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Evaluation Efficacy of Rhenium-188 loaded micro-particles for Radiotherapy in a Mouse Model of Hepatocellular Carcinoma Ying-Hsia Shih, Cheng-Liang Peng, Mao-Feng Weng, Ping-Fang Chiang, Tsai-Yueh Luo, and Xi-Zhang Lin Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b01083 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 19, 2019

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Molecular Pharmaceutics

Evaluation Efficacy of Rhenium-188 loaded micro-particles for Radiotherapy in a Mouse Model of Hepatocellular Carcinoma

Ying-Hsia Shih†, Cheng-Lian Peng†, Mao-Feng Weng†, Ping-Fang Chiang†, Tsai-Yueh Luo†, Xi-Zhang Lin‡

†Isotope

Application Division, Institute of Nuclear Energy Research, Taoyuan, Taiwan

‡Department

of Internal Medicine, National Cheng Kung University, Tainan, Taiwan

*Corresponding author Address reprint requests and correspondence to Tsai-Yueh Luo, PhD., Isotope Application Division, Institute of Nuclear Energy Research. No. 1000, Wenhua Road, Jiaan Village, Longtan District, Taoyuan City, 32546, Taiwan; Tel.: +886-3-4711400 ext. 7002; Fax: +886-3-4711416; E-mail: [email protected]. Cheng-Lian Peng, PhD., Isotope Application Division, Institute of Nuclear Energy Research. No. 1000, Wenhua Road, Jiaan Village, Longtan District, Taoyuan City, 32546, Taiwan; Tel.: +886-3-4711400 ext. 7298; Fax: +886-3-4711416; E-mail: [email protected]. Xi-Zhang Lin, MD., Department of Internal Medicine, College of Medicine, National Cheng Kung University. No.138, Sheng-Li Road, Tainan City, 704, Taiwan; Tel.:+886-6-2353535 ext. 3626; Fax: +886-6-2347270; E-mail: [email protected].

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Abstract Hepatocellular carcinoma (HCC) is one of the leading causes of mortality worldwide. The aim of the present study was to evaluate the distribution and the therapeutic effect of 188Re-Tin-colloid

micro-particles in subcutaneous HCC-bearing mice. The synthesis and

characterization of micro-particles labeled with

188Rhenium

isotope were performed. The

micro-particles were injected into the tumor site subcutaneously in the BNL HCC-bearing mice, with three treatment groups: normal saline, 188Re micro-particles, and 188Re-Tin-colloid micro-particles. The results of biodistribution showed that major radioactivity (188Re) of 188Re-Tin-colloid

compared with injection of

micro-particles (18.69 ± 4.28 %ID/g) remained at the tumor sites,

188Re

micro-particles (0.21 ± 0.12 %ID/g) post injection 24 h. Following the

188Re-Tin-colloid

micro-particles for 14 days, all BNL tumor in mice were

regression during the observation period. By contrast, all of the mice treated with normal 188Re

micro-particles had died by 24 and 28 days, respectively. The

188Re-Tin-colloid

micro-particles demonstrated high accumulation and therapeutic potential

saline or

in the subcutaneous HCC-bearing mice. Keywords: 188Re, colloid, micro-particles; Hepatocellular carcinoma


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1. Introduction Hepatocellular carcinoma (HCC) is one of the most common types of cancer and is associated with a poor prognosis worldwide.1-6 Surgery is usually the first-line treatment approach for removing HCC.6 Other treatment modalities are used for HCC. including chemotherapy,7,

8

transcatheter arterial embolization (TAE), transcatheter arterial

chemoembolization (TACE),9 radiation therapy,4 radiotherapy10 and radiofrequency ablation (RFA).10-12 HCC is the third common cause of cancer-associated mortality in the United States and other developing countries; survival rates are 3%-5% after initial diagnosis.13 Surgical resection (SR), liver transplantation (LT) and RFA of focal lesions are generally considered the preferred treatment options for HCC; however, the results remain unsatisfactory in many patients,14-16 and only 20%-35% of patients are eligible for this invasive procedure.17 TAE and TACE are usually considered for treating HCC when SR, LT, or RFA cannot be performed in patients. TAE and TACE are minimally invasive procedures performed using vascular photography, which can limit the oxygen and nutrition of blood supplying the tumor. Transcatheter arterial administration with therapeutic radionuclides can deliver a tumoricidal dose from radiation without harming the non-tumorous liver. Fortunately, precise catheterization skills and specialized equipment for the operators have been well developed. The majority of physicians committed to hepatoma treatment are familiar with local injection therapy. Brachytherapy, the treatment of cancer by inserting the maximum quantity of radioactive implants directly into the tumor, can minimize radiation dosage to surrounding normal tissues, thus fewer side effects.17,

18, 19

Brachytherapy is usually applied in the

treatment of breast cancer, prostate cancer, cervical cancer, and colorectal cancer.20-23 Iodine-125 (125I, t1/2 = 59.4 days) seed implantation24, 25 and yttrium-90 (90Y, t1/2 = 64.1 h) resin microsphere or glass microsphere radioembolization26, approaches for treating HCC.

125I

27

are commonly used

used as radiation therapy in brachytherapy emits -rays

with a maximum energy of 35 keV. However, iodine-126 (t1/2 = 13.1 days), an unwanted radioisotope, is an impurity (~0.2% atom fraction of the total iodine) in producing 125I, which interferes in dose calculations of brachytherapy. In addition, 90Y emits -rays with energy of 2.28 MeV to treat cancer as radiation therapy. The 90Y microsphere is considered promising and has been approved for the treatment of hepatoma in several countries.26, there have been reports that the accumulation of

90Y

27

However,

in the skeletal system causes bone



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marrow depression and complications that result from the radioactivity to surrounding structures.28,

29

The disadvantages of the currently available radioisotopes of

125I

and

90Y

described above limit their clinical potential. Rhenium-188 (188Re, t1/2 = 16.9 h) is emerging as a promising isotope in nuclear imaging and therapy for clinical use.30-34

188Re

has several advantageous features, including high

energy of -rays (2.1 MeV, 85%), favorable -ray energy (159 keV, 15%), shorter half-life, deep tissue penetration for local treatment (maximum = 11 mm, average = 3.8 mm),35 and in-house preparation of

188W/188Re

generator.36 This unique property of

188Re

is suitable for

therapeutics and diagnostics. The aim of the present study was to characterize and therapeutically evaluate 188Re-Tin-colloid

evaluated

a

loaded into micro-particles (188Re-Tin-colloid micro-particles).37 The study

delivery

188Re-Tin-colloid

system

for

(188Re-Tin-colloid

the

radioisotope-loaded

micro-particles),

for

local

micro-particles

drug,

radiotherapy.

BNL

subcutaneous xenograft-bearing mice were used as a model for in vivo biodistribution and therapeutic evaluation. It was expected that

188Re-Tin-colloid

micro-particles remain in the

tumor area consistently and destroy the majority of tumor cells in HCC-bearing mice.


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2. Materials and methods 2.1 Preparation of micro-particles Lipiodol, acetyl alcohol, glycol monostearate, stearyl acid, polycaprolactone, and cholesterol were used to prepare alginate-based micro-particles as described previously.37 All micro-particles were biodegradable and water insoluble. The sizes of the micro-particles were 100-150 μm. 2.2 Preparation of 188Re micro-particles and 188Re-Tin-colloid micro-particles 188ReO 4

was added to the micro-particles, and the reaction was performed on 300 rpm

shaker at 37 °C for 10-30 min to produce 188Re micro-particles, following which purification was performed with a centrifuge. For the

188Re-Tin-colloid

micro-particles,

188ReO 4

was

added to the micro-particles, following which the mixture was placed on a 300 rpm shaker at 37 °C for 10-30 min, and SnCl2 (15, 25, 35 mg/mL) was added to produce the 188Re-Tin-colloid

micro-particles (Scheme 1) for 10-30 min. Then, the samples were

centrifuged at 3000 rpm for 10 min. The supernatant liquid was removed, the particles was suspended in normal saline and repeated once. A single concentration of 35 mg/mL SnCl2 was added. As in our previous study38, preparation with 35 mg/mL SnCl2 showed the optimal release and was used for preparation. The 188Re micro-particles and 188Re-Tin-colloid micro-particles were used in characterization and evaluating the release profile, in vivo biodistribution, and antitumor efficacy. 2.3 Characterization of 188Re micro-particles, 188Re-Tin-colloid micro-particles The absorption rates of the

188Re

micro-particles and

188Re-Tin-colloid

micro-particles

(SnCl2 35 mg/mL) were determined by the radioactivity ratio of the micro-particles and 188Re by dose calibrator. The shape of the micro-particles was obtained using an optical microscope and scanning electron microscope (SEM; HITACHS-4800, Japan). The sizes of the two micro-particles were measured with a laser diffraction particle sizing analyzer (Beckman coulter LS 13 320, USA). 2.4 Release profile of 188Re micro-particles and 188Re-Tin-colloid micro-particles The release profile of

188Re

from

188Re

micro-particles and

188Re-Tin-colloid

micro-particles (SnCl2 35 mg/mL) were obtained by using the dialysis tube (molecular weight 5 ACS Paragon Plus Environment


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3.5 kDa) at 37 °C under 300 rpm shaking (pH = 7.4, PBS). The release solutions (200 μL) were collected at 0, 0.5, 1, 4, 16, 24, 40, and 48 h, respectively. The sample was added the same volume of PBS following each time point of sampling. The released 188Re

micro-particles and

188Re-Tin-colloid

188Re

from the

micro-particles was quantified using a γ

-counter (Cobra series Model 5003, USA). All experiments were performed in triplicate, and the results are expressed as in mean ± standard deviation..

2.5 Cell culture The BNL cell line was used to establish xenograft subcutaneous mice model of HCC. All BNL cells were cultured in DMEM (SIGMA-Aldrich, USA) supplemented with 10% FBS (GIBCO, USA) and 1% antibiotics (P/S (Pen Strep); GIBCO, USA). The cells were maintained with 5% CO2 at 37 C in an incubator. 2.6 Animal model of subcutaneous hepatocellular carcinoma Subcutaneous BNL tumor-bearing mice were established by the injection of 1 × 106 BNL cells into the right leg of 5-6 week old Balb/c mice, which were purchased from BioLASCO Taiwan (Ilan, Taiwan). After 7 days, subcutaneous BNL hepatoma tumor-bearing mice were established. 2.7 Biodistribution of 188Re micro-particles and 188Re-Tin-colloid micro-particles For the investigation of in vivo biodistribution, the distributions of

188Re

micro-particles

and 188Re-Tin-colloid micro-particles (equivalent to 37 MBq of 188Re) in mice were obtained by

using

nuclear

images

of

single-photon

emission

computed

tomography

(SPECT)/computed tomography (CT). The micro-particles were intratumorally injected when the subcutaneous tumors reached a volume of 150-200 mm3. The SPECT images and X-ray CT images were acquired using the Nano-SPECT/CT scanner system (XSPECT, Mediso, USA). Following administration of the micro-particles intratumorally, SPECT images were acquired at 1, 4, 24, 48, and 72 h using a low-energy, high-resolution collimator. Immobilization of the mice was ensured through inhalation of anesthetic isoflurane (Abbott Labs, England). Following capture of the SPECT images, CT images were acquired (X-ray source: 50 kV, 0.4 mA; 256 projections) at exactly the same position. For the investigation of biodistribution, the mice were sacrificed following intratumoral injection for 1, 4, and 24 h (n = 3). The radioactivities uptaken by the tumor and normal 6 ACS Paragon Plus Environment

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tissues were determined with a γ-counter (Cobra series model 5003, USA). All distribution data of tumors and tissues are expressed as the percentage of injected dose ratio to tissue weight (g) (%ID/g). 2.8 In vivo antitumor efficacy of 188Re micro-particles, 188Re-Tin-colloid micro-particles For in vivo antitumor efficacy experiments, an initial tumor size of 150-200 mm3 was required for each mouse. The subcutaneous HCC-bearing mice were randomly divided into three treatment groups (n = 6): control group (normal saline), and

188Re-Tin-colloid

188Re

micro-particles group,

micro-particles group. Normal saline (0.2 mL; 0.9% NaCl solution,

Taiwan Biotech Co., Ltd. Taoyuan City, Taiwan) was injected intratumorally, or 111 MBq/0.2 mL of

188Re

micro-particles or

188Re-Tin-colloid

micro-particles was injected

intratumorally. During the experimental observation period, the tumor sizes and body weights of the treated mice were measured at an interval of 3-4 days. Subcutaneous tumor volumes were calculated as [(length)(width)2]/2, where length and width are dimensions of tumor in millimeter. The tumor sizes and changes of body weight in each mouse were recorded for 60 days. In order to draw survival curve using Kaplan-Meier data analysis software (Small Stata 7.0, Stata Corporation, USA), the “dead dates” of the mice were determined in 90 days. The definition of the dead date was >30 mm in length or body weight loss >20% without recovery in few days. Survival assessment of the three experimental groups was analyzed with the log-rank test. p < 0.05 was considered to indicate a statistically significant difference. To evaluate the therapeutic responses of intratumorally injected normal saline, micro-particles, and

188Re-Tin-colloid

188Re

micro-particles, tumor tissues from antitumor efficacy

experiments were collected at the end of the observation period. Hematoxylin and eosin (H&E) staining, (NADPH)-diaphorase staining, and proliferating cell nuclear antigen (PCNA) immunohistochemical staining were used for pathology studies; staining methods were described in our previous report.38



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Scheme 1. Illustration of 188Re-Tin-colloid micro-particles. This radioisotope delivery system inhibited

HCC

and

enhanced

the

antitumor


effects

of

188Re.

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3. Results and discussions 3.1 Characterization of 188Re micro-particles and 188Re-Tin-colloid micro-particles The present study examined 188Re-Tin-colloid

188Re

188Re

micro-particles and

micro-particles (SnCl2 15, 25, 35 mg/mL), to determine the 188Re

percentage of micro-particles. The 188Re-Tin-colloid

loading rate of the loading rates of the

188Re

188Re

loading

micro-particles and

micro-particles (35 mg/mL) were was 14.92 ± 2.76% and 93.05 ± 0.95%,

respectively (Table 1). The

188Re

micro-particles had a relatively low

percentage (93%). The results of the

188Re-Tin-colloid

188Re

loading

micro-particles (SnCl2 35 mg/mL,

load rate demonstrated that

188Re

had loaded into the

micro-particles, which is caused by positively charged Sn ion (Sn2+) binding with negatively charged ions (ion exchange mechanism) in the material component of the micro-particles. The particle size of the 188Re-Tin-colloid

188Re

micro-particles was 161.2 ± 6.2 μm and that of the

micro-particles was 63.6 ± 1.8 μm (Supporting Information, Figure S1).

Furthermore, the particle size of Re-colloid was about 3-8 μm (Supporting Information, Figure S2) as in our previous study.38 The optical microscope images showed that the particle size of the

188Re

micro-particles were larger than that of the

micro-particles (Figure 1). The diameter of the

188Re-Tin-colloid

reduced about 40% (39.45%), compared with the

188Re

188Re-Tin-colloid

micro-particles was

micro-particles. This result was

similar to the commercially available DC bead (DC Bead Drug Delivery Embolisation System; Biocompatibles, UK), in which the diameter was found to be reduced to 25% following loading with doxorubicin.39 Table 1. 188Re loaded rate of the 188Re micro-particles and 188Re-Tin-colloid micro-particles. SnCl2 (mg/mL) 188Re

micro-particles

188Re-Tin-colloid

micro-particles 188Re

0

15

25

35

14.92±2.76%

--

--

--

--

90.89±2.38%

92.43±1.83%

93.05±0.95%

micro-particles (0 mg/mL SnCl2); 188Re-Tin-colloid micro-particles (15, 25, 35 mg/mL

SnCl2).



Page 10 of 24

The surface zeta potential of alginate-based micro-particles (-29.7 mV),

188Re

micro-particles (-32.2 mV), and 188Re-Tin-colloid micro-particles (-22.9 mV) were measured (as shown in Supporting Information, Figure S3). Results of surface zeta potential of 188Re-Tin-colloid

micro-particles demonstrated that Sn2+ cations of SnCl2 attractive to

micro-particles, which reduced the negative charge of micro-particles. Attraction between positive charge and negative charge or reduced negative charges maybe cause the structure of 188Re-Tin-colloid

micro-particles to become tight and have smaller particles size than

micro-particles. However,

188Re

micro-particles sucked

188ReO -, 4

which resulting in more

negative charges and loose structure of micro-particles. The SEM images showed the samilar results (Figure 2). The elemental analysis indicated that the content of Tin (Sn) were about 20.69 and 0 wt % in 188Re-Tin-colloid micro-particles and 188Re micro-particles, respectively (Supporting Information, Table S1, S2 and Figure S4, S5). Tin was existed in 188Re-Tin-colloid

micro-particles, but not in

experiments confirmed that

188Re

188Re-Tin-colloid

micro-particles. The results of the above micro-particles maintained

188Re

in

micro-particles for a long time through the formation of 188Re-Tin-colloids, which trapped in the pores of micro-particles.

Figure 1. Optical microscope images of micro-particles (A), 188Re-Tin-colloid

micro-particles (B) and

micro-particles (C) in ddH2O. (Scale bar = 100 μm)

Figure 2. SEM images of micro-particles (A), 188Re-Tin-colloid

188Re

188Re

micro-particles (B) and

micro-particles (C) after being lyophilized. (Scale bar = 100 μm) 10 ACS Paragon Plus Environment

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The release profiles of

188Re

from

188Re

micro-particles and

188Re-Tin-colloid

micro-particles (35 mg/mL SnCl2) were assessed (Figure 3). The release profiles revealed that 188Re-Tin-colloid micro-particles had slower release than 188Re micro-particles after 48 h. From the 188Re

188Re-Tin-colloid

from

188Re

188Re-Tin-colloid

micro-particles,

Evaluation Efficacy of Rhenium-188-Loaded Micro ... - ACS Publications

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