CN113262310A - Pullulan-modified ginsenoside Rg3Nanostructured lipid carriers and methods of making the same - Google Patents
Pullulan-modified ginsenoside Rg3Nanostructured lipid carriers and methods of making the same Download PDFInfo
- Publication number
- CN113262310A CN113262310A CN202110230216.7A CN202110230216A CN113262310A CN 113262310 A CN113262310 A CN 113262310A CN 202110230216 A CN202110230216 A CN 202110230216A CN 113262310 A CN113262310 A CN 113262310A
- Authority
- CN
- China
- Prior art keywords
- nlc
- pul
- ginsenoside
- modified
- group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Abstract
The invention discloses a pulullan modified ginsenoside Rg3A Nanostructured Lipid Carrier (NLC) is prepared by mixing ginsenoside Rg with ginsenoside carrier3Inside the coating, the outer side of the nano-structured lipid carrier is modified by Pullulan (PUL) and the adhesiveness of the nano-structured lipid carrier is increased. The invention constructs the pulullan Polysaccharide (PUL) modified ginsenoside Rg3The nanostructured lipid carrier can be retained in stomach tissue for a long time to promote ginsenoside Rg3Is absorbed mainly through the fine poresThe endocytosis pathway mediated by the caveolae-like pits promotes the intake of human gastric adenocarcinoma BGC-823 cells. The invention has the stomach adhesion property, can be retained in stomach tissues for a long time, promotes the absorption of gastric cancer cells to medicines and improves the anti-tumor effect.
Description
Technical Field
The invention relates to the field of pharmacy, in particular to pullulan modified ginsenoside Rg3Nanostructured lipid carriers and methods of making the same.
Background
Ginseng is the dry root of Panax ginseng of Panax of Araliaceae, is a traditional and famous Chinese medicinal material, has effects of invigorating primordial qi, invigorating qi, benefiting lung, tranquilizing mind and improving intelligence, has a long medicinal history, and is a reputation of Bai Cao Yao Wang, wherein ginsenoside is the main effective component in Ginseng radix. In recent years, researches show that the ginsenoside Rg in the chemical components of the ginsenoside Rg3Has obvious anti-tumor effect. Clinically, ginsenoside Rg3The monomer preparation, Shenyi Capsule, is widely applied to various tumors in combination with chemotherapy. However, ginsenoside Rg3The solubility in water is low, and the oral administration absorption effect is poor, so that the clinical application of the medicine is limited.
Disclosure of Invention
The invention aims to overcome the problems and provides a pulullan modified ginsenoside Rg3Nanostructured lipid carriers and methods of making the same. In order to achieve the purpose, the invention adopts the following technical scheme:
pullulan-modified ginsenoside Rg3Nanostructured lipid carrier, wherein the Nanostructured Lipid Carrier (NLC) converts ginsenoside Rg3Wrapped inside, the nanostructured lipid carrier is externally modified with Pullulan (PUL) and increases its adhesiveness.
The invention also discloses a pulullan modified ginsenoside Rg3The preparation method of the nano-structure lipid carrier comprises the following steps:
s1, glycerin monostearate and ginsenoside Rg3Adding phospholipid and linoleic acid into an absolute ethyl alcohol solvent, and heating and dissolving to obtain an organic phase;
s2, dissolving the PUL and the TPGS together in water;
s3, the organic phase obtained in S1 is at 1000-1400r/mSlowly injecting the PUL obtained in S2 and the TPGS mixed solution with the stirring of in; stirring at constant temperature of 60-70 deg.C to volatilize organic solvent, obtaining semitransparent emulsion after 2 hr, and performing ultrasonic mixing treatment to obtain PUL-Rg3-NLC dispersions.
As an improvement, the glycerin monostearate and the ginsenoside Rg in the S13The mass ratio of the phospholipid to the linoleic acid is 60:16:20: 19.
As an improvement, the ginsenoside Rg3The mass ratio to PUL was 8: 11.
The invention has the advantages that:
the invention constructs the pulullan Polysaccharide (PUL) modified ginsenoside Rg3The lipid carrier with nanostructure can improve ginsenoside Rg3The adhesive property of the cell can ensure that the cell can be retained in the stomach tissue for a long time, and the cell mainly promotes the intake of human gastric adenocarcinoma cell BGC-823 cells through an endocytosis pathway mediated by a cell cavernous depression.
The invention has the stomach adhesion property, can be retained in stomach tissues for a long time, promotes the absorption of gastric cancer cells to medicines and improves the anti-tumor effect.
Drawings
FIG. 1 shows Rg3-NLC (A) and PUL-Rg3-particle size distribution of nlc (b);
FIG. 2 shows Rg3-NLC (A) and PUL-Rg3-transmission electron microscopy of nlc (b);
FIG. 3 shows the fluorescence intensity observed on tissue sections of the stomach, small intestine and colon in the C6 group, C6-NLC group and PUL-C6-NLC group after 4,12,24 and 48 hours;
FIG. 4 shows fluorescence intensities of supernatant of homogenate of tissues in three parts of stomach, small intestine and colon in C6, C6-NLC and PUL-C6-NLC groups after 4h (A),12h (B), 24h (C), 48h (D)
FIG. 5 is a confocal observation and flow chart (D) of BGC-823 cell uptake by different experimental groups after incubation for 4h (A),8h (B), 12h (C);
FIG. 6 shows the different examples after 8h incubationTest component group for confocal observation (A), flow chart (B) and flow quantitative analysis chart of BGC-823 cell uptake
FIG. 7 shows Rg3(A)，Rg3-NLC (B) and PUL-Rg3Effect of NLC (C) on BGC-823 cell proliferation and IC 72h after each experimental group dosing50(D)；
FIG. 8 shows control group, Rg3Group, Rg3Group of-NLC and PUL-Rg3-flow chart (A) of BGC-823 cell apoptosis induction by NLC group and quantitative analysis (B) of apoptosis rate;
FIG. 9 shows a cell scratch experiment (A) and a scratch healing rate analysis (B) of BGC-823 cells after receiving different treatments;
FIG. 10 shows the cell invasion (A) and quantification (B) of BGC-823 cells after different treatments.
Detailed Description
The present invention will be described in detail and specifically with reference to the following examples so that the present invention may be better understood, but the following examples do not limit the scope of the present invention.
Example 1
The embodiment discloses a pulullan modified ginsenoside Rg3The preparation method of the nano-structure lipid carrier comprises the following steps:
s1, 60mg of glyceryl monostearate and 16mg of ginsenoside Rg3Adding 20mg of phospholipid and 19mg of linoleic acid into 10mL of absolute ethyl alcohol solvent, and heating and dissolving to obtain an organic phase;
s2, dissolving 22mgPUL and 100mgTPGS together in 10mL of water;
s6, slowly injecting the organic phase obtained in the step S1 into the mixed solution of PUL and TPGS obtained in the step S2 under the stirring of 1200 r/min; stirring at constant temperature of 60-70 deg.C to volatilize organic solvent, obtaining semitransparent emulsion after 2 hr, and performing ultrasonic mixing treatment to obtain PUL-Rg3-NLC dispersions.
In this embodiment, a comparison group is set, and the preparation method of the comparison group is as follows:
s1, glycerin monostearate and ginsenoside Rg3Adding phospholipid and linoleic acid into an absolute ethyl alcohol solvent, and heating and dissolving to obtain an organic phase;
s2, dissolving TPGS in water to obtain a water phase;
s3, slowly injecting the organic phase into the water phase under the stirring of 1200 r/min; stirring at constant temperature of 40-70 deg.C to volatilize organic solvent, and obtaining translucent emulsion after 2 hr;
s4, carrying out ultrasonic mixing treatment on the semitransparent emulsion to obtain Rg3-NLC dispersions.
Then, the control group was compared with PUL-Rg obtained in example 13-experimental comparison of NLC dispersions:
1. particle diameter and Zeta potential measurement
Under the condition of room temperature, respectively taking a proper amount of Rg3-NLC dispersion liquid and a proper amount of PUL-Rg3-NLC dispersion liquid, diluting the dispersion liquid to a proper concentration through deionized water, placing the dispersion liquid in a sample cell, and respectively measuring the particle size and the Zeta potential by using a laser particle size analyzer. The result shows that the particle size of Rg3-NLC is (157 +/-7.12) nm, the PDI is 0.145 +/-0.0187, and the Zeta potential is (-15.93 +/-0.91) mV; PUL-Rg3-NLC has particle size of (102 + -1.89) nm, PDI of 0.294 + -0.0465, and Zeta potential of (-11.57 + -0.87) mV.
The prepared Rg3-NLC and PUL-Rg3-NLC are both spherical and round, and the average particle sizes are respectively about 100 nm and 50nm, as shown in figure 2. Before observation of a transmission electron microscope, the Rg3-NLC and PUL-Rg3-NLC dispersion liquid is fully dried by a copper net, and a hydration layer is removed, so that the average particle size measured by the transmission electron microscope is about 50nm smaller than that measured by a laser particle size analyzer.
In vivo adhesion study of PUL-Rg3-NLC
(1) Preparation of fluorescence-labeled common nanostructured lipid carrier and fluorescence-labeled pullulan-modified nanostructured lipid carrier
Using C6 as a fluorescent probe, and the remaining adjuvants were kept unchanged, according to the preparation method in example 1, replacing the ginsenoside Rg3 component with 5mgC6, and the other steps were unchanged, to prepare a fluorescently-labeled ordinary nanostructured lipid carrier dispersion, labeled as C6-NLC. And adding PUL into the water phase according to the same method to prepare a fluorescence-labeled PUL-modified nanostructured lipid carrier dispersion liquid which is labeled as PUL-C6-NLC. The whole operation process is protected from light.
(2) In vivo adhesion
BALB/c mice (body mass 18-22 g), fasting for 12h before experiment, freely drinking water, 15min before anesthesia, gavage and feeding 0.5mL of PBS solution with pH7.4, and flushing gastrointestinal tract. The control group was intragastrically administered with a physiological saline suspension of C6, and the remaining 2 groups were intragastrically administered with a C6-NLC dispersion and a PUL-C6-NLC dispersion (the dosage of C6 was 5mg kg. multidot.kg)－1). The mice were anesthetized with ether 4,12,24,48h after administration, 3 mice were taken at each time point, the abdominal cavity was opened, the stomach, small intestine and colon parts of the mice were quickly cut off, cleaned with 37 ℃ Techno's solution, surface moisture was blotted with filter paper, and the mice were stored at-80 ℃ after precisely weighing the mass. Equal amount of tissue was taken, stained with DAPI for nuclei, and the fluorescence intensity of the drug in each tissue section was observed with a confocal laser microscope. The above experimental operations were repeated, and the supernatant was further homogenized from the tissue and organ and the fluorescence intensity of the supernatant was measured by a fluorescence microplate reader.
After 4h administration, the free C6 group was less absorbed by the stomach, small intestine, and colon, as shown in fig. 3. After C6-NLC is formed, the ingestion of C6 by stomach, small intestine and colon is obviously increased, which shows that the common nano-structure lipid carrier has absorption promoting effect on 3 parts when the loaded drug is absorbed by oral administration. After the PUL-modified nano-structure lipid carrier (PUL-NLC) is subjected to fluorescence labeling to form PUL-C6-NLC, the absorption promoting effect in the stomach, small intestine and colon is further enhanced when the loaded drug is subjected to oral absorption evaluation. After 12h administration, the accumulation of C6, C6-NLC and PUL-C6-NLC in the stomach peaked compared to the other groups. After 24 and 48h of administration, C6 in the C6 group and the C6-NLC group had weak accumulation in each tissue, while PUL-C6-NLC group still had some accumulation, indicating that PUL-NLC has adhesiveness and can stay in tissue and organ for a longer time.
After 4h of administration, the fluorescence intensity of supernatant of homogenate of stomach tissue of PUL-C6-NLC group is 1.74 times and 1.39 times of that of C6 group and C6-NLC group respectively, which shows that after PUL-C6-NLC is formed, the absorption of drug loaded in stomach tissue can be remarkably promoted, and the figure 4 shows that. After 12h, the uptake by gastric tissue was significantly reduced for the group C6, by 44.33% compared to 4h, probably due to gastric emptying; the counter-observation groups C6-NLC and PUL-C6-NLC, which are still more potent in gastric uptake, may be the result of the formation of nanostructured lipid carriers to facilitate uptake and increase retention. The fluorescence intensity of the tissue in the stomach was 1.47 times that of the group C6-NLC in the group PUL-C6-NLC. After 24h of administration, the fluorescence intensity of the C6 group, the C6-NLC group and the PUL-C6-NLC group in the stomach is obviously reduced, but compared with the C6 group and the C6-NLC group, the fluorescence intensity of the supernatant of the tissue homogenate of the stomach in the PUL-C6-NLC group is still strongest, which indicates that the adhesion nano-structure lipid carrier (PUL-NLC) has certain adhesiveness and can be remained in the stomach tissue for a longer time. The 48h results showed that the C6, C6-NLC and PUL-C6-NLC groups were not significantly different in each tissue. In addition, the ingestion and accumulation of PUL-C6-NLC in the stomach are significantly better than those in the small intestine and colon, so that the adhesion of PUL-C6-NLC under gastric acidic conditions is presumed to be stronger than that under alkaline conditions in the small intestine and colon. In conclusion, gastric adenocarcinoma cells were selected for further study.
(3) In-vitro uptake of PUL-C6-NLC by human gastric adenocarcinoma BGC-823 cells and mechanism evaluation thereof
Laser confocal as shown in fig. 5, human gastric adenocarcinoma BGC-823 cell had weak uptake ability of C6, and after incubation for 4h with the cells, it showed only weak green fluorescence (fig. 5A), and the fluorescence intensity gradually increased with the increase of incubation time (fig. 5B, 5C). The common Nanostructured Lipid Carrier (NLC) also shows the function of promoting the uptake of C6 by BGC-823 cells, and shows that the NLC has the absorption promoting effect on loaded drugs. Compared with common nanoparticles, the adhesion nanostructure lipid carrier (PUL-NLC) can remarkably promote the uptake of BGC-823 cells, and shows that PUL-NLC has a more remarkable absorption promoting effect on loaded drugs.
As shown in FIG. 5D, after the cells were incubated with the drug for 4h, the average fluorescence intensity of PUL-C6-NLC group was increased by 9.11 and 1.09 times, respectively, compared with those of C6 and C6-NLC groups; after 8 and 12h incubation, the fluorescence intensity of the PUL-C6-NLC group is 1411.34 and 1522.47 respectively, and the related result shows that the uptake of PUL-C6-NLC by BGC-823 cells is close to saturation. However, the fluorescence intensity of the C6 group and the C6-NLC group increased significantly with time, reaching 649.06 and 1081.37 respectively, but still lower than that of the PUL-C6-NLC group at the same time. The result shows that the PUL modified NLC obviously improves the amount of the medicine entering BGC-823 cells, increases the medicine intake, has a saturation phenomenon in the intake, and is nearly saturated after being incubated for 8 hours.
In order to further evaluate the cellular uptake mechanism of PUL-C6-NLC, hypertonic sucrose (clathrin-mediated endocytosis inhibitor), amiloride (inhibitor of the macropinocytic pathway) and nystatin (cytocave-like recess-mediated endocytosis inhibitor) were selected for experiments to study the uptake conditions.
In the laser confocal mode shown in FIG. 6A, after BGC-823 cells were pre-incubated for 8h with 3 inhibitors, the inhibition of cellular uptake occurred in 3 experimental groups to which the chemical inhibitor was added, compared to the PUL-C6-NLC group. Wherein the inhibitory effect of the group "PUL-C6-NLC + nystatin" is that of the group "PUL-C6-NLC + amiloride" or that of the group "PUL-C6-NLC + hypertonic sucrose". The result shows that 3 pathways all mediate BGC-823 cell uptake, wherein the endocytosis pathway mediated by the caveolae-like recess of the cell is superior to the endocytosis pathway mediated by the macropinocytic pathway and is superior to the endocytosis pathway mediated by the clathrin structure. The endocytosis pathway mediated by the caveolae is probably the main pathway for PUL-C6-NLC uptake by BGC-823 cells.
Flow quantitative analysis is shown in FIGS. 6B and 6C, compared with the PUL-C6-NLC group, after incubation with nystatin, the uptake of PUL-C6-NLC by BGC-823 cells can be significantly inhibited, the uptake inhibition rate of C6 is 48.8%, after incubation with amiloride, the uptake inhibition rate of C6 is 37.3%, and after incubation with hypertonic sucrose, the uptake inhibition rate of C6 is 22.8%. The related quantitative analysis result is consistent with the qualitative analysis result. Quantitative analysis further proves that the BGC-823 cell uptake mechanism of PUL-C6-NLC is related to hyperosmotic sucrose, amiloride and nystatin inhibition pathways, and a cell-cave-like depression-mediated pathway is probably the main pathway for the uptake of PUL-C6-NLC by BGC-823 cells.
Detection of cell proliferation by MTT method
Human gastric adenocarcinoma BGC-823 cells in logarithmic growth phase are taken, digested, counted and prepared into 5 cells104Cell suspension per mL, 100 μ L of cell suspension per well in a 96-well cell culture plate; the 96-well cell culture plate was placed at 37 ℃ in 5% CO2Culturing for 24h in an incubator; diluting the drug to the required concentration by using complete culture medium, adding 100 mu L of culture solution containing the drug into each hole of the experimental group, adding 100 mu L of culture medium containing 0.1% DMSO into each hole of the blank group, and respectively adding Rg3, Rg3-NLC or PUL-Rg3-NLC with different concentrations. The 96-well cell culture plate was placed at 37 ℃ in 5% CO2After 72 hours in the incubator, the 96-well plate was MTT-stained at λ 490nm, and the absorbance a was measured. Add 20. mu. LMTT (5 mg. mL) per well－1) Continuously culturing for 4h in the incubator; discard the medium, add 150 μ l of LDMSO solution per well, mix gently by shaking for 10 min. The microplate reader reads a per well at λ ═ 490nm and calculates the inhibition for each experimental group:
the inhibition rate was [ (a blank group-a experimental group)/a blank group ] × 100%.
Calculating the IC 72h after each experiment is added with medicine in groups50Calculating IC by probability unit weighted regression using SPSS (StaffticalPackagefor the social science)24.050。
MTT results show that after human gastric adenocarcinoma BGC-823 cells are subjected to different treatments of Rg3, Rg3-NLC and PUL-Rg3-NLC experimental groups respectively, as shown in FIGS. 7A-7C, Rg3, Rg3-NLC and PUL-Rg3-NLC, the cytotoxicity of BGC-823 cells is dose-dependent, namely, the higher the administration concentration is, the higher the inhibition rate of tumor cells is, and the lower the survival rate of tumor cells is. The inhibition effect on BGC-823 cells can be increased after the loading of the Rg3 by the NLC, and the inhibition effect on the BGC-823 cells is further enhanced after the modification by the pullulan. IC for Rg3 group for BGC-823 cells5050.629. mu.g/mL－1The Rg3-NLC group is 37.113 mu g/mL－1The PUL-Rg3-NLC group is 29.657 mu g/mL－1See fig. 7D. IC of PUL-Rg3-NLC group compared to Rg3 group and Rg3-NLC group50The reduction is 41.4% and 20.1% respectively, which shows that the pullulan modified nanostructured lipid carrier has the strongest antitumor activity compared with Rg3 group and Rg3-NLC group.
4. Apoptosis of cells
Get pairInoculating BGC-823 cells of human gastric adenocarcinoma in several growth stages into 6-well plates, adding non-medicated culture medium, Rg3, Rg3-NLC and PUL-Rg3-NLC culture medium according to group design the next day, incubating for 72h, collecting cells, washing cells with PBS for 2 times (1000 r.min.)－1Centrifugation for 5min) collection 5X 105Adding 500 mu LBindingBuffer suspension cells into the cells, adding 5 mu LannexinV-FITC, uniformly mixing, adding 5 mu LPropidiumIodide, uniformly mixing, reacting for 5-15 min at room temperature in a dark place, detecting apoptosis by using a flow cytometer, and calculating the apoptosis rate.
The BGC-823 cell apoptosis rate of the control group is only 2.19% + -0.31%, and no significant apoptosis exists, while after Rg3 administration, the BGC-823 cell apoptosis rate is significantly increased to 19.81% + -2.71%, the BGC-823 cell apoptosis rate of the Rg3-NLC group is 29.28% + -4.11%, and the BGC-823 cell apoptosis rate of the PUL-Rg3-NLC group is 39.39% + -4.95%, as shown in FIG. 8. The result shows that the Rg3 group can remarkably induce BGC-823 apoptosis; after the Rg3-NLC and the PUL-Rg3-NLC are formed, compared with the Rg3 group, the apoptosis induction effect is improved by 47.8 percent and 98.9 percent respectively, and the PUL modified nanostructure lipid carrier has a more remarkable apoptosis induction effect.
5. Cell scoring
Adding about 5X 10 to 6-well plate5The fusion rate of the individual gastric adenocarcinoma BGC-823 cells reaches 100% after overnight. On day 2, the cell layer was scratched with a tip perpendicular to the cell plane along the line drawn on the back of the plate. After the scratch is completed, the cells are washed for 3 times by using sterile PBS, nonadherent cells are washed away, gaps left after the scratch are clearly visible, the cells are placed under a microscope for photographing and recording (0h) and the width of the scratch is measured, and then the 6-hole plate is placed in a cell incubator for continuous culture. Adding fresh serum-free medium diluted medicine, dividing into 4 groups, respectively administering non-medicated culture medium, Rg3, Rg3-NLC and PUL-Rg3-NLC, placing cells at 37 deg.C and 5% CO2Culturing in an incubator, taking out cells after 24h, observing and measuring the width of the scratch by a microscope line, photographing, and calculating the average value of the distances between the cells to obtain the width of the scratch. The healing condition of the blank group and the administration group after cell scratching is observed. Calculating the healing rate of the scratch according to a formulaScar healing rate ═ 1- (24h scratch width/0 h scratch width)]X 100%. Compared with the control group, the scaring healing rates of the Rg3 group, the Rg3-NLC group and the PUL-Rg3-NLC group have the effect of inhibiting the healing of tumor cells, and the figure 9 shows; compared with the Rg3 group, the cell scratch healing rate of the Rg3-NLC group and the PUL-Rg3-NLC group is reduced by 30.3 percent and 47.8 percent respectively. Results show that the Rg3 group, the Rg3-NLC group and the PUL-Rg3-NLC group can obviously reduce the migration capacity of BGC-823 of human gastric adenocarcinoma cells, and the PUL modified nanostructured lipid carrier has the best inhibition effect on the migration of BGC-823 cells.
6. Cell invasion
The invasiveness of the BGC-823 cells was examined by the Transwell method. Cells were serum deprived and starved for 24h using incomplete medium. The Matrigel was thawed overnight at 4 ℃, the thawed Matrigel gel was diluted one-fold using incomplete medium, 30. mu.L of diluted Matrigel was added to the upper chamber of the Transwell, and incubated at 37 ℃ for 120min to polymerize the Matrigel to a gel. Taking human gastric adenocarcinoma BGC-823 cells in logarithmic growth phase according to 3 × 105Density of cells per well cells were seeded in 6-well plates and cultured for 24 h. After the cells were attached to the wall, the medium was aspirated and treated with Rg3, Rg3-NLC and PUL-Rg3-NLC, respectively, for 6 groups. After 24h of culture, the cells were digested, counted and the cell density was adjusted to 1X 10 using incomplete medium5one/mL, 100. mu.L of the cell suspension was added to a Transwell chamber, and 500. mu.L of a medium containing 20% FBS was added to the lower chamber. The 24-well cell culture plate was placed at 37 ℃ in 5% CO2Culturing for 72h in an incubator. The matrigel and cells in the upper chamber were wiped off with a cotton swab, the Transwell was removed, inverted, air dried, 500 μ L of 0.1% crystal violet was added to a 24-well plate, the chamber was placed in it, the membrane was immersed in the dye, taken out after 30min, washed with PBS, photographed in 3 fields in diameter (magnification 200) and counted.
Compared with the control group, the cell numbers of the Rg3 group, the Rg3-NLC group and the PUL-Rg3-NLC group are very different, and the figure 10 shows that the cell numbers of the two groups are different; compared with the group of Rg3-NLC, the group of PUL-Rg3-NLC also has very significant difference, and the number of cells is reduced by 48.6%. The results show that both Rg3-NLC and PUL-Rg3-NLC can reduce the invasion capacity of BGC-823 cells of human gastric adenocarcinoma, and the inhibition effect of PUL modified PUL-Rg3-NLC on the invasion capacity of BGC-823 cells is very obviously superior to that of Rg3-NLC which is not modified by PUL.
The embodiments of the present invention have been described in detail above, but they are merely exemplary, and the present invention is not equivalent to the above described embodiments. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, it is intended that all equivalent alterations and modifications be included within the scope of the invention, without departing from the spirit and scope of the invention.
Claims (4)
1. Pullulan-modified ginsenoside Rg3The nanostructured lipid carrier is characterized in that the Nanostructured Lipid Carrier (NLC) is used for carrying out ginsenoside Rg3Wrapped inside, the nanostructured lipid carrier is externally modified with Pullulan (PUL) and increases its adhesiveness.
2. A pullulan-modified ginsenoside Rg according to claim 13The preparation method of the nanostructured lipid carrier is characterized by comprising the following steps:
s1, glycerin monostearate and ginsenoside Rg3Adding phospholipid and linoleic acid into an absolute ethyl alcohol solvent, and heating and dissolving to obtain an organic phase;
s2, dissolving the PUL and the TPGS together in water;
s3, slowly injecting the organic phase obtained in the step S1 into the mixed solution of PUL and TPGS obtained in the step S2 under the stirring of 1000-1400 r/min; stirring at constant temperature of 60-70 deg.C to volatilize organic solvent, obtaining semitransparent emulsion after 2 hr, and performing ultrasonic mixing treatment to obtain PUL-Rg3-NLC dispersions.
3. A pullulan modified ginsenoside Rg according to claim 23The preparation method of the nanostructured lipid carrier is characterized in that the glyceryl monostearate and the ginsenoside Rg in the S13The mass ratio of the phospholipid to the linoleic acid is 60:16:20: 19.
4. A pullulan modified ginsenoside Rg according to claim 23The preparation method of the nanostructured lipid carrier is characterized in that the ginsenoside Rg is3The mass ratio to PUL was 8: 11.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110230216.7A CN113262310A (en) | 2021-03-02 | 2021-03-02 | Pullulan-modified ginsenoside Rg3Nanostructured lipid carriers and methods of making the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110230216.7A CN113262310A (en) | 2021-03-02 | 2021-03-02 | Pullulan-modified ginsenoside Rg3Nanostructured lipid carriers and methods of making the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113262310A true CN113262310A (en) | 2021-08-17 |
Family
ID=77228270
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110230216.7A Pending CN113262310A (en) | 2021-03-02 | 2021-03-02 | Pullulan-modified ginsenoside Rg3Nanostructured lipid carriers and methods of making the same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113262310A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4636381A (en) * | 1982-05-19 | 1987-01-13 | Eisai Co., Ltd. | Coated ubidecarenone-containing liposome |
| CN101254309A (en) * | 2008-04-11 | 2008-09-03 | 中国医学科学院生物医学工程研究所 | Folacin receptor mediated targeted acetyl pullulan polysaccharide nano granule and preparation thereof |
| WO2010111807A1 (en) * | 2009-04-03 | 2010-10-07 | 武汉大安制药有限公司 | A polysaccharide liposome, the preparation method and use of it |
| WO2014209246A1 (en) * | 2013-06-27 | 2014-12-31 | T.C. Ege Universitesi | Design of terbinafine hydrochloride loaded liposome included pullulan film system for ungual treatment of onychomycosis |
| CN105708801A (en) * | 2016-02-04 | 2016-06-29 | 大连民族大学 | Preparation method and application of 20 (R)-ginsenoside Rg3/soya bean lecithin/cholesterol/folic acid lipidosome medicine |
-
2021
- 2021-03-02 CN CN202110230216.7A patent/CN113262310A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4636381A (en) * | 1982-05-19 | 1987-01-13 | Eisai Co., Ltd. | Coated ubidecarenone-containing liposome |
| CN101254309A (en) * | 2008-04-11 | 2008-09-03 | 中国医学科学院生物医学工程研究所 | Folacin receptor mediated targeted acetyl pullulan polysaccharide nano granule and preparation thereof |
| WO2010111807A1 (en) * | 2009-04-03 | 2010-10-07 | 武汉大安制药有限公司 | A polysaccharide liposome, the preparation method and use of it |
| WO2014209246A1 (en) * | 2013-06-27 | 2014-12-31 | T.C. Ege Universitesi | Design of terbinafine hydrochloride loaded liposome included pullulan film system for ungual treatment of onychomycosis |
| CN105708801A (en) * | 2016-02-04 | 2016-06-29 | 大连民族大学 | Preparation method and application of 20 (R)-ginsenoside Rg3/soya bean lecithin/cholesterol/folic acid lipidosome medicine |
Non-Patent Citations (1)
| Title |
|---|
| 杨青等: "普鲁兰多糖修饰的人参皂苷Ｒg3 纳米结构脂质载体促吸收及其体外抗肿瘤评价", 中国中药杂志, vol. 45, no. 21, pages 5184 - 5192 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103705534A (en) | Preparation of natural active substance constructed polymer composite medicine and application thereof in inhibiting angiogenesis | |
| CN107412196B (en) | Orlistat nanoparticle and preparation method thereof and the application in antitumor drug | |
| CN114831931A (en) | Medicinal solution with anti-tumor synergistic attenuation effects and medicinal composition containing medicinal solution | |
| CN108042490A (en) | Nano medicament carrying system, its preparation method, pharmaceutical composition and the application in treating cancer | |
| CN109200021A (en) | A kind of pH sensitive nanoparticles and the preparation method and application thereof of lysosome targeting | |
| CN103524639B (en) | A kind of synthetic method of chitosan oligosaccharide/indograftin graftin and application thereof | |
| CN111249473B (en) | Delivery system and preparation method of polymerized chloroquine fluorene methyl carbonyl nanogel | |
| CN109953974B (en) | Preparation method of enzyme-reduction dual-responsiveness hyaluronic acid-polypropylene sulfide copolymer nanocapsule | |
| CN110559272B (en) | Anti-breast cancer nano-drug and preparation method thereof | |
| CN113262310A (en) | Pullulan-modified ginsenoside Rg3Nanostructured lipid carriers and methods of making the same | |
| CN106729746A (en) | To FAP α enzymes, the preparation method and applications of the tumor infiltrating nanosystems of the particle diameter shrinkage type of reducing environment sensitive | |
| CN104817608B (en) | Cordycepin salt of selenium-containing compound and its preparation method and application | |
| CN110801433A (en) | Targeted pharmaceutical composition loaded with amphotericin B and adriamycin together and application thereof | |
| CN105770912A (en) | Drug-loading ATP sensitive liposome with tumor near infrared fluorescence development function and preparation method of drug-loading ATP sensitive liposome | |
| CN110025792A (en) | A kind of preparation method of cisplatin nano drug that treating oophoroma | |
| Xu et al. | Revisiting the impacts of silica nanoparticles on endothelial cell junctions and tumor metastasis | |
| Ke et al. | Development of resveratrol with thiolated alginate as a supplement to prevent nonalcoholic fatty liver disease (NAFLD) | |
| CN108619528A (en) | A kind of cyclodextrin-mesoporous silicon multifunctional nano load medicine particle | |
| CN105476956B (en) | A kind of phycocyanin-polylactic acid-adriamycin micella and its preparation method and application inhibiting the cancer of the brain | |
| Liao et al. | Preparation of galactosyl nanoparticles and their targeting efficiency to hepatocellular carcinoma | |
| CN110200980B (en) | Oral nanoparticles of artemisinin-loaded citrus pectin | |
| CN109134694B (en) | Sulfated derivative of dendrobium nobile polysaccharide and preparation method and application thereof | |
| CN106727617B (en) | The nanometer formulation and preparation method of a kind of anticancer pharmaceutical composition and its application in treatment malignant tumour | |
| CN105769776B (en) | A kind of freeze-dried composition for treating non-Hodgkin lymphoma and preparation method thereof | |
| CN111249254A (en) | Preparation method and application of baicalin-entrapped folic acid coupled albumin nanoparticles |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |