Optimized Extraction of Amikacin from Murine Whole Blood

Amikacin (Amk) analysis and quantitation, for pharmacokinetics studies and other types of investigations, is conventionally performed after extraction from plasma. No report exists so far regarding drug extraction from whole blood (WB). This can represent an issue since quantification in plasma does not account for drug partitioning to the blood cell compartment, significantly underrating the drug fraction reaching the blood circulation. In the present work, the optimization of an extraction method of Amk from murine WB has been described. The extraction yield was measured by RP-HPLC-UV after derivatization with 1-fluoro-2,4-dinitrobenzene, which produced an appreciably stable derivative with a favorable UV/vis absorption. Several extraction conditions were tested: spiked Amk disulfate solution/acetonitrile/WB ratio; presence of organic acids and/or ammonium hydroxide and/or ammonium acetate in the extraction mixture; re-dissolution of the supernatant in water after a drying process under vacuum; treatment of the supernatant with a solution of inorganic salts. The use of 5% (by volume) of ammonium hydroxide in a hydro-organic solution with acetonitrile, allowed the almost quantitative (95%) extraction of the drug from WB.

Designing Sub-20 nm Nanocarriers for Small Molecule Delivery: Interplay among Structural Geometry, Assembly Energetics, and Cargo Release Kinetics

Biological constraints in diseased tissues have motivated the need for small nanocarriers (10-30 nm) to achieve sufficient vascular extravasation and pervasive tumor penetration. This particle size limit is only an order of magnitude larger than small molecules, such that cargo loading is better described by co-assembly processes rather than simple encapsulation. Understanding the structural, kinetic, and energetic contributions of carrier-cargo co-assembly is thus critical to achieve molecular-level control and predictable in vivo behavior. These interconnected set of properties were systematically examined using sub-20 nm self-assembled nanocarriers known as three-helix micelles (3HM). Both hydrophobicity and the “geometric packing parameter” dictate small molecule compatibility with 3HM’s alkyl tail core. Planar obelisk-like apomorphine and doxorubicin (DOX) molecules intercalated well within the 3HM core and near the core-shell interface, forming an integral component to the co-assembly, as corroborated by small angle X-ray and neutron-scattering structural studies. DOX promoted crystalline alkyl tail ordering, which significantly increased (+63%) the activation energy of 3HM subunit exchange. Subsequently, 3HM-DOX displayed slow-release kinetics (t1/2=40 h) at physiological temperatures, with ~50x greater cargo preference for the micelle core as described by two drug partitioning coefficients (micellar core/shell Kp1 ~24, and shell/bulk solvent Kp2 ~2). The geometric and energetic insights between nanocarrier and their small molecule cargos developed here will aid in broader efforts to deconvolute the interconnected properties of carrier-drug co-assemblies, and to understand nanomedicine behavior throughout all the physical and in vivo processes they are intended to encounter.

Partitioning of cancer therapeutics in nuclear condensates

The nucleus contains diverse phase-separated condensates that compartmentalize and concentrate biomolecules with distinct physicochemical properties. Here, we investigated whether condensates concentrate small-molecule cancer therapeutics such that their pharmacodynamic properties are altered. We found that antineoplastic drugs become concentrated in specific protein condensates in vitro and that this occurs through physicochemical properties independent of the drug target. This behavior was also observed in tumor cells, where drug partitioning influenced drug activity. Altering the properties of the condensate was found to affect the concentration and activity of drugs. These results suggest that selective partitioning and concentration of small molecules within condensates contributes to drug pharmacodynamics and that further understanding of this phenomenon may facilitate advances in disease therapy.

In Vitro Skin Retention of Crisaborole after Topical Application

Crisaborole, a nonsteroidal phosphodiesterase 4 inhibitor, represents the first nonsteroidal medication approved for the treatment of atopic dermatitis in over a decade. In this work, crisaborole skin permeation and retention was studied in vitro from a 2% ointment using porcine skin as barrier. Crisaborole was also characterized in terms of thermal behavior, solubility, and logP. Control experiments were performed also on tape stripped skin to clarify the role of stratum corneum in drug partitioning and permeation across the skin. The results obtained indicate that crisaborole accumulates into the skin in considerable amounts after application of a topical lipophilic ointment. Crisaborole shows more affinity for the dermis compared to the epidermis despite its relatively high value of partition coefficient; stratum corneum analysis revealed a low affinity of the drug for this skin layer. Skin penetration across hair follicles or sebaceous glands can be a reason for the high dermis retention and is worth further investigation. The comparison with data obtained from a solution in acetonitrile suggests that the formulation plays a certain role in determining the relative distribution of crisaborole in the skin layers and in the receptor compartment.

Chemometric Approaches for Developing Infrared Nanosensors to Image Anthracyclines

Generation, identification, and validation of optical probes to image molecular targets in a biological milieu remains a challenge. Synthetic molecular recognition approaches leveraging the intrinsic near-infrared fluorescence of single-walled carbon nanotubes is a promising approach for chronic biochemical imaging in tissues. However, generation of nanosensors for selective imaging of molecular targets requires a heuristic approach. Here, we present a chemometric platform for rapidly screening libraries of candidate single-walled carbon nanotube nanosensors against biochemical analytes to quantify fluorescence response to small molecules including vitamins, neurotransmitters, and chemotherapeutics. We further show this approach can be leveraged to identify biochemical analytes that selectively modulate the intrinsic near-infrared fluorescence of candidate nanosensors. Chemometric analysis thus enables identification of nanosensor-analyte ‘hits’ and also nanosensor fluorescence signaling modalities such as wavelength-shifts that are optimal for translation to biological imaging. Through this approach, we identify and characterize a nanosensor for the chemotherapeutic anthracycline doxorubicin, which provides an up to 17 nm fluorescence red-shift and exhibits an 8 µM limit of detection, compatible with peak circulatory concentrations of doxorubicin common in therapeutic administration. We demonstrate selectivity of this nanosensor over dacarbazine, a chemotherapeutic commonly co-injected with DOX. Lastly, we demonstrate nanosensor tissue compatibility for imaging of doxorubicin in muscle tissue by incorporating nanosensors into the mouse hindlimb and measuring nanosensor response to exogenous DOX administration. Our results motivate chemometric approaches to nanosensor discovery for chronic imaging of drug partitioning into tissues and towards real-time monitoring of drug accumulation.