A biomimetic nanomedicine carrier of neutrophil membrane fusion liposome and a preparation method and application thereof

Abstract

The application discloses a kind of neutrophil membrane fusion liposome biomimetic nanomedicine carrier and its preparation method and application, belong to medical field.The carrier includes liposome and the neutrophil membrane fused to liposome;Liposome raw materials include DOPE, DPPC and cholesterol, and mass ratio is 6:3:1 in turn;Liposome and membrane protein mass ratio is 1:1.The application establishes quantitative analysis strategy based on NanoFCM, can accurately evaluate the fusion efficiency and fusion uniformity of NM@lipos at single particle level.Based on DOPE, the hybridization efficiency of NM@lipos (D) reaches 92.0%, which is significantly higher than that of NM@lipos (L) based on lecithin (70.1%).In in vivo experiment, the tumor enrichment capacity of NM@lipos (D) is increased by 5.1 times compared with Lipos (D) in 24 hours, and shows significantly prolonged circulation time.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of medicine, and particularly relates to a neutrophil membrane fusion liposome biomimetic nanomedicine carrier and a preparation method and application thereof. BACKGROUND

[0002] Cell membrane biomimetic nanoparticles (CM@NPs) have attracted extensive attention in recent years as a kind of frontier drug delivery system. The CM@NPs utilize the biological functionality of the source cell membrane, can improve the biocompatibility of the nanocarrier, realize immune evasion and enhance the targeting specificity. As an important biomimetic membrane material, neutrophil membranes (NMs) are widely used in the construction of inflammation site and tumor targeted drug delivery systems due to their natural inflammation and tumor homing ability. Among the many construction strategies, the hybrid vesicles (CM@lipos) obtained by membrane fusion of natural cell membranes and synthetic liposomes have both the biological functionality of natural cell membranes and the high drug loading capacity and structural adjustability of liposomes. The multifunctional and efficient biomimetic drug delivery carrier has broad application prospects in the fields of precise drug delivery therapy, disease imaging and diagnosis.

[0003] Although the CM@lipos currently shows significant advantages in in vitro research, its actual performance in vivo is largely dependent on the efficiency and uniformity of the membrane fusion process. The membrane fusion efficiency not only affects the presentation state of the surface membrane protein, but also determines its immune camouflage ability and in vivo behavior. However, how the lipid composition regulates the fusion efficiency of NMs and liposomes, and the influence of the fusion level on its downstream biological behavior have not been systematically revealed, especially the functional differences between DOPE (unsaturated phospholipid) and DPPC (saturated phospholipid) in terms of fusion ability and structural stability [4, 5], but the specific influence of the combination ratio of the two on the performance of NM@lipos is still unclear. In addition, the commonly used evaluation methods (such as confocal microscopic imaging) are mostly limited to local qualitative observation, and lack the ability of single-particle quantitative analysis of fusion efficiency [1 , 2 3], which seriously restricts the systematic optimization of fusion parameters.

[0004] Therefore, it is of great significance to develop a neutrophil membrane fusion liposome biomimetic nanomedicine carrier with efficient fusion of NMs and liposomes and to establish an evaluation method for accurately evaluating the fusion efficiency.

[0005] REFERENCES

[0006] [1] Wu H, Jiang X, Li Y, et al. Engineering stem cell derived biomimetic vesicles for versatility and effective targeted delivery [J]. Advanced Functional Materials, 2020, 30(49): 2006169.

[0007] [2] Chen S, Lan H, Liu M, et al. Less is More: Biomimetic Hybrid Membrane Nanocarriers for Highly Efficient Tumor Targeted Drug Delivery [J]. Small, 2025: 2407245.

[0008] [3] Wu J, Ma T, Zhu M, et al. A pluripotential neutrophil-mimic nanovehicle modulates immune microenvironment with targeted drug delivery for augmented antitumor chemotherapy [J]. Acs Nano, 2024, 18(7): 5864.

[0009] [4] Kong H, Zheng C, Yi K, et al. An antifouling membrane-fusogenic liposome for effective intracellular delivery in vivo [J]. Nature Communications, 2024, 15(1): 4267.

[0010] [5] S, Moreira J N, Fonseca C, et al. On the formulation of pH-sensitive liposomes with long circulation times [J]. Advanced drug delivery reviews, 2004, 56(7): 947. SUMMARY

[0011] To solve the problems of the existing cell membrane-liposome hybrid nanoparticles (CM@lipos), a quantitative analysis strategy based on nano flow cytometry (NanoFCM) is established to accurately evaluate the fusion efficiency and uniformity of NM@lipos at the single particle level. On this basis, the key parameters such as the ratio of DOPE / DPPC, the ratio of phospholipid / cholesterol and the mass ratio of liposome / membrane protein are systematically optimized to determine the optimal conditions for promoting efficient fusion. Further in vivo and in vitro systems verify the pharmacokinetic behavior, inflammation targeting ability and anti-tumor efficacy of the optimized NM@lipos. The research finds that the fusion efficiency of NM@lipos (D) dominated by DOPE can reach 92.0%, which is significantly better than that of the lecithin system; it shows stronger tumor enrichment ability and more persistent in vivo circulation performance in the 4T1 mouse model, and realizes significant tumor inhibition effect (89.4%) and survival time extension on the basis of DOX loading. This research not only provides a technical framework for the quantitative construction of biomimetic hybrid nano systems, but also lays a foundation for the rational design and transformation application of precise nano drug delivery systems.

[0012] To achieve the above object, the present application adopts the following technical solutions:

[0013] In a first aspect, the present application provides a biomimetic nano drug carrier of neutrophil membrane fusion liposome.

[0014] The biomimetic nano drug carrier of neutrophil membrane fusion liposome comprises a liposome and a neutrophil membrane fused to the surface of the liposome.

[0015] The raw materials of the liposome include 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) and cholesterol, and the mass ratio of the three is 6:3:1.

[0016] The mass ratio of the liposome to the membrane protein in the neutrophil membrane is 1:1, and the mass of the liposome is calculated based on the total mass of 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) and cholesterol.

[0017] Preferably, the neutrophils are extracted and separated from mouse bone marrow cells by using a modified Percoll density gradient centrifugation method.

[0018] Preferably, the neutrophils need to be activated, and the specific method is to induce neutrophil activation by LPS to make the activated neutrophil membrane surface overexpress related adhesion molecules (such as CD11b).

[0019] Specifically, the unactivated neutrophil granulocyte is isolated from the bone marrow of BALB / c mice by using a modified Percoll density gradient centrifugation method; the neutrophil granulocyte is activated by using LPS (such as 200 ng / mL LPS); and the activated neutrophil granulocyte is subjected to hypotonic lysis, ultrasonic disruption by an ultrasonic cell disrupter, and differential centrifugation to obtain the neutrophil granulocyte membrane.

[0020] Further, the neutrophil granulocyte membrane is subjected to ultrasonic treatment by an ultrasonic cell disrupter in an ice bath to obtain neutrophil granulocyte membrane vesicles (NMVs).

[0021] Preferably, the ultrasonic treatment by the ultrasonic cell disrupter in the ice bath is performed at a power of 100 W, at a frequency of ultrasonic treatment for 2 s and resting for 4 s, and for a total time of 9 min.

[0022] Preferably, the liposome is prepared by a thin film hydration method.

[0023] Preferably, the liposome and the neutrophil granulocyte membrane are subjected to ultrasonic treatment to promote membrane fusion and achieve outer membrane fusion.

[0024] In a second aspect, the present application provides a preparation method of the biomimetic nanomedicine carrier of the neutrophil granulocyte membrane fusion liposome.

[0025] The preparation method of the biomimetic nanomedicine carrier of the neutrophil granulocyte membrane fusion liposome provided by the present application comprises the following steps:

[0026] 1) The neutrophil granulocyte is extracted and isolated from the bone marrow of mice by using a modified Percoll density gradient centrifugation method; the neutrophil granulocyte is activated by using LPS; the activated neutrophil granulocyte is subjected to hypotonic lysis, ultrasonic disruption, and differential centrifugation to obtain the neutrophil granulocyte membrane; and the neutrophil granulocyte membrane is subjected to ultrasonic treatment by an ultrasonic cell disrupter in an ice bath to obtain neutrophil granulocyte membrane vesicles (NMVs).

[0027] 2) The liposome is prepared by a thin film hydration method using 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol as raw materials; and the mass ratio of the DOPE, DPPC, and cholesterol is 6:3:1.

[0028] 3) The liposome and the neutrophil granulocyte membrane vesicles (NMVs) are mixed at a mass ratio of 1:1, placed in an ice bath, and then subjected to ultrasonic treatment by an ultrasonic cell disrupter to obtain the biomimetic nanomedicine carrier of the neutrophil granulocyte membrane coated liposome (NM@lipos (D) hybrid particles).

[0029] In the step 2) of the above method, the specific method for preparing the liposome by thin film hydration method is as follows: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol are dissolved in dichloromethane, and rotary evaporation is performed to form a lipid film; a PBS solution is added to the lipid film, and hydration is performed at 50℃ and 150 rpm for 2h; and the obtained suspension is treated in an ice bath using an ultrasonic disrupter (for example, 400W, intermittent mode: 2s on, 2s off) for 12min, to obtain the liposome.

[0030] In the step 3) of the above method, the mass of the liposome is calculated based on the total mass of DOPE, DPPC and cholesterol; and the mass of the membrane protein is the mass of the membrane protein in the neutrophil membrane vesicle.

[0031] In the step 3) of the above method, the ultrasonic treatment is performed using an ultrasonic disrupter at a power of 100W, at a frequency of ultrasonic treatment for 2s and rest for 4s, and the total ultrasonic treatment time is 9min.

[0032] In a third aspect, the present application provides a biomimetic nanodrug carrier of neutrophil membrane fusion drug-loaded liposome.

[0033] The biomimetic nanodrug carrier of neutrophil membrane fusion drug-loaded liposome provided by the present application comprises a drug-loaded liposome and a neutrophil membrane fused to the surface of the liposome.

[0034] The raw materials of the liposome comprise 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-hyaluronic acid (DSPE-HA2000), and the mass ratio of the four is 6:3:1:0.5, respectively.

[0035] The drug loaded in the drug-loaded liposome is doxorubicin (DOX).

[0036] The mass ratio of the drug-loaded liposome to the membrane protein in the neutrophil membrane is 1:1, and the mass of the drug-loaded liposome is calculated based on the total mass of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol and DSPE-HA2000.

[0037] Preferably, the neutrophil is extracted and separated from mouse bone marrow cells by using a modified Percoll density gradient centrifugation method.

[0038] Preferably, the neutrophils need to be activated, specifically by using LPS to induce neutrophil activation, so that the activated neutrophil membrane surface overexpresses related adhesion molecules (such as CD11b).

[0039] Specifically, the unactivated neutrophils are isolated from the bone marrow of BALB / c mice by using a modified Percoll density gradient centrifugation method; the neutrophils are activated by using LPS (such as 200 ng / mL LPS); the activated neutrophils are subjected to hypotonic lysis, ultrasonic fragmentation by an ultrasonic cell disrupter, and differential centrifugation to obtain neutrophil membranes.

[0040] Further, the neutrophil membranes are subjected to ice-bath ultrasonic treatment by an ultrasonic cell disrupter to obtain neutrophil membrane vesicles (NMVs).

[0041] Preferably, the ice-bath ultrasonic treatment by the ultrasonic cell disrupter is performed at a power of 100 W, with a frequency of 2 s of ultrasonic treatment and 4 s of rest, and a total ultrasonic treatment time of 9 min.

[0042] Preferably, the drug-loaded liposomes are prepared by a thin film hydration method.

[0043] Preferably, the mass ratio of the liposomes to doxorubicin in the drug-loaded liposomes is 10:1, and the mass of the liposomes is based on the total mass of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol, and DSPE-HA2000.

[0044] Preferably, the drug-loaded liposomes and the neutrophil membranes are subjected to ultrasonic treatment to promote membrane fusion.

[0045] In a fourth aspect, the present application provides a preparation method of the biomimetic nanomedicine carrier of the neutrophil membrane-fused drug-loaded liposomes.

[0046] The preparation method of the biomimetic nanomedicine carrier of the neutrophil membrane-fused drug-loaded liposomes provided by the present application comprises the following steps:

[0047] a) neutrophils are extracted and isolated from mouse bone marrow cells by using a modified Percoll density gradient centrifugation method; the neutrophils are activated by using LPS; the activated neutrophils are subjected to hypotonic lysis, ultrasonic fragmentation, and differential centrifugation to obtain neutrophil membranes; and the neutrophil membranes are subjected to ice-bath ultrasonic treatment by an ultrasonic cell disrupter to obtain neutrophil membrane vesicles (NMVs);

[0048] b) preparing drug-loaded liposomes by a thin film hydration method with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol, DSPE-HA2000 as raw materials; the mass ratio of the DOPE, DPPC, cholesterol, and DSPE-HA2000 is 6:3:1:0.5 in turn;

[0049] c) mixing the drug-loaded liposomes with neutrophil membrane vesicles (NMVs) at a liposome / membrane protein mass ratio of 1:1, placing in an ice bath, and then treating with an ultrasonic cell disruptor to obtain neutrophil membrane-coated liposomes, thereby obtaining a biomimetic nanomedicine carrier (NM@HA lipos(D, DOX) hybrid particles).

[0050] In the above method step b), the specific method for preparing the drug-loaded liposomes by the thin film hydration method is as follows: dissolving 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol, and DSPE-HA2000 in dichloromethane, and rotary evaporation to form a lipid film; adding a DOX solution and a PBS solution to the lipid film, hydrating at 50℃ and 150rpm for 2h; treating the obtained suspension in an ice bath with an ultrasonic cell disruptor (for example, 400W, intermittent mode: 2s on, 2s off) for 12min to obtain the drug-loaded liposomes; removing free DOX by dialysis in a 3kDa dialysis bag in PBS for 48h, and changing the buffer regularly.

[0051] Preferably, the mass ratio of the liposomes to doxorubicin in the drug-loaded liposomes is 10:1; and the mass of the liposomes is based on the total mass of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol, and DSPE-HA2000.

[0052] In the above method step c), the ultrasonic treatment condition by the ultrasonic cell disruptor is as follows: treating with the ultrasonic cell disruptor at a power of 100W for 2s, resting for 4s, and repeating for 3min, for a total of 9min.

[0053] In a fifth aspect, the present application provides an application of the biomimetic nanomedicine carrier of the neutrophil membrane-fused liposomes according to the first aspect.

[0054] The application is an application in the preparation of a targeted cancer treatment drug.

[0055] In a sixth aspect, the present application provides an application of the biomimetic nanomedicine carrier of the neutrophil membrane-coated drug-loaded liposomes according to the third aspect.

[0056] The application is the use of the drug in the preparation of a targeted therapy for cancer.

[0057] The cancer can be further breast cancer.

[0058] The present application establishes a quantitative evaluation method based on nano-flow cytometry (NanoFCM), which can accurately analyze the membrane hybridization efficiency and uniformity of NM@lipos at the single particle level, overcoming the limitations of the quantitative ability of traditional CLSM methods. By optimizing the lipid formula systemically, the optimal preparation conditions (the mass ratio of DOPE / DPPC / cholesterol is 6:3:1, and the mass ratio of liposome / membrane protein is 1:1) are determined. Under these conditions, the membrane hybridization efficiency of NM@lipos (D) based on DOPE reaches 92.0%, which is significantly better than that of NM@lipos (L) based on lecithin (70.1%). In vivo distribution experiments show that the enrichment of NM@lipos (D) at the tumor site is significantly enhanced, reaching 3.5 times and 1.4 times of lipos (D) and NM@lipos (L) respectively at 6h after injection, and increasing to 5.1 times and 1.87 times at 24h. Pharmacokinetic analysis further confirms that the plasma half-life of NM@lipos (D) is 54.2±2.9h, which is significantly better than that of lipos (D) (24.4±5.8h) and NM@lipos (L) (38.1±3.2h). In the 4T1 tumor model, NM@HAlipos (D, DOX) loaded with DOX and modified with HA achieves a tumor inhibition rate of 89.4%, which is significantly better than that of free DOX (41.3%), HAlipos (D, DOX) (66.5%) and NM@HAlipos (L, DOX) (81.0%). BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 Preparation and characterization of NM@lipos under different phospholipid / cholesterol ratios, DOPE / DPPC ratios and liposome / membrane protein mass ratios. a Wright-Giemsa staining shows neutrophils isolated from mouse bone marrow, scale bar: 50μm. b Neutrophils were labeled with Ly6G-FITC (green) and CD11b-PE (red) double antibodies and identified by flow cytometry. c LPS-activated neutrophils, flow analysis after labeling with CD11b-PE antibody. d Water particle size determination results of liposomes prepared under different phospholipid / cholesterol ratios. e Water particle size determination results of liposomes under different DOPE / DPPC ratios. f Evaluation of liposome membrane fluidity based on fluorescent probe PDA, with the ratio of excimer / monomer fluorescence intensity (I e / I m) As a quantitative indicator of membrane fluidity, the higher the value, the stronger the fluidity of the lipid bilayer. g-i Analysis of the hydrated particle size of NM@lipos constructed under different phospholipid / cholesterol ratios (h), DOPE / DPPC ratios (i), and lipidosome / membrane protein mass ratios (g). Data are expressed as mean ± standard deviation (n = 3). j Analysis of the effect of different lipidosome / membrane protein mass ratios on the particle size distribution of NM@lipos using nano-flow cytometry (Nano FCM).

[0060] Figure 2 Hydrated particle size (a) and zeta potential (b) of neutrophil membrane vesicles (NMVs).

[0061] Figure 3 Formulation screening and fusion level evaluation of neutrophil membrane hybrid liposomes (NM@lipos). a NM@lipos of different formulations were constructed by regulating the phospholipid / cholesterol ratio, DOPE / DPPC ratio, and lipidosome / membrane protein mass ratio. b The membrane hybridization efficiency of NM@lipos, including the overall hybridization rate and the single-particle hybridization degree, was quantitatively evaluated using the single-particle membrane fusion analysis method (SPACEMAN).

[0062] Figure 4To evaluate the hybridization rate and extent of NM@lipos based on single-particle cell membrane analysis (SPACEMAN). The evaluation covered both bulk and single-particle levels. a-c Histograms of NM@lipos hybridization rate under different phospholipid / cholesterol ratios (a), DOPE / DPPC ratios (b), and liposome / membrane protein mass ratios (c) based on NanoFCM analysis. d-f Comparison of hybridization rate under different phospholipid / cholesterol ratios (d), DOPE / DPPC ratios (e), and liposome / membrane protein mass ratios (f). g-i The proportion of free NMVs in total input NMVs in the system after hybridization, corresponding to different phospholipid / cholesterol ratios (g), DOPE / DPPC ratios (h), and liposome / membrane protein mass ratios (i), respectively. j-l The mean fluorescence intensity (MFI) of fused NMVs (DiD labeled) in hybrid particles, corresponding to different phospholipid / cholesterol ratios (j), DOPE / DPPC ratios (k), and liposome / membrane protein mass ratios (l), respectively. m-o The percentage distribution of NM@lipos particles according to the hybridization extent (0%-100%) under different formulation conditions (m phospholipid / cholesterol ratio; n DOPE / DPPC ratio; o liposome / membrane protein mass ratio). p-r The proportion of NM@lipos particles with medium-high hybridization extent (25%-75%) under different phospholipid / cholesterol ratios (p), DOPE / DPPC ratios (q), and liposome / membrane protein mass ratios (r). Data are expressed as mean ± standard deviation (n = 3), and statistical analysis was performed using one-way ANOVA, **p < 0.01, ****p < 0.0001.

[0063] Figure 5Lipso(D), NM@lipos(L) and NM@lipos(D) preparation and characterization. aHydrodynamic size analysis of the three carriers. bZeta potential analysis. cSDS-PAGE gel electrophoresis analysis. dWestern blot detection of protein markers. eTransmission electron microscopy (TEM) images, scale bar: 100 nm. fConfocal laser scanning microscopy (CLSM) images, red for DiD-labeled NMVs, green for DiO-labeled liposomes, scale bar: 25 pm. gHistogram of hybridization rate of NM@lipos(L) and NM@lipos(D). hComparison of hybridization rate of NM@lipos(L) and NM@lipos(D). iComparison of mean fluorescence intensity (MFI) of fused NMVs (DiD) in NM@lipos(L) and NM@lipos(D). jFluorescence intensity histogram of NM@lipos, four gating regions were set to represent different hybridization levels. k, 1Fluorescence intensity histograms of NM@lipos(L) (k) and NM@lipos(D) (1), respectively, the partition corresponds to the hybridization level of 0-25%, 25-50%, 50-75% and 75-100%. mSchematic diagram of the hybridization level (0-100%) of single-particle NM@lipos. nAnalysis of the proportion of particles with different hybridization levels (0-100%) in NM@lipos(L) and NM@lipos(D). oComparison of the proportion of NM@lipos particles with medium-high hybridization levels (25-75%) in L and D groups. Data are expressed as mean ± SD (n = 3).

[0064] Figure 6 Inflammatory targeting evaluation of Lipos(D), NM@lipos(L) and NM@lipos(D): aConfocal laser scanning microscopy (CLSM) images, HUVE cells were incubated with DiD-labeled lipos(D), NM@lipos(L) and NM@lipos(D) for 4 hours, respectively, red for carrier fluorescence, blue for cell nucleus, with or without the addition of TNF-a. Scale bar: 25 pm. bFlow cytometry quantitative analysis of the uptake of different carriers by HUVE cells (n = 3). cSchematic diagram of the in vitro blood vessel barrier model. d, eDetection of fluorescence signal changes in the lower chamber at the set time points under the condition of adding (d) or not adding (e) TNF-a and fMLP (n = 6). fTEM images of different carriers crossing the blood vessel model barrier (terminal time point), scale bar: 200 nm. All data are expressed as mean ± SD, statistical analysis using one-way or two-way ANOVA (ANOVA), significance is represented as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0065] Figure 7 Evaluation of tumor targeting ability of Lipos(D), NM@lipos(L) and NM@lipos(D).

