A platelet-loaded hsa-miR-143-3p interference plasmid-cerium dioxide nanoszyme complex, a preparation method thereof and application thereof in preparation of anti-inflammatory and antioxidant drugs
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- AFFILIATED HOSPITAL OF NANTONG UNIV
- Filing Date
- 2026-06-15
- Publication Date
- 2026-08-04
AI Technical Summary
[0006]然而，将二氧化铈纳米酶和miRNA干扰质粒联合负载于血小板载体中，实现“靶向递送-微环境改善-基因调控”的协同治疗，目前尚未有相关报道
1. 三重协同机制：
Smart Images
Figure CN122499321A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical and nanomedicine delivery technology, specifically relating to a platelet-loaded hsa-miR-143-3p interference plasmid-cerium dioxide nanoenzyme complex with targeted delivery function, its preparation method and its application in anti-inflammatory and antioxidant therapy, especially suitable for the treatment of liver fibrosis. Background Technology
[0002] Liver fibrosis is a common pathological outcome of various chronic liver injuries (including viral hepatitis, metabolic-associated fatty liver disease, alcoholic liver disease, and autoimmune hepatitis), and it easily progresses to cirrhosis or hepatocellular carcinoma, causing approximately 2 million deaths worldwide each year. Currently, there is a lack of radical treatments in clinical practice, and patients with advanced stages mainly rely on liver transplantation. The progression of liver fibrosis is closely related to inflammatory responses, oxidative stress, and the persistent activation of hepatic stellate cells (HSCs). Existing anti-inflammatory drugs have limited clinical application due to significant side effects from long-term use, poor targeting, and low bioavailability. Therefore, developing novel therapeutic strategies that combine anti-inflammatory, antioxidant, and targeted delivery functions has become an important research direction in this field.
[0003] In recent years, the role of miRNAs in the pathogenesis of liver fibrosis has attracted much attention. Studies have shown that hsa-miR-143-3p is significantly upregulated in fibrotic liver tissue, and it can promote the activation of hepatic stellate cells and collagen deposition by targeting negative regulators of the PI3K-Akt signaling pathway, thereby accelerating the fibrosis process. Therefore, inhibiting the overexpression of endogenous miR-143-3p has become a potentially effective strategy for anti-fibrotic therapy. However, small interfering RNAs (siRNAs) or interfering plasmids have limitations in in vivo application due to poor targeting, susceptibility to nuclease degradation, and low cellular uptake efficiency, which restrict their clinical translation.
[0004] Platelets, as a special type of blood cell, have become an ideal carrier for constructing novel drug delivery systems due to their unique biological characteristics. They not only possess a relatively long circulating half-life of approximately 30 hours (thanks to the immune escape function mediated by their surface CD47 protein), but also have the natural ability to actively target sites of vascular injury and inflammation. During liver inflammation and fibrosis, damaged hepatic sinusoidal endothelial cells and activated hepatic stellate cells highly express various adhesion molecules, which platelets can interact with through receptors such as P-selectin and GPIIb / IIIa on their surface, achieving specific homing.
[0005] Cerium dioxide nanozymes, with their excellent redox properties, can mimic the activity of superoxide dismutase (SOD) and catalase (CAT), efficiently scavenging reactive oxygen species (ROS), thereby alleviating liver damage caused by various drugs and toxins.
[0006] However, there are currently no reports on the synergistic therapy of "targeted delivery-microenvironment improvement-gene regulation" by loading cerium dioxide nanozymes and miRNA interference plasmids together into platelet vectors. Summary of the Invention
[0007] Purpose The purpose of this invention is to provide a platelet-loaded hsa-miR-143-3p interference plasmid-cerium dioxide nanozyme complex with targeting, antioxidant, anti-inflammatory and gene regulation functions, and its efficient and controllable preparation method, for the treatment of inflammatory diseases such as liver fibrosis.
[0008] Technical solution This invention first provides a platelet-loaded hsa-miR-143-3p interference plasmid-cerium dioxide nanozyme complex, which consists of thiolized platelets, hsa-miR-143-3p interference plasmid, and maleimide-functionalized cerium dioxide nanoparticles. The interference plasmid is loaded into the platelet cytoplasm to knock down the upregulated expression of profibrotic miR-143-3p in hepatic stellate cells. The cerium dioxide nanoparticles are coupled to the platelet surface via maleimide-thiol covalent bonds.
[0009] Its preparation methods include: S1: Preparation of cerium dioxide nanoparticles; S2: Surface maleimide functionalization of cerium dioxide nanoparticles; S3: Platelet extraction; S4: The hsa-miR-143-3p interference plasmid was loaded into platelets by ultrasound; S5: Platelets are modified with thiol and then linked with maleimide-functionalized cerium dioxide nanoparticles via a maleimide-thiol reaction to obtain a complex.
[0010] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. Triple Collaboration Mechanism: (1) Platelet-mediated targeted delivery enhances the enrichment of nanozymes and interfering plasmids in liver fibrosis lesions, breaking through the bottleneck of low passive targeting efficiency. (2) Cerium dioxide nanozymes remove local ROS, creating a low-oxidation, low-degradation delivery microenvironment for interfering plasmids, significantly improving plasmid transfection efficiency and gene silencing activity; (3) Interference plasmids silence fibrotic miR-143-3p, reducing the release of inflammatory factors from the source, reducing the continuous production of ROS, alleviating the clearance burden of nanozymes, and prolonging their effective action time.
[0011] The three work synergistically to achieve multi-level treatment of "targeted delivery, microenvironment improvement, and gene silencing".
[0012] 2. Innovative gene silencing strategy: Unlike the traditional strategy of overexpressing anti-fibrotic miRNAs, this invention directly targets and silences the pro-fibrotic miR-143-3p, blocking its pathogenic effect from the source, and has higher therapeutic specificity.
[0013] 3. Covalent bond connection, high stability: The nanozyme is anchored to the platelet surface through a stable maleimide-thiol covalent bond, which avoids premature drug detachment during in vivo circulation.
[0014] 4. The preparation method is mild and controllable: the plasmid is loaded with ultrasound to maintain platelet activity; the reaction conditions are mild and easy to scale up for production. Attached Figure Description
[0015] Figure 1 Transmission electron microscopy image of cerium dioxide nanoparticles.
[0016] Figure 2 Scanning electron microscope image of platelets.
[0017] Figure 3 Scanning electron microscope image of the composite of the present invention.
[0018] Figure 4 Particle size distribution of the complex (A: CeO2; B: platelets; C: complex) and zeta potential (D).
[0019] Figure 5 In vitro targeting validation.
[0020] Figure 6 In vivo targeting validation.
[0021] Figure 7 Protein assay for in vitro antifibrotic effect.
[0022] Figure 8 Flow cytometry was used to detect the effect of different formulations on the intracellular ROS levels of activated hepatic stellate cells.
[0023] Figure 9 In vivo evaluation of the efficacy and safety of antifibrotic drugs (A: serum liver function test; B: mouse liver H&E, Masson's red and Sirius red staining; C: Western blot detection of α-SMA, Collagen I and MMP1 mRNA expression; D: safety analysis of mouse heart, spleen, lung and kidney).
[0024] Figure 10This figure illustrates the preparation and mechanism of action of platelet-loaded hsa-miR-143-3p interference plasmid and cerium dioxide nanozyme complex (in-143 / CeO2@PLT): cerium dioxide nanoparticles are functionalized with maleimide, and platelets are loaded with the interference plasmid via ultrasound and modified with thiol. The two are then assembled into a complex via covalent bonds. This complex can target and activate hepatic stellate cells, synergistically exerting antioxidant and gene silencing effects, reversing the activated state of stellate cells, and achieving anti-fibrotic therapy. Detailed Implementation
[0025] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.
[0026] Example 1: Preparation of the complex 1.1 Preparation of cerium dioxide nanoparticles 108.5 mg of cerium nitrate hexahydrate and 100 mg of sodium citrate dihydrate were weighed and dissolved in 2 mL and 1 mL of deionized water, respectively, and mixed thoroughly. Then, 50 mL of 0.4 M ammonia was added as a precipitant, and the mixture was stirred at room temperature for 24 h. After the reaction was complete, the aggregated particles were removed by centrifugation, and the supernatant was dialyzed through a 3 kDa dialysis bag for 24 h. After dialysis, the mixture was centrifuged at 15000 g for 10 min, and the supernatant was freeze-dried to obtain cerium dioxide nanoparticles (CeO2 NPs), which were stored at 4 °C. Transmission electron microscopy (TEM) was performed. Figure 1 The particle shape is spherical, with a diameter of approximately 10 nm. Figure 4 A).
