CN115919799A - Gallium sulfide-based composite nanoparticle and preparation method and application thereof - Google Patents

Abstract

The invention provides a gallium sulfide-based composite nanoparticle and a preparation method and application thereof. The invention consists of metal gallium ions (Ga) ³⁺ ) Forming metal ion coordination complex solution with Bovine Serum Albumin (BSA), and then mixing with sodium sulfide (Na) ₂ S) reacting to generate coordination polymer nano particles, finally dialyzing, purifying, and freeze-drying to obtain the gallium sulfide-based composite nano particles with the average particle size of 10nm. The method has the characteristics of simple production process, low raw material cost, high yield, good reproducibility and the like, and can realize low-cost large-scale production under normal conditions at room temperature. Book (I)The invention reduces the toxic and side effects of pure gallium ions, improves the bioavailability of the pure gallium ions, further promotes the anti-tumor effect by combining with hydrogen sulfide, and has great application potential in the field of clinical anti-tumor.

Description

Gallium sulfide-based composite nanoparticle and preparation method and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a green and simple preparation method of gallium sulfide-based composite nanoparticles and application of the gallium sulfide-based composite nanoparticles in the field of antitumor medicines.

Background

Ovarian cancer is one of the most common female reproductive system malignancies, with about 70% of patients diagnosed at an advanced stage with mortality rates at the first of the gynecological malignancies. At present, the standard treatment mode of ovarian cancer at home and abroad is tumor cell debulking and chemotherapy assisted by paclitaxel and platinum drugs. However, 70-85% of patients with advanced ovarian cancer relapse within two years, even after standard treatment regimens. Patients will receive multi-line chemotherapy treatment after recurrence, and each treatment will further shorten the patient's disease-free interval, which results in nearly all ovarian cancer patients eventually progressing to platinum-resistant ovarian cancer. Therefore, overcoming platinum resistance in ovarian cancer is a key issue for improving survival of ovarian cancer patients, and the search for new drugs that can be used to overcome platinum resistance is urgent.

Gallium is the second metal element with anti-tumor activity discovered after platinum, gallium nitrate is discovered to have wide anti-tumor effect in 1971, and subsequent research discovers that gallium nitrate can be used for treating lung cancer, prostate cancer, breast cancer and the like. However, the single metal ion has short circulation time in vivo, low target selectivity and high systemic toxicity, and can cause wide adverse reactions, such as hepatotoxicity, nephrotoxicity, neurotoxicity, myelosuppression and the like. Hydrogen sulfide is a gaseous neurotransmitter which is second to carbon monoxide and nitric oxide and is involved in the regulation of physiological and pathological processes, has good biocompatibility, and is involved in the regulation of the occurrence and development of malignant tumors. High concentrations of hydrogen sulfide can act to block the cell cycle, inhibit cell proliferation, promote apoptosis, and inhibit cell migration and invasion. The metal sulfide nano material can degrade and release metal ions and hydrogen sulfide gas in an acidic tumor microenvironment, so that the metal sulfide nano material has the effects of single metal ions and hydrogen sulfide at the same time, and can show stronger synergistic antitumor characteristics. And the metal sulfide nano material can overcome the defects of short cycle time and high system toxicity of single metal ions. Therefore, the construction of the metal nanoparticles which release hydrogen sulfide gas in response to the tumor microenvironment has important significance for the treatment of malignant tumors. In this patent Ga is mixed with ³⁺ 、Na ₂ Synthesis of Ga from S and bovine serum albumin ₂ S ₃ BSA nanoparticles, simultaneous release of Ga in acidic tumor microenvironment ³⁺ And H ₂ S, combined with Ga ³⁺ And H ₂ The anti-tumor effect of S has a wide application prospect in the field of ovarian cancer treatment.

Disclosure of Invention

The invention aims to provide gallium sulfide nanoparticles, a preparation method and application thereof aiming at the defects of the prior art.

The technical scheme adopted by the invention is as follows:

a method for preparing gallium sulfide nanoparticles, comprising the steps of:

(1) Disposition of Ga ³⁺ -an aqueous solution of a BSA metal-organic complex; in the metal ion coordination complex solution, the mass ratio of bovine serum albumin to Ga ions is 20.7, and the concentration of Ga ions is 0.406mg/mL.

(2) Adding a sodium sulfide compound to Ga ³⁺ And (3) reacting in a BSA metal organic complex aqueous solution to generate a gallium sulfide polymer nanoparticle solution, and purifying to obtain a finished product. Wherein the molar ratio of the sodium sulfide to the Ga ions is 0.6:1. preferably, the sodium sulfide solution is added dropwise to Ga ³⁺ -stirring the aqueous BSA metal-organic complex solution for 1-3 hours. Finally, ga ₂ S ₃ The mass concentration of Ga ions in the BSA polymer nanoparticle solution is 0.382mg/mL, and the mass concentration of bovine serum albumin is 7.92mg/mL.

Further, in the step (1), bovine serum albumin and Ga ions are dissolved in water, respectively, and then mixed to form Ga ³⁺ -BSA metal ion coordination complex solution; ga ³⁺ The mass concentration of bovine serum albumin in the BSA metal ion coordination complex solution is 8.42mg/mL.

Preferably, the aqueous solution of Ga ions and the aqueous solution of bovine serum albumin may be mixed without a sequential order under light-shielding conditions.

Further, in the step (2), the mass concentration of the added sodium sulfide is 0.792mg/mL.

Further, the Ga ions are converted into Ga chloride (GaCl) ₃ ) Gallium nitrate (Ga (NO) ₃ ) ₂ ) One or two of the above-mentioned materials can be dissolved in water according to any proportion.

Further, in the step (2), a dialysis bag with a molecular weight of 15kd is used for purification, and the drying method is freeze drying.

The invention also provides Ga prepared by the preparation method ₂ S ₃ -BSA nanoparticles, said Ga ₂ S ₃ -BSA nanoparticles containing five elements Ga, C, O, N, S, containing electrostatic and coordination bonds, the particles having a size ranging from 5 to 15nm. The particle size distribution is uniform in the aqueous solution.

The gallium sulfide nano particles are applied to the preparation of anti-platinum drug-resistant ovarian cancer drugs for human and/or animal in vivo and/or in vitro cells.

The invention has the beneficial effects that: the invention makes metal gallium ion (Ga) ³⁺ ) Forming coordination polymer nanoparticles by a ligand combination method with biocompatible macromolecular albumin and a sodium sulfide compound in aqueous solution. The method has the advantages of simple operation, high yield, good repeatability and low cost and large-scale production, and the production process is carried out at room temperature. The prepared nano-particles can keep the antitumor effect of the Ga ions and the hydrogen sulfide, simultaneously play the effect of combined treatment effect, improve the bioavailability and the biocompatibility of the Ga ions, and have great promotion effect on the clinical transformation of the antitumor effect of the Ga ions and the sodium sulfide.

Drawings

FIG. 1: gallium sulfide nanoparticles (Ga) ₂ S ₃ -BSA) and characterization results. Wherein A and B are Ga respectively ₂ S ₃ TEM bright and dark field images of BSA nanoparticles (scale bar 20 nm). C. D is Ga ₂ S ₃ -elemental energy spectrum of BSA nanoparticles. E is Ga ₂ S ₃ -dynamic light scattering pattern of BSA nanoparticles. F is Ga ₂ S ₃ Zeta potential map of BSA nanoparticles. G is Ga ₂ S ₃ -fourier infrared spectroscopy of BSA nanoparticles. H-J are X-ray photoelectron spectroscopy (XPS) plots.

FIG. 2: ga ³⁺ -BSA、Na ₂ S、Ga ₂ S ₃ -BSA three drugs treated under different concentration conditions A2780-CIS (A) and SKOV3-CIS (B) two platinum-resistant ovarian cancer cell survival rate result chart and Ga ³⁺ -BSA、Na ₂ S、Ga ₂ S ₃ Graph showing the relative cell activity curves of A2780-CIS (C) and SKOV3-CIS (D) cells for three drugs in BSA under different time conditions.

FIG. 3: flow cytometry for Ga detection ³⁺ -BSA、Na ₂ S、Ga ₂ S ₃ A2780-CIS and SKOV3-CIS apoptosis results after BSA three-drug treatment are shown in a schematic diagram (A) and a statistical histogram (B).

FIG. 4: ga ³⁺ -BSA、Na ₂ S、Ga ₂ S ₃ Expression result graph (A) and statistical bar chart (B) of related proteins Bcl2 and cleared-caspase 3 in apoptosis process of A2780-CIS and SKOV3-CIS cells after BSA three-drug treatment.

FIG. 5: in vivo SKOV3-CIS model bioluminescence imaging technology under different time and treatment groups detects SKOV3-CIS-luc cell luciferase signal in vivo imaging graph (A) and fluorescence signal quantitative analysis result graph (B).

FIG. 6: digital camera images of tumor tissues from each group (4 groups) collected from different treatment groups of the in vivo SKOV3-CIS model.

