Preparation method and application of intestinal tract targeted DNA (deoxyribonucleic acid) nano origami

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a preparation method and application of intestinal tract targeted DNA (deoxyribonucleic acid) nano origame.The preparation method is technically characterized by comprising the following steps: S1, synthesizing planar DNA nano origametes (DONs); s2, assembling the DONs and the siRNA (small interfering ribonucleic acid); and S3, synthesizing the tubular DNA nano origami loaded with siRNA: uniformly mixing the purified DONs-siRNA origami with 10 times of excessive APE1 chain and cross-linked chain, annealing from 45 DEG C to 25 DEG C, and synthesizing the tubular DNA nano origami. According to the method disclosed by the invention, a nano delivery carrier which has intestinal targeting, is accurately loaded and can release siRNA as required is developed by utilizing a DNA (Deoxyribonucleic Acid) nano technology, so that efficient treatment of IBD (Infectious Bursal Disease) by siRNA is realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for preparing intestinal-targeted DNA nanoorigami and its application. Background Art

[0002] Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), is a multifactorial, chronic, and relapsing gastrointestinal disorder. IBD has become a global health burden, with an estimated 6.8 million patients worldwide in 2017. Although IBD has a low mortality rate directly attributable to the disease, patients often face a significant decline in quality of life due to its incurable nature, high morbidity, and lifelong recurrence. They may even develop complications such as intra-abdominal abscesses, intestinal fistulas, and anal fistulas, which can be life-threatening in severe cases. Although the pathogenesis of IBD remains incompletely elucidated, it is generally believed that its core cause lies in gastrointestinal inflammation triggered by an abnormal immune system. Therefore, current treatment strategies for IBD primarily focus on suppressing abnormal immune responses, including anti-inflammatory drugs, immunosuppressants, and biologics. Currently, biologics used clinically for the treatment of IBD primarily include TNF-α and antibodies targeting the α4β7 integrin. TNF-α antibodies alleviate IBD symptoms by suppressing inflammation and improving intestinal barrier function. Furthermore, lymphocyte homing to sites of intestinal inflammation is a key pathogenesis of IBD, a process that relies on the expression of integrin α4β7 on the lymphocyte surface. Blocking integrin α4β7 can inhibit lymphocyte migration across the endothelium to sites of intestinal inflammation, thereby slowing disease progression. However, these monoclonal antibody therapies are associated with patient non-response, with approximately 50% of IBD patients showing no significant response to TNF-α antibody therapy. Therefore, the development of novel drug therapies has significant clinical and scientific significance for the treatment of IBD.

[0003] Small interfering RNA (siRNA) therapy is a gene therapy technology based on RNA interference (RNAi). It treats diseases by specifically targeting and degrading the mRNA of disease-related genes, thereby inhibiting the expression of the corresponding proteins. Although siRNA therapeutics have been successful in animal models and have been approved for several indications, several key challenges remain regarding their efficacy and safety.

[0004] In summary, the existing technology has the following problems: (1) siRNA is easily degraded by RNA enzymes and cleared by the kidneys, and has a short blood circulation half-life. Although the stability of siRNA modified by chemical methods has been improved to a certain extent, the negative charge it carries makes it difficult to be taken up by cells, which seriously limits its silencing efficiency in cells. (2) After administration, siRNA is distributed throughout the body and has no tissue specificity. Increasing the drug concentration can easily cause serious toxic side effects, which limits the clinical use of siRNA. In order to expand the application of siRNA, a variety of drug delivery carriers, such as liposomes, polymers and inorganic nanoparticles, have been developed for siRNA delivery. Although lipid nanoparticles have been approved for siRNA delivery, these traditional siRNA nanodelivery carriers have difficulty in achieving precise loading of siRNA and releasing siRNA on demand in target cells. Precise loading of siRNA, targeted delivery to intestinal cells and release on demand to enhance the accumulation of siRNA in target cells are the key to efficient treatment of IBD. Therefore, there is an urgent need to develop a nanodelivery carrier with intestinal targeting, precise loading and on-demand release of siRNA to achieve efficient siRNA treatment of IBD.

[0005] To this end, the present application provides a method for preparing and applying intestinal-targeted DNA nanoorigami. Summary of the Invention

[0006] The purpose of the present invention is to solve the technical problems raised in the above-mentioned background technology and provide a preparation method and application of intestinal-targeted DNA nanoorigami.

[0007] The above-mentioned purpose of the present invention is achieved like this: The present invention provides a method for preparing intestinal-targeted DNA nanoorigami, comprising the following steps: S1. Synthesis of planar DNA nanoorigami (DONs): Synthesis of DONs with capture chains: In 1 x TAE-Mg 2+ In a buffer solution, backbone chain M13, staple chain, and capture chain were mixed evenly at a molar ratio of 1:5:10 and annealed from 95°C to 25°C to synthesize DONs with capture chain; Two sets of capture chains extend from the surface of DONs, one of which is complementary to the link chain of siTNFα, and the other to the link chain of single siIntegrin α4. By regulating the number of these two sets of capture chains, the assembly ratio of siTNFα and siIntegrin α4 in DONs can be controlled. Then, the DONs were ultrafiltered three times at 3000 g for 10 min in a 100 kD ultrafiltration tube to remove excess staple and capture chains. The purified DONs with capture chains were quantified by UV-visible absorption spectroscopy. Assembly of S2, DONs and siRNA: A 5-fold excess of siRNA was mixed with purified DONs with capture strands and annealed at 45°C in a PCR instrument until 25°C. Excess siRNA strands were removed by ultrafiltration three times at 3000g for 10 min using a 100 kD ultrafiltration tube. The morphologies before and after siRNA loading were characterized by AFM and TEM. S3. Synthesis of tubular DNA nanoorigami loaded with siRNA: The purified DONs-siRNA origami was mixed with a 10-fold excess of APE1 lock chains and cross-linked chains, and annealed from 45°C to 25°C to synthesize tubular DNA nanoorigami.

[0008] Furthermore, in step S1, two groups of capture chains, each with 20 strands, extend from the surface of the DONs.

[0009] Another aspect of the present invention provides an application of an intestinal-targeted DNA nanoorigami preparation method in the preparation of drugs for treating inflammatory bowel disease.

[0010] The difficulty and significance of the technical problem solved by the present invention are: DNA nanoorigami structures (DONs) possess precise addressability and programmability. Smart DNA nanodevices constructed based on these structures have been widely used to treat diseases such as cerebral thrombosis, arthritis, and cancer. These smart DNA nanodevices can respond to thrombin, pH, and GSH to change their conformation and achieve controlled drug release. This effectively prevents drug inactivation or clearance during delivery and allows for on-demand drug release within target organs or cells, significantly reducing drug toxicity and side effects. Furthermore, the nanoscale dimensions of these smart DNA nanodevices enable rapid cellular uptake and efficient intracellular delivery of siRNA. This study utilizes DNA nanotechnology to develop a nano-delivery vector that is intestinal-targeted, precisely loaded, and capable of on-demand release of siRNA, enabling highly effective siRNA treatment of IBD.

[0011] Compared with the prior art, the present invention has the following beneficial effects: 1. In the solution of the present invention, the drug-loaded tubular DNA nanoorigami can load nucleic acid drugs through base pairing; the tubular DNA nanoorigami includes M13mp 18DNA chains, staple chains, capture chains and cross-linking chains; 2. In the solution of the present invention, the tubular DNA nanoorigami can target the intestine and achieve significant intestinal enrichment; 3. The tubular DNA nanoorigami of the present invention can delay the degradation of nucleic acids; 4. The tubular DNA nanoorigami loaded with TNF-α and integrin α4 in the present invention effectively treats inflammatory bowel disease, including alleviating weight loss, intestinal epithelial damage, and inflammatory infiltration; In summary, the method of the present invention is developed based on DNA nanotechnology and can provide a nano-delivery carrier with intestinal targeting, precise loading and on-demand release of siRNA, so as to achieve efficient treatment of IBD with siRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the synthesis of siRNA-loaded planar DNA origami and tubular DNA origami in an embodiment of the present invention; Figure 2 is a characterization image of DNA origami AFM in an embodiment of the present invention; Figure 3 is a height diagram of the DNA origami according to an embodiment of the present invention; Figure 4 In the embodiments of the present invention, the fluorescence intensity of Cy5.5-labeled free DNA and Cy5.5-labeled DNA origami was detected in healthy mice and DSS-induced IBD mice using an in vivo imaging system; Figure 5 In the embodiment of the present invention, the heart, liver, spleen, lung, kidney, pancreas and intestine tissues of 24 mice were taken to detect the tissue fluorescence intensity; Figure 6 This is the therapeutic effect of the tubular DNA nanoorigami on IBD in the embodiment of the present invention. DETAILED DESCRIPTION

[0013] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0014] The implementation of the present invention is described in detail below with reference to specific embodiments.

[0015] Reference Figures 1-6 As shown, the following are preferred embodiments provided by the present invention.

[0016] Explanation of terms: IBD: inflammatory bowel disease; DONs: planar DNA nanoorigami.

[0017] Example: The present invention provides a method for preparing intestinal-targeted DNA nanoorigami, comprising the following steps: Step 1. Synthesis of planar DNA nanoorigami (DONs): Synthesis of DONs with capture strands: In 1 x TAE-Mg 2+ In a buffer solution, backbone chain M13, staple chain, and capture chain were mixed evenly at a molar ratio of 1:5:10 and annealed from 95°C to 25°C to synthesize DONs with capture chain; Two sets of capture chains (20 each) extend from the surface of DONs, one of which is complementary to the link chain of siTNFα, and the other to the link chain of single siIntegrin α4. By regulating the number of these two sets of capture chains, the assembly ratio of siTNFα and siIntegrin α4 in DONs can be controlled. Then, the DONs were ultrafiltered three times at 3000 g for 10 min in a 100 kD ultrafiltration tube to remove excess staple and capture chains. The purified DONs with capture chains were quantified by UV-visible absorption spectroscopy. Step 2: Assembly of DONs and siRNA: A 5-fold excess of siRNA was mixed with the purified DONs with capture strands, and the mixture was annealed at 45°C and then annealed to 25°C in a PCR instrument. The excess siRNA strands were removed by ultrafiltration three times at 3000g for 10 minutes using a 100 kD ultrafiltration tube. The morphology before and after siRNA loading was characterized by AFM and TEM. Step 3. Synthesis of tubular DNA nanoorigami loaded with siRNA: The purified DONs-siRNA origami was mixed with 10-fold excess APE1 chains and cross-linked chains, and annealed from 45°C to 25°C to synthesize tubular DNA nanoorigami.

[0018] The intestinal-targeted DNA nanoorigami preparation method of the above embodiment of the present invention can be used to prepare drugs for treating inflammatory bowel disease.

[0019] The following is a specific experimental implementation of the above embodiment of the present invention, including the following steps: 1. Synthesis of planar DNA nanoorigami (DONs): Synthesis of DONs with capture chains: In 1 x TAE-Mg 2+DONs with capture chains were synthesized by mixing backbone chain M13, staple chain, and capture chain at a molar ratio of 1:5:10 in buffer and annealing from 95°C to 25°C. Two sets of capture chains (20 each) extended from the DON surface: one set complementary to the link chain of siTNFα and the other to the link chain of single siIntegrin α4. The ratio of siTNFα and siIntegrin α4 assembled in the DONs could be controlled by adjusting the number of capture chains. Excess staple and capture chains were removed by ultrafiltration three times through a 100 kD ultrafiltration tube at 3000g for 10 minutes. The purified DONs with capture chains were then quantified by UV-visible absorption spectroscopy.

[0020] 2. Assembly of DONs and siRNA: A 5-fold excess of siRNA was mixed with purified DONs containing capture strands and annealed at 45°C in a PCR instrument until 25°C. Excess siRNA strands were removed by ultrafiltration three times at 3000g for 10 minutes using a 100 kD ultrafiltration tube. Morphologies before and after siRNA loading were characterized by AFM and TEM.

[0021] 3. Synthesis of siRNA-loaded tubular DNA nanoorigami: Purified DONs-siRNA origami were mixed with a 10-fold excess of APE1 lock chains and cross-linked chains and annealed from 45°C to 25°C to synthesize tubular DNA nanoorigami. The migration rates of M13, DONs, DONs-siRNA, and tubular DNA nanoorigami were characterized by 1% agarose gel electrophoresis. The morphology and yield of the tubular DNA nanoorigami were characterized by AFM and TEM.

[0022] 4. Distribution of tubular DNA nanoorigami in mouse tissues: Free DNA and tubular DNA nanoorigami labeled with the fluorescent group Cy5.5 were injected into healthy mice and IBD model mice via the tail vein at the same concentration (200 nM siRNA). An equal volume of PBS served as a blank control. Fluorescence distribution in various mouse tissues was observed using in vivo imaging 1, 6, 12, and 24 hours after the experiment. Twenty-four hours after the experiment, the mice were euthanized, and in vivo imaging of the heart, liver, spleen, lung, pancreas, and entire gastrointestinal tract was performed. Fluorescence intensity was compared across organs, and the distribution ratio of the DNA nanoorigami in the mouse intestine was calculated.

[0023] 5. The efficacy of tubular DNA nanoorigami in treating IBD: Mice were divided into five groups (8 mice per group): control, DSS+PBS, DSS+siRNA, DSS+tubular DNA nanoorigami, and DSS+tubular DNA nanoorigami-siRNA. 3% DSS was added to the mice's drinking water for 7 days. On days 1 and 4, mice were injected with 10 nM DONs-siRNA and an equal amount of free siRNA via the tail vein. The control group received an equal volume of PBS. Therapeutic efficacy and intestinal histopathological evaluation of mice were recorded daily starting on day 0. At the end of the experiment, the colorectum of each group was harvested and its length was measured. HE staining and histopathological scoring were performed.

[0024] Through the above embodiments and experiments of the present invention, the solution of the present invention solves the following defects in the prior art: (1) First, siRNA is easily degraded by RNase and cleared by the kidneys, and has a short blood circulation half-life. Although the stability of siRNA modified by chemical methods is improved to a certain extent, the negative charge it carries makes it difficult to be taken up by cells, limiting its silencing efficiency in cells. (2) After administration, siRNA is distributed throughout the body and has no tissue specificity. Increasing the administration concentration can easily cause serious toxic side effects, which limits the clinical use of siRNA. In order to expand the application of siRNA, a variety of drug delivery carriers, such as liposomes, polymers and inorganic nanoparticles, have been developed for siRNA delivery. Although lipid nanoparticles have been approved for siRNA delivery, these traditional siRNA nanodelivery carriers have difficulty in achieving precise loading of siRNA and on-demand release of siRNA in target cells. Based on DNA nanotechnology, the present invention has developed a nanodelivery carrier with intestinal targeting, precise loading and on-demand release of siRNA to achieve efficient siRNA treatment of IBD.

[0025] 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 scope of protection of the present invention.

Claims

1. A method for preparing intestinal-targeted DNA nanoorigami, characterized in that: The following steps are involved: S1. Synthesis of planar DNA nanoorigami (DONs): Synthesis of DONs with capture chains: In 1 x TAE-Mg 2+ In a buffer solution, backbone chain M13, staple chain, and capture chain were mixed evenly at a molar ratio of 1:5:10 and annealed from 95°C to 25°C to synthesize DONs with capture chain; Two sets of capture chains extend from the surface of DONs, one of which is complementary to the link chain of siTNFα, and the other to the link chain of single siIntegrin α4. By regulating the number of these two sets of capture chains, the assembly ratio of siTNFα and siIntegrin α4 in DONs can be controlled. Then, the DONs were ultrafiltered three times at 3000 g for 10 min in a 100 kD ultrafiltration tube to remove excess staple and capture chains. The purified DONs with capture chains were quantified by UV-visible absorption spectroscopy. Assembly of S2, DONs and siRNA: A 5-fold excess of siRNA was mixed with purified DONs with capture strands and annealed at 45°C in a PCR instrument until 25°C. Excess siRNA strands were removed by ultrafiltration three times at 3000g for 10 min using a 100 kD ultrafiltration tube. The morphologies before and after siRNA loading were characterized by AFM and TEM. S3. Synthesis of tubular DNA nanoorigami loaded with siRNA: The purified DONs-siRNA origami was mixed with a 10-fold excess of APE1 lock chains and cross-linked chains, and annealed from 45°C to 25°C to synthesize tubular DNA nanoorigami.

2. The method for preparing intestinal-targeted DNA nanoorigami according to claim 1, characterized in that: In step S1, two groups of capture chains, each containing 20 strands, extend from the surface of DONs.

3. Use of the intestinal-targeted DNA nanoorigami preparation method according to any one of claims 1-2 in the preparation of drugs for treating inflammatory bowel disease.

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