Lipid nano assembly loaded with hydrophilic antibacterial agent as well as preparation method and application of lipid nano assembly

Abstract

The invention discloses a lipid nano assembly loaded with a hydrophilic antibacterial drug as well as a preparation method and application of the lipid nano assembly, and belongs to the technical field of biological medicines. The lipid nano assembly loaded with the hydrophilic antibacterial drug is synthesized by combining phosphatidylcholine lipids and phenylboronic acid derivatives through dynamic covalent boric acid ester bonds, and lipid molecules can load hydrophilic antibacterial small molecule drugs. By screening Cip with different concentrations, high drug loading rate and encapsulation rate of Cip are realized. The lipid nano assembly has triple responsiveness of H < + >, H2O2 and ATP, the prepared lipid nano assembly can deliver hydrophilic drugs to a target part, enrichment is achieved at the target part, then controlled release of the drugs is achieved through the special microenvironment of the focus part, and the good treatment effect is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a lipid nanoassembly loaded with a hydrophilic antibacterial drug, a preparation method thereof, and an application thereof in the treatment of peritonitis. Background Art

[0002] In the biomedical field, the study of liposomes for drug delivery has received extensive attention and discussion. As a drug delivery system with excellent performance and a wide range of applications, lipid nanocarriers have been widely used in the treatment of various diseases such as cancer, infection, pain, acute and chronic inflammation. Despite the success, there are still some challenges in encapsulating and controlling the targeted release of the payload. Especially in the field of hydrophilic small molecule drugs. Non-targeted, uncontrolled release lipid nanocarriers can release a large amount of drugs immediately after administration, which may lead to local or systemic toxicity. In complex and harsh lesion environments, through their special structure, lipid nanocarriers have significant advantages in targeting cell membranes, biomembranes, and bioadhesion compared with other types of delivery methods, and can significantly improve the interaction with cells or pathogens in the targeted area. In addition, the composition structure of nanolipid molecules in this scheme can realize the responsive release of lipid molecules in complex lesion environments, can further regulate the release behavior of drugs, and increase the accumulation of drugs in the targeted area, thereby achieving accurate and efficient drug delivery, maximizing the efficacy, and reducing the adverse reactions caused by systemic administration.

[0003] Although lipid nanocarriers have shown significant advantages in drug delivery in the biomedical field, they still face challenges when loading hydrophilic therapeutic drugs. Broad-spectrum antibiotics are widely used in clinical practice, and systemic administration can lead to adverse gastrointestinal reactions and widespread drug resistance. The use of lipid nanocarriers for local delivery can solve the problem of systemic toxicity, but how to effectively encapsulate and deliver hydrophilic antimicrobial therapeutic drugs in lipid nanocarriers to achieve the accumulation of antimicrobial drugs at the target site and achieve the purpose of efficient treatment of biofilm infections is still a major challenge in the current technical field. Summary of the invention

[0004] To solve the above technical problems, the present invention proposes a lipid nanoassembly loaded with hydrophilic antibacterial drugs, a preparation method thereof, and an application in dissociating biofilms and treating peritonitis. The present invention utilizes a dynamic covalent boronate ester bond to connect phosphatidylcholine lipids and phenylboronic acid derivatives to form lipid molecules capable of self-assembly. These lipid molecules can self-assemble to form lipid nanocarriers, and achieve a high drug loading rate and encapsulation rate of hydrophilic drugs by adjusting the concentration of the hydrophilic drugs carried. In addition, due to protonation, redox reaction, and coordination reaction, triple responsive release of drugs at target sites can be achieved. Finally, a high loading rate of hydrophilic drugs is achieved, and the bioavailability of drugs is improved.

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

[0006] One of the technical solutions of the present invention:

[0007] A method for preparing a lipid nanoassembly loaded with a hydrophilic antibacterial drug comprises the following steps:

[0008] The phosphatidylcholine lipid and the phenylboronic acid derivative are respectively dissolved in a chloroform solution to obtain the corresponding mother solution, and the hydrophilic antibacterial drug is dissolved in ultrapure water;

[0009] The mother liquor of the above molecules is prepared by mixing phosphatidylcholine lipids and phenylboronic acid derivatives in a mass ratio of 10:1, performing rotary evaporation, vacuum drying, adding hydrophilic antibacterial drugs, ultrasonic vortexing, and extruding films to obtain lipid nanoassemblies loaded with hydrophilic antibacterial drugs;

[0010] The phenylboronic acid derivatives are obtained by the following method:

[0011] 1) A mixture of diphenyl phosphite (5.0 mmol, 1.0 equivalent), 1-n-decanol (10.0 mmol, 2.0 equivalent) and pyridine (0.5 mL) was distilled in a distillation apparatus at 140° C. under reduced pressure (4.10-2 Mbar) for 4 h, pyridine and phenol were removed by distillation, and dialkyl phosphite (yield> 94%) was obtained from the undistilled compound;

[0012] 2) N, N-dimethylethane-1,2-diamine (88.10 mg, 1 mmol, 1.0 equivalent) and (4-formylphenyl)boronic acid (149.94 mg, 1 mmol, 1.0 equivalent) were dissolved in methanol (3 mL), heated to 70° C. for 3 h under nitrogen protection, then evaporated at 30° C., didecyl phosphate (1 mmol, 1.0 equivalent) was added, and stirred at 100° C. for 1.5 h. The crude product was purified by silica gel column chromatography to obtain a brown solid phenylboronic acid derivative, whose structural formula is as shown in Formula I:

[0013]

[0014] Preferably, the phosphatidylcholine lipid is L-α-phosphatidylcholine, and the phenylboronic acid derivative is α-aminophosphonate containing phenylboronic acid, and the mother solution concentrations of both are 1 mg / mL.

[0015] Preferably, the hydrophilic antibacterial drug is ciprofloxacin with a concentration of 1 mg / mL.

[0016] Preferably, in the experimental steps, the mass ratio of L-α-phosphatidylcholine to the α-aminophosphonate containing phenylboronic acid is 10:1.

[0017] Preferably, the specific steps for preparing the lipid nanoassembly loaded with hydrophilic antibacterial drugs are as follows:

[0018] Use a 25mL round-bottom flask to absorb 1mL of PC and 100μL of PBA-Lipid. After mixing the two evenly, remove the chloroform by rotary evaporation at 50℃, and then place the round-bottom flask in a vacuum drying oven at 50℃ for 1h. Add 1mL of Cip (1mg / mL) under ultrasound, cover the bottle with a stopper, continue ultrasound-vortex-ultrasound for 30min, and then extrude 19 times with a 0.4μm membrane.

[0019] The second technical solution of the present invention:

[0020] The present invention also provides the use of the lipid nanoassembly loaded with hydrophilic antibacterial drugs in dissociating biofilms and treating peritonitis. The lipid nanoassembly loaded with hydrophilic antibacterial drugs of the present invention can be used to prepare drugs for preventing biofilm infection and treating peritonitis.

[0021] In clinical treatment, many broad-spectrum antibiotics are hydrophilic small molecule drugs. Oral administration has problems such as low oral bioavailability, severe gastrointestinal reactions, and systemic toxicity. At present, in the study of drug delivery in the field of biomedicine, hydrophilic small molecule drugs mainly have local drug delivery methods such as stents, spinning, and nanozymes. However, these methods all have the problem of low drug loading, so lipid nanocarriers with outstanding drug loading performance are selected. In summary, in the field of drug delivery, how to achieve efficient drug loading and targeted delivery of hydrophilic small molecule drugs is a key scientific problem that needs to be solved urgently. The focus of the present invention is on the high drug loading rate and responsive release treatment of hydrophilic small molecule drugs.

[0022] Compared with the prior art, the present invention has the following advantages and technical effects:

[0023] The present invention adopts dynamic covalent borate ester bonds to construct lipid molecules, and the lipid nanocarrier formed by hydrophilic and hydrophobic self-assembly has a unique structure that can efficiently encapsulate hydrophilic small molecule drugs. In addition, the lipid nanocarrier designed by the present invention is + Under the stimulation of a pathological environment with sufficient H2O2 and ATP, triple responsive release can be achieved, maximizing the therapeutic effect of antibacterial drugs and thus achieving efficient biofilm dissociation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0025] Figure 1 TEM images of the normal morphology and the triple-responsive morphology of the lipid nanoassembly containing α-aminophosphonate containing phenylboronic acid and capable of loading hydrophilic antibacterial drugs in Example 1 of the present invention.

