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CN-122005457-A - PDE 4B-targeted siRNA liposome nano-drug, preparation method and application

CN122005457ACN 122005457 ACN122005457 ACN 122005457ACN-122005457-A

Abstract

The invention discloses a PDE 4B-targeted siRNA liposome nano-drug, a preparation method and application thereof, belonging to the technical field of biological medicine and nano-drug. Cholesterol, DLin-MC3-DMA, DOPE, and DSPE-PEG2000 were dissolved in absolute ethanol as the ethanol phase, and siPDE B was dissolved in sodium citrate buffer as the aqueous phase. And then, rapidly mixing the ethanol phase and the water phase through a microfluidic device, and dialyzing the obtained mixture through PBS to promote liposome assembly and remove ethanol to obtain the PDE4B targeted siRNA liposome nano-drug. Compared with the prior art, the preparation method has the advantages of simple preparation flow, mass preparation, excellent anti-pulmonary fibrosis effect, low toxicity, inhalable administration and capability of effectively suppressing IPF.

Inventors

  • XU QINGWEN
  • YU CHENGGONG
  • LI BOWEI
  • SUN YUYAO
  • ZHANG CHAOCHAO

Assignees

  • 皖南医学院第一附属医院（皖南医学院弋矶山医院）

Dates

Publication Date
20260512
Application Date
20251230

Claims (8)

  1. 1. The preparation method of the PDE 4B-targeted siRNA liposome nano-drug is characterized by comprising the following steps: step (1), dissolving cholesterol, DOPE, DSPE-PEG2000 and DLin-MC3-DMA in absolute ethanol to prepare a lipid premix solution as an ethanol phase; Step (2), dissolving siPDE B in sodium citrate buffer solution to prepare nucleic acid premix solution as water phase, wherein the nucleotide sequence of the sense strand of siPDE B is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense strand of siPDE B is shown as SEQ ID NO. 2; And (3) mixing the ethanol phase and the water phase through a microfluidic device, and then carrying out dialysis and ultrafiltration purification and concentration to finally obtain the liposome nano-drug.
  2. 2. The method according to claim 1, wherein in the step (1), the mass ratio of DLin-MC3-DMA, cholesterol, DOPE, DSPE-PEG2000 is 10.7:5:2.5:1.
  3. 3. The method of claim 1, wherein in step (2), the sodium citrate buffer has a concentration of 0.1 m and a ph of 4.
  4. 4. The process according to claim 1, wherein in step (3), the molar ratio of N in the ethanol phase to P in the aqueous phase is (5-20): 1.
  5. 5. The method according to claim 1, wherein in the step (3), the volume ratio of the ethanol phase to the water phase in the microfluidic mixing step is 1:2.5.
  6. 6. The method of claim 1, wherein in step (3), the concentration method of dialysis and ultrafiltration purification comprises the steps of dialyzing a mixture of ethanol phase and water phase with PBS, obtaining MWCO of 3500 Da, centrifuging 10 min at 6500/rpm, and washing three times with deionized water to obtain the liposome nano-drug.
  7. 7. The PDE 4B-targeting siRNA liposome nano-drug prepared by the method of any one of claims 1 to 6.
  8. 8. Use of the PDE 4B-targeting siRNA liposome nano-drug prepared by the method of any one of claims 1 to 6 for the preparation of a therapeutic agent for idiopathic pulmonary fibrosis.

Description

PDE 4B-targeted siRNA liposome nano-drug, preparation method and application Technical Field The invention belongs to the technical field of biological medicines and nano medicines, and particularly relates to an siRNA liposome nano medicine targeting PDE4B, a preparation method and application thereof. Background Idiopathic Pulmonary Fibrosis (IPF) is a pulmonary interstitial disease of unknown etiology, progressive deterioration and irreversible, and its main pathological features are abnormal proliferation and activation of pulmonary fibroblasts, resulting in excessive deposition of extracellular matrix, eventually forming pulmonary fibrotic scars, destroying normal alveolar structures and severely affecting gas exchange functions. IPF patients have extremely poor prognosis, median survival after diagnosis is only 3 to 5 years, five-year mortality exceeds 60%, and clinical treatment approaches are extremely limited. At present, the U.S. FDA only approves pirfenidone and nidazole for IPF treatment, however, the two medicaments can only delay the disease progression of light and medium patients to a certain extent, have limited effect on improving the overall survival rate, especially have very little curative effect on middle and late patients, and cannot meet the urgent requirements of clinical high-efficiency and safe treatment schemes. In recent years, phosphodiesterase 4B (PDE 4B) has received widespread attention as a key target for anti-fibrotic therapy. For example, PDE4B small molecule inhibitors Nerandomilast developed by boilinginvahn have achieved positive results in phase III clinical trials, becoming a new therapy for IPF that reached the primary endpoint for the first decade. However, because of the high homology in the catalytic domains of the PDE4 family subtypes (PDE 4A-D), subtype selective inhibition is difficult to achieve with conventional small molecule drugs. The Nerandomilast with better PDE4B selectivity still cannot completely avoid toxic and side effects such as gastrointestinal reaction, depression and the like caused by cross inhibition, which severely limits the clinical application prospect. Based on this, small interfering RNA (siRNA) technology provides a new strategy for solving the selective problem in PDE4B targeted therapy. Through the sequence specificity recognition mechanism of the siRNA drug, the expression of PDE4B gene can be accurately silenced at the posttranscriptional level, and other PDE4 subtypes are hardly influenced, so that the toxic and side effects of the small molecular drug caused by off-target effect are radically avoided. At present, the siRNA technology platform is mature, a plurality of siRNA drugs are put on the market in batches, and the effectiveness and the safety of the siRNA drugs are fully verified. Especially in the field of respiratory diseases, the siRNA medicine can realize long-acting targeted intervention by local pulmonary administration, and has outstanding therapeutic potential. Against the background described above, there is still a lack of IPF therapies in the art that can target PDE4B efficiently, accurately and safely. Therefore, the development of the siRNA drug targeting PDE4B not only can break through the limitation of the existing small molecular drug in terms of selectivity and safety in terms of mechanism, but also is beneficial to constructing a technical system with independent intellectual property rights, and has important clinical value and strategic significance for promoting the crossing development of the field of innovative drugs for serious chronic pulmonary diseases. Disclosure of Invention The invention provides an siRNA liposome nano-drug targeting PDE4B, a preparation method and application thereof, and solves the problem that the prior art still lacks an IPF treatment means capable of targeting PDE4B efficiently, accurately and safely. An siRNA liposome nano-drug targeting PDE4B, comprising the steps of: (1) Dissolving cholesterol, 1, 2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), DSPE-PEG2000 and DLin-MC3-DMA in absolute ethanol to prepare a lipid premix solution as an ethanol phase; (2) siPDE4B is dissolved in sodium citrate buffer solution to prepare nucleic acid premix solution which is used as water phase; (3) Then, mixing the lipid premix solution (ethanol phase) and the nucleic acid premix solution (water phase) through a microfluidic device, and then, carrying out dialysis and ultrafiltration purification and concentration to finally obtain liposome nano-drugs (SNPs); Further, in the step (1), the mass ratio of DLin-MC3-DMA, cholesterol, DOPE and DSPE-PEG2000 is 10.7:5:2.5:1. Further, in step (2), the nucleotide sequence of the sense strand of siPDE B is shown as SEQ ID No.1, and the nucleotide sequence of the antisense strand is shown as SEQ ID No. 2. SEQ ID NO.1:GCGACAUCUUUCAGAAUCUTT(sense 5`~3`); SEQ ID NO.2:AGAUUCUGAAAGAUGUCGCTT(antisense 5`~3`)。 Further, in the step (2), the concent