New publication
August 20, 2024

Conventional drug and gene delivery nanoparticles typically employ a vehicle size below 200 nm to reduce loss to phagocytic cells, such as monocytes and macrophages. This is because phagocytosis, the specific particle uptake mechanism by these cells, is size dependent and has a higher efficiency when the cells encounter big particles particularly in the larger sub-micron to micron range. In recent years, monocytes and macrophages have become valuable therapeutic targets in gene therapy for cancer and auto-immune diseases. We had a great inspiration to ask an interesting question: Can size-dependent phagocytosis be exploited for targeted delivery of gene therapy to macrophages? While size-dependent phagocytosis has been well-characterized and documented in literature, the real challenge is to construct gene-loaded particles to a very large size. The most adopted stabilization methods for gene delivery nanoparticles, i.e., surface PEGylation (steric hindrance) and/or charge repulsion, limit the vehicle size around or below 100 nm. Particle assembly beyond 200 nm has been associated with undesired, uncontrollable aggregation behaviors, and instability in a suspension form.

We approached this challenge with knowledge gained throughout the years on the critical parameters governing the kinetic stability of nucleic acid/polycation complex nanoparticles. In particular, we invented a surface charge-dependent supramolecular assembly method that allowed us to assemble mRNA/poly(β-amino ester) nanoparticle "seeds" into superstructures, that can have a defined size kinetically tunable in the range of 100 to 1000 nm and fully programmable by controlling the solution quality. These supramolecular assemblies feature colloidal stability in physiological medium even after freeze-thaw, an extremely high payload capacity, and preferential uptake by and transfection of monocytes and macrophages in cell culture and after intravenous injection in mouse models. While we found that cellular uptake level monotonically increased with larger assembly size up until 1000 nm, meaning that monocytes and macrophages do always prefer larger particles, a size of 400 nm gave the highest efficiency in functionally delivering the mRNA to give gene expression. It was because it did not induce cytotoxicity due to phagocytosis of too much materials.

In this paper, we demonstrated the utility of this particle assembly system in delivering antigen mRNA and an adjuvant to monocytes, which mediated their subsequent differentiation into inflammatory dendritic cells that induce anti-tumor effects in a cancer model. Through this proof-of-concept study, we for the first time highlight the potential of circulating monocytes as a vaccine target, which was only predicted previously (Cheong et al., Cell, 2010). We expect this platform technology to find broader applications for in vivo cellular programming, such as stimulating tumor-associated macrophages (TAMs) and engineering chimeric antigen receptor (CAR)-functionalized monocytes and macrophages.

This study was co-led by Stephany Y. Tzeng, and supervised by Prof. Jordan J. Green, and Prof. Hai-Quan Mao at Johns Hopkins University. We appreciate collaborations with Prof. Erik Luijten from Northwestern University in understanding the supramolecular assembly mechanism. Our paper describing these findings has now been published with open access in Proceedings of the National Academy of Science of the United States of America (Hu and Tzeng, et al., PNAS, 2024).

Part of the image was generated using BioRender.com