Abstract
Background/Aim: In precision therapy, liposomal encapsulated chemotherapeutic drugs have been developed to treat cancers by achieving higher drug accumulation in the tumor compared to normal tissues/organs. Materials and Methods: We developed a novel chemoradiotherapeutic approach via nanoliposomes conjugated with vinorelbine (VNB) and 111In (111In-VNB-liposome) and examined their pharmacokinetics, biodistribution, maximum tolerance dose, and toxicity in a NOD/SCID mouse model. Results: Pharmacokinetic results showed that the area under the curve (AUC) of PEGylated liposomes was about 17-fold higher than that of the free radioisotope. Tumor growth inhibition by 111In-VNB-liposome was significantly higher than that of the control (p<0.05). Conclusion: The tumors in NOD/SCID mice bearing HT-29/tk-luc xenografts were significantly suppressed by 111In-VNB-liposomes. The study proposed repeated treatments with a novel liposome-mediated radiochemotherapy and validation of therapeutic efficacy via imaging.
- Biodistribution
- bioluminescence
- colorectal cancer
- PEG-liposome
- pharmacokinetics
- micro positron emission tomography
Colorectal cancer is a common malignant tumor in industrialized countries (1-3). From an epidemiological viewpoint, family inheritance, ageing and diabetes may all contribute to a higher risk of colorectal cancer (4-6). Clinically, colorectal cancer has a high mortality rate due to the fact that it remains undetected until later stages of the disease (7). The average five-year overall survival rate for all stages is about 65% based on surgery, adjuvant chemotherapy and radiotherapy (8).
Vinorelbine (5’-noranhydrovinblastine, VNB) is a drug widely used in cancer therapy (9, 10). It acts through microtubule disruption, thereby arresting cell division in mitosis. Also, it is less toxicity than the naturally occurring vinca alkaloids (11, 12). Today, patients receive chemotherapy mainly orally or intravenously. However, these means of administration result in the exposure of all organs of the body and the tumor to the same concentration of the drug, giving the drug a chance to damage the normal tissues. Thus, there is need to accurately and effectively deliver the drug to the lesion, as with radiation therapy.
The development of radiation therapy has advanced rapidly in recent years. Radionuclide therapy differs from traditional radiotherapy, since it delivers radioisotopes directly into the body's lesions. This methodology, is beneficial for the patients not only due to the shortening of the treatment time, but also due to the improved normal tissue/organ tolerance. Indium-111 (111In) is a radioactive isotope of indium (In) with an appropriate half-life (T1/2=2.8 days). During its decay by electron capture (EC) to 111Cd, the released gamma photons can be imaged using planar or SPECT (single photon emission computed tomography) gamma camera (γ-camera). Simultaneously the emitted Auger electrons produce a cancer therapeutic effect with high linear energy transfer (13). Several studies have reported that 111In-labeled drugs can be very effective in cancer therapy and can be used for real-time tracking of drug biodistribution (14-16).
Liposomes, usually 50-500 nm in diameter, are small artificial spherical vesicles with both hydrophobic and hydrophilic characteristics. Liposome-based chemotherapeutic drugs have been shown to increase the stability of the drug in vivo, extend the duration of drug retention in the blood circulation, and accumulate the drug at the tumor site. Therefore, liposomes can reduce the possible side effects of chemotherapy drugs on normal tissues and organs and improve therapeutic efficacies (17-19). The modification of the liposome phospholipids with polyethyleneglycol (PEG), usually named PEGylated liposomes, reduce the chance of the liposome to be captured by macrophages in the reticuloendothelial system (RES), and extend the time that the liposomes circulate in blood vessels (20-22). The characteristics and advantages of liposomes have been reported in several studies indicating that they significantly improve biodistribution and therapeutic efficacy of specific drugs (23, 24). In defining a therapeutic strategy, prospective anti-tumor drugs are designed to improve tumor growth control while reducing damage to normal tissues and organs as well (25-27). The enhanced permeability and retention (EPR) effects observed in many types of cancer are most commonly used for passive targeting. The main cause of EPR is the rapid growth of tumor tissue and vigorous angiogenesis. The liposomes can utilize the characteristics of tumor cells to carry radiotherapy and chemotherapeutic drugs in the tumor, where they enter the cells by endocytosis improving the therapeutic effects, protecting normal tissues while killing tumor cells (28-30).
