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Sérgio Scalzo,1 Anderson K Santos,1 Heloísa AS Ferreira,1 Pedro A Costa,1 Pedro HDM Prazeres,2 Natália JA da Silva,1 Lays C Guimarães,1 Mário de Morais e Silva,1 Marco TR Rodrigues Alves,1 Celso TR Viana,1 Itamar CG Jesus,1 Alice P Rodrigues,1 Alexander Birbrair,2 Anderson O Lobo,3 Frederic Frezard,1 Michael J Mitchell,4 Silvia Guatimosim,1 Pedro Pires Goulart Guimaraes1 1Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil; 2Department of General Pathology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil; 3Department of Materials Engineering, Federal University of Piauí, Teresina, PI, Brazil; 4Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USACorrespondence: Pedro Pires Goulart Guimaraes, Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil, Tel +55 031 3409-2948, Email ppiresgo@reitoria.ufmg.brIntroduction: Gene therapy is a promising approach to be applied in cardiac regeneration after myocardial infarction and gene correction for inherited cardiomyopathies. However, cardiomyocytes are crucial cell types that are considered hard-to-transfect. The entrapment of nucleic acids in non-viral vectors, such as lipid nanoparticles (LNPs), is an attractive approach for safe and effective delivery.Methods: Here, a mini-library of engineered LNPs was developed for pDNA delivery in cardiomyocytes. LNPs were characterized and screened for pDNA delivery in cardiomyocytes and identified a lead LNP formulation with enhanced transfection efficiency.Results: By varying lipid molar ratios, the LNP formulation was optimized to deliver pDNA in cardiomyocytes with enhanced gene expression in vitro and in vivo, with negligible toxicity. In vitro, our lead LNP was able to reach a gene expression greater than 80%. The in vivo treatment with lead LNPs induced a twofold increase in GFP expression in heart tissue compared to control. In addition, levels of circulating myeloid cells and inflammatory cytokines remained without significant changes in the heart after LNP treatment. It was also demonstrated that cardiac cell function was not affected after LNP treatment.Conclusion: Collectively, our results highlight the potential of LNPs as an efficient delivery vector for pDNA to cardiomyocytes. This study suggests that LNPs hold promise to improve gene therapy for treatment of cardiovascular disease.Graphical Abstract: Keywords: lipid nanoparticles, ionizable lipids, pDNA delivery, heart, cardiomyocytes