Nano-drug with dual targeting effect as well as preparation method and application of nano-drug

Abstract

The invention relates to the technical field of biological medicine, in particular to a nano-drug with a dual targeting effect and a preparation method and application thereof, and the nano-drug comprises exosome and ferritin; the surface of the exosome is modified with an azide group, the surface of the ferritin is modified with a cyclopentyl group, and the exosome and the ferritin are connected through a click chemical reaction between the azide group and the cyclopentyl group. According to the nano-drug, the drug carrying efficiency is improved through the exosome and the ferritin, meanwhile, the nano-drug can effectively penetrate through a cerebral blood barrier and is taken by glioma cells, and the killing effect of the drug on tumor cells is enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a nano drug with dual targeting effects, a preparation method and application thereof. Background Art

[0002] Glioblastoma Multiforme (GBM) is a highly malignant central nervous system tumor and a neuroepithelial tumor.

[0003] In the treatment of malignant glioblastoma, the limitation of blood-brain barrier (BBB) ​​is an important issue. BBB ensures normal brain function by strictly controlling the entry of substances from circulation into the brain. However, it also blocks about 98% of small molecule drugs and almost all macromolecule drugs from entering the brain. The physical (tight junctions and adhesion junctions) and biochemical (transporter-mediated efflux) barriers provided by BBB prevent brain penetration of anti-tumor agents and limit the accumulation of small molecule targeted therapy. Although studies have found that some new nanocarriers can cross BBB, most drug delivery systems have short circulation time in the body and poor targeting. The accumulated concentration in the tumor is far from the therapeutic concentration, resulting in poor efficacy. Therefore, it is urgent to find a delivery system with long circulation time, good biosafety and the ability to cross BBB-target GBM to improve the delivery efficiency of drugs.

[0004] Prior art, such as the invention patent with application number 2023108928046, provides a drug delivery carrier complex targeting the central nervous system, and its preparation method and application, the preparation method of which includes the following steps: azide-modifying the carrier cell to obtain an azide-modified carrier cell; modifying the thiol and cholesterol to the two ends of the therapeutic miRNA respectively, and then connecting the modified therapeutic miRNA with the propylene complex to form a miRNA complex; incubating the azide-modified carrier cell and the miRNA complex together, and forming the drug delivery carrier complex through a click chemistry reaction. However, the above method uses living cells as drug delivery carriers, which may induce an immune response or be cleared by the body's immune system, which will affect the therapeutic effect and safety.

[0005] Exosomes have a small particle size and good in vivo penetration. They can not only be enriched in the tumor site through EPR effect, chemotaxis, homing and other characteristics, but also pass through the blood-brain barrier for the treatment of brain diseases. Exosomes carry the genetic material, lipids and proteins of the mother cells inside and on the surface, and have functions such as immune regulation and tumor microenvironment regulation. They can inhibit tumor growth through various pathways.

[0006] Ferritin is a natural cage-type nanomaterial assembled from light chain subunits and heavy chain subunits. Its special structure and properties give it unique advantages as a nano-delivery carrier. Ferritin has cavities and a large surface area. Its cavities can not only load many molecular drugs, but also nanocrystals. Its large surface area allows many anti-tumor modifications, thereby achieving spatially differentiated loading of drugs, making it an ideal drug carrier. At the same time, studies have shown that ferritin receptors are overexpressed in a variety of malignant tumors, making heavy-chain ferritin an ideal system for anti-tumor drug delivery. Further studies have found that ferritin receptors are highly expressed on both BBB and glioma cells. Using heavy-chain ferritin to construct nanomedicines can successfully cross the BBB and enter the central nervous system. And through interaction with ferritin receptors, nanomedicines constructed based on ferritin can be effectively taken up by glioma cells, significantly enhancing the killing effect of drugs on tumor cells. Summary of the invention

[0007] In order to solve the problems in the above-mentioned background technology, the first object of the present invention is to provide a nano drug with dual targeting effect, including exosomes and ferritin; the surface of the exosomes is modified with an azide group, the surface of the ferritin is modified with a cyclized group, and the exosomes and the ferritin are connected by a click chemistry reaction between the azide group and the cyclized group.

[0008] The nanomedicine of the present application can effectively cross the brain-blood barrier and be taken up by glioma cells through exosomes and ferritin while improving the drug carrying efficiency, thereby enhancing the killing effect of the drug on tumor cells.

[0009] Preferably, the ferritin is heavy chain ferritin.

[0010] Preferably, the particle size of the heavy chain ferritin is 12 nm.

[0011] Preferably, the exosomes are one or more of M1 microglial exosomes, M1 macrophage exosomes or mesenchymal stem cell exosomes.

[0012] The present application uses exosomes with chemotaxis or homing functions to improve the targeting of nanomedicines through the above design.

[0013] Preferably, the exosome cavity can be loaded with temozolomide, cisplatin or small interfering RNA by electrotransfer.

[0014] Preferably, the ferritin cavity may be loaded with nano-manganese, nano-gold or nano-ferroferric oxide.

[0015] Through the above technical solutions, nanomedicines can synergistically treat glioblastoma in a multi-modal manner.

[0016] The second object of the present invention is to provide a method for preparing a nano drug with dual targeting effects, comprising the following steps:

[0017] S1, Preparation of exosomes with surface modified with azide groups

[0018] Azide choline was added to the complete cell culture medium, wherein the concentration of azide choline in the culture medium was 100 to 1600 μM, and then the cells were placed in a culture medium at a temperature of 37°C and CO 2 The cells were cultured in a constant temperature incubator with a concentration of 5% for 24 to 48 hours, and the cell supernatant was collected and then subjected to ultracentrifugation to obtain exosomes with azide groups modified on the surface, wherein the particle size of the exosomes was 30 to 150 nm;

[0019] S2, preparation of ferritin with surface modified cyclopentadienyl groups

[0020] Dibenzocyclooctyne-pentaethylene glycol-active ester (DBCO-PEG 5 -NHS) was added to the ferritin solution to form a mixed solution 1, in which ferritin and DBCO-PEG 5 -NHS molar ratio is 1:12 to 1:120;

[0021] Subsequently, the mixed solution was placed in a vertical suspension instrument at 20 r / min at 25-37°C for 1 h to 6 h, and then ultrafiltration centrifuged using a 10 kDa ultrafiltration tube to remove unreacted DBCO-PEG in the mixed solution. 5 -NHS, to obtain ferritin with surface modified with cyclopentadienyl groups;

[0022] S3, Preparation of nanoparticles with dual targeting effects

[0023] The exosomes modified with azide groups on the surface and the ferritin modified with cyclopentadiene groups on the surface are mixed together to form a second mixed solution, wherein the ratio of the number of the exosomes modified with azide groups on the surface to the number of the ferritin modified with cyclopentadiene groups on the surface is 1:10 to 1:100;

[0024] Subsequently, the mixed solution was placed in a vertical suspension instrument at 25-37° C. and vertically suspended at a speed of 20 r / min for 2 h to 6 h, and then centrifuged to obtain dual-targeting nanoparticles.

[0025] Preferably, the cells in step 1 are M1 microglial cells HMC3.

[0026] The third object of the present invention is to provide an application of a nano drug in a preparation for treating glioblastoma.

[0027] A fourth object of the present invention is to provide a drug delivery system.

[0028] In summary, the beneficial effects of the present invention are:

[0029] Using exosomes and ferritin to construct dual-targeting nanomedicines, the dual effects of ferritin-ferritin receptor and exosome homing / chemotaxis can cross the BBB and target GBM;

[0030] Anti-tumor drugs such as temozolomide, cisplatin or small interfering RNA are loaded into the exosome cavity by electroporation to improve drug stability and enhance drug efficacy;

[0031] Nanocrystals such as nanomanganese, gold, and ferroferric oxide are loaded into the ferritin cavity by in situ growth to construct nanomedicines with dual targeting effects and synergistic multi-modal treatment of glioblastoma;

[0032] At the same time, drugs can also be carried in ferritin to improve drug carrying efficiency and enhance treatment efficiency.

[0033] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the following specifically cites a preferred embodiment and describes it in detail with the accompanying drawings as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Detection of Exos and Exos-N for flow cytometric analysis 3 images;

[0035] Figure 2 The emission spectra of ferritin (HFn), DBCO-HFn and Cy5.5 fluorescent dye (N3-Cy5.5) with an azide group (-N3) after reaction;

[0036] Figure 3 Exos-N 3 , transmission electron microscopy images of DBCO-HFn and Exos-HFn;

[0037] Figure 4 Exos-N 3 , DBCO-HFn and Exos-HFn particle size test chart;

[0038] Figure 5 Confocal imaging of Exos, HFn, and Exos-HFn targeting GBM cells in vitro;

[0039] Figure 6In vivo imaging of Exos, HFn, and Exos-HFn targeting GBM in mice;

[0040] Figure 7 Exos(DIO): confocal imaging of Exos labeled with DIO; siMCT4(Cy5): confocal imaging of siMCT4 labeled with Cy5; Merge: confocal imaging of siMCT4 loaded with Exos;

[0041] Figure 8 The radio microscope photo and XPS spectrum of HFn of multivalent nano manganese oxide in situ grown in the cavity;

[0042] Fig. 9 Luminescent imaging of tumor-bearing mice treated with PBS, Exos@siMCT4-HFn, Exos-HFn@Mn, and Exos@siMCT4-HFn@Mn;

[0043] Fig.10 The inhibition rates of Exos@siMCT4-HFn, Exos-HFn@Mn and Exos@siMCT4-HFn@Mn on GBM. DETAILED DESCRIPTION

[0044] In order to make the content of the present invention more clearly understood, the present invention is further described below based on specific embodiments in conjunction with the accompanying drawings.

[0045] 1. Preparation of dual-targeted composite nanomedicine

[0046] 1. Preparation of exosomes modified with azide groups on the surface (Exos-N 3 )

[0047] S1, adding azidocholine to the complete medium cultured with human microglial cells HMC3, wherein the concentration of azidocholine is 800 μM;

[0048] S2, place the cells in a culture medium at 37°C and CO 2 The cells were cultured in a constant temperature incubator with a concentration of 5% for 24 h, and the cell supernatant was collected. The cell supernatant was then placed in a centrifuge for step-by-step centrifugation in the order of centrifugal force 300 g, centrifugal time 10 min → centrifugal force 3000 g, centrifugal time 10 min → centrifugal force 10000 g, centrifugal time 30 min → centrifugal force 110000 g, centrifugal time 70 min to obtain Exos-N. 3 .

[0049] Labeled with DCBO-Cy3, N 3 Whether the modification is successful, the result is Figure 1As shown, Exos-N 3 Preparation was successful.

[0050] 2. Preparation of ferritin modified with cyclopentadienyl groups (DBCO-HFn)

[0051] S1, dibenzocyclooctyne-pentaethylene glycol-active ester (DBCO-PEG 5 -NHS) was added to the ferritin (HFn) solution with a concentration of 10 mg / mL to form a mixed solution 1, in which HFn and DBCO-PEG 5 -NHS molar ratio is 1:48;

[0052] S2, at 25°C, the mixed solution was placed in a vertical suspension instrument and vertically suspended at a speed of 20r / min for 2h, then ultrafiltration centrifuged using a 10kDa ultrafiltration tube, and then washed three times with sterile phosphate buffered saline (PBS) to remove unreacted DBCO-PEG in the mixed solution. 5 -NHS, obtained DBCO-HFn.

[0053] HFn and DBCO-HFn with N 3 -Cy5.5 were incubated at 25°C for 1 h, then ultrafiltration and centrifugation were performed using a 10 kDa ultrafiltration tube, and then washed three times with PBS to remove unreacted N 3 -Cy5.5, and the emission spectrum was used to detect whether DBCO modification was successful. The results are as follows Figure 2 As shown, DBCO-HFn modification was successful.

[0054] 3. Preparation of Nanoparticles with Dual Targeting Effects (Exos-HFn)

[0055] S1, Exos-N 3 Mixed with DBCO-HFn to form mixed solution 2, in which the ratio of the number of exosomes modified with azide groups on the surface to the number of ferritin modified with cyclopentadiene groups on the surface is 1:20;

[0056] S2, then at 25°C, the mixed solution was placed in a vertical suspension instrument and vertically suspended at a speed of 20 r / min for 2 hours, and then centrifuged to obtain Exos-HFn.

[0057] After negative staining with 2% phosphotungstic acid solution, transmission electron microscopy (TEM) imaging was performed. The samples were Exos-N 3 , DBCO-HFn and Exos-HFn, the results are as follows Figure 3 As shown, Exos-HFn was successfully prepared. 3The particle sizes of DBCO-HFn and Exos-HFn are as follows: Figure 4 shown.

[0058] 2. Experimental Analysis

[0059] Experimental analysis (1)

[0060] An in vitro BBB model was constructed using Transwell. HCMEC / D3 cells, the brain vascular endothelial cells, were seeded in the upper inorganic membrane chamber of the chamber, and U87 cells, a human glioblastoma cell line, were seeded in the lower chamber.

[0061] When the upper layer of hCMEC / D3 grows into a monolayer and completely covers the inorganic membrane, the transendothelial resistance is 150Ω·cm 2 When the cells were treated with Cy5, Cy5-labeled Exos(Cy5), Exos(Cy5)-HFn, HFn(Cy5), and Exos-HFn(Cy5) were added.

[0062] After 24 hours, confocal imaging was performed on U87 cells to observe the uptake of different groups of nanoparticles by U87 cells.

[0063] The results are as follows Figure 5 As shown, the ability of Exos-HFn to target glioblastoma U87 cells in vitro was significantly enhanced

[0064] Experimental analysis (2)

[0065] Female Balb / c nude mice aged 4-6 weeks were selected and 3 μL of luc-U87 cells were injected into the mouse brain using a stereotaxic apparatus. 3×10^5 luc-U87 cells were injected each time to construct a glioblastoma U87 tumor model.

[0066] After the successful construction of the GBM model was confirmed by small animal bioluminescence imaging on the 14th day, the tumor-bearing mice were randomly divided into 4 groups and injected with 200 μg of Cy7-labeled Exos (Cy7), Exos (Cy7)-HFn, HFn (Cy7), and Exos-HFn (Cy7) through the tail vein. Fluorescence imaging of the mice was performed 4 hours later to observe the distribution of nanoparticles in different groups of mice.

[0067] As shown in 6, the ability of Exos-HFn to target glioblastoma in vivo was significantly enhanced.

[0068] Combining experimental analysis (1) and experimental analysis (2), it is shown that the nanoparticle Exos-HFn with dual targeting effect of the present application can promote the drug to cross BBB and target GBM.

[0069] Experimental analysis (3)

[0070] The collected Exos-N 3 After DIO labeling, it was mixed with Cy5-labeled siMCT4 at a ratio of 1:2, and then electroporated using an electroporator at 100 V, 200 Ω resistance, and 100 μF capacitance. It was then allowed to stand at 37°C for 30 min and purified using a NAP-5 column to remove unreacted free siMCT4 to obtain Exos loaded with siMCT4 (Exos@siMCT4).

[0071] Exos@siMCT4 was dropped on a glass slide coated with paraformaldehyde and incubated at 37°C for 1 h. The non-adherent nanoparticles were then washed away with PBS before confocal imaging.

[0072] The results are as follows Figure 7 As shown, siMCT4 can be successfully loaded into Exos-N by electroporation. 3 In the cavity.

[0073] Experimental analysis (4)

[0074] Add 20 mL of 0.1 M NaCl aqueous solution to a 50 mL three-necked flask, then drop 1 M NaOH into the flask until the pH is 8.5. Then, introduce nitrogen and deoxygenate for 15 min, then add 20 mg of ferritin (HFn). Raise the temperature of the solution in the three-necked flask to 65 °C, and add 50 mM MnCl at a rate of 20 μL / min while stirring. 2 ·4H 2 O, 50mMH 2 O 2 and 0.12M NaOH solutions.

[0075] After the addition is completed, the reaction is continued for 5 minutes. Nitrogen is passed throughout the reaction to ensure the absence of oxygen. After the reaction is completed, centrifuge at a centrifugal force of 10000g for 10 minutes to remove the precipitate, collect the supernatant, use a 10kD ultrafiltration tube, centrifuge at a centrifugal force of 6000g and perform ultrafiltration for 30 minutes to obtain concentrated ferritin grown with manganese oxide (HFn@Mn).

[0076] The manganese oxide in the HFn cavity was observed by TEM imaging, and the valence state of Mn was analyzed by X-ray photoelectron spectroscopy (XPS).

[0077] The results are as follows Figure 8 As shown, multivalent nano manganese oxide Mn was successfully grown in the HFn cavity by in situ growth.

[0078] Experimental analysis (5)

[0079] Female Balb / c nude mice aged 4-6 weeks were selected to construct a glioblastoma U87 tumor model. The tumor-bearing mice were randomly divided into 4 groups and injected with PBS, Exos@siMCT4-HFn, Exos-HFn@Mn and Exos@siMCT4-HFn@Mn through the tail vein, respectively. The drugs were given every 2 days, and small animal bioluminescence imaging was performed on the 10th and 30th days to study the inhibition rate of Exos@siMCT4-HFn, Exos-HFn@Mn and Exos@siMCT4-HFn@Mn on the tumor. The results are shown in Figure 2. Fig. 9 and 10 shown.

[0080] Combination Fig. 9 and Fig.10 The experimental results showed that the tumor inhibition effect of Exos@siMCT4-HFn@Mn was significantly stronger than that of Exos@siMCT4-HFn and Exos-HFn@Mn, and the dual-targeting nanoparticles Exos-HFn could synergistically inhibit the growth of GBM after drug loading.

[0081] The embodiments described above are only preferred implementation modes of the present invention and cannot be used to limit the protection scope of the present invention. Any non-substantial changes and modifications made by those skilled in the art on the basis of the invention shall fall within the protection scope of the present invention.

Claims

1. A nano drug with dual targeting effect, characterized in that: It includes exosomes and ferritin; the surface of the exosomes is modified with an azide group, the surface of the ferritin is modified with a cyclization group, and the exosomes and the ferritin are connected through a click chemistry reaction between the azide group and the cyclization group.

2. The nano drug with dual targeting effect according to claim 1, characterized in that: The ferritin is heavy chain ferritin.

3. The nano drug with dual targeting effect according to claim 2, characterized in that: The particle size of the heavy chain ferritin is 12 nm.

4. The nano drug with dual targeting effect according to claim 1, characterized in that: The exosomes are one or more of M1 microglial exosomes, M1 macrophage exosomes or mesenchymal stem cell exosomes.

5. The nano drug with dual targeting effect according to claim 1, characterized in that: The exosome cavity is loaded with temozolomide, cisplatin or small interfering RNA by electrotransfer.

6. The nano drug with dual targeting effect according to claim 1, characterized in that: The ferritin cavity is loaded with nano-manganese, nano-gold or nano-ferroferric oxide.

7. A method for preparing a nano drug with dual targeting effects according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1, Preparation of exosomes with surface modified with azide groups Adding choline azide to the complete culture medium of the cells, wherein the concentration of choline azide in the culture medium is 100 to 1600 μM, then placing the cells in a constant temperature incubator at a culture temperature of 37° C. and a CO2 concentration of 5% for 24 to 48 hours, then collecting the cell supernatant, and then ultracentrifuging the cell supernatant to obtain exosomes with surface modified with azide groups, wherein the particle size of the exosomes is 30 to 150 nm; S2, preparation of ferritin with surface modified cyclopentadienyl groups adding dibenzocyclooctyne-pentapolyethylene glycol-active ester (DBCO-PEG5-NHS) to the ferritin solution to form a mixed solution 1, wherein the molar ratio of ferritin to DBCO-PEG5-NHS is 1:12 to 1:120; Subsequently, the mixed solution 1 is placed in a vertical suspension instrument at 20 r / min for vertical suspension at 25-37° C. for 1 h to 6 h, and then ultrafiltration centrifugation is performed using a 10 kDa ultrafiltration tube to remove unreacted DBCO-PEG5-NHS in the mixed solution 1, thereby obtaining ferritin with a surface modified with a cyclopentane group; S3, Preparation of nanoparticles with dual targeting effects The exosomes modified with azide groups on the surface and the ferritin modified with cyclopentadiene groups on the surface are mixed together to form a second mixed solution, wherein the ratio of the number of the exosomes modified with azide groups on the surface to the number of the ferritin modified with cyclopentadiene groups on the surface is 1:10 to 1:100; Subsequently, the mixed solution was placed in a vertical suspension instrument at 25-37° C. and vertically suspended at a speed of 20 r / min for 2 h to 6 h, and then centrifuged to obtain nanoparticles with dual targeting effects.

8. The preparation method according to claim 7, characterized in that: The cells in step 1 are M1 microglial cells HMC3.

9. Use of a nano drug in the preparation of a preparation for treating glioblastoma, characterized in that: Use the nano drug as described in any one of claims 1-6.

10. A drug delivery system, characterized in that: Use the nano drug described in any of claims 1-6.

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