Spontaneous confocal Raman microscopy--a tool to study the uptake of nanoparticles and carbon nanotubes into cells
© Romero et al; licensee Springer. 2011
Received: 13 October 2010
Accepted: 16 June 2011
Published: 16 June 2011
Confocal Raman microscopy as a label-free technique was applied to study the uptake and internalization of poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) and carbon nanotubes (CNTs) into hepatocarcinoma human HepG2 cells. Spontaneous confocal Raman spectra was recorded from the cells exposed to oxidized CNTs and to PLGA NPs. The Raman spectra showed bands arising from the cellular environment: lipids, proteins, nucleic acids, as well as bands characteristic for either PLGA NPs or CNTs. The simultaneous generation of Raman bands from the cell and nanomaterials from the same spot proves internalization, and also indicates the cellular region, where the nanomaterial is located. For PLGA NPs, it was found that they preferentially co-localized with lipid bodies, while the oxidized CNTs are located in the cytoplasm.
The use of nanomaterials and nanoparticles (NPs) in medicine as drug delivery vectors, sensors or contrast agents is among the most promising areas in nanotechnology research. For the application of nanotechnology in medicine, 'in vitro' work is of paramount importance, especially regarding the assessment of possible toxicological consequences of the nanomaterials/NPs. It is a key issue to study the effects of 'nano' in the cellular machinery and to understand how the nanomaterials are processed in the cell, and their distribution and fate, after being taken up by the cells. Confocal laser scanning microscopy (CLSM) is often applied for uptake studies, but its application for NPs is not always easy, since the size of the NPs falls well below optical resolution. Also, a main drawback of CLSM is that, for most of the cases, both the NPs and cellular compartements must be fluorescently labelled, and this is not always an easy task. Besides that, labelling of NPs may in some cases require complex chemical routes including, for example, silanization, assembly of polymers, etc. As a result, the labelling can induce significant changes in the structure and properties of NPs, which may affect uptake and toxicity. Transmission electron microscopy (TEM) can be used to study the uptake and localization of NPs and nanomaterials, avoiding their labelling. The drawback of TEM for this application is that it requires complex and time-demanding preparations that also may affect the localization of the nanomaterials within the cell. Other label-free techniques for the study of the localization of nanostructures within cells are spontaneous Raman microscopy and coherent anti-stokes Raman (CARS) microscopy. In CARS, a single Raman band coming from the nanomaterial is scanned throughout the cell. A mapping of the cell is obtained showing the intensity distribution of the chosen Raman band . Drawbacks of CARS are that only selected bands can be mapped, and furthermore, spectral overlapping may cause problems.
Confocal Raman microscopy (CRM) combines spontaneous Raman emission with confocal detection. We will show here that CRM can be used to study the localization of nanomaterials in the cells, taking advantage of the fact that in every spot the whole Raman spectrum is recorded. The latter thus contains bands coming from the nanomaterials and from representative cell molecules: proteins, DNA and lipids, which allow to identify the region of the cell [2, 3], where the nanostructures are located. This article is among the first  to explore the use of the spontaneous Raman emission for the detection of nanomaterials inside cells and to assess the intracellular region from the spectra, where the nanomaterial is located. Previous study with CRM and cells has focused in the recognition of different cellular environment through their chemical fingerprints and the evaluation of changes in metabolism [5, 6]. Poly(lactide-co-glycolide) (PLGA) NPs and carbon nanotubes (CNTs) have been chosen as two representative and remarkably different systems that can be studied with CRM. To our knowledge, this is the first article where CRM is used for CNT detection at cellular level.
Materials and methods
PLGA (d,l-lactide 85,: glycolide 15, inherent viscosity within 0.55-0.,75 dL/g) was purchased from LACTEL®. Branched PEI, Mw 25 kDa, BSA, PIERCE BCATM Protein Assay Kit, Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum and penicillin-streptomycin were purchased from Sigma-Aldrich. All chemicals were used as received. Hepatocarcinoma human cell line (HepG2) was obtained from American Type Culture Collection (ATCC-HB-8065).
PLGA NPs were prepared by the O/W emulsion-solvent evaporation method . Size and shape of the PLGA NPs were characterized by TEM (JEOL JEM 2100F, Japan) . Multiwalled CNTs were purchased from Proforma (USA). Oxidation of CNTs was achieved as described in the literature by Zhang et al. .
Micro-Raman analyses were performed using a Renishaw inVia Raman Microscope. Measurements were performed using a 532-nm laser excitation wavelength with a grating of 1800 mm-1. Most measurements were conducted using a ×40 water immersion objective with a focal spot of approximately 1 μm in diameter. Spectra were recorded in the region 300-3600 cm-1 , with a resolution of approximately 7 cm-1. The system was calibrated to the spectral line of crystalline silicon at 520.7 cm-1. At least 8-15 accumulation scans, at different spots in the various cell compartments, lipid bodies (LB), cytoplasm and nucleus, were used to reduce the spectral noise. All spectra had a correction for the PBS solution and glass cover slip baseline. After CNTs or NPs exposure and repeated washings with PBS, the Raman spectra were taken only from cells, where no visible CNT aggregates could be observed.
Spontaneous CRM has been successfully applied to identify PLGA NPs and oxidized CNTs in single hepatocarcinoma cells, which had been co-cultivated with the NPs and CNTs. The data prove that CRM, being a label-free technique, is a valuable tool to study the uptake of nanomaterials into cells. For PLGA NPs, CRM confirms the observations from CLSM and proves internalization. The z-scanning of the cells with CNTs reveals that these are incorporated in the cytoplasm and are not co-associated with the LB.
coherent anti-stokes Raman
confocal laser scanning microscopy
confocal Raman microscopy
transmission electron microscopy.
This study was supported by the European Commission in the framework of FP7 Theme 4-NMP, Proposal No: CP-FP 228825-2 HINAMOX, as well as by the grant MAT2010-18995 from the Spanish Ministry of Science and Innovation. S.E. Moya is a Ramon y Cajal Fellow.
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