
==== Front
Asian J Pharm Sci
Asian J Pharm Sci
Asian Journal of Pharmaceutical Sciences
1818-0876
2221-285X
Shenyang Pharmaceutical University

S1818-0876(24)00046-1
10.1016/j.ajps.2024.100929
100929
VSI: Emerging Nanocarriers
Pulmonary fibroblast-specific delivery of siRNA exploiting exosomes-based nanoscaffolds for IPF treatment
Lu Haoyu a
Liu Xulu a
Zhang Mengjun a
Bera Hriday ab
Xu Wenwen a
Jiang Huiyang a
Zhao Xing a
Wu Lan debbiewu54@163.com
a⁎
Cun Dongmei cundongmei@163.com
a⁎
Yang Mingshi mingshi.yang@sund.ku.dk
ac⁎
a Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang 110016, China
b Dr. B.C. Roy College of Pharmacy & Allied Health Sciences, West Bengal 713212, India
c Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen DK-2100, Denmark
⁎ Corresponding authors. debbiewu54@163.comcundongmei@163.commingshi.yang@sund.ku.dk
11 6 2024
8 2024
11 6 2024
19 4 1009293 1 2024
1 5 2024
20 5 2024
© 2024 Published by Elsevier B.V. on behalf of Shenyang Pharmaceutical University.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Idiopathic pulmonary fibrosis (IPF) is a progressive pulmonary disease that leads to interstitial inflammation, lung damage, and eventually life-threatening complications. Among various pathologic factors, Smad4 is a pivotal molecule involved in the progression and exacerbation of IPF. It mediates nuclear transfer of Smad2/Smad3 complexes and initiates the transcription of fibrosis-promoting genes. Thus, the inhibition of Smad4 expression in pulmonary fibroblasts by small interfering RNAs (siRNAs) might be a promising therapeutic strategy for IPF. Herein, we engineered exosome membranes (EM) by cationic lipid (i.e., DOTAP) to load siRNAs against Smad4 (DOTAP/siSmad4@EM), and investigated their specific delivery to pulmonary fibroblasts for treating IPF in a mouse model via pulmonary administration. As reference nanoscaffolds, undecorated DOTAP/siSmad4 complexes (lipoplexes, consisting of cationic lipid DOTAP and siRNAs) and siSmad4-loaded lipid nanoparticles (DOTAP/siSmad4@lipo, consisting of lipoplexes fused with DPPC—Chol liposomes) were also prepared. The results showed that DOTAP/siSmad4@EM exhibited a higher cellular uptake and gene silencing efficacies in mouse pulmonary fibroblasts (viz., MLg2908) as compared to the two reference nanoscaffolds. Furthermore, the outcomes of the in vivo experiments illustrated that DOTAP/siSmad4@EM could significantly down-regulate the Smad4 expression with augmented anti-fibrosis efficiency. Additionally, the DOTAP/siSmad4@EM conferred excellent biocompatibility with low cytokine levels in bronchoalveolar lavage fluid and proinflammatory responses in the pulmonary area. Taken together, the outcomes of our investigation imply that specific inhibition of Smad4 expression in pulmonary fibroblasts by pulmonary administrated DOTAP/siSmad4@EM is a promising therapeutic strategy for IPF, which could safely and effectively deliver siRNA drugs to the targeted site of action.

Graphical abstract

Image, graphical abstract

Keywords

Idiopathic pulmonary fibrosis
siRNA delivery
Exosomes
Pulmonary administration
Pulmonary fibroblasts
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pmc1 Introduction

Idiopathic pulmonary fibrosis (IPF) is a debilitating and fatal lung disorder characterized by inflammation, progressive scarring, architectural disruption and irreversible loss of function [1]. Repeated injuries to the alveolar epithelium with abnormal tissue repair result in overaccumulation of extracellular matrix (ECM), ultimately leading to respiratory failure and death [2]. This disease influences over 3 million people worldwide, with a median survival of 2 – 3 years after diagnosis [3]. High-resolution CT patterns portray bilateral reticulation and honeycombing in the lungs of IPF patients, predominantly in the peripheral and lower lobes [4]. The pulmonary function of IPF patients exhibits ventilatory insufficiency, attributed to low respiratory and diffusing capacities [1]. Furthermore, the bronchoalveolar lavage fluid of such patients may present plenty of inflammatory cells, including lymphocytes, macrophages and others [5]. To date, the symptomatic treatment of IPF is the only available medical option with marginal therapeutic success [5,6]. Therefore, creating effective and safe anti-fibrotic drugs can address the currently unmet clinical need of treating and even curing IPF based on its underlying etiology.

IPF is an epithelial-driven disease in which the abnormally activated epithelial cells plenteously secrete cytokines and chemokines which further result in the proliferation, migration and differentiation of pulmonary fibroblasts [7]. Subsequently, the activated fibroblasts over-express the collagen, producing denser ECM and eventually leading to the remodeling of the alveolar structure [8]. Therefore, epithelial cell injury and fibroblast proliferation are the principal hallmarks of IPF pathology. Among various mediators of IPF, TGF-β1 is considered the most effective profibrotic cytokine [9]. However, directly targeting TGF-β1 may not be clinically ideal owing to its extensive sources and involvement in numerous physiological processes [10]. Alternatively, an array of molecular downstream regulators of the TGF-β1 signaling pathway can be exploited to more specifically retard the IPF progression [9]. Among such signaling mediators, Smad4 has been recognized as a key molecular target. Pulmonary fibroblast-expressed Smad4 regulates the nuclear translocation of Smad2/Smad3 complexes, which in turn initiate the transcription of profibrotic genes, such as α-smooth muscle actin (α-SMA), tissue inhibitor of matrix metalloproteinases (TIMP) and collagen [11,12]. These eventually induce the differentiation of fibroblasts into myofibroblasts, which are mainly responsible for excessive ECM deposition [13,14]. Consequently, the RNA interference to inhibit the Smad4 gene expression in pulmonary fibroblasts might reveal promising therapeutic outcomes for IPF [15].

Small interfering RNAs (siRNAs) are short duplex RNAs composed of around 20 nucleotides. Upon cellular entry, siRNAs split into the passenger strands and the guide strands. The guide strands are subsequently integrated into the RNA-induced silencing complexes (RISC), whereas the passenger strands are degraded. In RISC, the guide strands bind to sequence-complementary mRNAs, which trigger the cleavage causing post-transcriptional gene silencing [16,17]. Because of their unique features, siRNAs have been widely exploited for disease-relevant gene silencing, illustrating great potential in treating various life-threatening illnesses precisely [18,19]. However, free siRNAs are vulnerable to degradation by nuclease and their penetration into cell membranes is limited pertaining to high molecular weight, hydrophilicity and electronegativity [20]. Thus, various lipid- and polymer-based vectors have long been employed to achieve efficient siRNA delivery into the cells [20,21]. Regrettably, such approaches often provide elusive success accredited to their potential immunogenicity and non-specific targetability [22]. To confront this hurdle, bio-mimetic delivery vectors, such as extracellular vesicles (EVs), have recently gained immense research interest and have demonstrated siRNA delivery efficiency [23,24] with clinical potential [25,26].

EVs are cell-derived membranous nanoparticles secreted by a variety of cells. These vesicles mediate intercellular communications by exchanging their inner biomolecules like nucleic acids, proteins, lipids and small molecules [27]. Nowadays, EVs are modified with other cationic scaffolds to incorporate distinct siRNA delivery characteristics. For instance, Zhao et al. afforded cationic bovine serum albumin (CBSA) through acetylation reaction to electrostatically bind siRNAs. This conjugate was then coated with autogenous breast cancer cell-derived EVs to accomplish a biomimetic nano-delivery system (CBSA/siS100A4@Exosome). This system could effectively elicit gene silencing efficiency endowed with impressive anti-tumor potentials in vitro and in vivo [28]. However, there are several researches validated that the component loaded inside the fibroblasts derived EVs might result in undesired side effects such as fibroblast invasion and epithelial cell senescence in lung tissues [29,30], which hinder their application in drug delivery. In that case, we hypothesize that EVs membrane (EM) could preserve EVs original “home target” abilities by inheriting the membrane proteins while avoiding EVs adverse effects to some extent

Although most of the preclinical and clinical research examined the performances of EVs-based siRNA delivery systems through intravenous route [31], several current reports have highlighted comparatively enhanced therapeutic efficacies of bioactive molecules in the lungs via pulmonary delivery. Additionally, the pulmonary route ensures direct contact of the therapeutic molecules with the targeted lung cells, bypassing intricate barrier effects in the systemic circulation [32] and offering the advantage of dose minimization. Zhang and co-researchers engineered inhalable lipid nanoparticles that could selectively and efficiently deliver siRNAs to airway epithelial cells and exhibit outstanding potential in alleviating allergic asthma [33]. Zhu et al. studied inhalation of allogeneic human mesenchymal stem cell-derived EVs in patients with COVID-19. The outcomes of this preliminary study underlined EVs-mediated significant alleviation in lung damage without evidence of any adverse event during and after the treatment [34]. Precisely, pulmonary delivery of siRNAs and EVs is a current research spotlight to achieve optimal treatment options against various diseases [35,36].

In the present research endeavor, we isolated EVs from pulmonary fibroblasts (MLg2908) and collected their membranes (EM). The EM was further engineered using a cationic lipid 2,3-dioleoyloxy-propyl-trimethylammonium-chloride (DOTAP) to deliver Smad4-targeted siRNAs (siSmad4) via the pulmonary route in IPF mouse model (Scheme 1). It was hypothesized that the cationic lipid would improve the siRNA-loading efficiency, while the modification of lipoplexes with EM could circumvent the cytotoxicity of DOTAP and promote the cell internalization, fibroblasts-specific delivery, and ultimately, the gene silencing efficiency of siSmad4. The physicochemical properties of the constructed nanoscaffolds were characterized employing various techniques, and their cellular uptake behavior and the siRNA transfection efficiency were determined at cellular level. Furthermore, the biodistribution and anti-fibrotic potentials of the nanoscaffolds were assessed in bleomycin (BLM)-challenged IPF mice. Lastly, their safety profiles were systematically evaluated in healthy mice.Scheme 1 Schematic illustration of pulmonary fibroblasts specific delivery of DOTAP/siRNA@EM alleviates IPF via pulmonary administration.

Scheme 1

2 Materials and methods

2.1 Materials

Duplex siSmad4, negative-control siRNA (siNC), FAM-siRNA and Cy5-siRNA were purchased from GenePharma, (Suzhou, China). The sequences of the sense and anti-sense strands of siSmad4 were 5′-GAUGAAUUGGAUUCUUUAATT-3′ and 5′-UUAAAGAAUCCAAUUCAUCTT-3′, respectively. DOTAP, 1,2-dipalmitoyl-sn‑glycero-3-phosphocholine (DPPC) and cholesterol were received from AVT, (Shanghai, China). Lipofectamine 3000, HRP-conjugated secondary antibodies (goat anti-mouse IgG), agarose and TRIzol were procured from Thermo Fisher Scientific, (MA, USA). The primary antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calnexin (CANX), Smad4, α-SMA and HRP-conjugated secondary antibodies (goat anti-rabbit IgG) were obtained from ABclonal Technology, (Wuhan, China). The primary antibodies against CD63 and CD47 were purchased from Affinity Biosciences, (Liyang, China). 3-(4,5-dimethylthiazol-2)−2,5-diphenyl-tetrazolium bromide (MTT), filipin, chlorpromazine, EIPA, 4′,6-diamidino-2-phenylindole (DAPI), 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), 1,1-dioctadecyl-3,3,3,3- tetramethylindocarbocyanine iodide (DiI) and 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) were purchased from Dalian Meilun Biotech, (Dalian, China). Phenylmethanesulfonyl fluoride (PMSF), Coomassie blue fast staining solution, GelRed nucleic acid gel stain and Lyso-Tracker Red were received from Beyotime Biotechnology, (Shanghai, China). Bleomycin (BLM) and Diff-Quik stain were obtained from Solarbio Science & Technology, (Beijing, China). TGF-β1 cytokine was procured from PeproTech Inc, (NJ, USA). Mouse IL-6 ELISA kit and mouse TNF-α ELISA kit were purchased from Neobioscience, (Shenzhen, China) and Jijia Biotechnology, (Shenyang, China), respectively. All the other reagents utilized in this study were of analytical grade.

2.2 Cell lines and animals

Mouse lung fibroblast cells (MLg2908) and macrophages (RAW264.7) were received from Guangzhou Jennio Biotech, China and Dalian Meilun Biotech, China, respectively. Animal studies were performed on male C57BL/6 mice (5 - 6 weeks old) in accordance with the experimental protocols approved by the animal ethical committee, Shenyang Pharmaceutical University, China (No. SYPU-IACUC—C2021–12–03–107).

2.3 Cell culture and isolation of EVs and EM

MLg2908 cells were cultured in DMEM appended with 10 % FBS and 1 % penicillin-streptomycin at 37 °C in a 5 % CO2 incubator. To isolate EVs, the cells were grown in FBS-free DMEM for 24 h and the supernatant of the media was collected following the ultracentrifugation method as reported previously [37]. Briefly, the supernatant was centrifuged at 300 × g for 10 min, 2,000 × g for 10 min and 10,000 × g for 30 min at 4 °C to discard dead cells, broken organelles and apoptotic bodies, respectively. Subsequently, the supernatant was ultracentrifuged at 100,000 × g for 1.5 h at 4 °C. The pellets gathered in the bottom were then washed with PBS and ultracentrifuged again at 100,000 × g for 0.5 h at 4 °C. After that, EVs were resuspended in sterile PBS and the EM was extracted using hypoosmotic protocol [37]. Precisely, EVs were suspended in DEPC water containing 1 % PMSF for 12 h to remove their contents, followed by ultracentrifugation at 120,000 × g for 2 h at 4 °C. The aggregated EM was resuspended in sterile DEPC. Both EVs and EM were deposited at −80 °C.

The protein contents of EVs and EM were determined by BCA assay [38]. EVs marker and negative marker (viz., CD63 and calnexin respectively) were also detected via western blotting [39]. Besides, GAPDH was probed as a control to examine the content contamination of EVs [40]. Furthermore, dot blot assay was carried out to investigate the preservation of proteins on EVs membrane. EVs, EM and DOTAP/siRNA@EM were blotted onto NC membranes followed by air-drying for complete adsorption. After blocking with 5 % skim milk, the membranes were incubated with extracellular CD47 antibody overnight. Subsequently, the membranes were incubated with HRP-conjugated secondary antibody and then imaged using Gel Imager System (Bio-Rad, USA). Moreover, particle diameter, PDI values and zeta potentials of EVs and EM were measured using dynamic light scattering (DLS, Malvern, UK). Transmission electron microscope (TEM, JEM-2100, JEOL Ltd., Japan) analysis was performed to observe their morphological appearances.

2.4 Optimization of nanoscaffolds

To optimize DOTAP/siRNA@EM, lipoplexes and EM were labeled with DiO and DiI separately and DOTAP/siRNA@EM were prepared with different weight ratios of DOTAP and EM. Afterward, the dual-dye labeled DOTAP/siRNA@EM were examined for their fluorescence resonance energy transfer (FRET) abilities under a microplate reader (ThermoFisher, USA) [41]. The fluorescence emission spectra of DOTAP/siRNA@EM within 480 - 630 nm were recorded with the excitation wavelength of 460 nm. Moreover, the binding capabilities of DOTAP and siRNAs at different N/P ratios were detected through electrophoresis on agarose gels (2 %) [42].

2.5 Formulation of nanoscaffolds loading siRNA

The siRNA-DOTAP complexes (lipoplexes) were initially constructed according to the method reported previously [43]. Briefly, the DOTAP film evaporated at 37 °C was hydrated in 10 mM HEPES buffer for 30 min and then ultrasonicated to reduce the diameter of DOTAP dispersions. Thereafter, equal volumes of DOTAP dispersions and siRNA solution were mixed at different N/P ratios (10, 5, 2 and 1) and vortexed for 10 min to prepare lipoplexes. To accomplish DOTAP/siRNA@EM, lipoplexes and EM were mixed at different weight ratios, and the mixtures were then ultrasonicated (20 % amplitude, 3 s pulse on/off, for 2 min) under ice bath to ensure the complete fusion of two components. In addition, EM was replaced with DPPC—Chol (weight ratio 2:1) liposomes to form lipid nanoparticles (DOTAP/siRNA@lipo), which were used as the reference formulation.

2.6 Characterization of nanoscaffolds

The diameter, PDI values, and zeta potentials of lipoplexes, DOTAP/siRNA@lipo and DOTAP/siRNA@EM were evaluated by DLS. Their morphologies were examined under TEM. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was further performed to characterize the protein profiles of MLg2908, EVs, EM, lipoplexes, DOTAP/siRNA@lipo and DOTAP/siRNA@EM [44]. In brief, different samples were lysed by RIPA and quantified by BCA assay protocol. Equal amount of the total proteins (15 µg) was loaded onto the 10 % SDS-PAGE gels and electrophoresed. Subsequently, the gels were stained with Coomassie brilliant blue and washed overnight for imaging. Moreover, to confirm the successful encapsulation of siRNAs, DOTAP/siRNA@EM composed of DOTAP, FAM-siRNA and DiI labeled EM were scanned under confocal laser scanning microscopy (CLSM, TCS SP2/AOBS, Nikon) [45].

2.7 Storage stability and siRNA stability

The lipoplexes, DOTAP/siRNA@lipo and DOTAP/siRNA@EM were kept in PBS (pH 7.4) at 4 °C, and their particle sizes were detected by DLS for three weeks to investigate their storage stability. Furthermore, to evaluate the siRNA protection abilities of different nanoscaffolds, the free siRNAs (i.e., control), lipoplexes, DOTAP/siRNA@lipo and DOTAP/siRNA@EM were incubated in PBS (pH 7.4) containing 50 % FBS at 37 °C. At various planned times (0, 1, 2, 4, 8, 12 and 24 h), various samples were lysed by Triton X-100 to dissociate siRNAs and then were analyzed by electrophoresis on agarose gels (2 %).

2.8 Cytotoxicity

The in vitro cytotoxicity of different nanoscaffolds on MLg2908 cells was determined by MTT assay [46]. Briefly, MLg2908 cells were seeded into 96-well plates at a density of 2,000 cells/well and incubated overnight. MLg2908 were then treated with lipoplexes, DOTAP/siRNA@lipo and DOTAP/siRNA@EM with various concentrations of siRNAs (10, 20, 50, 100 and 200 nM), respectively, and incubated for another 24 or 48 h. Subsequently, the culture media was discarded and MTT solution (20 µl) was added into each well and further incubated for 4 h. Finally, the MTT was dumped and DMSO (150 µl) was added to solubilize formazan crystals. The absorbance of plates was subsequently recorded on a microplate reader (FLUOstar Omega, Germany) at 570 nm.

2.9 Cellular uptake

The cellular uptake efficacy of FAM-siRNA loaded nanoscaffolds was detected using flow cytometry [47]. Precisely, the cells (MLg2908 and RAW264.7) were seeded in 12-well plates at a density of 2 × 105 cells/well and incubated overnight. The culture media were then replaced by FBS-free media containing FAM-siRNA-loaded nanoscaffolds (siRNA concentration, 100 nM) and incubated for another 6 h. Thereafter, the cells were washed, collected and resuspended in 0.5 ml PBS. Subsequently, the intracellular fluorescence signals were measured using a flow cytometer (FACSAria™ Ⅲ, BD, USA).

CLSM observation was also applied to evaluate the uptake efficiency of different nanoscaffolds on MLg2908 cells. The cells were seeded on coverslips in 12-well plates at a density of 2 × 105 cells/well and incubated overnight. The cells were then treated with FAM-siRNA-loaded nanoscaffolds as mentioned above. After treatment, the cells were washed with PBS for three times and fixed with 4 % paraformaldehyde solution. The cell nuclei were then counterstained with DAPI. Subsequently, the prepared samples were imaged under CLSM (Olympus FV1000-IX81, Japan).

2.10 Endocytosis pathways

To investigate the cellular endocytosis pathways of various nanoscaffolds, the MLg2908 cells were treated with different inhibitors, including chlorpromazine (CPZ, inhibitor of clathrin-dependent endocytosis, 10 µg/ml), filipin (inhibitor of caveolae-dependent endocytosis, 5 µg/ml) [48] and EIPA (inhibitor of macropinocytosis-dependent endocytosis, 7.5 µg/ml) [49] for 1 h. The FAM-siRNA loaded nanoscaffolds (siRNA concentration, 100 nM) were then introduced and incubated with the cells for another 6 h. Thereafter, the cells were washed, collected, resuspended in 0.5 ml PBS, and the intracellular fluorescence signals were measured using flow cytometry as described in Section 2.9.

2.11 Endosomal escape

The intracellular distribution of FAM-siRNA-loaded nanoscaffolds was observed by CLSM imaging [50,51]. The MLg2908 cells were seeded on coverslips, incubated and treated with FAM-siRNA-loaded nanoscaffolds (siRNA concentration, 100 nM) for 6 h as described in Section 2.9. Afterwards, the cells were washed thrice with PBS and stained with Lyso-Tracker Red (1:3,000 dilution) for 1 h. After rinsing with PBS for three times, the cells were fixed with 4 % paraformaldehyde solution, dyed with DAPI and imaged under CLSM (Olympus FV1000-IX81, Japan). Mander's coefficient was calculated by Image J 2022.

2.12 In vitro gene silencing effects

To evaluate the relative expression levels of Smad4 mRNA, the MLg2908 cells pre-treated with various nanoscaffolds were analyzed by reverse transcriptase-PCR (RT-qPCR) protocol [52]. Briefly, the cells were seeded in 12-well plates at a density of 2 × 105 cells/well and placed at 37 °C in a 5 % CO2 incubator. After growing for overnight, the media were replaced with new FBS-free media containing one of the following agents: (1) free siSmad4; (2) nanoacaffolds loaded with siSmad4 or siNC (siRNA concentration, 100 nM); (3) PBS; (4) the mixture of siSmad4 and commercial transfection reagent Lipofectamine 3000 (Lipo3000). PBS and Lipo3000 were used as the blank and positive control, respectively. After incubating for 6 h, the cells were washed thrice with PBS to get rid of any uninternalized siRNAs and nanoscaffolds. Cells were cultured in fresh media for another 24 or 48 h. Total RNAs were extracted from MLg2908 using TRIzol as per the manufacturer's instructions. Reverse transcription was then implemented using the PrimeScript RT Reagent Kit (Takara, Japan). The real-time PCR amplification and valuation were carried out with TB Green Premix Ex Taq II (Takara, Japan) using PCR System (QuantStudio1, Thermo Fisher, USA). The target gene (Smad4) expression was normalized with internal control β-actin.

The gene silencing effects at the protein level were further tested by western blotting assay [53]. The cell culture and treatment were performed as described above. After 48-h incubation, MLg2908 were washed with PBS, collected by centrifugation (300 × g for 5 min), followed by re-suspended in 150 µl of 1 % PMSF-containing RIPA lysis buffer for 1 h at 4 °C. The cellular protein was then purified by centrifugation at 15,000 × g for 15 min at 4 °C and quantified by a BCA kit. After boiling them with protein loading buffer, various samples were loaded on 10 % SDS-PAGE gel, electrophoresed and transferred to PVDF membranes. Subsequently, the membranes were blocked with 5 % skimmed milk and incubated with the primary antibodies (1:2,000 dilution) overnight at 4 °C. After washing with TBST, the membranes were treated with HRP-conjugated secondary antibodies (1:5,000 dilution) at room temperature for 2 h and washed further with TBST. Finally, the membranes were imaged under Gel Imager System (Bio-Rad, USA). GAPDH was used as an internal standard.

2.13 Anti-migration assay

The migration ability of MLg2908 cells was estimated by wound healing assay on an in vitro IPF model [54]. In brief, the cells were seeded in the 12-well plates at a density of 2 × 105 cells/well and cultured up to 90 % confluency at 37 °C in a 5 % CO2 incubator. The cell monolayer was then scratched using a 10 µl pipette tip and gently washed with PBS to get rid of the floating cells and cell debris. Thereafter, the fresh media containing TGF-β1 cytokine (5 ng/ml) along with different nanoscaffolds was added. Images of cell migration were captured at designed timepoints (0, 24 and 48 h) using an inverted phase-contrast microscope (Olympus, Japan).

2.14 C57BL/6 pulmonary fibrosis model

The pulmonary fibrosis C57BL/6 mice model was established via a single intratracheal administration of 25 µl BLM solution (3 U/kg) to the anesthetized mice using an intratracheal microsprayer (HRH-MAG4, Yuyan instruments Co. Ltd., China) [55]. The mouse lungs were then harvested on the designed days (1, 3, 7, 14 and 21 d), excised and washed with saline. The interstitial pneumonia was examined via hematoxylin and eosin staining (H&E) staining while the collagen deposition was estimated through Masson staining of the lung tissues.

2.15 Biodistribution and pulmonary retention

On the third-day post-BLM challenge, the Cy5-siRNA loaded nanoscaffolds (25 µl, 4 µg siRNA/mouse) were administered intratracheally to the anesthetized mice. At pre-designed time points (1, 6, 12 and 24 h), the major organs were collected and the fluorescence images of organs were captured by IVIS Spectrum imaging system (PerkinElmer, USA) [56]. Subsequently, the mouse lungs at the last time point (24 h) were collected, immersed in 4 % paraformaldehyde solution and then sliced into 5 µm thick slices. Thereafter, the slices were incubated with anti-F4/80 antibody to spot macrophages and anti-α-SMA antibody to locate fibroblasts. After staining with fluorescence-labeled secondary antibody, the slices were imaged by CLSM.

2.16 In vivo anti-fibrotic efficiency

On the third-day post-BLM challenge, the mice were randomly separated into six groups (n = 3) and administrated one of the following agents: (1) PBS; (2) free siSmad4; (3) lipoplexes; (4) DOTAP/siSmad4@lipo; (5) DOTAP/siNC@EM or (6) DOTAP/siSmad4@EM intratracheally at every alternative day for three times. The amount of siRNA in different nanoscaffolds was 4 µg siRNA/mouse. After 48 h of the last administration, mice were sacrificed and their lungs were collected. The left lobes of the lungs were immersed in 4 % paraformaldehyde solution and assessed following H&E and Masson staining.

2.17 In vivo gene silencing effects

The right lobes of mouse lungs were homogenized to analyze Smad4 mRNA by RT-qPCR. Briefly, the lungs (around 100 mg) were sectioned and quickly ground into powders in a clean mortar precooled by liquid nitrogen. Subsequently, TRIzol (1 ml) was added to the mortar and the tissues were continuously ground until they became puree. Thereafter, the RNA extraction, reverse transcription and RT-qPCR were performed successively according to the steps described in Section 2.12. The target gene expression was normalized with GAPDH expression.

To evaluate the gene silencing effect at protein level, western blotting was then implemented. About 100 mg of the right lungs were homogenized in RIPA lysis buffer containing 1 % PMSF and centrifuged at 10,000 × g at 4 °C for 10 min to acquire tissue proteins. The protein concentration in various samples was then quantified by BCA analyses and western blotting was proceeded as per the protocol mentioned in Section 2.12. The GAPDH was used as an internal standard.

2.18 In vivo biocompatibility

Healthy male C57BL/6 mice were chosen to assess the safety profile of the delivery systems. Briefly, the mice were randomly separated into five groups (n = 6) and administrated PBS (viz., negative control), BLM (viz., positive control) or siNC-loaded nanoscaffolds (25 µl, 4 µg siRNA/mouse) as described in Section 2.15. After 48 h of the last administration, three mice from each group were anesthetized and their lungs were purged with PBS (1 ml) two times to gather lung bronchoalveolar lavage fluid (BALF). Subsequently, BALF was centrifuged at 500 × g at 4 °C for 10 min and the supernatant was collected to determine the inflammatory cytokine (TNF-α and IL-6) levels using enzyme-linked immunosorbent assay (ELISA) [57]. Meanwhile, the cell pellet was also collected, resuspended in 0.5 ml PBS, stained with Diff-Quik stain dye (Solarbio Science & Technology, China), and subjected to total cell counting and differential cell counting for the percentage of macrophages and neutrophils [57]. The other three mice of each group were sacrificed to harvest their major organs for H&E staining.

2.19 Statistical analyses

Different numerical data were expressed as mean ± standard deviation (SD). Statistical analyses of data were performed using the SPSS 27.0 software. The results among various groups were compared by one-way ANOVA and independent t-test. The P values lesser than 0.05 were considered statistically significant.

3 Results and discussion

3.1 Preparation and characterization of various nanoscaffolds

To achieve efficient and specific siRNA delivery to pulmonary fibroblasts, EM/cationic lipid hybrid nanoscaffolds (DOTAP/siSmad4@EM) were accomplished. In this context, EVs were initially isolated from mouse lung fibroblast cells (i.e., MLg2908) through ultracentrifugation process and then subjected to hypoosmotic treatment to purify EM [37]. Both EVs and EM consisted of lipid bilayers and displayed saucer-like architectures under TEM analyses (Fig. 1A). The hydrodynamic diameters of EVs and EM were 185.6 ± 2.3 nm and 192.7 ± 0.7 nm, respectively, with a narrow size distribution (PDI values, ∼ 0.2). The zeta potential of EVs was −16.8 ± 4.1 mV, while the value was dramatically increased for EM (−9.7 ± 1.6 mV) (Table S1), possibly ascribed to declined protein and glycosyl contents in EVs as well as the loss of ions such as Na+, K+, Ca2+ and H+ [58]. Western blotting analyses were further conducted to identify various protein markers in EVs and EM. Encouragingly, CD63 was detected in EVs but no CANX (a marker of the endoplasmic reticulum), signifying successful isolation of EVs. Furthermore, the GAPDH exhibited high expression in both MLg2908 and EVs but showed extremely poor signal in the case of EM, thus confirming the purity of EM (Fig. 1B).Fig. 1 Formulation and optimization of various nanoscaffolds. (A) TEM images of EVs, EM, lipoplexes, DOTAP/siRNA@lipo and DOTAP/siRNA@EM. (B) Western blotting analyses of different protein markers including CANX, CD63 and GAPDH in MgL2908, EVs and EM. (C) FRET analyses to invastigate optimal weight ratio of DOTAP and EM for the preparation of DOTAP/siRNA@EM. (D) The results of agarose gel electrophoresis to determine optimal combination ratio of DOTAP and siRNAs in different nanoscaffolds (EM, DPPC—Chol lipo, DOTAP/siRNA@lipo and DOTAP/siRNA@EM). (E) CLSM images of DOTAP/siRNA@EM composed of DiI-labeled EM (red) and FAM-siRNAs (green), scale bar: 5 µm. (F) Protein profiles of MLg2908, EVs, EM, lipoplexes, DOTAP/siRNA@lipo and DOTAP/siRNA@EM measured by SDS-PAGE electrophoresis. (G) Electrophoretic strips of free siRNAs, lipoplexes, DOTAP/siRNA@lipo and DOTAP/siRNA@EM obtained after their incubation in PBS (pH7.4) containing 50 % FBS.

Fig 1

Following isolation and purification of EVs and EM, the siRNAs were combined with cationic lipid DOTAP to afford lipoplexes, which depicted hydrodynamic diameters of 173.2 ± 3.0 nm and the zeta potential values of +38.2 ± 1.6 mV (Table S1). The agarose gel electrophoresis was employed to optimize the N/P ratio of DOTAP and siRNAs. At 5 and higher N/P ratios, the migration of the siRNAs in agarose gel was completely retarded, indicating electrostatic stability of the lipoplexes (Fig. S1D). Moreover, to identify the best hybridization efficiency between EM and DOTAP in accomplishing DOTAP/siRNA@EM, FRET was performed on different samples prepared using varying combination ratios of DiI-labelled DOTAP and DiO-labelled EM. A weight ratio of DOTAP to EM of 4:1 portrayed the highest FRET efficiency, representing the best hybridization potential between the two components. Similarly, the weight ratio of DOTAP to DPPC—Chol of 1:1 was chosen to formulate DOTAP/siRNA@lipo based on their superior FRET efficiency (Figs. 1C and S1C). Thereafter, the agarose gel electrophoresis was further carried out to investigate the influence of hybridization of two components on the complexing efficiency of DOTAP and siRNAs (Fig. 1D). On account of complete retardation of siRNAs migration, DOTAP to siRNAs N/P ratio of 10 was finally selected to prepare DOTAP/siRNA@EM and DOTAP/siRNA@lipo.

DOTAP/siRNA@EM and DOTAP/siRNA@lipo demonstrated hydrodynamic diameters of approximately 190 nm with homogeneous size distribution (PDI values, ∼0.2), and their zeta potential values ranged from +33 to +34 mV (Table S1). After hybridization with negatively charged EM, the size of DOTAP/siRNA@EM increased slightly, while its zeta potential value declined as compared to the corresponding lipoplexes. This data signified the successful insertion of EM into lipoplexes. Various nanoscaffolds revealed nearly spherical shapes under TEM imaging (Fig. 1A). The colocalization of red and green fluorescence signals of DiI-labeled EM and FAM-siRNA detected under CLSM further implied an excellent fusion of two components with ideal uniformity in appearances (Fig. 1E).

The outcomes of SDS-PAGE gel electrophoresis analyses confirmed well-preserved EM proteins in DOTAP/siRNA@EM (Fig. 1F), which might assist in eliciting potential biological functions. In addition, the levels of membrane protein CD47 in EVs, EM and DOTAP/siRNA@EM were studied by dot blot assay (Fig. S1E). The results showed that the CD47 levels of EVs, EM and DOTAP/siRNA@EM were almost the same, which indicated that the membrane contents of EVs were well reserved by EM, and the preparation process of DOTAP/siRNA@EM would not influence the membrane characteristics of EM. Therefore, the fibroblast-specific targeting ability of EVs could be inherited by DOTAP/siRNA@EM. Various nanoscaffolds depicted no significant changes in their particle sizes during 3 weeks of storage at 4 °C, demonstrating an ideal storage stability (Fig. S1F). The enzymatic stability of siRNAs in different nanoscaffolds was further investigated via agarose gel electrophoresis following their incubation with 50 % FBS-containing PBS (Fig. 1G). The results illustrated that all nanoscaffolds could protect the loaded siRNAs from the enzymatic degradation. It also indicated that most of siRNAs were encapsulated inside the cores of nanoscaffolds rather than on their surfaces. Overall, the results of various studies conferred the successful formulation of DOTAP/siRNA@EM.

3.2 Cytotoxicity of various nanoscaffolds

The cytotoxicity is a major concern for the targeted drug delivery systems. Previous studies suggested strong cytotoxic potentials of cationic DOTAP-based siRNA vectors [43]. Thus, the role of EM in reducing DOTAP's cytotoxicity was assessed through the MTT assay in mouse lung fibroblast cells (MLg2908), which were chosen as the target cells for siRNA delivery and as the parental cell line of EVs. The cells were treated with lipoplexes, DOTAP/siRNA@lipo and DOTAP/siRNA@EM in serial siRNA concentration and the cell viability at variable timepoints (24 and 48 h) were compared (Fig. 2F and 2G). Among different nanoscaffolds, DOTAP/siRNA@EM having highest siRNA concentration (200 nM) displayed the lowest cytotoxicity (cell viability, ∼80 %) following 48 h-treatment. With parallel siRNA content (200 nM) and exposure time (48 h), the DOTAP/siRNA@lipo depicted cell viability of about 70 %, although they were composed of DPPC, a surfactant lipid of the lung. On the other hand, lipoplexes exhibited highest cytotoxicity in an analogous condition, which was consistent with the previous reports [43]. The lower cytotoxicity of DOTAP/siRNA@EM could be attributed to the existence of an autogenous cellular component (i.e., EM) in the nanoscaffolds, reflecting excellent cytocompatibility.Fig. 2 Cytotoxicity and cellular uptake behavior of different nanoscaffolds. (A) Representative histograms of flow cytometry analyses of MLg2908 cells following treatment with FAM-siRNA loaded nanoscaffolds (*P < 0.05, **P < 0.01, *** P < 0.001, compared with PBS) and (B) their corresponding mean fluorescence intensities. (C) Representative histograms of flow cytometry analyses of RAW264.7 cells following treatment with FAM-siRNA loaded nanoscaffolds (*P < 0.05, ** P < 0.01, ***P < 0.001, compared with PBS) and (D) their corresponding mean fluorescence intensities. (E) Cellular uptake efficiency of FAM-siRNA loaded nanoscaffolds on MLg2908 cells following pretreatment with CPZ, Filipin and EIPA (*P < 0.05, **P < 0.01, ***P < 0.001, compared with Untreated). (F) and (G) In vitro cytotoxicity of lipoplexes, DOTAP/siRNA@lipo and DOTAP/siRNA@EM loading siNC in MLg2908 (Mean ± SD, n = 3) (DOTAP/siRNA@lipo, DOTAP/siNC@EM compared to lipoplexes at different concentrations) (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig 2

3.3 Cellular uptake efficiencies and mechanisms

Successfully targeted cellular delivery is a key determinant for siRNAs to exert efficient gene silencing effects [21]. It is well-known that macrophages are one of the most important components of the reticuloendothelial systems and are responsible for recognizing and phagocytosing foreign materials [59]. In IPF, particularly, the overexpressed and secreted cytokines and chemokines would recruit a vast number of alveolar macrophages (AMs). Taking this into account, the cellular uptake efficiency of DOTAP/siRNA@EM and other reference nanoscaffolds (viz., lipoplexes and DOTAP/siRNA@lipo) were examined in mouse lung fibroblasts (MLg2908) and macrophages (RAW264.7) to compare their cell selectivity. As shown in Fig. 2A and 2B, the fluorescence intensity of DOTAP/siRNA@EM was significantly higher in MLg2908 as compared to lipoplexes and DOTAP/siRNA@lipo (P < 0.01). However, the signals were not obviously different between lipoplexes and DOTAP/siRNA@lipo (P = 0.175). The improved cellular uptake ability of DOTAP/siRNA@EM within MLg2908 cells might be ascribed to the integrated cellular components (viz., EM) of such systems. Furthermore, DOTAP/siRNA@EM treated cells illustrated widely distributed green fluorescent dots, which confirmed the augmented intracellular delivery efficiency of such nanoscaffolds as compared with that of lipoplexes and DOTAP/siRNA@lipo in Fig. S2. This was consistent with the results of flow cytometery. Interestingly, the DOTAP/siRNA@EM depicted the lowest fluorescence signals in RAW264.7 as compared to other nanoscaffolds (Fig. 2B and 2C), implying that the introduction of EM in DOTAP/siRNA@EM might provide homologous targeting effects to pulmonary fibroblasts while reduce the recognition and clearance rate by macrophages. A similar phenomenon was reported earlier [60]. In contrast, among various nanoscaffolds, the lipoplexes exhibited superior fluorescence intensity on RAW264.7, conferring their enhanced recognition and clearance potential by the reticuloendothelial systems, which was well collaborated with the earlier report [43].

To further explore the cellular uptake mechanisms of various nanoscaffolds, the MLg2908 were pretreated with CPZ, filipin or EIPA (inhibitors of clathrin-dependent, caveolae-dependent and micropinocytosis-dependent endocytosis) respectively, and incubated with the nanoscaffolds subsequently. Compared with untreated group, the cellular uptake efficiencies of lipoplexes in cells pretreated with three endocytosis pathway inhibitors exhibited no significant differences, implying the nonselective cellular uptake mechanisms of lipoplexes. The DOTAP/siRNA@lipo demonstrated significantly decreased cellular uptake efficiency in cells pretreated with filipin, while no significant difference was detected in cells pretreated with EIPA. Interestingly, the cellular uptake efficiency of DOTAP/siRNA@lipo increased in cells pretreated with CPZ. The results suggested that the internalization of DOTAP/siRNA@lipo into MLg2908 cells was primarily carried out through caveolae-mediated endocytosis. The macropinocytosis-dependent endocytosis had no influence on the internalization of DOTAP/siRNA@lipo, while inhibition of clathrin-dependent endocytosis would lead to compensatory increase of cellular uptake. The mechanism of this phenomenon warrants further exploration. In contrast, the cellular uptake efficiency of DOTAP/siRNA@EM remarkably declined in the cells pretreated with CPZ (P < 0.01), with no significant differences in the cells pretreated with filipin or EIPA. These results indicated that clathrin-mediated endocytosis was the major pathway for the cellular internalization of DOTAP/siRNA@EM (Fig. 2E).

Different endocytic pathways could further influence the intracellular transportation of siRNAs [61]. For instance, following clathrin-mediated endocytosis, the nanoscaffolds would be entrapped within endosomes, which might degrade the contained substances and eventually compromise the therapeutic efficiency of siRNAs [51]. Taking this into consideration, the subcellular localization of nanoscaffolds was investigated (Fig. 3A). In this context, endosomes were labeled with Lyso-Tracker Red, and the intracellular colocalization of FAM-siRNA loaded nanoscaffolds were observed under CLSM. In addition, the Mander's coefficients of FAM-siRNA and Lyso-Tracker Red labeled endosome in different groups were calculated (Fig. 3B and 3C). After incubation for 1 h, the Mander's coefficients of all the nanoscaffolds were at the same level. However, after 6-h incubation, the Mander's coefficient of DOTAP/siRNA@lipo was the highest among three nanoscaffolds, following with lipoplexes, while the Mander's coefficient of DOTAP/siRNA@EM was the lowest among all the groups with statistical differences (P = 0.03 compared with lipoplexes and P < 0.01 compared with DOTAP/siRNA@lipo). The high Mander's coefficient indicates low endosome escape ability, which further lead to inefficient intracellular delivery of siRNA. The most profound endosome escape ability of DOTAP/siRNA@EM may be related to the unique function of EM by accelerating the Golgi body- and endoplasmic reticulum (ER)- mediated intracellular transportation and subsequently bypassing the endosomal degradation process [48]. And the endosome escape ability of lipoplexes was attributed to the “proton sponge effect” [51]. In contrast, the poor endosome escape ability of DOTAP/siRNA@lipo might be due to the disturbance of DPPC—Chol on lipoplexes. The negatively charged DPPC incorporated in the lipoplexes might form a complex lipid structure with DOTAP, weakening the interaction of DOTAP with the endosome membrane, leading to a limited endosome escape ability. The differences of endosome escape ability of the three nanoscaffolds may further lead to different gene silencing effects.Fig. 3 Endosome colocalization of different nanoscaffolds. (A) CLSM images of the intracellular colocalization of FAM-siRNA-loaded nanoscaffolds (green) and endosomes stained with lyso-traker (red), while cell nuclei were marked with DAPI (blue), scale bar: 50 µm, and (B) and (C) their measurements of Mander's coefficient (*P < 0.05, ** P < 0.01, *** P < 0.001).

Fig 3

3.4 Gene silencing and anti-migration efficiencies at cellular level

The therapeutic efficacies of various nanoscaffolds in vitro were evaluated by assessing their gene silencing potentials in MLg2908 cells using the RT-qPCR and western blotting techniques to quantitate the Smad4 mRNA and protein levels, respectively. As depicted in Fig. 4B, 4C and S3A, free siRNAs lacked gene silencing effects attributed to poor cell penetration [20]. Similarly, the siNC loaded nanoscaffolds (i.e., DOTAP/siNC@EM) exhibited no gene silencing potential. Therefore, any influence of free siRNAs and empty delivery vectors on Smad4 expression was ruled out. Lipoplexes exhibited a relatively ideal gene silencing effect with Smad4 mRNA knockdown efficiency up to 40 % at 24 h and 45 % at 48 h. On the contrary, DOTAP/siSmad4@lipo showed limited inhibitory effect on the mRNA expression of Smad4 (19 % at 24 h and 36 % at 48 h, respectively). Compared with lipoplexes and DOTAP/siRNA@lipo, the DOTAP/siSmad4@EM exhibited superior gene silencing potentials at 24 h and 48 h, with mRNA silencing efficiency of about 60 % and 50 %, respectively. Several nanoscaffolds demonstrated protein level gene silencing effects comparable to that at the mRNA level. The expression of Smad4 protein was dramatically declined (∼70 %) when the cells were treated with DOTAP/siSmad4@EM for 48 h. What's more, DOTAP/siSmad4@EM showed a remarkable inhibitory effect on α-SMA expression, which demonstrating that downregulating the expression of Smad4 could effectively inhibit differentiation of fibroblasts into myofibroblasts. The different gene silencing effects of several nanoscaffolds might be accredited to their different cellular uptake and endosomal escape abilities in MLg2908 cells. In general, the DOTAP/siSmad4@EM could effectively promote the siRNA transfection, resulting in a stronger gene silencing efficiency. The poor gene silencing effects of the positive control group (Lipo3000) may appertain to scant uptake efficacy for MLg2908 cells.Fig. 4 In vitro gene silencing and anti-migration efficiencies of different nanoscaffolds. (A) The migration abilities of MLg2908 cells after treatment with different formulations, scale bar: 200 nm. (B) Relative levels of Smad4 mRNA expression on MLg2908 cells detected by RT-qPCR technique, n = 3 (*P < 0.05, ** P < 0.01, *** P < 0.001, compared with PBS). (C) Relative levels of Smad4 and α-SMA expression on MLg2908 cells detected by Western blotting, n = 3 (*P < 0.05, ** P < 0.01, *** P < 0.001, compared with PBS). (D) Cell migration of MLg2908 expressed relative to the positive control (TGF-β1) (*P < 0.05, ** P < 0.01, *** P < 0.001).

Fig 4

One of the most prominent pathological characteristics of pulmonary fibrosis is the migration of fibroblasts to the fibrous foci [2]. The capacities of various nanoscaffolds to inhibit the migration of fibroblasts under an in vitro fibrotic environment were evaluated through wound healing assay and compared (Fig. 4A and 4D). With reference to the negative control group, MLg2908 cells showed significantly enhanced migration in the presence of TGF-β1 (5 ng/ml), a simulated fibrotic lung microenvironment. Possibly, TGF-β1 could amplify the differentiation of MLg2908 cells into myofibroblasts and, thereby, increase Smad4 and α-SMA protein expressions, facilitating cell migration [62]. The DOTAP/siNC@EM did not inhibit cell migration, which was consistent with their gene silencing effects. Contrarily, DOTAP/siSmad4@EM significantly attenuated the migration capability of MLg2908 cells, which further indicated that DOTAP/siSmad4@EM might portray in vivo anti-fibrotic potential by inhibiting the pulmonary fibroblast migration to the fibrous foci. In addition, lipoplexes exhibited the strongest anti-migration effect as demonstrated by a smaller migration area than the negative group (Control) (Fig. 4D). This may be ascribed to the high cytotoxicity of lipoplexes which could affect cell viability. In contrast, DOTAP/siSmad4@lipo could not effectively attenuate the migration capability of MLg2908 cells.

3.5 Lung retention of siRNAs

Murine (C57BL/6) model of pulmonary fibrosis was established by a single intratracheal administration of BLM solution (3 U/kg). Following H&E staining, the lung tissues were examined for their morphological changes, hyperplasia and inflammation. The alveolar and airway walls of the lungs of the control group were thin without interstitial hyperplasia and inflammation (Fig. S4A). However, the modeling group exhibited interstitial hyperplasia, which became gradually serious with airway walls thickening and inflammatory cell infiltration. The results of Masson staining also illustrated the muscle fibers and collagen deposition in the modeling group, which were progressively increased over time (Fig. S4B). The results confirmed that the pulmonary fibrosis occurred within 3 d after intratracheal administration of BLM. The mice with pulmonary fibrosis were then used for following study.

The biodistribution of the therapeutic molecules within the body may substantially influence their effectiveness [63]. To evaluate the biodistribution and retention abilities of siRNAs in the lungs, the Cy5-siRNA loaded nanoscaffolds were intratracheally administrated to mice. At pre-designed time points (1, 6, 12 and 24 h) post intratracheal administration, the biodistribution in different vital organs was investigated using IVIS imaging system. Free siRNA exhibited the highest fluorescence signal among all the groups at 1 h after intratracheal administration. However, its signal decreased sharply at 6 h post-administration. No fluorescence signal was detected at 12 h and 24 h post-administration (Fig. 5A). The results indicated that free siRNA experienced rapid clearance after entering the lungs [64]. In contrast, the siRNAs loaded in the nanoscaffolds exhibited significantly prolonged lung retention, which confirmed that the nanoscaffolds are conductive to the siRNA accumulation in the lungs and reduce the systemic exposure. The signals in the nanoscaffolds were lower than that of free siRNA at 1 h post administration, which might be attributed to the blocking of siRNA fluorescence by the nanoscaffolds. The lipoplexes exhibited comparable signal intensity as DOTAP/siRNA@EM at 1 h and 6 h post-administration; however, the signals decreased quickly at 12 h post-administration and no signals were detected at 24 h post-administration (Fig. 5A). The DOTAP/siRNA@EM exhibited comparable fluorescence intensity level with DOTAP/siRNA@lipo at 6 h and 12 h. However, the fluorescence intensity of DOTAP/siRNA@EM was significantly higher than that of DOTAP/siRNA@lipo at 24 h post- administration (Fig. 5A and 5C). Collectively, DOTAP/siRNA@EM demonstrated the best ability of prolonging the siRNA retention within the lungs, which might be attributed to the reduced recognition and clearance rate of DOTAP/siRNA@EM by AMs than that of lipoplexes and DOTAP/siRNA@lipo.Fig. 5 Biodistribution and lung retention of various nanoscaffolds after their pulmonary administration. (A) Representative ex vivo imaging of different vital organs captured at various time points of pulmonary administration of Cy5-siRNA loaded nanoscaffolds. (B) The images of dual immunofluorescence staining (α-SMA and F4/80) of lung tissues captured after 24 h of intratracheal administration of Cy5-siRNA loaded nanoscaffolds (pink). The cell nuclei were stained with DAPI (blue), scale bar: 50 µm. (C) Radiant efficiency of lungs measured by IVIS for different groups over time (mean ± SD, n = 3). (D) Measurements of Mander's coefficient in (B), Cy-5 siRNA (pink) with α-SMA fibroblasts (green) and Cy-5 siRNA (pink) with F4/80 macrophages (red), (*P < 0.05, **P < 0.01, ***P < 0.001, compared with DOTAP/siRNA@EM).

Fig 5

To further investigate the distribution of siRNAs in different lung cells, immunofluorescence staining of lung tissues was performed. The α-SMA was used to label pulmonary fibroblasts (green fluorescence) and F4/80 was exploited to mark macrophages (red fluorescence). As illustrated in Fig. 5B and 5D, DOTAP/siRNA@EM exhibited higher colocalization ratios with the fibroblasts and little association with macrophages. The modification of EM within DOTAP/siRNA@EM could hinder its AMs mediated phagocytosis and clearance while promoting internalization into the pulmonary fibroblasts (Fig. S5B).

3.6 Gene silencing and anti-fibrotic effects in animal model

The gene silencing efficiencies and anti-fibrotic potentials of various nanoscaffolds were evaluated on murine pulmonary fibrosis model. After 9-d treatment, mice in various groups were sacrificed and their lungs were carefully collected for further analyses. The Smad4 expression in the murine lungs was evaluated by RT-qPCR and western blotting assay. The lipoplexes, DOTAP/siRNA@lipo and DOTAP/siSmad4@EM could effectively suppress the Smad4 mRNA expression but without showing a significant difference (Fig. 6B). Interestingly, among several nanoscaffolds, the DOTAP/siSmad4@EM demonstrated the strongest ability to downregulate Smad4 protein expression, which further downregulate α-SMA expression effectively (Fig. 6C).Fig. 6 n vivo gene silencing and anti-fibrotic efficiencies of various nanoscaffolds. (A) Schematic illustration of the IPF model establishment and treatment protocol. (B) Relative levels of Smad4 mRNA expression detected by RT-qPCR, n = 3 (*P < 0.05, ** P < 0.01, *** P < 0.001, compared with PBS). (C) Relative protein expression of Smad4 and α-SMA measured by western blotting in mouse lung, n = 3 (*P < 0.05, ** P < 0.01, *** P < 0.001, compared with PBS). (D) and (E) Results of H&E and Masson staining of pathological sections of mouse lung tissues, scale bar: 100 µm.

Fig 6

The H&E and Masson stainings were utilized to assess the impact of the treatment on the degree of lung fibrosis (Fig. 6D and E). The pulmonary interstitial hyperplasia appeared prominent with highly thickened airway walls and a huge inflammatory cell infiltration in free siRNA and DOTAP/siNC@EM treated groups. In the case of the lipoplexes, DOTAP/siRNA@lipo and DOTAP/siSmad4@EM groups, interstitial hyperplasia was effectively inhibited with considerably preserved amounts of healthy alveolar areas. However, only DOTAP/siSmad4@EM treated group exhibited an eminently normal alveolar area, whereas lipoplexes and DOTAP/siRNA@lipo treatments revealed remarkably thickened airway walls. Correspondingly, Masson-stained lung tissues of free siRNA and DOTAP/siNC@EM treated mice showed no anti-fibrotic effects, as illustrated by a massive proliferation of muscle fibers and server collagen deposition. On the contrary, the lipoplexes, DOTAP/siRNA@lipo and DOTAP/siSmad4@EM treated groups could efficiently suppress the muscle fiber proliferation and collagen deposition.

3.7 Safety profiles

The safety of various nanoscaffolds was evaluated in healthy mice after being administered intratracheally. This assessment involved quantifying the proinflammatory factors (IL-6 and TNF-α) in the BALF, counting the infiltrated inflammatory cells and H&E staining. The IL-6 level was increased in the positive control group (i.e., BLM-challenged) as compared to the negative control group (viz., healthy mice), while various nanoscaffolds treated groups revealed IL-6 levels comparable to that of negative control group (Fig. 7A). The TNF-α levels of positive control, lipoplexes, and DOTAP/siNC@lipo groups were dramatically increased as compared to negative control and DOTAP/siNC@EM groups (Fig. 7B). Moreover, the total amount of inflammatory cells in BALF of positive control and lipoplexes groups was significantly higher than that in the other groups (P < 0.05), indicating their stronger inflammatory responses (Fig. 7C). DOTAP/siNC@EM group revealed the lowest total number of immune cells with minimal change in their proportion (Fig. 7C and 7E). This result underlined the importance of incorporating EM in the targeted nanoscaffolds to effectively reduce DOTAP toxicity, similar to previous investigations [65]. The results were corroborated by H&E staining of DOTAP/siNC@EM treated groups, which exhibited undetectable levels of systematic toxicity in various vital organs (Fig. 7F). Conversely, apparent inflammatory cell infiltration and airway wall thickening were observed in the mice in the lipoplexes and positive control groups. To sum up, these results implied an excellent biosafety of DOTAP/siNC@EM after multiple pulmonary administrations.Fig. 7 In vivo safety assessments of various nanoscaffolds. The levels of (A) IL-6, (B) TNF-α in the BALF measured by ELISA technique (*P < 0.05, ** P < 0.01, *** P < 0.001, compared with PBS). (C) Total cell counts in the BALF. (D) Neutrophil and (E) macrophage percentages in the BALF (Mean ± SD, n = 3) (*P < 0.05, ** P < 0.01, *** P < 0.001, compared with PBS). (F) Histological assessment of tissues of various vital organs including heart, liver, spleen, lung and kidney following H&E staining, scale bar: 100 µm.

Fig 7

4 Conclusions

This study demonstrates that pulmonary fibroblast derived EM can be used for the targeted delivery of siRNAs. Engineering EM using a cationic lipid DOTAP could enhance the siRNAs loading efficiency of the EM. The modification of DOTAP/siSmad4 (lipoplexes) using EM could circumvent the DOTAP cytotoxicity, while promoting cell internalization and fibroblast specific delivery. The novel EVs membrane/cationic lipid hybridized systems (DOTAP/siRNA@EM) could effectively deliver siRNAs to the pulmonary fibroblasts. These nanoscaffolds could efficiently preserve the proteins on the EM, protect siRNAs from degradation and confer excellent storage stability. The engineered nanoscaffolds demonstrated MLg2908 cells specific internalization and a remarkable gene silencing efficiency with significant anti-migration efficacy at cellular level. In addition, DOTAP/siSmad4@EM showed prolonged pulmonary retention abilities and augmented internalization rate by the pulmonary fibroblasts in vivo. Furthermore, the nanoscaffolds displayed outstanding gene silencing and anti-fibrotic potentials in C57BL/6 pulmonary fibrosis model with impressive biocompatibility. Overall, the newly fabricated nanoscaffolds could selectively and safely deliver siRNAs to the pulmonary fibroblasts and might be exploited as promising anti-fibrotic therapy for clinical applications. More importantly, this formulation strategy has great potential in delivering other nucleic acid drugs, such as mRNA, DNA and ASO, by electrostatic complexion between negatively charged nucleic acid and positively charged DOTAP, followed by fusion with EM derived from other parental cells that play a crucial pathophysiological role in various diseases.

Conflicts of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Appendix Supplementary materials

Image, application 1

Acknowledgements

This research work was financially supported by the Liaoning Pan Deng Xue Zhe Scholar (grant No. XLYC2002061), the 10.13039/501100001809 National Natural Science Foundation of China (grant No. 82173768 ), and the 10.13039/501100013313 Overseas Expertise Introduction Project for Discipline Innovation (“111 Project”) (grant No. D20029 ). M.Y. thanks Independent Research Fund Denmark for the financial support (Grant ID: 10.46540/3105-00249B). L.W. acknowledges the financial supports of 10.13039/501100001809 National Natural Science Foundation of China (grant No. 82204316 ), 10.13039/501100002858 China Postdoctoral Science Foundation (grant Nos. 2021TQ0219 and 2022MD713776 ). H.B. thanks the financial support from 10.13039/501100001809 National Natural Science Foundation of China , Govt. of China (grant No. 82050410448 ) and Fellowship of 10.13039/501100002858 China Postdoctoral Science Foundation , Govt. of China (grant No. 2021MD703857 ). D.C. acknowledges financial support from Nature Science Foundation of Liaoning Province (grant No. 2022-MS-241), and Ministry of Education Chunhui Program (2020).

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.ajps.2024.100929.
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