Release of siRNA from Liposomes Induced by Curcumin

1Department of Food and Nutrition, Faculty of Human Life Science, Senri Kinran University, Fujishirodai, Suita City, Osaka, Japan 2R&D Division, Katayama Chemical Industries, Co., Ltd., Ina, Minoh City, Osaka, Japan 3Department of Bioengineering Sciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya City, Aichi, Japan 4Department of Biomolecular Science and Reaction, The Institute of Scientific and Industrial Research (Sanken), Osaka University, Ibaraki City, Osaka, Japan 5Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama City, Okayama, Japan


Introduction
Liposomes are artificial vesicles with lipid bilayers formed spontaneously through self-organization by phospholipids dispersed in an aqueous media.They are spherical particles at nanometer or micrometer levels in diameter.As the membrane structure of liposomes mimics cell membranes, liposomes are believed to be excellent in respect of biocompatibility and biodegradability.Liposomes can retain a variety of substances, so they are presumed to be a potential candidate as a carrier in drug delivery systems (DDS) applicable for in vivo administration [1].In fact, liposomes containing anticancer drugs have been prepared for the purpose of resolving problems in conventional chemotherapy including cytotoxicity against normal cells and difficulties in achieving specific delivery to cancer cells [2,3].
Small interfering RNA (siRNA) consists of 21-to-23-basepair (bp) double strand RNA and functions to degrade mRNA through paring between siRNA and mRNA in an RNA sequence-specific manner.siRNA is thus expectedly applicable for the treatment of various diseases including cancer through the suppression of a targeted gene expression, so-called gene silencing [4].However, one of the major problems in the application of siRNA for in vivo use is its instability.Naked siRNA is rapidly degraded by, for example, RNAse in body fluids.In addition, negatively charged polynucleotides cannot pass through negatively charged cell membranes because of electrostatic repulsion.To overcome these problems, liposomes are expected as a proper carrier for siRNA.The lipid bilayer of liposomes can protect siRNA from RNAse or other decomposers during in vivo delivery.Furthermore, since liposomes can generally be incorporated into cells by mechanisms including endocytosis, they are thought to be suitable siRNA carriers [5].
After in vivo administration of liposomes, they are thought to be predominantly trapped by the reticuloendothelial system (RES) [2].To achieve a specific delivery to target cells in vivo without trapping by RES, various modifications have been made on liposomes.Although the fate of liposomes in cells after incorporation by endocytosis has not always been clarified, a current presumed pathway is as follows: endosomes incorporating liposomes are subsequently fused to lysosomes; liposomes are then degraded by lysosomal enzymes including phospholipases [6].In the case of liposomes containing siRNA, it is considered to be important to induce the release of siRNA from the liposomes properly after the liposomes are incorporated into cells.
In the case of small molecular weight compounds encapsulated in liposomes, controlled release has been realized by various techniques; for example, the lipid membrane of liposomes at around its gel-to-liquid crystalline phase transition temperature evidently increases its permeability and this causes the release of small molecules from the liposomes [7][8][9].As for siRNA, some techniques have been developed to achieve the release of siRNA in the endosomes before the liposomes are destroyed in the lysosomes [5,10].We think that it is important to find a proper molecule capable of controlling the release of siRNA from liposomes.
Curcumin is a major constituent of the spice turmeric (Curcuma longa), a yellow-colored polyphenol.As this compound has unique properties including the suppression of NF-B which is a nuclear factor playing a pivotal role in evoking immune responses, the induction of apoptosis, and antioxidant activities [11], various applications have been investigated.However its clinical application has been limited, because it has problems of low solubility, bioavailability, and stability.To resolve these problems, various approaches have been attempted [12], for example, synthesis of useful curcumin derivatives capable of inhibiting islet amyloid polypeptide aggregation [13] and creating nanoparticles containing curcumin applicable for in vivo administration [14].We have reported that nanosized liposomal curcumin is efficiently incorporated into macrophages and suppresses cytokine production in mice after in vivo administration [15].In the course of our study to search for a molecule to induce siRNA from liposomes, we found that curcumin has an ability to induce the release of siRNA from certain types of liposomes.In this paper, we would like to demonstrate that curcumin could induce siRNA from liposomes containing siRNA in a unique manner.

Preparation of Liposomes.
Liposomes containing siRNA were prepared using the minor modification method reported by Bailey and Sullivan [16].The lipids, 1,2-dioleoyl-snphosphatidylcholine (DOPC), 1,2-dioleoyl-sn-phosphatidylethanolamine (DOPE), and cholesterol, were purchased from NOF Corporation.Synthetic 21-nucleotide RNA duplex (rCUUrArCrGrCUrGrArGUrArCUUrCrGrATT) that can interfere with the expression of luciferase [17] was purchased from Sigma-Aldrich and used as siRNA.Briefly, the above lipids were dissolved in chloroform and the solution was evaporated in an eggplant shaped flask to form lipid films.Liposomal formation was done by ultrasonic treatment after a 10 mM Tris-HCl buffer (pH 7.6) was added to the lipid films.Encapsulation of siRNA into liposomes was performed in the presence of 40% ethanol and 2 mM CaCl 2 .Liposomes encapsulating siRNA suspended in phosphate-buffered saline (PBS) without Ca 2+ and Mg 2+ were obtained through dialysis (molecular weight: 1,000 kDa, Spectrum) or ultrafiltration (molecular weight: 500 kDa, Millipore).Three types of liposomes were used in this study: (1) liposomes composed of DOPC and DOPE at the molar ratio of 1 : 1 (DOPC/DOPE liposomes), (2) those of only DOPC (DOPC liposomes), and (3) those of DOPC and cholesterol at the molar ratio of 8 : 2 (DOPC/Cholesterol liposomes).The particle size and zeta potential of liposomes were measured through dynamic light scattering and electrophoretic light scattering, respectively, with Zetasizer Nano ZSP (Malvern).Lipid amounts of liposomes were determined with Phospholipid C-Test Wako (Wako Pure Chemical Industries) after the liposomes were lysed with 2% sodium dodecyl sulfate for 15 min at 90 ∘ C. Liposomes' contents of siRNA were quantified with Quant-iT6 RiboGreen RNA Assay Kit (Invitrogen).

Treatment of Liposomes with Curcumin.
We admixed liposomal suspension containing 250 ng siRNA, curcumin (Sigma-Aldrich) dissolved in 1 l of dimethyl sulfoxide (DMSO, Wako Pure Chemical Industries), and appropriate amounts of distilled water to obtain a total volume of 10 l in a 0.5 ml plastic tube (As One).As a positive control compound to make liposomes release siRNA, we used a detergent 1% Triton X-100 (Sigma-Aldrich).The mixture including liposomes and test compounds was incubated at various temperatures using PCR Thermal Cycler TP2000 (Takara Bio) for 30 min.

Agarose Gel Electrophoresis.
To estimate siRNA released from liposomes by curcumin, we performed agarose gel electrophoresis after treatment with curcumin.Electrophoretic analysis was useful because we could determine whether siRNA was intact at the same time to quantify siRNA.After a liposomal suspension was treated with test compounds, we added 2 l of a loading buffer containing 0.2% Orange G (Nacalai Tesque) and 5% glycerin (Wako Pure Chemical Industries) to the mixture.We loaded the resultant mixture for agarose gel electrophoresis.It was carried out in 2% Agarose 1200 (PH Japan) in TE buffer at 135 V for 15 min with an i-MyRun.NC electrophoretic system (Cosmo Bio).

Quantification of siRNA Released from Liposomes.
The electrophoresed siRNA was stained with a 1% ethidium bromide (EtBr) solution and then detected with a transilluminator (3UV6, UVP) at 302 nm wavelength.Images of the stained gels were captured through a 590 DF 100/49 filter (Kodak) with a digital camera SH-25MR (Olympus).The images were converted to black and white pictures using Viewer 2 software (Olympus).The resultant images were analyzed with Doc-It LS Image Analysis Software Ver.7.1 (UVP).The amount of siRNA detected in each sample was estimated from I-Max value in 1D gel analysis of the software, because this value was found to show good correlation with the amount of siRNA in preliminary experiments (data not shown).Relative amounts of siRNA released in each sample were calculated so that Triton X-100-treated liposomes

Quantification of Curcumin Absorbed into Liposomes.
To determine the amount of curcumin absorbed into DOPC/DOPE liposomes, we admixed liposomal suspension (47 mg/ml of lipids) containing siRNA (250 ng), curcumin (60 g/ml), and appropriate amounts of distilled water containing 10% DMSO to reach a total volume of 15 l in a 0.5 ml plastic tube.We incubated the mixture for 30 min at room temperature and then centrifuged the tube at 13,000 rpm by a microcentrifuge (Himac CT 13R; Hitachi) for 20 min at 4 ∘ C to precipitate the liposomes.To access nonspecific binding of curcumin to the tube, we prepared a mixture without liposomes in a tube as a blank and then treated the tube in the same manner as described above.After the centrifugation, resultant supernatants in the tubes were carefully removed, and then precipitates were dissolved in 15 l of DMSO, respectively.The amount of curcumin in the DMSO solution was determined by detecting the fluorescence from curcumin.Firstly, we obtained a calibration curve by measuring fluorescence from 5 l droplet of curcumin solution dissolved in DMSO at appropriate concentrations, respectively.The fluorescence of curcumin was determined in a similar manner as described in the part of "Quantification of siRNA Released from Liposomes" by using the transilluminator and software.We determined the concentration of curcumin in each test sample (5 l) based on the calibration curve.The amount of curcumin absorbed into the liposomes was calculated as the following calculation formula: the amount of curcumin in the tube with liposome − that in the blank tube.The assay was done in duplicate.

Results and Discussion
In this study, we examined whether curcumin could work to control the release of siRNA from liposomes.Because it has been reported that curcumin influences the cell membrane and destabilizes its structure [18], we thought that curcumin would be able to induce siRNA release from liposomes.To examine this hypothesis, we prepared liposomes containing siRNA and determined the effect of curcumin on siRNA release from liposomes.To determine the effect of curcumin, we employed DOPC/DOPE, DOPC, and DOPC/Cholesterol liposomes, because DOPC/DOPE liposomes have been well characterized to form small unilamellar vesicles capable of carrying polynucleotides [16].Representative data on the properties of these liposomes are shown in Table 1.It has also been reported that lipid membranes consisting of unsaturated lipids are not as rigid as those consisting of saturated lipids [19].DOPC/DOPE liposomes are thus expected to be an easier model to examine the controlled release of siRNA.To detect siRNA released from liposomes, we used agarose gel electrophoresis and ethidium bromide staining, because the release of siRNA is quickly detectable and the intactness of the released siRNA is also easily confirmable by this analytical method.A representative result of agarose gel electrophoresis after treatment of DOPC/DOPE liposomes with curcumin is shown in Figure 1.In comparison with control DOPC/DOPE liposomes [lane 2: untreated, lane 3: 10% DMSO-treated], evident release of siRNA was detected in those treated with curcumin at higher concentration more than 40 g/ml (lanes 6 to 10) for 30 min at room temperature.The results of repeated experiments are shown in Figure 2. Dose-response curve of curcumin showed a unique bell-shaped pattern.Significant release of siRNA from the liposomes was detected from curcumin concentration of 30 g/ml.The release gradually increased according to the curcumin concentrations and reached the maximal level at around 50 to 60 g/ml.Doses over 70 g/ml seemed to reduce the release of siRNA.We subsequently examined the effects of temperature and curcumin concentration on the release of siRNA from DOPC/DOPE, DOPC, and DOPC/Cholesterol liposomes, respectively.As shown in Figure 3, for DOPC/DOPE liposomes, the effect of curcumin at the range of 16 to 250 mg/ml on the release of siRNA did not apparently differ between room temperature and 37 ∘ C.However, in comparison, under conditions at 50 and 60 ∘ C, the release of siRNA was enhanced at 125 and 250 g/ml concentrations of curcumin.In addition, we examined the effect of curcumin on liposomes with DOPC and DOPC/Cholesterol lipid compositions.Although proper doses of curcumin efficiently promoted the release of siRNA from DOPC/DOPE liposomes even at room temperature, curcumin did not evidently induce the release of siRNA from both DOPC liposomes and DOPC/Cholesterol liposomes at the range of 16 to 250 g/ml under the same conditions (Figure 4).However, curcumin induced the release of siRNA from DOPC liposomes and DOPC/Cholesterol liposomes at doses ranging from 63 to 250 g/ml at 60 ∘ C.These results demonstrate that curcumin's ability to evoke the release of siRNA depends on both temperature and the lipid composition of the liposomes.In the absence of curcumin, hyperthermia treatment at 50 or 60 ∘ C solely did not show apparent effect on the release of siRNA from DOPC or DOPC/Cholesterol liposomes used in this study.It has been reported that heat treatment around the gelto-liquid crystalline phase transition temperature of lipids could promote the release of small molecules from liposomes [ [7][8][9].However, the phase transition temperature of DOPC is approximately −19 ∘ C [9].The temperature used in the heat treatment was far higher than the phase transition temperature of DOPC/liposomes.Our results demonstrated that the liposomes used in this study were very stable against the heat treatment.DOPE reportedly increases permeability and fusogenicity in DOPC bilayers, while cholesterol decreases these properties [20].Curcumin's siRNA-releasing ability (i.e., DOPC/DOPE > DOPC > DOPC/Cholesterol) seems to be well consistent with these reported changes in permeability and fusogenicity [20], suggesting that curcumin promotes these changes in liposomes and the resultant changes cause the release of siRNA.We estimated that about 10% of curcumin was absorbed into the liposomes (47 mg/ml) by the treatment of curcumin at 60 g/ml for 30 min at room temperature.Barry et al. have proposed an interaction model between liposomal lipid membrane and curcumin [18].According to their model, curcumin is inducible to disorder liposomal lipid bilayer.An interesting aspect in this model is that curcumin disorders the lipid bilayer more at lower concentrations (0.25-0.5 mole% in lipids) than at higher concentrations (≥1 mole%).This curcumin and liposome interaction model might explain our results that dose-response in the release of siRNA induced by curcumin showed the bell-shaped curve.However, curcumin absorbed into liposomes at 60 g/ml under our experimental conditions was estimated to be about 3 mole% in lipids.Although the amount of absorbed curcumin was approximately 10 times higher than that inducible high disorder reported by Barry et al., we think that it is due to different experimental conditions: in the model by Barry et al., curcumin was already incorporated into a lipid film before liposomal formation, while, in our experiments, curcumin was added to the liposomes containing siRNA.We speculate that, under our experimental conditions, greater amount of curcumin was required to achieve the similar interaction between lipid bilayer and curcumin proposed by Barry et al., though we could not rule out the possibility that in our experiments different mechanisms from Barry's model might work to induce the release of siRNA from DOPC/DOPE by curcumin and it remains to be clarified by future studies.Bicelles are recently used as an excellent membrane model system to analyze the interaction between the lipid bilayer and molecules integrated into the membrane, so that this system would be useful in clarifying the fine mechanism of curcumin-induced siRNA release from liposomes [21].

Conclusions
Curcumin is a safe food ingredient widely used in a long human history.As curcumin has unique molecular properties, it has been of interest in search for various applications.
In this study, we analyzed the effect of curcumin on liposomal functions and demonstrated that curcumin has an ability to induce the release of siRNA from liposomes encapsulating siRNA.We believe that this is the first report examining the influence of curcumin on the release of contents from liposomes.The effect of curcumin depends on at least the lipid constituents of liposomes, temperature, and curcumin concentrations.In particular, curcumin dose-dependency of siRNA release showed a unique bell-shaped pattern.Our results suggest that curcumin is useful for regulating the permeability of liposomes to release high molecular weight molecules.

Figure 2 :
Figure 2: Bell-shaped dose-response in the release of siRNA from DOPC/DOPE liposomes induced by curcumin.DOPC/DOPE liposomes containing siRNA were treated with curcumin at 25 to 125 mg/ml, and then agarose gel electrophoresis was done in the same manner as Figure 1.The amount of released siRNA was determined from photographic images of EtBr-stained agarose gels by using commercial software.Assays were done in triplicate and data were represented as mean values (e) and standard errors (vertical bars).Percentages of siRNA release were expressed as relative values with Triton X-100-treated samples defined as 100% and DMSO-treated samples as 0%.The results of Student's -test in comparison of each sample treated with curcumin to control are expressed as  values with symbols (&: <0.05; * : <0.01) in the figure, respectively.

Figure 3 :
Figure 3: Effect of temperature on the release of siRNA from DOPC/DOPE liposomes induced by curcumin.DOPC/DOPE liposomes were treated with curcumin at the indicated concentrations for 30 min at room temperature (e), 37 ∘ C (△), 50 ∘ C (◻), and 60 ∘ C (◊), respectively.Assays were done in triplicate and data were represented as mean values and standard errors (vertical bars).Percentages of siRNA released were expressed as relative values with Triton X-100-treated samples defined as 100% and DMSO-treated samples as 0%.The results of Student's -test in comparison of each sample treated with curcumin to control are expressed as  values with symbols (&: <0.05; * : <0.01) in the figure, respectively.

Figure 4 :
Figure 4: Effect of temperature on the release of siRNA from DOPC or DOPC/Cholesterol liposomes induced by curcumin.DOPC or DOPC/Cholesterol liposomes were treated with curcumin at the indicated concentrations for 30 min at room temperature or 60 ∘ C, respectively.Symbols indicate the following: e, DOPC liposomes treated at room temperature; I, DOPC liposomes treated at 60 ∘ C; △, DOPC/Cholesterol liposomes treated at room temperature; , DOPC/Cholesterol liposomes treated at 60 ∘ C. Assays were done in triplicate and data were represented as mean values and standard errors (vertical bars).Percentages of siRNA release were expressed as relative values with Triton X-100-treated samples defined as 100% and DMSO-treated samples as 0%.The results of Student's -test in comparison of each sample treated with curcumin to control are expressed as  values with symbols (&: <0.05; * : <0.01) in the figure, respectively.
a Liposomes were suspended in PBS.b Diameter and zeta potentials were determined with Zetasizer Nano ZSP.equaled 100% while DMSO-treated liposomes equaled 0%.Statistical analysis (Student's -test) was done with Microsoft Excel.