Single-molecule imaging of the biophysics of molecular interactions with precision and control, in cell-like conditions, and without tethers

Leslie, Sabrina; Berard, Daniel; Kamanzi, Albert; Metera, Kimberly; Scott, Shane; Shaheen, Cynthia; Shayegan, Marjan; Tahvildari, Radin; Zhang, Zhiyue

doi:10.1016/j.cobme.2019.10.004

Cited by 4 publications

(8 citation statements)

References 48 publications

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“…19,27 CLiC Microscopy. LNP fusion experiments were performed using CLiC microscopy 17,18,23 in combination with FRET. Briefly, a microfluidic chuck for sample exchange was used to hold the flow cell on a microscope sample plate, located between the objective and the CLiC pusher lens (see Figure 1 of Leslie et al 23 ).…”

Section: Methodsmentioning

confidence: 99%

Quantitative Visualization of Lipid Nanoparticle Fusion as a Function of Formulation and Process Parameters

Kamanzi,

Zhang,

et al. 2024

ACS Nano

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Lipid nanoparticles (LNPs) have proven to be promising delivery vehicles for RNA-based vaccines and therapeutics, particularly in LNP formulations containing ionizable cationic lipids that undergo protonation/deprotonation in response to buffer pH changes. These nanoparticles are typically formulated using a rapid mixing technique at low pH, followed by a return to physiological pH that triggers LNP− LNP fusion. A detailed understanding of these dynamic processes is crucial to optimize the overall performance and efficiency of LNPs. However, knowledge gaps persist regarding how particle formation mechanisms impact drug loading and delivery functions. In this work, we employ single-molecule Convex Lens-induced Confinement (CLiC) microscopy in combination with Forster resonance energy transfer (FRET) measurements to study LNP fusion dynamics in relation to various formulation parameters, including lipid concentration, buffer conditions, drug loading ratio, PEG-lipid concentrations, and ionizable lipid selection. Our results reveal a strong correlation between the measured fusion dynamics and the formulation parameters used; these findings are consistent with DLS and Cryo-TEM-based assays. These measurements offer a cost-effective method for characterizing and screening potential drug candidates and can provide additional insights into their design, with opportunities for optimization.

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Section: Methodsmentioning

confidence: 99%

Quantitative Visualization of Lipid Nanoparticle Fusion as a Function of Formulation and Process Parameters

Kamanzi,

Zhang,

et al. 2024

ACS Nano

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“…We demonstrate this capability both in the data presented in this response (Figures and ) by tracking polystyrene nanoparticles for 2000 frames and in our previously published measurements by tracking fluorescently labeled lipid nanoparticles for 2000 frames (see the size and loading measurements presented in Figures 2, 3, and 6 of ref ), and up to 6000 frames (in photobleaching measurements presented in Figures 4 and 5 of ref ). Our experimental methods do indeed allow for longer trajectories to be used, as the nanoparticles are fully confined within our imaging plane during measurements and are only allowed to escape in order to refresh the microwells with new particles. ,, The choice of number of frames to use was informed by our simulations, which showed no significant improvement in measurement errors using longer trajectories (see SI of ref for details).…”

Section: New Measurements Of Two Sizes Of Polystyrene Nanoparticles U...mentioning

confidence: 99%

“…Madison et al suggest that our results may be affected by artifacts intrinsic to imaging objectives. The advantage of convex lens-induced confinement (CLiC) microscopy , is that it is fairly independent of the lens setup. This technique allows us to avoid “aberration artifacts such as distortion and defocus”, by simply using an imaging objective whose depth of field is deeper than confinement micro wells.…”

Section: New Measurements Of Two Sizes Of Polystyrene Nanoparticles U...mentioning

confidence: 99%

“…Our experimental methods do indeed allow for longer trajectories to be used, as the nanoparticles are fully confined within our imaging plane during measurements and are only allowed to escape in order to refresh the microwells with new particles. 1,4,5 The choice of number of frames to use was informed by our simulations, which showed no significant improvement in measurement errors using longer trajectories (see SI of ref 1 for details).…”

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Response to “Letter to the Editor Concerning ‘Simultaneous, Single-Particle Measurements of Size and Loading Give Insights into the Structure of Drug-Delivery Nanoparticles’”

et al. 2023

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“…Typically, there is a need to suppress interactions with the surface since the molecules to be studied or detected will be (or should be) in the solution phase. This is the case in, for example, electrostatic traps, 6 nanochannels for DNA analysis, 7 convex lens-induced confinement, 8 and solid-state nanopore sensors. 9 Notably, all these devices have oxidized silicon as the material forming the interface to the biological solution and preventing biomolecule adsorption is key to successful operation in all cases.…”

Section: ■ Introductionmentioning

confidence: 99%

Polymer Brushes on Silica Nanostructures Prepared by Aminopropylsilatrane Click Chemistry: Superior Antifouling and Biofunctionality

Andersson

Järlebark

et al. 2023

ACS Appl. Mater. Interfaces

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In nanobiotechnology, the importance of controlling interactions between biological molecules and surfaces is paramount. In recent years, many devices based on nanostructured silicon materials have been presented, such as nanopores and nanochannels. However, there is still a clear lack of simple, reliable, and efficient protocols for preventing and controlling biomolecule adsorption in such structures. In this work, we show a simple method for passivation or selective biofunctionalization of silica, without the need for polymerization reactions or vapor-phase deposition. The surface is simply exposed stepwise to three different chemicals over the course of ∼1 h. First, the use of aminopropylsilatrane is used to create a monolayer of amines, yielding more uniform layers than conventional silanization protocols. Second, a cross-linker layer and click chemistry are used to make the surface reactive toward thiols. In the third step, thick and dense poly(ethylene glycol) brushes are prepared by a grafting-to approach. The modified surfaces are shown to be superior to existing options for silica modification, exhibiting ultralow fouling (a few ng/cm2) after exposure to crude serum. In addition, by including a fraction of biotinylated polymer end groups, the surface can be functionalized further. We show that avidin can be detected label-free from a serum solution with a selectivity (compared to nonspecific binding) of more than 98% without the need for a reference channel. Furthermore, we show that our method can passivate the interior of 150 nm × 100 nm nanochannels in silica, showing complete elimination of adsorption of a sticky fluorescent protein. Additionally, our method is shown to be compatible with modifications of solid-state nanopores in 20 nm thin silicon nitride membranes and reduces the noise in the ion current. We consider these findings highly important for the broad field of nanobiotechnology, and we believe that our method will be very useful for a great variety of surface-based sensors and analytical devices.

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Single-molecule imaging of the biophysics of molecular interactions with precision and control, in cell-like conditions, and without tethers

Cited by 4 publications

References 48 publications

Quantitative Visualization of Lipid Nanoparticle Fusion as a Function of Formulation and Process Parameters

Quantitative Visualization of Lipid Nanoparticle Fusion as a Function of Formulation and Process Parameters

Response to “Letter to the Editor Concerning ‘Simultaneous, Single-Particle Measurements of Size and Loading Give Insights into the Structure of Drug-Delivery Nanoparticles’”

Polymer Brushes on Silica Nanostructures Prepared by Aminopropylsilatrane Click Chemistry: Superior Antifouling and Biofunctionality

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