Layer-by-Layer Nanoparticles for Dexamethasone Targeted Delivery to Hematopoietic Progenitor Cells

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) have particularly dismal patient outcomes, with low 5-year survival rates in adult patients. Outcomes in pediatric patients are more optimistic, mainly due to their ability to withstand more aggressive treatment regimens. Glucocorticoids, although used as a standard treatment, can have severe adverse effects that limit dosing regimens. To address these challenges, directing drugs to hematopoietic progenitor cells implicated in these leukemias via targeted drug delivery therapies would allow for more aggressive and effective treatments, with reduced side effects. Layer-by-layer (LbL) nanoparticles (NPs) are a modular drug delivery system that enables the incorporation of a wide range of charged polymers on the surfaces of a drug-loaded NP core, to tune surface properties, targeting capacity, and biodistribution of NPs. The outer polymers can also be used as scaffolds to conjugate molecules, like antibodies, to aid in targeting drugs to cells of interest. In this work, we developed LbL NPs with dexamethasone loaded in the liposome core and functionalized them with polymers and antibodies to evaluate their targeting capacity to hematopoietic stem and progenitor cells (HSPCs) implicated in AML and ALL.<br/><br/>Dexamethasone palmitate (DXP) was incorporated as part of the lipid bilayer of liposomes using lipid hydration. Drug concentrations were measured via high-performance liquid chromatography (HPLC). Negatively-charged liposomes, empty or encapsulated with DXP, were layered with a cationic polypeptide, followed by a layer of anionic synthetic polymer on top. HSPC-targeting antibodies were then conjugated onto the top polymer layer. NP size and surface charge were monitored throughout the process via dynamic light scattering. NP concentration was determined via fluorescence intensity measurements of dye-conjugated lipids in the liposomal core. A bicinchoninic acid (BCA) assay was used to assess antibody concentration after purification. NP-cell association studies were conducted via fluorescence-activated cell sorting (FACS).<br/><br/>Antibodies were successfully conjugated to the NPs, with no notable impact on their physicochemical properties when measured over a 2-month period. Furthermore, antibody-functionalized LbL NPs demonstrated preferential association with a relevant myeloid progenitor-like cell line (ER-HOXB8), following 90-minute incubation. In fact, two candidate antibodies had a 1.5-fold increase in NP-cell association compared to unconjugated LbL NPs. For DXP-loaded NPs, it was found that the presence of the drug in the liposomal core had minimal impact on the stability of the NP throughout the layering process, with negligible changes in drug loading. Preliminary longitudinal studies found that the presence of the drug had no notable impact on the colloidal stability of the LbL NPs.<br/><br/>Overall, we have shown that LbL NPs can be functionalized with HSC-targeting antibodies, with a preferential association to relevant cell lines, and that the LbL construction of NPs is unhindered by the presence of DXP in the core. Future work would involve combining the drug-loaded NPs and functionalizing them with the candidate antibodies to ensure that similar preferential association is observed in relevant <i>in vitro</i> and <i>in vivo</i> models of acute leukemias.