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)<TabScrollLinktab="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)<TabScrollLinktab="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.
In the next phase, we chose to use a new LNP formulation, namely the <strong>LNP Starter Kit #2</strong><TabScrollLinktab="tab-delivery"num="5"scrollId="desc-5"/> of <aonClick={()=>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 <aonClick={()=>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><TabScrollLinktab="tab-delivery"num="5"scrollId="desc-5"/> of <aonClick={()=>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 <aonClick={()=>goToPagesAndOpenTab('kolonkofirst','/human-practices')}>Dr. Katharina Kolonko</a>, it was determined that capsaicin was not suitable in our way of pulmonary application.
Schematic view of LNP #2 from Corden Pharma with DOTAP as cationic lipid, DSPC as phospholipid and mPEG-200-DSPE as PEG lipid.
Schematic view of LNP #2 from Corden Pharma with DOTAP as cationic lipid, DSPC as phospholipid and mPEG-2000-DSPE as PEG lipid.
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@@ -1092,7 +1092,7 @@ export function Engineering() {
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<Collapsibleid="Col1"open={false}title="Ambivalence of PEG and our implementation">
<Collapsibleid="Col1"open={false}title="Ambivalence of PEG and our implementation">
<p>
<p>
<H4text="What is PEG and why is it important for LNPs?"id="text"/>
<H4text="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>
<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>
<H4text="Why is PEG relevant for LNPs in mRNA delivery?"id="text"/>
<H4text="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.
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.
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@@ -1107,11 +1107,11 @@ export function Engineering() {
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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.
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>
</p>
</Collapsible>
</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 <aonClick={()=>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 <aonClick={()=>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>By combining these components with the spray drying method from <aonClick={()=>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 <aonClick={()=>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>
<H5text="Storage"id="text"/>
<H5text="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>
<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>
</Collapsible>
<p>The final innovation for our LNP to become <strong>AirBuddy</strong> came through consultation with Benjamin Winkeljann from <aonClick={()=>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<TabScrollLinktab="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 <aonClick={()=>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 chitosancomplexing 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 <aonClick={()=>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<TabScrollLinktab="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 <aonClick={()=>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>
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>
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@@ -1150,7 +1150,7 @@ export function Engineering() {
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@@ -1150,7 +1150,7 @@ export function Engineering() {
<divclassName="box">
<divclassName="box">
<pid="del5">
<pid="del5">
<H3text="Outlook"id="del5head"/>
<H3text="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 lungspecificity 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<TabScrollLinktab="tab-delivery"num="9"scrollId="desc-9"/> and ionocytes<TabScrollLinktab="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<TabScrollLinktab="tab-delivery"num="9"scrollId="desc-9"/> and ionocytes<TabScrollLinktab="tab-delivery"num="10"scrollId="desc-10"/> as most CFTR-expressing lung epithelial cells, would round off our most important aspect of precision.
