diff --git a/src/contents/Human Practices/Further Engagement/SupMaterial.tsx b/src/contents/Human Practices/Further Engagement/SupMaterial.tsx
index 2a5f20df31b36d9bf4ba2b68c4ea2ceabe76bf77..f50b72a2b6f58f1659b5f3bff06a75cedd979faf 100644
--- a/src/contents/Human Practices/Further Engagement/SupMaterial.tsx
+++ b/src/contents/Human Practices/Further Engagement/SupMaterial.tsx
@@ -8,10 +8,10 @@ export function HPSupplement(){
<Section title="Supplementary Material" id="Supplementary Material">
<div className="row">
<div className="col">
- <p>Click here to see our raw data of our surverys.</p>
+ <p>Click here to see our raw data of our surverys</p>
</div>
<div className="col">
- <DownloadLink url="https://static.igem.wiki/teams/5247/pdfs/raw-data-surveys.pdf" fileName="raw-data-surveys.pdf"></DownloadLink>
+ <DownloadLink url="https://static.igem.wiki/teams/5247/pdfs/raw-data-survey.pdf" fileName="raw-data-survey.pdf"></DownloadLink>
</div>
</div>
</Section>
diff --git a/src/contents/description.tsx b/src/contents/description.tsx
index 6d930233f709b61d8f9a6891b7797a4be3608233..7c2c6e2677c141e0979bd6a7150207dd2bd4385a 100644
--- a/src/contents/description.tsx
+++ b/src/contents/description.tsx
@@ -273,7 +273,7 @@ export function Description() {
<figure>
<img src="https://ars.els-cdn.com/content/image/1-s2.0-S1773224724002156-gr3_lrg.jpg" alt="Aufnahme LNP" style={{maxHeight: "200pt"}}/>
<figcaption>
- <b>Figure. </b>
+ <b>Figure 2. </b>
Endosomal escape vs degradation of LNP cargo at endocytosis <SupScrollLink label="4"/> .
</figcaption>
</figure>
@@ -298,16 +298,16 @@ export function Description() {
</Collapsible>
<br/>
<div id="airbuddy-hook" className='row align-items-center'>
- <p>To optimize AirBuddy for pulmonary delivery, we collaborated extensively with several experts, including <a onClick={() => goToPagesAndOpenTab('weber', '/human-practices')}>Prof. Weber, Dr. Große-Onnebrink</a> and <a onClick={() => goToPagesAndOpenTab('kolonkofirst', '/human-practices')}>Dr. Kolonko</a> as medical experts, <a onClick={() => goToPagesAndOpenTab('kristian', '/human-practices')}>Prof. Dr. Müller</a>, <a onClick={() => goToPagesAndOpenTab('radukic', '/human-practices')}>Dr. Radukic</a>, <a onClick={() => goToPagesAndOpenTab('moorlach', '/human-practices')}>Benjamin Moorlach</a> and the <a onClick={() => goToPagesAndOpenTab('biophysik', '/human-practices')}>Physical and Biophysical Chemistry working group</a> as academic experts form Bielefeld University and FH Bielefeld as well as <a onClick={() => goToPagesAndOpenTab('corden', '/human-practices')}>Corden Pharma</a> and <a onClick={() => goToPagesAndOpenTab('rnhale', '/human-practices')}>RNhale</a> as industrial experts. Throughout the <a onClick={() => goToPageWithTabAndScroll({tabId:'tab-delivery', path: '/engineering', scrollToId: "delivery-header"})}>development process</a>, we tested two commercially available kits: the <strong>Cayman Chemical LNP Exploration Kit (LNP-102)</strong> and the <strong>Corden Pharma LNP Starter Kit #2</strong>. While the Cayman kit demonstrated limited transfection efficiency, the Corden Pharma formulation significantly enhanced cellular uptake in lung tissues. Building on this, we integrated the <strong>SORT LNP</strong> method based on Wang's research <SupScrollLink label="1"/> , making our nanoparticles lung-specific. Additionally, we employed the <strong>spray-drying technique</strong> in cooperation with RNhale <SupScrollLink label="2"/> to improve the stability of our LNP, ensuring that it withstands the inhalation process without degradation. This stability is crucial for the efficient delivery of mRNA into lung epithelial cells, where PrimeGuide can effectively perform genome editing.</p>
