Si-TGM2-loaded macrophage targeting nano material as well as preparation method and application thereof

Abstract

The invention discloses a si-TGM2-loaded macrophage targeting nano-material as well as a preparation method and application thereof, relates to the technical field of biological targeting, and aims at solving the problem that a macrophage targeting nano-drug for inhibiting severe acute pancreatitis aiming at a TGM2 target is lacked in the prior art. The technical key points of the invention are as follows: the si-TGM2-loaded macrophage targeting nano material is provided, the si-TGM2-loaded macrophage targeting nano material is a lactoferrin modified cationic lipid nucleic acid drug, and the cationic lipid nucleic acid drug is prepared by coating si-TGM2 as shown in SEQ.ID. NO.1 with a cationic liposome. The si-TGM2-loaded macrophage targeting nanometer material disclosed by the invention has a wide application prospect in preparation of an acute pancreatitis diagnostic kit and a medicine.

Description

Technical Field

[0001] This invention relates to the field of biotargeting technology, and more specifically, to a si-TGM2 macrophage-targeting nanomaterial, its preparation method, and its application. Background Technology

[0002] Acute pancreatitis (AP) is a common and severe digestive system disease characterized by an inflammatory response that triggers autodigestion, edema, hemorrhage, and necrosis of pancreatic tissue. Treatment for AP has limited efficacy and significant side effects. Transglutaminase 2 (TGM2) is a calcium-dependent acyltransferase involved in many biological processes, such as bone development, angiogenesis, wound healing, cell differentiation, chromatin modification, and cell death. As a novel tumor marker, TGM2 has been shown to promote the progression of pancreatic and liver cancer. Furthermore, it is also significantly highly expressed in resident macrophages of pancreatic tissue in patients with pancreatitis. However, the specific mechanism is not yet fully understood. Professor Xue Dongbo's research group at the First Affiliated Hospital of Harbin Medical University discovered through previous studies that in acute pancreatitis (AP), increased TGM2 directly inhibits STAT6 phosphorylation and nuclear translocation, reducing the transcriptional effect of pSTAT6 on GAS6, thereby weakening macrophage burial and promoting the severity of acute pancreatitis. Due to problems such as single therapeutic target, poor water solubility, short half-life, and limited pancreatic targeting specificity, the efficacy of existing drug therapies in the research and development or clinical trial stages is still not ideal. Novel nanomaterials possess good targeting, biosafety, sensitivity, and specificity, and have attracted widespread attention in the field of precision diagnosis and treatment of AP. One such material is an acid-responsive, neutrophil-membrane-encapsulated hollow mesoporous Prussian blue nanoparticle that can dually target inflammation and acinar cells by regulating Ca2+. 2+ Homeostasis and inhibition of pancreatic autodigestion in the treatment of acute pancreatitis Traditional antioxidants have limited clinical efficacy due to their inability to penetrate the blood-pancreas barrier and lack of targeting. Novel mitochondrial-targeting nano-antioxidants—tannic acid-modified tungsten-based heteropolyacid nanomedicines (mTWNDs)—can effectively treat acute pancreatitis (AP), significantly improving the precision and effectiveness of antioxidant therapy. A macrophage-targeting emodin nanomaterial can effectively treat acute pancreatitis (AP) by regulating the JNK pathway. In addition, lipid nanoparticles (LNPs) are also widely used to encapsulate chemical drugs for disease treatment. In recent years, in the field of gene therapy, LNPs have been used to encapsulate nucleic acids, such as mRNA and siRNA, forming nucleic acid lipid nanoparticles. LNPs have been shown to effectively and selectively deliver mRNA to the pancreas. Studies have shown that delivering FGF21 mRNA, APOA1 mRNA, or a fusion protein of both via LNP can alleviate pancreatic damage in a mouse model of acute pancreatitis. (PubMed: 39871339); However, no nanomaterials targeting TGM2 have been used to treat AP in existing studies. Summary of the Invention

[0003] The technical problem to be solved by this invention is:

[0004] There is a lack of macrophage-targeted nanomedicines that target TGM2 to inhibit the progression of acute pancreatitis to a severe stage.

[0005] Therefore, the present invention provides a si-TGM2 macrophage-targeting nanomaterial, its preparation method and application.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0007] This invention provides a macrophage-targeting nanomaterial loaded with si-TGM2, which is a lactoferrin-modified cationic lipid nucleic acid drug. The cationic lipid nucleic acid drug is prepared by encapsulating si-TGM2 as shown in SEQ.ID.NO.1 with cationic liposomes.

[0008] This invention provides a method for preparing si-TGM2 macrophage-targeting nanomaterials, comprising the following steps:

[0009] Step 1: Dissolve si-TGM2 as shown in SEQ.ID.NO.1 in buffer solution to prepare an aqueous phase. Quickly mix the pre-prepared lipid organic phase with the aqueous phase to allow LNP to fully self-assemble. Remove organic solvent and free small molecule compounds to obtain LNP@si-TGM2.

[0010] Step 2: Add the cross-linking agent to LNP@si-TGM2 for activation treatment, add lactoferrin to make it connect to the LNP surface through the cross-linking agent, remove unassembled lactoferrin, and obtain Lactoferrin-LNP@si-TGM2.

[0011] Furthermore, the crosslinking agent mentioned in step 2 is selected from a mixed crosslinking agent of NHS and EDC, with a mass ratio of NHS to EDC of 4:(5-7).

[0012] Further, the lipid organic phase described in step 1 is prepared by dissolving DSPE-TK-PEG2000-COOH, ALC-0315, ALC-0159, DSPC and CHOL in an organic solvent in a certain proportion.

[0013] Furthermore, in step 1, the volume ratio of the aqueous phase to the organic phase is (5-7):2.

[0014] This invention provides the application of the above-mentioned si-TGM2 macrophage-targeting nanomaterials in the preparation of diagnostic kits and drugs for acute pancreatitis.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] (1) In this invention, LNP is used as a lipid delivery carrier for si-TGM2. The interior of LNP is weakly acidic, which can better bind to the alkaline environment of the pancreas. The siRNA interacts with the outer lipid layer of LNP, and its lipid shell encapsulates si-TGM2 in its core, effectively shielding it from nucleases and preventing it from being degraded in vivo. This allows for efficient and safe delivery of si-TGM2.

[0017] (2) This invention utilizes lactoferrin to modify LNPs. The outer layer of LNPs, coated with lactoferrin, specifically binds to the highly expressed LRP-1 receptor on the surface of macrophages, achieving active targeting of M1 macrophages at the site of inflammation. This not only significantly improves the bioavailability and cellular uptake of si-TGM2, but also enhances the transcriptional activity of GAS6 by promoting STAT6 phosphorylation and nuclear translocation, thereby strengthening macrophage burial and inhibiting the progression of acute pancreatitis (AP) to severe stages. Furthermore, it can reduce pancreatic inflammation by directly downregulating pro-inflammatory factors such as IL-1β, IL-6, and TNF-α secreted by M1 macrophages. Thus, it exerts a precise and efficient therapeutic effect on AP.

[0018] (3) The raw materials prepared by the method of the present invention are readily available, low in cost and simple in process. The Lactoferrin-LNP@siRNA prepared has high stability, targeting, high efficiency and safety. The nanomaterial delivery platform can achieve targeted delivery of si-TGM2 in pancreatic tissue macrophages, and has broad clinical application prospects in the treatment of AP and prevention of AP severe disease. Attached Figure Description

[0019] Figure 1 This is an electron microscope image of Lactoferrin-LNP@siRNA in an embodiment of the present invention;

[0020] Figure 2 The images show H&E staining and pathological scoring of the mouse pancreas in this embodiment of the invention.

[0021] Figure 3 The images show immunohistochemical images and relative expression levels of IL1-β, IL-6, and TNF-α in mice in this embodiment of the invention. Detailed Implementation

[0022] To enable those skilled in the art to better understand the present invention, exemplary embodiments or examples of the present invention will be described below in conjunction with the accompanying drawings. Obviously, the described embodiments or examples are merely some, not all, of the embodiments or examples of the present invention. All other embodiments or examples obtained by those skilled in the art based on the embodiments or examples of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0023] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0024] Example 1

[0025] DSPE-TK-PEG2000-COOH, ALC-0315, ALC-0159, DSPC, and CHOL were dissolved in ethanol at a molar ratio of 0.5:50:1:10:38.5 to obtain the lipid organic phase. The siRNA based on SEQ.ID.NO.1 was dissolved in 100 mL of citrate buffer (pH 4-6) to obtain the aqueous phase. The aqueous and organic phases were rapidly mixed at a volume ratio of 3:1 and incubated at room temperature to allow LNPs to fully self-assemble. The mixture was purified by dialyzing in sterile PBS for 2 h to remove ethanol, yielding LNP@si-TGM2. 4 mg NHS and 6 mg EDC were added to LNP@si-TGM2, and the mixture was activated at 4°C for 2 h. Then, 5 mg of lactoferrin (LP) was added, and the reaction was carried out overnight at 4°C. Unassembled lactoferrin was removed by filtration, and the mixture was purified by dialyzing for 2 h to obtain Lactoferrin-LNP@si-TGM2. Figure 1 As shown, Lactoferrin-LNP@si-TGM2 exhibits a spherical structure. Using a BI-200 SM dynamic light scattering system (Brookhaven Instruments), the final particle size of Lactoferrin-LNP@si-TGM2 was 156.36 ± 37.43 nm, and the zeta potential was 8.12 ± 2.06 mV.

[0026] Table 1. si-TGM2 sequence list

[0027]

[0028] Comparative Example 1

[0029] TEOS, anhydrous ethanol, and deionized water were mixed thoroughly in a molar ratio of 1:4:4 to obtain a homogeneous solution. The pH of the solution was adjusted to 2-3 with dilute HCl to catalyze the hydrolysis reaction. The solution was stirred and refluxed at 60°C for 1-2 hours to obtain a TEOS sol. The citrate buffer solution containing the siRNA shown in SEQ.ID.NO.1 from Example 1 was slowly added to the TEOS sol and mixed thoroughly. Dilute ammonia (NH4OH) was slowly added dropwise to adjust the pH to 7-8 to initiate a condensation reaction. The mixture was allowed to stand to form a gel. The gel was sealed in a container and allowed to age for 24-48 hours to obtain a SiO2@si-TGM2 nanomaterial gel.

