Bioinspired theranostic coordination polymer nanoparticles for intranasal dopamine replacement in parkinson's disease

Bioinspired Theranostic Coordination Polymer Nanoparticles for Intranasal Dopamine Replacement in Parkinson’s Disease Javier García-Pardo, Fernando Novio, Fabiana Nador, Ivana Cavaliere, Salvio Suárez-García, Silvia Lope-Piedrafita, Ana Paula Candiota, Jordi Romero-Gimenez, Beatriz Rodríguez-Galván, Jordi Bové, Miquel Vila, Julia Lorenzo,*and Daniel Ruiz-Molina* Cite This: ACS Nano 2021, 15, 8592−8609 Read Online ACCESS Metrics & More Article Recommendations * sıSupporting Information ABSTRACT: Dopamine (DA) is one of the main neurotransmitters found in the central nervous system and has a vital role in the function of dopaminergic (DArgic) neurons. A progressive loss of this specific subset of cells is one of the hallmarks of age-related neurodegenerative disorders such as Parkinson’s disease (PD). Symptomatic therapy for PD has been centered in the precursor LDOPA administration, an amino acid precursor of DA that crosses the blood−brain barrier (BBB) while DA does not, although this approach presents mediumto long-term side effects. To overcome this limitation, DA-nanoencapsulation therapies are actively being searched as an alternative for DA replacement. However, overcoming the low yield of encapsulation and/or poor biodistribution/ bioavailability of DA is still a current challenge. Herein, we report the synthesis of a family of neuromelanin bioinspired polymeric nanoparticles. Our system is based on the encapsulation of DA within nanoparticles through its reversible coordination complexation to iron metal nodes polymerized with a bis-imidazol ligand. Our methodology, in addition to being simple and inexpensive, results in DA loading efficiencies of up to 60%. In vitro, DA nanoscale coordination polymers (DA-NCPs) exhibited lower toxicity, degradation kinetics, and enhanced uptake by BE(2)-M17 DArgic cells compared to free DA. Direct infusion of the particles in the ventricle of rats in vivo showed a rapid distribution within the brain of healthy rats, leading to an increase in striatal DA levels. More importantly, after 4 days of nasal administrations with DA-NCPs equivalent to 200 μgof the free drug per day, the number and duration of apomorphine-induced rotations was significantly lower from that in either vehicle or DA-treated rats performed for comparison purposes. Overall, this study demonstrates the advantages of using nanostructured DA for DA-replacement therapy. KEYWORDS: neuromelanin, dopamine, Parkinson’s disease, neurodegeneration, coordination polymers INTRODUCTION Parkinson’s disease (PD), the second most common neurodegenerative disorder in the world, affecting more than 10 million people, 1,2 is characterized by motor symptoms including muscular rigidity, rest tremor, posture instability, and bradykinesia. 1 The origin is associated with early prominent death of DArgic neurons typically of the substantia nigra pars compacta (SNpc), 3 which leads to diminished levels of dopamine (3,4-dihydroxyphenethylamine, DA) in the striatum of these patients. 4−7 Mainly due to restrictions for direct DA replacement, delivery of the precursor levodopa (L3,4-dihydroxyphenylalanine, L-DOPA) has been used in the clinic for more than 50 years to mitigate Parkinson symptoms. 1 However, long-term administration of this drug is associated with developing highly disabling complications such as drug-induced dyskinesia and motor fluctuations. 8−10 Both complications are in part due to pulsatile L-DOPA administration and can be minimized with continuous and sustainable L-DOPA delivery by duodenal infusion. 11 Evidence Received: January 17, 2021 Accepted: April 12, 2021 Published: April 22, 2021 Article www.acsnano.org © 2021 American Chemical Society 8592 https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 Downloaded via CSIC on March 17, 2022 at 08:54:05 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. suggests that up to 50% of PD patients develop these complications after five years of chronic treatment with levodopa. Therefore, the development of alternative strategies to overcome these complications is an important challenge in modern medicine: among them, finding approaches for the delivery of DA through its nanoencapsulation. Nanocarriers can help DA to avoid metabolism, cross the BBB, and promote a sustained release, reducing recurrent drug administration and, therefore, adverse effects. So far, different families of nanocarriers have been described with this aim, including chitosan 12 and transferrin 13 functionalized liposomes, (bio)hydrogels, 14,15 chitosan nanoparticles, 16,17 carbon nanotubes, 18 alginate/magnetite hybrid beads, 19 carbon dots, 20 carboxymethyl cellulose coated magnetic nanoparticles, 21 borneol and lactoferrin co-modified nanoparticles, 22 and a polyvinylpyrrolidone-poly(acrylic acid) nanogel. 23 The work by Gupta et al. deserves special mention, which reported DA-loaded poly lactic-co-glycolic acid (PLGA) nanoparticles able to reverse functional deficits in Parkinsonian rats. 24 However, despite these advances, most of the nanocarriers used for DA replacement therapy often suffer from low yield of encapsulation and/or poor biodistribution and drug bioavailability, so alternative encapsulation approaches are required. Neuromelanin (NM) particles, found in DArgic neurons from the SNpc, incorporate large amounts of DA and iron ions, which allow for their use as a biomarker for PD. Therefore, we hypothesize that melanin granular pigments found in nature may offer inspiration to develop more effective DA nanocarriers. 25 To mimic the synthesis of melanin-like nanoparticles in the lab, whose tentative formula is shown in Figure 1a, 26 three main approaches have been so far reported. In the first approach, self-oxidation of DA under alkaline conditions results in the formation of colloidal polyDA (PDA) nanoparticles, which subsequently are doped with the appropriate metal salt (postdoping approach). 27−30 Alternatively, one-pot polymerization of DA in the presence of transition-metal ions results in their inclusion in the final PDA (in situ doping). 31−33 And the third approach involves the preformation of Fe(DA)3monomeric complexes that systematically control the percentage of iron in the particles. 34−36 Following these approaches, synthetic nanoparticles showing excellent biocompatibility and biodegradability have already been prepared and successfully used in phototherapy/drug delivery 37 and imaging. 38−40 However, none of them were found to be useful for the delivery of DA, as the polymerization and formation of the nanoparticles always take place through irreversible covalent bonds. Figure 1. Bioinspired approach for dopamine (DA) replacement in Parkinson’s disease (PD). (a) Schematic composition of neuromelanin (NM) and the related nanoscale coordination polymer (DA-NCP) proposed herein. (b) Schematic representation of the synthesis of DANCPs from DA, Fe(AcO)2, and BIX. (c) Proposed model for DA-based replacement therapy in Parkinson’s disease using DA-NCPs. ACS Nano www.acsnano.org Article https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 8593 Herein we envision that this could be achieved with the formation of a tailor-made coordination polymer, where the central polymeric backbone is made of iron nodes linked to a bidentate ligand such as 1,4-bis(imidazol-1-ylmethyl)benzene (BIX) and DA is used as a counter ligand to complete the coordination sphere. The composition of the resulting nanoparticles (named from now on as DA-NCPs) is expected to have a structure inspired by NM, as shown in Figure 1a, but with the fundamental difference being that the incorporation of DA to the NP is reversible inside the cell. A schematic representation of the synthesis of the DA-NCPs nanoparticles and the expected biological function is shown in Figure 1b,c. RESULTS AND DISCUSSION Synthesis and Characterization of DA-NCPs. DANCPs were prepared following a procedure already described for related catechol-based nanoparticles. 41,42 In a typical synthesis, nanoparticles were obtained through a reaction of Fe(AcO)2with DA hydrochloride in the presence of the ditopic ligand BIX. After 2 h under constant stirring at room temperature, a dark precipitate was isolated by centrifugation and washed gently with ethanol to eliminate all the unbound ligands (see details in Methods and Supporting Information). Scanning electron microscopy (SEM) and negative-staining transmission electron microscopy (NS-TEM) images of the obtained material revealed the presence of spherical nanoparticles with an average diameter of 64 ±9.0 nm and 56 ± 9.0 nm, respectively (Figure 2a,c). These results were in agreement with dynamic light scattering (DLS) measurements of the DA-NCPs dispersed in ethanol, which showed a hydrodynamic diameter of 81 ±4.0 nm (PDI: 0.124) (Supporting Information, Figure S1). Energy-dispersive Xray analysis (EDX) profile of the TEM image clearly revealed the presence of iron homogeneously distributed along the whole nanoparticle (see Figure 2d), and Mossbauer spectroscopy studies confirmed that the iron was in the high-spin Fe(III) form (Supporting Information, Figure S2). This observation suggested that Fe(II) was oxidized to Fe(III) along with the nanoparticle synthesis. In agreement, the Figure 2. Chemical and morphological characterization of DA-NCPs. (a) SEM images of neuromelanin-inspired DA-NCPs. The inset shows a colloidal dispersion of the nanoparticles in ethanol. Scale bar, 200 nm. (b, c) Particle size distribution of DA-NCPs determined by (b) SEM and (c) negative-staining transmission electron microscopy (NS-TEM). (d) TEM image (left) and EDX profile of DA-NCPs along the dark red line marked (right). (e) UV−vis absorbance spectra of the particles (solid dark red line) and the corresponding chemical precursors: DA (black dashed line), BIX (solid blue line), and Fe(AcO)2(solid orange line) determined at a concentration of 100 μg/mL in water. ACS Nano www.acsnano.org Article https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 8594 absorbance spectrum of DA-NCPs in the 400−800 nm range exhibited a characteristic maximum at 562 nm, which was attributed to the formation of Fe-bis-catechol coordination bonds (Figure 2e). This electronic modification has been attributed to a redox interplay between the metal ion and electroactive catechol ligands in air, as previously reported. 43 The absence of diffraction peaks observed by powder X-ray diffraction (PXRD) indicated the amorphous nature of the nanoparticles (see Supporting Information, Figure S3). The chemical characterization of DA-NCPs was addressed using different complementary techniques. Fourier transform infrared spectroscopy (FT-IR) confirmed the presence of DA and a BIX ligand coordinated to the Fe(III) metal ions (see Supporting Information, Figure S4). The FT-IR spectrum shows the disappearance of the bands corresponding to the catechol −OH groups of DA in the range of ν= 3000−3500 cm−1,reflecting their deprotonation, the stretching and bending vibrations of the amine group (3340 and 1618 cm−1, respectively), and the stretching vibrations of C−H (3040 cm−1) and C−C of the aromatic ring (1550 cm−1). Moreover, the vibration band assigned to a C−O stretch for the catechol coordinated to the metal appeared in the frequency range 1200−1300 cm−1.Additionally,typical vibrational bands of the BIX ligand (ν= 1520, 1262, 1105 cm−1) were observed in the FT-IR spectra. The 1H NMR spectrum of DA-NCPs dissolved in a deuterated methanolHCl solution confirmed the presence of acetate counterions (see Supporting Information, Figure S5) and both ligands, although with a lower contribution of the BIX ligand than theoretically expected from the concentrations of the initial reactants. These results were quantitatively confirmed by highperformance liquid chromatography (HPLC) analysis of the product resulting upon treatment of DA-NCPs with an acidic solution; these analyses showed a content of 20% BIX (w/w) ligand. More interestingly, HPLC analysis using electrochemical detection (HPLC-ECD) confirmed the presence of 52.5 ±7.2% DA in DA-NCPs nanoparticles (see Supporting Information and Materials and Methods section for experimental details). This exceptionally high value was confirmed with absorbance measurements using DA hydrochloride as a standard (Supporting Information, Figure S6). These analyses also revealed a very high loading efficiency (LE) of 69.7 ±7.2%. For instance, this value is 6-fold higher compared with the LE of dopamine achieved with PLGA in previous reports. As far as iron is concerned, inductively coupled plasma-mass spectroscopy (ICP-MS) data confirmed a total amount of 10.2% in the nanoparticles. Finally, the combination of elemental analysis, ICP-MS, 1H NMR, and HPLC data yielded the following final tentative chemical formula for the nanoparticles: [Fe- (DA)1.6(BIX)0.5(AcO)0.2(H2O)1.9] (see Supporting Information, Table S1). This formula differs from that theoretically expected on the basis of the initial reagents added (1 Fe:1 BIX:2 DA), which is attributed to their synthesis under out-ofequilibrium conditions induced by the fast precipitation process, a common phenomenon for this family of amorphous NCPs. Another plausible explanation for such differences could be the encapsulation of free ligand/solvent molecules within the particles present in the reaction solution, as already demonstrated in related systems. 44,45 Such encapsulation ability has been confirmed by encapsulating, as a proof-ofconcept, the recombinant epidermal growth factor (EGF) with an excellent encapsulation efficiency (EE) of 89.6%, and loading capacity (LC) of almost 7.7 μg of EGF per milligram of particles (Supporting Information,Materials and Methods and Figure S7). Colloidal Stability and DA Release Kinetics under Physiological Conditions. To simulate the colloidal stability of DA-NCPs in physiological environments, the nanoparticles were dispersed in water and phosphate-buffered saline (PBS) solution (with and without bovine serum albumin, BSA) at physiological conditions (0.5 mM). 46,47 DLS measurements in water (Supporting Information, Table S2) showed higher hydrodynamic diameters (171 ±4.2 nm; PDI: 0.208) than those obtained in ethanol (81 ±4.0 nm; PDI: 0.124) or as a solid powder by SEM measurements (64 ±9 nm). Moreover, even though the ξ-potential value in water was close to +25 mV, the colloidal solution was not stable and started to aggregate and precipitate over a relatively short period of time (sedimentation observed after 1 h). In PBS (20 mM PBS buffer at pH 7.4), the mean size values for nanoparticles indicated the presence of large aggregates (Supporting Information, Table S2) and showed a ξ-potential of −13.1 mV. The addition of 0.5 mM BSA, extensively used for dispersing inorganic and polymeric nanoparticles, 48,49 successfully induced a disaggregation process and their stabilization without precipitation for several hours. The nanoparticles now showed a lower hydrodynamic diameter (103 ±16.2 nm; PDI: 0.198), similar to that observed using culture medium containing fetal bovine serum (FBS) (Supporting Information, Table S2). The increase in size (24−28 nm) with respect to the value found in ethanol could be attributed to the formation of a protein corona around the nanoparticles. We next investigated the release kinetics of DA from DANCPs nanoparticles after incubation for 24 h in a PBS−BSA buffer at 37 °C, using HPLC-ECD. At pH 7.4, a continuous release of DA, reminiscent of a standard saturation curve (Supporting Information, Figure S8), was observed with a cumulative release of 50% in the first 2 h (t1/2 = 2 h). Afterward, the release notably slowed down with an increase of 5% in the next 22 h. If the nanoparticles were dispersed at pH 5.5, the accumulative release of 50% was reached in less than 1 h. These results confirmed that the pH decrease induced a much faster release of the DA due to the lower stability of the coordinative bond between iron and catechol ligands. Finally, the release increased up to 85% in 10 h and 90% at 24 h. This pH dependence has been observed previously in mussel-inspired hydrogels with Fe-catechol complexes and in other Fe-containing polymeric nanoparticles. 30,50 Worth mentioning is that the FBS used in the previous experiments was heat-inactivated and does not fully represent the real physiological conditions found in the blood and plasma under in vivo conditions. For this reason, we also performed an additional experiment to study the colloidal stability of the DA-NCPs in the presence of nontreated human plasma. For this, an aliquot of DA-NCPs was suspended in 1 mL of ethanol or in 1 mL of nontreated human plasma. Afterward, the hydrodynamic diameter of the nanoparticles was evaluated at differenttimepointsof incubation at 37 °C. No relevant changes in the size distribution of the nanoparticles up to 6 h incubation under these conditions were detected. Only a small shift toward higher dimensions was observed, compatible with the progressive formation of the protein corona due to the high concentration of proteins in the human plasma. As expected, ACS Nano www.acsnano.org Article https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 8595 the presence of these proteins was visible as a secondary peak (peak 2, in Figure S9) with a size of ∼40 nm. Evaluation of the Cytotoxicity of DA-NCPs against DArgic Neurons. To evaluate the effect of DA-NCPs on DArgic cell survival, in vitro cytotoxicity assays with (BE)2M17 cells were performed. (BE)2-M17 is a neuroblastomaderived DArgic cell line that expresses large amounts of key proteins essential for the DArgic metabolism and, therefore, extensively used as a cellular model to study DA metabolism. 51 Treatment of the (BE)2-M17 cells with DANCPs did not lead to any appreciable cell viability reduction after 48 h of incubation at concentrations up to 100 μg/mL (expressed as DA concentration) (see Supporting Information, Figure S10). The same concentrations of free DA in the range from 0.01 to 100 μg/mL showed comparable results. However, slightly higher levels of the free neurotransmitter (i.e., 200 and 250 μg/mL) caused a dramatic death of DArgic cells with more than 85% reduction in the cell viability (Supporting Information, Figure S10). Interestingly, comparable concentrations of DA-NCPs caused lower cytotoxic effects. The toxicity found for the highest concentrations of DA was attributed to the intracellular metabolism of DA (conversion of DA to 3,4-dihydroxyphenylacetaldehyde, DOPAL) that generates hydrogen peroxide and, therefore, reactive oxygen species (ROS) effects. This process can take place both enzymatically (monoamine oxidase, MAO) or nonenzymatically. ROS can also be generated upon DA oxidation; when free DA cytosol levels exceed a certain threshold level, DA can spontaneously autoxidize, producing o-quinone molecules. 52 These species are very reactive and may contribute to increasing the levels of oxidative stress inside the cells. To confirm this fact, intracellular ROS formation in BE(2)-M17 cells was also measured after treatment with DA or DA-NCPs, through the 2′,7′- dichlorofluorescein diacetate (DCFDA) assay (see Materials and Methods). 53 After a 24 h treatment, the 300-fold DCF fluorescence increase found for the highest free DA concentration (see Supporting Information, Figure S11) confirmed the ROS production. 24 Ascorbic acid (AA), at physiological concentrations of 20 μg/mL, was required to drastically reduce ROS effects of H2O2. Interestingly, DANCPs showed a lower DCF fluorescence increase (200-fold) even considering the possible oxidative stress coming from iron ions (Supporting Information, Figure S12). On the basis of these findings, we can state that our nanoparticles did not show a reduction in cell viability at the assayed DA concentrations in the range 0.01 to 10 μg/mL 54 and that DA nanostructuring into DA-NCPs reduced the toxicity of the monoamine against DArgic cells. Similar observations were reported by Pahuja et al., 24 who Figure 3. Uptake kinetics of DA and DA-NCPs by DArgic (BE)2-M17 cells. (a) Intracellular DA levels determined by HPLC-ECD after incubation of (BE)2-M17 cells for 2, 6, and 24 h in the absence (0 μg/mL, shown in light pink) or in the presence of DA at two different concentrations (1 and 10 μg/mL of DA, shown in light red and red, respectively). (b) Rate of DA uptake after the treatment of the cells with DA at 1, 10, 25, and 50 μg/mL for 2 h. (c) Intracellular DA levels after incubation of (BE)2-M17 cells for 2, 6, and 24 h in the absence (0 μg/mL, shown in light pink) or in the presence of nanostructured DA (DA-NCPs) at two different concentrations (concentration equivalent to 1 and 10 μg/mL of DA, shown in light green and green, respectively). (d) Rate of DA uptake after the treatment of the cells with DA-NCPs at concentrations equivalent to DA concentrations of 1, 10, 25, and 50 μg/mL for 2 h. (e, f) Relative intracellular levels of DA after incubation for (e) 2 h and (f) 24 h in the absence (0 μg/mL, shown in light pink) or in the presence of DA or DA-NCPs at two different concentrations (concentration equivalent to 1 and 10 μg/mL). In all cases, values are mean ±SEM. In (a) and (c), *p< 0.05, compared to 0 μg/mL; #p< 0.05, compared to treatment with 1 μg/mL; a three-way ANOVA (full factorial) and Sidak correction was applied to the log-transformed data. In (e) and (f), *p< 0.05, compared to control; #p< 0.05, compared to DA (one-way ANOVA and Turkey’sposthoc test). ACS Nano www.acsnano.org Article https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 8596 demonstrated a reduction in the toxicity and oxidative damage induction of L-DOPA upon encapsulation into PLGA nanoparticles. Evaluation of the toxicity of the iron salt and the linker ligand was performed in (BE)2-M17 cell culture using concentrations ranging from 0 to 100 μg/mL for comparison purposes (see Supporting Information, Figure S13), with no cytotoxicity detected even at the highest concentration assayed (100 μg/mL).Finally,andfor comparison purposes, we also studied the cytotoxicity of DA and DA-NCPs in a non-DArgic cell line such as HeLa cells (Supporting Information, Figure S13), where as expected, IC50 was smaller for both cases (see Supporting Information, Table S3). Uptake of DA-NCPs by DArgic (BE)2-M17 Cells. The DA-NCPs’uptake kinetics by DArgic (BE)2-M17 cells was studied and compared to free DA. To perform the experiments, cells were exposed to increasing concentrations of DA or DA-NCPs and incubated for 2, 6, and 24 h. Immediately after incubation, cells were collected and the intracellular concentration of DA was measured by HPLCECD (for more details, see Supporting Information and Materials and Methods). Free DA. Incubation of (BE)2-M17 cells in the presence of 1μg/mL of free DA resulted in a rapid concentrationdependent increase in intracellular DA level after 2 h of incubation (Figure 3a). This fact could be attributed to a large amount of DA transporters (DATs) that DArgic neurons use to control extracellular DA levels by pumping the extracellular DA from the synaptic cleft to the cytosol. 55 As shown in Figure 3a, at longer incubations times (6 and 24 h), a progressive decrease in the total intracellular DA concentration was observed. This decrease was accompanied by a constant reduction in the extracellular DA levels (see Supporting Information, Figure S14). The rate of uptake of DA through DAT is assumed to follow characteristic saturation kinetics. 56 To confirm this, the experimental data obtained for the different DA concentrations used at 2 h were plotted and precisely fitted to Michaelis−Menten kinetics, Figure 4. Metabolism kinetics of DA and DA-NCPs in DArgic neurons. (a) Principal degradation pathways of DA in DArgic neurons and main products obtained. (b) Representative HPLC-ECD chromatograms showing those DA derivatives found in (BE)2-M17 cells after treatment with two different concentrations (1 and 10 μg/mL) of free DA or DA-NCPs for 2 h. The mix of standards (mix) contained 1: LDOPA; 2: DHBA, 3: DOPAC; 4: DA; 5: 5HIIA; 6: HVA, 7: 3-MT, and 8: 5HT. (c, d) Intracellular levels of DA and its metabolites (DOPAC and HVA) after 2 h of incubation with (c) 1 μg/mL or (d) 10 μg/mL of free DA or DA-NCPs. (e) Effect of deprenyl R, a specific monoamine oxidase inhibitor (MAOB-I), on DA metabolism. (f) Effects of chemical inhibition for selected endocytic pathways on DA uptake. Nys: nystatin; CytD: cytochalasin D. In all cases, values are mean ±SEM and n= 6 independent experiments, unless otherwise specified. In (c) and (d), *p< 0.05, compared to CNT; #p< 0.05, compared to treatment with DA (three-way ANOVA (full factorial) and Sidak correction was applied to the log-transformed data). In (e), *p< 0.0001, when compared between conditions with and without MAOB-I (two-tailed t-test). In (f), **p< 0.01, ***p< 0.001, ns (nonsignificant), when compared to CNT (two-tailed t-test); n=3 independent experiments. ACS Nano www.acsnano.org Article https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 8597 with approximate Kmand Vmax values of 0.1 μg/mL and 10.0 ng·mg prot−1·min−1, respectively (Figure 3b). All in all, the results suggested that DA was actively taken up and metabolized by the DArgic (BE)2-M17 cells due to the action of the intracellular DArgic mechanism. DA-NCPs. Incubation of cells with DA-NCPs accounting overall for the same concentration of DA showed different intracellular profiles (Figure 3c,d). At 2 h and the lowest concentration (1 μg/mL), the DA internalized amount was slightly lower than that of free DA. Incubation of cells with higher doses (10 μg/mL) for the same period of time induced an intracellular level of DA up to 6360 ng/mg. This concentration was 6-fold higher than that obtained for free DA (1156 ng/mg) under the same concentration and experimental conditions. Moreover, the plot of DA internalization vs the initial external concentration used showed a nonsaturable dose−response mechanism instead of the characteristic Michaelis−Menten kinetics (Figure 3d), suggesting the presence of another pathway for nanoparticle uptake. Similar results and differences were found at 6 h of incubation, in agreement with the presence of DA outside the cells at this incubation time (Figure S14). Note that the total intracellular DA determined by HPLCECD represents the sum of vesicular DA and the fraction of DA present in the cytoplasm. In fact, only the excess of DA accumulated in the cytoplasm of the cells is available and can be degraded by the MAO. Therefore, the overall effect is a progressive and slow decline of intracellular DA levels that do not fully correspond to the extracellular DA concentration. Finally, after 24 h of incubation in the presence of 1 μg/mL, DA-NCPs-exposed cells displayed a 4-fold amount of internalization compared to control and free DA-treated cells (Figure 3a and f), where depletion of the extracellular DA caused normalization of the intracellular levels of the neurotransmitter. This finding is of particular significance as demonstrated by the enhanced intracellular retention of the neurotransmitter for longer periods of time upon nanostructuring. Metabolism Kinetics of DA and DA-NCPs by DArgic Cells. The death of DArgic neurons in the substantia nigra leads to deficient striatal DA levels responsible for the cardinal motor symptoms of PD (i.e., including bradykinesia, tremor, and rigidity). 57 Under normal conditions, the stimulation of healthy DArgic neurons causes the release of the synaptic vesicles into the synaptic cleft, where the neurotransmitter interacts with the postsynaptic DA receptors or regulatory presynaptic DA autoreceptors. 58−62 When DA reaches the cytoplasm of DArgic neurons, this neurotransmitter is rapidly Figure 5. Biodistribution and metabolism of DA-NCPs in the brain after i.c.v. injection. (a) Schematic representation of the rat brain depicting the DA-NCPs injection site (red dot) and the location of the different regions analyzed (vide infra). (b, c) DA levels in the Str and SNpc (b) ipsilateral (right) or (c) contralateral (left) to the injected side after 2 h post-DA-NCPs administration. (d) DA levels in the Ctx and Cb after 2 h post-DA-NCPs injection. (e−j) Time-dependent metabolism of DA-NCPs after the i.c.v. injection of the nanoparticles. (e and f) DA, (g and h) dihydroxyphenylacetic acid (DOPAC), and (i and j) homovanillic acid (HVA) levels determined in the right and left striatum at different time points post-DA-NCPs injection. Data represent mean ±SEM. In all cases n= 3, unless otherwise indicated. ACS Nano www.acsnano.org Article https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 8598 stored back into the synaptic DA storage vesicles. However, an excess of DA in the cytoplasm of the cell is metabolized into 3,4-dihydroxyphenylacetic acid (DOPAC) by monoamine oxidase B enzyme (MAO-B) 58 and aldehyde dehydrogenase (ALDH). Then DOPAC is converted into homovanillic acid (HVA) by the action of catechol-O-methyltransferase (COMT) (see Figure 4a). 59 Besides, synaptic cleft DA can be taken up by surrounding glial cells and be degraded by MAO and also by COMT, which transforms DOPAC into HVA, one of the main degradation products of DA (Figure 4a). To study this metabolism and degradation kinetics, the intracellular concentration of DA and derived metabolites was determined using HPLC-ECD in (BE)2-M17 cells incubated with free DA and DA-NCPs for 2 h and at two concentrations (1 and 10 μg/mL). As shown in Figure 4b, in both cases levels of DA and metabolites increased though DOPAC and HVA levels were 5.3 times and 3.1 times, respectively, lower for DA-NCPs than for free DA (Figure 4b,c). In other words, at 1 μg/mL the DA/(DOPAC+HVA) ratios for free DA or DA-NCPs were 0.58 and 1.54, respectively (∼2.6-fold reduction for DA-NCPs). This difference was accentuated at 10 μg/mL (Figure 4d), where the DA/(DOPAC+HVA) ratios were 0.30 and 2.80 for DA and DA-NCPs, respectively (9.3fold increase). As anticipated, free DA exhibited a progressive reduction in DOPAC and HVA levels with time for the 1 μg/mL concentration (see Supporting Information, Figure S15), while a higher DOPAC production and less abrupt reduction were observed at 10 μg/mL (HVA levels remained quite homogeneous with time). A similar tendency was found for DA-NCPs at the lowest concentration, though at higher concentrations (10 μg/mL) the generation of metabolites remained constant and only fell after 24 h. These observations could be indicative of the DA-reservoir properties of DANCPs and a relatively slow release of free DA from the nanoformulation, which limits the formation of metabolites. Another essential factor to determine was how DA-NCPs (or DA released from them) could be metabolized by MAO-B. For this, we incubated (BE)2-M17 cells with the DA-NCPs in the presence of 2 μM deprenyl-R, a specific monoamine oxidase B inhibitor (MAOB-I). As shown in Figure 4e, the levels of DOPAC and HVA fell following treatment with this particular chemical inhibitor. Similar results were observed in the case of free DA-treated cells, suggesting that DA released from the nanoparticles could be actively processed by the oxidative DArgic mechanism. Finally, we evaluated the uptake of DA or DA-NCPs in the presence of specific inhibitors of endocytic pathways. As shown in Figure 4f, the presence of cytochalasin D (CytD) induced a small decrease in DA uptake for free DA, while both nystatin (Nys) and CytD caused an important reduction in the uptake of DA-NCPs. These results indicated that an essential fraction of the internalization of DA-NCPs was mediated by specific endocytic pathways. Moreover, actin/ dynamin interactions play a central role in the recycling of DAT via the bulk endocytic route. In fact, treatment of adult dopaminergic neurons with CytD (used to disrupt the actin Figure 6. Magnetic resonance imaging (MRI) experiments. (a) In vitro T1w and T2w MRI of phantoms with DA-NCPs in comparison with commercial GdDTPA at decreasing metal concentration. (b) Ex vivo DA-NCPs T1w (top) and T2w (bottom) MRI obtained after stereotactic injection of DA-NCPs. (c) Postprocessing of T1w and T2w images by an algebraic algorithm of imaging division (T1w/T2w). For the ex vivo experiments, DA-NCPs and GdDTPA were stereotactically injected in brain parenchyma. The dashed circles indicate the selection of ROIs for the RCE calculation. Color scales show the range of pixel values. ACS Nano www.acsnano.org Article https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 8599 filaments) significantly reduces DAT surface levels to 69.9 ± 5.1% of control levels. 63 This reduction in the number of receptors on the surface of the cells is compatible with the decrease in the DA uptake (63.1 ±2.8% compared to control) observed in our experiments upon CytD treatment. Figure 7. Intranasal administration of DA-NCPs and pharmacological effects on the 6-OHDA rat model of PD. (a) Schematic representation for the intranasal administration of the DA-NCPs. (b−f) Biodistribution of intranasally applied DA-NCPs. T1w MRI from the nasal cavity of a rat obtained (b) before (pretreatment) and (c) after (post-treatment) intranasal application of the nanoparticles. (d) DA levels detected in the plasma of healthy rats treated with vehicle or with a dose of DA-NCPs equivalent to 50 μg of free DA. (e) DA concentrations in the right striatum of healthy rats determined at two different time points (30 min and 2 h) post-DA-NCP treatment. Values are expressed as ng of DA/mg of protein. Data are presented as mean ±SEM. (f) Levels of DA in the right striatum of healthy rats. The test groups include animals treated with vehicle or intranasally administered with a dose of DA or DA-NCPs (dose equivalent to 50 μg of DA). Values are presented as percentage of DA compared to the vehicle-treated animals. Data are presented as mean ±SEM. (g) Apomorphine-induced rotational behavior of 6-OHDA-lesioned rats. Data are percentage of apomorphine-induced rotations determined 24 h postadministration. The net number of contralateral rotations was calculated as total left 360°−total right 360°turns, and data are presented as percentage of rotations in comparison with values achieved in basal conditions before treatment. Duration of the apomorphine-induced rotational behavior. Data are expressed as percentage of the time achieved in the basal experiment before treatment. The apomorphine-induced rotation test was performed in 6-OHDA-lesioned rats previously treated with repeated administrations of vehicle (n= 6), DA (n= 6), or DA-NCPs (n= 6). During the semichronic treatment, each animal received a total dose of DA or DA-NCPs equivalent to 800 μg of free DA, administered on 4 consecutive days (200 μg/day). In all cases, values are shown as mean ±SEM. In (d) and (e) ns (nonsignificant) when compared to vehicle; *p< 0.05, compared to vehicle (two-tailed t-test). In (f)−(h), ns, *p< 0.05, compared to vehicle; #p< 0.05, compared to treatment with DA-NCPs (one-way ANOVA and Tukey’sposthoc test). ACS Nano www.acsnano.org Article https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 8600 (15) Ren, Y.; Zhao, X.; Liang, X.; Ma, P. X.; Guo, B. 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