in Formulas 1-2, denoted as cRGD- AC The structure of r4-CPM, a polypeptide fragment 2 linked to a drug molecule, is shown in Formula 2-2 and is denoted as cRGD-r4-Cy5.

[0077]

[0078] Formula 1-2

[0079] Equation 2-2 cRGD- AC The synthesis method of r4-CPM is basically the same as that of cRGD in Example 2. AC The only difference with R4-CPM is that the amino acid raw materials with the corresponding configuration are selected according to Formula 1-2.

[0080] The synthesis method of cRGD-r4-Cy5 is basically the same as that of cRGD-R4-Cy5 in Example 2, except that the amino acid raw materials with the corresponding configuration are selected according to Formula 2-2.

[0081] This embodiment of cRGD- AC The activation and coupling method for r4-CPM and cRGD-r4-Cy5 is the same as in Example 2, that is, the two polypeptide fragments are dissolved in 10mM TCEP Tris buffer at pH 7.5 at a molar ratio of 1:1, and reacted for 30 min to obtain the R8 transmembrane peptide, denoted as R8. AC r4-r4-Cy5.

[0082] The cRGD- obtained in this embodiment AC See the mass spectrum of r4-CPM. Figure 6 The mass spectrum of the intermediate product r4-Cy5 (i.e., the product of step 4 in this embodiment) is shown in [reference needed]. Figure 13 For the mass spectrum of cRGD-r4-Cy5, please refer to [link / reference]. Figure 8 All of these studies confirmed the successful synthesis of the corresponding structural compounds.

[0083] Example 4 Drug delivery system (cRGD- AC Synthesis and activation of R4-CPM+cRGD-R4-CPT The drug delivery system includes peptide fragment 1 and peptide fragment 2 linked with a drug molecule.

[0084] Unlike Example 2, this example introduces the drug molecule camptothecin (CPT). The polypeptide fragment 1 of the drug delivery system in this example is the cRGD- obtained in Example 2. AC R4-CPM, the structure of the polypeptide fragment 2 linked to the drug molecule is shown in Formula 2-3, and is denoted as cRGD-R4-CPT.

[0085]

[0086] Equation 2-3 The synthesis method of cRGD-R4-CPT is as follows: (1) Synthesis of CR4K peptide by solid phase polypeptide synthesis method: same as step (1) of the synthesis process of cRGD-R4-Cy5 in Example 2.

[0087] (2) Removal of the Mtt protecting group from the side chain: Same as step (2) of the synthesis process of cRGD-R4-Cy5 in Example 2.

[0088] (3) Camptothecin-CPT-coupled peptide: Weigh 200 mg of CR4K resin (with Mtt protection removed) into a peptide reaction tube. Weigh CPT (117.5 mg, 2 eq.), PyBOP (136 mg, 2 eq.), HOBT (35 mg, 2 eq.), and DIPEA (67.7 mg, 4 eq.) and dissolve them in DMF. After complete dissolution, add the mixture to the peptide reaction tube and react under nitrogen bubbling in the dark at room temperature for 4 h. After the reaction, add DMF, DCM, and DMF in sequence to wash and dry the resin. After CPT coupling, remove the N-terminal protecting group as described above and continue coupling with a cysteine ​​residue (L-type) to obtain CR4K-CPT resin.

[0089] (4) The polypeptide is cleaved from the solid support: the same as step (4) of the synthesis process of cRGD-R4-Cy5 in Example 2, to obtain the R4-CPT polypeptide.

[0090] (5) Targeting peptide (cRGD) coupled with peptide R4-CPT: 100 mg of the cleaved R4-CPT peptide was weighed and dissolved in 5 ml of DMF, and 2,2'-dithiopyridine (33.8 mg, 2 eq.) was added and reacted overnight. After the reaction was completed, cRGD (44 mg, 1 eq.) was added and the reaction was continued for 4 h. Diethyl ether was added to precipitate the peptide, and the crude peptide was purified by HPLC.

