In situ continuous Dopa supply by responsive artificial enzyme for the treatment of Parkinson's disease

Abstract

Oral dihydroxyphenylalanine (Dopa) administration to replenish neuronal dopamine remains the most effective treatment for Parkinson's disease (PD). However, unlike the continuous and steady dopamine signaling in normal neurons, oral Dopa induces dramatic fluctuations in plasma Dopa levels, leading to Dopa-induced dyskinesia. Herein, we report a functional nucleic acid-based responsive artificial enzyme (FNA-Fe₃O₄) for in situ continuous Dopa production. FNA-Fe₃O₄ can cross the blood-brain barrier and target diseased neurons relying on transferrin receptor aptamer. Then, FNA-Fe₃O₄ responds to overexpressed α-synuclein mRNA in diseased neurons for antisense oligonucleotide treatment and fluorescence imaging, while converting to tyrosine aptamer-based artificial enzyme (Apt-Fe₃O₄) that mimics tyrosine hydroxylase for in situ continuous Dopa production. In vivo FNA-Fe₃O₄ treatment results in recovery of Dopa and dopamine levels and decrease of pathological overexpressed α-synuclein in PD mice model, thus ameliorating motor symptoms and memory deficits. The presented functional nucleic acid-based responsive artificial enzyme strategy provides a more neuron friendly approach for the diagnosis and treatment of PD.

Fig. 1. Schematic diagram of FNA-Fe₃O₄ for the treatment of PD.

a Catalytic mechanism of Fe₃O₄ nanozymes on the hydroxylation of tyrosine to produce Dopa (Phase I), illustration of the functions of Apt-Fe₃O₄ (Phase II) and FNA-Fe₃O₄ (Phase III). b Illustration of FNA-Fe₃O₄ for synergistic treatment of PD.

Fig. 2. The tyrosine hydroxylase-mimicking activity of Fe₃O₄ nanoparticles.

a Time-dependent UPLC-MS peak intensities changes of reactant tyrosine and product Dopa in the presence of 10 μg mL⁻¹ Fe₃O₄ nanoparticles, 100 μM tyrosine, 5 mM H₂O₂, and 5 mM AA. Peaks at m/z = 182.08 and m/z = 198.08 represent tyrosine and Dopa respectively. b UPLC-MS peak intensities of reactant tyrosine and product Dopa in systems with different reaction conditions. c Dopa generation in the reaction system (10 μg mL⁻¹ Fe₃O₄ nanoparticles, 100 μM tyrosine, 5 mM H₂O₂ and 5 mM AA) in the presence of 1 mM tert-butanol or ethanol. The results were expressed as mean ± SD (n = 3 independent experiments). d–h ESR spectra corresponding to the ·OH generated by Fe₃O₄ nanoparticles in the presence of H₂O₂ (d), no free radical generated by Fe₃O₄ nanoparticles in the presence of AA (e), the ·OOH generated by Fe₃O₄ nanoparticles in the presence of H₂O₂ and AA (f), no free radical generated in the mixture of H₂O₂ and AA (g), the ·OOH generated by Fe₃O₄ nanoparticles in the presence of H₂O₂ and AA with added tyrosine (h). i Time-dependent GC-MS peak intensities of MSM in the presence of 10 μg mL⁻¹ Fe₃O₄ nanoparticles, 1 mM DMSO, 5 mM H₂O₂ and 5 mM AA. Peak at m/z = 79.00 represent MSM formation. j Schematic suggested mechanism for the production of Fe(IV) = O intermediate by Fe₃O₄ nanoparticles in the presence of H₂O₂ and AA and the catalytic process of hydroxylation of tyrosine to Dopa. a, b, d–i one representative data was shown from three independently repeated experiments. Source data are provided as a Source Data file.

Fig. 3. Characterizations of artificial enzymes and their catalytic and fluorescence imaging performances.

a Schematic diagram of Apt-Fe₃O₄ catalysis. b, c Rates of hydroxylation of tyrosine to Dopa by Fe₃O₄, Apt-Fe₃O₄ and Ran-Fe₃O₄ in the presence of H₂O₂, AA and variable concentrations of tyrosine in aqueous solution (b) and in crowded solution (c). d Schematic diagram of strand displacement responsiveness and catalysis of FNA-Fe₃O₄. e PAGE analysis of the functional nucleic acids structure and the strand displacement reaction (red strand: tyrosine aptamer; green-yellow strand: block strand; blue strand: SNCA mRNA; green substrate: tyrosine). Gel was representative of n = 3 independent experiments. f, g Rates of hydroxylation of tyrosine to Dopa by FNA-Fe₃O₄ with or without mRNA in the presence of H₂O₂, AA and variable concentrations of tyrosine in aqueous solution (f) and in crowded solution (g). h The typical cyclic process of tyrosine capture and hydroxylation by 10 μg mL⁻¹ artificial enzymes in the presence of 100 μM tyrosine, 5 mM H₂O₂ and 5 mM AA. i Catalytic selectivity of 10 μg mL⁻¹ artificial enzymes for hydroxylation of 100 μM tyrosine and interference molecules in the presence of 5 mM H₂O₂ and 5 mM AA. b, c, f, g, h and i The results were expressed as mean ± SD (n = 3 independent experiments). b, c, f and g P-values were calculated by two-tailed t-test. Source data are provided as a Source Data file.

Fig. 4. Cellular availability of the artificial enzymes.

a Schematic of FNA-Fe₃O₄ crossing the in vitro BBB model. b Transport efficiency of the artificial enzymes across the BBB in vitro. c Flow cytometry analysis of cellular uptake of the artificial enzymes. d Confocal fluorescence images of the gradual cellular internalization of FNA-Fe₃O₄ at different time interval. e, Cell viability of BV2 cells incubated with the artificial enzymes at different concentrations. b, e The results were expressed as mean ± SD (n = 3 biologically independent samples). b P-values were calculated by two-tailed t-test. c, d One representative data was shown from three independently repeated experiments. Source data are provided as a Source Data file.

Fig. 5. Intracellular biological effects of the artificial enzymes.

a Schematic diagram of Cat-Fe₃O₄ Catalysis. b Schematic diagram of strand displacement of ASO-Fe₃O₄. c Artificial enzymes mediated changes in intracellular dopamine levels. d Dopa incubation induced dramatic fluctuations in intracellular dopamine levels. Arrows represent Dopa removal time point (adding fresh culture medium). e SNCA expression in diffierent treated SH-SY5Y cells. f, g Confocal imaging of normal cells (without EGFP fluorescence) and PD cells (express EGFP fluorescence) (f) and co-cultured cells by FNA-Fe₃O₄ (g). h Flow cytometry analysis corresponding to each panel in (f) and (g). c, d The results were expressed as mean ± SD (n = 3 biologically independent samples). c P-values were calculated by two-tailed t-test. e-h one representative data was shown from three independently repeated experiments. Source data are provided as a Source Data file.

Fig. 6. In vivo biological effects of the artificial enzymes.

a In vivo fluorescence imaging of FNA-Fe₃O₄ in normal mice and PD mice. b, c Artificial enzymes mediated changes in Dopa levels (b) and dopamine levels (c) in mice striatal microdialysates (arrows represent dosing time point, each line represent one mouse). d Dopa administration induced dramatic fluctuations in dopamine levels in mice striatal microdialysates (dash line represents the mean dopamine level of normal mice in (c), each line represent one mouse). e Schematic timeline of microdialysis, injections and behavioral tests. f The expression of SNCA in the brain tissue of differently treated mice. g Descent time spent by the mice in the pole test. h Wire suspension time of the mice in the wire suspension test. i Step-through latency time of the mice in the step-through passive avoidance test. a, f One representative data was shown from three independently repeated experiments. b–d each line represent one mouse, n = 3 biologically independent animals. g–i The results were expressed as mean ± SD (n = 3 biologically independent animals), P-values were calculated by two-tailed t-test. Source data are provided as a Source Data file.