DESIGN OF HEART TARGETED LIPID NANOPARTICLES FOR NUCLEIC ACID DELIVERY

Gene therapies, especially the use of therapeutic DNA, have made it possible to treat a wide range of diseases exploiting precision medicine. Over the last few years, advances have been made also in the design of therapeutic RNAs, including mRNA. The latter has certain advantages over DNA-based therapies: higher transfection efficiency and lower toxicity. Therapeutics nucleic acids need to be protected to avoid their degradation and to improve pharmacokinetic profile; the use of lipid nanoparticles as carriers for the administration of sensitive drugs, such as therapeutic nucleic acids, has proven to be effective and safe, thanks to biodegradability and biocompatibility of these carriers. In addition, the use of nanoparticles could increase patient compliance, thanks to reduction of the number of administrations, as well as the possibility of adopting various routes of administration. The success of mRNA based vaccines against the Sars Cov-2 virus has opened up new research opportunities in the field of gene therapy. Lipid-nanoparticles available on the market are mostly composed of 4 types of lipids, which confers them the lipophilicity suitable to interact with cell-membrane, penetrate it with endocytosis e release the cargo inside the cell: ionizable cationic lipid, cholesterol, two helper lipids. The lipids used in this thesis project were: ACL-0315 and KC2, as ICL; DSPC, mPEG2000-DMG and as helper lipids mPEG2000-DSPE; cholesterol as structural lipid. The entrapment of nucleic acid into a carrier also entails the advantage of exploiting it for specific targeting action. Targeted therapies could be the better way to treat patients affected by a specific cardiomyopathy, since the current treatments do not tackle the disease at its root. Cardiomyopathies fall into the category of cardiovascular diseases that affect about 350,000 people a year in Italy. The onset of cardiomyopathies is quite heterogeneous; they are distinguished as primary, if the predominant cause is a functional or structural alteration of the heart muscle; secondary, if the onset is caused by multisystem disorders. Primary cardiomyopathies are classified into genetic, acquired, or mixed. The final goal of the project is to design and optimize nucleic acids carrying targeted lipid nanoparticles, able to express a protein treating cardiomyopathies. The lipid nanoparticles were prepared by using a microfluidics technique and labelled with a lipophilic fluorescent dye. Characterisations of the formulations will mainly be done by DLS analysis to assess size and PDI. The particularity of the here proposed nanocarriers is the presence on the surface of a heart-targeting peptide, called PEP1, specific for the cardiomyocytes. The peptide was conjugated to DSPE-PEG2000NHS and following characterised by TNBS assay, to evaluate the presence of free peptide, and HPLC. In order to distinguish targeted-LNPs from those without targeting, evaluations will be carried out by means of UV-Visible analysis. Three targeting strategies have been investigated: post-insertion, pre-insertion, and post functionalization. Studying transfection efficiency was another goal of this project. The plasmid pCS2 eGFP, expressing the green fluorescent protein GFP, was encapsulated in the formulation of labelled LNPs for evaluating the ability of LNP to preserve the plasmid integrity, then the transfection efficiency and the biodistribution in Zebrafish larvae. The characterisation done involved DLS analysis to evaluate the LNPs size and polydispersity, TEM characterization to visualize the LNPs morphology, and agarose gel electrophoresis to evaluate the encapsulation efficiency and plasmid integrity. All the assessment about the different formulations were done in transgenics Zebrafish larvae at the Genetics of Development Laboratory of Prof. Argenton Francesco, Department of Biology (UNIPD), by Dr. Piersanti Angela and the Master's candidate Piovan Alberto.