Stabilization of Human Tyrosine Hydroxylase in Maltodextrin

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Stabilization of human tyrosine hydroxylase in maltodextrin nanoparticles for delivery to neuronal cells and tissue Maria Teresa Bezem, Fredrik Gullaksen Johannessen, Kunwar Jung-KC, Edvin Tang Gundersen, Ana Jorge-Finnigan, Ming Ying, Didier Betbeder, Lars Herfindal, and Aurora Martinez Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00807 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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Bioconjugate Chemistry

Stabilization of human tyrosine hydroxylase in maltodextrin nanoparticles for delivery to neuronal cells and tissue Maria T. Bezem†1, Fredrik G. Johannessen†1, Kunwar Jung-KC†, Edvin Tang Gundersen‡, Ana JorgeFinnigan†, Ming Ying†, Didier Betbeder§, Lars Herfindal‡, Aurora Martinez†,*

†

Department of Biomedicine and K.G. Jebsen Centre for Neuropsychiatric Disorders, University of

Bergen, 5009 Bergen, Norway. ‡Centre for Pharmacy, Department of Clinical Science, University of Bergen, Norway. §LIRIC – Lille Inflammation Research International Center – U995, University of Lille, and Inserm, CHU Lille, F-59000 Lille, France. 1

These authors have contributed equally to the work

*Address correspondence to: [email protected]

ABSTRACT Enzyme replacement therapy (ERT) is a therapeutic approach envisioned decades ago for the correction of genetic disorders, but ERT has been less successful for the correction of disorders with neurological manifestations. In this work, we have tested the functionality of nanoparticles (NP) composed of maltodextrin with a lipid core to bind and stabilize tyrosine hydroxylase (TH). This is a complex and unstable brain enzyme that catalyzes the rate-limiting step in the synthesis of dopamine and other catecholamine neurotransmitters. We have characterized these TH-loaded NPs to evaluate ACS Paragon Plus Environment

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their potential for ERT in diseases associated to TH dysfunction. Our results show that TH can be loaded onto the lipid core maltodextrin NPs with high efficiency, and that both stability and activity are maintained through loading and are preserved during storage. Binding to NPs also favored the uptake of TH to neuronal cells, both in cell culture and brain. The internalized NP-bound TH was active as we measured an increase in intracellular L-Dopa synthesis following NP uptake. Our approach seems promising for the use of catalytically active NPs in ERT to treat neurodegenerative and neuropsychiatric disorders characterized by dopamine deficiency, notably Parkinson’s disease.

KEYWORDS: Polymeric nanoparticles, enzyme replacement therapy, tyrosine hydroxylase, stabilization, protein delivery, cell uptake, mouse brain.

Parkinson’s disease (PD) is the second most common age related neurodegenerative disease and the lifetime risk has been estimated to be 2% for men and 1.3% for women1, although there are differences between geography, ethnicity and exposure to genetic, behavioral and environmental risk factors2. PD is characterized by the progressive death of dopaminergic neurons in the substantia nigra of the brain, leading to the loss of dopamine, a neurotransmitter that controls motor activity, hormone release and reward-motivated behavior3. The most effective treatment of PD is the symptom modifying oral administration of L-dihydroxyphenylalanine (L-Dopa), the precursor of dopamine4. Although the overall response to L-Dopa treatment is positive, there are many drawbacks related to its elevated peripheral degradation, loss of efficacy with time and dyskinesia, and extensive research is dedicated to the development of alternative treatments4. Dopamine deficiency is also associated with other disorders, including several neurodegenerative and neuropsychiatric diseases, such as Alzheimer’s disease5, depression6, schizophrenia7 and rare disorders caused by mutations in tyrosine hydroxylase (TH), such as TH deficiency (THD)8, or in enzymes associated with synthesis and regeneration of the TH cofactor tetrahydrobiopterin (BH4)9,10. TH catalyzes the rate-limiting step in the biosynthesis of dopamine and ACS Paragon Plus Environment

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other catecholamines by converting L-tyrosine into LDopa11. TH requires an enzyme-bound non-heme ferrous iron (Fe2+), BH4 and molecular oxygen (O2) as additional substrate for catalysis12. TH is thus a potential protein drug or biologic not only for PD, where the lack of TH leads to movement and cognition impairment, but also for dopamine deficiencies in general. Enzyme replacement therapy (ERT) is a therapeutic strategy where a functional enzyme is delivered to substitute the deficient endogenous enzyme. So far, ERT is the current standard in several metabolic diseases such as lysosomal storage diseases with no or little neuronal involvement, but advances in nanoparticle-driven therapy have opened for promising outcomes also for neuropathic lysosomal disorders13. ERT often implies the delivery of large therapeutic protein drugs, which typically have low bioavailability and rapid clearance. Circulation time can be increased by different strategies, such as PEGylation14, but nanocarriers are increasingly used due to their effectivity to increase the biodistribution of their cargo by protecting it from enzymatic degradation15. Nanocarriers enhance the effect of the drug by aiding in the delivery to the target site while keeping the drug in an active state. Polymeric nanoparticles (NPs) are well suited for carrying proteins as they increase the stability, have high biocompatibility, and have a high versatility in protein-drug formulation. The latter characteristic is an advantage above other nanoacarriers, as polymeric NPs have a good storage stability and the possibility to load the protein after the production stage, avoiding harsh conditions often needed in their synthesis16. Positively charged maltodextrin-based NPs are a type of polymeric NPs that have shown to be able to absorb large amounts of protein post-synthesis as they behave as flexible sponges, are biodegradable and when loaded with 70% (w/w) lipid do not induce complement activation17. Thus, ERT with highly functional TH represents a novel method for a controlled synthesis of dopamine locally in the brain. The aim of our work has been to investigate the applicability of TH bound to nanocarriers with the long-term goal of increasing life quality of patients with PD and other dopamine deficiencies. We have characterized the functionality of porous maltodextrin nanoparticles (NPs) with bound TH with regards to loading efficiency, stability, enzymatic activity, cellular uptake ACS Paragon Plus Environment

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and bio-distribution within the brain. Towards this aim, we have recently optimized TH expression and purification to obtain enzyme preparations with best possible stability and activity18. Here we present the potential of lipid-loaded cationic porous maltodextrin to deliver active TH, and demonstrate reduced in vitro aggregation, increased cellular uptake of fluorescently-labeled TH in neuroblastoma cells, and improved diffusion of TH into mouse brain tissue around the injection site. TH activity measurements using both neuroblastoma cells and mouse brain lysates show an increase in intracellular TH activity after TH uptake facilitated by the NPs, which indicates the potential of these catalytically active, THbound nanocarriers as possible therapeutic agents. RESULTS Preparation and stability of TH-loaded maltodextrin nanoparticles. TH-loaded NPs were titrated against recombinant TH that had been expressed in E. coli, purified and cleaved from the His6-MBPTH1 complex as described previously18. We applied dynamic light scattering (DLS) to characterize the binding of TH to maltodextrin NPs. Lipid insertion into the porous maltodextrin NPs (loaded with 70% lipid17) increased the NPs’ diameter from ∼65 nm (Supplementary Figure S1, green curve) to ∼100 nm (Figure 1A, green curve). Upon addition of TH to the lipid-modified NPs at a 2:1 (w/w; TH:NPs) or 1:1 ratios no peak of ∼15 nm in diameter, corresponding to free TH in solution (Figure 1A, red curve), was observed (Figure 1A, yellow curve and data not shown). On the other hand, if the ratio was increased to 3:1 (data not shown) or 4:1 (Figure 1A, light blue curve) non-encapsulated TH appeared in the samples. Moreover, an increase in NP size was observed at the 4:1 ratio, suggesting the presence of TH on the NP surface. The saturation of TH inclusion into the porous maltodextrin matrix thus occurs at a ratio close to 2:1 and, at these binding conditions, no increase of size of the NPs was observed. Binding to the slightly smaller non-lipidic NPs also occurred without size increase, but saturation with TH was already observed at 1:20 and 1:10 (w/w) ratios of TH:NPs (Figure S1), implying that a larger amount of TH absorbed into the porous matrix of the NPs with lipid-core. These lipid-core NPs with higher loading capacity were therefore selected for the rest of this work. ACS Paragon Plus Environment

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To investigate the stability of TH-loaded NPs at physiological temperature, they were incubated at 37 °C and the sizes were measured continuously by DLS (Figure 1B). The increase in the Z-average diameter indicates that TH rapidly aggregates, whereas loading into NPs prevents (at 1:1 ratio) or largely delays (at 2:1 ratio) the aggregation of (released) TH (Figure 1B). Aggregation was temperaturedependent and TH did not aggregate during 5 day-storage at 4 °C, neither did any of the TH-loaded NPs (Figure S2A).

Figure 1. TH loading into NPs with a lipid-core, and stability of the TH-NP complex monitored by dynamic light scattering (DLS). A) Intensity-based size distribution of NPs that were loaded with TH at different TH:NPs ratios. The data are presented as mean of three replicates of a representative loading experiment. B) Time-dependent stability of TH-loaded NPs at 37 °C presented as mean of three independent measurements of Z-average diameter, each performed in triplicates. ACS Paragon Plus Environment

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We also measured the functionality of the NPs by TH activity assays. In living cells TH activity is stabilized through regulated binding of the enzyme to partners in a phosphorylation dependent manner, as well as feed-back catecholamine inhibition19. However, recombinant TH is very unstable in vitro and its activity quickly decreases after incubation at 37 °C for 24 h18. The loading of TH into the NPs did not affect the enzymatic activity, as there were no significant differences between TH in solution and TH-loaded NPs (shown for selected TH:NP ratios in Figure S2B, at t = 0), but time dependent loss of enzyme activity at 37 °C was still observed. On the other hand, higher TH stability is encountered upon incubation at 4 °C, which allowed to measure the additional stabilization provided by TH binding to NPs, both below- and at saturation (1:1 and 2:1 TH:NPs ratios). These conditions preserved TH activity for 5 days, whereas the activity of the free enzyme and the TH-loaded NPs above saturation (3:1 and 4:1 TH:NPs ratios) were significantly decreased (Figure S2B).

Maltodextrin nanoparticles can deliver functional TH to neuronal and endocrine cells. Lipid-core maltodextrin NPs have been demonstrated to be effective for intracellular delivery of proteins into the cytoplasm20, and we next investigated the cellular uptake of functional TH loaded in NPs. In order to assess the internalization, neuroblastoma cells, which represent in vitro models of neuronal function, were treated with Alexa568-labeled TH-loaded NPs for 1 h and imaged with confocal microscopy. The NPs were loaded at a 1:3 TH:NPs ratio, which was found to provide optimal loading of the fluorescently labeled TH. The membranes were stained with Oregon Green®488-labeled wheat germ agglutinin (WGA) to evaluate the extent of TH localization within the plasma membrane. The results show that TH uptake is higher in cells treated with TH-loaded NPs than in cells treated with TH in solution (Figure 2A). The uptake was also remarkably high in PC12 cells, which represent models of neurosecretory cells that easily take up cargo from the environment21 (Figure S3). The use of an early endosome marker (EEA-1) shows that some internalized TH-loaded NPs co-distribute with early endosomes (Figure 2B).

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Figure 2. TH uptake mediated by NPs in SH-SY5Y neuroblastoma cells. A) Representative confocal images are shown of control cells (untreated) or treated with Alexa568-labeled TH (red) or Alexa568labeled TH-loaded NPs (1:3 TH:NPs ratio). Membranes were stained with WGA-Oregon Green®488 (green). Arrows indicate TH:NPs uptake. B) Co-distribution EEA-1 (early endosome marker shown in green) with Alexa568-labeled TH-loaded NPs. Arrows indicate signal co-distribution. In all cases nuclei were stained with DAPI (blue) and scale bars are 10 µm. ACS Paragon Plus Environment

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Detailed examination of the TH-NPs uptake was performed by live cell imaging of neuroblastoma cells. The cells were transfected with farnesylated-GFP to map the cytoplasmic side of the plasma membrane, and incubated with TH-Alexa568:NPs, which aided to identify signals from TH-loaded NPs located within the cell (Figure 3A). Furthermore, we investigated a possible targeting of the NPs to the lysosomal compartment and, as shown in Figure 3B, a large fraction of the TH-NPs was not associated with lysosomes even after 1 h treatment with NPs.

Figure 3. Localization of TH in live cells using confocal microscopy after 1h treatment of TH:NPs. A) Live cell imaging of SH-SY5Y neuroblastoma cells transfected with farnesylated GFP (green) mapping the cytoplasmic side of the membrane, and incubated with 1:3 TH-Alexa568:NP (red) for 1 h. In the 3D reconstruction, the yellow signal is a surface rendered based on co-localized labeled TH and GFP signals and the green signal was made transparent to better detect TH:NP within the cell. B) Live cell imaging of SH-SY5Y cells incubated with 1:3 TH-Alexa568:NP (red) for 1 h and stained with lysotracker DND-26 (green). In the 3D reconstruction, the co-localization between green (lysotracker) and red (TH-Alexa568:NP) is seen in yellow and show some co-localization of TH:NPs and lysosomes. All the confocal images shown are max projections of a z-stack, and the nuclei are dyed with Hoechst


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(blue). 3D reconstructions were made using Imaris x64 8.3.1. (Bitplane) for clearer visualization of the co-localizations. Scale bars are 5 µm. To evaluate the level and functionality of TH after intracellular delivery, lysates of neuroblastoma cells treated with TH-loaded NPs were analyzed by Western blot and assayed for TH activity (Figure 4). In control samples treated with high levels of NPs (410 µg), fewer cells could be collected. Since earlier studies of these maltodextrin NPs without lipidic modifications have shown no cytotoxicity but some indications of genotoxic effects at extremely high doses22, a toxic effect of the lipid-core maltodextrin NPs cannot be ruled out and must be investigated further. In any case, NP-levels up to 75 µg did not elicit any cytotoxicity, and this amount was used with TH-loaded NPs (at 2:1 TH:NPs ratio). At these conditions, we observed a significant increase in the intracellular TH content in neuroblastoma cells, to a much higher extent than treatment with TH alone (Figure 4A). TH delivered by NPs was well folded and regulated as it was detected by an antibody specific to a phosphorylated form of TH at Ser40, a post translational modification that leads to enzyme activation9 (Figure 4A). A lower fraction of TH (with respect to total internalized TH upon treatment with the control enzyme, without NPs) was intracellularly phosphorylated at Ser40 (Figure 4A). Uptake of TH-loaded NPs at saturating conditions (2:1 TH:NPs ratio) increased the intracellular TH activity significantly compared to the background activity of the untreated and TH controls (Figure 4B). Given dopamine’s chemical toxicity23, TH is a strictly regulated enzyme24,25 to keep dopamine concentrations at a low level in ‘resting situations’. Therefore, it is difficult to measure direct increases in intracellular levels of dopamine and other catecholamines that correlate with increases of total TH activity. To bypass this difficulty and be able to obtain a more direct measurement of TH intracellular activity, we have monitored the accumulation of L-Dopa, the direct product of TH, which is not subject of such intracellular homeostasis as dopamine. In order to do so, we have applied a method that includes the use of the inhibitors of L-Dopa decarboxylase (DDC) benserazide and 3-hydroxybenzylhydrazine dihydrochloride (NSD 1015) to avoid the conversion of L-Dopa to dopamine, and further permits the ACS Paragon Plus Environment

