Age Matters: in vivo Charged PEGDA Nanoparticle Phagocytotic Capacity of Aged Murine Pulmonary Antigen Presenting Cells via Direct Lung Delivery

Introduction: Natural aging leads to higher susceptibility to severe respiratory infection due to altered pulmonary immune cell functions. This dysfunction by immune cells leads to reduced clearance of inhaled debris and pathogens via cell-based mechanisms, which perpetuates a chronic low-grade pro-inflammatory microenvironment in the lung. Overall, this leads to reduced immunity and vaccine responses to traditional therapeutic and vaccination approaches. Pulmonary delivery offers an alternative method to directly target immune cells in the lung and overcome the challenges of needle-based routes of administration. In fact, pulmonary antigen-presenting cells (APCs) are abundant in lung tissue and are ripe targets for nanoparticle(NP)-based designs given their basic functionality to engulf local inhaled debris and activate global, mucosal immunity. However, to date there exists no known aged-specific parameters to design engineered immunomodulatory NPs for pulmonary delivery. Thus, there is clear motivation to study inhaled NP drug delivery platforms within an aged model that can promote effective, long-term systemic immunity via precise targeting of pulmonary APCs.
APCs are cells that are capable of processing pathogen material (“antigen”) and presenting it to T cells for specific cellular- and antibody-based forms of immunity. Within the lung there are four major classes of APCs, alveolar macrophages (AMs), interstitial macrophages (IMs), CD11b+ dendritic cells (DCs), and CD103+ DCs. Previous literature has established that AMs and DCs show unique and opposing charged-based preferential uptake of inhaled polyethylene-glycol diacrylate (PEGDA) NPs. Hence, we sought to determine if aged APCs demonstrated similar surface charge-based NP uptake to young adults.

Materials and

Methods: PEGDA hydrogel NPs were synthesized via a batch, reverse emulsion photopolymerization technique at 50 wt% solids. Charges were modified in the NP monomeric mixture via 10 wt% incorporation of either a co-monomer with an amine-presenting group (positive (+NP); pink) or a carboxyl-acid presenting group (negative (-NP), purple). A Cy5 maleimide molecule was also added to the monomeric mixture for fluorescence assays. Size, charge, and polydispersity index of the NPs was characterized using dynamic light scattering (DLS) and thermogravimetric analysis (TGA). Characterized PEGDA NPs demonstrated hydrodynamic sizes between 200-300 nm and absolute zeta potentials around 25 mV.

Female C57BL/6 mice (n=6) of two ages (2-months and 16-months) were dosed via orotracheal instillation with PEGDA NPs in PBS. Dosages of 100 ug of NPs were administered and then all studies had an endpoint of 72 hrs. Terminal bronchoalveolar lavage fluid (BALF) and whole lungs were obtained for imaging, histology, and flow cytometry studies. Whole lung digests were used to isolate the 4 cells of interest via antibody staining, which allowed for NP uptake and cell phenotype isolation.

GraphPad prism 9 was used to generate all quantitative figures and perform statistical analyses. Numerical data represents mean ± standard deviation for all studies representing at least 2 independent replicates except the histology samples (n=1).

Results, Conclusions, and Discussions: Untreated specimens from both age groups (2-months and 16-months) demonstrated intrinsic differences across the 4 APC populations of interest via flow cytometry comparison. Cell counts from the aged group showed decreases in the AM and CD103+ DC populations with subsequent increases in the IM and CD11b+ DC populations (Figure 1A). Furthermore, phenotype comparison of surface markers (CD45, MerTK, CD64, CD11c, SiglecF, CD103, CD11 b, MHC II) used for cell isolation also demonstrated changes between the 2-month and 16-month populations (Figure 1B-C). Further histological studies demonstrated that the overall CD45 expression in the lung tissue increased in the aged model (Figure 1D-E).

72 hr time point delivery assays demonstrated unique changes in NP uptake across both macrophage and DC populations utilizing median fluorescent intensity. Both macrophage types demonstrated preferential negative NP phagocytosis in the aged group, however reduced AM -NP uptake was countered by a dramatic increase by IMs (Figure 2). Furthermore, DC populations showed overall higher NP uptake in the aged group with only the CD11b+ DCs exhibiting +NP preference (Figure 2). Additional imaging assays of the bronchoalveolar lavage fluid (BALF) and lung tissue further confirmed these trends in cellular uptake of the two NPs across the macrophages and DCs.

The work here elucidates key NP interactions with aged APCs that have yet to be observed elsewhere and which can be applied for elderly-specific inhalable vaccines. The complimentary -NP uptake demonstrated by the macrophage populations agrees with hypotheses in literature about the role of IMs in the aged lung but until now has not been confirmed. Moreover, the increase in NP uptake by the DC populations in the aged model suggest that the decreased AM phagocytosis capacity led to DCs having a higher chance of encountering and engulfing an inhaled therapeutic. This work suggests that inhalable therapies for lung diseases and other vaccines may serve as an alternative strategy to target DCs that can leave the lung and generate systemic immune responses. Thus, as the elderly population grows it is critical to develop effective and specific methods to tailor therapeutics by patient ages for the continued health of our society.

Acknowledgements (Optional): The authors acknowledge Michael Trautmann-Rodriguez for assistance in experimental studies here.