Modularized pathogenic microorganism targeting fluorescent probe, antibacterial drug delivery oligopeptide, preparation method and application

Abstract

The invention discloses a modularized pathogenic microorganism targeting fluorescent probe, an antibacterial drug delivery oligopeptide and a preparation method and application thereof, the probe is composed of the following functional modules: a chemotactic module: formyl-D-Phe-Leu-D-Met, which realizes active migration to an infectious inflammation microenvironment by combining with a formyl peptide receptor; the targeting module comprises D-Trp-D-Trp-Arg and RWWR which are specifically combined with the surfaces of the pathogenic microorganisms through cationic interaction and hydrophobic interaction; the fluorescence activation module is a double Cy5.5 fluorophore which is covalently connected through lysine side chain amino; and the connection and stability module is a PEG4-GGSG flexible linker, has C-terminal amidation and key site D-type amino acid modification, and is used for enhancing the stability of the probe. The probe can actively migrate to an infectious inflammation microenvironment, is specifically combined with the surface of pathogenic microorganisms, and triggers fluorescence activation after combination, so that accurate imaging of an infected part is realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical detection, and particularly relates to a modular pathogenic microorganism targeting fluorescent probe, an antibacterial drug delivery short peptide, and a preparation method and application thereof. BACKGROUND

[0002] Early and accurate diagnosis of infectious diseases is crucial for treatment plan and prognosis. Traditional pathogenic microorganism detection methods such as culture method are time-consuming and low in sensitivity, while imaging detection means lack specificity and are difficult to accurately identify infection foci in complex inflammatory environment. In recent years, fluorescent probe technology has shown great potential in the field of biomedical imaging, but the existing probes generally have problems such as insufficient targeting, high fluorescence background, poor stability, etc. For example, most probes lack active chemotaxis ability to inflammatory microenvironment, resulting in low enrichment efficiency at the infection site; some fluorescent probes produce fluorescence signals before reaching the target site, causing background interference; in addition, the anti-degradation ability and safety of the probes also limit their clinical application. Therefore, it is of great clinical significance and application value to develop a pathogenic microorganism targeting fluorescent probe with chemotaxis, targeting, intelligent fluorescence activation, high stability and safety.

[0003] Bacterial and fungal infections are important factors threatening human health, especially the emergence of multiple drug-resistant bacteria, which significantly reduces the therapeutic effect of traditional antibacterial drugs. Currently, the clinical application of antibacterial drugs faces many problems: on the one hand, the systemic administration mode leads to insufficient drug concentration at the infection site, making it difficult to effectively kill pathogens, and the distribution of drugs in non-infection sites increases the risk of adverse reactions; on the other hand, a single antibacterial mechanism is easily evaded by pathogens, resulting in drug resistance.

[0004] Although some targeted antibacterial drug delivery systems have been developed, the existing systems still have obvious defects. For example, most targeted delivery systems lack active chemotaxis ability and cannot quickly accumulate at the infection site; the release of drugs lacks specific response to the infection microenvironment, resulting in premature release or insufficient release of drugs; in addition, a single antibacterial action mode is difficult to cope with complex drug resistance. Therefore, it is of great significance to develop a multifunctional antibacterial drug delivery system that can actively migrate to the infection site, specifically target pathogenic microorganisms, intelligently release drugs in the infection microenvironment, and has synergistic antibacterial effect, to solve the problem of drug-resistant bacterial infection. SUMMARY

[0005] The first object of the present application is to provide a modular pathogenic microorganism targeting fluorescent probe and its preparation method and application, which can actively migrate to an infectious inflammatory microenvironment, specifically bind to the surface of pathogenic microorganisms, trigger fluorescence activation after binding, and realize precise imaging of the infection site. At the same time, the probe has good stability, low cytotoxicity and simple synthesis process, meets the requirements of drug development, and provides an efficient tool for the diagnosis and treatment of infectious diseases.

[0006] To achieve the above object, the present application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a modular pathogenic microorganism targeting fluorescent probe.

[0008] A modular pathogenic microorganism targeting fluorescent probe has the following chemical structure:

[0009] Formyl-D-Phe-Leu-D-Met-PEG4-GGSG-D-Trp-D-Trp-Arg-Lys(Cy5.5)-RWWR-NH2

[0010] Composed of the following functional modules:

[0011] Chemotaxis module: including formyl-D-Phe-Leu-D-Met, used to achieve active migration to an infectious inflammatory microenvironment by binding to formyl peptide receptors;

[0012] Targeting module: including D-Trp-D-Trp-Arg and RWWR, used to specifically bind to the surface of pathogenic microorganisms through cationic action and hydrophobic interaction;

[0013] Fluorescence activation module: including double Cy5.5 fluorescent groups covalently linked through the lysine side chain amino group, used to realize fluorescence self-quenching-activation after binding to microbial membranes;

[0014] Connection and stability module: including PEG4-GGSG flexible linker, C-terminal amidation and key site D amino acid modification, used to enhance the stability of the probe.

