Although thousands of organ transplant operations happen worldwide each year, the efficacy of the transplants is limited by a reduction in graft survival over time due to acute and chronic immune responses. Immuno-suppressive therapies such as antibodies or small molecule drugs can reduce tissue rejection but at a cost of heightened risks of infections and cardiotoxicity. The ability to modify the immunogenicity of transplanted tissues would therefore provide an attractive alternative.

Capitalizing on this idea, Jiajia Cui and colleagues at Yale University have synthesized biodegradable poly(amine-co-ester) (PACE) nanoparticles containing small interfering RNA (siRNA) capable of reducing tissue rejection. Their work is recently published in Nature Communications.

The team fabricated PACE nanoparticles which encapsulate siRNA, thereby enhancing the stability and delivery of siRNA through the cell membranes. The researchers showed with both in vitro and in vivo systems that their nanoparticles reliably reduced expressions of major histocompatibility complex (MHC) which are cell surface proteins that enable the immune system to recognize foreign entities such as tissue grafts. The nanoparticles were also found to have minimal toxicity to the host cells.

The research team started out by synthesizing a range of PACE nanoparticles with a type of lactone known as 15-pentadecanolide (PDL). They found that PACE nanoparticles with different PDL composition exhibited burst (80%) release of siRNA in the first 2 days with gradual linear release over the next 5 days.

After several iterations and characterizations, Cui and co-workers decided to make use of PACE-70 nanoparticles with 70% PDL content, which had reasonably low toxicity (30%) to human umbilical vein endothelial cells, which are laboratory models of cells lining blood vessels. PACE-70 nanoparticles were also chosen as they suppressed >90% expression of MHC proteins which was significantly more than other PACE nanoparticle formulations.

Cui told MRS Bulletin that the PACE nanoparticles-siRNA is also relatively simple to fabricate and highly cost-effective. It takes about 9 days for each batch of nanoparticles. She also added that “the majority of the cost of making these nanoparticles comes from purchasing siRNA molecules (~$50 per batch), but one batch of nanoparticles can be used for multiple organ perfusions, so in the end the cost is not too high.”

The researchers demonstrated the utility of PACE nanoparticles-siRNA to suppress immune responses using several model systems. First, they showed that PACE nanoparticles-siRNA inhibited the expression of MHC class II proteins for more than 10 days with less than 10% of cells regaining their MHC expression at day 5. On the other hand, >70% of human umbilical vein endothelial cells treated with lipofectamine, the gold standard for transfection reagent, and siRNA regained their MHC expression at day 5.

The researchers also perfused solutions containing PACE nanoparticles through isolated blood vessels. They observed that endothelial cells harvested from veins perfused with PACE nanoparticles-siRNA had >90% reduction in MHC class II expression compared to endothelial cells harvested from veins perfused with nanoparticles without siRNA. Endothelial cells treated with PACE nanoparticles-siRNA also reduced the proliferation of co-cultured CD4⁺ memory T cells. This is a crucial step in immuno-suppression as this prevents CD4⁺ memory T cells from recognizing foreign entities, thus disabling their ability to initiate fast and potent immune responses to foreign substances.

Lastly, the researchers pretreated human arterial grafts with PACE nanoparticles-siRNA before transplanting them into severe immuno-compromised disease mice which are genetically engineered to contain human immune cells. They are used as laboratory animal models to study human immune response and tissue transplants. It was found that even after 6 weeks post-transplant, there was 20% suppression of MHC class II proteins. The research team also observed that such treatment conferred protective effects on grafts by reducing the infiltration of T cells that typically initiate graft destruction.

Cui hopes to see their technology being applied for in vivo therapeutic applications. However, she acknowledges that there is still a barrier to overcome. “We made our PACE nanoparticles for delivery of therapeutics to the vascular endothelium in the context of ex vivo perfusion. If we were to use these particles for in vivo therapeutic applications, we would need to add potential targeting molecules to improve delivery to desired organs and tissues.” 

Read the article in Nature Communications.