SiRNA delivery nanomaterial targeting m2 macrophages and applications thereof

Abstract

The present application provides siRNA delivery nanomaterials targeting M2 macrophages, which are mannosylated bovine serum albumin nanoparticles encapsulating siRNA interfering with KLK12. The mannose coating of the materials of the present application can provide targeting effect on M2 macrophages, and the siRNA released after endocytosis of the materials can silence the expression of KLK12 and its downstream MMPs genes, reduce the degradation of collagen fibers in lung tissues, thereby controlling the number and volume of pulmonary tuberculosis granulomas, preventing the occurrence of active tuberculosis, and providing a new option for the treatment of tuberculosis.

Description

Technical Field

[0001] This application pertains to the fields of tuberculosis treatment and pharmaceutics. Specifically, this application provides a siRNA delivery nanomaterial targeting M2 macrophages and its application. Background Technology

[0002] Tuberculosis (TB) is an infectious disease and one of the leading causes of disease and death worldwide. TB typically affects the lungs and has a high mortality rate (approximately 50%) if left untreated, killing about 1.5 million people annually. Currently, host-directed therapy (HDT) is used as an adjunct to TB treatment with three main strategies: enhancing host immunity; modulating inflammation to reduce lung tissue damage; and killing or inhibiting Mycobacterium tuberculosis. HDT methods can shorten treatment time, limit immunopathology by modulating abnormal immune responses, and improve treatment outcomes.

[0003] Matrix metalloproteinases (MMPs) are involved in the immunopathology of pulmonary tuberculosis (TB). MMP-9 is involved in the recruitment of monocytes and macrophages required for granuloma formation, while MMP-1 is involved in the progression of granulomas to high-bacterial-burden tuberculous cavities. These studies indicate that MMP-1 and MMP-9 are potential therapeutic targets for TB, and in fact, MMP inhibitors have long been investigated for adjuvant therapy in TB. Interestingly, our study shows that both MMP-1 and MMP-9 are regulated by tissue kallikrein (KLK)12. Therefore, blocking KLK12 may improve the efficacy of existing anti-TB drugs by preventing granuloma maturation, reducing matrix degradation, and cavitary lesions. To maximize the therapeutic potential of the KLK12 target, this strategy of silencing KLK12 needs to be delivered to the pathogenic cell population while preserving the immune protection and tissue homeostasis functions of other lung macrophages. Summary of the Invention

[0004] The applicant's research found that KLK12 is highly expressed primarily in M2 macrophages rich in mannose receptors (MRs). MRs are important pattern recognition and endocytic receptors in the innate immune system. In this study, we designed bovine serum albumin nanoparticles encapsulated with siRNA. To precisely target these nanoparticles to M2 macrophages expressing KLK12, we coated the nanoparticles with a layer of mannose. Utilizing the strong affinity between mannose and MRs, we targeted M2 macrophages and introduced the nanoparticles into the cells via mannose receptor-mediated endocytosis, improving overall transmembrane transport efficiency. After introduction into the cells, the nanoparticles silence the expression of KLK12 and its downstream MMP genes by releasing siRNA, reducing collagen fiber degradation in lung tissue, thereby controlling the number and volume of tuberculous granulomas, preventing the occurrence of active TB, and improving the efficacy of existing anti-tuberculosis drugs.

[0005] On the one hand, this application provides siRNA delivery nanomaterials targeting M2 macrophages, wherein the material is mannosylated bovine serum albumin nanoparticles, and the nanoparticles encapsulate siRNA that interferes with KLK12.

[0006] Furthermore, the mannose used in the mannosylation has the structure shown in Formula I, and the mannose is used to target M2 macrophages. The aldehyde group of the mannose is opened to react with the amino group of bovine serum albumin.

[0007]

[0008] Furthermore, the method for preparing the mannose used in the mannosylation includes dissolving D-mannose in an acetate-sodium acetate buffer solution with a pH of 4-5 and heating it at 55-65°C for 0.5-1.5 h; preferably, dissolving D-mannose in an acetate-sodium acetate buffer solution with a pH of 4.5 and heating it at 60°C for 1 h.

[0009] Furthermore, the sense strand sequence of the KLK12 interfering siRNA is 5'-UCAGAACCAUGAGCAUGAUTT-3' (SEQ ID NO.1), and the antisense strand sequence of the siRNA is 5'-AUCAUGCUCAUGGUUCUGATT-3' (SEQ ID NO.2).

[0010] On the other hand, this application provides a method for preparing the above-mentioned material, the method comprising the following steps:

[0011] (1) Dilute bovine serum albumin to obtain bovine serum albumin solution;

[0012] (2) Dissolve siRNA in DMSO solution and mix it with bovine serum albumin solution, then add anhydrous ethanol;

[0013] (3) Add glutaraldehyde solution, mix, and form bovine serum albumin nanoparticles;

[0014] (4) Centrifuge and collect the nanoparticles formed in step (3), then wash them;

[0015] (5) Resuspend the washed nanoparticles obtained in step (4) to obtain a nanoparticle solution;

[0016] (6) Dissolve D-mannose in a buffer solution with a pH of 4-5 and heat to obtain an active mannose solution;

[0017] (7) Add the active mannose solution obtained in step (6) to the nanoparticle solution obtained in step (5) and mix to form mannose / bovine serum albumin nanoparticles;

[0018] Furthermore, the method further includes: (8) centrifuging to collect the mannose / bovine serum albumin nanoparticles formed in step (7) and washing them; (8) resuspending the washed mannose / bovine serum albumin nanoparticles obtained in step (8).

[0019] Furthermore, the concentration of the bovine serum albumin solution obtained in step (1) is 15-25 mg / mL;

[0020] Further, in step (2), siRNA is dissolved in 5% v / v DMSO solution to form a 100 μM siRNA solution, and the siRNA solution is mixed with 10 times the volume of bovine serum albumin solution for 1 hour. Then, 3-4 times the volume of anhydrous ethanol is slowly added to the mixture while stirring.

[0021] Further, in step (3), 38 μL / mL of 25% glutaraldehyde solution is added and the mixture is stirred slowly overnight;

[0022] Furthermore, the concentration of the nanoparticle solution obtained in step (5) is 15-20 g / mL;

[0023] Further, in step (6), D-mannose is dissolved in an acetate-sodium acetate buffer solution with a pH of 4-5 and heated at 55-65°C for 0.5-1.5 h;

[0024] Further, in step (7), the active mannose solution obtained in step (6) is added to the nanoparticle solution obtained in step (5), and the volume ratio of the active mannose solution to the nanoparticle solution is 1:5, and the mixture is stirred overnight.

[0025] On the other hand, this application provides the use of the above-mentioned materials in the preparation of a drug for treating tuberculosis.

[0026] The drug described in this application can be in dosage forms such as tablets, granules, oral liquids, capsules, water injections, and powder injections. The design of the specific dosage form and the selection of excipients can be completed by those skilled in the art using conventional techniques in the pharmaceutical field as needed.

[0027] Furthermore, the drug is in the form of an injection or oral dosage form. An injection dosage form is preferred.

[0028] Furthermore, the drug downregulates the expression of the KLK12 and MMP genes in the pathogenic cell population.

[0029] This invention leverages the therapeutic potential of the KLK12 target. This strategy of silencing KLK12 is delivered to the pathogenic cell population while preserving the immune protection and tissue homeostasis functions of other lung macrophages. The nanomaterials used in this application exhibit rapid delivery, strong targeting, long half-life, and high safety. By targeting and downregulating the expression of the KLK12 gene in the pathogenic cell population, it reduces the degradation of collagen fibers by MMPs, thereby controlling the number and volume of tuberculous granulomas, reducing antibiotic use, shortening the treatment cycle, and providing a new option for the treatment of tuberculosis. Attached Figure Description

[0030] Figure 1 These are scanning electron microscope images of nanomaterials.

[0031] Figure 2 The results are from in vitro experiments on nanomaterials (RAW264.7 cell line).

[0032] Figure 3 The results are from in vivo experiments of nanomaterials (C57 / BL6 mice).

[0033] Figure 4 The therapeutic effects of nanomaterials in a mouse model of tuberculosis.

[0034] Figure 5 The therapeutic effect of nanomaterials in a mouse model of drug-resistant tuberculosis.

[0035] Figure 6 This is a schematic diagram of the preparation process and structure of nanomaterials. Detailed Implementation

[0036] Example 1: Preparation of siRNA delivery nanomaterials targeting M2 macrophages

[0037] Preparation of bovine serum albumin spherical nanoparticles:

[0038] Dilute bovine serum albumin (BSA) to 20 mg / mL with water free of endotoxins and mix thoroughly with a magnetic stirrer.

[0039] Slowly add anhydrous ethanol to the BSA solution at a ratio of 1:3.5 (BSA / ethanol, v / v), controlling the addition rate at 1 mL / min while stirring (Note: To encapsulate siRNA or FITC, first dissolve siRNA or FITC in 5% DMSO solution (100 μM), then mix thoroughly with the BSA solution (siRNA or FITC solution to BSA solution ratio is 1:10) for 1 hour, and then react with anhydrous ethanol).

[0040] Add 25% glutaraldehyde solution, 38 μL / mL (v / v), and place at 4°C with slow stirring overnight to form stable BSA nanoparticles.

[0041] The nanoparticles were collected by centrifugation at 15000g for 20 min and then resuspended and washed three times with the same volume of endotoxin-free water.

[0042] After the third wash, the particles were resuspended in a small amount of endotoxin-free water to form a high-concentration BSA nanoparticle solution (approximately 20 mg / mL), which was then stored at 4°C for a short period.

[0043] Preparation of mannosylated bovine serum albumin nanoparticles:

[0044] D-mannose was dissolved in sodium acetate buffer (pH 4.5) (80 mM) and heated at 60 °C for 1 h to open the mannose rings, exposing the aldehyde groups and facilitating further coupling to the BSA particle surface. The reaction conditions and molecular structure are as follows: Figure 6 As shown:

[0045] The cooled active mannose was added to the endotoxin-free BSA nanoparticles at a ratio of 1:5 (mannose / BSA nanoparticles, v / v) and stirred overnight at 4°C.

[0046] The nanoparticles were collected by centrifugation at 15000g for 20 min, and then resuspended and washed three times with the same volume of endotoxin-free water. After the third wash, the nanoparticles were resuspended in sterile PBS.

[0047] The aforementioned nanoparticles were dehydrated, dried, and sputter-coated with gold before being observed using a scanning electron microscope. Figure 1 As shown, the nanoparticles are regular spherical in shape, with a hydrodynamic diameter of 487 nm and a zeta potential of -25.6 mV.

[0048] Example 2: In vitro experiment of silencing KLK12 and its downstream MMP genes using siRNA delivery nanomaterials targeting M2 macrophages.

[0049] This embodiment uses an anti-mannose receptor blocking antibody (CD206 antibody) to verify the ability of nanoparticles to target mannose receptors, such as Figure 2 As shown in Part A, this antibody significantly reduced the uptake of nanoparticles by RAW264.7 cells in a concentration-dependent manner. Furthermore, as... Figure 2 As shown in Part B, in IL-4-induced differentiated M2 macrophages, the degree of nanoparticle internalization was higher than that in control M0 macrophages or LPS-induced M1 macrophages. These results suggest that the nanoparticles target M2 macrophages due to the affinity between mannose and its receptor.

[0050] To verify the biological functions of the nanoparticles, an in vitro release experiment was conducted in this embodiment. Figure 2 As shown in section C, the nanoparticles interfered with KLK12 gene expression in M2 macrophages 6 hours after introduction, and this interference effect continued to increase over 24 hours. Furthermore, since macrophages are also an important source of MMPs mediating extracellular matrix degradation and remodeling, this example detected MMP-1 and MMP-9 in M2 macrophages after nanoparticle introduction. The results are as follows... Figure 2 As shown in part D, when the expression of the KLK12 gene is interfered with by nanoparticles, the expression of MMPs is also significantly downregulated.

[0051] In vivo experiments

[0052] In this embodiment, FITC-labeled nanoparticles were injected into the peritoneal cavity of mice, and the in vivo dynamics of the nanoparticles were studied using a small animal in vivo imaging system. Figure 3 As shown in Part A, FITC-labeled nanoparticles can reach the liver and lungs one hour after injection, while the heart, spleen, and kidneys show almost no FITC signal. Figure 3 As shown in Part B, the signal intensity in the mouse lungs peaked 6 hours after injection and could last for more than 48 hours.

[0053] like Figure 3 As shown in section C, after the nanoparticles entered the mouse lungs, they rapidly downregulated the expression of KLK12, MMP-1, and MMP-9, and this interference effect could last for more than 48 hours. ELISA results are shown below. Figure 3 As shown in section D, the levels of KLK12, MMP-1, and MMP-9 in mouse serum were also significantly downregulated. These results indicate that intraperitoneal injection of nanoparticles inhibits the activation of KLK12 and its downstream signaling MMPs.

[0054] Example 3: Application of siRNA delivery nanomaterials targeting M2 macrophages in the treatment of tuberculosis.

[0055] To test the feasibility of using nanoparticles as an adjunct therapy for active tuberculosis in a mouse model of tuberculosis, nanoparticles encapsulated with KLK12 siRNA were synthesized and administered via intraperitoneal injection at 7 days (Early does, Ed) and 21 days (Latedoes, Ld) post-infection. The experiment was terminated at 28 days post-infection to observe the therapeutic effect of the nanoparticles on tuberculosis.

[0056] The results are as follows Figure 4 As shown in Part A, the weight loss in mice in the Ed and Ld groups was significantly improved, indicating that intraperitoneal injection of nanoparticles achieved the expected therapeutic effect. Mice in each group were sacrificed 28 days after infection, and serum was separated. ELISA results are shown below. Figure 4 As shown in Part B, intraperitoneal injection of nanoparticles significantly downregulated KLK12 secretion. Gross lung lesions and H&E staining results of paraffin sections are as follows... Figure 4 As shown in section C, the number of granulomas in the lungs of mice in the Ed and Ld groups was significantly reduced, and the area of ​​the inflammatory region was significantly reduced.

[0057] Results of acid-fast staining (Ziehl-Neelsen, ZNS) of lung tissue sections are as follows: Figure 4 As shown in section D, the number of bacteria in the lungs of mice in the intraperitoneal injection group was significantly reduced. In this example, colony-forming units (CFUs) were counted in the organs of each group of mice, and the results are as follows... Figure 4 As shown in section E, the bacterial load in the lungs and spleen of mice in the Ed and Ld groups was significantly reduced.

[0058] Example 4: Application of siRNA delivery nanomaterials targeting M2 macrophages in the treatment of drug-resistant tuberculosis.

[0059] In this embodiment, C57BL / 6 mice were infected with 200 CFU of drug-resistant strains to establish a drug-resistant tuberculosis model. One week after infection, mice were intraperitoneally injected with nanoparticles every 3 days. Rifampicin (RIF) and rifampicin + nanoparticles (RIF + MBNPs) groups were set up as parallel controls. Three weeks after infection, mice in each group were sacrificed, and serum was collected. ELISA results are shown below. Figure 5 As shown in Part A, the concentration of KLK12 in the serum of mice injected with nanoparticles was significantly decreased, while the injection of RIF had no significant effect on the concentration of KLK12 in the serum of mice. Weight monitoring results are as follows... Figure 5 Part B shows that injecting nanoparticles prevented weight loss in infected mice, while injecting RIF did not alleviate the weight loss. These results indicate that the nanoparticles in this embodiment also have a significant effect in treating drug-resistant tuberculosis.

