Microscopy: Science, Technology, Applications and Education A. Méndez-Vilas and J. Díaz (Eds.) ______________________________________________

Localizing cellular uptake of nanomaterials in vitro by transmission electron microscopy C.T. Ng1,2 , J.J. Li1,2 , R. Perumalsamy3, F. Watt3, L.Y.L. Yung2 and B.H. Bay1 1

Department of Anatomy, National University of Singapore, 4 Medical Drive, S 117 597, Singapore Department of Chemical & Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, S 117 576, Singapore 3 Department of Physics, National University of Singapore, 2 Science Drive 3, S 117 542, Singapore 2

Nanomaterials are increasingly used in biomedical applications as tools for research and drug delivery. Scanning electron microcopy (SEM) and transmission electron microscopy (TEM) have been employed to characterize the microstructure and investigate detailed surface morphology of nanoparticles and microspheres. Besides characterization of materials, electron microscopy has also been utilized to analyze material-biology interface. In this ultrastructural study, we show the internalization and localization of (a) gold nanoparticles in MRC 5 human embryonic lung fibroblasts and primary small airway epithelial cells, and (b) polystyrene microspheres in lung fibroblasts by TEM. The presence of gold nanoparticles in lung fibroblasts was confirmed by Energy Dispersive X-ray Analysis profiling. Although advance electron microscopy techniques, such as scanning transmission electron microscopy (STEM) are indispensable for characterizing nanomaterials, this study illustrates the use of conventional TEM in localizing nanomaterials in biological environments. Keywords: transmission electron microscopy; gold nanoparticles; microspheres; Energy Dispersive X-ray Analysis

1. Introduction The advent of advance microscopy techniques for characterization and manipulation of materials at the nanolevel has facilitated the expansion of nanotechnology for diagnosis and treatment of diseases and the evolution of a new field of medicine called nanomedicine [1]. Nanomaterials are defined as materials with at least one dimension between 1 and 100 nanonmeters. Scanning electron microcopy (SEM) is valuable for analyzing the detailed surface morphology of nanoparticles (NPs) and microspheres. Transmission electron microscopy (TEM) is a useful tool for characterizing the morphology, crystallinity and micro/nano structures of nanomaterials, which are critical for understanding the properties of these materials for the development of potential new biomedical applications [2, 3]. Using a high energy electron beam flooding onto a thin sample, TEM is able to image and analyse the microstructure of nanomaterials with atomic scale resolution [4]. In fact, TEM has been touted as the only tool which can correctly distinguish between a carbon nanotube and a carbon nanofiber [4]. TEM has enabled comprehensive identification of the nanobio interface which is important for enhancing the development of effective nanocarriers [1, 5]. In this respect, TEM can provide confirmatory evidence of bioactive materials encapsulated inside nanocarriers [1, 6, 7] and reveal interactions of nanocarriers with biological macromolecules [1, 8]. TEM has facilitated in depth analysis of the morphology and functional state of the cells following the uptake of nanomaterials. We have previously shown by TEM that gold nanoparticles (AuNPs) were taken up by lung fibroblasts in vitro [9]. The AuNPs were observed to generate oxidative stress culminating in DNA damage and inhibition of cell proliferation with alteration of cell cycle genes. Recently, we reported that uptake of AuNPs could also induce the process of autophagy [10]. TEM revealed the presence of autophagosomes containing cell membrane-like debris occurring concomitantly with the uptake of AuNPs in the lung fibroblasts. It is undeniable that conventional TEM continues to have a place in validating the cellular uptake and localization of nanomaterials. In this study, internalization of AuNPs in lung fibroblasts and small airway epithelial cells and polystyrene microspheres in lung fibroblasts will be shown.

2. Materials and Methods 2.1. Cell cultures MRC-5 human embryonic lung fibroblasts (ATCC CCL-171) were cultured in Roswell Park Memorial Institute medium (RPMI 1640) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin). Primary human Small Airway Epithelial Cells (SAEC Clone CC-2547) and cell culture reagents were purchased from Lonza (Walkersville, USA). The SAECs which are derided from respiartory tract epithelium, were cultured in Small Airway Epithelial Cell Basal Medium (SABM) containing SingleQuots® growth supplements. Both cell lines were maintained in an incubator at humidified atmosphere of 37°C and 5% carbon dioxide.

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Microscopy: Science, Technology, Applications and Education A. Méndez-Vilas and J. Díaz (Eds.) ______________________________________________

2.2. Exposure of cells to AuNPs and polystyrene nanospheres Cells were first seeded onto 6-well plate at a seeding density of 8,000 cells/well. MRC-5 lung fibroblasts and SAECs were treated with 1 nM AuNPs (prepared from a 10 nM sterile-filtered stock solution of AuNPs with culture media as the diluent). The 20 nm diameter AuNPs were synthesized from an aqueous chlorauric acid solution containing 5 mg of Au. The lung fibroblasts were also separately exposed to 20nM diameter polystyrene nanospheres (FluoSpheres® Fluorescent Microspheres, Invitrogen) at a concentration of 1nM for 72 h. 2.3. Transmission Electron Microscopy (TEM) Cells were washed three times with Phosphate Buffer Saline (PBS) prior to fixation with 2.5% glutaraldehyde for 1 hour. The specimen was then rinsed with PBS three times at a time interval of 5 minutes each followed by osmification with 1 % osmium tetroxide (OsO4) containing a few crystals of potassium ferrocyanide (K4Fe2(CN)6) for 1 h at room temperature. Samples were dehydrated in an ascending series of ethanol (25%, 50%, 75%, 95% and 100% twice) for 4 minutes each and embedded in araldite. Gradual infiltration with partially polymerized araldite was achieved using increasing concentrations of araldite till saturation (pure araldite). The araldite infiltration was initially carried out using ethanol and araldite in 1:1 ratio for 30 minutes, followed by a ratio of 1:4 lastly for another 30 minutes before addition of pure araldite and incubation at 40°C for 45 minutes. Fresh araldite was added to replace the previously used araldite before incubating at a higher temperature of 50°C for 30 minutes. Finally, fresh araldite was added and incubated at 55°C for 30 minutes. Ultrathin sections were cut and mounted onto copper grids which were pre-coated with Formvar. Sections were post-doubly stained with uranyl acetate and lead citrate before viewing with an Olympus EM208S transmission electron microscope. 2.4 Energy Dispersive X-ray (EDX) Analysis The elemental composition of the TEM specimen was analyzed by the CM120 BioTWIN electron microscope coupled with a Philips EDAX Microanalysis system.

3. Results and Discussion We observed that AuNPs which were taken up by the fibroblasts, appeared mainly as electron dense clusters located inside cellular vesicles (black arrows) with some AuNPs scattered in the cytosol (Fig. 1). It may sometimes be difficult to distinguish electron dense particles such as titanium dioxide NPs from glycogen granules or ribosomes in TEM specimens [11].

Fig. 1. TEM micrographs of a lung fibroblast treated with 1 nM AuNP for 72 h. (A) AuNPs appear as electron dense agglomerates inside vesicles (indicated by black arrows). Scale bar = 1 µm. (B) At higher magnification, electron deposits are also seen in the cytosol (black circle). Scale bar = 0.5 µm.

However, EDX analysis coupled with TEM has made possible detailed examination of the chemical composition of NPs [12]. The presence of two Au peaks corresponding to the AuM shell (2.2 KeV) and AuL shell (9.7 KeV) detected by EDX analysis has verified that the electron dense deposits in lung fibroblasts are internalized AuNPs (Fig. 2).

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Fig. 2. Elemental profiling of a AuNP-treated lung fibroblast. The high Cu content peak reflects the copper grid on which the TEM specimen was mounted and the osmium peak is due to the osmification process in the specimen preparation. Bar = 0.2 µm.

Similarly, AuNPs taken up by SAECs were mainly localized in vacuoles as shown in Fig. 3

Fig. 3. TEM micrographs of SAECs treated with 1 nM AuNP for 72 h. AuNPs appear as electron dense deposits (black arrows). (A) Scale bar = 2 µm. (B) Higher magnification of AuNPs in a different SAEC. Scale bar =1 µm.

Polystyrene nanospheres taken up by a lung fibroblast is shown in Fig.4. It must be borne in mind that NPs which are not electron dense such as those formulated from biocompatible and biodegradable polymers, poly(D, L-lactide-coglycolide) (PLGA) and poly(lactide) (PLA) may sometimes be confused with vesicular structures present in cells [13].

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Fig. 4. TEM micrograph of a lung fibroblast treated with 1 nM polystyrene nanospheres for 72 h. The internalized microspheres appear as electron lucent agglomerates in the cytosol (white arrows). Scale bar= 0.5 µm.

The distribution of NPs in cellular organelles, cells, tissues and organs can also be quantified based on stereology principles which allow for unbiased statistically valid comparisons [14, 15]. Quantitative electron microscopy would be useful to researchers who need to quantify NPs at the EM level, in order to obtain more scientific information. A vital key to successful TEM analysis is specimen preparation. It is imperative that specimens must be adequately and properly fixed before processing for TEM. The two methods used for fixing TEM specimens are chemical fixation and physical fixation or cryofixation. In addition, the specimens have to be correctly embedded in the embedding medium before ultrathin sections are cut. As a major drawback of TEM is that specimen thickness should be less than 100 nm to obtain high-resolution TEM images [1], occasionally extra sample preparation steps such as ion beam milling are required [16]. For instance, a focused ion beam instrument can be utilized to prepare TEM sections out of a specific region of the bulk sample. There is no question that the advancement of TEM systems hold immense potential for NP research. High resolution EM techniques, such as the ultrahigh vacuum molecular beam epitaxy transmission electron microscope system, have been employed to study the structure of Germanium NPs [17]. Energy filtered TEM which combines TEM with electron energy loss spectroscopy and imaging, allows for examination and spatial visualization of the elemental composition of a specific structure [11, 18]. The development of electron tomography and sophisticated software tools, have made possible high resolution 3D reconstruction based on a sequence of serial sections [11, 19]. Scanning transmission electron microscopy (STEM) is also indispensable for characterizing NPs. According to Liu [20], the versatility of the STEM instrument, which is equipped with various imaging, diffraction and spectroscopy techniques incorporated in a single instrument, has made “STEM the most powerful microscope for characterizing the physicochemical nature of nanoscale systems”. Three dimensional STEM imaging has also facilitated analysis of coreshell NPs used for DNA detection [21]. Data acquired would be useful in assisting the synthesis and optimization of core-shell NPs. Besides TEM and SEM, other commonly used microscopy imaging technologies for NP research include atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM), combined systems of multiple imaging technologies such as AFM-CLSM, SEM-Spectroscopy and correlative light and electron microscopy [1].

4. Conclusion High-resolution bio-imaging instrumentation has become a necessary tool for investigating the spatiotemporal distributions of ambient, biological, manufactured and engineered NPs in human cells, tissues and organs [12]. This chapter aims to provide a basis for NP researchers to integrate TEM analyses when designing experimental protocols. It is likely that TEM analyses will continue to be an important research tool in NP research.

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Acknowledgements. The authors are grateful to Ms Yee-Gek Chan, Ms Song-Lin Bay and Mr Chun-Peng Low for technical assistance. The work was supported by the Singapore Ministry of Education Academic Research Fund Tier 2 via grant MOE2008T2-1-046

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