<p>Ensuring the safety and thorough characterization of our lipid nanoparticles (LNPs) was a central part of our project, as these particles are intended for use in biological systems. We implemented a comprehensive range of assays and techniques to assess their biosafety and physical properties, ensuring their suitability for applications such as drug delivery and gene therapy. Below is an overview of the key steps we took in our assessment.</p>
<p>Ensuring the safety and thorough characterization of our LNPs was a central part of our project, as these particles are intended for use in biological systems. We implemented a comprehensive range of assays and techniques to assess their biosafety and physical properties, ensuring their suitability for applications such as drug delivery and gene therapy. Below is an overview of the key steps we took in our assessment.</p>
<H4text="MTT Assay"></H4>
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@@ -96,7 +96,7 @@ export function Methods() {
<Subesctiontitle="In-Depth Characterization of LNPs"id="In-Depth Characterization of LNPs">
<H4text="Dynamic Light Scattering (DLS) and Zeta Potential"></H4>
<p>The hydrodynamic radius (𝑅𝐻) of the vesicles and lipid nanoparticles (LNPs) was determined through angle-dependent photon correlation spectroscopy (PCS) at 𝑇=20∘C. Samples were measured in NMR tubes using a 3D LS Spectrometer Pro (LS Instruments, Fribourg, Switzerland), which was equipped with a HeNe Laser (632.8 nm, 1145P; JDSU, Milpitas, CA, USA), a decaline index-matching vat, an automated goniometer, and two detectors. Measurements were performed in a 3D cross-mode to eliminate multiple scattering effects, covering a scattering angle range of 30° to 120° in increments of 10°, with a measuring time of three intervals of 120 s per angle.The autocorrelation function of the scattered light intensity was generated using a multiple-τ digital correlator and analyzed via inverse Laplace transformation (CONTIN) to determine the mean relaxation rate (Γ). From these data, the hydrodynamic radius (𝑅𝐻) was calculated using the Stokes–Einstein equation:
<p>The hydrodynamic radius (𝑅𝐻) of the vesicles and LNPs was determined through angle-dependent photon correlation spectroscopy (PCS) at 𝑇=20°C. Samples were measured in NMR tubes using a 3D LS Spectrometer Pro (LS Instruments, Fribourg, Switzerland), which was equipped with a HeNe Laser (632.8 nm, 1145P; JDSU, Milpitas, CA, USA), a decaline index-matching vat, an automated goniometer, and two detectors. Measurements were performed in a 3D cross-mode to eliminate multiple scattering effects, covering a scattering angle range of 30° to 120° in increments of 10°, with a measuring time of three intervals of 120 s per angle.The autocorrelation function of the scattered light intensity was generated using a multiple-τ digital correlator and analyzed via inverse Laplace transformation (CONTIN) to determine the mean relaxation rate (Γ). From these data, the hydrodynamic radius (𝑅𝐻) was calculated using the Stokes–Einstein equation:
𝑅𝐻=𝑘𝐵⋅𝑇/6𝜋𝜂𝐷𝑇 where 𝑘𝐵 is the Boltzmann constant, T is the temperature, η is the solvent viscosity, and DT
is the translational diffusion coefficient. The value of 𝐷𝑇 was obtained from the slope of the linear relationship between the relaxation rate (Γ) and
the squared magnitude of the scattering vector (𝑞2) as defined by:Γ =𝐷𝑇⋅𝑞2Γ.
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@@ -106,9 +106,9 @@ Overall, the combination of PCS, DLS, and zeta potential measurements provided a
<H4text="SEM and Cryo-EM for Structural Analysis"></H4>
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<p>For the cryo-transmission electron microscopy (Cryo-TEM) analysis, samples were vitrified on holey carbon TEM grids (Lacey Carbon Film coated, 200 Mesh; Science Services, München, Germany) using a Leica blotting and plunging device (Leica EM GP, Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany). The grids were rapidly plunged into liquid ethane cooled by liquid nitrogen to ensure sufficiently fast cooling. After vitrification, the grids were transferred to a cryo transfer and tomography holder (Fischione Model 2550, E.A. Fischione Instruments, Pittsburgh, USA).
<p>For the cryogenic electron microscopy (Cryo-EM) analysis, samples were vitrified on holey carbon TEM grids (Lacey Carbon Film coated, 200 Mesh; Science Services, München, Germany) using a Leica blotting and plunging device (Leica EM GP, Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany). The grids were rapidly plunged into liquid ethane cooled by liquid nitrogen to ensure sufficiently fast cooling. After vitrification, the grids were transferred to a cryo transfer and tomography holder (Fischione Model 2550, E.A. Fischione Instruments, Pittsburgh, USA).
TEM images were acquired using a JEOL JEM-2200FS electron microscope (JEOL, Freising, Germany) equipped with a cold field emission electron gun, operated at an acceleration voltage of 200 kV. All images were captured digitally using a bottom-mounted camera (Gatan OneView, Gatan, Pleasanton, USA) and processed with a digital imaging processing system (Digital Micrograph GMS 3, Gatan, Pleasanton, USA).
In addition to Cryo-TEM, we employed scanning electron microscopy (SEM) to further characterize the morphology and surface structure of the lipid nanoparticles (LNPs). SEM provided high-resolution images that confirmed the spherical shape and uniformity of the LNPs. Cryo-electron microscopy (cryo-EM) was also used to investigate the internal structure of the LNPs, revealing the presence of lipid layers and encapsulated materials, which are essential for understanding their functionality in drug delivery applications. </p>
In addition to Cryo-EM, we employed scanning electron microscopy (SEM) to further characterize the morphology and surface structure of the LNPs. SEM provided high-resolution images that confirmed the spherical shape and uniformity of the LNPs.</p>