[0060] Gross lesions of mouse lungs and H&E staining results of paraffin sections are as follows: Figure 5 As shown in section C, the nanoparticles significantly reduced the number of nodules and decreased the inflammatory area. Acid-fast staining results of lung paraffin sections are shown below. Figure 5 As shown in section D, injecting nanoparticles significantly reduced the bacterial load in the lungs. These results suggest that the nanoparticles in this embodiment may be a safe and effective alternative treatment for drug-resistant tuberculosis that is difficult to treat with antibiotics.

[0061] As demonstrated by the above embodiments, this invention provides an siRNA delivery nanomaterial and a nanomedicine material targeting M2 macrophages, possessing advantages such as rapid delivery, strong targeting, long half-life, and high safety. The siRNA delivery nanomaterial and nanomedicine material of this invention reduce the degradation of collagen fibers by MMPs by targeting and downregulating the expression of the pathogenic cell population KLK12 gene, thereby controlling the number and volume of tuberculous granulomas and reducing the bacterial load in the lungs and spleen. This invention shows significant efficacy in the treatment of tuberculosis and drug-resistant tuberculosis, reducing or replacing antibiotic use, shortening the treatment cycle, and providing a new technology for host-guided therapy for inflammatory diseases such as tuberculosis.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of siRNA delivery nanomaterials targeting M2 macrophages in the preparation of drugs for treating tuberculosis, characterized in that, The siRNA delivery nanomaterial targeting M2 macrophages is mannosylated bovine serum albumin nanoparticles, which encapsulate KLK12 interfering siRNA; the sense strand sequence of the KLK12 interfering siRNA is 5'-UCAGAACCAUGAGCAUGAUTT-3', and the antisense strand sequence is 5'-AUCAUGCUCAUGGUUCUGATT-3'. The preparation method of the siRNA delivery nanomaterial targeting M2 macrophages includes the following steps: (1) Dilute bovine serum albumin to obtain bovine serum albumin solution; (2) Dissolve the siRNA that interferes with KLK12 in DMSO solution and mix it with bovine serum albumin solution, then add anhydrous ethanol; (3) Add glutaraldehyde solution, mix, and form bovine serum albumin nanoparticles; (4) Centrifuge and collect the nanoparticles formed in step (3), then wash them; (5) Resuspend the washed nanoparticles obtained in step (4) to obtain a nanoparticle solution; (6) Dissolve D-mannose in a buffer solution with a pH of 4-5 and heat to obtain an active mannose solution; (7) Add the active mannose solution obtained in step (6) to the nanoparticle solution obtained in step (5) and mix to form mannose / bovine serum albumin nanoparticles.

2. According to claim 1, the method for preparing the siRNA delivery nanomaterial targeting M2 macrophages further includes: (8) Centrifuge to collect the mannose / bovine serum albumin nanoparticles formed in step (7), and wash them; (9) Resuspend the washed mannose / bovine serum albumin nanoparticles obtained in step (8).

3. According to claim 1, the concentration of the bovine serum albumin solution obtained in step (1) is 15-25 mg / mL; And / or, in step (2), the siRNA interfering with KLK12 is dissolved in 5% ( v / v A 100 μM siRNA solution was prepared in DMSO solution. The siRNA solution was mixed with 10 times the volume of bovine serum albumin solution for 1 hour. Then, 3-4 times the volume of anhydrous ethanol was slowly added to the mixture while stirring. And / or, in step (3), add 38 μL / mL of 25% glutaraldehyde solution and stir slowly overnight; And / or, the concentration of the nanoparticle solution obtained in step (5) is 15-20 g / mL; And / or, in step (6), D-mannose is dissolved in an acetate-sodium acetate buffer solution with a pH of 4-5 and heated at 55-65°C for 0.5-1.5 h; And / or, in step (7), the active mannose solution obtained in step (6) is added to the nanoparticle solution obtained in step (5), with the volume ratio of the active mannose solution to the nanoparticle solution being 1:5, and the mixture is stirred overnight.

4. The application according to claim 3, wherein the drug is an injectable or oral dosage form.

5. The application according to claim 1, wherein the drug downregulates the expression of the pathogenic cell population KLK12 and MMP genes.

Images