Cytotoxic Effects of Pegylated Anti-EGFR Immunoliposomes Combined with Doxorubicin and Rhenium-188 Against Cancer Cells

ANTICANCER RESEARCH 35: 4777-4788 (2015)

Cytotoxic Effects of PEGylated Anti-EGFR Immunoliposomes Combined with Doxorubicin and Rhenium-188 Against Cancer Cells

WEI-CHUAN HSU1,2, CHU-NIAN CHENG2, TE-WEI LEE2 and JENG-JONG HWANG1

1Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan, R.O.C.; 2Division of Isotope Application, Institute of Nuclear Energy Research, Taoyuan, Taiwan, R.O.C.

Abstract. Background/Aim: We aimed to construct the treatment of a wide range of cancer types, including non- epidermal growth factor receptor (EGFR)-targeting Hodgkin’s and Hodgkin’s lymphoma, multiple myeloma, and cetuximab-immunoliposomes (IL-C225) for targeted delivery lung, ovarian, gastric, thyroid, breast, sarcoma, and pediatric of doxorubicin and rhenium-188 (Re-188) to EGFR+ cancer cancer (1, 2). Doxorubicin therapy is associated with a cells. Materials and Methods: Synthesized IL-C225 was variety of side-effects, including myelotoxicity, stomatitis, analyzed by dynamic light scattering, transmission electron fatigue, complete alopecia, and cardiotoxicity because it can microscopy, and matrix-assisted laser desorption/ionization also target normal cells (3). Several research groups have time-of-flight mass spectroscopy. Cell binding and attempted to improve the toxicity profile of doxorubicin (4). internalization were examined using doxorubicin-loaded IL- For example, the use of liposomal encapsulation technology, C225 (DXR-IL-C225) with confocal microscopy. IL-C225 which provides lipid carriers for targeting the drug, has combined with doxorubicin and Re-188 (188Re-DXR-IL- resulted in dramatic improvement of pharmacokinetics, C225) was synthesized, and the cytotoxic effects of 188Re- reduced toxicity, and improved therapeutic efficacy (5-7). DXR-IL-C225 were analyzed in EGFR+ cancer cells using An unspecific polyethylene glycol (PEG)ylated cell viability assays. Results: IL-C225 bound to EGFR on liposomal doxorubicin formulation (commercial name A431 cancer cells and was rapidly internalized. Furthermore, Doxil/CAELYX) has been developed and has different IL-C225 localized within the tumor cells efficiently. 188Re- pharmacokinetics and biodistribution from those of free DXR-IL-C225 exhibited outstanding cytotoxic effects against doxorubicin due to the enhanced permeability and retention EGFR+ cancer cells in vitro and showed superior cytotoxic (EPR) effect. Doxil has shown efficacy in the treatment of effects compared to DXR-IL-C225 or 188Re-IL-C225 alone. Kaposi’s sarcoma, metastatic breast cancer, ovarian cancer, Conclusion: The new formulation of 188Re-DXR-IL-C225 may multiple myeloma, and other malignancies (8). During the be a potential theranostic vehicle for delivery of drugs in the past decade, several approaches to further improve the treatment of EGFR-overexpressing human cancer. performance of PEGylated liposomal doxorubicin, such as antibody-coated liposomes [immunoliposomes (ILs)], have The anthracycline doxorubicin, a metabolite of Streptomyces been extensively studied in vitro and in vivo (9). Pre- peucetius var. caesius, is a chemotherapeutic agent used in clinical studies have shown that ILs bind specifically to receptors, subsequently internalizing encapsulated drugs and releasing them into the intracellular environment. Correspondence to: Professor Jeng-Jong Hwang, Department of Moreover, anti-epidermal growth factor receptor (EGFR) Biomedical Imaging and Radiological Sciences, National Yang- ILs containing doxorubicin (DXR-IL-C225) are under Ming University, No. 155, Sec. 2, Li-Nong Street, Taipei 112, evaluation in clinical trials (10). Taiwan (ROC). Tel: +886 228267064, Fax: +886 228201095, e- The current focus of nanomedicine is the development of mail: [email protected], and Dr. Te-Wei Lee, Division of Isotope combinational or multi-functional nanoplatforms combining Application, Institute of Nuclear Energy Research, 1000 Wenhua multiple drugs. Chemotherapy drug-loaded liposomes can be Road, Chiaan Village, Lungtan, Taoyuan 325, Taiwan (ROC). Tel: +886 34711400, Fax: +886 34711416, e-mail: [email protected] combined with radioisotopes and used for cancer therapy and diagnosis (i.e. theranosis) (11-13). Herein, we examined the Key Words: Immunoliposomes, 188Re-DXR-IL-C225, cytotoxicity, use of rhenium-188 (Re-188) combined with DXR-IL-C225 theranostic vehicle. for several reasons. Firstly, liposomes have become an

