<Subesctiontitle="In-Depth Characterization of LNPs"id="In-Depth Characterization of LNPs">
<H4text="Dynamic Light Scattering (DLS) and Zeta Potential"></H4>
<p>We used dynamic light scattering (DLS) to measure the size distribution and polydispersity index (PDI) of our LNPs. This technique allowed us to confirm that the LNPs had a consistent size distribution with minimal aggregation, which is essential for their stability. Additionally, we measured the zeta potential of the LNPs to assess their surface charge. A high zeta potential confirmed that the LNPs were stable in suspension, which is critical for their effectiveness in biological environments. </p>
<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:
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.
The viscosity of water was calculated based on the temperature to provide accurate measurements for the given conditions.
To complement the PCS analysis, dynamic light scattering (DLS) was used to determine the size distribution and polydispersity index (PDI) of the LNPs. DLS measurements confirmed that the LNPs had a consistent size distribution with minimal aggregation, which is crucial for their stability and effectiveness. Furthermore, we assessed the zeta potential of the LNPs to evaluate their surface charge. A high zeta potential value indicated that the LNPs were stable in suspension, a necessary condition for maintaining their functionality in biological environments.
Overall, the combination of PCS, DLS, and zeta potential measurements provided a comprehensive characterization of the LNPs, confirming their hydrodynamic properties, stability, and suitability for drug delivery applications. </p>
<H4text="SEM and Cryo-EM for Structural Analysis"></H4>
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<p>To further characterize the morphology and surface structure of the LNPs, we employed scanning electron microscopy (SEM). SEM provided high-resolution images that confirmed the spherical shape and uniformity of the LNPs. Additionally, cryo-electron microscopy (cryo-EM) allowed us to investigate the internal structure of the LNPs, revealing the presence of lipid layers and encapsulated materials, which are crucial for understanding their function in drug delivery. </p>
<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).
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>
