Active targeting nanoparticles are a new generation of drug and gene delivery systems with the potential for greatly improved therapeutics delivery compared to conventional nanomedicine approaches. Despite their potential, the translation of active targeting nanoparticles faces challenges in production scale-up and batch consistency. Accurate quality control methods for nanoparticle therapeutic payload and coating characterization are critical for attaining the desired levels of batch repeatability, drug/gene loading efficiency, targeting molecule coating effectiveness, and safety profiles. Current limitations in nanoparticle characterization technologies, such as relying on ensemble-average analysis, pose challenges in assessing the drug/gene content and surface modification heterogeneity, which can greatly affect therapeutic outcomes. Single-molecule analysis technologies have emerged as a promising alternative, offering rich information on heterogeneity and stochastic variations between nanoparticle batches. This review first evaluates and identifies the challenges of traditional nanoparticle characterization tools that rely on indirect, bulk solution quantification methods. Subsequently, newly emerging characterization technologies are introduced for the quantification of therapeutic loading and targeted moiety coating efficiencies with single-nanoparticle resolution, to help guide researchers towards advancing the translation of active targeting nanoparticles into the clinical setting.

Background/Objectives: Peripheral artery disease (PAD) is frequently asymptomatic, requiring non-invasive approaches for disease evaluation and therapy monitoring. This study demonstrates that multispectral optoacoustic tomography (MSOT) and laser speckle contrast imaging (LSCI) can non-invasively assess changes in tissue vascular oxygenation and perfusion, respectively, in a mouse hind-limb PAD model, enabling comparison of age-dependent vascular responses. Methods: PAD was induced by cauterization of the femoral artery in young (2 months) and old (18 months) mice, which were imaged using MSOT and LSCI at baseline (Day 0) and on Days 3, 7, and 14 post-surgery. Correlative histology including Hematoxylin and Eosin (H&E), Masson’s trichrome for collagen, and immunofluorescence for CD31 and Ki-67 were performed. Results: Reduced tissue oxygenation was observed by MSOT in the ischemic limb shortly after surgery and faster recovery occurred in young compared to old mice. LSCI revealed time-dependent perfusion recovery in both groups, with consistently better recovery in young mice. Histological analyses confirmed ischemic damage and demonstrated enhanced angiogenesis and cellular proliferation in young muscle tissues. The observations were consistent for each methodology. Conclusions: These results indicate that both MSOT and LSCI serve as effective, non-invasive tools for longitudinal monitoring of muscle injury, capable of revealing age-dependent vascular responses without the need for exogenous contrast agents.

The ultrasound-assisted transport of drugs or fluorophore-loaded nanoagents plays an important role in the desirable drug delivery and imaging contrasts. Unlike conventional ultrasound techniques that rely on thermal or cavitation effects, this study aims to conduct an experimental investigation into the dynamics of interstitial fluid streaming and tissue recovery in ex vivo chicken breast and porcine loin muscle tissues during and after ultrasound exposures, which has not been experimentally investigated in the literature. Biological tissues consist of both a fluid and a solid matrix, and an ultrasound beam compresses the tissues within a small focal volume from all directions, which generates macroscopic streaming of interstitial fluid and compression of the tissue's solid matrix. After the ultrasonic exposure, the solid matrix undergoes recovery, leading to a backflow of the fluid matrix. Temperature-insensitive sulforhodamine-101 encapsulated poly(lactic-co-glycolic acid) nanoparticles with an average diameter size of 175 nm were locally injected into ex vivo chicken breast and porcine loin muscle tissues to study the ultrasound-induced dynamics in the tissues during and after ultrasound exposure by analyzing the distribution of fluorescence. The changes in fluorescence over time caused by the streaming and backflow of interstitial fluid were studied with two ex vivo tissue models, and a faster recovery was observed in porcine tissues compared with chicken tissues. The ultrasound-induced transportability of the nanoagent in porcine muscle tissues was much higher (∼8.75 times) than in chicken breast tissue likely due to structural differences. The study reveals a promising, non-invasive strategy for enhancing drug delivery in dense tissues by leveraging mechanical ultrasound effects, potentially advancing therapeutic and diagnostic applications.

Background/Objectives: Peripheral artery disease (PAD) is frequently asymptomatic, requiring non-invasive approaches for disease evaluation and therapy monitoring. This study demonstrates that multispectral optoacoustic tomography (MSOT) and laser speckle contrast imaging (LSCI) can non-invasively assess changes in tissue vascular oxygenation and perfusion, respectively, in a mouse hindlimb PAD model, enabling comparison of age-dependent vascular responses. Methods: PAD was induced by cauterization of the femoral artery in young (2 months) and old (18 months) mice, which were imaged using MSOT and LSCI at baseline (Day 0) and on Days 3, 7, and 14 post-surgery. Correlative histology including Hematoxylin and Eosin (H&E), Masson’s Trichrome for collagen, and immunofluorescence for CD31 and Ki-67 were performed. Results: Reduced tissue oxygenation was observed by MSOT in the ischemic limb shortly after surgery and faster recovery occurred in young compared to old mice. LSCI revealed time-dependent perfusion recovery in both groups, with consistently better recovery in young mice. Histological analyses confirmed ischemic damage and demonstrated enhanced angiogenesis and cellular proliferation in young muscle tissues. The observations were consistent for each methodology. Conclusions: These results indicate that both MSOT and LSCI serve as effective, non-invasive tools for longitudinal monitoring of muscle injury, capable of revealing age-dependent vascular responses without the need for exogenous contrast agents.