[0066] Tumor targeting evaluation: In vivo fluorescence imaging was performed at multiple time points (0.5, 1, 2, 4, 6, 8, 12, 24 h) after tail vein injection of lipos(D), NM@lipos(L), NM@lipos(D) in g 4T1 tumor-bearing mice. h Major organs were isolated at 6 h and 24 h post-injection for ex vivo fluorescence imaging (n = 3), from left to right: heart, liver, spleen, lung, kidney and tumor. i, j Quantitative analysis of fluorescence intensity in each tissue at 6 h (i) and 24 h (j) post-injection, processed using Living Imaging software.

[0067] Pharmacokinetic evaluation: Plasma concentration-time curves after injection of lipos(D), NM@lipos(L), NM@lipos(D) in healthy rats (n = 4), blood sampling time points were 5 min, 15 min, 30 min, 1 h, 2 h and 4 h. 1 Analysis of plasma half-life (t1 / 2). m Analysis of area under the curve (AUC). n Analysis of clearance (CL). All data were expressed as mean ± SD, statistical analysis was performed using one-way or two-way analysis of variance (ANOVA), significance was represented as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0068] Figure 8 Preparation and characterization of HA@lipos(DOX), NM@HA@lipos(L, DOX) and NM@HA@lipos(D, DOX). (a) Hydrated particle size; (b) Zeta potential; (c) Standard curve of DOX in PBS, X axis is DOX concentration (μg / mL), Y axis is absorbance (a.u.); (d) Encapsulation efficiency; (e, f) Cumulative release percentage of DOX of the three carriers under physiological conditions (e, pH = 7.4) and tumor micro-acidic environment (f, pH = 6.5) within 24 h; (g, h) Stability of the three carriers in PBS within 7 days under physiological temperature (g) and low temperature conditions (h). Data were expressed as mean ± SD (n = 3).

[0069] Figure 9NM@lipos(D) and NM@HAlipos(D) in 4T1 cells: o Flow cytometry analysis of the uptake of DiD-labeled NM@lipos(D) and NM@HAlipos(D) by 4T1 cells (n = 3). p Confocal images showing the uptake of the two carriers by 4T1 cells after 4 h of co-incubation, with red fluorescence of DiD and blue fluorescence of the cell nucleus. q Evaluation of the toxicity of the carriers in different forms loaded with DOX to 4T1 cells. Cell viability was determined after 24 h using the CCK-8 method. Test groups included Lipos(D, DOX), HAlipos(D, DOX), NM@lipos(L, DOX), NM@HAlipos(L, DOX), NM@lipos(D, DOX), and NM@HAlipos(D, DOX), with different concentration gradients (n = 4).

[0070] Figure 10 Evaluation of the in vivo therapeutic effect of HAlipos(D, DOX), NM@HAlipos(L, DOX), and NM@HAlipos(D, DOX) in 4T1 tumor-bearing mouse models. a Schematic diagram of the treatment plan and modeling process (n = 6). b Tumor real image after the mice were sacrificed on day 15. c, d Tumor volume change curve (c) and tumor weight change curve (d) after the mice were injected with PBS, free DOX, HAlipos(D, DOX), NM@HAlipos(L, DOX), and NM@HAlipos(D, DOX) in the tail vein. e, f Tumor weight (e) and relative tumor weight (f) of the mice when they were sacrificed. g Tumor growth inhibition rate (TGI). h Survival curve analysis of the mice. i HE staining of the tumor tissue; j Ki67 immunofluorescence staining (red); k TUNEL cell apoptosis staining (green); blue represents the cell nucleus. l, m Quantitative analysis of the proportion of Ki67-positive cells (l) and TUNEL-positive cells (m) (n = 3).

[0071] Figure 11 HE staining of the main organs of 4T1 tumor-bearing BALB / c mice after injection of PBS, DOX, HAlipos(D, DOX), NM@HAlipos(L, DOX), and NM@HAlipos(D, DOX) in the tail vein for 15 days.

[0072] Figure 12The results of the detection of serum biochemical indicators related to liver function (ALT, AST), kidney function (UREA, CREA) and heart function (CK, LDH) after healthy BALB / c mice were injected with PBS, HAlipos (D), NM@HAlipos (L) and NM@HAlipos (D) via the tail vein for 24 h (n=3). The data are expressed as mean ± standard deviation.

[0073] Figure 13 Cell viability after HAlipos (D), NM@HAlipos (L) and NM@HAlipos (D) were used to treat HUVE cells at different concentrations for 24 h. The data are expressed as mean ± standard deviation (n=4).

[0074] Figure 14 The results of the detection of serum biochemical indicators related to liver function (ALT, AST), kidney function (UREA, CREA) and heart function (CK, LDH) after healthy BALB / c mice were injected with PBS, HAlipos (D), NM@HAlipos (L) and NM@HAlipos (D) via the tail vein for 24 h (n=3). The data are expressed as mean ± standard deviation. DETAILED DESCRIPTION

[0075] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the application.

[0076] The experimental methods in the following examples are all routine methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0077] The materials used in the following examples are as follows: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-hyaluronic acid (DSPE-HA2000) and cholesterol were purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd. Doxorubicin (DOX), 1-pyrenedodecanoic acid (PDA) and lecithin were purchased from China Macklin Biochemical Co., Ltd. Dulbecco's modified medium (DMEM), Roswell Park Memorial Institute 1640 medium (RPMI 1640), fetal bovine serum (FBS, certified level) and trypsin-EDTA digestion solution (0.25%) were purchased from Gibco, USA. Penicillin-streptomycin mixture (100x) was purchased from Hyclone, USA. Tumor necrosis factor-alpha (TNF-alpha) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) were purchased from MedChemExpress (MCE), USA. Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich, Germany. Percoll density gradient separation solution, Wright-Giemsa staining solution and phosphate buffered saline (PBS, 1x) were purchased from Solarbio, China. BCA protein concentration detection kit, cell counting kit-8 (CCK-8), 4% paraformaldehyde, 3,3'-dioctyloxy carbocyanine perchlorate (DiO), 1,1'-dioctyl-3,3,3',3'-tetramethylindocyanine perchlorate (DiD), 4',6-diamidino-2-phenylindole (DAPI) and coomassie brilliant blue staining solution were purchased from Beyotime, China. Rabbit anti-CXCR2, CD11b and CD62L antibodies, horseradish peroxidase (HRP) labeled goat anti-rabbit secondary antibody for Western blot detection, TUNEL and Ki67 detection kits were purchased from Servicebio, China. FITC labeled anti-Ly6G antibody and PE labeled anti-CD11b antibody were purchased from BioLegend, USA. Denatured protein precast gel buffer, 4-20% high resolution HEPES-Tris precast gel and protein molecular weight marker (245kDa) were purchased from Yeasen, China. Hematoxylin-eosin (H&E) staining reagent and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) kit were purchased from Beyotime, China. Unless otherwise specified, all reagents were used according to the original specifications without further purification. Double distilled water (ddH2O) was used for all solution preparation and experimental operation.

[0078] The structural formula of the DSPE-HA2000 is as follows:

[0079]

[0080] The cells and cell culture methods involved in the following examples are as follows: Human umbilical vein endothelial cells (HUVE cells) and 4T1 breast cancer cells were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). HUVE cells were cultured in DMEM medium, and 4T1 cells were cultured in RPMI 1640 medium, both supplemented with 10% fetal bovine serum (FBS), 100 μg / mL streptomycin and 100 U / mL penicillin. All cells were cultured in a constant temperature and humidity environment at 37°C with 5% CO2.

[0081] The animals and feeding methods involved in the animal experiments in the following examples are as follows: 6-8 week old female BALB / c mice and male Sprague-Dawley (SD) rats with a body weight of 210±10 g were purchased from Shandong Jinan Pengyue Experimental Animal Co., Ltd. All animals were raised in a standard environment (25±1°C, free access to food and water). All experimental procedures were approved by the China Ocean University Animal Ethics Committee (Ethical Number: OUC-SMP-2024-08-02).

[0082] The statistical analysis methods used in the following examples are as follows:

[0083] All experimental data are expressed as "mean ± standard deviation" (mean ± SD). Differences between groups were tested using one-way or two-way ANOVA, with appropriate post-hoc multiple comparison methods for significance analysis. Differences were considered statistically significant when p<0.05. Statistical significance levels are represented as follows: p<0.05: *, p<0.01: **, p<0.001: ***, p<0.0001: ****.

[0084] Example 1, Isolation of mouse neutrophils and membrane extraction

[0085] 1. Isolation of neutrophils

[0086] Adult BALB / c mice were sacrificed by cervical dislocation, and femurs and tibias were removed and cleaned of soft tissue and washed with pre-chilled PBS. Bone marrow cavities were flushed with 1 mL of RPMI 1640 using a 1 mL syringe (27G needle), and bone marrow cells were collected by filtration through a 100 pm cell strainer and centrifugation at 500 g for 5 min. Cells were separated by density gradient centrifugation (500 g for 30 min) using 45% and 62% Percoll, and the cells in the middle layer were collected and washed twice with PBS to remove residual Percoll. 3-5 mL of red blood cell lysis solution was added, and the cells were lysed for 10 min on ice, then washed with PBS and centrifuged at 500 g for 5 min. Cell purity and integrity were confirmed by Wright-Giemsa staining.

[0087] 2 Neutrophil purity detection

[0088] 1 x 10 6 Cells were suspended in PBS, and 1 pL of FITC-labeled anti-Ly6G antibody and 1 pL of PE-labeled anti-CD11b antibody were added, and incubated at 4°C in the dark for 30 min. After washing three times with PBS (300 g, 5 min) to remove free antibodies, the cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, USA).

[0089] 3 Neutrophil LPS stimulation activation

[0090] Neutrophils were stimulated with different concentrations of LPS (25, 50, 100, and 200 ng / mL) for 4 h, with a control group without LPS. After stimulation, the cells were collected by centrifugation (300 g, 5 min) and washed with PBS. For flow cytometry detection, 1 x 10 6 Cells were suspended in PBS, and 1 pL of FITC-labeled anti-Ly6G antibody and 1 pL of PE-labeled anti-CD11b antibody were added, and incubated at 4°C in the dark for 30 min. After washing three times with PBS (300 g, 5 min) to remove free antibodies, the cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, USA).