[0026] Figure 2 The particle sizes of the lipid nanoassemblies capable of loading hydrophilic antibacterial drugs with and without α-aminophosphonates containing phenylboronic acid in Example 1 of the present invention under different pH environmental conditions, ATP-rich environmental conditions, and H2O2-rich environmental conditions.

[0027] Figure 3 The zeta potential of the lipid nanoassemblies capable of loading hydrophilic antibacterial drugs and containing or not containing α-aminophosphonates containing phenylboronic acid in Example 1 of the present invention under different pH environmental conditions, ATP-rich environmental conditions, and H2O2-rich environmental conditions.

[0028] Figure 4 It is the Young's modulus of the lipid nanoassembly capable of loading hydrophilic antibacterial drugs in Example 1 of the present invention after triple responsive stimulation.

[0029] Figure 5 This is the CLSM experimental result of lipid nanoassemblies capable of loading hydrophilic antibacterial drugs targeting bacteria in Example 2 of the present invention.

[0030] Figure 6 This is a flow cytometric analysis of lipid nanoassemblies capable of loading hydrophilic antibacterial drugs targeting bacteria in Example 2 of the present invention.

[0031] Figure 7 This is the QCM experimental result of lipid nanoassemblies capable of loading hydrophilic antibacterial drugs targeting bacteria in Example 2 of the present invention.

[0032] Figure 8 The figures show the drug loading capacity and encapsulation efficiency of the lipid nanoassembly capable of loading hydrophilic antibacterial drugs for different concentrations of hydrophilic ciprofloxacin in Example 3 of the present invention.

[0033] Fig. 9 It is the triple responsive drug release percentage of the lipid nanoassembly loaded with ciprofloxacin in Example 4 of the present invention.

[0034] Fig.10 4 is the bactericidal effect of the lipid nanoassembly in the dissociated biofilm in Example 4 of the present invention, wherein A is a fluorescent confocal image of live-dead staining of bacteria under different groups of drug treatments, and B is a SEM image of the morphological changes of bacteria under different drug treatments.

[0035] Fig.11This is a study on the antibacterial mechanism of the drug-loaded lipid nanoassembly in Example 4 of the present invention.

[0036] Fig.12 This is an evaluation of the therapeutic effect of the drug-loaded nanolipid assembly in the treatment of peritonitis in Example 4 of the present invention. The figure shows the bioluminescent fluorescence imaging of peritonitis mice after drug treatment.

[0037] Fig.13 This is an evaluation of the hemolysis rate of mouse erythrocytes by the drug-loaded lipid nanoassembly in Example 4 of the present invention. DETAILED DESCRIPTION

[0038] 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.

[0039] It should be understood that the terms described in the present invention are only for describing special embodiments 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. Each smaller range between the intermediate value in any stated value or stated range and 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.

[0040] Unless otherwise indicated, all technical and scientific terms used in the present invention have the same meanings as those generally understood by those skilled in the art. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described in the present invention 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 related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0041] 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 those skilled in the art. The present invention description and examples are exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in the present invention are open-ended terms, meaning including but not limited to.

[0043] Unless otherwise specified, the room temperature in the present invention is 25±2°C.

[0044] All raw materials and reagents used in the examples of the present invention were purchased from commercial sources. For example, L-α-phosphatidylcholine (1 mg / mL) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; ciprofloxacin (1 mg / mL) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.; and chloroform was purchased from Beijing Bailingwei Technology Co., Ltd.

[0045] The method for preparing a lipid nanoassembly loaded with a hydrophilic antibacterial drug provided by the present invention comprises the following steps:

[0046] The phosphatidylcholine lipid and the phenylboronic acid derivative are respectively dissolved in a chloroform solution to obtain the corresponding mother solution, and the hydrophilic ciprofloxacin antibiotic is dissolved in ultrapure water;

[0047] The mother liquor of the above-mentioned molecule is prepared by mixing phosphatidylcholine lipids and phenylboronic acid derivatives in a mass ratio of 10:1, performing rotary evaporation, and then vacuum drying. The hydrophilic antibacterial drug is then added and ultrasonically vortexed, and then subjected to film extrusion treatment to obtain a lipid nanoassembly loaded with the hydrophilic antibacterial drug.

[0048] In the embodiment of the present invention, the phenylboronic acid derivative is obtained by the following method:

[0049] 1) distilling a mixture of 5.0 mmol, 1.0 equivalent of diphenyl phosphite, 10.0 mmol, 2.0 equivalent of 1-n-decanol and 0.5 mL of pyridine in a distillation apparatus at 140° C. under reduced pressure of 4.10 to 2 Mbar for 4 h, removing pyridine and phenol by distillation, and obtaining dialkyl phosphite from the undistilled compound;

[0050] 2) 1 mmol, 1.0 equivalent of N, N-dimethylethane-1,2-diamine and 1 mmol, 1.0 equivalent of (4-formylphenyl)boric acid were dissolved in 3 mL of methanol, heated to 70° C. for 3 h under nitrogen protection, then evaporated at 30° C., 1 mmol, 1.0 equivalent of the dialkyl phosphite obtained in step 1) was added, stirred at 100° C. for 1.5 h, and the crude product was purified by silica gel column chromatography to obtain a phenylboronic acid derivative, whose structural formula is as shown in Formula I:

[0051]

[0052] The phosphatidylcholine lipid is L-α-phosphatidylcholine PC, the phenylboronic acid derivative is α-aminophosphonate PBA-Lipid containing phenylboronic acid; the hydrophilic antibacterial drug is ciprofloxacin Cip; and the organic solvent is chloroform CHCl3.

[0053] The technical solution of the present invention is further illustrated by the following embodiments.

[0054] Example 1

[0055] A method for preparing a lipid nanoassembly capable of loading a hydrophilic antibacterial drug,

[0056] The steps are as follows:

[0057] (1) Preparation of C-PBA-LNPs liposomes:

[0058] First, prepare the stock solutions of PC 1 mg / mL and PBA 1 mg / mL dissolved in chloroform. Use a 25 mL round-bottom flask to draw 1 mL of PC and 100 μL of PBA-Lipid. After mixing the two evenly, remove the chloroform by rotary evaporation at 50 °C. Then place the round-bottom flask in a vacuum drying oven at 50 °C for 1 h. Add 1 mL of PBS (10 mM) under ultrasound, cover the bottle with a stopper, and continue ultrasound-vortex-ultrasound for 30 min. Then, extrude 19 times using a 0.4 μm membrane.

[0059] (2) Preparation of C-LNPs liposomes:

[0060] The preparation process is the same as the preparation process of C-PBA-LNPs liposomes, except that no PBA solution is added during the preparation process.

[0061] (3) C-LNPs and C-PBA-LNPs liposomes in H + , H2O2, ATP environment stimulation, the corresponding lipid morphology changes are shown Figure 1 , and the corresponding liposome particle size, see Figure 2 ; Zeta potential, see Figure 3 .besides, Figure 4 This is the change in Young's modulus of the C-PBA-LNPs lipid nanoassembly prepared in this example under triple responsiveness.

[0062] The electron microscopy processing steps for liposomes are as follows:

[0063] 100 μL of C-PBA-LNPs (1 mg / mL) was added to phosphate buffer (PB) at pH 6.5 to prepare for transmission electron microscopy (TEM) analysis. Subsequently, 10 μL of ATP (500 mM) and 10 μL of H2O2 (10 mM) were added and incubated for 8 hours in a 1.5 mL EP tube. After incubation, 10 μL of the solution was dropped onto a copper grid.

[0064] H + The specific steps for particle size and potential after H2O2 and ATP environmental stimulation are as follows:

[0065] ATP treatment. 100 μL C-LNPs or C-PBA-LNPs (4 mg / mL) were mixed with 890 μL phosphate buffer (PB, 0.1 M) at pH 7.4 and 10 μL ATP (500 mM). The final concentration of NPs was 400 μg / mL and the final concentration of ATP was 5 mM.

[0066] Hydrogen peroxide treatment. 100 μL of C-LNPs or C-PBA-LNPs (4 mg / mL) was mixed with 890 μL of phosphate buffer (PB, 0.1 M) at pH 7.4 and 10 μL of H2O2 (10 mM). The final NP concentration was 400 μg / mL and the H2O2 concentration was 100 μM.

[0067] H + Treatment. 100 μL of C-LNPs or C-PBA-LNPs (4 mg / mL) was mixed with 900 μL of phosphate buffer (PB, 0.1 M) with a pH range of 5.5 to 7.4. The final NP concentration was 400 μg / mL.

[0068] H + , H2O2, ATP triple stimulation (pH 6.5 / ATP / H2O2) treatment. 100 μL C-LNPs or C-PBA-LNPs (4 mg / mL) were mixed with 880 μL phosphate buffer (PB, pH 6.5, 0.1 M), 10 μL H2O2 (10 mM) and 10 μL ATP (500 mM). The final NP concentration was 400 μg / mL, the H2O2 concentration was 100 μM, and the ATP concentration was 5 mM.

[0069] Example 2

[0070] The CLSM study of the interaction between bacteria and lipid nanoassemblies is as follows: 10 μL of logarithmic phase Staphylococcus aureus (3×10 8 CFU / mL) was dropped into a laser confocal microplate and allowed to stabilize for 15 min. 200 μL of C-PBA-LNPs (2 mg / mL) and Nile red (20 μg / mL) were mixed at a volume ratio of 1:1, and then 100 μL of the mixture was dropped into the bacterial solution and observed on a Nikon confocal laser microscope to image the bacteria. Figure 5The results of confocal fluorescence images showed that within 1 h, the ability of liposome nanoparticles containing α-aminophosphonates containing phenylboronic acid to target the bacterial surface was significantly better than that of liposome nanoparticles without α-aminophosphonates containing phenylboronic acid. Then, flow cytometry was further used to verify the interaction between bacteria and lipid nanocarriers. First, 200 μL C-PBA-LNPs (2 mg / mL) was drawn and 2 μL DiR (20 μg / mL) was added. After mixing evenly, it was used as the C-PBA-LNPs+DiR experimental group, and 1 mL S. aureus*WH was added. GFP (1×109 CFU / mL) bacterial solution was incubated at 37°C for 2 hours. Finally, flow cytometry was used to evaluate the binding ability of lipid nanoassemblies to bacteria. Figure 6 The results showed that C-PBA-LNPs+DiR+Sa WH GFP After the bacteria in the group were incubated with liposomes for 2 hours, the fluorescence co-localized in the Q2 region, and 76.2% of the liposomes loaded with phenylboronic acid targeted bacteria. This result showed that compared with other groups, it had better bacterial targeting and binding ability. Figure 7 The QCM experimental results for detecting the adhesion of liposome nanoassemblies to bacteria show that the adhesion of lipid nanoassemblies rich in phenylboronic acid to bacteria is enhanced, and the affinity and binding ability are also improved accordingly. The ability of the C-PBA-LNPs group to target bacteria is significantly improved compared with the C-LNPs group. The specific QCM operation steps for verifying the adhesion of lipid nanoassemblies are as follows: 200 μL S. aureus (1×10 9 CFU / mL inPBS), dripped on the chip and left to stand for 30min, then removed the liquid by spin coater. First, PBS solution was passed to establish a stable baseline, then C-LNPs (0.1mg / mL) and C-PBA-LNPs (0.1mg / mL) liposome nanoassemblies were connected, and the peristaltic pump flow rate was (50μL / min). When the value was balanced, PBS solution was passed again.

[0071] Staphylococcus aureus strain: provided by the First Affiliated Hospital of Wenzhou Medical University.

[0072] Example 3

[0073] A method for preparing a lipid nanoassembly loaded with hydrophilic ciprofloxacin antibacterial drug

[0074] C-PBA-Cip-LNPs liposomes (lipid nanoassemblies loaded with hydrophilic ciprofloxacin antibacterial drugs):

[0075] The preparation process is the same as that of C-PBA-LNPs liposomes, except that 1 mL of PBS (10 mM) is replaced with Cip (0.05-2 mg / mL) during the preparation process. The optimal concentration of Cip is determined based on the drug loading rate and encapsulation efficiency. The results are shown in Figure 8 When the Cip concentration is 1 mg / mL, the drug loading rate and encapsulation efficiency of C-PBA-Cip-LNPs liposomes are most ideal.

