Liposome with basil essential oil for targeting breast cancer

Abstract

The invention discloses a tropical basil essential oil liposome for targeting breast cancer. The liposome is composed of a breast cancer targeting liposome skeleton and tropical basil essential oil. Wherein the breast cancer targeted liposome skeleton is composed of soybean lecithin, cholesterol, and vasoactive intestinal peptide modified distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000. The mass ratio of the tropical basil essential oil to the liposome skeleton in the liposome is 1: 50. The liposome carrying the tropical basil essential oil for targeting the breast cancer, prepared by the invention, has high affinity to breast cancer cells and high cell uptake, can remarkably inhibit proliferation, migration and invasion of triple-negative breast cancer cells, remarkably improves the effect of the tropical basil essential oil on resisting triple-negative breast cancer, reduces adverse reactions, and has a remarkable progress. The preparation method of the liposome is simple, the raw materials are easy to obtain, the anti-cancer effect is remarkable, and the liposome has good application value and market prospect.

Description

Technical Field

[0001] The invention relates to a liposome carrying tropical basil essential oil and targeting breast cancer, belonging to the technical field of liposomes. Background Art

[0002] Breast cancer is a common female malignancy worldwide. It accounts for 25% of all types of cancer and 12% of all new cancers. There are several biological subtypes of breast cancer, and triple-negative breast cancer (TNBC) is one of them. TNBC is characterized by the lack of progesterone receptors, estrogen receptors, and human epidermal growth factor receptor-2, and accounts for approximately 15-20% of all breast cancers. TNBC is aggressive, metastasizes widely at an early stage, and lacks effective targeted treatments, so patients have a poor survival prognosis. The main metastatic sites of TNBC are bones, lungs, brain, liver, soft tissues, and adrenal glands. Once TNBC metastasizes, the prognosis is very poor, with an average median overall survival of only 10-13 months. Therefore, early detection and early treatment are important means to improve the prognosis of TNBC.

[0003] Currently, the treatment options for TNBC are constantly expanding, including surgical resection of the tumor site or the entire breast, radiotherapy and chemotherapy, targeted therapy, endocrine immunotherapy, and the emerging "precision therapy" system. However, skin damage caused by radiotherapy and adverse reactions caused by chemotherapy drugs can cause damage to the patient's body. As a new treatment method, targeted therapy can target the site where the tumor occurs and use drugs that can specifically bind to that site to accurately and specifically attack tumor cells. Targeted therapy has become the most promising option for tumor treatment because of its high specificity, significant effect, and minimal damage to normal tissues.

[0004] Basil is native to India and other tropical regions of Asia. It is valued in traditional Indian medicine and is known as the "king of herbs." It is widely cultivated in southeastern my country. Its pungent, sweet, and warm nature are known for promoting the flow of qi and blood circulation, as well as reducing swelling and detoxifying. Basil essential oil is extracted from the Lamiaceae plant, Basil. It contains a large amount of essential oil and is a pale yellow, transparent liquid whose main chemical components are eugenol and propenylanisole. Basil essential oil also has certain pharmacological effects, including anti-inflammatory, analgesic, antioxidant, anti-thrombotic, blood sugar and lipid-lowering, disinfecting and insecticidal effects, and a significant inhibitory effect on tumor metastasis. As a drug carrier, liposomes can improve the stability and solubility of the encapsulated drug while also reducing the adverse reactions of the drug. Therefore, the development of a new liposome containing basil essential oil for breast cancer treatment is of great significance. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a liposome containing tropical basil essential oil targeting breast cancer and a preparation method thereof.

[0006] The technical solutions of the present invention are as follows:

[0007] The invention provides a liposome targeting breast cancer and carrying tropical basil essential oil. The liposome is composed of a breast cancer-targeted liposome skeleton and tropical basil essential oil. The breast cancer-targeted liposome skeleton is composed of soybean lecithin (SPC), cholesterol (Chol), and vasoactive intestinal peptide-modified distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG-VIP).

[0008] Preferably, the breast cancer-targeted liposome matrix is ​​composed of SPC, Chol, and DSPE-PEG-VIP in a mass ratio of 7:2:1. The mass ratio of tropical basil essential oil to liposome is 1:50.

[0009] The present invention provides a breast cancer-targeting liposome containing tropical basil essential oil, which has the following beneficial effects in treating triple-negative breast cancer:

[0010] 1. The present invention constructs a liposome targeting breast cancer. The liposome targets breast cancer through vasoactive intestinal peptide (VIP) and has long circulation characteristics. It can deliver anti-tumor drugs to breast cancer cells in a targeted manner, thereby improving anti-tumor activity and reducing its adverse reactions, which is a significant improvement.

[0011] 2. The present invention uniquely loads tropical basil essential oil into liposomes targeting breast cancer, achieving intracellular delivery of tropical basil essential oil targeted to breast cancer, greatly enhancing the anti-triple-negative breast cancer effect of tropical basil essential oil. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Transmission electron micrograph of breast cancer-targeting liposomes loaded with tropical basil essential oil.

[0013] Figure 2 Affinity of rhodamine B-loaded targeted liposomes to MDA-MB-231 cells.

[0014] Figure 3 Effects of breast cancer-targeted liposomes loaded with tropical basil essential oil on MDA-MB-231 cell viability. Compared with the BC group, *P < 0.01; compared with the NC group, #P < 0.01; compared with the same concentration of TB, &P < 0.01.

[0015] Figure 4Effects of breast cancer-targeted liposomes loaded with basil essential oil on the migration of MDA-MB-231 cells (A: representative images; B: quantitative results). *P < 0.01 compared with the BC group; &P < 0.01 compared with the NC group; #P < 0.01 compared with the TB group.

[0016] Figure 5 Effects of breast cancer-targeted liposomes loaded with basil essential oil on the invasion of MDA-MB-231 cells (A: representative images; B: quantitative results). *P < 0.01 compared with the BC group; &P < 0.01 compared with the NC group; #P < 0.01 compared with the TB group. DETAILED DESCRIPTION

[0017] The technical solutions of the present invention are further described below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention. The materials, reagents, etc. used in the examples of the present invention, unless otherwise specified, can be obtained from commercial sources.

[0018] Example 1 Preparation and Characterization of Liposomes Targeting Breast Cancer with Basil Essential Oil

[0019] 1. Experimental Materials

[0020] Soy lecithin (SPC) and cholesterol (Chol) were purchased from Shanghai Yien Chemical Technology Co., Ltd.; vasoactive intestinal peptide-modified distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG-VIP) was synthesized by Xi'an Ruixi Biosynthesis Co., Ltd.; tropical basil essential oil was purchased online and originated from India.

[0021] 2. Experimental Methods

[0022] (1) Soy lecithin, cholesterol, and vasoactive intestinal peptide-modified distearoylphosphatidylethanolamine-polyethylene glycol 2000 were mixed in a mass ratio of 7:2:1, dissolved in chloroform, placed in a dry and clean rotary evaporation bottle, and rotary evaporated under vacuum to form a lipid film;

[0023] (2) adding PBS buffer to the lipid film and slowly adding tropical basil essential oil at a mass ratio of 1:50 to liposomes;

[0024] (3) placing the film in a 65°C water bath until the film is completely hydrated, and filtering through a 200 nm microporous filter membrane to obtain liposomes loaded with tropical basil essential oil targeting breast cancer.

[0025] (4) The particle size, polydispersity index (PDI), and zeta potential of liposomes were measured by dynamic light scattering using a Zetasizer Nano ZS 90 laser particle size analyzer.

[0026] (5) Drop the liposomes onto the ultra-thin carbon film copper mesh. After air-drying, drop a drop of 3% phosphotungstic acid on the sample surface for negative staining. Blot dry. After the sample is completely dry, observe it under a transmission electron microscope.

[0027] 3. Experimental results:

[0028] The particle size of the prepared liposomes loaded with tropical basil essential oil targeting breast cancer was 115.9±0.97nm, PDI was 0.213±0.021, and Zeta potential was -22.7±0.519mV. Figure 1 As shown, the particles are in regular spherical shape with good dispersion, and the particle size is basically consistent with that measured by a laser particle size analyzer.

[0029] 4. Conclusion

[0030] The liposomes loaded with tropical basil essential oil targeting breast cancer constructed in this patent have a particle size of about 115 nm, are well dispersed, and are suitable for intracellular delivery.

[0031] Example 2 Affinity of liposomes loaded with tropical basil essential oil targeting breast cancer for triple-negative breast cancer cells

[0032] 1. Experimental Materials

[0033] Rhodamine B was purchased from Shanghai McLean Biochemical Technology Co., Ltd.; human triple-negative breast cancer cell line MDA-MB-231 was purchased from Shanghai Bolson Biotechnology Co., Ltd.; cell culture reagents were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; other materials were the same as above.

[0034] 2. Experimental Methods

[0035] In this experiment, liposomes were prepared using the fluorescent dye Rhodamine B instead of tropical basil essential oil to observe the affinity of the patented liposome drug delivery system to triple-negative breast cancer cells MDA-MB-231 cells.

[0036] 2.1 Preparation of liposomes:

[0037] 2.1.1 Preparation of Rhodamine B-loaded Liposomes

[0038] SPC, Chol and rhodamine B were accurately weighed in proportion, dissolved in chloroform, placed in a dry and clean rotary evaporation flask, and rotary evaporated under vacuum conditions to form a lipid film; PBS buffer was added to the lipid film, and the film was hydrated in a 65°C water bath until the film was completely hydrated. The film was filtered through a 200nm microporous membrane to obtain ordinary liposomes loaded with rhodamine B (LPs-RB).

[0039] 2.1.2 Preparation of Rhodamine B-loaded Targeted Liposomes

[0040] SPC, Chol, PEG2000-DSPE-VIP, and rhodamine B were accurately weighed in proportion, dissolved in chloroform, placed in a dry and clean rotary evaporation flask, and rotary evaporated under vacuum conditions to form a lipid film; PBS buffer was added to the lipid film, and the film was hydrated in a 65°C water bath until the film was completely hydrated. The film was filtered through a 200nm microporous membrane to obtain rhodamine B-loaded breast cancer-targeting liposomes (TLPs-RB).

[0041] 2.2 Qualitative observation of cellular uptake

[0042] Human triple-negative breast cancer cells MDA-MB-231 were digested with trypsin, centrifuged, resuspended, and then seeded into 24-well plates (1×10 5 When the cells reached a density of 70%, free rhodamine B (RB), LPs-RB, and TLPs-RB prepared in serum-free DMEM were added to the cells to a concentration of 0.01 mg / mL rhodamine B per well. After incubation at 37°C in a CO2 incubator for 4 hours, the cells were washed three times with PBS, digested, and fixed with 4% paraformaldehyde for 10 minutes. Images were captured using a fluorescence microscope to observe the red fluorescence of intracellular rhodamine B.

[0043] 3. Experimental Results

[0044] The red fluorescence in MDA-MB-231 cells Figure 2 As shown. The results showed that rhodamine B could enter MDA-MB-231 cells, and thus red fluorescence was observed in the cells. This fluorescence amount was significantly increased in the LPs-RB group, suggesting that ordinary liposomes as a drug delivery system can increase the affinity of cells for rhodamine B. Compared with the LPs-RB group, the intracellular fluorescence amount of the TLPs-RB group was significantly increased. The results suggest that the liposome drug delivery system targeting breast cancer constructed by the present invention has a significantly increased affinity for MDA-MB-231 cells, has targeting properties for triple-negative breast cancer cells, and is conducive to delivering drugs to triple-negative breast cancer cells to exert anti-tumor effects.

[0045] 4. Conclusion

[0046] The breast cancer-targeted liposome drug-carrying system constructed by the present invention has a high affinity for human triple-negative breast cancer cells and can be used for targeted delivery of triple-negative breast cancer cells.

[0047] Example 3 Inhibitory effect of breast cancer-targeted liposomes loaded with tropical basil essential oil on triple-negative breast cancer cells

[0048] 1. Experimental Materials

[0049] MTT, DMSO, tissue cell fixative, and crystal violet were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; other materials were the same as above.

[0050] 2. Experimental Methods

[0051] 2.1 Preparation of liposomes

[0052] 2.1.1 Preparation of liposomes loaded with tropical basil essential oil targeting breast cancer

[0053] According to the method of Example 1, liposomes loaded with tropical basil essential oil (TLPs-TB) targeting breast cancer were prepared.

[0054] 2.1.2 Preparation of blank liposomes

[0055] SPC and Chol were accurately weighed according to the ratio, dissolved in chloroform, and placed in a dry and clean rotary evaporation bottle. Under vacuum conditions, rotary evaporation was performed to form a lipid film. PBS buffer was added to the lipid film, and the film was hydrated in a 65°C water bath until the film was completely hydrated. The film was then filtered through a 200 nm microporous membrane to obtain blank liposomes.

[0056] 2.2 MTT assay

[0057] TLPs-TB and tropical basil essential oil (TB) were dissolved in serum-free DMEM complete culture medium, and the concentrations of TB were controlled at 0.5, 1, 2, 4, and 8 μg / mL, respectively. MDA-MB-231 cells were seeded in 96-well plates (1×10 cells per well). 4 cells), and cultured in a CO2 incubator at 37°C for 24 hours. 200 μL of the above sample was added to each well, and equal amounts of culture medium or blank liposome solution were added to the blank control group (BC) and negative control group (NC). After 24 hours of cell culture, the liquid in the wells was discarded, and 100 μL of MTT solution (5 mg / mL) was added to each well and cultured for 4 hours. The MTT solution in the well plate was discarded, 150 μL of DMSO was added to each well, and the plate was shaken for 5 minutes to dissolve the purple crystals in the living cells. The absorbance was measured at 490 nm using a microplate reader. Cell viability was calculated according to the following formula:

[0058] 2.3 Cell scratch assay

[0059] MDA-MB-231 cells were seeded in 6-well plates (1×10 4Cells were cultured for 48 hours until the cells adhered. A 200 μL sterile pipette tip was then used to scratch the well from one end to the other, perpendicular to the marked black line and perpendicular to the cell surface. 2 mL of TLPs-TB and TB solution were added to each well. The wells were incubated in a CO2 incubator at 37°C. The wells were observed and photographed under a microscope at 0 and 48 hours. Images were processed using Image J software to measure the scratch area. The 48-hour cell migration rate was calculated as follows: The blank control group was used for normalization.

[0060] 2.4 Transwell assay

[0061] The chambers were placed in a 24-well plate and 100 μL of serum-free DMEM was added to the chambers for 10 minutes. Matrigel was diluted 1:3 with serum-free DMEM and 100 μL was added to each chamber. The cells were incubated in a CO2 incubator at 37°C overnight to allow gelation. Cells (density 2×10 5 / mL), then add 200μL of serum-free DMEM containing TLPs-TB and TB respectively, add 750μL of high-concentration DMEM culture medium to the lower chamber, and place it in a CO2 incubator at 37°C for 48h. Aspirate the culture medium in the upper chamber, wipe off the cells in the upper chamber that have not migrated with a cotton swab, and wash the chamber twice in PBS. Add 500μL of 4% tissue cell fixative to each well and fix at room temperature for 30min. Remove the chamber and wash twice with PBS. Add 0.1% crystal violet and stain in the dark for 20min. Discard the staining solution and wash twice with PBS. Take pictures under a fluorescence microscope, observe, count the stained cells using Image J software, and normalize the blank control group to obtain the cell invasion rate.

[0062] 3. Experimental Results

[0063] Effects of basil essential oil-loaded liposomes targeting breast cancer on the viability of MDA-MB-231 cells Figure 3 As shown. It can be seen from the figure that TB and TLPs-TB can inhibit the viability of MDA-MB-231 cells (compared with BC and NC groups, P<0.01), and the inhibitory effect is enhanced with increasing doses. The half inhibitory concentrations (IC50) of TLPs-TB and TB are 0.6877μg / mL and 0.8024μg / mL, respectively. Compared with the TB group at the same dose, the viability of MDA-MB-231 cells in the TLPs-TB group was significantly decreased (P<0.01). The experiment also observed the effects of 8μg / mL of TB and TLPs-TB on the migration and invasion of MDA-MB-231 cells, as shown in Figure 5. Figure 4 and Figure 5As shown in the figure, both TB and TLPs-TB can inhibit the migration and invasion of MDA-MB-231 cells (compared with the BC and NC groups, P < 0.05-0.01), among which the inhibitory effect of TLPs-TB is significantly stronger than that of TB (P < 0.05). The results suggest that loading tropical basil essential oil into liposomes can target breast cancer cells and increase cellular uptake, thereby enhancing its inhibitory effect on MDA-MB-231 cell viability, cell migration and invasion. There was no significant difference between the NC group and the BC group, indicating that the carrier material is safe and non-toxic.

[0064] 4. Conclusion

[0065] The breast cancer-targeting tropical basil essential oil constructed in this patent can significantly inhibit the viability, cell migration and invasion of triple-negative breast cancer cells, and can be used to prepare anti-triple-negative breast cancer drugs.

Claims

1. A liposome containing tropical basil essential oil targeting breast cancer, characterized in that: The liposomes are composed of a breast cancer-targeted liposome skeleton and tropical basil essential oil; Among them, the breast cancer-targeted liposome skeleton is composed of soybean lecithin, cholesterol, and vasoactive intestinal peptide-modified distearoyl phosphatidylethanolamine-polyethylene glycol 2000.

2. The liposome containing tropical basil essential oil targeting breast cancer as claimed in claim 1, characterized in that: The breast cancer-targeted liposome skeleton material comprises soybean lecithin, cholesterol, and vasoactive intestinal peptide-modified distearoyl phosphatidylethanolamine-polyethylene glycol 2000 in a mass ratio of 7:2:

1.

3. The liposome containing tropical basil essential oil targeting breast cancer as claimed in claim 1, characterized in that: The mass ratio of tropical basil essential oil to liposome matrix is ​​1:50.

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