Gel of Denaturing RNA Gel Electrophoresis for mRNA synthesized from pcDNA 3.1 eYFP indicating successful RNA synthesis. Lane 1: Low Range Ribo Ruler, Lane 2: FLuc Control Template, Lane 3: Negative Control, Lane 4-9 mRNA from pcDNA 3.1 eYFP.
</figcaption>
</figure>
</div>
<divclassName="col">
<p>We began by synthesizing mRNA <i>in vitro</i> using a plasmid with a eYFP reporter from Addgene (pcDNA 3.1 eYFP) before proceeding with the synthesis of our approximately 6000 bp prime editing RNA. This was done to test the transfection efficiency and compatibility of our lipid nanoparticles (LNPs). The synthesis was successful, yielding an average of 1400 ng/µl of purified mRNA from 1 µg of plasmid DNA determined by Nanodrop measurement (data not shown). The size and integrity of the synthesized RNA were confirmed using a denaturing RNA gel, where we expected to see a product of 900 nucleotides. As anticipated, a strong and prominent band corresponding to this size was observed (Figure X). This mRNA was subsequently used in further LNP formulations with RNA.</p>
<p>We began by synthesizing mRNA <i>in vitro</i> using a plasmid with a eYFP reporter from Addgene (pcDNA 3.1 eYFP) before proceeding with the synthesis of our approximately 6000 bp prime editing RNA. This was done to test the transfection efficiency and compatibility of our lipid nanoparticles (LNPs). The synthesis was successful, yielding an average of 1400 ng/µl of purified mRNA from 1 µg of plasmid DNA determined by Nanodrop measurement (data not shown). The size and integrity of the synthesized RNA were confirmed using a denaturing RNA gel, where we expected to see a product of 900 nucleotides. As anticipated, a strong and prominent band corresponding to this size was observed (Figure 16). This mRNA was subsequently used in further LNP formulations with RNA.</p>
</div>
</div>
<H4text="Cayman LNP"/>
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<p>Next, we formulated the LNPs using the Cayman LipidLaunch™ LNP-102 Exploration Kit after the manufacturers protocol. The initial assembly attempt was unsuccessful, as no cloudy, bluish solution formed after mixing the lipids. Additionally, transfection of HEK293 cells with LNPs containing nucleic acids did not produce any fluorescence. After consulting with expert <aonClick={()=>goToPagesAndOpenTab('radukic','/human-practices')}>Dr. Marco Radukic</a> and adjusting our LNP formulation and transfection protocols, specifically by pre-acidifying the OptiMEM medium, we were able to successfully assemble and transfect the LNPs. We also got from him Minicircle DNA from <ahref="https://www.plasmidfactory.com/custom-dna/minicircle-dna/"title="PlasmidFactory">PlasmidFactory</a> as a small plasmid carrying an eYFP gene and easy to transform, by that serving as a positive control in our experiments. Upon pipetting the components together, the solution immediately turned cloudy and bluish, indicating successful LNP formation (Figure X).</p>
<p>Next, we formulated the LNPs using the Cayman LipidLaunch™ LNP-102 Exploration Kit after the manufacturers protocol. The initial assembly attempt was unsuccessful, as no cloudy, bluish solution formed after mixing the lipids. Additionally, transfection of HEK293 cells with LNPs containing nucleic acids did not produce any fluorescence. After consulting with expert <aonClick={()=>goToPagesAndOpenTab('radukic','/human-practices')}>Dr. Marco Radukic</a> and adjusting our LNP formulation and transfection protocols, specifically by pre-acidifying the OptiMEM medium, we were able to successfully assemble and transfect the LNPs. We also got from him Minicircle DNA from <ahref="https://www.plasmidfactory.com/custom-dna/minicircle-dna/"title="PlasmidFactory">PlasmidFactory</a> as a small plasmid carrying an eYFP gene and easy to transform, by that serving as a positive control in our experiments. Upon pipetting the components together, the solution immediately turned cloudy and bluish, indicating successful LNP formation (Figure 17).</p>
Cayman LNP Formation indicated by blue color and turbidity. Mini DNA = Minicircle DNA from PlasmidFactory.
</figcaption>
</figure>
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...