+ <p>To optimize AirBuddy for pulmonary delivery, we collaborated extensively with several experts, including <a onClick={() => goToPagesAndOpenTab('weber', '/human-practices')}>Prof. Weber, Dr. Große-Onnebrink</a> and <a onClick={() => goToPagesAndOpenTab('kolonkofirst', '/human-practices')}>Dr. Kolonko</a> as medical experts, <a onClick={() => goToPagesAndOpenTab('kristian', '/human-practices')}>Prof. Dr. Müller</a>, <a onClick={() => goToPagesAndOpenTab('radukic', '/human-practices')}>Dr. Radukic</a>, <a onClick={() => goToPagesAndOpenTab('moorlach', '/human-practices')}>Benjamin Moorlach</a> and the <a onClick={() => goToPagesAndOpenTab('biophysik', '/human-practices')}>Physical and Biophysical Chemistry working group</a> as academic experts from Bielefeld University and FH Bielefeld as well as <a onClick={() => goToPagesAndOpenTab('corden', '/human-practices')}>Corden Pharma</a> and <a onClick={() => goToPagesAndOpenTab('rnhale', '/human-practices')}>RNhale</a> as industrial experts. Throughout the <a onClick={() => goToPageWithTabAndScroll({tabId:'tab-delivery', path: '/engineering', scrollToId: "delivery-header"})}>development process</a>, we tested two commercially available kits: the <strong>Cayman Chemical LNP Exploration Kit (LNP-102)</strong> and the <strong>Corden Pharma LNP Starter Kit #2</strong>. While the Cayman kit showed good non-lung-specific transfection efficiency, the Corden Pharma formulation also proved not to be the right approach. Building on this, we integrated the <strong>SORT LNP</strong> method based on Wang's research<SupScrollLink label="1"/> , making our nanoparticles lung-specific. Additionally, we employed the <strong>chitosan-complexation of the therapeutic cargo</strong> to improve the stability of our LNP, ensuring that it withstands the inhalation process without degradation. Moreover, further stabilization approaches including the employment of the <strong>spray-drying technique</strong> in cooperation with RNhale<SupScrollLink label="2"/> are in the pipeline. This improved stability is crucial for the efficient delivery of mRNA into lung epithelial cells, where PrimeGuide can effectively perform genome editing.</p>
<img src="https://static.igem.wiki/teams/5247/delivery/big-plan-inhalation-teil-del.webp"/>
</div>
- <p>To evaluate the <strong>delivery efficiency</strong>, we transfected HEK292 and CFBE41o- cells using fluorescent cargo and quantified the results through FACS analysis. We also ensured that AirBuddy meets the necessary standards for safety and efficacy since we conducted extensive <a onClick={() => goToPageAndScroll ('In-Depth Characterization of LNPsH', '/materials-methods')}> characterization of the LNPs </a>using techniques such as Zeta potential analysis, Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), and Cryogenic Electron Microscopy (cryo-EM). These methods confirmed the uniformity, stability, and optimal size distribution of the nanoparticles. Furthermore, <strong>cytotoxicity assessments</strong> including MTT and proliferation assays demonstrated that our LNPs are biocompatible and do not impede cell growth or function by the incorporation of <a onClick={() => goToPageWithTabAndCollapsible({tabId:'tab-delivery', path: '/engineering', collapseId: "Col1"})}>PEG</a> and other ambivalent components. These findings reinforce AirBuddy's potential as a safe and effective tool for pulmonary delivery, with broad implications for gene therapies targeting lung diseases.</p>
+ <p>To evaluate the <strong>delivery efficiency</strong>, we transfected HEK293 and CFBE41o- cells using fluorescent cargo and quantified the results through flow cytometry analysis. We also ensured that AirBuddy meets the necessary standards for safety and efficacy since we conducted extensive <a onClick={() => goToPageAndScroll ('In-Depth Characterization of LNPsH', '/materials-methods')}> characterization of the LNPs </a>using physicochemical techniques such as Zeta potential analysis, Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), and Cryogenic Electron Microscopy (cryo-EM). These methods confirmed the stability and optimal size distribution of the nanoparticles. Furthermore, <strong>cytotoxicity assessments</strong> including MTT and proliferation assays demonstrated that our LNPs are biocompatible despite the incorporation of <a onClick={() => goToPageWithTabAndCollapsible({tabId:'tab-delivery', path: '/engineering', collapseId: "Col1"})}>PEG</a> and other ambivalent components. These findings reinforce AirBuddy's potential as a safe and effective tool for pulmonary delivery, with broad implications for gene therapies targeting lung diseases.</p>
