The intrinsic heterogeneity of many nanoformulations
is currently
challenging to characterize on both the single particle and population
level. Therefore, there is great opportunity to develop advanced techniques
to describe and understand nanomedicine heterogeneity, which will
aid translation to the clinic by informing manufacturing quality control,
characterization for regulatory bodies, and connecting nanoformulation
properties to clinical outcomes to enable rational design. Here, we
present an analytical technique to provide such information, while
measuring the nanocarrier and cargo simultaneously with label-free,
nondestructive single particle automated Raman trapping analysis (SPARTA).
We first synthesized a library of model compounds covering a range
of hydrophilicities and providing distinct Raman signals. These compounds
were then loaded into model nanovesicles (polymersomes) that can load
both hydrophobic and hydrophilic cargo into the membrane or core regions,
respectively. Using our analytical framework, we characterized the
heterogeneity of the population by correlating the signal per particle
from the membrane and cargo. We found that core and membrane loading
can be distinguished, and we detected subpopulations of highly loaded
particles in certain cases. We then confirmed the suitability of our
technique in liposomes, another nanovesicle class, including the commercial
formulation Doxil. Our label-free analytical technique precisely determines
cargo location alongside loading and release heterogeneity in nanomedicines,
which could be instrumental for future quality control, regulatory
body protocols, and development of structure–function relationships
to bring more nanomedicines to the clinic.