Quantitative imaging of doxorubicin diffusion and cellular uptake in biomimetic gels with human liver tumor cells

Abstract

Novel tumor-on-a-chip approaches are increasingly used to investigate tumor progression and potential treatment options. To improve the effect of any cancer treatment it is important to have an in depth understanding of drug diffusion, penetration through the tumor extracellular matrix and cellular uptake. In this study, we have developed a miniaturized chip where drug diffusion and cellular uptake in different hydrogel environments can be quantified at high resolution using live imaging. Diffusion of doxorubicin was reduced in a biomimetic hydrogel mimicking tissue properties of cirrhotic liver and early stage hepatocellular carcinoma (373 ± 108 µm²/s) as compared to an agarose gel (501 ± 77 µm²/s, p = 0.019). The diffusion was further lowered to 256 ± 30 µm²/s (p = 0.028) by preparing the biomimetic gel in cell media instead of phosphate buffered saline. The addition of liver tumor cells (Huh7 or HepG2) to the gel, at two different densities, did not significantly influence drug diffusion. Clinically relevant and quantifiable doxorubicin concentration gradients (1-20 µM) were established in the chip within one hour. Intracellular increases in doxorubicin fluorescence correlated with decreasing fluorescence of the DNA-binding stain Hoechst 33342 and based on the quantified intracellular uptake of doxorubicin an apparent cell permeability (9.00 ± 0.74 × 10^-4 µm/s for HepG2) was determined. Finally, the data derived from the in vitro model were applied to a spatio-temporal tissue concentration model to evaluate the potential clinical impact of a cirrhotic extracellular matrix on doxorubicin diffusion and tumor cell uptake.

Keywords: Cellular uptake; Doxorubicin; Drug delivery; Drug diffusion; Liver cancer; Miniaturized chip.

Fig. 1

a The combined drug diffusion and cellular uptake chip and b zoom in on one of five gel reservoirs flanked by donor and acceptor reservoirs. c Top view of a drug diffusion and cellular uptake experiment where a gel (with or without cells) is loaded (1), once the gel has solidified the acceptor and donor solution reservoirs are filled (2), and finally the region of interest (ROI) is imaged as the drug concentration gradient (shown in green) is established (3).

Fig. 2

a Representative examples of DOX fluorescence gradients (here shown in green) formed in LMPA gel (PBS) reservoirs at 10, 20, 30 and 40 min (scale bar; 200 µm). White boxes indicate the selected ROI where DOX fluorescence intensity was recorded and visualized in profiles exemplified in b. The light gray curve corresponds to the 10 min profile and the black curve to the 40 min profile, blue curves show the fitted fluorescence profiles (see Materials and Methods for additional information) at 10 and 20 min in order to determine the apparent DOX diffusion coefficient. The blue dotted lines correspond to the chosen lower and upper boundary of the gels length

Fig. 3

Scatterplot with mean values (horizontal line) for the determined apparent diffusion coefficients (µm²/s) of DOX in low-melting point agarose gels in PBS (LMPA, n = 8), cirrhotic gels containing no cells in PBS (n = 7) or no cells in DMEM cell media (n = 6) or in cirrhotic gels in DMEM cell media mixed with Huh7 or HepG2 cells (all n = 4). Low density (LD) and high density (HD) corresponds to one million and two million tumor cells per mL of cell media, respectively. An unpaired t-test for LMPA gel and Cirrhotic gel resulted in a p = 0.019 (*) and an unpaired t test with Welch's correction for Cirrhotic gel (PBS) and Cirrhotic gel (DMEM) resulted in a p = 0.028 (*)

Fig. 4

a The total recorded number of cells per gel reservoir (mean ± SD, n = 5) and b the distribution of all recorded cells (the y-positions in µm for each single cell along the gel axis is indicated) for the indicated control (CTRL) and experimental replicates (R1-R4) from chips with HepG2 tumor cells in cirrhotic gels. Low density (LD) and high density (HD) corresponds to 1 million and 2 million tumor cells per mL of cell media, respectively

Fig. 5

a Grayscale images from a representative drug diffusion and cellular uptake experiment highlighting the gel zones in the gel reservoirs and DOX fluorescence gradient formation over time. b DOX uptake and corresponding NucBlue reduction in representative cell images and c the corresponding quantifications of fluorescence intensities along a profile line. d Relative cellular fluorescence intensities (from n = 4 gels, mean ± SD) of NucBlue (blue) and DOX (green) in a high density of HepG2 tumor cells (2 million cells / mL of cell media) in cirrhotic gels at 0 and 180 min

Fig. 6

a Illustration of the employed spatio-temporal tissue concentration model used to simulate intracellular (red) and extracellular (black) DOX concentration–time curves b, c at 10 and 100 µm from the nearest blood vessel after an intravenous bolus dose of 50 mg/m². The simulations were generated using apparent DOX diffusion coefficients corresponding to those determined for the cirrhotic gel with a high density of HepG2 cells. In c the apparent cell permeability of DOX determined for a high density of HepG2 cells in a cirrhotic gel was utilized