MiR-29b-2-5p-loaded targeted kidney engineered exosome and application thereof in diabetic nephropathy

Abstract

The invention provides an engineered exosome loaded with miR-29b-2-5p targeting kidney and application of the engineered exosome in diabetic nephropathy, and relates to the technical field of biological pharmacy. It is found for the first time that miR-29b-2-5p has the effect of targeted negative regulation of GSK3beta-mediated DN renal tubule injury in DN, and a new idea is provided for targeted treatment of DN. The engineering exosome LT-Exo modified by LTH and miR-29b-2-5p is constructed through combination of 3D culture and targeted modification and miRNA loading technologies, and the stability of the structure and the function of the exosome can be maintained by the engineering exosome LT-Exo modified by the LTH and the miR-29b-2-5p. The LTH-Exo provided by the invention realizes high load to the miR-29b-2-5p and high targeting to the kidney, improves diabetes-related metabolic disorder and kidney injury by enhancing the targeting delivery capacity of the miR-29b-2-5p, plays a significant role in key pathological processes such as GSK3beta-mediated oxidative stress and ferroptosis, provides a new strategy for DN precise molecular therapy, and has a broad application prospect. And a theoretical basis is also laid for clinical application of the engineered exosome to other diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and particularly relates to an engineered exosome loaded with miR-29b-2-5p and targeted to the kidney and its application in diabetic nephropathy. Background Art

[0002] With the increasing prevalence of diabetes globally, the incidence of diabetic nephropathy (DN) has increased significantly and has become a public health challenge that urgently needs to be addressed. Currently, the treatment of DN faces many bottlenecks. Although classic renin-angiotensin system (RAAS) inhibitors (such as ACEI and ARB) can reduce proteinuria and delay the decline of renal function, they cannot prevent the progression of DN. New hypoglycemic drugs, such as GLP-1 receptor agonists and SGLT2 inhibitors, although showing renal protective effects in specific populations, still have limitations in indications and adverse reactions. In addition, DN lacks an individualized treatment strategy based on pathological mechanisms, and the current standardized treatment regimens have uneven efficacy in different patients. Limited anti-fibrotic and anti-inflammatory treatment methods, lack of early diagnostic markers, and limited treatment options for end-stage DN further exacerbate the challenges of clinical management. Future research should focus on precision medicine, individualized treatment, development of new targeted drugs, and optimization of early diagnostic strategies to improve the prognosis of patients.

[0003] Glycogen synthase kinase 3 beta (GSK3β) is a key serine / threonine protein kinase that plays an important role in the occurrence and progression of diabetes-related kidney injury. Studies have shown that the activity of GSK3β is enhanced under hyperglycemic conditions, leading to disorders of glucose and lipid metabolism, abnormal activation in diabetes-related tubular injury, especially a significant increase in expression in the renal proximal tubules. In addition, GSK3β can induce inflammation, oxidative stress, apoptosis, and renal interstitial fibrosis through signal pathways such as Wnt / β-catenin and PI3K / Akt, exacerbating kidney injury. Therefore, targeted regulation of GSK3β activity in the renal tubules of DN, inhibiting GSK3β can significantly reduce the inflammatory response, inhibit apoptosis and fibrosis, and has potential renal protective effects, and is expected to relieve diabetes-related kidney injury. Some small molecule GSK3β inhibitors (such as TDZD-8 and LY2090314) have shown potential in improving DN in preclinical studies. However, their oral or intravenous injection is often accompanied by systemic toxicity, and the drug has limited enrichment in the kidney. Although GSK3β has become a new target for the treatment of DN, the current specific inhibition of it in the kidney still faces challenges, and the targeted inhibition strategy is not yet perfect.

[0004] In recent years, exosomes have become a new strategy for the treatment of DN due to their role in intercellular communication, delivery of bioactive molecules (such as miRNAs, lncRNAs, proteins, etc.), and good biocompatibility. Exosomes can serve as drug carriers to target and deliver GSK3β inhibitors or RNA interference molecules, enhance the inhibitory effect of GSK3β, and reduce the progression of DN inflammation and fibrosis. Studies have shown that exosomes loaded with small molecule GSK3β inhibitors can improve their bioavailability and significantly alleviate renal fibrosis in diabetic model mice. In addition, exosomes mediate the delivery of miRNAs (such as miR-26a, miR-135b) to downregulate the expression of GSK3β. However, the existing efficiency of loading GSK3β inhibitors into exosomes is low, affecting their therapeutic effect, and the specific mechanism of exosome targeting of GSK3β is not yet clear. Summary of the Invention

[0005] (I) Technical problems to be solved

[0006] In view of the deficiencies of the prior art, the present invention provides an engineered exosome loaded with miR-29b-2-5p and targeting the kidney, as well as its application in diabetic nephropathy.

[0007] (II) Technical solutions

[0008] To achieve the above objectives, the present invention is realized through the following technical solutions:

[0009] In the first aspect, the present invention provides the application of miR-29b-2-5p in the preparation of a drug for negatively regulating GSK3β.

[0010] Specifically, the drug includes any one of a fusion protein, a recombinant plasmid, or an exosome overexpressing miR-29b-2-5p.

[0011] In the second aspect, the present invention provides an engineered exosome loaded with miR-29b-2-5p and targeting the kidney, which is an exosome with a targeting peptide LTH modified on its surface and loaded with miR-29b-2-5p.

[0012] Specifically, the amino acid sequence of the targeting peptide LTH is LTHVVWL.

[0013] In the third aspect, the present invention provides a method for preparing the above-mentioned engineered exosome, and the engineered exosome is prepared according to the following steps:

[0014] I. Preparation of exosomes

[0015] HEK293 cells were cultured using a 3D cell culture method. After 5 - 7 days of culture, the culture supernatant was collected and centrifuged at 2000×g for 10 min; the supernatant was collected and centrifuged at 10000×g for 30 min; the supernatant was collected and centrifuged at 200000×g for 2 h; the supernatant was discarded, and the exosome precipitate was collected. The exosome precipitate was resuspended in PBS solution and centrifuged at 10000×g for 30 min, and the supernatant was discarded. The precipitate was the exosome;

[0016] II. Targeted modification of exosomes

[0017] (1) Preparation of lipid - anchored targeting peptide solution: Lipid - anchored targeting peptide LTH was added to the lipid - anchored targeting peptide dissolution solution and dissolved thoroughly. Then, it was adjusted to a concentration of 1 μg / L with the lipid - anchored targeting peptide dissolution solution and stored at - 20°C in the dark for later use;

[0018] (2) Co - incubation of exosomes with lipid - anchored targeting peptide: 500 μg of exosomes and 100 μL of lipid - anchored targeting peptide solution were taken, added to the co - incubation reaction solution, and shaken and incubated at 25°C and 250 rmp for 3 h; then transferred to 4°C and statically incubated for 24 h to obtain targeted exosomes;

