Nanocomposites of lung surfactant and biodegradable cationic nanoparticles improve transfection efficiency to lung cells

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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l

Nanocomposites of lung surfactant and biodegradable cationic nanoparticles improve transfection efficiency to lung cells Juliane Nguyen a,b, Regina Reul a, Thomas Betz a, Eyas Dayyoub a, Thomas Schmehl b, Tobias Gessler b, Udo Bakowsky a, Werner Seeger b, Thomas Kissel a,⁎ a b

Department of Pharmaceutics and Biopharmacy, Philipps Universität, 35037 Marburg, Germany University of Giessen, Lung Center (UGLC), 35392 Gieβen, Germany

a r t i c l e

i n f o

Article history: Received 9 March 2009 Accepted 26 July 2009 Available online 8 August 2009 Keywords: Nanocomposites Biodegradable nanoparticles Pulmonary gene delivery AFM nanoindentation measurements Aerosolization

a b s t r a c t The objective of this study was to develop highly efficient ternary nanocomposites for aerosol gene therapy consisting of a biodegradable polymer core, poly[vinyl-3-(diethylamino)propylcarbamate-co-vinyl acetateco-vinyl alcohol]-graft-poly(D,L-lactide-co-glycolide), pDNA and a third component to alter surface properties, physicochemical characteristics and biological activity. The effects of the surface altering components lung surfactant, carboxymethyl cellulose (CMC) or poloxamer on nanocomposites were characterized with regard to size, zeta potential, cytotoxicity, biological activity and surface properties. With increasing concentrations of lung surfactant, CMC or poloxamer, sizes of nanocomposites increased. AFM nanoindentation measurements showed a significant increase in adhesion forces of nanocomposites compared to pure nanoparticles. Zeta potential values, cytotoxicity and intracellular uptake demonstrated a strong dependency on the surface altering component. While an excess of CMC led to a decreased uptake into cells due to the negative zeta potential, nanocomposites with lung surfactant displayed enhanced intracellular uptake. Transfection efficiency of nanocomposites with lung surfactant was 12-fold higher compared to pure nanoparticles and 30-fold higher compared to polyethylenimine in lung cells and could also be maintained after nebulization. Ternary nanocomposites prepared with lung surfactant proved to be a potent pulmonary gene delivery vector due to its high stability during aerosolization with a vibrating mesh nebulizer and favourable biological activity. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Nanocomposites or hybrid assemblies are an interesting class of multi-materials and offer a versatile platform for the delivery of macromolecules such as peptides, proteins and nucleic acids [1]. Consisting of dissimilar components different properties are combined in one system. Nanocomposites which are composed of a polymeric particle core surrounded by a lipid shell for example, combine mechanical stability of the nanoparticles with the amphiphilic character and biocompatibility of lipids. To change surface properties of nanocomposites, the outer shell can be substituted by other substances or coating materials. Attachment of the outer shell onto the nanoparticle surface can be achieved by adsorption, electrostatic interactions or chemical modification [2]. In general, exchanging components of nanocomposites or hybrid assemblies creates a new system with different properties. For the treatment of chronic lung diseases such as cystic fibrosis or pulmonary arterial hypertension, nanocomposites made of a biode⁎ Corresponding author. Department of Pharmaceutics and Biopharmacy, Ketzerbach 63, 35032 Marburg, Germany. Tel.: +49 6421 28 25880; fax: +49 6421 28 27016. E-mail address: [email protected] (T. Kissel). 0168-3659/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2009.07.017

gradable and biocompatible material would be advantageous. Degradation to renally excreted, non-toxic cleavage products could circumvent unwanted accumulation in the lung, which would lead to the development of lung fibrosis and lung cancer in the long term [3]. To deliver pDNA effectively to the lung, the nanocomposites should remain stable while protecting pDNA against high shear forces during aerosolization, overcome the numerous barriers in the lung and release the pDNA at the target site [4]. For effective lung deposition particle properties, i.e. aerodynamic diameter, surface charge and hygroscopicity play an important role. By incorporation of the nanoparticulate structures into aerosol droplets in a defined range of 1–5 μm, a deposition in the deeper airways can be achieved [5]. The delivery of nanoparticulate carriers in aerosol droplets is quite effective. A recent study has shown that one aerosol droplet of 3.5 μm comprises approximately 2930 superparamagnetic iron oxide nanoparticles with a core diameter of 50 nm [6]. The formulation of nanocomposites from biodegradable nanoparticles for pulmonary gene delivery can be approached by chemical modification, adsorption and electrostatic interactions. Depending on the functional group on the nanoparticle surface, chemical coupling of ligands, lipids or polysaccharides can be difficult in some cases [7]. Reaction conditions for surface modifications of inorganic or non-

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degradable nanoparticles are often too harsh and would lead to complete destruction of biodegradable polyesters or the encapsulated active ingredients, i.e. pDNA, siRNA, peptides or other sensitive drugs. Therefore several approaches have been undertaken to modify the surface of nanoparticles based on self-assembly processes. One example for surface modification without synthetical procedures are poly(lactic acid-glycolic acid) (PLGA) nanoparticles coated with polysaccharides. Chitosan has been reported to be attached on the surface of these nanoparticles by entanglement with the PLGA chains and electrostatic interactions, leading to higher stability of PLGA nanoparticles in the presence of lysozyme [8]. Using amphiphilic phenoxy dextran, coating of poly(lactid acid) (PLA) nanoparticles was enabled as a result of hydrophobic interactions between the phenoxy groups of the dextran and the hydrophobic surface of PLA nanospheres [9]. The objective of this study was to develop nanocomposites for pulmonary gene delivery and to investigate the impact of different surface altering components on physicochemical properties, cytotoxicity and biological activity. These nanocomposites consisted of biodegradable cationic nanoparticles, poly[vinyl-3-(diethylamino)propylcarbamate-co-vinyl acetate-co-vinyl alcohol]-graft-poly(D,L-lactideco-glycolide), pDNA and a surface altering component attached by hydrophobic, hydrophilic or electrostatic interactions. A lung surfactant, mainly composed of phospholipids, a non-ionic amphiphilic triblock copolymer and negatively charged carboxymethyl cellulose (CMC) were used to create hybrid assemblies with different properties. Atomic force microscopy (AFM) nanoindentation measurements served to prove the attachment of these surface altering components or coating materials on the outer surface of the nanocomposites by studying the adhesive properties of the different nanocomposites.

ZS (Malvern Instruments, Herrenberg, Germany). Measurements of the zeta potential were performed at 25 °C and the duration was set to automatic. Three independent measurements (fifteen runs each) were performed and experiments were carried out in triplicate. 2.4. Atomic force microscopy Nanoindentation experiments of the nanocomposites were carried out on a JPK NanoWizard™ (JPK Instruments, Berlin). Commercially available I-type cantilevers (Micromasch, Estonia) with spring constants of about 0.3 N/m and a length of 350 μm were treated with concentrated H2SO4 for 30 min to obtain a negatively charged surface then washed extensively with distilled water. While the cantilever was extended the deflection of the cantilever was recorded, indicating the force exerted on the tip of the AFM. A precleaned glass slide served as an incompressible surface to calibrate the setup. The sensitivity was determined taking the slope of the retracted part of the obtained spectroscopy curve. Calibration of the spring constant of the installed cantilever was performed by a thermal noise method. The AFM tip was indented into the nanocomposite surface with a force of 4 nN and a depth of 4 nm. The force needed to extract the AFM tip from the nanocomposite surface was measured and is proportional to the adhesive forces and the viscoelastic surface properties. Indentation time and extraction time was 5 s for all measurements. During each experiment 2048 data pairs were recorded. Measurements were performed under water and 20 particles of each nanoparticle formulation were investigated at least 5 times. The adhesion forces were calculated based on the Hooke's law according to Ping et al. [14]. 2.5. Cytotoxicity in A549 and Calu-3 cells

2. Materials and methods 2.1. Materials Biodegradable branched polyesters poly[vinyl-3-(diethylamino) propylcarbamate-co-vinyl acetate-co-vinyl alcohol]-graft-poly(D,Llactide-co-glycolide), abbreviated as P(68)-10, were synthesized and characterized as previously described [10,11]. pCMV-Luc plasmid was purchased from the Plasmid Factory (Bielefeld, Germany). PEI 25 kDa was a kind gift from BASF (Germany). Bovine lung surfactant was obtained from Boehringer Ingelheim (Alveofact™, Germany). It consists of 88% phospholipids, with phosphatidylcholine as the main component (72%) followed by phosphatidylglycerol (8%) and lysophosphatidylcholine (b1%). Additional components are cholesterol, free fatty acids and proteins [12]. The amphiphilic non-ionic triblock copolymer (Poloxamer F68) was purchased from BASF (Ludwigshafen, Germany) and carboxymethyl cellulose (Tylopur® C 600) was obtained from Clariant (Sulzbach, Germany). YOYO-1 iodide (491/ 509) was purchased from Invitrogen (Karlsruhe, Germany). 2.2. Preparation of nanocomposite suspension Nanocomposites were prepared as described by Oster et al. [13]. Briefly, 1.9 mg of polymer was dissolved in 250 μl acetone 48 h prior to use. To obtain an N/P ratio of 10, 25 μg pDNA was dissolved in 50 μl ultrapure water and mixed with the acetone/polymer solution. This mixture was injected into an aqueous solution either containing poloxamer (0.0025% to 0.1%), lung surfactant (0.0025% to 0.045%) or carboxymethyl cellulose (0.00075% to 0.03%). Acetone was removed from the nanocomposite suspensions by evaporation under laminar flow for 3 h. 2.3. Size and zeta potential Hydrodynamic diameter of nanocomposite formulations was determined by photon correlation spectroscopy (PCS) using a Zetasizer Nano

MTT assay was performed to determine the cytotoxicity of the different nanocomposite formulations. Human lung alveolar epithelial cell line (A549) was obtained from the German Collection of Microorganisms and Cell Cultures and was maintained according to the supplier's specifications. Cell passages between 8 and 15 were used for the experiments. The Calu-3 human bronchial epithelial cell line obtained from ATCC was used between passages 3 and 10. A549 cells were seeded into 96-well plates at a density of 8000 cells/well. Calu-3 cells were seeded into 96-well plates at a density of 30,000 cells/well. After 24 h the cell culture medium was replaced with increasing polymer concentration in 100 μl cell culture medium. After 24 h incubation, medium was replaced by DMEM without serum, containing 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma, Deisenhafen, Germany). After 4 h incubation at 37 °C, medium was aspirated and formazane crystals were dissolved in 200 μl DMSO per well. Measurement was performed using an ELISA reader (Titertek Plus Ms 212, ICN, Eschwege, Germany) at a wavelength of 570 nm and 690 nm. Relative viability was calculated using 0% (wells without cells) and 100% (wells with untreated cells) controls. Data are presented as a mean of four measurements. IC50 was calculated using the Boltzman sigmoidal function from Microcal Origin® v 7.0 (OriginLab, Northampton, USA). Experiments were repeated twice. 2.6. Uptake kinetics in A549 cells by FACS For uptake studies A549 cells were seeded on 48-well plates at a density of 30,000 cells/well 24 h prior to the experiment. pDNA was fluorescently labeled with YOYO-1 [15]. Cells were incubated with the nanocomposite formulations at different timepoints (0.5 h, 1 h, 2 h, 3 h and 4 h) at 37 °C. After washing the cells with PBS extracellular fluorescence was quenched by incubation with Trypan blue 0.4% for 5 min, followed by washing with PBS again. Cells were detached by trypsinization and were resuspended in a 1:1 mixture of FACSFlow (BD Biosciences, San Jose, CA) and 4% paraformaldehyde in PBS for cell

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fixation. The amount of nanocomposites taken up into A549 cells was measured by FACScan (BD Biosciences, San Jose, CA) with excitation at 488 nm and the emission filter set to 530/30 bandpass. 5000 cells were counted for each sample and experiments were performed in triplicate. Data acquisition and analysis were performed using CellQuest Pro (BD Biosciences, San Jose, CA) and FCS Express V3.00 (DeNovo Software, Thornhill, Canada).

