International Journal of Pharmaceutics 486 (2015) 259–267

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Curcumin delivery from poly(acrylic acid-co-methyl methacrylate) hollow microparticles prevents dopamine-induced toxicity in rat brain synaptosomes Krassimira Yoncheva a, * , Magdalena Kondeva-Burdina b , Virginia Tzankova b , Petar Petrov c, Mohamed Laouani d,e, Silvia S. Halacheva e, ** a

Department of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy, Medical University of Sofia, 1000 Sofia, Bulgaria Department of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Pharmacy, Medical University of Sofia, 1000 Sofia, Bulgaria c Institute of Polymers, Bulgarian Academy of Sciences, Acad. G. Bonchev, Block 103 A, 1113 Sofia, Bulgaria d ICAM Toulouse, 75 Avenue de Grande Bretagne, CS 97615 31 076 Toulouse Cedex 3, France e University of Bolton, Institute for Materials Research and Innovation, Deane Road, Bolton, BL3 5AB Greater Manchester, UK b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 20 January 2015 Received in revised form 24 March 2015 Accepted 27 March 2015 Available online 31 March 2015

The potential of poly(methyl methacrylate-co-acrylic acid) (PMMA-AA) copolymers to form hollow particles and their further formulation as curcumin delivery system have been explored. The particles were functionalized by crosslinking the acrylic acid groups via bis-amide formation with either cystamine (CYS) or 3,30 -dithiodipropionic acid dihydrazide (DTP) which simultaneously incorporated reversibility due to the presence of disulfide bonds within the crosslinker. Optical micrographs showed the formation of spherical hollow microparticles with a size ranging from 1 to 7 mm. Curcumin was loaded by incubation of its ethanol solution with aqueous dispersions of the cross-linked particles and subsequent evaporation of the ethanol. Higher loading was observed in the microparticles with higher content of hydrophobic PMMA units indicating its influence upon the loading of hydrophobic molecules such as curcumin. The in vitro release studies in a phosphate buffer showed no initial burst effect and sustained release of curcumin that correlated with the swelling of the particles under these conditions. The capacity of encapsulated and free curcumin to protect rat brain synaptosomes against dopamineinduced neurotoxicity was examined. The encapsulated curcumin showed greater protective effects in rat brain synaptosomes as measured by synaptosomal viability and increased intracellular levels of glutathione. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Hollow microspheres Poly(methyl methacrylate-co-acrylic acid) Disulphide crosslinking Curcumin Rat brain synaptosomes

1. Introduction Hollow particles have been used extensively for a large range of applications such as drug delivery (Yang et al., 2010), gene delivery (Zhu et al., 2011), and in regenerative medicine (Liu et al., 2011). Caruso et al. (1998) prepared hollow particles by layer-by-layer assembly of oppositely charged polymers onto colloidal silica templates. Removal of the template was achieved by the use of

* Corresponding author at: Department of Pharmaceutical Technology, Faculty of Pharmacy, 2 Dunav Str., 1000 Sofia, Bulgaria. Tel.: +359 29236544; fax: +359 29879874. ** Corresponding author at: University of Bolton, Institute for Materials Research and Innovation, Deane Road, Bolton, Greater Manchester BL3 5AB, UK. Tel.: +44 0 1204 903785. E-mail addresses: [email protected] (K. Yoncheva), [email protected] (S.S. Halacheva). http://dx.doi.org/10.1016/j.ijpharm.2015.03.061 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.

hydrofluoric acid, which is highly hazardous reagent. Calcium carbonate has also been utilized as a template (Addison et al., 2010). Other techniques for preparing hollow polymer particles involve double emulsions (Cayre and Biggs, 2009), polymer precipitation by phase separation (Yow and Routh, 2006), layerby-layer assembly (Kinnane et al., 2011), vesicle formation by spontaneous self-assembly of block copolymers (Du and Chen, 2004; Otsuka et al., 2001), etc. For example Wong et al. (2002) prepared macroporous capsules by organising colloids around liquid droplets, followed by evaporation of the droplets. Li et al. (2013) used a solvent evaporation technique to produce hollow particles. Hollow biodegradable particles based on a polylactide polymer terminated with pentadecafluoro-1-octanol (PFO-PLL) were prepared via dissolution of the polymer in a decane/ dichloromethane mixture, followed by emulsification of the solution (Lensen et al., 2011). Ultrasound triggered release of a drug from the hollow particles was then examined. Im et al. (2005)

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prepared hollow polystyrene particles with a degree of control over the formation of surface pores. Hollow poly(methyl methacrylate-co-methacrylic acid)-(PMMA-MAA) and poly(ethyl acrylate-co-methacrylic acid)-based (PEA-MAA) particles that are both pH-responsive and redox-sensitive were prepared via a solvent evaporation approach (Bird et al., 2011, 2012). These studies have also demonstrated the pH-triggered release of a model solute from the PMMA-MAA and PEA-MAA particles. Recently, Halacheva et al. (2013, 2014),) prepared novel poly(methyl methacrylate-comethacrylic acid), poly(ethylacrylate-co-methacrylic acid) and poly(methyl methacrylate-co-acrylic acid) biodegradable hollow particles via a solvent evaporation method. The hollow particles were formed shortly after emulsification due to the polymers’ precipitation at the dichloromethane droplet/water interface. The particles were crosslinked with the disulphide-containing diamines cystamine (CYS) or 3,30 -dithiodipropionic acid dihydrazide (DTP), swelled within the physiological pH range and formed physical gels from their concentrated dispersions. The gels had high porosity, high elasticity and were rapidly disassembled upon addition of glutathione. As the PMMA-AA gels also showed good biodegradability and biocompatibility profiles that were tuneable through variation of the particle composition, they were considered as potentially suitable for future use in minimally-invasive tissue repair. Herein we aim to expand our previous studies and to investigate the potential use of the PMMA-AA hollow particles for drug delivery applications. In particular, this research describes the preparation of curcumin-loaded PMMA-AA/CYS and PMMA-AA/ DTP hollow particles and will evaluate their protective effect against dopamine-induced oxidative neurodegeneration in isolated rat synaptosomes. Curcumin is a candidate for use in the prevention or treatment of major disabling age-related neurodegenerative diseases as shown by cell culture and animal model data (Monroy et al., 2013). However, clinical application of curcumin is hindered because of its low water solubility, instability (hydrolytic degradation at pH-values above 7.0) and poor oral bioavailability. In this view, the incorporation of such a labile active molecule into particulate drug delivery systems, like micro- and nanoparticles, could increase its stability against hydrolytic degradation and could enable cellular uptake. Dopamine, while an essential neurotransmitter, is also a known neurotoxin that potentially plays an etiologic role in several neurodegenerative disorders, such as Parkinson’s disease. Parkinson’s disease has been modeled in vitro through the specific neurotoxic effect of 6-hydroxydopamine (6-OHDA) (Beal, 2001). Taking into account the antioxidant properties of curcumin and the oxidative process involved in the toxicity induced by dopamine, we suppose that microencapsulated curcumin might be able to attenuate the damage induced by dopamine in isolated rat brain synaptosomes in vitro. 2. Materials and methods 2.1. Materials Tetrahydrofuran (Aldrich, anhydrous, inhibitor-free, 99.9%), dichloromethane (Aldrich, HPLC grade, 99.8%), methanol (Aldrich, HPLC grade, 99.9%), 2,20 -azobis(2-methylpropionitrile) (AIBN, Aldrich, 98%), methyl methacrylate (MMA, Aldrich, 98.5%), acrylic acid (AA, Aldrich, 99%), cystamine dihydrochloride (CYS, Aldrich, 96%), N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDC, Aldrich, 99%), N-hydroxysuccinimide (NHS, Aldrich, 98%), 3,30 -dithiodipropionic acid (Aldrich, 99%), hydrazine monohydrate (Fluka, purum, 98%), concentrated sulphuric acid (Aldrich, ACS reagent, 95–98%), poly(vinyl pyrrolidone) (PVP, Aldrich, average molecular weight 40,000 g mol1), curcumin (Aldrich, analytical standard grade, 98%), HEPES (Aldrich,

anhydrous 99.5%), Percoll (Aldrich, pH 8.5–9.5) and 3-[4,5dimethylthiazol-2-yl]-2,5diphenyl-tetrazolium bromide (Aldrich) were used as received. NaCl, KCl, D-glucose, 6-hydroxydopamine and 2,20 -dinitro-5,50 -dithiodibenzoic acid (DTNB) were supplied by Merck. Milli-Q water was used throughout, unless otherwise stated. Reactions requiring anhydrous conditions were performed in oven-dried glassware, with anhydrous solvents, under a positive pressure of nitrogen. 3,30 -Dithiodipropionic acid dihydrazide (DTP) was synthesized according to a literature method (Vercruysse et al., 1997). 2.2. Synthesis of copolymers The PMMA-AA copolymers were synthesised by free radical polymerization of mixtures of MMA with AA. The copolymer abbreviations used here indicated the molar percentages of each component. For example, PMMA-30AA contains 30 mol% AA and 70 mol% MMA (monomer units). The following synthesis of PMMA-30AA is representative of the procedure employed for all copolymers: An oven-dried 250 ml, two-necked round bottom flask, fitted with a condenser and gas inlet adapter, was purged with nitrogen. AIBN (0.25 g) and anhydrous THF (100 ml) were added to the flask and the resulting solution was magnetically-stirred and heated at reflux (66  C), under a steady flow of nitrogen, for one hour. A mixture of MMA (7.50 g, 74.9 mmol, 0.68 equiv.), AA (2.54 g, 35.2 mmol, 0.3 equiv.) and AIBN (0.03 g) was dissolved in anhydrous THF (20 ml) and added to the refluxing solution at a uniform rate over 2 h. The reaction mixture was maintained at reflux for a further 18 h, cooled to room temperature and concentrated under reduced pressure to a volume of approximately 100 ml before being poured into cold water (1000 ml). The precipitated polymer was filtered off under suction, washed with water (3  200 ml) and petroleum ether (2  200 ml) and air-dried overnight. Residual water was removed by freeze-drying. 2.3. Characterization of the copolymers Proton magnetic resonance spectra (1H NMR) were recorded using a 250 MHz Bruker WM 250 spectrometer. Chemical shifts (dH) are quoted in parts per million and are referenced to the residual solvent peak. Gel permeation chromatography (GPC) analysis was carried out with a Waters system consisted of four Styragel columns with nominal pore sizes of 100, 500, 500, and 1000 Å and a refractive index detector (R401). Tetrahydrofuran (THF) was used as an eluent at a flow rate of 1 ml/min at 40  C. Samples were dissolved in anhydrous, inhibitor-free THF (1 mg/ml) at room temperature. Potentiometric titration was performed using a Metrohm 716 DMS Titrino instrument. Measurements were performed on 40 ml of a 1 wt.% non-crosslinked dispersion using standardised NaOH solutions. All pKa values reported here are apparent values. Optical microscopy was conducted with a Carl Zeiss Jena Binocular Microscope and white transmitted light. 2.4. Particle preparation The non-crosslinked PMMA-AA particles were prepared by dissolving 10.00 g of copolymer in 440 ml of a mixed CH2Cl2–MeOH (84:16, v/v) solvent. A solution of poly(vinyl pyrrolidone) (PVP) (12.00 g, average MW 40,000 g/mol) in 1200 ml of water was cooled to 0  C and sheared at 10,000 rpm. The solution of copolymer was then added, at a uniform rate of 10 ml/min, to the PVP solution. Emulsification was continued for a further 30 s after addition of the polymer solution and the emulsion was then

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allowed to stir slowly overnight in order to remove CH2Cl2. The product was purified by repeated centrifugation and redispersion in water in order to remove excess PVP. The resultant suspension was then filtered through a 50 mm filter and stored at 2–4  C. The following procedure for the preparation of the crosslinked PMMA-28AA/2CYS is representative of the general procedure employed: 15.0 ml of the concentrated PMMA-30AA particle stock dispersion (4.3 wt.%, 1 mmol COOH groups) was diluted with 0.1 M pH 6.4 phosphate buffer solution to a volume of 28.0 ml and a final concentration of 1.5 wt.%. The pH of the buffer utilized depended upon the pKa of the particles. To the resulting stirred suspension, N-hydroxysuccinimide (NHS) (0.38 g, 1.22 equiv.) was added followed by ethylcarbodiimide hydrochloride (EDC) (0.41 g, 0.80 equiv.). Stirring was continued for a further fifteen minutes before cystamine dihydrochloride (CYS) (0.3 g, 0.50 equiv.) was added. The reaction mixture was then stirred at room temperature for 24 h and the crosslinked particles were isolated by centrifugation, followed by three cycles of redispersion in water and centrifugation. The DTP crosslinked particles were prepared in an analogous manner by substituting DTP for CYS dihydrochloride. The crosslinked particle abbreviation used here identifies the mol% non-functionalized MAA or AA groups and the mol% CYS or DTP incorporated into the particles. For example PMMA-28AA/2CYS contained 28 mol% AA and 2 mol% CYS. 2.5. Characterization of the particles The hydrodynamic diameter (Dh, in nm) and the zeta-potential of the particles were measured at 90 using a Malvern Zetasizer Nano ZS. The particle size measurements were carried out at pH values ranging from 5.4 to 8.0 and at a constant solution concentration of 0.1 wt.%. The dispersions were treated with 1.0 M NaOH solution until the desired pH value was reached, then equilibrated for 15 min at room temperature. At least 10 correlation functions were analysed per sample, at each pH value, in order to obtain an average measurement. The volume swelling ratio, Q, of the PMMA-AA particles, functionalised with either CYS or DTP, was estimated using Q = (Dh/Dh(collapse))3, where Dh and Dh(collapse) are the hydrodynamic diameters of the particles at a given pH (well below the particles’ pKa values) and in the collapsed non-swollen state, respectively (Halacheva et al., 2013). All Dh(collapse) values were measured at the lowest pH values tested. The zeta-potential of the microparticles dispersions was measured in purified water. The particle morphology was studied by SEM (Lyra/Tescan). Dispersions were deposited on SEM stubs by evaporation at room temperature.

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2.7. In vitro release studies For in vitro release studies, 1 mg of the drug-loaded hollow particles were incubated in 10 ml acidic or phosphate buffer (pH 1.2 or pH 6.8) containing 10% ethanol. The samples were placed into a shaking bath set at 37  C and 100 rpm. The entire dispersions were centrifuged at selected time intervals and the concentration of the released curcumin was determined by UV–vis spectrophotometry as described above. 2.8. Cell culture Human hepatoma cells (HepG2) were kept in culture and expanded at 37  C in a humidified atmosphere of 5% CO2 in culture medium DMEM (Dulbecco’s Modified Eagle’s Medium, Lonza, Bazel, CH), supplemented with 20% fetal bovine serum (FBS) (Gibco BRL) at 10%, penicillin/streptomycin 100 (Euroclone, Devon, UK), Glutamax 100 (Invitrogen) and non-essential amino acids 100 (Invitrogen). 2.9. MTT-dye reduction assay HepG2 cells were seeded in 96-well microplates at a density 2  105 cells/well and allowed to attach to the well surface for 24 h at 37  C in a humidified atmosphere with 5% CO2. After incubation, different concentrations of the hollow particles (50, 100, 200, 500 and 1000 mg/ml) were added to cells, and incubated for a period of 24 h, 48 h and 72 h. For each concentration a set of at least 8 wells were used. After the treatment, 10 ml MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (10 mg/ml in PBS) aliquots were added to each well. The microplates were further incubated for 4 h at 37  C and the obtained formazan crystals were dissolved by the addition of 50 ml/well of DMSO. The absorbance was measured at 570 nm on a Labexim LMR-1 microplate reader (Lengau, Austria). 2.10. Lactate dehydrogenase (LDH) leakage Stock dispersions of the microparticles were freshly prepared and diluted with growth medium to the concentrations of 50, 100, 200, 500 and 1000 mg/ml. Thereafter the microparticles were added to cells (2  105 cells/well), and incubated for periods of 24 h, 48 h and 72 h. Eight wells were used for each concentration. LDH leakage from the cells was determined using a commercial LDH cytotoxicity detection kit (Clontech, US) according to the manufacturer’s protocols. LDH activity was assessed in the conditioned media and the amounts detected were calculated as a percentage of the solvent-treated control (DMEM growth medium) (GraphPad Prizm Software).

2.6. Loading of crosslinked particles with curcumin 2.11. Isolation and incubation of synaptosomes An ethanol solution of curcumin (1 mg/ml) was added to an aqueous dispersion containing the pre-formulated crosslinked particles (5 mg/ml) and the mixture was incubated for 4 h. The ethanol was evaporated under reduced pressure (Buchi-144, Switzerland) and the resulting dispersion was centrifuged at 14,000 rpm for 30 min. The pellets obtained after centrifugation were dried under vacuum. The entrapment efficiency was determined by extraction of curcumin from hollow particles with 10 ml ethanol under stirring (700 rpm). After 4 h stirring, the dispersion was centrifuged (as described above) and the concentration of curcumin into the ethanol supernatant was measured by spectrophotometry at a wavelength of 428 nm (Hewlett Packard 8452A, US). The curcumin concentration was calculated according to a standard curve prepared in the range of 2–10 mg/ml (r > 0.994).

Male Wistar rats (body weight, 200–250 g) were used. Rats were housed in Plexiglas cages (3 per cages) in a 12/12 light/dark cycle, temperature 20  2  C. Food and water were provided ad libitum. Animals were purchased from the National Breeding Centre, Sofia, Bulgaria. All experiments were performed after at least one week of adaptation to this environment. The experimental procedures were approved by the Institutional Animal Care and Use Committee at the Medical University of Sofia, Bulgaria. The guidelines stated in the Principles of Laboratory Animal Care (NIH publication #85-23, revised in 1985) were followed strictly throughout the experiment. Synaptosomes were prepared from the brains of adult male Wistar rats, as previously described by Taupin et al. (1994). The brains were homogenized in 10 vol. of cold buffer 1, containing: 5 mM HEPES and 0.32 M sucrose (pH 7.4). The

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brain homogenate was centrifuged twice at 1000  g for 5 min at 4  C. The supernatant was collected and centrifuged 3 times at 10,000  g for 20 min at 4  C. The pellet was resuspended in icecold buffer 1. The synaptosomes were isolated by using Percoll reagent to prepare the gradient. Synaptosomes were resuspended and incubated in buffer 2, containing: 290 mM NaCl, 0.95 mM MgCl26H2O, 10 mM KCl, 2.4 mM CaCl2H2O, 2.1 mM NaH2PO4, 44 mM HEPES, 13 mM D-glucose. Incubations were performed in a 5% CO2 + 95% O2 atmosphere. The content of synaptosomal protein was determined according to the method of Lowry et al. (1951) using serum albumin as a standard. Isolated rat synaptosomes were exposed to 6-hydroxydopamnine (6-OHDA) (150 mM), as a model of dopamine-induced oxidative neurodegeneration; to free curcumin (10 mg/ml); or curcumin-loaded particles (final curcumin concentration 10 mg/ml).

in DMSO. The absorbance was measured spectrophotometrically at l = 580 nm. 2.13. Statistical analysis Results from the viability tests are expressed as means  SEM from at least three independent experiments. The cell survival data were normalized as percentage of the untreated control (set as 100% viability). The statistical analysis of the data was carried out using ANOVA, followed by Tukey’s HSD post hoc test. For the differences between untreated control and the positive control, Student's t-test for independent samples was used. The values of p < 0.05, p < 0.01 and p < 0.001 were considered as statistically significant. 3. Results and discussion

2.12. Glutathione (GSH) content and MTT-dye reduction assay 3.1. Copolymer synthesis and characterization The levels of glutathione (GSH) were determined in rat brain synaptosomes with the Ellman reagent (DTNB), which forms colored complexes with  SH groups at pH 8 with maximum absorbance at 412 nm, as described by Robyt et al. (1971). Viability of synaptosomes was measured by MTT-test as described by Mungarro-Menchaca et al. (2002). After incubation with the tested compounds, synaptosomes were treated with MTT solution (0.5 mg/ml) for 1 h at 37  C and were centrifuged at 15,000  g for 1 min. The formed formazan crystals were dissolved

PMMA-30AA and PMMA-50AA were synthesised by freeradical polymerisation of the appropriate mixture of MMA and AA monomers in the presence of 2,20 -azobis(2-methylpropionitrile). The weight average molecular weight and polydispersity of the copolymers were determined by GPC (Fig. 1(a)). The weight average molecular weights for PMMA-30AA and PMMA-50AA were 28.320 and 31.130 g mol1, respectively (Table 1). GPC analyses gave monomodal distributions with polydispersities

Fig. 1. GPC traces of PMMA-30AA and PMMA-50AA copolymers (a) and 1H NMR data of PMMA-50AA in d-DMSO (b).

K. Yoncheva et al. / International Journal of Pharmaceutics 486 (2015) 259–267 Table 1 Characterisation data of PMMA-AA copolymers. Composition

PMMA-30AA PMMA-50AA a b c

Mw (g mol1)a

PDIa

28.320 31.130

2.00 1.89

(approximately 0.6–1.0 mm) (Fig. S1, Supporting information). Similar particle morphology has been reported in our previous studies (Halacheva et al., 2013, 2014). These particles were formed shortly after emulsification of the aqueous dispersions of PMMAAA copolymers. The copolymers, which are insoluble in both the organic solvent and the aqueous phase, form shells around the dichloromethane droplets which subsequently collapse as the residual solvent evaporates. The molar percentages (mol%) of AA in the PMMA-30AA and PMMA-50AA non-crosslinked particles were measured by potentiometric titration (Fig. S2, Supporting information). These values were in reasonable agreement with those calculated from the 1H NMR spectra and with the theoretical values (Table 1). The pKa values decrease as the AA content increased (Table 1), in agreement with our previous work. To prepare the crosslinked PMMA-AA-based particles an earlier established 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) mediated coupling methodology was employed, utilising the diamino compounds CYS and DTP as crosslinkers (Halacheva et al., 2014) (Fig. 2). The present approach is faster and more facile as compared to the method based on solid core templates employed for hollow particle fabrication (Manju and Sreenivasan (2011)). The evaporation of organic solvent allowed formation of hollow particles in a very short time unlike the time-consuming dissolution of inorganic core particles. The compositions of the crosslinked particles were determined from elemental analyses. In all reactions, the same molar proportions of reagents (EDC, NHS, CYS/DTP) relative to the number of available COOH groups were used. For PMMA-AA, the proportion of available RCOOH groups which were successfully reacted with either CYS or DTP was 6–24% (Table 2). The higher extent of crosslinker incorporation observed for PMMA-AA/DTP

pKac

mol% MAA b

c

Theor.

Exper.

Exper.

32 55

30 50

33 49

7.7 6.6

Determined by GPC. Determined by 1H NMR. Determined from the potentiometric titration data.

ranging from 1.89 to 2.00. The copolymers’ structural compositions were determined from their 1H NMR spectra in DMSO-d6 (Fig. 1(b)). The compositions of the PMMA-AA samples were calculated by examining the relative ratios of signal intensities of the methoxyl protons of MMA groups and the methylene protons of both MMA and AA groups. The experimental values obtained by 1 H NMR were in good agreement with the theoretical values (Table 1). 3.2. Particle preparation and characterisation A sequential emulsification-solvent evaporation protocol was used for the preparation of the PMMA-30AA- and PMMA-50AAbased non-crosslinked particles dispersions (Fig. 2). The copolymer solution was added slowly to the aqueous PVP solution in order to facilitate the formation of homogeneous and colloidally stable (for at least three months after preparation) particle dispersions. The optical microscope images of the PMMA-30AA particle dispersion taken immediately after emulsification showed that the particles were spherical, hollow and with thin shells

HO2C

H Me

CO2Me

a

b

1. CH2Cl2-MeOH (84:16, v/v) 2. 1 wt% aq. PVP soln. (10,000 rpm, 0 oC) 3. Stir 18 h, rt PMMA-AA microparticles (collapsed, hollow)

1. EDC, NHS, pH 6.4, rt, 15 m 2. Cystamine dihydrochloride

HO

O Me

O

OMe O

OMe O Me O O

O

HN Me OMe

OH O

OH

HN S

O

MeO OMe

O HO

OH OH

S

MeO

Me

Me O MeO

263

O

PMMA-AA microparticles (crosslinked, hollow)

O

Fig. 2. Preparation of the PMMA-AA non-crosslinked and PMMA-AA/CYS crosslinked microparticles.

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Table 2 Composition and swelling of the crosslinked hollow microparticles. Copolymer

RCONHRa

Composition

PMMA-30AA

7 10

PMMA-28AA/2CYS PMMA-27AA/3DTP

1.9 3.7

PMMA-50AA

6 24

PMMA-47AA/3CYS PMMA-38AA/12DTP

6.0 12.1

Qmaxb

a

Percentage of RCONHR formed. Swelling ratio at pH 7.0. In each case this value of Q corresponded to the maximum swelling ratio. b

Table 3 Variation of the hydrodynamic diameter (Dh, in nm) with pH for non-crosslinked and crosslinked particles. Particle composition

pH 6.0

pH 6.5

pH 7.0 Dh (nm)

pH 7.5

pH 8.0

PMMA-30AA PMMA-28AA/2CYS

2324 3883

1965 4564 1.6a

1864 4844 1.9a

1582 4299 1.4a

583 451 0.002a

PMMA-27AA/3DTP

3604

5221 3.0a

5560 3.7a

4812 2.4a

1058 0.03a

PMMA-50AA PMMA-47AA/3CYS

3171 2835

2444 4293 3.5a

1883 5160 6.0a

793 4870 5.1a

587 2408 0.6a

PMMA-38AA/12DTP

2425

4925 8.4a

5560 12.1a

5460 11.4a

4474 6.3a

a The swelling ratios calculated from the ratio of the volume of the swelled particles at the given pH to the volume of the particles at the lowest pH tested.

was rationalised by the retainment of nucleophilicity by the terminal nitrogen atoms of hydrazides at acidic pH values (Halacheva et al., 2014; Bulpitt and Aeschlimann, 1999). The low percentage of RCONHR group formation was also observed for the analogous PMMA-MAA, PEA-MAA, PMMA-AA and PBA-MAA particles, which were also crosslinked with either CYS or DTP. This was attributed to the low level of carboxylic acid group activation at the reactions’ pH values. All the crosslinking reactions were performed at pH 6.4, which was well below the pKa of the non-crosslinked particles (Table 1), as the dispersions of noncrosslinked particles would dissolve if the solution pH reached their corresponding pKa value. The sizes of the non-cross-linked and cross-linked particles were investigated by DLS as a function of solution pH. The measurements were carried out at pH values ranging from 6.0 to 8.0 and the Dh values are summarized in Table 3. For clarity, only

Dh, nm

10000

Fig. 4. Optical micrographs of PMMA-47AA/3CYS aqueous hollow particle dispersions (pH 6.5).

the data from the major particles, with intensity more than 80% of the particles in each sample, are included. A gradual disassociation of any large particle presented was observed upon increasing pH. The particle disintegration process is attributed to the ionisation of COOH groups along the polymer chains which weaken their associative hydrophobic interactions and increase particle solvation. For PMMA-50AA, PMMA-47AA/3CYS and PMMA-38AA/12DTP the variations of the Dh of the dominant particles, Dhslow, with the solution pH are presented in Fig. 3. Compared to the noncrosslinked particles, dispersions of crosslinked particles showed pH-triggered swelling, rather than disintegration. The swelling process was attributed to electrostatic repulsion between negatively charged carboxylate groups in the polymers which are effectively held together by their crosslinks. As the pH increases, the particle dimensions are reduced. This is probably due to the hydroxide ion-mediated cleavage of some disulphide groups (which are present in CYS and DTP) at high pH values and subsequent removal of polymer chains from the particles under the conditions reported. The maximum swelling values (Qmax) for all crosslinked particles are presented in Table 2. In each case, Qmax was observed at pH 7.0 which correlates well with the pKa values of the corresponding (parent) non-crosslinked particles (see Table 1). In line with what has been previously reported (Halacheva et al., 2014), PMMA-AA/DTP particles have been found to swell to a larger extent than those which are crosslinked with a comparable proportion of CYS. The larger swelling ratios observed for DTPcontaining crosslinked particles have been related to the increased length of the DTP molecule compared to CYS. Optical micrographs (Fig. 4) of PMMA-47AA/3CYS cross-linked dispersions show spherical hollow objects which range in size from 1 to 7 mm.

1000

100

PMMA-50AA PMMA-47AA/3CYS PMMA-38AA/12DTP 6

7

8

9

10

pH Fig. 3. Variations of the Dh of the dominant particles, Dhslow, with the solution pH.

Fig. 5. Loading of curcumin into the different types of crosslinked hollow microparticles (mg curcumin/ mg hollow microparticles). Mean  SD (n = 3).

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265

Fig. 6. SEM images of PMMA-AA/CYS and PMMA-AA/DTP microparticles loaded with curcumin.

3.3. Encapsulation and release studies Drug loading was performed by simple incubation of aqueous dispersion of PMMA-AA/CYS and PMMA-AA/DTP particles with the solution of curcumin in ethanol. According to this procedure, loading levels between 44 and 65 mg/mg were obtained (Fig. 5). Higher loading was observed for PMMA-28AA/2CYS and PMMA27AA/3DTP, which indicated that the higher content of hydrophobic PMMA units played an important role in the loading of hydrophobic molecules such as curcumin. The surface morphologies of the PMMA-AA/CYS and PMMAAA/DTP particles loaded with curcumin (PMMA-AA/CYS/CUR and PMMA-AA/DTP/CUR, respectively) were evaluated by SEM (Fig. 6). The particles’ morphology was independent of the type of the cross-linking agent. However, the percentage of acrylic acid in the particles had a significant effect on the dried particle morphology. The cross-linked particles with higher acrylic acid content (PMMA47AA/3CYS/CUR and PMMA-38AA/12DTP/CUR) featured irregular shapes and rough surfaces (Fig. 6(a) and (c)). In contrast, the PMMA-28AA/2CYS/CUR and PMMA-27AA/3DTP/CUR particles

Fig. 7. Zeta-potential of the different types of crosslinked hollow microparticles loaded with curcumin. Mean  SD (n = 3)

with lower AA content were spherical with smooth surfaces (Fig. 6(b) and (d)). The PMMA-AA/CYS/CUR and PMMA-AA/DTP/ CUR particles were characterized with negative zeta-potential suggesting the presence of free, non-crosslinked acrylic acid groups on the hollow particles' surfaces which is in agreement with the elemental analysis results (Fig. 7,Table 2). The negative charges on particles’ surface ensure good colloidal stability of the

Fig. 8. In vitro release of curcumin from the PMMA-AA/CYS and PMMA-AA/DTP hollow microparticles in acid (pH 1.2) and phosphate buffer (pH 6.8). Mean  SD (n = 3).

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systems due to the strong electrostatic repulsion and diminish the tendency for agglomeration. The in vitro release studies were performed in acid and phosphate buffers with pH-values representing those in gastrointestinal tract. In the acid buffer, the curcumin release was slow probably due to the deprotonation of acrylic acid groups (Fig. 8). The in vitro release studies in a phosphate buffer showed no initial burst effect and sustained release of curcumin from the swollen hollow particles (Fig. 8). The burst effect is frequently associated with the surface location of the encapsulated drug. In our case, the absence of the initial burst release was probably due to the specific structure of the PMMA-AA/CYS and PMMA-AA/DTP particles, in particular curcumin was loaded into the internal hydrophobic rather than in the outer hydrophilic layer of particles. Compared to PMMA-AA/CYS/CUR, the PMMA-AA/DTP/CUR showed slower release of curcumin. In particular, between 14 and 23% curcumin was released after 12 h from PMMA-AA/DTP/CUR particles, whereas from PMMA-AA/CYS/CUR the released amount was 40–45%, respectively. This could be attributed to the higher swelling ratios of the PMMA-AA/DTP particles compared to those crosslinked with a comparable proportion of CYS (Table 2). The relatively higher Q values suggest that the extra amide bonds within the DTP molecule impart an increased degree of rigidity that will decrease the intraparticle elasticity and increase the density of the PMMA-AA/DTP (Halacheva et al., 2014). 3.4. In vitro cytotoxicity studies of PMMA-AA/CYS and PMMA-AA/DTP particles In vitro cytotoxicity of the PMMA-AA/CYS and PMMA-AA/DTP particles was investigated by monitoring mitochondrial function (MTT assay) and the cellular membrane integrity (LDH assay) in HepG2 cells (Fig. 9). The MTT tests revealed no cytotoxicity of the PMMA-AA/CYS and PMMA-AA/DTP particles at concentrations of 50 and 100 mg/ml after 24 h incubation. These results indirectly indicated that the formulated particles did not alter mitochondrial function. An increased cytotoxicity was observed only at concentrations higher than 500 mg/ml and longer incubation (48 and 72 h) (not shown). Regarding LDH studies, the PMMA-AA/CYS and PMMA-AA/DTP particles exhibited no apparent cytotoxicity to HepG2 cells at concentrations lower than 100 mg/ml.

Fig. 9. In vitro cytotoxicity of PMMA-AA/CYS and PMMA-AA/DTP microparticles on HepG2 cells measured by MTT test (24 h exposure) and LDH-leakage. Mean  SEM (n = 3).

Fig. 10. Effect of curcumin loaded PMMA-AA/CYS and PMMA-AA/DTP particles on synaptosomal viability in 6-OHDA treated isolated rat brain synaptosomes. Mean  SEM (n = 7). Values of ***p < 0.001 vs control group; #p < 0.05 and ##p < 0.01 (vs 6-OHDA group) were considered statistically significant.

3.5. Synaptosomal protection study Nowadays, it is well known that the mechanisms of neuroprotective effects include both direct and indirect antioxidant effects of curcumin. In particular, it scavenges reactive oxygen and nitrogen species (Barzegar and Moosavi-Movahedi, 2011; Trujillo et al., 2013) and induces cytoprotective enzymes such as glutathione-S transferase (GST), g-glutamyl cysteine ligase (g-GCL), heme oxygenase-1 (HO-1) (Dinkova-Kostova and Talalay, 2008; El-Agamy, 2010; Singh and Sharma, 2011; Reyes-Fermin et al., 2012). Since curcumin is characterized with poor absorption and low bioavailability, the present study evaluated the potential of encapsulated curcumin for protection against dopamine induced neurotoxicity in rat brain synaptosomes (Fig. 10). As shown, the incubation of rat brain synaptosomes with 150 mM 6-OHDA drastically reduced synaptosomal viability, which is in agreement with previous study (Stokes et al., 2002). However, the treatment with the encapsulated and free curcumin diminished the lesions caused by 6-OHDA in the synaptosomes. As shown, in the presence of curcumin loaded hollow particless, the decrease in synaptosomal viability induced by 6-OHDA was significantly attenuated. It is known that dopamine oxidative metabolism is accompanied by the formation of reactive oxygen species which triggers dopamine neurotoxicity and neurodegeneration. Glutathione (GSH) is the most abundant intracellular antioxidant which plays an important role in the protection of biological systems against oxidative stress damages. We show that in the presence of curcumin (10 mg/ml), the GSH depletion induced by 6-OHDA in rat brain synaptosomes was significantly reduced (Fig. 11). Moreover, this effect was most pronounced when curcumin was loaded into the PMMA-AA/CYS and PMMA-AA/DTP particles formulations. The increase in total GSH content was most pronounced for the PMMAAA/CYS (4-fold increase), compared to PMMA-AA/DTP particles (3-fold increase compared to 6-OHDA). The important finding was that these beneficial effects were achieved at concentration of the drug-loaded microspheres lower than cytotoxic concentration observed on HEP-G2 cells (Fig. 9). Our findings suggested that

Fig. 11. Effects of curcumin loaded hollow particless on 6-OHDA induced toxicity in isolated rat brain synaptosomes as measured by intra-synaptosomal total GSH. Mean  SEM (n = 7). Values of *p < 0.05 and ***p < 0.001 (vs control non-treated synaptosomes); ##p < 0.01 and ###p < 0.001 (vs 6-OHDA group) were considered statistically significant.

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curcumin counteracted the in vitro toxicity of 6-OHDA through its anti-oxidant properties (attenuate GSH depletion) and preserved synaptosomal viability. Thus, the incorporation of curcumin into the PMMA-AA/CYS and PMMA-AA/DTP particles significantly increased the protective effect against 6-OHDA dopamine induced neurotoxicity in rat brain synaptosomes as measured by synaptosomal viability and increased GSH levels. 4. Conclusions In the present study we have prepared and evaluated the properties of hollow PMMA-AA/CYS and PMMA-AA/DTP microparticles as curcumin delivery systems. Curcumin loading was achieved under mild conditions, registering higher loading capacity for the microparticles consisting of highest content of hydrophobic PMMA units. A sustained release of curcumin in a phosphate buffer (pH 6.8) correlated with the swelling of the microparticles at this pH-value. The particles did not alter the mitochondrial function and showed no cytotoxicity on HepG2 cells. The incubation of rat brain synaptosomes with PMMA-AA/CYS/CUR and PMMA-AA/DTP/CUR prevented the GSH depletion induced by 6-OHDA and preserved their viability. Thus, PMMA-AA/CYS and PMMA-AA/DTP particles are a promising prototype for curcumin delivery applications, especially in the treatment of neurodegenerative disorders, where oxidative stress is involved in general mechanisms of toxicity. Acknowledgements We thank to Prof. Maria Frosini and Prof. Massimo Valoti (Siena University, Italy) for the kindly provided human hepatoma cells (HepG2). Mrs. Teodora Atanassova (Department of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Pharmacy-Sofia) is acknowledged for technical assistance in in vitro cell experiments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2015.03.061. References Addison, T., Cayre, O.J., Biggs, S., Armes, S.P., York, D., 2010. Polymeric microcapsules assembled from a cationic/zwitterionic pair of responsive block copolymer micelles. Langmuir 26, 6281–6286. Barzegar, A.A., Moosavi-Movahedi, A., 2011. Intracellular ROS rotection efficiency and free radical-scavenging activity of curcumin. PLoS One 6, e26012. Beal, M.F., 2001. Experimental models of Parkinson’s disease. Nat. Rev. Neurosci. 2, 325–334. Bird, R., Freemont, T.J., Saunders, B.R., 2011. Hollow polymer particles that are pHresponsive and redox sensitive: two simple steps to triggered particle swelling gelation and disassembly. Chem. Commun. 47, 1443–1445. Bird, R., Freemont, T., Saunders, B.R., 2012. Tuning the properties of pH-responsive and redox sensitive hollow particles and gels using copolymer composition. Soft Matter 8, 1047–1057. Bulpitt, P., Aeschlimann, D., 1999. New strategy for chemical modification of hyaluronic acid: preparation of functionalized derivatives and their use in the formation of novel biocompatible hydrogels. J. Biomed. Mater. Res. A 47, 152–169. Caruso, F., Caruso, R.A., Möhwald, H., 1998. Nanoengineering of inorganic and hybrid hollow spheres by colloidal templating. Science 282, 1111–1114. Cayre, O.J., Biggs, S., 2009. Hollow microspheres with binary porous membranes from solid-stabilised emulsion templates. J. Mater. Chem. 19, 2724–2728.

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Curcumin delivery from poly(acrylic acid-co-methyl methacrylate) hollow microparticles prevents dopamine-induced toxicity in rat brain synaptosomes.

The potential of poly(methyl methacrylate-co-acrylic acid) (PMMA-AA) copolymers to form hollow particles and their further formulation as curcumin del...
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