PDE4B-targeted siRNA lipidosome nano-drug, preparation method and application of PDE4B-targeted siRNA lipidosome nano-drug
By preparing siRNA liposome nanomedicines targeting PDE4B, the problems of insufficient selectivity and safety of traditional small molecule drugs in the treatment of IPF have been solved. This has enabled precise targeted delivery of PDE4B and potent anti-fibrotic effects, reduced side effects, and provided a safe and efficient treatment option for IPF.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE FIRST AFFILIATED HOSPITAL OF WANNAN MEDICAL COLLEGE (YIJISHAN HOSPITAL OF WANNAN MEDICAL COLLEGE)
- Filing Date
- 2025-12-30
- Publication Date
- 2026-05-12
Smart Images
Figure CN122005457A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedicine and nanomedicine technology, specifically relating to a siRNA liposome nanomedicine targeting PDE4B, its preparation method, and its application. Background Technology
[0002] Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible interstitial lung disease of unknown etiology. Its main pathological feature is the abnormal proliferation and activation of pulmonary fibroblasts, leading to excessive extracellular matrix deposition and ultimately fibrotic scarring. This damages normal alveolar structure and severely impairs gas exchange. IPF patients have an extremely poor prognosis, with a median survival of only 3 to 5 years after diagnosis and a five-year mortality rate exceeding 60%. Clinical treatment options are extremely limited. Currently, the US FDA has only approved pirfenidone and nintedanib for IPF treatment. However, these two drugs can only delay disease progression to a certain extent in mild to moderate cases, with limited improvement in overall survival, especially for patients with advanced stages, failing to meet the urgent clinical need for efficient and safe treatment options.
[0003] In recent years, phosphodiesterase 4B (PDE4B) has received widespread attention as a key target for antifibrotic therapy. For example, Nerandomilast, a small molecule PDE4B inhibitor developed by Boehringer Ingelheim, achieved positive results in a phase III clinical trial, becoming the first novel IPF therapy to meet its primary endpoint in a decade. However, due to the high homology of catalytic domains among the various subtypes of the PDE4 family (PDE4A-D), traditional small molecule drugs struggle to achieve subtype-selective inhibition. Even with its superior PDE4B selectivity, Nerandomilast cannot completely avoid gastrointestinal reactions and toxic side effects such as depression caused by cross-inhibition, which severely limits its clinical application prospects.
[0004] Based on this, small interfering RNA (siRNA) technology offers a novel strategy for addressing the selectivity challenge in PDE4B targeted therapy. siRNA drugs, through their sequence-specific recognition mechanism, can precisely silence PDE4B gene expression at the post-transcriptional level, with minimal impact on other PDE4 subtypes, thus fundamentally avoiding the off-target toxicity of small molecule drugs. Currently, the siRNA technology platform is mature, and several siRNA drugs have been approved for marketing, with their efficacy and safety fully validated. Particularly in the field of respiratory diseases, siRNA drugs, administered locally to the lungs, can achieve long-term targeted intervention, demonstrating significant therapeutic potential.
[0005] Against this backdrop, current technologies still lack efficient, precise, and safe treatments for IPF that target PDE4B. Therefore, developing a PDE4B-targeting siRNA drug not only overcomes the limitations of existing small molecule drugs in terms of selectivity and safety at the mechanistic level, but also helps build a technological system with independent intellectual property rights. This has significant clinical value and strategic importance for promoting the leapfrog development of innovative drugs for major chronic lung diseases. Summary of the Invention
[0006] The existing technology still lacks an IPF treatment method that can efficiently, accurately and safely target PDE4B. The present invention provides a PDE4B-targeting siRNA liposome nanomedicine, its preparation method and application.
[0007] A siRNA liposome nanomedicine targeting PDE4B comprises the following steps:
[0008] (1) Cholesterol, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), DSPE-PEG2000 and DLin-MC3-DMA were dissolved in anhydrous ethanol to prepare a lipid premix solution as the ethanol phase;
[0009] (2) Dissolve siPDE4B in sodium citrate buffer to prepare a nucleic acid premix solution as the aqueous phase;
[0010] (3) Subsequently, the lipid premixed solution (ethanol phase) and the nucleic acid premixed solution (aqueous phase) were mixed by a microfluidic device, and then purified and concentrated by dialysis and ultrafiltration to finally obtain liposome nanomedicines (SNPs).
[0011] Furthermore, in step (1), the mass ratio of DLin-MC3-DMA, cholesterol, DOPE, and DSPE-PEG2000 is 10.7:5:2.5:1.
[0012] Furthermore, in step (2), the nucleotide sequence of the sense strand of the siPDE4B is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.2.
[0013] SEQ ID NO.1: GCGACAUCUUCAGAAUCUTT (sense 5`~3`);
[0014] SEQ ID NO.2: AGAUUCUGAAAGAUGUCGCTT (antisense 5`~3`).
