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{% extends "layout.html" %}
{% block title %}Human Practices{% endblock %}
{% block lead %}How the world affects our work and how our work affects the world.{% endblock %}
......@@ -290,7 +290,7 @@
<h5>
Account Manager in Promega
</h5>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/17.webp" alt="Faustin Mbuyi" class="profile-img">
<img src="https://static.igem.wiki/teams/5342/images/human-practices/18.webp" alt="Faustin Mbuyi" class="profile-img">
</div>
</div>
<div>
......@@ -367,18 +367,18 @@
<p>
</p>
<div class="center">
<h5>
iGEM 2024 team Linköping
</h5>
<h5>
iGEM 2024 team Bielefeld
</h5>
<h5>
iGEM 2024 team Patras
</h5>
<h5>
iGEM 2024 team Termosz, Hungary
</h5>
<h5>
iGEM 2024 team Linköping
</h5>
<h5>
iGEM 2024 team Bielefeld
</h5>
<h5>
iGEM 2024 team Patras
</h5>
<h5>
iGEM 2024 team Termosz, Hungary
</h5>
</div>
<div class="center"><img src="https://static.igem.wiki/teams/5342/images/human-practices/27.webp" alt="Lipid Delivery System Handbook" class="med-img"></div>
<p>
......@@ -402,4 +402,264 @@
</div>
{% endblock %}
<div>
<h2>Creating a project with an innovative research idea while meeting requirements of iGEM</h2>
</div>
<div>
<h3>
FINDING A PROJECT TOPIC (SEPTEMBER, 2023 - FEBRUARY, 2024)
</h3>
<p>
Brainstorming our project ideas was not easy, and to answer the question: “Which challenge is iGEM Radboud 2024 going to take on?” During the multiple brainstorm sessions, and getting feedback from <i><b> Dr. Luc-Jan Laarhoven, Dr. Tom Bloemberg, and ing. Frank Nelissen, we have cast away research topics such as</b></i> glowing UV mushrooms detector, milk spoiling detector, or mold detector, and stuck with the topic of Haemophilia.
When we first started our research, we wanted to target the research of aptamers in Haemophilia patience - an immune response of the people who start to get treated against Haemophilia disease.
</p>
</div>
<div>
<h5>
Sanna Rijpma
</h5>
<h5>
Clinical Chemist at Radboudumc
</h5>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/29.webp" alt=" Sanna Rijpma" class="profile-img">
<p>To answer the question: “Where do we start with the research on aptamer?”, we decided to consult with an expert from Radboud University Clinical Center on Haemophilia A (HemA) disease, Sanna Rijpma, and we have received some useful insight into HemA and the advice on the further project development.
As it turned out, a treatment to aptamers is to develop an immune resistance under constant control of the doctors, and since we wanted to create a patient friendly system with which they would not have to leave the comfort of their home, we focused on developing an innovative treatment method of the Haemophilia disease - InfinitiF∞.
</p>
<h3>
RESEARCH PLANNING AND BRAINSTORMING (APRIL, 2024 - JUNE, 2024)
</h3>
<p>
When it came to a step of brainstorming the research procedure, our team received a lot of feedback from Tom Bloemberg (Provided planning for the LNP part, and insights into analytical techniques regarding LNPs) who provided an insight of the importance of elaborate research planning. It took us several attempts to improve our research planning to get Tom’s and Frank Nelissen (Provided planning for the mRNA part, and insights into analytical techniques regarding mRNA) approvals and access into the student labs. The planning involved planning of the research timeline, procedures, and scientific model selection, since the budget of our team was limited. But in the end, we came up with the idea of the following research plan:
Radboud iGEM Team Research Planning.
<a href="https://static.igem.wiki/teams/5342/documents/human-practices/radboud-igem-team-research-planning.pdf" target="_blank"> Radboud iGEM Team Research Planning </a>
<!--Uploaded as PDF doc, can you link that one?-->
</p>
<h5>
Luuk van Summeren
</h5>
<h5>
Employee of the Molecular Sciences Practical Training at Radboud University
</h5>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/30.webp" alt=" Luuk van Summeren" class="profile-img">
<p>
In the midway of our research progress, we encountered a problem of our expected lipid materials for LNPs to be too expensive for our budget, for which we consulted Luuk van Summeren who gave us insight into our planning, as well as into our ionizable lipid synthesis results: he suggested to analyze NMR more precisely to make sure that the compound that we look for is correct, and if the results are not very precise to conduct MS analysis as well as elemental analysis. On the matter of lipid particles, he agreed with Tom Bloemberg that a model system of LNPs is necessary, and suggested that we contact Moussa Boujemaa on the following matter to get a better insight on the matter of LNP structure. After all of the acquired feedback, our team came up with the following alternative model compared to the LNPs from the original reference article [1]: LNP material choice.
<a href="https://static.igem.wiki/teams/5342/documents/human-practices/lnp-material-choice.pdf" target="_blank"> lipid materials for LNPs </a>
<!--Uploaded as PDF doc, can you link that one?-->
</p>
<h5>
Moussa Boujemaa
</h5>
<h5>
PhD candidate - Systems Chemistry Department
</h5>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/31.webp" alt=" Moussa Boujemaa" class="profile-img">
<p>
And so, the next person who improved our insight into LNP formulation was Moussa Boujemaa. With his vast knowledge in this field of research, he provided us with useful insight into the LNP preparation and practical tips when we still were setting up the research of lipid carriers.
</p>
<h5>
Dr. Kevin Neumann
</h5>
<h5>
Assistant Professor at Systems Chemistry Department, Radboud University
</h5>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/32.webp" alt=" Dr. Kevin Neumann" class="profile-img">
<p>
Kevin Neumann, also from the Systems Chemistry Department of our University, shared his insight into the LNP preparation, practical tips when we still were setting up the research of lipid carriers.
</p>
<h5>
Remi Peters
</h5>
<h5>
Intern at Radboud University
</h5>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/33.webp" alt=" Remi Peters" class="profile-img">
<p>
Remi Peters provided us with insight into the types of LNPS analysis and characterization, when we still were setting up the research of lipid carriers.
</p>
<h5>
Kevin Venrooij
</h5>
<h5>
PhD student at Synthetic Organic Chemistry, Radboud University
</h5>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/34.webp" alt="Kevin Venrooij" class="profile-img">
<p>
Kevin Venrooij, with his specialization being alternative treatment of autoimmune diseases, reactive b cells, t cells, chemically made peptides, and peptide targeting for certain receptors, provided us with practical tips when setting up research, insight into the cost calculation and time estimation for the LNP formulation procedure, when we still were setting up the research of lipid carriers. He especially made us consider potential immune responses of the patient on the external LNPs.
</p>
<p>
<b><i>Gert Jan Ettema and Robert de Boer</i></b>
</p>
<p>
Gert Jan Ettema and Robert de Boer provided us with an insight on our plan, and research tracking - they suggested a table tracking method which we applied for our project.
On the matter of research, they challenged our opinion, and made us decide on using cheap cells, like HeLa or HEK cells (which we used in the end), and not LSEC cells which are very rare and expensive.
They gave us an insight on the ethanol injection procedure which we used in the end, and suggested on the future plan of action if we were to do the nasal spray excipients: to work on its formulation, dosage, and apply a single use spray pack to avoid side-effects of the overdose.
They also suggested that we analyze LNPs with TEM, DLS machine (size and Z-potential), and encapsulation efficiency, all of which we applied in the end.
We got an insight from them which we applied that the mRNA degradation can be prevented by reducing pH, and storing them in solution with alcohol and aqueous phase.
</p>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/35.webp" alt="Gert Jan Ettema and Robert de Boer" class="profile-img">
<h5>
Zainab Javed
</h5>
<h5>
PhD candidate - Physical Organic Chemistry
</h5>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/36.webp" alt="Zainab Javed" class="profile-img">
<h5>
Dr. Wojciech Lipiński
</h5>
<h5>
Postdoctoral researcher at Radboud University
</h5>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/37.webp" alt="Wojciech Lipiński" class="profile-img">
<p>
<b><i>Zainab Javed and Wojciech Lipiński</i></b> provide us with an answer to the following topics (questions were brainstormed together with <b><i>Tom Bloemberg</i></b>): Cheapest and most accessible lipid stocks (we found DSPE PEG2000 to be the most suitable candidate), any common mistakes in the LNP synthesis procedure (sonication of lipids before use helps to break down the aggregates that they form), a possibility of not including mRNA in LNPs (it is possible not to add mRNA to the aqueous phase), common analysis of LNPs (DLS, potentially TEM), the length of procedure for LNP formulation (1 day), storage of LNPs (Lipid stock: -20; Lipids: RT; LNPs: -80).
Not only that, but they provided us with the necessary lipids from their lab for our experiments, which we are very grateful for.
</p>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/38.webp" alt="Wojciech and Zainab" class="profile-img">
<h5>
Merlijn van Haren
</h5>
<h5>
PhD candidate at Radboud University
</h5>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/39.webp" alt=" Merlijn van Haren" class="profile-img">
<p>
In out time of struggle with finding the right equipment for the LNP purification, we received invaluable help from Merlijn van Haren, who provided us with insight into dialysis membranes and LNP purification techniques, as well as invaluable help with provision of necessary lab equipment - dialysis tubes, all of which we applied in our further research procedure.
</p>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/40.webp" alt=" Merlijn van Haren" class="small-img">
</div>
<div>
<h3>
LABORATORY WORK (MAY - SEPTEMBER, 2024)
</h3>
<p>
During our lab work, we have met multiple complications and were puzzled by many questions such as: where do I store this material, how is it better to deal with this chemical, how should I do this analysis, “what do I see on the gel?!”. With these and many more things we have received a lot of help and support from <b><i>Frank Nelissen, our iGEM instructor</i></b>, who was there to support us by answering these questions,supervised wet lab work in the mRNA production part of our research, and provided critical feedback along the way.
We not only had the struggles with analysis of the results, but also with constantly getting access to university facilities and laboratories, for which we were lucky to address <b><i>our advisor, Tom Bloemberg</i></b>, who was there to not only give us access, but to also provide his critical thinking to support us on our planning and decision making regarding the acquisition of certain materials. Tom also helped us a lot by supervised wet lab work in the LNP production part of our research.
</p>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/41.webp" alt=" Tom Lab" class="profile-img">
<p>
We have received a lot of support not only from Frank and Tom, but also from the other staff members of the <b><i>Radboud University Student Lab facility</i></b>. They provided us with access to university facilities and helped us with multiple other things.
<b><i>Luuk van Summeren</i></b> was there to help us with the brainstorming of our laboratory work planning; technical training and insight in laboratory work, help with getting needed supplies for the lab. Luc provided us with an insight of how to use Fluorescence Plate Reader, which we used to do the analysis for mRNA encapsulation efficiency of our LNPs.
</p>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/42.webp" alt="Luuk Lab" class="small-img">
<h5>
Pepijn Geutjes
</h5>
<h5>
Amanuensis Molecular Sciences
</h5>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/43.webp" alt=" Pepijn Geutjes" class="profile-img">
<p>
Pepijn Geutjes provided us with his Insight in sonication procedure, as well as explaining to us the COSY-NMR analysis. He operated the NMR machine from which we received data required to analyze the ionizable lipids in our research.
</p>
<h5>
Michel Giesbers
</h5>
<h5>
Education assistant and warehouse employee
</h5>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/44.webp" alt=" Pepijn Geutjes" class="profile-img">
<p>
Michel provided us with the needed glassware for our research and gave us insight into the better option of glassware for a certain experiment.
</p>
<h5>
Ishani Bhattacharya
</h5>
<h5>
Supervisor, Synthetic Chemistry Labs
</h5>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/45.webp" alt=" Ishani Bhattacharya" class="profile-img">
<p>
Ishani provided us with the needed support and encouragement during the lab work, as well as answering our immediate questions in the lab which helped us to keep up the pace when performing the experiments in the lab.
