Bioinspired Theranostic Coordination Polymer Nanoparticles for Intranasal Dopamine Replacement in Parkinson’s Disease

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.


Materials
All chemical reagents were purchased from Sigma-Aldrich (unless otherwise specified) and used as received. Solvents were obtained from Scharlab and used as received. Cell culture media and culture media supplements were ordered from Life Technologies. 1,4-Bis(imidazol-1-ylmethyl)benzene (BIX) was synthesized as previously reported. 1 Self-assembled DA-based coordination polymer nanoparticles (DA-NCPs) were prepared under general aseptic conditions to ensure the suitability of the final material for biological experiments.
Accordingly, the sterility of the final samples used for cell culture was evaluated following standard microbiological procedures. 2 Cell lines were obtained from the American Type Culture Collection (ATCC).

HPLC Quantification of 1,4-Bis(imidazol-1-ylmethyl)benzene (BIX) in the DA-NCPs
The concentration of BIX present in the DA-NCPs was determined by HPLC-UV using an HPLC Waters 2695 separation module coupled to a Waters 2487 UV-Vis detector (suitable for dual detection). Analyses were performed using the Chromolith ® Performance RP-18e (100 mm x 4.6 mm) column. Samples of DA-NCPs were prepared by dissolving 1.5 mg of dried nanoparticles in 0.15 mL of a methanol/HCL mixture (50 µl of HCl were dissolved in 1 mL methanol), and then sonicated for 20 min in an ultrasonic bath until a precipitate is observed.
The initial samples were diluted to a final volume of 1.5 mL of Milli-Q water and centrifuged 10 min at 16000 x g. After centrifugation, the supernatants were further diluted in 0.1% (v/v)

ICP-MS Quantification of Iron in DA-NCPs
The total iron present in DA-NCPs was determined by ICP-MS using a Perkin Elmer NexION

In vitro Release Kinetics Under Physiological Conditions
In vitro release kinetics of DA from DA-NCPs was evaluated by the dialysis method. Briefly Dopamine concentration in samples was determined by HPLC-ECD as previously described.

Processing and Post-Processing of MR Data
Processing and post-processing of T1 and T2 maps were carried out with Bruker software Paravision (version 5.1) by using the image sequence analysis (ISA) tool package. T1 and T2 weighted MRI were analyzed with ImageJ 1.49V (National Institutes of Health, USA).
Three regions of interest (ROIs) were manually defined after visual inspection both in the area of maximum enhancement and equivalent area of contralateral parenchyma. The relative contrast enhancement (RCE) -injection site ROI vs. contralateral parenchyma -obtained in each case was used for calculations (see Equation S1 and S2). Only the slice with a betterdefined contrast-enhanced region in the best study (clear detection of individual RCE regions) was used for measurements. Slices of similar anatomical positions were used for T1 and T2 RCE measurements.

Equation (S1)
Where S (i) is the absolute signal intensity of the "ipsilateral" region with respect to the contrast administration and that visually shows contrast enhancement, and S (c) is the absolute signal intensity of the equivalent contralateral region, which serves as a control and that is defined as "100%". where y is the image signal intensity after t, A is the absolute bias, C is the estimated image signal intensity at t = 0 sec, and t stands for the different TR used in the sequence.

Equation (S3)
where y is the image signal intensity after t, A is the absolute bias, C is the estimated image signal intensity at t = 0 sec, and t stands for the different TE used in the sequence.
Finally, dual images reflecting both T1 and T2 RCE changes were generated with a postprocessing algorithm of imaging division (T1w image / T2w image) producing a colored image highlighting dual RCE (figure 6c of the main manuscript).

Encapsulation of Epidermal Growth Factor (EGF)
To quantify the amount of encapsulated EGF, a series of dilutions of pure EGF were analysed by HPLC ( Figure S7). The amount of loaded EGF has been calculated as the difference between the amount of EGF present in the initial reaction and the amount of protein determined from the supernatant after the synthesis. The equations used for calculation of the EE and LC are the following equations:            µL of EGF at a concentration of 10 µg/µL were mixed with iron acetate solution, which was then quickly added to DA/BIX solution. The reaction was then followed as usual. As control condition, the same reaction was carried out by replacing the volume of EGF solution with PBS. The supernatants of each reaction was collected and lyophilized. These samples were suspended in 1 mL of 0.1 % v/v of trifluoroacetic acid (TFA) water solution, and a dilution of 1:4 in the same buffer was made. The diluted samples, along with different dilutions of EGF as standards, were analyzed by HPLC-UV using a C18 column.         Values are mean ± SEM. Statistical analysis: three-way ANOVA (full factorial) and Sidak post-hoc correction was applied to the log-transformed data. *p < 0.05, compared to basal levels (0 µg/mL); #p < 0.05, compared to treatment with DA; a p < 0.05, compared to the levels found after 2 h; b p < 0.05, compared to levels found after 6 h;