A.L. Fussell

In situ dissolution analysis using coherent anti-Stokes Raman scattering (CARS) and hyperspectral CARS microscopy.

The solid-state form of an active pharmaceutical ingredient (API) in an oral dosage form plays an important role in determining the dissolution rate of the API. As the solid-state form can change during dissolution, there is a need to monitor the oral dosage form during dissolution testing. Coherent anti-Stokes Raman scattering (CARS) microscopy provides rapid, spectrally selective imaging to monitor the oral dosage form during dissolution. In this study, in situ CARS microscopy was combined with inline UV absorption spectroscopy to monitor the solid-state change in oral dosage forms containing theophylline anhydrate undergoing dissolution and to correlate the solid-state change with a change in dissolution rate. The results from in situ CARS microscopy showed that theophylline anhydrate converted to theophylline monohydrate during dissolution resulting in a reduction in the dissolution rate. The addition of methyl cellulose to the dissolution medium was found to delay the theophylline monohydrate growth and changed the morphology of the monohydrate. The net effect was an increased dissolution rate for theophylline anhydrate. Our results show that in situ CARS microscopy combined with inline UV absorption spectroscopy is capable of monitoring oral dosage forms undergoing dissolution and correlating changes in solid-state form with changes in dissolution rate.

Coherent anti-Stokes Raman scattering (CARS) microscopy visualizes pharmaceutical tablets during dissolution.

Traditional pharmaceutical dissolution tests determine the amount of drug dissolved over time by measuring drug content in the dissolution medium. This method provides little direct information about what is happening on the surface of the dissolving tablet. As the tablet surface composition and structure can change during dissolution, it is essential to monitor it during dissolution testing. In this work coherent anti-Stokes Raman scattering microscopy is used to image the surface of tablets during dissolution while UV absorption spectroscopy is simultaneously providing inline analysis of dissolved drug concentration for tablets containing a 50% mixture of theophylline anhydrate and ethyl cellulose. The measurements showed that in situ CARS microscopy is capable of imaging selectively theophylline in the presence of ethyl cellulose. Additionally, the theophylline anhydrate converted to theophylline monohydrate during dissolution, with needle-shaped crystals growing on the tablet surface during dissolution. The conversion of theophylline anhydrate to monohydrate, combined with reduced exposure of the drug to the flowing dissolution medium resulted in decreased dissolution rates. Our results show that in situ CARS microscopy combined with inline UV absorption spectroscopy is capable of monitoring pharmaceutical tablet dissolution and correlating surface changes with changes in dissolution rate.

Coherent anti-Stokes Raman scattering (CARS) microscopy driving the future of loaded mesoporous silica imaging

This study reports the use of variants of coherent anti-Stokes Raman scattering (CARS) microscopy as a novel method for improved physicochemical characterization of drug-loaded silica particles. Ordered mesoporous silica is a biomaterial that can be loaded to carry a number of biochemicals, including poorly water-soluble drugs, by allowing the incorporation of drug into nanometer-sized pores. In this work, the loading of two poorly water-soluble model drugs, itraconazole and griseofulvin, in MCM-41 silica microparticles is characterized qualitatively, using the novel approach of CARS microscopy, which has advantages over other analytical approaches used to date and is non-destructive, rapid, label free, confocal and has chemical and physical specificity. The study investigated the effect of two solvent-based loading methods, namely immersion and rotary evaporation, and microparticle size on the three-dimensional (3-D) distribution of the two loaded drugs. Additionally, hyperspectral CARS microscopy was used to confirm the amorphous nature of the loaded drugs. Z-stacked CARS microscopy suggested that the drug, but not the loading method or particle size range, affected 3-D drug distribution. Hyperspectral CARS confirmed that the drug loaded in the MCM-41 silica microparticles was in an amorphous form. The results show that CARS microscopy and hyperspectral CARS microscopy can be used to provide further insights into the structural nature of loaded mesoporous silica microparticles as biomaterials.

Hyperspectral coherent anti-Stokes Raman scattering microscopy for in situ analysis of solid-state crystal polymorphs

Hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy is quickly becoming a prominent imaging modality because of its many advantages over the traditional paradigm of multispectral CARS. In particular, recording a significant portion of the vibrational spectrum at each spatial pixel allows image-wide spectral analysis at much higher rates than can be achieved with spontaneous Raman. We recently developed a hyperspectral CARS method, the driving principle behind which is the fast acquisition and display of a hyperspectral datacube as a set of intuitive images wherein each material in a sample appears with a unique trio of colors. Here we use this system to image and analyze two types of polymorphic samples: the pseudopolymorphic hydration of theophylline, and the packing polymorphs of the sugar alcohol mannitol. In addition to these solid-state form modifications we have observed spectral variations of crystalline mannitol and diprophylline as functions of their orientations relative to the optical fields. We use that information to visualize the distributions of these compounds in a pharmaceutical solid oral dosage form.

Non-linear optical imaging – Introduction and pharmaceutical applications

Nonlinear optical imaging is an emerging technology with much potential in pharmaceutical analysis. The technique encompasses a range of optical phenomena, including coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and twophoton excited fluorescence (TPEF). The combined potential of these phenomena for pharmaceutical imaging includes chemical and solidstate specificity, high optical spatial and temporal resolution, nondestructive and non-contact analysis, no requirement for labels, and the compatibility with imaging in aqueous and biological environments. In this article, the theory and practical aspects of nonlinear imaging are briefly introduced and pharmaceutical and biopharmaceutical applications are considered. These include material and dosage form characterization, drug release, and drug and nanoparticle distribution in tissues and within live cells. The advantages and disadvantages of the technique in the context of these analyses are also discussed. Introduction to Nonlinear Optics Nonlinear optics deals with processes where two or more photons interact with the sample material simultaneously. This is only possible when a high enough number of photons is confined in a small volume, i.e. the intensity of the incident light has to be much higher when compared to linear effects (normal absorption, refractive index). In practice this is made possible using ultrashort laser pulses with durations from picoseconds to femtoseconds. The narrow temporal width limits the interaction time between laser pulses and the target medium. Therefore, they offer a way to precisely probe the medium with reduced risk of optically induced damage. Even more importantly, the energy confined within the narrow optical pulse gives rise to very high light intensities and, thus, ultrashort pulses constitute ideal means for provoking a variety of nonlinear optical phenomena. Since the signal is produced selectively at the focus where the intensity is highest, nonlinear methods offer intrinsic axial sectioning property wellsuited for three-dimensional imaging. In optical microscopy, the two most commonly exploited phenomena are TPEF and SHG. CARS microscopy is yet another emerging nonlinear optical technique which is suitable for imaging various biological and pharmaceutical samples. The energy level diagrams of these processes are illustrated in Fig. 1. Figure 1. Energy level diagrams of two-photon excited fluorescence (left), second harmonic generation (middle), and coherent anti-Stokes Raman scattering (right). Two-photon excitation occurs through the absorption of two photons via intermediate virtual energy levels (dashed line). In SHG, two photons are combined similarly, but the emission occurs directly from a short-lived virtual level without excitation to the higher electronic states. In the CARS process, two photons stimulate a selected vibrational mode. Probing of this level with the third excitation photon results in the CARS emission. In the TPEF process (Fig. 1, left) a fluorophore molecule is excited by the simultaneous absorption of two photons in the infrared spectral range. In the conventional one-photon fluorescence the same transition to the higher energy level requires more energetic photons in the ultraviolet or visible range. The same fluorophores can often be used with both techniques. However, due to the fundamentally different quantum-mechanical processes, the excitation efficiencies are typically not equivalent. The fluorescence emission spectra are essentially the same in both cases. In TPEF, the longer incident wavelength leads to improved depth penetration in tissues, with reduced potential for photolytic damage [1, 2]. This has led to its increasing popularity in the biomedical setting, and the technique is commonly referred to as multiphoton imaging (this is because it is the most established nonlinear optical imaging method, even though other nonlinear phenomena also involve multiple photons). SHG is a nonlinear process where two photons interact with the target medium so that the energy from the incident laser beam is transferred to a beam with precisely double the original frequency (half the wavelength). This process does not involve the absorption of the photons, but relies on so-called virtual energy levels (Fig. 1, middle). SHG can only occur in materials that exhibit non-centrosymmetric structure. In biology, one such example is collagen, and most pharmaceutical crystals are also examples (whereas the amorphous form is not). One benefit of the technique is that it is straightforward to spectrally separate the SHG signal from other emission sources such as autofluorescence. In CARS microscopy, the signal originates from vibrational motion of the molecules in the sample. The process is based on a nonlinear phenomenon called four-wave mixing. In this process, three beams are interacting with the target medium to produce the fourth at a different wavelength (Fig. 1, right). The frequency difference between two of the input beams is selected to match the frequency of a vibrational energy state of the target molecule. When this resonant condition is fulfilled, the stimulating beams enhance the selected vibrational mode (e.g. stretching of a C-H molecular bond). Using the third beam, this motion can be probed. The resulting CARS signal is generated at the blueshifted (antiStokes) spectral range when compared to the input beams. Stimulated Raman scattering (SRS) is another variant of coherent nonlinear processes used to selectively probe molecular vibrations. When the diff erence frequency between two input beams matches a vibrational resonance, a small fraction (about one millionth) of the incident energy is transferred from one beam to the other. In order to measure the subtle variation, one of the input beams is intensitymodulated at high frequency. This modulation is transferred to the other beam and can be measured using sensitive photodetection. The main benefit of SRS over CARS is the lack of non-resonant background. Like conventional Raman microscopy (based on spontaneous Raman scattering), CARS and SRS are mostly used for imaging different chemical components without the need for labels. The major advantage of CARS and SRS compared to conventional Raman microscopy is the much faster imaging speed (orders of magnitude). This allows video rate imaging of dynamic processes, not to mention dramatically increasing analytical throughput. On the other hand, the spontaneous Raman microscopy typically gives richer spectral information which facilitates analysis of complex mixtures. The development of coherent Raman imaging systems capable of rapidly collecting rich spectral information is gradually overcoming this limitation. As a result, it is entirely possible that Raman microscopy based on spontaneous Raman scattering will largely be replaced with coherent Raman imaging in the pharmaceutical setting in the coming years. Nonlinear optical microscopes capable of TPEF and SHG (multiphoton imaging microscopes) are relatively widespread. CARS and SRS microscopes, which are also capable of TPEF and SHG imaging, are technically more complex but capable of richer sample analysis, especially when multiple nonlinear phenomena are simultaneously used (multimodal imaging). To illustrate the value of nonlinear optical imaging in the pharmaceutical setting, various examples are presented below. These include imaging drugs and dosage forms during the lifecycle of the product, from manufacturing to their fate in the body. Only label-free imaging examples are presented. Imaging Drugs and Dosage Forms Nonlinear optical imaging is slowly gaining interest in the area of drug and formulation imaging (Table 1). Some of the earliest work focused on using CARS to image the composition of emulsions [3]. In this work, the authors utilized CARS microscopy to image based on the C-H stretch vibrational region (2800-3200 cm-1). Later work conducted by Day et al. [4] used oil-in-water emulsions and investigated lipid digestion with CARS microscopy. CARS microscopy allowed them to discriminate between undigested oil and lipolytic products without the need for labeling. Table 1. Some publications for nonlinear optical imaging of pharmaceutical formulations

In situ dissolution analysis of pharmaceutical dosage forms using coherent anti-Stokes Raman scattering (CARS) microscopy

A custom-built intrinsic flow-through dissolution setup was developed and incorporated into a home-built CARS microscope consisting of a synchronously pumped optical parametric oscillator (OPO) and an inverted microscope with a 20X/0.5NA objective. CARS dissolution images (512×512 pixels) were collected every 1.12s for the duration of the dissolution experiment. Hyperspectral CARS images were obtained pre- and postdissolution by rapidly imaging while sweeping the wavelength of the OPO in discrete steps so that each frame in the data stack corresponds to a vibrational frequency. An image-processing routine projects this hyperspectral data into a single image wherein each compound appears with a unique color. Dissolution was conducted using theophylline and cimetidine-naproxen co-amorphous mixture. After 15 minutes of theophylline dissolution, hyperspectral imaging showed a conversion of theophylline anhydrate to the monohydrate, confirmed by a peak shift in the CARS spectra. CARS dissolution images showed that monohydrate crystal growth began immediately and reached a maximum with complete surface coverage at about 300s. This result correlated with the UV dissolution data where surface crystal growth on theophylline compacts resulted in a rapidly reducing dissolution rate during the first 300s. Co-amorphous cimetidinenaproxen didn’t appear to crystallize during dissolution. We observed solid-state conversions on the compact’s surface in situ during dissolution. Hyperspectral CARS imaging allowed visual discrimination between the solid-state forms on the compact’s surface. In the case of theophylline we were able to correlate the solid-state change with a change in dissolution rate.