Revealing Population Heterogeneity in Vesicle-Based Nanomedicines Using Automated, Single Particle Raman Analysis

Abstract

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.

Enhanced Antimalarial and Antisequestration Activity of Methoxybenzenesulfonate-Modified Biopolymers and Nanoparticles for Tackling Severe Malaria

“…We next employed single-particle automated Raman trapping analysis (SPARTA), previously used to establish single-particle variation in synthetic nanoparticles, , to evaluate changes and distributions of polymer amount per particle incorporated in PLNs. The average Raman spectra across all of the single-particle traps (Figure e) show the characteristic PDLLA-AMBS signals (1032 and 1613 cm –1 ) together with the lipid signal (1439 cm –1 ), confirming coassembly of the polymer and lipid on a single-particle level.…”

Innovative Approaches for Drug Discovery: Quantifying Drug Distribution and Response with Raman Imaging

“…The authors successfully demonstrated the advanced capabilities of SPARTA by analyzing different nanoparticle formulations, including liposomes and polymersomes, as well as the functionalization of polystyrene particles. Since its initial demonstration, SPARTA has been used to study a multitude of nanoparticle systems in recent years. − Saunders et al adapted SPARTA to distinguish cargo location and loading heterogeneity within nanoparticle populations . They generated a theoretical model and showed a positive, linear correlation between the encapsulating polymer and cargo mass for membrane loading and a much lower cubic relationship for core loading.…”

“…Reprinted with permission from Saunders, C.; Foote, J. E. J.; Wojciechowski, J. P.; Cammack, A.; Pedersen, S. V.; Doutch, J. J.; Barriga, H. M. G.; Holme, M. N.; Penders, J.; Chami, M.; Najer, A.; Stevens, M. M. Revealing Population Heterogeneity in Vesicle-Based Nanomedicines Using Automated, Single Particle Raman Analysis. ACS Nano 2023 , 17 (12), 11713–11728 (ref ). Copyright 2023 American Chemical Society.…”

Application of Single Extracellular Vesicle Analysis Techniques

“…Researchers have employed automated SPARTA to demonstrate that this technology can accurately characterize single-vesicle cargo information and determine whether SEV is loaded with active ingredients. 78 Additionally, another study introduced a high-throughput method for the absolute quantification of liposomal nanomedicine particle size, drug content, drug encapsulation fraction, and particle concentration at the single-particle level using nFCM. 79 Utilizing single-vesicle technology is crucial for producing and evaluating therapeutic EVs, but further studies are still needed to explore its potential fully.…”