Abstract
Nanoliposome can be designed as a drug delivery carrier to improve the pharmacological and therapeutic properties of drug administration. 188Re-labeled nanoliposomes are useful for diagnostic imaging as well as for targeted radionuclide therapy. In this study, the in vivo nuclear imaging, pharmacokinetics and biodistribution of administered nanoliposomes were investigated as drug and radionuclide carriers for targeting solid tumor via intravenous (i.v.) administration. The radiotherapeutics (188Re-liposome) and radiochemotherapeutics (188Re-DXR-liposome) were i.v. administered to nude mice bearing human HT-29 colorectal adenocarcinoma xenografts. 188Re-liposome and 188Re-DXR-liposomes show similar biodistribution profile; both have higher tumor uptake, higher blood retention time, and lower excretion rate than 188Re-N,N-bis(2-mercaptoethyl)-N′,N′-diethylenediamine (BMEDA). In contrast to tumor uptake, the area under the curve (AUC) value of tumor for 188Re-liposome and 188Re-DXR-liposome was 16.5- and 11.5-fold higher than that of free 188Re-BMEDA, respectively. Additionally, 188Re-liposome and 188Re-DXR-liposome had a higher tumor-to-muscle ratio at 24 h (14.4±2 .7 and 17.14±4.1, respectively) than 188Re-BMEDA (1.6±0.1). The tumor targeting and distribution of 188Re-(DXR)-liposome (representing 188Re-DXR-liposome and 188Re-liposome) can also be acquired by signal photon-emission computed tomography/computed tomography images as well as whole body autoradiograph. These results suggest that 188Re-(DXR)-liposomes are potentially promising agents for passive targeting treatment of malignant disease.
Colorectal cancer is highly prevalent and a common cause of cancer in Taiwan, fourth most common malignancy in the United States, and is the second leading cause of cancer-related death (1, 2). It is currently very attractive to develop anticancer drug delivery system for cancer therapy. Nanoparticles can be designed as a drug delivery system to improve the pharmacological and therapeutic properties of drug administration through the enhanced permeability and retention effect (EPR) in tumor sites (3). Several types of carriers have been developed in the last few decades, these include nanoliposomes, carbon nanotubes, micelles, dendrimers, iron oxides and quantum dots (4, 5).
Liposomes are well known to the medical community, particularly as drug carriers for cancer treatment. Nanoliposomes alter the pharmacokinetics and biodistribution of free drugs and function as a reservoir for sustained drug release. Moreover, the leaky vasculature and lack of a well-defined lymphatic system allow intravenously (i.v.) administered nanoliposomes to achieve spontaneous accumulation via the EPR effect in tumor sites. The advantages of nanoliposome enable it to cause fewer side-effects than do free drugs alone. To minimize the rate of mononuclear phagocyte system or reticuloendothelial system (RES) uptake for developing a long blood circulation, the most commonly used strategy is to conjugate polyethylene glycol (PEG) polymer, which is a relatively inert hydrophilic polymer that provides good steric hindrance for preventing protein binding onto the surface of the liposome (6-8).
One application of nanoliposome as a carrier system is the encapsulation of therapeutic radionuclides for internal targeted radiotherapy. Chang et al. have reported the long retention of 188Re-liposome compared with that of unencapsulated 188Re in tumor following i.v. injection in C26 tumor-bearing mice (9). Bao et al. also reported that 99mTc- N,N-bis(2-mercaptoethyl)-N′,N′-diethylenediamine (BMEDA) pegylated liposomal doxorubicin and 186Re-BMEDA pegylated liposome have longer half-life in blood than that of unencapsulated 99mTc-BMEDA and 186Re-BMEDA after i.v. injection in normal mice (10, 11). These preclinical studies clearly indicate that radionuclides encapsulated in liposome are capable of improving the profile of biodistribution and pharmacokinetics for passive target cancer therapy. Moreover, liposome encapsulating γ-emission therapeutic radionuclides 111In, 123I, 188Re and 186Re are able to offer tools as signal photon-emission computed tomography (SPECT) imaging (5). Ogihara-Umeda et al. reported a higher accumulation of small-sized (80 nm) liposome-encapsulated 67Ga-NTA and 111In-NTA in tumor compared with that of free 67Ga, 111In and 99mTc (12). Additionally, 188Re 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 for imaging and its 2.12 MeV β emission, with maximum tissue penetration range of 11 mm for tumor therapeutics (9, 13, 14).
Currently, the combination of chemotherapeutic drugs with radiation has been shown to improve survival and locoregional control of various types of cancer compared with radiotherapy alone (15, 16). Several studies have shown significant increase in therapeutic efficacy and reduced toxicity in delivery of chemotherapeutics such as doxorubicin, paclitaxel, epirubicin, vinorelbine and topotecan (17). It is valuable to monitor the pharmacokinetics and biodistribution of nanoliposomes followed by imaging to understand and predict their efficacy and side-effects.
With this in mind, we employed 188Re-liposome (9, 14, 18) and 188Re-DXR-liposome (18, 19) as carriers to estimate the pharmacokinetics and therapeutic efficacy of radionuclide drug in murine C26 colon solid tumor or/and ascites model. However, the 188Re-radiolabeled Lipo-Dox (188Re-DXR-liposome), pharmacokinetics and imaging study of radiochemotherapeutics in human colorectal HT-29 solid tumor model has not been reported yet. Moreover, dual functional and dual modality 188Re-DXR-liposome is a novel nanocarrier for non-invasive simultaneous imaging and therapy (20, 21). In this study, the imaging, pharmacokinetics and biodistribution of administered nanotargeted 188Re-(DXR)-liposomes (representing 188Re-DXR-liposome and 188Re-liposome) were investigated as drug carriers for treating HT-29 solid tumor via i.v. administration. This dual functional design of 188Re-(DXR)-liposome can also be used to predict the pharmacological distribution via SPECT/CT imaging as well as whole-body autoradiography (WBAR).
Materials and Methods
Materials. Distearoylphosphatidylcholine (DSPC), cholesterol and polyethylene glycol (average M.W. 2000)-derived distearoyl-phosphatidylethanolamine (PEG-DSPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Cell culture materials were obtained from GIBCO BRL (Grand Island, NY, USA). PD-10 column and Sepharose 4 Fast Flow were purchased from GE Healthcare (Uppsala, Sweden). N,N-Bis(2-mercaptoethyl)-N′,N′-diethylethylenediamine (BMEDA) were purchased from ABX (Radeberg, Germany). All other chemicals were purchased from Merck (Darmstadt, Germany).
Cell line and animal model. The HT-29 human colorectal adenocarcinoma cell line was purchased from the Bioresource Collection and Research Center, Hsinchu, Taiwan. It was grown in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum and 2 mM L-glutamine at 37°C in 5% CO2. Cells were detached with 0.05% trypsin/0.53 mM EDTA in Hanks' balanced salt solution. Female nude mice (4 to 6 weeks old) were obtained from BioLASCO Taiwan Co. Ltd. (Taipei, Taiwan), with water and food being provided ad libitum in the animal house of the Institute of Nuclear Energy Research (Taoyuan, Taiwan). Nude mice were subcutaneously inoculated with 2×106 HT-29 cells in the right hind flank. Tumors were measured, individual tumor volumes were calculated by the formula: V=(length × width2)/2.
Nanoliposome preparation. Liposomes were prepared by a lipid film hydration-extrusion method using repeated freeze-thawing to hydrate the lipid films (22). Liposomes were composed of DSPC, cholesterol and methoxy polyethylene glycol (mPEG)-1,2-distearoyl-3-sn-glycerophosphoethanol-amine at a molar ratio of 3:2:0.3. Following hydration in ammonium sulfate solution at 60°C, liposomes were repeatedly extruded through polycarbonate membrane filters (0.2-, 0.1- and 0.05-μm pore sizes) (Costar, Cambridge, MA, USA). The extraliposomal salts were removed by a Sephadex G-50 column (22). Phospholipid concentration was measured by phosphate assay (23). The liposomes were finally analyzed at 14.14 μmol/ml phospholipids having an average particle size of 80.5±14.1 nm.
