Nanopharmaceutical composition, preparation method and use thereof

Abstract

A nanomedicine composition, preparation method, and use thereof, comprising: a negatively charged nanophospholipid disk and an antimicrobial compound; wherein the negatively charged nanophospholipid disk comprises a negatively charged phospholipid layer and a membrane scaffold protein, wherein the negatively charged phospholipid layer is formed by mixing a neutral phospholipid and a negatively charged phospholipid in a molar ratio of approximately 5:5-7:3. The nanomedicine composition provided herein reduces the toxicity of the antimicrobial compound while enhancing its antimicrobial efficacy, and has great potential for expansion into clinical applications.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceuticals, and in particular to a nanopharmaceutical composition, a preparation method thereof, and uses thereof. Background Art

[0002] Antibiotic resistance is an increasingly serious problem, and multidrug-resistant bacterial infections have become a serious global threat to human health. According to data from the U.S. Centers for Disease Control and Prevention (CDC), antibiotic-resistant bacterial infections currently kill over 700,000 people worldwide annually, and this number is projected to rise to 10 million by 2050. In 2017, the World Health Organization (WHO) released a list of priority pathogens in need of new antibiotics, categorizing carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, and carbapenem-resistant and extended-spectrum β-lactamase-producing Enterobacteriaceae as extremely important (Category 1 priority). To address these bacteria, which are difficult to treat with existing conventional antibiotics, the established drug polymyxin B has been revived due to its potent antibacterial activity against Gram-negative bacteria. However, its severe nephrotoxicity and neurotoxicity limit its clinical application. Summary of the Invention

[0003] To address the increasing prevalence of multidrug-resistant Gram-negative bacterial infections and the dwindling availability of available treatments, as well as the neurotoxicity and nephrotoxicity of polymyxin B, which limits its clinical application, this application provides a nano-bionic delivery system for polymyxin B, a compound used to treat Gram-negative bacterial infections. By designing and synthesizing negatively charged nanophospholipid disks to load polymyxin B, this system reduces its toxicity while enhancing its antibacterial efficacy.

[0004] In one aspect, the present application provides a nanopharmaceutical composition comprising:

[0005] Negatively charged nanophospholipid disks and antimicrobial compounds; wherein,

[0006] The negatively charged nanophospholipid disk comprises a negatively charged phospholipid layer and membrane scaffold proteins (MSP), wherein the negatively charged phospholipid layer is formed by mixing neutral phospholipid and negatively charged phospholipid in a molar ratio of about 5:5-7:3.

[0007] In another aspect, the present application also provides a method for preparing the nanopharmaceutical composition described herein, comprising:

[0008] 1) mixing a neutral phospholipid and a negatively charged phospholipid in a molar ratio of about 5:5-7:3 to form the negatively charged phospholipid layer;

[0009] 2) mixing the membrane scaffold protein with the negatively charged phospholipid layer prepared in step 1) at a molar ratio of about 1:50-100, and self-assembling to form a negatively charged nanophospholipid disk;

[0010] 3) mixing the antibacterial compound with the nanophospholipid disk at a molar ratio of about 3-5:1, so that the antibacterial compound is embedded on the surface of the nanophospholipid disk to form the nano drug composition.

[0011] On the other hand, the present application also provides use of the nanopharmaceutical composition described herein in the preparation of drugs for resisting bacterial or fungal infections.

[0012] On the other hand, the present application also provides use of the nanopharmaceutical composition described herein in the preparation of drugs for resisting Gram-negative bacterial infection.

[0013] This application provides Figure 1 The negatively charged nanophospholipid disks shown and Figure 2 The nanoparticle drug composition loaded with polymyxin B is shown. Phospholipid molecules DMPC (dimyristoylphosphatidylcholine) and DMPG (dimyristoylphosphatidylglycerol) are dissolved in 300 mM sodium cholate to form 25 mg / ml micelles. The two phospholipid micelles are then mixed in a proportionally uniform manner and then mixed with a membrane scaffold protein represented by SEQ ID NO: 1 at a mass ratio of one-third. After self-assembly through thermal cycling, the mixture is dialyzed to form negatively charged nanophospholipid disks. The polymyxin B compound is dissolved in PBS to 2 mg / ml and then mixed evenly with the prepared negatively charged nanophospholipid disks. After gentle shaking at 4°C, unbound polymyxin B is removed by ultrafiltration to obtain negatively charged nanophospholipid disks stably loaded with polymyxin B. This negatively charged nanophospholipid disk polymyxin B delivery system exhibits excellent antibacterial activity against a variety of Gram-negative bacteria, including sensitive and resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii, while reducing the cytotoxicity of the compound itself. The toxicity of polymyxin B loaded onto nanophospholipid disks against Vero cells (African green monkey kidney epithelial cells) was significantly lower than that of polymyxin B alone. The minimum inhibitory concentration (MIC) of polymyxin B loaded onto nanophospholipid disks against Escherichia coli was 0.13-0.25 μg / mL, and the MIC against Pseudomonas aeruginosa was 0.5-1 μg / mL, equivalent to that of the control drug polymyxin B. The MIC against Klebsiella pneumoniae was 0.13-0.5 μg / mL, and against Acinetobacter baumannii was 0.13-0.25 μg / mL, significantly superior to the control drug polymyxin B alone.

[0014] One of the primary factors limiting the clinical application of polymyxin B is its nephrotoxicity. The embodiments described in this application have demonstrated that polymyxin B loaded with nanophospholipid disks is significantly less toxic to mammalian kidney cells than polymyxin B alone, potentially significantly improving the drug's inherent nephrotoxicity. Similarly, with the high prevalence of carbapenem-resistant Gram-negative bacteria in clinical practice, the potent killing effect of negatively charged nanodisks as a delivery system for polymyxin B against these resistant bacteria holds the potential for expanded clinical application.

[0015] Other features and advantages of the present application will be described in the following description, and in part will become apparent from the description, or will be understood by practicing the present application. Other advantages of the present application can be realized and obtained by the solutions described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0017] Figure 1 Schematic diagram of the structure of a negatively charged nanophospholipid disk in one embodiment of the present application;

[0018] Figure 2 A schematic structural diagram of a nanomedicine composition according to one embodiment of the present application;

[0019] Figure 3 Figure 2 shows the cytotoxicity of polymyxin B loaded onto nanodiscs with different phospholipid ratios against Vero cells in one embodiment of the present application; the polymyxin B concentration was 150 μg / mL. (## and P2 indicate comparisons with the PXB control, ## indicates p < 0.01; ** and P1 indicate comparisons with the Nanodisc control, ** indicates p < 0.01).

[0020] Figure 4 The figure shows the particle size of a nanophospholipid disc (Nanodisc) and a nanophospholipid disc loaded with polymyxin B (Nanodisc-PXB) in one embodiment of the present application;

[0021] Figure 5 The zeta potential of the nanophospholipid disk (ND) and the nanophospholipid disk loaded with polymyxin B (ND-PXB) in one embodiment of the present application is shown, and * indicates p<0.05, which is significantly different;

[0022] Figure 6This is a transmission electron microscopy morphology of the nanophospholipid disk and the loaded polymyxin B in one embodiment of the present application;

[0023] Figure 7 The standard curve of the polymyxin B content in the nanomedicine composition according to one embodiment of the present application is shown using the BCA method;

[0024] Figure 8 The effects of different concentrations of polymyxin B and polymyxin B loaded on Vero cells in one embodiment of the present application are shown. DETAILED DESCRIPTION

[0025] Unless otherwise indicated, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this application belongs. When a certain amount, concentration or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper numerical limit and a preferred lower numerical limit, it should be understood that it is equivalent to specifically revealing any range by combining any pair of upper range limits or preferred numerical values ​​with any lower range limit or preferred numerical value, without considering whether the range is specifically revealed. Unless otherwise indicated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range.

[0026] The terms "about" and "approximately" when used in conjunction with a numerical variable generally refer to the value of that variable and all values ​​of that variable are within experimental error (e.g., within a 95% confidence interval for the mean) or within ±10% of the stated value, or wider.

[0027] The expression "comprising" or its synonyms "including," "containing," and "having" are open-ended and do not exclude additional unrecited elements, steps, or ingredients. The expression "consisting of excludes any elements, steps, or ingredients not specified. The expression "consisting essentially of" means that the scope is limited to the specified elements, steps, or ingredients, plus any optional elements, steps, or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of."

[0028] The expression "at least one" or "one or more" means 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0029] Nanodiscs share a similar structural structure to natural cell membranes, consisting of a phospholipid bilayer. Each phospholipid molecule has an active, hydrophilic head group and a hydrophobic tail. Like a typical cell membrane, nanodiscs are composed of two back-to-back layers of phospholipids. To maintain their flat surface, a ring of proteins (MSPs) is added to the nanodisc's exterior to constrain it. These MSPs are scaled-down versions of the apolipoprotein ApoA-I, wrapping around the lipid bilayer to form the disc-like structure. Nanodiscs have an inward-facing hydrophobic face and an outward-facing hydrophilic face. This structure gives them high solubility in aqueous solutions.

