Bionic nano-drug carrier of neutrophile granulocyte membrane fusion liposome as well as preparation method and application of bionic nano-drug carrier

Abstract

The invention discloses a bionic nano-drug carrier of neutrophile granulocyte membrane fusion liposome as well as a preparation method and application of the bionic nano-drug carrier, and belongs to the field of medicines. The vector comprises a liposome and a neutrophile granulocyte membrane fused in the liposome, the liposome raw materials comprise DOPE, DPPC and cholesterol in a mass ratio of 6: 3: 1; the mass ratio of the liposome to the membrane protein is 1: 1. According to the method, a quantitative analysis strategy based on NanoFCM is established, and the fusion efficiency and fusion uniformity of NM (at) lipos can be accurately evaluated at the single particle level. The hybridization efficiency of the NM (at) lips (D) constructed on the basis of DOPE reaches 92.0% and is obviously higher than that of NM (at) lips (L) (70.1%) based on lecithin. In in-vivo experiments, the tumor enrichment capacity of NM (at) lipos (D) in 24 hours is improved by 5.1 times compared with that of Lipos (D), and the NM (at) lipos (D) shows remarkably prolonged circulation time.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a bionic nano drug carrier of neutrophil membrane fusion liposomes and a preparation method and application thereof. Background Art

[0002] Cell membrane biomimetic nanoparticles (CM@NPs) have received widespread attention in recent years as a cutting-edge drug delivery system. They utilize the biological functionality of the original cell membrane to improve the biocompatibility of nanocarriers, achieve immune escape, and enhance targeting specificity. As an important type of biomimetic membrane material, neutrophil membranes (NMs) are widely used in the construction of inflammatory site and tumor-targeted drug delivery systems due to their natural inflammation and tumor homing abilities. Among the many construction strategies, 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 resulting multifunctional and efficient biomimetic drug delivery carriers have broad application prospects in the fields of precision drug delivery therapy, disease imaging, and diagnosis.

[0003] Although CM@lipos has shown significant advantages in in vitro studies, its actual performance in vivo depends largely on the efficiency and uniformity of the membrane fusion process. Membrane fusion efficiency not only affects the presentation state of surface membrane proteins, but also determines their immune camouflage ability and in vivo behavior. However, how lipid composition regulates the fusion efficiency of NMs and liposomes, and the impact of fusion level on their downstream biological behavior, has not yet been systematically revealed. In particular, DOPE (unsaturated phospholipids) and DPPC (saturated phospholipids) have functional differences in fusion ability and structural stability. [4, 5], but the specific effect of its combination ratio on the performance of NM@lipos is still unclear. In addition, the currently commonly used evaluation methods (such as confocal microscopy) are mostly limited to local qualitative observation and lack the ability to quantitatively analyze the fusion efficiency of single particles. [1 , 2 ,3], which seriously restricts the system optimization of fusion parameters.

[0004] Therefore, it is of great significance to study and develop a biomimetic nanodrug carrier of neutrophil membrane fusion liposomes that can efficiently fuse NMs with liposomes and to establish an evaluation method to accurately evaluate 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 of the Invention

[0011] To address the shortcomings of existing cell membrane-liposome hybrid nanoparticles (CM@lipos), we established a quantitative analysis strategy based on nanoflow cytometry (NanoFCM) to accurately assess the fusion efficiency and uniformity of NM@lipos at the single-particle level. Key parameters, including the DOPE / DPPC ratio, phospholipid / cholesterol ratio, and liposome / membrane protein mass ratio, were systematically optimized to identify optimal conditions for efficient fusion. The pharmacokinetic behavior, inflammation-targeting ability, and antitumor efficacy of the optimized NM@lipos were further validated in vitro and in vivo. The DOPE-dominated NM@lipos(D) achieved a fusion efficiency of 92.0%, significantly superior to that of the phosphatidylcholine system. In a 4T1 mouse model, it exhibited enhanced tumor accumulation and prolonged in vivo circulation. Furthermore, upon DOX loading, it achieved significant tumor suppression (89.4%) and prolonged survival. This study not only provides a technical framework for the quantitative construction of biomimetic hybrid nanosystems but also lays the foundation for the rational design and translational application of precision nano-drug delivery systems.

[0012] In order to achieve the above object, the present invention adopts the following technical solutions:

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

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

[0015] The raw materials of the liposomes include 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-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; the mass of the 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) and cholesterol.

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

[0018] Preferably, the neutrophils need to be activated, and the specific method is: using LPS to induce neutrophil activation, so that the activated neutrophils overexpress relevant adhesion molecules (such as CD11b) on the membrane surface.

[0019] Specifically, unactivated neutrophils are isolated from the bone marrow of BALB / c mice using a modified Percoll density gradient centrifugation method; LPS (e.g., 200 ng / mL LPS) is used to induce neutrophil activation; the activated neutrophils are subjected to hypotonic lysis, ultrasonic disruption using an ultrasonic disruptor, and differential centrifugation to obtain neutrophil membranes.

[0020] Furthermore, the neutrophil membrane is subjected to ultrasonic treatment in an ice bath using an ultrasonic disruptor to obtain neutrophil membrane vesicles (NMVs).

[0021] Preferably, the conditions for the ultrasonic treatment in an ice bath using an ultrasonic disruptor are: a power of 100 W, a frequency of ultrasonication for 2 seconds and rest for 4 seconds, and a total ultrasonic treatment time of 9 minutes.

[0022] Preferably, the liposomes are prepared by a thin film hydration method.

[0023] Preferably, the liposome and neutrophil membrane are promoted to fuse by ultrasonic treatment to achieve fusion of the outer membrane.

[0024] In a second aspect, the present invention provides a method for preparing the biomimetic nano-drug carrier of the neutrophil membrane fusion liposome described in the first aspect.

[0025] The method for preparing the biomimetic nano drug carrier of neutrophil membrane fusion liposome provided by the present invention comprises the following steps:

[0026] 1) Neutrophils are extracted and isolated from mouse bone marrow using a modified Percoll density gradient centrifugation method; the neutrophils are activated using LPS; the activated neutrophils are subjected to hypotonic lysis, ultrasonic disruption, and differential centrifugation to obtain neutrophil membranes; the neutrophil membranes are sonicated in an ice bath using an ultrasonic disruptor to obtain neutrophil membrane vesicles (NMVs);

[0027] 2) preparing liposomes 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; the mass ratio of DOPE, DPPC, and cholesterol being 6:3:1, respectively;

[0028] 3) The liposomes were mixed with neutrophil membrane vesicles (NMVs) at a liposome / membrane protein mass ratio of 1:1, placed in an ice bath, and then sonicated using an ultrasonic disruptor to obtain neutrophil membrane-coated liposome biomimetic nanodrug carriers (NM@lipos(D) hybrid particles).

[0029] In step 2) of the above method, the specific method for preparing liposomes by the 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 evaporated to form a lipid film; PBS solution is added to the lipid film and hydrated at 50°C and 150 rpm for 2 hours; the resulting suspension is treated in an ice bath using an ultrasonic disruptor (e.g., 400W, intermittent mode: 2s on, 2s off) for 12 minutes to obtain the liposomes.

[0030] In step 3) of the above method, the mass of the liposome is calculated as the sum of the masses of DOPE, DPPC, and cholesterol; the mass of the membrane protein is the mass of the membrane protein in the neutrophil membrane vesicles.

[0031] In step 3) of the above method, the ultrasonic treatment conditions using an ultrasonic disruptor are: a power of 100 W, a frequency of ultrasonication for 2 seconds and rest for 4 seconds, and a total ultrasonic treatment time of 9 minutes.

[0032] In a third aspect, the present invention provides a biomimetic nano drug carrier of neutrophil membrane fusion drug-loaded liposomes.

[0033] The bionic nano drug carrier of neutrophil membrane-fused drug-loaded liposome provided by the present invention comprises a drug-loaded liposome and a neutrophil membrane fused to the surface of the liposome;

[0034] The raw materials of the liposomes include 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 liposomes to the membrane proteins in the neutrophil membrane is 1:1; the mass of the drug-loaded liposomes 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 neutrophils are extracted and separated from mouse bone marrow cells using a modified Percoll density gradient centrifugation method.

[0038] Preferably, the neutrophils need to be activated, and the specific method is: using LPS to induce neutrophil activation, so that the activated neutrophils overexpress relevant adhesion molecules (such as CD11b) on the membrane surface.

[0039] Specifically, unactivated neutrophils are isolated from the bone marrow of BALB / c mice using a modified Percoll density gradient centrifugation method; LPS (e.g., 200 ng / mL LPS) is used to induce neutrophil activation; the activated neutrophils are subjected to hypotonic lysis, ultrasonic disruption, and differential centrifugation to obtain neutrophil membranes.

[0040] Furthermore, the neutrophil membrane is subjected to ultrasonic treatment in an ice bath using an ultrasonic disruptor to obtain neutrophil membrane vesicles (NMVs).

[0041] Preferably, the ultrasonic treatment conditions of the ultrasonic disruptor in an ice bath are: a power of 100 W, a frequency of ultrasonication for 2 seconds and rest for 4 seconds, and a total ultrasonic treatment time of 9 minutes.

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

[0043] Preferably, the mass ratio of liposomes to doxorubicin in the drug-loaded liposomes is 10:1; 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 neutrophil membranes are fused by promoting membrane fusion through ultrasonic treatment.

[0045] In a fourth aspect, the present invention provides a method for preparing the biomimetic nano drug carrier of the neutrophil membrane-fusion drug-loaded liposome described in the third aspect.

[0046] The method for preparing the biomimetic nano drug carrier of neutrophil membrane-coated drug-loaded liposome provided by the present invention comprises the following steps:

[0047] a) extracting and isolating neutrophils from mouse bone marrow cells using a modified Percoll density gradient centrifugation method; inducing activation of the neutrophils using LPS; subjecting the activated neutrophils to hypotonic lysis, ultrasonic disruption, and differential centrifugation to obtain neutrophil membranes; and sonicating the neutrophil membranes in an ice bath using an ultrasonic disruptor to obtain neutrophil membrane vesicles (NMVs);

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

[0049] c) mixing the drug-loaded liposomes with neutrophil membrane vesicles (NMVs) at a liposome / membrane protein mass ratio of 1:1, placing the mixture in an ice bath, and then sonicating the mixture using an ultrasonic disruptor to obtain neutrophil membrane-coated liposome biomimetic nanodrug carriers (NM@HAlipos(D,DOX) hybrid particles).

[0050] In step b) of the above method, the specific method for preparing the drug-loaded liposomes by the thin film hydration method is as follows: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol, and DSPE-HA2000 are dissolved in dichloromethane and rotary evaporated to form a lipid film; DOX solution and PBS solution are added to the lipid film, and hydrated at 50°C and 150 rpm for 2 hours; the resulting suspension is treated in an ice bath using an ultrasonic disruptor (e.g., 400W, intermittent mode: 2s on, 2s off) for 12 minutes to obtain the drug-loaded liposomes; dialyzed in PBS with a 3kDa dialysis bag for 48 hours to remove free DOX, and the buffer solution is replaced regularly.

[0051] Preferably, the mass ratio of liposomes to doxorubicin in the drug-loaded liposomes is 10:1; 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 step c) of the above method, the ultrasonic treatment using an ultrasonic disruptor is carried out under the following conditions: ultrasonic treatment with a power of 100 W for 2 seconds and a rest frequency of 4 seconds, for a total treatment time of 3 minutes, for a total treatment time of 9 minutes.

[0053] In a fifth aspect, the present invention provides an application of the biomimetic nano-drug carrier of the neutrophil membrane fusion liposome described in the first aspect.

[0054] The application is its application in preparing targeted cancer treatment drugs.

[0055] In a sixth aspect, the present invention provides an application of the biomimetic nano drug carrier of the neutrophil membrane-coated drug-loaded liposome described in the third aspect.

[0056] The application is its application in preparing targeted cancer treatment drugs.

[0057] The cancer may further be breast cancer.

[0058] The present invention establishes a quantitative evaluation method based on nanoflow 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 traditional CLSM method's insufficient quantitative capability. By systematically optimizing the lipid formulation, the optimal preparation conditions were determined (DOPE / DPPC / cholesterol mass ratio of 6:3:1, liposome / membrane protein mass ratio of 1:1). Under these conditions, the membrane hybridization efficiency of DOPE-based NM@lipos(D) reached 92.0%, significantly superior to the lecithin-based NM@lipos(L) (70.1%). In vivo distribution experiments showed that the enrichment of NM@lipos(D) at the tumor site was significantly enhanced, with the enrichment levels of NM@lipos(D) being 3.5-fold and 1.4-fold higher than those of lipos(D) and NM@lipos(L), respectively, at 6 hours after injection, and increasing to 5.1-fold and 1.87-fold at 24 hours. Pharmacokinetic analysis further confirmed that NM@lipos(D) had a plasma half-life of 54.2±2.9 hours, significantly superior to lipos(D) (24.4±5.8 hours) and NM@lipos(L) (38.1±3.2 hours). In the 4T1 tumor model, DOX-loaded and HA-modified NM@HAlipos(D,DOX) achieved an 89.4% tumor inhibition rate, significantly superior to free DOX (41.3%), HAlipos(D,DOX) (66.5%), and NM@HAlipos(L,DOX) (81.0%). BRIEF DESCRIPTION OF THE 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: 50 μm. b Neutrophils were labeled with Ly6G-FITC (green) and CD11b-PE (red) double antibodies and identified by flow cytometry. c Neutrophils activated by LPS stimulation and flow cytometry analysis after labeling with CD11b-PE antibody. d Determination of the hydrated particle size of liposomes prepared with different phospholipid / cholesterol ratios. e Determination of the hydrated particle size of liposomes under different DOPE / DPPC ratios. f Evaluation of liposome membrane fluidity based on the fluorescent probe PDA, using the excimer / monomer fluorescence intensity ratio (I e / I m) is used as a quantitative indicator of membrane fluidity; higher values indicate greater lipid bilayer fluidity. gi. Hydrated particle size analysis of NM@lipos constructed with different phospholipid / cholesterol ratios (h), DOPE / DPPC ratios (i), and liposome / membrane protein mass ratios (g). Data are presented as mean ± SD (n = 3). j. The effect of different liposome / membrane protein mass ratios on the particle size distribution of NM@lipos was analyzed using nanoflow cytometry (NanoFCM).

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

[0061] Figure 3 Formulation screening and fusion level assessment of neutrophil membrane hybrid liposomes (NM@lipos). a. NM@lipos with different formulations were constructed by manipulating the phospholipid / cholesterol ratio, DOPE / DPPC ratio, and liposome-to-membrane protein mass ratio. b. The single-particle membrane fusion assay (SPACEMAN) was used to quantitatively evaluate the membrane hybridization efficiency of NM@lipos, including the overall hybridization rate and the degree of single-particle hybridization.

[0062] Figure 4The hybridization rate and degree of NM@lipos were evaluated using the single-particle cell membrane analysis method (SPACEMAN). This evaluation covers both the overall and single-particle levels. ac Histograms of the hybridization rate of NM@lipos under different phospholipid / cholesterol ratios (a), DOPE / DPPC ratios (b), and liposome / membrane protein mass ratios (c), analyzed by NanoFCM. df Comparison of hybridization rates under different phospholipid / cholesterol ratios (d), DOPE / DPPC ratios (e), and liposome / membrane protein mass ratios (f). gi The ratio of free NMVs to total input NMVs in the hybridized system, corresponding to different phospholipid / cholesterol ratios (g), DOPE / DPPC ratios (h), and liposome / membrane protein mass ratios (i). jl Mean fluorescence intensity (MFI) of fused NMVs (DiD-labeled) in the hybridized particles, corresponding to different phospholipid / cholesterol ratios (j), DOPE / DPPC ratios (k), and liposome / membrane protein mass ratios (l). mo. Percentage distribution of NM@lipos particles by hybridization degree (0%–100%) under different formulation conditions (m. phospholipid / cholesterol ratio; n. DOPE / DPPC ratio; o. liposome / membrane protein mass ratio). pr. Comparison of the proportion of NM@lipos particles with intermediate to high hybridization levels (25%–75%) under different phospholipid / cholesterol ratios (p), DOPE / DPPC ratios (q), and liposome / membrane protein mass ratios (r). Data are expressed as mean ± SD (n = 3). Statistical analysis was performed using one-way analysis of variance (ANOVA). **p < 0.01, ****p < 0.0001.

[0063] Figure 5Preparation and characterization of Lipso(D), NM@lipos(L), and NM@lipos(D). a Hydration particle size analysis of the three carriers. b Zeta potential analysis. c SDS-PAGE gel electrophoresis analysis. d Western blot detection of protein markers. e Transmission electron microscopy (TEM) image, scale bar: 100 nm. f Confocal laser scanning microscopy (CLSM) image, red: DiD-labeled NMVs, green: DiO-labeled liposomes, scale bar: 25 μm. g Histogram of hybridization rates of NM@lipos(L) and NM@lipos(D). h Comparison of hybridization rates of NM@lipos(L) and NM@lipos(D). i Comparison of mean fluorescence intensity (MFI) of NMVs (DiD) fused to NM@lipos(L) and NM@lipos(D). j Fluorescence intensity histogram of NM@lipos, with four gated regions set to represent different degrees of hybridization. k, l show the fluorescence intensity histograms of NM@lipos(L)(k) and NM@lipos(D)(l), respectively, with the corresponding hybridization levels being 0%-25%, 25%-50%, 50%-75%, and 75%-100%. m Schematic diagram of the hybridization degree (0%-100%) of a single NM@lipos particle. n Analysis of the proportion of particles with different hybridization degrees (0%-100%) in NM@lipos(L) and NM@lipos(D). o Comparison of the proportion of NM@lipos particles with medium-to-high hybridization levels (25%-75%) in groups L and D. Data are presented as mean ± SD (n = 3).

[0064] Figure 6 Evaluation of the inflammatory targeting of Lipos(D), NM@lipos(L), and NM@lipos(D): a Confocal laser scanning microscopy (CLSM) images of HUVE cells incubated with DiD-labeled lipos(D), NM@lipos(L), and NM@lipos(D) for 4 hours, respectively. Red represents vector fluorescence, and blue represents cell nuclei. Conditions were varied with and without the addition of TNF-α. Scale bar: 25 μm. b Flow cytometric analysis of HUVE cell uptake of different vectors (n = 3). c Schematic diagram of the in vitro vascular barrier model. d, e Fluorescence signal changes in the lower chamber were measured at defined time points with (d) or without (e) the addition of TNF-α and fMLP (n = 6). f TEM images of different vectors after they crossed the vascular barrier model (final time point). Scale bar: 200 nm. All data are expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using one-way or two-way analysis of variance (ANOVA). Significance was indicated as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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

[0066] Tumor targeting assessment: g In vivo fluorescence imaging was performed at multiple time points (0.5, 1, 2, 4, 6, 8, 12, and 24 hours) after tail vein injection of lipos (D), NM@lipos (L), and NM@lipos (D) in 4T1 tumor-bearing mice. h Ex vivo fluorescence imaging of major organs was performed at 6 and 24 hours after injection (n = 3). From left to right, the images are heart, liver, spleen, lung, kidney, and tumor. i, j Quantitative analysis of fluorescence intensity in various tissues at 6 hours (i) and 24 hours (j) after injection, processed using Living Imaging software.

[0067] Pharmacokinetic Assessment: k Plasma concentration-time curves of lipos(D), NM@lipos(L), and NM@lipos(D) in healthy rats after injection (n=4). Blood was collected at 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, and 4 hours. l Plasma half-life (t1 / 2) analysis. m Area under the curve (AUC) analysis. n Clearance (CL) analysis. All data are expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using one-way or two-way analysis of variance (ANOVA). Significance is indicated as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0068] Figure 8 Preparation and characterization of HAlipos(DOX), NM@HAlipos(L,DOX), and NM@HAlipos(D,DOX). (a) Hydrated particle size; (b) Zeta potential; (c) DOX standard curve in PBS, with DOX concentration (μg / mL) on the X-axis and absorbance (au) on the Y-axis; (d) Encapsulation efficiency; (e, f) Cumulative DOX release percentages over 24 hours from the three carriers under physiological conditions (e, pH = 7.4) and the slightly acidic tumor environment (f, pH = 6.5); (g, h) Stability of the three carriers in PBS over 7 days at physiological temperature (g) and low temperature (h). Data are presented as mean ± SD (n = 3).

[0069] Figure 9Figure 4: 4T1 cell uptake and toxicity of NM@lipos(D) and NM@HAlipos(D). Flowcytometry analysis of DiD-labeled NM@lipos(D) and NM@HAlipos(D) by 4T1 cells (n=3). Confocal images show 4T1 cell uptake after 4 hours of co-incubation with the two vectors. Red represents DiD fluorescence, and blue represents cell nuclei. Toxicity assessment of DOX-loaded vectors in 4T1 cells. Cell viability was measured using the CCK-8 assay after 24 hours. The 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 a concentration gradient treatment (n=4).

[0070] Figure 10 In vivo evaluation of the therapeutic efficacy of HAlipos(D,DOX), NM@HAlipos(L,DOX), and NM@HAlipos(D,DOX) in a 4T1 tumor-bearing mouse model. a Schematic diagram of the treatment plan and modeling process (n = 6). b Images of tumors after mice were sacrificed on day 15. c, d Tumor volume change curves (c) and tumor weight change curves (d) in each group of mice after tail vein injection of PBS, free DOX, HAlipos(D,DOX), NM@HAlipos(L,DOX), and NM@HAlipos(D,DOX). e, f Tumor weight (e) and relative tumor weight (f) at the time of mouse sacrifice. g Tumor growth inhibition rate (TGI). h Mouse survival curve analysis. i HE staining of tumor tissue; j Ki67 immunofluorescence staining (red); k TUNEL apoptosis staining (green); blue represents cell nuclei. l, m Quantitative analysis of the proportion of Ki67-positive cells (l) and the proportion of TUNEL-positive cells (m) (n = 3).

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

[0072] Figure 12Serum biochemical parameters related to liver function (ALT, AST), renal function (UREA, CREA), and cardiac function (CK, LDH) were measured in BALB / c mice bearing 4T1 tumors 15 days after tail vein injection of PBS, DOX, HAlipos (D, DOX), NM@HAlipos (L, DOX), and NM@HAlipos (D, DOX) (n = 3). Data are expressed as mean ± SD.

[0073] Figure 13 Cell viability of HUVE cells after 24 h of treatment with HAlipos (D), NM@HAlipos (L), and NM@HAlipos (D) at different concentrations. Data are expressed as mean ± SD (n = 4).

[0074] Figure 14 Results of serum biochemical markers related to liver function (ALT, AST), renal function (UREA, CREA), and cardiac function (CK, LDH) in healthy BALB / c mice 24 hours after tail vein injection of PBS, HAlipos(D), NM@HAlipos(L), and NM@HAlipos(D) (n=3). Data are presented as mean ± SD. DETAILED DESCRIPTION

[0075] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0076] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[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., China. Doxorubicin (DOX), 1-pyrenedodecanoic acid (PDA), and lecithin were purchased from MacLean Biochemical Co., Ltd., China. Dulbecco's modified Eagle's medium (DMEM), Roswell Park Memorial Institute 1640 medium (RPMI 1640), fetal bovine serum (FBS, certified grade), and trypsin-EDTA digestion solution (0.25%) were purchased from Gibco, USA. Penicillin-streptomycin mixture (100×) was purchased from Hyclone, USA. Tumor necrosis factor-α (TNF-α) 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 medium, Wright-Giemsa staining solution, and phosphate-buffered saline (PBS, 1×) were purchased from Solarbio, China. The BCA protein concentration assay kit, cell counting kit-8 (CCK-8), 4% paraformaldehyde, 3,3'-dioctyloxycarbonyl cyanine 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 all purchased from Beyotime, China. Rabbit anti-CXCR2, CD11b, and CD62L antibodies, horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies for Western blot analysis, and TUNEL and Ki67 detection kits were purchased from Servicebio, China. FITC-conjugated anti-Ly6G antibody and PE-conjugated anti-CD11b antibody were purchased from BioLegend, USA. Denaturing protein precast gel buffer, 4-20% high-resolution HEPES-Tris precast gels, and a protein molecular weight marker (245 kDa) were purchased from Yeasen, China. Hematoxylin and eosin (H&E) staining reagent and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) kit were purchased from Beyotime, China. Unless otherwise stated, all reagents were used according to their original specifications without further purification. Double-distilled water (ddH2O) was used in all solution preparations and experimental procedures.

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

[0079]

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

[0081] The animals and husbandry methods used 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 weighing 210 ± 10 g were purchased from Shandong Jinan Pengyue Experimental Animal Co., Ltd. All animals were housed in a standard environment (25 ± 1°C, with free access to food and water). All experimental procedures were approved by the Animal Ethics Committee of Ocean University of China (Ethics Number: OUC-SMP-2024-08-02).

[0082] The statistical analysis method used in the following examples is as follows:

[0083] All experimental data are presented as mean ± standard deviation (SD). Differences between groups were tested using one-way or two-way analysis of variance (ANOVA), with appropriate post hoc multiple comparisons for significance. Differences were considered statistically significant when p < 0.05. The levels of statistical significance are indicated as follows: p < 0.05: *, p < 0.01: **, p < 0.001: ***, p < 0.0001: ****.

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

[0085] 1. Isolation of Neutrophils

[0086] Adult BALB / c mice were sacrificed by cervical dislocation, and the femurs and tibias were removed, the soft tissues stripped, and the cells washed with pre-chilled PBS. RPMI 1640 was injected into the bone marrow cavity using a 1 mL syringe (27G needle) to flush the bone marrow cells. The cells were filtered through a 100 μm cell strainer and centrifuged at 500 g for 5 minutes to collect the cells. A 45% and 62% Percoll density gradient was used for layered centrifugation (500 g, 30 minutes). The middle layer of cells was collected and washed twice with PBS to remove residual Percoll. 3-5 mL of red blood cell lysis buffer was added, and the cells were lysed in an ice bath for 10 minutes. The cells were then washed with PBS and centrifuged at 500 g for 5 minutes. Cell purity and integrity were confirmed by Wright-Giemsa staining.

[0087] 2 Neutrophil purity detection

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

[0089] 3 Neutrophil LPS stimulation and activation

[0090] Neutrophils were stimulated with different concentrations of LPS (25, 50, 100, and 200 ng / mL) for 4 h, and a no-LPS control group was set up. After stimulation, the cells were collected by centrifugation (300 g, 5 min) and washed with PBS. 1×10 6 Cells were added with 1 μL of PE-labeled anti-CD11b antibody, incubated at 4°C in the dark for 30 min, and then washed twice with PBS before detection.

[0091] 4 Neutrophil membrane extraction

[0092] Purified neutrophils were suspended in 10 mL of hypotonic lysis buffer (2 mM MgCl2, 10 mM KCl, 20 mM Tris-HCl, pH 7.4) and incubated at 4°C for 2 h. The cells were sonicated for 6 min using an ultrasonic disruptor (JY92-IIN, Xinzhi, Ningbo, China) at 100 W in discontinuous mode (2 s on, 10 s off). The lysate was then subjected to differential centrifugation (4°C) to remove cell debris and organelles in a stepwise manner.

[0093] First, centrifuge at 1,500 rpm for 5 minutes to remove unbroken cells and large particles, and retain the supernatant; sonicate the precipitate again and collect the supernatant; combine the supernatants and centrifuge at 3,600 rpm for 15 minutes to remove cell nuclei and large particle organs; finally, centrifuge the supernatant at 11,000 rpm for 30 minutes to obtain the cell membrane precipitate, wash three times with PBS, and resuspend for storage.

[0094] 5 Preparation and Characterization of Neutrophil Membrane Vesicles (NMVs)

[0095] The neutrophil membrane suspension extracted above was placed in an ice bath and ultrasonicated (100W, 9 minutes, 2 seconds on, 4 seconds off) to prepare NMVs. 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 the NMV suspension was added dropwise to a copper grid, allowed to stand for 1 minute, then rinsed three times with distilled water, negatively stained with 1% uranyl acetate, blotted dry with filter paper, and imaged using a TEM (JEM-2100, JEOL, Japan). Membrane protein concentration was quantified using the BCA assay.

[0096] 6 Results

[0097] To obtain high-purity neutrophils, the present invention 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), consistent with the morphological characteristics of neutrophils [3] , indicating that the separation process was successful. Flow cytometry further confirmed that Ly6G + / CD11b + The proportion of double-positive cells was 83.1% ( Figure 1 b), indicating that the purity of the obtained neutrophils was high. After stimulation with different concentrations of LPS (50, 100, 200 ng / mL), the expression level of CD11b on the surface of neutrophils was upregulated in a dose-dependent manner, among which the expression level of the 200 ng / mL treatment group increased by about 4.9 times ( Figure 1 c), and thus this concentration was used in the subsequent activation step.

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

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

[0100] 1. Preparation of phospholipid / cholesterol stock solution

[0101] Dissolve DOPE, DPPC, and lecithin in methanol to prepare a 10 mg / mL stock solution (200 mg in 20 mL). Store in a sealed brown glass bottle. Dissolve DSPE-HA2000 (10 mg) in 1 mL of methanol. Dissolve cholesterol (200 mg) in a mixture of 15 mL of methanol and 5 mL of dichloromethane to a concentration of 10 mg / mL. Store all stock solutions at -20°C in the dark until needed.

[0102] 2 Liposome preparation and particle size analysis

[0103] Lipids and cholesterol were weighed according to the specified phospholipid (DOPE+DPPC) / cholesterol mass ratios (7:3, 7.5:2.5, 8:2, 8.5:1.5, and 9:1) and DOPE / DPPC mass ratios (4.5:4.5, 5:4, 6:3, 7:2, and 8:1), dissolved in 3 mL of dichloromethane, and rotary evaporated (45°C) to form a lipid film. 4 mL of PBS was added to the dried film and hydrated at 50°C, 150 rpm, for 2 h. The resulting suspension was sonicated in an ice bath (400 W, intermittent mode: 2s on, 2s off) for 12 min to obtain liposomes. The particle size and zeta potential of the resulting liposomes were measured using a Zetasizer Nano ZS particle size analyzer.

[0104] Note: When screening the mass ratio of phospholipids (DOPE+DPPC) / cholesterol, the mass ratio of DOPE to DPPC was fixed at 4.5:4.5; when screening the mass ratio of DOPE / DPPC, the mass ratio of phospholipids (DOPE+DPPC) / cholesterol was fixed at 9:1. 3 Liposome membrane fluidity test (based on 1-pyrene dodecanoic acid, PDA)

[0105] Liposome samples were diluted to a lipid concentration of 0.1 mg / mL, and PDA was added to a final concentration of 1 μM. The cells were incubated at 37°C in the dark for 30 minutes. Fluorescence emission spectra were recorded using a microplate reader (excitation wavelength 360 nm, emission wavelength range 370-550 nm). The monomer peak (approximately 398 nm) and the excimer peak (approximately 475 nm) were recorded separately. Membrane fluidity was assessed by the fluorescence intensity ratio of the excimer peak to the monomer peak (E / M). A higher E / M value indicates greater membrane fluidity.

[0106] 4 Results

[0107] Liposomes were prepared by thin film hydration method, and the phospholipid / cholesterol (PL / Chol) ratio and DOPE / DPPC mass ratio were regulated. DLS analysis results showed that as the cholesterol content decreased, the liposome particle size gradually decreased. When the PL / Chol ratio was 9:1, the liposome particle size was 106.6±3.9nm and the PDI was 0.260±0.012( Figure 1 d). In terms of optimizing the DOPE / DPPC ratio, increasing DOPE in appropriate amounts can improve liposome uniformity, but excessive amounts can lead to significant aggregation. When the DOPE / DPPC ratio is 7:2, unstable large particles with a particle size of 401±18.6nm and a PDI of 0.506±0.020 are formed ( Figure 1 e). PDA-based membrane fluidity assays further demonstrated that either lowering cholesterol or increasing DOPE content could enhance lipid membrane fluidity ( Figure 1 f). DOPE is a cone-shaped phospholipid with a low phase transition temperature (Tm), which facilitates membrane fusion and the formation of membrane curvature. DPPC, on the other hand, has a Tm of approximately 41°C and imparts strong membrane rigidity at physiological temperatures. Therefore, rationally regulating the DOPE content is crucial for achieving efficient fusion while maintaining liposome stability.

[0108] Example 3: Construction of NM@lipos and evaluation of fusion efficiency

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

[0110] Liposomes prepared from different formulations were mixed with neutrophil membrane vesicles (NMVs) at a 1:1 liposome / membrane protein mass ratio, transferred to a 5 mL centrifuge tube, and placed in an ice bath to prevent high-temperature damage to the membrane structure. Subsequently, ultrasonication (100 W, 9 minutes, 2 seconds on, 3 seconds off) was used to promote membrane fusion, resulting in NM@lipos hybrid particles.

[0111] To further optimize membrane fusion conditions, the DOPE / DPPC / cholesterol mass ratio was fixed at 6:3:1, while varying the liposome / membrane protein mass ratio (1:0.05, 1:0.1, 1:0.2, 1:0.5, 1:1, and 1:2) was set, while all other conditions remained the same. The resulting hybrid particles were measured for size and zeta potential using a Zetasizer Nano ZS, and the size distribution and fusion efficiency were further analyzed using nanoflow cytometry (NanoFCM, Xiamen Fuliu Biotechnology Co., Ltd.).

[0112] Subsequently, liposomes were mixed with NMVs at different liposome / membrane protein mass ratios to prepare NM@lipos. When the optimized ratio was 1:1, the obtained NM@lipos had a particle size of 153.1±2.6nm and a PDI of 0.233±0.031( Figure 1 g), showed good stability under different lipid composition conditions ( Figure 1 h, 1i). As the membrane protein ratio increases, the particle size gradually decreases and reaches a plateau at 1:1, indicating that the fusion process has reached saturation. Continuing to increase the membrane protein ratio has little effect on the particle size, but may change the surface membrane properties. NanoFCM analysis results are consistent with the above trend, indicating that the particle size distribution of NM@lipos is consistent under different formulations ( Figure 1 j) When the membrane protein ratio is low (1:0.05-1:0.5), fusion is incomplete and many unfused liposomes exist in the system.

[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 beneficial for its performance in subsequent in vitro and in vivo applications.

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

[0115] To evaluate the membrane fusion efficacy of NM@lipos, liposomes and NMVs were labeled with green fluorescence (DiO) and red fluorescence (DiD), respectively: 4 mL of NMV suspension (2.5 mg / mL, BCA assay) was added to 50 μL of 1 mg / mL DiD (dissolved in DMSO) and incubated on ice for 2 hours. Liposomes (2.5 mg / mL) were similarly incubated with DiO (50 μL, 1 mg / mL). After dye incubation, the sample was ultrafiltered (8000 rpm, 30 min) to remove free dye, then resuspended in PBS and ultrafiltration repeated four times.

[0116] Finally, the double-fluorescent-labeled liposomes and NMVs were fused according to the standard process to prepare NM@lipos, and the fusion efficiency was analyzed by NanoFCM combined with SPACEMAN (Single Particle Analysis of Cell Membrane) method. Figure 3 As shown in 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] Here are the results:

[0120] At the overall level, flow cytometry was used to set gates for analysis ( Figure 4 a–c) It was found that the hybridization rate increased with decreasing cholesterol content and reached a peak of 80.7% at a PL / Chol ratio of 9:1 ( Figure 4 d), which may be attributed to the enhancement of membrane fluidity. At the same time, the increase in hybridization rate is accompanied by a decrease in the proportion of free NMVs in the system ( Figure 4 g), indicating that more NMVs successfully fused into the hybrid vesicles.

[0121] Similarly, the DOPE content has a bell-shaped effect on the fusion efficiency: when the DOPE / DPPC ratio is 6:3, the hybridization rate reaches a maximum of 92.0%, but as the DOPE content continues to increase, the hybridization rate decreases, accompanied by the instability of the liposome structure and the intensification of the aggregation tendency ( Figure 4 As a cone-shaped 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 accordingly, reaching 97.7% ( Figure 4 j). However, compared with 1:1, although the hybridization rate increased slightly (from 92.0% to 97.7%), the proportion of free NMVs increased significantly (about 1.9 times the original) ( Figure 4 i) suggests that doubling the NMV input only increases fusion efficiency to a limited extent, resulting in protein waste. Therefore, considering fusion efficiency, membrane resource utilization, and process scalability, a 1:1 ratio was ultimately determined to be the optimal ratio.

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

[0124] Quadrant gating analysis was further used to fine-tune the distribution of hybridization. The low hybridization (0–25%) particles dropped to 21.1% at a PL / Chol ratio of 9:1, while the high hybridization (50–75%) particles increased to 26.7% ( Figure 4 m); when DOPE / DPPC is 6:3, the proportion of low hybrid particles is reduced to 13.1%, and the proportion of high hybrid particles is 39.8%, of which the proportion of medium and high hybrid particles (25–75%) is as high as 81.6% ( Figure 4 n). A similar trend was observed with the increase in the ratio of membrane proteins. Under the 1:1 condition, the percentage of low hybrid particles decreased to 5.9%, while the percentage of high hybrid particles increased to 39.9% ( Figure 4 o).

[0125] The results indicate that the fusion efficiency and uniformity of NM@lipos are optimized when the DOPE / DPPC / Chol ratio is 6:3:1 and the liposome / membrane protein mass ratio is 1:1. This study establishes a quantitative evaluation system for NM@lipos formulation optimization, providing key guidance for the rational design of next-generation biomimetic drug delivery systems.

[0126] Example 4. Preparation and characterization of optimal formulation liposomes and NM@lipos(L / D)

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

[0128] Liposomes (D) were prepared at a DOPE / DPPC / cholesterol mass ratio of 6:3:1, and lipos (L) were prepared at a lecithin / cholesterol mass ratio of 9:1. Both were prepared using the standard liposome preparation process (see Example 2 for the preparation of liposomes 2). Liposomes (D) or lipos (L) were mixed with NMVs at a liposome / membrane protein mass ratio of 1:1, placed in an ice bath, and sonicated using an ultrasonic disruptor (100 W, 9 min, 2 s on, 3 s off) to achieve membrane fusion, yielding NM@lipos (D) and NM@lipos (L) for subsequent characterization and analysis.

[0129] 2 Dynamic Light Scattering (DLS)

[0130] The hydrated particle size and zeta potential of liposomes and hybrid particles were measured using a Zetasizer Nano ZS instrument.

[0131] 3SDS-PAGE analysis

[0132] Membrane proteins were extracted from NMVs, NM@lipos(L), and NM@lipos(D). Lysis was performed on ice for 30 minutes using a final concentration of 2% SDS. Protein quantification was performed using the BCA assay. Electrophoresis was performed at 150V for 45 minutes using a Bio-Rad gel electrophoresis system using SDS-PAGE, followed by visualization with Coomassie Brilliant Blue staining to analyze protein composition.

[0133] 4Western blot detection

[0134] Neutrophils, NMVs, NM@lipos(L), and NM@lipos(D) (with the same number of control particles) were lysed in 2% SDS and incubated on ice for 30 minutes. The cells were centrifuged at 11,000 rpm for 10 minutes, and the supernatant was collected. Protein concentration was determined using the BCA assay and adjusted to 30 μg / 20 μL. SDS-PAGE was performed at 150 V for 45 minutes. After electrophoresis, the proteins were transferred to a PVDF membrane (300 mA, wet transfer for 90 minutes).

[0135] The membrane was blocked in blocking buffer for 1 h at room temperature and then incubated with primary antibodies (CXCR2, CD11b, CD62L, 1:1000 dilution) overnight at 4°C. After washing with TBST, HRP-conjugated goat anti-rabbit secondary antibody (1:10,000, incubated at room temperature for 1 h) was added and imaged using ECL developer (Tanon Gel Imaging System, China).

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

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

[0138] CLSM colocalization analysis: Double-fluorescence-labeled NM@lipos (L) and NM@lipos (D) were dropped onto glass slides, fixed, and stained for nuclei. The colocalization of red and green fluorescence was observed using a confocal microscope (Zeiss LSM 700, Zeiss, Germany) to verify fusion uniformity.

[0139] 6 Nano-flow cytometry and Spaceman analysis

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

[0141] 7 Results

[0142] Based on the optimized formula, lipo(D), lecithin-based NM@lipos(L) and NM@lipos(D) were successfully prepared and characterized. DLS test results showed that the particle sizes of the three were 132.6±4.3nm, 154.6±1.4nm and 134.3±6.1nm respectively. Figure 5 a), and the corresponding Zeta potentials were -39.8±0.9mV, -43.6±2.4mV, and -52.7±0.3mV ( Figure 5 b). All formulations had suitable particle size distribution and surface charge, with NM@lipos(D) showing a slight decrease in zeta potential after membrane fusion. SDS-PAGE analysis showed that the protein bands of NM@lipos(D) and NM@lipos(L) were basically consistent with those of NMVs ( Figure 5 c), confirming that membrane proteins were successfully integrated 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 structural feature similar to "invagination" ( Figure 5 e). In confocal microscopy, DiO-labeled liposomes and DiD-labeled NMVs showed obvious co-localization ( Figure 5 f), further confirming the successful fusion of the two.

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

[0144] In terms of the single particle hybridization distribution, the proportion of low hybridization particles (0%-25%) in NM@lipos(D) is only 12.9%, significantly lower than the 39.1% of NM@lipos(L); while the proportion of high hybridization particles (50%-75%) increases significantly to 41.3%, much higher than the 9.8% of the L group ( Figure 5 n). In the medium to high hybridization level range (25%-75%), the proportion of NM@lipos(D) particles is as high as 81.1%, which is significantly better than the 60.5% of NM@lipos(L). Figure 5These results further confirm the key role of DOPE in promoting the efficient fusion of liposomes and cell membranes, and provide 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 an inflammatory endothelial model

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

[0147] DiD-labeled liposomes (D) were prepared at a DOPE:DPPC:cholesterol mass ratio of 6:3:1. 450 μL of DOPE, 450 μL of DPPC, 100 μL of cholesterol, and 100 μL of DiD solution were dissolved in 3 mL of dichloromethane and rotary evaporated at 45°C to form a lipid film. The mixture was then hydrated with 4 mL of PBS 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 form DiD-labeled liposomes. To remove free DiD, the liposomes were dialyzed against PBS in a 3 kDa dialysis bag for 48 h, with regular buffer changes.

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

[0149] DiD-lipos (D) or DiD-lipos (L) were then mixed with NMVs at a 1:1 liposome / membrane protein ratio and sonicated on ice (100 W, 9 min, 2 s on, 3 s off) to produce NM@lipos (D) and NM@lipos (L). The final concentration was adjusted to 2.5 mg lipid / mL and 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 (2.5×10 4 cells / dish) for CLSM observation, or seeded in 6-well plates (5×10 5 Cells (cells / well) were used for flow cytometry analysis and cultured in DMEM + 10% FBS + 1% antibiotics for 24 hours. The cells were then divided into two groups: an experimental group was stimulated with 10 ng / mL TNF-α for 4 hours, while a control group remained untreated. Following stimulation, various nanoparticles (50 μg lipid / mL) were added and incubated for 4 hours.

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

[0153] Flow cytometry group: After incubation, the cells were washed with PBS, digested with trypsin and collected, and the difference in nanoparticle uptake under TNF-α treatment and untreated conditions was quantitatively analyzed using CytoFLEX flow cytometer.

[0154] 3. Transwell model to evaluate the ability of carriers to penetrate the endothelium

[0155] HUVE cells (1×10 4 Cells / well) were seeded on the upper layer of the Transwell chamber (0.4 μm pore size, 24-well plate, Corning) and cultured overnight to form a monolayer. The lower chamber was divided into two groups: the inflammation group was added with DMEM containing TNF-α (100 ng / mL) and fMLP (10 nM); the control group contained only DMEM. DiD-labeled lipos (D), NM@lipos (L), and NM@lipos (D) (50 μg lipid / mL) were added to the upper chamber and incubated for 6 h, 12 h, and 24 h, respectively. The culture medium in the lower chamber was collected at each time point, and the DiD fluorescence intensity was detected using a microplate reader (TECAN, Switzerland) to evaluate the particle penetration efficiency. The nanoparticle samples in the lower chamber were used for TEM imaging to observe the penetration of the membrane.

[0156] 4 Results

[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-α stimulation was constructed. Flow cytometry results showed that under non-inflammatory conditions, the uptake of NM@lipos(D) by HUVEC was 1.7 times and 1.3 times that of Lipos(D) and NM@lipos(L), respectively. Figure 6 b). After TNF-α stimulation, the uptake of the three vectors was significantly increased, and NM@lipos(D) still showed the highest uptake level. The CLSM results were also consistent. The uptake of all vectors was significantly enhanced under inflammatory conditions, among which NM@lipos(D) had the strongest fluorescence signal ( Figure 6 a). The results showed that TNF-α could effectively enhance the uptake of nanoparticles, and NM@lipos(D) had the most significant inflammation homing ability.

[0158] Furthermore, an in vitro vascular crossing model was established, in which endothelial cells were activated by TNF-α and chemokine fMLP ( Figure 6c), simulated vascular penetration process under inflammatory conditions. Under non-stimulating conditions, the penetration ability of NM@lipos(D) at 6h, 12h and 24h was significantly better than that of the other two groups. At 12h, the penetration ability of NM@lipos(D) was 4.4 times and 1.8 times that of Lipos(D) and NM@lipos(L), respectively. Figure 6 d). Under the stimulation of TNF-α and fMLP, the penetration ability of each group was further improved, but NM@lipos(D) still maintained the highest level, which was 3.8 times and 1.9 times that of Lipos(D) and NM@lipos(L), respectively ( Figure 6 e). TEM imaging of the lower chamber sample of Transwell further confirmed that the number of NM@lipos(D) penetrations was significantly greater than that of NM@lipos(L) ( Figure 6 f), indicating its enhanced transendothelial migration ability, which may be associated with higher membrane fusion efficiency.

[0159] Example 6. Distribution and Pharmacokinetic Analysis of Liposomes and NM@lipos in 4T1 Tumor Mouse Model 1 Establishment of 4T1 Tumor Model

[0160] Eighteen female BALB / c mice were randomly divided into two experimental groups (n=9 / group), and each group was further divided into three groups (n=3). DiD-labeled lipos(D), NM@lipos(L), or NM@lipos(D) were injected respectively (same as Example 5). The orthotopic mammary tumor model was constructed by injecting 1×10 6 4T1 cells (100 μL PBS) were added, and the tumor volume was measured every two days, and the calculation formula was V = 0.5 × L × W 2 (L is the maximum diameter, W is the minimum diameter). When the tumor volume reaches about 200mm 3 Biodistribution experiments were carried out.

[0161] 2 24h in vivo distribution experiment

[0162] Mice were injected via the tail vein with DiD-labeled lipos (D), NM@lipos (L), or NM@lipos (D) at a dose of 60 mg 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, and 24 hours. Mice were sacrificed 24 hours later, and heart, liver, spleen, lung, kidney, and tumor tissues were harvested for ex vivo fluorescence imaging. Fluorescence signals were quantified using Living Image software.

[0163] 3 6h in vivo distribution experiment

[0164] According to the above-mentioned dosing regimen, mice were killed 6 hours after injection, and major organs were collected for in vitro fluorescence imaging and fluorescence intensity quantification to evaluate the distribution characteristics of nanoparticles in the body at the early stage.

[0165] 4 Pharmacokinetic experiments

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

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

[0168] Fluorescence intensity was detected using a microplate reader (excitation wavelength Ex = 640 nm, emission wavelength Em = 685 nm). The concentration of nanoparticles in plasma was calculated based on the standard curve (Fig. S12). The blood drug concentration-time curve was plotted and the main pharmacokinetic parameters were further calculated, including: half-life (t 1 / 2 ); Area under the curve (AUC 0-48 h); clearance rate (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 under inflammatory conditions, a 4T1 tumor-bearing mouse model was constructed, and different DiD-labeled vectors were injected into the tail vein. In vivo fluorescence imaging was performed from 0.5 to 24 hours. The results showed that NM@lipos(D) showed significant tumor site enrichment at all time points, which was significantly better than Lipos(D) and NM@lipos(L) ( Figure 7 g). In vitro organ imaging and quantitative analysis further confirmed that the tumor accumulation of NM@lipos(D) was the highest at 6h and 24h after injection ( Figure 7 h). At 6 h, 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), and increased to 5.1 times and 1.87 times respectively at 24 h ( Figure 7These results indicate that NM@lipos(D) possesses excellent targeting ability in the inflammatory tumor microenvironment, further highlighting the advantages of the hybrid membrane strategy in enhancing targeted drug delivery.

[0172] To evaluate its in vivo pharmacokinetic properties, a pharmacokinetic study was conducted in a rat model. Blood samples were collected at multiple time points (5 minutes to 48 hours), the fluorescence intensity in plasma was measured, and the blood drug concentration-time curve was plotted. Compared with Lipos(D) and NM@lipos(L), NM@lipos(D) showed a significantly prolonged systemic circulation time ( Figure 7 k), and its half-life (t1 / 2) was 54.2±2.9h, which was significantly longer than that of Lipos(D) (24.4±5.8h) and NM@lipos(L) (38.1±3.2h). Figure 7 l). In addition, the systemic exposure of NM@lipos(D) was significantly increased, with AUC 0-48 h reached 84.1±5.2, which were 1.5 times and 2.4 times that of NM@lipos(L) and Lipos(D), respectively ( Figure 7 m). In terms of clearance rate (CL), NM@lipos(D) was significantly reduced to only 0.12±0.01, which was significantly superior to NM@lipos(L) (0.18±0.02) and Lipos(D) (0.29±0.1) ( Figure 7 These data indicate that NM@lipos(D) possesses excellent pharmacokinetic properties, including a longer circulation time, higher bioavailability, and slower clearance, likely 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, mass ratio 6:3:1) and 50 μL of DSPE-HA2000 (10 mg / mL) were dissolved in 3 mL of dichloromethane and rotary evaporated at 45°C to form a uniform lipid film. 1 mL of DOX solution (1 mg / mL) and 3 mL of PBS were then added to the vial and hydrated at 50°C, 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 prepare HAlipos (D,DOX). The product was dialyzed against PBS using a 3 kDa dialysis tubing for 48 h to remove free DOX, with the buffer replaced regularly.

[0175] Preparation of HAlipos (L,DOX): Dissolve 1 mL of lecithin solution (containing 10 mg of lecithin) and 50 μL of DSPE-HA2000 (concentration 10 mg / mL) in 3 mL of dichloromethane. After rotary evaporation to form a lipid film, rehydrate with 1 mL of DOX solution (concentration 1 mg / mL) and 3 mL of PBS and sonicate. Subsequent treatment methods are the same as above.

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

[0177] 2 Particle 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 measured using a Zetasizer Nano ZS. To evaluate storage stability, samples were stored at 4°C and 37°C for 7 days, with particle size and turbidity measured periodically to assess physical stability.

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

[0180] Encapsulation efficiency (EE%): Determined by ultrafiltration combined with fluorescence spectrophotometry. Take 200 μL of sample (2.5 mg lipid / mL) and place it into a 10 kDa ultrafiltration tube. Centrifuge at 8000 rpm for 10 min and determine the free DOX content in the filtrate (W free ). Take another sample of equal volume and add 2% Triton X-100 to fully lyse it, and measure the total DOX content (W total ). Use a microplate reader (Ex = 480nm, Em = 590nm) to read the fluorescence intensity, and the calculation formula is as follows:

[0181]

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

[0183]

[0184] 4 Results

[0185] In order to further improve the tumor enrichment efficiency, HA2000 targeting ligands were modified on the above three carriers to enhance their targeting to 4T1 cells. Given that 4T1 cells highly express hyaluronic acid (HA) receptors, it is expected that HA2000 can effectively enhance the targeting ability. Doxorubicin (DOX) was encapsulated into various carriers to obtain HAlipos (D,DOX), NM@HAlipos (L,DOX) and NM@HAlipos (D,DOX). DLS detection showed that the particle sizes of the three were 106.2±1.1nm, 107.5±5.3nm and 120.6±0.7nm, respectively, and the PDI were 0.24±0.001, 0.31±0.038 and 0.23±0.008 ( Figure 8 a), indicating that DOX encapsulation has little effect on particle size and dispersibility. The DOX encapsulation efficiencies of the three were 64.1%, 61.2% and 70.1% respectively ( Figure 8 d), with good drug loading capacity. Drug release experiments showed that DOPE-based NM@HAlipos (D,DOX) has pH-responsive properties and exhibits controlled release behavior under both physiological pH 7.4 and tumor slightly acidic pH 6.5 conditions ( Figure 8 Stability experiments showed that the particles showed no significant aggregation or size change when stored at 4°C and 37°C for 7 days ( Figure 8 gh), and has good storage and in vivo application stability.

[0186] Example 8: Evaluation of 4T1 cell uptake and cytotoxicity of different nanocarriers

[0187] 1 Cellular uptake analysis

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

[0189] In the samples used for flow cytometry, 4T1 cells (5×10 5 Cells were seeded in 6-well plates (100 cells / well) under identical culture conditions. After 24 hours of incubation, NM@lipos(D,DOX) and NM@HAlipos(D,DOX) (same as in Example 8) were added at a concentration of 50 μg lipid / mL. After 4 hours of incubation, the cells were washed with PBS, trypsinized, and harvested. Flow cytometry (CytoFLEX) was used to quantitatively analyze the cellular uptake of the two nanocarriers.

[0190] NM@lipos(D,DOX) was prepared as follows: 10 mg of lipids (DOPE:DPPC:cholesterol, mass ratio 6:3:1) were dissolved in 3 mL of dichloromethane and rotary evaporated at 45°C to form a uniform lipid film. Subsequently, 1 mL of DOX solution (1 mg / mL) and 3 mL of PBS were added to the vial and hydrated at 50°C, 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 produce Lipos(D,DOX). The product was dialyzed against PBS using a 3 kDa dialysis tubing for 48 h to remove free DOX, with the buffer replaced regularly.

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

[0192] 2. Cytotoxicity assay (CCK-8)

[0193] The CCK-8 method was used to evaluate the inhibitory effect of different nanocarriers on 4T1 cells. 4 Cells were seeded at a density of 100 cells / well in a 96-well plate and incubated at 37°C, 5% CO2 for 24 hours. Subsequently, different treatment groups were added: lipos(DOX), HAlipos(DOX), NM@lipos(L,DOX), NM@HAlipos(L,DOX), NM@lipos(D,DOX), and NM@HAlipos(D,DOX), at concentrations ranging from 0 to 200 μg lipid / mL. After 24 hours of incubation, the culture medium was removed, and 100 μL of fresh culture medium and 10 μL of CCK-8 working solution were added, and incubation continued for 2-4 hours.

[0194] The absorbance (OD) was read at a wavelength of 450 nm and the cell viability was calculated using the following formula:

[0195]

[0196] Among them, As is the absorbance of the experimental well, A b is the absorbance of the blank well, A c is the absorbance of the control group.

[0197] 3 Results

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

[0199] The results of cytotoxicity experiments showed that HAlipos (D, DOX), NM@HAlipos (L, DOX) and NM@HAlipos (D, DOX) all had significant killing effects on 4T1 cells, and the effects were better than those of the corresponding groups without modified HA ( Figure 9 q). These results indicate that HA2000 modification not only improves the cellular uptake efficiency but also enhances the anti-tumor therapeutic effect, confirming that NM@HAlipos(D,DOX) is a chemotherapy nanocarrier with good tumor targeting potential.

[0200] Example 9. Evaluation of the anti-tumor efficacy of HAlipos and its hybrid carriers in 4T1 tumor-bearing mice

[0201] 1 Animal experiment design and dosing regimen

[0202] Thirty healthy female BALB / c mice were 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. 1×10 6 4T1 cells (100 μL PBS) were added to establish a breast tumor model.

[0203] When the tumor volume reaches about 100 mm 3 Treatment was initiated afterward. Each group of mice was injected via the tail vein with different therapeutic preparations (100 μL), with the liposome group receiving 60 mg lipid / kg and the free DOX group receiving 5 mg / kg. The dosing schedule was 1, 3, 5, 7, 8, 10, 12, and 14 days, for a total of 8 times.

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

[0205] 2 Histopathological analysis

[0206] HE staining

[0207] The harvested major organ and tumor tissues were rinsed with physiological saline and fixed in 10% neutral formaldehyde for 24 hours. The sections were then routinely paraffin-embedded and sectioned at a thickness of 4 μm. After dewaxing and rehydration, the sections were stained with hematoxylin for 5 minutes, differentiated, washed, and stained with eosin for 15 seconds. The sections were then dehydrated with graded ethanol and transparentized with xylene before mounting. Tissue morphology and damage were observed using an optical microscope.

[0208] Ki67 immunofluorescence staining

[0209] After dewaxing and antigen retrieval, paraffin sections were blocked with 10% donkey serum for 30 minutes at room temperature. The sections were then incubated with the primary antibody Ki67 (1:200, Abcam, USA) at 4°C overnight. The sections were washed the following day and incubated with a fluorescently labeled secondary antibody (1:500, incubated at room temperature in the dark for 50 minutes). Cell nuclei were stained with DAPI for 10 minutes. Confocal microscopy was used to observe and count the percentage of Ki67-positive cells, as indicated by red fluorescence (Ex = 530 nm, Em = 590 nm), to reflect the level of tumor cell proliferation.

[0210] TUNEL fluorescence staining

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

[0212] 3 Results

[0213] To evaluate the antitumor effect of NM@HAlipos(D,DOX) in vivo, a 4T1 tumor-bearing BALB / c mouse model was established and treated with PBS, free DOX, HAlipos(D,DOX), NM@HAlipos(L,DOX) and NM@HAlipos(D,DOX) ( Figure 10a). A comprehensive evaluation of its antitumor activity and biosafety was conducted by monitoring tumor volume, physiological body weight changes, and histological analysis. The results showed that NM@HAlipos (D, DOX) exhibited the most significant tumor inhibition effect, with a tumor inhibition rate of 89.4%, significantly superior to the DOX group (41.3%), HAlipos (D, DOX) group (66.5%), and NM@HAlipos (L, DOX) group (81.0%) ( Figure 10 g). Tumor growth curve and tumor weight measurement results ( Figure 10 cf) further verified the above conclusion. It is worth noting that the body weight of mice in the NM@HAlipos (D,DOX) treatment group remained stable throughout the treatment period ( Figure 10 d), suggesting that it has low systemic toxicity and good in vivo biocompatibility.

[0214] To further explore its therapeutic mechanism, histological analysis of tumor tissue was performed. HE staining results showed that the tumor tissue structure of the NM@HAlipos (D, DOX)-treated group was severely damaged, with large areas of necrosis, which was in sharp contrast to the compact structure of the other groups ( Figure 10 i). Immunostaining of the proliferation marker Ki67 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 10 j, l). TUNEL apoptosis staining further showed that the NM@HAlipos(D,DOX) group induced the highest level of apoptosis, with the proportion of TUNEL-positive cells reaching 84.2%, which was much higher than that of the PBS (4.0%), DOX (9.8%), HAlipos(D,DOX) (16.8%) and NM@HAlipos(L,DOX) (59.6%) groups ( Figure 10 k, m). These results indicate that the significant antitumor effect of NM@HAlipos(D,DOX) is derived from its ability to simultaneously inhibit tumor cell proliferation and promote cell apoptosis.

[0215] Example 10: In vivo safety evaluation of HAlipos and its hybrid carriers

[0216] After the animal treatment in Example 9, major organs such as the heart, liver, spleen, lungs, and kidneys were harvested and weighed, and the organ index (organ weight / body weight × 100%) was calculated. The tissues were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) to observe for pathological changes in their tissue structure. Blood was collected via the eye sockets for measurement of serum biochemical indicators such as ALT, AST (liver function), UREA, CREA (renal function), and CK and LDH (myocardial function) to comprehensively assess the systemic toxicity of the vector.

[0217] HE staining results of major organs are as follows Figure 11 As shown. Figure 11 At the end of treatment, HE staining of the main organs (heart, liver, spleen, lung, and kidney) showed no significant pathological changes compared to the healthy control group. The tissue structure was intact, with no obvious inflammation, necrosis, or other pathological abnormalities.

[0218] The test results of serum biochemical indicators related to liver function (ALT, AST), renal function (UREA, CREA) and cardiac function (CK, LDH) are as follows Figure 12 As shown. Figure 12 It can be seen that serological tests of mice in each treatment group showed that liver function indicators (ALT, AST), renal function indicators (BUN, CRE), and cardiac function (CK, LDH) were all within the normal range, with no significant differences compared with the control group, suggesting that the treatment regimen used did not cause obvious liver and kidney toxicity.

[0219] Example 11: Effects of HAlipos and its hybrid vectors on the survival of 4T1 tumor-bearing mice

[0220] Female BALB / c mice aged 6-8 weeks were randomly divided into five groups (n=6) and inoculated with 1×10 6 4T1 cells (100 μL PBS) were added to the right mammary fat pad to establish an orthotopic tumor model. 3 After about 7-9 days, the drug treatment was started.

[0221] Mice in each group were injected via the tail vein with PBS, free DOX (5 mg / kg), HAlipos (D,DOX), NM@HAlipos (L,DOX), or NM@HAlipos (D,DOX). Dosing was performed on days 1, 3, 5, 7, 8, 10, 12, and 14, for a total of eight doses. The survival status of the mice was recorded daily for a 60-day observation period. Survival curves were plotted using the Kaplan-Meier method, and statistical analysis was performed using the Log-rank (Mantel-Cox) test. P < 0.05 was considered significant.

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

[0223] The results showed that during the 60-day observation period, the survival rate of mice in the NM@HAlipos(D,DOX) group was 67%, which was significantly better than that in the DOX group (0%), HAlipos(D,DOX) group (0%) and NM@HAlipos(L,DOX) group (33%) ( Figure 10 h).

[0224] In summary, NM@HAlipos(D,DOX) effectively inhibited tumor growth and induced tumor cell apoptosis, while significantly prolonging the survival of mice with low systemic toxicity, fully demonstrating the great potential of hybrid membrane liposome nanocarriers in precision tumor therapy.

[0225] Example 12: In vitro and in vivo biocompatibility assessment

[0226] 1 In vitro biocompatibility evaluation

[0227] HUVEC cells were seeded in 96-well plates at a density of 1×10 4 Cells were cultured overnight at 37°C, 5% CO2. Different concentrations of various liposomes or hybrid carriers (HAlipos(D), NM@HAlipos(L), and NM@HAlipos(D)) were then added. After 24 hours of incubation, cell viability was assessed using CCK-8 reagent. 100 μL of fresh culture medium and 10 μL of CCK-8 working solution were added to each well. Incubation continued for 2-4 hours. Absorbance (450 nm) was measured using a microplate reader to calculate cell viability.

[0228] 2 In vivo biosafety evaluation

[0229] Female BALB / c mice aged 6-8 weeks were randomly divided into four groups (n=3). HAlipos(D), NM@HAlipos(L), and NM@HAlipos(D) were injected into the tail vein at a dose of 60 mg lipid / kg every two days for a total of three times. A PBS group was set as a control. Blood was collected 24 hours after the last administration, and serum biochemical indicators were measured using an automatic biochemical analyzer (Chemray 800, Shenzhen Ruituo Biotechnology), including: liver function: alanine aminotransferase (ALT), aspartate aminotransferase (AST); renal function: urea (UREA), creatinine (CREA); cardiac function: creatine kinase (CK), lactate dehydrogenase (LDH). The above indicators were used to comprehensively evaluate the in vivo systemic toxicity of the nanocarrier.

[0230] 3 Results

[0231] The cell viability results of HUVE cells after treatment with HAlipos(D), NM@HAlipos(L) and NM@HAlipos(D) at different concentrations for 24 h are shown in Figure 2. Figure 13 As shown. Figure 13 It can be seen that the in vitro cytotoxicity was evaluated by the CCK-8 method, and the results showed that within the tested concentration range, each treatment group showed no obvious toxicity to [cell type], the cell viability was above 90%, and there was no significant difference compared with the control group.

[0232] The test results of serum biochemical indicators related to liver function (ALT, AST), renal function (UREA, CREA) and cardiac function (CK, LDH) are as follows Figure 14 As shown. Figure 14 It can be seen that the test results show that there is no significant difference between the indicators of each treatment group and the control group, suggesting that the various carriers did not cause obvious liver, kidney or heart toxicity at the tested dose.

[0233] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A biomimetic nano-drug carrier of neutrophil membrane-fused liposomes, comprising a liposome and a neutrophil membrane fused to the liposome; The raw materials of the liposome include DOPE, DPPC and cholesterol, and the mass ratio of the three is 6:3:1; The mass ratio of the liposome to the membrane protein in the neutrophil membrane is 1:1; the mass of the liposome is calculated based on the total mass of DOPE, DPPC and cholesterol.

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

3. The biomimetic nano drug carrier according to claim 1 or 2, characterized in that: The neutrophil membrane preparation method is as follows: unactivated neutrophils are separated from the bone marrow of BALB / c mice using a modified Percoll density gradient centrifugation method; neutrophil activation is induced using LPS; the activated neutrophils are subjected to hypotonic lysis, ultrasonic disruption using an ultrasonic disruptor, and differential centrifugation to obtain neutrophil membranes; The neutrophil membrane was ultrasonically treated in an ice bath using an ultrasonic disruptor to obtain neutrophil membrane vesicles; the conditions for the ultrasonic treatment in an ice bath using the ultrasonic disruptor were: a power of 100 W, a frequency of ultrasonication for 2 seconds and rest for 4 seconds, and a total ultrasonic treatment time of 9 minutes.

4. A method for preparing the biomimetic nano-drug carrier of neutrophil membrane fusion liposomes according to any one of claims 1 to 3, comprising the following steps: 1) extracting and isolating neutrophils from mouse bone marrow using a modified Percoll density gradient centrifugation method; inducing activation of the neutrophils using LPS; subjecting the activated neutrophils to hypotonic lysis, ultrasonic disruption, and differential centrifugation to obtain neutrophil membranes; and sonicating the neutrophil membranes using an ultrasonic disruptor in an ice bath to obtain neutrophil membrane vesicles; 2) preparing liposomes using DOPE, DPPC, and cholesterol as raw materials by a thin film hydration method; the mass ratio of DOPE, DPPC, and cholesterol is 6:3:1; 3) mixing the liposomes with neutrophil membrane vesicles at a liposome / membrane protein mass ratio of 1:1, placing the mixture in an ice bath, and then sonicating the mixture using the ultrasonic disruptor to obtain a biomimetic nano-drug carrier of neutrophil membrane fusion liposomes.

5. The preparation method according to claim 4, characterized in that: In step 2), the thin film hydration method for preparing liposomes is as follows: DOPE, DPPC, and cholesterol are dissolved in dichloromethane and rotary evaporated to form a lipid film; PBS solution is added to the lipid film and hydrated at 50° C. and 150 rpm for 2 hours; the resulting suspension is treated in an ice bath using an ultrasonic disruptor for 12 minutes to obtain the liposomes; And / or, in step 3), the ultrasonic treatment is performed using an ultrasonic disruptor under the following conditions: a power of 100 W, a frequency of ultrasonication for 2 seconds and rest for 4 seconds, and a total ultrasonic treatment time of 9 minutes.

6. A biomimetic nano-drug carrier of neutrophil membrane-fused drug-loaded liposomes, comprising a drug-loaded liposome and a neutrophil membrane fused to the liposome; The raw materials of the liposome include DOPE, DPPC, cholesterol, and DSPE-HA2000, and the mass ratio of the four is 6:3:1:0.5; The drug loaded in the drug-loaded liposome is doxorubicin; The mass ratio of the drug-loaded liposomes to the membrane proteins in the neutrophil membrane is 1:1; the mass of the drug-loaded liposomes 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.

7. The biomimetic nano drug carrier according to claim 6, characterized in that: The neutrophil membrane preparation method is as follows: unactivated neutrophils are separated from the bone marrow of BALB / c mice using a modified Percoll density gradient centrifugation method; neutrophil activation is induced using LPS; the activated neutrophils are subjected to hypotonic lysis, ultrasonic disruption using an ultrasonic disruptor, and differential centrifugation to obtain neutrophil membranes; The neutrophil membrane was ultrasonically treated in an ice bath using an ultrasonic disruptor to obtain neutrophil membrane vesicles; the conditions for the ultrasonic treatment in an ice bath using the ultrasonic disruptor were: a power of 100 W, a frequency of ultrasonication for 2 seconds and rest for 4 seconds, and a total ultrasonic treatment time of 9 minutes.

8. A method for preparing the biomimetic nano drug carrier of neutrophil membrane fusion drug-loaded liposomes according to claim 6 or 7, comprising the following steps: a) extracting and isolating neutrophils from mouse bone marrow using a modified Percoll density gradient centrifugation method; inducing activation of the neutrophils using LPS; subjecting the activated neutrophils to hypotonic lysis, ultrasonic disruption, and differential centrifugation to obtain neutrophil membranes; and sonicating the neutrophil membranes using an ultrasonic disruptor in an ice bath to obtain neutrophil membrane vesicles; b) using DOPE, DPPC, cholesterol, DSPE-HA2000 and doxorubicin as raw materials, preparing drug-loaded liposomes by thin film hydration method; wherein, The mass ratios of DOPE, DPPC, cholesterol, and DSPE-HA2000 are 6:3:1:0.5, respectively; c) mixing the drug-loaded liposomes with neutrophil membrane vesicles at a liposome / membrane protein mass ratio of 1:1, placing the mixture in an ice bath, and then sonicating the mixture using an ultrasonic disruptor to obtain a biomimetic nano-drug carrier of neutrophil membrane-fused drug-loaded liposomes.

9. The preparation method according to claim 8, characterized in that: In step b), the specific method for preparing the drug-loaded liposomes by the thin film hydration method is as follows: DOPE, DPPC, cholesterol, and DSPE-HA2000 are dissolved in dichloromethane and rotary evaporated to form a lipid film; doxorubicin solution and PBS solution are added to the lipid film, and hydrated at 50° C. and 150 rpm for 2 hours; the resulting suspension is treated in an ice bath using an ultrasonic disruptor for 12 minutes to obtain the drug-loaded liposomes; and the free doxorubicin is removed by dialysis in PBS using a 3 kDa dialysis bag for 48 hours. And / or, in step c), the ultrasonic treatment using an ultrasonic disruptor is performed under the following conditions: a power of 100 W, ultrasonication for 2 seconds, rest for 4 seconds, ultrasonication for 3 minutes, and a total duration of 9 minutes.

10. Use of the biomimetic nano drug carrier of the neutrophil membrane-coated drug-loaded liposome according to claim 6 or 7 in preparing drugs for targeted cancer treatment.

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