CN109045305B - Preparation method of TPGS-modified hectorite nanoparticles - Google Patents
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Abstract
The invention discloses a preparation method of TPGS modified hectorite nanoparticles and application of the TPGS modified hectorite nanoparticles in tumor chemotherapy and anti-tumor multi-drug-resistant materials. The preparation method comprises the following steps: dispersing hectorite in ultrapure water, dropwise adding a silane coupling agent, stirring for reaction, cooling, dialyzing to obtain lithium amide saponite LM-NH2(ii) a Dissolving TPGS in solvent, adding CDI for activation, and adding dropwise into lithium amide saponite LM-NH2Reacting in the aqueous solution, and dialyzing to obtain TPGS modified hectorite nanoparticles LM-TPGS; and dropwise adding the adriamycin aqueous solution into the TPGS-modified hectorite nanoparticle LM-TPGS aqueous solution, carrying out a light-shielding reaction, and carrying out centrifugal washing to obtain LM-TPGS/DOX. The method is simple, and the prepared nanoparticles can be applied to inhibiting the proliferation of drug-resistant cells and have industrial and commercial potentials.
Description
Technical Field
The invention belongs to the field of nano drug-loaded materials and preparation and application thereof, and particularly relates to a preparation method of TPGS (Tetrakis-methyl-GS) -modified hectorite nano particles and application thereof in the aspects of anti-tumor multi-drug resistance.
Background
Chemotherapy has long received a great deal of attention as one of the important means of cancer therapy. However, many antitumor drugs have problems of low clinical efficacy, great toxic and side effects, etc., and tumor multidrug resistance (MDR) is the leading cause of failure of tumor chemotherapy, and in clinical treatment, the chemotherapy drugs often accompany the occurrence of MDR while killing cancer cells. The MDR phenomenon refers to the tolerance of tumor cells to various drugs under the stimulation of an anti-cancer drug, and is the self-protection behavior of the cancer cells after the stimulation of the drug. The increase in drug efflux phenomenon mediated by P-glycoprotein (P-gp) is the most important mechanism of multidrug resistance in tumors. Many anticancer drugs frequently used in clinic, such as taxol, adriamycin and the like, are substrates of P-gp action. To date, a number of approaches to inhibiting MDR have been reported in the literature either clinically or at the experimental stage: the combination therapy of various medicines, the improvement of the administration method, the development of novel anti-cancer medicines, the application of a P-gp inhibitor TPGS, gene silencing therapy and the like. However, these methods have drawbacks such as large side effects, poor efficacy, high price, etc., and the clinical treatment results are not satisfactory. Therefore, a new therapeutic strategy for treating drug-resistant tumors and avoiding the development of drug resistance is needed. The nano drug-loaded is a new treatment strategy for effectively delivering drugs into focus by using nano materials as carriers, and has the advantages of avoiding the loss of drug effect caused by the release of free drugs in the area which does not reach the tumor and the like.
As a layered inorganic nano-material hectorite LAP, the laponite LAP has good colloidal stability and biocompatibility, and can wrap anticancer drugs DOX between layers. TPGS is a P-gp inhibitor that disrupts mitochondrial function in cancer cells and thereby inhibits intracellular ATP production. Modification of TPGS on the LAP surface is expected to improve the enrichment of drugs in drug-resistant cells, enhance the chemotherapy effect and realize the effect of reversing tumor MDR.
The domestic and foreign documents are searched, and no relevant report about a preparation method of the polyethylene glycol 1000 vitamin E succinate (TPGS) modified hectorite nano-particles is found.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the preparation method of the TPGS modified hectorite nano-particles is simple in process, mild in reaction condition, easy to operate and low in cost; the prepared LM-TPGS/DOX nano-particles have good slow release effect and PH responsive release characteristic, have obvious inhibition effect on the proliferation of MCF-7/ADR cells (DOX-resistant human breast cancer cells), and can be used for the effective treatment of drug-resistant cells.
In order to solve the above problems, the present invention provides a method for preparing TPGS-modified hectorite nanoparticles, which is characterized by comprising the following steps:
step 1): dispersing hectorite in ultrapure water, dropwise adding a silane coupling agent, stirring, reacting at 45-60 ℃ for 12-16 hours, cooling, and dialyzing to obtain lithium amide saponite LM-NH2；
Step 2): dissolving TPGS in a solvent, adding CDI for activation for 5-7 hours, and then dropwise adding LM-NH of the lithium amide soap stone prepared in the step 1)2Reacting in the aqueous solution for 48-72 hours, and dialyzing to obtain TPGS modified hectorite nanoparticles LM-TPGS;
step 3): dropwise adding an adriamycin aqueous solution into the TPGS modified hectorite nanoparticle LM-TPGS aqueous solution prepared in the step 2), reacting in the dark, stirring for 12-24 hours, and centrifuging and washing to obtain LM-TPGS/DOX.
Preferably, the silane coupling agent in step 1) is (3-aminopropyl) dimethylethoxysilane (APMES).
Preferably, the concentration of the solution after the dispersion of the hectorite in the step 1) is 5-10 mg/mL; the mass ratio of the hectorite to the silane coupling agent is 5: 1-5: 4.
Preferably, the dialysis conditions in step 1) or 2) are: dialyzing with a dialysis bag with a cut-off molecular weight of 8000-14000 for 2-3 days.
Preferably, the molar ratio of the CDI to the TPGS in the step 2) is 10: 1-15: 1; the mass concentration of TPGS in LM-TPGS is 15-25 percent; lithium amide saponite LM-NH2The concentration of the aqueous solution of (a) is 6-8 mg/mL.
Preferably, the solvent used for TPGS in step 2) is DMSO.
Preferably, the mass ratio of LM-TPGS to DOX in the step 3) is 1: 1-4: 1; the concentration of the aqueous solution of the TPGS-modified hectorite nanoparticle LM-TPGS is 2-5 mg/mL; the concentration of the adriamycin aqueous solution is 1-2 mg/mL.
Preferably, the centrifugal washing in the step 3) is specifically: centrifuging at 8500-9000 rpm for 10-15 minutes, discarding the supernatant, redissolving with ultrapure water, and centrifuging at 8500-9000 rpm for 10-15 minutes; repeating the operation for 2-3 times.
The invention also provides application of the TPGS modified hectorite nano-particles prepared by the preparation method of the TPGS modified hectorite nano-particles in tumor chemotherapy and anti-tumor multi-drug resistant materials.
The invention adopts P-gp inhibitor TPGS to modify inorganic nano-material laponite LAP for loading anticancer drug adriamycin DOX. Compared with the hectorite nano drug-loaded particles modified by mPEG, the hectorite drug-loaded system modified by TPGS can effectively inhibit P-gp activity, prevent P-gp from pumping out a P-gp substrate DOX to the outside of a cell membrane, and effectively inhibit the proliferation of cancer cells with drug resistance by increasing the accumulation of anticancer drugs in the cancer cells, thereby showing excellent antitumor effect. The invention adopts silane coupling agent APMES to modify inorganic nano material laponite LAP with biocompatibility, and then modifies P-gp inhibitor TPGS on the surface of the laponite LAP to obtain the nano material with anti-tumor multi-drug resistance. The method is simple and easy to operate, and the cost of raw materials is low; the prepared nano-particles have good colloidal stability and biocompatibility, and have potential application value in the field of tumor reversal MDR. The invention uses nuclear magnetic resonance spectrum (1H NMR (NMR), infrared (FTIR), thermogravimetric analysis (TGA), Zeta potential and hydrated particle size (DLS), Ultraviolet (UV) and other methods characterize the physical and chemical properties of the prepared nano-material. The cytotoxicity of the nano material in MCF-7/ADR cells (DOX-resistant human breast cancer cells) is subsequently evaluated by a CCK-8 method, the endocytosis effect of the cells on the drug is measured by using a laser confocal microscope and a flow cytometer, and finally the effect of the material on reversing tumor MDR is evaluated by an ATP detection method and membrane potential measurement.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention adopts simple chemical reaction to prepare TPGS modified hectorite nano-particles with good biocompatibility, and then obtains LM-TPGS/DOX nano-particles to be applied to reversing tumor MDR by physically wrapping and loading anticancer drug adriamycin;
(2) the method has the advantages of simple operation process, low raw material cost, mild reaction condition, environmental protection and easy operation and separation. The prepared nano-particles have good colloidal stability and biocompatibility, and have the prospect of implementation commercialization.
Drawings
FIG. 1 is a schematic diagram of a method for preparing TPGS-modified hectorite nanoparticles according to the present invention;
FIG. 2 shows LM-NH in example 1 and comparative example 12(a) Comparison of nuclear magnetic resonance hydrogen spectra of LM-TPGS (b) and LM-mPEG (c);
FIG. 3 shows LAP and LM-NH in example 22LM-TPGS and LM-mPEG;
FIG. 4 shows LAP and LM-NH in example 32Thermogravimetric analysis of LM-TPGS and LM-mPEG;
FIG. 5 shows DOX, LM-NH in example 42LM-TPGS, LM-mPEG, LM-TPGS/DOX and LM-mPEG/DOX;
FIG. 6 is a graph comparing the in vitro slow release profiles of the drugs at different pH's for LM-TPGS/DOX (a) and LM-mPEG/DOX (b) of example 5;
FIG. 7 is a graph comparing the cell viability map of the nanoparticles LM-TPGS and LM-mPEG of example 6 after 24 hours of co-culture with MCF-7 cells (a) or MCF-7/ADR cells (c) with the cell viability map of DOX, LM-TPGS/DOX, and LM-mPEG/DOX after 24 hours of co-culture with MCF-7 cells (b) or MCF-7/ADR cells (d);
FIG. 8 is a comparative graph of confocal microscopy of laser light after co-culture of DOX, LM-TPGS/DOX, and LM-mPEG/DOX with MCF-7 cells (a) or MCF-7/ADR cells (b) for 4 hours in example 7;
FIG. 9 is a graph comparing fluorescence intensity profiles of flow cytometers after co-culturing DOX, LM-TPGS/DOX, and LM-mPEG/DOX with MCF-7 cells (a) or MCF-7/ADR cells (b) for different time periods (1h, 3h, 6h) in example 7;
FIG. 10 is a graph of the relative ATP content of LM-TPGS/DOX and LM-mPEG/DOX of example 8 after co-culturing with MCF-7/ADR cells for various time periods (1h, 3h, 6 h);
FIG. 11 is a graph showing the red-green ratio of mitochondrial membrane potential after cocultivation of LM-TPGS and LM-mPEG with MCF-7/ADR cells in example 8 for different periods of time (1h, 3h, 6 h).
Detailed Description
In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.
Example 1
A preparation method of TPGS modified hectorite nanoparticles comprises the following steps:
dissolving 50mg of laponite LAP powder in 5mL of ultrapure water, and magnetically stirring overnight at 50 ℃ in a water bath condition to completely dissolve the LAP to obtain a transparent solution; 1mL of an aqueous solution of APMES having a concentration of 20mg/mL was slowly added dropwise to the above aqueous solution of LAP, and the reaction was carried out while maintaining the water bath temperature at 50 ℃ for 16 hours. After the reaction, the reaction mixture was naturally cooled to room temperature, and the product was dialyzed for 3 days (2L of distilled water for each dialysis, 9 times in total) with a dialysis bag having a cut-off molecular weight of 8000-14000. After the dialysis is finished, transferring the product in the dialysis bag into a 50mL EP tube for storage at 4 ℃ to obtain the LM-NH modified by the silane coupling agent2。
10.28mg of TPGS was dissolved in a dimethylsulfoxide DMSO (5mL) solution and activated with CDI (11.02mg) for 6 hours. 3mL of LM-NH2 aqueous solution with the concentration of 6.12mg/mL is slowly and dropwise added into the activated TPGS solution, the reaction is carried out for 3 days by magnetic stirring, and after the reaction is finished, the product is dialyzed for 3 days by a dialysis bag with the cut-off molecular weight of 8000-14000 (2L of distilled water used in each dialysis, 9 times of water replacement). After the dialysis is finished, transferring the product in the dialysis bag into a 50mLEP tube for storage at 4 ℃ to obtain LM-TPGS.
6mL of LM-TPGS aqueous solution having a concentration of 3.3mg/mL was added to 6mL of DOX aqueous solution having a concentration of 1.1mg/mL, and the mixture was magnetically stirred in the dark for reaction for 24 hours. And after the reaction is finished, transferring the product into a 15mL centrifuge tube, centrifuging at 8500rpm for 10 minutes, discarding the supernatant, redissolving with ultrapure water, centrifuging at 8500rpm for 10 minutes, and repeating the operation for 3 times to obtain the drug-loaded nanoparticle LM-TPGS/DOX.
Comparative example 1
The surface modification polymer is a modification method of a common nano material, and the stability of the modified nano particles is improved. To compare the performance of TPGS with conventional polymer-modified nanoparticles, LM-NH was synthesized according to the method and procedure in example 12. Then the prepared LM-NH2Dropwise adding the mixture into mPEG-OH (DMSO solution) activated by CDI, magnetically stirring for reaction for 3 days, and dialyzing the product for 3 days by using a dialysis bag with the molecular weight cut-off of 8000-14000 after the reaction is finished. Finally, drug-loaded nanoparticles LM-mPEG/DOX were synthesized according to the procedure and method in example 1.
Example 2
The LAP, LM-NH obtained in example 1 and comparative example 1 were taken2LM-TPGS, LM-TPGS/DOX and LM-mPEG, LM-mPEG/DOX, etc., which are diluted with ultrapure water to an appropriate concentration, are used for measuring surface potential and hydrodynamic diameter. The experimental results show (Table 1) that LM-NH was prepared2The surface potentials of LM-TPGS, LM-TPGS/DOX, LM-mPEG and LM-mPEG/DOX nanoparticles are respectively-10.0 +/-1.39, -20.0 +/-1.56, -12.4 +/-0.30, -14.0 +/-3.86 and-11.3 +/-1.27 mV, and the experimental result shows that the potential value of the hectorite nanoparticles is increased due to the increase of surface amino groups after the modification of the silane coupling agent, which indicates that LM-NH2Successful synthesis of the compound; and the surface potential value is reduced after the TPGS and mPEG are modified due to the consumption of the amino group modified before, which indicates the successful modification of the TPGS and mPEG. Due to modification of substances such as APMES and TPGS, the hydrodynamic diameter of the nanoparticles is increased, but the hydrodynamic diameter is still maintained below 300nm, which indicates that the material size is uniform. Changes in both surface potential and hydrated particle size indicate that mPEG and TPGS have been modified to the surface of the hectorite.
TABLE 1 LAP, LM-NH2Hydrodynamic diameter and potential of LM-TPGS, LM-TPGS/DOX, LM-mPEG and LM-mPEG/DOX
Example 3
Example 1 and example 1 were weighed separatelyMaterial prepared in comparative example 1: LM-NH2The nuclear magnetic resonance spectra were measured at 3mg each for LM-TPGS and LM-mPEG (shown in FIG. 2). By analyzing the NMR spectrum (see FIG. 2), LM-NH was observed in the graph (a)2The peak appearing at 0-1 ppm proves that the silane coupling agent APMES can be connected to the hectorite in a magnetic stirring manner; as can be seen from the graph (b), characteristic peaks on TOS and PEG in TPGS appear around 0.9 and 3.8ppm respectively, which proves the successful modification of TPGS; as can be seen from the graph (b), a characteristic peak of hydrogen on PEG methoxyl appears around 3.8ppm, which proves the successful modification of mPEG.
The materials prepared in example 1 and comparative example 1 were weighed separately: LM-NH2Infrared spectroscopy (FIG. 3) and thermogravimetric analysis (FIG. 4) were carried out using 2mg each of LM-TPGS and LM-mPEG. By analytical infrared spectroscopy (see FIG. 3), LM-NH2Corresponding curve 1213cm-1The peak at (A) is the stretching vibration peak of the C-N bond on APMES, and is also shown in LM-TPGS and LM-mPEG. And curves corresponding to LM-TPGS and LM-mPEG, 2926cm-1And 2917cm-1The characteristic absorption peak is attributed to the stretching vibration of methylene on the PEG; at the same time, 1735cm-1And 1643cm-1Characteristic absorption peak of (A) is attributed to C ═ O bond stretching vibration on carbonyl group formed by reaction, and 3685cm-1The characteristic absorption peak is attributed to the stretching vibration of the N-H bond on the carbonyl group formed by the reaction. The presence of these characteristic peaks demonstrates the successful modification of APMES, TPGS and mPEG. In addition, the TGA test results show (as in FIG. 4), LM-NH2The weight loss of (A) was 1.62%, and the weight loss of LM-TPGS and LM-mPEG was 21.89% and 15.23%, respectively, thus demonstrating the successful modification of APMES, TPGS and mPEG, while the ratio of TPGS and mPEG to LAP was quantitatively analyzed as 20.27% and 13.61%.
Example 4
The materials LM-NH obtained in example 1 and comparative example 1 were taken2UV absorption was measured by LM-TPGS, LM-TPGS/DOX and LM-mPEG, LM-mPEG/DOX (as shown in FIG. 5). The LM-TPGS has a strong ultraviolet absorption value at 280nm, which is a characteristic peak on a TPGS benzene ring, and the analysis of an ultraviolet spectrum proves that the LM-TPGS has a strong ultraviolet absorption valueSuccessfully modifying; LM-TPGS/DOX and LM-mPEG/DOX have stronger ultraviolet absorption values at 480nm, which are characteristic absorption peaks of DOX and can indicate the successful load of the anti-cancer drug DOX.
Example 5
The materials LM-TPGS/DOX and LM-mPEG/DOX obtained in example 1 and comparative example 1 were dissolved in a buffer solution having a pH of 7.4, a pH of 6.5 and a pH of 5.0 to a concentration of 1mg/mL, and 1mL of the solution was fixed in a dialysis bag, placed in containers containing 9mL of buffers having different pH values, and shaken in a shaker at 37 ℃. Samples were taken every 2 hours for the first 12 hours and every 12 hours thereafter. Taking 1mL of liquid outside the dialysis bag each time, measuring the absorbance value at 480nm, and adding 1mL of corresponding buffer solution outside the dialysis bag. The release profiles of DOX from LM-TPGS/DOX and LM-mPEG/DOX at different pH values in vitro were obtained using this method. By analyzing the sustained-release profile (as shown in fig. 6), it can be seen that DOX can be released from LM-TPGS/DOX and LM-mPEG/DOX at an accelerated rate under an acidic tumor-like environmental condition (pH 5.0), but hardly released under an alkaline condition, which proves that the material has a slow release effect, and can prevent the drug from losing efficacy due to the release without reaching tumor cells.
Example 6
The influence of the prepared LM-TPGS, LM-mPEG, DOX, LM-TPGS/DOX and LM-mPEG/DOX on cell survival was evaluated using MCF-7 and MCF-7/ADR cells as model cells. PBS solutions of LM-TPGS, LM-mPEG, DOX, LM-TPGS/DOX and LM-mPEG/DOX nanoparticles prepared in example 1 and comparative example 1 were prepared at various concentrations. Collecting MCF-7 and MCF-7/ADR cells in logarithmic growth phase, inoculating at 10000 cells per well density on 96-well cell culture plate, adding 100 μ L DMEM medium, and placing the cell culture plate in CO2The culture was carried out at a concentration of 5% and a temperature of 37 ℃ for 24 hours. After discarding the medium, 90. mu.L of DMEM medium was replaced per well and 10. mu.L of LM-TPGS, LM-mPEG (at concentrations of 250, 500, 800, 1000 and 2000. mu.g/mL) was added at different concentrations, respectively; for MCF-7 cells, DOX, LM-TPGS/DOX, LM-mPEG/DOX (DOX concentrations of 6.25, 12.5, 25, 50, 75, 100 and 150 μ g/mL) and pure PBS groups (control groups) were added, with 5 replicates set at each concentration; for MCF-7/ADR cells, DOX, LM-TPGS/DOX, LM-mPEG/DOX (DOX concentrations of 25, 50, 100, 200, 400, 800 and 1000. mu.g/mL) and pure PBS groups (control groups) were added, with 5 replicates set at each concentration. The cell culture plate was placed on 5% CO2Incubation was continued for 24 hours at 37 ℃. The original medium was removed and washed 3 times with sterile PBS, 90. mu.L of DMEM medium was added to each well, 10. mu.L of CCK-8 solution was added, and incubation was continued for 4 hours at 37 ℃ in a dark environment. The absorbance of each well at 490nm excitation wavelength was measured on an MK3 microplate reader, and the magnitude of the absorbance reflected the number of viable cells (as shown in fig. 7). As shown in fig. 7(a) and (c), there was no significant difference in the effect of LM-TPGS and LM-mPEG on MCF-7 and MCF-7/ADR cell viability over the experimental concentration range compared to the PBS control material, which is a good indication of the biocompatibility of the material. According to FIGS. 7(b) and (d), there was no significant difference in the effect of the 3 drug-containing materials DOX, LM-TPGS/DOX and LM-mPEG/DOX on MCF-7 cell viability; however, compared with free DOX and LM-mPEG/DOX, LM-TPGS/DOX has a significant difference in the effect on the survival rate of MCF-7/ADR cells, which fully indicates that LM-TPGS/DOX can effectively inhibit the proliferation of MCF-7/ADR cells and reverse the MDR of tumors.
Example 7
The mechanism of tumor MDR is P-gp mediated drug efflux, so the endocytosis effect of prepared DOX, LM-TPGS/DOX and LM-mPEG/DOX is evaluated by using MCF-7 and MCF-7/ADR cells as model cells. Treated round slides of appropriate size were placed into 12-well plates at a density of 1 slide per well, and 1mL of media was added per well and soaked for 24 hours. Discarding the soaking medium, collecting MCF-7 and MCF-7/ADR cells in logarithmic growth phase, inoculating on 12-well cell culture plate at density of 1 × 105 cells/well, adding 1mL DMEM medium, placing the cell culture plate in CO2The culture was carried out at a concentration of 5% and a temperature of 37 ℃ for 24 hours. After discarding the medium, 900. mu.L of the medium was replaced per well and 100. mu.L of PBS solution containing DOX, LM-TPGS/DOX or LM-mPEG/DOX (DOX concentration 100. mu.g/mL) or PBS (control group) was added. Continuously adding into 5% CO2Incubation was carried out at 37 ℃ for 4 hours. Discarding the culture medium, washing with sterile PBS for 3 times, adding 2.5% glutaraldehyde PBS solution 0.5mL per well, standingThe immobilization was carried out at 4 ℃ for 30 minutes. The glutaraldehyde solution was discarded, washed 3 times with sterile PBS, covered with DAPI (1ug/ml), and left to stand to stain at 37 ℃ for 15 minutes. DAPI was aspirated, washed 3 times with sterile PBS, a drop of fluorescent blocking agent was added dropwise to the slide, the coverslip was lifted out of the 12-well plate, the side with cells was pressed onto the slide, and the morphology of the cells and the distribution of intracellular fluorescence were observed with a laser scanning confocal microscope (see fig. 8). As shown in FIG. 8, the fluorescence intensity of DOX, LM-mPEG/DOX and LM-TPGS/DOX in MCF-7 and MCF-7/ADR cells increased in this order, indicating that the addition of the inorganic nanocarriers LAP and the P-gp inhibitor TPGS effectively increases phagocytosis of anticancer drugs by cells.
Similarly, log phase MCF-7 and MCF-7/ADR cells were collected at 2X 105Cell/well density was plated on 12-well cell culture plates in 5% CO2Incubation was carried out at 37 ℃ for 24 hours. After discarding the medium, 900. mu.L of the medium was replaced per well and 100. mu.L of DOX, LM-TPGS/DOX or LM-mPEG/DOX (DOX concentration 100. mu.g/mL) in PBS or PBS (control) was added. Continuously adding into 5% CO2Incubation was carried out at 37 ℃ for 4 hours. The medium was discarded, washed 3 times with sterile PBS, digested for about 3 minutes by adding 100. mu.L of pancreatin, immediately aspirated and collected with about 1mL of PBS, transferred to a 10mL centrifuge tube, centrifuged at 1000rpm for 5 minutes, and resuspended in 0.5mL of PBS. Taking the fluorescence resolution as a parameter, adding the cell sap which is re-suspended by the filter screen filtration as a sample into a flow cytometer for detection, and obtaining the phagocytosis result of the cells to the medicine (as shown in fig. 9). As shown in FIG. 9, LM-TPGS/DOX has significant differences in fluorescence intensity in MCF-7 and MCF-7/ADR cells compared to DOX and LM-mPEG/DOX, indicating that TPGS is more effective in enhancing DOX phagocytosis by cells.
Example 8
P-gp is taken as a transmembrane transporter capable of mediating drug efflux and is an ATP-dependent drug efflux pump, and TPGS reduces the generation of ATP in cells by breaking the function of mitochondria, thereby inhibiting the activity of the function of P-gp efflux drugs, simultaneously causing the membrane potential of cell membranes to be reduced, and successfully reversing MDR of tumors. Collecting MCF-7/ADR cells in logarithmic growth phase according to 2X 105Density of cells/holeInoculating on 12-well cell culture plate, and placing in 5% CO2Incubation was carried out at 37 ℃ for 24 hours. After discarding the medium, 900. mu.L of the medium was replaced per well and 100. mu.L of LM-TPGS/DOX or LM-mPEG/DOX (DOX concentration 1 OO. mu.g/mL) in PBS or PBS (control) was added. Continuously adding into 5% CO2Incubation at 37 ℃ for various periods of time (1h, 3h, 6 h). The medium was discarded, washed 3 times with sterile PBS, 100 μ L ATP-assay lysate was added to each well under ice bath conditions, and the cells were lysed. After lysis 12000g were centrifuged at 4 ℃ for 5 minutes and 20. mu.L of the supernatant was collected for further use. Under the condition of keeping out of the light, 100 mu L of ATP detection working solution is added into each hole of a 96 blackboard, the operation is waited for several minutes to consume the interference of background ATP, then 20 mu L of the cell supernatant of LM-TPGS/DOX or LM-mPEG/DOX which is prepared before and is incubated for different time periods and PBS blank control are sequentially added, the cell supernatant and the PBS blank control are rapidly mixed by a pipette, and after at least 2 seconds of interval, the RLU value is detected by a multifunctional microplate reader with the function of detecting chemiluminescence (as shown in figure 10). As can be demonstrated from the results in FIG. 9, LM-TPGS/DOX reduced intracellular ATP production compared to LM-mPEG/DOX.
Collecting MCF-7/ADR cells in logarithmic growth phase according to 2X 105Cell/well density was plated on 12-well cell culture plates in 5% CO2Incubation was carried out at 37 ℃ for 24 hours. After discarding the medium, 900. mu.L of the medium was replaced per well and 100. mu.L of LM-TPGS or LM-mPEG (nanoparticle concentration 100. mu.g/mL) in PBS or PBS (control) was added. Continuously adding into 5% CO2Incubation at 37 ℃ for various periods of time (1h, 3h, 6 h). The medium was discarded, washed 3 times with sterile PBS, digested for about 3 minutes by adding 100. mu.L of pancreatin, immediately aspirated and collected with about 1mL of PBS, transferred to a 10mL centrifuge tube, centrifuged at 1000rpm for 5 minutes, and resuspended in 0.5mL DMEM. Adding 0.5mL JC-1 dyeing working solution, reversing for several times, mixing evenly, and placing in 5% CO2Incubating for 20 minutes at 37 ℃, then centrifuging for 3-4 minutes at 600g at 4 ℃, and discarding the supernatant. After washing twice with JC-1 staining buffer, 0.1mL of JC-1 staining buffer is added for resuspension, then the mixture is placed on a 96 blackboard, the fluorescence intensity of the mixture is detected by a multifunctional microplate reader, and the detection result is recorded as the red-green ratio in cells (shown in figure 11). Data according to FIG. 11As a result, compared with LM-mPEG, the intracellular red-green ratio of the LM-TPGS group is obviously reduced and has significant difference, which indicates that LM-TPGS can reduce the intracellular membrane potential. The ATP detection result and the MMP detection result show that LM-TPGS can reduce the generation of ATP in cells, thereby inhibiting the activity of the function of P-gp efflux drugs, simultaneously causing the membrane potential of cell membranes to be reduced, successfully reversing MDR of tumors, and having good application prospect.
Claims (5)
1. A preparation method of TPGS modified hectorite nanoparticles is characterized by comprising the following steps:
step 1): dispersing hectorite in ultrapure water, dropwise adding a silane coupling agent, stirring, reacting at 45-60 ℃ for 12-16 hours, cooling, and dialyzing to obtain lithium amide saponite LM-NH2(ii) a The silane coupling agent in the step 1) is (3-aminopropyl) dimethylethoxysilane; the concentration of the solution after the hectorite is dispersed is 5-10 mg/mL; the mass ratio of the hectorite to the silane coupling agent is 5: 1-5: 4;
step 2): dissolving TPGS in a solvent, adding CDI for activation for 5-7 hours, and then dropwise adding LM-NH of the lithium amide soap stone prepared in the step 1)2Reacting in the aqueous solution for 48-72 hours, and dialyzing to obtain TPGS modified hectorite nanoparticles LM-TPGS; the molar ratio of the CDI to the TPGS in the step 2) is 10: 1-15: 1; the mass concentration of TPGS in LM-TPGS is 15-25 percent; lithium amide saponite LM-NH2The concentration of the aqueous solution is 6-8 mg/mL;
step 3): dropwise adding an adriamycin aqueous solution into the TPGS modified hectorite nanoparticle LM-TPGS aqueous solution prepared in the step 2), reacting in the dark, stirring for 12-24 hours, and centrifuging and washing to obtain LM-TPGS/DOX; the mass ratio of LM-TPGS to DOX is 1: 1-4: 1; the concentration of the aqueous solution of the TPGS-modified hectorite nanoparticle LM-TPGS is 2-5 mg/mL; the concentration of the adriamycin aqueous solution is 1-2 mg/mL.
2. The method for preparing TPGS-modified laponite nanoparticles of claim 1, wherein the conditions of dialysis in step 1) or 2) are: dialyzing with a dialysis bag with a cut-off molecular weight of 8000-14000 for 2-3 days.
3. The method for preparing the TPGS-modified hectorite nanoparticles of claim 1, wherein the solvent used for TPGS in step 2) is DMSO.
4. The method for preparing the TPGS-modified hectorite nanoparticles as claimed in claim 1, wherein the centrifugal washing in the step 3) is specifically as follows: centrifuging at 8500-9000 rpm for 10-15 minutes, discarding the supernatant, redissolving with ultrapure water, and centrifuging at 8500-9000 rpm for 10-15 minutes; repeating the operation for 2-3 times.
5. Use of the TPGS-modified hectorite nanoparticles prepared by the method of preparing the TPGS-modified hectorite nanoparticles of any one of claims 1-4 in the preparation of tumor chemotherapy and anti-tumor multidrug resistant materials.
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