Inflammation environment macrophage targeting small activation nucleic acid nano-drug as well as preparation method and application thereof

Abstract

The invention discloses an inflammatory environment macrophage targeting small activation nucleic acid nano-drug as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The inflammatory environment macrophage targeting small activation nucleic acid nano-drug provided by the invention is composed of a small activation nucleic acid core and a liposome shell. After the inflammatory environment macrophage targeting small activation nucleic acid nano-drug is actively targeted to an inflammatory environment and enters cells through receptor recognition, a liposome coat is swelled and disintegrated, small activation RNA is released, gene expression is regulated and controlled, M1 type macrophages are reprogrammed into M2 type macrophages from the two aspects of phenotype and metabolic mode, anti-inflammatory factors are released, and the anti-inflammatory effect is achieved. The aim of inhibiting inflammation is fulfilled. Therefore, the invention has a very wide application prospect in gene therapy and immunotherapy of inflammation, infectious diseases and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a small activated nucleic acid nano drug targeting macrophages in an inflammatory environment, and a preparation method and application thereof. Background Art

[0002] Pneumonia refers to inflammation of the terminal airways, alveoli, and lung interstitium, and is a hallmark pathological feature of lung damage caused by exogenous or endogenous attacks on the lungs. As the main cellular component of the inflammatory response, macrophages gather in large numbers in the inflammatory microenvironment. After being activated by inflammatory mediators, they attack normal cells in the inflammatory area. As an innate immune cell, their 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 γ, lipopolysaccharide, and TNFα, and have the ability to enhance antigen presentation, produce nitric oxide, and secrete a large number of pro-inflammatory factors; M2 macrophages can be induced by CSF-1, IL1β, IL4, and IL10, which can inhibit the secretion of pro-inflammatory factors of M1 macrophages, 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 of treating inflammatory lung diseases.

[0003] Gene therapy refers to a new treatment method that treats diseases by introducing external genes at the genetic level to achieve changes such as replacement, substitution, knock-in / knock-out, activation / inhibition, etc. of genes in the body. Small activating RNA (saRNA) is a new type of gene therapy drug that can stimulate gene expression at the transcriptional level. It has the characteristics of small molecular weight, gene specificity and activation of gene expression. Studies have shown that upregulating PPARγ can reprogram M1 macrophages into M2 macrophages.

[0004] Like most nucleic acid drugs and other RNA drugs, saRNA still has limitations in specificity, stability and delivery methods. Although chemical modification can improve its stability and specificity, it will 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 easily modified with various ligands and functional molecules, which can greatly improve the targeting of RNA. How to reduce the nonspecific clearance of nucleic acid drug carriers and improve their tissue / cell targeting is a hot spot and difficulty in nucleic acid drug delivery. Summary of the invention

[0005] The present invention provides a preparation method of an inflammatory environment macrophage-targeted small activating nucleic acid nanomedicine, 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, and the organic solvent is removed by rotary evaporation. Subsequently, 1-3 mL of a buffer solution 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 ribonuclease-free water, and is complexed with the liposome solution according to a mass ratio of the liposome solution to saRNA of 38-42:0.8-1.2, and is left standing at room temperature and then centrifuged. The obtained precipitate is the inflammatory environment macrophage-targeted small activating nucleic acid nanomedicine;

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

[0009] In some embodiments of the present invention, 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 present invention, the organic solvent in the step (1) is a mixed liquid of chloroform and methanol.

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

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

[0013] In some embodiments of the present invention, 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 present invention, 1 μg of saRNA is diluted in 1 μL of ribonuclease-free water in the step (2).

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

[0016] The present invention also provides a macrophage-targeted small activating nucleic acid nanomedicine for the inflammatory environment prepared by the above preparation method.

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

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

[0019] The present invention uses liposomes modified with the M1 macrophage-targeting peptide TKPR to load small activating RNA of PPARγ (sa-PPARγ) to prepare a macrophage-targeted small activating nucleic acid nanomedicine for the inflammatory environment. The macrophage-targeted small activating nucleic acid nanomedicine for the inflammatory environment recognizes the Nrp-1 receptor under the mediation of the TKPR targeting peptide and enters M1 macrophages, releases sa-PPARγ, significantly up-regulates the expression of PPARγ, reprograms the phenotype and metabolic mode of M1 macrophages into M2 macrophages, thereby releasing anti-inflammatory factors and down-regulating the level of pro-inflammatory factors, and finally achieving the purpose of reducing inflammation. It can be seen that the present invention provides a new drug for the treatment of pneumonia. Description of the Drawings

[0020] Figure 1 It is the screening result of the nucleic acid sequence in Example 1, wherein A) Real-time fluorescence quantitative PCR was used to detect the expression of PPARγ mRNA in macrophages transfected with different sequences of saRNA. B) Western blotting was used to detect the expression of PPARγ protein in macrophages transfected with different sequences of saRNA.

[0021] Figure 2 It is the result of PPARγ regulating macrophage polarization in Example 1, wherein A) Optical microscopy was used to observe the cell morphology of M1 macrophages and cells transfected with saPPARγ, scale bar = 200 μm; B) Western blotting was used to detect the protein expression in M1 macrophages transfected with saPPARγ; C) Real-time fluorescence quantitative PCR was used to detect the mRNA expression in M1 macrophages transfected with saPPARγ; D) Real-time fluorescence quantitative PCR was used to detect the mRNA expression in M1 macrophages transfected with different concentrations of saPPARγ.

[0022] Figure 3Test results of TLP synthesis and characterization in Example 1, where A) Comparison of particle size and zeta potential of TLP with different mass ratios; B) Gel retardation of TLP with different mass ratios; C) Nucleic acid protection of TLP with different mass ratios; D) Encapsulation of TLP with different mass ratios; E) Cellular uptake of liposomes with different electricities, scale bar = 50 μm; F) Particle size distribution and morphology of TLP when the mass ratio = 40; G) Three-day stability of TLP in water when the mass ratio = 40; H) Drug release curves of TLP under different pH conditions.

[0023] Figure 4 Test results of TLP-mediated macrophage reprogramming in Example 1, where A) Cellular uptake of different formulations detected by laser confocal microscopy and flow cytometry, scale bar = 50 μm; B) Uptake of TLP in different cells detected by laser confocal microscopy and flow cytometry, scale bar = 50 μm; C) Lysosomal escape of TLP observed by laser confocal microscopy, scale bar = 50 μm; D) Cell viability of macrophages transfected with different formulations observed by laser confocal microscopy, scale bar = 200 μm; E) Effects of different formulations on macrophage viability detected by CCK8 assay; F) Effects of different concentrations of saPPARγ on macrophage viability detected by CCK8 assay; G) Protein expression in macrophages transfected with different formulations detected by Western blotting; H) mRNA expression in macrophages transfected with different formulations detected by real-time fluorescence quantitative PCR.

[0024] Figure 5 Test results of TLP in vivo distribution in Example 1, where A) Distribution and metabolism of different formulations in pneumonia mice observed by small animal in vivo imaging; B) Distribution of different formulations in major organs of pneumonia mice observed by small animal in vivo imaging; C) Comparison of fluorescence signals of different formulations in major organs of pneumonia mice.

[0025] Figure 6 Test results of TLP in the treatment of pneumonia in Example 1, where A) Histological analysis of lung tissue; B) Western blot 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 of the present invention and schematic diagram of the action principle of TLP. Detailed implementation mode

[0027] Example 1

[0028] I. Method

[0029] 1 Nucleic acid sequence screening

[0030] M1 macrophages were cultured overnight, transfected with saRNAs of different sequences using PEI, and then cultured for another 48 h. Total cellular RNA was extracted and the expression level of PPARγ was analyzed by real-time fluorescence quantitative PCR. The saRNA sequences are 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-PPARγ-1:

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

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

[0037] sa-PPARγ-2:

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

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

[0040] sa-PPARγ-3:

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

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

[0043] sa-PPARγ-4:

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

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

[0046] 2 PPARγ regulates macrophage polarization

[0047] 2.1 Macrophage morphological changes

[0048] M1 macrophages were cultured overnight, transfected with sa-PPARγ using PEI, and continued to be cultured for 48 h. The cell morphology was observed under an optical microscope.

[0049] 2.2 PPARγ regulates macrophage polarization

[0050] M1 macrophages were cultured overnight, transfected with sa-PPARγ using PEI, and continued to be cultured for 48 h. The total cellular proteins were extracted, and the expression levels of Arg1 and CD206 were analyzed by Western blotting. The total cellular RNA was extracted, and the expression levels of Arg1 and CD206 were analyzed by real-time fluorescence quantitative PCR.

[0051] M1 macrophages were cultured overnight, transfected with different concentrations of sa-PPARγ using PEI, and continued to be cultured for 48 h. The total cellular RNA was extracted, and the expression levels of PPARγ, CD206, and IL-10 were analyzed by real-time fluorescence quantitative PCR.

[0052] 3 Synthesis and characterization of TLP

[0053] 3.1 TL synthesis

[0054] The thin-film dispersion method was adopted: Liposomes were synthesized using five components, namely SM-102, DSPC, cholesterol, DMG-PEG2000, and TKPR-PEG2000-DSPE. Each component was weighed according to the molar ratio of 50 / 10 / 37 / 1.5 / 1.5 and dissolved in a chloroform / methanol (3 / 1, v / v) mixed solution. The organic solvent was removed by rotary evaporation on a rotary evaporator for 30 min. Subsequently, 2 mL of 10 nM citrate buffer (pH = 3) was added for hydration to make the total concentration of SM-102, DSPC, cholesterol, DMG-PEG2000, and TKPR-PEG2000-DSPE in the mixed solution 0.5 mg / mL. After extrusion back and forth with an extruder, a liposome solution (TL) was formed and stored at 4 °C for later use.

[0055] Using TLC and LP as controls. TL and saRNANC were complexed at a mass ratio (TL: saRNA) of 40:1 to form TLC. Liposome solution prepared with a molar ratio of SM-102, DSPC, cholesterol, and DMG-PEG2000 of 50 / 10 / 37 / 3 was combined with saRNA at a mass ratio (liposome solution: saRNA) of 40:1 to prepare nanoparticles without macrophage-targeting peptides, abbreviated as LP.

[0056] 3.2 TLC Quality Ratio Screening

[0057] Take 1 μg of saRNA and dilute it in 1 μL of nuclease-free water to a final concentration of 1 μg / μL. Complex it with liposome solution at mass ratios (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 to remove unencapsulated saRNA. After centrifugation, discard the supernatant, add 1 mL of PBS to resuspend, store at 4°C for later use. Dilute the complex solution with 1 mL of double-distilled water, and measure the particle size and zeta potential of the complex using a Malvern particle size analyzer.

[0058] Take 1 μg of saRNA and dilute it in 1 μL of nuclease-free water. Complex it with liposome solution at mass ratios (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 the supernatant, add 10*Loading buffer, mix well, and load the sample. Electrophorese on a 1% agarose gel at 120 V for 20 min, take a photo with a gel imager, and calculate the encapsulation efficiency.

[0059] 3.3 Drug Release

[0060] Prepare TLC with fluorescently labeled saRNA, place it in a dialysis bag with a molecular weight cut-off of 3500, and place it in PBS buffer solutions at pH 5.4 and pH 7.4 respectively. Take the dialysis fluid at 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 48, 72 h, measure the fluorescence intensity with a fluorescence spectrophotometer, and calculate the cumulative drug release rate.

[0061] 4 Macrophage Reprogramming Mediated by TLC

[0062] 4.1 Cell Uptake

[0063] Prepare TLC with saRNA labeled with a fluorescent dye. Incubate M1 macrophages overnight, then add PBS, saRNA, LP, and TLC culture media respectively, and incubate for 9 h. Detect the cell uptake of different preparations using a flow cytometer and a laser confocal fluorescence microscope.

[0064] 4.2 Cellular specific uptake

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

[0066] 4.3 Lysosomal escape

[0067] M1 macrophages were cultured overnight, and then TLP containing equal mass of fluorescently labeled saRNA (60 nM) was added and incubated for 3 h and 9 h respectively. The cells were gently washed twice with PBS, then a green lysosomal probe was added and incubated. The lysosomal escape of nanoparticles in the cells was observed by laser confocal microscopy.

[0068] 4.4 Cytotoxicity

[0069] M1 macrophages were cultured overnight, and then incubated with culture media containing PBS, TL, saRNA, TLC, LP, TLP (the latter four groups each containing 60 nM saRNA) for 8 h, then the media was changed and the cells were continued to be cultured until 48 h. The cells were stained with a cell viability / cytotoxicity staining kit, and the viability and cytotoxicity of different preparations were analyzed by observing the live and dead cells with a laser confocal microscope.

[0070] 4.5 Macrophage reprogramming efficiency

[0071] M1 macrophages were cultured overnight, and then incubated with culture media containing PBS, TL, saRNA, TLC, LP, TLP (the latter four groups each containing 60 nM saRNA) for 8 h, then the media was changed and the cells were continued to be cultured until 48 h. Total cellular proteins were extracted, and the expression levels of iNOS, CD80, CD206, and PPARγ were analyzed by Western blot to evaluate the efficiency of TLP in reprogramming macrophages.

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

[0073] 5.1 Establishment of a mouse pneumonia model

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

[0075] 5.2 In vivo distribution

[0076] Preparation of TLP with Cy7-saRNA. Four hours after intratracheal instillation of LPS (2 mg / kg), mice were randomly divided into 4 groups and injected intraorbitally with PBS, saRNA, LP, and TLP (the latter three groups each contained 20 μg Cy7-saRNA / 100 μL), and the injection dose for each group was 100 μL / mouse. The mice were imaged using a small animal imager at 2, 6, 12, 24, and 48 h to observe the distribution and metabolism of TLP in vivo. After 48 h, the mice were dissected to obtain the heart, liver, spleen, lungs, and kidneys, and imaging was performed to observe the distribution of TLP in each organ.

[0077] 5.3 Treatment of pneumonia

[0078] Mice were randomly divided into 6 groups. Four hours after intratracheal instillation of LPS (2 mg / kg), except for the normal control group, the remaining 5 groups were injected intraorbitally with normal saline, TL, saRNA, LP, and TLP (each of the latter three groups contained 20 μg saPPARγ / 100 μL), and the injection dose for each group was 100 μL / mouse. The drugs were administered once every 3 days for a total of two administrations. Three days after the last administration, the pulmonary function indexes of the mice were measured to evaluate the recovery effect of TLP on pulmonary function. The mice were dissected, and lung tissues were taken for hematoxylin and eosin (H&E) staining to evaluate the therapeutic effect of TLP; lung tissue proteins were extracted, and the expression levels of Arg1, CD80, CD206, and PPARγ were analyzed by Western blotting to evaluate the in vivo reprogramming efficiency of TLP.

[0079] II. Results

[0080] After M1 macrophages were transfected with saRNAs of different sequences, the results of real-time fluorescence quantitative PCR showed that the expression of PPARγ in cells transfected with sa-PPARγ-1 was upregulated most significantly ( Figure 1 A), and the results of Western blotting were consistent with it ( Figure 1 B), indicating that sa-PPARγ-1 could reprogram M1 macrophages well. Therefore, this sequence was used in subsequent experiments, namely:

[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 by optical microscopy. After transfection with saPPARγ, M1 macrophages became spindle-shaped M2 macrophages ( Figure 2A). After transfection of M1 macrophages with saPPARγ, the protein and mRNA levels of the M2 macrophage markers CD206 and Arg1 were upregulated ( Figure 2 B and Figure 2 C), indicating that PPARγ can regulate the polarization process of macrophages, polarizing M1 macrophages into M2 macrophages. M1 macrophages were transfected with different concentrations of saPPARγ, and it was found that when the concentration of saPPARγ reached 60 nM, the mRNA levels of PPARγ and the M2 macrophage markers CD206 and IL10 were upregulated to the peak, and then their expression levels did not change with the increase in the concentration of saPPARγ, indicating that the polarization of M1 macrophages could be well regulated at a saPPARγ concentration of 60 nM ( Figure 2 D). Therefore, saPPARγ with a concentration of 60 nM was used to synthesize TLP in subsequent experiments.

[0084] As the mass ratio increased, the particle size of the liposome delivery system (abbreviated as TLP) was at a relatively small value when the mass ratio was 40, and its zeta potential was about 4 mV ( Figure 3 A), and its encapsulation efficiency was 97.58%, which could well load saRNA ( Figure 3 D). TLPs with different mass ratios could well encapsulate saRNA in liposomes ( Figure 3 B), and could effectively protect saRNA after co-incubation with serum for 60 min ( Figure 3 C). Laser confocal microscopy was used to observe the uptake of M1 macrophages by liposomes with different zeta potentials. The results showed that when TLP was positively charged, M1 macrophages had the highest uptake ( Figure 3 E). Considering the particle size, zeta potential and cell uptake, a mass ratio of 40 was selected to prepare TLP. TLP was circular nanoparticles with an obvious liposome morphology. It could be observed in the electron micrograph that there were cavities formed by the complex of several SM-102 and saRNA in the liposome cavity, further indicating the successful preparation of TLP ( Figure 3 F). Subsequently, the stability of TLP in water for three days was observed. The results showed that there were no significant changes in its particle size and dispersion coefficient, indicating that TLP had a relatively stable structure ( Figure 3 G). The cumulative drug release of TLP reached 99% at pH 5.4 for 72 h, while the cumulative release was about 60% at pH 7.5 ( Figure 3 H).

[0085] M1 macrophages were transfected with pure saRNA, LP without targeting peptide and TLP. Compared with the other two groups, TLP showed stronger fluorescence signals in cells ( Figure 4A). TLP was co-cultured 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 types of cells was weaker than that in M1 macrophages, indicating that TLP could better recognize M1 macrophages and had good targeting ability to M1 macrophages. Figure 4 B). After co-incubating TLP with cells for 3 h, the fluorescence signal overlapped with the green fluorescence signal of lysosomes, indicating that it was captured by lysosomes at this time. After 9 h, the fluorescence of TLP separated from the lysosome fluorescence signal, and the lysosome fluorescence signal weakened, indicating that TLP escaped from lysosomes at this time. Figure 4 C). The cells of M1 macrophages were still positive for calcein after transfection with TLP for 24 h and 48 h. Figure 4 D), and the cells still had good viability after transfection. Figure 4 E), indicating that TLP had low cytotoxicity. At the same time, the CCK8 results showed that TLP containing different concentrations of saPPARγ had no obvious effect on cell viability within 24 h, and TLP containing high concentration of saPPARγ showed inhibition of cell viability after 48 h. Figure 4 F). After transfection of M1 macrophages with TLP, the expression of PPARγ was up-regulated, the M2 macrophage marker protein CD206 increased, while the M1 macrophage marker iNOS decreased. Figure 4 G), and the results of real-time fluorescence quantitative PCR were consistent with those of Western blotting. Figure 4 H). It shows that TLP can effectively reprogram M1 macrophages.

[0086] After TLP was injected into the orbital cavity of pneumonia mice, it gradually accumulated in the lung, and the lung fluorescence gradually increased. There was still a strong fluorescence signal after 48 h. Figure 5 A). After sacrificing the mice at 48 h and taking out the main internal organs for imaging again, the fluorescence was distributed in the liver, spleen and lung. Figure 5 B). The fluorescence of TLP group in the lungs of mice was the strongest among all groups. Figure 5 C). The above results indicate that TLP can effectively target to the lungs and prolong the in vivo circulation time.

[0087] Pneumonia mice were given drugs via the orbital cavity every 3 days. Three days after the last drug administration, the lung function indexes were measured and then the mice were dissected. After the lungs were fixed and sectioned, they were stained with hematoxylin and eosin (H&E). There was almost no therapeutic effect in the TL group, showing a significant increase in immune cells and red blood cells in the alveoli and interstitial spaces, the formation of hyaline membranes and thickening of the alveolar walls, which was the same as that of pneumonia mice treated with normal saline. The therapeutic effects of the Sa group and the LP group were weak, while the treatment in the TLP group showed thin alveolar walls, fewer inflammatory cells and red blood cells in the alveoli and interstitial regions, indicating that pneumonia was significantly alleviated. Figure 6A). TLP also showed an improvement in lung function, as demonstrated by the minute volume (MV) and inspiratory time (TI) of pneumonia mice, which were close to the levels of normal mice ( Figure 6 C and Figure 6 D), indicating that pneumonia was significantly alleviated and lung function was restored after TLP treatment. Tissue proteins were extracted for immunoblotting detection, and the M1 macrophage marker CD80 was significantly downregulated, while the markers of M2 macrophages, Arg1 and CD206, were significantly upregulated ( Figure 6 B), proving that TLP can reprogram macrophages well in vivo.

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

Claims

1. A preparation method of a macrophage-targeted small activating nucleic acid nanomedicine for an inflammatory environment, characterized in that, The preparation method includes the following steps: (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, and then 1-3 mL of buffer solution 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 μg of saRNA is diluted in 0.8-1.2 μL of nuclease-free water. According to the mass ratio of the liposome solution to saRNA of 38-42:0.8-1.2, it is complexed with the liposome solution, left standing at room temperature, and then centrifuged. The obtained precipitate is the macrophage-targeted small activating nucleic acid nanomedicine in an inflammatory environment; The sequences of the sense strand and the antisense strand of the saRNA are shown in SEQ ID NO.3 and 4 respectively.

2. The preparation method according to claim 1, characterized in that, In the step (1), the molar ratio of SM-102, DSPC, cholesterol, DMG-PEG2000, and TKPR-PEG2000-DSPE is 50:10:37:1.5:1.

5.

3. The preparation method according to claim 2, characterized in that, In the step (1), the organic solvent is a mixed liquid of chloroform and methanol.

4. The preparation method according to claim 3, characterized in that, In the step (1), the volume ratio of chloroform to methanol is 3:

1.

5. The preparation method according to claim 4, characterized in that, In the step (1), the buffer solution is 8-12 nM citrate buffer solution.

6. The preparation method according to claim 5, characterized in that, 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.

7. The preparation method according to claim 6, wherein In the step (2), 1 μg of saRNA is diluted in 1 μL of nuclease-free water.

8. The preparation method according to claim 7, characterized in that, In the step (2), the mass ratio of the liposome solution to saRNA is 40:

1.

9. The macrophage-targeted small activating nucleic acid nanomedicine in an inflammatory environment prepared by the preparation method according to any one of claims 1-8.

10. Use of the macrophage-targeted small activating nucleic acid nanomedicine in an inflammatory environment according to claim 9 in the preparation of an anti-inflammatory drug.

Images