Modified montmorillonite nanosheet capable of being used as cfDNA scavenger as well as preparation method and application of modified montmorillonite nanosheet

Abstract

The invention discloses a modified montmorillonite nanosheet capable of being used as a cfDNA scavenger as well as a preparation method and application of the modified montmorillonite nanosheet. The modified nanosheet is prepared by modifying the surface of a two-dimensional montmorillonite nanosheet with polylysine cations. The modified montmorillonite nanosheet can achieve the effect of inhibiting inflammation by removing cfDNA and adjusting the abundance of intestinal microorganisms, effectively relieves symptoms induced by IBD, has good stability, biocompatibility and biodegradability, and can be used for treating inflammatory bowel diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials, and specifically relates to a modified montmorillonite nanosheet that can be used as a cfDNA scavenger, and a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic and recurrent inflammation of the intestine caused by abnormal immune mechanisms due to multiple etiologies. Crohn's disease (CD) and ulcerative colitis (UC) are the two main forms of IBD, both of which significantly reduce the quality of life of patients. At the same time, the occurrence of IBD often causes a series of complications, including fistulas, abscesses, malignancies, colorectal cancer, and intestinal fibrosis, making IBD a major healthcare problem. Environmental, genomic, immune, and microbial factors are all associated with the occurrence and development of IBD, and microbial dysbiosis is caused by the oxidative nature of the intestinal inflammatory response. The recognition of DAMPs produced or released by damaged and dead cells promotes sterile inflammation, among which the released cfDNA is considered to be a potential receptor that mediates TLR9-dependent proinflammatory effects. Therefore, the clearance of cfDNA can also be used as a potential effective strategy for the treatment of IBD.

[0003] Polylysine has a certain inhibitory effect on the respiration of Escherichia coli, and is significantly different from typical respiratory pathway inhibitors. At the same time, polylysine also acts on the biomembrane system and protein synthesis system. When polylysine contacts the biomembrane, it can directly and quickly destroy the energy metabolism that depends on the integrity of the membrane structure and the selectivity of the substances on which cells and organelles survive, causing the intracellular lysosomal membrane to rupture and induce microorganisms to produce autolysis, ultimately leading to bacterial death. In addition, montmorillonite has a layered structure and non-uniform charge distribution. After being taken by the human body, it will not be absorbed by the human intestine, but will adhere to the surface of the intestinal wall, adsorb and fix the microorganisms in the intestine, and the toxins they produce, so that they lose their pathogenic effect. In addition, montmorillonite covers the digestive tract mucosa, and by combining with mucus glycoproteins, it can enhance the defense function of the digestive tract mucosa against attacking factors. The method of synergistic treatment of IBD based on polylysine and montmorillonite has not been disclosed. Summary of the invention

[0004] In order to solve the problem that conventional drugs currently used in clinical practice can only temporarily relieve IBD symptoms but cannot cure the damaged intestinal barrier and imbalance of flora, the present invention constructs a modified montmorillonite nanosheet and applies it to the treatment of inflammatory bowel disease, which can adsorb cfDNA, regulate intestinal flora, and accumulate at the site of inflammation, thereby achieving the effect of treating IBD.

[0005] In order to solve the technical problem, the present invention adopts the following technical solution: The present invention first discloses a modified montmorillonite nanosheet that can be used as a cfDNA scavenger, which is characterized in that: the modified montmorillonite nanosheet is a two-dimensional montmorillonite nanosheet as the core, and the surface of the two-dimensional montmorillonite nanosheet is modified with polylysine cations. The modified montmorillonite nanosheet has a thickness of 1 to 2 nm and a diameter of 200 to 300 nm.

[0006] The preparation method of the modified montmorillonite nanosheet of the present invention is as follows: after repeatedly freezing and thawing montmorillonite, deionized water is added, ice bath ultrasound is performed (ultrasound power is 200-300 W, time is 0.5-1 h), centrifugation is performed at 2000-2500 rpm for 15-30 min, and after collecting the supernatant, centrifugation is performed at 10000-13000 rpm for 15-20 min to obtain pure two-dimensional montmorillonite nanosheet. ε-polylysine is added to the aqueous solution of the pure two-dimensional montmorillonite nanosheet and ultrasonically dispersed uniformly (ultrasound power is 30-60 W, time is 5-10 min), and then stirred at room temperature for 4-12 h: the obtained product is centrifuged (the centrifugal speed is 10000-12000 rpm, and the centrifugal time is 10-15 min), and the modified montmorillonite nanosheet is obtained.

[0007] Furthermore, the mass ratio of ε-polylysine to pure two-dimensional montmorillonite nanosheets is 2 to 4:1. The modified montmorillonite nanosheet of the present invention can be used to prepare a cfDNA scavenger for treating cfDNA-related inflammation, wherein the cfDNA-related inflammation includes one of inflammatory bowel disease, arthritis, and sepsis. The modified montmorillonite nanosheet of the present invention shows excellent effects in the treatment of inflammatory bowel disease, has no significant toxicity to mammalian cells, has good biosafety, and can be used to prepare a new nanopharmaceutical for treating inflammatory bowel disease, and its implementation mechanism is: anchoring around the colon inflammation area to achieve precise drug delivery to improve the therapeutic effect of inflammatory bowel disease. The modification of polylysine cations improves the binding ability of positively charged nanosheets to cfDNA. The modified montmorillonite nanosheet can reduce cfDNA-induced TLR9 signal transduction and pro-inflammatory macrophage activation to restore the epithelial barrier and reduce inflammation by adsorbing cfDNA, and can also promote the polarization of pro-inflammatory macrophages M1 to anti-inflammatory macrophages M2. After oral administration, the modified montmorillonite nanosheet protects the intestinal barrier by reducing inflammation, removing intestinal bacteria, and regulating intestinal microbiota, thereby significantly reducing the symptoms of inflammatory bowel disease.

[0008] The beneficial effects of the present invention are embodied in: 1. In order to solve the problem that polylysine cations can still stimulate the TLR9 pathway to produce inflammation as a product of cell damage after directly combining with cfDNA, the present invention prepares polylysine cation-modified montmorillonite nanosheets to adsorb cfDNA produced after cell damage, directly blocking its connection with the TLR9 pathway, thereby weakening the ability of the TLR9 pathway to produce inflammation.

[0009] 2. In the modified montmorillonite nanosheets of the present invention, after polylysine kills harmful intestinal bacteria, montmorillonite can clean the intestines, thereby achieving the effect of treating IBD under the synergy of the two.

[0010] 3. The preparation process of the modified montmorillonite nanosheets of the present invention is simple, the reaction conditions are mild, and it is possible to produce on a large scale, and has the potential for industrial and practical applications.

[0011] 4. The modified montmorillonite nanosheets of the present invention have good dispersibility and stability, and have good biocompatibility, have no direct or indirect toxic effects on the human body, have no potential toxicity, and are conducive to clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the synthesis of modified montmorillonite nanosheets of the present invention.

[0013] Figure 2 This is a transmission electron microscopy image of the modified montmorillonite nanosheets prepared in Example 1.

[0014] Figure 3 This is an atomic force microscope image of the modified montmorillonite nanosheets prepared in Example 1.

[0015] Figure 4 Zeta potential diagram of pure two-dimensional montmorillonite nanosheets and modified montmorillonite nanosheets prepared in Example 1.

[0016] Figure 5 This is a particle size diagram of the modified montmorillonite nanosheets prepared in Example 1.

[0017] Figure 6 This is a graph showing the adsorption performance of modified montmorillonite nanosheets, pure two-dimensional montmorillonite nanosheets and polylysine prepared in Example 1 for ctDNA (calf thymus DNA).

[0018] Figure 7 This is a graph showing the performance of modified montmorillonite nanosheets, pure two-dimensional montmorillonite nanosheets and polylysine prepared in Example 1 on CpG-induced macrophage inflammatory factors.

[0019] Figure 8 Biocompatibility diagram of modified montmorillonite nanosheets with different concentrations prepared in Example 1.

[0020] Fig. 9 This is a graph showing the changes in body weight of mice in each group during the evaluation of colitis prevention and treatment performance.

[0021] Fig.10 Figure 2. Post-anatomical colon images of mice in each group for evaluation of colitis prevention and treatment performance.

[0022] Fig.11 This is a statistical chart of the colon length after dissection of each group of mice in the evaluation of the preventive treatment performance of colitis.

[0023] Fig.12 This is a graph showing the changes in body weight of mice in each group during the evaluation of colitis therapeutic performance.

[0024] Fig.13 Figure 2 shows the dissected colon of each group of mice used in the evaluation of colitis therapeutic performance.

[0025] Fig.14 This is a statistical chart of the colon length after dissection of each group of mice in the evaluation of colitis treatment performance.

[0026] Fig.15 The abundance of intestinal flora was measured in the feces collected after autopsy of each group of mice in the evaluation of colitis treatment performance, where (a) is Chao1 analysis and (b) is Shannon index analysis. DETAILED DESCRIPTION

[0027] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail in combination with the embodiments below. The following content is only an example and explanation of the concept of the present invention. The technicians in the relevant technical field make various modifications or supplements to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

[0028] Example 1 In this embodiment, montmorillonite nanosheets were prepared as follows: 1 g of montmorillonite powder was repeatedly frozen and thawed and added to 20 mL of deionized water, and then ultrasonicated in an ice bath (50% of the maximum power of the ultrasonicator, ≈300 W ultrasonic power) for 1 h, centrifuged at 2500 rpm for 30 min, and the supernatant was collected and centrifuged at 13000 rpm for 20 min. The resulting precipitate was the two-dimensional montmorillonite nanosheet (denoted as MMT NSs). ε-polylysine (the mass ratio of ε-polylysine to pure two-dimensional montmorillonite nanosheet was 4:1) was added to the aqueous solution of pure two-dimensional montmorillonite nanosheet and ultrasonically dispersed evenly (ultrasonic power was ≈60 W, time was 10 min), and then stirred at room temperature for 12 h: the resulting product was centrifuged at 12000 rm for 15 min, and the resulting precipitate was the modified montmorillonite nanosheet (denoted as MMT@PLLNSs).

[0029] The morphology and performance of the MMT@PLL nanosheets obtained in this example were characterized as follows: 1. Morphological Characterization Figure 2 This is a transmission electron microscope image of the MMT@PLL nanosheet obtained in this example. From the image, it can be seen that the diameter of the nanosheet is 200-300 nm.

[0030] Figure 3 This is an atomic force microscope image of the MMT@PLL obtained in this example. From the image, it can be seen that the thickness of the nanosheet is 1 to 2 nm.

[0031] Figure 4 The Zeta potential diagram of the MMT nanosheets and MMT@PLL nanosheets obtained in this example shows that the Zeta potential value of the pure two-dimensional montmorillonite nanosheets is -13 mV. After modification with polyamino acid molecules, the Zeta potential value increases to +31 mV, indicating that the polylysine cations are successfully loaded onto the surface of the two-dimensional montmorillonite nanosheets. The increase in the Zeta potential value may be due to the ion exchange between the polylysine cations and the interlayer cations of the montmorillonite nanosheets.

[0032] Figure 5 This is a diagram of the hydrated particle size of the MMT@PLL nanosheets obtained in this example. From the figure, it can be seen that the diameter of the nanosheets is 200-300 nm, which is consistent with the transmission electron microscopy results.

[0033] 2. Testing the ability to clear cfDNA Serum cfDNA levels are closely correlated with the expression levels of colon TLR9, TNF-α, iNOS, and F4 / 80. Higher serum cfDNA levels are jointly correlated with IBD severity and colon TLR9 expression, indicating that cfDNA-TLR9 signaling is a target for IBD treatment. In order to verify the ability of MMT@PLL nanosheets to clear cfDNA, this example uses calf thymus DNA (ctDNA) as a model, and uses MMT nanosheets and ε-polylysine PLL raw materials as comparisons. The specific characterization method is: add MMT@PLL NSs, MMT NSs or PLL materials to the ctDNA solution so that the mass ratio of the material to ctDNA is 1:1~7:1, and then add ethidium bromide (EtBr) to evaluate the binding ability of the nanoreagent to ctDNA. The results are as follows: Figure 6 As shown in Figure 3, the removal ability of cfDNA is best when the mass ratio of MMT@PLL NSs material to DNA is 6:1. Such a high removal efficiency should be attributed to the strong electrostatic attraction between PLL and DNA phosphate.

[0034] 3. In vitro inhibition of inflammation in the constructed macrophage inflammation model Figure 7 The ability of two-dimensional montmorillonite nanosheets to inhibit inflammation in vitro in constructing a macrophage inflammation model was interpreted. Because macrophages are important participants in the immune pathogenesis, the present invention speculates that cfDNA-mediated TLR9 activation is the key to M1 macrophage polarization, because macrophage polarization leads to the release of proinflammatory cytokines in cells. First, CpG 1826 was used as an in vitro substitute for cfDNA to induce macrophage inflammation, and then MMT@PLL NSs, MMTNSs or PLL materials were added to the cell culture dish and incubated with the stimulated macrophages for 12 hours, and then the level of inflammatory factors in the supernatant of the culture medium was detected. Through the significance comparison of the final data, it was found that MMT@PLLNSs had a good inhibitory effect on TNF-α, a polarization marker of M1 macrophages.

[0035] IV. In vivo biosafety testing of materials Figure 8The figure shows the biocompatibility of MMT-PLL nanosheets with different concentrations. The characterization method is as follows: dilute the MMT-PLL nanosheets to 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL in the dispersion of the whole system. 0.2 mL of the above solutions with different concentrations were mixed with 0.2 mL of treated blood (500 μL of fresh blood was added to 4.5 mL of normal saline and centrifuged for 5 to 8 times, the centrifugal speed was 3000 rpm, the centrifugal time was 10 min, and the supernatant was discarded after the blood supernatant was clear and transparent, and the volume was fixed to 5 mL with normal saline) and 0.6 mL of normal saline and incubated at 37 °C for 4 h, then centrifuged at 3000 rpm for 10 min, the supernatant was aspirated to measure the absorbance at OD541 nm, and the hemolysis rate was calculated. It can be seen from the figure that the hemolysis rate of MMT@PLL nanosheets with different concentrations is less than 5%, indicating that the material has good biocompatibility.

[0036] 5. Colitis prevention and treatment performance Figure 9-11 To evaluate the therapeutic performance of MMT@PLL nanosheets in treating colitis, 6-week-old female c57 mice were housed in groups of 8 mice per cage and acclimated for 1 week before inclusion in the study. In the preventive treatment of UC, healthy mice were randomly divided into 5 groups (n = 8): (1) PBS + water, (2) PBS + 2.5% DSS, (3) 5ASA (pentaaminosalicylic acid group, dosage 30 mg / kg) + 2.5% DSS, (4) MMT NSs (dosage 30 mg / kg) + 2.5% DSS, (5) MMT@PLLNSs (dosage 30 mg / kg) + 2.5% DSS. The treatment methods of each group are as follows: The first day of oral administration of DSS was designated as day 0, and ordinary drinking water was used to replace the drinking water containing DSS on day 7. Oral administration was performed on days 1, 3, 5, and 7. Body weight, visible stool consistency, and fecal bleeding were assessed daily during the 9-day experiment. The colon was collected after euthanasia of the mice on the ninth day, and the colon length was calculated. It can be seen from the figure that DSS caused the mice to continue to lose weight, but after treatment with MMT@PLL nanosheets, the weight loss trend was alleviated, and the weight after treatment could be restored to normal levels and the therapeutic effect was better than that of pentaaminosalicylic acid; DSS caused the colon of mice to be significantly shortened, and the colon length of the mice in the treatment group was alleviated differently. After treatment with MMT@PLL nanosheets, the colon length basically returned to normal levels. The results showed that the nanosheets obtained in this embodiment can achieve a therapeutic effect on inflammatory bowel disease.

[0037] 6. Colitis treatment performance The evaluation method was as follows: 6-week-old female c57 mice were housed in a cage of 8 mice and acclimated for 1 week before inclusion in the study. In the delayed treatment of UC, healthy mice were randomly divided into 8 groups (n = 8): (1) Control group; (2) 2.5% DSS control group, (3) PLL (dosage 30 mg / kg) + 2.5% DSS, (4) MMT NSs (dosage 30 mg / kg) + 2.5% DSS, (5) 5-ASA (i.e., pentaaminosalicylic acid group, dosing 30 mg / kg) + 2.5% DSS, (6) 10 mg / kg MMT@PLLNSs + 2.5% DSS, (7) 30 mg / kg MMT@PLL NSs + 2.5% DSS, (8) 50 mg / kg MMT@PLL NSs +2.5% DSS. The treatment methods of each group are as follows: The control group was fed with normal drinking water, and the other groups were fed with drinking water containing 2.5% DSS. The first day of oral administration of DSS was designated as day 0, and normal drinking water was used instead of DSS-containing drinking water on day 7. The mice were gavaged on days 7, 9, 11, and 13, with groups (1) and (2) receiving 100 μL of PBS solution, and groups (3) to (8) receiving 100 μL of PBS solution containing the drug according to the corresponding dosage. Body weight changes were recorded every 2 days. On day 15, the mice were euthanized and the colon was collected to calculate the colon length, and feces were collected for intestinal flora analysis.

[0038] Fig.12 The weight changes of each group during the experiment show that DSS caused the mice to lose weight continuously in the first 7 days, and the weight loss of untreated mice was not alleviated. However, after treatment with the prepared nanomaterials, the weight loss trend was alleviated and returned to normal levels. It can also be seen that the treatment effect is better than that of pentaaminosalicylic acid. Fig.13 Colon pictures and Fig.14 The colon length statistics showed that DSS caused the colon of mice to shorten significantly, and the colon length of mice in the treatment groups was relieved to varying degrees. After treatment with 30 mg / kg MMT@PLL NSs, the colon length basically returned to normal levels. Fig.15 It can be seen that the richness of intestinal microorganisms in mice induced by DSS was significantly reduced, while the richness of intestinal microorganisms in the treatment group of mice increased, which was basically consistent with the control group. The above results show that the nanomaterial obtained in this example can achieve a therapeutic effect on inflammatory bowel disease.

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A modified montmorillonite nanosheet that can be used as a cfDNA scavenger, characterized in that: The modified montmorillonite nanosheet is based on a two-dimensional montmorillonite nanosheet as a core, and the surface of the two-dimensional montmorillonite nanosheet is modified with polylysine cations.

2. The modified montmorillonite nanosheet according to claim 1, characterized in that: The modified montmorillonite nanosheet has a thickness of 1 to 2 nm and a diameter of 200 to 300 nm.

3. A method for preparing the modified montmorillonite nanosheets according to claim 1 or 2, characterized in that: ε-polylysine is added to an aqueous solution of pure two-dimensional montmorillonite nanosheets and dispersed uniformly by ultrasonication, and then stirred for reaction at room temperature for 4 to 12 hours. The obtained product is centrifuged to obtain the modified montmorillonite nanosheets.

4. The preparation method according to claim 3, characterized in that: The mass ratio of ε-polylysine to pure two-dimensional montmorillonite nanosheets is 2-4:

1.

5. The preparation method according to claim 3, characterized in that: The power of the ultrasound is 30-60 W and the time is 5-10 min.

6. The preparation method according to claim 3, characterized in that: The centrifugal speed is 10000-12000 rpm and the centrifugal time is 10-15 min.

7. An application of the modified montmorillonite nanosheet according to claim 1 or 2, characterized in that: Used for preparing cfDNA clearing agents for treating cfDNA-related inflammation.

8. The use according to claim 7, characterized in that: The cfDNA-related inflammation includes one of inflammatory bowel disease, arthritis, and sepsis.

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