(H-301) Viral Drug Screening Platform on a Vessel-on-a-chip Using Virus Mimicking Particles

Introduction::

A system platform of viral inspection is necessary due to increase of interest in viral diseases and drug screening after COVID-19. Development of viral medicine through rapid and efficient methods is also required. A 3D organ-on-a-chip technology may be chosen to recapitulate the infection of cells to particular viruses. Especially vessels are primarily exposed to invading viruses in many cases. A virus mimicking particle, Poly(I:C) is used and through imaging results of inflammatory markers such as ICAM-1 on a 3D vascular network the optimal concentration for candidate drugs are suggested. Furthermore, the inflammation reduction effect of the tested drug at each optimal concentration can be compared to suggest the most effective.

Materials and Methods::

A 3D microfluidic device with 2 media channels and a gel channel was made through softliftography with poly-dimethyl-siloxane (PDMS). Human umbilical vein endothelial cells (HUVEC)s were cultured in endothelial cell growth medium-2 (EGM-2) supplemented with 1% penicillin solution. After Polydopamine (PDA) coating, the HUVECs were seeded with fibrin gel in the gel channel and were cultured up to 4 days to form a vascular network. Interstitial flow mimicry was induced using pre-cut syringe tips to help grow the microvasculature. Several bromodomain4 inhibitors of NF-κB signaling and anti-inflammatory drugs PFI-1 (Pfizer), JQ-1 (MedChemExpress), Apabetalone (Selleckchem) were treated in the media in several concentrations for 18h to reduce inflammation response and vessel damage. To induce viral inflammation response, a respiratory virus mimicking particle polyinosinic-polycytidylic acid (Poly(I:C)) (InvivoGen) of 5μg/ml mixed with media was delivered for 8h. ICAM-1, VE-Cadherin, Rhodamine-Phalloidin, and DAPI were dyed with proper antibodies after fixing the cells and permeabilizing their membranes. For each ROI (Region of Interest) a fluorescence image was obtained, and the ICAM-1 expression image was output separately to analyze the mean intensity via imageJ.

Results, Conclusions, and Discussions::

Results and Discussions: From examination of inflammatory response (ICAM-1 mean intensity) according to Poly(I:C) concentration, the highest ICAM-1 expression intensity was shown at a concentration of 5μg/ml. Higher concentrations than 5μg/ml resulted in greater cytotoxicity and a tendency for ICAM-1 expression to decline, not due to reduction in inflammation but a dominant effect of cell death, similarly to actual viruses in high doses. Investigation of ICAM-1 mean intensity on PFI-1 concentration for the specified Poly(I:C) concentration shows that the ICAM-1 intensity seems to decrease at the 25, 50, 100nM case compared to the 0nM case. However examining the vascular wall status with VE-Cadherin, it appears that a high degree of cell death occurred at 100nM due to the inherent cytotoxicity of PFI-1 while at the 25nM and 50nM condition the vascular wall remains most intact. Combining, the effective dose of PFI-1 can be suggested to be 25nM~50nM in this platform. In the case of JQ-1, even in high concentrations it seemed to be relatively less cytotoxic than PFI-1. However for the low concentration cases of 20nM JQ-1 and 12.5nM PFI-1, both drugs seemed to be cytotoxic.

Conclusions: The virus mimicking particle Poly(I:C) can be used for rapid drug screening due to its easy treatment and accessibility (BSL2). Poly(I:C) is able to produce inflammation in the vascular network structure and the chosen drugs showed anti-inflammatory effects to it. The drug dose that shows the highest efficacy in preventing vascular damage or inflammation can be easily compared for each drug in this platform.

Acknowledgements (Optional): : This work was supported by the National Research Foundation of Korea (2020R1A2C1100471) and the Brain Korea 21 Plus project.

References (Optional): :

[1] Primiceri, Elisabetta, et al. "Cell chips as new tools for cell biology–results, perspectives and opportunities." Lab on a Chip 13.19 (2013): 3789-3802.

 [2] Salmon, Hugo. Mobile magnetic microrobots control and study in microfluidic environment: New tools for biomedical applications. Diss. Paris 11, 2014.

 [3] HinTang, Justin Jit, et al. "JAK/STAT signaling in hepatocellular carcinoma." Hepatic oncology 7.1 (2020): HEP18.

[4] Benam, KambezH., et al. "Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro." Nature methods 13.2 (2016): 151-157.

[5] Tian, Mingfu, et al. "HIF-1α promotes SARS-CoV-2 infection and aggravates inflammatory responses to COVID-19." Signal Transduction and Targeted Therapy 6.1 (2021): 1-13.

[6] Wang, Xiang, ZhaomiaoLiu, and Yan Pang. "Concentration gradient generation methods based on microfluidic systems." RSC advances 7.48 (2017): 29966-29984.

[7] Kim, Seunggyu, et al. "Microfluidic-based observation of local bacterial density under antimicrobial concentration gradient for rapid antibiotic susceptibility testing." Biomicrofluidics13.1 (2019): 014108.

[8] Shahid, Sidra, et al. "Small Molecule BRD4 Inhibitors Apabetalone and JQ1 Rescues Endothelial Cells Dysfunction, Protects Monolayer Integrity and Reduces Midkine Expression." Molecules 27.21 (2022): 7453.