Unraveling Interaction of Rhenium-188 Microspheres with Primary Hepatic Cancer Cell: A Breakthrough Study

Abstract

Introduction:

Hepatocellular carcinoma is a prevalent contributor to global mortality rates. The main palliative treatments are trans-arterial chemoembolization and selective intra-arterial radionuclide therapy.

Methods:

A novel freeze-dried nonradioactive microsphere kit formulation has been developed, and the behavior and therapeutic potential of ¹⁸⁸Re microspheres have been assessed. The microspheres were labeled with fluorescein isothiocyanate (FITC) and ¹⁸⁸ReO₄ ⁻. The uptake of FITC microspheres by HepG2 cells was examined at various time intervals. The impact of ¹⁸⁸Re microspheres on cell viability and the mode of cell death were investigated with HepG2 cells using MTT and Annexin FITC-V/propidium iodide (PI) apoptosis assay.

Results:

The labeling efficiency of microspheres was more than 99% with FITC and ¹⁸⁸ReO₄ ⁻. The maximum uptake of FITC microspheres by HepG2 cells was achieved at 6 h. The exposure to ¹⁸⁸Re microspheres has shown a decrease in cellular viability from 77.81% ± 0.015% to 42.03% ± 0.148% at 192 h of incubation (∼11 half-lives). The cellular uptake of ¹⁸⁸Re microspheres was 0.255–0.901 MBq. These values were concordant with Annexin FITC-V/PI apoptosis assay. At 192 h, 53.28% ± 0.01% of cells entered the apoptotic phase after treatment with ¹⁸⁸Re microspheres, and only 39.34% ± 0.02% of cells remained viable. However, in the cells treated with ¹⁸⁸ReO₄ ⁻ alone, 74.86% ± 0.005% of cells were viable, and only 24.75% ± 0.577% of cells were in the early apoptotic phase at 192 h.

Conclusion:

The data revealed that ¹⁸⁸Re microspheres treatment led to significant growth inhibition in HepG2 cells compared with ¹⁸⁸ReO₄ ⁻.

Introduction

Hepatocellular carcinoma (HCC) is the fourth highest cause of cancer deaths worldwide. HCC shows a variable geographic distribution with high incidence in Eastern and Northern Africa.¹ The prominent risk factor for HCC is hepatitis B virus infection and accounts for almost 50% of cases.² The risk attributed to hepatitis C virus (HCV) infection has substantially decreased owing to patients achieving sustained virological response with antiviral drugs.³ Patients having HCV clearance after cirrhosis are still considered to be at high risk for HCC incidence. The fastest growing etiology of HCC is non-alcoholic steatohepatitis, which is associated with metabolic syndrome or diabetes mellitus.⁴ The potential pathogenic cofactors in the development of HCC are aristolochic acid and tobacco.⁵

The management of HCC has markedly improved, but therapeutic options are limited. The mainstay curative treatments are hepatic resection and liver transplantation. In the initial treatment phase, the combined use of atezolizumab and bevacizumab has shown remarkable advancements in both overall survival and progression-free survival. Currently, it is the established standard for treating advanced HCC.⁶ Chemotherapy and external radiation therapy are ineffective because of poor hepatic tolerance.⁷ The liver has a dual blood supply. The normal liver is fed mainly by the portal vein, whereas the tumor is fed via the hepatic artery. This makes locoregional tumor therapy possible. The trans-arterial delivery of chemotherapeutic drugs (trans-arterial chemoembolization [TACE]) and therapeutic radionuclides (selective intra-arterial radionuclide therapy [SIRT]) are widely used for palliative treatments.⁸

SIRT is an important alternative when portal vein is thrombosed and TACE is contraindicated. The radiolabeled particles are injected via the hepatic artery to tumor vasculature. It delivers high radiation dose to the tumor and spares the normal liver parenchyma. The radionuclides yttrium-90 (⁹⁰Y), holmium-166 (¹⁶⁶Ho), and rhenium-188 (¹⁸⁸Re) have been often used for SIRT. ⁹⁰Y is a pure β⁻-emitter (βmax = 2.28 MeV), with a physical half-life of 64.1 h. ¹⁶⁶Ho emits several γ-photons, most of which are 81 keV (abundance 6.6%), 1379 keV (0.9%), or 1581 keV (0.2%).⁹ ¹⁸⁸Re also emits both β⁻-particles (βmax = 2.12 MeV, 85%) and γ-photons of 155 keV (15.1%) and has shown additional benefits in pre- and post-therapy dosimetry using single photon emission computed tomography/computed tomography.¹⁰

The half-lives of ¹⁸⁸Re and ⁹⁰Y are 16.9 and 64 h, respectively, and the mean tissue penetration values are 2.5 and 3.0 mm, respectively. ¹⁸⁸Re is cost-effective due to the availability of transportable ¹⁸⁸W/¹⁸⁸Re generator, could be housed in hospital radiopharmacy, and the shelf life is more than 6 months. ⁹⁰Y microspheres are commercially available as a ready to use formulation. However, the high cost of ⁹⁰Y microspheres is a major issue that limits the clinical usefulness of ⁹⁰Y. ¹⁸⁸Re in the form of perrhenate (¹⁸⁸ReO₄ ⁻) is eluted easily in normal saline for radiolabeling. The therapeutic β⁻-particles of ¹⁸⁸Re help in destroying tumor cells. Moreover, the patients do not require isolation due to the emission of low-energy γ-photons (Eγ-155 keV).

The rationale behind using cancer cell lines in this study is that the HepG2 cell lines retain the hallmark of the primary liver cancer cells and may help to study the behavior and interaction with indigenous prepared tin oxide microspheres of less than 20 μm. For this purpose, the microspheres were labeled with fluorescent molecule (fluorescein isothiocyanate [FITC]) as well as therapeutic radionuclide (¹⁸⁸ReO₄ ⁻) and incubated with cultured HepG2 cells.

Materials and Methods

Cell culture

Human hepatocellular cancer cell line (HepG2 cells) was maintained by culturing in Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific) and 100 IU/mL penicillin–streptomycin (MP Biomedicals). Cells were incubated in a CO₂ humidified incubator at 37°C (ESCO Life Sciences) with 5% CO₂. The study is exempt from IRB approval due to its in vitro nature.

Cellular uptake of FITC-labeled microspheres in liver cancer cell lines

To study the uptake in HepG2 cells, microspheres (200 mg) were labeled with FITC (0.2 mg) and ascorbic acid (0.5 mg) as stabilizer in water at 100°C for 1 h. The labeling efficiency of FITC-labeled microspheres was studied by flow cytometry (BD CANTO). The data were acquired based on forward and side scattering by flow cytometer and were analyzed by Kaluza Analysis Flow Cytometry Software version 2.1.1. Further to study the uptake of FITC-labeled microspheres, the cells were grown in monolayers on four-chambered slides coated with poly-d-lysine in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin in an atmosphere of 95% air and 5% CO₂ at 37°C. HepG2 cells (1 × 10⁶ cells per chamber) were seeded overnight for attachment.

Subsequently, the medium was removed, and the serum-free medium containing FITC-labeled microspheres was added into three chambers, and the fourth chamber of each slide was treated as control. The slides were incubated for 6, 12, and 24 h at 37°C in the dark. At each time point, the respective glass slide was washed three times with Dulbecco's phosphate-buffered saline (DPBS). The cells were then fixed with 4% paraformaldehyde for 15 min at 37°C, followed by washing with DPBS and mounting with dibutylphthalate polystyrene xylene. The three-dimensional (3D) image z-stacks of fixed cells were generated as the z-stack function of confocal microscopy (Olympus Fluoview 3000), which produces a series of images at various depths. The generated optical sections were used to quantify the intensities and uptake of fluorescent microspheres and were quantified using Fiji software.

Radiolabeling of nonradioactive microsphere kit formulation with ¹⁸⁸Re perrhenate (¹⁸⁸ReO₄ ⁻)

The nonradioactive microsphere kit formulation was prepared as described earlier and stored at −20°C.¹¹ The kit was thawed at room temperature before use. Subsequently, the freshly eluted ¹⁸⁸Re-perrhenate in 0.9% saline was added into nonradioactive kit and heated at 100°C for 30 min. The obtained radiolabeled formulation was cooled to room temperature, and pH was adjusted to 6.5–7.0 with the help of sterile buffer (pH 8). The pellet was collected by centrifugation at 3000 rpm for 10 min. Radioactivity in pellet and supernatant was measured and recorded using a precalibrated dose calibrator. The percent labeling (labeling efficiency) was calculated using the following equation: $% l a b e l i n g = [a c t i v i t y i n p e l l e t ∕ (a c t i v i t y i n p e l l e t + a c t i v i t y i n s u p e r n a t a n t)] * 100 .$

Quality control of ¹⁸⁸Re microspheres was performed. Parameters such as labeling efficiency and radiochemical purity were noted.

Cytotoxicity assay by calculating the uptake

A cytotoxicity assay was performed using MTT assay (HiMedia). The cells were seeded in 96-well plates (n = 4) at a density of 7000–8000 cells per well and incubated at 37°C and 5% CO₂ until 70%–80% confluency was achieved. Subsequently, the cells were treated with increasing radioactivity amounts of ¹⁸⁸Re microspheres and ¹⁸⁸ReO₄ ⁻ (0.074, 0.185, 0.37, 0.74, 1.48, and 2.96 MBq of each). The medium was removed after 6 h, and the radioactivity in the medium was measured with a precalibrated dose calibrator. The uptake of ¹⁸⁸Re microspheres and ¹⁸⁸ReO₄ ⁻ was calculated by subtracting the decay-corrected radioactivity in the medium (after 6 h) from the radioactivity initially added in each well.

The cells were washed with DPBS and replenished with the fresh complete medium. The plates were incubated for 24, 48, 72, and 192 h at 37°C and 5% CO₂, separately. At each time point, the freshly prepared MTT reagent was added and incubated for 4 h. Following the incubation, dimethyl sulfoxide (100 μL) was added to each well and incubated at room temperature for 15 min. The optical density of the formazan crystal formed in each well was measured at 570 nm, with a reference at 630 nm using an ELISA plate reader (ALTA).

Percent survival was calculated for each dose $% S = \frac{O D c - O D s}{O D c} * 100,$

% C = 100 - % S,

where %S represents the percent survival, %C represents the percent cytotoxicity, ODc represents the mean optical density of control, and ODs represents the mean optical density of the sample.

Annexin V-FITC/propidium iodide apoptosis detection assay

The mode of cell death was studied using Annexin V-FITC apoptosis detection kit (BD Pharmingen). Approximately 3 × 10⁶ cells per well were seeded in six-well plates (n = 5) and incubated at 37°C and 5% CO₂ until 70%–80% confluency was achieved. The cells were treated with ¹⁸⁸Re microspheres and ¹⁸⁸ReO₄ ⁻ for 6 h. The medium was removed, and the wells were washed with DPBS. The complete medium was added to each well, and the plates were incubated separately for 24, 48, 72, and 192 h. One plate remained untreated for control. The treated and untreated cells were harvested by centrifugation (1200 rpm for 3 min) and resuspended in 1 × binding buffer. The cells were stained with 1 μL Annexin V-FITC and 1 μL propidium iodide (PI) solution and were incubated for 10 min at room temperature in the dark. Induction of cell death was measured using a flow cytometer (BD CANTO), and the data were analyzed using Kaluza Analysis Flow Cytometry Software version 2.1.1.

Statistics

Statistical calculations were performed using GraphPad Prism 9.4.1 software (GraphPad Software, La Jolla, CA). All values are given as mean ± standard deviation.

Results

Labeling efficiency of microspheres with FITC and their uptake

Based on the forward and side scatter properties of flow cytometry, both the cells (HepG2) and microspheres were observed in the same region. A fluorophore FITC was conjugated to microspheres to differentiate HepG2 cells from microspheres. The labeling efficiency of FITC-labeled microspheres was more than 99% as determined by the flow cytometry gating principle. Figure 1A shows a single distinct peak for HepG2 cells and microspheres. After conjugation of microspheres with FITC, two peaks were visualized (Fig. 1A.4). Another histogram was plotted for only FITC-labeled microspheres (Fig. 1A.5). By overlaying the two histograms, that is, Figure 1A.4 and A.5, it was found that the microspheres were efficiently labeled (>99%) with FITC.

FIG. 1.

(A) A single distinct peak was observed for only cells (1) and only cells with microspheres (3); peak of only cells when treated with FITC microspheres shifted toward higher fluorescence (2). In (4), two peaks were observed: one at low fluorescence showing only cells and one at high fluorescence showing FITC-labeled microspheres; this can be overlayed on (5), which gives the labeling efficiency. (B) Images were taken for HepG2 cells showing the uptake of FITC-labeled microspheres by a confocal microscope (40 × oil immersion) at three different time points. (C) The montage captures on confocal showing the uptake of FITC-labeled microspheres in HepG2 cells. The in-depth sections of a Z series were taken using green filter (498) after 6 h (1), 12 h (2), and 24 h (3) of exposure at λ_Ex = 490 nm and λ_Em = 550 nm. (D) Radio-paper chromatogram showing radiochemical purity of ¹⁸⁸Re microspheres as more than 99%. Acetone is used as mobile phase and ITLC-SG plate as stationary phase with R_f value = 0. (E) Cytotoxicity effect of ¹⁸⁸ReO₄ ⁻ and ¹⁸⁸Re microspheres was studied for more than 10 half-lives of ¹⁸⁸Re. (F) Percent apoptosis versus different time points between ¹⁸⁸ReO₄ ⁻ and ¹⁸⁸Re microspheres were analyzed by Annexin V-FITC/PI assay. FITC, fluorescein isothiocyanate; PI, propidium iodide.

The confocal microscopy showed the uptake of FITC-labeled microspheres in HepG2 cells at 498 nm laser with Ex/Em of 490 nm/550 nm (Fig. 1B and Supplementary Video S1). The mean intensities were found to be 250.554 ± 30.108 at 6 h, 250.390 ± 30.301 at 12 h, and 251.008 ± 28.840 at 24 h from Z-axis profile (Fiji). The Z-series were difficult to represent as a two-dimensional (2D) image; therefore, a montage (single-grid image, Fig. 1C) has been shown to visualize 3D data set for best uptake time in the 2D plane. A 3D video is enclosed for better understanding (Supplementary Video S2).

Radiolabeling of nonradioactive microsphere kit formulation with ¹⁸⁸Re perrhenate

The nonradioactive microsphere kit formulation showed high radiolabeling yield (90%–98%) with ¹⁸⁸ReO₄ ⁻. Washed and pelleted ¹⁸⁸Re microspheres remained at origin of spot (R_f = 0) in ITLC strip (Fig. 1D) and free ¹⁸⁸ReO₄ ⁻ moved with the solvent front (0.9% saline; R_f = 1). The calculated radiochemical purity was greater than 99%.

Growth inhibition and viability

HepG2 cells showed maximum uptake of ¹⁸⁸Re microspheres at 6 h based on uptake studies (Confocal Microscopy). The MTT cytotoxicity assay exhibited different growth responses at different time points. After 24 h, the mean viability of cells, when exposed to 0.255–0.901 MBq, lies in the range of 61.1% ± 0.22% to 86.37% ± 0.025% with ¹⁸⁸Re microspheres and 66.77% ± 1.92% with ¹⁸⁸ReO₄ ⁻. However, after 48 h of incubation, the mean viability of cells was found to be further decreased from 85.5% ± 0.015% to 70.03% ± 0.036% with ¹⁸⁸Re microspheres and also from 88.77% ± 5.12% to 55.72% ± 1.06% in case of ¹⁸⁸ReO₄ ⁻.

After four half-lives of ¹⁸⁸Re, at 72 h, the mean viability of HepG2 cells was decreased from 75.4% ± 0.048% to 50.04% ± 0.082% in case of ¹⁸⁸Re microspheres, but the mean viability increased, 71.75% ± 5.3% (0.255 MBq) to 62.39% ± 2.51% (0.901 MBq), in case of ¹⁸⁸ReO₄ ⁻. The mean viability of cells after 11 half-lives of ¹⁸⁸Re (at 192 h) with ¹⁸⁸Re microspheres was calculated as 77.81% ± 0.015% to 42.03% ± 0.148%. However, in case of ¹⁸⁸ReO₄ ⁻, the cytotoxicity was not observed since the viability was noted at 83.78% ± 0.76% at 0.255 MBq activity and 63.64% ± 1.2% at 0.901 MBq activity. The results showed significant differences (p < 0.001) in cell viability (Fig. 1E).

Apoptosis assay

Annexin V-FITC/PI staining showed a dramatic reduction in the percentage of viable HepG2 cells as observed during flow cytometric analysis in a dose- and time-dependent manner. Statistically significant (p < 0.0001) increase in the number of apoptotic HepG2 cells was found when treated with ¹⁸⁸ReO₄ ⁻ and ¹⁸⁸Re microspheres. The observations at various time points are shown in subsequent sections.

Twenty-four hours of incubation

The mean viable HepG2 cells were 71% ± 1.367% when treated with ¹⁸⁸ReO₄ ⁻ compared with 31% ± 2.107% mean viable HepG2 cells in case of treatment with ¹⁸⁸Re microspheres. After ¹⁸⁸Re microsphere treatment, 54.43% ± 3.214% of cells were in the early apoptotic cell cycle phase, while only 19.6% ± 1.054% of cells were in this phase after treatment with ¹⁸⁸ReO₄ ⁻. The cells in the late apoptotic phase were almost same with both treatments.

Forty-eight hours of incubation

The mean viable HepG2 cells were found to be 75.6% ± 0.648% in case of ¹⁸⁸ReO₄ ⁻ and 51.7% ± 1.045% in case of ¹⁸⁸Re microspheres. The early apoptotic cells were almost double (41% ± 0.103%) after treatment with¹⁸⁸Re microspheres compared with ¹⁸⁸ReO₄ ⁻ treatment. In the late apoptotic phase, 5% ± 0.036% and 2.23% ± 0.012% of cells were observed with ¹⁸⁸Re microspheres and ¹⁸⁸ReO₄ ⁻, respectively.

Seventy-two hours of incubation

The mean viability of HepG2 cells, treated with ¹⁸⁸ReO₄ ⁻, was increased to 84% ± 0.006%. On the contrary, there was a continuous decrease in the mean viability of cells (39.3% ± 0.020%) treated with ¹⁸⁸Re microspheres. Of the cells treated with ¹⁸⁸Re microspheres, 50% ± 0.010% of cells entered the early apoptotic phase after 72 h of incubation, while only 12.467% ± 0.015% of cells were in the early apoptotic phase after treatment with ¹⁸⁸ReO₄ ⁻. In the ¹⁸⁸Re microsphere-treated cells, the late apoptotic cells were turned out to be doubled, from 5% ± 0.036% to 9.027% ± 0.025% compared with 48 h of incubation. However, the percentage of the late apoptotic cells decreased from 2.23% ± 0.012% to 1.687% ± 0.015% in case of ¹⁸⁸ReO₄ ⁻.

One hundred ninety-two hours of incubation

At 192 h, the mean HepG2 viable cells treated with ¹⁸⁸ReO₄ ⁻ was increased to 74.86% ± 0.005%. On the contrary, there was a continuous decrease in the mean viability of cells treated with ¹⁸⁸Re microspheres. After 192 h, only 36.5% ± 0.020% of mean viable cells were found. However, 60.59% ± 0.030% of cells treated with ¹⁸⁸Re microspheres entered the early apoptotic phase, and only 24.75% ± 0.577% of cells were in the early apoptotic phase when treated with ¹⁸⁸ReO₄ ⁻. The late apoptotic cells were 2.44% ± 0.016% in case of ¹⁸⁸Re microsphere-treated cells. Also, the necrotic cells were not recorded up to 192 h with both treatments (Fig. 1F).

Discussion

¹⁸⁸Re is eluted in the form of ¹⁸⁸ReO₄ ⁻ from the ¹⁸⁸W/¹⁸⁸Re generator, which is a convenient and cost-effective option for radionuclide therapy. It is crucial to gather preliminary data from cell line studies, as these elements are integral to understanding the molecular and cellular mechanisms involved in drug discovery. The rationale for using HepG2 cells as an experimental model of HCC is that it retains the hallmarks of the primary liver cancer cells.

Microspheres provide an innovative carrier system for precise drug delivery, enabling site-specific actions and reduced potential side effects.¹² The microspheres are used as radiation delivery device for locoregional therapy, SIRT, of primary HCC and liver cancers metastatic to other primary cancers such as neuroendocrine and bladder. The microspheres emerge as a noteworthy and significant treatment option not only for HCC but also for intrahepatic cholangiocarcinoma (ICC), which is a severe and rapidly progressive hepatic tumor. The use of ⁹⁰Y-labeled spheres in radioembolization has been identified as a promising therapeutic approach for ICC.¹³ Similarly, ¹⁸⁸Re microspheres could also be used for ICC.

The microspheres, used in this study, are small spherical particles, and probably due to their small size, enhanced permeability, and retention of tumor cell, these microspheres passively enter and accumulate within the tumor cell. The study demonstrated 6 h as optimum time for uptake of microspheres by HepG2 cells, which was supported by confocal microscopy. As a part of optical microscopy, confocal microscopy is one of the principal tools for analyzing cells, tissues, and complex models of cellular function.

To examine subcellular structures, visualization and analysis require the incorporation of high magnification objectives leading to high probability that portions of the image will be out of focus. This is due to an increase in higher power objectives. This issue can be resolved by stacking and projection of the collected images, which allows accurate visualization of the cells or tissue in the X- and Y-axis as well as it takes care of Z-axis. A projected image provides a clear picture of all the portions of the structure, which allows to draw precise conclusions. Based on the mean intensity data, and from the single-grid image (Fig. 1B, C), for all the three time points, 6 h was observed as the optimum time required for the good uptake of microspheres. These microspheres were found to be retained within the HepG2 cancer cells, as shown in the 2D and 3D videos (Supplementary Videos S1 and S2).

The high radiolabeling efficiency and radiochemical purity of ¹⁸⁸Re microspheres were determined with paper chromatography. Rhenium-188 has a half-life of 16.9 h. The prolonged cytotoxic effect of ¹⁸⁸ReO₄ ⁻ and ¹⁸⁸Re microspheres has been observed up to 192 h (almost 11 half-lives). Both ¹⁸⁸Re microspheres and ¹⁸⁸ReO₄ ⁻ alone showed cell toxicity up to 48 h (approximately three half-lives). In the 72 h study, that is, more than four half-lives of ¹⁸⁸Re, ¹⁸⁸Re microspheres showed toxicity. However, ¹⁸⁸ReO₄ ⁻ could not show further cell death beyond 48 h. The mean viability of HepG2 cells was assessed after the 11 half-lives of ¹⁸⁸Re (192 h), with a cumulative radioactivity range of 0.255–0.901 MBq, and it was calculated as 77.81% ± 0.015% to 42.03% ± 0.148% in case of ¹⁸⁸Re microspheres. On the contrary, at 192 h, good cell growth, 83.78% ± 0.76% at 0.255 MBq ¹⁸⁸ReO₄ ⁻ and 63.64% ± 1.2% at 0.901 Mbq ¹⁸⁸ReO₄ ⁻, was noted.

A similar trend was observed with the Annexin/PI flow cytometric results; ¹⁸⁸Re microspheres significantly increased apoptosis in HepG2 cells (p < 0.0001) at 48 h post-treatment. Overall, the cell proliferation was influenced by the effective radiation dose and exposure time. The mean viability of HepG2 cells was significantly decreased (p < 0.0001) when treated with ¹⁸⁸Re microspheres compared with ¹⁸⁸ReO₄ ⁻ (Fig. 1F). At 192 h after treatment, more than 50% of cells entered the early apoptotic phase with ¹⁸⁸Re microsphere treatment and 25% with ¹⁸⁸ReO₄ ⁻ treatment. This demonstrated high impact of delivery of radionuclide when delivered in the form of microspheres. The plausible justification is that the ¹⁸⁸Re microspheres were taken up by HepG2 cells and retained within the cells as indicated by Z-stack study (Supplementary Video S1).

The percentages of the late apoptotic and necrotic cells remained low (0.25% vs. 0.13%) after treatment with ¹⁸⁸Re microspheres and ¹⁸⁸ReO₄ ⁻ at all time points and doses. The reason for this may be that the necrotic cells were de-adhered and removed during pre-staining washing, and few apoptotic cells entered the late apoptotic phase. The duration of the current study was up to 192 h, as the long-term studies are not technically feasible with cell lines. But the results clearly indicate that all the cells in the early/late apoptosis will ultimately die in due course of time.

These observations are useful in understanding (i) better outcome of particulate ¹⁸⁸Re due to internalization and retention within the liver cancer cells compared with ¹⁸⁸Re alone (¹⁸⁸ReO₄ ⁻), and (ii) delayed cytotoxic response to radiation exposure. In this study, it has been observed that ¹⁸⁸ReO₄ ⁻ and ¹⁸⁸Re microspheres significantly affect the viability of HepG2 cells in a dose- and time-dependent manner. The long-term aim of this study was to use these microspheres for locoregional therapy of liver cancer and to save normal liver from radiation damage. ¹⁸⁸ReO₄ ⁻ may leach from the tumor to adjacent normal liver, vasculature that may damage thyroid and may cause gastric ulcers, whereas ¹⁸⁸Re microspheres are highly stable and showed precise delivery of high radiation doses to tumor for a longer duration. It may act as a permanent radiation implant within the tumor cells to deliver the required radiation dose.¹⁰

Conclusions

The radiolabeling of nonradioactive microsphere kit formulation with ¹⁸⁸ReO₄ ⁻ is simple and efficient. ¹⁸⁸Re microspheres are internalized within the tumor cell, triggering apoptosis to achieve the necessary cytotoxicity. Consequently, ¹⁸⁸Re microspheres hold the promise of becoming an economically efficient medical device for SIRT for primary or metastatic liver cancers.

Footnotes

Acknowledgments

The authors wish to thank the staff of the Department of Central Sophisticated Instrument Cell (CSIC), PGIMER, Chandigarh, India, for assistance in confocal microscopy.

Authors' Contributions

A.A.: Methodology, software, validation, investigation, data curation, writing—original draft preparation, project administration. G.K.: Formal analysis, investigation, writing—reviewing. R.S.J.: Resources, software. B.M.: Resources, supervision. B.R.M.: Resources, editing. J.S.: Conceptualization, resources, writing—reviewing and editing, supervision.

Disclosure Statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

Funding Information

This research was funded by the Indian Council of Medical Research, New Delhi, India (NM/JS/ICMR/19-20/02-03).

Supplementary Material

Supplementary Video S1

Supplementary Video S2

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Supplementary Material

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Unraveling Interaction of Rhenium-188 Microspheres with Primary Hepatic Cancer Cell: A Breakthrough Study

Abstract

Introduction:

Methods:

Results:

Conclusion:

Introduction

Materials and Methods

Cell culture

Cellular uptake of FITC-labeled microspheres in liver cancer cell lines

Radiolabeling of nonradioactive microsphere kit formulation with 188Re perrhenate (188ReO4 −)

Cytotoxicity assay by calculating the uptake

Annexin V-FITC/propidium iodide apoptosis detection assay

Statistics

Results

Labeling efficiency of microspheres with FITC and their uptake

Radiolabeling of nonradioactive microsphere kit formulation with 188Re perrhenate

Growth inhibition and viability

Apoptosis assay

Twenty-four hours of incubation

Forty-eight hours of incubation

Seventy-two hours of incubation

One hundred ninety-two hours of incubation

Discussion

Conclusions

Footnotes

Acknowledgments

Authors' Contributions

Disclosure Statement

Funding Information

Supplementary Material

References

Supplementary Material

Radiolabeling of nonradioactive microsphere kit formulation with ¹⁸⁸Re perrhenate (¹⁸⁸ReO₄ ⁻)

Radiolabeling of nonradioactive microsphere kit formulation with ¹⁸⁸Re perrhenate