Spatiotemporal-controllable delivery of superparamagnetic nanoparticle carriers of RNA therapeutics for targeting cardiac diseases

Background: Cardiovascular diseases are among the most common causes of death in the world, but their treatment options are limited. Clinical studies are already showing alternative treatment approaches based on antisense oligonucleotides (ASOs) for the inhibition of non-coding RNAs (ncRNAs), which have shown important regulatory functions in a variety of cellular processes and disease conditions. A major challenge, however, is the effective and safe delivery of these therapeutics to the heart. In this project, we therefore aimed to optimize the delivery of ncRNA-therapeutics and to enhance cardiac uptake by using superparamagnetic iron oxide-based nanoparticles (SPIONs) as delivery vehicles. 

Methods and Results: We designed nanoparticles (NPs) allowing coupling of an antisense oligonucleotide (ASO) to the SPION via a newly developed thermosensitive linker-conjugate. We showed that binding of the ASO to this conjugate keeps it in an inactive state and can be guided to an organ of interest through a magnetic field. Application of a fast alternating magnetic field (AMF) leads to heating of the SPION core, breakage of the thermos-cleavable linker and subsequent traceless release of the ASO in the desired organ. First, we compared different SPION subtypes with regard to their biocompatibility showing that the cellular morphology and viability of cardiac cells remained unaltered when treated with one specific subtype. In a next step, we evaluated the toxicology profile and performed LDH and caspase assays in human cardiac fibroblasts, cardiomyocytes as well as hepatocytes and renal cells as the main off-target organs. We could show that all components of this novel nanoparticle system are not toxic and well tolerated. Furthermore, application of an AMF and therefore heating up the NP core did not cause any harmful effects. We also examined the release kinetics in vitro showing that 80% of the ASO could be efficiently released via an AMF. To examine the biosafety in vivo, we performed a proof-of-principle mouse study in which we injected intravenously our nanoparticle system (without release of ASO) and compared it to injection of pure ASO and PBS as a control. The survival rate indicated that our NP system is well tolerated and measurement of plasma markers confirmed that there were no unwanted immune responses and no signs for liver and kidney damage. In a subsequent efficacy study, we injected our NP-ASO conjugate, pure ASO and PBS as a control and released the bound ASO via an AMF. To enhance cardiac uptake we attracted the SPION-ASO conjugate via an external magnet in the region of the heart prior to injection. Application of pure ASO led to a strong suppression of the responsive ncRNA in all organs, whereas the use of the SPION-ASO conjugate resulted in a more cardiac-specific knockdown and significantly reduced off-target effects up to 400%. In a next step, we evaluated the therapeutic effect in an angiotensin mouse model showing that the SPION-ASO conjugate efficiently reduced fibrosis and improved cardiac function by inhibiting a pro-fibrotic ncRNA.

Summary: In summary, our data suggests that SPIONs can be efficiently used as cardiac-targeted delivery tools for ncRNA inhibitors and that they might overcome the current hurdles towards clinical practice such as off-target effects and unwanted immune responses. This is translatable to other organs of interest.