Macrophage-polymer lipid nanoparticle composite drug delivery system, preparation method thereof and application of macrophage-polymer lipid nanoparticle composite drug delivery system in cryptococcus infection resistance

Abstract

The invention discloses a macrophage-polymer lipid nanoparticle composite drug delivery system, a preparation method thereof and application of the macrophage-polymer lipid nanoparticle composite drug delivery system in cryptococcus infection resistance. According to the drug delivery system, alpha-linolenic acid (ALA), high molecular weight chitosan oligosaccharide (HCOS) and amphotericin B (AmB) are used as raw materials to construct nanoparticles AmB-PLHNs, the nanoparticles are loaded in macrophages through the phagocytosis of the macrophages, and the AmB-PLHNs (at) M1 is constructed. AmB is a common medicine for clinically treating Cn infection, but has the problems of toxic and side effects on kidney and incapability of being transferred into the center. The AmB-PLHNs (at) M1 carries AmB-PLHNs to be transferred into the brain according to the blood-brain barrier penetrating characteristic of M1 type macrophages, and the brain entering efficiency of the medicine is improved. By combining the phagocytosis of macrophages on cryptococcus, the inflammation immune regulation function of the macrophages and the bacteriostatic action of the drug-loaded nanoparticles, the drug-loaded nano-particles are used for treating Cn central infection, and the toxic and side effects of AmB are reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of targeted delivery systems, and in particular to a macrophage-polymer lipid nanoparticle composite drug delivery system, a preparation method thereof, and application thereof in resisting cryptococcal infection. Background Art

[0002] Cryptococcus neoformans (Cn) is the primary pathogen causing cryptococcal meningitis and disseminated invasive fungal disease. Cn has a complex pathogenic mechanism, capable of escaping immune function through capsular polysaccharides and exploiting the macrophage "Trojan Horse" mechanism to cross the blood-brain barrier (BBB) and initiate central nervous system infection. Amphotericin B (AmB) is a commonly used drug for the clinical treatment of Cn infection, but it is associated with nephrotoxic side effects, hematologic toxicity, and a lack of central nervous system transport. Summary of the Invention

[0003] To address the above technical problems, the present invention provides a macrophage-polymer lipid nanoparticle composite drug delivery system, its preparation method, and its application in combating cryptococcal infection. The present invention utilizes polymer-lipid hybrid nanoparticles (PLHNs), a nanocarrier that combines the properties of liposomes and polymer nanoparticles. M1 macrophages are loaded with AmB-PLHNs to construct a living cell-nanoparticle composite drug delivery system. ① Utilizing the natural ability of M1 macrophages to cross the blood-brain barrier, nanoparticles are delivered to the brain in a targeted manner, reducing nonspecific distribution, increasing the effective drug concentration in the brain while lowering systemic toxicity. ② M1 polarization enhances the macrophage's ability to internalize Cryptococcus, resulting in intracellular AmB-PLHNs exhibiting superior antibacterial activity to AmB, effectively disrupting biofilm growth and inhibiting Cryptococcus proliferation. ③ M1 macrophages simultaneously upregulate proinflammatory cytokines to inhibit pathogen growth, recruit immune cells such as neutrophils to the site of infection, and modulate late-stage inflammatory responses, thereby enhancing the host's ability to combat Cryptococcus infection. Provide new ideas and experimental basis for the treatment of central fungal infections.

[0004] The first object of the present invention is to provide a method for preparing a macrophage-polymer lipid nanoparticle composite drug delivery system, comprising the following steps:

[0005] Amphotericin B, hydrogenated soybean phospholipids (HSPC), egg yolk phosphatidylcholine (EPC), cholesterol (CHOL), and α-linolenic acid (ALA) were dissolved in a mixed solvent, and then evaporation was performed in the dark, followed by addition of a buffer solution for hydration to obtain hydrated AmB liposomes, which were then filtered through a membrane to obtain ALA-Lips.

[0006] The high molecular weight chitosan oligosaccharide HCOS solution was added dropwise to the ALA-Lips, mixed and stirred to obtain AmB-PLHNs;

[0007] M1 macrophages were co-incubated with the AmB-PLHNs, and then washed to obtain the macrophage-polymer lipid nanoparticle composite drug delivery system (AmB-PLHNs@M1).

[0008] In some embodiments of the present invention, the mass ratio of amphotericin B, hydrogenated soybean lecithin, egg yolk lecithin, cholesterol and α-linolenic acid is (0.1-1): (1-5): (1-5): (0.5-3): (0.5-3).

[0009] In some embodiments of the present invention, the mixed solvent includes methanol and one or more of chloroform, ethanol, ether, and dichloromethane;

[0010] The temperature of rotary evaporation in the dark is 20-50℃ and the time is 0.5-3h;

[0011] The number of filtration times is 15-35 times, and the filter membrane is a 0.1-0.2 μm filter membrane.

[0012] In some embodiments of the present invention, the buffer solution includes a PBS buffer solution; the pH of the PBS buffer solution is 7.4; the concentration of the high molecular weight chitosan oligosaccharide HCOS solution is 0.1-0.5 mg / mL; and the molecular weight of the chitosan oligosaccharide HCOS in the high molecular weight chitosan oligosaccharide HCOS solution is 2kDa-30kDa.

[0013] In some embodiments of the present invention, the volume ratio of the ALA-Lips to the high molecular weight chitosan oligosaccharide HCOS solution is 2:1-5.

[0014] In some embodiments of the present invention, the mixing speed is 50-350 rpm and the time is 0.1-5 h.

[0015] In some embodiments of the present invention, the inoculation amount of M1 macrophages in the co-culture is 0.5×10 6 -5×10 6 cells / mL.

[0016] A second objective of the present invention is to provide a macrophage-polymer lipid nanoparticle composite drug delivery system comprising nanoparticles AmB-PLHNs and M1 macrophages. The nanoparticles are recognized and bound by macrophages via mannose receptors on their surfaces, activating their phagocytic function. Complete M1 macrophage morphology can be observed, and the nanoparticles are present in the cytoplasm of the M1 macrophages.

[0017] The third object of the present invention is to provide an antibacterial agent comprising the macrophage-polymer lipid nanoparticle composite drug delivery system.

[0018] In some embodiments of the present invention, the bacterial species in the antibacterial agent is Cryptococcus.

[0019] The above technical solution of the present invention has the following advantages over the prior art:

[0020] The present invention prepares PLHNs using alpha-linoleic acid (ALA) and high molecular weight chitosan oligosaccharide lactate (HCOS), which can be recognized and bound by macrophages through the mannose receptors on the surface of macrophages, thereby activating the phagocytic function of macrophages. In cell-mediated nanoparticle delivery systems (CMNDs), macrophages have become a commonly used cell carrier due to their good phagocytic function and immunoregulatory ability. M1 macrophages have a stronger uptake capacity and secrete cytokines such as TNF-α, IL-1β, and IL-6, which effectively regulate antibacterial immune responses. At the same time, macrophages increase BBB transport under fungal infection conditions. The mechanism is that pathogens trigger endothelial cell activation to express the adhesion molecule VCAM, and the macrophage surface receptor integrin VLA-4 binds to VCAM-1.

[0021] The present invention constructs an M1 macrophage-polymer lipid nanoparticle (AmB-PLHNs@M1) central nervous system targeted drug delivery system, prepares polymer lipid nanoparticles PLHNs with α-linolenic acid and chitosan oligosaccharide and loads AmB. M1 macrophages take up PLHNs, and the system combines the inflammatory immunoregulatory function of macrophages with the antibacterial effect of drug-loaded nanoparticles to treat Cn central nervous system infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0023] Figure 1 These are optical microscopic images of the macrophages of the present invention (Scale bar 100 μm); (A) M0 macrophages; (B) M1 macrophages.

[0024] Figure 2Flow cytometry was used to detect the expression of CD86 in LPS-induced RAW 264.7 cells (n=3); (A) Flow cytometric plot of CD11b and CD86 in RAW 264.7 cells; (B) Flow cytometric overlay histogram of CD86; (C) CD86 positivity rate ***P<0.001.

[0025] Figure 3 ELISA of the present invention detects the secretion of TNF-α and IL-6 in RAW264.7 cells induced by LPS (n=3) (A) Secretion of TNF-α; (B) Secretion of IL-6 ns P<0.05, * P<0.05, ** P<0.01, *** P<0.001.

[0026] Figure 4 Comparison of the particle size and Zeta potential of different nanoparticles of the present invention (n=3), (A) particle size; (B) Zeta potential *** P<0.001.

[0027] Figure 5 Transmission electron microscopy images of the nanoparticles of the present invention (Scale bar 100 nm): (A) AmB-Lips; (B) AmB-PLHNs.