[0030] Polymyxin compounds, taking polymyxin B as an example, have a compound structure consisting of a hydrophilic cyclic peptide portion and a hydrophobic fatty acyl chain portion, and are amphiphilic. The large number of amino groups in the compound molecule give it a strong positive charge. Structure-activity relationship studies on polymyxin compounds have shown that its positive charge is related to its antibacterial activity and cytotoxicity. In order to reduce its severe cytotoxicity, it was modified and transformed to reduce its positive charge, and it was found that its antibacterial activity also decreased. Therefore, how to enhance or not affect its antibacterial activity while reducing its toxicity is the focus of this application.

[0031] Nanolipid disks, as lipoprotein nanoparticles, differ from other lipid-based formulations such as liposomes in their exceptionally high solubility in aqueous environments and their hydrophobic interior, rather than the hydrophilic cavity of conventional liposomes. The amphiphilic nature of polymyxins allows them to be embedded on the surface of the nanolipid disks rather than encapsulated within them. To ensure that polymyxins are released at sites of strongly negatively charged bacterial infection, thereby achieving a bactericidal effect, while preventing their release from weakly negatively charged normal cells or tissues, thereby reducing their cytotoxicity, the nanolipid disks must possess a certain degree of negative charge to constrain the positively charged polymyxins. The negative charge of the nanolipid disks is provided by a certain proportion of negatively charged phospholipids. This ratio should be neither too high nor too low, as this would hinder the release of the polymyxins from bacteria, nor too low a proportion would reduce their cytotoxicity. The phospholipids in the nanophospholipid disk include neutral phospholipids that provide a scaffolding effect and negatively charged phospholipids that provide negative charge. Neutral phospholipids can be PE (phosphatidylethanolamine) and PC (phosphatidylcholine). Negatively charged phospholipids are PG (phosphatidylglycerol), including but not limited to DOPG, DMPG, POPG, DSPG, etc. In this application, DMPC (dimyristoylphosphatidylcholine) was selected as the neutral phospholipid and DMPG (dimyristoylphosphatidylglycerol) was selected as the negatively charged phospholipid. A series of different phospholipid ratios were tested to load polymyxin B, and it was finally determined that the optimal phospholipid ratio that reduced toxicity while ensuring antibacterial activity was DMPC:DMPG = 6:4 (see Figure 3 ).

[0032] Based on the structure of nanodiscs, in one aspect, the present application provides a nanomedicine composition comprising:

[0033] Negatively charged nanophospholipid disks and antimicrobial compounds; wherein,

[0034] The negatively charged nanophospholipid disk comprises a negatively charged phospholipid layer and a membrane scaffold protein. The negatively charged phospholipid layer is formed by mixing neutral phospholipids and negatively charged phospholipids in a molar ratio of about 5:5-7:3.

[0035] In some embodiments, the negatively charged phospholipid layer is formed by mixing a neutral phospholipid and a negatively charged phospholipid in a molar ratio of about 5:5, 6:4, or 7:3.

[0036] In some embodiments, the molar ratio of the antimicrobial compound to the negatively charged nanophospholipid disks is about 3-5: 1. In some embodiments, the molar ratio of the antimicrobial compound to the negatively charged nanophospholipid disks is about 3: 1, 3.5: 1, 4: 1, 4.5: 1, or 5: 1.

[0037] In some embodiments, the molar ratio of the membrane scaffold protein to the negatively charged phospholipid layer is about 1:50-100. In some embodiments, the molar ratio of the membrane scaffold protein to the negatively charged phospholipid layer is about 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95 or 1:100.

[0038] In some embodiments, the neutral phospholipid is selected from phosphatidylethanolamine or phosphatidylcholine, and the negatively charged phospholipid is selected from phosphatidylglycerol, including but not limited to DOPG, DMPG, POPG, DSPG, etc.; preferably, the neutral phospholipid is DMPC and the negatively charged phospholipid is DMPG.