[0015] In the chemotaxis module, formylation modification enhances the affinity to formyl peptide receptors, and D-phenylalanine prevents enzymolysis.

[0016] In the targeting module, arginine binds to the negatively charged membrane components of pathogenic microorganisms, and tryptophan inserts into the hydrophobic region of the microbial membrane.

[0017] In the fluorescence activation module, the double Cy5.5 fluorescent groups are quenched due to close spatial proximity when not bound to the microbial membrane, and the fluorescence is restored when the double Cy5.5 fluorescent groups are separated after binding.

[0018] In a second aspect, the present application provides a preparation method of a modular pathogenic microorganism-targeting fluorescent probe.

[0019] A preparation method of a modular pathogenic microorganism-targeting fluorescent probe, comprising the following steps:

[0020] Step 1, a short peptide backbone is synthesized by using a solid-phase synthesis method as follows:

[0021] Formyl-D-Phe-Leu-D-Met-PEG4-GGSG-D-Trp-D-Trp-Arg-Lys-RWWR;

[0022] Step 2, a Cy5.5 fluorescent group is covalently coupled to the side chain amino group of lysine in the short peptide by using a click chemistry method;

[0023] Step 3, the target fluorescent probe is obtained after purification.

[0024] In some preferred embodiments, the preparation method comprises the following steps:

[0025] (1) a short peptide backbone is synthesized by using a solid-phase synthesis method (SPPS), and the specific steps include: a first amino acid is connected to a solid-phase carrier by a covalent bond, amino acids are sequentially added, and the synthesis of the short peptide chain is completed by deprotection, coupling and other reactions;

[0026] (2) a Cy5.5 fluorescent group is covalently coupled to the side chain amino group of lysine (Lys) in the short peptide by using a click chemistry method, to obtain the target fluorescent probe.

[0027] In a third aspect, the present application provides an application of the modular pathogenic microorganism-targeting fluorescent probe.

[0028] The application of the modular pathogenic microorganism-targeting fluorescent probe in preparing a pathogenic microorganism detection reagent or an infectious inflammation site imaging reagent.

[0029] Preferably, the reagent is used for real-time marking of infection foci and precise diagnosis of drug-resistant bacterial infections in surgery.

[0030] A second object of the present application is to provide a modular antibacterial drug delivery short peptide and a preparation method and application thereof, which can actively chemotaxis to an infectious inflammation microenvironment, specifically bind to the surface of pathogenic microorganisms, realize intelligent release of antibacterial drugs under the stimulation of the infection microenvironment, and achieve the purpose of synergistic antibiosis by self-assembly to destroy microbial membranes. At the same time, the short peptide has low toxicity, high stability and good drug potential, which provides a new effective means for the treatment of drug-resistant bacterial infections.

[0031] To achieve the above object, the present application adopts the following technical solutions:

[0032] In a fourth aspect, the present application provides a modular antimicrobial drug delivery short peptide.

[0033] A modular antimicrobial drug delivery short peptide, whose chemical structure is:

[0034] Formyl-D-Phe-Leu-Met-PEG4-GSGSG-RRWR-Lys(hydrazone-PMB)-Pro-LLLHHH-NH2

[0035] Composed of the following functional modules:

[0036] Chemotactic module: including formyl-D-Phe-Leu-Met, for realizing active migration to infectious inflammatory microenvironment by binding formyl peptide receptor;

[0037] Targeting module: including RRWR, for specific binding to pathogenic microorganism surface by cationic effect and hydrophobic effect;

[0038] Drug loading and responsive release module: including lysine (Lys) side chain, for releasing drug by pH-sensitive hydrazone bond coupling antimicrobial drug under low pH of infectious microenvironment or esterase action;

[0039] Self-assembled membrane disruption module: including LLLHHH, for self-assembling to form transmembrane pore to disrupt microbial membrane under low pH environment of inflammation;

[0040] Connection and stability module: including PEG4-GSGSG flexible linker, C-terminal amidation, and key site D-type amino acid modification and proline, for enhancing short peptide stability.

[0041] Preferably, the antimicrobial drug is polymyxin B (PMB) or tigecycline (TGC) and other antimicrobial drugs.

[0042] Preferably, in the chemotactic module, active migration to the infection site is realized by binding to the formyl peptide receptor (FPR) on the surface of neutrophils / macrophages.

[0043] Preferably, in the targeting module, the arginine in the RRWR sequence electrostatically binds to the negative components of the microbial membrane, and the tryptophan inserts into the membrane hydrophobic region.

[0044] Preferably, in the self-assembled membrane disruption module, the hydrophobic region LLL inserts into the microbial membrane lipid bilayer, and the histidine buffer region HHH is protonated to trigger self-assembly under low pH.

[0045] In a fifth aspect, the present application provides a preparation method of a modular antimicrobial drug delivery short peptide.

[0046] A preparation method of a modular antimicrobial drug delivery short peptide, comprising the following steps:

[0047] Step a, the following short peptide chain is synthesized from C terminal to N terminal by solid phase synthesis method:

[0048] Formyl-D-Phe-Leu-Met-PEG4-GSGSG-RRWR-Lys-Pro-LLLHHH;

[0049] D-type amino acids and proline are introduced during the synthesis process.

[0050] Step b, polypeptide B (PMB) or tigecycline (TGC) is coupled to the lysine side chain by click chemistry method through hydrazone bond.

[0051] Step c, the synthesized short peptide is purified and freeze-dried to obtain the target short peptide.

[0052] In some preferred embodiments, the preparation method comprises the following steps:

[0053] (a) using Fmoc-protected amino acids as raw materials, the amino acids are sequentially connected to the solid phase carrier in the order from C terminal to N terminal by solid phase synthesis method (SPPS), and the following short peptide chain is synthesized:

[0054] Formyl-D-Phe-Leu-Met-PEG4-GSGSG-RRWR-Lys-Pro-LLLHHH;

[0055] During the synthesis process, D-type amino acids, proline are introduced at specific positions, and the lysine side chain is reserved for subsequent drug coupling.

[0056] (b) polypeptide B (PMB) or tigecycline (TGC) is coupled to the lysine side chain by click chemistry method through hydrazone bond to complete the loading of antibacterial drugs;

[0057] (c) the synthesized short peptide is cleaved, falls off from the solid phase carrier and removes the protecting group, and then purified by high performance liquid chromatography (HPLC), the target product is collected, and the modular antibacterial drug delivery short peptide is obtained after freeze-drying.

[0058] In a fifth aspect, the application provides the application of the modular antibacterial drug delivery short peptide.

[0059] The modular antibacterial drug delivery short peptide is used for preparing drugs for treating multiple drug-resistant gram-negative bacterial infections, other bacterial infections, fungal infections, antiviral drugs or deep tissue infections.

[0060] Preferably, the modular antibacterial drug delivery short peptide is used for preparing a carrier for precise delivery of various pathogenic microorganism anti-infection treatment drugs.

[0061] Preferably, the modular antibacterial drug delivery short peptide is used for preparing a delivery system of an anti-infective therapeutic drug for treating deep tissue infections (such as abdominal abscesses, pneumonia).

[0062] Preferably, the modular antibacterial drug delivery short peptide is used for various anti-infective therapeutic adjuvant therapies, which have the effects of destroying cell membranes and reducing adverse drug reactions.

[0063] Advantages:

[0064] The modular pathogenic microorganism targeting fluorescent probe of the present application has the following advantages:

[0065] Dual targeting mechanism: the chemotactic module guides the probe to the inflammation site, and the targeting module specifically binds to the pathogenic microorganism membrane, significantly reducing off-target effects and improving the enrichment efficiency of the probe at the infection site.

[0066] Intelligent fluorescence activation: the self-quenching-activated fluorescence design effectively reduces background signals and significantly improves the signal-to-noise ratio, which is suitable for high-resolution imaging of deep tissues.

[0067] Good drugability: the probe has a molecular weight of about 4.2 kDa, which meets the standard for rapid kidney clearance and can reduce the risk of systemic exposure; the preparation process using solid-phase synthesis and click chemistry is simple and has a high yield; after being prepared into a freeze-dried powder injection, the reconstitution pH is 7.4, and the storage stability at 4℃ is >1 year, which is convenient for storage and clinical application; and the short peptide structure has low immunogenicity and is suitable for clinical conversion.

[0068] The modular antibacterial drug delivery short peptide of the present application has the following advantages:

[0069] Precise and efficient delivery: the synergistic effect of the chemotactic module and the targeting module enables the short peptide to actively and specifically enrich at the infection site, significantly reducing the systemic exposure risk of the drug, increasing the drug concentration at the infection site, and enhancing the therapeutic effect.

[0070] Intelligent response release: based on the dual response mechanism of pH and esterase in the infection microenvironment, the antibacterial drug is precisely released at the infection site, effectively avoiding the premature release of the drug at non-infection sites, improving the local drug concentration, and enhancing the antibacterial activity.

[0071] Synergistic antibacterial mechanism: the synergistic effect of drug release and short peptide self-assembly destroying the microbial membrane can overcome the drug resistance of pathogens, has stronger killing ability for multi-drug resistant bacteria, and reduces the risk of drug-resistant bacteria.

[0072] Excellent drug-grade properties: The short peptide has a molecular weight of less than 5 kDa, which meets the criteria for rapid renal clearance and can reduce drug accumulation in the body; the mature solid-phase synthesis combined with click chemistry preparation process facilitates large-scale production; through various stability optimization strategies, the short peptide has high stability, and the dosage form is developed as a lyophilized powder for injection, which has a stability of more than 18 months after reconstitution and storage at 4°C; at the same time, the short peptide has low toxicity and low immunogenicity, making it suitable for clinical development. Attached Figure Description

[0073] Figure 1 This is a schematic diagram of the chemical structure of the modular pathogenic microorganism-targeting fluorescent probe of Example 1;

[0074] Figure 2 This is a schematic diagram of the conformation of the modular pathogenic microorganism-targeting fluorescent probe of Example 1;

[0075] Figure 3 This is a functional annotation diagram of the modular pathogenic microorganism-targeting fluorescent probe from Example 1;

[0076] Figure 4 This is a schematic diagram of the conformation of the modular antimicrobial drug delivery short peptide in Example 2;

[0077] Figure 5 This is a functional annotation diagram of the modular antimicrobial drug delivery short peptide in Example 2. Detailed Implementation

[0078] The present invention will be further explained below with reference to the accompanying drawings and embodiments.

[0079] Example 1

[0080] A modular fluorescent probe for targeting pathogenic microorganisms, such as Figure 1 As shown, its chemical structure is:

[0081] Formyl-D-Phe-Leu-D-Met-PEG4-GGSG-D-Trp-D-Trp-Arg-Lys(Cy5.5)-RWWR-NH2

[0082] It consists of the following functional modules:

[0083] Chemotaxis module: formyl-D-Phe-Leu-D-Met, used to achieve active migration to the infectious inflammatory microenvironment by binding to the formyl peptide receptor; wherein, formylation modification enhances the affinity for the formyl peptide receptor, and D-phenylalanine prevents enzymatic degradation;

[0084] Targeting modules: D-Trp-D-Trp-Arg and RWWR, used to specifically bind to the surface of pathogenic microorganisms through cationic and hydrophobic interactions; arginine binds to the negatively charged membrane components of pathogenic microorganisms, and tryptophan inserts into the hydrophobic region of the microbial membrane;

[0085] Fluorescence activation module: The dual Cy5.5 fluorescent groups covalently linked by amino groups on the lysine side chain are used to achieve fluorescence self-quenching-activation after binding to the microbial membrane; when not bound to the microbial membrane, the dual Cy5.5 fluorescent groups cause fluorescence quenching due to spatial proximity, and after binding, the dual Cy5.5 fluorescent groups separate to restore fluorescence, improving the signal-to-noise ratio by 5-10 times.

[0086] The linkage and stability module includes a PEG4-GGSG flexible linker, C-terminal amidation, and D-type amino acid modification at key sites to enhance probe stability.

[0087] The breakdown and molecular weight of each functional module are shown in Table 1.

[0088] Table 1

[0089]

[0090] The probe conformation is shown in the diagram. Figure 2 As shown, the function labels are as follows: Figure 3 As shown.

[0091] Construct description:

[0092] D-Phe-Leu-D-Met (hydrophobic core):

[0093] Conformation: D-type amino acids form rigid hydrophobic clusters, which are resistant to protease degradation and may drive self-assembly.

[0094] Function: Enhances structural stability and reduces non-specific enzymatic hydrolysis.

[0095] PEG4 (hydrophilic protective layer):

[0096] Conformation: Hydrophilic chains encapsulate a hydrophobic core, reducing recognition by the immune system and non-specific adsorption.

[0097] Function: Extends the half-life in vivo (predicted to be approximately 12 hours).

[0098] D-Trp-D-Trp-Arg (membrane permeation module):

[0099] Conformation: Tryptophan (W) forms a hydrophobic interface through π-π stacking, while arginine (R) provides a positive charge (+1). Function: Synergistically disrupts bacterial or tumor cell membranes.

[0100] Lys (Cy5.5) (Fluorescence Imaging Module):

[0101] Conformation: The side chain extension of the fluorescent dye may slightly interfere with local peptide chain folding.

[0102] Function: Real-time tracking of peptide distribution in vivo (e.g., tumor targeting).

[0103] RWWR (Hydrophobic / Charge Synergistic Module):

[0104] Conformation: Tryptophan (W) forms a hydrophobic nucleus, and arginine (R) provides a positive charge (+3).

[0105] Function: Enhances membrane penetration and targeting.

[0106] The key parameters of the probe and the experimental verification results of this embodiment are shown in Table 2.

[0107] Table 2

[0108]

[0109] This embodiment describes a method for preparing a modular pathogenic microorganism-targeting fluorescent probe, comprising the following steps:

[0110] (1) Solid-phase synthesis of short peptide backbones

[0111] The first amino acid (C-terminal amino acid) is covalently linked to RinkAmideMBHA resin (solid support);

[0112] Following the sequence of formyl-D-Phe-Leu-D-Met-PEG4-GGSG-D-Trp-D-Trp-Arg-Lys-RWWR, the Fmoc solid-phase synthesis strategy was used to gradually add amino acids through steps such as removing Fmoc protecting groups, activating amino acids, and coupling reactions to complete the synthesis of short peptide chains.

[0113] Finally, the peptides were cleaved using reagents such as trifluoroacetic acid (TFA) to remove them from the resin and eliminate the protecting groups, yielding free short peptides.

[0114] (2) Click on chemical coupling Cy5.5

[0115] The synthesized short peptide and the azide-modified Cy5.5 fluorescent group were dissolved in an organic solvent, wherein the organic solvent was dimethyl sulfoxide (DMSO).

[0116] A catalyst was added, and a click chemistry reaction was carried out at room temperature to covalently couple Cy5.5 with a short peptide via the amino group of the lysine side chain; the catalyst was a Cu(I) catalyst.