0250-7005/2015 $2.00+.40 4777 ANTICANCER RESEARCH 35: 4777-4788 (2015) excellent vehicle for drug delivery for radioisotopes, such as BRL (Grand Island, NY, USA). WST-1 assay kits were obtained In-111, Tc-99m, Re-188, Ga-67 and Ac-225. Unlike liposomes from Roche Diagnostics (Mannheim, Germany). PD-10 column and loaded with chemotherapeutic agents, fewer radioliposomes Sepharose 4B were purchased from GE Healthcare (Uppsala, Sweden). BMEDA was purchased from ABX (Radeberg, Germany). are under clinical trials, and no commercialized Traut’s Reagent and BCA Assay kits were purchased from Thermo radioliposomes have been developed. One example is the Scientific (Rockford, IL, USA). All other chemicals were purchased tumor contrast agent VesCan (In-111-liposomes), in which In- from Merck (Darmstad, Germany). 111 enters the liposome through ionophore binding to nitrilotriacetic acid, allowing In-111 to remain stably within the Preparation of PEGylated liposomes. Unilamellar liposomes were liposome. However, this drug failed to be commercialized due prepared by a lipid film hydration-extrusion method. A chloroform solution of lipids consisting of DSPC/cholesterol/DSPE-PEG to drug sensitivity, complexity, and competition with other (3:2:0.24 mol%), with DSPE-PEG-Mal (0.06 mol%) was dried into tumor contrast agents (14). Additionally, Re-188- and Re-186- a thin film in a round-bottom flask on a rotary evaporator and then labeled N,N-bis(2-mercaptoethyl)-N’,N’-diethylethylenediamine further dried for 2 h under nitrogen. The resulting thin lipid film (BMEDA)-embedded liposomes have been used in basic was hydrated in 5 ml of a solution of (NH4)2SO4 (250 mM, pH 5.0). research for external passive targeted-radiotherapy in animal The resulting suspension was subjected to six cycles of freezing and studies (15, 16). In recent years, a method for embedding α- thawing and then serially extruded 10-15 times through a track- particles of radioisotopes into liposomes has also been etched polycarbonate membrane (Costar, Cambridge, MA, USA) with pore sizes of 200, 100, and 50 nm using a Lipex Extruder developed (17). For example, Chang et al. published a (Northern Lipids, Vancouver, Canada) at 60˚C. After purification by method for enhancing the entry of Ac-225 into liposomes Sephadex G-50 chromatography, liposome size and zeta potential (18). Suna et al. used an amphipathic polychelating were determined by dynamic light scattering on a nano-ZX compound for surface labeling of liposomes to obtain labeled (Malvern, UK). The phospholipid content was measured using a products for effective, highly specific diagnostic capacity colorimetric assay (22). Liposomes were stored at 4˚C until use. (19). Thus, radiolabeled liposomes may have potential for Preparation of IL-C225. ILs constructed by conjugation with an cancer therapy and imaging. However, fewer reports have intact MAb (IgG, as a nontargeting control MAb, and C225, as the studied radiolabeled ILs. Secondly, Re-188 is a high-energy targeting MAb) were thiolated for 1 h at room temperature by β-emitting radioisotope obtained from a tungsten-188/Re- reacting with 5-fold excess of Traut’s reagent in degassed HEPES 188 (W-188/Re-188) generator, which has shown utility for a buffer (20 mM HEPES, 140 mM NaCl, 2 mM EDTA, pH 8.0) (23). variety of therapeutic applications in nuclear medicine, For direct coupling of MAb to liposomes, thiolated MAb was added oncology, and interventional radiology/cardiology (20, 21). to preformed liposomes (DSPC/cholesterol/DSPE-PEG/DSPE-PEG- Thus, Re-188 encapsulated within DXR-IL-C225 (188Re- Mal at 3:2:0.24:0.06 mol ratio) at 600 μg MAb per μmol lipids and then gently shaken at room temperature for 4 h under nitrogen. DXR-IL-C225) has the potential to enhance the cytotoxic Excess maleimide groups were quenched by incubation with 2- efficacy of DXR-IL-C225. Finally, theranostic agents contain mercaptoethanol (1 mM) for 30 min at room temperature. Unbound both diagnostic and therapeutic functions, enabling MAb and 2-mercaptoethanol were then removed by Sepharose CL- diagnosis, therapy, and monitoring of therapeutic responses 4B gel filtration. After purification, liposome size, zeta potential, simultaneously. Re-188 decay is accompanied by 155-keV and phospholipid content were measured, as previously described. γ-emission, which can be detected by γ-cameras for imaging, Lowry protein assays were carried out using BCA kits to determine biodistribution, or absorbed radiation dose studies. Therefore, the coupling efficiency. The purified IL-C225 was identified by matrix-assisted laser desorption/ionization time-of-flight mass 188Re-DXR-IL-C225 may have potential as a novel 188 spectroscopy (MALDI-TOF) as follows. The C225 standard or theranostic drug. Here, we synthesized Re-DXR-IL-C225 purified IL-C225 solution was mixed with a saturated sinapinic acid and examined the quality, targeting capacity, and efficacy of matrix solution at a volumetric ratio (Vsample/Vmatrix) of 1:10. The this formulation in cancer cells overexpressing EGFR. solution (5 μl) was then transferred to the MALDI target surface. After the solution was dried under air, the target was induced into Materials and Methods the MALDI ion source of a MALDI-TOF/TOF mass spectrometer (Bruker Daltonics Ultraflex III TOF/TOF) (Bruker Daltonics, GmbH, Bremen, Germany) operated in linear mode. The sample Reagents. 1,2-Distearoyl-distearoylphosphatidylcholine (DSPC), spot was irradiated with a pulsed nitrogen laser (337 nm) for cholesterol, and sn-glycero-3-phosphoethanolamine-N-[methoxy desorption and ionization. (poly(ethylene glycol))-2000] (DSPE-PEG) were purchased from Genzyme (Cambridge, MA, USA). 1,2-Distearoyl-sn-glycero-3- Transmission electron microscopy (TEM) analysis. Liposome- phosphoethanolamine-N-[maleimide-(poly(ethylene glycol))2000] conjugated C225 MAbs were examined using a conjugate of 10 nm (DSPE-PEG-Mal lipid) was purchased from NOF Corporation gold and an anti-mouse secondary antibody (Sigma, St. Louis, MO, (Tokyo, Japan). C225 (cetuximab), a human-mouse chimerized USA). Liposomes and IL-C225 (0.5-1 μmol) were incubated with monoclonal antibody (MAb) directed against EGFR, was kindly undiluted IgG gold conjugate (1×1012 gold particles) for 1 h in provided by Taipei Veterans General Hospital (Taipei, Taiwan, 0.018 M Tris-buffered saline, pH 8.0, with 0.9% bovine serum ROC). Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640 albumin and 17% glycerol in a total volume of 100 μL and then medium, and fetal bovine serum (FBS) were obtained from GIBCO examined directly by TEM.

4778 Hsu et al: Preparation and In Vitro Evaluation of 188Re-DXR-IL-C225

Cells. A431 human epidermoid carcinoma cells (overexpressing 188Re-IL-IgG, and 188Re-liposomes (phospholipid content: 19.8- EGFR), COLO 205 human colon adenocarcinoma cells, and MAD- 20.7 nmol). Cells were incubated for 2 h at 4˚C and 37˚C in MB-435 human breast adenocarcinoma cells (low expression of triplicate. For the blocking study, 200 μg C225/tube was added. The EGFR) were obtained from the National Health Research Institutes cells were washed with cold PBS twice to remove unbound ILs. (Taipei, Taiwan, ROC). A431 and MAD-MB-435 cells were grown After centrifugation at 179 × g for 5 min, we removed and collected in DMEM supplemented with 10% (v/v) FBS. COLO 205 cells the supernatant. Cellular uptake efficiency was calculated using the were grown in RPMI-1640 medium supplemented with 10% (v/v) following formula: FBS. All cells were cultured in a humidified atmosphere containing 5% CO2 at 37˚C. Cells were detached with 0.05% trypsin/0.53 mM activity of 188Re in the cells EDTA in Hanks’ balanced salt solution. pellet after 2 h incubation Cellular uptake = ×100 efficiency (%) Fluorescence confocal microscopy of IL-C225. To evaluate cell- total activity of 188Re added to the cells specific binding and internalization of IL-C225, fluorescence confocal microscopy was performed. Liposomes were prepared as described above with doxorubicin loading (135 μg doxorubicin/ Time-dependent cellular uptake of 188Re-IL-C225, 188Re-IL-IgG, μmol lipid). The amount of doxorubicin trapped inside the liposome and 188Re-liposomes was examined. Briefly, approximately 1×106 was determine with a spectrofluorometer (FP6200, JASCO) at an cells/tube were resuspended and incubated with radiolabeled excitation wavelength of 475 nm and an emission wavelength of 580 liposomes or ILs as described above. The cells were incubated at nm. A431 cells cultured as monolayers in T75 flasks were 4˚C and 37˚C for 0.083, 1, 2, 4, or 15 h. After incubation, the cells harvested, pelleted by centrifugation at 179 × g for 5 min, and were washed and counted, and the radioactivity was measured as resuspended in serum-supplemented media. Cells were seeded onto described. circular cover slips (12 mm in diameter) at a density of 1×106 cells/well and cultured for 4 h before the experiment. Cells were Cellular retention of 188Re-IL-C225 in A431 cells. A431 cells were then incubated with 5 μM DXR-loaded liposomes (DXR- resuspended in 1.5-ml Eppendorf tubes at a density of 1×106 liposomes), DXR-loaded nontargeted IL-IgG (DXR-IL-IgG), or cells/tube. 188Re-IL-C225 or 188Re-IL-IgG (22 ng/ml, 1.5 nM) DXR-IL-C225 for 2 h at 37 or 4˚C. To study the receptor-mediated were diluted in DMEM and added to prewashed cells for a total uptake kinetics, cells were further incubated for 2, 16, or 24 h with volume of 1 ml/tube. 188Re-IL-C225 or 188Re-IL-IgG were different liposome and IL preparations (1 μmol phospholipids/ml) incubated with cells at 37˚C for 2 h. All cells were then washed with doxorubicin. After incubation, cells were rinsed three times three times in cold PBS and further incubated with 1 ml fresh with cold PBS and fixed with 3.7% formaldehyde. Thereafter, medium for 0, 1, 2, 4, 8, 24, or 48 h. After incubation, the medium coverslips containing cells were mounted onto slides using glycerol was collected, cells were washed, and radioactivity was measured. and observed with an Olympus FLUOVIEW FV300 confocal The percentage of radioactivity retention was calculated using the microscope (Olympus, Tokyo, Japan). following formula: Preparation of nanotargeted 188Re-IL-C225 and 188Re-DXR-IL-C225. activity of 188Re retained in The radiolabeling of ILs using 188Re was carried out as follows. 188Re Retained the cells after incubation was obtained from an alumina-based 188W/188Re generator. Elution radioactivity (%) = ×100 of the 188W/188Re generator with normal saline provided solutions of total activity of 188Re in the carrier-free Re-188 as sodium perrhenate (NaReO ). Five milligrams 4 cell pellet and medium of BMEDA was pipetted into a new vial then 0.5 ml of 0.17 M sodium gluconate/0.17 M acetate solution was added, followed by 120 μl stannous chloride (10 mg/ml). After flushing the solution with Cytotoxicity of 188Re-DXR-IL-C225. The cytotoxic activity of 188Re- DXR-IL-C225 and other drug formulations was measured in A431 N2 gas, 0.2-0.5 m1 NaReO4 (0.37-1.85 GBq) was added. The mixtures were incubated at 80˚C for 1 h. The labeling efficiency of cells using WST-1 assays. In a 96-well plate, adherent A431 (8×103 188Re-BMEDA was analyzed by thin layer chromatography-silica gel cells/well) or MDA-MB-435 (4×103 cells/well) cells were treated 188 188 with medium containing 188Re-DXR-IL-C225, 188Re-IL-C225, using normal saline (Rf values: ReO4, 0.8-1.0; Re-BMEDA, 0.0-0.2) as developer. The PEGylated liposomes, IL-C225, IL-IgG, or DXR-IL-C225, 188Re-liposomes, DXR-liposomes, liposomes, or DXR-IL-C225 (1 ml) were added to the 188Re-BMEDA (50-250 normal saline at 37˚C for 2 h in a total volume of 200 μl/well. The MBq) solution and incubated at 60˚C for 30 min. The radiolabeled specific activity of 188Re-loaded liposomes or ILs was 4.4 MBq liposomes or ILs were separated from free 188Re-BMEDA using a (phospholipid content of liposomes or ILs ranged from 3 to PD-10 column (GE Healthcare) eluted with normal saline. The 4 μmol/ml) in 200 μl reaction medium. The concentration of labeling efficiency was determined by measuring the activity in doxorubicin was 0.1 μg/ml loaded in liposomes or ILs. After PEGylated liposomes or ILs after separation divided by the total incubation, the cells were washed three times with cold PBS, and activity before separation. treated cells were further incubated for 3 days with fresh medium. Next, 10% WST-1 reagent was added for 1 h. Formazan crystals Cellular uptake of 188Re-IL-C225. Cellular uptake experiments were were extracted and quantified in an enzyme-linked immunosorbent performed at both 4˚C and 37˚C to study the effects of receptor- assay (ELISA) reader at 450 nm. The number of living cells was mediated endocytosis of C225-conjugated ILs. Briefly, A431 and confirmed by trypan blue exclusion staining. Data are presented as COLO 205 cells were resuspended in culture medium at a density of mean±standard deviations (SDs). Unpaired t-test was used for 1×106 cells/1.5-ml Eppendorf tube. The medium was exchanged comparison between two experiments. A value of p<0.05 was with 1 ml culture medium containing 0.055 MBq 188Re-IL-C225, considered statistically significant.

4779 ANTICANCER RESEARCH 35: 4777-4788 (2015)

Figure 1. Scheme for the preparation of rhenium-188-doxorubibin-immunoliposome- cetuximab (188Re-DXR-IL-C225). BMEDA: N,N-bis(2- mercaptoethyl)-N’,N’-diethylethylenediamine; DXR: doxorubibin; IL: immunoliposome; ITLC-SG/sali: thin layer chromatography-silica gel using normal saline; C225: cetuximab; PEGylated: polyethylene glycolylated; MALDI-TOF-TOF-MS: matrix-assisted laser desorption/ionization time-of- flight mass; DSPC: 1,2-Distearoyl-distearoylphosphatidylcholine; DSPE-PEG: sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))- 2000]; DSPE-PEG-Mal lipid: 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide-(poly(ethylene glycol))2000]. QC: Quality control.

Table I. Characterization of doxorubibin-immunoliposome-cetuximab (DXR-IL-C225), rhenium-188-immunoliposome-cetuximab (188Re-IL-C225) and rhenium-188-doxorubibin-immunoliposome-cetuximab (188Re-DXR-IL-C225).

DXR-IL-C225 188Re-IL-C225 188Re-DXR-IL-C225

PL concentration (μmol/ml) 3.77±0.58 3.93±0.85 3.28±0.78 Antibodies/liposome 16.9±2.35 22.5±4.35 16.9±2.35 Particle size (nM) 79.59±23.09 88.59±29.70 81.29±28.17 Zeta potential (mV) −3.08 −1.71 −2.17 Loading efficiency of 188Re to IL-MAbs --- >75% >75% Specific activity (MBq/ml) --- 22.2-55.5 <55.5 Loading efficiency of DXR to IL-MAbs >95% --- >95% DXR concentration (μg/μmol PL ) 130±5 --- <130±5 Vesicle/ml 4.29×1013 3.62×1013 3.95×1013

DXR: Doxorubibin; IL: immunoliposome; C225: cetuximab; PL: phospholipid; MAbs: monoclonal antibodies. Data are the mean±SD obtained from at least three replications.

Results Thiolated antibodies (IgG and C225) were reacted with the maleimide group on the distal termini of PEG chains on Preparation and characterization of liposomes and IL-C225. these liposomes to form thioether linkages. The molar ratio Figure 1 summarizes the preparation scheme and of C225/Traut’s/liposome-Mal was examined in order to characterization assays used in the study. In order to directly determine the optimal conditions for preparation of IL-C225. conjugate C225 to the liposomes, the liposome formulation After purification of the ILs (Figure 2A), we measured the contained 0.06 mol% DSPE-PEG-Mal synthesized by a lipid liposome size (Figure 2B), phospholipid and protein film hydration-extrusion method. The mean diameter of concentrations of ILs, and number of MAbs (IgG and C225) liposomes containing DSPE-PEG-Mal was 75.43±20.66 nm. per liposome (Table I).

4780 Hsu et al: Preparation and In Vitro Evaluation of 188Re-DXR-IL-C225

Figure 2. A: Elution profiles of immunoliposomes (ILs) constructed with liposome-monoclonal antibodies (MAbs) and unbound MAbs. A Sepharose CL-4B gel filtration column was used. Fractions 5-7 contained immunoliposomes-cetuximab (IL-C225) or IL-IgG. Fractions 10-15 were free, unbound C225 and IgG. Each fraction had a volume of 0.5 ml. The protein content was measured by Lowry protein assay. B: Particle size analysis of IL-C225. C: Positive-ion matrix-assisted laser desorption/ionization (MALDI) mass spectra of standard C225 and IL-C225.

Figure 3. Transmission electron micrograph of polyethylene glycol (PEG)ylated immunoliposomes (PEGylated ILs) negatively stained with phosphotungstic acid solution. Liposomes (A) and cetuximab (C225)-conjugated liposomes (B, C) were imaged. The scale bars are 100 nm for (A), 80 nm for (B), and 20 nm for (C).

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Figure 4. Fluorescence micrographs of A431 cells treated with immunoliposomes (ILs). A: Internalization of immunoliposomes-cetuximab (IL-C225), nontargeted IgG-conjugated ILs, and liposomes in A431 cells. A431 cells were incubated with liposomes, C225, or IgG ILs containing doxorubibin (DXR) fluorescent dye for 4 h at 37˚C. Right: Cells visualized in the phase-contrast mode; left: cells visualized in the fluorescence mode: (i, ii) A431 cells treated with DXR-loaded liposomes; (iii, iv) A431 cells treated with DXR-loaded nontargeted IL-IgG; (v, vi) A431 cells treated with DXR-loaded IL-C225. B: Confocal micrographs of A431 cells treated with control DXR-liposomes (i, iv), non-targeted DXR-IL-IgG (ii, v), and targeted DXR-IL-C225 (iii, vi). Suspended cells were treated with drugs for 2 h at 4˚C (i-iii) or 37˚C (iv-vi).

4782 Hsu et al: Preparation and In Vitro Evaluation of 188Re-DXR-IL-C225

Figure 5. A: Confocal laser scanning microscopy of A431 cells incubated with doxorubibin (DXR)-loaded liposomes (A, D, G), immunoliposomes- IgG (IL-IgG) (B, E, H), or immunoliposomes-cetuximab (IL-C225) (C, E, I) for 2 (A-C), 16 (D-F), and 24 h (G-I).

Standard C225 and C225-conjugated liposomes were treated with fluorescent DXR-loaded liposomes or ILs were identified with MALDI-TOF/MS, and the positive ion-mode observed by confocal laser scanning microscopy. The loading MALDI mass spectra are shown in Figure 2C. A efficiency of doxorubicin was more than 95%. Fluorescence characteristic protein ion signal at m/z 147,680 was detected micrographs of A431 cells treated with DXR-loaded for standard C225. For C225-conjugated liposomes, the liposomes or ILs are shown in Figure 4A. additional increased ion signal at m/z 151,401 was detected. IL-C225 bound to the A431 cell surface (4˚C) and Moreover, signals at m/z 73,878 and m/z 75,125 were localized intracellularly (37˚C). However, the incubation of doubly-charged protein ion signals of standard C225 and A431 cells with DXR-liposomes or nontargeted DXR-IL-IgG C225-conjugated liposomes, respectively. PEGylated did not result in any detectable fluorescence (Figure 4B). The nanoparticles were generally spherical and of regular size fluorescent intensity of IL-C225 weakened at 16 and 24 h when examined by TEM (Figure 3). Figure 3A shows after incubation (Figure 5). liposomes treated with 10-nm gold and anti-mouse IgG. However, C225-conjugated nanoliposomes were visualized Preparation of nanotargeted 188Re-IL-C225 and 188Re-DXR- by binding to a conjugate of 10-nm gold and anti-mouse IgG IL-C225. The labeling efficiency of 188Re-BMEDA was greater (Figures 3B and 3C). than 95%. The loading efficiencies of 188Re-IL-C225 and Fluorescence confocal microscopy of IL-C225. In order to 188Re-DXR-IL-C225 were 82.3%±4.5% and 81.1%±5.1%, confirm the specific cellular binding and localization of IL- respectively. The radiochemical purities of 188Re-IL-C225 and C225 on EGFR-overexpressing cancer cells, A431 cells 188Re-DXR-IL-C225 were more than 95%.

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Figure 6. Cellular uptake of rhenium-188-immunoliposome-IgG (188Re-IL-IgG), rhenium-188-immunoliposome-cetuximab (188Re-IL-C225), and 188Re-liposomes by A431 and COLO 205 cells 2 h after incubation at 37˚C (A, B) and 4˚C (C, D) with (B, D) and without (A, C) 200 μg cetuximab. Data (means±SDs) were obtained from at least three experiments.

188 After Re-BMEDA encapsulation into IL-C225 and DXR- liposomes and 188Re-IL-IgG (Figures 6A and 6C). The IL-C225, the average particle size, zeta potential, phospholipid cellular uptake efficiencies of 188Re-IL-C225 were 15% (4˚C) 188 content, and protein concentration of Re-BMEDA- and 38% (37˚C) in A431 cells, respectively. However, no encapsulated IL-C225 or DXR-IL-C225 were measured (Table significant difference was observed for the cellular uptake I). According to the average particle size and phospholipid efficiencies of 188Re-IL-C225 in COLO 205 cells at 4˚C and 188 188 content, the numbers of Re-IL-C225 and Re-DXR-IL- 37˚C, respectively (Figures 6B and 6D). Moreover, analysis 13 13 C225 in solution is 3.62×10 and 3.95×10 vesicles/ml, of the kinetics of 188Re-IL-C225 binding and internalization 188 respectively. The specific activities of Re-IL-C225 and in A431 cells showed that 188Re-IL-C225 binding began 188 Re-DXR-IL-C225 both are lower than 59.2 MBq/ml. The within 1 h and plateaued after 4 h (Figures 7A and 7B). No ratios of Abs/liposome are ranged from 16.9 to 22.5. significant difference was observed in COLO 205 cells. Together, these data showed that IL-C225 efficiently localized Cellular uptake of 188Re-IL-C225 in A431 cells. In order to within cancer cells and that the cellular uptake of ILs was confirm the EGFR receptor-mediated cellular binding and associated with receptor-mediated endocytosis. internalization of the 188Re-ILs, temperature-dependent cellular uptake of 188Re was compared between liposomes Cellular retention of 188Re-IL-C225 in A431 cells. Figure 8 and ILs. 188Re-IL-C225 exhibited higher cellular uptake in shows the cellular retention of non-targeted 188Re-IL-IgG A431 cells at 4˚C and 37˚C, and than that of both 188Re- and targeted 188Re-IL-C225 on EGFR-overexpressing A431

