A modified montmorillonite nanosheet that can be used as a cfDNA scavenger, its preparation method and application

Abstract

This invention discloses a modified montmorillonite nanosheet that can be used as a cfDNA scavenger, its preparation method, and its application. The modified nanosheet is a two-dimensional montmorillonite nanosheet with polylysine cations modified on its surface. The modified montmorillonite nanosheet of this invention can inhibit inflammation by scavenging cfDNA and regulating the abundance of intestinal microorganisms, effectively alleviating symptoms induced by IBD. It also exhibits good stability, biocompatibility, and biodegradability, and can be used to treat inflammatory bowel disease.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, specifically relating to a modified montmorillonite nanosheet that can be used as a cfDNA scavenger, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory bowel disease mediated by abnormal immunity, caused by multiple etiologies. Crohn's disease (CD) and ulcerative colitis (UC) are two major forms of IBD, both of which significantly reduce patients' quality of life. Furthermore, IBD often leads to a range of complications, including fistulas, abscesses, malignancies, colorectal cancer, and intestinal fibrosis, making it a major healthcare concern. Environmental, genomic, immune, and microbial factors are all involved in the occurrence and development of IBD, with dysbiosis stemming from the oxidative nature of the intestinal inflammatory response. The recognition of DAMPs produced or released by damaged and dead cells promotes aseptic inflammation, with released cfDNA considered a potential receptor mediating TLR9-dependent pro-inflammatory effects. Therefore, cfDNA clearance could also be a potentially effective strategy for treating IBD.

[0003] Polylysine has a certain inhibitory effect on the respiration of *E. coli*, which is significantly different from typical respiratory pathway inhibitors. Simultaneously, polylysine also acts on biomembrane systems and protein synthesis systems. When polylysine comes into contact with biomembranes, it can directly and rapidly disrupt energy metabolism dependent on membrane structure integrity and the selectivity of substances essential for cell and organelle survival, leading to lysosomal membrane rupture and inducing autolysis in microorganisms, ultimately resulting in bacterial death. Furthermore, montmorillonite has a layered structure and non-uniform charge distribution. After ingestion, it is not absorbed by the human intestine but adheres to the intestinal wall, adsorbing and immobilizing intestinal microorganisms and their toxins, thus neutralizing their pathogenicity. In addition, montmorillonite, when coating the digestive tract mucosa, can enhance the mucosa's defense against aggressive factors by binding to mucus glycoproteins. Methods based on the synergistic treatment of IBD using polylysine and montmorillonite have not been publicly disclosed. Summary of the Invention

[0004] To address the issue that conventional drugs currently used in clinical practice can only temporarily relieve IBD symptoms without curing the damaged intestinal barrier and gut microbiota imbalance, this invention constructs a modified montmorillonite nanosheet and applies it to the treatment of inflammatory bowel disease. It can adsorb cfDNA, regulate gut microbiota, and accumulate at the site of inflammation, thereby achieving the effect of treating IBD.

[0005] To solve the technical problem, the present invention adopts the following technical solution:

[0006] This invention first discloses a modified montmorillonite nanosheet that can be used as a cfDNA scavenger. The modified montmorillonite nanosheet is characterized by having a two-dimensional montmorillonite nanosheet as its core, with polylysine cations modified on the surface of the two-dimensional montmorillonite nanosheet. The modified montmorillonite nanosheet has a thickness of 1–2 nm and a diameter of 200–300 nm.

[0007] The preparation method of the modified montmorillonite nanosheets of this invention is as follows: montmorillonite is repeatedly freeze-thawed and then added to deionized water. The mixture is then subjected to ultrasonication in an ice bath (ultrasonic power of 200-300 W for 0.5-1 h), centrifuged at 2000-2500 rpm for 15-30 min, and the supernatant is collected. The supernatant is then centrifuged at 10000-13000 rpm for 15-20 min to obtain pure two-dimensional montmorillonite nanosheets. ε-polylysine is added to the aqueous solution of the pure two-dimensional montmorillonite nanosheets and ultrasonically dispersed evenly (ultrasonic power of 30-60 W for 5-10 min). The mixture is then stirred at room temperature for 4-12 h. The resulting product is then centrifuged (centrifugation speed of 10000-12000 rpm for 10-15 min) to obtain the modified montmorillonite nanosheets.

[0008] Furthermore, the mass ratio of ε-polylysine to pure two-dimensional montmorillonite nanosheets is 2–4:1.

[0009] The modified montmorillonite nanosheets of this invention can be used to prepare cfDNA scavengers for treating cfDNA-related inflammation, including inflammatory bowel disease, arthritis, and sepsis. The modified montmorillonite nanosheets of this invention exhibit excellent efficacy in the treatment of inflammatory bowel disease and show no significant toxicity to mammalian cells, demonstrating good biocompatibility. They can be used to prepare novel nanomedicines for treating inflammatory bowel disease. The mechanism of action involves precise drug delivery by anchoring the nanosheets around the inflamed area of ​​the colon, thereby improving the therapeutic effect of inflammatory bowel disease. Modification with polylysine cations enhances the binding capacity of the positively charged nanosheets to cfDNA. By adsorbing cfDNA, the modified montmorillonite nanosheets can reduce cfDNA-induced TLR9 signaling and pro-inflammatory macrophage activation to restore the epithelial barrier and alleviate inflammation. Simultaneously, they can promote the polarization of pro-inflammatory macrophages M1 into anti-inflammatory macrophages M2. After oral administration, the modified montmorillonite nanosheets protect the intestinal barrier by reducing inflammation, clearing intestinal bacteria, and regulating the intestinal microbiota, thereby significantly alleviating the symptoms of inflammatory bowel disease.

[0010] The beneficial effects of this invention are reflected in:

[0011] 1. To address the issue that polylysine cations, even after directly binding to cfDNA, still stimulate the TLR9 pathway and generate inflammation as a product of cell damage, this invention prepares montmorillonite nanosheets modified with polylysine cations. These nanosheets adsorb cfDNA generated after cell damage, directly blocking its connection with the TLR9 pathway and thus weakening the ability of the TLR9 pathway to generate inflammation.

[0012] 2. In the modified montmorillonite nanosheets of the present invention, after polylysine kills harmful bacteria in the intestines, montmorillonite can cleanse the intestines, thereby achieving the effect of treating IBD through the synergy of the two.

[0013] 3. The modified montmorillonite nanosheets of this invention have a simple preparation process and mild reaction conditions, making them possible for large-scale production and possessing potential for industrial and practical applications.

[0014] 4. The modified montmorillonite nanosheets of the present invention have good dispersibility and stability, and excellent biocompatibility. They have no direct or indirect toxic effects on the human body and no potential toxicity, which is beneficial for clinical use. Attached Figure Description

[0015] Figure 1 This is a schematic diagram illustrating the synthesis of the modified montmorillonite nanosheets of this invention.

[0016] Figure 2 Transmission electron microscopy (TEM) image of the modified montmorillonite nanosheets prepared in Example 1.

[0017] Figure 3 An atomic force microscope image of the modified montmorillonite nanosheets prepared in Example 1.

[0018] Figure 4 Zeta potential diagrams of pure two-dimensional montmorillonite nanosheets and modified montmorillonite nanosheets prepared in Example 1.

[0019] Figure 5 The particle size distribution of the modified montmorillonite nanosheets prepared in Example 1 is shown.

[0020] Figure 6 The graph shows the adsorption performance of modified montmorillonite nanosheets and pure two-dimensional montmorillonite nanosheets and polylysine on ctDNA (calf thymus DNA) prepared in Example 1.

[0021] Figure 7 The graph shows the performance of modified montmorillonite nanosheets and pure two-dimensional montmorillonite nanosheets and polylysine on CpG-induced macrophage inflammatory factors prepared in Example 1.

[0022] Figure 8 Biocompatibility diagrams of modified montmorillonite nanosheets prepared at different concentrations for Example 1.

[0023] Figure 9 This is a graph showing the weight changes of mice in each group during the evaluation of the preventive and therapeutic effects of colitis.

[0024] Figure 10 This is a anatomical colon image of mice in each group during the evaluation of the preventive and therapeutic effects of colitis.

[0025] Figure 11 This is a statistical graph showing the length of the colon after dissection in mice from each group during the evaluation of the preventive and therapeutic effects of colitis.

[0026] Figure 12 This is a graph showing the changes in body weight of mice in each group during the evaluation of the therapeutic performance of colitis.

[0027] Figure 13 This is a anatomical colon image of mice in each group during the evaluation of the therapeutic performance of colitis.

[0028] Figure 14 This is a statistical graph showing the length of the colon after dissection in mice from each group during the evaluation of the therapeutic performance of colitis.

[0029] Figure 15 The abundance of gut-like flora was measured in feces collected after dissection of mice in each group during the evaluation of the therapeutic performance of colitis. (a) is the chao1 analysis and (b) is the shannon index analysis. Detailed Implementation

[0030] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to examples. The following content is merely an example and illustration of the concept of the present invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, as long as they do not depart from the inventive concept or exceed the scope defined by the claims, all of which should fall within the protection scope of the present invention.

[0031] Example 1

[0032] In this embodiment, montmorillonite nanosheets were prepared using the following method:

[0033] 1 g of montmorillonite powder was repeatedly freeze-thawed and added to 20 mL of deionized water. The mixture was then sonicated in an ice bath (50% of the maximum ultrasonic power, approximately 300 W) for 1 h, centrifuged at 2500 rpm for 30 min, and the supernatant was collected. The supernatant was then centrifuged again at 13000 rpm for 20 min, and the resulting precipitate was the two-dimensional montmorillonite nanosheets (denoted as MMT NSs). ε-polylysine (at a mass ratio of ε-polylysine to pure two-dimensional montmorillonite nanosheets of 4:1) was added to an aqueous solution of pure two-dimensional montmorillonite nanosheets and dispersed ultrasonically (ultrasonic power approximately 60 W for 10 min). The mixture was then stirred at room temperature for 12 h. The resulting product was centrifuged at 12000 rpm for 15 min, and the resulting precipitate was the modified montmorillonite nanosheets (denoted as MMT@PLLNSs).

[0034] The morphology and properties of the MMT@PLL nanosheets obtained in this embodiment were characterized as follows:

[0035] I. Morphological Characteristics

[0036] Figure 2 The image shows a transmission electron microscope (TEM) image of the MMT@PLL nanosheets obtained in this embodiment. The image shows that the nanosheets have a diameter of 200–300 nm.

[0037] Figure 3 The image shows an atomic force microscope image of the MMT@PLL obtained in this embodiment. The thickness of the nanosheet is 1–2 nm.

[0038] Figure 4 The figure shows the Zeta potential of the MMT nanosheets and MMT@PLL nanosheets obtained in this embodiment. As can be seen from the figure, the Zeta potential of the pure two-dimensional montmorillonite nanosheets is -13 mV. After modification with polyamino acid molecules, the Zeta potential increases to +31 mV, indicating that polylysine cations were successfully loaded onto the surface of the two-dimensional montmorillonite nanosheets. The increase in Zeta potential may be due to ion exchange between polylysine cations and interlayer cations of montmorillonite nanosheets.

[0039] Figure 5 The image shows the hydrated particle size of the MMT@PLL nanosheets obtained in this embodiment. As can be seen from the image, the diameter of the nanosheets is 200-300 nm, which is consistent with the results of transmission electron microscopy.

[0040] II. Test for cfDNA clearance ability

[0041] Serum cfDNA levels were closely correlated with the expression levels of colonic TLR9, TNF-α, iNOS, and F4 / 80. Higher serum cfDNA levels were co-correlated with IBD severity and colonic TLR9 expression, indicating that cfDNA-TLR9 signaling is a target for IBD treatment. To verify the cfDNA clearance ability of MMT@PLL nanosheets, this example used calf thymus DNA (ctDNA) as a model and MMT nanosheets and ε-polylysine PLL raw materials as controls. The specific characterization method was as follows: MMT@PLL NSs, MMT NSs, or PLL material were added to the ctDNA solution, with a material-to-ctDNA mass ratio of 1:1 to 7:1, and then ethidium bromide (EtBr) was added to evaluate the binding ability of the nanoreagent to ctDNA. The results are as follows: Figure 6 As shown, the removal efficiency of cfDNA was best when the mass ratio of MMT@PLL NSs material to DNA was 6:1. This high removal efficiency is attributed to the strong electrostatic attraction between PLL and DNA phosphate.

[0042] III. The in vitro anti-inflammatory ability of the constructed macrophage inflammation model

[0043] Figure 7 This study elucidated the in vitro anti-inflammatory ability of two-dimensional montmorillonite nanosheets in constructing a macrophage inflammation model. Since macrophages are crucial participants in immunopathogenesis, this invention hypothesizes that cfDNA-mediated TLR9 activation is key to M1 macrophage polarization, as macrophage polarization leads to the release of pro-inflammatory cytokines. First, CpG 1826 was used as an in vitro substitute for cfDNA to induce inflammation in macrophages. Then, MMT@PLL NSs, MMTNSs, or PLL materials were added to cell culture dishes and co-incubated with stimulated macrophages for 12 hours. The levels of inflammatory factors in the supernatant were then measured. Significance comparison of the final data revealed that MMT@PLLNSs exhibited a good inhibitory effect on TNF-α, a polarization marker of M1 macrophages.

[0044] IV. Biocompatibility Testing of Materials in Vivo

[0045] Figure 8The biocompatibility of MMT-PLL nanosheets at different concentrations is shown in the figure. The characterization method was as follows: MMT-PLL nanosheets were diluted in the overall dispersion to 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL. 0.2 mL of each concentration solution was mixed with 0.2 mL of treated blood (500 μL of fresh blood was added to 4.5 mL of physiological saline and centrifuged 5–8 times at 3000 rpm for 10 min until the blood supernatant was clear and transparent; the supernatant was then discarded, and the volume was adjusted to 5 mL with physiological saline) and 0.6 mL of physiological saline, respectively, and incubated at 37 ℃ for 4 h. Afterward, the mixture was centrifuged at 3000 rpm for 10 min, and the absorbance at OD541 nm was measured to calculate the hemolysis rate. The figure shows that the hemolysis rate of MMT@PLL nanosheets at different concentrations was less than 5%, indicating good biocompatibility of the material.

[0046] V. Performance in the prevention and treatment of colitis

[0047] Figure 9-11 To evaluate the therapeutic efficacy of MMT@PLL nanosheets for colitis, 6-week-old female c57 mice were housed in groups of 8 and acclimatized for 1 week prior to inclusion in the study. In the prophylactic treatment of UC, healthy mice were randomly assigned to 5 groups (n = 8): (1) PBS + water, (2) PBS + 2.5% DSS, (3) 5ASA (pentaaminosalicylic acid group, 30 mg / kg) + 2.5% DSS, (4) MMT NSs (30 mg / kg) + 2.5% DSS, (5) MMT@PLL NSs (30 mg / kg) + 2.5% DSS. The treatment methods for each group were as follows:

[0048] Day 0 was designated as the first day of oral administration of DSS, and regular drinking water was used instead of DSS-containing water on day 7. Oral administration was performed on days 1, 3, 5, and 7. Body weight, visible fecal consistency, and fecal bleeding were assessed daily throughout the 9-day experiment. After euthanasia on day 9, colons were collected and colon length was calculated. The figures show that DSS caused a continuous decrease in mouse body weight, but this trend was mitigated after treatment with MMT@PLL nanosheets, and the weight returned to normal levels after treatment, with better therapeutic effects than pentamisalicylic acid. DSS significantly shortened the colon in mice, and the colon length in the treatment group was alleviated to varying degrees. After treatment with MMT@PLL nanosheets, the colon length essentially returned to normal levels. These results indicate that the nanosheets obtained in this embodiment can achieve a therapeutic effect on inflammatory bowel disease.

[0049] VI. Performance in treating colitis

[0050] The evaluation method was as follows: Eight 6-week-old female c57 mice were housed in each cage and acclimatized for one 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 (30 mg / kg) + 2.5% DSS; (4) MMT NSs (30 mg / kg) + 2.5% DSS; (5) 5-ASA (i.e., pentamirrolic acid group, 30 mg / kg) + 2.5% DSS; (6) 10 mg / kg MMT@PLL NSs + 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 for each group were as follows:

[0051] The control group was fed with regular drinking water, while the other groups were fed with drinking water containing 2.5% DSS. Day 0 was the first day of oral administration of DSS, and regular drinking water was used instead of the DSS-containing water on day 7. Administered the medication by gavage on days 7, 9, 11, and 13. Groups (1) and (2) were administered 100 μL of PBS solution by gavage, while groups (3) to (8) were administered 100 μL of PBS solution containing the corresponding dosage by gavage. Body weight changes were recorded every two days. After euthanasia on day 15, the colon was collected, colon length was calculated, and feces were collected for intestinal flora analysis.

[0052] Figure 12 The study showed that DSS caused a continuous decrease in mouse weight during the first 7 days, and the weight loss in untreated mice was not alleviated. However, after treatment with the prepared nanomaterials, the weight loss trend was alleviated and the weight returned to normal levels. Furthermore, the treatment effect was better than that of pentamirric acid. Figure 13 Colon pictures and Figure 14 Statistical analysis of colon length showed that DSS significantly shortened the colon in mice. The treatment group experienced varying degrees of relief in colon length, with the colon length essentially returning to normal after treatment with 30 mg / kg MMT@PLL NSs. Figure 15 It was found that DSS-induced gut microbiota richness in mice was significantly reduced, while the gut microbiota richness in the treatment group recovered, basically consistent with the control group. These results indicate that the nanomaterials obtained in this embodiment can achieve a therapeutic effect on inflammatory bowel disease.

[0053] 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 within 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 nanosheets are based on two-dimensional montmorillonite nanosheets, with polylysine cations modified on the surface of the two-dimensional montmorillonite nanosheets; the thickness of the modified montmorillonite nanosheets is 1-2 nm and the diameter is 200-300 nm. The modified montmorillonite nanosheets are prepared by adding ε-polylysine to an aqueous solution of pure two-dimensional montmorillonite nanosheets and dispersing it evenly by ultrasonication, and then stirring the reaction at room temperature for 4 to 12 hours. The resulting product is then centrifuged to obtain the modified montmorillonite nanosheets, wherein the mass ratio of ε-polylysine to pure two-dimensional montmorillonite nanosheets is 2 to 4:

1.

2. The modified montmorillonite nanosheets that can be used as cfDNA scavengers according to claim 1, characterized in that: The ultrasound power is 30-60 W and the duration is 5-10 min.

3. The modified montmorillonite nanosheets that can be used as cfDNA scavengers according to claim 1, characterized in that: The centrifugation speed is 10000~12000 rpm and the centrifugation time is 10~15 min.

4. The application of the modified montmorillonite nanosheets as described in claim 1, which can be used as a cfDNA scavenger, in the preparation of a drug for treating colitis.

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