Article www.acsnano.org 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 * sı Supporting 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 L- DOPA administration, an amino acid precursor of DA that crosses the blood−brain barrier (BBB) while DA does not, although this approach presents medium- to 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 μg of 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 symptoms.1 However, long-term administration of this drug Parkinson’s disease (PD), the second most common neuro- is associated with developing highly disabling complications degenerative disorder in the world, affecting more than 10 such as drug-induced dyskinesia and motor fluctuations.8−10 million people,1,2 is characterized by motor symptoms Both complications are in part due to pulsatile L-DOPA including muscular rigidity, rest tremor, posture instability, administration and can be minimized with continuous and and bradykinesia.1 The origin is associated with early sustainable L-DOPA delivery by duodenal infusion.11 Evidence prominent death of DArgic neurons typically of the substantia nigra pars compacta (SNpc),3 which leads to diminished levels Received: January 17, 2021 of dopamine (3,4-dihydroxyphenethylamine, DA) in the Accepted: April 12, 2021 striatum of these patients.4−7 Mainly due to restrictions for Published: April 22, 2021 direct DA replacement, delivery of the precursor levodopa (L- 3,4-dihydroxyphenylalanine, L-DOPA) has been used in the clinic for more than 50 years to mitigate Parkinson © 2021 American Chemical Society https://doi.org/10.1021/acsnano.1c00453 8592 ACS Nano 2021, 15, 8592−8609 ACS Nano www.acsnano.org Article 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 DA- NCPs from DA, Fe(AcO)2, and BIX. (c) Proposed model for DA-based replacement therapy in Parkinson’s disease using DA-NCPs. suggests that up to 50% of PD patients develop these Neuromelanin (NM) particles, found in DArgic neurons complications after five years of chronic treatment with from the SNpc, incorporate large amounts of DA and iron levodopa. Therefore, the development of alternative strategies ions, which allow for their use as a biomarker for PD. to overcome these complications is an important challenge in Therefore, we hypothesize that melanin granular pigments modern medicine: among them, finding approaches for the found in nature may offer inspiration to develop more delivery of DA through its nanoencapsulation. effective DA nanocarriers.25 To mimic the synthesis of Nanocarriers can help DA to avoid metabolism, cross the melanin-like nanoparticles in the lab, whose tentative formula BBB, and promote a sustained release, reducing recurrent drug is shown in Figure 1a,26 three main approaches have been so administration and, therefore, adverse effects. So far, different far reported. In the first approach, self-oxidation of DA under families of nanocarriers have been described with this aim, alkaline conditions results in the formation of colloidal including chitosan12 and transferrin13 functionalized lip- polyDA (PDA) nanoparticles, which subsequently are doped osomes, (bio)hydrogels,14,15 chitosan nanoparticles,16,17 car- with the appropriate metal salt (postdoping approach).27−30 bon nanotubes,18 alginate/magnetite hybrid beads,19 carbon Alternatively, one-pot polymerization of DA in the presence of dots,20 carboxymethyl cellulose coated magnetic nano- transition-metal ions results in their inclusion in the final PDA particles,21 borneol and lactoferrin co-modified nanopar- (in situ doping).31−33 And the third approach involves the ticles,22 and a polyvinylpyrrolidone-poly(acrylic acid) nano- preformation of Fe(DA)3 monomeric complexes that system- gel.23 The work by Gupta et al. deserves special mention, atically control the percentage of iron in the particles.34−36 which reported DA-loaded poly lactic-co-glycolic acid (PLGA) Following these approaches, synthetic nanoparticles showing nanoparticles able to reverse functional deficits in Parkinso- excellent biocompatibility and biodegradability have already nian rats.24 However, despite these advances, most of the been prepared and successfully used in phototherapy/drug nanocarriers used for DA replacement therapy often suffer delivery37 and imaging.38−40 However, none of them were from low yield of encapsulation and/or poor biodistribution found to be useful for the delivery of DA, as the and drug bioavailability, so alternative encapsulation ap- polymerization and formation of the nanoparticles always proaches are required. take place through irreversible covalent bonds. 8593 https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 ACS Nano www.acsnano.org Article 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. Herein we envision that this could be achieved with the ditopic ligand BIX. After 2 h under constant stirring at room formation of a tailor-made coordination polymer, where the temperature, a dark precipitate was isolated by centrifugation central polymeric backbone is made of iron nodes linked to a and washed gently with ethanol to eliminate all the unbound bidentate ligand such as 1,4-bis(imidazol-1-ylmethyl)benzene ligands (see details in Methods and Supporting Information). (BIX) and DA is used as a counter ligand to complete the Scanning electron microscopy (SEM) and negative-staining coordination sphere. The composition of the resulting transmission electron microscopy (NS-TEM) images of the nanoparticles (named from now on as DA-NCPs) is expected obtained material revealed the presence of spherical nano- to have a structure inspired by NM, as shown in Figure 1a, particles with an average diameter of 64 ± 9.0 nm and 56 ± but with the fundamental difference being that the 9.0 nm, respectively (Figure 2a,c). These results were in incorporation of DA to the NP is reversible inside the cell. agreement with dynamic light scattering (DLS) measurements A schematic representation of the synthesis of the DA-NCPs of the DA-NCPs dispersed in ethanol, which showed a nanoparticles and the expected biological function is shown in hydrodynamic diameter of 81 ± 4.0 nm (PDI: 0.124) Figure 1b,c. (Supporting Information, Figure S1). Energy-dispersive X- ray analysis (EDX) profile of the TEM image clearly revealed RESULTS AND DISCUSSION the presence of iron homogeneously distributed along the Synthesis and Characterization of DA-NCPs. DA- whole nanoparticle (see Figure 2d), and Mössbauer spectros- NCPs were prepared following a procedure already described copy studies confirmed that the iron was in the high-spin for related catechol-based nanoparticles.41,42 In a typical Fe(III) form (Supporting Information, Figure S2). This synthesis, nanoparticles were obtained through a reaction of observation suggested that Fe(II) was oxidized to Fe(III) Fe(AcO)2 with DA hydrochloride in the presence of the along with the nanoparticle synthesis. In agreement, the 8594 https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 ACS Nano www.acsnano.org Article absorbance spectrum of DA-NCPs in the 400−800 nm range loading capacity (LC) of almost 7.7 μg of EGF per milligram exhibited a characteristic maximum at 562 nm, which was of particles (Supporting Information, Materials and Methods attributed to the formation of Fe-bis-catechol coordination and Figure S7). bonds (Figure 2e). This electronic modification has been Colloidal Stability and DA Release Kinetics under attributed to a redox interplay between the metal ion and Physiological Conditions. To simulate the colloidal stability electroactive catechol ligands in air, as previously reported.43 of DA-NCPs in physiological environments, the nanoparticles The absence of diffraction peaks observed by powder X-ray were dispersed in water and phosphate-buffered saline (PBS) diffraction (PXRD) indicated the amorphous nature of the solution (with and without bovine serum albumin, BSA) at nanoparticles (see Supporting Information, Figure S3). physiological conditions (0.5 mM).46,47 DLS measurements in The chemical characterization of DA-NCPs was addressed water (Supporting Information, Table S2) showed higher using different complementary techniques. Fourier transform hydrodynamic diameters (171 ± 4.2 nm; PDI: 0.208) than infrared spectroscopy (FT-IR) confirmed the presence of DA those obtained in ethanol (81 ± 4.0 nm; PDI: 0.124) or as a and a BIX ligand coordinated to the Fe(III) metal ions (see solid powder by SEM measurements (64 ± 9 nm). Moreover, Supporting Information, Figure S4). The FT-IR spectrum