Curcumin loaded nanostructured lipid carriers: In vitro digestion and release studies

Polyhedron 164 (2019) 113–122

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Curcumin loaded nanostructured lipid carriers: In vitro digestion and release studies Elham Sadati Behbahani a, Mehrorang Ghaedi a,⇑, Mohammadreza Abbaspour b, Kobra Rostamizadeh c, Kheibar Dashtian a a b c

Chemistry Department, Yasouj University, Yasouj 75918-74831, Iran Targeted Drug Delivery Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran Zanjan Pharmaceutical Nanotechnology Research Center, Department of Medicinal Chemistry, School of Pharmacy, Zanjan University of Medical Sciences, 45139-56184 Zanjan, Iran

a r t i c l e

i n f o

Article history: Received 10 October 2018 Accepted 6 February 2019 Available online 10 February 2019 Keywords: Nanostructured lipid carrier (NLC) Curcumin Simulated Gastric Medium (SGM) Drug loading In vitro release

a b s t r a c t This study aimed to design NLCs to investigate formulation composition contribution on curcumin-NLC characteristics by CCD, while its stability, digestion and release in SGM was evaluated. Tween 80 and pluronic F167 were employed as surfactants, and SA and CT selected as appropriate lipids. CurcuminNLC were prepared by microemulsion–sonication technique, the lipids ratio (SA/CT), surfactants ratio (tween 80/pluronic F167) and curcumin to lipid ratio were selected as independent variables and their effects on NLC physical characteristics such as PS and DL (%) were studied. The optimized curcumin-NLC formulation was determined relying upon statistical analysis which contained lipids ratio of 63.2/37.6, surfactant ratios of 34/66 and drug/lipid ratio of 33/100 and had particle size of 225.8 ± 2.3 nm and DL (%) of 49.9 ± 0.39%. Particle size and surface morphology of optimized curcumin-NLC formulation were characterized by FE-SEM and PCS. The interactions and the crystallinity of both curcumin and lipids were investigated by FT-IR, DSC and XRD analyses. The findings successfully confirm that 41% of the curcumin released from NLC during 2 h. Afterwards, the curcumin release kinetic studies were applied to find best fitting model. The results revealed acceptable NLC and curcumin stability (
95%) in SGM up to 2 h by in vitro digestion assay. Ó 2019 Elsevier Ltd. All rights reserved.

1. Introduction Curcumin (polyphenol extracted from the herb Curcuma longa L) is mainly applied in traditional Chinese medicine and some food industries, while supply unique biological and pharmacological activity including anti-cancer, anti-inflammatory and anti-oxidant properties [1–3]. Furthermore, it is able to enhance anti-tumor effects of several classic chemotherapeutic drugs such as doxorubicin, cis-platinum and paclitaxel [4,5]. Limited aqueous solubility, lower chemical stability in alkaline medium and rapid metabolism are drawbacks which extensively reduce therapeutic efficiency of

Abbreviations: NLC, nanostructured lipid carriers; CCD, central composite design; SGM, Simulated Gastric Medium; SA, stearic acid; CT, caprylic/capric triglycerides; PS, particle size; DL, drug loading efficiency; FE-SEM, field emission scanning electron microscopy; PCS, particle correlation spectroscopy; FT-IR, Fourier transformed infrared spectroscopy; DSC, differential scanning calorimetry; XRD, Xray diffraction analysis; DF, desirability function; GMS, glycerol monostearate. ⇑ Corresponding author. E-mail address: [email protected] (M. Ghaedi). https://doi.org/10.1016/j.poly.2019.02.002 0277-5387/Ó 2019 Elsevier Ltd. All rights reserved.

curcumin, however its solubility in physiologic medium could be enhanced by the formation of nanoparticulate systems. Nanoparticles are colloidal particles with sizes of approximately 10–1000 nm. These were first developed around 35 years ago, initially as carriers using biodegradable polymers for cancer chemotherapic agents and delivering vaccines [6]. These particles may be divided into nano-carriers and nano-drugs. Nano-carrier systems can generally be divided into two groups such as polymerand lipid-based systems. Lipid nanoparticles is alternative of other colloidal systems such as emulsions, liposomes and polymeric nanoparticles via their advantages combination and successfully lower their major disadvantages [7,8]. Lipid nanoparticles exhibit high drug encapsulation efficiency and high stability without utilization of organic solvents during manufacture which made this approach as inexpensive and easy method to scale-up [9,10]. Moreover, lipid nanoparticles are made of endogenous lipids or lipids similar to those existing in the human body, and accordingly known as biodegradable, biocompatible and nontoxic material [11].

