NANO delivery carrier for targeted tumor drug delivery and use thereof

Abstract

The present invention relates to a nano delivery carrier for targeted tumor drug delivery and the use thereof. A method for preparing the nano delivery carrier, which method comprises: mixing a nucleic-acid aptamer NucA targeting tumor, DSPE-PEG2000-NHS and DMF; then adding triethylamine to adjust the pH to 8-9; and reacting same at room temperature to obtain a nano delivery carrier for targeted tumor drug delivery. An anti-tumor drug based on the nano delivery carrier can accurately target a tumor to be killed, for example, an anti-tumor drug containing resveratrol based on the nano delivery carrier can target and kill ovarian cancer cells without damaging normal tissue.

Description

A nano-delivery carrier for targeted tumor drug delivery and its application Technical Field

[0001] The present invention belongs to the technical field of tumor-targeted drugs, and particularly relates to a nanometer delivery carrier for tumor-targeted drug administration and its application. Background Art

[0002] Cancer is a major disease that currently threatens human life and health. Currently, the main treatments for tumors are surgery, chemotherapy, and radiotherapy. Patients in the middle and late stages of the disease have lost the opportunity for surgery. Although surgery can remove the primary lesion, it cannot fundamentally prevent the recurrence and reproduction of cancer cells, and cancer is very likely to recur after surgery. The commonly used method currently is surgery combined with drug therapy, and drug therapy generally uses chemotherapy. Although chemotherapy can kill cancer cells, it also damages a large number of normal tissue cells and has significant toxic side effects. These side effects include body weakness, loss of appetite, hair loss, suppression of hematopoietic function, damage to liver, kidney and ovarian function, induction of gastrointestinal reactions, bone marrow suppression, and damage to liver, kidney and heart function.

[0003] Targeted drugs are drugs or their formulations that have been endowed with targeting capabilities. Their purpose is to enable the drug or its carrier to home in on a specific lesion and accumulate or release its active ingredient at that target site. Using targeted drugs in cancer treatment can reduce or even eliminate damage to tissues outside the tumor.

[0004] Therefore, it is of great significance to develop a carrier or drug that can deliver drugs to tumors in a targeted manner.

[0005] Summary of the Invention

[0006] In response to the above problems, the present invention aims to provide a nano-delivery carrier for tumor-targeted drug delivery and its application. The nano-delivery carrier is formed by connecting a nucleic acid aptamer that specifically targets tumors with DSPE-PEG2000-NHS. Based on the nano-delivery carrier for tumor-targeted drug delivery, a solvent replacement method can be used to replace NHS with an anti-tumor active ingredient to prepare an anti-tumor drug for targeted drug delivery.

[0007] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0008] On one hand, the present invention provides a nano-delivery carrier for tumor-targeted drug delivery. The preparation method thereof comprises: mixing a tumor-targeted nucleic acid aptamer NucA, DSPE-PEG2000-NHS and DMF; then adding triethylamine to adjust the pH to 8-9, and reacting at room temperature to obtain the nano-delivery carrier for tumor-targeted drug delivery.

[0009] Another aspect of the present invention provides an anti-tumor drug for targeted drug delivery, the preparation method of which comprises: mixing oleic acid-coated SPIO nanoparticles, an anti-tumor active ingredient, the above-mentioned targeted tumor drug delivery nano-delivery carrier, and chloroform to obtain a mixed solution; then gradually adding ethylene glycol to the mixed solution under an ultrasonic environment; removing chloroform, adding water, and removing ethylene glycol; then ultrasonicating, centrifuging, and removing the precipitate to obtain the anti-tumor drug for targeted drug delivery.

[0010] Another aspect of the present invention provides a preparation for treating tumors, comprising the above-mentioned anti-tumor drug for targeted administration and a pharmaceutically acceptable carrier.

[0011] The beneficial effects of the present invention include at least: anti-tumor drugs based on the nano-delivery carrier for targeted tumor administration provided by the present invention can be prepared to target tumors that need to be killed. For example, anti-tumor drugs containing resveratrol based on the nano-delivery carrier can target and kill ovarian cancer cells without damaging normal tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a two-dimensional structure diagram of resveratrol;

[0013] FIG2 is a transmission electron micrograph of PEG-SPIO nanoparticles prepared in Example;

[0014] FIG3 is a transmission electron micrograph of PEG-SPIO-RVT nanoparticles prepared in Example;

[0015] FIG4 is a diagram showing the results of the hydrated particle size test of PEG-SPIO and PEG-SPIO-RVT nanoparticles prepared in Example;

[0016] FIG5 is a graph showing the surface zeta potential of PEG-SPIO and PEG-SPIO-RVT nanoparticles prepared in Example;

[0017] FIG6 is a graph showing the UV / visible absorption spectra of PEG-SPIO and PEG-SPIO-RVT nanoparticles prepared in Example;

[0018] FIG7 is an in vitro magnetic resonance imaging image of PEG-SPIO and PEG-SPIO-RVT nanoparticles prepared in Example at different concentrations of iron ions;

[0019] FIG8 is a graph showing the effect of different concentrations of iron ions on the in vitro magnetic resonance imaging frequency of PEG-SPIO and PEG-SPIO-RVT nanoparticles prepared in Example;

[0020] FIG9 is a diagram showing the cell viability detection of RVT;

[0021] FIG10 is a diagram showing the cell viability detection of PEG-SPIO;

[0022] FIG11 is a diagram showing the cell viability detection of PEG-SPIO-RVT encapsulated with RVT. DETAILED DESCRIPTION

[0023] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0024] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context clearly has a different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, materials or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0025] The present invention provides a nano-delivery carrier for tumor-targeted drug delivery. The preparation method thereof comprises: mixing a tumor-targeted nucleic acid aptamer NucA, DSPE-PEG2000-NHS and DMF; then adding triethylamine to adjust the pH to 8-9, and reacting at room temperature to obtain the nano-delivery carrier for tumor-targeted drug delivery.

[0026] It should be noted that the aforementioned tumor-targeted nanodelivery vector is formed by linking the tumor-specific nucleic acid aptamer NucA with DSPE-PEG2000-NHS to form the tumor-targeted nanodelivery vector DSPE-PEG2000-NHS-NucA. Furthermore, during the preparation process, ultraviolet (UV) spectroscopy and infrared (FTIR) spectroscopy can be used to confirm the successful linkage of NucA to DSPE-PEG2000-NHS.

[0027] In some specific embodiments, the molar ratio of the nucleic acid aptamer NucA to DSPE-PEG2000-NHS can be 1:(2.5-3.5), such as 1:2.8, 1:3 or 1:3.2, among which 1:3 is preferred. The tumor-targeted drug delivery nanocarrier prepared at this molar ratio has the best performance.

[0028] In some specific embodiments, the tumor is ovarian cancer. It should be noted that the tumor can be any tumor known in the art, such as breast cancer, lung cancer, stomach cancer, liver cancer, colorectal cancer, or ovarian cancer. When different tumors are selected, different nucleic acid aptamers need to be selected.

[0029] In some specific embodiments, when the tumor is ovarian cancer, the nucleic acid aptamer NucA may preferably have a sequence as set forth in SEQ ID NO: 1, which is: "5'-GGTGGTGGTGGTTGTGGTGGTGGTGG- / 3ammc7-r / -3'". It should be noted that "3ammc7-r" is an amino group -NH2. The amino group can react with certain groups, such as -COOH and -NHS. The amino group is modified and can also be modified with other groups. That is, "3ammc7-r" represents a modification of the 3' hydroxyl end with an amino group.

[0030] In some specific embodiments, the reaction time at room temperature can be 24 hours. Of course, the reaction time can be adjusted according to the specific situation, as long as the nano-delivery vector can be prepared, for example, the reaction time can be 5 hours, 10 hours, 15 hours or 20 hours.

[0031] In some specific embodiments, after the preparation of the nano-delivery vector for tumor-targeted drug delivery is completed, it can be dialyzed for 24 hours using a dialysis membrane (molecular weight 3.5 kD) to remove unreacted NucA, DSPE-PEG2000-NHS and other organic solvents.

[0032] Another embodiment of the present invention provides an anti-tumor drug for targeted drug delivery, the preparation method of which comprises: mixing oleic acid-coated SPIO nanoparticles, an anti-tumor active ingredient, the above-mentioned nano-delivery carrier for targeted tumor delivery, and chloroform to obtain a mixed solution; then gradually adding ethylene glycol to the mixed solution under an ultrasonic environment; removing the chloroform, adding water, and removing the ethylene glycol; then ultrasonicating, centrifuging, and removing the precipitate to obtain the anti-tumor drug for targeted drug delivery.

[0033] It should be noted that the preparation method of the above-mentioned targeted anti-tumor drug is based on the solvent replacement method, which is to replace the original solution with solutions of gradually increasing polarity twice to prepare DSPE-PEG2000-RVT-NucA nanoparticles.

[0034] It should also be noted that in the above-mentioned method for preparing an anti-tumor drug for targeted drug delivery, the terms "ultrasound" and "centrifugation" have conventional meanings in the art and do not have any specific meanings. In addition, in some embodiments, the centrifugation condition can be 5000 rpm and the ultrasonic power can be 60 W.

[0035] In some specific embodiments, the anti-tumor active ingredient is resveratrol. Specifically, the chemical structure of resveratrol is shown below, and its two-dimensional structure is shown in Figure 1.

[0036] It should be noted that resveratrol (3,4',5-trihydroxy-trans-stilbene) is a natural anti-tumor active ingredient extracted from plants that can inhibit the occurrence and development of tumors, especially ovarian cancer. Based on the diversity of its chemical properties and its binding to ovarian cancer-related proteins, and the application of new nano-encapsulated resveratrol drug formulations in ovarian cancer, there are no reports. In the nano-delivery carrier for targeted tumor administration of the present invention, the trans isomer of resveratrol is retained, ensuring the specificity and stability of the binding of resveratrol to the target, thereby exerting an anti-cancer sensitization effect; with the help of molecular imaging methods, through the killing experiment of ovarian cancer cells, a method of retaining the physical and chemical properties of natural drugs for the acquisition of living biological information for the integrated diagnosis and treatment of ovarian cancer is achieved.

[0037] In some specific embodiments, the mass ratio of oleic acid-coated SPI0 nanoparticles, anti-tumor active ingredients and the above-mentioned nano-delivery carrier for targeted tumor administration can be (4.5-5.5): (0.5-1.5): (9.5-10.5), such as 4.7:0.8:9.7, 5:1:10 or 5.3:1.3:10.3, etc., among which 5:1:10 is preferred.

[0038] In some specific embodiments, the amount of ethylene glycol added can be 3-5 times, such as 4 times, the volume of the mixed solution.

[0039] It should also be noted that the oleic acid-coated SPIO nanoparticles can be prepared by methods known in the art. For example, an iron oleate complex is first prepared by a known method, and then coated with oleic acid to obtain oleic acid-coated SPIO nanoparticles.

[0040] In some specific embodiments, the preparation method of oleic acid-coated SPI0 nanoparticles includes: (1) synthesis of oleic acid iron complex: reacting FeCl·6H2O and sodium oleate in a mixture of ethanol, distilled water and n-hexane; then collecting the oleic acid iron complex and washing it with distilled water; finally removing the hexane in the oleic acid iron complex by a rotary evaporator to obtain the oleic acid iron complex; (2) synthesis of oleic acid-coated SPI0 nanoparticles: mixing the prepared oleic acid iron complex and oleic acid with 1-octadecene, heating to 110°C and vacuuming for 30 minutes; then, under argon protection, slowly heating the reactants to 320°C at a rate of 5°C per minute and maintaining for 30 minutes; finally, cooling the reactants to room temperature, taking them out, washing them with ethanol, and precipitating them in chloroform to obtain oleic acid-coated SPI0 nanoparticles.

[0041] Another embodiment of the present invention provides a preparation for treating tumors, comprising the aforementioned targeted anti-tumor drug and a pharmaceutically acceptable carrier. It should be noted that, depending on clinical needs, the aforementioned targeted anti-tumor drug can be added with a pharmaceutically acceptable carrier to prepare different dosage forms, such as an injectable or oral formulation.

[0042] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0043] Example 1 Nanomaterial Synthesis Method

[0044] (1) Synthesis of iron oleate complex

[0045] FeCl·6H2O (2.7 g) and sodium oleate (9.125 g) were reacted in a mixture of ethanol (20 mL), distilled water (15 mL) and n-hexane (35 mL) at 70°C for 4 hours. The iron oleate complex was then collected using a separatory funnel and washed with distilled water (50 mL). The hexane in the iron oleate complex was removed using a rotary evaporator and sealed in a round-bottom flask for storage under dry conditions at room temperature.

[0046] (2) Synthesis of oleic acid-coated SPIO nanoparticles (OA-SPIO)

[0047] The iron oleate complex (1.0 g) and oleic acid (0.25 g) prepared in (1) were mixed with 1-octadecene (5.5 g), heated to 110°C and vacuumed for 30 minutes; then, under argon protection, the reactants were slowly heated to 320°C at a rate of 5°C per minute and maintained for 30 minutes; finally, the reactants were cooled to room temperature, taken out, washed with ethanol, and precipitated in chloroform.

[0048] (3) Synthesis of DSPE-PEG2000-NHS-NucA (PEG-NucA)

[0049] NucA (a nucleic acid aptamer) and DSPE-PEG2000-NHS were added to a DMF solution in a molar ratio of 1:3, and triethylamine was added to adjust the pH to 8.5. The solution was reacted at room temperature for 24 hours and then dialyzed using a dialysis membrane (molecular weight 3.5 kD) for 24 hours to remove unreacted NucA, DSPE-PEG2000-NHS, and other organic solvents. Ultraviolet (UV) spectroscopy and infrared spectroscopy (FTIR) were used to determine whether NucA and DSPE-PEG2000-NHS were successfully linked.

[0050] (4) Synthesis of DSPE-PEG2000-SPIO-RVT-NucA (PEG-SPIO-RVT-NucA)

[0051] DSPE-PEG2000-RVT-NucA nanoparticles were prepared according to the solvent replacement method. The specific preparation process is as follows: OA-SPIO, RVT (resveratrol) prepared in the above (2) and PEG-NucA prepared in the above (3) were added to a round-mouth flask containing 0.5 mL of chloroform in a mass ratio of 10:2:20, and placed in an ultrasonic cleaner for brief ultrasonication to allow each component to be fully dissolved; then, 4 times the volume of ethylene glycol solvent was gradually added to the mixed solution under ultrasonic conditions; then, chloroform was evaporated to dryness using a rotary evaporator, an appropriate amount of deionized water was added to the solution, and the ethylene glycol solution was removed by multiple centrifugation using an ultrafiltration tube (100 mw); then, a cell ultrasonic disruptor was used to ultrasonicate at 60 W power for 5 minutes; finally, the solution was centrifuged at 5000 rpm to remove the precipitate to remove large aggregates to obtain PEG-SPIO-RVT-NucA.

[0052] The synthesis methods of PEG-SPIO and PEG-SPIO-RVT in the control group were the same as above.

[0053] Example 2 Nanomaterial Characterization

[0054] The PEG-SPIO nanoparticles and PEG-SPIO-RVT nanoparticles prepared in Example 1 were analyzed by transmission electron microscopy, and the results are shown in Figures 2 and 3 .

[0055] The PEG-SPIO nanoparticles and PEG-SPIO-RVT nanoparticles prepared in Example 1 were tested for hydrated particle size, and the results are shown in FIG4 ;

[0056] The surface zeta potential of the PEG-SPIO nanoparticles and PEG-SPIO-RVT nanoparticles prepared in Example 1 were analyzed, and the results are shown in FIG5 .

[0057] The PEG-SPIO nanoparticles and PEG-SPIO-RVT nanoparticles prepared in Example 1 were subjected to UV / visible absorption spectral analysis, and the results are shown in FIG6 .

[0058] The PEG-SPIO nanoparticles and PEG-SPIO-RVT nanoparticles prepared in Example 1 were subjected to in vitro magnetic resonance imaging analysis at different concentrations. The in vitro magnetic resonance imaging analysis graph is shown in FIG7 , and the in vitro magnetic resonance imaging frequency is shown in FIG8 .

[0059] Example 3 Cell Viability Detection

[0060] In order to verify whether resveratrol (RVT) can induce tumor cell death in the present embodiment, SKOV3 cells were treated with different concentrations of RVT for 24 hours, and then the cell viability was detected using CCK-8 detection reagent. The results are shown in Figure 9. As the RVT concentration increases, the viability of SKOV3 cells gradually shows a downward trend.

[0061] In addition, SKOV3 cells were treated with different concentrations of PEG-SPIO for 24 hours, and the cell viability was detected using CCK-8 detection reagent. The results are shown in Figure 10. When the concentration was as high as 200 μg / mL, it did not cause SKOV3 cell death, indicating that PEG-SPIO had no obvious cytotoxicity to cells.

[0062] Furthermore, SKOV3 cells were treated with RVT-encapsulated PEG-SPIO-RVT nanoparticles for 24 hours, and the cell viability was detected using CCK-8 detection reagent. The results are shown in Figure 11. The results are the same as those of RVT alone. With the increase of the concentration of PEG-SPIO-RVT nanoparticles, the cell viability decreased significantly.

[0063] The above results indicate that RVT is the real cause of SKOV3 cell death.

[0064] Example 4: Verification of ovarian cancer cell killing

[0065] [Corrected 27.06.2024 according to Rule 91] The experimental verification of ovarian cancer tumor cells in the examples of the present invention showed that the natural drug targeted delivery system has a killing effect on ovarian cancer cells without damaging normal tissues.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. A nano-delivery carrier for targeted tumor administration, characterized in that: The preparation method comprises: mixing tumor-targeting nucleic acid aptamer NucA, DSPE-PEG2000-NHS and DMF; then adding triethylamine to adjust the pH to 8-9, and reacting at room temperature to obtain a tumor-targeting drug delivery nanometer carrier.

2. The nano-delivery carrier for tumor-targeted drug delivery according to claim 1, characterized in that: The molar ratio of nucleic acid aptamer NucA to DSPE-PEG2000-NHS is 1:(2.5-3.5).

3. The nano-delivery carrier for tumor-targeted drug delivery according to claim 1 or 2, characterized in that: The tumor is ovarian cancer.

4. The nano-delivery carrier for tumor-targeted drug delivery according to claim 3, characterized in that: The nucleic acid aptamer NucA has a sequence as shown in SEQ ID NO:

1.

5. An anti-tumor drug for targeted drug delivery, characterized in that: The preparation method comprises mixing oleic acid-coated SPI0 nanoparticles, antitumor active ingredients, a nano-delivery carrier for targeted tumor administration according to any one of claims 1 to 4, and chloroform to obtain a mixed solution; then gradually adding ethylene glycol to the mixed solution under an ultrasonic environment; removing chloroform, adding water, and removing ethylene glycol; then ultrasonicating, centrifuging, and removing the precipitate to obtain an antitumor drug for targeted administration.

6. The anti-tumor drug for targeted drug delivery according to claim 5, characterized in that: The anti-tumor active ingredient is resveratrol.

7. The anti-tumor drug for targeted drug delivery according to claim 5 or 6, characterized in that: The mass ratio of oleic acid-coated SPI0 nanoparticles, antitumor active ingredients and nano-delivery carriers for targeted tumor administration is (4.5-5.5): (0.5-1.5): (9.5-10.5)。 8. The targeted anti-tumor drug according to claim 5 or 6, characterized in that: The amount of ethylene glycol added is 3-5 times the volume of the mixed solution.

9. A preparation for treating tumors, characterized in that: The invention comprises the anti-tumor drug for targeted administration according to any one of claims 5 to 8; and a pharmaceutically acceptable carrier.

10. The preparation for treating tumors according to claim 9, characterized in that: The pharmaceutically acceptable carrier is suitable for injection or oral administration.

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