(190bg) Electrospun Microfibers and Lipid-Based Nanoparticles: A Combination Delivery System for Resveratrol and siRNA

Combining two therapeutic molecules has gained attention with the possibility of reducing both the dosages when they act synergistically. Previously, we showed that when resveratrol, a polyphenolic compound, is added along with siRNA into cell culture medium, the effects were additive [1]. However, resveratrol has a short half-life (1-3 hours) in plasma and its oral bioavailability is low (<1%). In order to obtain effective therapeutic levels, controlled and targeted release of resveratrol from core-shell microfibers is explored in this project. Endogenous characteristics of siRNA such as negative charge, rigid structure, size, and stability, makes passive diffusion through cell membrane quite challenging. To address this challenge, delivering siRNA using endocytosis has become an effective mechanism that can be achieved using liposomes. We questioned whether a dual delivery is advantageous to control these components. In particular, we first developed resveratrol containing co-axial fibers to achieve a higher dosage. Next, we developed ligand-targeted liposomes. Others have studied each method individually, showing unprecedented opportunities in the field of cancer diagnosis and therapy. We tested the combination effect on K562 cells.

Poly(ε-caprolactone) (PCL) and gelatin (GT) blends were utilized to form fibers with resveratrol, using a method previous reported [2]. Controlled release profile of resveratrol from the fibers was monitored over five days and samples were analyzed using HPLC. Next, siRNA-containing holo-transferrin-conjugated PEG-liposomes were synthesized using established techniques. Holo-transferrin conjugation efficiency was analyzed using BCA assay. Particle size was analyzed using DLS and TEM, and samples were further purified using Sepharose CL-4B chromatography columns. Uptake of Alexa-Fluor 488 conjugated siRNA by K562 cells was monitored using flow cytometry over a five day period. Based on the release studies and uptake results, apoptotic and necrotic effects on K562 cells were evaluated using Annexin V and Propidium Iodide staining, and flow cytometry.

Fibers formed with 40 µM resveratrol had a loading efficiency of 88.3 (±8.06) %, and 95% release by day five. Liposomes’ average effective diameter was 123 (±6.65) nm. The loading efficiency of siRNA in the surface-modified liposomes was 94.78%, and K562 uptake reached 83.44 (±3.04) % after 4 days of incubation. Holo-transferrin conjugation efficiency was 85.91 (± 7.30) %. The presence of the delivery system combination showed increased death rates as well as higher uptake rates compared to bolus doses combinations.

Loading resveratrol into electrospun fibers provides a potential localized delivery method, and increases resveratrol’s apoptotic effect. Encapsulating siRNA into surface modified liposomes increases its uptake rate by K562. The combination delivery system shows higher death levels in cancer cells. This combination has a broader impact potential on different types of cancer.

Acknowledgements: we would like to acknowledge the Oklahoma Center for Advancement of Science and Technology (HR15-142) and the BP Endowment, for financial support. Thanks are extended to Dr. Abdu Khalf for sharing his expertise in electrospinning, Dr. Carrie German for her insightful guidance with HPLC analysis, and PhD candidate Nicholas Flynn for his help with DLS.

[1] Al-Attar T, Khalf A, Madihally SV. Targeted and Controlled Combination Therapy Using siRNA and Resveratrol for Inducing Leukemic Cell Apoptosis. 2017 AIChE Annual Meeting, Minneapolis, MN.

[2] Khalf A, Madihally SV. Modeling the Permeability of Multiaxial Electrospun poly(ε-caprolactone)- Gelatin Hybrid Fibers for Controlled Doxycycline Release. Materials Science & Engineering C: Materials for Biological Applications. 76:161-170, 2017