J Neural Transm (2013) 120:903–910 DOI 10.1007/s00702-013-0992-2
NEUROLOGY AND PRECLINICAL NEUROLOGICAL STUDIES - ORIGINAL ARTICLE
Interaction of selegiline-loaded PLGA-b-PEG nanoparticles with beta-amyloid fibrils Ipek Baysal • Samiye Yabanoglu-Ciftci • Yeliz Tunc-Sarisozen • Kezban Ulubayram Gulberk Ucar
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Received: 14 October 2012 / Accepted: 5 February 2013 / Published online: 19 February 2013 Ó Springer-Verlag Wien 2013
Abstract Alzheimer’s disease (AD) is an irreversible and progressive neurodegenerative disease that is caused by the irreversible loss of neurons in the hippocampus and cortex regions of the brain. Although the molecular mechanism of the disease is still unclear, the deposition of the amyloid beta proteins (senile plaques) in the extracellular synaptic spaces of the neocortex is suggested to play a major role in progress of AD. The increased activity of monoamine oxidase-B (MAO-B) in AD brains was suggested to cause oxidative damage, and MAO-B inhibitors have been reported to inhibit the neuronal degeneration. Selegiline, a selective MAO-B inhibitor, known to have beneficial effects in the brain regions which are rich by dopamine receptors, however, studies based on brain targeting of selegiline are limited. Since some recent studies showed the possible Ab-fibril destabilizing effects of MAO inhibitors, present study was designed to (1) prepare the selective MAO-B inhibitor selegiline-loaded Poly (lacticco-glycolic acid)-poly (ethylene glycol) (PLGA-b-PEG) nanoparticles (2) to investigate the in vitro Ab-fibril destabilizing effect of the loaded particles. Selegilineloaded PLGA-b-PEG nanoparticles were prepared by waterin-oil-in-water (W/O/W) emulsion solvent evaporation
I. Baysal S. Yabanoglu-Ciftci G. Ucar (&) Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, 06100 Ankara, Turkey e-mail:
[email protected] Y. Tunc-Sarisozen K. Ulubayram Department of Basic Pharmaceutical Sciences, Faculty of Pharmacy, Hacettepe University, 06100 Ankara, Turkey K. Ulubayram G. Ucar Nanotechnology and Nanomedicine Division, Institute of Science, Hacettepe University Beytepe, 06800 Ankara, Turkey
method. Destabilizing effect of these particles on the b-amiloid fibril (Ab 1-40 and Ab 1-42) formation was determined in vitro by evaluating the decrease in ThT fluorescence intensity and verified by AFM images. Nanoparticle prepared with 5 mg selegiline was found to be the one with highest encapsulation efficiency. Particle size and polydispersity index for this formulation were determined as 217 ± 15.5 nm and 0.321, respectively. For both fibril types, destabilizing effect were found to be increased by increasing incubation time until 6 h; and reached a plateau after the 6 h. Data showed that selegiline-loaded PLGA-b-PEG nanoparticles seem to be a promising drug carrier for destabilizing the b-amiloid fibrils in Alzheimer patients. Keywords PLGA-b-PEG Nanoparticles Selegiline b amyloid fibril Alzheimer’s disease
Introduction AD is a progressive neurodegenerative disorder and characterized by cognitive and memory deterioration, progressive impairment of activities of daily living, and a variety of neuropsychiatric symptoms and behavioral disturbances (Alzheimer 1907; Olson and Shaw 1969). The distinguishing characteristic neuropathologies of the disorder are: amyloidrich senile plaques (Selkoe 2000), neurofibrillary tangles, and neuronal degeneration (Spillantini and Goedert 1998). Molecular mechanisms of AD have been explained with two major hypotheses: the cholinergic hypothesis and the amyloid cascade hypothesis. Amyloid cascade hypothesis states that the neurodegenerative process observed in AD brains is series of events that are triggered by the abnormal processing of the amyloid precursor protein that causes production,
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aggregation, deposition and toxicity of Ab derivative (Checler and Vincent 2002). The increased activity of monoamine oxidase-B (MAO-B) in AD brains was suggested to cause oxidative damage, and MAO-B inhibitors have been reported to inhibit the neuronal degeneration. In the pathogenesis of AD, accumulation of Ab peptides play a major role hence, the treatment approaches intended mainly to prevent or destabilize fibril formation (Mattsson et al. 2009). Furthermore, some recent studies have shown the possible Ab-fibril destabilizing effects of MAO inhibitors (Ono et al. 2006). Selegiline, a selective MAO-B inhibitor, is known to have beneficial effects in the brain regions that are rich by dopamine receptors. The potential use of selegiline in AD as a neuroprotective agent was originated following reports of elevated MAO-B activity, compared to healthy older people, in patients with dementia of Alzheimer type. A Cochrane review analyzed 17 double-blind, randomized, placebocontrolled trials evaluating selegiline at a dosage of 10 mg per day for the treatment of Alzheimer disease. The authors concluded that cognition improved at four to 6 weeks in some trials; however, there were no differences after 6 weeks (Birks and Flicker 2003). The benefits were found primarily in two studies; other trials did not support these findings. Currently, there is not enough evidence to recommend selegiline for the treatment of Alzheimer disease thus this study will provide basic information on direct effect of selegiline for the treatment of Alzheimer disease. Nanoparticles such as liposomes, polymer therapeutics, dendrimers, micelles, nanocrystals and carbon nanotubes have been successfully used to prevent, diagnose and treat diseases in the field of nanomedicine (Duncan 2011). Polymeric nanoparticles play very important roles for the advancements of new approaches in drug delivery systems because of their superior abilities like; penetrating through biological barriers (i.e., such as blood–brain barrier), transporting the cargo molecules to their targets more precisely, delivering the drugs that have solubility problems without any issues, getting eliminated from the body after its work has done, lowering the toxicity risk of drug at other regions of the body (Medina et al. 2007). Among them, poly(lactic acid)-b-poly(ethylene glycol) (PLA-bPEG) nanoparticles appeared to be an excellent drug carrier due to its low tissue toxicity, few side effects and a controllable drug release rate (Bala et al.2004). However, selegiline-loaded nanoparticles and their destabilizing effect of these nanoparticles on the b-amiloid fibril (Ab 1-40 and Ab 1-42) formation have not been studied in literature. The purpose of the present study is to prepare the selective MAO-B inhibitor selegiline-loaded PLGA-b-PEG nanoparticle formulations and to investigate the in vitro Ab-fibril destabilizing effects of the prepared nanoparticles.
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Materials and methods Reagents Poly(D,L-lactide-co-glycolide) Resomer RG502H was purchased from Boehringer Ingelheim Pharma GmbH and Co. (Ingelheim, Germany). Poly(ethylene glycol) (COOHPEG24-NH2), 1-Ethyl-3-[3-dimethylaminopropyl]-carbodiimide and N-Hydroxysuccinimide were obtained from Thermo Scientific. Ab (1-40), Ab (1-42) peptides and other chemicals were purchased from Sigma-Aldrich (Germany). Synthesis of PLGA-b-PEG Poly(D,L-lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG) was synthesized by poly(D,L-lactide-coglycolide) (PLGA) with terminal carboxylate groups and COOH-PEG24-NH2. Briefly, PLGA-COOH in dichloromethane (at the polymer concentration of 3.7 wt. %) was incubated with excess N-hydroxysuccinimide (NHS) (20.7 mg) and 1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide (EDC) (34.5 mg). Next, the polymer, PLGA-NHS was precipitated with ethyl ether and washed twice with methanol. The resulting polymer was dissolved in chloroform and conjugated with COOH-PEG24-NH2 at room temperature for 20 h. The final product, PLGA-b-PEG copolymer was precipitated with ethyl ether and dried under vacuum. The chemistry of the product was verified by 1H-NMR analysis. The spectrum showed characteristic PLGA signals at 5.2, 4.8, and 1.5 ppm, which are assigned to the hydrogen of the lactide units (–O–CH*(CH3)–CO–), methylene hydrogen of the glycolide unit (–O–CH*2–CO–) and methyl hydrogen of the lactide units (O–CH(CH*3)– CO–), respectively, and the peak at 3.6 ppm belonged to the methane proton of PEG chain (–O–CH*2–CH*2–). Preparation of selegiline-loaded PLGA-b-PEG nanoparticles Selegiline-loaded PLGA-b-PEG nanoparticles were prepared by water-in-oil-in-water (W/O/W) emulsion solvent evaporation method (Sarı 2011). Briefly, the primary emulsion was formed by homogenizing 100 lL aqueous selegiline solution (stock solutions of selegiline at a concentration of 100 mg/mL, 75 mg/mL 50 mg/mL or 25 mg/mL were used for different formulations) in 1 ml PLGA-b-PEGCOOH polymer solution (20 mg/ml in dichloromethane) while homogenizing at 1,500 rpm for 1 min. This primary solution was immediately added dropwise with an injector into a secondary aqueous phase containing 2 wt. % Pluronic F68 (5 mL) and was homogenized at 15,000 rpm for 1 min. This second emulsion was added dropwise into 0.5 wt. % Pluronic F68 solution (50 mL) while stirring on a magnetic
Interaction of selegiline-loaded PLGA-b-PEG nanoparticles
stirrer. The obtained emulsion was stirred overnight at 4 °C to remove residual solvent. Obtained suspension was first centrifuged at 2,8009g for 5 min to remove the large microparticle/macroaggregate fractions and then nanoparticles were precipitated with 20,0009g centrifugation of nanoparticle solution for 20 min. Nanoparticles were rinsed two times to remove residual surfactant. Selegiline-free nanoparticles were prepared by the same method as a control group. Particle size, polydispersity index, and zeta potential measurements of the nanoparticles were performed by Malvern Zetasizer Nano ZS. Topological analysis of the nanoparticles was performed by atomic force microscopy (AFM). Nanoparticles were suspended in water, administered on mica plates and dried with nitrogen gas for AFM analysis. While observing AFM images, device was run in dynamic mode (tapping mode) using Buggetsensor Tap300-G model silicon tip (radius \10 nm) at resonance frequency of 300 kHz and constant force of 40 N/m. Tip angle was determined 20–25° throughout cantilever, 25–30° from sides and 10° from the top. Determination of selegiline loading efficiencies Selegiline loading efficiencies for the nanoparticles were determined indirectly by analysis of unloaded selegiline amount in the outer water phase. Quantification of selegiline was performed by modifying of the HPLC method reported earlier (Tzanavaras et al. 2008). 50:50 (v/v) acetonitrile/PBS (3.7 mM KH2PO4 and 4.4 mM K2HPO4. 3H2O, pH 7.0) mixture was used as mobile phase. Dionex C18 reverse phase column (250 9 4.6 mm, 5 mm) was used for analytic separation (25 °C). Selegiline detection was observed at k = 220 nm. Flow rate and injection volume were determined as 1 mL/min and 50 mL, respectively. Stock solution of selegiline [water:acetonitrile (v/v)] was prepared as 1 mg/mL. The second solution was prepared as 0.1 mg/mL from the stock solution. Eight different calibration points were prepared by dilution (100, 50, 25, 10, 5, 2.5, 1, 0.5 lg/mL) from the second solution. Selegiline release studies Selegiline-loaded nanoparticles (3 mg) were dispersed into 1.5 mL PBS solution and stirred at 37 °C. Samples from the selegiline-released medium were withdrawn (replaced with fresh medium) at certain time intervals and injected to the HPLC system to determine the released drug concentration. Preparation of Ab and b-amyloid fibril (fAb) solutions Ab (1-40) and Ab (1-42) were dissolved by brief vortexing in a 0.02 % ammonia solution at a concentration of
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500 lM (2.2 mg/mL) and 250 lM, respectively, at 4 °C and stored at -80 °C before assaying (fresh Ab (1-40) and Ab (1-42) solutions). fAb (1-40) and fAb (1-42) were formed from the fresh Ab (1-40) and Ab (1-42) solutions, respectively, sonicated, and stored at 4 °C as described elsewhere (Hasegawa et al. 1999). Fresh, non-aggregated fAb (1-40) and fAb (1-42) were obtained by extending sonicated fAb (1-40) or fAb (1-42) with fresh Ab (1-40) or Ab (1-42) solutions, respectively, just before the destabilization reaction (Ono et al. 2002). The reaction mixture was 600 lL and contained 10 lg/mL (2.3 lM) f Ab (1-40) or f Ab (1-42), 50 lM Ab (1-40) or Ab (1-42), 50 mM phosphate buffer, pH 7.5, and 100 mM NaCl. Measurement of the fluorescence of ThT showed that the extension reaction proceeded to equilibrium after incubation at 37 °C for 3–6 h under non-agitated conditions. Incubation of selegiline-loaded nanoparticles with fAb (1-40) and fAb (1-42) 500 lL reaction mixture was formed with 50 lM fresh fAb (1-40) or fAb (1-42), nanoparticle solutions containing 20, 40, 60, and 100 nM selegiline, 100 mM NaCl and 50 mM phosphate buffer pH 7.5. Solutions were incubated at 37 °C and samples were retrieved at specific time points. Measurement of ThT fluorescence Optimum fluorescence measurements of fAb (1-40) and fAb (1-42) were obtained at the excitation and emission wavelengths of 448 and 490 nm, respectively, with the reaction mixture containing 5 mM ThT and 50 mM of glycine–NaOH buffer, pH 8.5. Fibril formation and fibril destabilization by particules were followed by the measurement of ThT fluorescence. 100 lL of samples were taken from the incubation mixture of fAb and nanoparticles which were incubated for 0, 2, 4, and 6 h. Fluorescence measurement of ThT was carried out using Shimadzu (RF-5301PC) fluorescence spectrophotometer according to the method previously reported (Hiller-Sturmho¨fel and Swartzwelder 2004). Reduction in the fluorescence indicated destabilization of fibril formation by nanoparticules. Morphological analysis of incubation of fAb (1-40) with selegiline-loaded nanoparticules by AFM Fibril suspension was dropped on mica plates and dried with nitrogen gas. AFM device was run in dynamic mode (tapping mode) during the study using Buggetsensor Tap300-G model silicone tip (radius \10 nm), 300 kHz of resonance frequency, 40 M/m of constant force. Tip angle was kept as 20–25° throughout cantilever, 25–30° from
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sides and 10° from the top. Destabilizing effect of these particles on the fAb1-40 and fAb 1-42 formation was also determined in vitro by AFM images.
incubation time until 6 h; this effect reached a plateau after 6 h for incubation with both fAb (1-40) and fAb (1-42) [Fig. 2c, d, respectively]. Destabilization data were verified by their AFM images [Fig. 3].
Results Discussion Selegiline-loaded PLGA-b-PEG nanoparticles were prepared with different concentrations of selegiline in the range of 2.5–10 mg [Table 1]. Size and zeta potential measurements showed that the average size and the zeta potentials of the nanoparticles prepared by selegiline in the range of 2.5–7.5 mg were between 217 and 246 nm and -34.7 and -38 mV, respectively. On the other hand, the nanoparticles prepared by 10 mg selegiline showed some signs of emulsion instability during production process and therefore these measurements were not performed for this formulation. The polydispersity of the first three formulations was around 0.3 and did not seem to be affected by the amount of selegiline. Loading efficiencies of selegiline in these nanoparticles were determined from the quantitative analysis of unloaded selegiline amount in the outer aqueous phase of the emulsion and determined as in the range of 4.92–12.57 lg selegiline for 1 mg nanoparticles. The highest loading efficiency was observed for the nanoparticles prepared with 5 mg selegiline (NP 2). Therefore, the formulation with 5 mg selegiline (NP 2) was selected to be used in vitro experiments including drug release experiments. Representative AFM image taken from the most efficient formulation (NP 2) showed that the nanoparticles have a spherical geometry and the particle size determined in AFM image [Fig. 1a] was found to be in correlation with the size determined with nanosizer [Fig. 1b]. In the drug release studies performed by NP 2, it was observed that 70 % of the loaded selegiline released in the first 2 h and the release reached to a plateau after 10 h [Fig. 1c]. Destabilizing effect of the loaded particles on the b-amyloid fibril formation was determined for both fibril types (fAb (1-40) and fAb (1-42); Fig. 2a, b, respectively. Destabilizing effects of the loaded particles were found to be increased by increasing selegiline concentration and also by increasing
