An engineered exosome targeting proinflammatory macrophages and preparation method and application thereof

Abstract

The present application relates to an engineered exosome targeting proinflammatory macrophages and a preparation method and application thereof, and belongs to the field of biomedical technology. The membrane outer surface of the exosome is loaded with a targeting peptide, and the membrane inner surface is loaded with a marker protein. The engineered exosome can perform diagnostic operations such as in vivo tracing and imaging on proinflammatory macrophages. At the same time, because it can efficiently and specifically target proinflammatory macrophages, it can also deliver therapeutic media for proinflammatory macrophages, thereby providing an efficient, specific and safe treatment strategy for inflammatory diseases such as osteoarthritis and rheumatoid arthritis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an engineered exosome that targets pro-inflammatory macrophages, its preparation method, and its application. Background Technology

[0002] Pro-inflammatory macrophages are typically induced by TNF-γ, Toll-like receptors, and other receptors to produce a series of pro-inflammatory factors such as TNF-α, IL-6, and iNOS. They are target cells in various inflammatory diseases, including osteoarthritis, rheumatoid arthritis, and bone and cartilage injuries. Pro-inflammatory macrophages are closely related to the occurrence, development, and activity levels of these diseases; therefore, in vivo observation of the tissue distribution of pro-inflammatory macrophages can serve as an important basis for related disease research. Furthermore, in inflammatory diseases, HIF-1α is a regulatory signal for multiple pathophysiological processes, playing a crucial role in various life activities. For example, in articular chondrocytes, HIF-1α promotes chondrocyte survival and proliferation, and maintains the metabolic balance of the articular cartilage matrix; however, in macrophages, it promotes macrophage inflammatory responses, leading to increased synovial inflammation, joint pain, impaired joint movement, and even exacerbating bone and articular cartilage destruction in inflammatory diseases such as osteoarthritis and rheumatoid arthritis. Studies have shown that regulating HIF-1α levels can be used to treat inflammatory diseases; however, widespread intervention in this signaling pathway can produce serious side effects, thus limiting its clinical application. Targeted intervention is one of the common methods for regulating HIF-1α levels. Targeted intervention of pro-inflammatory macrophages for HIF-1α can avoid the potential adverse effects of indiscriminate intervention, thereby better exerting its therapeutic effect. Currently used targeting materials are nanoparticles or chemically synthesized materials. Compared with nanoparticles and chemically synthesized materials, engineered exosomes have advantages such as high efficiency, fewer unpredictable toxic side effects, and wide applicability. However, there are currently no reports on using engineered exosomes to achieve targeted manipulation of pro-inflammatory macrophages and delivery of therapeutic mediators.

[0003] Therefore, in order to perform in vivo tracing and imaging of pro-inflammatory macrophages to facilitate research on related diseases, and to fill the gap of the lack of engineered exosomes that target pro-inflammatory macrophages and thus minimize the side effects of intervening in pro-inflammatory macrophage HIF-1α, it is necessary to develop an engineered exosome that targets pro-inflammatory macrophages. Summary of the Invention

[0004] In view of this, one objective of the present invention is to provide an engineered exosome that targets pro-inflammatory macrophages; a second objective of the present invention is to provide a method for preparing engineered exosomes that target pro-inflammatory macrophages; and a third objective of the present invention is to provide an application of engineered exosomes that target pro-inflammatory macrophages in the in vivo tracing and imaging of pro-inflammatory macrophages and in the preparation of drugs for treating inflammatory diseases.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] 1. An engineered exosome that targets pro-inflammatory macrophages, wherein the exosome's outer membrane surface is loaded with a targeting peptide and its inner membrane surface is loaded with a labeled protein.

[0007] Preferably, the exosomes are derived from any one of tool cells, embryonic stem cells, perinatal stem cells, adult stem cells, or induced pluripotent stem cells.

[0008] Preferably, the tool cells are selected from any one of HEK 293, HeLa, NCI-60, CHOs, C3H10T1 / 2, SW1353, MC3T3-E1, RAW264.7, or C2C12; the perinatal stem cells are selected from any one of umbilical cord blood, umbilical cord, placenta, mesenchymal stem cells, or subpluripotent stem cells; and the adult stem cells are selected from any one of bone marrow, adipose tissue, nerve tissue, or skin.

[0009] Preferably, the amino acid sequence of the targeting peptide is FQHPSFI, SPLSQSA, LPSSGAA, TGSPELH, QMPEMKA, RQSHLKV, SGYPRHY, KYNTHHA, RGD, or TKPR.

[0010] Preferably, the marker protein is any one of green fluorescent protein, red fluorescent protein, yellow fluorescent protein, or blue fluorescent protein.

[0011] 2. The method for preparing engineered exosomes targeting pro-inflammatory macrophages, wherein the preparation method is as follows:

[0012] The DNA sequence encoding the recombinant protein is inserted into the PLVX-puro lentiviral vector to obtain a lentivirus containing the DNA sequence encoding the recombinant protein, wherein the recombinant protein contains the target peptide and the marker protein; then, cells that secrete exosomes are infected to obtain engineered cells that can secrete engineered exosomes, the engineered cells are collected and cultured, and the culture supernatant of the engineered cells is filtered and centrifuged to obtain engineered exosomes that can target pro-inflammatory macrophages.

[0013] Preferably, the recombinant protein comprises a signal peptide, a target peptide, LAMP2B, a labeling protein, and a linker peptide. The signal peptide, the three target peptides, and LAMP2B are sequentially linked by the linker peptide, and the labeling protein is linked to the end of LAMP2B. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 1, the amino acid sequence of LAMP2B is shown in SEQ ID NO: 2, the amino acid sequence of the labeling protein is shown in SEQ ID NO: 3, and the amino acid sequence of the linker peptide is shown in SEQ ID NO: 4.

[0014] 3. The application of the engineered exosomes targeting pro-inflammatory macrophages in in vivo tracing and imaging of pro-inflammatory macrophages and in the preparation of drugs for treating inflammatory diseases.

[0015] Preferably, the inflammatory disease is arthritis.

[0016] Preferably, the inflammatory disease treatment drug comprises engineered exosomes loaded with HIF-1α-siRNA, the sequence of which is: AAACAGAGACGAAGGACAATT.

[0017] The beneficial effects of this invention are as follows: This application provides an engineered exosome that targets pro-inflammatory macrophages. The outer surface of the exosome is loaded with a targeting peptide, and the inner surface is loaded with a marker protein. When loaded with HIF-1α-siRNA, it can target pro-inflammatory macrophages in inflamed joint tissues, downregulating the inflammatory response of macrophages. This can be used to treat knee joint inflammation, synovial swelling, and joint effusion, with significant therapeutic effects and minimal unpredictable toxic side effects. Furthermore, because the exosome contains marker proteins, it can also be used to visualize the aggregation of in vivo inflammatory macrophages in other inflamed tissues such as skin in real time.

[0018] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0019] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0020] Figure 1 This is a schematic diagram of the structure of a recombinant protein containing a targeting peptide and a labeled protein, as well as the DNA and amino acid sequences of the targeting peptide;

[0021] Figure 2A schematic diagram of the PLVX-puro lentiviral vector;

[0022] Figure 3 Image showing the detection of green fluorescent protein (eGFP) in HEK 293T cells infected with 10 recombinant chronic viruses containing targeting peptides;

[0023] Figure 4 A schematic diagram of the structure of engineered exosomes targeting pro-inflammatory macrophages and a schematic diagram of their targeted delivery of therapeutic siRNA to pro-inflammatory macrophages to downregulate macrophage inflammatory responses;

[0024] Figure 5 Transmission electron microscopy and Nanosight detection results of 10 engineered exosomes containing different targeting peptides;

[0025] Figure 6 A comparative diagram showing the targeting effects of 10 engineered exosomes containing targeting peptides on pro-inflammatory macrophages in vitro;

[0026] Figure 7 To evaluate the therapeutic effect of engineered exosomes #3 loaded with HIF-1α-siRNA on experimental arthritis;

[0027] Figure 8 This is a timeline diagram of engineered exosomes #3 targeting macrophages at inflammatory sites in vivo. Detailed Implementation

[0028] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0029] Example 1

[0030] The specific steps for preparing a stable HEK 293T cell line expressing recombinant proteins containing 10 target peptides are as follows:

[0031] according to Figure 1A recombinant protein containing a signal peptide, target peptides, LAMP2B, a labeling protein, and a linker peptide was constructed using this method. The signal peptide, the three target peptides, and LAMP2B are sequentially linked by linker peptides, with the labeling protein directly attached to the terminal of LAMP2B. The amino acid sequence of the signal peptide is: MVCFRLFPVPGSGLVLVCLVLGAVRSYA, as shown in SEQ ID NO: 1. The amino acid sequence of the target peptide is as follows: Figure 1As shown, the targeted peptides are: 1# Targeted peptide: FQHPSFI, 2# Targeted peptide: SPLSQSA, 3# Targeted peptide: LPSGAA, 4# Targeted peptide: TGSPELH, 5# Targeted peptide: QMPEMKA, 6# Targeted peptide: RQSHLKV, 7# Targeted peptide: SGYPRHY, 8# Targeted peptide: KYNTHHA, 9# Targeted peptide: RGD, and 10# Targeted peptide: TKPR. The amino acid sequence of LAMP2B is: LELNLTDSENATCLYAKWQMNFTVRYETTNKTYKTVTISDHGTVTYNGSICGDDQNGPKIAVQFGPGFSWIANFTKAASTYSIDSV SFSYNTGDNTTFPDAEDKGILTVDELLAIRIPLNDLFRCNSSLSTLEKNDVVQHYWDVLVQAFVQNGTVSTNEFLCDKDKTSTVAPTIHTTVPSPTTTPTP KEKPEAGTYSVNNGNDTCLLATMGLQLNITQDKVASVININPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNISMYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAQECSLDDDTILIPIIVGAGLSGLIIVIVIAYVIGRRKSYAGYQTL, as in SEQ ID NO:2. The amino acid sequence of the marker protein eGFP is MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK, as shown in SEQ ID NO: 3. The amino acid sequence of the linker peptide is GGGGS, as shown in SEQ ID NO: 4.

[0032] from Figure 1As can be seen, the targeting peptides have 10 different amino acid sequences (1# to 10#) and the DNA sequences encoding them. Therefore, DNA sequences encoding 10 recombinant proteins can be obtained, as shown in SEQ ID NO: 5-14. The obtained DNA sequences encoding the 10 recombinant proteins were then ligated into the PLVX-puro lentiviral vector (the structure of the PLVX-puro lentiviral vector is shown in Figure 1). Figure 2 The XhoI and BamHI restriction sites (as shown) are used to obtain lentiviral particles containing nucleic acid coding sequences of different recombinant proteins, which are then used to infect HEK 293T cells. Infected cells are seeded into DMEM complete medium and cultured at 37°C and 5% CO2 for 3-5 days. After selection with 2 μg / ml puromycin, lentivir-infected HEK293T cells can be initially obtained. Lentivirally infected cells are then selected for monoclonal culture under puromycin conditions for 14-21 days to obtain a stable HEK 293T cell line expressing recombinant proteins containing 10 target peptides. The expression of recombinant proteins in the cell line is detected by green fluorescent protein, and the experimental results are shown below. Figure 3 As shown. From Figure 3 As can be seen, the HEK293T cell line containing 10 target peptide recombinant proteins all showed green fluorescence after photoexcitation, indicating that the recombinant proteins were successfully expressed in the HEK 293T cell line.

[0033] Example 2

[0034] The preparation process of engineered exosomes targeting pro-inflammatory macrophages is as follows:

[0035] The HEK 293T cell line, which stably expresses recombinant proteins containing 10 target peptides, was expanded and cultured. The cell culture supernatant was collected and subjected to gradient centrifugation at 300g / 10min, 2000g / 10min, and 10000g / 10min to remove cells, cell debris, vesicles, and other precipitates. Larger extracellular vesicles were then removed by passing the supernatant through a 0.22μm filter membrane. Finally, engineered exosomes targeting pro-inflammatory macrophages were obtained by ultracentrifugation at 100000g.

[0036] The formation process of engineered exosomes targeting pro-inflammatory macrophages and the targeted delivery process to pro-inflammatory macrophages are as follows: Figure 4 As shown, when the exosomes are loaded with HIF-1α-siRNA, they can target pro-inflammatory macrophages and downregulate the inflammatory response of macrophages. Transmission electron microscopy and Nanosight detection revealed that the precipitate obtained after ultracentrifugation of the supernatant exhibited exosome characteristics. The experimental results are as follows: Figure 5As shown in the figure; the 10 obtained target peptide exosomes were co-incubated with pro-inflammatory macrophages. Comparison revealed that the engineered exosomes containing target peptide #3 had the best targeting effect on pro-inflammatory macrophages, and exhibited the strongest GFP fluorescence signal under the same incubation conditions. The experimental results are shown in the figure. Figure 6 As shown.

[0037] Example 3

[0038] The therapeutic effects of engineered exosomes loaded with HIF-1α-siRNA and targeting peptide #3 on experimental arthritis were investigated. The specific steps are as follows:

[0039] (1) Four experimental groups were set up: sham surgery group, arthritis model group, arthritis model group + HIF-1α-siRNA delivery by ordinary exosomes, and arthritis model group + HIF-1α-siRNA delivery by engineered exosomes. Among them, the arthritis model group was obtained by inducing an arthritis model through collagenase VII (CIOA);

[0040] (2) Construction of exosomes loaded with HIF-1α-siRNA: HIF-1α-siRNA was loaded onto engineered exosomes of the #3 target peptide and ordinary exosomes using the Exo-Fect exosome loading kit. Each 1*10^9 exosome was incubated with 20 pmol HIF-1α-siRNA + 10 μl Exo-Fect at 37°C for 10 min and then temporarily stored on ice. The sequence of HIF-1α-siRNA was: AAACAGAGACGAAGGACAATT.

[0041] (3) Treatment of arthritis in the model group: On days 4, 7, 10, and 13 after modeling, 1*10^9 engineered exosomes loaded with HIF-1α-siRNA #3 targeting peptide and 1*10^9 ordinary exosomes loaded with HIF-1α-siRNA were injected into the joint cavity, respectively. The therapeutic effects were as follows: Figure 7 As shown.

[0042] Figure 7 The images show MRI images of the four experimental groups. Evaluation of the experimental results showed that, compared with ordinary exosomes loaded with HIF-1α-siRNA, the engineered exosomes loaded with HIF-1α-siRNA and targeting peptide #3 were more effective in treating knee joint inflammation, synovial swelling, and joint effusion.

[0043] Furthermore, when intravenously injected with engineered exosomes containing the #3 targeting peptide, these exosomes can target macrophages in CIOA knee osteoarthritis tissue (experimental results are shown below). Figure 8 (As shown). LysM is used in this example. cre-tdTomato labels macrophages, producing a red signal; DID labels exosomes, producing a green signal. Over time, it was found that macrophages (red signal) and exosomes (green signal) completely overlapped, indicating that the engineered exosomes have a targeting and tracing effect on macrophages in arthritic tissues.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An engineered exosome targeting pro-inflammatory macrophages, characterized in that: This engineered exosome is secreted by cells expressing a recombinant protein. Its outer membrane surface is loaded with a targeting peptide, and its inner membrane surface is loaded with a labeling protein. The recombinant protein includes a signal peptide, a targeting peptide, LAMP2B, a labeling protein, and a linker peptide. The signal peptide, the three targeting peptides, and LAMP2B are sequentially linked by the linker peptide, and the labeling protein is attached to the terminal of LAMP2B. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 1, the amino acid sequence of LAMP2B is shown in SEQ ID NO: 2, the amino acid sequence of the labeling protein is shown in SEQ ID NO: 3, and the amino acid sequence of the linker peptide is shown in SEQ ID NO:

4. The amino acid sequence of the targeting peptide is FQHPSFI, SPLSQSA, LPSGAA, TGSPELH, QMPEMKA, RQSHLKV, SGYPRHY, KYNTHHA, RGD, or TKPR.

2. A method for preparing engineered exosomes targeting pro-inflammatory macrophages, characterized in that, The preparation method is as follows: The DNA sequence encoding the recombinant protein is inserted into the PLVX-puro lentiviral vector to obtain a lentivirus containing the DNA sequence encoding the recombinant protein. This lentivirus is then used to infect cells that secrete exosomes, resulting in engineered cells capable of secreting engineered exosomes. These engineered cells are collected and cultured. The culture supernatant is filtered and centrifuged to obtain engineered exosomes that can target pro-inflammatory macrophages. The recombinant protein comprises a signal peptide, a targeting peptide, LAMP2B, a labeling protein, and a linker peptide. The signal peptide, the three targeting peptides, and LAMP2B are sequentially linked by the linker peptide, with the labeling protein attached to the end of LAMP2B. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 1, the amino acid sequence of LAMP2B is shown in SEQ ID NO: 2, the amino acid sequence of the labeling protein is shown in SEQ ID NO: 3, and the amino acid sequence of the linker peptide is shown in SEQ ID NO:

4. NO:4; the amino acid sequence of the target peptide is FQHPSFI, SPLSQSA, LPSSGAA, TGSPELH, QMPEMKA, RQSHLKV, SGYPRHY, KYNTHHA, RGD or TKPR.

3. The application of the engineered exosomes targeting pro-inflammatory macrophages as described in claim 1 in the preparation of drugs for treating inflammatory diseases, characterized in that... The inflammatory disease treatment drug contains engineered exosomes loaded with HIF-1 α-siRNA, the sequence of which is: AAACAGAGACGAAGGACAATT.

4. The application according to claim 3, characterized in that, The inflammatory disease mentioned is arthritis.

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