</p>
<h5>
Rob Mesman
</h5>
<h5>
Staff Scientist - Microbiology
</h5>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/46.webp" alt=" Rob Mesman" class="profile-img">
<p>
Rob Mesman conducted the TEM analysis for our team, and provided his insight into what results we see after the analysis of the LNPs and transfection.
With his input we understood that it is important to add lipids into the aqueous phase upon mixing during the ethanol injection, and that it is important to have higher concentration of lipids, since otherwise they mostly form lipid clusters.
</p>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/47.webp" alt=" Rob Mesman2" class="small-img">
<h5>
Dustin van Doeselaar
</h5>
<h5>
PhD candidate at the Systems Chemistry department of Radboud University
</h5>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/48.webp" alt=" Dustin van Doeselaar" class="profile-img">
<p>
With invaluable help of Dustin van Doeselaar, we have received an insight into the DLS analysis method, since Dustin provided us with a special DLS machine training and helped us to get an access to the DLS machine booking system.
Dustin gave us his opinion on our analysis results, such as behavior of correlogram and the correct analysis mode, which helped us to reach a more certain conclusion about our research results.
</p>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/49.webp" alt=" Dustin van Doeselaar2" class="profile-img">
<h5>
Martin Emmaneel
</h5>
<h5>
PhD candidate - Biophysical Chemistry
</h5>
<img src="https://static.igem.wiki/teams/5342/images/human-practices/50.webp" alt=" Martin Emmaneel" class="profile-img">
<p>
Finally, with the invaluable help of Martin Emmaneel, we managed to perform transfection of our LNPs and mRNA, and with his reference GFP we received useful insight that the targeting and LNP procedure should be improved in the future.
</p>
</div>
<div>
<h4>
We are convinced that by performing multiple collaborations within, and from the people outside of Radboud University, we made the iGEM competition more renown not only among the students and administrations of Radboud, but also among multiple Radboud research groups. This, in the future of other Radboud iGEM Teams will make their path to success easier, since they will receive more help and support from all the members of Radboud community, which will also unite our university in a creative project on a way to develop innovative solutions for improving lives of many across the world!
</h4>
<div class="center">
<img src="https://static.igem.wiki/teams/5342/images/human-practices/52.webp" alt=" Team" class="med-img">
</div>
</div>
<div>
<h3>
References:
</h3>
<p>
1. M. Kim et al.,Engineered ionizable lipid nanoparticles for targeted delivery of RNA therapeutics into different types of cells in the liver.Sci. Adv.7,eabf4398(2021).DOI:10.1126/sciadv.abf4398
</p>
</div>
{% endblock %}
\ No newline at end of file
wiki/pages/measurement.html
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{% block lead %}Synthetic Biology needs great measurement approaches for characterizing parts, and efficient new methods for characterizing many parts at once. Describe your measurement approaches on this page.{% endblock %}
{% block page_content %}
<style>
h3 {
color: #730e04;
/* font-weight: bold; */
font-size: 36pt;
margin-top: 3vh;
}
h4 {
color: #e3000b;
font-size: 20pt;
}
h5 {
color: #e3000b;
font-size: 18pt;
margin-left:2vw;
}
p {
font-size: 15pt;
text-align: justify;
}
.list-empty li {
list-style: none;
font-size: 15pt;
}
.list-show li {
font-size: 15pt;
}
.med-img {
width:auto;
height:auto;
max-width: 60%;
}
.small-img {
width:auto;
height:auto;
max-width: 40%;
}
.profile-img {
width:auto;
height:auto;
max-width: max(20%,100px);
}
.center {
display: flex;
justify-content: space-evenly;
}
</style>
<div style="margin: 2vh auto; height: 1200px; width: 850px; max-height: 85vh; max-width:55%">
<iframe src="https://static.igem.wiki/teams/5342/documents/plan-b/best-measurement-2.pdf" title="Best Measurement" allow="fullscreen" style="height: 1200px; width: 800px; max-height:100%; max-width:100%"></iframe>
<div>
<h2>OVERVIEW</h2>
</div>
<div>
<h3>Measurements in our project</h3>
<p>The correct measurement is a proper recording of the data which can be applied to interpret the result. Any measurement rarely comes alone, but rather as a combination of measurements from different analysis methods.
