Double-enzyme cascade-gene regulation nano delivery system as well as preparation method and application thereof

Abstract

The invention relates to the technical field of gene regulation and control, in particular to a nano delivery system and a preparation method and application thereof.The nano delivery system comprises Mxene nano enzyme modified by a surface modifier, a regulation and control carrier and a delivery carrier, the surface modifier is PEGylated PEI, PEI in the surface modifier is used for being connected with the regulation and control carrier, and the delivery carrier is used for being connected with the regulation and control carrier. The Mxene nano enzyme-regulatory vector is used as a delivery vector to form an Mxene nano enzyme-regulatory vector compound, the regulatory vector comprises a CRISPR / dCas system, the CRISPR / dCas system comprises a coding sequence of sgRNA and a coding sequence of dCas protein, the sgRNA comprises a fragment combined with a target gene, and the delivery vector is probiotics for colonizing intestinal tracts. The nano delivery system can improve the intestinal inflammatory environment, promote the colonization of probiotics, restore the proportion of the beneficial flora in the intestinal tract, correct the alteration of the flora in the intestinal tract and establish a healthier and more stable intestinal micro-ecological environment.

Description

Technical Field

[0001] The present invention relates to the field of gene regulation technology, and in particular to a nano delivery system and a preparation method and application thereof. Background Art

[0002] At present, traditional treatments for inflammatory bowel disease (IBD) mainly include the following categories: ① Chemical drugs: such as 5-aminosalicylic acid (5-ASA) and glucocorticoids, which can relieve intestinal inflammation and alleviate IBD symptoms. However, the duration of action of chemical drugs is usually short, and patients face a high risk of side effects when using them for a long time. In addition, such drugs often fail to achieve long-term relief, leading to recurrence of IBD patients. ② Biological agents: including anti-TNF-α drugs, anti-integrin drugs (such as vedolizumab) and IL-12 / IL-23 inhibitors (such as ustekinumab). These biological agents reduce inflammation through specific immunosuppression and have certain efficacy. However, the cost of biological agents is high, and some patients do not respond well to them or develop drug resistance, resulting in unstable treatment effects. ③ Probiotic therapy: Probiotics (such as lactic acid bacteria) as natural intestinal regulators help restore microecological balance and improve the intestinal environment. However, due to the low colonization efficiency of probiotics in intestinal lesions and poor survival ability in oxidative stress environments, their therapeutic effects are limited, and it is difficult to exert a stable anti-inflammatory effect under high inflammation conditions.

[0003] In addition, current drug delivery systems for IBD often have the following problems: ① Difficulty in achieving targeted delivery: Most delivery systems lack specificity, and drugs tend to diffuse widely in the intestine, resulting in insufficient concentration of active ingredients in the lesion area, affecting the therapeutic effect. ② Low cell uptake efficiency: After entering the target cells, gene delivery systems and drugs often have difficulty effectively passing through the cell membrane and entering the cytoplasm. A large number of genes or drugs are degraded by phagosomes in cells, reducing the efficiency of treatment. ③ Lack of versatility and comprehensive regulatory capabilities: Most existing therapies can only work through a single mechanism, such as inhibiting inflammatory factors or maintaining the balance of intestinal microecology, and cannot achieve a comprehensive treatment strategy. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a nano delivery system and a preparation method and application thereof, so as to solve the problems in the prior art.

[0005] To achieve the above-mentioned purpose and other related purposes, the present invention first provides a nano-delivery system, which includes a Mxene nano-enzyme modified by a surface modifier, a regulatory vector and a delivery vector, wherein the surface modifier is PEGylated PEI, and the PEI in the surface modifier is used to connect with the regulatory vector to form a Mxene nano-enzyme-regulatory vector complex, and the regulatory vector is a CRISPR / dCas system, and the CRISPR / dCas system includes a coding sequence of sgRNA and a coding sequence of dCas protein, and the sgRNA includes a fragment that binds to a target gene, and the delivery vector is a probiotic that colonizes the intestine.

[0006] The present invention also provides a method for preparing a nano delivery system, the method comprising the following steps:

[0007] 1) mixing PEGylated PEI and Mxene nanozyme to prepare surface-modified Mxene nanozyme;

[0008] 2) mixing the regulatory carrier with the product prepared in step 1) to connect the regulatory carrier with PEI in the surface modifier to form a Mxene nanozyme-regulatory carrier complex;

[0009] 3) Mixing the product prepared in step 2) with a delivery carrier so that the Mxene nanozyme-regulatory carrier complex is connected to the delivery carrier by electrostatic adsorption to obtain a Mxene nanozyme-regulatory carrier-delivery carrier complex, i.e., a nano delivery system.

[0010] The present invention also provides an application of the nano delivery system in preparing a targeted preparation for treating diseases caused by oxidative stress.

[0011] As described above, a nano delivery system and a preparation method and application thereof of the present invention have the following beneficial effects:

[0012] 1) Relieve oxidative stress: Through the catalytic action of MXene nanozymes, the excess reactive oxygen species (ROS) in the intestine can be effectively removed, reducing the oxidative stress environment at the site of inflammation, thereby alleviating the inflammatory response and improving IBD symptoms;

[0013] 2) Regulate inflammatory factors and promote anti-inflammatory gene expression: Use the CRISPR / dCas system to upregulate the expression of anti-inflammatory genes, regulate the release of local inflammatory factors, further promote intestinal immune balance, and alleviate inflammatory responses;

[0014] 3) Repair the balance of intestinal microecology: by improving the intestinal inflammatory environment, promote the colonization of probiotics, restore the proportion of beneficial intestinal flora, correct the common intestinal flora imbalance in IBD patients, and establish a healthier and more stable intestinal microecological environment;

[0015] 4) Improve the safety and tolerance of treatment: Compared with traditional drugs and biological preparations, the present invention uses probiotics and gene editing technology to achieve therapeutic effects in a natural and safe way, reducing dependence on the immune system, reducing the risk of side effects, and improving patient tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shown is a schematic diagram of TEM and corresponding elemental mapping of the dual enzyme cascade-gene regulation nanodelivery system (LGG-M@P) of the present invention.

[0017] Figure 2 Shown is a schematic diagram of the fluorescence intensity of the ROS scavenging activity of LGG-M@P of the present invention.

[0018] Figure 3 Shown is a schematic diagram of Western blotting of LGG-M@P of the present invention.

[0019] Figure 4 Shown is a schematic diagram of the in vivo therapeutic effect of LGG-M@P of the present invention. Figure a is a schematic diagram of the treatment process of acute colitis induced by DSS; Figure b is a schematic diagram of the weight changes of mice in each group (healthy group, control group, LGG group, M group, M@P group, LGG-M group and LGG-M@P group) within 10 days; Figure c is a schematic diagram of the measurement of colon tissue length of mice in each group on the 10th day; Figure d is a schematic diagram of the changes in the disease activity index (DAI) within 10 days; Figures f, g, h, and i are respectively schematic diagrams of the levels of MPO, TNF-α, IL-6 and IL-1β in the colon tissue of mice on the 10th day after different treatments (healthy, control, LGG, M, M@P, LGG-M and LGG-M@P). Figure e is a schematic diagram of HE staining of colon sections of mice with acute colitis on the 10th day. DETAILED DESCRIPTION

[0020] The present invention first provides a nano-delivery system, which includes a Mxene nanozyme modified by a surface modifier, a regulatory vector and a delivery vector, wherein the surface modifier is PEGylated PEI, and the PEI in the surface modifier is used to connect with the regulatory vector to form a Mxene nanozyme-regulatory vector complex, and the regulatory vector is a CRISPR / dCas system, and the CRISPR / dCas system includes a coding sequence of sgRNA and a coding sequence of dCas protein, and the sgRNA includes a fragment that binds to a target gene, and the delivery vector is a probiotic that colonizes the intestine.

[0021] In certain embodiments of the present invention, the Mxene nanozyme is i-Mxene nanozyme.

[0022] In certain embodiments of the present invention, the i-Mxene nanozyme is Mo 1.33 C.

[0023] In certain embodiments of the present invention, the PEGylated PEI is PEG-d-PEI, mPEG-d-PEI or PEtOz-d-PEI.

[0024] In a preferred embodiment of the present invention, the PEGylated PEI is mPEG-d-PEI, namely mPEG-detachable PEI.

[0025] In certain embodiments of the present invention, the regulatory vector is a plasmid.

[0026] In certain embodiments of the present invention, the target gene is an anti-inflammatory gene.

[0027] In a specific embodiment of the present invention, the anti-inflammatory gene is NLRP12;

[0028] In certain embodiments of the present invention, the coding sequence of the sgRNA of the anti-inflammatory gene NLRP12 is shown in SEQ ID NO.1.

[0029] In certain embodiments of the present invention, the coding sequence of the sgRNA as shown in SEQ ID NO.1 includes a fragment that binds to the target gene and a conserved sequence required for binding to the Cas protein, namely, a Scaffold sequence.

[0030] In certain embodiments of the present invention, the dCas protein is a dCas 9 protein or a variant thereof.

[0031] In certain embodiments of the present invention, the Cas protein variant is a dCas 9 / VPR fusion protein.

[0032] In certain embodiments of the present invention, the dCas 9 / VPR fusion protein is a dCas 9 / VPR fusion protein formed by fusing the VPR (VP64-p65-Rta) protein to the C-terminus of the dCas 9 protein.

[0033] In certain embodiments of the present invention, the coding sequence of the dCas9 / VPR fusion protein is shown in SEQ ID NO.2.

[0034] In certain embodiments of the present invention, the probiotics that colonize the intestine are LGG probiotics (Lactobacillus rhamnosus GG, referred to as LGG).

[0035] The present invention also provides a method for preparing a nano delivery system, the method comprising the following steps:

[0036] 1) mixing PEGylated PEI and Mxene nanozyme to prepare surface-modified Mxene nanozyme;

[0037] 2) mixing the regulatory carrier with the product prepared in step 1) to connect the regulatory carrier with PEI in the surface modifier to form a Mxene nanozyme-regulatory carrier complex;

[0038] 3) Mixing the product prepared in step 2) with a delivery carrier so that the Mxene nanozyme-regulatory carrier complex is connected to the delivery carrier by electrostatic adsorption to obtain a Mxene nanozyme-regulatory carrier-delivery carrier complex, i.e., a nano delivery system.

[0039] In certain embodiments of the present invention, in step 1), the Mxene nanozyme is prepared by existing technology or purchased.

[0040] In certain embodiments of the present invention, in step 1), the Mxene nanozyme is i-Mxene nanozyme.

[0041] In certain embodiments of the present invention, the PEGylated PEI is PEG-d-PEI, mPEG-d-PEI or PEtOz-d-PEI.

[0042] In a preferred embodiment of the present invention, the PEGylated PEI is mPEG-d-PEI, namely mPEG-detachable PEI.

[0043] In certain embodiments of the present invention, the mPEG-d-PEI is commercially available mPEG-d-PEI.

[0044] In certain embodiments of the present invention, in step 1), the ratio of the Mxene nanozyme to the PEGylated PEI is 400-600 μg / ml: 1 mg / ml.

[0045] In a preferred embodiment of the present invention, in step 1), the mass ratio of the Mxene nanozyme to the PEGylated PEI is 500 μg / ml:1 mg / ml.

[0046] In certain embodiments of the present invention, in step 1), the mPEG-d-PEI is prepared by mixing mPEG-CHO and PEI and then dissolving the mixture in water.

[0047] In certain embodiments of the present invention, in step 2), the ratio of the regulatory vector to the product prepared in step 1) is 15-25 μg / ml: 250 μg / ml.

[0048] In a preferred embodiment of the present invention, in step 2), the mass ratio of the regulatory vector to the product prepared in step 1) is 20 μg / ml:250 μg / ml. In certain embodiments of the present invention, step 2) further comprises stirring after mixing.

[0049] Furthermore, the stirring time is 1 to 3 hours.

[0050] Furthermore, the stirring time is 2 hours.

[0051] In certain embodiments of the present invention, in step 3), the ratio of the delivery vector to the product prepared in step 2) is 8×10 7 ~2×10 8 CFU / ml:1mg / ml.

[0052] In certain embodiments of the present invention, in step 3), the mixing temperature is room temperature (20° C. to 30° C.).

[0053] In a preferred embodiment of the present invention, in step 3), the mixing temperature is 25°C.

[0054] In certain embodiments of the present invention, step 3) further comprises stirring after mixing.

[0055] In certain embodiments of the present invention, the stirring time is 3 to 5 hours.

[0056] In a preferred embodiment of the present invention, the stirring time is 4 hours.

[0057] The present invention also provides an application of the nano delivery system in preparing a targeted preparation for treating diseases caused by oxidative stress.

[0058] In certain embodiments of the present invention, the formulation is in the form of a capsule.

[0059] In certain embodiments of the invention, the disease is inflammatory bowel disease (IBD).

[0060] In certain embodiments of the invention, the inflammatory bowel disease (IBD) comprises ulcerative colitis (UC) or Crohn's disease (CD).

[0061] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0062] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0063] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0064] Example 1 Preparation of Nano-delivery System

[0065] (1) Preparation of i-MXene nanozyme (M):

[0066] Material preparation: Mo, Y, Al and C powders were mixed in a molar mass ratio of 133:133:100:100. The mixture was then heated to 1500°C in an alumina crucible and exposed to an argon flow for 20 hours. After cooling to room temperature, the product was ground into powder in an agate mortar and sieved with a 200-mesh sieve. The resulting (Mo2 / 3Y1 / 3)2AlC iMAX phase precursor fine powder was used for subsequent iMXene preparation.

[0067] Etching and intercalation: 20 mL of 40% HF was mixed with 1 g of (Mo2 / 3Y1 / 3)2AlC iMAX phase precursor powder and stirred at room temperature for 24 hours before centrifugation. The supernatant was filtered and resuspended with deoxygenated double distilled water. Washed repeatedly with deoxygenated double distilled water five times to remove any residual acid and / or reaction products, and then filtered. The powder of the filter membrane was dried at room temperature for 12 hours to obtain multilayer iMXene. Take 0.1 g of the dried multilayer iMXene, mix it with 1 mL of TBAOH and place it in a centrifuge tube. After manual shaking for 5 minutes, centrifuge at 6000 rpm for 5 minutes to obtain the supernatant. Deoxygenated double distilled water was injected into the centrifuge tube to remove residual TBAOH, and the rinse was repeated and centrifuged 3 times. Finally, deoxygenated double distilled water was added to the resulting precipitate and shaken for 5 minutes to obtain a single layer of iMXene.

[0068] Cleaning and drying: The etched nanosheets are centrifuged and purified, and then dried under vacuum conditions to form the final iMXene nanosheets.

[0069] (2) mPEG-d-PEI surface modified i-MXene nanozyme (M@):

[0070] Polyethyleneimine (PEI) (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.) and methoxy polyethylene glycol aldehyde (mPEG-CHO) (purchased from Xi'an Ruixi Biotechnology Co., Ltd.) were mixed in a mass ratio of 2:1 at pH 7.4 with deoxygenated double distilled water as solvent to form an mPEG-d-PEI solution. The prepared mPEG-d-PEI was mixed with the i-MXene nanozyme to make the final concentration of the i-MXene nanozyme 500 μg / ml and the final concentration of mPEG-d-PEI in the system 1 mg / ml, thereby obtaining the i-MXene nanozyme (M@) surface modified with mPEG-d-PEI.

[0071] (3) Preparation of nano-delivery system

[0072] The product prepared in step (2) was mixed with a plasmid containing the coding sequence of the sgRNA of the anti-inflammatory gene NLRP12 and the coding sequence of the dCas / VPR fusion protein (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd.) at a mass ratio of 1:12.5, so that the final concentration of the product prepared in step (2) was 250 μg / ml and the final concentration of the plasmid was 20 μg / ml. The mixture was stirred at room temperature for 2 hours to obtain a plasmid-nanozyme complex (M@P); 1 mg of the plasmid-nanozyme complex was mixed with 1 mL of LGG (containing 1 mL of PBS, 1×10 8 LGG) and stirred for 4 hours to finally obtain the nanozyme-plasmid-LGG complex, namely the nanodelivery system (M@P-LGG).

[0073] (4) Delivery and release mechanism of nanozyme-plasmid-LGG complex:

[0074] Intestinal release: LGG carries M@P into the intestine, and M@P is released from LGG under the high ion concentration and oxidative stress environment of IBD.

[0075] Cellular uptake: The released M@P is taken up by intestinal cells, enters the cells through the endocytosis mechanism, and then escapes into the lysosomes.

[0076] Upregulation of anti-inflammatory genes and regulation of oxidative stress: After M@P escapes from the lysosome, it upregulates the anti-inflammatory gene NLRP12 through the CRISPR / dCas system. At the same time, iMXene nanozymes clear ROS, effectively inhibiting the inflammatory response and restoring intestinal immune balance.

[0077] Example 2 Characterization of Nanodelivery System

[0078] TEM (FEI Tecnai G2 F20) was used to observe the structure and composition of LGG and LGG combined with M@P (i.e., LGG-M@P), and the corresponding element scanning was performed.

[0079] Depend on Figure 1 It can be seen that the nanosheets are unevenly attached to the LGG surface, and the Mo and P elements are co-localized with the nanosheets, indicating that the nanozyme-plasmid complex is stably attached to the LGG surface.

[0080] Example 3 Investigating the ROS scavenging activity and protein expression enhancement ability of the nanodelivery system

[0081] ROS scavenging activity: Raw 264.7 cells were cultured at 1×10 5 The density of cells was inoculated in a confocal specific culture dish and incubated at 37°C for 24 hours. The cells were first pretreated with LPS (1 μg / ml) for 30 minutes, and then the culture medium was replaced with M@P-LGG (100 μg / mL) for a total of 6 hours. The culture medium was then washed three times with PBS, and the cell nuclei were stained with DAPI for 20 minutes to further observe the intracellular fluorescence intensity and measure the fluorescence signal.

[0082] To enhance protein expression, CT26 cells were cultured at 1×10 5The cells were inoculated in 6-well plates at a density of 100 μg / dish, cultured overnight to allow the cells to adhere to the wall, and Western Blot was used to detect the expression of NLRP12. The cells were incubated with control, i-MXene nanozyme (M), i-MXene nanozyme (M@) modified with mPEG-d-PEI surface, and i-MXene nanozyme-plasmid complex (M@P) (100 μg / mL) for 24 hours. Each well of cells was rinsed twice with PBS, then dissolved on ice for 30 minutes and gently inverted to mix. After centrifugation (13680×g), the total protein concentration of the cell supernatant lysate was determined using the BCA protein detection kit. The total protein (30 μg) was analyzed by 10% SDS-PAGE and transferred to a PVDF membrane. The samples were incubated with blocking buffer at room temperature for 1 hour. The membrane was soaked in a glyceraldehyde-3-phosphate solution (1:10 000) containing β-actin anti-rabbit antibody (Abcam, Cat#ab8227) and NLRP12 rabbit monoclonal antibody for 12 h, and then soaked in a conjugated secondary antibody (Beyotime) at room temperature for 1 h. Finally, the PVDF membrane was washed 3 times (10 min each time) with 0.02% Tris buffer and Tween 20 buffer, and then washed with Pierce TM ECL Western blotting substrate was used to observe the target protein.

[0083] Depend on Figure 2 It can be seen that the fluorescence of the LPS-treated group was the highest, indicating an increase in ROS levels. In the group containing nanozymes (M), the fluorescence intensity decreased, indicating that the M@P dual enzyme gene regulation framework has excellent ROS scavenging ability.

[0084] Depend on Figure 3 These findings were further supported by Western blot and protein expression analysis, showing that NLRP12 protein levels were significantly increased only in the M@P group, and that NLRP12 protein levels in the non-M@P group were lower than those in the M@P group, indicating a significant effect of the M@P gene regulatory framework on NLRP12 expression. This significant upregulation was not negatively affected by adsorption to LGG.

[0085] Example 4 Investigating the in vivo therapeutic effect of LGG-M@P nanodelivery system

[0086] The mice were divided into 7 groups, 5 in each group. The mice in the 6 groups drank DSS aqueous solution (3%, w / w) for 7 consecutive days to induce acute colitis. The mice in the control group drank only plain water. From the 7th day to the 10th day, the DSS-treated mice orally took cold-dried enteric-coated capsules of different substances, including LGG, M, M@P, LGG-M and LGG-M@P (LGG = 1×108, M = 10 mg / kg), once a day for 4 days.

[0087] When comparing the efficacy with clinical drugs, the dosage was 40 mg / kg 5-aminosalicylic acid (5-asa) (MedChemExpress LLC.Cat#HY-15027) was orally administered into the microenvironment (mic), and 5 mg / kg vedolizumab (MedChemExpress LLC.Cat#HY-P9911), ustekinumab (MedChemExpress LLC.Cat#HY-P9909) and infliximab (MedChemExpress LLC.Cat#HY-P9970) were infused via the tail vein. The above clinical drugs were administered once every 2 days. The severity of colitis in mice was evaluated every day. The severity was assessed by the disease activity index (0-11), including weight loss (1, 1-5%; 2, 5-10%; 3, 10-15%; 4, 15-20%), rectal bleeding (0, normal; 1, semi-normal; 2, fecal occult blood positive; 3, gross bloody stool; 4, severe rectal bleeding), and stool consistency (0, normal; 1, soft stool; 2, mucous stool; 3. loose stool; 4, liquid stool adhering to the anus). The mice were then killed and colon tissues were collected for histological and ELISA analysis.

[0088] Figure 4 This suggests that while clinical interventions have shown some effectiveness in reducing inflammation, the LGG-M@P system significantly improved the overall health of UC mice, especially compared with 5-ASA. In addition, the relief of UC-related symptoms was more robust compared with other M@Ps ( Figure 4 bd). Histological analysis showed that the LGG-M@P group effectively repaired the pathological damage of the colon tissue of IBD mice, showing a greater therapeutic effect than the clinical medication group ( Figure 4 e). In addition, the myeloperoxidase (MPO) level in the LGG-M@P group was significantly lower than that in the 5-ASA group and the biological agent treatment group, indicating that it is more effective in inhibiting the inflammatory response ( Figure 4 f). The determination of inflammatory factors (including TNF-α, IL-6, and IL-1β) in mouse colon tissue further emphasized the anti-inflammatory efficacy of the LGG-M@P probiotic dual enzyme cascade repair system ( Figure 4 gi).

[0089] The sequences involved in this application are as follows:

[0090] SEQ ID NO.1

[0091]

[0092] SEQ ID NO.2

[0093]

[0094]

[0095]

[0096]

[0097] TGCATATCAG CACAGGACTG TCCATCTTCG ACACATCTCT GTTTGA

[0098] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A nano delivery system, characterized in that: The nano delivery system includes a Mxene nanozyme modified by a surface modifier, a regulatory vector and a delivery vector, wherein the surface modifier is PEGylated PEI, and the PEI in the surface modifier is used to connect with the regulatory vector to form a Mxene nanozyme-regulatory vector complex, and the regulatory vector is a CRISPR / dCas system, and the CRISPR / dCas system includes a coding sequence of sgRNA and a coding sequence of dCas protein, and the sgRNA includes a fragment that binds to the target gene, and the delivery vector is a probiotic that colonizes the intestine.

2. The nano delivery system according to claim 1, characterized in that Includes any one or more of the following conditions: A) the Mxene nanozyme is i-Mxene nanozyme; B) the PEGylated PEI is PEG-d-PEI, mPEG-d-PEI or PEtOz-d-PEI; preferably, the PEGylated PEI is mPEG-d-PEI; C) the regulatory vector is a plasmid; D) The probiotics that colonize the intestine are LGG probiotics.

3. The nano delivery system according to claim 1, characterized in that The target gene is an anti-inflammatory gene; preferably, the anti-inflammatory gene is NLRP12.

4. The nano delivery system according to claim 3, characterized in that The coding sequence of the sgRNA of the anti-inflammatory gene NLRP12 is shown in SEQ ID NO.

1.

5. The nano delivery system according to claim 1, characterized in that The dCas protein is a dCas 9 protein or a variant thereof.

6. The nano delivery system according to claim 5, characterized in that The dCas 9 protein variant is a dCas 9 / VPR fusion protein; preferably, the coding sequence of the dCas9 / VPR fusion protein is shown in SEQ ID NO.

2.

7. The method for preparing the nano delivery system according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: 1) mixing PEGylated PEI and Mxene nanozyme to prepare surface-modified Mxene nanozyme; 2) mixing the regulatory carrier with the product prepared in step 1) to connect the regulatory carrier with PEI in the surface modifier to form a Mxene nanozyme-regulatory carrier complex; 3) Mixing the product prepared in step 2) with a delivery carrier so that the Mxene nanozyme-regulatory carrier complex is connected to the delivery carrier by electrostatic adsorption to obtain a Mxene nanozyme-regulatory carrier-delivery carrier complex, i.e., a nano delivery system.

8. The method for preparing the nano delivery system according to claim 7, characterized in that: In step 1), the ratio of the Mxene nanozyme to the PEGylated PEI is 400-600 μg / ml: 1 mg / ml; And / or, in step 2), any one or more of the following conditions are included: 21) The ratio of the regulatory vector to the product prepared in step 1) is 15-25 μg / ml: 250 μg / ml; 22) stirring after mixing; preferably, the stirring time is 1 to 3 hours.

9. The method for preparing the nano delivery system according to claim 7, characterized in that: In step 3), any one or more of the following conditions are included: 31) The ratio of the delivery vector to the product obtained in step 2) is 8×10 7 ~2×10 8 CFU / ml: 1mg / ml; 32) The mixing temperature is 20-30°C; preferably, the mixing temperature is 25°C; 33) stirring after mixing; preferably, the stirring time is 3 to 5 hours.

10. Use of the nano delivery system according to any one of claims 1 to 6 in the preparation of targeted preparations for treating diseases caused by oxidative stress; preferably, the disease caused by oxidative stress is inflammatory bowel disease.

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