[0039] In some embodiments, the antibacterial compound is selected from polymyxin compounds (including polymyxin B and colistin), vancomycin or amphotericin B. In some embodiments, the antibacterial compound is polymyxin B or colistin; preferably, the antibacterial compound is polymyxin B. Depending on the type of antibacterial compound loaded in the nanomedicine composition, the nanomedicine composition in the present application can be used to prepare drugs for resisting different microbial infections. When loaded with vancomycin, it can be used to resist infection with drug-resistant Gram-positive bacteria; when loaded with amphotericin B, it can be used to resist infection with fungi; when loaded with polymyxin B and colistin, it can be used to resist infection with drug-resistant Gram-negative bacteria. Preferably, the nanomedicine composition in the present application contains polymyxin B or colistin; more preferably, the nanomedicine composition in the present application contains polymyxin B.

[0040] In some embodiments, the membrane scaffold protein is selected from apolipoprotein A1 (ApoA-1) mimetic protein. In some embodiments, the amino acid sequence of the membrane scaffold protein is as shown in SEQ ID NO: 1. In some embodiments, other types of apolipoprotein ApoA-1 mimetic proteins can also be used to achieve similar technical effects as the present application.

[0041] In some embodiments, the present application provides a nanoparticle drug composition comprising:

[0042] Negatively charged nanophospholipid disks and polymyxin B; wherein the molar ratio of polymyxin B to the negatively charged nanophospholipid disks is approximately 3-5:1; the negatively charged nanophospholipid disks comprise a negatively charged phospholipid layer and a membrane scaffold protein as shown in SEQ ID NO:1, wherein the molar ratio of the membrane scaffold protein to the negatively charged phospholipid layer is approximately 1:50-100, and the negatively charged phospholipid layer is formed by mixing DMPC and DMPG in a molar ratio of approximately 6:4.

[0043] In some embodiments, the nanolipid discs provided herein are negatively charged nanolipid discs having a zeta potential between -15 mV and -25 mV. The zeta potential of the negatively charged nanolipid discs provided herein is -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, or -25 mV. When the negatively charged nanolipid discs are embedded with positively charged polymyxin B, the zeta potential is between -10 mV and -15 mV. In some embodiments, the zeta potential of the negatively charged nanolipid discs embedded with positively charged polymyxin B provided herein is -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, or -15 mV.

[0044] In some embodiments, the average particle size of the empty and drug-loaded negatively charged nanophospholipid disks provided herein is between about 8 nm and 12 nm. In some embodiments, the average particle size of the empty and drug-loaded negatively charged nanophospholipid disks provided herein is about 8 nm, 8.5 nm, 9 nm, 9.5 nm, 10 nm, 10.5 nm, 11 nm, 11.5 nm, or 12 nm.

[0045] In another aspect, the present application also provides a method for preparing the nanopharmaceutical composition described herein, comprising:

[0046] 1) Mixing neutral phospholipids and negatively charged phospholipids at a molar ratio of about 5:5-7:3 to form a negatively charged phospholipid layer;

[0047] 2) mixing the membrane scaffold protein with the negatively charged phospholipid layer prepared in step 1) at a molar ratio of about 1:50-100 to self-assemble into negatively charged nanophospholipid disks;

[0048] 3) mixing the antibacterial compound with the nanophospholipid disk at a molar ratio of about 3-5:1, so that the antibacterial compound is embedded on the surface of the nanophospholipid disk to form a nano drug composition.

[0049] In this method, neutral phospholipids and negatively charged phospholipids are first self-assembled in a surfactant to form phospholipid micelles. The neutral phospholipids and negatively charged phospholipids are then uniformly mixed at a molar ratio of approximately 5:5-7:3. A membrane scaffold protein is then mixed with the phospholipid bilayer at a molar ratio of 1:50-100. The membrane scaffold is then self-assembled into nanophospholipid discs through a thermal cycling step. The surfactant is then removed through dialysis to obtain the negatively charged nanophospholipid discs for loading antimicrobial compounds.

[0050] In some embodiments, the method comprises:

[0051] 1) DMPC and DMPG were separately dissolved in a 300 mM sodium cholate solution by low-frequency ultrasound and self-assembled into approximately 25 mg / ml phospholipid micelles. The DMPC and DMPG phospholipid micelles were then mixed at a molar ratio of approximately 6:4 to form a negatively charged phospholipid layer;

[0052] 2) mixing the membrane scaffold protein with the negatively charged phospholipid layer prepared in step 1) at a molar ratio of about 1:50-100, and dialyzing to form negatively charged nanophospholipid disks after self-assembly through thermal cycling;

[0053] 3) mixing the antimicrobial compound with the nanophospholipid disks at a molar ratio of approximately 3-5:1, gently shaking at 4°C to load the antimicrobial compound onto the surface of the nanophospholipid disks, and ultrafiltration to remove the antimicrobial compound not bound to the negatively charged nanophospholipid disks to obtain a nanopharmaceutical composition stably loaded with the antimicrobial compound.