@@ -196,52 +196,52 @@ export function Results() {
</div>
<H5text="Transfection"/>
<p>To evaluate the efficiency of transfection, we performed fluorescence microscopy (Leica DMI6000 B at 20x magnification) on HEK293 cells transfected with LNP-formulated DNA and mRNA of pcDNA 3.1 eYFP, Minicircle DNA as technical positive control and LNP without cargo.</p>
<p>24 h, 48 h and 72 h post-transfection, we observed in the conditions with Lipofectamine alone, or combined with DNA or RNA, no fluorescence, indicating unsuccessful transfection. Similarly, no fluorescence was seen in cells treated with LNPs alone or in combination with DNA or RNA. When LNPs were combined with Minicircle DNA, clear fluorescence was observed, indicating successful transfection and expression of our eYFP reporter under this condition (figure X). However, a strong background fluorescence from the OptiMEM medium was observed, complicating the analysis.</p>
<p>Until 72 h post-transfection, we observed in the conditions with Lipofectamine alone or combined with RNA, no fluorescence, indicating unsuccessful transfection with RNA. Similarly, no fluorescence was seen in cells treated with LNPs alone or in combination with DNA or RNA. When LNPs were combined with Minicircle DNA or the cells were transfected with lipofectamine and Minicircle DNA, clear fluorescence was observed, indicating successful transfection and expression of our eYFP reporter under this condition (Figure 18). However, a strong background fluorescence from the OptiMEM medium was observed, complicating the analysis.</p>
<p>Overall, among all the tested conditions, the LNP formulation with Minicircle DNA was the only combination that resulted in noticeable fluorescence, suggesting it to be the most effective transfection method for HEK293 cells in this experiment.</p>
Figure X Fluorescence microscopic images of transfected HEK293 cells at 20x magnification after 72 h post-transfection with different Cayman LNP formulations recorded with Leica DMI6000 B.
<b>Figure 18. </b>
Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification after 72 h post-transfection with different Cayman LNP formulations recorded with Leica DMI6000 B. For Lipofectamine (Lipo) + Minicircle DNA (Mini DNA) only the fluorescence image is shown.
</figcaption>
</figure>
<H5text="SEM"/>
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<p>Scanning Electron Microscopy (SEM) (Phenom ProX, Thermo Fisher) was employed by us to examine the morphology and surface characteristics of Cayman LNPs. The SEM images revealed that the LNPs displayed a generally spherical morphology with a relatively smooth surface (Figure X). The average particle size was approximately 200 nm. However, a heterogeneous distribution of particle sizes was observed, with some larger, round structures present. These larger structures could potentially indicate aggregated LNPs.</p>
<p>Scanning Electron Microscopy (SEM) (Phenom ProX, Thermo Fisher) was employed by us to examine the morphology and surface characteristics of Cayman LNPs. The SEM images revealed that the LNPs displayed a generally spherical morphology with a relatively smooth surface (Figure 19). The average particle size was approximately 200 nm. However, a heterogeneous distribution of particle sizes was observed, with some larger, round structures present. These larger structures could potentially indicate aggregated LNPs.</p>
SEM image of Cayman LNPs (10,000x magnification) with Topography mode. </figcaption>
</figure>
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<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>Fluorescence microscopy with the Leica DMI6000 B microscope at 20x magnification was by us 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>
</figure>
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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>
<p>During the preparation of the LNPs, the solution became turbid and bluish, indicating successful nanoparticle formation (Figure 20). 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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</div>
<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>
<H5text="Transfection"/>
<p>Fluorescence microscopy with the Leica DMI6000 B microscope at 20x magnification was by performed us 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>
<p>Quantitatively, after 72 h none of the LNP-treated samples showed significant fluorescence, indicating a failure in transfection (Figure 21). The lack of fluorescence in all experimental groups, except lipofectamine and Minicircle DNA as the positive control, suggests either insufficient uptake of the LNPs by the cells or a failure in expression of the YFP reporter, indicating that the Corden LNP may not suited as our delivery system. Also the deformed morphology and decreased growth are indicators for negative effects of the Cayman LNP on HEK293, probably reasoned in the employment of more cytotoxic mPEG-DSPE compared to DMG-PEG in the Cayman and SORT LNP.</p>
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.
<b>Figure 21. </b>
Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification after 72 h post-transfection with different Corden LNP formulations recorded with Leica DMI6000 B.
<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>
<p>The images reveal the presence of spherical LNP structures with an approximate size of 100 nm (Figure 22). 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>
</div>
</div>
<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>
<H4text="Sort LNP"/>
<H4text="SORT LNP"/>
<H5text="Transfection"/>
<p>Text :D</p>
<p>Already at 48 h of 72 monitored hours post-transfection of HEK293 with the SORT LNP, clear and strong fluorescence was already observed when using LNPs combined with Minicircle DNA, as well as in the Lipofectamine and Minicircle DNA condition, confirming successful transfection and robust expression of the eYFP reporter (Figure 23). This early detection of fluorescence highlights the efficiency of SORT LNPs as delivery system. In contrast, no fluorescence was detected in any conditions involving Lipofectamine alone, Lipofectamine with RNA, or LNPs combined with DNA or RNA, further emphasizing the superior performance of the Minicircle DNA formulations and highlighting the need for improvement for our DNA choice and RNA synthesis.</p>
Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification after 48 h post-transfection with different SORT LNP formulations recorded with Leica DMI6000 B.
</figcaption>
</figure>
<p>Notably, the SORT LNP formulation with Minicircle DNA showed significant transfection efficiency within 48 hours, making it the most effective delivery method tested among all three LNPs. This early and robust expression demonstrates the clear advantage of this approach for HEK293 cells.</p>
<H5text="Flow cytometry"/>
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<p>We performed flow cytometries analysis 72 h post-transfection to evaluate the transfection efficiency of the SORT LNP in HEK293. The relative percentage of fluorescent cells was determined by measuring the percentage of FITC-A+ cells, followed by normalization to the negative control and fold change calculation.</p>
<p>We performed flow cytometry analysis 72 h post-transfection to evaluate the transfection efficiency of the SORT LNP in HEK293. The relative percentage of fluorescent cells was determined by measuring the percentage of FITC-A+ cells, followed by normalization to the negative control and fold change calculation.</p>
Percentage of fluorescent cells (FITC-A+) performed 72 h post-transfection of SORT LNP in HEK293. Mean +/- SEM for n=3. For statistics one-way ANOVA was performed.
</figcaption>
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<p>The SORT LNP-transfected sample carrying Minicircle DNA exhibited a significant increase in fluorescence compared to the lipofectamine transfection of Minicircle DNA, with approximately 14 times more fluorescent cells compared to the lipofectamine-transfected sample (Figure a). This substantial difference indicates that the transfection efficiency with LNPs is markedly higher than with lipofectamine, demonstrating the superior performance of our LNP formulation in delivering nucleic acids to HEK cells.</p>
<p>The SORT LNP-transfected sample carrying Minicircle DNA exhibited a significant increase in fluorescence compared to the lipofectamine transfection of Minicircle DNA, with approximately 14 times more fluorescent cells compared to the lipofectamine-transfected sample (Figure 24). This substantial difference indicates that the transfection efficiency with LNPs is markedly higher than with lipofectamine, demonstrating the superior performance of our LNP formulation in delivering nucleic acids to HEK cells.</p>
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</div>
<H5text="Zeta Potential"/>
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<p>We measured both the particle size distribution and the Zeta potential using the Nanotrack Wave II. We could assume that the particles exhibit a polarized Zeta potential, which is sufficient to provide electrostatic stabilization, thereby preventing aggregation and maintaining particle stability. For effective targeting of lung cells which have negatively charged surfaces, a negative polarity is desirable meaning the LNP is positively charged, so there can be electrostatic attraction to lung epithelial cells. We were able to show that our SORT LNP has these properties regardless of the load. Furthermore we could <ahref="https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Physical_Methods_in_Chemistry_and_Nano_Science_(Barron)/02%3A_Physical_and_Thermal_Analysis/2.05%3A_Zeta_Potential_Analysis"title="StabZeta">determine the stability via the Zeta potential</a>. In detail the mean of the Zeta potential lays at 16.2 mV for the SORT LNP with Minicircle DNA as cargo, indicating incipient stability, at 59.45 mV for the SORT LNP with pcDNA 3.1 eYFP as cargo, indicating good stability and at 88.22 mV for the SORT LNP without cargo indicating excellent stability (Figure z). The good stability of the SORT LNP with pcDNA 3.1 eYFP is crucial for our purposes, as it ensures effective delivery and performance. In contrast, the stability of the LNPs with Minicircle DNA can be considered secondary, as it primarily serves as a positive transfection control and is not central to our main objectives.</p>
<p>We measured both the particle size distribution and the Zeta potential using the Nanotrack Wave II. We could assume that the particles exhibit a polarized Zeta potential, which is sufficient to provide electrostatic stabilization, thereby preventing aggregation and maintaining particle stability. For effective targeting of lung cells which have negatively charged surfaces, a negative polarity is desirable meaning the LNP is positively charged, so there can be electrostatic attraction to lung epithelial cells. We were able to show that our SORT LNP has these properties regardless of the load. Furthermore we could <ahref="https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Physical_Methods_in_Chemistry_and_Nano_Science_(Barron)/02%3A_Physical_and_Thermal_Analysis/2.05%3A_Zeta_Potential_Analysis"title="StabZeta">determine the stability via the Zeta potential</a>. In detail the mean of the Zeta potential lays at 16.2 mV for the SORT LNP with Minicircle DNA as cargo, indicating incipient stability, at 59.45 mV for the SORT LNP with pcDNA 3.1 eYFP as cargo, indicating good stability and at 88.22 mV for the SORT LNP without cargo indicating excellent stability (Figure 25). The good stability of the SORT LNP with pcDNA 3.1 eYFP is crucial for our purposes, as it ensures effective delivery and performance. In contrast, the stability of the LNPs with Minicircle DNA can be considered secondary, as it primarily serves as a positive transfection control and is not central to our main objectives.</p>
Zeta potential of SORT LNP with different cargos measured with Nanotrack Wave II indicating varying degrees of stability but most important good stability for the SORT LNP loaded with pcDNA 3.1 eYFP (LNP DNA). Mean +/- SEM for n=5. For statistics one-way ANOVA was performed. </figcaption>
Size distribution for the SORT LNP with different cargos weighted by scattering intensity measured with Nanotrack Wave II.
</figcaption>
</figure>
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<p>The size distribution for all three samples shows a predominantly monomodal, yet broad, distribution with diameters ranging between 50 nm and 700 nm, with the peak of the distribution lying between 150 nm and 200 nm (Figure d). SORT LNPs without DNA exhibited larger radii, with a peak around 300 nm. The SORT LNP containing Minicircle DNA suggests the presence of larger aggregates with diameters exceeding 1 µm. The likely reason for this variable particle size distribution, despite loading with different types of DNA, could be attributed to the manufacturing method. Since the LNPs were not produced using an extruder but rather via dialysis, this is highly plausible.</p>
<p>The size distribution for all three samples shows a predominantly monomodal, yet broad, distribution with diameters ranging between 50 nm and 700 nm, with the peak of the distribution lying between 150 nm and 200 nm (Figure 26). SORT LNPs without DNA exhibited larger radii, with a peak around 300 nm. The SORT LNP containing Minicircle DNA suggests the presence of larger aggregates with diameters exceeding 1 µm. The likely reason for this variable particle size distribution, despite loading with different types of DNA, could be attributed to the manufacturing method. Since the LNPs were not produced using an extruder but rather via dialysis, this is highly plausible.</p>
</div>
</div>
<H5text="Cryo-EM"/>
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<p>Cryo-EM analysis was also performed of SORT LNPs by us with the same JEOL JEM-2200FS microscope at 200kV, allowing visualization of LNPs in their native hydrated state. The images show spherical LNP structures around 100 nm, with some larger aggregates also present (data not shown). These aggregates likely result from interactions between particles due to the non-extrusion-based preparation method, which may explain the variability in particle size. Additionally, particles potentially representing different LNP populations or overlapped structures with low contrast were observed (Figure X). The low sample concentration likely contributed to the limited number of visible particles.</p>
<p>Cryo-EM analysis was also performed of SORT LNPs by us with the same JEOL JEM-2200FS microscope at 200kV, allowing visualization of LNPs in their native hydrated state. The images show spherical LNP structures around 100 nm, with some larger aggregates also present (data not shown). These aggregates likely result from interactions between particles due to the non-extrusion-based preparation method, which may explain the variability in particle size. Additionally, particles potentially representing different LNP populations or overlapped structures with low contrast were observed (Figure 27). The low sample concentration likely contributed to the limited number of visible particles.</p>
Results for hydrodynamic radius determination by DLS Measurements for our SORT LNP, indicating a radius of approximately 100 nm.
