A nano-drug for treating sepsis and a preparation method and use thereof

Abstract

The application discloses a kind of nanomedicine for treating sepsis and its preparation method and purposes, belong to medical technology field.The nanomedicine is tetrahedral framework nucleic acid modified by N-acetyl proline-glycine-proline.The nanomedicine of the application can be transported to inflammatory site by following the chemotaxis of neutrophil to inflammatory site, to play the target regulation effect on sepsis, to increase the bioavailability of drug, improve the target treatment effect of drug.The nanomedicine of the application can be used not only for preparing the drug for treating sepsis, but also for preparing the drug for treating other diseases caused by neutrophil infiltration, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a nanomedicine for treating sepsis, its preparation method, and its uses. Background Technology

[0002] Sepsis is a life-threatening multi-organ dysfunction caused by a dysregulated host response to infection. Studies have shown that sepsis is an unbalanced immune-inflammatory response, with excessive inflammation and immunosuppression being the main causes of multi-organ failure and early death in the host. Therefore, intervening in excessive inflammation and immunosuppression in sepsis is an important means of improving treatment and prognosis.

[0003] Currently, treatment for sepsis remains largely supportive, including antibiotics, pain management, and supportive care to control infection, such as steroidal and nonsteroidal anti-inflammatory therapies. However, these methods lack specificity, and long-term use can lead to an increase in secondary infections. Recent research on targeted inflammation therapy has focused on achieving targeted treatment through intravenous injection of cells or monoclonal antibodies, such as stem cell therapy, cytokine blocking therapy, angiogenesis blocking therapy, and targeted immune cell blocking therapy. However, targeted inflammation therapy through intravenous injection of cells or monoclonal antibodies is prone to systemic side effects, including weakened hematopoietic function, secondary infections, immunotoxicity, and off-target effects. Furthermore, it also suffers from drawbacks such as insufficient efficacy.

[0004] Therefore, it is of great significance to find a drug that can not only effectively target and treat sepsis, but also reduce side effects. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide nanomedicines that can not only effectively target and treat sepsis but also reduce side effects, as well as their preparation methods and uses.

[0006] Specifically, the present invention provides a nanomedicine, which is a tetrahedral framework nucleic acid modified with N-acetylproline-glycine-proline.

[0007] Furthermore, the nanomedicine is obtained by self-assembly of a single DNA strand with the sequence shown in SEQ ID NO.1, a single DNA strand with the sequence shown in SEQ ID NO.2, a single DNA strand Ac-PGP-S3, and a single DNA strand with the sequence shown in SEQ ID NO.4, wherein the single DNA strand Ac-PGP-S3 is a product obtained by modifying the single DNA strand with the sequence shown in SEQ ID NO.3 with N-acetylproline-glycine-proline.

[0008] Furthermore, the preparation method of the nanomedicine includes the following steps:

[0009] (1) N-acetylproline-glycine-proline reacts with a single strand of DNA with the sequence shown in SEQ ID NO.3 to obtain a single strand of DNA Ac-PGP-S3;

[0010] (2) DNA single strands with sequences as shown in SEQ ID NO.1, DNA single strands with sequences as shown in SEQ ID NO.2, DNA single strand Ac-PGP-S3, and DNA single strands with sequences as shown in SEQ ID NO.4 are self-assembled to obtain nanomedicines.

[0011] Furthermore, in step (1), the reaction is carried out under the action of a condensing agent.

[0012] Further, in step (1), the condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine, and the molar ratio of N-acetylproline-glycine-proline, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, and the DNA single strand with the sequence shown in SEQ ID NO.3 is 2:2:2:(0.5-2), preferably 2:2:2:1.

[0013] Further, in step (2), the molar ratio of the DNA single strand with the sequence shown in SEQ ID NO.1, the DNA single strand with the sequence shown in SEQ ID NO.2, the DNA single strand Ac-PGP-S3, and the DNA single strand with the sequence shown in SEQ ID NO.4 is 1:1:1:1, and the self-assembly conditions are to first maintain at 95°C for 10 minutes, and then cool down to 4°C and maintain for 20 minutes.

[0014] The present invention also provides a method for preparing the above-mentioned nanomedicine, the method comprising the following steps:

[0015] (1) N-acetylproline-glycine-proline reacts with a single strand of DNA with the sequence shown in SEQ ID NO.3 to obtain a single strand of DNA Ac-PGP-S3;

[0016] (2) DNA single strands with sequences as shown in SEQ ID NO.1, DNA single strands with sequences as shown in SEQ ID NO.2, DNA single strand Ac-PGP-S3, and DNA single strands with sequences as shown in SEQ ID NO.4 are self-assembled to obtain nanomedicines.

[0017] The present invention also provides the use of the above-mentioned nanomedicine in the preparation of a drug for treating sepsis.

[0018] The present invention also provides the use of the above-mentioned nanomedicine in the preparation of drugs for diseases caused by neutrophil infiltration.

[0019] Furthermore, the disease caused by neutrophil infiltration is inflammation caused by neutrophil infiltration.

[0020] Experimental results show that the nanomedicine of the present invention can be targeted and transported to the site of inflammation by following the chemotactic movement of neutrophils to exert a targeted regulatory effect on sepsis, thereby increasing the bioavailability of the drug and improving the targeted therapeutic effect.

[0021] The nanomedicines provided by this invention can not only be used to prepare drugs for treating sepsis, but also for treating other diseases caused by neutrophil infiltration, showing broad application prospects.

[0022] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0023] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0024] Figure 1 The PAGE gel electrophoresis results of Ac-PGP-tFNAs confirm the successful synthesis of Ac-PGP-tFNAs.

[0025] Figure 2 : The uptake of Ac-PGP-tFNAs by neutrophils.

[0026] Figure 3 Distribution and metabolism of Ac-PGP-tFNAs in major organs of mice.

[0027] Figure 4 Colocalization of Ac-PGP-tFNAs with neutrophils in major organs of mice.

[0028] Figure 5 The protective effect of Ac-PGP-tFNAs on major organs in septic mice (H&E staining results). Detailed Implementation

[0029] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0030] N-acetylproline-glycine-proline (Ac-PGP) was purchased from Hefei Guotai Biotechnology Co., Ltd., and its structure is as follows:

[0031] Example 1: Preparation of Ac-PGP-tFNAs complex

[0032] (1) Preparation of Ac-PGP-S3

[0033] Linking via chemical reaction: Add HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 10 μL, 400 mM in DMA) and DIPEA (N,N-diisopropylethylamine, 10 μL, 400 mM in DMA) to Ac-PGP (20 μL, 200 mM in DMA) solution. React at 0 °C for 5 min, then add the mixture to borate buffer (200 μL, 250 mM, pH 9.4) containing 200 nmol, 1 equiv. of S3 DNA strand. Vortex at room temperature for 30 min. After LCMS monitoring, add NaCl aqueous solution (24 μL, 5 M) and anhydrous ethanol (792 μL), and vortex. Cool the mixture to -20 °C for 2 h, then centrifuge at 4 °C for 30 min at 12000 rpm. After removing the supernatant, the crude product was obtained by filtration through a 10K filter at 20°C (4000 rds) for 30–60 min. The crude product was purified by high performance liquid chromatography, and the eluent was filtered through a 10K filter. The residue was lyophilized to obtain Ac-PGP-S3, a white solid.

[0034] (2) Preparation of Ac-PGP-tFNAs complex

[0035] Four DNA single strands (S1, S2, Ac-PGP-S3, S4) were added to an equimolar amount of TM buffer composed of Tris-HCl and MgCl2, and then heated (95°C, 10 min) and cooled (4°C, 20 min) to obtain the Ac-PGP-tFNAs complex, abbreviated as APTs.

[0036] Table 1. Sequences S1, S2, S3, S4

[0037]

[0038] The following experimental examples demonstrate the beneficial effects of the present invention.

[0039] Experimental Example 1: Synthesis and Characterization

[0040] 1. Experimental Methods

[0041] The synthesis of Ac-PGP-tFNAs was verified by PAGE gel electrophoresis.

[0042] 2. Experimental Results

[0043] The PAGE gel electrophoresis results of Ac-PGP-tFNAs are as follows: Figure 1 As shown, this demonstrates the successful synthesis of Ac-PGP-tFNAs.

[0044] Experimental Example 2: In vitro uptake and release of Ac-PGP-tFNAs

[0045] 1. Experimental Methods

[0046] (1) The human promyelocytic leukemia cell line HL-60 was treated with DMSO for 72 hours and used as a substitute for neutrophils.

[0047] (2) HL-60 cells were treated with Ac-PGP-tFNAs and tFNAs with CY5 fluorescent groups, respectively, and their uptake and intracellular distribution were detected by flow cytometry and fluorescence confocal microscopy.

[0048] (3) HL-60 cells were pretreated with several common endocytosis inhibitors, and then treated with Ac-PGP-tFNAs complex carrying CY5 fluorescent group. The cell entry rate was detected by flow cytometry to explore the mechanism of cell entry.

[0049] (4) After treating HL-60 cells with Ac-PGP-tFNAs containing CY5 fluorescent group, the supernatant was removed, washed with PBS, and the culture medium without Ac-PGP-tFNAs was replaced and cultured again. The supernatant was taken, and the CY5 content in the supernatant was detected by microplate reader. The release of extracellular CY5-Ac-PGP-tFNAs was observed by confocal fluorescence microscopy.

[0050] 2. Experimental Results

[0051] The results are as follows Figure 2 As shown in the figure, the Ac-PGP-tFNAs complex can achieve targeted recognition and uptake of neutrophils.

[0052] Experimental Example 3: Targeted Therapeutic Effect of Ac-PGP-tFNAs in a Mouse Model of Sepsis

[0053] 1. Experimental Methods

[0054] (1) BALB / C mice weighing approximately 20-23g were selected, and a mouse sepsis model was established by intraperitoneal injection of LPS. At the same time, CY5-Ac-PGP-tFNAs solution (solution concentration of 1 μmol / L, dosage of 100 μL / 20g) was injected intraperitoneally.

[0055] (2) CY5-Ac-PGP-tFNAs solution (concentration of 1 μmol / L, dosage of 100 μL / 20g) was injected into normal mice and septic mice via tail vein. Whole blood was collected 0.5 h later, and the uptake rate and fluorescence intensity of the complex by neutrophils in the blood were detected by flow cytometry to investigate the targeting effect of Ac-PGP-tFNAs on neutrophils in vivo.

[0056] (3) In normal mice and septic mice, CY5-Ac-PGP-tFNAs solution (solution concentration of 1 μmol / L, dosage of 100 μL / 20g) was injected intraperitoneally. The fluorescence of major organs (heart, liver, spleen, lung, and kidney) at different time points was observed using a small animal in vivo imaging system. After 6 hours of treatment, the major organs (heart, liver, spleen, lung, and kidney) were collected for frozen sections, stained with DAPI, and the fluorescence uptake distribution in each organ tissue was observed using a confocal fluorescence microscope to explore the targeting effect of Ac-PGP-tFNAs on the inflammatory site.

[0057] (4) A mouse sepsis model was established by intraperitoneal injection of LPS. At the same time, CY5-Ac-PGP-tFNAs solution (solution concentration of 1 μmol / L, dosage of 100 μL / 20g) was injected intraperitoneally. After 24 hours, blood was collected from the eyeballs for routine blood tests to analyze changes in blood count. Serum was collected for blood biochemical analysis to observe the damage to major organs. Serum was collected and ELISA kits were used to detect the expression of IL-1β and TNF-α to observe the regulatory effect of the drug on systemic inflammation.

[0058] (5) Intraperitoneal injection of CY5-Ac-PGP-tFNAs solution (solution concentration of 1 μmol / L, dosage of 100 μL / 20g) for 24h, organs (heart, liver, spleen, lung, kidney) were taken for H&E staining, and the morphological changes in the major organs were observed to explore the therapeutic effect of the drug on sepsis.

[0059] (6) After intraperitoneal injection of CY5-Ac-PGP-tFNAs solution (solution concentration of 1 μmol / L, dosage of 100 μL / 20g) for 24 hours, organs (heart, liver, spleen, lung, kidney) were taken, tissue suspensions were extracted, cell surface antibody staining was performed, and then flow cytometry was used to analyze the changes in the content of immune cells (neutrophils, macrophages, T cells) in the tissues, so as to explore the regulatory effect of the drug on the immune response of sepsis.

[0060] 2. Experimental Results

[0061] from Figure 3-4 It can be seen that simple tFNAs, after systemic administration, are mainly taken up by macrophages and quickly excreted through liver and kidney metabolism, resulting in low in vivo utilization and low drug concentrations reaching the site of inflammation, thus exhibiting low bioavailability. The Ac-PGP-tFNAs complex, however, significantly alters the drug uptake pathway in vivo, changing it from macrophage uptake to neutrophil recognition and uptake. Neutrophils, with their chemotactic activity, actively aggregate towards the site of inflammation. Therefore, the drug can passively target the inflamed site in large numbers via neutrophils, greatly improving the bioavailability of the material. It also changes the target cells of the material (from macrophages to neutrophils). Compared to simple tFNAs, the Ac-PGP-tFNAs complex can target and aggregate more extensively at the site of inflammation and co-localize with neutrophils, significantly improving the material's targeting and therapeutic efficacy.

[0062] from Figure 5 It can be seen that, compared with tFNAs alone, the Ac-PGP-tFNAs complex can effectively protect the major organs such as heart, liver, spleen, lungs and kidneys in septic mice and effectively treat sepsis.

[0063] In summary, this invention provides a nanomedicine for treating sepsis, its preparation method, and its uses. The nanomedicine of this invention can target and transport to the site of inflammation by following the chemotactic movement of neutrophils, thereby exerting a targeted regulatory effect on sepsis, increasing drug bioavailability, and improving the targeted therapeutic effect. This nanomedicine can not only be used to prepare drugs for treating sepsis but also for treating other diseases caused by neutrophil infiltration, showing broad application prospects.

Claims

1. A nanomedicine for treating sepsis, characterized in that, It is a tetrahedral framework nucleic acid modified with N-acetylproline-glycine-proline; the nanomedicine is obtained by self-assembly of a single DNA strand with the sequence shown in SEQ ID NO.1, a single DNA strand with the sequence shown in SEQ ID NO.2, a single DNA strand Ac-PGP-S3, and a single DNA strand with the sequence shown in SEQ ID NO.4, wherein the single DNA strand Ac-PGP-S3 is a product obtained by modifying the single DNA strand with the sequence shown in SEQ ID NO.3 with N-acetylproline-glycine-proline.

2. The nanomedicine according to claim 1, characterized in that, The preparation method of the nanomedicine includes the following steps: (1) N-acetylproline-glycine-proline reacts with a single strand of DNA with the sequence shown in SEQ ID NO.3 to obtain a single strand of DNA Ac-PGP-S3; (2) DNA single strands with sequences as shown in SEQ ID NO.1, DNA single strands with sequences as shown in SEQ ID NO.2, DNA single strand Ac-PGP-S3, and DNA single strands with sequences as shown in SEQ ID NO.4 are self-assembled to obtain nanomedicines.

3. The nanomedicine according to claim 2, characterized in that, In step (1), the reaction is carried out under the action of a condensing agent.

4. The nanomedicine according to claim 3, characterized in that, In step (1), the condensing reagent is (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine, wherein the molar ratio of N-acetylproline-glycine-proline, (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, and the DNA single strands with the sequence shown in SEQ ID NO.3 is 2:2:2: (0.5-2)。 5. The nanomedicine according to claim 4, characterized in that, The molar ratio of the N-acetylproline-glycine-proline, (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, and DNA single strands with the sequence shown in SEQ ID NO.3 is 2:2:2:

1.

6. The nanomedicine according to any one of claims 2-5, characterized in that, In step (2), the molar ratio of the DNA single strand with the sequence shown in SEQ ID NO.1, the DNA single strand with the sequence shown in SEQ ID NO.2, the DNA single strand Ac-PGP-S3, and the DNA single strand with the sequence shown in SEQ ID NO.4 is 1:1:1:

1. The self-assembly conditions are to first maintain at 95°C for 10 minutes, and then cool down to 4°C and maintain for 20 minutes.

7. A method for preparing the nanomedicine according to any one of claims 1-6, characterized in that, The method includes the following steps: (1) N-acetylproline-glycine-proline reacts with a single strand of DNA with the sequence shown in SEQ ID NO.3 to obtain a single strand of DNA Ac-PGP-S3; (2) DNA single strands with sequences as shown in SEQ ID NO.1, DNA single strands with sequences as shown in SEQ ID NO.2, DNA single strand Ac-PGP-S3, and DNA single strands with sequences as shown in SEQ ID NO.4 are self-assembled to obtain nanomedicines.

8. Use of the nanomedicine according to any one of claims 1-6 in the preparation of a medicament for treating sepsis.

9. Use of the nanomedicine according to any one of claims 1-6 in the preparation of a medicament for inflammation caused by neutrophil infiltration.

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