Liposomal encapsulation improves numerous physiochemical and biological properties of curcumin. The aim of this work was to impart slow release and skin delivery of curcumin via liposomal encapsulation. Liposomes were made using egg yolk phosphatidylcholine as the staple lipid while incorporating polysorbate 80 and stearylamine to prepare hybrid liposomes and positively charged liposomes, respectively. Negatively charged liposomes exhibited the highest encapsulation efficiencies (
Curcumin is a natural compound that exhibits numerous important bioactivities such as antioxidant, antimicrobial, anti-inflammatory, and anticarcinogenic activities [
Liposomal encapsulation has improved numerous important properties of curcumin with potential applications in many different fields. For instance, curcumin encapsulated liposomes have been utilized in developing antimicrobial surfaces for the food industry [
The lipid composition of liposomes is well documented as one of the main factors that determines the properties of liposomes. Examples of properties of liposomes affected by the lipid composition include size, zeta-potential, stability, encapsulation efficiency, release properties, and skin permeation properties [
The present study describes the effect of charge (stearylamine) and surfactants (polysorbate 80) on the properties of curcumin encapsulated liposomes prepared using egg yolk phosphatidylcholine (PC) and cholesterol (CH). The properties of liposomes considered were size, zeta-potential, encapsulation efficiency, loading capacity,
PC (~60%, TLC), CH (purity ≥ 99%), P80, SA (assay 90%), curcumin (assay ≥ 65%, HPLC), ethanol (HPLC grade), methanol (HPLC grade), phosphoric acid (85% wt.% in water), and dialysis membrane (MWCO 12 000) were purchased from Sigma-Aldrich. Chloroform (reagent grade) was obtained from Fisher Scientific. Deionized water filtered through a 0.2
Thin-film hydration method was used to prepare curcumin encapsulated liposomes. The method described by Aditya and coworkers was followed, with modifications [
The amounts of chemicals used in the preparation of curcumin encapsulated liposomes.
Liposomal formulation | PC (mg) | CH (mg) | P80 (mg) | SA (mg) | Curc. (mg) |
---|---|---|---|---|---|
NL (negatively charged liposomes) | 200 | 50 | — | — | 10 |
NHL (negatively charged hybrid liposomes) | 200 | 25 | 25 | — | 10 |
PL (positively charged liposomes) | 200 | 25 | — | 25 | 10 |
PHL (positively charged hybrid liposomes) | 200 | 25 | 25 | 25 | 10 |
PC: egg yolk phosphatidylcholine; CH: cholesterol; P80: polysorbate 80; SA: stearylamine, Curc.: curcumin.
The EE and LC of curcumin encapsulated liposomes were determined using a HPLC method. First, unencapsulated curcumin was removed from liposomes by dialyzing the liposomal solutions against deionized water at 4°C for 3 days. Next, the amount of encapsulated curcumin remaining in the dialysis bag was freeze-dried and, then, determined by HPLC analysis after disrupting the liposomes in ethanol. The formulas used to calculate EE and LC are given in the following:
Particle sizes and zeta-potentials of liposomes were determined using a Malvern Zetasizer Nano ZS (Malvern instruments, UK) fitted with a red laser of 633 nm, using dynamic light scattering technique and laser Doppler electrophoresis technique, respectively. Liposome suspensions were diluted in deionized water and equilibrated at 25°C prior to analysis [
TGA was carried out using a thermogravimetric analyzer (TA Instruments SDTQ600). An alumina crucible was used to hold approximately 5 mg of sample. The sample was heated from room temperature to 800°C at a heating rate of 20°C/min under a high purity nitrogen flow of 100 mL/min [
DSC was performed on a Q200 DSC. A 4–6 mg portion of the sample was placed in crimped but vented aluminum pans and heated at a rate of 10°C/min in the temperature range of −40°C–+80°C. The sample was purged by a stream of dry nitrogen flowing at 50 mL/min [
Release profiles of different curcumin encapsulated liposomal formulations were fitted to six different drug release models: zero order, first-order, Higuchi, Hixon-Crowell, Korsmeyer-Peppas, and Gompertz. The model that exhibited the adjusted
(1) Zero order model is as follows:
(2) First-order model is as follows:
(3) Higuchi model is as follows:
(4) Hixson-Crowell model is as follows:
(5) Korsmeyer-Peppas model is as follows:
(6) Gompertz model is as follows:
HPLC determination of curcumin was carried out using a C18 column using an Agilent LCMS fitted with a 1100/1200 diode array detector, 1100/1200 quaternary pump, and 1100 autosampler. A phosphate buffer of pH 2.2 and acetonitrile were used for gradient elution and the retention time was 11.5 min. Detection was carried out using a diode array detector at 425 nm.
All data are presented as mean ± standard deviation (SD) of three parallel experiments (
The EE and LC values obtained for each type of liposomes are shown in Table
Encapsulation efficiencies (EEs) and loading capacities (LCs) of different liposomal formulations. Values are reported as mean ± SD (
Liposomal formulation | EE (%) | LC (%) |
---|---|---|
NL | 87.8 ± 4.3 |
3.4 ± 0.2 |
NHL | 77.8 ± 5.7 |
3.0 ± 0.2 |
PL | 57.5 ± 1.3 |
2.2 ± 0.1 |
PHL | 54.5 ± 2.2 |
1.9 ± 0.1 |
NL: negatively charged liposomes; NHL: negatively charged hybrid liposomes: PL: positively charged liposomes; PHL: positively charged hybrid liposomes.
The LCs ranged from
Although P80 and SA decrease the encapsulation of curcumin, it is unreasonable to generalize this effect over all surfactants and positively charged species. Surfactants and positively charged species incorporated in the lipid bilayers show great diversity in their structures so that their effects on the arrangement of lipids in the lipid bilayers also vary widely. Thus, it can be concluded that P80 and SA have a detrimental effect on the EE of curcumin in liposomes made of egg yolk PC and CH, and hence conventional liposomes (NL) are superior in terms of EE and LC in this context.
The particle size, polydispersity index, and zeta-potential of each type of liposomes are shown in Table
Diameter, polydispersity index, and zeta-potential of different types of curcumin encapsulated liposomes. Each value represents mean ± SD (
Liposomal formulation | Diameter (nm) | Polydispersity index | Zeta-potential (mV) |
---|---|---|---|
NL | 284.2 ± 6.3 |
0.296 ± 0.046 |
−57.4 ± 3.5 |
NHL | 225.7 ± 5.0 |
0.293 ± 0.020 |
−48.7 ± 1.7 |
PL | 226.8 ± 10.9 |
0.399 ± 0.038 |
+42.0 ± 0.3 |
PHL | 265.3 ± 3.3 |
0.363 ± 0.059 |
+48.2 ± 0.8 |
NL: negatively charged liposomes; NHL: negatively charged hybrid liposomes.
PL: positively charged liposomes; PHL: positively charged hybrid liposomes.
Although the incorporation of P80 had a significant effect on the size, it had no effect on the polydispersity index of liposomes. Thus, this surfactant can be used to prepare homogeneous populations of curcumin encapsulated liposomes. According to the polydispersity indices of the four types of liposomes, NLs and NHLs are more homogenous than PLs and PHLs. These results indicate that the incorporation of SA contributes to the formation of more dispersed populations of liposomes. Thus, if the application demands a narrow size distribution of liposomes, either negatively charged liposomes or other positively charged lipids should be utilized instead of SA.
Although the lipids, PC and CH, and the surfactant P80 bear no net charge, liposomes made using those species showed negative charge (NL was
The four types of curcumin encapsulated liposomes and their constituents were subject to TGA and their main degradation temperatures and the corresponding weight losses are given in Table
The main degradation temperatures and corresponding weight losses of curcumin encapsulated liposomes and their constituents.
Compound/formulation | Degradation temperature (°C) | Corresponding weight loss (%) |
---|---|---|
Phosphatidylcholine | 344.63 | 52.05 |
378.84 | 29.18 | |
Cholesterol | 352.03 | 69.95 |
439.90 | 19.36 | |
Polysorbate 80 | 413.54 | 96.92 |
Stearylamine | 262.57 | 95.88 |
Curcumin | 380.45 | 58.92 |
NL | 368.29 | 77.09 |
NHL | 368.06 | 85.18 |
PL | 353.64 | 75.59 |
PHL | 359.22 | 85.76 |
NL: negatively charged liposomes; NHL: negatively charged hybrid liposomes.
PL: positively charged liposomes; PHL: positively charged hybrid liposomes.
These results clearly indicate that the degradation of liposomes occur around the degradation temperatures of the staple lipid, phosphatidylcholine. Further, the incorporation of SA in the lipid bilayer of curcumin encapsulated liposomes results in a depression of the main degradation temperature by 10–15°C making the vesicles more susceptible to thermal degradation. In addition, although the main degradation temperature is unaffected by incorporating P80 in negatively charged liposomes, an increase by approximately 5°C was observed in positively charged liposomes. Thus, thermal stability of curcumin encapsulated positively charged liposomes may be increased by incorporating P80.
Despite these subtle variations of the main degradation temperatures, both negatively and positively charged liposomes are unlikely to undergo thermal degradation during manufacturing or storing because manufacturing and storing temperatures of liposomes are much lower than the degradation temperatures exhibited by those liposomal formulations.
The melting temperatures (
Melting temperatures (
Liposomal formulation |
|
|
---|---|---|
NL | 14.8 | 9.9 |
NHL | 14.2 | 9.7 |
PL | 40.6 | 21.0 |
PHL | 50.8 | 31.8 |
NL: negatively charged liposomes; NHL: negatively charged hybrid liposomes.
PL: positively charged liposomes; PHL: positively charged hybrid liposomes.
Further, according to our results, as the melting temperatures of freeze-dried NL and NHL are lower than room temperature, the lipid bilayer of negatively charged liposomes may exist mainly in the liquid state at room temperature. However, as the melting temperatures of freeze-dried PL and PHL are higher than room temperature, those liposomes may exist mainly in the solid state at room temperature. Therefore, incorporation of SA into the lipid bilayer may result in the ordering of lipids in the bilayer, thus favoring the solid state. Although the melting temperatures of freeze-dried NL and NHL are quite similar, those of freeze-dried PL and PHL are significantly different. In fact, the melting temperature of freeze-dried PHL is 10°C higher than that of freeze-dried PL. Accordingly, the crystallization temperature of freeze-dried PHL is, also, approximately 10°C higher than that of freeze-dried PL. These features may have a bearing on release properties and interaction of curcumin encapsulated liposomes with the skin.
The ability of liposomes to function as sustained release systems can be improved by incorporating charged lipids into the lipid content during liposome preparation. However, the effect of charge on sustained release properties is dependent on the nature of the encapsulated material [
Since surfactants usually play significant roles in enhancing skin delivery of encapsulated species of liposomes [
The
According to the
The release properties of curcumin encapsulated liposomes appear to correlate with their phase transition temperatures as well as the charge. The fact that the two types of positively charged liposomes (i.e., PL and PHL) exhibited higher melting and crystallizing temperatures than the two types of negatively charged liposomes (i.e., NL and NHL) sheds light on why PL and PHL remain mainly in the more ordered solid state at room temperature while NL and NHL remain in the less ordered liquid state. This greater degree of order in the lipid bilayers of PL and PHL may have contributed to the slower average release exhibited by these types of liposomes [
The slower average release of curcumin may be achieved by incorporating P80 in negatively charged liposomes (Figure
Drug release kinetic studies were carried out to select a model that best describes the release behavior of the four different curcumin encapsulated liposomal formulations. Six different models were tested and their adjusted
Adjusted
Type of liposome | Adjusted |
|||||
---|---|---|---|---|---|---|
Zero order | First-order | Higuchi | Hixson-Crowell | Korsmeyer-Peppas | Gompertz | |
NL | 0.9830 | 0.6830 | 0.8817 | 0.9917 | 0.9672 | 0.9987 |
NHL | 0.9917 | 0.5406 | 0.8218 | 0.9919 | 0.8961 | 0.9908 |
PL | 0.9832 | 0.6703 | 0.7937 | 0.9837 | 0.8796 | 0.9992 |
PHL | 0.9777 | 0.7740 | 0.7372 | 0.9766 | 0.9425 | 0.9998 |
NL: negatively charged liposomes; NHL: negatively charged hybrid liposomes.
PL: positively charged liposomes; PHL: positively charged hybrid liposomes.
Parameters for Gompertz model of release of curcumin encapsulated liposomes.
Formulation |
|
|
|
---|---|---|---|
NL | 26.13 | 0.19 | 7.25 |
NHL | 20.58 | 0.21 | 8.17 |
PL | 10.77 | 0.24 | 8.24 |
PHL | 8.97 | 0.20 | 9.60 |
NL: negatively charged liposomes; NHL: negatively charged hybrid liposomes.
PL: positively charged liposomes; PHL: positively charged hybrid liposomes.
Equation for Gompertz model is as follows:
According to
Percentage skin deposition and amount of deposition of curcumin from different types of liposomes at 25°C. Values are indicated as mean ± SD (
Liposomal formulation | Percentage of skin deposition (%) | Amount deposited ( |
---|---|---|
NL | 7.3 ± 0.6 | 25.7 ± 2.2 |
NHL | 2.9 ± 0.5 | 8.6 ± 1.4 |
PL | 1.9 ± 0.3 | 4.4 ± 0.6 |
PHL | 1.1 ± 0.2 | 2.5 ± 0.5 |
NL: negatively charged liposomes; NHL: negatively charged hybrid liposomes.
PL: positively charged liposomes; PHL: positively charged hybrid liposomes.
In our laboratory, the smaller analogues of curcumin, namely, ferulic acid (FA) and caffeic acid (CA), were found to show skin penetration. CA displayed 41% permeation and 2% deposition during 7 h and FA displayed 8–20% permeation and 2-3% deposition during 7 h depending on the charge of the liposomes [
Although no skin penetration occurred under the experimental conditions of this study, Chen and coworkers reported skin penetration of curcumin from liposomes made from either egg yolk phospholipids, soybean phospholipids, or hydrogenated phospholipids. In fact, they reported a percentage permeation of approximately 5% at 8 h. The difference in permeation results of our study and the study conducted by Chen et al. may be due to the smaller size of liposomes used by them (100 nm) and the use of rat abdominal skin as compared to the full thickness pig ear skin [
Curcumin containing liposomes were monitored by Berginc et al. (2012) for
In general, the negatively charged liposomes exhibited a higher percentage of skin deposition of encapsulated curcumin than the positively charged liposomes. Thus, skin delivery of liposomal curcumin is dependent on the charge of liposomes.
Edge activators and skin-penetration enhancers are substances that improve the skin delivery, including skin deposition, of liposomal bioactive agents, especially when incorporated in liposomes. P80 which is also called Tween 80 has shown to increase skin deposition of many substances [
This study reveals that the charge of liposomes and the presence of surfactants impart a pronounced effect on the properties of curcumin encapsulated liposomes.
Egg yolk PC and CH with or without P80 form negatively charged liposomes. Positively charged liposomes are formed by incorporating SA in liposomes made of egg yolk PC and CH with or without P80.
The EE depends on the lipid composition of curcumin encapsulated liposomes. Negatively charged liposomes were superior in terms of EE and LC to the positively charged liposomes. Moreover, incorporation of P80 resulted in a decrease of these effects.
P80 and SA if incorporated separately result in curcumin encapsulated liposomes of smaller size (approx. 226 nm). Incorporation of P80 results in an increase in the zeta-potential and thus it may be utilized for tuning of zeta-potential of curcumin encapsulated liposomes.
Incorporation of SA influences the liquidity of lipid bilayers of curcumin encapsulated liposomes. This decreased liquidity upon the incorporation of SA may affect properties such as release and skin delivery of liposomal curcumin.
The charge of liposomes has a significant effect on the release properties of liposomal curcumin. Positively charged liposomes show better average slow release properties of curcumin. Incorporation of P80 further decreases the rate of release.
Negatively charged liposomes show better skin deposition of liposomal curcumin than positively charged liposomes. Incorporation of P80 has a detrimental effect on skin delivery of liposomal curcumin.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The National Science Foundation, Sri Lanka, is acknowledged for providing financial assistance through Grant no. NSF/SCH/2013/01. Also, the University of Peradeniya, Sri Lanka, is acknowledged for providing financial assistance through Hilda Obeysekara Research Fellowship (AC/490/2010/2011/02) to KMGKP.