Introduction

1.1 Imaging application and limitation

1.1.1 Reporter system

The discovery of reporters such as green fluorescent protein (GFP) and the development of their applications opens a new era on biomedical imaging¹. By genetic modification, the fluorescent protein driven by specific promoters could address the cell fate². Furthermore, it could address promoter associating protein by using luminance-based promoter assay³. As the development progressing, Cre-loxp recombination system can provide more detail for the development and fate mapping^4,5. The advantage of cells carrying reporters can be tracked and monitored after cell therapy and get the knowledge of the location of the cells, and the interaction of cells. Moreover, the known

fluorescent molecules mediated by specific transporter can be applied for

6. The fluorescent and luminescent imaging modalities have high

sensitivity; however, the limitation of penetration depth is because of the nature of optics. Currently, more efforts have been focused on the infrared fluorescent dyes and other imaging modalities that have much deeper tissue visualization and higher resolution.

1.1.2 Cell tracking imaging application and limitation

Molecular imaging opened a new page for the explosion of intracellular or intercellular interaction in vivo. There are two major methods to address cell tracking: one is treating cells with dyes/contrasts which could be detected by imaging modalities, the other is manipulating the genome of cells to carry reporter genes^7,8. Short term cell tracking can be achieved by labeling fluorescent dyes, contrasts, and/or nanoparticles which represent magnetic resonance (MR)/computed tomography (CT)/positron emission tomography (PET) signals. However, there is a dilutional effect due to cell divisions and its metabolism^9,10. Moreover, inappropriate interpretation may be guided because dyes/contrasts/nanoparticles are intake by macrophages or other cells once labeled cells are dead¹¹. Thus, the better method to address long-term cell tracking is the genetic modification with reporter genes, such as traditional GFP, red fluorescent protein (RFP), and bioluminescence imaging (BLI)^12,13; herpes simplex virus type 1 thymidine kinase reporter, norepinephrine transporter, and dopamine transporter in PET^14,15; transferrin receptor, organic-anion-transporting polypeptide 1B1 (OATP1B1), and organic-anion-transporting polypeptide 1B3 (OATP1B3) in magnetic resonance imaging^16–18. Although Fluorescence and BLI have advantages in the sensitivity of the detection as compared with other imaging modality, the penetration depth is restricted due to the nature of optics and the copy number of reporter genes. Furthermore, the

manipulation of the genome may change the characteristics of cells such as uncontrollable proliferation, apoptosis, or dysfunction. Currently, the application of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 to genome engineering achieving more specific genomic editing decreases the risk by previous methods to insert reporter genes¹⁹. At present, the infrared fluorescent dyes have become the main development for the investigation in molecular imaging because of its deeper tissue visualization. Indocyanine green (ICG), one of the best candidates, has been applied widely in clinical research^20–23.

1.2 Indocyanine green (ICG)

Indocyanine green is the Food and Drug Administration (FDA) approved the near-infrared (NIR) compound. It has been widely made use of clinical or biomedical imaging applications since ICG has remarkable tissue penetration and the low background interference at the excitation/emission of 760nm/800nm compared with traditional NIR fluorescent dyes such as Cy5.5 which has the excitation/emission of 675nm/694nm^20,24–28. ICG has more extensive applications in cardiac output measurements²⁹, ophthalmic angiography³⁰, rheumatoid arthritis³¹, hepatic function test³², intraoperative angiography³³, and tumor detection and treatments³⁴. ICG has a fast binding rate with plasma proteins. Subsequently, the complexes of ICG combining with plasma protein enter into cells through transporters such as OATP1B3 and sodium

taurocholate cotransporting polypeptide (NTCP). Then, the efflux of ICG is major through multidrug resistance protein 3 (MDR3)³⁵ and minor through multidrug resistance protein 1 (MDR1)³⁶. Eventually, it excretes quickly through the liver into bile juice^37,38. The median lethal dose (LD50) of ICG is 50-80mg/kg in animals after intravenous injection³⁹. In the human liver function test, the dosage of ICG is 0.5mg/kg which is extremely low and far from 50-80mg/kg. Thus, the low toxicity of ICG makes

it the only one fluorescent dye served in clinical application and diagnostic medication

40. According to the benefit of ICG, establishing an imaging modality through

combining ICG and transporters such as OATP1B3 and NTCP as reporter genes will make in vitro and in vivo biomedical application more reliable and more extensive.

1.3 Membrane transporters

1.3.1 Survey

Membrane transporters respond to the exchanges of nutrients, metabolites, drugs, and toxic substances to decide the fate of these substances⁴¹. There are many classes of membrane transporters such as organic anion transporting polypeptide (OATP) family, P-glycoprotein (P-gp), organic cation transporters (OCTs), organic anion transporters (OATs), multidrug resistance protein (MRP) family, and the multidrug and toxin extrusion transporter (MATE) family⁴¹. Here, we focus on the transporters that could deliver gadolinium-ethoxybenzyl-diethylenetriaminepentaacetic acid (Gd-EOB-DTPA,

Primovist) and ICG for our study.

1.3.2 Organic-anion-transporting polypeptide 1B3 (OATP1B3)

OATP1B3 belongs to OATPs family, is expressed in the hepatocytes. It is a transmembrane glycoprotein responsible to transport bilirubin⁴², nutrients, drugs⁴³, certain MRI agents such as Primovist^17,18,44, and ICG^18,38. Because there are many components deliver into cells mediated by OATPs, it is very important to know the DDI through OATP1B3.

1.3.3 Sodium taurocholate cotransporting polypeptide (NTCP)

NTCP is sodium-dependent transporter and it belongs to the solute carrier family of transporters 10 (SLC10). It transports bile salts, sulfated compounds, thyroid hormones, drugs, and toxins^45,46. NTCP and OATPs are responsible for the intake of bile salts from plasma to the liver⁴⁷. Moreover, it can transport ICG into cells³⁸. Recent studies have revealed a new role of NTCP as an entry receptor of the hepatitis B virus (HBV) and hepatitis D virus (HDV)⁴⁸. Many inhibitors have developed for preventing HBV infection by blocking NTCP such as cyclosporin A, Myrcludex B, and oxysterols^48–50. Therefore, screening and verifying NTCP inhibitors could contribute to the prevention and therapy for hepatitis.

1.3.4 Apical sodium-dependent bile acid cotransporter (ASBT)

ASBT, mainly expressing in the intestine, kidney, cholangiocytes, and gallbladder,

belongs to the SLC10 family and responds for the transportation of bile salts, such as conjugated, unconjugated bile acids, and taurocholic acid⁵¹. Dysfunction of ASBT may cause more bile acids in the colon and leads to diarrhea, gallstone disease, hypertriglyceridemia, or even colon cancer⁵². Hypercholesterolemia could be treatable through using the inhibitors of bile acids transporters such as ASBT, NTCP, and so on⁵³. That’s a reason why investigating the drug-drug interaction of ASBT is valuable.

Currently, the known inhibitors of ASBT are dihydropyridine calcium channel blockers statins and hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors (statins)⁵⁴

1.4 Drug screening platform

The metabolism of drugs is an essential issue to discover for health. Many drug absorption is mediated by OATP1B1 and OATP1B3 mainly expressed in liver⁴³. It makes OATP1B1 and OATP1B3 as pioneers for the screening of drug candidates to select molecules that could serve as potential drugs. The fluorescence-based method is advantageous for investigating transport because of its convenience, safety, and optical characters⁵⁵. Screening molecules for fluorescence-based method to address OATP1B3 are Fluo-3, fluorescein-methotrexate (FMTX), and 8-fluorescein-cAMP (8-FcA)^56,57. Although three fluorescent molecules can be used for drug screening, they have some disadvantages, such as Fluo-3 depending on Ca²⁺, the toxicity of FMTX and 8-FcA.

Moreover, as mentioned before, NTCP is the entry of HBV and HDV virus⁴⁸. Recently, ICG is reported that it can be transported into cells mediated OATP1B3 and NTCP³⁸. We testified that whether ICG serves as a screening molecule for a fluorescence-based method to address OATP1B3 and NTCP.