A lipid prodrug nanoassembly based on dynamic covalent chemistry and its preparation method and application

Abstract

The present invention discloses a lipid prodrug nanoassembly based on dynamic covalent chemistry, and its preparation method and application, which belong to the fields of dynamic binding chemistry and nanotechnology. The present invention designs a dynamic covalent chemical lipid nano drug delivery system, which is mainly obtained by self-assembly of lipid molecules with a hydrophilic head, a hydrophobic tail and a dynamic covalent side chain. The molecule has an o-diphenol side chain group and can be combined with anti-cancer, antibacterial and antifungal drugs, and has good anti-tumor and antibacterial effects. The present invention constructs a lipid drug delivery platform through dynamic covalent chemistry for the first time, which has the advantages of responsiveness, fixed point, on-demand release, etc. in terms of function. It combines a variety of drugs such as anti-cancer, antibacterial and antifungal drugs with lipid molecules through dynamic covalent chemistry to improve the therapeutic effect of drugs.

Description

Technical Field

[0001] The present invention relates to the fields of dynamic binding chemistry and nanotechnology, and in particular to a lipid prodrug nanoassembly based on dynamic covalent chemistry, and a preparation method and application thereof. Background Art

[0002] Currently, a variety of major diseases, including microbial infections, cancer, and cardiovascular disease, pose a serious threat to people's physical and mental health. According to statistics, the main therapeutic drugs used to treat these diseases are small molecules. Due to their lack of targeting within the body, small molecule drugs often have unsatisfactory clinical therapeutic effects and are even associated with serious side effects. To address this, various nano-drug delivery systems have been developed to reduce drug toxicity and side effects. For example, nano-drugs such as doxorubicin liposomes and paclitaxel albumin have significantly reduced drug side effects. However, clinical data show that patient survival rates have not significantly improved, and the development of new and efficient delivery systems to improve disease treatment efficacy continues to attract significant attention.

[0003] With the continuous development of nanotechnology, the emergence of various environmentally responsive delivery systems has laid a solid foundation for the development of smart nanomedicines. These smart nanomedicines can achieve targeted delivery and responsive release of drugs based on the microenvironmental differences between the lesion site and normal tissue (such as pH, GSH, H2O2), and have attracted widespread attention in the field of disease diagnosis and treatment. Among them, lipid nanomedicines are mainly obtained by self-assembly of amphiphilic lipid molecules. They have the advantages of simple preparation methods, wide sources of raw materials, and low cost. They have achieved good research results and application transformation in many fields such as drug delivery, protein delivery, and gene transfection. However, at present, lipid nanosystems mainly carry out drug loading through hydrophilic / hydrophobic interactions and electrostatic interactions. During the delivery process, due to the complexity of the microenvironment in the body, the drug is suddenly released, resulting in huge safety risks and seriously limiting their practical application.

[0004] Dynamic covalent chemistry is a series of covalent chemical reactions based on reversible thermodynamic equilibrium. Its core concept is reversible covalent bonds, such as imine bonds, borate bonds, acylhydrazone bonds, and disulfide bonds. The reversible nature of these reactions enables intelligent responsiveness in the synthesis process of dynamic covalent chemistry. By introducing specific structures and modifying equilibrium conditions (such as pH, temperature, GSH, and H2O2), targeted drug delivery can be achieved. This provides new insights into the development of novel and efficient delivery systems to improve disease treatment outcomes. Summary of the Invention

[0005] The purpose of the present invention is to provide a lipid prodrug nanoassembly based on dynamic covalent chemistry, a preparation method and application thereof, in order to solve the problems existing in the above-mentioned prior art. By designing lipid molecules mainly having a hydrophilic head, a hydrophobic tail and a dynamic covalent side chain, a dynamic covalent chemistry lipid nano drug delivery system is obtained by self-assembly. The lipid nano drug delivery system has an o-diphenol side chain group and can be combined with anticancer, antibacterial and antifungal drugs, and has excellent anti-tumor and antibacterial effects.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a lipid molecule based on dynamic covalent chemistry, the structural formula of the lipid molecule is shown in the following formula I:

[0008]

[0009] Wherein, n is any integer between 10 and 14; R1 is methyl or ethyl; and R2 is any one of 2,3-catechol, 3,4-catechol, 2-phenylboronic acid, 3-phenylboronic acid or 4-phenylboronic acid.

