Optimization of Curcumin-Loaded PEG-PLGA Nanoparticles by GSH

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Optimization of curcumin loaded-PEG-PLGA nanoparticles by GSH functionalization. Investigation of the internalization pathway in neuronal cells. Ghislain Djiokeng Paka, and Charles Ramassamy Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00738 • Publication Date (Web): 15 Oct 2016 Downloaded from http://pubs.acs.org on October 16, 2016

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Molecular Pharmaceutics

Optimization of curcumin loaded-PEG-PLGA nanoparticles by GSH functionalization. Investigation of the internalization pathway in neuronal cells. Ghislain Djiokeng Pakaa,b and Charles Ramassamya,b* a

Institut National de la Recherche Scientifique-Institut Armand Frappier, Laval, Canada

b

Institut sur la Nutrition et les Aliments Fonctionnels, Laval University, Québec, Canada

*Correspondance to: Charles Ramassamy, PhD,

INRS- Institut Armand Frappier,

531, boul. des Prairies, Laval, Québec, H7V 1B7, Canada;

Tel/Fax: +001-450-687-5010, +001-450-687-5510;

E-mail: [email protected]

Keywords: GSH-functionalized nanopaticles, click chemistry, brain drug delivery, endocytosis, nanoneuropharmacology, oxidative stress, acrolein toxicity.

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Abstract One major challenge in the field of nanotherapeutics is to increase the selective delivery of cargo to targeted cells. Using Poly Lactic-co-Glycolic Acid (PLGA), we recently highlighted the importance of polymer composition in the biological fate of the nanodrug delivery systems. However the route of internalisation of polymeric nanoparticles (NPs) is another key component to consider in the elaboration of modern and targeted devices. For that purpose, herein, we effectively synthesized and characterised glutathionefunctionalized PLGA-nanoparticles (GSH-NPs) loaded with curcumin (GSH-NPs-Cur), using thiol-maleimide click reaction and determined their physicochemical properties. We found that GSH- functionalization did not affect the drug loading efficiency (DLE), the size, the polydispersity index (PDI), the zeta potential, the release profile and the stability of the formulation. While being non-toxic, the presence of GSH on the surface of the formulations exhibits a better neuroprotective property against acrolein. The neuronal internalisation of GSH-NPs-Cur was higher than with free curcumin. In order to track the intracellular localisation of the formulations, we used a covalently attached Rhodamine (PLGA-Rhod), into our GSH-functionalized matrix. We found that GSHfunctionalized matrix could easily be taken up by neuronal cells. Furthermore, we found that GSH-conjugation modifies the route of internalisation enabling them to escape the uptake through macropinocytosis and therefore avoiding the lysosomal degradation. Taken together, GSH-functionalization increases the uptake of formulations and modifies the route of internalization towards a safer pathway. This study shows that the choice of ideal ligand to develop NPs-targeting devices is a crucial step when designing innovative strategy for neuronal cells delivery.


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Introduction Alzheimer disease (AD) represents the main cause of dementia in elderly people with an important socio-economical and medical burden all over the world 1. Keys factors are implicated in the onset of AD such as the β-amyloid cascade, mitochondrial dysfunction,

oxidative

stress,

tau-hyperphosphorylation,

progressive disruption of cholinergic function in the brain

inflammation 2-6

and

the

. Actually, proposed

treatments for AD are associated with side-effects which often explained the discontinuation of these pharmacotherapies 7.

Moreover, their low efficacy is also

deplored, since less than 50% of patients respond to these therapies. Therefore, many efforts are given to the discovery of multitargeted drugs 7. Curcumin represents a promising compound for the treatment of AD. Since accumulating data have demonstrated its neuroprotective properties through antioxidant, antiinflammatory, anti-amyloid, anti-Tau hyperphosphorylation activities on several in vitro and in vivo models 8. However, the high hydrophobicity of curcumin, which endorses its poor oral brain bioavailability, explained its limitation as therapeutic agent. Taken together, proposed or promising compounds applicable to treat AD suffer from either their blind distribution or low brain availability. The common challenge is the presence of the blood brain barrier (BBB) which maintains the cerebral homeostasis, limiting the access to drugs. Several studies have shown that, the use of biodegradable polymer like PLGA, could enhance the stability and brain bioavailability of these compounds9-12. For example, we and others have previously found that curcumin-loaded into PLGA nanoparticles prolonged and enhanced the antioxidant, anti-inflammatory activity of curcumin while suppressing the Akt and Tau phosphorylation or upregulates survival 3 ACS Paragon Plus Environment


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genes under oxidative stress conditions

9, 13-16

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. Moreover, we have recently demonstrated

that polymer composition and architecture are of great importance since the ratio of lactic/glycolic acid and PEGylation parameters influence the physicochemical properties and the biological fate of NPs

17, 18

. Nanocurcumin formulations with PLGA 50:50

exhibited the greatest neuroprotective activity, suggesting a potential pharmacological application when designing devices to treat neurodegenerative disorders such as AD 17. Nowadays, it is well established that NPs' intracellular trafficking is a key factor when elaborating modern drug delivery systems. NPs should be able to escape the acidification and degradation by lysosomes since these processes would destroy a certain amount of the formulation. When designing a device, one can solve this by modulating the surface of NPs such as the sharp, the charge, the hydrophobicity, the flexibility, the presence of different type of ligands or peptides

19

. Also the density of a surface ligand can deeply

affect the mode of internalisation and the following appropriate intracellular transport, which is indispensable in order to deliver a therapeutic formulation to the targeted intracellular site 20. This can further be explained by the fact that the uptake mechanisms will need to easily escape the lysosomal degradation

21

. Several attempts have been

proposed using numerous types of ligands such as synthesized or endogenous peptides, some antibodies or some endogenous proteins with well-characterized receptors such as transferrin, lactoferrin, insulin and lipoprotein 22-29. GSH-PEGylated liposomes were found to enhance brain drug delivery in several animal models

30, 31

. Very recently, Geldenhus and coworkers synthesized doxorubicin PLGA-

NPs coated with GSH in order to enhance brain tropism of their formulations 32. Using an in vitro model of BBB, they found a greater permeability at 48 h, with constant rate 4 ACS Paragon Plus Environment

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suggesting a time-dependent transport when compared to free drug solution. In proof-ofconcept studies, it was demonstrated that increasing the amounts of GSH-PEGylated liposomes resulted in higher amounts of free drugs in the brain of rats

33

. Several

promising formulations using GSH as brain ligand-targeting for nanomedicine are undergoing clinical evaluation such as 2B3-101. This PEGylated formulation contains GSH as a brain targeting ligand for the treatment of glioma and is currently undergoing phase I/IIa trials

25

. However, the mechanisms underlying the cellular uptake and

particularly by neuronal cells, remain to be studied. A certain distribution of GSH transporters has been described on BBB namely EAC1 transporter

34

. Stringently, NPs uptake through transporters would face the main

limitation of size threshold. Several pathways have been cited for the internalisation of peptide modified nanoformulations with major route being endocytosis which includes the clathrin-dependent endocytosis, caveolae-dependent endocytosis or macropinocytosis 35, 36

. Modern drug delivery systems should be able to follow a safe endocytotic pathway

avoiding the lysosomal degradation. So in this study, we have synthesized and characterised GSH-covalently attached to curcumin-loaded PLGA-NPs (NPs-PLGAPEG-Mal-GSH-Cur or GSH-NPs-Cur) in order to investigate the effect of GSHfunctionalization on the uptake pathway using different pharmacological inhibitors specific for major cited endocytosis routes. Our results show that surface modification with both PEG and GSH increase the neuronal internalisation of formulation. Moreover, we found that the functionalization with GSH modifies the internalisation pathway by avoiding the micropinocytosis route towards caveolae and clathrin dependant endocytosis. However the detailed mechanisms of GSH as an eventual ligand mediating 5 ACS Paragon Plus Environment


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endocytosis need to be elucidated. Taken together, these results provide a new insight into the development of effective nanomedicine strategy able to escape lysosomal degradation, therefore increasing the therapeutic fate for potential application in the treatment of AD.

MATERIALS AND METHODS Materials Curcumin, PLGA (50-50, 30-60kDa), dialysis bag (12kDa and 50 kDa), Bovine serum albumin (BSA), hydrogen peroxide (H2O2), GSH, acrolein, minimal essential medium Eagle (MEM), foetal bovine serum (FBS), penicillin, streptomycin, sodium pyruvate, 2,2Diphenyl-1-picrylhydrazyl

(DPPH),

2-4

dinitrophenylhydrazine

Monochlorobimane (MCB), DL-buthionine-(S,R)-sulfoximine (BSO)

(DNPH), nocodazole,

chlorpromazine hydrochloride, genistein and Methyl-β-cyclodextrin were obtained from Sigma-Aldrich (Oakville, ON, Canada). PLGA-PEG2000, PLGA-PEG-Mal (60kDa3.4kDa) and PLGA-Rhod (30kDa) were purchased from Akina.Inc. Acetone was purchased from Fisher (Ottawa, ON, Canada). 2’,7’- dichlorofluorescein-diacetate (DCFDA) was from Invitrogen (Burlington, ON, Canada). MilliQ water was used for all the experiments.

