Hailong
Yu
,
Ji
Li
,
Ke
Shi
and
Qingrong
Huang
*
Department of Food Science, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, New Jersey 08901, USA. E-mail: qhuang@aesop.rutgers.edu; Fax: +732-932-6776; Tel: +732-932-7193
First published on 20th June 2011
The micelle structure of octenyl succinic anhydride modified ε-polylysine (M-EPL), an anti-microbial surfactant prepared from natural peptide ε-polylysine in aqueous solution has been studied using synchrotron small-angle X-ray scattering (SAXS). Our results revealed that M-EPLs formed spherical micelles with individual size of 24–26 Å in aqueous solution which could further aggregate to form a larger dimension with averaged radius of 268–308 Å. Furthermore, M-EPL micelle was able to encapsulate curcuminoids, a group of poorly-soluble bioactive compounds from turmeric with poor oral bioavailability, and improve their water solubility. Three loading methods, including solvent evaporation, dialysis, and high-speed homogenization were compared. The results indicated that the dialysis method generated the highest loading capacity and curcuminoids water solubility. The micelle encapsulation was confirmed as there were no free curcuminoid crystals detected in the differential scanning calorimetry analysis. It was also demonstrated that M-EPL encapsulation stabilized curcuminoids against hydrolysis at pH 7.4 and the encapsulated curcuminoids showed elevated cellular antioxidant activity compared with free curcuminoids. This work suggested that M-EPL could be used as new biopolymer micelles for delivering poorly soluble drugs/phytochemicals and improving their bioactivities.
Many drug candidates and bioactive phytochemicals are water insoluble, which leads to limited bioavailability. Among those compounds, curcuminoids are a group of curcumin-like compounds extracted from turmeric. They have been shown to have anti-inflammatory, anti-cancer, antioxidant and anti-microbial activities.3–6 Unfortunately, its oral bioavailability was greatly limited by its water insolubility.7
Recently, we synthesized a new amphiphilic polymer, modified ε-polylysine (M-EPL), using ε-polylysine (EPL, Fig. 1A) and octenyl succinic anhydride (OSA).8EPL is a biopolymer generated by bacteria Streptomyces albulus,9,10 and is a natural antimicrobial agent.11 After chemical modification, M-EPLs (or called OSA-g-EPLs, Fig. 1B) were amphiphilic and able to lower the surface tension of water and form polymer micelles. Meanwhile, they still retained the same antimicrobial activity of EPL, rendering them bifunctional (surface active and antimicrobial) molecules.8 However, although M-EPLs were shown to form micelles in water, the micelle structure still remains unclear.
Fig. 1 Chemical structures of epsilon polylysine (EPL) (A) and octenyl succinic anhydride modified ε- polylysine (OSA-g-EPL) (B). |
In this study, the micelle structures of M-EPLs were analyzed using synchrotron small-angle X-ray scattering (SAXS). Subsequently, three loading methods were compared to achieve the maximal water solubility of curcuminoids upon encapsulation in the M-EPL-based polymer micelles. Furthermore, the effects of polymer micelle encapsulation on the stability against alkaline hydrolysis and the cellular antioxidant activity of curcuminoids were also investigated.
HepG2 cells were generously provided by Dr Mou-Tuan Huang from Department of Chemical Biology, Rutgers, the State University of New Jersey. Minimum Essential Medium (MEM), Hank's buffered salt solution (HBSS), RPMI-1640 media, fetal bovine serum (FBS), phosphate buffered saline (PBS), 100X penicillin and streptomycin, and 0.25% trypsin with ethylenediaminetetraacetic acid (EDTA), L-glutamine, were all purchased from Thermo Scientific. Insulin, 2′,7′-dichlorfluorescein-diacetate (DCFH-DA), 2,2′-azobis (2-amidinopropane) (ABAP), Williams' Medium E (WME), hydrocortisone were purchased from Sigma-Aldrich.
To quantify the curcuminoids amount encapsulated in the M-EPL micelle, lyophilized curcuminoids in M-EPL micelles were dissolved in dH2O at the concentration of 1 mg mL−1. Two volumes of HPLC-grade acetonitrile was then added in and mixed before quantification using HPLC.
In all the following assays, M-EPL encapsulated curcuminoids were obtained using the dialysis method.
To calculate the CAA value of each treatment, the area-under-the-curve (AUC) for the plot of fluorescence intensity against time was calculated with trapezoidal method. Then the CAA value was calculated as
(1) |
Subsequently, Fa/Fu was plotted against the concentration of curcuminoids on double logarithmic scale, where Fa = CAA and Fu = 100 − CAA. EC50 was determined as the concentration where Fa/Fu = 1. By linear regression of the data on the plot, this value was able to be obtained mathematically. The molecular weight of curcumin was used to calculate the molar concentration of curcuminoids.
Fig. 2 Small-angle X-ray scattering profiles of modified ε-polylysine with different degrees of substitution. The solid line (OSA-g-EPL8.5), long dash line (OSA-g-EPL12.4), and short dash line (OSA-g-EPL20.5) are fitted curves obtained from inversed Fourier transform of pair distribution function (PDF) by Irena package in Igor Pro software. PDF curves were normalized for better comparison. |
Fig. 3 shows the two PDF curves of OSA-g-EPL12.4 solution generated from GNOM and Irena package. It is clear that the data obtained from GNOM and Irena package have negligible difference. Therefore, only one set of dimensional parameters from Irena package were selected herein for display. As shown in Fig. 4, the bell shape of all three PDF curves indicated the spherical shape of the large micelle aggregates. However, the dimension parameters of these three OSA-g-EPLs were not identical. Table 1 lists the dimension parameters of the micelle aggregates, including maximum dimension (Dmax), radius of gyration (Rg), peak 1&2 positions and micelle number within a single aggregate. It was found that Dmax, the largest dimension within size distribution, and Rg, radius of gyration, reached the lowest value for OSA-g-EPL12.4 are 752 Å and 268 Å, respectively. The dimensions of those three OSA-g-EPLs were similar with each other although their PDF curves were not exactly overlapping. There appeared two peaks in all three PDF curves of OSA-g-EPLs. From Table 1, the first peak located at ∼24 Å, was reasonably relevant to the size of individual micelle. The second peak, located at ∼330 Å with a 15–40 Å shift for large aggregates, corresponded to the average size of OSA-g-EPLs micelle aggregate. Therefore, it was thought that the large aggregation was composed of small global micelles tightly connected with each other within the large global micelle aggregate. The aggregation number naggregation of micelles in one aggregate can be calculated through the forward scattering I (Q = 0), which was reasonably used in other amphiphilic structures as well.16 The forward scattering I (Q = 0) was obtained through a Guinier fit of the scattering profile in the low Q range. It was shown that the aggregation number within one micelle aggregate at 5 mg ml−1 increased from 366 for OSA-g-EPL8.5 to 1242 for OSA-g-EPL20.5. It suggested that the increase of substitution degrees caused more micelles to aggregate and form larger particles, which can also be verified by the PDF curves' peak shift towards a large value when the degree of substitution was increased. This result was also consistent with the relatively large particle sizes determined by dynamic light scattering.8
Fig. 3 Comparison of pair distribution functions of OSA-g-EPL with 12.4% degree of substitution (DS) generated from either GNOM (empty circles) or Irena package (solid line). |
Fig. 4 Pair distribution function (PDF) curves of modified ε-polylysine with different degrees of substitution (DS): 8.5% (solid line); 12.4% (long dash line); and 20.5% (short dash line). |
Dmax (Å) | Rg (Å) | Peak 1 (Å) | Peak 2 (Å) | naggregation | |
---|---|---|---|---|---|
OSA-g-EPL8.5 | 778 | 276 | 24 | 326 | 366 |
OSA-g-EPL12.4 | 752 | 268 | 24 | 311 | 529 |
OSA-g-EPL20.5 | 800 | 308 | 26 | 357 | 1242 |
Loading water-insoluble compounds into the hydrophobic core of polymer micelles is usually controlled by kinetics. Different loading methods usually have different efficiencies.17 In this study, three loading methods (solvent evaporation, dialysis and homogenization) were compared to find the best way to achieve the highest loading capacity (and thus the water solubility) of curcuminoids in M-EPL micelles (Fig. 5). In the solvent evaporation method (Fig. 5A), chloroform with dissolved curcuminoids was homogenized to form a coarse emulsion in the M-EPL solution. As chloroform evaporated, a fraction of curcuminoids was trapped in the M-EPL micelles. In the dialysis method (Fig. 5B), M-EPL and curcuminoids were both dissolved in DMSO and dialyzed against dH2O. As the DMSO inside the dialysis bag was replaced by dH2O, curcuminoids were gradually encapsulated in M-EPL micelles. In the homogenization method (Fig. 5C), a simple high-speed homogenization was used. Under high shear, curcuminoids crystals were expected to be broken into smaller size, which, according to Ostwald-Freundlich equation, would have greater water solubility and thus be easier to be encapsulated into micelles. In our previous study, similar method was used to encapsulate curcumin into micelles formed by hydrophobically modified starch.12
Fig. 5 Scheme of three loading methods used to encapsulate curcuminoids into M-EPL micelles: (A) Solvent evaporation – curcuminoids were dissolved in chloroform and M-EPL was dissolved in distilled water (dH2O). Coarse emulsion was generated by high-speed homogenization. Subsequently, chloroform was removed by evaporation; (B) dialysis – curcuminoids and M-EPL were co-dissolved in DMSO and dialyzed again dH2O to remove DMSO; and (C) high-speed homogenization (HSH) – HSH was used to break down the curcuminoids crystals, and the high shear force facilitated curcuminoids dissolution and encapsulation. |
The encapsulated curcuminoids in the three methods were then quantified by HPLC. As clearly showed in Fig. 6, the dialysis method resulted in the highest loading capacity: in dried samples, curcuminoids accounted for 5.3 ± 1.9% (w/w), which was significantly higher than that in homogenization (1.1 ± 0.3%) and solvent evaporation methods (0.8 ± 0.4%). Accordingly, in 1 mg mL−1M-EPL micelle solution, the maximal solubilized curcuminoids concentration was 53 ± 19 μg/mL. Compared with the water solubility of curcuminoids (11 ng mL−1),18 this represented almost 5000-fold increase.
Fig. 6 Mass percentages of curcuminoids in freeze-dried M-EPL samples prepared by three loading methods described in Fig. 5. Data are presented as mean ± standard deviation (n = 3). * denotes statistically significant difference (P < 0.05). |
The percentages of the three curcuminoid compounds encapsulated in the micelles were also compared with that of curcuminoids raw materials (Table 2). It was found that the composition of curcuminoids prepared by dialysis was similar to that of original curcuminoids powder, while the composition of curcuminoids prepared by either solvent evaporation or high-speed homogenization had a slightly lower content of curcumin, suggesting that different loading methods may have weak preference toward selected curcuminoid components.
Curcuminoids compound | Raw materials | Dialysis | High-speed homogenization | Solvent evaporation |
---|---|---|---|---|
a Data are presented as mean ± standard deviation (n = 3, except for raw materials, n = 12). | ||||
Cur (%) | 82.1 ± 1.0 | 82.4 ± 1.3 | 77.7 ± 1.0 | 78.3 ± 5.4 |
D-Cur (%) | 14.8 ± 0.5 | 14.8 ± 0.9 | 15.8 ± 0.4 | 13.9 ± 1.1 |
BD-Cur (%) | 3.1 ± 0.5 | 2.8 ± 0.3 | 6.5 ± 0.6 | 7.8 ± 6.5 |
Additionally, dynamic light scattering was used to examine the particle size of the M-EPL micelles before and after encapsulation of curcuminoids: the hydrodynamic diameter of pure M-EPL micelle was 74.7 ± 1.0 nm with polydispersity of 0.424, while that of M-EPL micelle with curcuminoids was 135.5 ± 1.5 nm with polydispersity as 0.273. The results suggested that the encapsulation of curcuminoids can cause M-EPL micelles to associate and form larger micellar aggregates.
Fig. 7 (A) Differential scanning calorimetry (DSC) results of curcuminoids, curcuminoids/M-EPL simple mixture, and curcuminoids encapsulated in M-EPL micelle prepared by dialysis method. The arrow indicates the melting peaks of curcuminoids in the mixture; and (B) Zoom-in DSC curves of simple mixture of curcuminoids and M-EPL as well as curcuminoids encapsulated in M-EPL micelle through dialysis in the temperature from 160 to 200 °C. |
The DSC results also suggest that compared with other types of formulations, such as solid lipid nanoparticle and liposome, this micelle encapsulation system is ready to be lyophilized and reconstituted.
Fig. 8 Stability of curcuminoids at pH 7.4: (A) The stability of total curcuminoids for the free curcuminoids versus curcuminoids encapsulated in M-EPL micelle through dialysis; and (B) The stability of curcumin, demethoxycurcumin (DCur), and bisdemethoxycurcumin (BDCur) for their free forms versus each component encapsulated in M-EPL micelle through dialysis. Data are shown as mean ± standard deviation (n = 3). |
Since the curcuminoids used in this study contained curcumin, DCur and BDCur, the stability of each individual curcuminoid compound with and without encapsulation was examined simultaneously. As shown in Fig. 8B, different curcuminoid compounds had different hydrolysis rate at pH 7.4. Namely, curcumin underwent fastest hydrolysis, followed by DCur, while BDCur was relatively resistant to the hydrolysis. To the best of our best knowledge, this was the first time that different curcuminoids showed different stability against hydrolysis at weak alkaline conditions. Since BDCur was relatively stable and soluble at pH 7.4, its bioactivity and bioavailability compared with that of curcumin may need further examination.
Fig. 9 Measurement of the cellular antioxidant activity of curcuminoids: (A) Cellular antioxidant activity (CAA) of curcuminoids at different concentrations; and (B) Determination of the EC50 of curcuminoids. Data are shown as mean ± standard deviation (n = 3). |
Fig. 10 Comparison of the CAA values of free curcuminoids and encapsulated curcuminoids. CAA values were determined at the curcuminoids concentration of 2 μM. Data are presented as mean ± standard deviation (n = 4). ** denoted for very significant difference (p < 0.001, t-test). |
Two mechanisms may be used to explain the enhanced cellular antioxidant activity upon encapsulation. First, free curcuminoids were technically dispersed from DMSO solution into the cell media, which may form sub-micron sized particles and have limited solubility. Encapsulated curcuminoids, on the other hand, were originally in the dissolved form in the micelle core and may still largely remain soluble form upon dilution in the treatment media. Therefore, the concentration of dissolved curcuminoids was expected to be greater in curcuminoids micelle solution than in curcuminoids dispersion. The second possible mechanism may be the rapid hydrolysis of curcuminoids at weak basic condition. Treatment media were at pH 7.4, which may cause rapid degradation of curcuminoids, as shown in Fig. 8. On the other hand, encapsulation may stabilize curcuminoids against hydrolysis. Thus, the curcuminoids amount and the cellular antioxidant activity from the micelle encapsulation were expected to be greater. On the other hand, these interpretations did not exclude the possibility that M-EPL had specific interaction with the HepG2 cells which might facilitate the movement of curcuminoids into/onto the cells.
This journal is © The Royal Society of Chemistry 2011 |