Construction method and application of novel fat tissue targeting sulforaphene liposome

Abstract

The invention relates to the technical field of sulforaphene liposomes, in particular to a construction method and application of novel targeted adipose tissue sulforaphene liposomes, and the construction method comprises the following steps: S1, taking quantitative synthetic DSPE-PEG2k-CKGGRAKDC, egg yolk lecithin and sulforaphene powder, and jointly dissolving in a chloroform / acetonitrile solution to prepare a thin film; s2, putting the prepared film in preheated ddH2O, and sequentially performing hydration and ultrasonic treatment to obtain a liposome solution; s3, filtering by using a 0.22 mu m filter membrane, standing at room temperature for 2 hours, dialyzing at room temperature by using a PBS (Phosphate Buffer Solution), freeze-drying and storing reaction liquid in a dialysis bag, and detecting the embedding rate of the sulforaphene by using an HPLC (High Performance Liquid Chromatography) detection method; s4, after the freeze-dried product is redissolved through ddH2O, a TEM sample is prepared, the formation condition of the sulforaphene lipidosome is detected, and the sulforaphene lipidosome of the targeted adipose tissue is obtained.The water stability and fat targeting performance of sulforaphene can be effectively improved.

Description

Technical Field

[0001] The invention relates to the technical field of radish extract liposomes, and in particular to a construction method of a novel radish extract liposome targeting adipose tissue and an application thereof. Background Art

[0002] Sulforaphene, an active ingredient widely found in the seeds of radish, a cruciferous plant, is the most potent anti-cancer active ingredient found in vegetables, and has good detoxification, antibacterial, and antioxidant effects. Compared to sulforaphene (C6H11NOS2), another isothiocyanate active ingredient that has been studied extensively, sulforaphene (C6H9NOS2) has only one more unsaturated double bond on the hydrocarbon chain, but is 1.3-1.5 times more active. The conjugated double bonds and isothiocyanate groups (—N=C=S) of sulforaphene make it unstable in water, alkaline conditions, and high temperature environments, greatly reducing its scope of use and efficiency. Figure 1 The degradation pathway of radish extract attacked by water molecules in aqueous solution was shown. The study also showed that radish extract showed better downregulation of lipid synthesis than sulforaphane at the cellular level. However, since the water stability of radish extract has yet to be solved, its lipid metabolism regulation mechanism needs further exploration. Therefore, how to prepare a stable and efficient delivery system to improve the water stability of radish extract, increase the bioavailability of radish extract, and systematically explain the fat-reducing mechanism of radish extract has very important research significance and economic value for expanding the application scope of radish extract in fat-reducing functional foods.

[0003] The Chinese invention patent application with application publication number CN116115576A discloses a functionalized dual-drug liposome nanoparticle and its preparation method and application, which specifically comprises the following steps: S1: weighing DSPE-PEG2000-NHS and dissolving it in DMF, then adding CNNQKIVNLKEKVAQLEA peptide and triethylamine to dissolve completely, and reacting at room temperature; S2: transferring the reaction solution to a dialysis bag, dialyzing it in pure water, and collecting the dialysate for freeze-drying to obtain DSPE-PEG2000-γ3 ; S3: weigh phospholipids, carboxylated cholesterol and DSPE-PEG2000-γ3, and dissolve them in chloroform respectively; S4: dissolve curcumin in ethanol, and then mix it with the substance obtained in S3; S5: remove the organic solvent in the mixture, rotary evaporate to form a film, then add ammonium sulfide to hydrate, and then dialyze with PB solution; S6: after dialysis, add ciprofloxacin hydrochloride for incubation, and then dialyze again to remove the unencapsulated ciprofloxacin hydrochloride; S7: add an appropriate amount of lyophilizer to the dialyzed liquid, and collect the lyophilized powder after freeze-drying. The antibiotic cyclamate hydrochloride is loaded in the hydrophilic core of the liposome, and the hydrophobic shell is loaded with the natural drug curcumin with hydrophobic antibacterial and anti-inflammatory effects, so as to finally realize the active targeting and responsiveness of the infection microenvironment of the liposome. However, the above-mentioned research scheme is not the same as the problem of improving the water stability of radish extract and increasing the bioavailability of radish extract to be solved in this application. Summary of the invention

[0004] Technical purpose: In order to overcome the deficiencies in the prior art, the present invention provides a novel method for constructing radish extract liposomes targeting adipose tissue and its application.

[0005] Technical solution: To achieve the above purpose, the present invention discloses a novel method for constructing radish extract liposomes targeting adipose tissue, comprising the following steps:

[0006] S1: Take a certain amount of synthetic DSPE-PEG2k-CKGGRAKDC, egg yolk lecithin, and radish extract powder and dissolve them in chloroform / acetonitrile solution, mix them evenly, and evaporate the organic solvent to form a film;

[0007] S2: placing the film prepared in step S1 in preheated ddH2O, hydrating under vacuum conditions, and ultrasonically treating the film to obtain a liposome solution;

[0008] S3: filtering the liposome solution through a 0.22 μm filter membrane, leaving it at room temperature for 2 h, transferring it to an 8KD dialysis bag, dialyzing it with a PBS solution at room temperature, freeze-drying the reaction solution in the dialysis bag, and detecting the radish extract embedding rate by HPLC;

[0009] S4: After the freeze-dried product is reconstituted with ddH2O, a TEM sample is prepared to detect the formation of radish peroxidase liposomes and obtain radish peroxidase liposomes targeting adipose tissue.

[0010] Preferably, in step S1, the concentration of DSPE-PEG2k-CKGGRAKDC in the mixed solution is 2 mg / mL, the concentration of egg yolk lecithin in the mixed solution is 4 mg / mL, the concentration of radish extract in the mixed solution is 0.4 mg / mL, and the volume ratio of chloroform to acetonitrile in the chloroform / acetonitrile solution is 1:1.

[0011] Preferably, the evaporation temperature of the evaporated organic solvent and the preheated ddH2O temperature are both 45°C, the hydration time is 2 hours, the ultrasonic treatment time is 10 minutes, the room temperature dialysis time using PBS solution is 1 day, and the dialysate is replaced every 8 hours.

[0012] Preferably, the synthesis steps of DSPE-PEG2k-CKGGRAKDC in step S1 are as follows:

[0013] S101: Dissolve a certain amount of DSPE-PEG2k-NHS in DMF to make the concentration of DSPE-PEG2k-NHS 33.3 mg / mL, add CKGGRAKDC peptide and triethylamine until completely dissolved, and react at room temperature for 24 hours;

[0014] S102: The reaction solution obtained in step S101 was transferred to a dialysis bag with a cutoff separation capacity of 1000 Da, dialyzed in pure water for 24 hours, and the dialysate was collected and freeze-dried to obtain DSPE-PEG2k-CKGGRAKDC.

[0015] The present invention also provides a targeted application of raphanin liposomes obtained based on a novel method for constructing raphanin liposomes targeting adipose tissue, and the testing steps are as follows:

[0016] S201: taking a certain amount of synthesized DSPE-PEG2k-CKGGRAKDC and egg yolk lecithin and dissolving them in a chloroform / acetonitrile solution to form a mixed solution, adding Cy7 dye and mixing evenly, and evaporating the organic solvent to form a film;

[0017] S202: placing the film prepared in step S201 in preheated ddH2O, hydrating under vacuum conditions, and ultrasonically treating the film to obtain a liposome solution;

[0018] S203: filtering the liposome solution obtained in step S202 through a 0.22 μm filter membrane, leaving it at room temperature for 2 hours, transferring it to an 8KD dialysis bag, dialyzing it at room temperature using a PBS solution, and freeze-drying the solution in the dialysis bag to obtain radish liposomes targeting adipose tissue and encapsulating Cy7 dye;

[0019] S204: The adipose tissue-targeted radish liposomes encapsulating Cy7 dye were reconstituted with ddH2O and injected into mice to monitor the biodistribution of Cy7.

[0020] Preferably, in step S201, the concentration of DSPE-PEG2k-CKGGRAKDC in the mixed solution is 2 mg / mL, the concentration of egg yolk phosphatidylcholine in the mixed solution is 4 mg / mL, the amount of Cy7 dye used is 0.2% of the volume of the mixed solution, and the volume ratio of chloroform to acetonitrile in the chloroform / acetonitrile solution is 1:1.

[0021] Preferably, the evaporation temperature of the evaporated organic solvent and the preheated ddH2O temperature are both 45°C, the hydration time is 2 hours, the ultrasonic treatment time is 10 minutes, the room temperature dialysis time using PBS solution is 1 day, and the dialysate is replaced every 8 hours.

[0022] The present invention also provides a fat-reducing application of radish liposomes obtained based on a novel method for constructing radish liposomes targeting adipose tissue, and the test steps are as follows:

[0023] S301: Adopting the adipocyte differentiation model of 3T3-L1 preadipocytes, 3T3-L1 cells were differentiated into adipocytes by MDI induction, and radish extract liposomes were treated during the differentiation process;

[0024] S302: Oil red staining of adipocytes was performed to monitor the accumulation of lipid droplets in adipocytes.

[0025] The beneficial effects of the present invention are:

[0026] 1. In the process of forming liposomes using (DSPE-PEG2k-CKGGRAKDC) and egg yolk phospholipids, radish extract is embedded in the liposomes, the isothiocyanate group of radish extract that is vulnerable to attack by water molecules is protected, and a new type of nanoparticles targeting adipose tissue is formed, which can effectively improve the water stability of radish extract, improve the fat targeting of radish extract, and improve its fat-reducing bioavailability.

[0027] 2. The radish peroxidase liposomes obtained based on the construction method of a new type of radish peroxidase liposomes targeting adipose tissue have good fat targeting properties and can be used as a potential adipose tissue targeting carrier for the delivery of various fat-reducing drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the degradation pathway of radish extract in water described in the present invention;

[0029] Figure 2 This is a synthetic reaction diagram of DSPE-PEG2k-CKGGRAKDC in the present invention;

[0030] Figure 3 It is the nuclear magnetic hydrogen spectrum of DSPE-PEG2k-CKGGRAKDC in the present invention;

[0031] Figure 4 This is a TEM picture of the radish extract liposome in the present invention;

[0032] Figure 5 HPLC high performance liquid phase chromatogram (254nm) of radish extract in the present invention;

[0033] Figure 6 This is a fluorescent imaging photo of mice in the present invention;

[0034] Figure 7 This is the lipid droplet generation of 3T3-L1 adipocytes in each treatment group of the present invention;

[0035] Figure 8 These are pictures of the adipose tissue of mice in each treatment group in the present invention. DETAILED DESCRIPTION

[0036] The following is combined with Figure 2 To Attachment Figure 8 The principles and features of the present invention are described, and the examples given are only used to explain the present invention, rather than to limit the scope of the present invention.

[0037] Embodiment 1:

[0038] A novel method for constructing radish extract liposomes targeting adipose tissue comprises the following steps:

[0039] S1: Take a certain amount of synthetic DSPE-PEG2k-CKGGRAKDC, egg yolk lecithin, and radish extract powder and dissolve them in chloroform / acetonitrile solution, mix them evenly, put the obtained reaction solution into a round-bottom flask, and use a rotary evaporator to evaporate the organic solvent at 45°C to form a thin film;

[0040] The concentration of DSPE-PEG2k-CKGGRAKDC in the mixed solution is 2 mg / mL, the concentration of egg yolk lecithin in the mixed solution is 4 mg / mL, the concentration of radish in the mixed solution is 0.4 mg / mL, and the volume ratio of chloroform to acetonitrile in the chloroform / acetonitrile solution is 1:1. In this embodiment, the amount of DSPE-PEG2k-CKGGRAKDC is 10 mg, the amount of egg yolk lecithin is 20 mg, the amount of chloroform / acetonitrile solution is 5 mL, and the amount of radish is 2 mg.

[0041] S2: The film prepared in step S1 is hydrated in preheated 45°C ddH2O under vacuum for 2 hours, and then ultrasonically treated for 10 minutes after hydration. The ultrasonic treatment requires a power of 50W, 3 seconds of operation, and 5 seconds of rest to obtain a liposome solution;

[0042] S3: The liposome solution was filtered through a 0.22 μm filter membrane, and then transferred to an 8KD dialysis bag after standing at room temperature for 2 h. The dialysis was performed at room temperature using a PBS solution. The dialysis time was 1 day, and the dialysate was replaced every 8 hours. The reaction solution in the dialysis bag was freeze-dried and stored, and the radish extract embedding rate was detected by HPLC.

[0043] The unencapsulated radix glycosides in the dialysate during the preparation of radix glycosides liposomes were detected by HPLC. After the liquid outside the dialysis membrane was concentrated, the concentration of radix glycosides was detected by HPLC, and the unencapsulated radix glycosides were calculated. The unencapsulated radix glycosides were deducted from the total amount of radix glycosides added during the preparation of radix glycosides liposomes. Figure 5 As shown, the embedding rate of radish extract was 73%. The embedding rate of radish extract liposome prepared by the above method was relatively high.

[0044] S4: After the freeze-dried product is reconstituted with ddH2O, a TEM sample is prepared to detect the formation of radish peroxidase liposomes and obtain radish peroxidase liposomes targeting adipose tissue.

[0045] like Figure 4 As shown, nano-radish liposomes can be successfully prepared by the above method. The measurement results of the particle size analyzer show that the average particle size of the radish liposomes is 120.5nm and the PDI is 0.23, indicating that the particle size uniformity is good.

[0046] like Figure 2 As shown, the synthesis steps of DSPE-PEG2k-CKGGRAKDC in step S1 are as follows:

[0047] S101: Dissolve a certain amount of DSPE-PEG2k-NHS in DMF to make the concentration of DSPE-PEG2k-NHS 33.3 mg / mL, add CKGGRAKDC peptide and triethylamine until completely dissolved, and react at room temperature for 24 hours; in this embodiment, the amount of DSPE-PEG2k-NHS is 100 mg, and the amount of DMF is 3 mL, wherein the molar ratio of DSPE-PEG2k-NHS to CKGGRAKDC in the CKGGRAKDC peptide is 1:1, and the molar ratio of DSPE-PEG2k-NHS to triethylamine in the triethylamine is 1:3.

[0048] S102: The reaction solution obtained in step S101 was transferred to a dialysis bag with a cut-off separation capacity of 1000Da, and dialyzed in pure water for 24 hours. The dialysate was replaced every 8 hours, and the liquid in the dialysis bag was collected and freeze-dried to obtain the product, i.e., DSPE-PEG2k-CKGGRAKDC. The obtained product was tested, and the obtained nuclear magnetic hydrogen spectrum was as follows: Figure 3 shown.

[0049] Embodiment 2:

[0050] A targeted application of raphanin liposomes obtained based on the construction method of the novel raphanin liposomes targeting adipose tissue, the test steps are as follows:

[0051] S201: Take a certain amount of synthesized DSPE-PEG2k-CKGGRAKDC and egg yolk lecithin and dissolve them in chloroform / acetonitrile solution to form a mixed solution, add Cy7 dye and mix evenly, place the reaction solution in a round-bottom flask, and use a rotary evaporator to evaporate the organic solvent at 45° C. to form a thin film.

[0052] Wherein, the concentration of DSPE-PEG2k-CKGGRAKDC in the mixed solution is 2 mg / mL, the concentration of egg yolk lecithin in the mixed solution is 4 mg / mL, the amount of Cy7 dye is 0.2% of the volume of the mixed solution, and the volume ratio of chloroform to acetonitrile in the chloroform / acetonitrile solution is 1: 1. In this embodiment, the amount of DSPE-PEG2k-CKGGRAKDC is 10 mg, the amount of egg yolk lecithin is 20 mg, the amount of chloroform / acetonitrile solution is 5 mL, and the amount of Cy7 dye is 10 μL.

[0053] S202: the film prepared in step S201 is hydrated in preheated 45° C. ddH2O under vacuum for 2 h, and then subjected to ultrasonic treatment for 10 min after hydration. The ultrasonic treatment requires a power of 50 W, 3 s of operation, and 5 s of rest, to obtain a liposome solution;

[0054] S203: The liposome solution obtained in step S202 is filtered through a 0.22 μm filter membrane, and after standing at room temperature for 2 hours, it is transferred to an 8KD dialysis bag, and dialyzed with a PBS solution at room temperature for 1 day, and the dialysate is replaced every 8 hours. The solution in the dialysis bag is freeze-dried and stored, and is protected from light throughout the process to obtain radish liposomes targeting adipose tissue and encapsulating Cy7 dye;

[0055] S204: The adipose tissue-targeted radish liposomes encapsulating Cy7 dye were reconstituted with ddH2O and injected into mice to monitor the biodistribution of Cy7.

[0056] Specifically, in this example, the radish liposomes targeting adipose tissue and encapsulating Cy7 dye were reconstituted with ddH2O and injected into the inguinal fat area of ​​the left abdomen of shaved mice. The mice were placed under a fluorescent imaging system at 1, 2, 8, 24, and 36 hours after injection to monitor the biodistribution of Cy7. The results are as follows: Figure 6As shown in the figure, most of the Cy7 embedded in the fat-targeted liposomes is enriched in the left inguinal fat area, and a small amount of Cy7 is detected in the right inguinal fat area. Cy7 can stay in the fat area for up to 36 hours, which shows that the carrier has good fat targeting and can be used as an adipose tissue targeting carrier for the delivery of various fat-reducing drugs.

[0057] Embodiment 3:

[0058] A fat-reducing application of radish liposomes obtained based on the construction method of the novel radish liposomes targeting adipose tissue, the test steps are as follows:

[0059] S301: Adopting the adipocyte differentiation model of 3T3-L1 preadipocytes, 3T3-L1 cells were differentiated into adipocytes by MDI induction, and radish extract liposomes were treated during the differentiation process;

[0060] S302: Oil red staining of adipocytes was performed to monitor the accumulation of lipid droplets in adipocytes.

[0061] Specifically, in order to verify the fat-reducing effect of raphanin liposomes, the fat differentiation model of 3T3-L1 preadipocytes was used. 3T3-L1 cells were differentiated into adipocytes using MDI induction. During the differentiation process, raphanin liposomes (PE-Lipo-SE) and raphanin (SE) were treated respectively. The raphanin treatment concentration was 5 μM. After the treatment, the adipocytes were stained with oil red to observe the generation of oil droplets in each treatment group. Figure 7 As shown, the results showed that the radish liposome treatment group obtained by the new method of constructing radish liposomes targeting adipose tissue had the lowest lipid droplet content in adipocytes and the fat reduction effect was better than that of the radish liposome treatment group alone.

[0062] To verify the in vivo fat-reducing activity of radish extract liposomes, an obese mouse model was established by feeding mice with a high-fat diet for one month. The obese mice induced by a high-fat diet were treated with radish extract (SE) or a new type of radish extract liposomes targeting adipose tissue (PE-Lipo-SE) by tail vein injection. The radish extract treatment amount was 0.5 mg / kg BW, once a day. After 30 days of treatment, white adipose tissues from major parts of the mice were collected for comparison. Figure 8As shown in the figure, the main adipose tissues of mice were taken out (E-iWAT: epididymal white fat; P-WAT: perirenal white adipose tissue; I-iWAT: inguinal adipose tissue; S-BAT: scapular brown adipose tissue), and photos were taken for comparison. The results showed that the adipose tissues of obese mice induced by high-fat diet (HFD-induced obese mice) were heavier than those of mice not induced by high-fat diet (NCmice), while the adipose tissues of obese mice treated with fat-targeted radix liposomes were lighter than those of obese mice, and the effect was better than that of the radix liposome group treated alone. The radix liposomes obtained based on the construction method of the new radix liposome targeting adipose tissue can effectively improve the water stability of radix liposomes, improve the fat targeting of radix liposomes, and improve its fat-reducing bioavailability.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a novel radish extract liposome targeting adipose tissue, characterized in that: The following steps are involved: S1: Take a certain amount of synthetic DSPE-PEG2k-CKGGRAKDC, egg yolk lecithin, and radish extract powder and dissolve them in chloroform / acetonitrile solution, mix them evenly, and evaporate the organic solvent to form a film; S2: placing the film prepared in step S1 in preheated ddH2O, hydrating under vacuum conditions, and ultrasonically treating the film to obtain a liposome solution; S3: filtering the liposome solution through a 0.22 μm filter membrane, leaving it at room temperature for 2 h, transferring it to an 8KD dialysis bag, dialyzing it with a PBS solution at room temperature, freeze-drying the reaction solution in the dialysis bag, and detecting the radish extract embedding rate by HPLC; S4: After the freeze-dried product is reconstituted with ddH2O, a TEM sample is prepared to detect the formation of radish peroxidase liposomes and obtain radish peroxidase liposomes targeting adipose tissue.

2. The method for constructing a novel radish extract liposome targeting adipose tissue according to claim 1, characterized in that: In step S1, the concentration of DSPE-PEG2k-CKGGRAKDC in the mixed solution is 2 mg / mL, the concentration of egg yolk phosphatidylcholine in the mixed solution is 4 mg / mL, the concentration of radish extract in the mixed solution is 0.4 mg / mL, and the volume ratio of chloroform to acetonitrile in the chloroform / acetonitrile solution is 1:

1.

3. The method for constructing a novel radish extract liposome targeting adipose tissue according to claim 2, characterized in that: The evaporation temperature of the evaporated organic solvent and the preheated ddH2O temperature were both 45°C, the hydration time was 2 hours, the ultrasonic treatment time was 10 minutes, the room temperature dialysis time using PBS solution was 1 day, and the dialysate was replaced every 8 hours.

4. The method for constructing a novel radish extract liposome targeting adipose tissue according to claim 3, characterized in that: The synthesis steps of DSPE-PEG2k-CKGGRAKDC described in step S1 are as follows: S101: Dissolve a certain amount of DSPE-PEG2k-NHS in DMF to make the concentration of DSPE-PEG2k-NHS 33.3 mg / mL, add CKGGRAKDC peptide and triethylamine until completely dissolved, and react at room temperature for 24 hours; S102: The reaction solution obtained in step S101 was transferred to a dialysis bag with a cutoff separation capacity of 1000 Da, dialyzed in pure water for 24 hours, and the dialysate was collected and freeze-dried to obtain DSPE-PEG2k-CKGGRAKDC.

5. A targeted application of raphanin liposomes obtained by the construction method of any one of claims 1 to 4 for novel raphanin liposomes targeting adipose tissue, characterized in that: The test steps are as follows: S201: taking a certain amount of synthesized DSPE-PEG2k-CKGGRAKDC and egg yolk lecithin and dissolving them in a chloroform / acetonitrile solution to form a mixed solution, adding Cy7 dye and mixing evenly, and evaporating the organic solvent to form a film; S202: placing the film prepared in step S201 in preheated ddH2O, hydrating under vacuum conditions, and ultrasonically treating the film to obtain a liposome solution; S203: filtering the liposome solution obtained in step S202 through a 0.22 μm filter membrane, leaving it at room temperature for 2 hours, transferring it to an 8KD dialysis bag, dialyzing it at room temperature using a PBS solution, and freeze-drying the solution in the dialysis bag to obtain radish liposomes targeting adipose tissue and encapsulating Cy7 dye; S204: The adipose tissue-targeted radish liposomes encapsulating Cy7 dye were reconstituted with ddH2O and injected into mice to monitor the biodistribution of Cy7.

6. The targeted application of radish liposomes obtained by the method for constructing a novel radish liposome targeting adipose tissue according to claim 5, characterized in that: In step S201, the concentration of DSPE-PEG2k-CKGGRAKDC in the mixed solution is 2 mg / mL, the concentration of egg yolk phosphatidylcholine in the mixed solution is 4 mg / mL, the amount of Cy7 dye used is 0.2% of the volume of the mixed solution, and the volume ratio of chloroform to acetonitrile in the chloroform / acetonitrile solution is 1:

1.

7. The targeted application of raphanin liposomes obtained by the method for constructing the novel raphanin liposomes targeting adipose tissue according to any one of claims 1 to 4 according to claim 6, characterized in that: The evaporation temperature of the evaporated organic solvent and the preheated ddH2O temperature were both 45°C, the hydration time was 2 hours, the ultrasonic treatment time was 10 minutes, the room temperature dialysis time using PBS solution was 1 day, and the dialysate was replaced every 8 hours.

8. A fat-reducing application of radish liposomes obtained by the method for constructing a novel radish liposome targeting adipose tissue according to any one of claims 1 to 4, characterized in that: The test steps are as follows: S301: Adopting the adipocyte differentiation model of 3T3-L1 preadipocytes, 3T3-L1 cells were differentiated into adipocytes by MDI induction, and radish extract liposomes were treated during the differentiation process; S302: Oil red staining of adipocytes was performed to monitor the accumulation of lipid droplets in adipocytes.

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