[0054] In some embodiments, in step 2), the membrane scaffold protein is mixed with the negatively charged phospholipid layer prepared in step 1), incubated on ice for about 10 minutes, then heated to about 40°C, and incubated again for about 10 minutes. The above thermal cycling steps are repeated 3 times, and then dialyzed at 4°C using a dialysis bag to remove sodium cholate to form a negatively charged nanophospholipid disk.

[0055] In another aspect, the present application also provides use of the nanopharmaceutical composition described herein in the preparation of a drug for resisting bacterial or fungal infection.

[0056] In another aspect, the present application also provides the use of the nanopharmaceutical composition described herein in the preparation of a medicament for use against Gram-negative bacterial infection. In some embodiments, the Gram-negative bacteria include sensitive bacteria and drug-resistant bacteria. In some embodiments, the Gram-negative bacteria are selected from any one of sensitive or drug-resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii.

[0057] In some embodiments, the Gram-negative bacteria is a carbapenem-resistant Gram-negative bacteria. In some embodiments, the Gram-negative bacteria is carbapenem-resistant Klebsiella pneumoniae or carbapenem-resistant Acinetobacter baumannii.

[0058] In some embodiments, the Gram-negative bacteria is an extended-spectrum β-lactamase-producing Gram-negative bacteria. In some embodiments, the Gram-negative bacteria is an extended-spectrum β-lactamase-producing Klebsiella pneumoniae.

[0059] The nanopharmaceutical composition provided in this application not only reduces the toxicity of the antibacterial compound it carries, but also enhances its antibacterial activity, achieving the desired results in both cell and antibacterial experiments. It is highly suitable for clinical use in combating infections caused by drug-resistant Gram-negative bacteria.

[0060] This application describes multiple embodiments, but this description is exemplary rather than restrictive, and it will be apparent to those skilled in the art that more embodiments and implementations may be included within the scope of the embodiments described herein. Although many possible feature combinations are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature of any embodiment may be used in combination with any other feature in any other embodiment, or may replace any other feature in any other embodiment.

[0061] This application includes and contemplates combinations of features known to those of ordinary skill in the art. The embodiments and features disclosed in this application may also be combined with any conventional features to form a unique inventive solution defined by the claims. Any feature of any embodiment may also be combined with features from other inventive solutions to form another unique inventive solution defined by the claims. Therefore, it should be understood that any feature shown and / or discussed in this application may be implemented individually or in any appropriate combination. Therefore, the embodiments are not subject to other limitations except as provided in the appended claims and their equivalents. In addition, various modifications and changes may be made within the scope of protection of the appended claims.

[0062] In addition, when describing representative embodiments, the specification may have presented the method and / or process as a specific sequence of steps. However, to the extent that the method or process does not rely on the specific order of the steps described herein, the method or process should not be limited to the steps in the specific order described. As will be understood by those skilled in the art, other orders of steps are also possible. Therefore, the specific order of the steps set forth in the specification should not be interpreted as a limitation to the claims. In addition, the claims for the method and / or process should not be limited to performing their steps in the order written, and those skilled in the art can readily understand that these orders can be changed and still remain within the spirit and scope of the embodiments of the present application.

[0063] The experimental methods in the following examples, for which specific conditions are not specified, are generally determined in accordance with national standards. The experimental materials in the following examples, for which the sources are not specified, are all commercially available raw materials. The equipment used in each step in the following examples is all conventional equipment. If there are no corresponding national standards, the methods are carried out in accordance with general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise defined or indicated, all professional and scientific terms used in this application have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of this application.

[0064] The relevant materials used in the examples are as follows:

[0065] 1. Reagents and materials

[0066] DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); polymyxin B; kanamycin; sodium cholate; IPTG (isopropylthiogalactoside); PBS; isopropanol; LB medium; DMEM medium; FBS (fetal bovine serum); CAMH medium; BCA protein concentration assay kit; CCK-8 cell viability assay kit, etc.

[0067] 10KDa ultrafiltration tube; 30KDa ultrafiltration tube; 14KDa dialysis bag; centrifuge tube; round-bottom 96-well bacterial culture plate; flat-bottom 96-well ELISA plate; Ni-NTA agarose beads, etc.

[0068] 2. Instruments

[0069] Centrifuge; probe ultrasonic cell disruptor; microplate reader; transmission electron microscope; Malvern particle size analyzer; metal bath, etc.

[0070] Example 1: Preparation of negatively charged nanophospholipid disks and polymyxin B loaded nanoplates

[0071] 1. Prepare protein solution

[0072] The protein portion MSP1D1 in the nanophospholipid disc was obtained by induction expression and isolation and purification. First, Escherichia coli BL21 (DE3) expressing the protein (shown in SEQ ID NO: 1) was cultured in LB medium at 37°C and 220 rpm to an OD = 1.0. The temperature was then lowered to 25°C, and IPTG was added to a final concentration of 1 mM and cultured for another 4 hours. The cells were then harvested by centrifugation at 8000 g, resuspended, and ultrasonically disrupted. The supernatant was obtained by centrifugation at 12000 g for 10 minutes. The supernatant was filtered through 0.22 μm and purified using Ni-NTA agarose beads. The supernatant was further purified and the buffer exchanged by dialysis in PBS (0.01 M phosphate buffer) at 4°C for 36 hours using a 14 kDa dialysis bag. The supernatant was then concentrated to 5 mg / ml using a 10 kDa ultrafiltration tube and frozen at -80°C for future use.

[0073] 2. Prepare Phospholipid Solution

[0074] The phospholipids used in this application are DMPC (dimyristoylphosphatidylcholine) and DMPG (dimyristoylphosphatidylglycerol), both purchased from CordenPharma, Switzerland. Sodium cholate was first dissolved in PBS to a final concentration of 300 mM. DMPC and DMPG were then dissolved in sodium cholate PBS at these concentrations using low-frequency ultrasound and self-assembled into micelles. The phospholipid micelle concentration was 25 mg / ml and set aside.

[0075] 3. Preparation of Negatively Charged Nanophospholipid Disks with Different Phospholipid Ratios

[0076] Negatively charged nanophospholipid disks were prepared using a thermal cycling-self-assembly method. First, a mixture of MSP1D1 protein and phospholipid micelles (DMPC:DMPG = 5:5, 6:4, 7:3, 8:2, and 9:1) was uniformly mixed at a molar ratio of 1:75 and incubated on ice for 10 minutes. The mixture was then heated to 40°C in a metal bath and incubated again for 10 minutes. This thermal cycling step was repeated three times. The nanophospholipid disks containing different negatively charged phospholipid ratios were then dialyzed against PBS at 4°C for 36 hours using a 14 kDa dialysis bag to remove sodium cholate. The resulting mixture was then concentrated to 2 mg / ml (based on MSP1D1) using a 30 kDa ultrafiltration tube and set aside.

[0077] 4. Negatively charged nanophospholipid disks loaded with polymyxin B

[0078] Polymyxin B API was dissolved in PBS to a net concentration of 2 mg / ml. The mixture was uniformly mixed at a molar ratio of 3:1 between polymyxin B and nanolipid disks (calculated as MSP1D1). The mixture was gently shaken at 4°C for 30 minutes to allow polymyxin B to embed onto the surface of the negatively charged nanolipid disks, completing drug loading. Unbound polymyxin B was then removed by ultrafiltration three times using a 30 kDa ultrafiltration tube to obtain negatively charged nanolipid disks stably loaded with polymyxin B.

[0079] 5. Quantification and Characterization of Negatively Charged Nanophospholipid Disks Loaded with Polymyxin B

[0080] The nanophospholipid disk with phospholipid ratio of DMPC:DMPG=6:4 was selected and its particle size before and after drug loading ( Figure 4 ) and Zeta potential ( Figure 5 ) was measured by dynamic light scattering (DLS). The average particle size of the unloaded nanophospholipid disks was 9.441 nm and the Zeta potential was -20.63 mV; the average particle size of the drug-loaded nanophospholipid disks was 8.576 nm and the Zeta potential was -13.03 mV. The specific morphology was determined by transmission electron microscopy (TEM) ( Figure 6 ). The content of polymyxin B was determined by the BCA method. Specifically, three volumes of isopropanol were first added to the negatively charged nanodisc-loaded polymyxin system, and the MSP1D1 was completely precipitated by vortexing. MSP1D1 was then precipitated by centrifugation at a centrifugal force of 12,000 g for 5 minutes, and polymyxin B was present in the supernatant. The polymyxin B raw material was dissolved in 75% isopropanol + 25% PBS solution, and a BCA standard curve was prepared with a concentration gradient of 2000, 1500, 1000, 500, and 250 μg / mL ( Figure 7The polymyxin B to be tested and the standard curve were added to the same 96-well plate, and the corresponding polymyxin B content was calculated based on the standard curve.

[0081] Example 2: Cytotoxicity assay of negatively charged nanophospholipid disks loaded with polymyxin B on Vero cells

[0082] The toxicity of drugs to cells was determined by CCK-8 (Cell Counting Kit-8 cell counting). Vero cells were cultured using DMEM+10% FBS culture medium, planted in 96-well plates at a density of 9,000 cells per well, and cultured for 4 hours at 37°C and 5% carbon dioxide to allow complete attachment. Polymyxin B and polymyxin B loaded on nanophospholipid disks were diluted twice from 512 μg / mL to 2 μg / mL, added to 96-well plates seeded with cells, and incubated under the same conditions for 24 hours. The culture medium was then removed, and DMEM culture medium containing 10% CCK-8 was added back to the 96-well plates. After incubation under the same conditions for 2 hours, the absorbance was measured at a wavelength of 450 nm to determine the cytotoxicity of the drugs to Vero cells.

[0083] The results of the cytotoxicity of the pharmaceutical composition of negatively charged nanophospholipid disks loaded with polymyxin B prepared with different phospholipid ratios on Vero cells are shown in Figure 2. Figure 3 As shown in the figure, it can be seen that when DMPC:DMPG = 5:5, 6:4 and 7:3, the cytotoxicity to Vero cells is relatively low. It is preferred to use the negatively charged nanophospholipid disk with DMPC:DMPG = 6:4.

[0084] At the same time, the cytotoxicity of polymyxin B loaded with negatively charged nanophospholipid disks in a ratio of DMPC:DMPG=6:4 was compared with that of polymyxin B alone on Vero cells. Figure 8 As shown, the IC of polymyxin B alone was calculated. 50 The half inhibitory concentration (CI) value was 67.38 μg / mL, and the IC of polymyxin B loaded with nanophospholipid disks was 0. 50 The value was 341.4 μg / mL, from which it can be concluded that nanophospholipid disks loaded with polymyxin B can significantly reduce the toxicity of polymyxin B to Vero cells.

[0085] Example 3: Minimum inhibitory concentration of negatively charged nanophospholipid disks loaded with polymyxin B against different Gram-negative bacteria

[0086] Two days before the experiment, remove the test strain from the -80°C freezer and streak onto LB agar medium for 18 hours of recovery. The day before the experiment, select three colonies of consistent size and morphology and inoculate them into 4 mL of CAMH (Cation-Adjusted Mueller Hinton) broth. Incubate at 35°C overnight.

[0087] The minimum inhibitory concentration (MIC) of the drug against each test strain was determined by microdilution method. The specific method is as follows. The drug stock solution was diluted two-fold with CAMH broth in a 96-well plate to form a series of required concentration gradients, with final concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL. The overnight cultured test bacterial solution was adjusted to 0.5 McFarland concentration by turbidimetry, and then the 0.5 McFarland concentration bacterial solution was diluted 100 times (approximately 10 μg / mL) with CAMH broth. 6 CFU / ml), pipette 50 μL of the diluted bacterial solution into the aforementioned two-fold dilution series of drug-containing CAMH broth. A control tube without drug was also included. Gently tap the edge of the culture plate to mix thoroughly, and incubate at 37°C for 18 hours. Remove the 96-well plate and visually inspect the well bottoms for bacterial colonies. The minimum drug concentration in the wells showing no bacterial growth is the MIC (refer to CLSI (Clinical and Laboratory Standards Institute) standards).

[0088] The MICs of the negatively charged nanophospholipid disks loaded with polymyxin B against Escherichia coli ATCC25922 prepared using different phospholipid ratios are shown in Table 1:

[0089] Table 1

[0090]

[0091] At the same time, the minimum inhibitory concentrations of polymyxin B loaded with negatively charged nanophospholipid disks in a ratio of DMPC:DMPG = 6:4 were compared with those of polymyxin B alone against different Gram-negative bacteria. The experimental results are shown in Table 2 below. The minimum inhibitory concentration of polymyxin B loaded with nanophospholipid disks against Escherichia coli was 0.13-0.25 μg / mL, and the MIC against Pseudomonas aeruginosa was 0.5-1 μg / mL, which was equal to that of the control drug polymyxin; the MIC against Klebsiella pneumoniae was 0.13-0.5 μg / mL, and the MIC against Acinetobacter baumannii was 0.13-0.25 μg / mL, which was significantly better than the control polymyxin B alone.

