Measurement

Measurements in our project

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:

Materials used for LNPs

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.

LNP Preparation procedure

Prepare Nucleic Acid Solution

• Prepare 10 mL of 10 mM citrate buffer at pH 4.0.
• Dissolve 3 mg of nucleic acid (e.g., mRNA) in 3 mL of the citrate buffer.

Lipid Nanoparticle Formation

Ethanol Injection:

Figure 1. Ethanol injection procedure [2]

• 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.
• The rapid mixing of the ethanol-lipid solution with the aqueous phase leads to the spontaneous formation of lipid nanoparticles.

Equilibration:

• Continue stirring for 5-10 minutes to allow the system to equilibrate and ensure complete nanoparticle formation.

Equilibration:

• Use a dialysis machine to remove the ethanol solvent (procedure description: [3], see video)

Analysis

• Dynamic light scattering(DLS)
• Encapsulation efficiency
• Transmission Electron Microscopy (TEM)

Storage

• Without mRNA: RT
• LNPs with mRNA store at: -80 C

Produced LNP batches

Batches (see in Notebook section for the referenced experiments):

• Batch1 - trial LNPs containing torula yeast RNA from experiment RAD202
• Batch2 - trial LNPs containing torula yeast RNA from experiment RAD203
• Batch3 - trial LNPs containing torula yeast RNA from experiment RAD204
• Batch4 - model LNPs containing F8 and GFP mRNA from experiment RAD205
• Batch5 - model LNPs containing F8 and GFP mRNA from experiment RAD206

OUR MEASUREMENTS

Dynamic light scattering (DLS)

Background

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.

Results and Discussion

Batches 1 and 2

Figure 2. DLS analysis of Batch 1 (a) and Batch 2 (b). Size measurement; PEG2000 material mode; Dispersant PBSx10.

Table 1. Overview of size measurements of Batch 1 and Batch2 of LNPs; PEG2000 material mode; Dispersant PBSx10.

Figure 3. DLS analysis of Batch 1 (a,b) and Batch 2 (c,d). Z potential; PEG2000 material mode; Dispersant PBSx10.

Table 2. Overview of Batch 1 (a,b) and Batch 2 (c,d). Z potential; PEG2000 material mode; Dispersant PBSx10.

Analysis of Batches 1 and 2

Size (Z-average)

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.

Z potential

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.

Batch 3

Figure 4. DLS analysis of Batch 3. Size measurement; Liposome material mode; Dispersant PBSx1.

Table 3. Overview of size measurement of Batch 3; Liposome material mode; Dispersant PBSx1.

Figure 5. DLS analysis of Batch 3. Z potential; Liposome material mode; Dispersant PBSx1.

Table 4. Overview of Z-potential measurement of Batch 3; Liposome material mode; Dispersant PBSx1.

Analysis of Batch 3

Size (Z-average)

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].

Z potential

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.

Batch 4

Figure 6.Analysis of Batch 4. Size measurement; Liposome material mode; Dispersant PBSx10 (a) LNPs with GFP mRNA, (b) LNP with no mRNA

Table 5.Overview of size measurements of Batch 4 LNPs; Liposome material mode; Dispersant PBSx10.

Figure 7. 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

Table 6. Overview of Z-potential measurement of Batch 4; Liposome material mode; Dispersant PBSx10.

Analysis of Batches 1 and 2

Size (Z-average)

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).

Z potential

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.

Batch 5

Figure 8.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.

Table 7.Overview of size measurements of Batch 5; Liposome material mode; Dispersant PBSx10.

Analysis of Batch 5

Size (Z-average)

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.

Z potential

No Zeta potential was measured.

Encapsulation efficiency

Background

Encapsulation efficiency is defined as the concentration of the free material (mRNA in this case) over the entrapped material concentration inside the LNPs in our project. This is measured with a fluorescence assay.

Results and Discussion

Dilution series with stock RNA from sample GFP3 was made for analysis of encapsulation efficiency.

Table 8.Encapsulation efficiency. Standard curve of GFP3 mRNA, concentrations.

Figure 9.Encapsulation efficiency. Standard curve of GFP3 mRNA.

Analysis Encapsulation efficiency of Batch 5

The encapsulation efficiency of the last batch of LNPs was measured by making a dilution series with known concentrations of the same RNA that is encapsulated in the LNPs. By lysing the LNPs and measuring the fluorescence with SYBR® Gold Nucleic Acid Gel Stain, the concentration of mRNA encapsulated in the LNP could be measured. In Figure 9, it can be seen that the intensity of mRNAs in the standard curve is not correlated to the decreasing concentration values of mRNA which proved to us that the SYBR Gold dye in the end did not apply to this analysis. It is important to note that the SYBR Gold dye is also usually used for analysis of DNA and not RNA [2], and was used in this case due to the lack of budget and materials.

Transmission Electron Microscopy (TEM)

Background

Transmission Electron Microscopy is an analytical technique used to visualize nanoparticles using a high-energy electron beam directed to the particles. The resolution of this microscope is far greater than the conventional light microscope due to the short wavelength of electrons compared to visible light [6].

Results and Discussion

LNPs from the Batch 4 and Batch 5 were prepared for transfection to test the properties of our ionizable LNPs in vitro, LNPs were produced containing mRNA coding for GFP. LNPs were formulated with previously prepared mRNA: GFP1, GFP2, F8 1, 5xFV F8, and GFP control. Control GFP samples were borrowed from Martin Emmaneel.

TEM of Batch 4

Figure 10.TEM Figures: (c) TEM pictures of LNPs205 LNPs with GFP mRNA show that no encapsulation occurred; (d) TEM pictures of LNPs205 LNPs with no mRNA show LNP formation; white line at the bottom: 100 nm.

Analysis of DLS Batch 4

Correct LNPs without mRNA inside have formed (Figure 10d). However, analysis with transmission electron microscopy (TEM) also showed that the lipids formed clusters in the aqueous phase without incorporating any of the mRNA as the result of incorrect mixing (Figure 10c). It is important to note that lipids should always be added to the aqueous phase during mixing to prevent this. It is also recommended to use other mixing methods like microfluidics that are better suited for smaller volumes.

TEM of Batch 5

Figure 11.TEM pictures of F8 1 mRNA LNP; white line at the bottom: 100 nm

Analysis of DLS Batch 5

With TEM, some LNPs could be seen, although in extremely low abundance - in total 6-10 LNPs were observed in 3 samples (Figure 11). With this, we conclude that the formulation method is not efficient. LNPs almost do not form with the ethanol injection method when using low lipid concentrations. It is recommended to use a higher concentration of lipids and implement a pulsification method or microfluidic mixing.

Conclusion:

A study of measurements, and their interconnection with each other provides a possibility to reach a conclusion on how a particle, the studied subject, looks. In this specific examples, we managed to reach some important conclusion for future research: Ethanol injection method is not efficient, especially at lower lipid concentration in solution, so pulsification is recommended to be applied during mixing or, preferably, a more expensive microfluidics method is suggested instead. However, if ethanol injection is still used, it is crucial to use higher lipid concentrations and lipids to the aqueous solution UPON mixing.

In our opinion, this constitutes the true value of measurement - not only the correct numbers, values, and units, but the useful conclusions and insights which are combined together to receive a solid background for future researchers.

Sources:

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
2. Jung, Han Na, et al. “Lipid Nanoparticles for Delivery of RNA Therapeutics: Current Status and the Role of in Vivo Imaging.” Theranostics, vol. 12, no. 17, 2022, pp. 7509–7531, https://doi.org/10.7150/thno.77259.
3. “Dialysis Methods for Protein Research | Thermo Fisher Scientific - CA.” Thermofisher.com, 2024, www.thermofisher.com/nl/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/dialysis-methods-protein-research.html#gsttagged. Accessed 29 Sept. 2024.
4. Fisher, Thermo. “SYBRTM Gold Nucleic Acid Gel Stain (10,000X Concentrate in DMSO).” Thermofisher.com, 2022, www.thermofisher.com/order/catalog/product/S11494.
5. Sciencedirect.com. [cited 2024 Sep 29]. Available from: https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/dynamic-light-scattering
6. People O. Transmission electron microscopy [Internet]. Nanoscience Instruments. [cited 2024 Sep 29]. Available from: https://www.nanoscience.com/techniques/transmission-electron-microscopy/