</figcaption>
</figure>
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<p>The results showed a hydrodynamic diameter of SORT LNPs yielding an average radius of approximately 100 nm (Figure X). These findings are consistent with our previous applied size determination methods, such as Zeta potential and Cryo-EM, which also indicated similar particle dimensions in appropriate range for our research and medical applications.</p>
<p>The results showed a hydrodynamic diameter of SORT LNPs yielding an average radius of approximately 100 nm (Figure 28). These findings are consistent with our previous applied size determination methods, such as Zeta potential and Cryo-EM, which also indicated similar particle dimensions in appropriate range for our research and medical applications.</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>
</figure>
</div>
<divclassName="col">
{/* <p>In order to evaluate the <a onClick={() => goToPageAndScroll ('Biosafety2', '/safety')}>biosafety</a> of our lung-specific LNPs, particularly concerning the choice of <a onClick={() => goToPagesAndOpenTab({collapseId: 'Col1', path: '/engineering', tabId: 'delivery' })}>PEG</a> - known to cause cytotoxicity issues - we performed MTT assays using HEK293 cells with various LNP formulations. The results demonstrated that the Cayman LNP achieved 74.90% viability and SORT LNP showed 75.01% viability, exhibiting lower cytotoxicity due to the inclusion of DMG-PEG, a less cytotoxic PEG variant compared to mPEG-2000-DSPE, which resulted in 66.69% viability in the Corden LNP (Figure t). These findings prove we made the best decision by choosing the SORT LNP as the least cytotoxic LNPs.</p> */}
<p>In order to evaluate the <aonClick={()=>goToPageAndScroll ('Biosafety2','/safety')}>biosafety</a> of our lung-specific LNPs, particularly concerning the choice of PEG - known to cause cytotoxicity issues - we performed MTT assays using HEK293 cells with various LNP formulations. The results demonstrated that the Cayman LNP achieved 74.90% viability and SORT LNP showed 75.01% viability, exhibiting lower cytotoxicity due to the inclusion of DMG-PEG, a less cytotoxic PEG variant compared to mPEG-2000-DSPE, which resulted in 66.69% viability in the Corden LNP (Figure t). These findings prove we made the best decision by choosing the SORT LNP as the least cytotoxic LNPs.</p>
<p>In order to evaluate the <aonClick={()=>goToPageAndScroll ('Biosafety2','/safety')}>biosafety</a> of our lung-specific LNPs, particularly concerning the choice of PEG - known to cause cytotoxicity issues - we performed MTT assays using HEK293 cells with various LNP formulations. The results demonstrated that the Cayman LNP achieved 74.90% viability and SORT LNP showed 75.01% viability, exhibiting lower cytotoxicity due to the inclusion of DMG-PEG, a less cytotoxic PEG variant compared to mPEG-2000-DSPE, which resulted in 66.69% viability in the Corden LNP (Figure 29). These findings prove we made the best decision by choosing the SORT LNP as the least cytotoxic LNPs.</p>
</div>
</div>
<H4text="SORT LNP Chitosan-RNA Complex"/>
<p>We successfully transfected a well-established lung epithelial cell line CFBE41o- with our chitosan-RNA complexes using our synthesized pcDNA 3.1 eYFP mRNA, which were subsequently incubated with SORT LNPs for packaging. The goal was to encapsulate the chitosan-RNA complexes within the LNPs to enhance mRNA delivery to cells. After formulation, the LNP Chitosan-RNA complexes were transfected into CFBE41o- cells, to assess the efficiency of mRNA delivery and expression.</p>
<p>We tested two different chitosan and mRNA concentrations (always used both in the same concentrations), 50 ng/µl and 500 ng/µl, in combination with our LNP to assess their impact on transfection efficiency. Interestingly, both concentrations resulted in similarly high levels of transfection, as evidenced by the fluorescence microscopy images, further validation is necessary to confirm these results.</p>
<p>Fluorescence microscopy was performed 24 hours post-transfection to evaluate the expression of the YFP reporter gene encoded by the pcDNA 3.1 eYFP mRNA. This reporter gene serves as an indicator of successful transfection and translation of the mRNA into protein. The results of the fluorescence microscopy were highly positive and aligned with our expectations, indicating efficient delivery, uptake, and expression of the mRNA in the CFBE41o- cells.</p>
<H4text="Flow cytometry"/>
<p>We tested two different chitosan and mRNA concentrations (always used both in the same concentrations), 50 ng/µl and 500 ng/µl, in combination with our LNP to assess their impact on transfection efficiency. Interestingly, both concentrations resulted in similarly high levels of transfection, as evidenced by the fluorescence microscopy images, further validation is necessary to confirm these results.</p>
Overlay of phase contrast and fluorescence microscopic images of with SORT + chitosan 500 transfected CFBE41o- cells at 20x magnification after 48 h post-transfection recorded with Leica DMI6000 B.
</figcaption>
</figure>
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<p>The sample containing 500 ng/µl of chitosan and pcDNA 3.1 eYFP mRNA, encapsulated within SORT LNPs, exhibited fluorescence 24 hours post-transfection (Figure 30). This result indicates that the RNA was successfully delivered into the CFBE41o- cells, where it was transcribed and translated into the YFP protein. The presence of YFP fluorescence confirms not only successful transfection but also robust expression of the reporter gene.</p>
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<p>In the sample containing 50 ng/µl of chitosan and pcDNA 3.1 eYFP mRNA was used, which is tenfold lower than the chitosan and mRNA concentration used in the chitosan500 sample (see above). Despite the lower mRNA concentration, fluorescence was still observed (Figure 31) indicating that the mRNA was efficiently delivered and expressed in the CFBE41o- cells. This result suggests that even at lower mRNA doses, the system can achieve successful transfection and gene expression. We are planning to perform a more detailed comparison of fluorescence intensity between the chitosanRNA500 and chitosanRNA50 samples and even lower concentrations to assess the relationship between mRNA dose and expression level.</p>
Overlay of phase contrast and fluorescence microscopic images of with SORT + chitosan 50 transfected CFBE41o- cells at 20x magnification after 48 h post-transfection recorded with Leica DMI6000 B.
</figcaption>
</figure>
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</div>
<p>The samples containing only chitosan (Figure 32) and only SORT LNP (Figure 33) without any mRNA, did not exhibit any detectable fluorescence. This result is consistent with expectations, as chitosan alone and SORT LNP alone are not fluorescent. These samples served as important negative controls to confirm that the chitosan itself and the SORT LNP itself don't interfere with the fluorescence signal.</p>
Overlay of phase contrast and fluorescence microscopic images of with SORT transfected CFBE41o- cells at 20x magnification after 48 h post-transfection recorded with Leica DMI6000 B.
Overlay of phase contrast and fluorescence microscopic images of with chitosan transfected CFBE41o- cells at 20x magnification after 48 h post-transfection recorded with Leica DMI6000 B.
Overlay of phase contrast and fluorescence microscopic images of untransfected CFBE41o- cells at 20x magnification after 48 h post-transfection recorded with Leica DMI6000 B.
</figcaption>
</figure>
</div>
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<p>The untreated control, which did not receive any chitosan, RNA, or LNPs, did not show any fluorescence (Figure 34). This negative control confirms that the cells themselves do not express YFP under normal conditions, and that any observed fluorescence in the experimental groups is directly attributable to the transfection and expression of the delivered RNA.</p>
</div>
</div>
<p>The results demonstrate that chitosan-RNA complexes, when packaged with SORT LNPs, can efficiently deliver mRNA into CFBE41o- cells and facilitate the expression of a YFP reporter gene. Furthermore, the absence of fluorescence in the control samples validates the specificity of the system, ensuring that the fluorescence signal is solely due to the delivered mRNA and not from other components of the system. Additional testing, such as flow cytometry analysis, is planned to provide a more quantitative assessment of transfection efficiency. This would allow us to accurately determine whether there are subtle differences between the two concentrations that were not detectable by microscopy alone.</p>
<p>Overall, this experiment highlights the potential of chitosan-RNA complexes in combination with SORT LNPs as a promising platform for mRNA delivery and gene expression in airway epithelial cells. Further investigation could focus on optimizing the mRNA dose to maximize expression levels and transfection efficiency.</p>
</Subesction>
<Subesctiontitle="PreCyse"id="Results4">
<H4text="Goals"/>
...
