Nanoparticles for treating lung diseases as well as preparation method and application of nanoparticles

Abstract

The invention discloses nano-particles for treating lung diseases as well as a preparation method and application of the nano-particles. The nanoparticles for treating lung diseases provided by the invention comprise a nanoparticle carrier and nucleic acid molecules, the nano-particle carrier comprises the following raw materials: TM2 and DOPE in a molar ratio of 1: 2. The nucleic acid entrapped nano-particles provided by the invention can efficiently deliver nucleic acid molecules to the lung and down-regulate pathogenic protein expression; compared with the prior art, the preparation method has the advantages that deposition of the medicine in the lung can be obviously increased, the effect after administration is effectively guaranteed, meanwhile, the preparation method has the advantages of being simple and convenient in administration mode, free of trauma, high in safety and the like, is a high-innovativeness efficient lung deposition mode, and has a good application prospect in the aspect of treatment of related diseases of the lung.

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 attracted much attention in recent years, mainly because of its great potential in the treatment of respiratory diseases and systemic diseases. Compared with traditional drug delivery methods, nanoparticles can efficiently deliver drugs, especially gene drugs, through the lungs, providing higher bioavailability, more precise targeting, and prolonged drug release time. The lungs have a large gas exchange surface, a rich vascular network, and a thin air-blood barrier, making them an ideal target for nanoparticle drug delivery. In addition, the lungs as a drug delivery target can avoid the first-pass effect and significantly improve the efficiency of gene therapy. The advantage of nanoparticles in gene therapy is that they can protect easily degradable nucleic acids (such as DNA, mRNA, siRNA) from enzymatic hydrolysis in vivo, and achieve targeted delivery to specific cells through surface modification. Especially for diseases such as lung cancer, pulmonary fibrosis, cystic fibrosis, chronic obstructive pulmonary disease (COPD), nanoparticles can accurately deliver gene editing tools (such as CRISPR-Cas9) or RNA interference molecules to lesion cells, repair gene mutations or inhibit 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 and improve 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, showing great potential in the local treatment of respiratory diseases. Summary of the invention

[0004] The object of the present invention is to provide a nanoparticle containing a nucleic acid. The nanoparticle containing a nucleic acid is suitable for oral tracheal administration or delivery of RNA or therapeutic genes for targeted gene therapy to the lungs in the form of aerosol inhalation, 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 solution:

[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, the TM-1 compound and N-Boc-6-aminocaproic acid are subjected to an esterification reaction to obtain the 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 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, and the organic solvent 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] Further, the step 1) is specifically as follows: dissolving the TM-1 compound, N-Boc-6-aminocaproic acid and DMAP in THF, stirring for 30 minutes in argon; 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 vacuum 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, and the organic solvent 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 as follows: dissolve the compound TM-3 and S-

[12] aneN3 in DCM under argon, add HBTU and DIPEA at 0°C, and stir the reaction at 25°C for 10 hours; add an appropriate amount of DCM to dilute, wash with saturated brine, and remove 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 of hydrogen chloride.

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

[0028] The nanoparticle carrier provided by the present invention has raw materials including the 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 carrier 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 a siRNA targeting Ttc3, and the sequence of the siRNA is 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 nanoparticle to the mass of the nucleic acid molecule is (1-50):1.

[0035] Furthermore, the particle size of the nanoparticles encapsulating nucleic acid 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.

[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 comprises 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 administration method of the pharmaceutical preparation is preferably tracheal administration, such as aerosol inhalation or dry powder inhalation.

[0049] Furthermore, the drug preparation is a tracheal administration preparation, such as 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, and the like.

[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 via tracheal administration or atomization, thereby achieving specific targeted delivery to lung cancer cells.

[0054] When the drug is used to treat chronic obstructive pulmonary disease (COPD), it encapsulates siRNA targeting COPD-related genes and is delivered to the lesioned area of ​​the lungs by aerosol inhalation. Studies have shown that the system can efficiently deliver siRNA to the lungs and significantly reduce 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 lesioned area of ​​the lung by aerosol inhalation. The system can efficiently deliver siRNA to the lungs. 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 by aerosol inhalation. The 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 (siRNA) RNA for Ttc3 are administered to mice via the oral trachea. In the case of oral tracheal administration, the efficiency of Ttc3 silencing is high in the lungs (>70% knockdown), and can effectively prevent / eliminate the pathological alveolar septa of BLM-induced pulmonary fibrosis in mice, with obvious disordered thickening, collapse of alveolar spaces, and collagen deposition of tissue inflammatory infiltration and fibroblasts. The method of small animal in vivo imaging is used to detect the amount of deposition of nanoparticles containing nucleic acids into the lungs and 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 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-traumatic, 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 The synthetic route for the molecule TM2;

[0060] Figure 2 is a transmission electron microscope (TEM) image of the nanoparticle 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 nanoparticle TMD2 in Example 3;

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

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

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

[0066] Figure 8 This is an in vitro fluorescence image of the main organs after siTtc3@TMD2 lung perfusion in Example 6;

[0067] Fig. 9 This is a graph showing the weight changes of mice in Example 7;

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

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

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

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

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

[0073] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0074] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

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

[0076] The method for obtaining the above HE mice and WT mice is as follows:

[0077] Tc3 + / - The C57BL / 6J strain is a Ttc3 knockout mouse constructed based on C57BL / 6J mice using CRISPR-Cas9 technology, and was provided by the Institute of Model Organisms of Nanjing University. The specific preparation method is as follows: Cas9 protein is used to cut the 188bp fragment of Exon14 in the Ttc3 gene of the fertilized egg of C57BL / 6J mice mediated by sgRNA, resulting in DNA double-strand breaks, which in turn causes non-homologous end joining repair, and then causes subsequent gene translation time shift (knockout part contains a 124bp fragment in mRNA), causing translation to terminate prematurely, thereby achieving the knockout of the Ttc3 gene in the mouse body.

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

[0079] 2. The genomic DNA of each hybrid offspring obtained in step 1 was extracted and used 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 knockout fragment amplification) to obtain PCR amplification products, and then the following judgment was made: 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, named Ttc3 + / + mice; if a PCR amplification product contains a DNA fragment with a size of 224 bp, the hybrid offspring is a mouse with a 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 + / + The mice were crossed and two types of mice were obtained from the hybrid offspring, namely Ttc3 + / - mice (hereinafter named HE mice) and Ttc3 + / + Mice (hereinafter 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 contains two DNA fragments with sizes of 412bp and 224bp, respectively. The PCR amplification product of WT mice contains 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 2 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 flow chart shown in the synthesis of 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 for 30 minutes in argon. 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 obtain a light yellow solid product (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 compound: Compound TM-2 (0.20 g, 0.19 mmol) was dissolved in 10 mL of ethyl acetate saturated with hydrogen chloride (2N), stirred at 0°C for 1 h, and the organic solvent was removed in vacuo. The residue was purified by recrystallization (PE:EA=30:1, v / v) to obtain the product as a light 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 DCM under argon. HBTU (64 mg, 0.17 mmol) and DIPEA (45 mg, 0.35 mmol) were added at 0°C, stirred at 25°C for 10 h, diluted with an appropriate amount of DCM, washed with saturated brine, and removed in vacuo. Purified by column chromatography (EA: MeOH = 20: 1, v / v) to obtain 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 ethyl acetate saturated with hydrogen chloride (2N), stirred at 0°C for 1 h, and the organic solvent was removed in vacuo. The product was purified by recrystallization (PE:EA=30:1, v / v) to obtain 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 obtained 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 5 μM diluted TM2 solution. 10 μL of the diluted TM2 solution was dropped onto the amorphous carbon film copper mesh carriers, and after drying, the amorphous carbon film copper mesh carriers were observed using a transmission electron microscope (FEITalos F200S, Thermo Scientific). Figure 2-3 As shown, it can be observed that the TM2 nanoparticles have good dispersibility and a relatively uniform particle size, with an average diameter of 195 nm, and the PDI is within a reasonable range. Through transmission electron microscopy (TEM), it can be clearly seen that TM2 forms spherical nanoparticles with regular morphology and good dispersion, with a particle size of about 200 nm, which is consistent with the results of DLS.

[0097] Example 3: Preparation of Nanoparticles TMD2

[0098] Synthesis of TMD2: 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) is a synthetic analog of PE phospholipids, containing 18:1 fatty acids at the sn-1 and sn-2 positions. This auxiliary lipid can be used as an emulsifier to promote the transmembrane transport of RNA liposome complexes. It is used in combination with positively charged micelles as a non-viral gene delivery method to effectively improve the efficiency of nucleic acid transfection. According to the previous research experience of the research group, the transfection effect is best when the compound is combined with DOPE at a 1:2 (molar ratio). Here, TM2 and the auxiliary reagent DOPE are also selected to generate new nanoparticles TMD2 at a molar ratio of 1:2. The specific method is as follows: TM2 and DOPE are completely dissolved in CDCl3 at a molar ratio of 1:2, and the solvent is removed in vacuo to obtain a thin layer of DOPE / TMD film (TMD2); then the above DOPE / TMD film is hydrated with PBS (pH=7.4) to obtain a nanoparticle TMD2 solution. The size of the obtained nanoparticles TMD2 is 173.9 nm, which is slightly smaller than the size of the micelle nanoparticles formed by TM2. The addition of auxiliary reagents further makes the nanoparticle structure compact.

[0099] Test and characterize the prepared TMD2:

[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 obtained 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 on the amorphous carbon film copper mesh carrier, and after drying, it was observed by a transmission electron microscope (FEITalosF200S, Thermo Scientific). Figure 4-5 As shown, it can be observed that the TM2 nanoparticles have good dispersibility and a relatively uniform particle size, with an average diameter of 173.9 nm, and the PDI is within a reasonable range. Through transmission electron microscopy (TEM), it can be clearly seen that TMD2 forms spherical nanoparticles with regular morphology and good dispersion, with a particle size of about 180 nm, which is consistent with the results of DLS.

[0102] Example 4: siRNA packaging efficiency experiment

[0103] The results of condensation of siRNA by nanoparticles TM2 and TMD2 at different concentrations in DEPC water (mass ratio); [siRNA] = 500 nM, the concentrations of nanoparticles are 4, 11, 22, 33, 44 μM respectively. The siRNA used in this example is an siRNA (siTtc3) that interferes with the mouse Ttc3 gene, and the sequence of the siRNA is as follows: sense5'-AGGAUCUAAUUCAGCAGCAUGUAAA-3' (SEQ ID No. 3), antisense5'-UUUACAUGCUGCUGAAUUAGAUCCU-3' (SEQ ID No. 4).

[0104] The siRNA was complexed with nanoparticles TM2 (the mass of TM2 contained therein) and nanoparticles TMD2 (the mass of TM2+DOPE contained therein) 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 at 120V for 10 minutes in a 1% agarose gel. Markers (Sangon Biotech) were used as ladders. Finally, the gel was imaged using the ChemiDoc system (Bio-Rad, USA) and the bands were analyzed.

[0105] Results Figure 6 .Depend on Figure 6 It can be seen that the packaging efficiency of TMD2 is better than that of TM2. TMD2 can completely package siRNA at a mass ratio of 12:1. As the mass ratio of TMD2 to siRNA increases, the expression level of the Ttc3 gene continues to decrease. Therefore, TMD2 is an excellent siRNA carrier that can successfully bring the carried siRNA into the cell and successfully reduce the mRNA level of Ttc3.

[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 organs. The mice were imaged for whole body distribution observation on days 1, 3, 5, and 7 of administration, and the main organs were harvested. The fluorescence images are shown in Figure 2. Figure 7-8 As shown. The fluorescently labeled siTtc3@TMD2 used is cy5.5-labeled siTtc3 encapsulated in TMD2. The preparation method is the same as that in Example 4, except that the siRNA in Example 4 is replaced with cy5.5-labeled siRNA. In order to detect the biodistribution of nanoparticles, mice were anesthetized and imaged by 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 killed, and the organs were removed and imaged on the IVIS system.

[0110] Cy5.5-labeled siRNA and nanoparticles were administered to mice through tracheal intubation to observe their biodistribution in the lungs. The fluorescent signal mainly accumulated in the lungs. No enrichment was found in other major organs, and siRNA could still remain in the lungs within 7 days, which means that siTtc3@TMD2 can accumulate in the lungs and release siRNA.

[0111] Example 7: Effect of siTtc3@TMD2 nanoparticles on body weight changes 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 (BL2) 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 endotracheal 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 the 2nd to the 14th day after modeling, and the mice were killed 14 days after modeling (during these 14 days, the pulmonary fibrosis model treatment group and the pulmonary fibrosis model control treatment group were given nanoparticles every five days, that is, nanoparticles were given on the 5th and 10th days 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), and 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 Fig. 9It can be seen that after endotracheal intubation and instillation of PBS / BLM, the weight of mice in the WT-BLM group decreased significantly more than that in the HE-PBS group, and the weight recovery was slow during the 14-day modeling process. siTtc3@TMD2 significantly slowed down the rapid weight loss of mice with pulmonary fibrosis and improved the overall survival rate of mice with bleomycin-induced fibrosis. This shows that the nanoparticles can inhibit the expression of the Ttc3 gene and have an important effect on the changes in mouse weight and survival rate during pulmonary fibrosis.

[0115] Example 8: Effect of siTtc3@TMD2 nanoparticles on the progression of pulmonary fibrosis in mice

[0116] The lung tissues of the mice sacrificed in the above experiment were frozen and sectioned. The specific operation steps were as follows: (1) the lung tissues were fixed with 4% paraformaldehyde at 4°C for 24 h; (2) the lung tissues were dehydrated and precipitated in 10%, 20%, and 30% sucrose solutions in sequence; (3) the lung tissues were embedded in 0CT at -28°C and 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) The sections were baked in a 65°C oven for 1 hour and washed with ddH2O; (2) The nuclei were stained with hematoxylin stain and incubated at room temperature for 5 to 10 minutes; (3) The sections were washed with ddH2O for 5 minutes; (4) The sections were differentiated with 0.1% hydrochloric acid-ethanol for 10-30 seconds; (5) The sections were rinsed with ddH2O for 1 minute; (6) The sections were decolorized and blued in PBS for 30-60 seconds; (7) The sections were rinsed with ddH2O for 5 minutes; (8) The sections were washed with 95% ethanol for 5-10 seconds; (9) The sections were counterstained with eosin stain for 30 seconds to 2 minutes. Note: Eosin is highly soluble in water. If the sections are overstained, they can be rinsed with tap water; (10) The sections were dehydrated with 95% ethanol twice, each for 5 minutes; (11) The sections were cleared with xylene twice, each for 1-2 minutes, and sealed 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), left at room temperature overnight or placed in a 57°C-60°C incubator for 1 hour for mordant staining, and then rinsed with running water for 10 minutes; (2) The sections were dripped with lapis lazuli blue staining solution for 2-3 minutes, and washed with water twice, each time for 10-15 seconds; (3) The sections were dripped with Mayer hematoxylin staining solution 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 wash with water to terminate differentiation, and rinse with distilled water for 10 minutes; (5) Dye with Ponceau fuchsin staining solution for 10 minutes, and wash twice with distilled water, each time for 10-15 seconds; (6) Treat with phosphomolybdic acid solution for about 10 minutes; (7) Pour off the upper solution, do not wash the slices with water, and directly add aniline blue staining solution for 5 minutes; (8) After washing the aniline blue solution with weak acid solution, continue to add weak acid working solution to cover the slices for 2 minutes; (9) Dehydrate with 95% ethanol for 30 seconds. Dehydrate with anhydrous ethanol twice, the first time for 30 seconds, the second time for 1 minute; (10) Transparent with xylene twice, each time for 1-2 minutes. Seal with neutral gum.

[0119] Results Figure 10-11 . The WT-BLM group showed severe pulmonary fibrosis, characterized by loss of normal alveolar structure, obvious disordered thickening of alveolar septa, collapse of 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 alleviate BLM-induced pulmonary fibrosis in mice.

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

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

[0122] Results Fig.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. The mean fluorescence intensity was semi-quantitatively analyzed by image J software, and 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, using TMD2 nanoparticles to encapsulate siRNA targeting lung cancer-related genes and delivering it to the lungs through tracheal administration or aerosolization can achieve specific targeted delivery to lung cancer cells.

[0124] Example 10: Responsiveness of TM2 to NTR

[0125] Compound TM2 showed very weak fluorescence intensity in PBS buffer because the nitro group promoted the photoinduced energy transfer (PET) process and quenched the fluorescence emission. TM2 (10 μM) was analyzed after adding NADH (100 μg / mL) and various concentrations of NTR (0-20 μg / mL), and the fluorescence spectrum is shown in the figure. In order to monitor whether TM2 has high selectivity for nitroreductase. In PBS solution, TM2 (10 μM) was treated with various analytes, 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 TNN (10 μM) solution containing 100 μg / mL NADH, a good linear relationship was shown between the fluorescence intensity and NTR concentration (0-20 μg / mL), and the detection limit of TM2 for NTR was 14.07 ng / mL (signal-to-noise ratio was 3:1), indicating that TM2 micelles have good sensitivity to NTR. Fig.13 Figure C shows that only under the conditions of NADH and nitroreductase, the fluorescence at 480nm wavelength is significantly enhanced, and the treatment of other analytes has little effect on the fluorescence of the compound. In summary, TM2 has a higher selectivity for nitroreductase than other related biostimulants.

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

[0128] The cell counting kit-8 method was used to detect the cytotoxicity of the nanodrug. Cell Counting Kit-8, referred to as CCK8 (or WST-8) kit, is a rapid and highly sensitive detection kit based on WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazolyl monosodium salt) and is widely used in cell proliferation and cytotoxicity.

[0129] Working principle of WST-8: In the presence of electron coupling reagent, it can be reduced by dehydrogenase in mitochondria to generate highly water-soluble orange-yellow formazan. The depth of color is proportional to cell proliferation and inversely proportional to cytotoxicity. The OD value is measured at a wavelength of 450nm using an enzyme reader, which indirectly reflects the number of living cells.

[0130] 1. Prepare 100L of cell suspension in a 96-well plate. Pre-culture the culture 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, and then further incubate in complete medium for 20 hours. Then, starve NIH-3T3 cells in serum-free DMEM for 24 hours. The starved cells were then cultured in DMEM containing TGF-β1 (10ng / 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] Results Fig.14 . After testing, it was found that TGF-β1 treatment of cells will cause cell proliferation, while the addition of siTtc3@TMD2 and siNC@TMD2 has 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 implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended 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, the TM-1 compound and N-Boc-6-aminocaproic acid are subjected to an esterification reaction to obtain the TM-2 compound; 2) Under acidic conditions, deprotecting the BOC in the TM-2 compound to obtain the TM-3 compound; 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, the BOC in the TM-4 compound is deprotected to obtain the TM2 compound.

3. The preparation method according to claim 2, characterized in that: In step 1), the esterification reaction is carried out in an organic solvent, and the organic solvent is THF; And / or, in the 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 the 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 the 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 the 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 saturated ethyl acetate solution of hydrogen chloride.

4. A nanoparticle carrier, the raw materials of which include TM2 and DOPE as claimed in claim 1; Preferably, the molar ratio of TM2 to DOPE is 1:

2.

5. A nanoparticle encapsulating nucleic acid, 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 nanoparticle to the mass of the nucleic acid molecule is (1-50):1; And / or, the particle size of the nanoparticles encapsulating nucleic acid is 50 to 200 nm.

7. The method for preparing the nucleic acid-encapsulated 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 film 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.

9. The product for treating lung diseases according to claim 8, characterized in that: The product is a pharmaceutical preparation; And / or, the drug preparation is administered via tracheal administration; preferably, the tracheal administration is atomized inhalation or dry powder inhalation; And / or, the drug preparation is a tracheal administration preparation; preferably, the tracheal administration preparation is an aerosol inhalation or a dry powder inhalation; And / or, the product further comprises a pharmaceutically acceptable carrier.

10. The product for treating lung diseases according to claim 8 or 9, characterized in that: The pulmonary diseases include, but are not limited to: pulmonary fibrosis, lung cancer, chronic obstructive pulmonary disease, cystic fibrosis, pulmonary hypertension; Furthermore, the lung disease is pulmonary fibrosis, and the nucleic acid molecule is siRNA targeting a gene related to pulmonary fibrosis; Furthermore, the lung disease is lung cancer, and the nucleic acid molecule is siRNA targeting a gene related to lung cancer; Further, the pulmonary disease is chronic obstructive pulmonary disease, and the nucleic acid molecule is siRNA targeting a gene related to chronic obstructive pulmonary disease; Furthermore, the lung disease is pulmonary cystic fibrosis, and the nucleic acid molecule is siRNA targeting pulmonary cystic fibrosis-related genes; Furthermore, the lung disease is pulmonary hypertension, and the nucleic acid molecule is siRNA targeting genes related to pulmonary hypertension.

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