Drug delivery to a desired site is a method of delivering medication in a manner that increases the concentration of the medication in site of action relative to other regions of the body. Drug delivery also leads to concentrating the medication in the tissues of interest while reducing the relative concentration of the medication in other tissues. This is resulted in increased efficacy of the treatment and reduced the side effects of the drug. The most important advantage of nanoparticles over the other transport systems is their submicron sizes. This property enables an extravasation and prevents absorption from terminal blood vessels (Barratt 2003). Nanoparticles are also designed to overcome the aspects that free drugs fail such as unsuccessful therapies or weak therapeutic responses (MeriskoLiversidge et al. 2002). Polymeric drug transport systems are well developed (Hans and Lowman 2002) and they generate an attractive alternative for the therapeutic agents to be administered gradually or for a long-time period (Sanchez et al. 2003). Especially, the polymeric micelles such as block copolymers which contain PEG have gained more attention due to some attractive properties such as good stability, longevity, and ability to accumulate in the areas with an abnormal vasculature via the enhanced permeability and retention effect. These micelles are biocompatible, non-toxic and can be targeted by attaching specific targeting ligand molecules to the micelle surface or can be comprised of stimuli-responsive amphiphilic block copolymers. Addition of second component such as surfactant or another hydrophobic material to the main micelle forming material further improves the solubilizing capacity of micelles without compromising their stability.
Table 1 Loading efficiencies, average nanoparticle sizes, polydispersity indexes and zeta potentials of nanoparticle formulations with different selegiline concentrations (n = 3) Nanoparticle formulation NP 1
Weighed amount of selegiline to be loaded in nanoparticles (mg)
Amount of selegiline determined in nanoparticles (lg)
Loading efficiency (%)
Average size (nm)
Polydispersity index (PDI)
Zeta potential (mV)
236.00 ± 12.0
0.331 ± 0.2
38.00 ± 1.4
2.50
4.92 ± 3.4
11.70
NP 2
5.00
12.57 ± 3.0
13.00
217.00 ± 15.5
0.321 ± 0.1
34.80 ± 0.3
NP 3
7.50
4.92 ± 0.6
3.30
246.30 ± 47.6
0.295 ± 0.1
34.70 ± 4.2
NP 4
10.00
7.37 ± 2.9
5.20
ND
ND
ND
ND not determined
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Fig. 1 a AFM image of selegiline-loaded nanoparticles (NP2), b particle size distribution of selegiline-loaded nanoparticles (NP2) obtained by zetasizer and c in vitro drug release of NP2 (n = 4)
Selegiline, also known as l-deprenyl, is an irreversible inhibitor of the MAO-B. Since dopaminergic and noradrenergic system damages are observed in Alzheimer patients, it is suggested that MAO-B inhibitors reduce neuron damage (Foley et al. 2000) and slow down the progression of AD (Knoll 1998). Since it is previously reported that selegiline improves behavioral disorders in AD and Parkinson’s Disease (PD) by improving catecholamine levels (Alafuzoff et al. 2000) and it was recently suggested that MAO-B inhibitors may inhibit Ab fibril formation (Ono et al. 2006), in the present study, the destabilizing effect of selegiline which was loaded in PLGA-b-PEG nanoparticles on in vitro fibril formation was investigated. Selegiline-loaded nanoparticles were prepared with the double emulsion solvent evaporation method using PLGAb-PEG which is designed as a PLGA block (hydrophobic) and PEG block (hydrophilic) copolymer (Sarı 2011). Particle formulation (NP 2) with the highest efficiency is prepared with 5 mg of selegiline. The particles prepared with
higher amount of selegiline showed lower loading efficiencies. The decrease in loading with increasing amount of selegiline was thought to be coming from the decrease in emulsion system stability with increasing selegiline content above 5 mg in the preparation process. Despite its widespread use, preparation of nanoparticles by double emulsion systems has the disadvantage of delicate system stability. As it is seen from the experimental part, in the preparation of selegiline-loaded nanoparticles, selegiline was first dissolved in the primary aqueous phase and then emulsified in dichloromethane to form the primary emulsion. The double emulsion was then formed by dispersing the primary emulsion into an aqueous phase. The decrease in emulsion stability was thought to be occurred in the first emulsification step due to high selegiline content. Therefore, among the nanoparticle formulations (NP1-4), NP2 with the highest loading efficiency was selected to be used in further experiments. Average nanoparticle size, zeta potential, and polydispersity index for NP 2 were determined as 217 ± 15.5 nm, -34.80 ± 0.28 mV and 0.321 ± 0.036,
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Fig. 2 Fluorescence intensity-time graph showing a fAb (1-40) and b fAb (1-42) formation, concentration dependent destabilization effect of nanoparticles on c fAb (1-40) and d fAb (1-42) (n = 4) (filled square) nanoparticle containing 20 nM selegiline, (filled black
square) nanoparticle containing 40 nM selegiline, (filled triangle) nanoparticle containing 60 nM selegiline, (filled circle) nanoparticle containing 100 nM selegiline
Fig. 3 AFM images showing interaction of fAb (1-40) with nanoparticles loaded with selegiline (NP2) a just before the incubation period at zero point and b after 6-h incubation period. Asterisks
nanoparticles (showing their spherical morphology) and arrows fibril and nanoparticle interactions (aggregates)
respectively. Nanoparticles prepared from PLGA-b-PEG polymers have low negative zeta potential values because of the carboxylic acid end groups. Polydispersity values range from 0 to 1; a low PDI value indicates a homogeneous nanoparticle size distribution. The ability to control nanoparticle sizes is important for their use in systemic administration and brain transport. Generally, the size of brain delivery nanoparticles is controlled under 250 nm to facilitate the endocytosis by the brain capillary cells (Calvo et al. 2001; Olivier et al. 2002). The size of the prepared NP was
considered as favorable for brain transport. Prepared selegiline-loaded nanoparticles had small particle size, exhibited time-dependent release. NP2 formulation released 70 % of their selegiline content within the first 2 h. This burst release of selegiline depends on fast solubilization of surface-loaded selegiline. The release profiles of the nanoparticle formulation reach a plateau between 2 and 10 h and continue for about 72 h. Destabilizing effects of the loaded particles were found to be increased by increasing selegiline concentration and
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Interaction of selegiline-loaded PLGA-b-PEG nanoparticles
also by increasing incubation time until 6 h; this effect reached a plateau after 6 h for incubation with both fAb (1-40) and fAb (1-42). The inhibition of amyloid beta fibril formation by selegiline-loaded nanoparticles may involve different mechanisms, in inhibition of either a nucleation process or an elongation process. There are few reports concerning inhibition of amyloid fibril formation (Ono et al. 2004; Durairajan et al. 2008; Huong et al. 2010) In all these studies, inhibition mechanisms were found to be different thus consequent graph profiles varied. The different tendency of ThT fluorescence decrease of fAb (1-42) (Fig. 2c) and fAb (1-40) (Fig. 2d) is possibly due to different inhibition mechanisms of Ab (1-40) and Ab (1-42) fibril formation by selegiline-loaded nanoparticles. In order to clarify if selegiline-loaded nanoparticles inhibit a nucleation process or elongation process or both of these further experiments needed to be carried out. Our study concerns some preliminary results. Mechanisms that involved in inhibition processes in detail will be investigated with further experiments. It was concluded that selegiline-loaded PLGA-b-PEG nanoparticles were shown to destabilize the fAb formation in vitro. These findings suggested that selegiline-loaded PLGA-b-PEG polymeric nanoparticles could be promising nanosystems for Alzheimer’s disease by depending on concentration of selegiline-loaded nanoparticles. Of course, it is clear that further experiments are needed for investigating efficiency of the prepared nanoparticles in vivo. These data encouraged us to design future studies which will be focused on preparing novel nanoparticles carrying Ab antibodies on their surfaces and targeting them directly to fAbs in vivo. Thus concentration of selegiline in the fAb region will be increased and formation of fAb will be highly destabilized. Since it was previously reported that targeting selegiline minimize the central nervous system side effects of this drug (Antoniadesa et al. 2002), it is expected that distribution of selegiline in the body compartments also will be minimalized through targeting of selegiline-loaded PLGA-b-PEG polymeric nanoparticles directly to the brain.
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