CN107041876A - A kind of nano particle for killing cancer cell and preparation method thereof - Google Patents

Abstract

The invention discloses new method prepared by a kind of Acetylshikonin nano-particle for antitumaous effect.This method is that Acetylshikonin is loaded into graphene/nanometer composite, and carries out plug-hole with hyaluronic acid.Hyaluronic acid can be with target cancer cell, and medicine-carried nano particles are entered after cancer cell by endocytosis, and the hyaluronidase of overexpression can degrade hyaluronic acid in cancer cell, and release Acetylshikonin kills cancer cell.Meanwhile, chemotherapy/light heat synergetic action to cancer cell can be achieved under the irradiation of near infrared light.

Description

Nanoparticles for killing cancer cells and preparation method thereof

technical field

The invention relates to the field of nano biomedicine, in particular to a nano particle for killing cancer cells and a preparation method thereof.

Background technique

Most chemotherapeutic drugs currently used to treat malignant tumors do not kill cancer cells by inducing tumor cell apoptosis, but inhibit the synthesis of various nucleotides or direct cytotoxicity through competitive inhibition, so they have great side effects and damage The body's immune balance. Cell proliferation, apoptosis, differentiation, and senescence are all cell cycle-dependent. Therefore, choosing drugs that can disrupt the cell cycle and cause apoptosis, and regulating the cell cycle as a strategy will be a new approach for anti-tumor therapy in the future. Many studies have shown that traditional Chinese medicine inhibits the occurrence, development and metastasis of tumors in many aspects such as inducing tumor cell apoptosis, resisting tumor cell invasion and metastasis, inducing tumor cell differentiation, inhibiting the expression of oncogenes, and promoting the expression of oncogenes. , and many traditional Chinese medicines can act on the above-mentioned multiple links at the same time; however, traditional Chinese medicines have complex components and the mechanism of action is not clear enough, so finding natural, effective, and less side effect anti-tumor drugs is the key to tumor treatment. Comfrey extract, as a natural medicine derived from plants, can effectively induce tumor cell apoptosis, and is a potential and effective antitumor drug. The main anti-cancer active ingredients of comfrey are naphthoquinone pigment compounds, that is, shikonin and its derivatives, especially acetylshikonin (AS) and β,βʹ-dimethylacryloylshikonin. Anticancer ingredients. At present, the use of modern technology to extract the active ingredients of comfrey for the treatment of tumors has made great progress, which can increase the content of active ingredients and reduce its toxicity. However, current comfrey extracts still lack specificity for tumors, easily lead to underdose in diseased areas, and cause unexpected side effects on normal tissues.

Contents of the invention

Aiming at the above deficiencies, the present invention provides a nanoparticle for killing cancer cells and a preparation method thereof. In the present invention, acetylshikonin is loaded into graphene/mesoporous silicon nanocomposites, and hyaluronic acid is used for plugging Well, hyaluronic acid can target cancer cells. After drug-loaded nanoparticles enter cancer cells through endocytosis, the overexpressed hyaluronidase in cancer cells can degrade hyaluronic acid and release shikonin to kill cancer cells. At the same time, chemotherapy/photothermal synergistic therapy of cancer can be realized under the irradiation of near-infrared light.

The technical solution adopted by the present invention to solve its technical problems is:

The beneficial effects of the present invention are:

The use of nanotechnology for the delivery of its active ingredients and the use of enzymes overexpressed in tumor sites to control drug release can well solve the current lack of specificity of comfrey extracts to tumors, which can easily lead to insufficient dosage in diseased areas and damage to normal tissues. Problems causing unintended side effects.

The sandwich-like graphene/mesoporous silicon nanosheet (GS) has become a new type of drug carrier, which has excellent biocompatibility, hydrophilicity and dispersibility, and can combine the photothermal function of graphene with mesoporous Silicon's large pore size combined. Drugs can be loaded into the mesopores of GS, and the pores can be encapsulated with degradable molecules that respond to the microenvironment of the targeted site. In this way, an "on-demand" drug delivery platform can be constructed. Hyaluronic acid (HA), a biodegradable and nontoxic component of the extracellular matrix, has been widely used as a targeting and encapsulating agent for cancer therapy. It can specifically bind to the CD44 receptor and can be degraded by hyaluronidase, both of which are overexpressed in various cancer cells.

Description of drawings

Figure 1 is a typical scanning electron microscope (A) and transmission electron microscope (B) images of graphene/mesoporous silicon nanocomposites;

Figure 2 is the fluorescence microscope pictures of HeLa cells treated with GS@HA (A), GS@HA-AS (B), GS@HA+NIR (C) and GS@HA-AS+NIR (D). Cells were stained with Calcein AM, showing green, and dead cells were stained with PI, showing red;

Figure 3 is a graph of cell viability after HeLa cells were treated in different ways.

detailed description

Example 1

(1) Preparation of graphene/mesoporous silicon nanocomposites: First, 50 mg of graphene oxide was added to 50 mL of an aqueous solution containing 0.5 g of CTAB and 20 mg of sodium hydroxide, and ultrasonically dispersed for 1 hour; then the above solution was dissolved at 40 Stir at ℃ for 2 hours; then add 500 μL TEOS and continue the reaction for 12 hours. The obtained product was collected by centrifugation and washed three times with 20 mL of hot absolute ethanol; then the product was dispersed in 50 mL of acetone and stirred at 40 °C for 24 hours to remove the CTAB template; the obtained product was washed with ethanol and deionized water respectively, and dispersed Prepare in 10 mL deionized water, which is the graphene/mesoporous silicon nanoparticles.

