Abstract
Liposomes modified with a high concentration of polyethylene glycol (PEG) could significantly prolong the retention time of the carried drug in the circulation, thus improving the drug accumulation in the tumor. In this study, 6 mol% rather than 0.9 mol% PEGylated liposomes (100 nm in diameter) encapsulated with indium-111 were used in a human colorectal carcinoma HT-29/luc tumor-bearing mouse model for comparing the PEGylation effect. Pharmacokinetics, biodistribution, passive-targeted assay, bioluminescence imaging (BLI) and tumor growth measurements were used for the spatial and temporal distribution, tumor localization and therapeutic evaluation of the drug. Pharmacokinetic studies indicated that the terminal half-life (T½λz) and Cmax of 6 mol% PEG 111In liposomes were similar to those of 0.9 mol% PEG 111In liposomes. In the blood, the total body clearance (Cl) of 6 mol% PEG 111In liposomes was about 1.7-fold lower and the area under the curve (AUC) was 1.7-fold higher than those of 0.9 mol% PEG 111In liposomes. These results showed that the long-term circulation and localization of 6 mol% PEGylated liposomes was more appropriate for use in the tumor-bearing animal model. In addition, the biodistribution of 6 mol% PEG 111In liposomes showed significantly lower uptake in the liver, spleen, kidneys, small intestine and bone marrow than those of 0.9 mol% PEG 111In liposomes. The clearance rate of both drugs from the blood decreased with time, with the maximum at 24 h post intravenous (i.v.) injection. Prominent tumor uptake and the highest tumor/muscle ratios were found at 48 h post injection. Both AUC and relative ratio of the AUCs (RR-AUC) also showed that 6 mol% PEGylated liposomes significantly reduced the uptake of drugs in the reticuloendothelial system (RES), yet enhanced the uptake in the tumor. Gamma scintigraphy at 48 h post injection also demonstrated more distinct tumor uptake with 6 mol% PEG 111In liposomes as compared to that of 0.9 mol% PEGylated liposomes (p<0.01). BLI and in vivo tumor growth tracing showed that growth in tumor volume could largely be inhibited by 6 mol% PEG 111In liposomes. The results suggest that 6 mol% PEGylated liposomes might be a more suitable liposomal carrier for drug delivery than 0.9 mol% PEGylated liposomes, not only by reducing the drug accumulation in the RES or its related organs, but by prolonging drug circulation and eventually enhancing the targeting efficiency in the tumor to reach a better therapeutic index.
- Polyethylene glycol
- PEGylated liposome
- reticuloendothelial system
- indium-111
- HT-29/luc
- pharmacokinetics
- biodistribution
- bioluminescence imaging
Liposomes are polymeric nanoparticles which consist of one or more concentric phospholipid bilayers and are widely used as carriers for diagnostic and therapeutic agents (1-5). Liposomes comprise a promising drug delivery system owing to their slow drug release, and spatial and temporal distribution of drugs for targeted therapies. Liposomal carriers could be used to effectively encapsulate chemotherapeutic drugs for cancer therapy, antisense oligonucleotides for gene therapy, peptides for the treatment of infectious diseases, antigens to stimulate immune response, as well as radiopharmaceuticals for targeted imaging and radiotherapy (6-8). These encapsulated agents showed some improvement in pharmacokinetic stability, better biodistribution i.e. higher accumulation in target organs and lower normal tissue toxicity, and were delivered to areas which are usually inaccessible to non-encapsulated drugs. These properties have resulted in better therapeutic efficacy (9-11).
