siRNA and miRNA Cell Delivery Utilizing a Novel Nanoplatelet Platform, 01-R9736
Principal Investigators
Larry A. Cabell
Kent E. Coulter
Vicky Z. Poenitzsch
Joel J. Kampa
Lucy M. Kimmel
Ranjan Perera
Inclusive Dates: 07/01/07 – 12/01/10
Background — Chromosomal mutations often convert normal differentiated cells to undifferentiated, highly adaptive, stem cells that can re-differentiate into cancer tissues such as melanoma. These adaptive tissues often escape first-pass chemotherapies and become resistant to the first-pass chemotherapeutic agent in recurring tumors. New therapeutics such as siRNA (small interfering) and miRNA (micro interfering) are being synthesized to re-regulate intracell metabolic pathways that cancer cells depend on for their unregulated growth, immortality, metastatic invasion and other hallmarks exhibited by the specific cancer phenotype. Since siRNAs and miRNAs are unstable in the in vivo environment, they must be stabilized by nanoencapsulation in particles (100 to 200 nm) that avoid the immune system and travel through the tissue from the site of application into the region of the tumor where they are incorporated into the cytoplasm (endocytosed) and release the siRNA in a controlled fashion. Proper endocytosis requires narrow particle size distribution and a defined morphology that is limited by co-participation methods. These characteristics offer substantial impediments to introduction of these new therapies into the drug pipeline that is administered by the FDA. Thus, a new nanoparticle delivery system must be designed to be biocompatible, non-immunogenic and bioresorbable at the cytoplasm of the target cell where it will release its drug cargo in a controlled fashion (in this case siRNA and miRNA targeted for melanoma pathways). In addition, the delivery particle must be monodisperse in size and shape to ensure proper endocytosis. Finally, this delivery system must be amendable to cGMP manufacturing requirements.
Approach — The primary goal of this project is to develop a revolutionary generic drug delivery platform that employs physical deposition of drug, inorganic encapsulating and coating multilayers on nanopatterned substrates produced by photolithography. Functional, fluorescently labeled siRNA and later miRNA will be alternating current (AC) nanoelectrosprayed on an amorphous, magnetron-sputtered calcium phosphate (magnesium or fluorine-doped) layer. After sandwiching the drug layer with another calcium phosphate-based layer, the film will be fractured along the nanoembossed pattern on the substrate into 200 nm lateral dimension nanoplatelets that will be incorporated into a vehicle dispersion and functionalized by polyethylene glycol. Nanoplatelets will be characterized with respect to particle size, charge, surface area, solution aggregation properties and any perturbations in the double strand siRNA or miRNA structure induced by processing to optimize the manufacturing process. The endocytosis of the labeled siRNA loaded nanoplatelets into HeLa cells will be mapped as a function of time by a Nikon Eclipse TE2000E inverted microscope equipped for multicolor fluorescence imaging and siRNA transfection efficiency determined. Once the initial stages of the platelet production are completed, miRNA (specifically directed against the production of critical proteins used by melanoma cell lines) will be encapsulated in nanoplatelets and characterized and evaluated against well characterized melanoma cell line pathways using array mRNA expression analysis and other molecular biology techniques.
Accomplishments — E-beam deposition of a first bioresorbable calcium phosphate (CaP) layer on specially manufactured nanopatterned substrate was accomplished. This material was then subjected to AC electrospray deposition of a siRNA (silencing interfering RNA) second layer on the first CaP layer. A second e-beam deposition process of a CaP layer to seal in the siRNAs was also accomplished. This three-layer film was then fractured along the nanopattern. The nanoplatelets were unstable to dissolution and recrystallization in suspension with the surface treatments employed. In addition, there was incomplete evidence that the test electrosprayed siRNA was stable on the CaP surface (possible base hydrolysis). Finally, there was difficulty in fracturing the films along the nanopattern to generate the desired narrow particle distribution. The instability of the nanoplatelets, the difficulty in the fracturing process and in the nanoplatelet recovery from the fracturing process were believed to be the synergistic product of a state-of-the-art nanopatterned substrate that could not be produced to desired specifications to produce 200nm particles and particle solvent process requirements to free and isolate nanoplatelets. At this time of this project, a finer mask to produce the nanopatterned substrate could not be produced. Therefore a better understanding of the solvent requirements to fracture a coated nanopatterned substrate and recover particles without CaP crystal growth or recrystallization was undertaken. Solvent requirements to fracture a multi-coated nanopatterned CaP substrate and recover particles without CaP crystal growth or recrystallization was not accomplished by the end of this project. CaP solvent requirement investigations did produce a repeatable co-precipitation solution process to produce CaP-encapsulated isRNA and miRNA 100-200-nm particles.