Preparation of curcumin loaded nanoparticles Formulations were prepared by nanoprecipitation method. Briefly, curcumin and different polymers were dissolved in acetone (2.5 mL), according to the type of NPs to prepare (see description and composition in table 1). Then, the organic phase was added 6 ACS Paragon Plus Environment

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to an aqueous phase (12.5 mL) with a flow of 0.5mL/min. The mixture was stirred and then the organic phase was evaporated. PLGA-PEG with PEG2000 was used to create a certain furtiveness of the NPs and PLGA-PEG-Mal as a cross linker or functionalizing block with PEG3400 being a spacer. In order to follow the internalisation of the matrix, PLGA-Rhod (10% of matrix composition) was used to prepare autofluorecent PLGA matrix. These blocks of copolymers would self-assemble into the matrix with their hydrophobic cores loaded with curcumin. Blank or void NPs were also prepared for each type of formulations with the same procedure but without curcumin.

GSH-functionalization of NPs using click chemistry After nanoprecipitation, formulations were collected and resuspended in phosphate buffer (pH=6.5). The high reactivity of thiols with the maleimide moieties is well known. GSH (10mg/mL) was added to the formulation and stirred overnight in order to allow the formation of a covalent bond between the free –SH group of GSH and free maleimide group at the terminal distal ends of the cross linker PLGA-PEG-Mal. This click chemistry provides GSH-NPs or GSH-NPs-Cur corresponding to NPs-PLGA-PEG-Mal-GSH and NPs-PLGA-PEG-Mal-GSH-Cur, respectively. The formulations were thereafter purified by two series of dialysis; the first one using a cut-off of 12kDa for GSH, curcumin and salt elimination. For the second one, we used a dialysis bag of 50 kDa, for the elimination of non-matrixial polymer. The final formulation was washed by centrifugation and keep at 4°C.

Characterisation of GSH conjugated formulation



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Fourier transform infrared spectroscopy (FTIR)

FTIR spectroscopy was used to confirm the structural changes which occurred after GSH conjugation. In order to record FTIR spectra of our formulations, we used a Spectrum One spectrophotometer (Perkin-Elmer, Woodbridge, ON, Canada) equipped with an attenuated total reﬂectance device for solids analysis and a high-linearity lithium tantalate detector. Spectra were analyzed using Spectrum 10.3.9 software. Samples were placed onto a zinc selenide crystal and the analysis was performed within the spectral region of 650–4,000 cm-1, with 64 scans recorded at 4 cm-1 resolution. After attenuation of total reﬂectance and baseline correction, spectra were normalized with a limit ordinate of 1.5 absorbance units. Void formulations were analyzed for structural characterization by either the disappearance of conjugated bond of maleimide (3100-3000 cm-1) or the appearance of the thioether bond (700-600 cm-1). The resulting FTIR spectra of GSHNPs were compared to non GSH-NPs in order to characterise the click reaction based on the intensity or shift of the vibrational bands.

Spectrometric-based analysis of the conjugation using DPPH antiradical activity of remaining GSH or direct colorimetric detection of maleimide group

The reactivity between thiol and maleimide groups was also completed by analyzing the DPPH antiradical activity of the remaining GSH in the supernatant. Briefly, the DPPH scavenging activity of the remaining GSH of the supernatant was measured by a colorimetric method

37

. 20 µL of the supernatant were mixed with 200 µL of DPPH

solution (0.2 mM). The reaction mixture was incubated for 30 min in darkness at room temperature. The absorbance of the resulting solution was measured at 517 nm. The 8 ACS Paragon Plus Environment

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radical scavenging capacity of the initial concentration of GSH before the reaction was considered as 100%. Assays were carried out at least in triplicate and at least three independent experiments were performed.

The conjugation reaction was also directly monitored by following the decrease of maleimide group absorbance following the structural rearrangement created by the fixation of GSH. This assay was used for the determination of initial and final absorbance of maleimide group, respectively in void non GSH-NPs (NPs-PLGA-PEG-Mal) and GSH-NPs (NPs-PLGA-PEG-Mal-GSH), in curcumin-loaded non GSH-modified NPs (NPs-PLGA-PEG-Mal-Cur) and GSH-modified NPs (NPs-PLGA-PEG-Mal-GSH-Cur). This assay represents a direct analysis since native maleimide group possesses a maximal absorption at 302 nm

38

. A decrease of absorbance means its structural rearrangement

associated with the formation of the thioether bond.

Physicochemical characterization of nanoformulations Particle size and zeta potential Dynamic light scattering (DLS) was used for the measurement of average hydrodynamic diameters and the polydispersity index (PDI) of each formulations. Measurements were performed using a Zetasizer from Malvern Zetasizer Nano-ZS, Malvern Instruments, UK. Effective mean diameter of the NPs was obtained from 3 runs for 3 different measurements. Zeta potential data were measured through electrophoretic light scattering (ELS), in triplicate for each sample (Malvern Zetasizer Nano-ZS, Malvern Instruments, UK). For both DLS and ELS measurements, water was taken as the dispersant medium.



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Entrapment and Drug Loading Efficiency of curcumin-loaded formulations The percentage of curcumin incorporated during NPs preparation was determined using a UV-vis spectrophotometer at the wavelength of 420 nm. After centrifugation of curcumin-loaded formulations resuspended in acetonitrile, analyses were performed on supernatants. The efficiencies were calculated using the following equations: Entrapment Efficiency (EE) = (Weight of drug into NPs/initial weight of drug) X 100 Drug Loading Efficiency (DLE) = (Weight of drug into NPs/Weight of produced formulation) X 100 Mean values were reported from three individual experiments.

Transmission electron microscopy (TEM) The morphology and the size of each formulations were observed using TEM (Hitachi H7100) at 40,000× magnification. Briefly, a drop (100 µL) of each formulation was placed on a copper grid and air-dried. The grid was then immerged in water, air dried, and then stained by adding one drop of 3% (w/v) phosphotungstic acid (PTA). Then the grid was air-dried before loading on the microscope and photographed.

In vitro release studies For in vitro release study, we used a recently described protocol in regards to sink conditions

17

. Briefly, curcumin-loaded non GSH-NPs and GSH-NPs were freely

dispersed in sodium phosphate buffer (pH 7.4). The release medium was supplemented with 3% w/v BSA as a natural solubility enhancer in order to maintain sink conditions 39.


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All samples were kept at 37◦C under magnetic stirring and away from light. At various pre-determined endpoints, 1 mL was collected and centrifuged immediately (20,000 g for 30 min at 4◦C) to quantify the amount of curcumin in the supernatant at each time. The samples were then analyzed by using a UV-vis spectrophotometer at the wavelength of 436 nm; in the presence of BSA, which induces a shift of the absorbance from 420 to 436 nm

40

. The concentration of curcumin released from the formulation was expressed as a

percentage of the total curcumin loaded into the formulation and was plotted as a function of time. Culture assay SK-N-SH cells, a human neuroblastoma cell line from ATCC (Manassas, VA, USA), were maintained in Minimum Essential Medium (MEM) supplemented with 10% (v/v) foetal bovine serum (FBS), 1% penicillin/streptomycin and sodium pyruvate (1mM) in a humidified incubator at 37°C with 5% CO2. Cells were grown to 80% confluence and then seeded into multiwell cell culture plates for the experimental procedures.

Cell viability and neuroprotective property of the formulations SK-N-SH cells were plated at a density of 2.0 × 104 cells/well in 96-well plates and incubated for 24 h at 37◦C. Then, the media was completely removed and cells were kept in MEM. Cells were co-treated with different concentrations of nanoformulations with or without acrolein (10 µM). Cells survival was assessed 24 h after the treatment using the Tox-8 (Resazurin-based) following the manufacturer’s instructions. Values obtained from controls, untreated cells, were considered as 100%.



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Intracellular reactive oxygen/nitrogen species (ROS) Intracellular ROS accumulation was measured by following the oxidation of 2′, 7 dichlorofluorescein diacetate (DCF-DA). Briefly, SK-N-SH cells (2 × 104 /well) were plated into 96 well plates and allowed to attach for 24 h. After 24 h, cells were starved and co-treated with free curcumin, different formulations in presence or absence of 1.0 mM of H2O2 for 1 h. DCF-DA was added to a final concentration of 10 µM for 20 min. The fluorescence was then determined with the excitation/emission filters at 485/535 nm using a Synergy multidetection microplate reader.