[0092] Table 2

[0093]

[0094]

[0095]

[0096] Table Notes: ESBLs, extended-spectrum β-lactamases; CRE, carbapenem-resistant Enterobacteriales; ND, not detected.

Claims

1. A nanomedicine composition comprising: Negatively charged nanophospholipid disks and antimicrobial compounds; wherein, The negatively charged nanophospholipid disk comprises a negatively charged phospholipid layer and a membrane scaffold protein, wherein the negatively charged phospholipid layer is formed by mixing a neutral phospholipid and a negatively charged phospholipid in a molar ratio of 5:5-7:3; The neutral phospholipid is DMPC, the negatively charged phospholipid is DMPG, the membrane scaffold protein is apolipoprotein ApoA-I mimic protein, and the antibacterial compound is polymyxin B.

2. The nano drug composition according to claim 1, wherein The molar ratio of the antibacterial compound to the negatively charged nanophospholipid disk is 3-5:

1.

3. The nano drug composition according to claim 1, wherein The molar ratio of the membrane scaffold protein to the negatively charged phospholipid layer is 1:50-100.

4. The nano drug composition according to claim 1, wherein The amino acid sequence of the membrane scaffold protein is shown in SEQ ID NO:

1.

5. A method for preparing the nanopharmaceutical composition according to any one of claims 1 to 4, comprising: 1) mixing a neutral phospholipid and a negatively charged phospholipid in a molar ratio of 5:5-7:3 to form the negatively charged phospholipid layer; 2) mixing the membrane scaffold protein with the negatively charged phospholipid layer prepared in step 1) at a molar ratio of 1:50-100, and self-assembling to form the negatively charged nanophospholipid disk; 3) mixing the antibacterial compound with the negatively charged nanophospholipid disk at a molar ratio of 3-5:1, so that the antibacterial compound is embedded on the surface of the nanophospholipid disk to form the nano drug composition.

6. The preparation method according to claim 5, wherein The method comprises: 1) DMPC and DMPG were separately dissolved in a 300 mM sodium cholate solution by low-frequency ultrasound and self-assembled into 25 mg / ml phospholipid micelles, and then the DMPC and DMPG phospholipid micelles were uniformly mixed at a molar ratio of 6:4 to obtain the negatively charged phospholipid layer; 2) mixing the membrane scaffold protein with the negatively charged phospholipid layer prepared in step 1) at a molar ratio of 1:50-100, and performing self-assembly through thermal cycling and then dialyzing to form the negatively charged nanophospholipid disk; 3) mixing the antimicrobial compound with the nanophospholipid disk at a molar ratio of 3-5:1, gently shaking at 4°C to allow the antimicrobial compound to be embedded on the surface of the nanophospholipid disk, and ultrafiltration to remove the antimicrobial compound that is not bound to the negatively charged nanophospholipid disk to obtain the nanomedicine composition stably loaded with the antimicrobial compound.

7. The preparation method according to claim 6, wherein In step 2), the membrane scaffold protein is mixed with the negatively charged phospholipid layer prepared in step 1), incubated on ice for 10 minutes, then heated to 40°C and incubated again for 10 minutes. The above thermal cycling steps are repeated 3 times, and then dialyzed at 4°C using a dialysis bag to remove sodium cholate to form the negatively charged nanophospholipid disk.

8. Use of the nanopharmaceutical composition according to any one of claims 1 to 4 in the preparation of drugs for resisting bacterial or fungal infections.

9. Use of the nanopharmaceutical composition according to any one of claims 1 to 4 in the preparation of a drug for resisting Gram-negative bacterial infection.

10. The use according to claim 9, wherein The Gram-negative bacteria are carbapenem-resistant Gram-negative bacteria.

11. The use according to claim 10, wherein The Gram-negative bacteria are carbapenem-resistant Klebsiella pneumoniae or carbapenem-resistant Acinetobacter baumannii.

12. The use according to claim 9, wherein The Gram-negative bacteria are Gram-negative bacteria that produce extended-spectrum beta-lactamases.

13. The use according to claim 12, wherein The Gram-negative bacteria are Klebsiella pneumoniae that produces extended-spectrum β-lactamases.

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