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. 2019 May 1;4(5):7953–7962. doi: 10.1021/acsomega.8b00626

Dopamine Released from TiO2 Semicrystalline Lattice Implants Attenuates Motor Symptoms in Rats Treated with 6-Hydroxydopamine

Margarita Gómez-Chavarín †, Gina Prado-Prone ‡,§, Patricia Padilla ∥, Jesús Ramírez Santos ⊥, Gabriel Gutiérrez-Ospina ⊥,*, Jorge A García-Macedo §,*
PMCID: PMC6648478  PMID: 31459884

Abstract

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The motor dysfunction featured by patients aggrieved by Parkinson’s disease (PD) results from the reduction of dopamine (DA) availability in the caudate nucleus (CN). Restituting CN DA levels is therefore essential to ameliorate PD motor deficits. In this regard, nanotechnology may offer solutions to restore CN DA availability. DA, however, can be rapidly oxidized into toxic compounds if made available in situ, unprotected. Then, we tested whether a semicrystalline TiO2 lattice, implanted into the CN of 6-hydroxydopamine (6-OHDA)-lesioned, hemiparkinsonian rats, was able to release DA during a time window sufficient to attenuate motor symptoms while protecting it from the ongoing oxidation. Accordingly, implanted semicrystalline TiO2 lattices released incremental amounts of DA into the CN of lesioned rats. Motor symptoms were already attenuated by the 1st month and significantly reduced 2 months after implantation. These effects were specific since TiO2 lattices alone did not modify motor symptoms in lesioned rats. DA-unloaded or -loaded TiO2 lattices did not produce obvious symptoms of systemic or neurological toxicity nor significantly increased CN lipid peroxidation in implanted, lesioned rats at the time of sacrifice. Our results thus support that loaded TiO2 lattices are capable of releasing DA while protecting it from the ongoing oxidation when implanted into the brain. Their implantation does not cause noticeable systemic or local toxicity. On the contrary, they attenuated motor symptoms in hemiparkinsonian rats.

1. Introduction

Parkinson’s disease (PD) is a prevalent neurodegenerative disorder that typically becomes symptomatic in people over 40 years of age.1 It features a myriad of autonomic and somatic signs and symptoms that worsen with disease progression.2 At intermediate stages, most of the people afflicted by PD develop a motor syndrome characterized by tremor, akinesia, and rigidity.3 This syndrome arises, in part, as a result of a deficient modulation of the activity of the corticostriatal excitatory input by dopamine (DA);4 in parkinsonian patients, DA concentration gradually decreases in the caudate nucleus (CN) following the progressive degeneration of dopaminergic projection neurons located in the substantia nigra pars compacta (SNpc).5−7 Therefore, the restitution of DA concentrations in the depleted CN must alleviate PD-related motor dysfunction. Accordingly, the oral administration of l-3,4-dihydroxyphenylalanine (l-DOPA), a synthetic precursor of DA, attenuates motor dysfunction in both PD patients6 and experimental animal units that model PD, by increasing CN DA availability.8 Unfortunately, l-DOPA loses its pharmacological actions with time as dopaminergic neuron death progresses;9 this pharmacological agent requires its enzymatic conversion into DA by the corresponding enzymatic machinery located in the still living dopaminergic neurons.10 Hence, other dopaminergic agonizts and nondopaminergic medications (e.g., anticholinergic drugs) have been assessed. These agents have had a limited clinical success when used chronically.11

Based on the previous discourse line, the restitution of CN DA levels still is the most sensible way to therapeutically approach PD motor symptoms. Oxidative processes following their release from nerve terminals, nonetheless, rapidly inactivate DA. This process is not inert but potentially harmful since it gives rise to toxic metabolites that increase neural damage.12,13 Consequently, therapeutic approaches aimed at reducing PA motor symptoms must ensure providing DA in concentrations sufficient to achieve their modulatory actions while avoiding its toxicity. Transplantation of dopaminergic neurons obtained through different means has been offered as an alternative treatment to deliver DA into CN from natural sources. Even though cell grafts allow patients to recover some motor control in the short term, long-term success is rather limited because the implanted cells become diseased over time (reviewed14). Nanotechnological tools constitute another stronghold of therapeutic resources. Particularly, nanohybrid composites of dopamine, chitosan, and TiO2 have been synthesized by sol–gel method and their release of the neurotransmitter has been obtained by electrochemical determination and UV–vis absorbance techniques.15 Furthermore, attempts have been made to use nanostructured materials to deliver DA into the brain. Jain et al. (1998)16 were among the first to use DA entrapped in liposomes to ameliorate catatonia in rats, following intraperitoneal administration. This work, nonetheless, is short term, largely inferential, and provides no technical data on brain liposome diffusion, DA levels, toxicity, and place specificity. On a similar verge, Pichandy et al. (2010)17 used liposomes administered through the peritoneum to decrease PD-like symptoms induced by haloperidol. However, this study is acute, the specific entrance of dopamine into the CN is inferred and not confirmed, the experimental animal unit chosen models some PD-like motor symptoms with no association with neuronal death, DA oxidation is not evaluated, and DA/liposome-related toxicity is not assessed. The work conducted by Pillay et al. (2009),18 on the other hand, tested the ability of a “nano-enabled [cellulose acetate phthalate] scaffold device for the site-specific delivery of dopamine (DA) as a strategy to minimize the peripheral side effects of conventional forms of Parkinson’s disease therapy”. In this study, DA release was monitored for up to 1 month, following intraparenchymal implantation of the device into the frontal lobe of intact rats. Unfortunately, this study was conducted in an animal experimental unit that does not model PD, DA oxidation was not prevented or assessed, and the toxicity of the device was untested. Most of the limitations highlighted before with regard to previous studies were effectively overcome by a pair of studies conducted by Trapani et al. (2011),19 who used chitosan nanoparticles as systemic DA carriers, and by Pahuja et al. (2015),20 who used poly(lactic-co-glycolic acid) to entrap DA. In both cases, they succeeded in supplying in vivo DA in sufficient amount to the striatum with low, local, and systemic toxic effects, following intraperitoneal injections or intravenous infusions, respectively. Unfortunately, Trapani et al. (2011)19 did not test the therapeutic performance of DA/chitosan nanoparticles in an experimental animal unit modeling Parkinson. Pahuja et al. (2015),20 on the other hand, did it. In fact, this group reported, “These particles up-regulated and maintained the dopamine levels in the striatum of through slow and sustained release. This was accompanied by a decrease in dopamine receptor supersensitivity and reversal of neurobehavioral deficits”. Although both works undoubtedly represent significant advances in the field, they are short term, a circumstance that limits the applicability of their inferences on systemic and local toxicities, specificity, and therapeutic potential when considering their long-term usage.

Having all of these information in mind, the present work aimed at evaluating the use of titanium dioxide (TiO2)-crafted lattices as local DA storing/releasing implantable nanodevices. We tested the ability of these lattices to release DA in sufficient amount for a period appropriate to attenuate motor symptoms in 6-hydroxydopamine (6-OHDA)-lesioned, hemiparkinsonian rats, an accepted animal experimental unit to model PD motor symptoms. Because (1) unprotected, freed DA may be rapidly metabolized to form toxic oxidative derivatives and (2) TiO2 nanoparticles may cause oxidative damage, we monitor lipid peroxidation as an index of local toxicity for both DA-unloaded and -loaded matrices. Lastly, we preferred to test the effects of TiO2 lattices after local administration to limit body distribution, thus avoiding systemic toxicity and body accumulation over time. Also, a reduced initial dose can be used since neither organ-unspecific uptake is expected nor biological barriers must be passed through. Overall, our results showed that TiO2 lattices are physically stable and protect DA from oxidation. They also readily release it after being implanted into the CN of hemiparkinsonian rats. Motor deficits were greatly attenuated by DA release from the TiO2 lattices.

2. Experimental Section

2.1. Synthesizing and Loading Dopamine into the TiO2 Lattice

DA-unloaded or -loaded TiO2 lattices were synthesized at room temperature, following the conventional sol–gel method.21 Briefly, the working solution was prepared by stirring titanium(IV) butoxide (Fluka) and ethyl alcohol (1:14.1 molar ratio; Sigma-Aldrich) for 2 h at room temperature. A solution of ethyl alcohol in deionized water (1:1 molar ratio; Hycel) was then dropped, and, after 15 min, a mixture of 1.11 mL of tetra-ethyleneglycol (Sigma-Aldrich) and 0.578 mL of crown ether (15C5; Sigma-Aldrich) was finally added. The TiO2 solution was stirred for 4 h at room temperature. The addition of 15C5 provided an organic template,22 which, while binding TiO2, favors the formation of nanometric crystalline zones with anatase and rutile phases. In addition, we have previously found that 15C5 also reduces the rate of DA oxidation.23 For DA-loaded lattices, dopamine hydrochloride (Sigma-Aldrich) was dissolved (0.3 w/v %) in 10 mL of a TiO2 working solution (TiO2 lattice); this mixture was stirred for 4 h at room temperature in a light-protected jar. The final solution of DA-loaded TiO2 lattices (TiO2/DA complex) acquired a yellowish tint (Supporting Information, Figure S1A,B), a color that indicates that the DA’s enediol functional group networked with TiO2 surface through a ligand-to-metal charge-transfer interactions.24 No further treatment was performed to the lattices before their implantation.

2.2. Physicochemical Analyses of Dopamine Antioxidative Property of the TiO2 Lattice

2.2.1. Fourier Transform Infrared Spectroscopy (FTIR) Analyses

FTIR spectral analyses allowed us to evaluate the antioxidative property of the TiO2 lattice. DA can be rapidly oxidized to dopamine-quinone (DAq) and dopamine-chrome (DAcr) when unprotected by the lattice. We identified the presence of both oxidized DA species within the TiO2 lattice by comparing the vibrational modes of the bonds established among the atoms of the distinct DA species with those of the TiO2 lattice. FTIR spectra were obtained at different times following DA loading of the TiO2 lattice with the aid of an FTIR Spectrophotometer Bruker Tensor 27 FT-IR, equipped with an attenuated total reflection accessory using a range of 400–4000 cm–1 at room temperature. The measurements were directly taken from liquid samples.

2.3. Structural Analyses of Dopamine Antioxidative Property of the TiO2 Lattice

2.3.1. High-Resolution Transmission Electron Microscopy (HRTEM) and X-ray Diffraction (XRD) Analyses

We conducted HRTEM and XRD analyses to determine the structure of the TiO2/DA complex and the size of TiO2 lattice crystalline nanoparticles. The abilities of the TiO2/DA lattice to (1) protect DA from being oxidized and (2) release it following a concentration gradient partly depend upon its structural organization. In general, it is expected that a “relatively loose” lattice formed by amorphous and nanocrystalline phases combined would provide DA with satisfactory protection and diffusion possibilities. The local structure and crystal nanoparticle size of TiO2 lattices loaded or not loaded with DA were determined by using HRTEM conducted in a JEM2010 FEG with a point-to-point resolution of 0.25 nm. For HRTEM images, lattices were resuspended in ethanol and a drop of the mixture was deposited on a copper grid. The diameter of 70 particles randomly taken from three different samples was averaged to estimate the average particle size. The structures of TiO2 lattices, whether loaded or not loaded with DA, were evaluated by using XRD analyses. Filmed liquid samples of the TiO2 lattices were placed on glass wafers (2.5 cm × 2.5 cm) and sprayed seven times at a rate of 2700 rpm for 15 s using a spin coater (SCS G6P8). XRD diffraction patterns were obtained by using a Bruker AXS D8 Advance X-ray diffractometer under a Ni-filtered Cu Kα radiation. We used a step-scanning mode with 0.02° steps in the 10–80° range in 2θ and an integration time of 2 s.

2.4. Testing in Vivo Dopamine Release from the TiO2 Lattice

2.4.1. Animals

The experiments described below were conducted in young adult male Wistar rats (250–260 g) born and raised in the animal facility at the Instituto de Investigaciones Biomédicas (IIB), Universidad Nacional Autónoma de México (UNAM). Rats were caged individually in rooms having controlled illumination (12 h light/12 h dark cycles), temperature (23 °C), and relative humidity (50 ± 5%). Water and food were provided ad libitum. Protocols of animal handling and experimentation were revised and approved by the local animal rights committee at IIB, UNAM (CICUAL; ID-221).

2.4.2. Animal Groups and Experimental Procedures

Rats were subjected to stereotaxic surgeries aimed at unilaterally instilling, at the nigrostriatal pathway, ascorbic acid alone (AAc; 0.5 mg; Sigma-Aldrich; control group) or 6-hydroxydopamine (6-OHDA; 6 μg; Sigma-Aldrich; experimental group), each diluted in 5 μL of saline solution; 6-OHDA causes a retrograde degeneration of dopaminergic neurons located in the substantia nigra pars compacta (SNpc), leading to a motor dysfunction associated with DA depletion in the CN.25 Both AAc and 6-OHDA were administered (10 μL) using pressure injections through a Hamilton syringe at a rate of 1 μL/min. To reduce backflow, the syringe’s needle was kept in place at the injection site for 20 min. To evaluate the efficacy of 6-OHDA treatments, both AAc-treated (n = 4) and 6-OHDA-treated (n = 16) rats were subjected to the turning behavior test following an intraperitoneal administration of amphetamine (5 mg/kg of body weight; Sigma-Aldrich).25 For the case of 6-OHDA-treated rats, only those having ≥200 turns per hour and have TH immunoreactivity significantly reduced (see below) were included in the study. Turning behavior tests were conducted in a custom-made, Plexiglas 60 cm diameter/45 cm height cylinder placed under red light illumination; tests were all scheduled to proceed between 9 and 12 AM. Turning sessions were recorded and analyzed off-line. After ending the turning sessions, 6-OHDA-treated rats were further subgrouped into those carrying DA (3.25 μg/L)-unloaded (n = 8) or -loaded (n = 8) TiO2 lattices. From previous experiments, we determine that this concentration is adequate to observe good responses, as will be shown. DA-loaded or -unloaded TiO2 lattices were stereotaxically introduced (5 μL) into the anterodorsal aspect of the caudate nucleus at a rate of 1 μL/min through a 10 μL Hamilton syringe. Surgery was conducted under anesthesia induced after intramuscular injections of ketamine (60 mg/kg) and xylazine (8 mg/kg). Corneal dryness was prevented using artificial tears. The stereotaxic coordinates used for targeting the nigrostriatal pathway (AP = −4.5, ML = −1.5, DV = −8.25) and the anterodorsal caudate nucleus (AP = 0, ML = +3.2, DV = −4) were established based on the Rat Brain Stereotaxic Atlas published by Paxinos et al.26 After surgery, rats were caged individually in acrylic cages were they remained until further testing. Neither analgesics nor antibiotics were required after the surgery. Finally, two additional groups of animals were added to our experimental design. They were unlesioned but carried DA-unloaded (n = 8) or -loaded (n = 8) TiO2 lattices. In both cases, lattices were implanted as described above and kept in the brain for 1 (n = 4/group) or 2 months (n = 4/group). The goal of these groups was to test systemic and local implant toxicities through analyzing turning behavior and the number of SNpc dopaminergic neuron number and CN lipoperoxidation, respectively.

2.4.3. Immunocytochemistry

Immunocytochemical studies were carried out to verify the degree of dopaminergic denervation achieved after 6-OHDA treatments. The rats (n = 24) used for carrying out these experiments were anesthetized with pentobarbital (0.5 mg/kg of body weight) and perfused through the heart with saline solution followed by a buffered solution of 4% paraformaldehyde. After the perfusion, their brains were removed, cryopreserved in a buffered solution containing 30% sucrose until they sank, frozen in 2-methyl-butane prechilled with dry ice, and stored at −70 °C until use. Cryostat sagittal brain slices (40 μm thick) were cut and incubated with primary polyclonal antibodies raised in rabbits against tyrosine hydroxylase (TH; Cat. No. sc-14007; Santa Cruz Biotechnology), diluted 1:1000 in phosphate-buffered saline (0.1 M; pH 7.4) and supplemented with 0.3% Triton (PBSt) during 48 h at 4 °C. After a thorough wash, slices were incubated with biotin-conjugated, secondary polyclonal antibodies raised in goats against rabbit IgGs (Cat. No. BA-1000; Vector Laboratories), diluted 1:200 in PBSt during 2 h at room temperature. This step was followed by three washes in PBSt. Then, slices were incubated with avidin–peroxidase as recommended by the supplier (Standard Elite kit; Cat. No. PK-6100; Vector Laboratories) during 2 h at room temperature, washed in PBSt, and finally incubated with a solution containing 2,2-diaminobenzidine and hydrogen peroxide as recommended by the supplier (Peroxidase substrate kit DAB; Cat. No. SK-4100; Vector Laboratories). The slices were mounted onto gelatin-subbed slides, counterstained with cresyl violet, and coverslipped with Cytoseal 60 (Cat. No. 8310-4; ThermoScientific). Slides were observed under bright-field microscopy and representative digital photographs taken at 10× using a Leica EZ4D stereomicroscope. Additionally, TH immune-positive neurons were counted across six consecutive sections of the SNpc obtained from brains of unlesioned (n = 3), 6-OHDA lesioned (n = 4), unlesioned carrying DA-unloaded TiO2 lattice implants (n = 3), unlesioned carrying DA-loaded TiO2 lattice implants (n = 3), 6-OHDA lesioned carrying DA-unloaded TiO2 lattice implants (n = 8), and 6-OHDA lesioned carrying DA-loaded TiO2 lattice implant (n = 8) rats. In all cases, neurons counted always had clearly defined somatic boundaries and near-central nuclei. SNpc located in both sides of the mesencephalon were compared within and across groups.

2.4.4. In Vivo DA Release and Quantification

To assess whether TiO2 lattices indeed released DA into the denervated CN, we obtained tissue samples of this brain region from rats implanted or not with DA-unloaded (n = 4) or -loaded (n = 8) TiO2 lattices. Animals carried the implants during either 15 or 30 days. Rats were sacrificed by overdosing them with pentobarbital, and the tissue samples were rapidly obtained following decapitation. After removing the brains from the skull, each brain was hemisected longitudinally, and the CN was dissected, collected in centrifuge tubes, and sonicated in 200 μL of acetonitrile (Sigma-Aldrich). The homogenates were then centrifuged at 10 000 rpm during 5 min at room temperature. After centrifugation, the organic phase was evaporated and the pellet resuspended in 100 μL of 0.1 M perchloric acid supplemented with 0.1 mM ethylenediaminetetraacetic acid. Each sample was then stored at −70 °C until use. Protein quantification was done using the bicinchoninic acid protein quantification assay kit (cat 23225; Pierce). On the day of the experiment, 10 μL samples were injected into the chromatographic column (Phenomenex Hyper Clone ODS, 5 μm internal diameter and 120 Å pore, 4.6 mm × 150 mm; No. 00F-4361-EO). The separation was achieved using an aqueous mobile phase of 0.05 M ammonium acetate (supplemented with 0.66 M glacial acetic acid to adjust the pH to a value of 3.5) and an organic mobile, acetonitrile phase. The flow rate of the mobile phase was 1.0 mL/min at room temperature, and the separation was conducted following a gradient-based protocol. DA was detected using a variable wave ultraviolet (UV) light detector at λ = 270 nm; DA’s retention time is 3.36 min (±0.15 min). The data were captured by a Waters e-SAT/IN interface, processed using Empower 3 software and reported as ng/mg of protein.

2.4.5. In Vitro DA Release and Quantification

To assay DA release from TiO2 lattices in vitro, a series of DA-loaded TiO2 lattices (5 μL) were incubated in 500 μL Eppendorf tubes, each filled with 250 μL of artificial cerebrospinal fluid (CSF, 119 mM NaCl, 26.2 mM NaHCO3, 2.5 mM KCl, 1 mM NaH2PO4, 1.3 mM MgCl2, 10 mM glucose, and pH 7.4). The tubes were kept at 37 °C under gentle shaking. Individual tubes were removed from the incubator, one by one, at different times along 1 month. The concentration of DA was estimated by HPLC, as described previously.

2.4.6. Lipid Peroxidation

DA, DAq, DAcr, and TiO227 may induce oxidative stress. We then tested whether TiO2 matrices alone or loaded with DA increase polyunsaturated lipid peroxidation by using an assay (TBARS Assay Kit, Cat. No. KA1381AB, Abnova) designed to detect malondialdehyde (MDA), an end product of the lipid oxidative reaction chain. In this assay, lipid peroxidation is estimated by colorimetric (532 nm) measures following the formation of a pink product that results from the condensation of MDA and thiobarbituric acid (TBA), after a nucleophilic attack involving TBA’s carbon-5 and MDA’s carbon-1 of MDA. The concentration of MDA–TBA product is proportional to the MDA present. Hence, the intensity of the pink MDA–TBA product available in the solution indicates the extent of lipid peroxidation.

Male Wistar rats (250 g) were used to estimate lipid peroxidation in tissue samples obtained from animals stereotaxically implanted in the CN (AP = 0, ML = +3.2, DV = −4) with 5 μL of TiO2 matrices unloaded (n = 5) or DA-loaded (n = 5). After 2 months, rats of both groups were euthanized with pentobarbital and decapitated as previously described. Then, their brains were obtained and hemisected. The exposed CNs were dissected and processed precisely as recommended by the kit’s supplier. In brief, 25 mg of CN was homogenized through sonication in radioimmunoprecipitation assay buffer (250 μL, supplemented with the complete protease inhibitor cocktail (Cat. No. 4693124001, Roche) at 4 °C). The samples were centrifuged (1600 g) for 10 min at 4 °C. Supernatants were collected and used for estimating MDA concentration reported as μM/25 mg of the tissue.

2.4.7. Statistics

Behavioral, morphological, and biochemical results are all presented as mean values ± standard error and compared between groups by using one-way ANOVA tests followed by post hoc Tukey’s multiple comparison tests; all data passed normality tests. In all cases, the p-value was considered significant if reached <0.01. Graphs were generated, and the statistical analyses conducted, using Prism software 6.0 (GraphPad, Inc.).

3. Results

3.1. Physicochemical Analyses of DA-Unloaded or -Loaded TiO2 Lattices

3.1.1. DA Binds to the TiO2 Lattice

FTIR spectra of DA, of the TiO2 matrix, and of the TiO2/DA complex were obtained to establish the chemical interaction taking place between TiO2 and DA. Results are shown in Figure 1. The analyses of DA (Figure 1A) revealed peaks between 2959 and 3373 cm–1 associated with hydrogen bonds, at 1620 cm–1 related with N–H bond bending and at 1585 cm–1 associated with the C=C bond vibration modes within the aromatic ring, at 1319 and 1338 cm–1 produced by the C–N bond stretching modes, at 1284 cm–1 produced by C–H2 bonds vibrational mode, at 1190 cm–1 arising from the C–O bonds stretching vibration, and at 940 cm–1 corresponding to C–C–N bonds bending vibration. FTIR analyses of the TiO2 matrix (Figure 1B) showed the presence of Ti–O–C bonds resulting from the partial interaction between TiO2 with some carbons of the highly organized structure of 15C5. We also observed peaks at about 439 and 474 cm–1 that corresponded to the Ti–O–Ti stretching vibration bonds of rutile and anatase crystalline phases, respectively. As we expected, the presence of the organic template 15C5 during the synthesis of the TiO2 matrix generated its partial crystallization favoring the formation, at room temperature, of some anatase and rutile zones. Ti–O bonds vibration mode was represented by a peak at 551 cm–1; documenting the presence of Ti–O bonds is important since they decrease the metal ion biological toxicity.36 Spectra inflections at 761 and 839 cm–1 represent C–H bending vibration bonds from the 15C5 crown ether, whereas those observed at 1014 and 1080 cm–1 arise from the νC–O vibrations. Ti–O–C bonds are represented by the peak arising at 1160 cm–1; this peak results from the partial interaction between TiO2 with some carbons of 15C5 structure. Probably this interaction between TiO2 molecules with the highly ordinated structure of 15C5 gives rise to the semicrystallinity of the TiO2 once incorporated into the matrix.

Figure 1.

Figure 1

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Representative FTIR spectra of (A) DA, (B) TiO2 matrix, and (C) TiO2/DA complex. The vibrational modes corresponding to DA and TiO2 are identified with black dashed lines and black solid lines, respectively.

Finally, FTIR spectra of the TiO2/DA complex (Figure 1C) revealed remarkable differences when compared with the spectra obtained from the unloaded matrix. Most notably, the 15C5 structure-associated peaks are absent in the former. Also, in TiO2/DA complex spectra, two new peaks at 1067 and 1093 cm–1 appeared. These likely arise from the presence of C–O–C bonds, which is itself evidence suggesting that C–O groups from the 15C5 structure are interacting not only with TiO2 matrix but also with the CH groups of DA. In support of this notion, we also observed peaks at 2430 and 2864 cm–1 ascribed to hydrogen bonds. Furthermore, TiO2/DA complex spectra show the presence of two new peaks at 509 and 568 cm–1 that presumably represent the bonding between Ti and DA hydroxyl groups.37−39Table 1 depicts the whole set of FTIR spectral peaks obtained for DA and DA-unloaded and -loaded TiO2 matrices.

Table 1. FTIR Frequencies in cm–1 Corresponding to DA, TiO2 Matrix, and TiO2/DA Complex Spectra and Their Assignments.
DA TiO2 matrix TiO2/DA complex assignment reference
940 439 439 Ti–O–Ti rutile (28)
474 474 Ti–O–Ti anatase (29)
551 509, 568 Ti–O (30)
761   C–H (bending) from 15C5 exp.
839   C–H (bending) from 15C5 exp.
  940 C–C–N (bending) (30)
1014   νC–O from 15C5 exp.
1080   νC–O from 15C5 (31)
  1067 C–O–C exp.
  1093 C–O–C exp.
1160 1160 Ti–O–C (32)
1219 1219 C–O (stretching) (30)
1190 1190 1190 C–O (stretching) (32) and (33)
1284     C–H2 (30)
1319     C–N (stretching) exp.
1338     C–N (stretching) exp.
1585     aromatic C=C (33) and (34)
1620     N–H (bending) (35)
2959–3373   2864, 2430 OH (33)
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3.1.2. TiO2 Lattice Prevents DA Oxidation

The vibrational modes corresponding to the oxidation products DA-quinone (DAq) and DA-chrome (DAcr) were identified by means of oxidized dopamine FTIR spectrum and are shown in Figure 2A. DA oxidation process into the TiO2 matrix was evaluated by FTIR (Figure 2B). At room temperature, DAq traces were detected at time zero, as documented by the presence of low-amplitude peaks occurring at 1375 and 1510 cm–1. These inflections correspond to DAq’s C=O and C=C stretching bond modes.40 Thus, DA was lightly oxidized by the time it was introduced into the matrix. After 456 h, two low-amplitude peaks appeared at 1290 and 1456 cm–1. These peaks corresponded to DAq’s C–O bonds and DAcr’s C=C stretching vibration bonds,40 thus indicating that a small fraction of DA became oxidized.

Figure 2.

Figure 2

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Representative FTIR spectra of (A) oxidized dopamine and (B) TiO2/DA complex preserved at room temperature. The oxidation process of DA into TiO2 matrix was recorded as a function of time. The vibrational modes corresponding to DA-quinone (DAq) and DA-chrome (DAcr) are identified with black dotted lines.

Finally, a low-amplitude peak ascribed to DAq’s C=O stretching vibration bond modes was detected at 1652 cm–1.31 Interestingly, at all times recorded, the amplitude of all peaks remains similar, thus indicating that DA oxidation proceeds at a constant rate. Such a rate is temperature-sensitive since it is greatly reduced by keeping DA-loaded matrix at 4°C (Supporting Information, Figure S2); thus, the matrix can be stored for several months after preparation and before implantation.

3.1.3. TiO2 Lattice Crystallinity Is Preserved After DA Bonding

DA-unloaded or -loaded TiO2 matrices predominantly displayed an amorphous phase, as supported by the presence of an undefined broad band at 2θ = 22.5° under X-ray diffraction pattern analyses, as shown in Figure 3. However, crystallization is partially present (semicrystalline structure) in both DA-unloaded or -loaded TiO2 matrices with nanometric zones, as observed by HTEM in Figure 4A,D. At these zones, the nanocrystal is practically spherical because diameters Dmax and Dmin are almost equal (Figure 4G). In both cases, nanocrystals presented either rutile phase (an interplanar distance of 0.205–0.207 nm, which corresponds to the (210) reflection) (Figure 4B,E) or anatase phase (an interplanar distance of 0.184 nm, which correspond to the (200) reflection) (Figure 4C,F), as predicted by FTIR. The semicrystalline structures of DA-unloaded and -loaded TiO2 matrices were comparable. Hence, DA does not affect the semicrystalline morphology of TiO2 matrices, an observation that implies that DA binds to the surface of the TiO2. It has been reported that the interaction between DA and crystalline TiO2 may be established by bidentate modes (the molecule bonded to two Ti sites via its two oxygen sites), a chelated mode (i.e., the molecule bonded to one Ti site via its two oxygen sites), and a monodentate mode (i.e., the molecule bonded to one Ti site via one oxygen site). However, since the bidentate mode is the most favorable energetically,41,42 it is possible that the enediol ligands, interacting with the two OH groups of DA, bind DA to the surface of TiO2 matrix, resulting in a single surface-modified TiO2.

Figure 3.

Figure 3

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X-ray diffraction patterns of the TiO2 matrix and TiO2/DA complex showing a characteristic amorphous peak at 2θ = 22.5°.

Figure 4.

Figure 4

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HRTEM micrographs of (A) TiO2 matrix and (D) TiO2/DA complex samples showing populations of nanocrystals with an almost spherical shape. High-resolution micrographs of TiO2 matrix (B, C) and TiO2/DA complex (E, F) exhibited reflections corresponding to rutile and anatase crystalline phases of TiO2. The corresponding reflections are identified with white arrows. (G) Average of largest and smallest particle diameter values ± HRTEM (Dmax and Dmin) and shape factor aspect ratios (Q) of nanoparticles in TiO2 matrix and TiO2/DA complex.

3.2. Testing in Vivo TiO2 Lattices Loaded with Dopamine

3.2.1. 6-OHDA Treatment Readily Decreased CN TH-Positive Fiber Immunostaining and SNpc TH-Positive Neuron Number While Inducing Motor Deficits

TH immunohistochemistry was used to corroborate the efficiency of the CN dopaminergic denervation in lesioned rats. As shown in Figure 5, control rats displayed intense CN and SNpc TH immunoreactivity (Figure 5A), whereas the 6-OHDA-lesioned rats had a noticeable reduction of this labeling in both the CN and SNpC ipsilateral to the lesioned side (Figure 5B) but not in the opposite side (not shown). In agreement with this result, 6-OHDA-lesioned rats showed 10 times more contralateral turns after amphetamine treatment (Figure 5C; see also Supporting Information Video S1) and displayed a marked reduction of the number of SNpc TH-positive neurons ipsilateral to the lesioned side, regardless of whether they carried DA-unloaded or -loaded TiO2 lattices (Supporting Information, Figure S3). Unlesioned rats implanted with DA-unloaded or -loaded TiO2 lattices did not show altered numbers of TH-positive neurons, thus supporting that implants loaded or not with DA have no neurologic toxic effects for up to 2 months (Supporting Information, Figure S3). In addition, rats implanted with DA-unloaded or -loaded TiO2 lattices revealed CN TH immunostaining equivalent to that seen in nonimplanted, 6-OHDA-lesioned rats (Figure 5D), thus indicating that neither lattice implantation nor DA released from it promotes the regeneration of the nigrostriatal dopaminergic pathway. It was also observed that implants remained relatively intact in place at least after 2 months (Figure 5D), thus suggesting that systemic toxicity may be limited.

Figure 5.

Figure 5

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Photomicrographs that illustrate sagittal sections through the caudate nucleus (CN) and substantia nigra pars compacta (SNpc) immunostained for tyrosine hydroxylase in the brain of unlesioned (A) or 6-OHDA-lesioned (B) rats. Note the reduction of the immunohistochemical staining of both nuclei in the lesioned rat following the degeneration of the nigrostriatal pathway. LS = lesion site. (C) Bar graph that shows the number of turns per hour induced by amphetamine in unlesioned and 6-OH-lesioned rats. Differences among groups are highly significant: t = 5.065, df = 17, p < 0.0001. (D) Photomicrograph that illustrates a sagittal section through the CN and SNpc immunostained for tyrosine hydroxylase in the brain of a 6-OHDA-lesioned rat implanted with a DA-loaded, TiO2 lattice (asterisk) 2 months after implantation. Note the reduction of the immunohistochemical staining of both nuclei in the lesioned, implanted rat as compared with that in the unlesioned rat illustrated in (A). Implantation did not promote the regenerative processes of the nigrostriatal pathway. LS = lesion site. (E) Bar graph that shows DA concentration in the CN of unlesioned rats (control sham) and in 6-OHDA-lesioned rats carrying DA-unloaded (TiO2) and -loaded TiO2 (TiO2/DA) lattices after 15 or 30 days of implantation. ANOVA (F(3,11) = 51.59, p < 0.0001, r2 = 0.9336), (a) TiO2/DA groups versus control sham or TiO2; (b) TiO2/DA group at 30 days versus control sham, TiO2 or TiO2/DA group at 15 days. (F) Line graph that depicts the dynamics of DA release from TiO2 lattices in vitro for up to a month. (G) Bar graph that shows the concentration of malondialdehyde (MDA), a metabolite derived from lipoperoxidation, in CN homogenate samples obtained from unlesioned (control) rats and 6-OHDA-lesioned rats implanted with TiO2 lattices unloaded (TiO2) or loaded (TiO2/DA) with DA. No significant differences among groups were observed.

3.2.2. TiO2 Lattices Readily Release DA in Vivo and in Vitro

To evaluate whether TiO2 lattices release DA into the CN and under in vitro conditions, we measure DA content in CN homogenates and in liquid samples of tubes filled with ACSF (Figure 5F), respectively, through HPLC. DA content in the CN samples of 6-OHDA-lesioned rats implanted with DA-loaded TiO2 lattices, but not in those implanted with DA-unloaded lattices, increased steadily for up to 2 months following implantation (Figure 5E). In vitro, DA concentration rapidly increased during the first 15 days and then dropped by day 30 of incubation (Figure 5F). Clearly, TiO2 lattices are capable of releasing DA when tested under in vivo and in vitro conditions. Differences of the rate of DA release between conditions likely reflect the technical variations inherent to in vivo and in vitro testing.

3.2.3. TiO2 Lattices Unloaded or Loaded with DA Did Not Increase CN Lipid Peroxidation

Lipid peroxidation is an index used to estimate oxidative damage. DA, its metabolites, and TiO2 may induce oxidative stress. We then estimated the concentration of MDA to evaluate whether DA-loaded or -unloaded TiO2 lattices induce oxidative stress following implantation. Our results show that MDA concentration (Figure 5G) was similar in the CN of nonlesioned or 6-OHDA-lesioned, implanted rats regardless of whether the TiO2 lattices carried DA or not, at least after a month following implantation, telling us that the implant does not produce oxidative stress.

3.2.4. CN Implantation of DA-Loaded TiO2 Lattices Attenuates Motor Symptoms in 6-OHDA-Lesioned Rats

We tested the therapeutic effects of implanting DA-loaded TiO2 lattices into the CN of 6-OHDA-lesioned rats. Rats lesioned unilaterally with 6-OHDA in the nigrostriatal pathway, but implanted with DA-loaded TiO2 lattices, showed a significant reduction of the number of amphetamine-induced turns, as compared with 6-OHDA-lesioned rats carrying no implants (Figure 6; compare 6-OHDA and 6-OHDA/TiO2DA columns; also see Supporting Information, Video S2). The effect seems cumulative over time because rats implanted with DA-loaded TiO2 lattices displayed normalized amphetamine-induced turning behavior by the end of the 2nd month of sustained treatment (Figure 6; compare 6-OHDA/TiO2DA columns at 1 and 2 months following implantation). The beneficial effect of implanting DA-loaded TiO2 lattices seems specific since the implantation of DA-unloaded ones did not attenuate amphetamine-induced, abnormal turning behavior in 6-OHDA-lesioned rats (Figure 6; compare 6-OHDA/TiO2 columns versus 6-OHDA/TiO2DA column at 1 and 2 months following implantation). Lastly, we introduced two groups of control rats that carried one DA-unloaded (Figure 6; TiO2 columns) and the other DA-loaded (Figure 6; TiO2/DA columns) TiO2 lattices to evaluate implant toxicity. As shown in Figure 6, rats carrying implants loaded or not with DA showed no abnormal turning behavior following 1 or 2 months after implantation. These results suggest that neither neurological nor systemic toxicity is induced by the implant regardless of whether or not they carry DA, at least during the period evaluated.

Figure 6.

Figure 6

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6-OHDA-lesioned rats implanted with DA-loaded TiO2 lattices significantly attenuated amphetamine-induced turning behavior over a 2 month period following surgery. Bar graph showing the number of turns per hour in unlesioned (control), 6-OHDA-lesioned rats (6-OHDA), unlesioned rats implanted with DA-unloaded (TiO2) and -loaded TiO2 (TiO2/DA) lattices, and 6-OHDA-lesioned rats implanted with DA-unloaded (6-OHDA/TiO2) and -loaded TiO2 (6-OHDA/TiO2DA) lattices, as evaluated 1 and 2 months after lattice implantation. Average values ± standard error of the mean are depicted. Each circle represents an individual animal. ANOVA (F(11,80) = 68.52.07; α,βp < 0.0001; r2 = 0.9040).

4. Discussion and Conclusions

Parkinson’s disease (PD) is a prevalent neurodegenerative disorder characterized, at intermediate stages, by the expression of motor symptoms associated with the decrease of CN DA availability. The restitution of CN DA concentration must then be at the core of the therapeutic measures aimed at correcting PD-associated motor deficits. Many have been the strategies developed over the years that procure this goal. Unfortunately, most, if not all, have failed in providing a long-term, reliable source of DA to the depleted CN. We think that nanotechnological developments could improve our chances of accomplishing such a goal. Accordingly, in this work, we describe the assembly of a TiO2 semicrystalline lattice that readily releases DA when implanted into 6-OHDA-lesioned rats. The DA released by the implanted TiO2 lattice attenuated motor symptoms to various degrees in this group of rats in a specific way since implanted DA-unloaded TiO2 lattices did not attenuate the motor dysfunction. In addition, we did not observe systemic or local toxicity associated with the implantation of DA-loaded or -unloaded TiO2 lattices during the periods evaluated. Hence, the use of implantable devices designed to permit the frequent replacing of DA-loaded TiO2 lattices might prove to be an effective, long-term source of DA for PD patients in the future. Even though such a device is not available yet, we are currently working on the prototype.

DA, when released free and exposed to the extracellular space, is rapidly oxidized into the toxic metabolites DAq and DAcr that may lead to oxidative stress. It is imperative, therefore, to evade as much as possible DA oxidation when administering it into the brain from external, non-neural sources. Accordingly, in this work, we showed that TiO2 semicrystalline lattices reduce DA oxidation. Indeed, the formation of DAq and DAcr was significantly decreased when DA was contained into the TiO2 lattice. Even though some oxidation may occur, its rate was low and remained constant for months under in vitro conditions at room temperature. Although the molecular interactions underlying the ability of TiO2 lattices to protect DA from being oxidized are yet unclear, we think that the charge-transfer complex formed between the DA and TiO2 crystalline phases prevents DA from the ongoing oxidation; the structural stability of TiO2–DA linkages depends on the bidentate binding, as previously suggested.24 Furthermore, we believe that the crown ether is situated, electrostatically, around some of DA’s H atoms, also preventing DA to be oxidized. It has been shown that 15C5 enhances the fluorescence emission of Eu2+ because the coordinating macrocyclic ligand protects these ions of interactions with OH groups from water, which induce its nonradiative decay.43 In a similar way, we also consider that the 15C5 protects DA from interactions with external O2 ions, avoiding its oxidation.

DA must be readily available in the extracellular space to exert its modulatory actions. Its excess, nonetheless, might lead to toxic effects. In our in vivo experiments, TiO2 lattices implanted into the CN readily release, for up to a month, DA in amounts sufficient to attenuate the motor symptoms displayed by 6-OHDA-lesioned rats. During this time, however, DA concentration was several fold higher in homogenates of implanted CN than that in reference CNs. Given these concentrations, DA could have been toxic locally. We then tested this possibility by estimating lipoperoxidation in the CN of unlesioned rats and of rats implanted with TiO2 lattices loaded or not with DA. We did not find significant differences in CN lipoperoxidation among groups, thus suggesting that neither DA nor TiO2 lattices are toxic alone or conjoint under the experimental conditions assessed for up to 1 month. We complemented these results by evaluating turning behavior and SNpc neuron number after implanting DA-unloaded or -loaded TiO2 lattices into the CN of unlesioned rats. Both parameters were found fully comparable to those observed in intact rats, also suggesting that neither DA nor TiO2 lattices are toxic alone or conjoint at the systemic level for up to 2 months. Our results, nevertheless, cannot rule out DA and/or TiO2 lattice in vivo toxicity if they are available for time periods longer than those tested here. This is why, in the future, we advise that TiO2 lattice-containing devices must be capable of isolating lattices from the host tissue and carefully dosing the amount of DA released into the implanted CN. Also, since COX2 mediates DA conversion into its oxidizing metabolites,44 the device might be designed to corelease DA and an inhibitor of COX2. Also, the dietary consumption of antioxidants and/or the oral administration of COX2 inhibitors (e.g., meloxicam) could help prevent and/or attenuate potential side effects associated with the use of DA-loaded TiO2 lattice devices to locally and chronically supply DA in PD patients. Lastly, since DA released by the matrix likely diffuses widely through the caudate nucleus (volume transmission for DA has been estimated to be 14.7–9.3 nM),35 the final concentration at distinct points could be low enough to avoid toxicity since DA-buffering mechanisms could take care of it. Clearly, future experiments must assess the in vivo diffusion rate and ratio of the DA freed by the implanted TiO2 lattices by using radioactive DA. They may also establish the mechanism by which DA is freed from the TiO2 lattice. We think, nonetheless, that DA release from the TiO2 lattice might ensue following the “rupture” of hydrogen bonds occurring predominantly in the amorphous structure of the TiO2 lattice when placed in aqueous media. In any event, it is fair to state that TiO2 lattices stand as a promising tool to release DA readily following a concentration or electromotive gradient.

Finally, an intriguing aspect of this work relates to the basic aspects of brain function. The brain takes a great deal of time to form rather specific connections among neuronal assemblies. This is why we have a “connectivist” view of the brain morphofunctional operation. The fact that DA is not released at specific synaptic sites but rather freed widely in the extracellular space from the TiO2 lattices, and yet it remains capable of attenuating motor dysfunctions in 6-OHDA-lesioned rats, challenges the notion of a predominantly connectivist brain. In this case, it appears that volume transmission and the widespread activation of dopaminergic transmission with lack of topographic specificity are sufficient to overcome the functional morphological impairment associated with DA depletion of the CN in hemiparkinsonian rats. It would then be necessary to explore the long-term effects of volume transmission on the specificity of synaptic and circuit operations to foresee potential negative effects.

Acknowledgments

The authors thank Fís. Roberto Hernández, Arq. Diego Quiterio, and Dra. Ivonne More for technical advice and administrative support during the execution of the present work. This project was partly supported by the Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, CONACyT scholarship 443935, CONACyT 179607, PAPIIT IN 113917, and SECITI/053/2016.

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00626.

  • Photographs of TiO2 and TiO2/DA complex, FTIR spectra of TiO2/DA complex preserved at 4 °C, TH immunoreactivity of control rats and these lesioned with and without the implant loaded or not loaded with DA (PDF)

  • Behavior of rat no. 10 lesioned with 6-OHDA (AVI)

  • Behavior of rat no. 10 treated with TiO2/DA implant (AVI)

Author Contributions

# M.G.-C. and G.P.-P. contributed equally to this work; they are both first authors.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

Supplementary Material

ao8b00626_si_001.pdf (349.2KB, pdf)
ao8b00626_si_002.avi (2.7MB, avi)
ao8b00626_si_003.avi (3MB, avi)

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Supplementary Materials

ao8b00626_si_001.pdf (349.2KB, pdf)
ao8b00626_si_002.avi (2.7MB, avi)
ao8b00626_si_003.avi (3MB, avi)

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