Embedded Core-Shell 3D Printing (eCS3DP) with Low-Viscosity Polysiloxanes.

Flexible core-shell 3D structures are essential for the development of soft sensors and actuators. Despite recent advancements in 3D printing, the fabrication of flexible 3D objects with internal architectures (such as channels and void spaces) remains challenging with liquid precursors due to the difficulty to maintain the printed structures. The difficulty of such fabrication is prominent especially when low-viscosity polysiloxane resins are used. This study presents a unique approach to applying direct ink writing (DIW) 3D printing in a three-phase system to overcome this limitation. We performed core-shell 3D printing using a low-viscosity commercial polysiloxane resin (Ecoflex 10) as shell inks combined with a coaxially extruded core liquid (Pluronic F127) in Bingham plastic microparticulate gels (ethanol gel). In the process termed embedded core-shell 3D printing (eCS3DP), we highlighted the dependence of the rheological characteristics of the three fluids on the stability of the printed core-shell filament. With the core liquid with a sufficiently high concentration of Pluronic F127 (30 w/w%; σy = 158.5 Pa), the interfacial instability between the shell liquid and core liquid was suppressed; the removal of the core liquid permitted the fabrication of perfusable channels. We identified the printing conditions to ensure lateral attachments of printed core-shell filaments. Interestingly, judicious selection of the rheological properties and flow rates of three phases allowed the formation of droplets consisting of core liquids distributed along the printed filaments. eCS3DP offers a simple route to fabricate 3D structures of a soft elastomeric matrix with embedded channels and should serve as a useful tool for DIW-based fabrication of flexible wearable devices and soft robotic components.

ECM-based microfluidic gradient generator for tunable surface environment by interstitial flow.

We present an extracellular matrix (ECM)-based gradient generator that provides a culture surface with continuous chemical concentration gradients created by interstitial flow. The gelatin-based microchannels harboring gradient generators and in-channel micromixers were rapidly fabricated by sacrificial molding of a 3D-printed water-soluble sacrificial mold. When fluorescent dye solutions were introduced into the channel, the micromixers enhanced mixing of two solutions joined at the junction. Moreover, the concentration gradients generated in the channel diffused to the culture surface of the device through the interstitial space facilitated by the porous nature of the ECM. To check the functionality of the gradient generator for investigating cellular responses to chemical factors, we demonstrated that human umbilical vein endothelial cells cultured on the surface shrunk in response to the concentration gradient of histamine generated by interstitial flow from the microchannel. We believe that our device could be useful for the basic biological study of the cellular response to chemical stimuli and for the in vitro platform in drug testing.

ECM-based microchannel for culturing in vitro vascular tissues with simultaneous perfusion and stretch.

We present an extracellular matrix (ECM)-based stretchable microfluidic system for culturing in vitro three-dimensional (3D) vascular tissues, which mimics in vivo blood vessels. Human umbilical vein endothelial cells (HUVECs) can be cultured under perfusion and stretch simultaneously with real-time imaging by our proposed system. Our ECM (transglutaminase (TG) cross-linked gelatin)-based microchannel was fabricated by dissolving water-soluble sacrificial polyvinyl alcohol (PVA) molds printed with a 3D printer. Flows in the microchannel were analyzed under perfusion and stretch. We demonstrated simultaneous perfusion and stretch of TG gelatin-based microchannels culturing HUVECs. We suggest that our TG gelatin-based stretchable microfluidic system proves to be a useful tool for understanding the mechanisms of vascular tissue formation and mechanotransduction.

Synthesis and biological evaluation of 6-phenylpurine linked hydroxamates as novel histone deacetylase inhibitors.

A series of 6-phenylpurine based hydroxamates have been designed, synthesized and evaluated. Compound 3b and its analogs are potent histone deacetylase (HDAC) but weak PI3K/mTOR inhibitors. These compounds demonstrated broad anti-cancer activities against 38 cancer cell lines with leukemia, lymphoma, and the majority of liver cancer cell lines exhibiting the most sensitivity towards these compounds. Compound 3b demonstrated modulation of HDAC targets in vitro in a dose-dependent manner. It has good in vitro ADME profile that translated into a greatly improved pharmacokinetic profile. 3b also demonstrated modulation of HDACs in tumors in a PC-3 xenograft model. It was further evaluated in combination therapies in vitro. It exhibited additive or synergistic growth inhibition effect in HepG2 cells when combined with a number of approved drugs such as sorafenib, sunitinib, and erlotinib. Hence, 3b has the potential to be combined with the above to treat advanced liver cancer. As such, current data warrant further evaluation, optimization, and subsequent in vivo validation of the potential combination therapies.

Dual Sacrificial Molding: Fabricating 3D Microchannels with Overhang and Helical Features.

Fused deposition modeling (FDM) has become an indispensable tool for 3D printing of molds used for sacrificial molding to fabricate microfluidic devices. The freedom of design of a mold is, however, restricted to the capabilities of the 3D printer and associated materials. Although FDM has been used to create a sacrificial mold made with polyvinyl alcohol (PVA) to produce 3D microchannels, microchannels with free-hanging geometries are still difficult to achieve. Herein, dual sacrificial molding was devised to fabricate microchannels with overhang or helical features in PDMS using two complementary materials. The method uses an FDM 3D printer equipped with two extruders and filaments made of high- impact polystyrene (HIPS) and PVA. HIPS was initially removed in limonene to reveal the PVA mold harboring the design of microchannels. The PVA mold was embedded in PDMS and subsequently removed in water to create microchannels with 3D geometries such as dual helices and multilayer pyramidal networks. The complementary pairing of the HIPS and PVA filaments during printing facilitated the support of suspended features of the PVA mold. The PVA mold was robust and retained the original design after the exposure to limonene. The resilience of the technique demonstrated here allows us to create microchannels with geometries not attainable with sacrificial molding with a mold printed with a single material.

Design, Synthesis, and Preclinical Evaluation of Fused Pyrimidine-Based Hydroxamates for the Treatment of Hepatocellular Carcinoma.

Class I histone deacetylases (HDACs) are highly expressed and/or upregulated in hepatocellular carcinoma (HCC) and are associated with aggressiveness, spread, and increased mortality of HCC. Activation of phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway was involved in the development of HCC and acquired resistance to sorafenib. A series of purine or 5H-pyrrolo[3,2-d]pyrimidine based hydroxamates were designed and developed as multitarget drugs to modulate both HDACs and the PI3K/Akt/mTOR pathway. Among 39 cell lines screened, the molecules (e.g., 20e, 20f, and 20q) were the most selective against leukemia, lymphoma, and HCC cells; they also demonstrated target modulation in cancer cell lines and in mice bearing MV4-11 and HepG2 tumors. Compound 20f in particular showed significant single agent oral efficacy in hypervascular liver cancer models (e.g., HepG2, HuH-7, and Hep3B) and was well-tolerated. These encouraging results, along with its favorable target profile and tissue distribution, warrant further development of 20f.

Highly stretchable hydrogels for UV curing based high-resolution multimaterial 3D printing.

We report a method to prepare highly stretchable and UV curable hydrogels for high resolution DLP based 3D printing. Hydrogel solutions were prepared by mixing self-developed high-efficiency water-soluble TPO nanoparticles as the photoinitiator with an acrylamide-PEGDA (AP) based hydrogel precursor. The TPO nanoparticles make AP hydrogels UV curable, and thus compatible with the DLP based 3D printing technology for the fabrication of complex hydrogel 3D structures with high-resolution and high-fidelity (up to 7 µm). The AP hydrogel system ensures high stretchability, and the printed hydrogel sample can be stretched by more than 1300%, which is the most stretchable 3D printed hydrogel. The printed stretchable hydrogels show an excellent biocompatibility, which allows us to directly 3D print biostructures and tissues. The great optical clarity of the AP hydrogels offers the possibility of 3D printing contact lenses. More importantly, the AP hydrogels are capable of forming strong interfacial bonding with commercial 3D printing elastomers, which allows us to directly 3D print hydrogel-elastomer hybrid structures such as a flexible electronic board with a conductive hydrogel circuit printed on an elastomer matrix.

Editor's Highlight: Transgenic Zebrafish Reporter Lines as Alternative In Vivo Organ Toxicity Models.

Organ toxicity, particularly liver toxicity, remains one of the major reasons for the termination of drug candidates in the development pipeline as well as withdrawal or restrictions of marketed drugs. A screening-amenable alternative in vivo model such as zebrafish would, therefore, find immediate application in the early prediction of unacceptable organ toxicity. To identify highly upregulated genes as biomarkers of toxic responses in the zebrafish model, a set of well-characterized reference drugs that cause drug-induced liver injury (DILI) in the clinic were applied to zebrafish larvae and adults. Transcriptome microarray analysis was performed on whole larvae or dissected adult livers. Integration of data sets from different drug treatments at different stages identified common upregulated detoxification pathways. Within these were candidate biomarkers which recurred in multiple treatments. We prioritized 4 highly upregulated genes encoding enzymes acting in distinct phases of the drug metabolism pathway. Through promoter isolation and fosmid recombineering, eGFP reporter transgenic zebrafish lines were generated and evaluated for their response to DILI drugs. Three of the 4 generated reporter lines showed a dose and time-dependent induction in endodermal organs to reference drugs and an expanded drug set. In conclusion, through integrated transcriptomics and transgenic approaches, we have developed parallel independent zebrafish in vivo screening platforms able to predict organ toxicities of preclinical drugs.

Humanizing the zebrafish liver shifts drug metabolic profiles and improves pharmacokinetics of CYP3A4 substrates.

Understanding and predicting whether new drug candidates will be safe in the clinic is a critical hurdle in pharmaceutical development, that relies in part on absorption, distribution, metabolism, excretion and toxicology studies in vivo. Zebrafish is a relatively new model system for drug metabolism and toxicity studies, offering whole organism screening coupled with small size and potential for high-throughput screening. Through toxicity and absorption analyses of a number of drugs, we find that zebrafish is generally predictive of drug toxicity, although assay outcomes are influenced by drug lipophilicity which alters drug uptake. In addition, liver microsome assays reveal specific differences in metabolism of compounds between human and zebrafish livers, likely resulting from the divergence of the cytochrome P450 superfamily between species. To reflect human metabolism more accurately, we generated a transgenic "humanized" zebrafish line that expresses the major human phase I detoxifying enzyme, CYP3A4, in the liver. Here, we show that this humanized line shows an elevated metabolism of CYP3A4-specific substrates compared to wild-type zebrafish. The generation of this first described humanized zebrafish liver suggests such approaches can enhance the accuracy of the zebrafish model for toxicity prediction.

Peripherally acting novel lipo-endomorphin-1 peptides in neuropathic pain without producing constipation.

We previously described two novel analogues of endomorphin-1 (Tyr-Pro-Trp-Phe-NH2, 1), modified with an 8-carbon lipoamino acid (C8LAA) with or without replacement of Tyr(1) with 2,6-dimethyltyrosine (Dmt) at the N-terminus of the peptide (compounds 3 and 4, respectively). They were shown to be more stable and permeable, and acted as potent µ-opioid receptor agonists. In this study we report that the C8LAA modification resulted in successful systemic delivery of both analogues. They produced potent dose-dependent pain relief in a chronic constriction injury-rat model of neuropathic pain after intravenous administration with ED50 values obtained at 6.58 (±1.22) µmol/kg for 3 and 6.18 (±1.17) µmol/kg for 4. Using two different rat models of constipation that assess the effects of µ-opioid receptor agonists on stool hydration and gastro-intestinal motility, compound 3 produced insignificant constipation at 16 µmol/kg, whereas morphine elicited significant constipation at 2 µmol/kg. Compound 3 in contrast to morphine, did not attenuate the hypercapnic ventilatory response at 5 µmol/kg, a dose that fully alleviated hindpaw sensitivity at the time of peak effect in CCI-rats. This finding revealed the lack of respiratory depression effect at antinociceptive dose.