[0015] Furthermore, in step (2), the concentration of the sodium citrate buffer solution is 0.1 M and the pH value is 4.
[0016] Furthermore, in step (3), the molar ratio (N / P) of N in the ethanol phase to P in the water phase is (5-20):1.
[0017] Furthermore, in step (3), the volume ratio of the ethanol phase (lipid premixed solution) and the aqueous phase (nucleic acid premixed solution) in the microfluidic mixing step is 1:2.5.
[0018] Further, in step (3), the dialysis and ultrafiltration purification and concentration method is as follows: the mixture of lipid premixed solution (ethanol phase) and nucleic acid premixed solution (aqueous phase) is dialyzed with PBS with MWCO of 3500 Da, then centrifuged at 6500 rpm for 10 min, and washed three times with deionized water to obtain liposome nanomedicines (SNPs).
[0019] The application of nanomedicines prepared by the above method in the preparation of therapeutic agents for idiopathic pulmonary fibrosis.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] (1) High precision and specificity: Compared with traditional PDE4 inhibitors, this invention utilizes liposome nanocarriers to achieve lung-specific delivery of siPDE4B, significantly increasing the drug concentration at the lesion site. At the same time, siPDE4B only works against PDE4B subtypes, avoiding off-target effects on other subtypes, thus ensuring therapeutic precision at the molecular level.
[0022] (2) Strong anti-fibrotic and anti-inflammatory effects: By specifically silencing the PDE4B gene, SNPs can continuously increase the local cAMP level, effectively inhibiting the production of inflammatory mediators and fibroblast activation.
[0023] (3) Significantly reduced systemic side effects: Due to the local action characteristics and subtype selectivity of SNPs, their systemic exposure is extremely low, avoiding adverse reactions such as nausea and vomiting commonly seen with traditional PDE4 inhibitors. This greatly improves treatment safety and patient tolerability, making long-term IPF management possible.
[0024] (4) High technical feasibility and translational potential: This invention is based on mature liposome and siRNA technologies, with a simple preparation process, controllable cost, and the liposome composition can be adjusted to adapt to different administration routes. Compared with existing IPF treatment drugs, SNPs provide a modular and scalable new treatment strategy with broad clinical application prospects. Attached Figure Description
[0025] Figure 1 The particle size distribution diagram is shown for the nanomedicine prepared in Example 1.
[0026] Figure 2 The ultraviolet absorbance spectrum of the nanomedicine prepared in Example 1;
[0027] Figure 3 TEM image of the nanomedicine prepared in Example 1;
[0028] Figure 4 This is a comparison of the inhibitory effects of the nanomedicine prepared in Example 1 and the PDE4B inhibitor on PDE4B.
[0029] Figure 5 The image shows the anti-fibrotic effect of the nanomedicine prepared in Example 1 and the control group at the cellular level.
[0030] Figure 6 The image shows the evaluation of the antifibrotic effect of the nanomedicine and other treatment groups prepared in Example 1 at the animal level after inhalation administration.
[0031] Figure 7 The image shows the safety evaluation of the nanomedicine prepared in Example 1 and the control group after inhalation administration at the animal level. Detailed Implementation
[0032] The technical solution of the present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited to the embodiments described.
[0033] Example 1: Preparation of a PDE4B-targeting siRNA liposome nanomedicine (SNPs).
[0034] The preparation steps are as follows:
[0035] (1) 5 mg cholesterol (Annegi Chemical, 57-88-5), 10.7 mg DLin-MC3-DMA (Suzhou Aimaite Biotechnology, AS02573), 2.5 mg DOPE (Maclean's Reagent, 4004-05-1) and 1 mg DSPE-PEG2000 (Xi'an Ruixi Biotechnology, R-1028-2K) were dissolved in 3.3 mL of anhydrous ethanol to prepare a lipid premix solution. The liposome premix solution (1025 nmol, calculated as Dlin-MC3-DMA) was diluted to 1 mL of anhydrous ethanol solution as the ethanol phase.
[0036] (2) Subsequently, siPDE4B (1 OD) was dissolved in 2.5 mL of sodium citrate buffer (0.1 M, pH 4.0) as the aqueous phase.
[0037] (3) The ethanol and aqueous solutions were mixed thoroughly using a microfluidic system. The resulting mixture was immediately dialyzed with PBS (MWCO: 3500 Da) to promote liposome assembly and remove residual ethanol. Subsequently, it was centrifuged at 6500 rpm for 10 min and washed three times with deionized water to obtain liposomal nanomedicines (SNPs). At the same time, blank liposomes (NPs) without siPDE4B loading were prepared as a control by adjusting the composition.
[0038] The zeta potential and hydrodynamic diameter of the nanomedicine were measured using a particle size analyzer (Zetasizer Nano ZS, Malvern Corporation). UV-Vis absorption spectra were recorded using a Shimadzu UV-2500 UV-Vis spectrophotometer. SNP solutions were deposited onto a carbon support film (Beijing Keyi Technology Co., Ltd.) to prepare transmission electron microscopy (TEM) samples. After staining with 1% uranium acetate for 2 min, TEM images of the nanoparticles were acquired using a Hitachi HT 7700 TEM instrument with an accelerating voltage of 100 kV.
[0039] Figure 1 The particle size distribution diagram of the nanomedicine prepared in Example 1 shows that the prepared nanomedicine (SNPs) has good size uniformity with an average particle size of about 200 nm.
[0040] Figure 2 The UV-Vis absorption spectrum of the nanomedicine prepared in Example 1 shows the characteristic absorption peak of siPDE4B at 260 nm, further confirming the successful construction of SNPs.
[0041] Figure 3 TEM image of the nanomedicine prepared in Example 1, as shown. Figure 3 As shown, SNPs exhibit a characteristic phospholipid bilayer structure and are relatively uniform in size.
[0042] Example 2: Using Nerandomilast, a selective small molecule inhibitor of PDE4B, as a control, the inhibitory effect of SNPs on PDE4B was detected.
[0043] The experiment was divided into four groups, and the experimental steps for each group are as follows:
[0044] A549 cells were seeded in 12-well plates. When the cell density reached approximately 70%, the following procedures were performed:
[0045] Control group: The cell culture medium was replaced with a complete culture medium containing an equal volume of ddH2O, and incubation continued for 48 h.
[0046] TGF-β group: The cell culture medium of the TGF-β group was replaced with complete culture medium containing TGF-β (10 ng / mL), and incubation was continued for 48 h.
[0047] TGF-β+Nerandomilast group: The cell culture medium was first replaced with complete medium containing TGF-β (10 ng / mL) and incubated for 48 h. Then, the medium was discarded and replaced with complete medium containing Nerandomilast (1 μM) and incubated for another 30 min.
[0048] TGF-β+SNPs group: The cell culture medium was first replaced with complete medium containing SNPs (100 μM) and incubated for 6 h. Then, the medium was discarded and replaced with complete medium containing TGF-β (10 ng / mL) and cultured for another 48 h.
[0049] After incubation, the medium was replaced with complete medium containing 0.5 mM IBMX (phosphodiesterase inhibitor) and 10 mM Forskolin (adenylate cyclase activator), and incubated for 30 min to stabilize intracellular cAMP levels. Subsequently, cAMP quantification was performed using the Add&Read™ cAMP Detection kit to assess PDE4B activity.
[0050] Figure 4 This is a comparison of the inhibitory effects of the nanomedicine prepared in Example 1 and the PDE4B inhibitor on PDE4B; as shown. Figure 4 As shown, in the TGF-β-induced fibrotic cell model, the level of cAMP, a hydrolytic substrate of PDE4B, was significantly increased in cells after SNP treatment compared to Nerandomilast, indicating that SNPs have a stronger PDE4B inhibitory effect than PDE4B inhibitors.
[0051] Example 3: Detection of the inhibitory effect of SNPs on TGF-β-induced lung fibroblast activation.
[0052] The experiment was divided into four groups, and the experimental steps for each group are as follows:
[0053] Lung fibroblasts (MLg) were seeded into 12-well plates corresponding to the four experimental groups. Once the cell density reached approximately 70%, the next step was performed:
[0054] SNPs group: Replace the cell culture medium of the SNPs group with complete culture medium containing SNPs (100 μM) and incubate for 6 h.
[0055] TGF-β group: The cell culture medium of the TGF-β group was replaced with complete culture medium containing TGF-β (10 ng / mL) and incubated for 48 h.
[0056] TGF-β+SNPs group: The cell culture medium of the TGF-β+SNPs group was replaced with complete medium containing SNPs (100 μM) and incubated for 6 h. Subsequently, the medium was discarded and replaced with complete medium containing TGF-β (10 ng / mL) for another 48 h.
[0057] Control group: Add an equal volume of PBS buffer to the cells in the Control group.
[0058] Total RNA was extracted from cells using a kit and converted into cDNA. The expression levels of type I collagen (Col1a1) and myofibroblast marker α-SMA gene (Acta2) in cells were detected using RT-qPCR.
[0059] Figure 5 The images show the anti-fibrotic effects of the nanomedicine prepared in Example 1 and the control group at the cellular level; Figure 5 As shown, in the TGF-β-induced fibrosis cell model, the expression of the genes encoding fibroblast activation markers Col1a1 and Acta2 was significantly increased. Treatment with SNPs reversed this phenomenon, indicating that SNPs can significantly inhibit lung fibroblast activation at the cellular level and have good anti-fibrotic potential. Example 3 and... Figure 5 This confirms that SNPs successfully knocked down PDE4B.
[0060] Example 4: Evaluation of the antifibrotic effect of liposomal nanomedicines SNPs and other treatment groups at the animal level after administration.
[0061] The specific steps are as follows:
[0062] (1) C57BL / 6 mice were randomly divided into 4 groups, with 6 mice in each group. The groups were normal group, fibrosis group, Nerandomilast group and SNPs group.
[0063] (2) Modeling: Mice in the fibrosis group, Nerandomilast group and SNPs group were given bleomycin (2 mg / kg) via tracheal infusion for IPF modeling, while the normal group was given the same volume of physiological saline.
[0064] (3) Drug treatment:
[0065] SNPs group mice were administered the drug via tracheal drip on days 3, 6, 9, 12, 15, 18, and 21 after modeling, for a total of 7 administrations. Each administration dose was 150 μL of SNPs (containing 0.75 OD of siPde4b).
[0066] Nerandomilast mice were administered the drug via gavage once daily from the first day of modeling, at a dose of 5 mg / kg each time.
[0067] Mice in the normal group and the fibrotic group were given the same volume of physiological saline.
[0068] (4) Modeling: On day 21, mice were euthanized and lung tissue was removed and fixed with 4% paraformaldehyde solution. Masson staining was used to evaluate the therapeutic effects of SNPs and other treatment groups on pulmonary fibrosis.
[0069] Figure 6 The graph shows the evaluation of the antifibrotic effects of the nanomedicine and other treatment groups prepared in Example 1 after inhalation administration at the animal level; as shown. Figure 6 As shown, SNPs have the best anti-fibrotic and collagen degradation capabilities. Masson-stained lung tissue sections showed that collagen accumulation in the SNPs group was significantly improved compared to the pulmonary fibrosis group and other treatment groups, and the lung tissue structure was close to that of the normal group.
[0070] Example 5: Safety evaluation of liposomal nanomedicine SNPs and control group in animals after administration.
[0071] Health: The normal group in Example 4;
[0072] IPF+SNPs: SNP groups in Example 4.
[0073] On day 21 of modeling, a suitable amount of mouse blood was collected to test liver and kidney function indicators in order to preliminarily evaluate liver and kidney toxicity.
[0074] Figure 7 The figure shows the animal-level safety evaluation of the nanomedicine prepared in Example 1 and the control group after inhalation administration. As shown in Figure 7, compared with the control group, SNP inhalation treatment did not cause significant pathological changes in liver and kidney tissues, and the SNPs were preliminarily judged to be relatively safe.
[0075] These results support the ability of SNPs to significantly inhibit the progression of fibrosis, making them a highly promising therapeutic agent for IPF. The preparation process of this invention is simple and can be mass-produced. The prepared nanomedicine is an inhalable liposomal nanomedicine with excellent anti-pulmonary fibrosis effects, low toxicity, and can be administered via inhalation, effectively inhibiting IPF.
[0076] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention.
Claims
1. A method for preparing a PDE4B-targeting siRNA liposome nanomedicine, characterized in that, Includes the following steps: Step (1): Dissolve cholesterol, DOPE, DSPE-PEG2000 and DLin-MC3-DMA in anhydrous ethanol to prepare a lipid premix solution as the ethanol phase; Step (2): Dissolve siPDE4B in sodium citrate buffer to prepare a nucleic acid premix solution as the aqueous phase; the nucleotide sequence of the sense strand of siPDE4B is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.2; Step (3): The ethanol phase and the aqueous phase are mixed by a microfluidic device, and then purified and concentrated by dialysis and ultrafiltration to finally obtain liposome nanomedicine.
2. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of DLin-MC3-DMA, cholesterol, DOPE, and DSPE-PEG2000 is 10.7:5:2.5:
1.
3. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the sodium citrate buffer solution is 0.1 M and the pH value is 4.
4. The preparation method according to claim 1, characterized in that, In step (3), the molar ratio of N in the ethanol phase to P in the water phase is (5-20):
1.
5. The preparation method according to claim 1, characterized in that, In step (3), the volume ratio of the ethanol phase to the water phase in the microfluidic mixing step is 1:2.
5.
6. The preparation method according to claim 1, characterized in that, In step (3), the dialysis and ultrafiltration purification and concentration method is as follows: the mixture of ethanol phase and aqueous phase is dialyzed with PBS with MWCO of 3500 Da, then centrifuged at 6500 rpm for 10 min, and washed three times with deionized water to obtain liposome nanomedicine.
7. The siRNA liposome nanomedicine targeting PDE4B prepared by any of the preparation methods described in claims 1-6.
8. The use of the PDE4B-targeting siRNA liposome nanomedicine prepared by any of the preparation methods described in claims 1-6 in the preparation of therapeutic agents for idiopathic pulmonary fibrosis.