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Colloids and Surfaces B: Biointerfaces 101 (2013) 353– 360

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / c o l s u r f b

harmacokinetics of curcumin-loaded PLGA and PLGA–PEG blend nanoparticles fter oral administration in rats

ajeh Maissar Khalil , Thuane Castro Frabel do Nascimento , Diani Meza Casa , Luciana Facco Dalmolin , na Cristina de Mattos, Ivonete Hoss, Marco Aurélio Romano, Rubiana Mara Mainardes ∗

epartment of Pharmacy, Universidade Estadual do Centro-Oeste/UNICENTRO, Rua Simeão Camargo Varela de Sá 03, 85040-080 Guarapuava, PR, Brazil

r t i c l e i n f o

rticle history: eceived 2 March 2012 eceived in revised form 10 June 2012 ccepted 12 June 2012 vailable online 28 June 2012

eywords: urcumin C–MS/MS ioavailability anoparticles

a b s t r a c t

The aim of this study was to assess the potential of nanoparticles to improve the pharmacokinetics of curcumin, with a primary goal of enhancing its bioavailability. Polylactic-co-glycolic acid (PLGA) and PLGA–polyethylene glycol (PEG) (PLGA–PEG) blend nanoparticles containing curcumin were obtained by a single-emulsion solvent-evaporation technique, resulting in particles size smaller than 200 nm. The encapsulation efficiency was over 70% for both formulations. The in vitro release study showed that cur- cumin was released more slowly from the PLGA nanoparticles than from the PLGA–PEG nanoparticles. A LC–MS/MS method was developed and validated to quantify curcumin in rat plasma. The nanoparticles were orally administered at a single dose in rats, and the pharmacokinetic parameters were evaluated and compared with the curcumin aqueous suspension. It was observed that both nanoparticles formu- lations were able to sustain the curcumin delivery over time, but greater efficiency was obtained with the PLGA–PEG nanoparticles, which showed better results in all of the pharmacokinetic parameters ana- lyzed. The PLGA and PLGA–PEG nanoparticles increased the curcumin mean half-life in approximately 4 and 6 h, respectively, and the Cmax of curcumin increased 2.9- and 7.4-fold, respectively. The distribution

and metabolism of curcumin decreased when it was carried by nanoparticles, particularly PLGA–PEG nanoparticles. The bioavailability of curcumin-loaded PLGA–PEG nanoparticles was 3.5-fold greater than the curcumin from PLGA nanoparticles. Compared to the curcumin aqueous suspension, the PLGA and PLGA–PEG nanoparticles increased the curcumin bioavailability by 15.6- and 55.4-fold, respectively. These results suggest that PLGA and, in particular, PLGA–PEG blend nanoparticles are potential carriers for the oral delivery of curcumin.

. Introduction

Curcumin is a polyphenol compound extracted from the oot of Curcuma longa Linn, commonly known as turmeric. hemically, curcumin is 1,7-bis(4-hydroxy-3-methoxyphenyl)- ,6-heptadiene-3,5-dione, commonly called diferuloylmethane. urcumin has been used for centuries in Chinese and Indian edicine to treat a variety of disorders [1]. Several studies have

hown that curcumin presents anti-inflammatory [2,3], antioxidant 4–6] and antimicrobial activities [7], but the most important effect s its potential use against cancer due to its ability to suppresses the roliferation of a wide variety of tumor cells [8–10]. Curcumin is ble to modulate numerous targets including transcription factors,

eceptors, kinases, cytokines, enzymes and growth factors, affect- ng numerous molecular and biochemical cascades [1,11].

∗ Corresponding author. Tel.: +55 42 3629 8160; fax: +55 42 3629 8102. E-mail address: [email protected] (R.M. Mainardes).

927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. ttp://dx.doi.org/10.1016/j.colsurfb.2012.06.024

© 2012 Elsevier B.V. All rights reserved.

The great pharmacological potential of curcumin and its ther- apeutic applications are restricted because the molecule presents some drawbacks, including low aqueous solubility at acidic and physiological pH conditions, rapid hydrolysis in alkaline media and light instability, inherent to its chemical composition. The hydrophobic character of curcumin results in pharmacokinetic restrictions such as low absorption and bioavailability by oral route, extensive metabolism and rapid elimination [12,13]. The main strategies used to overcome the physicochemical limitations of cur- cumin and to increase its bioavailability are based on loading the compound in nanocarriers, such as liposomes [14], cyclodextrins [15], solid lipids [16,17] and polymeric nanoparticles [18–20].

