Two-stage targeting amphotericin B drug loading system as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a two-stage targeted amphotericin B drug loading system as well as a preparation method and application thereof. The drug loading system disclosed by the invention is obtained by modifying a lipid nanodisk sND with polypeptide cRGD, and is marked as cRGD-sND; wherein the polypeptide is specifically combined with integrin on the surface of a microglial cell, transcellular membrane transport of the nano-drug is mediated, and the affinity of the nano-drug and a fungal biological membrane is enhanced; according to the polypeptide modified lipid nanodisk, the BBB crossing capacity is obtained by adsorbing apolipoprotein in blood; the medicine carrying system is loaded with amphotericin B, and cRGD-sND / AmB is obtained and used for targeted therapy of CM; animal experiments show that brain drug delivery is achieved through a two-stage targeting mechanism, first-stage targeting is based on high-affinity adsorption of a lipid disc to plasma apolipoprotein, receptor-mediated cross-blood-brain-barrier transport is promoted, and second-stage targeting is achieved by utilizing cRGD-mediated receptor recognition to achieve accurate positioning of microglial cells and fungal biofilms; therefore, the composition shows a remarkable curative effect on CM.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a two-stage targeted amphotericin B drug delivery system and a preparation method and application thereof. BACKGROUND

[0002] Cryptococcal meningitis (CM) is a kind of clinical refractory deep fungal infectious encephalopathy with extremely high mortality and disability. Amphotericin B (AmB) has a strong killing effect on cryptococcus and is a first-line drug for anti-cryptococcus infection. However, due to the blood brain barrier (BBB), the host cell membrane (microglial cell membrane) where cryptococcus is located and the biofilm formed by cryptococcus, the drug concentration at the infection site in the brain is too low after intravenous administration of the existing preparation of AmB, and the clinical treatment effect on CM is poor.

[0003] Stealth lipid nanodiscs (sND) are a new type of nanocarrier with good biocompatibility and long blood circulation time. However, there is no report on the use of stealth lipid nanodiscs for loading amphotericin B. SUMMARY

[0004] The purpose of the present application is to provide a two-stage targeted amphotericin B drug delivery system and a preparation method and application thereof, so as to improve the clinical treatment effect on CM.

[0005] The two-stage targeted amphotericin B drug delivery system provided by the present application is obtained by modifying stealth lipid nanodiscs (sND) with a polypeptide: cyclic arginine-glycine-aspartic acid (cRGD), and is denoted as cRGD-sND; wherein:

[0006] The polypeptide (cRGD) can specifically bind to the integrin on the surface of microglial cells, mediate the transmembrane transport of nanomedicines, and enhance the affinity of the nanomedicines to fungal biofilms. The stealth lipid nanodiscs (sND) modified by the polypeptide can obtain the ability to cross the BBB by adsorbing apolipoprotein in blood, so as to have a two-stage targeting function for amphotericin B, thereby greatly improving the clinical treatment effect on CM.

[0007] The drug delivery system (cRGD-sND) provided by the present application is used for loading amphotericin B (AmB) and is denoted as cRGD-sND / AmB. The strategy of one-stage targeting the BBB and two-stage targeting the host cell membrane and the cryptococcus biofilm can improve the distribution of AmB at the infection site of CM, and achieve the treatment goal of synergistic effect and attenuation.

[0008] The application provides a preparation method of a drug-loaded system (cRGD-sND), and specific steps are as follows:

[0009] (1) Synthesis of cRGD-PEG-DSPE

[0010] The polypeptide cRGD (i.e., a targeting molecule) is covalently coupled to PEG3500-DSPE material in a weak alkaline environment; specific operations are as follows: cRGD-SH and Mal-mPEG3500-DSPE are dissolved in PBS (10 mM, pH 7.4) and anhydrous DMF respectively at a molar ratio of 1.5:1; the DMF solution containing Mal-mPEG3500-DSPE is added dropwise into the PBS solution of cRGD-SH under continuous stirring at room temperature; after the reaction is completed, the residual thiolated cRGD peptide is removed by dialysis; the obtained cRGD-PEG-DSPE conjugate is freeze-dried and stored at -20℃ for standby use.

[0011] (2) Preparation of cRGD-sND

[0012] The cRGD-sND is prepared by a thin film hydration and ultrasonic dispersion method; specific operations are as follows: POPC, CHO, mPEG2000-DSPE and cRGD-PEG-DSPE are dissolved in a mixed solution of chloroform and methanol at a molar ratio of 35:40:23:(0-2); the organic solvent is removed by a rotary evaporator to form a thin lipid film; after vacuum drying and hydration, the thin film is treated by probe ultrasonic in an ice bath to obtain the cRGD-sND with a homogeneous disc size.

[0013] In addition, POPC, CHO and mPEG2000-DSPE are prepared into common sND without cRGD modification by the same method at a ratio of 35:40:23.

[0014] The present application can also prepare cRGD-sND or sND with fluorescent dye label, the preparation method is similar, that is, a certain proportion of fluorescent dye is incorporated into the lipid mixture during film formation. Commonly used fluorescent dyes include 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD), and 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO).

[0015] The drug delivery system (cRGD-sND) provided by the present application can load amphotericin B (AmB) to obtain cRGD-sND / AmB, which is used for targeted treatment of cryptococcal meningitis (CM) and improves the treatment effect of CM.

[0016] The operation of loading AmB into the drug delivery system (cRGD-sND) to obtain cRGD-sND / AmB is as follows:

[0017] The lipid material for preparing cRGD-sND in step (2) of the preparation method of cRGD-sND is mixed with AmB in a certain mass ratio (such as 100: (5-20)), and the drug and the lipid material are fully dissolved in a chloroform-methanol mixture under ultrasonic treatment. The film formation, hydration, drying, and ultrasonic treatment are performed synchronously in the method in step (2), and a cRGD-sND / AmB mixture containing a small amount of unencapsulated free AmB is obtained. The unencapsulated free AmB is removed by Sephadex G50 column chromatography, and cRGD-sND / AmB (i.e., cRGD lipid nanodiscs loaded with AmB) is obtained. Here, the lipid material refers to POPC, CHO, mPEG2000-DSPE, and cRGD-PEG-DSPE, and the ratio of the four is: 35:40:23: (0.5-2). Among them, cRGD-PEG-DSPE is obtained according to the method in step (1) of the preparation method of cRGD-sND.

[0018] In addition, sND / AmB is prepared by the same method.

[0019] Animal model experiments show that the system can significantly improve the survival rate and reduce toxicity. The dual-targeting nano platform combines apolipoprotein-mediated BBB transport with cRGD-mediated intracellular delivery and biomembrane penetration, providing a new idea for breaking through the pharmacological obstacles of cryptococcal meningitis treatment, opening up a new path for designing safe and effective brain-targeting antifungal nano drugs, and providing an extremely promising innovative strategy for central nervous system fungal infection.

[0020] The application innovatively constructs a cRGD modified lipid nanodisc (drug delivery system), and uses it to load amphotericin B (cRGD-sND / AmB). The preparation has a dual-targeting function, can efficiently break through multiple barriers such as BBB, and has a novel construction mode and design idea. The cRGD-sND / AmB not only has superior efficacy in the CM model, but also has good safety. It can not only solve the bottleneck problem of AmB in the clinical treatment of CM, but also has potential clinical conversion prospects.

[0021] The cRGD-sND (drug delivery system) constructed by the application can realize brain drug delivery through a dual-targeting mechanism: the first targeting is based on the high affinity adsorption of the lipid disc to plasma apolipoprotein, which promotes receptor-mediated transport across the blood-brain barrier, and the second targeting utilizes cRGD-mediated receptor recognition to achieve precise positioning of microglial cells and fungal biofilms. The system exhibits significant efficacy in cryptococcal meningitis in vivo. The application can use the cRGD-sND drug delivery system to deliver all antibacterial drugs to the brain tissue to treat brain infectious diseases.

[0022] The application provides an ideal carrier for delivering antifungal drugs for treating cryptococcal meningitis. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The particle size, zeta potential and cryo-EM characterization of the lipid nanodisc are shown.

[0024] Figure 2 The stability experiment results of cRGD-sND / AmB are shown.

[0025] Figure 3 The dose optimization is shown.

[0026] Figure 4 The drug-time curve and area under the curve of cRGD-sND / AmB are shown.

[0027] Figure 5 The tissue distribution of cRGD-sND / AmB is shown.

[0028] Figure 6To study the delivery mechanism of cRGD-sND.

[0029] Figure 7 To show the enhanced binding affinity of cRGD-sND to fungal biofilm and microglial cells (scale: 50 pm).

[0030] Figure 8 To show the stronger affinity of cRGD-sND to free bacteria.

[0031] Figure 9 To determine the MIC of AmB by standard broth microdilution method.

[0032] Figure 10 To show the killing-time curve of each preparation against H99.

[0033] Figure 11 To study the in vitro efficacy of cRGD-sND / AmB.

[0034] Figure 12 To study the in vivo pharmacodynamics of cRGD-sND / AmB.

[0035] Figure 13 To evaluate the in vivo safety. DETAILED DESCRIPTION

[0036] The application is further described below with reference to the accompanying drawings

[0037] 1. Preparation and characterization of drug-loaded system

[0038] 1.1 Synthesis of cRGD-PEG-DSPE

[0039] The targeting molecule cRGD in the present application is covalently coupled to the PEG3500-DSPE material in a weakly basic environment. The specific steps are as follows: cRGD-SH and Mal-mPEG3500-DSPE are accurately weighed in a molar ratio of 1.5:1, and dissolved in PBS (10 mM, pH 7.4) and anhydrous DMF, respectively. While continuously stirring at room temperature, the DMF solution containing Mal-mPEG3500-DSPE is added dropwise to the PBS solution of cRGD-SH. After 4 hours of reaction, the residual thiolated cRGD peptide is removed by dialysis for 72 h (MWCO > 3500 kDa). The obtained cRGD-PEG-DSPE conjugate is freeze-dried and stored at -20°C for standby use.

[0040] 1.2 Preparation of cRGD-sND / AmB

[0041] The cRGD-sND / AmB in the present application is prepared by a thin film hydration and ultrasonic dispersion method. POPC / CHO / mPEG2000-DSPE / cRGD-PEG-DSPE (molar ratio 35:40:25:0.5 or 35:40:23:2) and AmB (lipid / AmB mass ratio 10:1) are accurately weighed and dissolved in a mixed solution of chloroform and methanol (volume ratio 1:1), and ultrasonic treatment is performed for 30 min to fully dissolve the drug and lipid material. A rotary evaporator is used to remove the organic solvent to form a thin lipid film. After vacuum drying of the thin film, physiological saline is added for hydration at 37°C for 20 min, followed by probe ultrasonic treatment in an ice bath for 45 min (20% power, ultrasonic treatment for 2 s and pause for 1 s) to homogenize the disc size. Unencapsulated free AmB is removed by Sephadex G50 column chromatography, and sND / AmB and cRGD-sND / AmB loaded with AmB are obtained. The preparation method of cRGD-sND or sND (containing 0.5% (molar ratio) DiI / DiD / DiO) with fluorescent dye labeling is similar, that is, the dye is incorporated into the lipid mixture during the thin film formation process.

[0042] 1.3 Characterization and results of cRGD-sND / AmB

[0043] The particle size, particle size distribution (PDI) and zeta potential of the lipid nanodiscs diluted 50 times in deionized water are measured using a laser particle size analyzer. The morphology of the lipid nanodiscs is characterized by freeze electron microscopy. The results, as shown in Figure 1 The present application successfully prepared the dual-stage targeted drug delivery system cRGD-sND / AmB. The dynamic light scattering analysis results show that the disc size is 67.59±2.22 nm, and the zeta potential is -25.14±0.87 mV. Freeze electron microscopy further confirms the disc structure of the nanodiscs: the top view shows a circular bilayer structure with a diameter of about 60 nm; and the side view shows a cord-like structure with a thickness of about 5-10 nm. As a control, the present application also prepared sND without cRGD modification. The characterization results show that the modification of cRGD does not significantly change the particle size or zeta potential.

[0044] The HPLC method was used to quantitatively analyze the AmB loaded in the lipid nanodisc. The specific method was as follows: AmB was used as the standard, dissolved with DMSO, and diluted with methanol to prepare a 1 mg / mL stock solution. The stock solution was diluted with methanol to prepare 7 gradient concentrations of standard solutions, vortexed for 10 s, and 40 μL was taken for HPLC analysis. The standard curve equation was fitted by linear regression of the AmB peak area (Y) versus the AmB concentration (C, μg / mL). The sND / AmB and cRGD-sND / AmB were diluted 5 times with methanol, vortexed, centrifuged (4℃, 12000 g, 10 min), and 40 μL of the supernatant was taken for HPLC determination. The peak area was brought into the standard curve equation, and the drug concentration of each was calculated. The corresponding encapsulation efficiency and drug loading were calculated according to the feeding ratio. The HPLC conditions were as follows: Diamonsil-C18 (4.6×150 mm, 5 μm), mobile phase was 35% acetonitrile (0.1% TFA)-65% 0.02 mol / L EDTA.2Na (0.1% TFA); UV=386 nm; flow rate 0.8 mL / min, injection volume 40 μL. The quantitative results showed that the drug loading of cRGD-sND was 5.00±0.55%, and that of sND was 4.83±0.35%. Other parameters are shown in Tables 1-3.

[0045] Table 1-3 Summary of cRGD-sND / AmB characterization results

[0046] .

[0047] The stability of cRGD-sND / AmB was investigated, and the results of the stability experiment of cRGD-sND / AmB are shown in Figure 2 . The results showed that the appearance of cRGD-sND / AmB remained light yellow and transparent after storage at 4℃ for 14 days, without obvious turbidity, precipitation, or layering. The particle size, zeta potential, and polydispersity index (PDI) did not change significantly, providing a good preparation guarantee for subsequent in vivo studies.

[0048] 2, Pharmacokinetics and tissue distribution of cRGD-sND / AmB

[0049] 2.1 Pharmacokinetic study of cRGD-sND / AmB

[0050] Drug dosage optimization was performed before the study: 20 mice were randomly divided into 4 groups, 5 mice in each group, including 1 group of blank control. Three doses of AmB (1 mg·kg -1 , 3 mg·kg -1 , 6 mg·kg -1), 7 times continuously, and the blank mice were injected with the same volume of normal saline as control. After the last administration, they were continuously fed for 21 days, see Figure 3 . The results showed that 3 mg / kg was the highest dose that did not cause death, so it was selected as the administration dose for subsequent experiments.

[0051] 15 mice were randomly divided into 3 groups, 5 mice in each group. The tail vein was injected with cRGD-sND / AmB, sND / AmB or AmB at a dose of 3 mg / kg. Blood was taken from the lateral canthus into 1.5 mL anticoagulant tubes at 5 min, 15 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after administration. The plasma was separated by centrifugation at 3000 rpm for 8 min. DMSO / methanol (1:2, v / v) solution was used to precipitate proteins to extract AmB. After subsequent centrifugation at 12,000 rpm for 10 min, the supernatant was analyzed by HPLC, using 4-nitro-1-naphthylamine as an internal standard, and the chromatographic conditions were as described in Section 1.5. The pharmacokinetic parameters were calculated using GraphPad Prism 8.0.1.

[0052] The results are shown in Figure 4 Compared with free AmB, both cRGD-sND / AmB and sND / AmB showed significantly improved blood concentrations. The area under the plasma concentration-time curve (AUC 0-24 h ) of cRGD-sND / AmB within 24 hours was 35.53 ± 4.6 μg·h / mL, which was 2.88 times higher than that of free AmB. The results showed that the nanodiscs could effectively prolong the blood circulation time of AmB. The other main pharmacokinetic parameters are shown in Table 2-2.

[0053] Table 2-2 Main pharmacokinetic parameters of cRGD-sND / AmB

[0054] .

[0055] 2.2 Tissue distribution study of cRGD-sND / AmB

[0056] 48 mice were randomly divided into 3 groups, 4 mice in each group. The tail vein was injected with cRGD-sND / AmB, sND / AmB or AmB at a dose of 3 mg / kg. Blood was taken at 0.5 h, 1 h, 4 h and 12 h after administration, and the brain, heart, liver, spleen, lung and kidney were taken after tritriobethanol anesthesia and normal saline perfusion, weighed and homogenized with 5% Triton X-100. The sample pretreatment and determination were the same as in Section 2.2.

[0057] The tissue distribution of cRGD-sND / AmB is shown in Figure 5The results show that the concentration of AmB in the plasma of the cRGD-sND / AmB group is always significantly higher than that of the free drug group. Free AmB mainly accumulates in the liver and spleen, while the AmB of the cRGD-sND / AmB and sND / AmB groups still preferentially distributes in the above reticuloendothelial system-enriched organs, but the accumulation in the liver and spleen is significantly reduced compared with the AmB group, and there is no statistically significant difference between the two nanodisc preparations. The prolonged blood circulation time of cRGD-sND / AmB and sND / AmB leads to higher AmB levels in the kidney than in the free AmB group.

[0058] The distribution of cRGD-sND / AmB in brain tissue is significantly better than that of free AmB. One hour after administration, its concentration in the brain (0.025 ± 0.010 μg / g) is about 8.3 times that of free AmB, and 12 hours later it still maintains the highest level among the three groups. The above results show that cRGD-sND / AmB not only efficiently crosses the BBB, but also has excellent brain retention capacity. Compared with the classic liposome dosage form, cRGD-sND also exhibits excellent brain targeting ability. Therefore, the dual-targeting cRGD-sND / AmB drug delivery system constructed in the present application has the ability to significantly improve the brain targeting of AmB and the potential to improve the treatment dilemma of central nervous system fungal infection.

[0059] 3. cRGD-sND delivery mechanism research

[0060] 3.1 Experimental method

[0061] 3.1.1 cRGD-sND serum protein crown research

[0062] After the nanodrug enters the blood circulation, it inevitably interacts with serum proteins, adsorbs serum proteins, and forms a protein crown on its surface. The present application studies the protein crown adsorbed by cRGD-sND by simulating the blood environment in vitro. The in vitro protein crown separation is carried out by affinity chromatography based on single-chain antibodies (scFv). The specific experimental steps are as follows: mix 20 μL of DiI-labeled lipid nanodiscs with an equal volume of fresh C57 mouse serum, pre-incubate at room temperature for 30 min. Then add PEG-scFv (50 μL, 0.5 mg / mL), incubate at 4°C for 30 min. Load the mixture into a 3 mL gravity column with Ni-NTA resin (HisSep Ni-NTA agarose resin 6FF, 400 μL), and equilibrate with 10 column volumes of PBS. Slowly load the mixture into the column, incubate with Ni-NTA resin at room temperature for 10 minutes to ensure that the Ni 2+His-tagged PEG-scFv was fully bound. The column was then washed with 10-fold column volume of buffer (PBS with 15 mM imidazole, pH 7.4) to remove unbound proteins. Finally, the protein-sND complex was eluted using PEG8000 solution (10 mg / mL in PBS). The protein concentration of the collected complex was determined by BCA kit, and the DiI fluorescence intensity was detected using a microplate reader with excitation / emission wavelength set at 540 nm / 580 nm.

[0063] The collected protein-sND complex was mixed with loading buffer (containing β-mercaptoethanol) after adjusting the fluorescence intensity to be consistent, and denatured at 95°C for 10 min. An equal volume of sample (10 μL) was loaded onto a 4-20% gradient polyacrylamide gel for SDS-PAGE separation. Then the proteins were visualized using a fast silver staining kit. For immunoblotting, the separated protein bands were transferred to a PVDF membrane by electrophoresis. The membrane was blocked with 5% (w / v) non-fat milk in TBST at room temperature for 1 h, and then incubated with primary antibody (4°C overnight) and HRP-labeled secondary antibody (room temperature for 2 h) in sequence. Chemiluminescence signals were collected, and gray-scale analysis was performed by Image J software for quantification.

[0064] After protein extraction and peptide enzymatic treatment, the polypeptides extracted from each sample were analyzed by data-independent mass spectrometry data acquisition (DIA) LC-MS / MS using a Thermo Scientific Orbitrap Astral mass spectrometer. Then, the protein signals were obtained by comparing with the UniProt mouse proteome database, and bioinformatics analysis was mainly performed for protein abundance analysis.

[0065] 3.1.2 Affinity of cRGD-sND with Cryptococcus biofilm and microglial cells

[0066] H99 was cultured to construct its biofilm using a 12-well polystyrene plate. The method was as follows: 5 x 10 5 CFU H99 was incubated at 30°C for 72 h, and the culture medium was carefully aspirated and washed with PBS to remove free fungi to form a biofilm. The biofilm was then incubated with DiI-labeled lipid nanodiscs at 37°C for 2 h, followed by three washes. Then the biofilm was stained with DAPI, and observed using a fluorescence microscope.

[0067] To explore the affinity of cRGD-sND for free Cryptococcus, DiD-labeled cRGD-sND and sND with consistent fluorescence intensity were added to 90 μl containing 5 x 10 7CFU H99 was incubated in YPD broth at 37°C for 2 h. After centrifugation (× 2000 g), H99 containing lipid nanodiscs was obtained. The precipitate was washed three times with PBS and resuspended in PBS. Analytical analysis was performed using flow cytometry.

[0068] Mouse microglia (BV2 cells) were digested with trypsin, resuspended in high-glucose DMEM medium, and quantified. Based on the cell count results, cells containing 3 × 10⁻⁶ cells were selected. 7 Cell samples were aliquoted into 1.5 mL centrifuge tubes (95 μL per tube) and divided into three experimental groups: control group, cRGD-sND group, and sND group (n = 5). Subsequently, 5 μL of pre-prepared DiO-labeled lipid nanodisks (cRGD-sND or sND) were added to each experimental tube, while the control tubes received an equal volume of culture medium. Cells were incubated at 37°C for 30 min, followed by washing three times with PBS, fixation with 4% paraformaldehyde at room temperature for 15 min, resuspending in PBS, and analysis by flow cytometry.

[0069] 3.2 Experimental Results

[0070] 3.2.1 The cRGD-modified nanodiscs still retain the protein crown-mediated brain-targeting function.

[0071] This invention investigated the protein corona of cRGD-sND in an in vitro simulated blood environment. The results are as follows: Figure 6 As shown in Figure A, after separation by SDS-PAGE, both cRGD-sND and sND showed two strongly stained bands in the 20-37 kDa range, presumably representing apolipoproteins ApoA1 and ApoE. This result was validated by Western blot analysis. Figure 6 B). Gray-scale analysis showed that cRGD modification did not significantly alter the binding ability of sND to ApoE or ApoA1. Figure 6 C, D). Quantitative analysis of the protein corona components by LC-MS / MS showed that cRGD-sND and sND mainly adsorbed apolipoproteins, with no significant difference in the main protein types. Figure 6 E). ApoA1 was the most abundant protein in both groups, accounting for 27.41% and 31.36%, respectively. The proportion of ApoE adsorbed by cRGD-sND was 2.53%, ranking fifth; while in the sND group, ApoE accounted for 3.29%, ranking fourth. Figure 6 F).

[0072] 3.2.2 cRGD-sND showed high affinity for Cryptococcus neoformans biofilm and microglia.

[0073] To evaluate the affinity of cRGD-sND for a novel Cryptococcus biofilm, this invention constructed a mature novel Cryptococcus biofilm in vitro and performed three-dimensional imaging analysis after co-incubation with fluorescently labeled lipid nanodisks. Results ( Figure 7 A) The established Cryptococcus biofilm exhibits a dense structural organization. Compared to sND, cRGD-sND displays a stronger and more widespread fluorescence signal within the biofilm. cRGD-sND not only adheres more readily to the biofilm surface but also penetrates deeper into the structure and exhibits a more uniform distribution, indicating that cRGD modification significantly enhances the affinity of the nanodiscs for the biofilm. This invention hypothesizes that its mechanism involves the negatively charged cell wall and biofilm of Cryptococcus: although the cRGD-modified nanodiscs remain anionic overall, the cRGD portion, as a cationic peptide, forms a localized positive charge density on the nanoparticle surface, thereby promoting initial electrostatic binding with the biofilm. This electrostatic attraction, combined with the structural similarity of the lipid nanocarrier to natural biofilm components and its inherent small size, synergistically enhances the binding affinity of cRGD-modified nanodiscs to fungal biofilms. Based on this theoretical framework, cRGD-sND should exhibit measurable affinity for novel Cryptococcus due to the inherent negative charge of the fungal cell wall. Therefore, this invention investigates this aspect. In the free Cryptococcus experiment, after co-incubation at 37°C for 2 hours, the flow cytometry results were as expected: the fluorescence signal of the cRGD-sND group was stronger ( Figure 8 This indicates that cRGD modification promotes its binding to free fungal cells.

[0074] To investigate the microglia targeting effect mediated by cRGD, this invention co-cultured BV2 microglia with fluorescein-labeled cRGD-sND and sND in vitro, followed by flow cytometry analysis. The results showed that compared with sND, the percentage of positive cells and the relative fluorescence intensity of cRGD-sND were significantly higher, indicating that microglia had enhanced uptake capacity for cRGD-sND. Figure 7 (B, C, D). The results above show that cRGD modification can enhance the targeting efficiency of lipid nanodiscs on microglia, laying the foundation for achieving secondary targeting of Cryptococcus in brain microglia by cRGD-sND / AmB and improving anti-infection efficacy.

[0075] In summary, after entering the bloodstream, cRGD-sND effectively adsorbs brain-targeting proteins such as ApoA1 and ApoE, while exhibiting stronger affinity for Cryptococcus H99 and its biofilm. Furthermore, cRGD functionalization enhances the uptake of lipid-based nanodiscs by microglia, achieving a secondary targeting effect, thus enabling cRGD-sND / AmB to achieve a two-stage targeted antibacterial effect in vivo.

[0076] 4. Pharmacodynamics evaluation of cRGD-sND / AmB

[0077] 4.1 Experimental methods

[0078] 4.1.1 MIC determination

[0079] MIC was determined by standard broth dilution method. After the frozen glycerol bacteria plate streak culture, single colony was picked to YPD medium, incubated at 30°C, 150 rpm until logarithmic growth phase, diluted to 1 x 10 7 CFU / mL for standby. cRGD-sND / AmB, sND / AmB and AmB were diluted to 8 μg / mL with YPD broth, then half-diluted to 0.125 μg / mL, 2 x 10 5 CFU H99 were inoculated in each tube. After 72 h incubation, the lowest concentration without turbidity was the MIC of the preparation.

[0080] 4.1.2 In vitro pharmacodynamics evaluation of cRGD-sND / AmB

[0081] Based on the MIC value of AmB (0.5 μg / mL), cRGD-sND / AmB, sND / AmB and AmB were diluted to 1 μg / mL, 0.5 μg / mL and 0.25 μg / mL respectively with YPD broth, 2 mL drug-containing medium was added to each culture tube (n = 3), and a drug-free control group was set. 2 x 10 5 CFU of Cryptococcus neoformans H99 strain were inoculated in each tube. After 15 min incubation at 30°C with 150 rpm shaking, free drugs were removed by centrifugation. Samples were taken at 0 h, 1 h, 2 h, 3 h and 4 h intervals, diluted and plated on YPD agar plates for 30 h incubation at 30°C for colony counting.

[0082] H99 biofilm was cultured by the method described in 3.2.2. Pre-diluted cRGD-sND / AmB, sND / AmB and AmB were added to the biofilm at a series of concentrations (n = 3), with concentrations set at 0.5 μg / mL, 1 μg / mL and 2 μg / mL. A blank control group was set. After 24 h incubation at 30°C, the culture medium was discarded, the biofilm was washed with PBS, fixed with methanol, stained with 0.5% crystal violet for 20 min, and then observed after washing and air-drying. To quantify the number of bacteria in the biofilm, 1 mL of 33% acetic acid solution was added to each well, and the crystal violet was completely dissolved after 30 min standing. The absorbance was measured at 595 nm using a microplate reader, and the results were normalized against the control group.

[0083] 4.1.3 Construction and verification of CM mouse model

[0084] Cryptococcal meningitis (CM) model was constructed using Cryptococcus neoformans H99-Luc strain. After the mice were anesthetized, they were fixed on a stereotaxic instrument. A hole was drilled at 3 mm lateral and 3 mm ventral to the bregma, and 1 x 10 6 CFU of Cryptococcus suspension in PBS was inoculated intracranially. The surgical site was sutured and disinfected. Three days after inoculation, the mice received intraperitoneal injection of D-luciferin potassium salt (150 mg / kg). Whole-body bioluminescence imaging was performed using an IVIS Spectrum imaging system, followed by ex vivo organ imaging. To quantify fungal load, model mice and control groups were euthanized under sterile conditions. Brain tissue was taken, washed, weighed, and homogenized in sterile saline at a ratio of tissue weight (g) to saline volume (ml) of 1:3 (w / v). The homogenate was serially diluted, and colony counting on YPD agar plates at 30°C for 48 h was used to verify whether the model was successfully constructed.

[0085] 4.1.4 In vivo pharmacodynamic evaluation of cRGD-sND / AmB

[0086] Forty CM mice were randomly divided into four groups, with 10 mice in each group, and were injected with cRGD-sND / AmB, sND / AmB, AmB (AmB 3 mg / kg), and the same volume of normal saline via the tail vein, respectively. The mice were given the drugs every other day for 7 times. On the 7th and 14th day after the first administration, 5 mice were randomly selected for small animal live imaging to monitor the brain infection in real time, and the data were collected and the bioluminescence signal intensity was semi-quantitatively analyzed using LivingImage software. All mice were bred until the 21st day after the first administration to investigate their survival rate.

[0087] To investigate the fungal load in the brain tissue of CM mice, 5 mice were randomly selected on the 7th and 14th day after the first administration, and the brain tissue was taken out under sterile conditions. The homogenate was diluted 10, 10 2 , 10 3 , 10 4 , 10 5 times with sterile saline and plated on YPD solid medium, which was incubated at 30°C for 48 h before colony counting. Part of the brain tissue on the 7th day was immersed in 4% paraformaldehyde fixative, paraffin-embedded, and subjected to PAS staining to directly evaluate the therapeutic effect of the formulations on infected mice.

[0088] 4.2 Experimental results

[0089] 4.2.1 Enhanced in vitro antibacterial effect of cRGD-sND / AmB on Cryptococcus neoformans.

[0090] MIC results showed that the MIC values of cRGD-sND / AmB, sND / AmB and AmB were all 0.5 µg / mL. See Figure 9 .

[0091] The in vitro pharmacodynamics of cRGD-sND / AmB showed that there were significant differences in the killing mode of different formulations. All formulations containing amphotericin B showed concentration- and time-dependent killing effect, but showed different killing kinetics. The two formulations of cRGD-sND / AmB and sND / AmB reached the maximum killing activity at 4 h after treatment, and the duration of their activity was longer than that of AmB, which was due to the gradual release of the drug from the nanocarrier.

[0092] The killing-time curves of each formulation against H99 are shown in Figure 10 .

[0093] As described in Section 3.3.2, cRGD-sND showed significant affinity for free C. neoformans. To verify whether this targeting property can improve the efficacy, the present application designed an experimental scheme in which the drug was first incubated with free bacteria, and then the unbound drug was removed. After statistical analysis, the number of colonies at each time point was divided by the initial number of colonies at 0 h to calculate the fungal growth fold. As shown in Figure 11 B, C, D, at the tested concentrations (0.25, 0.5 and 1 µg / mL), cRGD-sND / AmB showed the lowest growth fold, indicating that it had the strongest growth inhibition effect. It is worth noting that the growth fold of the cRGD-sND / AmB treatment group at a concentration of 0.25 µg / mL was significantly different from that of the AmB alone group at 3 h (p<0.01) and 4 h (p<0.05) after treatment. In addition, at the same concentration, cRGD-sND / AmB and sND / AmB formulations showed more significant differences at the 4 h time point (p<0.01). At the other two concentrations, the antibacterial effect of the cRGD-sND / AmB group also showed a significant advantage. The results showed that cRGD-sND / AmB achieved more AmB deposition by virtue of its targeted affinity to the surface of Cryptococcus, thus transforming into better pharmacodynamic performance.

[0094] To test the antibacterial effect of cRGD-sND / AmB on Cryptococcus biofilm, the present application cultured C. neoformans in a 12-well plate for 72 h to form mature biofilm. Then, using the minimum inhibitory concentration (0.5 µg / mL) of AmB as the starting concentration, the biofilm was treated with drugs for 24 h, and the crystal violet staining method was used to quantitatively evaluate the removal effect of each formulation on mature biofilm. The quantitative results (OD450) are shown in Figure 11F) showed that cRGD-sND / AmB exhibited the most significant anti-biofilm activity among the three groups, significantly disrupting biofilm structure even at an initial concentration of 0.5 µg / mL. In contrast, sND / AmB and AmB alone showed only slight scavenging effects at the same concentration. The scavenging effects of sND / AmB and AmB only improved when the concentration was increased to 2 µg / mL, but still were less than those of cRGD-sND / AmB at the same concentration. These quantitative results are consistent with the degree of biofilm morphological disruption observed under a microscope. Figure 11 E) The results are completely consistent, further demonstrating that cRGD modification significantly enhances the ability of nano-formulations to penetrate and remove Cryptococcus biofilms.

[0095] 4.2.2 CM Model Construction and Validation

[0096] A mouse CM model was established by intracerebroventricular injection of a novel Cryptococcus H99-Luc suspension. Three days after H99-Luc infection, mice were intraperitoneally injected with D-fluorescein potassium (150 mg / kg). Whole-body bioluminescence imaging using the IVIS Spectrum imaging system revealed strong intracranial luminescence signals, indicating active fungal proliferation in the brain. Quantitative culture of brain tissue homogenates consistently showed abundant Cryptococcus colonies, further confirming the successful establishment of a stable and reproducible CM model. Figure 12 A, B).

[0097] 4.2.3 cRGD-sND / AmB significantly enhanced the in vivo therapeutic effect of cryptococcal meningitis.

[0098] like Figure 12 As shown in Figure D, the bioluminescence intensity in the brains of mice in the blank control group increased significantly over time, and their condition deteriorated and they began to die after day 14, further validating the successful establishment of the CM model and the active proliferation of fungi in the brain. In contrast, the bioluminescence intensity in the brain of the free AmB treatment group remained basically the same as the baseline before treatment, or even showed an upward trend, indicating that its efficacy was limited. Both the sND / AmB and cRGD-sND / AmB groups significantly inhibited fungal growth, as reflected in the significant decrease in brain bioluminescence intensity 14 days after administration, and the quantitative fluorescence results ( Figure 12 C) provides strong support. Notably, the cRGD-sND / AmB group showed the most significant inhibitory effect, suggesting stronger fungal clearance and superior therapeutic activity.

[0099] To further quantitatively assess the fungal load in brain tissue, this invention involves collecting mouse brain tissue samples on days 7 and 14 post-treatment, followed by homogenization, YPD agar culture, and colony counting. Figure 12E, F). At day 7, the treatment effect of free AmB was the most limited, in contrast, the fungal load in sND / AmB and cRGD-sND / AmB groups decreased by 1.20 and 2.66 log 10 units, respectively. By day 14, the brain fungal load in the cRGD-sND / AmB group was further reduced by 4.41 log 10 units, significantly better than the sND / AmB group (2.90 log 10 ) and the free AmB group (1.50 log 10 ), thus confirming that the cRGD-modified nanodisc formulation has a stronger antifungal effect in vivo.

[0100] To visually demonstrate the extent of intracranial infection, the present application performed PAS staining on brain tissue samples taken at day 7 after infection and after completing three doses of treatment Figure 12 H). Compared with the normal saline control group, the brain sections of all treatment groups showed significantly reduced tissue cavities caused by Cryptococcus. In particular, the cRGD-sND / AmB group showed almost no aggregated fungal mass, indicating that it had the most prominent effect on fungal clearance.

[0101] Based on the reduction in brain fungal load, the present application further investigated the effect of cRGD-sND / AmB on mouse survival rate. Mice treated with cRGD-sND / AmB, sND / AmB, AmB or normal saline were monitored for 21 days to assess long-term treatment effects. The normal saline group began to die on day 8, and all mice died by day 17. The AmB and sND / AmB groups only showed slight survival improvement, while the survival time of the cRGD-sND / AmB group was significantly prolonged, with a survival rate of 60% on day 21, significantly higher than the other groups Figure 12 G). These results show that cRGD-sND / AmB, with its enhanced brain targeting ability and binding characteristics to biofilms, can effectively deliver AmB to the infection site, thus achieving better treatment effects and significantly prolonging survival.

[0102] 5. Safety evaluation

[0103] 5.1 Experimental methods

[0104] To evaluate the biosafety of cRGD-sND / AmB, 12 mice were randomly divided into 4 groups (3 mice in each group), and were injected with cRGD-sND / AmB, sND / AmB or AmB (dose 3 mg / kg) via tail vein, once every other day, for 7 times. The control group was injected with the same volume of normal saline. Blood samples were collected on the 14th day after the first administration, and were centrifuged at 3000 rpm for 8 min after standing at room temperature. The serum was collected and used to detect the four blood biochemical indicators of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CRE) and urea nitrogen (BUN) to preliminarily judge the influence of each preparation on liver and kidney function. Subsequently, one mouse in each group was randomly selected for perfusion, and the heart, liver, spleen, lung, kidney and other major organs were taken out and immersed in 4% paraformaldehyde fixing solution. After paraffin section, HE staining was performed to observe the histopathological changes. Finally, using the same processing procedure, the AmB content in each tissue after multiple administrations was determined to analyze the in vivo distribution of the drug.

[0105] 5.2 Experimental results

[0106] The dose-dependent toxicity of amphotericin B (AmB) is a key factor limiting its clinical application, especially in the treatment of meningitis, where it is difficult to simply increase the systemic dose to obtain sufficient drug concentration in the brain. Although the cRGD-sND / AmB preparation has made significant progress in achieving brain-targeted delivery, its safety still needs to be systematically evaluated. Therefore, using the same administration scheme as the in vivo pharmacodynamics study, the body weight of the mice was monitored, the biochemical indicators were detected, and the pathological analysis of the tissue sections was performed.

[0107] After the first administration, the body weight of all treatment groups decreased significantly compared with the blank control group. Subsequently, the body weight of the cRGD-sND / AmB group gradually recovered to the level comparable to the control group, although there was a slight fluctuation after multiple administrations, but the difference was not statistically significant. In contrast, the body weight of the AmB-treated mice decreased significantly, indicating that the cRGD-sND preparation significantly reduced the toxicity associated with AmB ( Figure 13 A).

[0108] The distribution of AmB in tissues after repeated administration further confirmed the safety of cRGD-sND / AmB. Compared with the free AmB group, the latter was mainly accumulated in the liver and spleen, and the distribution in the kidney was significantly increased, while the accumulation of cRGD-sND / AmB in the liver was only 15.8% of that of free AmB, and the accumulation in the spleen was only 42.0% of that of free AmB, and the distribution in the kidney was not significantly increased ( Figure 13 B). Biochemical detection showed that repeated administration of free AmB significantly increased the levels of liver ALT and AST ( Figure 13C, D), while no such increase was observed in the lipid nanodisc group. cRGD-sND / AmB slightly increased the levels of kidney CRE and BUN compared to the normal control group (P < 0.05) Figure 13 E, F), but showed no significant difference compared to the free AmB group. HE histopathological examination of major organs showed that no obvious histological lesions were observed in each group (G). In summary, these data suggest that cRGD-sND / AmB significantly reduces off-target toxicity in key metabolic and excretion organs, and has good overall safety. Figure 13 G). In summary, these data suggest that cRGD-sND / AmB significantly reduces off-target toxicity in key metabolic and excretion organs, and has good overall safety.

[0109] In summary, the present application successfully constructed a cRGD-modified lipid nanodisc (cRGD-sND) drug delivery system loaded with amphotericin B (AmB). The nanolipid disc of the drug delivery system can efficiently adsorb circulating apolipoprotein and significantly improve the accumulation of drugs in brain tissue through receptor-mediated transport across the blood-brain barrier (BBB). cRGD functionalization further enables precise positioning of microglial cells and fungal biofilms, promoting intracellular uptake and biofilm penetration, thereby achieving more thorough fungal clearance.

Claims

1. A two-stage targeting amphotericin B drug delivery system, characterized in that, It is obtained by modifying lipid nanodisks (sND) with a polypeptide: cyclic arginine-glycine-aspartic acid (cRGD), denoted as cRGD-sND; wherein: The peptide (cRGD) specifically binds to integrins on the surface of microglia, mediating the transmembrane transport of nanomedicines and enhancing their affinity for fungal biofilms. The lipid nanodiscs (sND) modified with this peptide gain trans-BBB ability by adsorbing apolipoproteins in the blood, giving them a dual-targeting function for amphotericin B, thereby greatly improving the clinical treatment effect of CM.

2. A method for preparing a dual-targeting amphotericin B drug delivery system as described in claim 1, characterized in that, The specific steps are as follows: (1) Synthesis of cRGD-PEG-DSPE The cRGD peptide was covalently coupled to PEG3500-DSPE material under weakly alkaline conditions. The specific procedure was as follows: cRGD-SH and Mal-mPEG3500-DSPE were mixed at a molar ratio of 1.5:

1. cRGD-SH was dissolved in PBS, and Mal-mPEG3500-DSPE was dissolved in anhydrous DMF. Under continuous stirring at room temperature, the DMF solution containing Mal-mPEG3500-DSPE was added dropwise to the PBS solution containing cRGD-SH. After the reaction was complete, dialyzing was performed to remove residual thiolized cRGD peptides. The resulting cRGD-PEG-DSPE conjugate was freeze-dried and stored at -20°C for later use. (2) Preparation of cRGD-sND cRGD-sND was prepared using a thin-film hydration and ultrasonic dispersion method. The specific operation was as follows: POPC, CHO, mPEG2000-DSPE, and cRGD-PEG-DSPE were dissolved in a mixture of chloroform and methanol at a molar ratio of 35:40:23:(0.5~2) to ensure complete dissolution of the lipid material. The organic solvent was removed using a rotary evaporator to form a thin lipid film. After vacuum drying and hydration, the film was ultrasonically treated with an ice bath probe to homogenize the disk size and obtain cRGD-sND.

3. The application of the dual-targeting amphotericin B drug delivery system as described in claim 1 in loading amphotericin B (AmB), a therapeutic drug for cryptococcal meningitis (CM), yields a cRGD-sND system loaded with AmB, denoted as cRGD-sND / AmB, for targeted treatment of cryptococcal meningitis (CM).

4. The application according to claim 3, characterized in that, The specific operation of loading amphotericin B (AmB) is as follows: In step (2) of the cRGD-sND preparation method, the lipid material to be prepared for cRGD-sND is mixed with AmB at a mass ratio of 100:(5-20). The mixture is then sonicated in a chloroform and methanol mixture to fully dissolve the drug and lipid material. The same process as in step (2) is followed to form a film, hydrate, dry, and sonicate the mixture to obtain a cRGD-sND / AmB mixture containing a small amount of unencapsulated free AmB. The unencapsulated free AmB is removed by Sephadex G50 column chromatography to obtain cRGD-sND / AmB, which is a cRGD lipid nanodisc loaded with AmB. The lipid material mentioned here refers to POPC, CHO, mPEG2000-DSPE, and cRGD-PEG-DSPE, with a molar ratio of 35:40:23:(0.5~2). cRGD-PEG-DSPE is obtained according to the method in step (1) of the cRGD-sND preparation method.

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