Therapies using small interfering RNA (siRNA) show significant potential for tackling conditions such as cancer and genetic diseases, but their success largely relies on effective delivery methods. A recent investigation revealed that the technique used to combine siRNA with lipid nanoparticles (LNPs) is crucial for achieving therapeutic success. By utilizing nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS), researchers discovered that differing preparation techniques influence the internal organization and distribution of siRNA within the LNPs, which in turn affects their therapeutic capabilities. Optimizing these techniques can boost the effectiveness of siRNA-encapsulated LNPs.
Small interfering RNA (siRNA) molecules are incredibly promising for treating diseases by targeting and silencing specific genes. When wrapped within lipid nanoparticles (LNPs), siRNA can be efficiently transported to intended cells. Nonetheless, the success of these treatments relies heavily on the inner structure of the LNPs, which significantly influences their ability to transport siRNA. Conventional methods often lack the detailed molecular understanding necessary to refine LNP design for maximum therapeutic benefit.
A study published in the Journal of Controlled Release on August 02, 2024, led by Assistant Professor Keisuke Ueda from Chiba University’s Graduate School of Pharmaceutical Sciences, presents a new method for enhancing siRNA-loaded LNPs. By using NMR to analyze molecular characteristics, the research explores how various siRNA blending methods impact the uniformity and molecular state of siRNA within LNPs. The study included contributions from Dr. Hidetaka Akita and Dr. Kenjirou Higashi from Chiba University and Tohoku University, and Dr. Kunikazu Moribe from Chiba University.
“NMR allowed us to look deeply into these nanoparticles at the molecular level, showing us the complex arrangement of siRNA inside the LNP core. This level of detail is vital for improving and optimizing LNP formulations,” Dr. Ueda stated.
The research team examined three different methods for preparing siRNA-loaded LNPs to assess their influence on molecular structure and gene-silencing effectiveness. The methods consisted of pre-mixing, where siRNA and lipids were combined using a microfluidic mixer; post-mixing (A), in which siRNA was added to empty LNPs under acidic conditions with ethanol; and post-mixing (B), where siRNA was incorporated into empty LNPs in an acidic environment without ethanol.
All three methods produced LNPs of similar size, around 50nm, maintaining a stable ratio of siRNA to lipid. However, the distribution of siRNA within these LNPs displayed notable differences. The pre-mixing technique led to a more even distribution of siRNA, while the post-mixing method resulted in uneven distribution with varying concentrations across the LNPs.
“This uneven distribution can greatly affect the siRNA’s silencing capability. LNPs with a more uniform siRNA arrangement are more effective in delivering their therapeutic content to targeted cells. This emphasizes the vital importance of refining preparation conditions to enhance treatment results,” Dr. Ueda elaborated.
The results suggest that pre-mixed LNPs have a more efficient gene-silencing ability. In these LNPs, the ionizable lipids were found to be more closely linked with siRNA, forming a stacked bilayer structure that strengthened the gene-silencing effect. On the other hand, post-mixed LNPs presented a more varied structure, likely hindering their interaction with cell membranes and reducing their therapeutic potential.
“This research has the potential to transform lives through the advancement of gene therapies and RNA-based treatments. By refining the delivery methods of siRNA through lipid nanoparticles (LNPs), therapies for conditions like cancer, genetic disorders, and viral infections could become significantly more potent. Additionally, this advancement could enhance the efficacy and safety of RNA vaccines, such as those developed for COVID-19, by improving their stability and minimizing side effects. Overall, this study may pave the way for more effective and safer patient treatments,” Dr. Ueda commented.
In the future, these innovations could facilitate the creation of personalized medicine, offering treatments tailored to individual patient needs. Improved drug delivery systems may also lower costs and increase access to cutting-edge therapies, benefiting a broader population.
