<p>The design path of our lipid nanoparticle (LNP) for mRNA delivery underwent multiple cycles of research and discussion, marked by important decision points and learnings along the way. By ongoing further improvement, we designed our lungs-specific LNP called AirBuddy with improved stability aspects, becoming more precise in the delivery of our therapeutic cargo LNP by LNP.</p>
<p>The design path of our lipid nanoparticle (LNP) for mRNA delivery underwent multiple cycles of research and discussion, marked by important decision points and learnings along the way. By ongoing further improvement, we designed our lungs-specific LNP called AirBuddy with improved stability aspects, becoming more precise in the delivery of our therapeutic cargo LNP by LNP.</p>
<p>Initially, this project part started with a discussion with <aonClick={()=>goToPagesAndOpenTab('kristian','/human-practices')}> Prof. Dr. Krisitan Müller</a>, PI of our team with expertise in Adeno-associated viruses (AAVs), focusing on whether to pursue LNPs or AAVs for mRNA delivery. The deciding factor leaned towards LNPs, as they offered a significant advantages including less immunogenic potential [1] and bigger loading capacity [2]. LNPs loading capacity depends on various factors, but in general they offer a bigger cargo size compared to 4.7 kb for AVVs [3]. This allows the delivery of bigger mRNA constructs compared to AAVs, which is needed for our Prime Editing construct.</p>
<p>Initially, this project part started with a discussion with <aonClick={()=>goToPagesAndOpenTab('kristian','/human-practices')}> Prof. Dr. Krisitan Müller</a>, PI of our team with expertise in Adeno-associated viruses (AAVs), focusing on whether to pursue LNPs or AAVs for mRNA delivery. The deciding factor leaned towards LNPs, as they offered a significant advantages including less immunogenic potential [1] and bigger loading capacity [2]. LNPs loading capacity depends on various factors, but in general they offer a bigger cargo size compared to 4.7 kb for AVVs [3]. This allows the delivery of bigger mRNA constructs compared to AAVs, which is needed for our Prime Editing construct.</p>
<p><aonClick={()=>goToPagesAndOpenTab('weber','/human-practices')}>Prof. Wolf-Michael Weber and Dr. Jörg Große-Onnebrink</a> from the UKM in Münster were our first point of contact for the development of our LNP for CFTR treatment. Moreover, <aonClick={()=>goToPagesAndOpenTab('radukic','/human-practices')}>Dr. Marco Radukic </a>form Bielefeld University provided us with a very useful cargo, namely minicircle DNA carrying the EYFP gene from <ahref="https://www.plasmidfactory.com/custom-dna/minicircle-dna/"title="PlasmidFactory">PlasmidFactory</a> as a positive control for our experiments. He also helped us establish protocols for LNP synthesis and LNP transfection in our lab.</p>
<p><aonClick={()=>goToPagesAndOpenTab('weber','/human-practices')}>Prof. Wolf-Michael Weber and Dr. Jörg Große-Onnebrink</a> from the UKM in Münster were our first point of contact for the development of our LNP for CFTR treatment. Moreover, <aonClick={()=>goToPagesAndOpenTab('radukic','/human-practices')}>Dr. Marco Radukic </a>form Bielefeld University provided us with a very useful cargo, namely minicircle DNA carrying the EYFP gene from <ahref="https://www.plasmidfactory.com/custom-dna/minicircle-dna/"title="PlasmidFactory">PlasmidFactory</a> as a positive control for our experiments. He also helped us establish protocols for LNP synthesis and LNP transfection in our lab.</p>
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<h3>Interation 2 - Cayman LNP</h3>
<H3text="Interation 2 - Cayman LNP"id="text"/>
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) [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) [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.
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<h3>Interation 3 - Corden LNP</h3>
<H3text="Interation 3 - Corden LNP"id="text"/>
In the next phase, we chose to use a new LNP formulation, namely the <strong>LNP Starter Kit #2</strong> [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> [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.
