CN101129335B - Application of a nanostructure lipid carrier drug delivery system - Google Patents

Abstract

The invention provides the application of a nanostructure lipid carrier drug delivery system in antitumor therapeutic drugs. The main purpose is to provide the application of a nanostructured lipid carrier drug delivery system in reversing the multidrug resistance of drug-resistant tumor cells. The drug delivery system is composed of solid lipid material, liquid lipid material and antitumor drugs, wherein the proportion of liquid lipid (such as oleic acid) can be 0-30wt%. The solid lipid is selected from monoglyceride, the liquid lipid is selected from oleic acid, and the antitumor drug is selected from paclitaxel or adriamycin. In the present invention, the nanostructured lipid carrier with high-efficiency cell uptake and cytoplasmic concentration functions encapsulates the antitumor drug whose molecular target is located in the cell, which can avoid the recognition of the antitumor drug by P-glycoprotein in the cytoplasm of drug-resistant cells, Reduce the efflux of drugs from cells, improve the efficacy of anti-tumor drugs, and reverse the drug resistance and multidrug resistance of drug-resistant tumor cells.

Description

Application of a nanostructure lipid carrier drug delivery system

technical field

The invention relates to the application of the nanostructure lipid carrier delivery system, and mainly relates to the application of the nanostructure lipid carrier delivery system in anti-tumor treatment and reversal of tumor multidrug resistance.

Background technique

Tumor has always been a major disease that directly threatens human health. Due to the lack of molecular targeting of drugs themselves, tumor chemotherapy has major treatment problems such as low cure rate, multidrug resistance, and huge toxic and side effects. One of the important reasons for the failure of clinical chemotherapy is the resistance of tumor cells to chemotherapy drugs. The cause of death of most cancer patients is directly or indirectly related to the resistance of tumor cells to anti-tumor drugs. Therefore, finding and studying drug delivery systems that can reverse the drug resistance of drug-resistant tumor cells is one of the important strategies and directions for the research and development of cancer chemotherapy.

Studies have found that there are many reasons that lead to the generation of tumor multidrug resistance, and it is related to P-glycoprotein (P-gp), glutathione S-transferase (GST-Л), and topoisomerase (TOPO -II) abnormal expression. P-glycoprotein (P-gp) is mainly distributed in the cell membrane, is a drug export pump dependent on ATPase, and is also a channel for Ca ²⁺ and Cl ^- . P-gp can combine with anti-tumor drugs that enter the cytoplasm, and use the energy released by ATP hydrolysis to directly or indirectly pump the drug out of the cell; or use the channel function of P-gp itself to pump the drug out of the cell. , leading to a decrease in drug concentration in tumor cells, weakening or loss of cytotoxicity, and thus drug resistance in tumors.

Studies have shown that polymer carriers can alleviate the multidrug resistance of tumors to a certain extent. Nonionic surfactant polyethylene oxide-polyphenylene ether-polyethylene oxide terpolymer (PEO-PPO-PEO), after wrapping anti-tumor drugs, when the polymer concentration is near or below the critical micelle concentration, It can exert the best anti-tumor effect and reflect the tendency of reversing multi-drug resistance. The polymer carrier can effectively reduce the amount of ATP in drug-resistant cells, but it will not cause changes in the amount of ATP in sensitive cells. The reduction of the amount of ATP in drug-resistant cells directly reduces the amount of tumor drugs pumped out by drug-resistant cells, which is manifested as the reversal of tumor drug resistance. As another example, poly-L-histidine (PolyHis), through the "proton sponge" mechanism of imidazole groups, can achieve endosomal membrane lysis activity. If the polymer micelles are designed to trigger release and internalization in early endosomes, followed by rupture of mature endosomal membranes, drugs can escape and be released in the cytoplasm and nucleus (potentially Such a drug delivery system will be an effective drug delivery model to overcome multidrug resistance. Therefore, polymer micelles with a poly-L-histidine core are also expected to be effective reversal agents of multidrug resistance in tumor cells.

The molecular targets of most anticancer drugs are located in cells. The existing cancer chemotherapy technology basically follows the non-targeted drug delivery mode. Through appropriate carrier technology, the drug is directly targeted to the diseased tissue (organ), cell and subcellular organelle, which is a solution to the low cure rate of cancer chemotherapy and the One of the important means of toxic side effects. At present, scientists at home and abroad have made some progress in the tissue (organ) and cell targeting of anti-tumor drugs through nanocarrier technology, but have not achieved breakthrough efficacy. One of the basic reasons is that the molecular targets of most antineoplastic drugs are located in cells. Therefore, the research and development of targeted nano-carrier material technology for drug molecular targets (subcellular organelles) in tumor cells is the key to breaking through the bottleneck technology of tumor chemotherapy.

The solid lipid nanoparticle drug delivery system is a new type of colloid drug delivery system developed in the early 1990s. It is a targeted controlled-release drug delivery system with great development potential after emulsions, liposomes, and polymer nanoparticles. system. The solid lipid nanoparticle drug delivery system uses natural or synthetic lipid materials, such as stearic acid, lecithin, monoglyceride, etc., as carriers, and encapsulates the drug in the lipid core to form a solid micelle drug delivery system. It not only has the advantages of controlled release of polymer drug delivery system and avoiding drug leakage, but also has the advantages of low toxicity, good biocompatibility and high bioavailability of emulsion and liposome. However, the solid lipid nanoparticle drug delivery system also has some potential limitations, such as limited drug loading capacity, drug exclusion during storage and other issues.

In solid lipid nanoparticles, liquid lipids with different shapes are added as a mixed lipid matrix to prepare a new nanostructured lipid carrier (NLC). The addition of liquid lipids can disturb the regular lattice structure of solid lipids, increase the proportion of irregular crystal forms in the nanoparticle structure, increase the space capacity of encapsulated drugs, and then improve the drug-loading capacity of the carrier. By controlling the ratio of liquid lipids, NLC can also maintain a solid skeleton structure at body temperature to achieve controlled release of NLC drugs.

Contents of the invention

The first object of the present invention is to provide the application of a nanostructured lipid carrier drug delivery system in antitumor therapeutic drugs.

Another object of the present invention is to provide the application of a nanostructured lipid carrier delivery system in reversing the multidrug resistance of drug-resistant tumor cells.

The nanostructure lipid carrier drug delivery system of the present invention is composed of solid lipid material, liquid lipid material and antitumor drug. The solid lipid is monoglyceride, the liquid lipid is oleic acid, and the antitumor drugs can be paclitaxel and doxorubicin. The proportion of oleic acid can be 0-30%, and the proportion of antitumor drugs can be 1-5%. The nanostructured lipid carrier drug delivery system is prepared by the aqueous solvent diffusion method. The composition and preparation method of the nanostructured lipid carrier in the present invention have been covered by the patent "A Nanostructured Lipid Carrier with Efficient Anti-tumor Activity" (Patent Application No. 200610155605.3).

The present invention utilizes nanostructured lipid carriers with efficient cell uptake and cytoplasmic concentration functions to encapsulate paclitaxel, doxorubicin and other antitumor drugs whose molecular targets are located in cells, while improving the antitumor efficacy of such drugs, Reversal of drug resistance and multidrug resistance in resistant tumor cells.

The benefit of the present invention is that the anti-tumor drug whose molecular target is located in the cell is encapsulated by the nano-structured lipid carrier with high-efficiency cell uptake and cytoplasmic concentration functions, which can significantly increase the uptake of anti-tumor drugs by tumor cells and improve The drug concentration at the target site of the drug molecule enhances the efficacy of anti-tumor drugs. Encapsulating drugs with nanostructured lipid carriers can avoid the recognition of anti-tumor drugs by P-glycoprotein in the cytoplasm of drug-resistant cells, reduce the efflux of drugs from cells, and reverse drug-resistant tumors while improving the efficacy of anti-tumor drugs Cellular resistance and multidrug resistance.

Description of drawings

Figure 1 is a fluorescent photo of the uptake of fluorescently labeled monoglyceride nanostructured lipid carriers in MCF-7 cells and MCF-7-adr cells.

Fig. 2 is a fluorescent photo of the uptake of fluorescently labeled monoglyceride nanostructured lipid carriers in SKOV3 cells and SKOV3-adr cells.

Detailed ways

Example 1: Preparation and Application of Doxorubicin Monoglyceride Nanostructured Carrier Delivery System

1) Preparation of Adriamycin Monoglyceride Nanostructured Carrier Delivery System

Take 60 mg of monoglyceride and 3 mg of doxorubicin respectively, weigh them accurately, add 6 mL of absolute ethanol, dissolve in a water bath at 70°C, and form an organic phase. Using distilled water as the dispersed phase, put it in a 70°C water bath, and under the condition of 400r·min ^-1 mechanical stirring, inject the organic phase into 60mL of the dispersed phase, and stir for 5min to prepare the doxorubicin monoglyceride nanostructured lipid carrier. Drug system dispersion liquid, the dispersion liquid was adjusted to pH 1.2 with 3mol·L ^-1 HCl solution, centrifuged at 20000r·min ^-1 for 10min, and the precipitate was re-dispersed with 0.1% Poloxamer (w/v). 1.0mol·L ^-1 NaOH solution was used to adjust the pH to 7.0 to obtain a doxorubicin monoglyceride nanostructured lipid carrier delivery system.

The particle size, surface potential and drug encapsulation efficiency of the doxorubicin monoglyceride nanostructured lipid carrier delivery system are shown in Table 1.

Table 1 Particle size, surface potential and drug encapsulation efficiency of doxorubicin monoglyceride nanostructured lipid carrier delivery system

2) Antitumor efficacy of monoglyceride nanostructured lipid carrier delivery system and reversal of drug resistance of drug-resistant tumor cells

Taking breast cancer cell MCF-7 cells and doxorubicin-resistant breast cancer cell MCF-7-adr cells as model cells, the antitumor efficacy of doxorubicin monoglyceride nanostructured lipid carrier delivery system, and the effect on drug resistance The reversal efficiency of tumor cell drug resistance is evaluated by the IC ₅₀ value (half lethal rate of cells) after co-incubation of the drug delivery system and cells. The cell viability test was determined by tetrazolium salt colorimetric method (MTT Assay). Pre-cultivate in 24-well plate for 24 hours, after the cells adhere to the wall, add different concentrations of blank monoglyceride nanostructured lipid carrier suspension, doxorubicin solution and doxorubicin monoglyceride nanostructured lipid carrier for administration system suspension. Control wells were set up in the experiment, and each group was repeated 3 times. After incubation for 48 hours, add 60 μL of MTT solution to each well, discard the supernatant after incubation for 4 hours, wash with PBS solution twice, and add 500 μL of DMSO solution to each well to terminate the reaction. Shake the culture plate horizontally for 10 min, measure the absorbance at 570 nm with an enzyme-linked detector, and calculate the cell survival rate according to formula (1):

Cell viability (%)=A ₅₇₀ (sample)/A ₅₇₀ (control)×100% (1)

Wherein A ₅₇₀ (sample) is the absorbance of cells added to the suspension, and A ₅₇₀ (control) is the absorbance of blank control cells.

See Table 2 for the measured IC ₅₀ values of the blank monoglyceride nanostructured lipid carrier, doxorubicin solution and doxorubicin monoglyceride nanostructured lipid carrier delivery system.

Table 2 _IC50 values of monoglyceride nanostructured lipid carrier, doxorubicin solution and doxorubicin monoglyceride nanostructured lipid carrier delivery system

The research results show that the monoglyceride nanostructured lipid carrier is a low-toxicity carrier material. After doxorubicin is encapsulated by the monoglyceride nanostructured lipid carrier, it can improve the anti-tumor effect on breast cancer sensitive cells and drug-resistant cells, respectively. The curative effect is 0.5 times and 5.3 times. The drug-resistant multiple of breast cancer drug-resistant cells is about 35.2 times, and doxorubicin can partially reverse the drug resistance of breast cancer drug-resistant cells after being encapsulated by the monoglyceride nanostructured lipid carrier.

Example 2: Preparation and Application of Doxorubicin Monoglyceride/Oleic Acid Nanostructured Carrier Delivery System

1) Preparation of doxorubicin monoglyceride/oleic acid nanostructure carrier drug delivery system

Take 60 mg of monoglyceride, 6 mg of oleic acid and 3 mg of doxorubicin, respectively, and weigh them accurately. Add 6 mL of absolute ethanol and dissolve in a water bath at 70°C. Using distilled water as the dispersed phase, put it in a water bath at 70°C, and inject the organic phase into 60 mL of the dispersed phase under the condition of 400 r·min ^-1 mechanical stirring, and stir for 5 min to obtain the doxorubicin monoglyceride/oleic acid nanostructured lipid Carrier drug delivery system dispersion, the dispersion is adjusted to pH 1.2 with 3mol L ^-1 HCl solution, centrifuged at 20000r min ^-1 for 10min, and redispersed after adding 0.1% Poloxamer (w/v) to the precipitate , adjust the pH to 7.0 with 1mol·L ^-1 NaOH solution, and obtain the doxorubicin monoglyceride/oleic acid nanostructured lipid carrier delivery system.

The particle size, surface potential and drug encapsulation efficiency of the doxorubicin monoglyceride/oleic acid nanostructured lipid carrier delivery system are shown in Table 3.

Table 3 Particle size, surface potential and drug encapsulation efficiency of doxorubicin monoglyceride nanostructure lipid carrier delivery system

2) Antitumor efficacy of monoglyceride/oleic acid nanostructured lipid carrier delivery system and reversal of drug resistance of drug-resistant tumor cells

Taking breast cancer cell MCF-7 cells and doxorubicin-resistant breast cancer cell MCF-7-adr cells as model cells, the antitumor efficacy of doxorubicin monoglyceride/oleic acid nanostructured lipid carrier delivery system, and The reversal efficiency of drug resistance to drug-resistant tumor cells is evaluated by the IC ₅₀ value (half lethal rate of cells) after co-incubation of the drug delivery system and cells. The cell viability test was determined by tetrazolium salt colorimetric method (MTT Assay). Pre-cultivate in 24-well plate for 24 hours, after the cells adhere to the wall, add different concentrations of blank monoglyceride/oleic acid nanostructured lipid carrier suspension, doxorubicin solution and doxorubicin monoglyceride/oleic acid Nanostructured lipid carrier drug delivery system suspension. Control wells were set up in the experiment, and each group was repeated 3 times. After incubation for 48 hours, 60 μL of MTT solution was added to each well. After incubation for 4 hours, the supernatant was discarded, washed twice with PBS solution, and 500 μL of DMSO solution was added to each well to terminate the reaction. Shake the culture plate horizontally for 10 min, measure the absorbance at 570 nm with an enzyme-linked detector, and calculate the cell survival rate according to formula (1).

See Table 4 for the measured IC ₅₀ values of the blank monoglyceride/oleic acid nanostructured lipid carrier, doxorubicin solution and doxorubicin monoglyceride/oleic acid nanostructured lipid carrier delivery system.

Table 4 _IC50 values of monoglyceride/oleic acid nanostructured lipid carrier, doxorubicin solution and doxorubicin monoglyceride/oleic acid nanostructured lipid carrier delivery system

The research results show that the monoglyceride/oleic acid nanostructured lipid carrier is a low-toxicity carrier material, and after doxorubicin is encapsulated by the monoglyceride/oleic acid nanostructured lipid carrier, it can inhibit breast cancer sensitive cells and drug-resistant cells. The anti-tumor efficacy can be increased by 5% and 7.7 times respectively. The drug-resistant multiple of breast cancer drug-resistant cells is about 35.2 times, and doxorubicin can partially reverse the drug resistance of breast cancer drug-resistant cells after being encapsulated by monoglyceride/oleic acid nanostructured lipid carrier.

Example 3: Preparation and Application of Paclitaxel Monoglyceride Nanostructured Carrier Delivery System

1) Preparation of paclitaxel monoglyceride nanostructure carrier drug delivery system

Take 60 mg of monoglyceride and 3 mg of paclitaxel respectively, weigh them accurately, add 6 mL of absolute ethanol, and dissolve in a water bath at 70°C. Using distilled water as the dispersed phase, put it in a 70°C water bath, and under the condition of 400 r·min ⁻¹ mechanical stirring, inject the organic phase into 60 mL of the dispersed phase, and stir for 5 min to obtain the dispersion of paclitaxel monoglyceride nanostructured lipid carrier drug delivery system. solution, the dispersion liquid was adjusted to pH 1.2 with 3mol·L ^-1 HCl solution, centrifuged at 20000r·min ^-1 for 10min, the precipitate was re-dispersed with 0.1% Poloxamer (w/v), and then washed with 1mol·L ^-1 NaOH solution to adjust the pH to 7.0 to obtain a paclitaxel monoglyceride nanostructured lipid carrier drug delivery system.

The particle size, surface potential and drug encapsulation efficiency of paclitaxel monoglyceride nanostructured lipid carrier delivery system are shown in Table 5.

Table 5 Particle size, surface potential and drug encapsulation efficiency of paclitaxel monoglyceride nanostructure lipid carrier delivery system

2) Antitumor efficacy of monoglyceride nanostructured lipid carrier delivery system and reversal of drug resistance of drug-resistant tumor cells

Breast cancer cell MCF-7 cells and doxorubicin-resistant breast cancer cell MCF-7-adr cells, ovarian cancer cell SKOV3 cells resistant to paclitaxel ovarian cancer cell SKOV3-adr cells were used as model cells, paclitaxel monoglyceride nanostructured lipids The anti-tumor efficacy of the carrier delivery system and the reversal efficiency of drug resistance to drug-resistant tumor cells are evaluated by the IC ₅₀ value (half lethality of the cells) after the delivery system is co-incubated with the cells. The cell viability test was determined by tetrazolium salt colorimetric method (MTT Assay). Pre-cultured in a 24-well plate for 24 h, after the cells adhered to the wall, different concentrations of blank monoglyceride nanostructured lipid carrier suspension and paclitaxel solution (solvent: Cremophor: absolute ethanol mixed solvent (1:1, v/v)) and paclitaxel monoglyceride nanostructure lipid carrier (oleic acid content is 0) delivery system suspension. Control wells were set up in the experiment, and each group was repeated 3 times. After incubation for 48 hours, 60 μL of MTT solution was added to each well. After incubation for 4 hours, the supernatant was discarded, washed twice with PBS solution, and 500 μL of DMSO solution was added to each well to terminate the reaction. Shake the culture plate horizontally for 10 min, measure the absorbance at 570 nm with an enzyme-linked detector, and calculate the cell survival rate according to formula (1).

See Table 6 for the measured IC ₅₀ values of the blank monoglyceride nanostructured lipid carrier, paclitaxel solution and paclitaxel monoglyceride nanostructured lipid carrier delivery system.

The _IC50 value of table 6 monoglyceride nanostructure lipid carrier, paclitaxel solution and paclitaxel monoglyceride nanostructure lipid carrier delivery system

The research results show that the monoglyceride nanostructured lipid carrier is a low-toxicity carrier material. After paclitaxel is encapsulated by the monoglyceride nanostructured lipid carrier (without oleic acid), it can be expressed on breast cancer sensitive cells and drug-resistant cells, respectively. Improve the anti-tumor efficacy by 2.2 times and 125.5 times. The multiple drug resistance of breast cancer drug-resistant cells to paclitaxel is about 29.7 times. After paclitaxel is encapsulated by monoglyceride nanostructured lipid carrier, it can completely reverse the multi-drug resistance of breast cancer drug-resistant cells. After paclitaxel is encapsulated by a monoglyceride nanostructured lipid carrier (without oleic acid), the anti-tumor efficacy can be increased by 0.8 times and 5.9 times on ovarian cancer sensitive cells and drug-resistant cells, respectively, and it can partially reverse ovarian cancer drug-resistant cells drug resistance.

Example 4: Preparation and Application of Paclitaxel Monoglyceride/Oleic Acid Nanostructured Carrier Delivery System

1) Preparation of paclitaxel monoglyceride/oleic acid nanostructure carrier drug delivery system

Take 60 mg of monoglyceride, 6 or 18 mg of oleic acid, and 3 mg of paclitaxel, weigh them accurately, add 6 mL of absolute ethanol, and dissolve in a water bath at 70°C. Using distilled water as the dispersed phase, put it in a water bath at 70°C, and under the condition of 400r·min ^-1 mechanical stirring, inject the organic phase into 60mL of the dispersed phase, and stir for 5min to obtain paclitaxel monoglyceride/oleic acid nanostructured lipid carrier. Drug system dispersion liquid, the dispersion liquid was adjusted to pH 1.2 with 3mol·L ^-1 HCl solution, centrifuged at 20000r·min ^-1 for 10min, and the precipitate was re-dispersed with 0.1% Poloxamer (w/v). 1mol·L ^-1 NaOH solution was used to adjust the pH to 7.0 to obtain the paclitaxel monoglyceride/oleic acid nanostructured lipid carrier drug delivery system.

The particle size, surface potential and drug encapsulation efficiency of paclitaxel monoglyceride/oleic acid nanostructured lipid carrier delivery system are shown in Table 7.

Table 7 Particle size, surface potential and drug encapsulation efficiency of paclitaxel monoglyceride nanostructured lipid carrier delivery system

2) Translocation of monoglyceride nanostructured lipid carrier to lung cancer A549 cells

The invention adopts a chemical graft containing fluorescein isothiocyanate (FITC) and stearylamine as a fluorescent marker for studying the translocation of lung cancer A549 cells as a monoglyceride nanostructured lipid carrier. The monoglyceride nanostructured lipid carrier containing fluorescein isothiocyanate and stearylamine chemical graft is prepared by the following method: take 27 mg of monoglyceride and 4.5 mg of the chemical graft of fluorescein isothiocyanate and stearylamine mg, accurately weighed, add 3mL of absolute ethanol, and dissolve in a water bath at 70°C. With distilled water as the dispersed phase, put it in a 70°C water bath. Under the condition of mechanical stirring at 400r·min ^-1 , inject the organic phase into 30mL of the dispersed phase, and stir for 5min to obtain a dispersion liquid of fluorescently labeled monoglyceride nanostructured lipid carrier, and adjust the pH of the dispersion liquid with 3mol·L ^-1 HCl solution to 1.2, centrifuge at 20000r·min ^-1 for 10min, add 0.1% poloxamer (Poloxamer) (w/v) to the precipitate and redisperse, adjust the pH to 7.0 with 1.0mol·L ^-1 NaOH solution, and use the dispersion for Translocation studies in lung cancer A549 cells.

The breast cancer cell MCF-7 and the doxorubicin-resistant breast cancer cell MCF-7-adr were respectively cultured in RPMI 1640 medium containing 10% calf serum (5% CO ₂ , 37°C incubator). Take cells in the logarithmic growth phase, rinse with PBS, add trypsin to digest and dilute with culture medium, inoculate in a 24-well culture plate at a density of 1× ¹⁰⁵ cells per well, and the cells in the 24-well culture plate grow adherently Afterwards, add fluorescently labeled monoglyceride nanostructured lipid carrier (final concentration: 100 μg·mL ^-1 ), incubate for 1, 2, 4, 12, and 24 h, wash the cells with PBS three times, observe and take pictures with a fluorescent inverted microscope , see Figure 1 for the results. Among them, the nanocarriers were co-incubated with the cells for 12 hours. A is the fluorescent photo taken by the fluorescently labeled monoglyceride nanostructured lipid carrier (containing 30% oleic acid) in MCF-7 cells, and B is the fluorescently labeled monoglyceride nanostructured lipid carrier. (Containing 30% oleic acid) Fluorescence photographs were taken in MCF-7-adr cells.

Breast cancer cells MCF-7 cells, breast cancer cells resistant to doxorubicin MCF-7-adr cells, ovarian cancer cells SKOV3 cells resistant to paclitaxel ovarian cancer cells SKOV3-adr cells were cultured in RPMI 1640 containing 10% calf serum Culture in liquid (5% CO ₂ , 37°C incubator). Take cells in the logarithmic growth phase, rinse with PBS, add trypsin to digest and dilute with culture medium, inoculate in a 24-well culture plate at a density of 1× ¹⁰⁵ cells per well, and the cells in the 24-well culture plate grow adherently Afterwards, add fluorescently labeled monoglyceride/oleic acid nanostructured lipid carrier (final concentration: 100 μg·mL ^-1 ), incubate for 1, 2, 4, 12, and 24 h, wash cells with PBS three times, and fluorescent inverted microscope Observe and take photos, the results are shown in Figure 2, where the nanocarriers were co-incubated with the cells for 12 hours, which is a fluorescent photo of the fluorescently labeled monoglyceride nanostructured lipid carrier (containing 30% oleic acid) in SKOV3 cells, B fluorescently labeled monoglyceride Fluorescent photographs of nanostructured lipid carriers (containing 30% oleic acid) in SKOV3-adr cells.

3) Antitumor efficacy of monoglyceride/oleic acid nanostructured lipid carrier delivery system and reversal of drug resistance of drug-resistant tumor cells

Breast cancer cell MCF-7 cells, adriamycin-resistant breast cancer cell MCF-7-adr cells, ovarian cancer cell SKOV3 cells resistant to paclitaxel ovarian cancer cell SKOV3-adr cells were used as model cells, paclitaxel monoglyceride/oleic acid nano The antitumor efficacy of the structured lipid carrier delivery system and the reversal efficiency of drug resistance to drug-resistant tumor cells are evaluated by the IC ₅₀ value (half lethality of the cells) after the delivery system is co-incubated with the cells. The cell viability test was determined by tetrazolium salt colorimetric method (MTT Assay). Pre-cultured in a 24-well plate for 24 hours, after the cells adhered to the wall, different concentrations of blank monoglyceride/oleic acid nanostructured lipid carrier suspension and paclitaxel solution (solvent: Cremophor: absolute ethanol mixed solvent (1: 1, v/v)) and paclitaxel monoglyceride/oleic acid nanostructured lipid carrier (containing 10 and 30% oleic acid) delivery system suspension. Control wells were set up in the experiment, and each group was repeated 3 times. After incubation for 48 hours, 60 μL of MTT solution was added to each well. After incubation for 4 hours, the supernatant was discarded, washed twice with PBS solution, and 500 μL of DMSO solution was added to each well to terminate the reaction. Shake the culture plate horizontally for 10 min, measure the absorbance at 570 nm with an enzyme-linked detector, and calculate the cell survival rate according to formula (1).

See Table 8 for the measured IC ₅₀ values of the blank monoglyceride/oleic acid nanostructured lipid carrier, paclitaxel solution and paclitaxel monoglyceride/oleic acid nanostructured lipid carrier delivery system.

_IC50 values of table 8 monoglyceride/oleic acid nanostructured lipid carrier, paclitaxel solution and paclitaxel monoglyceride/oleic acid nanostructured lipid carrier delivery system

The research results show that the monoglyceride/oleic acid nanostructured lipid carrier is a low-toxicity carrier material. After paclitaxel is encapsulated by the monoglyceride/oleic acid nanostructured lipid carrier (containing 30% oleic acid), it is effective in breast cancer sensitive cells. and doxorubicin-resistant drug-resistant cells, the anti-tumor efficacy can be increased by 3.5 times and 123.8 times, respectively. The multiple drug resistance of breast cancer drug-resistant cells to paclitaxel was 29.7 times. After paclitaxel was encapsulated by monoglyceride nanostructured lipid carrier, the multi-drug resistance of breast cancer drug-resistant cells could be completely reversed. After paclitaxel is encapsulated by a monoglyceride nanostructured lipid carrier (without oleic acid), the anti-tumor efficacy can be increased by 2.0 times and 175.4 times on ovarian cancer sensitive cells and drug-resistant cells, respectively, and it can completely reverse ovarian cancer drug-resistant cells drug resistance.

Claims (2)

1. the application of nano structured lipid carrier drug feeding system in the preparation antitumor drug, it is characterized in that this drug-supplying system is to be prepared by following steps: (1) gets monoglyceride 60mg, oleic acid 6mg and amycin 3mg respectively; (2) add the 6mL dehydrated alcohol, 70 ℃ of dissolvings of water-bath are decentralized photo with the distilled water, put in 70 ℃ of water-baths, at 400rmin ^-1Under the mechanical agitation condition, organic facies is injected in the 60mL decentralized photo, stirs 5min, obtain amycin monoglyceride/oleic acid nano structured lipid carrier drug feeding system dispersion liquid; (3) dispersion liquid 3molL ^-1HCl solution is regulated pH to 1.2, with 20000rmin ^-1Centrifugal 10min after precipitation adds 0.1% poloxamer (w/v) redispersion, uses 1molL ^-1NaOH solution is regulated pH to 7.0, obtains amycin monoglyceride/oleic acid nano structured lipid carrier drug feeding system.

2. the application of nano structured lipid carrier drug feeding system in the preparation antitumor drug, it is characterized in that this drug-supplying system is to be prepared by following steps: (1) gets monoglyceride 60mg, oleic acid 6 or 18mg and paclitaxel 3mg respectively; (2) add the 6mL dehydrated alcohol, 70 ℃ of dissolvings of water-bath are decentralized photo with the distilled water, put in 70 ℃ of water-baths, at 400rmin ^-1Under the mechanical agitation condition, organic facies is injected in the 60mL decentralized photo, stirs 5min, obtain paclitaxel monoglyceride/oleic acid nano structured lipid carrier drug feeding system dispersion liquid, (3) dispersion liquid 3molL ^-1HCl solution is regulated pH to 1.2, with 20000rmin ^-1Centrifugal 10min after precipitation adds 0.1% poloxamer (w/v) redispersion, uses 1molL ^-1NaOH solution is regulated pH to 7.0, obtains paclitaxel monoglyceride/oleic acid nano structured lipid carrier drug feeding system.

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