In vitro study
Vials (25 mg) of commercially available HS microsphere (Merit Medical), 30–60 µm in dry state, were used for all sample series. Clinical grade epirubicin Hydrochloride for injection (Pfizer) and nonionic Ultravist (370 mg I /mL) (Bayer) were used in this study.
Various solvent media composed of different ratio of normal saline (N/S, 0.9% NaCl), hypertonic saline (N/S, 10.0% NaCl) and nonionic contrast Ultravist (370 mg I /mL) were prepared. Commercially available epirubicin (30–50 mg) was then added to these solvent media. The subsequent solutions were then exposed to vials of HS microsphere. Each vail of HS microspheres was agitated briefly and allowed to incubate for 2 hours at room temperature. After completion of drug-loading, the mean diameter and the size distribution were evaluated using a digital microscope (Leica, GER). Supernatant (about 100 uL) was collected, from each sample and analyzed using a validated high-performance liquid chromatography (HPLC) method to quantify the loading efficacy of HS microsphere. Each method was repeated three times. The method which effectively reduced the diameter of microsphere after loading drugs was selected as a new loading method for further study.
In vivo study
This study was approved by the Committee on Animal Affairs in Zhongshan Hospital, Fudan University. All experiments in this study were performed according to the animal care guidelines of our institution.
Adult New Zealand white rabbits (weighting between 2.5 and 3.0 kg) were used for this study. The VX2 tumors were transplanted in the hind limb of a carrier rabbit. When the tumor size reached about 1cm in diameter the tissue was harvested and cut into small cubes (approximately 1mm3) for subsequent tests. The tumor pieces were implanted surgically into the left lobe of liver of each rabbit. The liver tumors with a diameter of 1.0 cm (2 to 3 weeks after implantation) were considered as an idea condition for embolization. Forty rabbits with VX2 liver tumors were divided into four groups: the control group (n = 8), the conventional TACE (cTACE) group (n = 8), the recommend drug-loading method TACE (dTACE) group (n = 12) and the expansion reduce drug-loading method TACE (ndTACE) group (n = 12).
Nothing was performed until euthanasia in the control group. Epirubicin emulsion reconstituted with 0.4 ml lipiodol, 4 mg epirubicin and contrast medium was used for hepatic artery chemoembolization in the cTACE group. For the dTACE group, 20 ml of epirubicin solution at a concentration of 2.5 mg/mL was used for drug-loading in a vial of HS microsphere. After loading completion, 8% of one vial microsphere (4 mg epirubicin) was used for embolization in each animal. For the ndTACE group, HS microspheres were prepared according to the results of the part of the in vitro study, and each animal was embolized with the HS microsphere solution containing 4 mg of epirubicin. For TACE group, the planned final dose of epirubicin for each animal was 4 mg. The epirubicin emulsion injection was terminated in advance, when reflux to gastric vessel occurred and the amount of epirubicin injected was recorded.
Procedure of transarterial chemoembolization
All procedures were performed by a certified interventional radiology physician. Right femoral artery was exposed, separated by surgical incision and a 4-F introducer sheath (Merit Medical) was introduced. A 4-F RH catheter was introduced and then a 1.9-F microcatheter (Merit Medical) was used to perform selective angiography of the celiac trunk. Branche of the left hepatic artery was super selectively catheterized and the tumor feeding artery were also reconfirmed by artery angiography. The treatment was performed in the different groups, taking care to avoid reflux.
Pharmacokinetics study
To measure serum epirubicin concentrations, 2 ml of blood was obtained before and at 10, 30, 60 and 180min after TACE. Each sample was centrifuged at 3,000 rpm for 10 min at 4 ℃ and the plasma was immediately frozen using liquid nitrogen and stored at -80 ℃. All the serum samples were processed for HPLC analysis.
Necropsy and liver harvest were performed in all groups, 7 days after embolization. Tumor and adjacent hepatic parenchyma were immediately weighted, frozen using liquid nitrogen and stored at -80 ℃ for epirubicin concentration measurements, the remained tumor pieces were fixed in 10% buffered formalin and stained with hematoxylin and eosin (H&E) to assess tumor necrosis. The rates of tumor necrosis were calculated as a percentage of the tumor necrosis area for each slice by an independent pathologist blinded to the treatments. Visual calculation was used to estimate the tumor necrosis ratio.
Epirubicin concentration was analyzed according to a previously published protocol 18,19. This was done through liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) as well as using a TSQ Quantum Ultra mass spectrometric detector (Thermo Fisher Scientific) and a Dionex UltiMate 3000 RSLC Nano (Thermo Fisher Scientific). To measure epirubicin concentration in the serum, blank serum samples (190 uL) were spiked with epirubicin standard solution (Shanghai Kewel Chemical Technology Co.) (10 uL) to make the standard curve in serum, ranging from 5 ng/mL to 5000 ng/mL. The lowest limit of quantity for epirubicin was set as 5 ng/mL. The plasmatic samples were pretreated with methanol for protein precipitation. A column (2.1×100 mm, 1.9 µm; Thermo Hypersil GOLD) was used and the flow rate was set as 0.5 ml/min. The injection volume of standards or samples was 5 µL.
Epirubicin concentration in the tumor regions and adjacent hepatic parenchyma were measured by using HPLC method as mentioned earlier. According to a previously published protocol 20, each tissue was mixed with PBS (1:5, v/v) and then homogenized. The subsequent pretreatment method was the same as that for plasma.
Statistics analysis
Statistical analyses were performed using SPSS Statistics software version 24.0 (SPSS Inc.). The plasma and tissue drug concentration data were analyzed using a one-way analysis of variance model (ANOVA), whereas multiple comparisons were determined by LSD test. The level of significance was set at p value < .05.