Engineered neutrophil membrane-camouflaged nanocomplexes for targeted siRNA delivery against myocardial ischemia reperfusion injury

Jiang, Yaohui; Jiang, Rongyan; Xia, Zequn; Guo, Meng; Fu, Yanan; Wang, Xiaocheng; Xie, Jun

doi:10.1186/s12951-025-03172-w

Research
Open access
Published: 22 February 2025

Engineered neutrophil membrane-camouflaged nanocomplexes for targeted siRNA delivery against myocardial ischemia reperfusion injury

Yaohui Jiang¹^na1,
Rongyan Jiang⁴^na1,
Zequn Xia¹,
Meng Guo¹,
Yanan Fu³,
Xiaocheng Wang² &
…
Jun Xie^1,3

Journal of Nanobiotechnology volume 23, Article number: 134 (2025) Cite this article

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Abstract

Small interfering RNA (siRNA) therapies hold great potential for treating myocardial ischemia-reperfusion injury (MIRI); while their practical application is limited by the low bioavailability, off-target effects, and poor therapeutic efficacy. Here, we present an innovative engineered neutrophil membrane-camouflaged nanocomplex for targeted siRNA delivery and effective MIRI therapy. A nanoparticle (NP)-based siRNA delivery system, namely MNM/siRNA NPs, is camouflaged with neutrophil membranes modified by hemagglutinin (HA) and integrins. Our comprehensive in vitro studies show that MNM/siRNA NPs effectively facilitate endosomal escape through HA, achieve excellent targeting via integrins, and significantly reduce integrin α9 expression. Furthermore, in MIRI mice, we identify integrin α9 as a potential target for MIRI therapy and demonstrate that MNM/siRNA NPs significantly decrease myocardial infarction area and improve cardiac function by reducing neutrophil recruitment, neutrophil extracellular trap (NET) and microthrombus formation. These findings highlight the engineered membrane-camouflaged NPs as a promising siRNA delivery platform, offering an effective treatment strategy for MIRI.

Introduction

Acute myocardial infarction (AMI) is a leading cause of mortality and morbidity globally. Although early reperfusion therapies, such as percutaneous coronary intervention (PCI), can salvage ischemic myocardium and improve survival, the sudden restoration of blood supply can induce myocardial ischemia-reperfusion injury (MIRI) [1]. Among the various cell types implicated in the pathogenesis of MIRI, neutrophils play a crucial role [2, 3], integrin α9 is essential for neutrophil-mediated inflammation and facilitating neutrophil extracellular trap (NET) formation through interactions with endothelial cells and platelets [4]. Studies have also shown that integrin α9 deficiency in mice leads to reduced neutrophil recruitment, decreased NET formation, and alleviation of thrombus burden [5], suggesting that it plays a potential role against MIRI. Precisely targeting neutrophils in ischemic injury sites with conventional oral drugs is a great challenge in clinical practice due to their poor targeting, potential for drug resistance, and adverse side effects. Small interfering RNA (siRNA) therapies, leveraging RNA interference mechanisms, represent a promising alternative with high gene silencing efficiency, enhanced specificity, and broad target applicability. siRNA therapies have demonstrated notable success in oncology and hold potential for treating cardiovascular conditions including MIRI [6]. However, their clinical application is limited by challenges such as poor bioavailability, instability, and immunogenicity [6, 7]. Thus, developing a novel delivery platform that enhances siRNA stability and therapeutic efficacy is critical for attenuating MIRI.

Herein, we propose engineered neutrophil membrane-camouflaged nanocomplexes for targeted siRNA delivery and effective MIRI therapy, as illustrated in Fig. 1(a), nanoparticle (NP)-based siRNA delivery system is designed to target integrin α9 and is camouflaged with modified neutrophil membranes. A novel cationic polymer, poly (CBA-co-4-amino-1-butanol) (pABOL), has been developed to improve siRNA delivery while reducing cytotoxicity compared to conventional polymers [8]. As we known, NPs are often sequestered by the mononuclear phagocyte system, limiting their circulation time. Cell membrane-camouflaged NPs have demonstrated the ability to evade immune detection, extend drug circulation, and improve accumulation at inflammation sites [9, 10]. Nevertheless, achieving efficient cytoplasmic delivery remains challenging due to lysosomal degradation [11]. Certain viruses, such as the influenza virus, utilize hemagglutinin (HA) proteins to escape endosomal degradation, offering a model for siRNA delivery [12, 13]. To improve the targeting efficacy of NPs, integrin α9 and β1 were overexpressed in neutrophil membrane, as they mediate neutrophils recruitment to ischemic injury sites by binding with vascular cell adhesion protein 1 (VCAM-1) on vascular endothelial cells (ECs) [14]. Given HA-mediated endosomal escape and integrins’ role in neutrophil recruitment, combining HA and integrins-modified neutrophil membranes with the pABOL-based siRNA systems (namely MNM/siRNA NPs, Fig. 1(a)) is anticipated to offer an effective strategy for targeted siRNA delivery against MIRI.

In this study, we fabricated the HA and integrins modified neutrophil membrane-camouflaged pABOL NPs loaded with siRNA targeting integrin α9 (namely MNM/siRNA NPs, Fig. 1(a)). We induced HA protein expression through adenovirus transfection and enriched integrins on neutrophil membranes via endothelin-1 (ET-1) stimulation [15, 16]. The engineered neutrophil membrane was then extracted to camouflage pABOL NPs loaded with siRNA targeting integrin α9. Compared to free siRNA, siRNA-loading pABOL NPs (siRNA NPs), unmodified neutrophil membrane-camouflaged siRNA NPs (NM/siRNA NPs), and MNM/siRNA NPs demonstrated superior endosomal escape and targeting (Fig. 1(b)), significantly reducing integrin α9 expression and showing enhanced therapeutic effects both in vitro and in vivo. More importantly, MNM/siRNA NPs significantly decreased myocardial infarction area and improved cardiac function in MIRI mice by reducing neutrophil recruitment, as well as the formation of NETs and microthrombus (Fig. 1(b)). These results underscored the potential of MNM/siRNA NPs for effective siRNA delivery, attributed to their excellent endosomal escape and targeting properties.

Results and discussion

Preparation and characterization of MNM/siRNA NPs

Neutrophils, as the most abundant leukocyte in the peripheral blood, are the first cells to be recruited to ischemic injury sites [2, 3]. The mouse neutrophil cell (MNHC) line was selected for cell membrane coating due to its rapid recruitment to ischemic sites and interaction with inflamed endothelial cells via increased integrins and chemokine receptors [17, 18]. Given the HA’s role in endosomal escape and integrins’ role in recruitment, we utilized adenovirus transfection to express HA and stimulated integrin α9/β1 expression on neutrophils using ET-1 (Fig. 2(a)). Flow cytometry analysis and immunofluorescence confirmed the expression of HA protein on neutrophils after adenovirus transfection by detecting HA-tagged green fluorescent protein (HA-GFP, Fig. 2(b-d)). Further ET-1 stimulation resulted in the overexpression of integrin α9/β1 on neutrophil cells, as detected by western blot (WB) and quantitative polymerase chain reaction (qPCR) (Fig. 2(e-i)). These results indicated that the neutrophils successfully acquired abundant HA and integrin membrane proteins after the treatment.

Subsequently, we synthesized pABOLs with a molecular weight of 8.1 kDa, chosen for their high transfection efficiency and low cytotoxicity based on previous studies [8, 19]. Additionally, the pABOLs were easier to prepare, reducing both processing time and complexity. These advantages made pABOLs a more practical and robust platform for siRNA delivery, offering an innovative and scalable solution for potential therapeutic applications. The siRNA NPs were then obtained by mixing pABOLs with siRNA. To prepare MNM/siRNA NPs, we isolated the engineered neutrophil membranes using a hypotonic lysis technique, followed by assembling them with the siRNA NPs through sonication (Fig. 3(a)). To validate the encapsulation of cell membranes on NPs, dynamic light scattering (DLS) measurements showed that the diameter of the formulated MNM/siRNA NPs was 30 nm greater than that of the siRNA NPs, indicating the addition of an extra bilayered cell membrane onto pABOLs (Fig. 3(b)). Previous studies have demonstrated that NPs with diameter sizes between 10 and 200 nm exhibit optimal blood circulation lifespans, with decreased uptake by non-phagocytic cells as the diameter increases [20, 21]. The surface zeta potential measurements revealed values of -21.63 ± 1.04 mV for MNM, 3.69 ± 0.96 mV for siRNA NPs, and − 6.47 ± 2.46 mV for MNM/siRNA NPs, suggesting the presence of a biomimetic surface charge (Fig. 3(c)). Additionally, DLS measurements confirmed that the formulated MNM/siRNA NPs maintained their size over 96 h in phosphate-buffered saline (PBS) and 10% fetal bovine serum (FBS) (Fig. 3(d)), highlighting the colloidal stability of MNM/siRNA NPs in biological conditions. Further confocal laser scanning microscopy (CLSM) images showed the co-localization of Dil-labeled MNM and Cy5-labeled pABOLs, providing visual confirmation of successful MNM encapsulation on pABOLs after extrusion (Fig. 3(e)). Transmission electron microscopy (TEM) imaging also revealed a cell membrane layer coating the siRNA NPs, which was absent in siRNA NPs alone (Fig. 3(f)). These results collectively indicated the successful membrane coating process of MNM onto pABOLs.

We next evaluated whether MNM/siRNA NPs retained key membrane proteins from MNM. Coomassie blue staining revealed that the total protein composition of MNM/siRNA NPs closely resembled that of MNM vesicles, confirming the successful transfer of most proteins to the surface of MNM/siRNA NPs during the coating process (Fig. 3(g)). WB analysis provided further validation by detecting the presence of key proteins on MNM/siRNA NPs derived from MNM vesicles, including integrin α9, integrin β1, lymphocyte function-associated antigen-1 (LFA-1), P-selectin glycoprotein ligand-1 (PSGL-1), HA, C-X-C chemokine receptor type 2 (CXCR2), and C-C chemokine receptor type 2 (CCR2), while no protein bands were observed in the uncoated siRNA NPs (Fig. 3(h)). These findings suggested that MNM successfully retained the critical physiological properties of neutrophils.

Immune escape and targeting ability of MNM/siRNA NPs in vitro

Cytotoxicity testing is essential for evaluating the safety of pharmaceuticals. We assessed the cytotoxicity of siRNA NPs and MNM/siRNA NPs using the Cell Counting Kit-8 (CCK-8) method prior to cellular experiments. The results (Fig. 4(a) and Figure S1(a, b)) demonstrated that concentrations up to 200 µg/ml of both siRNA NPs and MNM/siRNA NPs were safe for MNHCs, human umbilical vein endothelial cells (HUVECs), and smooth muscle cells (SMCs) after incubation for 24 h.

To evaluate immune evasion, we incubated free siRNA, siRNA NPs, NM/siRNA NPs, and MNM/siRNA NPs with RAW 264.7 cells for 4 h, using Cy5-labeled siRNA. CLSM and flow cytometry analyses revealed that NM/siRNA NPs and MNM/siRNA NPs significantly reduced macrophage phagocytosis compared to free siRNA and siRNA NPs (Fig. 4(b, c), and Figure S1(c, d)), demonstrating the effectiveness of cell membranes in immune evasion.

We then assessed the endosomal escape and degradation avoidance of MNM/siRNA NPs in vitro. MNHCs were treated with NM/siRNA NPs and MNM/siRNA NPs for varying durations following lipopolysaccharide (LPS) administration to simulate activated neutrophils. Cy5, Hoechst 33,342, and LysoTracker Red were used to stain siRNA, nuclei, and lysosomes, respectively, before CLSM analysis. After 1 h of incubation, both NM/siRNA NPs and MNM/siRNA NPs were primarily localized to the cell surface. At the 6-hour mark, colocalization with lysosomes indicated endocytosis. By 12 h, MNM/siRNA NPs exhibited increased NP signals and reduced lysosome signals, suggesting effective endosomal escape, whereas NM/siRNA NPs remained colocalized with lysosomes. At 24 h, MNM/siRNA NPs signals were detected in the cytosol, while no such signal was observed for NM/siRNA NPs (Fig. 4(d)). These findings suggested that the biomimetic neutrophil membrane effectively hindered macrophage phagocytosis, and the HA protein on the engineered neutrophil membrane prevented degradation within endosomal compartments.

Neutrophils are well-documented for their recruitment to inflammatory sites through the upregulation of integrins and chemokine receptors [17, 18]. We then tested the targeting ability of MNM/siRNA NPs to activated neutrophils at ischemic injury sites. MNHCs, stimulated by LPS to simulate activated neutrophils, were incubated with equal concentrations of free siRNA, siRNA NPs, NM/siRNA NPs, and MNM/siRNA NPs for 4 h, using Cy5-labeled siRNA. Flow cytometry analysis quantitatively revealed that LPS-stimulated neutrophils took up more siRNA compared to unstimulated neutrophils (Fig. 4(e), Figure S1(e-g)). Notably, MNM/siRNA NPs treated cell showed a significantly higher amount of intracellular siRNA compared to free siRNA, siRNA NPs, and NM/siRNA NPs treated (Fig. 4(e)). CLSM analysis further demonstrated the superior targeting capability of MNM/siRNA NPs to activated neutrophils (Fig. 4(f)). These findings confirmed the enhanced targeting ability of MNM/siRNA NPs, probably due to the increased expression of integrins on the MNM, which enhanced uptake by neutrophils.

Furthermore, we quantitatively compared the proposed MNM/siRNA NP delivery system with the siRNA transfection reagent riboFECT™CP in terms of immune evasion, delivery efficiency, and bioavailability (Figure S2). We found that the uptake of riboFECT™CP/siRNA by RAW 264.7 cells was similar to that of siRNA NPs but slightly higher than that of NM/siRNA NPs and MNM/siRNA NPs (Figure S2(a, b)), suggesting that the NM/siRNA NPs and MNM/siRNA NPs exhibited enhanced immune evasion properties compared to riboFECT™CP. Additionally, in LPS-stimulated MNHCs, the uptake of NM/siRNA NPs and MNM/siRNA NPs was significantly greater than that of free siRNA, riboFECT™CP/siRNA, and siRNA NPs after 4 h of incubation (Figure S2(c, d)). This result highlighted the superior delivery efficiency and bioavailability of NM/siRNA NPs and MNM/siRNA NPs, likely due to their specific surface modifications and the inherent properties of their nanostructure.

MNM/siRNA NPs inhibited the formation of NETs and release of inflammatory factors in vitro

Previous evidence indicates that integrin α9 plays a crucial role in the formation of NETs and neutrophil-mediated inflammation [4, 5]. Consequently, a reduction in integrin α9 expression can significantly inhibit the inflammatory response. Optimal suppression of integrin α9 expression was achieved with a pABOLs/siRNA ratio of 2:1 (w/w) (Fig. 5(a)). The efficacy of MNM/siRNA NPs in siRNA-mediated knockdown of integrin α9 was subsequently evaluated in vitro using WB and qPCR. MNHCs stimulated with LPS to simulate activated neutrophils were incubated with free siRNA, siRNA NPs, NM/siRNA NPs, and MNM/siRNA NPs for 24 h. Both NM/siRNA NPs and MNM/siRNA NPs significantly reduced integrin α9 expression compared to the control, free siRNA, and siRNA NPs groups (Fig. 5(b, c) and Figure S3(a)). Notably, the MNM/siRNA NPs group exhibited the lowest integrin α9 expression, indicating that the engineered neutrophil membrane on the surface of siRNA NPs enhanced efficacy.

The anti-NET effect of MNM/siRNA NPs was evaluated in MNHCs using WB and enzyme-linked immunosorbent assay (ELISA). Myeloperoxidase (MPO) and citrullinated histone H3 (CitH3), as main components of NETs, along with peptidylarginine deiminase 4 (PAD4), known as a key regulator of NET formation [22, 23], were both assessed. MNM/siRNA NPs treatment demonstrated a reduction in NET release in vitro, confirmed by WB analysis of PAD4, MPO, and CitH3 (Fig. 5(d-g)). Additionally, the levels of MPO-DNA, neutrophil elastase (NE)-DNA, and cathepsin G, all markers of NETs, were significantly lower in the MNM/siRNA NPs group than those of other groups (Fig. 5(h-j)), further confirming the anti-NET effects of MNM/siRNA NPs.

Several cytokines, including tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and IL-6 secreted by neutrophils, are involved in tissue injury induced by ischemia [24, 25]. In this study, we investigated the ability of MNM/siRNA NPs to neutralize inflammatory factors in vitro using receptors inherited from activated MNHCs, as assessed by ELISA. The results showed that MNM/siRNA NPs effectively neutralized the cytokines including TNF-α, IL-1β, and IL-6 (Fig. 5(k-m)). Overall, these findings suggested that MNM/siRNA NPs significantly inhibited the formation of NETs and the release of inflammatory factors in vitro, which could be attributed to the physiological function of the specialized protein HA and integrins on the modified membrane.

Targeting ability of MNM/siRNA NPs in vivo

The circulation time of NPs is critical for targeted drug delivery, as prolonged circulation can enhance their recruitment to the intended organ [26]. To assess the circulation time of MNM/siRNA NPs, pharmacokinetic studies were conducted by measuring the fluorescence intensity of Cy5-labeled siRNA at various time points (6, 12, 18, and 24 h) from blood and heart (Fig. 6(a)). We found that MNM/siRNA NPs both had a stronger fluorescence intensity at all time points compared to free siRNA, siRNA NPs, and NM/siRNA NPs (Fig. 6(b-d)). These findings demonstrated that siRNA NPs, with the aid of MNM components, exhibited a stronger sustained release and long circulation capabilities in blood and heart, making them ideal carriers for siRNA delivery.

Investigating potential off-target effects is essential for assessing the safety and specificity of the siRNA delivery system. To further evaluate the targeting efficiency of MNM/siRNA NPs in a mouse model of MIRI, equivalent doses (5 mg kg^− 1) of PBS, free siRNA, siRNA NPs, NM/siRNA NPs, and MNM/siRNA NPs were administered to MIRI mice, with Cy5-labeled siRNA used for tracking. Using an in vivo imaging system, fluorescence signals were detected in major organs, including the heart, liver, spleen, lungs, and kidneys, 24 h post-injection. Fluorescence signals were detected in non-target organs such as the liver and spleen, likely reflecting the natural biodistribution and clearance pathways of NPs. However, these signals did not necessarily correlate with functional off-target effects. Quantitative analysis revealed that the fluorescence intensity in the MNM/siRNA NPs group was higher in the heart compared to the free siRNA, siRNA NPs, and NM/siRNA NPs groups (Fig. 6(e, f)). However, the fluorescence intensity in the MNM/siRNA NPs group was lower in the liver, lungs, and kidneys compared to these other groups. This enhanced targeting in the heart is likely due to the presence of MNM proteins on the NPs.

Reduced NET formation and inflammation by MNM/siRNA NPs in MIRI mice

To evaluate the in vivo efficacy of MNM/siRNA NPs in knocking down integrin α9, we conducted qPCR and WB analyses using a MIRI mouse model (Fig. 7(a)). The results demonstrated that both NM/siRNA NPs and MNM/siRNA NPs significantly reduced integrin α9 expression compared to the control, free siRNA, and siRNA NPs groups 24 h after MIRI (Fig. 7(b-d)). Notably, the MNM/siRNA NPs group exhibited the most pronounced reduction in integrin α9 expression, highlighting the enhanced efficacy provided by the engineered neutrophil membrane-camouflaged siRNA NPs.

The inflammatory response is a pivotal pathological mechanism in MIRI. The increased activated neutrophils and NET formation in the heart were observed 24 h after MIRI [27, 28]. To our delight, the neutrophil infiltration in ischemic cardiac tissue 24 h after MIRI in the MNM/siRNA NPs group was significantly reduced, as confirmed by the immunohistochemistry and hematoxylin-eosin (H&E) staining (Fig. 7(e, f), and Figure S3(b)). Moreover, genes associated with neutrophil chemotaxis and infiltration (CCR2, CXCR2, matrix metalloproteinase-2(MMP2), and MMP9) were downregulated in this group (Figure S3(c-f)).

Additionally, the MNM/siRNA NPs group exhibited reduced levels of PAD4, MPO, and CitH3 in the injured myocardium 24 h after MIRI (Fig. 8(a-d)), indicating a reduction in NET release in these mice. Immunofluorescence analyses further confirmed the suppressed NET release by MNM/siRNA NPs in MIRI mice (Fig. 8(e, f)). Moreover, MNM/siRNA NPs significantly reduced the release of MPO-DNA, NE-DNA, and cathepsin G (Fig. 8(g-i)), further validating their anti-NET effects in vivo. Unlike conventional therapies that target specific cytokines with single antibodies, MNM/siRNA NPs functioned as decoys by interacting with MNM receptors to inhibit cytokine activity and neutrophil recruitment. This approach resulted in significant reductions in cytokines such as TNF-α, IL-1β, and IL-6, as demonstrated by in vivo studies (Figure S3(g-i)). Collectively, MNM/siRNA NPs with their enhanced sustained release and prolonged circulation capabilities, significantly reduced the inflammatory response in a mouse model of MIRI by inhibiting neutrophil infiltration and NET formation.

MNM/siRNA NPs suppressed the microthrombus formation and endothelial damage in vivo

Recent evidence implicates that NETs interact with platelets and red blood cells to form microthrombus [29, 30]. Here, we assessed thrombus density using CLSM with anti-thrombocyte and anti-CD31 antibodies. Our results showed that the administration of NM/siRNA NPs and MNM/siRNA NPs notably reduced microthrombus in the ischemic border zone (Fig. 9(a, b)). FITC-dextran staining further confirmed improved perfusion in the injured myocardium in these groups (Fig. 9(c, d)). Moreover, there were no significant differences in coagulation function, including prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen, and platelet counts, between the control and intervention groups (Figure S4(a-e)). In addition, we employed CLSM with anti-albumin and anti-CD31 antibodies to assess vascular integrity. MNM/siRNA NPs were found to preserve microcirculatory integrity. Notably, less albumin leakage was observed in the NM/siRNA NPs and MNM/siRNA NPs groups compared to the control, free siRNA, and siRNA NPs groups (Fig. 9(e-f)). Furthermore, we utilized WB analysis with an anti- zonula occludens-1 (ZO-1) antibody to evaluate endothelial connectivity, which revealed a corresponding reduction in ZO-1 levels in the NM/siRNA NPs and MNM/siRNA NPs groups (Figure S5(a, b)). Taken together, these findings compellingly demonstrated the superior efficacy of MNM/siRNA NPs in mitigating microthrombus formation and endothelial damage, highlighting their potential as a promising therapeutic strategy for ischemic heart conditions.

MNM/siRNA NPs decreased myocardial infarction area and improved cardiac function in vivo

To further validate the efficacy of MNM/siRNA NPs, a mouse model of MIRI was utilized (Fig. 10(a)). Mice were administered PBS, free siRNA, siRNA NPs, NM/siRNA NPs, or MNM/siRNA NPs, and randomly divided into short-term (3–7 days) and long-term (14 days) groups. The survival rate of mice in the MNM/siRNA NPs group post-MIRI was higher than that in the control, free siRNA, siRNA NPs and NM/siRNA NPs groups (Figure S6), indicating that neutrophil membrane encapsulation reduced mortality in both short-term and long-term scenarios. Notably, the MNM/siRNA NPs group exhibited the lowest mortality rate, suggesting that the engineered neutrophil membrane enhanced the therapeutic effect of the siRNA NPs.

By reducing NET and microthrombus formation, MNM/siRNA NPs could significantly decrease myocardial infarction area and improve cardiac function following MIRI. In a short-term assessment, the infarction size (IS) to area at risk (AAR) ratio was significantly smaller in the MNM/siRNA NPs group compared to the control, free siRNA, siRNA NPs and NM/siRNA NPs groups, which was evaluated 24 h after MIRI using Evan’s blue and triphenyltetrazolium chloride (TTC) staining (Fig. 10(b-d)). In a long-term evaluation, myocardial fibrosis was assessed 14 days post-MIRI using Masson’s trichrome staining, which revealed reduced myocardial fibrosis in the MNM/siRNA NPs group compared to the control and other intervention groups (Fig. 10(e, f)). Echocardiographic analysis further demonstrated that MNM/siRNA NPs significantly improved cardiac function, as evidenced by increased left ventricular ejection fraction (LVEF%) and fractional shortening (FS%), along with decreased left ventricle end-diastolic diameter (LVEDD) and left ventricle end-systolic diameter (LVESD) (Fig. 10(g-k)). These findings suggested that MNM/siRNA NPs could effectively mitigate myocardial infarction area, improve cardiac function, and delay the cardiac remodeling process, with the modified membrane structure contributing to enhanced preservation of cardiac function.

Biological safety evaluation

Given the potential hemolytic risk associated with drug delivery systems, evaluating the biocompatibility of NPs is crucial. Blood compatibility, a key parameter in this assessment, was determined via a hemolysis assay. Free siRNA, siRNA NPs, NM/siRNA NPs, and MNM/siRNA NPs were incubated with blood using a direct contact approach. The absence of irregularities or particulate sedimentation in the supernatant indicated no erythrocyte damage for all NPs (Fig. 11(a-e)).

To assess the potential adverse effects of NPs on major organs, including the lungs, liver, spleen, and kidneys, we evaluated organ edema and injury. The organ-to-body weight ratio was measured to determine major organ edema, and H&E staining was performed to detect tissue injury. No adverse immune responses were observed after the administration of MNM/siRNA NPs. Results showed no significant differences in major organ edema or injury between the control and intervention groups (Fig. 11(f, g)). Additionally, biochemical markers of liver and kidney function, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA), were monitored to assess potential hepatic and renal impairment. All marker levels remained within the normal range, indicating that the NP administration did not affect liver or kidney function (Figure S7(a-d)).

Conclusion

In conclusion, we presented HA- and integrins-modified neutrophil membrane-camouflaged pABOL NPs loaded with siRNA targeting integrin α9 (i.e., MNM/siRNA NPs) to enhance endosomal escape and targeting capabilities for treating MIRI. Our comprehensive in vitro studies demonstrated that MNM/siRNA NPs effectively facilitated endosomal escape through HA protein, achieved excellent targeting capabilities via integrins, and significantly reduced integrin α9 expression. Furthermore, we demonstrated in MIRI mice that integrin α9 was a potential target against MIRI, and that MNM/siRNA NPs effectively decreased myocardial infarction area and improved cardiac function by decreasing neutrophil recruitment, the formation of NETs and microthrombus.

In this study, we aim to develop a novel engineered siRNA NP-delivery system for improving delivery efficiency by enhancing endosomal escape and targeting, attenuating MIRI through integrin α9 inhibition. This system tackles key challenges of siRNA therapy, such as low bioavailability and off-target effects, by minimizing degradation and enhancing siRNA utilization. The pABOL-based NPs improve siRNA delivery performance, while engineered membrane-camouflaged NPs effectively evade macrophage-mediated phagocytosis. Furthermore, the incorporation of HA on the membrane surface facilitates endosomal escape, and integrins enhance targeting specificity. Altogether, these features make the engineered membrane-camouflaged NPs a promising siRNA delivery platform, offering an effective treatment strategy for MIRI. Such innovative siRNA NP system has substantial potential to advance targeted therapies and could be expanded to address a wider range of diseases beyond MIRI. Future research should focus on further modifying and optimizing this system for practical applications, with the goal of enabling personalized and precision medicine in cardiovascular and related conditions.

Experimental section

Modification of MNHCs

The HA and integrin protein overexpression on MNHCs (BFB, Shanghai, China) was achieved via adenovirus transfection and drug stimulation protocols. Gene sequences for HA were provided found in Table S1. Initially, mouse HA adenovirus (GENECHEM, Shanghai, China) was introduced to MNHCs, followed by a 48-hour incubation to induce HA protein overexpression. Since adenovirus expressed its own GFP, transfection efficiency was assessed using flow cytometry analysis (CYTEK, Delaware, USA) and confirmed with CLSM imaging (Leica, Germany). Subsequently, ET-1 (Yuanye, Shanghai, China) was administered to stimulate MNHCs for integrin α9/β1 modification [16]. Protein and gene expression levels of integrin α9/β1 were validated using WB and qPCR techniques. WB analysis employed anti-integrin α9 antibody (Abcam, ab140599) and anti-integrin β1 antibody (Abcam, ab179471) to assess protein levels. All related primers were summarized in Table S2.

Isolation of engineered MNHC membrane

To create a biologically functional membrane, we utilized a hypotonic lysis technique to disrupt MNHCs. Initially, the MNHCs were gathered and suspended in a hypotonic lysate solution containing 1 mM KCl, 1.5 mM MgCl2, 1 mM phenylmethanesulfonyl fluoride (PMSF), and 10 mM Tris-HCl at pH 8.0 for 15 min at 4 °C. Subsequently, the cell suspension underwent four to five cycles of freezing and thawing, followed by centrifugation at 900 × g for 5 min at 4 °C. The resulting supernatant was collected and underwent a second centrifugation at 15,000 × g for 20 min at 4 °C to obtain the engineered MNHC membrane fragments. The membrane protein concentration was determined using the BCA Protein Assay Kit (Thermo Fisher, Waltham, USA).

Formulation of MNM/siRNA NPs

Initially, pABOLs were prepared based on previous findings [8]. N, N′-cystaminebis (acrylamide) (221.0 mg, 0.848 mmol), 4-amino-1-butanol (78 µl, 0.840 mmol), and triethylamine (12 µl, 0.084 mmol) were added to a stirring bottle with a rotor under a nitrogen atmosphere. A mixed solvent of MeOH/water (176 µl, 4/1, v/v) was also included. The reaction occurred in the dark at 45 °C for 32 h, resulting in a highly viscous solution. Once the desired molecular weight was achieved, the reaction was stopped by adding MeOH (50 ml). The resulting mixture was acidified to pH ∼4 using 1.0 M HCl and then purified by dialysis (molecular weight cutoff = 3.5 kDa) using acidic water (4.0 L, pH ∼5, refreshed 6 times over 3 days). After freeze-drying, the polymer was obtained as a white solid in the form of its HCl salt.

The siRNA NPs were formulated using the direct mixing method in both in vitro and in vivo. First, 10 ul siRNA (20 nmol ml^− 1 diluted by sterile water) silencing integrin α9 (5’-GGACAGGTCACTGTCTACA-3’) (Ribo, Guangzhou, China) was added to 190 ul of 4-(2-hydroxyerhyl)piperazine-1-erhanesulfonic acid (HEPES) buffer (20mM HEPES, 5 wt% glucose aqueous solution, pH 7.4). 20 µl pABOLs (5ug ml^− 1 diluted by sterile water) was also added to 780 ul HEPES buffer, then, the pABOL buffer solution was rapidly added to the siRNA buffer solution, followed by vortexing for 20 s to form siRNA NPs. A series of siRNA NPs solutions were prepared with pABOLs to siRNA ratio of 2:1 (w/w). For siRNA NPs in vivo, 10 nmol siRNA freeze-dried powder in each mouse was dissolved in 50ul of PBS, and 20 µl pABOLs (50ug ml^− 1 diluted by sterile water) was diluted by 100 ul of PBS, and other steps remained the same. Nextly, the extracted MNM and siRNA NPs underwent sonication for 15 min, followed by extrusion through a 400 nm pore size membrane filter. Finally, the MNM vesicles were combined with siRNA NPs in at a weight ratio of 1:1 ratio and treated with ultrasound. The hydrodynamic diameter and zeta potential of MNM/siRNA NPs were measured by DLS (Malvern, Zetasizer). Dil (Biyuntian, Changsha, China) (0.1 wt%) and Cy5 (Biyuntian, Changsha, China) (0.1 wt%) were respectively incorporated into MNM and NPs to observe the co-localization using CLSM. The morphology of MNM/siRNA NPs was further observed using TEM (FEI Tecnai F20, America). The whole proteins of MNM/siRNA NPs were detected using Coomassie blue staining (Beyotime, Haimen, China). The antibodies against integrin α9, integrin β1, LFA-1 (Abcam, ab52920), PSGL-1 (Bioss, bs-10253R), HA (Cell Signaling Technology, 3724 S), CXCR2 (Proteintech, 20634-1-AP) and CCR2 (Proteintech, 16153-1-AP) were performed for WB to confirm the key proteins on MNM.

Cytotoxicity evaluation

MNHCs, HUVECs (Science Cell, Carlsbad, CA), and SMCs (Beinart Biotech, Beijing, China) were seeded in a 96-well plate at a density of 10,000 cells per well and incubated for 12 h. Subsequently, the cells were treated with varying concentrations of siRNA NPs and MNM/siRNA NPs for 24 h. Cell viability post-treatment was evaluated using a CCK-8 assay (Biyuntian, Changsha, China).

In vitro assessment of immune escape and targeting ability

RAW 264.7 cells (ZQXZ Biotechnology, Shanghai, China) were used to verify the immune escape of MNM/siRNA NPs in vitro. RAW 264.7 cells were plated in a 6-well format at a density of 10,000 cells per well overnight. The free siRNA, siRNA NPs, NM/siRNA NPs and MNM/siRNA NPs were incubated with RAW 264.7 cells for 4 h where Cy5 (Ribo, Guangzhou, China) labeled siRNA. Subsequently, the nuclei of the cells were stained using 4′,6-diamidino-2-phenylindole (DAPI, Solarbio, Beijing, China) and examined with CLSM and flow cytometry analysis.

To further verify the endosomal escape of MNM/siRNA NPs in vitro, MNHCs were cultured in a Dulbecco"s Modified Eagle Medium (DMEM) with 100 ng ml^− 1 of LPS, and then NM/siRNA NPs or MNM/siRNA NPs were added to each well and incubated for 1 h, 6 h, 12 h and 24 h where Cy5 labeled siRNA. After a PBS wash, the nuclei were stained by DAPI and the lysosomes were stained by LysoTracker™ Deep Red (Thermo Fisher, Waltham, the USA). The endosomal escape was determined by CLSM.

To assess the targeting ability of MNM/siRNA NPs in vitro, Cy5-labeled siRNA, siRNA NPs, NM/siRNA NPs and MNM/siRNA NPs were added to each well respectively where the MNHCs were cultured in a DMEM with or without 100 ng ml^− 1 of LPS for 4 h, flow cytometry analysis and CLSM observed the efficiency of MNHC uptake for NPs.

Detection of cytokines secreted by neutrophils

MNHCs were plated in a 6-well plate at a density of 10,000 cells each well and incubated overnight. Subsequently, free siRNA, siRNA NPs, NM/siRNA NPs, and MNM/siRNA NPs were added to each well, where the MNHCs were cultured in DMEM supplemented with 100 ng ml^− 1 of LPS for 24 h. After centrifugation of the culture media, the supernatant was collected, and the expression levels of NET markers (MPO-DNA (Hengyuan, Shanghai, China), NE-DNA (Meimian, Yancheng, China, and cathepsin G (Meilian, Shanghai, China)) and inflammatory markers (TNF-α (ELK, Wuhan, China), IL-1β (ELK, Wuhan, China), and IL-6 (ELK, Wuhan, China)) were determined by ELISA.

Establishment of a mouse I/R model

This study received approval from the Ethics Committee of Anhui Medical University (Anhui, China) under reference number LLSC20230846, and it was conducted in accordance with the guidelines established by the European Parliament Directive 2010/63/EU. The animals were provided with unrestricted access to water and food. Male C57 BL/6 mice (8-week-old, Jicui Pharmachem Biotechnology Co.) were used to establish the MIRI model. Chloral hydrate (3.5% concentration) was intraperitoneally injected to induce anesthesia before open-heart surgery was performed. The left anterior descending branch (LAD) was temporarily occluded using 7 − 0 silk sutures for 60 min. Subsequently, MIRI mice were injected with 5 mg kg^− 1 of PBS, free siRNA, siRNA NPs, NM/siRNA NPs, and MNM/siRNA NPs via the tail vein after 30 min. Hearts and blood samples were collected from the mice for further analysis.

Immunofluorescence analysis

Frozen heart tissue sections were subjected to three rounds of 10-minute PBS washes, followed by blocking with goat serum for 1 h at room temperature. The sections were then incubated overnight at 4 °C with the following antibodies respectively: anti-histone H3 (citrulline R2 + R8 + R17) antibody (Abcam, ab5103), anti- MPO antibody (Abcam, ab208670), anti-CD31 antibody (BD bioscience, 550274), anti-thrombocyte antibody (LSbio, LS-C348178) and anti-albumin antibody (Proteintech,16475-1-AP). Subsequently, fluorescent secondary antibodies were applied and incubated for 2 h at room temperature. The slides were then counterstained with DAPI for 5 min and coverslipped before data acquisition using CLSM. Image J software was used for data analysis.

Dextran prefusion staining

To visualize the vascular perfusion of the injured myocardium, mice were injected via the tail vein with approximately 5 mg of dextran-FITC in PBS (Sigma Aldrich, FD2000S). After 5 min, the mice were euthanized, and their hearts were removed, quick-frozen, and sectioned as previously described. Vascular structures were labeled using an anti-CD31 antibody, with nuclei counterstained using DAPI. CLSM was utilized to examine the vascular perfusion of the injured myocardium, and data analysis was performed using Image J software.

Histological analysis

Evan’s blue staining and triphenyltetrazolium chloride (TTC) staining were performed to assess IS at 24 h post-MIRI. Simply put, mice were anesthetized with chloral hydrate, and their chests were reopened to expose the heart. 1% Evan’s blue dye solution injection revealed rapid staining outside the supply area of LAD. After freezing and slicing the heart, 1.5% TTC staining identified ischemic and necrotic myocardium. Non-ischemic area was stained blue by both Evan’s blue dye and TTC. Myocardial ischemic area appeared red as it was stained by TTC but not by Evan’s blue dye. Myocardial infarction area appeared white because it was not stained by either Evan’s blue dye or TTC. Additionally, the AAR included both the red ischemic and the white infarction areas. Image quantification was carried out by segmenting the stained regions of each tissue section using ImageJ software. The size of infarction was then expressed as a percentage of the AAR (AAR area/total area of myocardium) and IS (IS area/AAR).

Masson’s trichrome staining was utilized to assess myocardial fibrosis in heart tissues 14 days after MIRI. The tissues were fixed in a 4% paraformaldehyde solution for 48 h and embedded in paraffin wax for further processing. The fibrotic areas, stained blue, indicated perivascular and interstitial collagen accumulation. Quantitative analysis was performed using ImageJ software, with the extent of fibrosis expressed as a percentage of the myocardial fibrosis (myocardial fibrosis area/total area of myocardium).

Statistical analysis

The data were presented as mean ± standard deviation. GraphPad Prism software (version 8.02) was utilized for data analysis. Students’ t test was applied for comparing 2 experimental groups. One-way analysis of variance (ANOVA) was performed for multiple experimental groups followed by Dunnett’s multiple comparisons test. Statistical significance was defined as P < 0.05.

Data availability

The data supporting the results of this research can be obtained from the corresponding author upon a reasonable request.

Abbreviations

siRNA:: Small interfering RNA
MIRI:: Myocardial ischemia-reperfusion injury
HA:: Hemagglutinin
NETs:: Neutrophil extracellular traps
AMI:: Acute myocardial infarction
PCI:: Percutaneous coronary intervention
NP:: Nanoparticle
pABOL:: Poly (CBA-co-4-amino-1-butanol)
VCAM-1:: Vascular cell adhesion protein 1
ECs:: Endothelial cells
ET-1:: Endothelin-1
siRNA NPs:: siRNA-loading pABOL NPs
NM/siRNA NPs:: Unmodified neutrophil membrane-camouflaged siRNA NPs
MNM/siRNA NPs:: Modified neutrophil membrane-camouflaged siRNA NPs
MNHC:: Mouse neutrophil cell
WB:: Western blot
qPCR:: Quantitative polymerase chain reaction
DLS:: Dynamic light scattering
PBS:: Phosphate-buffered saline
FBS:: Fetal bovine serum
CLSM:: Confocal laser scanning microscopy
TEM:: Transmission electron microscopy
LFA-1:: Lymphocyte function-associated antigen-1
PSGL-1:: P-selectin glycoprotein ligand-1
CCK-8:: Cell Counting Kit-8
HUVECs:: Human umbilical vein endothelial cells
SMCs:: Smooth muscle cells
LPS:: Lipopolysaccharide
ELISA:: ENZYME-Linked Immunosorbent Assay
MPO:: Myeloperoxidase
CitH3:: Citrullinated histone H3
PAD4:: Peptidylarginine deiminase 4
TNF-α:: Tumor necrosis factorα
IL:: Interleukin
TTC:: Triphenyltetrazolium chloride
ANOVA:: One-way analysis of variance

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Acknowledgements

YJ, RJ, and ZX contributed equally to this work. This work was supported by the Natural Science Foundation of China (grant numbers 92068116, 82370305, and 82470282).

Funding

This study was supported by the Natural Science Foundation of China (grant numbers 92068116, 82370305, and 82470282).

Author information

Yaohui Jiang, Rongyan Jiang, and Zequn Xia are equally to this work.

Authors and Affiliations

Department of Cardiology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China
Yaohui Jiang, Zequn Xia, Meng Guo & Jun Xie
Department of Rheumatology and Immunology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
Xiaocheng Wang
Department of Cardiology, National Cardiovascular Disease Regional Center for Anhui, The First Affiliated Hospital of Anhui Medical University, Hefei, China
Yanan Fu & Jun Xie
Department of Cardiology, Affiliated Bozhou Hospital of Anhui Medical University, Bozhou, Anhui, China
Rongyan Jiang

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Yaohui Jiang
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Meng Guo
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Yanan Fu
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Xiaocheng Wang
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Contributions

YJ, RJ, and ZX contributed equally to this work, and were the co-first authors. YJ, RJ, ZX, MG, YF, XW, and JX designed and completed experiments. YJ, RJ, ZX and MG collected and analyzed the data. YJ, RJ, ZX and MG wrote the manuscript.

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Correspondence to Yanan Fu, Xiaocheng Wang or Jun Xie.

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All animal experiments in this study were approved by the Ethics Committee of Anhui Medical University (Anhui, China) under reference number LLSC20230846.

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The authors have no conflicts of interest to disclose. Fig. 6(a), 7(a), 10(a), and 11(a) were created utilizing Figdraw (https://www.figdraw.com).

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Jiang, Y., Jiang, R., Xia, Z. et al. Engineered neutrophil membrane-camouflaged nanocomplexes for targeted siRNA delivery against myocardial ischemia reperfusion injury. J Nanobiotechnol 23, 134 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12951-025-03172-w

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Received: 05 November 2024
Accepted: 27 January 2025
Published: 22 February 2025
DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12951-025-03172-w

Engineered neutrophil membrane-camouflaged nanocomplexes for targeted siRNA delivery against myocardial ischemia reperfusion injury

Abstract

Introduction

Results and discussion

Preparation and characterization of MNM/siRNA NPs

Immune escape and targeting ability of MNM/siRNA NPs in vitro

MNM/siRNA NPs inhibited the formation of NETs and release of inflammatory factors in vitro

Targeting ability of MNM/siRNA NPs in vivo

Reduced NET formation and inflammation by MNM/siRNA NPs in MIRI mice

MNM/siRNA NPs suppressed the microthrombus formation and endothelial damage in vivo

MNM/siRNA NPs decreased myocardial infarction area and improved cardiac function in vivo

Biological safety evaluation

Conclusion

Experimental section

Modification of MNHCs

Isolation of engineered MNHC membrane

Formulation of MNM/siRNA NPs

Cytotoxicity evaluation

In vitro assessment of immune escape and targeting ability

Detection of cytokines secreted by neutrophils

Establishment of a mouse I/R model

Immunofluorescence analysis

Dextran prefusion staining

Histological analysis

Statistical analysis

Data availability

Abbreviations

References

Acknowledgements

Funding

Author information

Authors and Affiliations

Contributions

Corresponding authors

Ethics declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Additional information

Publisher’s note

Electronic supplementary material

Supplementary Material 1

Rights and permissions

About this article

Cite this article

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Keywords

Journal of Nanobiotechnology

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