Targeted antibacterial organism medicament as well as preparation method and application thereof

Abstract

The invention provides a targeted antibacterial organism medicament as well as a preparation method and application thereof. The targeted antibacterial organism medicament comprises a biological active part with targeting property, a medicament combined with the biological active part and the like. The medicament comprises a medicament body and a coating body which is used for coating antibiotics and is combined with a bioactive part, the coating body is used for dissolving and releasing antibiotics in a preset environment. Compared with the prior art, the method has the advantage that the killing or inhibiting effect on target bacteria can be improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a targeted antibacterial biological agent and a preparation method and application thereof. Background Art

[0002] Biofilm is an ordered aggregate formed by bacteria adhering to a specific carrier. The process is that planktonic bacteria migrate to the surface of the carrier to adhere and colonize, forming microcolonies, and then mature into a complex community structure. In the process, they secrete substances such as polysaccharides and DNA to form extracellular polymeric substances (EPS). Studies have shown that bacteria with "biofilm phenotype" are 10 to 1,000 times more resistant than those with "planktonic phenotype". EPS provides important structural support and protection for the surrounding microbial community and prevents antibiotics from killing them.

[0003] In the face of the crisis of multidrug-resistant (MDR) bacterial infection, phage therapy is a resurgent antibacterial biological therapy. The typical phage structure consists of two parts: the head and the tail. The head is composed of a protein capsid and genetic material, and the tail is composed of a tail sheath, a tail tube, a base plate, a spike, and a tail filament. Its spike and tail filament have receptor binding proteins that can specifically bind to bacteria and then inject their own genetic material into the host bacteria. In this process, the lytic phage hijacks the gene expression of the host bacteria to achieve the replication of its own genetic material and protein synthesis, and produces progeny phages. The progeny phages can attack neighboring hosts and achieve a new round of infection. Therefore, the bactericidal mechanism of phages is inconsistent with that of antibiotics. Compared with antibiotics, lytic phages can replicate themselves and are highly specific to the host, while effectively killing biofilm infections caused by multidrug-resistant bacteria. In addition, its feet carry enzymes (depolymerases) that specifically degrade extracellular polymers to disintegrate biofilms internally. However, when facing a simple phage infection, bacteria will evolve various anti-phage defense systems that can prevent being killed at various stages of infection and replication. For example, during the infection stage, the innate immune system will be quickly activated to prevent phage adsorption by changing receptors on the cell membrane, or use restriction enzyme systems to degrade foreign DNA; and when genetic material is replicated in the host, the adaptive immune response will be mobilized to provide highly specific and long-term anti-phage protection (such as the CRISPR-Cas system). Therefore, how to enhance the phage's defense system against bacteria is crucial to combat biofilm infection.

[0004] Therefore, how to provide a targeted antibacterial agent and a preparation method thereof to enhance the killing or inhibition effect on target bacteria is a technical problem that urgently needs to be solved in this field. Summary of the invention

[0005] The object of the present invention is to provide a targeted antibacterial biological agent, comprising: a biologically active portion with targeting properties, and an agent combined with the biologically active portion; wherein the agent comprises: an agent body, a coating body used to coat an antibiotic and combined with the biologically active portion; the coating body is used to dissolve and release the antibiotic under a preset environment.

[0006] Further preferably, the coating body is covalently coupled to the biologically active part.

[0007] Further preferably, the encapsulated body is a nano-sized liposome.

[0008] Further preferably, the average particle size of the coating is 24 nm to 70 nm.

[0009] Further preferably, the average particle size of the coating is 30.55 nm to 38.74 nm.

[0010] Further preferably, the nano-sized liposome is covalently coupled to the azidated organism via the DBCO functional group contained therein.

[0011] Further preferably, the biologically active portion is a bacteriophage modified with an azidation group.

[0012] Further preferably, the liposome comprises: liposome phospholipids, cholesterol, and polyethylene glycol phospholipids with covalent coupling groups;

[0013] The ratio of the liposome phospholipid, cholesterol and polyethylene glycol phospholipid is 10:(1-5):(0.1-1).

[0014] Further preferably, the ratio of the liposome phospholipid, cholesterol, and polyethylene glycol phospholipid is 10:(1-3.5):(0.1-1).

[0015] Further preferably, the ratio of the liposome phospholipid, cholesterol, and polyethylene glycol phospholipid is 10:(1-3.5):0.2.

[0016] Further preferably, the liposome phospholipid is 1-palmitoyl-2-oleoyl-phosphatidylcholine;

[0017] The polyethylene glycol phospholipid is distearoyl phosphatidylethanolamine-polyethylene glycol-diphenylcyclohexane.

[0018] Further preferably, the drug substance is an antibiotic;

[0019] The antibiotics include: water-soluble antibiotics and / or fat-soluble antibiotics;

[0020] Alternatively, the antibiotics include: bactericidal antibiotics and / or bacteriostatic antibiotics.

[0021] Further preferably, the drug body is an antibiotic; wherein the antibiotic includes: rifampicin and / or tigecycline.

[0022] Further preferably, the biologically active portion is a phage targeted for lysing a target bacterium; wherein the target bacterium is any one of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Propionibacterium acnes or Acinetobacter baumannii.

[0023] Further preferably, the biologically active portion is a targeted bacteriophage having a depolymerase gene, wherein the depolymerase gene is selected from ORF96.

[0024] Further preferably, the biologically active portion targets bacteriophage Sb-1; the bacterium targeted for lysis by the biologically active portion is Staphylococcus aureus.

[0025] Further preferably, the concentration of the antibiotic in the biologically active portion is 0.0002-9 μg / mL; or the titer of the bacteriophage in the biologically active portion is 1×10 4 PFU / mL or above.

[0026] Further preferably, the concentration of the antibiotic in the biologically active portion is 0.0001-2.6 μg / mL.

[0027] Further preferably, the encapsulation efficiency of the nano-liposome is greater than or equal to 45.59%;

[0028] The maximum cumulative release rate of the nano-liposomes in a PBS solvent at a neutral pH after 4 to 7 days is 6.31% to 6.70%;

[0029] Alternatively, when the antibiotic is a water-soluble antibiotic, the maximum cumulative release rate of the nano-sized liposomes after 4 to 7 days in a PBS solvent at a neutral pH is 6.31%;

[0030] Alternatively, when the antibiotic is a lipid-soluble antibiotic, the maximum cumulative release rate of the nano-sized liposomes after 4 to 7 days in a PBS solvent at neutral pH is 6.70%.

[0031] The present application also provides a method for preparing a targeted antibacterial biological agent, and the method is used to prepare the above-mentioned targeted antibacterial biological agent.

[0032] Further preferably, the preparation method comprises the following steps:

[0033] Extracting a biologically active portion capable of specifically lysing target bacteria and degrading its biofilm matrix, and preparing a biologically active portion solution;

[0034] by adding NHS-N3 linker to the biologically active part solution to obtain the azidated biologically active part;

[0035] Dissolving phospholipids, cholesterol, and polyethylene glycol phospholipids in chloroform according to a preset ratio, and after the chloroform evaporates, adding a preset drug body to prepare a lipid drug suspension, and then placing the lipid drug suspension in a microfluidizer to synthesize nano-scale liposomes encapsulating the drug body;

[0036] The coated body composed of nano-liposomes is mixed and cultured with the azidation biologically active part to obtain the targeted antibacterial biological agent.

[0037] Further preferably, the concentration of the NHS-N3 linker is 0.00009-0.0005 μg / mL.

[0038] The present application also provides a sterilization method, which is carried out by lysing the above-mentioned targeted antibacterial organism agent.

[0039] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0040] The targeted antimicrobial biological agent and preparation method thereof provided by the present invention improve the killing or inhibition effect on target planktonic bacteria and / or biofilms, while reducing the usage of antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is an antibacterial pattern diagram of the targeted antibacterial biological agent in the first embodiment of the present invention;

[0042] Figure 2 is a schematic diagram of liposome synthesis in the first embodiment of the present invention;

[0043] Figure 3 It is a schematic diagram of the effect of different modified concentrations of NHS-N3 on the activity of organisms of the present invention;

[0044] Figure 4 is a morphological diagram of liposomes under transmission electron microscopy in the present invention;

[0045] Figure 5 It is a morphological diagram of the biological agent under a transmission electron microscope in the present invention;

[0046] Figure 6is a schematic diagram of the particle sizes of rifampicin encapsulated by liposomes with different cholesterol concentrations in the present invention;

[0047] Figure 7 is a schematic diagram of the particle sizes of tigecycline encapsulated by liposomes with different cholesterol concentrations in the present invention;

[0048] Figure 8 is a schematic diagram of the particle size distribution of liposome-encapsulated rifampicin and tigecycline in the present invention;

[0049] Fig. 9 is a schematic diagram of the efficiency of liposomes with different cholesterol concentrations encapsulating rifampicin and tigecycline in the present invention;

[0050] Fig.10 is a schematic diagram of the efficiency of releasing rifampicin and tigecycline from liposomes with different cholesterol concentrations in the present invention;

[0051] Fig.11 It is a comparison diagram of the gene fragments of the bacteriophage Sb-1 of the present invention and the published bacteriophage K;

[0052] Fig.12 It is a 3D model diagram of the presence of biofilm EPS depolymerase in the foot of bacteriophage Sb-1 in the present invention;

[0053] Fig.13 It is a schematic diagram of the degradation of biofilm matrix at different time points observed by CLSM in the present invention;

[0054] Fig.14 is a schematic diagram of evaluating the successful coupling of drug-loaded liposomes to the surface of bacteriophages through azide-alkyne cycloaddition reaction by fluorescence intensity in the present invention;

[0055] Fig.15 The present invention uses an isothermal microcalorimeter to record the heat flow diagram during the mixed culture process of the targeted antibacterial biological agent coated with rifampicin and the bacterial solution;

[0056] Fig.16 The present invention uses an isothermal microcalorimeter to record the heat flow diagram during the mixed culture process of the targeted antibacterial biological agent coated with tigecycline and the bacterial solution;

[0057] Fig.17 It is a comparison diagram of heat flows generated during the mixed culture of the targeted antibacterial biological agent coated with rifampicin in the present invention, the mixture of bacteriophage and rifampicin liposome and the bacterial solution;

[0058] Fig.18 It is a comparison diagram of heat flows generated during the mixed culture of the targeted antibacterial biological agent encapsulating tigecycline in the present invention, a mixture of bacteriophage and tigecycline liposome and a bacterial solution;

[0059] Fig.19 It is a schematic diagram of the colony counting method for evaluating the killing time of planktonic bacteria by the liposome-bacteriophage complex in the present invention;

[0060] Fig. 20 It is a schematic diagram of the colony counting method for evaluating the time of killing planktonic bacteria by simple liposomes in the present invention;

[0061] Fig.21 It is a semi-quantitative evaluation of fluorescence intensity in the present invention for the content of tigecycline-cy3 entering bacteria after the interaction of phage, tigecycline-cy3 liposome, a mixture of tigecycline-cy3 liposome and phage, and tigecycline-cy3 liposome-phage complex with Staphylococcus aureus;

[0062] Fig. 22 The present invention uses fluorescence imaging technology to detect that the tigecycline liposome-phage complex can specifically kill Staphylococcus aureus;

[0063] Fig.23 The plate count in the present invention is used to evaluate the bactericidal amount of MRSA bacteria after the action of the tigecycline liposome-phage complex;

[0064] Fig.24 It is a schematic diagram of colony counting in the present invention to evaluate the number of bacteria in the Staphylococcus aureus biofilm killed by the rifampicin liposome-bacteriophage complex;

[0065] Fig.25 It is a schematic diagram of colony counting in the present invention to evaluate the number of bacteria in the Staphylococcus aureus biofilm killed by the tigecycline liposome-bacteriophage complex;

[0066] Fig.26 It is a schematic diagram of the colony count comparison of the residual bacteria count of the rifampicin liposome-bacteriophage complex and the bacteriophage after 24 hours of degradation of the biological enzyme matrix and then adding rifampicin liposomes to kill the biofilm in the present invention;

[0067] Fig. 27 It is a schematic diagram of the colony count comparison of the number of residual bacteria in the present invention after the tigecycline liposome-bacteriophage complex and the phage degraded the biological enzyme matrix for 24 hours and then added the tigecycline liposome to kill the biofilm;

[0068] Fig.28 It is a schematic diagram of evaluating the implant status of the distal tibia implant in an animal body by using X-ray in the present invention;

[0069] Fig.29 This is a schematic diagram of evaluating the decline time of inflammatory indicators in the process of treating plant infection by liposome-bacteriophage complexes in animals according to the present invention;

[0070] Fig.30It is a schematic diagram of evaluating the number of biofilms on the surface of plants eradicated by liposome-bacteriophage complexes in animals in the present invention;

[0071] Fig.31 Schematic diagram of evaluating the amount of bacteria eradicated from the soft tissue surrounding an implant by the liposome-bacteriophage complex in an animal in vivo according to the present invention. DETAILED DESCRIPTION

[0072] The present invention provides a targeted antibacterial biological agent and a preparation method thereof. In order to make the purpose, technical scheme and advantages of the present invention clearer and more specific, the present invention is further described in detail in conjunction with the accompanying drawings and the following reference examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Under the premise of no conflict, the embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0073] 1.1 Experimental equipment:

[0074] Microfluidizer high pressure homogenizer (NanoGenizer, Genizer, USA).

[0075] Transmission electron microscopy (Hitachi H 7500, Tokyo, Japan) was used to observe the morphology of phages and liposomes;

[0076] Malvern Panalytical Nano-Particle Size Potential Analyzer (Zetasizer, Nano-ZS90, Malvern Instruments) was used to measure the particle size of liposomes loaded with rifampicin and tigecycline;

[0077] ELISA reader ( 200PRO, Tecan, USA), used to determine the liposome encapsulation efficiency and the cumulative release at different time points;

[0078] Confocal laser scanning microscope (TCS SP8, Leica, Germany) was used to observe the degradation of biofilm matrix by bacteriophages;

[0079] Heat flow instrument, also known as isothermal microcalorimetry (Isothermal microcalorimetry, thermal activity monitor, model 3102TAMIII; TAInstruments, USA), is a technology that monitors heat production during bacterial growth in real time and is used to evaluate the efficiency of targeted antimicrobial agents in killing planktonic bacteria;

[0080] A small animal in vivo imager (ABL X5, Tanon, Shanghai, China) was used to observe the targeted killing of host S. aureus by targeted antimicrobial agents.

[0081] 1.2 Experimental reagents:

[0082] POPC: 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine;

[0083] Ch:Cholesterol, cholesterol;

[0084] DSPE-PEG(2000)-DBCO: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[dibenzocyclooctyl(polyethylene-glycol)-2000](ammonium salt, DBCO; PEG), distearoylphosphatidylethanolamine-polyethylene glycol-diphenylcyclooctyne);

[0085] NHS-N3: Table N-hydroxysuccinimide-azide compound;

[0086] Rifampicin, 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin;

[0087] Bacteriophage Sb-1;

[0088] Tigecycline;

[0089] Tigecycline-cy3;

[0090] The above reagents and phages can be purchased through commercial channels. 1.3 Specific Examples

[0092] Example 1

[0093] This embodiment provides a method for preparing a targeted antibacterial biological agent (refer to Figure 1-2 ), including the following steps:

[0094] Step 1: Mix 0.0001 μg / mL NHS-N3 with 1 mL of a 10 8 PFU / mL of purified phage (target phage Sb-1) were mixed and dialyzed to obtain azidized phage. The obtained azidized phage was titered to prove that it had not lost its biological activity ( Figure 3 );

[0095] Step 2: dissolving 1-palmitoyl-2-oleoyl phosphatidylcholine, cholesterol, and distearoyl phosphatidylethanolamine-polyethylene glycol-diphenylcyclooctyne in chloroform at a molar ratio of 10:3.5:0.2, and after the chloroform evaporates, adding the drug body (antibiotic), and preparing liposomes with an average particle size of 30 nm and coated with antibiotics by an ultra-high pressure microfluidizer as a drug for targeted use;

[0096] The antibiotic selected is rifampicin; the liposomes are mixed with methanol at a ratio of 1:10 by shaking to dissolve and release the antibiotics in the liposomes, and the content of the antibiotics is quantified by an ELISA instrument; wherein rifampicin is 3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin;

[0097] Step 3: Mix 10 μL of liposomes obtained in step 2 with 1 mL of liposomes obtained in step 1 with a unit titer of 10 8 PFU / mL of azidated phage (biologically active part) are mixed, and after removing unbound liposomes through a centrifugal column, phages carrying antibiotics are obtained, that is, targeted antibacterial biological agents. Among them, the phages are coupled with liposomes encapsulated with antibiotics; the phages are targeted phages used for extensive lysis of Staphylococcus aureus and treated with azidation, that is, targeted antibacterial biological agents.

[0098] The prepared liposomes and biological agents were analyzed by electron microscopy, and the Figure 4 and Figure 5 .

[0099] Step 4: Dilute the concentration of antibiotics in the targeted antimicrobial agent to 0.0002-9 μg / ml, where the titer of the bacteriophage is 1×10 4 PFU / mL or above.

[0100] Example 2

[0101] This embodiment provides a method for preparing a targeted antimicrobial biological agent, which is substantially the same as the above-mentioned embodiment 1, except that POPC, Ch, and DSPE-PEG(2000)-DBCO in this embodiment are dissolved in a molar ratio of 10:1.1:0.2.

[0102] The particle sizes of the liposomes prepared in Example 1 and Example 2 were measured. Figure 6 shown.

[0103] Example 3

[0104] This embodiment provides a method for preparing a targeted antibacterial biological agent, which is substantially the same as the above-mentioned embodiment 1, except that the antibiotic in this embodiment is preferably tigecycline.

[0105] Example 4

[0106] This example provides a method for preparing a targeted antibacterial biological agent, which is substantially the same as the above-mentioned Example 3, except that POPC, Ch, and DSPE-PEG(2000)-DBCO in this example are dissolved in a molar ratio of 10:1.1:0.2. The particle sizes of the liposomes prepared in Examples 3 and 4 were measured, and the results were as follows: Figure 7 shown.

[0107] The encapsulation efficiency and cumulative release rate of the liposomes prepared in Examples 1-4 were tested respectively, and the results were as follows: Fig. 9 and Fig.10 As shown, by analyzing the proportion of cholesterol lipids in targeted antibacterial biological agents, for example, the encapsulation rate after encapsulating antibiotics with cholesterol liposomes at a concentration of about 25% and the encapsulation rate after encapsulating antibiotics with cholesterol liposomes at a concentration of about 10% are compared and analyzed, it can be seen that as the proportion of cholesterol in the liposome increases, its encapsulation rate is higher. When the concentration of cholesterol is 25%, its encapsulation rate can be greater than or equal to 45.59%, and its leakage of antibiotics is the lowest, with a cumulative release rate of less than or equal to 6.5% after 5 days. When the concentration of cholesterol liposomes is 37.5%, it is difficult to synthesize liposomes that can encapsulate antibiotics.

[0108] Example 5

[0109] This embodiment provides a method for preparing a targeted antimicrobial biological agent. This embodiment is a further improvement of any of the above embodiments, and the improvement is that before step 1, the following steps are further included:

[0110] Step 01: Select the commercial bacteriophage Sb-1 that can widely lyse the target bacteria. In this embodiment, the target bacteria is Staphylococcus aureus as an example for illustration;

[0111] Step 02: Analyze and obtain the phage Sb-1 and draw the phage gene map. After dissolving the bacterial nucleic acid with DNaseI / RNaseA, protein kinase K is used to release the phage DNA, and its concentration and purity are determined by a micro-spectrophotometer (Nanodrop). Finally, sequence it on a high-throughput sequencing system (Illumina), and use the RAST server for gene annotation and analysis of the phage's depolymerase, and compare it with the published depolymerase sequence of phage K ( Fig.11 and Fig.12 );

[0112] Step 03: Use Blast to verify whether the phage contains biofilm matrix (EPS) degradation genes, and add biofilm matrix dye wheat germ agglutinin (WGA) and live cell nuclear dye SYTO 9 (green cyanine) solution to the 24h mature biofilm, and CLSM quantitatively observe the live bacteria marked with green fluorescence and the biofilm matrix marked with blue fluorescence ( Fig.13 ).

[0113] Example 6

[0114] This embodiment provides a targeted antimicrobial biological agent, including: a targeted antimicrobial biological agent prepared by the method for preparing the targeted antimicrobial biological agent described in any one of the above embodiments 1 to 4.

[0115] Example 7

[0116] This embodiment provides a method for detecting the sterilization of the targeted antimicrobial biological agent prepared in the fifth embodiment, comprising the following steps:

[0117] Step S10: In the dark, the targeted antimicrobial biological agent prepared in the above embodiment is placed in a fluorescent microplate reader to detect the fluorescence intensity of the targeted bacteriophage at the same concentration and the liposomes encapsulated with antibiotics and the targeted antimicrobial biological agent after centrifugation. This step is used to verify whether the liposomes are successfully coupled to the phage surface ( Fig.14 );

[0118] Step S20: Mix the targeted antimicrobial agent with 100 μL of bioluminescent Staphylococcus aureus and GFP Escherichia coli (~10 6 CFU / mL) were incubated at 37°C for 4 h. After the interaction, 100 μL of bacteria were resuspended in the plate, and animal imaging (ABL X5, Tanon, Shanghai) was used to observe the selective killing of bioluminescent Staphylococcus aureus by targeted antimicrobial agents ( Fig. 22 and Fig.23 );

[0119] Step S30: performing an in vitro antibacterial test on the bioactive agent;

[0120] Step S40: performing an in vivo implantation antibacterial test on the bioactive agent.

[0121] Comparative Example 1

[0122] This embodiment provides a method for preparing a targeted antimicrobial biological agent, which is substantially the same as the above-mentioned embodiment 1, except that POPC, Ch, and DSPE-PEG(2000)-DBCO in this embodiment are dissolved in a molar ratio of 10:6:0.2.

[0123] Comparative Example 2

[0124] This embodiment provides a method for preparing a targeted antimicrobial biological agent, which is substantially the same as the above-mentioned embodiment 3, except that POPC, Ch, and DSPE-PEG(2000)-DBCO in this embodiment are dissolved in a molar ratio of 10:6:0.2.

[0125] 1.4 Experimental data

[0126] Table 1: Comparative data of relevant parameters of the preparation method of the targeted antibacterial biological agent in each embodiment

[0127] Example Parameter 1 Parameter 2 Parameter 3 Parameter 4 Parameter 5 Parameter 6 Parameter 7 Parameter 8 Parameter 9 Example 1 Rifampin 30 10:3.5:0.2 <![CDATA[10 8 ]]> 9.11 2.44 0.000244 37.53 45.59％ Example 2 Rifampin 30 10:1.1:0.2 <![CDATA[10 8 ]]> 7.46 0.001 none 62.83 37.34％ Example 3 Tigecycline 30 10:3.5:0.2 <![CDATA[10 8 ]]> 9.78 2.6 0.0026 35.63 48.91％ Example 4 Tigecycline 30 10:1.1:0.2 <![CDATA[10 8 ]]> 8.18 0.001 none 67.17 40.92％ Comparative Example 1 Rifampin 30 10:6:0.2 <![CDATA[10 8 ]]> none none none none none Comparative Example 2 Tigecycline 30 10:6:0.2 <![CDATA[10 8 ]]> none none none none none

[0128] Note: Parameter 1-antibiotic; Parameter 2-parameter of target synthetic liposome particle size (mm); Parameter 3-molar ratio of POPC, Ch, and DSPE-PEG (2000) -DBCO; Parameter 4-titer of the diluted phage used to prepare the targeted antimicrobial agent (PFU / mL); Parameter 5-concentration of the antibiotic detected before centrifugation of the targeted antimicrobial agent (μg / mL); Parameter 6-concentration of the antibiotic detected after centrifugation of the targeted antimicrobial agent (μg / mL); Parameter 7-concentration of the antibiotic after multiple dilutions of the targeted antimicrobial agent (μg / mL); Parameter 8-particle size of the liposome measured after 14 days (mm); Parameter 9-encapsulation efficiency.

[0129] From the above Examples 1 to 4, Comparative Examples 1 to 2, Table 1 and related experimental data, it can be seen that the molar ratio of POPC, Ch, and DSPE-PEG (2000) -DBCO is preferably between 10: 1: 0.2 and 10: 5: 0.2, and is in accordance with the proportion of cholesterol in the lipid bilayer membrane of natural cells, for example, 10% to 50%, so as to avoid the proportion of cholesterol in the liposome being too low to well encapsulate the antibiotic, or the encapsulation rate being too low. If the proportion is too large, it is easy to cause the liposome to no longer be squeezed out by the microfluidizer, and it is difficult to prepare liposomes that can encapsulate antibiotics. Among them, the molar ratio of POPC, Ch, and DSPE-PEG (2000) -DBCO is more preferably 10: 1.1: 0.2 to 10: 3.5: 0.2.

[0130] Moreover, the average particle size of the liposomes with a cholesterol concentration of about 25% prepared by the above method can be maintained between 35.63nm nm and 37.53nm within 14 days, and the concentration of the targeted antibacterial biological agent antibiotic can be 0.001-2.44μg / mL, ensuring an encapsulation rate of more than 45%. It can be seen that within the effective shelf life, the antibiotics coated and retained can meet the use requirements, so as to facilitate its good effect in subsequent antibacterial tests, and the effective period can be more durable, for example, it is also effective within 28 days. The average particle size range of the liposomes with a cholesterol concentration of about 10% will increase, and the stability is relatively poor, but it can also be maintained in the range of about 62.83nm to 67.17nm, and the encapsulation rate can still reach more than 40.92%. Therefore, within 14 days, the antibiotics coated and retained can meet the use requirements, but as time goes on, more leakage may occur, resulting in a poorer therapeutic effect, for example, after 28 days, it may fail.

[0131] 1.5 In vitro antibacterial test method:

[0132] 1) In vitro antibacterial test

[0133] (1) Experiment on anti-planktonic bacteria

[0134] 0.9 mL of a specific diluted bacterial solution (10 5 CFU / mL), respectively, with the experimental group of 0.1mL of "liposome-phage" complexes encapsulating rifampicin or tigecycline diluted 10 times multiple times, that is, the liquid obtained after diluting 100μL of targeted antibacterial biological agents 10 times multiple times was placed in a culture dish for mixing, and then cultured in an isothermal microcalorimeter for 24 hours, and the heat flow diagram of the bacterial solution was measured and recorded by a heat flow meter. Among them, the concentration at which no heat flow curve is generated (heat flow value is 0) is the lowest concentration for inhibiting planktonic bacteria. The remaining experimental groups corresponded to the addition of phages, liposomes encapsulated with antibiotics, and liposomes + phages for mixing, placed in a culture dish for mixing, and then cultured in an isothermal microcalorimeter for 24 hours, and the heat flow diagram of the bacterial solution was measured and recorded by a heat flow meter for comparative analysis. The control group was PBS solution (phosphate buffer). (ND; nodetection; is not detected, LOD; limit ofdetection; is the lowest detection line).

[0135] (2) Anti-biofilm experiments

[0136] 1. Evaluate the sterilization rate of biofilm

[0137] The prepared experimental group of targeted antimicrobial biological agents were incubated with the biofilm of specific bacteria matured for 24 hours at 37°C, and the killing rate of the biofilm was calculated by the plate coating method to reflect the killing rate of the biofilm. The remaining experimental groups were bacteriophage, liposomes encapsulated with antibiotics, phages degraded the bioenzyme matrix for 24 hours, and then the antibiotic liposome group and the control group PBS (phosphate buffered saline). Among them, the following experimental groups in this embodiment are only used as an example to illustrate the targeted antimicrobial biological agent prepared by the bacteriophage Sb-1 of the target bacteria.

[0138] 2) In vivo treatment of implant infections

[0139] (1) Preparation of implant infection model

[0140] Fifteen healthy male SD rats (200 g) were anesthetized, and the skin around the knee joint was prepared and disinfected to expose the distal tibia. A Kirschner wire was used to make a hole between the tibial condyles and implants were implanted. After 7 days, 100 μL of 10 5 CFU of Staphylococcus aureus was injected into the rat's tibia for 48 hours to form a biofilm on the implant surface. The knee joint was treated with intra-articular injection for 14 consecutive days, twice a day.

[0141] (2) Experimental groups

[0142] The rats were divided into 5 groups, 3 rats in each group. The first control group was intervened with phosphate buffered saline (PBS) reagent, the second group was intervened with bacteriophage, the third group was intervened with targeted antimicrobial organism agents loaded with antibiotic rifampicin, the fourth group was intervened with tigecycline liposome-phage complex, and the fifth group was intervened with rifampicin liposome-phage complex.

[0143] (3) Inspection indicators

[0144] 2. Before euthanasia, collect serum at different time points and test C-reactive protein (CRP).

[0145] 3. The bacteria in the biofilm on the surface of the implant are dissociated by ultrasonic vibration and the soft tissue around the implant is ground, and then the number of biofilm colonies is counted on a plate (ND: no detection, LOD: limit of detection).

[0146] 4. Calculation method of the coupling rate between phage and liposome:

[0147] 4.1 Preliminarily, Tigecycline labeled with the fluorescent group Cy3 was synthesized into Tigecycline Cy3 liposomes by an ultra-high pressure microfluidizer, according to the method in Example 3, and the corresponding fluorescence intensity was measured and recorded;

[0148] 4.2 The Tigecycline Cy3 liposomes and the bacteriophages were diluted 1:100 and mixed, and then centrifuged to obtain the Tigecycline Cy3 liposome-bacteriophage complex, i.e., the targeted antibacterial biological agent, and the fluorescence intensity of the targeted antibacterial biological agent was measured and recorded.

[0149] 1.51 Experimental Results

[0150] Depend on Fig.14 By comparing the fluorescence intensities in the liposomes, that is, by calculating the ratio of the fluorescence intensity of the targeted antibacterial biological agent to the fluorescence intensity corresponding to the liposomes coated with tigecycline cy3, it can be calculated that the coupling rate between the liposomes and the bacteriophages in the above embodiment is 26.7%, thereby calculating the concentration of the antibiotics in the liposomes coated with antibiotics, and the concentration of the antibiotics is estimated to be 2.3μg / ml.

[0151] Fig.15 The heat flow diagram of the isothermal microcalorimeter is used to evaluate the efficiency of each experimental group of the targeted antibacterial biological agent coated with rifampicin in killing planktonic bacteria. By observing the growth rate corresponding to the heat generated by the bacteria during the proliferation process in the heat flow diagram, the growth rate of the bacteria can be better reflected. It can be seen that when the targeted antibacterial biological agent coated with rifampicin is diluted ten times multiple times, even with a lower antibiotic, such as 0.000244μg / mL, it can still inhibit the growth rate of heat caused by the proliferation of planktonic bacteria of Staphylococcus aureus, that is, it can better inhibit the proliferation process of planktonic bacteria of Staphylococcus aureus. Among them, A1 represents a solution with 0.9mL of a specific diluted bacterial solution (10 5 CFU / mL) of PBS buffer (phosphate buffered saline) was used as the control group to measure the heat flow data; B1 represents the liquid obtained by taking 0.1 mL of the targeted antimicrobial biological agent and then diluting the targeted antimicrobial biological agent coated with rifampicin 5 times by ten (the content of bacteriophage in the liquid is 10 3 PFU / mL) was used to treat the above experimental group, and the thermal flow diagram data were measured; C1 represents the liquid treatment experimental group obtained by diluting the liposomes coated with rifampicin six times ten-fold, and the thermal flow diagram data were measured; D1 represents the thermal flow diagram data obtained by using a single phage (10 8 PFU / mL) after the experimental group was treated with the thermogram data obtained by measurement; E1 represents the liquid obtained by diluting the targeted antibacterial agent coated with rifampicin 4 times by ten times (the content of bacteriophage in the liquid is 10 4PFU / mL) was used to treat the above experimental group, and the thermal flow diagram data were measured; F1 represents the liquid treatment experimental group obtained by diluting the liposomes coated with rifampicin 5 times, and the thermal flow diagram data were measured; it can be seen that: the rifampicin liposomes can inhibit the heat generated by the planktonic bacteria of Staphylococcus aureus after 5 ten-fold dilutions, while the rifampicin liposome-phage complex requires dilution and centrifugation of the liposomes due to the synthesis process, and after 4 ten-fold dilutions, it shows that the rifampicin antibiotics contained in the complex are lower than those in the rifampicin liposomes, and can also inhibit the heat generated by the planktonic bacteria of Staphylococcus aureus.

[0152] Fig.16 The heat flow diagram of the isothermal microcalorimeter is used to evaluate the efficiency of each experimental group of the targeted antibacterial biological agent coated with tigecycline in killing planktonic bacteria. By observing the growth rate corresponding to the heat generated by the bacteria during the proliferation process in the heat flow diagram, the growth rate of the bacteria can be better reflected. It can be seen that when the targeted antibacterial biological agent coated with tigecycline is diluted tenfold multiple times, even with a lower concentration of antibiotics, such as 0.0026μg / mL, it can still inhibit the growth rate of heat caused by the proliferation of planktonic bacteria of Staphylococcus aureus. That is to say, the proliferation process of planktonic bacteria of Staphylococcus aureus can be better inhibited. Among them, A2 represents the heat flow data obtained by measuring the untreated PBS buffer (phosphate buffered saline) as the control group; B2 represents the liquid treatment of the above experimental group (the content of bacteriophage is 10 4 PFU / mL), and the measured thermal flow diagram data; C2 represents the liquid treatment experiment group obtained by diluting the liposomes coated with tigecycline five times ten-fold, and the measured thermal flow diagram data; D2 represents the liquid treatment experiment group obtained by diluting the liposomes coated with tigecycline five times ten-fold, and the measured thermal flow diagram data; 8 / PFU / mL) treated with the experimental group; E2 represents the liquid obtained by diluting the targeted antibacterial agent coated with tigecycline three times tenfold to treat the above experimental group (the content of bacteriophage is 10 5 PFU / mL), and the thermal flow diagram data obtained by measuring; F2 is the liquid treatment experimental group obtained by diluting the liposomes coated with tigecycline four times by ten times, and the thermal flow diagram data obtained by measuring. It can be seen that the tigecycline liposomes can inhibit the heat generated by the floating bacteria of Staphylococcus aureus after 4 ten-fold dilutions, and the tigecycline liposome-phage complex contains lower tigecycline antibiotics after 3 ten-fold dilutions, and can also effectively inhibit the heat generated by the floating bacteria of Staphylococcus aureus. Therefore, the targeting of bacteriophages is used to deliver antibiotics to target bacteria, such as Staphylococcus aureus, thereby achieving the effect of active targeted synergistic antibacterial, and only a small dose of antibiotics is required during the sterilization process.

[0153] Fig.17The heat flow diagram of isothermal microcalorimeter is used to evaluate the efficiency of rifampicin liposome-phage complex in killing planktonic bacteria. Among them, A3 represents the heat flow data obtained by measuring the PBS buffer (phosphate buffered saline) as the control group without treatment; B3 represents the heat flow data obtained by using four ten-fold dilutions of phage (10 4 PFU / mL), and then mixed with liposomes coated with rifampicin six times ten-fold dilution, and then treated with the experimental group, and the thermal flow diagram data were measured; C3 represents the targeted antibacterial biological agent coated with rifampicin four times ten-fold dilution (the content of bacteriophage in the liquid is 10 4 PFU / mL) was used to obtain the liquid, and the experimental group was measured, and the thermal flow diagram data obtained by the measurement. It can be seen that: at the same phage low concentration, the direct mixing of liposomes containing six ten-fold dilutions of rifampicin has a higher antibiotic content, but still fails to inhibit the growth of bacteria.

[0154] Fig.18 The heat flow diagram of the isothermal microcalorimeter is used to evaluate the efficiency of the tigecycline liposome-phage complex in killing planktonic bacteria. A3 represents the heat flow data obtained by measuring the PBS buffer (phosphate buffered saline) as the control group without treatment; B3 represents the heat flow data obtained by using three ten-fold dilutions of phage (10 5 PFU / mL), and then mixed with liposomes coated with tigecycline five times by ten times, and the experimental group was treated with the measured thermal flow diagram data; C3 represents the liquid obtained by three times ten-fold dilution of the targeted antibacterial agent coated with tigecycline (the content of bacteriophage is 10 5 PFU / mL), the treatment experimental group was measured, and the thermal flow diagram data obtained by measurement. It can be seen that: at the same phage low concentration, the liposomes directly mixed with five ten-fold dilutions of rifampicin have a higher antibiotic content, but still fail to inhibit the growth of bacteria. Therefore, the targeted antibacterial biological agent has a better antibacterial ability.

[0155] Fig.19 A schematic diagram showing the killing time of planktonic bacteria by evaluating the combination of liposomes coated with different antibiotics and bacteriophages to obtain targeted antimicrobial agents for biological targets by colony counting method.

[0156] Fig. 20 A schematic diagram showing the killing time of planktonic bacteria by simple liposomes evaluated by colony counting method is shown.

[0157] Depend on Fig.15 Fig.19 It can be seen that the targeting of bacteriophages is used to deliver antibiotics to target bacteria, such as Staphylococcus aureus, thereby achieving the effect of active targeted synergistic antibacterial, and only a small dose of antibiotics is needed during the sterilization process.

[0158] From the above analysis, it can be seen that by mixing liposomes coated with antibiotics and bacteriophages, it only takes 4 hours to generate a liposome-phage complex, that is, a targeted antibacterial agent, while simple liposomes still need 24 hours to kill bacteria at a higher antibiotic content.

[0159] Fig.21 showed that the use of Staphylococcus aureus (10 8 The semi-quantitative evaluation of intracellular fluorescence intensity of phage, tigecycline-cy3 liposome, mixture of tigecycline-cy3 liposome and phage, and tigecycline-cy3 liposome-phage complex within 60 minutes was performed. It can be seen that through the trajectory tracking of tigecycline-cy3 liposome-phage complex, it can be known that after the targeted antibacterial biological agent acts on the host bacteria, under the targeted guidance of the phage, the tigecycline inside can be released and can act on the host bacteria.

[0160] Fig. 22 The antibacterial specificity of the tigecycline liposome-phage complex was detected by imaging of Staphylococcus aureus and GFP-Escherichia coli. The visible light image represents the mixture of Staphylococcus aureus and GFP-Escherichia coli, the color bioluminescence detection represents Staphylococcus aureus, and the green fluorescence excited by 488nm irradiation represents GFP-Escherichia coli.

[0161] Fig.23 The plate counts show that after 4 hours, the number of MRSA bacteria was below the detection limit.

[0162] Fig.24 Colony counts evaluating the killing of bacteria in biofilm by rifampicin are shown.

[0163] Fig.26 It was shown that the bactericidal effect of the rifampicin liposome-phage complex evaluated by colony count was better than that of the phage after 24h degradation of the bioenzyme matrix and then the addition of rifampicin liposomes, i.e., liposomes simply coated with rifampicin.

[0164] Fig.25 Colony counts evaluating the number of bacteria killed by tigecycline in biofilms are shown.

[0165] Fig. 27 The colony count evaluation showed that the bactericidal effect of the tigecycline liposome-phage complex was better than that of the tigecycline liposome added after the phage degraded the bioenzyme matrix for 24 hours.

[0166] Fig.28 This is a schematic diagram of using X-ray to evaluate the implant status of the distal tibia in animals.

[0167] Fig.29A schematic diagram showing the time of decrease of inflammatory indicators during the treatment of plant infection by liposome-phage complexes in animals using C-reactive protein levels.

[0168] Fig.30 The amount of biofilm on plant surfaces within each experimental group evaluated in vivo in animals is shown.

[0169] Fig.31 The number of bacteria in the soft tissue surrounding the eradicated implants in each experimental group evaluated in animals is shown.

[0170] Depend on Figures 22 to 31 It can be seen that the antibiotic-encapsulated liposome-phage complex, that is, the targeted antibacterial organism agent, can kill biofilm bacteria at a lower antibiotic content.

[0171] In summary: The present invention encapsulates antibiotics in liposomes and combines them with azide-modified targeted phages, and uses the targeting property of phages to deliver them to target bacteria, such as Staphylococcus aureus, thereby achieving an active targeted synergistic antibacterial effect. Regardless of whether the bacteria are currently in a planktonic state or a biofilm state, a good killing or inhibition effect can be achieved, and only a small dose of antibiotics is required during the treatment process.

[0172] Furthermore, the azidated bacteriophage used in the present invention carries a depolymerase that specifically degrades extracellular polymers, and can therefore degrade the biofilm matrix of the target bacteria well, and has a good release efficiency. While achieving the disintegration of the biofilm, the antibiotics can also be stably delivered to the interior of the target bacteria, and the generated bacteria, especially the targeted bacteria in the planktonic state, can be killed with a low dose of antibiotics, thereby improving the efficiency of killing bacteria while ensuring safety.

[0173] In addition, through the above Figures 14 to 31 , and combined with Table 1, we can see that:

[0174] The concentration of the targeted antimicrobial agent antibiotic prepared in the above embodiment, even after multiple dilutions, at a lower concentration, for example, between 0.0002 and 0.00026 μg / mL, also has a good effect of inhibiting the proliferation of Staphylococcus aureus planktonic bacteria.

[0175] In addition, it should be noted that the targeted antimicrobial biological agent in this embodiment can be used not only for in vivo treatment of humans or animals, but also for non-disease treatment, such as antibacterial or sterilization in food or the environment, etc., and no further specific description or limitation will be given here.

[0176] Thus, in view of the many possible embodiments that can be applied to the disclosed principles, it should be recognized that the above embodiments are merely examples and should not be considered limiting in scope. Therefore, we reserve all rights to the subject matter disclosed herein, including the right to claim any and all combinations of the subject matter disclosed herein, including but not limited to all that is within the scope and spirit of the claims.

Claims

1. A targeted antimicrobial agent, characterized in that: include: A targeted biologically active part and a drug combined with the biologically active part; wherein the drug comprises: a drug body, a coating body used to coat an antibiotic and combined with the biologically active part; the coating body is used to release the antibiotic after being dissolved.

2. The targeted antimicrobial agent according to claim 1, characterized in that The coating body is covalently coupled to the biologically active part.

3. The targeted antimicrobial agent according to claim 1, characterized in that The encapsulated body is a nano-sized liposome; wherein the particle size of the liposome is nano-sized; And / or, the average particle size of the coating is 24 nm to 70 nm.

4. The targeted antimicrobial agent according to claim 1, characterized in that The average particle size of the coating is 30.55nm to 38.74nm.

5. The targeted antimicrobial agent according to claim 2, characterized in that: The coating body at least includes: a DBCO functional group, which is used to form the covalent coupling with the biologically active part; And / or, the biologically active portion is a bacteriophage modified with an azidation group.

6. The targeted antimicrobial agent according to claim 1, characterized in that The liposome comprises: liposome phospholipid, cholesterol, and polyethylene glycol phospholipid with a group for forming a covalent coupling; The ratio of the liposome phospholipid, cholesterol and polyethylene glycol phospholipid is 10:(1-5):(0.1-1).

7. The targeted antimicrobial agent according to claim 6, characterized in that The ratio of the liposome phospholipid, cholesterol, and polyethylene glycol phospholipid is 10:(1-3.5):(0.1-1).

8. The targeted antimicrobial agent according to claim 6, characterized in that The ratio of the liposome phospholipid, cholesterol, and polyethylene glycol phospholipid is 10:(1-3.5):0.

2.

9. The targeted antimicrobial agent of claim 1, wherein: The liposome phospholipid is 1-palmitoyl-2-oleoyl-phosphatidylcholine; The polyethylene glycol phospholipid is distearoyl phosphatidylethanolamine-polyethylene glycol-diphenylcyclohexane.

10. The targeted antimicrobial agent of claim 1, wherein: The medicament body is an antibiotic; The antibiotics include: water-soluble antibiotics and / or fat-soluble antibiotics; Alternatively, the antibiotics include: bactericidal antibiotics and / or bacteriostatic antibiotics.

11. The targeted antimicrobial agent of claim 1, wherein: The antibiotics include: rifampicin; and / or, tigecycline.

12. The targeted antimicrobial agent according to claim 1 or 11, characterized in that: The antibiotics include: any one or a combination of vancomycin, penicillin G, daptomycin, gentamicin, linezolid, ampicillin sodium sulbactam, levofloxacin, ciprofloxacin, co-trimoxazole, doxycycline, gentamicin, fosfomycin, amoxicillin, amoxicillin-clavulanic acid, piperacillin-tazobactam, cefoperazone sulbactam, ceftazidime avibactam, cefazolin, and cefuroxime.

13. The targeted antimicrobial agent of claim 1, wherein: The biologically active portion is a bacteriophage targeted for lysing a target bacterium; wherein the target bacterium is any one of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Propionibacterium acnes or Acinetobacter baumannii.

14. The targeted antimicrobial agent of claim 13, wherein: The biologically active part is a targeted bacteriophage having a depolymerase gene, wherein the depolymerase gene is selected from ORF96.

15. The targeted antimicrobial agent of claim 13, wherein: The biologically active part is targeted to bacteriophage Sb-1; the bacteria targeted for lysis by the biologically active part is Staphylococcus aureus.

16. The targeted antimicrobial agent of claim 1, wherein: The concentration of the antibiotic in the biologically active portion is 0.0002 to 9 μg / mL; or the titer of the bacteriophage in the biologically active portion is 1×10 4 PFU / mL or above.

17. The targeted antimicrobial agent of claim 1, wherein: The concentration of the antibiotic in the biologically active portion is 0.001-2.6 μg / mL.

18. The method for preparing the targeted antibacterial biological agent according to claim 1, characterized in that: The encapsulation efficiency of the nano-liposome is greater than or equal to 45.59%; The maximum cumulative release rate of the nano-liposomes in a PBS solvent at a neutral pH after 4 to 7 days is 6.31% to 6.70%; Alternatively, when the antibiotic is a water-soluble antibiotic, the maximum cumulative release rate of the nano-sized liposomes after 4 to 7 days in a PBS solvent at a neutral pH is 6.31%; Alternatively, when the antibiotic is a lipid-soluble antibiotic, the maximum cumulative release rate of the nano-sized liposomes after 4 to 7 days in a PBS solvent at neutral pH is 6.70%.

19. A method for preparing a targeted antibacterial biological agent, characterized in that: The preparation method is used for preparing the targeted antimicrobial agent according to any one of claims 1 to 18.

20. The preparation method according to claim 19, characterized in that: The preparation method comprises the following steps: Extracting a biologically active portion capable of specifically lysing target bacteria and degrading its biofilm matrix, and preparing a biologically active portion solution; by adding NHS-N3 linker to the biologically active part solution to obtain the azidized biologically active part; Dissolving phospholipids, cholesterol, and polyethylene glycol phospholipids in chloroform according to a preset ratio, and after the chloroform evaporates, adding a preset drug body to prepare a lipid drug suspension, and then injecting the lipid drug suspension into a microfluidizer to synthesize nano-scale liposomes encapsulating the drug body; The coated body composed of nano-liposomes is mixed and cultured with the azidation biologically active part to obtain the targeted antibacterial biological agent.

21. The preparation method according to claim 19, characterized in that: The concentration of the NHS-N3 linker is 0.00009-0.0005 μg / mL.

22. A sterilization method, characterized in that: Lysis is performed by a targeted antimicrobial agent as claimed in any one of claims 1 to 21.

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