Neurotensin-polyplex nanoparticles provide efficient gene transfection of nigral dopaminergic neurons when intracerebrally injected in preclinical trials of Parkinson's disease because they do not cross the blood-brain barrier (BBB). Therefore, this study aimed to open BBB with focused ultrasound (FUS) on the substantia nigra to attain systemic and intranasal transfections and evaluate its detrimental effect in rats. Systemically injected Evans Blue showed that a two-pulse FUS opened the nigral BBB. Accordingly, 35 μL of neurotensin-polyplex nanoparticles encompassing the green fluorescent protein plasmid (79.6 nm mean size and + 1.3 mV Zeta-potential) caused its expression in tyrosine hydroxylase(+) cells (dopaminergic neurons) of both substantiae nigrae upon delivery via internal carotid artery, retro-orbital venous sinus, or nasal mucosa 30 min after FUS. The intracarotid delivery yielded the highest transgene expression, followed by intranasal and venous administration. However, FUS caused neuroinflammation displayed by infiltrated lymphocytes (positive to cluster of differentiation 45), activated microglia (positive to ionized calcium-binding adaptor molecule 1), neurotoxic A1 astrocytes (positive to glial fibrillary acidic protein and complement component 3), and neurotrophic A2 astrocytes (positive to glial fibrillary acidic protein and S100 calcium-binding protein A10), that ended 15 days after FUS. Dopaminergic neurons and axonal projections decreased but recuperated basal values on day 15 after transfection, correlating with a decrease and recovery of locomotor behavior. In conclusion, FUS caused transient neuroinflammation and reversible neuronal affection but allowed systemic and intranasal transfection of dopaminergic neurons in both substantiae nigrae. Therefore, FUS could advance neurotensin-polyplex nanotechnology to clinical trials for Parkinson's disease.