Nanoparticles uptake in SK-N-SH cells by fluorescence microscopy In order to characterize the cellular uptake of the GSH-NPs-Cur, SK-N-SH cells were cultured on cover slips coated with poly-D-lysine at a density of 1.5 × 104 cells/well in 24-well plates. Cells were incubated for 24 h at 37°C and then treated with 1 µM of free curcumin (used as positive control) or different formulations for 2 h. Cells were then fixed with methanol and the nuclei stained with 1 µg/mL 4', 6-diamidino-2-phenylindole (DAPI) for 15 min. In order to characterize the route of internalisation, cells were pre-incubated for 1 h with different pharmacological inhibitors at the following concentrations: nocodazole 20 µM, chlorpromazine hydrochloride 10µg/ml, genistein 200 µM and m-β-CD at 2.5 mM. In another experiments, cells were pre-incubated at 4°C for 1 h prior the exposure to the formulations (2 h at 4°C) in order to assess eventual energy dependence of the uptake. After this pre-incubation, formulations or free curcumin at the final concentration of 1


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µM were added and incubated for another 2 h. Untreated cells were used as negative controls. After 2 h, cells were washed in order to ensure particle removal. In order to track the intracellular localisation of the formulation, cells were incubated for 2 h at 37˚C with NPs prepared with a fluorescent coumpound rhodamine which was covalently attached to PLGA (PLGA-Rhod). The glass slides were mounted with prolong gold antifade reagent, protected from light and air-dried. For fluorescence microscopy analysis, images were captured using a camera (SensiCam high performance) connected to a Leica ECB microscopy with under the DAPI filter for DAPI detection and the FITC filters for the DAPI and curcumin signal detection, respectively. The mean of the intensity of fluorescence per surface unit in each condition was then quantified using the Image-Pro plus 5.0 software (Media Cybernetics, USA) and Log2 change over nontreated cells was calculated.

Fluorescence data analysis The mean of the whole cell fluorescence intensities in each condition was quantified and reported to the same arbitrary surface unit for normalization reasons. Thereby, hundreds of cells from several images, of at least three independent experiments, were used for quantification by the Image-Pro plus 5.0 software (Media Cybernetics, USA). Log2 change over the vehicle-treated cells, used as controls, was calculated. Results represent log2 change ± SEM.

Intracellular glutathione level determination using MCB



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Cellular level of GSH was measured using a cell permeable fluorescent dye MCB, a GSHspecific dye, that forms adducts with GSH. SK-N-SH cells were plated at the density of 20.000 cells/well in 96-well plates. After 24 h, cells were starved and treated with or without acrolein (10 µM) and different formulations or free curcumin (1 µM) for 30 minutes or buthionine sulfoximine (BSO, 200 µM) for 24 h (used as a positive control). Cells were then washed with PBS-Ca2+/Mg2+ then cells were incubated for 15 minutes at 37◦C in the dark containing 100 µM of MCB. Once the MCB was loaded in cells, they were washed again and the intensity of fluorescence of the bimane–glutathione conjugates was determined using a Synergy HT multidetection microplate reader at 360/480 nm. The values of control cells were considered as 100%.

Results and discussion NPs preparation using nanoprecipitation and characterisation of physicochemical properties of different formulations Targeting of cells type or active sites could be due to the inherent physicochemical properties of the NPs such as the size, the shape, the charge, the flexibility of NPs or the presence of a recruiting ligand as previously reported with polysorbate 80 or PEG41. Active targeting describes the mode of action using ligand-conjugated NPs to target a specific receptor. Conjugated ligand-NPs would probably improve the ability of NPs to specifically target the main therapeutic site, minimizing the adverse effects of off-target drugs and GSH was demonstrated to be such a cited ligand

30, 31

. So, after

nanoprecipitation and purification, functionalization was performed using click chemistry. For this, we used both PEGylation (PEG2000) and peptide surface modification 14 ACS Paragon Plus Environment

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using GSH. PEG is known to increase the widespread and the distribution of the formulation. Then, the functionalization of GSH to the maleimide group of NPs-PLGAPEG-Mal was characterized using both infrared and colorimetric measurements.

Fourier transform infrared spectroscopy (FTIR) GSH conjugation to NPs-PLGA-PEG-Mal was achieved using click chemistry in a reaction mechanism involving the thiol and maleimide groups. FTIR studies were carried out in order to characterize the thioether bond (C-S-C) between GSH and the maleimide groups or related structural modifications. The FTIR spectra of NPs-PLGA-PEG, NPsPLGA-PEG-Mal and NPs-PLGA-PEG-Mal-GSH are presented in Figure 1 A. A strong absorption band occurring at ~1700 cm-1, characteristic of PLGA C=O stretching, was observed in the spectra of all the formulations. Using PLA, Molinaro and coworkers also detected this absorption band around 1,747 cm-1 due to C=O stretching of that polymer 42. Spectrum also reveals the presence of low intensity bands characteristic of C–S and C-SC stretching between 650 and 775 cm-1. According to Fang and coworkers’ studies, those bands are supposed to be a quite weak stretches

43

. These stretching bands were not

observed for NPs-PLGA-PEG-Mal. Also, the fixation of GSH to maleimide group induced the disappearance of the stretch corresponding to conjugated bond of maleimide (3100-3000 cm-1) in NPs-PLGA-PEG-Mal-GSH spectrum which remains present in NPsPLGA-PEG-Mal spectrum. Another evidence is the lack of an -SH stretch at 2540 cm−1 in the infrared spectra of NPs-PLGA-PEG-Mal-GSH meaning that the formulation are pure with no free GSH 44.



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Spectrometric-based analysis of the conjugation using DPPH antiradical activity of remaining GSH or direct detection of maleimide group

DPPH assay was used to measure the activity of the remaining free GSH in the supernatant. As shown in Figure 1B, we found that the antiradical activity of GSH decreased to nearly 62%. This suggests that a certain amount (around 38%) of the initial GSH may have reacted with the maleimide groups. Void formulation was used in order to discard any interference with the strong antiradical activity of free curcumin remaining in the supernatant. In another experiment, we performed spectrophotometric measurement of the maleimide group from NPs-PLGA-PEG-Mal, NPs-PLGA-PEG-Mal-GSH, NPsPLGA-PEG-Mal-Cur and NPs-PLGA-PEG-Mal-GSH-Cur (Figure 1C). This assay was carried out in order to confirm the structural modification of maleimide group after conjugation, since native maleimide group is known to have a maximal absorption around 302 nm. As shown in the diagram of figure 1C, we found a reduction (around 30%) of maleimide absorbance after the addition of GSH to either void or curcumin loaded NPs. In the presence of curcumin loaded nanoparticles, we found a decrease of maleimide absorbance but this decrease was more important after the click reaction. This implies a possible attachment of GSH to maleimide group and also an eventual interference between curcumin loaded particles and maleimide. Taken together this confirmed that GSH was linked to the surface of our conjugated formulations.

During nanoprecipitation process and GSH conjugation, drug was incorporated into the PLGA matrix mixture and would be released gradually for sustainable pharmacological effect. Therefore, the drug loading efficiency directly influences the therapeutic fate of


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the NPs. The characteristics of prepared NPs in addition to their drug loading and entrapment efficiency are shown in Table 2. From the initial drug to polymer ratio of 10%, we found a drug loading mean of around 7 % for all the formulations, with about 56 % as the maximum EE for NPs-PLGA-PEG-Mal-Cur. As determined by DLS, particles had an average size between 149 to 180 nm and were also monodispersed as confirmed with their PDI values which were less than 0.3. Development of monodispersed NPs is essential for biomedical applications. All the formed NPs carried a slightly negatively charged zeta potential of about - 6mv. Zeta potential values were all negatives, irrespective of the drug loading or surface modification as concomitant with what is generally observed with PLGA matrices. So, we found that GSH-conjugation did not affect DLE, EE, size, PDI, and zeta potential. These imply a very good colloidal stability after nanoprecipitation and throughout the process of conjugation. As depicted in Figure 2, we found that the presence of GSH on the surface (Figure 2C) did not modify the initial distribution of particle (Figure 2A), whereas particles loaded with curcumin were more heterogeneous with regard to size distribution (Figure 2 D and fig 2E). Morphology of NPs is also another critical hallmark for exploiting their properties in nano-medecine. So, TEM was used to determine the morphology of the formulations. As shown in Figure 3, NPs-PLGA-PEG-Mal (Figure 3A), NPs-PLGAPEG-Mal-Cur (Figure 3B), NPs-PLGA-PEG-Mal-GSH (Figure 3C) and NPs-PLGAPEG-Mal-GSH-Cur (Figure 3D) displayed a spherical shape. The vast majority of NPs developed for drug delivery has a spherical shape

45

. In all cases, the size of NPs was

smaller with TEM (less than 109 nm) than the one reported with DLS data (bigger than 149 nm). This classical observation could be explained by the state of the polymeric 17 ACS Paragon Plus Environment


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matrices since TEM used dried NPs while DLS measure the hydrodynamic size of particles.

Release kinetics of curcumin from NPs-Cur Sustainable release is a key criterion to study for the application of nanotechnology in medicine. The in vitro release profiles of curcumin from non GSH-NPs and GSH-NPs loaded with curcumin were studied in phosphate buffer containing 3% BSA at pH 7.4. As depicted in Figure 2F we found that for both formulations, a moderate burst release was noted for the first 6 h, followed by a slower and stationary release over 24 h. The release of curcumin from GSH-NPs was not different to non GSH-NPs after 24 h, reaching 42 % and 37 %, respectively (Figure 2F). This implies that GSH conjugation does not affect the release profile of the formulation.

Nanoparticles uptake by SK-N-SH cells In order to determine the effect of GSH-conjugation on the uptake of curcumin by cultured neurons, SK-N-SH cells were incubated with 1µM of free curcumin (control), or different formulations for 2 h at 37◦C. As observed on Figure 4, control cells (Figure 4A) and cells treated with NPs-PLGA-PEG-Mal-GSH (Figure 4B) did not display any fluorescence while in the presence of free curcumin (Figure 4C) a clear fluorescent signal was observed in some cells. Cells treated with NPs-PLGA-PEG-Mal-Cur (Figure 4D) and NPs-PLGA-PEG-Mal-GSH-Cur (Figure 4E) display a strong green signal characteristic of curcumin accumulation inside the cells and within the cellular extensions. After a quantitative analysis, we found that the neuronal internalisation of curcumin with GSH-


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NPs and non GSH-NPs by SK-N-SH cells was higher than with free curcumin (Figure 4F). One would expect a better therapeutic index with the increase of the intracellular accumulation. Using PLGA 50:50 NPs loaded with 1 wt% Lumogen Red (NPs-Lumogen), we recently demonstrated that, the matrix PLGA was taken up by the cells 17. Here, in order to track the localisation of the PLGA-PEG-MAL-GSH conjugated matrix into neuronal cells, we used a covalently attached rhodamine (PLGA-Rhod) into our matrix. Rhodamine incorporation was confirmed using a spectrophotometrical scan of NPs-PLGA-PEG-MalCur (Figure 5A), NPs-PLGA-Rhod (Figure 5B) and NPs-PLGA-Rhod-PEG-Mal-GSHCur (Figure 5C). So, we found that the picks corresponding to the maximal absorption of curcumin (420 nm) and Rhodhamine (560nm) were both present on the labelled and conjugated NPs (NPs-PLGA-Rhod-PEG-Mal-GSH-Cur). When cells were exposed to NPs-PLGA-Rhod-PEG-Mal-GSH-Cur for 2 h, we found a partial co-localization of DAPI (Figure 5B), curcumin (Figure 5C) and Rhodamine (Figure 5D). This suggests that, after 2 h of incubation, they might be a certain amount of curcumin remaining into the matrix taken up by the cells. The isolated green or red signals correspond to free curcumin or void matrices, respectively. These results clearly demonstrated that the conjugated matrix could easily be taken up by SK-N-SH cells and that after 2 h, there is a remaining drug in the matrix for sustainable release. Also, the internalized curcumin was found localized in almost every cells and throughout the cell components meaning that the GSH surface-modification allow a better cellular trafficking of the formulation as compared to free curcumin. Several studies have pointed out the fact that the surfacic presence of some surfactants (PEG or polysorbate 80), ligands,



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antibodies or penetrating peptide could improve the internalization capacity of NPs

22-29

.

This could be explained by the fact that their eventual interaction may optimise the internalization through endocytic process according to the cell types. So, NPs internalisation could be through different endocytic process and this is known to be closely related to the biological activity fate. Recently, an increasing number of papers have pointed GSH as a potent enhancer of drug internalisation into neuronal cells

30, 31

.

Until now, there is no clear clue in the literature on how the presence of GSH could mediate the internalisation of NPs at cellular level. So in this part of the study, we proposed to elucidate the effect of GSH-conjugation on the cellular uptake mechanisms of GSH-NPs-Cur, using some well-known pharmacological inhibitors of endocytosis.

Cellular uptake mechanisms

In order to determinate if the mode of internalisation was energy dependent, firstly we incubated our cells at low temperature (4°C) for 1 h in presence of the formulations. We found that the green signal in SK-N-SH cells was reduced to 12.9% and 16.7% with non GSH-NPs (Figure 6H) and GSH-NPs (Figure 7H), respectively, as compared to the control performed at 37°C. This implies that the cellular uptake of NPs was mainly through an energy dependant process and only a minor fraction of them entry through a passive mechanism. Endocytosis is a well-known route of internalisation of NPs

46

. The

major mechanism during endocytic process of NPs involves caveolae-mediated endocytosis, clathrin-mediated endocytosis, macropinocytosis and clathrin and caveolaeindependent endocytosis dependent endocytosis

47, 48

35, 46

. Therefore, chlorpromazine was used to inhibit the clathrin

; Methyl-β-cyclodextrin as cholesterol depletory, genistein as


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the inhibitor for caveolae dependent endocytosis specifically block macropinocytosis

49

36, 46

. Nocodazole was used in order to

. All the pharmacological inhibitors were used at

non-toxic concentration on our cells (supporting data).

So, we have exposed SK-N-SH cells to the formulations in the presence of pharmacological inhibitors of endocytosis. Figure 6 presents the images obtained after exposure of non GSH-NPs-Cur to SK-N-SH cells pretreated with or without different inhibitors. Similar images were also captured with GSH-NPs-Cur as presented in figure 7. The signal in absence of inhibitors was set as control (100%).

In the presence of nocodazole, the uptake of NPs was significantly decreased to 47.8 % (P≤0.05) for non GSH-NPs-Cur (Figure 6) while being not significant with GSH-NPsCur (84.3%). This suggests that macropinocytosis was more important for non GSH-NPsCur than for GSH-NPs-Cur uptake. Macropinocytosis is described as an endocytic process, involving actin formation

50

, where cells can internalize NPs with the size of

around 200 nm through the formation of vesicles of around 0.2–5 µm 46, 51, 52. The fate of NPs internalized within macropinosomes varies according to cell types

45

and would

either escape lysosomal degradation and therefore have their major portion recycled out of the cells 53. In contrast, the mature macropinosomes could also merge with pre-existing lysosomes due to a proton-ATPase pump dependent acidification of the environment that activates endosomal/lysosomal degradative enzymes

54

. Moreover, this pathway may

serve as a non-specific entry route for nanodevices. This suggests that GSH-conjugation on the surface of NPs may help the formulations to escape the uptake through macropinocytosis and therefore avoiding the lysosomal degradation. Lysosomes play a



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key role in the therapeutic fate of NPs applicable for neurodegenerative diseases, since particles taking that route would be highly vulnerable to degradation.

Chlorpromazine was used to inhibit clathrin-coated vesicles formation

46, 48

and the

results witness a higher decrease of GSH-NPs-Cur uptake to 75.9 % versus a nonsignificant inhibition (85.9 %) in presence of non GSH-NPs. These suggest a greater mediation of clathrin pits in the uptake of GSH-NPs-Cur than non GSH-NPs-Cur. Clathrin-mediated endocytosis is mainly reported for its role in the selective uptake of molecules through specific receptors. Numerous ligands have been used for receptordependant clathrin-mediated endocytosis including low density lipoprotein (LDL), transferrin and epidermal growth factor

55

. Internalisation of NPs with a diameter lower

than 200 nm was also found to involve clathrin-mediated endocytosis

45

. However, this

path could also mediate the internalisation of nutrients and the degradation of recycle substances. When using genistein for caveolae-mediated endocytosis blocking 56, the only significant decreased was found with GSH-NPs-Cur uptake, while having no inhibition on the non GSH-NPs-Cur uptake, indicating that caveolae-associated endocytosis was also involved in the uptake of GSH-NPs but not for non GSH-NPs. Caveolae are particularly abundant on endothelial cells, where they can constitute 10-20% at the cell surface 52. More studies need to be performed to better understand caveolae mediated endocytosis uptake of particles around 200 nm 57. Ligands known to be taken up through this route include folic acid, albumin and cholesterol 55.


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Very recently, various clathrin and caveolae independent endocytosis pathway have been described. In this study, since chlorpromazine and genistein were the most effective inhibitor of the uptake, but not fully inhibit the uptake, we proposed to go further into the uptake mechanisms. We therefore treat cells in the presence of the mixture of chlorpromazine and genistein in order to better investigate the uptake route. Our results shown that, the mixture highly reduced the cellular uptake of GSH-NPs-Cur (Figure 7F) to 53.2% (P

Optimization of Curcumin-Loaded PEG-PLGA Nanoparticles by GSH

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