[0091] The mass spectrum of cRGD-R4-CPT obtained in this embodiment can be found in [reference needed]. Figure 10 This confirmed the successful synthesis of the corresponding structural compound.

[0092] This embodiment of cRGD- AC The activation and coupling method for R4-CPM and cRGD-R4-CPT is the same as in Example 2. The two polypeptide fragments are dissolved in 10 mM TCEP Tris buffer at a molar ratio of 1:1 and reacted for 30 min to obtain the R8 transmembrane peptide. This is denoted as... AC R4-R4-CPT Example 5 Drug delivery system (cRGD- AC Synthesis and activation of r4-CPM+cRGD-r4-CPT The drug delivery system includes peptide fragment 1 and peptide fragment 2 linked with a drug molecule.

[0093] Unlike Example 3, this example introduces the drug molecule camptothecin (CPT). The polypeptide fragment 1 of the drug delivery system in this example is the cRGD- obtained in Example 3. AC The structure of r4-CPM, a polypeptide fragment 2 linked to a drug molecule, is shown in Formula 2-4 and is denoted as cRGD-r4-CPT.

[0094]

[0095] Equation 2-4 The synthetic route of cRGD-r4-CPT is basically the same as that of cRGD-R4-CPT in Example 4, except that the amino acid raw materials with the corresponding configuration are selected according to Formula 2-4.

[0096] The mass spectrum of cRGD-r4-CPT obtained in this embodiment can be found in [reference needed]. Figure 11 This confirmed the successful synthesis of the corresponding structural compound.

[0097] This embodiment of cRGD- AC The activation and coupling method for r4-CPM and cRGD-r4-CPT is the same as in Example 2. The two polypeptide fragments are dissolved in 10 mM TCEP Tris buffer at a molar ratio of 1:1 and reacted for 30 min to obtain the R8 transmembrane peptide. This is denoted as... AC r4-r4-CPT.

[0098] Compare with Example 1 Synthesis of R8-Cy5, r8-Cy5 and r8-CPT The structural formula of R8-Cy5 is shown in Equation 4-1, the structural formula of r8-Cy5 is shown in Equation 4-2, and the structural formula of r8-CPT is shown in Equation 4-3.

[0099] Equation 4-1

[0100] Equation 4-2

[0101] Equation 4-3 The synthesis method of R8-Cy5 is as follows: (1) Synthesis of R8 peptide by solid-phase peptide synthesis: Rink Amide-MBHA resin was swollen in N,N-dimethylformamide (DMF) for 20 min. The Fmoc protecting group was removed using a deprotection solution (piperazine:DMF = 8:2, v / v). The peptide chain was assembled by standard Fmoc solid-phase synthesis, with each amino acid coupled twice in DMF under nitrogen atmosphere using HATU / DIPEA as the coupling system for 1 hour each time.

[0102] (2) N-terminal Fmoc removal: After the peptide sequence is synthesized, weigh 200 mg of R8 resin and place it in the peptide reaction tube. Add 10 ml of DMF to swell for 5 min. After removing the filtered DMF, add 2 ml of deprotection solution to remove the Fmoc protecting group. After completion, add DMF, DCM and DMF to wash the resin in sequence.

[0103] (3) Coupling Cy5: Weigh 200 mg of R8 resin that has been deprotected from Fmoc and place it in a peptide reaction tube. Weigh Cy5 (158.1 mg, 2 eq.), PyBOP (136 mg, 2 eq.), HOBT (35 mg, 2 eq.), and DIPEA (67.7 mg, 4 eq.) and dissolve them in DMF. After the mixture is fully dissolved, add it to the peptide reaction tube and react with nitrogen bubbling at room temperature in the dark for 4 h. After the reaction is completed, add DMF, DCM, and DMF in sequence to wash and dry the resin.

[0104] (4) The polypeptide is cleaved from the solid support: the same cleavage method as in Example 2.

[0105] The synthesis method of r8-Cy5 is as follows: (1) Synthesis of r8 peptide by solid-phase peptide synthesis: Rink Amide-MBHA resin was swollen in N,N-dimethylformamide (DMF) for 20 min. The Fmoc protecting group was removed using a deprotection solution (piperazine:DMF = 8:2, v / v). The peptide chain was assembled by standard Fmoc solid-phase synthesis, with each amino acid coupled twice in DMF under nitrogen atmosphere using HATU / DIPEA as the coupling system.

[0106] (2) N-terminal Fmoc removal: After the peptide sequence is synthesized, weigh 200 mg of r8 resin and place it in the peptide reaction tube. Add 10 ml of DMF to swell for 5 min. After removing the filtered DMF, add 2 ml of deprotection solution to remove the Fmoc protecting group. After completion, add DMF, DCM and DMF to wash the resin in sequence.

[0107] (3) Coupling Cy5: Weigh 200 mg of R8 resin that has been deprotected from Fmoc and place it in a peptide reaction tube. Weigh Cy5 (158.1 mg, 2 eq.), PyBOP (136 mg, 2 eq.), HOBT (35 mg, 2 eq.), and DIPEA (67.7 mg, 4 eq.) and dissolve them in DMF. After the mixture is fully dissolved, add it to the peptide reaction tube and react with nitrogen bubbling at room temperature in the dark for 4 h. After the reaction is completed, add DMF, DCM, and DMF in sequence to wash and dry the resin.

[0108] (4) The polypeptide is cleaved from the solid support: the same cleavage method as in Example 2.

[0109] The synthesis method of r8-CPT is as follows: (1) Synthesis of r8 peptide by solid-phase peptide synthesis: Rink Amide-MBHA resin was swollen in N,N-dimethylformamide (DMF) for 20 min. The Fmoc protecting group was removed using a deprotection solution (piperazine:DMF = 8:2, v / v). The peptide chain was assembled by standard Fmoc solid-phase synthesis, with each amino acid coupled twice in DMF under nitrogen atmosphere using HATU / DIPEA as the coupling system.

[0110] (2) N-terminal Fmoc removal: After the peptide sequence is synthesized, weigh 200 mg of r8 resin and place it in the peptide reaction tube. Add 10 ml of DMF to swell for 5 min. After removing the filtered DMF, add 2 ml of deprotection solution to remove the Fmoc protecting group. After completion, add DMF, DCM and DMF to wash the resin in sequence.

[0111] (3) Coupling with camptothecin CPT: Weigh 200 mg of deprotected R8 resin and place it in a polypeptide reaction tube. Weigh CPT (117.5 mg, 2 eq.), PyBOP (136 mg, 2 eq.), HOBT (35 mg, 2 eq.), and DIPEA (67.7 mg, 4 eq.) and dissolve them in DMF. After complete dissolution, add the mixture to the polypeptide reaction tube and react under nitrogen bubbling in the dark at room temperature for 4 h. After the reaction is completed, add DMF, DCM, and DMF in sequence to wash and dry the resin.

[0112] (4) The polypeptide is cleaved from the solid support: the same cleavage method as in Example 2.

[0113] See the mass spectrum of R8-Cy5. Figure 14 For the mass spectrum of r8-Cy5, see [link to mass spectrum]. Figure 15 For the mass spectrum of r8-CPT, please refer to [link / reference]. Figure 16 All of these studies confirmed the successful synthesis of the corresponding structural compounds.

[0114] Experimental Example 1 Cell penetration ability experiment To assess the ability of cells to take up peptide fragments, this study selected mouse melanoma cells (B16F10) as an in vitro uptake model and systematically analyzed the cellular uptake behavior of various Cy5-labeled peptides.

[0115] Experimental Methods: B16F10 cells were evenly seeded in 24-well plates and cultured at 37℃, 5% CO2, and 90% humidity for 24 h. The next day, the culture medium was discarded, and a peptide solution prepared with serum-free medium was added at a concentration of 10 μM to treat the cells for 6 h. The cells were washed three times with PBS buffer, and fixed with 4% paraformaldehyde solution at room temperature for 20 min. The liquid in the wells was aspirated, and the cells were washed three times with PBS buffer. 200 μL of DAPI solution was added to each well, and the cells were stained at room temperature for 20 min to stain the cell nuclei. The liquid in the wells was aspirated, and the cells were washed three times with PBS buffer. The uptake of peptides by cells was observed using a fluorescence microscope while maintaining constant exposure time and fluorescence intensity.

[0116] Experimental results: such as Figure 18 As shown, after incubating cells for 6 h, R8-Cy5 showed strong fluorescence, which is due to the presence of 8 positive charges that facilitate cell uptake and produce red fluorescence. R4-Cy5, due to insufficient positive charge, was difficult for cells to take up and only showed weak fluorescence. R4-Cy5 coupled with RGD peptide showed strong fluorescence because RGD can target the integrin αvβ3 receptor. After pretreating cells with RGD for 1 h, no obvious fluorescence was observed, which proves that R4-Cy5 coupled with RGD peptide is taken up by cells through RGD targeting ability.

[0117] Experiment Example 2 Cell deep penetration research To investigate the ability of conjugated peptide fragments to penetrate multiple cell layers, we performed a Transwell assay. Activated transmembrane peptides, after in situ conjugation within cells, generate the R8 transmembrane peptide, which possesses stronger penetrating power and can therefore penetrate multiple cell layers.

[0118] Experimental Methods: B16F10 cells were seeded in 12-well Transwell chambers and 12-well plates and cultured at 37°C, 5% CO2, and 90% humidity for 24 h. The culture medium in the chambers was discarded, and a polypeptide solution prepared with 10 μM serum-free medium was added and incubated for 6 h. The culture medium in the chambers was discarded, and the cells were washed three times with PBS buffer. The culture medium in the plates was discarded, and the cells were washed three times with PBS buffer. FBS-free medium was added to the plates, and the cells in the chambers and plates were co-incubated for 24 h. The culture medium in the plates was discarded, and the cells were washed three times with PBS buffer. Cells were fixed with 4% paraformaldehyde for 20 min. 400 μL of DAPI solution was added to each well, and staining was performed at room temperature for 20 min. The liquid in the wells was aspirated, and the cells were washed three times with PBS buffer.

[0119] Experimental results: such as Figure 19 As shown in the Transwell cell penetration assay, the activatable transmembrane peptides can be coupled intracellularly to generate the R8 transmembrane peptide, which has stronger penetrating ability, and can penetrate from one cell layer to the next. The excess penetrating ability of the L and D conformations of the peptides has little impact; R4-Cy5 cannot be taken up by cells because it lacks an RGD-targeting peptide; although R8-Cy5 can be taken up by cells, it cannot penetrate from one cell layer to the next.

[0120] Experimental Example 3 Cytotoxicity assay To investigate the cytotoxicity of peptide fragments and explore the optimal working concentration of peptide fragments, different peptide concentration gradients were used in the study.

[0121] Experimental Methods: 100 μL of 5kJ / well was seeded into 96-well plates and cultured for 24 h at 37℃, 5% CO2, and 90% humidity. Different concentration gradients of peptide solutions (3 μM, 6 μM, 12 μM, 25 μM, 50 μM, 100 μM, 200 μM) were prepared, with three replicates for each concentration, and cultured for 24 h at 37℃, 5% CO2, and 90% humidity. 100 μL of 10% CCK-8 solution was added to each well, and three blank wells (containing only 100 μL of 10% CCK-8 solution) were prepared and incubated for another 1 h. The absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated as [(OD experimental wells - OD blank wells) / (OD negative control wells - OD blank wells)] × 100%.

[0122] Experimental results: such as Figure 20 As shown, the cytotoxicity of the peptide sequence R8-Cy5 increased with increasing incubation concentration. At a concentration of 100 μM, the survival rate of B16F10 cells was 36.58%, which is attributed to the fact that R8 contains eight positively charged groups that disrupt cell membrane stability. Peptide fragment 2 (cRGD-R4-Cy5) did not produce significant cytotoxicity in B16F10 cells at a concentration of 100 μM. The conjugation of the two peptide fragments also did not produce significant cytotoxicity in B16F10 cells at a concentration of 100 μM, demonstrating that the peptide fragments have good biocompatibility. At the same time, splitting R8 into two functionalized R4 fragments greatly reduced cytotoxicity. Experiment Example 4 In vivo drug metabolism and distribution experiments that can activate membrane-penetrating peptides After validating the potential of activatable transmembrane peptides, the next question arises: how to ensure that these peptides precisely reach the tumor site for deep penetration. Given that many peptides remain in the mouse tail or fail to effectively enter the bloodstream after tail vein injection, developing delivery systems with tumor-targeting capabilities is crucial.

[0123] To investigate the tumor enrichment capacity of the activating transmembrane peptide after tail vein injection and its in vivo drug metabolism and distribution, we injected a Cy5-labeled peptide (100 μM, 200 μL) into C57BL / 6 mice via tail vein. In vivo imaging with IVScope 8000M was performed on anesthetized mice at different time points post-injection. Mice were sacrificed 24 h after injection, and ex vivo tissues were collected for fluorescence imaging.

[0124] Experimental methods: 1. Subcutaneously inject healthy B16F10 cells into the left axilla of nude mice (5.0 × 10⁵ cells per mouse, 100 μL DMEM serum-free medium).

[0125] 2. Observe the condition of the mice and the growth of the tumor. When the tumor volume reaches approximately 50 mm... 3 At that time, all tumor-bearing mice were divided into five groups: r8-Cy5, r4-Cy5, cRGD-r4-Cy5, cRGD- AC r4-CPM+cRGD-r4-Cy5, Cy5, 4 animals in each group, there was no significant difference in tumor volume among the groups (p>0.05).

[0126] 3. In vivo imaging was performed in B16F10 tumor-bearing mice. Five groups of tumor-bearing mice were injected once via tail vein with Cy5, r4-Cy5, r8-Cy5, peptide fragment 2 (cRGD-r4-Cy5), or peptide fragment 1 + peptide fragment 2 (cRGD- AC (r4-CPM+cRGD-r4-Cy5), and then the fluorescence signal intensity of the mice was observed and recorded by a fluorescence imaging system at 10 min, 1 h, 6 h, 12 h and 24 h.

[0127] Experimental results: such as Figure 21 As shown on the left, r4-Cy5 exhibits relatively low cellular uptake efficiency due to insufficient positive charge; while r8-Cy5 effectively enters cells, its distribution in vivo is highly nonspecific due to a lack of targeting, especially showing significant accumulation at injection sites (such as the tail). Peptide fragment 2 (cRGD-r4-Cy5), with the aid of a conjugated RGD targeting peptide, can specifically accumulate in tumor regions, but only a weak fluorescence signal was detected 12 hours after administration, indicating limited tumor retention capacity. In contrast, peptide fragment 1 (cRGD- ACAfter in situ conjugation of r4-CPM and peptide fragment 2 (cRGD-r4-Cy5), a significantly enhanced tumor penetration and retention effect was observed: strong fluorescence signals in the tumor area could be observed as early as 6 hours after injection, and the signals could still be detected up to 24 hours later. This result confirms that this conjugation strategy can effectively enhance the accumulation and retention of the probe in tumor tissue. After 24 hours of in vivo imaging, mice in each group were sacrificed and their major organs and tumor tissues were collected for ex vivo fluorescence imaging, as shown in Figure 6 (right). Peptide fragment 1 (cRGD-r4-Cy5) showed a significantly enhanced tumor penetration and retention effect: strong fluorescence signals in the tumor area could be observed as early as 6 hours after injection, and the signals could still be detected up to 24 hours later. AC The fluorescence intensity of peptide fragment 2 (cRGD-r4-Cy5) in tumor tissue after in situ conjugation with r4-CPM was higher than that of the control group, consistent with in vivo imaging results. Furthermore, fluorescence signals were also observed in the liver and kidneys, indicating that the peptides can be metabolized by the liver and kidneys. These results suggest that in situ conjugation of the peptides can achieve persistent enrichment in the tumor region, allowing for prolonged treatment after a single injection, thus enhancing therapeutic efficacy.