[0010] The present invention also provides a lipid prodrug nanoassembly based on dynamic covalent chemistry, comprising the lipid molecules and the drugs loaded by the lipid molecules.

[0011] Preferably, the drug comprises a drug or prodrug containing boronic acid or phenylboronic acid.

[0012] More preferably, the drug containing boronic acid is bortezomib; the prodrug containing phenylboronic acid is: (1) drugs containing secondary and / or tertiary amine groups, such as ciprofloxacin, voriconazole, clotrimazole and other phenylboronic acid derivatives; (2) drugs containing primary amine groups, such as doxorubicin, gemcitabine and other phenylboronic acid derivatives; (3) drugs containing hydroxyl groups, such as camptothecin, paclitaxel and other phenylboronic acid derivatives.

[0013] More preferably, the preparation method of the prodrug of the drug containing secondary amine and / or tertiary amine groups modified with phenylboronic acid is as follows: adding the drug containing secondary amine and / or tertiary amine groups to N,N-dimethylformamide and water solvent (the solvent volume ratio is 2:

[0014] 1), adding potassium carbonate, sonicating, and then adding 4-(bromomethyl)phenylboronic acid, wherein the ratio of drug to potassium carbonate to 4-(bromomethyl)phenylboronic acid is 1:3:1.2. Stirring at room temperature, after completion of the reaction, adjusting the pH with dilute hydrochloric acid solution until a precipitate forms, which is then filtered and dried to obtain a phenylboronic acid prodrug.

[0015] More preferably, the phenylboronic acid-modified prodrug containing a secondary amine and / or tertiary amine drug is ciprofloxacin and clotrimazole. The specific reaction process is as follows:

[0016]

[0017] In the above reaction, the reaction solvent for phenylboronic acid-modified ciprofloxacin is preferably a mixed solvent of N,N-dimethylformamide and water, with a volume ratio of N,N-dimethylformamide to water of 2:1, and the reaction temperature is room temperature. The reaction solvent for phenylboronic acid-modified clotrimazole is preferably a mixed solvent of tetrahydrofuran and methanol, with a volume ratio of tetrahydrofuran to methanol of 2:1, and the reaction temperature is preferably 45°C. The molar ratio of the drug to potassium carbonate to 4-(bromomethyl)phenylboronic acid is 1:3:1.2.

[0018] More preferably, the preparation method of the phenylboronic acid-modified prodrug of the primary amine-containing drug is as follows: 4-nitrobenzoyl chloride and 4-hydroxymethylphenylboronic acid pinacol ester are reacted in dichloromethane for 3 hours, the organic phase is dried over anhydrous magnesium sulfate, and passed through a column to obtain an intermediate product, a drug containing a primary amine group is added, the reaction is continued for 3 hours, and then passed through a column, and the product is reacted with diethanolamine for deprotection to obtain the phenylboronic acid-modified prodrug of the primary amine-containing drug.

[0019] More preferably, the prodrug of the phenylboronic acid-modified primary amine-containing drug is doxorubicin, and the specific reaction process is as follows: the molar ratio of doxorubicin to 4-nitrobenzoyl chloride and 4-hydroxymethylphenylboronic acid pinacol ester is 1:1.2:1.2.

[0020]

[0021] More preferably, the preparation method of a phenylboronic acid-modified prodrug of a hydroxyl-containing drug comprises reacting the hydroxyl-containing drug with triphosgene, followed by reaction with hydroxymethylphenylboronic acid pinacol ester, separating and purifying the intermediate by column separation, and then deprotecting the intermediate by reaction with diethanolamine. The hydroxyl-containing phenylboronic acid prodrug is extracted with dichloromethane, precipitated in glacial ether, and filtered and dried to obtain the prodrug. The molar ratio of the drug to triphosgene is 1:1.5, the molar ratio of the drug to hydroxymethylphenylboronic acid pinacol ester is 1:1.5, and the molar ratio of the drug to diethanolamine is 1:5.

[0022] More preferably, the prodrug of the hydroxyl-containing drug modified with phenylboronic acid is camptothecin, and the specific reaction process is as follows:

[0023]

[0024] Preferably, when the lipid molecule is 3,4-catechol, the catechol on the lipid molecule combines with the boric acid in the drug to form a lipid-drug molecule with a dynamic covalent bond of a boronate ester bond, which self-assembles into the lipid prodrug nanoassembly.

[0025] Preferably, the structural formula of the lipid nanoparticle drug is shown in Formula II below:

[0026]

[0027] Wherein, n is any integer between 10 and 14; R1 is a methyl group or an ethyl group; and R2 is a drug or prodrug containing boronic acid or phenylboronic acid.

[0028] The present invention also provides a method for preparing the lipid prodrug nanoassembly, comprising the following steps:

[0029] The lipid molecules are reacted with the drug in an organic solvent, concentrated, and then a phosphate buffer is added. Ultrasonic oscillation is performed until the organic solvent is completely volatilized to obtain a lipid prodrug nanoassembly; the molar ratio of the lipid molecules to the drug is 1:1, and the drug includes a drug or prodrug containing boric acid or phenylboronic acid.

[0030] Preferably, the organic solvent is chloroform, the reaction temperature is 40-50° C., and the reaction time is 12 hours.

[0031] Preferably, the ultrasonic oscillation temperature is 40-45° C., and the ultrasonic oscillation time is 10-30 minutes.

[0032] More preferably, the lipid molecule has a structure as shown in Formula I, and is synthesized by reacting formylcatechol or formylbenzoic acid with N,N-diethylethylenediamine or N,N-dimethylethylenediamine to synthesize an imine intermediate, which is then added to a dialkyl phospholipid, wherein the molar ratio of formylcatechol or formylbenzoic acid to N,N-diethylethylenediamine or N,N-dimethylethylenediamine is 1:1, and the molar ratio of the imine intermediate to the dialkyl phospholipid is 1.5:1. The lipid molecule is then filtered through a column, rotary evaporated, and dried to obtain catechol and phenylboronic acid lipid molecules with dynamic covalent chemical properties. The solvent used in the column separation and purification process is a methanol / dichloromethane mixture, with the methanol to dichloromethane ratios being 1:30, 1:20, and 1:10, respectively. The dialkyl phospholipid is synthesized by distilling diphenyl phosphite and an alkyl alcohol in pyridine under reduced pressure at 120-140°C.

[0033] More preferably, when the lipid molecules synthesized by the above lipid molecule synthesis method react with the drug to generate the lipid prodrug nanoassembly shown in Formula II, the synthesis route is as follows:

[0034]

[0035] The present invention also provides the application of the lipid molecule or the lipid prodrug nanoassembly in antibacterial aspects.

[0036] The present invention also provides the use of the lipid molecule or the lipid prodrug nanoassembly in preparing a drug for improving the therapeutic effect of tumors.

[0037] The present invention discloses the following technical effects:

[0038] This invention utilizes dynamic covalent chemistry to construct lipid prodrug nanoassemblies, achieving dynamic binding of drugs to lipid molecules and responsive drug release. Compared to traditional lipid nanoparticles, this system requires specific conditions for drug release through dynamic covalent chemical binding, thus avoiding the problem of sudden drug release in the bloodstream. Because dynamic covalent bonds exhibit acid- and ROS-responsive properties, drug release can be tailored to the microenvironmental differences between the lesion site and the normal tissue.