[0028] Figure 6 The UV-Vis graph and standard curve of AmB of the present invention (A) (a) Blank-PLHNs solution (b) AmB standard (c) AmB-PLHNs test sample; (B) UV-Vis graph of AmB; (C) Standard curve of the AmB UV-Vis analysis method.

[0029] Figure 7 Figure 3 shows the stability of AmB-PLHNs of the present invention (n=3). (A) Comparison of the solubility and dispersibility of AmB-PLHNs and AmB; (B) Particle size of AmB-PLHNs in 10% FBS (a) and RPMI-1640 (b) ns P>0.05.

[0030] Figure 8 The cytotoxic effects of different drug treatment groups of the present invention on RAW264.7 cells (n=3) are shown. * P<0.05, *** P<0.001, ns P>0.05.

[0031] Figure 9The effects of different drug treatment groups of the present invention on the hemolytic activity of RAW264.7 (n=3), (A) Hemolytic images of AmB and AmB-PLHNs at the same concentration; (B) Hemolytic activity analysis of AmB and AmB-PLHNs at the same concentration *** P<0.001.

[0032] Figure 10 Transmission electron microscopy images of AmB-PLHNs@M1 of the present invention (A) Scale bar 2 μm (white squares are the area in Figure B); (B) Scale bar 400 nm (white arrows indicate AmB-PLHNs).

[0033] Figure 11 HPLC chromatograms of AmB of the present invention, (A) Blank-PLHNs@M1 solution; (B) AmB standard; (C) AmB-PLHNs@M1 test sample.

[0034] Figure 12 is the standard curve of the AmB HPLC analysis method of the present invention (n=3).

[0035] Figure 13 This is the optimal prescription of AmB-PLHNs@M1 screened by the star-response surface methodology of the present invention.

[0036] Figure 14 This is the LSCM image of the fluorescence co-localization of AmB-PLHNs and M1 macrophages at different times of the present invention: blue: cell nucleus; green: AmB-PLHNs; red: M1 macrophages (Scale bar 50 μm).

[0037] Figure 15 In vitro release of AmB-PLHNs@M1 of the present invention (A) AmB released by M1 macrophages and the remaining intracellular content (n=3); (B) Microscopic images of the fluorescence expression of AmB-PLHNs in M1 macrophages at different times (Scalebar 200 μm).

[0038] Figure 16 The MIC of AmB-PLHNs@M1, AmB-PLHNs and free drug groups of the present invention is 80 (n=3).

[0039] Figure 17 Figure 2 is the time-growth curve and 24h colony count of different drug treatment groups of the present invention (n=3), (A) time-growth curve; (B) 24h colony count, ns P>0.05, ** P<0.01, *** P<0.001.

[0040] Figure 18 is the biofilm inhibition rate of the different drug treatment groups of the present invention (n=3), ns P>0.05, *** P<0.001.

[0041] Figure 19 is the bacterial load in the brain, kidney and liver tissues of the Cn infection model of the present invention (n=5).

[0042] Figure 20 These are the in vivo imaging and ex vivo organ imaging results of intravenously injected PLHNs and PLHNs@M1 of the present invention (n=3).

[0043] Figure 21 is the bacterial load in the brain, liver and kidney of C57 mice in different drug administration groups of the present invention (n=6), ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0044] Figure 22 Figure 2 is the weight change of C57 mice in different drug administration groups of the present invention (n=10), (A) weight curve; (B) weight change rate within 6 days; ns P>0.05, **P<0.01, ****P<0.0001.

[0045] Figure 23 These are PAS staining images of brain tissues after treatment in different drug administration groups of the present invention (Scale bar 1.25 mm, 200 μm).

[0046] Figure 24 The expression of cytokines in the blood of different drug administration groups of the present invention (n=5) ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0047] Figure 25 The expression of cytokines in brain tissue of different drug administration groups of the present invention (n=5) ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0048] Figure 26 The immunofluorescence assay of the present invention detected the expression of CD11b, CD86, and CD4 in brain tissue (Scale bar 2.5 mm, Scale bar 600 μm). DETAILED DESCRIPTION

[0049] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0050] Example

[0051] 1. Induction and identification of M1 macrophages

[0052] a. Induction of M1 macrophages: RAW 264.7 cells were taken at 2×10 5 The cells were seeded into 6-well plates and incubated for 24 hours. The supernatant was discarded, the cells were washed with PBS, and 2 mL of DMEM complete medium (containing 0.4 mL of LPS, 1 μg / mL) was added and incubated for 24 hours to obtain M1 macrophages.

[0053] b. Preparation of PLHNs: PLHNs were prepared by the thin-film dispersion-electrostatic adsorption method. Phosphatidylcholine, CHOL, and ALA were dissolved in a mixed solvent (methanol:chloroform = 1:1, v / v) at a mass ratio of 6:1:1. Rotary evaporation (60 rpm) was performed at 45°C to form a homogeneous lipid structure. Residual organic solvent was removed by thin-film nitrogen purge for 15 minutes or overnight in a fume hood. 5 mL of PBS was added, and the mixture was hydrated at 55°C for 45 minutes. Ultrasonication was performed at 200 W for 5 minutes. ALA liposomes were prepared by extrusion through a 0.1 μm filter membrane. HCOS solution (0.25 mg / mL to 1 mg / mL, pH 7.4) was mixed with the liposome suspension at a volume ratio of 1:1. The mixture was magnetically stirred at 300 rpm for 30 minutes to allow electrostatic adsorption of the positively charged chitosan oligosaccharide onto the liposome surface. PLHNs of three different particle sizes were prepared, sterilized through a 0.22 μm filter membrane, and stored at 4°C in the dark.

[0054] c. Preparation of PLHNs@M1 drug delivery system: M1 macrophages were cultured at 1.5×10 6 cells / mL were inoculated into 6-well plates. After 12 hours, the culture medium was aspirated and the cells were washed three times with PBS. PLHNs nanoparticles were incubated with the cells for 2 hours, the solution was aspirated, and the cells were washed three times with PBS to obtain PLHNs-loaded M1 macrophages (PLHNs@M1).

[0055] d. Flow cytometry was used to detect the expression of CD11b and CD86 on macrophages.

[0056] (1) Blocking: M0 macrophages (RAW264.7 cells in their original state without stimulation are called M0 macrophages), M1 macrophages, and PLHNs@M1 samples were washed three times with PBS. An appropriate amount of PBS was added to each well to blow off the cells and collect them into a centrifuge tube. Centrifuge at 300g for 5 minutes. The supernatant was discarded, the cells were resuspended in PBS and counted. 1×106 The cells were placed in a centrifuge tube, centrifuged and the supernatant was discarded. 0.1 mL PBS and 2 μL PEAnti-CD11b were added to each centrifuge tube, blocked at 4°C for 10 min, and washed with PBS three times.

[0057] (2) Extracellular staining: Add 0.1 mL PBS and 2.5 μL APCAnti-CD86 to a centrifuge tube and incubate at 4°C for 20 min. Wash 3 times with PBS, resuspend in 0.5 mL PBS, and detect using a flow cytometer. The above operations must be performed in the dark to prevent quenching of the fluorescent dye. The experimental results are shown in Figure 1 .

[0058] like Figure 1 As shown in the figure, M0 macrophages grow semi-adherently, are round or oval, with a cell body diameter of about 15 to 25 μm, uniform cytoplasm without obvious protrusions, a moderate nuclear-cytoplasmic ratio, and an oval or kidney-shaped nucleus. M1 macrophages grow semi-adherently, with a cell body diameter of about 30 to 40 μm and a polymorphic morphology, such as star-shaped, spindle-shaped or irregular pseudopodia extension, increased cytoplasmic particles, condensed nuclear chromatin and obvious nucleoli. Flow cytometry was used to analyze the expression of macrophage markers CD11b and M1 marker CD86, as shown in the figure. Figure 2 As shown, LPS-induced M1 macrophages showed a significant increase in CD86 expression (P < 0.001), accounting for 63.37 ± 3.16% of the total cell population. The PLHNs@M1 group showed an increase in CD86 expression of 84.27 ± 5.04%, significantly superior to LPS-induced M1 macrophages (P < 0.001). These results demonstrate that LPS successfully induced M1 macrophages and that PLHN uptake promotes M1 polarization of macrophages.

[0059] 2.ELISA detection of IL-6 and TNF-α secretion by M1 macrophages

[0060] a. Follow the instructions of the IL-6 ELISA kit:

[0061] (1) 100 μL of gradient standard was added to a 96-well plate. 100 μL of supernatant from the above-mentioned M0 macrophages, M1 macrophages, and PLHNs@M1 were added to a 96-well plate. Each sample was plated in triplicate and incubated at 37°C for 90 min.

[0062] (2) Discard the liquid, pat dry with absorbent paper, fill each well with washing solution, let it stand for 1 minute, shake off the washing solution, pat dry with absorbent paper, and repeat this washing process twice.

[0063] (3) Add 100 μL of biotin-antibody working solution to each well, incubate at 37°C for 60 min, and soak and wash the plate three times.

[0064] (4) Add 100 μL of HRP-labeled IL-6 antibody to each well, incubate at 37°C for 30 min, and soak and wash the plate 5 times.

[0065] (5) Add 90 μL of TMB chromogenic substrate to each well and incubate at 37°C in the dark for 15 min.

[0066] (6) Add 50 μL of the reaction stop solution to each well, measure the OD value of each well at a wavelength of 450 nm within 15 min, and draw a standard curve to calculate the IL-6 concentration of the sample.

[0067] b. TNF-α ELISA experimental method is the same as above.

[0068] The results are as follows Figure 3 As shown, the expression levels of IL-6 (P < 0.01) and TNF-α (P < 0.001) were significantly increased in the M1 macrophage group and the PLHNs@M1 group. The expression level of TNF-α (P < 0.05) was significantly increased in the PLHNs@M1 group compared to the M1 macrophage group. These results further confirmed the induction of M1 macrophages and that the ingestion of PLHNs enhanced the ability of M1 macrophages to secrete TNF-α.

[0069] 3. Preparation and Characterization of AmB-PLHNs

[0070] (1) Preparation

[0071] Preparation of AmB-PC-Lips: Accurately weigh 10 mg each of AmB, HSPC, EPC, and CHOL to prepare a stock solution. A volume of each stock solution was taken to ensure a concentration of 0.5 mg AmB, 3 mg EPC, 3 mg HSPC, and 1 mg CHOL. The solution was dissolved in 25 mL of a mixed solvent (methanol:chloroform = 1:1, v / v). The solution was rotary evaporated at 45°C in the dark for 1 h, followed by hydration with 5 mL of PBS (pH 7.4). The hydrated AmB liposomes were passed through a 0.1 μm filter membrane 25 times through an extruder to prepare AmB-PC-Lips.

[0072] Preparation of AmB-Lips: Accurately weigh 10 mg each of AmB, HSPC, EPC, CHOL, and ALA to prepare a stock solution of defined concentrations. A specific volume of each solution, containing 0.5 mg of AmB, 3 mg of EPC, 3 mg of HSPC, 1 mg of CHOL, and 1 mg of ALA, was mixed and dissolved in 25 mL of a mixed solvent (methanol:chloroform = 1:1, v / v). The solution was rotary evaporated at 45°C in the dark for 1 hour, followed by hydration with 5 mL of PBS (pH 7.4). The hydrated AmB liposomes were filtered 25 times through a 0.1 μm filter membrane using an extruder to prepare AmB-Lips.

[0073] Preparation of AmB-PLHNs: AmB-Lips were prepared using the same method as above. 10 mg of HCOS was accurately weighed and dissolved in 5 mL of deionized water. The volume was then brought to 10 mL with deionized water and diluted to 0.25 mg / mL to obtain an HCOS solution. The HCOS solution was slowly added dropwise to AmB-Lips (AmB-Lips:HCOS = 1:1, v / v) with magnetic stirring at 300 rpm for 5 minutes. The mixture was stirred for 0.5 hours to obtain AmB-PLHNs (polymer lipid nanoparticles, α-linolenic acid liposomes modified with HCOS) via electrostatic adsorption.

[0074] (2) Characterization

[0075] a. Particle size and Zeta potential determination: The particle size and Zeta potential of AmB-PC-Lips (without ALA), AmB-Lips, and AmB-PLHNs were measured using a Malvern particle size analyzer. The particle size and Zeta potential of AmB-PC-Lips (without ALA), AmB-Lips, and AmB-PLHNs are shown in Table 1. Figure 4 As shown in the results, the particle size of AmB-Lips increased significantly (P < 0.001) and the negative charge increased significantly (P < 0.001) with the addition of ALA. HCOS electrostatically adsorbed on the surface of AmB-Lips caused the particle size of AmB-PLHNs to increase significantly (P < 0.001) and become positively charged.

[0076] Table 1 Particle size and Zeta potential of different nanoparticles ( n=3)

[0077]

[0078] b. TEM observation: AmB-Lips and AmB-PLHNs were negatively stained with sodium phosphotungstate (pH 7.4) and observed under a transmission electron microscope. Figure 5 , AmB-Lips (A) are irregular elliptical liposomes, and AmB-PLHNs are irregular elliptical with a shell-like structure.

[0079] c. Determination of AmB content in AmB-PLHNs by UV spectrophotometry:

[0080] ① Ultraviolet spectrophotometry (UV-Vis) conditions

[0081] The UV spectrophotometer scanned the drug at all wavelengths, showing that the maximum absorption wavelength of AmB was 408 nm.

[0082] ② Preparation of solution

[0083] Standard stock solution: Accurately weigh 0.5 mg of AmB and add deionized water to make up to 10 mL to obtain an AmB standard stock solution with a concentration of 50 μg / mL.

[0084] Test solution: Prepare AmB-PLHNs by centrifuging the supernatant and filtering. The filtrate is used as the drug-loaded test solution 1. Add the AmB-PLHNs to a mixed solvent (methanol:chloroform = 1:1, v / v), vortex extract for 1 minute, and disrupt with ultrasonication. Centrifuge at 10,000 rpm for 5 minutes. Filter the supernatant and filter through a 0.22 μm polyethersulfone filter membrane to prepare test solution 2.

[0085] Preparation of blank polymeric lipid nanoparticles (PLHNs): 10 mg each of HSPC, EPC, CHOL, and ALA were precisely weighed and prepared into a stock solution of defined concentrations. A specific volume of each solution was mixed to yield 3 mg of EPC, 3 mg of HSPC, 1 mg of CHOL, and 1 mg of ALA. The solution was dissolved in 25 mL of a mixed solvent (methanol:chloroform = 1:1, v / v). The solution was then rotary evaporated at 45°C in the dark for 1 hour and then hydrated with 5 mL of PBS (pH 7.4). The hydrated AmB liposomes were filtered through a 0.1 μm filter membrane 25 times through an extruder to prepare ALA-Lips. 10 mg of HCOS was precisely weighed and dissolved in 5 mL of deionized water. The solution was then diluted to 10 mL with deionized water and diluted to 0.25 mg / mL to obtain the HCOS solution. Take the HCOS solution and slowly add ALA-Lips (ALA-Lips:HCOS=1:1, v / v) to mix, the magnetic stirring speed is 300 rpm, the addition time is 5 min, and the stirring reaction is 0.5 h. Blank-PLHNs (polymer lipid nanoparticles, which are α-linolenic acid liposomes modified with HCOS) are obtained by electrostatic adsorption.

[0086] Blank matrix solution: 1 mL of blank polymer lipid nanoparticles (PLHNs) was added to a mixed solvent (methanol:chloroform = 1:1, v / v). Vortex extraction was performed for 1 min, followed by ultrasonic disruption. Centrifugation was performed at 10,000 rpm for 5 min, and the supernatant was filtered and passed through a 0.22 μm polyethersulfone filter membrane as blank matrix solution 1.

[0087] ③Standard curve

[0088] Accurately weigh 0.5 mg of AmB reference substance, dissolve it completely in a small amount of DMSO, and then dilute to 10 mL with methanol to prepare a stock solution. Accurately aspirate the AmB stock solution and dilute it with methanol to a series of standard solutions with concentrations of 1.56, 3.13, 6.25, 12.50, 25.00, and 40.00 μg / mL. Pass each standard solution through a 0.45 μm microporous filter. Measure the absorbance (A) at 408 nm for each concentration using UV-visible light. Perform linear regression with the corresponding concentration (C) to calculate the regression equation.

[0089] ④Specificity inspection

[0090] Take 1 mL each of the above AmB standard solution, blank matrix solution 1, drug-loaded test solution 1, and drug-loaded test solution 2, and perform UV-vis full wavelength scanning to record the spectrum. The results are as follows Figure 6 As shown, Figure 6 As shown in (A), the above method has good specificity. The blank carrier solution has no absorption in the wavelength range of 350-450 nm and has no effect on the determination of AmB in AmB-PLHNs. Figure 6 (B), the linear regression equation is A=0.0352C+0.006, R 2 =0.9995, demonstrating a good linear relationship between absorbance and concentration within the concentration range of 1.56–50.00 μg / mL. (A) (a) Blank-PLHNs solution; (b) AmB standard; (c) AmB-PLHNs test sample; (B) UV-Vis spectrogram of AmB; (C) Standard curve for the AmB UV-Vis analysis method.

