Lcn2-targeted lipid nanoparticle for treating osteoporosis and preparation and application thereof

Abstract

The present application relates to a kind of Lcn2 targeting lipid nanoparticles for treating osteoporosis and its preparation and application, the Lcn2 targeting lipid nanoparticles is core-shell structure nanoparticles, wherein core includes nucleic acid drug;Shell includes lipid carrier;Wherein lipid carrier includes ionizable lipid, pegylated lipid, auxiliary phospholipid and cholesterol.The innovative dual-effect bone homeostasis regulation mechanism of the present application, overcome the defect of single existing drug target;Highly targeted specificity and excellent physical and chemical properties, high delivery efficiency;Excellent safety and metabolic neutrality, avoid the side effects of systemic intervention;Economic and social benefits are remarkable.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to an Lcn2-targeted lipid nanoparticle for treating osteoporosis, its preparation and application. Background Technology

[0002] Osteoporosis is a systemic bone metabolic disease characterized by low bone mass and destruction of bone microstructure, leading to increased bone fragility and susceptibility to fractures (Sing CW, et al. J Bone Miner Res, 2023, 38:1064-1075). Recent clinical observations have revealed that bone homeostasis is not only regulated by intrinsic cellular biology of the skeleton but also profoundly influenced by inter-organ communication (especially the liver-bone axis) (Yuan W, et al. Med Rev, 2022, 2:331-348; Deng AF, et al. Mil Med Res, 2024, 11:37). Almost all patients with chronic liver disease have abnormal bone metabolism (Ehnert S, et al. Int J Mol Sci, 2019, 20; Nussler AK, et al. Arch Toxicol, 2014, 88:997-1006), among whom patients with non-alcoholic fatty liver disease (NAFLD) have a significantly increased risk of low bone mineral density and high-turnover osteoporotic fractures (Li M, et al. J Clin Endocrinol Metab, 2012, 97:2033-2038; Vachliotis ID, et al. Diabetes Obes Metab, 2022, 24:1702-1720; Cui A, et al. Front Endocrinol, 2023, 14:1215790; Chen HJ, et al. Medicine, 2018, 97:e12835).

[0003] Currently, while conventional pharmacological strategies for osteoporosis in clinical practice (such as bisphosphonates, denosumab, romozomab, etc.) can effectively reduce fracture risk, these existing technologies have the following significant problems and limitations:

[0004] 1. Single target: Existing drugs usually only target and inhibit bone resorption or promote bone formation (Chen YJ, et al. Front Pharmacol, 2024, 15:1456796), and cannot restore bone homeostasis at both levels.

[0005] 2. Neglecting systemic metabolic drivers: Current medications fail to address the systemic metabolic and inflammatory background driving bone loss. For patients with comorbid metabolic liver disease (such as NAFLD), there is often a lack of therapeutic interventions targeting the pathogenic factors of the liver-bone axis.

[0006] 3. Lack of safe tissue-targeted interventions: Lipocalin-2 (LCN2) is a hepatokine primarily secreted by hepatocytes under metabolic and inflammatory stimulation (Xu MJ, et al. Hepatology, 2015, 61:692-702; Zhang Y, et al. PLoS One, 2014, 9:e96997), and its levels are significantly elevated in obese patients or those with non-alcoholic steatohepatitis (NASH) (Ye D, et al. J Hepatol, 2016, 65:988-997). Although studies have found that LCN2 is widely involved in cross-organ communication, there are currently no clinical drugs that target it as a therapeutic target for the liver-bone axis. In addition, previous studies have shown that non-specific systemic Lcn2 inhibition or knockout may cause side effects such as systemic lipid and energy metabolism disorders (Capulli M, et al. J Bone Miner Res, 2018, 33:1141-1153; Xu Y, et al. Hepatol Commun, 2019, 3:763-775).

[0007] It should be noted that siLcn2 is not a clinically known anti-osteoporosis drug. While existing research suggests that LCN2 participates in bone metabolism and has potential interventional value, related studies mainly focus on the association between LCN2 and bone phenotype, genetic knockout, or in vitro cellular level mechanism verification. To the extent that the inventors of this application possess the prior art, there is no publicly available pharmaceutical regimen for using siLcn2 with the specific double-stranded sequence described in this application to form liver-preferentially distributed lipid nanoparticles for the treatment of NAFLD-related osteoporosis.