188Re-(DXR)-liposome preparation. 188Re was obtained as an isotonic solution in the form of sodium perrhenate from an aluminum oxide column by elution with normal saline (24). 188Re-BMEDA was prepared following the method proposed by Bao et al. (11). Five mg of BMEDA were dissolved in 0.5 ml of 0.17 mol/l sodium gluconate (in acetate solution) and 120 μl of stannous chloride (10 mg/ml), followed by the addition of 0.2-0.5 ml of NaReO4 under a nitrogen atmosphere. The mixture was incubated at 80°C for 1 h. The labeling efficiency of the 188Re-BMEDA complex was confirmed by paper chromatography with normal saline as the eluent. 188Re-liposomes were prepared by adding 1 ml of liposomes to the 188Re-BMEDA solution and incubated at 60°C for 30 min. The free 188Re-BMEDA was removed using a PD-10 column (GE Healthcare) eluted with normal saline. The 188Re-BMEDA loading efficiency was determined by taking the radioactivity in pegylated liposomes after separation divided by the total radioactivity before separation. 188Re-DXR-liposome was prepared in the same way as 188Re-liposome, 1 ml of 14.14 μmol/ml of phospholipid Lipo-Dox (TTY Biopharm, Taipei, Taiwan) was mixed with 188Re-BMEDA solution and incubated at 60°C for 30 min (10).
In vitro stability. The in vitro labeling stabilities of 188Re-BMEDA with liposome and Lipo-Dox were studied comparably in normal saline and human plasma solution. After separation of 188Re-(DXR)-liposome from free 188Re-BMEDA complexes by PD-10 column, the in vitro labeling stability of 188Re-(DXR)-liposome was evaluated by incubating 188Re-(DXR)-liposome in normal saline (NS) (1:1 volume ratio) at room temperature and human plasma (1:19 volume ratio) at 37°C, respectively. At specific times after incubation, 150 μl of 188Re-(DXR)-liposome solution were removed and the mixture separated on a column of Sepharose 4 Fast Flow (GE Healthcare) packed in a Poly-Prep chromatography column (Bio-Rad) using normal saline as eluent. The 188Re-(DXR)-liposome was collected and counted using a Cobra Π Auto-Gamma counter (Packard, USA). The labeling stability was calculated by dividing the 188Re-(DXR)-liposome radioactivity by the total radioactivity (9, 11).
Biodistribution study. Nude mice were subcutaneously injected with HT-29 colorectal carcinoma cell line (2×106 cells) in the right hind flank. When tumor xenografts were fully established and had reached volumes of around 50 to 100 mm3, 1.85 MBq of 188Re-BMEDA or 188Re-(DXR)-liposome (phospholipid concentration 14.14 μmol/ml) were i.v. injected into each mice (n=5). At different times (1, 4, 16, 24 and 48 h) after i.v. injection, mice were sacrificed by CO2 asphyxiation. Blood samples were collected through cardiac puncture. Organs of interest were removed, washed and weighed with radioactivity measured by a Cobra Π Auto-Gamma counter. The results were expressed as the percentage of injected dose per gram of tissue (%ID/g).
Pharmacokinetic study. Pharmacokinetics of each blood sample were further calculated using WinNonlin software version 5.0.1 (Pharsight Corp., Mountain View, CA, USA). The parameters were calculated using noncompartmental analysis model 201 (i.v.-bolus input) with the log/linear trapezoidal rule. The pharmacokinetic parameters including area under the curve (AUC, h %ID/g), the maximum concentration (Cmax, %ID/g), clearance (Cl, ml/h), and mean residence time (MRT, h) were calculated.