[0019] III. Preparation of targeted engineered exosomes loaded with miR - 29b - 2 - 5p

[0020] (1) Encapsulation of miR - 29b - 2 - 5p

[0021] CY3 - labeled 5' end of has - miR - 29b - 2 - 5p was purchased from Shanghai Sangon Biotech Co., Ltd.; Reagents were added in the following order and amounts: 1000 pmol miRNA → 1000 μg targeted exosomes → 200 μL ETP → reaction solution accounting for 1 / 10 of the exosome volume; Incubated at 37°C in the dark with continuous shaking at 150 rpm for 2 h;

[0022] (2) Removal of free miR - 29b - 2 - 5p: The incubated sample was transferred to a 100 kDa ultrafiltration tube; Wash Buffer was added to make up to 4 mL, and the sample was gently resuspended; Centrifuged at 4000×g for 30 minutes at room temperature to concentrate the volume to about 250 μL;

[0023] (3) Washing: Repeat step (2) for two washes, and finally concentrate the sample to 250 μL to obtain targeted engineered exosomes loaded with miR - 29b - 2 - 5p.

[0024] Fourthly, the present invention provides the application of the above - mentioned engineered exosomes in the preparation of drugs for treating diabetic nephropathy.

[0025] (III) Beneficial effects

[0026] The present invention has the following advantages:

[0027] 1. We first discovered that miR-29b-2-5p was differentially downregulated in DN patients and high-glucose-induced HK-2 cells, and was negatively correlated with the expression of GSK3β, indicating that miR-29b-2-5p has a role in targeting and negatively regulating GSK3β-mediated renal tubular injury in DN, providing a new idea for targeted treatment of DN.

[0028] 2. The present invention constructed engineered exosomes LTH-Exo modified with LTH and miR-29b-2-5p by 3D culture combined with targeted modification and miRNA loading technology. The LTH-Exo provided by the present invention can better maintain the stability of the structure and function of exosomes, providing a basis for its therapeutic application in diseases.

[0029] 3. The LTH-Exo provided by the present invention significantly improved renal targeting in the db / db mouse model. In addition, natural miR-29b-2-5p is easily degraded by RNase in vivo, resulting in low bioavailability. Through engineering transformation, the present invention effectively encapsulated miR-29b-2-5p in exosomes with high renal targeting, avoiding its degradation in peripheral blood, thereby prolonging the half-life and increasing its bioavailability, and enhancing the application potential of miR-29b-2-5p in the treatment of DN. The LTH-Exo provided by the present invention can effectively increase the expression level of miR-29b-2-5p in renal tubules, providing an important basis for its role in DN-related pathological processes.

[0030] 4. The LTH-Exo provided by the present invention can improve diabetes-related metabolic abnormalities and kidney damage by enhancing the targeted delivery ability of miR-29b-2-5p, and play a significant role in key pathological processes such as oxidative stress and ferroptosis mediated by GSK3β. Compared with ordinary exosomes, LTH-Exo shows obvious advantages in treatment efficiency and targeting specificity. It provides a new strategy for precision molecular therapy of DN and also lays a theoretical foundation for the clinical application of engineered exosomes in other diseases. Brief Description of the Drawings

[0031] Figure 1 : Figure 1 A: Heat map drawn based on high-throughput gene chip data, showing the miRNA expression differences between DN and Con kidney tissues. The colors from blue to red represent the low to high expression levels of miRNAs respectively; Figure 1 B: Bioinformatics tools miRDB and mirmap were used to predict the potential target miRNAs of GSK3β, and intersection analysis was performed with the miRNAs with low expression in DN patients (DN-down) to screen the miRNAs related to GSK3β and with low expression in DN; Figure 1C: Detailed information of miRNA, including fold change of expression, chromosome location and its sequence.

[0032] Figure 2 represents the relative expression level of miR-29b-2-5p under different conditions; Figure 2 A: The expression level of miR-29b-2-5p in HK-2 cells was detected by RT-qPCR. The NG group was used for normalization analysis. **p < 0.01 compared with the NG group. Data are expressed as mean ± standard error. The t-test was used for comparison between two groups. The experiment was repeated 5 times; Figure 2 B: The expression level of miR-29b-2-5p in the renal tissue (Human Kidney) of DN patients and normal controls (Con) was detected by RT-qPCR. The Con group was used for normalization analysis. ***p < 0.001 compared with the Con group. Data are expressed as mean ± standard error. The t-test was used for comparison between two groups. The experiment was repeated 5 times; Figure 2 C: Mature sequence information of miR-29b-2-5p in Homo sapiens and Mus musculus, including organism, accession number and sequence; the sequence information was obtained from the miRbase website.

[0033] Figure 3 represents the prediction and functional verification of the binding site between miR-29b-2-5p and GSK3β; Figure 3 A: TargetScanHuman was used to predict the potential binding site between miR-29b-2-5p and the 3'UTR of GSK3β; Figure 3 B: Dual-luciferase reporter gene assay, ***P < 0.001; data are expressed as mean ± standard error. The t-test was used for comparison between two groups; the experiment was repeated 3 times. Ns indicates no statistical difference between two groups; Figure 3 C: Molecular interaction network diagram. The information was obtained from the website STRING (https: / / string-db.org / ). Each connecting line represents the possible interaction of these molecules in biological processes; Figure 3 D: Mature sequence information of miR-29b-2-5p in Homo sapiens and Mus musculus, including organism, accession number and sequence; the sequence information was obtained from the miRbase website.

[0034] Figure 4 represents the verification of the stable exosome function of LTH-Exo; Figure 4A: NTA was used to detect the concentration of exosomes. ***P < 0.001 compared with the General Cultured group. Data are presented as mean ± standard error. The t-test was used for comparison between the two groups. The experiment was repeated 3 times. Figure 4 B: Mean and median particle size values of exosomes from general culture (General Cultured Non-engineered Exo). Figure 4 C: Mean and median particle size values of exosomes from 3D culture (3D Cultured Non-engineered Exo). Figure 4 D: Mean and median particle size values of miR-29b-2-5p-loaded targeted engineered exosomes LTH-Exo (3D Cultured Engineered Exo) under 3D culture conditions. Figure 4 E: TEM was used to observe the morphology of exosomes, showing a typical cup-shaped vesicle structure (marked by red arrows). Scale bar: 200 nm. The inset is a magnified view of the local area.

[0035] Figure 4 F: Western blot was used to detect the expression levels of TSG101, CD81, HSP70, and Calnexin proteins in each group. Figure G: RT-qPCR was used to evaluate the expression level of miR-29b-2-5p, with normalization analysis performed using the Exo group. ***P < 0.001 compared with the Exo group. Data are presented as mean ± standard error. The t-test was used for comparison between the two groups. The experiment was repeated 3 times.

