Direct sampling from human liver tissue cross sections for electrophoretic analysis of doxorubicin

Abstract

After chemoembolization of the liver with doxorubicin (Dox), this drug and its metabolites are not homogeneously distributed in this organ. The distribution cannot be easily measured making it difficult to assess how the drug performs in different tissue regions. Here we report a technique for sampling tissue cross sections that can analyze the contents of micrometer size regions. The tissue cross sections were from the explanted liver of a hepatocellular carcinoma patient. Samples were directly aspirated from a 5 microm thick tissue cross section into a 50 microm i.d. capillary where the tissue was solubilized with a separation buffer containing sodium dodecyl sulfate. Upon sample dissolution, Dox and natively fluorescent compounds were separated and detected by micellar electrokinetic chromatography with laser-induced fluorescence detection. Sampling reproducibility and recovery were assessed using 10% (w/v) gelatin as tissue mimic. Sampling from gelatin slices containing Dox revealed a relative standard deviation of 13%, which was comparable to that of sampling from solution. Dox recovery was 82% +/- 16% (n = 5). When sampling tumor and nontumor tissue regions, samples could be taken from the same region 100 microm apart. Atomic force microscopy was used to determine that each sample was 8.4 +/- 1.0 pL in volume which made it possible to determine Dox concentrations in the ranges of 0.4-1.3 and 0.3-0.5 microM for the samples taken from tumor and nontumor regions, respectively. The results demonstrated the feasibility of sampling, detection, and quantification of Dox in micrometer size regions, which could be a useful resource for analyzing the Dox concentration and distribution in highly heterogeneous tissues.

Figure 1. Details relevant to direct tissue sampling

The tip of a capillary before (A) and after (B) HF etching. (C) Diagrams illustrating the position of a capillary before (i), at (ii) and after (iii) sampling from a tissue cross-section or gelatin slice.

Figure 2

Direct sampling from a 5 m thick gelatin slice. (A) Bright field image of a gelatin slice after six direct samplings. (B) Electropherograms of spots 2-6; those of spots 3-6 are x and y offset for clarity. Gelatin was sampled at 7.6 kPa for 2 s with an etched-tip capillary. Separations were performed in a 48.5 cm long, 50 μm i.d. fused silica capillary at 400 V/cm in BS-10 buffer. Analytes were excited at 488 nm and fluorescence was detected at 635 ± 27.5 nm.

Figure 2

Direct sampling from a 5 m thick gelatin slice. (A) Bright field image of a gelatin slice after six direct samplings. (B) Electropherograms of spots 2-6; those of spots 3-6 are x and y offset for clarity. Gelatin was sampled at 7.6 kPa for 2 s with an etched-tip capillary. Separations were performed in a 48.5 cm long, 50 μm i.d. fused silica capillary at 400 V/cm in BS-10 buffer. Analytes were excited at 488 nm and fluorescence was detected at 635 ± 27.5 nm.

Figure 3

Tapping-mode AFM images. Eight regions showing samplings from a 5-μm thick tissue cross-section. Tissues were sampled at 7.6 kPa for 2 s with an etched-tip capillary. AFM images were obtained with 1 Hz scan rate.

Figure 4

MEKC-LIF analysis of bulk liver tissue extract. Trace a: tissue extract only. Trace b: tissue extract spiked with Dox. Trace c: tissue extract spiked with doxorubicinol. Peak 2 and 3 are Dox and doxorubicinol, respectively; Peak 1 and 4 are endogenous fluorescence species in the tissue. Traces b and c are y-axis offset by 0.15 and 0.30 A.U. for clarity. Separations were performed in BS-CD buffer in a 39.2 cm long, 50 μm i.d. fused silica capillary at 400 V/cm after 3 s hydrodynamic injection of sample at 7.6 kPa. Analytes were excited at 488 nm. Detection of fluorescein (internal standard) was at 535 ± 17.5 nm (not shown) and red fluorescence (shown here) was at 635 ± 27.5 nm.

Figure 5

Direct tissue sampling from a tissue cross-section and MEKC-LIF analysis. Bright field images of the tissue cross-section before (A), during (B) and after (C) sampling using an etched-tip capillary and the corresponding electropherogram (D). Experimental conditions were the same as in Figure 4 except a 45.7 cm long capillary was used for separations and samples were injected for 2 s.

Figure 6

Direct tissue sampling from the non-tumor and tumor regions of a tissue cross-section and their respective MEKC-LIF analyses. Bright field images of the tissue cross-section after 3 samplings from non-tumor (A) and tumor (B) regions and corresponding electropherograms (C: non-tumor, D: tumor). The electropherograms of spots 1 and 2 are y-axis offset by 0.2 and 0.1 A.U. For clarity, the traces have been x-axis offset to align the internal standard, fluorescein (labeled with “+”). Peaks labeled with “*” and “#” are assigned to Dox and doxorubicinol after mobility corrections with Equations 4 and 5. Experimental conditions were as described in Figure 5.