Engineered tiny particles that control harmful molecules and suppress the immune system to improve lupus kidney inflammation

Uniformly sized Ce nanoparticles were synthesized via the reaction of cerium acetate hydrate and citric acid (Figure S1). X-ray photoelectron spectroscopy (XPS) analysis confirmed the coexistence of cerium in mixed valence states (Ce³⁺ and Ce⁴⁺) on the nanoparticle surface (Figure S2). The calculated Ce³⁺/Ce⁴⁺ ratio of approximately 1.30 indicates a high concentration of oxygen vacancies, which contributes to robust ROS scavenging capacity. Furthermore, we evaluated the potential ROS scavenging ability of Ce nanoparticles, specifically targeting superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂), which are key ROS that can cause oxidative stress and cell apoptosis when overproduced [30]. Electron paramagnetic resonance (EPR) measurements showing a scavenging rate of 78.76% for the O₂⁻ (Figure S3A). In the case of H₂O₂, spectral line broadening and reduced hyperfine resolution indicated an elevated oxygen concentration, confirming effective decomposition (Figure S3B). Then, Ce nanoparticles were covered with DSPE-PEG and DSPE-PEG-MAL to enable water dispersion and maleimide modification, producing Ce-Mal. Dynamic light scattering (DLS) analysis revealed that Ce-Mal had an average size of approximately 17.73 nm with a polydispersity index (PDI) of 0.3174 (Fig. 2A).

To obtain the MEXs, MSCs at passages 3 to 6 were cultured extensively in serum-free medium. The cell culture media were collected after 48 h and subjected to ultracentrifugation at 100,000 × g for MEX isolation [27]. The quality control parameters of exosomes obtained from different batches demonstrated consistent compliance with established standards (Table S1). According to transmission electron microscopy (TEM), the MEXs demonstrated a distinct bilayer membrane microstructure, characteristic of typical extracellular vesicles (Fig. 2B, and S4). The presence of exosome markers (CD81, TSG101 and Alix) and the cell contamination marker (calnexin and histone H1) was confirmed (Figure S5). These findings demonstrate that MEXs were successfully isolated from the supernatants of MSC cultures for subsequent experiments.

The disulfide bond on the MEX membrane was disrupted by Tris (2-carboxyethyl) phosphine (TCEP), exposing the sulfhydryl groups. The Ce nanoparticle-immobilized MEX (CEX) was then constructed by a Michael addition reaction, using Ce-Mal as the decorative component and thiol-functionalized MEX as the support. TEM images reveal that numerous Ce nanoparticles densely coated the surface of MEXs, leading to an increase in hydrodynamic diameter as they attached (Fig. 2C and S6).

To produce the drug-loaded CEX (CEX@Rapa), rapamycin was physically encapsulated into thiol-functionalized MEXs through ultrasonication, followed by conjugation with Ce-Mal via a thiol-maleimide reaction. The successful construction of CEX@Rapa was confirmed by examining the co-localization of Ce-Mal, MEXs, and rapamycin (Fig. 2D, F and G, and S7). After Ce nanoparticles bonded to the MEX surface, ζ-potential measurements of the CEX and CEX@Rapa indicated a reduction in negative charge (Fig. 2E). We then evaluated the drug loading content of rapamycin in CEX@Rapa, which was 23.66%, and the drug loading efficiency, which was 60.6%. UV spectra demonstrated that CEX encapsulation significantly enhanced the stability of free rapamycin, with only 50% degradation observed after 15 days of incubation in PBS at pH 7.4 (Figure S8). The release profile of rapamycin from CEX@Rapa exhibited sustained and slow kinetics under physiological conditions (PBS, pH 7.4), with approximately 40% of the drug released over 120 h. In contrast, under pathologically relevant oxidative conditions (1 mM H₂O₂ or 1 mM HOCl), release was significantly accelerated, reaching approximately 80% and 70% respectively within the same period—nearly twice that of the physiological control (Fig. 2H). This enhanced release under oxidative conditions is attributed to ROS-induced structural disruption of the exosomal membrane. Specifically, as confirmed by TEM imaging (inset, Fig. 2H), treatment with 1 mM H₂O₂ for 24 h resulted in clear loss of membrane integrity and vesicle deformation, consistent with lipid peroxidation-mediated breakdown [34 –36]. This mechanism enables targeted rapamycin release within inflammatory sites characterized by high ROS levels, while minimizing premature leakage under normal physiological conditions, highlighting its suitability for delivering drugs to the oxidative renal environment associated with LN [37].

