A neutrophil mimicking vesicle nano-preparation targeting colorectal cancer and a preparation method and application thereof
By modifying the surface of biomimetic vesicles of neutrophils with RGD cyclic peptides and encapsulating the photosensitizer dihydroporphyrin e6, a core-shell structured nano-formulation was formed, which solved the problems of poor targeting and high toxicity in the treatment of colorectal cancer, and achieved highly efficient photodynamic therapy within tumor cells with low toxicity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGSU UNIV
- Filing Date
- 2026-04-21
- Publication Date
- 2026-05-29
Smart Images
Figure CN122097579A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a neutrophil-inspired biomimetic vesicle nanoformation targeting colorectal cancer, its preparation method, and its application. Background Technology
[0002] Colorectal cancer is a common and highly malignant digestive tract tumor worldwide. Its aggressive nature and tendency to metastasize to distant sites pose major challenges to clinical treatment. Currently, conventional treatments for colorectal cancer include surgical resection, chemotherapy, molecular targeted therapy, and immune checkpoint inhibitors. While these methods have improved patient survival to some extent, their application still has significant limitations: chemotherapy drugs cause extensive damage to normal tissues while killing tumor cells; targeted therapy is prone to resistance due to tumor heterogeneity and the accumulation of gene mutations; and immunotherapy only responds well in some patients with microsatellite instability-high (MSI-H). Furthermore, even with comprehensive treatment, tumor recurrence remains a key challenge affecting prognosis.
[0003] Photodynamic therapy (PDT) utilizes photosensitizers to generate reactive oxygen species that kill tumors, but it faces challenges such as poor targeting and toxic side effects. Therefore, there is an urgent need for a nanoformulation that can effectively improve photosensitizer delivery. Summary of the Invention
[0004] The purpose of this invention is to provide a neutrophil-inspired biomimetic vesicle nanoformation targeting colorectal cancer, its preparation method, and its application. The nanoformation uses neutrophil-inspired biomimetic vesicles loaded with photosensitizers, which enhances targeting ability and biocompatibility, resulting in significant photodynamic therapy effects.
[0005] This invention provides a neutrophil-inspired biomimetic vesicle nanoformulation targeting colorectal cancer, wherein the neutrophil-inspired biomimetic vesicle nanoformulation comprises neutrophil-inspired biomimetic vesicles, a tumor-targeting peptide, and a photosensitizer; the tumor-targeting peptide is modified on the surface of the neutrophil-inspired biomimetic vesicles; and the photosensitizer is loaded within the neutrophil-inspired biomimetic vesicles. The photosensitizer includes dihydroporphyrin E6.
[0006] As a preferred embodiment, the photosensitizer is encapsulated in photosensitizing liposomes.
[0007] As a preferred embodiment, the mass ratio of the photosensitive liposome to the neutrophil biomimetic vesicle membrane protein is 1:1 to 1:2.
[0008] As a preferred embodiment, the mass ratio of the neutrophil biomimetic vesicles to the tumor-targeting peptide is 1:5~10.
[0009] As a preferred embodiment, the tumor-targeting peptide includes RGD cyclic peptide.
[0010] As a preferred embodiment, the RGD cyclic peptide is a thiolized RGD cyclic peptide; the surface of the neutrophil biomimetic vesicle is modified with maleimide groups.
[0011] As a preferred embodiment, the neutrophil biomimetic vesicle nanoformulation has a particle size of 100~200 nm, a polydispersity index of <0.15, and a zeta potential of -15~-30 mV.
[0012] The present invention also provides a method for preparing the neutrophil biomimetic vesicle nanoformation described above, comprising the following steps: modifying neutrophil biomimetic vesicles with tumor-targeting peptides to obtain tumor-targeting peptide-modified neutrophil biomimetic vesicles; The tumor-targeting peptide-modified neutrophil biomimetic vesicles were mixed with a photosensitizer and extruded to obtain the neutrophil biomimetic vesicle nanoformulation.
[0013] The present invention also provides the application of the neutrophil biomimetic vesicle nanoformulation described in the above scheme or the neutrophil biomimetic vesicle nanoformulation prepared by the above preparation method in the preparation of targeted drugs for colorectal cancer.
[0014] The present invention also provides a targeted drug for colorectal cancer, comprising the neutrophil biomimetic vesicle nanoformulation described in the above scheme or the neutrophil biomimetic vesicle nanoformulation prepared by the preparation method described above and a pharmaceutically acceptable carrier.
[0015] Beneficial Effects: This invention provides a neutrophil-inspired biomimetic vesicle nanoformulation targeting colorectal cancer. The neutrophil-inspired biomimetic vesicle nanoformulation comprises neutrophil-inspired biomimetic vesicles, a tumor-targeting peptide, and a photosensitizer. The tumor-targeting peptide is modified on the surface of the neutrophil-inspired biomimetic vesicles; the photosensitizer is loaded within the neutrophil-inspired biomimetic vesicles; the photosensitizer includes dihydroporphyrin E6 (Ce6). The nanoformulation of this invention has a stable core-shell structure. The outer shell is a neutrophil-inspired biomimetic vesicle modified with the tumor-targeting peptide, endowing the nanoformulation with long circulation, immune escape, inflammatory tropism, and active targeting capabilities. The inner shell is a photosensitizer capable of generating reactive oxygen species to kill tumor cells. The neutrophil-inspired biomimetic vesicle nanoformulation of this invention effectively protects the activity of the photosensitizer, preventing leakage and aggregation quenching, and specifically enriches the photosensitizer (Ce6) within tumor cells, achieving targeted drug delivery. Simultaneously, the initiation of photodynamic therapy is precisely controlled by an external laser, achieving "spatiotemporal controllability" of the treatment. This combination of "targeted delivery" and "spatiotemporal control" ensures efficient in-situ generation of singlet oxygen within tumor cells while minimizing damage to normal tissues and significantly reducing systemic toxicity. It solves the problems of poor targeting, high toxicity, short cycle time, and poor biocompatibility of existing photosensitizers and synthetic nanocarriers. Attached Figure Description
[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.
[0017] Figure 1 A schematic diagram illustrating the synthesis of RGD-NNV@CE6 nanoformulation; Figure 2 Characterization of the RGD-NNV@CE6 nanoformulation in Example 1; where a is the hydration particle size distribution of the RGD-NNV@CE6 nanoformulation; b is the zeta potential; c is the polydispersity index (PDI) plot; and d is the transmission electron microscopy result. Figure 3 The results show the evaluation of the photothermal / photodynamic properties of the RGD-NNV@CE6 nanoformulation in Example 1; in a, the left figure shows the ROS generation in MC38 cells detected by flow cytometry, and the right figure shows the average fluorescence intensity; b is the photothermal temperature rise graph. Figure 4 The results of the cell uptake experiment of RGD-NNV@CE6 in Example 3 are shown; where a is the flow cytometry detection of nanovesicle uptake; b is the fluorescence microscopy result. Figure 5 The results show the evaluation of RGD-NNV@CE6-induced immunogenic cell death (ICD) in tumor cells in Example 4; where a is the CRT / DAPI staining result; b is the HMGB1 / DAPI staining result; and c is the ATP release level. Figure 6 Example 5 shows the in vitro antitumor effect of RGD-NNV@CE6; where a is the apoptosis rate; b is the Calcein AM / PI live / dead cell fluorescence image. Figure 7 The in vivo antitumor effect of RGD-NNV@CE6 in Example 6 is shown; where a is the volume of the in situ tumor; b is the volume of the distal tumor; and c is the result of HE and TUNEL staining. Figure 8 The in vivo antitumor evaluation results of RGD-NNV@CE6 combined with Anti-PD-1 in Example 7 are shown; where a is tumor volume; b is mouse body weight; and c is HE and TUNEL staining results. In the figure, ns indicates that the data show no significant difference; express p <0.05; express p <0.001; express p <0.0001. Detailed Implementation
[0018] This invention provides a neutrophil-inspired biomimetic vesicle nanoformulation targeting colorectal cancer, wherein the neutrophil-inspired biomimetic vesicle nanoformulation comprises neutrophil-inspired biomimetic vesicles, a tumor-targeting peptide, and a photosensitizer; the tumor-targeting peptide is modified on the surface of the neutrophil-inspired biomimetic vesicles; and the photosensitizer is loaded within the neutrophil-inspired biomimetic vesicles. The photosensitizer includes dihydroporphyrin E6.
[0019] Unless otherwise specified, the raw materials described in this invention can all be obtained through conventional commercial methods in the field.
[0020] The neutrophil-inspired biomimetic vesicle nanoformulation of this invention comprises neutrophil-inspired biomimetic vesicles (NNV), tumor-targeting peptides, and photosensitizers. In one embodiment, the neutrophils are human neutrophils. The inherent inflammatory chemotaxis of the human neutrophil membrane allows it to actively migrate into the tumor microenvironment. Furthermore, the CD47 protein on the human neutrophil membrane can more effectively interact with signal regulatory protein α on the surface of human macrophages, transmitting a "don't eat me" signal, thereby significantly delaying clearance by the mononuclear phagocytic system and achieving an ultra-long circulating half-life, providing crucial time assurance for effective drug accumulation at the tumor site. Moreover, human cells minimize the immune rejection response that may be triggered by xenogeneic proteins, making their immune recognition and clearance behavior in the human body closer to that of autologous components, resulting in extremely high biosafety and paving the way for clinical translation. In one embodiment, the neutrophils are isolated from peripheral blood. In a specific embodiment of this invention, the neutrophils are high-purity primary neutrophils obtained from peripheral blood by density gradient centrifugation. The neutrophils isolated from peripheral blood in this invention belong to the traditional N1 type neutrophils and have a tumor-killing effect.
[0021] The tumor-targeting peptide of this invention is modified on the surface of a neutrophil biomimetic vesicle. As one embodiment, the tumor-targeting peptide includes an RGD cyclic peptide. The RGD cyclic peptide can specifically recognize and bind to tumor cells that highly express αvβ3 integrin, such as colorectal cancer cells, achieving precise "locking" at the cellular level. This invention does not specifically limit the source of the RGD cyclic peptide; it can be obtained using conventional commercially available methods in the art. In a specific embodiment of this invention, the RGD cyclic peptide includes c(RGDfK) (SEQ ID NO.1) and / or c(RGDyK) (SEQ ID NO.2) high-affinity cyclic peptides; the amino acid sequence of c(RGDfK) is Arg-Gly-Asp-D-Phe-Lys; and the amino acid sequence of c(RGDyK) is Arg-Gly-Asp-D-Tyr-Lys. As one embodiment, the mass ratio of the neutrophil biomimetic vesicle to the tumor-targeting peptide is 1:5~10. In a specific embodiment of the present invention, the mass ratio of the neutrophil biomimetic vesicles to the tumor-targeting peptide can be any ratio from 1:5 to 10, such as 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. The present invention modifies the surface of neutrophil biomimetic vesicles with RGD cyclic peptides, endowing the prepared nano-formulations with long circulation, immune escape, inflammatory chemotaxis, and active targeting capabilities. Specifically, the inherent inflammatory chemotaxis of the human neutrophil membrane can mediate the active migration of the nano-formulations into the tumor microenvironment. Simultaneously, the CD47 protein on the surface of the neutrophil membrane can evade recognition and clearance by the body's immune system, reducing phagocytosis and degradation by immune cells such as monocytes / macrophages. Furthermore, the RGD cyclic peptides modified on the surface of the biomimetic vesicles can specifically recognize and bind to αvβ3 integrin, which is highly expressed on the surface of colorectal cancer cells, achieving precise "locking" at the cellular level. This dual mechanism of "chemotactic navigation" and "molecular locking" creates a powerful synergistic effect, greatly increasing the enrichment concentration of nanoparticles at the tumor site and promoting the endocytosis efficiency of nanoparticles by tumor cells.
[0022] In one embodiment, the RGD cyclic peptide is a thiolized RGD cyclic peptide; the surface of the neutrophil biomimetic vesicle is modified with maleimide groups. In this invention, after the RGD cyclic peptide is thiolized (-SH), the thiol (-SH) groups on the RGD cyclic peptide can couple with the maleimide groups (Mal) on the surface of the neutrophil biomimetic vesicle through a click chemical reaction, achieving a stable covalent connection between the RGD cyclic peptide and the biomimetic vesicle, thus avoiding detachment caused by physical adsorption.
[0023] The RGD cyclic peptide-modified neutrophil biomimetic vesicles of this invention constitute the shell of the nano-formulation, used to encapsulate the photosensitizer and form a core-shell structure. This core-shell structure effectively protects the activity of the photosensitizer (Ce6), preventing leakage and aggregation quenching. Results show that the singlet oxygen yield generated by the nano-formulation under laser irradiation is significantly higher than that of free Ce6, exhibiting superior photodynamic therapy efficacy.
[0024] The photosensitizer of this invention includes dihydroporphyrin e6 (Ce6). In one embodiment, the photosensitizer is encapsulated in photosensitive liposomes. Dihydroporphyrin e6 (Ce6) is a highly efficient photosensitizer that, under irradiation with light of a specific wavelength, can generate a large amount of reactive oxygen species (ROS) through photodynamic reactions, thereby inducing tumor cell apoptosis and necrosis, and destroying tumor angiogenesis, achieving precise killing of tumor tissue. In one embodiment, the photosensitive liposomes include the photosensitizer, phospholipids, cholesterol, and distearylphosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-Mal). In one embodiment, the mass ratio of the photosensitizer, phospholipids, cholesterol, and DSPE-PEG-Mal is 1~2:1~2:1~2:1~2. In one embodiment, the phospholipids include at least one of dipalmitoylphosphatidylcholine, distearylphosphatidylcholine, and hydrogenated soybean lecithin. Phospholipids are the basic framework of lipid bilayers. This invention selects phospholipids with high phase transition temperatures, such as dipalmitoylphosphatidylcholine, distearate phosphatidylcholine, or hydrogenated soybean lecithin, to enhance the stability of liposomes in vivo. The cholesterol described in this invention can regulate the fluidity and mechanical strength of the phospholipid membrane, increasing stability and reducing leakage of contents. In the DSPE-PEG-Mal, DSPE is a hydrophobic phospholipid chain that can be anchored to the lipid membrane, while the PEG chain enhances steric stability and reduces protein adsorption. The terminal maleimide group provides an active site for subsequent covalent bonding with the shell.
[0025] In one embodiment, the mass ratio of the photosensitive liposomes to the neutrophil-inspired biomimetic vesicle membrane protein is 1:1 to 1:2. In this invention, the neutrophil-inspired biomimetic vesicle membrane protein is the natural membrane protein of neutrophil-inspired biomimetic vesicles. If the mass ratio of the photosensitive liposomes to the neutrophil-inspired biomimetic vesicle membrane protein is less than 1:1 (too few liposomes), the complex particle size increases, the structure becomes loose, and the photosensitizer delivery efficiency decreases; if the mass ratio is greater than 1:2 (too many liposomes), the membrane protein coating is insufficient, the liposomes are easily cleared by the mononuclear phagocyte system, the biomimetic targeting ability is weakened, and the precise enrichment and killing effect at the tumor site is affected.
[0026] In one embodiment, the neutrophil biomimetic vesicle nanoformulation has a particle size of 100-200 nm, a polydispersity index <0.15, and a zeta potential of -15 to -30 mV. In a specific embodiment of the present invention, the particle size of the neutrophil biomimetic vesicle nanoformulation can be any value within the range of 100-200 nm, such as 100, 110, 113.6, 116.8, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm. The zeta potential of the neutrophil-inspired biomimetic vesicle nanoformulation can be any value from -15 to -30 mV, such as -15, -16, -17, -18, -19, -19.3, -20, -20.4, -21, -21.5, -22, -23, -24, -25, -26, -27, -28, -29, or -30 mV. The neutrophil-inspired biomimetic vesicle nanoformulation of this invention has a stable core-shell structure. The outer shell is composed of RGD cyclic peptide-modified human neutrophil-inspired biomimetic vesicles, which endows the nanoformulation with long circulation, immune escape, inflammatory tropism, and active targeting capabilities. The outer shell material is derived from human cells, minimizing the immune rejection response that may be triggered by foreign proteins, making its immune recognition and clearance behavior in the human body closer to that of autologous components, resulting in extremely high biosafety and paving the way for clinical translation. The inner shell is a photosensitizing liposome carrying a photosensitizer (Ce6), serving as both the photosensitizer carrier and the singlet oxygen generation unit. The core-shell structure effectively protects the photosensitizer's activity, preventing leakage and aggregation-quenching. Furthermore, the neutrophil-inspired biomimetic vesicle nanoformulation of this invention specifically enriches the photosensitizer (Ce6) within tumor cells, achieving targeted drug delivery. Simultaneously, the initiation of photodynamic therapy is precisely controlled by an external laser, achieving "spatiotemporal controllability" in the treatment. This combination of "targeted delivery" and "spatiotemporal controllability" ensures efficient in-situ generation of singlet oxygen within tumor cells, while minimizing damage to normal tissues and significantly reducing systemic toxicity.
[0027] The present invention also provides a method for preparing the neutrophil biomimetic vesicle nanoformation described above, comprising the following steps: modifying neutrophil biomimetic vesicles with tumor-targeting peptides to obtain tumor-targeting peptide-modified neutrophil biomimetic vesicles; The tumor-targeting peptide-modified neutrophil biomimetic vesicles are mixed with a photosensitizer and extruded to obtain the neutrophil biomimetic vesicle nanoformulation. A schematic diagram of the preparation method described in this invention is shown below. Figure 1 As shown.
[0028] The source of neutrophils described in this invention has been discussed above and will not be repeated here. As one embodiment, the preparation of the neutrophil biomimetic vesicles includes: disrupting the neutrophils, then differentially centrifuging to obtain a cell membrane precipitate; resuspending and extruding the cell membrane precipitate to obtain the neutrophil biomimetic vesicles. As one embodiment, the disruption method includes at least one of hypotonic lysis, nitrogen cavitation, and repeated freeze-thaw cycles. As one embodiment, the differential centrifugation sequentially includes low-speed centrifugation and ultracentrifugation; the centrifugal force of the low-speed centrifugation is 500-1000 g; the low-speed centrifugation time is 10 min. In a specific embodiment of this invention, the centrifugal force of the low-speed centrifugation can be any value within the range of 500-1000 g, for example, 500, 600, 700, 800, 900, or 1000 g. In one embodiment, the centrifugal force of the ultracentrifugation is ≥100,000 g; the ultracentrifugation time is 30~60 min; in a specific embodiment of the present invention, the centrifugal force of the ultracentrifugation can be any value of ≥100,000 g, such as 100,000 or 150,000 g; the ultracentrifugation time can be any value of 30~60 min, such as 30, 35, 38, 40, 43, 45, 49, 50, 55, 58 or 60 min.
[0029] After obtaining the cell membrane precipitate, the present invention resuspends the cell membrane precipitate to obtain a resuspended solution; as one embodiment, resuspending is performed using PBS buffer. The present invention then extrudes the resuspended solution to obtain the neutrophil biomimetic vesicles. The present invention does not specifically limit the extrusion method; conventional extrusion methods in the art can be used. In one specific embodiment of the present invention, the extrusion is performed using a micro-extruder. As one embodiment, the extrusion includes: sequentially passing the vesicles through polycarbonate membranes with decreasing pore sizes, performing stepwise extrusion to obtain neutrophil biomimetic vesicles. As one embodiment, the pore sizes of the polycarbonate membranes are 1 μm, 400 nm, and 200 nm; as one embodiment, the number of extrusions is 10-15 times, for example, 10, 11, 12, 13, 14, or 15 times. The number of extrusions mentioned in the present invention refers to the number of extrusions per polycarbonate membrane with a pore size; for example, when passing through a polycarbonate membrane with a pore size of 1 μm, the number of extrusions is 10-15 times.
[0030] This invention utilizes tumor-targeting peptides to modify neutrophil biomimetic vesicles, obtaining tumor-targeting peptide-modified neutrophil biomimetic vesicles. The types and functions of the tumor-targeting peptides described in this invention have been discussed above and will not be repeated here. As one embodiment, the RGD cyclic peptide is a thiolized RGD cyclic peptide, denoted as RGD-SH. The thiolized RGD cyclic peptide of this invention involves introducing a free thiol group (-SH) into the RGD cyclic peptide. As one embodiment, the free thiol group can be derived from mercaptoethylamine, 2-iminothione, or N-succinimide-S-acetylthioacetate. In a specific embodiment of this invention, the preparation of the thiolized RGD cyclic peptide includes: mixing the RGD cyclic peptide with a crosslinking agent and reacting for 30-60 min; then reacting with mercaptoethylamine for 2-4 h to obtain the thiolized RGD cyclic peptide.
[0031] In this invention, RGD cyclic peptides are mixed with a cross-linking agent and reacted for 30-60 minutes to obtain an intermediate product. As one embodiment, the cross-linking agent is Sulfo-SMCC (sulfosuccinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate), purchased from Thermo Scientific™ 22122. Sulfo-SMCC is a heterobifunctional cross-linking agent, with one end of the molecule being an N-hydroxysuccinimide (NHS) ester and the other end being a maleimide group. As one embodiment, the amount of cross-linking agent added is 5-20 times, for example, 5, 6, 7, 8, 9, 10, 12, 14, 15, 17, 18, or 20 times the molar amount of the amino groups of the RGD cyclic peptide. In this invention, the RGD cyclic peptide is mixed with the cross-linking agent and reacted, where the NHS ester end of Sulfo-SMCC reacts with the primary amine of the lysine residue in the RGD cyclic peptide to form an amide bond.
[0032] After obtaining the intermediate product, the process further includes: removing excess cross-linking agent to obtain a purified intermediate product. As one embodiment, the removal of excess cross-linking agent includes using a desalting column method and / or dialysis. After obtaining the purified intermediate product, the present invention reacts the purified intermediate product with mercaptoethylamine for 2-4 h to obtain a mercapto-modified RGD cyclic peptide, denoted as thiolized RGD cyclic peptide (RGD-SH). As one embodiment, the amount of mercaptoethylamine added is 2-5 times the molar amount of maleimide groups in the intermediate product. As one embodiment, the reaction with mercaptoethylamine is carried out under an inert atmosphere. As one embodiment, obtaining the mercapto-modified RGD cyclic peptide further includes: purification; the purification method includes reversed-phase HPLC. The present invention thiolates the tumor-targeting peptide (RGD cyclic peptide) to obtain a tumor-targeting peptide (RGD-SH) with a mercapto group (-SH), which is beneficial for subsequent connection with neutrophil biomimetic vesicles.
[0033] In one embodiment, the surface of the neutrophil biomimetic vesicle is modified with maleimide groups. The maleimide (Mal) group used in this invention is selected from substances containing maleimide groups, such as DSPE-PEG-Mal. This invention uses DSPE-PEG-Mal to modify the surface of neutrophil biomimetic vesicles as an example to illustrate the preparation method of neutrophil biomimetic vesicles modified with maleimide groups, but this should not be construed as a limitation on the scope of protection. The preparation of the neutrophil biomimetic vesicle surface modified with maleimide groups according to this invention includes: co-incubating DSPE-PEG-Mal with neutrophil biomimetic vesicles to obtain incubated neutrophil biomimetic vesicles, denoted as PEG-Malized biomimetic vesicles. In one embodiment, the mass ratio of DSPE-PEG-Mal to neutrophil biomimetic vesicle membrane protein is 1:5 to 1:10, for example, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In one embodiment, the co-incubation time is 2 hours; the co-incubation temperature is 37°C. In this invention, DSPE-PEG2000-Mal is co-incubated with biomimetic vesicles, wherein the DSPE portion is anchored to the lipid membrane, the PEG chain enhances steric stability and reduces protein adsorption, and the terminal maleimide group provides an active site for subsequent covalent linkage with the thiol group in RGD-SH.
[0034] This invention utilizes RGD-SH to modify PEG-Malized biomimetic vesicles to obtain neutrophil biomimetic vesicles modified with tumor-targeting peptides. As one embodiment, the modification includes: mixing and incubating the RGD-SH and the PEG-Malized biomimetic vesicles to obtain neutrophil biomimetic vesicles modified with tumor-targeting peptides, i.e., RGD-modified biomimetic vesicles, denoted as RGD-NNV or RN. The mass ratio of the neutrophil biomimetic vesicles to the tumor-targeting peptides has been discussed above and will not be repeated here. As another embodiment, the molar ratio of the maleimide groups of the RGD-SH and the PEG-Malized biomimetic vesicles is 10:1. As one embodiment, the mixing and incubation is carried out under an inert atmosphere. As one embodiment, the inert atmosphere includes nitrogen. As one embodiment, the mixing and incubation time is 12 h. This invention involves mixing and incubating RGD-SH with PEG-Malized biomimetic vesicles. Through a highly efficient and specific click chemistry reaction between maleimide and thiol groups, the RGD cyclic peptide is covalently immobilized on the vesicle surface. This method results in strong attachment and controllable orientation. As one embodiment, after obtaining the RGD-modified biomimetic vesicles, the process further includes ultracentrifugation; the centrifugal force is 150,000 g; and the ultracentrifugation time is 60 min.
[0035] The photosensitizer described in this invention is encapsulated in photosensitive liposomes. As one embodiment, the preparation method of the photosensitive liposomes includes a thin-film hydration-extrusion method. This invention does not impose specific limitations on the operation of the thin-film hydration-extrusion method; conventional operations in the art can be used. In a specific embodiment of this invention, the thin-film hydration-extrusion method includes: dissolving the photosensitizer, phospholipids, cholesterol, and DSPE-PEG-Mal in an organic solvent to obtain a mixture; subjecting the mixture to vacuum rotary evaporation to form a uniform lipid film; drying the lipid film and then adding phosphate buffer for hydration to obtain multi-compartment liposomes; and sequentially passing the multi-compartment liposomes through polycarbonate membranes with decreasing pore sizes (400 nm, 200 nm, and 100 nm) to obtain photosensitive liposomes. The mass ratio of the photosensitizer, phospholipids, cholesterol, and DSPE-PEG-Mal in this invention has been discussed above and will not be repeated here. As one embodiment, the organic solvent includes a chloroform / methanol mixed solvent; the volume ratio of chloroform / methanol is 3:1 to 2:1. In one embodiment, phospholipids, cholesterol, and DSPE-PEG-Mal are used as lipids, and the mass-volume ratio of the lipids to the organic solvent can be any ratio from 1 mg:10 to 50 μL, such as 1 mg:10 μL, 1 mg:15 μL, 1 mg:20 μL, 1 mg:25 μL, 1 mg:30 μL, 1 mg:35 μL, 1 mg:40 μL, 1 mg:45 μL, or 1 mg:50 μL.
[0036] This invention involves mixing the RGD-modified biomimetic vesicles and the photosensitive liposomes to obtain a mixture. As one embodiment, the mass ratio of the RGD-modified biomimetic vesicle membrane protein to the photosensitive liposomes is 1:1 to 1:2. A suitable mass ratio ensures that the total drug content of the prepared nano-formulation reaches a sufficient level, thereby increasing the drug loading capacity.
[0037] After obtaining the mixture, the present invention extrudes the mixture to obtain a neutrophil biomimetic nanoparticle formulation, denoted as RNC. As one embodiment, the extrusion includes: extrusion using a 200 nm polycarbonate membrane; the number of extrusions is 10-15 times; in specific embodiments of the present invention, the number of extrusions can be any value within the range of 10-15 times, for example 10, 11, 12, 13, 14, or 15 times. A suitable number of extrusions ensures uniform particle size of the nanoparticle formulation; too few extrusions result in larger particle size; too many extrusions may lead to fragmentation of the nanoparticle formulation. The present invention repeatedly extrudes the mixture of RGD-modified biomimetic vesicles and photosensitive liposomes through a polycarbonate membrane with a specific pore size. Under shear force, the biomimetic vesicles are opened and re-encapsulated on the surface of the liposomes, forming a stable core-shell structure. The method described in this invention can effectively protect the activity of photosensitizers and prevent their leakage and aggregation quenching. Experiments have shown that the singlet oxygen yield generated by this nano-formulation under laser irradiation is much higher than that of free Ce6, demonstrating excellent photodynamic therapy efficacy.
[0038] This invention also provides the application of the neutrophil-inspired biomimetic vesicle nanoformulation described above, or the neutrophil-inspired biomimetic vesicle nanoformulation prepared by the aforementioned method, in the preparation of tumor-targeted drugs. The core-shell structure of the neutrophil-inspired biomimetic vesicle nanoformulation of this invention effectively protects the activity of the photosensitizer, preventing leakage and aggregation quenching. Furthermore, the nanoformulation can be targeted to the tumor site, specifically enriching the photosensitizer (Ce6) within tumor cells, achieving targeted drug delivery. This combination of "targeted delivery" and "spatiotemporally controllable" ensures efficient in-situ generation of singlet oxygen within tumor cells, while minimizing damage to normal tissues and significantly reducing systemic toxicity.
[0039] This invention also provides a tumor-targeting drug, comprising the neutrophil-inspired biomimetic vesicle nanoformulation described in the above-described scheme or the neutrophil-inspired biomimetic vesicle nanoformulation prepared by the aforementioned method, and a pharmaceutically acceptable carrier. As one embodiment, the pharmaceutically acceptable carrier includes, but is not limited to, physiological saline, glucose injection, buffer solution, etc. The drug is in the form of an injection and can be administered via intravenous injection, peritumoral injection, or intratumoral injection.
[0040] When applying the neutrophil-inspired biomimetic vesicle nanoformulation described in this invention, local laser irradiation of the tumor site is required at a specific time point after drug administration (usually 4-24 hours after administration, when the drug accumulation at the tumor site reaches its peak). The laser wavelength should match the maximum absorption peak of the photosensitizer; when the photosensitizer is Ce6, red light around 660 nm is preferred. The irradiation power density and treatment time need to be adaptively adjusted according to the size and location of the tumor to ensure sufficient singlet oxygen to kill tumor cells while minimizing thermal and photodamage to surrounding normal tissues.
[0041] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.
[0042] Example 1: Preparation and characterization of RGD-NNV@CE6 nanoformulation The materials and instruments involved in this embodiment are as follows: (1) Materials and reagents: Dihydroporphyrin e6 (Ce6), dipalmitoylphosphatidylcholine (DPPC), cholesterol, DSPE-PEG2000-Mal, c(RGDfK) cyclic peptide, Sulfo-SMCC, mercaptoethylamine, Tris-HCl, EDTA, and sucrose were all purchased from Xi'an Ruixi Biotechnology Co., Ltd.; human peripheral blood neutrophil separation medium (Solepro, P9040) and erythrocyte lysis buffer (Solepro, R1010) were all of analytical grade. Primary human neutrophils were provided by the Affiliated Hospital of Jiangsu University.
[0043] (2) Instruments and equipment: rotary evaporator (IKA RV 10 digital FLEX-0010005576), nitrogen blower (MIULAB-NDK200-2N), micro extruder and polycarbonate membrane (Avanti micro extruder-610000), dynamic light scattering instrument (Brookhaven-BI-200SM), transmission electron microscope (Hitachi-HT7800), high performance liquid chromatography system (Tongwei-EasySep-1020), ultracentrifuge (Eppendorf-CS150NX), fluorescence spectrophotometer (Titan-F9300).
[0044] The specific steps are as follows: (1) Preparation of Ce6-loaded liposomes (photosensitive liposomes) ① Weighing and dissolving: Accurately weigh DPPC (2 mg), cholesterol (2 mg), DSPE-PEG2000-Mal (2 mg), and Ce6 (2 mg), and dissolve them in 10 mL of chloroform / methanol mixed solvent to obtain a mixed solution. The volume ratio of chloroform to methanol is 3:1.
[0045] ② Film formation: The mixture from step ① is rotary evaporated at 45°C under reduced pressure until a uniform lipid film is formed, and then vacuum dried overnight.
[0046] ③ Hydration: Add 0.75 mL of preheated PBS (pH 7.4) to the homogeneous lipid film from step ②, and hydrate at 50°C for 1 h to obtain multi-compartment liposomes.
[0047] ④ Extrusion: Pass the multi-compartment liposomes from step ③ through 400 nm, 200 nm and 100 nm polycarbonate membranes in sequence, and extrude them 10 times each time to obtain single-compartment liposomes.
[0048] ⑤ Purification: The above single-compartment liposomes were subjected to Sephadex G-50 column chromatography to remove free Ce6. The blue fraction was collected, which is the Ce6-loaded liposome, and is referred to as photosensitive liposome.
[0049] (2) Preparation and RGD modification of human neutrophil biomimetic vesicles ① RGD cyclic peptide thiolation: A. Dissolve c(RGDfK) in PBS buffer (pH 7.2~7.5), add freshly prepared Sulfo-SMCC crosslinking agent, and react at 4℃ for 30 minutes to allow the ester end of NHS to react with the primary amine of lysine residues in c(RGDfK) to form an amide bond; wherein the amount of Sulfo-SMCC crosslinking agent added is 10 times in excess relative to the amino group of c(RGDfK). B. Immediately after the reaction, remove excess cross-linking agent using a desalting column or dialysis, and replace the buffer with a coupling buffer containing EDTA (pH 6.5–7.0), as described in Thermo Fisher Scientific's SMCC and Sulfo-SMCC User Guide (Pub. No. MAN0011295 C.0). C. Subsequently, add mercaptoethylamine to the purified intermediate c(RGDfK)-SMCC solution and react at 4°C for 2 h under an inert atmosphere to allow maleimide to specifically couple with the thiol group to form a stable thioether bond. The amount of mercaptoethylamine added is 2–5 times in excess of the maleimide group. D. Finally, purify the target product, the thiol-modified RGD cyclic peptide, by reversed-phase HPLC, denoted as RGD-SH.
[0050] ② Biomimetic vesicle preparation: Primary neutrophils were resuspended in pre-cooled hypotonic lysis buffer (purchased from Shanghai Shangbao Biotechnology Co., Ltd., T18245) and lysed in an ice bath for 30 min. Then, the cells were centrifuged sequentially at low speed (1,000g, 10 min) and ultracentrifuged at high speed (100,000g, 60 min) to collect the cell membrane precipitate. The cell membrane precipitate was resuspended in PBS to obtain a resuspended solution. The resuspended solution was extruded sequentially through 1 μm, 400 nm, and 200 nm polycarbonate membranes. Each polycarbonate membrane with a pore size was extruded 10 times to obtain vesicle suspensions.
[0051] ③RGD Modification: DSPE-PEG2000-Mal was mixed with biomimetic vesicles at a DSPE-PEG-Mal:membrane protein mass ratio of 1:5 to 1:10 and incubated for 2 h to obtain incubated neutrophil biomimetic vesicles, denoted as PEG-Malized biomimetic vesicles. The DSPE portion of DSPE-PEG2000-Mal is anchored to the lipid membrane, the PEG chain enhances steric stability and reduces protein adsorption, and the terminal maleimide group provides an active site for subsequent covalent bonding with the shell. RGD-SH was added to the PEG-Malized biomimetic vesicles, and the reaction was carried out under nitrogen protection for 12 h. The mixture was then purified by ultracentrifugation at 100,000 g for 60 min to obtain RGD-modified biomimetic vesicles, denoted as RN. The molar ratio of maleimide groups in RGD-SH to those in the PEG-Malized biomimetic vesicles was 10:1.
[0052] (3) Construction of core-shell structured nano-formulations The Ce6-loaded liposomes from step (1) and the RGD-modified vesicles from step (2) were mixed at a protein ratio of 1:1 to obtain a mixture. The mixture was extruded through a 200 nm polycarbonate membrane 15 times to obtain the RGD-NNV@CE6 nanoformulation, denoted as RNC.
[0053] The Ce6-loaded liposomes, RGD-modified vesicles, and RGD-NNV@CE6 nanoformulations prepared in this embodiment were physically characterized according to [Zhang J, et al. Engineered neutrophil-derived exosome-like vesicles for targeted cancer therapy. Sci Adv, 2022, DOI: 10.1126 / sciadv.abj8207.]. The results are as follows: Figure 2 As shown. According to Figure 2 It can be seen that the average hydrated particle size of the RGD-NNV@CE6 nanoformulation is 116.8±3.2 nm, PDI is 0.09, and Zeta potential is -20.4±1.1 mV. Figure 2(a~c) Transmission electron microscopy reveals a regular spherical core-shell structure. Figure 2 (d).
[0054] The encapsulation efficiency and drug loading of RGD-NNV@CE6 nanoformulation were determined by high performance liquid chromatography (HPLC), and the calculation formulas are as follows: Encapsulation efficiency (%) = ×100%; Drug loading (%) = ×100%; The results showed that the encapsulation efficiency of the RGD-NNV@CE6 nanoformulation was 86.5%±2.3%, and the drug loading was 4.1%±0.2%.
[0055] Example 2: Evaluation of the photothermal / photodynamic properties of RGD-NNV@CE6 (1) ROS detection Materials and reagents: Reactive oxygen species detection probe DCFH-DA (Beyotime, S0033S), phosphate buffer, flow cytometer.
[0056] The specific groups are as follows: RNC group: ① Dilute the RGD-NNV@CE6 nanoparticles prepared in Example 1 with PBS to a Ce6 concentration of 10 μg / mL; add to culture medium and incubate for 24 h; ② Add 10 μM ROS fluorescent probe (DCFH-DA) and mix well; ③ Use a 660 nm laser (power density: 0.2 W / cm²) to incubate. 2 Irradiate the sample for 5 min in step ②; ④ After 4 h, use flow cytometry to detect the fluorescence intensity of each group to quantify the ROS generation level.
[0057] RN group: The operation is the same as that of RNC group, except that step ① is to add the same mass of RGD modified vesicles prepared in Example 1 as the RGD-NNV@CE6 nanoformulation of RNC group; Ce6 group: The procedure is the same as the RNC group, except for step ①: Dilute the free Ce6 solution with PBS to a Ce6 concentration of 10 μg / mL.
[0058] PBS group: The procedure is the same as the RNC group, except that 10 μM ROS fluorescent probe is added to PBS.
[0059] The test results are shown in Table 1 and Figure 3 As shown in Figure a. The results show that after irradiation with a 660 nm laser, the fluorescence intensity of the RGD-NNV@CE6 group (RNC) was significantly higher than that of the free Ce6 group, indicating that the nano-formulation can generate ROS more efficiently and its photodynamic activity is significantly enhanced.
[0060] Table 1. Average fluorescence intensity of each treatment group
[0061] (2) Photothermal performance test The temperature changes of the PBS, RN, and RNC groups under near-infrared laser irradiation in step (1) were monitored using an infrared thermal imager, and the results are as follows: Figure 3 As shown in Figure b and Table 2, the results indicate that RGD-NNV@CE6 possesses a mild photothermal conversion capability, which can synergistically enhance the photodynamic therapy effect.
[0062] Table 2 Temperature changes in each treatment group
[0063] Example 3: Cellular uptake of RGD-NNV@CE6 Materials and cells used in this embodiment: MC38 mouse colon cancer cells, fluorescence microscope, flow cytometer.
[0064] (1) Cell preparation: MC38 cells in the logarithmic growth phase were digested with trypsin, counted, and seeded into confocal culture dishes (1×10⁻⁶ cells / year). 5 (pieces / dish) and 6-well plate (2×10) 5 Cells were cultured overnight at 37°C in a 5% CO2 incubator (cells / well) to allow them to adhere to the cell wall.
[0065] (2) The MC38 cells in the confocal culture dish and the 6-well plate were randomly divided into 4 groups, as follows: RNC Group: ① The RGD-NNV@CE6 nanoparticle formulation prepared in Example 1 was co-incubated with MC38 mouse colon cancer cells for 24 h; wherein, the amount of RGD-NNV@CE6 nanoparticle formulation added was 5 μg / mL based on Ce6 mass. ② After incubation, the drug-containing culture medium was discarded, and 1 mL of fresh complete culture medium was added to each well. A 660 nm laser (power density 0.2 W / cm²) was used. 2 Irradiate vertically for 5 minutes. After irradiation, return the cells to the incubator and continue incubation for 4 hours.
[0066] RN group: ① The RGD-modified vesicles prepared in Example 1 were co-incubated with MC38 mouse colon cancer cells for 24 h, wherein the amount of RGD-modified vesicles added was the same as the mass of vesicles used in the RGD-NNV@CE6 nano-formulation in the RNC group; ② After incubation, the drug-containing culture medium was aspirated, and 1 mL of fresh complete culture medium was added to each well, and incubation was continued for 4 h.
[0067] Ce6 group: ① Free Ce6 solution was co-incubated with MC38 mouse colon cancer cells for 24 h; the amount of free Ce6 solution added was 5 μg / mL based on the mass of Ce6; ② After incubation, the drug-containing culture medium was discarded, and 1 mL of fresh complete culture medium was added to each well, and incubation continued for 4 h.
[0068] PBS group: MC38 mouse colon cancer cells were co-incubated with PBS for 24 h, with the amount of PBS added being the same as that of the RGD-NNV@CE6 nano-formulation in the RNC group.
[0069] (3) Laser confocal microscopy observation: After incubation of each group, the culture medium was discarded and the samples were washed three times with PBS. 1 mL of serum-free culture medium was added to the confocal culture dish and observed under a laser confocal microscope. Images were randomly selected from 5 to 10 fields of view to be acquired, keeping the acquisition parameters consistent for each group, and the intensity of red fluorescence in each group was compared.
[0070] (4) Quantitative detection by flow cytometry: Cells from 6-well plates were digested with trypsin and collected into 1.5 mL EP tubes. The tubes were centrifuged at 350×g for 5 min at 4°C, and the supernatant was discarded. Cells were washed twice with PBS and resuspended in 500 μL PBS. The fluorescence intensity of the PE channel was detected using a CytoFLEX flow cytometer. At least 1×10⁻⁶ cells were collected from each tube. 4 Each cell was counted, and the average fluorescence intensity was recorded.
[0071] Test results as follows Figure 4 As shown, the results indicate that RGD modification significantly enhances the uptake of nano-formulations in MC38 cells, confirming its targeting and endocytic efficiency.
[0072] Example 4 Evaluation of RGD-NNV@CE6-induced immunogenic cell death (ICD) in tumor cells (1) Detection of calreticulin (CRT) membrane exposure Materials and reagents: MC38 mouse colon cancer cells, RGD-NNV@CE6 nanoformulation (RNC, prepared in Example 1), free Ce6, drug-free RGD-NNV (RN, prepared in Example 1), PBS, anti-CRT monoclonal antibody (Proteintech, 27298-1-AP), Alexa Fluor 594-labeled goat anti-mouse IgG (red, Thermo Fisher Scientific, R37121), DAPI staining solution (Beyotime, C1005), 4% paraformaldehyde (Beyotime, P0099-3L), and laser confocal microscope.
[0073] The experimental steps are as follows: ① MC38 cells were seeded in a confocal culture dish and cultured overnight until the cells adhered to the dish to obtain cultured cells; ② The cultured cells were randomly divided into 4 groups: RNC group: Based on Ce6 concentration, the RGD-NNV@CE6 nano-formulation prepared in Example 1 was added to the cells to make the Ce6 concentration 1 μg / mL, and incubated for 4h.
[0074] RN group: The RGD-modified vesicles prepared in Example 1 were added to the cells and incubated for 4 h. The amount of RGD-modified vesicles added was the same as that of the RGD-NNV@CE6 nanoformulation in the RNC group.
[0075] Ce6 group: Free Ce6 solution was added to cells and incubated for 4 h; the amount of free Ce6 solution added was 1 μg / mL based on the mass of Ce6.
[0076] PBS group: PBS was added to the cells and incubated for 4 h. The amount of PBS added was the same as that of the RGD-NNV@CE6 nanoformulation in the RNC group.
[0077] ③ Discard the supernatant, wash twice with PBS, add fresh culture medium, and then use a 660 nm laser (power density: 0.2 W / cm²). 2 Irradiate for 5 minutes.
[0078] ④ Continue culturing for 4 hours, then discard the culture medium and fix the cells with 4% paraformaldehyde at room temperature for 15 minutes.
[0079] ⑤ Wash 3 times with PBS, add 5% BSA and block at room temperature for 1 h.
[0080] ⑥ Add rabbit anti-CRT primary antibody (1:200 dilution) and incubate overnight at 4°C.
[0081] ⑦ Wash 3 times with PBS, label goat anti-mouse IgG with Alexa Fluor 594 (1:500 dilution), and incubate at room temperature in the dark for 1 h.
[0082] ⑧ Wash three times with PBS, then add DAPI staining solution to counterstain the cell nuclei for 5 minutes.
[0083] ⑨ Images were observed and acquired under a laser confocal microscope. CRT positive signals appeared as red fluorescence and were distributed on the cell membrane surface.
[0084] Test results as follows Figure 5 As shown in Figure a, laser confocal microscopy images show that the CRT red fluorescence signal on the surface of MC38 cells in the PBS group, free Ce6 group, and RN group is weak, while a clear red fluorescence ring is visible on the cell membrane surface of the RNC-treated group, indicating that CRT is significantly exposed, confirming that RNC can effectively induce CRT membrane translocation in tumor cells.
[0085] (2) Detection of release of high-mobility group box 1 (HMGB1) Materials and reagents: MC38 cells, anti-HMGB1 monoclonal antibody (Proteintech, Cat NO.82973-1-RR), Alexa Fluor 594-labeled goat anti-mouse IgG (red, Thermo Fisher Scientific, R37121), and other reagents as per CRT assay.
[0086] The experimental steps are as follows: ① Cell seeding, grouping, and laser irradiation conditions are the same as in CRT detection steps ①~③; ②After culturing for 4 hours, discard the culture medium and fix with 4% paraformaldehyde for 15 minutes.
[0087] ③ Wash 3 times with PBS, then permeabilize with 0.1% Triton X-100 for 10 min (HMGB1 is a nucleoprotein and requires permeabilization).
[0088] ④ Wash 3 times with PBS, then block with 5% BSA for 1 h.
[0089] ⑤ Add rabbit anti-HMGB1 primary antibody (1:200 dilution) and incubate overnight at 4°C.
[0090] ⑥ Add Alexa Fluor 594-labeled secondary antibody (1:500 dilution) and incubate at room temperature in the dark for 1 h.
[0091] ⑦ Counterstain cell nuclei with DAPI for 5 min, observe under a laser confocal microscope, and the results are as follows. Figure 5 As shown in Figure b, HMGB1 exhibits red fluorescence and co-localizes with blue DAPI when localized in the cell nucleus. After release, the red fluorescence in the nucleus weakens or disappears.
[0092] The results showed that bright red HMGB1 fluorescence was visible in the nuclei of MC38 cells in the PBS group, free Ce6 group, and RN group, and it co-localized well with DAPI. The red fluorescence in the nuclei of cells in the RNC treatment group was significantly reduced or disappeared, indicating that HMGB1 had been released from the nucleus to the extracellular space.
[0093] (3) ATP release level detection Materials and reagents: MC38 cells, ATP assay kit (Beyotime, S0026), white opaque 96-well plate, multi-functional microplate reader.
[0094] The experimental steps are as follows: ① Seed MC38 cells into 6-well plates and culture overnight.
[0095] ②The grouping and laser irradiation conditions are the same as those in CRT detection steps ②~③.
[0096] ③ After culturing for another 4 hours, collect the cell culture supernatant from each well.
[0097] ④ Prepare the ATP detection working solution according to the instructions of the ATP detection kit.
[0098] ⑤ Add 100 μL of ATP detection working solution to a white opaque 96-well plate and let it stand at room temperature in the dark for 5 minutes to consume background ATP.
[0099] ⑥ Add 50 μL of the collected cell supernatant to each well and mix quickly.
[0100] ⑦ Immediately use a multi-functional microplate reader to detect the chemiluminescence value (RLU).
[0101] ⑧ Calculate the ATP concentration in each sample based on the standard curve, and the results are as follows: Figure 5 As shown in c.
[0102] The results showed that the ATP release level in the supernatant of MC38 cells treated with RNC was significantly higher than that in the PBS group, the free Ce6 group, and the RN group, indicating that RNC can effectively induce ATP release. Example 5: In vitro antitumor effect of RGD-NNV@CE6 (1) Apoptosis detection MC38 cells were seeded in 6-well plates. Once the cell adhesion rate reached over 90%, the cells were randomly divided into 4 groups as follows: RNC group (also referred to as RGD-NNV@CE6+NIR group): ① The RGD-NNV@CE6 nano-formulation prepared in Example 1 was co-incubated with MC38 cells for 24 h; wherein, the amount of RGD-NNV@CE6 nano-formulation added was 1 μg / mL of culture medium based on Ce6 mass. ② After incubation for 24 h, the cells were irradiated with a 660 nm laser for 5 min (0.2 W / cm²). 2Continue culturing for 1 h. ③ Cell collection: Discard the culture medium and wash the cells once with pre-chilled PBS. Add 300 μL of EDTA-free trypsin (EDTA will affect Annexin V binding) to each well to digest the cells. After the cells become rounded, add 600 μL of complete culture medium to stop digestion. Collect the cell suspension into a 1.5 mL EP tube, centrifuge at 350×g for 5 min at 4℃, and discard the supernatant. ④ Washing: Resuspend the cells in 1 mL of pre-chilled PBS, centrifuge again at 350×g for 5 min at 4℃, and discard the supernatant. Repeat the washing once. ⑤ Staining: Add 100 μL of 1×Binding Buffer to each tube and gently resuspend the cells. Add 5 μL of Annexin V-FITC and 5 μL of PI staining solution, gently vortex to mix, and incubate at room temperature in the dark for 20 min. ⑥ Detection: After incubation, add 400 μL of 1×Binding Buffer to each tube and gently mix. Detect using flow cytometry within 1 h. Annexin V-FITC was detected using the FITC channel (excitation 488 nm, emission 525 nm), and PI was detected using the PE channel (excitation 488 nm, emission 585 nm). At least 1 × 10⁻⁶ samples were collected from each tube. 4 Cells were analyzed using FlowJo software to determine apoptosis rate (early apoptosis: Annexin V). + / PI - Late apoptosis: Annexin V + / PI - ).
[0103] RN group: The operation is the same as that of the RNC group, except that in step ①, the RGD modified vesicles prepared in Example 1 are co-incubated with MC38 cells for 24 h, wherein the amount of RGD modified vesicles added is the same as that of the RGD-NNV@CE6 nano-formulation in the RNC group; in step ②, no laser irradiation is performed.
[0104] Ce6 group: The operation is the same as that of RNC group, except that step ① is to co-incubate free Ce6 solution with MC38 cells for 24 h; wherein, the amount of free Ce6 solution added is 1 μg / mL of culture medium based on the mass of Ce6.
[0105] PBS group: The operation is the same as that of the RNC group, except that in step ①, MC38 cells are co-incubated with PBS for 24 h, and the amount of PBS added is the same as that of the RGD-NNV@CE6 nano-formulation in the RNC group; in step ②, no laser irradiation is performed.
[0106] Test results as follows Figure 6 As shown in Table a and Table 3, the results indicate that the apoptosis rate of the RGD-NNV@CE6+NIR group was significantly higher than that of the other groups.
[0107] Table 3 Apoptosis rate of each treatment group
[0108] (2) Live and dead staining experiment Materials and reagents: Calcein-AM (live cell staining), propidium iodide (PI, dead cell staining), fluorescence microscope.
[0109] The experimental steps are as follows: ① MC38 cells were seeded and grouped as in step (1) for apoptosis detection; ② After incubation for 24 hours, the culture medium was discarded, and 100 μL of PBS solution containing Calcein-AM (2 μM) and PI (4 μM) was added. The cells were incubated at 37°C in the dark for 20 minutes. ③ After washing with PBS, 200 μL of PBS was retained, and the cells were immediately observed and photographed under a fluorescence microscope. The results are as follows. Figure 6 As shown in b.
[0110] Calcein-AM excitation resulted in green fluorescence (in live cells), while PI excitation resulted in red fluorescence (in dead cells). The results showed that the RGD-NNV@CE6+NIR group exhibited abundant red fluorescence (in dead cells) and significantly reduced green fluorescence (in live cells), clearly demonstrating that the formulation has a strong cytotoxic effect, strong targeting, and low toxicity.
[0111] Example 6: In vivo antitumor evaluation of RGD-NNV@CE6 (1) Establishment of animal models Materials and Methods: C57BL / 6 mice (6-8 weeks old) were subcutaneously inoculated with MC38 mouse colon cancer cells (1×10⁻⁶) on the left posterior back on day 0. 6 MC38 mouse colon cancer cells (1×10⁶ cells / mouse) were subcutaneously injected into the right posterior back on day 5 to establish an orthotopic tumor model. 6 (Each tumor sample / animal) was used to establish a distant tumor model. The in situ tumor volume was allowed to grow to approximately 100 mm. 3 At that time, the mice were randomly divided into the following four groups (n=5 per group): RGD-NNV@CE6+NIR group: RGD-NNV@CE6 nanoparticle formulation (calculated based on Ce6 dose of 5 mg / kg) was injected via tail vein, followed by local tumor irradiation with 660 nm laser (power density 0.2 W / cm²) 24 h later. 2 Irradiate for 10 minutes, once every 3 days; Ce6 photosensitizer + NIR group: Each mouse was injected with a 5 mg / kg photosensitizer via tail vein, and 24 h later, the tumor site was irradiated with a 660 nm laser (power density 0.2 W / cm²). 2 Irradiate for 10 minutes, once every 3 days; RGD-NNV group: RGD-NNV@CE6 equivalent mass of RGD modified vesicles were injected via tail vein once every 3 days; Control group: The same volume of PBS as the RGD-NNV@CE6+NIR group was injected via tail vein once every 3 days.
[0112] (2) Treatment and monitoring: Administer the medication twice a week for a total of 2 weeks. Measure tumor volume every 3 days (Formula: V = length × width) 2 ×0.5), the result is as follows Figure 7 As shown in Figures a through b.
[0113] Mice were euthanized after treatment, and tumor tissue was collected for further analysis.
[0114] (3) Tumor tissue analysis: H&E staining was used to observe the morphology and necrosis of tumor cells, and the method is described in the manual. TUNEL staining was used to detect tumor cell apoptosis, and the method is described in the manual of the staining reagent.
[0115] Test results as follows Figure 7 As shown in Figure c, RGD-NNV@CE6-mediated photodynamic therapy not only effectively inhibits local tumor growth but also induces systemic anti-tumor immune memory, thereby achieving inhibitory effects on distant metastases. H&E staining and TUNEL staining of ex vivo tumor tissue further confirmed its ability to induce tumor cell apoptosis.
[0116] Example 7: In vivo antitumor evaluation of RGD-NNV@CE6 combined with Anti-PD-1 (1) Establishment of animal models Materials and Methods: C57BL / 6 mice (6-8 weeks old) were subcutaneously inoculated with MC38 colon cancer cells (1×10⁻⁶). 6 (Each mouse model was established using individual tumors). The tumor volume was approximately 100 mm². 3 At that time, the mice were randomly divided into the following four groups: Control group: PBS treatment PD-1 monoclonal antibody group: PBS of the same volume as the nano-formulation in the combination therapy group was injected via tail vein; at the same time, PD-1 antibody, 100 μg / animal, was injected intraperitoneally every 3 days.
[0117] RGD-NNV@CE6+NIR group: The nano-formulation (containing Ce6 5 mg / kg) was administered via tail vein injection, and simultaneously, an equivalent volume of PBS containing PD-1 antibody was injected intraperitoneally as in the combination therapy group; 24 h later, the tumor was irradiated with a 660 nm laser (power density: 0.2 W / cm²). 2 (Time: 10 minutes), once every 3 days.
[0118] Combined treatment group: The animal received a tail vein injection of a nano-formulation containing Ce6 5 mg / kg, and simultaneously, an intraperitoneal injection of PD-1 antibody, 100 μg / animal. Twenty-four hours later, the tumor was irradiated with a 660 nm laser (power density: 0.2 W / cm²). 2 (Time: 10 minutes), once every 3 days.
[0119] (2) Treatment and monitoring: Administer the medication twice a week for a total of 2 weeks. Measure tumor volume every 3 days (Formula: V = length × width). 2 (×0.5) and mouse body weight, results as follows Figure 8 As shown in a~b. After treatment, the mice were sacrificed, and tumor tissue was collected for further analysis.
[0120] (3) Tumor tissue analysis: The procedure is the same as step (3) in Example 6, and the detection results are as follows. Figure 8 As shown in c.
[0121] The results showed that tumor growth was significantly inhibited in the RGD-NNV@CE6+ Anti-PD-1 group, and the mice maintained stable body weight with no obvious systemic toxicity. H&E staining and TUNEL staining of ex vivo tumor tissue further confirmed its ability to induce tumor cell apoptosis.
[0122] In summary, the neutrophil biomimetic vesicle nanoformulation of the present invention has excellent biocompatibility, strong targeting, and significant photodynamic therapy effect, solving the problems of poor targeting, large toxic side effects, short cycle time, and poor biocompatibility of existing photosensitizers and synthetic nanocarriers.
[0123] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A neutrophil-inspired biomimetic vesicle nanoformation targeting colorectal cancer, characterized in that, The neutrophil biomimetic vesicle nanoformulation comprises neutrophil biomimetic vesicles, tumor-targeting peptides, and photosensitizers; the tumor-targeting peptides are modified on the surface of the neutrophil biomimetic vesicles; and the photosensitizers are loaded within the neutrophil biomimetic vesicles. The photosensitizer includes dihydroporphyrin E6.
2. The neutrophil biomimetic vesicle nanoformulation according to claim 1, characterized in that, The photosensitizer is encapsulated in photosensitizing liposomes.
3. The neutrophil biomimetic vesicle nanoformulation according to claim 2, characterized in that, The mass ratio of the photosensitive liposome to the neutrophil biomimetic vesicle membrane protein is 1:1 to 1:
2.
4. The neutrophil biomimetic vesicle nanoformulation according to claim 1, characterized in that, The mass ratio of the neutrophil biomimetic vesicles to the tumor-targeting peptides is 1:5~10.
5. The neutrophil biomimetic vesicle nanoformulation according to claim 1, characterized in that, The tumor-targeting peptides include RGD cyclic peptides.
6. The neutrophil biomimetic vesicle nanoformulation according to claim 5, characterized in that, The RGD cyclic peptide is a thiolized RGD cyclic peptide; the surface of the neutrophil biomimetic vesicle is modified with maleimide groups.
7. The neutrophil biomimetic vesicle nanoformulation according to claim 1, characterized in that, The neutrophil biomimetic vesicle nanoparticle formulation has a particle size of 100~200 nm, a polydispersity index of <0.15, and a zeta potential of -15~-30 mV.
8. The method for preparing the neutrophil biomimetic vesicle nanoformation according to any one of claims 1 to 7, characterized in that, The process includes the following steps: modifying neutrophil biomimetic vesicles with tumor-targeting peptides to obtain tumor-targeting peptide-modified neutrophil biomimetic vesicles; The tumor-targeting peptide-modified neutrophil biomimetic vesicles were mixed with a photosensitizer and extruded to obtain the neutrophil biomimetic vesicle nanoformulation.
9. The use of the neutrophil biomimetic vesicle nanoformulation according to any one of claims 1 to 7 or the neutrophil biomimetic vesicle nanoformulation prepared by the preparation method according to claim 8 in the preparation of targeted drugs for colorectal cancer.
10. A targeted drug for colorectal cancer, characterized in that, The formulation includes the neutrophil biomimetic vesicle nanoformulation according to any one of claims 1 to 7 or the neutrophil biomimetic vesicle nanoformulation prepared by the preparation method according to claim 8 and a pharmaceutically acceptable carrier.