Borneolum syntheticum modified erythrocyte membrane coated dendrimer bionic nano drug delivery system as well as preparation and application thereof

Abstract

The invention discloses a borneol modified erythrocyte membrane coated dendritic macromolecule bionic nano drug delivery system and preparation and application thereof, the bionic nano drug delivery system comprises an erythrocyte membrane and a drug loaded dendritic macromolecule coated in the erythrocyte membrane, the surface of the erythrocyte membrane is modified with targeted molecule borneol, the drug is an anti-tumor drug, such as bufotalin. In combination with a nasal drop administration mode, not only can the brain targeting efficiency of the glioma treatment drug be improved, but also the glioma treatment drug has immune escape and tumor microenvironment response capabilities, the in-vivo circulation time of the drug is effectively prolonged, and the glioma treatment effect is further improved. The design that the dendritic macromolecules are coated with the modified erythrocyte membranes combines the biocompatibility of natural cell membranes and the multifunctionality of nanoparticles, and the dendritic macromolecules have huge application potential in the fields of drug delivery, immune regulation, detoxification, targeted therapy and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a borneol-modified erythrocyte membrane-coated dendritic macromolecule biomimetic nano drug delivery system and its preparation and application. Background Art

[0002] Gliomas are the most common primary malignant tumors of the central nervous system. They are highly invasive, prone to recurrence, and have a poor prognosis. Their incidence is increasing year by year worldwide, making them a major health threat. In clinical treatment, surgical resection, radiotherapy, and chemotherapy are the mainstays. However, due to the strong infiltration of tumor cells and unclear boundaries, complete tumor resection is often difficult to achieve with surgical treatment alone, and the tumor responds poorly to radiotherapy and chemotherapy. Furthermore, single-agent chemotherapy shows limited efficacy and often induces drug resistance, leading to tumor recurrence and poor prognosis.

[0003] Erythrocytes (RBCs) are the most numerous cell population in the blood system and the cell type with the longest circulation cycle (living for approximately 100-120 days). They possess unique biological properties: their membrane structure gives them suspension stability, osmotic regulation, and deformability, enabling them to adapt to complex circulatory environments while fulfilling physiological functions such as oxygen transport and immune regulation. More importantly, RBCs transmit "self-recognition" signals to immune cells via CD47 molecules on their membrane surfaces, effectively circumventing phagocytic clearance by the reticuloendothelial system. This innate immune escape property gives RBC membranes excellent biocompatibility and low immunogenicity, and has been widely used in the surface modification of nanocarriers to enhance their long-circulation properties. However, in practical applications, RBC membranes suffer from limited internal space, making it difficult to efficiently load large molecule drugs or hydrophobic drugs. Furthermore, natural RBC membranes lack active targeting capabilities and require chemical modification (such as antibody or ligand conjugation) for targeted delivery.

[0004] The blood-brain barrier (BBB) is a crucial structure that protects the brain from harmful substances. However, its highly selective nature also limits the entry of most drugs into brain tissue, particularly small and large molecules. This property results in poor therapeutic efficacy for brain diseases such as gliomas, making it difficult to achieve effective drug concentrations, which in turn affects chemotherapy efficacy and may lead to cancer recurrence. To further enhance the ability of nanoparticles to cross the BBB, the surface of red blood cell membranes was modified with borneol. Borneol, a natural medicinal substance with low toxicity and good biocompatibility, is widely used as a "drug guide" due to its aromatic properties, according to Traditional Chinese Medicine (TCM) theory and clinical practice. Studies have shown that it can inhibit P-glycoprotein (P-gp) efflux, increase serotonin (5-HT) and nitric oxide (NO) levels, and widen the intercellular space.

[0005] In recent years, dendrimers have shown great potential in tumor treatment. Their unique structural and functional properties make them ideal drug delivery carriers and multifunctional therapeutic platforms. Dendrimers are a class of three-dimensional macromolecules with a highly branched structure. Their unique tree-like morphology and precise molecular structure make them show a wide range of application potential in nanotechnology, drug delivery, biomedicine and other fields. However, the cationic charge carried on the surface of dendrimers can cause problems such as cell membrane damage, hemolysis or inflammatory response. Although there are currently methods such as polyethylene glycol (PEG) modification, coupling targeting ligands (such as folic acid, RGD peptide) or nanocarrier encapsulation to functionalize dendrimers, such modification methods may reduce drug loading efficiency, and the binding efficiency of targeting ligands is affected by the tumor microenvironment.

[0006] In summary, the current drug delivery system still needs to be further improved. Summary of the Invention

[0007] The purpose of the present invention is to address the deficiencies of the prior art and to provide a borneol-modified erythrocyte membrane-coated dendrimer biomimetic nano-drug delivery system and its preparation and application.

[0008] In a first aspect, the present invention provides a method for preparing a borneol-modified erythrocyte membrane-coated dendrimer biomimetic nano-drug delivery system, comprising the following steps:

[0009] Step (1), DSPE-PEG 2000 -COOH (distearylphosphatidylethanolamine-polyethylene glycol 2000-carboxyl crosslinked product) reacts with borneol under a weak alkaline environment at room temperature and is freeze-dried to obtain DSPE-PEG 2000 -BO;

[0010] Step (2), add DSPE-PEG to the red blood cell membrane solution 2000 -BO, incubated in the dark, targeted modified erythrocyte membrane obtained by lipid intercalation;

[0011] Step (3), dissolving the drug and PAMAM (polyamidoamine) in a solvent, stirring, and freeze-drying to obtain a drug-loaded dendrimer (drug@PAMAM);

[0012] Step (4), mixing the drug-loaded dendrimer and the targeted modified erythrocyte membrane and co-extruded to obtain the borneol-modified erythrocyte membrane-coated dendrimer biomimetic nano drug delivery system.

[0013] Preferably, in step (1), DSPE-PEG 2000 The mass ratio of -COOH to borneol is 12.5-14:1.

[0014] Preferably, the stirring time in step (1) is 24 hours.

[0015] Preferably, in step (2), DSPE-PEG 2000 The mass ratio of -BO to red blood cell membrane is 1-1.5:8.

[0016] Preferably, the mass ratio of the drug to PAMAM in step (3) is 0.75-1.25:1, more preferably 1:1.

[0017] Preferably, in step (4), the mass ratio of the drug-loaded dendrimer to the targeted modified erythrocyte membrane is 1:1.5-2.

[0018] Preferably, the drug is an anti-tumor drug; more preferably, the drug is Gamabufotalin.

[0019] In the second aspect, the present invention provides a borneol-modified erythrocyte membrane-coated dendritic macromolecule biomimetic nano-drug delivery system, which is prepared by the above method and includes an erythrocyte membrane and a drug-loaded dendritic macromolecule wrapped in the erythrocyte membrane. The surface of the erythrocyte membrane is modified with a targeting molecule borneol, the drug is tofentazocine, and the dendritic macromolecule is PAMAM.

[0020] In a third aspect, the present invention provides the use of the borneol-modified erythrocyte membrane-coated dendrimer biomimetic nano-drug delivery system in the preparation of tumor-targeted drugs.

[0021] Preferably, the tumor is a glioma.

[0022] Preferably, the tumor-targeted drug is administered by nasal drops.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention modifies erythrocyte membranes with borneol through a lipid intercalation method. Leveraging borneol's "aromatically invigorating" properties, the blood-brain barrier is reversibly opened, increasing the efficiency of nanoparticle entry into the brain and thus improving the efficacy of glioma treatment. Furthermore, the invention introduces the dendritic macromolecule PAMAM, and through co-extrusion, creates a nano-drug delivery system with a dendritic macromolecule core and a borneol-modified erythrocyte membrane outer layer. This nano-drug delivery system can be loaded with bufotolin and used in glioma treatments, achieving immune evasion. The system exhibits excellent brain targeting, tumor microenvironment responsiveness, long-lasting circulation, and good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 These are transmission electron micrographs of different preparation groups, with a scale of 100 nm.

[0026] Figure 2 (A) Drug loading and (B) encapsulation efficiency of CS-6 in PAMAM-CS-6 at different CS-6 concentrations.

[0027] Figure 3 The drug release of (A) PAMAM-CS-6 group and (B) BO-RBCm@PAMAM-CS-6 group at different pH values.

[0028] Figure 4 Figure 2 shows the uptake of BO-RBCm@PAMAM-FITC by U87 and RAW264.7 cells.

[0029] Figure 5 Figure 3 shows the fluorescence distribution in various tissues and organs of nude mice after 24 hours of BO-RBCm@PAMAM-Cy5.5 administration via nasal drops and tail vein, respectively.

[0030] Figure 6 The fluorescence distribution in various tissues and organs of nude mice 96 hours after nasal administration of RBCm@PAMAM-Cy5.5 with or without borneol targeting modification.

[0031] Figure 7 (A) In vivo bioluminescence images, (B) relative bioluminescence intensity, and (C) weight changes of nude mice bearing brain glioma after intranasal administration of normal saline, CS-6, and BO-RBCm@PAMAM-CS-6. DETAILED DESCRIPTION

[0032] In order to make the technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] As described above, the present invention provides a borneol-modified erythrocyte membrane-coated dendrimer biomimetic nano-drug delivery system, comprising an erythrocyte membrane and a drug-loaded dendrimer encapsulated in the erythrocyte membrane, wherein the surface of the erythrocyte membrane is modified with a targeting molecule borneol, the drug is bufotolin, and the dendrimer is polyamidoamine (PAMAM). The borneol-modified erythrocyte membrane-coated dendrimer biomimetic nano-drug delivery system is prepared by the following method:

[0034] Step (1), DSPE-PEG 2000 -COOH and borneol reacted at a mass ratio of 12.5-14:1 under weak alkaline conditions with stirring at room temperature, and DSPE-PEG was obtained after freeze-drying. 2000 -BO;

[0035] Step (2), add DSPE-PEG to the red blood cell membrane solution 2000 -BO, incubated in the dark, targeted modified erythrocyte membrane was obtained by lipid intercalation; DSPE-PEG 2000 -The mass ratio of BO to erythrocyte membrane is 1-1.5:8;

[0036] Step (3), dissolving the drug and PAMAM in a solvent at a mass ratio of 0.75-1.25:1, stirring and freeze-drying to obtain a drug-loaded dendrimer;

[0037] Step (4), mixing the drug-loaded dendrimer and the targeted modified erythrocyte membrane in a mass ratio of 1:1.5-2 and co-extruded to obtain the borneol-modified erythrocyte membrane-coated dendrimer biomimetic nano drug delivery system.

[0038] In the preparation method provided by the present invention, the ratio of each raw material can be selected according to actual needs. For example, in some embodiments, DSPE-PEG 2000 The mass ratio of -COOH to borneol can be selected from 12.5:1, 13:1, 13.5:1, 14:1, or other values within the range, which is not limited here; 2000 The mass ratio of -BO to erythrocyte membrane can be specifically 1:8, 1.1:8, 1.2:8, 1.3:8, 1.4:8, 1.5:8, or other values within the range, which is not limited herein. In some embodiments, the mass ratio of the drug to PAMAM can be specifically selected from 0.75:1, 1:1, or 1.25:1, which can be arbitrarily selected within the range according to actual needs, which is not limited herein. The mass ratio of the drug-loaded dendrimer to the targeted modified erythrocyte membrane can be specifically selected from 1:1.5, 1:1.75, or 1:2, or other values within the range, which can be selected according to actual needs, which is not limited herein.

[0039] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0040] Example 1

[0041] A method for preparing a modified erythrocyte membrane-coated dendrimer nano-drug delivery system loaded with bufatoxin comprises the following steps:

[0042] (1) Preparation of DSPE-PEG by EDCI / DMAP reaction 2000 -BO, 15 mg DSPE-PEG 2000-COOH was dissolved in dichloromethane and activated by adding 1.5 mg of EDCI. 1.2 mg of borneol solution was slowly added dropwise, and then 0.2 mg of DMAP was added. The mixture was stirred with a magnetic stirrer under a weak alkaline and room temperature environment for 24 h. The product was dialyzed in 20% ethanol for 24 h and then immersed in ultrapure water for 24 h to remove unreacted substances. DSPE-PEG was obtained after freeze-drying. 2000 -BO;

[0043] (2) Fresh whole blood was obtained from mice by orbital blood sampling, and red blood cells were separated by centrifugation (3000 rpm, 20 min, 4°C), and washed three times with phosphate buffered saline. The washed red blood cells were gently mixed with an excess of 1 / 4× PBS, and the red blood cells were placed under low osmotic pressure and lysed on ice for 2 h to ensure that the red blood cells were fully ruptured. The resulting mixture was then centrifuged (13000 rpm, 15 min, 4°C) to collect red blood cell membranes (RBCm), and the protein concentration was determined by the BCA method to determine the membrane content, and the cells were stored at -80°C.

[0044] (3) Take 2 mg / mL red blood cell membrane solution, ultrasonicate it in an ice bath for 10 min, and take an appropriate amount of 250 μg / mL DSPE-PEG 2000 -BO was added to the red blood cell membrane, vortexed and mixed, incubated at 4°C in the dark for 30 minutes, and centrifuged at 13000 rpm for 5 minutes to remove free DSPE-PEG 2000 -BO, targeted modified erythrocyte membrane obtained by lipid intercalation;

[0045] (4) 1 mg / mL CS-6 (bufotolin) was added to 1 mg of PAMAM methanol solution and gently stirred at room temperature for 24 h. The mixture was then centrifuged at 4000 rpm for 10 min using a 3500 Da ultrafiltration centrifuge tube. This was repeated three times to remove free drug. The drug-loaded dendrimer PAMAM-CS-6 was obtained after freeze-drying.

[0046] (5) The obtained PAMAM-CS-6 was mixed with the modified red blood cell membrane in a mass ratio of 1:1.5, gently stirred for 10 min in an ice bath, and sequentially extruded through a polycarbonate porous membrane (pore size of 800 nm, 400 nm, and 200 nm) using a liposome extruder to form a uniform suspension to obtain biomimetic nanoparticles BO-RBCm@PAMAM-CS-6. The nanoparticles were uniformly dispersed in PBS and stored at 4°C.

[0047] 2 mg of samples from different groups (RBCm, PAMAM, PAMAM-CS-6, BO-RBCm@PAMAM-CS-6) were dissolved in 1 mL of aqueous solution and dropped onto a copper grid. After the samples dried naturally, they were negatively stained with phosphotungstic acid negative staining solution. The morphology of the nanoparticles was observed under a transmission electron microscope for morphological characterization.

[0048] Figure 1 Transmission electron microscopy images of different groups show that the particle size of red blood cell membrane vesicles is about 200 nm, PAMAM is uniformly distributed, and the drug-loaded PAMAM shows good monodispersity. The particle size of the BO-RBCm@PAMAM-CS-6 group coated with red blood cell membrane and modified with borneol is about 100 nm, and there is an obvious membrane structure on the surface.

[0049] Test Example 1: Analysis of drug loading and encapsulation efficiency of bufotolin

[0050] BO-RBCm@PAMAM-CS-6 was prepared according to the method in Example 1, except that the mass ratios of CS-6 and PAMAM were adjusted to 0.5:1, 0.75:1, 1:1, and 1.25:1, respectively.

[0051] The centrifugal supernatant was collected and the concentration of CS-6 in the supernatant with different mass ratios was determined by HPLC. The drug loading capacity and encapsulation efficiency of PAMAM for CS-6 were calculated. The results are shown in Figure 2 .from Figure 2 It can be seen that when the mass ratio of CS-6 to PAMAM is above 1:1, the drug loading rate and encapsulation efficiency remain basically unchanged and reach a saturated state.

[0052] Test Example 2: Investigation of the drug release performance of the material

[0053] In order to study whether the wrapping of modified red blood cell membranes affects the release of drugs, 1 mL of BO-RBCm@PAMAM-CS-6 nanoparticles was dissolved in 1 mL of PBS at pH = 7.4, pH = 6.5 and pH = 5.1, then transferred to a dialysis bag (3000Da) and placed in 30 mL of PBS at different pH values, and finally placed at 37 ° C, 180 rpm, and shaken. 1 mL of PBS was taken out at certain time points (15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h, 72h) and supplemented with the same volume of PBS of the corresponding pH value. The value of the maximum absorption wavelength of CS-6 at 296nm was measured by high performance liquid chromatography, and its cumulative release amount was calculated. The results are shown in Figure 3 The above results indicate that BO-RBCm@PAMAM-CS-6 has good acid-responsive release ability.

[0054] Test Example 3: Material Immune Escape Performance Investigation

[0055] In this example, fluorescein isothiocyanate (FITC) was used to label the nanoparticles.

[0056] U87 (human brain astroglioblastoma cells) and RAW264.7 (mouse mononuclear macrophage leukemia cells) cells were collected by digestion and plated at 3×10 5 Cells were seeded into each well of a 6-well plate and cultured for 24 hours. An equal amount of FITC@RBCm@PAMAM solution was then added to the plate and incubated in a 37°C cell culture incubator for 1, 2, 3, and 4 hours, respectively. The solution was then removed and the cells were washed three times with PBS. The cells were digested, centrifuged at 1000 rpm for 5 minutes, the supernatant discarded, and resuspended in PBS. The cell suspension density was adjusted and analyzed by flow cytometry to detect the fluorescence intensity of FITC in U87 and RAW264.7 cells. The results are shown in the figure. Figure 4 , it can be seen that FITC@RBCm@PAMAM is taken up more by macrophages, which proves that the nanoparticles can reduce the phagocytic ability of macrophages RAW264.7 after modification with red blood cell membrane.

[0057] Test Example 4: Investigation of the route of administration of the material

[0058] BO-RBCm@PAMAM nanoparticles were labeled with fluorescent dye and administered into nude mice via tail vein and nasal drops. After 24 hours, the mice were killed and their hearts, livers, spleens, lungs, kidneys, and brains were removed. In vitro fluorescence imaging was performed using a small animal in vivo fluorescence imaging system. Figure 5 According to the statistical graph, the proportion of fluorescence in the nude mouse brain to total tissue fluorescence after intranasal administration is much higher than that after tail vein injection. The results show that intranasal administration has better brain targeting ability.

[0059] Test Example 5: In vivo brain targeting performance evaluation of materials after nasal administration

[0060] Borneol-targeted and non-borneol-targeted RBCm@PAMAM nanoparticles were labeled with fluorescent dyes and administered to nude mice via nasal drops. After 96 hours, the mice were killed and their hearts, livers, spleens, lungs, kidneys, and brains were removed. In vitro fluorescence imaging was performed using a small animal in vivo fluorescence imaging system. Figure 6 The fluorescence intensity in the brain of the borneol-targeted modification group was significantly enhanced, showing a good brain-targeting effect.

[0061] Test Example 6: Investigation of the therapeutic effect of the material on nude mice with glioma

[0062] Luciferase-labeled U87 cells were injected into the striatum of nude mice using a stereotaxic device to establish an orthotopic glioma nude mouse model. Seven days after modeling, normal saline, CS-6, and BO-RBCm@PAMAM-CS-6 were administered intranasally, once every two days for a total of six doses. During this period, the size of the glioma was monitored using a small animal in vivo fluorescence imaging system. The results are shown in Figure 2. Figure 7 Compared with the saline and free CS-6 groups, the fluorescence of glioma cells in nude mice treated with BO-RBCm@PAMAM-CS-6 was significantly reduced, indicating its anti-glioma effect. During the treatment period, there was no significant change in body weight, confirming its good biosafety.

[0063] The embodiment described above is only a preferred solution of the present invention and does not limit the present invention in any form. Other variations and modifications are possible without exceeding the technical solution described in the claims.

Claims

1. A borneol-modified erythrocyte membrane-coated dendrimer biomimetic nano-drug delivery system, characterized in that: The invention comprises an erythrocyte membrane and a dendritic macromolecule loaded with drugs wrapped in the erythrocyte membrane. The surface of the erythrocyte membrane is modified with a targeting molecule borneol.

2. The borneol-modified erythrocyte membrane-coated dendrimer biomimetic nano-drug delivery system according to claim 1, characterized in that: The drug is bufalin, and the dendrimer macromolecule is polyamidoamine PAMAM.

3. A method for preparing the borneol-modified erythrocyte membrane-coated dendrimer biomimetic nano-drug delivery system according to any one of claims 1-2, characterized in that: The method comprises the following steps: Step (1), DSPE-PEG 2000 -COOH reacts with borneol under weak alkaline conditions and is stirred at room temperature to obtain DSPE-PEG after freeze-drying. 2000 -BO; Step (2), add DSPE-PEG to the red blood cell membrane solution 2000 -BO, incubated in the dark, targeted modified erythrocyte membrane obtained by lipid intercalation; Step (3), dissolving the drug and PAMAM in a solvent, stirring and freeze-drying to obtain a drug-loaded dendrimer; Step (4), mixing the drug-loaded dendrimer and the targeted modified erythrocyte membrane and co-extruded to obtain the borneol-modified erythrocyte membrane-coated dendrimer biomimetic nano drug delivery system.

4. The method according to claim 3, characterized in that DSPE-PEG in step (1) 2000 The mass ratio of -COOH to borneol is 12.5-14:

1.

5. The method according to claim 3, characterized in that DSPE-PEG in step (2) 2000 The mass ratio of -BO to red blood cell membrane is 1-1.5:

8.

6. The method according to claim 3, characterized in that The mass ratio of the drug to PAMAM in step (3) is 0.75-1.25:

1.

7. The method according to claim 3, characterized in that In step (4), the mass ratio of the drug-loaded dendrimer to the targeted modified erythrocyte membrane is 1:1.5-2.

8. Use of the borneol-modified erythrocyte membrane-coated dendrimer biomimetic nano-drug delivery system according to any one of claims 1 to 2 in the preparation of tumor-targeted drugs.

9. The use according to claim 8, characterized in that The tumor is a glioma.

10. The use according to claim 9, characterized in that The tumor-targeted drug is administered by nasal drops.

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