[0027] 1.2 Preparation of maleimide-functionalized cerium dioxide nanoparticles The CeO2 NPs prepared above were dissolved in chloroform at a mass ratio of 1:2:2 with distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) and dipalmitoylphosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-Mal). The mixture was ultrasonicated for 10 min to ensure homogeneity. Chloroform was removed by rotary evaporation, and the solution was dried in a vacuum oven at 60 °C for 3 h. Deionized water was added and ultrasonically hydrated to obtain a transparent colloidal suspension. Excess PEG was removed by centrifugation at 5000 g, and the solution was filtered through a 0.22 μm filter membrane to obtain a maleimide-functionalized cerium dioxide solution (Mal-CeO2).
[0028] 1.3 Construction of hsa-miR-143-3p interference plasmid Design inhibitor RNA oligonucleotide chains (or miRNA sponge sequences) targeting the mature hsa-miR-143-3p, clone them into inhibitor RNA expression vectors such as pGPU6 / GFP / Neo, and after confirming their correctness through sequencing, extract the plasmids using an endotoxin-free plasmid large-scale extraction kit for later use.
[0029] 1.4 Platelet extraction, sonication, and thiolation Fresh, anticoagulated whole blood from healthy individuals was centrifuged at 200 g for 10 min, and the supernatant (platelet-rich plasma) was collected. This process was repeated three times. Prostaglandin E1 (final concentration 1 μM) was added, and the mixture was centrifuged at 2200 g for 20 min. The precipitate was resuspended in PBS to obtain platelets. Scanning electron microscopy (SEM) was used to analyze the platelets. Figure 2 The results showed that the platelets were spherical, with a particle size of approximately 1.8 μm. Figure 4 B). The hsa-miR-143-3p interfering plasmid (final concentration 50 nM) was mixed with platelets (1 × 10⁻⁶). 8 The mixture (number of plasmids per mL) was sonicated at 4°C and 80 W for 10 min to allow the interfering plasmid to be loaded into the platelets. The platelets loaded with the interfering plasmid were then resuspended in Tyrode's buffer, and 2-iminothionane hydrochloride (Traut's) was added to a final concentration of 2 mM. The mixture was incubated at room temperature with stirring for 30 min. After the reaction, the platelets were washed three times with Tyrode's buffer to obtain thiolized platelets.
[0030] 1.5 Assembly of the complex Thiolized platelets were added to Mal-CeO2 solution (final CeO2 concentration 100 μg / mL) and gently stirred at room temperature for 3 h. After the reaction was complete, the platelets were washed three times with PBS to obtain the platelet-loaded hsa-miR-143-3p interference plasmid-cerium dioxide nanozyme complex (denoted as "in-143 / CeO2@PLT"). Scanning electron microscopy (SEM) Figure 3 The study showed that nanoparticles with a particle size of approximately 1.9 μm were attached to the surface of the composite. Figure 4 C), Zeta potential approximately -13 mV ( Figure 4 D).
[0031] 1.6 Specific molecular mechanisms and key advantages of the triple synergistic effect 1.6.1 Synergistic mechanism of platelet-targeted delivery on nanozymes and plasmids During the progression of liver fibrosis, activated hepatic stellate cells highly express P-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Platelets naturally express their ligands, P-selectin glycoprotein ligand-1 (PSGL-1), integrin αIIbβ3 (GPIIb / IIIa), and glycoprotein Ibα (GPIbα). This invention utilizes this natural ligand-receptor interaction to achieve active targeting of the complex to liver fibrotic lesions.
[0032] 1.6.2 The protective effect of cerium dioxide nanozymes on plasmid transfection efficiency by scavenging ROS High levels of reactive oxygen species (ROS) in the liver fibrosis microenvironment severely damage plasmid DNA integrity and inhibit intracellular transfection efficiency. In this complex, cerium dioxide nanozymes remove local ROS in situ, directly alleviating oxidative stress damage to hepatocytes and creating a low-oxidation, low-degradation intracellular delivery environment for the hsa-miR-143-3p interfering plasmid.
[0033] 1.6.3 Synergistic enhancement of the persistence of nanozymes by interfering plasmid silencing pro-fibrosis genes The hsa-miR-143-3p interfering plasmid reduces the release of pro-inflammatory and pro-fibrotic factors at the source by inhibiting the activation of hepatic stellate cells (downregulating fibrosis markers such as α-SMA and Collagen I), thereby synergistically reducing the continuous production of local ROS. This "gene silencing" effect significantly reduces the clearance burden of cerium dioxide nanozymes, preventing rapid inactivation of nanozymes due to excessive consumption. Example 2: In vitro targeting verification After co-incubating 3D tumor spheroids formed from rhodamine-labeled human hepatic stellate cell line LX-2 for 4 h, the results were observed using confocal microscopy. The results showed that strong fluorescent signals of GFP appeared in the 3D tumor spheroids of the in-143 / CeO2@PLT group, and the signal intensity was significantly higher than that of the interfering plasmid group. This indicates that the platelet vector can efficiently deliver the interfering plasmid and nanozyme together into target cells. Figure 5 ).