On the other hand, liposomes are also easily taken up by cells of the mononuclear phagocyte system (MPS) (12), primarily those located in the reticuloendothelial system (RES)-rich organs, such as liver, spleen and bone marrow. To overcome this problem, hydrophilic phosphatidylethanolamine (PE) derivatives of polyethylene glycol (PEG)-conjugated liposomes were initially developed to evade rapid liposomes clearance by the RES and ensure longer circulation of the drug for treatment (12, 13). PEGylated (i.e. coated with polyethylene glycol) liposomes exhibit their passive targeting property via the enhancement of permeability and retention (EPR) of nanoliposomes through leaky tumor vasculature in tumor xenografted animal models (6, 7, 14, 15). Pharmacokinetics and biodistribution of PEGylated liposomal formulations of doxorubicin (16-19) and daunorubicin (20, 21) have been applied to treat effectively against AIDS-related Kaposi's sarcoma (16, 22), ovarian (23), breast (24) and prostate cancer (25), and further gained regulatory approval for clinical use (26). Indium-111 has an appropriate half-life (T½=67.9 h) and energy characteristics for diagnosis with gamma-ray imaging and in therapeutic strategies with Auger electrons. It has been demonstrated to be very efficient for tumor cell killing due to its extremely short, sub-cellular range (≤1 μm) with high linear energy transfer (LET) and relatively biological effectiveness (RBE). These electrons generate double-strand breaks in DNA through internalization of radiopharmaceuticals into the cell nucleus and kill neighboring tumor cells via a bystander effect (27-29). The formulation of indium-111-labeled PEGylated liposomes has been used successfully to characterize prolonged retention in tumor-targeted radiotherapy. Therefore, prior to a preclinical therapeutic trial, the goal of this study was to investigate the difference of pharmacokinetics and biodistribution, effectiveness of passive targeting and tumor growth inhibition between 0.9 mol% and 6 mol% PEGylated liposomes encapsulated with indium-111 in human colorectal carcinoma (HT-29/luc) xenografted mice. Since our previous use of 0.9 mol% PEGylated liposomes caused the highest amount of radioactivity in the RES (44), this study was to investigate the reduction in uptake of radioactivity by the RES using 6 mol% rather than 0.9 mol% PEGylated liposomes. The results could provide useful information for using PEGylated liposomes encapsulated with indium-111 and an anticancer drug, such as vinorelbine (VNB), in a human tumor-bearing animal model.
Materials and Methods
Cell culture. The human colorectal carcinoma cell line (HT-29) was purchased from the Bioresource Collection and Research Center, Hsinchu, Taiwan, R.O.C. This cell line was transfected with the luciferase gene (luc) as reporter. HT-29/luc tumor cells were maintained in RPMI-1640 medium with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone, Utah, USA) and supplemented with L-glutamine, sodium bicarbonate, 100 units/ml penicillin and 100 μg/ml streptomycin. The stably transfected HT-29/luc cells were grown at 37°C in a humidified atmosphere containing 5% CO2 and contained 500 μg/ml G418 (Merck, USA) to maintain stable expression of the luc gene (8).
Tumor cell preparation. HT-29/luc tumor cells were harvested by brief incubation with trypsin at 37°C and a single-cell suspension was prepared in RPMI-1640 (FBS-free) medium. Male NOD/SCID mice were inoculated with 2×106/200 μl tumor cells subcutaneously in the dorsal region of the right thigh.
Preparation of liposomes. The preparation of PEGylated liposomes were carried out as previousely described by Tseng et al. (30). Small unilamellar vesicles (SUV, ~100 nm diameter) were prepared by a combination of the standard thin-film hydration method and repeated extrusion. PEG-distearoylphosphatidylethanolamine (DSPE) of 0.9 and 6 mol% was prepared using the following ratios: disteroyl-phosphatidylcholine (DSPC):cholesterol (Chol):DSPE covalently linked polyethylene glycol (PEG) of 2:1:0.027 for 0.9 mol% PEG-DSPE or 2:1:0.18 for 6 mol% PEG-DSPE. The above mixtures were dissolved in chloroform and placed in a round-bottomed flask. The solvent was removed by rotary evaporation under reduced pressure. The resulting dry lipid film was hydrated at 60°C in an aqueous solution of triethylammonium sucrose octasulfate (TEA-SOS; 0.6 M triethylammonium, pH 5.7-6.2) and dispersed by hand shaking at 60°C. The suspension was frozen and thawed 5 times followed by repeated extrusion through polycarbonate membrane filters (Costar, Cambridge, MA, USA) of 0.1 mm pore size 3 times and 0.05 mm 7 times using high pressure extrusion equipment (Lipex Biomembranes, Vancouver, BC, USA) at 60°C. After extrusion, the extra-liposomal salt was removed by a Sephadex G-50 column eluted with histidine-sucrose buffer (24 mM histidine hydrogen chloride, 90 g/l sucrose, pH adjusted to 6.0 with NaOH) (8).
Radiolabeling of 111In-oxine. Fifteen microliters of 68 mM 8-hydroxyquinoline (oxine; Sigma-Aldrich Co., St. Louis, MO, USA) in ethanol were added to 10 μl of 111In (indium chloride in 0.05 M HCl; 0.01-1 mCi; Perkin Elmer, Boston, MA, USA) in 400 μl of 0.1 M sodium acetate buffer (pH 5.5) and then incubated at 50°C for 20 min. The lipophilic components were extracted 3 times with 0.5 ml of chloroform and then dried using a rotary evaporator. The labeling efficiency with 111In-oxine was determined by an instant thin layer chromatography (ITLC) method. ITLC was performed on a silica gel-impregnated glass fiber sheet (ITLCTM SG; Pall Corporation, New York, NY, USA) using ethanol as the developing agent. In this system, free 111In-Cl3 remains at the origin (Rf=0) while 111In-oxine moves to the top of the strip (Rf=0.9-1.0). The radiochemical yield was generally greater than 90% 111In-oxine (8).
Preparation of 111In liposomes. The extracted 111In-oxine in chloroform was evaporated to dryness. This was followed by the addition of 20 μl of ethanol and 80 μl of distilled water into the vial, and then the mixture was incubated with 1.5 ml of liposomes for 30 min at 37°C. Two milligrams of EDTA were added to chelate any residual free indium-111 and to promote prompt excretion after i.v. injection. Liposomes were labeled by incubating with 2 mCi 111In-oxine for 30 min at room temperature. The entrapment of indium-111 within liposomes (6 mol% PEG) was assayed by loading a 100 μl sample onto a Sephadex™ G-50 Fine (40×8 mm) column, and eluted with normal saline. Eight consecutive 0.5 ml fractions were collected; the liposomes were eluted from the second and the third fractions. The radioactivity of each fraction was measured using either a dose calibrator (CRC-15R; Capintec; Bioscan, Washington DC, USA) or a gamma scintillation counter (Cobra II Autogamma; Packard, Downers Grove, IL, USA). The entrapment of indium-111 was more than 90% (8).
Pharmacokinetics and biodistribution of 111In liposomes in the HT-29/luc tumor-bearing mice. Pharmacokinetics and biodistribution studies were performed when tumor size reached 70±10 mm3. At this time, the tumor-bearing NOD/SCID mice were randomly divided into two groups and one group received intravenous administration of 70 μCi (2.59 MBq)/100 μl of 111In-labeled 0.9 mol% and 6 mol% PEGylated liposomes, respectively. In pharmacokinetic studies (n=6 for each group), blood samples (1 μl) were collected from mouse tail veins at various time points (1, 5, 15 and 30 min, and 1, 2, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 56, 64 and 72 h) after drug administration. The radioactivity of blood samples was measured using a gamma scintillation counter (Cobra II Auto-Gamma counter; Packard). Data were expressed as the percentage of the injected dose per milliliter (% ID/ml). Pharmacokinetic parameters were analyzed using WinNonlin software version 5.0.1 (Pharsight Corp., Mountain View, CA, USA). Noncompartmental analysis (NCA) model 201 (IV-Bolus Input) of plasma data was used with the log/linear trapezoidal rule. Parameters, including terminal half-life (T½λz), Cmax, total body clearance (Cl) and area under the curve (AUC) were determined. In biodistribution studies (n=4 for each group), mice were sacrificed at 1, 4, 24, 48 and 72 h post injection. Anatomization was carried out and the tissues/organs of interest including blood, heart, lung, liver, stomach, spleen, pancreas, large and small intestines, bladder, urine, kidneys, muscle, bone marrow and the tumor were excised and collected. The net tissue weights were obtained and radioactivity was measured using the same counter. The uptake of 111In-labeled PEGylated liposomes in various tissues/organs was expressed as counts per minute (cpm) with decay correction standards and was normalized as % ID per gram (% ID/g) according to the following formula:
% ID/g=(A0 × 1000)/[Injected dose (μCi) × 3.7 × 104 × 60 × Eff × tissue/organ weight]
where ln (A1/A0)=-0.693t/t½, in which A1 is the radioactivity (cpm) of tissue assayed with the gamma counter, A0 is the decay-corrected radioactivity (cpm) of tissue, Eff is the counting efficiency of the gamma scintillation counter (Eff=0.42), t½=half-life of radioisotope, t is time post injection, and the organ weight is in mg (31).
2D Planar gamma scintigraphy. Based on the biodistribution results, mice were anesthetized with isoflurane (Abbott Laboratories, Queenborough, Kent, UK) using a vaporizer system (A.M. Bickford, Wales Center, NY, USA); then 2D planar gamma scintigraphy was performed on the HT-29/luc tumor-bearing mice at 4, 24, 48 and 72 h post injection with 70 μCi (2.59 MBq)/100 μl of 0.9 mol% and 6 mol% PEG 111In liposomes, respectively. A dual head gamma camera (E.Cam Multiangle Cardiac; Siemens, Münich, Germany) equipped with a 4 mm pinhole collimator and ICON P computer system (Siemens) was used for the gamma imaging. The anesthetized mice were placed prone on the camera's pinhole collimator and the images were acquired as a 256×256 matrix for 20 min. Regions of interest (ROIs) of the tumor, liver, spleen and muscle areas were drawn and analyzed (8).
Bioluminescence imaging (BLI). In vivo BLI was carried out on the HT-29/luc tumor-bearing NOD/SCID mice using a cooled IVIS50® animal imaging system (Xenogen, Corp., Alameda, CA, USA). The IVIS50® system consists of a cooled CCD camera mounted in a light-tight specimen chamber, a cryogenic refrigeration unit, a camera controller and a computer system for data analysis. This system provides high signal-to-noise images of the luciferase signals emitted from within living animals. D-Luciferin is a chemical substrate of the firefly luciferase that creates the bioluminescence photon flux in the presence of oxygen and ATP. The photons emitted from the target site penetrate across the mammalian tissues and could be externally detected and quantified using a sensitive light-imaging system (31). Based on this property, the mice were anesthetized with 1-3% isoflurane using a vaporizer system and intraperitoneally injected (i.p.) with 150 mg/kg D-Luciferin 15 min prior to imaging. Image acquisition time was 5 s dependent on the bioluminescent intensity of the tumors. The photon flux emitted from the ROIs of tumor sites were detected by the IVIS50® imaging system and the displayed images of the tumor sites were drawn around and quantified in photons/s (ph/s) using Living Image software (Xenogen, Corp.) (8).
Tumor growth tracing. A total of 2×106 HT-29/luc tumor cells were subcutaneously implanted into the dorsal region of the right thigh of male NOD/SCID mice (n=8). Tumor growth curves in vivo were established using a digital caliper when the bulge was visible. Tumor volume was calculated as the following: 0.523 × (length × width × thickness).
Statistical analysis. Student's t-test for significant difference between control and drug-treated mice, or between 0.9 mol% and 6 mol% PEGylated liposomal drugs was performed. The significance was defined as p<0.05 (marked as *) and p<0.01 (marked as **).
Results
Pharmacokinetics of 0.9 mol% and 6 mol% PEG 111In liposomes in HT-29/luc tumor-bearing mice. The radioactivity in the blood of the HT-29/luc tumor-bearing mice was collected and calculated after intravenous (i.v.) administration of 0.9 mol% and 6 mol% PEG 111In liposomes, respectively. The concentration of radioactivity represented as % ID/ml is shown in Figure 1. The parameters obtained from the pharmacokinetic study are summarized in Table I. The terminal half-life (T½λz) of 6 mol% and 0.9 mol% PEG 111In liposomes was 16.2 h and 18.3 h, respectively. The maximum concentration of radioactivity (Cmax) in the blood was calculated as being from 49.37 to 50.84% ID/ml at 0.02 h by both treatments. The total body clearance of radioactivity in the 0.9 mol% PEG 111In liposomes group was 0.27 ml/h, and was 0.16 ml/h for the 6 mol% PEG 111In liposomes group. The AUC(o→∞) of 6 mol% PEGylated liposomes was also higher than that of 0.9 mol% PEGylated liposomes.
Biodistribution of 0.9 mol% and 6 mol% PEG 111In liposomes in HT-29/luc tumor-bearing mice. The accumulation of liposomal drugs in the tissues of the HT-29/luc tumor-bearing NOD/SCID mice (n=4) were measured at different time points after 70 μCi (2.59 MBq)/100 μl administration of 0.9 mol% and 6 mol% PEG 111In liposomes. The concentration of radioactivity in the blood decreased rapidly from 19.76±1.28% ID/g to 0.61±0.06% ID/g and from 20.19±2.21% ID/g to 0.29±0.04% ID/g after treatment with 6 mol% and 0.9 mol% PEG 111In liposomes, respectively (Table II and III). A rapid clearance of radioactivity from the blood at 24 h post injection was found. Prominent radioactivity accumulation was found in the liver, spleen and kidneys in all treatments, with maximum levels of 14.65±1.54 vs. 37.61±2.06, 25.27±3.22 vs. 164.16±16.35, and 23.72±2.76 vs. 28.36±3.72% ID/g, for 6 mol% vs. 0.9 mol% PEG 111In liposomes treatments. On the contrary, the maximum uptake of the tumor (70±10 mm3) was 15.48±2.21 vs. 8.93±1.76% ID/g at 48 h post-treatment. The AUCs of 6 mol% PEG 111In liposomes revealed a prolonged circulation of the drug in the plasma, with a significant decrease in the liver, spleen, small intestine and kidneys as well as bone marrow, and enhanced accumulation in the tumor, suggesting the EPR effect in the tumor was strengthened when PEG was increased from 0.9 mol% to 6 mol% (Table IV).
Gamma scintigraphy of xenografted mice. 2D Planar gamma scintigraphy of the HT-29/luc tumor-bearing NOD/SCID mice was performed at various time points post i.v. injection with 70 μCi (2.59 MBq)/100 μl of 0.9 mol% and 6 mol% PEG 111In liposomes. The values of gamma photon counts and tumor/muscle (T/M) ratios (Table V) from gamma scintigraphy were well correlated to that of the biodistribution, with the highest uptake at 48 h by 6 mol% PEG 111In liposomes treatment.
Tumor growth inhibition. Male NOD/SCID mice were transplanted with 2×106 HT-29/luc tumor cells in the subcutaneous dorsal region of the right thighs. Tumor growth monitoring was initiated on day 16 after tumor cell inoculation (tumor size~50±5 mm3). Multiple doses of 0.9 mol% and 6 mol% PEG 111In liposomes [70 μCi (2.59 MBq)/100 μl] were i.v. administrered once a week for one month. Both BLI and caliper measurements were performed twice a week (Figure 2A and 3). The tumor growth delay time was 10.63 days and the greater inhibition rate of tumor growth was 0.49 by 6 mol% PEG 111In liposomes treatment (p<0.01) (Table VI). The photon counts of luminescence (Figure 2B) were collected and measured from the ROIs of tumor sites (n=4). A significant inhibition of tumor growth was found by 6 mol% PEG 111In liposomes treatment, which corresponds with lower tendency of photon flux distribution. No significant difference was observed during days 44 to 55. This may have been caused by the characteristics of the tumor cell line or the induction of tumor necrosis as the tumor size reaches more than 500 mm3.
Discussion
Liposomal carriers modified with PEG has been used as a vehicle for therapeutic agent delivery, such as of doxorubicin and daunorubicin, and are in various stages of preclinical and clinical development (16-22). However, the degree of PEGylation may also influence the stability of nanoparticles. Excessive PEGylation would disturb the balance of hydrophilicity and hydrophobicity by disrupting the integrity of the surface lipid bilayer (32). On the other hand, a lower concentration of PEGylation would increase the RES uptake and renal elimination (31). Therefore, an optimal level of PEGylation is important for ideal liposomal nanoparticles as shown by Li and Huang with 5 mol% PEGylated liposomes (32). In this study, we also obtained better spatial and temporal distribution of 111In-labeled PEGylated liposomes using 6 mol% PEG significantly prolonging the drug circulation in the plasma, attenuating the injected dose (% ID/g) in the liver, spleen, kidneys, small intestine and bone marrow (p<0.05), while enhancing the accumulation in the tumor. The total body clearance of radioactivity was 1.7-fold lower and the AUC 1.7-fold higher in 6 mol% PEG 111In liposomes than in those of 0.9 mol% PEG (Table I). In mice treated with 6 mol% PEG 111In liposomes, the average amount of radioactivity in the liver and spleen was lower by 2.68- and 5.32-fold, respectively, over a 72 h period as compared to that in mice treated with 0.9 mol% PEG 111In liposomes (Table IV). The uptake in the small intestine, kidneys and the bone marrow decreased by 1.45-, 1.45- and 1.55-fold, respectively. These results suggested that PEGylation indeed affected the accumulation and distribution of liposomal drug. The tumor uptake with 6 mol% PEG 111In liposomes was also better. In both groups, the tumor uptake gradually increased with time, with maximum accumulation at 48 h post treatment (Tables II and III). Non-invasive and dynamic molecular imaging using animal positron-emission tomography (microPET), magnetic resonance imaging (microMRI), BLI, and single photon-emission computed tomography (SPECT) is often applied for efficacy evaluation in diagnosis and therapeutics, new drug discovery and preclinical studies (33-35). Molecular imaging also provides a faster and convenient approach for tumor growth and metastatic tracing, targeting efficiency and therapeutic response during the treatment course (36, 37). Since the animal model used in this study carried a luciferase reporter gene, both BLI and digital caliper assays were used to evaluate the diagnostic and therapeutic efficiency. Again, 6 mol% PEG 111In liposomes showed a better inhibition rate (p<0.01) than that of 0.9 mol% with these two approaches (Figures 2 and 3).
Using radiolabeled liposomal drugs as therapeutic agents, the absorbed radiation doses in critical organs should be considered for future application in clinical trials. An understanding of the distribution in these organs, such as the liver, spleen, kidneys and bone marrow, is crucial for the understanding normal tissue tolerance, such as myelotoxicity and secondary organ toxicities. Lower critical tissue toxicity may be achieved by various strategies including suitable PEGylation (5-10 mol% PEG) of the liposomal surface (13, 38), optimal particle size (~100 nm diameter) (39), neutral lipid structure (±10 mV for potential) (40, 41); receptor-mediating immunoliposome, pH-sensitive liposome (42), thermal-sensitive liposome (43), and prior administration of empty liposomes to occupy the RES-rich organs may also be considered to improve the bioavailability of nanomedicine.
Conclusion
Liposomes with 6 mol% PEG (100 nm in diameter and 5.06 mV for ζ potential) were shown to have higher tumor uptake but lower toxicity in organs such as the liver, spleen, kidneys, small intestine as well as bone marrow than those with 0.9 mol% PEG in mice with HT-29/luc human colorectal adenocarcinoma xenograft. Liposomes modified with 6 mol% PEG, instead of 0.9 mol%, showed longer drug circulation in the plasma, enhanced tumor targeting and improved the therapeutic efficiency.
Acknowledgements
This study was supported by grant 95A1-NMPPO1-007 from the National Health Research Institute, Taipei, Taiwan, R.O.C. The nanoliposomes were kindly provided by Taiwan Liposome Company, Taipei, Taiwan, R.O.C. The authors also appreciated the support from the MAGIC core of the National Research Program for Genomic Medicine, National Science Council, Taipei, Taiwan, R.O.C.
- Received December 16, 2008.
- Revision received February 24, 2009.
- Accepted April 2, 2009.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved