Enhanced brain-derived neurotrophic factor distribution in differentiated human neuroblastoma SH-SY5Y cells by PEG-ylated protein nanoparticles: model of application in neurodegenerative diseases CURRENT

Brain-derived neurotrophic factor (BDNF) is essential for the development and function of human neurons, therefore it is a promising target for neurodegenerative disorders treatment. Here, we studied BDNF-based electrostatic complex with dendrimer nanoparticles encapsulated in polyethylene glycol (PEG) in injured, differentiated neuroblastoma SH-SY5Y cells, a model of neurodegenerative mechanisms. PEG layer was adsorbed at dendrimer-protein core nanoparticles to decrease their cellular uptake and to reduce BDNF-serum proteins interaction for prolonged time. Cytotoxicity and confocal microscopy analysis revealed PEG-ylated BDNF-dendrimer nanoparticles can be used for continuous neurotrophic factor delivery enhancing its distribution into cells over 24 hours without toxic effect. We offer reliable electrostatic route for efficient encapsulation and controlled transport of fragile therapeutic proteins without any covalent cross-linker; this could be considered as safe drug delivery system. Understanding of polyvalent BDNF interactions with dendrimer core nanoparticles offers new possibilities for design of well-ordered protein drug delivery systems. the present study was designed to: (a) elucidate the BDNF desorption from well-characterized PEG-ylated PAMAM dendrimer nanoparticles, (b) investigate diffusion and cytotoxicity of PEG-ylated BDNF-PAMAM dendrimer electrostatic complex in differentiated neuroblastoma SH-SY5Y cells in real-time up to 24 hours after administration. We used neuroblastoma cells for different time lengths (2min, 5min, 10min, 30min, 1h, 4h and 24h) and subjected to examinations by spectrofluorimetry evaluation. The cellular internalization of BDNF-PAMAM-AF488 and BDNF-PAMAM-AF488-PEG by SH-5YSY neuroblastoma cells was investigated by determination of green fluorescence intensity. significant increase in their concentration level compared to the control groups (100 µM 6OHDA) for both type of PAMAM-based nanoparticles. We found that PEG-ylated PAMAM-BDNF nanoparticles-treated cells expressed significantly higher level of neuroprotective protein when compared with the PAMAM-BDNF nanoparticles-treated group. These difference in the BDNF levels in extracellular region in the PEG-ylated PAMAM-BDNF nanoparticles-treated group


Introduction
Nanostructures are a promising tool for efficient therapeutics delivery even to the difficult tissues, like brain, in which blood-brain barrier remains a fundamental challenge for drug delivery systems. Since brain-derived neurotrophic factor (BDNF) induces neuronal survival and tissue repair, it is a promising therapeutic agent for treatment of neurodegenerative diseases e.g. Alzheimer's disease in which cholinergic neurons are depleted 1 ; Parkinson's disease 2 in which dopaminergic neurons of the substantia nigra are lost and amyotrophic lateral sclerosis 3 (ALS) in which cerebral and spinal motor neurons degenerate. However, efficient BDNF delivery to the brain poses a few difficulties.
BDNF is the most abundant member of neurotrophic factors family in the mammalian central nervous system. BDNF homodimer has about 27 kDa, exerts biological activity in a dimeric state and has a common structural motif consisting of 120 amino acids and forms three disulfide bridges. The isoelectric point of fatty acid free BDNF is pH 10-10.9. The electric charge over BDNF molecules is heterogeneously distributed. As a result, the amino acids structural elements of BDNF molecule like Lysine 96, Arginine 97, Glutamine 84 are presented in the active site, which gives the largest 3 positively charged region over protein molecules. 4,5 BDNF binds with high affinity to tyrosine kinase B receptor to promote trophic signaling and apoptotic events. [6][7][8] Indeed, low BDNF levels are observed in brains of patients suffering from multiple pathologies of central nervous system and changes in BDNF concentration or its distribution have been linked with several neurodegenerative and psychiatric disorders, like depression and schizophrenia. 9,10 Neurotrophins are challenging candidates for drug development, because of their low bioavailability for therapeutic targets and insubstantial pharmacokinetic behavior. Synthesis, secreted concentration and half-life of BDNF in human body are limited. 11 BDNF is the diffusible factor secreted by neuronal development system and is not available for all neurons; therefore, its delivery from cells to tissues results in concentration gradients. 12 Accordingly, improved administration of exogenous BDNF and consequent neuroprotection as well as neuroregeneration have been considered potentially novel treatments for neurodegenerative diseases, including Parkinson disease. However, carrier-free administration of BDNF is relatively unstable because of rapid degradation in biological medium due to very short in vivo half-life (<2min) and low biodistribution that cause this side effect. The rational design of BDNF-based nanoparticles requires a good understanding of their interactions for controlled protein release in order to achieve higher delivery efficiency due to increased BDNF presence in the tissue as well as its improved bioavailability.
Nanocarriers such as dendrimer have been extensively studied in various BDNF drug delivery systems 13,14 to improve their therapeutic efficiency by increasing circulation time and bioavailability at the targeted site (dendrimers possess high biocompatibility and facile functionalization lead to responsiveness to specific stimuli). In our previous studies, on 7th day post application we observed improved administration of neurotrophin-4 using dendrimer nanoparticles in impaired retina tissue. 15 Physicochemical properties of densely branched dendrimer molecules with well-defined spherical geometry, enhance stability and surface functionality of neurotrophin delivery system. Size of dendrimers nanoparticles due to their higher curvature will have a fewer number of nanoparticles ligands that can interact with protein side chains. As the result, PAMAM surface area accessible to the 4 neurothrophin will be lower, this could potentially result in less proteins denaturation. Dendrimers present strong ability to escape from the uptake by non-specific ReticuloEndothelial System and consequently avoid long term toxicities effect [16][17][18][19] . In a localized manner, dendrimers, due to high degree of structural control (monodispersity and tunable chemical structure), can be administrated in vivo as a widely utilized biological functional nanocarriers for drug, 20, 21 biomacromolecule, gene delivery, [22][23][24] imagining agents, 25,26 and diagnostic product. 27 The polyvalent interaction of dendrimers with protein 28, 29 resembles a common type of interaction between biological entities such as receptors and ligands or virus and cell surface, etc. [30][31][32][33] Nano-sized carriers systems with dendrimer core are monodisperse therapeutic scaffolds that would possibly allow BDNF delivery to damaged cells, enhancing its local concentration and protein stability against enzymatic degradation.
Therefore, we thought to use dendrimers to improve the extracellular retaining of BDNF without the need of covalent chemistry. However, we decided to improve delivery system with PEG (poly(ethylene glycol)), what allows them prevent unspecific protein adsorption onto the nanoparticle's surface [34][35][36] as well as increasing in vivo blood circulation retention times. Protein resistant PEG layer is independent of the particular choice of PEG molecular weight and thicker polymer brush does not allow proteins to experience electrostatic and van der Waals attractions. PEG brushes are grafted onto dendrimer-protein surface to render them more biocompatible by making nanoparticles less visible to phagocytic cells and improving circulating half-life of nanocarriers. The overall size of negatively charged poly(amidoamine) (PAMAM) dendrimer 5.5 generation nanocarriers with PEG core could enable efficient diffusion of BDNF across the tissue.
The main goal of our study was to determine efficient encapsulation of BDNF by PAMAM nanoparticles as well as PEG-ylated -PAMAM drug delivery system and assess their usefulness in in vitro system using neuroblastoma model. Therefore, the present study was designed to: (a) elucidate the BDNF desorption from well-characterized PEG-ylated PAMAM dendrimer nanoparticles, (b) investigate diffusion and cytotoxicity of PEG-ylated BDNF-PAMAM dendrimer electrostatic complex in differentiated neuroblastoma SH-SY5Y cells in real-time up to 24 hours after administration. We used 5 this particular cell line 37-40 as a model for the degradation of dopaminergic neuron network in the substantia nigra parts compacta (SCs) observed in Parkinson's disease (PD) patients. 41

Materials And Methods
Adsorption/desorption transition of BDNF molecules at/from PAMAM dendrimers (Figure 1) was studied in PBS buffer using the dynamic light scattering, electrophoresis, solution depletion techniques, enzyme-linked immunosorbent assay and atomic force microscopy. This allowed us to precisely determine maximum loading of BDNF molecule at PAMAM-based nanoparticles under in situ conditions. Afterwards, we compared desorption kinetics of BDNF from PAMAM-based nanoparticles as well as PEG-ylated -PAMAM nanoparticles in PBS buffer and in neuron-like differentiated SH-SY5Y cells environment to quantitatively assess in real time cellular internalization by neuroblastoma cells PAMAM-based nanoparticles (using spectrofluorimetry and confocal microscopy evaluation). The suspension of PAMAM G5.5 ethylenediamine core five and a half generation dendrimers with sodium carboxylate surface groups, (536784, Sigma Aldrich, kraj) was used as colloid carrier for BDNF. The stock suspension was diluted prior to each adsorption experiment to a desired mass concentration, equal to 10 mg L -1 .

PAMAM-AF488 Conjugates:
For detection of protein -PAMAM nanoparticles in differentiated neuroblastoma SH-SY5Y cells by confocal microscopy, a detectable fluorescent molecule i.e. PAMAM-AF488 was needed. We Protein concentrations were determined using ELISA protein assay.
Suspensions of nanoparticles as well as protein diluted with 0.15M PBS buffer to a suitable concentration were measured in Zetasizer Nano ZS apparatus (Malvern Instruments, Malvern, UK) equipped with a laser of 633nm wavelengths. Data analysis was performed in automatic mode at 25 C. Measured size was presented as the average value of 20 runs, with triplicate measurements within each run. Particle size distributions were obtained from measured diffusion coefficients.

-surface
Ruby muscovite mica (Continental Trade, Poland) was used as a substrate for BDNF, PAMAM and BDNF based nanoparticles, adsorption measurements. Fresh, solid pieces of mica were cleaved into thin sheets prior to every experiment.
The AFM (atomic force microscopy) technique was used to obtain information about the size distribution of BDNF, PAMAM dendrimers and nanoparticles. The nanoparticle as well as BDNF protein were left to deposit on mica sheets placed in the diffusion cell over a controlled time, and then substrate was removed, rinsed for half an hour in ultrapure water. The samples were left for air-drying 8 until the next day. Next, the dry sample was placed under 7-10 nm AFM tip. The AFM measurements were carried out under ambient air conditions using the NanoWizard AFM (JPK Instruments AG, kraj).
The intermittent contact mode images were obtained in the air, using ultrasharp silicon cantilevers (NSC35/AlBS, MicroMash, Spain) and the cone angle of the tip was less than 20 o . The images were recorded at the scan rate of 1 Hz for the six randomly chosen places. The images were flattened using an algorithm provided with the instrument. We captured all images in random areas within the scan size of 0.5 x 0.5 µm or 1 x 1 µm. BDNF, PAMAM 5.5, BDNF-PAMAM and PEG-ylated BDNF-PAMAM surface dimensions were determined using ImageJ software by gathering the number and coordinates of single protein/nanoparticles molecules. Manual counting of protein/nanoparticles molecules was based on comparing the original image and the same picture altered by digital image filters by cutting off the picture background.

Nanoparticle zeta potential determination
The electrophoretic mobility of BDNF molecules, PAMAM, BDNF-PAMAM as well as PEG-ylated BDNF-PAMAM nanoparticles was measured at pH 7.4 and 0.15M ionic strength with Laser Doppler Velocimetry (LDV) technique with the aid of the abovementioned Malvern device. Electrophoretic mobility was recalculated to zeta potential using Henry equation valid for higher ionic strength where the polarization of the electric double layer is relevant (the double-layer thickness becomes smaller than the protein dimension). penicillin (100 U/mL), L-glutamine (2 mM) and 15% heat-inactivated fetal bovine serum (FBS) at 37•C in saturated humidity atmosphere containing 5% CO 2 . The proliferation medium was changed every 2-3 days, and the cells were passaged when they reached 80% confluence. After the proliferation 9 step, the cells were transferred into new culture plates and incubated 24h with MEM supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), L-glutamine (2mM) and 1% FBS. On the next day, the medium was changed to a differentiation medium consisting of MEM supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), L-glutamine (2mM), 1% FBS and Retinoic Acid (0.01µmol/mL) (RA, Sigma Aldrich, St.Louis, MO, USA). The differentiation was carried out for 5 days and the medium was changed every 2 days. which protocol is based on the conversion of water soluble 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide to an insoluble formazan product, which has a purple color. Cells were incubated with 50 μL of MTT reagent mixed with 50µl MEM for 3h, then 150µl of detergent solution was added to solubilize the colored crystals. Finally, absorbance was measured at OD590nm using Varioskan LUX Multimode Microplate Reader (Thermo Fisher, Waltham, MA, USA). Toxicity was calculated from the equation in the manufacturer's protocol.

BDNF Quantification -in PBS
The protein release kinetics from PAMAM as well as PEG-ylated PAMAM nanoparticles was assessed by using ultrafiltration method with a 30kDa cutoff membrane (Millipore, Billerica, MA, USA) in PBS at pH 7.4 and 0.15M ionic strength. It was done in two-stage procedure, where first BDNF adsorption process was carried out for 1 hour. The BDNF molecules released from nanoparticles were quantified with ELISA immunoassay method according to the manufacturer's protocol. Initially, the residual (unbound) BDNF concentration in the filtrate was determined immediately after adsorption at PAMAM nanoparticles by applying sandwich ELISA technique to monitor simultaneously the maximum concentration of unbound BDNF in the supernatant suspensions. Thus, it was possible to precisely determine concentration of non-adsorbed BDNF molecules at PAMAM as well as PEG-ylated PAMAM nanoparticle surface. These measurements were utilized for determining the maximum coverage of neurotrophin under various protein bulk condition (0.002-1 mg L -1 ). Afterward, the concentration of desorbed BDNF for different time increment (20 min, 2h, 3h, 5h, 8h, 10h, 24h) was quantified by UV-VIS spectroscopy and calculated according to ELISA standard curve.

-in neuroblastoma cell culture
Concentration of releasing BDNF molecules was determined by exposing differentiated human neuroblastoma cells SH-SY5Y to 6-hydroxydopamine (6-OHDA) as well as nanoparticles with different BDNF concentrations for 24h at 37 o C. At the end of the treatments /incubation, the medium was discarded and collected to quantify BDNF concentration using UV-VIS spectroscopy calculated according to ELISA standard curve.

Internalization of PAMAM-based nanoparticles
In addition, the cellular internalization of nanoparticles with BDNF by SH-5YSY neuroblastoma cells was further investigated by determination of green fluorescence of PAMAM-AF488 conjugates.

Immunofluorescent labeling-nanoparticles imaging in vitro
For immunofluorescence analysis SH-SY5Y cells were plated in 4-well chamber slides at density 5x10 4 /well. After the differentiation and 6OHD treatment (protocols described in sections 2.4 and 2.5, respectively) cells were incubated with BDNF-PAMAM-AF488 and BDNF-PAMAM-AF488-PEG nanoparticles (0.1 μg/mL protein loading) for different time lengths (5min, 10min, 30min, 1h and 24h). At designated time points, cells were washed with PBS and fixed with 70% ethanol for 15min. To visualize surface glycoproteins, cells were stained with wheat germ agglutinin conjugated to Texas Red-X (WGA-Texas Red-X, Thermo Fisher, Waltham, MA, USA) in HEPES buffer for 30min. After DAPI counterstain, the slides were mounted and subjected to z-stack analysis using a LSM700 confocal system (Carl Zeiss, Jena, Germany).

Statistical analysis
All presented data are expressed as means +/-SD from at least three independent experiments.
Statistical analysis among each study group was performed using Kruskal-Wallis test. Two-Way ANOVA was used for analysis between experimental groups. p < 0.05 was considered statistically significant. The data are presented as the mean ± standard deviation (SD).

BDNF release from PAMAM-based nanoparticles in PBS buffer
In order to determine the possibility of desorption from dendrimers-based nanoparticles, once adsorption reached equilibrium, before the in vitro toxicity test was performed, we studied the BDNF release in PBS buffer.
Initially, adsorption of slightly positively charged BDNF molecules to negatively charged PAMAM dendrimers core surfaces was precisely determined to control the concentration of unbounded protein molecule and protein-laden nanoparticles structure. For every system the saturation concentration of 13 protein has to be determined empirically. PAMAM nanoparticle is characterized with significant changes in its apparent zeta potential during adsorption, which can be efficiently monitored by LDV method. Importantly, at pH 7.4 BDNF molecule carry a net positive charge adsorb onto negatively charged uniform surface. After loading of BDNF into PAMAM-based nanoparticles, we determined the dependence of the zeta potential of nanoparticles on the initial concentration of BDNF in the PAMAM suspension (after mixing). As is depicted in Figure 1  For both type of nanoparticles, first phase in releasing profile is characterized by a fast release of BDNF molecules from their surfaces, which probably results from the solubilization of protein adjoining dendrimer surface. In all cases, for loaded concentration of BDNF 0.02 mg L -1 the spontaneous electrostatic interaction led to release less than 10 ng L -1 of BDNF from 5 h up to 24 hours, what gives less than 0,01% of loaded protein. One can see that for BDNF concentration less than 0.02 mg L -1 at dendrimer surface, desorption of protein molecule was negligible, indicating that its adsorption onto 10 mg L -1 PAMAM nanoparticles was almost completed. In this way, we found that PAMAM molecules for laden BDNF concentration less than 0.02 mg L -1 are likely to form irreversibly adsorbed BDNF layer therefore effect of substrate remains significant, making the desorption process less efficient than in case of densely packed layers. postulating an irreversible adsorption of the protein governed by the random sequential adsorption.

Nanoparticle toxicity
In order to determine the possibility of the toxicity of PAMAM-based nanoparticles in biomedical applications, under increased concentration of BDNF, we examined cell viability in modified 15 neuroblastoma SH-SY5Y cancer cells.
At the beginning we differentiated SH-SY5Y cells by combination of RA treatment and lowering FBS in cell culture according to reports [44][45][46][47] . We observed, that extension of neurites, a typical neuronal phenotype, was visible 48 h after application of retinoic acid and retained till 7 days according to Ref. 44 We observed change in SH-SY5Y cell morphology during the differentiation process, the induction of extensive neurites outgrowth as early as 48h of treatment.
Systemic administration of 6-OHDA toxin is known to selectively impair the dopaminergic neurons, resulting in cell death and selectively kills neurons in substantia nigra and striatum in animals model Ref. 48 To establish experimental dosage for testing toxicity of PAMAM-based nanoparticles, first we determined their responsiveness to 6-OHDA dose-response plot (Fig.5).
We examined differentiated SH-SY5Y cells responsiveness to various concentrations of 6-OHDA for 24h and drug neurotoxicity was assessed by MTT assay. Reduction of cell viability was employed here as an indicator of cell proliferation and toxicity. We established that exposure to 100 µMol/L 6-OHDA resulted in a significant 70% decline in cell viability. After exploring the differences in toxicity upon exposure to varying concentrations of 6-OHDA, we chose 100 µMol/L 6-OHDA for further studies and used this concentration to determine the cytotoxicity of PAMAM-based nanoparticles with different loading BDNF concentration on differentiated SH-SY5Y cells after 24h of incubation (Fig. 6).
For neuroblastoma cell line, the critical PAMAM dendrimer concentration, above which significant decrease in cell viability (cytotoxicity) occurs is ∼20 mg L -1 (Supporting information). The MTT assay demonstrated that no observable toxicity was detected for nanoparticles with 10 mg L -1 concentration of PAMAM 5.5 dendrimers. The data presented in Figure 6 further shows that after 24h of nanoparticles incubation with SH-SY5Y, the cell viability increases from initial value obtained only for BDNF without any carriers (90%) to PAMAM-based nanoparticles (150%). Less pronounced cytotoxicity in SH-SY5Y cells by PAMAM-based nanoparticles than BDNF without nanocarriers may reflect differences in the relative susceptibility of BDNF internalization. Moreover, with increasing concentration of protein loading, a decrease in toxicity was observed only for PAMAM-BDNF nanoparticles reversely to PEG-ylated one. There was a significant statistical difference between the effect of BDNF, dendrimers and PEG-ylated dendrimers nanoparticles.
A marked increase in SH-SY5Y cell viability in the presence of PAMAM-BDNF and PEG-PAMAM-BDNF nanoparticles for high protein concentration (41 and 52%, respectively), compared to the BDNF without carrier, indicates that PEG layer markedly decreased in vitro cellular toxicity level of employed BDNF.

SH-SY5Y
To understand whether neural-like cells are able to take up PAMAM-BDNF and PEG-PAMAM-BDNF nanoparticles we used confocal microscopy as well as spectrofluorimetry. Confocal analysis was conducted to qualitatively assess the localisation of BDNF-PAMAM-AF488 and BDNF-PAMAM-AF488-PEG nanoparticles and cellular uptake up to 24h.
As shown in Figure 7, differentiated neuron-like SH-5YSY neuroblastoma cells exhibited kineticdependent internalization of both types of nanoparticle. We found that from 5 min after the addition of PAMAM based nanoparticles with 0,1 mg L -1 BDNF concentration up to 24h, there was no observable cellular internalization likely because of inefficient endocytosis. These results were more apparent for BDNF-PAMAM-AF488-PEG nanoparticles which strongly suggest that protein molecules encapsulated in polyelectrolyte with their appropriate sizes are well protected, and effectively adsorbed by cells membrane. PEG's floppy chains and their charge neutrality can prevent non-specific adsorption and prevent or enhance linking chemistry via electrostatic repulsion or attraction, what involves nanoparticles interactions with an appropriate number of cell surface sites, which are necessary to produce an adequate binding energy.
In line with the confocal microscopy results, significantly more pronounced green fluorescence was observed within the cells treated with BDNF-PAMAM-AF488, when compared to those of the cells incubated with BDNF-PAMAM-AF488-PEG. This confirms significantly hampered cellular internalization, likely because of inefficient endocytosis of nanoparticles by cells.
As shown in Figure 8, upon incubation of SH-5YSY neuroblastoma cells with BDNF-PAMAM-AF488, increase in green fluorescence was evident after 24h, in a dramatic contrast to that of 2 min-treated cells where little green fluorescence was observed, suggesting an efficient interaction with cell membrane for both type of nanoparticles. In fact, given enough time, more BDNF molecules could be released from PAMAM-based nanoparticles in immediate vicinity and subsequently enter nucleus.
Cellular uptake in serum-free media is greatly reduced for PEG-ylated PAMAM-BDNF nanoparticles compared to the non-PEGy-lated.

BDNF release from PAMAM-based nanoparticles in vitro
The sustained delivery of proteins from dendrimer nanoparticles was determined to assess a therapeutic level of the neurotrophin over prolonged periods. We investigated the suitability of PAMAM nanoparticles to the in vitro transportation of BDNF to differentiated neuroblastoma cells exposed to treatment of 6-hydroxydopamine. This study was designed to quantitatively identify penetration of BDNF within damaged neuron-like cells. To assess the distribution of injected dendrimer-neurotrophin conjugates, we analyzed the BDNF concentrations after administration of BDNF and nanoparticles, using ELISA after 24 hours post-treating. The cells exposed to RA and 25µMol/L 6OHDA served as controls. The results are summarized in Figure 9.
We observed that BDNF concentration in the PAMAM-based nanoparticles-treated groups significantly increased compared to the control at 24h. Released BDNF from PAMAM based nanoparticles to extracellular region led to a significant increase in their concentration level compared to the control groups (100 µM 6OHDA) for both type of PAMAM-based nanoparticles. We found that PEG-ylated PAMAM-BDNF nanoparticles-treated cells expressed significantly higher level of neuroprotective protein when compared with the PAMAM-BDNF nanoparticles-treated group. These difference in the BDNF levels in extracellular region in the PEG-ylated PAMAM-BDNF nanoparticles-treated group compared to the PAMAM-BDNF nanoparticles-treated group are in line with releasing protein profile presented in PBS. For BDNF release kinetics from PEG-BDNF-PAMAM nanoparticles in PBS after 24h of treating, we observed significant desorption of BDNF from nanoparticle surfaces compared to PAMAM-BDNF nanoparticles of laden protein 1 mg L -1 and 2 mg L -1 equal to 39 ng L -1 and 42 ng L -1 respectively. Treating differentiated human neuroblastoma cell line SH-SY5Y with PAMAM-based nanocarriers intensifies the secretion of BDNF. These data strongly suggest that there is some interaction between surface TrkB cell receptors and BDNF nanoparticles in that system. Thus, the local secretion of BDNF may then have exerted its action locally, through its stimulatory effect on nerve regeneration acting as retrograde signal for survival neurons via the TrkB receptor. The increase in size, colloidal stability and reduction of surface charge density for PEG-ylated nanoparticles could lead to less efficient cellular internalization by differentiated SH-SY5Y cells through hampering interactions with their TrkB receptors.

Conclusion
In the design of nanocarriers for neurodegenerative diseases treatment the sustained administration of neuroprotective protein is required. Therefore, in this study we introduced a versatile nanoparticles

Competing interests
The authors declare no competing interests.

Consent for publication
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Data availability
The data required to reproduce these findings are availability for any research.    Immunofluorescence assessment of dendrimers-based nanoparticles internalization by neuron-like differentiated human neuroblastoma cell line SH-SY5Y upon treatment for 0, 2-60min, 4h and 24h. Data were presented as the mean +/-SD (n = 12).