[0039] The small molecule nanomaterials synthesized in this invention require inexpensive and readily available raw materials, and the synthesis and assembly methods are simple. This method has a wide range of applications, including the assembly of a variety of drugs containing primary, secondary, and tertiary amines, hydroxyl groups, and diols, in areas such as antibacterial, antifungal, anticancer, diabetes, and cardiovascular disease, enabling the construction of a new intelligent nanoplatform.

[0040] The dynamic covalent chemical lipid nanomedicine constructed in this invention can serve as a novel drug delivery platform, enabling targeted delivery and on-demand, responsive release of drugs. In vitro and animal studies have shown that the material is significantly effective in treating bacterial infections, cancer, and fungal infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0042] Figure 1 Schematic diagram of the assembly of the lipid nanomedicine prepared in the present invention;

[0043] Figure 2 The H-NMR spectrum and P-NMR spectrum of the 3,4-catechol lipid molecule prepared by the present invention;

[0044] Figure 3 This is the lipid-ciprofloxacin nuclear magnetic resonance of Example 1 of the present invention 1 H-NMR (400 MHz, CDCl3) and infrared spectra (FT-IR);

[0045] Figure 4 This is a transmission electron microscopy (TEM) image of the particle size distribution of ciprofloxacin lipid nanoparticles prepared in Example 1 of the present invention;

[0046] Figure 5This is a comparison chart of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ciprofloxacin lipid nanoparticles prepared in Example 1 of the present invention;

[0047] Figure 6 This is a diagram of a mouse model of intraperitoneal infection treated with ciprofloxacin lipid nanoparticles prepared in Example 1 of the present invention;

[0048] Figure 7 The bortezomib lipid nanoparticles prepared in Example 2 of the present invention are subjected to nuclear magnetic resonance imaging. 1 H-NMR (400 MHz, CDCl3) and infrared spectra (FT-IR);

[0049] Figure 8 This is a transmission electron microscopy (TEM) image of the particle size distribution of bortezomib lipid nanoparticles prepared in Example 2 of the present invention;

[0050] Figure 9 Figure 2 is a graph showing the in vitro and in vivo anti-tumor effects of bortezomib lipid nanoparticles prepared in Example 2 of the present invention;

[0051] Figure 10 This is a transmission electron microscopy (TEM) image of the particle size distribution of clotrimazole lipid nanoparticles prepared in Example 3 of the present invention;

[0052] Figure 11 This is a comparison chart of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of clotrimazole lipid nanoparticles prepared in Example 3 of the present invention for antifungal activity in vitro. DETAILED DESCRIPTION

[0053] 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0054] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0055] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents of this specification shall prevail.

[0056] 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 without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0057] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0058] Example 1

[0059] (1) Preparation method of catechol lipid molecules, the synthesis route is as follows:

[0060]

[0061] The specific synthesis steps are as follows:

[0062] 0.667 mL of diphenyl phosphite and 1.59 g of tetradecyl alcohol were placed in a flask, followed by the addition of 0.35 mL of pyridine. After a 4-hour reaction, the pyridine and byproducts were removed by vacuum distillation, yielding 2.1 g of a white solid, tetradecyl phosphate, with a yield of 98%. 140.5 mg of N,N-diethylethylenediamine was mixed with 138.2 mg of 3,4-dihydroxybenzaldehyde under nitrogen atmosphere, followed by the addition of 3 mL of formaldehyde. The mixture was reacted at 70°C for 3 hours, then dried on a rotary evaporator at 30°C. 474.75 mg of tetradecyl phosphate was then quickly added and reacted at 100°C for 1.5 hours. A 1:30, 1:20, and 1:10 methanol:dichloromethane solution was then passed through the column. Finally, the desired product appeared at a 1:10 ratio, and the remaining liquid was completely evaporated to yield 316 mg of a brown solid with a yield of 41.9%.

[0063] like Figure 2The following are the H-NMR and P-NMR spectra of the prepared catechol. The characteristic peak of tetradecanol (3.74 ppm) in the H-NMR spectrum completely disappears, while the characteristic peak of phospholipid (4.05 ppm) appears, indicating the successful synthesis of ditetradecanoic acid phosphate. In the subsequent reaction, the characteristic peaks of catechol (6.84-6.88 ppm, 7.05 ppm) appear, indicating that catechol is successfully branched onto the phospholipid molecule and 3,4-catechol lipid molecules are successfully synthesized. At the same time, the characteristic peaks of the raw material and intermediate product (0.1 ppm and 8.5 ppm) in the P-NMR spectrum completely disappear in the product, and a new characteristic peak of the product (23.5 ppm) appears, again confirming the successful synthesis of catechol lipid molecules.

[0064] (2) A method for preparing a ciprofloxacin phenylboronic acid prodrug, comprising the following steps:

[0065] Ciprofloxacin (0.25 g, 0.76 mmol) was added to a mixture of DMF and water (4 mL and 2 mL, respectively). Potassium carbonate (0.32 g, 2.28 mmol, 3.0 equiv) was added and sonicated to dissolve the mixture. 4-(Bromomethyl)phenylboronic acid (0.2 g, 0.91 mmol, 1.2 equiv) was then added and stirred overnight. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction, the pH of the system was adjusted to neutral with 1N hydrochloric acid solution to precipitate the product. The product was filtered and dried to obtain 0.21 g of a white solid, the ciprofloxacin derivative (intermediate Cip-BPA), with a yield of 58%. The specific synthesis process is as follows:

[0066]

[0067] (3) A method for preparing ciprofloxacin lipid nanoparticles based on dynamic covalent chemistry, comprising the following steps:

[0068] 2.32 mg of ciprofloxacin phenylboronic acid prodrug and 3.56 mg of 3,4-catechol lipid molecules were dissolved in 0.5 mL of chloroform, mixed thoroughly, and reacted at 45°C for 12 hours. The reaction solution was concentrated to 200 mL, 10 mL of phosphate buffer (pH 7.4) was added, and ultrasonication was performed at 45°C until the chloroform in the system was completely evaporated. Ultrasonication was continued for 10 minutes to obtain ciprofloxacin lipid nanoparticles.

[0069] like Figure 3 As shown, the NMR spectra of ciprofloxacin lipid molecules prepared in this example are shown. 1H-NMR and infrared spectroscopy (FT-IR) showed that after the phospholipid molecules reacted with ciprofloxacin phenylboronic acid in the hydrogen spectrum, the characteristic peaks of catechol on the phospholipid molecules (6.84-6.88ppm, 7.05ppm) were passivated and shifted to the high field under the action of the boron atom, indicating that ciprofloxacin phenylboronic acid reacted with catechol. At the same time, a series of characteristic peaks of ciprofloxacin phenylboronic acid appeared, proving that the ciprofloxacin lipid molecule was successfully prepared. The infrared spectrum results showed that the characteristic peak of the hydroxyl group in the boric acid (3500cm -1 ) and the characteristic peak of hydroxyl groups in catechol (3000-3500cm -1 ) disappeared, indicating that the two reacted to form a phenylboronic acid ester bond, and the ciprofloxacin lipid molecule was successfully prepared.

[0070] like Figure 4 The figure shows the particle size distribution of the ciprofloxacin lipid nanoparticles prepared in this example by transmission electron microscopy (TEM) and elemental mapping. The dynamic light scattering (DLS) results show that the particle size of the ciprofloxacin lipid nanoparticles is about 250 nm, the PDI is 0.031, and the particle size distribution is uniform. The TEM results confirm that the nanoparticles are solid spheres of about 230 nm, which is consistent with the DLS results. In the elemental mapping, boron, carbon, oxygen, nitrogen and other elements all appear and are distributed uniformly, indicating that the nanoparticles composited with lipid molecules and ciprofloxacin were successfully prepared.

[0071] Test Example 1

[0072] This test example is to verify the in vitro antibacterial effect of the material prepared in Example 1, using the following method:

[0073] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the nanoparticles (Lipid-CIP), lipid molecules (Lipid), ciprofloxacin (CIP) and ciprofloxacin phenylboronic acid (CIP-PBA) in Example 1 were measured respectively.

[0074] Each sample was diluted to the corresponding concentration with PBS (phosphate buffer solution), 100 μL of each concentration of sample was added to a 96-well plate, and then 100 μL of 2*10 5 CFU / mL of the prepared bacterial solution S. aureus Xen36 (bioluminescent gene-transfected Staphylococcus aureus). After 24 hours, the absorbance at 600 nm was measured using a microplate reader.

[0075] The results are as follows Figure 5As shown in Table 1, the MIC and MBC of nanoparticle Lipid-CIP against S. aureus Xen36 were 0.125 μg / mL and 0.25 μg / mL, respectively, which were lower than 0.5 μg / mL and 1.0 μg / mL of ciprofloxacin monomer itself and 0.25 μg / mL and 0.5 μg / mL of ciprofloxacin phenylboronic acid CIP-PBA.

[0076] Table 1 Minimum inhibitory concentration and minimum bactericidal concentration of different samples against S. aureus Xen36

[0077] sample MIC (μg / mL) MBC (μg / mL) Lipid ＞32 ＞32 CIP 0.5 1.0 CIP-PBA 0.25 0.5 Lipid-CIP 0.125 0.25

[0078] Test Example 2

[0079] In order to verify the in vivo effect of the material prepared in Example 1, a mouse abdominal infection model was established for verification, and the method was as follows:

[0080] All healthy and active mice were divided into 4 groups, namely blank group (PBS), lipid molecule group (Lipid), ciprofloxacin group (Cip) and ciprofloxacin lipid nanoparticle group (Lipid-CIP), with 7 mice in each group. 300μL 5*108CFU / mL pre-prepared PBS bacterial solution S.aureus Xen36 was injected into the abdominal cavity of each mouse. Two hours later, in vivo imaging was taken (as the starting point of bioluminescence for each mouse) and the drug was immediately administered. The dosage was calculated based on the amount of Cip 0.2mg per mouse. In vivo imaging (IVIS) was then taken at regular intervals to observe the changes in bioluminescence intensity in the mice. 24 hours after administration, the mice were killed, and ascites was collected for plate count and statistical bacterial concentration.

[0081] The results are as follows Figure 6 As shown in the in vivo imaging images, the bacterial concentration in all components showed a downward trend in the mice. The bacterial content in the ascites of the PBS and Lipid groups was similar after 24 hours of treatment, and was significantly decreased after 10 8 CFU / mL, indicating that the lipid molecules themselves have no bactericidal effect. One day after the administration of ciprofloxacin, the bioluminescence intensity of bacteria can still be detected, and after the plate count of ascites, there are still more than 10 3 The presence of bacteria at CFU / mL indicated that the current dose of ciprofloxacin was not able to kill all the bacteria after one treatment. However, in mice treated with the assembly, almost no bacteria were detected at the 8th hour, and this was maintained until the 24th hour. After taking the ascites for plate counting, it was found that the bacteria in the ascites had dropped to 10 2 CFU / mL or less, indicating that the assembly can effectively kill bacteria in mice.

[0082] Example 2 A method for preparing a bortezomib lipid nanoparticle drug based on dynamic covalent chemistry, comprising the following steps:

[0083] 1.94 mg of bortezomib and 3.56 mg of 3,4-catechol lipid molecules prepared in Example 1 were dissolved in 0.5 mL of chloroform, thoroughly dissolved and mixed, and reacted at 45° C. for 12 hours. The reaction solution was concentrated to 200 mL, 10 mL of phosphate buffer (pH 7.4) was added, and ultrasonication was performed at 45° C. until the chloroform in the system was completely evaporated. Ultrasonication was continued for 10 minutes to obtain bortezomib lipid nanoparticles.

