SiRNA delivery material for targeting M2 type macrophages as well as preparation method and application of siRNA delivery material
By using engineered polyethylene glycol PEG-Pery-M2, luteolin and ZrCl4 assembled siRNA delivery materials, the problem of existing vectors being inefficient in siRNA delivery and easy inducing immune responses is solved, and efficient targeted delivery of M2 macrophages is achieved, with superior biocompatibility and application prospects.
Patent Information
- Application Number
- CN202411795238.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-09
- Publication Date
- 2025-03-14
AI Technical Summary
Existing siRNA delivery vectors have low efficiency in delivering siRNA, have high metabolic toxicity, and are prone to induce immune responses, making it difficult to effectively target M2 macrophages, especially in the treatment of idiopathic pulmonary fibrosis.
The siRNA delivery material targeting M2 type macrophages is used assembled by engineered polyethylene glycol PEG-Pery-M2, luteolin and ZrCl4. By adding these components to the DEPC aqueous solution of siRNA, efficient loading and targeted delivery of siRNA are achieved.
It has achieved efficient targeting of siRNA to M2 macrophages, with superior macrophage targeting and lesion targeting in vivo pulmonary fibrosis mice, which is safe and reliable, and has broad application prospects.
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Figure CN119607007A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to an siRNA delivery material targeting M2 macrophages and a preparation method and application thereof, belonging to the technical field of biomedical materials. Background Art
[0002] The direct cause of idiopathic pulmonary fibrosis (IPF) is that a large number of lung fibroblasts are activated and proliferate into myofibroblasts, which secrete a large amount of extracellular matrix (ECM), such as fibronectin and collagen. The activation and proliferation of fibroblasts from various sources in lung tissue are regulated by the lung immune environment. Therefore, starting from changing the immune microenvironment, it can provide a new idea for the treatment of idiopathic pulmonary fibrosis. The latest report found that the inflammatory immune environment dominated by M2 macrophages plays a vital role in the occurrence and development of idiopathic pulmonary fibrosis. M2 macrophages specifically and highly express osteopontin (OPN), and OPN further activates M2 macrophages through a positive feedback loop. In addition, OPN can also promote epithelial-mesenchymal transition (EMT) of alveolar epithelial cells. This process is closely related to the transformation of epithelial cells into fibroblasts. EMT triggers the release of a large number of extracellular matrix components, growth factors and inflammatory mediators, which together promote the occurrence and development of pulmonary fibrosis.
[0003] RNA interference (RNAi) changes cell fate in a sequence-specific manner and achieves post-transcriptional regulation of protein expression. By specifically silencing undruggable target genes, it provides new potential for disease treatment. Small interfering RNA (siRNA) is a class of double-stranded oligonucleotides that can induce complementary mRNA sequence-specific silencing through RNA interference (RNAi). siRNA-based therapeutic methods have attracted widespread attention due to their extremely high development potential. However, due to its negative charge, hydrophilicity and nuclease sensitivity, the clinical application of siRNA requires a safe and efficient carrier to transport it to the lesion.
[0004] Currently approved clinical siRNA drugs (such as N-acetylgalactosamine-conjugated siRNA) are mainly used for the treatment of liver diseases due to their natural accumulation in the liver after systemic administration. In order to improve the in vivo distribution of siRNA after systemic administration, the development of carriers for targeted delivery of siRNA is crucial. In recent years, nanocarriers carrying biomacromolecules are being continuously developed for the protection and targeted delivery of active molecules. Nano cationic liposomes are common small interfering RNA delivery carriers. However, common nanoliposomes have low siRNA delivery efficiency, high metabolic toxicity and are prone to induce immune responses. Therefore, the development of carriers with efficient targeted delivery capabilities of siRNA is the key to solving the problem of clinical application of siRNA.
[0005] Metal-Phenolic Networks (MPNs) are supramolecular structures that combine metal ions and plant polyphenols, which are connected by coordination bonds. MPNs have stability in body fluid environments and can achieve the synergistic release of natural polyphenols and metal ions in cells. In recent years, biomacromolecule assembly systems based on polyethylene glycol and metal-polyphenol networks have been reported for the encapsulation and delivery of macromolecules such as proteins and nucleic acids. The delivery system constructed by MPN and PEG provides more possibilities for metal loading of carriers and modification of targeted substances.
[0006] Luteolin (Lut) is a flavonoid compound with multiple biomedical functions, especially good anti-inflammatory and anti-cancer properties. However, luteolin has low water solubility and low bioavailability. How to better increase the bioavailability of luteolin and improve its pharmacokinetics is a biomedical problem that needs to be solved urgently. Summary of the invention
[0007] The purpose of the present invention is to provide a siRNA delivery material targeting M2 macrophages, and verify the targeted accumulation in IPF lesions highly expressed in macrophages.
[0008] The technical solution adopted in the present invention is as follows:
[0009] A siRNA delivery material targeting M2 macrophages is assembled from engineered polyethylene glycol PEG-Pery-M2, luteolin and ZrCl4 in a DEPC aqueous solution of siRNA. The material is coupled with M2 macrophages M2Pep and has M2 macrophage targeting properties.
[0010] The molecular formula of luteolin is C 15 H 10 O6, chemical structure as shown in formula (I):
[0011]
[0012] Preferably, the mass ratio of PEG-Pery-M2, luteolin and ZrCl4 added during assembly is 4:0.17:0.14, the mass ratio of Zr element in the material is 3.24%-3.68%, and the atomic ratio on the surface of the material is 0.11%.
[0013] Preferably, the hydrated particle size (DLS) of the material is 113.28 nm-113.31 nm, and appears as sticky particles under the SEM field of view.
[0014] Preferably, by 89 Zr labeling reaction makes some Zr in the material 4+ for 89 Zr 4+.through 89 After Zr labeling, its distribution and metabolic behavior in the body can be detected by PET-CT.
[0015] Preferably, the engineered polyethylene glycol PEG-Pery-M2 is prepared by polycondensation of NH2-PEG-Mal and 3,9-perylene dicarboxylic acid, followed by modification of M2Pep.
[0016] The present invention also discloses a method for preparing the above-mentioned siRNA delivery material, the steps of which include: adding engineered polyethylene glycol PEG-Pery-M2, ZrCl4 and luteolin to a DEPC aqueous solution of siRNA, stirring the reaction at room temperature, and centrifuging to obtain a precipitate to obtain the siRNA delivery material.
[0017] Preferably, the preparation method of engineered polyethylene glycol PEG-Pery-M2 is:
[0018] (1) NH2-PEG-Mal (230 mg) and 3,9-perylene dicarboxylic acid (16 mg) were dissolved in DMSO / pyridine (9 / 1), EDC (19.2 mg) was added, and stirred at room temperature for 24 h;
[0019] (2) The product obtained in step 1 was dialyzed and purified (MW: 3500), and then freeze-dried after purification for 36 hours to obtain PEG-Pery;
[0020] (3) PEG-Pery (50 mg) obtained in step 2 was dissolved in DMSO (5 mL), M2Pep (30 mg) and TCEP (5 mg) were added and dissolved by ultrasound, and stirred for 24 h;
[0021] (4) The product obtained in step 3 was dialyzed and purified (MW: 3500), and then freeze-dried after purification for 36 hours to obtain PEG-Pery-M2;
[0022] (5) PEG-Pery-M2 (20 mg / mL, 200 μL), ZrCl4 (4.3 mg / mL, 32 μL), and luteolin (16.94 mg / mL, 10 μL) were added to the DEPC aqueous solution of siRNA (2.5 μM) in sequence. The mixture was stirred at room temperature for 30 min, centrifuged at 1200 rpm, washed with DEPC water for 3 times, and the product was finally dispersed in DEPC water.
[0023] In the preparation process, part of 89 Zr 4+ Replacement of Zr 4+ , which can be used for image detection.
[0024] The present invention also discloses the use of the siRNA delivery material in preparing a drug targeting M2 macrophages.
[0025] The invention also discloses the use of the siRNA delivery material in preparing a medicine targeting fibrotic lungs.
[0026] The siRNA delivery material targeting M2 macrophages synthesized by the present invention has excellent siRNA carrying capacity, stable in vivo targeting capacity, ideal microscopic size, and good biocompatibility. The siRNA delivery material targeting M2 macrophages has strong advantages in carrying and targeting siRNA, and provides a theoretical basis for the subsequent development of nanopharmaceuticals for delivering biomacromolecules.
[0027] Compared with the prior art, the present invention has the following advantages:
[0028] (1) The present invention uses Lut and Zr 4+ The stable coordination of the engineered polyethylene glycol enhanced the cross-linking between the engineered polyethylene glycol and the siRNA, and achieved efficient siRNA loading and targeting peptide modification. The siRNA delivery material targeting M2 macrophages with stable properties was obtained. The design method is simple and easy to operate, and can efficiently synthesize the targeted delivery material.
[0029] (2) The siRNA delivery material targeting M2 macrophages synthesized by the present invention has excellent macrophage targeting and in vivo lesion targeting in mice with pulmonary fibrosis. It is safe and reliable and has broad application prospects in the field of biomacromolecule delivery.
[0030] (3) The siRNA delivery material targeting M2 macrophages of the present invention is the first to simultaneously achieve the functions of siRNA loading and M2 macrophage targeted delivery, which has not been reported in domestic and foreign literature. It can be seen that the siRNA delivery material targeting M2 macrophages obtained by the method of the present invention is novel in design and has potential application value.
[0031] (4) The siRNA delivery material targeting M2 macrophages described in the present invention can achieve customized modification of surface targeting molecules through PEG engineering, providing a theoretical basis for the subsequent targeted delivery of biological macromolecules for other diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 Schematic diagram of the synthesis route of si@LuZ-M2.
[0033] Figure 2 It is the hydrated particle size of si@LuZ-M2 of the present invention measured by dynamic light scattering (DLS).
[0034] Figure 3The material morphology of si@LuZ-M2 of the present invention was measured using a scanning electron microscope (SEM).
[0035] Figure 4 This is the PAGE electrophoresis analysis of siRNA in different components during the synthesis of si@LuZ-M2 of the present invention.
[0036] Figure 5 This is the X-ray photoelectron spectroscopy (XPS) spectrum of si@LuZ-M2 of the present invention.
[0037] Figure 6 This is the peak spectrum of Zr 3d in the XPS spectrum of si@LuZ-M2 of the present invention.
[0038] Figure 7 is the H of PEG-Pery in the si@LuZ-M2 synthesis module of the present invention 1 NMR Characterization.
[0039] Figure 8 is the H of PEG-Pery-M2 in the si@LuZ-M2 synthesis module of the present invention. 1 NMR Characterization.
[0040] Fig. 9 This is the cytotoxicity analysis of si@LuZ-M2 of the present invention on macrophages.
[0041] Fig.10 This is the in vitro fluorescence imaging of the main organs of IPF model mice by si@LuZ-M2 of the present invention.
[0042] Fig.11 This is the PET-CT imaging of IPF model mice by si@LuZ-M2 of the present invention.
[0043] Fig.12 Analysis of lung CT values between IPF model mice and healthy mice used in the present invention.
[0044] Fig.13 This is a correlation analysis between the lung uptake rate and CT value of si@LuZ-M2 of the present invention in IPF model mice. Specific implementation methods
[0046] The present invention is further described below in conjunction with embodiments, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.
[0047] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention, and the terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In addition, although demonstrations of parameters including specific values may be provided herein, it should be understood that the parameters do not need to be exactly equal to the corresponding values, but may be approximated to the corresponding values within an acceptable error tolerance or design constraint.
[0048] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.
[0049] Example 1 Synthesis of si@LuZ-M2
[0050] (1) NH2-PEG-Mal (230 mg) and 3,9-perylene dicarboxylic acid (16 mg) were dissolved in DMSO / pyridine (9 / 1), EDC (19.2 mg) was added, and the mixture was stirred at room temperature for 24 h.
[0051] (2) The product obtained in step 1 was dialyzed and purified (MW: 3500). After purification for 36 hours, it was freeze-dried to obtain PEG-Pery.
[0052] (3) The PEG-Pery (50 mg) obtained in step 2 was dissolved in DMSO (5 mL), and M2Pep (30 mg) and TCEP (5 mg) were added and dissolved by ultrasound, and stirred for 24 h.
[0053] (4) The product obtained in step 3 was dialyzed and purified (MW: 3500). After purification for 36 hours, it was freeze-dried to obtain PEG-Pery-M2.
[0054] (5) In the DEPC aqueous solution of siRNA (2.5 μM), add PEG-Pery-M2 (20 mg / mL, 200 μL), ZrCl4 (4.3 mg / mL, 32 μL), and luteolin (16.94 mg / mL, 10 μL) in sequence, stir at room temperature for 30 min, centrifuge at 1200 rpm, discard the supernatant, wash with DEPC water three times, and the product is finally dispersed in DEPC water.
[0055] Example 2 Engineering Polyethylene Glycol PEG-Pery and PEG-Pery-M2 1 H NMR analysis
[0056] (1) PEG-Pyer and PEG-Pyer-M2 were synthesized according to the method described in Example 1.
[0057] (2) The synthesized PEG-Pyer and PEG-Pyer-M2 were dissolved in deuterated DMSO and 1 H NMR analysis. The results showed that the CH characteristic peak on the aromatic ring of perylene-3,9 dicarboxylic acid appeared in the PEG-Pyer spectrum ( Figure 7 ); The CH characteristic peaks on the aromatic ring of perylene-3,9 dicarboxylic acid and the CH characteristic peaks on the benzene ring of tryptophan (Try) and tyrosine (Tyr) residues in M2Pep appeared in the PEG-Pyer-M2 spectrum ( Figure 8 ).
[0058] Example 3 Analysis of the biosafety of si@LuZ-M2 on macrophages
[0059] (1) PEG-Pyer and PEG-Pyer-M2 were synthesized according to the method described in Example 1. Different concentration groups (0nM, 20nM, 60nM, 100nM, 150nM, 250nM, 300nM) were set according to the siRNA content in si@LuZ-M2. After incubation with macrophages for 24h, the cell activity was tested with the CCK-8 kit. The results showed that ( Fig. 9 )si@LuZ-M2 has good biosafety against macrophages.
[0060] Example 4: In vitro fluorescence imaging of major organs of IPF mice by si@LuZ-M2
[0061] The material si@LuZ-M2 obtained according to the steps of Example 1 was used to test its targeting ability to the lung macrophages of IPF mice.
[0062] (1) Establishment of IPF mouse model: Male C57BL / 6 mice (6-7 weeks old) were anesthetized with pentobarbital (3 mg / kg) and injected slowly into the trachea with a single injection of bleomycin (2.5 U / kg) dissolved in 50 μL of saline using a microsprayer. Imaging experiments were performed 14 days after bleomycin administration.
[0063] (2) In vitro fluorescence imaging analysis of the main organs of IPF mice: IPF mice were injected with si@LuZ-M2 material into the tail vein and used as the model group (Bleomycin), and healthy C57BL / 6 mice were injected with si@LuZ as the control group (Control). Three mice in each group were killed at 0.5h, 5h, and 24h after injection, and the main organs (heart, liver, spleen, lung, and kidney) were taken for in vitro fluorescence imaging ( Fig.10 A). ROI analyzes the relative fluorescence intensity of the image. ROI performs statistics on the average fluorescence intensity of the main organs in the image ( Fig.10 B). The results showed that si@LuZ-M2 rapidly accumulated in the lung tissue after injection. At 5 h, the lung tissue signal of the model group (1.72×109 ±8.28×10 8 ) was significantly higher than that of the control group (3.74×10 8 ±1.30×10 8 ), there was still some accumulation in the lung tissue at 24 hours.
[0064] Example 5 PET-CT imaging analysis of IPF mice with si@LuZ-M2
[0065] (1) Establishment of IPF mouse model: Male C57BL / 6 mice (6-7 weeks old) were anesthetized with pentobarbital (3 mg / kg) and injected slowly into the trachea with a single injection of bleomycin (2.5 U / kg) dissolved in 50 μL of saline using a microsprayer. Imaging experiments were performed 14 days after bleomycin administration.
