A macrophage membrane-coated bionic DNA nanomedicine and its preparation method and application

Abstract

The present invention belongs to the field of nucleic acid drug loading and drug delivery, and relates to a bionic DNA nanodrug, in particular to a macrophage membrane-coated bionic DNA nanodrug and its preparation method and application. The bionic DNA nanodrug includes a drug-loaded DNA nanocage, cholesterol-modified TNF‑α siRNA and a macrophage membrane, wherein the drug-loaded DNA nanocage is coated with a macrophage membrane, and the cholesterol-modified TNF‑α siRNA is inserted on the surface of the macrophage membrane. The bionic DNA nanodrug obtained by the present invention utilizes two nucleic acid drugs to jointly regulate the TNF‑α and NF‑κB inflammatory pathways, significantly downregulates the expression levels of inflammatory factors and their transcriptional genes in inflammatory macrophages and adjuvant-induced arthritis mice, reduces the clinical arthritis index and improves joint damage, and is a new strategy for the combined treatment of rheumatoid arthritis.

Description

Technical Field

[0001] The invention belongs to the field of nucleic acid drug loading and drug delivery, and relates to a bionic DNA nano drug, in particular to a macrophage membrane-coated bionic DNA nano drug and a preparation method and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease that is currently difficult to cure. In the past two decades, a series of anti-cytokine biologics have been successfully marketed and applied, greatly improving anti-RA treatment. Unlike traditional anti-RA therapeutic drugs, biologics are mostly monoclonal antibodies, which can neutralize inflammatory cytokines in RA patients after injection, inhibit inflammatory reactions, and reduce inflammation levels. Tumor necrosis factor-α (TNF-α) is one of the most critical inflammatory cytokines in the pathological process of RA, and anti-TNF-α monoclonal antibodies are also the most effective class of biologics. However, anti-TNF-α biologics are only effective for no more than half of the patients, which is related to the high heterogeneity of RA disease, indicating that TNF-α is not the most critical factor causing RA in patients with poor efficacy; this is also related to the poor stability and poor targeting of monoclonal antibodies themselves, which make it difficult to reach the inflamed joint tissue in an effective form; in addition, after injection, anti-TNF-α biologics widely inhibit TNF-α in the body, which is easy to induce infection and malignant tumors. Therefore, there is an urgent need for optimized treatment strategies based on anti-TNF-α to enhance the efficacy and reduce the toxicity of anti-TNF-α therapy.

[0003] The pathological mechanism of RA is relatively complex, involving the interaction of multiple immune cells and somatic cells, accompanied by the disorder and dysregulation of many inflammatory pathways. Among them, the NF-κB pathway is one of the most important inflammatory pathways in the occurrence and development of RA. Inhibition of the NF-κB pathway can downregulate the expression of multiple inflammatory cytokines, chemokines, and adhesion factors, and is a highly effective therapeutic strategy to inhibit inflammation. Combined with the above-mentioned anti-TNF-α therapy, it is expected to overcome the heterogeneity of RA, reduce the dose of anti-TNF-α therapy, and improve the general effectiveness of the therapy.

[0004] Compared with monoclonal antibodies, nucleic acid drugs have lower production costs and weaker immunogenicity, making them an ideal alternative to monoclonal antibodies. For example, after small interfering RNA (siRNA) targeting TNF-α enters cells, it binds to the multiprotein complex in the cytoplasm to form an RNA-induced silencing complex (RISC), causing the mRNA that translates TNF-α to be degraded by RISC endonucleases, resulting in the silencing of TNF-α translation genes and downregulating TNF-α expression. After entering cells, NF-κB decoy oligodeoxyribonucleotides (NF-κB decoy dODNs) can specifically bind to the P50 / P65 complex of NF-κB, blocking NF-κB from entering the cell nucleus, inhibiting the expression of multiple inflammatory cytokines, chemokines, and adhesion factors, and reducing inflammatory responses. The combination of siRNA targeting TNF-α and NF-κB decoy dODNs is expected to enhance the efficacy of anti-TNF-α therapy by inhibiting the NF-κB pathway. However, similar to monoclonal antibodies, TNF-α siRNA and NF-κB decoy dODNs have poor in vivo stability, targeting, and low intracellular efficiency, which limits their in vivo application and co-delivery.

[0005] In order to solve the difficult-to-overcome technical problems in the application and delivery of NF-κB decoy dODNs and TNF-α siRNA, our research group conducted long-term research and exploration. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a macrophage membrane-coated bionic DNA nanomedicine and a preparation method and application thereof.

[0007] The technical solution of the present invention is achieved in this way:

[0008] A macrophage membrane-coated bionic DNA nanomedicine comprises a drug-loaded DNA nanocage, cholesterol-modified TNF-α siRNA and a macrophage membrane, wherein the drug-loaded DNA nanocage is coated by the macrophage membrane, the cholesterol-modified TNF-α siRNA is inserted into the surface of the macrophage membrane, and the macrophage membrane is derived from RAW 264.7 macrophages.

[0009] This application is the first to use a combination of NF-κB inhibitors and TNF-α silencers to block inflammatory responses and treat RA, and uses biofilm coating to enhance their targeting to inflamed joint tissues in vivo and their anti-RA efficacy.

[0010] The DNA nanocage of the present invention has the advantages of good biocompatibility, highly controllable structure and easy synthesis, and can accurately load nucleic acid drugs through base complementary pairing, while having the advantages of small and uniform particle size.

[0011] The present invention relates to a method for preparing a macrophage membrane-coated bionic DNA nanomedicine, the steps of which are as follows:

[0012] (1) Extract RAW264.7 macrophage membrane to obtain pure cell membrane:

[0013] The specific operation is as follows: the harvested macrophages are divided into 2-3×10 7 The cells were resuspended in TM (Tris∙HCl and MgCl2) buffer solution at a concentration of 100 / mL and homogenized at 20000 rpm in a homogenizer at 4°C for 1 min. The cell homogenate was then mixed with 1 M sucrose solution to a final concentration of 0.25 M sucrose solution, centrifuged at 2000 g and 4°C for 10 min, and the supernatant was centrifuged at 3000 g and 4°C for 30 min. The cell membrane was washed with ice-cold TM buffer solution containing 0.25 M sucrose and centrifuged at 3000 g and 4°C for 30 min. The obtained macrophage membrane was resuspended in PBS and passed through porous polycarbonate membranes with pore sizes of 1 µm, 400 nm, and 200 nm in sequence to obtain macrophage membrane vesicles by physical extrusion.

[0014] (2) Assemble the DNA nanocage through a one-step one-pot annealing process. The cube is composed of 8 short chains and 5 long chains.

[0015] The specific operation is as follows: First, 100 pmol of each long chain and 200 pmol of each short chain are placed in a PCR tube. Second, 10 µL of 10×TAMg (Tris and MgCl2) buffer is added to the PCR tube, and then ultrapure water is added to make the final volume of the solution reach 100 µL (1×TAMg). Again, vortex the PCR tube to mix evenly, place it in a PCR instrument, heat the sample to 95°C, cool to 80°C within 5 min, and then cool to 4°C at a rate of -1°C / 5 min (6.5 h). The final concentration of the DNA nanocage sample prepared in this protocol is 1 µM;

[0016] (3) NF-κB decoy dODNs were connected to the DNA nanocage through an extended chain (8 thymines) to prepare Cage-dODN;

[0017] (5) Dissolve the 3'-end cholesterol-modified TNF-α siRNA positive strand (3'-Chol TNF-α siRNA) in enzyme-free sterile water to a final concentration of 20 µM;

[0018] (6) The macrophage membrane obtained in step (1) was incubated with the 3'-Chol TNF-α siRNA in (5) and then mixed with the Cage-dODN in step (3) at a mass ratio of 20:1 (the final concentration of dODN was 1 µM and the final concentration of siRNA was 100 nM). The macrophage membrane-coated biomimetic DNA nanomedicine was successfully prepared by ice-bath sonication at 42 kHz / 100 W / 5 min.

[0019] The above two nucleic acid drugs are NF-κB decoy dODNs, whose sequences are: the sense chain nucleotide sequence is shown in SEQ ID No.1, and the antisense chain nucleotide sequence is shown in SEQ ID No.2; the sense chain nucleotide sequence of TNF-α siRNA is shown in SEQ ID No.3, and the antisense chain nucleotide sequence is shown in SEQ ID No.4, and the 2' position of the nucleotide ribose groups at positions 8, 10, 12, 14, 16, 18, 20, 22, 24, 25 and 26 is methylated.

[0020] The present invention has the following beneficial effects:

[0021] 1. The present invention uses macrophage membrane-coated DNA nanocages as carriers to load two nucleic acid drugs, thereby improving the stability of nucleic acid drugs and utilizing cell membrane coating to achieve their effective accumulation at inflammatory sites. After inflammatory cells were treated with siRNA@M (Cage-dODN), the expression of TNF-α and IL-6 in the cells was significantly downregulated, which is consistent with the trend of the potent anti-inflammatory drug dexamethasone (Dex). siRNA@M (Cage-dODN) can also effectively control the symptoms of adjuvant induced arthritis (AIA) mice, which is comparable to the potent anti-inflammatory drug DEX, indicating that the drug has good anti-inflammatory efficacy both in vivo and in vitro.

[0022] 2. This invention adopts the biomimetic strategy based on cell membrane-coated DNA nanomedicine for the first time to improve the stability and inflammation targeting of DNA nanomedicine; siRNA@M (Cage-dODN) can accumulate at high concentrations in the hind feet of mice. The in vivo distribution results show that free Cy5.5 accumulates less in the hind feet of mice; DNA Cage can accumulate in the hind feet of mice, but the concentration is not high, and it may be difficult to reach an effective concentration in actual use; siRNA@M (Cage-dODN) can accumulate at high concentrations in the inflamed hind feet of mice, and has strong inflammation targeting.

[0023] 3. The present invention adopts a dual inflammatory pathway blocking strategy to treat rheumatoid arthritis, proving the high efficiency of dual pathway blocking in treating rheumatoid arthritis, and is a new strategy for anti-inflammatory treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 Schematic diagram of the preparation of DNA nanocage (DNA Cage) and Cage-dODN.

[0026] Figure 2 Schematic diagram of the preparation of bionic DNA nanomedicine.

[0027] Figure 3 The following are the 6% polyacrylamide gel electrophoresis characterization results of (A) DNA Cage (lane 1: 50 bp DNA Marker; lane 2: PP1; lane 3: PP1+PP2; lane 4: PP1+PP2+PP3; lane 5: PP1+PP2+PP3+PP4; lane 6: 5PP; lane 7: 5PP+4X; lane 8: DNA Cage), (B) NF-κB decoy dODNs (lane 1: 50 bp DNA Marker; lane 2: ODN-sense chain; lane 3: ODN-antisense chain; lane 4: dODN), and (C) Cage-dODN (lane 1: Cage; lane 2: Cage-ODN-sense chain; lane 3: Cage-dODN).

[0028] Figure 4 The 6% polyacrylamide gel electrophoresis characterization results of siRNA@M(Cage-dODN) (lane 1: 50 bp DNA Marker; lane 2: MMV; lane 3: 5PP; lane 4: DNA Cage; lane 5: Cage-ODN-sense chain; lane 6: Cage-dODN; lane 7: siRNA@M(Cage-dODN)).

[0029] Figure 5The results of characterizing the membrane protein composition of siRNA@M(Cage-dODN) using sodium dodecyl sulfate-polyacrylamide gel (lane 1: total macrophage protein; lane 2: protein ladder; lane 3: macrophage membrane; lane 4: siRNA@M(Cage-dODN)).

[0030] Figure 6 (A) Agarose gel electrophoresis of DNA Cage after storage at 4°C for different time periods and (B) Agarose gel electrophoresis of DNA Cage after incubation with 10% (v / v) fetal bovine serum at 37°C for different time periods.

[0031] Figure 7 Denaturing urea-polyacrylamide gel electrophoresis of siRNA@M(Cage-dODN) after incubation with 10% (v / v) fetal bovine serum at 37 °C for different time periods (lane 1: Cage-dODN, lane 2: TNF-α siRNA, lanes 3-9: siRNA@M(Cage-dODN) after incubation with fetal bovine serum for 0 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h, respectively).

[0032] Figure 8 (A) Particle size and (B) Zeta potential of macrophage membrane vesicles (MMV), DNA Cage, and siRNA@M (Cage-dODN).

[0033] Fig. 9 This is a transmission electron photo of siRNA@M(Cage-dODN).

[0034] Fig.10 The cytotoxicity graphs of (A) TAMg buffer, (B) DNA Cage, and (C) siRNA@M (Cage-dODN) on RAW 264.7 macrophages.

[0035] Fig.11 Effects of different control groups and siRNA@M (Cage-dODN) on the gene expression levels of TNF-α and IL-1β in inflammatory RAW 264.7 macrophages (Compared with the LPS group, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns P > 0.05, N=3).

[0036] Fig.12Effects of different control groups and siRNA@M (Cage-dODN) on the expression of TNF-α and IL-6 in inflammatory RAW 264.7 cells (Compared with the LPS group, ****P < 0.0001, ***P < 0.001, **P < 0.01, N=3).

[0037] Fig.13 Photos of inflammatory RAW 264.7 cells taking up single-stranded DNA, DNA Cage, and siRNA@M (Cage-dODN).

[0038] Fig.14 Flow cytometry graphs showing the uptake of single-stranded DNA, DNA Cage, and siRNA@M (Cage-dODN) by inflammatory RAW 264.7 cells at (A) 2 hours and (B) 4 hours, respectively.

[0039] Fig.15 (A) Photos of the feet of mice with adjuvant-induced arthritis (AIA) 12 days after modeling, and (B) Photos of the feet of healthy mice.

[0040] Fig.16 (A) Schematic diagram of AIA mouse modeling and treatment plan, (B) photos of the hind feet of each group of mice after the treatment, (C) changes in the body weight of mice during AIA mouse modeling and treatment, (D) changes in the foot volume of each group of mice during treatment, (E) thickness of the hind feet of each group of mice after the treatment, and (F) changes in the arthritis index score of AIA mice during treatment.

[0041] Fig.17 Figure 2 shows the gene expression levels of (A) TNF-α and (B) IL-6 in the ankle joints of mice in each group after treatment (Compared with the LPS group, ****P < 0.0001, ***P < 0.001, **P < 0.01, N=3)

[0042] Fig.18 Figure 2 shows the levels of (A) TNF-α and (B) IL-6 in the serum of mice in each group after treatment (Compared with the LPS group, **P < 0.01, *P < 0.05, ns P > 0.05, N=3).

[0043] Fig.19 H&E stained pathological sections of ankle joints of mice in each group.

[0044] Fig. 20 The blood routine test results of mice in each group after treatment. (A) Blood cell count (red blood cells, white blood cells, platelets), (B) White blood cell count (monocytes, lymphocytes, granulocytes), (C) Hemoglobin concentration (hemoglobin, mean hemoglobin concentration).

[0045] Fig.21 Photos showing the distribution of DNA Cage and siRNA@M (Cage-dODN) in mice within 24 hours. DETAILED DESCRIPTION

[0046] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Before describing the present invention, the terms used in this article are defined as follows:

[0048] The term "siRNA" refers to: Small interfering RNA.

[0049] The term "dODN" refers to: NF-κB decoy oligodeoxyribonucleotide, double-stranded DNA.

[0050] The term "Cage" refers to: DNA nanocage, a self-assembled 5-prism hollow three-dimensional nucleic acid carrier.

[0051] The term "Cage-dODN" refers to: a DNA nanocage loaded with dODN.

[0052] The term "siRNA@M(Cage-dODN)" refers to a biomimetic DNA nanomedicine in which the macrophage membrane is loaded with TNF-α siRNA and coated with Cage-dODN. Example

[0053] 1. The preparation method of the macrophage membrane-coated biomimetic DNA nanomedicine of this embodiment comprises the following steps:

[0054] 1. Preparation of macrophage membrane: RAW 264.7 macrophages were harvested according to the cell culture method and cultured at 2.5×10 7 Resuspend in Tris-Mg 2+The cells were broken at 4 °C, and the cell fragments were homogenized in a homogenizer at 20,000 rpm for 1 min. The cell homogenate was then mixed with 1 M sucrose to a final concentration of 0.25 M sucrose and centrifuged at 2000 g and 4 °C for 10 min. The supernatant was then centrifuged at 3000 g and 4 °C for 30 min and washed with ice-cold Tris-MgSO containing 0.25 M sucrose. 2+ The cell membrane was washed with buffer and centrifuged at 3000 g and 4 °C for 30 min. The obtained macrophage membrane was resuspended in PBS and passed through porous polycarbonate membranes with pores of 1 μm, 400 nm, and 200 nm, respectively, to form macrophage membrane vesicles by physical extrusion.

[0055] 2. Preparation of DNA nanocages: DNA nanocages were assembled by a one-step one-pot annealing process. The nanocages consisted of 8 short chains and 5 long chains, as shown in the table below:

[0056] DNA nanocage-related nucleotide sequences

[0057]

[0058] The m in the above TNF-α siRNA antisense strand indicates that the 2' position of the ribose group of the nucleotide is methylated, the purpose of which is to increase the stability of the sequence without affecting the complementary base pairing.

[0059] First, add 100 pmol of each long chain and 200 pmol of each short chain to the PCR tube. Second, add 10 µL of 10×TAMg buffer to the PCR tube, and then add ultrapure water to make the final volume of the solution reach 100 µL (1×TAMg). Third, vortex the PCR tube to mix the solution evenly, place it in a PCR instrument, heat the sample to 95 °C, cool it to 80 °C within 5 min, and then cool it to 4 °C at a rate of -1 °C / 5 min (6.5 h) for annealing. The process of preparing DNA nanocages in this protocol is as follows Figure 1 As shown, the final concentration of the prepared DNA nanocages was 1 µM.

[0060] 3. Preparation of siRNA@M (Cage-dODN): First, extend the ends of X1, X3, X6, and X8 by 8 thymines (T), and then connect a single-stranded sequence of dODN. Use these four chains to replace X1, X3, X6, and X8 in Cage synthesis, respectively. Use the same method to synthesize Cage-dODN-sense chain. Mix the Cage-dODN-sense chain and the dODN-antisense chain in a molar ratio of 1:4, dilute with TM (Tris∙HCl and MgCl2) buffer to a final concentration of 0.5 μM, and vortex at a low speed until mixed evenly. In a PCR instrument, cool from 80 ℃ to 25 ℃ for 2 h to obtain Cage-dODN. Its preparation process is as follows: Figure 1 As shown; secondly, the cholesterol-modified TNF-α siRNA (3'-Chol TNF-α siRNA) was dissolved in sterile water with a final concentration of 20 µM. The obtained macrophage membrane was mixed with 3'-Chol TNF-α siRNA in a certain ratio and incubated to prepare siRNA@M. Finally, siRNA@M and Cage-dODN were mixed at a mass ratio of 20:1 (the final concentration of dODN was 1 µM, and the final concentration of siRNA was 100 nM), and the macrophage membrane-coated biomimetic DNA nanomedicine was successfully prepared by ice bath ultrasound (42 KHz, 100 W, 5 min). The preparation process is shown as follows Figure 2 shown.

[0061] 2. The characterization method of the macrophage membrane-coated biomimetic DNA nanomedicine of this embodiment is as follows:

[0062] 1. Characterization of the structures of Cage, Cage-dODN, and siRNA@M (Cage-dODN) by 6% polyacrylamide gel electrophoresis (PAGE): Prepare 6% polyacrylamide gel, add 5 μL of DNA Cage, Cage-dODN, and siRNA@M (Cage-dODN) to the sample wells, respectively, use 1×TAMg as the electrophoresis solution, set the electrophoresis voltage to 250 V, and run the electrophoresis for 30 min. After the electrophoresis, remove the gel, stain it, and analyze it using a gel imager.

[0063] Figure 3 Lanes 1 to 8 in A correspond to the annotated sequences or structures, respectively. As the molecular weight of the structure increases, the band migration rate slows down, indicating successful self-assembly between DNA chains. Figure 3 Lane 2 and lane 3 in B are two complementary single strands of dODN, respectively. After base complementary pairing, the dODN in lane 4 is obtained, and its migration rate is significantly slowed down, indicating the successful synthesis of dODN. Figure 3 In C, lane 1 is a DNA nanocage, lane 2 is Cage-dODN-sense chain with only one of the dODN chains, and lane 3 is Cage-dODN loaded with complete dODN. As the molecular weight of the DNA structure increases, its mobility slows down, indicating the successful self-assembly of the structure. Figure 4 The difference in the migration rate of different DNA structures in the gel is shown again. As shown in lane 7, after being coated by MMV, there is no free Cage-dODN, and the corresponding electrophoresis band of Cage-dODN disappears in the gel, indicating that Cage-dODN is successfully coated by MMV.

[0064] 2. Characterization of RAW 264.7 whole protein, MMV protein, and siRNA@M (Cage-dODN) protein by 10% sodium dodecyl sulfate (SDS)-PAGE: Prepare SDS-polyacrylamide gel consisting of 10% separation gel and 5% stacking gel, add 20 µg of RAW 264.7 whole protein, MMV protein, and siRNA@M (Cage-dODN) protein to the sample wells, and first perform electrophoresis at 80 V until the sample is at the junction of the stacking gel and the separation gel, then adjust the voltage to 100 V, and perform electrophoresis for about 60 min. After the electrophoresis, stain with Coomassie brilliant blue dye, then add 10% acetic acid decolorizing solution for decolorization, replace the decolorizing solution in time until the bands are clear, and analyze with a gel imager.

[0065] like Figure 5 As shown, lane 1 is the whole protein of RAW 264.7 macrophages, lane 3 is the membrane protein of extracted MMV, and lane 4 is the membrane protein of siRNA@M(Cage-dODN). The results show that the membrane proteins of extracted macrophages and siRNA@M(Cage-dODN) have similar protein spectra with the whole protein of RAW 264.7 macrophages, indicating that the main cell membrane proteins of macrophages are retained during the preparation process.

[0066] 3. Characterization of in vitro stability by agarose gel electrophoresis: The prepared Cage was mixed with an equal volume of 20% fetal bovine serum, and incubated at 4°C and 37°C for 0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h, respectively. Then, 4% agarose gel, 1×TAMg electrophoresis buffer, voltage 100 V, and ice bath electrophoresis for 1.5 h were used to characterize the incubated Cage. Figure 6 As shown in A, at 4 °C, within 48 h, the electrophoresis bands of Cage were relatively clear and in constant position, indicating good stability; Figure 6B shows that at 37 °C, after 8 h of incubation, the electrophoretic band began to move forward, indicating that the structure of the Cage was gradually degraded. The prepared siRNA@M (Cage-dODN) was mixed with an equal volume of 20% fetal bovine serum, incubated at 37 °C for 0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h, and then mixed with 3× formamide loading buffer at a ratio of 1:2 (v / v). 15% denatured urea PAGE was used to characterize the incubated siRNA@M (Cage-dODN). After Cage-dODN was coated with the cell membrane, as shown in Figure 7 As shown in the figure, under the same incubation conditions, no corresponding electrophoretic bands appeared within 24 h, indicating that Cage-dODN can be stably coated by the cell membrane.

[0067] 4. Particle size and potential characterization by particle size potentiometer: Take appropriate amount of MMV, Cage, siRNA@M (Cage-dODN) solution, dilute with ultrapure water, disperse by ultrasound, and place in a measuring cup. Use particle size potentiometer to measure the particle size and Zeta potential of each group. The results are shown in Figure 8 As shown in the figure, the hydrated particle size of MMV is 205.4±6.789 nm, and the Zeta potential is -15.970±0.513 mV; the hydrated particle size of Cage is 11.84±2.575 nm, and the Zeta potential is -4.370±1.555 mV; the particle size of siRNA@M(Cage-dODN) is 63.14±15.70 nm, and the Zeta potential is -20.70±3.843 mV. After MMV is coated with Cage-dODN, the particle size becomes smaller and the surface potential increases, indicating that more stable nanoparticles are formed.

[0068] 5. Characterization of the morphology of siRNA@M (Cage-dODN) by transmission electron microscopy: Take an appropriate amount of siRNA@M (Cage-dODN) solution, dilute it with ultrapure water, use ultrasonic dispersion, and drop a small amount of the solution onto the copper mesh with a support membrane. Let it stand for 5 minutes, and use filter paper to absorb the excess liquid on the edge of the copper mesh. After sufficient drying, observe the particle size and morphology of siRNA@M (Cage-dODN) under a transmission electron microscope. The results are as follows Fig. 9 As shown in Figure 2, the particle size of siRNA@M(Cage-dODN) is about 50 nm, which is similar to Figure 8 The results were consistent. The shape of siRNA@M(Cage-dODN) was spherical or quasi-spherical, with relatively uniform size and good dispersion.

[0069] Application Examples

[0070] 1. In vitro studies

[0071] 1. Cytotoxicity study: Raw 264.7 cells were cultured at 1.5×10 4 The cells were inoculated in a 96-well plate at a density of 100 μL of cell culture medium per well. The plates were cultured in an incubator at 37 °C and 5% CO2 for 24 h. The old culture medium was discarded and 100 μL of cell culture medium containing different concentrations of TAMg buffer, Cage or siRNA@M (Cage-dODN) was added respectively. Five replicate wells were set for each concentration, and a zero adjustment group and a blank control group were also set. The plates were cultured in an incubator for 24 h, and then the old culture medium was discarded. Under light-proof conditions, 200 μL of culture medium containing CCK-8 (5%, v / v) was added to each well. The plates were cultured for another 4 h. The absorbance values ​​of different groups at 450 nm were detected by an enzyme-labeled instrument, and the inhibition rates of different concentrations of TAMg buffer, Cage or siRNA@M (Cage-dODN) on the cells were calculated. The results are shown in Fig.10 As shown in the data, Cage had no significant inhibitory effect on RAW 264.7 cells at all tested concentrations, while TAMg buffer and siRNA@M (Cage-dODN) had some inhibitory effect on cells with increasing concentrations. However, the concentrations of TAMg buffer and siRNA@M (Cage-dODN) used in the study were low, had little effect on cell proliferation, and had better safety.

[0072] 2. Study on anti-inflammatory efficacy at the cellular gene level: RAW 264.7 cells were inoculated in 6-well plates and cultured in an incubator at 37°C and 5% CO2 for 24 h. The old culture medium was discarded and fresh culture medium containing 1 µg / mL bacterial lipopolysaccharide (LPS) was added and cultured for another 24 h to establish an inflammatory cell model. Different wells were divided into LPS stimulation model group, dexamethasone (Dex) group, free dODN group, free TNF-α siRNA group, and siRNA@M (Cage-dODN) group. A blank control group (normal cells) was also set up. Then the old culture medium was discarded, and 2 mL of fresh culture medium was added to the blank control group and LPS stimulation model group. 2 mL of culture medium containing the corresponding drugs was added to each well, so that the final concentration of dODN was 1 μM, the final concentration of TNF-α siRNA was 100 nM, the final concentration of siRNA@M(Cage-dODN) was 250 nM (corresponding to 1 μM dODN and 100 nM TNF-α siRNA), and the final concentration of Dex was 0.1 mg / mL. After incubation for 24 h, the expression levels of TNF-α mRNA and IL-1β mRNA in the cells were detected by qRT-PCR. The results are shown in Figure 2. Fig.11As shown, after inflammatory cells were treated with siRNA@M(Cage-dODN), the gene expression of TNF-α and IL-1β in the cells was significantly downregulated, which was consistent with the trend of the potent anti-inflammatory drug Dex.

[0073] 3. Study on anti-inflammatory efficacy at the cellular protein level: Raw264.7 cells were cultured at 1.5×10 4 The cells were inoculated into a 96-well plate at a density of 100 μL of culture medium per well, and cultured in an incubator at 37 °C and 5% CO2 for 24 h. The same modeling method and experimental grouping were used, with 3 replicate wells in each group. After the experiment, the cell supernatant was collected and centrifuged at 300 g for 10 min. The supernatant was collected and the ELISA kit was used to detect the content of IL-6 and TNF-α in the cell supernatant. The results are shown in Fig.12 As shown, after inflammatory cells were treated with siRNA@M(Cage-dODN), the expression of TNF-α and IL-6 in the cells was significantly downregulated, which was consistent with the trend of the potent anti-inflammatory drug Dex.

[0074] 4. Qualitative study of cellular uptake: Raw264.7 cells were cultured at 1.0×10 5 The cells were inoculated at a density of 100 μL / well in a 24-well plate with a cell slide, with each well containing 500 μL of culture medium, and cultured in an incubator at 37 °C and 5% CO2 for 24 h. A similar modeling method was used to establish an inflammatory cell model. DNA chains or structures were labeled with FITC fluorescence, and free FITC-DNA chains, FITC-DNA Cage, and FITC-siRNA@M (Cage-dODN) were added to different wells, with a final concentration of 250 nM. After incubation for 0.5 h, 1 h, 2 h, or 4 h, the drug-containing culture medium was discarded, and 500 μL of pre-cooled PBS buffer was added to each well to terminate the cell uptake of drugs. After washing the cells twice with PBS buffer, 400 μL of 4% paraformaldehyde solution was added to the cells, and the cells were incubated for 10 min to fix the cells. The paraformaldehyde was discarded, and after washing twice with PBS buffer, the slides were removed from the culture plate and covered on a slide with anti-fluorescence quenching blocking solution. The slides were sealed along the edges with nail polish and placed under a confocal microscope to observe the cell uptake of each group of drugs. The results are shown in Figure 2. Fig.13 As shown, single-stranded DNA is hardly taken up by cells, and the green fluorescence signal in the cells is weak; the cell uptake of Cage increases slightly over time, with partial green fluorescence; and the cell uptake of siRNA@M (Cage-dODN) is significantly higher than that of other groups, with strong green fluorescence at different time points, indicating that macrophage membrane coating can significantly enhance the macrophage uptake of the preparation.

[0075] 5. Quantitative study of cell uptake: Using the experimental method in 4, the 6-well plate was taken out after 2 h and 4 h of culture, the drug-containing culture medium was discarded, and 1 mL of PBS buffer was added to each well to wash the cells each time, and washed three times in total to terminate cell uptake and remove free drugs; then 500 µL of trypsin was added to each well, and the plate wall was gently tapped until the cells became round and were about to fall off, and 1 mL of complete culture medium was immediately added to terminate digestion, and the cells were collected with a cell spatula and centrifuged at 1000 rpm for 5 min; the obtained cell pellet was gently washed twice with PBS, and finally 200 µL of PBS was added to mix the cells, and the cell uptake of free FITC-DNA chains, FITC-DNA Cage, and FITC-siRNA@M (Cage-dODN) was detected by flow cytometry. The results are shown in Figure 2. Fig.14 The cells shown in the figure have a small uptake of free FITC-DNA chains, a certain uptake of Cage, and the highest uptake of siRNA@M (Cage-dODN), which is consistent with Fig.13 The qualitative uptake results were consistent with those in

[0076] 2. In vivo studies

[0077] 1. Establishment of AIA model: Purchase male Kunming mice, and allow them to eat and drink freely for one week. Then, disinfect the toes of the mice with 75% alcohol, and then inject 80 µL of complete Freund's adjuvant subcutaneously into the plantar part of the right hind paw of the mice. At the same time, inject the same volume of saline into the blank control group. The day of injection is recorded as day 0. One week later, inject 40 µL of complete Freund's adjuvant into the plantar part of the right paw of the mice as a booster dose. After the injection, observe the redness, swelling, stiffness and physiological state of the hind paw of the mice every other day. Fig.15 As shown, on the 12th day of modeling, the hind feet of mice in the model group were significantly swollen and stiff, and the glossiness of their hair was reduced compared with those in the healthy control group.

[0078] 2. In vivo anti-inflammatory effect study: The AIA mice with successful modeling were randomly divided into five groups, namely the model control group, Dex treatment group, dODN treatment group, TNF-α siRNA treatment group, siRNA@M (Cage-dODN) treatment group, and a healthy control group. The dosage of the dODN group was 0.12 mg / mouse, the dosage of the siRNA group was 0.02 mg / mouse, the dosage of the siRNA@M (Cage-dODN) group was 0.55 mg / mouse (containing dODN 0.12 mg, siRNA 0.02 mg), and the dosage of the Dex group was 0.02 mg / mouse. The healthy control group and the model control group were injected with the same volume of normal saline, administered every other day, 200 µL each time, for a total of 6 times (administered on the 13th, 15th, 17th, 19th, 21st, and 23rd days respectively). The experiment was terminated on the 24th day, and samples were collected for biological evaluation. The mice were dissected from day 0 to day 24 of modeling, and their body weights were monitored every other day. The thickness of the hind toes was measured with a vernier caliper, and the swelling of the toes was measured with a toe volume meter. The scores were scored according to the following scoring table, and the hind foot scores of each mouse were combined, with a maximum of 8 points.

[0079]

[0080] like Fig.16 As shown in the figure, after the treatment, the swelling of the toes of mice in each treatment group was reduced compared with that in the model control group, among which the Dex treatment group and the siRNA@M(Cage-dODN) treatment group had the largest reduction. During the experiment, the weight of mice generally increased, but the weight of mice in each treatment group and the model group fluctuated. This may be because AIA affected the physiological state of mice. After the treatment, the weight of the siRNA@M(Cage-dODN) group was closest to that of normal mice, indicating that siRNA@M(Cage-dODN) treatment had the least effect on the weight of mice and was relatively safe. Fig.16 D shows that during the modeling process from day 0 to day 12, the swelling of the mouse toes increased steadily. After the treatment every other day starting from day 13, the swelling of the toes of each treatment group gradually decreased, among which the Dex treatment group and the siRNA@M (Cage-dODN) treatment group decreased the fastest. Fig.16 E and Fig.16 F shows that the Dex-treated group and the siRNA@M(Cage-dODN)-treated group had the smallest foot thickness and the lowest joint score. These data indicate that siRNA@M(Cage-dODN) can effectively control the symptoms of AIA in mice, which is comparable to the potent anti-inflammatory drug Dex.

[0081] 3. Study on in vivo anti-inflammatory biochemical indicators

[0082] 1) Determination of ankle joint inflammatory factor mRNA levels: After the administration was completed on the 24th day, the hind ankle joints of the mice were collected and stored in RNAsolid tissue RNA stabilization solution. The total RNA in the ankle joints of the mice was extracted using an RNA extraction kit, and the expression levels of TNF-α mRNA and IL-1β mRNA were detected by qRT-PCR. The results are shown in Fig.17 As shown, after treatment with siRNA@M(Cage-dODN), the gene expression of TNF-α and IL-6 in the joints was significantly downregulated, which was consistent with the trend of the potent anti-inflammatory drug Dex.

[0083] 2) Determination of inflammatory factors in serum: After administration on the 24th day, 0.5 mL of mouse whole blood sample was collected by eyeball blood sampling and placed in a heparin-rinsed centrifuge tube. The sample was centrifuged at 4000 rpm for 15 min, and serum was collected. The ELISA method was used to detect the concentrations of TNF-α and IL-6 in the serum. The results are shown in Figure 2. Fig.18 As shown, after treatment with siRNA@M(Cage-dODN), the levels of TNF-α and IL-6 in serum were significantly downregulated, which is consistent with the trend of the potent anti-inflammatory drug Dex.

[0084] 4. In vivo anti-inflammatory pathological study: After the mice were killed, the hind feet of the mice were fixed with 4% paraformaldehyde, and then the paws were peeled and soaked in 10% EDTA solution for decalcification. After rinsing, they were dehydrated, waxed, embedded and sliced. The slice direction was as consistent as possible and the integrity of the tissue was ensured. Finally, hematoxylin and eosin (H&E stain) was used to complete the slice preparation. The joint slices of each group of mice were observed under a microscope. Fig.19 As shown, tissue debris was visible in the joint cavity of the model group, Dex group, dODN group and siRNA group, while the joint surface of the siRNA@M (Cage-dODN) group was smooth and there was no foreign matter in the joint cavity, which was closest to the healthy control group.

[0085] 5. In vivo safety evaluation: After the administration on the 24th day, an appropriate amount of mouse whole blood was collected by eyeball blood sampling into EDTA anticoagulant tubes. Three mice were randomly selected from each group for routine blood tests to examine the number of blood cells, white blood cells, hemoglobin concentration, etc. to preliminarily evaluate the in vivo safety of the drug. Fig. 20 As shown in the results, siRNA@M (Cage-dODN) had no significant effect on the detected indicators, which were close to the values ​​of the healthy control group, and the differences between different mice in the group were small. However, the powerful anti-inflammatory drug Dex had a certain effect on the routine blood indicators of mice, significantly reducing the number of granulocytes in the blood of mice, and the differences between different mice in the group were large. In actual treatment, some potential toxic side effects may occur.

[0086] 6. In vivo distribution study: AIA mice were randomly divided into 3 groups, 3 mice in each group, and the drugs in each group were diluted with physiological saline to a Cy5.5 concentration of 15 μM. 160 μL of free Cy5.5 (Free Cy5.5), DNA Cage (DNACage-Cy5.5) and siRNA@M(Cage-dODN) (siRNA@M(Cage-dODN)-Cy5.5) were injected intravenously, and the dynamic fluorescence distribution in the mice was observed by in vivo imaging at 1 h, 4 h, 8 h, 12 h, and 24 h. The results are shown in Figure 2. Fig.21 As shown in the figure, after injection, siRNA@M(Cage-dODN) can reach the hind feet of mice in large quantities and quickly. Within 1 to 12 hours, the fluorescence intensity of the hind feet of mice is always strong, indicating that siRNA@M(Cage-dODN) can accumulate at high concentrations in the hind feet of mice. Free Cy5.5 accumulates less in the hind feet of mice, and DNA Cage can accumulate in the hind feet of mice, but the concentration is always low, and it may be difficult to reach an effective concentration in actual treatment. The in vivo distribution results show that siRNA@M(Cage-dODN) has strong inflammatory targeting and can accumulate at high concentrations in the inflamed hind feet of mice.

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A macrophage membrane-coated biomimetic DNA nanomedicine, characterized in that: It includes a drug-loaded DNA nanocage, cholesterol-modified TNF-α siRNA and a macrophage membrane, wherein the drug-loaded DNA nanocage is coated by the macrophage membrane, and the cholesterol-modified TNF-α siRNA is inserted into the surface of the macrophage membrane; The DNA nanocage is prepared by dissolving 8 short chains and 5 long chains in ultrapure water respectively, then adding 10×TAMg buffer, and self-assembling through a PCR program after constant volume with ultrapure water. The ends of 4 of the 8 short chains in the DNA nanocage are connected to one of the chains of NF-κB decoy oligodeoxyribonucleotides (NF-κB decoydODNs) through an extended chain of 8 thymines, and then the complementary chain of NF-κB decoy dODNs is connected to the end of the short chain through base complementary pairing.

2. The biomimetic DNA nanodrug according to claim 1, characterized in that: The nucleotide sequence of the sense chain of the NF-κB decoy dODNs is shown in SEQ ID No.1, and the nucleotide sequence of the antisense chain is shown in SEQ ID No.2; the nucleotide sequence of the sense chain of the TNF-α siRNA is shown in SEQ ID No.3, and the nucleotide sequence of the antisense chain is shown in SEQ ID No.4, and the 2' position of the ribose groups of the nucleotides at positions 8, 10, 12, 14, 16, 18, 20, 22, 24, 25 and 26 are methylated.

3. The biomimetic DNA nanodrug according to claim 2, characterized in that: The macrophage membranes were derived from RAW264.7 macrophages.

4. Use of the bionic DNA nanomedicine according to any one of claims 1 to 3 in the preparation of a combined anti-rheumatoid arthritis drug.

5. The method for preparing the biomimetic DNA nanomedicine according to claim 3, characterized in that: Here are the steps: (1) RAW 264.7 macrophages were cultured and collected, resuspended in Tris-MgCl2 buffer, centrifuged, and disrupted. The macrophage membranes were extracted by differential centrifugation. After resuspending in PBS solution, the macrophage membrane vesicles were formed by physical extrusion through a porous polycarbonate membrane. (2) Eight short chains and five long chains were dissolved in ultrapure water, and then 10×TAMg buffer was added. After the volume was fixed with ultrapure water, DNA nanocages were self-assembled by PCR. (3) The ends of four of the eight short chains in step (2) are connected to one of the chains of NF-κB decoy dODNs through an extended chain of eight thymines, and then the complementary chain of NF-κB decoy dODNs is connected to the end of the short chain through base complementary pairing to prepare Cage-dODN; (4) preparing a sterile aqueous solution of cholesterol-modified TNF-α siRNA, and then vortexing and mixing with the macrophage membrane vesicles in step (2), sonicating in an ice bath, and then mixing and incubating at a constant temperature to obtain siRNA@M; (5) The siRNA@M prepared in step (4) is mixed with the Cage-dODN prepared in step (3), and the mixture is ultrasonically treated in an ice bath to obtain a biomimetic DNA nanodrug, namely, siRNA@M (Cage-dODN).

6. The preparation method according to claim 5, characterized in that: The concentration of RAW 264.7 macrophages after resuspension in Tris-MgCl2 buffer in step (1) is 2-3×10 7 Pieces / mL.

7. The preparation method according to claim 6, characterized in that: In the step (1), the liquid is passed through porous polycarbonate membranes with pore diameters of 1 µm, 400 nm, and 200 nm in sequence.

8. The preparation method according to claim 5, characterized in that: The nucleotide sequences of the eight short chains in step (2) are shown as SEQ ID No.5-SEQ ID No.12; the nucleotide sequences of the five long chains are shown as SEQ ID No.13-SEQ ID No.17; the amount of each short chain is 200 pmol, and the amount of each long chain is 100 pmol; the volume of 10×TAMg buffer is 10 μL; and the volume after constant volume is 100 μL.

9. The preparation method according to claim 5, characterized in that: The final concentration of the sterile aqueous solution of cholesterol-modified TNF-α siRNA in step (4) is 20 µM, ultrasonicated on ice for 3 min, and mixed at a constant temperature of 4°C for 5 min.

10. The preparation method according to claim 5, characterized in that: In step (5), the mass ratio of siRNA@M to Cage-dODN is 20:1, wherein the final concentration of siRNA is 100 nM, and the final concentration of dODN is 1 µM; the parameters of the ultrasonic treatment are 42 KHz / 100 W / 5 min.

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