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<h3>Interation 4 - Spray-dried SORT LNP called Airbuddy</h3>
<H3text="Interation 4 - Spray-dried SORT LNP called Airbuddy"id="text"/>
The next design iteration incorporated the insights from Wang's LNP work for building upon SORT principles to make the nanoparticles lung-specific [6]. The main components include DMG-PEG 2000, cholesterol, DOPE and DOTAP as phospholipids and cationic lipids such as 4A3-SC8. In our LNP development, we carefully considered the use of PEG. While PEG can improve stability, it can also reduce cellular uptake and induce immune responses, necessitating a balanced approach to its inclusion [7].
The next design iteration incorporated the insights from Wang's LNP work for building upon SORT principles to make the nanoparticles lung-specific [6]. The main components include DMG-PEG 2000, cholesterol, DOPE and DOTAP as phospholipids and cationic lipids such as 4A3-SC8. In our LNP development, we carefully considered the use of PEG. While PEG can improve stability, it can also reduce cellular uptake and induce immune responses, necessitating a balanced approach to its inclusion [7].
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<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.
<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"/>
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.
<H4text="Cytotoxicity and mPEG-2000-DSPE"id="text"/>
One challenge with PEGylation is the potential for immune responses, such as the <i>accelerated blood clearance</i> (ABC) phenomenon, where repeated exposure to PEGylated particles leads to faster clearance by the immune system. There are also risks of hypersensitivity reactions like <i>complement activation-related pseudoallergy</i> (CARPA). Thus, selecting the right PEG-lipid type is essential to mitigate these risks.
<p>We collaborated with <aonClick={()=>goToPagesAndOpenTab('corden','/human-practices')}>Corden Pharma</a>, a specialist in LNP technologies, to address these concerns. Based on their recommendations, we opted for <strong>mPEG-2000-DSPE</strong> as our PEG-lipid of choice. This variant minimizes cytotoxicity while providing excellent stability and circulation time. It has also proven effective in reducing immune-related side effects while preserving the integrity and performance of our nanoparticles. </p>
<H4text="DMG-PEG2000 vs mPEG-2000-DSPE"id="text"/>
While mPEG-2000-DSPE has traditionally been used for stabilizing LNPs and enhancing delivery efficiency, we decided to incorporate DMG-PEG2000 into our SORT LNP-based AirBuddy due to its superior properties. DMG-PEG2000 offers better biodegradability and enhanced stability in pulmonary applications. Unlike mPEG-2000-DSPE, which tends to accumulate in the body and may lead to immune activation over time, DMG-PEG2000 is known for its faster clearance and reduced potential for long-term toxicity. For lung-specific delivery, where stability and safety are critical, DMG-PEG2000 ensures that the nanoparticles remain stable long enough to deliver the therapeutic material effectively, but also degrade at a rate that minimizes unwanted immune responses. This makes DMG-PEG2000 a more suitable choice for therapies targeting CFTR-related diseases, where precise and safe delivery to the lungs is essential for treatment success.
<H4text="Conclusion"id="text"/>
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.
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<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 Physical and Biophysical Chemistry working group of Bielefeld University 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 physical properties in cooperation with the Physical and Biophysical Chemistry working group of Bielefeld University to characterize the LNPs. More details about the composition of the SORT LNPs and function of the components can be read below.</p>
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<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, helped improve stability and eco-friendliness of the product [8]. The spray-dried SORT LNPs demonstrated lower cytotoxicity, and the technique proved effective in maintaining particle integrity.</p>
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Schematic view of our lung-specific SORT LNP called AirBuddy.
Schematic view of our lung-specific SORT LNP called AirBuddy.
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<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, helped improve stability and eco-friendliness of the product [8]. The spray-dried SORT LNPs demonstrated lower cytotoxicity, and the technique proved effective in maintaining particle integrity.</p>
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<h3>Outlook</h3>
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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, antibody conjugation as a surface modification of our LNP for cell type-specific administration, more specifically club cells [9] and ionocytes [11] as most CFTR-expressing lung epithelial cells, would round off our most important aspect of precision. In addition, the discussion with Benjamin Moorlach, chitosan expert working at FH Bielefeld, provided new ideas for improvement by complexing the mRNA with chitosan to improve the stability of the cargo during spray drying and nebulization.
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 [9] and ionocytes [11] as most CFTR-expressing lung epithelial cells, would round off our most important aspect of precision. In addition, the discussion with Benjamin Moorlach, 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.
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