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accumulation of L-Dopa in the cytoplasm26-28. Using this strategy, we were able to demonstrate the intracellular functionality of TH by measuring the increased amounts of L-Dopa in cytoplasmic extracts following the incubation of neuroblastoma cells with TH-NPs. In order to ensure sufficient BH4 cofactor availability for additional TH activity, we added dihydrobiopterin (BH2) to the medium, both for untreated cells (control) and for cells treated with TH-NPs, as BH2 is effectively reduced to BH4 intracellularly by dihydrofolate reductase, whereas BH4 is toxic when added directly to the cell medium29. L-Dopa production in neuroblastoma cells was increased after 2 and 4 h of incubation with TH loaded NPs with respect to control cells (Figure 4C).

C

Figure 4. Protein level and activity of TH taken up by SH-SY5Y neuroblastoma cells alone or bound to NPs. A) Representative Western blots of total TH protein (upper immunoblot) and TH protein phosphorylated at Ser40 (THSer40) (lower immunoblot) in SH-SY5Y cell lysates after treatment with untreated (just PBS added) (lane 1) or treated cells with either 72 µg NPs (lane 2), 410 µg NPs (lane 3), 145 µg TH alone (lane 4) or TH-loaded NPs at 2:1 TH:NPs ratio, with 145 µg TH and 72 µg NPs (lane 5). B) TH activity in neuroblastoma cell lysates after treatment with TH-loaded NPs or controls. Data is presented as the mean ± combined SD of three independent experiments each performed in triplicates, *p≤0.01) compared to both untreated and TH control, using the Holm-Sidak method in a one-way ANOVA. C) Intracellular L-Dopa production in neuroblastoma cells after 2 and 4 h treatment with THloaded NPs. The amount of L-Dopa is determined through a NaIO4 treatment that converts L-Dopa to dopachrome, which absorbs at 475 nm. Data is presented as the mean ± combined SD of 3 independent ACS Paragon Plus Environment

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experiments each performed in duplicates, with two-tailed p-values *p≤0.05 ***p≤0.001 compared to the corresponding untreated sample (incubated at otherwise identical conditions; see Methods), using student t-test.

Biodistribution of TH-loaded NPs in mouse brain. We next studied if the TH-loaded NPs could internalize TH in the main organ affected in PD, namely the brain. Fluorescent NPs (labeled with Atto488) or TH-Alexa568-loaded NPs (2:1 TH:NPs) were injected intracerebrally in the caudate putamen of adult mice. Mice were sacrificed 24 h after treatment and their brains cryo-sectioned and imaged with fluorescence and confocal imaging. Fluorescent maps of each whole brain section per mouse show that both non-loaded and TH-loaded NPs accumulate close to the injection site (Figure 5AC), showing some ability to penetrate the tissue surrounding the injection site (Figure 5D-F). Also, the injection itself did not produce autofluorescence caused by tissue damage (Figure 5A and D), supporting that the green (Figure 5B, E, and I) or red (Figure 5C, F, and H) fluorescence observed indeed is from the injected NPs or TH-loaded NPs, respectively. An important observation was that the NPs could be detected within the borderline of the WGA membrane marker, suggesting that they are internalized, possibly into neuronal cells (Figure 5G-L).


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Figure 5. Representative imaging showing the distribution of empty and TH-loaded NPs in mouse brain after intracerebral injection. A-C) Composite images of horizontal section of the mouse brain 3-4 mm below/ventral of bregma. A) nanoparticle suspension medium (PBS), B) non-loaded Atto488ACS Paragon Plus Environment

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labeled NPs, C) Alexa568-labeled TH-loaded NPs at 2:1 ratio, with 10 µg total TH. D-F) Confocal images of the injection site and surrounding tissue of corresponding brain section above. G-L) Images of the cellular localization of Atto488-labeled NPs in a brain section stained with TexasRed-labeled WGA to mark membranes. Bright field (G), confocal (H,I) and merged (J). The scale bar represents 25 µm. The blue color in panels D-F and J-L corresponds to the DNA-specific dye DAPI, for nuclear labeling. K,L are expansions of the areas marked in J. Five mice were used for these analyses.

After injection, the fluorescence from both TH-Alexa568 and NP-Atto488 could be observed above and below the injection site. Figure S4 is a collage of images showing fluorescence surrounding the injection site. The images taken at 3.3 mm dorsal to ventral depth of a horizontal section of the brain serve to illustrate the biodistribution of NP-Atto488 within the tissue. A majority of the fluorescence was found within an area of approximately 500 µm in length and 800 µm in width. Mouse brains injected with TH-loaded NPs or controls (TH alone or NPs) were snap-frozen in liquid N2, and assayed for TH activity. The measurements show a significantly higher specific TH activity in lysates of mouse brains injected with either TH-loaded NPs or with free TH in solution (Figure 6), suggesting that the NP-encapsulated TH exhibits high activity also in the brain. The trend that TH-NPs have higher median than TH alone points towards a protective function of the NPs also in the brain, which might be more evident in subsequent studies.


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TH activity (pmol L-DOPA / min / total protein)


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*

5 4 3 2 1 0

Untreated

TH

NPs

TH:NPs

Figure 6. TH activity of mouse brain lysates after injection of TH-loaded NPs (at 1:1 TH:NPs ratio, with about 10 µg TH, which was similar in the TH control). Data are presented as dot-plot where each dot represents the value from one mouse. The black lines represent the median value. *, significance at p ≤ 0.05 compared to untreated control, using the Holm-Sidak method in a one-way ANOVA.

DISCUSSION Investigations on stabilization of enzyme activity upon binding to NPs are scarce, but some proteins such as β-N-acetyl-glucosaminidase have been found to be protected from loss of thermal and long-term stability by immobilization on linoleic acid-modified carboxymethyl chitosan NPs30. Insulin also retains its activity by encapsulation into alginate–dextran sulfate-based NPs31. The monoclonal antibody bevacizumab (Avastin) has been released from porous silicon nanostructures retaining its functional antigen binding properties32. On the contrary, we have earlier observed that TH activity is reduced upon ACS Paragon Plus Environment

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binding of the enzyme to gold NPs33 and negatively charged liposomes34,35. TH is an inherently unstable protein with respect to activity and conformation18,36. We have recently shown that TH binding to phospholipid liposomes induces protein misfolding and aggregation, with concomitant disruption of the membrane integrity37. Thus, the results presented in this work, demonstrating the stabilization and prevention of aggregation of TH upon its loading into lipid-core maltodextrin NPs, are remarkable in this context. TH activity is maintained during loading (Fig. 2) and preserved during storage (Fig. S2B), and this is, to our knowledge, the first report on the use of NPs to stabilize TH. Maltodextrin NPs thus show large potential for applications with TH and other unstable enzymes in biotechnology, research, and even medical applications, as these polymeric NPs facilitate the uptake of functional TH by neuronal cells (Figures 2-4) and represent promising nanocarriers for ERT with this important enzyme. TH-associated diseases are complex neurological disorders that often require treatments that target the central nervous system (CNS), and there have been attempts to restore TH activity by gene therapy and cell transplantations38. Although these treatments have a large potential for sustained therapeutic effects they have failed either to show convincing clinical efficacy or to obtain regulatory approval39. ERT with TH-NPs would be a valuable therapeutic option and the potential of an ERT in dopamine deficiencies has been shown, among other, in experiments using systemic supplement of a TH construct that entered neurons in mouse brain and induced an anti-depressant effect40. ERT with TH-loaded NPs appears as a novel approach, which in addition opens up for controlled delivery. In the animal study included here, the TH-loaded NPs and controls were injected directly into the brain, and it was therefore not surprising that the injection of TH alone, not bound to NPs, gave a rather similar increase in the specific TH activity of the mouse brain lysates compared to TH-loaded NPs (Fig. 6). This direct injection must be seen as a proof-of-concept for the delivery of TH to brain cells, and future experiments are expected to show that NPs are critical for the delivery of TH to the brain. Thus, the maltodextrin NPs used in the initial loading experiments have been shown to cross an in vitro model of the blood-brain barrier (BBB) before lipidic modifications41. The TH-loaded NPs could be functionalized to be suited for intravenous injection, and decorated to target and cross the BBB. ACS Paragon Plus Environment

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Interestingly, reduction of dopamine levels in striatum precedes neuronal degeneration and death, and correction of this deficit is expected to diminish the progression of PD, as seen in optogenetic studies42. An TH-NP-based ERT strategy might contribute to this endeavor since improvement of biomarkerbased diagnoses is reducing the detection point of the disease, and it is expected that in a near future PD can be diagnosed before the onset of neurodegeneration of dopaminergic neurons43. In addition, TH delivery to the brain to restore dopamine levels does not necessarily require the dopaminergic neurons of the substantia nigra, which in normal conditions are the responsible neurons for the production of dopamine. It is likely to be sufficient to deliver TH into other type of neurons than those in the nigrostriatal pathway of the brain. It is remarkable how surrounding cells take over the dopamine production as an adaptive measure to compensate for the degeneration of dopaminergic neurons in PD patients44-46. Indeed, there are several neurons that express aromatic amino acid decarboxylase (AADC), the enzyme that converts L-Dopa to dopamine, such as the monoenzymatic AADC-neurons, and catecholaminergic, serotoninergic and histaminergic fibers. These can capture L-Dopa from the extracellular space for dopamine synthesis46. A further improvement of this ERT approach might include brain-implantable biodegradable hydrogels including these maltodextrin-NPs for sustained delivery of TH47.

CONCLUSION Maltodextrin NPs with a lipid core seem well suited for carrying catalytically-active TH, as our results show that both stability and activity is maintained during loading and uptake in neuronal cells and tissue. These NPs show potential interest for ERT for diseases with dopamine deficiencies.

METHODS Expression and purification of recombinant TH. TH was expressed and purified as a fusion protein with maltose binding protein (MBP), cleaved with tobacco etch virus (TEV) protease as described18 and snap frozen in aliquots and stored in liquid nitrogen. Before all experiments, a TH aliquot was thawed, ACS Paragon Plus Environment

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diluted to 1-5 mg/ml, centrifuged 15 min at 4 °C and 20 000 x g. The concentration of TH in the supernatant was then measured using Direct Detect (Bio-Rad). Preparation of nanoparticles. Porous lipid-core maltodextrin nanoparticles were prepared and functionalized as described previously41. Briefly, maltodextrin was dissolved in 2 N sodium hydroxide with

magnetic

stirring

at

room

temperature.

Addition

of

epichlorhydrin

and

glycidyltrimethylammonium yielded a cationic polysaccharide gel that was neutralized with acetic acid and crushed using a high-pressure homogenizer. The nanoparticles obtained were purified by tangential flow ultra-filtration using a 300 kDa membrane and mixed with dipalmitoylphosphatidylglycerol above the gel-to-liquid phase transition temperature to produce porous maltodextrin nanoparticles with a lipid core which were stored at 5 mg/ml in 15 mM NaCl and at 4 °C. Dynamic light scattering. TH loading into NPs and the subsequent stability was evaluated by dynamic light scattering (DLS) experiments on a Zetasizer Nano ZS (Malvern) using a HeNe laser at 633 nm, a fixed scattering angle of 173° (back scatter) and normal resolution mode of three measurements per sample consisting of 10 runs of 10 sec each, at either 4, 23 or 37 °C. Samples were measured in either a 12 µl quartz cuvette (Malvern) or a 70 µl disposable cuvette (Brand) containing 0.5 mg/ml NPs and 0.1 – 2 mg/ml TH (= 1.8 - 36 µM subunit) in 20 mM Na-HEPES pH 7.0, 200 mM NaCl. Cell culture and treatment. Neuroblastoma (SH-SY5Y, ATCC: CRL-2266) cells were grown in DMEM (Sigma) supplemented with 10% fetal bovine serum (Sigma), 2 mM Glutamine (Sigma) 10 U/ml streptomycin (Sigma) and 10 U/ml penicillin (Sigma) at 37 °C in a humidified atmosphere of 95% air and 5% CO2. For confocal imaging, 20 000 neuroblastoma cells were seeded on glass coverslips coated with 3 mg/ml PureCol (Inamed Biomaterials) and grown 24 h before being treated for 1 h with 1:3 TH:NPs (3.5 µg Alexa568-labeled TH were loaded into 10 µg NPs in PBS and incubated 10 min on ice). Treatment with appropriate controls, cells treated with only TH (3.5 µg) or only NPs (10 µg), at 37 °C ACS Paragon Plus Environment

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in serum-free media were also included. After the incubation, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 30 min at 37 °C and membrane stained by 2 min incubation with 5 µg/ml Oregon Green®488-conjugated wheat germ agglutinin (Molecular Probes) at room temperature. For endosomal staining, cells were permeabilized and blocked with PBS supplemented with 0.3% saponin and 5% FBS PBS for 30 min, incubated for 30 h at RT °C with mouse-anti-EEA-1 antibody (1:100; Transduction laboratories) and Alexa-647-goat-anti-mouse (1:200; Life Technologies) before mounting with Prolong Gold with DAPI (Life technologies). For live cell imaging 100 000 cells were seeded in a total of 300 µl media in the wells of an 8-well Ibidi chamber slide that was coated for 30 min with 1:1 laminin:collagen. At 80% confluency, 1:3 TH:NPs were added as described for the confocal imaging. After 1 h incubation the media was removed and 100 nM lysotracker DND-26 (ThermoFisher Scientific) was added for 15 min. The cells were incubated with 0.3 mg/ml Hoechst 33342 (Sigma-Aldrich) for 5 min and washed 3 times in PBS before Opti-MEM (Fisher scientific) with 2 mM glutamine was added. To study NP uptake, neuroblastoma cells were transiently transfected overnight with 0.2 µg farnesylated-green fluorescent protein (GFP) plasmid (kindly provided by Dr. Ivan Rios-Mondragon, UiB) and lipofectamine LTX with PLUS reagent (Life Technologies) following the manufacturer’s instructions, before subjecting it to TH:NPs treatment 1h prior to confocal imaging. For TH activity assays, P100 plates (Sarstedt) with neuroblastoma cells at confluence in 6 ml medium were washed with PBS and treated for 1 h with 2:1 TH:NPs or controls in serum-free medium at 37 °C. The cell treatment was prepared by loading 145 µg TH into 72 µg NPs and incubating minimum 10 min on ice before diluting each treatment in 6 ml warm PBS. Cells were collected with a cell scraper and harvested by 5 min centrifugation at 1000 rpm and 4 °C. Cell pellet was washed three times with 1 ml PBS and 5 min centrifugation at 100 g and 4 °C, then frozen in liquid nitrogen and kept at -80 °C until analyzed. Cell lysates were prepared immediately before activity assay, by thawing pellets and resuspending in PBS containing 200 µM PMSF, protease inhibitor cocktail (Roche). Cells were lysed by ACS Paragon Plus Environment

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adding 0.1% Triton X-100 and incubated for 15 min on ice. Extracts were clarified by 15 min centrifugation at 20 000 x g at 4 °C. Low molecular weight components from the supernatant were removed by using a Zeba Spin Desalting Columns (Thermo Scientific) equilibrated in 20 mM NaHEPES pH 7.0, 200 mM NaCl. Protein concentration was measured by Bradford assay (Bio-Rad) or by Direct Detect instrument (Millipore). Cell lysates were snap frozen and stored in liquid nitrogen until analyses. For Western blot analysis, the cell lysates were incubated for 5 min at 95 °C in Laemmli sample buffer. SDS-PAGE was then performed at 200 V, and proteins were transferred onto a nitrocellulose membrane. The membranes were blocked and incubated with antibody against total TH (Thermo Scientific, OPA1-04050) or TH-p40 (PhosphoSolutions, p1580-40) and GAPDH (Abcam, ab9485) for loading control. Membranes were developed by chemiluminescence. For determination of intracellular L-Dopa production, SH-SY5Y cells were seeded in P60 plates and grown until 80% confluency in DMEM (Sigma-Aldrich). Opti-MEM was supplemented with BH2 (Dr B. Schircks Laboratory, Jona, Switzerland), 20 µM benserazide hydrochloride (Sigma-Aldrich) and 100 µM 3-hydroxybenzylhydrazine dihydrochloride (NSD 1015; Sigma-Aldrich), and then added to the cells 1 h before TH:NP treatment. Treatment included incubation with 72 µg TH-loaded NP or suspension medium with or without BH2, benserazide and NSD 1015 as controls, for 2 and 4 h in supplemented Opti-MEM. The cells were next harvested and collected in 1 ml of original media and centrifuged at 10 000 rpm for 10 min at 4 °C. The supernatant was removed and the pellet snap frozen in liquid N2, and then stored at -80 °C until use. The cells were resuspended by adding 50 µl PBS with EDTA-free cOmplete protease inhibitor cocktail (Roche) to the pellet. After the resuspension the cells were lysed by a freezing and thawing cycle, in liquid N2. The lysate was diluted 1:1 with 50 µl 2% acetate in ethanol, and then centrifuged at 10 000 rpm for 10 min at 4 °C. For the determination of LDopa the supernatant was immediately diluted with 100 µM (final concentration) sodium periodite (NaIO4, Sigma) to oxidize L-Dopa to the chromophore dopachrome48. The samples were prepared on a ACS Paragon Plus Environment

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Greiner UV-Star® 96 well half area microplate, and the dopachrome absorbance in each well was measured at 475 nm at RT in a Tecan Spark 20M spectrophotometer. Animal study. All experiments including mice were approved by the Norwegian Animal Research Authority and conducted according to the European Convention for the Protection of Vertebrates Used for Scientific Purposes. Wild-type Bl-6 mice of both sexes, weighing 18-25 g and aged 2-12 months old were kept at 22±1 °C and a 12-hour light–dark cycle and they were provided food and water ad libitum. For visualization studies, both sexes were used, for analyses of TH activity, only males between three and nine months were used. Before the intracranial injection, the mice were anesthetized with isoflurane (induction dose, 3 ml/l O2, maintenance dose, 1.5 ml/l O2). The head was fixed on a stereotaxic frame, and 100 µL local anesthetics (Temgesic, 0.3 mg/ml, Indivior UK Ltd. Slough, UK) were injected subcutaneous in the scalp. After one minute, an incision was made in the scalp sagittally along the sutura sagittalis and the skull exposed. A hole of one mm diameter was made one mm lateral (right) and 1.5 mm frontal to the bregma in the skull using a dental drill. The meninges were perforated with a needle before the tip of a Hamilton syringe with a flat needle was inserted into the brain 3.5 mm below bregma by first lowering needle 4 mm and the retracting it 0.5 mm up to make space for the injected liquid. 3 µl of sample was injected during two minutes, with a one-minute rest when half of the volume was injected. The needle was then retracted while observing that no liquid leaked from the hole in the scull, and the skin was sealed with suture. The mouse’s condition was closely monitored during the first 4 h after surgery. All mice undergoing surgery had normal behavior the day after the operation. After 24 h, the animals were euthanized by CO2 and the brains excised. Brains were either immediately fixed for cryosectioning and imaging, or snap frozen in liquid nitrogen and stored at -80 °C for homogenization and activity measurements. For imaging, mouse brains were fixed for 48 h in 2% paraformaldehyde in PBS at 4 °C in the dark, before dehydration in 30% sucrose at 4 °C until subsidence. They were next snap frozen in liquid ACS Paragon Plus Environment

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nitrogen and stored at -80 °C mouse brains until they were mounted with TissueTek (Sakura Finetek, USA), and sliced horizontally into 12 µm thick sections on a Leica CM3050S cryomicrotome. The sections were placed on glass slides and left to dry on a microscope slide overnight in the dark at room temperature before storage at -20 °C. Before imaging, samples were mounted with Prolong Gold with DAPI and left to solidify in the dark and at room temperature overnight. For membrane staining, samples were incubated 5 min with 200 µl 4.5 µg/ml TexasRed-conjugated wheat germ agglutinin (Molecular Probes) at room temperature and rinsed three times with water before mounting. For TH activity assays, lysates were prepared by homogenization of mouse brains on ice with a pellet pestle (Kimble Chase) in lysis buffer containing 50 mM Tris pH 7.5, 100 mM KCl, 1 mM DTT, 200 µM PMSF, 500 µM benzamidine and protease inhibitor cocktail (Roche). Extracts were clarified by 15 min centrifugation at 20 800 g and 4 °C. Low molecular weight components were removed from the supernatants and buffer exchanged using Zeba Spin Desalting Columns (Thermo Scientific) equilibrated in 20 mM Na-HEPES pH 7.0, 200 mM NaCl. Protein concentration was measured by Direct Detect (Bio-Rad). Lysates were assayed for TH activity immediately after preparation. Fluorescence and confocal microscopy. Mouse brain sections were imaged on a fluorescent microscope (Zeiss Axioplan 2). Multiple images were combined using the panorama function in Photoshop CS4 (Adobe). Confocal imaging of the cell cultures was performed on a Leica microscope TCS SP5 in the resonant scanner mode (Leica Microsystems GmbH) using a pinhole airy 1 and a 63x 1. 4 NA immersion oil lens. For each sample a stack of images encompassing the complete height of the cell was taken, with a 130 nm step-size and using the LasAF software from Leica. Each confocal plane was 512x512 pixels with a line-average of 20. Stack images were processed in batch using Fiji (freeware)49 with minimum adjustments of brightness and background.


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TH activity assays. Enzymatic activity of recombinant TH was measured with minor modifications from earlier described50. Briefly, TH or TH-loaded nanoparticles were pre-incubated with 1 % BSA (w/v) in 20 mM Na-HEPES pH 7.0, 200 mM NaCl at 0.1 mg/ml (= 1.8 µM subunit) TH and 37 °C. Aliquots of 5 µl were taken out and incubated for 1 min in a standard reaction mixture and then assayed for 5 min. The amount of L-DOPA was measured by high-performance liquid chromatography analysis with fluorescence detection. TH activity of cell or mouse brain lysates was measured using a radioactive assay as described51 with some modifications. Three parallels of 50 µl lysate were pre-incubated at 30 °C for 1 min in an reaction volume of 100 µl containing 100 mM NaHepes, pH 7.0, 50 µM L-[3,5-3H]-tyrosine, 0.05 mg/mL catalase and 20 µM ferrous ammonium sulphate and the reaction was started by the addition of 500 µM BH4 in 5 mM DTT, and stopped after 15 or 30 min incubation at 30 °C with 1 ml of 7.5% activated charcoal suspension in 1 M HCl. After centrifugation at 10,000 g, the radioactivity in the supernatant was measured in a scintillation counter. Blanks and totals were prepared using water instead of lysate and 1 M HCl without charcoal, respectively. Statistical analysis was performed by Holm-Sidak tests in one-way ANOVA using SigmaPlot (Systat Software, San Jose, CA).

Acknowledgements: We thank Ali Javier Sepulveda Muñoz for expert help with purification of TH. Marte I. Flydal for preparation of plasmids for expression and purification of TH, and Ercan Mutlu for demonstrating intracranial injections. We thank The Norwegian Research Council, The Kristian Gerhard Jebsen Foundation and the Meltzer Fund for financial support. A.J.F. was supported by grants from the EC, Marie Curie IF program (PIEF-GA-2011-299972) and the K.G. Jebsen foundation, L.H. from the Norwegian Cancer Society, and E.T.G. from the Western Norway Regional Health Authorities (Helse-Vest). The confocal imaging was performed at the Molecular Imaging Center, Dept. of Biomedicine, University of Bergen, Norway. ACS Paragon Plus Environment

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Stabilization of Human Tyrosine Hydroxylase in Maltodextrin

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