Method for preparing liposome loaded with bacteriophage and modifying TAT

Abstract

The invention provides a preparation method of a bacteriophage liposome, a bacteriophage-loaded liposome obtained according to the method, a method for further carrying out TAT modification on the obtained bacteriophage-loaded liposome, and the obtained TAT-modified bacteriophage-loaded liposome. The modified liposome obtained by the method disclosed by the invention has an excellent intracellular bactericidal effect, and the clearance rate is increased by about 2-4 times compared with that of a free bacteriophage.

Description

Technical Field

[0001] This invention discloses a method for modifying phage liposomes and TAT, belonging to the field of pharmaceutical formulation. Background Technology

[0002] Intracellular parasites (hereinafter referred to as intracellular bacteria) are one of the main culprits causing chronic, persistent, and latent infections, seriously threatening public health. Intracellular bacteria can not only evade the host's immune system, but host cells can also become a haven for them to avoid antibiotic damage, making them difficult to eradicate. Furthermore, intracellular bacteria can spread through host cells, causing new foci of infection and even systemic infection. For example, Staphylococcus aureus (… Staphylococcus aureus, S. aureus (This is a class of important opportunistic pathogens, primarily residing on the skin or mucous membranes. Once the body experiences trauma, they can invade various cells in the skin and connective tissue. Recent research has found that...) S. aureus As a facultative intracellular bacterium, it can enter various cells such as keratinocytes and endothelial cells through internalization, and can even invade phagocytes that respond to clear the bacteria, causing persistent intracellular infection. Hematogenous phagocytes can act as "Trojan horses" for intracellular S. aureus, spreading the bacteria from the initial site of infection to different tissues, causing meningitis, osteomyelitis, lung infections, endocarditis, etc., which greatly endanger human health.

[0003] Antibiotics struggle to effectively penetrate cells and kill intracellular bacteria, and inappropriate use of high doses can exacerbate bacterial resistance. Currently, the main clinical strategy for treating intracellular bacterial infections is long-term, high-dose antibiotic use. However, more than two-thirds of known antibiotics are ineffective against intracellular bacteria. Furthermore, factors such as cell metabolism, efflux, and drug distribution result in weak intracellular drug accumulation and low drug activity, leading to a situation where even large doses are insufficient to eliminate intracellular pathogens.

[0004] Bacteriophages are a class of viruses capable of lysing bacteria, naturally possessing promising applications in combating bacterial infections. In today's world, with increasingly severe antibiotic resistance, it has become a global consensus that humanity cannot rely solely on antibiotics in the fight against infection. Compared to traditional antibiotics, bacteriophages have the following advantages: ① High specificity, they do not kill bacteria other than the target species and have no impact on normal flora; ② After clearing the target bacteria in the body, they die along with the host bacteria, preventing accumulation and poisoning; ③ They only infect bacteria, do not disrupt human cell metabolism, and have no toxic side effects; ④ They can co-evolve with the host, avoiding bacterial resistance through high-frequency mutations. However, simple bacteriophage preparations are easily recognized as foreign antigens by the body's immune system and rapidly eliminated, and bacteriophages have difficulty directly entering eukaryotic cells to eliminate intracellular bacteria. Therefore, constructing safe and efficient vectors to effectively deliver bacteriophages into cells is a crucial factor in leveraging bacteriophages to kill intracellular MRSA.

[0005] The purpose of this invention is to prepare stable and uniform phage-loaded liposomes and TAT-modified phage-loaded liposomes using microfluidic technology, thereby providing new methods and products for enhancing the intracellular bactericidal activity of phages. Summary of the Invention

[0006] To achieve the above objectives, the present invention first provides a method for preparing liposomes from bacteriophages, the method comprising the following steps: S1. Prepare an organic phase containing DSPC and cholesterol, wherein the mass ratio of DSPC to cholesterol is 4-6:0.5-1.5; S2. Prepare an aqueous phase containing bacteriophage, wherein the bacteriophage titer is 10. 9 -10 11 PFU / mL; S3. Input the organic phase obtained in S1 and the aqueous phase obtained in S2 into the microfluidic chip at a volume ratio of 0.5-2:3, a total flow rate of 0.5-1.5 ml / h, and a flow rate ratio of 2:1-1:2 between the organic phase and the aqueous phase, respectively. S4. Harvest the liquid output from the microfluidic chip and perform dialysis using a 90-110 kDa dialysis bag; S5. Purify the dialysate obtained in S4 to obtain liposomes loaded with phage.

[0007] In a preferred embodiment of the present invention, in S1, the mass ratio of DSPC (1,2-distearate-sn-glycerol-3-phosphocholine) to cholesterol is 5:1. During the implementation of the present invention, a dye may be selectively added to S1 to facilitate localization in cells or tissues during liposome characterization and functional evaluation. Preferably, the cyanine dye Cy5 may be selected; more preferably, the Cy5 may be set to have the same mass composition as cholesterol.

[0008] In one specific embodiment of the present invention, the selected bacteriophage is vB_SauS_PHM, which has a broad lytic activity against MRSA strains obtained through screening. Bacteriophage fluids with lytic activity against other pathogenic bacteria can also be used in the method of the present invention. The usage parameters of bacteriophage vB_SauS_PHM in the present invention are also applicable to other bacteriophages.

[0009] In a preferred embodiment of the present invention, in S2, the titer of the bacteriophage is 10. 10 PFU / mL.

[0010] In another preferred embodiment of the present invention, in S3, the organic phase obtained in S1 and the aqueous phase obtained in S2 are input into the microfluidic chip at a volume ratio of 1:3, a total flow rate of 1 ml / h, and a flow rate ratio of 1-1 between the organic phase and the aqueous phase.

[0011] In a preferred embodiment of the present invention, in step S4, dialysis is performed using a 100 kDa dialysis bag.

[0012] In another preferred embodiment of the present invention, the purification described in S5 is a size exclusion chromatography column purification method.

[0013] Secondly, the present invention provides a phage-loaded liposome prepared according to the above method. In the present invention, the liposome is named "Lip@PHM". The hydrated particle size of the phage-loaded liposome is 725-750 nm, and the Zeta potential is -0.710 to -0.700 mV. In an actual measurement of the present invention, the hydrated particle size is 733.9 nm and the Zeta potential is -0.707 mV.

[0014] Furthermore, the present invention provides a method for TAT modification of the above-mentioned liposomes, the method comprising mixing the phage-loaded liposomes with 1 μM of TAT-PEG. 2000 -DSPE was incubated at 37 °C for 0.5-1.5 h, followed by separation and purification using an ultrafiltration tube with a molecular weight cutoff of 90-110 kDa. In the implementation of this invention, the phage-loaded liposomes were mixed with TAT-PEG in a sufficient or excessive ratio. 2000-DSPE ratio to ensure the technical effect of the modification.

[0015] In one specific embodiment of the present invention, the incubation time is 1 h, and then separation and purification are performed using an ultrafiltration tube with a molecular weight cutoff of 100 kDa.

[0016] Fourth, the present invention provides liposomes loaded with phage modified by the above method. In the present invention, the liposomes are named "TAT-Lip@PHM". The average hydrated particle size of the liposomes loaded with phage modified by TAT is 630-640 nm and the Zeta potential is 3.85-3.95 mV. In an actual measurement of the present invention, the hydrated particle size was 636.1 nm and the Zeta potential was 3.91 mV.

[0017] Finally, the present invention provides the application of the above-mentioned phage-loaded liposomes or TAT-modified phage-loaded liposomes in the preparation of intracellular bactericides.

[0018] Lip@PHM and TAT-Lip@PHM were constructed using the method of this invention. Compared with high concentrations of free phage, Lip@PHM and TAT-Lip@PHM containing low concentrations of phage showed better clearance effects against intracellular MRSA infection, indicating that liposome encapsulation and TAT modification enhanced the phage's ability to resist intracellular bacterial infection. Furthermore, with prolonged exposure time, the clearance rate of intracellular MRSA in the TAT-Lip@PHM group gradually increased, reaching 94.05% at 24 h, which demonstrated superior bactericidal ability compared to the free phage vB_SauS_PHM group (21.24%) and the Lip@PHM group (44.90%). Attached Figure Description

[0019] Figure 1 Phage vB_SauS_PHM (A) lysis region and (B) plaque; Figure 2 Transmission electron microscopy morphology of bacteriophage vB_SauS_PHM; Figure 3 Genome map of bacteriophage vB_SauS_PHM; Figure 4 A phylogenetic tree was constructed based on the whole genome sequence of bacteriophage vB_SauS_PHM. Figure 5 Optimal multiplicity of infection for bacteriophage vB_SauS_PHM; Figure 6 One-step growth curve of bacteriophage vB_SauS_PHM; Figure 7Thermal stability of bacteriophage vB_SauS_PHM; Figure 8 pH stability of bacteriophage vB_SauS_PHM; Figure 9 In vitro antibacterial effects of different MOI phages vB_SauS_PHM; Figure 10 Schematic diagram of Lip@PHM synthesis using microfluidic method; Figure 11 . Hydration particle size of Lip@PHM; Figure 12 Zeta potential of Lip@PHM (pH=7); Figure 13 Cryo-transmission electron microscopy image of Lip@PHM; Figure 14 . Hydration particle size of TAT-Lip@PHM; Figure 15 Zeta potential of TAT-Lip@PHM (pH=7); Figure 16 Fluorescence emission spectrum of TAT-Lip@PHM; Figure 17 Cytotoxicity of different final concentrations of TAT-Lip@PHM; Figure 18 Laser confocal microscopy images of TAT-Lip@PHM taken in RAW 264.7 at different time points (scale bar is 50 μm). Figure 19 Photographs of intracellular MRSA plating plates after different treatment times with TAT-Lip@PHM and Lip@PHM; Figure 20 Plate colony counts of intracellular MRSA at different treatment times with TAT-Lip@PHM and Lip@PHM. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0021] 1. Main reagents used in the embodiments of the present invention Nutrient agar medium (plates) (Guangdong Huankai), SM buffer (Shanghai Yuanye), polyethylene glycol PEG 8000 (Beijing Solarbio), DNase I (Tiangen Biochemical), RNase (Tiangen Biochemical), Mung Bean Nuclease (TaKaRa), Sodium chloride (Shanghai Maclean), tryptone (Oxoid), yeast extract (Oxoid), agar powder (BioDee), sterile syringe (Huachu), 0.22 μm filter (Jinmingcheng), sterile shaking tubes (Bioyijie), centrifuge tubes (CORNING). 1,2-Distearate-sn-glycerol-3-phosphocholine (DSPC) (Shanghai Maclean), Cholesterol (Avanti), Triton X-100 (Beijing Solarbio), DSPE-PEG2000 (Ruixibio), Cyanide Cy5 (Beijing Bailingwei), Ethylenediaminetetraacetic acid (EDTA) (Shanghai Maclean), Ultrafiltration tubes (Merck Millipore), SuperEV ultrapure size exclusion column (Liaoning Runji).

[0022] 2. The main instruments used in the embodiments of the present invention are shown in Table 1.

[0023] Table 1. Main instruments and models used in the embodiments of the present invention

[0024] Example 1. Isolation and purification of bacteriophage vB_SauS_PHM.

[0025] 1. Preliminary treatment of wastewater Take the untreated wastewater sample into a 50 mL centrifuge tube, centrifuge at 10,000 rpm for 10 min, and use a 10 mL sterile syringe to collect the supernatant and filter it through 0.45 μm and 0.22 μm filters in sequence to obtain coarsely treated wastewater, which is then stored at 4 ℃.

[0026] 2. Resuscitation of the strain The MRSA strain stored at -80 ℃ was brought to room temperature and thawed into a solution. Then, it was streaked onto a nutrient agar solid medium using a sterile inoculation loop. The plate was placed in a 37 ℃ incubator and incubated overnight. The next day, a single colony was picked and added to 6 ml of LB medium and incubated in a 37 ℃ shaker for 8-10 h to obtain the revived bacterial solution.

[0027] 3. Enrichment and isolation of bacteriophages Add 10 mL of LB broth and 1 mL of logarithmic-phase bacterial culture to 1 mL of coarsely treated wastewater. Incubate at 37 °C in a shaker for at least 8 h, centrifuge at 8000 rpm for 10 min, collect the supernatant and filter through a 0.22 μm filter. Repeat three times to obtain the phage stock solution. For the double-layer agar drop test, mix 100 μL of logarithmic-phase host bacteria with 5 mL of melted and incubated semi-solid culture medium at 50 °C, pour the mixture onto the bottom agar plate, and after solidification, add 3 drops of 5 μL of phage stock solution. After the droplets have completely dried, incubate the plate overnight at 37 °C. Observe the plate the next day for the appearance of clear or turbid lysis zones.

[0028] 4. Concentration and purification of bacteriophages Phages were concentrated and purified using PEG precipitation. 3-5 mL of logarithmic-phase host bacteria and 1 mL of stock phage solution were added to 800 mL of liquid culture medium and incubated at 37 °C for at least 8 h in a shaker to obtain amplified phage solution. This solution was aliquoted into 50 mL centrifuge tubes and centrifuged at 9807 rpm for 20 min at 4 °C. The solution was filtered through a 0.22 μm filter to remove bacteria and debris, and the supernatant was collected. DNase I and RNase A were added to the supernatant to a final concentration of 1 μg / mL and incubated at room temperature for 30 min. NaCl was then added to a final concentration of 1 mol / L, mixed thoroughly, and incubated on ice for 1-2 h. The mixture was then centrifuged at 4 °C and 9807 rpm for 15-20 min, and the supernatant was collected. PEG-8000 was added at a rate of 10 g per 100 mL of medium, gently stirred to dissolve the phage, and incubated on ice overnight to allow the phage to precipitate under the action of PEG-8000. After overnight incubation, centrifuge the culture at 4 °C and 9807 rpm for 15-20 min, discard the supernatant, and invert the centrifuge tube for 5 min to allow the residual liquid to drain. Gently resuspend the precipitate with 2 mL of SM buffer, add an equal volume of chloroform for extraction: shake for 30 s, then centrifuge at 4 °C and 7297 rpm for 15 min, recover the upper aqueous phase containing phage, add an equal volume of chloroform for extraction, repeat 3-5 times until the upper phase is clear, and finally store the recovered concentrated phage purification solution at 4 °C.

[0029] 5. Results This invention uses a laboratory-preserved clinical isolate of MRSA as the host bacteriology to isolate a bacteriophage from untreated wastewater in a military hospital in Beijing, and names it as Staphylococcus phage vB_SauS_PHM. Through a double-layer plate drop experiment, a clear lysis zone appeared on the MRSA bacterial colony. Figure 1 (A). After concentration and purification using PEG precipitation, the phage titer can reach 10. 10 PFU / mL; such as Figure 1As shown in Figure B, the phage plaques are uniform in size and shape, without halos, with neat edges, and a diameter of approximately 0.5 mm.

[0030] 6. Observe the morphology of bacteriophage vB_SauS_PHM using transmission electron microscopy.

[0031] Use a pipette to draw 20 μL of concentrated and purified phage solution onto a 200-mesh carbon membrane copper grid and let it stand for 3-5 min. Then, blot away excess liquid with filter paper. Add 2% phosphotungstic acid to the carbon-supported copper grid and let it stand for 1-2 min. Blot away excess liquid with filter paper and allow it to dry at room temperature. Observe under a transmission electron microscope and acquire images for analysis.

[0032] like Figure 2 As shown, bacteriophage vB_SauS_PHM has an ellipsoidal head and a long, non-retractable tail without a tail sheath. The head has a long diameter of approximately 103 nm and a short diameter of approximately 52 nm, while the tail is approximately 314 nm long. Based on morphological observation, bacteriophage vB_SauS_PHM belongs to the order Caudata and the family Pleurophagoptera.

[0033] 7. Bioinformatics analysis of bacteriophage vB_SauS_PHM.

[0034] Paired-end (PE) sequencing of sample DNA was performed using the Illumina next-generation sequencing platform. De Novo assembly was performed using SOAPdenovo37, assembling reads sequentially into contigs and scaffolds to obtain the final genome sequence. Sequence correction was then performed using CLC Genomics Workbench 3. Open reading frames (ORFs) were identified using the RAST online server (https: / / rast.nmpdr.org / rast.cgi) and the GeneMarks server (https: / / topaz.gatech.edu / gene mark / gene marks.cgi), and functional genes were predicted and annotated. The functions of proteins with amino acid sequences were predicted using the non-redundant (NR) protein database on the blastp online server, and the annotated functional genes were manually corrected. The genome information was uploaded to the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). Staphylococcus The GenBank accession number for phage vB_SauS_PHM is OR206057. A genomic map of the PHM was created using SnapGene.

[0035] To identify any virulence factors or antibiotic resistance genes in the *Staphylococcus phage* vB_SauS_PHM genome, a BLAST search was performed on the Comprehensive Antibiotic Resistance Database (CARD: https: / / card.mcmaster.ca / ) and the virulence factor database (VFDB: http: / / www.mgc.ac.cn / VFs / main.htm). The PhaBOX online server (https: / / phage.ee.cityu.edu.hk / ) was used to predict the phage's lifestyle or life cycle. Potential tRNA genes were identified using tRNA scan-SE v.2.0 (https: / / trna.ucsc.edu / software / trnascan-se-2.0.3.tar.gz). Nucleotide sequence similarity was performed using the blastn online server (blast.nebi.nlm.nih.gov), along with full-length genome sequence comparison analysis, to preliminarily predict taxonomic status. Informal guidelines published by members of the Bacterial and Archaeal Viruses Subcommittee (BAVS) of the International Committee on the Taxonomy of Viruses (ICTV) were used as a reference for phage nomenclature and classification. Whole genome sequences were aligned using mafftv 7.037 software, followed by Gblock alignment to eliminate misaligned positions and divergent regions. A maximum likelihood phylogenetic tree was constructed using IQTree v1.6.12 software with a bootstrap value of 1000.

[0036] Gene sequencing analysis showed that the full-length genome of bacteriophage vB_SauS_PHM was 42634 bp (G / C content was 33.79%), consisting of 59 open reading frames (ORFs). Most ORFs had the same orientation, and several genes overlapped. Figure 3 (GenBank accession number OR206057). No putative tRNA genes were found throughout the genome, indicating that phage protein synthesis is entirely dependent on host tRNA.

[0037] Gene function prediction results showed that most ORFs of phage vB_SauS_PHM were predicted as hypothetical proteins, with only 23 ORFs predicted to encode functional proteins (Table 2). The functional proteins encoded by these 23 ORFs could be divided into five modules: structural composition, DNA synthesis, cleavage, regulation, and metabolism. This included 6 ORFs encoding structural proteins; 8 ORFs encoding proteins involved in DNA replication; and 2 ORFs (ORF38 and ORF39) encoding proteins crucial for cleavage (holin and lysin). Furthermore, screening with VFDB and CARD confirmed that the genome of phage vB_SauS_PHM did not contain any known virulence factors or antibiotic resistance genes.

[0038] Table 2. Predicted functional open reading frames of bacteriophage vB_SauS_PHM

[0039] Phylogenetic trees reveal the phylogenetic relationships between bacteriophage vB_SauS_PHM and other staphylococcal bacteriophages. Figure 4 (Note: The numbers in parentheses in the phylogenetic tree represent GenBank accession numbers, and the scale represents genetic distance.) The most closely related phages are SAP11 (NCBI accession number: MK801681.1) and SMSAP5 (NCBI accession numbers: NC_019513.1, JQ779023.1). Based on ICTV guidelines and morphological characteristics, phage vB_SauS_PHM belongs to... Duplodnaviria Outline, Caudovirales Head, Siphoviridae Family: Triavirus; Genus: Triavirus.

[0040] Example 2. Biological characteristics analysis of vB_SauS_PHM.

[0041] 1. Host profile determination The host spectrum of bacteriophages was determined using the double-layer plate method, and the efficiency of plating (EOP) was calculated to compare the differences in plaque-forming ability of bacteriophages against different bacteria. The concentrated and purified bacteriophage solution (10⁻⁶) was diluted with SM buffer. 9 (PFU / mL) diluted to different concentration gradients (10-10) 8 Take 100 μL of overnight cultured bacterial solution and mix it with 100 μL of phage solution of different concentration gradients. Incubate at 37 °C for 15 min and then incubate overnight using the double-layer plate method. Observe whether phage plaques are formed and the number of phage plaques at each concentration gradient. Count the number of phage plaques (30-300 is appropriate).

[0042] The phage lysis profile is shown in Table 3. The lysis rate of phage vB_SauS_PHM on MRSA was 67% (8 / 12), indicating that phage vB_SauS_PHM has a wide range of lysis effects on MRSA strains.

[0043] Table 3. Lysis spectrum of bacteriophage vB_SauS_PHM

[0044] Note: - : No plaques; * : Original host bacterium MRSA 1; EOP = phage titer against test strain / phage titer against host bacterium MRSA 1 2. Optimal Multiplicity of Infection (MOI) Prepare logarithmic phase host bacterial culture medium in advance (10 7 (CFU / mL) Phages were serially diluted 10-fold with SM buffer in advance according to multiplicity of infection (MOI) ratios of 0.001, 0.01, 0.1, 1, 10, and 100. 100 μL of the phage and 100 μL of logarithmic-phase host bacterial culture were added to 6 mL of liquid culture medium and incubated overnight at 37 ℃ in a shaker. After centrifugation at 4000 rpm for 15 min, the supernatant was filtered through a 0.22 μm filter for sterilization. The titer of the phage was determined using the double-layer plate method, and the MOI with the highest titer in each experimental group was the optimal MOI.

[0045] like Figure 5 As shown (a, b, c, and d represent differences between treatment groups. Different groups indicate statistically significant differences (P≤0.05), while identical groups indicate no statistically significant differences (P>0.05)), the phage titer was highest at a multiplicity of infection of 0.01, reaching 6.2 × 10⁻⁶. 8 The PFU / mL indicates that the optimal multiplicity of infection for bacteriophage vB_SauS_PHM is 0.01, demonstrating its strong lytic ability against the host bacteria.

[0046] 3. One-step growth curve determination 10 9 CFU / mL host bacterial culture and bacteriophage were mixed at the optimal multiplicity of infection (MOI) of 0.01, incubated at 37 °C for 5 min, and centrifuged at 15,000 rpm for 5 min. The supernatant was discarded, and the precipitate was resuspended in 900 μL of liquid culture medium. The resuspended precipitate was transferred to 9 mL of liquid culture medium and cultured at 37 °C with shaking at 180 rpm. Samples were taken every 10 min, and the titer of the bacteriophage was determined using the double-layer plate method, continuously for up to 180 min.

[0047] like Figure 6As shown, the titer of bacteriophage vB_SauS_PHM did not change significantly within 20 min after infecting the host bacteria. During the period of 20-150 min, the titer of the bacteriophage maintained an increasing trend and then tended to stabilize. This indicates that the latency period of bacteriophage vB_SauS_PHM is 20 min, the lysis period is 130 min, the lysis amount is about 119.4 PFU / cell, and it enters the stationary phase after 150 min.

[0048] 4. Thermal stability determination Take 1 mL of bacteriophage (3 × 10⁻⁶) from each 9 Phage titers were determined by incubating PFU / mL in 1.5 mL centrifuge tubes at 4, 25, 37, 40, 50, 60, 70, 80, 90 and 100 °C for 1 h, and then by the double-layer plate method.

[0049] like Figure 7 The results showed that (a, b, and c represent differences between treatment groups. Different groups indicated statistically significant differences (P≤0.05), while the same groups did not indicate statistically significant differences (P>0.05)). Phage vB_SauS_PHM maintained stable titers (approximately 10) after 1 h of treatment at 4-40 ℃. 9 PFU / mL; after incubation at 50 ℃ and 60 ℃ for 1 h, the titer decreased by approximately one order of magnitude and five orders of magnitude, respectively, and the phage titer was 0 at 70-100 ℃. These results indicate that phage vB_SauS_PHM exhibits the best stability within the range of 4-40 ℃.

[0050] 5. pH stability determination Take 100 μL of phage (3 × 10⁻⁶) from each 9 900 μL of SM buffer with pH values ​​of 2.0, 4.0, 6.0, 7.0, 8.0, 10.0 and 12.0 was added to 1.5 mL centrifuge tubes, respectively. After incubation at 37 °C for 1 h, the phage titer was determined by the double-layer plate method.

[0051] like Figure 8 The results showed that (a, b, c, and d represent differences between treatment groups. Different representations were statistically significant (P ≤ 0.05), while the same representations were not statistically significant (P > 0.05)), the titer of bacteriophage vB_SauS_PHM remained stable at pH 4-8, approximately 10. 9 PFU / mL; when pH 10, the phage titer decreased significantly, by about 5 orders of magnitude; when pH ≥ 12 or pH ≤ 2, the phage titer was 0. The results indicate that phage vB_SauS_PHM exhibits the best stability at pH 4–8.

[0052] 6. In vitro antibacterial effect determination Prepare logarithmic phase host bacterial culture medium in advance (10 7 CFU / mL), the phages to be tested were serially diluted 10-fold with SM buffer in advance according to the multiples of infection (MOI) ratios of 0.01, 0.1, 1, 10, and 100. 1 mL of the phage to be tested and 1 mL of logarithmic-phase host bacterial culture were added to 6 mL of liquid culture medium as the experimental group; 1 mL of PBS and 1 mL of logarithmic-phase host bacterial culture were added to 6 mL of liquid culture medium as the control group. The culture was incubated at 37 ℃ with shaking at 180 rpm, and 200 μL samples were taken every 2 h to measure the OD. 600 value.

[0053] like Figure 9 The results showed that in the first 4 hours, the OD values ​​of each group were... 600 The values ​​are basically the same, but as time increases, the OD values ​​for MOI=0.01 and 0.1 groups... 600 The value continued to rise, but remained lower than that of the PBS control group, indicating that low-titer phages had a certain inhibitory effect on the host bacterium MRSA1; when MOI=1 and 10, OD 600 The value first increases and then decreases, possibly because the phage proliferates and then inhibits the growth of the host bacterium MRSA1; when MOI=100, OD 600 The value remained consistently around 0.05, indicating that high-titer phages could significantly inhibit the growth of the host bacterium MRSA1. In summary, the in vitro antibacterial effect of phages became more significant with increasing multiplicity of infection, suggesting that the highest possible multiplicity of infection should be used in subsequent intracellular bacterial treatments.

[0054] Example 3. Preparation of liposomes Lip@PHM loaded with vB_SauS_PHM 1. Synthesis of Lip@PHM via microfluidic method (illustrated) Figure 10 ): (1) Preparation of organic phase A solution of 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC) and cholesterol with a concentration of 20 mg / mL was prepared in advance using ethanol solution and mixed at a ratio of 5:1 to obtain 0.6 mL; 2 mg of cyanine dye Cy5 was added and mixed thoroughly to form the organic phase.

[0055] (2) Preparation of aqueous phase The aqueous phase consisted of 1.8 mL of concentrated and purified phage solution with a titer of 10. 10 PFU / mL.

[0056] (3) The organic phase solution and the aqueous phase solution were filtered through a 0.22 μm filter membrane respectively.

[0057] (4) Draw the organic phase solution into a 1 mL syringe, draw the aqueous phase solution into a 3 mL syringe, and expel the air from the syringe.

[0058] (5) Connect the syringe outlet and sample inlet tube and fix them to the syringe pump. Run the pump at a total flow rate of 1 mL / h and a flow rate ratio of 1:1 for the aqueous and organic phases. After the organic and aqueous phases are mixed, the DSPCs will experience a highly polar environment, causing them to self-assemble into spherical liposomes. This results in the phages in the surrounding aqueous phase being captured into the hydrophilic layer inside the liposomes. In addition, due to the hydrophobicity of the cyanine dye Cy5, it will be captured into the hydrophobic layer inside the liposomes.

[0059] (6) Collect the sample and dialyze it continuously for three days in a 100 kDa dialysis bag and ultrapure water at 4 degrees Celsius, changing the water every 12 hours.

[0060] (7) Collect the dialysate, concentrate it with an ultrafiltration tube, and store it at 4 °C.

[0061] 2. Size exclusion column purification of Lip@PHM The final product synthesized by microfluidic chromatography is a mixture of free phage, empty liposomes, and Lip@PHM. Since the free phage and empty liposomes differ in size from the latter, they are purified using a size exclusion chromatography column. When the solution passes through a stationary phase composed of porous resin particles, molecules are separated according to size. Particles smaller than the stationary phase pores enter the pores to achieve a longer transport distance, while larger particles that cannot enter the pores move directly around the resin. This results in particles of different sizes exhibiting different retention times. Therefore, after purification by a size exclusion chromatography column, Lip@PHM, empty liposomes, and free phage elute sequentially.

[0062] Operating steps: (1) Remove the chromatographic column from 4 °C and let it stand at room temperature for at least 30 min.

[0063] (2) Activate the column: Add 5 mL of ultrapure water and 15 mL of PBS to rinse the column bed repeatedly, about 10 times or more.

[0064] (3) Load 1 mL of the mixture, remove the bottom cap, wait for the liquid to completely immerse the column bed, add 1 mL of ultrapure water, and collect the outflowing liquid.

[0065] (4) Add 20 mL of ultrapure water to rinse the column.

[0066] 3. Optimization of the Lip@PHM purification method using size exclusion columns After several attempts, the main optimization steps were determined as follows: Reducing the flow rate ratio of the aqueous phase to the organic phase (1:1) and the total flow rate (1 mL / h) to increase the time and opportunity for interaction between the aqueous and organic phases, thereby producing liposomes with larger and more uniform particle size. In addition, increasing the phage titer in the initial aqueous phase (10⁻⁶) was also beneficial. 10 The PFU / mL ratio was used to increase the encapsulation efficiency (20.3%).

[0067] Example 4. Characterization and evaluation of Lip@PHM.

[0068] 1. Hydrated particle size and zeta potential were measured using a nanoparticle size and zeta potential analyzer.

[0069] (1) Turn on the instrument and preheat and calibrate it.

[0070] (2) Rinse the cuvette and potential testing device in turn with a wash bottle containing ultrapure water and the sample solution.

[0071] (3) Add the sample. The height of the sample solution poured into the test device is about 2 / 3 of the total height of the device.

[0072] (4) Place the quartz cuvette with the frosted side facing you into the clamping groove, avoiding touching the non-frosted side; when adding the sample to the potentiometer, avoid generating air bubbles (you can use a 1 mL syringe to draw the sample solution and inject it into the potentiometer), and do not touch the copper plates on both sides with your hands.

[0073] (5) Wipe the surface of the device, place it in the measuring chamber, close the cover, and measure its hydrated particle size and Zeta potential respectively.

[0074] like Figure 11 As shown, the unpurified system (A) had a mean hydrated particle size of 262 nm, with two peaks at 630.4 nm (area 69.2%, representing Lip@PHM or empty liposomes) and 141.8 nm (area 30.8%, representing free phages). After purification, the system (B) exhibited a more uniform particle size distribution and a single peak (peak value 500.9 nm, PDI: 0.315).

[0075] After method optimization, the hydrated particle size of the unpurified system (C) was mainly distributed at around 761.4 nm (peak area 97.5%), while the hydrated particle size of the purified system (D) was a single peak (peak value 733.9 nm, PDI: 0.135). The comparison shows that the size and uniformity of Lip@PHM were significantly improved after the method optimization. Figure 11 In the Chinese dictionary: (A) Before optimization - before purification (B) Before optimization - after purification (C) After optimization - before purification (D) After optimization - after purification

[0076] After microfluidic assembly, the resulting Lip@PHM system had a Zeta potential of -2.20 mV before purification (A), compared to -10.6 mV in the unassembled component mixture. Figure 12 Compared to C), the Zeta potential shifted positively and became more neutral. This is because during the microfluidic self-assembly of liposomes, some negatively charged phages were encapsulated, and neutrally charged Lip@PHMs were generated, resulting in a smaller system potential value.

[0077] After purification by size exclusion chromatography (B), the Zeta potential of Lip@PHM was -0.707 mV. Compared with before purification (-2.20 mV), the Zeta potential of the system shifted positively and became more neutral. This is because the size exclusion chromatography column separated the free phage in the system, resulting in a smaller system potential value. Figure 12 In the middle: (A) System before purification (B) System after purification (C) Component mixture without microfluidic assembly.

[0078] 2. Cryo-transmission electron microscopy observation of Lip@PHM.

[0079] After purification, Lip@PHM is treated with a cryoprotectant, and the sample solution is then dripped onto a specially designed grid to form a thin layer. Following rapid freezing, the sample is transferred to a cryotransfer device. In an electron microscope, a high-energy electron beam irradiates the frozen sample. The electron beam interacts with the atoms in the sample, generating transmitted electron signals. Transmission electron microscopy (TEM) mode is selected to acquire and analyze the images.

[0080] like Figure 13 As shown, Lip@PHM exhibits a circular or elliptical shape with a diameter of approximately 750 nm, which is consistent with the particle size detection results. Loaded phages (black arrows) are visible inside the liposomes.

[0081] Example 5. Preparation and characterization of TAT-Lip@PHM 1. TAT-PEG 2000 -DSPE synthesis Take 1 mmol DSPE-PEG 2000 Distearate phosphatidylethanolamine-polyethylene glycol 2000 was dissolved in 200 μL of borate buffer (pH=8.4), and 0.1% EDTA and 1 mmol of cell-penetrating peptide TAT were added. The mixture was reacted overnight at room temperature with shaking at 180 rpm. The next day, the product was desalted using a size exclusion column, separated by high performance liquid chromatography, and the peak product (TAT-PEG) at 30 min was collected. 2000 -DSPE), freeze-dried and stored at -20 ℃ for later use.

[0082] 2. Preparation of TAT-Lip@PHM Lip@PHM with 1 μM TAT-PEG 2000 -DSPE was incubated at 37 °C for 1 h, and then purified by ultrafiltration with a 100 kDa molecular weight cutoff tube to remove unbound TAT-PEG. 2000 -DSPE, obtain TAT-Lip@PHM, and store at 4 ℃ for later use.

[0083] 3. Characterization of TAT-Lip@PHM 3.1 Characterization of hydrated particle size and Zeta potential The hydration particle size and zeta potential of TAT-Lip@PHM were determined using the same method as in Example 4.

[0084] like Figure 14 As shown, after TAT modification, the average hydrated particle size of TAT-Lip@PHM is 636.1 nm (PDI: 0.148).

[0085] like Figure 15 As shown, after TAT modification, the Zeta potential of TAT-Lip@PHM is 3.91 mV. Compared with the unmodified state (-0.707 mV), the Zeta potential of the system changes from negative to positive. This is because TAT is a cationic polymer with a positive charge. When it is inserted into the surface of Lip@PHM, it causes its potential to reverse, indicating that TAT has successfully modified Lip@PHM.

[0086] 3.2 Fluorescence Spectroscopy Characterization The fluorescence intensity of TAT-Lip@PHM was detected using a fluorescence spectrophotometer with emission wavelengths ranging from 670 to 860 nm.

[0087] The fluorescence intensity of TAT-Lip@PHM varies with emission wavelength as follows: Figure 16 As shown, the emission wavelength corresponding to the peak is about 690 nm, which is consistent with the Cy5 dye, indicating that the Cy5 dye has been encapsulated inside the liposomes and can be used for subsequent intracellular fluorescence localization of TAT-Lip@PHM.

[0088] 4. Cytotoxicity of TAT-Lip@PHM The MTT assay was used to investigate the toxicity of different concentrations of TAT-Lip@PHM to RAW 264.7 mouse mononuclear macrophages. Adherent RAW 264.7 cells were resuspended in complete culture medium and seeded into 96-well plates at a density of 3000-10000 cells / well, with 100 μL per well and cultured overnight. After cell adhesion, 100 μL of TAT-Lip@PHM was added to each well, resulting in final concentrations of 0, 150, 300, 600, and 1200 µg / mL, with three replicates for each concentration. The cells were then cultured at 37 ℃ in a 5% CO2 incubator for 24 hours. The liquid in the wells was aspirated, and the cells were slowly washed three times with PBS. 90 µL of complete culture medium and 10 µL of MTT solution were added to each well, and the cells were cultured for 4 hours. The liquid in the wells was then aspirated, and 110 μL of Formazan lysate was added to each well. Place the plate on a shaker and shake at 300 rpm for 10 minutes to dissolve all the crystals. Measure the OD using a microplate reader. 490 The cell viability was calculated as follows: (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0089] Cytotoxicity results showed ( Figure 17 RAW 264.7 cells were treated with TAT-Lip@PHM at final concentrations of 150, 300, 600, and 1200 µg / mL for 24 h, and the cell viability rates were 94.19%, 87.98%, 79.84%, and 77.13%, respectively. This shows that TAT-Lip@PHM has no significant cytotoxicity at concentrations of 300 μg / mL and below, and the cell viability rate is greater than 85%, indicating that TAT-Lip@PHM has good cell compatibility.

[0090] 5. Intracellular fluorescence localization of TAT-Lip@PHM After resuspending adherent RAW 264.7 cells in complete culture medium, at approximately 10... 6 Seed 1.5 mL of TAT-Lip@PHM (final concentration 300 μg / mL) at a density of 1 cell / mL into a confocal culture dish and incubated overnight. After the cells adhered, add 0.5 mL of TAT-Lip@PHM to the culture dish. Observe the fluorescence status under a confocal microscope at 2, 4, 6, 12 and 24 h and calculate the cell infiltration ratio = number of fluorescent cells / total number of cells.

[0091] like Figure 18As shown, the cell penetration rates at 2, 4, 6, 12, and 24 h were 5.33%, 43.30%, 67.37%, 92.98%, and 94.38%, respectively. It can be seen that the intracellular fluorescence quantity increases with the increase of incubation time, reaching more than 90% after 12 h. This indicates that most TAT-Lip@PHM cells penetrate the cells at around 12 h, and the cell penetration rate can reach more than 90%.

[0092] Example 6. Evaluation of the intracellular bactericidal effect of TAT-Lip@PHM and Lip@PHM.

[0093] An in vitro model of intracellular MRSA infection was constructed. The experimental groups were as follows (Note: all concentrations shown are final concentrations): 1 mL of free bacteriophage vB_SauS_PHM (1.5 × 10⁻⁶). 9 PFU / mL) and 3 mL of complete culture medium, 1 mL of Lip@PHM (300 µg / mL, containing approximately 5.4 × 10⁻⁶ PFU / mL) 8 Phage (PFU / mL) and 3 mL of complete culture medium, 1 mL of TAT-Lip@PHM (300 µg / mL, containing approximately 3.7 × 10⁻⁶ PFU / mL) 8 The control group consisted of 1 mL PBS buffer and 3 mL complete culture medium (PFU / mL phage). After culturing in a cell culture incubator for 12, 18, and 24 hours, the liquid in the wells was aspirated and the cells were slowly rinsed once with PBS. 2 mL of 0.5% Triton X-100 was added to lyse the cells, and the cells were incubated for 30 minutes followed by pipetting. The number of intracellular bacteria and the clearance rate were calculated as (number of bacteria in the PBS control group - number of bacteria in the experimental group) / number of bacteria in the PBS control group × 100%.

[0094] Figure 19 Images of intracellular MRSA plating plates after different treatment times with TAT-Lip@PHM and Lip@PHM. Figure 20 For plate colony counting. For example... Figure 20 As shown in the figure (Note: a, b, c, d, e, f, g, h represent differences between treatment groups. Different groups indicate statistically significant differences (P≤0.05), while the same groups indicate no statistically significant differences (P>0.05)), the free phage vB_SauS_PHM group showed inhibition of intracellular MRSA in the initial 12 h (clearance rate of 19.92%). However, as the treatment time was extended to 18 h and 24 h, the number of intracellular MRSA gradually increased, while the clearance rate did not change significantly (21.85% and 21.24%, respectively). This indicates that the free phage vB_SauS_PHM cannot achieve long-term effective bactericidal efficacy against intracellular MRSA infection.

[0095] In contrast, the intracellular MRSA count in the TAT-Lip@PHM group was lower than that in the free phage vB_SauS_PHM group and the Lip@PHM group at every time point. This indicates that the vector system containing a relatively high concentration of free phage and a low concentration of phage has a better clearance effect on intracellular MRSA infection, suggesting that liposome encapsulation and TAT modification enhance the phage's ability to resist intracellular bacterial infection. Furthermore, with prolonged exposure time, the clearance rate of intracellular MRSA in the TAT-Lip@PHM group gradually increased, reaching 94.05% at 24 h, which is significantly higher than that in the free phage vB_SauS_PHM group (21.24%) and the Lip@PHM group (44.90%), demonstrating superior bactericidal ability.

Claims

1. A method for preparing phage-loaded liposomes, characterized in that, The method includes the following steps: S1. Prepare an organic phase containing DSPC and cholesterol, wherein the mass ratio of DSPC to cholesterol is 4-6:0.5-1.5; S2. Prepare an aqueous phase containing bacteriophage, wherein the bacteriophage titer is 10. 9 -10 11 PFU / mL; S3. Input the organic phase obtained in S1 and the aqueous phase obtained in S2 into the microfluidic chip at a volume ratio of 0.5-2:3, a total flow rate of 0.5-1.5 ml / h, and a flow rate ratio of 2:1-1:2 between the organic phase and the aqueous phase, respectively. S4. Harvest the liquid output from the microfluidic chip and perform dialysis using a 90-110 kDa dialysis bag; S5. Purify the dialysate obtained in S4 to obtain liposomes loaded with phage.

2. The method according to claim 1, characterized in that, In S1, the mass ratio of DSPC to cholesterol is 5:

1.

3. The method according to claim 1, characterized in that, In S2, the titer of the bacteriophage is 10. 10 PFU / mL.

4. The method according to claim 1, characterized in that, In S3, the organic phase obtained in S1 and the aqueous phase obtained in S2 are input into the microfluidic chip at a volume ratio of 1:3, a total flow rate of 1 ml / h, and a flow rate ratio of 1:1 between the organic phase and the aqueous phase.

5. The method according to claim 1, characterized in that, In S4, dialysis is performed using a 100 kDa dialysis bag.

6. The method according to claim 1, characterized in that, In S5, the purification is performed using size exclusion chromatography column purification.

7. The phage-loaded liposomes prepared by the method according to any one of claims 1-6, characterized in that, The liposomes loaded with the phage have a hydrated particle size of 730-750 nm and a zeta potential of -0.710 to -0.700 mV.

8. A method for TAT modification of the phage-loaded liposomes according to claim 7, characterized in that, The method includes reacting the phage-loaded liposomes with 1 μM TAT-PEG. 2000 -DSPE was incubated at 37 ℃ for 0.5-1.5 h, followed by separation and purification using an ultrafiltration tube with a molecular weight cutoff of 90-110 kDa.

9. The TAT-modified phage-loaded liposomes obtained by the method according to claim 8, characterized in that, The liposomes loaded with the TAT-modified phage had an average hydrated particle size of 630-640 nm and a Zeta potential of 3.91 mV.

10. The use of the liposomes according to claim 7 or 9 in the preparation of intracellular bactericides.

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