(F-203) Impact of nanoparticles characteristics on gene-silencing siRNA delivery

Introduction:: Small interfering RNA (siRNA) are noncoding double stranded RNAs with important roles in gene regulation that can exert gene silencing effects at the post-transcriptional level by targeting messenger RNA (mRNA). SiRNA can be used as a therapeutic tool to silence pro-inflammatory signals that can become aberrant in disease and injury models, thereby reducing inflammation. One potential application is spinal cord injury, which results in intense inflammation that causes further damage to the spinal cord, known as secondary injury. Although siRNA has a high silencing effect, it can be rapidly eliminated from the bloodstream and has poor transport across cell membranes. One approach to improving the delivery and efficacy of siRNA involves the use of nanoparticles (NPs). NPs work as a protective carrier which helps to avoid clearance mechanisms and increase payload stability. In this work, poly (lactic-co-glycolic acid) (PLGA) NPs were used to encapsulate siRNA with the goal of silencing inflammatory signals associated with traumatic injury.


Materials and Methods:: PLGA NPs were fabricated using a water-in-oil-in water double (w/o/w) emulsion technique. Sonicator speed and the solvent were tuned to optimize size and surface charge of NP formations, respectively – both factors that impact siRNA release and cellular engagement. NPs were evaluated with dynamic light scattering (DLS) to assess NP size, surface charge, and polydispersity. A fluorometric assay was performed to quantify fluorescent-conjugated siRNA encapsulation within the NPs. To do this assay, lyophilized NPs were resuspended in dichloromethane (DCM) until fully dissolved. Water was added to precipitate the siRNA from the polymer, after which the fluorescent siRNA was retrieved from the water layer and evaluated against a standard curve to identify the quantity of loaded siRNA that was encapsulated. Similarly, fluorophore-conjugated siRNA release was quantified through evaluation of the siRNA fluorescent signal. siRNA loaded NPs incubated in saline were continuously shaken at 37 °C degrees for 192 hours, with supernatants collected at regular intervals Supernatants were stored at –20°C until all time point samples were collected, at which time the fluorescent signal was measured to determine the amount siRNA released versus the original amount encapsulated. Ongoing experiments with cultured macrophages will test the gene silencing efficacy of soluble vs NP-encapsulated siRNA through use of a siRNA encoding for a fluorescent reporter gene. SiRNA silencing of pro-inflammatory pathways will also be tested through quantitative polymerase chain reaction and Western blot analysis. All experiments are repeated a minimum of 3 times with a minimum of 3 technical replicates.


Results, Conclusions, and Discussions:: siRNA-encapsulating PLGA-NP were successfully fabricated using a w/o/w double emulsion with an average size of 213.4 ±0.4 nm. As storage of sensitive biologics can be challenging, the impact of storage processing, namely lyophilization, was evaluated using DLS. Non-lyophilized vs lyophilized NPs did not result in differences in PLGA NP size, polydispersity, or surface charge (data not shown). However, NPs stored in saline at 4°C for 1 week showed higher polydispersity compared to lyophilized NPs stored at room temperature for 1 week (Fig. 1A-B). As lower polydispersity means less heterogeneity within the NP batch, all NPs were lyophilized to ensure storage stability and increase reproducibility for cellular assays. Lyophilized NPs were evaluated further for siRNA loading and release encapsulation efficiency of siRNA in PLGA NPs. Of the loaded siRNA, approximately 80% was released within 24 hrs from the NPs. (Fig. 1C).


siRNA can be efficiently encapsulated in PLGA NPs. However, siRNA burst release occurs within 24 hours. Burst release to target acute inflammation may be appropriate, however, slower degrading NP formulations are being evaluated for more sustained siRNA release for targeting chronic inflammation. This work also found that lyophilizing the NPs improves storage longevity without adversely changing the NP characteristics, including size and surface charge. Based on these findings, we are currently developing additional NP formulations and evaluating NP uptake and gene silencing in cultured macrophages using siRNA for reporter genes (EGFP) and pro-inflammatory genes (TNF). Together, these data suggest that the use of NPs to deliver siRNA is a promising next-generation drug delivery approach for the treatment of different injuries such as spinal cord injury.


Acknowledgements (Optional): : NeuSTEM laboratories, University of Miami Undergraduate Research Office, University of Miami Office of Academic Enhancement, The Miami Project and Florida-Georgia Louis Stokes Alliance for Minority Participation (FGLSAMP) program.

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