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Polyethyleneimine and DNA nanoparticles-based gene therapy for acute lung injury
BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 9 (2013) 1293 – 1303
Research Article
nanomedjournal.com
Polyethyleneimine and DNA nanoparticles-based gene therapy for acute lung injury Erh-Hsuan Lin, PhD a, b, c , Hsiang-Yi Chang, MS a, b, c , Shauh-Der Yeh, MD, PhD d , Kuang-Yao Yang, MD, PhD e, f , Huei-Sin Hu, MS b , Cheng-Wen Wu, MD, PhD a, b, c, d,⁎ a
Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taiwan b Institute of Clinical Medicine, National Yang-Ming University, Taiwan c Institute of Microbiology and Immunology, National Yang-Ming University, Taiwan d Institute of Biomedical Sciences, Academia Sinica, Taiwan e Department of Chest Medicine, Taipei Veterans General Hospital, Taiwan f School of Medicine, National Yang-Ming University Received 10 November 2012; accepted 9 May 2013
Abstract Acute lung injury (ALI) is a devastating clinical syndrome causing a substantial mortality, but to date without any effective pharmacological management in clinic. Here, we tested whether nanoparticles based on polyethylenimine (PEI) and DNA could be a potential treatment. In mouse model of ALI induced by lipopolysaccharide (LPS) (10 mg/kg), intravenous injection of PEI/DNA mediated a rapid (in 6 h) and shortlived transgene expression in lung, with alveolar epithelial cells as major targets. When β2-Adrenergic Receptor (β2AR) was applied as therapeutic gene, PEI/β2AR treatment significantly attenuated the severity of ALI, including alveolar fluid clearance, lung water content, histopathology, bronchioalveolar lavage cellularity, protein concentration, and inflammatory cytokines in mice with pre-existing ALI. In highdose LPS (40 mg/kg)-induced ALI, post-injury treatment of PEI/β2AR significantly improved the 5-day survival of mice from 28% to 64%. These data suggest that PEI/DNA nanoparticles could be an effective agent in future clinical application for ALI treatment. From the Clinical Editor: In this novel study, PEI/DNA nanoparticles are presented as an effective agent for the treatment of the devastating and currently untreatable syndrome of acute lung injury, using a rodent model system. © 2013 Elsevier Inc. All rights reserved. Key words: β2-Adrenergic receptor; Gene therapy; Lung epithelium; Nanoparticle; LPS-induced acute lung injury
Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome associated with inflammatory injury to lung epithelium/endothelium and the passage of protein-rich edema into air spaces, leading to noncompliant lungs that function poorly in gas exchange. 1 Only moderate advances have been made in the treatment of We are grateful to the animal housing and technical assistance of Taiwan Mouse Clinic (funded by the National Research Program for Biopharmaceuticals (NRPB) at NSC) and Pathology Core Lab (Institute of Biomedical Sciences, Academia Sinica). We appreciate the technical support and fruitful discussions coming from Dr. Patrick Erbacher and Dr. Jean-Luc Coll. We clarify that there is no conflict of interest with any financial organization regarding the material and method used and discussed in the manuscript. This work is supported by National Science Council (NSC) grants 1002325-B-010-011 and 100-2321-B-010-021, and Aim for the Top University Plan of National Yang-Ming University (101ADP902). ⁎Corresponding author: National Yang Ming University, Taipei 112, Taiwan. E-mail address: [email protected] (C.-W. Wu).
ALI/ARDS in past decades, and the mortality remains very substantial at 30%-50%. 1-3 Clinical recovery depends mainly on the use of lung-protective ventilation with low tidal volumes. A number of promising pharmacologic therapies, including β2AR agonists, have been evaluated in Phase II/III clinical trials. Unfortunately, these treatments to date have had limited success in improving outcomes. 1,2,4,5 Gene therapy is a promising approach for treatments of a variety of chronic and acute disease. However, gene therapy has not been sufficiently applied in ALI/ARDS. The vector could be a major obstacle for this because both viral and non-viral vectors have serious drawbacks that limit their clinical applications. Ideally, the vector should be safe and perform the gene delivery in a rapid, efficient, and transient manner, with the major targets in pathologic loci. Viral vectors are typically efficient in gene delivery, but bearing the risk of mutational insertions, carcinogenesis, and the induction of strong inflammatory responses. 6 Non-viral vectors have become widespread because they are
1549-9634/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nano.2013.05.004 Please cite this article as: Lin E-H, et al, Polyethyleneimine and DNA nanoparticles-based gene therapy for acute lung injury. Nanomedicine: NBM 2013;9:1293-1303, http://dx.doi.org/10.1016/j.nano.2013.05.004
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relatively safe, less inflammatory, and capable of transferring large genes, yet the low delivery efficiency and poor transgene expression hampered their use in clinic. 6 PEI is a cationic polymer with repeating units composed of an amine group and a two-carbon aliphatic spacer, with the molecular formula (C2H5N)n. PEI can be in linear or branched forms, and both are efficient for gene delivery in vivo, with alveolar epithelial cells as major targets. 7-10 However, PEI has never been tested for ALI/ARDS treatment by therapeutic gene delivery. β2AR is a G protein-coupled receptor present throughout the lung. Activation of β2AR regulates important factors needed for alveolar ion and fluid transport, 11 decreases neutrophil-related inflammation, 12,13 and may improve alveolar epithelial repair. 14 Overexpression of β2AR in mouse lung via adenovirus infection or in transgenic model has shown to increase alveolar fluid clearance and protect animals from the later induced lung injury. 15-18 These research models, however, were not applicable in subsequent pre-clinical or clinical therapeutic treatment of pre-existing ALI/ARDS. In the current study, we tested the gene therapy in mouse model of ALI based on nanoparticles formed by PEI and β2AR gene.
kD PEI (PolyPlus Transfection, Illkirch, France) (4.8 μl, in case of 30 μg of DNA) was diluted in 5% glucose in a final volume of 100 μl. The solution containing PEI was added to that containing DNA, and mixed thoroughly by a vortex of 10 s, and left to stand at room temperature for 15 min. Healthy or LPS-injured mice were randomly grouped (n = 5 for each group), and injected with PEI/DNA nanoparticles through lateral tail-vein in waking state. The sizes and zeta potentials of PEI/DNA were measured and provided in the Electronic Supplementary Material (ESM). The ratio of PEI to DNA is expressed as N/P ratio (the molar ratio between PEI Nitrogen and DNA Phosphate), which was maintained at 8 for in vivo gene delivery in this study. The nanoparticles complexed by PEI and pT3-luc, pT3-lacZ, and pcDNA3-flag-β2AR were indicated as PEI/luc, PEI/lacZ, and PEI/β2AR, respectively, in the article. Non-invasive bioluminescent imaging Non-invasive bioluminescent imaging was described previously, 19 and is also described in detail in the ESM. Histological examination
Methods Plasmids Luciferase and lacZ expression vectors pT3-luc and pT3-lacZ were kindly provided by Dr. Coll (INSERM-UJF U823, France). 19 pcDNA3-flag-β2AR is purchased from Addgene (#14697). Plasmids were purified using Mega-prep endotoxinfree kit (Qiagen, Hilden, Germany).
The lung section, X-gal staining, and haematoxylin/eosin (HE) staining were performed as previously described, 19 and are described in detail in the ESM. Real-time PCR RNAs extraction and real-time PCR were described previously, 20 and are described in detail in the ESM. Measurement of alveolar fluid clearance rate in live mice
Mouse Model of ALI Five-week-old Bltw:CD1(ICR) mice were purchased from BioLasco Taiwan Co., Ltd. and maintained in Taiwan Mouse Clinic in Institute of Biomedical Sciences, Academia Sinica. All animal experiment protocols are approved by Academia Sinica Institutional Animal Care and Utilization Committee. Mice were maintained in controlled environmental conditions of temperature (22 ± 2 °C) and humidity (60% ± 5%) with a strict 12 h light–dark cycle, and given free access to food and water. Mice were subjected to experiments and sacrificed within 1 week after receiving, with an average body weight around 19 ± 2 g. For ALI induction, mice were randomly grouped (n = 5 for each group), anesthetized by intraperitoneal injections with Tiletamine/Zolazepam (25 mg/kg) and Xylazine (10 mg/kg), and intratracheally instilled in lung with 70 μl of PBS or PBS containing 10 mg/kg body weight of LPS (E. coli serotype 055: B5, Sigma-Aldrich, St. Louis, MO, US) via a 20-gauge catheter. The mice were then maintained on spontaneous breathing at room air for later PEI/DNA nanoparticle treatments (see below). In vivo gene delivery in mouse lung In vivo delivery of PEI/DNA has been described previously. 19 The generation of nanoparticles was based on the electric charges between PEI (positive) and DNA (negative). Different quantities of DNA (generally 30 μg in therapeutic approach) were diluted in 5% glucose in a final volume of 100 μl. In another tube, linear 22-
The Evans Blue-labeled bovine serum albumin (EB-BSA) was freshly prepared before experiment by mixing EB (0.15 mg/ ml) in 5% BSA in Ringer's Lactate solution. The healthy or LPSinjured mice treated with PEI/DNA nanoparticles were anesthetized, maintained at 37 °C, and the trachea was cannulated with a 20-gauge catheter. A total of 400 μl of EB-BSA was instilled into the lung, and the mouse was ventilated for 20 min with a tidal volume of 10 ml/kg at a frequency of 90 breaths per minute with a ventilator (SAR-830 Small Animal Ventilators, CWEInc., Ardmore, PA, US). At the end of experiment, mouse chest was opened to allow aspiration of fluid from the tracheal catheter. The density of EB in aspirate was measured, and the alveolar fluid clearance rate was expressed as the percentage of cleared volume in 20 min, calculated with the equation: alveolar fluid clearance (%) = 100 × (1 − C0/C20), where C0 is the EBBSA concentration before instillation, and C20 is the EB-BSA concentration in the aspirate at the end of 20-min ventilation. Measurement of lung water content The level of lung water content was assessed by the ratio between wet lung to dry lung weight (wet-to-dry ratio), following a previous publication. 21 After irreversible anesthesia, mice were exsanguinated by laceration on heart. The lungs were removed and determined for wet lung weight. The lungs were then placed in an incubator at 70 °C for 72 h to remove all moisture, and dry lung weight was determined.
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Figure 1. PEI-mediated reporter gene delivery in healthy and injured lungs. PEI/DNA nanoparticles were injected through lateral tail-vein into healthy mice or mice with pre-existing ALI (A). Different amounts of luciferase expression vectors (0 to 50 μg, as indicated on the figure) were complexed with 22-kD linear PEI in a constant N/P ratio of 8. Luciferase reporter gene signal was followed and quantified using bioluminescent imaging at different time points after PEI/luc injection (B) (n = 5 for each group, error bars indicate Standard Deviation (SD)), and 2 mice were illustrated as samples of imaging (C). To determine the cell types targeted by PEI/DNA delivery, mice were injected with PEI/lacZ and sacrificed 1 day later. Lungs were stained with X-Gal and HE for histochemical examinations in × 400 (D) or × 1000 (E) magnification. Arrows indicate the X-Gal stained (blue) cells. Bar = 100 μm (D) or 20 μm (E).
Lung injury score The lung injury score was evaluated according to the criteria addressed. 22 The lung injury score was assessed on histopathol-
ogy by 2 clinicians (S-D Yeh and K-Y Yang) independently and averaged. For each condition, 15 fields (× 400 magnification, including a total of N 300 alveoli) of lung sections from 5 mice were analyzed.
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Bronchioalveolar lavage analysis The bronchioalveolar lavage fluid was collected on 48 h postinjury. The collection was performed according to a previous publication, 21 and is described in detail in the ESM. Survival analysis For survival analysis, mice were intratracheally instilled with 70 μl of PBS containing 40 mg/kg of LPS, and then randomly grouped (n = 5 for each group) to receive PEI, PEI/luc, or PEI/ β2AR treatment 1 h later, or not treated. The deaths of mice were recorded since 6 h after LPS instillation to day 5. Five independent experiments were performed, and the results were combined in one graph. Statistic analysis The statistical significances of the experimental results were assessed by independent-samples t test (Two-tailed) using SPSS Statistics v.20 (IBM, Armonk, NY, US). For survival analyses, Gehan–Breslow Test was used. P b 0.05 is considered significant. Results PEI/DNA-mediated gene delivery in mouse lung under healthy condition or ALI The particles complexed by PEI and DNA were analyzed, which showed average sizes around 60 nm and zeta potentials around 30 mV (Figure S1). After intravenous injection of PEI/ DNA in mice, the kinetics and target tissues of PEI/DNAmediated gene delivery were analyzed. The reporter gene expression was found mainly in lung, where it was approximately 100-fold higher than in other organs (Figure S2). The reporter gene signal in lung was also followed and quantified using non-invasive bioluminescent imaging (Figure 1, A to C). The results showed that PEI/luc induced a rapid and efficient transgene expression in lung as soon as 6 h post-injection, which then declined and became nearly undetectable on day 4. The transgene expression levels were positively related to the amounts of DNA delivered. We then administered PEI/luc in mice with pre-existing ALI induced by LPS. PEI/luc was injected in mice 1 h after LPS instillation, and we found that however, delivery of a dose of 50 μg of DNA resulted in a high mortality (90%, data not shown). A lower dose of DNA (30 μg), in contrast, did not result in any death. The luciferase expression kinetics in mice with ALI were similar to those in healthy ones, although the level was reduced by around 50% (Figure 1, B and C). PEI/lacZ was administered to identify the target cells of gene delivery in lung, and the results showed that alveolar epithelial cells were the major targets (Figure 1, D and E), and the pre-existing ALI did not block or alter the delivery. The identities of target cells were further confirmed by immuno-histochemical staining against Aquaporin 5 and Surfactant Protein C, which are specific markers of alveolar epithelial cells type 1 and 2, respectively. The cells co-stained with lacZ and these markers were observed (Figure S3).
Figure 2. Exogenous and endogenous β2AR gene expressions in mice lungs with ALI. ALI was induced in mice by intratracheal instillation of 10 mg/kg of LPS. The mice were then injected with PEI/β2AR 1 h post-injury (A and B), or not injected (C). Mice were sacrificed at indicated time points after PEI/ β2AR injection, and the lungs were harvested for RNA extraction. PBS was instilled into mice lungs as non-injury control (PBS). Exogenous (human) (A) and endogenous (mouse) (B and C) β2AR gene expressions were detected using Real-Time PCR with specific primers, and presented as ratios relative to PBS group. Error bars indicate SD, and n = 5 for each group.
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Figure 3. PEI/β2AR treatment improved alveolar fluid clearance and reduced lung water content in mice with pre-existing ALI. PEI/β2AR treatment was administered in mice 24 h post-injury (A). For control, mice were not treated, or treated with PEI/luc or PEI. PBS was instilled into mice lungs as non-injury control. Alveolar fluid clearance was measured in vivo and shown as percentage of total instilled volume cleared in 20 min (B). Lung water content was assessed by the measurement of wet-to-dry ratio (C). Error bars indicate SD, and n = 5 for each group.
Exogenous and endogenous β2AR gene expressions in mice lungs with ALI The human β2AR gene was applied as therapeutic gene in this study. PEI/β2AR was injected in mice with pre-existing ALI (Figure 1, A), and the lung proteins/mRNAs were extracted. The β2AR protein expression was verified using western blot analysis (Figure S4). The kinetics of exogenous (human) β2AR expression (Figure 2, A) were similar to those of luciferase observed in bioluminescent imaging (Figure 1, B). Strikingly, we found that at any time point after gene delivery, endogenous (mouse) β2AR expression was substantially repressed (Figure 2, B). In mice with LPS-induced ALI but without PEI/β2AR treatment (Figure 2, C), we found that the expression of endogenous β2AR was also significantly repressed up to 4 days. PEI/β2AR treatment improved alveolar fluid clearance and reduced lung water content in mice lungs with pre-existing ALI In our pilot study, it showed that LPS in a dose of 10 mg/kg efficiently induced an ALI syndrome in mice (Figure S5), and the pulmonary inflammation lasted for at least 48 h (Figure S6). We thus designed the therapeutic model by administering the PEI/β2AR treatment 24 h post-injury, and analyzing the outcome at 48 h (Figure 3, A). We first assessed the influence of PEI/β2AR treatment in alveolar fluid clearance. As illustrated on the figure, LPS-induced ALI led to a significant loss of alveolar fluid clearance activity (Figure 3, B). Treatment with PEI/β2AR recovered the alveolar fluid clearance activity to the level corresponding to the non-injury control. In contrast, PEI/
luc or PEI showed no effect. We then analyzed the level of lung water content by measuring the ratio of wet lung to dry lung (wet-to-dry) weights. In accordance with alveolar fluid clearance, ALI induced an increase in wet-to-dry ratio, and PEI/β2AR treatment reduced it to a nearly normal level (Figure 3, C), while treatment with PEI/luc or PEI showed no effect. PEI/β2AR treatment improved histopathology and bronchioalveolar lavage indexes in mice lungs with pre-existing ALI We then examined the lung histopathology. As expected, LPS instillation caused extensive morphological damages including edema, hemorrhage, thickness of alveolar walls, and infiltration of neutrophils in alveolar and interstitial spaces (Figure 4). These morphological changes were much less pronounced after PEI/ β2AR treatment. We also investigated the bronchioalveolar lavage indexes. In accordance with histopathology, ALI induced dramatic increases in cell number, protein concentration, and inflammatory cytokines TNF-α and IL-6 in bronchioalveolar lavage, as compared to non-injury control (Figure 5). Once more, PEI/β2AR treatment significantly attenuated the severity of these indexes, while PEI/luc or PEI treatment showed no effect. PEI/β2AR treatment improved the survival of mice in high-dose LPS-induced ALI In our pilot study, LPS in a dose higher than 40 mg/kg induced a high mortality in mice (Figure S7). In survival assay performed by intratracheal instillation of 40 mg/kg of LPS, the survival rate of mice dropped rapidly to 60% on 6 h, and to 40% on day 1 (Figure 6). PEI/β2AR treatment was administered 1 h
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Figure 4. PEI/β2AR treatment improved histopathology in mice with pre-existing ALI. PEI/β2AR treatment was administered as presented in Figure 3, A. For control, mice were not treated, or treated with PEI/luc or PEI. PBS was instilled into mice lungs as non-injury control. Histopathology was investigated in lung sections (5 μm, paraffin embedded) after HE staining. The lung injury scores were assessed in 15 fields (× 400 magnification, including a total of N300 alveoli) of lung sections, taken from 5 mice for each group. Bar = 100 μm. Error bars indicate SD.
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Figure 5. PEI/β2AR treatment improved bronchioalveolar lavage indexes in mice with pre-existing ALI. PEI/β2AR treatment was administered as presented in Figure 3, A. For control, mice were not treated, or treated with PEI/luc or PEI. PBS was instilled into mice lungs as non-injury control. The cell numbers (A), protein concentrations (B), and the levels of pro-inflammatory cytokines TNF-α (C) and IL-6 (D) in bronchioalveolar lavage were measured. Error bars indicate SD, and n = 5 for each group.
post-injury, which provided a significant improvement in survival rate, which was 92% on 6 h and 80% on day 1, respectively. No more death was observed after day 4, and the 5day survival rate was 64%, 15%, 5%, and 28% in PEI/β2AR-, PEI/luc-, PEI-, and non-treated groups, respectively. PEI/β2AR treatment showed statistically significant survival benefit (P b 0.01) as compared to any other group; while no statistical difference was found between the 3 other groups.
Discussion This is the first study testing the use of PEI/DNA nanoparticles for the delivery of a therapeutic gene in the treatment of LPS-induced ALI in an animal model. Our findings suggest that the treatment with PEI/β2AR is safe in the context of LPS-induced ALI, and provides rapid and timely benefits in mice. Like previous studies, 7-10 our reporter gene assay showed that systemic administration of PEI/DNA nanoparticles in vivo mediated an efficient gene delivery mainly in alveolar epithelial cells (Figure 1 and S3). The transgene expression was rapid (as soon as in 6 h post-injection) and short-lived (b 4 days). Despite a lower efficiency, PEI/DNA delivery is attainable in alveolar epithelial cells in lungs with pre-existing ALI. In the approach of therapeutic treatment, PEI/β2AR was administered in mice 24 h post-injury, a time point where ALI syndrome was already obvious (Figure 3, A and S6), to mimic a therapeutic, but not
prevention model. The results showed that PEI/β2AR treatment significantly attenuated the ALI severities, including alveolar fluid clearance, lung water content, histopathology, bronchioalveolar lavage cell number, protein concentration, and inflammatory cytokines (Figures 3 to 5). In survival analysis, because of the rapid mortality induced by high-dose LPS within in 24 h (the survival rate dropped to 40% in the absence of therapeutic treatment), PEI/β2AR treatment was administered 1 h postinjury. The result showed that PEI/β2AR provided a rapid and timely survival benefit in mice with ALI induced by high-dose LPS, with a 5-day survival rate of 64% in comparison with control groups, which showed a survival rate of b 28% (Figure 6). Despite of extensive researches in pathophysiology, ALI/ ARDS remains a devastating syndrome with a high mortality. β2AR signaling has gained considerable interest in ALI/ARDS therapy because of its ability to improve the resolution of pulmonary edema. 11 Unfortunately, 2 large-scale randomized controlled trials have recently been terminated because of futility and concerns about safety. 4,5 The failure could be attributed to adverse effects including cardiac effects and vasodilatation because of the systemic administration of β2AR agonist, and the desensitization/down-regulation of β2AR after prolonged agonist stimulation. 23-26 Different from past researches that focused on the development of β2AR agonists, our study here presented a PEI/DNA-nanoparticle based gene therapy, and the results showed important clinical implications. First, although the
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Figure 6. PEI/β2AR treatment improved the survival of mice in high-dose LPS-induced ALI. For survival assay, mice were intratracheally instilled with highdose LPS (40 mg/kg), and then treated with PEI/β2AR 1 h post-injury. For control, mice were not treated, or treated with PEI/luc or PEI. The survival of mice was recorded since 6 h post-injury. No more death was noticed after day 4. N = 25 for LPS and LPS + PEI/β2AR, and n = 20 for LPS + PEI/luc and LPS + PEI groups. P value was calculated by Gehan–Breslow statistic analysis.
administration of nanoparticles was systemic, the major targets of gene delivery were alveolar epithelial cells (the pathological loci of ALI), and the reporter gene signal in lung was approximately 100-fold higher than in heart or other organs (Figure 1 and S2). Such distribution would minimize the adverse effects like cardiac effect observed in the use of agonist. Second, in this study we found that the expression of endogenous (mouse) β2AR was significantly repressed in the presence of ALI (Figure 2, C). This finding would reflect a previous investigation that pulmonary edema in ALI patients is due not only to the increased fluid flux in airspace, but also the impairment of alveolar fluid clearance mechanism. Alveolar fluid clearance could still be impaired despite high levels of endogenous catecholamines, and the treatment of exogenous pharmaceuticals (e.g. albuterol) did not correlate with the rate of alveolar fluid clearance. 27 Hence, the repressed endogenous β2AR expression could be an undescribed reason of impaired alveolar fluid clearance mechanism and the failure of β2AR agonist to improve the clinical outcome. Accordingly, the PEI/ β2AR gene therapy model presented here could address such problem since it activates β2AR signaling by direct overexpression of β2AR, but not by agonists that need the endogenous β2AR expressed on cell surface.
Although gene therapy is a promising approach for treatments of various diseases, it has not been well-developed in ALI. Early researches have shown that the overexpression of β2AR in mouse lung increased alveolar fluid clearance and protected animals from the later-induced ALI. 15-18 These research models based on adenovirus-mediated gene delivery, however, were not applicable in subsequent clinical therapeutic treatment because of the safety concerns of viral vector. A unique research performing the feasible post-injury gene therapy in animal model was reported in 2007, 21 in which the Na +,K+-ATPase genes were delivered in lung by electroporation. Improvements in alveolar fluid clearance and respiratory mechanics were observed after gene delivery in mice with pre-existing ALI induced by LPS. However, the treatment was not able to ameliorate the syndrome or rescue the survival when severe ALI was induced by high-dose LPS. The authors thus concluded that although the delivery of therapeutic genes can be an effective and logical treatment for ALI, its application still needs refinement. 21 In our study, we used PEI/DNA-nanoparticles for β2AR gene delivery. The sizes of nanoparticles play a key role in their biodistribution and final target in vivo. Particles less than 5 nm are rapidly cleared from the circulation through extravasation or renal clearance, and particles as large as 15 μm or more
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accumulate in liver, spleen, and bone marrow. Smaller microparticles (roughly, from 1 to 10 μm) are mainly internalized by phagocytosis in special cell types such as macrophage and neutrophil, a condition that is undesired for most drug delivery. 29,30 Therefore, keeping the particles small (between 10 and 100 nm) facilitates their diffusion in the blood and tissues and the cellular internalization through endocytosis, and has become a foundation for current definition of nanomedicine. 30 In this study, we showed that nanoparticles formed by 22-kD linear PEI and DNA in non-ionic solution (5% glucose), like previous studies, 8,31-33 possessed sizes b 100 nm (Figure S1). These nanoparticles were able to diffuse across the endothelial lining and to target alveolar epithelial cells (Figure 1). Although the exact mechanism of PEI/DNA internalization in epithelial cells remains controversial (as reviewed by Sahay et al), 30 our study provides the first evidence that this kind of nanoparticles can be used in ALI treatment by therapeutic gene delivery in a postinjury model in animals. The gene delivery was rapid, efficient and short-lived (Figure 1), and did not further aggravate the lung inflammation, while showing therapeutic benefits (Figures 3 to 5). Importantly, PEI/β2AR was able to rescue the survival of mice in high-dose LPS-induced ALI (Figure 6). These properties potentiate PEI/β2AR as a proper reagent for the clinical treatment of ALI, because it is effective and minimizes the risks of mutational insertions, carcinogenesis, and the induction of strong inflammatory responses that may arise when using viral vectors. Although PEI-mediated gene delivery in vivo has been sufficiently addressed in many references, it has never been applied to deliver a therapeutic gene for ALI/ARDS treatment. A possible reason can be the safety concern. An early study showed that increasing the dose of PEI/DNA resulted in a marked augmentation of transgene expression, but liver necrosis and mouse death were reported when a high-dose DNA (over 100 μg) and high N/P ratio (= 10) were administrated. 34 The improper doses of DNA used in that research were, however, to evaluate the potential adverse effects of PEI/DNA, but not actually applied in common research models or clinical trials. 10 In our study, we did observe a high mortality (90%, data not shown) when a higher-than-proper dose of DNA (50 μg) was injected in mice with pre-existing ALI. Nevertheless, a moderate dose of DNA (30 μg) was able to transfect mouse lung with preexisting ALI without inducing any death, while showing therapeutic benefits. It thus suggests that in an acute and devastating syndrome like ALI/ARDS, an optimized protocol of gene delivery, which may not necessarily be the one showing the highest transfection efficiency, has to be carefully defined. For an effective treatment, a balance between efficiency and adverse effects has to be found, and here we show that it can be achieved in this gene therapy model. Inflammatory responses attributed to PEI/DNA administration have also been reported. 35-41 PEIs associated with these adverse effects, however, were uniformly in branched, but not linear forms. 35,37 A recent study using linear 22-kD PEI clearly showed no major production of proinflammatory cytokines or hepatic enzymes after systemic injection of PEI/DNA or PEI/siRNA. 10 In our study, we did not observe any additional pro-inflammatory effect other than LPS induction when using such linear PEI/DNA for treatment. Instead, PEI/β2AR treatment attenuated the inflammatory
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response as evidenced by the histopathology and cytokine measurements (Figures 4 and 5). Therefore, PEI/DNA nanoparticle-based gene therapy, at least in the context of endotoxemiaassociated ALI, is safe and advantageous. This study still has some limitations. First, the reason of the high mortality (90%, data not shown) induced by a higher-thanproper dose of DNA (50 μg) in mice with LPS-induced ALI remains unclear. Although the PEI in linear form, as described above, does not induce inflammatory response, PEI is known to interact with LPS based on electric charge. 42,43 Therefore, it is possible that PEI/DNA administration in the presence of LPS induced accumulations of macro-aggregations that led to in vivo toxicity. The exact mechanism of such PEI/DNA-induced in vivo toxicity, however, must be clarified before PEI/DNA moves toward clinical applications. Second, concerning the clinical application of gene delivery in lung, systemic injection with PEI/ DNA may face the problems of consumptions (in proportion, over 100 mg of DNA should be used in a 65-kg human adult according to this study) and other unexpected adverse effects that are not predicted in mouse model. Indeed, the report on the feasibility of systemic delivery of PEI/DNA in large animal or human is currently absent, although a clinical trial that applies nanoparticles containing PEI/DNA/siRNA 44 in multiple myeloma patients by intravenous infusion has been approved very recently (NCT01435720 on ClinicalTrials.gov). Local administration like intratracheal/aerosol delivery could be considered more preferable. However, in our gene delivery system with linear 22-kD PEI, we found so far that intravenous injection was the most efficient for gene expression in mice lungs, while intratracheal administration was insufficient (data not shown). The successful aerosol delivery of PEI/DNA found in literatures thus far depends on the use of branched PEI. Nevertheless, as mentioned above, branched PEI has been mentioned in many references to induce inflammatory response. Therefore, the optimization of PEI-based gene delivery in clinical application is still awaiting more studies. Third, although our finding suggested that β2AR expression was repressed in ALI, it's not clear whether the repression was a result of lung epithelial cell death or LPS-induced modulations on gene expressions. Since LPS has been known to influence gene expression profile through multiple transcription factors, 45 it is possible that β2AR repression was a consequence of LPS-modulated gene transcriptions in alveolar epithelial cells. The exact mechanisms associated with the repression, however, need more studies in the future. Fourth, although alveolar epithelial cells are the major targets of gene delivery, endothelial cells could still be transfected and contribute to some beneficial effect, which was not dissected in our current works. Finally, protective mechanical ventilation has been proven to improve survival and becomes a standard management on ALI/ARDS patients. Whether PEI/β2AR will provide additive benefits on patients that are already subjected to protective ventilation is uncertain, and thus needs further pre-clinical/clinical studies to clarify. In conclusion, our research here presents a simple, safe, and effective gene therapy for ALI in mouse model. PEI/DNA nanoparticle-mediated gene delivery in mouse lung is rapid, efficient, and short-lived. PEI/β2AR treatment significantly attenuated the severity of ALI and improved the survival of mice,
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without a major adverse effect observed. The PEI/DNA nanoparticle-based gene therapy could have great potential in future clinical applications. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2013.05.004.
References 1. Matthay MA, Zemans RL. The acute respiratory distress syndrome: pathogenesis and treatment. Annu Rev Pathol 2011;6:147-63. 2. Raghavendran K, Pryhuber GS, Chess PR, Davidson BA, Knight PR, Notter RH. Pharmacotherapy of acute lung injury and acute respiratory distress syndrome. Curr Med Chem 2008;15:1911-24. 3. Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, et al. Incidence and outcomes of acute lung injury. N Engl J Med 2005;353:1685-93. 4. Gao SF, Perkins GD, Gates S, Young D, McAuley DF, Tunnicliffe W, et al. Effect of intravenous beta-2 agonist treatment on clinical outcomes in acute respiratory distress syndrome (BALTI-2): a multicentre, randomised controlled trial. Lancet 2012;379:229-35. 5. Matthay MA, Brower RG, Carson S, Douglas IS, Eisner M, Hite D, et al. Randomized, placebo-controlled clinical trial of an aerosolized beta-2 agonist for treatment of acute lung injury. Am J Respir Crit Care Med 2011;184:561-8. 6. Boulaiz H, Marchal JA, Prados J, Melguizo C, Aranega A. Non-viral and viral vectors for gene therapy. Cell Mol Biol 2005;51:3-22. 7. Goula D, Benoist C, Mantero S, Merlo G, Levi G, Demeneix BA. Polyethylenimine-based intravenous delivery of transgenes to mouse lung. Gene Ther 1998;5:1291-5. 8. Zou SM, Erbacher P, Remy JS, Behr JP. Systemic linear polyethylenimine (L-PEI)-mediated gene delivery in the mouse. J Gene Med 2000;2:128-34. 9. Bragonzi A, Boletta A, Biffi A, Muggia A, Sersale G, Cheng SH, et al. Comparison between cationic polymers and lipids in mediating systemic gene delivery to the lungs. Gene Ther 1999;6:1995-2004. 10. Bonnet ME, Erbacher P, Bolcato-Bellemin AL. Systemic delivery of DNA or sirna mediated by linear polyethylenimine (L-PEI) does not induce an inflammatory response. Pharm Res 2008;25:2972-82. 11. Mutlu GM, Factor P. Alveolar epithelial beta2-adrenergic receptors. Am J Respir Cell Mol Biol 2008;38:127-34. 12. Perkins GD, Nathani N, McAuley DF, Gao F, Thickett DR. In vitro and in vivo effects of salbutamol on neutrophil function in acute lung injury. Thorax 2007;62:36-42. 13. Maris NA, de Vos AF, Dessing MC, Spek CA, Lutter R, Jansen HM, et al. Antiinflammatory effects of salmeterol after inhalation of lipopolysaccharide by healthy volunteers. Am J Respir Crit Care Med 2005;172:878-84. 14. Perkins GD, Gao F, Thickett DR. In vivo and in vitro effects of salbutamol on alveolar epithelial repair in acute lung injury. Thorax 2008;63:215-20. 15. McGraw DW, Fukuda N, James PF, Forbes SL, Woo AL, Lingrel JB, et al. Targeted transgenic expression of beta(2)-adrenergic receptors to type ii cells increases alveolar fluid clearance. Am J Physiol Lung Cell Mol Physiol 2001;281:L895-903. 16. Factor P, Adir Y, Mutlu GM, Burhop J, Dumasius V. Effects of beta2adrenergic receptor overexpression on alveolar epithelial active transport. J Allergy Clin Immunol 2002;110:S242-6. 17. Dumasius V, Sznajder JI, Azzam ZS, Boja J, Mutlu GM, Maron MB, et al. Beta(2)-adrenergic receptor overexpression increases alveolar fluid clearance and responsiveness to endogenous catecholamines in rats. Circ Res 2001;89:907-14.
18. Mutlu GM, Dumasius V, Burhop J, McShane PJ, Meng FJ, Welch L, et al. Upregulation of alveolar epithelial active NA + transport is dependent on beta2-adrenergic receptor signaling. Circ Res 2004;94: 1091-100. 19. Lin EH, Keramidas M, Rome C, Chiu WT, Wu CW, Coll JL, et al. Lifelong reporter gene imaging in the lungs of mice following polyethyleneimine-mediated sleeping-beauty transposon delivery. Biomaterials 2011;32:1978-85. 20. Hong CF, Chou YT, Lin YS, Wu CW. MAD2B, a novel TCF4-binding protein, modulates TCF4-mediated epithelial–mesenchymal transdifferentiation. J Biol Chem 2009;284:19613-22. 21. Mutlu GM, Machado-Aranda D, Norton JE, Bellmeyer A, Urich D, Zhou R, et al. Electroporation-mediated gene transfer of the NA +,K+ATPpase rescues endotoxin-induced lung injury. Am J Respir Crit Care Med 2007;176:582-90. 22. Matute-Bello G, Downey G, Moore BB, Groshong SD, Matthay MA, Slutsky AS, et al. An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals. Am J Respir Cell Mol Biol 2011;44:725-38. 23. Bassford CR, Thickett DR, Perkins GD. The rise and fall of beta-agonists in the treatment of ARDS. Crit Care 2012;16:208. 24. Thompson BT. Beta-agonists for ARDS: the dark side of adrenergic stimulation? Lancet 2012;379:196-8. 25. Perkins GD, McAuley DF. Pro: beta-agonists in acute lung injury—the end of the story? Am J Respir Crit Care Med 2011;184:503-4. 26. Papazian L. Con: beta2-adrenergic agonists in ALI/ARDS—not recommended or potentially harmful? Am J Respir Crit Care Med 2011;184:504-6. 27. Ware LB, Matthay MA. Alveolar fluid clearance is impaired in the majority of patients with acute lung injury and the acute respiratory distress syndrome. Am J Respir Crit Care Med 2001;163:1376-83. 28. Petros RA, DeSimone JM. Strategies in the design of nanoparticles for therapeutic applications. Nat Rev Drug Discov 2010;9:615-27. 29. Champion JA, Walker A, Mitragotri S. Role of particle size in phagocytosis of polymeric microspheres. Pharm Res 2008;25:1815-21. 30. Sahay G, Alakhova DY, Kabanov AV. Endocytosis of nanomedicines. J Control Release 2010;145:182-95. 31. Goula D, Remy JS, Erbacher P, Wasowicz M, Levi G, Abdallah B, et al. Size, diffusibility and transfection performance of linear PEI/ DNA complexes in the mouse central nervous system. Gene Ther 1998;5:712-7. 32. Bragonzi A, Dina G, Villa A, Calori G, Biffi A, Bordignon C, et al. Biodistribution and transgene expression with nonviral cationic vector/ DNA complexes in the lungs. Gene Ther 2000;7:1753-60. 33. Goula D, Becker N, Lemkine GF, Normandie P, Rodrigues J, Mantero S, et al. Rapid crossing of the pulmonary endothelial barrier by polyethylenimine/DNA complexes. Gene Ther 2000;7:499-504. 34. Chollet P, Favrot MC, Hurbin A, Coll JL. Side-effects of a systemic injection of linear polyethylenimine–DNA complexes. J Gene Med 2002;4:84-91. 35. Jeong GJ, Byun HM, Kim JM, Yoon H, Choi HG, Kim WK, et al. Biodistribution and tissue expression kinetics of plasmid DNA complexed with polyethylenimines of different molecular weight and structure. J Control Release 2007;118:118-25. 36. Regnstrom K, Ragnarsson EG, Fryknas M, Koping-Hoggard M, Artursson P. Gene expression profiles in mouse lung tissue after administration of two cationic polymers used for nonviral gene delivery. Pharm Res 2006;23:475-82. 37. Kawakami S, Ito Y, Charoensit P, Yamashita F, Hashida M. Evaluation of proinflammatory cytokine production induced by linear and branched polyethylenimine/plasmid DNA complexes in mice. J Pharmacol Exp Ther 2006;317:1382-90. 38. Regnstrom K, Ragnarsson EG, Koping-Hoggard M, Torstensson E, Nyblom H, Artursson P. PEI — a potent, but not harmless, mucosal immuno-stimulator of mixed T-helper cell response and FASL-mediated cell death in mice. Gene Ther 2003;10:1575-83.
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Research Article
nanomedjournal.com
Polyethyleneimine and DNA nanoparticles-based gene therapy for acute lung injury Erh-Hsuan Lin, PhD a, b, c , Hsiang-Yi Chang, MS a, b, c , Shauh-Der Yeh, MD, PhD d , Kuang-Yao Yang, MD, PhD e, f , Huei-Sin Hu, MS b , Cheng-Wen Wu, MD, PhD a, b, c, d,⁎ a
Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taiwan b Institute of Clinical Medicine, National Yang-Ming University, Taiwan c Institute of Microbiology and Immunology, National Yang-Ming University, Taiwan d Institute of Biomedical Sciences, Academia Sinica, Taiwan e Department of Chest Medicine, Taipei Veterans General Hospital, Taiwan f School of Medicine, National Yang-Ming University Received 10 November 2012; accepted 9 May 2013
Abstract Acute lung injury (ALI) is a devastating clinical syndrome causing a substantial mortality, but to date without any effective pharmacological management in clinic. Here, we tested whether nanoparticles based on polyethylenimine (PEI) and DNA could be a potential treatment. In mouse model of ALI induced by lipopolysaccharide (LPS) (10 mg/kg), intravenous injection of PEI/DNA mediated a rapid (in 6 h) and shortlived transgene expression in lung, with alveolar epithelial cells as major targets. When β2-Adrenergic Receptor (β2AR) was applied as therapeutic gene, PEI/β2AR treatment significantly attenuated the severity of ALI, including alveolar fluid clearance, lung water content, histopathology, bronchioalveolar lavage cellularity, protein concentration, and inflammatory cytokines in mice with pre-existing ALI. In highdose LPS (40 mg/kg)-induced ALI, post-injury treatment of PEI/β2AR significantly improved the 5-day survival of mice from 28% to 64%. These data suggest that PEI/DNA nanoparticles could be an effective agent in future clinical application for ALI treatment. From the Clinical Editor: In this novel study, PEI/DNA nanoparticles are presented as an effective agent for the treatment of the devastating and currently untreatable syndrome of acute lung injury, using a rodent model system. © 2013 Elsevier Inc. All rights reserved. Key words: β2-Adrenergic receptor; Gene therapy; Lung epithelium; Nanoparticle; LPS-induced acute lung injury
Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome associated with inflammatory injury to lung epithelium/endothelium and the passage of protein-rich edema into air spaces, leading to noncompliant lungs that function poorly in gas exchange. 1 Only moderate advances have been made in the treatment of We are grateful to the animal housing and technical assistance of Taiwan Mouse Clinic (funded by the National Research Program for Biopharmaceuticals (NRPB) at NSC) and Pathology Core Lab (Institute of Biomedical Sciences, Academia Sinica). We appreciate the technical support and fruitful discussions coming from Dr. Patrick Erbacher and Dr. Jean-Luc Coll. We clarify that there is no conflict of interest with any financial organization regarding the material and method used and discussed in the manuscript. This work is supported by National Science Council (NSC) grants 1002325-B-010-011 and 100-2321-B-010-021, and Aim for the Top University Plan of National Yang-Ming University (101ADP902). ⁎Corresponding author: National Yang Ming University, Taipei 112, Taiwan. E-mail address: [email protected] (C.-W. Wu).
ALI/ARDS in past decades, and the mortality remains very substantial at 30%-50%. 1-3 Clinical recovery depends mainly on the use of lung-protective ventilation with low tidal volumes. A number of promising pharmacologic therapies, including β2AR agonists, have been evaluated in Phase II/III clinical trials. Unfortunately, these treatments to date have had limited success in improving outcomes. 1,2,4,5 Gene therapy is a promising approach for treatments of a variety of chronic and acute disease. However, gene therapy has not been sufficiently applied in ALI/ARDS. The vector could be a major obstacle for this because both viral and non-viral vectors have serious drawbacks that limit their clinical applications. Ideally, the vector should be safe and perform the gene delivery in a rapid, efficient, and transient manner, with the major targets in pathologic loci. Viral vectors are typically efficient in gene delivery, but bearing the risk of mutational insertions, carcinogenesis, and the induction of strong inflammatory responses. 6 Non-viral vectors have become widespread because they are
1549-9634/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nano.2013.05.004 Please cite this article as: Lin E-H, et al, Polyethyleneimine and DNA nanoparticles-based gene therapy for acute lung injury. Nanomedicine: NBM 2013;9:1293-1303, http://dx.doi.org/10.1016/j.nano.2013.05.004
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relatively safe, less inflammatory, and capable of transferring large genes, yet the low delivery efficiency and poor transgene expression hampered their use in clinic. 6 PEI is a cationic polymer with repeating units composed of an amine group and a two-carbon aliphatic spacer, with the molecular formula (C2H5N)n. PEI can be in linear or branched forms, and both are efficient for gene delivery in vivo, with alveolar epithelial cells as major targets. 7-10 However, PEI has never been tested for ALI/ARDS treatment by therapeutic gene delivery. β2AR is a G protein-coupled receptor present throughout the lung. Activation of β2AR regulates important factors needed for alveolar ion and fluid transport, 11 decreases neutrophil-related inflammation, 12,13 and may improve alveolar epithelial repair. 14 Overexpression of β2AR in mouse lung via adenovirus infection or in transgenic model has shown to increase alveolar fluid clearance and protect animals from the later induced lung injury. 15-18 These research models, however, were not applicable in subsequent pre-clinical or clinical therapeutic treatment of pre-existing ALI/ARDS. In the current study, we tested the gene therapy in mouse model of ALI based on nanoparticles formed by PEI and β2AR gene.
kD PEI (PolyPlus Transfection, Illkirch, France) (4.8 μl, in case of 30 μg of DNA) was diluted in 5% glucose in a final volume of 100 μl. The solution containing PEI was added to that containing DNA, and mixed thoroughly by a vortex of 10 s, and left to stand at room temperature for 15 min. Healthy or LPS-injured mice were randomly grouped (n = 5 for each group), and injected with PEI/DNA nanoparticles through lateral tail-vein in waking state. The sizes and zeta potentials of PEI/DNA were measured and provided in the Electronic Supplementary Material (ESM). The ratio of PEI to DNA is expressed as N/P ratio (the molar ratio between PEI Nitrogen and DNA Phosphate), which was maintained at 8 for in vivo gene delivery in this study. The nanoparticles complexed by PEI and pT3-luc, pT3-lacZ, and pcDNA3-flag-β2AR were indicated as PEI/luc, PEI/lacZ, and PEI/β2AR, respectively, in the article. Non-invasive bioluminescent imaging Non-invasive bioluminescent imaging was described previously, 19 and is also described in detail in the ESM. Histological examination
Methods Plasmids Luciferase and lacZ expression vectors pT3-luc and pT3-lacZ were kindly provided by Dr. Coll (INSERM-UJF U823, France). 19 pcDNA3-flag-β2AR is purchased from Addgene (#14697). Plasmids were purified using Mega-prep endotoxinfree kit (Qiagen, Hilden, Germany).
The lung section, X-gal staining, and haematoxylin/eosin (HE) staining were performed as previously described, 19 and are described in detail in the ESM. Real-time PCR RNAs extraction and real-time PCR were described previously, 20 and are described in detail in the ESM. Measurement of alveolar fluid clearance rate in live mice
Mouse Model of ALI Five-week-old Bltw:CD1(ICR) mice were purchased from BioLasco Taiwan Co., Ltd. and maintained in Taiwan Mouse Clinic in Institute of Biomedical Sciences, Academia Sinica. All animal experiment protocols are approved by Academia Sinica Institutional Animal Care and Utilization Committee. Mice were maintained in controlled environmental conditions of temperature (22 ± 2 °C) and humidity (60% ± 5%) with a strict 12 h light–dark cycle, and given free access to food and water. Mice were subjected to experiments and sacrificed within 1 week after receiving, with an average body weight around 19 ± 2 g. For ALI induction, mice were randomly grouped (n = 5 for each group), anesthetized by intraperitoneal injections with Tiletamine/Zolazepam (25 mg/kg) and Xylazine (10 mg/kg), and intratracheally instilled in lung with 70 μl of PBS or PBS containing 10 mg/kg body weight of LPS (E. coli serotype 055: B5, Sigma-Aldrich, St. Louis, MO, US) via a 20-gauge catheter. The mice were then maintained on spontaneous breathing at room air for later PEI/DNA nanoparticle treatments (see below). In vivo gene delivery in mouse lung In vivo delivery of PEI/DNA has been described previously. 19 The generation of nanoparticles was based on the electric charges between PEI (positive) and DNA (negative). Different quantities of DNA (generally 30 μg in therapeutic approach) were diluted in 5% glucose in a final volume of 100 μl. In another tube, linear 22-
The Evans Blue-labeled bovine serum albumin (EB-BSA) was freshly prepared before experiment by mixing EB (0.15 mg/ ml) in 5% BSA in Ringer's Lactate solution. The healthy or LPSinjured mice treated with PEI/DNA nanoparticles were anesthetized, maintained at 37 °C, and the trachea was cannulated with a 20-gauge catheter. A total of 400 μl of EB-BSA was instilled into the lung, and the mouse was ventilated for 20 min with a tidal volume of 10 ml/kg at a frequency of 90 breaths per minute with a ventilator (SAR-830 Small Animal Ventilators, CWEInc., Ardmore, PA, US). At the end of experiment, mouse chest was opened to allow aspiration of fluid from the tracheal catheter. The density of EB in aspirate was measured, and the alveolar fluid clearance rate was expressed as the percentage of cleared volume in 20 min, calculated with the equation: alveolar fluid clearance (%) = 100 × (1 − C0/C20), where C0 is the EBBSA concentration before instillation, and C20 is the EB-BSA concentration in the aspirate at the end of 20-min ventilation. Measurement of lung water content The level of lung water content was assessed by the ratio between wet lung to dry lung weight (wet-to-dry ratio), following a previous publication. 21 After irreversible anesthesia, mice were exsanguinated by laceration on heart. The lungs were removed and determined for wet lung weight. The lungs were then placed in an incubator at 70 °C for 72 h to remove all moisture, and dry lung weight was determined.
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Figure 1. PEI-mediated reporter gene delivery in healthy and injured lungs. PEI/DNA nanoparticles were injected through lateral tail-vein into healthy mice or mice with pre-existing ALI (A). Different amounts of luciferase expression vectors (0 to 50 μg, as indicated on the figure) were complexed with 22-kD linear PEI in a constant N/P ratio of 8. Luciferase reporter gene signal was followed and quantified using bioluminescent imaging at different time points after PEI/luc injection (B) (n = 5 for each group, error bars indicate Standard Deviation (SD)), and 2 mice were illustrated as samples of imaging (C). To determine the cell types targeted by PEI/DNA delivery, mice were injected with PEI/lacZ and sacrificed 1 day later. Lungs were stained with X-Gal and HE for histochemical examinations in × 400 (D) or × 1000 (E) magnification. Arrows indicate the X-Gal stained (blue) cells. Bar = 100 μm (D) or 20 μm (E).
Lung injury score The lung injury score was evaluated according to the criteria addressed. 22 The lung injury score was assessed on histopathol-
ogy by 2 clinicians (S-D Yeh and K-Y Yang) independently and averaged. For each condition, 15 fields (× 400 magnification, including a total of N 300 alveoli) of lung sections from 5 mice were analyzed.
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Bronchioalveolar lavage analysis The bronchioalveolar lavage fluid was collected on 48 h postinjury. The collection was performed according to a previous publication, 21 and is described in detail in the ESM. Survival analysis For survival analysis, mice were intratracheally instilled with 70 μl of PBS containing 40 mg/kg of LPS, and then randomly grouped (n = 5 for each group) to receive PEI, PEI/luc, or PEI/ β2AR treatment 1 h later, or not treated. The deaths of mice were recorded since 6 h after LPS instillation to day 5. Five independent experiments were performed, and the results were combined in one graph. Statistic analysis The statistical significances of the experimental results were assessed by independent-samples t test (Two-tailed) using SPSS Statistics v.20 (IBM, Armonk, NY, US). For survival analyses, Gehan–Breslow Test was used. P b 0.05 is considered significant. Results PEI/DNA-mediated gene delivery in mouse lung under healthy condition or ALI The particles complexed by PEI and DNA were analyzed, which showed average sizes around 60 nm and zeta potentials around 30 mV (Figure S1). After intravenous injection of PEI/ DNA in mice, the kinetics and target tissues of PEI/DNAmediated gene delivery were analyzed. The reporter gene expression was found mainly in lung, where it was approximately 100-fold higher than in other organs (Figure S2). The reporter gene signal in lung was also followed and quantified using non-invasive bioluminescent imaging (Figure 1, A to C). The results showed that PEI/luc induced a rapid and efficient transgene expression in lung as soon as 6 h post-injection, which then declined and became nearly undetectable on day 4. The transgene expression levels were positively related to the amounts of DNA delivered. We then administered PEI/luc in mice with pre-existing ALI induced by LPS. PEI/luc was injected in mice 1 h after LPS instillation, and we found that however, delivery of a dose of 50 μg of DNA resulted in a high mortality (90%, data not shown). A lower dose of DNA (30 μg), in contrast, did not result in any death. The luciferase expression kinetics in mice with ALI were similar to those in healthy ones, although the level was reduced by around 50% (Figure 1, B and C). PEI/lacZ was administered to identify the target cells of gene delivery in lung, and the results showed that alveolar epithelial cells were the major targets (Figure 1, D and E), and the pre-existing ALI did not block or alter the delivery. The identities of target cells were further confirmed by immuno-histochemical staining against Aquaporin 5 and Surfactant Protein C, which are specific markers of alveolar epithelial cells type 1 and 2, respectively. The cells co-stained with lacZ and these markers were observed (Figure S3).
Figure 2. Exogenous and endogenous β2AR gene expressions in mice lungs with ALI. ALI was induced in mice by intratracheal instillation of 10 mg/kg of LPS. The mice were then injected with PEI/β2AR 1 h post-injury (A and B), or not injected (C). Mice were sacrificed at indicated time points after PEI/ β2AR injection, and the lungs were harvested for RNA extraction. PBS was instilled into mice lungs as non-injury control (PBS). Exogenous (human) (A) and endogenous (mouse) (B and C) β2AR gene expressions were detected using Real-Time PCR with specific primers, and presented as ratios relative to PBS group. Error bars indicate SD, and n = 5 for each group.
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Figure 3. PEI/β2AR treatment improved alveolar fluid clearance and reduced lung water content in mice with pre-existing ALI. PEI/β2AR treatment was administered in mice 24 h post-injury (A). For control, mice were not treated, or treated with PEI/luc or PEI. PBS was instilled into mice lungs as non-injury control. Alveolar fluid clearance was measured in vivo and shown as percentage of total instilled volume cleared in 20 min (B). Lung water content was assessed by the measurement of wet-to-dry ratio (C). Error bars indicate SD, and n = 5 for each group.
Exogenous and endogenous β2AR gene expressions in mice lungs with ALI The human β2AR gene was applied as therapeutic gene in this study. PEI/β2AR was injected in mice with pre-existing ALI (Figure 1, A), and the lung proteins/mRNAs were extracted. The β2AR protein expression was verified using western blot analysis (Figure S4). The kinetics of exogenous (human) β2AR expression (Figure 2, A) were similar to those of luciferase observed in bioluminescent imaging (Figure 1, B). Strikingly, we found that at any time point after gene delivery, endogenous (mouse) β2AR expression was substantially repressed (Figure 2, B). In mice with LPS-induced ALI but without PEI/β2AR treatment (Figure 2, C), we found that the expression of endogenous β2AR was also significantly repressed up to 4 days. PEI/β2AR treatment improved alveolar fluid clearance and reduced lung water content in mice lungs with pre-existing ALI In our pilot study, it showed that LPS in a dose of 10 mg/kg efficiently induced an ALI syndrome in mice (Figure S5), and the pulmonary inflammation lasted for at least 48 h (Figure S6). We thus designed the therapeutic model by administering the PEI/β2AR treatment 24 h post-injury, and analyzing the outcome at 48 h (Figure 3, A). We first assessed the influence of PEI/β2AR treatment in alveolar fluid clearance. As illustrated on the figure, LPS-induced ALI led to a significant loss of alveolar fluid clearance activity (Figure 3, B). Treatment with PEI/β2AR recovered the alveolar fluid clearance activity to the level corresponding to the non-injury control. In contrast, PEI/
luc or PEI showed no effect. We then analyzed the level of lung water content by measuring the ratio of wet lung to dry lung (wet-to-dry) weights. In accordance with alveolar fluid clearance, ALI induced an increase in wet-to-dry ratio, and PEI/β2AR treatment reduced it to a nearly normal level (Figure 3, C), while treatment with PEI/luc or PEI showed no effect. PEI/β2AR treatment improved histopathology and bronchioalveolar lavage indexes in mice lungs with pre-existing ALI We then examined the lung histopathology. As expected, LPS instillation caused extensive morphological damages including edema, hemorrhage, thickness of alveolar walls, and infiltration of neutrophils in alveolar and interstitial spaces (Figure 4). These morphological changes were much less pronounced after PEI/ β2AR treatment. We also investigated the bronchioalveolar lavage indexes. In accordance with histopathology, ALI induced dramatic increases in cell number, protein concentration, and inflammatory cytokines TNF-α and IL-6 in bronchioalveolar lavage, as compared to non-injury control (Figure 5). Once more, PEI/β2AR treatment significantly attenuated the severity of these indexes, while PEI/luc or PEI treatment showed no effect. PEI/β2AR treatment improved the survival of mice in high-dose LPS-induced ALI In our pilot study, LPS in a dose higher than 40 mg/kg induced a high mortality in mice (Figure S7). In survival assay performed by intratracheal instillation of 40 mg/kg of LPS, the survival rate of mice dropped rapidly to 60% on 6 h, and to 40% on day 1 (Figure 6). PEI/β2AR treatment was administered 1 h
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Figure 4. PEI/β2AR treatment improved histopathology in mice with pre-existing ALI. PEI/β2AR treatment was administered as presented in Figure 3, A. For control, mice were not treated, or treated with PEI/luc or PEI. PBS was instilled into mice lungs as non-injury control. Histopathology was investigated in lung sections (5 μm, paraffin embedded) after HE staining. The lung injury scores were assessed in 15 fields (× 400 magnification, including a total of N300 alveoli) of lung sections, taken from 5 mice for each group. Bar = 100 μm. Error bars indicate SD.
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Figure 5. PEI/β2AR treatment improved bronchioalveolar lavage indexes in mice with pre-existing ALI. PEI/β2AR treatment was administered as presented in Figure 3, A. For control, mice were not treated, or treated with PEI/luc or PEI. PBS was instilled into mice lungs as non-injury control. The cell numbers (A), protein concentrations (B), and the levels of pro-inflammatory cytokines TNF-α (C) and IL-6 (D) in bronchioalveolar lavage were measured. Error bars indicate SD, and n = 5 for each group.
post-injury, which provided a significant improvement in survival rate, which was 92% on 6 h and 80% on day 1, respectively. No more death was observed after day 4, and the 5day survival rate was 64%, 15%, 5%, and 28% in PEI/β2AR-, PEI/luc-, PEI-, and non-treated groups, respectively. PEI/β2AR treatment showed statistically significant survival benefit (P b 0.01) as compared to any other group; while no statistical difference was found between the 3 other groups.
Discussion This is the first study testing the use of PEI/DNA nanoparticles for the delivery of a therapeutic gene in the treatment of LPS-induced ALI in an animal model. Our findings suggest that the treatment with PEI/β2AR is safe in the context of LPS-induced ALI, and provides rapid and timely benefits in mice. Like previous studies, 7-10 our reporter gene assay showed that systemic administration of PEI/DNA nanoparticles in vivo mediated an efficient gene delivery mainly in alveolar epithelial cells (Figure 1 and S3). The transgene expression was rapid (as soon as in 6 h post-injection) and short-lived (b 4 days). Despite a lower efficiency, PEI/DNA delivery is attainable in alveolar epithelial cells in lungs with pre-existing ALI. In the approach of therapeutic treatment, PEI/β2AR was administered in mice 24 h post-injury, a time point where ALI syndrome was already obvious (Figure 3, A and S6), to mimic a therapeutic, but not
prevention model. The results showed that PEI/β2AR treatment significantly attenuated the ALI severities, including alveolar fluid clearance, lung water content, histopathology, bronchioalveolar lavage cell number, protein concentration, and inflammatory cytokines (Figures 3 to 5). In survival analysis, because of the rapid mortality induced by high-dose LPS within in 24 h (the survival rate dropped to 40% in the absence of therapeutic treatment), PEI/β2AR treatment was administered 1 h postinjury. The result showed that PEI/β2AR provided a rapid and timely survival benefit in mice with ALI induced by high-dose LPS, with a 5-day survival rate of 64% in comparison with control groups, which showed a survival rate of b 28% (Figure 6). Despite of extensive researches in pathophysiology, ALI/ ARDS remains a devastating syndrome with a high mortality. β2AR signaling has gained considerable interest in ALI/ARDS therapy because of its ability to improve the resolution of pulmonary edema. 11 Unfortunately, 2 large-scale randomized controlled trials have recently been terminated because of futility and concerns about safety. 4,5 The failure could be attributed to adverse effects including cardiac effects and vasodilatation because of the systemic administration of β2AR agonist, and the desensitization/down-regulation of β2AR after prolonged agonist stimulation. 23-26 Different from past researches that focused on the development of β2AR agonists, our study here presented a PEI/DNA-nanoparticle based gene therapy, and the results showed important clinical implications. First, although the
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Figure 6. PEI/β2AR treatment improved the survival of mice in high-dose LPS-induced ALI. For survival assay, mice were intratracheally instilled with highdose LPS (40 mg/kg), and then treated with PEI/β2AR 1 h post-injury. For control, mice were not treated, or treated with PEI/luc or PEI. The survival of mice was recorded since 6 h post-injury. No more death was noticed after day 4. N = 25 for LPS and LPS + PEI/β2AR, and n = 20 for LPS + PEI/luc and LPS + PEI groups. P value was calculated by Gehan–Breslow statistic analysis.
administration of nanoparticles was systemic, the major targets of gene delivery were alveolar epithelial cells (the pathological loci of ALI), and the reporter gene signal in lung was approximately 100-fold higher than in heart or other organs (Figure 1 and S2). Such distribution would minimize the adverse effects like cardiac effect observed in the use of agonist. Second, in this study we found that the expression of endogenous (mouse) β2AR was significantly repressed in the presence of ALI (Figure 2, C). This finding would reflect a previous investigation that pulmonary edema in ALI patients is due not only to the increased fluid flux in airspace, but also the impairment of alveolar fluid clearance mechanism. Alveolar fluid clearance could still be impaired despite high levels of endogenous catecholamines, and the treatment of exogenous pharmaceuticals (e.g. albuterol) did not correlate with the rate of alveolar fluid clearance. 27 Hence, the repressed endogenous β2AR expression could be an undescribed reason of impaired alveolar fluid clearance mechanism and the failure of β2AR agonist to improve the clinical outcome. Accordingly, the PEI/ β2AR gene therapy model presented here could address such problem since it activates β2AR signaling by direct overexpression of β2AR, but not by agonists that need the endogenous β2AR expressed on cell surface.
Although gene therapy is a promising approach for treatments of various diseases, it has not been well-developed in ALI. Early researches have shown that the overexpression of β2AR in mouse lung increased alveolar fluid clearance and protected animals from the later-induced ALI. 15-18 These research models based on adenovirus-mediated gene delivery, however, were not applicable in subsequent clinical therapeutic treatment because of the safety concerns of viral vector. A unique research performing the feasible post-injury gene therapy in animal model was reported in 2007, 21 in which the Na +,K+-ATPase genes were delivered in lung by electroporation. Improvements in alveolar fluid clearance and respiratory mechanics were observed after gene delivery in mice with pre-existing ALI induced by LPS. However, the treatment was not able to ameliorate the syndrome or rescue the survival when severe ALI was induced by high-dose LPS. The authors thus concluded that although the delivery of therapeutic genes can be an effective and logical treatment for ALI, its application still needs refinement. 21 In our study, we used PEI/DNA-nanoparticles for β2AR gene delivery. The sizes of nanoparticles play a key role in their biodistribution and final target in vivo. Particles less than 5 nm are rapidly cleared from the circulation through extravasation or renal clearance, and particles as large as 15 μm or more
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accumulate in liver, spleen, and bone marrow. Smaller microparticles (roughly, from 1 to 10 μm) are mainly internalized by phagocytosis in special cell types such as macrophage and neutrophil, a condition that is undesired for most drug delivery. 29,30 Therefore, keeping the particles small (between 10 and 100 nm) facilitates their diffusion in the blood and tissues and the cellular internalization through endocytosis, and has become a foundation for current definition of nanomedicine. 30 In this study, we showed that nanoparticles formed by 22-kD linear PEI and DNA in non-ionic solution (5% glucose), like previous studies, 8,31-33 possessed sizes b 100 nm (Figure S1). These nanoparticles were able to diffuse across the endothelial lining and to target alveolar epithelial cells (Figure 1). Although the exact mechanism of PEI/DNA internalization in epithelial cells remains controversial (as reviewed by Sahay et al), 30 our study provides the first evidence that this kind of nanoparticles can be used in ALI treatment by therapeutic gene delivery in a postinjury model in animals. The gene delivery was rapid, efficient and short-lived (Figure 1), and did not further aggravate the lung inflammation, while showing therapeutic benefits (Figures 3 to 5). Importantly, PEI/β2AR was able to rescue the survival of mice in high-dose LPS-induced ALI (Figure 6). These properties potentiate PEI/β2AR as a proper reagent for the clinical treatment of ALI, because it is effective and minimizes the risks of mutational insertions, carcinogenesis, and the induction of strong inflammatory responses that may arise when using viral vectors. Although PEI-mediated gene delivery in vivo has been sufficiently addressed in many references, it has never been applied to deliver a therapeutic gene for ALI/ARDS treatment. A possible reason can be the safety concern. An early study showed that increasing the dose of PEI/DNA resulted in a marked augmentation of transgene expression, but liver necrosis and mouse death were reported when a high-dose DNA (over 100 μg) and high N/P ratio (= 10) were administrated. 34 The improper doses of DNA used in that research were, however, to evaluate the potential adverse effects of PEI/DNA, but not actually applied in common research models or clinical trials. 10 In our study, we did observe a high mortality (90%, data not shown) when a higher-than-proper dose of DNA (50 μg) was injected in mice with pre-existing ALI. Nevertheless, a moderate dose of DNA (30 μg) was able to transfect mouse lung with preexisting ALI without inducing any death, while showing therapeutic benefits. It thus suggests that in an acute and devastating syndrome like ALI/ARDS, an optimized protocol of gene delivery, which may not necessarily be the one showing the highest transfection efficiency, has to be carefully defined. For an effective treatment, a balance between efficiency and adverse effects has to be found, and here we show that it can be achieved in this gene therapy model. Inflammatory responses attributed to PEI/DNA administration have also been reported. 35-41 PEIs associated with these adverse effects, however, were uniformly in branched, but not linear forms. 35,37 A recent study using linear 22-kD PEI clearly showed no major production of proinflammatory cytokines or hepatic enzymes after systemic injection of PEI/DNA or PEI/siRNA. 10 In our study, we did not observe any additional pro-inflammatory effect other than LPS induction when using such linear PEI/DNA for treatment. Instead, PEI/β2AR treatment attenuated the inflammatory
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response as evidenced by the histopathology and cytokine measurements (Figures 4 and 5). Therefore, PEI/DNA nanoparticle-based gene therapy, at least in the context of endotoxemiaassociated ALI, is safe and advantageous. This study still has some limitations. First, the reason of the high mortality (90%, data not shown) induced by a higher-thanproper dose of DNA (50 μg) in mice with LPS-induced ALI remains unclear. Although the PEI in linear form, as described above, does not induce inflammatory response, PEI is known to interact with LPS based on electric charge. 42,43 Therefore, it is possible that PEI/DNA administration in the presence of LPS induced accumulations of macro-aggregations that led to in vivo toxicity. The exact mechanism of such PEI/DNA-induced in vivo toxicity, however, must be clarified before PEI/DNA moves toward clinical applications. Second, concerning the clinical application of gene delivery in lung, systemic injection with PEI/ DNA may face the problems of consumptions (in proportion, over 100 mg of DNA should be used in a 65-kg human adult according to this study) and other unexpected adverse effects that are not predicted in mouse model. Indeed, the report on the feasibility of systemic delivery of PEI/DNA in large animal or human is currently absent, although a clinical trial that applies nanoparticles containing PEI/DNA/siRNA 44 in multiple myeloma patients by intravenous infusion has been approved very recently (NCT01435720 on ClinicalTrials.gov). Local administration like intratracheal/aerosol delivery could be considered more preferable. However, in our gene delivery system with linear 22-kD PEI, we found so far that intravenous injection was the most efficient for gene expression in mice lungs, while intratracheal administration was insufficient (data not shown). The successful aerosol delivery of PEI/DNA found in literatures thus far depends on the use of branched PEI. Nevertheless, as mentioned above, branched PEI has been mentioned in many references to induce inflammatory response. Therefore, the optimization of PEI-based gene delivery in clinical application is still awaiting more studies. Third, although our finding suggested that β2AR expression was repressed in ALI, it's not clear whether the repression was a result of lung epithelial cell death or LPS-induced modulations on gene expressions. Since LPS has been known to influence gene expression profile through multiple transcription factors, 45 it is possible that β2AR repression was a consequence of LPS-modulated gene transcriptions in alveolar epithelial cells. The exact mechanisms associated with the repression, however, need more studies in the future. Fourth, although alveolar epithelial cells are the major targets of gene delivery, endothelial cells could still be transfected and contribute to some beneficial effect, which was not dissected in our current works. Finally, protective mechanical ventilation has been proven to improve survival and becomes a standard management on ALI/ARDS patients. Whether PEI/β2AR will provide additive benefits on patients that are already subjected to protective ventilation is uncertain, and thus needs further pre-clinical/clinical studies to clarify. In conclusion, our research here presents a simple, safe, and effective gene therapy for ALI in mouse model. PEI/DNA nanoparticle-mediated gene delivery in mouse lung is rapid, efficient, and short-lived. PEI/β2AR treatment significantly attenuated the severity of ALI and improved the survival of mice,
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without a major adverse effect observed. The PEI/DNA nanoparticle-based gene therapy could have great potential in future clinical applications. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2013.05.004.
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