Screening oral drugs for their interactions with the intestinal transportome via porcine tissue explants and machine learning.

In vitro systems that accurately model in vivo conditions in the gastrointestinal tract may aid the development of oral drugs with greater bioavailability. Here we show that the interaction profiles between drugs and intestinal drug transporters can be obtained by modulating transporter expression in intact porcine tissue explants via the ultrasound-mediated delivery of small interfering RNAs and that the interaction profiles can be classified via a random forest model trained on the drug-transporter relationships. For 24 drugs with well-characterized drug-transporter interactions, the model achieved 100% concordance. For 28 clinical drugs and 22 investigational drugs, the model identified 58 unknown drug-transporter interactions, 7 of which (out of 8 tested) corresponded to drug-pharmacokinetic measurements in mice. We also validated the model's predictions for interactions between doxycycline and four drugs (warfarin, tacrolimus, digoxin and levetiracetam) through an ex vivo perfusion assay and the analysis of pharmacologic data from patients. Screening drugs for their interactions with the intestinal transportome via tissue explants and machine learning may help to expedite drug development and the evaluation of drug safety.

Computationally guided high-throughput design of self-assembling drug nanoparticles.

Nanoformulations of therapeutic drugs are transforming our ability to effectively deliver and treat a myriad of conditions. Often, however, they are complex to produce and exhibit low drug loading, except for nanoparticles formed via co-assembly of drugs and small molecular dyes, which display drug-loading capacities of up to 95%. There is currently no understanding of which of the millions of small-molecule combinations can result in the formation of these nanoparticles. Here we report the integration of machine learning with high-throughput experimentation to enable the rapid and large-scale identification of such nanoformulations. We identified 100 self-assembling drug nanoparticles from 2.1 million pairings, each including one of 788 candidate drugs and one of 2,686 approved excipients. We further characterized two nanoparticles, sorafenib-glycyrrhizin and terbinafine-taurocholic acid both ex vivo and in vivo. We anticipate that our platform can accelerate the development of safer and more efficacious nanoformulations with high drug-loading capacities for a wide range of therapeutics.

Robotically handled whole-tissue culture system for the screening of oral drug formulations.

Monolayers of cancer-derived cell lines are widely used in the modelling of the gastrointestinal (GI) absorption of drugs and in oral drug development. However, they do not generally predict drug absorption in vivo. Here, we report a robotically handled system that uses large porcine GI tissue explants that are functionally maintained for an extended period in culture for the high-throughput interrogation (several thousand samples per day) of whole segments of the GI tract. The automated culture system provided higher predictability of drug absorption in the human GI tract than a Caco-2 Transwell system (Spearman's correlation coefficients of 0.906 and 0.302, respectively). By using the culture system to analyse the intestinal absorption of 2,930 formulations of the peptide drug oxytocin, we discovered an absorption enhancer that resulted in a 11.3-fold increase in the oral bioavailability of oxytocin in pigs in the absence of cellular disruption of the intestinal tissue. The robotically handled whole-tissue culture system should help advance the development of oral drug formulations and might also be useful for drug screening applications.

Cell-geometry-dependent changes in plasma membrane order direct stem cell signalling and fate.

Cell size and shape affect cellular processes such as cell survival, growth and differentiation1-4, thus establishing cell geometry as a fundamental regulator of cell physiology. The contributions of the cytoskeleton, specifically actomyosin tension, to these effects have been described, but the exact biophysical mechanisms that translate changes in cell geometry to changes in cell behaviour remain mostly unresolved. Using a variety of innovative materials techniques, we demonstrate that the nanostructure and lipid assembly within the cell plasma membrane are regulated by cell geometry in a ligand-independent manner. These biophysical changes trigger signalling events involving the serine/threonine kinase Akt/protein kinase B (PKB) that direct cell-geometry-dependent mesenchymal stem cell differentiation. Our study defines a central regulatory role by plasma membrane ordered lipid raft microdomains in modulating stem cell differentiation with potential translational applications.

Preventing tissue fibrosis by local biomaterials interfacing of specific cryptic extracellular matrix information.

Matrix metalloproteinases (MMPs) contribute to the breakdown of tissue structures such as the basement membrane, promoting tissue fibrosis. Here we developed an electrospun membrane biofunctionalized with a fragment of the laminin ß1-chain to modulate the expression of MMP2 in this context. We demonstrate that interfacing of the ß1-fragment with the mesothelium of the peritoneal membrane via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor ß1 and rescues tissue integrity ex vivo and in vivo in a mouse model of peritoneal fibrosis. Importantly, our data demonstrate that the membrane inhibits MMP2 expression. Changes in the expression of epithelial-to-mesenchymal transition (EMT)-related molecules further point towards a contribution of the modulation of EMT. Biomaterial-based presentation of regulatory basement membrane signals directly addresses limitations of current therapeutic approaches by enabling a localized and specific method to counteract MMP2 release applicable to a broad range of therapeutic targets.

Flexible piezoelectric devices for gastrointestinal motility sensing.

Improvements in ingestible electronics with the capacity to sense physiological and pathophysiological states have transformed the standard of care for patients. Yet, despite advances in device development, significant risks associated with solid, non-flexible gastrointestinal transiting systems remain. Here, we report the design and use of an ingestible, flexible piezoelectric device that senses mechanical deformation within the gastric cavity. We demonstrate the capabilities of the sensor in both in vitro and ex vivo simulated gastric models, quantify its key behaviours in the gastrointestinal tract using computational modelling and validate its functionality in awake and ambulating swine. Our proof-of-concept device may lead to the development of ingestible piezoelectric devices that might safely sense mechanical variations and harvest mechanical energy inside the gastrointestinal tract for the diagnosis and treatment of motility disorders, as well as for monitoring ingestion in bariatric applications.

Sparse feature selection methods identify unexpected global cellular response to strontium-containing materials.

Despite the increasing sophistication of biomaterials design and functional characterization studies, little is known regarding cells' global response to biomaterials. Here, we combined nontargeted holistic biological and physical science techniques to evaluate how simple strontium ion incorporation within the well-described biomaterial 45S5 bioactive glass (BG) influences the global response of human mesenchymal stem cells. Our objective analyses of whole gene-expression profiles, confirmed by standard molecular biology techniques, revealed that strontium-substituted BG up-regulated the isoprenoid pathway, suggesting an influence on both sterol metabolite synthesis and protein prenylation processes. This up-regulation was accompanied by increases in cellular and membrane cholesterol and lipid raft contents as determined by Raman spectroscopy mapping and total internal reflection fluorescence microscopy analyses and by an increase in cellular content of phosphorylated myosin II light chain. Our unexpected findings of this strong metabolic pathway regulation as a response to biomaterial composition highlight the benefits of discovery-driven nonreductionist approaches to gain a deeper understanding of global cell-material interactions and suggest alternative research routes for evaluating biomaterials to improve their design.

High resolution Raman spectroscopy mapping of stem cell micropatterns.

We report on the use of high resolution Raman spectroscopy mapping combined with a micro-engineered stem cell platform. This technique obtains quantitative information about the concentration of individual intracellular molecules such as proteins, lipids, and other metabolites, while tightly controlling cell shape and adhesion. This new quantitative analysis will prove highly relevant for in vitro drug screening applications and regenerative medicine.

Comparative assessment of the stability of nonfouling poly(2-methyl-2-oxazoline) and poly(ethylene glycol) surface films: an in vitro cell culture study.

Poly(ethylene glycol) (PEG) has been the most frequently reported and commercially used polymer for surface coatings to convey nonfouling properties. PEGylated surfaces are known to exhibit limited chemical stability, particularly due to oxidative degradation, which limits long-term applications. In view of excellent anti-adhesive properties in the brush conformation and resistance to oxidative degradation, poly(2-methyl-2-oxazoline) (PMOXA) has been proposed recently as an alternative to PEG. In this study, the authors systematically compare the (bio)chemical stability of PEG- and PMOXA-based polymer brush monolayer thin films when exposed to cultures of human umbilical vein endothelial cells (HUVECs) and human foreskin fibroblasts (HFFs). To this end, the authors used cell-adhesive protein micropatterns in a background of the nonfouling PEG and PMOXA brushes, respectively, and monitored the outgrowth of HUVECs and HFFs for up to 21 days and 1.5 months. Our results demonstrate that cellular micropatterns spaced by PMOXA brushes are significantly more stable under serum containing cell culture conditions in terms of confinement of cells to the adhesive patterns, when compared to corresponding micropatterns generated by PEG brushes. Moreover, homogeneous PEG and PMOXA-based brush monolayers on Nb2O5 surfaces were investigated after immersion in endothelial cell medium using ellipsometry and x-ray photoelectron spectroscopy.

Extracellular vesicles derived from preosteoblasts influence embryonic stem cell differentiation.

Embryonic stem cells (ESCs) can differentiate into all cell types of the body and, therefore, hold tremendous promise for cell-based regenerative medicine therapies. One significant challenge that should be addressed before using ESCs in the clinic is to improve methods of efficiently and effectively directing the differentiation of this heterogeneous cell population. The work presented here examines the potential of harnessing naturally derived extracellular vesicles to deliver genetic material from mature cells to undifferentiated ESCs for the purpose of manipulating stem cell fate. Vesicles were isolated from preosteoblast cells and were found to be ∼170 nm in diameter and to express the CD40 surface marker. Multiple interactions were visualized between vesicles and ESCs using confocal microscopy, and no significant difference in cell viability was noted. Incubation with vesicles caused significant changes in ESC gene expression, including persistence of pluripotent gene levels as well as increased neurectoderm differentiation. Genetic cargo of the vesicles as well as the cells from which they were derived were examined using a small microRNA (miRNA) gene array. Interestingly, ∼20% of the examined miRNAs were increased more than twofold in the vesicles compared with preosteoblast cells. Together, these results suggest that extracellular vesicles may be utilized as a novel method of directing stem cell differentiation. Future work examining methods for controlled delivery of vesicles may improve the clinical potential of these physiological liposomes for therapeutic applications.

Correlative Light-Ion Microscopy for biological applications.

Here we report a new technique, Correlative Light-Ion Microscopy (CLIM), to correlate SEM-like micrographs with fluorescence images. This technique presents significant advantages over conventional methods in enabling topographical and biochemical information to be correlated with nanoscale resolution without destroying the fluorescence signal. We demonstrate the utility of CLIM for a variety of investigations of cell substrate interactions validating its potential to become a routine procedure in biomedical research.

Formation and characterization of DNA-polymer-condensates based on poly(2-methyl-2-oxazoline) grafted poly(L-lysine) for non-viral delivery of therapeutic DNA.

Successful gene delivery systems deliver DNA in a controlled manner combined with minimal toxicity and high transfection efficiency. Here we investigated 15 different copolymers of poly(l-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-g-PMOXA) of variable grafting densities and PMOXA molecular weights for their potential to complex and deliver plasmid DNA. PLL(20)g(7)PMOXA(4) formed at N/P charge ratio of 3.125 was found to transfect 9 ± 1.6% of COS-7 cells without impairment of cell viability. Furthermore these PLL-g-PMOXA-DNA condensates were internalized 2 h after transfection and localized in the perinuclear region after 6 h. The condensates displayed a hydrodynamic diameter of ∼100 nm and were found to be stable in serum and after 70 °C heat treatment, moreover the condensates protected DNA against DNase-I digestion. The findings suggest that DNA-PMOXA-g-PLL condensate formation for efficient DNA-delivery strongly depends on PMOXA grafting density and molecular weight showing an optimum at low grafting density between 7 and 14% and medium N/P charge ratio (3.125-6.25). Thus, PLL(20)g(7)PMOXA(4) copolymers might be promising as alternative to PLL-g-PEG-DNA condensates for delivery of therapeutic DNA.