Reporter gene technology is widely used in molecular biology. The herpes simplex virus-1-thymidine kinase (HSV1-tk) gene has been used as a suicide gene in cancer therapy for more than 25 years. However, it is more known for its contribution to non-invasive imaging, both clinically and preclinically, using positron emission tomography (PET) for tracking the growth of tumors (31-33). Another renowned reporter gene is the luciferase (luc) gene. Firefly luciferase reacts with firefly luciferin (LH2) (a substrate of luciferase) in the presence of ATP-Mg2+ to produce inorganic pyrophosphate and intermediates, luciferyl-adenylate (LH2-AMP), followed by LH2-AMP oxidative decarboxylation that produces oxyluciferin, CO2, AMP, and green biological luminescence, which can be used to track tumor cell growth and metastasis (34-37). The current study will use human colorectal cancer cell line (HT-29) transfected the double-reporter gene of herpes simplex virus-1-thymidine kinase (HSV1-tk) and firefly luciferase (luc). The human colorectal cancer cell line HT-29/tk-luc, stably expressing this double-reporter gene, will be implanted into NOD/SCID mice to establish an animal model for examining the therapeutic efficacy evaluation of a novel combination of VNB plus 111In.
In addition, to validate the established therapeutic efficacy evaluation system, we will examine the efficacy of the novel radiochemotherapy of VNB plus 111In, and investigate its pharmacokinetics, biodistribution, maximum tolerated dose, radiation dose and drug toxicity of 111In-VNB-liposome. The evaluation platform will be used to compare the efficacy of the combined treatment with radiopharmaceuticals plus VNB versus radiopharmaceutical alone or VNB alone as well as the efficacy of the repeated dose with that of the single dose. The therapeutic efficacy evaluation platform, the combination treatment and repeated treatments may be used concurrently with radiotherapy in clinical practice against colorectal carcinomas.
Materials and Methods
Tumor cell preparation. The human colorectal cancer tumor cells (HT-29/tk-luc colorectal carcinoma cell line) were maintained in RPMI-1640 medium (Gibco, Grand Island, NY, USA) with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and supplemented with L-glutamine, sodium bicarbonate, 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C with 5% CO2 in the presence of 500 μg/ml G418 (Merck, Branchburg, NJ, USA) in order to maintain stable expression of the reporter genes.
Tumor xenografted animal model. Tumor cells (2×106 cells/200 μl) were transplanted subcutaneously into the dorsal region of the right thigh of 6-week-old male NOD/SCID (with T- and B-cell deficiency, purchased from National Taiwan University Hospital Animal Center, Taipei, Taiwan) and this was followed by daily measurement using a digital caliper once the tumor bulge caused by the tumor cells at the injection site was visible. Fourteen days after inoculation tumors were assessed twice per week for one month, and when tumor size reached about 100±5 mm3, they were treated with or without 111In-VNB-liposomes. Tumor volume was calculated using the formula: =0.523×(length×width×thickness).
Transfection of the double reporter gene tk-luc into human colorectal cancer cell line. The HT-29 parental cells were cultured in 10 cm culture dishes. Five μg pC1-tk-IRES-luc plastid and 10 ml jetPEI reagent (Polyplus-transfection, Strasbourg, France) were added to 250 ml NaCl solution (150 mM). The two solutions were then mixed and placed at room temperature for 30 min to give a DNA-cationic complex. This mixture was added to HT-29 parental cells and cultured for 24 h for transfection. The transfected cells were selected using 500 μg/ml G418 (Merck) for two weeks. The expression of the luc gene was measured in a Wallac 1420 Multilabel Counters (PerkinElmer Life and Analytical Sciences, Wallac, Turku, Finland). The transfected cells (HT-29/tk-luc) were sub-cultured for 10 generations to confirm that the reporter gene was stably expressed. HT-29/tk-luc cells were cultured under the same conditions as the parental HT-29 cells in medium containing 200 μg/ml G418 (Merck) to maintain the stable expression of the tk/luc gene.
Preparation of PEGylated liposomes. The preparation of the PEGylated liposomes was performed as previously described by Tseng et al. (38). Briefly, 0.9 mol% of PEG-distearoylphosphatidylethanolamine (DSPE) were prepared at the following ratios: disteroylphosphatidylcholine (DSPC): cholesterol (Chol): DSPE covalently linked polyethylene glycol (PEG) of 2:1:0.027. Small unilamellar vesicles (SUV, ~100 nm diameter) were produced by a combination of the standard thin-film hydration method, the freeze and thaw method and repeated extrusion. The extra-liposomal salt was removed by a Sephadex G-50 column and elution with histidine-sucrose buffer (24 mM histidine hydrogen chloride, 90 g/l sucrose, pH adjusted to 6.0 with NaOH).
Anticancer drugs encapsulation. After removing the extra-liposomal salt by a Sephadex G-50 column, the anti-cancer agent, VNB was added immediately into the solution at a concentration of 3.5 mg VNB per 10 μmol phospholipid, and incubated at 60°C for 30 min on a hot plate magnetic stirrer (Corning) agitated with a stir bar and setting at 100 rpm. After loading, liposomal VNB was sterilized by 0.2-micron filtration and stored at 4-6°C until use. The PEGylated liposomes were characterized as follows: pH=6.1, osmolality=361 mmol/kg, mean particle size=95.2 nm, phospholipids=6.19 μmol/ml, VNB=2.08 mg/ml.
Radiolabeling of 111In-oxine. The labeling of oxine with 111In was performed as previously described by Chow et al. (39). Briefly, 15 μl 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; 3.7-74 MBq; 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 with chloroform and then evaporated. The labeling efficiency of 111In-oxine was determined by an instant thin layer chromatography (ITLC) method. The radiochemical yield was generally greater than 90% 111In-oxine.
Preparation of PEGylated 111In-VNB-liposomes. The extracted 111In-oxine residue was dissolved in 20 μl of ethanol and added to 80 μl of distilled water. The mixture was incubated with 1.5 ml of liposomes for 30 min at 37°C. EDTA (2 mg) was then added to chelate any residual free indium-111 and to promote prompt excretion after i.v. injection. The labeling of 111In within the liposomes (0.9 mol% PEG) was assayed by loading a 100 μl sample onto a column (40×8 mm; Bio-Rad; Hercules, CA, USA) containing Sephadex™ G-50 fine gel and elution was with normal saline. The labeling efficiency was determined by dividing the radioactivity in VNB-liposome fractions by the total amount loaded. The radioactivity of each fraction was measured using either a dose calibrator (CRC-15R, Capintec, Bioscan, Ramsey, NJ, USA) or a gamma scintillation counter (Cobra II Auto-Gamma counter; Packard). The entrapment of indium-111 was more than 90% (39).
Pharmacokinetics of 111In-VNB-liposomes in HT-29/tk-luc tumor-bearing mice. Six-week-old normal mice were randomly divided into experimental group (111In-VNB-liposom) and control group (unencapsulated 111In-DOTA). After intravenous (i.v.) administration of radiopharma-ceuticals, blood was collected at 1, 5, 15, 30, 60 min, and 2, 4, 8 h, and then every 4 h until 72 h thereafter. Using a lancet (Assistant, Germany) to make a stab into the mouse cheek, approximately halfway between the ear and the mandible with enough pressure to produce a small incision. Drops of blood excluded from the stick point and were collected into a 1 μl glass capillary. The radioactivity was measured by a gamma scintillation counter, and the amount of drug remaining in the blood at each time point was calculated by the following formula and expressed in terms of injection activity per ml (% ID/ml).
where ln(A/A0)=−0.693t/t1/2, A=radioactivity of blood sample by γ-counter, A0=decay-corrected radioactivity of blood sample, t1/2=half-life of radioisotope, t=time after injection and Eff is the counting efficiency of the gamma scintillation counter (Eff=0.42).
Biodistribution of 111In-VNB-liposomes in the HT-29/tk-luc tumor-bearing mice. The tumor-bearing mice (tumor size~ 100 mm3) were intravenously (i.v.) administered 3.7 MBq of 0.9 mol% PEG 111In- VNB-liposomes. For the biodistribution studies (n=4 per time point), mice were sacrificed at 1, 4, 24, 48 and 72 h post injection. Anatomization was carried out and tissues/organs of interest including blood, heart, lungs, liver, stomach, spleen, pancreas, large and small intestines, bladder, urine, kidneys, muscle, bone marrow and the tumor were excised, the net tissue weights were obtained and the level of radioactivity in each tissue was measured using a gamma scintillation counter at 72 h after injection. The results were expressed as counts per min (cpm) with decay correction standards, and were normalized as percentage of the injected dose per gram of tissue (%ID/g) (40).
Bioluminescence imaging (BLI). BLI was performed using an IVIS50® animal imaging system (Xenogen, Corp., Alameda, CA, USA.). The photons emitted from the target site penetrate through the mammalian tissue and can be externally detected and quantified using a sensitive light-imaging system (39). On day 17 after inoculation (tumor size ~100 mm3), the mice were anesthetized with 1-3% isoflurane using a vaporizer system and intraperitoneally injected with 150 mg/kg D-Luciferin 15 min prior to imaging. The image acquisition time was 2 sec. The displayed images of the tumor sites were drawn around and quantified in photons/second (ph/sec) using Living Image software (Xenogen, Corp.).
Single photon emission computed tomography (SPECT). SPECT dynamic images at 4, 24, 48 and 72 h after intravenous injection of 111In-VNB were obtained from three NOD/SCID mice with HT-29/tk-luc tumors (with a tumor size of 100 mm3). Mice were anesthetized with 1-2% isoflurane gas and e.Cam Multiangle Cardiac (Siemens, Munich, Germany) equipped with a 4 mm pinhole for 20 min. The region of interest (ROI) of the tumor and the contralateral thigh muscle of the same size was circled, and the %ID/g of the ROI and the ratio of the radioactivity on both sides were calculated.
Whole-body autoradiography of mice. Mice were subjected to whole-body autoradiography after 48 h of intravenous injection of 111In-VNB-liposome. The mice were sacrificed with carbon dioxide, fixed posture and then rapidly immersed in isopentane (Nacalai Tesque, Kyoto, Japan) as a medium for liquid nitrogen freezing. After the mouse ears, hands, feet and tail were cut off in about 3 to 5 min, they were placed on a cryostat holder (7×5 cm) and the mice were fully covered with 4% carboxylmethycellulose (CMC). The coated sample was quickly placed in cryostat (Bright Instrument, Huntingdon, UK) frozen (−40°C) for about 30 min. Frozen sections of about 30 μm were prepared and immediately placed on a pre-cooled adhesive tape (Deer brand, Four Pillars Company, Taipei, Taiwan, ROC). Then, they were placed on an imaging plate (IPs) (BAS-SR2040, Fuji Photo Film, Tokyo, Japan) in an imaging plate cassette (2040, Fuji Photo Film) for imaging. The imaging time was three days, and the IPs were finally analyzed with the FLA5000 reader (Fuji Photo Film).
Body weight and survival assessment. Six-week-old NOD/SCID male mice were treated with tumor cells under the skin of the right thigh. When the tumor grew to a size of about 100 mm3, the animals were divided into four groups of 10 mice each: control group (liposome), radiotherapy group (111In-liposome), chemotherapy group (VNB-liposome), and the combined treatment group (111In- VNB-liposome). The body weight loss and surviving fractions were estimated after a single dose and multiple doses (once a week, for one month) of drug were administered. The body weight change of each mouse was measured twice per week for one month. Survival was monitored daily and continued until all mice were dead post injection. The results were then subjected to Kaplan–Meier survival analysis.
Micro-PET. At the end of the treatment process (forty-eight days after tumor implantation), a total of twelve mice from the control group and each experimental group of three mice were analyzed. Twenty-four h before imaging, each animal was injected with about 50 μCi (1.85 MBq) of 124I-FIAU via the tail vein; 1 h before imaging, each animal was injected with 50 μCi (1.85 MBq) of 18F-FEAU via the tail vein. Animals were scanned using a PET R4 scanner (Concorde Microsystems, Knoxville, TN, USA) for 10 min and photographed with a steady supply of oxygen and maintained under anesthesia with 2% isofluorane. The 3D stereo image was taken at an energy of 350 to 650 keV. The images of the micro-PET of 124I-FIAU and 18F-FEAU were analyzed by software ASIPro (Concorde Microsystems, Knoxville, TN, USA). In each image, the ROI of the tumor location was circled, and the radioactivity of the region and the injection dose per gram (%ID/g) were calculated.
Biochemistry and hematology analyses. At the completion of the experiments (at the 50th day after tumor implantation), the mice were sacrificed. Blood samples were collected and tested as follows: alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate amino-transferase (AST), creatinine (CRE), blood urine nitrogen (BUN) and albumin (ALB). The above measurements were performed on a BECKMAN CX-5 PRO (Diamond Diagnostics, Holliston, MA, USA).
Tissue preparation for histopathology. At the end of the experiments, samples of the tumor mass and the major organs including lungs, liver, spleen and kidneys were fixed in cold 4% paraformaldehyde, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin (H&E staining).
Statistical analysis. The Student's t-test was used to examine the significance of differences. Statistically significant differences were identified at p<0.05 (marked as *) and p<0.01 (marked as **).
Results
Evaluation of the stability of HT-29/tk-luc reporter gene expression. First, the expression of tk-luc was examined. As shown in Figure 1A and B, the number of cells correlated well with the number of photons detected (R2=0.99) (Figure 1A and B). In addition, the FIAU uptake assay of the thymidine analog labeled with radioactive iodine 131 (Figure 1C) indicated that longer drug treatments were associated with higher accumulation of radioactivity within the cells. At the same time, no absorption of the drug was detected in cells without the tk gene, and there was a significant difference between the two groups (p<0.05).
Pharmacokinetics of 111In-VNB-liposome in HT-26/tk-luc tumor-bearing mice. Twelve six-week-old healthy SCID mice were randomized into two groups, one group was injected with 111In-VNB-liposome and another group with 111In-DOTA. The detecting time points after administration and %ID/ml are shown in the horizontal and vertical axis, respectively, of Figure 1D. It is clear that the radioisotope in the group without the liposomes is rapidly metabolized in the body compared with that in the group with the liposomes (Figure 1D). The pharmacokinetics were calculated by software Winalin and are presented in Figure 1D and in Table I. The parameters include half-life (T1/2), maximum activity time (Tmax), highest activity (Cmax), area under the curve (AUC(0-∞)), and Clear rate (CL). The clearance rate of the experimental group (111In-VNB-liposome) was significantly lower than that of the control group (111In-DOTA), and the results were also reflected in the area under curve (AUC). The signal of the experimental group was about 17-fold higher than that of the control group (Table I).
Biodistribution of 111In-VNB-liposomes in HT-26/tk-luc tumor-bearing mice. The biodistribution experiments were performed when the tumors were grown up to 100 mm3 in NOD/SCID mice. Five mice at each time point were sacrificed with carbon dioxide at 1, 4, 24, 48 and 72 h after administration of 111In-VNB-liposome. The radioactivity measured after removal of various organs was calculated as the injection dose per gram (%ID/g) as presented in Figure 2 and summarized in Table II. It can be observed that the amounts of 111In-VNB-liposome in the blood decreased rapidly and time-dependently, and after 24 h, the small intestine, spleen, and liver had a significant accumulation of 111In-VNB-liposome (Figure 2). The highest tumor/muscle ratio fell at 48 h after 111In-VNB-liposome treatment (Table II).
Liposomes passively target the tumor as detected with a γ-camera. Compared with other tissues, high radioactivity accumulation in tumors and the liver were observed. We circled and accessed the number of gamma photons per minute (cpm) in Figure 3A, calculated the cumulative radioactivity by the half-life formula and presented the results in Figure 3B. The results showed that the amount of 111In-VNB-liposome gradually increased, reaching a maximum at 48 h after administration (Figure 3B). Similarly, the number of gamma photons of the same ROI size on the opposite side of the tumor was calculated, and the ratio was obtained. The tumor/muscle ratio (T/M ratio) reached its highest value also at 48 h after administration (Figure 3C).
Biodistribution of 111In-VNB-liposomes assayed by whole-body autoradiography. In the previous biodistribution experiments, we obtained the highest ratio of tumor site accumulation to general tissue at 48 h after the administration of the liposome-encapsulated drug. Biodistribution was also examined by whole-body autoradiography at each time point (Figure 4). Optical imaging with a digital camera indicated that the drug obviously accumulated in the liver and small mesentery, which is consistent with the previous biodistribution (Figure 2). At the same time, it was also noticed that the drug accumulated in the tumor location. On the outside of the tumor, the activity near the blood vessels was higher, but not limited to the periphery of the tumor (Figure 4).
111In-VNB-liposome maximum tolerated dose in NOD/SCID mice. To determine the maximum dose that can be administered during treatment, 111In-VNB-liposomes of different radioactivity (0, 300, 400, 500 and 1000 μCi) were injected into the tail vein of NOD/SCID mice. The changes in body weight and survival in mice are depicted in Figure 5A. On the 8th day after drug treatment, the first dead mouse appeared in the group of 1000 μCi, and the best survival rate after four weeks was observed in the 300 μCi group (Figure 5B). Based on the results of survival, it was concluded that the maximum radioactivity that NOD/SCID mice can tolerate for 111In-VNB-liposome (VNB concentration at 3 mg/kg) is 300 μCi.
Weight and tumor size tracking during single-dose therapeutic efficacy assessment. The single-dose treatment began when the tumor grew to a size of 100 mm3. After three weeks of intravenous drug administration, the tumor size and the body weight of the mice were measured twice per week (Figures 6A and B). The weight loss of each mouse in each group was not less than 20% of the initial value. In terms of tumor growth, compared with the control group, the order of therapeutic efficacy was the combined group (p<0.01), and the chemotherapy group (p<0.01), followed by the radiotherapy group.
Weight and tumor size tracking during multi-dose therapeutic efficacy assessment. In order to evaluate the efficacy of multi-dose, treatment was started when the tumor size grew to 115 mm3. During the course of treatment, the body weight and tumor size were assessed twice a week (Figures 6C and D). The weight loss of each mouse in each group was not less than 20% of the initial value. In terms of tumor growth, compared with the control group, the order of efficacy was the combined group (p<0.01), and the chemotherapy group (p<0.05), followed by the radiotherapy group. Tumor growth delays are listed in Table III, and the combination index was 1.045. The formula is as follows (41):
Bioluminescence to track tumor growth as an assessment of therapeutic efficacy. In the established animal model, tumor cells (HT-29/tk-luc) were implanted subcutaneously in NOD/SCID mice, and the location and growth of tumors was tracked by the bioluminescence (BLI) produced by the cells. On the 20th day after tumor cell implantation, tumor growth in each mouse was assessed when the tumor size grew to about 100 mm3. Bioluminescence image tracking was performed twice a week until the end of the treatment process. As shown in Figure 7A, the number of photons was positively correlated with the tumor size, so the size of each group of tumors was successfully traced and accessed by imaging. The ROI of the tumor site is shown in Figure 7B. Obviously, the number of photons detected in the combined group was the lowest, which indicated that the growth of tumor was greatly suppressed in the combination group, followed by the groups of chemotherapy alone and radiotherapy alone. In the control group, tumor growth was not suppressed at all; the highest number of photons per sec were detected. The tumors in the control group were larger than those in any other group. It was obvious that the biological luminescence range was wider, followed by the radiotherapy group, while the chemotherapy and the combined group did not show any difference when observed by naked eye (Figure 7C).
Therapeutic efficacy evaluation with 124I-FIAU micro-PET. At the end of the treatment courses, three mice were taken from each group and were intravenously administered with 124I-FIAU. Since the tk gene expressed by tumor cells allows specific FIAU binding to the tumor, micro-PET was performed 24 h after FIAU injection (Figure 8A). The cumulative radioactivity of the combined group was the lowest, which indicated that the growth of tumor was greatly suppressed in the combination group, followed by the groups of chemotherapy alone and radiotherapy alone (Figure 8B). The cumulative radioactivity was positively correlated with the tumor size (Figure 8C).
Therapeutic efficacy evaluation with 18F-FEAU micro-PET. At the end of treatment courses, three mice were taken from each group and were intravenously administered with 18F-FEAU. Since the tk gene expressed by tumor cells allows specific binding of FEAU to tumor, micro-PET was performed 1 h after FEAU injection (Figure 9A). As in the case of 124I-FIAU, the cumulative radioactivity of the 18F-FEAU plus VNB group was the lowest, which indicated that the growth of tumor was greatly suppressed, followed by the groups of VNB alone and 18F-FEAU alone (Figure 9B). The cumulative radioactivity was positively correlated with the tumor size (Figure 9C).
Biochemical analysis of liver and kidney function in mice. At the end of the treatment process and on the 50th day after the tumor was implanted, 5 mice in each group were sacrificed, and their blood was subject to blood biochemical analysis. All groups administered with a liposome-encapsulated drug, whether it was the radiotherapy group, the chemotherapy group, or the combined group, the levels of the liver function indexes alanine aminotransferase (ALT) and aspartate amino-transferase (AST) were both higher than those of normal mice (Table IV). The results indicated that the mice had liver inflammation.
Discussion
The first aim of the current study was the application of a liposome-encapsulated drug delivery system, which can achieve targeted therapy via its enhanced permeability and retention (EPR) effects on tumor cells. The diameter of the liposomes was about 100 nm, which is the most suitable size. The liposomes used in this experiment were of 100 nm in their diameter, which has been shown to allow entrance in the cancer cells via endocytosis, delivering the encapsulated drug to them and inducing cytotoxicity (42-45).
The second aim of the study was the application of a molecular imaging system to follow up the biodistribution of the PEG-liposome-encapsulated antitumor drugs. The human reticuloendothelial system and immune cells have been found capable of capturing the circulated liposomes (46) and, at the same time, the biodistribution of these liposome-encapsulated drugs among the organs are very important in clinical practice but not easy to follow-up. In 2007, Papahadjopoulos et al., found that the cumulative amounts of conventional liposomes in the liver and the spleen can reach a significant level at 2 h after intravenous injection, while the amounts of PEG-liposomes were relatively low. It has been hypothesized that PEG-liposomes may reduce the detection of macrophages in the liver and spleen (42). Similarly, Ogawa et al. have also found that the biodistribution of PEG-modified 111In-labeled phosphatidyl serine liposomes in the liver was low (47, 48). In the current study, we found that the biodistribution of PEG-liposome was low in the liver and high in the spleen (Figure 1 and Table I). The differential distributions in liver and spleen have also been reported by Lee et al. using the same liposome composition (16). In 2009, Chou et al. reported that high concentrations of PEG can reduce the uptake of liposomes by reticuloendothelial tissues (24). In 2010's, radio oncologists have applied radionuclide 111In to bind with liposomes and investigate their biodistribution by molecular imaging, however, these pilot studies had not investigated the cytotoxicity of radionuclides to cancer cells (47-49). 111In is a γ-emitting isotope capable of being tracked by a radio-imaging system and killing cancer cells by inducing DNA double strand breaks (50, 51).
The highlights of the study are that we have not only validated the application of the combination of VNB plus 111In in rodent models (16), but also extended the efficacy of VNB plus 111In by multiple low dose treatments. In 2008, our team loaded liposomes with the radioactive 111In and the chemotherapeutic VNB and compared the therapeutic effects of them with other potential combinations (39). In 2012, the overall efficacies of the 111In-VNB-liposomes were investigated in a rat model with a perfect correlation between the pharmacokinetics and biodistribution (52). In the current study, we made a further modification by reducing the dose of the chemotherapeutic drugs from 5 mg/kg to 3 mg/kg and re-evaluated the overall efficacy from single dose to multiple doses. The multi-dose treatment showed a much better therapeutic effect than the single-dose treatment mode with regard to tumor growth inhibition (Figure 9 and Table III). Although the detail mechanisms and intracellular signaling networks are still largely unknown, our study indicated that the 111In-VNB-liposome could serve as a colorectal cancer therapeutic drug, while the efficacies of multiple low doses were much better than that of a single dose.
Conclusion
Assessing the therapeutic effect of 111In-VNB-liposomes on tumors, we obtained a good pharmacokinetic and biodistribution profile indicating that liposomes are ideal drug carriers for the treatment of cancer. The application of 111In-VNB-liposomes has a synergistic effect in the treatment of colorectal cancer, and its toxicity is within an acceptable range. Most valuably, we have demonstrated that 111In-VNB-liposomes could be administered to rodent models for the treatment of colorectal cancer and can be applied in multiple doses with a lower dose per fraction, i.e. smaller fraction size, instead as a single dose of a larger fraction size. Again, we demonstrated that the current multimodalities of an imaging system capable of tracking tumor growth and drug distribution simultaneously is indeed a convenient and powerful evaluating therapeutic system for translating research results in clinical practice in the era of precision and personalized medicine.
Footnotes
↵* These Authors contributed equally to this study.
Authors' Contributions
Research Design: Chien YC, Chou YH and Hwang JJ; Experiment Performance: Wang WH, Chen CH, Chang WS and Tsai CW; Statistical Analysis: Chien YC and Chou YH; Manuscript Writing: Bau DT and Hwang JJ; Reviewing and Revising: Bau DT, Chang WS and Tsai CW.
This article is freely accessible online.
Conflicts of Interest
No conflicts of interest exist regarding this study.
- Received September 30, 2019.
- Revision received November 4, 2019.
- Accepted November 5, 2019.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved