Short peptide-sound-sensitive agent conjugate capable of destroying bacterial cell membrane as well as preparation method and application of short peptide-sound-sensitive agent conjugate

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an oligopeptide-sound-sensitive agent conjugate capable of destroying bacterial cell membranes as well as a preparation method and application of the oligopeptide-sound-sensitive agent conjugate. The oligopeptide-sound sensitive agent conjugate provided by the invention is formed by coupling a sound sensitive agent and oligopeptide through an amido bond. The conjugate has double antibacterial mechanisms: on one hand, the structure of the conjugate can destroy bacterial cell membranes to cause bacterial structure damage and content leakage; on the other hand, active oxygen can be efficiently generated under ultrasonic excitation, and the killing effect on bacteria is further enhanced. Experiments show that the conjugate has remarkable antibacterial activity on gram-negative bacteria. In addition, the conjugate also shows a good inhibition effect on colon tumor cells. The oligopeptide-sonosensitizer conjugate provided by the invention has potential industrial value in preparation of antibacterial and anti-tumor materials.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a short peptide-sound sensitizer conjugate that disrupts bacterial cell membranes, its preparation method, and its application. Background Technology

[0002] Pathogenic bacterial infections pose a serious threat to global health care. Despite various treatment options, including antibiotics, the number of deaths from bacterial infections continues to rise annually, and the development of antibiotics lags significantly behind the rapid evolution of antibiotic resistance. Therefore, developing strategies for treating bacterial infections has significant social implications.

[0003] In recent years, researchers have increasingly focused on the killing of microorganisms through exogenous stimulation therapy. These therapies are suitable for treating multidrug-resistant bacterial infections due to their non-invasiveness, spatiotemporal controllability, and low likelihood of inducing drug resistance. Among them, ultrasound, with its excellent tissue penetration (approximately 10 cm), can reach deep organs with minimal energy attenuation. It can utilize the highly toxic reactive oxygen species (ROS) generated by sonosensitive agents under ultrasound to eliminate bacteria. Therefore, sonodynamic therapy has been adopted as a mature antibacterial treatment strategy.

[0004] While sonodynamic therapy shows promise in treating bacterial infections, most sonosensitive agents are only effective against Gram-positive bacteria, with limited efficacy against Gram-negative bacteria. This limitation stems from the structural differences between Gram-positive and Gram-negative bacteria. Both have a phospholipid inner membrane, but Gram-negative bacteria possess a unique outer membrane structure composed of tightly packed lipopolysaccharides, which acts as a strong permeation barrier, preventing sonosensitive agent molecules from penetrating the interior of Gram-negative bacteria. Furthermore, the extremely short lifetime (3.5 μs) and narrow diffusion radius (<0.3 μm) of reactive oxygen species (ROS) prevent sonodynamic therapy from damaging Gram-negative bacteria. Therefore, researchers urgently seek to develop sonosensitive agents that can effectively disrupt the outer membrane of Gram-negative bacteria, generating ROS within the bacteria to enhance antibacterial efficiency. Summary of the Invention

[0005] The first technical problem to be solved by the present invention is to provide a short peptide-sonic sensitizer conjugate that disrupts bacterial cell membranes, so as to overcome the shortcomings of existing sonodynamic therapy in killing Gram-negative bacteria.

[0006] The second technical problem to be solved by the present invention is to provide a method for preparing the short peptide-sound-sensitizer conjugate.

[0007] The third technical problem to be solved by the present invention is to provide the application of the short peptide-sound-sensitizer conjugate.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] A short peptide-sound sensitizer conjugate that disrupts bacterial cell membranes, wherein the short peptide-sound sensitizer conjugate is prepared by coupling the carboxyl group of a sound sensitizer to the amino group at the N-terminus of a short peptide via an amide bond; The structure of the sound-sensitive agent is shown in Formula I: (I); The amino acid sequence of the short peptide is any one of NH2-WWWR-CONH2, NH2-WWRR-CONH2, NH2-WRRR-CONH2 or NH2-RRRR-CONH2, preferably NH2-RRRR-CONH2.

[0010] In some embodiments, the preparation method of the short peptide-sound-sensitizer conjugate includes the following steps: (1) Solid-phase synthesis of short peptide resin: Using Rink resin as a carrier, the Fmoc solid-phase peptide synthesis method is adopted to sequentially link amino acids according to the amino acid sequence of the short peptide to obtain short peptide resin. (2) Coupling of the sound-sensing agent: The short peptide resin, sound-sensing agent, condensing agent and organic base obtained in step (1) are dissolved in an organic solvent to carry out an amide condensation reaction. After the reaction is completed, the resin is washed and dried. (3) Pyrolysis and purification: Pyrolysis agent is added to the resin obtained in step (2) for pyrolysis. After solid-liquid separation, precipitation, purification and freeze drying, the short peptide-sound sensitizer conjugate is obtained.

[0011] In some embodiments, step (1) specifically includes the following steps: (1a) After swelling and washing the Rink amide resin, the Fmoc protecting group was removed, and the resin was obtained after washing. (1b) The short peptide resin is obtained by solid-phase synthesis according to the amino acid sequence of the short peptide. Specifically, the single Fmoc-protected amino acid, condensing agent, organic base and the resin are dissolved in DMF to carry out amide condensation reaction. After the reaction is completed, the resin is washed to obtain a resin with Fmoc-protected amino acid. Then, the Fmoc protecting group is removed and the resin is washed.

[0012] In some embodiments, in step (2), the condensing agent is any one of benzotriazole-1-yl-oxytripyrrolidine phosphorus hexafluorophosphate, (7-azabenzotriazole-1-oxy)tripyrrolidine phosphorus hexafluorophosphate, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, O-benzotriazole-tetramethylurea hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid, N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate, N,N'-diisopropylcarbodiimide, or N,N'-dicyclohexylcarbodiimide; the organic base is any one of N,N-diisopropylethylamine, triethylamine, 4-dimethylaminopyridine, or N-methylimidazole; and / or, the organic solvent is N,N-dimethylformamide.

[0013] In some embodiments, in step (2), the amount of the sound-sensitive agent and the condensing agent is 1 to 4 equivalents of the number of moles of the reactive amino groups at the top end of the short peptide resin, and the amount of the organic base is 5 to 50 equivalents of the number of moles of the reactive amino groups at the top end of the short peptide resin; and / or, the amide condensation reaction is carried out at a temperature of 0 to 40 °C for a time of 1 to 12 h; and / or, the washing is carried out by washing with N,N-dimethylformamide 2 to 5 times, followed by washing with methanol 2 to 3 times.

[0014] In some embodiments, in step (3), the pyrolysis agent is a mixed solution of trifluoroacetic acid, phenol and water, with a volume ratio of 85~95:2.5~7.5:2.5~7.5; and / or, the amount of pyrolysis agent added is calculated based on the mass-volume ratio of the Rink resin to the pyrolysis agent in step (1) being 1g:10~30 mL; and / or, the pyrolysis is carried out at a temperature of 0~40 ℃ for a time of 1~4 h.

[0015] In some embodiments, in step (3), the solid-liquid separation is filtration; the precipitation is performed using diethyl ether, wherein the volume ratio of diethyl ether to the pyrolysis agent is 1:5~20; and / or, the purification is performed using high performance liquid chromatography; and / or, the freeze drying is performed at a temperature of -20~-80 ℃, a pressure of 0.1~50 Pa, and a time of 12~48 h.

[0016] In some embodiments, the use of the short peptide-sound-sensitizing agent conjugate in the preparation of medicaments for the prevention and / or treatment of bacterial infections is also within the scope of protection of this invention; and / or, the bacterial infection is caused by Gram-positive and / or Gram-negative bacteria, preferably Staphylococcus aureus and / or Staphylococcus epidermidis and / or methicillin-resistant Staphylococcus aureus and / or Pseudomonas aeruginosa and / or Escherichia coli and / or multidrug-resistant Escherichia coli and / or anti-nucleated Fusobacterium nucleatum, further preferably Gram-negative bacteria, and most preferably Fusobacterium nucleatum.

[0017] In some embodiments, the short peptide-sound sensitizer conjugate exerts its antibacterial effect by disrupting the bacterial cell membrane.

[0018] In some embodiments, the use of the short peptide-sound sensitizer conjugate in the preparation of antitumor drugs is also within the scope of protection of this invention; the tumor is preferably a colon tumor, and most preferably CT26 cells.

[0019] Beneficial effects:

[0020] (1) Currently, sonodynamic therapy kills bacteria by generating a large amount of ROS under the action of ultrasound using sonosensitive agents. However, most sonosensitive agents are only effective against Gram-positive bacteria and have limited therapeutic effects on Gram-negative bacteria with unique outer membranes. Under the same conditions, the short peptide-sonic sensitive agent conjugate provided by this invention has a much higher effect on destroying bacterial cell membranes than traditional sonosensitive agents. It can penetrate into the interior of Gram-negative bacteria and generate a large amount of ROS under the action of ultrasound, which can be used for highly effective antibacterial treatment.

[0021] (2) The short peptide-sound sensitizer conjugate (I) provided by the present invention has potential industrial value in preparation as an antibacterial drug.

[0022] (3) The short peptide-sound sensitizer conjugate developed in this invention has a significant inhibitory effect on tumor cells under ultrasound and can be used efficiently for anti-tumor treatment.

[0023] (4) The short peptide-sound sensitizer conjugate provided by this invention has broad application prospects. In the medical field, it is expected to be developed into a new drug to combat drug-resistant bacterial infections and related inflammations; in the cosmetic field, this technology provides a new and efficient product solution for the precise treatment of bacterial infectious skin diseases such as acne. Attached Figure Description

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0025] Figure 1 The UV-Vis absorption spectrum of the short peptide-sound sensitizer conjugate;

[0026] Figure 2 The fluorescence emission spectrum of the short peptide-sound sensitizer conjugate;

[0027] Figure 3 Characterization of the reactive oxygen species generation capacity of short peptide-sound sensitizer conjugates;

[0028] Figure 4 The change in PI fluorescence intensity of Fusobacterium nucleatum after ultrasonic treatment with a short peptide-sound sensitizer conjugate;

[0029] Figure 5 OD of the supernatant of *Fusobacterium nucleatum* after sonication with a short peptide-sound-sensitizing agent conjugate 260 Value change;

[0030] Figure 6 OD of Fusobacterium nucleatum after ultrasonic treatment with short peptide-sound-sensitizing agent conjugate 600 Value change;

[0031] Figure 7 This diagram illustrates the inhibition of tumor cell activity by a short peptide-sound sensitizer conjugate. Detailed Implementation

[0032] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0033] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0034] The short peptide-sound sensitizer conjugate provided by this invention is structurally designed to mimic amphiphilic antibacterial lipopeptides. By amide condensation, the amino group of the hydrophilic short peptide is linked to the carboxyl group of the hydrophobic sound sensitizer, thereby optimizing the short peptide-sound sensitizer conjugate with highly efficient ability to disrupt bacterial cell membranes, achieving highly efficient sonodynamic therapy against bacteria.

[0035] The acoustic sensitizer used in this invention was purchased from Shanghai Dibai Biotechnology Co., Ltd., CAS: 24533-72-0.

[0036] Example 1: Short peptide-sound sensitizer conjugate PSC 1R Preparation

[0037] (1) Weigh 200 mg of Rink Amide Resin (200 mesh) with a loading of 1 mmol / g (corresponding to a theoretical loading of 0.2 mmol), place it in a peptide synthesis reaction tube, add dichloromethane (20 mL / g resin) to swell, place it in a shaker and shake at room temperature for 2 h, then wash the resin with N,N-dimethylformamide (DMF, 10 mL each time, 3 times in total); add DMF solution containing 20% ​​piperidine (10 mL) to the resin, place it in a shaker and react at room temperature for 20 min to remove the Fmoc protecting group, wash the resin with DMF (15 mL each time, 5 times in total), take a small amount of resin for ninhydrin test to confirm complete deprotection; add Fmoc-Arg(pbf)-OH (0.4 mmol, 259.6 mg), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.4 mmol, 208.2 mg) and N,N-diisopropylethylamine (4 Fmoc-Trp(boc)-OH (0.4 mmol, 516.96 mg) was dissolved in DMF and added to the resin. The mixture was placed in a shaker and reacted at room temperature for 2 hours to carry out the amide condensation reaction. After the reaction, the resin was washed with DMF (15 mL each time, 3 times in total). Then, 10 mL of DMF solution containing 20% ​​piperidine was added to the resin, and the mixture was placed in a shaker and reacted at room temperature for 20 minutes to remove the Fmoc protecting group. The resin was washed with DMF (15 mL each time, 5 times in total). A small amount of resin was taken for ninhydrin test to confirm complete deprotection. Then, Fmoc-Trp(boc)-OH (0.4 mmol, 210.6 mg), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.4 mmol, 208.2 mg), and N,N-diisopropylethylamine (4 mmol, 516.96 mg) were dissolved in DMF and added to the resin. The mixture was placed in a shaker and reacted at room temperature for 2 hours to carry out the amide condensation reaction of the second amino acid. After the reaction, the resin was washed with DMF (15 mL each time, 5 times in total). The resin was washed with DMF solution (10 mL, 3 times in total) and then 20 mL of piperidine was added to the resin. The mixture was placed in a shaker and reacted at room temperature for 20 min to remove the Fmoc protecting group. The resin was washed with DMF (15 mL each time, 5 times in total). A small amount of resin was taken for ninhydrin test to confirm complete deprotection. The amino acid coupling and deprotection operations were performed sequentially from C-terminus to N-terminus (Arg → Trp → Trp → Trp) to finally synthesize the short peptide chain NH2-WWWR-CONH2 on the resin.

[0038] (2) Short peptide-sound sensitizer conjugate PSC 1RSynthesis: A sonic sensitizer (0.4 mmol, 213.9 mg), benzotriazol-1-yl-oxytripyrrolidine phosphorus hexafluorophosphate (0.4 mmol, 208.2 mg), and N,N-diisopropylethylamine (4 mmol, 516.96 mg) were added to the Rink resin connected to NH2-WWWR-CONH2 and dissolved in DMF. The mixture was placed in a shaker and reacted at room temperature for 2 h to carry out the amide condensation reaction. After the reaction was completed, the resin was washed with N,N-dimethylformamide (5 times), washed twice with methanol, and then dried under vacuum.

[0039] (3) Short peptide-sound sensitizer conjugate PSC 1R Purification: A mixed lysis reagent of trifluoroacetic acid, phenol, and water (volume ratio 90:5:5) (15 mL / g resin) was added to the dried resin and the mixture was shaken at room temperature for 3 h. After the reaction was completed, the short peptide-sound sensitizer conjugate PSC was added. 1R Separate from the resin by filtration, and precipitate the pyrolysis solution using diethyl ether (added at 10 times the amount of pyrolysis agent) to obtain PSC. 1R Solid precipitate was obtained by preparative HPLC (solvents: water and acetonitrile, both containing 0.1% trifluoroacetic acid) for PSC. 1R After purification, freeze-drying (pressure 50 Pa, temperature -78 °C, time 24 h) yielded a blackish-green solid PSC. 1R The yield was 61.6%. MALDI-TOF MS: Calculated value C 72 H 77 N 15 O6[M+H] + : 1248.51, test value 1248.37.

[0040] Example 2: Short peptide-sound sensitizer conjugate PSC 2R Preparation

[0041] (1) Weigh 200 mg of Rink Amide Resin (200 mesh) with a loading of 1 mmol / g (corresponding to a theoretical loading of 0.2 mmol), place it in a peptide synthesis reaction tube, add dichloromethane (20 mL / g resin) to swell, place it in a shaker and shake at room temperature for 2 h, then wash the resin with N,N-dimethylformamide (DMF, 15 mL each time, 3 times in total); add DMF solution (10 mL) containing 20% ​​piperidine to the resin, place it in a shaker and react at room temperature for 20 min to remove the Fmoc protecting group, wash the resin with DMF (15 mL each time, 5 times in total), take a small amount of resin for ninhydrin test to confirm complete deprotection; add Fmoc-Arg(pbf)-OH (0.4 mmol, 259.6 mg), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.4 mmol, 208.2 mg) and N,N-diisopropylethylamine (4 Fmoc-Arg(pbf)-OH (0.4 mmol, 516.96 mg) was dissolved in DMF and added to the resin. The mixture was placed in a shaker and reacted at room temperature for 2 hours to carry out the amide condensation reaction. After the reaction, the resin was washed with DMF (15 mL each time, 3 times in total). Then, 10 mL of DMF solution containing 20% ​​piperidine was added to the resin, and the mixture was placed in a shaker and reacted at room temperature for 20 minutes to remove the Fmoc protecting group. The resin was washed with DMF (15 mL each time, 5 times in total). A small amount of resin was taken for ninhydrin test to confirm complete deprotection. Then, Fmoc-Arg(pbf)-OH (0.4 mmol, 259.6 mg), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.4 mmol, 208.2 mg), and N,N-diisopropylethylamine (4 mmol, 516.96 mg) were dissolved in DMF and added to the resin. The mixture was placed in a shaker and reacted at room temperature for 2 hours to carry out the amide condensation reaction of the second amino acid. After the reaction, the resin was washed with DMF (15 mL each time, 5 times in total). The resin was washed with DMF solution (10 mL, 3 times in total) and then 20 mL of piperidine was added to the resin. The mixture was placed in a shaker and reacted at room temperature for 20 min to remove the Fmoc protecting group. The resin was washed with DMF (15 mL each time, 5 times in total). A small amount of resin was taken for ninhydrin test to confirm complete deprotection. The amino acid coupling and deprotection operations were performed sequentially from C-terminus to N-terminus (Arg → Arg → Trp → Trp) to finally synthesize the short peptide chain NH2-WWRR-CONH2 on the resin.

[0042] (2) Short peptide-sound sensitizer conjugate PSC 2RSynthesis: A sonic sensitizer (0.4 mmol, 213.9 mg), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.4 mmol, 208.2 mg), and N,N-diisopropylethylamine (4 mmol, 516.96 mg) were added to the Rink resin connected to NH2-WWRR-CONH2 and dissolved in DMF. The mixture was placed in a shaker and reacted at room temperature for 2 h to carry out the amide condensation reaction. After the reaction was completed, the resin was washed with N,N-dimethylformamide (5 times), washed twice with methanol, and then dried under vacuum.

[0043] (3) Short peptide-sound sensitizer conjugate PSC 2R Purification: A mixed lysis reagent of trifluoroacetic acid, phenol, and water (volume ratio 90:5:5) (15 mL / g resin) was added to the dried resin and the mixture was shaken at room temperature for 3 h. After the reaction, the short peptide-sound sensitizer conjugate PSC1R was separated from the resin by filtration, and the lysis buffer was settled using diethyl ether (10 times the amount of lysis reagent) to obtain PSC. 2R Solid precipitate was obtained by preparative HPLC (solvents: water and acetonitrile, both containing 0.1% trifluoroacetic acid) for PSC. 2R After purification, freeze-drying (pressure 50 Pa, temperature -78 °C, time 24 h) yielded a blackish-green solid PSC. 2R The yield was 55.4%. MALDI-TOF MS: Calculated value C 67 H 79 N 17 O6[M+H] + : 1218.48, test value 1218.76.

[0044] Example 3: Short peptide-sound sensitizer conjugate PSC 3R Preparation

[0045] (1) Synthesis of the short peptide NH2-WRRR-CONH2: 200 mg of Rink Amide Resin (200 mesh) with a loading of 1 mmol / g (corresponding to a theoretical loading of 0.2 mmol) was weighed and placed in a peptide synthesis reaction tube. Dichloromethane (20 mL / g resin) was added to swell the resin, and the mixture was shaken at room temperature for 2 h. The resin was then washed with N,N-dimethylformamide (DMF, 15 mL each time, 3 times in total). A DMF solution containing 20% ​​piperidine (10 mL) was added to the resin, and the mixture was reacted at room temperature for 20 min to remove the Fmoc protecting group. The resin was washed with DMF (15 mL each time, 5 times in total). A small amount of resin was tested for ninhydrin to confirm complete deprotection. Fmoc-Arg(pbf)-OH (0.4 mmol, 259.6 mg) and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.4 mmol, 208.2 mg) were added to the resin. Fmoc-Arg(pbf)-OH (0.4 mmol, 259.6 mg), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.4 mmol, 208.2 mg), and N,N-diisopropylethylamine (4 mmol, 516.96 mg) were dissolved in DMF and added to the resin. The mixture was placed in a shaker and reacted at room temperature for 2 h to carry out the amide condensation reaction. After the reaction, the resin was washed with DMF (15 mL each time, 3 times). Then, a DMF solution containing 20% ​​piperidine (10 mL) was added to the resin, and the mixture was placed in a shaker and reacted at room temperature for 20 min to remove the Fmoc protecting group. The resin was washed with DMF (15 mL each time, 5 times). A small amount of resin was taken for ninhydrin testing to confirm complete deprotection. Then, Fmoc-Arg(pbf)-OH (0.4 mmol, 259.6 mg), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.4 mmol, 208.2 mg), and N,N-diisopropylethylamine (4 mmol, 516.96 mg) were added to the resin. The Fmoc protecting group was removed by dissolving a 15 mL DMF solution in DMF and adding it to the resin. The mixture was then placed in a shaker and reacted at room temperature for 2 hours to carry out the amide condensation reaction of the second amino acid. After the reaction was completed, the resin was washed with DMF (15 mL each time, for a total of 3 times). Then, a DMF solution containing 20% ​​piperidine (10 mL) was added to the resin and reacted in a shaker at room temperature for 20 minutes to remove the Fmoc protecting group. The resin was washed with DMF (15 mL each time, for a total of 5 times). A small amount of resin was taken for ninhydrin testing to confirm that the deprotection was complete. The amino acid coupling and deprotection operations were carried out sequentially from the C-terminus to the N-terminus (Arg → Arg → Arg → Trp). Finally, the short peptide chain NH2-WRRR-CONH2 was synthesized on the resin.

[0046] (2) Short peptide-sound sensitizer conjugate PSC 3RSynthesis: A sonic sensitizer (0.4 mmol, 213.9 mg), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.4 mmol, 208.2 mg), and N,N-diisopropylethylamine (4 mmol, 516.96 mg) were added to the Rink resin linked with NH2-WRRR-CONH2 and dissolved in DMF. The mixture was placed in a shaker and reacted at room temperature for 2 h to carry out the amide condensation reaction. After the reaction was completed, the resin was washed with N,N-dimethylformamide (5 times), washed twice with methanol, and then dried under vacuum.

[0047] (3) Short peptide-sound sensitizer conjugate PSC 3R Purification: A mixed lysis reagent of trifluoroacetic acid, phenol, and water (volume ratio 90:5:5) (15 mL / g resin) was added to the dried resin and the mixture was shaken at room temperature for 3 h. After the reaction was completed, the short peptide-sound sensitizer conjugate PSC was added. 3R Separate from the resin by filtration, and precipitate the pyrolysis solution using diethyl ether (added at 10 times the amount of pyrolysis agent) to obtain PSC. 3R Solid precipitate was obtained by preparative HPLC (solvents: water and acetonitrile, both containing 0.1% trifluoroacetic acid) for PSC. 3R After purification, freeze-drying (pressure 50 Pa, temperature -78 °C, time 24 h) yielded a blackish-green solid PSC. 3R The yield was 44.8%. MALDI-TOF MS: Calculated value C 62 H 81 N 19 O6[M+H] + : 1188.46, test value 1188.73.

[0048] Example 4: Short peptide-sound sensitizer conjugate PSC 4R Preparation

[0049] (1) Synthesis of the short peptide NH2-RRRR-CONH2: 200 mg of Rink Amide Resin (200 mesh) with a loading of 1 mmol / g (corresponding to a theoretical loading of 0.2 mmol) was weighed and placed in a peptide synthesis reaction tube. Dichloromethane (20 mL / g resin) was added to swell the resin, and the mixture was shaken at room temperature for 2 h. The resin was then washed with N,N-dimethylformamide (DMF, 15 mL each time, 3 times in total). A DMF solution containing 20% ​​piperidine (10 mL) was added to the resin, and the mixture was reacted at room temperature for 20 min to remove the Fmoc protecting group. The resin was washed with DMF (15 mL each time, 5 times in total). A small amount of resin was tested for ninhydrin to confirm complete deprotection. Fmoc-Arg(pbf)-OH (0.4 mmol, 259.6 mg) and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.4 mmol, 208.2 mg) were added to the resin. Fmoc-Arg(pbf)-OH (0.4 mmol, 259.6 mg), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.4 mmol, 104.1 mg), and N,N-diisopropylethylamine (2 mmol, 258.48 mg) were dissolved in DMF and added to the resin. The mixture was placed in a shaker and reacted at room temperature for 2 h to carry out the amide condensation reaction. After the reaction, the resin was washed with DMF (15 mL each time, 3 times). Then, 10 mL of DMF containing 20% ​​piperidine was added to the resin and the mixture was placed in a shaker and reacted at room temperature for 20 min to remove the Fmoc protecting group. The resin was washed with DMF (15 mL each time, 5 times). A small amount of resin was taken for ninhydrin testing to confirm complete deprotection. Then, Fmoc-Arg(pbf)-OH (0.4 mmol, 259.6 mg), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.4 mmol, 104.1 mg), and N,N-diisopropylethylamine (2 mmol, 258.48 mg) were added to carry out the amide condensation reaction of the second amino acid. After the reaction, the resin was washed with DMF (15 mL each time, 10 mL) and reacted at room temperature for 20 min to remove the Fmoc protecting group. The resin was washed with DMF solution (10 mL, 3 times in total) and then 20 mL of piperidine was added to the resin. The mixture was placed in a shaker and reacted at room temperature for 20 min to remove the Fmoc protecting group. The resin was washed with DMF (15 mL each time, 5 times in total). A small amount of resin was tested for ninhydrin to confirm that the deprotection was complete. The amino acid coupling and deprotection operations were performed sequentially from the C-terminus to the N-terminus (Arg → Arg → Arg → Arg). Finally, the short peptide chain NH2-RRRR-CONH2 was synthesized on the resin.

[0050] (2) Short peptide-sound sensitizer conjugate PSC 4RSynthesis: A sonic sensitizer (0.4 mmol, 213.9 mg), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.4 mmol, 208.2 mg), and N,N-diisopropylethylamine (4 mmol, 516.96 mg) were added to Rink resin linked with NH2-RRRR-CONH2 and dissolved in DMF. The mixture was placed in a shaker and reacted at room temperature for 2 h to carry out the amide condensation reaction. After the reaction was completed, the resin was washed with N,N-dimethylformamide (5 times), washed twice with methanol, and then dried under vacuum.

[0051] (3) Short peptide-sound sensitizer conjugate PSC 4R Purification: A mixed lysis reagent of trifluoroacetic acid, phenol, and water (volume ratio 90:5:5) (15 mL / g resin) was added to the dried resin. The mixture was shaken at room temperature for 3 h. After the reaction, the short peptide-sound sensitizer conjugate PSC was added. 4R Separate from the resin by filtration, and precipitate the pyrolysis solution using diethyl ether (added at 10 times the amount of pyrolysis agent) to obtain PSC. 4R Solid precipitate was obtained by preparative HPLC (solvents: water and acetonitrile, both containing 0.1% trifluoroacetic acid) for PSC. 4R After purification, freeze-drying (pressure 50 Pa, temperature -78 °C, time 24 h) yielded a blackish-green solid PSC. 4R The yield was 46.39%. MALDI-TOF MS: Calculated value C 57 H 83 N 21 O6[M+H] + : 1158.43, test value 1158.87.

[0052] Example 5: Optical property characterization of short peptide-sound-sensitizer conjugates

[0053] To characterize the optical properties of the short peptide-sound-sensitizer conjugate described in this invention, its UV-Vis absorption and fluorescence emission spectra were measured at room temperature according to the following steps; all measurements were performed using optical-grade quartz cuvettes (optical path 1.0 cm). Baseline correction was performed using the same solvent before each measurement.

[0054] PegSC preparation: to PEG 550A sonosensitive agent (0.4 mmol, 213.9 mg), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.4 mmol, 208.2 mg), and N,N-diisopropylethylamine (4 mmol, 516.96 mg) were added to DMF and the mixture was stirred overnight at room temperature to carry out the amide condensation reaction. After the reaction, DMF was removed by rotary evaporation, and the mixture was purified by preparative HPLC (solvents: water and acetonitrile, both containing 0.1% trifluoroacetic acid). After freeze-drying (pressure 50 Pa, temperature -40 °C, time 24 h), a black-green solid, PegSC, was obtained, with a yield of 67.2%. MALDI-TOF MS: Calculated value C 59 H 73 N 15 O7[M+H] + The value was 1104.58, and the test showed a clear PEG characteristic peak near 1100.

[0055] The short peptide-sound-sensitizer conjugate (PSC prepared in Example 1) was used. 1R PSC prepared in Example 2 2R PSC prepared in Example 3 3R PSC prepared in Example 4 4R The PEG is prepared by dissolving it in a selected solvent (one or more combinations of degassed water, methanol, acetonitrile, tetrahydrofuran, or mixtures thereof, depending on the specific implementation) to form a solution (50 μM concentration), and a sound-sensing agent is selected to link the PEG. 550 As a control (PegSC), blank samples were prepared with solvent and baseline subtraction was performed. The absorption spectra of the samples were recorded using a UV-Vis spectrophotometer in the wavelength range of 350 nm–800 nm, and the absorbance was normalized. The fluorescence emission spectra were measured using a fluorescence spectrometer. During measurement, the excitation wavelength was fixed at the sample absorption peak (…). λ ex=405 nm, slit width is 2 nm).

[0056] Figure 1 The image shows the UV-Vis absorption spectrum of the short peptide-sound sensitizer conjugate, with distinct characteristic absorption peaks at 380 nm and 680 nm, which originate from the sound sensitizer.

[0057] Figure 2 The image shows the fluorescence emission spectrum of the short peptide-sound sensitizer conjugate, with the main fluorescence emission peaks in the range of 630-800 nm.

[0058] Example 6: Acoustodynamic performance characterization of short peptide-sound-sensitizer conjugates

[0059] Total ROS generation capacity was detected using 2,7-dichlorodihydrofluorescein (DCFH) as an indicator.

[0060] The specific operating steps are as follows: DCFH-DA (0.5 mL, 1 mM) and NaOH aqueous solution (2 mL, 1 mM) are reacted at room temperature for 30 min, and then diluted with 7.5 mL PBS to obtain a DCFH stock solution with a concentration of 50 μM; the above stock solution is then diluted, and 1 mL of PBS containing 10 μM DCFH is reacted with 1 mL of PegSC and PSC respectively. 1R PSC 2R PSC 3R and PSC 4R Mix (final concentration 2 μM, prepared with PBS), set the sonication frequency to 1.0 MHz and the power to 1 W cm⁻¹. −2 The duty cycle was 50%, the sonication time was 5 min, and the fluorescence spectrum of the system was tested every 1 min. The excitation wavelength was 488 nm and the emission wavelength range was 500-600 nm. The fluorescence intensity at 525 nm was recorded at different sonication times to detect the total ROS generation capacity.

[0061] The reactive oxygen species generation capacity of different short peptide-sound sensitizer conjugates, such as Figure 3 As shown in the figure, the fluorescence intensity of the short peptide-sound sensitizer conjugate gradually increases at 525 nm with the extension of ultrasound time. The connection of different short peptides does not have a significant effect on the ROS generation effect of the short peptide-sound sensitizer conjugate. The above data indicate that the short peptide-sound sensitizer conjugate has a certain ROS generation effect under ultrasound.

[0062] Example 7: Characterization of the ability of short peptide-sound sensitizer conjugates to disrupt the cell membrane of Gram-negative bacilli

[0063] The ability of short peptide-sound-sensitizer conjugates to disrupt bacterial cell membranes was evaluated using the following experimental procedures:

[0064] 1. Assess bacterial cell membrane integrity using propidium iodide (PI) staining: Dilute a suspension of Fusobacterium nucleatum (Gram-negative bacillus) to 10⁻⁶. 8 CFU / mL, 1 mL of bacterial suspension was mixed with 1 mL of PBS (negative control), PegSC, and PSC respectively. 1R PSC 2R PSC 3R and PSC 4R (Final concentration 20 μM, prepared with PBS) Incubate at 37 ℃ in the dark for 30 min, then divide each group of samples into two equal parts and perform the following treatments:

[0065] (1) Dark treatment group: Place the sample in the dark and let it stand for 1 min;

[0066] (2) Ultrasonic treatment group: The sample was subjected to ultrasound under light-protected conditions, with the ultrasonic frequency set to 1.0 MHz and the power to 1 W cm⁻¹. −2 The duty cycle is 50%, and the ultrasonic treatment lasts for 1 minute.

[0067] After treatment, propidium iodide (PI) staining solution was added to all samples to achieve a final PI concentration of 20 μg / mL. Staining was performed for 15 min, followed by washing with PBS by centrifugation and resuspending the bacteria to 10⁻⁶ ppm. 8 CFU / mL, data were recorded using flow cytometry.

[0068] The intracellular fluorescence intensity of PI, a DNA staining agent that cannot permeate membranes, is used as an indicator to detect bacterial membrane integrity. Figure 4 As shown: Under conditions without sonication, compared with the PBS group, the fluorescence intensity of *Fusobacterium nucleatum* treated with PegSC did not change significantly, while that treated with PSC... 2R PSC 3R and PSC 4R The fluorescence intensity of the PegSC group increased; however, after ultrasonic treatment, the fluorescence intensity of the PegSC group did not change significantly. 2R PSC 3R and PSC 4R The fluorescence intensity of the group was further enhanced compared to the group without ultrasound.

[0069] The above results indicate that PSC 2R PSC 3R and PSC 4R It can effectively destroy the cell membrane of Fusobacterium nucleatum (Gram-negative bacillus), and its destructive effect is significantly enhanced under acoustic dynamic conditions.

[0070] 2. Assess bacterial cell membrane permeability using a nucleic acid leakage test: Dilute a suspension of *Fusobacterium nucleatum* (a Gram-negative bacillus) to 10⁻⁶. 8 CFU / mL, 1 mL of bacterial suspension was mixed with 1 mL of PegSC and PSC respectively. 1R PSC 2R PSC 3R and PSC 4R (Final concentration 20 μM, prepared with PBS) Incubate at 37 ℃ in the dark for 30 min, then divide each group of samples into two equal parts and perform the following treatments:

[0071] (1) Dark treatment group: Place the sample in the dark and let it stand for 1 min;

[0072] (2) Ultrasonic treatment group: The sample was subjected to ultrasound under light-protected conditions, with the ultrasonic frequency set to 1.0 MHz and the power to 1 W cm⁻¹. −2 The duty cycle is 50%, and the ultrasonic treatment lasts for 1 minute.

[0073] After processing, all samples were centrifuged at 10,000 rpm for 5 min, and the bacterial supernatant was collected. The absorbance at 260 nm was measured using a nano-300 micro spectrophotometer.

[0074] Detection of bacterial supernatant OD 260 Value changes assess the ability of short peptide-sound-sensitizer conjugates to promote the leakage of bacterial contents (including DNA and RNA), such as... Figure 5 As shown: Under conditions without sonication, no release of contents was detected in the bacterial supernatant after PegSC treatment compared to the PBS group. 2R PSC 3R and PSC 4R The release of bacterial contents was detected in the supernatant after co-incubation with *Fusobacterium nucleatum* for 30 min; after sonication, the OD values ​​of each group were... 260 The increase was significantly greater than that in the group without ultrasound.

[0075] The above data indicate that the permeability of the cell membrane of *Fusobacterium nucleatum* is enhanced after treatment with the short peptide-sound sensitizer conjugate, among which PSC... 4R The group can effectively disrupt the cell membrane of Fusobacterium nucleatum and increase membrane permeability. 3R and PSC 2R Secondly, PegSC did not show a significant ability to disrupt bacterial cell membranes.

[0076] Example 8: In vitro antibacterial performance characterization of short peptide-sound sensitizer conjugates

[0077] The in vitro antibacterial properties of the short peptide-sound sensitizer conjugate were evaluated using the following experimental procedures:

[0078] Fusobacterium nucleatum was inoculated into liquid thioglycolate medium and placed in an anaerobic culture bag at 37 °C for static incubation. When the bacterial OD... 600 When the pH value reaches 0.8-1.2, collect the bacteria by centrifugation at 10000 rpm for 5 min, wash three times with PBS, and dilute the bacterial suspension to 10. 6 CFU / mL; 1 mL of bacterial suspension was mixed with 1 mL of PegSC and PSC at different concentrations. 1R PSC 2R PSC 3R and PSC 4R Mix (0.1, 0.5, 1, 5, 10 μM, prepared with PBS), incubate at 37 ℃ in the dark for 30 min, and divide each group of samples into two equal parts for the following treatment:

[0079] (1) Untreated group: The samples were incubated statically for 2 min;

[0080] (2) Ultrasonic treatment group: The sample was subjected to ultrasound for 2 min, with the ultrasonic frequency set to 1.0 MHz and the power to 1 W cm. −2 The duty cycle is 50%.

[0081] After processing, the samples were transferred to liquid thioglycolate medium and placed in an anaerobic culture bag at 37 °C. Bacterial OD was measured after 48 h. 600 The value change was used to detect its antibacterial properties.

[0082] The in vitro antibacterial properties of different short peptide-sound sensitizer conjugates, such as Figure 6 As shown, the OD values ​​of different concentrations of short peptide-sound-sensitizer conjugates after ultrasonic treatment of *Fusobacterium nucleatum* were determined using a turbidimetric method. 600 The value changes as the number of arginine residues in the short peptide sequence increases, OD... 600 The value gradually decreased. At a concentration of 10 μM without ultrasonic treatment, the PSC value... 1R PSC 2R PSC 3R and PSC 4R OD of treated Fusobacterium nucleatum 600 The value decreased slightly; after ultrasonic treatment, the OD values ​​of each group of samples decreased. 600 The value decreased significantly, and it showed a clear concentration-dependent killing effect, among which PSC 4R Among this series of short peptide-sound-sensitizing agent conjugates, the best antibacterial effect was observed. In contrast, at a concentration of 10 μM, the bacterial OD of PegSC-treated bacteria was significantly lower. 600 The value remained unchanged, and no significant change occurred even after ultrasonic treatment.

[0083] The above data indicate that the short peptide-sound sensitizer conjugate exhibits a certain sonodynamic antibacterial effect against Fusobacterium nucleatum, among which PSC... 4R PSC has the best antibacterial effect. 3R and PSC 2R The effect was secondary, and PegSC did not show a significant acoustic antibacterial effect.

[0084] Example 9: In vitro antitumor properties characterization of short peptide-sound sensitizer conjugates

[0085] Mouse colon cancer cell line (CT26 cells) in logarithmic growth phase were divided into groups of 5 × 10⁻⁶ cells per well. 3 Cells were seeded at a density of 100 µL per well in 96-well plates (RPMI-1640 medium containing 10% fetal bovine serum) and incubated at 37°C and 5% CO2 for 24 hours to allow cell adhesion. The medium was then discarded, and 100 µL of PBS, PegSC, or PSC were added to each well.1R and PSC 4R The culture medium (final concentration 10 μM) was incubated with the cells at 37 ℃ for 4 hours. Each sample was then divided into two equal parts and treated as follows:

[0086] (1) Untreated group: The samples were incubated statically for 2 min;

[0087] (2) Ultrasonic treatment group: The sample was subjected to ultrasound for 2 min, with the ultrasonic frequency set to 1.0 MHz and the power to 1 W cm. −2 The duty cycle is 50%.

[0088] After treatment, the culture medium in each well was removed, and a pre-prepared thiazolyl blue (MTT) solution (final concentration 0.5 mg / mL) was added to each well. The wells were then incubated at 37 °C for 4 hours. After incubation, the solution in the wells was aspirated, and 150 μL of dimethyl sulfoxide was added to each well. The absorbance at 490 nm was read using a microplate reader.

[0089] Using wells containing only culture medium and reagents as blanks for zeroing, cell viability was calculated using the following formula: Viability (%) = (OD) 实验组 - OD 空白组 ) / (OD) PBS对照组 - OD 空白组 ) × 100%;

[0090] The in vitro antitumor properties of different short peptide-sound sensitizer conjugates, such as Figure 7 As shown, PegSC showed no significant cytotoxicity to cells, with cell viability decreasing only after ultrasound, while PSC... 4R The group itself has a certain killing effect on cells, and the cell survival rate continued to decrease after ultrasound application. The above data indicate that, due to PSC... 4R Its excellent ability to disrupt cell membranes and generate ROS under ultrasound enables it to efficiently kill tumor cells.

[0091] This invention provides a short peptide-sound-sensitizing agent conjugate that disrupts bacterial cell membranes, its preparation method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A short peptide-sound-sensitizer conjugate that disrupts bacterial cell membranes, characterized in that, The short peptide-sound sensitizer conjugate is prepared by coupling the carboxyl group of the sound sensitizer to the amino group at the N-terminus of the short peptide via an amide bond. The structure of the sound-sensitive agent is shown in Formula I: （Ⅰ）； The amino acid sequence of the short peptide is any one of NH2-WWWR-CONH2, NH2-WWRR-CONH2, NH2-WRRR-CONH2, or NH2-RRRR-CONH2.

2. The method for preparing the short peptide-sound-sensitizer conjugate according to claim 1, characterized in that, Includes the following steps: (1) Solid-phase synthesis of short peptide resin: Using Rink resin as a carrier, the Fmoc solid-phase peptide synthesis method is adopted to sequentially link amino acids according to the amino acid sequence of the short peptide to obtain short peptide resin. (2) Coupling of the sound-sensing agent: The short peptide resin, sound-sensing agent, condensing agent and organic base obtained in step (1) are dissolved in an organic solvent to carry out an amide condensation reaction. After the reaction is completed, the resin is washed and dried. (3) Pyrolysis and purification: Pyrolysis agent is added to the resin obtained in step (2) for pyrolysis. After solid-liquid separation, precipitation, purification and freeze drying, the short peptide-sound sensitizer conjugate is obtained.

3. The preparation method according to claim 2, characterized in that, The specific operation of step (1) includes the following steps: (1a) After swelling and washing the Rink amide resin, the Fmoc protecting group was removed, and the resin was obtained after washing. (1b) The short peptide resin is obtained by solid-phase synthesis according to the amino acid sequence of the short peptide. Specifically, the single Fmoc-protected amino acid, condensing agent, organic base and the resin are dissolved in DMF to carry out amide condensation reaction. After the reaction is completed, the resin is washed to obtain a resin with Fmoc-protected amino acid. Then, the Fmoc protecting group is removed and the resin is washed.

4. The preparation method according to claim 2, characterized in that, In step (2), the condensing agent is any one of benzotriazole-1-yl-oxytripyrrolidine phosphorus hexafluorophosphate, (7-azabenzotriazole-1-oxy)tripyrrolidine phosphorus hexafluorophosphate, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, O-benzotriazole-tetramethylurea hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid, N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate, N,N'-diisopropylcarbodiimide, or N,N'-dicyclohexylcarbodiimide; and / or, the organic base is any one of N,N-diisopropylethylamine, triethylamine, 4-dimethylaminopyridine, or N-methylimidazole; and the organic solvent is N,N-dimethylformamide.

5. The preparation method according to claim 2, characterized in that, In step (2), the amount of the sound-sensitive agent and the condensing agent added is 1 to 4 equivalents of the number of moles of the reactive amino groups at the top end of the short peptide resin, and the amount of the organic base added is 5 to 50 equivalents of the number of moles of the reactive amino groups at the top end of the short peptide resin; and / or, the amide condensation reaction is carried out at a temperature of 0 to 40 °C for a time of 1 to 12 h; and / or, the washing is carried out by washing with N,N-dimethylformamide 2 to 5 times, followed by washing with methanol 2 to 3 times.

6. The preparation method according to claim 2, characterized in that, In step (3), the pyrolysis agent is a mixed solution of trifluoroacetic acid, phenol and water, with a volume ratio of 85~95:2.5~7.5:2.5~7.5; and / or, the amount of the pyrolysis agent added is calculated based on the mass-volume ratio of the Rink resin to the pyrolysis agent in step (1) being 1g:10~30 mL; and / or, the pyrolysis is carried out at a temperature of 0~40 ℃ for a time of 1~4 h.

7. The preparation method according to claim 2, characterized in that, In step (3), the solid-liquid separation is filtration; the precipitation is performed using diethyl ether, with the volume ratio of diethyl ether to the pyrolysis agent being 1:5~20; and / or, the purification is performed using high performance liquid chromatography; and / or, the freeze drying is performed at a temperature of -20~-80 ℃, a pressure of 0.1~50 Pa, and a time of 12~48 h.

8. The use of the short peptide-sound sensitizer conjugate of claim 1 in the preparation of a medicament for the prevention and / or treatment of bacterial infections; and / or, wherein the bacterial infection is caused by Gram-positive and / or Gram-negative bacteria.

9. The application according to claim 8, characterized in that, The short peptide-sound sensitizer conjugate exerts its antibacterial effect by disrupting the bacterial cell membrane.

10. The use of the short peptide-sound sensitizer conjugate according to claim 1 in the preparation of an antitumor drug; wherein the tumor is preferably a colon tumor.

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