CN109276720B - Metal-organic complex nano material and preparation method and application thereof - Google Patents

Abstract

The invention discloses a metal-organic complex nano material and a preparation method and application thereof, belonging to the technical field of preparation of antitumor drugs. The preparation method comprises the following steps: firstly, adding bovine serum albumin into an alkaline solution, then simultaneously dropwise adding a copper nitrate solution and a 5-nitro-8-hydroxyquinoline solution, reacting at room temperature, and self-assembling a product to form the metal-organic complex nano material. Through a simple and green preparation route, the prepared nano material has the advantages of good stability, long in-vivo circulation time, targeting property, good biocompatibility, small toxic and side effects and the like. The nano material provided by the invention can be applied to tumor treatment as a nano medicament, is enriched in tumor tissues through EPR effect and receptor-mediated endocytosis, improves the treatment effect of the metal-organic medicament on tumors, and realizes the systemic administration of the metal-organic medicament.

Description

Metal-organic complex nano material and preparation method and application thereof

Technical Field

The invention relates to the technical field of preparation of antitumor drugs, and particularly relates to a nano material based on a 5-nitro-8-hydroxyquinoline copper complex, and a preparation method and application thereof.

Background

In recent years, the incidence and mortality of malignant tumors are on the rise, and cancer has become the first cause of death of urban and rural residents in China. Small molecule drug therapy remains one of the major clinical approaches to tumor therapy, but there are several negative features, such as: the solubility in water is low, and the direct utilization is difficult; the half-life of the plasma is too short and must be repeated continuously; poor targeting property and toxic and side effects on organisms; drug resistance of tumors to chemotherapeutic drugs, and the like.

The transition metal element, especially platinum based antitumor drug such as cisplatin has obvious effect in cancer clinical treatment, but cisplatin has poor water solubility, short remission period and high toxicity to kidney. In recent years, the development of anti-tumor drugs based on other metals, which can overcome the defects of cisplatin dosage limitation and drug resistance, becomes a new research hotspot, wherein the copper complex shows good development prospect.

The copper ion ligand anti-tumor medicine is a complex containing copper ions in the structure, and the existing research shows that various binary metal copper complexes have the cytotoxic or enzyme inhibition function and can induce the apoptosis of tumor cells. For example, the complex formed by the bis-Schiff base and the copper can inhibit the growth of tumor and the metastasis of tissue tumor, and prolong the life cycle of tumor-bearing animals; the complex formed by the bivalent copper and the adriamycin has stronger cytotoxic effect, and can induce apoptosis or generate cell cycle arrest effect.

5-Nitro-8-hydroxyquinoline (NQ), an antibiotic approved by the Food and Drug Administration (FDA) in the United states, has been widely used for clinical medication since 1960. Recently, many studies have shown that NQ has potential anti-tumor activity against a variety of tumor cells, including ovarian cancer cells, bladder cancer cells, breast cancer cells, pancreatic cancer cells, brain glioma cells, and the like. Particularly, the cytotoxicity of the NQ chelate divalent copper complex is greatly improved after the NQ chelate divalent copper complex is formed. The more cytotoxic the complex is than the ligand alone, and the more harmful the tumor cells are correspondingly. However, how to release the insoluble complex NQ + cu (ii) to the tumor site by systemic administration is a great challenge, which severely limits the transformation for its clinical application.

Therefore, the development of a metal-organic protein complex nano-drug with long circulation, targeting property and water solubility meets the basic requirement of clinical application of nano-drugs, which is a problem to be solved by the technical personnel in the field.

Disclosure of Invention

The invention aims to provide a novel metal-organic complex nano material which has the characteristics of good stability, long in-vivo circulation time, small toxic and side effects and the like, can be effectively endocytosed by tumor cells, quickly releases a metal-organic complex, improves the treatment effect of a metal-organic complex medicament on tumors, and realizes the systemic administration of the metal-organic complex medicament.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for preparing a metal-organic complex nano material comprises the following steps: firstly, adding bovine serum albumin into an alkaline solution, then simultaneously dropwise adding a copper nitrate solution and a 5-nitro-8-hydroxyquinoline solution, reacting at room temperature, and self-assembling a product to form the metal-organic complex nano material.

The invention utilizes albumin as a delivery carrier of the copper complex, and the albumin has the advantages of safety, no toxicity, no immunogenicity, biodegradability, good biocompatibility and the like. The existing research shows that the surfaces of various tumor cells have high-expression albumin receptors, such as SPARC, FcRn, GP60 and the like, so that the albumin-based nano-drug can be passively targeted to the tumor through an EPR effect and can also be actively targeted to the tumor through receptor-mediated endocytosis.

In the preparation method of the present invention, nanoparticles are formed by spontaneous assembly between Bovine Serum Albumin (BSA), 5-nitro-8-hydroxyquinoline (NQ) and copper, specifically: copper and NQ form a metal-organic complex, and bovine serum albumin utilizes a large number of exposed active groups (such as amino, carboxyl, sulfydryl and the like) to wrap the metal-organic complex through intermolecular interaction force to form a spherical nano-scale material. Wherein the sample adding sequence of each material is a determining factor for forming the effective nano material, firstly BSA exposes active groups under alkaline conditions, and then spontaneous assembly is carried out between the BSA and the simultaneously dropwise added 5-nitro-8-hydroxyquinoline (NQ) and copper.

In the self-assembly process of the nano-particles, the alkaline strength of a reaction system is a main factor influencing the particle size, and researches show that when the pH value is more than 11, a large number of active groups are exposed out of BSA (bovine serum albumin), the nano-particles are easy to aggregate, and particles with larger particle sizes are formed; when the pH is less than 9, the BSA has fewer exposed active groups, so that part of the metal-organic matter is incompletely encapsulated to form particles with larger particle size.

Preferably, the alkaline solution is an aqueous sodium hydroxide solution having a concentration of 4 mM.

According to the invention, the water-soluble nano material is prepared by an extremely simple one-pot method, spherical nano particles with the particle size range of 70-300nm and narrow distribution can be obtained by adjusting the concentrations of BSA, copper nitrate and NQ, and can be enriched in tumor tissues through EPR effect and receptor-mediated endocytosis, so that the treatment effect of the complex NQ + Cu (II) on tumors is improved.

Preferably, the concentration of bovine serum albumin in the reaction system is 2-8 mg/mL. Researches find that when the amount of bovine serum albumin is insufficient, all metal-organic complexes cannot be loaded, and the particle size of the prepared nano-particles is larger; when the amount of bovine serum albumin is too large, the nanoparticles are easy to aggregate in the self-assembly process, and particles with larger particle size are formed.

Preferably, the feeding molar ratio of the copper nitrate to the 5-nitro-8-hydroxyquinoline is 1:1 to 1: 3. Researches show that when the molar ratio of the metal-organic complex to the organic complex is 1:1, the prepared nanoparticles have good particle size and distribution, but the drug-loading rate of the metal-organic complex is low; when the molar ratio of the two is 1:3, the prepared nanoparticles have large particle size and poor particle size distribution.

Specifically, the feeding ratio of bovine serum albumin, copper nitrate and 5-nitro-8-hydroxyquinoline in each 1mL reaction system is 2-8 mg: 2. mu. mol: 2-6 mu mol.

More preferably, the feeding ratio of bovine serum albumin, copper nitrate and 5-nitro-8-hydroxyquinoline in each 1mL reaction system is 4 mg: 2. mu. mol: 5. mu. mol.

The molecular formula of the 5-nitro-8-hydroxyquinoline is as follows: c₉H₆N₂O₃Molecular weight: 190.16, cas number: 4008-48-4, the structural formula is shown in formula (I):

the 5-nitro-8-hydroxyquinoline solution is a 5-nitro-8-hydroxyquinoline acetone solution with the concentration of 50 mM.

The research of the invention shows that the simultaneous and uniform dropping of the copper nitrate solution and the 5-nitro-8-hydroxyquinoline solution into the albumin solution is a key factor for forming the nano-particles with uniform size, and preferably, the copper nitrate solution and the 5-nitro-8-hydroxyquinoline solution are dropped at the speed of 100-.

Research shows that when the volume of water is more than 5mL, the nano material with better monodispersity is easier to form.

Preferably, the reaction time is 2-4h at room temperature. The room temperature is 20-24 ℃. More preferably, the reaction time is 3 hours.

After the reaction is finished, the metal-organic complex nano material is intercepted by a dialysis membrane, and then freeze drying or ultrafiltration concentration is carried out.

The invention also provides a metal-organic complex nano material prepared by the preparation method, wherein the mass percentage content of the 5-nitro-8-hydroxyquinoline copper complex in the nano material is 10-15%. The metal-organic complex drug content of the nano material needs to reach a certain amount to achieve the treatment effect.

The nano material prepared by the invention can stably exist in deionized water, PBS (pH7.4) and DMEM culture medium for more than two weeks, the particle size is 70-80nm, and the color is light yellow. Diluting with deionized water by 2-18 times, and the nanometer material has particle size of 70-90 nm. The metal-organic complex nano material prepared by the invention has good water solubility and stable particle size.

The invention also provides the application of the metal-organic complex nano material in the preparation of antitumor drugs.

Research shows that when the nano material is acted on tumor cells, the nano material can be quickly endocytosed by the tumor cells, and the metal-organic complex drug is released into the tumor cells, so that the proliferation of the tumor cells can be obviously inhibited, and the nano material can be used as an anti-tumor drug.

The invention has the following beneficial effects:

(1) according to the invention, bovine serum albumin, copper nitrate and 5-nitro-8-hydroxyquinoline are self-assembled to form the water-soluble nano material under an alkaline condition through a simple and green preparation route, and the prepared nano material has the advantages of good stability, long in-vivo circulation time, targeting property, good biocompatibility, small toxic and side effects and the like.

(2) The nano material provided by the invention can be applied to tumor treatment as a nano medicament, is enriched in tumor tissues through EPR effect and receptor-mediated endocytosis, improves the treatment effect of the metal-organic medicament on tumors, and realizes the systemic administration of the metal-organic medicament.

Drawings

FIG. 1 is a graph showing the dynamic light scattering of the nano-drug in water in the examples.

FIG. 2 is an electron microscope image of the nano-drug in the example.

FIG. 3 is a graph showing the variation of particle size of the nano-drugs in deionized water, PBS (pH7.4) and DMEM media, respectively, in the examples.

FIG. 4 is a graph showing the variation of particle sizes of the nano-drug diluted by deionized water in different times in the examples.

FIG. 5 is a graph showing the effect of the nano-drug on the proliferation of 4T1 cells, A549 cells and HeLa cells in the examples.

Fig. 6 is a graph of blood circulation of the nano-drug in the example.

FIG. 7 is a graph showing the distribution of the nano-drug in the tissues of the 4T1 breast cancer cell-bearing Balb/c mouse in the example.

FIG. 8 is a tumor growth curve diagram of the nano-drug in the experiment for inhibiting the tumor of a Balb/c mouse with breast cancer cells of 4T 1.

FIG. 9 is a graph showing the change of body weight of Balb/c mice in the experiment process of inhibiting tumors of Balb/c mice with breast cancer cells of 4T1 by using nano-drugs in the example.

Detailed Description

The present invention will be further described with reference to the following specific examples.

Example 1

1. Preparation of nano medicine

(1) Solution preparation

Copper nitrate aqueous solution: 0.75g of copper nitrate was weighed and dissolved in 40mL of deionized water to prepare a 100mM aqueous solution of copper nitrate.

5-nitro-8-hydroxyquinolineacetone solution: 0.38g of 5-nitro-8-quinolinol was weighed out and dissolved in 40mL of acetone to prepare a 50mM 5-nitro-8-quinolinolatone acetone solution.

(2) 20mg of bovine serum albumin was added to 5mL of deionized water, and 20. mu. mol of sodium hydroxide was added to make the concentration of sodium hydroxide in the system 4 mM. Then, 0.1mL of an aqueous copper nitrate solution and 0.5mL of a 5-nitro-8-quinolinolatone acetone solution were simultaneously added dropwise at a rate of 100. mu.L/min with stirring. After the addition, the reaction mixture was reacted at room temperature for 3 hours.

(3) Dialyzing (Mw 10000) for 48-72 hours.

(4) Freeze-drying or ultra-filtering, centrifuging and concentrating to obtain the nanometer medicine.

2. Performance analysis of Nanoparticulates

As shown in fig. 1, the average particle size of the nano-drug was 70nm (distribution coefficient PDI of 0.164) as measured by Dynamic Light Scattering (DLS).

As shown in fig. 2, the particle size of the nano-drug was observed to be around 60nm by Transmission Electron Microscopy (TEM), which is consistent with the particle size results measured by DLS.

As shown in FIG. 3, after incubation of the nano-drug in deionized water, PBS (pH7.4) and DMEM medium for 7 days, the particle size change is small, which indicates that the nano-drug is relatively stable.

As shown in fig. 4, after the nano-drug is diluted by deionized water by different times, the particle size of the nano-drug is not changed much, which once again illustrates the stability of the nano-drug.

3. Inhibition of tumor cells by nano-drugs

3.1 cytotoxicity

The in vitro toxicity of the nano-drug on A549 cells, HeLa cells and 4T1 cells is detected by adopting an MTT method. Cells in logarithmic growth phase were cultured in 96-well plates at a cell density of 5000 cells/well in 100. mu.L of RPMI-1640 medium per well. Cells were placed in 5% CO₂The culture is carried out in a 37 ℃ constant temperature and humidity incubator with the content and the relative humidity of 95 percent for 24 hours. After 24 hours, 200 mu L of RPMI-1640 medium containing different concentrations of BSA/Cu/NQ NP nano-drug was used to replace the original medium. The blank group was replaced with 200. mu.L of fresh RPMI-1640 mediumAnd (4) a culture medium. After the cells were cultured for another 48 hours, the relative viability of the cells was measured by a microplate reader. As shown in figure 5, the nano-drug has significant inhibition capability on the proliferation of three tumors, and the nano-drug is proved to have the potential of treating the tumors.

3.2 plasma Elimination

Female ICR white mice weighing about 20g were randomly divided into 2 groups of 3 mice each, and 30mg/kgBSA/Cu/NQ nano-drug and PBS solution were injected into the tail vein, respectively. At specific time points, 50 μ L of blood was collected from the posterior orbital, weighed, and then nitrated in aqua regia (concentrated nitric acid: concentrated hydrochloric acid: 3: 1). Diluting the nitrified solution by a certain multiple, filtering, and determining the content of copper in the sample by using an inductively coupled plasma mass spectrometer. As shown in fig. 6, the circulation profile of the nano-drug in blood over 24 hours. According to a quadratic nonlinear fitting equation, the first phase blood circulation half-life period and the second phase blood circulation half-life period of the nano-drug are respectively 0.45 hour and 3.8 hours, and the relatively long circulation time of the nano-drug in vivo is proved.

3.3 in vivo distribution

3 female BALB/c white mice were selected for in situ inoculation of 5X 10⁵One week after 4T1 murine breast cancer cells, the tumor volume was about 200mm³Then, 10mg/kg BSA/Cu/NQ NP nano-drugs are respectively injected. After 24 hours of injection, the rats were sacrificed by dislocation of cervical vertebrae, and major organs including heart, liver, spleen, lung, kidney and tumor were excised, washed with physiological saline, dried, weighed, and then nitrified with aqua regia (concentrated nitric acid: concentrated hydrochloric acid ═ 3: 1). Diluting the nitrified solution by a certain multiple, filtering, and determining the content of copper in the sample by using an inductively coupled plasma mass spectrometer. As shown in fig. 7, the distribution of the nano-drug in different tissues, especially in tumor, heart and spleen, was more enriched. The nano-drug is enriched in the heart and spleen, which is beneficial to removing foreign nano-particles out of the body; the nanometer medicine is enriched in tumor, and is favorable for inhibiting tumor growth.

3.4 tumor inhibition

Female BALB/c white mice were inoculated 5X 10 in situ⁵4T1 murine mammary carcinoma cells, until their average volume reaches 60mm³Thereafter, the samples were randomly divided into 3 groups (5 per group): NQ group (1.81)mg/kg), BSA/Cu/NQ NP group (NQ equivalent 1.81mg/kg) and blank control group (PBS). Administration was started simultaneously, once every 2 days for a total of 3 administrations. Nude mice body weight and tumor volume were recorded before each dose. As shown in FIG. 8, after three treatments, the tumors of both control groups (PBS group, 5-nitro-8-hydroxyquinoline group) showed the same tumor growth rate, indicating that 5-nitro-8-hydroxyquinoline alone did not inhibit tumor growth. The tumor of the experimental group obviously inhibits the growth rate of the tumor, which indicates that the nano-drug can be used as a drug for treating the tumor. As shown in fig. 9, the body weight of the mice in all three groups did not change significantly during the treatment period (18 days), indicating that the nano-drug was not significantly toxic to the mice.

Example 2

The nano-drug was prepared according to the reaction conditions of table 1, the process flow refers to example 1, and the particle size results of the prepared product are shown in table 1.

TABLE 1

Note: in the above table, BSA: bovine serum albumin; NaOH: sodium hydroxide; cu (NO)₃)₂: copper nitrate; NQ: 5-nitro-8-hydroxyquinoline.

As can be seen from the above table, when the amount of BSA or NaOH is too large, the nano-drug has a problem of being too large in size. The oversized nanoparticles cannot be enriched in tumor tissues through an EPR effect and are easily trapped by a reticuloendothelial system, so that the nanoparticles are not suitable for being used as the metal-organic complex nano-drug and tumor treatment.

Comparative example 1

Preparation of nano medicine

1. Each solution was prepared under the conditions of example 1.

2. 20mg of bovine serum albumin was added to 5mL of deionized water, and 20. mu. mol of sodium hydroxide was added to make the concentration of sodium hydroxide in the system 4 mM. Then 0.1mL of an aqueous solution of copper nitrate was added dropwise at a rate of 100. mu.L/min, and then 0.5mL of a 5-nitro-8-quinolineacetone solution was added dropwise, whereby the solution became cloudy. This is because the copper ions are first bound to bovine serum albumin, which results in no complex precipitation of 5-nitro-8-hydroxyquinoline.

Comparative example 2

Preparation of nano medicine

1. Each solution was prepared under the conditions of example 1.

2. 20mg of bovine serum albumin was added to 5mL of deionized water, and 20. mu. mol of sodium hydroxide was added to make the concentration of sodium hydroxide in the system 4 mM. Then, 0.5mL of 5-nitro-8-quinolinolato acetone solution was added dropwise at a rate of 100. mu.L/min, and then 0.1mL of copper nitrate aqueous solution was added dropwise, whereby the solution became cloudy. The reason is that the 5-nitro-8-hydroxyquinoline acetone solution is directly precipitated immediately after being dripped into the aqueous solution and can not be complexed with copper added later.

Comparative example 3

Preparation of nano medicine

1. Each solution was prepared under the conditions of example 1.

2. 20mg of bovine serum albumin was added to 5mL of deionized water, and then 0.5mL of a 5-nitro-8-hydroxyquinolineacetone solution and 0.1mL of an aqueous copper nitrate solution were simultaneously dropped at a rate of 100. mu.L/min. Further, 20. mu. mol of sodium hydroxide was added to adjust the concentration of sodium hydroxide in the system to 4 mM. The solution is unstable and tends to aggregate into large particles. This is because the bovine serum albumin has fewer exposed active functional groups, and cannot effectively entrap the metal-organic complex, resulting in easy aggregation and sedimentation of particles.

Claims (10)

1. A preparation method of a metal-organic complex nano material is characterized by comprising the following steps: firstly, adding bovine serum albumin into an alkaline solution, then simultaneously dropwise adding a copper nitrate solution and a 5-nitro-8-hydroxyquinoline solution, reacting at room temperature, and self-assembling a product to form the metal-organic complex nano material.

2. The method according to claim 1, wherein the alkaline solution is an aqueous solution of sodium hydroxide having a concentration of 4 mM.

3. The method according to claim 1, wherein the concentration of bovine serum albumin in the reaction system is 2 to 8 mg/mL.

4. The method according to claim 1, wherein the molar ratio of copper nitrate to 5-nitro-8-hydroxyquinoline is 1: 1-3.

5. The method according to claim 1 or 4, wherein the 5-nitro-8-quinolinol solution is a 5-nitro-8-quinolinolatone acetone solution having a concentration of 50 mM.

6. The method according to claim 1, wherein the copper nitrate solution and the 5-nitro-8-hydroxyquinoline solution are added dropwise at a rate of 100-.

7. The method of claim 1, wherein the reaction time is 2 to 4 hours at room temperature.

8. The method according to claim 1, wherein after the reaction, the metal-organic complex nanomaterial is entrapped by a dialysis membrane, and then freeze-dried or concentrated by ultrafiltration.

9. The metal-organic complex nano material prepared by the preparation method of any one of claims 1 to 8, wherein the mass percentage content of the 5-nitro-8-hydroxyquinoline copper complex in the nano material is 10 to 15 percent.

10. The use of the metal-organic complex nanomaterial of claim 9 in the preparation of an anti-tumor medicament, wherein the tumor is lung cancer, cervical cancer or breast cancer.

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