[0091] ⑤Precision experiment

[0092] Accurately weigh 0.5 mg of AmB reference solution, dissolve it in a small amount of DMSO, and dilute it with methanol to sample solutions with concentrations of 12.50, 25.00, and 40.00 μg / mL, respectively. Three consecutive measurements were performed, and the absorbance at 408 nm was recorded and the precision was calculated. The experimental results are shown in Table 2. Both the intra-day and inter-day RSDs were less than 2.0%, meeting the precision requirements.

[0093] Table 2 Precision of AmB UV-Vis analysis method ( n=3)

[0094]

[0095]

[0096] ⑥ Recovery rate experiment

[0097] Accurately weigh AmB standard and add it to the prescribed amount of PLHNs. Dissolve completely in DMSO and dilute with methanol to concentrations of 12.50, 25.00, and 40.00 μg / mL, respectively. Inject the sample under UV-visible conditions for detection. Calculate the recovery using the following formula 2-1 based on the measurement results and the amount added.

[0098]

[0099] The experimental results are shown in Table 3. The recoveries met the requirements of 90-110%, and the RSD values were all less than 2.0%. The above results show that the liquid phase method is accurate and reliable and can be used for the quantitative detection of AmB.

[0100] Table 3 Recovery of AmB UV-Vis analysis method ( n=3)

[0101]

[0102] ⑦Stability test

[0103] A methanol solution of AmB with a concentration of 40 μg / mL was prepared, and samples were taken at 0, 2, 4, 6 h and 1, 3, 5, and 7 days, and the content of AmB in the solution was determined by UV-vis. The AmB solution and AmB-PLHNs were stored at 4°C, and the particle size of the two solutions was measured at different times to detect the long-term stability of the nanoparticles. The AmB and AmB-PLHNs solutions were stored at room temperature, and photographed after 24 h to observe the dispersion in the solution. AmB-PLHNs were incubated with 10% FBS and RPMI 1640 culture medium at room temperature in a 1:1 ratio to evaluate their biological stability. The experimental results are shown in Tables 4 and Figure 7 As shown, the RSD values are all less than 2.0%, indicating that the samples are basically stable in the long term and short term. Figure 7 As shown in (A), after standing at room temperature for 24 hours, the AmB-PLHNs solution was still evenly dispersed, and AmB was clearly precipitated at the bottom of the bottle. Figure 7 As shown in (B), the particle size of AmB-PLHNs did not change significantly within 24 h in 10% FBS (a) and RPMI-1640 (b) culture media (P>0.05, P>0.05).

[0104] Table 4 Short-term and long-term stability of AmB-PLHNs ( n=3)

[0105]

[0106]

[0107] ⑧Establishment of encapsulation efficiency and drug loading determination methods

[0108] Take test solution 1 and test solution 2, determine the AmB content therein, and calculate the encapsulation efficiency and drug loading according to the following formula.

[0109]

[0110] The supernatant of AmB-PLHNs was collected and the AmB content was determined. The calculated AmB encapsulation efficiency was (85.58±3.98)%, and the drug loading was (2.78±0.07)%.

[0111] Cytotoxicity investigation of AmB-PLHNs:

[0112] ① M1 macrophage proliferation ability detection: CCK-8 method was used to detect the effect of AmB-PLHNs on the proliferation ability of M1 macrophages. M1 macrophages (5×10 3 cells / well), and AmB-PLHNs containing different AmB concentrations (0.25, 0.25×10 -1 , 0.25×10 -2 The cells were incubated with 4% paraformaldehyde (P<0.05) at 4% paraformaldehyde (P<0.05) for 24 hours (n=3). The drug-containing culture medium was discarded, and 10 μL of CCK8 solution in DMEM complete medium was added to each well. A blank control was set up and incubated at 37°C in the dark for 0.5-2 hours. The absorbance at 450 nm (Abs) was measured using a microplate reader, and the relative cell survival rate was calculated according to the following formula.

[0113] Relative survival rate (%) = (Abs test - Abs blank) / (Abs control - Abs blank) × 100%

[0114] Where Abs test represents the absorbance value of the sample group, Abs control represents the absorbance value of the control group, and Abs blank represents the absorbance value of the blank group.

[0115] CCK-8 kit was used to detect the effects of AmB and AmB-PLHNs on cell proliferation. Figure 8 As shown in the figure, 0.25 μg / mL AmB was highly toxic to RAW 264.7 cells, with a relative survival rate below 60%. As the drug concentration decreased, the cytotoxicity weakened, while AmB-PLHNs and Blank-PLHNs at the same concentration had almost no toxicity to RAW264.7 cells (relative survival rate > 90%). -1 At 4 μg / mL, there were significant differences in the toxicity of AmB, AmB-PLHNs and Blank-PLHNs to RAW264.7 cells (P < 0.001), and the cell proliferation rate in the Blank-PLHNs group reached 120% (P < 0.001).

[0116] ② Hemolytic Activity Assay: 5% rat erythrocytes were washed three times with equal volumes of PBS (pH 7.2). Equal volumes of AmB and AmB-PLHNs were incubated with rat erythrocytes at 37°C for 30 minutes. The cells were centrifuged at 5000 rpm for 5 minutes. The absorbance of the supernatant was measured at 405 nm using a microplate reader. The hemolytic activity of the drugs was calculated according to the following formula.

[0117] Hemolysis rate (%) = (Ax-A_N) / (Aw-A_N) × 100%

[0118] Ax is the absorbance of the sample-treated erythrocyte supernatant, Aw is the absorbance of erythrocytes treated with deionized water, representing 100% hemolysis, and AN is the absorbance of untreated erythrocytes, representing 0% hemolysis. The hemolytic activity of AmB-PLHNs on 5% rat erythrocytes was tested. Compared with the AmB group, the hemolytic activity of the AmB-PLHNs group on rat erythrocytes was significantly reduced (P < 0.001). Figure 9 As shown. When the drug concentration was greater than 12.50 μg / mL, the AmB group exhibited significant hemolytic effects on rat erythrocytes compared with the AmB-PLHNs and PBS groups (P < 0.001). The AmB-PLHNs group (AmB, 25.00 μg / mL) showed no hemolytic effect on erythrocytes, with no difference from the PBS group (P > 0.05). These results demonstrate that AmB-PLHNs overcome the hemolytic disadvantage of AmB and reduce its toxic side effects on erythrocytes.

[0119] 4. Preparation and Characterization of AmB-PLHNs@M1

[0120] (1) Preparation

[0121] M1 macrophages were counted at 1.5×10 6 cells / mL were seeded in a 6-well plate. After 12 hours, the culture medium was aspirated and the cells were washed three times with PBS. AmB-PLHNs were incubated with the cells for 2 hours, the solution was aspirated and the cells were washed three times with PBS to obtain AmB-PLHNs@M1.

[0122] (2) Characterization

[0123] TEM observation: AmB-PLHNs@M1 samples were fixed in 2.5% glutaraldehyde at 4°C for 24 hours and in 1% osmium sulfate fixative at 4°C for 2 hours. The samples were rinsed three times with 0.1M phosphate buffer (PBS) for 15 minutes each, then dehydrated in 30%, 50%, 70%, and 90% ethanol at 4°C for 15 minutes each, followed by two dehydrations in 100% ethanol for 20 minutes each. The dehydrated samples were washed three times in pure propylene oxide for 15 minutes each. The samples were then placed in a mixture of pure propylene oxide / embedding solution (1:1) for 3 hours, a mixture of acetone / embedding solution (1:3) for 4 hours, and pure embedding solution at 37°C overnight. The embedded samples were then placed in a 70°C oven and allowed to polymerize for 12 to 48 hours. Prepare ultrathin sections, spread them on a copper grid with a support film, stain with uranyl acetate for 8-15 minutes, stain with lead citrate for 5-10 minutes, and observe and photograph them under a microscope after drying. Figure 10 The complete morphology of M1 macrophages can be observed, and nanoparticles are present in the cytoplasm of M1 macrophages, indicating that M1 macrophages can take up AmB-PLHNs.

[0124] HPLC determination of AmB content in AmB-PLHNs@M1:

[0125] ① High-performance liquid chromatography (HPLC) conditions: the chromatographic column was a Diamonsil C18 column (250 mm × 4.6 mm, 5 μm), the mobile phase was acetonitrile-20 mM disodium EDTA solution (35:65), the flow rate was 1.0 mL / min, the detection wavelength was 410 nm, the injection volume was 20 μL, and the column temperature was 30°C.

[0126] Experimental results: The AmB content in AmB-PLHNs@M1 was determined by HPLC to be (6.23±0.05)μg / 3×10 6 cells.

[0127] ② Preparation of solution

[0128] Standard stock solution: Accurately weigh 0.5 mg of AmB and add deionized water to make up to 10 mL to obtain an AmB standard stock solution with a concentration of 50 μg / mL.

[0129] Test solution: Accurately weigh 10 mg each of AmB, HSPC, EPC, CHOL, and ALA to prepare a stock solution of defined concentrations. A specific volume of each solution was mixed to obtain a stock solution containing 0.5 mg of AmB, 3 mg of EPC, 3 mg of HSPC, 1 mg of CHOL, and 1 mg of ALA. The solution was dissolved in 25 mL of a mixed solvent (methanol:chloroform = 1:1, v / v). The solution was rotary evaporated at 45°C in the dark for 1 h, followed by hydration with 5 mL of PBS (pH 7.4). The hydrated AmB liposomes were filtered through an extruder 25 times with a 0.1 μm membrane to prepare AmB-Lips. Accurately weigh 10 mg of HCOS and dissolve it in 5 mL of deionized water. The volume was then adjusted to 10 mL with deionized water. Dilute to 0.25 mg / mL, take the HCOS solution and slowly add AmB-Lips (AmB-Lips:HCOS=1:1, v / v), magnetic stirring speed is 300 rpm, the addition time is 5 min, stirring reaction is carried out for 0.5 h, and AmB-PLHNs are obtained by electrostatic adsorption. M1 macrophages are 1.5×10 6 cells / mL were inoculated into a 6-well plate. After 12 hours, the culture medium was aspirated and the cells were washed three times with PBS. AmB-PLHNs were incubated with the cells for 2 hours, the solution was aspirated, and the cells were washed three times with PBS to obtain AmB-PLHNs@M1. All samples were added to the chloroform mixed solvent, vortex-extracted for 1 minute, freeze-thawed three times, and disrupted by ultrasonication. After centrifugation at 10,000 rpm for 5 minutes, the supernatant was filtered and passed through a 0.22 μm polyethersulfone filter membrane to serve as test solution 3.

[0130] Blank matrix solution: 1 mL of PLHNs@M1 was centrifuged to precipitate, then resuspended in 0.1 mL of deionized water and incubated at 37°C and 120 rpm for 4 h. The supernatant was centrifuged and filtered, and passed through a 0.22 μm membrane as blank matrix solution 2.

[0131] ③ Standard curve: 100 μL of PLHNs@M1 cell homogenate was added to 100 μL of AmB standard stock solution at various concentrations to prepare AmB standards at concentrations of 0.39 μg / mL, 0.78, 1.56, 3.13, 6.25, 12.50, and 25.00 μg / mL. All samples were added to a mixed solvent (methanol:chloroform = 1:1, v / v), vortexed for 1 minute, freeze-thawed three times, and disrupted by ultrasonication. Centrifugation was performed at 10,000 rpm for 5 minutes, and 20 μL of the supernatant was injected. Detection conditions were the same as those described for the HPLC method above.

[0132] ④ Specificity investigation: Take 20 μL of the above AmB standard solution, blank matrix solution 2 and drug-loaded test solution 3 and inject them into HPLC for determination, and record the chromatogram. Figure 11As shown in the results, the above method has good specificity. The blank carrier solution has no impurity peak at 8.5 min and has no effect on the determination of AmB content in AmB-PLHN.

[0133] ⑤Precision experiment: Accurately weigh 0.5 mg of AmB reference substance, dissolve it in a small amount of DMSO, and dilute it with methanol to sample solutions with concentrations of 5, 15, and 20 μg / mL, respectively. Measure three times continuously, record the peak area, and calculate the precision. Figure 12 As shown in the figure, AmB has a good linear relationship between concentration (C) and peak area (A) in the concentration range of 0.31 to 25.00 μg / mL. The standard curve of the AmB HPLC analysis method is A = 0.051C-0.0097, R 2 =0.999. As shown in Table 5, the intra-day and inter-day RSD values were both less than 2.0%, meeting the precision requirements.

[0134] Table 5 Precision of AmB HPLC analysis method ( n=3)

[0135]

[0136] ⑥ Recovery Experiment: 100 μL of PLHNs@M1 cell homogenate was precisely added to 100 μL of AmB stock solution at different concentrations to prepare AmB solutions at three concentrations (20 μg / mL, 10 μg / mL, and 5 μg / mL). The same procedures were followed as for the standard sample. The recovery was calculated using the same formula 2-1 as above. The results, shown in Table 6, showed recoveries meeting the required range of 90% to 110%, with RSDs of less than 2.0%. These results demonstrate that this liquid phase method is accurate and reliable for the quantitative detection of AmB.

[0137] Table 6 Recovery of AmB HPLC analysis method ( n=3)

[0138]

[0139] ⑦ Establishment of the cell drug loading determination method: Take the test solution 3, determine the AmB content, and calculate the encapsulation efficiency and drug loading according to Formula 2-2 and Formula 2-3.

[0140] The star-point effect surface methodology was used to screen the prescriptions of AmB-PLHNs@M1: The preliminary prescription screening found that ALA quality, HCOS quality, and incubation time were the three most important factors affecting the antibacterial efficiency. The star-point effect surface methodology was used to conduct a three-factor three-level prescription screening design to further screen and optimize the AmB-PLHNs@M1 prescription. With the antibacterial efficiency as the evaluation index, the corresponding prescriptions were prepared and compared to select the optimal prescription. With the inhibition ratio as the index, the star-point effect surface methodology was used to screen the prescriptions of AmB-PLHNs@M1. The results are shown in the figure. Figure 13 The optimal formulation was 0.5 mg of α-linolenic acid, 0.25 mg / mL of chitosan oligosaccharide, and an incubation time of 2 h.

[0141] LSCM detection of fluorescence distribution of AmB-PLHNs and M1 macrophages: Take an appropriate amount of DiO and add it to phospholipids (1:100, w / w) to prepare DiO-PLHNs labeled with fluorescent probes. M1 macrophages were incubated with DiO-PLHNs, and AmB-PLHNs@M1 were prepared according to the above method. LSCM was used to observe the uptake of nanoparticles by macrophages. Laser confocal microscopy was used to observe the distribution of AmB-PLHNs in RAW264.7 cells. Figure 14 As shown in the figure, after 2 hours of co-incubation, AmB-PLHNs were distributed in the cytoplasm of M1 macrophages and had stronger fluorescence than at other time points, while the fluorescence of AmB-PLHNs in the cells was lower after 0.5 hours of incubation. The results showed that when the incubation time was between 5 and 120 minutes, the uptake of AmB PLHNs by M1 macrophages increased with the extension of incubation time.

[0142] In vitro release study of AmB in AmB-PLHNs@M1: AmB-PLHNs@M1 was dispersed in complete culture medium and placed in an incubator at 37°C. The samples were placed in a 24-well culture plate with ultra-low adsorption. At 0, 4, 8, 12, 24, and 48 hours, the samples were collected and centrifuged, and the supernatant and cell pellet were measured. The supernatant and cell samples were processed according to the method in the standard curve, and the samples were injected for detection to calculate the cumulative release of AmB. The in vitro drug release results are shown in Figure 2. Figure 15 As shown in (A), the release rate reached 20% after 4h and 75% after 48h. Figure 15 As shown in (B), the fluorescence intensity of intracellular AmB-PLHNs decreased with time, indicating that AmB can be released from the interior of M1 macrophages.

[0143] M1 macrophages were cultured at a rate of 1.5 × 10 6Cells were seeded / well in a culture dish and incubated with AmB-PLHNs in a constant-temperature incubator for 2 hours. The culture medium was then replaced and culture continued. The culture medium was discarded at 0, 24, and 48 hours, and the cells were washed with PBS. Fluorescence intensity changes were observed under an inverted fluorescence microscope. After each observation, the culture medium was replaced and culture continued.

[0144] 5. In vitro antibacterial efficacy of AmB-PLHNs@M1 drug delivery system

[0145] 5.1. Determination of Minimum Inhibitory Concentration:

[0146] (1) Preparation of bacterial suspension: dilute the bacterial suspension with culture medium to prepare a bacterial suspension with a concentration of 1×10 3 CFU / mL.

[0147] (2) Sample preparation: Prepare different concentration gradients of AmB-PLHNs@M1, AmB-PLHNs, and AmB solutions, and dilute them serially from high to low concentrations. In a 96-well cell culture plate, the drug concentrations were 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 1.25×10 -1 μg / mL, 6.25×10 -2 μg / mL, 3.13×10 -2 μg / mL, 1.57×10 -2 μg / mL, 7.81×10 -3 μg / mL, 3.91×10 -3 μg / mL, 1.95×10 -3 μg / mL, with 3 replicate wells for each concentration.

[0148] (3) Set up positive control wells without drug and negative control wells without bacterial solution, with three replicates.

[0149] (4) Place the 96-well plate at 37°C and incubate for 72 hours.

[0150] (5) Use a microplate reader to measure the optical density (OD) of each well of fungi at 630 nm 630 The OD of the negative control group 630 The value is 100%, and the antibacterial rate calculation formula is as follows:

[0151]

[0152] The results of the minimum inhibitory concentration test were as follows Figure 16 MIC of AmB group 80 The MIC of the physical mixture of AmB and ALA 0.2 mg / mL was 2.50 μg / mL, and the antibacterial effect was relatively weak when used alone. 80was (0.36±0.19)μg / mL, which was lower than the MIC of AmB alone group. 80 The value is reduced by about 10 times. AmB and HCOS physical mixing group MIC 80 When AmB, ALA and HCOS were physically mixed, the MIC of AmB was (1.67±0.59)μg / mL. 80 The MIC values of AmB-PLHNs group and AmB-PLHNs@M1 group were significantly decreased to (0.11±0.07) μg / mL, indicating that the physical mixture of the three has a better antibacterial effect. 80 The MICs of the AmB group were (0.83±0.29) μg / mL, which were significantly higher than the MICs of the AmB group alone. 80 The difference between the two groups was not statistically significant (P<0.0001), indicating that both the AmB-PLHNs group and the AmB-PLHNs@M1 group exerted a better antibacterial effect than AmB.

[0153] 5.2. Time-growth curve:

[0154] (1) Preparation of bacterial suspension: dilute the bacterial suspension with culture medium to prepare a bacterial suspension concentration of 2×10 6 CFU / mL.

[0155] (2) Sample preparation: Prepare AmB-PLHNs@M1, AmB-PLHNs, and AmB solutions with different concentration gradients, and the dilution concentrations are 2.5, 5, and 10 μg / mL, respectively.

[0156] (3) Culturing the sample with H99 Cryptococcus neoformans: Set up a positive control well without drug and a negative control well without bacterial solution. Place the test tube in a 37°C shaking incubator for shaking culture.

[0157] (4) At different time points (0, 4, 8, 12, and 24 h), 50 μL of fungal culture solution was taken from each group, diluted appropriately, and plated on SDA medium. After incubation at 37 °C for 48 h, the number of single colonies was counted, and the bacterial concentration at different culture time points of each group was calculated and the log 10 The values were plotted using Graphpad Prism 8.

[0158] The results of the time-growth curve experiment are as follows Figure 17(A) As shown. The colony count in the Control group increased significantly over time, indicating that H99 grew logarithmically without drug intervention. The colony count growth rate in the 2.5μg / mLAmB group slowed down, indicating that this concentration of AmB had a certain inhibitory effect on the growth of H99, but the effect was relatively weak. The 5μg / mLAmB group had no obvious antibacterial effect in the early stage, and the colony count began to decline after 8h, showing a strong antibacterial activity. The colony count in the 2.5μg / mL AmB-PLHNs group began to fall below the initial value after 4h, and remained at a low level within 12h. After 12h, H99 showed an increasing trend. The colony count in the AmB-PLHNs@M1 group fluctuated greatly at different time points. The colony count decreased significantly at 8h, and it still showed the same antibacterial activity as the 5μg / mLAmB group at 24h. The results of the 24h plate colony counting method showed that ( Figure 17 (B) The colony count of M1 macrophage group was (6.36±0.11)log 10 CFU / mL, which was not statistically significant compared with the Control group (P>0.05), indicating that under this experimental condition, the inhibitory effect of M1 macrophages on the growth of H99 fungi was not significant. The colony count of the AmB group was (5.73±0.09) log 10 The CFU / mL was significantly lower than that of the Control group (P < 0.001), indicating that the AmB group (2.5 μg / mL) had a certain antibacterial effect. At the same time, the colony count of the AmB-PLHNs group was (4.63 ± 0.05) log 10 CFU / mL, AmB-PLHNs@M1 group was (4.26±0.04)log 10 The CFU / mL of AmB-PLHNs and AmB-PLHNs@M1 were significantly lower than those of the AmB group (P < 0.001, P < 0.001), indicating that both the AmB-PLHNs and AmB-PLHNs@M1 groups exerted superior antibacterial effects compared to the AmB group (2.5 μg / mL). In addition, the colony count of the AmB-PLHNs@M1 group was significantly lower than that of the AmB-PLHNs group (P < 0.01), indicating that AmB-PLHNs@M1 exerted a superior antibacterial effect compared to the AmB-PLHNs group (P < 0.01).

[0159] 5.3. Investigation of biofilm formation inhibition:

[0160] (1) Biofilm adhesion: The bacterial suspension was diluted with culture medium until the final bacterial suspension concentration reached 1×10 6 CFU / mL. Incubate in a 37°C incubator for 1.5 h. Aspirate the supernatant and wash three times with PBS.

[0161] (2) Sample preparation: AmB and ALA physical mixture solutions, AmB and HCOS physical mixture groups, AmB-PLHNs@M1, AmB-PLHNs, and AmB solutions with different AmB concentration gradients were prepared and serially diluted from high concentration to low concentration. In a 96-well cell culture plate, the AmB drug concentrations were 1, 0.5, 0.25, and 1.25 × 10 -1 , 6.25×10 -2 , 3.13×10 -2 , 1.57×10 -2 , 7.81×10 -3 , 3.91×10 -3 and 1.95×10 -3 The concentrations of ALA and HCOS were both 0.2 mg / mL.

[0162] (3) Sample and biofilm culture: Set up a positive control well without drug and a negative control well without bacterial solution. Place the test tube in a 37°C shaking incubator at 100 rpm and incubate for 24 h.

[0163] (4) After the incubation period, discard the liquid in the wells and wash three times with PBS. Add 100 μL of MTT solution (5 mg / mL) to each well and place the culture plate back in a 37°C constant temperature incubator for 3 hours in the dark.

[0164] (5) Transfer 80 μL of supernatant from each well to a new 96-well blank plate. Measure the absorbance at 492 nm using a microplate reader to calculate the inhibition rate of biofilm formation and analyze the inhibitory effect of AmB-PLHNs@M1 on H99 biofilm formation. The biofilm inhibition rate calculation formula is as follows:

[0165]

[0166] (6) Compared with the negative control group, OD 492 The lowest drug concentration at which the value is reduced by more than 80% is the SMIC of the drug. 80 value.

[0167] The experimental results are as follows Figure 18 As shown, the SMIC of the AmB group, the AmB and ALA physical mixture group, and the AmB and HCOS physical mixture group 80 The SMIC of AmB-PLHNs group was (2.50±0.00) μg / mL, but the physical mixing of AmB with HCOS and ALA did not enhance its anti-biofilm effect. 80 was (0.32±0.24)μg / mL, that of AmB-PLHNs@M1 group was (0.94±0.44)μg / mL, and the SMIC of AmB-PLHNs group was (0.32±0.24)μg / mL.80 The values were slightly lower than those in the AmB-PLHNs@M1 group and there was no significant difference (P>0.05). 80 The SMIC values of the free drug groups were significantly lower than those of the AmB group (P < 0.001, P < 0.001), the AmB and ALA physical mixture group (P < 0.001, P < 0.001), and the AmB and HCOS physical mixture group (P < 0.001, P < 0.001). 80 The values were 7.81 and 2.66 times that of the AmB-PLHNs group and the AmB-PLHNs@M1 group, respectively, further confirming the superiority of the AmB-PLHNs group in anti-biofilm.

[0168] 6. In vivo pharmacodynamic evaluation of the AmB-PLHNs@M1 drug delivery system

[0169] 6.1. Construction and validation of Cn infection model:

[0170] (1) Preparation of bacterial suspension: Take the H99 from above and inoculate it into YPD medium. Cultivate it at 37℃ with shaking for 18 hours until the logarithmic growth phase. Centrifuge at 5000 rpm for 5 minutes. Wash with PBS three times. Adjust the concentration of bacterial suspension to 1×10 6 CFU / mL.

[0171] (2) Infection model establishment: 0.1 mL of bacterial suspension (1×10 5 CFU / mouse. After infection, the mice's mental state, activity, and hair gloss were observed daily, and changes in body weight were recorded.

[0172] (3) Model verification: Mice were killed on the 7th day after infection, and brain, kidney, and spleen tissues were homogenized and inoculated into SDA medium after gradient dilution. The bacterial load (log 10 CFU / mL).

[0173] On the third day after infection, the animals began to show symptoms such as lethargy, piloerection, decreased activity, and continued weight loss. On the seventh day after infection, the animals were euthanized according to animal ethics requirements, and brain, kidney, and liver tissue homogenates were graded and diluted and inoculated into SDA medium. The bacterial loads in brain, kidney, and liver tissues were (4.03±0.17) log 10 CFU / mL, (3.52±0.17)log 10 CFU / mL, (4.00±0.09)log 10 CFU / mL. Mice developed typical infection symptoms and significantly increased bacterial loads in their organs, indicating that this infection dose can stably establish a systemic Cn infection model in mice.

[0174] 6.2. In vivo targeting investigation of AmB-PLHNs@M1:

[0175] (1) Nanoparticle labeling: AmB-PLHNs@M1 was labeled with DiR (10 μg / mL) fluorescent probe, incubated in the dark for 2 h, and then dialyzed for purification.

[0176] (2) Dosage regimen: A Cn-infected mouse model was established as described above, and mice were randomly divided into the AmB-PLHNs@M1 group and the AmB-PLHNs group, with 3 mice in each group. Starting from day 1 after infection, DiR-labeled AmB-PLHNs@M1 and AmB-PLHNs were injected into the tail vein.

[0177] (3) In vivo imaging: The intensity of brain fluorescence signals was detected using an IVIS system at 2, 4, 8, and 12 h after administration to analyze the accumulation of nanoparticles in brain tissue.

[0178] DiR-labeled PLHNs and PLHNs@M1 were injected into the tail vein to evaluate the in vivo distribution of the PLHNs@M1 drug delivery system in the Cn infection model. DiR-labeled AmB-PLHNs@M1 began to accumulate in the brain 4 hours after administration. Figure 20 As shown, brain fluorescence intensity gradually increased over time in all groups, and at the same time point, the PLHNs@M1 group exhibited higher fluorescence intensity than the PLHNs group. Comparison of major organs across the groups 12 hours after injection revealed significant enrichment of fluorescence in brain tissue in the PLHNs@M1 group compared to the PLHNs group. Fluorescence intensity was highest in the brain, liver, and kidneys, suggesting that PLHNs@M1 was primarily distributed in these infection sites. This is consistent with the bacterial load in major organs in the Cn infection model described above, further demonstrating that the drug delivery system reduces non-target organ distribution and potential toxicity to normal tissues. This demonstrates that PLHNs@M1 possesses BBB transport properties under infection.

[0179] 6.3. Investigation of Organ Bacterial Load

[0180] 6.3. Investigation of Organ Bacterial Load: (1) Dosing and grouping: constructed according to the above method Cn Infected mice were randomly divided into the control group, the AmB group, the AmB-PLHNs group, the AmB-PLHNs@M1 group, and the M1 macrophage group, with six mice in each group. The mice were administered with a dose of 0.25 mg / kg at 24-h intervals for five consecutive doses.

[0181] (2) Sample collection: On the 7th day after infection, animals were euthanized according to animal ethics requirements, and brain, liver, and kidney tissues were removed, weighed, and homogenized in 1 mL of PBS.

[0182] (3) Determination of bacterial load: The homogenate was diluted 10 -1 , 10 -2 , 10 -3 Take 100 μL of each strain and spread it on SDA medium. Culture at 37℃ for 48 h and then count the colonies formed.

[0183] The anti-Cn infection efficacy of AmB-PLHNs@M1 was evaluated by detecting the fungal load in brain, kidney, and liver tissues. Figure 21 The results showed that the bacterial load in the AmB-PLHNs@M1 group in brain tissue was lower than that in the Control group, AmB group, and M1 macrophage group (P<0.0001, P<0.001, P<0.01), and was better than that in the AmB-PLHNs group (P<0.05). In kidney tissue, the bacterial load in the AmB-PLHNs@M1 group was lower than that in the Control group and AmB group (P<0.0001, P<0.01), but there was no statistical difference between the AmB-PLHNs group and the AmB-PLHNs group (P>0.05). In liver tissue, the fungal load in the AmB-PLHNs@M1 group was significantly lower than that in the Control group, AmB group, and AmB-PLHNs group (P<0.001, P<0.001, P<0.001). Compared with the AmB group, the bacterial load in the brain, kidney, and liver of AmB-PLHNs@M1 was significantly reduced (P<0.001, P<0.0001, P<0.0001). AmB-PLHNs@M1 effectively inhibited Cn colonization in the brain, kidney, and liver tissues.

[0184] 6.4. Examination of weight curve:

[0185] A Cn-infected mouse model was established using the methods described above. Mice were randomly divided into a control group, an AmB group, an AmB-PLHNs group, an AmB-PLHNs@M1 group, and an M1 macrophage group, with 10 mice per group. The dose was 0.25 mg / kg, administered 24 hours apart for five consecutive doses. Mice were weighed daily at the same time after infection. When weight loss exceeded 20%, they were euthanized in accordance with animal ethics standards. The rate of weight change was calculated using Formula 4-1 as follows:

[0186]

[0187] Analyze the weight curve ( Figure 22) As can be seen, the body weight of the Control group showed a continuous and rapid decline after infection. Although the AmB group and the M1 macrophage group had a certain intervention effect, the body weight still decreased significantly. Before drug withdrawal, that is, within 6 days of infection, the body weight fluctuations of the AmB-PLHNs@M1 group and the AmB-PLHNs group were more stable (Δ% = 0.98% and 0.58%), and there was an upward trend. The comparison of Δ% within 6 days showed that the M1 macrophage group (P < 0.01), the AmB-PLHNs group (P < 0.0001), and the AmB-PLHNs@M1 group (P < 0.0001) were significantly different from the AmB group. This result indicates that the AmB-PLHNs@M1 drug delivery system inhibited the proliferation and spread of Cn in the body, reduced infection, and alleviated weight loss. After drug withdrawal, the weight loss trend of AmB-PLHNs@M1 mice increased (Δ% > 25%), similar to the terminal stage of the Control group.

[0188] 6.5. Examination of survival curves:

[0189] A Cn-infected mouse model was established using the methods described above. Mice were randomly divided into a control group, an AmB group, an AmB-PLHNs group, an AmB-PLHNs@M1 group, and an M1 macrophage group, with 10 mice in each group. The mice were administered at a dose of 0.25 mg / kg, administered every 24 hours for five consecutive doses. Survival time was recorded, and Kaplan-Meier curves were plotted. Differences between groups were compared using the log-rank test.

[0190] like Figure 23 As shown, PAS staining revealed numerous cavities of varying sizes and acellular structures in brain tissue sections from the control group. Purple-red Cn were densely distributed within these cavities, with diameters >200 μm. Cn-infected brain tissue exhibited cavitation, suggesting damage to brain tissue after fungal invasion of the central nervous system. In the AmB and M1 macrophage groups, the number of cavities in brain tissue was significantly reduced, but cavities were still visible and >200 μm in diameter, indicating that AmB and M1 macrophage groups have a certain inhibitory effect on Cn infection in the brain. Furthermore, cavities were only occasionally observed in AmB-PLHNs tissue, with diameters <200 μm, suggesting that Cn burden in the brain was further reduced compared to the AmB and M1 macrophage groups. In AmB-PLHNs@M1, cavities were almost absent in brain tissue, with diameters <50 μm, indicating that AmB-PLHNs@M1 exhibited superior antibacterial activity to other groups, inhibiting Cn colonization in the central nervous system.

[0191] 6.6. PAS staining of brain tissue:

[0192] 1) Grouping and Sampling: A Cn-infected mouse model was established as described above. Mice were randomly divided into the control group, the AmB group, the AmB-PLHNs group, the AmB-PLHNs@M1 group, and the M1 macrophage group. Mice were administered with a dose of 0.25 mg / kg at 24-hour intervals for five consecutive doses. On day 7 post-infection, mice were deeply anesthetized by intraperitoneal injection of sodium pentobarbital solution and systemically perfused with PBS.

[0193] (2) Sample processing: Fresh brain tissue was fixed with 4% paraformaldehyde for 24 h, dehydrated with graded ethanol, embedded in paraffin, and sliced to a thickness of 5 μm.

[0194] (3) Staining steps: Sections were dewaxed and hydrated, oxidized with periodic acid for 10 min, stained with Schiff reagent for 15 min, and counterstained with hematoxylin for nuclei. The purple-red staining of the fungal cell wall was observed under a microscope to assess the extent of Cn invasion into brain tissue.

[0195] Determination of cytokines in blood Figure 24 As shown. On the 7th day of infection, the serum TNF-α and IL-6 concentrations in the AmB-PLHNs@M1 group were significantly lower than those in the Control group (P<0.0001), but through the analysis of the rate of change (Δ%), IL-6 decreased the most (P<0.0001), and the TNF-α concentration was also significantly lower than that in the AmB group (P<0.01), indicating that the drug delivery system can more effectively inhibit the release of pro-inflammatory factors in the late stage of infection. Through Δ% analysis, the decrease in TNF-α (P<0.05) and IL-6 (P<0.0001) in the AmB-PLHNs@M1 group was significantly better than that in the AmB-PLHNs group, indicating that it effectively alleviated the inflammatory cascade caused by infection. On the first day, the IL-6 concentration in the AmB-PLHNs@M1 group was significantly higher than that in the Control group (P<0.0001), which may activate the early immune response. On the 7th day, the IL-6 concentration decreased, which had the effect of suppressing inflammation. The results of cytokine analysis showed that compared with the first day of infection, the levels of TNF-α (P<0.05) and IL-6 (P<0.0001) in the blood of the AmB-PLHNs@M1 group on the 7th day of infection were significantly lower than those in the AmB-PLHNs group, alleviating the inflammatory response caused by infection.

[0196] 6.7. ELISA determination of cytokine secretion in blood and brain tissue:

[0197] (1) Sample processing: The grouping, dosing, and sampling methods were the same as above. The serum and brain tissue homogenate supernatants of mice were collected on the 1st and 7th days after infection, and the supernatants were centrifuged at 12000 rpm at 4°C for 10 min. The supernatants were aliquoted and stored at -80°C.

[0198] (2) The levels of TNF-α, IL-1β, IL-6, and IL-17A were determined using the same method as described above.

[0199] Western Blot detection of iNOS expression in brain tissue:

[0200] (1) Sample processing: The grouping, dosing, and sampling methods were the same as above. The mouse brain tissues were collected on the 1st and 7th day after infection and stored at -80°C.

[0201] (2) Protein extraction: 0.1 g of brain tissue was collected and added to RIPA lysis buffer (containing 1% PMSF). The mixture was homogenized on ice and allowed to stand for 30 min. The mixture was centrifuged at 12,000 rpm for 15 min at 4°C and the supernatant was collected. The protein concentration was adjusted to 5 μg / μL by BCA assay.

[0202] (3) Western Blot: Total protein from brain tissue was extracted and the concentration was determined by BCA method. Then SDS-PAGE electrophoresis was performed. After transfer to the membrane, iNOS antibody (1:1000) and HRP-labeled secondary antibody (1:5000) were incubated in sequence. ECL color development and exposure were performed.

[0203] The results of cytokine determination in brain tissue are as follows Figure 25 As shown. On the first day of infection, the TNF-α concentration in the AmB-PLHNs@M1 group was higher than that in the Control group (P<0.01), and the IL-1β concentration was higher than that in the AmB and AmB-PLHNs groups (P<0.001, P<0.01). This is consistent with the increased IL-6 concentration in the AmB-PLHNs@M1 group, indicating the activation of an early immune response. AmB-PLHNs@M1 significantly inhibited inflammatory factors in the brain. On the seventh day of infection, the concentrations of TNF-α, IL-1β, IL-6, and IL-7A in the brain of this group were lower than those in the Control group (P<0.01, P<0.01, P<0.01, P<0.05). The secretion of TNF-α, IL-6, and IL-17A was lower than that in the AmB group (P<0.01, P<0.05, P<0.05), demonstrating its potent inhibitory effect on the proinflammatory response in the brain during the late stages of infection. Through Δ% analysis, AmB-PLHNs@M1 inhibited TNF-α and IL-1β in the brain better than AmB-PLHNs (P<0.05, P<0.001), proving that its inhibitory effect on brain inflammation in the late stage of infection was better than that of the AmB-PLHNs group.

[0204] 6.8. Immunofluorescence analysis of CD11b, CD86, and CD45 expression in brain tissue:

[0205] (1) Sample processing: The grouping, administration, and sampling methods were the same as above. On the 7th day after infection, mice were anesthetized by intraperitoneal injection of avertin solution and perfused with normal saline. Fresh brain tissue was obtained, fixed with 4% paraformaldehyde for 24 h, dehydrated with gradient ethanol, embedded in paraffin, and sliced into 5 μm thickness.

[0206] (2) Antigen retrieval: After dewaxing the sections, soak the samples in 0.01 M citrate buffer (pH 6.0), heat in a microwave oven on low heat for 5 min, and wash with PBST three times, 5 min each time.

[0207] (3) Blocking: Incubate cells with PBST containing 1% BSA and 22.52 mg / mL glycine for 30 min, and wash with PBST three times, each time for 5 min.

[0208] (4) Primary antibody incubation: CD11b, CD86, and CD4 primary antibodies were diluted at a ratio of 1:500 and incubated with different sections at 4°C overnight. The sections were then washed with PBST three times for 5 minutes each time.

[0209] (5) Secondary antibody incubation: Fluorescent secondary antibody was diluted at a ratio of 1:500 and incubated with different sections at room temperature in the dark for 2 h. The sections were then washed three times with PBST for 5 min each time.

[0210] (6) Nuclear staining: Add DAPI staining solution, incubate at room temperature in the dark for 5 minutes, and wash with PBST three times, each time for 5 minutes.

[0211] (7) Sealing: Add 5 μL of anti-fluorescence quencher and perform fluorescence imaging after sealing.

[0212] To evaluate the immune regulation of brain inflammatory response after drug administration, the expression of CD11b, CD86, and CD45 was examined. Figure 26 As shown, the control group showed the highest expression of CD11b, CD86, and CD45, followed by the AmB group. The expression of these three proteins was downregulated in the AmB-PLHNs group and the M1 macrophage group compared to the AmB group. The M1 macrophage group showed a more significant downregulation of these three proteins compared to the AmB-PLHNs group. The expression levels of the three proteins in the AmB-PLHNs@M1 group were close to those in the Shame group. The downregulation of CD45 expression in the AmB-PLHNs@M1 group and the M1 macrophage group is speculated to be related to blood-brain barrier repair or inhibition of peripheral immune cell chemotactic signals, thereby reducing inflammatory damage. High expression of CD86 marks the transformation of macrophages to the M1 phenotype. Combined with the above results, this may alleviate brain inflammation by clearing pathogens through the NO pathway. Low expression of CD86 in the AmB-PLHNs@M1 group suggests that AmB-PLHNs@M1 effectively inhibits Cn brain infection, reduces brain inflammation (CD86), and thus promotes recovery.

[0213] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for preparing a macrophage-polymer lipid nanoparticle composite drug delivery system, characterized in that: The following steps are involved: Amphotericin B, hydrogenated soybean phospholipids (HSPC), egg yolk phosphatidylcholine (EPC), cholesterol (CHOL), and α-linolenic acid (ALA) were dissolved in a mixed solvent, and then evaporation was performed in the dark, followed by addition of a buffer solution for hydration to obtain hydrated AmB liposomes, which were then filtered through a membrane to obtain ALA-Lips. The high molecular weight chitosan oligosaccharide HCOS solution was added dropwise to the ALA-Lips, mixed and stirred to obtain AmB-PLHNs; The M1 macrophages are co-incubated with the AmB-PLHNs, and then washed to obtain the macrophage-polymer lipid nanoparticle composite drug delivery system.

2. The preparation method according to claim 1, characterized in that The mass ratio of amphotericin B, hydrogenated soybean lecithin, egg yolk lecithin, cholesterol and α-linolenic acid is (0.1-1): (1-5): (1-5): (0.5-3): (0.5-3).

3. The preparation method according to claim 1, characterized in that The mixed solvent includes one or more of methanol and chloroform, ethanol, ether, and dichloromethane; The temperature of rotary evaporation in the dark is 20-50℃ and the time is 0.5-3h ； The number of filtration times is 15-35 times, and the filter membrane is a 0.1-0.2 μm filter membrane.

4. The preparation method according to claim 1, characterized in that The buffer solution comprises a PBS buffer solution; the concentration of the high molecular weight chitosan oligosaccharide HCOS solution is 0.1-0.5 mg / mL; and the molecular weight of the chitosan oligosaccharide HCOS in the high molecular weight chitosan oligosaccharide HCOS solution is 2kDa-30kDa.

5. The preparation method according to claim 1, characterized in that The volume ratio of the ALA-Lips to the high molecular weight chitosan oligosaccharide HCOS solution is 2:1-5.

6. The preparation method according to claim 1, characterized in that The mixing speed is 50-350 rpm and the time is 0.1-5 h.

7. The preparation method according to claim 1, characterized in that The inoculum size of M1 macrophages in co-culture was 0.5×10 6 -5×10 6 cells / mL.

8. A macrophage-polymer lipid nanoparticle composite drug delivery system according to any one of claims 1 to 7, characterized in that: It includes nanoparticles AmB-PLHNs and M1 macrophages, which are recognized and bound by macrophages through the surface mannose receptors of M1 macrophages.

9. An antibacterial agent, characterized in that The invention comprises the macrophage-polymer lipid nanoparticle composite drug delivery system according to claim 8.

10. The antibacterial agent according to claim 9, characterized in that The bacterial species in the antimicrobial agent is Cryptococcus.

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