We used a combination of 3 analysis methods to get a clear picture of the behavior of our ionizable lipid nanoparticles (LNPs): dynamic light scattering (DLS), and encapsulation efficiency.
The LNPs described by Kim et al [1] had a size of 60 - 100 nm, and composed of the following materials:
</p>
<h4>Materials used for LNPs
</h4>
<table>
<tr>
<td>Compound</td>
<td>Total mass of all compounds, mg</td>
</tr>
<tr>
<td>Ionisable lipids</td>
<td>85</td>
</tr>
<tr>
<td>1,4-Bis(3-aminopropyl) piperazine</td>
<td>17</td>
</tr>
<tr>
<td>1,2-epoxydecane</td>
<td>68</td>
</tr>
<tr>
<td>DOPE</td>
<td>0.12</td>
</tr>
<tr>
<td>Cholesterol</td>
<td>67</td>
</tr>
<tr>
<td>DSPE-PEG 2000 </td>
<td>0.03</td>
</tr>
</table>
<p>
were analyzed for their size, zeta potential, encapsulation efficiency, while checking their integrity with Transmission Electron Microscopy. This combination of analysis is what provides a full insight on how a single LNP and an LNP solution composition looks.
</p>
</div>
<div>
<h3>LNP Preparation procedure</h3>
<h4>Prepare Nucleic Acid Solution
</h4>
<ul>
<li> Prepare 10 mL of 10 mM citrate buffer at pH 4.0.
<li> Dissolve 3 mg of nucleic acid (e.g., mRNA) in 3 mL of the citrate buffer.
</ul>
<h4>Lipid Nanoparticle Formation
</h4>
<h5>
Ethanol Injection:
</h5>
<img src="https://static.igem.wiki/teams/5342/images/best-measurement/1-1.webp" alt="Fig 1" class="med-img">
<p>
<b>Figure 1.</b> Ethanol injection procedure [2]
</p>
<ul>
<li> Slowly inject the 1 mL ethanol-lipid solution into 3 mL of the citrate buffer containing the nucleic acid (1:3 volume ratio, which can be adjusted for the experiment's needs), while stirring the buffer rapidly.
<li> The rapid mixing of the ethanol-lipid solution with the aqueous phase leads to the spontaneous formation of lipid nanoparticles.
</ul>
<h5>
Equilibration:
</h5>
<ul>
<li> Continue stirring for 5-10 minutes to allow the system to equilibrate and ensure complete nanoparticle formation.
</ul>
<h4>
Equilibration:
</h4>
<ul>
<li> Use a dialysis machine to remove the ethanol solvent (procedure description: [3], see video)
</ul>
<h4>
Analysis
</h4>
<ul>
<li> Dynamic light scattering(DLS)
<li> Encapsulation efficiency
<li> Transmission Electron Microscopy (TEM)
</ul>
<h4>
Storage
</h4>
<ul>
<li> Without mRNA: RT
<li> LNPs with mRNA store at: -80 C
</ul>
<h4>
Produced LNP batches
</h4>
<p>Batches (see in Notebook section for the referenced experiments):</p>
<ul>
<li>Batch1 - trial LNPs containing torula yeast RNA from experiment RAD202</li>
<li>Batch2 - trial LNPs containing torula yeast RNA from experiment RAD203</li>
<li>Batch3 - trial LNPs containing torula yeast RNA from experiment RAD204</li>
<li>Batch4 - model LNPs containing F8 and GFP mRNA from experiment RAD205</li>
<li>Batch5 - model LNPs containing F8 and GFP mRNA from experiment RAD206</li>
</ul>
</div>
<div>
<h2>OUR MEASUREMENTS</h2>
</div>
<div>
<h3>Dynamic light scattering (DLS)</h3>
<h4>Background
</h4>
<p>
DLS is a technique used in molecular sciences to measure the average size and size distribution of nanoparticles. It is quick, non-invasive, and requires minimal sample preparation. The spectrometer machine produces a laser beam targeted toward the measured sample. Due to the Brownian motion, particles will move at different rates, and the light from the laser beam will be scattered at different intensities, while the spectrometer measures these fluctuations.[5]
Zeta potential is a measure of the dispersion of the particles in a solution. A high (positive or negative) zeta potential means that the particles are dispersed in solution, while a potential close to zero signifies that particles are likely to aggregate. Positive Zeta potential indicates that the particles in solution are positively charged, and negative Zeta indicates vice-versa.
</p>
<h4>Results and Discussion
</h4>
<h4>Batches 1 and 2
</h4>
<img src="https://static.igem.wiki/teams/5342/images/best-measurement/2-2.webp" alt="fig 2" class="med-img">
<p>
<b>Figure 2.</b> DLS analysis of Batch 1 (a) and Batch 2 (b). Size measurement; PEG2000 material mode; Dispersant PBSx10.
</p>
<table>
<tr>
<td>Z-Average (nm)</td>
<td></td>
<td></td>
</tr>
<tr>
<td>LNP Batch</td>
<td>Batch 1 (30/08/24)</td>
<td>Batch 2 (11/09/24)</td>
</tr>
<tr>
<td>Measurement 1</td>
<td>150.5</td>
<td>208.9</td>
</tr>
<tr>
<td>Measurement 2</td>
<td>133.1</td>
<td>232.4</td>
</tr>
<tr>
<td>Measurement 3</td>
<td>125.8</td>
<td>243.5</td>
</tr>
<tr>
<td>Average</td>
<td>136.5</td>
<td>223.5</td>
</tr>
</table>
<p>
<b>Table 1.</b> Overview of size measurements of Batch 1 and Batch2 of LNPs; PEG2000 material mode; Dispersant PBSx10.
</p>
<img src="https://static.igem.wiki/teams/5342/images/best-measurement/3-1.webp" alt="Fig 3" class="med-img">
<p><b>Figure 3.</b> DLS analysis of Batch 1 (a,b) and Batch 2 (c,d). Z potential; PEG2000 material mode; Dispersant PBSx10.</p>
<table>
<tr>
<td>Z-Average (nm)</td>
<td></td>
<td></td>
</tr>
<tr>
<td>LNP Batch</td>
<td>Batch 1 (30/08/24)</td>
<td>Batch 2 (11/09/24)</td>
</tr>
<tr>
<td>Measurement 1</td>
<td>8.062</td>
<td>-9.37</td>
</tr>
<tr>
<td>Measurement 2</td>
<td>-0.5397</td>
<td>3.651</td>
</tr>
<tr>
<td>Measurement 3</td>
<td>0.009314</td>
<td>6.353</td>
</tr>
<tr>
<td>Average</td>
<td>2.51</td>
<td>0.2119</td>
</tr>
</table>
<p><b>Table 2.</b> Overview of Batch 1 (a,b) and Batch 2 (c,d). Z potential; PEG2000 material mode; Dispersant PBSx10.</p>
<h4>Analysis of Batches 1 and 2
</h4>
<h5>Size (Z-average)</h5>
<p>
Every measurement should be evaluated. So did we with our measurement of Batch 1 and Batch 2. Both LNP Batches had a size of just over 100 nm (Figure 2a-b, Table 1), which is larger than their size in the reference [1]. This can be attributed to our mixing method, which was less precise than their microfluidics. Interestingly, Batch 2 (Figure 2b) shows an increase in LNPs with a diameter of 10,000nm. This was the result of LNP aggregation, which happens if LNPs are not sonicated before analysis.
</p>
<h5>Z potential</h5>
<p>
The zeta potential described in the article by Kim was -0.997 mV [1]. We measured a different, highly positive zeta potential (Figure 3, Table 2). A zeta potential that deviates from the literature would mean that our LNPs have different interactions with each other which could also change how they interact with cells.
</p>
</div>
<!-- Stopped here -->
<div>
<h4>Batch 3 </h4>
<!--Figure4-->
<p>
<b>Figure 4.</b> DLS analysis of Batch 3. Size measurement; Liposome material mode; Dispersant PBSx1.
</p>
<!--Table4-->
<p>
<b>Table 3.</b> Overview of size measurement of Batch 3; Liposome material mode; Dispersant PBSx1.
</p>
<!--Figure5-->
<p>
<b>Figure 5.</b> DLS analysis of Batch 3. Z potential; Liposome material mode; Dispersant PBSx1
</p>
<!--Table5-->
<p>
<b>Table 4.</b> Overview of Z-potential measurement of Batch 3; Liposome material mode; Dispersant PBSx1.
</p>
<h4>Analysis of Batch 3 </h4>
<h5>Size (Z-average)</h5>
<p>
Evaluation for Batch 3 gave the following results. Again, a size of just over 100 nm (Figure 4, Table 3) was observed. It is assumed that it not only can be attributed to our mixing method but also to the lipid composition which is different from the reference [1].
</p>
<h5>Z potential</h5>
<p>
To address the unusual Zeta potential of Batches 1 and 2, Batch 3 of LNPs was made in 1x PBS buffer instead of 10X PBS. The average zeta potential was still measured at 7.156 mV, and it was concluded that a change in buffer does not impact the zeta potential (Figure 5, Table 4). Rather, the difference in zeta potential from the source was due to the component substitutions we made.
</p>
<h4>Batch 4 </h4>
<!--Figure6-->
<p>
<b>Figure 6.</b>Analysis of Batch 4. Size measurement; Liposome material mode; Dispersant PBSx10 (a) LNPs with GFP mRNA, (b) LNP with no mRNA
</p>
<!--Table6-->
<p>
<b>Table 5.</b>Overview of size measurements of Batch 4 LNPs; Liposome material mode; Dispersant PBSx10.
</p>
<!--Figure7-->
<p>
<b>Figure 7.</b> Analysis of Batch 4. DLS analysis of Batch 4. Z potential; Liposome material mode; Dispersant PBSx10 (a) LNPs with GFP mRNA, (b) LNP with no mRNA
</p>
<!--Table7-->
<p><b>Table 6.</b> Overview of Z-potential measurement of Batch 4; Liposome material mode; Dispersant PBSx10.
</p>
<h4>Analysis of Batches 1 and 2
</h4>
<h5>Size (Z-average)</h5>
<p>
Evaluation for Batch 4 gave the following results. DLS revealed that the size of the LNPs containing GFP mRNA was 1000nm, way higher than it should be (Figure 6a, Table 5). The negative control LNPs without RNA had a slightly larger size, again a result of aggregation since the sample had not been sonicated before analysis (Figure 6b, Table 5).
</p>
<h5>Z potential</h5>
<p>
The average zeta potential was still measured at high (Figure 7, Table 6), and the conclusion on why this is the case was received during the previous Batch 3 measurement.
</p>
<h4>Batch 5</h4>
<!--Figure8-->
<p>
<b>Figure 8.</b>DLS size measurement of Batch 5, Liposome material mode; Dispersant PBSx10. (a) negative control LNPs, no mRNA; (b) positive control LNPs, control GFP mRNA; (c) GFP2 LNPs; (d) GFP1 LNPs; (e) 5xVF8-2 LNPs; (f) F8-1 LNPs.
</p>
<!--Table7-->
<p>
<b>Table 7.</b>Overview of size measurements of Batch 5; Liposome material mode; Dispersant PBSx10.
</p>
<h4>Analysis of Batch 5</h4>
<h5>Size (Z-average)</h5>
<p>
DLS size analysis showed that the prepared LNPs did not have the desired size, most had a size between 1 and 10 nm or a size between 100 and 5000 nm (Figure 8, Table 7).
Thus, it was assumed that the mixing procedure for making the LNPs was not suitable for forming right LNPs of the needed size, this will be further checked with the TEM measurement.
</p>
<h5>Z potential</h5>
<p>
No Zeta potential was measured.
</p>
</div>
{% endblock %}
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