[0084] like Figure 7 As shown, the bortezomib lipid molecule NMR prepared in this example 1 H NMR (400MHz, CDCl3) and infrared spectra (FT-IR). In the hydrogen spectrum, after the phospholipid molecule reacted with bortezomib, the characteristic peaks of catechol on the phospholipid molecule (6.84-6.88ppm, 7.05ppm) were passivated and shifted to the high field under the action of the boron atom, indicating that the boronic acid of bortezomib reacted with catechol. At the same time, the characteristic peak of hydrogen on the carbon connected to the boronic acid in bortezomib (5.02ppm) disappeared, and the characteristic peak of hydrogen on the carbon connected to the boric acid ester (5.45ppm) disappeared, proving that the bortezomib lipid molecule was successfully prepared. The infrared spectrum results showed that the characteristic peak of the carbon-oxygen bond of the boronic acid in bortezomib (1340cm -1 ) disappearance and the borate carbon-oxygen bond (1200 cm -1 ) indicates that the two reacted to form a boronate ester bond, and the bortezomib lipid molecule was successfully prepared.

[0085] like Figure 8 The following are the particle size distribution diagrams, transmission electron microscopy (TEM) and elemental mapping diagrams of the bortezomib lipid nanoparticles prepared in this example. The dynamic light scattering results show that the particle size of the bortezomib lipid nanoparticles is about 230 nm, the PDI is 0.114, and the particle size distribution is uniform. The TEM results confirm that the nanoparticles are solid spheres of about 220 nm, which is consistent with the DLS results. In the elemental mapping diagram, boron, carbon, oxygen, nitrogen and other elements all appear and are distributed consistently, indicating that nanoparticles composited with lipid molecules and bortezomib are successfully prepared.

[0086] Test Example 3

[0087] To verify the anticancer effect of the material prepared in Example 2, the method is as follows:

[0088] Three types of cancer cells were selected for verification, including breast cancer cells 4T1, human ovarian cancer cells A2780 and Hela cells. The cells were added to a 96-well plate at a concentration of 5,000 cells / well. After 24 hours of growth, different concentrations of bortezomib nanoparticles and an equal dose of bortezomib were added as a control. After incubation for 24 hours, culture medium containing 5% CCK-8 was added. After two hours of incubation, the absorbance at 425 nm was detected using a microplate reader.

[0089] The results are as follows Figure 9 The results showed that the killing effect of bortezomib nanoparticles on cancer cells was slightly lower than that of bortezomib, but the IC 50 Both were lower than 2μg / mL and still had strong anti-cancer effects.

[0090] Example 3 A method for preparing clotrimazole lipid nanoparticles based on dynamic covalent chemistry, comprising the following steps:

[0091] 2.4 mg of clotrimazole phenylboronic acid and 3.56 mg of 3,4-catechol lipid molecules prepared in Example 1 were dissolved in 0.5 mL of chloroform, mixed thoroughly, and reacted at 45°C for 12 hours. The reaction solution was concentrated to 200 mL, 10 mL of phosphate buffer (pH 7.4) was added, and ultrasonication was performed at 45°C until the chloroform in the system was completely evaporated. Ultrasonication was continued for 10 minutes to obtain clotrimazole lipid nanoparticles.

[0092] like Figure 10 As shown in the transmission electron microscopy (TEM) diagram of the particle size distribution of the nanoparticles prepared in this example, the dynamic light scattering results show that the particle size of the clotrimazole lipid nanoparticles is about 350 nm, the PDI is 0.592, and the TEM results confirm that the nanoparticles are solid particles of about 330 nm, which is consistent with the DLS results.

[0093] Test Example 4

[0094] This test example is to verify the in vitro antifungal effect, using the following method:

[0095] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the nanoparticles (Lipid-CTZ), lipid molecules (Lipid), clotrimazole (CTZ) and clotrimazole phenylboronic acid (CTZ-PBA) in Example 3 were measured respectively.

[0096] Each sample was diluted to the corresponding concentration with PBS (phosphate buffer solution), 100 μL of each concentration of sample was added to a 96-well plate, and then 100 μL of 2*10 5 CFU / mL of the prepared bacterial solution (Candida albicans). After 24 hours, the absorbance at 600 nm was measured using a microplate reader.

[0097] The results are as follows Figure 11 As shown in Table 2, the MIC and MBC of Lipid-CTZ against Candida albicans were 1 μg / mL and 2 μg / mL, respectively, which were lower than 2 μg / mL and 4 μg / mL of clotrimazole itself, indicating that clotrimazole nanoparticles have a good antifungal effect.

[0098] Table 2 Minimum inhibitory concentration and minimum bactericidal concentration of different samples against Candida albicans

[0099]

[0100]

[0101] As shown in the experimental results of the above examples, the present invention provides nanoparticles with antibacterial, antifungal, and anticancer activities synthesized and assembled using dynamic covalent chemistry of lipid molecules. The dynamic covalent chemistry of the nanoparticle's side chains is achieved through boronate ester bonds. These lipid-drug dynamic covalent molecules are formed by binding the catechol side chain of the lipid molecule to the boronic acid group of the drug or the phenylboronic acid of the prodrug, or by binding the phenylboronic acid side chain of the lipid molecule to the diol structure of the drug molecule. These molecules can then self-assemble into nanoparticles in an aqueous phase.

[0102] This invention constructs a lipid drug delivery platform through dynamic covalent chemistry for the first time, which has the advantages of responsiveness, fixed-point, and on-demand release in terms of function. By combining drugs with lipid molecules through dynamic covalent chemistry, it can be used for the delivery of multiple drugs and improve the therapeutic effect of drugs.

[0103] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A lipid prodrug nanoassembly based on dynamic covalent chemistry, characterized in that: Comprising lipid molecules, and drugs loaded by the lipid molecules; The structural formula of the lipid molecule is shown in Formula I below: Formula I Wherein, n is any integer between 10 and 14; R1 is methyl or ethyl; R2 is 3,4-catechol; The drug is a ciprofloxacin prodrug modified with bortezomib or phenylboronic acid; The catechol on the lipid molecule combines with the boric acid in the drug to form a lipid-drug molecule with a dynamic covalent bond of a boronate ester bond, which self-assembles into the lipid prodrug nanoassembly.

2. A method for preparing the lipid prodrug nanoassembly according to claim 1, characterized in that: The following steps are involved: The lipid molecules and the drug are reacted in an organic solvent, concentrated, and then a phosphate buffer is added. Ultrasonic oscillation is performed until the organic solvent is completely volatilized to obtain a lipid prodrug nanoassembly; the molar ratio of the lipid molecules to the drug is 1:

1.

3. The preparation method according to claim 2, wherein The organic solvent is chloroform, the reaction temperature is 40-50° C., and the reaction time is 12 hours.

4. The preparation method according to claim 2, wherein The ultrasonic oscillation temperature is 40-45° C., and the ultrasonic oscillation time is 10-30 minutes.

5. Use of the lipid prodrug nanoassembly according to claim 1 in the preparation of antibacterial drugs, characterized in that: The antibacterial method is to inhibit Candida albicans, and the drug in the nanoassembly is a ciprofloxacin prodrug modified with phenylboronic acid.

6. Use of the lipid prodrug nanoassembly according to claim 1 in preparing a drug for improving the therapeutic effect of tumors, characterized in that: The tumors are breast cancer cells 4T1, human ovarian cancer cells A2780 and Hela cells, and the drug in the nanoassembly is bortezomib.

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