Preparation method, product and application of calixarene engineered liposome

Abstract

The present invention relates to the technical field of liposome preparation, and in particular to a preparation method, product and application of calixarene engineered liposome. The preparation method of calixarene engineered liposome of the present invention comprises the following steps: dissolving a membrane material and guanidinocalixarene in an organic solvent, then drying the mixture by spin drying, and adding water to obtain the calixarene engineered liposome. The present invention proposes a preparation method of calixarene engineered liposome with high efficiency against bacterial biofilm, that is, preparing calixarene engineered liposome (Lipo / GA) by embedding amphiphilic guanidinocalixarene (GA) into a liposome bilayer membrane. Antibiotics are loaded through host-guest interaction to generate drugs (Lipo / GA / CFZ) against bacterial biofilm infection. Since GA has an ATP-responsive drug release function, Lipo / GA / CFZ can achieve site-specific controlled release of antibiotics.

Description

Technical Field

[0001] The invention relates to the technical field of liposome preparation, and in particular to a preparation method, product and application of calixarene engineered liposome. Background Art

[0002] Bacterial biofilm is a three-dimensional structured group composed of bacterial cells and their secreted extracellular polymers (EPS). Compared with planktonic bacteria, bacteria in biofilms have a specific microenvironment, including weak acidity, hypoxia, adenosine triphosphate (ATP), and enzyme overexpression ( Nat. Rev. Microbiol. 2020, 18, 571–586; Biomacromolecules 2023, 24, 1003−1013). This makes the bacteria in the biofilm 1,000-fold more resistant to antibiotics ( Adv. Mater. 2023, 2301623). Therefore, developing methods to combat bacterial biofilm infections is an important area of ​​research.

[0003] Liposomes are called "artificial biofilms" and show great potential for drug delivery. However, there are many challenges when using traditional liposomes as antibiotic delivery systems to combat bacterial biofilm infections, including low biofilm penetration efficiency and difficulty in achieving specific release of antibiotics. Therefore, it is necessary to provide a highly effective anti-bacterial biofilm liposome to combat bacterial biofilm infections. Summary of the invention

[0004] Based on the above content, the present invention provides a preparation method, product and application of calixarene engineered liposomes.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a method for preparing calixarene engineered liposomes, comprising the following steps:

[0007] Dissolving the membrane material and guanidinocalixarene in an organic solvent, then drying by rotation, and adding water to prepare the calixarene engineered liposome;

[0008] The membrane material is 1,2-dipalmitoyl- s - at least one of glycerol-3-phosphorylcholine, LA-phosphoryl-DL-glycerol palmitoyl sodium, 1,2-dioleoyl-3-trimethylpropylammonium chloride, and distearoylphosphatidylethanolamine-polyethylene glycol 2000;

[0009] The structural formula of the guanidinocalixarene is .

[0010] The second technical solution of the present invention is a calixarene engineered liposome prepared according to the above-mentioned preparation method.

[0011] The third technical solution of the present invention is the use of the above-mentioned calixarene engineered liposome in the preparation of drugs for resisting bacterial biofilm infection.

[0012] A fourth technical solution of the present invention is a drug for resisting bacterial biofilm infection, comprising the above-mentioned calixarene engineered liposome and an antibiotic.

[0013] A fifth technical solution of the present invention is a method for preparing the above-mentioned drug for resisting bacterial biofilm infection, comprising mixing and shaking the calixarene engineered liposome with the antibiotic to obtain the drug for resisting bacterial biofilm infection.

