CN107693487B - Anthracycline antitumor antibiotic compound micelle and preparation method and application thereof - Google Patents
Anthracycline antitumor antibiotic compound micelle and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a compound nano micelle simultaneously carrying anthracycline antitumor antibiotics and salvianolic acid A, and a preparation method and application thereof. The compound micelle comprises effective dose of anthracycline antitumor antibiotic, salvianolic acid A and amphiphilic high molecular polymer, and two medicines are stably wrapped in the nano micelle together in a simple physical wrapping mode through the combined action of electrostatic attraction between a basic group of the anthracycline antitumor antibiotic and an acid group of the salvianolic acid A and self-assembly of the amphiphilic high molecular polymer in an aqueous medium, so that the obtained compound micelle is uniform in particle size, the wrapping rate is more than 95%, and the drug loading rate and the stability are obviously superior to those of single-medicine micelle.
Description
Technical Field
The invention relates to a compound nano micelle simultaneously carrying anthracycline antitumor antibiotics and salvianolic acid A based on an amphiphilic high molecular polymer material, and provides a method for preparing the nano micelle and application thereof, belonging to the technical field of biological medicines.
Background
Cancer is a serious disease threatening human life and health, and traditional chemotherapy drugs are the main subjects of cancer clinical treatment. The anthracycline antitumor antibiotic is an antitumor drug which takes DNA chain breakage and structural damage as a main action mechanism, has the characteristics of wide antitumor spectrum and effectiveness on hypoxic cells, is a radiosensitizer, plays an important role in tumor treatment, is a core drug of a first-line treatment scheme of acute leukemia, lymphoma, breast cancer, lung cancer, liver cancer and various other solid tumors, and mainly comprises adriamycin (ADM, DOX), Epirubicin (EPI), Daunorubicin (DNR), pyrarubicin (THrarubicin, P-ADM), anamycin (Aclainomycin, ACM), mitoxantrone (MTT, DHAD) and the like. Because the oral bioavailability of the anthracycline antitumor antibiotic is low, intravenous drip is generally carried out in clinic, but the traditional injection preparation is adopted for administration, the medicine is quickly distributed on the whole body after intravenous drip, the effect efficiency at the focus part is low, the medicine is influenced by the multidrug resistance of the tumor, and the curative effect is improved by increasing the medicine dosage. On the other hand, the drugs induce dose-limiting toxicity mainly caused by cardiotoxicity, and limit the increase of the dose in the clinical treatment of malignant tumors. Therefore, the key to improving the clinical effect of the anthracycline antitumor antibiotics lies in: (1) overcoming multidrug resistance of tumor cells; (2) effectively antagonize drug-induced cardiotoxicity; (3) changes the in vivo process of the medicine, improves the tissue distribution selectivity, and realizes the attenuation while enhancing the efficacy.
Multidrug resistance (MDR) of tumor cells refers to a broad spectrum drug resistance phenomenon that tumor cells generate cross resistance to drugs with different mechanisms and independent structures while generating drug resistance to one drug, and is one of the main reasons for chemotherapy failure in current cancer treatment. Clinical tumor treatment practices prove that the combined application of the medicines with different action mechanisms can reduce the total medicine dosage, generate complementary action and multiple effects and is an effective way for overcoming multi-medicine resistance. However, since the generation of multidrug resistance in tumor cells involves multiple factors and multiple gene abnormalities, the mechanism is very complex, and it is difficult to effectively reverse the multidrug resistance of tumor cells by using a single mode of multidrug resistance regulator or immunotherapy. The active ingredients which have the advantages of multi-target comprehensive action and high safety on the occurrence, development, metastasis and drug resistance of tumors are found from the traditional Chinese medicines and natural products for combined use, a new solution is provided for improving the curative effect of the traditional chemotherapeutic drugs, and the method becomes a new hotspot for research on antitumor drugs.
Salvianolic acid A, also known as salvianolic acid A and Salvianolic Acid A (SAA), is a water-soluble phenolic acid component extracted from Saviae Miltiorrhizae radix, and is first separated and identified in 1984 by professor Riyeniae Rosenh of institute of pharmaceutical and biological technology of Chinese academy of science and sciences. A series of subsequent research results of the subject group show that SAA is an important functional component of the salvia miltiorrhiza for promoting blood circulation and removing blood stasis.
In conclusion, the SAA has good application prospect of realizing synergy and attenuation through multiple ways when being combined with anthracycline antitumor antibiotics, and the combined drug-loaded micelle carrier capable of simultaneously loading two different drugs has potential advantages in the aspect of constructing an antitumor drug combined delivery system. However, since there are significant differences in the physicochemical properties of the anthracycline antitumor antibiotic and SAA, how to achieve simultaneous combined delivery of the two becomes the biggest difficulty and challenge, and is also the key to achieving clinical combinations thereof. At present, no report is found on a compound preparation combining SAA and anthracycline antitumor antibiotics, and a compound micelle drug-loading system carrying the anthracycline antitumor antibiotics and SAA is still blank.
Disclosure of Invention
In view of the above, the present invention aims to provide a compound micelle carrying an anthracycline antitumor antibiotic and salvianolic acid a simultaneously, and a preparation method and a use thereof.
The invention provides a compound micelle simultaneously carrying anthracycline antitumor antibiotics and salvianolic acid A, which comprises therapeutically effective amounts of anthracycline antitumor antibiotics, salvianolic acid A and an amphiphilic high molecular polymer;
the anthracycline antitumor antibiotic is selected from one or more of doxorubicin, epirubicin, daunorubicin, epidaunorubicin, pyrane doxorubicin, idamycin, anastamomycin and mitoxantrone existing in a free radical or salt form;
the amphiphilic high polymer is an A-B type diblock copolymer, wherein the A block is polyethylene glycol (PEG) or polyethylene glycol monomethyl ether (mPEG) with the number average molecular weight of 400-20000; the B block polyester is a polymer of levorotatory lactide, dextrorotatory lactide, racemic lactide, glycolide or epsilon-caprolactone with the number average molecular weight of 500-100000, or a random copolymer of one or two of the monomers;
the mass ratio of the anthracycline antitumor antibiotic to the high molecular polymer to the salvianolic acid A is 1 to (100-5) to (0.2-10).
Preferably, the anthracycline antitumor antibiotic is doxorubicin existing in a free radical or salt form.
Preferably, the amphiphilic high polymer is an A-B type diblock copolymer consisting of polyethylene glycol monomethyl ether (mPEG) -Polycaprolactone (PCL).
The compound micelle carrying the anthracycline antitumor antibiotic and the salvianolic acid A simultaneously is preferably characterized in that the mass ratio of the anthracycline antitumor antibiotic to the amphiphilic high molecular polymer is 1: 15-25.
The compound micelle carrying the anthracycline antitumor antibiotic and the salvianolic acid A simultaneously is preferably characterized in that the mass ratio of the anthracycline antitumor antibiotic to the salvianolic acid A is 1 to (3-5).
The invention also provides a preparation method of the compound micelle simultaneously carrying the anthracycline antitumor antibiotic and the salvianolic acid A, and the technical idea of the method is that the anthracycline antitumor antibiotic and the salvianolic acid A are jointly wrapped in a micelle core formed by self-assembly of an amphiphilic high molecular polymer in a physical wrapping manner in an aqueous medium to prepare the compound micelle simultaneously carrying the anthracycline antitumor antibiotic and the salvianolic acid A.
The preparation method of the compound micelle simultaneously carrying the anthracycline antitumor antibiotic and the salvianolic acid A comprises the following steps:
(1) dissolving a hydrophilic high molecular polymer and salvianolic acid A in an organic solvent, adding 1-50 mg of the hydrophilic high molecular polymer and 1-10 mg of the salvianolic acid A in per 1 mL of the organic solvent, and removing the organic solvent by rotary evaporation at 55 ℃ to obtain a polymer film containing the salvianolic acid A, wherein the organic solvent is selected from any one or more of acetone, methanol, ethanol, chloroform, dichloromethane, tetrahydrofuran and acetonitrile;
(2) adding an aqueous medium into the polymer film obtained in the step (1), and hydrating at 25-70 ℃ for 1-60 min to obtain salvianolic acid A drug-loaded micelle with the concentration of salvianolic acid A being (0.2-20) mg/mL;
(3) dissolving anthracycline antitumor antibiotic in a stable soluble salt form in an aqueous medium to prepare an aqueous solution with the concentration of the anthracycline antitumor antibiotic being (1-10) mg/mL;
(4) sequentially adding a weakly alkaline buffer solution with the pH of 7-9 and an anthracycline antitumor antibiotic aqueous solution obtained in the step (3) into the salvianolic acid A drug-loaded micelle obtained in the step (2) according to the volume ratio of 1: 0.05-0.5: 0.1-1, stirring at the temperature of (25-70) ℃ for 15-30 min, and mechanically filtering to obtain the compound nano micelle which is distributed in the particle size range of 10-200 nm and simultaneously carries the anthracycline antitumor antibiotic and the salvianolic acid A;
the aqueous medium in the steps (2) and (3) is any one or more of water, normal saline, glucose injection, buffer solution, tissue culture solution or body fluid.
In the above method for preparing the compound micelle carrying the anthracycline antitumor antibiotic and the salvianolic acid a, the organic solvent is preferably selected from acetone.
The compound nano micelle simultaneously carrying the anthracycline antitumor antibiotic and the salvianolic acid A adopts the well-known preparation technology of pharmacy, is added with pharmaceutically acceptable auxiliary materials, and is prepared into freeze-dried powder for injection, oral administration, mucous membrane or external use.
The invention also aims to provide the application of the compound nano-micelle in resisting tumors.
Compared with the prior art, the method has the advantages that,
the drug delivery system based on the nanotechnology can overcome the defects of poor stability, low specificity, high toxicity and the like of the traditional chemotherapeutic drugs, and the unique size effect of the drug delivery system can selectively enrich the chemotherapeutic drugs loaded on the nano drug carrier in tumor tissues through high permeability and retention effect (EPR effect), so that the drug delivery system is an important strategy for realizing the synergy and attenuation of the traditional chemotherapeutic drugs.
In addition, the invention utilizes the amphiphilic high molecular polymer with biocompatibility as the polymer micelle carrier prepared by self-assembly of the basic material, and the preparation method has the advantages of simple preparation process, slow release of the medicine, targeting, long circulation and the like. In addition, the combined drug-loaded micelle carrier simultaneously entrapping two drugs with different physicochemical properties can not only make up the defect of low drug-loading capacity of the micelle, but also simultaneously play respective effects and synergistic effects of the drugs, and has potential advantages in the aspect of constructing an anti-tumor drug combined delivery system. The carrier compound can simultaneously transmit the anthracycline antitumor antibiotic and the salvianolic acid A to the tumor focus part in a combined manner, can realize intelligent controlled release, realizes the passive targeting effect of the medicament on the tumor part, and reduces the damage to normal tissues.
The detailed technical thought and innovation points are as follows:
the amphiphilic high molecular polymer is used as a carrier material of the compound nano micelle, and the electrostatic attraction between the basic group of the anthracycline antitumor antibiotic and the acid group of the salvianolic acid A and the self-assembly combined action in an aqueous medium are utilized to realize the simultaneous combined delivery of the basic group of the anthracycline antitumor antibiotic and the acidic group of the salvianolic acid A, so that the obtained compound nano micelle carrying the anthracycline antitumor antibiotic and the salvianolic acid A together has uniform particle size and high entrapment rate, and the drug loading rate and the stability are obviously superior to those of a single-drug micelle, thereby providing a feasible method and technology for effectively overcoming and preventing cardiotoxicity and multi-drug resistance of tumor cells induced by the anthracycline antitumor antibiotic so as to improve the tumor treatment effect, and having the application prospect of antitumor treatment.
The invention adopts amphiphilic diblock copolymer which is biocompatible and biodegradable as the high molecular polymer carrier material, in the self-assembly process of the aqueous medium, a hydrophilic chain segment consisting of polyethylene glycol or polyethylene glycol monomethyl ether outwards forms a hydrophilic micelle shell, a hydrophobic chain segment consisting of polyester entraps salvianolic acid A and anthracycline antitumor antibiotics to jointly form a hydrophobic micelle inner core, plays an effective isolation and protection role on the drug, is beneficial to improving the stability of the drug, and simultaneously accumulates at the tumor part through the enhanced permeation and retention Effect (EPR) in the blood circulation, realizes the passive targeting effect of the drug on the tumor part, reduces the damage to normal tissues, and the compound anti-tumor composition is beneficial to overcoming the multi-drug resistance of tumor cells, effectively improving the utilization efficiency of the drug and bringing the compound drug components into play with the comprehensive treatment effects of synergistic anti-tumor effect and attenuation.
The test results prove that: compared with single-drug micelles, the compound nano-micelle prepared by using the amphiphilic high molecular polymer as the carrier material has the advantages that the basic pharmaceutical characteristics are obviously improved, the particle size and the particle size distribution are smaller, the absolute numerical values of the drug loading rate, the encapsulation rate and the surface Zeta potential are obviously increased, the solubility, the dilution resistance and the in-vivo stability of the drug in a water-based solvent can be ensured, and the system bioavailability of the drug is effectively improved.
The invention adopts a physical rather than chemical loading mode to simultaneously load two drugs, avoids the problem that the macromolecule bonding drug prepared by a chemical loading method needs to be dissociated from a polymer carrier, has the advantages of simple drug loading process and definite drug release mechanism, and has the advantages of simple and easy preparation method, good controllability, low cost, convenient popularization and good industrial application prospect.
Drawings
Fig. 1 is a schematic view of a preparation process of the compound nano-micelle loaded with doxorubicin and salvianolic acid a in example 1 of the present invention;
FIG. 2 shows the particle size variation of the co-loaded doxorubicin and salvianolic acid A compound nano-micelle incubated at 37 ℃ in 1% and 10% BSA solutions;
FIG. 3 shows Zeta potential changes of co-loaded doxorubicin and salvianolic acid A compound nano-micelle incubated at 37 ℃ in 1% and 10% BSA solutions;
FIG. 4 is a comparison of the cTnT serum levels in rats 24 hours after treatment with different drugs;
FIG. 5 is a graph showing the change of tumor volume in nude mice bearing tumor of multiple myeloma doxorubicin drug-resistant cells RPMI8226/DOX in different drug-treated groups;
FIG. 6 is a graph showing the body weight change of nude mice bearing tumor of multiple myeloma doxorubicin drug-resistant cells RPMI8226/DOX in different drug treatment groups.
Detailed Description
In order to facilitate understanding of the present invention, examples and test examples are specifically set forth to further illustrate the present invention, but not to limit the present invention in any way.
Example 1 preparation and characterization of Compound Nano-micelle Co-loaded with Adriamycin and Salvianolic acid A
Preparing the compound nano micelle: dissolving 20 mg of salvianolic acid A and 100 mg of block copolymer mPEG-PCL in 5mL of acetone, removing the organic solvent by rotary evaporation at 55 ℃ to obtain a salvianolic acid A-loaded polymer film, adding 5mL of ultrapure water, hydrating for 30-60 min, continuously adding 0.5 mL of PBS (10X) buffer solution with pH 7.4 and 2 mL of adriamycin aqueous solution (concentration of 3 mg/mL), electromagnetically stirring at room temperature (350 rpm is multiplied by 20 min) until the solution is completely clear, and filtering through a 0.22 mu m sterilizing membrane to obtain the compound nano micelle loaded with the adriamycin and the salvianolic acid A together. Simultaneously preparing adriamycin single-drug micelle without containing salvianolic acid A under the same condition; 3mg of doxorubicin was dissolved in 1 mL of ultrapure water to prepare an doxorubicin aqueous solution having a doxorubicin concentration of 3 mg/mL. The preparation process is shown in figure 1.
Characterization observation: the drug loading and encapsulation efficiency of the micelles were determined by high performance liquid chromatography, and the particle size, particle size distribution (polydispersity, PDI) and potential of the micelles were determined by a Nano-particle size and potentiodynamic analyzer (malvern Nano ZS 90), with the results shown in table 1.
TABLE 1 comparison of characterization and observation results of doxorubicin and salvianolic acid A co-loaded compound nano-micelle (I) and doxorubicin single-drug micelle (II)
| Micellar class | Particle size (nm) | PDI | Zeta potential (mV) | Doxorubicin drug loading (%) | Encapsulation efficiency (%) |
| Ⅰ | 26.1 | 0.117 | -4.16 | 19.0 | 99.5 |
| Ⅱ | 50.9 | 0.160 | -1.05 | 2.9 | 69.4 |
As a result, it was found that: compared with the adriamycin single-drug micelle, the compound nano micelle carrying the adriamycin and the salvianolic acid A has smaller particle size and more uniform particle size distribution; the directions of the surface potentials are consistent, but the absolute numerical value is obviously increased, so that particle coagulation is not easy to occur, and the stability is improved; the encapsulation efficiency and the drug-loading rate are both obviously improved, which shows that the compound nano micelle loaded with the adriamycin and the salvianolic acid A has better comprehensive basic pharmaceutical characteristics.
Example 2 preparation of Compound Nano micelle lyophilized powder Co-loaded with Adriamycin and Salvianolic acid A
Dissolving 20 mg of salvianolic acid A and 100 mg of block copolymer mPEG-PCL in 5mL of acetone, removing the organic solvent by rotary evaporation at 55 ℃, adding 5mL of ultrapure water, and hydrating for 30-45 min to dissolve the medicinal film. Meanwhile, preparing adriamycin single-drug micelles without salvianolic acid A under the same condition, and dissolving 1-10 mg of adriamycin in 1 mL of ultrapure water to prepare an adriamycin water solution with adriamycin concentration of (1-10) mg/mL. Then, 0.5 mL of PBS buffer solution (10X) with the pH value of 7.4 and 2 mL of adriamycin aqueous solution (the concentration is 3 mg/mL) are added continuously, electromagnetic stirring is carried out at room temperature (300 rpm is multiplied by 30 min) until the solution is completely clear, and then filtration is carried out through a 0.22 mu m sterilization membrane, so that the compound nano micelle loaded with the adriamycin and the salvianolic acid A is obtained.
Or electromagnetically stirring at room temperature (300 rpm × 30 min) until the solution is completely clarified, adding 100 mg mannitol, filtering the solution with 0.22 μm sterilizing membrane, and lyophilizing to obtain compound nanometer micelle lyophilized powder loaded with adriamycin and salvianolic acid A. Through determination, the compound micelle freeze-dried powder is re-dissolved into a solution containing 5 mg/mL of salvianolic acid A and 1.5 mg/mL of adriamycin by normal saline, and the solution is placed for 24 hours at room temperature, the content of the medicine in a dissolved state is more than 95 percent, and the change of the particle size and the change of the Zeta potential is less than 5 percent.
Test example 3 stability study of Doxorubicin and Salvianolic acid A Co-loaded Compound Nano-micelle
With the compound nano-micelle co-loaded with doxorubicin and salvianolic acid a obtained in example 1 as an object, the particle size and Zeta potential changes of the compound nano-micelle in 1% and 10% fetal Bovine Serum Albumin (BSA) solutions incubated at 37 ℃ for 24 hours were measured, respectively, to examine the in vivo stability of the compound nano-micelle, and the results are shown in fig. 2 and fig. 3.
The results show that: incubation of the compound nano-micelle in a medium containing 1% BSA and 10% BSA at 37 ℃, wherein the particle size of the micelle is not obviously changed within 12 hours compared with that before incubation (1%; BSA and 10%p>0.05), increase in particle size was observed by 24 hours (. about.p<0.01), therefore, the compound nanomicelle remains stable for at least 12 hours under simulated humoral circulation conditions (fig. 2); the Zeta potential of the compound nano-micelle after being incubated for 12 hours at 37 ℃ in a medium containing 1% BSA and 10% BSA is negative, and is remarkably increased in absolute value compared with a control group of the compound nano-micelle solution without incubation and with equal concentration (i.e. the Zeta potential is not negative) containing BSAp<0.05, **p<0.01) and BSA with isoelectric point of 4.7 exists in the form of negative charge under the condition of neutral pH, so that the larger negative Zeta potential is one of the key factors for ensuring the stability of the compound nano-micelle provided by the invention in vivo (figure 3).
Experimental example 4 Effect of compound nano micelle carrying doxorubicin and salvianolic acid A on rat cardiotoxicity
Healthy male SD rats 18 with body weight of 250 +/-20 g were randomly divided into 3 groups (n = 6), the compound nano-micelle carrying doxorubicin and salvianolic acid A, the doxorubicin-salvianolic acid A physical mixture and doxorubicin obtained in example 1 were administered by tail vein injection, the dose of doxorubicin administered was 20 mg/kg, animal sera were collected 24 hours after administration treatment, the content of cardiac troponin T (cTnT), a serum marker of specific cardiac myocyte injury, was measured by ELISA, and the injury and influence of drug intervention on the cardiac muscle of rats were observed according to the procedures specified by the kit (Shanghai enzyme research Biotech Co., Ltd.). The results are shown in FIG. 4.
As a result, it was found that: the cTnT content of the serum of the animal in the dry group of the compound nano-micelle co-loaded with the adriamycin and the salvianolic acid A and the adriamycin-salvianolic acid A physical mixture with the same dosage as the compound nano-micelle is obviously lower than that of the adriamycin group, and the difference has obvious significance (x;)p<0.01), which shows that the salvianolic acid A has protective effect on rat myocardial damage induced by adriamycin; and the compound nano micelle carrying the adriamycin and the salvianolic acid A has obviously stronger protection effect compared with physical mixture with equal dosage (the pannier)p<0.05）。
Test example 5 experiment of inhibiting tumor cell proliferation in vitro by using compound nano micelle carrying doxorubicin and salvianolic acid A
The compound nano micelle carrying the doxorubicin and the salvianolic acid A obtained in example 1 is taken as an object, the doxorubicin is taken as a control, and the anti-tumor activity is examined on a human lung adenocarcinoma cell A549 in vitro culture model by using an MTT method.
Taking A549 cell suspension in logarithmic growth phase at 5 × 103Number per well seeded in 96 well cell culture plates in 5% CO2The cells were cultured in a cell culture chamber at 37 ℃ for 24 hours, 10. mu.l of each drug was administered to the cells for intervention treatment, and the cells were incubated at 37 ℃ in 5% CO2The culture was continued for 48 h under the conditions. Blank and control experiments were performed simultaneously. Detecting cell survival rate with MTT cytotoxicity detection kit (Nanjing institute of bioengineering), measuring Optical Density (OD) at 570 nm with enzyme labeling instrument (Shanghai Co., Ltd., Versamax, Meygen molecular instruments), calculating cell survival rate according to (OD value of cell subjected to drug intervention/OD value of blank control cell) x 100%, and determining cell survival rate according to cellThe survival rate is related to the change of the drug concentration, and the half Inhibition Concentration (IC) of the drug to the in vitro cell proliferation is calculated50）。
As a result, it was found that: IC of compound nano micelle carrying adriamycin and salvianolic acid A for in-vitro proliferation of A549 cells50IC of adriamycin on A549 cell proliferation in vitro at 17.3. mu. mol/L (calculated as adriamycin)50Is 22.1 mu mol/L (calculated by adriamycin), and shows that the compound nano micelle maintains and even enhances the in-vitro proliferation activity of the antitumor cells of the adriamycin.
Test example 6 experiment of overcoming drug resistance of A549/DOX cells by using compound nano micelle carrying doxorubicin and salvianolic acid A together
The compound nano micelle carrying the doxorubicin and the salvianolic acid A obtained in example 1 is taken as an object, the doxorubicin is taken as a control, and the proliferation inhibition activity of the drug on drug-resistant tumor cells is examined by using an MTT method on a human lung adenocarcinoma cell doxorubicin drug-resistant A549/DOX in vitro culture model.
To maintain cell resistance, a549/DOX cells were deprived of drug and cultured in culture for one week prior to MTT assay. Adjusting the concentration of A549/DOX cells at 5 × 103Number per well seeded in 96 well cell culture plates in 5% CO2The cells were cultured at 37 ℃ for 24 hours in a cell culture chamber, 10. mu.L of each drug (dissolved in physiological saline) was administered to the cells, and the cells were incubated at 37 ℃ with 5% CO2The culture was continued for 48 h under the conditions. Detecting cell survival rate with MTT cytotoxicity detection kit (Nanjing institute of bioengineering), measuring Optical Density (OD) at 570 nm with enzyme labeling instrument (Shanghai Co., Ltd., Versamax, Meygen molecular instruments), calculating cell survival rate according to (OD value of cell subjected to drug intervention/OD value of blank control cell) x 100%, and calculating half Inhibition Concentration (IC) of drug on cell proliferation in vitro according to the relationship of cell survival rate with drug concentration50）。
As a result, it was found that: the compound nano micelle carrying the adriamycin and the salvianolic acid A still has stronger dose-dependent inhibition effect on the in-vitro proliferation of A549 cells, and IC5076.7 mu mol/L (calculated as adriamycin), and the in vitro proliferation inhibition effect of the adriamycin on A549/DOX cells is better than that of non-drug-resistant cytomineShows a decrease in IC50The concentration of the compound nano micelle is 1.11 mmol/L, which shows that the compound nano micelle can overcome the drug resistance of tumor cells to adriamycin and improve the sensitivity of the tumor cells to the adriamycin.
Experimental example 7 experiment of inhibiting growth of xenograft tumor in nude mouse with multiple myeloma doxorubicin drug-resistant cell RPMI8226/DOX by using compound nano micelle loaded with doxorubicin and salvianolic acid A
Male BALB/c mice were inoculated ventrally subcutaneously at 5X 106And RPMI8226/DOX cells. One week later, 30 tumor-bearing mice with uniform size and good growth were selected and randomly divided into 3 groups, i.e., an I-blank solvent (physiological saline) group, an II-compound nano micelle group (derived from example 1, DOX: 20 mg/kg; SAA: 20 mg/kg), an III-doxorubicin and salvianolic acid A mixture group (DOX: 20 mg/kg; SAA: 20 mg/kg), and 10 mice per group. Injecting different preparations via tail vein on days 0, 4 and 8, weighing body weight of mice every other day, measuring tumor size with vernier caliper (a and b are long and short diameters of tumor), and calculating tumor volume (ab)2/2), all animals were sacrificed at day 14 after the initial dose, tumors were stripped, weighed, and tumor volume and tumor inhibition rate were calculated.
As a result, it was found that: compared with a blank solvent group (I), the compound nano micelle group (II) and the compound group (III) of adriamycin and salvianolic acid A which carry adriamycin and salvianolic acid A together at equal doses have inhibition effects on the growth of mouse transplantation tumor (figure 5), but the compound nano micelle group has stronger inhibition effects on the growth of adriamycin drug-resistant tumor cells in vivo, and the tumor inhibition rates of the two groups are respectively 80.6% and 43.2%, which shows that the compound nano micelle can overcome the drug resistance of the tumor cells to adriamycin in vivo and improve the sensitivity of the tumor cells in vivo to adriamycin; the animal body weight of the compound nano micelle group (II) carrying both the adriamycin and the salvianolic acid A has no obvious change, but the body weight of the mice of the compound group (III) carrying both the adriamycin and the salvianolic acid A is obviously reduced (figure 6), which shows that the compound of the adriamycin and the salvianolic acid A has certain inhibition effect on the in-vivo growth of adriamycin-resistant tumors and also has certain toxicity, and the compound nano micelle carrying both the adriamycin and the salvianolic acid A can effectively overcome the drug resistance of tumor cells to the adriamycin while reducing the drug toxicity, improve the sensitivity of the tumor cells to the adriamycin and generate good comprehensive curative effect.
Claims (4)
1. A compound nano micelle simultaneously carrying anthracycline antitumor antibiotics and salvianolic acid A is characterized in that: the compound nano micelle simultaneously contains effective treatment amount of anthracycline antitumor antibiotics, salvianolic acid A and amphiphilic high molecular polymer, wherein the anthracycline antitumor antibiotics are selected from one or more of adriamycin, epirubicin, daunorubicin, epidaunorubicin, pyrarubicin, idarubicin, anastamomycin and mitoxantrone existing in a free radical or salt form, the amphiphilic high molecular polymer is an A-B type diblock copolymer, and an A block is polyethylene glycol or polyethylene glycol monomethyl ether with the number average molecular weight of 400-20000; the B block polyester is a polymer of levorotatory lactide, dextrorotatory lactide, racemic lactide, glycolide or epsilon-caprolactone with the number average molecular weight of 500-100000, or a random copolymer of one or two of the monomers; the mass ratio of the anthracycline antitumor antibiotic to the amphiphilic high molecular polymer to the salvianolic acid A is 1 to (100-5) to (0.2-10).
2. The compound nano-micelle carrying the anthracycline antitumor antibiotic and the salvianolic acid A simultaneously as claimed in claim 1, wherein the anthracycline antitumor antibiotic is doxorubicin existing in a free radical or salt form, and the amphiphilic high molecular polymer is an A-B type diblock copolymer consisting of polyethylene glycol monomethyl ether (mPEG) -Polycaprolactone (PCL).
3. The compound nano-micelle carrying the anthracycline antitumor antibiotic and the salvianolic acid A simultaneously according to claim 1, wherein the mass ratio of the anthracycline antitumor antibiotic, the amphiphilic polymer and the salvianolic acid A is 1: 15-25: 3-5.
4. The preparation method of the compound nano micelle carrying the anthracycline antitumor antibiotic and the salvianolic acid A simultaneously as claimed in claim 1, is characterized by comprising the following steps:
(1) dissolving a hydrophilic high molecular polymer and salvianolic acid A in an organic solvent, adding 1-50 mg of the hydrophilic high molecular polymer and 1-10 mg of the salvianolic acid A in per 1 mL of the organic solvent, and removing the organic solvent by rotary evaporation at 55 ℃ to obtain a polymer film containing the salvianolic acid A, wherein the organic solvent is selected from any one or more of acetone, methanol, ethanol, chloroform, dichloromethane, tetrahydrofuran and acetonitrile;
(2) adding an aqueous medium into the polymer film obtained in the step (1), and hydrating at 25-70 ℃ for 1-60 min to obtain salvianolic acid A drug-loaded micelle with the concentration of salvianolic acid A being (0.2-20) mg/mL;
(3) dissolving anthracycline antitumor antibiotics existing in a stable soluble salt form in an aqueous medium to prepare an aqueous medium solution with the concentration of the anthracycline antitumor antibiotics being (1-10) mg/mL;
(4) sequentially adding a weakly alkaline buffer solution with the pH of 7-9 and the anthracycline antitumor antibiotic aqueous medium solution obtained in the step (3) into the salvianolic acid A drug-loaded micelle obtained in the step (2) according to the volume ratio of 1: 0.05-0.5: 0.1-1, stirring for 15-30 min at the temperature of 25-70 ℃, and mechanically filtering to obtain the compound nano micelle which is distributed in the particle size range of 10-200 nm and simultaneously carries the anthracycline antitumor antibiotic and the salvianolic acid A;
the aqueous medium in the steps (2) and (3) is any one or more of purified water, normal saline, glucose injection, buffer solution, tissue culture solution or body fluid.
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