Patent Application
20240342089
The present invention relates generally to a technology of using liposomes, and more particularly to an aquatic liposome encapsulating a natural compound and a manufacturing method thereof.
In general, a “liposome” is mainly applied as a carrier for a medication or a nutrient and are mainly adapted to encapsulate the medication or the nutrient and transport the medication or the nutrient to a target organ. The structure of the liposome could prevent the liposome from being removed by phagocytosis of macrophages during absorption and could slowly release the medication or the nutrient encapsulated.
Conventional research on the preparation of liposomes shows that aquatic products are rarely used as extraction sources. The process of the aquatic products emerges aquatic waste, such as fish skin and fish head. The phospholipids extracted from such aquatic waste typically contain a large amount of DHA, EPA, DPA, and ARA, which could relieve the cellular inflammation of the animal. However, there are currently no relevant studies exploring the use of aquatic product extraction to prepare liposomes to coat specific drugs to the anti-inflammatory effect.
Tetrahydrocurcumin (THC) is a metabolite of curcumin (CUR). In addition to having higher water solubility, chemical stability, and bioavailability compared to CUR, it also possesses many of the beneficial biological activities of CUR. Therefore, how to extract and prepare a liposome from an aquatic product for encapsulating tetrahydrocurcumin (THC), is a problem to be solved of the present invention.
Given the above, the primary objective of the present invention is to provide an aquatic liposome encapsulating a natural compound and a manufacturing method thereof, wherein the aquatic liposome encapsulates the natural compound into specific particle sizes that could efficiently enter cells.
The present invention provides an aquatic liposome encapsulating a natural compound, wherein an average particle size (a median particle size) of the aquatic liposome encapsulating the natural compound ranges from 80 nm to 200 nm.
In an embodiment, the natural compound includes tetrahydrocurcumin (THC) or quercetin, and an effective amount of the natural compound encapsulated in the aquatic liposome ranges from 10 μM to 60 μM.
The present invention further provides a manufacturing method of an aquatic liposome encapsulating a natural compound, including: step S1: providing an aquatic liposome and mixing the aquatic liposome with the natural compound, wherein a mixed concentration of the aquatic liposome ranges from 0.05 mg/ml to 1.0 mg/ml; step S2: performing an ultrasonic oscillation after mixing the aquatic liposome and the natural compound, so that the natural compound is encapsulated in the aquatic liposome; and step S3: measuring the aquatic liposome, wherein an average particle size (a median particle size) of the aquatic liposome encapsulating the natural compound ranges from 80 nm to 200 nm.
The experiments conducted prove that the aquatic liposome encapsulating the natural compound (tetrahydrocurcumin (THC)) could effectively enter the microglia and the retinal pigment epithelium cells. When the microglia produce the inflammatory response due to the induction of P. aeruginosa, the aquatic liposome encapsulating the natural compound (tetrahydrocurcumin (THC)) entered the microglia could effectively reduce the inflammatory mediator levels of the inflammatory response. When the retinal pigment epithelium cells produce the lesion due to the induction of sodium iodate, the aquatic liposome encapsulating the natural compound (tetrahydrocurcumin (THC)) entered the retinal pigment epithelium cells could effectively reduce the reactive oxygen species level. Accordingly, the aquatic liposome encapsulating the natural compound (tetrahydrocurcumin (THC)) could suppress the inflammatory response and relieve the apoptosis of the retinal pigment epithelium cells, thereby preventing or treating eye-related inflammatory diseases or symptoms.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The present invention will be best understood by referring to the following detailed description of some illustrative embodiments in conjunction with the accompanying drawings, in which
The present invention provides an embodiment of an aquatic liposome encapsulating a natural compound, wherein an average particle size (a median particle size) of the aquatic liposome encapsulating the natural compound ranges from 80 nm to 200 nm. In the current embodiment, the natural compound includes tetrahydrocurcumin (THC) or quercetin, and an effective amount of the natural compound encapsulated in the aquatic liposome ranges from 10 μM to 60 μM, but not limited thereto; the natural compound could be other drugs that could not directly enter cells. More specifically, in an embodiment, the aquatic liposome is obtained by extracting from fish skin of Acipenser sinensis, so that aquatic waste produced by processing the Acipenser sinensis could be consumed, thereby reducing the environmental pollution due to the aquatic waste. A process for the aquatic liposome to encapsulate the natural compound is to mix the aquatic liposome and the natural compound and then perform an ultrasonic oscillation on the aquatic liposome and the natural compound, wherein a mixed concentration of the aquatic liposome ranges from 0.05 mg/ml to 1.0 mg/ml. In an embodiment, the mixed concentration of the aquatic liposome ranges from 0.1 mg/ml to 0.5 mg/ml. In another embodiment, the mixed concentration of the aquatic liposome ranges from 0.2 mg/ml to 0.4 mg/ml. The aquatic liposome is recombined and polymerized due to the ultrasonic oscillation, so that the natural compound is encapsulated in the aquatic liposome during the recombination and polymerization of the aquatic liposome, thereby encapsulating the natural compound in the aquatic liposome. In the current embodiment, when the natural compound is tetrahydrocurcumin (THC), the average particle size (the median particle size) of the aquatic liposome encapsulating the tetrahydrocurcumin (THC) as measured ranges from 100 nm to 150 nm, but not limited thereto. In other embodiments, the aquatic liposome could encapsulate quercetin, and the average particle size (the median particle size) of the aquatic liposome encapsulating quercetin ranges from 100 nm to150 nm.
The current embodiment proves that the aquatic liposome with different concentrations affects an encapsulation efficiency of the aquatic liposome in encapsulating the natural compound. Use 0.05 to 1.0 mg/ml as the mixed concentration of the aquatic liposome, mix with tetrahydrocurcumin (THC) (50 μM) by ultrasonic oscillation, and the detect the encapsulation efficiency of the aquatic liposome. When the mixed concentration of the aquatic liposome ranges from 0.1 mg/ml to 0.5 mg/ml, the encapsulation efficiency of the aquatic liposome measured after the mixing and the ultrasonic oscillation of the aquatic liposome and the natural compound is greater than or equal to 50%, and the effective amount of the natural compound encapsulated in the aquatic liposome ranges from 12.5 μM to 50 μM. When the mixed concentration of the aquatic liposome ranges from 0.2 mg/ml to 0.4 mg/ml, the encapsulation efficiency of the aquatic liposome measured after the mixing and the ultrasonic oscillation of the aquatic liposome and the natural compound is greater than or equal to 65%, and the effective amount of the natural compound encapsulated by the aquatic liposome ranges from 25 μM to 50 μM. In an embodiment, the encapsulation efficiency of the aquatic liposome measured is greater than or equal to 80%.
A manufacturing method of the aquatic liposome encapsulating the natural compound of the aforementioned embodiment is explained below. Referring to
The term “effective amount” as referred to herein is an amount of an active ingredient in a composition which is sufficient to produce a desired physiological response. The effective amount of the active ingredient does not necessarily cure a disease or a symptom but could slow, stop, prevent, or relieve the development of the symptom. The actual effective amount of the active ingredient depends on many factors, such as particular the condition of experiment, the physiological condition of subjects (e.g., weight, age, gender), the type of subjects undertaking the experiments, the duration of the experiments, the actual formula used. For example, the effective amount of the active ingredient could be expressed in concentration, such as molar concentration, weight concentration, volume concentration, mole fraction, mass fraction, and mixing ratio. One of ordinary skill in the art could calculate the human equivalent dose (HED) for human medication based on doses determined from animal models.
The concentration ratio in the manufacturing method of the aquatic liposome encapsulating the natural compound in the current embodiment is not a limitation of the present invention. The parameters that could be appropriately adjusted by one of ordinary skill in the art after referring the present invention should still fall within the scope of the present invention.
In order to demonstrate the purpose, the features, and the effects of the present invention, characteristic analysis of the aquatic liposome before and after encapsulating tetrahydrocurcumin (THC) according to the current embodiment of the present invention, measurement of the encapsulation efficiency of the aquatic liposome, determination of the anti-inflammatory effect of tetrahydrocurcumin (THC) with different effective amounts, and determination of the effect of tetrahydrocurcumin (THC) with different effective amounts in suppressing the apoptosis are conducted.
Tetrahydrocurcumin (THC) (no.: sc-391609) is from Santa Cruz (US). Cell-permeable fluorescent probe H2DCFH-DA (2′,7′-dichlorodihydro-fluorescein diacetate) (no.: 15204) is from AAT bioquest (US). Cell Counting Kit-8 (CCK-8) is from Dojindo Molecular Technologies (US). Annexin V and propidium iodide (PI) are from Thermo Fisher (US).
The aquatic liposome (abbreviation: Asl-lipo) is obtained from 50 g of Acipenser sinensis skin in the market; the Acipenser sinensis skin and 950 mL of 95% ethanol are placed in a blender for evenly blending over 5 minutes to form a mixture; the mixture is placed in an ultrasonic machine (D200H) at 70° C. for oscillation over 1 hour; then, ethanol and most of the impurities of the Acipenser sinensis skin in the mixture are filtered by a filter funnel; ethanol is removed and subsequent drying is performed by a rotary evaporator; then 50 mL of acetone is added to form an acetone mixture; the acetone mixture is left to react overnight at −20° C.; the rotary evaporator is from Buchi Rotavapor Rii (Australia); after the reaction is completed, centrifugation at 3000 g is performed on the acetone mixture at 0° C. for 5 minutes; then a supernatant is removed; another 50 mL of acetone is added for another rinsing and another centrifugation is performed; another supernatant is removed; after residual acetone is evaporated, a precipitate is dissolved in an anhydrous ethanol to form an anhydrous ethanol mixture; the anhydrous ethanol mixture is stored a −20° C. to prevent oxidation of lipid; after ethanol in the anhydrous ethanol mixture is evaporated, a dried phospholipid is weighted; the dried phospholipid is redissolved in phosphate buffered saline (PBS) or double-distilled water (d2H2O); then an aggregated phospholipid precipitate is broken down by a tube shaker (model: Vortex-Genie 2, SI-0235), wherein the tube shaker (model: Vortex-Genie 2, SI-0235) is from Scientific Industries (US); afterwards, the ultrasonic oscillation is performed again (an oscillation frequency is 40 kHz) to obtain the aquatic liposome.
How an oscillation time of the ultrasonic oscillation correspondingly affects a distribution of a particle size of the aquatic liposome (Asl-lipo) is determined. The particle size of the aquatic liposome is measured by NanoSight NS3000. The measuring procedure includes the following steps: perform a device calibration by using standardized samples of 100 nm and 200 nm; then a 1 ml syringe is filled with a sample of the aquatic liposome and no bubble is produced; the syringe is connected to a transferring tube and a micro-injection pump; the micro-injection pump is set to transfer the sample of the aquatic liposome at 25 μL/min at 25° C.; the aforementioned step is performed three times for measuring the particle size of the aquatic liposome and each time last 60 seconds. NanoSight NS3000 is to detect nanoparticles of the aquatic liposome passing through the transferring tube to enter the 0.25 ml chamber with the setting of the absorption wavelength of 635nm and the shutter speed of 15 ms. The detection result of the nanoparticles of the aquatic liposome is analyzed by NTA2.1 software (Nanosight). An average particle size and a median particle size of the aquatic liposome (Asl-lipo) are calculated, and the number of particles at different concentrations is calculated based on the dilution factor.
The current experiment is to perform the ultrasonic oscillation at different oscillation times on the aquatic liposome (Asl-lipo), wherein the oscillation times of the ultrasonic oscillation (the oscillation frequency is set 40 kHz) are set 0 min, 5 min, 10 min, 20 min, 40 min, and 60 min, respectively. The average particle size (nm) and the polydispersity index (PDI) of the aquatic liposome (Asl-lipo) in correspondence with the different oscillation times are measured.
The result of the experiment is shown in
The result of the experiment shows that when the oscillation time of the ultrasonic oscillation is above 20 min, the polydispersity index (PDI) of the aquatic liposome (Asl-lipo) is lowered to be less than 0.1, and the average particle size of the aquatic liposome (Asl-lipo) is less than 100 nm, indicating that the oscillation time of the ultrasonic oscillation affects the average particle size (nm) and the polydispersity index (PDI) of the aquatic liposome (Asl-lipo).
Different letters (a-c) superscripted in the statistical chart and the description indicate that there is statistical significance between groups (p<0.05); on the contrary, being marked with the same letter indicates that there is no statistical significance.
The influence of the oscillation time of the ultrasonic oscillation on the particle distribution of the aquatic liposome (Asl-lipo) is determined. The current experiment is to similarly perform the ultrasonic oscillation on the aquatic liposome (Asl-lipo), wherein the oscillation times of the ultrasonic oscillation (the oscillation frequency is set 40 kHz) are set 0 min, 5 min, 10 min, 20 min, 40 min, and 60 min, respectively. The difference in the distribution of the aquatic liposome (Asl-lipo) with the particle size less than 200 nm in correspondence with different oscillation times are analyzed. The current experiment divides the average particle size (nm) of the aquatic liposome (Asl-lipo) into four groups, including 0-50 nm, 50-100 nm, 100-150 nm, and 150-200 nm. The distribution of the aquatic liposome (Asl-lipo) within the four groups are summarized.
The result of the experiment is shown in
It should be noted that when the oscillation time of the ultrasonic oscillation on the aquatic liposome (Asl-lipo) was 5 min, the measured particle size of the aquatic liposome was 40.69% with a particle size of 50-100 nm, and the amount with the particle size of 100-150 nm is 55.16%. When the oscillation time of the ultrasonic oscillation on the aquatic liposome (Asl-lipo) was 10 min, the measured particle size of the aquatic liposome was 37.67% with a particle size of 50-100 nm, and the amount with the particle size of 100-150 nm is 53.9% When the oscillation time of the ultrasonic oscillation on the aquatic liposome (Asl-lipo) is increased to 20 min or above (40 min and 60 min), the polydispersity index (PDI) of the aquatic liposome (Asl-lipo) in each group of the particle size is decreased. The particle size of the aquatic liposome (Asl-lipo) in each group of the particle size steadily falls within 50-100 nm, wherein the measured number of the aquatic liposome Asl-lipo ranges from 65.6% to 84.04%.
Moreover, the number of particles formed in correspondence with different oscillation times of the ultrasonic oscillation is analyzed. The result of the experiment is shown in
The aforementioned experiment shown that the oscillation time of the ultrasonic oscillation significantly influences the particle size, the particle size distribution, and the number of particles formed of the aquatic liposome (Asl-lipo). When the oscillation time of the ultrasonic oscillation on the aquatic liposome (Asl-lipo) is 10-60 min, the stability of the particle size distribution of the aquatic liposome (Asl-lipo) could be effectively enhanced, so that the average particle size of the aquatic liposome (Asl-lipo) is within 50-100 nm and the number of particles formed of the aquatic liposome (Asl-lipo) with the particle size of 50-100 nm is relatively increased.
The current experiment includes the following steps: the aquatic liposome (Asl-lipo) is prepared to have a mixed concentration of 0.05 mg/ml, 0.1 mg/ml, 0.2 mg/ml, 0.4 mg/ml, 0.6 mg/ml, 0.8 mg/ml, and 1.0 mg/ml respectively; an effective amount of tetrahydrocurcumin (THC) is 50 μM; each group of the aquatic liposome (Asl-lipo) (0.05 mg/ml, 0.1 mg/ml, 0.2 mg/ml, 0.4 mg/ml, 0.6 mg/ml, 0.8 mg/ml, and 1.0 mg/ml) is mixed with the same volume of tetrahydrocurcumin (THC); the ultrasonic oscillation is performed for 60 min (the oscillation frequency is 40 kHz) to evenly mix the aquatic liposome (Asl-lipo) and tetrahydrocurcumin (THC); an initial particle number is calculated by NTA.
Calculation with NTA is to firstly withdraw 500 μl of the mixed solution and add the mixed solution to 10 ml of d2H2O; after mixing evenly, ultracentrifugation at 100000 rpm is performed for 90 min; the supernatant is removed and then 500 μl of d2H2O is added for redissolving; the liposome is collected and the particle number collected is calculated by NTA; the collection rate of the liposome is calculated based on [the particle number collected/the initial particle number]. Afterwards, a polyphenolic compound content in the liposome is analyzed by Folin-Ciocalteu method. A relative content is calculated by comparing the standard curve of tetrahydrocurcumin (THC) through interpolation. An encapsulation concentration of tetrahydrocurcumin (THC) is calculated based on [the relative content of tetrahydrocurcumin (THC)/the collection rate]. A drug encapsulation efficiency of the tetrahydrocurcumin (THC) is calculated based on [the encapsulation concentration of tetrahydrocurcumin (THC)/an initial tetrahydrocurcumin (THC) concentration (50 μM)].
In the current experiment, the quantitation of the polyphenolic compound is to obtain 50 μL of samples and the samples are respectively added to a glass tube; after evenly mixing with 1 mL of IN Folin-Ciocalteu's phenol regent and 1 mL of 7.5% sodium carbonate solution, a resulted solution is left at room temperature for 5 min; afterward, 4,500 μL of 20% sodium carbonate solution is added; after ultrasonic oscillation is performed, the resulted solution is left at room temperature for 10 min; centrifugation at 3000 rpm is performed for 10 min; the supernatant is withdrawn and the 735 nm absorbance of the supernatant is measured by UV-visible spectrophotometer (T-90 Spectrophotometer from PG Instruments Limited). The greater the absorbance, the greater the phenolic compound content. To determine the drug encapsulation efficiency, the phenolic compound content of the current experiment is calculated by calculating a relative content of tetrahydrocurcumin (THC) in the sample while comparing the standard curve of tetrahydrocurcumin (THC).
The result of the experiment is shown in
The actual particle numbers of the aquatic liposome (Asl-lipo) encapsulating tetrahydrocurcumin (THC) and with the particle size of 50-100 nm and 100-150 nm are calculated. The result of the experiment is shown in
Moreover, to determine that the ability of the aquatic liposome (Asl-lipo) in encapsulating tetrahydrocurcumin (THC), the polyphenolic compound content of the aquatic liposome (Asl-lipo) (0.05 mg/ml, 0.1 mg/ml, 0.2 mg/ml, 0.4 mg/ml, 0.6 mg/ml, 0.8 mg/ml, and 1.0 mg/ml) encapsulating tetrahydrocurcumin (THC) is measured by Folin-Ciocalteu method. After comparing with the standard curve of tetrahydrocurcumin (THC; 50 μM), the relative tetrahydrocurcumin (THC) content and the encapsulation efficiency (EE) of encapsulating tetrahydrocurcumin (THC) are calculated.
The result of the experiment is shown in
The current experiment is to determine the aquatic liposome's (Asl-lipo) capability in encapsulating other drugs. The current experiment includes the following steps: the aquatic liposome (Asl-lipo) is dissolved in phosphate buffered saline (PBS); then two dyes that cannot directly enter cells, including Annexin V and Propidium iodide (PI), are respectively added to the aquatic liposome (Asl-lipo); mixing is performed through the ultrasonic oscillation, while no ultrasonic oscillation is performed on a control group; after adding Annexin V and Propidium iodide (PI) to the aquatic liposome (Asl-lipo) for reacting over 2 hours, a mixture is observed by using a fluorescence microscope. In the experiment of the permeability of the aquatic liposome (Asl-lipo), it can be seen from the aforementioned experiment and analysis of the encapsulation efficiency that when a mass ratio of the aquatic liposome (Asl-lipo) (the mixed concentration is 0.2 mg/ml) to tetrahydrocurcumin (THC) (50 μM, MW=374.12) is 10:1, the maximum encapsulation efficiency of the aquatic liposome (Asl-lipo) could be achieved. The cells of the current experiment are retinal pigment epithelium cells (ARPE-19) provided by professors in Chung Shan Medical University.
More specifically, the current experiment is to perform Annexin V and Propidium iodide (PI) test on three groups of the retinal pigment epithelium cells (ARPE-19): (1) Mock: only 20 μg/ml of PI is added (
Referring to the result of the experiment shown in
The influence of the aquatic liposome (Asl-lipo) encapsulating tetrahydrocurcumin (THC) on the induced inflammatory response of the microglia (BV-2) is determined. The current experiment includes the following steps: the aquatic liposomes (Asl-lipo) with the mixed concentration of 0 mg/ml, 0.1 mg/ml, and 0.2 mg/ml and tetrahydrocurcumin (THC) with the effective amount of 0 μM, 12.5 μM, 25 μM, and 50 μM are provided; each of the aquatic liposomes (Asl-lipo) is mixed with the tetrahydrocurcumin (THC) with different effective amounts and then the ultrasonic oscillation is performed; each of the aquatic liposomes (Asl-lipo) encapsulating tetrahydrocurcumin (THC) is transported into the microglia (BV-2); afterwards, Pseudomonas aeruginosa (P. aeruginosa) Lipopolysaccharide (LPS) is added for reaction over 24 hours; the levels of inflammatory mediators related to the inflammation is analyzed, wherein the inflammatory mediators include NO (
The result of the experiment is shown in
Referring to
The current experiment makes use of the mock, the control group, and the experimental group processed with the microglia (BV-2) as aforementioned. In the experimental group, the aquatic liposome (Asl-lipo) (0.2 mg/ml) encapsulating tetrahydrocurcumin (THC) (12.5 μM, 25 μM, and 50 μM) is prepared. The mock, the control group, and the experimental group are processed with the growth medium actinodaphnine for 1 hour; afterwards, 10 μM of DCFH-DA is added for reaction over 30 min; rinsing with phosphate buffered saline (PBS) is performed, and then 500 μl of trypsin/0.01% EDTA is added and the Petri dish is left at 37° C. for reaction over 5 min; the cells are detached from the Petri dish; then a culture fluid is added to neutralize trypsin; centrifugation is performed and a supernatant is removed; the reactive oxygen species (ROS) contents of the microglia (BV-2) of the mock, the control group, and the experimental group are measured by the flow cytometer.
The result of the experiment is shown in
5. Determination of the Effect of the Aquatic Liposome (Asl-lipo) Encapsulating Tetrahydrocurcumin (THC) on the Reactive Oxygen Species (ROS) Production and Apoptotic Rate of the Retinal Pigment Epithelium Cells (ARPE-19) With Lesion Due to the Induction of Sodium Iodate (NaIO3)
In the current experiment, the mixed concentrations of the aquatic liposomes (Asl-lipo) are 0 mg/ml, 0.1 mg/ml, 0.2 mg/ml, and 0.4 mg/ml, and the effective amounts of tetrahydrocurcumin (THC) are 0 μM, 12.5 μM, 25 μM, and 50 μM. In the mock, phosphate buffered saline (PBS) is used. In the control group, the mixed concentration of the aquatic liposome (Asl-lipo) is 0 mg/ml and no tetrahydrocurcumin (THC) is provided. In the experimental group, the aquatic liposome (Asl-lipo) (0 mg/ml, 0.1 mg/ml, 0.2 mg/ml, and 0.4 mg/ml) encapsulating tetrahydrocurcumin (THC) (0 μM, 12.5 μM, 25 μM, and 50 μM) is prepared, and the ultrasonic oscillation at 40 kHz is performed for 20 min. The combination of the aquatic liposome (Asl-lipo) with the mixed concentration of 0-0.4 mg/ml and tetrahydrocurcumin (THC) with the effective amount of 0-50 μM is to determine the performance of the aquatic liposome (Asl-lipo) when only the aquatic liposome (Asl-lipo) is applied.
The retinal pigment epithelium cells (ARPE-19) are incubated in DMEM (Dulbecco's Modified Eagle Medium) at 37° C. and 5% CO2 until the cells are attached to the Petri dish; The microculture tetrazolium test includes the following steps: the retinal pigment epithelium cells (ARPE-19) are incubated at 2.5×105 cell/well in a 12-well plate; the mock, the control group, and the experimental group are respectively injected into the 12-well plate containing the retinal pigment epithelium cells (ARPE-19); the 12-well plate is left for reaction over 1.5 hours; afterwards, 6 mM of sodium iodate (NaIO3) is respectively added to the 12-well plate of the experimental group and the 12-well plate of the control group for induction over 24 hours;
The result of the experiment is shown in
The reactive oxygen species (ROS) is analyzed by DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate), a chemical fluorescence substance commonly used to measure H2O2. DCFH-DA could freely pass through the cell membrane. After DCFH-DA enters the cell, DCFH-DA is converted by esterase in the cell to DCFH; at that time, DCFH could not freely pass through the cell membrane and is remained within the cell. DCFH is oxidized by H2O2 in the cell to DCF (dichlorofluorescein) Upon the activation by the wavelength of 450-490 nm, fluorescence at the wavelength of 515-550 nm is emitted. The amount of H2O2 produced in the cells can be deduced by measuring the fluorescence content within the cells using flow cytometry.
The current experiment makes use of the mock, the control group, and the experimental group processed with the retinal pigment epithelium cells (ARPE-19) as aforementioned. In the experimental group, the aquatic liposome (Asl-lipo) (0 mg/ml, 0.1 mg/ml, and 0.2 mg/ml) encapsulating tetrahydrocurcumin (THC) (0 μM, 12.5 μM, 25 μM, and 50 μM) is prepared. The combination of the aquatic liposome (Asl-lipo) with the mixed concentration of 0-0.2 mg/ml and tetrahydrocurcumin (THC) with the effective amount of 0-50 μM is to determine the performance of the aquatic liposome (Asl-lipo) when only the aquatic liposome (Asl-lipo) is applied.
The result of the experiment is shown in
The apoptosis experiment is to observe tetrahydrocurcumin (THC) entering the cells and the apoptosis of the retinal pigment epithelium cells (ARPE-19) by using Annexin V/Propidium iodide (PI). As aforementioned, the apoptosis experiment uses the mock, control, and experimental groups observed by using Annexin V/Propidium iodide (PI). Flow cytometer (FACSCalibur flow cytometer) detects green fluorescence wavelength (Annexin V-FITC) at 490 nm˜525 nm and red fluorescence wavelength (PI) at 590 nm. Fluorescence quantitation is performed on the mock, the control group, and the experimental group. In the experimental group, the aquatic liposome (Asl-lipo) (0 mg/ml, 0.1 mg/ml, and 0.2 mg/ml) encapsulating the tetrahydrocurcumin (THC) (0 μM, 12.5 μM, 25 μM, and 50 μM) is prepared. The combination of the aquatic liposome (Asl-lipo) with the mixed concentration of 0-0.2 mg/ml and tetrahydrocurcumin (THC) with the effective amount of 0-50 μM is to examine the performance of the aquatic liposome (Asl-lipo) when only the aquatic liposome (Asl-lipo) is applied.
The cells of the mock, the control group, and the experimental group are classified into four quadrants based on the fluorescence performance of the standard sample: [Q3-1=Annexin V(−), PI(+): Q3-2=Annexin V(+), PI(+); Q3-3=Annexin V(−), PI(−); Q3-4=Annexin V(+), PI(−)]. The apoptotic rate is a relative percentage of late apoptotic cells Q3-2 to early apoptotic cells Q3-4.
The result of the experiment is shown in
It must be pointed out that the embodiments described above are only some preferred embodiments of the present invention. All equivalent structures which employ the concepts disclosed in this specification and the appended claims should fall within the scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 112108673 | Mar 2023 | TW | national |
