Adenovirus delivery system wrapped by magnetic-guided palmitic acid modified cell membrane

Abstract

The invention provides an adenovirus delivery system wrapped by a magnetic-guided palmitic acid modified cell membrane, and relates to the field of biomedicine, the technical key points are as follows: the system comprises a magnetized adenovirus and a macrophage cell membrane, the magnetized adenovirus is located inside, and the capsid protein of the magnetized adenovirus is connected with Fe3O4 magnetic nanoparticles with the diameter of 10nm through DP-PEG-MAL; the macrophage membrane wraps the adenovirus, and the surface protein of the macrophage membrane is covalently connected with palmitic acid through an MAL-PEG linker. The invention mainly aims to provide a magnetic-guided palmitic acid modified cell membrane-coated adenovirus delivery system (AdV-Fe3O4-coated PACM), so as to solve the problems that in the prior art, adenovirus lacks effective targeting after intravenous injection, the blood vessel penetrating efficiency is low, the enrichment capacity in target tissues is poor and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to a magnetically guided palmitic acid-modified cell membrane-encapsulated adenovirus delivery system. Background Technology

[0002] In the fields of targeted gene therapy and viral vector drug development, adenoviruses are widely used in the treatment of various diseases (including tumors) due to their high infection efficiency and strong gene delivery capabilities. However, when adenoviruses are delivered via intravenous injection, the virus has difficulty effectively crossing the vascular endothelial barrier, resulting in poor accumulation in target tissues, rapid peripheral clearance, and low therapeutic efficiency. This has become one of the major technical bottlenecks limiting its clinical application.

[0003] To address this issue, existing research has attempted to employ methods such as surface modification, physical permeation enhancement, magnetic field guidance, or liposome encapsulation (PMID: 38505170). Existing methods, such as using magnetic nanoparticles to guide viruses to tumor sites, completely block the adenovirus capsid protein through non-covalent encapsulation. However, adenovirus infection of cells requires direct contact between its own fibrous protrusion proteins and the adenovirus receptor proteins on the cell surface; therefore, complete blockage hinders adenovirus infection. Furthermore, using magnetic nanoparticles alone cannot effectively assist viruses in penetrating blood vessel walls.

[0004] Another approach is to use cell membrane encapsulation to disguise adenoviruses, thereby prolonging their circulation time in vivo and reducing immune clearance. For example, encapsulating adenoviruses with macrophage membranes, erythrocyte membranes, or cancer cell membranes can improve their biocompatibility and immune evasion capabilities to some extent. However, cell membrane encapsulation alone cannot endow the virus with good active targeting, and due to the lack of transendothelial promotion mechanisms, its efficiency in penetrating blood vessel walls and entering target tissues remains low.

[0005] The natural properties of the cell membrane are insufficient to breach the tight junctions of the vascular endothelium, leading to viral aggregation within the blood vessel or phagocytosis by non-target tissues, thereby reducing treatment efficiency. Furthermore, cell membrane camouflage primarily relies on passive targeting and "homology recognition," failing to cope with the dynamic and complex internal environment.

[0006] Therefore, single cell membrane encapsulation technology still has significant shortcomings in terms of targeting and vascular permeability in adenovirus delivery, and further introduction of controllable guidance and vascular penetration enhancement mechanisms is needed to improve treatment efficacy. Summary of the Invention

[0007] The purpose of this invention is to solve the problem that the existing technology has not yet achieved the dual functions of precise in vivo localization of the virus and effective crossing of the vascular barrier. A new virus delivery strategy that has both targeting and guidance capabilities and can enhance vascular permeability is needed.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A magnetically guided palmitic acid-modified cell membrane-encapsulated adenovirus delivery system includes a magnetized adenovirus and a macrophage membrane, wherein the magnetized adenovirus is located inside, and its capsid protein isomorphic DP-PEG-MAL is linked to Fe3O4 magnetic nanoparticles with a diameter of 10 nm; the macrophage membrane encapsulates the adenovirus outside, and the macrophage membrane surface protein is covalently linked to palmitic acid (PA) via MAL-PEG linkers.

[0010] Preferably, the adenovirus delivery system is constructed as follows:

[0011] 10nm Fe3O4 magnetic nanoparticles were modified with DP-PEG-MAL and then covalently linked to the thiol groups of adenovirus capsid protein via maleimide; the macrophage cell membrane was modified with MAL-PEG-PA and then extracted.

[0012] Finally, the modified adenovirus and the modified cell membrane are encapsulated using membrane extrusion or microfluidic methods.

[0013] Preferably, the specific steps of constructing the adenovirus delivery system are as follows:

[0014] S1: Connecting adenovirus to Fe3O4 magnetic nanoparticles;

[0015] S2: Connects the macrophage membrane to palmitic acid;

[0016] S3: Palmitic acid-modified cell membrane encapsulates magnetized adenovirus.

[0017] Preferably, the specific steps of S1 are as follows:

[0018] The adenovirus was resuspended in PBS solution, and TCEP was added. The mixture was then stirred at 37°C to reduce the disulfide bonds in the viral capsid protein.

[0019] Then add PBS and concentrate the total volume using an ultrafiltration centrifuge tube.

[0020] Take DP-PEG-MAL modified Fe3O4, add PBS and use a magnet to attract it to settle, discard the supernatant; repeat once to fully remove the original solute;

[0021] The mixture was stirred at 37°C until fully induced. The adenovirus linked to Fe3O4 was precipitated by magnetic attraction. The supernatant was discarded, and the adenovirus linked to Fe3O4 was obtained after washing twice with PBS.

[0022] Preferably, the ultrafiltration centrifuge tube in S1 has a specification of 30 kDa, and the centrifugation conditions using the ultrafiltration centrifuge tube are 4000 rpm for 15 min.

[0023] Preferably, the specific steps of S2 are as follows:

[0024] RAW264.7 cells were collected, washed with PBS, and then TCEP was added before reacting at 37°C using a mixer.

[0025] The cells were washed away by centrifugation, then resuspended in PBS, and then palmitic acid (PA)-PEG-MAL was added. The mixture was stirred at 37°C for 1 hour.

[0026] After the reaction is complete, centrifuge, discard the supernatant, and wash twice with PBS.

[0027] Resuspend in 1 mL of PBS containing 1% PMSF, and perform all subsequent steps on ice to prevent membrane degradation;

[0028] Cells were broken up using an ultrasonic disruptor to extract cell membranes;

[0029] Add sucrose solution, centrifuge and collect the supernatant to remove cell nuclei and undisturbed cells, and repeat once to improve purity;

[0030] The supernatant was centrifuged at 4°C to obtain the membrane precipitate;

[0031] Resuspend in 1 mL of PBS containing 1% PMSF, and centrifuge again to remove cytoplasmic proteins; repeat washing twice, and resuspend in 1 mL of PBS containing 1% PMSF.

[0032] Preferably, the conditions for the ultrasonic disruptor in S2 are: ultrasonic 4s interval 30s, total 6min, and power 0.2kw.

[0033] Preferably, the specific steps of S3 are as follows: Take 1 mg of palmitic acid membrane, resuspend it by pipetting and vortex thoroughly, then place it in ice water for ultrasonic dispersion for 15 min, taking it out every 5 min and vortexing for 30 s to fully disperse the cell membrane; add 1 x 10 9 Ifu contains Fe3O4-linked adenovirus in S1. After thorough mixing, it is extruded 20 times using an Avanti liposome extruder through 1μm and 400nm filter membranes, respectively.

[0034] This application also provides the application of a magnetically guided palmitic acid-modified cell membrane-encapsulated adenovirus delivery system in the preparation of a tumor treatment drug, wherein the magnetically guided palmitic acid-modified cell membrane-encapsulated adenovirus delivery system is the adenovirus delivery system described above.

[0035] Compared with the prior art, this application has the following beneficial effects:

[0036] The main objective of this invention is to provide a magnetically guided palmitic acid-modified cell membrane-encapsulated adenovirus delivery system (AdV-Fe3O4@PACM) to address the problems in the prior art, such as the lack of effective targeting of adenovirus after intravenous injection, low efficiency in penetrating blood vessels, and poor accumulation ability in target tissues.

[0037] By covalently binding magnetic nanomaterials to the surface of adenovirus and constructing an encapsulation structure using palmitic acid modification of the cell membrane, this invention aims to achieve:

[0038] 1. Magnetic fields can controllably guide the accumulation of viruses in the target area, improving the spatial positioning accuracy of in vivo delivery;

[0039] 2. Palmitic acid-modified membranes enhance the virus's ability to penetrate blood vessel walls and improve transvascular transport efficiency;

[0040] 3. Improve the virus's ability to be effectively delivered and infect target tissues such as tumors.

[0041] This invention provides a new solution for the targeted delivery of viral vectors in clinical systemic drug administration, based on both structural design and functional synergy. Attached Figure Description

[0042] Figure 1 This is a construction strategy for palmitic acid-modified macrophage membrane-encapsulated magnetized adenovirus (AdV-Fe3O4@PACM) in one embodiment of the present invention.

[0043] Figure 2 This invention relates to palmitic acid modification of cell membranes in one embodiment, wherein (A) the structure of maleimide-polyethylene glycol-palmitic acid (MAL-PEG-PA) is shown. (B) 1x10 6 Mouse breast cancer cells (4T1) or macrophages (RAW264.7) were modified with different concentrations of PA-PEG-MAL. After modification, the palmitic acid content of the cells was detected by flow cytometry after BODIPY staining. **p<0.01, ***p<0.001, compared with no palmitic acid added.

[0044] Figure 3 This represents the yield of cell membrane extract from different cells after palmitic acid modification, according to one embodiment of the present invention. Specifically, it is 1x10⁻⁶. 8 Mouse macrophages (RAW264.7), human T cells (Jurkat), mouse breast cancer cells (4T1), mouse colon cancer cells (MC38), human lung cancer cells (A549), and human liver cancer cells (LM3), with or without 100 μM PA-PEG-MAL modification, had their cell membranes extracted. Cell membrane extraction yield was assessed by analyzing cell membrane protein content. *p<0.05, **p<0.01, compared to unmodified cells.

[0045] Figure 4 This illustrates a covalent link between Fe3O4 and adenovirus (AdV) in one embodiment of the present invention. Wherein, (A) 1x10 9 Different doses of DP-PEG-MAL modified Fe3O4 were added to the ifu AdV solution for ligation. After successful modification, the AdV titers in the supernatant and magnetized precipitate were determined by magnetic separation. (B) 1x10 9 The adenovirus of ifu, after being modified with different concentrations of Fe3O4, was used to determine its infection and replication capacity in tumor cells (human lung cancer cells A549 and human glioblastoma cells U87). *p<0.05, ***p<0.001; ns, no significant difference.

[0046] Figure 5 Particle characteristics of palmitic acid-modified macrophage cell membrane-encapsulated magnetized adenovirus (AdV-Fe3O4@PACM) in one embodiment of the present invention. (A) Transmission electron microscopy observation of unmodified adenovirus (AdV), palmitic acid-modified cell membrane (PACM), PACM-encapsulated adenovirus (AdV@PACM), Fe3O4, and AdV-Fe3O4@PACM. (B) Laser confocal imaging of AdV-Fe3O4@PACM nanoparticles. Adenovirus nucleic acid, Hoechst 33342; cell membrane, DilC18(3); palmitic acid, BODIPY. Particle size (C) and charge (D) of adenovirus (AdV), PACM-encapsulated adenovirus (AdV@PACM), unmodified cell membrane-encapsulated magnetized adenovirus (AdV-Fe3O4@CM), and AdV-Fe3O4@PACM.

[0047] Figure 6 The magnetic characteristics of AdV-Fe3O4@PACM are shown in one embodiment of the present invention. (A) Hysteresis loops of Fe3O4 and AdV-Fe3O4@PACM measured at room temperature. (B) AdV-Fe3O4@PACM was used to infect HEK293 cells in the presence of a magnetic field, and the adenovirus infection status on the magnetic and non-magnetic sides was detected 24 h later. Fluorescence represents AdV infection.

[0048] Figure 7 This illustrates the in vivo distribution of AdV-Fe3O4@PACM after intravenous injection under the influence of a magnetic field, according to one embodiment of the present invention. Panc02 cells were subcutaneously inoculated into C57 mice, and tumors were allowed to reach a volume of 200 mm². 3 At that time, administer 2 x 10 via tail vein injection. 8ifu adenovirus (AdV), magnetized adenovirus (AdV-Fe3O4), RAW264.7 cell membrane-encapsulated magnetized adenovirus (AdV-Fe3O4@CM), or palmitic acid-modified RAW264.7 cell membrane-encapsulated magnetized adenovirus (AdV-Fe3O4@PACM). All adenoviruses carried green fluorescent protein (GF). A 0.2T magnetic field was applied to the tumor site. (A) After 48 hours, mouse heart, liver, spleen, lung, kidney, and tumor tissues were collected for GFP fluorescence imaging analysis. (B) The fluorescence imaging results were quantitatively analyzed to compare the proportion of adenovirus in each tissue and organ. (C) Panc02 cells were subcutaneously inoculated into C57 mice until the tumor volume reached 200 mm². 3 At that time, administer 2 x 10 via tail vein injection. 8 Ifu adenovirus (AdV) or palmitic acid-modified RAW264.7 cell membrane-encapsulated magnetized adenovirus (AdV-Fe3O4@PACM). Forty-eight hours later, mouse heart, liver, spleen, lung, kidney, and tumor tissues were collected for GFP fluorescence imaging analysis. The fluorescence imaging results were quantitatively analyzed to compare the adenovirus percentage in each tissue / organ. *p<0.05, **p<0.01, ***p<0.001.

[0049] Figure 8 This invention describes the in vivo antitumor effect of AdV-Fe3O4@PACM intravenous injection under magnetic field in one embodiment. (A) Panc02 cells were subcutaneously inoculated into C57 mice until the tumor volume reached 100 mmHg. 3 At that time, 2x10g of the drug was administered via tail vein injection on days 0, 2, 4, 6, and 8. 8 ifu adenovirus (AdV), magnetized adenovirus (AdV-Fe3O4), RAW264.7 cell membrane-encapsulated magnetized adenovirus (AdV-Fe3O4@CM), or palmitic acid-modified RAW264.7 cell membrane-encapsulated magnetized adenovirus (AdV-Fe3O4@PACM). A 0.2T magnetic field was applied to the tumor site, and the tumor volume in mice was monitored. (B) Balb / c nude mice were subcutaneously inoculated with MDA-MB-231 cells until the tumor volume reached 100 mm². 3 At that time, 2x10g of the drug was administered via tail vein injection on days 0, 2, 4, 6, and 8. 8 ifuAdV, AdV-Fe3O4, AdV-Fe3O4@CM, or AdV-Fe3O4@PACM were used. A 0.2T magnetic field was applied to the tumor site, and the tumor volume in mice was monitored. *p<0.05, **p<0.01, ***p<0.001. Detailed Implementation

[0050] The present invention will be further described in detail below with reference to specific embodiments.

[0051] Please see Figure 1 A palmitic acid-modified magnetized adenovirus encapsulated in a macrophage cell membrane comprises an adenovirus and a macrophage membrane. The adenovirus is located internally, and its capsid protein is linked to 10 nm diameter Fe3O4 magnetic nanoparticles via bisphosphonate-polyethylene glycol-maleimide (DP-PEG-MAL). The macrophage membrane encapsulates the adenovirus externally, and the macrophage membrane surface protein is covalently linked to palmitic acid via a MAL-PEG linker.

[0052] The construction method is as follows: 10 nm Fe3O4 magnetic nanoparticles are modified with bisphosphonate-polyethylene glycol-maleimide (DP-PEG-MAL), and then maleimide is covalently linked to the thiol groups of the adenovirus capsid protein. Macrophage cell membranes are modified with maleimide-polyethylene glycol-palmitic acid (MAL-PEG-PA), and the cell membranes are extracted. Finally, the modified adenovirus and the modified cell membrane are encapsulated using membrane extrusion or microfluidic methods.

[0053] The specific steps are as follows:

[0054] S1: Connection between adenovirus and Fe3O4 magnetic nanoparticles

[0055] 1 x 10 10 Ifu adenovirus was resuspended in 0.5 mL PBS solution, and 10 mM TCEP was added. The mixture was then stirred at 37 °C for 1 h to reduce the disulfide bonds on the virus.

[0056] After adding 5 mL of PBS, centrifuge at 4000 rpm for 15 min using a 30 kDa ultrafiltration centrifuge tube to concentrate the total volume to 0.5 mL.

[0057] Take 5 μL of Fe3O4-PEG-Mal (25 mg / mL), add 1 mL of PBS, and use a magnet to allow it to settle. Discard the supernatant. Repeat once to ensure complete removal of the original solute.

[0058] The treated adenovirus and MAL-PEG-DP modified Fe3O4 microspheres were co-incubated, and the mixture was stirred evenly by inverting the mixer at a constant speed. The reaction was carried out at 37°C for 1 hour. The Fe3O4-linked adenovirus was precipitated by magnetic adsorption, the supernatant was discarded, and the adenovirus was obtained by washing twice with PBS.

[0059] In the above steps, Fe3O4-PEG-Mal refers to Fe3O4 microspheres modified with MAL-PEG-DP, and its construction method is shown below.

[0060] 100 mg of oleic acid-modified iron(III) oxide was dispersed in 100 mg of tetrahydrofuran solution and heated to reflux at 70 °C. After the temperature stabilized, 15 mg of DP-PEG-Mal was added, and the mixture was heated to reflux for another 5 hours. The mixture was then cooled and magnetically separated. The mixture was washed twice with ethanol and water, respectively, to obtain the final product, MAL-PEG-DP-modified Fe3O4 microspheres (Fe3O4-PEG-Mal).

[0061] S2: The connection between the cell membrane and palmitic acid:

[0062] Collect 1.5 x 10 8 RAW264.7 cells were washed twice with 1 mL PBS, and then reacted with 10 mM TCEP at 37°C for 1 h using a mixer.

[0063] Centrifuge at 400g for 3 min, discard the supernatant, and repeat once to wash away TCEP. Resuspend the cells in 1 mL PBS, add 100 μM palmitic acid-PEG-Mal, and mix thoroughly at 37°C for 1 h.

[0064] After the reaction was complete, centrifuge at 400g for 3 minutes, discard the supernatant, and wash twice with PBS to remove unbound palmitic acid.

[0065] Resuspend the membrane in 1 mL of PBS containing 1% PMSF. All subsequent steps should be performed on ice to prevent membrane degradation.

[0066] Cells were disrupted using an ultrasonic disruptor to extract cell membranes. The ultrasonic process lasted 6 minutes (4 seconds with 30 seconds intervals) at a power of 0.2 kW. 200 μL of a 1 M sucrose solution was added, and the mixture was centrifuged at 2000 g for 15 minutes at 4°C. The supernatant was collected to remove cell nuclei and undisrupted cells, and this process was repeated once to improve purity.

[0067] The supernatant was centrifuged at 14000g for 30 min at 4°C to obtain a membrane precipitate. The precipitate was resuspended in 1 mL of PBS containing 1% PMSF and centrifuged again at 14000g for 30 min to remove cytosolic proteins. The washing process was repeated twice, and the precipitate was resuspended in 1 mL of PBS containing 1% PMSF. Membrane protein concentration was determined using the BCA method.

[0068] S3: Palmitic acid-modified cell membrane encapsulation of magnetized adenovirus:

[0069] Take 1 mg of palmitic acid membrane, resuspend by pipetting and vortex thoroughly, then sonicate in ice water for 15 min, vortexing for 30 s every 5 min to ensure thorough dispersion of the cell membrane. Add 1 x 10 9 Ifu contains Fe3O4-linked adenovirus in S1. After thorough mixing, it is extruded 20 times using an Avanti liposome extruder through 1μm and 400nm filter membranes, respectively.

[0070] The above content will be explained in conjunction with specific verification experiments:

[0071] Experimental materials and their sources:

[0072] 1. Adenovirus

[0073] Adenoviruses were obtained by homologous recombination of shuttle plasmids and backbone plasmids (preserved in our laboratory), followed by packaging, amplification, and purification in 293T cells.

[0074] 2. Fe3O4 particles with a diameter of 10nm were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0075] 3. DP-PEG-MAL (bisphosphonic acid-polyethylene glycol-maleimide) was purchased from Suzhou Xinying Biomedical Technology Co., Ltd.

[0076] 4. MAL-PEG-PA (maleimide-polyethylene glycol-palmitic acid) was purchased from Shanghai Tuoyang Biotechnology Co., Ltd.

[0077] 5. RAW264.7 mouse macrophages and DMEM culture medium were purchased from Wuhan Pronosai Life Science Technology Co., Ltd.

[0078] Example 1: Establishing a method for palmitic acid-modified cell membranes

[0079] The cell membrane is mainly composed of a lipid bilayer and membrane proteins. Because palmitic acid is a hydrophobic molecule, if anchored to the lipid membrane, it tends to embed within the hydrophobic core region of the lipid bilayer, making it difficult to expose to the cell membrane surface. Therefore, modifying the surface of hydrophilic membrane proteins with palmitic acid is a preferred strategy.

[0080] To add an active group to palmitic acid that can be covalently linked to membrane proteins, a palmitic acid-polyethylene glycol-maleimide (PA-PEG-MAL) molecule was constructed in this embodiment to enable covalent linking of MAL to the thiol groups of membrane proteins. Figure 2 A). After incubating cells with different doses of PA-PEG-MAL, the palmitic acid content of the cells was analyzed by BODIPY staining. The results showed that treatment with 100 μM PA-PEG-MAL significantly increased the palmitic acid content of the cells, and further increasing the concentration of PA-PEG-MAL did not increase the palmitic acid content of the cells. Figure 2 B).

[0081] This demonstrates that this method can effectively attach palmitic acid to the cell membrane, and that a concentration of 100 μM can saturate the modification of 1 x 10⁻⁶ cells. 6 Each cell.

[0082] Example 2: Screening for cell types with high palmitic acid-modified cell membrane production

[0083] To evaluate the extraction yield of different cell membranes after PA-PEG-MAL modification, mouse macrophages (RAW264.7), human T cells (Jurkat), mouse breast cancer cells (4T1), mouse colon cancer cells (MC38), human lung cancer cells (A549), and human liver cancer cells (LM3) were selected for experiments in this example.

[0084] Without PA-PEG-MAL treatment, the extracted cell membrane content was comparable.

[0085] After PA-PEG-MAL treatment, the extractable amounts of cell membranes from 4T1, MC38, A549, and LM3 cells decreased significantly, while the extractable amounts of cell membranes from RAW264.7 and Jurkat cells did not decrease significantly. Figure 3 ).

[0086] The above results suggest that RAW264.7 and Jurkat cells can be used for cell membrane extraction after PA-PEG-MAL modification.

[0087] Example 3: Establishing a method for modifying Adv with Fe3O4

[0088] In order to enable the adenovirus to acquire magnetic drive capability, in this embodiment, Fe3O4 modified with DP-PEG-MAL is attached to the surface of the adenovirus capsid protein.

[0089] Through the reaction of different doses of Fe3O4 and Adv (AdV), it was found that with increasing Fe3O4 dose, the amount of Adv in the supernatant gradually decreased, while the amount of Adv that could be magnetically attracted gradually increased. Specifically, 0.125 mg Fe3O4 enabled more than 80% of the Adv to acquire magnetic driving ability. When the Fe3O4 dose was 15.625 mg, although the amount of Adv in the supernatant decreased significantly, the Adv titer in the precipitate also decreased significantly, suggesting that this dose may have caused Adv toxicity, leading to a decrease in its activity. Figure 4 A).

[0090] Further, magnetized AdV modified with Fe3O4 was used to infect tumor cells, and the viral replication ability in tumor cells was detected. The results showed that 0.125 mg of Fe3O4 modification had almost no effect on AdV replication in tumor cells, while after modification with 0.625 mg of Fe3O4, the replication ability of AdV in tumor tissue was maintained at over 85%. Figure 4 B).

[0091] These results demonstrate that the Fe3O4 modification method for AdV was successfully established in this embodiment, enabling AdV to acquire magnetic driving capability without affecting its infection and replication activity.

[0092] Example 4: Preparation of palmitic acid-modified cell membrane-encapsulated adenovirus AdV-Fe3O4@PACM

[0093] In this example, AdV-Fe3O4@PACM was successfully prepared by encapsulating magnetized adenovirus in a cell membrane modified with palmitic acid.

[0094] Transmission electron microscopy results showed that the AdV-Fe3O4@PACM nanoparticles were surrounded by cell membranes, and the AdV surface contained Fe3O4 components. Figure 5 A).

[0095] Laser confocal imaging results showed that the Adv-Fe3O4@PACM nanoparticles contained adenovirus, cell membrane, and palmitic acid components. Figure 5 B).

[0096] The diameter of AdV-Fe3O4@PACM is approximately 320 nm, which is three times that of the uncoated AdV, and its surface charge is -3.5 mV. Figure 5 CD).

[0097] Example 5: The prepared Adv-Fe3O4@PACM in vitro responsive magnetic field driven

[0098] To verify the magnetic response capability of AdV-Fe3O4@PACM, its magnetism was first evaluated in this embodiment.

[0099] The hysteresis loop results indicate that AdV-Fe3O4@PACM exhibits extremely low coercivity and good superparamagnetic behavior, with a saturation magnetization of approximately 54 emu / g Fe( Figure 6 A).

[0100] Under the presence of a 0.2T magnetic field, AdV-Fe3O4@PACM can be driven, thus favoring infection of cells on the magnet side. Figure 6 B).

[0101] These results demonstrate that AdV-Fe3O4@PACM possesses excellent magnetic response capabilities.

[0102] Example 6: After intravenous injection, the prepared AdV-Fe3O4@PACM was enriched at the tumor site under the guidance of a magnetic field.

[0103] In this embodiment, tumor-bearing mice were grouped, and different groups of tumor-bearing mice were injected via tail vein with adenovirus (AdV), magnetized adenovirus (AdV-Fe3O4), magnetized adenovirus encapsulated in RAW264.7 cell membrane (AdV-Fe3O4@CM), or palmitic acid-modified magnetized adenovirus encapsulated in RAW264.7 cell membrane (AdV-Fe3O4@PACM).

[0104] Under the influence of a magnetic field, the enrichment of both AdV-Fe3O4@CM and AdV-Fe3O4@PACM in tumor tissues was significantly increased. Figure 7 AB).

[0105] AdV-Fe3O4 was highly enriched in the kidneys of mice, suggesting possible nephrotoxicity.

[0106] Notably, under the influence of a magnetic field, AdV-Fe3O4@PACM showed superior tumor accumulation compared to AdV-Fe3O4@CM, indicating that palmitic acid-modified cell membranes have a better effect on promoting AdV entry into solid tumor tissues than unmodified cell membranes. However, in the absence of a magnetic field, the distribution of AdV-Fe3O4@PACM in vivo was similar to that of unmodified AdV, and no tumor accumulation effect was observed. Figure 7 C),

[0107] The results indicate that the enrichment of AdV-Fe3O4@PACM at the tumor site is magnetically driven. These results demonstrate that the AdV-Fe3O4@PACM of this application can significantly accumulate at the tumor site under the guidance of a magnetic field after intravenous injection.

[0108] Example 7: The prepared AdV-Fe3O4@PACM, after intravenous injection, exhibited significant antitumor activity under the guidance of a magnetic field.

[0109] To verify the antitumor effect of AdV-Fe3O4@PACM in vivo, a mouse pancreatic cancer model (Panc02) and a human breast cancer model (MDA-MB-231) were selected in this embodiment.

[0110] Mice were divided into groups and injected via tail vein with adenovirus (AdV), magnetized adenovirus (AdV-Fe3O4), RAW264.7 cell membrane-encapsulated magnetized adenovirus (AdV-Fe3O4@CM), or palmitic acid-modified RAW264.7 cell membrane-encapsulated magnetized adenovirus (AdV-Fe3O4@PACM). A 0.2T magnetic field was simultaneously applied to the tumor site.

[0111] The results showed that in both tumor models, the antitumor effect of AdV-Fe3O4@PACM was significantly greater than that of AdV, AdV-Fe3O4, or AdV-Fe3O4@CM. Figure 8 AB).

[0112] Compared to the Vehicle control group, AdV-Fe3O4@PACM achieved a tumor inhibition rate of 70.88% in the Panc02 model and 73.49% in the MDA-MB-231 model. These results suggest that AdV-Fe3O4@PACM has a good anti-tumor effect after intravenous injection under the influence of a magnetic field.

[0113] In summary, this application provides a magnetically guided palmitic acid-modified cell membrane-encapsulated adenovirus delivery system (AdV-Fe3O4@PACM), which can effectively solve the problems of adenovirus lacking effective targeting after intravenous injection, low efficiency in penetrating blood vessels, and poor accumulation ability in target tissues in the prior art.

[0114] In this application, magnetic nanomaterials are covalently bonded to the surface of adenovirus, and a cell membrane is modified with palmitic acid to construct an encapsulation structure. This enables the magnetic field to controllably guide the virus to target the target area, improving the spatial localization and virulence reduction of in vivo delivery. In addition, the palmitic acid-modified membrane enhances the virus's ability to penetrate blood vessel walls, thereby improving the efficiency of transvascular transport. It also improves the effective delivery and infection capabilities of the virus in target tissues such as tumors.

Claims

1. A magnetically guided palmitic acid-modified cell membrane-encapsulated adenovirus delivery system, characterized in that: The invention comprises a magnetized adenovirus and a macrophage membrane, wherein the magnetized adenovirus is located inside, and its capsid protein is linked to Fe3O4 magnetic nanoparticles with a diameter of 10 nm via DP-PEG-MAL; the macrophage membrane surrounds the adenovirus, and the macrophage membrane surface protein is covalently linked to palmitic acid via MAL-PEG linkers.

2. The magnetically guided palmitic acid-modified cell membrane-encapsulated adenovirus delivery system according to claim 1, characterized in that: The adenovirus delivery system is constructed as follows: 10nm Fe3O4 magnetic nanoparticles were modified with DP-PEG-MAL and then covalently linked to adenovirus capsid protein via the thiol group of maleimide; the macrophage cell membrane was modified with MAL-PEG-PA and then extracted. Finally, the modified adenovirus and the modified cell membrane are encapsulated using membrane extrusion or microfluidic methods.

3. The magnetically guided palmitic acid-modified cell membrane-encapsulated adenovirus delivery system according to claim 2, characterized in that: The specific steps for constructing the adenovirus delivery system are as follows: S1: Connecting adenovirus to Fe3O4 magnetic nanoparticles; (Fe3O4 on the outside of the adenovirus) S2: Connects the macrophage membrane to palmitic acid; (palmitic acid is on the outer surface of the cell membrane) S3: Palmitic acid-modified cell membrane encapsulates magnetized adenovirus.

4. The magnetically guided palmitic acid-modified cell membrane-encapsulated adenovirus delivery system according to claim 3, characterized in that: The specific steps of S1 are as follows: The adenovirus was resuspended in PBS solution, and TCEP was added. The mixture was then stirred at 37°C to reduce the disulfide bonds on the virus. Then add PBS and concentrate the total volume using an ultrafiltration centrifuge tube. Take DP-PEG-MAL modified Fe3O4, add PBS and use a magnet to attract it to settle, discard the supernatant; repeat once to fully remove the original solute; The mixture was stirred at 37°C until fully induced. The adenovirus linked to Fe3O4 was precipitated by magnetic attraction. The supernatant was discarded, and the adenovirus linked to Fe3O4 was obtained after washing twice with PBS.

5. The magnetically guided palmitic acid-modified cell membrane-encapsulated adenovirus delivery system according to claim 3, characterized in that: The ultrafiltration centrifuge tube in S1 has a specification of 30 kDa, and the centrifugation conditions for using the ultrafiltration centrifuge tube are 4000 rpm for 15 min.

6. The magnetically guided palmitic acid-modified cell membrane-encapsulated adenovirus delivery system according to claim 3, characterized in that: The specific steps of S2 are as follows: RAW264.7 cells were collected, washed with PBS, and then TCEP was added before reacting at 37°C using a mixer. The cells were washed away by centrifugation, then resuspended in PBS, and then palmitic acid-PEG-MAL was added. The mixture was then stirred at 37°C for 1 hour. After the reaction is complete, centrifuge, discard the supernatant, and wash twice with PBS. Resuspend in 1 mL of PBS containing 1% PMSF, and perform all subsequent steps on ice to prevent membrane degradation; Cells were broken up using an ultrasonic disruptor to extract cell membranes; Add sucrose solution, centrifuge and collect the supernatant to remove cell nuclei and undisturbed cells, and repeat once to improve purity; The supernatant was centrifuged at 4°C to obtain the membrane precipitate; Resuspend in 1 mL of PBS containing 1% PMSF, and centrifuge again to remove cytoplasmic proteins; repeat washing twice, and resuspend in 1 mL of PBS containing 1% PMSF.

7. The magnetically guided palmitic acid-modified cell membrane-encapsulated adenovirus delivery system according to claim 3, characterized in that: The conditions for the ultrasonic disruptor in S2 are: ultrasonic waves 4 seconds apart with a 30-second interval, a total duration of 6 minutes, and a power of 0.2 kW.

8. The magnetically guided palmitic acid-modified cell membrane-encapsulated adenovirus delivery system according to claim 6, characterized in that: The specific steps of S3 are as follows: Take 1 mg of palmitic acid membrane, resuspend it by pipetting and vortex thoroughly, then place it in ice water for ultrasonic dispersion for 15 min, removing it every 5 min and vortexing for 30 s to fully disperse the cell membrane; add 1 x 10 9 Ifu contains Fe3O4-linked adenovirus in S1. After thorough mixing, it is extruded 20 times using an Avanti liposome extruder through 1μm and 400nm filter membranes, respectively.

9. The application of a magnetically guided palmitic acid-modified cell membrane-encapsulated adenovirus delivery system in the preparation of antitumor drugs, characterized in that: The magnetically guided palmitic acid-modified cell membrane-encapsulated adenovirus delivery system is the adenovirus delivery system according to any one of claims 1-8.

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