Biodegradable polymeric nanoparticles are extensively used to improve the therapeutic properties of various drugs and bioac- tive compounds. Nanoencapsulation protects the molecules from premature degradation, improves their solubility, and promotes

controlled drug release and drug targeting. Nanoparticles present low risk of toxicity, and the drug efficacy, specificity, tolerabil- ity and the therapeutic index are enhanced with their use. The pharmacokinetic parameters of the drug are modified when it

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54 N.M. Khalil et al. / Colloids and Surfa

s loaded in nanoparticles. Specifically, there are improvements n absorption, bioavailability, and plasma circulation time, with eduction of clearance, consequently increasing the drug’s mean alf-life [21–25]. The physicochemical parameters of nanoparticles uch as particle size, surface modification charge, and hydropho- icity influence the drug’s pharmacokinetics, impacting the drug’s ioavailability and biodistribution in particular. It is well docu- ented that nanoparticles presenting a hydrophobic surface, like

he surface of poly (lactide-co-glycolide) (PLGA) nanoparticles, resent short circulation times because they are rapidly recog- ized by plasmatic opsonin and cleared by cells of the mononuclear hagocytic system (MPS). The process of opsonization is one of he most important biological barriers to nanoparticle-based con- rolled drug delivery. Coating of the surface of nanoparticles with ydrophilic polymers, such as polyethylene glycol (PEG), polysor- ates or poloxamers, sterically stabilizes the particles, i.e., they re able to repel the absorption of opsonin proteins via steric epulsion forces, and thus, the particles become “invisible” to MPS ells, increasing their plasmatic circulation time and resulting in an mprovement in drug bioavailability and half-life. Also, longer plas-

atic circulation times increase the probability of the nanoparticles eaching their target [26–29].

Some recent works have demonstrated that PLGA nanoparticles re able to improve the bioavailability of curcumin after oral admin- stration [18,19,30]. PLGA–PEG nanoparticles have been developed ecause of their great potential for having long circulation times. lso, the potential advantage provided by the hydrophilic charac-

er of PEG can improve the biocompatibility of the delivery system 31]. However, to the best of our knowledge, there has not yet been

demonstrated report about the use of PLGA–PEG nanoparticles s carriers for curcumin. Thus, polymeric nanoparticles, especially ong-circulating nanoparticles, were evaluated as potential carriers or curcumin oral delivery.

In this work, PLGA and PLGA–PEG blend nanoparticles were btained for curcumin loading. An analytical method based on C–MS/MS was developed and validated to quantify curcumin in at plasma. The nanoparticles were orally administered at a single ose in rats, and the pharmacokinetic parameters were evaluated nd compared with those of a curcumin aqueous suspension.

. Materials and methods

.1. Materials

Curcumin (code C1386), PLGA (Resomer RG 50:50 H; Mw 0–75 kDa, inherent viscosity 0.45–0.6 dl/g), PEG (Mw 10 kDa) and olyvinyl alcohol (PVA, 31 kDa, 88% hydrolyzed) were purchased rom Sigma–Aldrich (USA). The internal standard, salbutamol 99%), was obtained from European Pharmacopeia. Methylene hloride and ethyl acetate were purchased from FMaia® (Brazil). nalytical HPLC-grade ethanol, acetonitrile, methanol and acetic cid were purchased from J.T. Baker (USA). All other solvents and hemicals were analytical or HPLC grade.

.2. Preparation of curcumin-loaded PLGA and PLGA–PEG blend anoparticles

The nanoparticles were obtained by the single-emulsion olvent-evaporation technique, as previously described [32]. riefly, curcumin (5 mg) and PLGA (50 mg) were dissolved in a ixture of ethyl acetate (1.5 mL) and methylene chloride (0.5 mL)

ith or without PEG (10 mg) at room temperature. This organic hase was rapidly poured into 10 mL of PVA aqueous solution 0.5%, w/v) and emulsified by sonication for 5 min (35% of 500 W, nique® Ultrasonic Mixing, Brazil), resulting in an oil-in-water

Biointerfaces 101 (2013) 353– 360

(O/W) emulsion. Next, the organic solvent was rapidly eliminated by evaporation under vacuum (20 min) at 37 ◦C. The particles were then recovered by centrifugation (19,975 × g, 30 min, 4 ◦C, Cien- tec CT-15000R centrifuge, Brazil) and washed twice with water to remove the surfactant. The nanoparticles were dispersed in the cry- oprotectant sucrose (5%, w/v), and the resulting nanosuspension was cooled to −18 ◦C and freeze-dried (Terroni®, Brazil).

2.3. Particle size

The mean particle size and polydispersity index were deter- mined by dynamic light scattering (BIC 90 plus – Brookhaven Instruments Corp., USA). The analyses were performed at a scat- tering angle of 90◦ and a temperature of 25 ◦C. For each sample, the mean particle diameter, polydispersity and standard deviation of ten measurements were calculated.

2.4. Drug entrapment efficiency

A Waters 2695 Alliance HPLC system (Milford, MA, USA) was used for curcumin quantitation. The chromatographic analysis was performed in isocratic mode using a reverse phase C18 column (VertiSep GES,Vertical Chromatography Co) with a 5 �m particle size, 4.6 mm internal diameter and 250 mm length. The mobile phase consisted of a mixture of ethanol, acetonitrile and water (80:10:10), pumped at a flow rate of 0.8 mL/min. The sample injec- tion volume was 20 �L, and the fluorescence detector was operated at an excitation wavelength of 365 nm and an emission wavelength of 512 nm.

The amount of curcumin incorporated into the nanoparticles was determined directly after complete dissolution of nanoparti- cles in acetonitrile. The solutions were centrifuged and supernatant was collected. After the appropriate dilutions in ethanol, 20 �L of the sample was injected into the HPLC system, and the drug concen- tration was obtained by comparison with a previously constructed analytical curve. Before injection, all of the solutions were filtered through a PVDF membrane filter (0.22-�m pore size, Millipore). The entrapment efficiency (%) was estimated by comparing the amount of curcumin extracted from nanoparticles with the initial amount used for the nanoparticles preparation.

2.5. In vitro release profile

The release of curcumin from nanoparticles was conducted by suspending the nanoparticles (containing 1.5 mg of curcumin) in 12 mL of phosphate saline buffer (PBS, 0.01 M, pH 7.4), and the sus- pension was divided in eight Eppendorf tubes. The experiments were performed in triplicate and under sink conditions. The tubes were kept in a shaker at 37 ◦C at 150 rpm. At predetermined time intervals, the suspension was centrifuged at 19,975 × g for 15 min to separate the released curcumin from the nanoparticles [33,34]. The resulting precipitate in each tube was dispersed in 1.5 mL of phosphate saline buffer and incubated until the next sampling. The released curcumin present in the supernatant (1.5 mL in each tube) was diluted in ethanol, and 20 �L of this solution was injected into the HPLC to determine the amount of curcumin released at different time intervals.

2.6. Chromatograph system and conditions for curcumin quantitation in plasma

The LC–MS/MS analysis was conducted in positive ion ESI

mode on a Quattro Micro API–Waters hexapole mass spectrom- eter connected to a liquid chromatograph (Waters Alliance). The analysis was conducted on a Phenomenex Luna C18(2) 100A col- umn (250 mm × 4.6 mm, 5 �m). The mobile phase consisted of

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N.M. Khalil et al. / Colloids and Surfa

ethanol and 0.05% acetic acid solution (80:20, v/v) at a flow rate f 1.0 mL/min. The sheath gas and auxiliary gas were tuned to give n optimum response as necessary. The needle voltage was 4.5 kV. albutamol was used as internal standard (IS) [35]. Argon was used s the collision gas at collision energy of 15 eV (curcumin) and 18 eV salbutamol). The collision energy was individually tuned for each nalyte to obtain an optimum value. The analytes were quantified sing selected ion reaction monitoring (SRM). The ion transitions /z 369.3→285.0 and m/z 240.0→147.7 were used for the determi- ation of curcumin and salbutamol, respectively. The autosampler ooler was maintained at 4 ◦C.

.7. Preparation of curcumin standards and quality control

A concentrated stock standard of curcumin and salbutamol (IS) ere prepared by dissolving 4 mg of each compound in 20 mL

f methanol, generating a 200 �g/mL stock solution. Eight point alibration curves were prepared by serial dilution of the cur- umin stock solution (200 �g/mL in methanol) in the range of .5–500 ng/mL. The calibration curve was prepared daily using .45 mL of blank plasma with 50 �L of the appropriate working olution, resulting in concentrations of 0.5, 10, 25, 50, 100, 200, 350 nd 500 ng/mL. Three quality controls (QC) were prepared at 1.5 low concentration), 225 (medium concentration) and 450 ng/mL high concentration).

.8. Plasma sample preparation

To 100 �L of rat plasma sample (or a calibration standard or a QC ample) were added 100 �L of salbutamol (IS) and 100 �L of 0.5 M odium hydroxide (to assist in the extraction of curcumin). The mix- ure was vortexed for 1 min. After the curcumin was extracted with 300 �L of ethyl acetate (liquid–liquid extraction), followed by agi- ation in a shaker (10 min), it was centrifuged at 10,000 rpm at 4 ◦C or 10 min. The supernatant was evaporated using nitrogen gas in a ample concentrator. The obtained residue was reconstituted with he mobile phase and vortexed for 20 s. The samples were subjected o LC–MS/MS analysis.

.9. Bioanalytical method validation

The specificity of the method was investigated by comparing the hromatogram of blank plasma with the blank plasma spiked with tandard solutions and with the samples collected from rats after urcumin administration.

The linearity of the bioanalytical assay was evaluated with a otal of eight calibration standards over the concentration range of .5–500 ng/mL. Calibration curves were constructed by linear least- quares regression analysis by plotting the peak-area ratios versus he drug concentrations.

The limit of quantitation (LOQ) was defined as the lowest con- entration of the analyte in the calibration curve that could be etected with a variation of less than 15%.The intra-day preci- ion and accuracy were determined within one day by analyzing en replicates of the QC samples at concentrations of 1.5, 225 and 50 ng/mL of curcumin. The inter-day precision and accuracy were etermined on two separate occasions using replicates (n = 10) of ach concentration used. The intra- and inter-day precision was efined as the relative standard deviation (R.S.D.). The accuracy was xpressed using the following equation (1):

measured concentration ]

× 100 (1)

nominal spiked concentration

The freeze–thaw stability of the plasma samples was evaluated y exposing QC samples at low and high concentrations to four reeze–thaw (−20 ◦C to room temperature) cycles before sample

Biointerfaces 101 (2013) 353– 360 355

preparation. The stability of the samples in the autosampler was evaluated by analyzing the extracted QC samples after being placed in the autosampler at 20 ◦C for 6 h, at which time the samples were analyzed. The long-term stability was verified by freezing (−20 ◦C) the QC samples for 250 days. Freshly processed standard samples were used to quantitate all of the QC samples. The analyses were performed in quintuplicate.

2.10. Pharmacokinetic study

Male adult Wistar rats with a mean body weight of 200–300 g were fasted overnight prior to the experiments, with free access to water. The experimental protocol was approved by the Institutional Animal Ethics Committee of the Universidade Estadual de Ponta Grossa, Brazil (Registration no. 06/2010). The rats were divided randomly into three groups (n = 5). The formulations (curcumin aqueous suspension, dispersion of the curcumin-loaded PLGA nanoparticles and dispersion of the curcumin-loaded PLGA–PEG blend nanoparticles) were administered by oral gavage at a single dose of 50 mg/kg. The nanoparticles were dispersed in ultrapure water.

Blood samples (500 �L) were withdrawn from the tail vein into heparinized microtubes at the following times: 0.25, 0.5, 1, 1.5, 2, 4, 8, 12 and 24 h after dosing. The blood samples were centrifuged at 3020 × g for 10 min. The supernatant was collected, transferred to tightly sealed plastic tubes and stored at −20 ◦C until analysis by LC–MS/MS. After each sampling the same volume removed was replaced with saline solution.

2.11. Data analysis and statistics

All of the in vitro results were expressed as the mean ± standard deviation (S.D.) of three replicates. The in vivo results were presented as the mean ± S.D. of five replicates. Pharmacokinetic parameters were estimated using the model-independent method. The terminal elimination rate constant (Ke) was estimated by a linear regression analysis of the terminal portion of the log- linear blood concentration–time profile of curcumin. The terminal elimination half-life (t1/2) was calculated from Ke using the for- mula t1/2 = 0.693/Ke. The maximum observed plasma concentration (Cmax) and the time taken to reach it (Tmax) were obtained from the curve plotting curcumin concentration vs. time. The area under each drug concentration time curve (AUC, ng/mL h) to the last data point was calculated by the linear trapezoidal rule and extrapolated to time infinity by the addition of CLast/Ke, where CLast is the con- centration of the last measured plasma sample. The apparent body clearance (Cl) was calculated using the equation Cl = dose/AUC. The apparent volume of distribution (Vd) was calculated by the equation Vd = dose/Ke AUC. Statistical analysis of the data was performed via one-way analysis of variance (ANOVA). The results were considered statistically significant if p < 0.05.

