Protein-loaded lipid nano assembly as well as preparation method and application thereof

Abstract

The invention discloses a protein-loaded lipid nano assembly as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The protein-loaded lipid nano-assembly is spongy lipid molecules generated by combining catechol-containing alpha-amino phosphonate derivative lipid and phenylboronic acid derivatives through dynamic covalent borate bonds, the lipid molecules are self-assembled to form a lipid nano-carrier, and the lipid nano-assembly comprises a water phase region and an oil phase region, the water-phase region can load protein with high loading efficiency, and the oil-phase region can load hydrophobic small-molecule drugs with high drug loading capacity through dynamic covalent bonds. By screening different phenylboronic acid derivatives, the structure and the property of the lipid nano-carrier are optimized, the interaction between the lipid nano-carrier and the protein is regulated and controlled, and the loading capacity and the protection efficiency of the protein are remarkably improved. Under a slightly acidic environment and oxidative stimulation, the lipid nano assembly prepared by the invention can controllably release different drug molecules and protein drugs to achieve the purpose of collaborative treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a protein-loaded lipid nanoassembly, a preparation method and an application thereof. Background Art

[0002] As a highly adjustable drug delivery system, lipid nanocarriers have received extensive attention in the biomedical field in recent years and have been widely used in the treatment of various diseases such as cancer, neurological diseases, infectious diseases and uncontrolled inflammation. The carrier is composed of lipid molecules and can form a stable nanostructure. By carefully designing its structure, lipid nanocarriers can significantly enhance the ability to penetrate biological barriers (such as cell membranes or bacterial biomembranes), thereby improving the interaction with target cells or pathogens. In addition, by adjusting the morphology of the nanostructure, the release behavior of the drug, the efficiency of cell uptake and the ability to penetrate the biomembrane can be further regulated, thereby achieving more accurate and effective drug delivery.

[0003] Although lipid nanocarriers have shown significant advantages in many aspects, they still face challenges when loading protein therapeutics. Protein therapeutics need to be used in combination with chemotherapeutic drugs in the treatment of many diseases. However, due to the lipophilicity of lipid molecules, these nanostructures usually form micelles or solid nanoparticles, and their hydrophobic cores are not conducive to the stable loading of protein drugs. Protein drugs need to maintain their structural stability and functionality in an aqueous environment, while the hydrophobic properties of lipid nanocarriers may cause proteins to denature, aggregate or degrade during the loading process, weakening their therapeutic effects. In addition, the size and structure of lipid nanocarriers may make it difficult to provide sufficient space or suitable conformations for larger protein molecules, resulting in low loading efficiency and may cause the protein to lose activity after encapsulation. Therefore, how to effectively encapsulate and deliver protein therapeutics in lipid nanocarriers to achieve synergistic treatment of biofilm infections remains a major challenge in the current technical field. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a protein-loaded lipid nanoassembly and its preparation method and application. The present invention uses a dynamic covalent boronate ester bond to connect catechol-containing α-aminophosphonate-derived lipids and phenylboronic acid derivatives to form lipid molecules capable of self-assembly. These lipid molecules can self-assemble to form lipid nanocarriers, and the porous structure and related properties (charge, critical filling parameters and interface area) of the lipid nanocarriers can be regulated by screening different phenylboronic acid derivatives. With the help of electrostatic interaction, π-π stacking, N→B coordination, and cation π interaction, the lipid nanocarrier can efficiently load protein drugs, thereby improving the encapsulation effect and protection efficiency of protein drugs, and ultimately achieving synergistic treatment.

[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 protein-loaded lipid nanoassembly comprises the following steps:

[0008] The lipid molecule is formed by combining the catechol-containing α-aminophosphonate-derived lipid and the phenylboronic acid derivative through a dynamic covalent borate ester bond. The structural formula of the lipid molecule is shown in Formula I:

[0009]

[0010] In Formula I, R is a nitrogen-containing heterocyclic compound, including 1-ethyl-1H-imidazole, pyridine, ketoconazole, ciprofloxacin or itaconazole;

[0011] The lipid molecules are dissolved in an organic solvent, concentrated, a protein therapeutic drug dissolved in a phosphate buffer is added, ultrasonic vibration is performed, centrifugation is performed, the supernatant is discarded, and the solid is resuspended with a phosphate buffer to obtain a protein-loaded lipid nanoassembly.

[0012] The protein-loaded lipid nanoassembly of the present invention is a sponge-like lipid molecule generated by combining a catechol-containing α-aminophosphonate-derived lipid with a phenylboronic acid derivative through a dynamic covalent boronate ester bond. The lipid molecules self-assemble to form a lipid nanocarrier, which includes two regions, an aqueous phase and an oil phase. The aqueous phase region can load proteins with high loading efficiency, while the oil phase region can load hydrophobic small molecule drugs with high drug loading through dynamic covalent bonds. By screening different phenylboronic acid derivatives, optimizing the structure and properties of lipid nanocarriers, regulating the interaction between them and proteins, and significantly improving the loading amount and protection efficiency of proteins. Under a slightly acidic environment and oxidative stimulation, the lipid nanoassembly prepared by the present invention can controllably release different drug molecules and protein drugs to achieve the purpose of synergistic treatment.

[0013] Preferably, the step of combining the catechol-containing α-aminophosphonate-derived lipid and the phenylboronic acid derivative through a dynamic covalent borate ester bond to form a lipid molecule is: dissolving the catechol-containing α-aminophosphonate-derived lipid and the phenylboronic acid derivative in chloroform, and reacting them at 45°C for 12 hours, and obtaining the lipid molecule through the connection of the dynamic covalent borate ester bond.

[0014] Preferably, the molar ratio of the catechol-containing α-aminophosphonate-derived lipid to the phenylboronic acid derivative is 1:1.

[0015] Preferably, the protein therapeutic drug comprises DNase protein (deoxyribonuclease), RNase protein (ribonuclease), HRP protein (horseradish peroxidase) or BSA protein (bovine serum albumin).

[0016] Preferably, the method for preparing the catechol-containing α-aminophosphonate-derived lipid comprises the following steps:

[0017] Dissolve diphenylphosphite and 1-decanol in pyridine, react at 130-150°C for 4 hours to obtain decanyl phosphate; mix N,N-diethylethylenediamine and 3,4-dihydroxybenzaldehyde in methanol under a nitrogen atmosphere, react at 65-75°C for 3 hours, and then add the decanyl phosphate after vacuum drying, and react at 100-110°C for 1.5 hours to obtain the catechol-containing α-aminophosphonate-derived lipid, whose structural formula is shown in Formula II:

[0018]

[0019] Preferably, in the method for preparing catechol-containing α-aminophosphonate-derived lipids, the molar ratio of diphenylphosphite to 1-decanol is 1:2;

[0020] The molar ratio of the N,N-diethylethylenediamine to 3,4-dihydroxybenzaldehyde is 1:1.

[0021] More preferably, the preparation method of the catechol-containing α-aminophosphonate-derived lipid having an alkyl chain length of 10 is:

[0022] 50mmol of diphenylphosphite, 100mmol of 1-decanol and 50mL of pyridine were added to a round-bottom flask and reacted at 140°C for 4h. After the reaction, pyridine and by-product phenol were removed by reduced pressure distillation to obtain a white solid decanyl phosphate. Subsequently, 2mmol of N,N-diethylethylenediamine and 2mmol of 3,4-dihydroxybenzaldehyde were mixed under nitrogen protection, 6mL of methanol was added, and the reaction was carried out at 70°C for 3h. After the reaction was completed, the solvent was dried under reduced pressure at 30°C, and then 2mmol of decanyl phosphate was quickly added and reacted at 100°C for 1.5h. Finally, the product was separated and purified by a silica gel chromatography column, and a mixed solution of methanol and dichloromethane with a volume ratio of 1:30, 1:20, and 1:10 was used as an eluent for elution, and finally a catechol-containing α-aminophosphonate-derived lipid with an alkyl chain length of 10 was obtained.

[0023] Preferably, the structural formula of the phenylboronic acid derivative is as follows:

[0024]

[0025] Preferably, the preparation method of ketoconazole phenylboronic acid derivative comprises the following steps:

[0026] 1 mmol of ketoconazole and 1 mmol of 4-(bromomethyl)phenylboronic acid were dissolved in 15 mL of acetonitrile, and the mixture was reacted at 73° C. overnight. After the reaction was completed, the reaction solvent was removed by reduced pressure distillation to obtain a crude product. Finally, the crude product was purified by silica gel column chromatography to obtain a ketoconazole phenylboronic acid derivative, i.e., phenylboronic acid-modified ketoconazole (Ket-PBA), which was a white solid. The synthesis route is as follows:

[0027]

[0028] Preferably, the preparation method of ciprofloxacin phenylboronic acid derivative comprises the following steps:

[0029] 1 mmol of ciprofloxacin was added to 8 mL of a mixed solution of DMF (dimethylformamide) and water (the volume ratio of DMF to water was 2:1), 3 mmol of potassium carbonate was added and dissolved by ultrasound, and then 1.2 mmol of 4-(bromomethyl)phenylborane was added, and stirred overnight. The reaction progress was monitored by thin layer chromatography. After the reaction was completed, the pH of the system was adjusted to neutral with 1 M hydrochloric acid solution to precipitate the product, which was filtered and dried to obtain a white solid, which was a ciprofloxacin phenylboronic acid derivative. The synthesis route is as follows:

[0030]

[0031] Preferably, the preparation method of the pyridinephenylboronic acid derivative comprises the following steps:

[0032] 1 mmol of pyridine and 1 mmol of 4-(bromomethyl)phenylboronic acid were dissolved in 15 mL of acetonitrile and reacted at 70°C overnight. The precipitate obtained by the reaction was filtered, washed with dimethyl ether, and dried to finally obtain a pyridinephenylboronic acid derivative as a white solid. The synthetic route is as follows:

[0033]

[0034] Preferably, the organic solvent includes chloroform, ethanol, methanol or acetonitrile, more preferably chloroform;

[0035] The pH of the phosphate buffer is 7.2 to 8.2, and more preferably the pH is 7.4.

[0036] Preferably, the temperature of the ultrasonic oscillation is 35-45° C., and the time of the ultrasonic oscillation is 5-20 min. More preferably, the temperature of the ultrasonic oscillation is 45° C., and the time of the ultrasonic oscillation is 10 min.

[0037] The second technical solution of the present invention:

[0038] The present invention also provides a protein-loaded lipid nanoassembly prepared according to the above preparation method. The protein-loaded lipid nanoassembly of the present invention can effectively load and protect DNase protein, and can realize responsive release of drugs under the stimulation of a slightly acidic / oxidative environment.

[0039] The third technical solution of the present invention:

[0040] The present invention also provides the use of the protein-loaded lipid nanoassembly in dissociating biofilms. The protein-loaded lipid nanoassembly of the present invention can be used to prepare drugs for treating biofilm infections.

[0041] In clinical treatment, protein drugs are the first choice for treating multiple drugs. In the field of drug delivery, how to achieve the co-delivery of protein drugs and small molecule drugs is a key scientific problem that needs to be solved urgently. The focus of the present invention is on the synergistic loading and treatment of small molecules and protein drugs. In biofilms, eDNA and extracellular proteins can serve as bacterial connectors to form biofilm aggregates. Dissociation of eDNA can effectively dissociate EPS (extracellular polymer matrix) and promote the therapeutic effect of antibiotics.

[0042] The fourth technical solution of the present invention:

[0043] The present invention also provides the use of the protein-loaded lipid nanoassembly in the preparation of antifungal drugs.

[0044] Compared with the prior art, the present invention has the following advantages and technical effects:

[0045] 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 porous structure, and the porous structure and related properties of the lipid nanocarrier can be regulated by screening different phenylboronic acid derivatives. The porous structure formed by this lipid nanocarrier can load different types of proteins by means of electrostatic interaction, π-π stacking, N→B coordination, and cationic π interaction, and also enhances the interaction force between the lipid nanocarrier and the protein, greatly improving the encapsulation effect and protection efficiency of the protein. In addition, the lipid nanocarrier designed by the present invention can achieve responsive release under slightly acidic environment and oxidative stimulation, so that antibacterial drugs and protein drugs can work synergistically and effectively dissociate biofilms. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] 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:

[0047] Figure 1Schematic diagrams of the assembly of liposomes in Example 1 of the present invention, A is a schematic diagram of the assembly of liposomes in an aqueous phase, and B is a schematic diagram of the assembly of liposomes in an aqueous phase containing protein.

[0048] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the catechol-containing α-aminophosphonate-derived lipid in Example 1 of the present invention.

[0049] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of ketoconazole (Ket-PBA) modified with phenylboronic acid in Example 1 of the present invention.

[0050] Figure 4 This is the NMR boron spectrum of lipid molecule I formed by ketoconazole modified with phenylboronic acid in Example 1 of the present invention.

[0051] Figure 5 This is a schematic diagram of the synthesis of lipid molecule I formed by ketoconazole modified with phenylboronic acid in Example 1 of the present invention.

[0052] Figure 6 is the hydrated particle size of the assemblies formed by different nitrogen-containing heterocyclic compounds 1-ethyl-1H-imidazole, pyridine, ketoconazole, ciprofloxacin and itaconazole.

[0053] Figure 7 This is a data diagram of particle size distribution of lipid nanoassemblies formed by lipid molecules I loaded with different proteins in the present invention.

[0054] Figure 8 It is the encapsulation rate of different proteins of the lipid nanocarrier formed by self-assembly of lipid molecules I in the present invention.

[0055] Fig. 9 This is a transmission electron microscopy image of the D-LPNs prepared in Example 1 of the present invention.

[0056] Fig.10 Schematic diagram of the structure of D-LPNs prepared in Example 1 of the present invention.

[0057] Fig.11 The relative activity data of DNase before and after protease treatment in Example 1 of the present invention.

[0058] Fig.12 The graph is a drug release curve of the D-LPNs prepared in Example 1 of the present invention under different stimulation conditions, wherein A is the release rate of ketoconazole under different stimulation conditions, and B is the release rate of DNase under different stimulation conditions.

[0059] Fig.13The figure is the effect of D-LPNs prepared in Example 1 of the present invention on dissociating biofilms, wherein A is a laser confocal 3D imaging image of biofilms with different treatments after live or dead staining, B is a fluorescence photo of bacteria at 6 μm from the bottom of the biofilm, and different colors show different fluorescence intensities, C is the fluorescence intensity result at different biofilm thicknesses according to the laser confocal imaging results, D is the biomass content after different treatments according to the laser confocal imaging results, E is the average biofilm thickness after different treatments according to the laser confocal imaging results, F is the roughness coefficient after different treatments according to the laser confocal imaging results, G is the colony count of surviving bacteria in the biofilm after different treatments, and H is the relative biomass in the biofilm calculated by crystal violet staining.

[0060] Fig.14 The gel electrophoresis images of eDNA in the biofilm of the D-LPNs prepared in Example 1 of the present invention after the biofilm was dissociated, wherein A is a photo of the gel electrophoresis after different treatments, and B is the statistical result of the gel electrophoresis intensity after different treatments. DETAILED DESCRIPTION

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0066] Unless otherwise specified, the room temperature in the present invention is 25±2°C.

[0067] All raw materials and reagents used in the examples of the present invention were purchased from commercial sources. For example, DNase solution (1 mg / mL) was purchased from Beyotime Biotechnology Co., Ltd.; RNase solution (1 mg / mL) was purchased from Beyotime Biotechnology Co., Ltd.; HRP solution (1 mg / mL) was purchased from Beyotime Biotechnology Co., Ltd.; and BSA solution (1 mg / mL) was purchased from Beyotime Biotechnology Co., Ltd.

[0068] The technical solution of the present invention is further illustrated by the following embodiments.

[0069] Example 1

[0070] A method for preparing a lipid nanoassembly loaded with DNase protein, the assembly diagram of the liposome is as follows Figure 1 As shown (A is a schematic diagram of liposome assembly in an aqueous phase, and B is a schematic diagram of liposome assembly in an aqueous phase containing protein), the steps of the method are as follows:

[0071] 1) Preparation of catechol-containing α-aminophosphonate-derivatized lipids

[0072] 50mmol of diphenylphosphite, 100mmol of 1-decanol and 50mL of pyridine were added to a round-bottom flask and reacted at 140℃ for 4h. After the reaction, pyridine and by-product phenol were removed by vacuum distillation to obtain a white solid decanyl phosphate with a yield of 94%. Subsequently, 2mmol of N,N-diethylethylenediamine and 2mmol of 3,4-dihydroxybenzaldehyde were mixed under nitrogen protection, 6mL of methanol was added, and the reaction was carried out at 70℃ for 3h. After the reaction was completed, the solvent was vacuum-dried at 30℃, and then 2mmol of decanyl phosphate was quickly added and reacted at 100℃ for 1.5h. Finally, it was separated and purified by silica gel chromatography, and a mixed solution of methanol and dichloromethane with a volume ratio of 1:30, 1:20, and 1:10 was used as the eluent for elution, and finally a catechol-containing α-aminophosphonate-derived lipid with an alkyl chain length of 10 was obtained, which was a brown solid with a yield of 50%. The H NMR spectrum of catechol-containing α-aminophosphonate-derivatized lipids is shown in Figure 2 It can be seen that the present invention successfully synthesized catechol-containing α-aminophosphonate-derived lipids. The specific synthesis route is as follows:

[0073]

[0074] 2) Preparation of phenylboronic acid derivatives

[0075] The preparation method of ketoconazole phenylboronic acid derivatives comprises the following steps:

[0076] 1 mmol of ketoconazole and 1 mmol of 4-(bromomethyl)phenylboronic acid were dissolved in 15 mL of acetonitrile and reacted at 73 ° C overnight. After the reaction was completed, the reaction solvent was removed by reduced pressure distillation to obtain a crude product. Finally, the crude product was purified by silica gel column chromatography to finally obtain a ketoconazole phenylboronic acid derivative, namely phenylboronic acid-modified ketoconazole (Ket-PBA), which was a white solid with a yield of 63.2%. The nuclear magnetic resonance hydrogen spectrum of Ket-PBA is shown in Figure 3 , it can be seen that the present invention successfully synthesized ketoconazole phenylboronic acid derivatives. The specific synthesis route is as follows:

[0077]

[0078] The preparation method of ciprofloxacin phenylboronic acid derivatives comprises the following steps:

[0079] 1mmol of ciprofloxacin was added to 8mL of a mixed solution of DMF (dimethylformamide) and water (the volume ratio of DMF to water was 2:1), 3mmol of potassium carbonate was added and ultrasonically dissolved, and then 1.2mmol of 4-(bromomethyl)phenylborane was added, stirred overnight, and the reaction progress was monitored by thin layer chromatography. After the reaction was completed, the pH of the system was adjusted to neutral with 1M hydrochloric acid solution to precipitate the product, which was filtered and dried to obtain a white solid with a yield of 60.0%. The specific synthetic route is as follows:

[0080]

[0081] The preparation method of pyridine phenylboronic acid derivatives comprises the following steps:

[0082] 1 mmol of pyridine and 1 mmol of 4-(bromomethyl)phenylboronic acid were dissolved in 15 mL of acetonitrile and reacted at 70°C overnight. The precipitate obtained by the reaction was filtered, washed with dimethyl ether, and dried to finally obtain a pyridinephenylboronic acid derivative as a white solid with a yield of 57.3%. The specific synthetic route is as follows:

[0083]

[0084] 3) Preparation of lipid molecules

[0085] 5 μmol of ketoconazole phenylboronic acid derivative and 5 μmol of catechol-containing α-aminophosphonate-derived lipid with an alkyl chain length of 10 were dissolved in 1 mL of chloroform and reacted at 45°C for 12 h. Lipid molecule I (ketoconazole-liposome) with an alkyl chain length of 10 was obtained through the connection of dynamic covalent boronate bonds. Figure 4 The NMR boron spectrum of provides a basis for the formation of lipid molecule I. The specific synthesis diagram is as follows Figure 5 shown.

[0086] 5 μmol of ciprofloxacin phenylboronic acid derivative and 5 μmol of catechol-containing α-aminophosphonate-derived lipid with an alkyl chain length of 10 were dissolved in 1 mL of chloroform and reacted at 45°C for 12 h. Lipid molecule II (ciprofloxacin-liposome) with an alkyl chain length of 10 was obtained through the connection of dynamic covalent boronate bonds.

[0087] 5 μmol of pyridine phenylboronic acid derivative and 5 μmol of catechol-containing α-aminophosphonate-derived lipid with an alkyl chain length of 10 were dissolved in 1 mL of chloroform and reacted at 45°C for 12 h. Lipid molecule III (pyridine-liposome) with an alkyl chain length of 10 was obtained through the connection of dynamic covalent boronate bonds.

[0088] Liposomes formed by nitrogen-containing heterocyclic compounds 1-ethyl-1H-imidazole, itaconazole and triethylamine were also prepared in the above manner to obtain liposomes (respectively denoted as 1-ethyl-1H-imidazole-liposomes, itaconazole-liposomes and triethylamine-liposomes), and their hydration radii were measured using a particle size analyzer, as shown in FIG. Figure 6 As shown, it is proved that each of the above nitrogen-containing heterocyclic compounds can form liposomes with a particle size of about 200 nm.