To evaluate the therapeutic efficacy of CEX@Rapa, its catalytic performance was investigated first. In managing inflammatory diseases, a primary objective is the rapid removal of these species to alleviate inflammation and cytokine release. Owing to the outstanding ROS-scavenging abilities of the Ce nanoparticles, CEX@Rapa demonstrated effective catalase (CAT) and superoxide dismutase (SOD) mimicking activities. (Fig. 2I and J). Notably, MEX showed reduced activity levels, probably because MSCs secrete SOD [38]. Additionally, the antioxidant capabilities of Ce and Ce-Mal nanoparticles showed minimal differences. These results suggest that the chemical modification with MEX did not affect the catalytic activities of the Ce nanoparticles.

We then investigated if CEX@Rapa retains the properties of MEXs, which are known for their anti-inflammatory and immunomodulatory properties [39]. The presence of key immunomodulatory elements (IL-10, TGF-β, and COX-2) in CEX@Rapa confirmed that these therapeutic agents were maintained during manufacturing (Fig. 2K). Additionally, we observed reduced levels of pro-inflammatory genes (IL-6, CCL-2, CCL-5, and HMOX), indicating that the physical encapsulation of rapamycin and the chemical immobilization of Ce nanoparticles had a synergistic anti-inflammatory effect. These results suggest that CEX@Rapa can exert immunomodulatory properties and perform catalytic functions through transporting therapeutic molecules to specific cells.

RAW 264.7 macrophages were utilized for assessing the biocompatibility of CEX@Rapa prior to biological evaluation, and no apparent toxicity was found over 72 h of co-incubation (Figure S9). In contrast, only 60% of cells survived in the free rapamycin group, indicating that MEX encapsulation significantly reduced the cytotoxicity of rapamycin.

We performed a macrophage uptake assay to further assess the effectiveness of the prepared nanohybrids. Macrophages are essential in the secretion of inflammatory mediators and are plentifully found at sites of inflammation [40]. Hence, assessing the uptake of CEX@Rapa by activated macrophages is essential for determining its effectiveness in treating LN. Minimal free rapamycin was endocytosed by both LPS activated and non-activated macrophages (Fig. 3A). In contrast, significant cellular uptake of CEX@Rapa was observed in activated macrophages (Fig. 3B), indicating that exosomes enhance drug ingestion, enabling macrophages to effectively take up the drugs and exert their therapeutic effects.

To elucidate the mechanistic role of the loaded rapamycin, we evaluated its impact on mTOR pathway inhibition and autophagy induction in LPS-activated RAW 264.7 macrophages via immunofluorescence. As shown in Figure S10 and S11, treatment with CEX@Rapa markedly reduced the fluorescence signals of phosphorylated S6K (p-S6K) and 4EBP1 (p-4EBP1), key downstream effectors of mTORC1, confirming effective suppression of mTOR signaling. Concurrently, we observed a significant increase in LC3B expression, indicative of autophagosome formation, accompanied by a notable decrease in p62 expression, reflecting enhanced autophagic flux and robust autophagy activation.

Given that ROS overproduction primarily triggers macrophage activation, we explored the ability of nanohybrids to scavenge ROS in vitro. The findings from confocal microscopy indicated that CEX@Rapa successfully eliminated the excess ROS in activated macrophages, with ROS intensity levels comparable to those of Ce nanoparticles and CEX alone, highlighting the Ce-driven ROS removal capability (Fig. 3C and F). The combination of mTOR-dependent autophagy activation and Ce-mediated ROS clearance creates a coordinated intracellular environment conducive to anti-inflammatory polarization.

Considering this, we further evaluated the polarization of macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype under inflammatory conditions. M1 macrophages, characterized by high iNOS expression, exacerbate SLE progression through the secretion of inflammatory cytokines [37]. Transitioning to the M2 phenotype (marked by CD206) represents a promising therapeutic strategy for LN. In the CEX@Rapa treatment group, we observed significant enrichment of CD206 and downregulation of inflammation-related proteins compared to the PBS control (Fig. 3D and E). Flow cytometry analysis confirmed a pronounced shift from M1 to M2 polarization (Fig. 3G, H and Figure S12). Although individual treatments with Ce nanoparticles, MEX, or rapamycin alone exhibited anti-inflammatory effects, CEX@Rapa demonstrated superior efficacy, suggesting synergistic cooperation among the immunomodulatory cargo of MEX, rapamycin-mediated immunosuppression (via mTOR inhibition and autophagy induction), and Ce nanoparticle-driven catalytic activity in modulating innate immune responses. The MEX and rapamycin addresses the inflammatory signaling cascades from within, while the Ce nanoparticles mitigates the external oxidative stress that fuels inflammation, resulting in a more robust and sustained anti-inflammatory outcome.

Podocytes and their foot processes play a crucial role in glomerular filtration. Damage to these structures is crucial for the development of nephrotic syndrome associated with various glomerular injuries [41]. The biocompatibility of CEX@Rapa was assessed using MPC-5 cells, and no evident toxicity was observed (Figure S13). Given that nanohybrids can enter the bloodstream and target damaged kidney tissues for drug delivery due to the inherent ability of stromal cell-derived exosomes to target inflamed sites [22]. We investigated the uptake efficiency of CEX@Rapa using the podocyte cell line MPC-5. For this, puromycin amino nucleotide (PAN) was administered at a concentration of 15 µg/mL to establish a podocyte injury model. As shown in Fig. 4A, we observed a substantial accumulation of fluorescent nanohybrids in PAN-induced damaged podocytes. Quantitative data further confirmed an increase in fluorescence intensity in the MPC-5 injury model, as determined by flow cytometry (Fig. 4B), indicating that CEX@Rapa is readily phagocytized by podocytes, thereby facilitating its therapeutic effects.

Intracellular ROS levels in PAN-induced podocytes were analyzed and quantified using the DCFH-DA probe. The results revealed that PAN significantly elevated ROS levels in podocytes, while Ce nanoparticles and CEX effectively reduced these levels (Fig. 4C and D). Notably, free rapamycin could partially reverse the oxidative damage in podocytes, likely by activating autophagy. Among the treatments, CEX@Rapa exhibited the highest scavenging capacity in renal cells, attributed to the synergistic antioxidant effects of Ce nanoparticles and the released rapamycin. This discovery aligns with the in vitro release behavior of CEX@Rapa, which facilitates the cleavage and release of rapamycin under conditions of high ROS concentration (Fig. 1H).

We next assessed the damage-repair of PAN-induced podocytes to determine whether CEX@Rapa could repair cell tissue. Our results indicated that PAN induced podocyte injury in a time-dependent manner, as demonstrated by cell actin staining and the CCK-8 assay (Fig. 4E and F). The increase in cell area further confirmed the anti-damage effects of CEX@Rapa on MPC-5, leading to the rearrangement of the podocyte actin cytoskeleton and enhanced adhesion of the repaired podocytes. It is noted that rapamycin alone significantly improved or even reversed PAN-induced podocyte injury, likely through the rapamycin-mediated inhibition of the PI3K/AKT/mTOR signaling pathway and autophagy [42, 43]. In summary, these data highlight the anti-damage and antioxidant activities of CEX@Rapa, which are essential for podocyte and renal tissue repair.

A live animal imaging system was employed to evaluate the targeting efficacy of CEX@Rapa. DiD-tagged CEX@Rapa was administered intravenously (i.v.) to MRL/lpr mice, and its distribution was monitored using the live animal imaging system. Compared to free rapamycin, CEX@Rapa showed a marked buildup in the kidneys at 2, 4, and 24 h following administration (Fig. 5A). Notably, the near-infrared fluorescence (NIRF) signal in CEX@Rapa remained robust 24 h after administration, while the NIRF intensity treated with free rapamycin decreased considerably during the same period. This finding highlights the enhanced targeting accuracy and retention conferred through exosome encapsulation (Fig. 5B, C, and S14).

Furthermore, tissue sections immunofluorescence analysis verified the selective renal targeting capability of CEX@Rapa. The inflamed kidneys showed high fluorescence intensity, whereas the heart, liver, spleen, and lungs of MRL/lpr mice had minimal fluorescence accumulation (Fig. 5D). This targeted delivery to damaged kidneys is probably attributed to the increased permeability of renal blood vessels and the inherent capacity of stromal cell-derived exosomes to localize at inflamed sites. Overall, these results demonstrate CEX@Rapa's promise as a therapeutic agent for renal injury by utilizing both its targeted delivery and sustained retention in affected tissues.

CEX@Rapa's therapeutic potential in vivo was evaluated by intravenously administering it to MRL/lpr mice, recognized for SLE. The treatment plan included weekly i.v. administration of saline, Ce nanoparticles, MEX, CEX, rapamycin, and CEX@Rapa over a four-week period, focusing on LN (Fig. 6A). Notably, the group treated with CEX@Rapa showed a significant reduction in facial lesions area (mean ± SEM: 3.39% ± 0.68%) compared with alternative treatment groups (Fig. 6B and S15, S16). Additionally, this group showed considerably reduced spleen and lymph node sizes, supported by weight measurement comparisons (Fig. 6C and S17). Throughout the treatment period, MRL mice exhibited stable body weight maintenance with consistent growth trends (Figure S18).

To assess kidney dysfunction-an important indicator in LN-we analyzed the effects of CEX@Rapa on essential markers of glomerular filtration in MRL/lpr mice, such as proteinuria, blood urea nitrogen (BUN) and creatinine levels (Fig. 6D, E and F). Our findings revealed that CEX@Rapa nanohybrids outperformed all other groups by significantly reducing proteinuria, creatinine and BUN levels. In SLE, affected animals typically exhibit elevated levels of anti-dsDNA antibodies, which are strongly linked to glomerulonephritis. According to Fig. 6G, MRL/lpr mice given either Control, Ce nanoparticles, MEX, CEX, or rapamycin alone displayed higher anti-dsDNA antibody levels. Conversely, mice that received CEX@Rapa showed a marked decrease in anti-dsDNA Abs levels. In addition, ELISA findings revealed that the CEX@Rapa group exhibited notably decreased levels of inflammatory cytokines TNF-α and IL-6 after treatment, compared with alternative groups (Fig. 6H and I), indicating a notable reduction in the production of inflammatory cytokines in MRL/lpr mice. These outcomes corporately affirm the sustained synergistic effect of CEX@Rapa hybrid system in the progression of LN and highlight its potential for treating other conditions associated with immune dysregulation.

Spontaneous renal complications, like membranoproliferative glomerulonephritis (MPGN), commonly occur in MRL/lpr mice and are characterized by the accumulation of C3 and IgG in the glomeruli. According to the histological analysis, CEX@Rapa effectively lowered glomerular issues, evidenced by limited cell invasion and decreased mesangial hyperplasia (Fig. 7A and B). Additionally, the CEX@Rapa treatment effectively diminished the accumulation of immune complexes, as evidenced by lower levels of C3 and IgG staining (Fig. 7C-E). While the clinical application of rapamycin and Ce nanoparticles is often limited by their intrinsic toxicity, the challenge lies in mitigating these adverse effects without compromising their efficacy. Notably, the CEX@Rapa not only attenuated LN progression effectively but also exhibited minimal organ toxicity in vivo. Specifically, serum levels of ALP, ALT, and AST in treated MRL/lpr mice remained within normal physiological ranges, indicating an absence of hepatotoxicity-a finding consistent with prior in vitro biosafety assessments (Figure S19 and S20).

These findings highlight the synergistic effects achieved through the physical encapsulation of rapamycin, the chemical conjugation of Ce nanoparticles and MEX in the CEX@Rapa hybrid systems. This unique combination harnesses the inflamed renal homing ability and immunoregulatory properties of MEX, the immunosuppressive effects of rapamycin, and the ROS scavenging capability of Ce nanoparticles. As a result, CEX@Rapa enhances immune modulation and alleviates renal complications in MRL/lpr mice. Collectively, our data demonstrate that intravenous administration of CEX@Rapa in the MRL/lpr mouse model significantly decreased both the occurrence and intensity of the disease, highlighting the probability of CEX@Rapa as a prophylactic therapy for LN.