Sonodynamic antibacterial sound-sensitive agent synthesized based on hematoporphyrin monomethyl ether and antibacterial peptide
By combining hematoporphyrin monomethyl ether with antibacterial peptides, the new sound-sensitizer HMME@AMP was synthesized, which solved the problems of poor antibacterial effects and weak broad spectrum of traditional sound-sensitizers, and effectively inhibited a variety of bacteria.
Patent Information
- Application Number
- CN202411908699.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-23
- Publication Date
- 2025-04-25
AI Technical Summary
Traditional sonic sensitizers have shortcomings in antibacterial effects, and can only target a single bacteria, with weak broad spectrum.
By combining hematoporphyrin monomethyl ether with antimicrobial peptides, the new sound-sensitive agent HMME@AMP is synthesized by dehydration and condensation method to enhance its antibacterial effect and broad spectrum.
HMME@AMP significantly improves the inhibitory effect on Staphylococcus aureus and Pseudomonas aeruginosa and has good broad-spectrum antibacterial properties.
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Abstract
Description
Technical field:
[0001] The present invention relates to the fields of acoustics, materials and medicine, and specifically designs a new type of synthesized sonosensitizer HMME@AMP to achieve the purpose of inhibiting Staphylococcus aureus and Pseudomonas aeruginosa through the synergistic effect with ultrasound. Background technology:
[0002] Over the past decade, the rapid evolution and spread of multidrug-resistant (MDR) bacteria has become a threatening public health problem. These pathogens, called "ESKAPE" and including Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter, have the ability to render antibiotics ineffective in a short period of time. To address this problem, researchers are working to find alternative solutions to eliminate infections not only in medical and veterinary settings, but also to reduce the microbial load in industrial and environmental applications. In agricultural settings, the use of antibiotics plays a key role in the development of bacterial resistance through the spread of resistance through foodborne microorganisms and wastewater. Physical treatment methods such as irradiation, high pressure, and high temperature are popular because they produce less resistance and have greater potential for large-scale application. However, the industrial application and commercialization of these treatment methods are hampered by several limitations, including low penetration, high investment costs, and sophisticated equipment requirements.
[0003] Ultrasound has been explored as an antimicrobial tool since 1927. Wood et al. provided the first evidence that ultrasound can induce lethal effects on microorganisms, thus opening the door to ultrasound antimicrobial research. Since then, it has been found that ultrasound, whether used alone or in combination with other strategies, has the ability to destroy a variety of microorganisms, ranging from bacteria to viruses. This antimicrobial property of ultrasound has led to its use in various antimicrobial strategies, such as in the food sterilization industry for the treatment of microbial infections.
[0004] Sonodynamic antimicrobial therapy is a new antimicrobial treatment method developed on the basis of sonodynamic therapy (SDT). Similar to SDT, sonodynamic antimicrobial therapy uses low-intensity focused ultrasound (LIFU) to stimulate sonosensitizers to produce cytotoxic reaction substances that are toxic to microorganisms. LIFU is less expensive, can act on microorganisms deep in the tissue, and can be focused to a small area to activate sensitizers. These advantages allow ultrasound antimicrobial therapy to show synergistic effects on a variety of microorganisms, including bacteria, rootless biofilms, and yeast.
[0005] Although these synergistic effects of sonodynamic antimicrobial have been studied in many biological models in vitro and in vivo, their exact mechanisms remain unclear and there are differences. Chen et al. believed that the interference of sonodynamic antimicrobial with these microbial lives mainly depends on the biological model and experimental system, the type of combined sonosensitizers, and ultrasound parameters such as frequency and intensity. Although it is difficult to form a universal mechanism of sonodynamic antimicrobial action, some general characteristics have emerged, such as the production of ROS through sonoluminescence and pyrolysis, and cytotoxicity that is independent of ROS. These sonodynamic events lead to damage to microbial membranes, proteins, and DNA / RNA, ultimately leading to cell death.
[0006] As the understanding of the basic mechanism of sonodynamic antibacterial continues to deepen, more and more researchers have come to realize that sonosensitizers are one of the key factors. A variety of sonosensitizers have been used in sonodynamic antibacterial, including antibiotics, natural product extracts, nanoparticles / nanocomposites, and heteroanthracene dyes. Since Liu et al. first pointed out the antibacterial potential of ultrasound antibacterial, this prediction has been continuously confirmed by experimental studies and eventually entered medical applications and industrial process hygiene.
[0007] Although most studies on sonic antibacterial technology focus on its medical applications, by killing pathogens or tumor cells, some reports briefly mention the potential of sonic antibacterial technology in food safety and sewage purification. These studies demonstrate the application prospects of ultrasound antibacterial technology in non-medical fields and provide new directions for future research.
[0008] Sonodynamic antimicrobial technology has gained increasing popularity in recent years as a therapeutically useful modality. However, its application in decontamination and disinfection is still in its infancy. Most of the information on the mechanism of sonodynamic antimicrobial comes from SDT research. SDT has been extensively studied in medicine, but its mechanism of action has not yet been fully elucidated.
[0009] Although the antibacterial mechanism of sonodynamic therapy is not yet fully understood, many researchers have great interest in sonodynamic antibacterial therapy in view of its characteristic of not leading to enhanced bacterial resistance. In the process of sonodynamic antibacterial therapy, sonosensitizers play a vital role. However, the sonosensitizers currently used generally have the disadvantages of unsatisfactory antibacterial effect, only targeting a single bacterial species, and weak broad-spectrum antibacterial ability. In order to overcome the above-mentioned defects of traditional sonosensitizers, the present invention designs a new sonosensitizer HMME@AMP. The sonosensitizer is synthesized by combining hematoporphyrin monomethyl ether with antimicrobial peptides, which significantly makes up for the deficiencies of hematoporphyrin monomethyl ether and antimicrobial peptides in broad-spectrum antibacterial ability, and further enhances the efficacy of inhibiting bacterial activity on this basis. Summary of the invention:
[0010] The problem to be solved by the present invention is that the traditional sonosensitizer has a poor antibacterial effect, can only target a single bacterium, and has a weak broad-spectrum.
[0011] To solve the above problems, the present invention is based on hematoporphyrin monomethyl ether and antimicrobial peptides, and the two are combined by a dehydration condensation method to synthesize a new sonosensitizer that makes up for the shortcomings of hematoporphyrin monomethyl ether and antimicrobial peptides in antibacterial effect and broad spectrum.
[0012] In the synthesis process of sonosensitizer HMME@AMP, 2-Chlorotrityl Chloride Resin must first be placed in a reaction tube, followed by the addition of DMF (dosage of 15 ml / g) and oscillation for 60 minutes to complete swelling. Next, the antimicrobial peptide must be connected. This process requires the removal of the solvent by sand core filtration, followed by the addition of a 3-fold molar excess of Fmoc-protected amino acid (i.e., the first amino acid at the C-terminus), and the addition of a 10-fold molar excess of DIEA. Finally, it is dissolved in DMF and oscillated for 30 minutes, and the capping is performed with methanol. It takes 30 minutes to complete the connection between the antimicrobial peptide and the resin.
[0013] When removing the Fmoc protective layer, DMF must be removed first, and then a 20% piperidine DMF solution is added and allowed to act for 5 minutes, and then the solution is added again and allowed to act for 15 minutes. Before proceeding to the next step of synthesis, the deprotection situation must be tested, and this process is verified by the color change reaction of ninhydrin. During the test, the piperidine solution must be removed first, and then a small amount of resin is taken, washed with ethanol 3 times, and then 1 drop of ninhydrin, KCN and phenol solution are added, and heated at 105°C to 110°C for 5 minutes. If the solution turns dark blue, it indicates that the deprotection is complete. After the test is completed, it needs to be washed twice with DMF, methanol and DMF.
[0014] After the connection and deprotection of the antimicrobial peptide and the resin are completed, a dehydration condensation reaction between amino acids is required. This process requires the addition of 3 times molar excess of HMME, 3 times molar excess of HBTU and 10 times molar excess of DIEA, and finally dissolving with DMF and shaking for 45 minutes. After the synthesis of the antimicrobial peptide and the synthesis of the antimicrobial peptide and hematoporphyrin monomethyl ether are completed, ninhydrin is used for detection. If the result is colorless, it indicates that the synthesis of the crude material has been completed.
[0015] After the synthesis of the crude material is completed, the resin needs to be washed and drained, and then the first amino acid and the resin are cut. When washing the resin, it needs to be washed twice with DMF, three times with DCM and four times with methanol, and the draining time is 10 minutes. When cutting, a cutting liquid needs to be prepared, and its composition is TFA (95%), water (2%), EDT (2%) and TIS (1%), and the cutting time is 180 minutes. When blowing and washing, the cleavage solution needs to be blown dry as much as possible with nitrogen, and then precipitated with ether, centrifuged to remove the supernatant, the precipitate is washed 6 times with ether, and evaporated at room temperature.
[0016] The obtained crude product needs to be purified. During purification, a small amount of crude product is taken, dissolved in H2O / ACN, and analyzed on an HPLC analyzer to determine the peak time corresponding to the target peak. Subsequently, a C18 reverse phase chromatography preparation system (wavelength: 220nm; flow rate: 15ml / min; injection volume: 20mL; column temperature: 25°C; buffer A: 0.1% TFA aqueous solution; buffer B: 0.1% TFA acetonitrile solution) is used to collect the target peak solution, such as Figure 1 Finally, take a small amount of the target peak solution in a 1.5 ml centrifuge tube for mass spectrometry confirmation, as shown in Figure 2 After all purification and detection are completed, the powdered sonosensitizer HMME@AMP is sealed and packaged and stored at -20°C. Effect of the invention:
[0017] The present invention uses solid culture medium observation and bacterial weighing to evaluate the efficacy of the synthesized sonosensitizer HMME@AMP in inhibiting Staphylococcus aureus and Pseudomonas aeruginosa. The evaluation results show that the sonosensitizer HMME@AMP exhibits excellent inhibitory effects on both Staphylococcus aureus and Pseudomonas aeruginosa. Figures and descriptions of the figures: Figure 1 It is a diagram of purifying solution and detecting solution purity using C18 reverse phase chromatography preparation system. Figure 2 This is a graph showing the detection results of the collected materials using a mass spectrometer. Figure 3 This is a graph of the antibacterial ability against Staphylococcus aureus evaluated using solid culture medium. Figure 4 This is a graph of the antibacterial ability against Pseudomonas aeruginosa evaluated using solid culture medium. Specific implementation method:
[0018] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.
[0019] In order to explain the technical content, structural features and effects of the present invention in detail, the following will be described in detail in combination with the implementation and the accompanying drawings. Example 1: A highly effective sonosensitizer HMME@AMP based on hematoporphyrin monomethyl ether and antimicrobial peptide LL-37
[0020] Step S1: 2-Chlorotrityl Chloride Resin is placed in a reaction tube, followed by injection of DMF (15 ml per gram of resin), and oscillation for 60 minutes to achieve full swelling. Next, the antimicrobial peptide connection step is performed. After removing the solvent by sand core filtration, three times the molar amount of Fmoc protected amino acid (i.e., the first amino acid at the C-terminus) is added, and ten times the molar amount of DIEA is continued to be added, and finally dissolved in DMF and oscillated for 30 minutes, and methanol is used for end-capping treatment for 30 minutes to ensure that the antimicrobial peptide is successfully connected to the resin. When removing the Fmoc protective layer, first remove DMF, then add a 20% piperidine DMF solution for 5 minutes, and then repeat the addition of this solution and continue to act for 15 minutes. Before proceeding to the next step of synthesis, the deprotection state must be detected, and this process is confirmed by the ninhydrin color change reaction. During the test, first remove the piperidine solution, then take a small amount of resin, wash it three times with ethanol, then add one drop each of ninhydrin, KCN and phenol solution, and heat it at 105℃ to 110℃ for 5 minutes. If the solution turns dark blue, it indicates that the deprotection process is complete. After the test, it needs to be washed twice with DMF, methanol and DMF respectively.
[0021] Step S2: After the connection and deprotection of the antimicrobial peptide and the resin are completed, a dehydration condensation reaction between amino acids is required. This process requires the addition of 3 times molar excess of HMME, 3 times molar excess of HBTU and 10 times molar excess of DIEA, and finally dissolving with DMF and shaking for 45 minutes. After the synthesis of the antimicrobial peptide and the synthesis of the antimicrobial peptide and hematoporphyrin monomethyl ether are completed, ninhydrin is used for detection. If the result is colorless, it indicates that the synthesis of the crude material has been completed. Example 2: Purification and characterization of crude product HMME@AMP
[0022] The obtained crude product needs to be purified. During purification, a small amount of crude product is taken, dissolved in H2O / ACN, and analyzed on an HPLC analyzer to determine the peak time corresponding to the target peak. Subsequently, a C18 reverse phase chromatography preparation system (wavelength: 220nm; flow rate: 15ml / min; injection volume: 20mL; column temperature: 25°C; buffer A: 0.1% TFA aqueous solution; buffer B: 0.1% TFA acetonitrile solution) is used to collect the target peak solution, such as Figure 1 Finally, take a small amount of the target peak solution in a 1.5 ml centrifuge tube for mass spectrometry confirmation, as shown in Figure 2 After all purification and detection are completed, the powdered sonosensitizer HMME@AMP is sealed and packaged and stored at -20°C. Example 3: Evaluation of the antibacterial properties of HMME@AMP
[0023] In the process of evaluating the antibacterial efficacy of HMME@AMP, the test strains selected were Staphylococcus aureus and Pseudomonas aeruginosa, which represent Gram-positive bacteria and Gram-negative bacteria, respectively, and are highly representative in terms of bacterial structure. During the implementation of sonodynamic therapy, the ultrasound parameters set were a power density of 0.5w / cm 2 , frequency 1MHz, duty cycle 60%, and ultrasound duration of 10 minutes. The selected sonosensitizers were HMME, antimicrobial peptide LL-37 and HMME@AMP, with concentrations of 30ug / ml, 60ug / ml and 120ug / ml, respectively. After the ultrasonic treatment, the solution was transferred to a cell culture bottle, and 50ul of liquid culture medium was added, and then placed in a constant temperature incubator at 37 degrees Celsius for overnight culture. The next day, 10ul was taken out from the overnight culture solution, 5ml of PBS was added for dilution, and the diluted solution was used for coating and inoculation of solid culture medium. After inoculation, it was placed in a constant temperature incubator for overnight culture again. The antibacterial efficacy of sonosensitizers under the synergistic effect of ultrasound was evaluated by observing the growth of bacteria on solid culture medium. Figure 3 and Figure 4 The data showed that compared with HMME and LL-37, the synthetic sonosensitizer HMME@AMP exhibited better broad-spectrum antibacterial activity and showed good inhibitory effects on both Staphylococcus aureus and Pseudomonas aeruginosa.
[0024] In summary, the sonosensitizer HMME@AMP synthesized based on hematoporphyrin monomethyl ether and antimicrobial peptide LL-37, although its inhibitory effect against Staphylococcus aureus is not as good as that of hematoporphyrin monomethyl ether, and its inhibitory effect against Pseudomonas aeruginosa is not as good as that of antimicrobial peptide LL-37, but it shows good bacterial activity inhibition ability against both bacteria and has good broad-spectrum antibacterial properties.
Claims
1. A highly efficient sonosensitizer HMME@AMP based on hematoporphyrin monomethyl ether and antimicrobial peptide LL-37, characterized in that: The HMME@AMP sonosensitizer is composed of hematoporphyrin monomethyl ether (HMME) and antimicrobial peptide LL-37. The HMME@AMP sonosensitizer needs to be prepared in two steps: first, the antimicrobial peptide and the resin are connected; finally, a condensation agent and hematoporphyrin monomethyl ether are added to synthesize the hematoporphyrin monomethyl ether and the antimicrobial peptide through a condensation reaction.
2. The preparation method for connecting the antimicrobial peptide and the resin according to claim 1, characterized in that: The method is as follows: Step S1: Connection of antimicrobial peptide and resin: The resin (2-Chlorotrityl Chloride Resin) was placed in a reaction tube, followed by addition of DMF (dosage of 15 ml / g), and oscillation for 60 minutes to complete swelling. Next, the antimicrobial peptide needs to be connected. This process requires the removal of the solvent by sand core filtration, and then a 3-fold molar excess of the first C-terminal antimicrobial peptide is added, and a 10-fold molar excess of DIEA is continued. Finally, it is dissolved in DMF and oscillated for 30 minutes, and methanol is used for capping treatment. It takes 30 minutes to complete the connection of the antimicrobial peptide and the resin.
3. The method for preparing HMME@AMP according to claim 1, characterized in that: The method is as follows: Step S2: Synthesis of hematoporphyrin monomethyl ether and antimicrobial peptide: After the antimicrobial peptide is connected to the resin and deprotected, a dehydration condensation reaction between the antimicrobial peptide and hematoporphyrin monomethyl ether is required. This process requires the addition of hematoporphyrin monomethyl ether, HBTU and DIEA, and finally dissolving with DMF and shaking.
4. The method for preparing HMME@AMP according to claim 2, characterized in that: The specific method for synthesizing the antimicrobial peptide and hematoporphyrin monomethyl ether in step S2 is as follows: After the connection and deprotection of the antimicrobial peptide and the resin are completed, a dehydration condensation reaction is required. This process requires the addition of 3 times molar excess of HMME, 3 times molar excess of HBTU and 10 times molar excess of DIEA, and finally dissolving with DMF and shaking for 45 minutes.
5. The method for preparing HMME@AMP according to claim 3, characterized in that: The specific deprotection method for the synthesis of the antimicrobial peptide and hematoporphyrin monomethyl ether in step S2 is as follows: When removing the Fmoc protective layer, DMF must be removed first, and then a 20% piperidine DMF solution is added and allowed to act for 5 minutes, and then the solution is added again and allowed to act for 15 minutes. Before proceeding to the next step of synthesis, the deprotection situation must be tested, and this process is verified by the color change reaction of ninhydrin. During the test, the piperidine solution must be removed first, and then a small amount of resin is taken, washed with ethanol 3 times, and then 1 drop of ninhydrin, KCN and phenol solution are added, and heated at 105°C to 110°C for 5 minutes. If the solution turns dark blue, it indicates that the deprotection is complete. After the test is completed, it needs to be washed twice with DMF, methanol and DMF.
6. The method for preparing HMME@AMP according to claim 4, characterized in that: After the synthesis of the antimicrobial peptide and hematoporphyrin monomethyl ether in step S2 is completed, the specific method for detecting the result is as follows: To test the condensation results, take a small amount of resin, wash it with ethanol three times, then add 1 drop each of ninhydrin, pyridine and phenol solution, and heat it at 105℃ to 110℃ for 5 minutes. If the solution is colorless, it means that there is no deprotected amino acid in the solution, that is, the dehydration condensation between the two amino acids has been completed. When washing, it is necessary to wash it with DMF once, and then wash it with methanol and DMF twice each.
7. The method for preparing HMME@AMP according to claim 4, characterized in that: The specific method for synthesizing the antimicrobial peptide and hematoporphyrin monomethyl ether in step S2 is as follows: After the synthesis of the crude material is completed, the resin needs to be washed and drained, and then the antimicrobial peptide and the resin are cut. When washing the resin, it needs to be washed twice with DMF, three times with DCM, and four times with methanol, and the draining time is 10 minutes. When cutting, a cutting liquid needs to be prepared, and its composition is TFA (95%), water (2%), EDT (2%) and TIS (1%), and the cutting time is 180 minutes. When blowing and washing, the cleavage solution needs to be blown dry as much as possible with nitrogen, and then precipitated with ether, centrifuged to remove the supernatant, and the precipitate is washed 6 times with ether and evaporated at room temperature.
8. The method for preparing HMME@AMP according to claim 7, characterized in that: In step S2, the crude product synthesized from the antimicrobial peptide and hematoporphyrin monomethyl ether is completed, and after cutting, the crude material is purified. The specific method is as follows: During purification, take a small amount of crude product, dissolve it with H2O / ACN, and analyze it on the HPLC analyzer to determine the peak time corresponding to the target peak. Subsequently, the target peak solution was collected using a C18 reverse phase chromatography preparation system (wavelength: 220nm; flow rate: 15ml / min; injection volume: 20mL; column temperature: 25°C; buffer A: 0.1% TFA aqueous solution; buffer B: 0.1% TFA acetonitrile solution). Finally, take a small amount of the target peak solution in a 1.5ml centrifuge tube for mass spectrometry confirmation. After all purification and detection are completed, the powdered sonosensitizer HMME@AMP is sealed and packaged and stored at -20°C.