</Subesction>
</Section>
<Section title="Our Achievement" id="Our Achievement">
<p>We have successfully demonstrated a <b>proof of concept</b> for our gene therapy approach targeting cystic fibrosis. In initial experiments, HEK cells carrying a 3-base deletion analogous to the <i>F508del</i> mutation were transfected with our prime editing complex. The results met our expectations, confirming the viability of our approach for precise gene correction. Based on these findings, we optimized the prime editing complex, leading to the creation of <i>PrimeGuide</i>, a more compact and efficient editing tool. </p>
- <p>Central to our <b>delivery system</b> is <b>AirBuddy</b>, a lung-specific lipid nanoparticle designed to stabilize and protect the prime editing complex during transport to lung ionocytes. <i>AirBuddy</i> ensures that the protein complex is delivered specifically to lung cells, enhancing the efficiency of the gene-editing process. By modifying the lipid nanoparticle with protective features, we achieved increased stability, ensuring effective delivery to the target cells. </p>
+ <p>Central to our <b>delivery system</b> is <b>AirBuddy</b>, a lung-specific lipid nanoparticle designed to stabilize and protect the prime editing complex during transport to lung epithelial cells. <i>AirBuddy</i> ensures that the protein complex is delivered specifically to lung cells, enhancing the efficiency of the gene-editing process. By modifying the lipid nanoparticle with protective features, we achieved increased stability, ensuring effective delivery to the target cells. </p>
<p>We further optimized the prime editing fusion protein, <b>PrimeGuide</b>, to streamline its components, resulting in a smaller and more efficient prime editing complex. This improvement significantly enhances the precision of the gene editing process, reducing off-target effects and increasing the overall success of mutation correction. </p>
<p>In subsequent experiments, <b>HEK cells</b> carrying the CFTR <i>F508del</i> mutation were successfully <b>transfected</b> with the optimized prime editing complex. Our results indicated successful correction of the mutation, confirming the potential of our approach for treating cystic fibrosis. </p>
<p>Additionally, we explored <b>downstream applications</b>. Primary cell cultures were treated with lipid nanoparticles to introduce a reporter RNA. We also established 2D cultures transfected with YFP, a sodium-sensitive reporter protein, to assess ion channel functionality. Finally, in CFTR-deficient organoids, our system facilitated repair of the CFTR channel, evidenced by an increase in organoid volume upon treatment. This suggests successful functional restoration of CFTR activity. </p>
diff --git a/src/contents/engineering.tsx b/src/contents/engineering.tsx
index 707e73f88ed3a55027e7a0ea97ea71073c8281b4..ff090de3c7dc1a0e26ae4fd71576c8e41c8ec2aa 100644
--- a/src/contents/engineering.tsx
+++ b/src/contents/engineering.tsx
@@ -1062,26 +1062,26 @@ export function Engineering() {
<div className="box" >
<p id="del2">
<H3 text="Interation 2 - Cayman LNP" id="del2head" />
- In the first experimental phase, LNPs from <strong>Cayman Chemical LNP Exploration Kit (LNP-102)</strong> consisting of SM-102, 1,2-DSPC, cholesterol, and DMG-PEG(2000)<TabScrollLink tab="tab-delivery" num="4" scrollId="desc-4"/> were tested with mRNA encoding fluorescent protein to evaluate their transfection efficiency. However, the results showed low transfection efficiency, and the particles did not show specificity for the lungs, which was a critical requirement for the project. This led the team to reconsider the choice of the LNP.