[0008] Therefore, there is an urgent need in the field for a novel osteoporosis treatment agent that can target the pathogenic factors in the liver, inhibit bone resorption and promote bone formation, and does not cause systemic metabolic disorders. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide an Lcn2-targeted lipid nanoparticle for the treatment of osteoporosis, and its preparation and application, overcoming the shortcomings of existing osteoporosis treatment drugs, such as single action, inability to take into account the background of systemic metabolic diseases (such as NAFLD), and lack of specific intervention methods targeting key pathogenic targets of the liver-bone axis.

[0010] This invention provides an Lcn2-targeting lipid nanoparticle, wherein the Lcn2-targeting lipid nanoparticle is a core-shell structured nanoparticle, wherein the core includes a nucleic acid drug and the shell includes a lipid carrier;

[0011] Lipid carriers include ionizable lipids, polyethylene glycol-modified lipids, cofactor phospholipids, and cholesterol.

[0012] Preferably, the nucleic acid drug is siLcn2, wherein the double-stranded sequence of siLcn2 is:

[0013] The justice chain is 5′-CGAUGUACAGCACCAUCUAUGTT-3′; as shown in SEQ ID NO.1;

[0014] The antisense chain is 5′-CAUAGAUGGUGCUGUACAUCGTT-3′, as shown in SEQ ID NO.2.

[0015] This invention combines the specific siLcn2 sequence with lipid nanoparticles preferentially distributed in the liver, thereby releasing siLcn2 within hepatocytes and silencing Lcn2, reducing circulating LCN2 at its source, and thus simultaneously inhibiting bone resorption and promoting bone formation.

[0016] Preferably, the lipid carrier is composed of DLin-MC3-DMA, DMG-PEG2000, DSPC, and cholesterol, wherein the lipid carrier contains 45-55 mol% DLin-MC3-DMA, 8-12 mol% DSPC, 35-42 mol% cholesterol, and 1-3 mol% DMG-PEG2000.

[0017] More preferably, the lipid carrier contains 50 mol% DLin-MC3-DMA, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG2000.

[0018] Preferably, the nitrogen-to-phosphorus ratio of lipids to siRNA in the Lcn2-targeted lipid nanoparticles is 4-8.

[0019] More preferably, the nitrogen-to-phosphorus ratio of lipid to siRNA in the Lcn2 targeted lipid nanoparticles is 6, and the prepared lipid nanoparticles have a uniform spherical morphology, a hydrodynamic particle size (Z-average) of about 100 nm, a polydispersity index (PDI) of about 0.225, a zeta potential of about -9.3 mV, and an encapsulation efficiency of siRNA of over 90%.

[0020] The unique feature of this application's experimental verification lies in the fact that the four lipids were formulated in a molar ratio of DLin-MC3-DMA:DSPC:cholesterol:DMG-PEG2000 = 50:10:38.5:1.5, and encapsulated with the specific siLcn2 under an N / P ratio of 6. This resulted in particles with a particle size of approximately 100 nm, a PDI of approximately 0.225, a Zeta potential of approximately -9.3 mV, and an encapsulation efficiency of approximately 90%. In vivo, these particles exhibited a continuous technical effect of preferential distribution in the liver, effective silencing of Lcn2, and improvement in bone microstructure.

[0021] Under the existing experimental conditions of this application, the validated ionizable lipid is DLin-MC3-DMA. If other ionizable lipids are substituted, their key quality properties, liver selective distribution, Lcn2 silencing efficiency, and bone microstructure improvement effects should be further compared under the same siLcn2 loading and administration conditions. Without such comparative verification, it cannot be presumed that the substituted formulation can achieve the same technical effects as the preferred formulation of this invention.

[0022] This invention provides a method for preparing any of the Lcn2-targeted lipid nanoparticles, comprising:

[0023] (1) The lipid carrier component was dissolved in anhydrous ethanol to obtain the lipid phase;

[0024] (2) Dissolve the nucleic acid drug in an acidic buffer solution to obtain an aqueous phase;

[0025] (3) The lipid phase and aqueous phase are mixed and self-assembled by a microfluidic mixing system, and then post-processed to obtain Lcn2 targeted lipid nanoparticles.

[0026] In step (2), the acidic buffer solution is a citrate buffer solution with a pH of 4.0.

[0027] The set N / P ratio is achieved in step (2).

[0028] In step (3), the lipid phase and the aqueous phase are rapidly mixed using a microfluidic mixing system, which promotes the uniform and rapid self-assembly of lipids and nucleic acids under fluid shear force to form lipid nanoparticles encapsulating siRNA.

[0029] The post-processing in step (3) is purification and replacement, which includes: using ultrafiltration to remove residual ethanol from the system and completely replacing the buffer with neutral phosphate buffer PBS.

[0030] The ultrafiltration technology uses an ultrafiltration membrane or ultrafiltration tube with a molecular weight cutoff of approximately 30 kDa.

[0031] This invention provides the application of any of the Lcn2-targeting lipid nanoparticles in the preparation of drugs that inhibit bone resorption and promote bone formation.

[0032] This invention provides the use of any of the Lcn2-targeting lipid nanoparticles in the preparation of medicaments for the prevention and / or treatment of osteoporosis. The osteoporosis includes one or more of the following: metabolic liver disease-related osteoporosis, non-alcoholic fatty liver disease (NAFLD)-related osteoporosis, non-alcoholic steatohepatitis (NASH)-related osteoporosis, hepatic LCN2 elevation-related bone loss, high-turnover osteoporosis, and bone metabolic diseases with abnormal liver-bone axis communication.

[0033] Furthermore, the osteoporosis is osteoporosis associated with metabolic liver disease or osteoporosis associated with non-alcoholic fatty liver disease.

[0034] In the application of this invention in the preparation of drugs for the prevention or treatment of NAFLD-related osteoporosis, the lipid nanoparticles are delivered to the liver via intravenous injection. In a preferred embodiment, a twice-a-week injection frequency is adopted for continuous administration to achieve stable and efficient knockdown of liver-derived Lcn2 without interfering with the body's normal systemic glucose or liver lipid metabolism.

[0035] The technical solution of this invention is based on an innovative "liver-bone axis" cross-organ regulatory mechanism: the lipid formulation such as DLin-MC3-DMA endows the nanoparticles with high hepatocyte-targeting. When LNP-siLcn2 is taken up by hepatocytes, the released siRNA specifically degrades Lcn2 mRNA, cutting off the secretion of pathogenic LCN2 protein at its source. As the level of circulating LCN2 decreases, a bidirectional osteoprotective effect is generated: firstly, in osteoclasts, transferrin receptor (TFRC)-mediated iron uptake is downregulated, thereby blocking osteoclast differentiation (reducing bone resorption); secondly, in osteoblasts, the binding and shielding of LCN2 to the Wnt co-receptor LRP6 is released, restoring the typical Wnt / β-catenin osteogenic signaling pathway (increasing bone formation). Through dual-effect synergistic remodeling of bone homeostasis, the destruction of bone microstructure caused by metabolic abnormalities is fundamentally reversed.

[0036] Beneficial effects

[0037] (1) Innovative dual-effect bone homeostasis regulation mechanism, overcoming the shortcomings of existing drugs with single targets: The LNP-siLcn2 of this invention can target and downregulate the expression of liver-derived LCN2, thereby producing a dual osteoprotective effect: In osteoclasts, it significantly inhibits TFRC-mediated iron uptake, blocking osteoclast formation to reduce bone resorption; in osteoblasts, it relieves the binding inhibition of LCN2 to LRP6, restoring the Wnt / β-catenin signaling pathway to promote bone formation. This dual-effect mechanism of simultaneously inhibiting bone resorption and promoting bone formation fills the gap in current clinical single-target anti-osteoporosis drugs.

[0038] (2) High targeting specificity and excellent physicochemical properties, with high delivery efficiency: This invention uses an ionizable lipid formulation based on DLin-MC3-DMA to prepare nanoparticles with uniform morphology (hydrodynamic particle size of about 100 nm), good dispersibility (PDI of about 0.225), and high encapsulation efficiency (about 90%). In vivo distribution experiments confirmed that the formulation has extremely strong hepatocyte targeting, and after intravenous injection, it is highly enriched in the liver, while it is almost not distributed in non-target organs such as the heart, spleen, lungs, kidneys, and bones, significantly reducing the risk of off-target toxicity.

[0039] Furthermore, under the same lipid composition, siRNA dosage, and administration conditions, this invention used LNP-NC encapsulated with non-targeting siRNA as a control. Compared with the HFD-LNP-NC group, the HFD-LNP-siLcn2 group not only significantly reduced liver tissue Lcn2 mRNA and serum LCN2 levels, but also increased Ct.Th, cortical bone vBMD, cancellous bone vBMD, BV / TV, Tb.Th, and Conn.D, and decreased Ct.Po(tot), Tb.Sp, and SMI (all P < 0.05, see Table 1). This control indicates that the above-mentioned osteoprotective effect is not produced by conventional LNP vectors alone, but originates from the functional combination of specific siLcn2 sequences and liver delivery vectors, forming a continuous technical effect of "liver-targeted delivery - Lcn2 silencing - bidirectional restoration of bone homeostasis".

[0040] (3) Excellent safety and metabolic neutrality, avoiding the side effects of systemic intervention: Existing global knockout or systemic inhibition of Lcn2 can lead to severe systemic energy and lipid metabolism disorders. The liver-targeted LNP-siLcn2 of this invention effectively rescues NAFLD-induced bone loss and improves bone microstructure (increasing bone volume fraction and cortical bone thickness) without affecting the body's weight gain, blood glucose level, glucose tolerance, or lipid deposition status in the liver, demonstrating extremely high metabolic safety and clinical application prospects.

[0041] (4) Significant economic and social benefits: With the soaring global incidence of NAFLD, the patient population of metabolic-driven osteoporosis is enormous. This invention is the first to propose a treatment strategy targeting the "liver-bone axis". The LNP carrier system used has a mature process and is easy to scale up for production. It provides a novel, safe, and convertible nucleic acid nano-targeted therapy drug for the large patient population with metabolic liver disease and osteoporosis, and has great market development value. Attached Figure Description

[0042] Figure 1 Knocking down Lcn2 in the liver significantly alleviated bone loss and improved bone remodeling indices induced by a high-fat diet (HFD) in obese mice. (A) shows the experimental design, with the left side displaying the chemical components for preparing LNP-siLcn2 (DLin-MC3-DMA, DMG-PEG2000, DSPC, cholesterol, and siLcn2); the right side shows the mouse grouping and administration timeline: mice were fed a normal diet (NCD) or a high-fat diet (HFD) from week 8 to week 25. The HFD group received periodic injections of LNP-NC nanoparticles encapsulating negative controls or LNP-siLcn2 nanoparticles targeting Lcn2 from week 20 to week 25, and samples were collected for analysis at week 25. (BC) Real-time quantitative PCR (RT-qPCR) was used to detect the relative expression level of Lcn2 mRNA in the tissues of each group of mice, and enzyme-linked immunosorbent assay (ELISA) was used to detect LCN2 levels in the serum of each group of mice. The protein content (C) was used to verify the knockdown effect; (D) were representative micro-CT three-dimensional reconstruction images of the femur of mice in the NCD-LNP-NC group, HFD-LNP-NC group and HFD-LNP-siLcn2 group, from top to bottom: distal femoral cancellous bone, cortical bone shaft and longitudinal section of femur; (EJ) were the quantitative results of bone metrology parameters obtained by micro-CT analysis, including the ratio of trabecular bone volume to total bone volume (Trabecular BV / TV) (E), trabecular bone mineral density (Trabecular vBMD) (F), structural model index (SMI) (G), cortical bone thickness (Ct.Th) (H), cortical bone mineral density (Cortical vBMD) (I) and cortical bone total porosity (Ct.Po) (tot) (J); (KL) were the levels of bone formation marker PINP (K) and bone resorption marker CTX-1 (L) in the serum of mice in each group detected by ELISA.

[0043] Figure 2Characterization, in vivo targeting, and knockdown efficiency evaluation of lipid nanoparticles targeting Lcn2 (LNP-siLcn2); (AB) Particle size distribution histograms (left) and representative transmission electron microscopy (TEM) images (right, scale bar 50 nm) of empty lipid nanoparticles LNP (A) and lipid nanoparticles encapsulated with siLcn2 LNP-siLcn2 (B); (CF) Physicochemical characterization of LNP and LNP-siLcn2, including hydrodynamic particle size (C), polydispersity index (PDI) (D), surface potential (E), and small interfering RNA encapsulation efficiency (F) of LNP-siLcn2; (G) Representative confocal microscopy image showing the morphology or uptake of LNP (FAM-Green) encapsulated with green fluorescent labeling FAM-siRNA in vitro; (H) Real-time quantitative PCR (RT-qPCR) detection of Lcn2 mRNA in cells or tissues after treatment with LNP and LNP-siLcn2 groups. The relative expression levels of LNPs were analyzed. Among them, (I) representative in vivo fluorescence imaging images at different time points (0.5, 1, 4, 8, 24, 48 hours) after intravenous injection of Cy5.5-labeled LNPs in mice, showing the spatiotemporal distribution and targeted aggregation of LNPs in vivo; and (JK) representative fluorescence imaging images of major organs (heart, liver, spleen, lung, kidney, femur) in mice at different time points (4, 24, 48 hours) after intravenous injection of PBS or Cy5.5-LNPs in mice (J) and quantitative analysis of the corresponding tissue fluorescence intensity (K). Detailed Implementation

[0044] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0045] Unless otherwise stated, the reagents, materials, cell cultures, animal experiments, nucleic acid detection, ELISA, Micro-CT, dynamic light scattering, transmission electron microscopy, and fluorescence imaging described below can all be performed according to conventional methods in the art or the instructions for commercial reagents and instruments. Those skilled in the art can make equivalent substitutions for lipid ratios, buffer systems, nucleic acid modification methods, dosages, and dosing frequencies without departing from the inventive concept. Any resulting solutions that can target and reduce hepatocyte-derived Lcn2 / LCN2 and improve osteoporosis are all within the scope of this invention.

[0046] Example 1

[0047] Composition of LNP-siLcn2 targeted lipid nanoparticles

[0048] This embodiment provides an Lcn2-targeting lipid nanoparticle for treating osteoporosis, denoted as LNP-siLcn2. The LNP-siLcn2 consists of a nucleic acid drug and a lipid carrier, wherein the nucleic acid drug is a small interfering RNA (siLcn2) that specifically targets and silences Lcn2 / LCN2 gene expression, and the lipid carrier is used to encapsulate and deliver siLcn2 to hepatocytes.

[0049] The siLcn2 uses the following double-stranded sequence:

[0050] The chain of justice is 5′-CGAUGUACAGCACCAUCUAUGTT-3′;

[0051] The antisense strand is 5′-CAUAGAUGGUGCUGUACAUCGTT-3′. The terminal “T” can serve as a 3′ deoxythymidine dangling base.

[0052] As a negative control, a non-targeting siRNA can be used, with the sense strand being 5′-UUCUCCGAACGUGUCACGUTT-3′, as shown in SEQ ID NO.3; and the antisense strand being 5′-ACGUGACACGUUCGGAGAATT-3′, as shown in SEQ ID NO.4.

[0053] The lipid carrier comprises an ionizable lipid DLin-MC3-DMA, a helper phospholipid DSPC, cholesterol, and a PEGylated lipid DMG-PEG2000. The proportions of the lipid carrier can be finely adjusted while maintaining liver delivery capability and siRNA encapsulation capability. In an optional embodiment, DLin-MC3-DMA is 45-55 mol%, DSPC is 8-12 mol%, cholesterol is 35-42 mol%, and DMG-PEG2000 is 1-3 mol.

[0054] Specifically, the preferred molar ratio of the four lipids used in this embodiment is DLin-MC3-DMA:DSPC:cholesterol:DMG-PEG2000 = 50:10:38.5:1.5. In other words, when expressed in the order of DLin-MC3-DMA, DMG-PEG2000, DSPC, and cholesterol, the molar ratio is 50:1.5:10:38.5.

[0055] Example 2

[0056] Preparation method of LNP-siLcn2:

[0057] Step (1) Preparation of the lipid phase:

[0058] According to the molar ratio described in Example 1, DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000 were weighed, and each lipid component was fully dissolved in anhydrous ethanol to obtain a homogeneous, transparent, or substantially transparent lipid ethanol solution.

[0059] Step (2) Preparation of the aqueous phase:

[0060] The siLcn2 is dissolved in an acidic buffer solution, specifically, in this embodiment, a citrate buffer solution with pH 4.0 is preferred. The N / P ratio is calculated based on the molar ratio of protonable nitrogen atoms in the lipid to phosphate groups of siRNA. In optional embodiments, the N / P ratio can be 4-8, with the aim of obtaining nanoparticles with uniform particle size, high encapsulation efficiency, and stable Lcn2 silencing effect. Specifically, in this embodiment, an N / P ratio of 6 is preferred.

[0061] Step (3) Microfluidic mixing:

[0062] The lipid phase obtained in step (1) and the aqueous phase obtained in step (2) are respectively introduced into a microfluidic mixing system, so that the two are rapidly and uniformly mixed within the microfluidic chip, and self-assembled under fluid shearing to form lipid nanoparticles encapsulating SiLcn2. Preferably, this embodiment uses a Nano S microfluidic mixing system; if other microfluidic devices are used, the total flow rate and the organic phase / aqueous phase flow ratio can be adjusted according to the device instructions to maintain the hydrodynamic particle size of the obtained particles at approximately 70-150 nm and the polydispersity index below 0.30.

[0063] Step (4), purification and buffer replacement:

[0064] The mixture obtained in step (3) is purified by ultrafiltration. Specifically, in this embodiment, an ultrafiltration membrane or ultrafiltration tube with a molecular weight cutoff of approximately 30 kDa is preferably used to remove residual ethanol and unencapsulated small molecule components. The system buffer is then replaced with neutral phosphate buffer (PBS) to obtain the LNP-siLcn2 formulation. The concentration of the obtained formulation can be adjusted according to subsequent uses, and it can be stored under aseptic conditions or used for animal administration, cell experiments, and quality testing.

[0065] Example 3

[0066] Physicochemical characterization and quality control of LNP-siLcn2:

[0067] Dynamic light scattering (DLS) was used to detect the hydrodynamic particle size, polydispersity index (PDI), and zeta potential of LNP-siLcn2 at 25 °C; transmission electron microscopy (TEM) was used to observe the particle morphology; and the siRNA encapsulation efficiency was detected using the Quant-iT™ RiboGreenRNA Assay Kit, and the encapsulation efficiency was calculated as “(total siRNA amount - free siRNA amount) / total siRNA amount × 100%”.

[0068] The test results showed that both the blank LNP and LNP-siLcn2 prepared according to Examples 1 and 2 exhibited a uniform spherical morphology. The hydrodynamic particle size of LNP-siLcn2 was approximately 100 nm, the PDI was approximately 0.225, the Zeta potential was approximately -9.3 mV, and the siRNA encapsulation efficiency was approximately 90%. These results indicate that the encapsulation of siLcn2 did not significantly disrupt the stability of the lipid nanoparticles, and the resulting formulation possessed nanoscale and dispersibility suitable for in vivo liver delivery.

[0069] Example 4

[0070] In vitro hepatocyte uptake and Lcn2 silencing validation of LNP-siLcn2

[0071] To verify the delivery capability of the lipid nanoparticles to hepatocytes, 5′ FAM-labeled siRNA was encapsulated in LNPs, yielding FAM-siRNA-LNPs. Primary hepatocytes were incubated with FAM-siRNA-LNPs at 37 °C for 24 h, followed by washing three times with PBS, fixation with 4% paraformaldehyde, nuclear counterstaining with DAPI, and observation using confocal microscopy. The results showed that FAM fluorescence signal was clearly detectable within hepatocytes, indicating that the lipid nanoparticles could effectively deliver siRNA into hepatocytes.

[0072] To verify the Lcn2 silencing activity, primary hepatocytes were treated for 24 h with blank LNP, LNP-NC encapsulated with non-targeting siRNA, and LNP-siLcn2, respectively. Total RNA was extracted from the cells, and the Lcn2 mRNA expression level was detected by RT-qPCR. 18S rRNA was used as an internal control, and the relative expression level was calculated using the 2^-ΔΔCt method. Compared with blank LNP or LNP-NC, LNP-siLcn2 significantly reduced the Lcn2 mRNA level in hepatocytes, indicating that the formulation of this invention can release effective siRNA in hepatocytes and achieve target gene silencing.

[0073] Example 5

[0074] In vivo liver-targeted distribution validation of LNP-siLcn2 lipotransmitter:

[0075] To verify the in vivo tissue distribution characteristics of the lipid nanoparticles, LNPs were labeled with the near-infrared fluorescent dye Cy5.5 to prepare Cy5.5-LNPs. Eight-week-old male C57BL / 6J mice were injected with Cy5.5-LNPs via the tail vein; PBS-injected mice served as negative controls. Whole-body imaging was performed using an in vivo fluorescence imaging system at 0.5 h, 1 h, 4 h, 8 h, 24 h, and 48 h after administration.

[0076] In vivo imaging results showed that Cy5.5-LNP was mainly enriched in the abdominal liver region after administration, reaching a strong fluorescence signal at 4-8 h. Further, mice were sacrificed at 4 h, 24 h, and 48 h post-administration, and key tissues such as the heart, liver, spleen, lung, kidney, and femur were isolated for in vitro fluorescence imaging. In vitro imaging results showed that the fluorescence signal was mainly retained in the liver, while the signal in the heart, spleen, lung, kidney, and femur was low or close to background levels. These results demonstrate that the DLin-MC3-DMA lipid formulation used in this invention can achieve highly liver-selective in vivo delivery, thereby reducing the risk of exposure to non-target organs.

[0077] Example 6

[0078] Animal studies of LNP-siLcn2 in treating NAFLD-related osteoporosis:

[0079] Male C57BL / 6J mice were fed either a normal diet (NCD) or a high-fat diet (HFD) for 12 weeks to establish a NAFLD-related bone loss model. While continuing the HFD diet, the mice were randomly divided into an LNP-NC group and an LNP-siLcn2 group. The LNP-siLcn2 group received LNP-siLcn2 via tail vein injection at a dose of 1 mg / kg siRNA, administered every 3 days for a total of 10 doses. The LNP-NC group received lipid nanoparticles encapsulating non-targeting siRNA. Mice were sacrificed 24 hours after the last administration, and liver, serum, and femur samples were collected.

[0080] The level of Lcn2 mRNA in liver tissue was detected by RT-qPCR, and the level of LCN2 in serum was detected by ELISA. The results showed that compared with the HFD-LNP-NC group, the levels of Lcn2 mRNA in liver tissue and LCN2 in serum were significantly reduced in the HFD-LNP-siLcn2 group, indicating that the formulation of the present invention can effectively knock down liver-derived Lcn2 in vivo.

[0081] Micro-CT analysis was further used to analyze the microstructure of the distal femur. Compared with the HFD-LNP-NC group, mice in the HFD-LNP-siLcn2 group showed increased cancellous bone volume fraction (BV / TV) and cancellous bone volume mineral density (vBMD), and decreased structural model index (SMI); cortical bone thickness (Ct.Th) and cortical bone vBMD were increased, while total cortical porosity (Ct.Po(tot)) was decreased. Serum bone turnover marker detection showed that LNP-siLcn2 treatment increased the bone formation marker PINP and decreased the bone resorption marker CTX-I, suggesting that this agent can simultaneously inhibit bone resorption and promote the restoration of bone homeostasis related to bone formation.

[0082] Table 1. Effects of LNP-siLcn2 on bone microstructural parameters in HFD-induced osteoporosis mice (mean ± SEM)

[0083]

[0084] The above results demonstrate that LNP-siLcn2 can effectively reduce liver-derived LCN2 levels, improve the microstructure of cancellous and cortical bone, and reshape the bone turnover state in a direction conducive to bone mass recovery in the NAFLD-related osteoporosis model.

[0085] Example 7

[0086] Mechanism verification of the dual-effect osteoprotective effect of LNP-siLcn2

[0087] To verify the effect of LCN2 on osteoclasts, primary osteoclasts or osteoclast precursor cells were treated with recombinant mouse LCN2 protein at a preferred concentration of 200 ng / mL under RANKL induction conditions. Results showed that LCN2 upregulated osteoclast differentiation-related genes and proteins, including Nfatc1, Mitf, Pu.1, Dc-stamp, and Ctsk, and increased the number of TRAP-positive mature osteoclasts. Further analysis of total cellular iron content revealed that LCN2 treatment increased intracellular iron levels in osteoclasts. Intervention with the iron chelator deferoxamine (DFO, preferably 10 μM) or Tfrc siRNA significantly weakened the LCN2-induced enhancement of osteoclast differentiation, indicating that LCN2 enhances osteoclastogenesis by upregulating the transferrin receptor TFRC and promoting iron uptake.

[0088] To verify the effect of LCN2 on osteoblasts, MC3T3-E1 osteoblastic progenitor cells were treated with recombinant mouse LCN2 protein at a preferred concentration of 200 ng / mL under osteogenic induction conditions. Results showed that LCN2 reduced the expression of osteogenic-related genes or proteins such as Runx2, Col1a1, and Osx, and decreased the positivity of ALP staining and Alizarin Red mineralization staining. Immunoprecipitation experiments showed that LCN2 specifically bound to the Wnt co-receptor LRP6, but did not show significant binding to LRP5, AXIN1, or AXIN2. LCN2 treatment also promoted cytoplasmic β-catenin phosphorylation and inhibited β-catenin nuclear translocation; overexpression of β-catenin partially restored osteogenic differentiation inhibited by LCN2. These results indicate that LCN2 inhibits osteoblast differentiation by binding to LRP6 and suppressing the classical Wnt / β-catenin signaling pathway.

[0089] As can be seen from Examples 4-6, the LNP-siLcn2 of the present invention reduces circulating LCN2 from the source by targeting and reducing the expression of hepatocyte-derived Lcn2 / LCN2. On the one hand, it blocks the LCN2-TFRC-iron uptake axis to reduce osteoclast formation and bone resorption, and on the other hand, it relieves the inhibition of LRP6-Wnt / β-catenin osteogenic pathway by LCN2 to promote osteoogenesis, thereby producing a dual osteoprotective effect that simultaneously inhibits bone resorption and promotes bone formation.

[0090] Example 8

[0091] Pharmaceutical formulation, administration method and indications

[0092] The LNP-siLcn2 of the present invention can be prepared into an injectable formulation with pharmaceutically acceptable carriers or excipients. Preferably, the formulation uses PBS or other physiologically compatible buffers as a dispersion medium and is administered intravenously to achieve liver-targeted delivery. In animal experiments, the preferred dosage is 1 mg / kg siRNA, administered once every 3 days for a total of 10 times. In optional embodiments, the siRNA dosage can be adjusted to 0.1-3 mg / kg, preferably 0.5-2 mg / kg, based on the subject's weight, disease severity, dosing cycle, and safety evaluation results; the dosing frequency can be once every 2-4 days or 1-3 times per week.

[0093] The LNP-siLcn2 of this invention can be used to prepare drugs for the treatment or prevention of osteoporosis, particularly suitable for osteoporosis associated with metabolic liver disease, osteoporosis associated with non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH), bone loss associated with elevated LCN2 in the liver, high-turnover osteoporosis, and bone metabolic diseases accompanied by abnormal liver-bone axis communication. For different species or populations, siRNAs with the same silencing function can be designed based on the corresponding Lcn2 / LCN2 mRNA sequences, and encapsulated and delivered to the liver using the lipid nanoparticle system of this invention.

[0094] Explanation of English Abbreviations and Chinese Names

[0095]

Claims

1. An Lcn2-targeting lipid nanoparticle, characterized in that, The Lcn2-targeted lipid nanoparticles are core-shell structured nanoparticles, wherein the core includes a nucleic acid drug and the shell includes a lipid carrier; Lipid carriers include ionizable lipids, polyethylene glycol-modified lipids, cofactor phospholipids, and cholesterol.

2. The Lcn2-targeting lipid nanoparticle of claim 1, wherein, The nucleic acid drug is siLcn2, wherein the double-stranded sequence of siLcn2 is: 5′-CGAUGUACAGCACCAUCUAUGTT-3′ for the sense strand and 5′-CAUAGAUGGUGCUGUACAUCGTT-3′ for the antisense strand.

3. The Lcn2-targeting lipid nanoparticle of claim 1, wherein, The lipid carrier is composed of DLin-MC3-DMA, DMG-PEG2000, DSPC, and cholesterol, wherein the lipid carrier contains 45-55 mol% DLin-MC3-DMA, 8-12 mol% DSPC, 35-42 mol% cholesterol, and 1-3 mol% DMG-PEG2000. The nitrogen-to-phosphorus ratio of lipids to siRNA in the Lcn2-targeted lipid nanoparticles is 4-8.

4. The Lcn2-targeting lipid nanoparticle of claim 3, wherein, The lipid carrier contained 50 mol% DLin-MC3-DMA, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG2000. The nitrogen-to-phosphorus ratio of lipids to siRNA in the Lcn2-targeted lipid nanoparticles is 6.

5. A method for preparing Lcn2-targeted lipid nanoparticles according to any one of claims 1-4, comprising: (1) The lipid carrier component was dissolved in anhydrous ethanol to obtain the lipid phase; (2) Dissolve the nucleic acid drug in an acidic buffer solution to obtain an aqueous phase; (3) The lipid phase and aqueous phase are mixed and self-assembled by a microfluidic mixing system, and then post-processed to obtain Lcn2 targeted lipid nanoparticles.

6. The preparation method according to claim 5, characterized in that, In step (2), the acidic buffer solution is a citrate buffer solution with a pH of 4.

0.

7. The preparation method according to claim 5, characterized in that, The post-processing in step (3) is purification and replacement, which includes: using ultrafiltration to remove residual ethanol from the system and completely replacing the buffer with neutral phosphate buffer PBS.

8. The use of the Lcn2-targeting lipid nanoparticles according to any one of claims 1-4 in the preparation of a drug for inhibiting bone resorption and promoting bone formation.

9. The use of the Lcn2-targeting lipid nanoparticles according to any one of claims 1-4 in the preparation of a medicament for the prevention and / or treatment of osteoporosis.

10. The application according to claim 9, characterized in that, The osteoporosis mentioned includes one or more of the following: osteoporosis associated with metabolic liver disease, osteoporosis associated with non-alcoholic fatty liver disease (NAFLD), osteoporosis associated with non-alcoholic steatohepatitis (NASH), bone loss associated with elevated LCN2 in the liver, high-turnover osteoporosis, and bone metabolic diseases with abnormal liver-bone axis communication.

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