[0030] Example 2

[0031] To verify the efficacy of Lactoferrin-LNP@si-TGM2 in treating acute pancreatitis (AP) as described in Example 1, an acute pancreatitis mouse model induced by Lactoferrin was constructed in this example. Twenty C57BL / 6 mice were randomly divided into four groups (n=4): blank control group, AP model group, AP+SiO2@si-TGM2 (AP+Nanomaterial) group, and AP+Lactoferrin-LNP@si-TGM2 group.

[0032] One week after mice acclimatized to their environment, they were injected via tail vein into the veins using Lactoferrin-LNP@si-TGM2 and SiO2@si-TGM2 nanomaterials (10 ml / kg), while the control group received an equal volume of saline via tail vein injection (10 mL / kg). One week later, an acute pancreatitis (AP) model was established by intraperitoneal injection of 50 μg / kg Lactoferrin, once per hour for 12 hours, while the control group received an equal volume of saline. Mice were sacrificed 24 hours later, and pancreatic tissue was collected for H&E staining and immunohistochemical experiments. Figure 2 As shown, compared with the AP group, the pancreatic damage in the AP+Lactoferrin-LNP@si-TGM2 group was significantly reduced, and the pathological score was significantly lower (p<0.001). Although the AP+SiO2@si-TGM2 group could inhibit pancreatic hemorrhage and necrosis in AP mice to some extent, its therapeutic effect was still worse than that of the AP+Lactoferrin-LNP@si-TGM2 group (p<0.01).

[0033] Example 3

[0034] To investigate the role of Lactoferrin-LNP@si-TGM2 in the pancreatic inflammatory microenvironment, pancreatic tissues from each treatment group were collected for immunohistochemical experiments. This was to verify the expression of inflammatory factors associated with severe pancreatitis, such as TNF-α, IL-1-β, and IL-6.

[0035] like Figure 3As shown, the expression of inflammatory factors IL1-β, IL-6, and TNF-α was significantly decreased in the AP+Lactoferrin-LNP@si-TGM2 group (p<0.001). Although the AP+SiO2@si-TGM2 group could inhibit pancreatic inflammation in AP mice to some extent, its therapeutic effect was still worse than that of the AP+Lactoferrin-LNP@si-TGM2 group (p<0.05). This indicates that the macrophage-targeting nanomaterial loaded with si-TGM2 of the present invention can be delivered to macrophages in pancreatic tissue and released in a targeted manner after intravenous administration to mice. The released si-TGM2 can alleviate acute pancreatitis; this nanomedicine can effectively reduce pancreatic hemorrhage, necrosis, and inflammation in AP model mice; and can effectively alleviate acute pancreatitis and prevent the progression of acute pancreatitis to a severe stage.

[0036] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

[0037] The documents cited in this invention include:

Claims

1. A macrophage-targeting nanomaterial loaded with si-TGM2, characterized in that: It is a lactoferrin-modified cationic lipid nucleic acid drug, which is prepared by encapsulating si-TGM2 as shown in SEQ.ID.NO.1 with cationic liposomes.

2. A method for preparing si-TGM2 macrophage-targeting nanomaterials, characterized in that: Includes the following steps: Step 1: Dissolve si-TGM2 as shown in SEQ.ID.NO.1 in buffer solution to prepare an aqueous phase. Quickly mix the pre-prepared lipid organic phase with the aqueous phase to allow LNP to fully self-assemble. Remove organic solvent and free small molecule compounds to obtain LNP@si-TGM2. Step 2: Add the cross-linking agent to LNP@si-TGM2 for activation treatment, add lactoferrin to make it connect to the LNP surface through the cross-linking agent, remove unassembled lactoferrin, and obtain Lactoferrin-LNP@si-TGM2.

3. The method for preparing the si-TGM2 macrophage-targeting nanomaterial according to claim 2, characterized in that: The crosslinking agent mentioned in step 2 is selected from a mixture of NHS and EDC, with a mass ratio of NHS to EDC of 4:(5-7).

4. The method for preparing the si-TGM2 macrophage-targeting nanomaterial according to claim 3, characterized in that: The lipid organic phase described in step 1 was prepared by dissolving DSPE-TK-PEG2000-COOH, ALC-0315, ALC-0159, DSPC, and CHOL in an organic solvent in a specific ratio.

5. The method for preparing the si-TGM2 macrophage-targeting nanomaterial according to claim 4, characterized in that: In step 1, the volume ratio of the aqueous phase to the organic phase is (5-7):

2.

6. The application of the si-TGM2 macrophage-targeting nanomaterials described in claim 1 in the preparation of diagnostic kits and drugs for acute pancreatitis.

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