Engineered TAT and RVG dual-targeting peptide modified exosome loaded microRNA inhibitor as well as preparation method and application thereof

Abstract

The invention provides an engineered TAT and RVG dual-targeting peptide modified exosome loaded microRNA inhibitor as well as a preparation method and application thereof, and belongs to the technical field of medicines. According to the engineered TAT and RVG peptide exosome, the exosome is extracted from a supernatant of an MSC cell culture medium, the DiR-labeled exosome, FITC-labeled CP05-TAT peptide and 5-TAMRA-labeled CP05-RVG peptide are mixed and incubated together, the TAT and RVG dual-targeting peptide modified exosome is obtained, a miR-15b-5p inhibitor is loaded through ultrasound, and Exo-TATamp loaded with the miR-15b-5p inhibitor is prepared; the TAT and the RVG are used for co-modifying the MSC-EXOs, so that blood-brain barrier penetration and neuronal targeting can be remarkably enhanced, the delivery effect of a microRNA-15b-5p inhibitor is enhanced, and the injury induced by cerebral ischemia reperfusion is relieved by inhibiting neuronal apoptosis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an engineered TAT and RVG dual-targeting peptide modified exosome loading microRNA inhibitor, its preparation method, and its application. Background Technology

[0002] Stem cell exosomes (MSC-EVs) are extracellular vesicles with a diameter of 30-150 nm. They are natural carrier systems with endogenous cell tropism, capable of delivering drugs into cells for better therapeutic molecule delivery. However, natural exosomes suffer from insufficient targeting and delivery efficiency. After systemic administration, they are easily captured by the liver and spleen (>60%), with only a small amount reaching brain tissue (<1%). Their ability to penetrate the blood-brain barrier is limited, relying on passive diffusion or non-specific uptake. Trans-activator of transcription peptide (TAT) is a cell-penetrating peptide (CPP) derived from HIV-1 virus. Its highly efficient transmembrane delivery capability is widely used for intracellular delivery of drugs, nucleic acids (such as siRNA / miRNA inhibitors), and proteins. Extracellular vesicles modified with TAT peptide can penetrate the plasma membrane and blood-brain barrier, significantly improving drug delivery across the blood-brain barrier. Rabies virus glycoprotein (RVG) is a key protein on the envelope of rabies virus (RABV) and is crucial for the virus to invade the nervous system. Through modification, it is widely used to target drugs across the blood-brain barrier and specifically bind to acetylcholine receptor neurons.

[0003] Ischemic stroke accounts for approximately 80% of all strokes. Intravenous thrombolysis and mechanical thrombectomy are the main clinical treatments, with the core objective of restoring blood flow perfusion as early as possible. Cerebral ischemia-reperfusion injury (I / R) can worsen brain damage. I / R injury promotes inflammatory responses, increases blood-brain barrier permeability and oxidative stress, leading to neuronal damage and deterioration of neurological function.

[0004] MicroRNAs are a class of non-coding single-stranded RNA molecules, approximately 18-25 nucleotides in length, that participate in various physiological and pathological processes by regulating gene expression. They regulate protein expression by binding to the 3' untranslated region (3'-UTR) of target mRNAs, leading to mRNA degradation or translational repression. Early studies found that hsa-miR-15b-5p, hsa-miR-184, and hsa-miR-16-5p are specifically associated with ischemic stroke (IS) and are highly expressed in serum exosomes of IS patients and in newly resected human stroke brain tissue. miR-184 has been reported to promote brain injury by targeting ppap2b-mediated neuronal apoptosis following brain injury / reperfusion injury. miR-16-5p exacerbates brain injury / reperfusion injury by enhancing neuronal apoptosis. The role of miR-15b-5p in brain injury / reperfusion injury remains uncertain. Summary of the Invention

[0005] To address the aforementioned issues, this invention enhances the ability of MSC-Exos modified with both TAT and RVG peptides to penetrate the blood-brain barrier and specifically target neurons in the brain. Using this dual-targeting peptide to modify exosomes for loading miR-15b-5p inhibitors allows for more efficient delivery to the brain, enabling targeted treatment of cerebral ischemia-reperfusion injury.

[0006] The technical solution of this invention is implemented as follows:

[0007] This invention provides a method for preparing an engineered microRNA inhibitor loaded onto exosomes modified with TAT and RVG dual-targeting peptides, comprising the following steps:

[0008] S1. Link CP05 to TAT peptide and label it with FITC to prepare FITC-labeled CP05-TAT;

[0009] S2. CP05 was linked to the RVG peptide and labeled with 5-TAMRA to obtain 5-TAMRA-labeled CP05-RVG;

[0010] S3. The exosomes were mixed with FAM-CP05-TAT peptide and 5-TAMRA-CP05-RVG peptide and incubated to obtain the modified exosomes;

[0011] S4. The modified exosomes were loaded with miR-15b-5p inhibitors by ultrasound to prepare engineered TAT and RVG dual-targeting peptide modified exosomes loaded with microRNA inhibitors.

[0012] As a further improvement of the present invention, the amino acid sequence of the FITC-labeled CP05-TAT in step S1 is shown in SEQ ID NO.1.

[0013] As a further improvement of the present invention, the amino acid sequence of the 5-TAMRA-labeled CP05-RVG in step S2 is shown in SEQ ID NO.2.

[0014] As a further improvement of the present invention, the incubation conditions in step S3 are 2-6°C and the incubation time is 5-7 hours.

[0015] As a further improvement of the present invention, the mass ratio of exosomes, FAM-CP05-TAT peptide and 5-TAMRA-CP05-RVG peptide in step S3 is 50:1:1.

[0016] As a further improvement of the present invention, the ultrasonic loading condition in step S4 is to perform 6 cycles of processing, with each cycle being 30 seconds of on / off switching and a 30-second cooling time between each cycle.

[0017] As a further improvement of the present invention, the amount of miR-15b-5p inhibitor and modified exosomes used in step S4 is 250-350 nM miR-15b-5p inhibitor and 80-120 ng modified exosomes.

[0018] The present invention further protects an engineered TAT and RVG dual-targeting peptide modified exosome loading microRNA inhibitor prepared by the above-described preparation method.

[0019] This invention further protects the use of the above-described engineered TAT and RVG dual-targeting peptide modified exosome loaded with microRNA inhibitor in the preparation of a drug for treating cerebral ischemia-reperfusion injury.

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

[0021] This invention proposes and prepares an engineered exosome-loaded miR-15b-5p inhibitor modified with TAT and RVG peptides, which can deliver it to the brain more efficiently and be targeted and absorbed by neurons for targeted treatment of cerebral ischemia-reperfusion injury. Compared with existing technologies, it has the following beneficial effects:

[0022] The exosomes involved in this invention are secreted by mesenchymal stem cells and can be preserved for a long time and maintain their activity through ultra-high-speed centrifugation. The surface modification of exosomes with TAT and RVG dual-targeting peptides enhances their ability to penetrate the blood-brain barrier and specifically target neurons in the brain. Exosomes with TAT and RVG dual-targeting peptide surface modification can efficiently deliver miR-15b-5p inhibitors; by inhibiting the significant upregulation of miR-15b-5p after cerebral ischemia-reperfusion injury, they suppress neuronal apoptosis, thus providing targeted therapy for cerebral ischemia-reperfusion injury. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 (A) Scanning electron microscopy of exosomes derived from MSCs, (B) Nanoparticle tracking analysis of MSCs exosomes, (C) Western blot analysis of the expression of TSG101, CD81 and CD63 proteins in MSCs cell culture medium and isolated exosomes.

[0025] Figure 2 To enable confocal microscopy analysis of the cellular distribution of DIR (red) stained exosomes in hippocampal HT22 cells, the exosomes were modified with FiFC-labeled CP05-TAT peptide and / or 5-tamra-labeled CP05-RVG peptide.

[0026] Figure 3 (A) Cell viability detection of mouse hippocampal HT22 cells after 24 hours of exosome treatment (n=3), (B) qRT-PCR analysis of miR-15b-5p levels in hippocampal HT22 cells after 24 hours of exosome treatment in each group (n=3).

[0027] Figure 4 (A) Fluorescence images of mouse brains, (B) Statistical analysis of fluorescence intensity in mouse brains, (C) qRT-PCR analysis of miR-15b-5p expression levels in the ischemic penumbra of each group of brain I / R injured mice (n=3). Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1: Isolation and Identification of MSC-EXO

[0030] The exosomes provided by this invention are secreted by mesenchymal stem cells. The cell culture supernatant was collected, filtered, and concentrated by ultrafiltration. Finally, the concentrated supernatant was ultracentrifuged at 4°C, 120,000g, for 2 hours to collect the exosomes. The morphology and size distribution of the isolated exosomes were analyzed by transmission electron microscopy (TEM) and nanoparticle flow cytometry. Western blot analysis was performed to analyze the expression of TSG101, CD81, and CD63 proteins in the MSC cell supernatant and isolated exosomes.

[0031] TEM showed that the isolated exosomes were membrane-closed vesicles. Figure 1 A). Exosomes isolated by nanoparticle flow cytometry have a diameter range of 40-120 nm. Figure 1 B). Western blot analysis confirmed the presence of exosome biomarkers CD63, TSG101, and CD81 in the isolated products. Figure 1 C) This confirms that MSC-exos extraction was successful.

[0032] Example 2: Preparation of Exo-TAT & RVG

[0033] Utilizing the specific binding ability of CP05 (amino acid sequence: CRHSSQMTVTSRL) to CD63, which is highly expressed on the surface of exosomes, CP05 was linked to a TAT peptide (amino acid sequence: YGRKKRRQRRR) and labeled with FITC. CP05 was also linked to an RVG peptide (amino acid sequence: YTIWMPENPRPGTPCDIFTNSRGKRASNG) and labeled with 5-TAMRA. FITC-labeled CP05-TAT (amino acid sequence: FAM-CRHSQMTVTSRL-YGRKKRRQRRR, SEQ ID NO. 1) and 5-TAMRA-labeled CP05-RVG (amino acid sequence: 5-TAMRA-CRHSQMTVTSRL-YTIWMPENPRPGTPCDIFTNSRGKRASNG, SEQ ID NO. 2) were prepared. Exosomes were incubated with FAM-CP05-TAT peptide and 5-TAMRA-CP05-RVG peptide at a mass ratio of 3:1 at 4°C for 6 h. Obtain Exo-TAT&RVG.

[0034] like Figure 2 As shown, exosomes, RVG-exosomes, TAT-exosomes, and TAT&RVG-exosomes were effectively internalized by neurons and localized in the cytoplasm, demonstrating their potential in the development of treatments for neurological diseases. More importantly, the TAT&RVG-exosomes group exhibited the highest fluorescence intensity in neurons, indicating that through effective modification with RVG and TAT, TAT&RVG-exosomes effectively target and enhance uptake by neurons in vitro.

[0035] Example 3: miRNA inhibitor loading into exosomes

[0036] Exosomes were sonicated to load miR-15b-5p inhibitor. 300 nM miR-15b-5p inhibitor and 100 ng Exo-TAT & RVG were mixed and sonicated at 20% amplitude using a Model 505 Sonic Dismembrator for 6 cycles, each with a 30-second on / off cycle and a 30-second cooling interval between cycles. The mixture was incubated at 37°C for 60 minutes to restore exosome membrane integrity. Ultracentrifugation for 2 hours was then performed to remove free miRNA inhibitor.

[0037] Example 4: DiR labeling and cell tracking of exosomes

[0038] MSCs-derived exosomes were labeled with the lipid membrane dye DiR. 50 μg of MSC-Exos was incubated with 10 μM DiR at room temperature in the dark for 30 min. Unbound dye was removed by ultracentrifugation, and the DiR-labeled MSC-Exos were purified and resuspended in PBS. The DiR-labeled exosomes were then incubated with FAM-CP05-TAT peptide and 5-TAMRA-CP05-RVG peptide at a mass ratio of 3:1 at 4 °C for 6 h. Subsequently, these DiR-labeled MSC-Exos, along with TAT-Exo, RVG-Exo, or Exo-TAT&RVG, were incubated with HT22 cells for 12 h, and fluorescence imaging was performed using confocal microscopy.

[0039] By using ultrasound, miR-15b-5p inhibitors or NC inhibitors are loaded into exosomes or TAT&RVG-exosomes, resulting in Exo+NC inhibitors, Exo+miR-15b-5p inhibitors, and TAT&RVG-Exo+miR-15b-5p inhibitors, respectively. Figure 3 Treatment with Exo+NC inhibitor, Exo+miR-15b-5p inhibitor, or TAT&RVG-Exo+miR-15b-5p inhibitor had no significant effect on neuronal viability. Compared with the control group, treatment with Exo+NC, Exo+miR-15b-5p inhibitor, and TAT&RVG-Exo+miR-15b-5p inhibitor significantly reduced miR-15b-5p expression, indicating that exosomes co-modified with TAT and RVG can effectively deliver miR-15b-5p inhibitors and inhibit miR-15b-5p expression in neurons. Figure 3 B).

[0040] Example 5: Biodistribution analysis of dir-labeled MSC-Exos

[0041] MSC-Exos were labeled with the lipid membrane dye DiR, and their biodistribution in mice was detected. 100 μg of MSC-Exo was stained with 50 μM DiR for 30 min at room temperature in the dark, along with miRNA inhibitor controls (Exo + NC inhibitor), miR-15b-5p inhibitor (Exo + miR-15b-5p inhibitor), or Exo-TAT & RVG and miR-15b-5p inhibitor (Exo-TAT & RVG + miR-15b-5pinhibitor). DiR-labeled exosomes were then separated by ultracentrifugation at 120,000 × g for 2 h, unbound dye was removed, and the exosomes were resuspended in PBS. Two hours after brain intubation / reduction (I / R), MCAO / R model mice were injected via the tail vein with 200 μL of PBS, Exo+NC inhibitor, Exo+miR-15b-5p inhibitor, or tat&rsg-Exo+miR-15b-5p inhibitor (1000 μg / mL). Mice were sacrificed 24 hours after injection, and the brains were isolated. DiR biodistribution was analyzed using an AUY220 in vivo imaging system. DiR fluorescence signal intensity was measured, and quantitative fluorescence analysis was performed using the system's built-in software.

[0042] Exosomes stained with DiR were administered intravenously to mice. Figure 4 As shown in Figures A and B, fluorescence analysis and quantification revealed that, compared to other exosome groups, the TAT&RVG-Exo + miR-15b-5p inhibitor group exhibited higher DiR fluorescence accumulation in the brain, indicating that TAT&RVG-Exo has stronger permeability in the BBB and can more effectively target the infarcted area within the brain. Furthermore, the TAT&RVG-Exo + miR-15b-5p inhibitor group showed the most significant inhibitory effect on MCAO / r-induced miR-15b-5p upregulation, further confirming that TAT&RVG-Exo can effectively penetrate the BBB and target the infarcted area. Figure 4 C).

[0043] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an engineered exosome loaded with a microRNA inhibitor modified with TAT and RVG dual-targeting peptides, characterized in that, Includes the following steps: S1. Link CP05 to TAT peptide and label it with FITC to prepare FITC-labeled CP05-TAT; S2. CP05 was linked to the RVG peptide and labeled with 5-TAMRA to obtain 5-TAMRA-labeled CP05-RVG; S3. The exosomes were mixed with FAM-CP05-TAT peptide and 5-TAMRA-CP05-RVG peptide and incubated to obtain the modified exosomes; S4. The modified exosomes were loaded with miR-15b-5p inhibitors by ultrasound to prepare engineered TAT and RVG dual-targeting peptide modified exosomes loaded with microRNA inhibitors.

2. The preparation method according to claim 1, characterized in that, The amino acid sequence of the FITC-labeled CP05-TAT described in step S1 is shown in SEQ ID NO.

1.

3. The preparation method according to claim 1, characterized in that, The amino acid sequence of the 5-TAMRA-labeled CP05-RVG described in step S2 is shown in SEQ ID NO.

2.

4. The preparation method according to claim 1, characterized in that, The incubation conditions described in step S3 are 2-6℃ and the incubation time is 5-7h.

5. The preparation method according to claim 1, characterized in that, The mass ratio of exosomes, FAM-CP05-TAT peptide and 5-TAMRA-CP05-RVG peptide in step S3 is 50:1:

1.

6. The preparation method according to claim 1, characterized in that, The ultrasonic loading conditions described in step S4 are 6 cycles of treatment, each with a 30-second on / off cycle, and a 30-second cooling time between each cycle.

7. The preparation method according to claim 1, characterized in that, In step S4, the amount of miR-15b-5p inhibitor and modified exosomes used is 250-350 nM miR-15b-5p inhibitor and 80-120 ng modified exosomes.

8. An engineered TAT and RVG dual-targeting peptide modified exosome loading microRNA inhibitor prepared by the preparation method according to any one of claims 1-7.

9. The use of an engineered TAT and RVG dual-targeting peptide modified exosome loaded with a microRNA inhibitor as described in claim 8 in the preparation of a medicament for treating cerebral ischemia-reperfusion injury.

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