[0076] Example 4

[0077] Application of lipid nanoassemblies loaded with hydrophilic ciprofloxacin antibiotics

[0078] 1) A lipid nanoassembly loaded with hydrophilic ciprofloxacin antibacterial drug in H + Responsive release under / H2O2 / ATP environmental stimulation, the relevant method steps and effect verification are as follows:

[0079] The release of Cip from C-PBA-Cip-LNPs was determined by UV-visible absorption spectroscopy. First, a standard curve was established. The absorbance values ​​of Cip solutions (PH 7.4) with different concentrations at 280nm were measured to draw a standard curve.

[0080] The standard curve equation is: Y = 93.174*X-0.0534 (R 2 =0.9998,280nm)

[0081] To study the release of Cip from C-PBA-Cip-LNPs, a C-PBA-Cip-LNPs suspension (1 mL, 3 mg / mL) was placed in a dialysis bag (cut-off molecular weight: 500 Da) and soaked in PBS buffer (4 mL, 10 mM, pH 7.4 or 6.4, with or without 100 μM H2O2, 5 mM ATP). At different time intervals, 3.0 mL of the dialysate was collected and an equal amount of dialysate was added. The absorbance at 280 nm was recorded.

[0082] The equation for the zero-order model is

[0083] Mt=M0+K0t(1)

[0084] The first-order model considering burst release is expressed as

[0085] ln(Mt)=ln(M0)+K1t(2)

[0086] The Higuchi equation, which is simplified from Fick's law, is the best description of the diffusion-controlled transport system and is expressed as

[0087] Mt=KHt1 / 2(3)

[0088] Where Mt is the amount of drug released in time t, M0 is the initial amount of drug in the solution, K0 is the zero-order release rate constant, K1 is the first-order release rate constant, KH is the Higuchi model rate constant, and t is the release time. For the zero-order, first-order, and Higuchi models, the rate constant (K) and R2 were calculated from the plots of Mt / t, In(Mt) / t, or Mt / t1 / 2, respectively.

[0089] Cip release Fig. 9 As shown in the figure, when the C-PBA-Cip-LNPs suspension was in PBS (pH 7.4, containing only 100μM H2O2, or 5mM ATP), the release of Cip was only about 20%. However, when the C-PBA-Cip-LNPs suspension was in PBS (pH 6.5, containing 100μM H2O2, 5mM ATP), the release of Cip reached 60%, and the release amount was significantly improved. These results show that the lipid nanoassemblies loaded with hydrophilic ciprofloxacin antibacterial drugs have a good release rate in H + / H2O2 / ATP environment stimulation to release drugs responsively.

[0090] 2) The bactericidal effect of a lipid nanoassembly loaded with a hydrophilic ciprofloxacin antibacterial drug in a dissociated biomembrane, and the relevant method steps and effect verification are as follows:

[0091] Staphylococcus aureus (1×10 8 CFU / mL) were treated for 8 h according to the experimental groups. TM The samples were stained with 9 / PI fluorescent dye and observed on a Nikon confocal laser scanning microscope. SYTO was excited with a 488 nm argon ion laser TM 9 (green fluorescence), propidium iodide (red fluorescence) excited by 543 nm helium ion laser, and collected at 500-535 nm (SYTO TM 9) and fluorescence at 583-688 nm (PI). Fig.10 The results showed that the C-PBA-Cip-LNPs treatment group had the best bactericidal effect, with a bactericidal rate of up to 85%. At the same time, SEM was used to observe the morphological changes of bacteria in the corresponding treatment groups. The specific operation steps were as follows: a silicon wafer (L×W×H, 5×5×0.45mm 3 ) were placed in a 48-well plate and immersed in a bacterial suspension for 8 hours after various treatments to allow bacteria to adhere. They were then fixed with 4% paraformaldehyde fixative for 40 minutes and dehydrated with ethanol solutions of different concentrations for 20 minutes, with the ethanol concentrations being 30%, 50%, 70%, 90%, and 100% in sequence. The silicon wafers were dried at 4°C, sprayed with gold, and observed under SEM. The results are shown in Fig.10As shown in B, the bacterial surface of the C-PBA-Cip-LNPs group collapsed and shrank significantly, indicating that this group had the best bactericidal effect compared with other treatment groups, with bacterial membrane rupture, cell water loss or cytoplasm leakage, resulting in bacterial shrinkage. This experimental result is consistent with the bacterial live-dead staining results.

[0092] 3) A study on the bactericidal mechanism of a lipid nanoassembly loaded with hydrophilic ciprofloxacin antibacterial drug in dissociated biomembrane. The relevant method steps and effect verification are as follows:

[0093] 500 μL S.Xen36 (2×10 8 CFU / mL) was incubated with 500μl of different treatment group material solutions for 8h. PBS (pH=6.5,10mM), C-Cip-LNPs, Cip, C-PBA-Cip-LNPs (10μg / mL) were aspirated, and 10μL ATP (500mM) and 10μL H2O2 (10mM) were added. Four detection experiments of β-GAL, BCA, GPx, and DNase were carried out respectively. The experimental detection steps of β-GAL and BCA are as follows: centrifuge the bacterial solution of different components at 15000g, 4℃ for 10min, take the supernatant and place it on ice for testing, and then detect it with β-galactosidase detection kit and BCA detection kit respectively.

[0094] The experimental detection steps of GPx and DNase are as follows: the bacterial solution is ultrasonically disrupted with a 200w ultrasonic device for 3s, with an interval of 10s, repeated 30 times, 15000g, 4℃, centrifuged for 10min, the supernatant is placed on ice for testing, and then the glutathione peroxidase detection kit and DNase activity fluorescence detection kit are used for corresponding detection and analysis. The experimental results are as follows Fig.11 As shown in the figure, the results of the β-GAL and BCA detection experiments showed that after treatment with C-PBA-Cip-LNPs, the bacterial cell wall would be destroyed and the bacterial contents of β-GAL and BCA would leak out. When the bacteria were treated with C-PBA-Cip-LNPs, the bacteria would activate the protection mechanism accordingly. The results of the GPx detection kit showed that after being stimulated by liposome nanocarriers, the bacteria would produce oxidative stress damage and the GPx content would increase. The results of the DNase detection showed that the C-PBA-Cip-LNPs lipid nanoassembly would inhibit bacterial DNA replication and interfere with bacterial biosynthesis. The DNase content in the C-PBA-Cip-LNPs lipid nanoassembly treatment group was significantly lower than that in other treatment groups.

[0095] 4) A study on the application of a lipid nanoassembly loaded with hydrophilic ciprofloxacin antibacterial drug in vivo, using peritonitis as an in vivo research model. The relevant method steps and effect verification are as follows:

[0096] Peritonitis model establishment: ICR mice (female, 24-28g) were selected, and 300μL S.aureus Xen36 (1×10^8CFU / mL) was injected intraperitoneally to establish a peritonitis model. The mice were randomly divided into PBS, C-Cip-LNPs, Cip, and C-PBA-Cip-LNPs. After 2h, 100μL of the corresponding liquid was injected into the peritoneal cavity of the mice according to the group. The fluorescence changes of the bacteria from 0 to 24h were then detected by a mouse in vivo fluorescence imager. The experimental results are shown in Figure 2. Fig.12 As shown, after 24 hours of treatment, the fluorescence intensity of the C-PBA-Cip-LNPs treatment group was significantly lower than that of the other groups. After 8 hours of treatment, the fluorescence intensity of the C-PBA-Cip-LNPs treatment group decreased by 80%, and at 24 hours, basically no bioluminescence was detected. The results of this in vivo experiment show that the lipid nanoassembly loaded with hydrophilic ciprofloxacin antibiotics has a good in vivo anti-inflammatory effect. The lipid nanoassembly has a good in vivo antibacterial therapeutic effect on the one hand, and has good biosafety on the other hand. The biosafety evaluation of the lipid nanoassembly was carried out by hemolysis experiment, and the specific operation steps are as follows:

[0097] Take 1 mL of fresh mouse blood and add it to 9 mL of PBS and centrifuge at 4500 rpm for 5 min. After removing the supernatant, add 10 mL of PBS and gently blow and wash by centrifugation for 3 times until the supernatant becomes clear to separate the red blood cells. Then, resuspend the red blood cells in PBS (10 mL) to prepare a 4% red blood cell suspension and store it in a refrigerator at 4°C.

