Nanoparticles for treating lung diseases and preparation method and application thereof

Abstract

The present invention discloses a nanoparticle for treating lung diseases, and its preparation method and application. The nanoparticle for treating lung diseases provided by the present invention comprises a nanoparticle carrier and a nucleic acid molecule; the raw materials of the nanoparticle carrier include the TM2 and DOPE, and the molar ratio of the two is 1:2. The nucleic acid-encapsulated nanoparticles provided by the present invention can efficiently deliver nucleic acid molecules to the lungs and downregulate the expression of pathogenic proteins; they can significantly increase the deposition of drugs in the lungs, effectively ensuring the effect after administration, and at the same time have the advantages of simple administration, non-invasiveness, and high safety. It is a highly innovative and efficient lung deposition method with good application prospects in the treatment of lung-related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to nanoparticles for treating lung diseases, and a preparation method and application thereof. Background Art

[0002] The development of nanoparticles for pulmonary drug delivery has garnered significant attention in recent years, primarily due to their enormous potential for treating respiratory and systemic diseases. Compared to traditional drug delivery methods, nanoparticles enable efficient pulmonary drug delivery, particularly for gene therapy, offering higher bioavailability, more precise targeting, and prolonged drug release. The lungs' large gas exchange surface area, rich vascular network, and thin air-blood barrier make them an ideal target for nanoparticle drug delivery. Furthermore, the lungs as a drug delivery target can avoid the first-pass effect, significantly improving the efficiency of gene therapy. Nanoparticles offer advantages in gene therapy because they can protect sensitive nucleic acids (such as DNA, mRNA, and siRNA) from enzymatic degradation in vivo and enable targeted delivery to specific cells through surface modification. In particular, for diseases such as lung cancer, pulmonary fibrosis, cystic fibrosis, and chronic obstructive pulmonary disease (COPD), nanoparticles can precisely deliver gene editing tools (such as CRISPR-Cas9) or RNA interference molecules to diseased cells, repairing genetic mutations or inhibiting the expression of pathogenic genes. In addition, the size, drug loading and surface modification of nanoparticles can be controlled, and they can pass through biological barriers, thereby improving the cellular uptake and gene expression efficiency of gene drugs.

[0003] With the advancement of nanotechnology, pulmonary nanoparticle delivery systems are becoming a powerful supporter of gene therapy and showing great potential in the local treatment of respiratory diseases. Summary of the Invention

[0004] The present invention aims to provide a nanoparticle containing a nucleic acid. The nanoparticle containing the nucleic acid is suitable for oral tracheal administration or aerosol inhalation to deliver RNA or therapeutic genes for targeted gene therapy to the lungs, so that the nucleic acid drug can effectively exert its function after inhalation.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a lipid molecule TM2.

[0007] The lipid molecule TM2 has a structural formula as shown in Formula I:

[0008]

[0009] In a second aspect, the present invention provides a method for preparing the lipid molecule TM2 described in the first aspect.

[0010] The preparation method of TM2 provided by the present invention comprises the following steps:

[0011] 1) Under the catalytic action of DMAP and EDCI, TM-1 compound and N-Boc-6-aminocaproic acid are subjected to an esterification reaction to obtain TM-2 compound;

[0012]

[0013] 2) Under acidic conditions, deprotecting the BOC in the TM-2 compound to obtain the TM-3 compound;

[0014] 3) In the presence of HBTU and DIPEA, TM-3 compound is subjected to a condensation reaction with S-

[12] aneN3 to obtain TM-4 compound;

[0015]

[0016] 4) Under acidic conditions, the BOC in the TM-4 compound is deprotected to obtain the TM2 compound.

[0017] In step 1) of the above method, the esterification reaction is carried out in an organic solvent, which may be THF.

[0018] In step 1) of the above method, the molar ratios of the TM-1 compound, N-Boc-6-aminocaproic acid, DMAP, and EDCI are 1:1:0.5:1.2, respectively.

[0019] In step 1) of the above method, the reaction temperature of the esterification reaction is 0-25°C, and the reaction is stirred at 25°C for 4 hours.

[0020] Furthermore, the step 1) is specifically as follows: dissolving the TM-1 compound, N-Boc-6-aminohexanoic acid and DMAP in THF and stirring under argon for 30 minutes; adding EDCI at 0°C; stirring the mixture at 25°C for 4 hours; adding DCM, washing with saturated brine, and removing the organic solvent in vacuo to obtain the TM-2 compound.

[0021] In step 2) of the above method, the acidic condition can be provided by a saturated ethyl acetate solution with hydrogen chloride.

[0022] In step 3) of the above method, the condensation reaction is carried out in an organic solvent, which may be dichloromethane (DCM).

[0023] In step 3) of the above method, the molar ratios of the compounds TM-3, S-

[12] aneN3, HBTU, and DIPEA are 1:1:1.21:2.5, respectively.

[0024] In step 3) of the above method, the reaction temperature of the condensation reaction is 0-25°C, and the reaction is stirred at 25°C for 4 hours.

[0025] Furthermore, the step 3) is specifically as follows: dissolving compound TM-3 and S-

[12] aneN3 in DCM under argon, adding HBTU and DIPEA at 0°C, and stirring the reaction at 25°C for 10 hours; adding an appropriate amount of DCM to dilute, washing with saturated brine, and removing the organic solvent in vacuo to obtain the TM-4 compound.

[0026] In step 4) of the above method, the acidic condition can be provided by a saturated ethyl acetate solution with hydrogen chloride.

[0027] In a third aspect, the present invention provides a nanoparticle carrier.

[0028] The nanoparticle carrier provided by the present invention; its raw materials include TM2 and DOPE.

[0029] Furthermore, the molar ratio of TM2 to DOPE may be 1:2.

[0030] In a fourth aspect, the present invention provides a nanoparticle encapsulating nucleic acid.

[0031] The nanoparticles encapsulating nucleic acid drugs include the nanoparticle carriers and nucleic acid molecules described in the third aspect of the present invention.

[0032] Furthermore, the nucleic acid molecule includes one or more of a messenger nucleic acid molecule, a small interfering nucleic acid molecule, a micronucleic acid molecule, a small activating nucleic acid molecule, an antisense oligonucleotide molecule or an aptamer.

[0033] According to an embodiment of the present invention, the nucleic acid molecule is siRNA targeting Ttc3, and the sequences of the siRNA are as follows: sense 5'-AGGAUCUAAUUCAGCAGCAUGUAAA-3' (SEQ ID No. 1), antisense 5'-UUUACAUGCUGCUGAAUUAGAUCCU-3' (SEQ ID No. 2).

[0034] Furthermore, the mass ratio of the sum of the masses of TM2 and DOPE in the nanoparticles to the mass of the nucleic acid molecule is (1-50):1.

[0035] Furthermore, the particle size of the nucleic acid-encapsulated nanoparticles may be 50 to 200 nm.

[0036] In a fifth aspect, the present invention provides a method for preparing the nucleic acid-encapsulated nanoparticles described in the fourth aspect.

[0037] The method for preparing nucleic acid-encapsulated nanoparticles provided by the present invention comprises the following steps:

[0038] 1) dissolving the TM2 and DOPE in an organic solvent, and removing the solvent in vacuo to obtain a DOPE / TM2 film;

[0039] 2) hydrating the DOPE / TM2 film to obtain a nanoparticle TMD2 solution;

[0040] 3) Mix the nucleic acid molecule solution with the nanoparticle TMD2 solution and incubate at room temperature to obtain the product.

[0041] Furthermore, the TM2 and DOPE are mixed in a molar ratio of 1:2.

[0042] Furthermore, the organic solvent may be CDCl3.

[0043] Furthermore, the hydration medium used in the hydration may be PBS buffer (pH=7.4).

[0044] Furthermore, the incubation time may be 15-30 minutes.

[0045] In a sixth aspect, the present invention provides a product for treating lung diseases.

[0046] The product for treating lung diseases provided by the present invention includes the nucleic acid-encapsulated nanoparticles described in the fourth aspect of the present invention.

[0047] The product may be a pharmaceutical preparation.

[0048] Furthermore, the pharmaceutical preparation is preferably administered via tracheal administration, such as aerosol inhalation or dry powder inhalation.

[0049] Furthermore, the pharmaceutical preparation is a tracheal administration preparation, specifically a nebulizer inhaler or a dry powder inhaler.

[0050] Furthermore, the product also includes a pharmaceutically acceptable carrier.

[0051] The pulmonary diseases include but are not limited to pulmonary fibrosis, lung cancer, chronic obstructive pulmonary disease, cystic fibrosis, pulmonary hypertension, etc.

[0052] According to different lung diseases, the nucleic acid-encapsulated nanoparticles can encapsulate different nucleic acid molecules to achieve the purpose of treatment.

[0053] When the drug is used to treat lung cancer, the sensitivity of the nanoparticles to the concentration of nitroreductase is utilized to encapsulate siRNA targeting lung cancer-related genes, and the siRNA is delivered to the lungs through tracheal administration or atomization, thereby achieving specific targeted delivery to lung cancer cells.

[0054] When used to treat chronic obstructive pulmonary disease (COPD), the drug encapsulates siRNA targeting COPD-related genes and is delivered to the affected areas of the lungs via aerosol inhalation. Studies have shown that this system can efficiently deliver siRNA to the lungs, significantly reducing inflammatory responses and airway obstruction symptoms in COPD models.

[0055] When the drug is used to treat cystic fibrosis (CF), it encapsulates siRNA targeting CF-related genes and is delivered to the diseased area of ​​the lungs through aerosol inhalation. This system can efficiently deliver siRNA to the lungs, and the targeted siRNA inhibits excessive inflammatory response and reduces lung damage and infection caused by mucus accumulation.

[0056] When the drug is used to treat pulmonary arterial hypertension (PAH), it encapsulates siRNA targeting PAH-related genes and is delivered to the diseased area of ​​the lungs through aerosol inhalation. This system can target genes related to the balance of vascular dilation and contraction, helping to restore normal vascular function.

[0057] The present invention demonstrates that large nanoparticles (200 nm in diameter) loaded with silencing RNA (siRNA) targeting Ttc3 were administered orally to mice. Upon oral tracheal administration, Ttc3 silencing efficiency was high in the lungs (>70% knockdown) and effectively prevented / eliminated BLM-induced pulmonary fibrosis in mice, with pathological features of significant disordered thickening of alveolar septa, collapse of alveolar spaces, accompanied by tissue inflammatory infiltration and collagen deposition by fibroblasts. In vivo imaging of small animals was used to detect the entry of nucleic acid-loaded nanoparticles into the lungs and the amount of deposition in the lungs.

[0058] The nucleic acid-encapsulated nanoparticles provided by the present invention can efficiently deliver nucleic acid molecules (such as siRNA) to the lungs, downregulating the expression of pathogenic proteins; can significantly increase the deposition of drugs in the lungs, effectively ensuring the effect after administration; and at the same time have the advantages of simple administration, non-invasiveness, and high safety. It is a highly innovative and efficient lung deposition method and has good application prospects in the treatment of lung-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is the synthetic route of the molecule TM2;

[0060] Figure 2 is a transmission electron microscope (TEM) image of the nanoparticles TM2 in Example 2;

[0061] Figure 3The stability dynamic light scattering (DLS) graph of the nanoparticles TM2 in Example 2;

[0062] Figure 4 is a transmission electron microscope (TEM) image of the nanoparticles TMD2 in Example 3;

[0063] Figure 5 The stability dynamic light scattering (DLS) graph of the nanoparticles TMD2 in Example 3;

[0064] Figure 6 This is a graph showing the siRNA encapsulation efficiency of TM2 / TMD2 nanoparticles in Example 4;

[0065] Figure 7 Whole-body imaging at different time points of siTtc3@TMD2 lung perfusion in Example 6;

[0066] Figure 8 These are in vitro fluorescence images of major organs after siTtc3@TMD2 lung perfusion in Example 6;

[0067] Figure 9 Figure 7 is a graph showing changes in body weight of mice in Example 7;

[0068] Figure 10 HE and Masson staining images of lung tissue sections of mice in each group in Example 8;

[0069] Figure 11 The lung tissue pathological scores of each group of mice in Example 8;

[0070] Figure 12 This is a graph showing the fluorescence recovery experiment of TMD2 nitroreductase in Example 9;

[0071] Figure 13 is the responsiveness result of TM2 to NTR in Example 10;

[0072] Figure 14 This is a diagram of the cytotoxicity test results in Example 11. DETAILED DESCRIPTION

[0073] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0074] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0075] The mice used in the present invention are male C57BL / 6 mice (Model Organisms Institute, Nanjing University), which are generally recognized in the art. + / - Heterozygous offspring obtained by crossing founder mice with wild-type mice, WT represents wild-type control mice from the same littermates. The bleomycin (BLM)-induced mouse pulmonary fibrosis model was a classic modeling method.

[0076] The methods for obtaining the above HE mice and WT mice are as follows:

[0077] Ttc3 + / - The C57BL / 6J strain, a Ttc3 knockout mouse strain generated from C57BL / 6J mice using CRISPR-Cas9 technology, was provided by the Institute of Model Organisms at Nanjing University. The specific preparation method is as follows: sgRNA-mediated Cas9 protein cleaves the 188-bp fragment of Exon 14 in the Ttc3 gene of C57BL / 6J mouse fertilized eggs, causing DNA double-strand breaks, which trigger non-homologous end-joining repair and subsequently cause a frameshift in subsequent gene translation (the knockout portion contains a 124-bp fragment in the mRNA), leading to premature translation termination, thereby achieving systemic Ttc3 gene knockout in mice.

[0078] 1. Change Ttc3 + / - The C57BL / 6J strains were hybridized to obtain hybrid offspring.

[0079] 2. Extract the genomic DNA of each hybrid offspring obtained in step 1 and use it as a template to perform PCR amplification using a primer pair consisting of primer TTC3-F: 5'-CAGGCGTGGGACGTTTTC-3' and primer TTC3-R: 5'-AAGGTTGCCTGATGTGGGAG-3' (primer TTC3-F and primer TTC3-R are based on the knockout fragment amplification) to obtain PCR amplification products. Then, make the following judgment: If a PCR amplification product contains two DNA fragments with sizes of 412 bp and 224 bp, respectively, the hybrid offspring is a heterozygote and is named Ttc3. + / - mice; if a PCR amplification product contains a DNA fragment with a size of 412 bp, the hybrid offspring is a wild-type mouse of the same litter and is named Ttc3 + / + If a PCR amplification product contains a DNA fragment with a size of 224 bp, the hybrid offspring is a mouse with complete knockout of the Ttc3 gene, that is, a homozygous Ttc3 knockout mouse, named Ttc3 - / - mouse.

[0080] 3. Ttc3 obtained in step 2 + / - Mice and Ttc3 + / + Mice were hybridized and two types of mice were obtained from the hybrid offspring, namely Ttc3 + / - mice (subsequently named HE mice) and Ttc3 + / + Mice (subsequently named WT mice). The genomic DNA of HE mice and WT mice were extracted respectively and used as templates, and PCR amplification was performed using a primer pair consisting of primer TTC3-F: 5'-CAGGCGTGGGACGTTTTC-3' and primer TTC3-R: 5'-AAGGTTGCCTGATGTGGGAG-3' to obtain PCR amplification products. The PCR amplification product of HE mice contained two DNA fragments with sizes of 412bp and 224bp, respectively. The PCR amplification product of WT mice contained one DNA fragment with a size of 412bp. The difference between HE mice and WT mice is that the Ttc3 gene in WT mice has two copies, while the Ttc3 gene in HE mice has a single copy, that is, HE mice are mice with Ttc3 gene knockdown, and WT mice are mice with normal Ttc3 gene.

[0081] Example 1: Synthesis of TM2

[0082] according to Figure 1 The reaction scheme shown in the figure synthesizes TM2, (i) N-Boc-6-aminohexanoic acid, EDCI, DMAP, THF, 0 to 25°C, 4h, 80%; (ii) HCl / EA, 0°C, 1h, 81%; (iii) S-

[12] aneN3, HBTU, DIPEA, DCM, 0 to 25°C, 4h, 40%; (iv) HCl / EA, 0°C, 1h, 75%.

[0083] The specific steps are as follows:

[0084] Synthesis of TM-2 compound: 0.30 g of TM-1 compound (0.34 mmol), N-Boc-6-aminocaproic acid (69 mg, 0.34 mmol) and DMAP (21 mg, 0.17 mmol) were dissolved in 20 ml of THF and stirred under argon for 30 minutes. 78 mg of EDCI (0.41 mmol) was added at 0°C. The mixture was stirred at 25°C for 4 hours. 50 ml of DCM was added, washed with saturated brine, and the organic solvent was removed in vacuo. The residue was separated and purified by column chromatography (PE:EA=4:1, v / v) to give the product as a light yellow solid (0.28 g, 80%).

[0085] The structural identification data are as follows:

[0086] 1H NMR (400MHz, CDCl3) δ8.24(d,J=8.2Hz,2H),7.60(d,J=6.9Hz,2H),7.21(d,J=6.6Hz,2H),7.13 -6.80(m,14H),6.65-6.55(m,4H),5.26(d,J=4.0Hz,2H),5.14(s,2H),5.03(s,2H),4.54(d,J= 8.8Hz,2H),3.85(t,J=6.4Hz,2H),3.07(t,J=6.8Hz,2H),2.36-2.26(m,5H),1.72(p,J=6.5Hz, 2H),1.62(p,J=6.5Hz,2H),1.48-1.39(m,13H),1.31-1.24(m,18H),0.87(d,J=7.9Hz,3H).13C NMR (101MHz, CDCl3) δ173.2,168.7,157.7,156.0,153.5,147.7,144.2,144.1,140.3,13 9.1,137.7,135.9,134.9,132.6,132.5,132.3,132.0,131.9,131.4,129.5,128.6,127.7 ,127.6,126.3,123.9,113.8,113.7,113.6,79.1,75.6,67.8,65.4,62.1,61.1,40.4,34. 1,31.9,29.8,29.7,29.6,29.4,29.3,28.4,26.3,26.1,24.5,22.7,20.8,14.1.ESI-HRMS calcd.for C67H80N2O11(M+H+)1089.5835,found1089.5840(M+H+).

[0087] Synthesis of TM-3: Dissolve TM-2 (0.20 g, 0.19 mmol) in 10 mL of 2N ethyl acetate saturated with hydrogen chloride. Stir at 0°C for 1 h. Remove the organic solvent in vacuo. The residue is purified by recrystallization (PE:EA = 30:1, v / v) to afford the product as a pale yellow solid (0.15 g, 81%).

[0088] The structural identification data are as follows: 1H NMR (600 MHz, DMSO-d6) δ8.05 (d, J = 8.1 Hz, 2H), 7.87 (s, 3H), 7.51 (d, J = 8.5 Hz, 2H), 7.02 (d, J = 4.0 Hz, 2H), 6.95-6.82 (m, 6H), 6.79-6.79 (m, 4H), 6.65-6.55 (m, 4H), 6.50-6.35 (m, 4H), 4.99 (d, J = 2.9 Hz, 2H), 4.89 (s, 2H), 4.8 4(s,2H),4.53(d,J=9.0Hz,2H),3.61(t,J=6.5Hz,2H),2.29(t,J=2.1Hz,2H),2.10(t,J=8.0Hz,2H),2.06(s,3 H),1.41(t,J=7.3Hz,2H),1.37-1.25(m,4H),1.15-1.09(m,2H),1.08-0.95(m,18H),0.62(t,J=7.0Hz,3H).13C NMR(151MHz,DMSO-d6)δ172.6,168.6,157.2,155.9,153.0,147.1,144.9,143.8,143.7,13 9.6,139.0,136.5,135.4,134.0,131.9,131.1,130.8,129.4,128.8,128.1,127.8,127.7,1 26.4,126.3,123.6,113.9,113.8,113.7,113.6,79.3,74.9,74.6,67.3,64.6,61.5,60.8,3 8.5,33.2,31.3,29.1,29.0,28.8,28.7,26.6,25.6,25.3,23.9,22.1,20.4,14.0.ESI-HRMS calcd.for C62H72N2O9(M+H+)989.5311, found 989.5308(M+H+).

[0089] Synthesis of TM-4: Compound TM-3 (0.14 g, 0.14 mmol) and S-

[12] aneN3 (0.15 g, 0.14 mmol) were dissolved in 20 mL of DCM under argon. HBTU (64 mg, 0.17 mmol) and DIPEA (45 mg, 0.35 mmol) were added at 0°C and stirred at 25°C for 10 h. The mixture was diluted with an appropriate amount of DCM, washed with saturated brine, and removed in vacuo. Purification by column chromatography (EA:MeOH = 20:1, v / v) afforded a white solid (0.11 g, 40%).

[0090] The structural identification data are as follows: 1H NMR (600 MHz, CDCl3) δ8.23 (d, J = 7.5 Hz, 2H), 7.70 (s, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.32 (s, 1H), 7.24-7.17 (m, 2H), 7.12-6.96 (m, 12H), 6.95-6.85 (m, 4H), 6.64-6.55 (m, 4H), 5.52 (s, 4H), 5.26 (d, J = 6.4 Hz, 2H), 5.13 (s, 2H), 5.03 (s, 2H), 4.54 ( d,J=14.0Hz,2H),4.12(q,J=7.1Hz,2H),3.86(t,J=6.6Hz,3H),3.37-3.27(m,18H),2.62(s,8H),2.32-2.29(m,5H),1.9 3-1.81(m,14H),1.72(p,J=6.9Hz,2H),1.66-1.56(m,4H),1.44(s,36H),1.28-1.23(m,16H),0.87(t,J=7.1Hz,3H).13C NMR (151MHz, CDCl3) δ173.2,168.7,166.2,157.7,156.4,156.0,153.5,147.7,144.3,144.1,140.3,139.1 ,137.7,136.9,136.4,135.9,134.9,132.6,132.5,132.0,131.9,131.4,131.3,130.0,129.4,128.6,127. 7,127.7,127.6,127.0,126.3,123.9,113.8,113.7,113.6,79.5,75.6,67.8,65.4,62.0,61.2,53.4,49.5 ,47.3,45.5,44.3,40.0,34.1,31.9,29.7,29.6,29.4,28.5,26.4,26.0,24.5,22.7,20.8,14.1.ESI-HRMS calcd.for C115H156N14O18([M+2H+] / 2)1011.5934, found 1011.5939([M+2H+] / 2).

[0091] Synthesis of TM2: Compound TM-4 (0.10 g, 0.05 mmol) was dissolved in 10 mL of 2N ethyl acetate saturated with hydrogen chloride, stirred at 0°C for 1 h, and the organic solvent removed in vacuo. Purification by recrystallization (PE:EA = 30:1, v / v) afforded a white solid (67 mg, 75%).

[0092] The structural identification data are as follows: 1H NMR (600 MHz, DMSO-d6) δ9.36 (br, 8H), 8.46 (s, 1H), 8.24 (d, J = 8.6 Hz, 2H), 8.18 (s, 1H), 7.79 (s, 2H), 7.72 (d, J = 8.5 Hz, 2H), 7.30 (s, 1H), 7.21 (d, J = 2.7 Hz, 2H), 7.15-7.04 (m, 6H), 6.98-6.92 (m, 4H), 6.88-6.81 (m, 4H), 6.70-6.63 (m, 4H), 5.64 (s, 4H), 5.20 (d, J = 3.6 Hz, 2H), 5.11 (s, 2H), 5. 05(s,2H),4.72(d,J=12.2Hz,2H),4.23(s,12H),3.86(dt,J=6.6,3.3Hz ,2H),3.24-3.16(m,10H),3.10(s,8H),2.72(br,4H),2.32(t,J=7.5Hz, 2H),2.28(s,3H),2.10(s,4H),1.91(s,8H),1.68-1.61(m,2H),1.56-1. 48(m,4H),1.39-1.33(m,2H),1.32-1.22(m,18H),0.88-0.84(m,3H).13C NMR(101MHz,DMSO-d6)δ172.8,168.6,165.3,157.2,156.0,153.0,147.1,144.9,143.8,139 .7,139.1,136.5,135.8,135.5,134.1,131.9,131.1,130.8,129.5,128.8,128.1,127.8,126 .9,126.4,123.7,113.9,113.8,113.7,74.9,67.3,64.6,61.6,60.8,52.6,48.5,46.4,41.0 ,33.4,31.4,29.1,29.0,28.8,26.0,25.6,24.2,22.2,20.4,17.5,14.0.ESI-HRMScalcd.for C95H124N14O10([M+2H+] / 2)811.9902,found 811.9910([M+2H+] / 2).

[0093] The obtained compound was confirmed to be the target compound by structural identification.

[0094] Example 2: Test characterization experiment of TM2

[0095] The morphology of TM2 was characterized by transmission electron microscopy (TEM) (FEITalos F200S, Thermo Scientific). The particle size and polydispersity index (PDI) of TM2 in PBS (pH = 7.4) solution were determined by dynamic light scattering (DLS) (Malvern Zeta sizer NanoS90, UK).

[0096] The TM2 solution prepared in Example 1 was dispersed in a PBS (pH = 7.4) buffer system to obtain a diluted TM2 solution with a concentration of 5 μM. 10 μL of the diluted TM2 solution was dropped onto the amorphous carbon film copper mesh support. After drying, the solution was observed using a transmission electron microscope (FEITalos F200S, Thermo Scientific). Figure 2-3 As shown, the TM2 nanoparticles exhibited good dispersion and a relatively uniform particle size, with an average particle diameter of 195 nm and a PDI within a reasonable range. Transmission electron microscopy (TEM) clearly revealed that TM2 formed spherical nanoparticles with a regular morphology and good dispersion, approximately 200 nm in size, consistent with the DLS results.

[0097] Example 3: Preparation of Nanoparticles TMD2

[0098] Synthesis of TMD2: 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) is a synthetic analog of phospholipid PE, containing an 18:1 ratio of fatty acids at the sn-1 and sn-2 positions. This auxiliary lipid acts as an emulsifier to facilitate transmembrane transport of RNA-liposome complexes. Its use in combination with positively charged micelles as a non-viral gene delivery method effectively improves nucleic acid transfection efficiency. Previous research by our group has shown that transfection is most effective when the compound is combined with DOPE at a 1:2 molar ratio. Here, TM2 and the auxiliary reagent DOPE were similarly combined at a 1:2 molar ratio to generate new nanoparticles, TMD2. The specific method is as follows: TM2 and DOPE were completely dissolved in CDCl3 at a 1:2 molar ratio. The solvent was removed in vacuo to obtain a thin DOPE / TMD film (TMD2). The DOPE / TMD film was then hydrated with PBS (pH 7.4) to obtain a TMD2 nanoparticle solution. The diameter of the obtained nanoparticles TMD2 is 173.9 nm, which is slightly smaller than the micelle nanoparticles formed by TM2. The addition of auxiliary reagents further makes the nanoparticle structure compact.

[0099] The prepared TMD2 was tested and characterized:

[0100] The morphology of TMD2 was characterized by transmission electron microscopy (TEM) (FEITalos F200S, Thermo Scientific). The particle size and polydispersity index (PDI) of TMD2 in PBS (pH = 7.4) solution were determined by dynamic light scattering (DLS) (Malvern Zeta sizer NanoS90, UK).

[0101] 10 μL of the TMD2 solution prepared above was dispersed in 1 mL of PBS to obtain a diluted TMD2 solution. 10 μL of the diluted TMD2 solution was dropped onto the amorphous carbon film copper mesh support. After drying, the solution was observed using a transmission electron microscope (FEITalos F200S, Thermo Scientific). Figure 4-5 As shown, the TM2 nanoparticles exhibited good dispersion and a relatively uniform particle size, with an average particle diameter of 173.9 nm and a PDI within a reasonable range. Transmission electron microscopy (TEM) clearly revealed that TMD2 formed spherical nanoparticles with a regular morphology and good dispersion, with a particle size of approximately 180 nm, consistent with the DLS results.

[0102] Example 4: siRNA packaging efficiency experiment

[0103] Aggregation results of siRNA by different concentrations of TM2 and TMD2 nanoparticles in DEPC water (mass ratio); [siRNA] = 500 nM, nanoparticle concentrations of 4, 11, 22, 33, and 44 μM, respectively. The siRNA used in this example is a murine Ttc3 gene interfering siRNA (siTtc3), with the following sequences: sense 5'-AGGAUCUAAUUCAGCAGCAUGUAAA-3' (SEQ ID No. 3), antisense 5'-UUUACAUGCUGCUGAAUUAGAUCCU-3' (SEQ ID No. 4).

[0104] siRNA was complexed with nanoparticles TM2 (mass of TM2 contained) and nanoparticles TMD2 (mass of TM2 + DOPE contained) at a weight ratio of 1:2 to 24 and incubated at room temperature for 30 minutes. The samples were then mixed with loading buffer (TaKaRa) and electrophoresed on a 1% agarose gel at 120V for 10 minutes. Markers (Sangon Biotech) were used as ladders. Finally, the gels were imaged and bands analyzed using the ChemiDoc system (Bio-Rad, USA).

[0105] See the results Figure 6 .Depend on Figure 6 As shown, TMD2 exhibited superior encapsulation efficiency compared to TM2. At a mass ratio of 12:1, TMD2 completely encapsulated the siRNA. As the TMD2-to-siRNA mass ratio increased, Ttc3 gene expression levels decreased. Therefore, TMD2 is an excellent siRNA carrier, successfully delivering the siRNA into cells and reducing Ttc3 mRNA levels.

[0106] Example 5: Preparation of siTtc3@TMD2 nanoparticles

[0107] The siRNA solution (same as in Example 4) was mixed with the nanoparticle TMD2 solution obtained in Example 3 (so that the mass ratio of siRNA to nanoparticle TMD2 (the sum of the mass of TM2 and DOPE) was 1:24), and incubated at room temperature for 15 minutes to obtain siTtc3@TMD2 nanoparticles.

[0108] Example 6: Systemic distribution of siTtc3@TMD2 nanoparticles in mice

[0109] Based on the good siRNA encapsulation ability of the nanoparticle carrier prepared in Example 5, we observed the distribution of the nanoparticle system in mice. TMD2@Ttc3 was administered via the oral cavity. The distribution of the mice was observed by whole-body imaging on days 1, 3, 5, and 7 after administration. The main organs were harvested, and the fluorescence images were as follows: Figure 7-8 As shown. The fluorescently labeled siTtc3@TMD2 used was Cy5.5-labeled siTtc3 encapsulated in TMD2. The preparation method was the same as in Example 4, except that the siRNA in Example 4 was replaced with Cy5.5-labeled siRNA. To examine the biodistribution of the nanoparticles, mice were anesthetized and imaged using an in vivo imaging system (IVIS Spectrum, PerkinElmer, USA) 1, 3, 5, and 7 days after inhalation of Cy5.5-labeled siTtc3@TMD2. Afterwards, the mice were sacrificed, and their organs were removed and imaged on the IVIS system.

[0110] Cy5.5-labeled siRNA and nanoparticles were administered to mice via endotracheal intubation, and their biodistribution in the lungs was observed. Fluorescent signal accumulated primarily in the lungs. No enrichment was observed in other major organs, and siRNA remained in the lungs for 7 days, indicating that siTtc3@TMD2 is capable of accumulating in the lungs and releasing siRNA.

[0111] Example 7: Effect of siTtc3@TMD2 Nanoparticles on Body Weight Change in Mice

[0112] Eight-week-old C57BL / 6 mice were randomly divided into 6 groups (n=5-7): (1) control group (WT-PBS): PBS was instilled into wild-type mice by endotracheal intubation; (2) experimental control group (HE-PBS): PBS was instilled into heterozygous mice by endotracheal intubation; (3) pulmonary fibrosis model group (WT-BLM): bleomycin (BLM: 2.5 mg / kg) was instilled into wild-type mice by endotracheal intubation to induce pulmonary fibrosis; (4) pulmonary fibrosis model experimental group (HE-BLM): bleomycin (BLM: 2.5 mg / kg) was instilled into heterozygous mice by endotracheal intubation to induce pulmonary fibrosis; (5) pulmonary fibrosis model treatment group (WT-BLM-siTtc3@TMD2): TMD2@Ttc3 nanoparticles (siRNA: 0.75 mg / kg) and bleomycin (BL) were instilled into wild-type mice by endotracheal intubation. M: 2.5 mg / kg) to induce pulmonary fibrosis; (6) Pulmonary fibrosis model control treatment group (WT-BLM-TMD2@NC): TMD2@NC nanoparticles (NC-siRNA: 0.75 mg / kg) and bleomycin (BLM: 2.5 mg / kg) were instilled into wild-type mice by tracheal intubation to induce pulmonary fibrosis; free drinking water and diet were allowed, and the weight of mice was monitored from three days before modeling, and weighed continuously for 21 days. Nanoparticles were given on the fourth day (pulmonary fibrosis model treatment group and pulmonary fibrosis model control treatment group). Three days later, all groups were given BLM / PBS modeling and free drinking water and diet. Free drinking water and diet were allowed from day 2 to day 14 after modeling, and mice were killed 14 days after modeling (during these 14 days, nanoparticles were given every five days in the pulmonary fibrosis model treatment group and the pulmonary fibrosis model control treatment group, i.e., nanoparticles were given on day 5 and day 10 of modeling, respectively).

[0113] The preparation method of the "TMD2@NC nanoparticles" used in the above experiment is basically the same as that of the siTtc3@TMD2 nanoparticles in Example 5, except that the siRNA in Example 5 is replaced by NC-siRNA (negative control siRNA). The sequence of the NC-siRNA is as follows: sense 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID No. 3), antisense 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID No. 4).

[0114] Depend on Figure 9As can be seen, after endotracheal intubation and instillation of PBS / BLM, the WT-BLM group mice lost significantly more weight than the HE-PBS group, and weight recovery was slow during the 14-day modeling process. siTtc3@TMD2 significantly mitigated the rapid weight loss in mice with pulmonary fibrosis and improved the overall survival rate of mice with bleomycin-induced fibrosis. This suggests that the nanoparticles can inhibit Ttc3 gene expression and have a significant impact on mouse weight changes and survival during pulmonary fibrosis.

[0115] Example 8: Effect of siTtc3@TMD2 Nanoparticles on the Progression of Pulmonary Fibrosis in Mice

[0116] Lung tissues from mice sacrificed in the above experiment were frozen and sectioned. The specific steps were as follows: (1) Lung tissues were fixed with 4% paraformaldehyde at 4°C for 24 h; (2) Lung tissues were dehydrated and precipitated in 10%, 20%, and 30% sucrose solutions, respectively; (3) Lung tissues were embedded in OCT at -28°C, and then sectioned at 8 μm and mounted on glass slides.

[0117] The sections were stained with hematoxylin and eosin. The specific steps were as follows: (1) Bake the sections in a 65°C oven for 1 hour and rinse with ddH2O; (2) Stain the nuclei with hematoxylin solution and incubate at room temperature for 5 to 10 minutes; (3) Wash with ddH2O for 5 minutes; (4) Differentiate with 0.1% hydrochloric acid-ethanol for 10-30 seconds; (5) Rinse with ddH2O for 1 minute; (6) Decolorize and blue in PBS for 30-60 seconds; (7) Rinse with ddH2O for 5 minutes; (8) Wash with 95% ethanol for 5-10 seconds; (9) Counterstain with eosin solution for 30 seconds to 2 minutes. Note: Eosin is highly soluble in water. If it is overstained, rinse with tap water; (10) Dehydrate with 95% ethanol twice, each for 5 minutes; (11) Clear with xylene twice, each for 1-2 minutes, and mount with neutral resin.

[0118] The sections were taken for Masson staining. The specific operation steps were as follows: (1) The sections were immersed in mordant solution (covered), and mordantized at room temperature overnight or placed in a 57℃-60℃ incubator for 1 hour, and then rinsed with running water for 10 minutes; (2) The sections were dripped with lapis lazuli blue stain for 2-3 minutes, and washed with water twice, each time for 10-15 seconds; (3) The sections were dripped with Mayer's hematoxylin stain for 2-3 minutes, and washed with distilled water twice, each time for 10-15 seconds; (4) Differentiation was performed with acidic ethanol differentiation solution. After a few seconds, the tissue turns completely red, then the differentiation is terminated by washing with water, and the tissue is rinsed with distilled water for 10 minutes. (5) The tissue is stained with Ponceau fuchsin solution for 10 minutes, and then washed twice with distilled water, each time for 10-15 seconds. (6) The tissue is treated with phosphomolybdic acid solution for about 10 minutes. (7) The supernatant is discarded, and the sections are not washed with water. Aniline blue staining solution is added directly for 5 minutes. (8) After washing the aniline blue solution with a weak acid solution, the sections are covered with a weak acid working solution for 2 minutes. (9) Dehydrate with 95% ethanol for 30 seconds. Dehydrate twice with anhydrous ethanol, the first time for 30 seconds, and the second time for 1 minute. (10) Clear with xylene twice, each time for 1-2 minutes. Seal with neutral gum.

[0119] See the results Figure 10-11 . The WT-BLM group showed severe pulmonary fibrosis, which was characterized by the loss of normal alveolar structure, obvious disordered thickening of the alveolar septa, collapse of the alveolar spaces, accompanied by tissue inflammatory infiltration and collagen deposition of fibroblasts. Compared with the BLM or siNC@TMD2 control group, the siTtc3@TMD2 treatment group significantly reduced the severity of BLM-induced fibrosis by preserving the alveolar epithelial structure. Masson staining showed that collagen deposition and parenchymal destruction were significantly reduced after inhalation of siTtc3@TMD2. At the same time, data analysis was performed to judge the degree of fibrosis by the proportion of air in the alveoli. The results also showed that the air proportion of siTtc3@TMD2 was high and the degree of pulmonary fibrosis was low. It can be seen that siTtc3@TMD2 can reduce BLM-induced pulmonary fibrosis in mice.

[0120] Example 9: Sensitivity of TMD2 Nanoparticles to Nitroreductase

[0121] It has been reported that the concentration of nitroreductase in cancer cells is approximately 400 pg / mL, which is indeed higher than that in normal cells. Using HEK293T cells as a control, fluorescence images of HeLa and A549 cells incubated with TMD2 prepared in Example 3 for different times (2, 4, 6, and 8 hours) were studied.

[0122] See the results Figure 12The results showed that the fluorescence signal in HeLa and A549 cells gradually increased with the extension of culture time, while there was only a slight change in HEK293T. Semi-quantitative analysis of the mean fluorescence intensity using Image J software showed that the complex showed an intensity difference of almost 3 times in HeLa and A549 compared with HEK293T (Figure B). This indicates that TMD2 releases tetraphenylethylene fragments by breaking in HeLa and A549 cells overexpressing nitroreductase, leading to fluorescence recovery.

[0123] Based on this, TMD2 nanoparticles are used to encapsulate siRNA targeting lung cancer-related genes and delivered to the lungs through tracheal administration or aerosolization, which can achieve specific targeted delivery to lung cancer cells.

[0124] Example 10: Responsiveness of TM2 to NTR

[0125] Compound TM2 exhibits very weak fluorescence intensity in PBS buffer because the nitro group promotes photoinduced energy transfer (PET) and quenches fluorescence emission. TM2 (10 μM) was analyzed after the addition of NADH (100 μg / mL) and various concentrations of NTR (0-20 μg / mL). The fluorescence spectra are shown in the figure. To examine the high selectivity of TM2 for nitroreductase, TM2 (10 μM) was treated with various analytes in PBS, including glutathione (GSH), dithiothreitol (DTT), cysteine ​​(Cys), ascorbic acid (Vc), NaClO, H2O2, ·OH, ONOO-, O2 (100 μM), and artificial hypoxia (nitroreductase (20 μg / mL) with NADH (100 μg / mL)).

[0126] After adding NTR to a TNN (10 μM) solution containing 100 μg / mL NADH, a good linear relationship was shown between the fluorescence intensity and the NTR concentration (0-20 μg / mL). The detection limit of TM2 for NTR was 14.07 ng / mL (signal-to-noise ratio of 3:1), indicating that TM2 micelles have good sensitivity to NTR. Figure 13 Figure C shows that fluorescence at 480 nm is significantly enhanced only in the presence of NADH and nitroreductase, while treatment with other analytes has little effect on the compound's fluorescence. In summary, TM2 exhibits higher selectivity for nitroreductase than other relevant biostimulants.

[0127] Example 11: Cytotoxicity test of siTtc3@TMD2 nanoparticles

[0128] The cytotoxicity of the nanomedicine was detected using the Cell Counting Kit-8 method. The Cell Counting Kit-8, also known as CCK8 (or WST-8), is a rapid, highly sensitive assay for cell proliferation and cytotoxicity based on WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylphenyl)-2H-tetrazolium monosodium salt).

[0129] WST-8 works by reducing the product to form a highly water-soluble orange-yellow formazan in the presence of an electron-coupling reagent. The color intensity is directly proportional to cell proliferation and inversely proportional to cytotoxicity. The OD value is measured at a wavelength of 450 nm using a microplate reader, indirectly reflecting the number of viable cells.

[0130] 1. Prepare 100 μL of cell suspension in a 96-well plate. Pre-incubate the plate in an incubator overnight (37°C, 5% CO). Incubate the cells with siTtc3@TMD2 and siNC@TMD2 in serum-free DMEM for 4 hours, followed by a further incubation in complete medium for 20 hours. NIH-3T3 cells were then starved in serum-free DMEM for 24 hours. The starved cells were then cultured in DMEM supplemented with TGF-β1 (10 ng / ml) and 1% FBS for 24 hours.

[0131] 2. Add 10 mL of CCK solution to each well, taking care not to create bubbles in the wells.

[0132] 3. Incubate the culture plate in an incubator for 1-4 hours.

[0133] 4. Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0134] See the results Figure 14 After testing, it was found that TGF-β1 treatment of cells caused cell proliferation, while the addition of siTtc3@TMD2 and siNC@TMD2 had no obvious toxicity to cell proliferation, indicating that an appropriate amount of nanoparticles TMD2 carrying siRNA has no toxic effect on cells.

[0135] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A lipid molecule TM2, whose structural formula is shown in Formula I: 。 2. The method for preparing the lipid molecule TM2 according to claim 1, comprising the following steps: 1) Under the catalytic action of DMAP and EDCI, TM-1 compound and N-Boc-6-aminocaproic acid are subjected to esterification reaction to obtain TM-2 compound; 2) Under acidic conditions, deprotect the BOC in TM-2 to obtain TM-3; 3) In the presence of HBTU and DIPEA, TM-3 compound is subjected to condensation reaction with S-[12]aneN3 to obtain TM-4 compound; 4) Under acidic conditions, deprotecting the BOC in the TM-4 compound to obtain the TM2 compound.

3. The preparation method according to claim 2, wherein: In step 1), the esterification reaction is carried out in an organic solvent, and the organic solvent is THF; And / or, in step 1), the molar ratio of the TM-1 compound, N-Boc-6-aminocaproic acid, DMAP, and EDCI is 1:1:0.5:1.2, respectively; And / or, in step 1), the reaction temperature of the esterification reaction is 0-25°C, and the reaction is stirred at 25°C for 4 hours; And / or, in step 3), the condensation reaction is carried out in an organic solvent, and the organic solvent is dichloromethane; And / or, in the step 3), the molar ratios of the compound TM-3, S-[12]aneN3, HBTU, and DIPEA are 1:1:1.21:2.5 respectively; And / or, in step 3), the reaction temperature of the condensation reaction is 0-25°C, and the reaction is stirred at 25°C for 4 hours; And / or, in step 2) and step 4), the acidic condition is provided by a hydrogen chloride saturated ethyl acetate solution.

4. A nanoparticle carrier, the raw materials of which include TM2 and DOPE according to claim 1; The molar ratio of TM2 to DOPE is 1:

2. 5 . A nucleic acid-encapsulated nanoparticle comprising the nanoparticle carrier according to claim 4 and a nucleic acid molecule.

6. The nucleic acid-encapsulated nanoparticle according to claim 5, characterized in that: The nucleic acid molecule includes one or more of a messenger nucleic acid molecule, a small interfering nucleic acid molecule, a micronucleic acid molecule, a small activating nucleic acid molecule, an antisense oligonucleotide molecule or an aptamer; and / or, the mass ratio of the sum of the masses of TM2 and DOPE in the nanoparticles to the mass of the nucleic acid molecule is (1-50):1; And / or, the particle size of the nucleic acid-encapsulated nanoparticles is 50 to 200 nm.

7. The method for preparing the nucleic acid-loaded nanoparticles according to claim 5 or 6, comprising the following steps: 1) dissolving the TM2 and DOPE in an organic solvent, and removing the solvent in vacuo to obtain a DOPE / TM2 film; 2) hydrating the DOPE / TM2 membrane to obtain a nanoparticle TMD2 solution; 3) Mix the nucleic acid molecule solution with the nanoparticle TMD2 solution and incubate at room temperature.

8. A product for treating lung diseases, comprising the nucleic acid-encapsulated nanoparticles according to claim 5 or 6; the product is a pharmaceutical preparation; and the lung disease is pulmonary fibrosis or lung cancer.

9. The product for treating lung diseases according to claim 8, characterized in that: The pharmaceutical preparation is administered through the trachea. And / or, the pharmaceutical preparation is a tracheal administration preparation; And / or, the product further comprises a pharmaceutically acceptable carrier.

10. The product for treating lung diseases according to claim 9, characterized in that: The tracheal administration is by aerosol inhalation or dry powder inhalation; And / or, the tracheal administration preparation is an aerosol inhalation or a dry powder inhalation.

11. The product for treating lung diseases according to any one of claims 8 to 10, characterized in that: The nucleic acid is siRNA targeting pulmonary fibrosis-related genes; Alternatively, the nucleic acid is siRNA targeting lung cancer-related genes.

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