Electrojetted Biodegradeable Polymer Nanoparticles for drug delivery and cell targeting applications.
Preparation of protein-releasing micro and nanoscale particles by two-phase electrojetting of protein in the biodegradeable polymer PLGA using surfactants as emulsifying agents.Sejal Tailor, Sarah Richardson-Burns, Kyung-Ho Roh, Joerg Lahann, and David C. Martin
Current drug treatments for many ailments are invasive, unspecific, and uncomfortable, so studies involve developing implantable drug delivery methods. A new method for drug delivery involves electrojetted proteins emulsified in nanospheres made of a polymer. We employed a two-phase electrojetting/electrospinning technique in order to generate micro and nanoscale spheres and fibers comprised of 50:50 poly(lactic-co-glycolic acid) (PLGA, dissolved in chloroform) into which the protein bovine serum albumin (BSA, diluted in phosphate buffered saline; PBS) was mixed using the surfactant Triton X-100. The surfactant acted as an emulsifying agent to maintain the protein in suspension within the polymer. We first conducted solubility tests on a variety of surfactants including Triton X-100, Tween-20, Tween-80, Pluronic, Span-80, polyethylene glycol to determine which surfactant allowed PBS to remain in suspension in the PLGA the longest. Triton X-100, Span-80, and Tween-80 showed the best results with Triton X-100 showing the most complete, longest lasting emulsion. Therefore for future experiments the electrojetting solution used was comprised of .323 % (w/v) BSA (fluorescently labeled with AlexaFluor 594), .323% (v/v) Triton X, 9.68% (w/v) PLGA, 2.90 % PBS, 86.774% (v/v) Chloroform. After electrojetting/electrospinning of the PLGA-protein-surfactant emulsion, we assessed the morphology of the resulting micro/nanospheres using optical microscopy and used fluorescence microscopy to detect the presence of the protein within the PLGA nanospheres. Distinct globules of protein were detectable within both the micro/nanospheres and the nanofibers. PLGA is a biodegradable protein therefore, we next measured the amount of protein released from the micro/nanospheres incubated in PBS at 37oC over a two month timecourse. Data points were taken at 0h, 24h, 5 days, 12 days, 28 days, and 56 days and the BCA protein assay (Pierce Biochemicals) was used to quantify protein release. For the first four days an average of 8 µg of protein was released, and eight days later an average of 6 µg of protein released, and after 28 days of incubation an average of 13 µg of protein was released. A second experiment performed over a two week timecourse revealed that for the first two days an average of 18 µg of protein was released, and five days later an average of 18 µg of protein was released and at the end of the two-week period an average of 6 µg of protein had been released. We are currently performing cytotoxicity tests on PLGA-protein-surfactant spheres prepared using the surfactant Triton X-100 exposed to SH-SY5Y human neuroblastoma cells. We are also assessing whether two biologically-derived phospholipids, Dimodan (distilled monoglyceride) and Phosphotidylcholine (plasma membrane phospholipid) can act as emulsifying agents to replace the Triton X-100 in the PLGA-protein-surfactant mixture. We will also be using the biologically-active protein Nerve Growth Factor (NGF) to replace the BSA in the electrojetting mixture to generate NGF-containing PLGA micro/nanoparticles to deliver NGF to PC-12 neural cells. When exposed to NGF, PC-12 cells undergo differentiation characterized by specific morphological changes such as neurite extension. The utility of the PLGA micro/nanospheres as possible protein/drug-delivery vehicles will be assessed by detecting whether the PC-12 cells undergo differentiation following exposure to the NGF-containing particles.