On March 2020, the World Health Organization (WHO) declared the coronavirus disease a global pandemic. The immense impact of the pandemic on the global health and economics led to a massive resource¿s involvement in development of vaccine against SARS-Cov2. Thanks to this, big companies were able to produce efficiently and quickly, mRNA vaccines encapsulated in lipid nanoparticles (LNPs) which were never been approved before. Despite mRNA vaccines safety and effectiveness there are limits in their use. In fact, they need a cold chain to be maintained and delivered leading to logistical problems in view of mass vaccination. Furthermore, mRNA vaccines are quite expensive in respect to other most traditional vaccines. Considering this, DNA vaccines may represent a good alternative to mRNA vaccines as they do not require extremely low temperature for the storage and are relatively inexpensive. Importantly, the plasmid DNA containing the viral protein sequence can be easily modified to make the vaccine specific for the viral variants. pDNA needs to be conveyed into the target cells and to reach the nucleus to be transcribed and then translated into protein. Electroporation is the most diffused way to achieve the pDNA internalization however, it is an invasive procedure that may cause several side effects. For these reasons, the use of LNPs is preferable. Our group aims to produce a library of lipid-nanoparticles through microfluidic mixing technique, which offer the advantage to yield highly reproducible and low size nanoparticles. Different multicomponent lipid formulations will be characterized and tested in vitro on keratinocyte cell lines to evaluate the transfection efficiency (TE) and toxicity. Computational analysis of the LNPs features will allow their further optimization. Finally, the most performing LNPs will be functionalized using specific proteins improving the targeting to keratinocytes and dendritic cells, in view of subcutaneous vaccine administration.
The breakthrough in vaccinology is the development of genetic vaccines based on nucleic acids delivery. In fact, the absence of the virus (inactivated or attenuated) or viral protein subunit confer them higher safety profile compared to all the other vaccines. pDNA can be engineered using Spike protein sequence as it is an immunogenic protein of SARSCov2 which can be recognized by immune cells. Interestingly, the sequence can be modified using the spike sequence isolated from the new viral lineages, to meet the needs of specific vaccines effective against SARSCov2 variants. The current used mRNA vaccines are encapsulated in LNPs containing ionizable or cationic lipids along with helper and PEGylated lipids. PEGylation of lipids was introduced with the aim to reduce the cationic lipids toxicity. In fact, is thought to shield the charges and increase the permanence of LNPs in bloodstream (1). However, PEGylation is not able to avoid the BC formation which may affect the cellular uptake. Actually, polyethylene glycol (PEG) reduces the transfection efficiency of the lipid-based nanocarriers and triggers a specific immune response mediated by the production of anti-PEG antibodies (IgM). This leads to a fast PEGylated LNPs clearance after the first exposure. Furthermore, the immune response to PEG caused anaphylactic events after vaccination (1, 2). For this reason, the use of PEG can be replaced by the development of a stable preformed PC on the LNPs surface. The establishment of the PC may avoid unspecific biomolecular adsorption on the LNPs, reduce charges repulsion and can even increase the specific targeting to certain cell populations in vivo (3). We will synthesize both PEGylated and correspondent non-PEGylated formulations, to understand its impact on cellular uptake. Importantly, we will perform specific protein functionalization to overcome the use of PEGylated lipids. The delivery of nucleic acids by decorated LNPs is a groundbreaking technology never used in clinics and poorly discussed in literature until now. For this reason, this project may offer several hints to improve the effectiveness of gene delivery in vivo.
1. Schoenmaker L, Witzigmann D, Kulkarni JA, et al. mRNA-lipid nanoparticle COVID-19 vaccines¿: Structure and stability. Int J Pharm. 2021;601(April):120586. doi:10.1016/j.ijpharm.2021.120586
2. Palchetti S, Colapicchioni V, Digiacomo L, et al. The protein corona of circulating PEGylated liposomes. Biochim Biophys Acta - Biomembr. 2016;1858(2):189-196. doi:10.1016/j.bbamem.2015.11.012
3. Caracciolo G. Liposome-protein corona in a physiological environment: Challenges and opportunities for targeted delivery of nanomedicines. Nanomedicine Nanotechnology, Biol Med. 2015;11(3):543-557. doi:10.1016/j.nano.2014.11.003