[0091] 4 Neutrophil membrane extraction

[0092] Purified neutrophils were suspended in 10 mL of hypotonic lysis solution (2 mM MgCl2, 10 mM KCl, 20 mM Tris-HCl, pH 7.4) and 1% protease inhibitor cocktail was added, and incubated at 4°C for 2 h. The lysate was sonicated using an ultrasonic disrupter (JY92-IIN, Xinzhi, Ningbo, China) at 100 W in intermittent mode (2 s on, 10 s off) for 6 min. The lysate was centrifuged at different speeds (4°C) to remove cell debris and organelles:

[0093] First, unbroken cells and large particles were removed by centrifugation at 1,500 rpm for 5 min, and the supernatant was reserved; the precipitate was treated again by ultrasonication, and the supernatant was collected; after the supernatants were combined, the nuclei and large particles were removed by centrifugation at 3,600 rpm for 15 min; finally, the supernatant was centrifuged at 11,000 rpm for 30 min to obtain a cell membrane precipitate, which was washed with PBS three times and resuspended for preservation.

[0094] 5 Preparation and characterization of neutrophil membrane vesicles (NMVs)

[0095] The neutrophil membrane suspension obtained by extraction was placed in an ice bath, and NMVs were prepared using an ultrasonic disrupter (100 W, 9 min, 2 s on, 4 s off). The particle size and Zeta potential were measured using a Zetasizer Nano ZS particle size analyzer (Malvern Nano, UK). Transmission electron microscopy (TEM) observation: an appropriate amount of NMV suspension was added dropwise to a copper mesh, washed with distilled water three times after standing for 1 min, negatively stained with 1% uranyl acetate, and then imaged using TEM (JEM-2100, Japan JEOL) after being absorbed with filter paper. The membrane protein concentration was quantitatively analyzed by BCA method.

[0096] 6 Results

[0097] To obtain neutrophils with high purity, the present application uses a modified Percoll density gradient centrifugation method to isolate primary neutrophils from the bone marrow of 6-week-old female BALB / c mice. Wright-Giemsa staining shows that the isolated cells have a typical multinucleated morphology, and the cytoplasm is colorless Figure 1 a), which is consistent with the morphological characteristics of neutrophils [3] , indicating that the isolation process was successful. Flow cytometry further confirmed that Ly6G + / CD11b + double-positive cells accounted for 83.1% ( Figure 1 b), showing that the obtained neutrophils have high purity. After stimulation with different concentrations of LPS (50, 100, and 200 ng / mL), the expression level of CD11b on the surface of neutrophils was up-regulated in a dose-dependent manner, with the expression level of the 200 ng / mL treatment group increasing by about 4.9 times Figure 1 c), so this concentration was used for the subsequent activation step.

[0098] Subsequently, neutrophil membrane vesicles (NMVs) were prepared, and the membrane protein concentration was measured by BCA method to be 2.5 mg / mL. Transmission electron microscopy (TEM) observation showed that NMVs had uniform morphology and were regular spherical Figure 5 e); dynamic light scattering (DLS) measurement showed that the average particle size was 140 ± 2.1 nm, and the Zeta potential was -23 ± 1.5 mV Figure 2 , indicating that it has good colloidal stability.

[0099] Example 2, Preparation of DOPE / DPPC liposomes with different ratios of phospholipid / cholesterol

[0100] 1 Preparation of phospholipid / cholesterol stock solution

[0101] DOPE, DPPC and egg phospholipid were dissolved in methanol respectively to prepare 10 mg / mL stock solution (200 mg in 20 mL), and stored in a brown glass bottle after sealing; DSPE-HA2000 (10 mg) was dissolved in 1 mL methanol; cholesterol (200 mg) was dissolved in a mixture of 15 mL methanol and 5 mL dichloromethane, with a concentration of 10 mg / mL. All stock solutions were stored at -20°C in the dark for later use.

[0102] 2 Preparation of liposomes and particle size analysis

[0103] According to the set phospholipid (DOPE + DPPC) / cholesterol mass ratio (7:3, 7.5:2.5, 8:2, 8.5:1.5, 9:1) and DOPE / DPPC mass ratio (4.5:4.5, 5:4, 6:3, 7:2, 8:1), the lipids and cholesterol were weighed and dissolved in 3 mL dichloromethane, and rotary evaporation (45°C) was performed to form a lipid film. 4 mL PBS was added to the dry film, and the hydration was carried out at 50°C and 150 rpm for 2 h. The obtained suspension was treated in an ice bath using an ultrasonic disrupter (400W, intermittent mode: 2s on, 2s off) for 12 min to obtain liposomes. The particle size and Zeta potential of the obtained liposomes were determined using a Zetasizer Nano ZS particle size analyzer.

[0104] Note: When screening the mass ratio of phospholipid (DOPE + DPPC) / cholesterol, the mass ratio of DOPE to DPPC is fixed at 4.5:4.5; when screening the mass ratio of DOPE / DPPC, the mass ratio of phospholipid (DOPE + DPPC) / cholesterol is fixed at 9:1.

[0105] The liposome sample was diluted to a lipid concentration of 0.1 mg / mL, and 1 μM PDA was added to a final concentration. The sample was incubated at 37°C in the dark for 30 min. The fluorescence emission spectrum was recorded using a microplate reader (excitation wavelength 360 nm, emission wavelength range 370-550 nm), and the monomer peak (about 398 nm) and the excimer peak (about 475 nm) were recorded respectively. The membrane fluidity was evaluated by the fluorescence intensity ratio of excimer peak to monomer peak (E / M), and the higher the E / M value, the stronger the membrane fluidity.

[0106] 4 Results

[0107] Liposomes were prepared by thin-film hydration method, and the ratio of phospholipid / cholesterol (PL / Chol) and the mass ratio of DOPE / DPPC were adjusted. The results of DLS analysis showed that the particle size of liposomes gradually decreased with the decrease of cholesterol content, and the particle size of liposomes was 106.6±3.9 nm and the PDI was 0.260±0.012 when the PL / Chol was 9:1 Figure 1 d). In terms of the optimization of DOPE / DPPC ratio, appropriate increase of DOPE could improve the uniformity of liposomes, but excessive amount would lead to obvious aggregation. When the ratio of DOPE / DPPC was 7:2, unstable large particles with a particle size of 401±18.6 nm and a PDI of 0.506±0.020 were formed Figure 1 e). The membrane fluidity detection based on PDA further showed that the decrease of cholesterol or the increase of DOPE content could enhance the fluidity of the lipid membrane Figure 1 f). DOPE is a conical phospholipid with a lower phase transition temperature (Tm), which is helpful for membrane fusion and the formation of membrane curvature; while the Tm of DPPC is about 41℃, which can impart strong rigidity to the membrane at physiological temperature. Therefore, reasonable regulation of the content of DOPE is crucial for achieving efficient fusion while maintaining the stability of liposomes.

[0108] Example 3, Construction and Fusion Efficiency Evaluation of NM@lipos

[0109] 1. Preparation of NM@lipos under different phospholipid / cholesterol, DOPE / DPPC, and liposome / membrane protein ratios

[0110] The liposomes prepared with different formulations were mixed with neutrophil membrane vesicles (NMVs) at a liposome / membrane protein mass ratio of 1:1, and transferred to 5 mL centrifuge tubes, which were placed in an ice bath to prevent high temperature from damaging the membrane structure. Then, an ultrasonic disrupter was used for ultrasonic treatment (100 W, 9 min, 2 s on, 3 s off) to promote membrane fusion, and NM@lipos hybrid particles were obtained.

[0111] To further optimize the membrane fusion conditions, the mass ratio of DOPE / DPPC / cholesterol was fixed at 6:3:1, while different liposome / membrane protein mass ratios (1:0.05, 1:0.1, 1:0.2, 1:0.5, 1:1, 1:2) were set, and the remaining conditions were kept unchanged. The obtained hybrid particles were measured for particle size and Zeta potential by Zetasizer Nano ZS, and further analyzed for particle size distribution and fusion efficiency by nano flow cytometry (Nano FCM, Xiamen Fuli Biotechnology Co., Ltd.).

[0112] Subsequently, liposomes were mixed with NMVs at different liposome / membrane protein mass ratios to prepare NM@lipos. When the optimal ratio was 1:1, the particle size of the resulting NM@lipos was 153.1 ± 2.6 nm, and the PDI was 0.233 ± 0.031. The NM@lipos showed good stability under different lipid compositions (h, i). Figure 1 Figure 1 With the increase of membrane protein ratio, the particle size gradually decreased and tended to be flat at 1:1, suggesting that the fusion process reached saturation. Continuing to increase the membrane protein ratio had little effect on the particle size, but it could change the surface membrane properties. The NanoFCM analysis results were consistent with the above trend, indicating that the particle size distribution of NM@lipos was consistent under different formulations (j). When the membrane protein ratio was low (1:0.05-1:0.5), the fusion was not complete, and there were more un-fused liposomes in the system. Figure 1

[0113] In summary, when the mass ratio of liposomes to membrane proteins is 1:1, NM@lipos with moderate particle size and good stability can be obtained, which is conducive to its performance in subsequent in vitro and in vivo applications.

[0114] 2Evaluation of NM@lipos membrane fusion efficiency (hybridization rate and hybridization degree)

[0115] To evaluate the membrane fusion effect of NM@lipos, liposomes and NMVs were labeled with green fluorescence (DiO) and red fluorescence (DiD), respectively: 4 mL of NMV suspension (concentration 2.5 mg / mL, BCA determination) was added to 50 μL of 1 mg / mL DiD (dissolved in DMSO) and incubated for 2 h in an ice bath; liposomes (2.5 mg / mL) were incubated with DiO (50 μL, 1 mg / mL) in the same way. After dye incubation, the sample was removed by 3 kDa ultrafiltration centrifugation (8000 rpm, 30 min) to remove free dye, then resuspended with PBS, and repeated the ultrafiltration step 4 times.

[0116] Finally, the double-fluorescence-labeled liposomes and NMVs were prepared into NM@lipos according to the standard fusion process, and the fusion efficiency was analyzed by NanoFCM combined with SPACEMAN (Single Particle Analysis of Cell Membrane), as shown in Figure 3 b.

[0117] The hybridization rate is defined as the proportion of successfully hybridized NM@lipos particles in the total amount of input liposomes;

[0118] The hybridization degree is defined as the percentage of the surface area of a single NM@lipos particle covered by NMVs.

[0119] The results are as follows:​​

[0120] At the bulk level, the hybridization rate was determined by flow cytometry with gating analysis( Figure 4 a-c) and found to increase with decreasing cholesterol content, reaching a peak of 80.7% at PL / Chol 9:1( Figure 4 d), which can be attributed to the enhanced membrane fluidity. Meanwhile, the increase in hybridization rate was accompanied by a decrease in the proportion of free NMVs in the system( Figure 4 g), indicating that more NMVs successfully fused into hybrid vesicles.

[0121] Similarly, the DOPE content showed a bell-shaped trend on the fusion efficiency: the hybridization rate reached a peak of 92.0% at DOPE / DPPC 6:3, but decreased with further increase in DOPE, accompanied by instability of the liposome structure and increased aggregation tendency( Figure 4 e, 4h). As a conical phospholipid with a low phase transition temperature, DOPE can promote membrane curvature and fusion, but its excessive use can lead to impaired structural integrity due to excessive membrane fluidity.

[0122] In terms of membrane protein content, the hybridization rate increased, reaching 97.7% at a liposome / membrane protein mass ratio of 1:2( Figure 4 j). However, compared to 1:1, although the hybridization rate improved marginally (from 92.0% to 97.7%), the proportion of free NMVs increased significantly (about 1.9 times) Figure 4 i), suggesting that the fusion efficiency improved marginally with a doubling of NMV input, leading to protein waste. Therefore, considering the fusion efficiency, membrane resource utilization, and process scalability, 1:1 was ultimately determined to be the optimal ratio.

[0123] At the single-particle level, the SPACEMAN method can quantify the hybridization degree based on fluorescence intensity. The average fluorescence intensity (MFI) of DiO-labeled NMVs in NM@lipos increased with increasing PL / Chol ratio, reaching 675 ± 11.9 at 9:1( Figure 4 j); it showed a bell-shaped trend with changes in DOPE / DPPC ratio, reaching a peak of 1426 ± 29.5 at 6:3( Figure 4 k), further verifying that moderate DOPE content is beneficial to membrane fluidity and fusion, while excessive amounts lead to decreased stability. The average fluorescence intensity (MFI) also increased with increasing membrane protein content, reaching a peak of 1779 ± 42.1 at 1:1( Figure 4 l).

[0124] Further quadrant gating analysis was used to finely divide the distribution of hybridization degree. The low hybridization (0-25%) particles decreased to 21.1% when PL / Chol was 9:1, while the high hybridization particles (50-75%) increased to 26.7% (a) when PL / Chol was 6:3. Figure 4 m) When DOPE / DPPC was 6:3, the low hybridization particles decreased to 13.1%, and the high hybridization particles accounted for 39.8%, of which the particles with medium-high hybridization degree (25-75%) accounted for as high as 81.6% (b). Figure 4 n) With the increase of membrane protein ratio, similar trends were also observed. When the ratio of liposome to membrane protein was 1:1, the low hybridization particles decreased to 5.9%, and the high hybridization particles increased to 39.9% (c). Figure 5 o).

[0125] In summary, the results showed that when the mass ratio of DOPE / DPPC / Chol was 6:3:1 and the mass ratio of liposome to membrane protein was 1:1, the fusion efficiency and fusion uniformity of NM@lipos reached the optimal. This study constructed a quantitative evaluation system for the optimization of NM@lipos formulation, which provided key guidance for the rational design of the new generation of biomimetic drug delivery system.

[0126] Example 4, Preparation and characterization of optimal prescription liposome and NM@lipos (L / D)

[0127] 1 Preparation of liposome (lipos) and hybrid liposome (NM@lipos)

[0128] Liposomes (lipos) were prepared when the mass ratio of DOPE / DPPC / Chol was 6:3:1 (D), and liposomes (lipos) were prepared when the mass ratio of lecithin / cholesterol was 9:1 (L). Both of them used the standard liposome preparation process (refer to the preparation of liposome 2 in Example 2). Lipos (D) or lipos (L) was mixed with NMVs at a mass ratio of liposome to membrane protein of 1:1, and placed in an ice bath. The membrane fusion was achieved by ultrasonic treatment (100W, 9min, 2s on, 3s off) to obtain NM@lipos (D) and NM@lipos (L), which were used for subsequent characterization analysis.

[0129] 2 Dynamic light scattering (DLS)

[0130] The Zetasizer Nano ZS instrument was used to determine the hydration particle size and Zeta potential of liposomes and hybrid particles.

[0131] 3 SDS-PAGE analysis

[0132] Membrane proteins in NMVs, NM@lipos(L), and NM@lipos(D) were extracted, respectively, and 2% SDS was added for lysis on ice for 30 min. Protein quantification was performed using the BCA method. The electrophoresis condition was 150 V for 45 min using a Bio-Rad gel electrophoresis system, followed by protein composition analysis using Coomassie brilliant blue staining.

[0133] 4Western blot detection

[0134] Neutrophils, NMVs, NM@lipos(L), and NM@lipos(D) (control particle number was the same) were lysed in 2% SDS, respectively, and incubated on ice for 30 min. Centrifugation was performed at 11,000 rpm for 10 min, and the supernatant was collected. The protein concentration was determined using the BCA method, and was uniformly adjusted to 30 μg / 20 μL. The SDS-PAGE condition was 150 V for 45 min, and after electrophoresis, the protein was transferred to a PVDF membrane (300 mA, wet transfer for 90 min).

[0135] The membrane was blocked in blocking solution at room temperature for 1 h, followed by the addition of primary antibody (CXCR2, CD11b, CD62L, 1:1000 dilution) for overnight incubation at 4°C. After washing with TBST, HRP-labeled goat anti-rabbit secondary antibody (1:10,000, incubation at room temperature for 1 h) was added, and ECL developing solution was used for imaging (Tanon gel imaging system, China).

[0136] 5Transmission electron microscopy (TEM) and laser confocal microscopy (CLSM)

[0137] TEM observation: The nanoparticles were added to a copper grid, negatively stained with 1% uranyl acetate, and dried, and then the particle morphology was observed using a JEM-2100 TEM (Japan JEOL).

[0138] CLSM co-localization analysis: Double-fluorescence-labeled NM@lipos(L) and NM@lipos(D) were added to a glass slide, fixed, and stained, and then the red-green fluorescence co-localization was observed using a confocal microscope (Zeiss LSM 700, Germany Zeiss) after nuclear staining to verify the uniformity of fusion.

[0139] 6Nano flow cytometry (NanoFCM) and SPACEMAN analysis

[0140] NanoFCM combined with SPACEMAN strategy was used to quantitatively evaluate the fusion rate and fusion degree of hybrid particles. The fusion rate represents the percentage of successfully fused NM@lipos in all liposomes, and the fusion degree represents the coverage ratio of NMVs on the surface of each particle, reflecting the uniformity of fusion.

[0141] 7Results

[0142] Based on optimized formulations, lipo(D), lecithin-based NM@lipos(L), and NM@lipos(D) were successfully prepared and characterized. DLS analysis showed that the particle sizes of the three were 132.6±4.3 nm, 154.6±1.4 nm, and 134.3±6.1 nm, respectively. Figure 5 a) The corresponding Zeta potentials were -39.8±0.9mV, -43.6±2.4mV and -52.7±0.3mV, respectively. Figure 5 b). All formulations exhibited suitable particle size distribution and surface charge, with NM@lipos(D) showing a slight decrease in zeta potential after membrane fusion. SDS-PAGE analysis revealed that the protein bands of NM@lipos(D) and NM@lipos(L) were essentially consistent with those of NMVs. Figure 5 c) confirmed the successful integration of membrane proteins into the hybrid particles. Western blot further verified the presence of membrane marker proteins ( Figure 5 d). Transmission electron microscopy revealed that NM@lipos exhibited a "depression-like" structural feature. Figure 5 e). In confocal microscopy imaging, DiO-labeled liposomes and DiD-labeled NMVs exhibited significant co-localization. Figure 5 f), further confirming the successful fusion of the two.

[0143] To quantitatively assess the fusion effect, the SPACEMAN method was used for analysis at the single-particle level. Figure 5 The results of the streaming gating strategy show that the hybridization rate of NM@lipos(L) is 70.1%, while that of NM@lipos(D) is significantly increased to 92.0%. Figure 5 The fluorescence intensity of NM@lipos(D) was 1318±62.5, significantly higher than that of NM@lipos(L) (396.7±15.7 gh), indicating that DOPE-based liposomes have superior fusion ability. MFI analysis of fused NMVs (DiD) also showed that the fluorescence intensity of NM@lipos(D) was 1318±62.5, significantly higher than that of NM@lipos(L) (396.7±15.7 gh). Figure 5 i) reflects its higher membrane integration efficiency.

[0144] Regarding the distribution of hybridization degree among single particles, the proportion of low-hybridization particles (0%-25%) in NM@lipos(D) was only 12.9%, significantly lower than the 39.1% in NM@lipos(L); while the proportion of high-hybridization particles (50%-75%) significantly increased to 41.3%, far higher than the 9.8% in the L group. Figure 6 In the medium to high hybridization level range (25%-75%), NM@lipos(D) particles accounted for as high as 81.1%, significantly better than NM@lipos(L)'s 60.5%. Figure 6o). The above results further confirmed the key role of DOPE in promoting the efficient fusion of liposomes with cell membranes, providing a solid experimental basis for the rational design of liposome-cell membrane hybrid nanocarriers.

[0145] Example 5, Evaluation of the uptake and penetration ability of liposomes and NM@lipos in the inflammatory endothelial model

[0146] 1. Preparation of DiD-labeled liposomes and NM@lipos

[0147] DiD-labeled liposomes (D) were prepared according to the DOPE:DPPC:cholesterol mass ratio of 6:3:1. 450 μL of DOPE, 450 μL of DPPC, 100 μL of cholesterols, and 100 μL of DiD solution were dissolved in 3 mL of dichloromethane, and rotary evaporation was performed at 45°C to form a lipid film. Then, 4 mL of PBS was added, and the resulting suspension was hydrated at 50°C and 150 rpm for 2 h. The resulting suspension was then sonicated in an ice bath using an ultrasonic cell disruptor (400 W, 12 min, 2 s on, 2 s off) to form DiD-labeled liposomes. To remove free DiD, the liposomes were placed in a 3 kDa dialysis bag and dialyzed in PBS for 48 h, with the buffer being changed regularly.

[0148] The preparation process of DiD-lipos (L) was the same, except that DOPE / DPPC was replaced by lecithin.

[0149] Then, DiD-lipos (D) or DiD-lipos (L) was mixed with NMVs at a liposome / membrane protein ratio of 1:1, and NM@lipos (D) and NM@lipos (L) were prepared by ultrasonic cell disruptor (100 W, 9 min, 2 s on, 3 s off) in an ice bath. The final concentration was adjusted to 2.5 mg of lipids / mL, and the samples were stored at 4°C in the dark.

[0150] 2. Uptake of different liposomes by HUVEC cells (± TNF-α stimulation)

[0151] HUVE cells were seeded in 20 mm glass bottom dishes (2.5 x 10 4 cells / dish) for CLSM observation or in 6-well plates (5 x 10 5 cells / well) for flow cytometry, and were cultured in DMEM + 10% FBS + 1% antibiotic solution for 24 h. Then, they were divided into two groups: the experimental group was stimulated with 10 ng / mL TNF-α for 4 h, and the control group was not treated. After the stimulation, different nanoparticles (50 μg of lipids / mL) were added, and incubation was performed for 4 h.

[0152] CLSM group: washed with PBS, fixed with 4% paraformaldehyde for 10 min, stained with DAPI for 10 min, and observed the fluorescence distribution in cells using confocal microscope;

[0153] Flow cytometry group: after incubation, cells were washed with PBS, trypsinized and collected, and the difference in nanoparticle uptake under TNF-a treatment and non-treatment conditions was quantitatively analyzed using CytoFLEX flow cytometer.

[0154] 3Transwell model to evaluate the penetration ability of carriers across the endothelial layer

[0155] HUVE cells (1 x 10 4 Cells were seeded on the upper chamber of Transwell chambers (0.4 pm pore size, 24-well plate, Corning) at a density of 1 x 10

[0156] 4Results

[0157] To evaluate the inflammation targeting ability of Lipos(D), NM@lipos(L) and NM@lipos(D), a human umbilical vein endothelial cell (HUVEC) model with and without TNF-a stimulation was constructed. Flow cytometry results showed that under non-inflammatory conditions, the uptake of NM@lipos(D) by HUVECs was 1.7 times and 1.3 times that of Lipos(D) and NM@lipos(L), respectively Figure 6 b). After TNF-a stimulation, the uptake of the three carriers was significantly increased, and NM@lipos(D) still showed the highest uptake level. CLSM results were also consistent, and the uptake of all carriers was significantly enhanced under inflammatory conditions, with NM@lipos(D) showing the strongest fluorescence signal Figure 6 a). The results showed that TNF-a could effectively enhance the uptake of nanoparticles, and NM@lipos(D) had the most significant inflammation homing ability.

[0158] An in vitro blood vessel crossing model was further established to activate endothelial cells with TNF-a and chemotactic factor fMLP Figure 6c) The process of vascular penetration under simulated inflammatory conditions. Under non-stimulated conditions, the penetration ability of NM@lipos(D) was significantly better than the other two groups at 6h, 12h and 24h time points, 4.4 and 1.8 times of Lipos(D) and NM@lipos(L) at 12h, respectively Figure 6 d) Under TNF-a and fMLP stimulation, the penetration ability of each group was further improved, but NM@lipos(D) remained the highest level, 3.8 and 1.9 times of Lipos(D) and NM@lipos(L), respectively Figure 7 e) TEM imaging of Transwell lower chamber samples further confirmed that the number of NM@lipos(D) penetration was significantly more than NM@lipos(L) Figure 7 f) indicating its stronger transendothelial migration ability, which may be related to higher membrane fusion efficiency.

[0159] Example 6, Liposome and NM@lipos in vivo distribution and pharmacokinetic analysis in 4T1 tumor mouse model

[0160] Eighteen female BALB / c mice were randomly divided into two experimental systems (n = 9 / group), and each group was further divided into three groups (n = 3), and injected with DiD-labeled lipos(D), NM@lipos(L) or NM@lipos(D) (same as Example 5). Using the method of constructing an orthotopic breast tumor model, 1 x 10 6 4T1 cells (100 μL PBS) were injected into the right fourth mammary fat pad, and the tumor volume was measured every two days, and the formula was V = 0.5 x L x W 2 (L is the maximum diameter, and W is the minimum diameter). When the tumor volume reached about 200 mm 3 , the biodistribution experiment was carried out.

[0161] 2 24h in vivo distribution experiment

[0162] Mice were injected with DiD-labeled lipos(D), NM@lipos(L) or NM@lipos(D) via the tail vein, with a dose of 60 mg of lipid / kg. In vivo imaging was performed using a small animal fluorescence imaging system (IVIS Spectrum, PerkinElmer, USA) at 0.5, 1, 2, 4, 8, 12, 24 h, respectively. After 24 h, the mice were sacrificed, and the heart, liver, spleen, lung, kidney and tumor tissues were collected for ex vivo fluorescence imaging, and the fluorescence signal was quantitatively analyzed using Living Image software.

[0163] 3 6h in vivo distribution experiment

[0164] According to the above administration schedule, mice were sacrificed at 6h post-injection, and major organs were collected for ex vivo fluorescence imaging and fluorescence intensity quantification to evaluate the distribution characteristics of nanoparticles in the early stage in vivo.

[0165] 4 Pharmacokinetics experiment

[0166] Male SD rats (body weight 250-300 g) were selected and randomly divided into 3 groups (n = 4), and injected with DiD-labeled lipos (D), NM@lipos (L) or NM@lipos (D) via tail vein, with a dose of 60 mg lipid / kg. Before injection, the DiD fluorescence intensity of the three samples was uniformly calibrated using a microplate reader.

[0167] At 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h and 48 h, 500 μL of blood samples were collected from the orbital venous plexus, immediately centrifuged at 3000 g for 10 min (4°C), and the plasma was separated and stored at -80°C for later use.

[0168] The fluorescence intensity was detected using a microplate reader (excitation wavelength Ex = 640 nm, emission wavelength Em = 685 nm), and the nanoparticle concentration in the plasma was calculated according to the standard curve (Fig. S12) to draw the blood concentration-time curve and further calculate the main pharmacokinetic parameters, including: half-life (t 1 / 2 ); area under the curve (AUC 0-48 h); clearance (CL).

[0169] To comprehensively evaluate the in vivo circulation behavior and plasma clearance characteristics of different nanoparticles.

[0170] 5、Results

[0171] To verify the tumor targeting ability in the inflammatory environment, 4T1 tumor-bearing mouse models were constructed, and different carriers labeled with DiD were injected via tail vein, and in vivo fluorescence imaging was performed within 0.5 to 24 hours. The results showed that NM@lipos (D) exhibited significant tumor site enrichment at all time points, which was significantly better than Lipos (D) and NM@lipos (L) Figure 7 g). Ex vivo organ imaging and quantitative analysis further confirmed that the tumor accumulation of NM@lipos (D) was the highest at 6h and 24h post-injection Figure 7 h). At 6h, the fluorescence intensity of NM@lipos (D) in the tumor was 3.5 times and 1.4 times that of Lipos (D) and NM@lipos (L), respectively, and it increased to 5.1 times and 1.87 times at 24h Figure 7These results demonstrate that NM@lipos(D) possesses excellent targeting capabilities in the inflammatory tumor microenvironment, further highlighting the advantages of hybrid membrane strategies in enhancing targeted drug delivery.

[0172] To evaluate its in vivo pharmacokinetic properties, pharmacokinetic studies were conducted in a rat model. Blood samples were collected at multiple time points (5 min to 48 h), plasma fluorescence intensity was measured, and plasma drug concentration-time curves were plotted. Compared with Lipos(D) and NM@lipos(L), NM@lipos(D) exhibited a significantly prolonged systemic circulation time. Figure 7 The half-life (t1 / 2) of NM@lipos(L) was 54.2 ± 2.9 h, significantly longer than that of Lipos(D) (24.4 ± 5.8 h) and NM@lipos(L) (38.1 ± 3.2 h). Figure 7 Furthermore, the system exposure of NM@lipos(D) also increased significantly, with its AUC... 0-48 h reached 84.1±5.2, which was 1.5 times and 2.4 times that of NM@lipos(L) and Lipos(D), respectively. Figure 8 Regarding clearance rate (CL), NM@lipos(D) showed a significant reduction, at only 0.12±0.01, which was significantly better than both NM@lipos(L) (0.18±0.02) and Lipos(D) (0.29±0.1). Figure 8 These data indicate that NM@lipos(D) possesses excellent pharmacokinetic characteristics, with longer cycle times, higher bioavailability, and slower clearance rates, possibly due to its good membrane fusion efficiency and biomimetic properties, which reduce immune clearance and improve particle stability.

[0173] Example 7. Preparation and Characterization of HA-Modified, DOX-Loaded Liposomes and Their NM@HAlipos Hybrid Carriers 1. Preparation of DOX-Loaded HA-Modified Liposomes (HAlipos) and NM@HAlipos

[0174] Preparation of HAlipos(D,DOX): 10 mg of lipids (DOPE:DPPC:cholesterol in a mass ratio of 6:3:1) and 50 μL of LD50-HA2000 (10 mg / mL) were dissolved in 3 mL of dichloromethane and rotary evaporated at 45 °C to form a homogeneous lipid film. Then, 1 mL of DOX solution (1 mg / mL) and 3 mL of PBS were added to the flask, and the mixture was hydrated at 50 °C and 150 rpm for 2 h. The resulting suspension was sonicated in an ice bath (400 W, 12 min, 2 s on, 2 s off) to obtain HAlipos(D,DOX). Free DOX was removed by dialyzing in PBS using a 3 kDa dialysis bag for 48 h, with the buffer solution changed periodically.

[0175] Preparation of HAlipos(L, DOX): 1 mL lecithin solution (containing 10 mg lecithin) was mixed with 50 μL DSPE-HA2000 (concentration 10 mg / mL) in 3 mL dichloromethane, and after forming a lipid film by rotary evaporation, 1 mL DOX solution (concentration 1 mg / mL) was hydrated with 3 mL PBS and ultrasonicated, and the subsequent processing method was the same as above.

[0176] Preparation of NM@HAlipos(L, DOX) and NM@HAlipos(D, DOX): HAlipos(L, DOX) or HAlipos(D, DOX) was mixed with NMVs at a lipid / membrane protein mass ratio of 1:1, and ultrasonicated (100 W, 9 min, 2 s on, 3 s off) in an ice bath to achieve membrane fusion. The final concentration was adjusted to 2.5 mg lipid / mL, and stored at 4°C in the dark for standby use.

[0177] 2Particle size and stability characterization

[0178] The hydrated particle size and Zeta potential of HAlipos(D, DOX), NM@HAlipos(L, DOX), and NM@HAlipos(D, DOX) were determined using a Zetasizer Nano ZS. To evaluate their storage stability, the samples were stored at 4°C and 37°C for 7 days, respectively, and the particle size and turbidity were measured regularly to assess their physical stability.

[0179] 3DOX encapsulation efficiency (EE%) and drug release performance (DR%) determination

[0180] Encapsulation efficiency (EE%): ultrafiltration combined with fluorescence spectrophotometry was used for determination. 200 μL sample (2.5 mg lipid / mL) was placed in a 10 kDa ultrafiltration tube, centrifuged at 8000 rpm for 10 min, and the free DOX content (W free ) in the filtrate was determined. An equal volume of sample was added to 2% Triton X-100 for complete lysis, and the total DOX content (W total ) was determined. The fluorescence intensity was read using a microplate reader (Ex = 480 nm, Em = 590 nm), and the calculation formula was as follows:

[0181]

[0182] Drug release (DR%): 1 mL sample (2.5 mg lipid / mL) was placed in a 10 kDa dialysis bag, and placed in pH 7.4 (physiological conditions) and pH 6.5 (tumor microenvironment) PBS buffer, respectively, and shaken at 37°C and 100 rpm. At set time points (0.5, 1, 2, 4, 8, 12, 24 h), 200 μL dialysate was taken out and supplemented with an equal amount of PBS. The amount of DOX released (W released(Ex = 480nm, Em = 590nm), calculate the cumulative release rate using the following formula:

[0183]

[0184] 4 Results

[0185] To further enhance tumor enrichment efficiency, HA2000 targeting ligands were modified on the three vectors mentioned above to enhance their targeting of 4T1 cells. Given that 4T1 cells highly express hyaluronic acid (HA) receptors, HA2000 is expected to effectively enhance targeting ability. Doxorubicin (DOX) was encapsulated into various vectors to obtain HAlipos(D,DOX), NM@HAlipos(L,DOX), and NM@HAlipos(D,DOX), respectively. DLS analysis showed that the particle sizes of the three were 106.2±1.1 nm, 107.5±5.3 nm, and 120.6±0.7 nm, respectively, and the PDIs were 0.24±0.001, 0.31±0.038, and 0.23±0.008, respectively. Figure 8 a) indicates that DOX encapsulation has a relatively small impact on particle size and dispersibility. The DOX encapsulation efficiencies of the three are 64.1%, 61.2%, and 70.1%, respectively. Figure 8 d) It has good drug loading capacity. Drug release experiments showed that DOPE-based NM@HAlipos(D,DOX) exhibits pH-responsive characteristics, demonstrating controlled release behavior under both physiological pH 7.4 and tumor-microacidic pH 6.5 conditions. Figure 9 Stability tests showed that after 7 days of storage at 4℃ and 37℃, there was no significant aggregation or change in particle size. Figure 9 (gh), exhibiting good stability during storage and in vivo application.

[0186] Example 8: Evaluation of uptake and cytotoxicity of different nanocarriers by 4T1 cells.

[0187] 1. Cellular uptake analysis

[0188] 4 T1 cells (2.5 × 10⁴) were collected. 4 Cells (10 cells / dish) were seeded in 20 mm glass dishes and cultured in RPMI-1640 medium supplemented with 10% FBS and 1% antibiotic for 24 h. Then, NM@lipos(D,DOX) and NM@HAlipos(D,DOX) (same as in Example 8) were added at a concentration of 50 μg lipid / mL and incubated for 4 h. After incubation, the cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min, and stained with DAPI for 10 min. Intracellular fluorescence distribution was observed using a confocal microscope (CLSM) to compare the uptake capacity of different carriers.

[0189] For flow cytometry, 4T1 cells (5 x 10 5

[0190] The preparation method of NM@lipos(D, DOX) is as follows: 10 mg of lipid (DOPE:DPPC:cholesterol at a mass ratio of 6:3:1) is dissolved in 3 mL of dichloromethane, and a uniform lipid film is formed by rotary evaporation at 45°C. Then 1 mL of DOX solution (concentration 1 mg / mL) and 3 mL of PBS are added to the bottle, and the hydration is carried out at 50°C and 150 rpm for 2 h. The resulting suspension is ultrasonicated in an ice bath (400 W, 12 min, 2 s on, 2 s off) to prepare Lipos(D, DOX). The free DOX is removed by dialysis in PBS for 48 h using a 3 kDa dialysis bag, and the buffer is replaced regularly.

[0191] Lipos(D, DOX) and NMVs are mixed at a lipid / membrane protein mass ratio of 1:1, and ultrasonicated in an ice bath (100 W, 9 min, 2 s on, 3 s off) to achieve membrane fusion. The final concentration is adjusted to 2.5 mg of lipid / mL, and the prepared product is stored at 4°C in the dark.

[0192] 2 Cell cytotoxicity detection (CCK-8)

[0193] The CCK-8 method is used to evaluate the inhibitory effect of different nanocarriers on 4T1 cells. 4T1 cells are seeded in a 96-well plate at a density of 1 x 10 4

[0194] The absorbance (OD) is read at 450 nm wavelength, and the cell survival rate is calculated, with the calculation formula as follows:

[0195]

[0196] wherein, A​​s Absorbance of experimental wells, A b Absorbance of blank wells, A c Absorbance of control wells.

[0197] 3Results

[0198] Cell uptake experiment showed that the uptake amount of 4T1 cells to NM@HAlipos(D, DOX) was 1.9 times of NM@lipos(D, DOX) Figure 9 , and CLSM images also verified the conclusion Figure 10 p).

[0199] Cytotoxicity experiment results showed that HAlipos(D, DOX), NM@HAlipos(L, DOX) and NM@HAlipos(D, DOX) all had significant killing effect on 4T1 cells, and the effect was better than that of the corresponding group without HA modification Figure 10 q). These results showed that HA2000 modification not only improved the cell uptake efficiency, but also enhanced the anti-tumor treatment effect, confirming that NM@HAlipos(D, DOX) was a chemotherapy nanocarrier with good tumor targeting potential.

[0200] Example 9, Anti-tumor efficacy evaluation of HAlipos and its hybrid carrier in 4T1 tumor-bearing mice in vivo

[0201] 1Animal experiment design and drug administration scheme

[0202] Thirty healthy female BALB / c mice were selected and randomly divided into five groups (n = 6): PBS control group, free DOX group, HAlipos(D, DOX) group, NM@HAlipos(L, DOX) group and NM@HAlipos(D, DOX) group. Through in situ injection, 1 × 10 6 4th pair of right breast fat pad was inoculated with 4T1 cells (100 μL PBS) to establish a breast tumor model.

[0203] When the tumor volume reached about 100 mm 3 , the treatment was started. The mice in each group were injected with different treatment preparations (100 μL) through the tail vein, among which the dose of liposome was 60 mg lipid / kg, and the dose of free DOX was 5 mg / kg. The drug administration schedule was on the 1st, 3rd, 5th, 7th, 8th, 10th, 12th and 14th day, a total of 8 times.

[0204] During the experiment, the body weight and tumor volume of the mice were recorded every two days, and the tumor volume was calculated according to the formula V = 0.5 × L × W 2 (L is the longest diameter, and W is the shortest diameter). The mice were sacrificed on the 15th day, and the tumors and major organs (heart, liver, spleen, lung, kidney) were isolated and weighed for subsequent analysis.

[0205] 2 Histopathology analysis

[0206] HE staining

[0207] The harvested major organs and tumor tissues were washed with normal saline and fixed in 10% neutral formaldehyde solution for 24 h. Subsequently, they were routinely paraffin-embedded, sectioned at 4 μm, deparaffinated, rehydrated, stained with hematoxylin for 5 min, differentiated, washed, stained with eosin for 15 s, and sequentially dehydrated with ethanol and cleared with xylene before being mounted. The morphological changes and damage of the tissues were observed using an optical microscope.

[0208] Ki67 immunofluorescence staining

[0209] After deparaffination and antigen retrieval, the paraffin sections were blocked with 10% donkey serum at room temperature for 30 min, and then incubated with the primary antibody Ki67 (1:200, Abeam, USA) at 4 °C overnight. The next day, after washing, the fluorescently labeled secondary antibody (1:500, room temperature, incubation in the dark for 50 min) was added, and the nuclei were stained with DAPI for 10 min. The confocal microscope was used to observe and count the proportion of Ki67-positive cells represented by red fluorescence (Ex = 530 nm, Em = 590 nm), which reflected the level of tumor cell proliferation.

[0210] TUNEL fluorescence staining

[0211] After deparaffination and rehydration, the paraffin sections were treated with proteinase K (37 °C, 20 min) and then permeated with 0.1% Triton X-100 for 20 min. Subsequently, the TUNEL reaction solution (TdT enzyme:dUTP:buffer = 1:5:50) was added, and the sections were incubated in a humidified box at 37 °C for 1 h, and the nuclei were stained with DAPI for 10 min. Under fluorescence microscopy, the DAPI-positive nuclei were shown in blue (Ex = 350 nm, Em = 420 nm), and the TUNEL-positive apoptotic nuclei were shown in red (TMR labeling, Ex = 530 nm, Em = 590 nm), and thus the proportion of apoptotic cells in the tumor tissue was calculated.

[0212] 3 Results

[0213] To evaluate the in vivo anti-tumor effect of NM@HAlipos(D, DOX), a 4T1 tumor-bearing BALB / c mouse model was established, and the mice were treated with PBS, free DOX, HAlipos(D, DOX), NM@HAlipos(L, DOX), and NM@HAlipos(D, DOX), respectively. Figure 10a) The anti-tumor activity and biosafety were comprehensively evaluated by monitoring tumor volume, physiological weight change and histological analysis. The results showed that NM@HAlipos(D, DOX) exhibited the most significant tumor inhibition effect, with a tumor inhibition rate as high as 89.4%, which was significantly better than that of the DOX group (41.3%), the HAlipos(D, DOX) group (66.5%) and the NM@HAlipos(L, DOX) group (81.0%) Figure 10 g) The tumor growth curve and tumor weight determination results( Figure 10 c-f) further verified the above conclusion. It is worth noting that the mice in the NM@HAlipos(D, DOX) treatment group maintained stable body weight throughout the treatment cycle( Figure 10 d), suggesting that it has low systemic toxicity and good in vivo biocompatibility.

[0214] To further explore its treatment mechanism, histological analysis was performed on the tumor tissue. The HE staining results showed that the tumor tissue structure in the NM@HAlipos(D, DOX) treatment group was severely damaged, with a large area of necrosis, which was in sharp contrast to the tight structure in other groups( Figure 11 i) Ki67 immunostaining showed that the Ki67 positive rate in the NM@HAlipos(D, DOX) group was the lowest, only 4.4%, which was significantly lower than that in the PBS (78.6%), DOX (55.1%), HAlipos(D, DOX) (45.5%) and NM@HAlipos(L, DOX) (27.8%) groups( Figure 11 j, l) TUNEL apoptosis staining further showed that the NM@HAlipos(D, DOX) group induced the highest level of apoptosis, with a TUNEL positive cell ratio of 84.2%, which was much higher than that in the PBS (4.0%), DOX (9.8%), HAlipos(D, DOX) (16.8%) and NM@HAlipos(L, DOX) (59.6%) groups( Figure 12 k, m) These results indicated that the significant anti-tumor effect of NM@HAlipos(D, DOX) was derived from its ability to inhibit tumor cell proliferation and promote cell apoptosis.

[0215] Example 10, In vivo safety evaluation of HAlipos and its hybrid carrier

[0216] After the end of the animal experiment treatment in Example 9, the main organs such as heart, liver, spleen, lung, kidney, etc. were collected, weighed and the organ index (organ weight / body weight x 100%) was calculated. After the tissues were fixed with 4% paraformaldehyde, paraffin-embedded, sectioned and HE stained, whether the pathological changes of the tissue structure occurred was observed. At the same time, the blood was taken from the orbit, the serum biochemical indexes such as ALT, AST (liver function), UREA, CREA (kidney function) and CK, LDH (cardiac function) were detected, and the systemic toxicity of the vector was comprehensively evaluated.

[0217] The results of HE staining of the main organs are shown in Figure 12 From Figure 10 it can be seen that, at the end of the treatment, the main organs (heart, liver, spleen, lung, kidney) were observed by HE staining, and the results showed that there was no obvious pathological change compared with the healthy control group. The tissue structure was complete, and no obvious inflammation, necrosis or other pathological abnormalities were observed.

[0218] The detection results of serum biochemical indexes related to liver function (ALT, AST), kidney function (UREA, CREA) and heart function (CK, LDH) are shown in Figure 13 From Figure 13 it can be seen that the serological detection of mice in each treatment group showed that the liver function indexes (ALT, AST), kidney function indexes (BUN, CREA), and heart function (CK, LDH) were all within the normal range, and there was no significant difference compared with the control group, indicating that the treatment regimen used did not cause obvious hepatotoxicity and nephrotoxicity.

[0219] Example 11, Effect of HAlipos and its hybrid vector on the survival of 4T1 tumor-bearing mice

[0220] 6-8 week old female BALB / c mice were selected and randomly divided into five groups (n = 6), and 1 x 10 6 4T1 cells (100 μL PBS) were inoculated into the right mammary fat pad to establish an orthotopic tumor model. After the tumor volume reached about 100 mm 3 (about 7-9 days), the drug treatment was started.

[0221] Each group of mice was injected with PBS, free DOX (5 mg / kg), HAlipos (D, DOX), NM@HAlipos (L, DOX), and NM@HAlipos (D, DOX) via the tail vein, respectively. The drug administration scheme was on the 1st, 3rd, 5th, 7th, 8th, 10th, 12th and 14th day, a total of 8 times. The observation period was 60 days, and the survival status of the mice was recorded daily. The Kaplan-Meier method was used to draw the survival curve, and the Log-rank (Mantel-Cox) test was used for statistical analysis. The difference was significant when p < 0.05.

[0222] The criteria for euthanasia include: weight loss of more than 25%, tumor volume of more than 2000mm 3 , obvious cachexia, persistent anorexia or tumor ulceration / infection, etc. After the end of the experiment, the surviving mice were sacrificed, and the tumor and major organs were collected for further analysis.

[0223] The results show that the survival rate of NM@HAlipos(D, DOX) group mice is 67% in the 60-day observation period, which is significantly better than that of DOX group (0%), HAlipos(D, DOX) group (0%) and NM@HAlipos(L, DOX) group (33%) Figure 14 h).

[0224] In summary, NM@HAlipos(D, DOX) significantly prolongs the survival of mice while effectively inhibiting tumor growth and inducing tumor cell apoptosis, and has low systemic toxicity, fully demonstrating the great potential of hybrid membrane liposome nanocarriers in precise tumor therapy.

[0225] Example 12, in vivo and in vitro biocompatibility evaluation

[0226] 1 In vitro biocompatibility evaluation

[0227] HUVEC cells were seeded in a 96-well plate at a density of 1×10 4 cells / well and cultured overnight at 37°C, 5% CO2. Then different concentrations of each type of liposome or hybrid carrier (HAlipos(D), NM@HAlipos(L) and NM@HAlipos(D)) were added, and after 24h incubation, cell viability was detected by CCK-8 reagent. 100μL of fresh culture medium and 10μL of CCK-8 working solution were added to each well, and incubation was continued for 2-4h. The absorbance was measured at 450nm using a microplate reader, and the cell survival rate was calculated.

[0228] 2 In vivo biocompatibility evaluation

[0229] Six to eight-week-old female BALB / c mice were selected and randomly divided into four groups (n=3), and were injected with HAlipos(D), NM@HAlipos(L), NM@HAlipos(D) via the tail vein, with a dose of 60mg lipid / kg, and injection was performed every two days for a total of three times. The PBS group was set as a control. Blood was collected 24h after the last administration, and an automatic biochemical analyzer (Chemray 800, Shenzhen Ruituo Biology) was used to detect the serum biochemical indicators, including: liver function: alanine aminotransferase (ALT), aspartate aminotransferase (AST); kidney function: urea (UREA), creatinine (CREA); heart function: creatine kinase (CK), lactate dehydrogenase (LDH). The systemic toxicity of the nanocarrier in vivo was comprehensively evaluated by the above indicators.

[0230] 3 Results

[0231] Cell viability of HUVE cells after treatment with different concentrations of HAlipos(D), NM@HAlipos(L), and NM@HAlipos(D) for 24 hours is shown in the figure. Figure 14 As shown. By ​ As can be seen, the in vitro cytotoxicity was assessed by the CCK-8 assay. The results showed that within the tested concentration range, none of the treatment groups showed significant toxicity to any cell type, and the cell viability was above 90%, with no significant difference compared to the control group.

[0232] The results of serum biochemical markers related to liver function (ALT, AST), kidney function (UREA, CREA), and cardiac function (CK, LDH) are as follows: ​ As shown. By ​ The test results show that the indicators of each treatment group were not significantly different from those of the control group, indicating that the various carriers did not cause significant liver, kidney or cardiotoxicity at the test dose.

[0233] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A biomimetic nanodrug carrier of neutrophil membrane fused liposome, comprising a liposome and a neutrophil membrane fused to the liposome; raw materials of the liposome consist of DOPE, DPPC and cholesterol, and the mass ratio of the three is 6:3:1 in turn; the mass ratio of the liposome to the membrane protein in the neutrophil membrane is 1:1, and the mass of the liposome is based on the total mass of DOPE, DPPC and cholesterol.

2. The bionic nanopharmaceutical carrier according to claim 1, characterized in that: The neutrophils are extracted and separated from mouse bone marrow cells by using a modified Percoll density gradient centrifugation method; The neutrophils need to be activated, and the specific method is to induce neutrophil activation by LPS, so that the activated neutrophil membrane surface overexpresses related adhesion molecules.

3. The nanocarrier according to claim 1 or 2, wherein: The preparation method of the neutrophil membrane is as follows: neutrophils are separated from the bone marrow of BALB / c mice by using a modified Percoll density gradient centrifugation method; neutrophils are activated by LPS; and the activated neutrophils are subjected to hypotonic lysis, ultrasonic wave disruptor ultrasonic crushing and differential centrifugation extraction to obtain neutrophil membranes; The neutrophil membrane is subjected to ultrasonic wave disruptor ice bath ultrasonic treatment to obtain neutrophil membrane vesicles; the ultrasonic wave disruptor ice bath ultrasonic treatment conditions are as follows: the power is 100 W, the frequency is ultrasonic 2 s and resting 4 s, and the total ultrasonic treatment time is 9 min. 4.A preparation method of the biomimetic nanodrug carrier of neutrophil membrane fused liposome according to any one of claims 1-3, comprising the following steps: 1) neutrophils are extracted and separated from mouse bone marrow cells by using a modified Percoll density gradient centrifugation method; the neutrophils are activated by LPS; the activated neutrophils are subjected to hypotonic lysis, ultrasonic crushing and differential centrifugation extraction to obtain neutrophil membranes; and the neutrophil membranes are subjected to ultrasonic wave disruptor ice bath ultrasonic treatment to obtain neutrophil membrane vesicles; 2) liposomes are prepared by using DOPE, DPPC and cholesterol as raw materials by a thin film hydration method; and the mass ratio of the DOPE, DPPC and cholesterol is 6:3:1 in turn; 3) the liposomes and the neutrophil membrane vesicles are mixed according to a liposome / membrane protein mass ratio of 1:1, placed in an ice bath, and then subjected to ultrasonic treatment by using the ultrasonic wave disruptor to obtain the biomimetic nanodrug carrier of neutrophil membrane fused liposome.

5. The method of claim 4, wherein: In the step 2), the specific method for preparing the liposomes by the thin film hydration method is as follows: DOPE, DPPC and cholesterol are dissolved in dichloromethane, and rotary evaporation is performed to form a lipid film; PBS solution is added to the lipid film, and hydration is performed at 50°C and 150 rpm for 2 hours; and the obtained suspension is treated by using an ultrasonic wave disruptor in an ice bath for 12 min to obtain the liposomes; and / or, in the step 3), the ultrasonic treatment conditions by using the ultrasonic disruptor are as follows: the power is 100 W, the frequency is ultrasonic 2 s and resting 4 s, and the total ultrasonic treatment time is 9 min.

6. A biomimetic nanodrug carrier of neutrophil membrane-fused drug-loaded liposomes, comprising drug-loaded liposomes and neutrophil membranes fused to the liposomes; raw materials of the liposomes are composed of DOPE, DPPC, cholesterol and DSPE-HA2000, and the mass ratio of the four is 6:3:1:0.5 in turn; the drug loaded in the drug-loaded liposomes is doxorubicin; the mass ratio of the drug-loaded liposomes to the membrane proteins in the neutrophil membranes is 1:1; the mass of the drug-loaded liposomes is based on the total mass of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol and DSPE-HA2000.

7. The bionic nanopharmaceutical carrier according to claim 6, characterized in that: The preparation method of the neutrophil membranes is as follows: unactivated neutrophils are separated from the bone marrow of BALB / c mice by using a modified Percoll density gradient centrifugation method; the neutrophils are activated by using LPS; the activated neutrophils are subjected to hypotonic lysis, ultrasonic disruption by an ultrasonic cell disruptor, and differential centrifugation to obtain neutrophil membranes; The neutrophil membrane vesicles are obtained by ice-bath ultrasonic treatment of the neutrophil membranes by an ultrasonic cell disruptor; the ice-bath ultrasonic treatment of the ultrasonic cell disruptor is performed at a power of 100 W, at a frequency of ultrasonic treatment for 2 s and resting for 4 s, and for a total ultrasonic treatment time of 9 min.

8. A preparation method of the biomimetic nanodrug carrier of neutrophil membrane-fused drug-loaded liposomes according to claim 6 or 7, comprising the following steps: a) neutrophils are extracted and separated from the bone marrow of mice by using a modified Percoll density gradient centrifugation method; the neutrophils are activated by using LPS; the activated neutrophils are subjected to hypotonic lysis, ultrasonic disruption, and differential centrifugation to obtain neutrophil membranes; the neutrophil membranes are subjected to ice-bath ultrasonic treatment by an ultrasonic cell disruptor to obtain neutrophil membrane vesicles; b) preparing the drug-loaded liposome by using DOPE, DPPC, cholesterol, DSPE-HA2000, and doxorubicin as raw materials through the thin film hydration method; wherein, the mass ratio of the DOPE, DPPC, cholesterol and DSPE-HA2000 is 6:3:1:0.5 in turn; c) the drug-loaded liposomes and the neutrophil membrane vesicles are mixed at a mass ratio of liposomes to membrane proteins of 1:1, and placed in an ice bath, followed by ultrasonic treatment by an ultrasonic cell disruptor to obtain the biomimetic nanodrug carrier of neutrophil membrane-fused drug-loaded liposomes.

9. The method of claim 8, wherein: In step b), the drug-loaded liposomes are prepared by the following specific method of thin film hydration method: DOPE, DPPC, cholesterol and DSPE-HA2000 are dissolved in dichloromethane, and rotary evaporation is performed to form a lipid film; doxorubicin solution and PBS solution are added to the lipid film, and hydration is performed at 50°C and 150 rpm for 2 h; the obtained suspension is treated by an ultrasonic cell disruptor in an ice bath for 12 min to obtain the drug-loaded liposomes; 3kDa dialysis bags are used for dialysis in PBS for 48 h to remove free doxorubicin; And / or, in the step c), the condition of ultrasonic treatment by ultrasonic cell disrupter is: 100 W power, 2 s ultrasonic treatment by ultrasonic cell disrupter, 4 s rest, 3 min ultrasonic treatment, and 9 min total time.

10. The application of the biomimetic nanopharmaceutical carrier of the neutrophil membrane fusion drug-loaded liposome according to claim 6 or 7 in the preparation of a targeted cancer treatment drug; the cancer is breast cancer.

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