The antitumor effect of a thermosensitive polymeric hydrogel containing paclitaxel in a peritoneal carcinomatosis model
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- Yu, J., Lee, H., Hur, K. et al. Invest New Drugs (2012) 30: 1. doi:10.1007/s10637-010-9499-y
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The prognosis of peritoneal carcinomatosis is regarded as poor because safe, effective therapeutic modalities are lacking. Intraperitoneal chemotherapy is one treatment option, involving the delivery of a high concentration of chemotherapeutic drugs into the abdominal cavity, but the severe side effects associated with such treatment are a major obstacle in clinical application. We evaluated the anti-cancer effects of intraperitoneal delivery of a thermosensitive polymeric hydrogel containing chemotherapeutics in an animal model of carcinomatosis. The progress of peritoneal carcinomatosis, introduced by injecting a luciferase-transfected human gastric cancer cell line (HSC44Luc) into the peritoneal cavity of nude mice, was quantitatively evaluated by in vivo bioluminescence imaging. Three days after intraperitoneal (IP) injection of HSC44Luc cells, treatment solutions were injected into the peritoneal cavity. Mice were categorized into four groups depending on treatment method; these were (1) a control PBS group (n = 5), (2) a hydrogel-only group (n = 5), (3) a paclitaxel solution (30 mg/kg) group (n = 3), and (4) a hydrogel-with-paclitaxel (15 mg/kg) group (n = 5). Quantitative photon counting was performed weekly in each animal. Mice were sacrificed on the 5th or 28th day after treatment, for pathologic evaluation. In vivo bioluminescence imaging showed that photon counts in the hydrogel-with-paclitaxel and paclitaxel solution groups were significantly lower than in the PBS group over the entire experimental period. Although neither group of responding mice showed any peritoneal nodules on the 28th day after treatment, only the paclitaxel solution group exhibited dilated edematous changes in the intestine; these side effects were absent in animals treated with hydrogel-with-paclitaxel group. In conclusion, a thermosensitive hydrogel containing paclitaxel may be a safe and effective treatment option for peritoneal carcinomatosis.
KeywordsThermosensitive polymeric hydrogelBioluminescence imagingPeritoneal disseminationGastric cancerPaclitaxel
Peritoneal carcinomatosis, a malignancy found in the abdominal cavity, is one of the most intractable forms of metastasis. Currently, no standard treatment for peritoneal carcinomatosis is available. Several reports have indicated that intraperitoneal chemotherapy with or without hyperthermia, and peritonectomy, can prolong survival in patients with peritoneal carcinomatosis [1–4]. However, intraperitoneal chemotherapy does not offer any long-term survival benefit and can be associated with severe complications . Therefore, several novel local delivery techniques have been investigated in efforts to overcome such side effects [6–8]. Among these approaches, a drug delivery system using a polymeric hydrogel containing chemotherapeutic agents targeting tumor cells has been developed, and such drugs are maintained around tumor tissue at therapeutic concentrations over a long period of time, with a reduction in the level of systemic side effects [9, 10]. Hydrogels containing drugs have been used for localized drug delivery and controlled release of such drugs over a period of months. Hydrogels are thermosensitive and exhibit temperature-dependent reversible gel-to-solution transitions [11–14].
Paclitaxel (PTX) is one of the most widely used anticancer drugs. This agent shows significant activity against various solid tumors, including ovarian carcinoma, breast cancer, non-small cell lung cancer, and head and neck carcinomas [15, 16]. The drug interferes with mitosis and causes cell death by disrupting cell division [17, 18].
In subcutaneous xenografted mouse models, tumor volume can be easily measured with calipers [19, 20]. However, in peritoneal tumor dissemination models, it is impossible to measure tumor status or to evaluate a response to a drug using calipers. Therefore, we used photon counting to yield highly sensitive and quantitative measurements of several tumor-related variables in mice, and to evaluate peritoneal tumor dissemination. Photon emission data were converted into images using a non-invasive bioluminescent in vivo imaging system (IVIS). This method is based on expression of luciferase, a light-emitting enzyme of the firefly. After administration of the luciferin substrate, living cells containing luciferase emit photons owing to internal photochemical reactions and the particles are counted by a camera with a cooled charge-coupled device (CCD) [21, 22]. It is widely accepted that analysis of photon emission allows quantification of tumor cells transfected with luciferase.
Previously, we showed that when a hydrogel containing an anticancer agent was subcutaneously injected into the mouse, cancer was effectively suppressed and the bioluminescent imaging system allowed the results to be accurately evaluated . In the present study, we investigated the tumor suppressive effect of a hydrogel mixed with paclitaxel in mouse model of peritoneal carcinomatosis as a potential alternative treatment for gastric cancer metastasis in the peritoneal cavity.
Materials and methods
Biodegradable thermosensitive hydrogels
Hydrogels were obtained from the Korea Institute of Science and Technology. Preparation of a hydrogel physically mixed with paclitaxel was performed as described previously . Briefly, 10% (w/w) of aqueous polymer solution was dissolved in phosphate-buffered saline (PBS) and stirred continuously at 4°C for 4 days. After the hydrogel-forming material was dissolved, the solutions were filtered, sterilized by exposure to UV light, and next mixed with paclitaxel by stirring for 3 more days at 4°C.
The HSC44Luc human gastric cancer cell line
Luciferase-transfected human gastric cancer cells of the HSC44Luc were provided by the National Cancer Center Research Institute, Tokyo, Japan. Cells were cultured in RPMI1640 medium (Gibco BRL Life Technologies, Carlsbad, CA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco BRL) and 5% (v/v) of a mixture of penicillin (100 U/mL) and streptomycin (100 μg/mL) (Antibiotic-Antimycotic solution; Gibco BRL). Cells were grown at 37°C under an atmosphere of 5% CO2-95% air (v/v).
Quantification of luciferase activity
Twenty-four hours before transfection, cells were plated in 96-well plates with 1 × 104, 2 × 104, 4 × 104, 8 × 104, 1.6 × 105, or 3.2 × 105 cells per well. After 24 h of growth, cells were harvested using a lysis solution (100 mM potassium phosphate [pH 7.8] with 0.2% [v/v] Triton X-100) and a luciferase substrate (Applied Biosystems, Bedford, MA) was added to cell extracts followed by incubation at 37°C for 10 min. The intensity of luciferase activity was measured by luminometry (TR717; Applied Biosystems). All experiments were performed at least three times.
Sensitivity of HSC44Luc cells to paclitaxel
HSC44Luc cells were dispensed into wells of 96-well tissue culture plates. After incubation at 37°C for 24 h, paclitaxel solutions of various concentrations were added, and incubation continued for a further 72 h. The effects of treatment are presented as percentages of growth inhibition with respect to untreated control cells and as IC50 values (the drug concentrations inducing 50% reduction in cell survival), as calculated from dose–response curves.
Treatment of xenografted mice
Six-week-old female Balb/c nude mice were each intraperitoneally injected with 1 x 106 HSC44Luc cells. The animals were randomly divided into four groups and treated as follows: 1) intraperitoneal (IP) injection of PBS (PBS group, n = 5), 2) IP injection of hydrogel without paclitaxel (hydrogel-only group, n = 5), 3) IP injection of 30 mg/kg paclitaxel solution without hydrogel (paclitaxel solution group, n = 3), 4) IP injection of hydrogel physically mixed with 15 mg/kg paclitaxel (thus half the drug level used in group 3; group 4 is the hydrogel-with-paclitaxel group). These treatments were administered once three days after cell injection. On the 5th day after treatment, one mouse from each group was randomly selected and sacrificed for gross and microscopic examination of intraperitoneal organs and suspicious tumor nodules. On the 28th day after cell injection, the remaining animals were sacrificed.
In vivo bioluminescence imaging
Bioluminescence imaging was carried out as previously described . Each mouse was anesthetized with isoflurane and received an intraperitoneal injection of 100 μL of a 7.5 mg/mL solution of D-luciferin (Xenogen, Alameda, CA). Ten minutes after injection, bioluminescence imaging employing a CCD camera (IVIS, Xenogen) was performed with exposure times of 1–60 s, depending on the level of luciferase activity. Intraperitoneal tumor status was assessed by expressing the luciferase activity within each animal as a photon flux (photons/sec/cm2/steradian). Imaging was conducted before treatment to obtain background values. In the first 2 weeks after injection, luciferase activity was measured twice weekly and once weekly thereafter.
After sacrifice, peritoneal nodules were immediately removed, fixed in 10% (v/v) phosphate-buffered formalin, and embedded in paraffin. Formalin-fixed, paraffin-embedded sections were cut at a thickness of 4 μm, mounted on poly-L-lysine-coated slides (Sigma, St. Louis, MO), and dried overnight at 37°C. Sections were next dewaxed in xylene, rehydrated according to standard histopathologic procedures, and stained with H&E. A pathologist examined all specimens.
The independent t-test was used for comparison of photon count means among groups. P < 0.05 was considered to be statistically significant. Data analysis was performed using SPSS version 17.0 (SPSS, Chicago, IL).
In vitro luciferase activity assay
The intensity of luciferase activity in HSC44Luc cells was examined. As previously reported , activity increased as the number of transfected cells increased (data not shown). The cell line was thus confirmed to be appropriate for use in bioluminescence imaging.
Chemosensitivity of HSC44Luc cells to paclitaxel
The cytotoxicity of paclitaxel in HSC44Luc cells was determined using the MTT assay. The IC50 for paclitaxel was 0.007 μg/mL, and HSC44Luc cells were thus more sensitive to paclitaxel than were SNU gastric cancer cell lines (data not shown).
Local delivery of paclitaxel suppressed peritoneal tumor dissemination in a mouse xenograft model
Regarding adverse effects, intestinal dilatation and liver swelling were observed in some animals of the paclitaxel solution group on day 5 (arrowheads in Fig. 3a), but such changes were not observed in the PBS, hydrogel-only, or hydrogel-with-paclitaxel groups.
Peritoneal dissemination in patients with cancer recurrence or far advanced cancer is known to be an intractable problem. Therefore, many efforts have been made to develop treatments for peritoneal carcinomatosis, including systemic chemotherapy, peritonectomy, intraperitoneal chemotherapy, and thermotherapy. However, systemic chemotherapy is known to be ineffective in peritoneal carcinomatosis patients because drugs do not attain effective concentrations in the peritoneal cavity. Intraperitoneal chemotherapy may result in bone marrow suppression and other complications because chemotherapeutic agents are transiently present at high doses. Thus, compared with such methods, our hydrogel system may be safer and more effective for the treatment of peritoneal carcinomatosis, with a reduction in side effects.
To determine parameters for the experiment, we evaluated the optimal dosages of several chemotherapeutic agents when mixed with hydrogel (data not shown). Paclitaxel was the most effective drug in terms of both mixing capacity and cytotoxic effect when a thermosensitive hydrogel was used. Therefore, we chose paclitaxel as the model drug, and have shown here that the agent is effective in the treatment of peritoneal carcinomatosis arising from gastric cancer.
In a previous study, we reported that a thermosensitive hydrogel containing doxorubicin effectively permitted drug delivery into a tumor site and suppressed tumor growth . In the current study, we have shown that a hydrogel combined with paclitaxel was a more effective treatment for peritoneal carcinomatosis than was PBS or hydrogel alone in a mouse model. A paclitaxel solution had a similar antitumor effect, but such treatment was associated with significant side effects, which were absent when the drug was mixed with hydrogel. Also, the dose of paclitaxel (15 mg/kg) in the hydrogel was half of that in animals treated with paclitaxel solution (30 mg/kg).
With respect to the accuracy of the luciferase assay, we observed that luciferase photon counts were well correlated with macroscopic and microscopic findings in tumor seeding nodules. In the PBS and hydrogel-only groups, which showed higher luciferase activities during the entire observation period, malignant seeding nodules were observed on both day 5 and day 28. However, no such nodules were seen in animals treated with paclitaxel solution or hydrogel-with-paclitaxel on day 28, similar to the luciferase activity data. Therefore, the bioluminescence method can be effectively used to evaluate peritoneal tumor dissemination in mice.
The principal limitation of our study was that individual groups contained relatively few mice. In particular, the paclitaxel solution group initially contained only three animals and, after 1 week, one mouse was sacrificed for autopsy. Thus, we followed changes in luciferase activity in only two animals until day 28 post-injection. Similarly, the hydrogel-only group contained few animals owing to two early unexpected deaths (on days 2 and 6), the causes of which were not clear at autopsy.
In conclusion, we have shown that a thermosensitive controlled-release hydrogel system can be used to treat carcinomatosis in a peritoneal dissemination mouse model with minimal side effects. A polymeric hydrogel mixed with an anti-cancer drug may be a safe and effective treatment modality for patients with peritoneal carcinomatosis.
This study was supported by the Ministry of Education, Science and Technology (2009K001618) in Korea.