[0117] After the reaction was completed, the target product was purified by high performance liquid chromatography (HPLC), collected, and freeze-dried to obtain a modular pathogenic microorganism-targeted fluorescent probe.

[0118] Probe performance verification

[0119] (1) Chemotaxis detection

[0120] Transwell assay was used: culture medium containing chemokines was added to the lower chamber of the Transwell chamber, and probe solution was added to the upper chamber.

[0121] After a certain period of cultivation, the probes in the lower chamber were collected, and the fluorescence intensity was measured using a fluorescence spectrophotometer. The migration efficiency of the probes was calculated, with a target migration rate >60%.

[0122] (2) Target-specific detection

[0123] Confocal microscopy observation: The probe is co-incubated with Escherichia coli or Staphylococcus aureus, and the co-localization of the probe and bacteria is observed by confocal microscopy. The binding rate is calculated, with a target binding rate of >90%.

[0124] Competitive inhibition experiment: An excess of free RWWR sequence was added to the probe-bacterial incubation system, and the binding of the probe to the bacteria was observed to verify the binding specificity.

[0125] (3) Fluorescence activation detection

[0126] Liposome-mimicking membrane experiment: Liposomes simulating microbial membranes were prepared, and probes were co-incubated with liposomes;

[0127] The change in fluorescence intensity of Cy5.5 before and after the probe binds to liposomes was detected by fluorescence spectrophotometer, with the target being an 8-fold increase in signal after activation;

[0128] Infected mouse model experiment: An infected mouse model was established. After injecting the probe into the tail vein, the enrichment of fluorescence signals at the infection site was observed using an in vivo imaging system (IVIS) and compared with normal tissue.

[0129] (4) Stability and toxicity testing

[0130] Serum stability: The probe was incubated in human serum at 37°C, and samples were taken at different time points. The remaining content of the probe was analyzed by HPLC, and the half-life was calculated. The target half-life is >6 hours.

[0131] Hemolysis test: Mix the probe with a fresh red blood cell suspension, incubate and centrifuge, take the supernatant and measure the absorbance, calculate the hemolysis rate, the target is hemolysis rate <5%;

[0132] Cytotoxicity: The probe was co-cultured with primary human hepatocytes, and cell viability was detected by methods such as the MTT assay, with a target cell viability of >95%.

[0133] The modular pathogenic microorganism-targeting fluorescent probe of the present invention has the following application scenarios:

[0134] (1) Tumor targeted therapy and imaging

[0135] Mechanism: Cy5.5 fluorescent labeling allows for real-time tracking of peptide accumulation at the tumor site; RWWR disrupts the tumor cell membrane; and PEG4 prolongs the treatment time.

[0136] Advantages: Integrated diagnosis and treatment, precisely killing tumor cells.

[0137] (2) Treatment of drug-resistant bacterial infections

[0138] Mechanism: D-Trp-D-Trp-Arg disrupts bacterial membranes, RWWR synergistically enhances permeability, and PEG4 reduces immune clearance.

[0139] Applicable pathogens: Methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa.

[0140] (3) Inflammatory microenvironment response

[0141] Mechanism: The acidic microenvironment (pH<6.5) triggers the self-assembly of hydrophobic modules, enhancing local drug concentration.

[0142] Applications: Targeted therapy for rheumatoid arthritis and atherosclerotic plaques.

[0143] Example 2

[0144] A modular antimicrobial drug delivery short peptide, with the following chemical structure:

[0145] Formyl-D-Phe-Leu-Met-PEG4-GSGSG-RRWR-Lys(hydrazone bond-PMB)-Pro-LLLHHH-NH2

[0146] It consists of the following functional modules:

[0147] Chemotaxis module: Includes formyl-D-Phe-Leu-Met, a formylated fMLF sequence in which phenylalanine is replaced by D-Phe. By binding to the formyl peptide receptor (FPR) on the surface of neutrophils / macrophages, it guides the short peptide to actively migrate to the site of infection, while the introduction of D-Phe enhances the protease resistance of the short peptide;

[0148] Targeting module: Includes RRWR, composed of cationic arginine and hydrophobic tryptophan. Arginine binds to negatively charged components of the microbial membrane (such as lipopolysaccharide) through electrostatic interactions, while tryptophan inserts into the hydrophobic region of the membrane. The synergistic effect of both allows the short peptide to be specifically enriched on the surface of pathogenic microorganisms. Compared to simple polyarginine, this sequence has stronger penetrability and lower cytotoxicity.

[0149] Drug loading and response release module: Includes a lysine (Lys) side chain, through which antibacterial drugs such as polymyxin B (PMB) or tigecycline (TGC) are covalently coupled using pH-sensitive linkers (hydrazone bonds). In the infection microenvironment, the low pH (~5.5) at the site of inflammation or the cleavage of hydrazone bonds by esterases on the microbial membrane surface can achieve specific release of the antibacterial drug;

[0150] The self-assembled membrane disruption module includes LLLHHH, which contains a hydrophobic region (LLL) and a histidine buffer domain (HHH). The hydrophobic region inserts into the lipid bilayer of the microbial membrane. Under the low pH conditions of an inflammatory environment, histidine is protonated, triggering the self-assembly of short peptides to form transmembrane pores, disrupting the integrity of the microbial membrane, and producing a synergistic antibacterial effect with the released antimicrobial drugs.

[0151] The connectivity and stability module comprises a PEG4-GSGSG flexible linker, C-terminal amidation, and key site D-amino acid modifications and proline additions to enhance short peptide stability. The PEG4-GSGSG flexible linker connects the various functional modules, ensuring relative independence and flexibility between them. Simultaneously, C-terminal amidation, the introduction of D-amino acids at key sites (such as D-Phe in the chemotactic module and D-Trp in the targeting module), and proline insertion (such as inserting Pro before the self-assembly membrane disruption module) enhance the short peptide's resistance to degradation and conformational stability.

[0152] Among them, the antibacterial drugs are polymyxin B (PMB) or tigecycline (TGC) and other antibacterial drugs.

[0153] The chemical structure of this short peptide contains the sequence shown in Table 3.

[0154] Table 3

[0155]

[0156] In the modular antimicrobial drug delivery short peptide of this embodiment, the breakdown and molecular weight of each functional module are shown in Table 4.

[0157] Table 4

[0158]

[0159] The conformational diagram of the short peptide in this embodiment is shown below. Figure 4 As shown, the function labels are as follows: Figure 5 As shown.

[0160] D-Phe-Leu-Met (hydrophobic core):

[0161] Conformation: D-type amino acids form rigid hydrophobic clusters, which are resistant to protease degradation.

[0162] Function: Stabilizes peptide structure and drives self-assembly.

[0163] PEG4 (hydrophilic protective layer):

[0164] Conformation: Hydrophilic chains encapsulate hydrophobic cores, reducing non-specific adsorption.

[0165] Function: Prolongs the circulation time in the body (predicted half-life ≈ 12 hours).

[0166] RRWR (Membrane Permeation Module):

[0167] Conformation: Tryptophan (W) π-π stacking forms a hydrophobic interface, and arginine (R) provides a positive charge (+3). Function: Synergistically disrupts bacterial or cell membranes.

[0168] Lys (Hydroxybond-PMB):

[0169] Conformation: The hydrazone bond breaks at pH < 6.0, releasing PMB (p-methoxybenzoic acid).

[0170] Function: Acidic microenvironments (such as tumors or sites of inflammation) trigger drug release.

[0171] LLLHHH (pH Response Module):

[0172] Conformation: Histidine (H) is protonated (+3) at pH 6.0, inducing self-assembly of β-sheets.

[0173] Function: Enhances local drug accumulation. The amino acid (R) provides a positive charge (+3).

[0174] Function: Enhances membrane penetration and targeting.

[0175] The key parameters and experimental verification results of the short peptide in this embodiment are shown in Table 5.

[0176] Table 5

[0177]

[0178] This embodiment describes a method for preparing a modular antimicrobial drug delivery short peptide, comprising the following steps:

[0179] (1) Solid-phase synthesis of short peptide chains

[0180] Rink Amide MBHA resin was placed in a reaction vessel and swollen with N,N-dimethylformamide (DMF) for 30 minutes.

[0181] Add 20% piperidine DMF solution to remove the Fmoc protecting group. After reacting for 15 minutes, wash the resin three times with DMF.

[0182] Fmoc-protected C-terminal amino acid (Fmoc-NH2), N,N-diisopropylethylamine (DIEA), and benzotriazole hexafluorophosphate (HBTU) were dissolved in DMF at a molar ratio of 1:2:1.2. After activation for 10 minutes, the solution was added to the resin and reacted at room temperature for 2 hours. The reaction was determined to be complete by ninhydrin detection. If the reaction was incomplete, the coupling step was repeated.

[0183] Following the sequence formyl-D-Phe-Leu-Met-PEG4-GSGSG-RRWR-Lys-Pro-LLLHHH, deprotection, amino acid activation, and coupling reactions were carried out sequentially, with amino acids added stepwise. During the synthesis, D-type amino acids (such as D-Phe and D-Trp) and proline were introduced at the appropriate positions, while the lysine side chain was retained.

[0184] After synthesis, the resin was treated with a lysis buffer containing 95% trifluoroacetic acid (TFA), 2.5% water and 2.5% triisopropylsilane (TIS) and reacted at room temperature for 2 hours to remove the short peptide from the resin and remove the protecting group.

[0185] TFA in the lysis buffer was removed by rotary evaporation, then cold diethyl ether was added to precipitate the short peptides, the precipitate was collected by centrifugation, and the crude short peptide chains were obtained by vacuum drying.

[0186] (2) Antimicrobial drug conjugation

[0187] The crude short peptide chain is dissolved with polymyxin B (PMB) or tigecycline (TGC) in a suitable organic solvent (such as dimethyl sulfoxide, DMSO), and an appropriate amount of condensing agent (such as N,N'-dicyclohexylcarbodiimide, DCC) and catalyst (such as 4-dimethylaminopyridine, DMAP) are added. The reaction is carried out at room temperature for 12-24 hours, and the antibacterial drug is coupled to the lysine side chain through hydrazone bonds.