2.9. Statistics

2.7. Transfection experiments

3.1. Hydrodynamic diameter and zeta potential

A549 cells were cultured in DMEM supplemented with 10% fetal calf serum. Transfection experiments were performed as described by Oster et al. [13]. 24 h before transfection cells were plated in 24-well plates at a density of 25000 cells/well. Cells were incubated with 100 μl of the nanocomposite suspension containing 2 μg pCMV-Luc for 4 h in DMEM supplemented with 10% FCS, before exchanging the medium. Cells were harvested after a further 44 h. Luciferase gene expression was measured according to the protocol provided by Promega (Madison, WI, USA) as previously described [16]. All experiments were performed in triplicate and data were expressed in ng luciferase per mg protein (±SD). Experiments were repeated twice. The Calu-3 cells were seeded on 12-Transwell cell culture supports under air-interfaced conditions at a density of 1 × 105 cells/cm2 as previously described [16]. Prior to transfection experiments TEERmeasurements were performed on 3 control wells using chopstick electrodes and an EVOM volt ohmmeter (STX-2 and Evom G. World Precision Instruments, Berlin, Germany) to ensure cell integrity. After 14 days of culturing the Calu-3 cells 200 μl polyplex solution, containing 4 μg pCMV-Luc were added or directly nebulized onto the apical chamber of the transwells. The nanocomposite suspensions were aspirated after 4 h and medium in the basolateral chambers was exchanged. The cells were allowed to grow for additional 44 h and luciferase expression was quantified. Data were expressed in ng of luciferase per mg protein (±SD). TEER values of all transwells were measured after 48 h of transfection. At this time no significant differences between the TEER values of control wells and transfected wells could be observed. Experiments were performed in quadruplicate and are representative of three independent experiments.

To investigate the impact of different surface altering components and concentrations on physicochemical characteristics of nanocomposites, hydrodynamic diameter and zeta potential of the different formulations were investigated. Dependent on the surface altering components and concentrations used changes in size or zeta potential should occur. As depicted in Fig. 1A hydrodynamic diameter of nanocomposites increased with increasing concentrations of poloxamer. Nanocomposites prepared with the lowest concentration of poloxamer (0.0025%) were with 186 nm smaller than nanocomposites containing 0.1% poloxamer (198 nm). This is in good agreement with findings obtained by Redhead et al. and Dunn et al., who reported coating layer thicknesses of poloxamer between 2 nm and 10 nm [17,18]. Similar results were obtained for nanocomposites containing lung surfactant (Fig. 1B). With 210 nm nanocomposites in 0.0025% lung surfactant displayed significantly smaller particle sizes than those prepared in the highest concentration of lung surfactant (230 nm). Using carboxymethyl cellulose (CMC) as the third component significant changes in size and zeta potential of the nanocomposites were observed (Fig. 1C). With increasing concentrations of CMC particle sizes increased from 170 nm to 446 nm and zeta potential values decreased successively from + 40 mV to − 40 mV. The large particle sizes of the nanocomposites prepared in high concentrations of CMC (0.015% and 0.03%) were probably not only caused by an additional coating layer but also caused by interactions between the positively charged P(68)-10 polymer and the negatively charged CMC. These electrostatic interactions could have weakened the binding affinity of the P(68)-10 polymer to pDNA leading to larger particle sizes. The negligible changes in size when prepared with the lowest concentrations of CMC are most likely due to the strong entanglement of the negatively charged CMC with the positively charged tertiary amines of the nanocomposites. Overall, the increase in particle size and change in zeta potential can be most likely attributed to the adsorption of these coating materials on the surface of the nanocomposites. Based on the hydrodynamic diameter and zeta potential values one concentration from each nanocomposite formulation was chosen for further investigation. As there was no large difference in size and zeta potential in the case of nanocomposites prepared in poloxamer and lung surfactant, the concentrations with the highest transfection efficiency were further studied (data not shown). In addition, two different CMC formulations with similar sizes but different surface charges served to study the effect of a negative zeta potential on cytotoxicity and transfection efficiency. For further investigations the following nanocomposite formulations were selected using abbreviations as listed below.

