(647b) Control over the Temporal Profile and Sequence of Anticancer Therapeutics from Magnetically Responsive Hydrogels

CONTROL OVER THE TEMPORAL PROFILE AND SEQUENCE OF ANTICANCER THERAPEUTICS FROM MAGNETICALLY RESPONSIVE HYDROGELS

Tania T. Emi¹, Tanner Barnes², Emma Orton², Anne Reisch¹, Zachary Silveira², Miranda Mitchell², Celia Dunn², Anita Tolouei¹, Stephen M. Kennedy^1,2

¹ Department of Chemical Engineering, ² Department of Electrical, Computer and Biomedical Engineering, University of Rhode Island, Kingston, RI 02881

Problem: Cancer is the second leading cause of death in the US. It is estimated that, in 2017, 1.7 million American patients will be diagnosed with new cancer cases. Traditional chemotherapies involve systemic deliveries which can have difficulties in maintaining therapeutic drug concentration at tumor sites and can cause off-target side effects. Biomaterials-based chemotherapeutic strategies can circumvent these issues by providing localized and sustained delivery at tumor sites; however, the temporal drug delivery profiles provided by traditional biomaterial implants may not be optimal. Moreover, many current and emerging anticancer treatments involve the delivery of multiple therapeutics. Traditional biomaterials cannot coordinate temporally complex sequences of multiple therapeutics. We hypothesized that hydrogels designed to deform in response to magnetic stimuli could provide control over more optimized temporal profiles and sequences of anticancer drugs. Additionally, magnetically responsive hydrogels would provide real-time, remote control over drug delivery regimen. This flexibility would be critical in clinical settings where altering the course of therapy according to updated patient prognoses and patient history are critical. Here, we demonstrate the ability to magnetically control the temporal profile and sequence of anticancer deliveries from magnetically responsive hydrogels. Furthermore, we demonstrate that specific temporal delivery profiles and sequences generated by these magnetically responsive hydrogels are more effective at killing melanoma cells than deliveries associated with traditional hydrogels.

Methods: Magnetically responsive hydrogels were made by casting 7 wt% Fe₃O₄ in 1 wt% alginate with 2.5 mM Adipic acid dihydrazide crosslinker in the presence of a magnet to yield biphasic constructions. Gels of 8 mm diameter were cut using a biopsy punch, rinsed for 3 days in deionized water, frozen at -20⁰C overnight and lyophilized to yield porous, magnetically deformable structures. Gels were soaked with 125 ug of chemotherapeutic drugs (mitoxantrone, dacarbazine, 5-fluorouracil, or irinotecan) for 24 hours, rinsed to get rid of the excess drug, and placed in 1 mL of PBS. Drug-loaded gels were placed in a magnetic piston system and stimulated for various durations at various stimulation frequencies. During this magnetic stimulation, samples were taken periodically by collecting and replacing PBS. The concentration of collected samples was quantified using BioTek Cytation3 microplate reader to measure optical absorbance at different wavelengths, depending on the chemotherapeutic. In cell studies, B16-F10 mouse melanoma cells were seeded at 500 cells per cm² and allowed to grow for 1 to 3 days before treatment. In treatments that involved exposing melanoma cells to various chemotherapeutic temporal profiles, each treatment schedule used the same integrated amount of drug (666 ng per mL per day of mitoxantrone). In treatments that involved exposing melanoma cells to multiple drugs, cells were exposed to different sequences of 5-fluorouracil (5FU) and irinotecan at 10 and 25 micrograms per mL, respectively. To assess viability, both endpoint LIVE/DEAD viability staining was used as well as real-time cell indexing using an xCELLigence system. For statistical analyses, ANOVA with Tukey’s post-hoc tests was used with p < 0.05 being the benchmark of significance.

Results: Our in vitro cell studies demonstrated that pulsatile temporal delivery profiles significantly enhance the toxicity of mitoxantrone exposure to melanoma cells when compared to constant delivery profiles produced by traditional hydrogels (p < 0.01). Real-time cell viability analyses revealed that melanoma cell populations were most affected during drug pulsing, suggesting that increasing the number of pulses may be more effective. Critically, these more effective pulsatile mitoxantrone delivery profiles were capable of being reproduced using our magnetically responsive hydrogels. These hydrogels were capable of producing a wide range of pulsatile deliveries where pulse height was controlled through the frequency of magnetic stimulation and pulse timing/duration through the timing/duration of magnetic stimulation. Additional in vitro cell studies demonstrated that when delivering sequences of irinotecan and 5FU (a common combination cancer treatment), that initial delivery of 5FU followed by irinotecan yielded more efficient melanoma death than irinotecan followed by 5FU (p < 0.01). Experiments revealed several strategies for magnetically controlling sequences of 5FU and irinotecan. Compartmentalization of our magnetically responsive hydrogels into (1) a cylindrical shaped inner compartment nested inside (2) a ring-shaped outer compartment enabled independent deformations of inner and outer compartments when stimulating using cylindrical- and ring-shaped magnets, respectively. Individual drugs could be released at specific times by using appropriately shaped magnetics to trigger the compartment containing the desired drug. Additionally, by analyzing the release of 5FU and irinotecan as a function of magnetic stimulation frequency, it was discovered that 5FU could be released in earnest at early time points without magnetic stimulation (i.e., it diffused out of our alginate gels) whereas irinotecan could be retained in the gel until magnetically triggered to release. These 5FU and irinotecan release characteristics enable the sequenced release of 5FU followed by irinotecan, as shown to be optimal in our in vitro studies.

Conclusions: We successfully developed a hydrogel system that can deliver chemotherapeutics in a pulsatile manner through the use of externally applied magnetic fields. The temporal profiles of these deliveries were remotely controlled by the timing, duration, and frequency of the applied magnetic fields. In vitro cell studies demonstrated that the pulsatile deliveries made possible by these magnetically responsive hydrogels were significantly more effective in killing melanoma cells than the relatively continuous delivery profiles produced by traditional hydrogels. Additional in vitro studies demonstrated that delivery of 5FU followed by irinotecan was more effective than the delivery of irinotecan followed by 5FU. Further experiments performed on our magnetically responsive gels revealed several promising strategies for coordinating sequences of 5FU and irinotecan deliveries. We believe these magnetically responsive hydrogels will provide useful tools for treating solid tumors by enabling localized deliveries of more advanced drug temporal profiles and sequences. Their ability to be regulated remotely also enables real-time control over treatments, which adds greatly to their potential clinical utility.

Contact Information: Name: Stephen M. Kennedy, Institution: University of Rhode Island, Department: Electrical, Computer, & Biomedical Engineering and Chemical Engineering, Phone #401-874-5295, Email: smkennedy@uri.edu.