Impact of Different Packaging Configurations on A Topical Cream Product.

PURPOSE: The objective of this study was to investigate whether different dispensing processes can alter the physicochemical and structural (Q3) attributes of a topical cream product, and potentially alter its performance. METHODS: Acyclovir cream, 5% (Zovirax®) is sold in the UK and other countries in a tube and a pump packaging configurations. The structural attributes of the cream dispensed from each packaging configuration were analyzed by optical microscopy, confocal Raman microscopy and cryo-scanning electron microscopy. Rheological behavior of the products was also evaluated. Product performance (rate and extent of skin delivery) was assessed by in vitro permeation tests (IVPT) using heat-separated human epidermis mounted in static vertical (Franz-type) diffusion cells. RESULTS: Differences in Q3 attributes and IVPT profiles were observed with creams dispensed from the two packaging configurations, even though the product inside each packaging appeared to be the same in Q3 attributes. Visible globules were recognized in the sample dispensed from the pump, identified as dimethicone globules by confocal Raman microscopy. Differences in rheological behaviour could be attributed to these globules as products not dispensed through the pump, demonstrated a similar rheological behaviour. Further, IVPT confirmed a reduced rate and extent to delivery across human epidermis from the product dispensed through a pump. CONCLUSIONS: Different methods of dispensing topical semisolid products can result in metamorphosis and Q3 changes that may have the potential to alter the bioavailability of an active ingredient. These findings have potential implications for product developers and regulators, related to the manufacturing and comparative testing of reference standard and prospective generic products dispensed from different packaging configurations.

Analysis of Milk Microstructure Using Raman Hyperspectral Imaging.

Optical spectroscopic analysis of the chemical composition of milk in its natural state is complicated by a complex colloidal structure, represented by differently sized fat and protein particles. The classical techniques of molecular spectroscopy in the visible, near-, and mid-infrared ranges carry only bulk chemical information about a sample, which usually undergoes a destructive preparation stage. The combination of Raman spectroscopy with confocal microscopy provides a unique opportunity to obtain a vibrational spectrum at any single point of the sample volume. In this study, scanning confocal Raman microscopy was applied for the first time to investigate the chemical microstructure of milk using samples of various compositions. The obtained hyperspectral images of selected planes in milk samples are represented by three-dimensional data arrays. Chemometric data analysis, in particular the method of multivariate curve resolution, has been used to extract the chemical information from complex partially overlaid spectral responses. The results obtained show the spatial distribution of the main chemical components, i.e., fat, protein, and lactose, in the milk samples under study using intuitive graphical maps. The proposed experimental and data analysis method can be used in an advanced chemical analysis of natural milk and products on its basis.

Application of Confocal Raman Microscopy for the Characterization of Topical Semisolid Formulations and their Penetration into Human Skin Ex Vivo.

PURPOSE: The quality testing and approval procedure for most pharmaceutical products is a streamlined process with standardized procedures for the determination of critical quality attributes. However, the evaluation of semisolid dosage forms for topical drug delivery remains a challenging task. The work presented here highlights confocal Raman microscopy (CRM) as a valuable tool for the characterization of such products. METHODS: CRM, a laser-based method, combining chemically-selective analysis and high resolution imaging, is used for the evaluation of different commercially available topical acyclovir creams. RESULTS: We show that CRM enables the spatially resolved analysis of microstructural features of semisolid products and provides insights into drug distribution and polymorphic state as well as the composition and arrangement of excipients. Further, we explore how CRM can be used to monitor phase separation and to study skin penetration and the interaction with fresh and cryopreserved excised human skin tissue. CONCLUSION: This study presents a comprehensive overview and illustration of how CRM can facilitate several types of key analyses of semisolid topical formulations and of their interaction with their biological target site, illustrating that CRM is a useful tool for research, development as well as for quality testing in the pharmaceutical industry.

Versatile Confocal Raman Imaging Microscope Built from Off-the-Shelf Opto-Mechanical Components.

Confocal Raman microscopic (CRM) imaging has evolved to become a key tool for spatially resolved, compositional analysis and imaging, down to the µm-scale, and nowadays one may choose between numerous commercial instruments. That notwithstanding, situations may arise which exclude the use of a commercial instrument, e.g., if the analysis involves toxic or radioactive samples/environments; one may not wish to render an expensive instrument unusable for other uses, due to contamination. Therefore, custom-designed CRM instrumentation-being adaptable to hazardous conditions and providing operational flexibility-may be beneficial. Here, we describe a CRM setup, which is constructed nearly in its entirety from off-the-shelf optomechanical and optical components. The original aim was to develop a CRM suitable for the investigation of samples exposed to tritium. For increased flexibility, the CRM system incorporates optical fiber coupling to both the Raman excitation laser and the spectrometer. Lateral raster scans and axial profiling of samples are facilitated by the use of a motorized xyz-translation assembly. Besides the description of the construction and alignment of the CRM system, we also provide (i) the experimental evaluation of system performance (such as, e.g., spatial resolution) and (ii) examples of Raman raster maps and axial profiles of selected thin-film samples (such as, e.g., graphene sheets).

Overview of Popular Techniques of Raman Spectroscopy and Their Potential in the Study of Plant Tissues.

Raman spectroscopy is one of the main analytical techniques used in optical metrology. It is a vibration, marker-free technique that provides insight into the structure and composition of tissues and cells at the molecular level. Raman spectroscopy is an outstanding material identification technique. It provides spatial information of vibrations from complex biological samples which renders it a very accurate tool for the analysis of highly complex plant tissues. Raman spectra can be used as a fingerprint tool for a very wide range of compounds. Raman spectroscopy enables all the polymers that build the cell walls of plants to be tracked simultaneously; it facilitates the analysis of both the molecular composition and the molecular structure of cell walls. Due to its high sensitivity to even minute structural changes, this method is used for comparative tests. The introduction of new and improved Raman techniques by scientists as well as the constant technological development of the apparatus has resulted in an increased importance of Raman spectroscopy in the discovery and defining of tissues and the processes taking place in them.

Molecular targeting of bioconjugated graphene oxide nanocarriers revealed at a cellular level using label-free Raman imaging.

Two-dimensional materials as graphene oxide (GO) are able to accommodate labels as well as toxins for diagnostics and therapy, respectively. The transmembrane protein carbonic anhydrase (CA IX) is one of the molecules selectively expressed by tumor cells. Here, we demonstrate bioconjugation of GO to biotinylated M75 antibody highly selective towards CA IX. Based on a model system, binding between the bioconjugated GO-M75 and Madin-Darby Canine Kidney (MDCK) cells was evaluated. As proven by fluorescence-activated cell sorting, higher intake was observed for GO-M75 towards MDCK cells ectopically expressing CA IX protein on their surface when compared to control MDCK. In particular, we were able to localize GO nanocarrier crossing the membrane during endocytosis, thanks to the optical cross-sectioning of living cells in real-time employed the label-free confocal Raman microscopy. The increased affinity of the prepared GO-M75 molecular complexes validates the use of two-dimensional materials for future strategies of targeted cancer treatment.

Biomolecular Functionalization of a Nanomaterial To Control Stability and Retention within Live Cells.

Noncovalent hybrids of single-stranded DNA and single-walled carbon nanotubes (SWCNTs) have demonstrated applications in biomedical imaging and sensing due to their enhanced biocompatibility and photostable, environmentally responsive near-infrared (NIR) fluorescence. The fundamental properties of such DNA-SWCNTs have been studied to determine the correlative relationships between oligonucleotide sequence and length, SWCNT species, and the physical attributes of the resultant hybrids. However, intracellular environments introduce harsh conditions that can change the physical identities of the hybrid nanomaterials, thus altering their intrinsic optical properties. Here, through visible and NIR fluorescence imaging in addition to confocal Raman microscopy, we show that the oligonucleotide length controls the relative uptake, intracellular optical stability, and retention of DNA-SWCNTs in mammalian cells. Although the absolute NIR fluorescence intensity of DNA-SWCNTs in murine macrophages increases with increasing oligonucleotide length (from 12 to 60 nucleotides), we found that shorter oligonucleotide DNA-SWCNTs undergo a greater magnitude of spectral shift and are more rapidly internalized and expelled from the cell after 24 h. Furthermore, by labeling the DNA with a fluorophore that dequenches upon removal from the SWCNT surface, we found that shorter oligonucleotide strands are displaced from the SWCNT within the cell, altering the physical identity and changing the fate of the internalized nanomaterial. Finally, through a pharmacological inhibition study, we identified the mechanism of SWCNT expulsion from the cells as lysosomal exocytosis. These findings provide a fundamental understanding of the interactions between SWCNTs and live cells as well as evidence suggesting the ability to control the biological fate of the nanomaterials merely by varying the type of DNA wrapping.

The non-homogenous distribution and aggregation of carotenoids in the stratum corneum correlates with the organization of intercellular lipids in vivo.

The human stratum corneum (SC) contains an abundant amount of carotenoid antioxidants, quenching free radicals and thereby protecting the skin. For the precise measurements of the depth-dependent carotenoid concentration, confocal Raman microscopy is a suitable method. The quantitative concentration can be determined by the carotenoid-related peak intensity of a Gaussian function approached at ≈1524 cm-1 using non-linear regression. Results show that the carotenoid concentration is higher at the superficial layers of the SC then decreases to a minimum at 20% SC depth and increases again towards the bottom of the SC. In the present work, two carotenoid penetration pathways into the SC are postulated. The first pathway is from the stratum granulosum to the bottom of the SC, while in the second pathway, the carotenoids are delivered to the skin surface by sweat and/or sebum secretion and penetrate from outside. The carotenoids are aggregated at the superficial layers, which are shown by high correlation between the aggregation states of carotenoids and the lateral organization of lipids. At the 30%-40% SC depths, the ordered and dense lipid molecules intensify the lipid-carotenoid interactions and weaken the carotenoid-carotenoid interaction and thus exhibit the disaggregation of carotenoids. At 90%-100% SC depths, the carotenoid-lipid interaction is weakened and the carotenoids have a tendency to be aggregated. Thus, the molecular structural correlation of carotenoid and SC lipid might be reserved in the intercellular space of the SC and also serves as the skeleton of the intercellular lipids.

Novel confocal Raman microscopy method to investigate hydration mechanisms in human skin.

BACKGROUND: Skin hydration is essential for maintaining stratum corneum (SC) flexibility and facilitating maturation events. Moisturizers contain multiple ingredients to maintain and improve skin hydration although a complete understanding of hydration mechanisms is lacking. The ability to differentiate the source of the hydration (water from the environment or deeper skin regions) upon application of product will aid in designing more efficacious formulations. MATERIALS AND METHODS: Novel confocal Raman microscopy (CRM) experiments allow us to investigate mechanisms and levels of hydration in the SC. Using deuterium oxide (D2 O) as a probe permits the differentiation of endogenous water (H2 O) from exogenous D2 O. Following topical application of D2 O, we first compare in vivo skin depth profiles with those obtained using ex vivo skin. Additional ex vivo experiments are conducted to quantify the kinetics of D2 O diffusion in the epidermis by introducing D2 O under the dermis. RESULTS: Relative D2 O depth profiles from in vivo and ex vivo measurements compare well considering procedural and instrumental differences. Additional in vivo experiments where D2 O was applied following topical glycerin application increased the longevity of D2 O in the SC. Reproducible rates of D2 O diffusion as a function of depth have been established for experiments where D2 O is introduced under ex vivo skin. CONCLUSION: Unique information regarding hydration mechanisms are obtained from CRM experiments using D2 O as a probe. The source and relative rates of hydration can be delineated using ex vivo skin with D2 O underneath. One can envision comparing these depth-dependent rates in the presence and absence of topically applied hydrating agents to obtain mechanistic information.

Confocal Raman microscopy in life sciences.

Microscopy techniques are widely used in life sciences to study cells and tissues. Fluorescence microscopy, for example, is a very common method in many laboratories. While reliable and strong fluorescence signals are a clear advantage of this method, the labelling procedure with fluorescent dyes, the availability of required antibodies or the potentially necessary genetic modifications of the studied organism all introduce potential complications. By contrast, confocal Raman microscopy is a label-free and non-destructive imaging technique. In contrast to infrared microscopy, it is easily applicable in aqueous environments. Different microscope setups and combinations allow for the examination of various solid and liquid samples, even in their typical environments. The article demonstrates the analyzing capability of confocal Raman microscopy and correlative techniques through application examples of eukaryotic and prokaryotic cells, and cancerous and normal tissues and shows how confocal Raman microscopy provides valuable information for a more comprehensive understanding of the investigated sample.

Label-Free Confocal Raman Mapping of Transportan in Melanoma Cells.

Cell-penetrating peptides (CPPs) are promising vectors for the intracellular delivery of a variety of membrane-impermeable bioactive compounds. The mechanisms by which CPPs cross the cell membrane, and the effects that CPPs may have on cell function, still remain to be fully clarified. In this work, we employed confocal Raman microscopy (CRM) and atomic force microscopy (AFM) to study the infiltration and physiological effects of the amphipathic CPP transportan (Tp) on the metastatic melanoma cell line SK-Mel-2. CRM enabled the detection of label-free Tp within the cells. Raman maps of live cells revealed rapid entry (within 5 min) and widespread distribution of the peptide throughout the cytoplasm and the presence of the peptide within the nucleus after ∼20 min. Principal component analysis of the CRM data collected from Tp-treated and untreated cells showed that Tp Raman bands were not positively correlated with lipid Raman bands, indicating that Tp entered the cells via a nonendocytic mechanism. Analysis of intracellularly recovered Tp by mass spectrometry showed that Tp remained intact in SK-Mel-2 cells for up to 24 h. The Raman spectroscopic data also showed that, although Tp was predominantly unstructured (random coil) in aqueous solution, it accumulated to high densities within the cells with mostly ß-sheet and α-helical structures. AFM was employed to measure the effect of Tp treatment on cell stiffness. These data showed that Tp induced a significant increase in cell stiffness within the first hour of treatment, which was partially abated after 2 h. It is hypothesized that the increase in cell stiffness was the result of cytoskeletal changes triggered by Tp.

An Expandable Mechanopharmaceutical Device (3): a Versatile Raman Spectral Cytometry Approach to Study the Drug Cargo Capacity of Individual Macrophages.

PURPOSE: To improve cytometric phenotyping abilities and better understand cell populations with high interindividual variability, a novel Raman-based microanalysis was developed to characterize macrophages on the basis of chemical composition, specifically to measure and characterize intracellular drug distribution and phase separation in relation to endogenous cellular biomolecules. METHODS: The microanalysis was developed for the commercially-available WiTec alpha300R confocal Raman microscope. Alveolar macrophages were isolated and incubated in the presence of pharmaceutical compounds nilotinib, chloroquine, or etravirine. A Raman data processing algorithm was specifically developed to acquire the Raman signals emitted from single-cells and calculate the signal contributions from each of the major molecular components present in cell samples. RESULTS: Our methodology enabled analysis of the most abundant biochemicals present in typical eukaryotic cells and clearly identified "foamy" lipid-laden macrophages throughout cell populations, indicating feasibility for cellular lipid content analysis in the context of different diseases. Single-cell imaging revealed differences in intracellular distribution behavior for each drug; nilotinib underwent phase separation and self-aggregation while chloroquine and etravirine accumulated primarily via lipid partitioning. CONCLUSIONS: This methodology establishes a versatile cytometric analysis of drug cargo loading in macrophages requiring small numbers of cells with foreseeable applications in toxicology, disease pathology, and drug discovery.

Radical-Scavenging Activity of a Sunscreen Enriched by Antioxidants Providing Protection in the Whole Solar Spectral Range.

BACKGROUND/AIM: The main reason for extrinsic skin aging is the negative action of free radicals. The formation of free radicals in the skin has been associated with ultraviolet (UV) exposure and also to visible (VIS) and near-infrared (NIR) irradiations. The aim of the present study was to evaluate the efficacy of a sunscreen in the whole solar range. METHODS: The radical-scavenging activity of a sunscreen in the UV, VIS, and NIR ranges was evaluated using electron paramagnetic resonance spectroscopy. Ex vivo penetration profiles were determined using confocal Raman microscopy on porcine ear skin at different time points after application. RESULTS: Compared to the untreated skin, the sunscreen decreased the skin radical formation in the UV and VIS regions. Additional protection in the VIS and NIR ranges was observed for the sunscreen containing antioxidants (AO). The penetration depth of the cream was less than 11.2 ± 3.0 µm for all time points. CONCLUSION: A sunscreen containing AO improved the photoprotection in the VIS and NIR ranges. The sunscreen was retained in the stratum corneum. Therefore, these results show the possibility of the development of effective and safer sunscreen products.

Cryogenic transmission electron microscopy study: preparation of vesicular dispersions by quenching microemulsions.

We previously showed that long-lived nanoemulsions, seeming initially vesicular, might be prepared simply by diluting and cooling (quenching) warm microemulsions with n-hexadecane with precooled water. In this paper, we confirm that these systems are vesicular dispersions when fresh, and they can be made with similar structures and compositional dependence using alkanes with chain lengths ranging from octane to hexadecane. The nanostructures of fresh nanoemulsions are imaged with cryogenic transmission electron microscopy (cryo-TEM). We confirm that water-continuous microemulsions give simple dispersions of vesicles (sometimes unilamellar), typically less than 100 nm in diameter; these systems can avoid separation for over 2 months. Selected samples were also prepared using halogenated alkanes to create additional contrast in the cryo-TEM, allowing us to confirm that the oil is located in the observed vesicular structures.

Structural Insight into Cell Wall Architecture of Micanthus sinensis cv. using Correlative Microscopy Approaches.

Structural organization of the plant cell wall is a key parameter for understanding anisotropic plant growth and mechanical behavior. Four imaging platforms were used to investigate the cell wall architecture of Miscanthus sinensis cv. internode tissue. Using transmission electron microscopy with potassium permanganate, we found a great degree of inhomogeneity in the layering structure (4-9 layers) of the sclerenchymatic fiber (Sf). However, the xylem vessel showed a single layer. Atomic force microscopy images revealed that the cellulose microfibrils (Mfs) deposited in the primary wall of the protoxylem vessel (Pxv) were disordered, while the secondary wall was composed of Mfs oriented in parallel in the cross and longitudinal section. Furthermore, Raman spectroscopy images indicated no variation in the Mf orientation of Pxv and the Mfs in Pxv were oriented more perpendicular to the cell axis than that of Sfs. Based on the integrated results, we have proposed an architectural model of Pxv composed of two layers: an outermost primary wall composed of a meshwork of Mfs and inner secondary wall containing parallel Mfs. This proposed model will support future ultrastructural analysis of plant cell walls in heterogeneous tissues, an area of increasing scientific interest particularly for liquid biofuel processing.

Graphene multilayer as nanosized optical strain gauge for polymer surface relief gratings.

In this paper, we show how graphene can be utilized as a nanoscopic probe in order to characterize local opto-mechanical forces generated within photosensitive azobenzene containing polymer films. Upon irradiation with light interference patterns, photosensitive films deform according to the spatial intensity variation, leading to the formation of periodic topographies such as surface relief gratings (SRG). The mechanical driving forces inscribing a pattern into the films are supposedly fairly large, because the deformation takes place without photofluidization; the polymer is in a glassy state throughout. However, until now there has been no attempt to characterize these forces by any means. The challenge here is that the forces vary locally on a nanometer scale. Here, we propose to use Raman analysis of the stretching of the graphene layer adsorbed on top of polymer film under deformation in order to probe the strength of the material transport spatially resolved. With the well-known mechanical properties of graphene, we can obtain lower bounds on the forces acting within the film. Upon the basis of our experimental results, we can deduce that the internal pressure in the film due to grating formation can exceed 1 GPa. The graphene-based nanoscopic gauge opens new possibilities to characterize opto-mechanical forces generated within photosensitive polymer films.

Multiple microscopic approaches demonstrate linkage between chromoplast architecture and carotenoid composition in diverse Capsicum annuum fruit.

Increased accumulation of specific carotenoids in plastids through plant breeding or genetic engineering requires an understanding of the limitations that storage sites for these compounds may impose on that accumulation. Here, using Capsicum annuum L. fruit, we demonstrate directly the unique sub-organellar accumulation sites of specific carotenoids using live cell hyperspectral confocal Raman microscopy. Further, we show that chromoplasts from specific cultivars vary in shape and size, and these structural variations are associated with carotenoid compositional differences. Live-cell imaging utilizing laser scanning confocal (LSCM) and confocal Raman microscopy, as well as fixed tissue imaging by scanning and transmission electron microscopy (SEM and TEM), all demonstrated morphological differences with high concordance for the measurements across the multiple imaging modalities. These results reveal additional opportunities for genetic controls on fruit color and carotenoid-based phenotypes.

Combining confocal Raman microscopy and freeze-drying for quantification of substance penetration into human skin.

In the area of dermatological research, the knowledge of rate and extent of substance penetration into the human skin is essential not only for evaluation of therapeutics, but also for risk assessment of chemicals and cosmetic ingredients. Recently, confocal Raman microscopy emerged as a novel analytical technique for analysis of substance skin penetration. In contrast to destructive drug extraction and quantification, the technique is non-destructive and provides high spatial resolution in three dimensions. However, the generation of time-resolved concentration depth profiles is restrained by ongoing diffusion of the penetrating substance during analysis. To prevent that, substance diffusion in excised human skin can instantly be stopped at defined time points by freeze-drying the sample. Thus, combining sample preparation by freeze-drying with drug quantification by confocal Raman microscopy yields a novel analytical platform for non-invasive and quantitative in vitro analysis of substance skin penetration. This work presents the first proof-of-concept study for non-invasive quantitative substance depth profiling in freeze-dried excised human stratum corneum by confocal Raman microscopy.

In situ real-time pathway to study the polyethylene long-term degradation process by a marine fungus through confocal Raman quantitative imaging.

Since plastic waste has become a worldwide pollution problem, studying the ability of marine microorganisms to degrade plastic waste is important. However, conventional methods are unable to in situ real-time study the ability of microorganisms to biodegrade plastics. In recent years, Raman spectroscopy has been widely used in the characterization of plastics as well as in the study of biological metabolism due to its low cost, rapidity, label-free, non-destructive, and water-independent features, which provides us with new ideas to address the above limitations. Here, we have established a method to study the degradation ability of microorganisms on plastics using confocal Raman imaging. Alternaria alternata FB1, a recently reported polyethylene (PE) degrading marine fungus, is used as a model to perform a long-term (up to 274 days) in situ real-time nondestructive inspection of its degradation process. We can prove the degradation of PE plastics from the following two aspects, visualization and analysis of the degradation process based on depth imaging and quantification of the degradation rate by crystallinity calculations. The findings also reveal unprecedented degradation details. The method is important for realizing high-throughput screening of microorganisms with potential to degrade plastics and studying the degradation process of plastics in the future.

Structural Defects on Graphene Generated by Deposition of CoO: Effect of Electronic Coupling of Graphene.

Understanding the interactions in hybrid systems based on graphene and functional oxides is crucial to the applicability of graphene in real devices. Here, we present a study of the structural defects occurring on graphene during the early stages of the growth of CoO, tailored by the electronic coupling between graphene and the substrate in which it is supported: as received pristine graphene on polycrystalline copper (coupled), cleaned in ultra-high vacuum conditions to remove oxygen contamination, and graphene transferred to SiO2/Si substrates (decoupled). The CoO growth was performed at room temperature by thermal evaporation of metallic Co under a molecular oxygen atmosphere, and the early stages of the growth were investigated. On the decoupled G/SiO2/Si samples, with an initial low crystalline quality of graphene, the formation of a CoO wetting layer is observed, identifying the Stranski-Krastanov growth mode. In contrast, on coupled G/Cu samples, the Volmer-Weber growth mechanism is observed. In both sets of samples, the oxidation of graphene is low during the early stages of growth, increasing for the larger coverages. Furthermore, structural defects are developed in the graphene lattice on both substrates during the growth of CoO, which is significantly higher on decoupled G/SiO2/Si samples mainly for higher CoO coverages. When approaching the full coverage on both substrates, the CoO islands coalesce to form a continuous CoO layer with strip-like structures with diameters ranging between 70 and 150 nm.