[0188] (3) Purification

[0189] (4) After the reaction is completed, the product is purified by high performance liquid chromatography (HPLC). A C18 reversed-phase column is used, and gradient elution is performed with acetonitrile and water (containing 0.1% trifluoroacetic acid) as the mobile phase. The eluent corresponding to the target product peak is collected.

[0190] The collected eluent was concentrated using a rotary evaporator and then freeze-dried to obtain modular antimicrobial drug delivery short peptides.

[0191] Performance validation of short peptides

[0192] (1) Validation of chemotaxis and targeting

[0193] Transwell migration assay: In a Transwell chamber, culture medium containing chemokines was added to the lower chamber, and a short peptide solution was added to the upper chamber. After incubating the chambers at 37°C in a 5% CO2 incubator for a certain period of time, the short peptide solution in the lower chamber was collected, and the short peptide content was determined by high performance liquid chromatography (HPLC). The migration efficiency of the short peptides under the chemokine gradient was calculated, with a target migration efficiency greater than 70%.

[0194] Confocal localization assay: Fluorescently labeled short peptides were co-incubated with microorganisms (such as E. coli) under suitable conditions, and unbound short peptides were removed by washing with PBS. The binding of the short peptides to the microorganisms was observed using a confocal microscope, and the binding rate was calculated, with a target binding rate greater than 90%.

[0195] (2) Drug release verification

[0196] pH response experiment: The short peptide was placed in a buffer solution with pH 5.5, and samples were taken at different time points (e.g., 0 hours, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours). The release amount of PMB was detected by high performance liquid chromatography (HPLC), and the release kinetic curve was plotted. The target was that the release amount of PMB was greater than 80% within 24 hours.

[0197] Esterase activation experiment: Bacterial esterase was added to a short peptide solution and incubated under suitable conditions. Samples were taken at different time points, and the drug release efficiency was detected by HPLC to evaluate the effect of esterase on drug release.

[0198] (3) Verification of membrane damage capability

[0199] Liposome leakage experiment: Liposomes encapsulating calcein were prepared, and short peptides were mixed with the liposomes and incubated under suitable conditions. The leakage rate of the liposomes was calculated by measuring the fluorescence intensity of calcein in the solution. The target leakage rate was greater than 60% to demonstrate that the short peptides could disrupt the liposome membrane structure.

[0200] Electron microscopy observation experiment: The short peptide was co-cultured with bacteria, and the morphological changes on the surface of the bacterial membrane were observed by scanning electron microscopy (SEM) to verify whether the short peptide could form pores on the bacterial membrane.

[0201] (4) Synergistic antibacterial validation

[0202] Minimum inhibitory concentration (MIC) determination: The MIC values ​​of free PMB and short peptides were determined by microdilution method, and the difference between the two was compared. The goal was to reduce the MIC value by 4-8 times due to the synergistic effect of short peptides.

[0203] Time-bacterial kill curve determination: In the infection model, after adding the short peptide, samples were taken at different time points, and the number of bacteria was determined by colony counting method. Time-bacterial kill curves were plotted to evaluate the bactericidal kinetics of the short peptide in the infection model.

[0204] (5) Stability and toxicity verification

[0205] Serum half-life determination: The short peptide was placed in human serum at 37°C, and samples were taken at different time points (e.g., 0 hours, 1 hour, 2 hours, 4 hours, 6 hours, and 8 hours). The remaining content of the short peptide was analyzed by HPLC, and the half-life of the short peptide in serum was calculated. The target half-life is greater than 8 hours.

[0206] Hemolysis test: Short peptides of different concentrations were mixed with fresh red blood cell suspensions and incubated at 37°C for 1 hour. After centrifugation, the supernatant was collected, and the hemolysis rate was calculated by measuring the absorbance of the supernatant at 540 nm. The hemolysis rate was required to be less than 3% when the peptide concentration was ≤100 μM.

[0207] Cytotoxicity assay: Human primary kidney cells were co-cultured with different concentrations of short peptides, and cell viability was detected by the MTT assay. Cell survival rate was calculated with a target of greater than 95%.

[0208] The modular antimicrobial drug delivery short peptide of the present invention has the following applications:

[0209] This is used to prepare carriers for the precise delivery of anti-infective drugs for treating various pathogenic microorganisms.

[0210] Delivery systems for preparing anti-infective drugs for treating deep tissue infections (such as abdominal abscesses and pneumonia).

[0211] It is used as an adjunct therapy in various anti-infective treatments and has the effect of disrupting cell membranes and reducing adverse drug reactions.

[0212] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A modular fluorescent probe for targeting pathogenic microorganisms, characterized in that: Its chemical structure is: Formyl-D-Phe-Leu-D-Met-PEG4-GGSG-D-Trp-D-Trp-Arg-Lys(Cy5.5)-RWWR-NH2 It consists of the following functional modules: Chemotaxis module: including formyl-D-Phe-Leu-D-Met, used to achieve active migration to the infectious inflammatory microenvironment by binding to formyl peptide receptors; Targeting modules: including D-Trp-D-Trp-Arg and RWWR, used to specifically bind to the surface of pathogenic microorganisms through cationic and hydrophobic interactions; Fluorescence activation module: includes two Cy5.5 fluorescent groups covalently linked by amino groups on the lysine side chain, used to achieve fluorescence self-quenching-activation after binding to the microbial membrane; The linkage and stability module includes a PEG4-GGSG flexible linker, C-terminal amidation, and D-type amino acid modification at key sites to enhance probe stability.

2. The modular pathogenic microorganism-targeting fluorescent probe according to claim 1, characterized in that: In the chemotactic module, formylation enhances the affinity for the formyl peptide receptor, and D-phenylalanine prevents enzymatic degradation. In the targeting module, arginine binds to the negatively charged membrane components of pathogenic microorganisms, and tryptophan inserts into the hydrophobic region of the microbial membrane. In the fluorescence activation module, when the two Cy5.5 molecules are not bound to the microbial membrane, their fluorescence is quenched due to their spatial proximity. After binding, the two Cy5.5 molecules separate, which restores the fluorescence.

3. A method for preparing a modular pathogenic microorganism-targeting fluorescent probe as shown in any one of claims 1-2, characterized in that: Includes the following steps: Step 1: The following short peptide backbone was synthesized using a solid-phase synthesis method: Formyl-D-Phe-Leu-D-Met-PEG4-GGSG-D-Trp-D-Trp-Arg-Lys-RWWR; Step 2: The Cy5.5 fluorescent group is covalently coupled to the amino group of the lysine side chain in the short peptide using click chemistry. Step 3: After purification, the target fluorescent probe is obtained.

4. The application of the modular pathogenic microorganism-targeting fluorescent probe according to any one of claims 1-2 in the preparation of pathogenic microorganism detection reagents or infectious inflammatory site imaging reagents.

5. The application according to claim 4, characterized in that: The reagent is used for real-time marking of infection foci during surgery and for accurate diagnosis of drug-resistant bacterial infections.

6. A modular antimicrobial drug delivery short peptide, characterized in that: Its chemical structure is: Formyl-D-Phe-Leu-Met-PEG4-GSGSG-RRWR-Lys(hydrazone bond-PMB)-Pro-LLLHHH-NH2 It consists of the following functional modules: Chemotaxis module: including formyl-D-Phe-Leu-Met, which is used to achieve active migration to the infectious inflammatory microenvironment by binding to formyl peptide receptors; Targeting module: includes RRWR, used to specifically bind to the surface of pathogenic microorganisms through cationic and hydrophobic interactions; Drug loading and response release module: includes a lysine (Lys) side chain for coupling antibacterial drugs via pH-sensitive hydrazone bonds, releasing the drugs in the low pH of the infection microenvironment or under the action of esterases; Self-assembly membrane disruption module: including LLLHHH, used to self-assemble into transmembrane pores to disrupt microbial membranes under inflammatory low pH conditions; The linker and stability module includes the PEG4-GSGSG flexible linker, C-terminal amidation, and D-type amino acid modifications and proline at key sites to enhance the stability of short peptides.

7. The modular antimicrobial drug delivery short peptide according to claim 6, characterized in that: The antibacterial drugs are polymyxin B (PMB) or tigecycline (TGC) and other antibacterial drugs.

8. The modular antimicrobial drug delivery short peptide according to claim 6, characterized in that: In the chemotactic module, active migration to the site of infection is achieved by binding to the formyl peptide receptor (FPR) on the surface of neutrophils / macrophages; In the target module, arginine in the RRWR sequence electrostatically binds to the negatively charged components of the microbial membrane, and tryptophan inserts into the hydrophobic region of the membrane. In the self-assembled membrane disruption module, the hydrophobic region LLL is inserted into the microbial membrane lipid bilayer, and the histidine buffer domain HHH is protonated at low pH to trigger self-assembly.

9. A method for preparing a modular antimicrobial drug delivery short peptide according to any one of claims 6-8, characterized in that: Includes the following steps: Step a: The following short peptide chains were synthesized sequentially from the C-terminus to the N-terminus using a solid-phase synthesis method: Formyl-D-Phe-Leu-Met-PEG4-GSGSG-RRWR-Lys-Pro-LLLHHH; D-type amino acids and proline are introduced during the synthesis process; Step b involves using click chemistry to couple polymyxin B (PMB) or tigecycline (TGC) to the lysine side chain via hydrazone bonds. Step c: Purify the synthesized short peptide and freeze-dry it to obtain the target short peptide.

10. The use of the modular antimicrobial drug delivery short peptide according to any one of claims 6-8 in the preparation of drugs for treating multidrug-resistant Gram-negative bacterial infections, other bacterial infections, fungal infections, antiviral drugs, or drugs for deep tissue infections.

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