2.8. Nebulization of nanocomposite suspensions Nanocomposite suspensions were nebulized under aseptic conditions using the sterilized vibrating mesh nebulizer Aeroneb™. For transfection experiments in A549 cells the aerosol was collected and condensed into a 1.5 ml Eppendorf-cup. For transfection experiments in Calu-3 cells, cultured under air-interfaced conditions, nanocomposite suspension was directly nebulized onto the cells. A 2.5 cm spacer was attached to the Aeroneb and placed directly onto the transwell. Nanocomposite suspension was nebulized onto the Calu-3 cells for a duration of 2 min, which is equivalent to the amount of nanocomposites contained in 200 μl of nanocomposite suspension. This has been shown by collecting and mass weighing the samples after freeze drying at different time points (see Suppl. Fig. 1 for setup of the nebulization apparatus). Experiments were performed in quadruplicate and are representative of three independent experiments. pDNA stability before and after nebulization was investigated using gel electrophoresis. Briefly, 30 μl nanoparticle suspensions were degraded by addition of NaOH at a pH 10 for 30 min. To completely release the pDNA from the nanoparticles 50 I.U. of heparin was added and incubated for 15 min. 30 μl of nanoparticle suspension was loaded onto a 1% agarose gel supplemented with ethidium bromide. One hundred volts were applied to the gel for 60 min using TAE pH 7.4 as a running buffer. Degraded pDNA can be seen as a smear band, whereas intact pDNA show opencircular, linear and supercoiled bands. Experiments were performed three times and representative images are shown.

Significance between the mean values was calculated using oneway Anova analysis with Dunnett's post test or two-way Anova with Bonferroni post test. 3. Results

P(68)-10 pure: nanoparticle without coating materials P(68)-10 lung surfactant: nanocomposites with 0.0075% lung surfactant P(68)-10 CMC 1: nanocomposites with 0.00075% CMC P(68)-10 CMC 2: nanocomposites with 0.0075% CMC P(68)-10 poloxamer: nanocomposites with 0.1% poloxamer 3.2. Atomic force microscopy: AFM nanoindentation measurements As the preparation method allows encapsulation of the coating materials into the nanocomposites as well as coating of the nanocomposite surfaces, AFM was not only applied to gain information about the

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directly correlates with the properties caused by the coating materials. These coatings influenced the adhesive forces of the cantilever to the cell surface as well as the mechanical properties of the nanocomposites. Measurements of the adhesion properties of the different nanocomposite formulations would provide the information whether the coating materials are just dispersed or solved in the nanoparticle suspension, or whether they are directly attached to the nanoparticle surface. Hence, the adhesive properties of nanocomposites prepared in lung surfactant, CMC and poloxamer should differ from those of the pure nanoparticles. As depicted in Fig. 2, the forces needed to extract the tip from the surface of the nanoparticles prepared with different coatings were significantly different from the force applied when investigating the pure nanoparticles. With 3.69 × 10− 9 N the pure nanoparticles displayed the lowest adhesion force of all nanocomposite formulations. When testing the nanoparticles with different coating materials, P(68)-10 poloxamer displayed a 1.5-fold, P(68)-10 lung surfactant a 5.7 fold and P(68)-10 CMC 1 a 7-fold increase in the forces applied compared to the pure nanoparticles. This was most likely due to the additional layer on the pure nanoparticles. Creating a so-called softshell, the adhesion surface increased leading to enhanced interactions with the AFM tip. An excess of CMC on the nanoparticle surface, as it was the case with P(68)-10 CMC 2, led to such high repulsion with the negatively charged cantilever, that the adhesion force measured was negligible. This is in agreement with the negative surface charge, which was confirmed by the zeta potential values. These data suggest that surface properties of nanocomposites change, when using different coatings materials.

Fig. 1. Hydrodynamic diameter and zeta potential of P(68)-10/pDNA nanocomposite formulations in poloxamer (A), in lung surfactant (B) and in CMC (C). Significant differences compared to pure nanoparticles are noted with asterisks, ⁎p b 0.5, ⁎⁎p b 0.01.

morphology but also about the surface properties of the nanocomposites. With all formulations tested distinct and spherical nanocomposites with a smooth surface could be generated (Suppl. Fig. 2). Mechanical properties of materials, such as stiffness, elasticity or adhesive properties, can be investigated by AFM nanoindentation measurements [19]. Neu et al. have shown that mechanical stiffness of PEI/pDNA polyplexes significantly increased after crosslinking, thereby providing higher resistance against shear forces in the blood stream [20]. The force needed to extract the AFM tip from a nanoparticle surface

Fig. 2. (A) Schematic of a cantilever deflection vs piezo height curve (Dc vs Zc). (B) Adhesion force measurements by AFM on P(68)-10/pDNA nanocomposites with different surface coatings. Significant differences compared to pure nanoparticles are noted with asterisks, ⁎p b 0.05, ⁎⁎pb 0.01.

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Fig. 4. Uptake kinetics of YOYO-1 labelled pDNA in nanocomposites with different surface coatings and PEI/pDNA polyplexes over 0.5 h, 1 h, 2 h, 3 h and 4 h. Significant differences compared to pure nanoparticles are noted with asterisks, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001.

Calu-3 cells. In both cell lines P(68)-10 CMC 2 nanocomposites displayed such a low cytotoxicity that IC50 values could not be determined (Table 1). PEI 25 kDa served as a comparison.

3.4. Nanocomposite stability during nebulization

Fig. 3. Cytotoxicity of P(68)-10/pDNA nanocomposite formulations studied by MTT assay in A549 cells (A) and Calu-3 cells (B).

As ultrasonic energy is known to alter aerosolized drug and gene delivery formulations [4], nanocomposite suspensions were characterized with regard to size, zeta potential and integrity of pDNA before and after nebulization. No statistically significant changes in particle sizes and zeta potential were found for all nanocomposite formulations (unpaired Student's test p b 0.05) (see Table 1). While naked pDNA was completely degraded after aerosolization, visualized as a smear band in gelectrophoresis, nanocomposites provided effective pDNA protection against the destructive forces generated during nebulization (Suppl. Fig. 3).

3.3. Cytotoxicity in A549 and Calu-3 cells Cytotoxicity of the different nanocomposite formulations was evaluated by an MTT assay. Coating of the nanoparticles with different coating materials did not only alter surface properties of nanoparticles, as shown by AFM-measurements, but also had an effect on the cytotoxicity. For negatively charged nanocomposites, formulated in an excess of CMC, a cell viability of ~80% at a polymer concentration of 1.5 mg/ml could be observed (Fig. 3). Pure nanoparticles and nanocomposite formulations prepared in lung surfactant, poloxamer and CMC 1 however, displayed a higher cytotoxicity. This was most likely due to their positive zeta potential resulting in higher cell interactions. All nanocomposite formulations showed less toxic effects than PEI 25 kDa. IC50 values of pure nanoparticles, P(68)-10 lung surfactant, P(68)-10 poloxamer and P(68)-10 CMC 1 were between 0.33 mg/ml and 0.41 mg/ml in A549 cells and ~ 1 mg/ml in

3.5. Uptake kinetics in A549 by FACS and CLSM Cellular uptake of the different nanoparticles was investigated by FACS analysis and Confocal Laser Scanning Microscopy (CLSM). The highest uptake could be observed for the nanocomposite formulation with lung surfactant (Fig. 4). With a mean fluorescence intensity of 110 MFI, nanocomposites in lung surfactant were taken up twice as much as the pure nanoparticles and the nanocomposites formulated with poloxamer or CMC 1. The negatively charged nanocomposites P(68)-10 CMC 2 showed significantly less uptake than pure P(68)-10 nanoparticles. Uptake after a 30 min incubation was negligible and was only slightly increasing to 30 MFI after a 4 h incubation. CLSM images support the data assessed by FACS measurements (Suppl. Fig. 4). PEI 25 kDa served as comparison.

Table 1 Physicochemical properties of P(68)-10/pDNA nanocomposite formulations before and after nebulization.

P(68)-10 pure P(68)-10 lung surfactant P(68)-10 poloxamer P(68)-10 CMC 1 P(68)-10 CMC 2 PEI 25 kDa nn = non-nebulized. n = nebulized.

Hydrodynamic diameter (nm)

Zeta potential (mV)

nn

n

nn

n

A549 cells

IC50 values (mg/ml) Calu-3 cells

174.7 ± 4.0 214.8 ± 3.4 198.1 ± 4.2 170.1 ± 3.1 203.8 ± 4.8 102.3 ± 3.3

168.2 ± 4.0 210.2 ± 5.9 208.5 ± 6.1 190.1 ± 12.3 221.1 ± 10.1 109.1 ± 10.2

44.3 ± 1.1 38.0 ± 0.4 42.2 ± 0.9 39.8 ± 0.4 − 21.6 ± 0.4 27.8 ± 2.8

45.2 ± 3.1 37.5 ± 5.1 40.4 ± 1.0 40.2 ± 3.6 − 23.7 ± 2.9 29.3 ± 4.2

0.33 ± 0.01 0.39 ± 0.03 0.34 ± 0.04 0.41 ± 0.04 n.d. 0.02 ± 1E-3

1.05 ± 0.13 1.02 ± 0.01 1.03 ± 0.07 1.00 ± 0.01 n.d. 0.03 ± 1E-3

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3.6. Transfection experiments in A549 cells Surface coating had not only an effect on surface properties and uptake but also upon the transfection efficiency of the nanocomposite formulations in A549 cells. Nanocomposites with a lung surfactant layer showed the highest luciferase expression. With 5.5 ng luciferase/ mg protein it displayed a 12-fold increase in transfection efficiency compared to the pure nanoparticles and 30 times higher transfection efficiency than PEI 25 kDa (Fig. 5). In accordance to the data obtained by FACS and CLSM the negatively charged nanocomposites, P(68)-10 CMC 2, showed only little uptake into A549 cells. As a result the transfection efficiency in vitro was negligible. Pure nanoparticles and nanocomposites prepared in poloxamer or CMC 1 exhibited a slightly higher expression than PEI 25 kDa but were far lower than that of the nanocomposites coated with lung surfactant. For PEI 25 kDa an N/P ratio of 10 was chosen. This has been shown to be an optimal ratio for PEI with regard to transfection efficiency and cytotoxicity [21]. Though PEI 25 kDa displayed a higher intracellular pDNA uptake, its transfection efficiency was lower than that of P(68)-10 lung surfactant nanocomposites. These data indicate that a high intracellular uptake does not necessarily correlate with superior transfection efficiency. We assume that the degradation behaviour of the nanocomposites might be advantageous for effective nucleic acid delivery as reported earlier [10]. 3.7. Bioactivity in A549 and Calu-3 (AIC) before and after nebulization For further in vitro characterization the nanocomposite formulation with the highest bioactivity in A549 cells was chosen. pDNA integrity could be maintained in all nanocomposite formulations after aerosolization. Here, the nebulization effects on the transfection efficiency of nanocomposites coated with lung surfactant were investigated in A549 cells and Calu-3 cells. To mimic the in vivo situation, Calu-3 cells were cultured under air-interfaced conditions for 14 days [16]. Nanocomposite suspensions or polyplex solutions were directly nebulized onto the cells at the apical side. As can be seen in Fig. 6 the high level of transfection efficiency could be maintained even after nebulization. Aerosolization did not have a significant effect on the transfection efficiency in both cell lines, showing good stability of the P(68)-10 lung surfactant formulation against the high shear forces. This is in good agreement with the data obtained by gel electrophoresis (Suppl. Fig. 3). 4. Discussion The objective of this study was to develop highly efficient nanocomposite systems for pulmonary gene delivery, whose properties

Fig. 5. Transfection efficiency of P(68)-10/pDNA nanocomposite formulations with different surface coatings. Significant differences compared to P(68)-10 pure and PEI 25 kDa are marked with asterisks, ⁎⁎p b 0.01.

Fig. 6. Transfection efficiency of P(68)-10/pDNA nanocomposites with lung surfactant in (A) A549 cells and (B) Calu-3 cells before and after nebulization. Significant differences compared to PEI 25 kDa are noted with asterisks, ⁎⁎p b 0.01.

could be controlled by self-assembly surface modifications. Using negatively charged CMC, an amphiphilic lung surfactant and a nonionic copolymer poloxamer comprising a hydrophobic polyoxypropylene part flanked by two hydrophilic polyoxyethylene chains, adsorption and interaction with the positively charged nanoparticles can follow hydrophilic, hydrophobic or electrostatic processes. The polyanion carboxymethyl cellulose has previously been used as a coating material in nanoparticle formulations to control size and zeta potential, thereby enhancing the stability after nebulization [22–24]. Nanoparticles coated with poloxamer were reported to improve the anti-tumor effect of doxorubicin [25]. In the case of insulin encapsulating nanoparticles poloxamer led to a sustained release of insulin combined with a prolonged reduction of blood glucose levels in streptozotocin induced diabetic rats [26]. A bovine derived commercially available lung surfactant (Alveofact™) used for the treatment of premature newborns with surfactant deficiency, served as a third coating material [12]. It consists of 88% phospholipids, with phosphatidylcholine (PC) as the main component (72%), followed by phosphatidylglycerol (8%) and lysophosphatidylcholine (b1%). Additional components are cholesterol, free fatty acids and proteins. In Fig. 7 a schematic pattern of the possible formations and interactions of the coating materials with the nanoparticles is shown. Phosphatidylcholine comprises a hydrophilic zwitterionic headgroup and a hydrophobic part consisting of fatty acid chains. Hence, hydrophilic, hydrophobic and electrostatic interactions with the P(68)-10/pDNA nanoparticles are possible. Poloxamer might be attached with the hydrophobic PLGA chains of the nanoparticles via the hydrophobic polyoxypropylene anchor, while leaving the hydrophilic chains sticking out into the surrounding medium [27]. The negatively charged CMC might be electrostatically bound to the positively charged Diethylamino-1-propylamine (DEAPA) groups of the P(68)-10/pDNA nanoparticles. AFM nanoindentation measurements in combination with changes in zeta potential values and hydrodynamic

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Fig. 7. Schematic pattern of the structural organization of nanocomposites prepared with P(68)-10/pDNA nanoparticles and lung surfactant, CMC or poloxamer.

diameter could prove that nanocomposite surfaces were altered when prepared with different coating materials. As the adhesive forces between the AFM tip and the nanoparticle surface is dependent on surface properties such as charge, roughness or elasticity, the measured changes in adhesive properties of the nanocomposites suggests that the added coating materials formed an additional layer around the pure nanoparticles [19]. Creating a so-called soft-shell the adhesion surface increased leading to enhanced interactions with the AFM tip. The effect of the coatings on the nanoparticles was not only displayed in an increase in particle size, changes of zeta potential and adhesive forces, but also in the cytotoxic effects measured by a MTT assay. When coated with a high amount of CMC (P(68)-10 CMC 2) still 80% of the cells remained intact at the highest polymer concentration. This is most likely due to the negative zeta potential, which showed less interaction with the negatively charged cell membrane. All other nanocomposite formulations with positive surface charges displayed higher toxicity profiles. Furthermore, surface coating had a significant impact upon the intracellular uptake and biological activity of the ternary nanocomposites. While the P(68)-10 CMC 2 formulation showed only very low transfection efficiency induced by an excess of anionic CMC followed by a limited cellular uptake, nanocomposites with a lung surfactant layer showed an enhanced cellular uptake and a 12-fold increase in luciferase expression compared to the pure nanoparticles in A549 cells. Significantly higher transfection efficiency could also be achieved in Calu-3 cells, which were cultured under airinterface conditions to mimic the in vivo situation in the human lung. Composed of 80% phosphatidylcholine and phosphatidylglycerol the addition of lung surfactant most likely led to the formation of a lipid layer around the nanoparticles, which might have enhanced the

cellular uptake. The mechanisms of increased intracellular uptake and transfection efficiency after surfactant coating of nanoparticles remain to be investigated in detail. Enhanced cellular uptake by membrane fusion [28] as it has been suggested for liposomes, or accelerated and improved endosomal escape are possible explanations for the increase in transfection efficiency [29]. In good agreement with our results Fenart et al. have reported an increase in intracellular uptake of polysaccharide nanoparticles into endothelial cells after coating with phospholipids [30]. The P(68)-10 lung surfactant formulation also displayed remarkably good stability during nebulization. Transfection efficiency could be maintained after nebulization with a vibrating mesh nebulizer (Aeroneb™) showing that P(68)-10/pDNA nanocomposites prepared in lung surfactant are suitable carriers for inhalative pulmonary gene delivery. Additional investigations are required to fully elucidate the structural organization of these highly dynamic nanocomposite systems. In conclusion we have successfully demonstrated how physicochemical characteristics and biological activity of biodegradable nanocomposites can be influenced by exchanging the surface altering component. From the different formulations tested the nanocomposites prepared with lung surfactant proved to be most suitable for pulmonary gene delivery, as it combined superior transfection efficiency with good nebulization properties, when aerosolized with a mesh nebulizer. Acknowledgements We would like to thank Katherine Millen (Cambridge University) for carefully revising this manuscript. We thank Eva Mohr for her

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