Validation of a Caco-2 microfluidic Chip model for predicting intestinal absorption of BCS Class I-IV drugs.

Oral delivery is considered the most patient preferred route of drug administration, however, the drug must be sufficiently soluble and permeable to successfully formulate an oral formulation. There have been advancements in the development of more predictive solubility and dissolution tools, but the tools that has been developed for permeability assays have not been validated as extensively as the gold-standard Caco-2 Transwell assay. Here, we evaluated Caco-2 intestinal permeability assay in Transwells and a commercially available microfluidic Chip using 19 representative Biopharmaceutics Classification System (BCS) Class I-IV compounds. For each selected compound, we performed a comprehensive viability test, quantified its apparent permeability (Papp), and established an in vitro in vivo correlation (IVIVC) to the human fraction absorbed (fa) in both culture conditions. Permeability differences were observed across the models as demonstrated by antipyrine (Transwell Papp: 38.5 ± 6.1 × 10-8 cm/s vs Chip Papp: 32.9 ± 11.3 × 10-8 cm/s) and nadolol (Transwell Papp: 0.6 ± 0.1 × 10-7 cm/s vs Chip Papp: 3 ± 1.2 × 10-7 cm/s). The in vitro in vivo correlation (IVIVC; Papp vs. fa) of the Transwell model (r2 = 0.59-0.83) was similar to the Chip model (r2 = 0.41-0.79), highlighting similar levels of predictivity. Comparing to historical data, our Chip Papp data was more closely aligned to native tissues assessed in Ussing chambers. This is the first study to comprehensively validate a commercial Gut-on-a-Chip model as a predictive tool for assessing oral absorption to further reduce our reliance on animal models.

Engineering the ChlorON Series: Turn-On Fluorescent Protein Sensors for Imaging Labile Chloride in Living Cells.

Beyond its role as the "queen of electrolytes", chloride can also serve as an allosteric regulator or even a signaling ion. To illuminate this essential anion across such a spectrum of biological processes, researchers have relied on fluorescence imaging with genetically encoded sensors. In large part, these have been derived from the green fluorescent protein found in the jellyfish Aequorea victoria. However, a standalone sensor with a turn-on intensiometric response at physiological pH has yet to be reported. Here, we address this technology gap by building on our discovery of the anion-sensitive fluorescent protein mNeonGreen (mNG). The targeted engineering of two non-coordinating residues, namely K143 and R195, in the chloride binding pocket of mNG coupled with an anion walking screening and selection strategy resulted in the ChlorON sensors: ChlorON-1 (K143W/R195L), ChlorON-2 (K143R/R195I), and ChlorON-3 (K143R/R195L). In vitro spectroscopy revealed that all three sensors display a robust turn-on fluorescence response to chloride (20- to 45-fold) across a wide range of affinities (Kd ≈ 30-285 mM). We further showcase how this unique sensing mechanism can be exploited to directly image labile chloride transport with spatial and temporal resolution in a cell model overexpressing the cystic fibrosis transmembrane conductance regulator. Building from this initial demonstration, we anticipate that the ChlorON technology will have broad utility, accelerating the path forward for fundamental and translational aspects of chloride biology.

Rational Design of the ß-Bulge Gate in a Green Fluorescent Protein Accelerates the Kinetics of Sulfate Sensing.

Detection of anions in complex aqueous media is a fundamental challenge with practical utility that can be addressed by supramolecular chemistry. Biomolecular hosts such as proteins can be used and adapted as an alternative to synthetic hosts. Here, we report how the mutagenesis of the ß-bulge residues (D137 and W138) in mNeonGreen, a bright, monomeric fluorescent protein, unlocks and tunes the anion preference at physiological pH for sulfate, resulting in the turn-off sensor SulfOFF-1. This unprecedented sensing arises from an enhancement in the kinetics of binding, largely driven by position 138. In line with these data, molecular dynamics (MD) simulations capture how the coordinated entry and gating of sulfate into the ß-barrel is eliminated upon mutagenesis to facilitate binding and fluorescence quenching.

Supramolecular Reinforcement of a Large-Pore 2D Covalent Organic Framework.

Two-dimensional covalent organic frameworks (2D-COFs) are a class of crystalline porous organic polymers that consist of covalently linked, two-dimensional sheets that can stack together through noncovalent interactions. Here we report the synthesis of a novel COF, called PyCOFamide, which has an experimentally observed pore size that is greater than 6 nm in diameter. This is among the largest pore size reported to date for a 2D-COF. PyCOFamide exhibits permanent porosity and high crystallinity as evidenced by the nitrogen adsorption, powder X-ray diffraction, and high-resolution transmission electron microscopy. We show that the pore size of PyCOFamide is large enough to accommodate fluorescent proteins such as Superfolder green fluorescent protein and mNeonGreen. This work demonstrates the utility of noncovalent structural reinforcement in 2D-COFs to produce larger and persistent pore sizes than previously possible.

Excitation ratiometric chloride sensing in a standalone yellow fluorescent protein is powered by the interplay between proton transfer and conformational reorganization.

Natural and laboratory-guided evolution has created a rich diversity of fluorescent protein (FP)-based sensors for chloride (Cl-). To date, such sensors have been limited to the Aequorea victoria green fluorescent protein (avGFP) family, and fusions with other FPs have unlocked ratiometric imaging applications. Recently, we identified the yellow fluorescent protein from jellyfish Phialidium sp. (phiYFP) as a fluorescent turn-on, self-ratiometric Cl- sensor. To elucidate its working mechanism as a rare example of a single FP with this capability, we tracked the excited-state dynamics of phiYFP using femtosecond transient absorption (fs-TA) spectroscopy and target analysis. The photoexcited neutral chromophore undergoes bifurcated pathways with the twisting-motion-induced nonradiative decay and barrierless excited-state proton transfer. The latter pathway yields a weakly fluorescent anionic intermediate , followed by the formation of a red-shifted fluorescent state that enables the ratiometric response on the tens of picoseconds timescale. The redshift results from the optimized π-π stacking between chromophore Y66 and nearby Y203, an ultrafast molecular event. The anion binding leads to an increase of the chromophore pK a and ESPT population, and the hindrance of conversion. The interplay between these two effects determines the turn-on fluorescence response to halides such as Cl- but turn-off response to other anions such as nitrate as governed by different binding affinities. These deep mechanistic insights lay the foundation for guiding the targeted engineering of phiYFP and its derivatives for ratiometric imaging of cellular chloride with high selectivity.

A neutral porous organic polymer host for the recognition of anionic dyes in water.

Neutral hosts for the recognition of anionic guests in water remain underdeveloped due to the inherent thermodynamic barrier for desolvation. To address this challenge, we have repurposed crosslinked porous organic polymers (POPs) as hosts. This polymer architecture affords a hydrophobic environment with a densely packed array of urea hydrogen bond donors to cooperatively promote anion desolvation and recognition in water. Using the principles of supramolecular design, we demonstrate through adsorption assays that the resulting Urea-POP-1 can recognize structurally different dyes containing phosphonate, sulfonate, and carboxylate anions in water. Moreover, when compared to Methyl-POP-1, a control POP lacking hydrogen bond donors, we find that the driving force for desolvation and adsorption of each dye is achieved through hydrophobic interactions with the POP backbone and, more importantly, cooperative hydrogen bonding interactions with the urea sidechains. This starting point sets the stage to exploit the modularity of our design to build a family of neutral polymer hosts with tunable pore sizes and anion preferences for fundamental investigations and targeted applications.