An inflammation environment macrophage-targeting small activating nucleic acid nanodrug, a preparation method and application thereof

Abstract

The application discloses an inflammation environment macrophage targeting small activating nucleic acid nano-drug, a preparation method and application thereof, and belongs to the technical field of biological medicine. The inflammation environment macrophage targeting small activating nucleic acid nano-drug is composed of a small activating nucleic acid inner core and a liposome shell. After the inflammation environment macrophage targeting small activating nucleic acid nano-drug of the application is actively targeted to an inflammation environment, the liposome shell is swollen and disintegrated after entering a cell through receptor recognition, small activating RNA is released, the expression of a gene is regulated, M1 type macrophages are reprogrammed into M2 type from two aspects of a phenotype and a metabolic mode, anti-inflammatory factors are released, and the purpose of inhibiting inflammation is achieved. Therefore, the application has a very broad application prospect in gene therapy, immunotherapy and the like of inflammation and infectious diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to an inflammation environment macrophage targeted small activating nucleic acid nano-drug, a preparation method and application thereof. BACKGROUND

[0002] Pneumonia refers to inflammation of the terminal airway, alveolus and interstitial lung, and is a pathological characteristic of lung damage caused by exogenous or endogenous attack on the lung. Macrophages, as the main cell component of inflammatory response, are massively aggregated in the inflammatory microenvironment. After being activated by inflammatory mediators, they attack normal cells in the inflammatory area. As a kind of innate immune cells, the polarization process plays an important role in affecting the lung environment. Macrophages activated by different cytokines and chemokines can be divided into M1 pro-inflammatory macrophages and M2 anti-inflammatory macrophages, which can further produce different effects: M1 macrophages can be induced by interferon gamma, lipopolysaccharide and TNF alpha, and have the ability to enhance antigen presentation, produce nitric oxide and secrete a large amount of pro-inflammatory factors; M2 macrophages can be induced by CSF-1, IL1 beta, IL4 and IL10, and can inhibit the secretion of M1 macrophage pro-inflammatory factors, promote the healing of damaged lung tissue and regulate the functional balance of macrophages. Studies have shown that regulating the M1 / M2 balance of macrophages and reprogramming macrophages are important means for treating lung inflammatory diseases.

[0003] Gene therapy refers to a new treatment method for treating diseases by introducing external genes at the gene level to replace, replace, knock in / knock out, activate / inhibit and change the genes in the body. Small activating RNA (saRNA) is a new type of gene therapy drug that can stimulate gene expression at the transcription level, has the characteristics of small molecular weight, gene specificity and activating gene expression. Studies have shown that up-regulating PPAR gamma can reprogram M1 macrophages into M2 macrophages.

[0004] Like most nucleic acid drugs, saRNA has limitations in specificity, stability and delivery methods, similar to other RNA drugs. Chemical modification can improve its stability and specificity, but it may be accompanied by material toxicity, so the delivery system of saRNA needs further research. Liposomes show great advantages in the systemic or local delivery of RNA: due to their low toxicity and immunogenicity, the high biocompatibility and biodegradability of components such as phospholipids can increase the concentration of drugs in the body while protecting the drugs from degradation; secondly, liposomes are easy to modify with various ligands and functional molecules, which can greatly improve the targeting of RNA. How to reduce the non-specific clearance of nucleic acid drug carriers and improve their tissue / cell targeting is a hot and difficult point in nucleic acid drug delivery. SUMMARY

[0005] The application provides a preparation method of an inflammation environment macrophage targeted small activating nucleic acid nanodrug, and the preparation method comprises the following steps:

[0006] (1) SM-102, DSPC, cholesterol, DMG-PEG2000 and TKPR-PEG2000-DSPE are dissolved in an organic solvent according to a molar ratio of 45-55:8-12:30-42:1-2:1-2, the organic solvent is removed by rotary evaporation, then 1-3 mL of a buffer is added for hydration, so that the total concentration of SM-102, DSPC, cholesterol, DMG-PEG2000 and TKPR-PEG2000-DSPE in the mixed solution is 0.4-0.6 mg / mL, and a liposome solution is formed;

[0007] (2) 0.8-1.2 μg of saRNA is diluted in 0.8-1.2 μL of nuclease-free water, and is compounded with the liposome solution according to a mass ratio of the liposome solution to the saRNA of 38-42:0.8-1.2, and is left at room temperature, and then centrifuged, so that the obtained precipitate is the inflammation environment macrophage targeted small activating nucleic acid nanodrug;

[0008] The sequences of the sense strand and the antisense strand of the saRNA are shown in SEQ ID NO. 3 and 4 respectively.

[0009] In some embodiments of the application, the molar ratio of SM-102, DSPC, cholesterol, DMG-PEG2000 and TKPR-PEG2000-DSPE in the step (1) is 50:10:37:1.5:1.5.

[0010] In some embodiments of the application, the organic solvent in the step (1) is a mixed liquid of chloroform and methanol.

[0011] In some embodiments of the application, the volume ratio of chloroform and methanol in the step (1) is 3:1.

[0012] In some embodiments of the application, the buffer in the step (1) is 8-12 nM citrate buffer.

[0013] In some embodiments of the application, the total concentration of SM-102, DSPC, cholesterol, DMG-PEG2000 and TKPR-PEG2000-DSPE in the mixed solution in the step (1) is 0.5 mg / mL.

[0014] In some embodiments of the application, 1 μg of saRNA is diluted in 1 μL of nuclease-free water in the step (2).

[0015] In some embodiments of the present application, the mass ratio of the liposome solution to the saRNA in step (2) is 40:1.

[0016] The present application also provides the inflammation environment macrophage targeted small activating nucleic acid nanodrug prepared by the above preparation method.

[0017] The present application also provides the use of the above inflammation environment macrophage targeted small activating nucleic acid nanodrug in the preparation of anti-inflammatory drugs.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] The present application uses M1 type macrophage targeted peptide TKPR modified liposome to load PPAR gamma small activating RNA (sa-PPAR gamma), and prepares an inflammation environment macrophage targeted small activating nucleic acid nanodrug. The inflammation environment macrophage targeted small activating nucleic acid nanodrug recognizes Nrp-1 receptor into M1 type macrophage under the mediation of TKPR targeted peptide, releases sa-PPAR gamma, significantly up-regulates the expression of PPAR gamma, reprograms the phenotype and metabolic mode of M1 type macrophage into M2 type macrophage, thereby releasing anti-inflammatory factors and down-regulating the level of pro-inflammatory factors, and finally achieving the purpose of relieving inflammation. It can be seen that the present application provides a new drug for the treatment of pneumonia. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is the screening result of the nucleic acid sequence in Example 1, wherein A) real-time fluorescent quantitative PCR detects the PPAR gamma mRNA expression of macrophages after being transfected by different sequences of saRNA; and B) immunoblotting detects the PPAR gamma protein expression of macrophages after being transfected by different sequences of saRNA.

[0021] Figure 2 It is the result of PPAR gamma regulating macrophage polarization in Example 1, wherein A) optical microscope observes the morphology of M1 type macrophage and the cell morphology after being transfected by saPPAR gamma, the scale = 200 um; B) immunoblotting detects the protein expression of M1 macrophage after being transfected by saPPAR gamma; C) real-time fluorescent quantitative PCR detects the mRNA expression of M1 macrophage after being transfected by saPPAR gamma; and D) real-time fluorescent quantitative PCR detects the mRNA expression of M1 macrophage after being transfected by different concentrations of saPPAR gamma.

[0022] Figure 3Results of the tests for the synthesis and characterization of TLPs in Example 1, in which A) particle size and potential of TLPs with different mass ratios; B) gel retardation of TLPs with different mass ratios; C) nucleic acid protection of TLPs with different mass ratios; D) encapsulation of TLPs with different mass ratios; E) cell uptake of liposomes with different electricities, scale = 50 μm; F) particle size distribution and morphology of TLPs with a mass ratio of 40; G) three-day stability of TLPs in water with a mass ratio of 40; H) drug release curves of TLPs under different pH conditions.

[0023] Figure 4 Results of the tests for the TLP-mediated reprogramming of macrophages in Example 1, in which A) laser confocal microscopy and flow cytometry detection of cell uptake of different preparations, scale = 50 μm; B) laser confocal microscopy and flow cytometry detection of the uptake of different intracellular TLPs, scale = 50 μm; C) laser confocal microscopy observation of lysosomal escape of TLPs, scale = 50 μm; D) laser confocal microscopy observation of cell viability of macrophages transfected with different preparations, scale = 200 μm; E) CCK8 detection method for detecting the effect of different preparations on the viability of macrophages; F) CCK8 detection method for detecting the effect of different concentrations of saPPARγ on the viability of macrophages; G) Western blotting for detecting protein expression of macrophages transfected with different preparations; H) real-time fluorescent quantitative PCR for detecting mRNA expression of macrophages transfected with different preparations.

[0024] Figure 5 Results of the tests for the in vivo distribution of TLPs in Example 1, in which A) small animal live imaging observation of the distribution and metabolism of different preparations in mice with pneumonia; B) small animal live imaging observation of the distribution of different preparations in the main organs of mice with pneumonia; C) comparison of the fluorescence signals of different preparations in the main organs of mice with pneumonia.

[0025] Figure 6 Results of the tests for the treatment of pneumonia by TLPs in Example 1, in which A) histological analysis of lung tissue; B) Western blotting analysis of CD80, PPARγ, Arg1, and CD206; C) lung function tests, including inspiratory time (TI) and D) minute volume (MV).

[0026] Figure 7 Technical route and principle diagram of the action of TLPs. DETAILED DESCRIPTION

[0027] Example 1

[0028] I. Methods

[0029] 1. Nucleic acid sequence screening

[0030] M1 macrophages were cultured overnight, transfected with different sequences of saRNA using PEI, and then cultured for another 48 h. Total RNA was extracted and the expression level of PPARy was analyzed by real-time fluorescent quantitative PCR. The saRNA sequences were as follows:

[0031] saRNANC:

[0032] sense: 5'-UUCUCCGAACGUGUCACGUdTdT-3' (SEQ ID NO. 1)

[0033] anti-sense: 5'-ACGUGACACGUUCGGAGAAdTdT-3' (SEQ ID NO. 2)

[0034] sa-PPARy-1:

[0035] sense: 5'-CCAAUAGUCUAACUUAAAAdTdT-3' (SEQ ID NO. 3)

[0036] anti-sense: 5'-UUUUAAGUUAGACUAUUGGdTdT-3' (SEQ ID NO. 4)

[0037] sa-PPARy-2:

[0038] sense: 5'-GAGAUGAAAAGCACAUCUAdTdT-3' (SEQ ID NO. 5)

[0039] anti-sense: 5'-UAGAUGUGCUUUUCAUCUCdTdT-3' (SEQ ID NO. 6)

[0040] sa-PPARy-3:

[0041] sense: 5'-CUCUCCCAAAUAUUUGAAAdTdT-3' (SEQ ID NO. 7)

[0042] anti-sense: 5'-UUUCAAAUAUUUGGGAGAGdTdT-3' (SEQ ID NO. 8)

[0043] sa-PPARy-4:

[0044] sense: 5'-GGAGUUUCAACCAAAGAUAdTdT-3' (SEQ ID NO. 9)

[0045] anti-sense: 5'-UAUCUUUGGUUGAAACUCCdTdT-3' (SEQ ID NO. 10)

[0046] 2 PPARy regulates macrophage polarization

[0047] 2.1 Macrophage morphological changes

[0048] M1 macrophages were cultured overnight, transfected with sa-PPARy using PEI, and cultured for another 48 h. Cell morphology was observed under an optical microscope.

[0049] 2.2 PPARy regulates macrophage polarization

[0050] M1 macrophages were cultured overnight, transfected with sa-PPARy using PEI, and cultured for another 48 h. Total protein was extracted, and the expression levels of Arg1 and CD206 were analyzed by Western blotting. Total RNA was extracted, and the expression levels of Arg1 and CD206 were analyzed by real-time fluorescent quantitative PCR.

[0051] M1 macrophages were cultured overnight, transfected with sa-PPARy using PEI, and cultured for another 48 h. Total RNA was extracted, and the expression levels of PPARy, CD206, and IL-10 were analyzed by real-time fluorescent quantitative PCR.

[0052] 3 TLP synthesis and characterization

[0053] 3.1 TL synthesis

[0054] Thin film dispersion method: five components, SM-102, DSPC, cholesterol, DMG-PEG2000, and TKPR-PEG2000-DSPE, were used to synthesize liposomes. Each component was weighed according to the molar ratio of 50 / 10 / 37 / 1.5 / 1.5, dissolved in a chloroform / methanol (3 / 1, v / v) mixed solution, and rotary evaporated on a rotary evaporator for 30 min to remove the organic solvent. Then, 2 mL of 10 nM citrate buffer (pH = 3) was added for hydration, and the total concentration of SM-102, DSPC, cholesterol, DMG-PEG2000, and TKPR-PEG2000-DSPE in the mixed solution was 0.5 mg / mL. After being extruded back and forth with an extruder, a liposome solution (TL) was formed and placed at 4°C for standby.

[0055] TLC and LP as control. TL was mixed with saRNA at a mass ratio (TL:saRNA) of 40:1 to form TLC. The solution of liposome prepared with SM-102, DSPC, cholesterol, DMG-PEG2000 at a molar ratio of 50 / 10 / 37 / 3 was mixed with saRNA at a mass ratio (solution of liposome:saRNA) of 40:1 to prepare nanoparticles without macrophage targeting peptide, referred to as LP.

[0056] 3.2 TLP mass ratio screening

[0057] Take saRNA 1 μg diluted in 1 μL of nuclease-free water, and mix with the solution of liposome at a mass ratio (TL:saRNA) of 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1. After standing at room temperature for 15 min, centrifuge at 13000 rpm for 30 min. Take 20 μL of supernatant, add 10*Loading buffer, mix well, and load onto a 1% agarose gel for electrophoresis at 120V for 20 min. Take a photo of the gel using a gel imager, and calculate the encapsulation efficiency.

[0058] Take saRNA 1 μg diluted in 1 μL of nuclease-free water, and mix with the solution of liposome at a mass ratio (TL:saRNA) of 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1. After standing at room temperature for 15 min, centrifuge at 13000 rpm for 30 min. Take 20 μL of supernatant, add 10*Loading buffer, mix well, and load onto a 1% agarose gel for electrophoresis at 120V for 20 min. Take a photo of the gel using a gel imager, and calculate the encapsulation efficiency.

[0059] 3.3 Drug release

[0060] Prepare TLP with fluorescently labeled saRNA, and load into a dialysis bag with a molecular weight cutoff of 3500. Place the dialysis bag in PBS buffer solution at pH 5.4 and pH 7.4, respectively. At 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 48, and 72 h, take the dialysate, and measure the fluorescence intensity using a fluorescence spectrophotometer. Calculate the cumulative drug release rate.

[0061] 4 TLP-mediated macrophage reprogramming

[0062] 4.1 Cell uptake

[0063] Prepare TLP with fluorescently labeled saRNA. Add PBS, saRNA, LP, and TLP culture solution to M1 type macrophages cultured overnight, and incubate for 9 h. Use flow cytometry and laser confocal fluorescence microscopy to detect the cell uptake of different preparations.

[0064] 4.2 Cell-specific uptake

[0065] Mouse alveolar epithelial cells (MLE), mouse embryonic fibroblasts (3T3), M0 macrophages and M1 macrophages were cultured overnight and incubated with TLP containing equal mass of fluorescently labeled saRNA (60 nM) for 9 h, respectively. Flow cytometry and confocal fluorescence microscopy were used to detect the uptake of different cells.

[0066] 4.3 Lysosomal escape

[0067] M1 macrophages were cultured overnight and added with TLP containing equal mass of fluorescently labeled saRNA (60 nM) for 3 h and 9 h, respectively. The cells were gently washed twice with PBS and added with green lysosome probe for incubation. Laser confocal microscopy was used to observe the lysosomal escape of nanoparticles in cells.

[0068] 4.4 Cytotoxicity

[0069] M1 macrophages were cultured overnight and incubated with PBS, TL, saRNA, TLC, LP, TLP (the last four groups each containing 60 nM saRNA) for 8 h, then the solution was changed and the cells were cultured for another 48 h. Cell viability and death staining kit was used to stain the cells, and laser confocal microscopy was used to observe the viability and death of cells to analyze the cytotoxicity of different preparations.

[0070] 4.5 Reprogramming efficiency of macrophages

[0071] M1 macrophages were cultured overnight and incubated with PBS, TL, saRNA, TLC, LP, TLP (the last four groups each containing 60 nM saRNA) for 8 h, then the solution was changed and the cells were cultured for another 48 h. Total protein was extracted and Western blot was used to analyze the expression levels of iNOS, CD80, CD206 and PPARγ to evaluate the reprogramming efficiency of TLP on macrophages.

[0072] 5 Anti-inflammatory effect of TLP in vivo

[0073] 5.1 Construction of mouse pneumonia model

[0074] C57BL / 6J mice were intratracheally instilled with LPS (2 mg / kg) to establish a mouse pneumonia model.

[0075] 5.2 In vivo distribution

[0076] TLP was prepared with Cy7-saRNA. After 4h of intratracheal instillation of LPS (2mg / kg), mice were randomly divided into 4 groups, and injected with PBS, saRNA, LP, TLP (the latter three groups each contained 20μg Cy7-saRNA / 100μL) into the orbit, with an injection dose of 100μL per mouse. The distribution and metabolism of TLP in vivo were observed by small animal imaging at 2, 6, 12, 24, 48h. The mice were dissected to take out the heart, liver, spleen, lung and kidney at 48h, and the distribution of TLP in each organ was observed by imaging.

[0077] 5.3 Treatment of pneumonia

[0078] Mice were randomly divided into 6 groups. After 4h of intratracheal instillation of LPS (2mg / kg), except for the normal control group, the other 5 groups were injected with normal saline, TL, saRNA, LP, TLP (the latter three groups each contained 20μg saPPARγ / 100μL) into the orbit, with an injection dose of 100μL per mouse. The mice were given drugs every 3 days, a total of twice, and the lung function indicators of the mice were detected 3 days after the last administration to evaluate the lung function recovery effect of TLP. The mice were dissected to take out the lung tissue for hematoxylin and eosin (H&E) staining to evaluate the treatment effect of TLP; lung tissue protein was extracted, and the expression levels of Arg1, CD80, CD206 and PPARγ were analyzed by Western blot to evaluate the in vivo reprogramming efficiency of TLP.

[0079] II. Results

[0080] After transfection of M1 macrophages with different sequences of saRNA, the results of real-time fluorescence quantitative PCR showed that the PPARγ expression of cells transfected with sa-PPARγ-1 was most obviously up-regulated (A), and the results of Western blot were consistent with it (B), indicating that sa-PPARγ-1 could better reprogram M1 macrophages, so this sequence was used in the subsequent experiments, i.e.: Figure 1 Figure 1 B), indicating that sa-PPARγ-1 could better reprogram M1 macrophages, so this sequence was used in the subsequent experiments, i.e.:

[0081] sense: 5'-CCAAUAGUCUAACUUAAAAdTdT-3' (SEQ ID NO. 3)

[0082] anti-sense: 5'-UUUUAAGUUAGACUAUUGGdTdT-3' (SEQ ID NO. 4)

[0083] The morphology of macrophages before and after transfection was observed under an optical microscope. The M1 macrophages transfected with saPPARγ became long spindle-shaped M2 macrophages (A), and the M2 macrophages transfected with saPPARγ became long spindle-shaped M1 macrophages (B). Figure 2 ​A). The protein and mRNA levels of CD206 and Arg1, the markers of M2 macrophages, were up-regulated in M1 macrophages transfected with saPPARy Figure 2 B and Figure 2 C), indicating that PPARy can regulate the polarization process of macrophages, and M1 macrophages can be polarized into M2 macrophages. M1 macrophages were transfected with different concentrations of saPPARy, and it was found that the mRNA levels of PPARy and the markers of M2 macrophages CD206 and IL10 reached a peak when the concentration of saPPARy reached 60 nM, and then their expression levels did not change with the increase of the concentration of saPPARy, indicating that the polarization of M1 macrophages can be well regulated when the concentration of saPPARy is 60 nM Figure 2 D), so the subsequent experiment uses saPPARy with a concentration of 60 nM to synthesize TLP.

[0084] With the increase of mass ratio, the particle size of liposome delivery system (TLP for short) is at a relatively small value when the mass ratio is 40, its potential is about 4 mV Figure 3 A), and its encapsulation rate is 97.58%, which can well load saRNA Figure 3 D). Different mass ratios of TLP can well wrap saRNA in liposomes Figure 3 B), and can still effectively protect saRNA after 60 min of co-incubation with serum Figure 3 C). Laser confocal microscopy was used to observe the uptake of M1 macrophages to different electric liposomes, and the results showed that when TLP is positively charged, M1 macrophages take up the most Figure 3 E). The mass ratio of 40 was selected to prepare TLP by combining particle size, potential and cell uptake. TLP is a circular nanoparticle with obvious liposome morphology. Electron microscopy can observe that there are several cavities of SM-102 complex saRNA in the liposome cavity, which further proves that TLP is successfully prepared Figure 3 F). Then observe the stability of TLP in water for three days, and the results show that the particle size and dispersion coefficient have no great change, indicating that TLP has a relatively stable structure Figure 3 G). The cumulative release of TLP is about 99% in pH 5.4 environment for 72 h, and about 60% in pH 7.5 environment Figure 3 H).

[0085] Using pure saRNA, LP without targeting peptide and TLP to transfect M1 macrophages, compared with the other two groups, TLP shows a strong fluorescence signal in the cells Figure 4A). TLP was incubated with mouse alveolar epithelial cells (MLE), mouse embryonic fibroblasts (3T3), M0 macrophages and M1 macrophages overnight. The fluorescence intensity of TLP in the first three cells was weaker than that in M1 macrophages, indicating that TLP could better recognize M1 macrophages and had good M1 macrophage targeting property Figure 4 B). The fluorescence signal of TLP overlapped with the fluorescence signal of lysosomes after 3h of co-incubation with cells, indicating that the lysosomes were captured at this time. After 9h, the fluorescence signal of TLP separated from the fluorescence signal of lysosomes, and the fluorescence signal of lysosomes weakened, indicating that TLP escaped from the lysosomes at this time Figure 4 C). M1 macrophages were transfected with TLP for 24h and 48h, and the cells still showed positive signals of calcein Figure 4 D), the cells still had good viability after transfection Figure 4 E), indicating that TLP had low cytotoxicity. Meanwhile, the CCK8 results showed that TLP containing different concentrations of saPPARγ had no significant effect on cell viability within 24h, and TLP containing high concentrations of saPPARγ showed cell viability inhibition after 48h Figure 4 F). After M1 macrophages were transfected with TLP, the expression of PPARγ was up-regulated, the M2 macrophage marker protein CD206 was increased, and the M1 macrophage marker iNOS was decreased Figure 4 G), which was consistent with the results of Western blotting Figure 4 H). It was indicated that TLP could effectively reprogram M1 macrophages.

[0086] After TLP was injected into the orbit of mice with pneumonia, it gradually accumulated in the lung, and the fluorescence in the lung gradually increased. There was still strong fluorescence signal after 48h Figure 5 A). The main internal organs were removed from the mice after 48h and imaged again. The fluorescence was distributed in the liver, spleen and lung Figure 5 B). The fluorescence of TLP group in the lung of mice was the strongest among all groups Figure 5 C). The above results indicated that TLP could effectively target the lung and prolong the circulation time in vivo.

[0087] Pneumonia mice were given drugs every 3 days through the orbit, and the lung function indicators were detected 3 days after the last administration, and then the mice were dissected. After fixation, the lung slices were stained with hematoxylin and eosin (H&E). The TL group had almost no therapeutic effect, showing the same increase in immune cells and red blood cells in the alveolar and interstitial space as the pneumonia mice treated with normal saline, and the formation of transparent membrane and thickening of alveolar wall. The Sa group and the LP group had weak therapeutic effect, while the TLP group showed thin alveolar wall, fewer inflammatory cells and red blood cells in the alveolar and interstitial regions, which meant that the pneumonia was significantly reduced Figure 6A). TLP also showed improvement in lung function, which was evidenced by minute volume (MV) and inspiratory time (TI) of the mice with pneumonia, which were close to the levels of normal mice Figure 6 C and Figure 6 D), indicating that lung inflammation was significantly reduced and lung function was restored after TLP treatment. The tissue proteins were extracted for immunoblotting detection, and the M1 macrophage marker CD80 was significantly down-regulated, while the M2 macrophage markers Arg1 and CD206 were significantly up-regulated Figure 6 B), proving that TLP can well reprogram macrophages in vivo.

[0088] The above-described embodiments are only to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A preparation method of an inflammation environment macrophage targeted small activating nucleic acid nanodrug, characterized in that, The preparation method comprises the following steps: (1) SM-102, DSPC, cholesterol, DMG-PEG2000, TKPR-PEG2000-DSPE are dissolved in an organic solvent according to a molar ratio of 50:10:37:1.5:1.5, rotary evaporation is performed to remove the organic solvent, then 1-3 mL of buffer is added for hydration, so that the total concentration of SM-102, DSPC, cholesterol, DMG-PEG2000 and TKPR-PEG2000-DSPE in the mixed solution is 0.4-0.6 mg / mL, and a liposome solution is formed; (2) 0.8-1.2 μgsaRNA is diluted in 0.8-1.2 μL nuclease-free water, and is compounded with the liposome solution according to a mass ratio of the liposome solution to the saRNA of 38-42:0.8-1.2, is placed at room temperature, and is centrifuged, so that a macrophage-targeted small activating nucleic acid nanomedicine in an inflammatory environment is obtained; The sequences of the sense strand and the antisense strand of the saRNA are shown in SEQ ID NO. 3 and 4, respectively; SEQ ID NO. 3: sense: 5'-CCAAUAGUCUAACUUAAAAdTdT-3'; SEQ ID NO. 4: anti-sense: 5'-UUUUAAGUUAGACUAUUGGdTdT-3'; In the step (1), the organic solvent is a mixed liquid of chloroform and methanol; In the step (1), the volume ratio of chloroform to methanol is 3:1; In the step (1), the buffer is 8-12 nM citrate buffer; In the step (1), the total concentration of SM-102, DSPC, cholesterol, DMG-PEG2000 and TKPR-PEG2000-DSPE in the mixed solution is 0.5 mg / mL; In the step (2), 1 μg of saRNA is diluted in 1 μL of nuclease-free water; In the step (2), the mass ratio of the liposome solution to the saRNA is 40:

1.

2. The macrophage-targeted small activating nucleic acid nanomedicine in an inflammatory environment prepared by the preparation method of claim 1.

3. The use of the macrophage-targeted small activating nucleic acid nanomedicine in an inflammatory environment of claim 2 in the preparation of an anti-pneumonia drug.

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