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ANTICANCER RESEARCH 27: 2217-2226 (2007)

Biodistribution, Pharmacokinetics and MicroSPECT/CT Imaging of 188Re-BMEDA-Liposome in a C26 Murine Colon Carcinoma Solid Tumor Animal Model

YA-JEN CHANG1, CHIH-HSIEN CHANG1, TSUI-JUNG CHANG1, CHIA-YU YU1, LIANG-CHENG CHEN1, MEEI-LIN JAN1, TSAI-YUEH LUO1, TE-WEI, LEE1 and GANN TING2

1Institute of Nuclear Energy Research, Taoyuan, Taiwan; 2National Health Research Institutes, Miaoli, Taiwan, R.O.C.

Abstract. Nanoliposomes are useful carriers in . retention (EPR) effect on leaky tumor sites (1, 2). Since Radiolabeled nanoliposomes have potential applications in both the inside and outside regions of liposomes contain radiotherapy and diagnostic imaging. In this study, the hydrophilic phosphoric end groups, various drugs or biodistribution and pharmacokinetics of 188Re-BMEDA-labelled bioactive molecules could be encapsulated in the internal pegylated liposomes (RBLPL) and unencapsulated 188Re- aqueous compartment of liposomes and circulated in the BMEDA administered by the i.v. route in murine C26-colon blood, and the liposomes would be removed from the tumour-bearing mice were investigated. MicroSPECT/CT circulation by the reticulo-endothelial system (RES) after images were performed to evaluate the distribution and tumor binding with (3-5). Nanoliposome has been widely targeting of RBLPL in mice. For the biodistribution study, the studied as an important carrier in controlling the highest uptake of liposome in tumors was 3.62%±0.73% at 24 localization and concentration of drug molecules or other h after RBLPL administration, and the tumor to muscle ratio bioactive molecules for passive targeted therapy (6-12). of RBLPL was 7.1-fold higher than that of 188Re-BMEDA. Changing the pharmacokinetic properties of drug- With image analysis, the highest SUV in tumor was 2.81±0.26 encapsulated pegylated nanoliposomes extends the at 24 h after of RBLPL. The Pearson correlation retention of drugs in tumor or inflamed tissue (13), and analysis showed a positive correlation of tumor targeting or therefore promotes therapeutic efficacy with reduced uptake of RBLPL between biodistribution and microSPECT cellular toxicity and side-effects. The structure and size semi-quantification imaging analysis (r=0.633). The results of advantage of the liposome -bilayer membrane enables it the pharmacokinetics revealed that the area under the tissue to be degraded in the body with minimum toxicity and does concentration-time curve (AUC) of RBLPL was 4.7-fold higher not cause any immune response, unlike proteins (3), so that than that of unencapsulated 188Re-BMEDA. These results it can be applied for multi-dose treatments. suggested the potential benefit and advantage of 188Re-labeled N,N-bis(2-mercaptoethyl)-N’,N’-diethylethylenediamine nanoliposomes for imaging and treatment of malignant diseases. (BMEDA) is an SNS pattern ligand with a tridentate structure that has one nitrogen and two sulfur atoms (14, Nanoliposomes, double-membrane lipid vesicles with a 15). These three atoms are able to offer electrons to particle size from 10 nm to 100 nm, are important carriers rhenium-186, rhenium-188 and technium-99 resulting in a capable of packaging drugs in various drug delivery lipophilic complex in a neutral state. 99mTc-SNS/S has been applications through the enhanced permeability and studied for the possibility of brain imaging (16). Rhenium- 188 is a radionuclide for imaging and therapeutic dual applications due to its short physical half-life of 16.9 h with 155 keV gamma emission, and its 2.12 MeV ‚ emission, with Correspondence to: Dr. Gann Ting, National Institute of Cancer a maximum tissue penetration range of 11 mm. In addition, Research, National Health Research Institutes A191Ward, 188Re can be obtained from a generator, which makes it Veterans General Hospital-Taipei 201, Shih-Pai Rd., Sec.2, Beitou convenient for routine research and clinical use. Although Taipei 112, Taiwan, ROC. Fax: +886228716467, e-mail: 186Re-BMEDA-labeled pegylated liposomes have been [email protected] studied in normal rats, preclinical studies on the applications 188 Key Words: Biodistribution, nanoliposome, microSPECT/CT, of Re-BMEDA-labeled pegylated nanoliposomes in pharmacokinetics, rhenium-188, 188Re. tumor-bearing mice have not yet been reported.

0250-7005/2007 $2.00+.40 2217 ANTICANCER RESEARCH 27: 2217-2226 (2007)

Functional imaging of small animals, such as mice and ligand to make 188Re-SNS/S complexes. Five mg of BMEDA were rats, using positron emission tomography (PET) and single- pipetted into a fresh vial. A volume of 0.5 mL of 0.17 mol/L photon emission computed tomography (SPECT), is glucohepatonate dissolved in a 10% acetate was added, becoming a valuable tool for studying animal models of followed by the addition of 120 ÌL (10 Ìg/ÌL) of stannous chloride. After flushing the solution with N2 gas, highly specific activity of human diseases (17). Drug-containing liposomes have been 188Re-sodium perrhenate was added. The vial was sealed and identified by labeling them with photon-emitting heated in an 80ÆC water-bath for 1 h. The labeling efficiency of radionuclide for pharmacokinetics and biodistribution 188Re-BMEDA complexes was confirmed using paper studies (18-24), which allows longitudinal assessment with chromatography with normal saline as the eluent. non-invasive imaging in the same animal. Although the use of quantitative PET imaging in small animals has been Preparation of liposome. Liposomes were prepared as described reported (25), only a few types of quantitative SPECT/CT elsewhere (12, 27). DSPC, cholesterol, and DSPE2000-PEG (molar imaging were used in research applications. In this study, ratio is 3:2:0.3) were dissolved in 8 mL chloroform. The chloroform was evaporated to dryness under vacuum with a rotary evaporator semi-quantitative microSPECT/CT imaging, biodistribution at 60ÆC, the lipid film was hydrated at 60ÆC in ammonium sulfate 188 and pharmacokinetics of RBLPL and Re-BMEDA via solution (250 mM, pH 5.0, 530 mOs), and a containing intravenous injection were investigated in a C26 colon multilamellar vesicles was obtained. Liposomes were obtained by carcinoma mouse model. The major objective of this study freezing and thawing repeatedly using nitrogen and 60ÆC was to demonstrate the potential advantages of nano- water six times. The nanoliposomes were then extruded through targeted RBLPL applications for dual-functional imaging polycarbonate membranes (Costar, Cambridge, MA, USA) of pore and radiotherapy of an animal tumor model. sizes 0.1 and 0.05 Ìm consecutively using high-pressure extraction equipment at 55ÆC to make nanoliposomes of smaller size. The extra-liposomal salt was removed by a Sephadex G-50 column Materials and Methods (Pharmacia, Uppsala, Sweden) equilibrated with normal saline. The final concentration of nanoliposomes was estimated using a Materials. Distearoylphosphatidylcholine (DSPC), cholesterol and phosphate assay (28). It was found to contain 14 Ìmole/ml (average M.W. 2000)-derived distearoyl- . The pegylated nanoliposomes had an average (PEG-DSPE) were purchased from particle size of 74.2 nm ±9.1 nm, determined using an 830 nm UV- Avanti Polar (Genzyme, ª∞, USA). Cell culture materials VIS spectrophotometer (Jasco V-530, Tokyo, Japan). were obtained from GIBCO BRL (Grand Island, NY, USA). PD- 10 column and Sepharose 4 Fast Flow were purchased from GE Liposome labeling with 188Re-BMEDA. The labeling method for Healthcare (Uppsala, Sweden). N,N-bis(2-mercaptoethyl)-N’,N’- liposome with 188Re-BMEDA was as previously described by Bao diethylethylenediamine (BMEDA) were purchased from ABX et al. (14), with some modifications. Briefly, 188Re-BMEDA (Radeberg, Germany). All other chemicals were purchased from solution was adjusted to a pH of 7.0 prior to liposome labeling. The Merck (Darmstad, Germany). 188 liposomes encapsulating (NH4)2SO4 were mixed with Re- Cell line and animal model. The C26 murine colon carcinoma cell BMEDA solution and incubated in a 60ÆC water-bath for 1 h. PD- line was obtained from the American Type Culture Collection 10 column (G-25, Amersham Pharmacia Biotech, Sweden) (Manassas, VA, USA). It was grown in RPMI-1640 medium chromatography with normal saline was used to separate 188 supplemented with 10% (v/v) fetal bovine serum (FBS) and 2 mM radiolabeled liposomes from free Re-BMEDA. The labeling efficiency was determined from the 188Re activity before and after L-glutamine at 37ÆC in 5% CO2. Cells were detached with 0.05% trypsin/0.53 mM EDTA in Hanks’ Balanced Salt Solution (HBSS). separation using a Radioisotope Calibrator CRC-15R (Capintec, Four-week-old male BALB/c mice were obtained from the USA). The labeling efficiency of RBLPL was calculated as: National Animal Center of Taiwan (Taipei, Taiwan, ROC.), with food and water being provided ad libitum in the animal house of Radioactivity of fractions the Institute of Nuclear Energy Research (INER), Taoyuan with RBLPL (Taiwan). Animal protocols were approved by the Institutional Labeling efficiency (%)=100x Animal Care and Use Committee (IACUC) at the Institute of Total fraction radioactivity + Nuclear Energy Research. Seventy-two mice were subcutaneously column residue inoculated with 2x105 tumor cells in the right hind flank. The animals developed tumors of about 750 mm3 in size after tumor In vitro stability study of RBLPL. In vitro stability of RBLPL was cell inoculation. studied by incubating RBLPL (100 ÌL, 25 ÌCi) in rat plasma (1:19) at 37ÆC and in normal saline (1:1) at room temperature Preparation of 188Re-BMEDA. 188Re was obtained from an alumina- (RT), respectively. At desired times (1, 4, 8, 12, 24, 48 and 72 h), based 188W/188Re generator (26). Elution of the 188W/188Re 200 ÌL of RBLPL plasma solution were removed using a generator with normal saline provided of carrier-free micropipette to a column that was packed with SepharoseTM 4 188 Re as sodium perrhenate (NaReO4). The labeling method for Fast Flow (Amersham Pharmacia Biotech, Uppsala, Sweden) for BMEDA radiolabeled with 188Re was as previously described by separation of RBLPL from free 188Re-BMEDA complexes in Bao et al. (14), with some modifications. Briefly, BMEDA (ABX, plasma. At the same time-points, 200 ÌL of RBLPL solution were Germany), stannous chloride was used as the reductant and transferred to a column that was packed with Sephadexì G-50 glucoheptonate (GH) (Sigma, USA) was used as an intermediate (Amersham Pharmacia Biotech, Uppsala, Sweden) for the

2218 Chang et al: Biodistribution, Pharmacokinetics and MicroSPECT/CT Imaging of 188Re-Labeled Pegylated Liposomes separation of RBLPL from free 188Re-BMEDA complexes in Table I. In vitro stability of 188Re-BMEDA-labeled pegylated nanoliposomes normal saline. The 188Re activity was counted using a Cobra II at different times after incubation in normal saline at room temperature or Auto-Gamma counter (Hewlett-Packard, USA). The percentage rat plasma at 37ÆC (mean±SD, n=3). This stability study was performed of 188Re associated with pegylated nanoliposomes was determined up to 72 hours. by dividing the activity in pegylated nanoliposomes after separation by the total activity. Incubation time (h) Normal saline (%) Rat plasma (%)

Biodistribution studies. Mice (5 in number at each time-point) were 1 98.34±0.85 94.40±2.16 4 97.98±1.06 93.38±2.14 i.v. injected with 65 ÌCi of 188Re-BMEDA or RBLPL ( 16 97.20±1.08 91.22±1.34 concentration 0.17 Ìmol/mL) at 14 days after the implantation of 750 24 96.35±1.17 90.48±1.77 mm3-sized tumors. At various time-points (1, 4, 24, 48, 72, 96 h), mice 48 93.73±0.75 88.56±2.88 were sacrificed by CO2 asphyxiation. Blood samples were collected 72 92.01±1.31 82.40±1.64 through cardiac puncture. Organs of interest were removed, washed and weighed, and the radioactivity was measured with a Cobra II Auto-Gamma counter. The results were expressed as the percentage injected dose per gram of tissue (%ID/g). The correlation of the tumor uptake of RBLPL by biodistribution Pharmacokinetic studies. Concentration in whole blood samples was studies and microSPECT/CT semi-quantificative imaging analysis normalized to the percentage of injected dose per gram (% ID/g) at was analyzed using the Pearson correlation coefficient (r) using SPSS various time-points after intravenous injection of 188Re-BMEDA or version 11.0 software (SPSS Inc., Chicago, IL, USA) (29, 30). RBLPL (1, 4, 24, 48, 72, 96 and 200 h). Pharmacokinetic parameters were determined using the WinNonlin software version 5.0.1 (Pharsight Corp., Mountain View, CA, USA). Noncompartmental Results analysis model 201 (IV- input) was used with the log/linear trapezoidal rule. The pharmacokinetic parameters including area 188 under the tissue concentration-time curve (AUC), Cmax (the Re-BMEDA and RBLPL labeling. The labeling efficiency of maximum concentration), Cl (clearance) (mL/h), and MRT (mean 188Re-BMEDA complex was determined using ITLC-SG paper residence time) were calculated. chromatography eluted in normal saline and the experimental result showed that the labeling efficiency of 188Re-BMEDA MicroSPECT/CT imaging studies. SPECT images and X-ray CT complex was 99%±1.73% (n=3). The labeling efficiency of images were acquired using a microSPECT/CT scanner system (XSPECT, Gamma Medica, Northridge, CA, USA). SPECT RBLPL was approximately 80% (n=3) after incubating the 188 Imaging was performed using low-energy, high-resolution nanoliposomes with Re-BMEDA at 60ÆC for 1 h. collimators at 1, 4, 24, 48, 72 h after the intravenous injection of RBLPL (n=2). The source and detector are mounted on a circular In vitro labeling stability. In vitro labeling stability of RBLPL gantry allowing it to rotate 360Æ around the subject (mouse) is shown in Table I. The labeling stability was positioned on a stationary bed. The radius of rotation (ROR) was 93.75%±0.75% (n=3) at 48 h and 92.01%±1.31% at 72 h 1.0 cm with a field of view (FOV) of 1.37 cm. The imaging (n=3) in normal saline, respectively. RBLPL showed lower acquisition was accomplished using 64 projections at 120 sec per projection. For calculating standardized tumor uptake value stability after incubation in rat plasma at 37ÆC; the stability (SUV), 10-50 ÌCi radioactivity of Re-188 was used as a reference was 88.56%±2.88% (n=3) at 48 h and 82.40%±1.64% at sources. The energy window was set at 155 KeV±10~15%. The 72 h (n=3). SPECT imaging was followed by CT image acquisition (X-ray source: 50 kV, 0.4 mA; 256 projections) with the animal in exactly Biodistribution and pharmacokinetics of 188Re-BMEDA and the same position. The COBRA Exxim software was used for the RBLPL. Biodistribution of 188Re-BMEDA and RBLPL at 1, CT image reconstruction, the LumaGEM software was used for the 4, 24, 48 and 72h after intravenous injection are listed in Tables SPECT image reconstruction and the IDL 6.0 software was used for SPECT/CT imaging fusion. The SPECT images were II and III, respectively. The distribution profiles of the RBLPL 188 reconstructed to produce image sizes of 56x56x56 with an image are very different from that of Re-BMEDA. The results of 188 resolution of 0.2 mm. The CT images were also reconstructed to Re-BMEDA showed no significant accumulation in tumor, produce image sizes of 512x512x512 with an image resolution of liver, spleen or other organs. The fast blood clearance, and fast 0.3 mm. The SUV was determined from the radioactivities in the excretion from the liver, lung, bowel, kidneys and urine were regions of interest (ROI) on the tumor after intravenous injection observed (Table II). High levels of radioactivity were found in of RBLPL, the location of the edge of the ROI was the contour for the feces before 24 h. This indicated that the radioactivity was 70% of maximum intensity. The SUV was calculated according to the following standard formula: excreted rapidly in the feces within 24 h. The levels of radioactivity within the tumor peaked at 1 h and then declined 188 Mean ROI activity (ÌCi/g) rapidly. The highest tumor/muscle ratio (Tu/Mu) of Re- SUV= BMEDA of 2.01%±0.30% appeared at 1 h after injection, and Total injected activity (ÌCi)/mouse body weight (g) decreased progressively to 1.26%±0.21%, 0.76%±0.12%,

2219 ANTICANCER RESEARCH 27: 2217-2226 (2007)

Table II. Biodistribution of 188Re-BMEDA in murine C26-colon tumor-bearing BALB/c mice at 1, 4, 24, 48 and 72 hours after i.v. injection of 65 ÌCi of 188Re-BMEDA.

188Re-BMEDA (%ID/g) 1 h 4 h 24 h 48 h 72 h

Whole blood 19.85±7.88 8.32±5.10 0.33±0.17 0.28±0.03 0.15±0.02 Brain 0.50±0.19 0.22±0.12 0.18±0.01 0.02±0.00 0.02±0.00 Skin 0.75±0.09 0.52±0.11 0.23±0.06 0.27±0.05 0.50±0.35 Muscle 0.52±0.08 0.88±0.49 0.24±0.03 0.14±0.02 0.09±0.01 Bone 2.16±0.77 1.69±1.13 0.69±0.09 0.23±0.05 0.16±0.04 Spleen 4.67±1.80 3.50±1.75 0.80±0.30 1.53±0.74 0.35±0.06 Small intestine 4.34±1.34 6.58±3.92 0.63±0.15 0.37±0.10 0.35±0.14 Large intestine 1.24±0.38 1.86±0.49 1.39±0.33 0.32±0.05 0.16±0.03 Kidney 6.31±0.58 5.43±1.85 1.66±0.37 1.29±0.10 2.06±1.11 Lung 6.14±1.57 4.12±1.18 0.96±0.30 0.73±0.04 0.54±0.17 Heart 2.77±0.63 2.23±0.93 2.01±0.14 0.51±0.09 0.29±0.03 Liver 12.33±0.07 8.64±0.12 4.09±0.08 2.70±0.64 2.22±0.13 Bladder 3.70±0.83 1.76±0.85 0.35±0.14 0.30±0.05 0.25±0.10 Tumor 1.06±0.23 0.89±0.47 0.51±0.06 0.10±0.02 0.07±0.01 Pancreas 2.16±0.24 1.25±0.34 0.76±0.18 0.89±0.13 0.41±0.12 Feces 1.02±0.26 30.33±12.20 14.37±7.11 3.95±1.77 5.01±4.05

Tu / Mu 2.01±0.30 1.80±0.60 1.26±0.21 0.76±0.12 0.71±0.07

Data were expressed as percentage of injected dose per gram (%ID/g±standard error , n=5 at each time-point). Tu(tumor)/Mu(muscle).

Table III. Biodistribution of RBLPL in murine C26-colon tumor-bearing BALB/c mice at 1, 4, 24, 48 and 72 hours after i.v. injection of 65 ÌCi of RBLPL.

RBLPL (%ID/g) 1 h 4 h 24 h 48 h 72 h

Whole blood 36.09±1.34 29.17±0.52 11.31±1.56 3.60±0.39 2.50±1.16 Brain 0.75±0.13 0.61±0.04 0.27±0.04 0.12±0.01 0.07±0.03 Skin 0.49±0.04 0.54±0.03 0.58±0.04 0.72±0.12 0.62±0.12 Muscle 0.45±0.05 0.46±0.11 0.42±0.11 0.36±0.06 0.24±0.04 Bone 2.57±0.10 2.29±0.75 1.19±0.24 0.81±0.05 0.81±0.21 Spleen 4.80±0.50 10.10±0.63 12.56±3.19 9.24±1.84 10.15±2.60 Small intestine 3.27±0.95 9.58±1.95 9.78±2.35 6.07±1.17 3.63±1.24 Large intestine 1.62±0.74 1.74±0.36 1.25±0.15 1.09±0.32 1.15±0.52 Kidney 7.13±0.45 6.51±0.16 3.74±0.36 2.50±0.10 2.10±0.48 Lung 11.28±0.90 8.70±0.61 4.51±0.59 1.67±0.20 1.33±0.36 Heart 3.48±0.38 3.07±0.07 1.60±0.20 0.82±0.07 0.83±0.22 Liver 10.41±0.13 11.43±0.14 12.41±0.95 7.79±0.31 7.18±0.07 Bladder 1.08±0.12 0.94±0.16 0.74±0.08 0.62±0.09 0.42±0.11 Tumor 1.87±0.22 2.10±0.21 3.62±0.73 2.49±0.35 2.31±0.62 Pancreas 1.82±0.24 1.77±0.14 0.81±0.06 0.64±0.12 0.54±0.16 Feces 0.43±0.07 24.56±8.56 20.46±11.71 14.37±5.27 36.65±18.75

Tu / Mu 4.41±0.71 6.06±1.66 8.99±0.92 8.14±1.75 8.12±1.76

Data were expressed as percentage of injected dose per gram (%ID/g±standard error, n=5 at each time-point). Tu(tumor)/Mu(muscle).

0.71%±0.07% at 24, 48, 72 h after injection, respectively. In found to be 3.62%±0.73% at 24 h after RBLPL contrast, the highest uptake of RBLPL in the liver and spleen administration. The tumor uptake of RBLPL was steadily reached 12.41%±0.95% and 12.56%±3.19%, respectively, at maintained until 72 h after administration (2.31%±0.62%). 24 h after administration. Very low levels of nanoliposome The highest Tu/Mu ratio of RBLPL reached 8.99±0.92 at 24 h uptake were observed in the organs of the central nervous and after injection. The Tu/Mu ratios were 8.14±1.75 and musculoskeletal systems. The highest uptake in tumors was 8.12±1.76 at 48 and 72 h, respectively.

2220 Chang et al: Biodistribution, Pharmacokinetics and MicroSPECT/CT Imaging of 188Re-Labeled Pegylated Liposomes

Table IV. Pharmarcokinetic parameters of 188Re-BMEDA and RBLPL uptake after intravenous injection in BALB/c mice bearing C26 tumors.

Parameters 188Re-BMEDA RBLPL

Cmax (%ID/g) 19.85 36.09 Cl (mL/h) 0.58 0.12 AUC(0→∞) (%ID/gh) 172.59 800.60 MRT(0→∞) (h) 10.87 18.51

The pharmacokinetic parameters are presented in Table IV. The maximum concentration of 188Re-BMEDA and RBLPL was 19.85%ID/mL and 36.09%ID/mL, respectively. 188 The mean residence time (MRT0→∞) of Re-BMEDA was 10.87 h, which is shorter than that of RBLPL (18.51 h). The Figure 1. Tumor uptake of 188Re-BMEDA and RBLPL after 188 area under the time curve (AUC0→∞) of Re-BMEDA administration in BALB/c mice bearing C26 tumors. RBLPL or 188Re- and RBLPL was 172.59%ID/gh and 800.60%ID/gh, BMEDA containing 0.5 mCi of 188Re was administered to each mouse by respectively. The clearance rate (Cl) of 188Re-BMEDA was intravenous injection (%ID/g±SEM, n=5 at each time-point). 0.58 mL/h, which is 4.8-fold higher than that of RBLPL (0.12 mL/h), and may partially explain the lower AUC of 188Re-BMEDA compared with that of RBLPL. Compared with the results of tumor uptake after 188Re-BMEDA and Discussion RBLPL administration, RBLPL exhibited significantly higher uptake and concentration in tumor lesions than those Nanoliposomes encapsulating weak acid or (NH4)2SO4 of 188Re-BMEDA injected mice at each time-point (Figure (ammonium gradient liposome) have been used in the 1). The MRT of RBLPL was 1.8-fold greater than that of entrapment of chemotherapeutic agents for drug delivery 188Re-BMEDA. The AUC of RBLPL was 4.7-fold greater (13). The chemotherapeutic agents containing an amine than that of 188Re-BMEDA. group can be trapped in the lower pH environment of the liposome inner space. Bao et al. (14, 19) have MicroSPECT/CT imaging and standardised tumor uptake reported that liposomes can be radiolabeled with 186Re value (SUV). The microSPECT/CT imaging of 188Re- or 99mTc using BMEDA in normal rat studies. The BMEDA indicated no significant uptake in the tumors at 1 ammonium gradient is formed by removing ammonium and 4 h after intravenous injection (Figure 2a). The sulfate from the external liposome medium by + longitudinal microSPECT/CT imaging showed the filtration. Intraliposomal NH4 dissociates into NH3, accumulation of RBLPL in the liver, spleen and tumor which easily escapes from the liposomes, and H+ is then targeting at 1, 4, 24, 48 and 72 h after intravenous injection retained in the aqueous phase of the liposome. The (Figure 2b), and the images revealed a higher uptake in radiolabeling method of 188Re-nanoliposome utilized the tumors at 24, 48 and 72 h after intravenous injection. The lipophilic region of 188Re-BMEDA crossing the uptake in spleen and liver were the common features of lipophilic double-membrane of liposome. After 188Re- liposome distribution after intravenous injection into mice. BMEDA complexes enter the liposome’s inner space, the The accumulated activity of RBLPL in the tumor at the amine group of the 188Re-BMEDA complex is time-points was calculated from the images created by protonized and the complex becomes hydrophilic and drawing the region of interest (ROI) using the standard forms a gel-like precipitate followed by trapping of the 2– source as a point of reference. The SUV of RBLPL in SO4 anions in liposomes. The BMEDA is an ideal tumors was 1.94±0.10 at 1 h after injection, peaking at 24 h intermediate for radio-nanoliposome labeling. With our after injection (2.81±0.26). The trend of uptake analyzed by modification, the 188Re loading efficiency of nano- imaging is similar to the results of the biodistribution study liposome can reach higher than 80%. (Table V). A positive correlation (r=0.663) of tumor uptake 188Re-BMEDA showed fast blood clearance, fast of RBLPL by biodistribution and microSPECT semi- excretion from the bowel and kidney, and no significant quantification image analysis was obtained using Pearson accumulation in the liver or spleen. Our results showed a correlation analysis. significantly higher accumulation of RBLPL in liver and

2221 ANTICANCER RESEARCH 27: 2217-2226 (2007)

Figure 2. Comparisons of MicroSPECT/CT images of RBLPL or 188Re-BMEDA targeting C26 tumors bearing in BALB/c mice. The RBLPL and 188Re-BMEDA containing 0.5 mCi of 188Re was administered to each mouse by intravenous injection. The images were acquired at 1, 4, 24, 48 and 72 h after injection. The energy window was set at 155 KeV±10~15%, the image size was set at 64x64 pixels. The color map shows the SPECT pixel values from 0 to the maximum expressed with an arbitrary value of 100. (a) Images of mice at 1 and 4 h after i.v. injection of 188Re- BMEDA. (b) Images of mice at 1, 4, 24, 48 and 72 h after injection of RBLPL.

2222 Chang et al: Biodistribution, Pharmacokinetics and MicroSPECT/CT Imaging of 188Re-Labeled Pegylated Liposomes spleen compared with that of 188Re-BMEDA at all time- Table V. Comparisons of tumor uptake of RBLPL analyzed by points after administration (Tables II and III). The typical microSPECT imaging and biodistribution. distribution pattern of nanoliposomes via intravenous Time (h) microSPECT Biodistribution injection also has a slow blood clearance, and a relatively (SUV) (% ID/g) high spleen and liver accumulation (12-14, 18, 24). High accumulation of RBLPL was found in the lungs; this may be 1 1.94±0.10 1.87±0.22 due to the small tracheal diameter in the lungs of mice 4 2.72±0.27 2.10±0.21 causing accumulation of liposome in the lungs. The 24 2.81±0.26 3.62±0.73 targeting, concentration and accumulation of RBLPL in the 48 2.17±0.14 2.49±0.35 tumor is higher than that of 188Re-BMEDA and steadily persists in the tumor during the study period. In this study, Data were expressed as mean±standard error, n=2 and 5 for the tumor uptake and localization of RBLPL was microSPECT imaging and biodistribution, respectively. approximately 1.8-, 2.4-, 7.1-, 25- and 33-fold larger than the uptake of 188Re-BMEDA at 1, 4, 24, 48 and 72 h after administration, respectively. Safra et al. (31) report that the tumor size is an important correlation for tumor biodistribution studies (%ID/g) with prognostic factor for therapeutic response to Doxil in microSPECT semi-quantification imaging analysis (SUV) of ovarian cancer, which suggests that the tumor volume is tumor uptake from 1 to 48 h (r=0.663). This result clinically relevant for liposome uptake. The trend is that the suggested that microSPECT/CT imaging with dual- liposome uptake is higher in smaller tumors (32, 33), which functional 188Re-BMEDA-liposome is a good tool for is consistent with Harrington’s findings in animal tumor studying the tumor targeting, distribution and real-time models (34). The hypovascular areas and increased therapeutic response of 188Re-therapeutics in vivo. interstitial pressure in large tumors will interfere with Radiotherapy has proven to be highly effective against extravascular convective transport and decrease liposome primary tumors, preventing the development of uptake (35). In our current study, the tumor uptake of metastatic disease. Radioimmunotherapy uses anti-CD20 RBLPL in smaller tumors (200 mm3) was found to be target-specific radioimmunoconjugates with I-131 or Y- approximately two-fold higher than that in larger tumors 90 radionuclides that are successfully applied for clinical (750 mm3) (data not shown). treatment of non-Hodgkin’s lymphoma (37). Theragnostic In the pharmacokinetic study, the clearance rate of imaging will take advantage of molecular imaging for RBLPL was smaller than that of 188Re-BMEDA, and the biology-driven target-radiotherapy planning and 188 MRT(0→∞) of RBLPL was greater than that of Re- monitoring. The recent research and development of BMEDA. These data demonstrated prolonged retention of passive and active nano-targeted diagnostics and radiolabelled pegylated nanoliposomes, which was therapeutics will accelerate the progress and consistent with the results of Tu/Blood ratio. The slow achievements of these goals. Rhenium-188 has a half-life clearance rate of RBLPL may partially explain a greater of 17 h and a maximum beta energy of 2.12 MeV. The AUC than that of 188Re-BMEDA suggesting that the short physical half-life of 188Re may allow for higher pegylated nanoliposomes have higher than doses or multi-doses for tumor radiotherapy. Moreover, that of 188Re-BMEDA, and the transportation of passive 188Re could be obtained by daily elution from a nano-targeted liposomes loaded with radionuclides on 188W/188Re generator, and is very convenient and suitable tumor sites was achieved. for routine clinical use. MicroSPECT/CT imaging is a non-invasive imaging modality that can longitudinally monitor the behavior of Conclusion radiotherapeutics in the same animal across different time- points. Bao et al. (36) used 99mTc-liposomes for imaging Drug delivery through passive and active targeted studies via direct intratumoral injection. In this study, a creates a new opportunity and challenge for dual-functional imaging and therapeutic radionuclide of novel anticancer diagnostics and therapeutics development. 188Re which has longer half-life (17 h) than that of 99mTc (6 The results of the biodistribution, pharmacokinetics and in h) was selected, and the prepared passive nano-targeted vivo nuclear imaging studies here revealed the potential therapeutics of 188Re-BMEDA-liposome has dual function advantage and benefit of our developed dual-functional for imaging and radiotherapy on tumors at the same time. nano-targeted RBLPL for site-specific tumor imaging and Figure 2 shows clear tumor targeting and localization of therapeutics. The therapeutic efficacy and toxicology data nano-targeted RBLPL in vivo imaged with microSPECT/CT of this novel nano-targeted radiopharmaceuticals will be after 1 to 72 h of administration. Table V reveals a positive studied in future investigations.

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31 Safra T, Groshen S, Jeffers S, Tsao-Wei DD, Zhou L, 35 Jain RK: Delivery of molecular and cellular medicine to solid Muderspach L, Roman L, Morrow CP, Burnett A and Muggia tumors. Adv Drug Deliv Rev 26: 71-90, 1997. FM: Treatment of patients with ovarian carcinoma with 36 Bao A, Phillips WT, Goins B, Zheng X, Sabour S, Natarajan M, pegylated liposomal doxorubicin: analysis of toxicities and Ross Woolley F, Zavaleta C and Otto RA: Potential use of drug- predictors of outcome. Cancer 91: 90-100, 2001. carried-liposomes for cancer therapy via direct intratumoral 32 Harrington KJ, Mohammadtaghi S, Uster PS, Glass D, Peters injection. Int J Pharm 316: 162-169, 2006. AM, Vile RG and Stewart JS: Effective targeting of solid 37 Macklis RM and Pohlman B: Radioimmunotherapy for non- tumors in patients with locally advanced cancers by radiolabeled Hodgkin's lymphoma: a review for radiation oncologists. Int J pegylated liposomes. Clin Cancer Res 7: 243-254, 2001. Radiat Oncol Biol Phys 66: 833-841, 2006. 33 Lim HJ, Masin D, McIntosh NL, Madden TD and Bally MB: Role of drug release and liposome-mediated drug delivery in governing the therapeutic activity of liposomal mitoxantrone used to treat human A431 and LS180 solid tumors. J Pharmacol Exp Ther 292: 337-345, 2000. 34 Harrington KJ, Rowlinson-Busza G, Syrigos KN, Abra RM, Uster PS, Peters AM and Stewart JS: Influence of tumour size Received January 30, 2007 on uptake of 111ln-DTPA-labelled pegylated liposomes in a Revised April 27, 2007 human tumour xenograft model. Br J Cancer 83: 684-688, 2000. Accepted May 8, 2007

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