Raman Spectroscopy in Nanomedicine

Abstract and Introduction

Abstract

Raman spectroscopy is a branch of vibration spectroscopy that is capable of probing the chemical composition of materials. Recent advances in Raman microscopy have significantly added to the range of applications, which now extend from medical diagnostics to exploring the interfaces between biological organisms and nanomaterials. In this review, Raman is introduced in a general context, highlighting some of the areas in which the technique has been successful in the past, as well as some of the potential benefits it offers over other analytical modalities. The subset of Raman techniques that specifically probe the nanoscale, namely surface- and tip-enhanced Raman spectroscopy, will be described and specific applications relevant to nanomedical applications will be reviewed. Progress in the use of traditional label-free Raman for investigation of nanoscale interactions will be described, and recent developments in coherent anti-Stokes Raman scattering will be explored, particularly its applications to biomedical and nanomedical fields.

Introduction

Nanomedicine can be defined as the medical applications of nanotechnology,^[1] ranging from the use of nanomaterials in regenerative medicine, drug delivery strategies, medical diagnostics and therapeutics, and includes the potential negative impacts of nanomaterials to human health, commonly encompassed under the term nanotoxicology. In the context of this review, nanomedicine is viewed from the perspective of how Raman spectroscopy (and its variants) can be used in the assessment of the beneficial, as well as the potential negative, impacts of nanomaterials on human health. Nanomaterials have already found uses in a wide range of applications, including antimicrobial paint coatings,^[2] textile finishing^[3] and novel applications in the electronics industry.^[4] Notably, biomedical applications are rapidly emerging, ranging from nanoparticle-coated stents for angioplasty,^[5] contrast agents for diagnostic imaging,^[6,7] and also potential drug and gene delivery vehicles.^[8–10] These applications are largely dependent on the particular characteristics that nanomaterials and nanoparticles possess. These include properties such as increased surface-to-mass ratio, which in turn results in an increase in surface reactivity, while novel optical properties associated with some classes of nanoparticles are important for applications in theranostic imaging and subsequent monitoring of drug delivery. However, while these technologies show promise, it is important to be able to visualize how the materials behave in situ, particularly in the biological context, to be able to characterize their interactions and toxicological effects – whether they are in vitro or in vivo. While it has been highlighted that comprehensive characterization of the physicochemical properties of nanoparticles is imperative, changes to these properties, such as aggregation state and effective surface chemistry, can play a critical role in their modes of interaction and action.^[11] Equally, to understand the modes of action and optimize efficacies, monitoring and understanding changes to the biological environment is critical, not only on a cellular level, but also when considering the systemic responses.

Considering the system as a whole, one must be able to track a particle or material from initial exposure or administration through to the site of action and on to assimilation, degradation or excretion. At each step in this process, one must be able to access and visualize the efficacy by which the particles can overcome certain barriers to successful administration. These can vary from the route of exposure, assessing whether the particle causes toxicity, particle retention (e.g., via the enhanced permeability and retention effect), removal from the circulation via uptake by the reticuloendothelial system, accumulation of the nanoparticles over time, nonspecific interactions and the efficacy with which the particle reaches its desired location, among others.

Ideally, a method that can successfully characterize these processes is required, first in fundamental in vitro cytological and ex vivo histological studies, and, ultimately, in more realistic in vivo applications. This method should be capable of identifying the particle or material of interest, while simultaneously being able to access the surrounding environment while measuring the efficacy of the probe or nanocarrier and/or the physiological response of the organism.

A large range of analytical methods exists that can be used in the classification and characterization of nanomaterials. These include scanning and transmission electron microscopy, atomic force microscopy (AFM), other label-free optical methods such as differential interference contrast and dark-field microscopy, and fluorescent microscopy methods based on intrinsic nanoparticle or external label fluorescence, to name but a few. However, these methods are not without certain drawbacks that limit, to some extent, their applicability and effectiveness.

First, both AFM and scanning electron microscopy can be considered as primarily surface-sensitive techniques, while, when transmission electron microscopy is coupled with serial sectioning and ultramicrotomy, it has been used for 3D reconstructions and tomography.^[12,13] However, these processes are time consuming, costly and laborious. In addition, electron microscopy requires a particle to have a contrasting electron density compared with its environment in order for it to be visualized, which renders electron microscopy ineffective for many 'softer' polymeric nanoparticles. Electron microscopy does not allow live cells to be imaged and, as it requires extensive sample processing, it provides only a limited scope for rapid or routine investigation of nanomaterials in vitro. What electron microscopy and AFM do provide is the capability of imaging beyond the optical diffraction limit. More recently developed optical based methods, so-called super-resolution microscopy, have become available, which allow for imaging beyond this limit.^[14–16] However, their use has been limited in the field of nanomedical sciences.

By contrast, standard fluorescent-based microscopy has been used extensively in nanoparticle studies.^[16–20] Confocal laser scanning (fluorescence) microscopy has become a standard in the toolbox of techniques for in vitro cytometry.^[21] Although the technique is limited in resolution to hundreds of nanometers, it can potentially detect fluorescence emission from, and, therefore, the location of, individual nanoparticles. Penetration depths in vivo can be extended through two photon excitation techniques and/or near-infrared (IR) fluorophores.^[22,23] In the visible region, a range of fluorescent assays and labels are commercially available to probe a range of physiological processes in vitro, such as lyso- and mito-trackers used for labeling lysosomes and mitochondria.^[201] Intrinsically, fluorescent nanoparticles, such as inorganic semiconductor quantum dots, have been developed for similar applications,^[202] and surface functionalization of these types of materials has contributed to understanding the dependence of uptake and intracellular trafficking on surface chemistry.^[24] Many similar studies have been performed with fluorescently labeled nanoparticles,^[25,26] which are commercially available in a range of sizes and surface functionalities. However, not all nanoparticles can easily be fluorescently labeled. Furthermore, it is not clear whether the transport mechanisms of smaller nanoparticles, fluorescently labeled with anionic moieties, are the same as their unlabeled counterparts.^[27] Critically, there have been reports that labeled nanoparticles can release the dye into the surrounding biological environment, and, therefore, the distribution of fluorescence within the cell does not necessarily represent the presence or subcellular distribution of the nanoparticles.^[28–30] Other label-free optical microscopy techniques are also limited by the type of particle that can be visualized (i.e., only metal-based particles are effective for dark-field and differential interference contrast microscopy).^[31]

Raman spectroscopy has been proposed as a method for monitoring nanomaterials in biological systems, as it potentially provides a label-free, noninvasive probe of the nanoparticle itself, the local environment and the physiology of the organism.^[32] Over the past decade, Raman spectroscopy has been applied to a range of biomedical areas, including cancer diagnostics,^[33] toxicity studies,^[34] atherosclerosis^[35] and skin investigations.^[36,37] Importantly, Raman not only provides a method for differentiation between a diseased and nondiseased state, but also indicates the (bio)chemical nature of a sample, based on the characteristic vibrations of the molecular bonds of the constituent components. Raman is a form of vibrational spectroscopy, which in itself is a subset of the more general umbrella term of spectroscopy. The vibrations are characteristic of the molecular structure and, in polyatomic molecules, give rise to a spectroscopic 'fingerprint'. The spectrum of vibrational energies can, thus, be employed to characterize a molecular structure, or changes to it caused by the local environment or external factors. The Raman spectrum is, therefore, a truly label-free signature of the nanoparticle. Vibrational energies typically fall in the mid-IR region of the electromagnetic spectrum and are quite commonly probed using IR absorption spectroscopy. In many ways, Raman can be viewed as a complementary technique to IR spectroscopy; whereas IR involves the absorption of radiation, Raman is an inelastic scattering technique whereby the incident radiation couples with the vibrating polarization of the molecule and, thus, generates or annihilates a vibration. For a vibration to be active in IR spectroscopy, a change in dipole is required; whereas to be Raman active, a change in polarizability is required. As a rule of thumb, vibrations of asymmetric, polar bonds tend to be strong in the IR spectra, whereas Raman is particularly suitable as a probe of symmetric, nonpolar groups. Importantly, this means that the O-H bonds of water are strong absorbers in IR spectroscopy, whereas they are relatively weak Raman scatterers. This allows samples to be investigated in an aqueous environment and, therefore, the technique of Raman spectroscopy more readily lends itself to live cells in vitro^[38] or in vivo.^[39] As the vibrational spectrum is measured as a frequency (or energy) shift from that of the incident radiation, Raman spectroscopy can be performed across the UV, visible or near-IR spectral regions, and thus can benefit from the technologies available and advances made for confocal optical microscopy.

A number of variants that are based around the physical principle of Raman spectroscopy exist. Spontaneous Raman can take the form of Stokes Raman scattering and anti-Stokes Raman scattering. The former results from the creation of a vibration in a material, characterized by a decrease in the incident photon energy (frequency), while the latter results from the annihilation of vibration, characterized by an increase in the incident photon energy. If the incident radiation is resonant with an electronic absorption of the analyte, the Raman signal can be resonantly enhanced by several orders of magnitude. The use of resonant Raman spectroscopy in biomedical systems has been limited, however, owing to associated photochemical degradation phenomena and the generation of fluorescence, which can swamp the Raman signal of the overall sample.

Other variants of these two techniques with increased sensitivities for more molecularly specific characterization have been developed. These include resonant Raman spectroscopy, coherent anti-Stokes Raman spectroscopy (CARS), tip-enhanced Raman spectroscopy (TERS) and surface-enhanced Raman spectroscopy (SERS). The majority of these techniques have been applied to nanomedical applications; however, TERS and SERS deal inherently with the nanoscale. Although Raman is fundamentally an optical technique and is, therefore, similar to confocal optical microscopy, limited to spatial resolution of the order of hundreds of nanometers, nanometer resolution can be obtained through localized enhancement processes. This localized enhancement led to the initial interest in the use of Raman spectroscopy to probe the specific environment of the nanoparticle.

This article will outline the applications of the various Raman spectroscopy-based techniques in the broad area of nanomedicine. As they are nanospecific, the use of SERS and TERS techniques will be presented initially. The increasing interest in the use of truly label-free spontaneous Raman and CARS in nanomedical applications will then be explored. In Raman spectroscopy, the sensitivity, spatial resolution, penetration depth and required scan rates depend on the technique employed, resonance conditions and even the instrumental set-up (microscope objective, grating and laser power). In the sections describing each modality, examples of the state of the art nanomedical applications are provided. The 'Future perspective' section attempts to address routes beyond the current state of the art. A more detailed description of the historical origin and basic principles of the Raman scattering process can be found in numerous excellent text books^[40–44] and review articles.^[45–47] A comparison of Raman and IR spectroscopies for biomedical applications can be found in.^[48]