even though the ξ-potential value in water was close to +25 shows the disappearance of the bands corresponding to the mV, the colloidal solution was not stable and started to catechol −OH groups of DA in the range of ν = 3000−3500 aggregate and precipitate over a relatively short period of time cm−1, reflecting their deprotonation, the stretching and (sedimentation observed after 1 h). In PBS (20 mM PBS bending vibrations of the amine group (3340 and 1618 buffer at pH 7.4), the mean size values for nanoparticles cm−1, respectively), and the stretching vibrations of C−H indicated the presence of large aggregates (Supporting (3040 cm−1) and C−C of the aromatic ring (1550 cm−1). Information, Table S2) and showed a ξ-potential of −13.1 Moreover, the vibration band assigned to a C−O stretch for mV. The addition of 0.5 mM BSA, extensively used for the catechol coordinated to the metal appeared in the dispersing inorganic and polymeric nanoparticles,48,49 success- frequency range 1200−1300 cm−1. Additionally, typical fully induced a disaggregation process and their stabilization vibrational bands of the BIX ligand (ν = 1520, 1262, 1105 without precipitation for several hours. The nanoparticles now cm−1) were observed in the FT-IR spectra. The 1H NMR showed a lower hydrodynamic diameter (103 ± 16.2 nm; spectrum of DA-NCPs dissolved in a deuterated methanol- PDI: 0.198), similar to that observed using culture medium HCl solution confirmed the presence of acetate counterions containing fetal bovine serum (FBS) (Supporting Information, (see Supporting Information, Figure S5) and both ligands, Table S2). The increase in size (24−28 nm) with respect to although with a lower contribution of the BIX ligand than the value found in ethanol could be attributed to the theoretically expected from the concentrations of the initial formation of a protein corona around the nanoparticles. reactants. These results were quantitatively confirmed by high- We next investigated the release kinetics of DA from DA- performance liquid chromatography (HPLC) analysis of the NCPs nanoparticles after incubation for 24 h in a PBS−BSA product resulting upon treatment of DA-NCPs with an acidic buffer at 37 °C, using HPLC-ECD. At pH 7.4, a continuous solution; these analyses showed a content of 20% BIX (w/w) release of DA, reminiscent of a standard saturation curve ligand. More interestingly, HPLC analysis using electro- (Supporting Information, Figure S8), was observed with a chemical detection (HPLC-ECD) confirmed the presence of cumulative release of 50% in the first 2 h (t1/2 = 2 h). 52.5 ± 7.2% DA in DA-NCPs nanoparticles (see Supporting Afterward, the release notably slowed down with an increase Information and Materials and Methods section for of 5% in the next 22 h. If the nanoparticles were dispersed at experimental details). This exceptionally high value was pH 5.5, the accumulative release of 50% was reached in less confirmed with absorbance measurements using DA hydro- than 1 h. These results confirmed that the pH decrease chloride as a standard (Supporting Information, Figure S6). induced a much faster release of the DA due to the lower These analyses also revealed a very high loading efficiency stability of the coordinative bond between iron and catechol (LE) of 69.7 ± 7.2%. For instance, this value is 6-fold higher ligands. Finally, the release increased up to 85% in 10 h and compared with the LE of dopamine achieved with PLGA in 90% at 24 h. This pH dependence has been observed previous reports. As far as iron is concerned, inductively previously in mussel-inspired hydrogels with Fe-catechol coupled plasma-mass spectroscopy (ICP-MS) data confirmed complexes and in other Fe-containing polymeric nano- a total amount of 10.2% in the nanoparticles. Finally, the particles.30,50 combination of elemental analysis, ICP-MS, 1H NMR, and Worth mentioning is that the FBS used in the previous HPLC data yielded the following final tentative chemical experiments was heat-inactivated and does not fully represent formula for the nanoparticles: [Fe- the real physiological conditions found in the blood and (DA)1.6(BIX)0.5(AcO)0.2(H2O)1.9] (see Supporting Informa- plasma under in vivo conditions. For this reason, we also tion, Table S1). This formula differs from that theoretically performed an additional experiment to study the colloidal expected on the basis of the initial reagents added (1 Fe:1 stability of the DA-NCPs in the presence of nontreated BIX:2 DA), which is attributed to their synthesis under out-of- human plasma. For this, an aliquot of DA-NCPs was equilibrium conditions induced by the fast precipitation suspended in 1 mL of ethanol or in 1 mL of nontreated process, a common phenomenon for this family of amorphous human plasma. Afterward, the hydrodynamic diameter of the NCPs. Another plausible explanation for such differences nanoparticles was evaluated at different time points of could be the encapsulation of free ligand/solvent molecules incubation at 37 °C. No relevant changes in the size within the particles present in the reaction solution, as already distribution of the nanoparticles up to 6 h incubation under demonstrated in related systems.44,45 Such encapsulation these conditions were detected. Only a small shift toward ability has been confirmed by encapsulating, as a proof-of- higher dimensions was observed, compatible with the concept, the recombinant epidermal growth factor (EGF) progressive formation of the protein corona due to the high with an excellent encapsulation efficiency (EE) of 89.6%, and concentration of proteins in the human plasma. As expected, 8595 https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 ACS Nano www.acsnano.org Article 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’s posthoc test). the presence of these proteins was visible as a secondary peak place both enzymatically (monoamine oxidase, MAO) or (peak 2, in Figure S9) with a size of ∼40 nm. nonenzymatically. ROS can also be generated upon DA Evaluation of the Cytotoxicity of DA-NCPs against oxidation; when free DA cytosol levels exceed a certain DArgic Neurons. To evaluate the effect of DA-NCPs on threshold level, DA can spontaneously autoxidize, producing DArgic cell survival, in vitro cytotoxicity assays with (BE)2- o-quinone molecules.52 These species are very reactive and M17 cells were performed. (BE)2-M17 is a neuroblastoma- may contribute to increasing the levels of oxidative stress derived DArgic cell line that expresses large amounts of key inside the cells. To confirm this fact, intracellular ROS proteins essential for the DArgic metabolism and, therefore, formation in BE(2)-M17 cells was also measured after extensively used as a cellular model to study DA treatment with DA or DA-NCPs, through the 2′,7′- metabolism.51 Treatment of the (BE)2-M17 cells with DA- dichlorofluorescein diacetate (DCFDA) assay (see Materials NCPs did not lead to any appreciable cell viability reduction and Methods).53 After a 24 h treatment, the 300-fold DCF after 48 h of incubation at concentrations up to 100 μg/mL fluorescence increase found for the highest free DA (expressed as DA concentration) (see Supporting Informa- concentration (see Supporting Information, Figure S11) tion, Figure S10). The same concentrations of free DA in the confirmed the ROS production.24 Ascorbic acid (AA), at range from 0.01 to 100 μg/mL showed comparable results. physiological concentrations of 20 μg/mL, was required to However, slightly higher levels of the free neurotransmitter drastically reduce ROS effects of H2O2. Interestingly, DA- (i.e., 200 and 250 μg/mL) caused a dramatic death of DArgic NCPs showed a lower DCF fluorescence increase (200-fold) cells with more than 85% reduction in the cell viability even considering the possible oxidative stress coming from (Supporting Information, Figure S10). Interestingly, compa- iron ions (Supporting Information, Figure S12). rable concentrations of DA-NCPs caused lower cytotoxic On the basis of these findings, we can state that our effects. The toxicity found for the highest concentrations of nanoparticles did not show a reduction in cell viability at the DA was attributed to the intracellular metabolism of DA assayed DA concentrations in the range 0.01 to 10 μg/mL54 (conversion of DA to 3,4-dihydroxyphenylacetaldehyde, and that DA nanostructuring into DA-NCPs reduced the DOPAL) that generates hydrogen peroxide and, therefore, toxicity of the monoamine against DArgic cells. Similar reactive oxygen species (ROS) effects. This