阅读说明：本技术 一种抗肿瘤纳米药物及其制备方法与应用 (Anti-tumor nano-drug and preparation method and application thereof ) 是由 赵静 黄秀玉 邱妙娟 李宾宾 刘鹏 何裕隆 于 2021-07-28 设计创作，主要内容包括：本发明涉及肿瘤化疗技术领域,尤其涉及一种抗肿瘤纳米药物及其制备方法与应用。本发明的抗肿瘤纳米药物包含一种双磷酸盐、一种肿瘤化疗药物和至少一种金属离子。本发明将双磷酸盐与另一种抗肿瘤药物加入盐溶液中,组装成纳米颗粒。本发明的抗肿瘤纳米药物可制备成纳米颗粒冻干粉及悬浊液,可用于肿瘤患者腹腔灌注化疗,静脉注射化疗,口服,灌肠等使用给药方法。本发明的抗肿瘤纳米药物具有抗血管生成和促肿瘤凋亡双重作用,且可以与其它化疗药物或者抗体同时给药。(The invention relates to the technical field of tumor chemotherapy, in particular to an anti-tumor nano-drug and a preparation method and application thereof. The anti-tumor nano-drug comprises a diphosphate, a tumor chemotherapeutic drug and at least one metal ion. The nano-particles are assembled by adding the diphosphate and another anti-tumor drug into a salt solution. The anti-tumor nano-drug can be prepared into nano-particle freeze-dried powder and suspension, and can be used for the administration methods of intraperitoneal perfusion chemotherapy, intravenous injection chemotherapy, oral administration, enema and the like of tumor patients. The anti-tumor nano-drug has the dual effects of anti-angiogenesis and promoting tumor apoptosis, and can be simultaneously administered with other chemotherapeutic drugs or antibodies.)

1. An anti-tumor nano-drug, which is characterized by comprising a bisphosphonate, a tumor chemotherapeutic drug and at least one metal ion.

2. The anti-tumor nano-drug according to claim 1, wherein the bisphosphonate is alendronate, etidronate, clodronate, ibandronate, or zoledronic acid.

3. The nanomedicine according to claim 1, wherein the chemotherapeutic agent is an alkylating agent, an antimetabolite, an anticancer antibiotic, a traditional Chinese medicine drug or a hormone drug.

4. The anti-tumor nano-drug according to claim 1, wherein the preparation method of the anti-tumor nano-drug comprises: adding alendronate sodium water solution and THZ1 hydrochloride water solution into calcium chloride solution, adjusting pH value to neutral or alkaline, stirring, centrifuging to obtain nanometer medicinal preparation, and washing with water solution and ethanol to remove free medicinal preparation to obtain ALN-Ca-THZ1 nanometer medicinal preparation for treating tumor.

5. The anti-tumor nano-drug according to claim 1, wherein the preparation method of the anti-tumor nano-drug comprises: adding alendronate sodium water solution and 5 fluorouracil water solution into calcium chloride solution, adjusting pH value to be neutral or alkaline, stirring, centrifuging to obtain nano-drug, and washing free drug with water solution and ethanol to obtain ALN-Ca-5Fu anti-tumor nano-drug.

6. The anti-tumor nano-drug according to claim 1, wherein the anti-tumor nano-drug can further modify a group targeting tumor or a group targeting normal tissue on the surface of the nanoparticle.

7. The anti-tumor nano-drug according to claim 6, wherein the tumor targeting group is RGD polypeptide, folic acid or transferrin.

8. The anti-tumor nano-drug according to claim 7, wherein the RGD polypeptide is NHS-PEG-RGD, and the molecular weight of the NHS-PEG-RGD is 500-15000.

9. The use of the anti-tumor nano-drug of any one of claims 1 to 8 in the preparation of a medicament for the treatment of cancer.

10. The use of claim 9, wherein the cancer is ovarian cancer.

Technical Field

The invention relates to the technical field of tumor chemotherapy, in particular to an anti-tumor nano-drug and a preparation method and application thereof.

Background

The chemotherapy medicine is a medicine for treating tumor. The chemotherapy medicine can kill tumor cells. The medicines can act on different links of growth and reproduction of tumor cells to inhibit or kill the tumor cells. Chemotherapy is one of the main methods for treating tumors at present. The existing defects of tumor chemotherapy are low bioavailability and nonspecific targeting, and various drug resistances are easy to appear in the treatment process, so that tumor patients are relapsed and move to the life end point. Therefore, the development of novel antitumor drugs has great clinical significance.

The drug factors with different anti-tumor mechanisms are combined and packaged into single nano particles, so that the anti-tumor effects of the two drugs can be exerted simultaneously, the drug effect is enhanced, the drug resistance of the drugs is reduced, and the strong anti-tumor effect is achieved. Recently, researchers at the national academy of labor and technology of Massachusetts have devised a novel nanoparticle that can deliver two drugs simultaneously to the brain tumor site to enhance the therapeutic effect on glioblastoma multiforme, and have been successful in animal experiments. The study was published in journal of Nature Communications in the uk, and researchers found that the addition of transferrin to liposome nanoparticles enabled the particles to pass through the blood-brain barrier smoothly and reach the tumor site accurately while avoiding normal cells. Liposomes are understood to be hollow artificial spherical microparticles, the shell being a lipid bilayer. Researchers load the chemotherapy drug temozolomide inside liposomes, responsible for destroying the DNA of tumor cells (deoxyribonucleic acid); the outer shell is loaded with an experimental drug named "JQ-1" which is responsible for preventing tumor cells from repairing DNA damage. The two components act together to reduce drug resistance.

However, in the prior art, two drugs are loaded through liposome or nanoparticle carriers, and the method causes low drug loading rate, and the carriers also bring biological safety and immunogenicity problems.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an anti-tumor nano-medicament and a preparation method and application thereof.

The invention forms nano particles by self-assembling the medicine in aqueous solution, does not need a carrier and has strong anti-tumor activity.

In order to achieve the purpose, the invention adopts the technical scheme that: an anti-tumor nano-drug is provided, comprising a bisphosphonate, a tumor chemotherapeutic and at least one metal ion.

The nano-particles are assembled by adding the diphosphate and another anti-tumor drug into a salt solution. The anti-tumor nano-drug can be prepared into nano-particle freeze-dried powder and suspension, and can be used for the administration methods of intraperitoneal perfusion chemotherapy, intravenous injection chemotherapy, oral administration, enema and the like of tumor patients. The anti-tumor nano-drug has the dual effects of anti-angiogenesis and promoting tumor apoptosis, and can be simultaneously administered with other chemotherapeutic drugs or antibodies.

As a preferred embodiment of the anti-tumor nano-drug of the present invention, the bisphosphonate is alendronate, etidronate, clodronate, ibandronate or zoledronic acid.

As a preferred embodiment of the anti-tumor nano-drug of the present invention, the tumor chemotherapeutic is an alkylating agent, an anti-metabolite, an anti-cancer antibiotic, a Chinese medicine drug or a hormone drug.

As a preferred embodiment of the anti-tumor nano-drug of the present invention, the preparation method of the anti-tumor nano-drug comprises: adding alendronate sodium water solution and THZ1 hydrochloride water solution into calcium chloride solution, adjusting pH value to neutral or alkaline, stirring, centrifuging to obtain nanometer medicinal preparation, and washing with water solution and ethanol to remove free medicinal preparation to obtain ALN-Ca-THZ1 nanometer medicinal preparation for treating tumor.

As a preferred embodiment of the anti-tumor nano-drug of the present invention, the preparation method of the anti-tumor nano-drug comprises: adding alendronate sodium water solution and 5 fluorouracil water solution into calcium chloride solution, adjusting pH value to be neutral or alkaline, stirring, centrifuging to obtain nano-drug, and washing free drug with water solution and ethanol to obtain ALN-Ca-5Fu anti-tumor nano-drug.

As a preferred embodiment of the anti-tumor nano-drug of the present invention, the anti-tumor nano-drug may further modify a group targeting a tumor or a group targeting a normal tissue on the surface of the nanoparticle.

The surface of the nano-particle of the invention is provided with amino and can react with a plurality of groups, thus various tumor or normal tissue targeted groups can be further modified on the surface of the nano-particle.

As a preferred embodiment of the anti-tumor nano-drug of the present invention, the tumor targeting group is RGD polypeptide, folic acid, or transferrin.

As a preferred embodiment of the anti-tumor nano-drug of the invention, the RGD polypeptide is NHS-PEG-RGD, and the molecular weight of the NHS-PEG-RGD is 500-15000.

The invention also provides the application of the anti-tumor nano-medicament in preparing a medicament for treating cancer.

As a preferred embodiment of the use of the invention, the cancer is ovarian cancer.

The invention has the beneficial effects that:

(1) compared with other inventions, the invention does not need a drug carrier, and the diphosphate and the chemotherapeutic drug are self-assembled to form the nano-drug. The diphosphate selected by the invention has the activity of inhibiting the migration of tumor cells and can inhibit the diffusion of tumors. The drug effect can be further improved by combining with other chemotherapeutic drugs.

(2) Compared with the nano-drugs prepared by other methods, the nano-drug prepared by the invention has a large amount of amino groups on the surface, and can react with various groups, so that other functional groups are modified on the surface of the nano-drug.

(3) The invention discloses a method for self-assembling two small molecular drugs to form nano particles, and the nano drug prepared by the method is used for anti-tumor treatment, and the method comprises the following steps: has the functions of targeting tumor and effectively inhibiting tumor proliferation and metastasis. The invention constructs a mouse human ovarian cancer abdominal cavity tumor model, after solid tumors are formed in the mouse abdominal cavity, nano-drugs are injected into the mouse abdominal cavity through the abdominal cavity, the tumor development condition is monitored through a mouse living body imager, and the antitumor effect is evaluated through the mouse survival curve and the tumor size.

Drawings

FIG. 1: transmission electron microscopy pictures of sample 2; from the transmission results, the prepared sample was uniformly dispersed, and the size of the single particle was about 20 nm.

FIG. 2: x-ray photoelectron spectroscopy (XPS) pictures of sample 2; from the X-ray photoelectron spectrum, the nano-drug consists of four elements of C, O, P and Ca.

FIG. 3: ALN-Ca-THZ1 inhibits ovarian cancer cell proliferation time-concentration-dependently (n-6).

FIG. 4: a, cell scratch test result (n ═ 3); b, cell scratch experimental mobility statistical plot (n-3) p < 0.001.

FIG. 5: bioluminescence images in SKOV3 tumor-bearing mice treated with different drug groups.

Detailed Description

To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention.

Example 1

Dissolving 0.66g of alendronate sodium in 50mL of deionized water, and marking as a solution A; dissolving 0.2g of anhydrous calcium chloride in 50mL of deionized water, and marking as a solution B; 0.033g of doxorubicin hydrochloride was dissolved in 10mL of deionized water and labeled as solution C. The solutions A, B and C were added together and labeled as solution D, and the pH of the solution D was adjusted to 5.0 with NaOH and reacted at room temperature for 2 hours. Samples were collected by centrifugation, washed 1 time with deionized water, 2 times with alcohol (100% absolute ethanol), and lyophilized to give a powder labeled ALN-Ca-DOX (sample 1).

Example 2

Dissolving 0.66g of alendronate sodium in 50mL of deionized water, and marking as a solution A; dissolving 0.2g of anhydrous calcium chloride in 50mL of deionized water, and marking as a solution B; 0.022g of THZ1 hydrochloride was dissolved in 10mL of deionized water and labeled as solution C. The solutions A, B and C were added together and labeled as solution D, and the pH of the solution D was adjusted to 5.0 with NaOH and reacted at room temperature for 2 hours. Samples were collected by centrifugation, washed 1 time with deionized water and 2 times with alcohol. And made into lyophilized powder, labeled ALN-Ca-THZ1 (sample 2).

Example 3

Dissolving 0.66g of alendronate sodium in 50mL of deionized water, and marking as a solution A; dissolving 0.2g of anhydrous calcium chloride in 50mL of deionized water, and marking as a solution B; 0.03g of 5-fluorouracil (5FU) was dissolved in 10mL of deionized water and labeled as solution C. The solutions A, B and C were added together and labeled as solution D, and the pH of the solution D was adjusted to 5.0 with NaOH and reacted at room temperature for 2 hours. Samples were collected by centrifugation, washed 1 time with deionized water and 2 times with alcohol. And made into lyophilized powder, labeled ALN-Ca-5FU (sample 3).

Example 4

Dissolving 0.66g of alendronate sodium in 50mL of deionized water, and marking as a solution A; dissolving 0.2g of anhydrous calcium chloride in 50mL of deionized water, and marking as a solution B; 0.02g oxaliplatin was dissolved in 10mL deionized water and labeled solution C. The solutions A, B and C were added together and labeled as solution D, and the pH of the solution D was adjusted to 5.0 with NaOH and reacted at room temperature for 2 hours. Samples were collected by centrifugation, washed 1 time with deionized water and 2 times with alcohol. And made into lyophilized powder, labeled ALN-Ca-Oxa (sample 4).

Example 5

10mg of ALN-Ca-THZ1 lyophilized powder prepared in example 2 was dispersed in 10mL of anhydrous ethanol and labeled as solution A, 1mg of NHS-PEG-RGD was added to solution A, and reacted for 24 hours, and the sample was collected by centrifugation and lyophilized and labeled as ALN-Ca-THZ1-RGD (sample 5).

Example 6

10mg of RGD polypeptide-polyethylene glycol-N-hydroxysuccinimide (English name: RGD-PEG2000-NHS) is dissolved in 10mL of DMSO, then 100mg of PAMAM/CaPO is added and dispersed in the solution, and the reaction is carried out for 24 hours in the dark at normal temperature. Centrifuging, dialyzing and freeze-drying to obtain the RGD modified calcium phosphate nano material, namely RGD-PEG-CaPO.

100mg of 5-fluorouracil was dissolved in 10mL of DMSO, dissolved sufficiently, and 1mg of the above RDG-PEG-CaPO was dispersed in this solution. The reaction was carried out in the dark at room temperature for 48 hours. Namely, the RGD-PEG-CaPO nano-particles loaded with 5-Fu drugs are called RGD-PEG-CaPO/5Fu for short.

Example 7ALN-Ca-THZ1 cell killing experiment

Recovering and culturing human ovarian cancer cells SKOV3 to logarithmic phase, collecting cells, adjusting cell suspension concentration after cell counting, adding 100ul McCoys 5A complete culture medium (containing 10% FBS) containing cell suspension into each well of the middle area of a 96-well plate, and ensuring that the number of cells in each well is in the range of 5000-8000 (the marginal wells are filled with sterile double distilled water). Place 96-well plate in 5% CO₂Incubating at 37 deg.C until cell monolayer is fully coated on the bottom of 96-well plate, adding ALN-Ca-THZ1 with concentration gradient, and concentratingThe degrees are respectively: 100. 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78 mg/L. After the treated cells are respectively incubated for 24 hours, 48 hours and 72 hours, 10uL of CCK8 solution is added into each hole, after the cells are kept still for 2 to 4 hours, the transmission light intensity of each hole under the wavelength of 470nm is measured by a microplate spectrophotometer, the survival rate of the cells in each hole is calculated according to the measurement result, and the corresponding ALN-Ca-THZ1 concentration is plotted.

The results are shown in figure 3, ovarian cancer cells are respectively treated with ALN-Ca-THZ1 at different concentrations for 24 hours, 48 hours and 72 hours, the proliferation activity of the cells is detected by a CCK8 method, and the inhibition rate of ALN-Ca-THZ1 on the cell proliferation is increased along with the increase of the concentrations under the condition of the same intervention time; the inhibition rate of ALN-Ca-THZ1 on cell proliferation increased with time at the same intervention concentration.

Example 8 inhibition of tumor cell migration assay

Collecting SKOV3 cells in logarithmic growth phase, digesting with pancreatin, and treating with 1 × 10⁴The density per well was seeded in 12-well plates. At 37 ℃ and 5% CO₂And (5) standing and culturing in a constant-temperature incubator with saturated humidity. When the cells are cultured in a conventional way until the cells are in a monolayer and the cell fusion degree reaches 85 percent, 2 longitudinal lines in a straight line shape with the same width are scribed at the bottom of the pore plate in parallel by a 100-mu-L sterile gun head, and the cells and cell fragments are washed by PBS for 3 times immediately so as to wash off the suspended cells and cell fragments. The relative distance of the scratched area was recorded by taking a photograph under a microscope (0 hour). After sucking all PBS under negative pressure, 1mL of 2% serum culture medium containing 2mg/L ALN-Ca-THZ1 was added, and the culture was continued in an incubator with the 2% serum culture medium as a control group without drug addition. Observing under a microscope, taking a picture for 24 hours, placing the picture in an incubator, and continuing culturing at a magnification of 200 x. Each scratch was photographed at 6 different locations. Measuring the widths of different grouped scratches at different times in Adobe Illustrator (AI) software; cell mobility ═ (0 hr intercellular space-24 hr intercellular space)/0 hr intercellular space 100%.

The result of the cell scratching experiment is shown in fig. 4, and shows that the scratching distance of the ALN-Ca-THZ1 group is obviously larger than that of the control group, which indicates that the cell activity and the migration capability are weakened, and the ALN-Ca-THZ1 plays a role in obviously inhibiting the migration of ovarian cancer cells.

Example 9 animal experiments

SKOV3 cells were dispersed in PBS and cell density was adjusted to 2X 10⁷Per mL, 0.2mL of cell suspension is extracted by a 1mL syringe, 5-6 weeks old female nude mice abdominal skin is disinfected by 75% alcohol, and the cells with the number of 4 multiplied by 10 are injected into the nude mice abdominal cavity after the left lower abdomen is back-extracted without blood⁶And (4) respectively. After 5 days from the injection of tumor cells, nude mice were randomly divided into a control group and three treatment groups, the control group was intraperitoneally injected with PBS 0.2mL, and the treatment groups were intraperitoneally injected with NaALN 0.16 mg/mouse, THZ10.02mg/mouse, ALN-Ca-THZ10.2 mg/mouse, once every 7 days, and three consecutive injections were performed. The mental condition, skin, diet, activity and the like of the nude mice were observed every day, the abdominal circumference and body weight of the nude mice were measured from the seventh day after tumor transplantation, the growth of the abdominal cavity transplantation tumor was observed with a mouse living body imager, and the number of days of survival of each group of mice was calculated. The data processing is carried out by using SPSS12.0 statistical software, and the experimental data are all expressed by mean +/-standard deviation.

The results are shown in fig. 5, and the four groups of tumor-bearing nude mice all form transplantation tumor in the abdominal cavity, and the tumor formation rate is 100%. The fluorescence value of the abdominal cavity tumor of the nude mice of the ALN-Ca-THZ1 treatment group is smaller than that of the control group, and the number of the metastasis spots is not increased, which indicates that the ALN-Ca-THZ1 can inhibit the growth and the metastasis of the tumor in vivo, and has potential value for treating the abdominal cavity metastasis of the ovarian cancer.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.