Using the Fluorescence Red Edge Effect to Assess the Long-Term Stability of Lyophilized Protein Formulations

Nanosecond relaxation processes in sugar matrices are causally linked through diffusional processes to protein stability in lyophilized formulations. Long-term protein degradation rates track mean-squared displacement (⟨<i>u</i>²⟩) of hydrogen atoms in sugar glasses, a parameter describing dynamics on a time scale of picoseconds to nanoseconds. However, measurements of ⟨<i>u</i>²⟩ are usually performed by neutron scattering, which is not conducive to rapid formulation screening in early development. Here, we present a benchtop technique to derive a ⟨<i>u</i>²⟩ surrogate based on the fluorescence red edge effect. Glycerol, lyophilized trehalose, and lyophilized sucrose were used as model systems. Samples containing 10^–6 mole fraction of rhodamine 6G, a fluorophore, were excited at either 532 nm (main peak) or 566 nm (red edge), and the ⟨<i>u</i>²⟩ surrogate was determined based the corresponding Stokes shifts. Results showed reasonable agreement between ⟨<i>u</i>²⟩ from neutron scattering and the surrogate from fluorescence, although deviations were observed at very low temperatures. We discuss the sources of the deviations and suggest technique improvements to ameliorate these. We expect that this method will be a valuable tool to evaluate lyophilized sugar matrices with respect to their ability to protect proteins from diffusion-limited degradation processes during long-term storage. Additionally, the method may have broader applications in amorphous pharmaceutical solids

Surrogate for Debye–Waller Factors from Dynamic Stokes Shifts

We show that the short-time behavior of time-resolved fluorescence Stokes shifts (TRSS) are similar to that of the intermediate scattering function obtained from neutron scattering at <i>q</i> near the peak in the static structure factor for glycerol. This allows us to extract a Debye–Waller (DW) factor analogue from TRSS data at times as short as 1 ps in a relatively simple way. Using the time domain relaxation data obtained by this method, we show that DW factors evaluated at times ≥ 40 ps can be directly influenced by α relaxation and thus should be used with caution when evaluating relationships between fast and slow dynamics in glass-forming systems

Imaging the Molecular Structure of Polyethylene Blends with Broadband Coherent Raman Microscopy

Polyethylene (PE) has been widely used in a myriad of consumer products and critical infrastructure products such as underground gas and water pipes. These products are often made of blends of multiple types of PE with different molecular architectures. Although the long-term performance of these products is largely dictated by their local molecular structure, it has been studied mostly by indirect and bulk-averaging methods such as calorimetry and neutron scattering due to lack of chemical contrast for conventional imaging techniques. We demonstrate that broadband coherent anti-Stokes Raman scattering (CARS) microscopy can acquire images of the chemical composition and molecular orientation of a miscible semicrystalline PE blend with two different molecular architectures. We discuss the detailed crystal structure observed at different length scales and new insights it provides into polymer crystal morphology

Enhanced Stabilization in Dried Silk Fibroin Matrices

Preliminary studies have shown that silk fibroin can protect biomacromolecules from thermal degradation, but a deeper understanding of underlying mechanisms needed to fully leverage the stabilizing potential of this matrix has not been realized. In this study, we investigate stabilization of plasma C-reactive protein (CRP), a diagnostic indicator of infection or inflammation, to gain insight into stabilizing mechanisms of silk. We observed that the addition of antiplasticizing excipients that suppress β-relaxation amplitudes in silk matrices resulted in enhanced stability of plasma CRP. These observations are consistent with those made in sugar-glass-based protein-stabilizing matrices and suggest fundamental insight into mechanisms as well as practical strategies to employ with silk protein matrices for enhanced stabilization utility

DataSheet2_Identifying lipid particle sub-types in live Caenorhabditis elegans with two-photon fluorescence lifetime imaging.pdf

Fat metabolism is an important modifier of aging and longevity in Caenorhabditis elegans. Given the anatomy and hermaphroditic nature of C. elegans, a major challenge is to distinguish fats that serve the energetic needs of the parent from those that are allocated to the progeny. Broadband coherent anti-Stokes Raman scattering (BCARS) microscopy has revealed that the composition and dynamics of lipid particles are heterogeneous both within and between different tissues of this organism. Using BCARS, we have previously succeeded in distinguishing lipid-rich particles that serve as energetic reservoirs of the parent from those that are destined for the progeny. While BCARS microscopy produces high-resolution images with very high information content, it is not yet a widely available platform. Here we report a new approach combining the lipophilic vital dye Nile Red and two-photon fluorescence lifetime imaging microscopy (2p-FLIM) for the in vivo discrimination of lipid particle sub-types. While it is widely accepted that Nile Red staining yields unreliable results for detecting lipid structures in live C. elegans due to strong interference of autofluorescence and non-specific staining signals, our results show that simple FLIM phasor analysis can effectively separate those signals and is capable of differentiating the non-polar lipid-dominant (lipid-storage), polar lipid-dominant (yolk lipoprotein) particles, and the intermediates that have been observed using BCARS microscopy. An advantage of this approach is that images can be acquired using common, commercially available 2p-FLIM systems within about 10% of the time required to generate a BCARS image. Our work provides a novel, broadly accessible approach for analyzing lipid-containing structures in a complex, live whole organism context.</p

DataSheet1_Identifying lipid particle sub-types in live Caenorhabditis elegans with two-photon fluorescence lifetime imaging.ZIP

Fat metabolism is an important modifier of aging and longevity in Caenorhabditis elegans. Given the anatomy and hermaphroditic nature of C. elegans, a major challenge is to distinguish fats that serve the energetic needs of the parent from those that are allocated to the progeny. Broadband coherent anti-Stokes Raman scattering (BCARS) microscopy has revealed that the composition and dynamics of lipid particles are heterogeneous both within and between different tissues of this organism. Using BCARS, we have previously succeeded in distinguishing lipid-rich particles that serve as energetic reservoirs of the parent from those that are destined for the progeny. While BCARS microscopy produces high-resolution images with very high information content, it is not yet a widely available platform. Here we report a new approach combining the lipophilic vital dye Nile Red and two-photon fluorescence lifetime imaging microscopy (2p-FLIM) for the in vivo discrimination of lipid particle sub-types. While it is widely accepted that Nile Red staining yields unreliable results for detecting lipid structures in live C. elegans due to strong interference of autofluorescence and non-specific staining signals, our results show that simple FLIM phasor analysis can effectively separate those signals and is capable of differentiating the non-polar lipid-dominant (lipid-storage), polar lipid-dominant (yolk lipoprotein) particles, and the intermediates that have been observed using BCARS microscopy. An advantage of this approach is that images can be acquired using common, commercially available 2p-FLIM systems within about 10% of the time required to generate a BCARS image. Our work provides a novel, broadly accessible approach for analyzing lipid-containing structures in a complex, live whole organism context.</p

Pronounced Microheterogeneity in a Sorbitol–Water Mixture Observed through Variable Temperature Neutron Scattering

In this study, the structure of concentrated d-sorbitol–water mixtures is studied by wide- and small-angle neutron scattering (WANS and SANS) as a function of temperature. The mixtures are prepared using both deuterated and regular sorbitol and water at a molar fraction of sorbitol of 0.19 (equivalent to 70% by weight of regular sorbitol in water). Retention of an amorphous structure (i.e., absence of crystallinity) is confirmed for this system over the entire temperature range, 100–298 K. The glass transition temperature, Tg, is found from differential scanning calorimetry to be approximately 200 K. WANS data are analyzed using empirical potential structure refinement, to obtain the site–site radial distribution functions (RDFs) and coordination numbers. This analysis reveals the presence of nanoscaled water clusters surrounded by (and interacting with) sorbitol molecules. The water clusters appear more structured compared to bulk water and, especially at the lowest temperatures, resemble the structure of low-density amorphous ice (LDA). Upon cooling to 100 K the peaks in the water RDFs become markedly sharper, with increased coordination number, indicating enhanced local (nanometer-scale) ordering, with changes taking place both above and well below the Tg. On the mesoscopic (submicrometer) scale, although there are no changes between 298 and 213 K, cooling the sample to 100 K results in a significant increase in the SANS signal, which is indicative of pronounced inhomogeneities. This increase in the scattering is partly reversed during heating, although some hysteresis is observed. Furthermore, a power law analysis of the SANS data indicates the existence of domains with well-defined interfaces on the submicrometer length scale, probably as a result of the appearance and growth of microscopic voids in the glassy matrix. Because of the unusual combination of small and wide scattering data used here, the present results provide new physical insight into the structure of aqueous glasses over a broad temperature and length scale, leading to an improved understanding of the mechanisms of temperature- and water-induced (de)­stabilization of various systems, including proteins, pharmaceuticals, and biological objects

Multicomponent Chemical Imaging of Pharmaceutical Solid Dosage Forms with Broadband CARS Microscopy

We compare a coherent Raman imaging modality, broadband coherent anti-Stokes Raman scattering (BCARS) microscopy, with spontaneous Raman microscopy for quantitative and qualitative assessment of multicomponent pharmaceuticals. Indomethacin was used as a model active pharmaceutical ingredient (API) and was analyzed in a tabulated solid dosage form, embedded within commonly used excipients. In comparison with wide-field spontaneous Raman chemical imaging, BCARS acquired images 10× faster, at higher spatiochemical resolution and with spectra of much higher SNR, eliminating the need for multivariate methods to identify chemical components. The significant increase in spatiochemical resolution allowed identification of an unanticipated API phase that was missed by the spontaneous wide-field method and bulk Raman spectroscopy. We confirmed the presence of the unanticipated API phase using confocal spontaneous Raman, which provided spatiochemical resolution similar to BCARS but at 100× slower acquisition times