Prodrugs for use against infections and uses thereof

Abstract

The present application relates to a kind of for anti-infection prodrug and its application. Specifically, the present application provides a meropenem prodrug, the structure of the meropenem prodrug is as shown below. The meropenem prodrug of the present application has excellent therapeutic effect on bacterial biofilm-related infection.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, and more specifically to a prodrug for anti-infection and its application. Background Technology

[0002] Infections are a common clinical disease that endangers human health. If infections are not treated effectively, they often lead to many complications such as fever and pain, and in severe cases, they can cause shock and threaten life and health.

[0003] Clinically, infections such as bacterial infections are often treated with medications such as the antibiotic meropenem. While existing anti-infective drugs have achieved good results in treating infections, they still have many shortcomings. For example, anti-infective drugs have difficulty effectively targeting and accumulating at the site of infection and effectively inhibiting bacteria at the infection site. This limits the therapeutic effect of anti-infective drugs and reduces patient compliance with anti-infective drugs, especially for bacterial biofilm-related infections. The physical barrier composed of extracellular polymeric substances (EPS) in bacterial biofilms can resist antibiotic treatment. Therefore, developing drugs and methods for effectively treating infections remains a hot topic in current medical research.

[0004] Therefore, there is a need in this field to develop a drug that can effectively treat infections. Summary of the Invention

[0005] The purpose of this invention is to provide a drug for the effective treatment of infections such as bacterial infections.

[0006] In a first aspect, the present invention provides a meropenem prodrug, the structure of which is shown below:

[0007]

[0008] A second aspect of the present invention provides a liposome comprising a drug for preventing and / or treating infection.

[0009] Preferably, the infection includes a bacterial infection.

[0010] Preferably, the infection includes bacterial biofilm-related infections.

[0011] Preferably, the infection includes biofilm-related surgical site infections.

[0012] Preferably, the infection includes surgical site infection associated with bacterial biofilm.

[0013] Preferably, the bacteria include Pseudomonas aeruginosa.

[0014] Preferably, the bacteria include Pseudomonas aeruginosa PAO1.

[0015] Preferably, the liposomes are loaded with the drug for preventing and / or treating infection.

[0016] Preferably, the drug includes a pharmaceutically active ingredient, a prodrug, or a drug conjugate.

[0017] Preferably, the drug includes meropenem.

[0018] Preferably, the drug comprises meropenem prodrug as described in the first aspect of the invention.

[0019] Preferably, the drug includes drugs that are retained and / or degraded by lysosomes.

[0020] Preferably, the degradation includes degradation by lysosomal enzymes.

[0021] Preferably, the drug comprises a drug that is degraded by lysosomal enzymes.

[0022] Preferably, the drug targets the cytoplasm or the nucleus.

[0023] Preferably, the drug comprises a small molecule drug, a gene, or a protein.

[0024] Preferably, the gene is selected from the group consisting of DNA, RNA, or a combination thereof.

[0025] Preferably, the meropenem prodrug is a lipid bilayer.

[0026] Preferably, the meropenem prodrug is a lipid bilayer of a liposome.

[0027] Preferably, the liposomes are loaded with the meropenem prodrug.

[0028] Preferably, the liposomes comprise perfluoropentane.

[0029] Preferably, the liposomes are loaded with perfluoropentane.

[0030] Preferably, the lipid bilayer of the liposome is loaded with perfluoropentane.

[0031] Preferably, the liposomes further include a VCAM1 ligand.

[0032] Preferably, the VCAM1 ligand includes a ligand that targets VCAM1.

[0033] Preferably, the VCAM1 ligand modifies the liposome.

[0034] Preferably, the VCAM1 ligand is modified on the outer surface of the liposome.

[0035] Preferably, the VCAM1 ligand is modified onto the lipid material of the liposome.

[0036] Preferably, the modification includes physical modification and / or chemical modification.

[0037] Preferably, the modification includes physical adsorption, chemisorption, and / or coupling.

[0038] Preferably, the VCAM1 ligand is adsorbed on the surface of the liposome.

[0039] Preferably, the adsorption includes physical adsorption and / or chemical adsorption.

[0040] Preferably, the VCAM1 ligand is coupled to the lipid material of the liposome.

[0041] Preferably, the VCAM1 ligand comprises a polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0042] Preferably, the liposomes comprise dihydroporphyrin E6.

[0043] Preferably, the liposomes are loaded with dihydroporphyrin E6.

[0044] Preferably, the liposomes comprise lipid materials.

[0045] Preferably, the lipid material is a lipid bilayer.

[0046] Preferably, the lipid material comprises one or more of the following: 1,2-dispalmitoyl-sn-glycerol-3-phosphocholine, 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with VCAM1 ligand, 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with dihydroporphyrin E6, and 3β-[N-(N,N-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride.

[0047] Preferably, the lipid material comprises 1,2-dispalmitoyl-sn-glycerol-3-phosphocholine, 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with VCAM1 ligand, 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with dihydroporphyrin E6, and 3β-[N-(N,N-dimethylamine ethyl)]

[0048] [Carbamoyl] cholesterol hydrochloride.

[0049] Preferably, the liposomes comprise 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine.

[0050] Preferably, the liposomes comprise 1,2-distearate-sn-glycerol-3-phosphorylethanolamine-N-[polyethylene glycol-2000] coupled with dihydroporphyrin E6.

[0051] Preferably, the liposome comprises 1,2-distearate-sn-glycerol-3-phosphorylethanolamine-N-[polyethylene glycol-2000] coupled with VCAM1 ligand.

[0052] Preferably, the liposomes comprise 3β-[N-(N,N-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride.

[0053] Preferably, the liposomes comprise one or more of the following: 1,2-dispalmitoyl-sn-glycerol-3-phosphocholine; 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with VCAM1 ligand; 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with dihydroporphyrin E6; 3β-[N-(N,N-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride; and meropenem prodrugs as described in the first aspect of the invention.

[0054] Preferably, the liposomes comprise 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with VCAM1 ligand, 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with dihydroporphyrin E6, 3β-[N-(N,N-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride, and meropenem prodrug as described in the first aspect of the invention.

[0055] Preferably, the drug (such as meropenem prodrug) is 0.5-10 parts by weight, more preferably 0.8-8 parts by weight, even more preferably 1-5 parts by weight, even more preferably 2-4 parts by weight, even more preferably 2.5-3.5 parts by weight, even more preferably 2.8-3.2 parts by weight, and most preferably 3.0 parts by weight.

[0056] Preferably, the 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine is 0.5-10 parts by weight, more preferably 0.8-8 parts by weight, more preferably 1-5 parts by weight, more preferably 2-4 parts by weight, more preferably 2.5-3.5 parts by weight, more preferably 2.8-3.2 parts by weight, and most preferably 3.0 parts by weight.

[0057] Preferably, the 1,2-distearate-sn-glycerol-3-phosphorylethanolamine-N-[polyethylene glycol-2000] coupled to the VCAM1 ligand is 0.5-10 parts by weight, more preferably 0.8-8 parts by weight, more preferably 1-5 parts by weight, more preferably 2-4 parts by weight, more preferably 2.5-3.5 parts by weight, more preferably 2.8-3.2 parts by weight, and most preferably 3.0 parts by weight.

[0058] Preferably, the dihydroporphyrin E6-coupled 1,2-distearate-sn-glycerol-3-phosphorylethanolamine-N-[polyethylene glycol-2000] is 1-15 parts by weight, more preferably 2-10 parts by weight, more preferably 3-8 parts by weight, more preferably 5-7 parts by weight, more preferably 5.5-6.5 parts by weight, more preferably 5.8-6.2 parts by weight, and most preferably 6.0 parts by weight.

[0059] Preferably, the 3β-[N-(N,N-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride is 0.5-10 parts by weight, more preferably 0.8-8 parts by weight, more preferably 1-5 parts by weight, more preferably 2-4 parts by weight, more preferably 2.5-3.5 parts by weight, more preferably 2.8-3.2 parts by weight, and most preferably 3.0 parts by weight.

[0060] Preferably, the weight ratio of 1,2-dispalmitoyl-sn-glycerol-3-phosphocholine, 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with VCAM1 ligand, 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with dihydroporphyrin E6, 3β-[N-(N,N-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride, and the drug is (0.8-1.2):(0.8-1.2):(1.8-2.2):(0.8-1.2):(0.8-1.2), more preferably 1:1:2:1:1.

[0061] Preferably, the perfluoropentane is 0.01-0.5 parts by weight, more preferably 0.02-0.2 parts by weight, even more preferably 0.05-0.15 parts by weight, even more preferably 0.08-0.12 parts by weight, and most preferably 0.1 parts by weight.

[0062] Preferably, the mass-to-volume ratio (mg:μl) of the 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine to the perfluoro-n-pentane is (1-5):(70-130), more preferably (2-4):(80-120), even more preferably (2.5-3.5):(90-110), even more preferably (2.8-3.2):(95-105), and even more preferably 3:100.

[0063] Preferably, the liposomes further include water and / or a buffer solution.

[0064] Preferably, the liposomes are loaded with water and / or a buffer solution.

[0065] Preferably, the lipid bilayer of the liposome is coated with water and / or a buffer solution.

[0066] Preferably, the buffer solution comprises a buffer aqueous solution.

[0067] Preferably, the buffer solution comprises glycerol phosphate buffer.

[0068] Preferably, the glycerol phosphate buffer contains 5-15% glycerol by volume, more preferably 8-12%, and even more preferably 10%.

[0069] Preferably, the concentration of the glycerol phosphate buffer is 5-15 mM, more preferably 8-12 mM, and even more preferably 10 mM, based on the concentration of phosphate.

[0070] Preferably, the pH of the glycerol phosphate buffer solution is 7.2-7.6, more preferably 7.4.

[0071] Preferably, the lipid bilayer is coated with perfluoropentane and / or a buffer solution.

[0072] Preferably, the volume ratio of the perfluoropentane to the buffer solution is 1:40-60, more preferably 1:45-55, even more preferably 1:48-52, and even more preferably 1:50.

[0073] Preferably, the liposomes have a particle size of 130-300 nm, more preferably 150-270 nm, even more preferably 160-250 nm, even more preferably 170-240 nm, and most preferably 180-230 nm.

[0074] Preferably, the potential of the liposome is -35mV to -4mV, more preferably -30mV to -6mV, even more preferably -25mV to -8mV, and even more preferably -22mV to -9mV.

[0075] A third aspect of the present invention provides a method for preparing liposomes as described in the second aspect of the present invention, the method comprising the steps of:

[0076] (1) The lipid material and the meropenem prodrug as described in the first aspect of the present invention are dissolved in an organic solvent, and the organic solvent is removed to obtain a lipid membrane;

[0077] (2) After immersing the lipid membrane in perfluoropentane, a buffer solution was added for hydration and stirring to obtain liposomes.

[0078] Preferably, in step (1), the organic solvent is selected from the group consisting of chloroform, dichloromethane, or combinations thereof.

[0079] Preferably, in step (1), the mass-to-volume ratio (mg:ml) of the lipid material to the organic solvent is 3-8:1, more preferably 4-6:1, even more preferably 4.5-5.5:1, even more preferably 4.8-5.2:1, and even more preferably 5:1.

[0080] Preferably, in step (1), the mass-volume ratio (mg:ml) of 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine to the organic solvent is 0.2-2:1, more preferably 0.5-1.5:1, even more preferably 0.8-1.2:1, and even more preferably 1:1.

[0081] Preferably, in step (1), the mass-to-volume ratio (mg:ml) of the meropenem prodrug to the organic solvent is 0.2-2:1, more preferably 0.5-1.5:1, even more preferably 0.8-1.2:1, and even more preferably 1:1.

[0082] Preferably, in step (1), the organic solvent is removed by rotary vacuum evaporation.

[0083] Preferably, in step (1), the organic solvent is removed by rotary vacuum evaporation at 20-30°C.

[0084] Preferably, the mass-to-volume ratio (mg:μl) of the 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine to the perfluoro-n-pentane is (1-5):(70-130), more preferably (2-4):(80-120), even more preferably (2.5-3.5):(90-110), even more preferably (2.8-3.2):(95-105), and even more preferably 3:100.

[0085] Preferably, in step (2), the volume ratio of the perfluoropentane to the buffer solution is 1:40-60, more preferably 1:45-55, more preferably 1:48-52, and even more preferably 1:50.

[0086] Preferably, in step (2), the lipid membrane is immersed in perfluoropentane at low temperature.

[0087] Preferably, in step (2), the hydration is carried out at a low temperature.

[0088] Preferably, in step (2), the stirring includes the following steps:

[0089] Stir at a low temperature first, then stir again after raising the temperature.

[0090] Preferably, the low temperature is 2-10°C, more preferably 2-6°C, and even more preferably 4°C.

[0091] Preferably, the stirring time at low temperature is 20-40 min, more preferably 25-35 min, and even more preferably 30 min.

[0092] Preferably, the temperature rise is 20-40°C, more preferably 25-35°C, and even more preferably 28-32°C.

[0093] Preferably, the stirring time after the temperature is increased is 0.5-1.5h, more preferably 0.8-1.2h, and even more preferably 1h.

[0094] Preferably, during the stirring process after the temperature is raised, the container holding the stirring liquid is in an open state.

[0095] Preferably, the liposomes are in the form of a liposome dispersion.

[0096] Preferably, the encapsulation efficiency of the liposomes is ≥90%, more preferably ≥95%, more preferably ≥99%, and most preferably 100%.

[0097] Preferably, the drug loading of the liposomes is 5-10 wt%, more preferably 7-8 wt%.

[0098] Preferably, the method includes the following steps:

[0099] (1) 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine, 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with VCAM1 ligand, 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with dihydroporphyrin E6, 3β-[N-(N,N-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride and meropenem prodrug as described in the first aspect of the present invention are dissolved in chloroform in a round-bottom flask, and the solvent is removed by rotary vacuum evaporation to form a lipid film in the round-bottom flask;

[0100] (2) Cool the lipid membrane to 2-6℃, add perfluoropentane to immerse the lipid membrane, then add a buffer solution for hydration, stir at 2-6℃ for 25-35 min, and then stir in a round-bottom flask at 25-35℃ for 0.8-1.2 h to obtain liposomes.

[0101] Preferably, the method includes the following steps:

[0102] (1) 2.8-3.2 mg of 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, 2.8-3.2 mg of 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with VCAM1 ligand, 5.8-6.2 mg of 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with dihydroporphyrin E6, 2.8-3.2 mg of 3β-[N-(N,N-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride and 2.8-3.2 mg of meropenem prodrug as described in the first aspect of the present invention are dissolved in chloroform in a round-bottom flask, and the solvent is removed by rotary vacuum evaporation to form a lipid film in the round-bottom flask;

[0103] (2) Cool the lipid membrane to 2-6℃, add 90-110μL of perfluoron-pentane to immerse the lipid membrane, then add 4.5-5.5ml of buffer solution for hydration, stir at 2-6℃ for 25-35min, and then stir in a round-bottom flask at 25-35℃ for 0.8-1.2h with the mouth open to obtain liposomes.

[0104] In a fourth aspect, the present invention provides a VCAM1 ligand-modified medicament, said medicament comprising a medicament for the prevention and / or treatment of infection.

[0105] Preferably, the VCAM1 ligand includes a ligand that targets VCAM1.

[0106] Preferably, the VCAM1 ligand comprises a polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0107] Preferably, the infection includes a bacterial infection.

[0108] Preferably, the infection includes bacterial biofilm-related infections.

[0109] Preferably, the infection includes biofilm-related surgical site infections.

[0110] Preferably, the infection includes surgical site infection associated with bacterial biofilm.

[0111] Preferably, the bacteria include Pseudomonas aeruginosa.

[0112] Preferably, the bacteria include Pseudomonas aeruginosa PAO1.

[0113] Preferably, the drug includes a pharmaceutically active ingredient, a prodrug, or a drug conjugate.

[0114] Preferably, the drug includes meropenem.

[0115] Preferably, the drug comprises meropenem prodrug as described in the first aspect of the invention.

[0116] Preferably, the drug includes drugs that are retained and / or degraded by lysosomes.

[0117] Preferably, the degradation includes degradation by lysosomal enzymes.

[0118] Preferably, the drug comprises a drug that is degraded by lysosomal enzymes.

[0119] Preferably, the drug targets the cytoplasm or the nucleus.

[0120] Preferably, the drug comprises a small molecule drug, a gene, or a protein.

[0121] Preferably, the gene is selected from the group consisting of DNA, RNA, or a combination thereof.

[0122] Preferably, the VCAM1 ligand-modified drug comprises VCAM1 ligand-modified liposomes.

[0123] Preferably, the VCAM1 ligand-modified drug comprises liposomes, the liposomes comprising 1,2-dispalmitoyl-sn-glycerol-3-phosphocholine, 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with VCAM1 ligand, 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with dihydroporphyrin E6, 3β-[N-(N,N-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride, and meropenem prodrug as described in the first aspect of the invention.

[0124] Preferably, the liposomes comprise perfluoropentane.

[0125] Preferably, the liposomes are loaded with perfluoropentane.

[0126] Preferably, the lipid bilayer of the liposome is loaded with perfluoropentane.

[0127] Preferably, the liposomes are as described in the second aspect of the present invention above.

[0128] A fifth aspect of the present invention provides a composition comprising a meropenem prodrug as described in the first aspect of the present invention, a liposome as described in the second aspect of the present invention, or a drug modified with a VCAM1 ligand as described in the fourth aspect of the present invention.

[0129] Preferably, the composition is a composition for the prevention and / or treatment of infection.

[0130] Preferably, the infection includes a bacterial infection.

[0131] Preferably, the infection includes bacterial biofilm-related infections.

[0132] Preferably, the infection includes biofilm-related surgical site infections.

[0133] Preferably, the infection includes surgical site infection associated with bacterial biofilm.

[0134] Preferably, the bacteria include Pseudomonas aeruginosa.

[0135] Preferably, the bacteria include Pseudomonas aeruginosa PAO1.

[0136] Preferably, the composition further includes a reactive oxygen species generator.

[0137] Preferably, the reactive oxygen species generating agent includes a reagent that generates reactive oxygen species under ultrasonic irradiation conditions.

[0138] Preferably, the composition is a pharmaceutical composition.

[0139] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0140] Preferably, the dosage form of the composition is a solid dosage form, a liquid dosage form, or a semi-solid dosage form.

[0141] Preferably, the dosage form of the composition is an injectable formulation, an oral formulation, or a topical formulation.

[0142] Preferably, the injectable formulation includes an intravascular injection formulation.

[0143] Preferably, the injectable preparation is an intravenous injection preparation.

[0144] A sixth aspect of the present invention provides the use of a meropenem prodrug as described in the first aspect of the present invention, a liposome as described in the second aspect of the present invention, a VCAM1 ligand-modified medicament as described in the fourth aspect of the present invention, or a composition as described in the fifth aspect of the present invention, for the preparation of a formulation for the prevention and / or treatment of infection.

[0145] Preferably, the infection includes a bacterial infection.

[0146] Preferably, the infection includes bacterial biofilm-related infections.

[0147] Preferably, the infection includes biofilm-related surgical site infections.

[0148] Preferably, the infection includes surgical site infection associated with bacterial biofilm.

[0149] Preferably, the bacteria include Pseudomonas aeruginosa.

[0150] Preferably, the bacteria include Pseudomonas aeruginosa PAO1.

[0151] Preferably, the infection includes infection in humans or non-human mammals.

[0152] Preferably, the preparation is administered to humans or non-human mammals.

[0153] Preferably, the non-human mammals include mice, rats, dogs, rabbits, sheep, or cattle.

[0154] Preferably, the non-human mammal includes the BALB / c nude mouse.

[0155] Preferably, the preparation is a pharmaceutical preparation.

[0156] Preferably, the formulation further includes a pharmaceutically acceptable carrier.

[0157] Preferably, the formulation is a solid formulation, a liquid formulation, or a semi-solid formulation.

[0158] Preferably, the preparation is an injectable preparation, an oral preparation, or a topical preparation.

[0159] Preferably, the injectable formulation includes an intravascular injection formulation.

[0160] Preferably, the injectable preparation is an intravenous injection preparation.

[0161] Preferably, during the administration of meropenem prodrug as described in the first aspect of the invention, liposomes as described in the second aspect of the invention, VCAM1 ligand-modified drugs as described in the fourth aspect of the invention, or compositions as described in the fifth aspect of the invention for the prevention and / or treatment of infection, the infected site is subjected to ultrasonic irradiation.

[0162] Preferably, only the infected area is subjected to ultrasonic irradiation treatment.

[0163] A seventh aspect of the present invention provides a method for preventing and / or treating infection, the method comprising the steps of administering to a desired subject a meropenem prodrug as described in the first aspect of the present invention, a liposome as described in the second aspect of the present invention, a VCAM1 ligand-modified drug as described in the fourth aspect of the present invention, or a composition as described in the fifth aspect of the present invention, thereby preventing and / or treating infection.

[0164] Preferably, the infection is as described in the sixth aspect of the present invention.

[0165] Preferably, the object is a human or a non-human mammal.

[0166] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0167] Preferably, the object includes a BALB / c nude mouse.

[0168] Preferably, the application is by injection, oral administration, or topical application.

[0169] Preferably, the administration is by intravascular injection.

[0170] Preferably, the injection is administered intravenously.

[0171] Preferably, the method includes the following steps:

[0172] After administering meropenem prodrug as described in the first aspect of the invention, liposomes as described in the second aspect of the invention, VCAM1 ligand-modified drugs as described in the fourth aspect of the invention, or compositions as described in the fifth aspect of the invention to the desired subject, the site of infection is subjected to ultrasonic irradiation to prevent and / or treat infection.

[0173] Preferably, during the administration of meropenem prodrug as described in the first aspect of the invention, liposomes as described in the second aspect of the invention, VCAM1 ligand-modified drugs as described in the fourth aspect of the invention, or compositions as described in the fifth aspect of the invention for the prevention and / or treatment of infection, the infected site is subjected to ultrasonic irradiation.

[0174] Preferably, only the infected area is subjected to ultrasonic irradiation treatment.

[0175] An eighth aspect of the present invention provides a system or apparatus for preventing and / or treating infections, said system or apparatus comprising a meropenem prodrug as described in the first aspect of the present invention, a liposome as described in the second aspect of the present invention, a VCAM1 ligand-modified drug as described in the fourth aspect of the present invention, or a composition as described in the fifth aspect of the present invention; and an ultrasound device.

[0176] Preferably, the system or device further includes a manual or label, which states:

[0177] During the administration of meropenem prodrug as described in the first aspect of the invention, liposomes as described in the second aspect of the invention, VCAM1 ligand-modified drugs as described in the fourth aspect of the invention, or compositions as described in the fifth aspect of the invention for the prevention and / or treatment of infection, the infected site is subjected to ultrasonic irradiation.

[0178] Preferably, the ultrasonic device includes an ultrasonic transducer.

[0179] Preferably, the infection is as described in the sixth aspect of the present invention.

[0180] Preferably, the object includes a human or a non-human mammal.

[0181] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0182] Preferably, the application is by injection, oral administration, or topical application.

[0183] Preferably, the administration is by intravascular injection.

[0184] Preferably, the injection is administered intravenously.

[0185] A ninth aspect of the invention provides the use of a system or apparatus as described in the eighth aspect of the invention for preparing a device for preventing and / or treating infections.

[0186] Preferably, the device further includes an instruction manual or label, which states:

[0187] During the administration of meropenem prodrug as described in the first aspect of the invention, liposomes as described in the second aspect of the invention, VCAM1 ligand-modified drugs as described in the fourth aspect of the invention, or compositions as described in the fifth aspect of the invention for the prevention and / or treatment of infection, the infected site is subjected to ultrasonic irradiation.

[0188] Preferably, the ultrasound device performs ultrasound irradiation treatment on the infected site.

[0189] Preferably, ultrasound irradiation is performed only on the infected site.

[0190] Preferably, the infection is as described in the sixth aspect of the present invention.

[0191] Preferably, the object includes a human or a non-human mammal.

[0192] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0193] Preferably, the application is by injection, oral administration, or topical application.

[0194] Preferably, the administration is by intravascular injection.

[0195] Preferably, the injection is administered intravenously.

[0196] In a tenth aspect, the present invention provides an application of an ultrasonic device for manufacturing equipment, the device being used for the following purposes:

[0197] The preventive and / or therapeutic effects of meropenem prodrugs as described in the first aspect of the invention, liposomes as described in the second aspect of the invention, VCAM1 ligand-modified drugs as described in the fourth aspect of the invention, or compositions as described in the fifth aspect of the invention, applied to the target subject, are enhanced by ultrasonic irradiation of the infected site.

[0198] Preferably, the ultrasonic device includes an ultrasonic transducer.

[0199] Preferably, the infected site is treated with ultrasonic irradiation using an ultrasonic device.

[0200] Preferably, the infection is as described in the sixth aspect of the present invention.

[0201] Preferably, the object includes a human or a non-human mammal.

[0202] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0203] Preferably, during the administration of meropenem prodrug as described in the first aspect of the invention, liposomes as described in the second aspect of the invention, VCAM1 ligand-modified drugs as described in the fourth aspect of the invention, or compositions as described in the fifth aspect of the invention for the prevention and / or treatment of infection, the infected site is subjected to ultrasonic irradiation.

[0204] Preferably, the infected site is treated with ultrasonic irradiation using an ultrasonic device (such as an ultrasonic instrument).

[0205] Preferably, ultrasound irradiation is performed only on the infected site.

[0206] Preferably, the application is by injection, oral administration, or topical application.

[0207] Preferably, the administration is by intravascular injection.

[0208] Preferably, the injection is administered intravenously.

[0209] In an eleventh aspect, the present invention provides a use of a VCAM1 ligand for preparing a modifier, said modifier being used for one or more uses selected from the group consisting of: (a) modifying a drug; and / or (b) enhancing the efficacy of a drug against an infection;

[0210] The drugs mentioned include those for the prevention and / or treatment of infections.

[0211] Preferably, in (b), the VCAM1 ligand-modified drug.

[0212] Preferably, (b) includes enhancing the drug's efficacy against infection by modifying the drug with the VCAM1 ligand.

[0213] Preferably, the VCAM1 ligand includes a ligand that targets VCAM1.

[0214] Preferably, the VCAM1 ligand comprises a polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0215] Preferably, the infection includes a bacterial infection.

[0216] Preferably, the infection includes bacterial biofilm-related infections.

[0217] Preferably, the infection includes biofilm-related surgical site infections.

[0218] Preferably, the infection includes surgical site infection associated with bacterial biofilm.

[0219] Preferably, the bacteria include Pseudomonas aeruginosa.

[0220] Preferably, the bacteria include Pseudomonas aeruginosa PAO1.

[0221] Preferably, the drug includes a pharmaceutically active ingredient, a prodrug, or a drug conjugate.

[0222] Preferably, the drug includes meropenem.

[0223] Preferably, the drug comprises meropenem prodrug as described in the first aspect of the invention.

[0224] Preferably, the drug includes drugs that are retained and / or degraded by lysosomes.

[0225] Preferably, the degradation includes degradation by lysosomal enzymes.

[0226] Preferably, the drug comprises a drug that is degraded by lysosomal enzymes.

[0227] Preferably, the drug targets the cytoplasm or the nucleus.

[0228] Preferably, the drug comprises a small molecule drug, a gene, or a protein.

[0229] Preferably, the gene is selected from the group consisting of DNA, RNA, or a combination thereof.

[0230] According to a twelfth aspect of the present invention, a method for enhancing the efficacy of a drug against infection is provided, the method comprising the steps of:

[0231] Drugs can be modified with VCAM1 ligands to enhance their efficacy against infection.

[0232] The drugs mentioned include those for the prevention and / or treatment of infections.

[0233] Preferably, the method includes non-diagnostic and non-therapeutic methods.

[0234] Preferably, the VCAM1 ligand includes a ligand that targets VCAM1.

[0235] Preferably, the VCAM1 ligand comprises a polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0236] Preferably, the drug is as described in the eleventh aspect of the present invention.

[0237] Preferably, the infection is as described in the eleventh aspect of the present invention.

[0238] According to a thirteenth aspect of the present invention, a medicine box is provided, the medicine box comprising:

[0239] (1) Medications for the prevention and / or treatment of infection; and

[0240] (2) VCAM1 ligand.

[0241] Preferably, the medicine box includes a medicine box for the prevention and / or treatment of infection.

[0242] Preferably, the medicine box further includes an instruction manual, which states:

[0243] Drugs can be modified with VCAM1 ligands to enhance their efficacy against infection.

[0244] Preferably, the VCAM1 ligand includes a ligand that targets VCAM1.

[0245] Preferably, the VCAM1 ligand comprises a polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0246] Preferably, the drug is as described in the eleventh aspect of the present invention.

[0247] Preferably, the infection is as described in the eleventh aspect of the present invention.

[0248] In a fourteenth aspect of the present invention, there is a use of a medicine box as described in a seventh aspect of the present invention for preparing a medicine box for preventing and / or treating infections.

[0249] Preferably, the medicine box further includes an instruction manual, which states:

[0250] Drugs can be modified with VCAM1 ligands to enhance their efficacy against infection.

[0251] Preferably, the infection is as described in the eleventh aspect of the present invention.

[0252] Within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below can be combined with each other to form new or preferred technical solutions. Attached Figure Description

[0253] Figure 1 This is the synthetic route for OTM prodrugs.

[0254] Figure 2 The proton NMR spectrum of an OTM prodrug 1 H-NMR spectrum.

[0255] Figure 3 This is a matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) spectrum of an OTM prodrug.

[0256] Figure 4 This is the synthetic route for the OM prodrug.

[0257] Figure 5 The proton nuclear magnetic resonance spectrum of OM prodrug ( 1 H-NMR spectrum.

[0258] Figure 6 This is a matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) spectrum of the OM prodrug.

[0259] Figure 7 This is a cryo-transmission electron microscope (cryo-TEM) image of LPCOTML liposomes.

[0260] Figure 8 Immunofluorescence staining of VCAM1 (vascular cell adhesion molecule-1) in tissues infected with bacterial biofilms from BALB / c nude mice with deep surgical site infection and tissues from surgical sites in sham-operated BALB / c nude mice, scale bar = 100 μm.

[0261] Figure 9 Immunofluorescence staining of VEGFR1 (vascular endothelial growth factor receptor 1) in tissues infected with bacterial biofilms from BALB / c nude mice with deep surgical site infection and tissues from surgical sites in sham-operated BALB / c nude mice, scale bar = 100 μm.

[0262] Figure 10 Western blot analysis was conducted to determine the expression of VCAM1 (vascular cell adhesion molecule-1) in bacterial biofilm-infected tissues from BALB / c nude mice with deep surgical site infection and in surgical site tissues from sham-operated BALB / c nude mice. A p < 0.01 was considered statistically significant.

[0263] Figure 11 Western blot analysis was conducted to determine the expression of VEGFR1 (vascular endothelial growth factor receptor 1) in bacterial biofilm-infected tissues from BALB / c nude mice with deep surgical site infection and in surgical site tissues from sham-operated BALB / c nude mice. "ns" indicates no significance.

[0264] Figure 12 The percentage of meropenem (MEM) cumulatively released at different release time points in a methanol / H2O (1 / 1, v / v) mixed solution release medium of 0.5 mM H2O2.

[0265] Figure 13 The amount of meropenem (MEM) cumulatively released at a 1-hour release time point in a methanol / H2O (1 / 1, v / v) mixed solution release medium containing different concentrations of H2O2 is defined as the amount of meropenem (MEM) released by an OTM prodrug or an OM prodrug.

[0266] Figure 14 The content of meropenem (MEM) released from LPCOTML liposome dispersions (all liposomes contain 10 μM equivalent of meropenem) at 37°C with or without ultrasonic irradiation is given. "US" refers to ultrasonic irradiation (sound intensity: 2 W / cm²). 2 (Frequency: 3MHz, Duty Cycle: 50%, Duration: 10min) Processing.

[0267] Figure 15 VCAM1 receptor expression in HUVEC cells and inflammatory vascular endothelial cells (IVEC) was measured by Western blotting. GAPDH was used as a control protein in the Western blotting method. **p<0.01 indicates statistical significance.

[0268] Figure 16The mean fluorescence intensity (MFI) of Cy5-labeled COTML, PCOTML, LCOTML, or LPCOTML liposomes was measured by flow cytometry after incubation for 1 h with HUVEC (human umbilical vein endothelial cells) or inflammatory vascular endothelial cells (IVEC). A p < 0.01 was considered statistically significant.

[0269] Figure 17 To determine the subcellular distribution of Cy5-labeled LPCOTML liposomes in inflammatory vascular endothelial cells (IVEC) or HUVEC (human umbilical vein endothelial cells) cells after incubation for 0.5 h using confocal laser scanning microscopy (CLSM), cell nuclei were stained with Hoechst 33342, cell membranes were stained with DIO, and LPCOTML liposomes were labeled with Cy5. Scale bar = 25 μm.

[0270] Figure 18 To determine the subcellular distribution of Cy5-labeled LPCOTML liposomes in inflammatory vascular endothelial cells (IVEC) or HUVEC (human umbilical vein endothelial cells) after 1 h of incubation with Cy5-labeled LPCOTML liposomes using confocal laser scanning microscopy (CLSM), cell nuclei were stained with Hoechst 33342, cell membranes were stained with DIO, and LPCOTML liposomes were labeled with Cy5. Scale bar = 25 μm.

[0271] Figure 19 To determine the subcellular distribution of Cy5-labeled LPCO™ L liposomes in inflammatory vascular endothelial cells (IVEC) after 1 h of incubation with Cy5-labeled LPCOTML liposomes using confocal laser scanning microscopy (CLSM), cell nuclei were stained with Hoechst 33342, lysosomes were stained with LysoTracker Green DND26, and LPCOTML liposomes were labeled with Cy5. Scale bar = 25 μm.

[0272] Figure 20 This is a schematic diagram of the Transwell system.

[0273] Figure 21The mean fluorescence intensity (MFI) of different Cy5-labeled liposomes in the basal-side medium of the Transwell system was measured after incubating HUVEC cells and IVC cells pretreated with the exocytosis inhibitor EXO1 for 2 h. "EXO1" refers to the incubation of IVC cells with the exocytosis inhibitor EXO1 for 2 h before incubating the different Cy5-labeled liposomes with EXO1-pretreated IVC cells. **p<0.01 indicates statistical significance, and "ns" indicates no significance.

[0274] Figure 22 After pretreatment of HUVEC or IVEC cells with different endocytosis inhibitors, Cy5-labeled LPCOTML (LPCOTML) in the basal-side medium of the Transwell system Cy5 The mean fluorescence intensity (MFI) of liposomes, where p < 0.01 indicates statistical significance.

[0275] Figure 23 Images of bacteria in *Pseudomonas aeruginosa* PAO1 biofilms on agar plates were obtained after incubation with different drugs and subsequent ultrasonic irradiation, with or without the treatment. "US(+)" indicates incubation with different drugs followed by ultrasonic irradiation (sound intensity: 2 W / cm²). 2 (Frequency: 3MHz, duty cycle: 50%) for 5 minutes; "US(-)" means that different drugs were incubated with Pseudomonas aeruginosa PAO1 biofilm without ultrasonic irradiation treatment.

[0276] Figure 24 The number of bacterial colony-forming units (CFU) in the *Pseudomonas aeruginosa* PAO1 biofilm was obtained after incubation with different drugs and with or without ultrasonic irradiation. "US(+)" indicates ultrasonic irradiation (sound intensity: 2 W / cm²) after incubation with different drugs and the *Pseudomonas aeruginosa* PAO1 biofilm. 2 (Frequency: 3MHz, duty cycle: 50%) for 5 min; "US(-)" means that different drugs were incubated with Pseudomonas aeruginosa PAO1 biofilm without ultrasonic irradiation treatment; *p<0.05, ***p<0.001 indicate statistical significance, "ns" indicates no significance.

[0277] Figure 25 Different drugs were incubated with Pseudomonas aeruginosa PAO1 biofilm and then subjected to ultrasonic irradiation (sound intensity: 2W / cm²). 2The bacteria were treated with a frequency of 3 MHz, a duty cycle of 50%, and a time of 5 min. After incubation for 1 h, the extracellular polymer matrix barrier of the bacterial biofilm in each treatment group was observed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM).

[0278] Figure 26 To obtain live / dead bacterial staining and 3D reconstructed images of *Pseudomonas aeruginosa* PAO1 biofilms after incubation with different drugs and treatment with or without ultrasonic irradiation, SYTO9 staining was used to stain all bacteria in the biofilm, and PI staining was used to stain dead bacteria in the biofilm. Scale bar = 100 μm. "US(+)" indicates that the *Pseudomonas aeruginosa* PAO1 biofilm was irradiated with ultrasonic irradiation (sound intensity: 2 W / cm²) after incubation with different drugs. 2 (Frequency: 3MHz, duty cycle: 50%) for 5 minutes; "US(-)" means that different drugs were incubated with Pseudomonas aeruginosa PAO1 biofilm without ultrasonic irradiation treatment.

[0279] Figure 27 BALB / c nude mice with deep surgical site infections were injected via the tail vein with COTML containing the same fluorescence intensity in each group. Cy5 PCOTML Cy5 LCOTML Cy5 or LPCOTML Cy5 Six hours after liposome dispersion, the sample was irradiated with ultrasound (sound intensity: 2 W / cm²). 2 (Frequency: 3MHz, Duty Cycle: 50%, Duration: 10min) After treatment, mice were in vivo imaged 12h after administration. Bioluminescence and fluorescence imaging of BSSI tissue, heart, liver, spleen, lung, kidney and small intestine were obtained by dissection and isolation. Among them, 1 is BSSI (biofilm-associated surgical site infection) tissue, 2 is heart, 3 is liver, 4 is spleen, 5 is lung, 6 is kidney and 7 is small intestine.

[0280] Figure 28 To use Living Image-4.5 software to Figure 27 Quantitative analysis was performed on the fluorescence intensity of BSSI (biofilm-associated surgical site infection) tissues, heart, liver, spleen, lung, kidney, and small intestine isolated from each treatment group. **p<0.01, ***p<0.001 indicate statistical significance, and "ns" indicates no significance.

[0281] Figure 29 BALB / c nude mice with deep surgical site infections were injected via the tail vein with COTML containing the same fluorescence intensity in each group. Cy5 PCOTML Cy5 LCOTMLCy5 or LPCOTML Cy5 Six hours after liposome dispersion, the sample was irradiated with ultrasound (sound intensity: 2 W / cm²). 2 (Frequency: 3MHz, Duty Cycle: 50%, Duration: 10min) After treatment with intravenous FITC-Lectin for 12 hours and cardiac perfusion, the dissected BSSI (biofilm-associated surgical site infection) tissue was frozen in OCT embedding medium for tissue frozen sections. After frozen sectioning, the BSSI tissue was imaged by confocal laser scanning microscopy (CLSM) at a scale bar of 200μm.

[0282] Figure 30 BALB / c nude mice infected with P. aeruginosa PAO1 in the abdomen were injected via the tail vein with COTML, which has the same fluorescence intensity. Cy5 PCOTML Cy5 LCOTML Cy5 or LPCOTML Cy5 Confocal laser scanning microscopy (CLSM) imaging of bacterial infection sites at 0 min, 10 min and 30 min after liposome dispersion, scale bar = 100 μm.

[0283] Figure 31 In order to be in Figure 30 The mean integrated fluorescence intensity (MFI) of the selected region (i.e., the circled region away from the blood vessel) in the 30-minute CLSM image was quantitatively analyzed, where "ns" indicates no significance.

[0284] Figure 32 BALB / c nude mice infected with P. aeruginosa PAO1 in the abdomen were injected via the tail vein with COTML, which has the same fluorescence intensity. Cy5 PCOTML Cy5 LCOTML Cy5 or LPCOTML Cy5 Thirty minutes after dissection of the liposome dispersion, the bacterial-infected tissue was imaged by cryo-transmission electron microscopy (cryo-TEM), in which transvascular endothelial cell transporter vesicles are indicated by arrows, scale bar = 200 nm.

[0285] Figure 33 Photographs and colony counts of bacterial biofilm areas isolated from BALB / c nude mice with deep surgical site infections in different treatment groups at the end of day 7 of treatment.

[0286] Figure 34Colony-forming units (CFU) were counted in BALB / c nude mice with deep surgical site infections at the end of day 7 of treatment in different treatment groups. *p<0.05, **p<0.01, ***p<0.001 were considered statistically significant.

[0287] Figure 35 Hematoxylin-eosin (H&E) or Masson staining was performed on the bacterial biofilm areas of BALB / c nude mice with deep surgical site infections at the end of day 7 of treatment in different treatment groups.

[0288] Figure 36 Bioluminescence imaging of biofilm-associated surgical site infection areas in BALB / c nude mice with deep surgical site infections in different treatment groups was measured using the In Vivo Imaging System (IVIS) on days 0, 1, 3, 5, and 7 of treatment.

[0289] Figure 37 for Figure 36 The changes in bioluminescence intensity of the surgical site infection area in each treatment group on days 0, 1, 3, 5 and 7 of treatment.

[0290] Figure 38 The mean colony-forming units (CFU) count of the corresponding bacterial biofilm area tissue in BALB / c nude mice with deep surgical site infection in different treatment groups was calculated by colony counting method at the end of day 7 of treatment. Statistical significance was indicated by **p<0.01 and ***p<0.001. Detailed Implementation

[0291] This invention develops a meropenem prodrug (OTM prodrug), which is a ROS (reactive oxygen species) sensitive prodrug. ROS can catalyze the rapid and significant release of the active ingredient meropenem (MEM) from the OTM prodrug. This invention also develops a VCAM1 ligand-modified drug, comprising drugs for the prevention and / or treatment of infections such as bacterial biofilm-associated infections. After modification, the VCAM1 ligand binds to VCAM1 receptors highly expressed in bacterial biofilm-associated infection tissue cells and vascular endothelial cells at the infection site. This allows the VCAM1 ligand-modified drug to effectively cross vascular endothelial cells at the bacterial biofilm-associated infection site to enter the infected tissue and achieve highly efficient targeted accumulation at the site. Furthermore, it is rapidly and effectively taken up by the infected tissue cells, thereby enhancing the drug's efficacy against bacterial biofilm-associated infections.

[0292] the term

[0293] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only open-ended definitions but also semi-closed and closed definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0294] As used herein, the terms “PBS,” “phosphate buffer,” and “PBS buffer” are used interchangeably and refer to an aqueous solution of phosphate buffer.

[0295] As used in this article, the term "VCAM1" refers to vascular cell adhesion molecule-1, and is used interchangeably with "VCAM-1".

[0296] As used herein, the terms “VCAM1 ligand” and “vascular cell adhesion molecule-1 ligand” are used interchangeably and refer to ligands that bind to vascular cell adhesion molecule-1.

[0297] As used in this article, the term "perfluoropentane" is translated as perfluoropentane.

[0298] As used in this article, the terms “ultrasound irradiation” and “ultrasound stimulation” are used interchangeably.

[0299] As used in this article, the term "reactive oxygen species" is abbreviated as ROS.

[0300] As used in this article, the term "biofilm-associated surgical site infection" is abbreviated as BSSI.

[0301] As used in this article, the term "acoustic intensity" refers to acoustic intensity.

[0302] As used in this article, the term "frequency" refers to frequency.

[0303] As used in this article, the term "duty cycle" refers to the duty cycle.

[0304] As used in this article, the English term for "meropenem" is meropenem.

[0305] As used in this article, the term "DPPC" refers to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.

[0306] As used in this article, the term "Ce6" refers to dihydroporphyrin e6, also known as Chlorin e6.

[0307] As used herein, the term "DSPE-mPEG2000" refers to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].

[0308] As used herein, the term "DSPE-PEG2000-Ce6" refers to 1,2-distearate-sn-glycerol-3-phosphorylethanolamine-N-[polyethylene glycol-2000] coupled with dihydroporphyrin e6, also known as Chlorin e6-conjugated DSPE-PEG2000.

[0309] As used herein, the term “DSPE-PEG2000-VCAM1 ligand” refers to 1,2-distearate-sn-glycerol-3-phosphorylethanolamine-N-[polyethylene glycol-2000] coupled to VCAM1 ligand.

[0310] As used herein, the term “DC-Chol” refers to 3β-[N-(N,N-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride, CAS Registry Number: 166023-21-8.

[0311] As used in this article, the terms “OTM prodrug” and “OTM” are used interchangeably.

[0312] As used in this article, the terms "OM prodrug" and "OM" are used interchangeably.

[0313] As used herein, the term "glycerol phosphate buffer" refers to a phosphate buffer containing glycerol, where the concentration (e.g., mM) refers to the concentration of phosphate.

[0314] In this invention, the term "prevention" means a method of preventing the onset of a disease and / or its accompanying symptoms or protecting a subject from acquiring a disease.

[0315] In this invention, the term "treatment" includes inhibiting, reducing, alleviating, reversing, or eradicating the progression of disease, and does not require 100% inhibition, eradication, or reversal. In some embodiments, the drugs of this invention reduce, inhibit, and / or reverse bacterial infections by, for example, at least about 50%, or at least about 80%, or at least about 90%, or at least about 95%, or about 100%, compared to levels observed in the absence of the drugs described in this invention.

[0316] VCAM1 ligand

[0317] The VCAM1 ligand described in this invention is a ligand capable of binding to the VCAM1 receptor. Typically, the VCAM1 ligand described in this invention is a polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0318] drug

[0319] The drugs described in this invention include, but are not limited to, active pharmaceutical ingredients, prodrugs, or drug conjugates, such as meropenem.

[0320] Typically, the drugs described in this invention include meropenem prodrugs as described in the first aspect of this invention.

[0321] In a preferred embodiment of the invention, the drug comprises a drug that is retained and / or degraded by lysosomes.

[0322] Preferably, the degradation includes degradation by lysosomal enzymes.

[0323] Preferably, the drug comprises a drug that is degraded by lysosomal enzymes.

[0324] Preferably, the drug targets the cytoplasm or the nucleus.

[0325] Preferably, the drug comprises a small molecule drug, a gene, or a protein.

[0326] Preferably, the gene is selected from the group consisting of DNA, RNA, or a combination thereof.

[0327] Specifically, the drug described in this invention is as described in the second aspect of this invention above.

[0328] Infect

[0329] The infections described in this invention include, but are not limited to, bacterial infections.

[0330] In a preferred embodiment of the invention, the infection includes bacterial biofilm-related infections.

[0331] Preferably, the infection includes biofilm-related surgical site infections.

[0332] Preferably, the infection includes surgical site infection associated with bacterial biofilm.

[0333] In a preferred embodiment of the invention, the bacteria include Pseudomonas aeruginosa, such as Pseudomonas aeruginosa PAO1.

[0334] Specifically, the infection described in this invention is as described in the sixth aspect of this invention above.

[0335] Meropenem prodrug

[0336] This invention provides a meropenem prodrug, and representatively, the structure of the meropenem prodrug is shown below:

[0337]

[0338] Liposomes and their preparation methods

[0339] The present invention provides a liposome comprising a drug for preventing and / or treating infection.

[0340] In a preferred embodiment of the invention, the liposomes comprise perfluoropentane.

[0341] In a preferred embodiment of the invention, the liposomes comprise dihydroporphyrin E6.

[0342] The lipid material of the liposomes described in this invention may include, but is not limited to, one or more of the following: 1,2-dispalmitoyl-sn-glycerol-3-phosphocholine, 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with VCAM1 ligand, 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[polyethylene glycol-2000] coupled with dihydroporphyrin E6, and 3β-[N-(N,N-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride.

[0343] Specifically, the liposomes described in this invention are as described in the second aspect of this invention above.

[0344] Specifically, the method for preparing the liposomes is as described in the third aspect of the present invention above.

[0345] use

[0346] This invention provides the use of the meropenem prodrug, the liposome, the VCAM1 ligand-modified drug, or the composition thereof, for the preparation of formulations for the prevention and / or treatment of infections.

[0347] The present invention also provides a use of a VCAM1 ligand for the preparation of a modifier, said modifier being used for one or more uses selected from the group consisting of: (a) modifying a drug; and / or (b) enhancing the efficacy of a drug against an infection;

[0348] The drugs mentioned include those for the prevention and / or treatment of infections.

[0349] The present invention also provides a method for enhancing the efficacy of a drug against infection, the method comprising the steps of:

[0350] Drugs can be modified with VCAM1 ligands to enhance their efficacy against infection.

[0351] The drugs mentioned include those for the prevention and / or treatment of infections.

[0352] Compositions and dosage forms

[0353] The present invention also provides a composition or formulation, preferably a pharmaceutical composition or formulation. The composition or formulation of the present invention may further include a pharmaceutically acceptable carrier.

[0354] As used in this article, "pharmaceutically acceptable carrier" refers to one or more compatible solid, semi-solid, liquid, or gel fillers that are suitable for human or animal use and must have sufficient purity and sufficiently low toxicity.

[0355] It should be understood that, in this invention, there are no particular limitations on the pharmaceutically acceptable carriers used. Materials commonly used in the art can be selected, or they can be prepared using conventional methods or purchased from the market. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium dodecyl sulfate), buffers, chelating agents, thickeners, pH adjusters, transdermal penetration enhancers, colorants, flavoring agents, stabilizers, antioxidants, preservatives, antibacterial agents, pyrogen-free water, etc.

[0356] In this invention, the dosage forms of the compositions or preparations described herein include, but are not limited to, oral preparations, injectable preparations, and topical preparations.

[0357] Typically, the dosage form of the compositions or preparations described in this invention is an intravascular injection preparation, such as an intravenous injection preparation.

[0358] Pharmaceutical formulations should be matched to the route of administration, whereby a therapeutically effective amount of the drug is administered to the intended recipient (e.g., human or non-human mammal). As used herein, the term "therapeutically effective amount" refers to an amount that produces a functional or active effect in humans and / or animals and is acceptable to them. Those skilled in the art will understand that the "therapeutically effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs, all of which are within the scope of the skill of a skilled physician.

[0359] The main superior technical effects of this invention include:

[0360] 1. This invention develops a meropenem prodrug (OTM prodrug), which is a ROS (reactive oxygen species) sensitive prodrug. ROS can catalyze the rapid and significant release of the active ingredient meropenem (MEM) from the OTM prodrug. The ROS sensitivity of the OTM prodrug has significant application value. For example, due to hypoxia, the ROS level in infected tissues at deep surgical sites is significantly higher than in normal tissues. Therefore, the OTM prodrug can significantly release the active ingredient meropenem (MEM) in infected tissues at deep surgical sites, while it is difficult to release the active ingredient meropenem (MEM) in normal tissues. Thus, OTM prodrug administration can achieve significant targeted treatment of deep surgical site infections while reducing the killing effect on normal tissues, avoiding the side effects such as dysbiosis caused by the overuse of meropenem (MEM) antibiotics, thereby exhibiting excellent biocompatibility. For example, OTM prodrugs can be co-prepared into drugs with substances that generate ROS under ultrasound irradiation (e.g., OTM prodrugs and substances that generate ROS under ultrasound irradiation are co-loaded in liposomes). After administration, only the bacterial infection site is subjected to ultrasound irradiation. The ROS (reactive oxygen species) generated by the substances that generate ROS under ultrasound irradiation can catalyze the rapid and significant release of the active ingredient meropenem (MEM) from the OTM prodrug at the bacterial infection site. In contrast, OTM prodrugs distributed in normal tissues have difficulty releasing the active ingredient meropenem (MEM). This not only achieves significant targeted therapy to the bacterial infection site but also reduces the side effects of OTM prodrugs on normal tissue cells and avoids dysbiosis caused by the overuse of meropenem (MEM) antibiotics, thus exhibiting excellent biocompatibility.

[0361] 2. This invention unexpectedly discovers for the first time that VCAM1 is overexpressed in bacterial biofilm-associated infected tissue cells and vascular endothelial cells at the site of infection. Therefore, VCAM1 can serve as a good receptor for targeted therapy of bacterial biofilm-associated infections. By modifying drugs with VCAM1 ligands, the targeted therapeutic effect of drugs on bacterial biofilm-associated infections (such as BSSI) can be significantly enhanced, thereby improving the efficacy of drugs against bacterial biofilm-associated infections and reducing drug side effects.

[0362] 3. This invention develops a drug modified with VCAM1 ligand, the drug including drugs for preventing and / or treating infections such as bacterial biofilm-associated infections. The VCAM1 ligand can bind to VCAM1 receptors highly expressed in bacterial biofilm-associated infected tissue cells and vascular endothelial cells at the infection site, thereby enabling the VCAM1 ligand-modified drug to effectively cross the vascular endothelial cells at the bacterial biofilm-associated infection site to enter the bacterial biofilm-associated infected tissue and to efficiently target and accumulate at the bacterial biofilm-associated infected tissue site, and to be rapidly and effectively taken up by the bacterial biofilm-associated infected tissue cells, thereby enhancing the drug's efficacy against bacterial biofilm-associated infections.

[0363] 4. This invention provides the use of a VCAM1 ligand in modifying drugs and enhancing the efficacy of drugs against bacterial biofilm-associated infections, wherein the drugs include those for the prevention and / or treatment of bacterial biofilm-associated infections. This invention also provides a method for enhancing the efficacy of drugs against bacterial biofilm-associated infections, the method comprising modifying the drugs for the prevention and / or treatment of bacterial biofilm-associated infections with a VCAM1 ligand, thereby enhancing the therapeutic efficacy of the drugs against bacterial biofilm-associated infections. After the drug is modified with the VCAM1 ligand, the VCAM1 ligand can bind to VCAM1 receptors highly expressed in bacterial biofilm-associated infected tissue cells and vascular endothelial cells at the infection site, thereby enabling the VCAM1 ligand-modified drug to effectively cross vascular endothelial cells at the bacterial biofilm-associated infection site to enter the bacterial biofilm-associated infected tissue and to efficiently target and accumulate at the bacterial biofilm-associated infected tissue site, and to be rapidly and effectively taken up by the bacterial biofilm-associated infected tissue cells, thereby enhancing the efficacy of the drug against bacterial biofilm-associated infections.

[0364] 5. This invention develops an LPCOTML liposome, which comprises a DSPE-PEG2000-VCAM1 ligand, a DSPE-PEG2000-Ce6, and a meropenem prodrug (OTM prodrug). The VCAM1 ligand binds to the VCAM1 receptor, which is highly expressed in bacterial biofilm-associated infected tissue cells and vascular endothelial cells at the infection site. This allows the LPCOTML liposome to effectively cross the vascular endothelial cells at the bacterial biofilm-associated infection site and enter the bacterial biofilm-associated infected tissue, where it efficiently and targetedly accumulates. Furthermore, it is rapidly and effectively taken up by the bacterial biofilm-associated infected tissue cells, thereby enhancing the drug's efficacy against bacterial biofilm-associated infections. In addition, the LPCOTML liposome of this invention also possesses the following excellent properties:

[0365] (1) LPCOTML liposomes can significantly release the antibacterial active ingredient meropenem under ultrasound irradiation, but it is difficult to release the antibacterial active ingredient meropenem without ultrasound irradiation. Therefore, after LPCOTML liposome administration, only the infection sites related to bacterial biofilms are treated with ultrasound irradiation. Ultrasound irradiation can significantly catalyze and promote the rapid and effective release of the active ingredient meropenem by LPCOTML liposomes at the infection sites related to bacterial biofilms. LPCOTML liposomes distributed in normal tissue sites are difficult to release the active ingredient meropenem (MEM) because they have not been treated with ultrasound irradiation. This not only achieves significant targeted therapy to the infection sites related to bacterial biofilms, but also reduces the side effects of LPCOTML liposomes loaded with meropenem (MEM) on normal tissue cells, avoids the dysbiosis caused by the abuse of meropenem (MEM) antibiotics, and has excellent biosafety.

[0366] (2) Under ultrasound irradiation, LPCOTML liposomes can effectively destroy the extracellular polymeric substance (EPS) matrix barrier of bacterial biofilm, overcome the limitation of the extracellular polymeric substance matrix barrier of bacterial biofilm on the penetration of antibacterial drugs into the biofilm, and allow antibacterial drugs to penetrate into the deep part of the bacterial biofilm and kill the bacteria located in the deep part of the bacterial biofilm. Therefore, ultrasound irradiation can significantly promote and enhance the inhibitory effect of LPCOTML liposomes on bacteria in bacterial biofilm, thereby effectively inhibiting bacterial infection.

[0367] (3) The LPCOTML liposomes described in this invention have excellent targeting aggregation and distribution capabilities in bacterial biofilm-related infected tissue sites, and their distribution in bacterial biofilm-related infected tissue sites is significantly higher than that in other major organs such as the heart, liver, spleen, lungs, kidneys and small intestine. Therefore, LPCOTML liposomes can achieve effective targeted treatment of bacterial biofilm-related infections while reducing the toxic side effects of drugs on other organs and tissues such as the heart, liver, spleen, lungs, kidneys and small intestine, thereby improving the efficacy and safety of drug treatment.

[0368] (4) The LPCOTML liposomes described in this invention have good lysosomal avoidance properties, thereby preventing the LPCOTML liposomes and the drug loaded on them from being degraded by enzymes in lysosomes, effectively protecting the drug from degradation and destruction by lysosomes, enhancing the intracellular stability of LPCOTML liposomes and the drug loaded on them, thereby improving the therapeutic effect of the drug.

[0369] (5) The LPCOTML liposomes described in this invention have excellent biocompatibility.

[0370] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that the following specific embodiments are based on the present technical solution and provide detailed implementation methods and specific operation processes, but the scope of protection of the present invention is not limited to these embodiments.

[0371] Example 1

[0372] 1. Reagents and Materials

[0373] BSSI refers to biofilm-associated surgical site infection.

[0374] VCAM1 refers to vascular cell adhesion molecule-1, and it can be used interchangeably with VCAM-1.

[0375] VEGFR1 refers to vascular endothelial growth factor receptor 1.

[0376] MEM refers to meropenem, CAS Registry Number: 96036-03-2.

[0377] PAO1 refers to Pseudomonas aeruginosa PAO1.

[0378] PAO1-GFP refers to Pseudomonas aeruginosa PAO1 strain that expresses green fluorescent protein (GFP).

[0379] PAO1-Luc refers to P. aeruginosa PAO1 (PAO1-Luc) that expresses luciferase (Luc).

[0380] DPPC stands for 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.

[0381] Ce6 refers to dihydroporphyrin e6 (Chlorin e6).

[0382] DSPE-mPEG2000 refers to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]).

[0383] DSPE-PEG2000-Ce6 refers to 1,2-distearate-sn-glycerol-3-phosphorylethanolamine-N-[polyethylene glycol-2000] coupled with dihydroporphyrin e6, also known as Chlorin e6-conjugated DSPE-PEG2000.

[0384] DSPE-PEG2000-VCAM1 ligand refers to 1,2-distearate-sn-glycerol-3-phosphorylethanolamine-N-[polyethylene glycol-2000] coupled with VCAM1 ligand. VCAM1 ligand is a polypeptide with the amino acid sequence VHPKQHRGGSKGC (SEQ ID NO:1). VCAM1 ligand is coupled to DSPE-PEG2000-NHS via the carbonyl reaction chemistry of N-hydroxysuccinimide ester to form DSPE-PEG2000-VCAM1 ligand.

[0385] DC-Chol refers to 3β-[N-(N,N-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride, CAS Registry Number: 166023-21-8.

[0386] Cy5 refers to Cyanine 5.

[0387] The fluorescent probe DIO stands for 3,3'-dioctadecyloxacarbocyanine perchlorate.

[0388] FITC refers to fluorescein isothiocyanate.

[0389] TNF-α refers to tumor necrosis factor-α.

[0390] IVEC refers to inflammatory vascular endothelial cells.

[0391] HUVEC refers to human umbilical vein endothelial cells.

[0392] 2. Material Synthesis

[0393] 2.1 Synthesis of OTM prodrug

[0394] The structure of the OTM prodrug is as follows:

[0395]

[0396] The synthetic route of OTM prodrug is as follows: Figure 1 As shown, the specific synthesis method is as follows:

[0397] (2,2'-(propane-2,2-diylbis(sulfadiyl))bis(ethane-1-amine) (PDSE, 0.97 g, 5 mmol) and triethylamine (0.765 mL, 5.5 mmol) were dissolved in 25 mL of dichloromethane. 10 mL of oleoyl chloride (1.82 mL, 5 mmol) dichloromethane solution was added, and the mixture was stirred at 4 °C for 2 h. Then, CDI (triphosgene, 0.81 g, 5 mmol) and 0.5 mL of pyridine were added, and the mixture was stirred for 8 h. The reaction solution was washed three times with brine, dried over anhydrous Na₂SO₄, and then 20 mL of meropenem (1.915 g, 5 mmol) dichloromethane solution was added. The mixture was stirred under N₂ protection for 12 h. The resulting reaction solution was evaporated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, 10 / 1, v / v) to obtain the OTM prodrug (1.90 g, yield 43.8%).

[0398] 1H NMR spectrum of the synthesized OTM prodrug 1The spectra obtained by H-NMR (400 MHz, CDCl3) and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) are as follows: Figure 2 and Figure 3 As shown

[0399] 2.2M Prodrug Synthesis

[0400] The structure of the OM prodrug is as follows:

[0401]

[0402] The synthetic route of OM prodrug is as follows: Figure 4 As shown, the specific synthesis method is as follows:

[0403] Meropenem (1.03 g, 2.7 mmol) and triethylamine (0.45 mL, 3.25 mmol) were dissolved in 20 mL of dichloromethane. 10 mL of oleoyl chloride (1.07 mL, 3.23 mmol) solution in dichloromethane was added. After stirring at 4 °C under N2 protection for 4 h, the reaction solution was washed three times with brine and dried over anhydrous Na2SO4. The resulting reaction solution was evaporated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, 5 / 1, v / v) to obtain the OM prodrug (1.32 g, yield 75.1%).

[0404] 1H NMR spectrum of the synthesized OM prodrug 1 The spectra obtained by H-NMR (400 MHz, CDCl3) and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) are as follows: Figure 5 and Figure 6 As shown.

[0405] 3. Preparation of liposomes

[0406] 3.1 Preparation of LPCOTML liposome dispersion

[0407] (1) DPPC (3.0 mg), DSPE-PEG2000-VCAM1 ligand (3.0 mg), DSPE-PEG2000-Ce6 (6 mg), DC-Chol (3 mg) and OTM prodrug (3 mg) were dissolved in 3 mL of chloroform in a round-bottom flask. The organic solvent was removed by rotary evaporation under reduced pressure at 25 °C, and a lipid film was formed in the round-bottom flask.

[0408] (2) Cool the lipid membrane to 4°C, add 100 μL of perfluoropentane to immerse the lipid membrane, and then add 5 mL of glycerol phosphate buffer (10 mM, pH = 7.4, glycerol volume fraction of 10 v / v%) for hydration. Stir at 4°C for 30 min, and then stir at 30°C for 1 h in an open round-bottom flask to obtain LPCOTML liposome dispersion.

[0409] The encapsulation efficiency of meropenem (MEM) was determined to be 100% and the drug loading was 7.36% by dialysis. The formulas for calculating the encapsulation efficiency and drug loading are as follows:

[0410] "Encapsulation efficiency = mass of meropenem in liposomes / total mass of meropenem × 100%".

[0411] “LR = Mass of liposomal meropenem / Total mass (lipid + OTM) × 100%”.

[0412] The particle size of LPCOTML liposomes was determined to be 207±23.3 nm using a dynamic light scattering analyzer (Nano-ZS 90, Malvern), and the zeta potential was -15.4±6.2 mV. Cryo-transmission electron microscopy (cryo-TEM) images of the LPCOTML liposomes are shown below. Figure 7 As shown. Figure 7 The cryo-TEM images show that LPCOTML liposomes have a uniform monolayer lipid membrane with a distinct perfluoropentane ice cloud shadow in the center.

[0413] 3.2 Preparation of LCOTL ML liposome dispersion

[0414] (1) DPPC (3.0 mg), DSPE-PEG2000-VCAM1 ligand (3.0 mg), DSPE-PEG2000-Ce6 (6 mg), DC-Chol (3 mg) and OTM prodrug (3 mg) were dissolved in 3 mL of chloroform in a round-bottom flask. The organic solvent was removed by rotary evaporation under reduced pressure at 25 °C, and a lipid film was formed in the round-bottom flask.

[0415] (2) Cool the lipid membrane to 4°C, add 5 mL of glycerol phosphate buffer (10 mM, pH = 7.4, glycerol volume fraction of 10 v / v%) for hydration, stir at 4°C for 30 min, and then stir at 30°C for 1 h in a round-bottom flask under open conditions to obtain LCOTML liposome dispersion.

[0416] 3.3 Preparation of PCOTML liposome dispersion

[0417] (1) DPPC (3.0 mg), DSPE-mPEG2000 (3.0 mg), DSPE-PEG2000-Ce6 (6 mg), DC-Chol (3 mg) and OTM prodrug (3 mg) were dissolved in 3 mL of chloroform in a round-bottom flask. The organic solvent was removed by rotary evaporation under reduced pressure at 25 °C, and a lipid film was formed in the round-bottom flask.

[0418] (2) Cool the lipid membrane to 4°C, add 100 μL of perfluoropentane to immerse the lipid membrane, and then add 5 mL of glycerol phosphate buffer (10 mM, pH = 7.4, glycerol volume fraction of 10 v / v%) for hydration. Stir at 4°C for 30 min, and then stir at 30°C for 1 h in an open round-bottom flask to obtain PCOTML liposome dispersion.

[0419] 3.4 Preparation of COTML liposome dispersion

[0420] (1) DPPC (3.0 mg), DSPE-mPEG2000 (3.0 mg), DSPE-PEG2000-Ce6 (6 mg), DC-Chol (3 mg) and OTM prodrug (3 mg) were dissolved in 3 mL of chloroform in a round-bottom flask. The organic solvent was removed by rotary evaporation under reduced pressure at 25 °C, and a lipid film was formed in the round-bottom flask.

[0421] (2) Cool the lipid membrane to 4°C, add 5 mL of glycerol phosphate buffer (10 mM, pH = 7.4, glycerol volume fraction of 10 v / v%) for hydration, stir at 4°C for 30 min, and then stir at 30°C for 1 h in an open round-bottom flask to obtain COTML liposome dispersion.

[0422] 3.5 Preparation of LPL C1 liposome dispersion

[0423] (1) DPPC (6.0 mg), DSPE-PEG2000-VCAM1 ligand (3.0 mg), DSPE-PEG2000-Ce6 (6 mg) and DC-Chol (3 mg) were dissolved in 3 mL of chloroform in a round-bottom flask. The organic solvent was removed by rotary evaporation under reduced pressure at 25 °C, and a lipid film was formed in the round-bottom flask.

[0424] (2) Cool the lipid membrane to 4°C, add 100 μL of perfluoropentane to immerse the lipid membrane, and then add 5 mL of glycerol phosphate buffer (10 mM, pH = 7.4, glycerol volume fraction of 10 v / v%) for hydration. Stir at 4°C for 30 min, and then stir at 30°C for 1 h in an open round-bottom flask to obtain blank LPCL liposome dispersion.

[0425] 3.6 Preparation of Cy5-labeled liposomes

[0426] Cy5-labeled LPCOTML liposomes (LPCOTML) Cy5 The preparation method of liposomes is the same as that of "3.1 Preparation of LPCOTML liposome dispersion", except that in step (1), 0.3 mg of Cy5-labeled DSPE-PEG2000 replaces an equal weight of DSPE-PEG2000-Ce6 in the component.

[0427] Cy5-labeled LCOTML liposomes (LCOTML) Cy5 The preparation method of liposomes is the same as that of “3.2 LCOTML liposome dispersion preparation”, except that in step (1), 0.3 mg of Cy5-labeled DSPE-PEG2000 replaces an equal weight of DSPE-PEG2000-Ce6 in the component.

[0428] Cy5-labeled PCOTML liposomes (PCOTML) Cy5 The preparation method of liposomes is the same as that of "3.3 PCOTML liposome dispersion preparation", except that in step (1), 0.3 mg of Cy5-labeled DSPE-PEG2000 is used to replace an equal weight of DSPE-mPEG2000 in the component.

[0429] Cy5-labeled COTML liposomes (COTML) Cy5 The preparation method of liposomes is the same as that of "3.4 Preparation of COTML liposome dispersion", except that in step (1), 0.3 mg of Cy5-labeled DSPE-PEG2000 is used to replace an equal weight of DSPE-mPEG2000 in the component.

[0430] Cy5-labeled LPCL liposomes (LPCL) Cy5 The preparation method of liposomes is the same as that of "3.5LPCL liposome dispersion preparation", except that in step (1), 0.3 mg of Cy5-labeled DSPE-PEG2000 replaces an equal weight of DSPE-PEG2000-Ce6 in the component.

[0431] 4. Bacterial culture, cell culture, and animal models

[0432] Pseudomonas aeruginosa PAO1 (PAO1-GFP) expressing green fluorescent protein (GFP) and Pseudomonas aeruginosa PAO1 (PAO1-Luc) expressing luciferase (Luc) were purchased from BeNa Culture Collection.

[0433] Human umbilical vein endothelial cells (HUVECs) were purchased from ATCC and cultured at 37°C in DMEM medium (+P / S) containing 10% FBS.

[0434] Inflammatory vascular endothelial cells (IVECs) were constructed by culturing HUVECs in serum-free medium containing TNF-α (50 ng / mL) for 12 h.

[0435] 5. Construction of animal models

[0436] 5.1 Construction of a BALB / c nude mouse model of deep surgical site infection:

[0437] Pseudomonas aeruginosa PAO1 (PAO1-Luc) expressing luciferase was incubated at 37°C in a shaking incubator at 200 rpm to reach the logarithmic growth phase.

[0438] Male BALB / c nude mice (6-8 weeks old) were anesthetized, and a 10 mm diameter wound was made on the skin. 50 μL of PAO1-Luc cell suspension (1 × 10⁻⁶ cells) was added to the wound. 8 The bacterial biofilm was formed after incubation for 3 days (CFU / ml). The mature bacterial biofilm was then dissected and cut into small pieces. These small pieces were implanted into another anesthetized mouse through a horizontal surgical incision. After suturing and incubation for 3 days, a BALB / c nude mouse model with deep surgical site infection was constructed.

[0439] 5.2 Construction of the sham-operated BALB / c nude mouse model:

[0440] Male BALB / c nude mice (6-8 weeks old) were anesthetized, and a 10 mm diameter wound was made on the abdominal skin without implanting any object. After suturing and incubation for 3 days, a sham-operated BALB / c nude mouse model was constructed.

[0441] 6. Immunofluorescence analysis of tissues infected with bacterial biofilms

[0442] Tissue samples infected with bacterial biofilms were collected from a BALB / c nude mouse model with deep surgical site infection. Additionally, tissue samples from the surgical site of a sham-operated BALB / c nude mouse model were used as controls. The collected tissues were fixed in 4% paraformaldehyde at room temperature. The tissue samples were stained with CD31 (platelet-endothelial cell adhesion molecule) to identify blood vessels, and stained with the vasculitic factors VCAM1 (vascular cell adhesion molecule-1) or VEGFR1 (vascular endothelial growth factor receptor 1) to determine the overexpression of VCAM1 or VEGFR1 in vascular endothelial cells at the bacterial biofilm infection site. The specific methods are as follows:

[0443] Tissue sections were dewaxed in xylene and rehydrated in ethanol, followed by heat-induced epitope retrieval in citrate buffer (pH 6.0). Antigen retrieval was performed in a microwave oven for 15 min in 10 mM citrate buffer (pH 6.0), blocking endogenous peroxidase activity by adding 3% hydrogen peroxide at room temperature. Sections were then blocked in 5% goat serum for 30 min and incubated overnight at 4°C with rabbit anti-human VCAM1 specific primary antibody (1:250) or rabbit anti-human VEGFR1 (vascular endothelial growth factor receptor 1) specific primary antibody (5 μg / mL). After washing three times with PBS 7.4 buffer, the sections were... The slides were incubated with 647 goat anti-rabbit IgG (H+L) (1:200) for 1 h, then washed three times with PBS 7.4 buffer, stained with DAPI (4',6-diamidinyl-2-phenylindole), and analyzed using a confocal laser scanning microscope (CLSM).

[0444] Immunofluorescence staining of bacterial biofilm-infected tissues from BALB / c nude mice with deep surgical site infection and tissues from surgical sites in sham-operated BALB / c nude mice, as shown in the figures. Figure 8 and Figure 9 As shown.

[0445] from Figure 8 and Figure 9 The results show that, compared with the sham-operated group, the expression of VCAM1 (vascular cell adhesion molecule-1) receptor in vascular endothelial cells at bacterial biofilm-associated surgical site infections was significantly increased, while the expression of VEGFR1 (vascular endothelial growth factor receptor 1) receptor in vascular endothelial cells at bacterial biofilm-associated surgical site infections was not significantly different from that in the sham-operated group. Therefore, VCAM1 can serve as a good receptor for targeted therapy of bacterial biofilm-associated surgical site infections.

[0446] Furthermore, Western blot analysis revealed no significant difference in VCAM1 expression between vascular endothelial cells at the surgical site in sham-operated BALB / c nude mice and vascular endothelial cells at the same site in untreated normal BALB / c nude mice; both showed low expression.

[0447] 7. Western blotting analysis of tissues infected by bacterial biofilm

[0448] Tissue samples infected with bacterial biofilms were collected from a BALB / c nude mouse model with deep surgical site infection. Additionally, tissue samples from the surgical site in a sham-operated BALB / c nude mouse model were used as controls. The collected tissues were lysed using RIPA protein extraction reagent supplemented with benzyl sulfonyl fluoride (PMSF), milled, and the lysates were centrifuged to obtain the supernatant. An equal amount of protein (30 μg) from each sample was electrophoresed on a 4–20% SDS-polyacrylamide gel (SDS-PAGE), then transferred to a polyvinylidene fluoride (PVDF) membrane, blocked with 5% BSA (Bovine Serum Albumin) solution for 2 h at room temperature, and incubated with the following specific primary antibodies: rabbit anti-human VCAM1 antibody (1:1000), rabbit anti-human VEGFR1 antibody (1:1000), and rabbit anti-human GAPDH antibody (1:2000). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a loading control protein. The HRP (horseradish peroxidase) conjugated secondary antibody was goat anti-rabbit IgG (1:5000). Pre-stained color protein standard markers (10-250 kDa) were used as molecular markers. Specific bands were detected using an ultrasensitive ECL chemiluminescence kit, and the PVDF membrane was analyzed using an Azure c600 Imager.

[0449] Western blot analysis was performed on the expression of VCAM1 (vascular cell adhesion molecule-1) and VEGFR1 (vascular endothelial growth factor receptor 1) in bacterial biofilm-infected tissues from BALB / c nude mice with deep surgical site infection and tissues from surgical sites from sham-operated BALB / c nude mice. Figure 10 and Figure 11 As shown.

[0450] from Figure 10 and Figure 11The results show that, compared with the sham-operated group, the expression of VCAM1 (vascular cell adhesion molecule-1) receptor was significantly increased in surgical site infections associated with bacterial biofilms, while the expression of VEGFR1 (vascular endothelial growth factor receptor 1) receptor in vascular endothelial cells at the site of bacterial biofilm-associated surgical site infections was not significantly different from that in the sham-operated group. Therefore, VCAM1 can serve as a good receptor for targeted therapy of bacterial biofilm-associated surgical site infections.

[0451] Furthermore, Western blot analysis revealed no significant difference in VCAM1 expression between the surgical site tissues of sham-operated BALB / c nude mice and the same site tissues of untreated normal BALB / c nude mice; both tissues showed low expression.

[0452] 8. ROS (Reactive Oxygen Species) responsive drug release

[0453] 8.1 Prodrug Release

[0454] The release of OTM or OM prodrugs (with meropenem equivalents of 10 μM in each prodrug) was carried out by adding them to a methanol / H2O (1 / 1, v / v) mixed solution containing 0.5-10 mM H2O2. The mixture was stirred at 37 °C. At different release time points, 100 μL of the release solution sample was taken, and the meropenem content in the release solution sample was determined by high performance liquid chromatography (HPLC) to calculate the amount of meropenem released at different release times.

[0455] In a methanol / H2O (1 / 1, v / v) mixed solution release medium of 0.5 mM H2O2, the cumulative amount of meropenem (MEM) released by the OTM or OM prodrug at different release time points is as follows: Figure 12 As stated above.

[0456] In a methanol / H2O (1 / 1, v / v) mixed solution release medium containing different concentrations of H2O2, the cumulative amount of meropenem (MEM) released by the OTM or OM prodrug at the 1-hour release time point is as follows: Figure 13 As stated above.

[0457] from Figure 12 As can be seen, the reactive oxygen species (H2O2) environment can rapidly catalyze the release of the active ingredient meropenem (MEM) from the OTM prodrug, while the OM prodrug is hardly catalyzed by the reactive oxygen species (H2O2) to release meropenem (MEM). From... Figure 13As can be seen, with the increase of the concentration of reactive oxygen species (H2O2) in the release medium, the amount of meropenem (MEM) released by the OTM prodrug is accelerated. 5 mM H2O2 can catalyze the release of more than 90% of the meropenem (MEM) from the OTM prodrug within 1 hour, while the OM prodrug is hardly catalyzed by reactive oxygen species (H2O2) to release meropenem (MEM).

[0458] Therefore, from Figure 12 and Figure 13 As can be seen, OTM prodrugs are ROS (reactive oxygen species) sensitive prodrugs. ROS can catalyze the rapid and significant release of the active ingredient meropenem (MEM) from OTM prodrugs. The ROS sensitivity of OTM prodrugs has significant application value. For example, due to hypoxia, the ROS level in infected tissues at deep surgical sites is significantly higher than in normal tissues. Therefore, OTM prodrugs can significantly release the active ingredient meropenem (MEM) in infected tissues at deep surgical sites, while it is difficult to release the active ingredient meropenem (MEM) in normal tissues. Thus, OTM prodrug administration can achieve significant targeted treatment of deep surgical site infections while reducing the killing effect on normal tissues, avoiding the side effects such as dysbiosis caused by the overuse of meropenem (MEM) antibiotics, thereby exhibiting excellent biocompatibility. For example, OTM prodrugs can be co-prepared into drugs with substances that generate ROS under ultrasound irradiation (e.g., OTM prodrugs and substances that generate ROS under ultrasound irradiation are co-loaded in liposomes). After administration, only the bacterial infection site is subjected to ultrasound irradiation. The ROS (reactive oxygen species) generated by the substances that generate ROS under ultrasound irradiation can catalyze the rapid and significant release of the active ingredient meropenem (MEM) from the OTM prodrug at the bacterial infection site. In contrast, OTM prodrugs distributed in normal tissues have difficulty releasing the active ingredient meropenem (MEM). This not only achieves significant targeted therapy to the bacterial infection site but also reduces the side effects of OTM prodrugs on normal tissue cells and avoids dysbiosis caused by the overuse of meropenem (MEM) antibiotics, thus exhibiting excellent biocompatibility.

[0459] 8.2. Liposome release

[0460] LPCOTML liposome dispersions (with meropenem equivalents of 10 μM in all liposomes) were subjected to ultrasonic irradiation at 37°C with or without irradiation (sound intensity: 2 W / cm²). 2 After treatment (frequency: 3MHz, duty cycle: 50%, duration: 10min), the amount of meropenem (MEM) released in the sample was determined by high performance liquid chromatography (HPLC), and the results are as follows: Figure 14 As shown.

[0461] from Figure 14As can be seen, LPCOTML liposomes essentially do not release meropenem (MEM) without ultrasound irradiation treatment, but significantly release meropenem (MEM) after ultrasound irradiation treatment. This indicates that ultrasound irradiation can significantly catalyze and promote the release of meropenem from LPCOTML liposomes, which has significant application value. For example, after LPCOTML liposome administration, ultrasound irradiation treatment only on bacterial biofilm-related infection sites can significantly catalyze and promote the rapid and effective release of the active ingredient meropenem from LPCOTML liposomes at bacterial biofilm-related infection sites. However, LPCOTML liposomes distributed in normal tissue sites are difficult to release the active ingredient meropenem (MEM) due to the lack of ultrasound irradiation treatment. This not only enables significant targeted therapy to bacterial biofilm-related infection sites, but also reduces the side effects of meropenem (MEM)-loaded LPCOTML liposomes on normal tissue cells, avoids dysbiosis caused by the overuse of meropenem (MEM) antibiotics, and has excellent biocompatibility.

[0462] 9. Cellular uptake

[0463] Cy5-labeled COTML, PCOTML, LCOTML, or LPCOTML liposome dispersions (each equivalent to 50 μg / mL fluorescent lipid, 20 μL) were mixed with 1 mL of serum-free DMEM medium to obtain a mixture of Cy5-labeled liposomes and culture medium.

[0464] Lipid uptake by HUVEC cells

[0465] HUVEC cells (human umbilical vein endothelial cells, 1×10⁶) were used. 5 The cells were seeded in multi-well plates at 1 ml / ml and cultured for 12 h. The culture medium was removed, and an equal amount of the above-mentioned Cy5-labeled liposomes and culture medium mixture was added. After incubation at 37 °C for 1 h, the cells were washed three times with heparin sodium solution (2 mg / mL), digested, and the cells were collected. The cells were analyzed by flow cytometry to determine the average fluorescence intensity of Cy5-labeled liposomes taken up in HUVECs.

[0466] Lipid uptake in IVEC cells with high expression of VCAM1 receptor

[0467] HUVEC cells (human umbilical vein endothelial cells, 1×10⁶) were used. 5HUVEC endothelial cells were seeded in multi-well plates (1 ml / mL) and cultured for 12 h. The culture medium was removed, and serum-free medium containing TNF-α (tumor necrosis factor-α, 50 ng / mL) was added and cultured for another 12 h. Western blot analysis showed that HUVEC endothelial cells were induced to highly express the VCAM1 receptor and grew into IVC cells, transforming HUVEC cells into VCAM1-highly expressing inflammatory vascular endothelial cells (IVECs). The culture medium was then removed, and an equal volume of the above-mentioned Cy5-labeled liposomes and culture medium mixture was added. After incubation at 37°C for 1 h, the cells were washed three times with heparin sodium solution (2 mg / mL), digested, and collected. Flow cytometry was used to analyze the cells, measuring the average fluorescence intensity of the Cy5-labeled liposomes taken up by the IVC cells.

[0468] Western blotting was used to determine the expression of VCAM1 receptor in HUVEC and IVEC cells. Figure 15 As shown. From Figure 15 As can be seen, VCAM1 receptor expression was significantly increased in inflammatory vascular endothelial cells (IVECs) compared to HUVEC cells.

[0469] The mean fluorescence intensity (MFI) of Cy5-labeled COTML, PCOTML, LCOTML, or LPCOTML liposomes was measured by flow cytometry after incubation with HUVEC or IVEC cells for 1 h. Figure 16 As stated. From Figure 16 It can be seen that there is no significant difference in the uptake of COTML, PCOTML, LCOTML, or LPCOTML liposomes by HUVEC cells. However, the uptake capacity of VCAM1 receptor-highly expressed ...

[0470] 10. Subcellular distribution

[0471] Preparation of IVEC cells: HUVEC cells (human umbilical vein endothelial cells, 1×10⁶) were prepared. 5HUVEC endothelial cells were seeded in multi-well plates (1 ml / ml) and cultured for 12 h. The culture medium was then removed, and serum-free medium containing TNF-α (tumor necrosis factor-α, 50 ng / mL) was added and cultured for another 12 h. Western blot analysis showed that HUVEC endothelial cells were induced to highly express VCAM1 receptor (e.g., 1 ml / ml). Figure 15 As shown in the figure, they grow into IVC cells, and HUVEC cells transform into inflammatory vascular endothelial cells (IVECs) with high VCAM1 expression.

[0472] Cy5-labeled LPCO™ liposome dispersion (equivalent to 50 μg / mL fluorescent lipid, 20 μL) was mixed with 1 mL of serum-free DMEM medium to obtain a mixture of Cy5-labeled LPCO™ liposomes and culture medium. HUVEC or IVEC cells (1 × 10⁻⁶) were then added. 5 Cells were cultured in confocal culture dishes (1 ml, 1 cytidine / ml) for 24 h. Cell nuclei were stained with Hoechst 3342 (2 drops) for 20 min, and cell membranes were stained with fluorescent probe DIO (2.5 μL) for 5 min, or lysosomes were stained with LysoTracker Green DND26 (0.2 μL) for 30 min. Cells were then washed twice with PBS 7.4 buffer, and 1 mL of the above mixture of Cy5-labeled LPCOTML liposomes and culture medium was added. After incubation at 37°C for 0.5 h or 1 h, the subcellular distribution of Cy5-labeled LPCOTML liposomes in HUVEC or IVEC cells was photographed using a confocal laser scanning microscope (CLSM, using 405 nm, 488 nm, and 640 nm wavelength channels).

[0473] Subcellular distribution of Cy5-labeled LPCOTML liposomes after incubation with inflammatory vascular endothelial cells (IVECs) or HUVECs (human umbilical vein endothelial cells) for 0.5 h or 1 h is as follows: Figure 17 , Figure 18 and Figure 19 As shown.

[0474] from Figure 17 and Figure 18 As can be seen, when LPCOTML liposomes were incubated with IVC cells for 0.5 h, most LPCOTML liposomes were rapidly distributed on the IVC cell membrane. Subsequently, after 1 h of incubation, most LPCOTML liposomes were rapidly introduced into IVC cells. However, when LPCOTML liposomes were incubated with HUVEC cells for 0.5 h, only a small number of LPCOTML liposomes were introduced into HUVEC cells. This indicates that the high expression of VCAM1 receptor in IVC cells can rapidly mediate the incorporation of VCAM1 ligand-modified LPCOTML liposomes into IVC cells.

[0475] from Figure 19 As can be seen, after LPCOTML liposomes are taken up into inflammatory vascular endothelial cells (IVECs), they are distributed in large quantities in the cytoplasm and are not captured by lysosomes. This indicates that LPCOTML liposomes have good lysosomal avoidance properties, thereby preventing the LPCOTML liposomes and the drug they carry from being degraded by enzymes in lysosomes. This effectively protects the drug from degradation and destruction by lysosomes and enhances the stability of LPCOTML liposomes and the drug they carry in IVEC cells.

[0476] 11. Transendothelial transport

[0477] The Transwell system (3μm microporous polyester membrane and 24mm diameter built-in dish) is used to study ligand / receptor-mediated transport of liposomes in HUVEC or IVEC cells. A schematic diagram of the Transwell system is shown below. Figure 20 As shown.

[0478] Transport in HUVEC cells:

[0479] HUVEC cells (human umbilical vein endothelial cells, 1×10⁻⁶) 5 After incubating HUVEC cells (1 ml / ml) in the top chamber for 4 days to form a dense cell layer, they were then incubated with serum-free medium for 12 hours. COTML with the same fluorescence intensity was then applied. Cy5 LCOTML Cy5 PCOTML Cy5 or LPCOTML Cy5 The liposomes were added to the top chamber and incubated in the culture medium for 2 hours. The fluorescence intensity of the culture medium on the outside of the substrate was measured using an ELISA reader.

[0480] Transport in IVC cells:

[0481] HUVECs (human umbilical vein endothelial cells) (1×10) 5 HUVEC cells were incubated in the top chamber for 4 days (1 ml / ml) to form a dense cell layer. Then, they were incubated for 12 hours in serum-free medium containing TNF-α (tumor necrosis factor-α, 50 ng / mL). Western blot analysis showed that HUVEC endothelial cells were induced to overexpress the VCAM1 receptor and grow into IVC cells. HUVEC cells transformed into inflammatory vascular endothelial cells (IVECs) with high VCAM1 expression. Then, COTML with the same fluorescence intensity was used... Cy5 PCOTML Cy5 LCOTML Cy5 or LPCOTML Cy5Liposomes were added to the head chamber and incubated in culture medium for 2 hours. The fluorescence intensity of the medium on the outer side of the substrate was measured using a microplate reader. Furthermore, the fluorescence intensity of COTML (with the same fluorescence intensity) was investigated after IVEC cell blood vessels were pretreated with the exocytosis inhibitor EXO1 for 2 hours. Cy5 PCOTML Cy5 LCOTML Cy5 or LPCOTML Cy5 The liposomes were added to the top chamber and incubated in the culture medium for 2 hours. The fluorescence intensity of the culture medium on the outside of the substrate was measured using an ELISA reader.

[0482] Different Cy5-labeled liposomes were incubated for 2 hours with HUVEC cells and IVEC cells pretreated with or without the exocytosis inhibitor EXO1. The mean fluorescence intensity (MFI) of the different Cy5-labeled liposomes in the basal-side medium of the Transwell system was as follows: Figure 21 As shown. From Figure 21 It can be seen that VCAM1 ligand-modified LPCOTML and LCOTML liposomes have excellent transvascular transport capabilities across VCAM1 receptor-highly expressed IVC cells, significantly stronger than PCOTML and COTML liposomes. The transvascular transport capabilities of LPCOTML and LCOTML liposomes across IVC cells were significantly inhibited by exocytosis inhibitors, while the transvascular transport capabilities of different liposomes across HUVEC cells were low and showed no significant difference. This indicates that VCAM1 ligand-modified liposomes can significantly cross IVC cell vessels through VCAM1 receptor-mediated endocytosis and exocytosis. Therefore, VCAM1 ligand-modified liposomes can significantly cross the blood vessels of VCAM1 receptor-highly expressed cells at surgical sites associated with bacterial biofilms to enter the infection site, thereby effectively exerting the therapeutic effect of drugs on bacterial infections.

[0483] In addition, chlorpromazine (50 μM, clathrin-mediated endocytosis inhibitor), genistein (200 μM, cell wall-mediated endocytosis inhibitor), wortmannin (5 μM, phosphatidylinositol 3-kinase-mediated macropinocytosis inhibitor), and cytochalasin D (5 μM, actin polymerization inhibitor) were added to serum-free medium in the head chamber for 2 h before pre-incubating HUVEC or IVEC cells, and then LPCOTML was added. Cy5 Liposomes were added to the head chamber and incubated in culture medium for 1 hour. The fluorescence intensity of Cy5-labeled liposomes in the basal-side medium was measured using a microplate reader to study the endocytic pathway. After pretreatment of HUVEC or IVEC cells with different endocytosis inhibitors, the fluorescence intensity of Cy5-labeled LPCOTML in the basal-side medium of the Transwell system was measured. Cy5The mean fluorescence intensity (MFI) of liposomes is as follows: Figure 22 As shown. From Figure 22 It can be seen that IVC cells mainly take up and transport LPCOTML liposomes through endocytosis mediated by the cell membrane pit initiated by the VCAM1 receptor.

[0484] 12. In vitro antibacterial biofilm activity

[0485] 12.1 Colony Count Determination and Investigation of the Effects of Different Drug Treatments on the Extracellular Polymer Matrix Barrier of Bacterial Biofilms

[0486] 12.1.1 Colony counting is used to assess viable bacteria in biofilms after different treatments.

[0487] Pseudomonas aeruginosa PAO1 was inoculated into multi-well plates at 37°C and cultured for 72 h (Luria Bertani broth was replaced every 24 h). The culture medium was then removed to obtain a biofilm containing P. aeruginosa PAO1. PBS, blank LPCL liposomes, meropenem (MEM), COTML liposomes, PCOTML liposomes, LCOTTML liposomes, or a mixture of LPCOTML liposomes and LB broth were then added (where the doses of meropenem (MEM), COTML liposomes, PCOTML liposomes, LCOTTML liposomes, and LPCOTML liposomes were all 41.7 μM, based on meropenem equivalents). The biofilm was then subjected to ultrasound irradiation (sound intensity: 2 W / cm²) with or without. 2 (Frequency: 3MHz, Duty Cycle: 50%) Treatment for 5 min, followed by washing the biofilm with PBS 7.4 buffer, and then sonicating. The bacterial suspensions from each treatment group were serially diluted 10-fold, and 100 μl of each suspension was spread onto agar plates. After incubation for 24 h, the number of colony-forming units (CFU) in each treatment group was counted, and photographs were taken (e.g., frequency: 3MHz, duty cycle: 50%). Figure 23 and Figure 24 (As shown).

[0488] 12.1.2 Colony Count Determination and Investigation of the Effects of Different Drug Treatments on the Extracellular Polymer Matrix Barrier of Bacterial Biofilms

[0489] Pseudomonas aeruginosa PAO1 was inoculated into multi-well plates at 37°C and cultured for 72 h (Luria Bertani broth was replaced every 24 h). After removing the culture medium, a biofilm containing P. aeruginosa PAO1 was obtained. Then, a mixture of PBS, meropenem (MEM) or LPCOTML liposomes and LB broth was added (where the dose of meropenem (MEM) and LPCOTML liposomes was 20 μM, based on meropenem equivalents). The biofilm was then subjected to ultrasonic irradiation (sound intensity: 2 W / cm²).2 After treatment with a frequency of 3 MHz and a duty cycle of 50% for 5 min and incubation for 1 h, the biofilm was washed with PBS 7.4 buffer. A portion of the biofilm was then immersed in 2.5% glutaraldehyde solution for 12 h, followed by staining with 2% tannin and 1% osmium tetroxide for 2 h. The biofilm was then dehydrated with ethanol, transferred to isoamyl acetate for critical point drying, and treated with gold spraying. The biofilm was then observed using a scanning electron microscope (SEM). Figure 25 (As shown); another portion of the biofilm was immersed in PBS buffer and then stained with a biofilm matrix staining agent (FilmTracer). TM SYPRO TM Ruby, #F10318, Invitrogen Inc.) was stained for 10 min and observed using a confocal laser scanning microscope (CLSM) (e.g. Figure 25 (As shown).

[0490] Different drugs were incubated with Pseudomonas aeruginosa PAO1 biofilms, and the biofilms were treated with or without ultrasonic irradiation. The resulting images of bacteria in the P. aeruginosa PAO1 biofilms on agar plates and the number of bacterial colony-forming units (CFU) in the P. aeruginosa PAO1 biofilms were obtained as follows: Figure 23 and Figure 24 As shown. From Figure 23 and Figure 24 As can be seen, without ultrasonic irradiation treatment, there was no significant difference in the number of bacterial colony-forming units (CFU) in the P. aeruginosa PAO1 biofilm among the different treatment groups after incubation with different drugs. This means that there was no significant difference in the antibacterial effect of the treatment groups on the P. aeruginosa PAO1 biofilm. This is mainly because the extracellular polymeric substances (EPS) matrix barrier of the P. aeruginosa PAO1 biofilm restricts the penetration of antibacterial drugs into the biofilm, thereby protecting the bacteria from the inhibitory effect of antibacterial drugs and thus hindering the antibacterial effect of antibacterial agents. However, compared with the case where different drugs were incubated with Pseudomonas aeruginosa PAO1 biofilms without ultrasonic irradiation, the number of bacterial colony-forming units (CFU) in Pseudomonas aeruginosa PAO1 biofilms treated with LPCOTML liposomes was significantly reduced after ultrasonic irradiation, and was significantly lower than that in the meropenem (MEM), COTML liposome, PCOTML liposome, and LPCOTML liposome treatment groups.

[0491] Different drugs were incubated with Pseudomonas aeruginosa PAO1 biofilms, followed by ultrasonic irradiation. After 1 hour of incubation, the extracellular polymer matrix barrier of the bacterial biofilms in each treatment group was observed using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Figure 25 As shown. From Figure 25 As can be seen, the extracellular polymeric substances (EPS) matrix barrier of bacterial biofilms is densely distributed. Under ultrasound irradiation treatment following incubation with different drugs on *Pseudomonas aeruginosa* PAO1 biofilms, the EPS matrix barrier of the meropenem (MEM) treatment group remained unchanged. However, LPCOTML liposomes significantly disrupted the EPS matrix barrier, enhancing drug penetration throughout the deep biofilm. Therefore, ultrasound irradiation enables LPCOTML liposomes to effectively disrupt the EPS matrix barrier of *P. aeruginosa* PAO1 biofilms, overcoming the limitation of antimicrobial drug penetration by the EPS matrix barrier. This allows antimicrobial drugs to penetrate deep into the bacterial biofilm and kill bacteria located deep within it, thus significantly promoting and enhancing the inhibitory effect of LPCOTML liposomes on bacteria in *P. aeruginosa* PAO1 biofilms, effectively inhibiting bacterial infection.

[0492] 12.2 Staining of live / dead bacteria

[0493] Pseudomonas aeruginosa PAO1 was inoculated into glass dishes and cultured in Luria Bertani broth for 72 h (the broth was changed every 24 h). The culture medium was then removed to obtain a biofilm containing P. aeruginosa PAO1. PBS, blank LPCL liposomes, meropenem (MEM), COTML liposomes, PCOTML liposomes, LCOTTML liposomes, or a mixture of LPCOTML liposomes and LB broth were then added (where the dose of meropenem (MEM), COTML liposomes, PCOTML liposomes, LCOTTML liposomes, and LPCOTML liposomes was 41.7 μM, based on meropenem equivalents). The biofilm was then subjected to ultrasound irradiation (sound intensity: 2 W / cm²) with or without. 2The bacterial biofilm was treated with a frequency of 3 MHz, a duty cycle of 50%, and a duration of 5 min. After washing the biofilm with PBS 7.4 buffer, it was stained with a combination of SYTO9 and propidium iodide (PI) from the live / dead bacteria viability kit (L7012, Invitrogen) (SYTO9:PI molar ratio of 1:1) at room temperature and in the dark for 20 min. After washing the biofilm three times with PBS 7.4 buffer, the stained biofilm was imaged using a confocal laser scanning microscope (CLSM).

[0494] Different drugs were incubated with Pseudomonas aeruginosa PAO1 biofilms, followed by ultrasonic irradiation treatment. Live / dead bacterial staining and 3D reconstructed images of the biofilms were obtained for each treatment group. Figure 26 As shown. Figure 26 Fluorescent staining results showed that the bacterial mortality rate of *Pseudomonas aeruginosa* PAO1 biofilms obtained after incubation with different drugs and subsequent ultrasonic irradiation treatment was consistent with the results of the "colony counting determination" described above. Figure 26As can be seen, after different drugs were incubated with Pseudomonas aeruginosa PAO1 biofilms without ultrasonic irradiation, a small number of dead bacteria were observed in the P. aeruginosa PAO1 biofilms in each treatment group. This is mainly because the extracellular polymeric substances (EPS) matrix barrier of P. aeruginosa PAO1 biofilms restricts the penetration of antimicrobial drugs into the biofilm, thereby protecting the bacteria from the inhibitory effect of antimicrobial drugs and thus hindering the antimicrobial effect of antimicrobial agents. However, compared with the cases where different drugs were incubated with P. aeruginosa PAO1 biofilms without ultrasonic irradiation, the P. aeruginosa PAO1 biofilms treated with different drugs and then subjected to ultrasonic irradiation showed a small number of dead bacteria in the meropenem (MEM), COTML liposome, and LPCOTML liposome treatment groups, with the dead bacteria mainly located on the surface of the P. aeruginosa PAO1 biofilm. In contrast, the P. aeruginosa PAO1 biofilms treated with PCOTML and LPCOTML liposomes showed a large number of dead bacteria throughout the entire thickness. In particular, compared with other treatment groups, the P. aeruginosa PAO1 biofilms treated with LPCOTML liposomes showed the highest number of dead bacteria throughout the entire thickness, indicating that LPCOTML liposomes had the strongest antibacterial effect. Therefore, ultrasound irradiation can significantly disrupt the extracellular polymeric substance (EPS) matrix barrier of Pseudomonas aeruginosa PAO1 biofilm by LPCOTML liposomes, overcoming the limitation of the bacterial biofilm's EPS matrix barrier on the penetration of antimicrobial drugs into the biofilm. This allows the antimicrobial drugs to penetrate deep into the bacterial biofilm and kill bacteria located deep within it. Thus, ultrasound irradiation can significantly promote and enhance the inhibitory effect of LPCOTML liposomes on bacteria in Pseudomonas aeruginosa PAO1 biofilm, thereby effectively inhibiting bacterial infection.

[0495] 13. Biodistribution and permeability of liposomes in mice infected at deep surgical sites.

[0496] BALB / c nude mice infected with a deep surgical site constructed by infecting *Pseudomonas aeruginosa* PAO1 (PAO1-Luc) expressing luciferase were randomly divided into groups. Each group of mice was injected via tail vein with COTML containing the same fluorescence intensity. Cy5 PCOTML Cy5 LCOTML Cy5 or LPCOTML Cy5 Liposome dispersions were administered, and 6 hours after injection, the deep surgical sites of infected nude mice in each group were irradiated with ultrasound (sound intensity: 2 W / cm²). 2The mice were treated with an IVIS (excitation / emission wavelength, 640 / 670 nm, PerkinElmer) system (frequency: 3 MHz, duty cycle: 50%, duration: 10 min). Twelve h after administration, each group underwent in vivo imaging using the IVIS system. Each mouse was then intravenously injected with FITC-labeled tomato lectin (FITC-Lectin, 0.05 mg / mouse). Five min after injection, the heart was perfused with 4% paraformaldehyde solution. BSSI (biofilm-associated surgical site infection) tissues, heart, liver, spleen, lung, kidney, and small intestine were then dissected and isolated. Bioluminescence and fluorescence imaging of the dissected BSSI tissues, heart, liver, spleen, lung, kidney, and small intestine were performed using the IVIS system. The fluorescence intensity of the dissected BSSI tissues from each treatment group was quantitatively analyzed using Living Image-4.5 software. In addition, the dissected BSSI tissue was frozen in tissue OCT-Freeze Medium, and after being sectioned into 10μm thick sections, the infected tissue at the surgical site was imaged using a confocal laser scanning microscope (CLSM).

[0497] Six hours after intravenous injection of different liposomes into BALB / c nude mice with deep surgical site infections, the mice were subjected to ultrasound irradiation. In vivo imaging and bioluminescence and fluorescence imaging of dissected tissues were performed 12 hours after drug administration. Quantitative analysis of fluorescence intensity in isolated BSSI tissues, heart, liver, spleen, lung, kidney, and small intestine was also conducted. Figure 27 and Figure 28 As shown. From Figure 27 and Figure 28 As can be seen, there was no significant difference in the aggregation ability of COTML and PCOTML liposomes in BSSI tissue. Compared with COTML and PCOTML liposomes, LCOTML and LPCOTML liposomes were mainly distributed in BSSI (biofilm-associated surgical site infection) tissue. In particular, LPCOTML liposomes showed significantly superior fluorescence intensity in BSSI tissue. This is because the high expression of VCAM1 receptor on the blood vessels of BSSI tissue can mediate the transport of VCAM1 ligand-modified LPCOTML liposomes into BSSI tissue. Furthermore, ultrasound irradiation can effectively break the extracellular polymeric substance (EPS) matrix barrier of bacterial biofilms, overcoming the limitation of the bacterial biofilm EPS matrix barrier on the penetration of antibacterial drugs into the biofilm. This allows antibacterial drugs to penetrate deep into the bacterial biofilm and kill bacteria located deep in the bacterial biofilm. As a result, VCAM1 ligand-modified LPCOTML liposomes have the best targeted aggregation ability in BSSI tissue, thus effectively exerting the therapeutic effect of drugs on bacterial infections.

[0498] Six hours after intravenous injection of different liposomes into the tail vein of BALB / c nude mice with deep surgical site infections, the mice were treated with ultrasound irradiation. Twelve hours after administration, they received intravenous injection of FITC-Lectin and cardiac perfusion. Dissected BSSI tissue was then frozen in OCT embedding medium for tissue frozen sections. After sectioning, the BSSI tissue was imaged using a confocal laser scanning microscope (CLSM). Figure 29 As shown. From Figure 29 The co-localization of blood vessels and liposomes shows that COTML and PCOTML liposomes have almost no chance of entering the BSSI tissue through blood vessels at the surgical site of infection. LCOTML liposomes are mainly distributed around the blood vessels in the BSSI tissue. However, LPCOTML liposomes can effectively penetrate the BSSI tissue through blood vessels and distribute deep within the BSSI tissue. This indicates that LPCOTML liposomes have excellent ability to penetrate from blood vessels into the BSSI tissue and into its deep layers, thus giving them excellent antibacterial activity.

[0499] 14. Real-time extravasation within the body's blood vessels

[0500] BALB / c nude mice were subcutaneously injected with P. aeruginosa PAO1 near the abdominal blood vessels. 72 hours later, the bacterial biofilm-infected area was fixed onto a microscope slide using a dorsal skinfold chamber. The blood vessels surrounding the bacterial biofilm infection were clearly identified using confocal laser scanning microscopy (CLSM). Then, each group of mice was injected via tail vein with COTML, which has the same fluorescence intensity. Cy5 PCOTML Cy5 LCOTML Cy5 or LPCOTML Cy5 Liposome dispersions were used to image bacterial-infected tissues using confocal laser scanning microscopy (CLSM) at regular intervals. After measuring the corresponding fluorescence intensity from blood vessels to the infected tissue, bacterial-infected tissues were dissected and isolated 30 minutes after intravenous injection. The tissues were then fixed at 4°C in 10 volumes of 4% paraformaldehyde for 24 hours. The samples were then cut into 5 mm sections, immersed in 7% agarose solution, and sliced. The slices were washed with sodium carbonate buffer (100 mM) and fixed in 1% osmium tetroxide for 2 hours. After staining with uranyl acetate at 37°C for 48 hours and dehydrating with a gradient of acetone, the tissues were fixed with epoxy resin, sliced, stained with lead citrate, and observed by transmission electron microscopy (TEM).

[0501] In BALB / c nude mice infected with P. aeruginosa PAO1 in the abdomen, confocal laser scanning microscopy (CLSM) imaging and mean integrated fluorescence intensity (MFI) of the bacterial infection site were performed at 0 min, 10 min, and 30 min after intravenous injection of liposomes with different Cy5-labeled dispersions with the same fluorescence intensity. Figure 30 and Figure 31 As shown, this was used to observe the leakage of different liposomes from blood vessels at the biofilm infection site into the biofilm-infected tissue. Figure 30 and Figure 31 As can be seen, after intravenous injection of different liposomes, LCOTML and LPCOTML liposomes can rapidly adhere to the vascular endothelial cells of BSSI (biofilm-associated surgical site infection) within 30 minutes and leak from the blood vessels of BSSI into the BSSI tissue, while COTML and PCOTML liposomes have difficulty leaking from the blood vessels of BSSI into the BSSI tissue.

[0502] Cryo-transmission electron microscopy (cryo-TEM) imaging of BALB / c nude mice infected with P. aeruginosa PAO1 via the tail vein, 30 min later, of liposomes labeled with different Cy5-labeled molecules and having the same fluorescence intensity, was performed on dissected bacterial-infected tissues. Figure 32 As shown, this was used to observe and analyze the ultrastructure of blood vessels in BSSI tissue sites after different liposome treatments. Figure 32 As can be seen, in the COTML and PCOTML treatment groups, the blood vessels at the BSSI (biofilm-associated surgical site infection) site had well-tissued and tightly connected endothelial cells with almost no vesicle distribution. However, in the LCOTML and LPCOTML liposome-treated BSSI tissues, a large number of vesicles (such as...) were observed in the vessel walls. Figure 32 (As indicated by the arrows) These vesicles are typical transvascular endothelial cell transporters. Therefore, LCOTML and LPCOTML are transported across the blood vessels of BSSI tissue sites into the BSSI tissue sites via VCAM1 ligand / VCAM1 receptor-mediated endocytosis and exocytosis transport pathways.

[0503] 15. In vivo antibacterial biofilm activity

[0504] BALB / c nude mice infected with Pseudomonas aeruginosa PAO1 (PAO1-Luc), which expresses luciferase, were randomly divided into 7 groups. Each group of mice received a tail vein injection of PBS 7.4 buffer, meropenem (MEM) in PBS 7.4 dispersion, blank LPCL liposome dispersion, COTML liposome dispersion, PCOTML liposome dispersion, LCOTML liposome dispersion, or LPCOTML liposome dispersion (the dosage of meropenem (MEM), COTML liposome, PCOTML liposome, LCOTML liposome, or LPCOTML liposome was 10 mg / kg, based on the meropenem equivalent). Six hours after the tail vein injection, the deep surgical site infection area of ​​each group of mice was irradiated with ultrasound (sound intensity: 2 W / cm²). 2 Treatment involved administering the drug via tail vein injection every two days, with ultrasound irradiation (frequency: 3MHz, duty cycle: 50%, duration: 10min) to the infected areas of deep surgical sites in nude mice of each group 6 hours after administration (sound intensity: 2W / cm²). 2 Treatment with a frequency of 3 MHz, a duty cycle of 50%, and a duration of 10 min was administered to each group three times (the first administration was counted as day 0). On day 7, mice were sacrificed, and the bacterial biofilm area was dissected and photographed (e.g., [missing information]). Figure 33 (As shown), the dissected bacterial biofilm area tissue was then divided into two parts. One part was homogenized using an ultrasonic homogenizer and used for colony counting to obtain the number of colony-forming units (CFUs) (e.g., ...). Figure 34 As shown), the biofilm inhibition rate was calculated using the formula: Inhibition rate = (CFU of PBS 7.4 treatment group - CFU of other treatment groups) / CFU of PBS 7.4 treatment group × 100%; another part was fixed in a neutral 4% paraformaldehyde buffer solution and embedded in paraffin. 5μm thick tissue sections were placed on glass slides and stained with hematoxylin-eosin (H&E) or Masson stain, and examined using an optical microscope (e.g., ...). Figure 35 (As shown).

[0505] from Figure 33As can be seen from the macroscopic photographs at the end of the experiment, except for LPCOTML, the sutured skin at the surgical sites of all groups began to suppurate and ulcerate, indicating that biofilm infection at the abscess site caused a severe inflammatory response. Conversely, the surgical sites in the LPCOTML group almost healed, and no abscesses or wound ulcers were observed. To detect the presence of viable bacteria at the surgical wound site after treatment, the surgical wound tissue of each group was dissected, and its solution was applied to LB agar plates after thorough shaking. After 24 hours of incubation, the number of CFUs in each group was counted. Regarding viable bacteria, the average biofilm inhibition rate of LPCOTML was 61.2% compared to the PBS group, significantly higher than that of MEM, COTML, PCOTML, and LCOTML (e.g., MEM, COTML, PCOTML, and LCOTML). Figure 34 Histological analysis of BSSI tissue was performed to investigate the mechanism of its anti-biofilm activity. Figure 35 As can be seen from the hematoxylin and eosin (H&E) staining images, there was a large number of neutrophil infiltrations in the PBS, LPCL, COTML, LCOTOML, and PCOTML treatment groups, indicating a severe inflammatory response to the infection. However, the hair follicle morphology in the LPCOTML group was normal, without obvious neutrophil infiltration. Furthermore, Masson staining was used to observe collagen deposition and tissue repair in subcutaneous tissue. The LPCOTML group promoted subcutaneous collagen growth to facilitate wound healing after 7 days of treatment, while this trend was very weak in the other groups. In addition, in liposome-treated mice, H&E staining images of major organs (such as heart, liver, spleen, lungs, and kidneys) did not show significant pathological changes or adverse reactions, demonstrating good biocompatibility and safety.

[0506] 16. In vivo antibacterial biofilm activity comparison experiment with clinically commercial drugs

[0507] The efficacy and clinical application potential of LPCOTML liposomes were further investigated by comparing them with Amikacin liposome (AL), a commercially available antibiotic used to treat Pseudomonas aeruginosa, and Gentamicin, another antibiotic.

[0508] BALB / c nude mice infected with a deep surgical site constructed by infecting *Pseudomonas aeruginosa* PAO1 (PAO1-Luc) expressing luciferase were randomly divided into four groups. Each group of mice received a tail vein injection of either Amikacin liposome (AL) in PBS 7.4 dispersion (10 mg / kg, based on amikacin), gentamicin (Gen) in PBS 7.4 dispersion (20 mg / kg, based on gentamicin), LPCOTML liposome dispersion (10 mg / kg, based on meropenem equivalent), or PBS 7.4 buffer. Six hours after the tail vein injection, the deep surgical site infection area of ​​each group of mice was irradiated with ultrasound (sound intensity: 2 W / cm²). 2 Treatment involved administering the drug via tail vein injection every two days, with ultrasound irradiation (frequency: 3MHz, duty cycle: 50%, duration: 10min) to the infected areas of deep surgical sites in nude mice of each group 6 hours after administration (sound intensity: 2W / cm²). 2 Treatment with a frequency of 3 MHz, a duty cycle of 50%, and a duration of 10 min was administered to each group three times (the first administration was counted as day 0). During the treatment period, the size of the bacterial biofilm and the bioluminescence count (e.g., frequency: 3 MHz, duty cycle: 50%, duration: 10 min) were measured. Figure 36 and Figure 37 (As shown), on day 7, mice were sacrificed, and the bacterial biofilm area was dissected. The dissected bacterial biofilm area was then homogenized using an ultrasonic homogenizer and used for colony counting to obtain the number of colony-forming units (CFUs). Figure 38 As shown), the biofilm inhibition rate was calculated using the formula: Inhibition rate = (CFU of PBS 7.4 treatment group - CFU of other treatment groups) / CFU of PBS 7.4 treatment group × 100%.

[0509] from Figure 36 and Figure 37 As can be seen, the bacterial bioluminescence signal in the gentamicin (Gen) treatment group and the amikacin liposome (AL) treatment group steadily increased over time, while the bacterial bioluminescence signal in the LPCOTML liposome treatment group gradually decreased and even became undetectable. This indicates that LPCOTML liposomes have significantly superior in vivo anti-biofilm activity compared to the gentamicin (Gen) treatment group and the amikacin liposome treatment group. Figure 38As can be seen from the results, when the number of CFUs in each group was calculated based on the number of viable bacteria surviving in the bacterial biofilm area, the average biofilm inhibition rate of the LPCOTML liposome treatment group was 63.7%, which was much higher than that of the Amikacin liposome (AL) treatment group (16.9%) and the gentamicin (Gen) treatment group (11.3%). This indicates that LPCOTML liposomes have significant and excellent in vivo anti-biofilm activity, thus having good potential for clinical application.

[0510] Statistical analysis

[0511] All experiments were repeated at least three times. Data are presented as mean ± standard deviation and analyzed using Student's t-test and one-way ANOVA. GraphPad Prism 9.0 was used for plotting and statistical analysis in all tests, with statistical significance defined as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001; "ns" indicates no significance.

[0512] The above description is an implementation scheme designed for one case of the present invention. It should be noted that for those skilled in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A meropenem prodrug, characterized in that, The structure of the meropenem prodrug is shown below:

2. A liposome, characterized in that, The liposomes include drugs for the prevention and / or treatment of infections; the drugs include meropenem prodrug as described in claim 1.

3. The liposomes as described in claim 2, characterized in that, The liposomes comprise dihydroporphyrin E6; and The liposomes are loaded with perfluoropentane.

Images