(L-477) Characterization of a cost-effective lipid nanoparticle synthesis system to enable research and education

Nanotechnology has made significant progress in recent decades and gained prominence in the scientific community, especially with its application in COVID-19 vaccines. These vaccines utilize lipid nanoparticles (LNPs) as carriers to deliver the virus's genetic information, enabling the body to mount an effective defense against the virus. LNPs consist of a lipid mixture containing an oligonucleotide cargo. The lipid mixture comprises an ionizable cationic lipid and three helper lipids, typically including a glycerophospholipid, a PEGylated lipid, and a sterol lipid. The ionizable lipid serves the crucial role of avoiding cytotoxicity since its near-neutral charge at physiological pH allows cargo delivery without harmful effects. The glycerophospholipid enhances the efficiency of membrane fusion, aiding in the effective delivery of the cargo. On the other hand, the PEG prevents serum protein adsorption and hinders uptake by the mononuclear phagocyte system. Lastly, sterol is employed to fill lipid membrane packing defects and ensure structural integrity. The development of a standardized, cost-efficient, and accessible LNP production protocol would have significant implications for future research in education. Various methods exist for creating lipid nanoparticles, with the most common approaches involving microfluidic chip-based techniques, vortex mixing, or pipette mixing. In the microfluidic chip method, individual streams of the lipid and DNA mixtures are rapidly combined, often using a syringe pump to control flow rate and ratio. Pipette mixing entails adding the lipid mixture into the DNA mixture and employing rapid pipetting. Vortex mixing involves mixing the lipid and DNA mixtures using a vortex instead of rapid pipetting.

The syringe pump and microfluidic chip method were selected over other techniques due to their superior controllability, reproducibility, homogeneity, and high encapsulation efficiency. Despite being more costly compared to vortex or pipette mixing methods, these advantages justified the decision to use this approach. . Three lipid nanoparticles (LNPs) were created using a lipid component consisting of an ionizable lipid and helper lipids, along with a DNA component.

The helper lipids DSPC, DMG-PEG2000, and Cholesterol were consistently used for all three LNPs as the glycerophospholipid, PEGylated lipid, and sterol lipid, respectively. However, for the ionizable lipid in the lipid component, three different variants were employed: MC3, LP01, and SM102. Triplicates were prepared for each LNP variant. The GFP DNA was obtained through a midi prep kit and served as the DNA component. The syringe pump and microfluidic chip system were set up and operated to produce the LNPs. During the process, the resulting LNPs were collected in an Eppendorf tube through the outlet. Subsequently, the LNPs underwent a two-hour dialysis process. After dialysis, the LNPs were diluted as 1:1 with PBS (phosphate-buffered saline) and subjected to various characterization tests. The Picogreen test was conducted to measure encapsulation efficiency, the Nanosight technique verified the nanoparticles' correct size, DLS (dynamic light scattering) was employed to test size and PDI (Polydispersity index), and finally, the zeta potential test determined the charge for each nanoparticle using the DLS machine with a dip cell inserted into the cuvette.

The results of the Picogreen test reveal high encapsulation efficiencies for each LNP variant. MC3 exhibited an impressive 99.9% encapsulation efficiency, LP01 showed 95.6%, and SM102 had 97.2%. These values indicate minimal unencapsulated DNA cargo within the lipid nanoparticles. Similarly, the DLS test demonstrated favorable average sizes for the LNPs. MC3 had an average size of 147.7 nm, LP01 measured 154.5 nm, and SM102 showed 137.3 nm. These results infer that the LNPs are in the nanometer region which is a crucial characteristic for their optimal functionality and efficient cellular uptake. Furthermore, the PDI values obtained from the DLS test are commendably low for all three LNP variants. An average PDI of 0.1499 for MC3, 0.1371 for LP01, and 0.1267 for SM102 indicates a relatively uniform size distribution among the LNPs, enhancing their stability and consistency. However, the zeta potential results from the zeta test were not as expected. MC3 exhibited an average charge of -20.8 mV, LP01 showed -27.9 mV, and SM102 displayed -21.2 mV. The LNPs should ideally exhibit a positive charge at low pH to enable DNA complexation and then shift to a neutral charge at physiological pH to reduce potential toxicity. Unfortunately, the measured zeta potentials show negative values, which indicate an undesired charge state. Further testing is necessary to evaluate transfection efficiency and explore methods to adjust the zeta potential to a more neutral state.

In conclusion, the overall results show promising attributes for the LNPs, with high encapsulation efficiency, appropriate size in the nano range, and uniformity. The issue of zeta potential requires further investigation and optimization to achieve the desired charge profile for safe and efficient delivery of genetic cargo. Additional testing, including transfection efficiency assessment, will contribute to refining and advancing these lipid nanoparticles for potential therapeutic applications.