3. Results and discussion

3.1. Preparation of the curcumin-loaded PLGA and PLGA–PEG blend nanoparticles

The nanoparticles containing curcumin were successfully obtained by the single-emulsion solvent-evaporation method. The choice of a nanoencapsulation method is based on the drug solubil- ity, and because curcumin is hydrophobic, the method of reducing the size of the emulsion oil-in-water (O/W) is adequate for this

molecule. The ultrasonication was crucial to reduce the emulsion globules to nanometer size. Table 1 illustrates the size characteris- tics of the obtained nanoparticles. Both formulations, the PLGA and PLGA–PEG blend nanoparticles containing curcumin, presented

356 N.M. Khalil et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 353– 360

Table 1 Curcumin nanoparticle characteristics.

Polymer Particle size (nm)a Polydispersity indexa Size distributiona Encapsulation efficiency (%)b

PLGA 161.93 ± 6.7 0.042 ± 0.01 146.2–200.7 nm (100%) 77.07 ± 8.16 PLGA–PEG 152.37 ± 4.5 0.077 ± 0.01 109.9–185.1 nm (100%) 73.22 ± 9.77

Values reported as mean ± S.D.

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onodisperse profiles and narrow size distributions. The presence f PEG did not influence the mean particle size, but the polydis- ersity index was superior (p < 0.05) than that obtained from PLGA anoparticles, while maintaining a monomodal profile.

The encapsulation efficiency was determined directly, and the esults are presented in Table 1. The method used for nanoencapsu- ation resulted in significant enclosure of curcumin, and the process

as found to be highly reproducible. The PEG did not influence the ncapsulation, as the values between batches were not significantly ifferent (p > 0.05). Indeed, the hydrophilic character of PEG kept it irected to aqueous phase, while the hydrophobic core of PLGA can ntrap the hydrophobic drugs.

.2. In vitro curcumin release profile

The in vitro release of curcumin from nanoparticles was evalu- ted simulating physiological conditions (37 ◦C, PBS buffer pH 7.4). he in vitro release profiles of curcumin were obtained by graph- ng the cumulative percentage of the drug released with respect o the amount of curcumin encapsulated as a function of the time. he experiment was performed over nine days. Fig. 1 illustrates he curcumin release profiles from the PLGA and PLGA–PEG blend anoparticles and indicates that there was a pronounced time pro-

ongation of the drug release. It is evident that the PEG influenced the curcumin release

ecause there was a great difference between the release profiles f curcumin from nanoparticles of different compositions. A bipha- ic release pattern of curcumin was observed from the PLGA–PEG anoparticles, where the initial 24 h period released approximately 1% of drug, followed by a sustained release to a total of 56.9% over

ine days of observation. This initial burst release may be due to rug desorption from the particle surface, and the sustained release an be characterized by the drug diffusion through the polymeric atrix and subsequent diffusion/erosion of the polymeric matrix.

ig. 1. In vitro release profile of curcumin from PLGA and PLGA–PEG blend nanopar- icles in PBS (0.01 M, pH 7.4) at 37 ◦ C. Values reported as the mean ± S.D. (n = 3).

Curcumin release from the PLGA nanoparticles was slower than from the PLGA–PEG blend nanoparticles (p < 0.05), and the release was progressive because it did not have a biphasic profile. After 24 h, only 5.8% of the drug had been released, and in nine days, 37% of the curcumin had been released.

In general, it can be affirmed that the drug release depends upon the solubility, diffusion and biodegradation of the matrix mate- rials. Thus, the drug release mechanisms can be modified by the choice of polymer matrices. Drug release also depends upon the loading efficiency of the drug and the size of the nanoparticles [24]. In our case, because the particle size and curcumin loading are sim- ilar for the PLGA and PLGA–PEG nanoparticles, we can attest that the difference between the amount of drug released from the two nanoparticles is due to the presence of PEG, as it has a hydrophilic character and can enhance the water permeation and drug diffu- sion through the polymeric matrix [36]. It is possible that curcumin strongly interacts with the PLGA matrix, thus retarding the release capability, and that the PEG can increase the wettability of the polymeric surface and matrix, contributing to the increase in drug release. The results show that the PLA-PEG nanoparticles released more curcumin than the PLGA nanoparticles (by approximately 1.5-fold; p < 0.05) during the period analyzed.

3.3. Bioanalytical method development and validation

A LC–MS/MS method for the determination and quantitation of curcumin in rat plasma has been developed and validated. Initial runs were conducted with mobile phases composed of acetoni- trile:0.2% formic acid solution (40:60, v/v), acetonitrile:1% formic acid solution (70:30, v/v), acetonitrile:0.1% acetic acid solution (70:30, v/v) and acetonitrile:0.005% acetic acid solution (70:30, v/v). In all of these combinations, the curcumin peak resulted in tailing, and the signal was slow.