FIG. 7: detection of Ga by HE staining and Ki67 immunohistochemical staining of tumor tissues ₂ S ₃ Graph showing the results of the antitumor activity of BSA.

FIG. 8: digital camera images (A) and H & E pathological section images (B) of major organs collected from different treatment groups of an in vivo SKOV3-CIS model.

FIG. 9: in vivo SKOV3-CIS model blood routine (a) and blood biochemical outcome map (B) after treatment of different treatment groups.

FIG. 10: in vivo SKOV3-CIS model Ga ₂ S ₃ Digital camera shots (A) and H after 3 days of BSA continuous treatment&E pathological section picture (B).

FIG. 11: in vivo SKOV3-CIS model Ga ₂ S ₃ Graph of the results of blood routine (a) and blood biochemistry (B) after 3 days of BSA continuous treatment.

Detailed Description

The invention is further illustrated with reference to the following examples and figures; in this example, ga is used ₂ S ₃ The preparation technology of BSA nano-particles and the anti-tumor effect on ovarian cancer in vivo are illustrated.

Example 1 Ga ₂ S ₃ Synthesis and characterization of BSA nanoparticles

First of all, ga is prepared ³⁺ -BSA metal organic complex solutions, in particular: 160mg of bovine serum albumin was dissolved in 16mL of water, stirred, and then 3mL of a 37mM gallium chloride solution was added and stirred for 1 hour. Subsequently, ga stabilized in BSA ³⁺ -adding slowly dropwise 1.2mL of a 55.3mM sodium sulfide solution to a BSA metal-organic complex solution, and finally, ga ₂ S ₃ -the mass concentration of Ga ions in the BSA polymer nanoparticle solution is 0.382mg/mL, and the mass concentration of bovine serum albumin is 7.92mg/mL; stirring for 1-3 hours at normal temperature in the dark, purifying by using a dialysis bag with the molecular weight of 15kd, and freeze-drying to prepare Ga ₂ S ₃ -BSA nanoparticles. The morphology and particle size of the prepared nanoparticles were studied using a transmission electron microscope. As shown in FIGS. 1A and B, ga ₂ S ₃ -the BSA nanoparticles are spherical monodisperse nanoparticles with an average particle size of about 10nm. The elemental spectra indicate the presence of Ga, C, O, N, S elements (FIG. 1C, D). FIG. E shows Ga ₂ S ₃ Dynamic light scattering pattern of BSA nanoparticles, ga ₂ S ₃ The hydrodynamic radius of the BSA nanoparticles is about 15nm. FIG. F shows Ga ₂ S ₃ The Zeta potential of the BSA nanoparticles is about 10mV. Panel G demonstrates the presence of albumin and gallium sulfide in the nanoparticle component. Ga has been investigated by X-ray photoelectron spectroscopy (XPS) ₂ S ₃ -chemical composition of BSA nanoparticles. As shown, ga ₂ S ₃ The XPS spectrum of BSA nanoparticles comprises the peaks C1S, O1S, N1S and Ga3d, wherein the analysis of the peaks of Ga element proves the presence of Ga ions and the peaks of the spectrum of S element. The above results indicate the successful preparation of gallium sulfide nanoparticles.

Example 2 Ga ₂ S ₃ Characterization of the in vitro antitumor Properties of BSA nanoparticles

To test Ga ₂ S ₃ Anticancer Activity of BSA self-assembled nanoparticles against platinum-resistant ovarian cancer cell lines, this example with Ga formulated separately ³⁺ -aqueous BSA metal-organic Complex solution, na ₂ S and Ga ₂ S ₃ BSA treatment of human platinum-resistant ovarian cancer cells A2780-CIS and SKOV3-CIS cells cell viability was studied (FIGS. 2A-D). Ga used in all experiments ³⁺ Ga in BSA ³⁺ Concentration and Na ₂ S in S ^2- In a concentration of Ga ₂ S ₃ Corresponding Ga in BSA ³⁺ Concentration and S ^2- And (4) concentration. With Ga in different concentrations ³⁺ -BSA、Na ₂ S or Ga ₂ S ₃ Both cell lines showed similar dose-dependent cytotoxic propensity 48 hours after BSA treatment. Ga compared with the other two groups ₂ S ₃ The BSA group showed stronger killing ability of cancer cells, half Inhibitory Concentration (IC) of the three drugs ₅₀ ) As shown in table 1. In the use of Ga ₂ S ₃ IC of BSA ₅₀ Concentration and corresponding Ga ³⁺ -BSA and Na ₂ S concentration is subjected to drug adding culture, and Ga is added at 48h, 72h and 96h after drug adding ₂ S ₃ BSA self-assembled nanoparticles showed stronger anti-cancer capacity for both cell lines. Notably, compare Ga ³⁺ -BSA、Na ₂ S two drugs, A2780-CIS and SKOV3-CIS, both human platinum resistant ovarian cancer cells, to Ga ₂ S ₃ BSA nanoparticles are more sensitive (fig. 2C and D). Flow cytometry for apoptosis of A2780-CIS and SKOV3-CIS cells after different treatment modes, as shown in FIG. 3, ga ³⁺ -BSA and Na ₂ The S drug treatment group showed a small apoptosis rate, while Ga ₂ S ₃ -the rate of apoptosis induced by BSA nanoparticles is about Ga ³⁺ -BSA and Na ₂ 3-5 times of the S drug treatment group. Furthermore, the expression of the relevant proteins (Bcl 2 and cleared-caspase 3) during apoptosis is shown in FIG. 4. Ga compared with control group ³⁺ -BSA、Na ₂ Clear-caspase 3/caspase 3 and Bcl2 protein levels of the S-plus group did not change significantly, but Ga ₂ S ₃ Significant increase in clear-caspase 3/caspase 3 levels and Bcl2 protein levels after treatment with the group of BSA nanoparticlesIs significantly reduced. In summary, ga ₂ S ₃ BSA nanoparticles possibly by binding Ga ³⁺ And S ^2- The effect of (1) enhancing Ga ³⁺ -BSA or Na ₂ S single drug has the function of inhibiting the growth of tumor cells.

Table 1: IC50 value

Example 3 preliminary evaluation of antitumor Effect and biological safety of SKOV3 model in vivo

This example investigated Ga ₂ S ₃ Anti-tumor effects of BSA nanoparticles in SKOV3-CIS animal models. Firstly, platinum drug-resistant ovarian cancer cell SKOV3-CIS (SKOV 3-CIS-luc) marked by luciferase is injected into a nude mouse in an abdominal cavity to establish an ovarian cancer in-vivo SKOV3-CIS model, and after the cells are randomly divided into 4 groups according to in-vivo biological signals, sterile-filtered pure water (control) and 300 mu g/ml Ga are respectively used on the 1 st, 2 nd and 3 th days and the 14 th, 15 th and 16 th days after the grouping ³⁺ -BSA、Na ₂ S and Ga ₂ S ₃ BSA was injected into nude mice tail vein at a dose of 200. Mu.L/day. During treatment, luciferase signals from SKOV3-CIS-luc cells were detected by non-invasive bioluminescent imaging to assess treatment efficacy. Fig. 5A and 5B are quantitative statistical plots of bioluminescence intensity at day 0 (day of randomization) and day 22, with bioluminescence intensity for each group (n = 3) remaining substantially consistent at day 0, but a different bioluminescence intensity phenomenon occurred between the four treatment groups at day 22: wherein Ga ₂ S ₃ The lowest bioluminescence intensity of the BSA nanoparticle treatment group indicates the best therapeutic effect; and Ga ³⁺ -BSA and Na ₂ S has limited tumor inhibition effect on SKOV3-CIS cancer cells. Mice were then sacrificed and groups of tumor tissue were collected and photographed with a digital camera, as shown in figure 6, ga compared to the other three treatment groups ₂ S ₃ BSA nanoparticle therapy showed enhanced antitumor properties. H&The results of E staining for antitumor activity are shown in FIG. 7, ga ₂ S ₃ Tumor structures of the BSA nanoparticle-treated group were more numerous than those of the other three groupsSimultaneous immunohistochemical detection of Ki-67 (cell proliferation marker) expression for loosening, ga ₂ S ₃ Downregulation of Ki-67 expression in the BSA nanoparticle treatment group (FIG. 7), indicating that Ga ₂ S ₃ BSA nanoparticles have a marked proliferation-inhibiting effect on tumors. Finally, to better meet the needs of clinical transformation, ga is required ₂ S ₃ BSA nanoparticles for in vivo safety assessment. FIG. 8 is a digital camera image and pathological section H of major organs collected from different treatment groups of the SKOV3-CIS model in vivo&E staining pattern, gross images of major organs (heart, liver, spleen, lung and kidney) of each group were substantially identical (FIG. 8A), and H of each organ&E staining results show that Ga ₂ S ₃ Normal organ tissue structure without significant damage in the BSA nanoparticle treated group (FIG. 8B), indicating Ga ₂ S ₃ BSA nano-particles do not cause other damages to organisms and have no toxic or side effect. In addition, the blood routine suggests Ga ₂ S ₃ The increase of the white blood cells of the BSA nanoparticle group compared with the control group is consistent with the inflammation caused by the reaction of the hydrogen sulfide, and the conventional and biochemical results of the blood of the three groups have no significant difference, indicating the good biocompatibility (figure 9). Furthermore, ga is also evaluated ₂ S ₃ Acute toxic side effects of BSA nanoparticles, ga3 days continuously on nude mice ₂ S ₃ BSA nanoparticle injection, blood and major organs collected on day 4, FIG. 10 general view of major organs of each group (FIG. 10A) and organ H of each group&E staining pattern (FIG. 10B), the results showed that there was no significant change in each organ, ga ₂ S ₃ BSA nanoparticles have no acute toxic side effects. FIG. 11 shows the results of blood routine and blood biochemical analysis of each group, which show that there is no significant difference between the results of blood routine and blood biochemical analysis of each group, indicating that Ga ₂ S ₃ BSA nanoparticles do not cause acute toxic side effects in the body.

In summary, the present invention designs a gallium ion (Ga) composed of metal ³⁺ ) And reacting the hydrogen sulfide with the bovine serum albumin to generate the coordination polymer nanoparticles. The nanoparticles have low side effects and high bioavailability. Can be efficiently gathered to tumor cells in a targeted way, and has efficient anti-tumor effect.

Ga prepared ₂ S ₃ BSA nanoparticles can inhibit cell proliferation and promote apoptosis, and have a strong killing effect on platinum-resistant ovarian cancer cells.

Novel Ga developed by the present invention ₂ S ₃ The BSA nano-particles can keep the anti-tumor effect of Ga ions and hydrogen sulfide, simultaneously play the effect of combined treatment effect, and improve the bioavailability and biocompatibility of the Ga ions.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should all embodiments be exhaustive. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.

Claims (8)

1. A preparation method of gallium sulfide-based composite nanoparticles is characterized by comprising the following steps:

(1) Disposition of Ga ³⁺ -an aqueous solution of a BSA metal-organic complex; in the metal ion coordination composite solution, the mass ratio of bovine serum albumin to Ga ions is 20.7, and the concentration of the Ga ions is 0.406mg/mL;

(2) Adding a sodium sulfide compound to Ga ³⁺ Reacting in a BSA metal organic complex aqueous solution to generate a gallium sulfide polymer nanoparticle solution, and purifying and drying to obtain a finished product; wherein the molar ratio of the sodium sulfide to the Ga ions is 0.6:1.

2. the method according to claim 1, wherein in the step (1), the bovine serum albumin and the Ga ion compound are dissolved in water to obtain an aqueous solution of bovine serum albumin and an aqueous solution of trivalent Ga ion, respectively, and then the two solutions are mixed to form Ga ³⁺ -BSA metal ion coordination complex solution; ga ³⁺ The mass concentration of bovine serum albumin in the BSA metal ion coordination complex solution is 8.42mg/mL.

3. The production method according to claim 1, wherein in the step (2), a sodium sulfide compound is added to Ga ³⁺ In the BSA metal-organic complex aqueous solution, specifically, the sodium sulfide compound is prepared into a sodium sulfide solution with the concentration of 55.3mM, and then 1.2mL to Ga is dripped ³⁺ -aqueous solutions of BSA metal-organic complexes.

4. The method according to claim 1, wherein the Ga ions are obtained by gallium chloride (GaCl) ₃ ) Gallium nitrate (Ga (NO) ₃ ) ₂ ) One or two of them are dissolved in water according to any proportion to obtain.

5. The method according to claim 1, wherein in the step (2), the purification is carried out by using a dialysis bag having a molecular weight of 15kd, and the drying method is freeze-drying.

6. The production method according to claim 1, wherein in the step (2), the sodium sulfide solution is dropwise added to Ga ³⁺ -stirring the aqueous BSA metal-organic complex solution for 1-3 hours.

7. A gallium sulfide-based composite nanoparticle prepared by the preparation method of any one of claims 1 to 6, wherein the composite nanoparticle comprises five elements of Ga, C, O, N and S, and comprises an electrostatic bond and a coordination bond, and the particle size of the nanoparticle is in a range of 5-15nm.

8. Use of the gallium sulfide-based composite nanoparticle of claim 7 in the preparation of an anti-ovarian cancer medicament.

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