From Pulmonary Surfactant, Synthetic KL4 Peptide ... - ACS Publications

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From pulmonary surfactant – synthetic KL4 peptide as effective siRNA delivery vector for pulmonary delivery Yingshan Qiu, Michael Y. T. Chow, Wanling Liang, Wing Yan Chung, Judith Mak, and Jenny K.W. Lam Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00725 • Publication Date (Web): 09 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017

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Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Molecular Pharmaceutics

From pulmonary surfactant – synthetic KL4 peptide as effective siRNA delivery vector for pulmonary delivery

Yingshan Qiua, Michael Y. T. Chowa, Wanling Lianga, Winnie W.Y. Chunga, Judith C.W. Mak a,b, Jenny K.W. Lama*

a

Department of Pharmacology & Pharmacy, Li Ka Shing Faculty of Medicine, The

University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong

b

Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong,

21 Sassoon Road, Pokfulam, Hong Kong

Keywords: endocytosis; peptide; pulmonary surfactant, siRNA delivery; surfactant protein B.

ACS Paragon Plus Environment


Abstract Graphic

KL4/siRNA complexes

KL4 peptide (KLLLLKLLLLKLLLLKLLLLK-NH2)

siRNA

Effective in mediating siRNA transfection in the presence siRNA transfection efficiency in the presence of pulmoanry surfactant of pulmonary surfactant ***

remaining GAPDH expression

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OptiMEM

***

1.5

Infasurf (0.5mg/ml)

****

Infasurf (1mg/ml)

***

1.0

0.5

0.0 Lipo 2k(2:1)

Lipo 2k(4:1)

KL4 (20:1)


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Abstract

Pulmonary delivery of small interfering RNA (siRNA) has huge potential for the treatment of a wide range of respiratory diseases. The ability of naked siRNA to transfect cells in the lungs without a delivery vector has prompted the investigation of whether an endogenous component is at least partially responsible for the cellular uptake of siRNA, and whether a safe and efficient delivery system could be developed from this component to further improve the transfection efficiency. Surfactant protein B (SP-B), a positively charged protein molecule found in lung surfactant, is one of the possible candidates. While the role of SP-B in siRNA transfection remains to be determined, the SP-B mimic, synthetic KL4 peptide was investigated in this study as a potential siRNA carrier. KL4 is a 21-residue cationic peptide that was able to bind to siRNA to form nano-sized complexes. It mediated siRNA transfection effectively in vitro on human lung epithelial cells, A549 and BEAS-2B cells, that was comparable to Lipofectamine 2000. When commercial pulmonary surfactant (Infasurf) was added in the transfection medium, the gene silencing effect of siRNA in cells transfected with Lipofectamine 2000 was completely abolished, whereas those transfected with KL4 remained unaffected. At 4°C, KL4 failed to deliver siRNA into the cells, indicating that an energydependent process was involved in the uptake of the complexes. Chlorpromazine (inhibitor of chathrin-mediated endocytosis), but not nystatin (inhibitor of caveolae-mediated endocytosis), inhibited the uptake of KL4/siRNA complexes, suggesting that they entered cells through clathrin-mediated endocytosis. There was no sign of cytotoxicity or immune response caused by KL4 and KL4/siRNA complexes. Overall, this study demonstrated that synthetic KL4 peptide is a promising candidate for siRNA carrier for pulmonary delivery and could be a potential platform for delivering other types of nucleic acid therapeutics.



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Introduction

Small interfering RNA (siRNA) have potential for the treatment of various respiratory diseases including lung cancer, asthma, chronic obstructive pulmonary disease (COPD), tuberculosis and influenza 1, 2. Compared to systemic administration, pulmonary delivery of nucleic acids has several advantages 3, 4. Inhalation is non-invasive and easily accepted by patients. It avoids the serum interaction and nuclease degradation in the blood circulation before reaching the site of action. High local concentration could be achieved by direct delivery of the nucleic acid therapeutics to their target site, thereby reducing the dose required and minimizing systemic adverse effects. Moreover, the lungs are highly vascularized, offering a potential route of systemic delivery of siRNA for the treatment of other diseases.

The pulmonary lining fluid in the airways is often considered to interact with the pulmonary gene delivery systems5, 6. The composition of the pulmonary lining fluid and hence its effect varies along the airways. In the alveolar region, the pulmonary lining fluid contains a mixture of lipids (~90%) and proteins (~10%). This layer of fluid is known as the pulmonary surfactant, and its major function is to lower the surface tension at the respiratory air-liquid interface 7. It is synthesized and secreted into the alveolar space by type II epithelial cells. It has been suggested that the lipid components of the pulmonary surfactant destabilise lipoplexes, leading to the inhibition of gene transfection 6. Nucleic acids are negatively charged macromolecules that are incapable of crossing the cell membrane unassisted. However, gene-silencing effect of naked siRNA (without the use of delivery vector) following pulmonary delivery is evident as demonstrated in various in vivo studies as well as clinical trials 8-12. This leads to a hypothesis that an endogenous component may serve as a ‘natural carrier’ that promotes the cellular uptake of nucleic acids in the airway epithelium. Nevertheless, the complete reliance on the ‘yet to be identified’ endogenous components for cellular uptake of siRNA may not be ideal as this may lead to variation in transfection efficiency, and the gene silencing effect may not be easily reproduced as the dose of siRNA could be difficult to control. Recent publications have suggested that pulmonary surfactants could in fact facilitate the delivery of polymer-based delivery systems 13, 14. A study showed that by coating pulmonary surfactant on siRNA-loaded nanogels, the transfection efficiency in alveolar macrophages was improved in mice following pulmonary administration 15. Based on these observations, the investigation of the role of pulmonary surfactants on nucleic acids


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delivery in the airway is paramount, and a well-designed nucleic acid carrier could potentially be derived from the pulmonary surfactants in order to achieve a robust and reproducible transfection effect.

Back in 1994, Baatz et al. explored the modification of surfactant protein B (SP-B) for the delivery of DNA to the airway cells 16. By conjugation of poly(lysine) with SP-B, the DNA transfection efficiency was enhanced in cell culture. SP-B is one of the major surface-active components in the pulmonary surfactant. It has a net positive charge and promotes membrane fusion 17. This protein may bind to the negatively charged nucleic acids, contributing to the uptake of nucleic acids through membrane fusion mechanism. Based on the structural characteristics of SP-B, a model peptide KL4 was developed 18. KL4 is a 21-residue cationic peptide containing repeating KLLLL sequences. It was originally designed to simulate the overall ratio of cationic to hydrophobic amino acids in native SP-B. It lowers the surface tension at air-liquid interface effectively and was proved be a potent SP-B mimic in vitro as well as in vivo 18-20. KL4 is one of the active components in lucinactant (Surfaxin), a FDA approved intratracheal suspension of pulmonary surfactant indicated for the prevention of respiratory distress syndrome (RDS) in premature infants, and is safe for pulmonary administration. Due to the cationic nature of KL4, it is anticipated that this peptide can form complexes with nucleic acids and mediate effective transfection.

The overall aim of this study was to evaluate the potential of KL4 as a new siRNA carrier for pulmonary delivery. The physicochemical properties such as nucleic acid binding, particle size distribution and morphology of KL4/siRNA complexes were characterised. The siRNA transfection efficiency and the cellular uptake mechanism of the KL4 system were also investigated on lung epithelial cells. To examine the safety of KL4 as siRNA carrier, cytotoxicity study was carried out in lung epithelial cells, and the level of pro-inflammatory cytokines were measured in macrophage cell line.

Experimental Section

Material KL4 peptide (KLLLLKLLLLKLLLLKLLLLK-NH2) was purchased from ChinaPeptides (Shanghai, China) with >90% purity. KL4 stock solution was prepared at 1 mg/ml in 1%



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(v/v) DMSO. Silencer® Select GAPDH Positive Control siRNA and Silencer® Select negative control siRNA were purchased from ThermoFisher Scientific (Waltham, Massachusetts, USA). Fluorescently labeled siRNA (siGLO Cyclophilin B Control siRNA) was purchased from GE Dharmacon (Lafayette, CO, USA). siRNA stock solutions were prepared at 0.5 – 1 mg/ml in ultrapure DEPC-treated water. Dulbecco’s modified eagle medium (DMEM), Keratinocyte-SFM, OptiMEM® I reduced serum medium, Trypsin-EDTA (0.25%), Fetal Bovine Serum (FBS), Antibiotic-Antimycotic (100X), Lipofectamine 2000, Lysotracker Green DND-26, Hoechst 33258, DNA Gel Loading Dye (6X), MTT (3-(4, 5dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) were purchased from ThermoFisher Scientific (Waltham, Massachusetts, USA). GelRed™ nucleic acid stain was purchased from Biotium (Hayward, CA, USA). Anti-GAPDH antibody and anti-beta actin antibody were purchased from abcam (Cambridge, UK). Secondary antibody and Amersham ECL western blotting detection reagents were purchased from GE Health-care (Amersham, UK). Pulmonary surfactant Infasurf (calfactant) was kindly provided by ONY Inc. (Amherst, NY, USA). Mouse tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). Others reagents were obtained from Sigma Aldrich (Saint Louis, MO, USA) as analytical grade or better.

Gel retardation assay The gel retardation assay was carried out to examine the siRNA binding affinity of KL4. KL4/siRNA complexes were prepared at 5:1, 10:1, 15:1, 20:1, 25:1, 30:1 peptide to siRNA ratio (w/w), with 0.2 µg siRNA in 10 µl TAE buffer. The complexes were incubated for 30 min, followed by the addition of 2 µl gel loading dye (1x). The complexes were loaded into an 2% (w/v) agarose gel stained with GelRed™. Electrophoresis was run in TAE buffer at 125 V for 25 min. The gel was visualized under the UV illumination. To study the stability of the KL4/siRNA complexes in the presence of pulmonary surfactant, KL4/siRNA complexes were prepared at 20:1 ratio (w/w). Lipofectamine 2000/siRNA complexes prepared at 2:1 ratios (v/w) were used as comparison. At 30 min after complexes formation, Infasurf (0.5 or 1 mg/ml) were added and the mixtures were incubated for 30 min. The samples were loaded in an agarose gel and electrophoresis was performed as described above.

Particle size and zeta potential measurement


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For particle size measurement, KL4/siRNA complexes were prepared at 5:1 to 30:1 ratio (w/w) with 4 µg siRNA in 100 µl ultrapure water. At 30 min after complexes formation, the hydrodynamic size of the complexes were measured by dynamic light scattering (Delsa-Nano C, Beckman Coulter, CA, USA). For zeta potential measurement, the KL4/siRNA complexes were prepared at 20:1 (w/w) with 20 µg siRNA in 500 µl of 2% PBS. At 30 min after complexes formation, the zeta potential was measured in a flow cell using electrophoretic light scattering (Delsa-Nano C, Beckman Coulter, CA, USA).

Transmission electron microscopy KL4/siRNA complexes were prepared at 5:1 to 30:1 ratio (w/w) with 4 µg siRNA in 200 µl ultrapure water, and the samples were incubated for 30 min. A drop of sample was added to a discharged copper grid coated with carbon-Formvar. After 20 min, a filter paper was used to blot the excess liquid on the grid, followed by staining with 2% (w/v) uranyl acetate. The grid was washed with distilled water twice and air-dried. The morphology of the complexes was visualised by the transmission electron microscope (TEM) (Philips CM100 TEM, Philips Electron Optics, Eindhoven, Nether-lands) at a voltage of 100 kV. Micrographs were taken using a digital camera (Olympus, TENGRA 2.3 x 2.3K bottom mounted camera system with iTEM acquisition software)

Cell culture A549 cells (human alveolar epithelial adenocarcinoma), BEAS-2B cells (human bronchial epithelial cells) and RAW264.7 (mouse macrophage-like cells) were obtained from ATCC (Manassas, VA, USA). A549 cells and RAW264.7 cells were cultured in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) antibiotic-antimycotic. BEAS-2B cells were cultured in Keratinocyte-SFM supplemented with human recombinant Epidermal Growth Factor (rEGF), Bovine Pituitary Extract (BPE) and 1% (v/v) antibiotic-antimycotic. All the cells were maintained at 5% CO2, 37°C, and subcultured according to ATCC instruction.

siRNA transfection A549 cells and BEAS-2B cells were seeded in 6-well plates at a density of 1.5 x 105 and 2 x 105 cells per well, respectively, one day before transfection. The cells were transfected with KL4/siRNA complexes containing 50 pmol GAPDH siRNA or negative control siRNA per well (50 nM). The complexes were prepared in OptiMEM I reduced serum medium at 5:1 to 30:1 ratio (w/w). Lipofectamine 2000 was used as control. The complexes were added to the



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cells and incubated for 4 h at 5% CO2, 37°C before being washed with PBS. The transfection medium was removed and replaced with serum supplemented cell culture medium. At 72 h post-transfection, the cells were washed and lysed with cell lysis buffer. Western blotting assay was performed to analyse the level of GAPDH protein as previously described 21. To study the effect of pulmonary surfactant on transfection efficiency, Infasurf (0.5 or 1 mg/ml) was added to the cells during the 4 h incubation with the transfection medium. Lipofectamine 2000/siRNA complexes prepared 2:1 and 4:1 ratio (w/w) were used for comparison. The GAPDH expression was analysed by densitometry of Western blots using Image J software (Version 1.49). The remaining GAPDH expression was the density of GAPDH band of positive control (normalised with beta-actin band of the corresponding sample) divided by the GAPDH band of the negative control (normalised with the beta-actin band of the corresponding sample).

Confocal imaging A549 cells were seeded in a 35 mm Mattek glass bottom culture dish (Mattek Corp. Ashland, MA, USA) at a density of 1.5 x 105 cells per well one day before imaging. KL4/siRNA complexes at 20:1 ratio (w/w) were prepared with fluorescently labelled siRNA in OptiMEM I reduced serum medium. The complexes containing 150 pmol siRNA were added to the cells in the absence or presence of Infasurf (at a final concentration of 1 mg/ml). After 3 h of incubation, the transfection medium was removed and replaced with fresh culture medium. The Hoechst stain (5 µg/ml) was added to the cells for nuclei staining and incubated for 30 min. At 2 min prior to imaging, Lysotracker (50 nM) was added to the cells to locate the lysosomes. The cells were visualized at 4 h post-transfection by the confocal laser scanning microscope (Zeiss LSM 780 inverted microscope, Jena, Germany) with diode laser (405 nm), argon laser (488 nm), and diode-pumped solid state (DPSS) laser (561 nm).

Flow cytometry study Flow cytometry was used to investigate the cellular uptake of KL4/siRNA complexes. A549 cells were seeded in 6-well plates at a density of 2.5 x 105 cells per well one day before the experiment. The cells were transfected with KL4/siRNA complexes at 10:1 to 30:1 ratios (w/w) containing 150 pmol fluorescently labelled siRNA in Opti-MEM I reduced serum medium per well. To study the effect of pulmonary surfactant on cellular uptake, Infasurf (0.5 or 1 mg/ml) was added to the cells and co-incubated with KL4/siRNA complexes at 20:1 ratio (w/w). The transfection medium was removed after 4 h of incubation and the cells were


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washed with PBS once. The cells were trypsinized by 0.25% (w/v) Trypsin/EDTA and suspended in culture medium. The extracellular florescence signal was quenched with 0.04% (w/v) trypan blue solution. After 2 min of incubation, the cells were washed with PBS thrice. The cells were re-suspended in 500 µl of PBS and sieved with a sterile 40 µm cell strainer (BD Biosciences, CA, USA). The fluorescence intensity was analysed by flow cytometry (BD FACSCantoII Analyzer, BD Biosciences, CA, USA). At least 10,000 single cells were analysed for each sample. To investigate the cellular uptake mechanism, the experiments were carried as described above, except that the cells were either pre-treated with chlorpromazine (10 µg/ml), nystatin (25 µg/ml) or cooled at 4°C for 30 min before the addition of the complexes 22-24. The inhibitors were present in the transfection medium during the 4 h of incubation, and the pre-cooled cells were incubated at 4°C during the transfection. After 4 h, the cells were prepared for flow cytometry study as described.

MTT cytotoxicity assay A549 cells were seeded in 96-well plates at a density of 2 x 104 cells per well the day before experiment. KL4/siRNA complexes were prepared at various ratios from 10:1 up to 30:1 (w/w), each containing 10 pmol of GAPDH siRNA in OptiMEM® I reduced serum medium per well (50 nM), the same siRNA concentration used in the transfection study. The complexes were added to the cells, followed by incubation at 37°C for 4 h and the transfection medium was replaced. MTT assay was carried out at 4 h or 24 h posttransfection. MTT solution (0.8 mg/ml) was added to the cells. After 2 h, the insoluble formazan was dissolved in isopropanol and the absorbance at 595 nm was measured. Cell viability was expressed as the percentage of the absorbance from cells treated with KL4/siRNA complexes against the absorbance from the cells in Opti-MEM I reduced serum medium .

Measurement of pro-inflammatory cytokines RAW264.7 cells were seeded in 6-well plates at 2 x 105 cells per well. Before the experiment, the cells were starved overnight with DMEM supplemented with 1% FBS. The cells were then incubated with KL4/siRNA complexes prepared at 20:1 ratio w/w or KL4 peptide alone at different concentrations in 1 ml of OptiMEM I reduced serum medium. After 4 h, the medium was replaced with DMEM supplemented with 10% FBS. The secreted TNF-α, MCP-1 and IL-6 in cell supernatants were measured by ELISA after 4 h and 24 h of



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incubation. Untreated cells and LPS (10 and 100 ng/ml) were used as negative and positive control, respectively.

Statistical analysis Statistical test was carried out using Prism software version 6 (GraphPad Software Inc., San Diego, CA) and analyzed by one-way analysis of variance (ANOVA). Differences were considered as statistically significant at p < 0.05.

Results

Gel retardation assay The binding affinity of the KL4 to siRNA was evaluated by the gel retardation assay (Fig. 1). There was a gradual decrease in the intensity of siRNA band as the KL4 to siRNA ratio (w/w) increased. The disappearance of the siRNA band at 20:1 ratio indicated that complete binding of siRNA occurred around this ratio. When KL4/siRNA complexes were incubated in pulmonary surfactant (Fig. 2), the complexes (prepared at 20:1 w/w ratio) remained stable as siRNA band could barely been seen. On the other hand, the Lipofectamine 2000/siRNA complexes (prepared at 2:1 v/w ratio) dissociated in the presence of pulmonary surfactant, leading to the release of siRNA.


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KL4 / siRNA complexes siRNA

5:1

10:1

15:1

20:1

25:1

30:1

Figure 1. siRNA binding study by gel retardation assay. KL4/siRNA complexes were prepared at 5:1, 10:1, 15:1, 20:1, 25:1 and 30:1 ratio (w/w). After 30 min of incubation, the samples were loaded into a 2% (w/v) agarose gel stained with GelRed. Unbound siRNA was used as control. Electrophoresis was carried out at 125 V for 25 min and the gel was visualised under the UV illumination.

siRNA

siRNA

KL4/siRNA complexes Lipo2k/siRNA complexes

Infasurf (0.5 mg/ml)

-

-

-

-

+

-

-

+

-

Infasurf (1 mg/ml)

-

+

+

-

-

+

-

-

+

Figure 2. Stability study of siRNA complexes in pulmonary surfactant by gel retardation assay. KL4/siRNA complexes were prepared at 20:1 ratio (w/w) and Lipofectamine 2000 (Lipo2k)/siRNA complexes were prepared at 2:1 ratio (v/w). After 30 min, the complexes were incubated with Infasurf (0.5 or 1 mg/ml) for 30 min. The samples were loaded into a 2% (w/v) agarose gel stained with GelRed. Unbound siRNA, Infasurf, and siRNA+ Infasurf were used as controls. Electrophoresis was carried out at 125 V for 25 min and the gel was visualised under the UV illumination.

Physicochemical properties of KL4/siRNA complexes The hydrodynamic diameter of KL4/siRNA complexes was measured by dynamic light scattering (Table 1). Complexes prepared at different w/w ratios exhibited different particle size. At 5:1 ratio, the mean diameter of the complexes was above 600 nm and the particle size decreased gradually as the ratio increased. At 20:1 ratio, the mean diameter was around 280 nm. At a higher ratio of 30:1, the complexes became larger in size with a mean diameter of around 460 nm. The polydispersity index of the complexes formed at different ratios was similar, all lied between the range of 0.24 to 0.31. The zeta potential of the complexes formed at 20:1 ratio was also measured, and the value was +27.2 ± 1.8 mV (mean ± standard deviation; n=3). The morphology of KL4/siRNA complexes was examined by TEM (Fig. 3).



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At 5:1 ratio, large aggregates were observed. Incomplete siRNA binding could also be seen at low ratios. The complexes formed at 20:1 and 30:1 ratios appeared to be smaller in size and more compact.

Table 1. Particle size of KL4/siRNA complexes measured by dynamic light scattering. The complexes were prepared at different ratios (w/w). The data was presented as mean ± standard deviation (n=3). KL4:siRNA

Hydrodynamic diameter

Polydispersity Index

± standard deviation (nm)

± standard deviation

5:1

655.0 ± 82.7

0.24 ± 0.01

10:1

308.7 ± 11.9

0.25 ± 0.06

20:1

283.3 ± 7.9

0.28 ± 0.02

30:1

460.0 ± 13.7

0.31 ± 0.01

Figure 3. Transmission electron microscopy (TEM) images of KL4/siRNA complexes prepared at different ratios (w/w): (A) 5:1; (B) 10:1; (C) 20:1; and (D) 30:1. The complexes were stained with 2% (w/v) uranyl acetate before imaging. Scale bar = 200 nm.


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siRNA transfection The transfection efficiency of KL4 was performed on A549 cells and BEAS-2B cells using siRNA targeting GAPDH (Fig. 4). At 72 h post-transfection, the GAPDH protein was downregulated by the KL4/siRNA complexes on both cell lines. On A549 cells (Fig. 4A), over 50% knockdown of GAPDH protein was achieved in cells transfected with KL4/siRNA at ratio 15:1 and onwards, and there was no significant difference between Lipofectamine 2000 and KL4/siRNA complexes formed at different ratios. For BEAS-2B cells (Fig. 4B), KL4/siRNA complexes formed at 10:1 to 25:1 ratios had similar knockdown effects with Lipofectamine 2000, with over 50% of GAPDH protein expression was inhibited. Significant improvement can be observed in KL4/siRNA complexes prepared at 30:1 ratio compared to Lipofectamine 2000, with around 80% of GAPDH protein suppression achieved. The transfection efficiency of Lipofectamine 2000/siRNA and KL4/siRNA complexes in the presence of pulmonary surfactant was compared on A549 cells by adding Infasurf in the transfection medium (Fig. 5). The transfection efficiency of Lipofectamine 2000 was abolished after the addition of Infasurf. On the contrary, the transfection efficiency of KL4 was not affected by the presence of pulmonary surfactant. There was no significant difference in GAPDH protein level when the transfection was carried out in the absence or presence of Infasurf.



A remaining GAPDH expression

1.0

0.8

0.6

0.4

0.2

0.0

Lipo 2k

10:1

15:1

-

-

20:1

25:1

30:1

-

-

KL4 : siRNA

-

+

+

-

+

+

+

+

GAPDH β-actin

B

1.0


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0.8

*

0.6

0.4

0.2

0.0

Lipo 2k

10:1

15:1

20:1

25:1

30:1

KL4 : siRNA

-

+

-

+

-

+

-

+

-

+

-

+

GAPDH β-actin

Figure 4. siRNA transfection on (A) A549 cells and (B) BEAS-2B cells. KL4/siRNA complexes were prepared at 10:1 to 30:1 ratios (w/w) with 50 pmol of GAPDH siRNA (+) or negative control siRNA (-) per well in a 6-well plate (50 nM of siRNA). Lipofectamine 2000 (Lipo 2k)/siRNA at 2:1 ratio (v/w) was used as positive control. Western blot analysis of GAPDH protein was performed at 72 h post-transfection, with β-actin used as internal control. Densitometry results were shown as the mean ± standard deviation of three independent repeats (n=3). The data were analyzed by one-way ANOVA followed by Tukey’s post-hoc test. *(p < 0.05).


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A

-

+

-

+

-

+ GAPDH

Lipo2k (2:1)

β-actin

GAPDH

Lipo2k (4:1)

β-actin GAPDH

KL4 (20:1)

β-actin Infasurf (0.5 mg/ml)

Infasurf (1 mg/ml)

B ***


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OptiMEM

***

1.5

Infasurf (0.5mg/ml)

****

Infasurf (1mg/ml)

***

1.0

0.5

0.0 Lipo 2k(2:1)

Lipo 2k(4:1)

KL4 (20:1)

Figure 5. siRNA transfection in the presence of pulmonary surfactant (Infasurf) on A549 cells. KL4/siRNA complexes were prepared at 20:1 ratio (w/w) with 50 pmol of GAPDH siRNA (+) or negative control siRNA (-) per well in a 6-well plate (50 nM of siRNA). Lipofectamine 2000 (Lipo 2k)/siRNA at 2:1 and 4:1 ratio (v/w) were used for comparison. (A) Western blot analysis of GAPDH protein was performed at 72 h post-transfection, with β-actin used as internal control. (B) Densitometry results were shown as the mean ± standard deviation of three independent repeats (n=3). The data were analyzed by one-way ANOVA followed by Tukey’s post-hoc test. ***(p < 0.001); **** (p

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