MicroSPECT/CT imaging. The SPECT images and CT images were acquired using a microSPECT/CT scanner (X-SPECT, Gamma Medica, Northridge, CA, USA). Mice were anesthetized with 1.5% isoflourine at 1, 4, 16, 24 and 48 h after i.v. injection of 18.5 MBq/200 ml of 188Re-BMEDA and 188Re-(DXR)-liposome. The source and detector are mounted on a circular gantry, allowing it to rotate 360° around the subject (mouse) positioned on a stationary bed. The radius of rotation was 1.0 cm with a field of view of 1.37 cm. The images were acquired using 64 projections at 90 s per projection. The energy window was set at 155 keV±10-15%. The SPECT imaging was followed by CT image acquisition (X-ray source: 50 kV, 0.4 mA; 256 projections) with the animal in exactly the same position.
WBAR imaging. After SPECT/CT imaging at 72 h, mice were sacrificed by CO2 euthanasia and were immediately dipped into liquid nitrogen. The frozen carcasses were then embedded with 2.5% carboxymethyl cellulose (CMC). The frozen CMC block was attached to the sample stage in the cryochamber (−20°C). After 2 h, the frozen sample was then sectioned (40-μm-thick slices) using a cryomicrotome (CM 3600; Leica Instruments, Germany) at −20°C. These samples were placed in contact with an imaging plate (BASMS 2040; Fuji Photo Film Co., Tokyo, Japan) for five days. After complete exposure, the imaging plate was analyzed with an FLA-5100 reader (Fuji Photo Film Co.) and Multi Gauge V3.0 software (Fuji Photo Film Co.).
Results
Preparation of 188Re-BMEDA and 188Re-(DXR)-liposome. The labeling efficiency of 188Re-BMEDA complex was determined using ITLC-SG paper chromatography and was found to exceed 99%. The after-loading efficiency of 188Re-liposome BMEDA in nanoliposome (188Re-liposome) and Lipo-Dox (188Re-DXR-liposome) were approximately 80±0.6% and 85.3±0.15% (n=3), respectively.
In vitro stability of 188Re-(DXR)-liposome. In vitro stability of 188Re-liposome and/or 188Re-DXR-liposome at certain times after incubation in NS buffer at room temperature and 5% human serum-NS buffer at 37°C are shown in Figure 1a and b, respectively. The stability of 188Re-liposome (n=3) and 188Re-DXR-liposome (n=3) was 92.6±0.2 % and 77.5±2.3 % at 72 h, respectively in NS (Figure 1a), and 72.3±4.6 % and 60.2±9 % at 72 h, respectively, in 5% human serum (Figure 1b).
Biodistribution study. The %ID/g of 188Re-BMEDA and 188Re-(DXR)-liposome in blood, spleen, heart, liver, kidney, lung, tumor, feces and urine are presented in Figure 2, and the tumor to muscle (T/M) ratios are shown in Figure 3. The profiles of radiotherapeutics of 188Re-liposome are similar to those of 188Re-DXR-liposome, except that in spleen, but a significantly different profile was observed for 188Re-BMEDA. The nanoliposomal drug formulation resulted in significantly higher uptake in blood, liver, spleen, tumor and lung than free did that of 188Re-BMEDA. 188Re-BMEDA exhibits fast blood clearance, and fast excretion from feces, urine and kidneys in 4 h after i.v. injection. In contrast, the uptake in tumor shows the tumor concentration for free 188Re-BMEDA at 1 h after injection did not increase thereafter. 188Re-liposome and 188Re-DXR-liposome accumulation in the tumor was higher compared with free 188Re-BMEDA, and resulted in the highest tumor to muscle uptake ratio at 14.4±2.7% and 17.1±4.1% at 24 h after injection, respectively.
Pharmacokinetic study. The area under the concentration—time curves in blood, liver, spleen, kidneys, heart and lungs after i.v. injection of 188Re-liposome, 188Re-DXR-liposome and free 188Re-BMEDA are presented in Table I, and the pharmacokinetic parameters of drugs in blood are listed in Table II. 188Re-liposome and 188Re-DXR-liposome displayed a much greater systemic circulation time than did free 188Re-BMEDA, which also showed rapid clearance kinetics and lower maximum concentration. The calculated AUCs of 188Re-liposome and 188Re-DXR-liposome were 10.2- and 7.8-fold higher than those of free 188Re-BMEDA in blood, respectively. In addition, the nanoliposomal drug formulation also significantly increased AUC in liver, spleen, kidneys and lungs. However, a similar AUC was observed in heart for the three formulations. It is noteworthy that the mice treated with 188Re-DXR-liposome had lower AUC values in blood, but showed significantly higher AUC values in spleen compared with those treated with 188Re-liposome. The AUC value of tumor for 188Re-liposome and 188Re-DXR-liposome was 16.5- and 11.5-fold higher than that of free 188Re-BMEDA, respectively.
MicroSPECT/CT and WBAR imaging. The SPECT/CT imaging of 188Re-BMEDA indicates no significant uptake in tumor and other organs after i.v. injection, as shown in Figure 4. 188Re-BMEDA was rapidly cleared and excreted from feces and urine in 4 h. However, the imaging of 188Re-liposome and 188Re-DXR-liposome showed accumulation in the liver, spleen and tumor after i.v. injection. Moreover, tumor uptake can be clearly seen at 16, 24 and 48 h. The autoradiography imaging was performed after the SPECT/CT image at 48 h (Figure 5). The WBAR obtained from coronal sections showed biodistribution of radiopharmaceutical similar to that obtained by SPECT/CT imaging. The tumor, spleen, liver and feces revealed the highest apparent accumulation of radioactivity at 48 h with 188Re-(DXR)-liposome delivery. The WBAR can be employed to distinguish between the relative concentrations in each organ.
Discussion
To achieve nanoliposome labeling, radioisotopes can be attached to the surface of a liposome, embedded in double membrane of liposomes or encapsulated within the inner hydrophilic space of liposomes. An ideal liposome labeling method is the trapping of radioisotopes into the inner space of liposomes with high labeling efficiency and high specific activity using liposomes prepared before the radiolabeling procedure or radionuclide after-loading techniques (22, 25). The passively nanotargeted 188Re-(DXR)-liposomes were prepared with similar after-loading techniques as reported previously (9, 11, 14, 19).
Liposome nanoparticles may represent the most effective nanocarriers for cancer chemotherapy. It has been shown that more than 98% of the drug is in liposome-encapsulated form after i.v. injection, indicating that the pharmacokinetics of liposomal doxorubicin are dictated by the liposome carrier and most of the drug is delivered to the tissue in liposome-associated form (26). These nanoparticles can be surface-grafted with PEG to prolong their systemic circulating half-life and enhance their tumor accumulation and therapeutic efficiency (27). Our results indicated that accumulation of 188Re-liposomes and 188Re-DXR-liposomes is 16.5- and 11.5-fold higher than that of 188Re-BMEDA in tumor of HT-29 human solid tumor-bearing mice (Table I, Figure 2). The pharmacokinetics of 188Re-(DXR)-liposome in the blood shows prolonged blood circulation, reduced clearance, an increased AUC, and an increased MRT of these passively nanotargeted radio/radiochemotherapeutics (Table II). In comparison with our previous studies (9, 14), the AUC ratios of nanotargeted radiotherapeutics of 188Re-liposome to 188Re-BMEDA was 10.2-fold (see Table II), which was larger than those seen in C26 solid tumor (4.6-fold) (9) and C26 ascites (6.8-fold) (14) mouse models. The imaging efficiency of these nanoliposomes in tumors was evaluated by accumulation and tumor to blood ratio obtained after administration of radio/radiochemotherapeutics to the mice (12), which was consistant with the tumor to muscle ratio obtained in this study (Figure 3). These results suggest that 188Re-(DXR)-liposome may have better pharmacokinetics and higher bioavailability than 188Re-BMEDA in human HT-29 xenografts, thus enhancing the level of tumor delivery via the EPR effect. Moreover, a high accumulation of passively nanotargeted therapeutics often results in enhanced therapeutic efficacy, minimal toxicity and side-effects (7).
The applicaions of radionuclides encapsulated in nanoliposomes for imaging and internal radiotherapy have been discussed in previous reports (5, 28, 29). For diagnostic imaging, we have reported the bifunctional imaging and bimodality radiochemotherapeutic efficacy of 111In-VNB-liposome in HT-29/luc-bearing mice (21, 30). Bao et al. used 99mTc-labeled Doxil to study non-invasive in vivo pharmacokinetics by gamma camera imaging (10, 31). The passively nanotargeted 188Re-(DXR)-liposome tumor targeting was also confirmed by microSPECT/CT imaging (Figure 4) and validated by WBAR (Figure 5). The microSPECT/CT imaging provides faster dynamic non-invasive information for in vivo therapeutics tumor targeting and therapeutic response (Figure 4). 188Re-(DXR)-liposome imaging revealed relatively long circulation in blood followed by retention in reticuloendothelial system of spleen and liver (Figure 5). The information of SPECT/CT imaging and WBAR correlated well with that obtained from biodistribution.
Our results showed that passively nanotargeted 188Re-DXR-liposome has similar profile to that of 188Re-liposome in mouse organs (Figure 2), and with significant uptake in the RES of spleen and liver. The enhanced uptake in liver and spleen is largely attributed to the macrophages residing in the tissues which are responsible for clearing liposome in the blood (7). Nanotargeted 188Re-liposome and 188Re-DXR-liposome at the 100-nm size range can passively accumulate in the tumor tissue site through the EPR effect. Following i.v. administration of the nanoliposomes, these predominantly accumulate in the interstitial fluid of extracellular and perivascular space of the tumor (32). Biodistribution and therapeutic index may be improved via an increase in polyethylene glycol (PEG) from 0.9% to 6% on passively nanotargeted 111In-liposome in an HT-29/luc-xenografted mouse model (33). The nanoliposomal formulation of therapeutics diffused into the interstitial fluid of the tumor and RES may heavily affect the liposomal AUC in the blood stream. In comparsion with our previous results on biodistribution, imaging and pharmacokinetics of 188Re-liposome in the C26 tumor mouse model (9), similar findings were also obtained in the human HT-29 tumor-bearing animal models. In Taiwan, more information on the clinical application of 188Re-liposome is needed. Translational research of passively nanotargeted radio/radiochemotherapeutics of 188Re-(DXR)-liposome will be made in future therapeutic efficacy studies.
Conclusion
In vivo nuclear imaging, pharmacokinetics and biodistribution of passively nanotargeted radiotherapeutics of 188Re-liposome and radiochemotherapeutics of 188Re-DXR-liposome show that the high-energy β-emitters of 188Re-labeled nanoliposomes have potential as a drug delivery system for improving the pharmacological and targeting properties of radionuclides and drugs in the nude mouse model of human HT-29 solid colorectal adenocarcinoma. These results suggest that 188Re-(DXR)-liposomes are potentially promising agents for use in treatment of malignant diseases.
Acknowledgements
The authors thank Dr. Tsai-Yueh Luo and Mr. Ching-Jun Liu for providing the rhenium-188, Mr. Wei-Chuan Hsu and Mr. Chung-Li Ho for their technical support in the animal biodistribution experiment, and Mr. Wei-Neng Liao for assistance in preparing the manuscript.
Footnotes
- Received July 25, 2009.
- Revision received November 16, 2009.
- Accepted November 25, 2009.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved