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Targeting of the prostacyclin specific IP1 receptor in lungs with molecular conjugates comprising prostaglandin I2 analogues
Biomaterials 31 (2010) 2903–2911
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Targeting of the prostacyclin specific IP1 receptor in lungs with molecular conjugates comprising prostaglandin I2 analogues Johannes Geiger a, b, Manish K. Aneja a, Gu¨nther Hasenpusch a, Gu¨lnihal Yu¨ksekdag a, Grit Kummerlo¨we c, Burkhard Luy c, Tina Romer d, Ulrich Rothbauer d, Carsten Rudolph a, b, * a
Department of Pediatrics, Ludwig-Maximilians-University Munich, 80337 Munich, Germany Department of Pharmacy, Free University of Berlin, 14166 Berlin, Germany ¨t Mu ¨ nchen, 85747 Garching, Germany Department Chemie, LSOCII, Technische Universita d Department of Biology II, Ludwig-Maximilians-University Munich, 82152 Martinsried, Germany b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 September 2009 Accepted 14 December 2009 Available online 31 December 2009
Molecular conjugates comprising targeting ligands hold great promise for site-specific gene delivery to distant tumors and individual organs including the lung. Here we show that prostaglandin I2 analogues can be used to improve gene transfer efficiency of polyethylenimine (PEI) gene vectors on bronchial and alveolar epithelial cells in vitro and lungs of mice in vivo. Prostacyclin (IP1) receptor expression was confirmed in pulmonary epithelial cell lines by western blot. Iloprost (ILO) and treprostinil (TRP), two prostaglandin I2 analogues, were conjugated to fluorescein-labeled BSA (FLUO-BSA) and compared for IP1 receptor binding/uptake in different lung cell lines. Binding of FLUO-BSA-ILO was 2–4-fold higher than for FLUO-BSA-TRP and could be specifically inhibited by free ILO and IP1 receptor antagonist CAY10449. Internalization of FLUO-BSA-ILO was confirmed by confocal microscopy. Molecular conjugates of PEI and ILO (PEI-g-ILO) were synthesized with increasing coupling degree (FILO (ILO:PEI) ¼ 2, 5, 8, 16) and analyzed for DNA binding, particle formation and transfection efficiency. At optimized conditions (N/P 4, FILO ¼ 5), gene expression using PEI-g-ILO was significantly up to 46-fold higher than for PEI gene vectors and specifically inhibited by CAY10449. Gene expression in the lungs of mice after aerosol delivery was 14-fold higher with PEI-g-ILO FILO ¼ 5 than for PEI. We suggest that targeting of IP1 receptor using ILO represents a promising approach to improve pulmonary gene transfer. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Gene transfer Gene expression Lung Iloprost Polyethylenimine Aerosol
1. Introduction Gene transfer holds great promise for the treatment of acquired and inherited lung diseases and may further offer new perspectives for vaccination [1,2]. One great advantage of gene delivery to the lung is its relatively noninvasive accessibility by using well-developed delivery technologies such as aerosol or dry powder inhalation. These technologies allow uncomplicated repeated dosing which is a precondition for the treatment of chronic pulmonary diseases such as cystic fibrosis [3]. Numerous studies have demonstrated successful gene transfer to the lungs using a variety of viral and non-viral gene transfer agents after aerosol delivery to the lungs of mice [4], rabbits [5] and sheep [6]. As of yet, only one
* Corresponding author. Department of Pediatrics, Ludwig-Maximilians-University, Lindwurmstr. 2a 80337 Munich, Germany. Tel.: þ49 89 5160 7711; fax: þ49 89 5160 4421. E-mail address: [email protected] (C. Rudolph). 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.12.035
non-viral gene transfer agent, a cationic lipid formulation based on Genzyme lipid 67, has been successfully aerosolized to the lungs of patients in a phase I clinical trial [7]. Among non-viral vectors branched polyethylenimine 25 kDa (PEI) has been shown to be effective both in cell culture and in vivo [8]. PEI is a highly polycationic molecule which efficiently condenses plasmid DNA (pDNA) into nanoparticles [9] and protects DNA from nuclease degradation [8]. Nevertheless, its low gene transfer efficiency compared to viral vectors and its high toxicity due to high polymer doses needed for optimal gene transfer, limit the use of PEI, especially in terms of in vivo application. A variety of ligands including transferrin [10], folate [11], lactoferrin [12], clenbuterol [13], and growth factors such EGF [14], have been investigated to enhance PEI-mediated gene delivery in terms of cell specificity and reduction of cell toxicity. The prostacyclin (IP1) receptor is a seven transmembrane G-protein coupled receptor, identified in many tissues including the lungs [15–17]. Binding of IP1 receptor agonists leads to endosomal internalization of receptor/ligand complexes via clathrin-mediated
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process [18,19] and thus, iloprost (ILO) may serve as suitable targeting ligand to enhance gene delivery in a receptor-specific manner. Various IP1 receptor agonists like ILO and treprostinil (TRP) are used in patients for the treatment of pulmonary arterial hypertension via intravenous and aerosol application [20]. In the present study, IP1 receptor was investigated as novel target for receptor-mediated gene transfer into lung epithelial cells. We analyzed IP1 receptor expression in lung cells by western blot and further examined if ILO and TRP were capable of mediating cellular binding and uptake of model cargo conjugates. For this purpose, ILO and TRP were conjugated to fluorescein-labeled bovine serum albumin (FLUO-BSA) and their binding and cellular uptake were examined on various lung cell lines by flow cytometry and confocal laser scanning microscopy. In the next step, ILO was conjugated to PEI and the resulting PEI-g-ILO conjugates were characterized for pDNA binding and transfection of various lung cells in vitro. Finally optimized formulations were investigated for gene delivery to the lungs of mice after aerosol administration. 2. Materials and methods 2.1. Chemicals and plasmids Iloprost, treprostinil and CAY10449 were purchased from Cayman Chemical (Michigan, USA), branched polyethylenimine (average molecular weight of 25 kDa), N-hydroxysulfosuccinimide (sulfo-NHS), bovine serum albumin (BSA), sodium phosphate, picrylsulfonic acid solution, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) and heparan sulfate were obtained from Sigma Aldrich (Schnelldorf, Germany). PEI was diluted in double-distilled water (water for injection, B. Braun Melsungen AG, Melsungen, Germany), and adjusted to pH 7 with hydrochloric acid. Sodium phosphate was dissolved in double-distilled water to a concentration of 0.5 mM and adjusted to pH 7.5 with sodium hydroxide. HEPES was dissolved in distilled water to a concentration of 0.1 M and adjusted to pH 7.4 with sodium hydroxide. Heparan sulfate was dissolved in double-distilled water to a concentration of 5 mg/ml. Ethanol p.a. and 3-(N-morpholino)propanesulfonic acid (MOPS) were purchased from Merck (Darmstadt, Germany). MOPS was dissolved in double-distilled water to a concentration of 0.1 M and adjusted to pH 6 with hydrochloric acid. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 5-(and 6-)carboxyfluorescein succinimidyl ester (fluorescein-NHS) were obtained from Pierce (Rockford, USA). Dithiothreitol (DTT) was purchased from Amersham Biosciences (South San Francisco, USA). D-luciferin was purchased from Synchem OHG (Flensberg/Altenburg, Germany). The plasmid pCMV-luc containing the Photinus pyralis luciferase gene under the control of the cytomegalovirus immediate early promotor (CMV) was kindly provided by Prof. E. Wagner (Department of Pharmacy, Ludwig-Maximilians-University Munich, Germany). pCpG-luc was constructed by Manfred Ogris (Department of Pharmacy, Ludwig-MaximiliansUniversity Munich, Germany). Both plasmids were propagated in Escherichia coli and provided in a highly purified form (LPS content 0.1 E.U./mg DNA) by PlasmidFactory GmbH (Bielefeld, Germany). The amount of supercoiled pDNA was 90% ccc (covalently closed circular) for pCMV-luc and greater than 98% ccc grade for pCpGluc. 2.2. Cell lines A549 (human alveolar epithelial) cells were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). BEAS-2B (human bronchial epithelial), H441 (human bronchiolar epithelial) cells were purchased from the ATCC (American Type Culture Collection). 16HBE14o- (human bronchial epithelial) cells were kindly provided by Prof. D. C. Gruenert (University of Vermont, Burlington, USA). A549, BEAS-2B and 16HBE14o- cell lines were grown in Minimum Essential Media (MEM, Gibco-BRL, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS, Gibco-BRL, Karlsruhe, Germany) at 37 C in a 5% CO2 humidified air atmosphere. H441 cell line was grown in Roswell Park Memorial Institute media 1640 (RPMI 1640, Gibco-BRL, Karlsruhe, Germany) supplemented with 10% FCS at 37 C in a 5% CO2 humidified air atmosphere. 2.3. Animals Fourteen-week-old female BALB/c mice were obtained from Charles River Laboratories (Sulzfeld, Germany) and maintained under specific pathogen-free conditions. Mice were acclimatized to the environment of the animal facility for at least seven days prior to the experiments. All animal procedures were approved and controlled by the local ethics committee and carried out according to the guidelines of the German law of protection of animal life.
2.4. Western blot analysis A549, BEAS-2B and 16HBE14o- cells were washed with PBS and lysed on ice in lysis buffer containing 20 mM Tris/HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 0.05% sodium deoxycholate. Protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) and 1 mM DTT were added fresh before use. Protein concentrations were determined using BioRad Protein Assay (BioRad, Munich, Germany). For each cell line, 40 mg of protein was diluted in SDS sample loading buffer (62.5 mM Tris/HCl (pH 6.8), 2% SDS, 10% glycerol, 2% DTT, 0.001% bromphenol blue) boiled for 5 min, separated on a 7.5% Tris/HCl gel (BioRad, Munich, Germany) and transferred to PVDF membrane (Millipore, Schwalbach, Germany). Membranes were blocked with TBS-T (20 mM Tris/HCl (pH 7.6), 137 mM NaCl, 0.1% Tween-20) containing 5% skimmed milk powder (Sigma Aldrich, Deisenhofen, Germany) for 1 h at RT. The primary polyclonal antibody (diluted 1:500) for IP1 receptor (Cayman Chemical, Michigan, USA) was incubated overnight in 0.5% skimmed milk. Membranes were washed with TBS-T and incubated with an antirabbit HRP-conjugated secondary antibody (diluted 1:15,000; BioRad, Munich, Germany) for 1.5 h at RT in 0.5% skimmed milk. After several wash steps with TBS-T, chemiluminescence detection was done using ECL detection kit (PIERCE, Rockford, USA) following manufacturer’s instructions.
2.5. Synthesis of fluorescein-BSA-iloprost (FLUO-BSA-ILO) and fluorescein-BSAtreprostinil (FLUO-BSA-TRP) 20 mg (0.3 mmol) of BSA was diluted in 2.5 ml of sodium phosphate buffer pH 7.5 and mixed with a 10-fold molar excess of fluorescein-NHS. After stirring 1 h at RT, the mixture was purified on a PBS equilibrated Sephadex G-25 M PD-10 column (GE Healthcare, Uppsala, Sweden). Either 0.7 mg (1.8 mmol) of ILO or 0.8 mg (1.8 mmol) of TRP were dissolved in 130 ml ethanol p.a. and mixed with 370 ml of MOPS buffer 0.1 M pH 6. 0.5 mg (5 mM) of sulfo-NHS (in MOPS buffer) and 0.2 mg (2 mM) of EDC (in MOPS buffer) were added and stirred for 15 min at RT. Afterwards 5 ml (20 mM) of DTT (in distilled water) was added and immediately 3 mg (45.2 nmol) of FLUO-BSA in 190 ml and 210 ml of phosphate buffer 0.5 M were pipetted to the reaction mixture. After stirring for 2 h at RT the mixture was purified on a PBS equilibrated Sephadex G-25 M PD-10 column (GE Healthcare, Uppsala, Sweden). BSA amounts were quantified by BioRad Protein Assay using a BSA standard curve. Coupling efficiencies of the final and intermediate products were determined by TNBS-Assay [21] and measuring the absorbance at 495 nm. Coupling degree of BSA-ILO and BSA-TRP resulted in 10 mol ILO or TRP per mol BSA.
2.6. Synthesis of PEI-graft-iloprost polymers (PEI-g-ILO) Different coupling degrees of PEI-g-ILO were synthesized by variation of EDC amounts given to the reaction mixture. 1 mg (2.8 mmol) of ILO was diluted in 100 ml ethanol p.a., mixed with 68 nmol PEI in 900 ml HEPES buffer 0.1 M pH 7.4 and 1 mg (5 mM) sulfo-NHS. Different amounts of EDC to a final concentration of 25 mM, 50 mM, 60 mM and 100 mM were added and incubated under stirring for 4 h at RT. The reaction mixture was purified on a double-distilled water equilibrated Sephadex G-25 M PD-10 column (GE Healthcare, Uppsala, Sweden). Concentration of PEI was determined with CuSO4 assay according to Ungaro et al. [22]. 1H–1D NMR spectra of PEI-g-ILO were recorded in D2O on a Bruker AV 250 MHz spectrometer. Coupling degrees of PEI-g-ILO were calculated via integration of the broad multiplett of PEI (CH2–CH2–NH–) at d (1H) ¼ 2.5–3.1 ppm and the singulett of the terminal methyl group of ILO (–CXC–CH3) at d (1H) ¼ 1.73 ppm. Covalent conjugation of ILO to PEI resulted in four different coupling degrees (FILO (mol ILO per mol PEI) ¼ 2, 5, 8, 16). PEI-g-ILO constructs were divided into small aliquots, snap-frozen in liquid nitrogen and kept at 80 C until further use.
2.7. Incubation experiment of FLUO-BSA-ILO and FLUO-BSA-TRP Receptor binding/uptake of FLUO-BSA-ILO was investigated in A549, H441, 16HBE14o- and BEAS-2B cells. For FACS measurement experiments, 100,000 cells per well were seeded in 24-well plates (TPP, Trasadingen, Switzerland) 24 h prior to addition of the conjugates. FLUO-BSA-ILO, FLUO-BSA-TRP and FLUO-BSA conjugates, respectively, were diluted in MEM to a concentration of 0.5 mM and incubated on the cells for 4 h at 37 C. After washing the cells with PBS, cells were dislodged from the wells by trypsin treatment and FACS measurements were performed using a Becton Dickinson FACS Scan (San Jose, USA). For confocal laser scanning microscopy, experiments were performed in 4-chamber BD Falcon Culture Slides (BD Biosciences San Jose, USA) with 25,000 cells per chamber. Incubation of FLUO-BSA-ILO and FLUO-BSA was performed as described before. Cells were rinsed and fixed in 4% paraformaldehyde, followed by staining of cell nuclei with 0.33 mM DAPI (40 ,6-diamidino-2-phenylindole) and F-actin with Alexa FluorÒ 568 phalloidin (Invitrogen GmbH, Karlsruhe, Germany) using standard protocols. The slides were covered with mounting media (Vectashield, Vector Laboratories Inc., Burlingame, USA) and images were taken with a confocal laser scanning microscope (Leica, Solms, Germany).
J. Geiger et al. / Biomaterials 31 (2010) 2903–2911 2.8. Inhibition experiment of FLUO-BSA-ILO with CAY10449 Inhibition of receptor binding/uptake of FLUO-BSA-ILO was investigated on 16HBE14o- cells. 24-well plates were prepared as described earlier. CAY10449 was diluted in MEM to concentrations of 15 mM, 30 mM and 150 mM and incubated for 15 min at 37 C. Immediately, FLUO-BSA-ILO and FLUO-BSA were added to a final concentration of 25 nM and incubated on the cells for 4 h at 37 C. Binding/uptake was measured using FACS. 2.9. Preparation of gene vector particles Luciferase reporter gene containing plasmid (pCMV-luc), PEI and PEI-g-ILO, respectively, were diluted separately in 25 ml of double-distilled water. Different N/P ratios (molar ratio of PEI nitrogen to DNA phosphate) were tested. The pCMV-luc solution was added to an equal volume of the polymer solution and gently mixed by pipetting up and down 8 times, resulting in particles with a concentration of 20 mg pCMV-luc per ml. Gene transfer particles were incubated for 20 min at room temperature. 2.10. Particle size measurements Particle size (determined by dynamic light scattering) was measured using a ZetaPALS/Zeta Potential Analyzer (Brookhaven Instruments Corporation, Vienna, Austria). Gene vector particles were generated as described above. The following settings were used: five runs of 1 min measurement per sample; viscosity for water 0.89 cP; Ref. Index 1330; temperature 25 C. 2.11. DNA retardation assay PEI/pCMV-luc and PEI-g-ILO/pCMV-luc gene vector particles with different coupling degrees at N/P 4 were prepared in double-distilled water as described above. 5 ml of each particle solution were mixed either with 2 ml of double-distilled water or 2 ml of a heparan sulfate solution (5 mg/ml). After 45 min of incubation, samples were mixed with 1 ml loading buffer (0.25% bromphenol blue, 0.25% xylene cyanol FF, 30% glycerol in water), loaded into individual wells of a 0.8% agarose gel and separated by agarose gel electrophoresis at 125 V for 1 h. Staining of the gel was performed with ethidium bromide and DNA bands were visualized under UV light. 2.12. In vitro transfection studies A549, 16HBE14o- and BEAS-2B cells were seeded in 24-well plates (TPP, Trasadingen, Switzerland) 24 h prior to transfection with a density of 100,000 cells per well and grown in MEM containing 10% FCS supplemented with 1% (v/v) penicillin/ streptomycin. Before transfection, cells were washed with PBS and 450 ml of fresh serum-free medium was added per well. Subsequently 50 ml of the gene vector particles, corresponding to 1 mg of pCMV-luc, were pipetted onto the cells. For inhibition experiments, CAY10449 was added to fresh media with a concentration of 150 mM 15 min before addition of gene vector particles. After 4 h of incubation, the transfection medium was replaced with MEM including 10% FCS supplemented with 1% (v/v) penicillin/streptomycin. Twenty-four hours post-transfection, luciferase activity was measured using a Wallac Victor2 1420 Multilabel Counter (Perkin Elmer, Boston, USA) according to Huth et al. [23]. Results were normalized to total cell protein using BioRad Protein Assay and BSA as protein standard. 2.13. In vivo gene delivery studies To prepare gene vector particles for aerosol delivery to mice, pCpG-luc and PEI or PEI-g-ILO FILO ¼ 5 were each diluted in 4.0 ml of water for injection (B. Braun Melsungen AG, Melsungen, Germany) resulting in concentrations of 250 mg/ml pCpG-luc and 130.4 mg/ml PEI, respectively (corresponding to an N/P ratio of 4). The pCpG-luc solution was pipetted to the PEI solution, mixed by pipetting up and down 8 times, to yield a final DNA concentration of 125 mg/ml. The particles were incubated for 20 min at RT before use. Particles were nebulized using a PARI Turbo BoyÒ N inhalation device with a PARI LC Plus nebulizer (PARI GmbH, Starnberg, Germany)
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connected to a vertical whole body aerosol device according to Rudolph et al. [24]. After 24 h mice were anaesthetized and pulmonary application of D-luciferin substrate (1.5 mg/50 ml PBS per mouse) was done via sniffing [25]. After 10 min, bioluminescence was measured (IVIS 100 imaging system; Xenogen, Alameda, USA) using camera settings field of view 10, f1 f-stop, high resolution binning, and exposure time of 10 min. To confirm reporter gene expression levels in the lungs, mice were euthanized after in vivo bioluminescent imaging by cervical dislocation. After opening the peritonea by midline incisions, lungs were dissected from animals and perfused with PBS. Lungs were snap-frozen in liquid nitrogen and homogenized in the frozen state. After addition of 400 ml of lysis buffer (250 mM Tris pH 7.8, 0.1% Triton X-100, Roche Complete Protease Inhibitor Cocktail Tablets) and incubation for 20 min on ice, luciferase activity in the supernatant was measured using a Lumat LB9507 tube luminometer (EG&G Berthold, Munich, Germany). Recombinant luciferase (Roche Diagnostics GmbH, Mannheim, Germany) was used as a standard to calculate the amount of luciferase expressed in the lung tissue. 2.14. MTT-based assay Toxicity of PEI/pCMV-luc or PEI-g-ILO FILO ¼ 5/pCMV-luc particles was evaluated on 16HBE14o- cells with an N/P ratio of 4. Cells were seeded 24 h prior to the experiment with a density of 80,000 cells per well into a 24-well plate. Transfection was done as described earlier. After 4 h, transfection mixture was replaced with 400 ml media and MTT based assay was done using the Cell Proliferation Kit I (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. Untreated cells were used as a reference by setting the corresponding absorption to 100% viable cells. 2.15. Serum collection and analysis of cytokine concentration Twenty 4 h after aerosol delivery, blood samples were taken from mice and stored at 4 C over night. After coagulation blood was centrifuged and serum was collected. Quantification of Interleukin 12 (IL-12) and Interferon-g (INF-g) was done using the Mouse IL-12 (P40/P70) and the Mouse INF-g ELISA Kit (RayBiotech, Norcross, USA) according to the manufacturer’s instructions. Untreated mice were used as a reference by setting the corresponding concentration to 1. 2.16. Statistical analysis Results are reported as mean values standard deviation. Statistically significant differences were evaluated by a non paired Student’s t-test. p < 0.01 was considered as significant.
3. Results 3.1. Confirmation of IP1 receptor expression in lung cells by western blot Expression of IP1 receptor in human alveolar (A549) and bronchial (BEAS-2B, 16HBE14o-) epithelial cells was confirmed by western blot analysis. A protein band at 67 kDa could be detected (Fig. 1), which represents the glycosylated form of the IP1 receptor protein expressed on the cell membrane [26]. Therefore, we further investigated targeting of IP1 receptor for protein and gene delivery approaches. 3.2. Targeting of lung cells with different IP1 receptor ligands To investigate targeting of the IP1 receptor for receptor-mediated gene delivery, TRP and ILO were chemically coupled to
Fig. 1. Western blot showing the expression of IP1 receptor protein at 67 kDa in human alveolar (A549) and bronchial (BEAS-2B, 16HBE14o-) epithelial cells. Each lane was loaded with 40 mg of protein extract.
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fluorescein-labeled bovine serum albumin (FLUO-BSA) which served as a model cargo. Whereas incubation of A549 and 16HBE14o- cells with FLUO-BSA resulted in unspecific background binding, incubation with FLUO-BSA-TRP and FLUO-BSA-ILO resulted in 5.5 0.5% and 39.3 0.6% positive A549 and 51 1.8% and 76.1 1.4% positive 16HBE14o- cells, respectively (Fig. 2). The mean fluorescent intensity (MFI) of A549 and 16HBE14o- cells was significantly higher after incubation with FLUO-BSA-ILO than after incubation with FLUO-BSA-TRP. These results illustrate that TRP and ILO are capable of mediating successful binding of the model cargo FLUO-BSA to lung cells, but ILO is the more potent targeting ligand.
obtained in competition experiments with excess of unconjugated ILO (data not shown). FACS measurements together with inhibition experiments provide evidence for cell type-dependent cell surface expression of IP1 receptor on pulmonary epithelial cells. To further test if ILO mediates intracellular uptake of FLUO-BSA-ILO, additional experiments were performed using confocal laser scanning microscopy. 16HBE14o- cells were incubated with either 0.5 mM FLUO-BSA or FLUO-BSA-ILO. Visualization of the cells by confocal microscopy showed clear cell surface binding and subsequent intracellular uptake of FLUO-BSA-ILO conjugates (Fig. 3c), whereas no uptake of FLUO-BSA could be observed.
3.3. Specificity of FLUO-BSA-ILO binding to different lung cell lines
3.4. Characterization of PEI and PEI-g-ILO gene vector particles
ILO was further investigated as a targeting ligand on additional lung cell lines due to its higher cell binding/uptake property when compared with TRP. In addition to A549 and 16HBE14o- cells, incubation of H441 and BEAS-2B cells with FLUO-BSA-ILO resulted in significantly higher (p < 0.01) number of positive cells and MFI than the control FLUO-BSA (38.0 1.8% and 82.7 1.6% vs. 9.1 1.9% and 13.7 1.2% respectively, Fig. 3a). This effect was more pronounced on human bronchial epithelial cells (16HBE14o-, BEAS2B) compared to Clara (H441) or alveolar (A549) epithelial cells. These results indicate the differential cell surface expression of IP1 receptor in human lung cell types. To confirm the receptor specificity of the observed binding of FLUO-BSA-ILO in lung cells, 16HBE14o- cells were incubated with FLUO-BSA-ILO in the presence of increasing amounts of CAY10449. This compound has been previously reported to be a highly specific and potent antagonist of human IP1 receptor [27,28]. 16HBE14ocells were incubated with 25 nM FLUO-BSA-ILO together with increasing concentrations of CAY10449. Addition of CAY10449 resulted in a significant dose-dependent decrease (p < 0.01) of both number of fluorescence-positive cells and MFI (Fig. 3b). At the highest used concentration of CAY10449, the number of fluorescence-positive cells decreased from 95.7 0.7% to 7.4 0.9%. Cells incubated with FLUO-BSA conjugates were used as controls and showed no effect of CAY10449 addition. Similar results were
ILO was coupled to PEI via a carbodiimide chemistry with increasing coupling degree FILO ¼ 2, 5, 8 and 16 and the size of the resulting gene vector particles was measured by dynamic light scattering (Table 1). Particles with coupling degree FILO ¼ 2 and 5 at N/P ratios 4–8 showed hydrodynamic diameters of 50–100 nm which were comparable to PEI gene vectors. Particles prepared at PEI N/P 2, PEI-g-ILO FILO ¼ 2 N/P 2–3 and PEI-g-ILO FILO ¼ 16 N/P 4 were not stable and precipitated. Particles smaller than 150 nm showed a polydispersity of less than 0.2. We next characterized the PEI-g-ILO constructs for their DNA binding affinity. Particles were prepared at N/P 4 and DNA release assay was performed (Fig. 4). For PEI, PEI-g-ILO FILO ¼ 2 and PEI-gILO FILO ¼ 5 there was complete release of DNA upon addition of heparan sulfate. For higher coupling degrees, only partial release of DNA could be observed indicating stronger binding of the polymers to the plasmid. 3.5. In vitro transfection efficiency Increased binding and uptake of FLUO-BSA-ILO by different lung cells and the possibility to form PEI-g-ILO/pCMV-luc particles, prompted us to explore ILO as a ligand to improve gene transfer in vitro. 16HBE14o- cells were transfected with PEI-g-ILO gene vectors and compared with unmodified PEI as control. Gene
Fig. 2. Targeting of IP1 receptor with TRP and ILO on alveolar (A549) and bronchial (16HBE14o-) epithelial cell lines. Incubation of FLUO-BSA, FLUO-BSA-TRP and FLUO-BSA-ILO was performed with a concentration of 0.5 mM (n ¼ 4): FACS measurements. Results are reported as means standard deviation. ** represent statistical significance with p < 0.01, respectively.
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Fig. 3. Distribution of IP1 receptor and receptor binding in alveolar (A549), bronchoalveolar (H441) and bronchial (16HBE14o-, BEAS-2B) epithelial cells. Incubation of FLUO-BSA-ILO and FLUO-BSA was performed with a concentration of 0.5 mM (n ¼ 4): FACS measurements (a). 16HBE14o- cells were incubated with 25 nM FLUO-BSA-ILO and FLUO-BSA together with increasing concentrations of CAY10449 (n ¼ 4): FACS measurements (b). For confocal laser scanning microscopy 16HBE14o- cells were incubated with 0.5 mM FLUO-BSA-ILO and FLUO-BSA (c). Results are reported as means standard deviation. ** represent statistical significance with p < 0.01, respectively.
transfer efficiency increased in accordance with N/P ratio. The highest level of gene expression was found with N/P 4 and FILO ¼ 5. At these optimized conditions gene expression was significantly 46-fold higher than for PEI (Fig. 5a). Particle formation at higher N/P ratios (>4) resulted in no additional increase in gene expression. PEI-g-ILO conjugates with other coupling degrees resulted in either lower or similar transfection levels when compared to PEI. Competitive inhibition experiments with CAY10449 were performed to confirm receptor-mediated gene transfer of PEI-g-ILO
gene vectors. 16HBE14o- cells were transfected with either PEI or PEI-g-ILO FILO ¼ 5 gene vectors at N/P 4 in the presence or absence of 150 mM CAY10449. Gene expression observed with PEI-g-ILO FILO ¼ 5 was significantly (p < 0.01) reduced 33-fold to levels obtained with PEI (Fig. 5b). No effect of CAY10449 was observed on cells transfected with PEI. In addition, PEI-g-ILO FILO ¼ 5 was also tested on A549 and BEAS-2B cells. At optimized conditions PEI-g-ILO FILO ¼ 5 mediated significantly 45- and 14-fold higher expression levels on A549 and BEAS-2B cells than PEI, respectively (Fig. 5c).
Table 1 Physical characterization of PEI/pCMV-luc and PEI-g-ILO/pCMV-luc gene vectors using PEI or PEI-g-ILO with different coupling degrees (FILO ¼ 2, 5, 8, 16) at different N/P ratios: Particle size and polydispersity (in brackets) measurements. Results are reported as means standard deviation (n ¼ 3). N/P 2 PE1 FILO ¼ FILO ¼ FILO ¼ FILO ¼
1258 787 2 1197 1729 5 149 16 8 207 137 16 141 26
N/P 3 (0.34 (0.37 (0.17 (0.19 (0.18
0.04) 69 11 (0.15 0.23) 604 804 (0.25 0.04) 169 35 (0.10 0.10) 366 31 (0.25 0.02) 292 73 (0.22
N/P 4
0.03) 61 10 0.13) 97 20 0.02) 82 4 0.06) 261 13 0.05) 2314 946
N/P 5 (0.17 (0.17 (0.11 (0.12 (0.26
0.01) 60 11 0.07) 77 19 0.03) 75 15 0.03) 165 43 0.14) 418 156
N/P 6 (0.19 (0.15 (0.13 (0.08 (0.17
0.02) 53 8 0.03) 73 17 0.02) 74 11 0.04) 106 7 0.13) 258 61
N/P 8 (0.16 (0.15 (0.13 (0.08 (0.12
0.05) 51 3 0.08) 70 17 0.01) 76 16 0.01) 87 12 0.03) 213 48
(0.14 (0.16 (0.14 (0.12 (0.08
0.01) 0.05) 0.04) 0.03) 0.02)
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Fig. 4. DNA retardation assay of PEI and different PEI-g-ILO constructs at N/P 4. Polymers complexed with pCMV-luc were incubated with (þ) and without () heparan sulfate, resolved on agarose gel and visualized under UV light after ethidium bromide staining.
3.6. In vivo gene delivery studies PEI-g-ILO FILO ¼ 5 and PEI gene vector particles were delivered to the lungs of BALB/c mice via aerosol application and gene expression was analyzed 24 h post gene delivery. Measurement of luciferase gene expression via in vivo bioluminescence imaging showed a strong signal in the lungs of mice treated with PEI-g-ILO FILO ¼ 5 gene vectors but was at the limit of detection for PEI gene vectors (Fig. 6a). To quantify luciferase per g of lung tissue, mice were euthanatized and lungs were isolated. Luciferase expression measured in the homogenized lung tissue was significantly 14-fold higher for PEI-g-ILO FILO ¼ 5 gene vectors than for PEI gene vectors (Fig. 6b). 3.7. Toxicity in vitro and in vivo In vitro cell viability after application of gene vector particles (PEI-g-ILO FILO ¼ 5/pCMV-luc or PEI/pCMV-luc) was measured using MTT assay (Fig. 7a). Compared to PEI, no increase in cytotoxicity could be observed (86.0 10.1% cell viability for PEI-g-ILO FILO ¼ 5 vs. 89.2 3.2% for PEI). For in vivo toxicity and inflammation, serum was obtained from the treated mice and inflammatory cytokines including Interleukin-12 (IL-12) and Interferon-g (INF-g) were measured by ELISA (Fig. 7b). Similar to in vitro MTT results, no significant increase in either of the cytokines 24 h post gene delivery could be detected. 4. Discussion In this study we investigated if the prostaglandin I2 analog ILO, an IP1 receptor agonist, can be used as a targeting ligand for improving gene transfer of cationic polymers such as PEI in lung cells in vitro and in vivo. In our study, we demonstrate that reporter gene expression was significantly increased in human alveolar (A549) and bronchial epithelial cells (16HBE14o-, BEAS-2B) up to 46-fold. In addition, luciferase activity in lungs of mice after aerosol treatment was significantly (14-fold) higher compared to PEI.
Fig. 5. Transfection efficiencies in vitro. Transfection of 16HBE14o- cells with pCMV-luc complexed to different PEI-g-ILO constructs at various N/P ratios (n ¼ 4): Measurement of luciferase gene expression (a). Inhibition experiment of PEI/pCMV-luc and PEI-g-ILO FILO ¼ 5/pCMV-luc particles at an N/P ratio of 4 with CAY10449 (n ¼ 3): Measurement of luciferase gene expression (b). Transfection of A549, 16HBE14o- and BEAS-2B with PEI/pCMV-luc and PEI-g-ILO FILO ¼ 5/pCMV-luc at an N/P ratio of 4 (n ¼ 6): Measurement of luciferase gene expression (c). Luciferase gene expression was measured as relative light units (RLU) luminescence during 10 s/mg of cellular protein. Results are reported as means standard deviation. ** Represent statistical significance with p < 0.01, respectively.
ILO and TRP are agonists of the human IP1 receptor [29]. Both of them are approved for the treatment of pulmonary arterial hypertension via aerosol inhalation and i.v. application [20,30,31]. Furthermore, it has been previously shown that IP1 receptors are
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Fig. 6. In vivo luciferase gene expression obtained with PEI/pCpG-luc and PEI-g-ILO FILO ¼ 5/pCpG-luc particles at an N/P ratio of 4 in lungs of BALB/c mice after aerosol delivery (n ¼ 5). Bioluminescence images with an exposure time of 10 min after 24 h (a). Amount of luciferase expressed in the lung homogenates of mice (b). Results are reported as vertical point plot with median. ** represent statistical significance with p < 0.01, respectively.
Fig. 7. Metabolic activity of 16HBE14o- cells after treatment with PEI/pCMV-luc and PEI-g-ILO FILO ¼ 5/pCMV-luc particles at an N/P ratio of 4 (n ¼ 6). Values of untreated cells were taken to be 100% (a). Serum cytokine levels 24 h after aerosol delivery of PEI/ pCpG-luc and PEI-g-ILO FILO ¼ 5/pCpG-luc particles at an N/P ratio of 4 measured by ELISA (n ¼ 5). Values of untreated mice were taken to be 1. Results are reported as means standard deviation.
expressed in the human and murine lungs [15,32–34]. Moreover, in context with non-viral gene delivery it is important to note that the IP1 receptor/ligand complex is internalized into the cell [35,36]. In our study, we could confirm IP1 receptor expression in various lung cell types by western blot. In order to characterize IP1 receptor expression on the cell surface of lung cells in more detail, we synthesized fluorescein-labeled BSA-conjugates coupled with either ILO or TRP. We then incubated both constructs on alveolar (A549) and bronchial (16HBE14o-) epithelial cell lines and analyzed cell binding by flow cytometry. It should be mentioned, that it is relevant to measure both the number of positive cells and the MFI. The percentage of fluorescein-positive cells gives information about the receptor distribution, whereas the MFI contributes either to the receptor density or the kinetic of particle uptake. Our results demonstrate that IP1 receptors are present on each of the tested cell lines. However, ILO showed higher cell surface binding than TRP and we therefore used ILO as targeting ligand in all subsequent experiments. Specificity of binding to the IP1 receptor was shown by inhibition experiments with the specific IP1 receptor antagonist CAY10449 [27,28,32] and excess of free ILO (data not shown). In order to confirm these observations confocal laser scanning microscopy was performed which demonstrated binding of FLUOBSA-ILO to the cell surface and intracellular uptake on 16HBE14o-
cells. These results therefore demonstrate that ILO can be used as a targeting ligand which mediates binding and intracellular uptake of conjugated cargo model such FLUO-BSA, which represents a precondition for receptor-mediated gene transfer. For transfection studies, ILO was conjugated to branched PEI 25 kDa via amide bond formation. Synthesis resulted in conjugates with coupling degrees of FILO ¼ 2, 5, 8 and 16. PEI-g-ILO/pCMV-luc particles were screened on 16HBE14o- cells and highest transfection efficiency was observed with at N/P 4 FILO ¼ 5, whereas higher coupling degrees 8–6 resulted in lower transfection rates. This could be due to the incomplete release of pCMV-luc at higher coupling degrees as observed by DNA release assay. Release of pDNA from PEI/pDNA particles has been previously shown to be a critical parameter for successful gene transfer [37]. One may speculate that additional hydrophobic interaction of ILO to pDNA could increase pDNA binding. Size measurement of different particles showed that increasing amount of ligand led to large hydrodynamic diameters of PEI-g-ILO/pCMV-luc particles up to 1 mm. Similar results were obtained by Elfinger et al when clenbuterol was coupled to PEI [13]. Particles with PEI-g-ILO FILO ¼ 5 showed hydrodynamic diameters of less than 100 nm. In a previous study, particles of similar size have been shown to be internalized more efficiently than larger particles [38]. Transfection of alveolar
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(A549) and bronchial (16HBE14o-, BEAS-2B) epithelial cells with PEI-g-ILO FILO ¼ 5/pCMV-luc particles at N/P 4 resulted in up to 46-fold increase of reporter gene expression compared to PEI/ pCMV-luc particles at the same N/P ratio in all cell lines tested. The observed enhanced gene expression resulted in no significant increase of metabolic toxicity measured by MTT assay. In addition the hypothesis of receptor-mediated gene transfer was further supported by specific antagonist mediated inhibition experiments in 16HBE14o- cells. Addition of CAY10449 decreased gene expression to levels comparable with PEI. As IP1 receptors are also present on the endothelial cells, we are of the opinion that systemic application of PEI-g-ILO/pDNA particles will most likely result in extensive transfection of the endothelium. Keeping this fact in mind, we selected aerosol as a local route of application to the lungs. For animal experiments, a CpG-free luciferase expression plasmid (pCpG-luc) was used. CpG-free plasmids have been shown to be less inflammatory compared to their CpG containing counterparts. They have also been shown to result in higher and sustained pulmonary gene expression [39]. Prior to animal experiments, we nebulized PEIg-ILO FILO ¼ 5/pCpG-luc and PEI/pCpG-luc gene vectors, respectively, and collected different fractions (nebulized, non-nebulized) in order to test for the stability of the particles. Both gel retardation assay and particle size measurements indicated no effect of nebulization on the particles (data not shown). These observations confirmed that particles were not negatively affected by the aerosolization procedure. Similar results have been reported previously [40]. After aerosol gene delivery to the lungs of mice, gene expression was significantly 14-fold higher for PEI-g-ILO FILO ¼ 5/ pCpG-luc than for PEI/pCpG-luc gene vectors. Measurement of Interleukin-12 (IL-12) and Interferon-g (INF-g) in the murine serum revealed no significant increase in their levels. These observations are in accordance to Gautam et al [41], who showed that aerosol delivery of PEI-DNA particles does not induce high cytokine response. To summarize, in this study we investigated the potential of ILO as a novel ligand for non-viral aerosol gene delivery to the lung. Using fluorescein-based molecular conjugates, we could demonstrate that IP1 receptor is a suitable candidate for receptor-mediated gene delivery to lung cells. Receptor specific binding and uptake of molecular conjugates could be demonstrated in both alveolar and bronchial epithelial and Clara cells. In agreement with these observations, conjugation of ILO led to a specific significant increase (46-fold) of gene expression in vitro. In vivo aerosol delivery to the mice resulted in a 14-fold increase of luciferase expression in the lungs. With the observed more than 10-fold increase in gene expression it may be possible to reduce the amount of pDNA and gene carrier required for a particular amount of expression. This would further reduce both DNA and carrier mediated toxicity and inflammation. Our pre-experiments in 16HBE14o- cells lend support to this assumption. Equal levels of gene expression were observed with half of the amount of PEIg-ILO FILO ¼ 5/pCMV-luc compared to PEI/pCMV-luc gene vector particles (data not shown). Studies are under way to conjugate ILO to biodegradable polymers. Unlike previous ligands, for example lactoferrin and clenbuterol which target bronchial and alveolar epithelial cells respectively [12,13], ILO as a ligand increases gene expression in both cell types. Thus, ILO presents itself as a highly promising ligand for topical PEI-based aerosol application for pulmonary gene transfer. Acknowledgements This work was supported by the BMBF BioFuture programme (FKZ0311898), the programme 13N9182, Magselectofection (LSHB-
CT-2006-019038) and LMUexcellent (Investitionsfonds). G.K. and B.L. acknowledge funding by the ‘‘Fonds der Chemischen Industrie’’ and the ‘‘Deutsche Forschungsgemeinschaft’’ (LU 835/2,3,4). Appendix Figures with essential color discrimination. Figs. 2, 3, 5, 6 in this article have parts that are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10. 1016/j.biomaterials.2009.12.035. References [1] Gill DR, Davies LA, Pringle IA, Hyde SC. The development of gene therapy for diseases of the lung. Cell Mol Life Sci 2004;61(3):355–68. [2] Gurunathan S, Klinman DM, Seder RA. DNA vaccines: immunology, application, and optimization*. Annu Rev Immunol 2000;18:927–74. [3] Davies L, Hyde SC, Gill DR. Plasmid inhalation: delivery to the airways. In: Schleef Martin, editor. DNA pharmaceuticals: formulation and delivery in gene therapy, DNA vaccination and immunotherapy. Wiley-VCH Publishing GmbH & Co. KGaA; 2005. p. 145–59. 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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Targeting of the prostacyclin specific IP1 receptor in lungs with molecular conjugates comprising prostaglandin I2 analogues Johannes Geiger a, b, Manish K. Aneja a, Gu¨nther Hasenpusch a, Gu¨lnihal Yu¨ksekdag a, Grit Kummerlo¨we c, Burkhard Luy c, Tina Romer d, Ulrich Rothbauer d, Carsten Rudolph a, b, * a
Department of Pediatrics, Ludwig-Maximilians-University Munich, 80337 Munich, Germany Department of Pharmacy, Free University of Berlin, 14166 Berlin, Germany ¨t Mu ¨ nchen, 85747 Garching, Germany Department Chemie, LSOCII, Technische Universita d Department of Biology II, Ludwig-Maximilians-University Munich, 82152 Martinsried, Germany b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 September 2009 Accepted 14 December 2009 Available online 31 December 2009
Molecular conjugates comprising targeting ligands hold great promise for site-specific gene delivery to distant tumors and individual organs including the lung. Here we show that prostaglandin I2 analogues can be used to improve gene transfer efficiency of polyethylenimine (PEI) gene vectors on bronchial and alveolar epithelial cells in vitro and lungs of mice in vivo. Prostacyclin (IP1) receptor expression was confirmed in pulmonary epithelial cell lines by western blot. Iloprost (ILO) and treprostinil (TRP), two prostaglandin I2 analogues, were conjugated to fluorescein-labeled BSA (FLUO-BSA) and compared for IP1 receptor binding/uptake in different lung cell lines. Binding of FLUO-BSA-ILO was 2–4-fold higher than for FLUO-BSA-TRP and could be specifically inhibited by free ILO and IP1 receptor antagonist CAY10449. Internalization of FLUO-BSA-ILO was confirmed by confocal microscopy. Molecular conjugates of PEI and ILO (PEI-g-ILO) were synthesized with increasing coupling degree (FILO (ILO:PEI) ¼ 2, 5, 8, 16) and analyzed for DNA binding, particle formation and transfection efficiency. At optimized conditions (N/P 4, FILO ¼ 5), gene expression using PEI-g-ILO was significantly up to 46-fold higher than for PEI gene vectors and specifically inhibited by CAY10449. Gene expression in the lungs of mice after aerosol delivery was 14-fold higher with PEI-g-ILO FILO ¼ 5 than for PEI. We suggest that targeting of IP1 receptor using ILO represents a promising approach to improve pulmonary gene transfer. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Gene transfer Gene expression Lung Iloprost Polyethylenimine Aerosol
1. Introduction Gene transfer holds great promise for the treatment of acquired and inherited lung diseases and may further offer new perspectives for vaccination [1,2]. One great advantage of gene delivery to the lung is its relatively noninvasive accessibility by using well-developed delivery technologies such as aerosol or dry powder inhalation. These technologies allow uncomplicated repeated dosing which is a precondition for the treatment of chronic pulmonary diseases such as cystic fibrosis [3]. Numerous studies have demonstrated successful gene transfer to the lungs using a variety of viral and non-viral gene transfer agents after aerosol delivery to the lungs of mice [4], rabbits [5] and sheep [6]. As of yet, only one
* Corresponding author. Department of Pediatrics, Ludwig-Maximilians-University, Lindwurmstr. 2a 80337 Munich, Germany. Tel.: þ49 89 5160 7711; fax: þ49 89 5160 4421. E-mail address: [email protected] (C. Rudolph). 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.12.035
non-viral gene transfer agent, a cationic lipid formulation based on Genzyme lipid 67, has been successfully aerosolized to the lungs of patients in a phase I clinical trial [7]. Among non-viral vectors branched polyethylenimine 25 kDa (PEI) has been shown to be effective both in cell culture and in vivo [8]. PEI is a highly polycationic molecule which efficiently condenses plasmid DNA (pDNA) into nanoparticles [9] and protects DNA from nuclease degradation [8]. Nevertheless, its low gene transfer efficiency compared to viral vectors and its high toxicity due to high polymer doses needed for optimal gene transfer, limit the use of PEI, especially in terms of in vivo application. A variety of ligands including transferrin [10], folate [11], lactoferrin [12], clenbuterol [13], and growth factors such EGF [14], have been investigated to enhance PEI-mediated gene delivery in terms of cell specificity and reduction of cell toxicity. The prostacyclin (IP1) receptor is a seven transmembrane G-protein coupled receptor, identified in many tissues including the lungs [15–17]. Binding of IP1 receptor agonists leads to endosomal internalization of receptor/ligand complexes via clathrin-mediated
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process [18,19] and thus, iloprost (ILO) may serve as suitable targeting ligand to enhance gene delivery in a receptor-specific manner. Various IP1 receptor agonists like ILO and treprostinil (TRP) are used in patients for the treatment of pulmonary arterial hypertension via intravenous and aerosol application [20]. In the present study, IP1 receptor was investigated as novel target for receptor-mediated gene transfer into lung epithelial cells. We analyzed IP1 receptor expression in lung cells by western blot and further examined if ILO and TRP were capable of mediating cellular binding and uptake of model cargo conjugates. For this purpose, ILO and TRP were conjugated to fluorescein-labeled bovine serum albumin (FLUO-BSA) and their binding and cellular uptake were examined on various lung cell lines by flow cytometry and confocal laser scanning microscopy. In the next step, ILO was conjugated to PEI and the resulting PEI-g-ILO conjugates were characterized for pDNA binding and transfection of various lung cells in vitro. Finally optimized formulations were investigated for gene delivery to the lungs of mice after aerosol administration. 2. Materials and methods 2.1. Chemicals and plasmids Iloprost, treprostinil and CAY10449 were purchased from Cayman Chemical (Michigan, USA), branched polyethylenimine (average molecular weight of 25 kDa), N-hydroxysulfosuccinimide (sulfo-NHS), bovine serum albumin (BSA), sodium phosphate, picrylsulfonic acid solution, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) and heparan sulfate were obtained from Sigma Aldrich (Schnelldorf, Germany). PEI was diluted in double-distilled water (water for injection, B. Braun Melsungen AG, Melsungen, Germany), and adjusted to pH 7 with hydrochloric acid. Sodium phosphate was dissolved in double-distilled water to a concentration of 0.5 mM and adjusted to pH 7.5 with sodium hydroxide. HEPES was dissolved in distilled water to a concentration of 0.1 M and adjusted to pH 7.4 with sodium hydroxide. Heparan sulfate was dissolved in double-distilled water to a concentration of 5 mg/ml. Ethanol p.a. and 3-(N-morpholino)propanesulfonic acid (MOPS) were purchased from Merck (Darmstadt, Germany). MOPS was dissolved in double-distilled water to a concentration of 0.1 M and adjusted to pH 6 with hydrochloric acid. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 5-(and 6-)carboxyfluorescein succinimidyl ester (fluorescein-NHS) were obtained from Pierce (Rockford, USA). Dithiothreitol (DTT) was purchased from Amersham Biosciences (South San Francisco, USA). D-luciferin was purchased from Synchem OHG (Flensberg/Altenburg, Germany). The plasmid pCMV-luc containing the Photinus pyralis luciferase gene under the control of the cytomegalovirus immediate early promotor (CMV) was kindly provided by Prof. E. Wagner (Department of Pharmacy, Ludwig-Maximilians-University Munich, Germany). pCpG-luc was constructed by Manfred Ogris (Department of Pharmacy, Ludwig-MaximiliansUniversity Munich, Germany). Both plasmids were propagated in Escherichia coli and provided in a highly purified form (LPS content 0.1 E.U./mg DNA) by PlasmidFactory GmbH (Bielefeld, Germany). The amount of supercoiled pDNA was 90% ccc (covalently closed circular) for pCMV-luc and greater than 98% ccc grade for pCpGluc. 2.2. Cell lines A549 (human alveolar epithelial) cells were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). BEAS-2B (human bronchial epithelial), H441 (human bronchiolar epithelial) cells were purchased from the ATCC (American Type Culture Collection). 16HBE14o- (human bronchial epithelial) cells were kindly provided by Prof. D. C. Gruenert (University of Vermont, Burlington, USA). A549, BEAS-2B and 16HBE14o- cell lines were grown in Minimum Essential Media (MEM, Gibco-BRL, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS, Gibco-BRL, Karlsruhe, Germany) at 37 C in a 5% CO2 humidified air atmosphere. H441 cell line was grown in Roswell Park Memorial Institute media 1640 (RPMI 1640, Gibco-BRL, Karlsruhe, Germany) supplemented with 10% FCS at 37 C in a 5% CO2 humidified air atmosphere. 2.3. Animals Fourteen-week-old female BALB/c mice were obtained from Charles River Laboratories (Sulzfeld, Germany) and maintained under specific pathogen-free conditions. Mice were acclimatized to the environment of the animal facility for at least seven days prior to the experiments. All animal procedures were approved and controlled by the local ethics committee and carried out according to the guidelines of the German law of protection of animal life.
2.4. Western blot analysis A549, BEAS-2B and 16HBE14o- cells were washed with PBS and lysed on ice in lysis buffer containing 20 mM Tris/HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 0.05% sodium deoxycholate. Protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) and 1 mM DTT were added fresh before use. Protein concentrations were determined using BioRad Protein Assay (BioRad, Munich, Germany). For each cell line, 40 mg of protein was diluted in SDS sample loading buffer (62.5 mM Tris/HCl (pH 6.8), 2% SDS, 10% glycerol, 2% DTT, 0.001% bromphenol blue) boiled for 5 min, separated on a 7.5% Tris/HCl gel (BioRad, Munich, Germany) and transferred to PVDF membrane (Millipore, Schwalbach, Germany). Membranes were blocked with TBS-T (20 mM Tris/HCl (pH 7.6), 137 mM NaCl, 0.1% Tween-20) containing 5% skimmed milk powder (Sigma Aldrich, Deisenhofen, Germany) for 1 h at RT. The primary polyclonal antibody (diluted 1:500) for IP1 receptor (Cayman Chemical, Michigan, USA) was incubated overnight in 0.5% skimmed milk. Membranes were washed with TBS-T and incubated with an antirabbit HRP-conjugated secondary antibody (diluted 1:15,000; BioRad, Munich, Germany) for 1.5 h at RT in 0.5% skimmed milk. After several wash steps with TBS-T, chemiluminescence detection was done using ECL detection kit (PIERCE, Rockford, USA) following manufacturer’s instructions.
2.5. Synthesis of fluorescein-BSA-iloprost (FLUO-BSA-ILO) and fluorescein-BSAtreprostinil (FLUO-BSA-TRP) 20 mg (0.3 mmol) of BSA was diluted in 2.5 ml of sodium phosphate buffer pH 7.5 and mixed with a 10-fold molar excess of fluorescein-NHS. After stirring 1 h at RT, the mixture was purified on a PBS equilibrated Sephadex G-25 M PD-10 column (GE Healthcare, Uppsala, Sweden). Either 0.7 mg (1.8 mmol) of ILO or 0.8 mg (1.8 mmol) of TRP were dissolved in 130 ml ethanol p.a. and mixed with 370 ml of MOPS buffer 0.1 M pH 6. 0.5 mg (5 mM) of sulfo-NHS (in MOPS buffer) and 0.2 mg (2 mM) of EDC (in MOPS buffer) were added and stirred for 15 min at RT. Afterwards 5 ml (20 mM) of DTT (in distilled water) was added and immediately 3 mg (45.2 nmol) of FLUO-BSA in 190 ml and 210 ml of phosphate buffer 0.5 M were pipetted to the reaction mixture. After stirring for 2 h at RT the mixture was purified on a PBS equilibrated Sephadex G-25 M PD-10 column (GE Healthcare, Uppsala, Sweden). BSA amounts were quantified by BioRad Protein Assay using a BSA standard curve. Coupling efficiencies of the final and intermediate products were determined by TNBS-Assay [21] and measuring the absorbance at 495 nm. Coupling degree of BSA-ILO and BSA-TRP resulted in 10 mol ILO or TRP per mol BSA.
2.6. Synthesis of PEI-graft-iloprost polymers (PEI-g-ILO) Different coupling degrees of PEI-g-ILO were synthesized by variation of EDC amounts given to the reaction mixture. 1 mg (2.8 mmol) of ILO was diluted in 100 ml ethanol p.a., mixed with 68 nmol PEI in 900 ml HEPES buffer 0.1 M pH 7.4 and 1 mg (5 mM) sulfo-NHS. Different amounts of EDC to a final concentration of 25 mM, 50 mM, 60 mM and 100 mM were added and incubated under stirring for 4 h at RT. The reaction mixture was purified on a double-distilled water equilibrated Sephadex G-25 M PD-10 column (GE Healthcare, Uppsala, Sweden). Concentration of PEI was determined with CuSO4 assay according to Ungaro et al. [22]. 1H–1D NMR spectra of PEI-g-ILO were recorded in D2O on a Bruker AV 250 MHz spectrometer. Coupling degrees of PEI-g-ILO were calculated via integration of the broad multiplett of PEI (CH2–CH2–NH–) at d (1H) ¼ 2.5–3.1 ppm and the singulett of the terminal methyl group of ILO (–CXC–CH3) at d (1H) ¼ 1.73 ppm. Covalent conjugation of ILO to PEI resulted in four different coupling degrees (FILO (mol ILO per mol PEI) ¼ 2, 5, 8, 16). PEI-g-ILO constructs were divided into small aliquots, snap-frozen in liquid nitrogen and kept at 80 C until further use.
2.7. Incubation experiment of FLUO-BSA-ILO and FLUO-BSA-TRP Receptor binding/uptake of FLUO-BSA-ILO was investigated in A549, H441, 16HBE14o- and BEAS-2B cells. For FACS measurement experiments, 100,000 cells per well were seeded in 24-well plates (TPP, Trasadingen, Switzerland) 24 h prior to addition of the conjugates. FLUO-BSA-ILO, FLUO-BSA-TRP and FLUO-BSA conjugates, respectively, were diluted in MEM to a concentration of 0.5 mM and incubated on the cells for 4 h at 37 C. After washing the cells with PBS, cells were dislodged from the wells by trypsin treatment and FACS measurements were performed using a Becton Dickinson FACS Scan (San Jose, USA). For confocal laser scanning microscopy, experiments were performed in 4-chamber BD Falcon Culture Slides (BD Biosciences San Jose, USA) with 25,000 cells per chamber. Incubation of FLUO-BSA-ILO and FLUO-BSA was performed as described before. Cells were rinsed and fixed in 4% paraformaldehyde, followed by staining of cell nuclei with 0.33 mM DAPI (40 ,6-diamidino-2-phenylindole) and F-actin with Alexa FluorÒ 568 phalloidin (Invitrogen GmbH, Karlsruhe, Germany) using standard protocols. The slides were covered with mounting media (Vectashield, Vector Laboratories Inc., Burlingame, USA) and images were taken with a confocal laser scanning microscope (Leica, Solms, Germany).
J. Geiger et al. / Biomaterials 31 (2010) 2903–2911 2.8. Inhibition experiment of FLUO-BSA-ILO with CAY10449 Inhibition of receptor binding/uptake of FLUO-BSA-ILO was investigated on 16HBE14o- cells. 24-well plates were prepared as described earlier. CAY10449 was diluted in MEM to concentrations of 15 mM, 30 mM and 150 mM and incubated for 15 min at 37 C. Immediately, FLUO-BSA-ILO and FLUO-BSA were added to a final concentration of 25 nM and incubated on the cells for 4 h at 37 C. Binding/uptake was measured using FACS. 2.9. Preparation of gene vector particles Luciferase reporter gene containing plasmid (pCMV-luc), PEI and PEI-g-ILO, respectively, were diluted separately in 25 ml of double-distilled water. Different N/P ratios (molar ratio of PEI nitrogen to DNA phosphate) were tested. The pCMV-luc solution was added to an equal volume of the polymer solution and gently mixed by pipetting up and down 8 times, resulting in particles with a concentration of 20 mg pCMV-luc per ml. Gene transfer particles were incubated for 20 min at room temperature. 2.10. Particle size measurements Particle size (determined by dynamic light scattering) was measured using a ZetaPALS/Zeta Potential Analyzer (Brookhaven Instruments Corporation, Vienna, Austria). Gene vector particles were generated as described above. The following settings were used: five runs of 1 min measurement per sample; viscosity for water 0.89 cP; Ref. Index 1330; temperature 25 C. 2.11. DNA retardation assay PEI/pCMV-luc and PEI-g-ILO/pCMV-luc gene vector particles with different coupling degrees at N/P 4 were prepared in double-distilled water as described above. 5 ml of each particle solution were mixed either with 2 ml of double-distilled water or 2 ml of a heparan sulfate solution (5 mg/ml). After 45 min of incubation, samples were mixed with 1 ml loading buffer (0.25% bromphenol blue, 0.25% xylene cyanol FF, 30% glycerol in water), loaded into individual wells of a 0.8% agarose gel and separated by agarose gel electrophoresis at 125 V for 1 h. Staining of the gel was performed with ethidium bromide and DNA bands were visualized under UV light. 2.12. In vitro transfection studies A549, 16HBE14o- and BEAS-2B cells were seeded in 24-well plates (TPP, Trasadingen, Switzerland) 24 h prior to transfection with a density of 100,000 cells per well and grown in MEM containing 10% FCS supplemented with 1% (v/v) penicillin/ streptomycin. Before transfection, cells were washed with PBS and 450 ml of fresh serum-free medium was added per well. Subsequently 50 ml of the gene vector particles, corresponding to 1 mg of pCMV-luc, were pipetted onto the cells. For inhibition experiments, CAY10449 was added to fresh media with a concentration of 150 mM 15 min before addition of gene vector particles. After 4 h of incubation, the transfection medium was replaced with MEM including 10% FCS supplemented with 1% (v/v) penicillin/streptomycin. Twenty-four hours post-transfection, luciferase activity was measured using a Wallac Victor2 1420 Multilabel Counter (Perkin Elmer, Boston, USA) according to Huth et al. [23]. Results were normalized to total cell protein using BioRad Protein Assay and BSA as protein standard. 2.13. In vivo gene delivery studies To prepare gene vector particles for aerosol delivery to mice, pCpG-luc and PEI or PEI-g-ILO FILO ¼ 5 were each diluted in 4.0 ml of water for injection (B. Braun Melsungen AG, Melsungen, Germany) resulting in concentrations of 250 mg/ml pCpG-luc and 130.4 mg/ml PEI, respectively (corresponding to an N/P ratio of 4). The pCpG-luc solution was pipetted to the PEI solution, mixed by pipetting up and down 8 times, to yield a final DNA concentration of 125 mg/ml. The particles were incubated for 20 min at RT before use. Particles were nebulized using a PARI Turbo BoyÒ N inhalation device with a PARI LC Plus nebulizer (PARI GmbH, Starnberg, Germany)
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connected to a vertical whole body aerosol device according to Rudolph et al. [24]. After 24 h mice were anaesthetized and pulmonary application of D-luciferin substrate (1.5 mg/50 ml PBS per mouse) was done via sniffing [25]. After 10 min, bioluminescence was measured (IVIS 100 imaging system; Xenogen, Alameda, USA) using camera settings field of view 10, f1 f-stop, high resolution binning, and exposure time of 10 min. To confirm reporter gene expression levels in the lungs, mice were euthanized after in vivo bioluminescent imaging by cervical dislocation. After opening the peritonea by midline incisions, lungs were dissected from animals and perfused with PBS. Lungs were snap-frozen in liquid nitrogen and homogenized in the frozen state. After addition of 400 ml of lysis buffer (250 mM Tris pH 7.8, 0.1% Triton X-100, Roche Complete Protease Inhibitor Cocktail Tablets) and incubation for 20 min on ice, luciferase activity in the supernatant was measured using a Lumat LB9507 tube luminometer (EG&G Berthold, Munich, Germany). Recombinant luciferase (Roche Diagnostics GmbH, Mannheim, Germany) was used as a standard to calculate the amount of luciferase expressed in the lung tissue. 2.14. MTT-based assay Toxicity of PEI/pCMV-luc or PEI-g-ILO FILO ¼ 5/pCMV-luc particles was evaluated on 16HBE14o- cells with an N/P ratio of 4. Cells were seeded 24 h prior to the experiment with a density of 80,000 cells per well into a 24-well plate. Transfection was done as described earlier. After 4 h, transfection mixture was replaced with 400 ml media and MTT based assay was done using the Cell Proliferation Kit I (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. Untreated cells were used as a reference by setting the corresponding absorption to 100% viable cells. 2.15. Serum collection and analysis of cytokine concentration Twenty 4 h after aerosol delivery, blood samples were taken from mice and stored at 4 C over night. After coagulation blood was centrifuged and serum was collected. Quantification of Interleukin 12 (IL-12) and Interferon-g (INF-g) was done using the Mouse IL-12 (P40/P70) and the Mouse INF-g ELISA Kit (RayBiotech, Norcross, USA) according to the manufacturer’s instructions. Untreated mice were used as a reference by setting the corresponding concentration to 1. 2.16. Statistical analysis Results are reported as mean values standard deviation. Statistically significant differences were evaluated by a non paired Student’s t-test. p < 0.01 was considered as significant.
3. Results 3.1. Confirmation of IP1 receptor expression in lung cells by western blot Expression of IP1 receptor in human alveolar (A549) and bronchial (BEAS-2B, 16HBE14o-) epithelial cells was confirmed by western blot analysis. A protein band at 67 kDa could be detected (Fig. 1), which represents the glycosylated form of the IP1 receptor protein expressed on the cell membrane [26]. Therefore, we further investigated targeting of IP1 receptor for protein and gene delivery approaches. 3.2. Targeting of lung cells with different IP1 receptor ligands To investigate targeting of the IP1 receptor for receptor-mediated gene delivery, TRP and ILO were chemically coupled to
Fig. 1. Western blot showing the expression of IP1 receptor protein at 67 kDa in human alveolar (A549) and bronchial (BEAS-2B, 16HBE14o-) epithelial cells. Each lane was loaded with 40 mg of protein extract.
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fluorescein-labeled bovine serum albumin (FLUO-BSA) which served as a model cargo. Whereas incubation of A549 and 16HBE14o- cells with FLUO-BSA resulted in unspecific background binding, incubation with FLUO-BSA-TRP and FLUO-BSA-ILO resulted in 5.5 0.5% and 39.3 0.6% positive A549 and 51 1.8% and 76.1 1.4% positive 16HBE14o- cells, respectively (Fig. 2). The mean fluorescent intensity (MFI) of A549 and 16HBE14o- cells was significantly higher after incubation with FLUO-BSA-ILO than after incubation with FLUO-BSA-TRP. These results illustrate that TRP and ILO are capable of mediating successful binding of the model cargo FLUO-BSA to lung cells, but ILO is the more potent targeting ligand.
obtained in competition experiments with excess of unconjugated ILO (data not shown). FACS measurements together with inhibition experiments provide evidence for cell type-dependent cell surface expression of IP1 receptor on pulmonary epithelial cells. To further test if ILO mediates intracellular uptake of FLUO-BSA-ILO, additional experiments were performed using confocal laser scanning microscopy. 16HBE14o- cells were incubated with either 0.5 mM FLUO-BSA or FLUO-BSA-ILO. Visualization of the cells by confocal microscopy showed clear cell surface binding and subsequent intracellular uptake of FLUO-BSA-ILO conjugates (Fig. 3c), whereas no uptake of FLUO-BSA could be observed.
3.3. Specificity of FLUO-BSA-ILO binding to different lung cell lines
3.4. Characterization of PEI and PEI-g-ILO gene vector particles
ILO was further investigated as a targeting ligand on additional lung cell lines due to its higher cell binding/uptake property when compared with TRP. In addition to A549 and 16HBE14o- cells, incubation of H441 and BEAS-2B cells with FLUO-BSA-ILO resulted in significantly higher (p < 0.01) number of positive cells and MFI than the control FLUO-BSA (38.0 1.8% and 82.7 1.6% vs. 9.1 1.9% and 13.7 1.2% respectively, Fig. 3a). This effect was more pronounced on human bronchial epithelial cells (16HBE14o-, BEAS2B) compared to Clara (H441) or alveolar (A549) epithelial cells. These results indicate the differential cell surface expression of IP1 receptor in human lung cell types. To confirm the receptor specificity of the observed binding of FLUO-BSA-ILO in lung cells, 16HBE14o- cells were incubated with FLUO-BSA-ILO in the presence of increasing amounts of CAY10449. This compound has been previously reported to be a highly specific and potent antagonist of human IP1 receptor [27,28]. 16HBE14ocells were incubated with 25 nM FLUO-BSA-ILO together with increasing concentrations of CAY10449. Addition of CAY10449 resulted in a significant dose-dependent decrease (p < 0.01) of both number of fluorescence-positive cells and MFI (Fig. 3b). At the highest used concentration of CAY10449, the number of fluorescence-positive cells decreased from 95.7 0.7% to 7.4 0.9%. Cells incubated with FLUO-BSA conjugates were used as controls and showed no effect of CAY10449 addition. Similar results were
ILO was coupled to PEI via a carbodiimide chemistry with increasing coupling degree FILO ¼ 2, 5, 8 and 16 and the size of the resulting gene vector particles was measured by dynamic light scattering (Table 1). Particles with coupling degree FILO ¼ 2 and 5 at N/P ratios 4–8 showed hydrodynamic diameters of 50–100 nm which were comparable to PEI gene vectors. Particles prepared at PEI N/P 2, PEI-g-ILO FILO ¼ 2 N/P 2–3 and PEI-g-ILO FILO ¼ 16 N/P 4 were not stable and precipitated. Particles smaller than 150 nm showed a polydispersity of less than 0.2. We next characterized the PEI-g-ILO constructs for their DNA binding affinity. Particles were prepared at N/P 4 and DNA release assay was performed (Fig. 4). For PEI, PEI-g-ILO FILO ¼ 2 and PEI-gILO FILO ¼ 5 there was complete release of DNA upon addition of heparan sulfate. For higher coupling degrees, only partial release of DNA could be observed indicating stronger binding of the polymers to the plasmid. 3.5. In vitro transfection efficiency Increased binding and uptake of FLUO-BSA-ILO by different lung cells and the possibility to form PEI-g-ILO/pCMV-luc particles, prompted us to explore ILO as a ligand to improve gene transfer in vitro. 16HBE14o- cells were transfected with PEI-g-ILO gene vectors and compared with unmodified PEI as control. Gene
Fig. 2. Targeting of IP1 receptor with TRP and ILO on alveolar (A549) and bronchial (16HBE14o-) epithelial cell lines. Incubation of FLUO-BSA, FLUO-BSA-TRP and FLUO-BSA-ILO was performed with a concentration of 0.5 mM (n ¼ 4): FACS measurements. Results are reported as means standard deviation. ** represent statistical significance with p < 0.01, respectively.
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Fig. 3. Distribution of IP1 receptor and receptor binding in alveolar (A549), bronchoalveolar (H441) and bronchial (16HBE14o-, BEAS-2B) epithelial cells. Incubation of FLUO-BSA-ILO and FLUO-BSA was performed with a concentration of 0.5 mM (n ¼ 4): FACS measurements (a). 16HBE14o- cells were incubated with 25 nM FLUO-BSA-ILO and FLUO-BSA together with increasing concentrations of CAY10449 (n ¼ 4): FACS measurements (b). For confocal laser scanning microscopy 16HBE14o- cells were incubated with 0.5 mM FLUO-BSA-ILO and FLUO-BSA (c). Results are reported as means standard deviation. ** represent statistical significance with p < 0.01, respectively.
transfer efficiency increased in accordance with N/P ratio. The highest level of gene expression was found with N/P 4 and FILO ¼ 5. At these optimized conditions gene expression was significantly 46-fold higher than for PEI (Fig. 5a). Particle formation at higher N/P ratios (>4) resulted in no additional increase in gene expression. PEI-g-ILO conjugates with other coupling degrees resulted in either lower or similar transfection levels when compared to PEI. Competitive inhibition experiments with CAY10449 were performed to confirm receptor-mediated gene transfer of PEI-g-ILO
gene vectors. 16HBE14o- cells were transfected with either PEI or PEI-g-ILO FILO ¼ 5 gene vectors at N/P 4 in the presence or absence of 150 mM CAY10449. Gene expression observed with PEI-g-ILO FILO ¼ 5 was significantly (p < 0.01) reduced 33-fold to levels obtained with PEI (Fig. 5b). No effect of CAY10449 was observed on cells transfected with PEI. In addition, PEI-g-ILO FILO ¼ 5 was also tested on A549 and BEAS-2B cells. At optimized conditions PEI-g-ILO FILO ¼ 5 mediated significantly 45- and 14-fold higher expression levels on A549 and BEAS-2B cells than PEI, respectively (Fig. 5c).
Table 1 Physical characterization of PEI/pCMV-luc and PEI-g-ILO/pCMV-luc gene vectors using PEI or PEI-g-ILO with different coupling degrees (FILO ¼ 2, 5, 8, 16) at different N/P ratios: Particle size and polydispersity (in brackets) measurements. Results are reported as means standard deviation (n ¼ 3). N/P 2 PE1 FILO ¼ FILO ¼ FILO ¼ FILO ¼
1258 787 2 1197 1729 5 149 16 8 207 137 16 141 26
N/P 3 (0.34 (0.37 (0.17 (0.19 (0.18
0.04) 69 11 (0.15 0.23) 604 804 (0.25 0.04) 169 35 (0.10 0.10) 366 31 (0.25 0.02) 292 73 (0.22
N/P 4
0.03) 61 10 0.13) 97 20 0.02) 82 4 0.06) 261 13 0.05) 2314 946
N/P 5 (0.17 (0.17 (0.11 (0.12 (0.26
0.01) 60 11 0.07) 77 19 0.03) 75 15 0.03) 165 43 0.14) 418 156
N/P 6 (0.19 (0.15 (0.13 (0.08 (0.17
0.02) 53 8 0.03) 73 17 0.02) 74 11 0.04) 106 7 0.13) 258 61
N/P 8 (0.16 (0.15 (0.13 (0.08 (0.12
0.05) 51 3 0.08) 70 17 0.01) 76 16 0.01) 87 12 0.03) 213 48
(0.14 (0.16 (0.14 (0.12 (0.08
0.01) 0.05) 0.04) 0.03) 0.02)
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Fig. 4. DNA retardation assay of PEI and different PEI-g-ILO constructs at N/P 4. Polymers complexed with pCMV-luc were incubated with (þ) and without () heparan sulfate, resolved on agarose gel and visualized under UV light after ethidium bromide staining.
3.6. In vivo gene delivery studies PEI-g-ILO FILO ¼ 5 and PEI gene vector particles were delivered to the lungs of BALB/c mice via aerosol application and gene expression was analyzed 24 h post gene delivery. Measurement of luciferase gene expression via in vivo bioluminescence imaging showed a strong signal in the lungs of mice treated with PEI-g-ILO FILO ¼ 5 gene vectors but was at the limit of detection for PEI gene vectors (Fig. 6a). To quantify luciferase per g of lung tissue, mice were euthanatized and lungs were isolated. Luciferase expression measured in the homogenized lung tissue was significantly 14-fold higher for PEI-g-ILO FILO ¼ 5 gene vectors than for PEI gene vectors (Fig. 6b). 3.7. Toxicity in vitro and in vivo In vitro cell viability after application of gene vector particles (PEI-g-ILO FILO ¼ 5/pCMV-luc or PEI/pCMV-luc) was measured using MTT assay (Fig. 7a). Compared to PEI, no increase in cytotoxicity could be observed (86.0 10.1% cell viability for PEI-g-ILO FILO ¼ 5 vs. 89.2 3.2% for PEI). For in vivo toxicity and inflammation, serum was obtained from the treated mice and inflammatory cytokines including Interleukin-12 (IL-12) and Interferon-g (INF-g) were measured by ELISA (Fig. 7b). Similar to in vitro MTT results, no significant increase in either of the cytokines 24 h post gene delivery could be detected. 4. Discussion In this study we investigated if the prostaglandin I2 analog ILO, an IP1 receptor agonist, can be used as a targeting ligand for improving gene transfer of cationic polymers such as PEI in lung cells in vitro and in vivo. In our study, we demonstrate that reporter gene expression was significantly increased in human alveolar (A549) and bronchial epithelial cells (16HBE14o-, BEAS-2B) up to 46-fold. In addition, luciferase activity in lungs of mice after aerosol treatment was significantly (14-fold) higher compared to PEI.
Fig. 5. Transfection efficiencies in vitro. Transfection of 16HBE14o- cells with pCMV-luc complexed to different PEI-g-ILO constructs at various N/P ratios (n ¼ 4): Measurement of luciferase gene expression (a). Inhibition experiment of PEI/pCMV-luc and PEI-g-ILO FILO ¼ 5/pCMV-luc particles at an N/P ratio of 4 with CAY10449 (n ¼ 3): Measurement of luciferase gene expression (b). Transfection of A549, 16HBE14o- and BEAS-2B with PEI/pCMV-luc and PEI-g-ILO FILO ¼ 5/pCMV-luc at an N/P ratio of 4 (n ¼ 6): Measurement of luciferase gene expression (c). Luciferase gene expression was measured as relative light units (RLU) luminescence during 10 s/mg of cellular protein. Results are reported as means standard deviation. ** Represent statistical significance with p < 0.01, respectively.
ILO and TRP are agonists of the human IP1 receptor [29]. Both of them are approved for the treatment of pulmonary arterial hypertension via aerosol inhalation and i.v. application [20,30,31]. Furthermore, it has been previously shown that IP1 receptors are
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Fig. 6. In vivo luciferase gene expression obtained with PEI/pCpG-luc and PEI-g-ILO FILO ¼ 5/pCpG-luc particles at an N/P ratio of 4 in lungs of BALB/c mice after aerosol delivery (n ¼ 5). Bioluminescence images with an exposure time of 10 min after 24 h (a). Amount of luciferase expressed in the lung homogenates of mice (b). Results are reported as vertical point plot with median. ** represent statistical significance with p < 0.01, respectively.
Fig. 7. Metabolic activity of 16HBE14o- cells after treatment with PEI/pCMV-luc and PEI-g-ILO FILO ¼ 5/pCMV-luc particles at an N/P ratio of 4 (n ¼ 6). Values of untreated cells were taken to be 100% (a). Serum cytokine levels 24 h after aerosol delivery of PEI/ pCpG-luc and PEI-g-ILO FILO ¼ 5/pCpG-luc particles at an N/P ratio of 4 measured by ELISA (n ¼ 5). Values of untreated mice were taken to be 1. Results are reported as means standard deviation.
expressed in the human and murine lungs [15,32–34]. Moreover, in context with non-viral gene delivery it is important to note that the IP1 receptor/ligand complex is internalized into the cell [35,36]. In our study, we could confirm IP1 receptor expression in various lung cell types by western blot. In order to characterize IP1 receptor expression on the cell surface of lung cells in more detail, we synthesized fluorescein-labeled BSA-conjugates coupled with either ILO or TRP. We then incubated both constructs on alveolar (A549) and bronchial (16HBE14o-) epithelial cell lines and analyzed cell binding by flow cytometry. It should be mentioned, that it is relevant to measure both the number of positive cells and the MFI. The percentage of fluorescein-positive cells gives information about the receptor distribution, whereas the MFI contributes either to the receptor density or the kinetic of particle uptake. Our results demonstrate that IP1 receptors are present on each of the tested cell lines. However, ILO showed higher cell surface binding than TRP and we therefore used ILO as targeting ligand in all subsequent experiments. Specificity of binding to the IP1 receptor was shown by inhibition experiments with the specific IP1 receptor antagonist CAY10449 [27,28,32] and excess of free ILO (data not shown). In order to confirm these observations confocal laser scanning microscopy was performed which demonstrated binding of FLUOBSA-ILO to the cell surface and intracellular uptake on 16HBE14o-
cells. These results therefore demonstrate that ILO can be used as a targeting ligand which mediates binding and intracellular uptake of conjugated cargo model such FLUO-BSA, which represents a precondition for receptor-mediated gene transfer. For transfection studies, ILO was conjugated to branched PEI 25 kDa via amide bond formation. Synthesis resulted in conjugates with coupling degrees of FILO ¼ 2, 5, 8 and 16. PEI-g-ILO/pCMV-luc particles were screened on 16HBE14o- cells and highest transfection efficiency was observed with at N/P 4 FILO ¼ 5, whereas higher coupling degrees 8–6 resulted in lower transfection rates. This could be due to the incomplete release of pCMV-luc at higher coupling degrees as observed by DNA release assay. Release of pDNA from PEI/pDNA particles has been previously shown to be a critical parameter for successful gene transfer [37]. One may speculate that additional hydrophobic interaction of ILO to pDNA could increase pDNA binding. Size measurement of different particles showed that increasing amount of ligand led to large hydrodynamic diameters of PEI-g-ILO/pCMV-luc particles up to 1 mm. Similar results were obtained by Elfinger et al when clenbuterol was coupled to PEI [13]. Particles with PEI-g-ILO FILO ¼ 5 showed hydrodynamic diameters of less than 100 nm. In a previous study, particles of similar size have been shown to be internalized more efficiently than larger particles [38]. Transfection of alveolar
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(A549) and bronchial (16HBE14o-, BEAS-2B) epithelial cells with PEI-g-ILO FILO ¼ 5/pCMV-luc particles at N/P 4 resulted in up to 46-fold increase of reporter gene expression compared to PEI/ pCMV-luc particles at the same N/P ratio in all cell lines tested. The observed enhanced gene expression resulted in no significant increase of metabolic toxicity measured by MTT assay. In addition the hypothesis of receptor-mediated gene transfer was further supported by specific antagonist mediated inhibition experiments in 16HBE14o- cells. Addition of CAY10449 decreased gene expression to levels comparable with PEI. As IP1 receptors are also present on the endothelial cells, we are of the opinion that systemic application of PEI-g-ILO/pDNA particles will most likely result in extensive transfection of the endothelium. Keeping this fact in mind, we selected aerosol as a local route of application to the lungs. For animal experiments, a CpG-free luciferase expression plasmid (pCpG-luc) was used. CpG-free plasmids have been shown to be less inflammatory compared to their CpG containing counterparts. They have also been shown to result in higher and sustained pulmonary gene expression [39]. Prior to animal experiments, we nebulized PEIg-ILO FILO ¼ 5/pCpG-luc and PEI/pCpG-luc gene vectors, respectively, and collected different fractions (nebulized, non-nebulized) in order to test for the stability of the particles. Both gel retardation assay and particle size measurements indicated no effect of nebulization on the particles (data not shown). These observations confirmed that particles were not negatively affected by the aerosolization procedure. Similar results have been reported previously [40]. After aerosol gene delivery to the lungs of mice, gene expression was significantly 14-fold higher for PEI-g-ILO FILO ¼ 5/ pCpG-luc than for PEI/pCpG-luc gene vectors. Measurement of Interleukin-12 (IL-12) and Interferon-g (INF-g) in the murine serum revealed no significant increase in their levels. These observations are in accordance to Gautam et al [41], who showed that aerosol delivery of PEI-DNA particles does not induce high cytokine response. To summarize, in this study we investigated the potential of ILO as a novel ligand for non-viral aerosol gene delivery to the lung. Using fluorescein-based molecular conjugates, we could demonstrate that IP1 receptor is a suitable candidate for receptor-mediated gene delivery to lung cells. Receptor specific binding and uptake of molecular conjugates could be demonstrated in both alveolar and bronchial epithelial and Clara cells. In agreement with these observations, conjugation of ILO led to a specific significant increase (46-fold) of gene expression in vitro. In vivo aerosol delivery to the mice resulted in a 14-fold increase of luciferase expression in the lungs. With the observed more than 10-fold increase in gene expression it may be possible to reduce the amount of pDNA and gene carrier required for a particular amount of expression. This would further reduce both DNA and carrier mediated toxicity and inflammation. Our pre-experiments in 16HBE14o- cells lend support to this assumption. Equal levels of gene expression were observed with half of the amount of PEIg-ILO FILO ¼ 5/pCMV-luc compared to PEI/pCMV-luc gene vector particles (data not shown). Studies are under way to conjugate ILO to biodegradable polymers. Unlike previous ligands, for example lactoferrin and clenbuterol which target bronchial and alveolar epithelial cells respectively [12,13], ILO as a ligand increases gene expression in both cell types. Thus, ILO presents itself as a highly promising ligand for topical PEI-based aerosol application for pulmonary gene transfer. Acknowledgements This work was supported by the BMBF BioFuture programme (FKZ0311898), the programme 13N9182, Magselectofection (LSHB-
CT-2006-019038) and LMUexcellent (Investitionsfonds). G.K. and B.L. acknowledge funding by the ‘‘Fonds der Chemischen Industrie’’ and the ‘‘Deutsche Forschungsgemeinschaft’’ (LU 835/2,3,4). Appendix Figures with essential color discrimination. Figs. 2, 3, 5, 6 in this article have parts that are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10. 1016/j.biomaterials.2009.12.035. References [1] Gill DR, Davies LA, Pringle IA, Hyde SC. The development of gene therapy for diseases of the lung. Cell Mol Life Sci 2004;61(3):355–68. [2] Gurunathan S, Klinman DM, Seder RA. DNA vaccines: immunology, application, and optimization*. Annu Rev Immunol 2000;18:927–74. [3] Davies L, Hyde SC, Gill DR. Plasmid inhalation: delivery to the airways. In: Schleef Martin, editor. DNA pharmaceuticals: formulation and delivery in gene therapy, DNA vaccination and immunotherapy. Wiley-VCH Publishing GmbH & Co. KGaA; 2005. p. 145–59. 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