+ In the first experimental phase, LNPs from <strong>Cayman Chemical LNP Exploration Kit (LNP-102)</strong> consisting of SM-102, 1,2-DSPC, Cholesterol, and DMG-PEG(2000)<TabScrollLink tab="tab-delivery" num="4" scrollId="desc-4"/> were tested with mRNA encoding fluorescent protein to evaluate their transfection efficiency. However, the results indicated good non-lung-specific transfection efficiency, which was a critical factor for the project. This led the team to reconsider their choice of this LNP.
</p>
<figure>
<img src="https://static.igem.wiki/teams/5247/delivery/cayman-lnp-freigestellt.webp" alt="Cayman LNP"/>
<figcaption>
- <b>Figure 1.</b>
- Schematic view of LNP-102 from Cayman Chemical
+ <b>Figure 1. </b>
+ Schematic view of LNP-102 from Cayman Chemical.
</figcaption>
</figure>
</div>
<div className="box" >
<p id="del3">
<H3 text="Interation 3 - Corden LNP" id="del3head" />
- In the next phase, we chose to use a new LNP formulation, namely the <strong>LNP Starter Kit #2</strong><TabScrollLink tab="tab-delivery" num="5" scrollId="desc-5"/> of <a onClick={() => goToPagesAndOpenTab('corden', '/human-practices')}>Corden Pharma</a>, because it offered several advantages over the initial option. The key benefit of this new LNP lies in the use of DOTAP, a cationic lipid that enhances interaction with negatively charged cell membranes in the lungs, improving cellular uptake efficiency. While SM-102 in the Cayman LNP-102 is effective for systemic delivery, it lacks the same specificity for lung tissue. Additionally, Corden Pharma’s plant-based BotaniChol® prevents animal-sourced contamination and helps address the global lipid shortage for vaccine production. mPEG-2000-DSPE provides superior stability and reduces immune system activation over time, making it particularly suitable for pulmonary delivery. This made the new formulation a better choice for safely and effectively targeting lung tissue, especially in delivering therapies for CFTR-related diseases. During this time, the team encountered a paper on capsaicin-chitosan nanoparticles, which explored its use in targeted delivery and higher transfection efficiency. However, after further investigation and consultation of <a onClick={() => goToPagesAndOpenTab('kolonkofirst', '/human-practices')}>Dr. Katharina Kolonko</a>, it was determined that capsaicin was not suitable for our planned pulmonary application.
+ In the next phase, we chose to use a new LNP formulation, namely the <strong>LNP Starter Kit #2</strong><TabScrollLink tab="tab-delivery" num="5" scrollId="desc-5"/> of <a onClick={() => goToPagesAndOpenTab('corden', '/human-practices')}>Corden Pharma</a>, because it offered several advantages over the initial option. The key benefit of this new LNP lies in the use of DOTAP, a cationic lipid that enhances interaction with negatively charged cell membranes in the lungs, improving cellular uptake efficiency. While SM-102 in the Cayman LNP-102 is effective for systemic delivery, it lacks the same specificity for lung tissue. Additionally, Corden Pharma’s plant-based BotaniChol® prevents animal-sourced contamination and helps address the global lipid shortage for vaccine production. mPEG-2000-DSPE provides superior stability and reduces immune system activation over time, making it particularly suitable for pulmonary delivery. This made the new formulation a better choice for safely and effectively targeting lung tissue, especially in delivering therapies for CFTR-related diseases. During this time, the team encountered a paper on capsaicin-chitosan nanoparticles, which explored its use in targeted delivery and higher transfection efficiency. However, after further investigation and consultation of <a onClick={() => goToPagesAndOpenTab('kolonkofirst', '/human-practices')}>Dr. Katharina Kolonko</a>, it was determined that capsaicin was not suitable in our way of pulmonary application.
</p>
<figure>
<img src="https://static.igem.wiki/teams/5247/delivery/corden-lnp-freigestellt.webp" alt="Corden LNP"/>
<figcaption>
- <b>Figure 2.</b>
- Schematic view of LNP #2 from Corden Pharma with DOTAP as cationic lipid, DSPC as phospholipid and mPEG-200-DSPE as PEG lipid.
+ <b>Figure 2. </b>
+ Schematic view of LNP #2 from Corden Pharma with DOTAP as cationic lipid, DSPC as phospholipid and mPEG-2000-DSPE as PEG lipid.
</figcaption>
</figure>
</div>
@@ -1092,7 +1092,7 @@ export function Engineering() {
<Collapsible id="Col1" open={false} title="Ambivalence of PEG and our implementation">
<p>
<H4 text="What is PEG and why is it important for LNPs?" id="text" />
- Polyethylene glycol (PEG) is an essential component in the formulation of lipid nanoparticles (LNPs), which are widely used in drug delivery systems, particularly for mRNA-based therapies like vaccines. PEG-lipids are hybrid molecules consisting of a hydrophilic PEG chain attached to a hydrophobic lipid anchor. This unique structure enables PEG-lipids to interact effectively with both aqueous environments and lipid structures, such as cell membranes and lipid nanoparticles themselves.
+ Polyethylene glycol (PEG) is an essential component in the formulation of LNPs, which are widely used in drug delivery systems, particularly for mRNA-based therapies like vaccines. PEG-lipids are hybrid molecules consisting of a hydrophilic PEG chain attached to a hydrophobic lipid anchor. This unique structure enables PEG-lipids to interact effectively with both aqueous environments and lipid structures, such as cell membranes and lipid nanoparticles themselves.
<p>PEGylation—attaching PEG to lipids—provides numerous benefits. It increases the stability of LNPs by forming a protective outer layer, preventing aggregation, extending circulation time in the bloodstream, and reducing immune system detection. These advantages are critical in ensuring that the LNPs reach their target cells and deliver the therapeutic payload effectively. </p>
<H4 text="Why is PEG relevant for LNPs in mRNA delivery?" id="text" />
PEG improves the pharmacokinetics of LNPs by extending their systemic circulation time, which is crucial for therapies like mRNA vaccines, where the nanoparticles must remain in the bloodstream long enough to reach their target cells. Additionally, PEG-lipids can reduce the size of LNPs, enhancing their ability to penetrate cell membranes and deliver the therapeutic material efficiently. However, a balance must be struck. Increasing PEG content can lead to smaller, more stable particles, but it may also reduce intracellular delivery and protein expression. Therefore, while PEG boosts circulation and stability, too much can hinder therapeutic effectiveness.
@@ -1107,12 +1107,12 @@ export function Engineering() {
We use DMG-PEG2000 in our SORT LNP-based AirBuddy because of its superior biodegradability, enhanced stability, and reduced risk of immune system activation. By building on insights from experts and incorporating principles from Wang’s LNP work, we’ve tailored our nanoparticles for lung-specific delivery. This choice ensures that our formulations remain stable long enough to deliver the therapeutic payload effectively while minimizing potential long-term toxicity. This balance is crucial for pulmonary applications, where DMG-PEG2000 outperforms alternatives like mPEG-2000-DSPE, making it the ideal choice for treating CFTR-related lung diseases.
</p>
</Collapsible>
- <p>DMG-PEG2000 of the SORT LNP offers better biodegradability and enhanced stability in pulmonary applications - it is known for its faster clearance and reduced potential for long-term toxicity. To ensure we addressed this issue, cytotoxicity tests were performed in addition to the determination of physical properties in cooperation with the <a onClick={() => goToPagesAndOpenTab('biophysik', '/human-practices')}>Physical and Biophysical Chemistry working group of Bielefeld University</a> to characterize the LNPs. More details about the composition of the SORT LNPs and function of the components can be read below.</p>
+ <p>DMG-PEG2000 of the SORT LNP offers better biodegradability and enhanced stability in pulmonary applications - it is known for its faster clearance and reduced potential for long-term toxicity. To ensure we addressed this issue, cytotoxicity tests were performed in addition to the determination of physicochemical properties in cooperation with the <a onClick={() => goToPagesAndOpenTab('biophysik', '/human-practices')}>Physical and Biophysical Chemistry working group of Bielefeld University</a> to characterize the LNPs. More details about the composition of the SORT LNPs and function of the components can be read below.</p>
<figure>
- <img src="https://static.igem.wiki/teams/5247/delivery/sort-lnp-freigestellt.webp" alt="AirBuudy"/>
+ <img src="https://static.igem.wiki/teams/5247/delivery/airbuddy-svg.svg" alt="AirBuddy" style={{maxHeight: "350pt"}} />
<figcaption>
- <b>Figure 3.</b>
- Schematic view of our lung-specific SORT LNP called AirBuddy.
+ <b>Figure 3. </b>
+ Schematic view of our lung-specific SORT LNP called AirBuddy carrying chitosan-protected pegRNA and mRNA for the assembly of our prime editing technology named PrimeGuide.
</figcaption>
</figure>
<Collapsible id="Col2" open={false} title="Composition of our SORT LNP called Airbuddy // LNP Handbook Cooperation">
@@ -1131,17 +1131,17 @@ export function Engineering() {
<p>Click this Button to gain the LNP Handbook</p>
<DownloadLink url="https://static.igem.wiki/teams/5387/liposomes-handbook.pdf" fileName="liposomes-handbook.pdf" />
<H5 text="Spray drying Procedure" id="text" />
- <p>By combining these components with the spray drying method from <a onClick={() => goToPageAndScroll ('rnhale', '/human-practices')}> RNhale </a> we offer a versatile and efficient method for delivering mRNA therapeutics to the lung, paving the way for gene therapy, especially our Prime Guide. The effective delivery of the prime editing complex is a crucial point in our project. </p>
+ <p>By combining these components with the spray drying method from <a onClick={() => goToPageAndScroll ('rnhale', '/human-practices')}> RNhale </a> we offer a versatile and efficient method for delivering RNA therapeutics to the lung, paving the way for gene therapy, especially our Prime Guide. The effective delivery of the prime editing complex is a crucial point in our project. </p>
<H5 text="Storage" id="text" />
<p>The final LNP solution can be stored at 4 °C for a few days. It is recommended to use the formulated LNPs as soon as possible to maintain consistent results. Storage at RT is not recommended. Storage at freezing temperatures is also not recommended unless optimized cryoprotectants are used.</p>
</Collapsible>
- <p>The final innovation for our LNP to become <strong>AirBuddy</strong> came through consultation with Benjamin Winkeljann from <a onClick={() => goToPagesAndOpenTab('rnhale', '/human-practices')}> RNhale</a>, where the use of spray-drying techniques was discussed. Spray-drying the LNPs, instead of using traditional methods, helps improve stability and eco-friendliness of the product<TabScrollLink tab="tab-delivery" num="8" scrollId="desc-8"/>. Unfortunately, we were not in time for our samples to be delivered to RNhale for spray-drying before the wiki freeze, but we have already scheduled the transfer. Instead, the discussion with <a onClick={() => goToPagesAndOpenTab('moorlach', '/human-practices')}>Benjamin Moorlach</a>, chitosan expert working at FH Bielefeld, provided new ideas for improvement by <strong>complexing the mRNA with chitosan</strong> to improve the stability of the cargo during spray drying and nebulization. The positive effect of RNA chitosan complexing for opimized LNP delivery could be confimed in our lab. In conclusion, we created a stable LNP for efficient delivery of mRNA therapeutics to the lungs since the successful delivery of the prime editing complex via inhalation is key to our project. </p>
+ <p>The final innovation for our LNP to become <strong>AirBuddy</strong> came through consultation with Benjamin Winkeljann from <a onClick={() => goToPagesAndOpenTab('rnhale', '/human-practices')}> RNhale</a>, when the use of spray-drying techniques was discussed. Spray-drying the LNPs, instead of using traditional methods, helps improve stability and eco-friendliness of the product<TabScrollLink tab="tab-delivery" num="8" scrollId="desc-8"/>. Our samples are set for transfer to RNhale for spray-drying, with scheduling aligned to resume promptly after the wiki freeze. Meanwhile, the discussion with <a onClick={() => goToPagesAndOpenTab('moorlach', '/human-practices')}>Benjamin Moorlach</a>, chitosan expert working at FH Bielefeld, provided new ideas for improvement by <strong>complexing the cargo with chitosan</strong> to improve the stability of the cargo during spray drying and nebulization. The positive effect of chitosan-complexing for opimized LNP delivery could be confirmed in our lab. In conclusion, we created a stable LNP for efficient delivery of RNA therapeutics to the lungs since the successful delivery of the prime editing complex via inhalation is key to our project. </p>
<figure>
<img src="https://static.igem.wiki/teams/5247/delivery/big-plan-inhalation-teil-del.webp" alt="Flow DEL"/>
<figcaption>
<b>Figure 4. </b>
- Application stategy - AirBuddy is inhaled by the patient, enabling uptake of PrimeGuide in lung epithelial cells via endocytosis.
+ Application stategy - AirBuddy is inhaled by the patient, enabling uptake of PrimeGuide RNA in lung epithelial cells via endocytosis.
</figcaption>
</figure>
@@ -1150,7 +1150,7 @@ export function Engineering() {
<div className="box" >
<p id="del5">
<H3 text="Outlook" id="del5head" />
- Ultimately, through continuous cycles of experimentation, feedback, and optimization, a LNP formulation called AirBuddy was designed using SORT LNPs and a spray-drying process, achieving lung specificity and improved safety. We also want to state that for our LNP is further room for improvement. Intensive research led us to the realization that, among other modifications, <strong>antibody conjugation</strong> as a surface modification of our LNP for cell type-specific administration, more specifically club cells<TabScrollLink tab="tab-delivery" num="9" scrollId="desc-9"/> and ionocytes<TabScrollLink tab="tab-delivery" num="10" scrollId="desc-10"/> as most CFTR-expressing lung epithelial cells, would round off our most important aspect of precision.
+ Ultimately, through continuous cycles of experimentation, feedback, and optimization, our LNP formulation called AirBuddy was designed using SORT LNPs incorporating chitosan-complexation of the cargo and processing via spray-drying, achieving lung-specificity and improved stability suited for inhalation strategies. We also want to state that for our LNP is further room for improvement. Intensive research led us to the realization that, among other modifications, <strong>antibody conjugation</strong> as a surface modification of our LNP for cell type-specific administration, more specifically club cells<TabScrollLink tab="tab-delivery" num="9" scrollId="desc-9"/> and ionocytes<TabScrollLink tab="tab-delivery" num="10" scrollId="desc-10"/> as most CFTR-expressing lung epithelial cells, would round off our most important aspect of precision.
</p>
</div>
<Section title="References" id="references">
diff --git a/src/contents/methods.tsx b/src/contents/methods.tsx
index 7c46a2df7b2c57bad0ff08424fd65752e1b01b0e..657dfb385b7df188efc1f8784434abebfe2cfa33 100644
--- a/src/contents/methods.tsx
+++ b/src/contents/methods.tsx
@@ -22,14 +22,14 @@ export function Methods() {
<p>Patch clamp recording involves the use of a glass micropipette which is manufactured from a glass capillary through the use of a Micropipette Puller. The micropipette is then filled with an electrolyte solution, which is subsequently brought into contact with the cell membrane. By applying gentle suction, a high-resistance seal called giga seal is formed between the pipette tip and the membrane patch. This enables the measurement of ionic currents with minimal noise interference [3]. <strong>Whole-Cell Configuration</strong> records currents from the entire cell by rupturing the membrane patch, accessing the intracellular environment, and is useful for analysing overall ion channel activity and cellular responses. <strong>Single-Channel Recording</strong> measures currents through individual ion channels without rupturing the membrane, enabling high-resolution study of channel conductance, gating, and selectivity [2].</p>
<figure>
<iframe title="Bielefeld-CeBiTec: Patch Clamp Measurement (2024)" width="560" height="315" src="https://video.igem.org/videos/embed/0d948e57-5997-430a-a2df-815b71a2fc67?autoplay=1" frameBorder="0" allowFullScreen={true} sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
- <figcaption> <b>Figure 1.</b> Microscopic recording of micropipette sealing of a HEK293 cell </figcaption>
+ <figcaption> <b>Figure 1. </b> Microscopic recording of micropipette sealing of a HEK293 cell. </figcaption>
</figure>
