2. Materials and Methods2.1. Chemic

2. Materials and Methods

2.1. Chemicals

Hydrogenated L-α-phosphatidylcholine from egg yolk (HEPC), soybean oil, polysorbate 80 (polyoxyethylene (20) sorbitanmonooleate, Tween 80), and chloroform were purchased from NacalaiTesque Inc., Kyoto, Japan. In HEPC, the phospholipid content was more than 99% and the phosphatidylcholine content was approximately 70%. Lecithin from soybean was purchased from WAKO Pure Chemical Industries, Osaka, Japan. Curcumin from Curcuma Longa Linn (Turmeric) was purchased from Sigma-Aldrich, St. Louis, MO, USA. Polyoxyethylene hydrogenated castor oil 60 (Cremophor-HR60, HCO-60) was supplied by BASF, Ludwigshafen, Germany. Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 and penicillin-streptomycin were purchased from GIBCO Invitrogen (Grand Island, NY, USA). Fetal bovine serum (FBS) was obtained from Biochrom AG (Berlin, Germany). Ethanol was purchased from Fluka Chemicals (Buchs, Switzerland).

2.2. Preparation of Curcumin-Containing Lipid Nanoemulsion

Curcumin-containing lipid nanoemulsions were prepared by a modified thin-film hydration method at room temperature (24°C) as previously reported [32, 33]. Briefly, the emulsions consisted of soybean oil or lecithin (phosphatidylcholine), water, curcumin, HEPC, and an appropriate co-surfactant. The oil and HEPC were dissolved in 2 mL of chloroform. Curcumin was dissolved in 4 mL of chloroform. Co-surfactant (HCO-60 or Tween 80) was dissolved in 2 mL of chloroform (HCO-60) or distilled water (Tween 80). The mixture of the 3 solutions was dried by rotary evaporation and subjected to subsequent vacuum desiccation for 3–5 h to generate the dried thin film. The dried thin film was hydrated with 30 mL of distilled water warmed at 55–60°C in a bath-type sonicator (BRANSON-Yamato 2510, BRANSONIC, Emerson-Japan, Kanagawa, Japan), followed by vigorous mixing and sonicating for 5 min to create coarse lipid emulsions. The fine lipid emulsions were prepared by 30–60 min sonication under N2 atmosphere with a bath-type sonicator, which was thermostated at 55–60°C. The sonication was performed as follows: 3 min sonication and subsequent 2 min cooling, which were repeated for 30–60 min. Excess curcumin was removed by centrifugation at 3000 rpm for 15 min. The supernatant was collected as curcumin lipid nanoemulsion sample. Three different batches of lipid nanoemulsions were prepared with each formulation. The prepared curcumin lipid nanoemulsions were evaluated regarding particle size, curcumin concentration, and percent incorporation efficiency (% IE) described below.

2.3. Particle Size Measurement

The particle diameter of the nanoemulsions was measured by a dynamic light scattering method using Zetasizer
3000HS
A
(Malvern Instrument, UK) at room temperature. Particle size data were expressed as the mean of the Z-average of 3 independent batches of the nanoemulsions.

2.4. Determination of Curcumin Concentration and Incorporation Efficiency

Curcumin in lipid nanoemulsions was quantified using a simple colorimetric assay at 450 nm as described previously [25]. Briefly, a standard curve was generated from known concentrations of curcumin in HBSE-Triton X-100 (10 mM HEPES, 140 mM NaCl, 4 mM EDTA, and 1% Triton X-100). After centrifugation, 4 mL of HBSE-Triton X-100 was added to 25 μL of the curcumin lipid nanoemulsion to determine the curcumin concentration in the nanoemulsion. The absorbance was measured on a spectrophotometer (UV-150-02, SHIMADZU Corporation, Japan). The amount of curcumin in the lipid nanoemulsion was calculated from the final concentration of curcumin after preparation. The curcumin incorporation efficiency (% IE) in the lipid nanoemulsion was calculated by the following equation:
%IE=(Measuredamountofcurcumininlipidnanoemulsion)
(Totalamountofcurcuminappliedinpreparinglipidnanoemulsion)×100.(1)

2.5. Solubility of Curcumin

To determine the solubility of curcumin, 3 different solutions, including soybean oil, 1.25% (w/v) Tween 80 in distilled water, and 1.25% Tween 80 with 0.83% HEPC in distilled water were tested in this study. For determining the solubility of curcumin in soybean oil, 3.5 mg of curcumin was added to a test tube containing 1 mL of soybean oil. The mixture solutions were vortexed, allowed to cool, and kept at 24°C overnight. Thereafter, they were centrifuged at 3000 rpm for 15 min at 24°C. Undissolved curcumin, which was observed at the bottom of the test tube, was eliminated by centrifugation. Then, the amount of curcumin in soybean oil was determined by spectrophotometry at an absorbance of 450 nm.

For determining the solubility of curcumin in the Tween 80 aqueous solutions without (Tween 80 solution) or with HEPC (Tween 80-HEPC solution), 1.0 mg of curcumin was added to a test tube containing 1 mL of the aqueous solutions, followed by vortexing. The solutions were warmed and vortexed every 5 min in a water bath (60°C) for 1 h. After being allowed to cool and stand at 24°C overnight, they were centrifuged at 3000 rpm for 15 min at 24°C. The concentrations of curcumin in the supernatant of both Tween 80 and Tween 80-HEPC solutions were measured spectrophotometrically.

2.6. Cells and Cell Culture Conditions

B16F10 (mouse melanoma cell line) was cultured in DMEM containing 10% fetal calf serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. Four types of leukemic cell lines, including HL60 (promyelocytic leukemia), K562 (chronic myelocytic leukemia), Molt4 (lymphoblastic leukemia), and U937 (monocytic leukemia), were cultured in RPMI-1640 medium containing 10% fetal calf serum, 1 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. These cell lines were maintained in a humidified incubator with an atmosphere of 95% air and 5% CO2 at 37°C. When the cells reached confluency, they were harvested and plated for consequent passages or for nanoemulsion curcumin treatments.

2.7. MTT Assay

Cell viability was determined by the MTT test method. MTT (5 mg/mL) was dissolved in PBS. B16F10 and leukemic cells were cultured in 96-well plates (
1.0×10
4
and
3.0×10
3
 cells/well, resp.) containing 100 μL medium prior to treatment with curcumin and curcumin-loaded nanoemulsion at 37°C for 24 h. Subsequently, 100 μL fresh medium containing various concentrations (4.23, 8.47, 16.97, 33.93, and 67.86 μM) of curcumin or the corresponding curcumin-loaded nanoemulsion were added to each well, and incubated for another 48 h. Diluted curcumin solutions were freshly prepared in DMSO prior to each experiment. The final concentration of DMSO in culture medium was 0.2% (v/v). Diluted curcumin-loaded nanoemulsion was prepared in completed RPMI-1640 medium. The curcumin concentrations from curcumin-loaded nanoemulsion were determined by the colorimetric assay as previously described [24] using the HBSE buffer containing Triton X-100. The amount of curcumin in nanoemulsion was determined by comparing to the standard curcumin curve. Then, curcumin concentrations were calculated and diluted for the equal concentrations with equimolar of conventional curcumin to test cytotoxicity by MTT assay. The nanoemulsion without curcumin was used as vehicle control. The metabolic activity of each well was determined by MTT assay and compared to those of untreated cells. After removal of 100 μL medium, MTT dye solution was added (15 μL/100 μL medium) and the plates were incubated at 37°C for 4 h in a humidified 5% CO2 atmosphere. After that, 200 μL of DMSO were added to each well, and mixed thoroughly to dissolve the dye crystals. The absorbance was measured using an ELISA plate reader (Biotek EL 311) at 570 nm with a reference wavelength of 630 nm. High optical density readings corresponded to a high intensity of dye color, that is, to a high number of viable cells able to metabolize MTT salts. The fractional absorbance was calculated by the following formula:
%Cellsurvival=Meanabsorbanceintestwells
Meanabsorbanceincontrolwells×100.(2)

The average cell survival obtained from triplicate determinations at each concentration was plotted as a dose response curve. The experiment was carried out in 3 batches of nanoemulsion preparations in 3 time-independent experiments. The 50% inhibition concentration (IC50) of the active substances was determined as the lowest concentration which reduced cell growth by 50% in treated compared to untreated culture or vehicle control culture (0.4% DMSO in culture medium). The IC50s were mean ± standard error (SE) and compared for their activities.

2.8. In Vitro Release Kinetics of Curcumin-Loaded Nanoemulsion

Curcumin-loaded nanoemulsion at the concentration of 7 mg in 25% human serum in PBS was put into dialysis bag with pore size of 50°A (5 nm). The dialysis bag was kept in dark bottle containing 25% human serum in PBS with total volume of 150 mL. The condition was controlled under constant stirring in water bath set as 37°C. Sample (1 mL) was collected at certain time intervals of 2, 4, 6, 12, 24, 36, 48, 60, and 72 h. The fresh medium was added after withdrawal. The curcumin concentration in the sample was determined by colorimetric assay measured at 450 nm as compared to standard curcumin as described previously [25].

2.9. Statistical Analysis

Statistical analysis was performed using the SPSS software (version 10.0). All experiments were repeated at least 3 times. The data were expressed as the mean (standard deviation and standard error; SD and SE). Statistical differences between the means were tested by one-way ANOVA. Probability values

Kết quả (Việt) 1:

2. tài liệu và phương pháp2.1. hóa chấtDầu L-α-phosphatidylcholine từ lòng đỏ trứng (HEPC), đậu tương dầu, polysorbate 80 (polyoxyethylene (20) sorbitanmonooleate, Tween 80), và clorofom đã được mua từ NacalaiTesque Inc, Kyoto, Nhật bản. Trong HEPC, nội dung phospholipid đã là nhiều hơn 99% và nội dung phosphatidylcholine đã là khoảng 70%. Lecithin từ đậu tương đã được mua từ WAKO tinh khiết hóa học công nghiệp, Osaka, Nhật bản. Curcumin từ Curcuma Longa Linn (nghệ) đã được mua từ Sigma-Aldrich, St. Louis, MO, Mỹ. Polyoxyethylene dầu castor dầu 60 (Cremophor-HR60, HCO-60) được cung cấp bởi BASF, Ludwigshafen, Đức. Dimetyl sulfôxít (DMSO), 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyl tetrazolium bromua (MTT) đã được mua từ Sigma-Aldrich (St. Louis, MO, Mỹ). RPMI-1640 và penicillin-streptomycin đã được mua từ GIBCO Invitrogen (Grand Island, NY, Hoa Kỳ). Huyết thanh bò thai nhi (FBS) nhận được từ Biochrom AG (Berlin, Đức). Ethanol đã được mua từ Fluka hóa chất (Buchs, Thuỵ Sỹ).2.2. chuẩn bị của Curcumin có chứa Lipid NanoemulsionCurcumin có chứa chất béo nanoemulsions đã được chuẩn bị bởi một lần màng mỏng hydrat hóa phương pháp ở nhiệt độ phòng (24° C) như trước đây báo cáo [32, 33]. Một thời gian ngắn, các nhũ tương bao gồm đậu tương dầu hoặc lecithin (phosphatidylcholine), nước, curcumin, HEPC, và một hợp chất thích hợp. Dầu và HEPC được hòa tan trong cloroform 2 mL. Curcumin hòa tan trong cloroform 4 mL. Hợp chất (HCO-60 hoặc Tween 80) được hòa tan trong cloroform (HCO-60) 2 mL hoặc chưng cất nước (Tween 80). Hỗn hợp của các giải pháp 3 được sấy khô bằng máy bay hơi và phải chịu sự khô chân không tiếp theo cho 3-5 h để tạo ra bộ phim mỏng khô. Màng mỏng khô ngậm nước với 30 mL nước cất ấm ở 55-60° C trong một sonicator tắm kiểu (BRANSON-Yamato 2510, BRANSONIC, Emerson-Nhật bản, Kanagawa, Nhật bản), theo sau là mạnh mẽ trộn và sonicating cho 5 phút để tạo ra thô lipid nhũ tương. Nhũ tương chất béo tốt đẹp đã được chuẩn bị bởi 30-60 phút sonication dưới bầu khí quyển N2 với một tắm kiểu sonicator, mà là thermostated ở 55-60° C. Sonication đã được thực hiện như sau: sonication 3 phút và sau đó 2 phút làm mát, đã được lặp đi lặp lại cho 30-60 phút vượt quá curcumin đã được gỡ bỏ bởi số 3000 rpm trong 15 phút. Supernatant đã được thu thập như curcumin lipid nanoemulsion mẫu. Ba lô khác nhau của lipid nanoemulsions đã được chuẩn bị với công thức mỗi. Chuẩn bị sẵn sàng curcumin lipid nanoemulsions được đánh giá liên quan đến kích thước hạt, nồng độ curcumin, và hiệu quả kết hợp phần trăm (% IE) được mô tả dưới đây.2.3. hạt kích thước đo lườngĐường kính hạt của các nanoemulsions được đo bằng một phương pháp tán xạ ánh sáng năng động bằng cách sử dụng Zetasizer 3000HSA (Malvern cụ, UK) ở nhiệt độ phòng. Hạt kích thước dữ liệu đã được biểu thị dưới dạng trung bình của Z-trung bình của 3 lô độc lập của các nanoemulsions.2.4. xác định nồng độ Curcumin và kết hợp hiệu quảCurcumin trong lipid nanoemulsions được định lượng bằng cách sử dụng một khảo nghiệm colorimetric đơn giản tại 450 nm như mô tả trước đó [25]. Một thời gian ngắn, một đường cong tiêu chuẩn đã được tạo ra từ được biết đến nồng độ curcumin trong HBSE-Triton X-100 (10 mM HEPES, 140 mM NaCl, 4 mM EDTA, và 1% Triton X-100). Sau khi số, 4 mL của HBSE-Triton X-100 đã được thêm vào 25 μL curcumin lipid nanoemulsion để xác định nồng độ curcumin trong nanoemulsion. Sự hấp thu được đo trên một phối (UV-150-02, Tổng công ty SHIMADZU, Nhật bản). Số tiền của curcumin trong lipid nanoemulsion được xác định từ cuối cùng nồng độ curcumin sau khi chuẩn bị. Curcumin kết hợp hiệu quả (% IE) trong lipid nanoemulsion được tính bằng phương trình sau đây:%IE=(Measuredamountofcurcumininlipidnanoemulsion)(Totalamountofcurcuminappliedinpreparinglipidnanoemulsion) × 100. (1)2.5. độ hòa tan của CurcuminTo determine the solubility of curcumin, 3 different solutions, including soybean oil, 1.25% (w/v) Tween 80 in distilled water, and 1.25% Tween 80 with 0.83% HEPC in distilled water were tested in this study. For determining the solubility of curcumin in soybean oil, 3.5 mg of curcumin was added to a test tube containing 1 mL of soybean oil. The mixture solutions were vortexed, allowed to cool, and kept at 24°C overnight. Thereafter, they were centrifuged at 3000 rpm for 15 min at 24°C. Undissolved curcumin, which was observed at the bottom of the test tube, was eliminated by centrifugation. Then, the amount of curcumin in soybean oil was determined by spectrophotometry at an absorbance of 450 nm.For determining the solubility of curcumin in the Tween 80 aqueous solutions without (Tween 80 solution) or with HEPC (Tween 80-HEPC solution), 1.0 mg of curcumin was added to a test tube containing 1 mL of the aqueous solutions, followed by vortexing. The solutions were warmed and vortexed every 5 min in a water bath (60°C) for 1 h. After being allowed to cool and stand at 24°C overnight, they were centrifuged at 3000 rpm for 15 min at 24°C. The concentrations of curcumin in the supernatant of both Tween 80 and Tween 80-HEPC solutions were measured spectrophotometrically.2.6. Cells and Cell Culture ConditionsB16F10 (mouse melanoma cell line) was cultured in DMEM containing 10% fetal calf serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. Four types of leukemic cell lines, including HL60 (promyelocytic leukemia), K562 (chronic myelocytic leukemia), Molt4 (lymphoblastic leukemia), and U937 (monocytic leukemia), were cultured in RPMI-1640 medium containing 10% fetal calf serum, 1 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. These cell lines were maintained in a humidified incubator with an atmosphere of 95% air and 5% CO2 at 37°C. When the cells reached confluency, they were harvested and plated for consequent passages or for nanoemulsion curcumin treatments.2.7. MTT AssayCell viability was determined by the MTT test method. MTT (5 mg/mL) was dissolved in PBS. B16F10 and leukemic cells were cultured in 96-well plates (1.0×104 and 3.0×103 cells/well, resp.) containing 100 μL medium prior to treatment with curcumin and curcumin-loaded nanoemulsion at 37°C for 24 h. Subsequently, 100 μL fresh medium containing various concentrations (4.23, 8.47, 16.97, 33.93, and 67.86 μM) of curcumin or the corresponding curcumin-loaded nanoemulsion were added to each well, and incubated for another 48 h. Diluted curcumin solutions were freshly prepared in DMSO prior to each experiment. The final concentration of DMSO in culture medium was 0.2% (v/v). Diluted curcumin-loaded nanoemulsion was prepared in completed RPMI-1640 medium. The curcumin concentrations from curcumin-loaded nanoemulsion were determined by the colorimetric assay as previously described [24] using the HBSE buffer containing Triton X-100. The amount of curcumin in nanoemulsion was determined by comparing to the standard curcumin curve. Then, curcumin concentrations were calculated and diluted for the equal concentrations with equimolar of conventional curcumin to test cytotoxicity by MTT assay. The nanoemulsion without curcumin was used as vehicle control. The metabolic activity of each well was determined by MTT assay and compared to those of untreated cells. After removal of 100 μL medium, MTT dye solution was added (15 μL/100 μL medium) and the plates were incubated at 37°C for 4 h in a humidified 5% CO2 atmosphere. After that, 200 μL of DMSO were added to each well, and mixed thoroughly to dissolve the dye crystals. The absorbance was measured using an ELISA plate reader (Biotek EL 311) at 570 nm with a reference wavelength of 630 nm. High optical density readings corresponded to a high intensity of dye color, that is, to a high number of viable cells able to metabolize MTT salts. The fractional absorbance was calculated by the following formula:%Cellsurvival=MeanabsorbanceintestwellsMeanabsorbanceincontrolwells×100.(2)The average cell survival obtained from triplicate determinations at each concentration was plotted as a dose response curve. The experiment was carried out in 3 batches of nanoemulsion preparations in 3 time-independent experiments. The 50% inhibition concentration (IC50) of the active substances was determined as the lowest concentration which reduced cell growth by 50% in treated compared to untreated culture or vehicle control culture (0.4% DMSO in culture medium). The IC50s were mean ± standard error (SE) and compared for their activities.2.8. In Vitro Release Kinetics of Curcumin-Loaded NanoemulsionCurcumin-loaded nanoemulsion at the concentration of 7 mg in 25% human serum in PBS was put into dialysis bag with pore size of 50°A (5 nm). The dialysis bag was kept in dark bottle containing 25% human serum in PBS with total volume of 150 mL. The condition was controlled under constant stirring in water bath set as 37°C. Sample (1 mL) was collected at certain time intervals of 2, 4, 6, 12, 24, 36, 48, 60, and 72 h. The fresh medium was added after withdrawal. The curcumin concentration in the sample was determined by colorimetric assay measured at 450 nm as compared to standard curcumin as described previously [25].2.9. Statistical AnalysisStatistical analysis was performed using the SPSS software (version 10.0). All experiments were repeated at least 3 times. The data were expressed as the mean (standard deviation and standard error; SD and SE). Statistical differences between the means were tested by one-way ANOVA. Probability values