New Approach in Characterize siRNA Delivery Platform for Clinical Translation

Study Objectives A cationic peptide‐based nanotechnology system for siRNA delivery recently developed in our lab not only overcomes limitations of serum instability, endosomal entrapment and vascular constraint at the disease sites of interest, but also exhibits an exceptional safety profile (Hou et al, ACS Nano and Biomaterials; 2014). This delivery system has been applied in mechanistic and preclinical studies in the fields of cardiovascular diseases (Vendrov AE, J Am Heart Assoc. 2017; Lozhkin A, J Mol Cell Cardiol 2017) and arthritis (Yan, PNAS 2016; Zhou JCI 2014) with ongoing anti‐cancer applications on more than 6 different malignancies. Here we sought to leverage biophysical approaches to delineate certain critical physical properties responsible in part for the success of this siRNA delivery system. Methods and Results Total reflectance Fourier‐transform infrared spectroscopy (ATR‐FTIR) was used for the first time to demonstrate efficient packing of siRNA into peptide‐based nanoparticles. siRNA and p5RHH have different characteristic absorbance bands within the near IR spectrum, 1700 – 1500 cm −1 for p5RHH and 1250 −1000 cm −1 for siRNA. A net increase in these regions' band intensities corresponds to changes in local concentration of p5RHH/RNA on the FTIR‐ATR crystal. At 6°C and 400:4 μM p5RHH:siRNa ratios, nanoparticles of ~ 355 ± 141 nm radius form and then sediment to the bottom of test tubes (DLS measurements, data not shown). The local increased concentration on the FTIR crystal is due to the slow settling of nanoparticles where the measured spectrum represents a true read out of the nanoparticles composition with minimal contribution from the bulk material. Figure 1 demonstrates dominant bands in the spectrum in the 1700 −1500 cm −1 region (Amide I and II), while siRNA shows low (due to low concentration used) intensity (but distinguishable) peaks in the 1250 −1000 cm −1 with an additional small peak around 1710 cm −1 , corresponding to the C=O vibration of guanine. Absorbance bands within these regions exhibit 3 (p5RHH) and 8 (siRNa) fold augmented peaks and the ~1710 cm −1 peak emerges upon nanoparticle formation and settling down on the ATR crystal, strongly indicating a high RNA content in the nanoparticles. Conclusion This approach confirms siRNA‐peptide condensation and provides evidence‐based guidance for optimizing these and other nanoparticle formulations for more quantitative real time monitoring of siRNA‐peptide complex formation. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .