Inhalation Toxicology, 2009; 21(S1): 104–109

R E S E A R C H A R T I C L E

Low cytotoxicity of solid lipid nanoparticles in in vitro and ex vivo lung models

Matthias Nassimi1,2, Carsten Schleh1,3, Hans-Dieter Lauenstein1,3, Riem Hussein2, Katrin Lübbers2, Gerhard Pohlmann1, Simone Switalla1, Katherina Sewald1, Meike Müller1, Norbert Krug1, Christel C. Müller-Goymann2, and Armin Braun1

1Fraunhofer-Institut für Toxikologie und Experimentelle Medizin ITEM, Hannover, Germany, 2Institut für Pharmazeutische Technologie, Technische Universität Braunschweig, Braunschweig, Germany, and 3Medizinische Hochschule Hannover, Hannover, Germany

Christel C. Müller-Goymann and Armin Braun contributed equally to this work.Address for Correspondence: Armin Braun, Fraunhofer-Institut für Toxikologie und Experimentelle Medizin ITEM, Nikolai-Fuchs-Strasse 1, 30625 Hannover, Germany. E-mail: armin.braun@item.fraunhofer.de. Tel: +495115350263; Fax: +495115350155

(Received 23 April 2009; accepted 27 April 2009)

ISSN 0895-8378 print/ISSN 1091-7691 online © 2009 Informa UK LtdDOI: 10.1080/08958370903005769

AbstractThe aim of this study was to investigate the potential cytotoxicity of solid lipid nanoparticles (SLN) for human lung as a suitable drug delivery system (DDS). Therefore we used a human alveolar epithelial cell line (A549) and murine precision-cut lung slices (PCLS) to estimate the tolerable doses of these particles for lung cells. A549 cells (in vitro) and precision-cut lung slices (ex vivo) were incubated with SLN20 (20% phospholipids in the lipid matrix of the particles) and SLN50 (50% phospholipids in the lipid matrix of the particles) in increasing concentrations. The cytotoxic effects of SLN were evaluated in vitro by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Vitality of lung slices was controlled by staining with calcein AM/ethidium homodimer 1 using confocal laser scanning microscopy and followed by quantitative image analy-sis with IMARIS software. A549 cell line revealed a middle effective concentration (EC

50) for MTT assay for SLN20

of 4080 µg/ml and for SLN50 of 1520 µg/ml. The cytotoxicity in terms of LDH release showed comparable EC50

values of 3431 µg/ml and 1253 µg/ml for SLN20 and SLN50, respectively. However, in PCLS we determined only SLN50 cytotoxic values with a concentration of 1500 µg/ml. The lung slices seem to be a more sensitive test sys-tem. SLN20 showed lower toxic values in all test systems. Therefore we conclude that SLN20 could be used as a suitable DDS for the lung, from a toxicological point of view.

Keywords: Solid lipid nanoparticles; drug delivery; lung toxicity; precision cut lung slices

http://www.informapharmascience.com/iht

Introduction

Solid lipid nanoparticles (SLN) are part of aqueous nanoscale suspensions prepared from at body temperature solid lipids. SLN are a new form of particulate carriers besides the more conventional carriers such as liposomes, lipid emulsions and polymeric nanoparticles (Müller et al., 1995). Improved bioavailability, retention of drug release, and protection of sensitive drugs are proposed properties for SLNs.

Growing attention has been given to the pulmonary route as an alternative non-invasive route for both local and systemic drug delivery using lipid nanoparticles (Agu et al., 2001). The large inner surface of the lung and the thin alveolar epithelium allows rapid drug absorption (Videira

et al., 2006). The lung deposition efficiency is highly size dependent. The alveoli can be effectively targeted for drug absorption by delivering the vehicle as an aerosol, with mass median aerodynamic diameter less than 5 µm (Videira et al., 2006). Local inflammatory respiratory diseases are prime candidates for inhalation therapy, such as asthma and chronic obstructive pulmonary disease (COPD). According to Melani et al., inhalation therapy is the most effective, saf-est, and most cost-effective of all therapy (Melani, 2007). Treating lung diseases locally avoids first-pass metabolism and deposits directly at the disease site. This specific type of topical drug administration to the lung also minimizes potential side effects from high systemic concentrations

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Cytotoxicity of solid lipid nanoparticles 105

that are characteristics of conventional drug delivery (Bailey et al., 2009). Using radiolabelled lipid nanoparticles it was demonstrated that a few minutes after aerosolization, the inhaled material was translocated to the regional lymph nodes where a significant accumulation was found. The major fraction of the radiolabelled solid lipid nanoparticles was detected in the para-aortic, axillary, and inguinal lymph nodes (Videira et al., 2002). Lymphatic uptake after pulmo-nary administered lipid nanoparticles indicates that these vehicles are useful colloidal drug carriers for therapeutic purposes; therefore, inhalation could be an effective route to deliver immunomodulatory drugs directly into the local lymphnodes by lipid nanoparticles (Videira et al., 2006).

Peptides and proteins are susceptible to enzymatic degra-dation in the gastrointestinal tract (GT) in terms of denatura-tion (Bosquillon et al., 2004). Although metabolic enzymes can also be found in the lungs, the metabolic activities and pathways differ from those observed in the GT, which makes pulmonary administration for many protein drugs very promising. For example, Shen et al. (1999) found that pro-teolytic enzyme activities in lungs are lower than in the GT.

Considering the future potential of SLN as a drug deliv-ery system (DDS) for the lung, the acute cytotoxicity of SLN is an essential parameter (Schubert et al., 2005). The aim of the present study was to provide an in vitro and ex vivo approach of the toxic potential of solid lipid nano-particles. The SLN consisted of Softisan 154 (triglyceride mixture), Phospholipon 90G (soy lecithin), and Solutol HS15 (macrogol-15-hydroxystearate, Ph.Eur) used as a stabilizer. For the in vitro approach, human type II pneu-mocytes-like cells (A549 cell line) were exposed to different doses of nanoparticle suspension. The cytotoxicity of these nanosuspensions was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assay. For the ex vivo approach, cytotoxicity of the nanoparticles was determined using precision-cut lung slices (PCLS) and live/dead staining for confocal microscopy. PCLS represents an ex vivo model that closely resembles the morphology and functionality of the respiratory tract (Henjakovic et al., 2008b). Previous stud-ies have shown that PCLS can be successfully employed in assessments of pulmonary pharmacology and toxicology (Henjakovic et al., 2008a; Parrish et al., 1995). Therefore, it was chosen as a useful system for the evaluation of cyto-toxicity of SLN.

Methods

Preparation of solid lipid nanoparticles (SLN)The procedure started with the manufacture of the lipid matrices (LM). Softisan 154 (S154, Condea, Witten, Germany) and Phospholipon 90G (P90G, Phospholipid GmbH, Cologne, Germany) were mixed at 70°C until a trans-parent yellowish solution was obtained and tis was further stirred at room temperature until solidification. The P90G content of the binary mixtures was either 20% or 50%. The SLN dispersions contained 15% LM, 3% Solutol HS15 (BASF

AG, Ludwigshafen, Germany), and 82% double-distilled water. The compounds were heated up to 65–70°C. Hot pre-emulsions were produced by using an Ultra Turrax (Ika, Staufen, Germany) at 13,000 rpm for 5 min. The hot preemul-sion were homogenized at a pressure of 1000 bar and a temperature of about 60°C with an EmulsiFlex-C5 (Avestin, Ottawa, Canada) high-pressure homogenizer for 20 cycles. Afterward the dispersions were allowed to recrystallise at room temperature (Müller et al., 1996).

Particle size and zeta potential measurementThe hydrodynamic diameter (z-average) and polydispersity index (PDI) of the nanosuspensions (SLN20 and SLN50) were investigated by photon correlation spectroscopy (PCS) using a Zetasizer ZS Nano (Malvern Instruments, Herrenberg, Germany), equipped with an He/Ne laser (4 mW). The sam-ples were diluted with filtered double-distilled water until the appropriate concentration of particles was achieved to avoid multiscattering events and then measured in polycarbonate cells (Sarstedt AG & Co., Nuremberg, Germany) at 20°C. The detection of the scattered light was performed at an angle of 173° (NIBS, non-invasive backscatter detection) to reduce the path length of the scattered light from the samples and to minimize the risk of multiscattering. Each approach was performed in triplicate.

The zeta potential was measured by laser Doppler anemometry (LDA) using a Zetasizer ZS Nano (Malvern Instruments, Herrenberg, Germany). The analysis was per-formed at a temperature of 20°C using samples appropriately diluted. All measurements were carried out in triplicate.

Exposure of A549 to SLNA549 cells were seeded into 96-well plates at a density of 1.0 × 105 cells per well in 200 µl Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Wuppertal, Germany) with 5% foetal bovine serum (Sigma Aldrich, Munich, Germany) and allowed to attach overnight. The culture medium was refreshed the next day and the cells were exposed to 0– 15 mg/ ml solid lipid nanosuspension in 200 µl final vol-ume/well for 24 h.

Cell viability assayCytotoxicity of SLN on human A549 cells was deter-mined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay (Sigma Aldrich, Steinheim, Germany) (Mosmann, 1983). Mitochondrial dehydrogenases of viable cells reduce the yellowish water-soluble MTT to water-insoluble formazan crystals, which can be solubilized with a mixture of 0.01% hydrochloric acid in isopropyl alcohol. After exposure of A549 to SLN. MTT solu-tion (20 µl of 0.5 mg/ml stock solution) was added to each well and incubated at 37°C for 2 h. The cell culture medium was aspirated cautiously, and 200 µl HCl/isopropanol was added to each well and mixed thoroughly. Optical density (OD) was read by enzyme-linked immunosorbent assay (ELISA) (Dynatech Laboratories, Inc., Chantilly, VA) at 555 nm. The results were calculated in relation to the untreated control.

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Lactate dehydrogenase (LDH) determinationLDH leakage, which accompanies membrane integrity damage, was determined using a commercial LDH Kit (Roche, Mannheim, Germany) according to the manufac-turer’s protocols. Released LDH catalyzed the oxidation of lactate to pyruvate with simultaneous reduction of NAD+ to NADH. The rate of NAD+ reduction was measured as an increase in absorbance at 340 nm. The proportion of NAD+ reduction was directly proportional to LDH activ-ity in the cell medium. After incubation of A549 with nanosuspensions for 24 h, 100 µl of cell culture medium was collected for LDH measurement and remaining SLN particles were removed by centrifugation at 1000 × g for 5 min. The supernatant was incubated with 100 µl of reac-tion mixture for 30 min at room temperature in the dark. The optical density of the solution was measured on an ELISA plate reader (Dynatech Laboratories Inc., Chantilly, VA). The LDH activity in the incubation medium was nor-malized to the negative control (A549 incubated with 1% Triton X-100).

Preparation of precision-cut lung slices (PCLS)Preparation of PCLS was performed as previously described (Henjakovic et al., 2008; Held et al., 1999; Martin et al., 1996). Briefly, 8- to 12-wk-old female BALB/c mice (Charles River, Sulzfeld, Germany) were sacrificed with an ip over-dose of pentobarbital sodium (Narcoren, Pharmazeutische Handelsgesellschaft GmbH, Garbsen, Germany). Extraction of lung tissue was performed directly post mortem to con-serve vitality of the tissue. Lungs were filled in situ with 1.5% low-melting agarose medium solution (Sigma Aldrich, Munich, Germany). Lungs were cooled on ice, and lobes were separated and cut into approximately 200-µm-thick slices using a Krumdieck tissue slicer (Alabama Research and Development, Muniford, AL). Tissue slices were washed with Dulbecco’s modified Eagle’s medium (DMEM) for 2 h. PCLS were cultured in DMEM/nutrient mixture F-12 Ham with L-glutamine and 15 mM HEPES (Sigma Aldrich, Munich, Germany), penicillin (100 U/ml), and streptomy-cin (100 µg/ml) (Sigma Aldrich, Munich, Germany) for 24 h at 37°C, 5% CO

2, and 100% air humidity under cell culture

conditions.

Viability of PCLSViability of tissue slices was investigated by calcein ace-toxymethyl/ethidium homodimer-1 (calcein AM/EthD-1) staining (Invitrogen, Karlsruhe, Germany) using confocal laser scanning microscopy (CLSM, Carl Zeiss AG, Jena, Germany) (Henjakovic et al., 2008). Live cells were distin-guished by enzymatic conversion of calcein AM to intensely yellow fluorescent calcein. EthD-1 produces intracellular red fluorescence in nuclei of dead cells. After incubation of lung slices with SLN the tissue was incubated with 4 µM calcein AM and 4 µM EthD-1 for 45 min at room temperature. PCLS were washed and investigated by CLSM (40× water immer-sion objective, excitation wavelengths 488 nm and 543 nm, emission filters BP 505-550 nm and LP 560 nm, thickness

20 µm). Image stacks of a defined volume were analyzed with Bitplane IMARIS 4.5.2. The ratio of numbers of EthD-1-labeled cell nuclei to the volume of calcein in the cytoplasm of live cells was determined. Cell nuclei of dead cells were counted as spots ≥5 µm diameters. Thresholds were set once for each channel and used for all data sets. Viability of PCLS is expressed as quantity of nuclei (spots) in 105 µm3 yellow tissue volume.

Results

Nanoparticle characteristicsThe physicochemical characteristics of the SLNs are sum-marized in Table 1. SLN20 display the following charac-teristics: a hydrodynamic diameter of 143.67 ± 5.34 nm, a polydispersity index of 0.225 ± 0.06, and a zeta potential of −12.3 ± 0.6 mV. SLN50 exhibit a hydrodynamic diameter of 93.13 ± 4.38 nm and a polydispersity index of 0.157 ± 0.02, and a zeta potential of −22.6 ± 1.7 was measured.

Cytotoxicity of SLN on A549For the prediction of a non-toxic in vivo nanoparticle concentration the toxic effects of SLN20 and SLN50 were estimated on A549 cells. Viability of A549 was determined after incubation of cells for 24 h with SLN20 and SLN50 in serum-supplemented medium. SLN reduced the metabolic activity of A549 in concentration-dependent manner. In order to compare the results of both formulations, the mid-dle effective concentrations (EC

50) of SLN were determined.

The EC50

of SLN20 is 4080 µg/ml and of SLN50 is 1520 µg/ml (Figure 1A).

Quantitative analysis of the released LDH confirmed a progressive cellular damage induced by increasing SLN con-centrations. The results are in accordance with the MTT data. The cell viability decreased to 50% for SLN20 at a particle concentration of 3431 µg/ml and for SLN50 at 1253 µg/ ml (Figure 1B).

Cytotoxicity of SLN on murine PCLSPCLS were incubated for 24 h with 1 to 1500 µg/ml of SLN (Figure 2). The assessment of SLN50 induced acute toxicity displayed a critical issue for in vivo studies (Figure 3A). In the case of SLN20 the toxic dose was not reached for a parti-cle concentration up to 1500 µg/ml, providing a reasonably wide therapeutic window (Figure 3B).

Discussion and conclusion

Toxicological studies of SLN are essential in the develop-ment of drug delivery systems prior to in vivo tests. In this

Table 1. Characteristics of solid lipid nanoparticles: mean size, polydispersity index, and zeta potential of SLN20 and SLN50 (n = 3).

Nanoparticle Mean size Polydispersity index Zeta potential (Mv)

SLN20 143.67 ± 5.34 0.225 ± 0.06 −12.3 ± 0.6

SLN50 93.13 ± 4.38 0.157 ± 0.02 −22.6 ± 1.7

Note. Values are presented as the mean ± SD.

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Cytotoxicity of solid lipid nanoparticles 107

study, we examined the acute respiratory cytotoxicity of SLN with 20% and 50% phospholipid content in the LM in vitro and ex vivo.

Determination of mitochondrial activity by the MTT assay, which reflects cellular metabolic activity, showed clearly that the exposure of SLN20 and SLN50 to A549 cells leads to different cytotoxic profiles. The EC

50 value in A549

for SLN20 was 4080 µg/ml and for SLN50 1520 µg/ml. The cytotoxicity in terms of LDH release showed comparable EC

50 values of 3431 µg/ml and 1253 µg/ml for SLN20 and

SLN50, respectively. However, Yuan et al. (2008) tested SLN composed of different lipid materials on A549 cells and found IC

50 values between 300 µg/ml to 500 µg/ml. Yuan

et al. exposed the SLN for 48 h to A549 cells, while in the present study the incubation time was 24 h. This could be an explanation for the higher level of cytotoxicity reported by Yuan et al. compared to our results.

In the next step an ex vivo lung model was used for a closer approximation to the in vivo situation. The PCLS model allows one to assume respiratory cytotoxic con-centrations of substances after incubation of lung tissue. In PCLS, incubation with SLN20 did not lead to cytotoxic effects up to a concentration of 1500 µg/ml, in contrast to SLN50 incubation.The reason for the lower cytotoxicity in PCLS versus A549 cells may be attributed to the fact that PCLS contain all cell

Figure 2. Image analysis of representatives of concentration-dependent cell death in PCLS. Tissue slices were stained with 4 µM calcein AM and 4 µM EthD-1 after 24 h of cultivation with 100 µg SLN20 (A), 500 µg SLN20 (B), 1500 µg SLN20 (C), 100 µg SLN50 (D), 500 µg SLN50 (E), or 1500 µg (F). The images were examined by two-color immunofluorescence microscopy (40× water immersion objective fold, excitation wavelengths 488 nm and 543 nm, emission filters BP 505-550 nm and LP 560 nm, thickness 20 µm, grid spacing = 20 µm) and analyzed with IMARIS 4.5.2. Red color shows cell nuclei (diameter 5 µm) of dead cells and yellow color the cytoplasm of viable cells.

SLN20 100µgA SLN20 500µgB SLN20 1500µgC

SLN50 100µgD SLN50 500µgE SLN50 1500µgF

Figure 1. (A) MTT viability assay for human A549 cells exposed to solid lipid nanoparticles with 20% phospholipid content (SLN20; black squares) and to solid lipid nanoparticles with 50% phospholipid content (SLN50; gray rectangle) for 24 h. The MTT assay measures the activity of mitochondrial dehy-drogenases in living cells. Values are the means of six experiments (± SEM). (B) Cytotoxicity detection with lactate dehydrogenase assay (LDH) in A549 cells treated with different concentrations of SLN20 and SLN50 for 24 h. Values represent the means of four experiments (± SEM).

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types that can be found in vitro in the lung: e.g., epithelial and endothelial cells, macrophages, dendritic cells, lym-phocytes, fibroblasts, and basal cells (Goris et al., 2009). The presence of, and potential interaction among, different cell types may lead to the different sensitivity of PCLS to SLNs.

All assays revealed that SLN20 were much better tolerated in lung cells than SLN50. These findings indicate a positive correlation of cytotoxicity with the content of phospholipids in the SLN (Schubert and Muller-Goymann, 2005), which may be explained by structural differences between the two particle types. Higher amounts of phospholipids lead to higher amounts of surfactant groups on the particle surface, which may cause damage to cells. Both Solutol HS15 and the phospholipids might interact with the surrounding area and exhibit a synergistic effect on the cytotoxicity of the systems (Schubert and Muller-Goymann, 2005), but the effect may be greater for SLN50. Additionally, the SLN50 are approxi-mately 50 nm smaller compared to SLN20 and therefore have a larger surface area per mass. It has been reported that smaller particles have a higher toxicity (Valavanidis et al., 2008; Worle-Knirsch et al., 2007). Further, our experiments were performed with concentrations in micrograms per millilitre. This results in a higher particle number for SLN50 versus SLN20, which may also explain its greater toxicity (Wittmaack, 2007).

In conclusion, a concentration range for possible applications of SLN20 and SLN50 in vivo was determined. According to this result, an estimation of the relative toxic-ity of SLN20 and SLN50 can be made. The absolute acute toxicity was not determined. PCLS were more sensitive to toxicity of the particles than A549 lung epithelial cells, pos-sibly because the PCLS contain many different cell types. The lower acute cytotoxicity values of the SLN20 make these nanosuspensions more promising for further in vitro animal studies, compared to SLN50. Cytotoxicity and immuno-modulatory effects of SLN should be tested prior to clinical trials.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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Figure 3. Concentration-dependent changes in PCLS after incubation with 1 µg to 1500 µg (A) SLN20 and (B)SLN50 for 24 h. Viability was determined by live/dead staining with calcein AM/EthD-1 and subsequent image analysis with IMARIS 4.5.2. Values represent the mean of seven experiments (± SEM).

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