Testing several ratios of methanol and acetic acid, the combina- tion that resulted in a sharp peak with a sufficient response area was using methanol and 0.05% acetic acid solution (80:20, v/v) as the mobile phase at a flow rate of 1 mL/min. The retention times were approximately 1.08 min and 0.82 min for curcumin and IS, respectively, and the total run time was 2 min.

The specificity of the method was evaluated by comparing the chromatograms of curcumin in plasma (standard and sample) and those of potentially interfering plasma components. Representa- tive chromatograms are shown in Fig. 2, including a blank plasma sample (Fig. 2A), plasma containing curcumin and salbutamol stan- dard (Fig. 2B) and a plasma sample obtained 30 min after the oral administration of 50 mg/kg of curcumin-loaded PLGA nanoparticles (Fig. 2C). The resulting chromatograms show the assay specificity, as there were no endogenous plasma components eluted at the retention time of curcumin or IS.

The method was validated over a wide concentration range, and

the results were directly obtained and extrapolated on the calibra- tion curve. The calibration lines were shown to be linear from 0.5 to 500 ng/mL (r2 = 0.9941). The method was sensitive, and the LOQ was low (0.5 ng/mL). Other LC–MS/MS methods described in the

N.M. Khalil et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 353– 360 357

F n stan c

l 1

a r

ig. 2. Representative chromatograms of (A) a blank plasma sample, (B) a curcumi urcumin, while that the lower are indicative of the peak of the internal standard.

iterature for curcumin determination in plasma showed a LOQ of

0 ng/mL [16] and 2.5 ng/mL [37].

Table 2 shows a summary of intra- and inter-day precision and ccuracy for curcumin detection in rat plasma. The intra-day accu- acy of curcumin for rat plasma samples was 101.72–110.54% for

dard, and (C) a curcumin sample. The upper peak of the figure is representative of

QC samples with a R.S.D. of less than 6.70%. The inter-day accu-

racy of curcumin for rat plasma samples ranged from 96.34% to 107.94% for QC samples with an R.S.D. of less than 4.34%. These results were within the limits established by the FDA guidelines for the validation of bioanalytical methods [38].

358 N.M. Khalil et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 353– 360

Table 2 Intra-day and inter-day precision and accuracy of curcumin in rat plasma (n = 10).

Nominal concentration (ng/mL)

Measured concentration (ng/mL)

R.S.D. (%) Accuracy (%)

Intra-daya

1.5 1.66 ± 0.04 2.33 110.54 225 224.66 ± 8.00 3.56 99.85 450 457.76 ± 30.66 6.70 101.72 Inter-dayb

1.5 1.62 ± 0.05 3.39 107.94 225 216.77 ± 9.41 4.34 96.34 450 436.59 ± 18.74 4.29 97.02 a

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The analyses were performed in the same day; R.S.D. = relative standard devia- ion.

b The analyses were performed in two different days within one month.

Table 3 lists the data from the stability tests. No significant loss f curcumin (≤1.4%) was observed after storage of the plasma at oom temperature on the bench top for at least 6 h. The plasma amples were stable over at least four freeze/thaw cycles and were table at −20 ◦C for at least 250 days, with no significant loss of urcumin (≤1.8%). These results suggested that the plasma sam- les could be stored at −20 ◦C for long periods, could be thawed nd refrozen and could be maintained at room temperature for 6 h ithout compromising the integrity and accuracy of the samples.

The sensitivity of this LC–MS/MS method offered advantages ver other LC–MS/MS methods and conventional HPLC–UV meth- ds applied for curcumin pharmacokinetics. Also, the excellent pecificity and short run time analysis make this method efficient or curcumin pharmacokinetic applications.

.4. Pharmacokinetics study

The mean curcumin plasma concentration–time profiles after ral administrations of 50 mg/kg of curcumin in different for- ulations, curcumin aqueous suspension, curcumin-loaded PLGA

anoparticles and curcumin-loaded PLGA–PEG blend nanoparti- les, are expressed in Fig. 3. The Table 4 summarizes the relevant harmacokinetic parameters.

After the oral administration of a curcumin aqueous suspen- ion, the drug was absorbed quickly, and a maximum plasma oncentration (Cmax) of approximately 4.066 ± 0.564 ng/mL was eached in 30 min. Thereafter, the curcumin plasma concentra- ion decreased abruptly, as the drug was distributed and rapidly

etabolized, resulting in a high Ke and short t1/2, approximately .1 h. The curcumin was detected up to 8 h after administration.

sustained release of curcumin over 24 h was observed when it as carried by the two nanoparticles formulations. Thirty minutes

fter oral administration of the curcumin-loaded PLGA nanoparti-

les, the mean plasma concentration was 4.57 ± 0.35 ng/mL, and or the curcumin-loaded PLGA–PEG nanoparticles, the plasma oncentration was 9.1 ± 0.95 ng/mL. There was a significant ncrease (p < 0.01) in curcumin absorption from the PLGA–PEG

able 3 tability of curcumin in rat plasma.

Sample condition Curcumin nominal concentration

1.5 ng/mL

Concentration measured (ng/mL)a

R.S.D. (%)b Acc

6 h at room temperature 1.62 ± 0.1 1.42 108 Freeze–thaw four cycles 1.63 ± 0.04 0.7 109 250 days at −20 ◦ C 1.6 ± 0.05 1.1 107 a Values reported as mean ± S.D. (n = 5). b Relative standard deviation, calculated comparing with CQ freshly prepared.

Fig. 3. Comparison of in vivo plasma concentration vs. time profiles of the different curcumin formulations. All values reported are the mean ± S.D. (n = 5).

nanoparticles in the first 30 min compared to free curcumin and curcumin from the PLGA nanoparticles. The curcumin concentra- tion increased to 11.783 ± 0.454 ng/mL, Cmax, after 2 h (Tmax), and to 29.778 ± 4.632 ng/mL, Cmax, after 3 h (Tmax), with the PLGA and PLGA–PEG nanoparticles, respectively. Compared to free curcumin, the Cmax of curcumin from PLGA nanoparticles and PLGA–PEG nanoparticles was increased 2.9- and 7.4-fold, respectively. The increase in Cmax indicates that the nanoparticles were effective in increasing drug absorption, and the delayed Tmax demon- strates an obvious sustained release of curcumin. The distribution and metabolism of curcumin were decreased when it was car- ried by nanoparticles (p < 0.01). The clearance of curcumin from the PLGA and PLGA–PEG nanoparticles was 16.3- and 61.6-fold lower than that of free curcumin, respectively. The PLGA–PEG nanoparticles and PLGA nanoparticles decreased the curcumin volume of distribution by 11.6- and 4.6-fold compared to free curcumin. Thus, the t1/2 of curcumin from the PLGA increased to 4 h, and that from the PLGA–PEG nanoparticles was increased to 6 h, while for free curcumin the t1/2 was 1 h. There was a significant difference in the AUC0–inf between the curcumin aqueous suspension, the curcumin–PLGA nanoparticles and the curcumin–PLGA–PEG nanoparticles (p < 0.01). Between the two nanoparticle formulations, the curcumin from PLGA–PEG pre- sented a relative bioavailability 3.5-fold superior to that of the curcumin from PLGA nanoparticles. Compared to the curcumin aqueous suspension, the PLGA and PLGA–PEG blend nanoparticles increased the curcumin bioavailability 15.6- and 55.4-fold, respec- tively.

Recently, Shaikh et al. [19] demonstrated that the PLGA nanoparticles were able to increase the curcumin bioavailability at least 9-fold when compared to curcumin administered with an absorption enhancer. Tsai et al. [20] developed curcumin-loaded

450 ng/mL

uracy (%) Concentration measured (ng/mL)a

R.S.D. (%)b Accuracy (%)

.31 474.50 ± 13.36 0.47 105.44

.00 481.07 ± 18.52 0.56 106.9

.25 483.99 ± 5.6 1.8 107.55

N.M. Khalil et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 353– 360 359

Table 4 Pharmacokinetic parameters of curcumin following single oral administration of curcumin aqueous solution, curcumin-loaded PLGA nanoparticles and curcumin-loaded PLGA–PEG nanoparticles, in rats (n = 5).

Pharmacokinetic parameters Formulations

Curcumin aqueous suspension Curcumin PLGA nanoparticles Curcumin PLGA–PEG nanoparticles

Dose (mg/kg) 50 50 50 AUC0–t (h ng/mL) 8.695 ± 1.872 134.251 ± 3.446* , # 447.80 ± 64.028* AUC0–inf (h ng/mL) 8.762 ± 1.862 137.162 ± 3.694* , # 485.941 ± 54.663* Cmax (ng/mL) 4.066 ± 0.564 11.783 ± 0.454* , # 29.778 ± 4.632* Tmax (h) 0.5 2* , # 3*

Ke (1/h) 0.631 ± 0.072 0.178 ± 0.021* , # 0.119 ± 0.021* t1/2 (h) 1.109 ± 0.124 3.929 ± 0.451* , # 5.979 ± 1.126* Vd (L/kg) 9432.536 ± 2511.617 2073.664 ± 352.612* , # 900.544 ± 225.772* Cl (L/h/kg) 5859.700 ± 1399.927 365.191 ± 37.351* , # 103.679 ± 10.903*

Values reported as mean ± S.D. (n = 5). AUC: area under the plasma concentration–time curve; Cmax : peak concentration; Tmax : time to reach peak concentration; Ke : constant of elimination; t1/2 : mean half-life; Vd : apparent volume of distribution; Cl: clearance.

P a m u a c h [ e r s P

p c P p l p P b c u d f e t t n t [ p T r C t d t t i

n n r w m t

[

[

* Significantly different of free curcumin (p < 0.01). # Significantly different of curcumin from PLGA–PEG nanoparticles (p < 0.01).

LGA nanoparticles. When these particles were intravenously dministrated in rats, a significant amount of curcumin was found ainly in the spleen due to phagocytic cell uptake in the retic-

loendothelial system. Xie et al. [30] showed that after the oral dministration of curcumin-loaded PLGA nanoparticles, the cur- umin had a 5.6-fold higher relative bioavailability and had a longer alf-life than that of native curcumin. In a similar work, Anand et al. 18] demonstrated that curcumin-loaded PLGA nanoparticles have nhanced cellular uptake, increased bioactivity in vitro and supe- ior bioavailability in vivo relative to free curcumin. To date, no tudy has compared the pharmacokinetics of curcumin loaded in LGA and PLGA–PEG blend nanoparticles.

In our study, the significant difference in pharmacokinetic arameters, mainly bioavailability and half-life, between the free urcumin aqueous suspension and the curcumin-loaded PLGA and LGA–PEG nanoparticle dispersions is explained by the inherent roperties of colloidal nanoparticles in biological media, which pro-

ong drug release and its in vivo trajectory. The in vitro release rofile demonstrated that curcumin is released more rapidly from LGA–PEG nanoparticles than from PLGA nanoparticles and could e more quickly available in blood. It is well supported that pharma- okinetic parameters are altered depending upon the nanoparticles sed, and their surface composition plays an important role in rug bioavailability [39,40]. PEG is frequently used for the sur- ace modification of various polymeric nanoparticles because it xhibits excellent biocompatibility and is able to improve the long- erm systemic circulation of the nanoparticles. The PEG coating on he surface of the polymer reduces the interactions between the anoparticles and the enzymes of the digestive fluids and increases he uptake of the drug in the blood stream and lymphatic tissue 41]. This effect can explain the difference between the curcumin harmacokinetics from PLGA and PLGA–PEG blend nanoparticles. he ability of the PEG to make the coated nanoparticles invisible to ecognition by MPS cells gives the particles long circulation time. onsequently, the drug half-life and bioavailability are higher than hose of a drug carried in uncoated nanoparticles [27]. We recently emonstrated that the presence of PEG in PLA nanoparticles con- aining the antiretroviral zidovudine was essential in promoting he increase in drug bioavailability after intranasal administration n rats [42].

The increased curcumin bioavailability obtained with the anoparticulate systems confirms the excellent abilities of the anoparticles to modulate the physicochemical properties of drugs,

esulting in improved pharmacokinetics profiles. Because the poor ater solubility and low oral bioavailability of curcumin are the ajor drawbacks in its medicinal application, the studied nanopar-

icles represent an important initial step in the development of

[

[ [

a medicine containing curcumin, using the nanotechnology as a tool.

4. Conclusions

In this study, nanoparticles coated or not with PEG were suc- cessfully prepared by the emulsion solvent-evaporation method. Also, an analytical method for determining curcumin in plasma was optimized. We demonstrated that all curcumin pharmacokinetic parameters were improved by nanoparticles, especially PLGA–PEG nanoparticles. The curcumin Cmax, Tmax, t1/2 and AUC were signifi- cantly increased by nanoparticles, while distribution and clearance were decreased. The PLGA–PEG nanoparticles were able to increase the curcumin bioavailability in 3.5-fold compared to curcumin from PLGA nanoparticles. Compared to curcumin aqueous suspen- sion, PLGA and PLGA–PEG nanoparticles increased the curcumin bioavailability in 15.6 and 55.4-fold, respectively. These results demonstrate the great potential of PLGA and mainly PLGA–PEG blend nanoparticles as carriers for the oral delivery of curcumin.

Acknowledgments

This study was supported by Conselho Nacional de Desenvolvi- mento Científico Tecnológico (CNPq) (577183/2008-7), Fundaç ão Araucária (462/2010) and FINEP (01.08.0211.00).

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