process can take observations were reported by Pahuja et al., 24 who 8596 https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 ACS Nano www.acsnano.org Article 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: L- DOPA; 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. demonstrated a reduction in the toxicity and oxidative damage intracellular concentration of DA was measured by HPLC- induction of L-DOPA upon encapsulation into PLGA ECD (for more details, see Supporting Information and nanoparticles. Evaluation of the toxicity of the iron salt and Materials and Methods). the linker ligand was performed in (BE)2-M17 cell culture Free DA. Incubation of (BE)2-M17 cells in the presence of using concentrations ranging from 0 to 100 μg/mL for 1 μg/mL of free DA resulted in a rapid concentration- comparison purposes (see Supporting Information, Figure dependent increase in intracellular DA level after 2 h of S13), with no cytotoxicity detected even at the highest incubation (Figure 3a). This fact could be attributed to a large concentration assayed (100 μg/mL). Finally, and for amount of DA transporters (DATs) that DArgic neurons use comparison purposes, we also studied the cytotoxicity of DA to control extracellular DA levels by pumping the extracellular and DA-NCPs in a non-DArgic cell line such as HeLa cells DA from the synaptic cleft to the cytosol.55 As shown in (Supporting Information, Figure S13), where as expected, Figure 3a, at longer incubations times (6 and 24 h), a IC50 was smaller for both cases (see Supporting Information, progressive decrease in the total intracellular DA concen- Table S3). tration was observed. This decrease was accompanied by a Uptake of DA-NCPs by DArgic (BE)2-M17 Cells. The constant reduction in the extracellular DA levels (see DA-NCPs’ uptake kinetics by DArgic (BE)2-M17 cells was Supporting Information, Figure S14). The rate of uptake of studied and compared to free DA. To perform the DA through DAT is assumed to follow characteristic experiments, cells were exposed to increasing concentrations saturation kinetics.56 To confirm this, the experimental data of DA or DA-NCPs and incubated for 2, 6, and 24 h. obtained for the different DA concentrations used at 2 h were Immediately after incubation, cells were collected and the plotted and precisely fitted to Michaelis−Menten kinetics, 8597 https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 ACS Nano www.acsnano.org Article 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 inf ra). (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. with approximate Km and Vmax values of 0.1 μg/mL and 10.0 DA present in the cytoplasm. In fact, only the excess of DA ng·mg prot−1·min−1, respectively (Figure 3b). All in all, the accumulated in the cytoplasm of the cells is available and can results suggested that DA was actively taken up and be degraded by the MAO. Therefore, the overall effect is a metabolized by the DArgic (BE)2-M17 cells due to the progressive and slow decline of intracellular DA levels that do action of the intracellular DArgic mechanism. not fully correspond to the extracellular DA concentration. DA-NCPs. Incubation of cells with DA-NCPs accounting Finally, after 24 h of incubation in the presence of 1 μg/mL, overall for the same concentration of DA showed different DA-NCPs-exposed cells displayed a 4-fold amount of intracellular profiles (Figure 3c,d). At 2 h and the lowest internalization compared to control and free DA-treated concentration (1 μg/mL), the DA internalized amount was cells (Figure 3a and f), where depletion of the extracellular slightly lower than that of free DA. Incubation of cells with DA caused normalization of the intracellular levels of the higher doses (10 μg/mL) for the same period of time induced neurotransmitter. This finding is of particular significance as an intracellular level of DA up to 6360 ng/mg. This demonstrated by the enhanced intracellular retention of the concentration was 6-fold higher than that obtained for free neurotransmitter for longer periods of time upon nano- DA (1156 ng/mg) under the same concentration and structuring. experimental conditions. Moreover, the plot of DA internal- Metabolism Kinetics of DA and DA-NCPs by DArgic ization vs the initial external concentration used showed a Cells. The death of DArgic neurons in the substantia nigra nonsaturable dose−response mechanism instead of the leads to deficient striatal DA levels responsible for the cardinal characteristic Michaelis−Menten kinetics (Figure 3d), sug- motor symptoms of PD (i.e., including bradykinesia, tremor, gesting the presence of another pathway for nanoparticle and rigidity).57 Under normal conditions, the stimulation of uptake. Similar results and differences were found at 6 h of healthy DArgic neurons causes the release of the synaptic incubation, in agreement with the presence of DA outside the vesicles into the synaptic cleft, where the neurotransmitter cells at this incubation time (Figure S14). interacts with the postsynaptic DA receptors or regulatory Note that the total intracellular DA determined by HPLC- presynaptic DA autoreceptors.58−62 When DA reaches the ECD represents the sum of vesicular DA and the fraction of cytoplasm of DArgic neurons, this neurotransmitter is rapidly 8598 https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 ACS Nano www.acsnano.org Article 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. stored back into the synaptic DA storage vesicles. However, were observed at 10 μg/mL (HVA levels remained quite an excess of DA in the cytoplasm of the cell is metabolized homogeneous with time). A similar tendency was found for into 3,4-dihydroxyphenylacetic acid (DOPAC) by monoamine DA-NCPs at the lowest concentration, though at higher oxidase B enzyme (MAO-B)58 and aldehyde dehydrogenase concentrations (10 μg/mL) the generation of metabolites (ALDH). Then DOPAC is converted into homovanillic acid remained constant and only fell after 24 h. These observations (HVA) by the action of catechol-O-methyltransferase could be indicative of the DA-reservoir properties of DA- (COMT) (see Figure 4a).59 Besides, synaptic cleft DA can NCPs and a relatively slow release of free DA from the be taken up by surrounding glial cells and be degraded by nanoformulation, which limits the formation of metabolites. MAO and also by COMT, which transforms DOPAC into Another essential factor to determine was how DA-NCPs (or HVA, one of the main degradation products of DA (Figure DA released from them) could be metabolized by MAO-B. 4a). For this, we incubated (BE)2-M17 cells with the DA-NCPs in To study this metabolism and degradation kinetics, the the presence of 2 μM deprenyl-R, a specific monoamine intracellular concentration of DA and derived metabolites was oxidase B inhibitor (MAOB-I). As shown in Figure 4e, the determined using HPLC-ECD in (BE)2-M17 cells incubated levels of DOPAC and HVA fell following treatment with this with free DA and DA-NCPs for 2 h and at two concentrations particular chemical inhibitor. Similar results were observed in (1 and 10 μg/mL). As shown in Figure 4b, in both cases the case of free DA-treated cells, suggesting that DA released levels of DA and metabolites increased though DOPAC and from the nanoparticles could be actively processed by the HVA levels were 5.3 times and 3.1 times, respectively, lower oxidative DArgic mechanism. for DA-NCPs than for free DA (Figure 4b,c). In other words, Finally, we evaluated the uptake of DA or DA-NCPs in the at 1 μg/mL the DA/(DOPAC+HVA) ratios for free DA or presence of specific inhibitors of endocytic pathways. As DA-NCPs were 0.58 and 1.54, respectively (∼2.6-fold shown in Figure 4f, the presence of cytochalasin D (CytD) reduction for DA-NCPs). This difference was accentuated at induced a small decrease in DA uptake for free DA, while 10 μg/mL (Figure 4d), where the DA/(DOPAC+HVA) ratios both nystatin (Nys) and CytD caused an important reduction were 0.30 and 2.80 for DA and DA-NCPs, respectively (9.3- in the uptake of DA-NCPs. These results indicated that an fold increase). essential fraction of the internalization of DA-NCPs was As anticipated, free DA exhibited a progressive reduction in mediated by specific endocytic pathways. Moreover, actin/ DOPAC and HVA levels with time for the 1 μg/mL dynamin interactions play a central role in the recycling of concentration (see Supporting Information, Figure S15), DAT via the bulk endocytic route. In fact, treatment of adult while a higher DOPAC production and less abrupt reduction dopaminergic neurons with CytD (used to disrupt the actin 8599 https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 ACS Nano www.acsnano.org Article 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’s posthoc test). filaments) significantly reduces DAT surface levels to 69.9 ± 5.1% of control levels.63 This reduction in the number of decrease in the DA uptake (63.1 ± 2.8% compared to receptors on the surface of the cells is compatible with the control) observed in our experiments upon CytD treatment. 8600 https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 ACS Nano www.acsnano.org Article In Vivo Biodistribution and Metabolism of DA-NCPs based ligands to maximize second-sphere interactions with within the Brain. To examine the in vivo brain water molecules and therefore enhance the T1w MRI signal biodistribution of DA-NCPs, a single dose was infused in through hydrogen bonding with the oxygen atoms of the Fe− the lateral ventricle of healthy adult male Sprague−Dawley O−R linkages.71 Moreover, DA-NCPs probably contained rats (Figure 5a) through a surgical cannula installed in the water molecules coordinated to the metal sites, as well as free right ventricle. The i.c.v. infusion of DA and DA agonist has water molecules, which probably exchange with surrounding been attempted in the past in rodents, primate models, and water molecules, diffusing through the polymeric matrix.72 human patients of PD as an alternative to conventional Encouraged by these exceptional results and considering that treatments.64 Venna and co-workers reported a good tolerance phantom results are not always fully comparable with contrast to continuous i.c.v. DA infusion in patients treated over 1 enhancement in real tissues,73−75 ex vivo studies with DA- year.65 This continuous DA delivery resulted in a more stable NCPs were conducted following a methodology previously treatment effect without the “on−off” fluctuations of tradi- reported.76 For this, PBS−BSA dispersions of DA-NCPs and tional oral therapy. commercial GdDTPA (for comparison purposes) were The materials were well tolerated by the animals, as no intracranially administered post mortem to euthanized mice. adverse effects nor increased mortality was observed during T1w and T2w MRI was subsequently acquired, and relative nanoparticle administration, in agreement with the low in vitro contrast enhancement (RCE) calculated (see Table S4) toxicity values and the absence of systemic toxicity previously (details about calculations and protocol can be found in the reported. The animals were then euthanized to measure the Supporting Information and Materials and Methods section). DA and DA metabolite levels of different brain regions using Three different regions of interest (ROIs) were selected in HPLC-ECD at different time points after intracerebroven- T1w and T2w images, corresponding to the CA administration tricular (i.c.v.) injection. As shown in Figure 5, distribution of points (Figure 6b). DA-NCPs presented higher RCE T1 in DA-NCPs from the right lateral ventricle to the surrounding comparison with the commercial CA GdDTPA and neuronal tissues occurred mostly within the first 2 h, with a comparable RCE T2. The postprocessing of T1w and T2w notable increase in DA levels observed in the striatum and images by an algebraic algorithm of imaging division (T1w/ SNpc (Figure 5b,c and Figure S16). This observation T2w) showed a clear brightness area when DA-NCPs were suggested that DA released from the nanoparticles was readily injected (Figure 6c) into the brain, confirming the excellent bioavailable and could be efficiently incorporated by behavior of DA-NCPs for MRI monitoring. nigrostriatal DArgic neurons in vivo. Intranasal Administration of DA-NCPs. The direct It is worth mentioning that higher amounts of DA could be infusion of DA-NCPs demonstrated rapid biodistribution and detected in the right striatum ipsilateral to the site of pharmacokinetic potential. However, this administration route administration, in comparison with the levels detected for the is not an appropriate choice for the continued treatment of left striatum. In addition to the nigrostriatal track, we PD patients since it is invasive and requires a surgical identified small amounts of DA in other regions of the procedure for the insertion of the i.c.v. device. So, the brain, such as the cortex or the cerebellum (Figure 5d). potential application of DA-NCPs for nose-to-brain delivery of Striatal DA concentration recovered its basal levels after 24 h DA was then evaluated (Figure 7a) in rats. First, we aimed to postinjection, indicating that DA-NCPs were completely track the biological pathway followed by the nanoparticles cleared and/or metabolized in the brain by DArgic neurons after their intranasal administration using in vivo MRI. As or by surrounding glial cells (Figure 5e,f). Thus, we further shown in Figure 7a and b, a strong T1w signal was clearly investigated the DA-NCPs metabolism in the brain by observed in the right nasal cavity of the rat, near the measuring the levels of the two main metabolites, DOPAC respiratory epithelia. T1 values measured within ROIs from and HVA. As shown in Figure 5g−j, DOPAC and HVA levels three consecutive sections were significantly decreased after showed an increase in the right striatum after 2 and 6 h different preadministration of DA-NCPs (T1 = 707 ± 119 ms) postadministration, in agreement with the in vitro metabolism compared to postadministration values (1631 ± 91 ms, t-test: experiments using DArgic cells. p = 0.003). Previous intranasal studies demonstrate that drugs DA-NCPs Display Magnetic Resonance Imaging gain direct access to the central nervous system (CNS) via Properties. Longitudinal (r1) and transversal (r2) relaxivity direct pathways from the nasal cavity.77 A more detailed values were determined at 7 T by measuring corresponding analysis of different longitudinal and coronal sections of the relaxation rates in phantoms containing different concen- anterior rat head and nasal cavities (Figure S18a,b) elucidated trations of DA-NCPs dispersed in PBS/agarose 1% solution at the presence of a strong DA-NCPs signal in the right duct of pH 7.4 (T1 and T2 map sequences, see detailed protocols in the vomeronasal organ. These results clearly showed that DA- the Supporting Information; representations of R1, R2 vs metal NCPs were in contact with the mucosa and the epithelia of concentration are shown in Supporting Information, Figure the nasal cavity immediately before they were transferred into S17). As presented in Figure 6a, samples showed increased the CNS via the olfactory bulb.78 T1w enhancement (brighter signal) with the increase of the Plasma DA levels of healthy rats after 2 h of administration metal ion concentration. The r1 value was determined to be did not show relevant differences between samples taken from 5.1 ± 0.6 mM−1 s−1 for DA-NCPs, which was comparable rats treated with DA-NCPs (dose equivalent to 50 μg of free with related iron-based NCPs (i.e., r1 = 4.4 mM−1 s−1 for Fe- DA) or only with the vehicle (Figure 7d), indicating that DA NCPs),66,67 previously reported iron-based coordination was not transferred to systemic circulation after this time. By polymers,68,69 paramagnetic molecular complexes (3.0 mM−1 contrast, significantly higher amounts of striatal DA were s−1),70 or even commercial gadolinium diethylenetriamine- detected in the ipsilateral side of the rat brains after 30 min pentacetate (GdDTPA) contrast agents (CAs) (Magnevist, and 2 h of unilateral DA-NCPs nasal administration (Figure 4.4 ± 0.2 mM−1 s−1) (Figure 6b). Such an exceptional 7e). The maximum DA concentration was achieved after 30 response could be directly related to the ability of catechol- min with a 3.5-fold increase in comparison with basal striatal 8601 https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 ACS Nano www.acsnano.org Article DA levels, consistent with observations noted in earlier Finally, we studied direct delivery of the nanoparticles to intranasal investigations.78 Time evolution analysis of HVA the brain via nasal administration. Our findings showed an and DOPAC metabolites in the same samples nicely efficient delivery of DA to the nigrostriatal pathway capable of correlated with that of DA, showing a clear increase of attenuating motor alterations in a 6-OHDA-induced animal these two metabolites associated with the simultaneous model of PD. On top of that, the presence of iron ions was activation of the DA catabolism in the right striatum. used to track the nanoparticles by MRI after postprocessing of Interestingly, no significant increase in DA was observed in the T1w and T2w images. All in all, these results position this the contralateral striatum after unilateral administration of last generation of DA-based nanoparticles in the field of DA DA-NCPs (Figure S18c). Similar to DA-NCPs, unilateral replacement therapy with nanoparticles. intranasal administration of an equivalent dose of free DA into the right nostril also led to a clear increase of the striatal DA, MATERIALS AND METHODS reaching comparable levels to those observed in the case of the nanoparticles (Figure 7f). One-Pot Synthesis of DA-NCPs. DA-NCPs were prepared according to our previously published method for the synthesis of Pharmacological Effect in 6-HydroxyDA (6-OHDA)- NCPs with some modifications.41,44,79 Briefly, a mixture of DA Lesioned Rats. To determine if these increased levels of DA hydrochloride (0.5 mmol, 95 mg) and BIX (0.25 mmol, 60 mg) was in the striatum of DA-NCPs-treated rats are physiologically dissolved in 12.5 mL of ethanol. Under constant magnetic stirring relevant, we examined the pharmacological effect of the (900 rpm), an ethanolic solution of Fe(CH3COO)2 (0.25 mmol, 44 nanoparticles in 6-hydroxyDA (6-OHDA)-lesioned rats, a PD mg in 2.5 mL of ethanol) was added to the DA−BIX solution. The model characterized by a typical unilateral drug-induced reaction mixture was stirred at room temperature for 2 h, and the degeneration of the nigrostriatal DArgic neurons on the same suspension rapidly turned from colorless to black, which indicates the side as neurotoxin injection. Measurements following intra- formation of a fine precipitate. After 2 h, the precipitate was peritoneal injection of apomorphine, a potent DArgic agonist, centrifuged at 8000 rpm for 10 min. The pellet obtained was resuspended and washed three times with ethanol. The sample was in the 6-OHDA-lesioned rats revealed a characteristic divided into two batches; in one of them, the solvent was removed contralateral turning behavior (average of rotations in all the and the solid dried under vacuum and fully characterized by UV−vis, groups analyzed in the basal test: 249 ± 146 contralateral FT-IR, ICP-MS, elemental analysis, 1H NMR, and HPLC. The turns; average duration of the rotational behavior: 57 ± 11 remaining batch was kept under solution to avoid problems with min) in response to the stimulation of the DA receptors. After aggregation of the sample during the drying procedure and was 4 days of nasal administrations with DA-NCPs equivalent to characterized by DLS, zeta potential, TEM/STEM, and HPLC. For 200 μg of the free drug per day, the number and duration of in vivo experiments, the final pellet containing the nanoparticles was apomorphine-induced rotations were significantly lower from resuspended in ethanol (to reach a final DA-NCPs concentration of that in either vehicle- or DA-treated rats performed for ≥20.0 mg/mL (w/v)) and stored under solution at 4 °C until use. comparison purposes (Figure 7g,h). This shortening in the Size and Zeta-Potential Measurements. Mean particle size and zeta potential of the DA-NCPs were determined by DLS using a motor response is compatible with the continuous presence of Zetasizer Nano 3600 instrument (Malvern Instruments, UK). DA in the nigrostriatal pathway, indicating desensitization in Samples of the nanoparticles were dispersed in five different solvents: unilateral 6-OHDA-lesioned rats continuously treated with the ethanol absolute, 70% ethanol, PBS, PBS with 30 mg/mL (∼0.5 nanoparticles. Compared to DA-treated animals, the different mM) of BSA, or Opti-MEM culture medium containing 10% heat- therapeutic response observed in DA-NCPs might be inactivated FBS. In order to study the physical stability of DA-NCPs attributed to their more sustained release potential. over time, an aliquot of nanoparticles was dispersed in 70% ethanol, PBS−BSA, or culture medium with FBS, and each suspension was CONCLUSIONS analyzed immediately or after 10, 30, 60, and 120 min of incubation at 25 °C. In all cases, the nanoparticles were dispersed at a Inspired by natural NM, we have reported a family of DA- concentration of 100 μg/mL. Measurements were performed in based coordination polymer nanoparticles as stable colloidal automatic mode, as an average of 14−24 runs and in triplicate. suspensions with FBS or BSA and high encapsulation yields TEM and EDX Analysis. Samples of DA-NCPs were suspended (close to 60%). The encapsulated DA exhibited a reduced in ethanol, diluted to 200 μg/mL, and placed onto carbon-coated toxicity against DArgic cells by comparison with free DA and grids. After 2 min, grids were dried carefully and negatively stained a decreased ROS production even with the incorporation of with a 2% (w/v) uranyl acetate solution for 2 min. Micrographs were iron ions. In fact, the nanoparticles proved safe in vivo, with no acquired under a Hitachi H-7000 TEM with a 75 kV accelerating significant weight loss or toxicity symptoms for the 30-day voltage. The diameter of the particles was measured using ImageJ software.80 For EDX analysis, DA-NCPs were diluted in ethanol, period after the last injection administration. The nano- layered over carbon-coated grids, and then preparations washed twice structuring as DA-NCPs also favors (in comparison with the with distilled water. An EDX spectrum of the nonstained nano- incubation of DArgic cells with free DA) enhanced intra- particles was obtained in a Jeol JEM-1400 microscope operating at cellular retention of the neurotransmitter intracellularly 120 kV, by collecting X-rays emitted from the local volume probed released for extended periods of residence. This fact is mainly by the electron beam. attributed to both a very effective incorporation of the SEM Analysis. The structural properties (shape, surface nanoparticles by DArgic cells mediated by specific endocytic morphology, and particle size distribution) of the DA-NCPs were pathways and to slower metabolism kinetics into DA studied by a high-resolution field emission SEM in a Hitachi S-570 metabolites (i.e., DOPAC and HVA) due to the restrictions SEM. Samples of nanoparticles were diluted in ethanol and placed over an aluminum tape. Following solvent evaporation, samples were imposed by the DA supramolecular assembly. The i.c.v. coated with platinum under an argon atmosphere by using an infusion of the nanoparticles into the living brain of Sprague− Emitech K550 sputter coater (Emitech Ltd., Ashford, Kent, UK). Dawley rats was sufficient to promote an increase in striatal SEM micrographs were acquired at an accelerating voltage of 15 kV and SNpc DA levels as soon as 2 h after administration. DA under a high-vacuum mode and a distance of the sample of 5 mm. metabolites also increased accordingly, suggesting an efficient UV−Vis Spectroscopy and Spectroscopic DA Quantitation. clearance of the excess of DA in these living tissues. The absorbance spectra of DA-NCPs, as well as spectra of its 8602 https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 ACS Nano www.acsnano.org Article polymeric precursors (DA, and BIX), were recorded using a Cary data acquisition, and monoamine determinations were carried out 4000 spectrophotometer (Agilent), in the 200−800 nm wavelength using EZChrom Elite version 3.1.7 software. range, and using a matched pair of quartz cuvettes of 1 cm optical Cell Culture. The human BE(2)-M17 (ATCC CRL-2267) and path length. Immediately before analysis, samples were diluted in HeLa (ATCC CCL-2) cell lines were maintained in Opti-MEM or filtered Milli-Q water to a final concentration of 100 μg/mL. For the minimum essential medium α (MEM-α) medium, respectively. Both spectroscopic DA quantitation, 5 mg of dried DA-NCPs was media were further supplemented with 10% (v/v) FBS. Cells were dissolved in 5 mL of 1 M HCl, sonicated until the complete grown under a highly humidified atmosphere of 95% air with 5% dissolution of the nanoparticles, and diluted up to a concentration of CO2 at 37 °C. 0.1 mg/mL in 1 M HCl. Standards with different DA concentrations In Vitro Cytotoxicity Experiments. The cytotoxicity of the free (ranging from 0 to 0.4 mM) were prepared by diluting a stock ligands (DA, BIX, and iron acetate) and DA-NCPs toward BE(2)- solution (1 mM) of DA hydrochloride in 1 M HCl solution. The M17 and HeLa cells was evaluated by a resazurin-based assay using absorbance of the different samples was determined at 278 nm using the PrestoBlue cell viability reagent. Briefly, BE(2)-M17 cells were quartz cuvettes (1 cm optical path length) in a Cary 4000 seeded in 96-well plates at a concentration of 3 × 103 cells per well spectrophotometer (Agilent). The amount of loaded DA was and incubated for 24 h. After incubation, cells were treated with DA, calculated from the calibration curve (adjusted to a linear regression BIX, iron acetate, and DA-NCPs at different concentrations (ranging model with R2 > 0.99) (see Supporting Information, Figure S6). The from 0 to 100 μg/mL (w/v) for BIX and iron acetate or from 0 to loading efficiency was determined using the following equation: LE 250 μg/mL (w/v) for DA and DA-NCP). After 48 h of incubation, in DA-NCPs (%) = (WeightDA)/(WeightNCPs) × 100. aliquots of 10 μL of the PrestoBlue cell viability reagent solution FT-IR Spectroscopy. FT-IR spectra of the nanoparticles and were added to each well and cells were incubated for 1 h at 37 °C in precursors were recorded by transmission FT-IR on a Bruker Tensor the absence of light and in the presence of a highly humidified 27 (Bruker Optics) spectrophotometer. Pellets were prepared by atmosphere of 95% air with 5% CO2. Fluorescence (ex/em = 531/ mixing approximately 1 mg of the test sample with 100 mg of KBr, 572 nm) of each well was measured using a fluorescence microplate followed by subjection to high pressure using a manual pellet marker. reader (PerkinElmer Victor 3V). Cell cytotoxicity was evaluated in Each spectrum was acquired between 400 and 4000 cm−1 at a terms of BE(2)-M17 cell growth inhibition in treated samples and spectral resolution of 2 cm−1. expressed as percent of the control assay. All cytotoxicity experiments 1 H NMR Measurements. The chemical composition of DA- were performed at least in triplicate. NCPs was further investigated by 1H NMR. In a typical experiment, Reactive Oxygen Species Determination. For ROS determi- 5 mg of dry DA-NCPs were dispersed in deuterated methanol. To nation, BE(2)-M17 cells were seeded in a 96-well plate at a density ensure complete dissolution of the nanoparticles and the liberation of of 10 × 104 cells/well. After 24 h, DCFDA was added at a the ligands by demetalation, two drops of deuterated hydrochloric concentration of 25 μM, and the cells were incubated at 37 °C (5% acid were added to the suspension. After incubation for 30 min at 25 CO2), in the absence of light, for 30 min. DCFDA is a °C, 1H NMR spectra were recorded using a Bruker Avance DRX 250 nonfluorescent and permeable dye that, after cleavage and oxidation by ROS or intracellular esterases, generates dichlorofluorescein NMR spectrometer equipped with a 5 mm broad band inverse (DCF), a fluorescent nonpermeable compound. All conditions probe-head working at 250 MHz for 1H. Chemical shifts were were tested in the presence or absence of a natural antioxidant, referenced to the trace signals of acetone. The reference sample was AA, which, additionally, would avoid DA autoxidation. After prepared by mixing 10 mL of acetone into 0.5 mL of deuterated incubation, the media was replaced, and cells were exposed for 24 methanol in a WILMAD coaxial insert tube, which was immersed in h to DA, DA-NCPs (with an equivalent DA concentration of 100, 10, the 5 mm NMR sample tube (acetone chemical shift: 2.15 ppm). As and 1 μg/mL), and iron acetate (at concentrations corresponding to a reference, the different ligands (DA, BIX) were dissolved in the same as the iron present in DA-NCPs). Hydrogen peroxide deuterated methanol, acidified with deuterated hydrochloric acid, and (H2O2) was used as a positive control at a concentration of 250 μM. characterized by 1H NMR spectroscopy. Cell culture medium was supplemented or not with AA at 20 μg/mL. Simultaneous Determination of Monoamines Using HPLC- The fluorescence of each well was measured at 535 nm with a ECD. An HPLC-ECD method for the simultaneous quantification of Victor3 microplate reader (PerkinElmer), after excitation at 485 nm. monoamines (including DA, DOPAC, and HVA) was adapted from Results are reported as an average of two independent experiments. previously described methods.81 In brief, analyses were performed The analysis of variance was evaluated with one-way ANOVA using a Chromolith Performance RP-18e (100 mm × 4.6 mm, performed for ROS assays. The comparison between different groups Merck-Millipore, Darmstadt, Germay) column in an Elite LaChrom was assessed by applying the Tukey-HSD test. Significance level was system from Hitachi (Tokyo, Japan) coupled to a Coulochem 5100A set to 0.05. electrochemical detector from ESA (Chelmsford, MA, USA). The Cellular Uptake of DA and DA-NCPs and Metabolism detector was equipped with a 5011 dual-electrode analytical cell with Experiments. To study the uptake and metabolism of free DA and porous graphite electrodes set at +5 and +400 mV (for electrodes 1 DA-NCPs, human DArgic neuroblastoma BE(2)-M17 cells were and 2, respectively). Before the analysis, the column was pre- seeded at a density of 2.5 × 105 cells/plate onto 60 mm culture equilibrated in the mobile phase containing 99% v/v of a buffered plates and grown for 24 h (to reach about 60−70% confluence). aqueous solution (0.1 M citric acid, 0.05 mM EDTA, 1.2 mM After incubation, the medium was removed, and cells were treated sodium octyl sulfate) with 1% acetonitrile. The final pH of the with a control medium or with a medium containing the specified solution was adjusted to pH = 2.7 with triethylamine. Elution of the concentrations of free DA or DA-NCPs for 2, 6, and 24 h. To monoamines was performed with an isocratic elution of the mobile prevent DA oxidation during treatments, media were further phase, at 25 °C and a flow rate of 1 mL/min. Before analysis, supplemented with 20 μg/mL AA. samples of nanoparticles, culture medium, or cell lysates were For DA quantification, immediately before cell recovery, aliquots centrifuged at 15000g for 10 min at 4 °C and diluted in of 90 μL of each culture medium supernatant were collected, mixed homogenization buffer (0.25 M perchloric acid, 100 μM sodium with 10 μL of a 10× homogenization solution containing 2.5 M bisulfite, 250 μM EDTA), and 20 μL of each sample was injected perchloric acid, 1 mM sodium bisulfite, and 2.5 mM EDTA, and into the HPLC system. Calibration curves of DA, DOPAC, HVA, L- sonicated for 10 s on ice. For intracellular monoamine determi- DOPA, NA, DHBA, 5HIIA, 3-MT, and 5-HT were prepared by nations, treated cells were washed three times with cold PBS, injecting different concentrations from 50 to 1000 pg/mL. All scraped, and centrifuged at 1000g for 5 min. The resultant pellets samples and standards were prepared and analyzed in triplicate. The were resuspended in a 1× homogenization solution containing 0.25 amount of each monoamine present in the samples was calculated M perchloric acid, 100 μM sodium bisulfite, and 250 μM EDTA, and from the corresponding calibration curve adjusted to a linear samples were sonicated for 15 s on ice. The samples were centrifuged regression model (in all cases, with R2 > 0.99). Instrument control, for 40 min at 4 °C. The pellets were dispersed in PBS, and the total 8603 https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 ACS Nano www.acsnano.org Article amount of protein was determined by the BCA or Bradford protein enhancement (RARE) with variable repetition time (VTR) sequence assay kits (Thermo Fisher Scientific, Waltham, MA, USA). All was used. Echo time (TE) was 7.5 ms acquiring at 18 different samples were kept at −20 °C until HPLC-ECD analysis. Uptake rates repetition times (TR) ranging from 30 to 5000 ms; field of view were determined by measuring the intracellular levels of DA over (FOV), 90 × 60 mm; matrix size (MTX), 128 × 128 (700 μm × 470 time. In the case of DA, data were fitted to the following Michaelis− μm/pixel); number of averages (NA), 1; RARE factor 2; and total d[DA] V acquisition time (TAT) 21 min 22 s. Parameters for T2 maps were Menten equation: dt = Kmmax , where Vmax is the maximal velocity +1 multislice multiecho sequence (MSME); TR, 3 s; TE ranged from 10 [DA] as a function of the density of the transporters and Km is the value of to 600 ms with 10 ms echo spacing; NA, 1; same geometry [DA]0 in which the rate of uptake is half of the Vmax. parameters as in T1 maps; and TAT, 6 min 24 s. Preclinical Evaluation. Animal studies were approved by the For the ex vivo post mortem MRI studies, a 72 mm inner diameter corresponding local ethics committees, according to the regional and volume coil was used in combination with a dedicated mouse brain state legislation (protocol CEA-OH-9685/CEEAH-3665). The surface receiver coil. The DA-NCPs were dispersed in 2 mL of PBS animals used for the tolerability and MRI experiments were added with 0.5 mM BSA. The amount finally used for each animal C57BL/6J female mice of 12 weeks of age, weighing 21.5 ± 0.4 g was 5 nmol of metal dissolved in 4 μL of PBS−BSA solution (n = 3). (n = 9). These animals were purchased from Charles River The commercial solution of CA-based in GdDTPA (n = 3) was used Laboratories (l’Abresle, France) and housed in the animal facility as a standard to compare the enhancement obtained with the NCPs of the Universitat Autònoma de Barcelona (UAB). MRI studies were synthesized, and the vehicle was also injected for comparison performed at the joint nuclear magnetic resonance facility of the purposes (n = 3). Three injection points were used in each mouse. UAB and Centro de Investigació n Bioméd ica en Red − All administrations were performed post mortem using a stereotactic Bioingenieria,́ Biomateriales y Nanomedicina (CIBER-BBN) (Cerda- holder, as already described.76 High-resolution T1w and T2w MR nyola del Vallès, Spain), Unit 25 of NANBIOSIS, www.nanbiosis.es. images were obtained for each mouse for morphological character- The 7T Bruker BioSpec 70/30 USR spectrometer (Bruker BioSpin ization of the investigated tissue. T2w images (TR/TEeff = 4200/−36 GmbH, Ettlingen, Germany) equipped with a mini-imaging gradient ms) were acquired using a rapid acquisition with relaxation set (400 mT/m) was used for magnetic resonance acquisitions. The enhancement (RARE) sequence. Parameters for T2w images were in vivo biodistribution and behavioral experiments with Sprague− RARE factor = 8; FOV, 19.2 × 19.2 mm; MTX, 256 × 256 (75 μm Dawley rats were performed at the laboratory of animal service × 75 μm/pixel); NA, 4; TAT, 6 min 43 s. After this, a T1w image within the Vall d’Hebron Research Institute (VHIR). (TR/TE 350/10 ms) was acquired with an MSME sequence with the Tolerability of Systematically Administered DA-NCPS. same FOV and MTX; NA, 8; TAT, 11 min 56 s. Three ROIs Tolerability experiments were done following a previously reported corresponding to the injection coordinates were manually defined protocol.82 In a typical experiment, increasing amounts of DA-NCPs after visual inspection in both the area of maximum enhancement (0.003−0.1 mmol Fe kg−1) were intravenously (i.v.) administered and equivalent area of contralateral parenchyma using Paravision 5.1. (via tail vein) to n = 3 mice with a 2-day rest period between The RCEinjection site ROI vs contralateral parenchyma injections. An additional group of n = 3 wild-type (wt) mice was obtained in each case was assessed. Only the slice with a better injected with the vehicle as control (PBS with 0.5 mM mouse serum defined contrast-enhanced region was measured. For dual enhance- albumin (MSA)). Mice body weight and other welfare parameters ment images (Figure 6c), T1w and T2w images were processed with were followed for 30 days after the last administration. In these the algebraic algorithm (MR signal T1/MR signal T2) provided by conditions, a dose of 0.1 mmol kg−1 of DA-NCPs proved safe, and Paravision 5.1. no significant weight loss or toxicity symptoms were observed during To examine the local distribution of DA-NCPs following intranasal injections and for the 30-day period after the last administration. administration, in vivo MRI was performed using a transmit volume This value perfectly matches safety administration ranges for related resonator (72 mm inner diameter) for excitation and a dedicated rat systems such as Fe-based nanoparticles.83,84 brain surface coil as a receiver. Sprague−Dawley male rats (275−300 Intracerebroventricular Administration of the DA-NCPs. g) were anesthetized with isoflurane 1−2% in O2 at 1 L/min. The in vivo distribution of the nanoparticles in the nigrostriatal Breathing (60−80 breaths/min) and body temperature (37−38 °C) system was assessed in Sprague−Dawley male rats (275−300 g) after intracerebroventricular administration. A permanent cannula were constantly monitored (SA Instruments, Inc., New York, USA) (C313G/SPC 3.5 mm, Plastics One) was placed in the right lateral during the MRI experiments. Rats were placed into a holder with ventricle (AP: −0.8; L: −1.6; DV: −3.6) of the rats under isoflurane bite-bar and ear-bars for optimal head immobilization. A series of T1w anesthesia using a Kopf stereotaxic frame. A dummy cannula was coronal images and T1 mapping in the axial plane, covering the nasal placed in the guide cannula to prevent the entry of material. Three cavity from the external naris to the olfactory bulb, were acquired weeks later, DA-NCPs (a total dose equivalent to 50 μg of free DA) before and immediately after intranasal administration of the were infused into the lateral ventricle with an internal cannula with nanoparticles. Following the initial MRI scanning, DA-NCPs (40 tubing attached to a 2.5 mL Hamilton syringe placed in a peristaltic μL, with a total dose equivalent to 200 μg of free DA dispersed in pump (KDS 220). A 20 μL amount of a DA-NCP suspension was PBS-MSA) were applied to the right nostril of the animal placed in a infused at a 1 μL/min rate. The dummy cannula was placed back supine position by slow administration of drops within 10 min. into the guide cannula after the administration. Rats were euthanized Immediately after DA-NCP administration, a second MRI scanning at 2, 6, and 24 h after the i.c.v. administration of the nanoparticles. was carried out under identical experimental conditions. T1w images The brains were removed, and both right and left ventral midbrains from seven coronal 1.5 mm slices were acquired with an MSME and striata were dissected and kept separately at −80 °C until sequence with TR, 300 ms; TE, 10 ms; FOV, 40 × 35 mm; MTX, analysis. 256 × 256 (156 μm × 137 μm/pixel); NA, 4; and TAT, 3 min 50 s. In Vitro, ex Vivo, and in Vivo MRI Studies with DA-NCPs. For T1 maps in the axial plane were obtained using a RAREVTR the in vitro quantitative magnetic resonance imaging phantoms sequence with 7 ms TE and acquisition at 10 different TR values (MRI-Phantoms), a 7.2 cm inner diameter volume coil was used for (450, 525, 650, 750, 950, 1200, 1500, 1900, 2500, and 5000 ms). A excitation and reception of NMR signals. The samples of DA-NCPs total of 24 contiguous axial 1 mm slices were acquired with FOV, 35 were dispersed using agarose (1%) in PBS at pH = 7.4. Five samples × 35 mm; MTX, 128 × 128 (273 μm × 273 μm/pixel); RARE at decreasing concentrations (0.6, 0.3, 0.1, 0.05, and 0 mM of metal factor, 1; and TAT, 32 min 54 s. Data were fitted to an exponential mL−1) were prepared in 1.5 mL Eppendorf tubes. The total volume curve using the ISA tool in ParaVision 5.1 in order to estimate T1 was adjusted up to 1 mL with agarose in PBS. The suspensions were values, as described in the Supporting Information. Mean T1 values dispersed in an ultrasound bath just before the solidification of pre- and postadministration of DA-NCPs were obtained from ROIs medium and acquisition sequences for assessing longitudinal of three consecutive sections. ROIs were manually defined in areas relaxation (T1). For T1 maps, a rapid acquisition with relaxation along the right nasal conduit showing high enhancement on T1w 8604 https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 ACS Nano www.acsnano.org Article images postadministration, and afterward, the equivalent region from treatment was also determined for each of the animals and compared the preadministration data set was selected. with the duration of the rotational behavior before the treatment. 6-HyroxyDA-Lesioned Rat Model of PD. The 6-OHDA rat model of PD was prepared as described previously.85 In brief, for ASSOCIATED CONTENT stereotaxic surgery, Sprague−Dawley male rats (250−275 g) were * sı Supporting Information anesthetized with isoflurane (0.5−1.5% in O2). The animals were The Supporting Information is available free of charge at positioned on a stereotaxic frame (Kopf Instruments, Tujunga, CA, USA), and 6-OHDA was unilaterally injected in the right hemisphere https://pubs.acs.org/doi/10.1021/acsnano.1c00453. of the brain (2 μL of freshly dissolved 6-OHDA hydrochloride Details including Materials and Methods, HPLC prepared with 0.02% ascorbate, Sigma-Aldrich, St. Louis, MO, USA) quantification of bix in the DA-NCPs, DA content of using a 33-G needle Hamilton syringe (Hamilton Bonaduz AG., DA-NCPs determined by HPLC, ICP-MS quantifica- Bonaduz, Switzerland). The injection of the neurotoxin was tion of iron in DA-NCPs, in vitro release kinetics under performed into the medial forebrain bundle, with the needle placed physiological conditions and processing, postprocessing 4.0 mm anterior to the interaural line, 1.3 mm lateral to the midline, of MR data and encapsulation of EGF; additional tables and 8.4 mm ventral to the surface of the skull, according to the (Table S1−S4) and figures (Figure S1−S18) (PDF) reference coordinates from the Paxinos and Watson rat brain atlas.86 After stereotaxic injection, the animals were placed in their cages and monitored until recovery. AUTHOR INFORMATION Intranasal Administration of DA-NCPs. For the pharmacoki- Corresponding Authors netic study, 18 healthy Sprague−Dawley rats (275−300 g) were Julia Lorenzo − Institut de Biotecnologia i de Biomedicina randomly divided into two experimental groups and intranasally (IBB), Universitat Autònoma de Barcelona, 08193 administered with vehicle (PBS−MSA solution) or with DA-NCPs Bellaterra, Barcelona, Spain; Departament de Bioquímica i dispersed in PBS with 0.25 mM MSA. To perform the intranasal administrations, the animals were anesthetized with isoflurane (0.5− Biologia Molecular, Unitat de Bioquímica de Biociències, 1.5% in O2) and put in a supine position. The vehicle (20 μL) or Edifici C, Universitat Autònoma de Barcelona, 08193 DA-NCPs (20 μL, with a total dose equivalent to 50 μg of free DA) Cerdanyola del Vallès, Spain; Email: julia.lorenzo@uab.cat were slowly applied to the right and left nostrils using an Eppendorf Daniel Ruiz-Molina − Catalan Institute of Nanoscience and pipet. The animals were euthanized at 30 and 120 min after the Nanotechnology (ICN2), CSIC and BIST, 08193 Bellaterra, intranasal administration of the nanoparticles. Then, the brains were Barcelona, Spain; orcid.org/0000-0002-6844-8421; removed, and both right and left ventral midbrains and striata were Email: dani.ruiz@icn2.cat dissected and kept separately at −80 °C until HPLC analysis. To evaluate the performance of DA-NCP administration in comparison Authors with free DA, an additional experiment was performed. In this Javier García-Pardo − Catalan Institute of Nanoscience and experiment, nine healthy Sprague−Dawley rats (275−300 g) were Nanotechnology (ICN2), CSIC and BIST, 08193 Bellaterra, randomly divided into three groups and intranasally administered Barcelona, Spain; Institut de Biotecnologia i de Biomedicina with either vehicle, DA-NCPs, or free DA. These animals were (IBB), Universitat Autònoma de Barcelona, 08193 sacrificed 120 min administration postadministration, and the brains Bellaterra, Barcelona, Spain; Departament de Bioquímica i dissected as above. The levels of DA present in the samples were Biologia Molecular, Unitat de Bioquímica de Biociències, determined by HPLC-ECD as described hereafter. For behavioral testing, 18 6-OHDA-lesioned Sprague−Dawley rats Edifici C, Universitat Autònoma de Barcelona, 08193 were divided into three treatment groups. The animals were first Cerdanyola del Vallès, Spain allowed to habituate to the room and manipulation for at least 1 Fernando Novio − Catalan Institute of Nanoscience and week before treatments. Two months after the surgery, one group of Nanotechnology (ICN2), CSIC and BIST, 08193 Bellaterra, 6-OHDA-lesioned rats received a semichronic intranasal admin- Barcelona, Spain; Departament de Química, Universitat istration of DA-NCPs at doses equivalent to 800 μg of free DA (four Autònoma de Barcelona (UAB), 08193 Cerdanyola del daily doses of 50 μg, between 8 a.m. and 15 p.m.) per day, for 4 days. Vallès, Barcelona, Spain; orcid.org/0000-0002-1517- A second group of PD rats received equivalent doses of free DA. The 3612 third group of animals was treated with vehicle (PBS−MSA Fabiana Nador − Catalan Institute of Nanoscience and solution), as a control condition. After semichronic drug admin- Nanotechnology (ICN2), CSIC and BIST, 08193 Bellaterra, istration, rats were subjected to behavioral analyses. Barcelona, Spain Apomorphine-Induced Rotation Test. Rats with unilateral 6- OHDA lesions of the nigrostriatal pathway exhibit contralateral Ivana Cavaliere − Catalan Institute of Nanoscience and rotational behavior when administered with DA agonists such as Nanotechnology (ICN2), CSIC and BIST, 08193 Bellaterra, apomorphine.85 This phenomenon can be explained by the Barcelona, Spain hypersensitization of the injured striatum. For assessment of circling Salvio Suárez-García − Catalan Institute of Nanoscience and behavior, the 6-OHDA-lesioned rats were challenged with 0.5 mg/kg Nanotechnology (ICN2), CSIC and BIST, 08193 Bellaterra, of apomorphine (Sigma-Aldrich, St. Louis, MO, USA) 6 weeks after Barcelona, Spain; orcid.org/0000-0002-4156-0579 striatal 6-OHDA injections and after semichronic administration of Silvia Lope-Piedrafita − Centro de Investigacion Biomédica the nanoparticles. Note that the first evaluation was carried out to en Red en Bioingeniería, Biomateriales y Nanomedicina determine the effect of the unilateral 6-OHDA lesion on each animal. (CIBER-BBN), 08193 Cerdanyola del Vallés, Spain; Servei To perform the rotation tests, rats were introduced inside individual de Ressonància Magnètica Nuclear, Universitat Autònoma de circular cages and connected to an automated rotometer (LE 902 Barcelona, 08193 Cerdanyola del Vallès, Spain rotameter, Harvard Apparatus, Holliston, MA, USA). The animals were then habituated to the environment for 15 min. Afterward, a Ana Paula Candiota − Institut de Biotecnologia i de subcutaneous injection of apomorphine was given to each of the Biomedicina (IBB), Universitat Autònoma de Barcelona, animals, and the rotational activity was recorded for 1 h. The 08193 Bellaterra, Barcelona, Spain; Departament de rotational activity was determined by calculating the total number of Bioquímica i Biologia Molecular, Unitat de Bioquímica de net contralateral complete (360°) rotations after subtraction of the Biociències, Edifici C, Universitat Autònoma de Barcelona, contralateral turns. The total duration of the rotational behavior after 08193 Cerdanyola del Vallès, Spain; Centro de Investigacion 8605 https://doi.org/10.1021/acsnano.1c00453 ACS Nano 2021, 15, 8592−8609 ACS Nano www.acsnano.org Article Biomédica en Red en Bioingeniería, Biomateriales y M.V. received funding from “la Caixa” Foundation (ID 1178 Nanomedicina (CIBER-BBN), 08193 Cerdanyola del 100010434, under the agreement LCF/PR/HR17/52150003) Vallés, Spain 1179, Ministry of Economy and Competitiveness (MINECO, Jordi Romero-Gimenez − Neurodegenerative Diseases Spain) (SAF2016-77541-R) and The Michael J. Fox Research Group, Vall d’Hebron Research Institute (VHIR)- Foundation for Parkinson’s Research 1180 (Grant ID: Center for Networked Biomedical Research on 15291.01). A.P.C. received funding from the ATTRACT Neurodegenerative Diseases (CIBERNED), Edifici Collserola project funded by the EC under Grant Agreement 777222. Hospital Universitari Vall d’Hebron, 08035 Barcelona, The authors thank the support from COST Action CA17121. Spain We thank Susana Benítez Andreu and the Department of Beatriz Rodríguez-Galván − Neurodegenerative Diseases Biochemistry and Molecular Biology at the Universitat Research Group, Vall d’Hebron Research Institute (VHIR)- Autonoma de Barcelona for the exceptional technical support Center for Networked Biomedical Research on and assistance with HPLC-ECD determinations and data Neurodegenerative Diseases (CIBERNED), Edifici Collserola analysis. Special thanks to Karl-Richard Reutter (instagram: Hospital Universitari Vall d’Hebron, 08035 Barcelona, NIR.io_art) and Javier Saiz from 3D Science Visuals for Spain contributing to graphic design and scientific illustrations. We Jordi Bové − Neurodegenerative Diseases Research Group, also thank Prof. Azzedine Bousseksou and Dr. Gábor Molnár Vall d’Hebron Research Institute (VHIR)-Center for for Mössbauer experiments. Networked Biomedical Research on Neurodegenerative Diseases (CIBERNED), Edifici Collserola Hospital REFERENCES Universitari Vall d’Hebron, 08035 Barcelona, Spain (1) Kalia, L. V.; Lang, A. E. Parkinson’s Disease. Lancet 2015, 386 Miquel Vila − Neurodegenerative Diseases Research Group, (9996), 896−912. Vall d’Hebron Research Institute (VHIR)-Center for (2) Pringsheim, T.; Jette, N.; Frolkis, A.; Steeves, T. D. 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