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Tea polyphenol nanoparticles enable targeted siRNA delivery and multi-bioactive therapy for abdominal aortic aneurysms

Journal of Nanobiotechnology volume 22, Article number: 471 (2024) Cite this article

Abstract

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease, while there is a lack of pharmaceutical interventions to halt AAA progression presently. To address the multifaceted pathology of AAA, this work develops a novel multifunctional gene delivery system to simultaneously deliver two siRNAs targeting MMP-2 and MMP-9. The system (TPNs-siRNA), formed through the oxidative polymerization and self-assembly of epigallocatechin gallate (EGCG), efficiently encapsulates siRNAs during self-assembly. TPNs-siRNA safeguards siRNAs from biological degradation, facilitates intracellular siRNA transfection, promotes lysosomal escape, and releases siRNAs to silence MMP-2 and MMP-9. Additionally, TPNs, serving as a multi-bioactive material, mitigates oxidative stress and inflammation, fosters M1-to-M2 repolarization of macrophages, and inhibits cell calcification and apoptosis. In experiments with AAA mice, TPNs-siRNA accumulated and persisted in aneurysmal tissue after intravenous delivery, demonstrating that TPNs-siRNA can be significantly distributed in macrophages and VSMCs relevant to AAA pathogenesis. Leveraging the carrier’s intrinsic multi-bioactive properties, the targeted siRNA delivery by TPNs exhibits a synergistic effect for enhanced AAA therapy. Furthermore, TPNs-siRNA is gradually metabolized and excreted from the body, resulting in excellent biocompatibility. Consequently, TPNs emerges as a promising multi-bioactive nanotherapy and a targeted delivery nanocarrier for effective AAA therapy.

Graphical Abstract

Introduction

Abdominal aortic aneurysm (AAA) is a prevalent vascular disorder characterized by localized, full-layer enlargement of the abdominal aorta and degenerative impairment of the arterial wall [1, 2]. AAA, which typically occurs in the infrarenal abdominal aorta, can suddenly rupture, posing serious risks and potentially causing death [3, 4]. Clinical tests are used to diagnose AAA, which is classified as an abdominal aorta with a maximum diameter of ≥ 3 cm or an aorta that has expanded to 1.5 times the normal diameter [5, 6]. Most individuals with AAA remain asymptomatic during the early stages and are often diagnosed incidentally through routine physical examinations or during treatment of unrelated conditions. The most critical complication of AAA is aneurysm rupture, which is associated with a high mortality rate (up to 90%) [7,8,9]. Surgical intervention is the primary approach for treating AAA, however, its application is restricted to patients that meet specific surgical criteria (diameter > 5 cm) [10,11,12]. Patients with smaller AAAs (3–5 cm) follow a “watchful waiting” strategy, incurring substantial psychological and economic burdens [13,14,15]. The expansion of aneurysms is unpredictable, increasing the risk of rupture. Unfortunately, no effective pharmacological treatments are currently available to manage and slow the progression of AAA [16,17,18].

The key pathological features of AAA include inflammatory cell infiltration, oxidative stress responses, degradation and remodelling of the extracellular matrix (ECM), vascular smooth muscle cell (VSMC) calcification, and apoptosis; however, the aetiology and pathogenesis of AAA are not completely understood [19,20,21]. Recent studies suggest that matrix metalloproteinases (MMPs) play crucial roles in AAA pathogenesis, and their dysregulation, which leads to excessive production, is implicated in disease progression [22]. In an inflammatory milieu, MMPs contribute to the degradation of collagen and elastic fibres in the intima and adventitia of the abdominal aorta, resulting in weakened arterial wall elasticity and compromised integrity [23, 24]. Studies on AAA in humans and rodents have revealed that MMPs play a pivotal role in breaking down the ECM throughout the development of AAA [25, 26]. Various MMP types are expressed in AAA tissue, with MMP-2 and MMP-9 being particularly prominent [27]. Targeting the synthesis, secretion, and function of MMP-2 and MMP-9 has been a prospective approach for treating AAA [28]. In animal models, therapeutic efficacy has been achieved by systemic administration of MMP inhibitors [29]. However, the successful translation of these inhibitors into clinical applications has been hindered by several challenges, such as limited effectiveness at lower doses and systemic side effects at higher doses.

The use of small interfering RNA (siRNA) in targeted therapy has gained widespread application for treating various diseases owing to its notable specificity, high efficiency, and favourable safety profile compared to small molecule inhibitors. [30, 31]. The siRNA exerts a specific silencing effect on target genes, offering a novel approach for treating AAA by interfering with the expression of the MMP-2 and MMP-9 genes. Unlike MMP inhibitors that target cell surfaces or circulate throughout the bloodstream, siRNAs operate at the intracellular mRNA level, enabling more specific downregulation of MMP-2 and MMP-9 protein expression. However, before reaching target cells, siRNAs are susceptible to degradation by endogenous plasma nucleases. Additionally, free siRNA cannot easily traverse the cell membrane to exert intracellular affects because the molecules are hydrophilic, have a large molecular weight, and are negatively charged [32, 33]. Therefore, the development of specific delivery vehicles is crucial for transporting siRNA to AAA lesion cells, enabling MMPs silencing and achieving effective disease treatment.

This study developed a multifunctional siRNA delivery vehicle, known as tea polyphenols nanoparticles (TPNs), to address the complex pathological processes involved in AAA progression. TPNs, a nanomaterial recently reported by our group, are formed through the oxidation, polymerization and self-assembly of epigallocatechin gallate (EGCG), a tea polyphenol compound, and exhibits broad-spectrum free radical scavenging capabilities [34, 35]. Herein, we demonstrated that TPNs efficiently encapsulate anti-MMP-2/MMP-9 siRNA during the self-polymerization process, enhancing siRNA stability under physiological conditions and facilitating siRNA transfection into cells. Upon intracellular delivery, TPNs enable rapid escape from lysosomes, effectively silencing MMP-2 and MMP-9. Importantly, as a multi-active biomaterial, TPNs was found to effectively modulate local inflammation and oxidative stress at the AAA site, promote macrophage M1-to-M2 repolarization, and inhibit VSMC calcification and apoptosis. These activities collectively contribute to alleviating AAA disease and synergistically enhancing the efficacy of anti-MMP2/MMP9 siRNA. In a mouse AAA model, the study confirmed the targeting, in vivo efficacy, and safety of this nanomedicine, presenting a novel strategy for developing therapeutic drugs for AAA.