[0014] The present invention discloses the following technical effects:

[0015] The present invention proposes a method for preparing a highly effective anti-bacterial biofilm calixarene engineered liposome, that is, preparing a calixarene engineered liposome (Lipo / GA) by embedding an amphiphilic guanidinocalixarene (GA) into a liposome bilayer membrane. Subsequently, an antibiotic (cefazolin sodium (CFZ)) is loaded through host-guest interaction to generate a drug (Lipo / GA / CFZ) for anti-bacterial biofilm infection. Since GA has an ATP-responsive drug release function, Lipo / GA / CFZ can achieve site-specific controlled release of antibiotics. Lipo / GA / CFZ can not only destroy the mature biofilm formed by the Gram-positive bacterium Staphylococcus aureus, but also enhance the bactericidal effect of antibiotics on biofilm-related drug resistance. The present invention provides an effective method for constructing hybrid macrocyclic engineered liposomes, broadens the application potential of liposomes in anti-bacterial biofilm infection, and provides a new strategy for reducing antibiotic resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 is the average hydrated particle size of Lipo, Lipo / GA, and Lipo / GA / CFZ.

[0018] Figure 2 TEM images of Lipo, Lipo / GA, and Lipo / GA / CFZ.

[0019] Figure 3This is the curve of the cumulative release of CFZ from Lipo / GA / CFZ in the presence / absence of ATP.

[0020] Figure 4 Interaction of Lipo or Lipo / GA / CFZ with planktonic Staphylococcus aureus Xen36.

[0021] Figure 5 3D-CLSM images of Staphylococcus aureus biofilm formation after various treatments.

[0022] Figure 6 Quantitative statistics of biofilm biomass in 3D-CLSM images (results are expressed as mean ± SD, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001).

[0023] Figure 7 Quantitative statistics of biofilm biomass in crystal violet stained images.

[0024] Figure 8 3D-CLSM images of Staphylococcus aureus biofilms after treatment under different conditions.

[0025] Fig. 9 Quantitative statistics of biofilm biomass in 3D-CLSM images (results are expressed as mean ± SD, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001).

[0026] Fig.10 SEM images of biofilms after various treatments (yellow arrows indicate EPS in the biofilms).

[0027] Fig.11 Quantitative statistics of biofilm biomass in crystal violet stained images.

[0028] Fig.12 Experimental procedures for S. aureus Xen36 infection and treatment of mice; mice were treated with PBS, Lipo (850 μM), CFZ (100 μM), Lipo / GA (850 / 100 μM), or Lipo / GA / CFZ (850 / 100 / 100 μM) every other day for peritoneal infection.

[0029] Fig.13 Bioluminescence images of mice receiving different treatments.

[0030] Fig.14 Quantification results of bioluminescence intensity in bioluminescence images of mice receiving different treatments.

[0031] Fig.15The relative infection areas of mice after various treatments.

[0032] Fig.16 Images of remaining bacterial colonies in subcutaneous infection of mice after different treatments.

[0033] Fig.17 Quantification of CFUs collected from the subcutaneous infected area of ​​mice after various treatments; results are expressed as mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] The first aspect of the present invention provides a method for preparing a calixarene engineered liposome, comprising the following steps:

[0040] Dissolving the membrane material and guanidinocalixarene in an organic solvent, then drying by rotation, and adding water to prepare the calixarene engineered liposome;

[0041] The membrane material is 1,2-dipalmitoyl- s - at least one of glycerol-3-phosphorylcholine, LA-phosphoryl-DL-glycerol palmitoyl sodium, 1,2-dioleoyl-3-trimethylpropylammonium chloride, and distearoylphosphatidylethanolamine-polyethylene glycol 2000;

[0042] The structural formula of the guanidinocalixarene is .

[0043] In a preferred embodiment of the present invention, the membrane material is 1,2-dipalmitoyl- s - A mixture of glycerol-3-phosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol 2000 in a mass ratio of (0-13.16):(0-2.96).

[0044] In a preferred embodiment of the present invention, the mass ratio of the membrane material to the guanidinocalixarene is (0-16.5): (0-3.88).

[0045] In a preferred embodiment of the present invention, the organic solvent is a mixture of methanol:chloroform in a volume ratio of 1:1.

[0046] In a preferred embodiment of the present invention, after adding water, the step of ultrasonication at 80°C for 5 minutes is also included.

[0047] The second aspect of the present invention provides a calixarene engineered liposome prepared according to the above preparation method.

[0048] The third aspect of the present invention provides a use of the calixarene engineered liposome in the preparation of a drug for resisting bacterial biofilm infection.

[0049] The fourth aspect of the present invention provides a drug for resisting bacterial biofilm infection, comprising the above-mentioned calixarene engineered liposome and an antibiotic.

[0050] In a preferred embodiment of the present invention, the antibiotic is cefazolin sodium.

[0051] The fifth aspect of the present invention provides a method for preparing the above-mentioned drug for resisting bacterial biofilm infection, comprising mixing and shaking the calixarene engineered liposomes with the antibiotic to obtain the drug for resisting bacterial biofilm infection.

[0052] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0053] The guanidinocalixarene (GA) used in the present invention was synthesized and purified according to the literature method ( J. Am. Chem. Soc. , 2018, 140, 4945–4953). The specific synthesis route is as follows:

[0054]

[0055] In the above synthetic routes, (i) HNO3, AcOH, dry CH2Cl2, stirring at room temperature; (ii) SnCl2·2H2O, C2H5OH / AcOEt (1:1, v:v), reflux; (iii) 1,3-di(tert-butyloxycarbonyl)-2-methyl-2-isothiourea, triethylamine, AgNO3, dry CH2Cl2, stirring at room temperature; (iv) SnCl4, AcOEt, stirring at room temperature.

[0056] The hydrogen spectrum, carbon spectrum and mass spectrum of the GA are as follows:

[0057] 1 H NMR (400 MHz, CD3OD, δ ): 6.91 (s, 10H, ArH), 4.51 (d, J = 13.86 Hz,5H, Ar-CH2-Ar), 3.81 (t, J = 7.48 Hz, 10H, CH2-O-Ar), 3.38 (d, J = 13.63 Hz,5H, Ar-CH2-Ar), 1.93 (m, 10H, -C H 2-CH2-O-Ar), 1.22 (m, 90H, alkyl CH2), 0.81(t, J = 6.07 Hz, 15H, -CH3).

[0058] 13 C NMR (100 MHz, CD3OD, δ ): 156.7, 154.7, 135.6, 129.5, 126.5, 74.5,31.9, 30.4, 30.2, 30.0, 29.9, 29.7, 29.4, 26.4, 22.5, 13.1.

[0059] ESI-FTMS mass spectrum: m / z: [M+H-5HCl] + The calculated value is C 100 H 166 N15 O5 + 1658.3224; the detected value is 1658.3188.

[0060] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0061] Example 1

[0062] Weigh 1,2-dipalmitoyl- s 13.16 mg of 3-glycerol-3-phosphorylcholine (DPPC), 2.96 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000), and 3.88 mg of GA were dissolved in 4 ml of methanol / chloroform (v / v=1:1), then vacuum-dried at 50 °C, 10.4 ml of distilled water was added, and ultrasonicated at 80 °C for 5 minutes to obtain Lipo / GA. After Lipo / GA was prepared, 1.0 mg of cefazolin sodium (CFZ) was added and shaken for 5 minutes to obtain Lipo / GA / CFZ.

[0063] Comparative Example 1

[0064] The only difference from Example 1 is that the addition of GA and CFZ is omitted, and the remaining steps and parameters are the same as those of Example 1. Lipo is prepared.

[0065] Comparative Example 2

[0066] The only difference from Example 1 is that the addition of CFZ is omitted, and the remaining steps and parameters are the same as those of Example 1. Lipo / GA is prepared.

[0067] Characterization of Lipo / GA / CFZ:

[0068] The morphology of the prepared Lipo / GA / CFZ was characterized by dynamic light scattering (DLS), and Lipo and Lipo / GA were used as controls. Figure 1 As shown in Figure 2, the average hydrated particle sizes of Lipo, Lipo / GA, and Lipo / GA / CFZ are approximately 206 nm, 101 nm, and 108 nm, respectively. High-resolution transmission electron microscopy (TEM) images ( Figure 2 ) showed that Lipo / GA / CFZ, similar to Lipo and Lipo / GA, exhibited spherical morphological characteristics and were hollow vesicle structures.

[0069] Lipo / GA / CFZ responsively releases CFZ in the presence of ATP. The specific test method is as follows:

[0070] 250 μL of Lipo / GA / CFZ was encapsulated in a dialysis membrane (molecular weight cutoff 1000 Da) and immersed in 4750 μL of PBS (10 mM), pH 7.4, with or without ATP (2 mM). At designated time intervals (0, 0.5, 1, 2,4, 6, 8, 12, and 24 hours (h)), 100 μL of the solution was removed for subsequent analysis. Subsequently, an equal volume of new PBS was added to maintain a constant volume. Subsequently, the absorbance of the solution was evaluated at a wavelength of 270 nm by UV-visible spectrometer.

[0071] The results are as follows Figure 3 As shown, Figure 3 In the equation, "-ATP" indicates an environment without ATP, and "+ATP" indicates an environment with ATP. Figure 3 It can be seen that after 6 h of incubation in the absence of ATP, the release of CFZ in the Lipo / GA / CFZ system was 15%. In contrast, in the presence of ATP, the release of CFZ increased significantly to 25% after the same incubation time. Further extending the incubation time to 24 h, the release of CFZ in the ATP environment increased significantly to 43%. In sharp contrast, in the absence of ATP, the release of CFZ remained at a relatively stable level. These experimental results show that Lipo / GA / CFZ has ATP-responsive drug release capabilities.

[0072] Interaction between Lipo / GA / CFZ and planktonic bacteria:

[0073] In order to explore the interaction between Lipo / GA / CFZ and planktonic bacteria, the red fluorescent dye Nile Red (NR) was first embedded in Lipo / GA / CFZ and Lipo, respectively. The specific method was as follows: NR was dissolved in methanol, added to Lipo / GCA / CFZ or Lipo, ultrasonicated for 5 minutes, and the insoluble matter was removed after centrifugation to obtain the fluorescently labeled complex Lipo / GA / CFZ / NR or Lipo / NR. Subsequently, this complex was co-incubated with planktonic Staphylococcus aureus Xen36, and the bacteria were stained with the green fluorescent dye acridine orange (AO). The incubated samples were observed by confocal laser scanning microscopy (CLSM), and the results are shown in Figure 2. Figure 4As shown. As a control group, the red fluorescence of Lipo / NR did not show obvious overlap with the green fluorescence of Staphylococcus aureus, suggesting a lack of obvious interaction between Lipo / NR and bacteria. In contrast, the red fluorescence of Lipo / GA / CFZ / NR overlapped with the green fluorescence of bacteria, forming a yellow fluorescence image. In addition, through quantitative analysis, we found that there was a significant co-localization phenomenon between Lipo / GA / CFZ / NR and Staphylococcus aureus, with a Pearson correlation coefficient of up to 0.85, which was much higher than the Pearson correlation coefficient of Lipo / NR of 0.01. It is worth noting that Staphylococcus aureus showed obvious aggregation after contact with Lipo / GA / CFZ / NR, which further confirmed the strong interaction between Lipo / GA / CFZ and bacteria.

[0074] Lipo / GA / CFZ inhibits bacterial biofilm formation:

[0075] The present invention studies the inhibitory effect of Lipo / GA / CFZ on the biofilm formation of Staphylococcus aureus. 8 / mL of bacteria (1 mL) were added to the confocal culture dish, and then the resulting suspension was replaced with a culture medium (2 mL) containing different materials, with concentrations of Lipo (900 µM), CFZ (100 µM), Lipo / GA (900 / 100 µM) or Lipo / GA / CFZ (900 / 100 / 100 µM), PBS as the control group, and cultured at 37 °C for 24 h. Afterwards, the resulting suspension was replaced with 2 mL of culture medium and cultured for another 48 h. Through CLSM imaging, CFZ, Lipo / GA, and Lipo / GA / CFZ can effectively inhibit the formation of biofilms (such as Figure 5 Qualitative analysis of fluorescence intensity showed that the volume of biofilms pretreated with Lipo / GA was significantly lower than that of biofilms treated with Lipo, suggesting that the co-assembly of GA may enhance the ability of liposomes to inhibit biofilm formation (as shown in Figure 2A). Figure 6 CFZ, Lipo / GA, and Lipo / GA / CFZ all showed concentration-dependent biofilm inhibition effects, resulting in 58%, 80%, and 85% reductions in biofilm biomass at 100 µM concentration, respectively, compared with the PBS-treated group (as shown in Figure 2A). Figure 7 as shown).

[0076] Although inhibiting the formation of bacterial biofilms is of great significance in preventing infections, it is usually only taken seriously when mature biofilms cause infections. Therefore, it is particularly important to eliminate mature biofilms when treating infections associated with bacterial biofilms. Subsequently, the present invention studied the effects of Lipo / GA and Lipo / GA / CFZ in eradicating biofilms. The mature biofilms of Staphylococcus aureus were treated with PBS, Lipo (900 µM), CFZ (100 µM), Lipo / GA (900 / 100 µM) or Lipo / GA / CFZ (900 / 100 / 100 µM) for 24 h and then observed using CLSM. 3D CLSM images (such as Figure 8 ) showed that the biofilms treated with PBS, Lipo, and CFZ still maintained a dense and thick structure. In contrast, the bacterial density and thickness of the biofilms were significantly reduced after treatment with Lipo / GA and Lipo / GA / CFZ. Further quantitative analysis showed that ( Fig. 9 As shown in Figure 2 ), Lipo and CFZ had little effect on the biofilm volume, while Lipo / GA and Lipo / GA / CFZ treatments significantly reduced the biofilm volume, and the biofilm volume of the Lipo / GA / CFZ treatment was lower than that of the Lipo / GA treatment (** p < 0.01).

[0077] To further analyze the effects of different treatments on the structure of mature biofilms, scanning electron microscopy (SEM) was used to observe the treated biofilms. Fig.10 It was shown that in the biofilm treated with PBS, the bacteria were densely wrapped by EPS. In the biofilm treated with Lipo / GA and Lipo / GA / CFZ, the EPS matrix was almost invisible, and the number of bacteria was significantly reduced after Lipo / GA / CFZ treatment. The morphology of the remaining bacterial cells changed, with irregular boundaries and loss of complete membrane structure. Therefore, Lipo / GA / CFZ not only has the antibacterial activity of CFZ, but also has the biofilm destruction effect of Lipo / GA.

[0078] In order to verify this conclusion, the present invention used crystal violet staining to analyze the residual biomass in the biofilm after different treatments (such as Fig.11 The results showed that free CFZ had almost no ability to disperse mature biofilms, while Lipo / GA showed concentration-dependent biofilm dispersion ability, with a maximum dispersion of 65% of biofilms at a concentration of 100 μM. In contrast, Lipo / GA / CFZ had a higher biofilm dispersion efficiency and was able to remove 76% of S. aureus biofilms, a result consistent with previous speculation.

[0079] In the in vitro experiment, the significant removal effect of Lipo / GA / CFZ on biofilm was encouraging. Subsequently, the present invention further evaluated the ability of Lipo / GA / CFZ to remove Staphylococcus aureus Xen36 biofilm in vivo. The experimental design process is as follows Fig.12 As shown, 100 μL of 1×10 9 / mL of luminescent Staphylococcus aureus Xen36, the bio-optical imaging system was used to record the luminescence intensity and area of ​​the cocci, and the maximum value was reached on the second day after injection, indicating that the subcutaneous infection model of mice was successfully established. The mice were randomly divided into five groups. Starting from the second day after infection, the mice were given in situ subcutaneous injections of the corresponding materials every other day: (i) 100 μL PBS, (ii) 100 μL Lipo, (iii) 100 μL CFZ, (iv) 100 μL Lipo / GA, (v) 100 μL Lipo / GA / CFZ, and the equivalent concentrations of GA and CFZ were both 100 µM. The results showed that the bioluminescence intensity of the infection site of mice in the PBS and Lipo treatment groups first increased and then gradually weakened. The CFZ, Lipo / GA and Lipo / GA / CFZ treatment groups significantly reduced the bioluminescence intensity of the infection site (such as Fig.13 Quantitative analysis results showed that after 7 days of Lipo / GA / CFZ treatment, the bioluminescence intensity decreased by 95%, which was 35% more than that of the Lipo / GA treatment group (as shown in Fig.14 At the same time, the analysis of relative infection area (such as Fig.15 As shown in Figure 2, after treatment with PBS, Lipo, CFZ, or Lipo / GA, the infected area of ​​mice became 290%, 238%, 47%, and 47% of the original area, respectively, on day 7. However, the infected area of ​​the Lipo / GA / CFZ-treated group was reduced to 0.2%, showing a higher therapeutic effect (as shown in Figure 2). Fig.16 In addition, the viable bacteria counts at the subcutaneous infection sites of mice in different treatment groups were measured (as shown in Fig.17 The results (shown in Figure 2) showed that the CFUs in the Lipo / GA / CFZ-treated group were reduced most significantly, with a 3.6-log reduction compared with the PBS-treated group. This indicates that Lipo / GA / CFZ has excellent biofilm removal and bactericidal abilities in vivo.

[0080] The present invention develops an ATP-responsive calixarene engineered liposome (Lipo / GA), which aims to inhibit the formation of bacterial biofilms and eradicate mature biofilms that have been formed by bacteria. By encapsulating the antibiotic (CFZ) in Lipo / GA and utilizing the overexpression of ATP in the biofilm microenvironment, the precise release of the antibiotic is achieved. In a mouse model of Staphylococcus aureus infection, Lipo / GA / CFZ exhibited a significant synergistic therapeutic effect. Guanidino-modified calixarene endows liposomes with the ability to clear biofilms, while achieving site-specific release of antibiotics through an ATP-responsive mechanism, providing a general strategy for reducing drug resistance. In addition, the design of amphiphilic macrocyclic supramolecules as liposome materials provides a potential improvement approach for liposome-based biofilm delivery systems.

[0081] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing calixarene engineered liposomes, characterized in that: The following steps are involved: Dissolving the membrane material and guanidinocalixarene in an organic solvent, then drying by rotation, and adding water to prepare the calixarene engineered liposome; The structural formula of the guanidinocalixarene is ; The membrane material is 1,2-dipalmitoyl- s - a mixture of glycerol-3-phosphorylcholine and distearoylphosphatidylethanolamine-polyethylene glycol 2000 in a mass ratio of (0-13.16): (0-2.96); The mass ratio of the membrane material to the guanidinocalixarene is (0-16.5): (0-3.88).

2. The method for preparing calixarene engineered liposomes according to claim 1, characterized in that: The organic solvent is a mixture of methanol:chloroform in a volume ratio of 1:

1.

3. The method for preparing calixarene engineered liposomes according to claim 1, characterized in that: After adding water, the method further includes a step of ultrasonication at 80 °C for 5 minutes; the mass ratio of the membrane material to the water is (16-17): (10000-11000).

4. A calixarene engineered liposome prepared according to the preparation method according to any one of claims 1 to 3.

5. Use of the calixarene engineered liposome as claimed in claim 4 in the preparation of drugs for resisting bacterial biofilm infection.

6. A drug for resisting bacterial biofilm infection, characterized in that: It comprises the calixarene engineered liposome according to claim 4 and an antibiotic.

7. The drug for resisting bacterial biofilm infection according to claim 6, characterized in that: The antibiotic is cefazolin sodium.

8. A method for preparing a drug for resisting bacterial biofilm infection according to claim 6 or 7, characterized in that: The calixarene engineered liposomes and the antibiotics are mixed and shaken to obtain the drug for resisting bacterial biofilm infection.

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