CN113018453A - Targeted drug co-delivery nano system and preparation method and application thereof - Google Patents

Targeted drug co-delivery nano system and preparation method and application thereof Download PDF

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CN113018453A
CN113018453A CN202110296579.0A CN202110296579A CN113018453A CN 113018453 A CN113018453 A CN 113018453A CN 202110296579 A CN202110296579 A CN 202110296579A CN 113018453 A CN113018453 A CN 113018453A
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msn
ptx
gnr
alpha
pgnr
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CN113018453B (en
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郭玉琪
王雪琴
张川
靳林
贺红英
熊田
崔苗
任志帅
马沈倩
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Henan Provincial Peoples Hospital
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Abstract

The invention discloses a targeted drug co-delivery nano system, which is characterized in that mesoporous silica is coated on gold nanorods to form a nano co-carrier with strong drug loading capacity, and the targeted co-delivery of a chemotherapeutic drug PTX and a gene drug miR let-7 alpha is realized by combining the modification of amino, PEG and HA. The invention also discloses a preparation method of the targeted drug co-delivery nano system, which is simple to operate, and the size of the prepared product is controllable. The invention also discloses application of the targeted drug co-delivery nano system in preparation of a drug for treating ovarian cancer. The co-delivery nano system realizes effective transfer of chemotherapeutic drugs and gene drugs, can target tumor tissues, increases the permeability of tumor parts, promotes the uptake of drugs by cancer cells, contributes to improving the exertion of drug effect, reverses the multi-drug resistance of ovarian cancer tissues, enhances the treatment effect of PTX, and thus improves the treatment efficiency. Provides a new idea for treating ovarian cancer.

Description

Targeted drug co-delivery nano system and preparation method and application thereof
Technical Field
The invention relates to the technical field of pharmaceutical preparations, and relates to a targeted drug co-delivery nano system, and a preparation method and application thereof.
Background
Ovarian cancer is one of the most serious cancers which endanger the life health of women, and the incidence rate and the death rate of ovarian cancer are high and are the first of gynecological malignant tumors. At present, the main strategy for clinically treating ovarian cancer is based on Paclitaxel (PTX), platinum and other therapeutic drugs, and the treatment is carried out by combining with surgery. However, during chemotherapy, cancer cells often generate multidrug resistance (MDR) to chemotherapy drugs, which results in patients being insensitive to the treatment drugs and poor chemotherapy effect, and finally results in failure of chemotherapy and even disease recurrence. Statistically, 50-75% of ovarian cancer patients eventually relapse due to MDR. The high expression of the multidrug resistance related gene in ovarian tumor cells is the main reason of multidrug resistance of ovarian cancer. P-glycoprotein (P-glycoprotein, P-gp) is a transmembrane glycoprotein with the molecular weight of 170kDa and coded by a multidrug resistance gene family, and is also called P-170, and the P-gp can continuously exclude drugs from cancer cells and reduce the drug concentration in the cancer cells, thereby causing multidrug resistance. Adenosine Triphosphate (ATP) provides energy to rapidly pump chemotherapeutic drugs out of cancer cells, reducing cytotoxicity of anticancer drugs, and causing drug resistance. Previous studies have shown that MDR1 inhibitors can inhibit the expression of MDR1 in cancer cells, reversing MDR. Although the use of MDR1 inhibitors has been in history for decades, their clinical efficacy has not been sufficient to effectively reverse MDR and improve prognosis in ovarian cancer.
In recent years, nanotechnology-based targeted drug delivery systems have been widely used for the prevention, diagnosis, and treatment of various cancers. The use of specific micrornas (mirs) in combination with chemotherapeutic drugs to overcome multi-drug resistance of cancer is a new strategy to achieve effective treatment of cancer. MicroRNAs (mirs) are small endogenous single-stranded non-coding RNAs with the length of about 20-24 nucleotides, regulate the expression of a plurality of genes in a body, participate in almost all life processes such as cell proliferation, differentiation, apoptosis and metabolism and play an important role in researching the pathology of ovarian cancer. Some miRs have been found to have anti-ovarian effects, such as lethal (let-7) and miR-199a-3 p. However, miRs have the problems that the miRs are not expressed enough in ovarian cancer cells, cannot effectively inhibit the growth and apoptosis of tumors and weaken the killing effect of chemotherapeutic drugs on the tumor cells, and the like. Therefore, the search for a more effective method for reversing the multidrug resistance of ovarian cancer is the focus of current research, and the method has important and profound significance for treating ovarian cancer and other cancers.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a targeted drug co-delivery nano system, which can provide protection for drug transportation, maintain the stability of combination of chemotherapeutic drugs and gene drugs, and help the co-delivery drugs to exert a synergistic effect to effectively inhibit the growth of ovarian tumors.
The second purpose of the invention is to provide a preparation method of the targeted drug co-delivery nano system.
The invention also aims to provide the application of the targeted drug co-delivery system in preparing the drugs for treating ovarian cancer.
One of the purposes of the invention is realized by adopting the following technical scheme:
a targeted drug co-delivery nano system comprises gold nanorods, wherein the surfaces of the gold nanorods are coated with amino-modified mesoporous silica, the amino-modified mesoporous silica and the gold nanorods form a co-carrier loaded with a targeted drug, and the amino-modified mesoporous silica is sequentially modified by PEG and hyaluronic acid;
the targeted medicine is chemotherapeutic medicine and gene medicine.
Furthermore, the chemotherapeutic drug is paclitaxel, and the gene drug is microRNA lethal-7 alpha.
Further, the nucleotide sequence of the microRNA lethal-7 alpha is UGAGGUAGUAGGUUGUAUAGUU.
The second purpose of the invention is realized by adopting the following technical scheme:
the preparation method of the targeted drug co-delivery nano system comprises the following steps:
1) preparing a gold nanorod solution, namely a GNR solution, by adopting a seed growth method;
2) adjusting the pH value of the GNR solution obtained in the step 1) to 10-11, adding TEOS (tetraethyl orthosilicate) for reaction to obtain a mesoporous silica nanoparticle-coated gold nanorod composite, namely GNR @ MSN;
3) mixing the GNR @ MSN obtained by repeating the step 2) twice with a 3-aminopropyltriethoxysilane solution, and reacting to obtain an amino-modified GNR @ MSN compound, namely GNR @ MSN-NH2；
4) Preparing a mixed solution containing paclitaxel, and then adding the mixed solution to GNR @ MSN-NH obtained in the step 3)2In the solution, the GNR @ MSN-NH of the load paclitaxel is obtained by reaction2Namely PTX-GNR @ MSN;
5) preparing the PTX-GNR @ MSN obtained in the step 4) to obtain a solution, then adding guanidine hydrochloride and microRNA (miR let-7 alpha) into the solution to react to obtain the GNR @ MSN loaded with paclitaxel and microRNA (miR let-7 alpha) and the GNR @ MSN (PTX/miR let-7 alpha-GNR @ MSN);
6) dissolving NH2-PEG-COOH, then adding N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride into a mixed solution obtained by dissolving in 2- (N-morpholinyl) ethanesulfonic acid buffer solution, reacting to obtain activated PEG, then adding the activated PEG into the PTX/microRNA lethal-7 alpha-GNR @ MSN prepared in the step 5), and reacting to obtain PEG-modified PTX/microRNA lethal-7 alpha-GNR @ MSN (marked as PTX/miR let-7 alpha-pGNR @ MSN);
7) adding hydrated hyaluronic acid into a 2- (N-morpholino) ethanesulfonic acid buffer solution containing N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride to obtain a mixture, adjusting the pH of the mixture to obtain activated hyaluronic acid, mixing the activated hyaluronic acid with the PTX/microRNA lethal-7 alpha-pGNR @ MSN obtained in the step 6), and reacting to obtain the hyaluronic acid modified PTX/microRNA lethal-7 alpha-pGNR @ MSN (recorded as HA-PTX/miR let-7 alpha-pGNR @ MSN).
Further, the mass ratio of Au to TEOS contained in the GNR solution in step 2) was 5.28: 1.
Further, the mass ratio of 3-aminopropyltriethoxysilane to Au contained in GNR @ MSN in step 3) was 0.125: 1.
Further, PTX and GNR @ MSN-NH in step 4)2The mass ratio of the microRNA to the PTX-GNR @ MSN is 1:1, and the ratio of the microRNA to 7 alpha in the step 5) to the PTX-GNR @ MSN is 80 mu mol/L:1 mg.
Further, in the step 6), the mass ratio of PEG to PTX/microRNA lethal-7 alpha-pGNR @ MSN in the PTX/microRNA lethal-7 alpha-GNR @ MSN is 1:1, the mass ratio of HA to PTX/microRNA lethal-7 alpha-pGNR @ MSN in the step 7) is 1:1, and the redundant high molecular PEG and HA are removed by dialysis and centrifugation.
Further, the step 1) of preparing the gold nanorods by a seed growth method comprises the following steps:
separately adding HAuCl to CTAB solution4And NaBH4Preparing seed solution, adding HAuCl into CTAB solution4、AgNO3And preparing growth solution with hydroquinone, mixing the seed solution with the growth solution, and reacting to obtain the gold nanorods.
The third purpose of the invention is realized by adopting the following technical scheme:
application of targeted drug co-delivery nano system in preparation of drugs for treating ovarian cancer.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a targeted drug co-delivery nano-system HA-PTX/miR let-7 alpha-pGNR @ MSN, which can effectively reverse the multi-drug resistance of ovarian cancer. The co-delivery nano system is a co-carrier formed by gold nanorods and amino-modified mesoporous silica nanoparticles, has a larger specific surface area, and is beneficial to improving the loading capacity of drugs; and the co-carrier has good stability and biocompatibility and has low toxicity to organisms. The co-carrier is modified by PEG after carrying drugs, so that the circulation time of the co-delivery system in blood is prolonged, a good protection effect on drug delivery is achieved, the stable combination of PTX and miR let-7 alpha is maintained, and the co-delivery system can be absorbed by cancer cells; the co-delivery nano system is further modified by hyaluronic acid, and can be used for treating ovarian cancer SKOV3/SKOV3 after HA modificationTRThe CD44 receptor with high expression in the cell is specifically combined, so that more effective cell uptake can be realized, the permeability of the medicine at a tumor part is improved, and the medicine effect can be exerted to the maximum extent.
The co-delivery nano system comprises a chemotherapeutic drug, a gene drug and a co-carrier nano system, realizes co-delivery of the chemotherapeutic drug PTX and the gene drug miR let-7 alpha, and achieves the purpose of cooperative treatment. The invention adopts an exogenous gene supplement drug miR let-7 alpha, promotes the apoptosis of tumor cells, effectively enhances the drug effect of chemotherapy drug PTX on ovarian cancer, weakens the drug resistance of ovarian cancer on PTX, and achieves the purpose of treating ovarian cancer.
The invention also provides a preparation method of the targeted drug co-delivery nano system, the preparation process is simple, the size of the synthesized nano compound is controllable, and the nano compound is beneficial to reducing the phagocytosis of cancer cells caused by overlarge size or the clearance of the nano delivery system from the body by the filtration of glomeruli caused by undersize, thereby ensuring the exertion of the drug effect.
The invention also provides the application of the targeted drug co-delivery nano system in the preparation of drugs for treating ovarian cancer, the targeted drug co-delivery nano system realizes the effective transfer of chemotherapeutic drugs and gene drugs, can target tumor tissues, increases the permeability of tumor parts, promotes the uptake of drugs by cancer cells, is beneficial to improving the exertion of drug effect, reverses the multi-drug resistance of the ovarian cancer tissues, and enhances the treatment effect of PTX, thereby improving the treatment efficiency. Provides a new idea for treating ovarian cancer.
Drawings
FIG. 1 is an XRD spectrum of gold nanorods of the invention;
FIG. 2 is a transmission electron micrograph of gold nanorods of the present invention, wherein FIG. 2A is a TEM image of GNRs, and FIG. 2B is a HRTEM image of GNRs;
FIG. 3 is a TEM, HAADF and EDS-mapping of GNR @ MSN of the present invention, wherein FIG. 3A is a TEM of GNR @ MSN, FIG. 3B is a close-up view of the TEM of GNR @ MSN, and FIG. 3C is an HAADF and EDS-mapping view of the TEM of GNR @ MSN;
FIG. 4 shows the GNRs, GNR @ MSN-NH of the present invention2FT-IR spectra of pGNR @ MSN and HA-pGNR @ MSN;
FIG. 5 shows N of GNR @ MSN according to the present invention2Absorption-desorption isotherms and corresponding pore size distribution curves;
FIG. 6 is a graph of the particle size distribution (FIG. 6A) and zeta potential (FIG. 6B) of the nanomaterials of the invention;
FIG. 7 shows the biological safety of the HA-pGNR @ MSN co-vector nanosystem of the present invention: cell viability after 24h treatment with different concentrations of HA-pGNR @ MSN;
FIG. 8 shows the biological safety of the HA-pGNR @ MSN co-vector nanosystem of the present invention: hemolytic profile of HA-pGNR @ MSN (FIG. 8A), digital photographs of RBCs before and after 2 hours incubation at different concentrations of HA-pGNR @ MSN (FIG. 8B);
FIG. 9 shows the fluorescence signature of the HA-pGNR @ MSN co-vector nanosystem of the present invention: fluorescent RBITC-labeled HA-pGNR @ MSN (FIG. 9A), fluorescent RBITC-labeled pGNR @ MSN (FIG. 9B), and the fluorescence spectra of the corresponding fluorescent RBITC-labeled HA-pGNR @ MSN, pGNR @ MSN (FIG. 9C) were analyzed by Image-Pro Plus software;
FIG. 10 is a diagram of the structure of the cellular uptake assay of the present invention: wherein FIG. 10A is a plan view of a liquid crystal display deviceRBITCpGNR @ MSN andRBITCfluorescence image of SKOV3 cells treated with HA-pGNR @ MSN for 12h (concentration 50. mu.g/mL); FIG. 10B shows a schematic view of a processRBITCpGNR @ MSN andRBITCcorresponding quantitative data analysis of fluorescence images of SKOV3 cells treated by HA-pGNR @ MSN for 12h (concentration 50 mug/mL); FIG. 10C shows HA-FAMmiR let-7α-RBITCSKOV3/SKOV3 after 6, 12, and 24 hours of pGNR @ MSN treatmentTRFluorescence image of cells: FAM (green), RBITC (red) and DAPI (blue);
FIG. 11 is a drug loading analysis graph of the HA-PTX/miR let-7 alpha-GNR @ MSN co-delivery nano-system, wherein FIG. 11A is a PTX specific peak area in an experimental group HA-PTX/miR let-7 alpha-GNR @ MSN obtained by high performance liquid chromatography test, and FIG. 11B is a standard curve equation obtained by high performance liquid chromatography test of a PTX standard solution;
FIG. 12 is a gel blocking electrophoretogram of HA-pGNR @ MSN and miR let-7 a of the present invention under conditions of different mass ratios;
FIG. 13 is a graph showing the results of in vitro anti-proliferative capacity and therapeutic effect of the HA-PTX/miR let-7 alpha-GNR @ MSN co-delivery nanosystems of the present invention (where FIG. 13A is the cellular activity of targeted HA-PTX/miR let-7 alpha-pGNR @ MSN and non-targeted PTX/miR let-7 alpha-pGNR @ MSN treated SKOV3 cells; FIG. 13B is targeted HA-PTX/miR let-7 alpha-pGNR @ MSN and non-targeted PTX/miR let-7 alpha-pGNR @ MSN treated SKOV3TRThe cellular activity of the cell; FIG. 13C is a pair of different therapeutic nanocomplexes SKOV3TRTherapeutic effects of cell proliferation; FIG. 13D shows the determination of cell viability by PI and FDA double staining; FIG. 13E is a graph of the corresponding quantitative analysis of FIG. 13D; wherein a, a control group; b, HA-miR let-7 alpha-pGNR @ MSN; c, HA-PTX-pGNR @ MSN; d, PTX/miR let-7 alpha-pGNR @ MSN; e, HA-PTX/miR let-7 alpha-pGNR @ MSN);
FIG. 14 shows P-gp of the present invention at SKOV3TRAnalysis of expression levels in cells: FIG. 14A is proteinThe expression graph of P-gp is determined by blotting, and FIG. 14B is the corresponding quantitative analysis graph after the expression of P-gp is determined by Western blotting;
FIG. 15 is SKOV3 treated with HA-PTX/miR let-7 alpha-pGNR @ MSN of P-gp of the present invention at different concentrationsTRAnalysis of expression levels in cells;
FIG. 16 is a graph of the process of nuclear division of apoptotic cells of the invention: FIG. 16A is SKOV3 and SKOV3 stained with Hoechst H33258TRA cell fluorescence image; FIG. 16B is SKOV3 and SKOV3 treated with various therapeutic nanocomplexesTRCorresponding quantitative analysis of the apoptosis rate; FIG. 16C is SKOV3 and SKOV3 treated with different concentrations of HA-PTX/miR let-7 α -pGNR @ MSNTRA quantitative analysis chart corresponding to Hoechst 33258;
FIG. 17 shows SKOV3 treated with different therapeutic nanocomplexes in accordance with the present inventionTRWestern blot of apoptosis-related proteins: wherein FIG. 17A is a Western blot and FIG. 17B is a statistical analysis of protein levels (a, control; B, HA-miR let-7 α -pGNR @ MSN; c, HA-PTX-pGNR @ MSN; d, PTX/miR let-7 α -pGNR @ MSN; e, HA-PTX/miR let-7 α -pGNR @ MSN); FIGS. 17C and 17D are SKOV3 and SKOV3 treated with different concentrations of HA-PTX/miR let-7 α -pGNR @ MSNTRA western blot and corresponding quantitative analysis of (a);
FIG. 18 is a graph of an apoptosis assay of the invention: FIGS. 18A1-A5, B1-B5 are SKOV3 and SKOV3TRAfter the cells are treated by a-e group therapeutic nano-composite, the cell apoptosis is analyzed by flow cytometry by annexin V-FITC/PI staining;
FIGS. 18C, 18D are SKOV3 and SKOV3, respectively, for various therapeutic nanocomplex treatmentsTRCorresponding quantitative analysis graphs of the percentage of viable cells, early apoptotic cells, late apoptotic cells and necrotic cells after the cells;
FIG. 19 is a graph of Acridine Orange (AO) stain of the present invention vs. SKOV3 and SKOV3 treated with various therapeutic nanocomplexesTRFluorescence effect profile after staining cells (a, control group; b, HA-miR let-7 alpha-pGNR @ MSN; c, HA-PTX-pGNR @ MSN; d, PTX/miR let-7 alpha-pGNR @ MSN; e, HA-PTX/miR let-7 alpha-pGNR @ MSN);
FIG. 20 depicts SKOV3 and SKOV3 after treatment with various therapeutic nanocomplexes of the inventionTRCarrying out rhodamine 123 staining on the cells, wherein the figure 20A is a fluorescence effect graph, and the figure 20B is a quantitative analysis graph; a, a control group; b, HA-miR let-7 alpha-pGNR @ MSN; c, HA-PTX-pGNR @ MSN; d, PTX/miR let-7 alpha-pGNR @ MSN; e, HA-PTX/miR let-7 alpha-pGNR @ MSN;
FIG. 21 shows SKOV3 and SKOV3 after treatment with various therapeutic nanocomplexes of the inventionTRROS profile of cells in which FIGS. 21A1-A5, B1-B5 are SKOV3 and SKOV3TRFIG. 21C is a graph of the flow cytometric analysis of intracellular ROS levels in cells, SKOV3 and SKOV3TRA graph of flow cytometric quantification of intracellular ROS levels of cells; a, a control group; b, HA-miR let-7 alpha-pGNR @ MSN; c, HA-PTX-pGNR @ MSN; d, PTX/miR let-7 alpha-pGNR @ MSN; e, HA-PTX/miR let-7 alpha-pGNR @ MSN;
FIG. 22 shows SKOV3 and SKOV3 after treatment with various therapeutic nanocomplexes of the inventionTRThe expression level of different factor-related proteins of cells is shown in the figure 22A, in which the electrophoresis diagram of the expression levels of mTOR, stat3 and EZH2 factor-related proteins is shown in the figure 22B, and the quantitative analysis diagram of the expression levels of mTOR, stat3 and EZH2 factor-related proteins is shown in the figure 22B;
figure 23 is a graph of the in vivo therapeutic effect of various therapeutic nanocomplexes of the invention: wherein FIG. 23A is an experimental flow chart of the in vivo treatment of each therapeutic nanocomposite, and FIG. 23B is a graph of a mouse and an anatomic tumor after the in vivo treatment of each therapeutic nanocomposite;
FIG. 24 is a graph of the in vivo therapeutic effect of various therapeutic nanocomplexes of the invention: wherein figure 24A is a time-dependent tumor growth curve after in vivo treatment of each therapeutic nanocomposite object, and figure 24B is tumor weight after 15 days of in vivo treatment of each therapeutic nanocomposite;
figure 25 is an in vivo therapeutic pathology analysis of each therapeutic nanocomposite of the invention: FIG. 25A is a graph of P-gp immunohistochemical staining and TUNEL staining for pathological changes and FIG. 25B is a graph of H & E staining and Ki-67 immunohistochemical staining for tumor tissue cell proliferation after 15 days of treatment;
FIG. 26 is a representative image of H & E staining of mouse major organs (heart, liver, spleen, lung and kidney) after 15 days of in vivo treatment of each therapeutic nanocomplex of the present invention;
FIG. 27 shows that the HA-PTX/miR let-7 alpha-GNR @ MSN targeting drug co-delivery nano system of the invention is applied to ovarian cancer SKOV3/SKOV3TRSchematic diagram of targeted drug delivery and therapeutic mechanism in cell lines and tumor tissues.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
Example 1
A targeted drug co-delivery nano system comprises gold nanorods, wherein the surfaces of the gold nanorods are coated with amino-modified mesoporous silica, the amino-modified mesoporous silica and the gold nanorods form a co-carrier to load targeted drugs, and the amino-modified mesoporous silica is sequentially modified by PEG and hyaluronic acid.
The preparation process of the targeted drug co-delivery nano system comprises the following steps:
1) 5mL of 0.1M cetyltrimethylammonium bromide (CTAB) was added thereto 125. mu.L of HAuCl with a mass fraction of 1%4After sufficiently stirring, 300. mu.L of NaBH 0.01M was added4After the solution color is changed from golden yellow to brown, the solution is placed for 2 hours at the temperature of 30 ℃ to obtain a seed solution; 7mL of 0.1M CTAB and 300. mu.L of 1% HaCl4The solutions were mixed, and 10. mu.L of 0.1M AgNO was added to the mixture3. Then, 120 mu L of 0.33M hydroquinone is added into the mixed solution, and the solution is quickly colorless, thus obtaining a growth solution; adding 100 mu L of seed solution into the growth solution, uniformly mixing, reacting at 30 ℃ for 12h to obtain the gold nanorods, centrifuging for 3min at 5000 r, dispersing the precipitate in deionized water to obtain a GNR (gold nanorod dispersion solution), and storing at 4 ℃ until use.
2) 100 μ L of 28 wt% NH was used3·H2O adjusting the pH of the GNR solution obtained in the step 1) to 10-11, adding 17 μ L TEOS every 30min for 4 times to make the Au and TEOS contained in the GNR solutionThe mass ratio was 5.28: 1. Then reacted in a water bath at 30 ℃ for 12 h. Washing with deionized water and methanol, and centrifuging the reaction product for 5min at 5000 r to obtain a mesoporous silica nanoparticle coated gold nanorod composite, namely GNR @ MSN;
3) mixing the GNR @ MSN obtained by repeating the step 2) twice with 15 mu L of 3-aminopropyl triethoxysilane (APTES) and 85 mu L of methanol according to the adding proportion that the mass ratio of the 3-aminopropyl triethoxysilane to Au contained in the GNR @ MSN is 0.125:1, refluxing for 6h at 75 ℃ in a methanol solution, then centrifuging for 10min after heating methanol for 5000 revolutions to obtain an amino modified GNR @ MSN compound, namely GNR @ MSN-NH2；
4) Dissolving 1mg of paclitaxel in 1mL of dimethyl sulfoxide to obtain a mixed solution, and then adding to GNR @ MSN-NH obtained in step 3)2In the solution, continuously shaking to react overnight according to the mass ratio of 1:1, centrifuging for 10 minutes at 10000 rpm to obtain the GNR @ MSN loaded with paclitaxel, namely PTX-GNR @ MSN;
5) 0.25mg of PTX-GNR @ MSN obtained in step 4) was dissolved in 40. mu.L of ethanol to obtain a solution, to which 10. mu.L of 4mol/L guanidine hydrochloride and 20. mu.mol/L microRNA lethal-7. alpha. (brand: shanghai gima pharmaceutical technologies, ltd). Shaking at normal temperature for 2h, centrifuging at 10000 r/min for 10min to obtain GNR @ MSN loaded with paclitaxel and microRNA lethal-7 alpha, namely PTX/microRNA lethal-7 alpha-GNR @ MSN;
6) adding 10mg of NH2-PEG-COOH was dissolved in 8mL DMSO, then a mixed solution (pH 6.0) prepared by dissolving 38mg of N-hydroxysuccinimide (NHS) and 68mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in 2- (N-morpholino) ethanesulfonic acid (MES) buffer was added thereto, and stirred and activated for 24 hours at room temperature to obtain activated PEG, and then the activated PEG was added to PTX/microRNA lethal-7 α -GNR @ MSN prepared in step 5) at a mass ratio of 1:1 to obtain a mixture, stirring the mixture at room temperature for 24h, centrifuging at 10000 r/min to obtain PEG modified PTX/microRNA lethal-7 alpha-GNR @ MSN solution, namely PTX/microRNA lethal-7 alpha-pGNR @ MSN;
7) adding 20mg of hydrated HA to MES buffer (pH 6.0) containing 10mL of 10mg/mL of EDC/NHS for activation, and adjusting the pH of the mixture to 8.3 using Tris buffer to obtain activated hyaluronic acid; mixing the activated hyaluronic acid with the PTX/microRNA lethal-7 alpha-pGNR @ MSN obtained in the step 6) according to the mass ratio of 1:1 of HA to PTX/microRNA lethal-7 alpha-pGNR @ MSN, stirring for reaction for 24h at room temperature, and centrifuging for 10min at 10000 r/min after the reaction is finished to obtain the PTX/microRNA lethal-7 alpha-pGNR @ MSN modified by hyaluronic acid, namely HA-PTX/microRNA lethal-7 alpha-NR @ MSN.
Comparative example 1
Comparative example 1 differs from example 1 in that: the synthesis was carried out without adding PTX and was otherwise identical to example 1. And finally obtaining HA-miR let-7 alpha-pGNR @ MSN.
Comparative example 2
Comparative example 2 differs from example 1 in that: the gene drug miR let-7 alpha is not added in the synthesis process, and the rest is the same as that in the embodiment 1, so that HA-PTX-pGNR @ MSN is finally obtained.
Comparative example 3
Comparative example 3 differs from example 1 in that: HA is not added in the synthesis process for modification, and the rest is the same as that in the embodiment 1, and PTX/miR let-7 alpha-pGNR @ MSN is finally obtained.
Experimental example 1
The morphology, components, chemical bonds and microstructure of the product obtained in example 1 are systematically studied by modern nano-test analysis techniques such as XRD, HAADF-STEM, HRTEM, FTIR, zeta, BET and the like, and the results are as follows:
the crystal structures of GNRs obtained in the examples are analyzed by X-ray diffraction (XRD; X' Pert-PRO-MPD, Hol-land-Panalytical) with monochromatic X-ray beams and nickel-filtered Cu-Ka radiation, and the results are shown in figure 1, and the diffraction of the gold nanorods is clearly shown in an XRD pattern to appear at positions with 2 theta of 38.2 degrees, 44.4 degrees, 64.6 degrees, 77.6 degrees and 81.7 degrees and respectively correspond to crystal faces (111), (200), (220), (311) and (222), which indicates that the gold nanorods are successfully synthesized.
The present invention uses a high-resolution transmission electron microscope (HRTEM) of a high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) and a JEM-2100 analytical electron microscope (JEOL ltd, tokyo, japan) to characterize GNRs and GNR @ MSN obtained in the examples, and the results are shown in fig. 2 and 3. FIG. 2A (FIG. 2A, schematic diagram of gold nanorods in the upper right corner) shows that the average length and width of GNRs prepared by the example of the present invention are 75nm + -5 nm and 5nm + -5 nm, respectively, and the aspect ratio is about 7: 1. The gold nanorods prepared by the method have uniform size and controllable size. FIG. 2B is a schematic diagram of a gold nanorod, in which the GNRs are further magnified by HRTEM and the bright and dark stripes of the lattice spacing on the surface of the gold nanorod are clearly visible. FIGS. 3A and 3B show that mesoporous silica is uniformly coated on the surface of gold nanorods, and the average particle size of GNR @ MSN is 135nm +/-5 nm. FIG. 3C shows an elemental mapping pair GNR @ MSN analysis using dark-field detector and EDS-mapping in HAADF mode, showing that GNRs are mainly distributed in the bright regions. The Au and Si elements are uniformly distributed, and the position distribution of the Au and Si elements shows that the gold nanorod is arranged in the middle, and the mesoporous silicon dioxide is coated on the surface of the gold nanorod.
The present invention employs Fourier transform infrared Spectrometer (FTIR Spectrometer) (Nicolet Is 50 Continuum; Thermo Fisher Scientific, Waltham, Mass., USA) to detect GNRs, GNR @ MSN-NH obtained by each step of the embodiments of the present invention2pGNR @ MSN and HA-pGNR @ MSN, wherein the pGNR @ MSN and the HA-pGNR @ MSN are different from PTX/miR let-7 alpha-pGNR @ MSN and HA-PTX/miR let-7 alpha-pGNR @ MSN in that PTX and miR-let-7 are omitted in the preparation process, and the rest parts are the same. The detection result is shown in FIG. 4, and is at 1081cm-1And 798cm-1The characteristic absorption peak appears at the position of (A) is the vibration absorption peak of Si-O-Si, which shows the successful wrapping of mesoporous silica, GNR @ MSN-NH2FITR spectrum at 1560cm-1Has an absorption peak, indicating that GNR @ MSN-NH2The presence of an N-H bond demonstrates the successful modification of GNR @ MSN by an amino group. PITR spectrum of HA-pGNR @ MSN showing characteristic band of amide bond at amide N-H stretch (3680 cm)–1) And amide C ═ O stretching (1690 cm)–1) Here, the successful modification of pGNR @ MSN by HA is indicated.
The Barrett-Joyner-Halenda (BJH) and Brunauer-Emmett-Teller (BET) methods of the invention are carried out by the N2The pore size and specific surface area of the GNR @ MSN obtained in the examples were determined by absorption-desorption technique (TriStar II 3020 system (Micromeritics, usa)). The results are shown in the figureAs shown in FIG. 5, it is understood that the specific surface area of GNR @ MSN is 77.00m2The/g shows that the average pore diameter is 34.22nm, and the high specific surface area and pore diameter are beneficial to the mesoporous silica to realize effective adsorption of PTX and miR let-7 alpha drugs.
The zeta potential and particle size of the samples obtained in the various steps of the examples were determined using a Zetasizer Nano ZS90 Analyzer (Malvern Panalytical, Malvern, Netherland). The results are shown in FIG. 6, where FIG. 6A shows GNRs, GNR @ MSN-NH2pGNR @ MSN and HA-pGNR @ MSN hydrated particle size distributions of 255.7, 398.7, 488.9, 802.2 and 929.7nm, respectively, and FIG. 6B shows GNRs, GNR @ MSN-NH2The potentials of pGNR @ MSN and HA-pGNR @ MSN were +13.00, -6.94, +21.70, -3.55 and-37.33 mV, respectively. Therefore, the successful modification of each component in the synthetic process of the embodiment of the invention can be known.
The characterization and analysis results show that the amino-modified mesoporous silica-coated gold nanorod is firstly synthesized to be a co-carrier nano system, and the gold nanorod has a large specific surface area, so that functional modification and further coupling of a targeting ligand can be performed on the surface of the gold nanorod. And then the surface of the gold nanorod is coated with a layer of mesoporous silica, the mesoporous silica has large aperture, strong attraction and good morphological characteristics, and the mesoporous silica is combined with the gold nanorod to obtain a nano compound to form a co-carrier nano system, so that the improvement of the drug loading capacity of the carrier is facilitated. Then modifying polyethylene glycol on the surface of the nano system by an EDC/NHS crosslinking method to enhance the biocompatibility of the nano system, grafting targeted molecular hyaluronic acid on the surface through amido bond, and improving the enhanced carrier targeting property through the specific combination with CD44 receptor overexpressed on the surface of the tumor.
Experimental example 2
Evaluation of biological safety and fluorescence performance of HA-pGNR @ MSN co-carrier nano system
2.1 in vitro cytotoxicity test
Cell culture: the invention selects the ovarian cancer SKOV3 cell line which is purchased from Shanghai cell bank of Chinese academy of sciences (Shanghai, China). SKOV3 cells were cultured in RPMI 1640 medium containing 10% FBS, 100. mu.g/mL penicillin and 100. mu.g/mL streptomycin at 37 deg.C、5％CO2Humidifying the air. SKOV3TRThe cell line (PTX-resistant SKOV3 cells) was maintained in 10. mu.L of PTX solution at a concentration of 2mg/mL in DMSO as a solvent in RPMI 1640 medium.
Low cytotoxicity is crucial for the construction of targeted drug co-delivery nanosystems. The invention adopts a 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazole ammonium bromide (MTT) method to detect the cytotoxicity of HA-pGNR @ MSN (HA-pGNR @ MSN is different from the embodiment 1 in that PTX and miR-let-7 are omitted in the preparation process, and the rest is the same). The specific process is as follows: SKOV3 cells were plated in a 96-well plate at a density of 3000 cells/well and cultured for 12 h. Different concentrations of HA-pGNR @ MSN solutions were then injected into the above 96-well plates at HA-pGNR @ MSN concentrations of 0, 25, 50, 100, 200 and 400. mu.g/mL, respectively. After 24h incubation at 37 ℃, the medium was removed and 200 μ L of MTT solution at 0.5mg/mL was added per well, followed by 4h further incubation with 150 μ L DMSO per well and vortexing to obtain the samples to be tested. Finally, the absorbance of the sample to be tested at a wavelength of 570nm was measured using a microplate spectrophotometer (BioTek Instruments inc., Winooski, VT, USA).
As shown in FIG. 7, SKOV3 cells did not show significant cytotoxicity after 24 hours of treatment with HA-pGNR @ MSN at 0, 25, 50, 100, 200, and 400. mu.g/mL.
2.2 hemolysis experiment
The invention evaluates the blood compatibility of HA-pGNR @ MSN by a hemolysis experiment. The specific process is as follows: fresh human blood was centrifuged at 3000 rpm for 15min, the supernatant was removed, and the pellet was washed with 0.9% saline to obtain Red Blood Cells (RBCs). The erythrocytes were then diluted with the appropriate amount of 0.9% physiological saline, and 1mL of a 2% strength suspension of erythrocytes was added to different concentrations of HA pGNR @ MSN solutions, at concentrations of 50, 100, 200 and 400. mu.g/mL, respectively. Saline and deionized water were used as negative (-) and positive control (+) respectively. The sample was stabilized at room temperature for 2 hours and centrifuged at 3000 rpm, the supernatant was collected and the absorbance was measured at a wavelength of 540nm, and from the image of the obtained mixture, the hemolysis rate was calculated.
As shown in FIG. 8, FIG. 8A shows that the hemolysis of erythrocytes in HA-pGNR @ MSN solutions of different concentrations showed low levels, even at a maximum concentration of 400. mu.g/mL, with a hemolysis rate of only 4.85% (< 5%). FIG. 8B is a digital photograph of RBCs before and after incubation for 2 hours at various concentrations of HA-pGNR @ MSN. The above results indicate that HA-pGNR @ MSN can be used in subsequent in vivo and in vitro experiments.
2.3 cellular uptake assay
The invention adopts HA-pGNR @ MSN and pGNR @ MSN marked by fluorescent RBITC (pGNR @ MSN is different from the embodiment 1 of the invention in that PTX, miR-let-7 and HA are omitted in the preparation process, and the rest are the same) to carry out cell uptake analysis. The method comprises the following specific steps: HA-pGNR @ MSN and pGNR @ MSN are firstly marked by fluorescent RBITC, 10mg of vector is respectively dissolved in boric acid buffer solution with pH of 8.5, 20 mu L of RBITC (dissolved in DMSO) with the concentration of 1mg/mL is added, the mixture is shaken for 4h at normal temperature, and is washed by PBS for 3 times and then observed under a fluorescent microscope. Results as shown in fig. 9, fig. 9A and 9B are fluorescent RBITC labeled HA-pGNR @ MSN, respectively, and fig. 9C is a corresponding fluorescence map analyzed by Image-Pro Plus software, showing that the RBITC labeled HA-pGNR @ MSN shows stronger red fluorescence compared to pGNR @ MSN, indicating that the RBITC labeling method can be effectively used for cell uptake analysis of targeted drug co-delivery systems.
The process of the cell uptake experiment is as follows: SKOV3 cells were inoculated onto 24-well plates, incubated for 48h, added with fluorescent RBITC-labeled HA-pGNR @ MSN and pGNR @ MSN, and cultured again in the dark for 4 h. After the completion of the culture, the cells were washed 3 times with phosphate buffer (PBS, pH 7.4) and photographed under an inverted eclipse te2000-U fluorescence microscope (nikon, japan). The invention also detects the transfection efficiency of RNA through carboxyl Fluorescein (FAM) labeled miR let-7 alpha, and the cellular uptake of the FAM labeled miR let-7 alpha is the same as the processes of fluorescence RBITC labeled HA-pGNR @ MSN and pGNR @ MSN. Wherein the FAM-labeled miR let-7 alpha vector is purchased from Shanghai Jima pharmaceutical technology Co., Ltd, and HA-FAMThe miR let-7 alpha-pGNR @ MSN is marked by RBITC to prepare the fluorescent double-label HA-FAM miR let-7α-RBITC pGNR@MSN。
As a result, as shown in FIG. 10, FIGS. 10A and 10B show that the NPs of the RBITC mark can be markedThe effective endocytosis is realized by targeting HA, and the SKOV3/SKOV3 is effectively identified and combinedTRThe CD44 receptor with high expression in the cell improves the cellular uptake efficiency, wherein the cellular uptake efficiency of HA-pGNR @ MSN is about 150%. FIG. 10C shows fluorescent dual-labeled HA-FAM miR let-7α-RBITCpGNR @ MSN further confirmed HA-pGNR @ MSN and SKOV3/SKOV3TRTransfection effects after cell co-incubation for 6, 12 and 24 hours, HA-pGNR @ MSN can more effectively convey miR let-7 alpha to SKOV3/SKOV3TRCells, and reached maximum transfection efficiency after 12 hours of treatment.
Experimental example 3
Drug loading capacity of HA-PTX/miR let-7 alpha-pGNR @ MSN co-delivery nano system
As is known, paclitaxel, as a clinical classical antitumor drug, can lose the dynamic balance of synthesis and depolymerization of tubulin, induce and promote tubulin polymerization, microtubule assembly, and prevent depolymerization, thereby stabilizing microtubules, inhibiting mitosis of cancer cells and triggering apoptosis of cells, further effectively preventing proliferation of cancer cells, and playing a role in resisting ovarian cancer. The paclitaxel used as the anti-ovarian cancer chemotherapeutic drug for preparing the co-delivery nano system has the following problems: 1. hydrophobic drugs are difficult to combine with gold nanoparticles; 2. the gene medicine RNA is easy to degrade and is difficult to effectively deliver to an action target. Therefore, the invention utilizes mesoporous silicon dioxide with super-large pores to coat the surface of the gold nanorod so as to achieve the purpose of enhancing drug adsorption. pGNR @ MSN is PEG modified compound nano-particles, and PEG can prolong the blood circulation time of the medicament. The targeting ligand HA can specifically recognize ovarian cancer SKOV3/SKOV3TRHigh expression of CD44 receptor in cell line, resulting in high medicine releasing efficiency and high curative effect.
The invention respectively measures the drug loading capacity of PTX and miR let-7 alpha in an HA-PTX/miR let-7 alpha-pGNR @ MSN co-delivery nano system by High Performance Liquid Chromatography (HPLC) and agarose gel electrophoresis, and the specific steps are as follows: the mobile phase (water and methanol) was first filtered and sonicated for 0.5h to remove bubbles. Preparing standard solutions of paclitaxel by using a mobile phase, wherein the standard solutions are respectively 2.5 mu g/mL, 5 mu g/mL, 10 mu g/mL, 20 mu g/mL, 40 mu g/mL and 80 mu g/mL, dissolving 1mg of HA-PTX/miR let-7 alpha-pGNR @ MSN in the mobile phase, performing ultrasonic treatment until the drug is completely released, taking supernatant, and performing detection on a machine. Setting the wavelength of the detection parameters to be 227 nm; the sample injection amount is 10 mu L; the column temperature was 25 ℃; the ratio of mobile phase is 50: 50; the flow rate was 1 mL/min. Leveling a base line, carrying out sample injection detection, and obtaining the substance quantity of the nano material adsorbed medicine according to a standard curve drawn by the peak area and the concentration of the standard solution. The calculation formula is as follows:
Figure BDA0002984582460000121
Figure BDA0002984582460000122
wherein Wt is the weight of PTX in HA-PTX/let-7 α -GNR @ MSN, Ws is the weight of HA-PTX/let-7 α -GNR @ MSN, W0Is the initial weight of the PTX input.
The results are shown in fig. 11, where fig. 11A shows the specific peak area of PTX in the experimental group HA-PTX/miR let-7 α -GNR @ MSN obtained by hplc analysis, fig. 11B shows the standard curve obtained from the paclitaxel standard solution, and the standard curve equation is y-7.8 × 10, where the concentration of the analyte is abscissa and the peak area of the analyte is ordinate5+5.1×105And x (r is 0.9909), and substituting the peak area of the paclitaxel measured in the experimental group into a standard curve equation to obtain the HA-PTX/miR let-7 alpha-GNR @ MSN with the drug loading rate of the paclitaxel of 3.82% and the encapsulation rate of 38.2%.
In order to obtain the optimal combination rate of the miR let-7 alpha and a co-carrier system, the invention sets a series of GNR @ MSN-NH by taking 20 mu M as the final concentration of the miR let-7 alpha2And the proportional gradient of miR let-7 alpha, and the gel retardation method is adopted to determine GNR @ MSN-NH2And optimal ratio of miR let-7 alpha. Wherein the proportion gradient is respectively 0, 40:1, 80:1, 120:1, 160:1, 200:1, 240:1 and 280:1(w/w), and the proportion gradient is from a group a to a group h in sequence from small to large.
The results are shown in FIG. 12, along with GNR@MSN-NH2And the proportion of miR let-7 alpha is increased, the more fuzzy the bands in the agarose gel electrophoresis pattern are, and even the bands disappear. At a ratio of 200:1, no free miR let-7 alpha is found in the supernatant, which indicates that at the ratio, miR let-7 alpha and GNR @ MSN-NH are present2The binding was complete.
Experimental example 4
In-vitro anti-proliferation capacity and treatment effect of HA-PTX/miR let-7 alpha-GNR @ MSN co-delivery nano system
4.1MTT method for detecting in-vitro anti-proliferation capacity of HA-PTX/miR let-7 alpha-GNR @ MSN co-delivery nano system
The invention adopts MTT method to detect the anti-proliferation ability of various therapeutic nano-composites, and the experimental process is the same as 2.1 part of MTT method in the experimental example 2. Results are shown in FIG. 13, FIGS. 13A, 13B show targeting HA-PTX/miR let-7 α -GNR @ MSN pairs SKOV3 and SKOV3TRThe therapeutic effect of the cells is better than that of non-target PTX/miR let-7 alpha-GNR @ MSN, but compared with SKOV3 cells, the PTX treated SKOV3TRThe sensitivity of cells to targeted and non-targeted drugs is low, SKOV3TRThe cell survival rate is slightly higher than that of SKOV3 cells. The reason for this may be SKOV3TRThe high expression of P-gp (P-glycoprotein) in the cell can induce the active discharge of partial medicine. FIG. 13C shows targeted HA-PTX/miR let-7 alpha-pGNR treated SKOV3TRThe proliferation capacity of the cells is obviously lower than that of SKOV3 processed by non-targeting PTX/miR let-7 alpha-pGNR @ MSNTRAnd cells show that the HA-PTX/miR let-7 alpha-GNR @ MSN co-delivery nano system can effectively enhance the treatment effect.
4.2 evaluation of Activity of HA-PTX/miR let-7 alpha-GNR @ MSN Co-delivery nanosystem transfected cells by double staining method
The present invention also uses double FDA (fluorescein diacetate) and PI (propidium iodide) staining to assess cell viability. The specific process is as follows: treatment of SKOV3 and SKOV3 with PBS-washed various therapeutic nanocomplexesTRCells were incubated with 1. mu.g/mL FDA and 20. mu.g/mL PI for 5 min. Cells were then washed with PBS and photographed under an inverted Eclipse TE2000-U fluorescence microscope. Experimental setup group a was a control group of the same amount of physiological saline added, and the various therapeutic nanocomposites are example 1 and the pairThe products obtained in ratios 1 to 3 HA-miR let-7 alpha-pGNR @ MSN (group b), HA-PTX-pGNR @ MSN (group c), PTX/miR let-7 alpha-pGNR @ MSN (group d), HA-PTX/miR let-7 alpha-pGNR @ MSN (group e). The FDA/PI double staining results show (FIGS. 13D, 13E) that targeting HA-PTX/let-7 α -pGNR @ MSN HAs a better therapeutic effect than that of non-targeting PTX/let-7 α -pGNR @ MSN.
4.3 Western blotting of P-gp at SKOV3TRExpression analysis in cells
The invention also adopts a western blot method to detect P-gp at SKOV3TRExpression in cells, the process of western blotting treatment is: in the experiment, except for the concentration gradient, the concentration of nanoparticles of 100 mug/mL is used as the experimental concentration, and SKOV3 and SKOV3 treated by various therapeutic nano-composites obtained in example 1 and comparative examples 1 to 3 of the application are usedTRThe cells were lysed in lysis buffer and then boiled in a constant temperature metal bath at 100 ℃ for 5min to obtain total protein extract. Mu.g of total protein extract was then separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Burlington, MA, USA) and blocked for 1h in 0.05% phosphate buffered saline-Tween 20(PBST) buffer containing 8% (w/v) skim milk. The PVDF membrane was washed with PBST buffer and then incubated at 4 ℃ for 1h at room temperature with a primary antibody (diluted 1:5000 or 1:10000 with primary anti-diluent) and a secondary antibody conjugated with horseradish peroxidase (HRP) ensuring complete immersion of the PVDF membrane (diluted 1:10000 or 1:20000 with secondary anti-diluent). Finally, an immunoreactive band was generated using Pierce-ECL immunoblotting substrate (Thermo-Fisher-Scientific) and the relative protein amount was normalized to glyceraldehyde 3-phosphate dehydrogenase (GADPH) level.
Wherein each therapeutic nanocomposite is the product obtained in example 1 of the present invention and comparative examples 1 to 3: group c HA-miR let-7 alpha-pGNR @ MSN, group d HA-PTX-pGNR @ MSN, group e PTX/miR let-7 alpha-pGNR @ MSN, and group f HA-PTX/miR let-7 alpha-pGNR @ MSN. The buffer solution comprises the following components: 50mM Tris-HCl solution at pH 7.4, 150mM NaCl, 1% NP-40, 0.5% sodium D-cholate, 0.1% Sodium Dodecyl Sulfate (SDS), 2mM phenylmethylsulfonyl fluoride (PMSF).
The results are shown in FIG. 14, and compared with the HA-PTX-pGNR @ MSN group, the expression of P-gp in the targeted HA-miR let-7 alpha-pGNR @ MSN group is low (FIG. 14A), which indicates that let-7 alpha can effectively reduce the expression of P-gp, thereby further reducing SKOV3TRResistance of the cells to PTX. P-gp was minimally expressed in the group of targeted co-delivery HA-PTX/miR let-7 α -pGNR @ MSN (FIG. 14A), indicating that miR let-7 α further enhances the chemotherapeutic effect of PTX and promotes SKOV3TRAnd (4) apoptosis. FIG. 14B is a bar graph of the relative amounts of P-gp protein in each group corresponding to FIG. 14A.
In addition, on the basis of the conclusion, the HA-PTX/miR let-7 alpha-pGNR @ MSN solutions of 25, 50, 100, 200 and 400 mu g/mL are respectively prepared for processing SKOV3TRCells, and the SKOV3 when HA-PTX/miR let-7 alpha-pGNR @ MSN is added at different concentrations is researchedTRExpression of P-gp in the cell. The results are shown in FIG. 15, wherein FIG. 15A is a P-gp expression level electrophoretogram, and it can be seen that the P-gp expression level is reduced with the increase of the concentration of HA-PTX/miR let-7 alpha-pGNR @ MSN. FIG. 15B is a bar graph of the relative amounts of P-gp protein in each group corresponding to FIG. 15A. Therefore, the HA-PTX/miR let-7 alpha-pGNR @ MSN targeted drug co-delivery nano system provided by the invention effectively reverses SKOV3TRMDR of cells.
Experimental example 5
Analysis of apoptosis mechanism
5.1 Nuclear division Process of apoptotic cells
In order to observe the nuclear division process of apoptotic cells, the invention adopts Hoechst 33258 to detect SKOV3 and SKOV3 of different nano-composite therapeutic agentsTRThe effects of apoptosis. The specific experimental process is as follows: SKOV3 and SKOV3TRThe cells were cultured in 24-well plates for 12h, followed by addition of 100. mu.g/mL HA-miR let-7. alpha. -pGNR @ MSN (group b), HA-PTX-pGNR @ MSN (group c), PTX/miR let-7. alpha. -pGNR @ MSN (group d), and HA-PTX/miR let-7. alpha. -pGNR @ MSN (group e), obtained in example 1 and comparative examples 1 to 3, respectively, in a group A, which was set by addition of an equal amount of physiological saline as a control group. After 24h of treatment, the drug and stock culture were removed and the once treated cells were washed with PBS. Then 500. mu.L of 0.5mg/mL Hoechst 33258 stain was added to each well in the darkCulturing for 20 min. And washing the cells twice by using PBS (phosphate buffer solution), and removing unreacted staining solution to obtain a sample to be detected. The sample to be detected was placed under an inverted Eclipse-TE2000-U fluorescence microscope for observation.
The results are shown in FIG. 16, and SKOV3 and SKOV3 stained with Hoechst 33258 were obtainedTRTypical changes in apoptotic cells were chromatin condensation, perinuclear aggregation and nuclear fragmentation (FIGS. 16A, 16B), and as the added concentration of HA-PTX/miR let-7 α -pGNR @ MSN obtained in example 1 of the present invention increased, SKOV3 and SKOV3TRNuclear fragmentation and chromatin condensation of cells increased dramatically and the rate of apoptosis increased (fig. 16C).
5.2 SKOV3 treated with different therapeutic nanocompositesTRStatistical analysis of western blot and relative protein levels of apoptosis-related proteins
Myeloid leukemia factor-1 (Myeloid leukemia factor-1, Mcl-1) is an anti-apoptotic member of the B-cell lymphoma 2(B-cell lymphoma 2, Bcl-2) family, and is highly expressed in cancer cells. The expression of Mcl-1 not only promotes the generation and development of tumors, but also can make cancer cells generate drug resistance to chemotherapeutic drugs. SKOV3 treated with different therapeutic nanocomposites for this experimentTRThe procedure of the cells was the same as that of Experimental example 5.1 except that the Hoechst 33258 staining treatment was not performed.
Results as shown in figure 17, Mcl-1 expression was significantly reduced in the targeted co-delivery HA-PTX/miR let-7 α -pGNR @ MSN group, indicating SKOV3TRThe sensitivity of the cells to PTX was significantly increased and further induced an apoptotic map (17A). FIG. 17B SKOV3 treated with different therapeutic nanocomplexesTRStatistical analysis of relative protein levels of apoptosis-related proteins concluded the same as in fig. 17A.
As with Experimental example 4.3, the present invention explores the SKOV3 pair by adding HA-PTX/miR let-7 alpha-pGNR @ MSN solutions with different concentrations of 25, 50, 100, 200 and 400 mu g/mLTRInfluence of the expression level of Mcl-1 in the cell. Results As shown in FIG. 17, Mcl-1 expression also decreased with increasing HA-PTX/miR let-7 α -pGNR @ MSN concentration (FIGS. 17C and 17D). SKOV3 in the targeted co-delivery HA-PTX/miR let-7 alpha-pGNR @ MSN group compared to the other groupsTRIn cells, expression of a pro-apoptotic Bcl-2-associated X protein (Bcl-2-associated X protein, Bax) is increased, and expression of anti-apoptotic oversize B cell lymphoma (Bcl-XL) is reduced, which indicates that the HA-PTX/miR let-7 alpha-pGNR @ MSN targeted drug co-delivery system obtained by the invention can realize SKOV3TRReversal of MDR in cells.
5.3V-FITC/PI staining method for detecting apoptosis by flow cytometry
The invention adopts Annexin V-FITC and PI double staining method to detect SKOV3 and SKOV3TRAnd (4) apoptosis. The specific experimental process is as follows: SKOV3 and SKOV3 were first digested with 0.25% trypsinTRThe cells were then centrifuged for 5 minutes at 1000 rpm, and the supernatant was discarded to collect the treated cells. The cells were resuspended in 500. mu.L of PBS and then 5. mu.L of annexin V-FITC and PI, respectively, were added. And culturing the mixture in the dark at room temperature for 10min to obtain the sample cells to be detected. The cells to be detected were analyzed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) and Cell Quest software (BD Biosciences).
The mechanism of MDR reversal was further examined using Annexin V-FITC and PI staining. The results are shown in fig. 18, where the treated cells were divided into four quadrants according to the fluorescence intensity: viable cells (live), early apoptotic cells (early apoptosis), late apoptotic cells (late apoptosis) and necrotic cells (necrotize) (FIG. 18A1-A5, B1-B5). FIGS. 18C and 18D respectively carried out quantitative analysis on cell populations in four quadrants of each group, and the results showed that SKOV3 and SKOV3 were obtained after HA-PTX/miR let-7 alpha-pGNR @ MSN acted for 24 hoursTRThe cell survival rates are respectively 29.1% and 42.8%, and the results show that the HA-PTX/miR let-7 alpha-pGNR @ MSN targeted drug co-delivery nano system obviously promotes apoptosis. And the number of early apoptotic cells is significantly higher than late apoptotic cells, probably due to the reversion of Phosphatidylserine (PS) from the inside to the surface of the cell membrane and exposure to the extracellular environment.
Experimental example 6
Effect of therapeutic Nanocompositions on related organelles and mTOR-mediated Signaling pathways
6.1 Effect of various therapeutic Nanocomplexes on vesicles
To determine the effect of the various therapeutic nanocomplexes obtained in example 1 of the present invention and comparative examples 1 to 3 on vesicles, Acridine Orange (AO) staining solution was used to treat SKOV3 and SKOV3TRThe cells were stained. The experimental process is as follows: SKOV3 and SKOV3 were treated with various therapeutic nanocomplexes obtained in example 1 and comparative examples 1 to 3TRCells were washed twice with PBS for 24h, and resuspended in 300. mu.L of PBS. Then, 300. mu.L of 0.01% AO staining solution was added thereto, and the mixture was treated in the dark at room temperature for 15 min. After treatment was complete, the cells were washed twice again with PBS and imaged under an inverted fluorescent Eclipse TE2000-U microscope.
Results as shown in FIG. 19, HA-PTX/miR let-7 α -pGNR @ MSN targeted drug co-delivery system treated SKOV3 and SKOV3 compared to the other groupsTRThe number of red fluorescent spots induced in the cytoplasm of the cells increased significantly, indicating a decrease in acidic compartments such as lysosomes and autophagosomes.
6.2 mitochondrial membrane potential detection
Electrochemical potential energy is stored in the inner mitochondrial membrane. If the concentration of protons and other ions is asymmetrically distributed across the membrane, the mitochondrial membrane potential tends to rise, called "MMP". The invention adopts rhodamine 123 dyeing method to detect Mitochondrial Membrane Potential (MMP), and the specific process is as follows: SKOV3 and SKOV3T cells were treated with each of the therapeutic nanocomplexes obtained in example 1, comparative example 1 to comparative example 3 for 24 hours, washed twice with PBS, stained with 50 μ g/mL rhodamine 123, and cultured in the dark for 30 min. Next, the cells were washed twice again with PBS and imaged under an inverted eclipse TE2000-U fluorescence microscope.
The results are shown in fig. 20, HA-PTX/miR let-7 α -pGNR @ MSN induces an increase in intracellular green fluorescent aggregates, indicating that the mitochondrial inner membrane depolarizes (fig. 20A and 20B), and the depolarization process of the mitochondrial membrane potential leads to the release of a large amount of cytochrome c into the cytoplasm by the mitochondria, causing the apoptosis process. Compared with the control group and other therapeutic nano-composites, the apoptosis rate of the HA-PTX/miR let-7 alpha-pGNR @ MSN group cells is more obvious.
6.3 active oxygen analysis
SKOV3 and SKOV3 treated with different nanocomposites obtained from example 1, comparative examples 1 to 3TRCells were washed twice with PBS, treated with 50 μ M DCFH-DA dye in FBS-free medium, and cultured for 15min in the dark. Next, the cells were washed twice again with PBS and analyzed for Reactive Oxygen Species (ROS) with a flow cytometer.
The results are shown in fig. 21, compared with the control group and other therapeutic nanocomplexes, the cellular ROS level of the HA-PTX/miR let-7 α -pGNR @ MSN group is significantly increased, which indicates that ROS generation caused by mitochondrial function change is more significant, and is positively correlated with cancer cell apoptosis rate.
6.4 Effect of therapeutic Nanocompolexes on mTOR-mediated Signaling pathways
The target protein of rapamycin (mTOR) is an important regulator of cell growth and proliferation. Abnormal regulation of the mTOR signaling pathway is closely associated with cell proliferation. Signal Transducers and Activators of Transcription (STATs) are a family of highly conserved transcription factors whose complete phosphorylation and translocation in the cytoplasm is stimulated by extracellular signals. STAT3 is one of seven members of the STAT family, involved in cell cycle, apoptosis regulation, tumor angiogenesis, tumor cell invasion, metastasis, and immune escape. EZH2(enhancer of zeste homolog 2) is highly expressed in tumor drug-resistant cells, has histone methyltransferase activity, and participates in X chromosome inactivation, cell differentiation and embryonic development regulation.
Treatment with various therapeutic nanocomplexes for 24h, SKOV3 and SKOV3TRThe mTOR-related protein in the cells was subjected to western blot analysis and corresponding quantitative analysis. As a result, as shown in fig. 22, mTOR expression decreased, resulting in a rapid decrease in STAT3 levels and decreased phosphorylation of its regulatory protein, decreasing histone methyltransferase activity of EZH2 (fig. 22A, 22B). These cascades help to demonstrate that HA-PTX/miR let-7 alpha-pGNR @ MSN HAs the ability to reverse MDR and inhibit SKOV3TRThe ability of a cell to proliferate.
Experimental example 7
In vivo study of MDR reversal in ovarian cancer
7.1SKOV3TREstablishment of tumor xenograft model
The mice used in the experiments of the present invention were obtained from Wuhan laboratory animals GmbH (Wuhan, China) as 4-week-old male BALB/c-nu mice and placed under specific pathogen-free (SPF) conditions. All animal experiments were conducted in accordance with the guidelines of the university of zhengzhou animal care and use committee (zhengzhou, china).
The invention adopts SKOV3 in exponential growth phaseTRCells 100. mu.L of cell suspension (density 8X 10) was prepared6Individual cells). The cell suspension was inoculated subcutaneously into the back of mice subcutaneously. Mice were observed daily for health and behavior, and tumor volumes and body weights were recorded every 2 days. The tumor volume is calculated as V-1/2 × a × b2Wherein a and b are the major and minor diameters of the tumor, respectively. The tumor inhibition rate is calculated by the formula of R (%) ═ 1-Vt/V0) X 100, where Vt and V0Tumor volumes for the treatment group and control group, respectively. When the average tumor volume reaches 30mm3In this case, various therapeutic nanocomposites prepared in example 1 of the present invention, comparative example 1 to comparative example 3 were injected.
7.2 pharmaceutical intervention and histopathological analysis
SKOV3 to be successfully establishedTRBALB/c-nu mice in tumor xenograft models were randomly divided into 5 groups, treated with the following therapeutic nanocomposites prepared in inventive example 1, comparative example 1 to comparative example 3, respectively: HA-miR let-7 alpha-pGNR @ MSN, HA-PTX/miR let-7 alpha-pGNR @ MSN, PTX/miR let-7 alpha-pGNR @ MSN and HA-PTX/miR let-7 alpha-pGNR @ MSN. The control group was injected with an equal amount of physiological saline. The above nanocomposites were injected intratumorally at an injection rate of 15mg/kg (200. mu.L) every 2 days, respectively. After drug treatment (day 15), mice were sacrificed and tumor and major organs (liver, heart, lung, kidney, spleen) were taken for hematoxylin-eosin (H) treatment&E) Histopathological analysis was performed by staining, terminal deoxynucleotide transfer dUTP nick end labeling (TUNEL) staining, and antigen Ki-67 staining. Finally, the corresponding tumors were analyzed by Western blot assayAnd detecting the expression level of P-gp.
Results as shown in fig. 23, fig. 23A is a schematic experimental flow chart of the nanocomposite therapy. After 15 days of treatment, mice were sacrificed and major organs and tumor tissues were collected. The results found no significant change in body weight for all mice (fig. 23B), indicating that the HA-pGNR @ MSN nanocarrier did not produce significant in vivo toxicity during treatment. However, compared with the non-targeting PTX/miR let-7 alpha-pGNR @ MSN group, the targeting co-delivery HA-PTX/miR let-7 alpha-pGNR @ MSN group HAs smaller tumor volume in the mice and better treatment effect. In addition, co-delivery of PTX/miR using HA-pGNR @ MSN as a nanocarrier can effectively inhibit SKOV3 following intratumoral administrationTRCell growth and synergistic inhibition of tumor growth (FIGS. 24A-24B).
P-gp levels in cancer tissues were significantly reduced after HA-PTX/miR let-7 α -pGNR @ MSN treatment, consistent with in vitro experiments (fig. 25A). TUNEL staining results for the targeted co-delivery HA-PTX/miR let-7 α -pGNR @ MSN group showed decreased staining signals, suggesting increased cancer cell apoptosis compared to the other groups. In the targeted co-delivery HA-PTX/miR let-7 α -pGNR @ MSN group, H & E staining clearly showed significant solid tumor destruction (fig. 25B). A decrease in Ki-67 levels in tumor tissues also indicates a decrease in tumor proliferation capacity and malignancy following HA-PTX/miR let-7 α -pGNR @ MSN treatment (FIG. 25B). In addition, the targeted co-delivery HA-PTX/miR let-7 α -pGNR @ MSN group had no significant damage to major organs, further demonstrating the low toxicity and good safety of the nanocarriers (fig. 26).
Fig. 27 is a schematic diagram showing the targeting administration and treatment mechanism of the HA-PTX/miR let-7 α -GNR @ MSN targeting drug co-delivery nano system in ovarian cancer tumor tissues, and the schematic diagram shows that the co-delivery nano system can be specifically combined with a CD44 receptor to target tumor cells and release chemotherapeutic drug PTX and gene drug miR let-7 α after administration after ovarian cancer tumors are formed in mice. After the medicine acts, the change of related influencing factors in tumor cells is consistent with the description of the results of experimental example 6 and experimental example 7, and finally the expression level of P-gp is reduced, thereby achieving the purpose of treating ovarian cancer.
In conclusion, the invention provides a targeted drug co-delivery nano system, HA-PTX/miR let-7 alpha-pGNR @ MSN, which can effectively reverse the multi-drug resistance of ovarian cancer. The co-delivery nano system is a co-carrier nano system formed by gold nanorods and amino-modified mesoporous silica nanoparticles, has a larger specific surface area, and is beneficial to improving the loading capacity of drugs; and the co-carrier nano system has good stability and biocompatibility and has low toxicity to organisms.
The nano system realizes the co-delivery of miR let-7 alpha and PTX: the co-carrier nano system is modified by PEG after carrying drugs, so that the residence time of the co-delivery system in blood is prolonged, a good protection effect on drug delivery is achieved, and the miR let-7 alpha/PTX compound can be effectively co-transmitted to SKOV3/SKOV3 through the nano systemTRIn cells and ovarian cancer tissues, HA can be modified with SKOV3/SKOV3TRThe highly expressed CD44 receptor in cells specifically binds, enabling more efficient cellular uptake and permeability at the tumor site. Moreover, miR let-7 alpha can reduce P-gp expression and reverse SKOV3TRDrug resistance of cells and ovarian cancer tissues enhances the treatment effect of PTX, thereby improving the treatment efficiency.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
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Claims (10)

1. A targeted drug co-delivery nano system is characterized by comprising gold nanorods, wherein the surfaces of the gold nanorods are coated with amino-modified mesoporous silica, the amino-modified mesoporous silica and the gold nanorods form a co-carrier to load targeted drugs, and the amino-modified mesoporous silica is sequentially modified by PEG and hyaluronic acid;
the targeted medicine is chemotherapeutic medicine and gene medicine.
2. The targeted drug co-delivery nanosystem of claim 1, wherein the chemotherapeutic drug is paclitaxel and the genetic drug is microRNA lethal-7 α.
3. The targeted drug co-delivery nanosystem of claim 2, wherein the nucleotide sequence of microRNA lethal-7 a is UGAGGUAGUAGGUUGUAUAGUU.
4. The method for preparing a targeted drug co-delivery nanosystem according to any of claims 1 to 3, comprising the steps of:
1) preparing a gold nanorod solution, namely a GNR solution, by adopting a seed growth method;
2) adjusting the pH value of the GNR solution obtained in the step 1) to 10-11, adding TEOS (tetraethyl orthosilicate) for reaction to obtain a mesoporous silica nanoparticle-coated gold nanorod composite, namely GNR @ MSN;
3) mixing the GNR @ MSN obtained in the step 2) with a 3-aminopropyltriethoxysilane solution, and reacting to obtain an amino-modified GNR @ MSN compound, namely GNR @ MSN-NH2；
4) Preparing a mixed solution containing paclitaxel, and then adding the mixed solution to GNR @ MSN-NH obtained in the step 3)2In the solution, the GNR @ MSN-NH of the load paclitaxel is obtained by reaction2Namely PTX-GNR @ MSN;
5) preparing the PTX-GNR @ MSN obtained in the step 4) to obtain a solution, then adding guanidine hydrochloride and microRNA lethal-7 alpha into the solution, and reacting to obtain the GNR @ MSN loaded with paclitaxel and microRNA lethal-7 alpha, namely PTX/microRNA lethal-7 alpha-GNR @ MSN;
6) dissolving NH2-PEG-COOH, then adding N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride into a mixed solution obtained by dissolving in 2- (N-morpholinyl) ethanesulfonic acid buffer solution, reacting to obtain activated PEG, then adding the activated PEG into the PTX/microRNA lethal-7 alpha-GNR @ MSN prepared in the step 5), and reacting to obtain PEG-modified PTX/microRNA lethal-7 alpha-GNR @ MSN, namely PTX/microRNA lethal-7 alpha-pGNR @ MSN;
7) adding hydrated hyaluronic acid into a 2- (N-morpholino) ethanesulfonic acid buffer solution containing N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride to obtain a mixture, adjusting the pH of the mixture to obtain activated hyaluronic acid, mixing the activated hyaluronic acid with the PTX/microRNA lethal-7 alpha-pGNR @ MSN obtained in the step 6), and reacting to obtain the hyaluronic acid modified PTX/microRNA lethal-7 alpha-pGNR @ MSN, namely HA-PTX/microRNA lethal-7 alpha-pGNR @ MSN.
5. The targeted drug co-delivery nanosystem of claim 4, wherein the weight ratio of Au to TEOS contained in the GNR solution in step 2) is 5.28: 1.
6. The targeted drug co-delivery nanosystem of claim 4, wherein the mass ratio of 3-aminopropyltriethoxysilane to Au contained in GNR @ MSN in step 3) is 0.125: 1.
7. The targeted drug co-delivery nanosystem of claim 4, wherein in step 4) PTX and GNR @ MSN-NH2The mass ratio of the microRNA to the PTX-GNR @ MSN is 1:1, and the ratio of the microRNA to 7 alpha in the step 5) to the PTX-GNR @ MSN is 80 mu mol/L:1 mg.
8. The targeted drug co-delivery nanosystem of claim 4, wherein the mass ratio of PEG to PTX/microRNA lethal-7 α -GNR @ MSN in step 6) is 1:1 and the mass ratio of HA to PTX/microRNA lethal-7 α -pGNR @ MSN in step 7) is 1: 1.
9. The targeted drug co-delivery nanosystem of claim 4, wherein the step 1) of preparing the gold nanorods by a seed growth method comprises the steps of: separately adding HAuCl to CTAB solution4And NaBH4Preparing seed solution, adding HAuCl into CTAB solution4、AgNO3And preparing growth solution with hydroquinone, mixing the seed solution with the growth solution, and reacting to obtain the gold nanorods.
10. Use of the targeted drug co-delivery nanosystems of any of claims 1 to 3 in the manufacture of a medicament for the treatment of ovarian cancer.
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CN115430832B (en) * 2022-09-06 2024-05-03 河南大学 Preparation method and application of core-shell type gold nanomaterial and chemotherapy-thermal therapy type core-shell type gold nano drug targeted delivery system

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