[0128] These results show that the activatable transmembrane peptide not only possesses excellent tumor-targeting ability but also achieves deep penetration at the tumor level. We infer that this characteristic can still be attributed to the high efficiency of in situ conjugation, avoiding off-target effects.

[0129] 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, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A membrane-penetrating peptide that can be activated, characterized in that, Includes peptide fragment 1 and peptide fragment 2; The polypeptide fragment 1 includes a peptide chain 1, the N-terminus of which is acetylated and linked with a cyclic peptide RGD, and the peptide chain 1 is also linked with a cyano group. The polypeptide fragment 2 includes a peptide chain 2, the N-terminus of which is also connected to a cyclic peptide RGD, and the peptide chain 2 contains cysteine. The polypeptide fragment 1 and the polypeptide fragment 2 can be reactively coupled under activation conditions to form a membrane-penetrating peptide with cell-penetrating ability.

2. The membrane-penetrating peptide as described in claim 1, characterized in that, The activation condition is an alkaline reaction environment.

3. The membrane-penetrating peptide as described in claim 1 or 2, characterized in that, The sequence of peptide chain 1 is CWRRRRK, and the sequence of peptide chain 2 is CRRRRK.

4. The membrane-penetrating peptide as described in claim 3, characterized in that, The structural formula of polypeptide fragment 1 is shown in Formula 1 or Formula 2, and the structural formula of polypeptide fragment 2 is shown in Formula 3 or Formula 4. Formula 1 Formula 2 Formula 3 Formula 4 Wherein, R1 is selected from , , , , , , , , , Any one of the following.

5. The membrane-penetrating peptide as described in claim 3, characterized in that, The structural formula of the polypeptide fragment 1 is shown in Formula 5 or Formula 6: Formula 5 Formula 6.

6. The method for synthesizing the membrane-penetrating peptide as described in any one of claims 1 to 5, characterized in that, The method for synthesizing polypeptide fragment 1 includes the following steps: firstly, polypeptide chain 1 is synthesized by solid-phase polypeptide synthesis, then a cyano group is attached to polypeptide chain 1 by SN2 nucleophilic substitution reaction, and a cyclic peptide RGD is attached to polypeptide chain 1 by disulfide bond exchange reaction. The method for synthesizing polypeptide fragment 2 includes the following steps: firstly, polypeptide chain 2 is synthesized by solid-phase polypeptide synthesis, and then the cyclic peptide RGD is linked to polypeptide chain 2 by disulfide bond exchange reaction.

7. A drug delivery method, characterized in that, The method utilizes the membrane-penetrating peptide as described in any one of claims 1 to 5, comprising the following steps: (1) During the synthesis of the polypeptide fragment 2, drug molecules are attached to the polypeptide fragment 2; (2) The polypeptide fragment 1 and the polypeptide fragment 2 linked with the drug molecule are simultaneously delivered into the diseased tissue. The polypeptide fragment 1 and the polypeptide fragment 2 react and couple in the diseased tissue environment to form a membrane-penetrating peptide with cell-penetrating ability and carry the drug molecule into the diseased cell.

8. The use of the membrane-penetrating peptide as described in any one of claims 1 to 5 in the preparation of a delivery system for delivering a drug into cells.

9. A drug delivery system, characterized in that, It includes an activatable membrane-penetrating peptide as described in any one of claims 1 to 5 and a drug molecule, wherein the drug molecule is attached to the polypeptide fragment 2 of the activatable membrane-penetrating peptide.

10. The drug delivery system as claimed in claim 9, characterized in that, The structural formula of the polypeptide fragment 2 linked with the drug molecule is shown in Formula 7 or Formula 8: Formula 7 Formula 8 Wherein, R2 is the drug molecule, which is selected from any one of cyanine dye Cy5, camptothecin, paclitaxel, doxorubicin, oxaliplatin, protein degradation targeting chimera, and gemcitabine.

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