[0089] 4) Preparation of lipid nanoassemblies loaded with DNase protein

[0090] 5 μmol of lipid molecule I with an alkyl chain length of 10 was dissolved in 1 mL of chloroform, and then further concentrated to 200 μL, and 5 mL of DNase solution (1 mg / mL) dissolved in pH = 7.4 phosphate buffer was added, and ultrasonic oscillation was performed at 45°C for 10 min. Subsequently, centrifugation was performed (20000g, 2h), and the supernatant was discarded and resuspended in pH = 7.4 phosphate buffer to obtain lipid nanoassemblies (D-LPNs) loaded with DNase protein. Figure 7 As shown in Figure 1, dynamic light scattering results show that the particle size is around 266nm. Figure 8 As shown, the encapsulation efficiency of the lipid nanoassembly loaded with DNase protein was 65%. Fig. 9 This is a transmission electron micrograph of the lipid nanoassembly loaded with DNase protein prepared in this example. Fig.10 Schematic diagram of the structure of lipid nanoassembly loaded with DNase protein.

[0091] Example 2

[0092] A method for preparing a lipid nanoassembly loaded with RNase protein

[0093] The preparation process of Example 2 is the same as that of Example 1, except that the DNase solution (1 mg / mL) is replaced by the RNase solution (1 mg / mL) to obtain the RNase protein-loaded lipid nanoassemblies (R-LPNs). Figure 7 As shown in Figure 1, dynamic light scattering results show that the particle size is around 255nm. Figure 8 As shown, the encapsulation efficiency of the lipid nanoassembly loaded with RNase protein was 52%.

[0094] Example 3

[0095] A method for preparing lipid nanoassembly loaded with HRP protein

[0096] The preparation process of Example 3 is the same as that of Example 1, except that the DNase solution (1 mg / mL) is replaced by the HRP solution (1 mg / mL) to obtain lipid nanoassemblies (H-LPNs) loaded with HRP protein. Figure 7 As shown in Figure 1, dynamic light scattering results show that the particle size is around 295nm. Figure 8 As shown, the encapsulation efficiency of the HRP protein-loaded lipid nanoassemblies was 60%.

[0097] Example 4

[0098] A method for preparing lipid nanoassembly loaded with BSA protein

[0099] The preparation process of Example 2 is the same as that of Example 1, except that the DNase solution (1 mg / mL) is replaced by the BSA solution (1 mg / mL) to obtain BSA protein-loaded lipid nanoassemblies (B-LPNs). Figure 7 As shown in Figure 1, dynamic light scattering results show that the particle size is around 220nm. Figure 8 As shown, the encapsulation efficiency of BSA was 62%.

[0100] Example 5

[0101] Application of lipid nanoassemblies loaded with DNase protein

[0102] 1) A lipid nanoassembly loaded with DNase protein effectively loads and protects DNase protein. The relevant methods and effects are verified as follows:

[0103] The DNase protein-loaded lipid nanoassemblies (D-LPNs) and DNase alone were exposed to proteinase K (10 μg / mL) for different time periods (0.5 h, 1 h, and 2 h), and then the DNase in the D-LPNs was stimulated and released responsively by adding H2O2 (100 μM). Finally, the DNase enzyme activity in the two groups was detected using a DNase enzyme activity fluorescence kit.

[0104] The results are as follows Fig.11 As shown, when D-LPNs were treated with protease, the activity of DNase proteinase retained was significantly higher than that of free DNase proteinase, indicating that this lipid molecule self-assembly can be used to form lipid nanocarriers to achieve effective loading and protection of DNase protein.

[0105] 2) A lipid nanoassembly loaded with DNase protein is responsively released under the stimulation of a slightly acidic / oxidative environment. The relevant method steps and effect verification are as follows:

[0106] 5 mL of lipid nanoassembly (D-LPNs) loaded with DNase protein was mixed with 5 mL of phosphate buffer (pH=7.4), phosphate buffer (pH=6.5), phosphate buffer (pH=7.4) containing H2O2 (100 μM), and phosphate buffer (pH=6.5) containing H2O2 (100 μM). 0.5 mL of suspension was taken out at different time points of 1, 2, 3, 4, 8, 12, 16, 20, and 24 h, and the volume of the solution was supplemented to 10 mL with the corresponding buffer after each taking out. The absorbance value at 245 nm of the taken out suspension was measured by ultraviolet spectrophotometer to detect the release of ketoconazole. At the same time, the release of DNase protein was detected by BCA protein assay kit.

[0107] The results are as follows Fig.12 As shown in the figure, when D-LPNs were exposed to phosphate buffer (pH = 7.4), the release of ketoconazole and DNase was relatively slow, with a release rate of less than 20% within 24 hours. When D-LPNs were exposed to a slightly acidic / oxidative environment, the release of ketoconazole and DNase increased significantly. In particular, when D-LPNs were exposed to a phosphate buffer (pH = 6.5) containing H2O2 (100μM), the release rate of ketoconazole and DNase was the highest, with more than 90% of ketoconazole and DNase released. These results indicate that lipid nanoassemblies loaded with DNase proteins can responsively release drugs under the stimulation of a slightly acidic / oxidative environment.

[0108] 3) The effect of lipid nanoassemblies loaded with DNase protein on dissociating biomembranes, the relevant steps and effect verification are as follows:

[0109] 800 μL of Candida albicans suspension (2×10 8CFU / mL) was added to the confocal dish and incubated at 37°C for 1h to ensure that Candida albicans could effectively attach to the bottom of the dish. Subsequently, the supernatant was gently removed and washed 3 times with PBS. Next, 800μL BHI medium was added and the dish was placed in a 37°C incubator for 24h. After the incubation, it was replaced with a new medium and continued to be incubated in a 37°C incubator for 24h to obtain a mature Candida albicans biofilm. After that, the obtained Candida albicans biofilm was washed 3 times with PBS, and then 800μL of different materials (PBS, DNase, ketoconazole, LPNs, D-LPNs; ketoconazole concentration was 0.45mg / mL or / and DNase concentration was 0.5mg / mL) were added to different dishes. After continuing to incubate in a 37°C incubator for 6h, the supernatant was removed and gently washed 3 times with PBS. Then, AO (400 μL, 1 mg / mL) and EB (400 μL, 1 mg / mL) were added and stained for 10 min. The thickness and growth of the biofilm of Candida albicans were observed by laser confocal microscopy. At the same time, after the corresponding treatment, crystal violet solution (100 μL, 0.20 mg / mL) was added to each well and incubated at room temperature for 20 min. After that, the crystal violet solution was removed and washed 3 times with PBS. Then, 90% ethanol (100 μL) was added to dissolve the purple solid, and the ultraviolet absorbance at 570 nm was measured by an enzyme marker to quantify the biomass of Candida albicans biofilm by crystal violet staining. In addition, the biofilms treated with different materials were collected in a 1.5 mL centrifuge tube, ultrasonically treated for 5-6 s, and centrifuged at 5000 rpm for 10 min. 10 μL of the supernatant was taken to quantify the eDNA in the biofilms of different treatment groups by agarose gel electrophoresis.

[0110] Candida albicans strains: provided by the First Affiliated Hospital of Wenzhou Medical University.

[0111] The results are as follows Fig.13 As shown, compared with other treatment groups, the Candida albicans biofilm treated with D-LPNs showed significantly reduced fluorescence intensity, reduced biomass, thinner biofilm thickness and increased roughness, and the remaining microbial cells in the film were significantly reduced. CFU counts further confirmed that free DNase, ketoconazole, and physically mixed DNase and ketoconazole had little effect on the viability of Candida albicans biofilms, mainly due to their poor penetration into the biofilm matrix. In contrast, LPNs treatment resulted in a 99% sterilization rate of Candida albicans beads in the biofilm, which was further increased to 99.9% after D-LPNs treatment. At the same time, crystal violet staining showed that more than 75% of the Candida albicans biofilms were eradicated after D-LPNs treatment, which was significantly better than other treatment groups. In addition, as Fig.13As shown in the figure, Ket and LPNs treatment had no significant effect on the eDNA fragmentation of Candida albicans biofilm, while the eDNA of Candida albicans biofilm treated with D-LPNs was significantly reduced, indicating that D-LPNs can effectively fragment the eDNA in the biofilm, reduce the adhesion of the biofilm, promote the penetration of ketoconazole in the Candida albicans biofilm and exert an antibacterial effect. Overall, these results show that protein-loaded lipid nanoassemblies (D-LPNs) can combine ketoconazole and DNase to achieve a synergistic therapeutic effect on Candida albicans biofilm.

[0112] To further verify that liposome D-LPNs loaded with DNase and ketoconazole can effectively dissociate eDNA in biofilms, thereby achieving a synergistic therapeutic effect of dissociating C. albicans biofilms. Mature C. albicans biofilms grown for 48 hours in 24-well plates were washed three times with PBS 200 μL and then further incubated with 200 μL Ket, DNase I, LPN, DNase+Ket (D+K) and D-LPNs, with a Ket concentration of 0.45 mg / mL and a DNase concentration of 0.50 mg / mL for 6 h. Afterwards, they were washed three times with PBS 200 μL to remove the nanospheres and dissociated biofilms. The biofilm on the left was broken and separated with PBS 200 μL and collected in a 1.5 mL centrifuge tube. Then, they were briefly exposed to ultrasonic treatment for 5-6 seconds and centrifuged at 5000 rpm for 10 minutes. Then, 10 μL of the supernatant was extracted and deposited into the wells of a 1% agarose gel (composed of 0.75 g agarose and 750 mL TAE buffer). The gel was then run at 220 V for 30 minutes and observed using a UV transilluminator.

[0113] The results are as follows Fig.14 As shown, the C. albicans biofilm treated with D-LPNs showed significantly reduced eDNA content compared with other treatment groups, demonstrating that loading DNase in liposomes can effectively reduce eDNA in biofilms. Free DNase, as well as physically mixed DNase and Ket, had limited effects on reducing the content of eDNA in biofilms, demonstrating that the co-loading of eDNA and Ket in the assembly is key to the synergistic treatment of biofilm infections.

[0114] 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 protein-loaded lipid nanoassembly, characterized in that: The following steps are involved: The lipid molecule is formed by combining the catechol-containing α-aminophosphonate-derived lipid and the phenylboronic acid derivative through a dynamic covalent borate ester bond. The structural formula of the lipid molecule is shown in Formula I: In Formula I, R is a nitrogen-containing heterocyclic compound, including 1-ethyl-1H-imidazole, pyridine, ketoconazole, ciprofloxacin or itaconazole; The lipid molecules are dissolved in an organic solvent, concentrated, a protein therapeutic drug dissolved in a phosphate buffer is added, ultrasonic vibration is performed, centrifugation is performed, the supernatant is discarded, and the solid is resuspended with a phosphate buffer to obtain a protein-loaded lipid nanoassembly.

2. The method for preparing a protein-loaded lipid nanoassembly according to claim 1, characterized in that: The method for preparing the catechol-containing α-aminophosphonate-derived lipid comprises the following steps: Dissolve diphenylphosphite and 1-decanol in pyridine, react at 130-150°C for 4 hours to obtain decanyl phosphate; mix N,N-diethylethylenediamine and 3,4-dihydroxybenzaldehyde in methanol under a nitrogen atmosphere, react at 65-75°C for 3 hours, and then add the decanyl phosphate after vacuum drying, and react at 100-110°C for 1.5 hours to obtain the catechol-containing α-aminophosphonate-derived lipid, whose structural formula is shown in Formula II:

3. The method for preparing a protein-loaded lipid nanoassembly according to claim 2, characterized in that: The molar ratio of the diphenyl phosphite to 1-decanol is 1:2; The molar ratio of the N,N-diethylethylenediamine to 3,4-dihydroxybenzaldehyde is 1:

1.

4. The method for preparing a protein-loaded lipid nanoassembly according to claim 1, characterized in that: The structural formula of the phenylboronic acid derivative is as follows:

5. The method for preparing a protein-loaded lipid nanoassembly according to claim 1, characterized in that: The molar ratio of the catechol-containing α-aminophosphonate-derived lipid to the phenylboronic acid derivative is 1:

1.

6. The method for preparing a protein-loaded lipid nanoassembly according to claim 1, characterized in that: The organic solvent includes chloroform, ethanol, methanol or acetonitrile; The pH of the phosphate buffer is 7.2-8.

2.

7. The method for preparing a protein-loaded lipid nanoassembly according to claim 1, characterized in that: The temperature of the ultrasonic oscillation is 35-45° C., and the time of the ultrasonic oscillation is 5-20 minutes.

8. A protein-loaded lipid nanoassembly, prepared according to the preparation method according to any one of claims 1 to 7.

9. Use of the protein-loaded lipid nanoassembly according to claim 8 in dissociating biological membranes.

10. Use of the protein-loaded lipid nanoassembly according to claim 8 in the preparation of antifungal drugs.

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