Materials and methods

Materials

MMP-2 siRNA, MMP-9 siRNA and GP-transfect-Mate reagent were purchased from GenePharma (Shanghai, China). The FAM- or Cy5.5-labelled counterparts (random sequence, nonfunctional) were purchased from Bioegene (Shanghai, China). The primers for MMP-2, MMP-9, iNOS, Arg-1, CD80, CD206 and GAPDH were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). EGCG was purchased from Huagao Biological Products (Chengdu, China). Manganese chloride tetrahydrate (MnCl2·4H2O) was purchased from Aladdin (Shanghai, China). 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) was purchased from Solarbio (Beijing, China). Ethylenediamine tetraacetic acid dipotassium salt (EDTA·2 K) was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). RPMI-1640 cell culture medium and FBS were purchased from Gibco (Grand Island, NY, USA). Lipopolysaccharide (LPS) was purchased from Biosharp (Hefei, China). 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Shanghai Biological Technology Development Co., Ltd. (Shanghai, China). 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and dihydroethidium (DHE) were purchased from Macklin Co., Ltd. (Shanghai, China). Inhibition and produce superoxide anion assay kit, hydroxyl free radical assay kit, alanine aminotransferase kit (ALT), aspartate aminotransferase kit (AST), creatinine kit (Cre), and blood urea nitrogen kit (BUN) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). A nitric oxide detection kit, H2O2, a cell counting kit-8 (CCK-8), a nitric oxide assay kit and 4-amino,5-aminomethyl-2′,7′-difluorescein diacetate (DAF-FM DA) were purchased from Beyotime Biotechnology (Shanghai, China). An oxidative stress probe (ROS-ID hypoxia/oxidative stress detection kit) was purchased from Enzo Life Science (New York, USA). A 2′,7′-dichlorodihydrofluorescein diacetate (DCHF-DA) probe and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H tetrazolium bromide (MTT) were purchased from Sigma‒Aldrich (St. Louis, USA). Hydroxyphenyl fluorescein (HPF) was obtained from Maokang Biotechnology Co., Ltd. (Shanghai, China). Annexin V-FITC Apoptosis Detection Kit was provided by BD biosciences Co., Ltd. (San Jose, CA, USA). The antibodies of MMP-2 and MMP-9 were from Abcam (USA). The anti-iNOS antibody, anti-Arg-1 antibody and TUNEL Apoptosis Assay Kit was purchased from Boster (Wuhan, China). An MMP-2 ELISA kit was purchased from Huamei Biotechnology Co., Ltd. (Wuhan, China). The anti-CD31 antibody, anti-CD68 antibody, anti-α-SMA antibody, MMP-9 ELISA kit and IL-6 ELISA kit were purchased from Protein tech (Wuhan, China). The TNF-𝛼 and IL-1 ELISA kits were purchased from Jianglai Biotechnology Co., Ltd. (Shanghai, China).

Preparation of TPNs-siRNA

EGCG forms TPNs through Mn2+-catalysed polymerization under alkaline conditions. First, 2 mM MnCl2 was mixed with 2.5 mM EGCG in HEPES buffer (10 mM, pH 8.0) and vigorously stirred in a water bath with a constant temperature of 37 °C. After 1 h of reaction, the TPNs were collected by centrifugation (16000 rpm, 10 min), washed twice with HEPES buffer (10 mM, pH 7.4), sonicated for dispersal and stored at 4 °C for further use.

To prepare TPNs-siRNA, EGCG solution was added to HEPES buffer at pH 8.0, and the same volumes of siMMP-2 and siMMP-9 solutions were added for 30 min. Then, the MnCl2 solution was slowly added dropwise and left to react for 2 h at a constant temperature of 37 °C. TPNs-siRNA nanoparticles were collected and resuspended as described above. FAM- or Cy5.5-labelled nanoparticles were obtained by encapsulating FAM-siRNA or Cy5.5-siRNA using the same experimental method.

Characterization of TPNs-siRNA

A ZetaSizer Nano ZS (Malvern Instruments, UK) was used to determine the particle size and ζ potential. Transmission electron microscopy (TEM) (Titan G2-F20, FEI, USA) was used to observe the morphology of the nanoparticles. The FAM-siRNA loading capacity was quantified by collecting the unloaded FAM-siRNAs in the supernatant and performing PAGE. The percentage of encapsulated siRNA was quantified by ImageJ. The stability in different media (10 mM pH 7.4 HEPES, 10 mM pH 7.4 PBS, H2O, saline, and cell culture media) was studied by monitoring the change in particle size over time. The protective effect of the siRNAs was investigated by incubating the naked siRNA or TPNs-FAM-siRNA with various concentrations of FBS for 3 h at 37 °C, followed by visualization by PAGE.

DPPH• scavenging

Different concentrations of TPNs-siRNA (0, 5, 10, 25, 50 and 100 µg/mL, 20 µL) were mixed with DPPH ethanol solution (0.3 mM, 200 µL) and incubated in the dark. Absorbance at 517 nm was recorded every 5 min using a microplate reader and UV–visible spectra within 400–800 nm was recorded post 40 min.

ABTS+• scavenging

The ABTS+• working solution (800 µL) was added to 200 µL of various concentrations of TPNs-siRNA and incubated in the dark. The UV absorbance at 734 nm was recorded every 5 min using a microplate reader and UV–visible spectra within 400–850 nm was recorded post 30 min.

•OH scavenging

To assess •OH scavenging capacity, •OH was generated by a Fenton-like reaction with the Mn2+/H2O2 system. Simultaneously, different concentrations of TPNs-siRNA (5, 10, 25, 50, 100 and 200 µg/mL) and methylene blue (10 µg/mL) were added. After incubation at 37 °C for 30 min, the UV–vis spectra within 400–800 nm was recorded using a microplate reader.

•O2 − scavenging

To evaluate the •O2− scavenging capacity, •O2− was generated by xanthine and xanthine oxidase. TPNs-siRNA at various concentrations (5, 10, 25, 50, 100 and 200 µg/mL) was added. After 40 min of incubation at 37 °C, Griess reagent was added to begin the chromogenic reaction. A microplate reader was used to record the UV–vis spectra within 400–800 nm.

•NO scavenging

A nitric oxide assay kit was used to assess the ability to scavenge •NO, •NO was generated with sodium nitrate. Simultaneously, different concentrations of TPNs-siRNA were added. After incubation for 10 min, Griess reagent was added for •NO detection. The UV − vis spectra at 400–800 nm were recorded by a microplate reader.

Cell culture

Murine macrophages (RAW264.7) cells and Murine abdominal aorta smooth muscle cells (VSMCs) were obtained from Xiangya Cell Centre (Changsha, China) and cultured at 37 °C in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin–streptomycin solution in a 5% CO2 atmosphere.

MTT assay

RAW264.7 cells and VSMCs were seeded in 96-well plates at a density of 1 × 104 cells per well. After overnight culture, the cells were incubated with different concentrations of TPNs-siRNA for 24 h. Then, the medium was replaced with 100 µL of MTT solution (0.5 mg/mL), followed by a 4 h incubation. Finally, 100 µL of DMSO was added to dissolve the formazan crystals, and the absorbance at 570 nm was measured to calculate the cell viability.

Cellular uptake study

RAW264.7 cells seeded in confocal dishes (2 × 104 cells per dish) were treated for 48 h with or without LPS (10 µg/mL), incubated with FAM-labelled TPNs-siRNA (25 µg/mL) for 4 h and washed three times with PBS. Subsequently, 4% paraformaldehyde was added for 20 min to fix the cells, and DAPI (1 µg/mL) was added for 10 min to stain the cell nuclei. Finally, the cells were imaged by confocal laser scanning microscopy (CLSM). To obtain quantitative results, RAW264.7 cells were seeded in 6-well plates (2 × 105 cells per well) and incubated with or without LPS (10 µg/mL) for 48 h. Afterwards, FAM-labelled TPNs-siRNA was added for 4 h of incubation. Finally, the cells were collected and washed with PBS three times. Flow cytometry was used to analyse the fluorescence intensity of the cell resuspension.

Endo/lysosome escape

RAW264.7 cells were seeded in confocal dishes at a density of 5 × 104 cells per dish and incubated overnight. Then, FAM-labelled TPNs-siRNA (25 µg/mL) was added and incubated for 2, 4–8 h. After washing three times with PBS, Lysosome Tracker Red was added to stain lysosomes for 50 min, and DAPI (1 µg/mL) was added to stain the cell nuclei for 10 min. Finally, the colocalization of siRNA and lysosomes was observed by CLSM.

Anti-MMPs activity

RAW264.7 cells were seeded in 6-well plates and stimulated with LPS (10 µg/mL) for 48 h. The TPNs or TPNs-siRNA (TPNs equivalent concentration 25 µg/mL) were separately added for 24 h of incubation. Afterwards, total RNA was extracted, and the mRNA expression levels of MMP-2 and MMP-9 were quantified by RT‒qPCR (CFX-Connect, Bio-Rad, USA).

Intracellular RONS scavenging activity

RAW264.7 cells were seeded in 24-well plates (5 × 104 cells per well) and incubated with or without LPS (10 µg/mL) for 48 h. Afterwards, TPNs-siRNA (25 µg/mL) was added for 24 h of incubation. Then, each type of RONS probe (including ROS-ID for general ROS, DCFH-DA for H2O2, DHE for •O2−, HPF for •OH/ONOO−, and DAF-FM DA for •NO) was added for 45 min of incubation at 37 °C. The cells were harvested and analysed by flow cytometry for fluorescence quantification.

VSMCs were seeded in 24-well plates (1 × 105 cells per well) and incubated with or without H2O2 (200 µmol/L) for 6 h. Afterwards, TPNs-siRNA (25 µg/mL) was added and incubated for 24 h. Then, the above RONS probes were added separately and incubation at 37 °C. The cells were harvested and analysed by flow cytometry for fluorescence quantification.

Anti-inflammatory activity

RAW264.7 cells were seeded in 6-well plates and stimulated with LPS (10 µg/mL) for 48 h. The TPNs or TPNs-siRNA (TPNs equivalent concentration 25 µg/mL) were separately added for 24 h of incubation. After the above treatments, total protein was extracted from the cells, and the protein expression of IL-1𝛽, TNF-𝛼 and IL-6 was examined by ELISA.

Macrophage repolarization

For the immunofluorescence assay, RAW264.7 cells were seeded in 24-well plates with coverslips and incubated overnight. After treatment for 48 h with LPS (10 µg/mL), the TPNs or TPNs-siRNA (TPNs equivalent concentration 25 µg/mL) were added for 24 h of incubation. Afterwards, 4% paraformaldehyde was added to fix the cells, followed by incubation with iNOS and Arg-1 primary antibodies at 4 °C overnight. Then, the cells were stained with fluorescence-labelled secondary antibodies (Protein tech, China) for 1 h, followed by staining the nuclei with DAPI (1 µg/mL). Finally, the cells were imaged through fluorescence microscopy.

For qPCR, RAW264.7 cells were seeded in 6-well plates and incubated overnight. After treatment for 48 h with LPS (10 µg/mL), TPNs or TPNs-siRNA was added. Total RNA was extracted from cells incubated for 24 h with TRIzol reagent, and mRNA (including CD206, Arg-1, CD80, and iNOS) was extracted.

Detection of calcification in VSMCs

The degree of cell calcification was assessed by staining with Alizarin Red. Specifically, VSMCs were seeded in a 24-well plate at a density of 1 × 104 cells per well and incubated in 1 mL of growth medium for 12 h. TPNs and TPNs-siRNA (TPNs equivalent concentration 25 µg/mL) were separately added, and the cells were incubated. After 24 h, the cells were incubated in elevated Ca/Pi (containing 2 mM CaCl2 and a 2.7 mM mixture of NaH2PO4 and Na2HPO4, pH 7.4) for 1 day. The cells were washed with PBS three times, fixed with 4% paraformaldehyde, and stained with 0.2% (w/v) Alizarin Red for 30 min at room temperature (25 °C). Subsequently, the cells were thoroughly washed with PBS and observed by optical microscopy. The Alizarin Red-stained area is proportional to the extent of calcium mineral deposition in the culture well. For quantitative analysis, the deposited calcium was completely dissolved in 10% cetylpyridinium chloride (Sigma‒Aldrich, U.S.A.), and the absorbance at 405 nm was quantified with a microplate reader (Infinite M200 Pro, Tecan). Additionally, cell viability after different treatments was quantified by CCK-8 assay.

Anti-apoptosis activity

VSMCs were seeded in a 12-well plate at a density of 4 × 105 per well and incubated in 1 mL of growth medium for 12 h. The medium was then replaced with fresh growth medium containing TPNs and TPNs-siRNA (TPNs equivalent concentration 25 µg/mL). After 24 h of incubation, the cells were treated with 600 µmol/L H2O2 solution for 6 h. The cells were subsequently washed twice with cold cell staining buffer, collected by centrifugation, and resuspended in Annexin V binding buffer containing Annexin V-APC (Annexin V) and propidium iodide (PI) solution. Subsequently, the cells were gently vortexed and incubated at room temperature (25 °C) for 15 min in the dark, followed by flow cytometry analysis. The data were analysed using FlowJo V.10 software. Additionally, cell viability after different treatments was quantified by CCK-8 assay.

Animals

Male C57BL/6 mice (6 − 8 weeks old) were provided by Silaike Jingda Laboratory Animal Company (SJA, Hunan, China). The mice were fed in a sterile environment with an unlimited diet. All protocols were approved by the Experimental Animal Ethics Committee of Central South University with the assigned approval/accreditation number of CSU-2023-0299.

Establishment of a mouse model of AAA

AAA in mice was established in mice according to previously reported methods [36]. In brief, after the mice were anaesthetized, the abdominal cavity was opened, and the infrarenal abdominal aorta was exposed. The infrarenal abdominal aorta was treated by placing a piece of sterile cotton gauze (0.5 cm × 0.5 cm) soaked in CaCl2 (0.5 mol/L) on the aorta for 20 min. The treatment area was subsequently washed with warm saline and sutured. The abdominal incision was disinfected with iodine cotton balls. Mice in the sham group were surgically treated with saline instead of CaCl2 following similar procedures.

In vivo tissue biodistribution

The AAA mice were randomly divided into two groups and administered with Free-Cy5.5-siRNA, or TPNs-Cy5.5-siRNA by intravenous injection (Cy5.5-siRNA 1 mg/kg, TPNs-Cy5.5-siRNA 25 mg/kg). Mice in the sham group received an equivalent dose of TPNs-Cy5.5-siRNA intravenously. The aortas (thoracic aorta, abdominal aorta, and bilateral iliac arteries) and major organs (heart, liver, spleen, lung, and kidneys) of the mice were harvested at 8 h post the injection and imaged using in vivo imaging system. The fluorescence intensities were quantitatively analysed using the software provided by the manufacturer.

In a separate study, the AAA mice were euthanized at 3, 12, 24 and 48 h after i.v. injection of TPNs-Cy5.5-siRNA at 25 mg/kg, respectively, and the above organs were collected and imaged with a vivo imaging system.

Histological analysis in aorta

AAA mice and sham mice were treated with TPNs-Cy5.5-siRNA as described above. At 8 h after an intravenous injection was administered, the midsection of the abdominal aorta was harvested from each mouse, frozen directly in O.C.T. Compound, and sectioned at a thickness of 8 μm. Additionally, the obtained cryosections (10 μm) were formed and separately stained with an anti-CD31 antibody, anti-CD68 antibody and anti-α-smooth muscle actin antibody to further evaluate the tissue distribution of TPNs-siRNA. Subsequently, the samples were incubated at 4 °C overnight, and the nuclei were stained with DAPI. Fluorescence images were acquired after the samples were stained with a FITC-labelled secondary antibody.

Therapeutic efficacy in vivo

The male C57BL/6 mice (6 − 8 weeks old) were randomly assigned to four groups (n = 6). The model group, TPNs group and TPNs-siRNA group mice were used to establish AAA models by surgical intervention. On day 28 after CaCl2-induced injury, these three groups of mice were injected with saline, 25 mg/kg of TPNs, or 25 mg/kg of TPNs-siRNA through the tail vein, respectively. The mice without any treatment were injected with the same volume of saline as normal control. Treatment lasts 3 weeks, twice a week. On day 22 after different treatments, the mice were anesthetized. The maximal diameter of each abdominal aorta was measured by a Vevo 2100 micro-ultrasound imaging system (Fujifilm VisualSonics, Canada). After ultrasound examination, the mice were euthanized. The sub-renal abdominal aortic segment was exposed and acquired, and the mid-abdominal aortic segment was made into paraffin sections. The sections were separately subjected to TUNEL, haematoxylin and eosin (H&E), Verhoeff-van Gieson (EVG), and Alizarin Red staining. Additionally, the obtained sections were separately stained with anti-CD31 antibody, anti-CD68 antibody, anti-MMP-2 antibody, or anti-MMP-9 antibody.

Moreover, the remaining abdominal aortas were homogenized and lysed. The concentration of soluble proteins in the cell lysate was determined by a BCA protein assay. The contents of hydrogen peroxide and calcium were quantified by hydrogen peroxide and calcium kits, respectively. The levels of interleukin (IL)-1β, tumour necrosis factor (TNF)-α, interleukin (IL)-6, matrix metalloproteinase (MMP)-2, and matrix metalloproteinase (MMP)-9 were analysed by ELISA.

Safety evaluation

Blood was collected from C57BL/6 mice, placed in anticoagulant tubes and centrifuged at 1000 rpm for 5 min. The erythrocytes were collected and washed with saline several times until the supernatant became clear. Then, the supernatant was discarded, and saline was added to obtain a 2% erythrocyte suspension. Different concentrations of TPNs-siRNA (0–400 µg/mL) were added for 3 h of incubation, with saline used as a negative control and H2O used as a positive control. Afterwards, the state of the cell suspension was observed (no haemolysis, partial haemolysis, complete haemolysis, or agglutination), and the absorbance of the supernatant at 576 nm was measured by a microplate reader (Infinite M200 PRO, Tecan, Austria).

Body weight was recorded during the treatments, and the general behaviours of mice were observed for any signs or symptoms of illness each day. At the termination of the in vivo experiments, the blood was collected, and the serum was separated by centrifugation. The serum levels of ALT, AST, BUN, and Crea were measured by standard kits. Major organs (heart, liver, spleen, lung, and kidney) were harvested, weighed, fixed with 4% paraformaldehyde and subjected to H&E staining.

Statistical analysis

All the quantitative data are expressed as the mean ± standard deviation (SD). GraphPad Prism 8 was used for graphing and statistical analysis. Student’s t test and one-way analysis of variance (ANOVA) were used to assess the differences between two groups and among multiple groups, respectively. Significance was defined as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Results and discussion

Preparation and characterization of TPNs-siRNA

In this study, siRNA sequences targeting MMP-2 and MMP-9 were designed and validated for their silencing efficiency through real-time PCR experiments, with specific siRNA sequences achieving greater than 70% silencing efficacy (Fig. S1A, B). Notably, the first segment of siMMP-2 (5′-CAUACAGGAUCAUUGGUUATT-3′, 5′-UAACCAAUGAUCCUGUAUGTT-3′) and the third segment of siMMP-9 (5′-GCACUGGGCUUAGAUCAUUTT-3′, 5′-AAUGAUCUAAGCCCAGUGCTT-3′) exhibited the most significant silencing effects and were thus chosen for subsequent experiments.

The ability of TPNs to efficiently load siRNA was confirmed by synthesizing FAM-siRNA at varying concentrations (1, 2, 4 µmol/L). The encapsulation rate of the resulting TPNs-siRNA exceeded 80%, and no free nucleic acids were detected in the supernatant (Fig. 1A, B). TPNs have demonstrated efficacy as nucleic acid delivery carriers. In subsequent experiments, a siRNA concentration of 2 µmol/L was utilized (with a molar concentration ratio of 1:1 for siMMP-2 and siMMP-9). The nanoparticles exhibited a uniform particle size distribution, with a hydrated particle size of approximately 160 nm (Fig. 1C) and a negatively charged surface of approximately − 32 mV (Fig. 1D). The concentration of TPNs-siRNA after freeze-drying was approximately 500 µg/mL. Meanwhile, the particle size of TPNs-siRNA did not change significantly before and after freeze-drying. Transmission electron microscopy confirmed the spherical morphology of both TPNs and TPNs-siRNA (Fig. 1E), while colloidal stability studies demonstrated consistent particle size over 48 h, indicating potential biomedical applications (Fig. 1F).

Fig. 1
figure 1

(A) Gel electrophoresis image depicting FAM fluorescently labelled siRNA to calculate the loading capacity. Left side: control lane for FAM-siRNA solution; Right side: gel electrophoresis lane for the supernatant after TPNs-siRNA solution centrifugation. (B) Quantitative results obtained using ImageJ software. (C) Particle size distribution, (D) ζ potential, and (E) TEM transmission electron microscopy images of TPNs and TPNs-siRNA. Scale bar = 100 nm. (F) DLS examination of the colloidal stability of TPNs-siRNA in different media. (G) Gel electrophoresis experiment examining siRNA degradation. UV‒vis absorption spectra of TPNs-siRNA incubated with different concentrations of (H) DPPH•, (I) ABTS+•, (J) •OH, (K) •O2−, and (L) •NO solutions

Full size image

Maintaining siRNA stability under physiological conditions is crucial for successful targeted delivery. Gel electrophoresis experiments revealed that TPNs-siRNA effectively prevented siRNA degradation even at a 20% FBS concentration, whereas free siRNA was completely degraded (Fig. 1G). This study further investigated the antioxidative efficacy of TPNs-siRNA, which are crucial for addressing excessive free radicals in AAA pathology. Characterization of representative free radicals (DPPH•, ABTS+•, •OH, •O2−, and •NO) through UV‒visible absorption demonstrated that TPNs-siRNA induces a concentration-dependent reduction in free radical levels (Fig. 1H-L). The kinetic curves detailed in Figure S1 C and D confirmed the ability of TPNs-siRNA to clear various free radicals in vitro. Therefore, TPNs loaded with siRNA maintain antioxidative efficacy and exhibit the ability to clear free radicals in vitro.

Cellular uptake of TPNs-siRNA and effect on MMP silencing

As a gene delivery system, cellular uptake was studied to evaluate the transfection efficiency. To do this, we first evaluated the cytotoxicity of TPNs-siRNA using RAW264.7 and VSMCs. At concentrations ranging from 10 to 100 µg/mL, the cell viability exceeded 90% (Fig. 2A, B). Even at 200 µg/mL, TPNs-siRNA exhibited minimal toxicity, maintaining VSMC viability above 50%. Therefore, TPNs-siRNA demonstrated biocompatibility, supporting its potential application in cellular studies. To simulate the AAA cellular environment, RAW264.7 cells were stimulated with LPS to induce M1 macrophage polarization. The cellular uptake of TPNs-siRNA by LPS-stimulated and normal RAW264.7 cells was evaluated. Confocal microscopy revealed that siRNA uptake by the cells was efficient, with green fluorescence co-localizing around the cell nuclei (Fig. 2C). Interestingly, intensity quantification by flow cytometry confirmed increased intracellular green fluorescence in LPS-stimulated RAW264.7 compared to normal cells (Fig. 2D, E), indicating enhanced uptake by M1-polarized macrophages, potentially attributed to their heightened phagocytic capacity. Similarly, VSMCs were stimulated with H2O2 to mimic oxidative stress in AAA. Confocal microscopy revealed substantial green fluorescence inside the VSMCs, indicating that siRNA uptake via TPNs delivery was successful (Fig. S3A). Flow cytometry results depicted increased nanoparticle uptake by VSMCs under H2O2 stimulation (Fig. S3B, C). Therefore, the nanoparticle showed enhanced uptake by diseases macrophages and VSMCs.

Fig. 2
figure 2

(A) Viability of RAW264.7 cells and (B) VSMCs after 24 h of incubation with various TPNs-siRNA concentrations; ****P < 0.0001 compared to the control group (0 µg/mL). (C) Confocal image depicting TPNs-siRNA uptake. Scale bar = 20 μm. (D) Flow cytometry results and (E) quantitative analysis of the average fluorescence intensity. (F) Behaviour trajectory of TPNs-siRNA in RAW264.7 cells. Scale bar = 20 μm. (Blue: cell nucleus; Green: TPNs-siRNA; Red: lysosomes). RT‒qPCR analysis of (G) MMP-2 and (H) MMP-9 gene expression. **P < 0.01, ***P < 0.001, ****P < 0.0001

Full size image

To investigate lysosomal escape, lysosomal probes were used to co-localize TPNs-siRNA with lysosomes. Confocal microscopy revealed that TPNs-siRNA was predominantly located in lysosomes after 4 h and then translocated into the cell cytoplasm after 8 h in RAW264.7 cells and VSMCs (Fig. 2F, Fig. S3D), demonstrating effective lysosomal escape. Having confirmed the cellular delivery, we then studied the effect of gene silencing on MMP-2 and MMP-9. RT‒qPCR analysis of RAW264.7 cells and VSMCs demonstrated that TPNs exhibited a modest anti-MMP-2 and anti-MMP-9 effect (Fig. 2G, H, Fig. S3E, F), possibly associated with their ability to remove free radicals. In contrast, TPNs-siRNA significantly downregulated MMP-2 and MMP-9 at the cellular level, affirming the role of siRNA delivered through TPNs in intracellular gene silencing. The protein level results were consistent with the RT‒qPCR findings (Fig. S2A, B, Fig. S3G). We also evaluate effect of TPNs-siRNA treatment on related genes (p-ERK, ERK, p-JNK, JNK) both on VSMCs and RAW246.7 cells, results suggest that mitogen-activated protein kinase (MAPK) signalling pathway may be involved in the regulation (Fig. S2C, Fig. S3H).

Intracellular RONS scavenging activities of TPNs-siRNA

VSMCs within the aneurysmal site of AAA contribute significantly to the generation of reactive oxygen and nitrogen species (RONS), which induce cellular oxidative stress. To assess the scavenging effect of TPNs-siRNA on intracellular RONS (total ROS, H2O2, •O2−, •NO, and •OH/ONOO−), five probes, including ROS-ID, DCFH-DA, DHE, DAF-FM DA, and HPF, were employed. In the RAW264.7 model group treated with LPS, heightened fluorescence indicated increased free radical production and severe oxidative stress. However, upon treatment with TPNs-siRNA, a notable reduction in the fluorescence signal was observed (Fig. S4A). Flow cytometry analysis and quantification demonstrated that the levels of total ROS, H2O2, •O2−, •NO, and •OH/ONOO− increased following LPS stimulation (Fig. 3A-E). Nevertheless, TPNs-siRNA treatment significantly mitigated the levels of each RONS in the cells, underscoring the capacity of TPNs-siRNA to effectively clear free radicals and alleviate oxidative stress in LPS-stimulated RAW264.7 cells.

Fig. 3
figure 3

(A-E) Flow cytometry results and fluorescence quantification illustrating TPNs-siRNA-mediated removal of cellular RONS in RAW264.7 cells. (F-J) Flow cytometry results and fluorescence quantification demonstrating TPNs-siRNA removal of cellular RONS in VSMCs. ***P < 0.001, ****P < 0.0001

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Similarly, VSMCs treated with hydrogen peroxide (H2O2) exhibited intensified fluorescence (Fig. S4B), which is indicative of oxidative stress. Treatment with TPNs-siRNA led to a significant decrease in the fluorescence signal, indicating that free radicals were effectively cleared. Flow cytometry analysis and quantification revealed a notable increase in the corresponding free radicals upon H2O2 stimulation (Fig. 3F-J), while TPNs-siRNA treatment effectively reduced the excess free radicals in VSMCs. Collectively, these findings affirm that TPNs loaded with siRNA maintain their antioxidant capacity, effectively clearing intracellular free radicals and mitigating oxidative stress in RAW264.7 cells and VSMCs.

Ability of TPNs-siRNA to suppress inflammation and promote M1-to-M2 polarization

In the context of AAA, macrophages are recruited, leading to the release of inflammatory factors that, in turn, attract inflammatory cells, initiating a cascade of inflammatory reactions and exacerbating damage to the abdominal aorta [37,38,39]. Experimental findings revealed that LPS-pretreated RAW264.7 cells exhibited elevated production of inflammatory cytokines (Fig. 4A-C). Notably, TPNs-siRNA and TPNs effectively downregulated the secretion of IL-1β, IL-6, and TNF-α by RAW264.7 cells, indicating the anti-inflammatory activity of nanocarriers with free radical-clearing capacity. Interestingly, compared with TPNs, TPNs-siRNA exhibited a more pronounced inhibitory effect on inflammatory factors, suggesting that the synergistic effect of siRNA-mediated MMP silencing enhances the anti-inflammatory properties of nanoparticles.

Fig. 4
figure 4

Levels of the inflammatory factors IL-1β (A), IL-6 (B), and TNF-α (C) in RAW264.7 cells; (D) Immunofluorescence images showing iNOS and Arg-1 in RAW264.7 cells. Scale bar = 100 μm. (E) RT‒qPCR analysis of CD206, Arg-1, CD80, and iNOS gene expression; (F) Schematic diagram of the polarization of M1 to M2. *P < 0.05, ****P < 0.0001

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Under different stimuli, macrophages can polarize into pro-inflammatory M1 or anti-inflammatory M2 phenotypes. Given the crucial role of RONS in macrophage polarization, previous studies have demonstrated that TPNs can promote the repolarization of M1 to M2 macrophages by clearing RONS [40, 41]. To ascertain whether TPNs-siRNA elicits similar effects, we investigated the phenotypic changes in macrophages through immunofluorescence staining for iNOS (M1 marker) and Arg-1 (M2 marker). Immunofluorescence results indicated that LPS-stimulated RAW264.7 cells exhibited a heightened red fluorescence signal for iNOS, indicating polarization towards the M1 phenotype. However, treatment with TPNs-siRNA and TPNs led to a significant reduction in iNOS fluorescence and an enhancement in Arg-1 fluorescence, suggesting that RAW264.7 cells repolarized from the M1 to M2 phenotype (Fig. 4D). This effect was further confirmed by RT‒qPCR (Fig. 4E), which revealed that iNOS expression was inhibited and Arg-1 expression was increased at the mRNA level, consistent with the immunofluorescence findings (Fig. 4F).

Anti-calcification and anti-apoptosis activities of TPNs-siRNA

VSMCs, the primary cell type in the aortic media, play a pivotal role in maintaining the structural and functional integrity of the aortic wall, including a role in extracellular matrix (ECM) synthesis [42,43,44,45]. In the context of AAA, VSMCs are implicated in intimal and inner vascular wall calcification, elevating the risk of vascular rupture [46, 47]. In this study, VSMC calcification was induced using Ca/Pi to assess the in vitro anti-calcification effect of TPNs-siRNA. Compared to those in the normal group, VSMCs in the model group exhibited significant calcium deposition (Fig. 5A). Treatment with TPNs and TPNs-siRNA led to a noticeable reduction in the intensity of the red colour observed in the model group cells, which is indicative of decreased calcification. Quantitative analysis further substantiated the anti-calcification efficacy of TPNs and TPNs-siRNA (Fig. 5B). Notably, the anti-calcification ability of TPNs-siRNA may result from the capacity of TPNs to inhibit oxidative stress reactions in VSMCs, as suggested by previous studies [48, 49].

Fig. 5
figure 5

(A) Optical microscope image depicting VSMC calcium deposition. Scale bar = 100 μm. (B) Quantification of VSMC calcium deposition. (C) Cell viability of VSMCs after induction of calcium deposition. (D) Flow cytometry image illustrating VSMC apoptosis. (E) Quantification of VSMC apoptosis. (F) Cell viability of VSMCs after induced apoptosis. *P < 0.05, ****P < 0.0001

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Concurrently, the impact of the formulations on cell apoptosis was evaluated. Cell viability tests demonstrated that TPNs and TPNs-siRNA mitigated Ca/Pi-induced damage in VSMCs, with minimal observed cell death (Fig. 5C). Since the loss of mid-layer VSMCs in the aortic wall due to apoptosis is an early sign of AAA development, the effect of TPNs carriers on cell apoptosis was investigated [50,51,52]. Compared to the normal control group, the model group had a greater number of apoptotic and necrotic cells, as revealed by flow cytometry. Treatment with TPNs and TPNs-siRNA alleviated the degree of cell apoptosis to varying extents (Fig. 5D-F), underscoring the inhibitory effect of TPNs carriers on calcification and their anti-apoptotic properties.

Targeting and accumulation of TPNs-siRNA in mice AAA site

Animal models are essential for elucidating the pathophysiological characteristics of AAA and evaluating preclinical candidate drugs. In this study, we established a mouse model of AAA by wrapping the abdominal aorta with a CaCl2 solution and recapitulating several key pathological features observed in human AAA, including significant aortic calcification, inflammation, oxidative stress reactions, matrix metalloproteinases (MMPs) activity, elastin degradation, and VSMC apoptosis [53,54,55]. Leveraging this model, we investigated the targeting behaviour of TPNs-siRNA to target the lesion site of mouse AAA, laying the foundation for subsequent assessments of in vivo efficacy.

To assess the in vivo behaviour of the tracer nanoparticles, siRNA was labelled with Cy5.5 fluorescence. After fluorescently labelled Cy5.5-siRNA and TPNs-Cy5.5-siRNA were injected in the tail veins of mice, the distribution of TPNs-siRNA in the abdominal aortas of mice was evaluated using live imaging technology. The results demonstrated that, compared to Free-Cy5.5-siRNA, TPNs-Cy5.5-siRNA exhibited a robust fluorescence signal in the infrarenal aorta segment of the model mice, indicating the enhanced enrichment of nanocarriers at the AAA lesion site through passive targeting, thereby improving drug delivery efficiency (Fig. 6A). Notably, TPNs-Cy5.5-siRNA effectively distinguished between sham-operated mice and model mice. Fluorescence at the disease lesion site was three times higher than that in normal mice (Fig. 6B), validating the passive retention effect of nanoparticles at the AAA lesion site.

Fig. 6
figure 6

(A) Ex vivo fluorescence imaging of the mouse abdominal aorta; (B) quantification of average fluorescence intensity in the abdominal aorta; (C) ex vivo fluorescence imaging of the mouse abdominal aorta at various timepoints; (D) quantification of average fluorescence intensity; (E) distribution of major organs in mice at 8 h after various treatments; and (F) dynamic monitoring of the biodistribution of TPNs-siRNA. **P < 0.01,***P < 0.001, ****P < 0.0001

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Fig. 7
figure 7

(A) Fluorescence microscopy image illustrating the distribution of TPNs-Cy5.5-siRNA in the abdominal aortic tissue of the sham surgery group; (B) colocalization immunofluorescence analysis of CD31+ endothelial cells, CD68+ macrophages, and α-SMA+ vascular smooth muscle cells in the aneurysm with TPNs-Cy5.5-siRNA. Scale bar = 50 μm

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Subsequent investigations into the retention time of nanoparticles at the lesion site revealed that TPNs-Cy5.5-siRNA accumulation gradually decreased in the mouse abdominal aorta with prolonged administration time. After 48 h, nearly no fluorescence aggregation was absorbed in the abdominal aorta (Fig. 6C, D), suggesting gradual drug clearance. Tissue distribution of TPNs-Cy5.5-siRNA in major organs was evaluated, revealing that TPNs-Cy5.5-siRNA predominantly accumulates in the liver and kidneys, with some fluorescence signals in the lungs (Fig. 6E). This finding implies that the nanocarriers are primarily metabolized through hepatic and renal pathways. Further pharmacokinetic analysis demonstrated that the fluorescence signals in various organs gradually decreased over time (Fig. 6F), confirming the in vivo clearance of the TPNs-siRNA formulation.

Cellular localization analysis of TPNs-siRNA in AAA tissue

Previous experiments have established the ability of nanodrugs to effectively target and penetrate local lesion sites in AAA. To assess the distribution of TPNs-Cy5.5-siRNA in the abdominal aortas of the mice, we conducted immunofluorescence staining of key cell types in the tissue, including endothelial cells (CD31+), macrophages (CD68+), and vascular smooth muscle cells (α-SMA+). Subsequently, co-localization analysis was performed with TPNs-Cy5.5-siRNA to elucidate its specific cellular interactions.

The immunofluorescence results demonstrated that in the sham surgery group, minimal TPNs-Cy5.5-siRNA (red fluorescence) was detected in the Cy5.5 channel (Fig. 7A). This observation indicates that infiltration of the drug in abdominal aorta tissue is limited, consistent with the outcomes of in vivo animal experiments. Conversely, in the model group, a substantial accumulation of red fluorescence was evident on the arterial wall in the Cy5.5 channel. Colocalization analysis of vascular smooth muscle cells labelled with α-SMA+ and macrophages labelled with CD68+ revealed a clear association between TPNs-siRNA and these cellular populations (Fig. 7B). This finding suggested that TPNs-siRNA effectively entered vascular smooth muscle cells and macrophages within the diseased tissue of AAA. Therefore, TPNs-siRNA can accumulate effectively at the AAA site and successfully permeate afflicted cells.

Evaluation of the therapeutic efficacy of TPNs-siRNA in AAA mice

Subsequently, we evaluated the therapeutic effects of the nanomedicine at the animal level. Using ultrasound imaging technology, we scrutinized the morphological characteristics of blood vessels in the coronal and sagittal planes of the abdominal aorta. Compared with the normal group, the model group exhibited significant dilation of the infrarenal aorta segment (Fig. 8A). Treatment with different drug formulations resulted in varying degrees of reduction in vessel diameters, with the TPNs-siRNA treatment group demonstrating the most significant decrease. Analysis of vessel diameter measurements were consistent with the image results (Fig. 8B).

Fig. 8
figure 8

(A) Ultrasound images of transverse (top) and sagittal (bottom) sections of the abdominal aorta; (B) colour Doppler image of the maximum diameter of the mouse abdominal aorta; (C) IL-1β, (D) IL-6, and (E) TNF-α contents; (F) calcification and (G) H2O2 in abdominal aortic tissue after treatment with different formulations; (H) MMP-2 and (I) MMP-9 in abdominal aortic tissue; (J) TUNEL staining of abdominal aortic tissue slices at 40x magnification; (K) H&E, EVG, and alizarin red staining of abdominal aortic tissue slices at 40x magnification; (L) positive expression of CD31, CD68, MMP-2, and MMP-9 in abdominal aortic tissue. **P < 0.01, ***P < 0.001, ****P < 0.0001

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Fig. 9
figure 9

(A) In vitro haemolysis test of TPNs-siRNA; (B) Mouse weight change curve and changes in major organ indices (C); (D) H&E staining images of major organs in mice; (E) Biochemical indicators ALT, AST (F), BUN (G), and CR (H). **P < 0.01, ***P < 0.001, ****P < 0.0001

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To validate these mechanisms, we measured inflammatory factor levels, MMP levels, vascular calcification, and oxidative stress indicators in AAA tissue homogenates. The results demonstrated that the expression of these markers reduced after treatment with different formulations (Fig. 8C-I, Fig. S5A). Notably, there were no significant differences between TPNs and TPNs-siRNA in terms of their anti-inflammatory, anti-calcification, or antioxidant effects. However, TPNs-siRNA significantly suppressed the expression of MMP-2 and MMP-9, which was attributed to the efficacy of the siRNAs (Fig. 8F, G). These results suggest that the nanocarrier and siRNA exert a synergistic effect in treating AAA, in which TPNs contribute to free radical clearance, inhibition of local inflammation and vascular calcification, while siRNA reduces MMP levels.

Immunofluorescence staining of abdominal aortic tissue slices revealed increased iNOS fluorescence and weak Arg-1 fluorescence in the model group, indicating that macrophages polarized towards the M1 phenotype (Fig. S5B, C). Treatment with TPNs-siRNA and TPNs resulted in reduced iNOS fluorescence and increased Arg-1 fluorescence, indicating that the macrophages repolarized from the M1 to M2 phenotype. TUNEL staining revealed that VSMC apoptosis in the medial layer of the artery in the model group was inhibited by treatment with TPNs and TPNs-siRNA, with a more pronounced effect in the TPNs-siRNA group (Fig. 8J).

Pathological staining of AAA tissue revealed the effectiveness of both TPNs and TPNs-siRNA in improving AAA pathological changes (Fig. 8K). EVG staining demonstrated alleviation of elastic fiber degradation in both treatment group. Alizarin red staining indicated the degree of tissue calcification, with severe calcification in the model group, while both nanodrugs exhibited effective anti-calcification effects. Immunohistochemical analysis revealed a reduction in the positive expression of CD31, CD68, MMP-2, and MMP-9 in abdominal aortic tissue after treatment with both TPNs and TPNs-siRNA (Fig. 8L), indicating that siRNA continuously silences MMP-2 and MMP-9 by in vivo under the protection of TPNs. These results validate the multifaceted mechanisms by which TPNs-siRNA exerts activity in treating AAA.

Safety assessment of TPNs-siRNA

Next, we thoroughly evaluated the safety profile of TPNs-siRNA. Given the systemic administration through intravenous injection, we assessed the haemolytic potential of the nanoparticles. The haemolysis rate of TPNs-siRNA at various concentrations remained below 5% (Fig. 9A), indicating negligible haemolysis. This finding underscores the safety of TPNs-siRNA and TPNs, indicating their compatibility with red blood cells and confirming their suitability for intravenous administration. The in vivo safety evaluation following intravenous administration revealed no discernible differences in body weight among the experimental mouse groups and the physiological saline group (Fig. 9B). Mice across all groups exhibited consistent fur conditions, mental states, food intake, excrement, and activity levels. Notably, the organ indices did not significantly differ among the experimental groups, indicating the high safety margin of TPNs-siRNA and TPNs for major organs (Fig. 9C). Notably, a slight decrease in liver function was observed in the model group of mice, potentially due to the AAA condition. Subsequent histological examination through H&E staining of major organs revealed a normal tissue architecture, confirming the safety of TPNs-siRNA and TPNs in vivo (Fig. 9D). Furthermore, blood tests for hepatic and renal function were performed (Fig. 9E-H), and a significant decrease in the ALT and BUN levels was detected. However, all these parameters, including ALT, AST, BUN, and CR, were restored to normal levels after treatment with TPNs-siRNA and TPNs. These findings collectively affirm that nanomedicine minimally impacts body health, reinforcing the high safety profile of TPNs-siRNA and TPNs within the biological system.

Conclusion

In conclusion, TPNs-siRNA is a promising therapeutic approach for the treatment of AAA and shows versatile efficacy in vitro and in vivo. Functioning as a gene carrier, TPNs adeptly and efficiently load siRNAs, shielding siRNAs from biological degradation, facilitating intracellular delivery, and promoting endo/lysosome escape to unleash siRNAs for effective MMP silencing. As a multi-active material, TPNs can modulate diverse cellular processes, including inflammation, oxidative stress, apoptosis, and calcification. The integration of these two functionalities positions TPNs-siRNA as a potent intervention to mitigate the progression of AAA. Following intravenous administration, TPNs-siRNA effectively accumulated within AAA mice aneurysmal tissue with prolonged retention, specifically targeting macrophages and VSMCs crucial to AAA pathogenesis. Consequently, TPNs-siRNA exhibits robust efficacy in combating AAA while maintaining excellent biosafety, underscoring its translational potential as a viable therapeutic modality to address the intricate pathophysiology of AAA.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

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Funding

This work was supported by grants from the National Natural Science Foundation of China (82170501).

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  1. Zhen Wu and Peng Zhang co-first authors

Authors and Affiliations

  1. Department of Vascular and Interventional Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, 150001, Heilongjiang, China

    Zhen Wu & Haiyang Wang

  2. Department of Vascular Surgery, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510120, Guangdong, China

    Peng Zhang, Jie Yue, Qingshan Wang, Peipei Zhuang, Shah Jehan, Liyuan Fan & Jiarun Xue

  3. Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, 410013, Hunan, China

    Wenhu Zhou

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Contributions

Conceived and designed the experiments: ZW and PZ. JY, QW, and PZ carried out the experiment. SJ, LF, and JX contributed to analyze the experimental results. WZ: methodology, visualization. PZ: methodology. HW and ZW wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Haiyang Wang.

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Wu, Z., Zhang, P., Yue, J. et al. Tea polyphenol nanoparticles enable targeted siRNA delivery and multi-bioactive therapy for abdominal aortic aneurysms. J Nanobiotechnol 22, 471 (2024). https://doi.org/10.1186/s12951-024-02756-2

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Journal of Nanobiotechnology

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