Clodronate-loaded liposomal and fibroblast-derived exosomal hybrid system for enhanced drug delivery to pulmonary fibrosis

Lingna Sun a, Mingrui Fan a, Dong Huang a, Bingqin Li a, Ruoting Xu a, Feng Gao a,b,**, Yanzuo Chen a,b,*

Abstract

Pulmonary fibrosis is a rapidly progressive and fatal fibrotic lung disease with high mortality and morbidity. However, pulmonary fibrosis therapy in the clinic has been limited by poor selectivity and inefficiency of drug delivery to fibroblasts. Herein, a clodronate (CLD)-loaded liposome and fibroblast-derived exosome (EL-CLD) hybrid drug delivery system with non-specific phagocytosis inhibition and fibroblast homing properties, was designed for the treatment of pulmonary fibrosis. EL-CLD effectively depleted Kupffer cells via apoptosis by passive targeting after intravenous injection, and thus significantly reduced accumulation in the liver. Notably, the EL-CLD hybrid system preferentially accumulated in the fibrotic lung, and significantly increased penetration inside pulmonary fibrotic tissue by targeted delivery due to the specific affinity for fibroblasts of the homologous exosome. Nintedanib (NIN), an anti-fibrotic agent used to treat pulmonary fibrosis, was loaded in the EL-CLD system, and achieved a remarkable improvement in curative effects. The enhanced therapeutic efficacy of NIN was a result of enhanced pulmonary fibrotic tissue accumulation and delivery, combined with a diminished macrophage-induced inflammatory response. Hence, the EL-CLD hybrid system acts as an efficient carrier for pulmonary anti-fibrotic drug delivery and should be developed as an efficient fibroblast specific therapy.

Keywords:
Pulmonary fibrotic therapy
Exosomes
Liposomes
Macrophages depletion Fibrosis penetration

1. Introduction

Pulmonary fibrosis is an unpredictable, progressive, and fatal fibrosing lung disease with a median survival of 2–5 years [1]. With persistent, unremitting lung parenchymal injury, pulmonary fibrosis ultimately leads to the destruction of the lung architecture, fulminant respiratory failure, and death. To date, pulmonary fibrosis therapies mainly depend on antifibrotic drugs, but due to their poor selectivity, these agents cannot effectively halt the progression of fibrosis [2]. Thus, the development of effective nano-drug delivery systems to improve drug accumulation and homing to pulmonary fibrosis tissue is highly desirable. The existence of an enhanced permeability and retention (EPR) effect has been reported in pulmonary fibrosis, which benefits the passive targeting of nanoparticles [3]. However, nanoparticles for pulmonary fibrosis treatment still face biological obstacles that severely limit drug delivery and thus fail to achieve the expected therapeutic outcomes. One of the main disadvantages is the nonspecific uptake by Kupffer cells [4,5]. Over 90% of systemically injected nanoparticles accumulate in the liver, and less than 1% reach the intended location, resulting in inadequate drug accumulation at fibrotic sites [6]. In addition, the high density of crosslinked extracellular matrix (ECM) and excessive fibrosis collagen deposits further weaken the penetration of nanoparticles at fibrotic sites [7].
Multifunctional nanoparticles are designed to reduce clearance by Kupffer cells including pegylation and “don’t eat-me” marker CD47 modification [8], yet they have achieved negligible improvement [9]. Recently, a strategy of harnessing macrophage functions that diminish nanoparticles uptake has been proposed. Clodronate (CLD) was the first generation of bisphosphonates, used for the treatment of osteoporosis and osteolytic metastasis [10]. Because osteoclasts originate from the mononuclear-macrophage system, encapsulation of clodronate into liposomes (clodrolip) resulted in the depletion of in macrophage via apoptosis once they were recognized and engulfed by Kupffer cells [11]. It has been reported that after macrophage depletion by clodrolip, nanoparticle tumor delivery is enhanced and improved therapeutic effects can be achieved [12,13]. We hypothesized that a CLD-loaded liposome would deplete macrophages at the liver and thus diminish hepatic uptake, resulting in the improvement of nanoparticle-based drug delivery to fibrotic tissue in the lung. Despite these attempts, the CLD-loaded liposome was still insufficient for effective pulmonary anti-fibrotic drug delivery, due to the limited penetration of nanoparticles across the ECM.
Exosomes are small membrane vesicles with diameters ranging from 40 to 150 nm, which are naturally secreted by most cell types and stably exist in bodily fluids [14]. After secretion, the lipid bilayer of the exosome protects its cargo from the plasma and immune components and directs it to specific recipient cells, properties that present great potential for exploiting exosomes as targeted drug delivery vehicles [15,16]. Since exosomes are phospholipid structures with particular surface proteins like tetraspanin inherited from parental cells, they exhibit specific homing properties to their original cells, thus reducing off-target effects in vivo [17,18]. In addition, exosomes can potentially overcome biological barriers [19] and efficiently accumulate and penetrate into the dense fibrotic stroma [20]. However, the low drug-loading efficiency of exosomes limits their potential application as a drug delivery platform [21]. An additional property is that the composition of the exosome bilayer membrane resembles that of liposomes [22]. Hence, the preparation of an exosomal and liposomal hybrid system by membrane fusion to enhance drug-loading and fibroblast-specific delivery has been proposed.
In order to improve nano-drug delivery to fibrotic tissue in the lung, a CLD-loaded liposome and fibroblast-derived exosome (EL-CLD) hybrid was designed. Given CLD released by the liposome depletes Kupffer cells and a homologous exosome favors homing to fibroblast, we hypothesized that intravenous injection of the EL-CLD hybrid would display enhanced pulmonary fibrosis-specific accumulation and penetration (Scheme 1). In vitro and in vivo experiments were performed to evaluate the effects of EL-CLD on macrophages and fibroblasts. Based on the favorable fibrotic-specific drug delivery-potential of EL-CLD, nintedanib (NIN), a triple angiokinase inhibitor approved by the United States Food and Drug Administration (FDA) as an anti-fibrotic agent for the clinical treatment of pulmonary fibrosis, served as the model drug. NIN was encapsulated in the EL-CLD hybrid to verify the potentially enhanced anti-fibrotic effects. In addition, the mechanisms of the improved anti- fibrotic efficacy of the NIN-based EL-CLD hybrid delivery system were elucidated.

2. Materials and methods

2.1. Materials, animals, and antibodies

Clodronate disodium (CLD) was purchased from Hubei Norna Technology Co., Ltd (Wuhan, China). 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was purchased from Corden Pharma Switzerland LLC (Liestal, Switzerland). 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG 2000) was purchased from A.V.T. Pharmaceutical Co., Ltd. (Shanghai, China). Cholesterol was purchased from Sigma-Aldrich (USA.). Phospholipid- conjugated L-α-phosphatidylethanolamine-N- (4-nitrobenzo-2-oxa-1, 3- diazole) (ammonium salt) (PE-NBD) and L-α-phosphatidylethanolamine-N-(lissamine rhodamine-B sulfonyl) (ammonium salt) (PE-RhB) were purchased from Avanti Polar Lipids, Inc. (Alabaster, USA.). Bleomycin sulfate (BLM, 1.5–2.0 units/mg) was obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). NIN was purchased from Nanjing Core Tech Biomaterial Co., Ltd. (Nanjing, China). All other chemicals were purchased from Titan Scientific Co. Ltd. (Shanghai, China), unless specifically mentioned.
Hoechst 33342 and the Bicinchoninic acid protein assay (BCA) kit were provided by the Beyotime Institute of Biotechnology Co., Ltd. (Shanghai, China); 1, 1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiD) was purchased from US Everbright Inc. (Suzhou, China); coumarin 6 (C6) was purchased from Thermo Fisher Scientific (Massachusetts, USA.); and 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) and 4, 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (USA.). The TUNEL assay kit was purchased from Wuhan Saville Biotechnology Corporation (Wuhan, China). Assay kits for measurement of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), total bilirubin (TBIL), and blood urea nitrogen (BUN) were purchased from Nanjing Jiancheng Bioengineering Institute. (Nanjing, China). The enzyme-linked immunosorbent assay (ELISA) kit for detection of matrix metallopeptidase 7 (MMP-7) and hydroxyproline (Hyp) were purchased from Shanghai Enzyme-linked Biotechnology Corporation (Shanghai, China).
The murine macrophage cell line RAW 264.7 and murine fibroblast cell line L-929 were obtained from the Chinese Academy of Sciences Cell Literary (Shanghai, China). RAW 264.7 cells were cultivated in RPMI 1640 medium, and L-929 cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with fetal bovine serum (FBS, 10%, v/v, Gibco, USA.) and an antibiotic-antimycotic solution (1%, v/v, Gibco, USA.) at 37 ◦C and 5% CO2 in a humidified atmosphere. All experiments were performed with cells in the logarithmic phase of growth. Eight-week-old male C57BL/6 mice were obtained from Slaccas Experimental Animal Co. Ltd. (Shanghai, China). All animal handling procedures adhered to the East China University of Science and Technology Ethics Committee guidelines. Antibodies used for Western blot and immunofluorescence staining are listed in Table S1.

2.2. Preparation and characterization of EL-CLD

2.2.1. Isolation of exosomes

Exosomes were harvested from L-929 cells using the ultracentrifugation method. L-929 cells were cultured for 48 h in exosome-free DMEM containing FBS and antibiotic-antimycotics, which had been centrifuged at 100,000×g before use. The culture supernatants were collected, centrifuged at 300×g for 10 min and then at 2000×g for 20 min, and finally at 10,000×g for 30 min to remove cells, cellular debris, and broken organelles, and was ultimately followed by filtration through a 0.22 μm filter. Exosomes were pelleted by ultracentrifugation at 120,000×g for 60 min at 4 ◦C. The pellets were rinsed with PBS, ultracentrifuged again, and resuspended in sterile PBS. The protein concentrations of the exosome suspensions were determined using the BCA assay, and concentrations were adjusted to 200 μg/mL. Isolated exosomes were stored at − 20 ◦C. Exosome markers and negative markers, HSP70, CD9, and calnexin were detected by western blotting analysis.

2.2.2. Preparation of the EL-CLD hybrid

CLD-loaded liposomes (L-CLD) were prepared by the reverse-phase evaporation method described previously with modifications due to the higher degree of CLD loading [23]. Briefly, 85 mg of L-CLD with a lipid composition of cholesterol: DOPC: DSPE-PEG 2000 at a molar ratio of 2: 10: 1 was prepared. The lipids were dissolved in chloroform, dried into a thin film on a rotary evaporator and then re-suspended in chloroform to prepare a homogeneous dispersion of the drug. A 2 mL volume of CLD solution (20 mg/mL) was gradually added into the organic phase and further emulsified under magnetic stirring for 0.5 h. Afterwards, an organic solvent was removed by a rotary evaporator at 25 ◦C. Next, the residual lipid membrane adhering to the bottle wall was hydrated with 3 mL PBS followed by homogenization using sonication (40% of amplitude, 2/3 s pulse on/off, for 5 min) to obtain L-CLD. Free CLD was removed by gel filtration through Sepharose (CL-4B, Solarbio, China). The exosomes and L-CLD (EL-CLD) hybrid was prepared using a membrane extrusion method. A 0.2 mg protein equivalent of exosomes and 1 mg of lipid film were mixed to a final volume of 1 mL by vortexing and sonication (30% amplitude, 30 s pulse on/off, for 2 min) and then extruded through 400 and 200 nm polycarbonate membranes 10 times (Mini-Extruder, Avanti Polar Lipoids, USA.), respectively. To prepare the C6-loaded liposomes, DiD-loaded liposomes and NIN-loaded liposomes (L-CLD-NIN and EL-CLD-NIN), the same lipid composition of the L-CLD or EL-CLD, and 2% C6, DiD or NIN were mixed in chloroform. Unentrapped dyes and NIN were removed by filtration through 0.22 μm membrane. Additionally, blank liposome and blank exosomes hybrid liposome (EL) were prepared in the same way.

2.2.3. Characterization

The size and surface charge of the exosomes and liposomes were determined by a Zetasizer (Nano ZS90, Malvern, UK) dynamic light scattering (DLS) instrument. Transmission electron microscopy (TEM, JEM-1400, JEOL, Japan), was used to observe the morphology of exosomes and liposomes. TEM samples were stained with 1% phosphotungstic acid before being dropped on a copper net. The concentration of CLD was determined by ultraviolet spectrophotometer (Evolution 220, Thermo Scientific, USA.) [11]. L-CLD was stored at 4 ◦C for 4 weeks, and particle size was monitored.

2.2.4. Elucidation of the hybridization mechanism

Hybridization of the liposome and exosome was verified by a fluorescence resonance energy transfer (FRET) study [24]. For FRET analysis, the liposome and FRET EL preparation, 2% (w/w) DOPC was replaced by PE-NBD acting as an electron donor and PE-RhB acting as an electron acceptor, in equal amounts. FRET samples were analyzed by fluorescence spectroscopy (F-4600, Hitachi, Japan) by exciting samples at 470 nm and measuring the emission spectra between 500 and 700 nm. FRET efficiency was calculated using the following equation: where Fa = emission fluorescence of acceptor (PE-RhB) and Fd = emission fluorescence of donor (PE-NBD).

2.3. In vitro studies

2.3.1. Cellular uptake

RAW 264.7 cells and L-929 cells were seeded in 24-well plates at densities of 1 × 104 cells/well in 1 mL of growth medium, respectively. After incubating for 24 h, the medium was refreshed with 500 μL of FBS- free medium containing C6-loaded liposomal solution (1 μg/mL) for 0.5 h or 4 h; fluorescence images were recorded immediately using a fluorescent microscope (Ti–S, Nikon, Japan). Five randomly selected microscopic fields were quantitatively analyzed using Image J software.

2.3.2. Cell viability assay

RAW 264.7 cells and L-929 cells were cultured at 5 × 103 cells per well in 96-well plates for 24 h, respectively. The medium was replaced with culture medium containing various concentrations of exosomes, ELs, and blank liposomes and cultures were incubated for 72 h. CLD, L- CLD and EL-CLD were incubated for 48 h. After incubation, the supernatant was discarded. Then 20 μL of MTT (5 mg/mL) solution was added to each well for 4 h, and absorbance was measured at 490 nm using a microplate reader (TecanSafire 2, Switzerland).

2.3.3. Nuclear staining by hoechst 33342

Nuclear morphology of RAW 264.7 cells and L-929 cells exposed to different treatments were evaluated using by Hoechst 33342 staining. Cells were seeded in 6-well plates at the density of 5 × 105 cells per well and cultured for 24 h. Cells were then incubated with medium containing free CLD or liposomes at a final CLD concentration of 20 μg/mL for RAW 264.7 cells, and 200 μg/mL for L-929 cells. After 24 h incubation, cells were then fixed with paraformaldehyde, stained with 10 μg/mL Hoechst 33342, and then photographed using a Nikon fluorescent microscope.

2.4. Biodistribution in a murine model of pulmonary fibrosis

Pulmonary fibrosis was induced by a single intratracheal injection of 2 mg/kg BLM solution in mice, whereas mice in the control group received saline intratracheally [25]. In detail, BLM was dissolved in saline solution at a concentration of 0.8 mg/mL. The BLM solution was loaded into a MicroSprayer™ aerosolizer (Huironghe Technology Co., Ltd. Beijing, China) and a dose of 50 μL was intratracheally sprayed into anesthetized mice. Body weight, micro-computed tomography (CT), and pulmonary function were monitored. In addition, the Ashcroft score, semi-quantitative alveolar air area, and collagen content of lung sections by Masson staining were also evaluated [26,27]. Three weeks post-BLM insult, the DiD-loaded liposome, EL and EL-CLD (CLD dose: 15 mg/kg) were intravenously injected into mice. The distribution of DiD-loaded liposomes in fibrotic mice and in major organs was detected by an IVIS kinetics optical system (PerkinElmer, USA.). Lungs and livers were fixed in 4% paraformaldehyde, then sectioned (5 μm) for immunofluorescence analysis.

2.5. Anti-fibrotic efficiency in vivo

The anti-fibrotic efficacies of NIN, L-NIN, EL-NIN, L-CLD-NIN, and EL-CLD-NIN were evaluated in the pulmonary fibrosis mouse model. Liposomes were administered via intravenous injection at a 3 mg/kg dose of NIN on days 1, 4, 8, and 11, respectively, and NIN was administered gastrointestinally, as it is an orally administered medication in the clinic. The weights of mice were monitored every other day. Three days after the last injection (day 14), mice were subjected to micro-CT to determine pulmonary function, and then blood samples were collected and centrifuged. Serum ALT, AST, CRE, TBIL, and BUN levels were measured according to International Federation of Clinical Chemistry (IFCC) primary reference procedures. Mice were then euthanized and their lungs and livers were harvested, weighed, and homogenized for ELISA. Lungs, livers, and other organs were also fixed in 4% paraformaldehyde, then sectioned for immunofluorescence, immunohistochemistry (IHC), hematoxylin and eosin (H&E) staining, Masson staining and TUNEL assay. Serum MMP-7 levels and Hyp levels in the lung were detected by ELISA using a commercial kit following the manufacturer’s instructions.

2.6. Micro-CT

Pulmonary fibrosis-induced mice were anesthetized and lung imaging was performed using a micro-CT scanner (NEMO NMC-100, PINGSENG, China), according to the manufacturers’ protocol. Images were analyzed using the three-dimensional finite element algorithms (AVATAR 1.5.0, PINGSENG, China).

2.7. Determination of pulmonary function

Mice were placed in the plethysmography chambers of a whole-body plethysmograph (WBP-4MR, TOW, China), after 30 min-acclimation in the cavity. Over a period of 20 min, unrestrained mice were monitored. Tidal volume, breath frequency, inspiration time (Ti), expiration time (Te), minute volume (MV), midexpiratory tidal flow (EF50), and Penh were determined by the software (ResMass 1.4.2., TOW, China). Mice were then anesthetized, tracheostomized, and placed in a forced pulmonary maneuver system (PFT, TOW, China). An average breathing frequency of 150 breaths/min was imposed on the mice. Forced vital capacity (FVC), inspiratory resistance (RI) and respiratory dynamic compliance (Cdyn) were recorded.

2.8. Tissue section staining

Immunofluorescence staining was performed on paraffin embedded tissue slices, by deparaffinization, antigen retrieval, permeabilization, and blocking in 5% goat serum albumin. Fluorescent-conjugated primary antibodies were incubated overnight at 4 ◦C. After staining nuclei with prolong diamond anti-fade mounting solution with DAPI for an additional 10 min. The sections were observed under fluorescent microscopy. Lung and liver tissue samples from pulmonary fibrotic mice after treatment were evaluated by H&E and Masson staining. The expressions of IL-1β and TGF-β in the lung were evaluated by IHC. The slides were observed using a Nikon microscope.

2.9. Statistical analysis

All results are expressed as the mean ± standard deviation (SD). The analysis of variance was performed using two-tailed paired Student’s t- test for comparison between two groups or non-parametric one-way analysis of variance (ANOVA) for comparison of three or more groups. A p-value less than 0.05 indicated a statistically difference.

3. Results and discussion

3.1. Preparation and characterization of EL-CLD

Isolation of exosomes by ultracentrifugation often contains impurities such as microvesicles [28,29]. Therefore, the term “exosome” in this study defines exosomes and small microvesicles with sizes below 200 nm. These vesicles are constituted of the skeleton of the phospholipid bilayer and can be pictured as a typical liposome [30]. In this study, exosomes were harvested from conditioned media of L-929 cells using ultracentrifugation and filtration. The isolated exosome population was initially characterized by TEM and DLS (Fig. 1A and B). TEM analysis showed a round-shaped morphology of exosomes with visible lipid layer. The hydrodynamic dimension of the exosome was found to be ~120 nm, with polydispersity index (PDI) of 0.22, and the ζ potential was − 10.8 mV. To further confirm the identity of exosomes, proteins were extracted from exosomes secreted by L-929 cells. The western blotting analysis confirmed the presence of exosomal marker proteins, such as CD9 and HSP70 [31,32]. Calnexin, a marker of the endoplasmic reticulum, was absent (Fig. 1C). The CLD-loaded liposome (L-CLD) was prepared by reverse evaporation technique to achieve a hydrodynamic diameter of ~89 nm, and a ζ potential of − 3.8 mV. The encapsulation efficiency of L-CLD was 70.6 ± 2.7%. After storage for four weeks at 4 ◦C, there were negligible changes in appearance, particle size, ζ potential, or encapsulation efficiency of L-CLD, indicating the liposomes possessed good stability.
The ratio of the liposome (based on lipid weights) to exosome (based on protein contents) was optimized to 5:1. A 0.2 mg protein equivalent of exosome and 1 mg lipid equivalent of liposome were dispersed in 1 mL PBS, and then extrusion resulted in the formation of the exosome hybridized liposome as shown in Fig. 1D. The hybridized EL-CLD exhibited a spherical structure with uniform particles and obvious bilayer membrane, with a mean diameter of ~125 nm (PDI 0.19) and ζ potential of − 7.1 mV (Fig. 1E and F). Compared to the exosomes and L- CLD (Fig. S1), the diameter of the EL-CLD hybrid was moderately larger, likely due to the insertion of exosomes into the L-CLD bilayer which increased its interaction with water molecules thereby increasing the hydration layers [30]. The reduction in the PDI of the EL-CLD hybrid indicated better homogeneity of the size distribution following extrusion through the 200 nm polycarbonate membrane. The ζ potential of EL-CLD fell between that of the L-CLD and exosome, and confirmed the successful hybridization of exosomes with liposomes. The efficiency of the exosome and liposome hybridization was investigated by FRET assay, a technique widely used to study membrane fusion [22,24]. The FRET liposome was prepared with fluorescence donor (PE-NBD) and a fluorescent acceptor (PE-RhB) as the building blocks of the liposome. Energy transfer can occur from donor to the acceptor when they are in close proximity, thus minimizing the donor energy. The energy transfer in the FRET liposome was monitored before and after hybridization as shown in Fig. 1G, and the calculated FRET efficiency is shown in Fig. 1H. Exosome infusion with the FRET liposome showed significantly diminished FRET activity, which is only possible when the distance between the two fluorophores increases. The results indicated the insertion of the exosome in the lipid bilayer of liposome, validating successful hybridization.

3.2. Dual properties of the EL-CLD hybrid on macrophages and fibroblasts in vitro

It has been reported that clodrolip is recognized and engulfed by macrophages, and thereby inhibits the survival of macrophages [33]. Thus, cellular uptake, cytotoxicity, and nuclear fluorescent morphology studies were performed to evaluate the depletion effects of EL-CLD on RAW 264.7 macrophages. C6 was employed to label the liposomes for the cellular uptake study. As shown in Fig. 2A and B, three groups of liposome all exhibited time-depended cellular internalization. The green fluorescence of the C6 in the blank liposome was stronger than in the CLD-loaded liposomes at the 4 h time point. Such results may be attributed to the depletion effect of CLD in liposomes. Exosome hybridization did not alter the CLD-loaded liposomes uptake by RAW 264.7 cells. EL-CLD and L-CLD also exhibited similar stronger cytotoxic effects than free CLD (Fig. 2C, p < 0.001). The IC50 values were 7.48 μg/mL, 6.09 μg/mL, and 85.3 μg/mL for EL-CLD, L-CLD, and CLD, respectively. The ability of the CLD-loaded liposomes to induce RAW 264.7 cell apoptosis was examined by nuclear staining (Fig. 2D). The nuclei of untreated RAW 264.7 cells showed homogenous fluorescence with no evidence of segmentation or fragmentation. Slight morphological changes were observed in the CLD group, while the nuclei of cells treated with L-CLD and EL-CLD induced more severe fragmentation, which could be attributed to the enhanced liposomal phagocytosis potency of the macrophage [34] and was in accordance with the results of in vitro cytotoxicity study.
The in vitro anti-fibrotic efficiency of EL-CLD was evaluated using L- 929 fibroblast cells. The effects of exosomes, EL, and the combined solution of exosomes and blank liposomes (1: 5) on cell viability were examined for 72 h. As shown in Fig. 2E, the cell viability after these treatments neither increased nor decreased. Although there have been reports of cancer cell- or cancer-associated fibroblasts derived-exosomes contributing to tumor proliferation and metastasis [35,36], the formulation of EL in this study showed good biocompatibility without inducing fibroblast growth. Cytotoxic effects of EL-CLD on L-929 cells were determined after a 48-h incubation. As shown in Fig. 2F, there was no significant difference in cell viability between CLD and L-CLD. While EL-CLD exhibited much stronger cytotoxicity than CLD and L-CLD (p < 0.001), with IC50 values decreasing ~2.2-fold. The L-929 cellular uptake of EL-CLD at 0.5 h and 4 h was ~1.3-fold higher than that of L-CLD (Fig. 2G and H, p < 0.001). The concentration of CLD in C6-loaded liposomes used in the cellular uptake study was merely 10 μg/mL. Therefore, the results of L-929 cellular uptake were not influenced by CLD in liposomes. The increased L-929 cellular accumulation of EL-CLD may be due to the homing ability of homologous exosomal hybridization. Moreover, EL-CLD treatment also induced more severe nuclear fragmentation than CLD and L-CLD, and further generation of apoptotic bodies (Fig. 2I). The enhanced L-929 cellular apoptosis and cytotoxicity of EL-CLD may be attributed to the enhanced cellular accumulation induced by hybridized with homologous cell derived exosomes. Although EL-CLD could cause L-929 cytotoxicity and apoptosis at higher concentrations, EL-CLD is still far from independent use as anti-fibrotic therapy. EL-CLD exerted different effects on L-929 and RAW 264.7 cells. RAW 264.7 macrophages uptake was not influenced by exosome hybridization, due to their much stronger phagocytic capacity [34,37]. While L-929 fibroblasts possessed weaker phagocytic ability compared to macrophages [38,39], resulting in a higher IC50 value than that of RAW 264.7. All of these in vitro results indicated that EL-CLD exhibited dual-functional potency on macrophage inhibition and fibroblast accumulation.

3.3. The tissue distribution of DiD-loaded liposomes in pulmonary fibrosis mice

A pulmonary fibrosis mouse model was established using a single, high-dose, intratracheal BLM injection in C57BL/6 mice. Body weight was monitored throughout the model establishment period. Although one week after intratracheal BLM injection, significant loss of body weight was observed, body weight stabilized after day 6 and resumed after day 8 (Fig. S2A). On day 22, the body weight of the BLM-treated mice remained lower than in the control group (p < 0.01). Micro-CT was used to identify the morphological hallmarks of pulmonary fibroblasts. Reticular opacities and honeycombing-like cysts with a spatially heterogeneous distribution were identified in the peripheral regions of the lung in BLM-treated mice (Fig. 3A). Fibrosis characterized by high levels of α-smooth muscle actin (α-SMA), was clearly distinct in lungs treated with saline solution (Fig. S2B). Masson staining and pulmonary function were used to stage fibrosis progression. On day 0, mice showed normal lung architecture with no fibrosis masses in the parenchyma (Fig. 3B). Single fibrotic masses were observed from day 7 and evolved into confluent conglomerates of substitutive collagen from day 14. It has been reported that an initial inflammatory phase transits to a more fibrogenic phase [40]. We observed significant incremental collagen deposition, thickened alveolar walls, damage of alveolar structure in lesion regions, and decreased alveolar air area in the BLM-treated mice, compared with those receiving saline treatment (Fig. 3B and Fig. S3, p < 0.001). The Ashcroft score was higher in BLM-treated mice throughout the three week-treatment duration than in the saline group (p < 0.001). Whole-body barometric plethysmography (WBP) was used to monitor pulmonary function during the course of pulmonary fibrosis development and progression. Tidal volume was maximal at day 7 but returned to within basal levels by day 21 (Fig. 3C). There was a significant difference between saline and BLM-treated mice for breathing frequency (p < 0.001, Fig. S4), and by day 7 MV began to recover. On day 7, the Ti, Te, and Ti/Te ratio were significantly different than those of the saline and BLM-treated mice (p < 0.05). Prior to treatment, mice had a symmetrical Ti/Te ratio close to 1.0, with Ti and Te of essentially equal duration. While the Ti/Te ratio of BLM-treated mice reduced to below 0.5 from day 7–21, indicating an asymmetric breathing cycle. Overall, these results showed that three weeks after the intratracheal BLM injection, serious alveolar epithelial damage and collagen deposition were induced and pulmonary function declined. Therefore, studies to evaluate effects on pulmonary fibrosis in the mouse model began three weeks after the intratracheal BLM injection in this study.
To investigate the biodistribution of EL-CLD in the pulmonary fibrosis mouse model, DiD dye was loaded in liposomes, and the liposomal accumulation, penetration, and distribution in major organs were studied. At the 24 h time point after injection, pulmonary DiD signals in the EL and EL-CLD groups were 1.8- and 3.4-fold stronger than in the blank liposome (p < 0.01, Fig. 3D–F). Although the EPR effect has previously been reported in inflammation-related diseases, including fibrosis [3], the liposome distribution to fibrotic pulmonary tissue by a passive targeting effect was still modest. The high density of crosslinked ECM constitute an obstacle for liposomal delivery and further limit their penetration [41]. By means of fibroblast cell-derived exosomes hybridization, greater amounts of liposomes could accumulate in the fibrotic lung tissue. Therefore, the pulmonary DiD signals were still strong in the EL-CLD group both in vivo and ex vivo even 48 h after injection, while the lung accumulation of DiD signals in the EL group obviously decreased (Fig. 3E left and F). EL-CLD exhibited obviously weaker hepatic and splenic accumulation at the 24-h and 48-h time points (p < 0.001, Fig. 3F and Fig. S5). The results clearly indicated that CLD-loaded liposomes had the advantage of decreased deposition in filtration organs, liver and spleen, where a large amount of macrophages reside.
It has been reported that 48 h post-intravenous injection, clodrolip can deplete ~98% of Kupffer cells, hence subsequent nanoparticles can achieve reduced liver accumulation [42]. A single administration of EL-CLD also effectively inhibited liver accumulation, with a correspondingly enhanced pulmonary accumulation, even so, most of the first dose of the liposomes entered and were deposited in the liver, which probably had an impact on their distribution to the lung. Therefore, a second dose of DiD-loaded liposomes was administered 48 h after the first dose, and was expected to improve pulmonary accumulation based on the Kupffer cells depletion. As shown in Fig. 3E (right) and Fig. 3F, the second dose in the EL-CLD group exhibited stronger DiD fluorescence in the lung at both 24 h and 48 h time points, with 1.3-fold and 1.7-fold higher fluorescence intensity than that of first dose EL-CLD group, respectively. A second dosage of EL also achieved enhanced pulmonary accumulation by virtue of the homing affinity of exosomes to the fibrotic lung. Conversely, a second dose of EL-CLD significantly reduced hepatic accumulation by 1.5-fold, while hepatic accumulation showed no differences between the first and second doses in other groups. Staining of liver and lung sections with DAPI were also examined, and the largest DiD fluorescence areas were identified in the EL-CLD group (Fig. 3G). Overall, the biodistribution results demonstrated that EL-CLD could effectively reduce hepatic uptake and enhance accumulation in the fibrotic lung, due to the dual effects of macrophage inhibition by CLD and fibroblast homing of the exosome component of the EL-CLD hybrid.

3.4. EL-CLD distribution in liver and lung tissue of pulmonary fibrosis mice

DiD-loaded liposome distribution in the liver and lung of pulmonary fibrosis mice was further evaluated. Immunofluorescence staining of liver sections was performed (Fig. 4A). Most DiD red fluorescence in the blank liposome and EL groups was localized in the same field as the green fluorescence marking F4/80-positive macrophages, indicating that the liposomes deposited in the liver were phagocytized by Kupffer cells. Instead, fewer F4/80-positive cells were detected in the EL-CLD group and these did not colocalize with DiD fluorescence, which suggested that the EL-CLD hybrid induced depletion of Kupffer cells and the EL-CLD hybrid was not deposited in the liver but rather was distributed outside of Kupffer cells. The CLD dose of 15 mg/kg used in our work was lower than that used in previous studies. It has been reported that following exposure to CLD-loaded liposomes (40 mg/kg), approximately 98% of Kupffer cells were depleted on day 2, which then reappeared on day 5, and recovered to normal levels on day 14 [42]. A limited degree of liver toxicity was verified using histological sections and hematological parameters. Therefore, the lower dose of CLD used in our study would not interfere with Kupffer cell function in the liver nor with the body’s defense and immunity.
Macrophages in the liver were depleted to a large extent, inducing lesser EL-CLD hepatic accumulation and more pulmonary accumulation. Further details of liposomal accumulation and penetration in fibrotic pulmonary tissue were revealed by co-localization with fibrosis sites and blood vessels in the lung. Fibrosis present in lung sections was stained by a green fluorescence-labeled α-SMA antibody. EL displayed greater fibrotic accumulation than the blank liposome, and the EL-CLD group exhibited the largest areas of overlapping yellow fluorescence, indicating that more EL-CLD could accumulate at the fibrotic site than the other treatments (Fig. 4B), which is an advantage for fibrosis treatment. Furthermore, neither accumulation of SPC-positive alveolar epithelial cells II nor of CD68-positive pulmonary macrophages was observed in either of the three liposomal groups (Fig. S6). These results demonstrated that EL-CLD could penetrate deeply into pulmonary fibrosis sites by means of enhanced distribution in pulmonary fibrosis tissue and exosome homing to the fibroblast.
Blood vessels in the lung were labeled by a CD31-fluorescent antibody to analyze the penetration of EL-CLD. The fluorescence of the DiD- loaded blank liposome was largely associated with the green fluorescence visible in blood vessels (Fig. 4C). DiD fluorescence could be detected within 20 μm from the vessel (Fig. 4D), indicating the limited penetration in fibrotic tissue. EL merely increased liposomal penetration up to ~30 μm, indicating exosomes hybridization exerted positive effects on promoting liposomal penetration. Specific membrane proteins served as the exosome-ligand and directed hybridized liposome homing to the homologous cell, which resulted in deeper penetration [20]. In marked contrast, when treated with EL-CLD, DiD signals in the lung became much stronger and more diffuse, and penetrated into regions distant more than 50 μm from the blood vessels, indicating a greatly enhanced pulmonary accumulation and enhanced interstitial penetration. Three main reasons may explain this result: (1) increased lung accumulation with the EL-CLD hybrid due to combination of Kupffer cell depletion and the EPR effect in fibrosis; (2) enhanced vascular permeability of EL-CLD due to inhibition of proliferative and angiogenic activity of fibroblast growth factor 2 (FGF-2) [43]; and (3) enhanced distribution in lung fibrosis due to the inhibition of pulmonary macrophages. The enhanced accumulation in pulmonary fibrotic tissue and interstitial penetration of EL-CLD suggested the possibility of using EL-CLD for more effective pulmonary fibrosis drug delivery.

3.5. Effects of the NIN-loaded EL-CLD hybrid system on pulmonary fibrosis therapy

Considering the favorable pulmonary fibrotic accumulation and penetration characteristics of the EL-CLD hybrid, it can be expected to function as a drug delivery system with enhanced efficiency of delivery of anti-fibrotic agents. NIN is a triple tyrosine kinase receptor inhibitor that targets the platelet-derived growth factor, FGF-2, and endothelial vascular growth factor receptors. NIN is used clinically to treat pulmonary fibrosis. However, the tissue or cellular distribution of NIN is non- specific and side-effects such as diarrhea and nausea were reported to occur frequently during treatment [44]. In this study, NIN was selected as a model drug and was loaded in the EL-CLD hybrid (EL-CLD-NIN), to determine whether this system could improve its anti-fibrotic efficiency further. The EL-CLD-NIN hybrid system was 119 nm in diameter and possessed a − 8.2 mV surface charge, which was the same as that of the empty EL-CLD hybrid. The NIN encapsulation efficiency was ~90%. The anti-fibrotic efficacy of the EL-CLD-NIN hybrid system was investigated in the pulmonary fibrosis mouse model. Three days after the fourth administration, micro-CT scan images of NIN, L-NIN, EL-NIN, L-CLD-NIN, and EL-CLD-NIN used to treat pulmonary fibrotic mice are shown in Fig. 5A and Fig. S7. The lungs of mice treated with PBS, free NIN, L-NIN, EL-NIN, and L-CLD-NIN showed obvious increased signals of fibrotic tissue density, accompanied by severe interstitial septal and bronchial wall thickening with honeycomb appearance, and focal fibrotic nodules on pathology assessment. Instead, in mice treated with the EL-CLD-NIN hybrid system, the lung showed a significant reduction in high density areas and a significant improvement on pathology assessment, indicating the severity of fibrosis was greatly alleviated. The Lung Index is the ratio of lung weight to the body weight, and is an important indicator of the degree of pulmonary injury. In the pulmonary fibrosis mouse model, the lung mass increased mainly due to cell swelling, capillary congestion, and collagen deposition; while the body weight declined due to the state of the disease, which directly contributed to an increase in the Lung Index [45]. As shown in Fig. 5B, the Lung Index values of NIN-treated groups were significantly lower than in the PBS-treated group. Among the liposomal treatment groups, EL-CLD-NIN exhibited the lowest Lung Index and showed no differences compared to healthy mice. The lung morphology of the EL-CLD-NIN group was similar to that of the healthy lung with less inflammatory edema and collagen deposits.
Multiple pulmonary function tests were carried out using WBP and PFT to evaluate the impact of treatments on the treated mice (Fig. 5C and Fig. S8). Pulmonary function was improved by the EL-NIN, L-CLD- NIN, or EL-CLD-NIN treatment, compared to treatment with free NIN and L-NIN, with a significant decline of Penh and RI (p < 0.01) and rise in the EF50, Ti/Te ratio, MV, FVC, and Cdyn (p < 0.01), which indicated that airway obstruction decreased and bronchoconstriction and compliance improved. Compared to all other liposomal treatments, the EL-CLD-NIN hybrid system resulted in sharp improvement in multiple pulmonary functions (p < 0.01). Respiratory function showed significant recovery following EL-CLD-NIN treatment, and also alleviated the persistent lung damage caused by fibrosis, resulting in reduced tissue stiffness and elastic recoil. EL-CLD-NIN treatment also reduced fibrosis by preserving alveolar epithelial structures and reduced collagen deposition (Fig. 5D–H). Collagen content, alveolar air area, and the Ashcroft score of Masson staining all revealed that EL-CLD-NIN was able to reduce the fibrotic area and reverse the alveolar epithelial damage back to a nearly healthy level. Major biomarkers of pulmonary fibrosis, Hyp and MMP-7 levels, were also decreased after treatment with EL- CLD-NIN compared to other treatments (Fig. 5I and J), suggesting that EL-CLD-NIN had the ability to diminish ECM deposition and retard fibrotic progression. The development of pulmonary fibrosis ultimately
Liver sections (A-C) and lung sections (D-I) of mice with pulmonary fibrosis after the indicated treatments. (A) Immunofluorescence double staining for F4/80- positive macrophages (red), TUNEL (green) and apoptotic macrophages (yellow). (B) Quantitation of TUNEL-positive cells and F4/80-positive macrophages. (C) Quantitation of apoptotic macrophages expressed as the percentage of total apoptotic cells and macrophages. (D) Immunofluorescence double staining for CD68- positive macrophages (red), TUNEL-positive (green) and apoptotic macrophages (yellow). (E) Quantitation of TUNEL-positive cells and CD68-positive macrophages. (F) Quantitation of apoptotic macrophages expressed as the percentage of total apoptotic cells and macrophages, *p < 0.05, **p < 0.01, ***p < 0.001. (G) IL- 1β immunohistochemistry. (H) TGF-β immunohistochemistry. (I) α-SMA immunofluorescence, *p < 0.001, vs. the PBS group, #p < 0.05, vs. the NIN group; $ p < 0.001, vs. the EL-NIN group. n = 5, bar: 50 μm. causes acute respiratory failure and leads to death. The median survival time of pulmonary fibrosis mice treated with PBS was only 36 days, and the survival rate within 60 days after intratracheal BLM injection was 12.5% (Fig. 5K). While the EL-CLD-NIN therapy notably prolonged survival with 87.5% survival at 60 days, only 25% survival was observed in the L-NIN treated group (p < 0.001). Overall, these results fully supported the application of EL-CLD as a drug delivery system to enhance the efficiency of NIN in pulmonary anti-fibrotic therapy.

3.6. Mechanisms of the EL-CLD-NIN hybrid system as pulmonary anti- fibrotic therapy

The mechanism involved in the improved anti-fibrotic efficacy of NIN using the EL-CLD hybrid delivery system was investigated. Apoptosis of liver macrophages was evaluated by double-staining with TUNEL assay and the F4/80 antibody (Fig. 6A–C). The results showed that EL-CLD-NIN and L-CLD-NIN treatments caused a dramatic increase in the F4/80-positive macrophage apoptosis rate compared to NIN treatment without the CLD formulation (p < 0.001). The EL-NIN treatment induced ~2% apoptosis in the liver, which was less than that observed with L-NIN treatment. We concluded that the increased macrophage apoptosis may be attributed to the macrophage-depletion effect of the CLD by the liposomes, and the decreased apoptosis of the EL-NIN treatment could be attributed to fibroblast-derived exosomal hybridization, which tended to localize mainly at the pulmonary fibrotic sites. Kupffer cells in the liver and spleen sequester up to 90% of systemically injected nanoparticles, hence become the major barrier for site-specific delivery to sites characterized by pulmonary fibrosis [46]. Given the depletion of Kupffer cells, additional EL-CLD-NIN is available to reach pulmonary fibrotic tissue, and the exosomal homing effect would further increase of liposomal accumulation and penetration into fibroblasts. Furthermore, approximately 86% of apoptotic cells in the CLD-loaded liposomal groups were F4/80 positive, which was 84% higher than those in non-CLD-loaded liposomes. The results indicated that most of the CLD-loaded liposomes were phagocytosed by Kupffer cells, thereby reducing the toxicity to normal liver tissue. All treatments induced neither obvious histological abnormalities on H&E staining of liver sections nor abnormal changes in serum ALT, AST, and TBIL levels, which suggested that CLD and NIN caused no obvious damage to hepatic function (Fig. S9). The slight increase in ALT and AST levels observed in L-CLD-NIN and EL-CLD-NIN groups may have resulted from Kupffer cell inhibition, which decreased the clearance of hepatic enzymes [47]. Moreover, a steady growth of body weight was observed during the EL-CLD-NIN treatment (Fig. S10). Overall, these results strongly confirmed the excellent macrophage depletion potency and safety of the EL-CLD-NIN hybrid system.
Pulmonary macrophage apoptosis after EL-CLD-NIN treatment was also evaluated (Fig. 6D–F). CLD-loaded liposomes induced more macrophage apoptosis than other groups (p < 0.001), similar results were observed in the liver. EL-CLD-NIN induced apoptosis in 39% of CD68-positive macrophage, which was less than that induced by treatment with L-CLD-NIN and was likely due to the exosomal homing effect causing decreased EL-CLD-NIN uptake by pulmonary macrophages. Notably, more than 5% of cells were apoptotic after CLD-loaded liposomes treatment, among which more than 53% apoptotic cells were CD68-positive macrophages. This difference suggested that pulmonary macrophages had a greater tendency to be depleted than normal lung cells or fibroblasts. Macrophages reportedly serve as a central contributor to recruiting inflammatory cells and initiating fibrosis via the production of various cytokines. Among the numerous fibrogenic factors, IL-1β and TGF-β are pivotal molecules involved in fibroblast proliferation, differentiation, migration, and aberrant ECM deposition with higher levels of α-SMA [48,49]. Immunohistochemical analysis of fibrotic lung sections showed that EL-CLD-NIN treatment significantly suppressed the secretion of the proinflammatory cytokine IL-1β (Fig. 6G). The anti-inflammatory properties of EL-CLD-NIN are likely to be the result of the capacity for macrophage depletion induced by the EL-CLD delivery system. TGF-β expression in the lung after different treatments is shown in Fig. 6H. Compared to other treatments, EL-CLD-NIN and L-CLD-NIN treatment significantly decreased the expression of TGF-β, indicating that CLD in liposomes had the capacity to reduce fibrosis. The expression of the crucial biomarker of ECM deposition, α-SMA, decreased after treatment with different NIN formulations in fibrotic pulmonary tissue (Fig. 6I). Both L-NIN and EL-NIN did not show any superiority over free NIN, as NIN could target crucial tyrosine kinase receptors on the membrane of the fibroblast, while most L-NIN and EL-NIN were likely be deposited in the liver as revealed by the tissue distribution studies. CLD-loaded NIN liposomes exhibited stronger potency on fibroblast suppression, especially by the exosomal hybrids. The pulmonary immunofluorescence results suggested that the EL-CLD drug delivery system could achieve obvious pulmonary macrophage depletion, anti-inflammatory effects, and enhanced anti-fibrotic effects by increased exposure to NIN.

4. Conclusion

In summary, the EL-CLD hybrid was explored as a potential approach to depletion of Kupffer cells and enhanced drug delivery to pulmonary fibrotic lesions. The EL-CLD hybrid was prepared by combining hybridized fibroblast-derived exosomes with a CLD-loaded liposome, with a particle size of approximately 125 nm. In in vitro studies, EL-CLD was effective at depletion of macrophages by virtue of CLD and affinity to fibroblasts presumably due to the homologous exosome formulation. In contrast to the reduced liver distribution, the DiD-loaded EL-CLD accumulation and penetration in pulmonary fibrosis was more significant and showed greater co-localization with fibrotic tissue. The EL-CLD hybrid further enhanced the suppressive effect of NIN on fibrosis, and improved the efficacy of pulmonary fibrosis treatment. Kupffer cell- depletion and homologous affinity by CLD and fibroblast-derived exosomes in EL-CLD significantly enhanced pulmonary fibrotic drug delivery. Further, the pulmonary macrophage-depletion suppressed the release of fibrogenic cytokines, and promoted the anti-fibrotic effects of NIN. Hence, the EL-CLD hybrid system developed in this study may become a promising fibrotic-specific drug delivery platform for anti- fibrotic treatment of pulmonary tissue.

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