In vivo hematopoietic stem cell targeting with layer-by-layer nanoparticles

Introduction:: Ex vivo cell therapies for hematopoietic stem cell (HSC) modification present high therapeutic potential for many diseases. Although promising, these strategies carry high costs through cell harvesting, transduction, and reinfusion, as well as the need for specialized personnel and equipment. Developing a system that can reach HSCs in vivo is a drug delivery challenge that needs to be overcome in order to create more translatable and accessible treatments [1].
Nanoparticle (NP)-based drug delivery systems have demonstrated huge therapeutic impact, due to their tunability and capacity to deliver a variety of drugs [2]. Nevertheless, delivery to the very low numbers of HSCs is hampered by non-specific NP uptake by off-target circulating immune cells, which rapidly clear NPs from circulation. In this context, the layer-by-layer (LbL) technique enables incorporation of a wide range of charged polymers on the surfaces of the NPs to tune their surface properties, targeting capacity, and biodistribution [3].
The objective of this work has been to develop a delivery carrier based on LbL NPs functionalized with HSC-targeting antibodies, that will allow for an effective HSC targeting in vivo.

Materials and Methods: Fluorescently-labeled liposomes were prepared by film hydration, and layered with oppositely charged polymers to form LbL NPs. Dynamic light scattering was used to track size and surface charge. Antibodies were covalently conjugated to the outer most layer of the NP. Uptake in vitro was analyzed by FACS, and trafficking by confocal microscopy. C57Bl/6J mice received one intravenous injection of NPs, and interactions with blood cells and HSCs was quantified 1.5 hours after administration using flow cytometry.

Results, Conclusions, and Discussions: Our first goal was to decrease the clearance rate of the NPs by circulating myeloid cell. To do this, we designed a library of LbL NPs with three outer layer polymers, with known different cell targeting capacities. After intravenous injection of fluorescently-labeled LbL NPs, we observed a significant difference in NP uptake by circulating myeloid cells, with a 5-fold decrease in NP uptake when switching from a polysaccharide as outer layer to a synthetic acrylic polymer. These results further support that the tunability of the surface properties of LbL NPs makes them a unique drug delivery platform.
Based on these results, we then conjugated a selection of HSC-targeting antibodies onto the surface of the LbL NPs with lower myeloid uptake. To better understand the changes in the targeting capacity of these NPs, we compared NP uptake in three relevant cell lines: an immortalized bone marrow myeloid cell line, freshly harvested mouse bone marrow cells, and progenitor mouse bone marrow cells. Significant differences in uptake were detected among the different antibody candidates, with a stronger difference after 90 minutes of incubation (Figure 1A). Microscopy analysis showed that the different antibody candidates modified the NP uptake pathways, with some of them associating only to the cell membrane, and others increasing NP internalization (Figure 1B). In vivo administration of the targeted LbL NPs showed that, after one single injection, ~10% of the bone marrow resident HSCs had been targeted by the best candidate selected in vitro. Lastly, the conjugation of the humanized version of the antibody improved NP uptake by human CD34⁺ cells in vitro, confirming the translatability of the carrier.

In conclusion, we designed a drug delivery system that is able to efficiently target HSCs in vivo over circulating myeloid cells. This work further demonstrates the tunability of LbL NPs can be leveraged for a variety of drug delivery applications. Indeed, understanding the nano-bio interactions between nanoparticles and the targeted cells, and the mechanisms driving them, is key to design the next generation of drug delivery carriers.

Acknowledgements (Optional): :
Funding was provided by the Bill and Melinda Gates Foundation. Authors thank the Koch Institute's Robert A. Swanson (1969) Biotechnology Center for technical support, specifically the High Throughput Sciences, Microscopy and Flow Cytometry cores.

References (Optional):
[1] Tucci, F.; Scaramuzza, S.; Aiuti, A.; Mortellaro, A. Update on Clinical Ex Vivo Hematopoietic Stem Cell Gene Therapy for Inherited Monogenic Diseases. Molecular Therapy 2021, 29 (2), 489–504. https://doi.org/10.1016/j.ymthe.2020.11.020.
[2] Anselmo, A. C.; Mitragotri, S. Nanoparticles in the Clinic: An Update Post COVID ‐19 Vaccines. Bioeng Transl Med 2021, 6 (3). https://doi.org/10.1002/btm2.10246.
[3] Correa, S.; Dreaden, E. C.; Gu, L.; Hammond, P. T. Engineering Nanolayered Particles for Modular Drug Delivery. Journal of Controlled Release 2016, 240, 364–386. https://doi.org/10.1016/j.jconrel.2016.01.040.