MMP-Activated Liposomal Nanoplatform Co-loaded with Kaempferol and ICG for Targeted Imaging and Therapy of Abdominal Aortic Aneurysm

Abstract

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disorder characterized by excessive oxidative stress, chronic inflammation, and extracellular matrix (ECM) degradation. Despite its high prevalence and poor clinical outcomes, no effective pharmacological therapies currently exist. Here, we developed a theranostic liposomal nanoplatform co-loaded with kaempferol (KPF), a natural flavonoid with potent anti-inflammatory and antioxidant activity, and indocyanine green (ICG), a photoacoustic imaging agent. The platform was surface-modified with an activatable cell-penetrating peptide (ACPP), a protease-cleavable cell-penetrating peptide that becomes active upon exposure to MMP-9 in AAA lesions, thereby enabling targeted delivery. The resulting formulation (ICG-KPF@ALNPs) exhibited selective accumulation in aneurysmal tissues and produced strong photoacoustic signals in vitro and in vivo. Therapeutically, ICG-KPF@ALNPs markedly reduced reactive oxygen species (ROS), suppressed inflammatory cytokines, and preserved ECM integrity. Mechanistically, kaempferol exerted a dual protective effect by activating the Nrf2/HO-1 pathway to scavenge ROS and inhibiting the NLRP3 inflammasome to limit macrophage pyroptosis, collectively mitigating oxidative stress and inflammation. These findings highlight ICG-KPF@ALNPs as a promising nanotheranostic strategy for noninvasive imaging and targeted treatment of AAA.

1. Introduction

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disorder defined by irreversible dilation of the abdominal aorta due to progressive thinning and structural failure of the vessel wall. Typically asymptomatic until rupture, AAA may be complicated by intraluminal thrombosis, dissection, or catastrophic hemorrhage, with postrupture mortality approaching 65–85%. Current management relies exclusively on open surgical or endovascular repair, as no pharmacological agents have been shown to halt or reverse disease progression. , Early intervention for small aneurysms offers limited survival benefits, while routine imaging surveillance imposes economic and psychological burdens, underscoring the need for effective drug-based therapies.

Macrophage infiltration is closely associated with the initiation and progression of AAA. Within the inflammatory milieu, macrophages adopt a proinflammatory state and release cytokines such as TNF-α and IL-1β, ROS, and matrix metalloproteinases, causing vascular smooth muscle cell dysfunction, extracellular matrix degradation, and progressive vessel-wall failure. NLRP3 is a cytosolic NOD-like receptor that senses danger signals and assembles the inflammasome, activating caspase-1 to drive IL-1β/IL-18 maturation and gasdermin-D-dependent pyroptosis. , This pathway is a key driver of AAA. ROS functions upstream to trigger NLRP3 activation, amplifying oxidative stress and sustaining an inflammation-oxidative stress-pyroptosis loop. , Elevated ROS levels simultaneously trigger the activation of Nuclear factor erythroid 2-related factor 2 (Nrf2), leading to the upregulation of the antioxidant defense system, which in turn attenuates ROS accumulation and restores redox homeostasis. Thus, inhibiting NLRP3 inflammasome activation in lesion-resident macrophages is a rational strategy to curb AAA progression.

Kaempferol, a dietary flavonoid abundant in fruits and medicinal plants, exerts antioxidant, anti-inflammatory, and metabolic regulatory effects. It exerts its effects by suppressing inflammatory gene expression, inhibiting MMPs, and attenuating ROS/NF-κB signaling. , Structurally related flavonoids, such as naringenin, have been shown to prevent AAA formation by downregulating NLRP3 activity. Accordingly, we hypothesize that kaempferol could restrain AAA progression through comparable mechanistic routes. However, the poor aqueous solubility of kaempferol limits its bioactivity and pharmacological efficacy in vitro and in vivo, and conventional free-drug administration lacks lesion specificity, thereby diminishing therapeutic outcomes.

Nanoparticle-based delivery systems can address these limitations by improving solubility, stability, pharmacokinetics, and targeted accumulation. − Advances in nanomedicine over the past few years have intensified research into its potential advantages for AAA. − In our previous work, we developed a multifunctional nanoparticle platform integrating active targeting ligands with microbubble-assisted ultrasound delivery, which significantly enhanced drug stability, prolonged circulation time, and promoted lesion-specific accumulation via both passive (EPR) and active targeting while enabling spatiotemporally controllable release. This strategy effectively improved therapeutic efficacy and biosafety, underscoring the potential of advanced nanocarrier systems for precision drug deliver.

Hence, building on the above background, we developed an ACPP-modified liposomal nanoplatform (ICG-KPF@ALNP) co-loaded with kaempferol and ICG (Figure ). ACPPs comprise a polyanionic inhibitory segment, a linker specifically cleavable by MMP-9, and a cationic cell-penetrating peptide (CPP). Accumulating evidence indicates high MMP-9 expression in AAA tissues. In this design, ACPPs serve as MMP-9-cleavable molecular switches that expose a cationic cell-penetrating segment upon proteolysis, enabling site-specific activation and controlled intracellular delivery of kaempferol. − Simultaneously, ICG, a clinically validated photoacoustic contrast agent imparts imaging capability for lesion localization and image-guided therapy. , Following intravenous administration, this innovative dual-drug delivery system demonstrates rapid and selective accumulation in AAA lesions. Mechanistically, released kaempferol concurrently attenuated oxidative stress and inflammation via a dual regulatory effect: activating the Nrf2/HO-1 pathway to scavenge ROS, and suppressing NLRP3 inflammasome to limit macrophage pyroptosis, thus reducing inflammatory burden. In vivo, ICG-KPF@ALNP significantly outperformed controls in ameliorating AAA progression as well as providing real-time photoacoustic imaging capability, underscoring its potential as a precise theranostic strategy for AAA management.

1.

Schematic diagram displaying the ICG-KPF@ALNP fabrication, targeting, and its anti-AAA properties.

2. Materials and Methods

2.1. Preparation of ICG-KPF@ALNPs

ICG-KPF@ALNPs were prepared via thin-film hydration. Briefly, 9.9 mg of DSPC, 9.6 mg of DSPE-PEG2000, 4.9 mg of DSPE-PEG2000-ACPP, 2.0 mg of KPF, and 1.0 mg of ICG were dissolved in chloroform/methanol (15:5, v/v) in a round-bottom flask. The solvent was evaporated at 60 °C for 1 h to form a lipid film, followed by vacuum drying for 1 h. The film was hydrated with 5 mL of PBS at 60 °C for 30 min, followed by bath sonication at 40 kHz for 5 min at 25 °C. Subsequently, the mixture was further processed using a probe sonicator on ice (200 W, 3 s on/3 s off cycles for 5 min). Unencapsulated agents were removed by ultrafiltration using 100 kDa MWCO filters (Merck) at 5000×g for 10 min. The final formulation was stored in sterile vials and sealed with a rubber lid at 4 °C. Nontargeted liposomes (ICG-KPF@LNP) were prepared identically but without DSPE-PEG2000-ACPP.

2.2. Characterization of ICG-KPF@ALNPs

Particle size, polydispersity index (PDI), and zeta potential were measured at room temperature by dynamic light scattering (DLS, Malvern Nano-ZS, U.K.). Morphology was examined using transmission electron microscopy (TEM, Hitachi HT-7800, Japan). Ultraviolet-Visible (UV–vis) spectroscopy (Shimadzu UV-2600) was employed to confirm the KPF/ICG encapsulation and characteristic peaks. The concentration of KPF was measured by HPLC (LC 2010A, Shimadzu, Japan). The HPLC instrument was equipped with an Agilent C18 column (250 × 4.6 mm², 5 μm). The mobile phase included 80% acetonitrile and 20% water, and KPF was detected by a UV detector at 366 nm. To demonstrate whether ICG-KPF@ALNPs successfully loaded ACPP, the lipophilic dye DiI (Invitrogen) was incorporated into the lipid mixture at 0.5–1.0 mol % during film formation, and ACPP was FITC-labeled (Sigma-Aldrich) via standard isothiocyanate-amine coupling, with unbound dye removed by dialysis. All of the fluorescence images were taken by confocal laser scanning microscopy (CLSM, Leica Microsystems, Wetzlar, Germany). In vitro photoacoustic imaging was conducted by using a Vevo LAZR photoacoustic imaging system (Vevo 3100, Canada). ICG-KPF@ALNPs were diluted to different concentrations to obtain the corresponding PA images.

2.3. Cell Culture, Cytotoxicity, and Cellular Uptake

RAW264.7 macrophages (ATCC) were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO₂ incubator. To mimic the inflammatory microenvironment of abdominal aortic aneurysm (AAA), cells were stimulated with lipopolysaccharide (LPS, 1.0 μg/mL) for 24 h with or without ATP (5.0 μM, Sigma) to induce pyroptosis. Control cells were cultured under identical conditions without LPS treatment.

For intervention studies, kaempferol, ICG-KPF@LNPs, or ICG-KPF@ALNPs (20 μM) were administered at the indicated concentrations 2 h prior to stimulation. Cell viability was evaluated by using the CCK-8 assay. Briefly, RAW264.7 cells (1.0 × 10⁴ per well in 96-well plates) were treated with the indicated formulations for 24 h, followed by incubation with CCK-8 solution (10 μL/well, 1 h). Absorbance was recorded at 450 nm on a Spark microplate reader (Tecan, Switzerland).

For uptake studies, RAW264.7 cells (1.0 × 10⁵ per well in 24-well plates) were pretreated for 24 h with LPS or vehicle in the presence or absence of GM6001­(a matrix metalloproteinase inhibitor, 20 μM) and then incubated with DiI-labeled ICG-KPF@LNPs or ICG-KPF@ALNPs for 4 h. After PBS washing and nuclear counterstaining with DAPI (10 min), intracellular localization was visualized by confocal laser scanning microscopy (CLSM).

2.4. Transcriptomic Analysis

To characterize transcriptional changes in macrophages following kaempferol exposure, we performed RNA sequencing. Macrophages were treated with kaempferol or left untreated (control). Total RNA was extracted with TRIzol reagent according to the manufacturer’s instructions. RNA quantity and integrity were assessed, and only high-quality RNA was used for library construction. Libraries passing quality control were sequenced on an Illumina NovaSeq 6000. Raw reads underwent quality assessment and adapter/low-quality trimming, followed by alignment with the reference genome and gene-level quantification. Differential expression between kaempferol-treated and control samples was then computed, and functional enrichment analyses were performed.

2.5. Intracellular ROS-Scavenging

LPS-stimulated RAW264.7 cells were treated with kaempferol, ICG-KPF@LNPs or ICG-KPF@ALNPs (20 μM) for 24 h, and then incubated with DCFH-DA (10 μM, 30 min). ROS levels were quantified via CLSM and flow cytometry (BD FACSCelesta). Mean fluorescence intensity (MFI) was analyzed by using ImageJ.

2.6. RNA Interference

Two groups were used: RAW264.7 cells transfected with a nontargeting control siRNA or with an Nrf2-targeting siRNA. Cells were seeded 1 day prior to reach 50–70% confluence (1 × 10⁶ per well in 6-well plates). 2.5 μL amount of siRNA (20 nM) and 3.75 μL of Lipofectamine 3000 (Invitrogen, Carlsbad, CA) were each diluted in 125 μL of Opti-MEM, combined, and incubated for 15 min to form complexes. Subsequently, 250 μL of complex and 750 μL of Opti-MEM were added per well. After 6 h, the medium was optionally replaced with the complete medium and the cells were maintained at 37 °C, 5% CO₂. The sequence of Nrf2 siRNA: Forward: 5′-AUACUUCUCGACUUACUCCAA-3′. Reverse: 5′-GGAGUAAGUCGAGAAGUAUUU-3′.

2.7. AAA Mouse Model and In Vivo Imaging

All animal experiments were approved by the Animal Ethics Committee of Guangdong Provincial People’s Hospital, Southern Medical University (KY2025–440–01), and conducted in accordance with the ARRIVE guidelines and the NIH Guide for the Care and Use of Laboratory Animals. Male ApoE^–/– mice (8 weeks old) were maintained on a high-fat diet for 4 weeks and subsequently implanted with osmotic minipumps delivering Ang II (1,000 ng/kg/min) to induce abdominal aortic aneurysm (AAA). AAA formation was confirmed by ultrasound imaging (Vevo 2100, Canada) when the aortic diameter exceeded 50%.

For pharmacokinetic analysis of ICG-KPF@ALNPs, mice (n = 3) received tail-vein injections of free kaempferol or ICG-KPF@ALNPs at 20 mg of KPF/kg. Plasma was collected at 2, 4, 6, 8, 12, 24, and 48 h. For sample preparation, 100 μL plasma was mixed with 300 μL ethyl acetate, vortexed for 2 min, and centrifuged at 3,000 g for 15 min at 4 °C. The supernatant was collected, evaporated to dryness under vacuum, and reconstituted in 100 μL methanol. Kaempferol concentrations were then determined by high-performance liquid chromatography. Relative fluorescence of blood samples in the ICG-KPF@ALNPs group was measured using an in vivo imaging system (IVIS, PerkinElmer).

For in vivo photoacoustic imaging, mice were intravenously injected with ICG-KPF@LNPs or ICG-KPF@ALNPs (3.6 mg of ICG/kg) via the tail vein. After 24 h, imaging was performed using a Vevo 3100 LAZR system to assess the photoacoustic properties of the nanoplatforms. The Vevo 3100 LAZR system equipped with a MX250 transducer; the nominal axial resolution of this transducer is 75 μm (manufacturer’s specification).

For biodistribution studies, AAA mice received intravenous injections of saline, ICG-KPF@LNPs, or ICG-KPF@ALNPs (3.6 mg of ICG/kg). After 24 h, major organs and aortas were harvested and imaged using an IVIS Spectrum system.

2.8. Therapeutic Efficacy Assessment

Male mice were randomly assigned to five experimental groups: PBS (control), Angiotensin II (Ang II), Ang II plus kaempferol, Ang II plus ICG-KPF@LNPs, and Ang II plus ICG-KPF@ALNPs (20 mg KPF/kg). Treatments were administered via intravenous injection twice weekly for 4 consecutive weeks. Abdominal aortic diameters were measured longitudinally by high-frequency ultrasound imaging, including both transverse and longitudinal planes to accurately assess aneurysm size. At the study endpoint, animals were euthanized, and aortic tissues were harvested for histological and molecular analyses. Hematoxylin and Eosin (H&E), elastic Van Gieson (EVG), and Masson’s trichrome staining were performed to evaluate vascular morphology, elastic fiber integrity, and collagen deposition, respectively. EVG staining was semiquantitatively scored based on elastic fiber disruption as follows: Score 0: Intact elastic lamellae with continuous and well-organized fibers; Score 1: Mild fragmentation or waviness of elastic fibers affecting < 25% of the media; Score 2: Moderate fragmentation involving 25–50% of the media; Score 3: Severe fragmentation or loss of elastic fibers affecting > 50% of the media. Reactive oxygen species (ROS) levels were assessed by dihydroethidium (DHE) staining. Immunofluorescence staining was conducted to detect NLRP3 inflammasome. Immunohistochemistry (IHC) was performed to evaluate the expression of matrix metalloproteinase-9 (MMP-9) and gasdermin D (GSDMD).

2.9. Biosafety Evaluation

Whole blood was collected from mice into anticoagulant tubes and centrifuged at 1000 rpm for 5 min. The erythrocyte pellet was rinsed repeatedly with saline until the supernatant was clear; the supernatant was discarded, and a 2% red blood cell suspension was prepared in saline. Subsequently, the 2% erythrocyte suspension was incubated with ICG-KPF@ALNPs and ICG-KPF@LNPs at concentrations of 0 to 160 μg/mL for 2 h at 37 °C. The suspensions were inspected for hemolysis, and the supernatant absorbance at 576 nm was read on a microplate reader.

Blood was analyzed for complete blood count (CBC), liver/kidney function markers (AST, ALT, BUN, creatinine), and complement components (C3a, C5a). Major organs were H&E-stained for histopathology.

2.10. Statistical Analysis

Data are expressed as the mean ± standard deviation (SD). Unpaired Student’s t test was used for quantitative comparisons between two groups. One-way ANOVA or two-way ANOVA followed by a posthoc Bonferroni test was used for comparisons among multiple groups. Kaplan–Meier analysis was used for survival analysis. Significance: *P < 0.05, ** P < 0.01, ***P < 0.001, **** P < 0.0001.

3. Results and Discussion

3.1. Preparation and Characterization of ICG-KPF@ALNPs

ICG-KPF@ALNPs were synthesized via the thin-film hydration method (Figure A). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) confirmed that both ICG-KPF@LNPs and ICG-KPF@ALNPs exhibited uniformly distributed, quasi-spherical morphologies consistent with typical liposomes, with mean hydrodynamic diameters of approximately 115 and 125 nm, respectively (Figure B,C). The corresponding zeta potentials were −35 and −30 mV (Figure D). Dynamic monitoring of particle size and polydispersity index (PDI) in PBS at 37 °C over 7 consecutive days revealed no significant changes, and storage at 4 °C maintained PDI for approximately 2 weeks, together indicating excellent colloidal stability of the nanoparticles (Figures E, S1). Ultraviolet–visible absorption spectroscopy (200–900 nm) showed two characteristic peaks at 366 and 784 nm, corresponding to kaempferol and ICG respectively, confirming successful coencapsulation of both agents within ICG-KPF@ALNPs (Figure F). Given that ICG displays a characteristic absorption maximum at 784 nm in its UV–vis spectrum, the ICG content was quantified by constructing a standard calibration curve at 784 nm, while the kaempferol content was determined by high-performance liquid chromatography (HPLC, Figure S2A,B). The formulated nanoparticles showed satisfactory encapsulation efficiency (EE: 69.1% for ICG, 61.1% for KPF) and drug loading capacity (DLC: 1.7% for ICG, 4.9% for KPF). To verify successful conjugation of ACPP to nanoparticles, FITC-labeled ACPP and DiI-labeled liposomes were coincubated and visualized by confocal laser scanning microscopy, which revealed clear fluorescence colocalization, indicating efficient ACPP modification (Figure G). ICG exhibits strong absorption in the near-infrared (NIR) region, making it well suited for photoacoustic imaging. , Furthermore, its use as a photoacoustic contrast agent has been widely reported. Accordingly, we formulated ICG-KPF@ALNPs as a photoacoustic contrast agent. Using PBS as the control, in vitro photoacoustic measurements demonstrated a concentration-dependent and linear increase in the signal intensity for ICG-KPF@ALNPs, confirming their potential as an effective photoacoustic imaging contrast agent (Figure H–J).

2.

Fabrication and characterization of ICG-KPF@ALNP. (A) Schematic illustration of ACPP-modified liposomes co-loading ICG and kaempferol (ICG-KPF@ALNPs). (B–D) TEM image, size distribution, and zeta potential of the ICG-KPF@LNPs and ICG-KPF@ALNPs (scale bars = 100 nm). (E) PDI of the ICG-KPF@ALNPs over a 7-d period at 37 °C. (F) UV–vis absorption spectra (200–900 nm) of free ICG, free kaempferol, blank LNPs, and ICG-KPF@ALNPs. Characteristic maxima: kaempferol at 366 nm and ICG at 784 nm. (G) CLSM of ICG-KPF@ALNPs shows successful loading of ACPP (green) onto liposome naonoparticals (red). (H) Schematic illustration of PA images. (I) PA images and (J) quantitative PA intensities of ICG-KPF@ALNPs at different concentrations in vitro.

3.2. Targeting and Endocytosis of ICG-KPF@ALNPs

The pathogenesis of AAA involves pronounced infiltration of inflammatory cells, with M1-polarized macrophages playing a critical role. Accordingly, we first assessed the biocompatibility of ICG-KPF@ALNPs in macrophages. CCK-8 assays demonstrated that free kaempferol, ICG-KPF@LNPs, and ICG-KPF@ALNPs maintained cell viability above 80% across concentrations ranging from 5 μM to 20 μM (Figure A–C). These results indicate that ICG-KPF@ALNPs exhibit good biocompatibility without inducing significant cytotoxic effects.

3.

Endocytosis of ICG-KPF@ALNPs and cell viability in LPS-induced macrophages. (A) Viability of RAW264.7 cells treated with kaempferol, (B) ICG-KPF@LNPs, or (C) ICG-KPF@ALNPs at varying concentrations. (D) Fluorescence images of M1 macrophages incubated with ICG-KPF@ALNPs in the presence or absence of GM6001. (E) Quantitative analysis of the mean fluorescence intensity. (F) Schematic illustration of different LNP cellular uptake. (G) Fluorescence images of RAW264.7 cells coincubated with the ICG-KPF@LNPs and ICG-KPF@ALNPs. Cell nuclei were stained with DAPI (blue), LNPs were stained with DiI (red). (H) Quantitative analysis of the mean fluorescence intensity (n = 3). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = no significance).

Subsequently, to assess both the MMP responsiveness of ICG-KPF@ALNPs and their targeting specificity toward AAA-associated macrophages, we established an in vitro model. In this model, LPS stimulation was used to induce M1 polarization in native macrophages, recapitulating the key inflammatory conditions of AAA. M1 macrophages were pretreated with or without the broad-spectrum MMP inhibitor GM6001 (20 μM) for 2 h prior to incubation with DiI-labeled ICG-KPF@ALNPs. Cell viability remained > 80% under this concentration (Figure S3). CLSM revealed a markedly attenuated intracellular red fluorescence signal in the inhibitor-treated group compared with the uninhibited control. This result confirms that the enhanced internalization of ICG-KPF@ALNPs into M1 macrophages is dependent on MMP-mediated activation of the nanoplatform (Figure D,E). Subsequently, the in vitro targeting specificity was assessed. DiI-labeled ICG-KPF@ALNPs (targeted) and ICG-KPF@LNPs (untargeted) were incubated with DAPI-stained M1 macrophages for 4 h (Figure F). CLSM revealed substantially higher DiI (red) fluorescence in M1 macrophages treated with ICG-KPF@ALNPs than in those treated with ICG-KPF@LNPs, consistent with ACPP-mediated enhancement of cellular uptake. Quantitative analysis corroborated this observation, showing a 2.5-fold increase in DiI signal for ICG-KPF@ALNPs relative to ICG-KPF@LNPs. Conversely, coincubation of DiI-labeled ICG-KPF@ALNPs with DAPI-stained M0 macrophages resulted in almost no detectable DiI (red) fluorescence, confirming selective internalization by the proinflammatory M1 phenotype. Consistent with this, quantitative fluorescence analysis showed that the DiI signal intensity in M1 macrophages was approximately 4.5-fold higher than that in M0 macrophages (Figure G,H). Abundant MMP-9 released by M1-polarized macrophages specifically processes ACPP to unmask the CPP, which then binds to membrane components and mediates uptake, enabling selective internalization of the nanoparticle carrier and delivery of its therapeutic payload. , In line with this mechanism, our findings demonstrate that ICG-KPF@ALNPs preferentially accumulate in M1 macrophages and are subsequently efficiently internalized by phagocytosis.

However, how kaempferol-loaded targeted liposomes exert their effects after internalization by M1-polarized macrophages remains to be fully elucidated. To obtain an unbiased, quantitative view of kaempferol-induced changes, we profiled the transcriptomes of M1 macrophages before and after treatment by RNA-seq.

3.3. Transcriptomics Analysis

Transcriptomics provides a comprehensive framework for profiling gene expression programs and inferring gene function. Accordingly, we performed RNA-seq to quantify treatment-induced expression changes and clarify the mechanisms by which kaempferol modulates the M1 macrophage behavior. The principal component analysis demonstrated that the kaempferol-treated group exhibited significant differences from the control group (Figure A). Our analysis revealed 1278 differentially expressed genes (DEGs) between the control and treatment groups. Of these, 872 genes were significantly upregulated, while 406 genes were significantly downregulated (Figure B). Among these, genes associated with macrophage inflammation, including CCL-2, IL-6, IL-1β, Nos2, exhibiting significant downregulation, the increased expression of the Sphk1 gene suggests a potential inhibition of inflammatory mediator release. All of these are indicative of possible suppression of inflammation in M1 macrophages induced by kaempferol treatment. − Additionally, the upregulation of certain antioxidant genes (e.g., Prdx1, Gpx1, Gclm, and Nqo1) suggests their potential roles in ameliorating oxidative stress. − Heatmap analysis revealed differentially expressed genes between the kaempferol-treated and the control groups (Figure C). Gene Ontology (GO) enrichment classified these genes into molecular function (MF), biological process (BP), and cellular component (CC) categories (Figure S4). MF terms were enriched for ATP binding, cytokine binding, and tubulin binding; BP terms for cytokine-mediated signaling; and CC terms for centrioles and other structural elements, implicating kaempferol in the modulation of inflammatory signaling. KEGG pathway analysis further revealed prominent enrichment in inflammation-associated signaling pathways, including the TNF signaling pathway and the NF-kappa B signaling pathway, with significant enrichment of the NOD-like receptor signaling pathway (Figure D). Gene set enrichment analysis (GSEA) showed negative enrichment of NLRP3 inflammasome complex assembly and inflammatory response gene sets, whereas positive enrichment of antioxidant activity was observed in the treatment group, compared with the control group (Figure E,F).

4.

Transcriptomic analysis of kaempferol on M1 Macrophages. (A) PCA plot of RNA-seq data. (B) Volcano plot showing significantly upregulated (red) and downregulated (blue) genes, with a bar chart of their counts. (C) Heatmap of differentially expressed genes (DEGs). (D) KEGG pathway analysis for DEGs. (E–G) Results of gene set enrichment analysis (GSEA). Gene sets related to NLRP3 inflammasome complex assembly and the inflammatory response are negatively enriched, whereas antioxidant activity is positively enriched. NES, normalized enrichment score.

3.4. Kaempferol Attenuates Macrophage Pyroptosis via NLRP3 Inflammasome Signaling Pathway

Based on the above transcriptomic analyses, kaempferol appears to boost antioxidant and anti-inflammatory defenses in M1 macrophages by attenuating the NLRP3 inflammasome signaling pathway, which is part of the NOD-like receptor signaling pathway. Nominal correlations were observed between kaempferol treatment and expression of NLRP3 inflammasome signaling pathway constituents, among which were positive for nine genes and negative for one (Figure A). The corresponding FPKM values are listed in Figure B.

5.

Kaempferol regulates NLRP3 inflammasome signaling and pyroptosis. (A) Schematic of kaempferol’s effect on NLRP3 inflammasome and cell pyroptosis. (B) Expression of genes in the NOD-like receptor signaling pathway. (C–J) Western blot and quantification of NLRP3, pro-caspase-1, cleaved-caspase-1, F-GSDMD, N-GSDMD, pro-IL-1β, and IL-1β. Protein levels were normalized to β-actin. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = no significance, n = 3).

Subsequently, Western blotting confirmed functional alteration of the pathway, showing marked suppression of NLRP3 activation by all kaempferol formulations in M1 macrophages, with ICG-KPF@ALNPs achieving maximal inhibition. Furthermore, protein levels of the pro- and cleaved isoforms of caspase-1, GSDMD, and IL-1β decreased following exposure to free kaempferol, as well as kaempferol-encapsulated liposomes. Consistently, the strongest downregulation occurred in the ICG-KPF@ALNPs treatment group (Figure C–J). Collectively, these results underscore the suppression of the NLRP3 inflammasome signaling pathway as a central mechanism underlying the therapeutic action of ICG-KPF@ALNPs, thereby limiting macrophage pyroptosis and cytokine release to attenuate inflammation.

3.5. Antioxidant and Anti-Inflammatory Effects of ICG-KPF@ALNPs

Oxidative stress plays a pivotal role in the pathogenesis of AAA, with excessive ROS driving vascular inflammation, endothelial injury, and upregulation of MMPs. Therefore, mitigating the ROS production is essential to impede AAA progression. In our nanodelivery system, kaempferol exhibits potent antioxidant properties. DCFH-DA staining revealed intense fluorescence in LPS-stimulated macrophages, indicative of elevated ROS levels. Treatment with free kaempferol, ICG-KPF@LNPs, and ICG-KPF@ALNPs produced a stepwise decrease in ROS-associated fluorescence in M1 macrophages, with the greatest reduction observed for ICG-KPF@ALNPs. Compared with ICG-KPF@LNPs, ICG-KPF@ALNPs produced an approximately 4-fold reduction in fluorescence intensity and restored ROS levels toward baseline controls. All reported differences were statistically significant (Figure A,B). These results were corroborated by flow cytometry and quantitative analyses, confirming the robust ROS-scavenging capacity of kaempferol formulations and enhanced efficacy following ACPP modification (Figure S5A,B). Mechanistically, as previously reported, oxidative stress induces nuclear translocation of Nrf2, which upregulates antioxidant enzymes such as HO-1. , Western blot analysis demonstrated increased Nrf2 and HO-1 expression in LPS-stimulated macrophages pretreated with free kaempferol, ICG-KPF@LNPs, and ICG-KPF@ALNPs, with ICG-KPF@ALNPs producing the most significant upregulation (Figure C–E). Nrf2 knockout markedly attenuated the ROS-scavenging capacity of KPF, further demonstrating that KPF exerts its antioxidant effect via Nrf2 upregulation (Figure S6).

6.

Dual effects of ICG-KPF@ALNPs. (A, B) Fluorescence images and quantification of ROS levels. (C–E) Western blot and quantification of Nrf2 and HO-1. (F–I) Western blot and (J–M) RT-qPCR analysis of inflammatory markers (iNOS, CD86, MMP-9, IL-1β, IL-6, MCP-1). Data are normalized to β-actin. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = no significance, n = 3).

In AAA lesions, infiltrating macrophages orchestrate inflammatory cascades that exacerbate local inflammation and vascular injury. Macrophages pretreated with free kaempferol, ICG-KPF@LNPs, or ICG-KPF@ALNPs for 2 h, followed by 24 h LPS stimulation, exhibited decreased expression of M1 markers (CD86 and iNOS) by Western blotting, with the most pronounced suppression observed in the ICG-KPF@ALNPs group compared to LPS-only controls (Figure F–H). Moreover, LPS-induced overexpression of MMP-9 in macrophages was likewise markedly reduced by all kaempferol formulations. The reduction was significantly greater with ICG-KPF@ALNPs than with ICG-KPF@LNPs, which can be attributed to ACPP-mediated enhancement of selective cellular internalization (Figure I). qPCR analysis further confirmed significant downregulation of proinflammatory genes IL-1β, IL-6, CCL2, and iNOS (Figure J–M). These results indicate that kaempferol effectively inhibits M1 polarization and attenuates the inflammatory cytokine production.

Collectively, these data demonstrate that kaempferol activated the Nrf2/HO-1 pathway, exhibited potent ROS scavenging activity, and concurrently suppressed macrophage M1 polarization and proinflammatory cytokine release, thereby ameliorating inflammation. The targeting and cellular uptake of ICG-KPF@ALNPs further augment this effect independent of any intrinsic activity of the liposomal carrier (Figure S7). These findings, in line with our sequencing results, suggest that kaempferol mediates antioxidant and anti-inflammatory actions via activation of Nrf2/HO-1 coupled with suppression of the NLRP3 inflammasome signaling pathway. Owing to active targeting, the ICG-KPF@ALNPs group exhibited the most pronounced antioxidant and anti-inflammatory effects compared with the other groups (free kaempferol and ICG-KPF@LNPs), thereby supporting subsequent in vivo evaluation.

3.6. Targeting Capability and Biodistribution of ICG-KPF@ALNPs

Animal models are indispensable for elucidating the pathophysiology of AAA and for evaluating preclinical therapeutics. Here, an angiotensin II (Ang II)-induced AAA mouse model was established, which recapitulated key pathological hallmarks of human AAA, including inflammatory cell infiltration, elevated MMP expression, and extracellular matrix degradation/remodeling. , This model was subsequently used for therapeutic assessment. In vivo targeting capability of ICG-KPF@ALNPs was then evaluated in this AAA model using photoacoustic and fluorescence imaging. We first investigated the pharmacokinetics of the ICG-KPF@ALNPs. High-performance liquid chromatography from 2 to 48 h showed a terminal half-life of approximately 10 h and about 8.9% of the injected dose remaining in blood at 48 h for ICG-KPF@ALNPs, whereas free kaempferol was nearly cleared by 12 h (Figure S8A). Serial blood fluorescence measured on an in vivo imaging system mirrored the HPLC profiles, supporting prolonged circulation and increased exposure with liposomal encapsulation (Figure S8B,C). Healthy and AAA mice received tail-vein injections of ICG-KPF@LNPs or ICG-KPF@ALNPs, followed by imaging at 24 h (Figure A). No photoacoustic signal was detected in the arterial walls of healthy mice for either formulation. In contrast, AAA mice exhibited aneurysm-wall signals for both with markedly higher intensities in the ICG-KPF@ALNPs group (Figure B,C). Consistently, fluorescence imaging revealed strong, lesion-localized signals in the ICG-KPF@ALNPs group, whereas only weak signals were observed with ICG-KPF@LNPs (Figure D,E). Further immunofluorescence analysis confirmed the colocalization of DiI-labeled nanoparticles with CD86-positive M1 macrophages in the aneurysm tissue (Figure S9). Collectively, these findings confirm that ICG-KPF@ALNPs selectively accumulate in AAA lesions. Organ biodistribution analysis further revealed fluorescence predominantly in the liver, kidneys, and spleen, identifying these organs as primary clearance sites (Figures F, S10A). Relative to ICG-KPF@LNPs, administration of ICG-KPF@ALNPs decreased hepatic and renal signals and produced a significantly higher aortic %ID/g versus nontarget tissues, consistent with reduced off-target accumulation in clearance organs (Figure S10B,C).

7.

In vivo targeting and biodistribution of ICG-KPF@ALNPs in AAA mice. (A) Schematic of targeting and biodistribution workflow. (B, C) PA imaging and quantification of ICG-KPF@ALNPs accumulation in aneurysm sites. (D, E) Ex vivo fluorescence imaging and quantification in the abdominal aorta. (F) Distribution in major organs 24 h postintravenous injection. (*P < 0.05, **P < 0.01, ***P < 0.001, ns = no significance, n = 3).

3.7. Therapeutic Efficacy against AAA

On the basis of the above encouraging in vitro results, we next assessed the therapeutic efficacy of nanotherapeutics in angiotensin II (Ang II)-induced AAA mice. Following 4 weeks of concurrent high-fat diet and subcutaneous Ang II infusion, mice received weekly intravenous injections of free kaempferol, ICG-KPF@LNPs, or ICG-KPF@ALNPs (Figure A). Vascular morphology was evaluated by ultrasonography and gross anatomical inspection (Figure B,D). Untreated AAA mice exhibited significant aortic diameter expansion compared with healthy controls, confirming successful model induction. All kaempferol formulations reduced aortic dilation, with kaempferol and ICG-KPF@LNPs producing moderate effects, whereas ICG-KPF@ALNPs restored diameters to levels closest to healthy controls, demonstrating that targeted nanoencapsulation markedly enhances kaempferol’s therapeutic potency. Histological analyses further supported these findings. H&E and EVG staining showed that ICG-KPF@ALNPs best preserved vascular wall thickness and elastic fiber integrity compared with untreated AAA mice (Figure E–G). Masson staining further showed that ICG-KPF@ALNPs preserved collagen fiber organization in the vascular wall, whereas extensive collagen disruption was observed in untreated AAA (Figure H and I). Survival analysis revealed the highest survival rate in the ICG-KPF@ALNPs group, accompanied by the lowest aneurysm incidence, highlighting its protective effect against rupture and disease progression (Figure S11A,B). MMP-9 expression, markedly elevated in AAA lesions, was most strongly suppressed by ICG-KPF@ALNPs (Figure J,K). Oxidative stress analysis by DHE staining revealed intense red fluorescence in AAA tissues, indicative of excessive ROS production. ICG-KPF@ALNPs treatment produced the greatest attenuation of ROS levels among all groups (Figure L,M). Given the established link between aberrant ROS elevation and NLRP3 inflammasome activation, we examined the downstream inflammatory signaling. Immunofluorescence and immunohistochemistry staining demonstrated that all treatments reduced NLRP3 and GSDMD expression relative to untreated AAA mice, with ICG-KPF@ALNPs producing the most pronounced suppression (Figure N–Q). These results indicate that kaempferol attenuates ROS generation, inhibits NLRP3 inflammasome activation, and suppresses downstream inflammatory mediator release. With superior lesion-targeting and drug delivery, ICG-KPF@ALNPs achieve more effective AAA treatment.

8.

In vivo therapeutic efficacy of ICG-KPF@ALNPs in AAA mice. (A) Schematic of the treatment plan. (B) Gross morphology of AAA formation under the indicated treatments (scale bar = 5 mm). Ultrasound images of the abdominal aorta in (C) transverse and (D) longitudinal views after the respective treatments. (E–Q) Histological and immunostaining analyses of aneurysm lesions: HE (E), EVG (F, G), Masson (H, I), MMP-9 (J, K), DHE (L, M), NLRP3 (N, O), and GSDMD (P, Q), with corresponding quantitative analyses (n = 4). Data are expressed as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = no significance).

Compared with passive or antibody-only targeting designs reported for AAA nanomedicines, the MMP-9-activated ACPP in ICG-KPF@ALNPs drives lesion-restricted uptake and intracellular release, thereby enhancing specificity and on-target delivery. , Moreover, under our experimental conditions, treatment with ICG-KPF@ALNPs led to a lower incidence of AAA than that reported for Lipo-MM/SEI (Chen et al.) and Mn-UiO-66-NH₂@HA (Lie et al.), suggesting enhanced protective efficacy. , In contrast to studies focused solely on diagnosis or therapy, coloading ICG provides high-contrast photoacoustic imaging alongside kaempferol treatment, enabling image-guided dosing and real-time monitoring of therapeutic response. Moreover, our study presents a modular theranostic nanoplatform that can be tailored to different indications by altering either the loaded drug or the targeting and activation module. On the therapeutic side, the drug cargo can be exchanged to match disease biology, for example, statins for atherosclerosis. On the delivery side, the MMP-9 responsive ACPP can be replaced with pH-responsive triggers to achieve acid-MMP-9-activated delivery in inflammatory vascular disease, collectively highlighting strong potential for translation across cardiovascular disorders. While the platform shows preclinical promise, future work will clarify ICG photostability under clinically relevant imaging, address immunogenicity, and establish long-term safety to enable translation

3.8. Biosafety Assessment

The biosafety of ICG-KPF@ALNPs was comprehensively assessed over a 4-week (Figure ) and 8-week (Figure S13) study. Hemolysis rates for ICG-KPF@ALNPs and ICG-KPF@LNPs were below 5% at every tested concentration (Figure S12A,B), demonstrating good hemocompatibility and suitability for intravenous administration. No significant differences in body weight were observed among groups, indicating good systemic tolerance following intravenous administration (Figure S12C). Histopathological examination of major organs (heart, liver, spleen, lungs, kidneys) by H&E staining revealed intact architecture without pathological alterations in any treatment group (Figures A, S14). Serum biochemical analyses confirmed normal hepatic (ALT, AST) and renal (BUN, CREA) function markers; complement markers (C3a, C5a) showed no acute activation, and complete blood count parameters remained within physiological ranges (Figures B–I, S13, and S14). Together, these results demonstrate the favorable biosafety profile of the ICG-KPF@ALNPs nanoplatform.

9.

Biosafety assessment. (A) H&E staining of major organs (scale bars = 100 μm). (B–I) Blood biochemistry and complete blood count.

4. Conclusion

In this study, we developed an ACPP-modified liposomal nanoplatform co-loaded with kaempferol and ICG (ICG-KPF@ALNPs) for targeted theranostics of AAA. The MMP-9-responsive ACPP facilitates selective delivery of kaempferol to aneurysmal lesions, while ICG enables concurrent photoacoustic imaging. Mechanistically, kaempferol exerts a dual protective effect by activating the Nrf2/HO-1 pathway to scavenge ROS and inhibiting the NLRP3 inflammasome to limit macrophage pyroptosis, collectively mitigating oxidative stress, inflammation, and matrix degradation. Both in vitro and in vivo results confirm that ICG-KPF@ALNPs effectively suppress aneurysm progression with favorable biosafety, highlighting its potential as a promising candidate for future clinical translation in AAA therapy.

Glossary

Abbreviations

AAA:

abdominal aortic aneurysm

CPP:

cationic cell-penetrating peptide

ACPP:

activatable cell-penetrating peptide

KPF:

kaempferol

ICG:

indocyanine green

Ang II:

angiotensin II

DHE:

dihydroethidium probe

ECM:

extracellular matrix

ROS:

reactive oxygen species

HO-1:

heme oxygenase 1

Nrf2:

nuclear factor erythroid 2-related factor 2

NLRP3:

NOD-like receptor thermal protein domain associated protein 3

Data will be made available on request

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.5c26135.

• Materials and methods; primer sequences; size and PDI stability of ICG-KPF@ALNPs; standard curves for ICG (UV–vis) and kaempferol (HPLC); RAW264.7 viability after GM6001 treatment; GO functional enrichment analysis; flow cytometry and quantification of intracellular ROS scavenging by ICG-KPF@ALNPs; Nrf2 knockdown validation and intracellular ROS measurement (Western blot/quantification and fluorescence/quantification); in vivo therapeutic comparison of ICG-KPF@ALNPs versus ICG@ALNPs (iNOS/CD86 Western blot and ROS fluorescence/quantification); kaempferol pharmacokinetics and blood circulation/fluorescence of ICG or ICG-KPF@ALNPs; immunofluorescence of CD86 and DiI in aneurysmal and normal aortae; organ fluorescence quantification, KPF calibration curve, and biodistribution (%ID/g) at 24 h; Kaplan–Meier survival and aneurysm incidence; in vitro hemolysis and body-weight monitoring; serum complement activation (C3a and C5a); biosafety evaluation (major-organ H&E, blood biochemistry, and complete blood count); and uncropped Western blot data (PDF)

§.

A.C., Z.H., and W.Z. contributed equally to this work. conceptualization, H.F. and Y.L.; data curation, A.C., Z.H., and W.Z.; formal analysis, A.C.; funding acquisition, Y.L. and H.F.; investigation, A.C., Y.L., and G.W.; methodology, H.F.; project administration, H.F.; resources, A.C., Z.H., W.Z., Y.L., G.W., J.Y., Y.G., and X.J.; software, A.C., Z.H., and W.Z.; supervision, H.F.; validation, A.C., Y.L., and H.F.; visualization, A.C., Z.H., and W.Z.; writingoriginal draft, A.C.; writingreview and editing, H.F.. All authors have read and agreed to the published version of the manuscript.

The authors declare no competing financial interest.