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@@ -371,6 +443,7 @@ export function Results() {
<H4text="Conclusion"/>
<p>text</p>
</Subesction>
<Subesctiontitle="Patch Clamp"id="Results5">
<p>To validate our gene editing approach by prime editing of CFTR F508del delivered to lung cells via SORT LNPs, we planned to use <aonClick={()=>goToPageAndScroll ('Patch Clamp','/materials-methods')}>Patch Clamp</a> as a downstream method. Our goal was to detect the restored conductance of the repaired CFTR by this electrophysiological method. This was made possible through the assistance of the <aonClick={()=>goToPagesAndOpenTab('patchclamp','/human-practices')}>Cellular Neurophysiology research group</a> at our university.</p>
Current density of HEK293, HEK293T CFTR WT and HEK293T CFTR F508del showing significant differences of both HEK293T cell lines compared to HEK293 but no significant differences between them. For statistics one-way ANOVA was performed.
</figcaption>
</figure>
</div>
<divclassName='col'>
<p>In our first set of experiments, we measured current density in <aonClick={()=>goToPageAndScroll ('Cell Culture','/materials-methods')}>HEK293T CFTR wild-type (WT) and HEK293T F508del</a> cell lines, comparing them with regular HEK293. The results demonstrated significant differences in chloride ion conductance, with the CFTR-expressing cell lines showing enhanced conductivity compared to HEK293 (Figure 1). However, a drawback was that we did not observe any significant differences between the HEK293T CFTR WT and F508del cell line. This was unexpected, as the F508del mutation typically leads to a knockdown of the CFTR protein, impairing chloride ion transport through the CFTR channel.</p>
<p>In our first set of experiments, we measured current density in <aonClick={()=>goToPageAndScroll ('Cell Culture','/materials-methods')}>HEK293T CFTR wild-type (WT) and HEK293T F508del</a> cell lines, comparing them with regular HEK293. The results demonstrated significant differences in chloride ion conductance, with the CFTR-expressing cell lines showing enhanced conductivity compared to HEK293 (Figure 35). However, a drawback was that we did not observe any significant differences between the HEK293T CFTR WT and F508del cell line. This was unexpected, as the F508del mutation typically leads to a knockdown of the CFTR protein, impairing chloride ion transport through the CFTR channel.</p>
</div>
</div>
<H4text="Further Validation and Challenges"/>
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<p>In light of these results, we improved our experimental setup and performed additional validation experiments. Unfortunately, the repeated measurements yielded similar outcomes, confirming the absence of a significant difference between the two CFTR-expressing cell lines (Figure 2). This finding led us to consult with the research group at <aonClick={()=>goToPagesAndOpenTab('mattijsvisit','/human-practices')}>KU Leuven</a>, who established these cells lines. Although they had not conducted similar Patch Camp measurements, they suggested an alternative approach using Ussing Chamber measurements. This technique, unlike Patch Camp, does not rely on single-cell measurements but rather examines the ion currents across the entire cell monolayer, which may provide a more comprehensive view of CFTR functionality.</p>
<p>In light of these results, we improved our experimental setup and performed additional validation experiments. Unfortunately, the repeated measurements yielded similar outcomes, confirming the absence of a significant difference between the two CFTR-expressing cell lines (Figure 36). This finding led us to consult with the research group at <aonClick={()=>goToPagesAndOpenTab('mattijsvisit','/human-practices')}>KU Leuven</a>, who established these cells lines. Although they had not conducted similar Patch Camp measurements, they suggested an alternative approach using Ussing Chamber measurements. This technique, unlike Patch Camp, does not rely on single-cell measurements but rather examines the ion currents across the entire cell monolayer, which may provide a more comprehensive view of CFTR functionality.</p>
Repeated validation of current density measurements in HEK293T CFTR WT and HEK293T CFTR-F508del, showing consistent results with the initial experiment. For statistics one-way ANOYA was performed.
