Poster W18 - Elucidating nanoparticle shape requirements to maximize lymphatic and lymph node targeted drug delivery

Introduction: Nanoparticle (NP) shape plays a pivotal role in dictating their interactions with biological systems, influencing cellular uptake, tissue penetration, and ultimately therapeutic efficacy. Understanding the impact of NP shape on lymphatic transport and accumulation within lymph nodes (LNs) is crucial for optimizing targeted drug delivery strategies, particularly in the context of immunotherapy. We have recently established formulation characteristics and requirements, such as size and surface chemistry, to optimize NP lymphatic transport. However, our understanding of how NP shape specifically affects transport across lymphatic endothelial cells (LECs) remains limited. I here investigated shape as a crucial material property modulating NP transport via lymphatics and within the LNs.
We hypothesized that anisotropic NPs will be more efficiently transported across LECs compared to isotropic NPs due to their enhanced interaction with cells. We also hypothesize that anisotropic shape will enhance nanoparticle accumulation in the lymph nodes and distribution throughout the lymph node parenchyma due to their improved diffusion through extracellular spaces, which was previously shown1–4. We found that both of our hypotheses were true and thus have laid the foundation for more effective design criteria NPs tailored to specific therapeutic targets within the lymphatic system. Ultimately this work will improve treatment outcomes of immune modulatory therapies such as vaccines and cancer immunotherapies.

Materials and

Methods: Nanoparticle formulation:
Carboxylate-modified polystyrene microspheres were conjugated with 5kDa PEG using EDC/NHS chemistry. After dissolving particles in borate buffer, amine-terminated PEG was conjugated using 0.02 mM EDC and 7 mM NHS, and left to react for four hours. The particles were then washed to purify the PEGylated nanoparticles. To obtain rod, worm, and sphere shapes, nanoparticles were suspended in polyvinyl alcohol (PVA) with glycerol, cast into films, and either heated in oil or submerged in toluene for stretching. Nanoparticles were recovered by dissolving the films in 30% isopropanol/water at 65ºC and stored in deionized water.

In vitro lymphatic transport model:
Human primary LECs were cultured on transwell membranes to confluence, confirmed by transepithelial resistance (TEER) measurements and fluorescence imaging. Fluorescent nanoparticles were added to the apical side, and transport to the basolateral side was assayed over time (0h, 3h, 6h, 9h, 12h, 18h, 24h).

Nanoparticle accumulation:
An in vivo imaging system (IVIS) measured fluorescence intensity at the injection site and in draining LNs over time to assess accumulation and migration. LNs were harvested and homogenized at 8h and 12h to quantify nanoparticle content using a standard curve and plate reader.

Nanoparticle distribution in the LNs:
LNs were collected and frozen in OCT compound at 4h, 8h, and 12h post-injection. Sections were fixed and stained for various cell markers (B cells, T cells, macrophages, dendritic cells, fibroblastic reticular cells, lymphatic endothelial cells), and nanoparticle distribution was visualized via confocal microscopy.

Results, Conclusions, and Discussions: We successfully created rod-shaped NPs and characterized their surface chemistry, focusing on PEG density as well as their size and shape (Figure 2). We investigated the transport mechanisms of 100 nm PSPEG NPs across LECs and found that rod-shaped NPs exhibit significantly enhanced transport compared to spherical NPs. Specifically, our data suggest that rod-shaped NPs are predominantly transported via paracellular mechanisms and may also utilize macropinocytosis, whereas micropinocytosis does not appear to play a significant role. In vitro experiments showed a notably higher rate of transport for rod-shaped NPs across LECs (Figure 2). Furthermore, our in vivo studies indicated that rod-shaped NPs accumulate more effectively in LNs compared to spherical counterparts, with rod-shaped NP accumulation being approximately 2.5-fold higher (Figure 2). Rod-shaped NPs were observed to penetrate deeper into the LN parenchyma, while spherical NPs primarily localized in the capsular sinuses of the lymph nodes. This deeper penetration is significant because it suggests that rod-shaped NPs could more effectively reach T and B cells, which reside within the parenchyma. Enhanced delivery to these immune cells could improve the efficacy of vaccines and immunotherapies by ensuring more direct and substantial interaction with the target cells, thereby potentially enhancing the immune response.

Our study successfully engineered rod-shaped NPs and conducted a comprehensive characterization of their properties. Through in vitro experiments comparing the transport of NPs with rod or spherical morphology across LECS, we observed a notable increase in NP transport with the rod-shaped formulation. This finding suggests that the rode shape enhances the efficiency of NP transport across LECs. Moreover, the data indicate that rod-shaped NPs exhibit superier accumulatoin in LNs, with potential penetration into the LN parennchyma.

Nanoparticle morphology is crucial for interactions with LECs and transport across barriers. Future studies should explore a wider range of shapes to unveil their effects on cellular uptake, transport kinetics, and biodistribution in the lymphatic system, optimizing drug delivery. Additionally, our research lays the groundwork for investigating the protein corona's role in NP behavior, refining nanomedicine design for tailored immunotherapies. Advancing these questions will enhance our understanding and clinical applications of nanoparticle-based lymphatic therapies.

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