Purpose: Neuroblastoma is the most common extracranial solid tumor of childhood tumor and treatment requires surgical resection and multi-drug chemotherapy. Etoposide is an effective drug for the treatment of neuroblastoma; however, systemic delivery of etoposide is limited due to low water solubility and high toxicity. We hypothesized that an injectable, sustained release gel formulation, loaded with etoposide could suppress tumor growth after injecting into the tumor while limiting systemic toxicity. We prepared and characterized etoposide loaded silk gels and evaluated the formulations for in vitro release and in vitro cytotoxicity.
Methods: Silk fibroin was isolated from Bombyx mori cocoons as previously described (1). Seven percent silk solutions were prepared by diluting the 30 min extracted (30mE) silk stock. Etoposide was dispersed in ethanol before mixing with 7%, 30MB silk solution (1:1) and then the mixture was vortexed until it formed gels. The structural features of the gels were evaluated using Fourier Transform Infrared (FTIR) Spectroscopy. The gels were placed into 1 mL phosphate buffered saline (PBS) and incubated at 37°C for in vitro drug release studies (n=3). Release medium was collected and replaced with fresh PBS at time points and etoposide in the samples was quantified by UV/Vis spectroscopy at 285 nm. Blank silk gels were used for background correction. Cytotoxicity of etoposide gel formulations on human neuroblastoma cells (KELLY) and metastatic neuroblastoma cells (SK-N-AS) were evaluated using AlamarBlue® assay. Cells were seeded in 96-well plates at 10,000 cells per well and allowed to adhere overnight at 37°C in a 5% CO2 atmosphere. Etoposide gel release samples were added to the cells and incubated 48 hours before 10% AlamarBlue® solution was added to the plates. Four hours later the fluorescence was read on a plate reader using excitation at 570 nm and emission at 600 nm to evaluate cell viability.
Results: FTIR measurements were used to determine the crystalline structure of silk induced by ethanol treatment during gel preparation. The FTIR data showed that the β-sheet content of blank and etoposide loaded gels were 35.8% and 34.2%, respectively. The β-sheet content of the formulations indicated the stability of the silk due to the crystallization, and etoposide loading did not have a significant effect on the β-sheet content of the gels. In vitro release studies showed that 66.52% of the drug was released in the first 24 hours, then the release continued slowly for 24 days where the total drug release was 99.85% (Fig. 1). Alamar blue analysis showed that the etoposide gel formulations killed 95.77% of the KELLY cells and 97.55% of the SK-N-AS cells, respectively, in the first 2 hours. KELLY cell viability was 1.69%, 26.38%, 73.73% and 49.07% following the treatments at 1, 3, 9 and 22 days release. SK-N-AS cell viability was 5.58%, 26.64%, 27.88% and 77.10% following the treatments at 1, 3, 9 and 22 days release.
Conclusion: Based on our prior studies, biodegradable silk gels can be easily injected within solid tumors. We prepared and characterized a controlled release silk gel formulation to deliver etoposide to tumors for neuroblastoma treatment. We plan to test the in vivo efficacy of the gels in an orthotopic neuroblastoma mouse model.
1) Rockwood et.al. Nat. Protoc., 6 (2011), pp. 1612-1631.
Kristin Harrington– Research Technician, Tufts University
Jeannine M. Coburn– Assistant Professor, Worcester Polytechnic Institute
Bill Chiu– Associate Professor, Stanford University
David L. Kaplan– Professor, Tufts University