The Accumulation and Effects of Liposomal Doxorubicin in Tissues Treated by Radiofrequency Ablation and Irreversible Electroporation in Liver: In Vivo Experimental Study on Porcine Models

Abstract

Purpose

To compare the accumulation and effect of liposomal doxorubicin in liver tissue treated by radiofrequency ablation (RFA) and irreversible electroporation (IRE) in in vivo porcine models.

Materials and Methods

Sixteen RFA and 16 IRE procedures were performed in healthy liver of two groups of three pigs. Multi-tined RFA parameters included: 100 W, target temperature 105°C for 7 min. 100 IRE pulses were delivered using two monopolar electrodes at 2250 V, 1 Hz, for 100 µsec. For each group, two pigs received 50 mg liposomal doxorubicin (0.5 mg/kg) as a drip infusion during ablation procedure, with one pig serving as control. Samples were harvested from the central and peripheral zones of the ablation at 24 and 72 h. Immunohistochemical analysis to evaluate the degree of cellular stress, DNA damage, and degree of apoptosis was performed. These and the ablation sizes were compared. Doxorubicin concentrations were also analyzed using fluorescence photometry of homogenized tissue.

Results

RFA treatment zones created with concomitant administration of doxorubicin at 24 h were significantly larger than controls (2.5 ± 0.3 cm vs. 2.2 ± 0.2 cm; p = 0.04). By contrast, IRE treatment zones were negatively influenced by chemotherapy (2.2 ± 0.4 cm vs. 2.6 ± 0.4 cm; p = 0.05). At 24 h, doxorubicin concentrations in peripheral and central zones of RFA were significantly increased in comparison with untreated parenchyma (0.431 ± 0.078 µg/g and 0.314 ± 0.055 µg/g vs. 0.18 ± 0.012 µg/g; p < 0.05). Doxorubicin concentrations in IRE zones were not significantly different from untreated liver (0.191 ± 0.049 µg/g and 0.210 ± 0.049 µg/g vs. 0.18 ± 0.012 µg/g).

Conclusions

Whereas there is an increased accumulation of periprocedural doxorubicin and an associated increase in ablation zone following RFA, a contrary effect is noted with IRE. These discrepant findings suggest that different mechanisms and synergies will need to be considered in order to select optimal adjuvants for different classes of ablation devices.

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Acknowledgments

This study was supported by Ministry of Health of the Czech Republic, Grant No. 15-32484a, and supported by funds from the Faculty of Medicine MU to junior researcher Tomáš Andrašina.

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Correspondence to Tomáš Jůza.

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Author Nahum S. Goldberg is a consultant to Angiodynamics, Inc. and Cosman Medical Inc.

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Appendices

Appendix 1: Supplementary Animal Information

The pigs were housed in stone hutches under controlled environmental conditions (20–22 °C room temperature, 50–60% relative humidity, 12 h light and 12 h dark cycle). The whole procedure was carried out under general anesthesia with deep muscle relaxation, and the animals were intubated and artificially ventilated. The following combinations of substances were applied for general anesthesia. Premedication: i.m. Tiletamine 2 mg/kg + zolazepam 2 mg/kg (Zoletil 100, Virbac) + ketamine 2 mg/kg (Narketan, Vetoquinol) + xylazine 2 mg/kg (Sedazine, Fort Dodge) i.v. cannulation + Buprenorphine (Temgesic, Schering-Plow) 0.01 mg/kg i.m. Induction: Norofol (propofol, Fresenius) to effect (1–2 mg/kg) i.v. Lateral ear vein was used for catheterization. Conduction: Inhalation anesthesia of O2–air mixtures- Isoflurane (Aerrane, Baxter) Monitoring: HR, RR, SpO2, ETCO2. Atracrium (1.5 mg/kg, Tracurium, GlaxoSmithKline, Italy) was used for muscle relaxation. Each animal underwent median laparotomy whereby the dominant surface of liver was exposed. After surgery, the animals were awakened and pain was efficiently controlled by meloxicam (0.2 mg/kg, Metacam 5 mg/mL, Boehringer Ingelheim, Germany). The animals were euthanized 24 and 72 h after surgery (Thiopental, T61 Intervet International GmbH). In each pig, an en bloc specimen of liver was excised.

Appendix 2: Supplementary Laboratory Information

Briefly, specified tissues were harvested, weighed, and homogenized with a MagNA Lyser (Roche, Basel, CH) in acid alcohol (0.3 N hydrochloric acid, 70% ethyl alcohol). Doxorubicin was extracted for 24 h at 5 °C. The extracted doxorubicin supernatant from all tissue homogenate samples was quantified with fluorescence photometry using an excitation wavelength of 485 nm while measuring the intensity of the emission at 535–595 nm. The obtained concentrations were plotted on a standard curve of liposomal doxorubicin serially diluted likewise in acid alcohol. Considering fluorescence photometry analysis, we note that the absolute values of concentration of doxorubicin in liver tissue homogenate were influenced by autofluorescent properties of physiologically presented molecules such as cytochromes in liver tissue. Nevertheless, these natural backgrounds could be statistically distinguished from tissue treated with liposomal doxorubicin.

Appendix 3: Supplementary Laboratory Information

Representative tissue samples of each treated area from central and peripheral zone harvested after 24 h were embedded in OCT compound (Leica), snap-frozen, and stored at −20 °C. The “central zone” was defined as the 1 cm most central part of the treated area, and the “peripheral zone” was defined as the tissue 2–3 mm nearest to “apparently normal surrounding liver parenchyma.” Tissues were sectioned on a Leica Cryostat (7 µm) and placed on treated slides for fluorescent analysis. Sections were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) and mounted with 80% glycerol mounting medium for fluorescent microscopic analysis using TissueFax (TissueGnostics). A two-step immunohistochemical protocol was performed: primary antibodies for specific antigens included HSP70 (Assay Designs, Ann Arbor, Mich) to assess for cellular stress; γH2AX (Cell Signaling Technologies, Danvers, Mass) as a marker of DNA damage; and cleaved caspase-3 (Cell Signaling Technologies) as a marker of apoptosis and secondary staining with fluorochrome. Images of whole sections were automatically acquired with 10x/0.45 objective for two channels (405/450 nm excitation/emission, 488/535 nm ex/em). Quantitative analysis was performed by measuring thickness of the rim of staining, from each central and peripheral ablation zone, and four random fields were analyzed.

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Andrašina, T., Jaroš, J., Jůza, T. et al. The Accumulation and Effects of Liposomal Doxorubicin in Tissues Treated by Radiofrequency Ablation and Irreversible Electroporation in Liver: In Vivo Experimental Study on Porcine Models. Cardiovasc Intervent Radiol 42, 751–762 (2019). https://doi.org/10.1007/s00270-019-02175-z

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Keywords

  • Liposomal doxorubicin
  • Radiofrequency ablation
  • Irreversible electroporation