4784 Hsu et al: Preparation and In Vitro Evaluation of 188Re-DXR-IL-C225

Figure 7. Time-dependent uptake of rhenium-188-immunoliposome-IgG (188Re-IL-IgG), rhenium-188-immunoliposome-cetuximab (188Re-IL-C225), and 188Re-liposomes in A431 (A, B) and COLO 205 cells (C, D) at 37 (A, C) and 4˚C (B, D). Data (means±SDs) were obtained from at least three experiments.

cells. The remaining cell-associated radioactivity after 48 h overexpressing cancer cells. However, the multi-functional of incubation was 72% for 188Re-IL-C225, while almost all formulation of 188Re-DXR-IL-C225 showed additional cell-associated radioactivity had disappeared after 8 h of significant cytotoxic effects (about 65% growth inhibition incubation with 188Re-IL-IgG. when compared with the control group) for EGFR+ cancer cells (Figure 9), demonstrating the superior cytotoxic effects Cytotoxicity of 188Re-IL-C225 and 188Re-DXR-IL-C225. In of 188Re-DXR-IL-C225 compared with DXR-IL-C225, and order to evaluate the cytotoxic effects of 188Re-DXR-IL- 188Re-IL-C225 alone. C225 in EGFR-overexpressing cancer cells, we performed a preliminary dose-dependent cytotoxicity study using free Re- Discussion 188 or doxorubicin against A431 and MDA-MB-435 cells. 188 The IC50 of Re-IL-C225 was 3.70-5.55 MBq/ml when the Combinational nanodrug formulations are emerging as new cells were incubated at 37˚C for 2 h. The IC50 of DXR-IL- promising nanomedicines for drug delivery systems due to C225 in A431 cells was about 0.1 μg/ml when the cells were their multiple functions. In this study, 188Re-DXR-IL-C225 incubated at 37˚C for 2 h. DXR-IL-C225 and 188Re-IL-C225 was synthesized with the aim of characterizing the caused a significant decrease (50-60%) in growth when physicochemical features of the novel combination drug and compared to the control group (normal saline) in EGFR- evaluating its bioactivity and cytotoxic efficacy. Many

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Figure 8. Cellular retention of non-targeted rhenium-188- Figure 9. Effects of liposomes and cetuximab (C225)-conjugated immunoliposome-IgG (188Re-IL-IgG) and targeted rhenium-188- immunoliposomes (ILs) containing various drugs on growth inhibition of immunoliposome-cetuximab (188Re-IL-C225). Data (means±SDs) were A431 and MDA-MB-435 cells. Cell growth was analyzed by WST-1 obtained from at least three experiments. assay. Data (means±SDs) were obtained from at least three experiments. **p<0.01.

methods have been used for evaluating manufactured surface EGFR of tumor cells (i.e. in A431 cells) and was nanomaterials, including techniques in optical spectroscopy, rapidly internalized. IL-C225 was also efficiently localized electron microscopy, surface scanning, light scattering, within tumor cells, implying the capacity for superior circular dichroism, magnetic resonance, mass spectrometry, cytotoxic efficacy in EGFR+ cancer cells. Furthermore, the X-ray scattering and spectroscopy, and zeta potential rapid elimination of 188Re-IL-IgG suggests that 188Re-IL- measurements, as well as thermal techniques, centrifugation, IgG associates with the liposome surface in a nonspecific chromatography, and electrophoresis (24-26). Although the manner. These results imply that 188Re-loaded 'active incorporation of antibodies into liposomes is a well- targeting' ILs may increase retention of the drug within established technique, the efficacy of IL-C225 must be tumor cells and avoid washout from tumor tissue in vivo. confirmed by determining its bioactivity and physical The principles of combination chemotherapy, i.e. the characteristics. Herein, we demonstrated the physical combination of therapeutic efficacy for drugs with different characteristics of IL-C225 with dynamic light scattering, mechanisms of action and non-overlapping side-effects, can be TEM, and MALDI-TOF. Mass spectrometry is a well-known applied to the development of nanomedicines. A variety of approach for highly sensitive and selective detection of nano- different combinations have been use in recent years, with at and biomaterials. In the analysis of C225 conjugated with least additive increases in therapeutic outcomes for the liposomes by MALDI-TOF/MS, the ion signal at m/z combinations compared to individual therapies (28-30). 151,401 did not correspond to the molecular weight of the Although the present study was an in vitro study, we showed an C225-conjugated liposomes, and no other ion signals at addition of cytotoxic combinational therapy when combining higher m/z values were observed. The positive-ion MALDI Re-188 and DXR-IL-C225. Certain studies have shown that mass spectra revealed that the m/z value of standard C225 Re-188 combined with liposomal or peptide-linked liposomal (m/z 147,680) and C225-conjugated liposomes (m/z 151,401) doxorubicin may have additive or synergistic effects in animal differed by the molecular weight of DSPE-PEG-Mal. models (31, 32). The bioactivity of IL-C225 was evaluated with The design of this formulation may represent a novel doxorubicin or Re-188-loaded IL-C225. DXR-loaded anti- multifunctional/theranostic radionanomedicine. Some lipo- EGFR ILs have been widely studied (27), and a phase I somal theranostic nanomedicines have been studied; however, clinical study using ILs loaded with a chemotherapy drug is the primary focus of these studies was vinorelbine combined currently underway. In our study, DXR-IL-C225 and 188Re- with In-111. Animal studies have shown that the formulation IL-C225 showed specific binding and internalization in of liposomes encapsulated with vinorelbine and In-111 has a EGFR+ cancer cells. Moreover, doxorubicin and Re-188 synergistic effect (33). In-111 is an auger electron radionuclide were retained within the EGFR+ cancer cells for 48 h after used as an imaging and therapeutic agent. However, in therapy delivery. Additionally, 188Re-IL-C225 bound to the cell with In-111, high doses are necessary. Recently, In-111 was

4786 Hsu et al: Preparation and In Vitro Evaluation of 188Re-DXR-IL-C225 also used in clinical trials. For example, 111In-pentetreotide has 8 Duggan ST and Keating GM: Pegylated liposomal doxorubicin: been evaluated in a phase II clinical study; patients are required a review of its use in metastatic breast cancer, ovarian cancer, to be treated with a total dosage ranging from 6,660 to 46,620 multiple myeloma and AIDS-related Kaposi's sarcoma. Drugs 71: 2531-2558, 2011. MBq (34, 35) Re-188 is a β-emitter and may therefore be more 9 Gao J, Chen H, Song H, Su X, Niu F, Li W, Li B, Dai J, Wang suitable for therapy in the clinical setting, with a requirement H and Guo Y: Antibody-targeted immunoliposomes for cancer 188 for relatively lower doses. Therefore, Re-DXR-IL-C225 treatment. Mini Rev Med Chem 13: 2026-2035, 2013. may be a better vehicle for theranostic agents in cancer therapy. 10 Mamot C, Ritschard R, Wicki A, Stehle G, Dieterle T, However, while several multifunctional or theranostic Bubendorf L, Hilker C, Deuster S, Herrmann R and Rochlitz C: radionanomedicines have been reported in the literature (30, Tolerability, safety, pharmacokinetics and efficacy of DXR- 31, 36), no studies have examined radiochemical immuno- loaded anti-EGFR immunoliposomes in advanced solid tumours: a phase 1 dose-escalation study. Lancet Oncol 13: 1234-1241, liposomal multifunctional/theranostic radionano-medicines. 2012. Because the immunoliposomal vehicle has been proven to be 11 Lee DS, Im HJ and Lee YS: Radionanomedicine: Widened 188 superior to liposomal vehicles in vitro and in vivo, Re-DXR- perspectives of molecular theragnosis. Nanomedicine 11: 795- IL-C225 may have great potential as an improved theranostic 810, 2015. radionanomedicine in the treatment of cancer. 12 Muthu MS and Wilson B: Multifunctional radionanomedicine: a novel nanoplatform for cancer imaging and therapy. Conclusion Nanomedicine (Lond) 5: 169-171, 2010. 13 Ryu JH, Koo H, Sun IC, Yuk SH, Choi K, Kim K and Kwon IC: Tumor-targeting multi-functional nanoparticles for theragnosis: Multi-functional, nanoactive targeted 188Re-DXR-IL-C225 + new paradigm for cancer therapy. Adv Drug Deliv Rev 64: 1447- showed outstanding cytotoxic effects against EGFR cancer 1458, 2012. cells in vitro, with superior cytotoxic effects compared to 14 Jensen GM and Bunch TH: Conventional liposome performance DXR-IL-C225 and 188Re-IL-C225 alone. This multi- and evaluation: lessons from the development of Vescan. J functional liposomal formulation may have great potential as Liposome Res 17: 121-137, 2007. a theranostic radionanomedicine. 15 Chang YJ, Chang CH, Chang TJ, Yu CY, Chen LC, Jan ML, Luo TY, Lee TW and Ting G: Biodistribution, pharmacokinetics and Acknowledgements microSPECT/CT imaging of 188Re-BMEDA-liposome in a C26 murine colon carcinoma solid tumor animal model. Anticancer The Authors thank Hung LM, Yu CY and Chang TJ for their Res 27: 2217-2225, 2007. 16 Chen LC, Chang CH, Yu CY, Chang YJ, Hsu WC, Ho CL, Yeh technical assistance. CH, Luo TY, Lee TW and Ting G: Biodistribution, pharmacokinetics and imaging of (188)Re-BMEDA-labeled pegylated References liposomes after intraperitoneal injection in a C26 colon carcinoma ascites mouse model. Nucl Med Biol 34: 415-423, 2007. 1 Speth PA, van Hoesel QG and Haanen C: Clinical pharmacokinetics 17 Borchardt PE, Yuan RR, Miederer M, McDevitt MR and of doxorubicin. Clin Pharmacokinet 15: 15-31, 1988. Scheinberg DA: Targeted actinium-225 in vivo generators for 2 Keizer HG, Pinedo HM, Schuurhuis GJ and Joenje H: therapy of ovarian cancer. Cancer Res 63: 5084-5090, 2003. Doxorubicin (adriamycin): a critical review of free radical- 18 Chang MY, Seideman J and Sofou S: Enhanced loading dependent mechanisms of cytotoxicity. Pharmacol Ther 47: 219- efficiency and retention of 225Ac in rigid liposomes for potential 231, 1990. targeted therapy of micrometastases. Bioconjug Chem 19: 1274- 3 Barrett-Lee PJ, Dixon JM, Farrell C, Jones A, Leonard R, 1282, 2008. Murray N, Palmieri C, Plummer CJ, Stanley A and Verrill MW: 19 Erdogan S, Roby A and Torchilin VP: Enhanced tumor Expert opinion on the use of anthracyclines in patients with visualization by gamma-scintigraphy with 111In-labeled advanced breast cancer at cardiac risk. Ann Oncol 20: 816-827, polychelating-polymer-containing immunoliposomes. Mol 2009. Pharm 3: 525-530, 2006. 4 Rivankar S: An overview of doxorubicin formulations in cancer 20 Pillai MR, Dash A and Knapp FF Jr.: Rhenium-188: availability therapy. J Cancer Res Ther 10: 853-858, 2014. from the (188)W/(188)Re generator and status of current 5 Mufamadi MS, Pillay V, Choonara YE, Du Toit LC, Modi G, applications. Curr Radiopharm 5: 228-243, 2012. Naidoo D and Ndesendo VM: A review on composite liposomal 21 Argyrou M, Valassi A andreou M and Lyra M: Rhenium-188 technologies for specialized drug delivery. J Drug Deliv 2011: production in hospitals, by w-188/re-188 generator, for easy use 939851, 2011. in radionuclide therapy. Int J Mol Imaging 2013: 290750, 2013. 6 Madni A, Sarfraz M, Rehman M, Ahmad M, Akhtar N, Ahmad 22 Bartlett GR: Phosphorus assay in column chromatography. 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24 Kim YP, Shon HK, Shin SK and Lee TG: Probing nanoparticles 32 Chen LC, Chang CH, Yu CY, Chang YJ, Wu YH, Lee WC, Yeh and nanoparticle-conjugated biomolecules using time-of-flight CH, Lee TW and Ting G: Pharmacokinetics, micro-SPECT/CT secondary ion mass spectrometry. Mass Spectrom Rev 34: 237- imaging and therapeutic efficacy of (188)Re-DXR-liposome in 247, 2015. C26 colon carcinoma ascites mice model. Nucl Med Biol 35: 25 Lammers T, Kiessling F, Hennink WE and Storm G: Drug 883-893, 2008. targeting to tumors: principles, pitfalls and (pre-) clinical 33 Chow TH, Lin YY, Hwang JJ, Wang HE, Tseng YL, Pang VF, progress. J Control Release 161: 175-187, 2012. Wang SJ, Whang-Peng J and Ting G: Diagnostic and therapeutic 26 Lin PC, Lin S, Wang PC and Sridhar R: Techniques for evaluation of 111In-vinorelbine-liposomes in a human colorectal physicochemical characterization of nanomaterials. Biotechnol carcinoma HT-29/luc-bearing animal model. Nucl Med Biol 35: Adv 32: 711-726, 2014. 623-634, 2008. 27 Milla P, Dosio F and Cattel L: PEGylation of proteins and 34 Graham MM and Menda Y: Radiopeptide imaging and therapy liposomes: a powerful and flexible strategy to improve the drug in the United States. J Nucl Med 52(Suppl 2): 56s-63s, 2011. delivery. Curr Drug Metab 13: 105-119, 2012. 35 Anthony LB, Woltering EA, Espenan GD, Cronin MD, Maloney 28 Patel NR, Rathi A, Mongayt D and Torchilin VP: Reversal of TJ and McCarthy KE: Indium-111-pentetreotide prolongs multidrug resistance by co-delivery of tariquidar (XR9576) and survival in gastroenteropancreatic malignancies. Semin Nucl paclitaxel using long-circulating liposomes. Int J Pharm 416: Med 32: 123-132, 2002. 296-299, 2011. 36 Chen MH, Chang CH, Chang YJ, Chen LC, Yu CY, Wu YH, Lee 29 Loi M, Marchio S, Becherini P, Di Paolo D, Soster M, Curnis F, WC, Yeh CH, Lin FH, Lee TW, Yang CS and Ting G: Brignole C, Pagnan G, Perri P, Caffa I, Longhi R, Nico B, MicroSPECT/CT imaging and pharmacokinetics of 188Re- Bussolino F, Gambini C, Ribatti D, Cilli M, Arap W, Pasqualini (doxorubicin)-liposome in human colorectal adenocarcinoma- R, Allen TM, Corti A, Ponzoni M and Pastorino F: Combined bearing mice. Anticancer Res 30: 65-72, 2010. targeting of perivascular and endothelial tumor cells enhances anti-tumor efficacy of liposomal chemotherapy in neuroblastoma. J Control Release 145: 66-73, 2010. 30 Liu Y, Lu WL, Guo J, Du J, Li T, Wu JW, Wang GL, Wang JC, Zhang X and Zhang Q: A potential target associated with both cancer and cancer stem cells: a combination therapy for eradication of breast cancer using vinorelbine stealthy liposomes plus parthenolide stealthy liposomes. J Control Release 129: 18- 25, 2008. 31 Chang YJ, Chang CH, Yu CY, Chang TJ, Chen LC, Chen MH, Lee TW and Ting G: Therapeutic efficacy and microSPECT/CT Received April 21, 2015 imaging of 188Re-DXR-liposome in a C26 murine colon Revised May 29, 2015 carcinoma solid tumor model. Nucl Med Biol 37: 95-104, 2010. Accepted June 6, 2015

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