As a high-resolution, single-particle approach to nanoparticle analysis, Tunable Resistive Pulse Sensing (TRPS) is used across a wide range of developments in nanomedicine. Here, we highlight 4 examples that demonstrate the versatility of TRPS across different particle types.
1. Measuring anti-cancer liposomes designed for targeted delivery and controlled release
Vascular disruptive agents, like combretastatin A4 phosphate (CA4P), have shown great potential as cancer treatments, as they bind to the immature and unstable tumour vasculature. Thébault et al (2020) proposed to minimise adverse effects to the vasculature presented by these agents by improving drug delivery at the tumour site. This aim was tested by using magnetic nanoparticles-loaded liposomes called Ultra Magnetic Liposomes (UML) as the CA4P-targeting strategy, and High Intensity Focused Ultrasound (HIFU) as the drug-releasing trigger.
UML and CA4P-loaded UML particles were characterised with TRPS (nanopore size NP200), confirming similar overall particle sizes of 230 nm and 209 nm, respectively. While in vitro CA4P passive release from CA4P-UML occurred at steady increments from 3.5% to 9.4% during the first 60 minutes, HIFU-triggered CA4P release reached 17%.
MRI analysis of anti-tumour efficacy in vivo showed that free CA4P drug injected intraperitoneally in mice (100 mg CA4P/kg) resulted in a similar reduction in tumour growth compared to CA4P-loaded UMLs injected at 150 times lower concentration (0.65 mg CA4P/kg) when used with both magnetic targeting on one side of the body and HIFU. In addition, the combined therapy was effective after 24 hours and a single dose, indicating promising potential.1
2. TRPS used to characterise a siRNA-nanoparticle therapy for COVID-19
The emergence of new SARS-CoV-2 variants constantly challenges the effectiveness of some previously successful vaccines. While combinations of steroids and broad-spectrum antivirals like Dexamethasone and Remdesivir can benefit patients with COVID-19, currently there are no therapeutics specifically targeting SARS-CoV-2. As the RNA genome in coronaviruses presents a great opportunity for therapeutic strategies based on RNA interference, Idris et al. (2021) screened several small interfering RNAs (siRNA) delivered with lipid nanoparticle formulations.
Of the 18 tested siRNAs targeting conserved SARS-CoV-2 genomic regions, five were identified which demonstrated high potency and dose-dependent repression of virus expression in in vitro infection assays. Previously optimised liposome-based lipid nanoparticle (LNP) formulations that showed good stability in serum, long circulation times and siRNA protection, were further modified in this study with different lipid composition to ameliorate LNP-siRNA immune stimulation.
Overall, the authors made a number of observations which led them to conclude that the selection of LNP formulation containing the high cationic ionisable lipid MC3 helped facilitate the endosomal release of siRNAs and high 1,2-dioleoyl-3-trimethylammonium-Propane (DOTAP) helped to target the lungs. Among the LNP formulation assessments were TRPS-enabled (NP150 nanopores) measurements of particle concentration, as well as comprehensive assessments of LNP storage, confirmation of resistance to enzymatic degradation, and no alteration to cell viability (primary human macrophages). On the in vivo side, virus-infected mice treated with the new LNP formulation and siHel2 presented survival advantage, less weight loss and a lower clinical score compared to virus-infected mice treated with control LNP-siRNAs.2
Read study summary in more detail.
3. Investigating therapeutics delivered by ultrasound
Developing ways to accurately deliver therapeutics to a desired location is a priority for the medical nanotechnology field. Although acoustofluidic manipulation of microbubbles (MB) has been studied, what happens to clusters of MBs after acoustic waves stop has not been investigated. AlSadiq et al. (2022) therefore studied the interplay of different forces that are at hand, by assessing the clustering behaviour of macula-targeted (part of the retina) MBs and gas-filled echogenic liposome (ELIPs) drug-delivery systems.
TRPS enabled zeta potential measurements of MBs and ELIPs, and showed mean values of −2.43 mV and −3.63 mV, respectively. In addition, both particle types exhibited the same range of zeta potential variance across different particle diameters, indicating that the zeta potential of MBs and ELIPs is not strongly dependent on their size. This finding was important to the diffusion behaviour analysis, and confirmed that the effect of particle movement due to buoyancy was minimal in comparison to the effect of electrophoresis. Sonication at 10 kPa and 6 MHz caused five pairs of ELIP particles, ranging from 12 to ~26 μm apart, to quickly come into contact at the 1-1.5 seconds mark with no signs of repulsion after sonication stopped at 8 seconds. The same sonication parameters were applied to MBs, with the bubbles making contact at ~3.5 seconds and remaining as such for 12 seconds. Large clusters of MBs and ELIPs were indeed formed during sonication at 10 kPa and 5MHz, however they started to diffuse after sonication stopped.
These results indicate that the cluster diffusion behaviour must be taken into consideration in drug delivery applications that rely mostly on LNP shell degradation, particularly important in organs of low circulation, since the diffusion of the clusters may result in a reduction in drug concentration at the target site before LNP shell degradation. The authors also suggest that the pressure used to initiate the acoustofluidic MB/ELIP translation through different types of viscose fluids (other than water) without causing tissue damage also requires further exploration.3
4. Characterising virus-like particles to better understand process development
Biophysical characterisation is critical in assisting the process of vaccine development of virus-like particles (VLPs), for example in the assessment of VLP integrity and stability. It has been proposed to include a comprehensive analytical toolbox for Influenza VLPs, including biochemical, biophysical, and immunological assays, as opposed to only traditional methods that are currently utilised. In this publication, a set of biochemical and biophysical analyses were performed on six different Influenza VLPs (groups A and B, subtypes H1 and H3, and mono and penta valences) which were isolated from the same cell and expression vector system, to determine the applicability of analyses for process monitoring.
Biophysical and biochemical characterisation revealed firstly and as expected, that the ratio of hemagglutinin protein (in VLPs) to impurities (like DNA, total protein or expression vectors) increased throughout the purification process in all six VLPs analysed. Secondly, it was found that particle concentration, size distribution, and zeta potential, measured with TRPS and NP200 nanopores, showed no significant differences between all different VLPs. Nevertheless, hemagglutinin protein content, cell/VLP purification efficiency (dictated by cell viability and cell-released impurities) and VLPs thermal stability had a VLP-type dependency. Overall, results from this study suggest analytical methods provided here can be beneficial for in-process monitoring of VLP production in general, especially with complex enveloped and multi-protein ones like Influenza VLPs.4