[0036] Figure 5 This shows the distribution of LTH-Exo in mice and the kidneys. Figure 5 A: In vivo fluorescence imaging of small animals. The right side shows the intensity of the fluorescence signal. Figure 5 B: Fluorescence imaging of ex vivo kidneys. The lower side shows the intensity of the fluorescence signal.

[0037] Figure 6 This shows the targeted delivery of miR-29b-2-5p by LTH-Exo to damaged kidneys. Figure 6 A: Distribution of exosomes (Exo, yellow signal) and miR-29b-2-5p (red signal) in kidney tissues. Nuclear staining was performed using DAPI (blue signal). The scale bar is as shown. Figure 6 B: Relative quantitative analysis of exosome fluorescence signals (n = 3, ***P < 0.001). Figure 6 C: RT-qPCR was used to measure the expression level of miR-29b-2-5p (n = 6, ***P < 0.001). Data are presented as mean ± standard error. The t-test was used for comparison between the two groups.

[0038] Figure 7 LTH-Exo treatment improved the deterioration of blood glucose and weight gain in db / db mice; Figure 7 A: Schematic diagram of animal experiment design: db / db mice were treated with PBS, Exo, and LTH-Exo respectively starting from 8 weeks of age, and blood samples (marked as red water droplets) were collected regularly and body weight was measured. The experiment continued until 20 weeks of age, and urine was collected (marked as yellow water droplets); Figure 7 B: Dynamic changes in fasting blood glucose levels of mice, n = 6, compared with the db / db + Exo group, ***P < 0.001; Figure 7 C: Dynamic changes in body weight of mice, n = 6, compared with the db / db + Exo group, *P < 0.05, ***P < 0.001. Data are expressed as mean ± standard error, and the t-test was used for comparison between the two groups.

[0039] Figure 8 LTH-Exo improved the related indexes of kidney function in db / db mice; Figure 8 A: Results of urine albumin / creatinine ratio (uACR) measurement. n = 6, **P < 0.01; Figure 8 B: SDS-PAGE and Western blot were used to detect the expression levels of urinary albumin and urinary NGAL; glomerular filtration rate (GFR) of mice ( Figure 8 C), serum creatinine (SCr) ( Figure 8 D), and blood urea (BUN) ( Figure 8 E) values, n = 6, *P < 0.05, **P < 0.01. Data are expressed as mean ± standard error, and the t-test was used for comparison between the two groups.

[0040] Figure 9 LTH-Exo improved the pathological changes of renal tissue in db / db mice; PAS staining: The renal tissue of db / db mice showed exfoliation of renal tubular epithelial cells, vacuolar degeneration of renal tubules, and dilation of renal tubules (indicated by red arrows); Masson staining: The degree of fibrosis in the renal tissue of db / db mice increased significantly (collagen deposition was shown in blue), scale bar: 50 μm.

[0041] Figure 10 LTH-Exo promoted the expression of miR-29b-2-5p in the renal tissue of db / db mice; Immunofluorescence staining of renal tissue: LTL (green), miR-29b-2-5p (red), and DAPI (blue, nucleus). Scale bar: 50 μm.

[0042] Figure 11 LTH-Exo improved renal tubular injury in db / db mice by inhibiting GSK3β; Figure 11A: Western blot showed the expression of GSK3β in renal tissues; Figure 11 B: Quantitative analysis of gray values. n = 3, *P < 0.05; Figure 11 C: Immunofluorescence staining, LTL (green), GSK3β (red), and DAPI (blue, nucleus); Zoomed-in local view; Immunohistochemical staining of GSK3β, scale bar as shown; Figure 11 D: Figure 11 Quantitative immunofluorescence analysis of C, normalized to the db / m group, n = 3, **P < 0.01. Data are expressed as mean ± standard error. t-test was used for comparison between two groups.

[0043] Figure 12 For LTH-Exo to downregulate the expression of KIM1 and NGAL; Figure 12 A: Western Blot showed the expression of KIM1 and NGAL; Figure 12 B: Quantitative analysis results of gray values of KIM1 and NGAL proteins, n = 3, **P < 0.01; Figure 12 C: Immunohistochemical staining analysis of KIM1, scale bar: 100μm. Data are expressed as mean ± standard error. t-test was used for comparison between two groups.

[0044] Figure 13 For LTH-Exo to increase the expression of Nrf2; Figure 13 A: Western Blot was used to detect the expression of Nrf2 protein; Figure 13 B: Quantitative analysis results of Nrf2 gray values, n = 3, **P < 0.01; Figure 13 C: Immunohistochemical staining results of Nrf2 in renal tissues. The zoomed-in view showed nuclear positivity (red arrow), scale bar: 100μm. Data are expressed as mean ± standard error. t-test was used for comparison between two groups.

[0045] Figure 14 For LTH-Exo to increase the expression of GPX4; Figure 14 A: Western Blot was used to detect the expression of GPX4 protein; Figure 14 B: Quantitative analysis of GPX4 gray values, n = 3, **P < 0.01; Figure 14 C: Immunohistochemical staining results of GPX4 in renal tissues, scale bar: 100μm. Data are expressed as mean ± standard error. t-test was used for comparison between two groups.

[0046] Figure 15 For LTH-Exo to alleviate ferroptosis in db / db mice; Figure 15 A: IRON content value, n = 6, **P < 0.01. Figure B: GSH-PX activity, n = 6, **P < 0.01;Figure 15 C: MDA value, n = 3, **P < 0.01; Figure 15 D: Transmission electron microscopy (TEM) showed ultrastructural changes of renal mitochondria. Scale bar: 200 nm. Data are expressed as mean ± standard error. The t-test was used for comparison between two groups. Specific implementation manners

[0047] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0048] Example 1

[0049] Application of miR-29b-2-5p in negatively regulating GSK3β in DN

[0050] 1 Predicting key miRNAs negatively regulating GSK3β in DN

[0051] Using high-throughput gene expression profiles to screen miRNAs related to DN ( Figure 1 A). Based on the bioinformatics databases miRDB and mirmap, predicting miRNA targets interacting with GSK3β ( Figure 1 B). Performing intersection analysis on the predicted miRNAs and gene chip data of renal tissues of DN patients to screen miRNAs that are lowly expressed in renal tissues of DN patients and are closely related to GSK3β, namely miR-29b-2-5p and miRNA-199a-5p. Performing differential fold analysis on the miRNAs ( Figure 1 C), and the expression change fold of miR-29b-2-5p is more significant (-2.48-fold). Therefore, we took miR-29b-2-5p as the research core, focused on its molecular mechanism mediated by regulating GSK3β, and explored its regulatory role and potential therapeutic target value in DN renal tubular injury.

[0052] 2 miR-29b-2-5p is lowly expressed in high glucose-induced HK-2 cells and db / db mice

[0053] We used real-time fluorescence quantitative PCR (RT-qPCR) to detect the relative expression level of miR-29b-2-5p in HK-2 cells cultured under normal glucose concentration (NG) and high glucose concentration (HG) conditions. The RT-qPCR results showed that the relative expression level of miR-29b-2-5p under HG was lower than that under NG conditions (**P < 0.01,Figure 2 A), suggesting that a high-glucose environment can inhibit the expression of miR-29b-2-5p in HK-2 cells.

[0054] To further verify this result, we detected the relative expression levels of miR-29b-2-5p in the renal tissues of DN patients and normal individuals. The RT-qPCR results showed that the expression level of miR-29b-2-5p in the renal tissues of DN patients was lower than that in normal individuals (***P<0.001, Figure 2 B).

[0055] In summary, the low expression of miR-29b-2-5p is closely related to the high-glucose environment and diabetes-related kidney lesions. Comparing the mature sequences of miR-29b-2-5p in humans and mice ( Figure 2 C), showing a high degree of homology between the two, provides an important theoretical basis and experimental foundation for subsequent cross-species functional studies.

[0056] 3 miR-29b-2-5p inhibits the expression of GSK3β

[0057] The TargetScanHuman website predicted the potential binding sites of miR-29b-2-5p in the 3'UTR region of GSK3β ( Figure 3 A). The results showed that the 3'UTR of GSK3β contains multiple highly conserved potential binding sites for miR-29b-2-5p. Dual-luciferase reporter gene assays verified the binding of miR-29b-2-5p to GSK3β. Under the condition of wild-type GSK3β 3'UTR (GSK3β-WT), miR-29b-2-5p decreased luciferase activity (***P<0.001, Figure 3 B); under the condition of mutant GSK3β 3'UTR (GSK3β-MUT), no significant change in luciferase activity was observed, demonstrating that miR-29b-2-5p inhibits the expression of GSK3β by directly binding to its 3'UTR. The STRING database predicted the molecular interaction network among GSK3β, NFE2L2, and GPX4 in different species (humans and mice) ( Figure 3 C). In summary, miR-29b-2-5p may play an important role in the protein regulatory network of GSK3β, Nrf2, and GPX4.

[0058] We found that there was differential low expression of miR-29b-2-5p in DN patients and high-glucose-induced HK-2 cells, and it was negatively correlated with the expression of GSK3β. The above results indicate that miR-29b-2-5p has a role in targeting and negatively regulating GSK3β-mediated renal tubular injury in DN.

[0059] Example 2

[0060] 1. Preparation of engineered exosomes loaded with miR-29b-2-5p targeting the kidney (hereinafter referred to as LTH-Exo), which is specifically carried out according to the following steps:

[0061] 1. Preparation of exosomes

[0062] HEK293 cells were cultured using a 3D cell culture method. After 5 - 7 days of culture, the culture supernatant was collected and centrifuged at 2000×g for 10 min; the supernatant was collected and centrifuged at 10000×g for 30 min; the supernatant was collected and centrifuged at 200000×g for 2 h; the supernatant was discarded, and the exosome precipitate was collected. The exosome precipitate was resuspended in PBS solution and centrifuged at 10000×g for 30 min, and the supernatant was discarded. The precipitate was the exosome.

[0063] 2. Targeted modification of exosomes

[0064] (1) Preparation of lipid-anchored targeting peptide solution: Lipid-anchored targeting peptide was added to the lipid-anchored targeting peptide dissolution solution and dissolved thoroughly. Then, it was adjusted to a concentration of 1 μg / L with the lipid-anchored targeting peptide dissolution solution and stored at -20°C in the dark for later use.

[0065] (2) Co-incubation of exosomes with lipid-anchored targeting peptide: 500 μg of exosomes and 100 μL of lipid-anchored targeting peptide solution were taken, and co-incubation reaction solution was added. The mixture was shaken and incubated at 25°C and 250 rmp for 3 h; then it was transferred to 4°C and incubated statically for 24 h to obtain targeted exosomes.

[0066] 3. Preparation of LTH-Exo

[0067] (1) Encapsulation of miR-29b-2-5p

[0068] CY3-labeled 5' end of has-miR-29b-2-5p was purchased from Shanghai Sangon Biotech Co., Ltd. Reagents were added in the following order and amounts: 1000 pmol miRNA → 1000 μg of targeted exosomes → 200 μL of transport peptide ETP (Exosome-Transit Peptide) → reaction solution accounting for 1 / 10 of the exosome volume; incubated at 37°C in the dark with continuous shaking at 150 rpm for 2 h.

[0069] (2) Removal of free miR-29b-2-5p: The incubated sample was transferred to a 100 kDa ultrafiltration tube; Wash Buffer was added to make up to 4 mL, and the sample was gently resuspended; centrifuged at 4000×g for 30 minutes at room temperature to concentrate the volume to approximately 250 μL.

[0070] (3) Washing: Repeat step (2) for two washes, and finally concentrate the sample to 250 μL to obtain LTH-Exo.

[0071] II. Verification of the Functional Stability of LTH-Exo

[0072] Collect equal volumes of culture supernatants from general culture and 3D culture, extract exosomes by ultracentrifugation, and detect the particle size distribution and concentration of exosomes by nanoparticle tracking analysis (NTA) technology. The results show that the concentration of exosomes in 3D culture is increased compared with that in general culture (***General Cultured group, ***P<0.001, Figure 4 A). Under general culture conditions, the average particle size of exosomes is 85.22 nm and the median particle size is 80.75 nm ( Figure 4 B). The average particle size of exosomes in 3D culture is 85.04 nm and the median particle size is 82.25 nm ( Figure 4 C), indicating that 3D culture significantly improves the exosome secretion amount without affecting the exosome particle size. In addition, the average particle sizes (85.04 nm and 85.62 nm) and median particle sizes (82.25 nm and 82.25 nm) of non-engineered exosomes (Exo) and LTH-Exo in 3D culture are similar ( Figure 4 D), indicating that the engineering modification method provided by the present invention does not affect the exosome particle size. In summary, 3D culture not only improves the exosome secretion efficiency but also maintains the stability of particle size distribution, provides an efficient optimization strategy for large-scale preparation and functional research of exosomes, and lays a foundation for its clinical transformation.

[0073] Transmission electron microscopy (TEM) shows ( Figure 4 E) that both non-engineered exosomes (Non-engineered Exo, Exo) and engineered exosomes (Engineered Exo, LTH-Exo) present typical cup-shaped vesicle morphology (marked by red arrows) and have a uniform particle size distribution. In addition, the Western Blot results show that the marker proteins (HSP70, CD81, TSG101) of Exo and LTH-Exo are positively expressed and Calnexin (endoplasmic reticulum marker protein) is negatively expressed ( Figure 4 F). It shows that LTH-Exo is consistent with Exo in terms of morphology, particle size and marker protein expression, and has stable structure and function. The RT-qPCR results show that the expression level of miR-29b-2-5p in Exo is low, and the expression level of miR-29b-2-5p in LTH-Exo is up-regulated and the enrichment efficiency is improved (***Exo, ***P<0.001, Figure 4 G). Engineering modification enhances the ability of exosomes to carry specific miRNAs while maintaining good controllability and stability.

[0074] The above results indicate that the 3D culture environment improves the yield of exosomes, providing advantages for the efficient preparation of engineered exosomes. Through the combination of 3D culture, targeted modification, and miRNA loading techniques, the present invention constructs engineered exosomes LTH and miR-29b-2-5p-modified LTH-Exo, which can better maintain the stability of the structure and function of exosomes, providing a basis for their therapeutic application in diseases.

[0075] Example 3

[0076] Application of miR-29b-2-5p-loaded and kidney-targeted engineered exosomes (hereinafter referred to as LTH-Exo) in diabetic nephropathy

[0077] 1 Experimental methods

[0078] The experimental animals were male db / db mice aged 6 - 8 weeks, randomly divided into four groups of 6 mice each, including: control group (db / m group), DN group (db / db group), DN normal exosome treatment group (db / db + Exo group), and DN engineered exosome treatment group (db / db + LTH-Exo group). The mice were housed under standard animal room conditions (constant temperature, constant humidity, 12-hour light / dark cycle, free access to water and food). After one week of adaptive feeding, PBS (db / m group, db / db group) or exosomes (db / db + Exo group, db / db + LTH-Exo group; 100 μg) were injected via the tail vein once every 2 weeks, and fasting blood glucose was measured and mouse body weight was monitored every 4 weeks. At 20 weeks, random urine samples were collected from mice in each group, and then all mice were euthanized at 20 weeks, and kidney and blood specimens were collected rapidly.

[0079] 2 Detection indicators

[0080] (1) Detect the urinary albumin-to-creatinine ratio (uACR) in the urine samples of mice in each group.

[0081] (2) Detect the relative expression level of miR-29b-2-5p in the renal tissues of mice in each group by real-time fluorescence quantitative PCR (RT-PCR).

[0082] (3) Analyze the urinary protein composition of mice in each group by SDS-PAGE combined with Coomassie Brilliant Blue staining.

[0083] (4) Perform PAS staining, Masson staining, immunohistochemistry (IHC) staining, immunofluorescence (IF) staining, and RNA fluorescence in situ hybridization (FISH) staining on paraffin sections of renal tissues of mice in each group

[0084] (5) Observe the renal tissues of mice in each group by transmission electron microscopy.

[0085] (6) Protein immunoblotting (Western blot) was used to detect the expression levels of GSK3β, Nrf2, GPX4, KIM-1, and NGAL in the kidney tissues of each group of mice.

[0086] (7) The contents of glutathione peroxidase (GSH-PX), malondialdehyde (MDA), and iron ions in the kidney tissues of each group of mice were measured.

[0087] (8) Serum creatinine (SCr) and blood urea nitrogen (BUN) in the sera of each group of mice were measured.

[0088] 3 In vivo imaging of mice

[0089] (1) 16-week-old db / db mice and db / m mice were selected, adaptively fed for 7 days to ensure good health, and grouped according to the experimental requirements. There were 5 mice in each group.

[0090] (2) Exosome injection: Experimental groups: Group 1: db / m mice + injected with PBS solution, 100 μL; Group 2 (db / db mice + injected with PBS solution, 100 μL); Group 3: db / db mice + group of DIR-fluorescently labeled ordinary exosomes (injected with 100 μg of DIR-fluorescently labeled ordinary exosomes, Exo, 100 μL); Group 4: db / db mice + group of DIR-fluorescently labeled engineered exosomes (injected with 100 μg of DIR-fluorescently labeled engineered exosomes, LTH-Exo, 100 μL).

[0091] Primary injection: According to the allocation of each group, PBS or DIR-fluorescently labeled exosome solution was injected via the tail vein, with a dose of 100 μg / mouse, and a total of 100 μL of solution was injected into each group.

[0092] Second injection: 6 hours after the primary injection, the same dose (100 μg / mouse) of PBS or DIR-fluorescently labeled exosome solution (according to the grouping) was injected again via the tail vein.

[0093] (3) Pre-experiment of in vivo fluorescence imaging: Small animal in vivo fluorescence imaging was performed every 12 hours after each injection. Before imaging, the small animals were anesthetized, their skin was prepared, and disinfected. An appropriate fluorescence imaging device was used, and the excitation wavelength and emission wavelength of the DIR dye were set to 754 nm and 778 nm, respectively, to observe the distribution of exosomes and the change in fluorescence intensity in the bodies of each group of mice.

[0094] (4) Incision to expose the kidney: During the formal experiment, at the time of the maximum fluorescence intensity. The small animals were anesthetized, their skin was prepared, and disinfected. At 1 cm on the left and right sides, 1 cm below the costal margin angle on the back of the mouse, a 1-cm-long longitudinal incision was made. Sterile surgical instruments (such as forceps and scissors) were used to carefully separate the skin and muscle to expose the kidney.

[0095] (5) Data analysis: Quantitative analysis was performed on the in vivo imaging results to compare the fluorescence signal intensity and distribution between different groups. The distribution differences of exosomes in mice of different groups in vivo and the effect of time after injection on fluorescence intensity were analyzed by statistical methods.

[0096] 4 Statistical analysis

[0097] The Image J software was used to analyze the protein expression levels of semi-quantitative immunohistochemistry, immunofluorescence and Western blot bands. The GraphPad Prism 8.0 software was used for analysis. The independent samples t-test was used for comparison between two groups. Before performing the t-test, the data were first subjected to normality test (Kolmogorov-Smirnov or Shapiro-Wilk test) and homogeneity of variance test (Levene’s Test). For two groups of data that conform to normal distribution and homogeneity of variance, the unpaired t-test (Student’s t-test) was used for comparison; if the variances are not homogeneous, the Welch corrected t-test was used. For data that do not conform to normal distribution, the non-parametric test of Mann-Whitney U test was used. All normally distributed data were expressed as mean ± standard error (Mean ± SEM); non-normally distributed data were expressed as median (interquartile range). The statistical test was a two-sided test, and when P < 0.05, the difference was considered statistically significant.

[0098] 5 Results

[0099] 5.1 High targeting and high loading capacity of LTH-Exo

[0100] 5.1.1 LTH-Exo targets damaged kidneys

[0101] Sixteen-week-old db / m and db / db mice were injected via the tail vein twice (with a 6-hour interval) with PBS, DIR fluorescently labeled exosomes (Exo, 100 μg / mouse), and DIR fluorescently labeled engineered exosomes (LTH-Exo, 100 μg / mouse). In the preliminary experiment, the fluorescence intensity was observed every 12 hours, and the results showed that the fluorescence intensity was the strongest 24 hours after injection. To avoid the interference of the liver, spleen and skin on the fluorescence intensity of the kidneys, 24 hours after the exosome injection, a 1-cm long incision was made longitudinally 1 cm below the costal margin angle on the back of the mouse, 1 cm to the left and right, and the kidneys were exposed for imaging. In vivo imaging of small animals showed ( Figure 5A), There was no fluorescence signal in db / m and db / db mice injected with PBS. Ordinary exosomes were enriched in the kidneys of db / db mice, but the signal was weak. This indicates that the renal targeting of ordinary exosomes is limited. LTH-Exo showed a high-density aggregation in the kidneys of db / db mice, and the fluorescence signal was enhanced. The fluorescence imaging results of mouse kidney tissues showed ( Figure 5 B), The fluorescence signal of ordinary exosomes in the kidneys was weak and distributed dispersedly. The fluorescence signal of LTH-Exo was enhanced and aggregated in specific regions of the kidneys. In summary, LTH-Exo has stronger targeting in db / db mice.

[0102] 5.1.2 Delivery of miR-29b-2-5p to damaged kidneys by LTH-Exo

[0103] Fluorescence localization was performed on fluorescently labeled exosomes. The immunofluorescence results of kidney tissue sections showed that there was no fluorescence signal in mice injected with PBS. The signal of fluorescently labeled ordinary exosomes in the kidney tissues of db / db mice was weak; after treatment with engineered exosomes, the fluorescence signal was significantly enhanced ( Figure 6 A). Quantitative analysis verified that the fluorescence intensity of the LTH-Exo treatment group was higher than that of other groups ( Figure 6 B, ***P<0.001). In summary, the engineered exosomes have excellent targeting.

[0104] FISH results showed that the fluorescence intensity of miR-29b-2-5p in db / db mice was weaker than that in db / m. After treatment with engineered exosomes, the fluorescence signal was significantly enhanced ( Figure 6 A). RT-qPCR results showed ( Figure 6 C), Compared with the db / m group, the expression of miR-29b-2-5p in the kidneys of the db / db untreated group and the simple exosome treatment group was significantly decreased. In addition, there was no difference between the db / db untreated group and the simple exosome treatment group, indicating that simple exosome treatment did not restore the expression level of miR-29b-2-5p. However, LTH-Exo treatment increased the expression level of miR-29b-2-5p (Figure C, ***P<0.001). In summary, the present invention provides that LTH-Exo can accurately target and deliver miR-29b-2-5p to damaged kidneys.

[0105] 5.5 Improvement of kidney injury in db / db mice by LTH-Exo treatment

[0106] 5.2.1 Improvement of blood glucose deterioration and weight gain in db / db mice by LTH-Exo treatment

[0107] 8-week-old db / db mice were randomly assigned to the PBS group, the normal exosome treatment group (Exo), and the engineered exosome group (LTH-Exo), and were injected via the tail vein once a week for 12 weeks. db / m mice were injected with PBS once a week as a control. Fasting blood glucose and body weight were measured every 4 weeks. At 20 weeks of age, the mice were euthanized ( Figure 7 A).

[0108] Compared with the db / m group at 20 weeks of age, the blood glucose of the mice in the db / db group and the Exo group deteriorated, while the blood glucose of the mice in the LTH-Exo group was significantly improved (*** db / db + Exo group, P < 0.001). Compared with the db / m group, the body weight of the mice in the db / db group and the Exo group increased, while the body weight gain of the mice in the LTH-Exo group was improved (*** db / db + Exo group, P < 0.001). It is shown that the treatment with engineered exosomes can not only improve blood glucose control, but also regulate the accompanying symptoms of diabetes ( Figure 7 B-C).

[0109] 5.2.2 LTH-Exo treatment improves the renal function of db / db mice

[0110] The urinary creatinine / urinary albumin ratio (uACR) is a sensitive indicator of tubulo-glomerular injury. Compared with the db / m group, the uACR values in the db / db group and the Exo group were significantly increased. The uACR value in the LTH-Exo group was significantly improved (** P < 0.01, Figure 8 A). Coomassie brilliant blue staining of urinary protein ( Figure 8 B) showed that the staining intensity of urinary protein in the mice of the db / db group and the Exo group was significantly stronger than that in the db / m group. The urinary protein in the LTH-Exo group was significantly decreased compared with the db / db group and the Exo group. It is suggested that LTH-Exo treatment can reduce kidney injury and urinary protein excretion in db / db mice.

[0111] The glomerular filtration rate (GFR) of the mice in the db / db group and the Exo group at 20 weeks of age decreased, and LTH-Exo treatment improved GFR (** P < 0.01, Figure 8 C). The serum creatinine (SCr) levels of the mice in the db / db group and the Exo group at 20 weeks of age increased, indicating impaired renal function, while LTH-Exo treatment decreased the SCr level (* P < 0.05, Figure 8 D). In addition, the blood urea nitrogen (BUN) of the mice in the db / db group and the Exo group at 20 weeks of age increased, and LTH-Exo treatment decreased BUN (** P < 0.01, Figure 8 E). In summary, LTH-Exo treatment improves kidney injury and functional indexes in DN mice, showing potential renal protective effects.

[0112] 5.2.3 LTH-Exo treatment improves renal pathological damage in db / db mice

[0113] PAS results showed that obvious tubular damage occurred in the kidneys of mice in the db / db group and the Exo group, including phenomena such as exfoliation of tubular epithelial cells, vacuolar degeneration of renal tubules, and dilation of renal tubules. The tubular damage in the LTH-Exo treatment group was reduced, and the renal tubular morphology was relatively normal. Masson staining results showed that obvious renal tubular fibrosis changes occurred in the kidneys of the db / db group and the Exo group, with increased deposition of collagen fibers, and significant fibrosis characteristics were also shown in the renal interstitium. The degree of renal tubular fibrosis in LTH-Exo decreased, and collagen deposition decreased. Engineered exosomes alleviated the pathological changes of DN by regulating the renal fibrosis pathway. The results are as Figure 9 shown.

[0114] 5.3 LTH-Exo regulates GSK3β to improve renal tubular injury in db / db mice

[0115] 5.3.1 LTH-Exo treatment increases the expression level of miR-29b-2-5p in renal tubules

[0116] LTL (Lotus Tetragonolobus Lectin) is a renal tubular specific marker, showing the overall structure and distribution of renal tubules. Immunofluorescence showed that the renal tubular morphology of db / m mice was intact, and the LTL signal was strong and uniform; while the renal tubular structure of mice in the db / db group and the Exo group was disordered, and the LTL signal was weakened. The LTL signal of renal tubules in the LTH-Exo group of mice was enhanced, suggesting a reduction in renal tubular injury. miR-29b-2-5p was weakly expressed in db / m mice and less expressed in the db / db group and the Exo group. LTH-Exo treatment enhanced the expression of miR-29b-2-5p. Immunofluorescence co-staining showed that LTH-Exo enhanced the expression of miR-29b-2-5p in specific regions of renal tubules, highly overlapping with the renal tubular structure. In summary, engineered exosomes promoted the delivery of miR-29b-2-5p. The results are as Figure 10 shown.

[0117] 5.3.2 LTH-Exo inhibits GSK3β to improve renal tubular injury

[0118] The activation of GSK3β is related to apoptosis, fibrosis, and inflammatory responses in DN. Western blot( Figure 11 A) and quantitative analysis showed that compared with the db / m group, the expression of GSK3β in the db / db group and the Exo group increased, while LTH-Exo treatment inhibited GSK3β (*P<0.05, Figure 11 B). Engineered exosomes regulated the expression of GSK3β. The results of immunofluorescence in renal tissues showed( Figure 11C), The renal tubular structure of db / m mice was intact, with strong and uniform LTL signals and weak GSK3β expression; the renal tubular structure of db / db group and Exo group mice was disordered, LTL signals were weakened, and GSK3β expression was enhanced; LTL signals in the LTH-Exo group were restored and GSK3β signals were weakened, suggesting a reduction in renal tubular injury. GSK3β immunohistochemistry showed that the expression of GSK3β in the renal tubules of the db / db group and Exo group was enhanced and the distribution range was wider; LTH-Exo downregulated the expression of GSK3β and the staining intensity was weakened. The results of GSK3β / LTL immunofluorescence quantitative analysis were consistent (**P<0.01, Figure 11 D). In summary, LTH-Exo improves the injury of renal tubules in db / db mice by inhibiting GSK3β and restores the integrity of renal tubular structure.

[0119] 5.3.3 LTH-Exo reduces renal tubular injury proteins in db / db mice

[0120] KIM1 and NGAL are kidney injury markers. Western Blot showed that ( Figure 12 A), compared with the db / m group, the expressions of KIM1 and NGAL in the db / db group and Exo group were increased; however, the expressions of KIM1 and NGAL in the LTH-Exo group were decreased. The results of the gray value analysis of KIM1 / β-actin and NGAL / β-actin proteins were consistent (**P<0.01, Figure 12 B). Immunohistochemical staining showed that KIM1 was widely expressed in db / db mice and Exo mice. LTH-Exo treatment significantly reduced the expression of KIM1 ( Figure 12 C). In summary, LTH-Exo downregulates the expression of renal tubular injury proteins in DN.

[0121] 5.4 LTH-Exo improves renal tubular ferroptosis in db / db mice

[0122] 5.4.1 LTH-Exo increases the expression of Nrf2 in db / db mice

[0123] Nrf2 (Nuclear factor erythroid 2-related factor 2) is an important molecular marker related to oxidative stress. Western Blot ( Figure 13 A) and the gray value analysis results showed that Nrf2 in db / db mice and Exo mice was slightly increased. However, LTH-Exo treatment significantly increased the expression of Nrf2 (**P<0.01, Figure 13 B). The results of Nrf2 immunohistochemical staining of paraffin sections of renal tissue were consistent with the WB experimental results ( Figure 13C). The nuclear positivity in the kidney tissues of db / db mice and Exo mice increased slightly, while that in the LTH-Exo group increased significantly ( Figure 13 C, red arrow). In summary, LTH-Exo protects against kidney injury by regulating the antioxidant pathway and enhancing Nrf2 activity.

[0124] 5.4.2 LTH-Exo increases the expression of GPX4 in db / db mice

[0125] GPX4 (Glutathione peroxidase 4) is closely related to ferroptosis and inhibits ferroptosis by reducing the accumulation of peroxides. WB ( Figure 14 A) and the gray scale analysis results showed that GPX4 in db / db mice was significantly decreased. There was no improvement in GPX4 in db / db mice and Exo mice. In contrast, GPX4 in the LTH-Exo group increased significantly (**P<0.01, Figure 14 B). The results of GPX4 immunohistochemical staining of paraffin sections of kidney tissues were consistent with those of the WB experiment. In summary, LTH-Exo can relieve the pathological role of ferroptosis in DN by regulating the expression of GPX4.

[0126] 5.4.3 LTH-Exo alleviates ferroptosis in db / db mice

[0127] IRON (divalent iron), GSH-Px (glutathione peroxidase), and MDA (malondialdehyde) are important indicators of ferroptosis injury. Compared with the db / m group, IRON in db / db mice and Exo-treated mice increased significantly. LTH-Exo treatment significantly reduced IRON (**P<0.01, Figure 15 A). GSH-Px is one of the important antioxidant enzymes that inhibits ferroptosis by reducing the accumulation of peroxides. The expression of GSH-Px in the kidney tissues of db / db was significantly decreased. The expression of GSH-Px in the Exo-treated group was still low and did not return to the normal level. The expression of GSH-Px in the LTH-Exo treatment group increased significantly (**P<0.01, Figure 15 B). MDA is a lipid peroxidation product that reflects the accumulation of lipid peroxides in cells. The level of MDA in the db / db group increased significantly, and the level of MDA in the Exo-treated group was still high. The level of MDA in the LTH-Exo treatment group decreased significantly (**P<0.01, Figure 15 C).

[0128] Transmission electron microscopy (TEM) findings ( Figure 15D), The mitochondria in the renal tissues of db / db mice and Exo-treated mice were significantly damaged, showing mitochondrial atrophy, disappearance of the highly folded cristae in the inner membrane, dilation of the inner cavity, and partial vacuolization. The mitochondrial damage in the LTH-Exo treatment group was significantly improved. The mitochondrial morphology was regular, the structure of the mitochondrial cristae was restored, and there was no obvious dilation or vacuolization in the inner cavity of the mitochondria. In summary, LTH-Exo alleviates iron overload, regulates the activity of antioxidant enzymes, reduces lipid peroxidation and improves mitochondrial damage, inhibits ferroptosis, and improves kidney damage in db / db mice.

[0129] 6 Conclusions

[0130] As one of the most common and severe microvascular complications of diabetes, DN significantly reduces the quality of life of patients and increases the risks of ESRD and cardiovascular diseases. As a novel treatment strategy for DN, exosome therapy has potential. However, exosomes injected via the tail vein mainly accumulate in the liver and spleen, with a small amount distributed in the lungs and kidneys, which limits their therapeutic effects in target organs. Improving the targeting of exosomes is crucial for reducing non-target organ distribution and side effects.

[0131] The LTH-Exo provided by the present invention significantly improves the targeting to the kidneys in the db / db mouse model. In addition, natural miR-29b-2-5p is easily degraded by RNase in vivo, resulting in its low bioavailability. Through engineering transformation, the present invention effectively encapsulates miR-29b-2-5p in exosomes with high targeting to the kidneys, avoiding its degradation in peripheral blood, thereby prolonging the half-life and improving its bioavailability. The results show that the LTH-Exo provided by the present invention can effectively increase the expression level of miR-29b-2-5p in the renal tubules of db / db mice, providing an important basis for its role in DN-related pathological processes. The present invention provides a new idea for exosome-mediated miR-29b-2-5p targeted therapy and a potential application prospect for the clinical treatment of DN.

[0132] GSK3β plays an important role in the progression of DN, and its activation is closely related to apoptosis, fibrosis, and inflammatory responses. We found that GSK3β is a key target of miR-29b-2-5p. The expression of GSK3β in the renal tubules of db / db mice was significantly upregulated compared with that in db / m mice, and the expression of GSK3β in the renal tubules of db / db mice could be significantly reduced after treatment with LTH-Exo.

[0133] Diabetes-related hyperglycemia and metabolic disorders are important driving factors for the progression of DN. After 12 weeks of LTH-Exo treatment, the blood glucose of db / db mice decreased significantly, accompanied by a slowdown in the body weight gain rate, indicating that LTH-Exo has a beneficial effect on the accompanying symptoms of diabetes by improving insulin sensitivity and regulating metabolic pathways. After LTH-Exo treatment, the urinary protein excretion of db / db mice decreased, the uACR decreased significantly, and the degree of renal tubular injury was significantly alleviated, indicating that LTH-Exo provided by the present invention has the effect of improving the renal function of DN.

[0134] Oxidative stress and ferroptosis play important roles in the progression of DN. Hyperglycemia can lead to an increase in the level of oxidative stress, which in turn causes iron accumulation in the body. Iron generates highly reactive hydroxyl radicals through the Fenton reaction in cells, and these radicals damage cell lipids, especially the unsaturated fatty acids of phospholipid membranes, triggering lipid peroxidation reactions, which is one of the core mechanisms of ferroptosis. Studies have found that in diabetic patients and animal models, the iron content in the kidneys increases significantly, and iron overload is closely related to renal function decline. GPX4 is the main antioxidant enzyme that prevents lipid peroxidation, inhibiting GPX4 promotes ferroptosis, and Nrf2 is an antioxidant stress transcription factor. We found that in the renal tissue of db / db mice, Nrf2 was slightly elevated, suggesting the existence of a stress-induced antioxidant response. However, the oxidative stress markers (such as increased MDA levels and decreased GSH-Px activity) and the decreased expression of GPX4 indicated that the oxidative damage and ferroptosis in the renal tubules of db / db mice were exacerbated, and the stress-induced antioxidant response was insufficient to counteract it. After LTH-Exo treatment, the expression of Nrf2 in the renal tubules of db / db mice was significantly upregulated, indicating enhanced antioxidant capacity; the expression of GPX4 was restored, indicating that LTH-Exo inhibits ferroptosis by reducing lipid peroxidation damage. In addition, LTH-Exo also has a protective effect on mitochondrial function. LTH-Exo provided by the present invention may alleviate oxidative stress and ferroptosis by regulating the Nrf2-GPX4 axis, achieving the effect of improving the progression of DN.

[0135] The present invention provides cutting-edge evidence for the application of engineered exosomes in the treatment of DN. LTH-Exo provided by the present invention can improve diabetes-related metabolic abnormalities and kidney damage by enhancing the targeted delivery ability of miR-29b-2-5p, and plays a significant role in key pathological processes such as oxidative stress and ferroptosis. Compared with ordinary exosomes, LTH-Exo shows obvious advantages in treatment efficiency and targeting specificity. In summary, LTH-Exo provided by the present invention inhibits GSK3β-mediated ferroptosis and oxidative stress by targeting the kidney to deliver miR-29b-2-5p, improves the kidney function of db / db mice, provides a new strategy for the precise molecular treatment of DN, and also lays a theoretical foundation for the clinical application of engineered exosomes in other diseases.

[0136] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of miR-29b-2-5p in the preparation of drugs that negatively regulate GSK3β.

2. The use of miR-29b-2-5p in the preparation of a drug for negatively regulating GSK3β according to claim 1, characterized in that: The drug comprises any one of a fusion protein, a recombinant plasmid or an exosome that overexpresses miR-29b-2-5p.

3. An engineered exosome loaded with miR-29b-2-5p targeting the kidney, characterized in that: The engineered exosomes are exosomes loaded with miR-29b-2-5p and surface-modified with the targeting peptide LTH.

4. The method for preparing a kidney-targeting engineered exosome loaded with miR-29b-2-5p as claimed in claim 3, characterized in that: The engineered exosomes are prepared according to the following steps:

1. Preparation of Exosomes HEK293 cells were cultured using a 3D cell culture method. After 5-7 days of culture, the culture supernatant was collected and centrifuged at 2000×g for 10 min; the supernatant was collected and centrifuged at 10000×g for 30 min; the supernatant was collected and centrifuged at 200000×g for 2 h; the supernatant was discarded, the exosome precipitate was collected, PBS solution was added to resuspend the exosome precipitate, and the supernatant was discarded. The precipitate is the exosome; 2. Targeted modification of exosomes (1) preparing lipid anchor targeting peptide solution: adding lipid anchor targeting peptide dissolving solution to LTH-lipid anchor targeting peptide to fully dissolve it, then adjusting the concentration to 1 μg / L with lipid anchor targeting peptide dissolving solution, and storing it at -20°C away from light for later use; (2) Co-incubation of exosomes with lipid-anchored targeting peptides: 500 μg of exosomes and 100 μL of lipid-anchored targeting peptide solution were added to the co-incubation reaction solution, and incubated at 25°C, 250 rpm for 3 h with shaking; then the mixture was transferred to 4°C and incubated for 24 h to obtain targeted exosomes; 3. Preparation of targeted engineered exosomes loaded with miR-29b-2-5p (1) Encapsulation of miR-29b-2-5p The 5′ end of CY3-tagged has-miR-29b-2-5p was purchased from Shanghai Sangon Biotechnology Co., Ltd.; Reagents were added in the following order and dosage: 1000 pmol miRNA → 1000 μg targeted exosomes → 200 μL ETP → reaction solution accounting for 1 / 10 of the volume of exosomes; incubated at 37°C in the dark, with continuous shaking at 150 rpm, for 2 h; (2) Removal of free miR-29b-2-5p: Transfer the incubated sample to a 100 kDa ultrafiltration tube; add the washing buffer to make up to 4 mL and gently resuspend the sample; centrifuge at 4000 × g for 30 minutes at room temperature to concentrate the volume to about 250 μL; (3) Washing: Repeat step (2) for two washes, and finally concentrate the sample to 250 μL to obtain targeted engineered exosomes loaded with miR-29b-2-5p.

5. Use of the engineered exosomes loaded with miR-29b-2-5p targeting the kidney as claimed in claim 3 in the preparation of a drug for treating diabetic nephropathy.

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