mRNA vaccines have become excellent vaccine candidates during the COVID-19 pandemic because of their unique favorable characteristics, including their ability to generate nontoxic, effective immune responses and the potential for rapid design and scale-up production. More and more domestic and foreign companies are committed to the development of mRNA-LNP vaccines.
1. The antigen is selected according to the indication and cultured to obtain the circular plasmid. Subsequently. The linearized plasmid is obtained by enzyme digestion, and mRNA (chemically modified and purified) is synthesized by in vitro transcription, and finally the mRNA concentration is determined.
2. LNP components were screened and LNP formulations were optimized. mRNA-LNP was synthesized using microfluidic technology.
LNP generally consists of cationic lipids, cholesterol, adjuvant lipids and excipients such as PEGylated phospholipids. The composition ratio of LNP formulations was optimized to improve the cyclic stability of mRNA-LNP drugs.
Figure 1. Schematic diagram of mRNA-LNP process development.
mRNA-LNP morphology was identified using cryo-TEM. The polydispersity index PDI, zeta potential, mRNA identification and concentration, and mRNA encapsulation efficiency (%) were also determined. The quality of mRNA-LNP was characterized. (Refer to Figure 2)
Figure 2. Characterization of mRNA-LNP.1
Cellular uptake tests can be used to study endocytosis pathway. Cy5-mRNA, a model mRNA, was utilized to prepare Cy5-mRNA-LNP. Different inhibitors were co-incubated with cell, then the inhibitors were replaced with Cy5-mRNA-LNP and kept for certain hours. Finally, cells were collected and analyzed by flow cytometry. The commonly used inhibitors include:
1. Filipin to block fovea-mediated endocytosis.
2. Cytochalasin-D to block actin-associated cytosolic effluent.
3. Wortmannin to block phosphatidylinositol 3 kinase-associated cytosolic effluent.
4. Chloropromazine to block reticulin-mediated endocytosis.
Figure 3. Cellular uptake, intracellular trafficking, and lysosomal escape of LNP.2
Cy5 staining allows real-time tracking of mRNA cellular uptake, cellular internalization, and lysosomal escape at different times, which helps to study the mechanism behind mRNA delivery. It can also further optimize the formulation of LNP by exploring the release of mRNA in vivo. (Refer to Figure 3)
DC2.4 cells were selected as the model cells for the contagion experiment. GFP-mRNA-LNP was contagious to the cells and the intracellular GFP signals were observed under the fluorescence microscope. And the transfection efficiency was analyzed by flow cytometry.
CD Bioparticles can assist customers with immunostaining studies, which not only examine the efficiency of mRNA transmission, but also help to identify specific cell types that are transfected to a particular type of organ. (Refer to Figure 4)
Figure 4. Identification of transfected liver cell types 5 h after IV eGFP mRNA-LNP injection.3
To visualize LNP distribution observation in organisms, fluorescent dyes (such as DiD and DiO) and luciferase were used for labeling. Typically, we used luciferase mRNA (Luc-mRNA) as a model mRNA for tracking the in vivo distribution of the expressed protein. Following intravenous (IV) injection of Luc-mRNA-LNP, heart, liver, spleen, lung, kidney, and lymph node tissues were isolated. The tissues were visualized using the IVIS Spectral Live Image System. This will help to visualize the study of organ distribution and duration of protein activity production. (Refer to Figure 5)
Figure 5. Visualization of the expression by an in vivo imaging system.4
An experimental animal model (e.g., mice, rabbits, pigs, etc.) was established with cells expressing the relevant antigens and the experimental animals were immunized intravenously every few days. The experimental animals were administered and tested daily for body weight and tumor volume. Subsequently, the experimental animals were executed to isolate the heart, liver, spleen, lung and kidney tissues for weighing. Spleen, lymph nodes and tumors were collected to determine the maturation of DCs and the number of CTLs respectively. (Refer to Figure 6)
Figure 6. Determination of body weight and tumor volume in mice.4
Sections of organs were taken and stained for pathologic observation. The commonly used pathologic staining methods include:
1. H&E staining method. (Using two dyes, Hematoxylin and Eosin, the former stains the nucleus and some acidic cytoplasm, the latter stains the cytoplasm and collagen fibers, and the tissue sections stained to show blue and pink morphology).
2. PAS staining method. (Using two kinds of dyes, Periodicacid and Schiff'sreagent, the tissue sections were stained with purplish-red color).
3. Masson staining method. (Mainly used to show connective tissues such as collagen fibers, skeletal muscle fibers, etc. Using two dyes, Anilineblue and Picro-fuschin, the tissue sections show red and blue coloration after staining).
For example, H&E stained for pathological observation. Briefly, stained with Gill's Hematoxylin, blued with ammonia, washed in ethanol, stained with 0.25% Eosin Y. Then it observed under bright-field microscope. Recovery of organ function can be detected by observation of immunologic tissue. (Refer to Figure 7)
Figure 7. Tissue section staining studies.4
Blood was taken from the eyeballs to determine alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), uric acid (UA), urinary anhydride (UREAL) and other biochemical indexes to detect toxicity of mRNA-LNP to the liver and kidney. (Refer to Figure 8)
The assay was performed using the kit according to manufacturer's method. Briefly, 10μL of serum was mixed with the provided reagent mixture at 37℃ and readings were measured at 5 nm every 1 minute for 3 minutes using a multi-mode enzyme marker.
Figure 8. Determination of parameters related to mRNA-LNP administration therapy.5
A hormonal mouse model was established and injected with a therapeutic vaccine. Mice were executed and spleen, lymph and tumor were collected for detection of antigen-presenting and antigen-specific T cells. Cells were labeled with appropriate markers and analyzed and detected by flow cytometry.
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