<p>The success of patch clamp experiments heavily depends on the composition of the solutions used. Typically, two main types of solutions are employed: The <strong>Pipette Solution</strong> in the micropipette mimics the intracellular environments, while the <strong>Bath Solution</strong> surrounds the cell and usually contains components that replicate the extracellular environment. Both solutions are meticulously designed to reflect the physiological conditions under which the cells operate, thereby ensuring that the measurements accurately reflect ion channel activity in a natural setting [2].</p>
<figure>
<img src="https://static.igem.wiki/teams/5247/photos/for-wiki-texts/meth-patch-clamp/bild-meth-patch-clamp.png" alt="Patch clamp setup"/>
- <figcaption><b>Figure 2.</b> Patch clamp setup</figcaption>
+ <figcaption><b>Figure 2. </b> Patch clamp setup.</figcaption>
</figure>
</Subesction>
<Subesction title="Application in CFTR gene prime editing validation" id="Patch Clamp3">
@@ -42,7 +42,7 @@ export function Methods() {
<p>For testing our prime editing approach, we needed an easy-to-handle cell line with a measurable high expression of CFTR and the CFTR F508del mutation. When talking to Mattijs Bulcaen from the Laboratory of Molecular Virology and Gene Therapy at KU Leuven, he recommended to use HEK293T cell lines overexpressing CFTR they had used. HEK293 cells are a very common immortalized human cell line derived from the kidneys of a female embryo. They are particularly suited to research due to their convenient handling and transfection properties. Basic HEK293 cells were provided to us by the Cellular and Molecular Biotechnology working group at Bielefeld University led by Prof. Dr. Kristian Müller, who is also one of the Principal Investigators of our team. HEK293T cells express an additional tsA1609 allele of the SV40 large T-antigen, allowing for replication of vectors containing the SV40 origin of replication[5]. Besides the native CFTR gene, which is not expressed in HEK cells, the HEK293T cell lines used in Leuven carry another copy of the gene embedded in an expression cassette. The cassette includes a CMV promoter, which is a standard promoter used for gene overexpression in human cells derived from the human Cytomegalovirus[6], as well as a puromycin resistance co-expressed with the CFTR allowing for continuous selection of CFTR expressing cells. The whole construct was stably inserted into the genome using lentiviral transduction[7][8]. </p>
<figure>
<img src="https://static.igem.wiki/teams/5247/photos/for-wiki-texts/meth-used-cells/mikroskopie-hek293t.png" alt="Phase contrast image of HEK293T at 20x magnification"/>
- <figcaption> <b>Figure 3.</b>Phase contrast image of HEK293T at 20x magnification</figcaption>
+ <figcaption> <b>Figure 3. </b>Phase contrast image of HEK293T at 20x magnification.</figcaption>
</figure>
</Subesction>
<Subesction title="CFBE41o- cell line" id="Cell Culture2">
@@ -79,7 +79,7 @@ export function Methods() {
<img src="https://static.igem.wiki/teams/5247/integrated-human-practices/mttassay.webp" alt="PC1" style={{maxHeight: "200pt"}}/>
<figcaption>
<b>Figure 6. </b>
- MTT Assay: formation of purple formazan crystals by living cells.
+ MTT Assay: Formation of purple formazan crystals by living cells.
</figcaption>
</figure>
</div>
@@ -115,7 +115,7 @@ In addition to Cryo-EM, we employed scanning electron microscopy (SEM) to furthe
<img src="https://static.igem.wiki/teams/5247/delivery/plasmatem.webp" alt="PC1" style={{maxHeight: "200pt"}}/>
<figcaption>
<b>Figure 7. </b>
- Sample preparation for SEM: sputtering in Argon plasma.
+ Sample preparation for SEM: Sputtering in Argon plasma.
</figcaption>
</figure>
</div>