Cationic nanoparticles directly bind angiotensin-converting enzyme 2 and induce acute lung injury in mice
- Yang Sun†1,
- Feng Guo†1,
- Zhen Zou†1,
- Chenggang Li†1,
- Xiaoxu Hong†1, 2, 3,
- Yan Zhao†1,
- Chenxuan Wang4,
- Hongliang Wang1,
- Haolin Liu1,
- Peng Yang1,
- Zongsheng Han1,
- Kangtai Liu1,
- Keiji Kuba5,
- Bin Song1,
- Jinming Gao6,
- Ziyao Mo7,
- Dangsheng Li8,
- Bo Li9,
- Qihan Li2,
- Nanshan Zhong7,
- Chen Wang4,
- Josef M Penninger5Email author and
- Chengyu Jiang1, 6, 10Email author
© Sun et al.; licensee BioMed Central. 2015
Received: 3 August 2014
Accepted: 27 January 2015
Published: 7 March 2015
Nanoparticles have become a key technology in multiple industries. However, there are growing reports of the toxicity of nanomaterials to humans. In particular, nanomaterials have been linked to lung diseases. The molecular mechanisms of nanoparticle toxicity are largely unexplored.
Acute lung injury was induced in wild-type mice and angiotensin-coverting enzyme 2 (ACE2) knockout mice by the intratracheal instillation of cationic polyamidoamine dendrimer (PAMAM) nanoparticles. For rescue experiments, losartan (15 mg/kg in PBS) was injected intraperitoneally 30 min before nanoparticle administration.
Some PAMAM nanoparticles, but not anionic PAMAM nanoparticles or carbon nanotubes, triggered acute lung failure in mice. Mechanistically, cationic nanoparticles can directly bind ACE2, decrease its activity and down-regulate its expression level in lung tissue, resulting in deregulation of the renin-angiotensin system. Gene inactivation of Ace2 can exacerbate lung injury. Importantly, the administration of losartan, which is an angiotensin II type I receptor antagonist, can ameliorate PAMAM nanoparticle-induced lung injury.
Our data provide molecular insight into PAMAM nanoparticle-induced lung injury and suggest potential therapeutic and screening strategies to address the safety of nanomaterials.
Nanoparticles have become key materials in the pharmaceutical, information, and communication industries as well as in multiple other areas that require strong, light materials . However, several concerns have been raised about the safety of the widespread use of nanoparticles [2,3]. Among these concerns, the potential toxicity of nanoparticles to humans is among the most distressing. Nanomaterials have been reported to be harmful at the cellular, subcellular, and protein levels, and they have been found to elicit injurious responses in various organisms [4-6]. Many of these studies have focused on lung diseases, including clinical studies of workers who developed pulmonary disease from nanoparticle exposure [7-11]. A worldwide moratorium on nanomaterials has even been called until the safety issues have been resolved . Nanoparticle safety in the respiratory system is important not only for workers who have high exposure to the particles but also in pulmonary drugs where nanoparticles serve as a vehicle directly to the lung . Therefore, it is of critical importance to elucidate the molecular mechanisms by which nanoparticles induce lung injury.
Polyamidoamine (PAMAM) dendrimers are a family of dendritic polymers that are based on an ethylenediamine core and repeated amidoamine branching. As PAMAM dendrimers grow through generations 1–10 (G1-G10, G represents the generation of the dendrimers, which are based on an ethylenediamine core and repeated amidoamine branching), their size increases from 1.1–12.4 nm [14,15]. PAMAM dendrimers are commercially available as either whole (cationic) or half (anionic) generation polymers. The low polydispersity index and the ability to precisely control their surface chemistry make dendrimers useful for biomedicine applications such as drug or gene delivery and diagnostic imaging [6,14]. For example, PAMAM dendrimers can be used to inhibit inflammation , improve therapeutic responses against cancer , and suppress HIV-1 infection . In addition, PAMAM dendrimers can also be used as nanocarriers for pulmonary drug delivery [19,20] or as an effective vector for pulmonary gene transfer . Because many PAMAM nanoparticles have been considered carriers for pulmonary drug delivery, in addition, a study reported that the PAMAM can be found in major organs and have highest levels in lungs in B16 melanoma and DU145 human prostate cancer mouse tumor model , the safety of PAMAM nanoparticles should be under rigorous assessment and evaluation .
Previous reports have indicated that PAMAM dendrimers are toxic, and their toxicity is significantly influenced by size and surface charge . Specifically, the charge of nanoparticles can also affect the oral toxicity of PAMAM dendrimers . Furthermore, PAMAM dendrimers have been demonstrated to aggressively initiate blood clot formation and disrupt key platelet functions [26,27]. Although the systemic and oral toxicity of PAMAM nanoparticles has been investigated, there are few studies that have focused on the safety of PAMAM nanoparticles in the respiratory system. Considering the potential biomedical applications of PAMAM nanoparticles, especially the applications in pulmonary disease, it is paramount to investigate the underlying molecular mechanisms that may be involved in PAMAM-induced lung injury in vivo.
G5 of PAMAM nanoparticles induce acute lung injury in mice
The result of acute toxicity of G5 PAMAM nanoparticles in mice
Number of animals
We first examined the survival rate of mice after the administration of G5 PAMAM dendrimers to the lungs. Almost all mice treated with G5 PAMAM nanoparticle died within 48 hrs, whereas all control mice and mice instilled with anionic G5.5 PAMAM dendrimers survived (Figure 1B). The cause of death was rapid development of severe lung injury characterized by decreased blood oxygenation (Figure 1C), increased lung tissue wet-to-dry weight ratios (Figure 1D), and massively altered elastance (Figure 1E). (Lung elastance is a measure of lung function, i.e., the change in pressure per unit change in volume, and indicates the stiffness of the lungs .) The histopathology of G5 PAMAM-treated lungs showed increased bleeding and inflammatory cell infiltration (Figure 1F). Moreover, G5 PAMAM administration resulted in increased vascular permeability (Figure 1G), another hallmark of acute lung injury/respiratory distress syndrome in humans [31,32]. These results indicate that the inhalation of G5 PAMAM nanoparticles induces acute lung injury in mice, resulting in pathologies that resemble acute respiratory distress syndrome in humans.
ACE2 controls G5 PAMAM nanoparticle-induced lung injury
To further elucidate the mechanism of ACE2 down-regulation, we first explored whether the loss of ACE2 activity was caused by the direct binding of ACE2 to nanoparticles. We assessed the binding affinity of ACE2 for various nanoparticles using surface plasmon resonance (SPR). Interestingly, most cationic PAMAMs could bind ACE2 protein directly, whereas the anionic PAMAMs failed to bind ACE2 (Figure 2D, Additional file 3: Figure S2). This is predictable since the theoretical isoelectric point of protein ACE2 calculated by Expasy (http://www.expasy.org) is 5.36 and the SPR assay is performed in neutral pH. The dynamic binding constants and equilibrium dissociation constants for the binding between the examined nanoparticles and ACE2 were also measured, demonstrating that G5 PAMAM nanoparticles, in particular, strongly bind to ACE2 (Additional file 4: Table S2). We next monitored ACE2 enzyme activity by measuring AngII levels after mixing recombinant ACE2 protein and various nanoparticles in vitro. The enzymatic activity of ACE2 was decreased after incubation with cationic G5 PAMAM nanoparticles, but its activity was not affected after incubation with anionic G5.5 PAMAM in vitro (Figure 2E). These data show that G5 PAMAM nanoparticles can directly associate with ACE2 and alter its enzymatic activity, thereby resulting in activation of the RAS.
Losartan alleviates G5 PAMAM nanoparticle-induced lung injury in mice
Since G5 PAMAM dendrimers can down-regulate the expression of ACE2 in mRNA and protein levels significantly after 3 hrs of instillation (Figure 2 B,C), and death of mice occurred about 12 hours after instillation of PAMAM G5 (Figure 1B), we choose time-point 10 hrs after instillation of PAMAMs to evaluate the change of lung function. Our data provide in vivo molecular insight into the acute lung failure that can be induced by nanomaterials. Our data indicate that G5 PAMAM dendrimers down-regulate ACE2 expression in lung tissue, resulting in increased AngII production and, consequently, acute lung failure. Our SPR data show that PAMAM G5 and protein ACE2 have the highest binding affinity (Additional file 4: Table S2), however, PAMAM G7 have the most amino surface groups (Additional file 1: Table S1), suggesting that the nanoparticles surface charge is not the only determinant. Further studies of conformation characteristic and stability of PAMAM G5-ACE2 complex are needed.
In our study, PAMAM nanoparticles of G4-G7 could induce lung edema (Figure 1A), whereas lower generation cationic and anionic PAMAM dendrimers exhibited no acute lung toxicity. This result is consistent with previous findings that state nanoparticle size significantly influences the toxicology of PAMAM nanoparticles . It is interesting to note that not all nanoparticles have such effects on the ACE2 and AngII levels; for example, G5.5 PAMAM dendrimers appear to be less toxic than G5 PAMAM dendrimers. Additional studies regarding the entry of nanoparticles into pulmonary epithelial cells and/or local resident macrophages are required to explain these differences. Nevertheless, minor modifications of nanomaterials appear to significantly reduce their pulmonary toxicity, suggesting a possible approach to enable a safer use of nanomaterials. We have previously shown that G3-8 cationic PAMAM nanoparticles, COOH single-walled carbon nanotube, and copper oxide nanoparticles induce autophagy in vitro in A549 cell models or in vivo in mouse models [9,39,40]. Thus, the severity of the lung injury triggered by PAMAM nanoparticles may be due to the activation of multiple signaling pathways that collectively cause severe lung disease. A previous study showed that AngII could increase autophagy levels via activation of the AT1 receptor and NADPH oxidase in vascular muscle cells ; whether this signaling transduction pathway is involved in the pathogenesis of lung failure induced by G5 PAMAM nanoparticles needs further study. Our new data now clearly establish that ACE2 and the RAS play key roles in G5 PAMAM nanoparticle-induced lung injury. Interestingly, these mechanisms also apply in severe acute lung injury associated with acid aspiration, sepsis, or infection with SARS-CoV, H5N1 and H7N9 influenza [33-35,42]. Thus, these parallel mechanisms suggest that ACE2 and RAS share a common disease mechanism for acute lung injury and respiratory distress syndrome. Modulating the RAS may have a wide range of therapeutic applications.
The development of nanotechnology has rapidly accelerated, with an estimated market of approximately one trillion US dollars within the next few years . Safety problems associated with nanomaterials have recently attracted attention, highlighting the urgent need for protocols that protect workers, consumers, and the environment . We propose here a valuable strategy using ARB drug to address potential acute lung injury might be induced by application of PAMAMs.
We show that cationic PAMAM dendrimer nanoparticles can trigger acute lung failure in mice. Mechanistically, cationic nanoparticles bind to ACE2 and down-regulate its function and expression in lung tissue, resulting in deregulation of the RAS. Importantly, the administration of losartan ameliorated the nanoparticle-induced lung injury.
Our new insight into the molecular pathogenesis of cationic PAMAM nanoparticle-induced lung failure might therefore contribute to a better understanding of nanoparticle toxicity and the development of improved safety procedures for nanomaterials. Because ARB drugs have a long historical application for hypertension, our results not only uncover an important pathway of cationic PAMAM nanoparticle-induced lung toxicity, but may allow translation of our findings to novel therapies using classical ARB drugs.
All PAMAM dendrimers, carbon nanotubes, and inorganic nanoparticle powders were purchased from Sigma-Aldrich PAMAM nanoparticles were air-dried on a super-clean bench for 24 hrs to remove the methanol before an equal volume of PBS was added to the tube. The samples were mixed and stored at 4°C before use. The anti-ACE antibody (2E2) was purchased from Santa Cruz Biotechnology, Inc. The anti-ACE2 antibody (clone 460502) and recombinant human ACE2 protein were purchased from R&D systems. The anti-β-actin antibody (clone AC-15), angiotensin II (human), losartan and Evans blue were purchased from Sigma-Aldrich. HS-PEG-COOH (Mw = 2000 Da), HS-PEG-NH2 (Mw = 2000 Da), and HS-(CH2)11-EG6-OH (Mw = 468 Da) were purchased from Shanghai Yare Biotechnology, Inc. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), ethanol, NaOH, and ethanolamine were purchased from Sigma-Aldrich. All these reagents were used without further purification. Milli-Q water was obtained from a Millipore − ELIX water purification system. All buffers and reagents used were degassed and filtered prior to use in the SPR experiments.
All mice had a C57BL/6 J background. The development of the Ace2-knockout mouse has been previously described . C57BL/6 J mice were purchased from Vital River, Beijing. All mice were 4–6 weeks old. All animal experiments were conducted in the animal facility at the Institute of Basic Medical Sciences of the Peking Union Medical College in accordance with local and institutional guidelines.
Induction of lung injury
Lung injury was induced in mice by the intratracheal instillation of nanoparticles at a concentration of 15 μg/g using a technology that we have previously described for SARS spike protein- and acid-induced lung injury . For rescue experiments, losartan (15 mg/kg in PBS) was injected intraperitoneally 30 min before nanoparticle administration.
Acute toxicity study
A total of 48 male mice were divided into six groups of six mice each and randomly allotted to the control and G5 PAMAM nanoparticle groups. PBS was used in Group I as a control. The mice in Groups II, III, IV, V and VI were intratracheally injected with G5 PAMAM nanoparticles with the concentration of 7.35, 10.5, 15, 21.43, 30.61 μg/g, respectively. The animals were observed for 48 hrs after administration with nanoparticles and the mortality rate was recorded. The LD50 value (95% CL) was calculated using the improved Karber's method. The LD50 was 11.22 μg/g and the 95% confidence limit was 9.89-12.74 μg/g.
Assessment of pulmonary edema
Mice were instilled intratracheally with nanoparticles at a concentration of 15 μg/g before assessing the extent of pulmonary edema 10 hrs later when the lung wet-to-dry weight ratios were calculated. In brief, after the blood was drained from the excised lungs, the wet weight of the lungs was measured. Lungs were then heated to 68°C in an oven for 24 hrs and weighed to determine the baseline dry mass of the lungs.
Mice were intratracheally injected with either the vehicle or nanoparticles (15 μg/g). The survival of each group was recorded for 48 hrs and plotted using Kaplan-Meier survival curves. In the rescue group, losartan was intraperitoneally injected 30 min before nanoparticle administration.
Assessment of blood oxygenation
Mice were intratracheally injected with nanoparticles at a concentration of 15 μg/g. Ten hours later, blood samples were obtained from the common carotid artery, and the PaO2 was measured (Radiometer ABL720 blood gas analyzer, Diamond Diagnostics-USA) to assess arterial blood oxygenation as an indicator of respiratory failure.
Mice were anesthetized with sodium pentobarbital, and then either the vehicle (control) or nanoparticles (15 μg/g) dissolved in the same vehicle were administered intratracheally. Lung elastance was then measured by BUXCO Pulmonary Function Testing (PFT) every 30 min during spontaneous breathing periods for 4 hrs. In the rescue group, losartan (15 mg/kg) was intraperitoneally injected 30 min before nanoparticle administration.
Ten hours after the administration of nanoparticles (15 μg/g), mice were sacrificed and their body weights determined. The lungs were removed from the thoracic cavity and placed in a glass vial containing approximately 50 mL of fixative. Each glass vial was assigned a number unknown to the pathologists. The lungs were fixed for at least 48 hrs in formalin, embedded in paraffin, thin-sectioned coronally, and mounted on glass slides using standard histopathological techniques. The sections were stained with hematoxylin and eosin. For each mouse, lung sections were examined independently by three pathologists.
Pulmonary vascular permeability
Following the induction of lung injury, pulmonary vascular permeability was assessed by measuring the pulmonary extravasation of Evans blue. Evans blue (20 μg/g) was injected into the tail vein 10 hrs after the intratracheal injection of nanoparticles. Ten minutes later, the animals were sacrificed and their lungs were removed from the thoracic cavity and perfused with ice-cold PBS via injection into the right ventricle. Then, the lung tissue was immersed in formamide and analyzed to determine the Evans blue content. The OD value of the Evans blue formamide solution was read using a microplate spectrophotometer at 620 nm. A standard concentration regression curve was created and used to determine the level of Evans blue in the lung tissue.
Angiotensin II peptide levels
Mice were intratracheally injected with nanoparticles (15 μg/g) and blood was collected 3 hrs later into 1.5 mL tubes containing a mixture of peptidase inhibitors (0.30 M ethylenediaminetetraacetic acid, 0.32 M dimercaprol, and 0.34 M 8-hydroxyquinoline sulfate). Radioimmunoassays to determine the levels of angiotensin peptide in the plasma extracts were performed as described previously . In the rescue group, losartan (15 mg/kg) was intraperitoneally injected 30 min before nanoparticle administration.
Real-time quantitative PCR analysis
For real-time quantitative PCR, lung tissue was obtained 3 hrs after the intratracheal injection of the nanoparticles (15 μg/g) and then homogenized using TRIzol reagent (Invitrogen). The RNA was extracted according to manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 1.5 μg of total RNA with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR amplification assays were performed with the LightCycler 480 SYBR Green I Master (Roche Applied Science) on a LightCycler 480 PCR System (Roche Applied Science). PCR products were confirmed by sequencing. Lung tissue samples were normalized to β-actin levels. The specific primers used were listed as follows:
mouse β-actin forward primer: 5'- CTCTCCCTCACGCCATCC-3'
mouse β-actin reverse primer: 5'- CGCACGATTTCCCTCTCAG-3'
mACE2 forward primer: 5'- GGCTACAACTATAACCGTAAC-3'
mACE2 reverse primer: 5'- TATCCATCAACTTCCTCCTC-3'.
Lung samples were obtained 3 hrs after the intratracheal injection of the nanoparticles (15 μg/g) and homogenized in ice-cold lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1.0% Triton X-100, 20 mM EDTA, 1 mM Na3VO4, 1 mM NaF, and protease inhibitors). Tissue lysates were separated by SDS-PAGE and proteins were transferred to NC membranes. The membranes were incubated with antibodies to ACE, ACE2, and β-actin, followed by incubation with a horseradish peroxidase–conjugated secondary antibody. The band intensity was analyzed using Quantity One software.
Surface plasmon resonance (SPR) experiments
To immobilize analytes with an amino terminus, HS-PEG-COOH and HS-(CH2)11-EG6-OH (molar ratio, 1:10) at a total concentration of 1 mM were printed on a bare gold SPRi chip. To immobilize analytes with a carboxyl terminus, HS-PEG-NH2 and HS-(CH2)11-EG6-OH (molar ratio, 1:10) were printed on a bare gold SPRi chip at a total concentration of 1 mM. Chips were incubated in wet air at −4°C for 12 hrs to allow the formation of a uniform self-assembled mono-layer. Chips were washed with deionized water and ethanol and dried in a stream of N2. After activation with EDC/NHS for 10 min (0.1 M NHS, 0.4 M EDC), 1 mg/mL analyte (G1, G2, G3, G4, G5, G6, G7) was printed on the chip’s surface and the chips were incubated for 2 hrs at −4°C. The un-reacted esters were deactivated by exposing the chips to 1 M ethanolamine aqueous solution (pH = 8.5) for 10 min. For carboxyl terminal analytes, the unreacted area of the SPRi chip’s surface was deactivated by exposure to 1 M ethanolamine aqueous solution (including EDC/NHS solution) for 30 min. The surface was washed with deionized water and ethanol and then dried with nitrogen.
Real-time detection of protein adsorption was performed using a Plexera Κχ5 V2 SPRi apparatus (Plexera Bioscience LLC, USA). The entire SPRi chip surface was imaged during the angular scan. The immobilized analyte spots with 100 μm diameters on the SPRi chip were selected. The working optical position was automatically calculated according to the plasma curve. The auto-injection system controlled the flow at a rate of 2 μL⋅s−1. PBS (pH = 7.4) was used as the running buffer. When baseline was obtained, proteins at different concentrations (0.03 μM, 0.06 μM, 0.12 μM, and 0.23 μM) were injected into the flow cell. NaOH (20 mM) was used as the regeneration solution. In the kinetic analysis, the Plexera evaluation software was used to fit the SPR curves to the Langmuir equation.
Enzymatic activity assays
Recombinant human ACE2 protein (20 ng/mL) was incubated with or without 8 mg/mL different nanoparticles (10 μL or 30 μL) at 37°C in assay buffer (75 mM Tris, 1 M NaCl, pH = 7.5) for 10 min before adding angiotensin II (400 pg/mL) for an additional 30 min at 37°C, except for the blank control sample. Radioimmunoassays to determine the level of angiotensin II peptide in the assay buffer were performed as described previously .
Bronchoalveolar lavage fluid (BALF) was obtained 10 hrs after the intratracheal injection of the nanoparticles (15 μg/g). The concentration of IL-6 in the BALF was determined by ELISA (R&D systems, DY406) according to the manufacturer’s instructions. In the rescue group, losartan (15 mg/kg) was intraperitoneally injected 30 min before nanoparticle administration.
All data are shown as the mean values ± SEM. Measurements at single time points were analyzed by ANOVA, and if they demonstrated significance, they were further analyzed using a two-tailed t-test. Time courses were analyzed using repeated measures (mixed model) ANOVA with Bonferroni post-hoc analyses. All statistical tests were conducted using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). A value of p < 0.05 was used to indicate statistical significance.
This work was supported by the Ministry of Science and Technology of China (2015CB553406), the National Natural Science Foundation of China (81230002 and 81490531), and Ministry of Education (IRT1121 and B08007). D.S.L. acknowledges the support of the Science and Technology Commission of Shanghai Municipality (07pj14096). J.M.P. is supported by IMBA and the European Community’s Seventh Framework Program (FP7/2007-2013) under ERC grant agreement no. 233407.
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