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There are two essential types of lipid nanoparticles including solid lipid nanoparticles (SLN) and nanostructures lipid carriers (NLC), while SLNs only contains solid lipids which supply perfect crystallinity, that lead to lower drug encapsulation efficiency, which emerged from few vacant spaces for compound incorporation [8]. It also leads to drug’s expulsion during long-term storage due to the changes in lipid packaging [12]. NLCs are new generation of lipid nanoparticles and overcome the obstacles of SLN, while NLC (solid and liquid lipids) has lower crystallinity, higher incidence of defects in the matrix and less dense lipid packaging. Afterwards, higher drug encapsulation efficiency and stability achieved during long-term storage in comparison with SLNs [13,14]. Drug release is the procedure by which a drug leaves a drug product and is subjected to absorption, distribution, metabolism and excretion (ADME) and finally supply them for pharmacological action. Drug release is described in various methods including immediate, modified, delayed, extended, controlled and pulsatile release [15]. In vitro dissolution has been identified as major element in drug development, under certain conditions as substitute material for evaluation of bioequivalence. Various theories/kinetics models describe drug dissolution from immediate and modified release dosage forms such as zero-order, first-order, Higuchi and Hixson–Crowell. The nature of drug, its polymorphic form, particle size, solubility, crystallinity and their amount of pharmaceutical dosage form significantly influence the release kinetic [16]. CCD with a minimum number of experiments supply the best optimal composition for achieving the presetting target and simultaneously represent interaction among variables [17–20]. In this paper, curcumin-NLC was prepared to improve drug loading and release of curcumin following optimization variables such as solid/liquid lipid ratios, surfactant composition and drug/ lipid ratios by CCD. Additionally, the optimized curcumin-NLCs were characterized by drug release test, FTIR, DSC and XRD. NLC digestion and curcumin stability in SGM also was evaluated by in vitro assay.

2. Experimental 2.1. Materials and instruments The description of all chemical materials, reagents and apparatuses were presented in more detail in the ‘‘Supplementary Materials” file (Section S-1.1). 2.2. Preparation of curcumin-NLCs Curcumin-NLC and bare NLC were prepared using microemulsion method in the presence of sonication [20]. Briefly, lipids (SA and CT) were heated to 75 °C and surfactants (tween 80 and pluronic F127) were dispersed in the melted lipid until clear. 1 mL of distilled water and non-ionic surfactants were heated to 75 °C and were added to the melted lipid containing curcumin under stirring. The emulsion was subjected to sonication (Elmasonic E60 H) for 5 min and immediately dispersed in cold distilled water (2–4 °C) and following stirring was solidified and passed through a homogenizer (IKA RW16 basic) at 8000 rpm for 5 min. The schematic processes of the preparation of curcumin-NLC is displayed in (Fig. 1). 2.3. Statistical experimental design CCD was applied for process optimization of the variables and their statistical significance and accordingly variables such as stearic acid/caprylic-capric acid ratio (X1), tween 80/pluronic F127 (X2) and curcumin/lipid ratio (X3) at five levels in 18 experiments randomly designed to reduce errors and minimize the uncontrolled effects of the variables (Table S1). The effect of critical factors, interactions and model efficiency was evaluated using analysis of variance (ANOVA) [21,22] to establish the statistical validation of the polynomial equations generated by STATISTICA Software. Optimization was performed by desirability function to obtain the optimal points concerning the predetermined constraints in which the

Fig. 1. Preparation of curcumin-NLC procedure.

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DL and PS are in maximum and minimum levels, respectively [23,24].

The first-order describes the release from system where release rate is concentration dependent (Eq. (3)) [16].

2.4. Determination of drug loading efficiency

log C ¼ log C 0 

DL (%) of nanoparticles was determined by analyzing the concentration of free curcumin in the dispersion medium [25] via subsequently filtration of aqueous dispersion by syringe filter (pore size: 0.22 lm) and non-loaded curcumin content were quantified at 427 nm using UV–Vis spectrophotometer. DL (%) was calculated using Eq. (1):

where, C 0 is the initial concentration of drug and k1 is first order constant. Higuchi described the release of drugs from insoluble matrix as square root of time dependent process based on Fickian diffusion Eq. (4).

 DL ¼


 Wi  Wf  100 Wi

ð1Þ

where W i and W f are the amounts of initial and amount of free curcumin detected in the aqueous supernatant, respectively. 2.5. In vitro lipid digestion assay in SGM The stability of the fabricated NLCs in SGM were determined by NLCs subjecting to simulated digestion at pH 2.0 in the presence of pepsin at 37 °C for 2 h. 2.5.1. Simulated Gastric Digestion SGM which virtually mimics the conditions in the stomach was constituted as described earlier with slight modification [25]. A total of 15 mL of blank NLCs was mixed with 15 mL of SGM (0.32% w/v pepsin, 2 g of NaCl and 7 mL of HCl dissolved in 1 L of water and its pH adjusted to 2 using 1 mol L1 HCl) and incubated at 37 °C with continuous shaking in a water bath for 2 h. Subsequently, samples were taken for measuring size distributions with PCS. 2.6. In vitro stability of curcumin in SGM To investigate the stability of curcumin incorporated in NLCs in SGM without enzymes, curcumin-loaded NLCs were added to mediums and incubated in 37 °C in water bath at 250 strokes/ min for 2 h. After that, curcumin solution was diluted with ethanol (1:10 (v/v)) and centrifuged at 10 000 rpm for 20 min and the curcumin content in supernatant solution was quantified using spectrophotometry based on calibration plotted at the same conditions.

k1 t 2:303

Q ¼ kH t 1=2

ð3Þ

ð4Þ

where, kH is the constant reflecting the design variables of the system. The Hixson–Crowell cube root law describes the release from systems where change in surface area and diameter of particles or tablets (Eq. (5)) [26].

Q 1=3  Q 1=3 ¼ kHC t t 0

ð5Þ

where, Q t is the amount of drug released in time t, Q 0 is the initial amount of the drug in tablet and kHC is the rate constant for Hixson– Crowell rate equation. The following plots were made: cumulative (%) curcumin release versus time (zero-order kinetic model); log cumulative (%) of curcumin remaining vs. time (first-order kinetic model); cumulative (%) curcumin release vs. square root of time (Higuchi model) and cube root of drug (%) remaining in matrix vs. time (Hixson–Crowell cube root law). The linear form of each equation (y = at + b) was fitted to the pure or transformed data (according to each equation) and the squared correlation coefficient (R2) between time and y parameter (related to release data based on each equation) was determined in each case. The R2 was used as a critical parameter to evaluate strength of correlation in models and also to compare each model with others [27].

3. Results and discussion 3.1. Characterization of curcumin-NLC

2.7. In vitro release in SGM The release studies of curcumin from the NLCs were carried out using dialysis membrane bags with 12 000 Da pore size. The membrane bags were soaked in water overnight ahead of use. The bags were filled with 10 mL of dispersion containing 0.4 mg of curcumin in loaded NLC. The bags were immersed in 50 mL of enzyme free SGM in ethanol (50% v/v) and rotated at 50 rpm at 37 °C. The curcumin amount diffused through the dialysis bag was determined as follow: samples (2 ml) were taken at fixed time points and replaced with a same volume of fresh dissolution medium and its curcumin content was measured via spectrophotometry at 427 nm [20]. 2.8. Drug release kinetics In vitro release data was checked and examined upon several kinetic models as follow. Zero-order rate is independent of its concentration (Eq. (2)) [26].

C ¼ k0 t

ð2Þ

where, k0 is zero-order rate constant expressed in units of concentration/time and t is the time.

3.1.1. FT-IR analysis The FT-IR spectra of curcumin, SA and curcumin-NLC are shown (Fig. 1). Presence of functional groups such as hydroxyl (AOH), carbonyl (C@O) and ethylene groups (C@C) are shown as peaks at 3498–3417 cm1, 1627 cm1 and 1512 cm1, respectively (Fig. 2a). Regarding to the curcumin-NLC, the peaks correspond to these functional groups were displayed at 3544–3417 cm1 (becomes wider due to hydrogen bonding), 1619 cm1 and 1511 cm1 (respectively) demonstrate which are characteristic peaks of curcumin sustained in curcumin-NLC [28,29]. Absence of these marker peaks in SA suggests the presence of curcumin inside the NLC. The strong peaks at 2925 cm1 and 2855 cm1 in SA and curcumin-NLC could be due to stretching and deformation of methyl groups, while the strong peak at 1740 cm1 in SA is due to C@O adsorption (Fig. 2b, c) [30–32]. As shown (Fig. 2a, b) the peaks at 968 cm1, 809 cm-1 and 620 cm1 is emerged from bending vibrations of ACH bond of alkene group [33,34] and curcumin-NLC supply peaks in the same region. Both curcumin and curcumin-NLC indicate peak around 1280 cm1 which may correspond to the CAO stretching frequency of ether group in curcumin [35,36] which is in good agreement with and similar to curcumin-NLC and pure curcumin.

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Fig. 2. FT-IR study of curcumin (a), curcumin-NLC (b) and SA (c).

3.1.2. Shape and morphology Shape and surface morphology of the curcumin-NLC was checked by FE-SEM. The photograph (Fig. 3) of optimized formulation demonstrates that curcumin-NLCs in a semi-spherical shape with a smooth surface. 3.1.3. Particle size determination The results of PCS of optimized curcumin-NLCs (Fig. 4) reveals narrow distribution width and considerable narrow particle size (220–231 nm) for NLCs prepared by microemulsion method and confirming the uniformity of dispersion.

3.2. Statistical analysis of experimental data Variables such as lipid and surfactant ratios and drug/lipid ratios affect the size of particles and drug loading efficiency which were optimized by CCD and statistical significance of the factors was checked by STATISTICA software and accordingly the effect of critical factors, interactions and model efficiency were evaluated by ANOVA analysis [21,37]. RSM allows evaluation of relative significance and interaction of all variables based on 18 random experiments (three times replicated) to minimize the effect of uncontrolled variables (Table S1).

Fig. 3. FE-SEM image of curcumin-NLCs with 40.00 KX resolution.

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Fig. 4. PCS images of optimized curcumin-NLC.

The influence of critical factors and the model efficiency was checked by ANOVA according to Fisher’s statistical analysis. Criterion for significant contribution of each variable is p-value less than 0.05 and F-value more than 0.05 which confirm their significant contribution on the model (Table S2) [38,39]. The lack of fit (LOF) (variation of the data around the fitted model) gives information about adequacy of fitting data [40]. As shown in Table S2, a p-value of LOF for PS and DL (%) was 0.41 and 0.06, respectively and indicates high ability of model for well-fitting experimental data. The quality of fitting the polynomial model equations for PS and DL (%) was expressed by the coefficient of determination (R2 = 0.9956 and adjusted R2 = 0.9906) and (R2 = 0.9996 and adjusted R2 = 0.9993), respectively. R2 as measure of the amount of variation around the mean explained by the model and large adjusted R2 values show good relationship between the experimental and data assign to fitted model (Fig. S1) [41–43]. Analysis of results by RSM for plotting PS and DL (%) versus significant variables was investigated and results are shown in Figs. S2 and S3.

3.2.1. Particle size of NLCs Based on the analyses using STATISTICA software and the related modeling, the following equation was obtained which shows the effect of each factor on the PS (Eq. (6)):

PS ¼ 21:0046 þ 61:9X 1 þ 1:15X 2  7:52X 3  0:1X 1 X 2 þ 0:15X 2 X 3  0:35X 21

ð6Þ

PS of NLCs dispersions affects both curcumin release pattern and curcumin absorption [44]. The 3D response plots for PS show positive relation among increment of particle size with fraction of solid lipid which is related to raising viscosity in the dispersion and lead to higher surface tension and larger particle size, while based on Stokes’ law this fact is due to difference in density between the internal and external phases [45]. The size of lipid nanoparticles is highly depends on lipid content and lipid tendency to coalesce at high lipid concentration. Furthermore, increasing PS at higher content of solid lipid may be related to higher collision and aggregation of the nanoparticles [46] or non-sufficiency of

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surfactant for capping nanostructure [46], while higher lipid content lead to raising mean PS of NLCs (Fig. S2a). As indicated in Fig. S2b, X2 shows positive effects on particle size, while the intensity of contribution was relatively low. The negative and reverse trend of amount of drug (X3) on PS is shown in (Fig. S2c). 3.2.2. Drug loading efficiency The drug loading efficiency of different formulations reveals that DL (%) correlated with significant variables according to Eq. (3) based on their p-values which are given in Table S2.

DLð%Þ ¼ 3:051  0:76X 1 þ 0:01X 2 þ 2:5X 3  0:02X 1 X 3  0:01X 21 ð7Þ As shown (Fig. S3a), DL (%) has reverse trend with ratio of solid to liquid lipid and experimental results confirm that drug loading efficiency of NLC were increased from 10.20 to 52.22% by raising CT percentage [47]. Incorporation of liquid lipid to solid lipid generally cause to massive crystal order disturbance and great imperfections in the crystal lattice and supply more space to accommodate drug molecules and improve drug loading efficiency

Fig. 5. DSC thermographs of Curcumin (a), SA (b), NLC (c) and Curcumin-NLC (d).

Fig. 6. XRD pattern of Curcumin-NLC, NLC, SA and Curcumin.

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Fig. 7. Particle size of blank NLCs before incubation in pH = 6.8 (a), after incubation in SGM (b).

3.2.3. Optimization and validation by DF The DF used for optimization of prescriptive criteria for achievement of low PS and maximum DL (%) (Fig. S4). the composition of optimum formulation was determined as: lipids ratio of 63.2/37.6, tween 80 to Pluronic F127 ratio of 34/66 and drug to lipid ratio of 33/100. The predicted values of PS and DL (%) were 227.2 nm and 50.2%, respectively. The observed optimized formulation has parti-

cle size of 227.2 ± 2.3 nm and DL (%) of 50.2 ± 0.39%, and extensively has good agreement with the predicted values.

110 100

Curcumin Stability (%)

[48]. Surfactant ratios play a major role in the solubility of drug in external phase and due to lipophilic nature of curcumin able to entrap in lipid core (Fig. S3b, c). The drug content in delivery system depends on its physicochemical properties and preparation process. A significant positive effect of drug to lipid ratio (X3) on DL (%) (Fig. S3c) confirm high solubility of the drug in the melted lipid which supply more space to accommodate excessive drugs and accordingly a positive trend among DL (%) and curcumin content could be related to higher solubility of curcumin in lipid phase [20].

90 80 70 60 50 0

20

40

60

80

100

120

140

Time (min) Fig. 8. Curcumin stability after incubation in enzyme free SGM in pH 2.0.

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3.3. DSC analysis DSC thermograms of curcumin and SA (Fig. 5a, b) represent that SA and curcumin have sharp melting endotherm at 60 °C and 173.17 °C, which indicate their crystallinity. In case of NLC, the reduction in enthalpy of SA from 127.12 J/g to 112.04 J/g indicates change in the polymorphic state of lipid from crystalline to amorphous with more defects in the crystal lattice which can accommodate space for drug entrapment (Fig. 5c) [49]. Furthermore, in loaded-NLC, SA enthalpy decreased from 112.04 J/g to 64.42 J/g, while for curcumin its enthalpy reduced from 45.28 J/g to

17.53 J/g, which strongly confirms conversion of crystalline curcumin to amorphous form in the NLC formulation due to incorporation of curcumin into melted lipid matrix (Fig. 5d). Presence of small endothermic peak at 173.17 °C in loaded-NLC could be attributed to un-entrapped curcumin present in the aqueous phase. The NLC and curcumin-NLC formulations has single endothermic peaks with lower melting point in comparison to SA and accordingly this fact was emerged from interference of liquid lipid in the crystallization of solid lipid and its nanometric size which supply high specific surface area of the lipid nanoparticles [50]. 3.4. X-ray diffraction analysis

45

Comulative Release %

40 35 30

25 20 15 10 5 Time(min) 0 0

10

20

30

40

50

60

70

80

90

100 110 120

Fig. 9. In vitro release profile of curcumin from NLCs in enzyme free SGM by dialysis membrane method under sink condition (50% ethanol).

Table 1 The R2 values from in vitro release kinetics and the k values or release rate constant. Function

R2

k

Zero-order First-order Higuchi Hixson–Crowell

0.9944 0.9251 0.9951 0.9716

12.956 0.049 69.221 0.792

The XRD pattern of SA and curcumin are composed of sharp peaks at 2h scattered angles 7.01, 11.72, 21.54 and 23.84 which in comparison to sharp peak at 2h scattered angles 9 and 17.4 confirm their crystalline nature [20]. Furthermore, reduction in intensity of SA and curcumin characteristic peaks in NLC and loadedNLC indicates the reduction in crystallinity of curcumin (Fig. 6) which may be related to incorporation of curcumin in lipid matrix of SA and as seen is correlated with DSC analysis. Moreover, diffraction pattern of NLC and loaded-NLC exhibit no significant difference in the peak pattern which represent that curcumin addition did not change the nature of NLC. 3.5. In vitro lipid digestion assay in SGM To evaluate the protection of curcumin from degradation while passing through unfavorable environment in the gastrointestinal tract the suitability and stability of fabricated NLCs were evaluated in SGM [51]. Size measurement was used to measure the aggregation or degradation of NLCs since if particle degradation occurs then particle size decreases in the beginning due to loss of surfactant coating on the surface and later size increases due to aggregation of particles owing to lack of surfactants to protect the

Fig. 10. Zero-order model (a), First-order model (b), Higuchi model (c) and Hixson–Crowell cube-root model release of curcumin-NLCs.

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aggregation [52]. In SGM, NLCs were stable and no aggregation or degradation occurred during 2 h of incubation (Fig. 7). This stability of NLCs in acidic pH and hydrolysis by pepsin may be due to steric stabilization effect of nonionic surfactant tween 80. The nonionic surfactants has resistance to flocculation and coalescence in low pH due to their molecular structure [53]. It has been shown that acid stable GMS is a good candidate for construction of wall material for the fabrication of NLCs which might have also contributed to the stability of NLCs in SGM [54].

121

Appendix A. Supplementary data Materials and instruments, figures of 3D response surface plots, the plot of predicted versus observed values, desirability function and tables of CCD with experimental results, and the analysis of variance (ANOVA) for the model. Supplementary data to this article can be found online at https://doi.org/10.1016/j.poly.2019.02.002.

References 3.6. In vitro stability of curcumin in SGM Both in vitro and in vivo stability is one of the major drawbacks that affects the therapeutic efficiencies of curcumin. To study the stability of curcumin loaded NLCs they were incubated in SGM without enzymes for up to 2 h. Curcumin under these conditions was 100% stable up to 2 h (Fig. 8). These results indicates that our formulation increased the stability of curcumin in SGM by protecting the encapsulated curcumin against hydrolysis and biotransformation [55]. 3.7. In vitro release in SGM In this study, the release of curcumin from NLCs was investigated in vitro in SGM free enzyme for 2 h at 37 °C using a dialysis bag (Fig. 9). Over a period of 2 h approximately 41% of curcumin was released from loaded-NLCs. The release kinetics was evaluated by fitting the data into zeroorder, first-order, Higuchi and Hixon–Crowell models and drug release kinetic correspond to loaded-NLC formulation was depicted in Table 1. In Zero-order plot (Fig. 10a) the R2 value obtained is 0.9944 and first-order (Fig. 10b) gave 0.9251 describing the drug release rate relationship with concentration of drug. The best linearity was found in Higuchi’s equation plot (Fig. 10c) (R2 = 0.9951) indicating the release of drug from matrix depends to the square root of time so the possible mechanisms for the curcumin release might be diffusion of the drug from the matrix and matrix erosion resulting from degradation of lipids [34,56]. Faster drug release from smaller particles could be attributed to the larger surface area of these particles, while the process may be mainly controlled by drug diffusion through the lipid matrix. 4. Conclusion In this study, curcumin-NLC prepared by microemulsion–ultrasonication were optimized, characterized and also assessed for digestion and release in SGM. The formulated curcumin-NLCs were in nanometric range with semi-spherical structure. The results indicated that 41% of the curcumin released from NLC in SGM up to 2 h. The release of curcumin from NLCs was best-fitted Higuchi equation and the possible mechanisms for the drug release might be diffusion of the curcumin from the matrix. Furthermore, the stability of NLCs in SGM is over 95%. The minimum PS of NLCs (227.2 nm) and maximum DL (%) (50.2%) were found at SA/CT ratio: 63.2/37.6, tween 80/pluronic F127 ratio: 34/66 and curcumin amount: 33 mg. The DSC and XRD analyses confirm that curcumin was mostly entrapped into the NLC which is related to its amorphous structure. Acknowledgments The authors acknowledge the instructors of the Graduate School, Research Council of the University of Yasouj and School of Pharmacy at Ahvaz Jundishapur University of Medical Sciences.

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