Inhalable Gene Delivery System Using a Cationic RAGE-Antagonist

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Inhalable gene delivery system using a cationic RAGEantagonist peptide for gene delivery to inflammatory lung cells Chunxian Piao, Gyeungyun Kim, Junkyu Ha, and Minhyung Lee ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.9b00004 • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 30, 2019

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ACS Biomaterials Science & Engineering

Inhalable gene delivery system using a cationic RAGE-antagonist peptide for gene delivery to inflammatory lung cells

Chunxian Piao, Gyeungyun Kim, Junkyu Ha, and Minhyung Lee* Department of Bioengineering, College of Engineering, Hanyang University, Seoul 04763, Korea

*Corresponding author: Minhyung Lee, Ph.D. Tel: +82-2-2220-0484, Fax: +82-2-2220-4454, E-mail: [email protected]

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Abstract Acute lung injury (ALI) is a severe lung inflammatory disease. In ALI, the receptor for advanced glycation end-products (RAGE) is over-expressed in lung epithelial cells and involved in inflammatory reactions. A previous report showed that a RAGE-antagonist peptide (RAP), from high-mobility group box-1, bound to RAGE and reduced inflammatory reactions. RAP has high levels of positive amino acids, which suggests that RAP may form a complex with plasmid DNA (pDNA) by charge interactions. Because the charge density of RAP is lower than polyethylenimine (25 kDa, PEI25k), it may be able to avoid capture by the negatively-charged mucus layer more easily and deliver pDNA into RAGE-positive lung cells of ALI animals by RAGEmediated endocytosis. To prove this hypothesis, RAP was evaluated as a delivery carrier of adiponectin plasmid (pAPN) in lipopolysaccharide (LPS)-induced ALI animal models. In vitro transfection assays showed that RAP had lower transfection efficiency than PEI25k in L2 lung epithelial cells. However, in vivo administration to ALI animal models by inhalation showed that RAP had higher gene delivery efficiency than PEI25k. Particularly, due to a higher expression of RAGE in lung cells of ALI animals, the gene delivery efficiency of RAP was higher in ALI animals than that in normal animals. Delivery of the pAPN/RAP complex had anti-inflammatory effects, reducing proinflammatory cytokines. Hematoxylin and eosin staining confirmed that pAPN/RAP decreased inflammation in ALI models. Therefore, the results suggest that RAP may be useful as a carrier of pDNA into the lungs for ALI gene therapy.

Keywords: Acute lung injury, adiponectin, gene delivery, RAGE-antagonist peptide, receptor for advanced glycation end-products 2 ACS Paragon Plus Environment

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INTRODUCTION Acute lung injury (ALI) is an inflammatory disease caused by various direct and indirect injury factors.1 Sepsis, infections, ischemia/reperfusion, and massive blood transfusions induce ALI. ALI is characterized by increased permeability of endothelium and epithelium and loss of vascular integrity. This leads to a leak of protein-rich fluids into the alveolar space.2-3 As a result, the damage causes inefficient gas exchange and cell death of type I epithelial cells.3 Therapeutic options for ALI are limited and mechanical respiratory aid with a ventilator is a main clinical option.4 However, mechanical aid cannot reduce inflammation in the lungs and furthermore, high oxygen supply with a ventilator may induce hyperoxia, increasing cell death in the lungs.5 Steroid antiinflammatory drugs may help reduce inflammatory reactions in the lungs. However, the therapeutic effects of the anti-inflammatory drugs are controversial and not applicable to clinical settings.6-8 Therefore, more effective therapeutic options should be developed for ALI treatment. Many studies for the development of effective therapies for ALI have been conducted, including gene therapy. Previous reports showed that gene delivery using polymeric carriers or stem cells reduced inflammatory reactions and ameliorated ALI in animal models, suggesting that gene therapy may be a promising therapeutic option for ALI.9-14 For clinical applications of gene therapy against ALI, safety and efficiency of the gene therapy system should be achieved. In this viewpoint, one of the main problems in ALI gene therapy is to develop a safe and efficient gene delivery carrier. In previous studies, polymeric gene carriers were evaluated for gene delivery into the lungs of ALI animal models. The carriers include reducible poly(oligo-D-arginine) (rPOA), 3 ACS Paragon Plus Environment


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dexamethasone-conjugated polyethylenimine (PEI-Dexa), dexamethasone conjugated polyamidoamine (PAM-Dexa), and R7L10 peptide micelles.13,

15-17

Studies of gene

delivery into ALI animal models indicated that the polymeric carriers delivered genes into the lungs more efficiently than polyethylenimine (25 kDa, PEI25k). Furthermore, the toxicities of the carriers were lower than PEI25k. However, the carriers did not have specificity of gene delivery for higher transfection into the lung cells. In addition, the carriers were not free from cytotoxicities, since they had high positive charge densities. It was previously reported that the high charge density might be the cause of cytotoxicity.18 Considering this, a carrier with lower charge density and high delivery efficiency to the lung cells in ALI is desirable. Taken together, peptides from nuclear proteins may be more appropriate candidates for gene delivery. First, nuclear proteins such as histones and high mobility group box-1 (HMGB1) have 20~30% arginine and lysine amino acid composition. Therefore, charge densities of nuclear proteins are lower than synthetic polymeric carriers such as PEI25k and polyamidoamine (PAMAM). Indeed, peptides from histone 1 and HMGB1 were reported as gene carriers and they delivered genes into cells in vitro.19-21 Second, nuclear proteins are endogenous proteins, which are free from immune reactions. Recently, a peptide from HMGB1 was developed as an antagonist peptide against the receptor for advanced glycation end-products (RAGE).22 RAGE is a typical patternrecognition receptor23-24 that binds to endogenous damage-associated molecular patterns (DAMP) such as high-mobility group box-1 (HMGB1), -amyloid, and S100 proteins as well as pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS).24-27 On activation of RAGE by its ligands, it activates mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathways, inducing 4 ACS Paragon Plus Environment

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nuclear translocation and activation of nuclear factor-B (NF-B).27-28 This results in the increase of pro-inflammatory cytokines. It was previously reported that RAGE is a mediator of inflammation in ALI.29-30 The RAGE-antagonist peptide (RAP) was a recombinant peptide from the RAGE-binding domain of HMGB1. Notably, inhalation of RAP into ALI animal models had anti-inflammatory effects.14, 22, 26 In the view of gene delivery, RAP has an interesting peptide with high levels of lysines and arginines. In the current study, it was hypothesized that RAP might bind to plasmid DNA (pDNA) by charge interaction and deliver it into cells. Furthermore, RAP may be able to deliver pDNA into RAGE-positive cells, due to specific interactions with RAGE. Therefore, RAP may be a useful gene carrier for inflammatory diseases such as ALI, since RAGEs are over-expressed in inflammatory cells.31 In the current study, RAP was evaluated as a carrier of the adiponectin (APN) gene into the lungs of ALI animal models. Adiponectin (APN) is a signaling molecule that is an adipose tissue-derived factor and mainly secreted from adipocytes.32 APN possesses a variety of biological effects including anti-inflammation, vascular protection, cardio-protection, and metabolic actions.33-36 Previously, it was shown that APN gene delivery had cytoprotective effects in ALI animal models. In this study, physical characterization was performed to identify the complex formation between RAP and pDNA. Gene delivery into the LPS-induced ALI animal models was performed to evaluate gene delivery efficiency and therapeutic effects of the pAPN/RAP complex. The results suggest that RAP is a useful gene carrier for the treatment of ALI.

MATERIALS AND METHODS 5 ACS Paragon Plus Environment


Materials PEI25k, LPS (Escherichia coli 055:B5), (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from Sigma–Aldrich (Saint Louis, MO). Phenylmethyl sulfonyl fluoride (PMSF) and bicinchoninic acid (BCA) assay reagents were purchased from Thermo Fisher Scientific (Waltham, MA). The L2 and Raw264.7 cell lines were obtained from the Korea Cell Line Bank (Seoul, Korea). Roswell Park Memorial Institute (RPMI) 1640, Dulbecco’s Modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Welgene (Seoul, Korea). The luciferase assay kit, reporter lysis buffer, and pEmpty (pCI) were purchased from Promega (Madison, WI). Tumor necrosis factor (TNF)-α and interleukin (IL)-6 enzyme-linked immunosorbent assay (ELISA) kits were purchased from eBioscience (San Diego, CA). The APN ELISA kit was obtained from Komabiotech (Seoul, Korea). Endofree Plasmid Maxi kits and Ni-NTA were purchased from Qiagen (Valencia, CA). APN and RAGE antibodies were purchased from Abcam (Cambridge, MA). Anti-GFP antibody was purchased from Invitrogen (California, US). In situ BrdU-Red DNA fragmentation TUNEL assay kit was obtained from Abcam (Cambridge, MA).

RAP expression pET21a-RAP was constructed previously.22 pET21a-RAP transformed BL21 bacteria were cultured in 20 ml LB medium for 4 h at 37C. The bacterial culture was added to 1 L of LB medium and cultured until the optical density reached between 0.6 and 0.8 at the wavelength of 600 nm. Then, IPTG was added to the medium at a concentration of 500 M. The bacteria were further cultured in a shaking incubator at 6 ACS Paragon Plus Environment

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26°C overnight. The bacteria were harvested and resuspended in lysis buffer (6 M urea, 50 mM NaH2PO4, 10 mM imidazole, and 300 mM NaCl, pH 8.0). Then, PMSF and lysozyme were added to the samples. The cells were subjected to sonication (8  25 bursts on ice) for lysis. The cell debris was removed by centrifugation at 7000  g for 45 min. The supernatant was applied to column chromatography for purification.

Nickel-affinity chromatography The expressed RAP in bacteria was purified by nickel-affinity chromatography. A nickel-affinity column was washed with equilibration buffer (6 M urea, 20 mM imidazole, 50 mM NaH2PO4, and 300 mM NaCl, pH 8.0). The cell lysate was loaded onto the column. The unbound proteins were eluted with equilibration buffer. The bound RAP proteins were eluted with a imidazole step gradient (50 - 200 mM) in elution buffer (6 M urea, 50 mM NaH2PO4, and 300 mM NaCl, pH 8.0). The protein concentrations of the eluted fractions were measured by the BCA assay kit. The eluent was dialyzed against distilled water containing 0.15 mM PMSF using a dialysis membrane (MWCO, 2000 Da) at 4°C overnight. The proteins were analyzed by SDS-PAGE.

Size and zeta potential of pDNA/RAP complexes The pAPN/RAP complexes were prepared at various weight ratios. The pAPN/PEI25k complex optimal ratio was 1:1, based on previous studies. The complexes were fixed at a concentration of 5 μg/ml pAPN in distilled water or 78 mM NaCl solution. The particle size and zeta potential of the complexes were measured by Zetasizer Nano ZS system (Malvern Instruments, Worcestershire, UK).



Scanning electron microscopy (SEM) imaging pAPN/RAP complexes were prepared and mounted on a silicon wafer. The complexes were dried overnight under vacuum. The dried complexes were coated for 50 s with silver under vacuum. The images of the complexes were obtained by SEM (NANO SEM 450, FEI, Hillsboro, OR).

Gel retardation assays pAPN/RAP and pAPN/PEI25k complexes were prepared at various weight ratios and incubated for 30 min at room temperature. After incubation, the complexes were electrophoresed on 1% agarose gels for 30 min. pDNAs were visualized on a UV transilluminator.

Heparin competition assays pAPN/RAP and pAPN/PEI25k complexes were prepared at 1:9 and 1:1 weight ratios, respectively. After 30 min of incubation, the complexes were incubated with increasing amounts of heparin for an additional 30 min. The complexes were analyzed on 1% agarose gel.

DNase I protection assay pDNA/RAP complex was prepared with 8 g pLuc in 250 l PBS at a 1:5 weight ratio. Naked pLuc was a control. The complex solutions were incubated with 4 units of DNase I for 15, 30, or 60 min at 37 °C. Fifty microliters of the samples were collected and the reaction was quenched with 50 l of 2 stop solution (80 mM EDTA and 2% SDS). To dissociate pDNA from the complexes, 1 l of heparin solution (20 mg/ml) was 8 ACS Paragon Plus Environment

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added to the mixtures and incubated for 30 min at room temperature. Then, pDNA was analyzed by 1% agarose gel electrophoresis.

Transfection and luciferase assays Rat lung epithelial L2 cells and Raw 264.7 cells were cultured in DMEM containing 10% FBS, respectively. They were propagated at 37°C in a humidified 5% CO2 incubator. L2 cells were seeded on 12-well cell culture plates at a density of 1105 cells/well and incubated 24 h before transfection. The p-Luc/RAP complex was prepared at various weight ratios (1:1, 1:3, 1:5, and 1:7). The p-Luc/PEI25k complex was prepared at a weight ratio of 1:1, based on previous studies. The amount of pDNA was 1 g in 20 l 5% glucose. Just prior to transfection, the medium was replaced with fresh RPMI without FBS. For competition assays, free RAP (5 or 20 g) was added and incubated with the cells before transfection. Then, the complexes were added to the wells and the cells were incubated for 4 h at 37°C. After incubation, the transfection mixture media were replaced with fresh RPMI containing 10% FBS and the cells were incubated for an additional 20 h. Then, the cells were washed twice with 1 ml DPBS and lysed in 120 μl reporter lysis buffer. The samples were centrifuged at 12,000 × g for 7 min to eliminate debris. Then, the supernatants were subjected to luciferase assay. The protein concentrations of the samples were measured using the BCA assay kit. Luciferase activities of the samples were measured using a luminometer (Berthold Detection System GmbH, Pforzheim, Germany). The results are presented as RLU/mg protein.

MTT assays The cultured L2 cells were seeded on 24-well cell culture plates at a density of 9 ACS Paragon Plus Environment


3104 cells/well and incubated 24 h before transfection. The complexes of naked pAPN, PEI25k alone, pAPN/PEI25k complexes, RAP alone, and pAPN/RAP complexes were prepared at their optimal ratios for transfection. The amount of pDNA was fixed at 1 g/well. Transfection was performed as described above for the luciferase assay. After 24 h transfection, 40 l of 5 mg/ml MTT solution were added to the wells, and the cells were incubated for an additional 4 h at 37°C. Then, the medium was removed, and 500 l of DMSO were added to the cells. The absorbance at 570 nm was measured using a microplate reader. Cell viability (%) was calculated as follows: Cell viability (%) = (OD570(sample) / OD570(control)) × 100.

Transfection and ELISAs Transfection of pAPN/carrier complexes to L2 cells was performed as described above. Naked pAPN, pAPN/PEI25k complexes, and pAPN/RAP complexes were prepared at their optimal ratios. After 24 h of incubation, the culture medium was harvested and analyzed by the APN ELISA according to the manufacturer’s manual. RAW264.7 murine macrophage cells were seeded in 12-well cell culture plates at a density of 1105 cells/well and incubated 24 h before transfection. Then, 10 ng/well of LPS were added to the cells and incubated for 4 h to activate the cells. The amount of plasmid DNA was fixed at 1 g/well. After the incubation, pAPN alone, pAPN/PEI25k complexes, RAP alone, or pAPN/RAP complexes were added to the cells and incubated for 4 h. pEmpty/PEI25k and pEmpty/RAP complexes were also transfected to RAW264.7 cells. The transfection mixture media were replaced with fresh RPMI containing 10% FBS and the cells were incubated for an additional 20 h in a CO2 incubator. The media 10 ACS Paragon Plus Environment

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were collected and the TNF- and IL-6 levels in the media were analyzed by ELISA according to the manufacturer’s instructions.

LPS-induced ALI animal models All animal experimental procedures were approved by the IACUC at Hanyang University (Accreditation number: 2018-0147A) and were performed in accordance with the institutional guidelines of the IACUC at Hanyang University. Male BALB/C mice were raised under special pathogen-free conditions. Mice (21 g) were anesthetized and subjected to intratracheal injection of 20 g of LPS in 50 l saline. pAPN/PEI25k and pAPN/RAP complexes were prepared at their optimal ratios (1/1 for PEI25k and 1/5 for RBP) in 100 l saline. The amount of pDNA was fixed at 5 g/mouse. The concentration of pDNA was 0.05 mg/ml. The complexes were administered into the lungs by intratracheal injection after 2 h of the LPS challenge. The animals were sacrificed at 24 h after the injection to harvest tissues and bronchoalveolar (BAL) fluid for further analysis.

Quantification of EGFP mRNA level (Realtime RT-PCR) Naked pEGFP, pEGFP/PEI25k and pEGFP/RAP complexes were administrated into ALI model mice and normal mice, respectively. After 24 h, lung tissue samples were harvested and embedded in paraffin. Total RNAs in paraffin embedded lung sections were extracted using miRNeasy PPFE kit according to the manufacturer's manual. Then, cDNA was synthesized using iScript cDNA synthesis kit according to the manufacturer’s manual. Reverse transcription was performed using SensiFAST SYBR No-ROX Kit and analyzed using an Applied Biosystems 7500 Real-time PCR System (Applied Biosystems, Waltham, MA). The sequences of the EGFP primers were as follows: forward primer, 5’11 ACS Paragon Plus Environment


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TTTAGTGAACCGTCAGATC-3’; reverse primer, 5’-AACAGCTCCTCGCCCTTG- 3’. The sequences of the GAPDH primers were as follows: forward primer, 5’AGACAGCCGCATCTTCTTGT-

3’;

reverse

primer,

5’-

CTTGCCGTGGGTAGAGTCAT- 3’.

TUNEL assay The pEGFP/PEI25k and pEGFP/RAP complexes were administrated into normal mice. After 24 h, lung tissues were harvested and fixed in 4% paraformaldehyde solution. The tissue samples were embedded in paraffin, cut into sections (5 μm thick). The TUNEL assay was performed using in situ BrdU-Red DNA fragmentation TUNEL assay kit, following the manufacturer's instructions. The nuclei were stained with 4′,6diamidino-2-phenylindole (DAPI). The samples were observed by fluorescence microscopy.

Immunohistochemistry (IHC) The lungs were fixed in 4% paraformaldehyde solution. The tissue samples were embedded in optimal cutting temperature (OCT) compound and cut into frozen sections (8 m thick). Immunohistochemical staining was performed using antibodies against APN, RAGE, and EGFP. The tissue sections were observed by fluorescence microscopy.

Preparation and ELISAs of BAL fluids and tissue extracts BAL fluids of lungs were lavaged twice with 1 ml of DPBS including 0.05 M EDTA. The harvested BAL fluids were centrifuged at 12,000×g for 10 min and the supernatants were stored at −80°C until analysis. Lung extracts were prepared by 12 ACS Paragon Plus Environment

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homogenization of the tissues in reporter lysis buffer. The homogenized tissues were transferred to microtubes and centrifuged at 12,000×g for 10 min. The supernatants were transferred to fresh microtubes. BAL fluids and homogenization samples were analyzed by TNF- and IL-6 ELISA.

Hematoxylin and eosin (H&E) staining Lung tissues were fixed in 4% paraformaldehyde solution. The samples were embedded in paraffin and cut into sections (5 m thick). The tissue sections were stained with H&E staining. The tissue sections were analyzed using a light microscope.

Statistical Analysis Data were analyzed for statistical differences using ANOVA, followed by the Newman-Keuls test. P-values less than 0.05 were considered statistically significant. Data are presented as mean ± standard deviation.

RESULTS Physical characterization of plasmid DNA/RAGE-binding peptide complex RAP was produced by recombinant DNA technology in bacteria, based on the sequence of the RAGE-binding domain of HMGB1. In the previous study, RAP bound to RAGE and inhibited RAGE-mediated signal transduction, decreasing inflammatory reactions in ALI animal models. The sequence of RAP is as follows: NH2MASMTGGQQMGRGSEFKLKEKYEKDIAAYRAKGKPDAAKKGVVKAEKSKKK KECTRLEHHHHHH-COOH (MW 7,273 Da). The content of lysines and arginines is 26% of total amino acids. The positive charge density of RAP is lower than that of PEI25k. 13 ACS Paragon Plus Environment


RAP was expressed in bacteria and purified by nickel affinity chromatography (Fig. 1A). The purification of RAP was confirmed by SDS-PAGE (Fig. 1B). To verify the complex formation between pDNA and RAP, pAPN/RAP complexes were prepared at various weight ratios. The size and surface charge of the complexes were measured. The size of the pAPN/RAP complex in water was around 190 nm at the range of weight ratio from 1:1 to 1:5. The size increased as the ratio of the pAPN/RAP complex increased up to a 1:9 weight ratio (Fig. 2A). The size in NaCl solution was also measured. The results showed that the sizes of pAPN/PEI25k and pAPN/RAP complexes increased, compared with the sample in water (Fig. 2A). The surface charge of the pAPN/RAP complex was around +14 mV at every weight ratio (Fig. 2B). In particular, this surface charge of pAPN/RAP complexes was lower than that of pAPN/PEI25k complexes (Fig. 2B). This may be due to the lower charge density of RAP than PEI25k. The morphology of pAPN/RAP complexes at a 1:5 weight ratio was investigated by SEM. The results showed that the pAPN/RAP complex had a spherical shape (Fig. 2C). However, pDNA alone did not show any definable shape. Formation of pDNA/RAP complexes was confirmed by gel retardation assays. A fixed amount of pDNA was mixed with increasing amounts of RAP. The results showed that pDNA was completely retarded at a 1:3 weight ratio (Fig. 3A). In contrast, PEI25k retarded pDNA at a 1:1 weight ratio (Fig. 3A), and this may be due to the higher charge density of PEI25k than RAP. The higher charge density of PEI25k may contribute not only to pDNA/PEI25k complexes at a lower weight ratio than pDNA/RAP, but also to the stability of pDNA/PEI25k complexes. To evaluate the stabilities of pDNA/RAP and pDNA/PEI25k complexes, heparin competition assays were performed. pDNA/RAP and pDNA/PEI25k complexes were prepared at 1:5 and 1:1 weight ratios, respectively. Then, 14 ACS Paragon Plus Environment

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increasing amounts of heparin were added to the complex. The results confirmed that pDNA/RAP complexes released pDNA with lower amounts of heparin than did pDNA/PEI25k, suggesting that pDNA/RAP complex had less stable than pDNA/PEI25k complex (Fig. 3B). DNase I protection assay was performed to evaluate the DNA protection ability of RAP. The results showed that naked pDNA was completely degraded by DNase I after 15 min incubation (Fig. 3C). However, pDNA in pDNA/RAP complex was protected from DNase I attack up to 60 min (Fig. 3C).

In vitro transfection and cytotoxicity to cultured L2 cells In vitro gene delivery efficiency of RAP was evaluated by transfection assays with the luciferase plasmid (pLuc). The transfection efficiencies of pLuc/RAP complexes at various weight ratios were measured by luciferase assays. The results showed that the pLuc/RAP complex had the highest transfection efficiency at a 1:5 weight ratio (Fig. 4A). Therefore, the ratio between pDNA and RAP was fixed at a 1:5 weight ratio. At this ratio, the transfection efficiency of the pLuc/RAP complex was lower than the pLuc/PEI25k complex (Fig. 4A). The cytotoxicity of pLuc/RAP complexes was compared with that of pLuc/PEI25k complexes. The results showed that PEI25k had higher toxicity to L2 cells, but naked pLuc, RAP alone, and pLuc/RAP complexes did not have any cytotoxicity to cells (Fig. 4B). To confirm the RAP-mediated transfection, the competition assays were performed with free RAP. The cells were pre-incubated with free RAP and then, transfection was performed with pLuc/RAP complex. pLuc/PEI25k complex was used as 15 ACS Paragon Plus Environment


a control. The results showed that the transfection with pLuc/RAP complex was decreased by the pre-incubation with RAP (Fig. 4C). However, the transfection efficiency of pLuc/PEI25k complex was not decreased as much as pLuc/RAP complex (Fig. 4C). This suggests that pLuc/RAP transfected cells by specific interaction between RAP and its receptor, RAGE. Since RAP has an antagonist effect on RAGE-mediated signal transduction, the RAGE-antagonist effect of pLuc/RAP complexes was also evaluated in LPS-activated RAW264.7 macrophage cells. RAW264.7 cells were activated with LPS treatment, and then pAPN/PEI25k, pAPN, RAP alone, or pAPN/RAP complexes were added to the cells. After 20 h, the pro-inflammatory cytokine levels were measured by ELISA to evaluate anti-inflammatory effects of RAP or pAPN/RAP complexes. The results showed that pAPN/PEI25k complexes and naked pAPN did not decrease the TNF- and IL-6 levels significantly (Fig. 5A and 5B). Since the transfection efficiency of naked pDNA was very low, the naked pAPN did not show any anti-inflammatory effects. However, pAPN/PEI25k complexes did not have anti-inflammatory effects (Fig. 5A and 5B), although PEI25k might deliver pDNA into the cells efficiently and express APN relatively high compared with naked pAPN. This low anti-inflammatory effect of the pAPN/PEI25k complex may be due to the cytotoxicity of PEI25k. Due to cytotoxicity of PEI25k, the cells might undergo necrosis and apoptosis. During the cell death process, HMGB1 might be released from dying cells and induce pro-inflammatory responses. To investigate the effects of PEI25k and RAP on cytokine expression, pEmpty/PEI25k and pEmpty/RAP complexes were transfected into the cells. As a result, pEmpty/PEI25k increased the expression of TNF-, as compared with control and pEmpty/RAP complex (Fig. 5C). In contrast, RAP alone and pAPN/RAP complexes reduced the cytokine levels 16 ACS Paragon Plus Environment

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effectively compared with pAPN/PEI25k complexes and naked pAPN (Fig. 5A and 5B). Furthermore, the pAPN/RAP complex reduced TNF- levels more efficiently than RAP alone (Fig. 5A). This tendency was also observed in the IL-6 levels (Fig. 5B), although the difference between RAP alone and pAPN/RAP complexes was not statistically significant. These results suggest that the pAPN/RAP complex may have additive antiinflammatory effects of APN and RAP, and the additive effects of pAPN and RAP may be useful for the treatment of ALI.

Gene delivery efficiency and therapeutic effects of pDNA/RAP complexes in LPSinduced ALI animal models To evaluate in vivo gene delivery efficiency of RAP, the pEGFP/RAP complex was administrated into the lungs by intratracheal injection. After 24 h, the lungs were observed by fluorescence microscopy to measure the EGFP expression. A pEGFP/PEI25k complex was administrated as a positive control. The results showed that EGFP expression was enhanced by the pEGFP/RAP complex, compared with the pEGFP/PEI25k complex (Fig. 6A). This enhanced gene delivery by RAP may be due to the lower charge density of RAP than PEI25k, which might be beneficial to escaping capture by negatively charged mucin in the mucus layer. The gene delivery efficiency of RAP in the lungs was further enhanced in the LPS-induced ALI animal models. It was previously reported that RAGE was over-expressed in the inflammatory lung epithelial cells (Fig. 6A). Therefore, RAP that is a RAGE-binding peptide might have higher chances of binding to the lung epithelial cells. As a result, the delivery efficiency of RAP was higher in the LPS-treated mice than in the normal mice. Realtime RT-PCR was performed to evaluate EGFP mRNA level. The results 17 ACS Paragon Plus Environment


showed that pEGFP/RAP complex had higher EGFP mRNA level than pEGFP/PEI25k complex (Fig. 6B). In particular, EGFP mRNA level by pEGFP/RAP complex was higher in the LPS treated lungs than in the normal lungs (Fig. 6B). The cytotoxicities of the complexes were evaluated by TUNEL assay. pEGFP/PEI25k and pEGFP/RAP complexes were administrated into the lungs of normal mice. After 24 h, the lungs were subjected to TUNEL assay. The results showed that pEGFP/PEI25k group had higher level of apoptotic cells than pEGFP/RAP and control group (Fig. 6C). The results suggest that pEGFP/RAP complex was less toxic to cells than pEGFP/PEI25k complex. pAPN/RAP complexes were administrated into the LPS-induced ALI mice to evaluate gene delivery and therapeutic effects of the complex. Naked pAPN and pAPN/PEI25k complexes were injected as controls. As a result, the expression of APN was induced in the pAPN/RAP complex group, compared with the naked pAPN and pAPN/PEI25k groups (Fig. 7). This result suggests that RAP is an efficient carrier of pDNA into the inflammatory lungs. A therapeutic effect of RAP is down-regulation of RAGE. RAGE, when it is activated by its ligand, activates signal-transduction pathways that induce the activity of NF-B. In turn, NF-B upregulates the expression of the RAGE gene, which is a positive feedback. Therefore, if RAGE-mediated signal transduction is inhibited, RAGE expression in the cells is also down-regulated. To evaluate this effect, RAGE was immuno-stained after injection of the complexes. The results suggest that RAGE expression was induced by the treatment of LPS compared with normal mice (Fig. 8). However, RAGE levels were down-regulated by treatment with pAPN/RAP complexes (Fig. 8). It seems that pAPN/PEI25k complexes also reduced RAGE levels in the LPS18 ACS Paragon Plus Environment

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treated mouse lungs. This down-regulation of RAGE by the pAPN/PEI25k complex may be due to the anti-inflammatory effects of APN. The therapeutic effects of APN gene delivery were measured by cytokine ELISAs. BALF from the lungs were subjected to TNF- and IL-6 ELISAs. The results showed that TNF- was reduced by delivery of pAPN/PEI25k, naked pAPN, RAP alone, and pAPN/RAP complexes. In particular, the TNF-a levels were reduced most effectively by the pAPN/RAP complex (Fig. 9A). Similar results were obtained from the IL-6 ELISAs. The IL-6 levels in the pAPN/RAP complex group were reduced compared with other groups. This suggests that the pAPN/RAP complex had the highest antiinflammatory effects in the tested groups (Fig. 9B). The lung tissues were also extracted and subjected to cytokine ELISAs. The results also suggest that the pAPN/RAP complex group had the highest anti-inflammatory effects, compared with naked pAPN, pAPN/PEI25k complexes, and RAP alone (Fig. 9C and D). The therapeutic effects of the pAPN/RAP complex in ALI was demonstrated by H&E staining of the lung tissues (Fig. 10). The results showed that pathophysiological changes such as hemolysis and infiltration of monocytes were reduced by the administration of pAPN/PEI25k complexes, naked pAPN, RAP alone, and pAPN/RAP complexes, compared with the LPS treatment group (Fig. 10). In particular, the pAPN/RAP complex reduced hemolysis and infiltration of monocytes most effectively (Fig. 10).

DISCUSSION Gene therapy requires development of a gene carrier with high delivery efficiency and low cytotoxicity. In this study, we have shown that RAP delivered pAPN 19 ACS Paragon Plus Environment


into ALI lungs more efficiently than PEI25k. In addition to higher gene delivery efficiency than PEI25k, RAP has anti-inflammatory effects by reducing RAGE expression. Therefore, RAP may be an efficient gene carrier for the treatment of ALI. RAP is an anti-inflammatory agent via binding to RAGE and down-regulation of RAGE-mediated NF-B activation as suggested in the previous report.22 Pulmonary RAGE expression is localized in the type I lung epithelial cells and alveolar macrophages.37 RAGE activation induced production of pro-inflammatory cytokines.3839

Therefore, RAP may interact with the RAGEs in type I epithelial cells and alveolar

macrophages for anti-inflammatory effects. However, RAP has not been evaluated as a gene carrier. With the positive charge, RAP formed stable complexes with pDNA. Gene delivery efficiency of RAP to the lungs was higher than PEI25k in the RAGE-induced animal models, although RAP had lower transfection efficiency in vitro than PEI25k. This may be due to the lower charge density of RAP compared with PEI25k. In the airways, epithelial cells are covered with a negatively charged mucus layer. Thus, positively charged molecules were captured by this layer and cleared though the gastrointestinal track. pDNA/polymer complexes for gene delivery usually contain a positive charge on the surface, since the complexes were prepared with excess cationic polymers for tight complex formation. The positive surface charge is useful in that the complexes can interact with negatively charged cell membranes easily, which may increase the chances of cellular uptake. However, excessive positive charge is a kind of barrier in lung gene delivery, due to clearance by the mucus layer. In previous reports, polyethylene glycol (PEG) was conjugated to gene carriers to shield the excessive positive charges of gene carriers.40 As a result, PEG-conjugated polymers had higher transfection efficiency than carriers without PEG. In another study, a positively charged 20 ACS Paragon Plus Environment

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anti-inflammatory peptide was complexed with negatively charge heparin to reduce capture by the mucus layer.29 These reports suggest that reducing the positive surface charge of nanoparticles is helpful to increase the delivery efficiency of positively charged reagents into the lungs. pDNA/RAP complex had lower positive charge than pDNA/PEI25k (Fig. 2B). Due to lower positive charge, the interaction between pDNA/RAP complex and mucus layer may be less than that between pDNA/PEI25k complex and mucus layer. Therefore, RAP may have higher transfection efficiency than PEI in the lungs in vivo, unlike in vitro results. The second possibility of higher transfection efficiency of RAP, compared with PEI25k, may be due to RAGE-binding activity. It was previously reported that RAP reduced RAGE in LPS-induced macrophages.22 In the current study, RAP had higher gene delivery efficiency to the cells in LPS-induced ALI animals compared with that of normal animals (Fig. 6). These findings suggest that RAP may bind to RAGE that is upregulated in inflammatory cells. Another advantage of RAP as a gene carrier is low cytotoxicity. RAP originated from an endogenous protein, HMGB1. First, RAP has a low charge density, whereas a high charge density is closely related to cytotoxicity. In the previous study, complexes with a high positive charge, such as the pDNA/PEI25k complex, aggregated on the surface of negatively charged cell membranes and ruptured the membranes, resulting in cell death.18 Therefore, a lower charge density reduces the possibility of aggregation on the cell membrane, reducing toxicity. In Fig. 4B, PEI25k alone or pLuc/PEI25k complexes had higher toxicity than control. However, RAP alone or pLuc/RAP did not show any toxicity to cells. In the current study, the APN gene was evaluated as a therapeutic gene. APN gene delivery using RAP reduced inflammatory reactions in ALI animal lungs. RAP had 21 ACS Paragon Plus Environment


an anti-inflammatory effect, but pAPN/RAP had a higher anti-inflammatory effect than RAP alone. These may be due to combinatorial effects of gene and peptide delivery. RAP reduced the expression of RAGE by inhibition of positive-feedback of RAGE-mediated signal transduction in the lungs in the ALI animal models.22 Therefore, RAP itself has a biological effect as an anti-inflammatory drug. APN was reported to inhibit the synthesis of pro-inflammatory cytokines by inhibition of NF-B activation.41-42 Therefore, RAP and pAPN may have additive effects in anti-inflammatory therapy. The possibility of RAP as a gene carrier to various organs or tissues other than lungs was not evaluated. Thus, further research should be performed to assess RAP as a gene carrier to other organs. However, RAP had a higher transfection efficiency to lungs in vivo and may be useful for the treatment of various lung inflammatory diseases such as asthma and cystic fibrosis as well as ALI. Further evaluation may be required to assess the usability of RAP in these lung diseases.

CONCLUSIONS RAP was evaluated as a gene carrier to the lungs of ALI animal models. The results showed that RAP had higher gene delivery efficiency to the lungs in vivo than PEI25k, especially in the lungs of ALI animal models. Furthermore, RAP had antiinflammatory effects, reducing the expression of RAGE. Together, the results suggest that RAP may be a useful carrier of anti-inflammatory genes such as the APN gene to the lungs for the treatment of ALI.

References 22 ACS Paragon Plus Environment

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FIGURE LEGENDS Fig. 1. Purification of RAP protein and SDS-PAGE. (A) Nickel chelate affinity 28 ACS Paragon Plus Environment

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chromatography. RAP was purified using nickel chelate affinity chromatography with increasing concentrations of imidazole gradient. RAP was eluted at 150 mM imidazole concentration. (B) SDS-PAGE with purified RAP. 1: Crude extracts, 2: Fraction from purified RAP, M: Molecular weight markers.

Fig. 2. Particle size, surface charge, and morphology of the pAPN/RAP complex. (A) Particle size and (B) zeta potential. pAPN/RAP complexes were prepared in water or 78 mM NaCl solution. The particle size and zeta potential were measured. The data are expressed as a mean value ± standard deviation of triplicate experiments. (C) SEM. pAPN/RAP complex was prepared at a weight ratio of 1:5 and the size and morphology were evaluated by SEM.

Fig. 3. Complex formation and stability assays. (A) Gel retardation assay. pAPN/RAP and pAPN/PEI25k complexes were prepared at various ratios. The complexes were analyzed by agarose gel electrophoresis. (B) Heparin competition assay. pAPN/RAP and pAPN/PEI25k complexes were prepared at weight ratios of 1:5 and 1:1. After 30 min, increasing amounts of heparin were added to the complexes. The complexes were analyzed by electrophoresis on a 1% agarose gel. (C) DNase I protection assay. pLuc/RAP complex was prepared at a 1:5 weight ratio. Naked pLuc was used as a control. DNase I were added to the complex solutions and the reaction mixtures were incubated for 15, 30, or 60 min at 37 °C. The reaction was quenched with stop solution. pDNA was analyzed by 1% agarose gel electrophoresis.



Fig. 4. Transfection efficiency and cytotoxicity of RAP in vitro. (A) Luciferase assay. pLuc/RAP complexes were prepared at various weight ratios and transfected into L2 lung epithelial cells. The transfection efficiencies were measured by luciferase assays. (B) MTT assay. Naked pLuc, RAP, pLuc/RAP, PEI25k, and pLuc/PEI25k were added to L2 cells and incubated for 4 h. After an additional 20 h, the toxicities of the samples were measured by MTT assays. (C) Competition assays. L2 cells were preincubated with free RAP before transfection. pLuc/RAP and pLuc/PEI25k were transfected into the cells. The transfection efficiencies were measured by luciferase assays. The data are presented as a mean value ± standard deviation of quadruplicate experiments. *P < 0.05 compared with the weight ratios of naked pLuc, 1/1, 1/3 and 1/5, but there was no statistical significance compared with 1/7. **P < 0.05 compared with the other groups. ***P < 0.05 compared with PEI25k and pAPN/PEI25k. ****P < 0.05 compared with control.

Fig. 5. The anti-inflammatory effects of pAPN/RAP and pEmpty/RAP in vitro. RAW264.7 cells were activated by LPS and pAPN/PEI25k, pAPN, RAP, and pAPN/RAP were added to the cells. The pro-inflammatory cytokine levels were measured by (A) TNF- and (B) IL-6 ELISAs. (C) TNF- ELISA after pEmpty/RAP transfection. pEmpty/PEI25k and pEmpty/RAP complexes were transfected into the RAW264.7 cells. The TNF- level was measured by ELISA. The data are presented as a mean ± standard deviation of quadruplicate experiments. *P < 0.05, **P < 0.05 compared with the other groups. #P

Inhalable Gene Delivery System Using a Cationic RAGE-Antagonist

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