[0066] (2) 89 Synthesis of Zr-labeled si@LuZ-M2: When synthesizing siOPN@LuZ-M2, ZrCl4 solution (3.4 mg / mL) and 89 Zr (300 μCi) was mixed and added to the reaction system instead of the non-radioactive ZrCl4 solution. The product was separated by centrifugation at 12,000 rpm and washed three times with DEPC water to obtain 89 Zr-labeled si@LuZ-M2. The radioactivity counter (RM-905A) measured the radioactivity of the starting material and the final product.
[0067] (3) PET-CT imaging analysis of IPF mice: using Super PET / CT scanner (Ping Seng Scientific) performed micro PET-CT scans of mouse lungs. 89 Zr-labeled si@LuZ and 89Zr-labeled si@LuZ-M2 (60-70 μCi per mouse) were injected, and PET-CT imaging was performed at 2, 8, 24, and 48 hours after injection. The images were reconstructed after scanning, and the correlation between the relative uptake rate (ID% / g) of the lung and the CT value of the lung was analyzed.
[0068] In most cases, CT image analysis can independently provide reliable diagnosis of early fibrosis without invasive sampling. In the mouse IPF model, Micro CT can effectively monitor the progression of fibrosis, and the CT value under filling state is highly correlated with the severity of the disease. Micro CT lung scans were performed on mice participating in PET imaging ( Fig.11 ), the results showed that the lung CT value of the fibrosis model group (-243.23±130.25) was significantly higher than that of healthy mice (-372.52±26.68) ( Fig.12). Pearson correlation analysis was performed between lung CT values and lung uptake at different time points ( Fig.13 ), the results showed that there was a strong positive correlation between the lung uptake of siOPN@LuZ-M2 and the degree of fibrosis. In contrast, in healthy mice, a reliable correlation between uptake and CT values was only observed at the 2nd and 4th hours.
[0069] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it accordingly. They cannot be used to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.
Claims
1. A siRNA delivery material targeting M2 macrophages, characterized in that , assembled by engineered polyethylene glycol PEG-Pery-M2, luteolin and ZrCl4 in a DEPC aqueous solution of siRNA.
2. The siRNA delivery material according to claim 1, characterized in that The mass ratio of Zr element in the material is 3.24%-3.68%.
3. The siRNA delivery material according to claim 1, characterized in that The hydrated particle size of the material is distributed in the range of 113.28nm-113.31nm, and appears as sticky particles under the SEM field of view.
4. The siRNA delivery material according to claim 1, characterized in that The material is coupled with M2 macrophage M2Pep and has M2 macrophage targeting properties.
5. The siRNA delivery material according to claim 1, characterized in that Part of Zr in the material 4+ for 89 Zr 4+ , the marking rate was 26.10%-28.25%.
6. The siRNA delivery material according to any one of claims 1 to 5, characterized in that The engineered polyethylene glycol PEG-Pery-M2 is obtained by polycondensing NH2-PEG-Mal with 3,9-perylene dicarboxylic acid and then modifying M2Pep.
7. The method for preparing the siRNA delivery material according to any one of claims 1 to 6, characterized in that The steps include: (1) Dissolve NH2-PEG-Mal and 3,9-perylene dicarboxylic acid in a mixed solution of DMSO and pyridine, add EDC, and stir at room temperature; (2) The product obtained in step (1) is dialyzed and purified, and then freeze-dried to obtain PEG-Pery; (3) The PEG-Pery obtained in step (2) was dissolved in DMSO, M2Pep and TCEP were added and dissolved by ultrasound, and the reaction was stirred at room temperature; (4) The product obtained in step (3) is dialyzed and purified, and then freeze-dried to obtain engineered polyethylene glycol PEG-Pery-M2; (5) The PEG-Pery-M2 obtained in step (4) is added to the DEPC solution of siRNA, ZrCl4 and luteolin, stirred at room temperature for reaction, and centrifuged to obtain the precipitate, thereby obtaining the siRNA delivery material.
8. Use of the siRNA delivery material according to any one of claims 1 to 6 in the preparation of nanomedicines targeting M2 macrophages.
9. Use of the siRNA delivery material according to any one of claims 1 to 6 in the preparation of nanomedicines targeting fibrotic lungs.
10. An engineered polyethylene glycol PEG-Pery-M2, characterized in that It is made by the condensation reaction of NH2-PEG-Mal and 3,9-perylene dicarboxylic acid, followed by modification of M2Pep.