<p>While many particles retained their structural integrity, the presence of these aggregates suggests that, under certain conditions, the LNPs may tend to cluster. It is important to note that for SEM analysis, the samples were dried and observed under vacuum, which probably have affected the structure and shape of the LNPs. This preparation process can introduce artifacts that would not typically be present in solution and should be considered when interpreting the results. Additionally, the contrast under vacuum conditions was too low to reliably distinguish the LNPs with sufficient detail. It provided a useful initial glimpse into the world of nanoparticles. Further complementary techniques will be needed for a more accurate and detailed characterization.</p>
<H4text="Corden LNP"/>
<H5text="Transfection"/>
<p>Text :D</p>
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<p>Fluorescence microscopy with the Leica DMI6000 B microscope at 20x magnification was conducted on HEK293 cells transfected with LNPs containing pcDNA 3.1 eYFP DNA and mRNA. Minicircle DNA served as the positive control, while LNPs without cargo acted as the negative control. Cells were imaged at 24 h, 48 h, and 72 h post-transfection.</p>
Turbidity after components of the Corden LNP have been pipetted together indicates particle formation. </figcaption>
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<p>During the preparation of the LNPs, the solution became turbid and bluish, indicating successful nanoparticle formation (Figure X). This was further confirmed by cryo-EM analysis, which revealed the presence of well-formed LNPs. Despite the successful formation of LNPs, no detectable fluorescence was observed in the cells treated with LNPs containing pcDNA 3.1 eYFP DNA or mRNA at any of the measured time points, indicating that transfection did not occur under these conditions.</p>
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<p>Quantitatively, none of the LNP-treated samples showed significant fluorescence, indicating a failure in transfection. The lack of fluorescence in all experimental groups, except the positive control, suggests either insufficient uptake of the LNPs by the cells or a failure in expression of the YFP reporter. </p>
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<b>Figure X.</b>
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<b>Figure X.</b>
Fluorescence microscopic images of transfected HEK293 cells at 20x magnification after 48 h post-transfection with different Corden LNP formulations recorded with Leica DMI6000 B.
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<H5text="cryo-TEM"/>
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<H5text="Cryo-EM"/>
<p>Cryo-EM (cryogenic electron microscopy) as a form of transmission electron cryomicroscopy was performed using a JEOL JEM-2200FS electron microscope (JEOL, Freising, Germany) operating at 200kV, equipped with a cold field emission electron gun. The sample preparation and imaging were carried out at cryogenic temperatures, which allowed for the visualization of LNPs in their native hydrated state.</p>
<p>The images reveal the presence of spherical LNP structures with an approximate size of 100 nm (Figure X). The LNPs appear well-formed, with uniform morphology, indicating successful nanoparticle formation. In addition to individual particles, some larger, round structures were also observed, which could represent aggregated LNPs. These aggregations are a common phenomenon in LNP systems and could be attributed to interactions between particles under certain conditions.</p>
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<p>While Cryo-EM provides valuable insights into the morphology and size distribution of the LNPs, there are some limitations to this technique. The imaging process involves cryogenic freezing and exposure to high-energy electron beams, which can potentially induce minor structural artifacts. Furthermore, the thinness of the sample may limit contrast, making it difficult to fully distinguish between different LNP populations or their internal structures. Despite these limitations, Cryo-EM still offers a high-resolution view of the LNPs in their near-native state, providing essential information about their size and shape.</p>
MTT Assay of LNPs from all iterations performed on HEK293 including Triton as negative control and untreated cells as positive control. Mean +/- SEM for n=6. For statistics one-way ANOVA was performed. </figcaption>