[0034] Example 3: In vivo targeting verification Platelet membranes were labeled with FITC, and Cy5-in-143 / CeO2 and Cy5-in-143 / CeO2@PLT were prepared according to the method in Example 1 and assembled into FITC-PLT / Cy5-in-143 / CeO2 and free Cy5-in-143 / CeO2 complexes.
[0035] A liver fibrosis model was established using C57BL / 6J mice by intraperitoneal injection of CCl4 (20%, 2 mL / kg) twice weekly for 6 weeks. Normal mice served as controls. Fluorescence images (Cy5 excitation / emission wavelengths: 649 / 670 nm) were acquired using a small animal in vivo imaging system 2 h after injection. Figure 6 Mice were subsequently euthanized, and major organs (heart, liver, spleen, lung, and kidney) were harvested for in vitro imaging. Liver tissue was collected after drug administration, embedded in OCT, and frozen sections (10 μm) were prepared. The following staining was performed: nuclear staining with DAPI; anti-Desmin or anti-α-SMA antibody (HSC marker) + Alexa Fluor 647-labeled secondary antibody; direct observation of the distribution of Cy5-in-143 / CeO2 (red) and FITC-PLT (green). Confocal microscopy was used to observe and analyze the co-localization of the complex with HSCs.
[0036] Example 4: In vitro anti-fibrotic effect LX-2 cells were divided into the following treatment groups: control group, TGF-β1 (5 ng / mL) stimulation group, TGF-β1+ free CeO2 group, TGF-β1+ free interfering plasmid group, and TGF-β1+in-143 / CeO2@PLT group. After 48 h of incubation, the protein ( Figure 7 The expression of α-SMA, Collagen I, TGF-β1, and MMP1 was detected. Results showed that the in-143 / CeO2@PLT group exhibited significantly better inhibitory effects on these fibrosis markers than any single-drug group. P The result (< 0.001) confirms the synergistic effect of the three.
[0037] Example 5: In vitro ROS scavenging ability LX-2 cells were seeded in 12-well plates. Once the cell density reached 60%-70%, TGF-β1 (5 ng / mL) was added for 24 h to establish a liver fibrosis cell model. Then, PBS (model group), free CeO2, free interference plasmid, and in-143 / CeO2@PLT were added (CeO2 concentration was 100 μg / mL, and interference plasmid concentration was 50 nM in each group). After co-incubation for 24 h, intracellular ROS levels were detected using the DCFH-DA probe. Flow cytometry results (…) Figure 8 The results showed that the ROS fluorescence intensity in the model group was significantly increased; all treatment groups could reduce ROS, with the in-143 / CeO2@PLT group showing the lowest ROS level and the best clearance effect, proving that the complex has excellent antioxidant synergistic effect.
[0038] Example 6: Evaluation of the efficacy and safety of antifibrotic drugs in vivo 7.1 Animal Models and Drug Administration Male C57BL / 6J mice aged 6-8 weeks were intraperitoneally injected with CCl4 (20%, dissolved in olive oil, 2 mL / kg) twice a week for 6 consecutive weeks to establish a liver fibrosis model. After successful model establishment, the mice were randomly divided into two groups (n=3): the model group (CCl4) and the in-143 / CeO2@PLT group. Each mouse was injected via the tail vein with the corresponding preparation (platelet dose of 1×10⁻⁶). 8 Each animal was administered 2 mg / kg of CeO2 and 0.5 mg / kg of interfering plasmid every 3 days for a total of 4 weeks. A normal control group (n=3) was also included.
[0039] 7.2 Detection Indicators Forty-eight hours after the last administration, the mice were sacrificed and their serum and liver were collected.
[0040] Liver function tests: Detect serum ALT, AST, ALP, and Cr levels. Figure 9 A).
[0041] Histopathology: Liver tissue sections were stained with H&E (to assess inflammation and necrosis) and Masson's Red and Sirius Red (to assess collagen deposition). Figure 9 B).
[0042] Fibrosis-related genes: Western blot analysis of α-SMA, Collagen I, and MMP1 mRNA expression. Figure 9 C).
[0043] Safety: Mouse weight changes were recorded; heart, spleen, lungs, and kidneys were collected for H&E staining ( Figure 9 D).
[0044] 7.3 Results Compared with the fibrosis group, mice in the in-143 / CeO2@PLT group showed significantly lower serum ALT, AST, and ALP levels (p < 0.001); Sirius red staining showed a reduction of approximately 70% in collagen area percentage; miR-143-3p expression levels in liver tissue were reduced by approximately 75%, confirming the in vivo gene silencing effect; and the expression of fibrosis-related genes was significantly downregulated. H&E staining showed significantly improved liver tissue structure in this group, with only a small amount of inflammatory cell infiltration. Mice in all treatment groups showed no significant decrease in body weight, and no obvious pathological damage was observed in major organs, indicating that the complex has good biocompatibility.
[0045] in conclusion This invention successfully constructed a platelet-loaded hsa-miR-143-3p interfering plasmid-cerium dioxide nanozyme complex targeting liver fibrosis. This complex is stably linked by maleimide-thiol covalent bonds, possessing a triple function of active platelet targeting, antioxidant activity of cerium dioxide nanozyme, and silencing of pro-fibrotic miRNAs by the interfering plasmid. In vitro and in vivo experiments confirmed its significant synergistic anti-inflammatory, antioxidant, and anti-fibrotic effects, with good biocompatibility, providing a new strategy for the treatment of inflammatory diseases such as liver fibrosis.
Claims
1. A platelet-loaded hsa-miR-143-3p interference plasmid-cerium dioxide nanozyme complex, characterized in that: The complex comprises thiolized platelets, an hsa-miR-143-3p interference plasmid, and maleimide-functionalized cerium dioxide nanoparticles; the hsa-miR-143-3p interference plasmid is loaded onto platelets to inhibit the expression of endogenous hsa-miR-143-3p in hepatic stellate cells; the maleimide-functionalized cerium dioxide nanoparticles are coupled to the surface of thiolized platelets via maleimide-thiol covalent bonds.
2. The platelet-loaded hsa-miR-143-3p interference plasmid-cerium dioxide nanozyme complex according to claim 1, characterized in that: The hsa-miR-143-3p interference plasmid is an inhibitor RNA expression plasmid targeting hsa-miR-143-3p.
3. The platelet-loaded hsa-miR-143-3p interference plasmid-cerium dioxide nanozyme complex according to claim 1, characterized in that: The platelets are derived from mammals, specifically humans, mice, or rats.
4. The platelet-loaded hsa-miR-143-3p interference plasmid-cerium dioxide nanozyme complex according to claim 1, characterized in that: The cerium dioxide nanoparticles have a particle size of 5-15 nm.
5. A method for preparing the platelet-loaded hsa-miR-143-3p interference plasmid-cerium dioxide nanozyme complex according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Prepare cerium dioxide nanoparticles and modify them with maleimide functionalization; (2) Platelets were extracted, and the hsa-miR-143-3p interference plasmid was mixed with the platelets and subjected to sonication to load the interference plasmid into the platelets. (3) Platelets loaded with interfering plasmids were modified by thiolation using Traut's reagent to obtain thiolated platelets; (4) Add maleimide-functionalized cerium dioxide nanoparticles, stir the reaction at room temperature, and purify to obtain the complex.
6. The method according to claim 5, characterized in that: In step (1), the preparation of the cerium dioxide nanoparticles is specifically as follows: 50-200 mg of cerium nitrate hexahydrate and 50-200 mg of sodium citrate dihydrate are dissolved in deionized water, mixed, and then 20-80 mL of 0.4 M ammonia water is added. The mixture is stirred for 6-24 h, centrifuged, dialyzed, and freeze-dried.
7. The method according to claim 5, characterized in that: In step (1), the specific method for functionalizing maleimide is as follows: Cerium dioxide nanoparticles, distearate phosphatidylethanolamine-polyethylene glycol (DSPE-PEG) and dipalmitoylphosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-Mal) are dissolved in chloroform at a mass ratio of 1:2:2, ultrasonically treated for 10 min, hydrated after solvent removal, and centrifuged and filtered.
8. The method according to claim 5, characterized in that: In step (2), the conditions for ultrasonic treatment are: power 50-100 W, time 5-15 min, and temperature 2-6℃.
9. The method according to claim 5, characterized in that: In step (3), the thiolization modification specifically involves incubating platelets with 2-5 mM Traut's reagent at room temperature for 30-60 min, followed by washing with Tyrode's buffer.
10. The use of the platelet-loaded hsa-miR-143-3p interference plasmid-cerium dioxide nanozyme complex according to any one of claims 1-4 in the preparation of a drug for treating liver fibrosis.