Antibody-functionalized
nanoparticles (NPs) are commonly used to
increase the targeting selectivity toward cells of interest. At a
molecular level, the number of functional antibodies on the NP surface
and the density of receptors on the target cell determine the targeting
interaction. To rationally develop selective NPs, the single-molecule
quantitation of both parameters is highly desirable. However, techniques
able to count molecules with a nanometric resolution are scarce. Here,
we developed a labeling approach to quantify the number of functional
cetuximabs conjugated to NPs and the expression of epidermal growth
factor receptors (EGFRs) in breast cancer cells using direct stochastic
optical reconstruction microscopy (dSTORM). The single-molecule resolution
of dSTORM allows quantifying molecules at the nanoscale, giving a
detailed insight into the distributions of individual NP ligands and
cell receptors. Additionally, we predicted the fraction of accessible
antibody-conjugated NPs using a geometrical model, showing that the
total number exceeds the accessible number of antibodies. Finally,
we correlated the NP functionality, cell receptor density, and NP
uptake to identify the highest cell uptake selectivity regimes. We
conclude that single-molecule functionality mapping using dSTORM provides
a molecular understanding of NP targeting, aiding the rational design
of selective nanomedicines.
Nanosized artificial antigen-presenting cells (aAPCs),
synthetic
immune cell mimics that aim to activate T cells
ex
or
in vivo
, offer an effective alternative to cellular
immunotherapies. However, comprehensive studies that delineate the
effect of nano-aAPC topology, including nanoparticle morphology and
ligand density, are lacking. Here, we systematically studied the topological
effects of polymersome-based aAPCs on T cell activation. We employed
an aAPC library created from biodegradable poly(ethylene glycol)-
block
-poly(
d
,
l
-lactide) (PEG-PDLLA) polymersomes
with spherical or tubular shape and variable sizes, which were functionalized
with αCD3 and αCD28 antibodies at controlled densities.
Our results indicate that high ligand density leads to enhancement
in T cell activation, which can be further augmented by employing
polymersomes with larger size. At low ligand density, the effect of
both polymersome shape and size was more pronounced, showing that
large elongated polymersomes better activate T cells compared to their
spherical or smaller counterparts. This study demonstrates the capacity
of polymersomes as aAPCs and highlights the role of topology for their
rational design.
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