Ultrasonic microbubble functionalized exosome as well as preparation method and application thereof

Abstract

The invention provides an ultrasonic microbubble functionalized exosome as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. According to the ultrasonic microbubble functionalized exosome provided by the invention, by adopting the RGD peptide lipid microbubbles, visual delivery can be realized under ultrasonic guidance, and the exosome uptake efficiency of damaged kidney cells is remarkably enhanced, so that the treatment effect of acute kidney injury is improved, and the problem that the exosome delivery effect is limited in the prior art is solved. According to the invention, the ultrasonic microbubble functionalized exosome can better target the local part of the kidney by utilizing the combination principle of RGD peptide and integrin, so that the delivery efficiency of the local part of the kidney by applying the exosome is improved, and the targeting of drug delivery is enhanced. Under ultrasonic guidance, the ultrasonic microbubble functionalized exosome provided by the invention is injected to a specified position, so that the visualization of percutaneous intrarenal injection is realized, and the accuracy and safety of injection are improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to ultrasound microbubble functionalized exosomes and a preparation method and application thereof. Background Art

[0002] Acute kidney injury is a common clinical syndrome characterized by a sudden increase in serum creatinine levels, decreased urine output, or both. Acute kidney injury can be caused by a variety of factors, including renal ischemia, sepsis, drug toxicity, and damage to myoglobin or hemoglobin. As a major global public health problem, acute kidney injury affects approximately 13.3 million people each year, and this number continues to grow. However, the current treatment options for acute kidney injury are very limited. For patients with severe acute kidney injury, clinical practice mainly relies on dialysis to relieve symptoms, but dialysis cannot promote kidney repair and regeneration. Therefore, there is an urgent need to develop new therapies that can promote kidney tissue repair and functional recovery.

[0003] Exosomes are lipid bilayer vesicles that can be secreted by almost all cells for intercellular communication. Their particle size is usually between 30-200 nm. Exosomes derived from mesenchymal stem cells have become an important research direction for the treatment of acute kidney injury due to their unique potential in tissue repair, anti-inflammation and immune regulation. Although current studies have shown that exosomes have significant therapeutic effects in the treatment of acute kidney injury, their clinical transformation still faces many challenges, among which low delivery efficiency is one of the key issues limiting efficacy.

[0004] In order to improve the delivery efficiency of exosomes, existing studies have proposed the use of local treatment instead of traditional intravenous infusion, which significantly increases the aggregation of exosomes in the kidneys. However, the clinical transformation of local injection methods is still subject to many limitations, especially due to the invisibility of local injection operations under non-open surgery, which leads to high technical difficulty and operational risks in the injection process. At the same time, current research mainly focuses on increasing the proportion of exosomes reaching renal target cells, and pays less attention to the uptake capacity of damaged renal cells for exosomes. In fact, the uptake capacity of renal tubular cells for exosomes is significantly reduced due to damage in the state of acute renal injury, which further limits the treatment effect. The existing local exosome treatment schemes for the kidneys have the problems of lack of visibility of the injection operation and low exosome delivery efficiency. Summary of the invention

[0005] The purpose of the present invention is to provide an ultrasound microbubble functionalized exosome and a preparation method and application thereof, which can achieve visualized delivery and significantly enhance the uptake efficiency of exosomes by damaged kidney cells, thereby improving the therapeutic effect of acute kidney injury.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides an ultrasound microbubble functionalized exosome, wherein the structure of the ultrasound microbubble functionalized exosome contains ultrasound microbubbles and exosomes; The ultrasound microbubbles are modified with RGD peptides, and the ultrasound microbubbles are connected to integrins on the surface of exosomes through the RGD peptides.

[0007] The present invention also provides a method for preparing the above-mentioned ultrasound microbubble functionalized exosomes, comprising the following steps: The ultrasound microbubbles modified with RGD peptides are mixed and incubated with exosomes to obtain ultrasound microbubbles functionalized exosomes, wherein the structure of the ultrasound microbubbles functionalized exosomes contains ultrasound microbubbles and exosomes; The ultrasound microbubbles are modified with RGD peptides, and the ultrasound microbubbles are connected to integrins on the surface of exosomes through the RGD peptides.

[0008] Preferably, the ultrasound microbubbles modified with RGD peptide are obtained by a preparation method according to the following steps: The Labeler R ultrasound microbubble contrast agent was subjected to an oscillation treatment to obtain ultrasound microbubbles modified with RGD peptides.

[0009] Preferably, the shaking treatment is performed by an amalgamator.

[0010] Preferably, the oscillation treatment time is 40 to 60 seconds.

[0011] Preferably, the particle number ratio of the ultrasonic microbubbles modified with RGD peptide and the exosomes mixed is 1:50~200.

[0012] Preferably, the incubation time is 20 to 40 minutes.

[0013] Preferably, the exosomes are extracted from the culture supernatant of mesenchymal stem cells by ultrafiltration concentration and exclusion chromatography purification.

[0014] Preferably, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0015] The present invention also provides the use of the ultrasound microbubble functionalized exosomes in the preparation of acute kidney injury drugs.

[0016] Beneficial effects of the present invention: The ultrasound microbubble functionalized exosomes provided by the present invention can achieve visual delivery under ultrasound guidance by combining RGD peptide lipid microbubbles, and significantly enhance the uptake efficiency of damaged kidney cells to exosomes, thereby improving the therapeutic effect of acute kidney injury. The present invention utilizes the combination principle of RGD peptide and integrin, and the ultrasound microbubble functionalized exosomes can better target the kidney, improve the delivery efficiency of exosomes in the kidney, and enhance the targeting of drug delivery.

[0017] Under ultrasound guidance, the ultrasound microbubble functionalized exosomes provided by the present invention are injected to the designated location, realizing the visualization of percutaneous intrarenal injection and improving the accuracy and safety of injection. The results of in vivo and in vitro experiments showed that ultrasound microbubble functionalized exosomes have higher delivery efficiency in the kidneys than simple exosomes or a mixture of ultrasound microbubbles and exosomes, and significantly improve renal function and reduce kidney damage. By improving the delivery efficiency of exosomes and enhancing the uptake capacity of damaged kidney cells for exosomes, ultrasound microbubble functionalized exosomes help promote the repair and functional recovery of renal tissue, and the visualization of local injection operations under non-open surgery reduces the technical difficulty and operational risks, making the treatment safer.

[0018] The development of ultrasound microbubble functionalized exosomes in the present invention provides a new method for improving the therapeutic effect of acute kidney injury. Through ultrasound guidance and targeted delivery, visualization, controllable delivery and enhanced therapeutic effect of exosome therapy are achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the expression analysis diagram of characteristic proteins of mesenchymal stem cell exosomes (positive proteins: CD81, CD9, Alix, negative protein: Calnexin); Figure 2 Characterization diagram of mesenchymal stem cell exosomes, where: A is an electron micrograph of exosomes; B is an exosome particle size distribution diagram; C is an ultrasound microbubble electron micrograph; D is an ultrasound microbubble particle size distribution diagram; E is an ultrasound microbubble functionalized exosome electron micrograph (the circled area is the exosome); F is an ultrasound microbubble functionalized exosome particle size distribution diagram; Figure 3 This is the fluorescence co-localization analysis of ultrasound microbubbles functionalized exosomes, where: A is the fluorescence co-localization image of ultrasound microbubbles and exosomes, with green marking microbubbles and red marking exosomes; B is the quantitative analysis of the degree of fluorescence co-localization; Figure 4 The figure is a graph for investigating the in vitro delivery efficiency of ultrasound microbubble functionalized exosomes, where: A is a graph showing the fluorescence distribution of exosomes after ultrasound excitation (simple exosome group); B is a graph showing the fluorescence distribution of exosomes after ultrasound excitation (ultrasound microbubble mixed exosome group); C is a graph showing the fluorescence distribution of exosomes after ultrasound excitation (ultrasound microbubble functionalized exosome group) Figure 5 This is an ultrasound image of transcutaneous intrarenal injection of ultrasound microbubble functionalized exosomes; Figure 6 This is a graph for investigating the in vivo delivery efficiency of ultrasound microbubble functionalized exosomes, where: A is a graph showing the fluorescence distribution of exosomes in different groups of kidney cells; B is a graph showing the comparison of fluorescence quantitative analysis of exosomes in different groups of kidney cells; Figure 7 This is a graph evaluating the effect of ultrasound microbubble functionalized exosomes in the treatment of acute kidney injury, where: A is a graph showing changes in serum creatinine (SCr); B is a graph showing changes in urea nitrogen (BUN); Figure 8 PAS staining images of the morphological and structural changes of kidneys in each group of rats. DETAILED DESCRIPTION

[0020] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention. Example

[0021] 1. Preparation and characterization of mesenchymal stem cell exosomes 1. Preparation of Mesenchymal Stem Cell Exosomes Human umbilical cord mesenchymal stem cells were inoculated in DMEM / F12 medium containing 10% fetal bovine serum and cultured every two days. When the cell confluence reached about 60%, the medium was replaced with DMEM / F12 medium containing 10% exosome-free fetal bovine serum. After 24 hours of continuous culture, the cell culture supernatant was collected and only the supernatant of the 6th to 8th passage cells was used.

[0022] Exosomes were extracted from the cell supernatant by ultrafiltration concentration and exclusion chromatography purification. The steps are as follows: the collected culture supernatant was first centrifuged at 1000×g and 3000×g for 10 minutes at 4°C, and the supernatant was taken to remove cell debris. The supernatant was then filtered with a 0.22μm filter and concentrated using a 100kDa ultrafiltration tube at 4000×g for 30 minutes at 4°C. The concentrate was loaded into a chromatographic column, eluted with PBS, and the exosome-containing fraction was collected.

[0023] 2. Characterization of Mesenchymal Stem Cell Exosomes Western blot was used to detect the expression of exosome-specific proteins (CD81, CD9, Alix) and negative proteins (Calnexin). The results were as follows Figure 1 The structural morphology of exosomes was observed by transmission electron microscopy, confirming their typical double-layer membrane structure. Figure 2 A. Nanoflow cytometry was used to measure the particle size and distribution of exosomes. The results are shown in Figure 2 As shown in B.

[0024] 2. Preparation and characterization of ultrasound microbubble functionalized exosomes 1. Preparation of Ultrasound Microbubble Functionalized Exosomes The Labeler R ultrasound microbubble contrast agent was vigorously shaken in an amalgamator for 50 seconds to obtain RGD peptide-microbubbles. Subsequently, the RGD peptide-microbubbles were diluted to 2×10 9The concentration of particles / mL was 2 × 10 11 The diluted EVs were mixed with MBs or RGD-MBs at a particle number ratio of approximately 100:1 (volume ratio of 1:1) and incubated at room temperature for 30 min to form ultrasound microbubble-functionalized exosomes.

[0025] 2. Characterization of Ultrasound Microbubble Functionalized Exosomes The vacuolar structure of the microvesicles and the double-layer membrane structure of the attached exosomes were observed by transmission electron microscopy. Figure 2 C, 2E. The particle size and size distribution of exosomes functionalized with pre-RGD peptide lipid microbubbles and post-ultrasound microbubbles were measured using dynamic light scattering. Figure 2 D, 2F. By marking lipid microvesicles and exosomes with green and red fluorescence, respectively, and performing immunofluorescence co-localization analysis, the different situations of the two under binding and non-binding can be distinguished. Figure 3 A,3B. Experimental example

[0026] Study on the effect of ultrasound microbubble functionalized exosomes on controlled release-assisted delivery in vitro In order to verify the effect of ultrasound microbubbles and exosomes on improving delivery efficiency after connecting with integrins through RGD peptide, the results were first verified in an in vitro model. HK-2 cells (immortalized human renal tubular cell line) were cultured in vitro until the confluence reached 80%, and the culture medium containing cisplatin (5μg / mL) was replaced and incubated for 12 hours to simulate the damaged renal tubular cell model. Subsequently, the exosomes were labeled with PKH26 dye (red) in advance, and ultrasound microbubble functionalized exosomes were added to the culture medium, and ultrasound was used for excitation (MI=0.751, duration 5 seconds, interval 5 seconds, repeated 6 times), and the fluorescence distribution of exosomes was observed after 4 hours. The ultrasound conditions of the simple exosome group and the exosome and microbubble mixture group were the same, and the results are shown in the table. Figure 4 A, 4B. In vitro studies further confirmed that ultrasound microbubble functionalized exosomes had higher delivery efficiency to damaged renal tubular cells than simple exosomes and a mixture of ultrasound microbubbles and exosomes (directly mixed under the same conditions but without RGD-integrin connection).

[0027] Ultrasound-guided percutaneous intrarenal injection of ultrasound-microbubble functionalized exosomes for visualization and in vivo controlled release 1. Visualization of percutaneous intrarenal injection under ultrasound guidance Ultrasound coupling agent was applied to the back of the anesthetized rat, and the largest section of the kidney was located by ultrasound probe. In contrast imaging mode, the injection needle was inserted into the renal cortex along the ultrasound scanning plane, and 200 μL of ultrasound microbubble functionalized exosomes were injected. The high echo at the injection point was clearly visible. The results are shown in Figure 5 .

[0028] 2. Investigation of the effect of controlled release-assisted delivery in vivo After the ultrasound microbubble functionalized exosomes were injected into the designated location in the kidney, the microbubbles were excited using the microbubble excitation key (MI = 1.111, exposure time 5 seconds, interval 5 seconds, repeated 10 times). After the excitation, the high echo shadow was significantly weakened to disappear, indicating that the excitation was successful. The results are shown in Figure 5 .

[0029] The present invention uses PKH26 dye to label exosomes in advance, and compares the differences between different injection drugs (exosomes alone, exosomes and microbubbles, ultrasound microbubbles functionalized exosomes). All groups use the same ultrasound conditions. After injection and stimulation for 8 hours, the distribution and intracellular quantity of exosome fluorescence in renal cells are observed after immunofluorescence staining of rat kidney sections. The results are shown in Figure 6 A, 6B. In vivo studies confirmed that ultrasound microbubble-functionalized exosomes had higher delivery efficiency in the kidneys than simple exosomes and a mixture of ultrasound microbubbles and exosomes.

[0030] Effect of ultrasound-microbubble functionalized exosomes on the treatment of acute kidney injury Twenty-four male SD rats at week 8 were evenly and randomly divided into different treatment groups (sham operation group: normal rats were subjected to ultrasound-guided percutaneous intrarenal injection of PBS; model group: rats with cisplatin acute kidney injury were subjected to ultrasound-guided percutaneous intrarenal injection of PBS; exosome (EVs) group: rats with cisplatin acute kidney injury were subjected to ultrasound-guided percutaneous intrarenal injection of exosomes; ultrasound microbubble functionalized exosomes (MB-EVs) group: rats with cisplatin acute kidney injury were subjected to ultrasound-guided percutaneous intrarenal injection of ultrasound microbubble functionalized exosomes.). 5 mg / kg cisplatin was infused into the tail vein to establish the model. On the first day after modeling, the drug was injected under ultrasound guidance and ultrasound excitation was performed. All groups used the same ultrasound parameters for excitation. Subsequently, samples were collected on the 4th day to detect renal function and injury. The present invention measured the changes in serum creatinine (SCr) and urea nitrogen (BUN) of each rat by separating serum from the tail vein blood every day. The results are shown in Table 1. Figure 7 A, 7B. The kidney sections of rats on the 4th day were sliced ​​and PAS staining was performed to observe the changes in kidney morphology and structure. It was found that the pathological damage of the ultrasound microbubble functionalized exosome group was significantly alleviated. Figure 8 The above results all confirmed that ultrasound microbubble functionalized exosomes not only significantly improved renal function and alleviated renal damage compared with the model group, but also had a more significant improvement than the exosome group.

[0031] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An ultrasound microbubble functionalized exosome, characterized in that: The structure of the ultrasound microbubble functionalized exosomes contains ultrasound microbubbles and exosomes; The ultrasound microbubbles are modified with RGD peptides, and the ultrasound microbubbles are connected to integrins on the surface of exosomes through the RGD peptides.

2. The method for preparing ultrasound microbubble functionalized exosomes according to claim 1, characterized in that: The following steps are involved: The ultrasound microbubbles modified with RGD peptides are mixed and incubated with exosomes to obtain ultrasound microbubbles functionalized exosomes, wherein the structure of the ultrasound microbubbles functionalized exosomes contains ultrasound microbubbles and exosomes; The ultrasound microbubbles are modified with RGD peptides, and the ultrasound microbubbles are connected to integrins on the surface of exosomes through the RGD peptides.

3. The preparation method according to claim 2, characterized in that: The ultrasonic microbubbles modified with RGD peptide are obtained by the preparation method according to the following steps: The Labeler R ultrasound microbubble contrast agent was subjected to an oscillation treatment to obtain ultrasound microbubbles modified with RGD peptides.

4. The preparation method according to claim 3, characterized in that: The shaking treatment is performed by an amalgamator.

5. The preparation method according to claim 4, characterized in that: The oscillation treatment time is 40 to 60 seconds.

6. The preparation method according to claim 2, characterized in that: The particle number ratio of the ultrasonic microbubbles modified with RGD peptide and the exosomes mixed is 1:50-200.

7. The preparation method according to claim 2, characterized in that: The incubation time is 20 to 40 minutes.

8. The preparation method according to claim 2, characterized in that: The exosomes are extracted from the culture supernatant of mesenchymal stem cells by adopting ultrafiltration concentration method and exclusion chromatography purification method.

9. The preparation method according to claim 8, characterized in that: The mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

10. Use of the ultrasound microbubble functionalized exosomes according to claim 1 in the preparation of acute kidney injury drugs.

Images