[0098] Evaluation of the hemolytic performance of the material: Add 500 μL of 4% red blood cell suspension to a 1.5 mL EP tube, then add 500 μL of the treatment group solution of different concentrations, mix, incubate at 37°C for 1 hour; centrifuge at 4500 rpm for 15 minutes, and measure the absorbance value at 545 nm using an enzyme reader. Red blood cells treated with Triton-X and PBS were used as positive and negative controls, respectively.

[0099] The results of hemolysis test were as follows Fig.13 As shown, the hemolysis rate was less than 5% at a concentration of 250 μg / mL, indicating that no serious hemolysis reaction would occur below this concentration and the drug had relatively good biosafety.

[0100] Based on the above description, Examples 1 and 3 of the present invention respectively provide C-LNPs liposomes, C-Cip-LNPs liposomes, C-PBA-LNPs liposomes, and methods for preparing C-PBA-Cip-LNPs liposomes, and verify the performance of the obtained liposomes and their effects on bacteria. The results show that the drug loading rate and encapsulation rate of the C-PBA-Cip-LNPs liposomes obtained by the present invention are the most ideal, and the bactericidal effect in the dissociated biofilm is more excellent.

[0101] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A method for preparing a lipid nanoassembly loaded with a hydrophilic antibacterial drug, characterized in that: The following steps are involved: The phosphatidylcholine lipid and the phenylboronic acid derivative are respectively dissolved in a chloroform solution to obtain the corresponding mother solution, and the hydrophilic ciprofloxacin antibiotic is dissolved in ultrapure water; The mother liquor of the above molecules is prepared by mixing phosphatidylcholine lipids and phenylboronic acid derivatives in a mass ratio of 10:1, performing rotary evaporation, vacuum drying, adding hydrophilic antibacterial drugs, ultrasonic vortexing, and extruding films to obtain lipid nanoassemblies loaded with hydrophilic antibacterial drugs; The phenylboronic acid derivatives are obtained by the following method: 1) distilling a mixture of 5.0 mmol, 1.0 equivalent of diphenyl phosphite, 10.0 mmol, 2.0 equivalent of 1-n-decanol and 0.5 mL of pyridine in a distillation apparatus at 140° C. under reduced pressure of 4.10 to 2 Mbar for 4 h, removing pyridine and phenol by distillation, and obtaining dialkyl phosphite from the undistilled compound; 2) 1 mmol, 1.0 equivalent of N, N-dimethylethane-1,2-diamine and 1 mmol, 1.0 equivalent of (4-formylphenyl)boric acid were dissolved in 3 mL of methanol, heated to 70° C. for 3 h under nitrogen protection, then evaporated at 30° C., 1 mmol, 1.0 equivalent of the dialkyl phosphite obtained in step 1) was added, stirred at 100° C. for 1.5 h, and the crude product was purified by silica gel column chromatography to obtain a phenylboronic acid derivative, whose structural formula is as shown in Formula I:

2. The method for preparing a lipid nanoassembly loaded with a hydrophilic antibacterial drug according to claim 1, characterized in that: The phosphatidylcholine lipid is L-α-phosphatidylcholine PC, the phenylboronic acid derivative is α-aminophosphonate PBA-Lipid containing phenylboronic acid; the hydrophilic antibacterial drug is ciprofloxacin Cip; and the organic solvent is chloroform CHCl3.

3. The preparation method according to claim 1, characterized in that: The mother solution concentration of the phosphatidylcholine lipid is 1 mg / mL.

4. The preparation method according to claim 1, characterized in that: The concentration of the mother solution containing the phenylboronic acid derivative is 1 mg / mL.

5. The preparation method according to claim 1, characterized in that: The mother solution concentration of the hydrophilic antibacterial drug is 1 mg / mL.

6. The preparation method according to claim 1, characterized in that: The rotary evaporation temperature was 50°C, the vacuum drying time was 1 h, and the temperature was 50°C.

7. The preparation method according to claim 1, characterized in that: The ultrasonic vortexing time was 30 min.

8. The preparation method according to claim 1, characterized in that: The membrane used for extruding the liposomes was 0.4 μm and the number of extrusions was 19.

9. A lipid nanoassembly loaded with a hydrophilic antibacterial drug, prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the lipid nanoassembly loaded with a hydrophilic antibacterial drug according to claim 9 in the preparation of a drug for dissociating biofilms and treating peritonitis.

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