(2) Dissolve 20 mg of graphene/mesoporous silicon nanoparticles and 20 mg of acetylshikonin obtained in the previous step (purified and prepared by high-speed countercurrent chromatography in our research group, and related papers have been published) in PBS buffer (10 mM , pH 7.4), stirred for 24 hours; then the precipitate was collected by centrifugation at 12000 rpm, the above steps were repeated three times, and the collected precipitate was graphene/mesoporous silicon nanoparticles loaded with acetylshikonin.

(3) Add 50 mg EDC and 30 mg NHS to 100 mg HA, activate in MES buffer (pH 5.5) for 2 hours, and then mix acetylshikonin-loaded graphene/mesoporous silicon nanoparticles with it , and continue stirring for 6 hours; then the obtained product was washed 3 times with PBS to obtain the final product of the present invention, a nanoparticle that kills cancer cells.

Cancer cell death imaging, as shown in Figure 2

HeLa cells were placed in DMEM cell culture medium containing 10% fetal bovine serum, 100 μg/mL penicillin and streptomycin, and cultured at 37°C in a cell culture incubator containing 5% _CO2 ; Visualize cell death by staining with a fluorescent indicator to distinguish live from dead cells. HeLa cells were cultured in 100 μL of RPMI cell culture medium in a 24-well plate at a density of ¹ × 104 cells per well for 24 h. After the cells were washed, the graphene/mesoporous silicon nanoparticles wrapped with hyaluronic acid (GS@HA) and the graphene/mesoporous silicon nanoparticles loaded with acetylshikonin (GS@HA-AS) (GS concentration of 200 μg/mL) were added to the wells and incubated for 2 hours. For the photothermal group, irradiate with an 808 nm laser at an intensity of 6 W/cm for ⁵ min. Culture was continued for 12 hours after replacement of fresh DMEM cell culture medium. Finally, the cells were stained with live-dead cells. The live cells were stained with Calcein AM, showing green, and the dead cells were stained with propidium iodide (PI), showing red. The results obtained are shown in Figure 2. From the figure, we can see Results: The nanoparticles loaded with acetylshikonin or near-infrared light both have a certain ability to kill cancer cells, and when the two act together, they have the highest cell death rate.

In vitro cancer cell killing experiment (as shown in Figure 3)

MTT method was used to measure the survival rate of cancer cells under different treatments. After HeLa cells with a density of 1×10 ⁵ cells/well were cultured in a 96-well cell culture plate for 24 hours, nanoparticles GS@HA and GS@HA-AS (GS concentration of 200 μg/mL) were added to the wells for total Incubate for 2 hours. The photothermal experiment group was irradiated with an 808 nm laser at an intensity of 6 W/ _cm2 for 5 minutes. The supernatant culture medium was removed and washed 3 times with PBS solution, and fresh DMEM medium was added, and then culture was continued for 24 hours. Add 10 μL of MTT (5 mg/mL) and continue to incubate for 4 hours. Finally, carefully aspirate the supernatant, add 100 μL DMSO to each well and shake gently on a shaker. Cell viability was calculated by recording the absorbance at 570 nm. The formula for calculating the relative activity of cell growth is: % = (1-[OD] _test /[OD] _control ) × 100, and the results are shown in Figure 3. From the figure, we can see that the nanoparticles themselves have little toxicity to cancer cells. When the concentration of nanoparticles is 400 μg/mL, the HeLa cells still maintain a survival rate of 94%. The drug-loaded system can realize chemotherapy/photothermal synergistic therapy. The cell survival rate of 100 μg/mL GS@HA-AS was 80.6%. When adding μg/mL GS@HA-AS nanoparticles, only 25.6% of the cells could survive.

Of course, the above descriptions are not intended to limit the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or replacements made by those skilled in the art within the scope of the present invention shall also belong to the present invention. protection scope of the invention.

Claims (5)

1. A nanoparticle for killing cancer cells, characterized in that: the structure of the particle is that the inner layer is acetylshikonin loaded in the graphene/mesoporous silicon nanocomposite material, and the outer layer is encapsulated with hyaluronic acid. 2. a kind of preparation method of the nanoparticle of killing cancer cell as claimed in claim 1, is characterized in that, comprises the following steps: (1) Preparation of graphene/mesoporous silicon nanocomposites; (2) Acetylshikonin loaded into graphene/mesoporous silicon nanocomposites; (3) Hyaluronic acid sealing package. 3. the preparation method of a kind of nanoparticle that kills cancer cells according to claim 2, is characterized in that, described graphene/mesoporous silicon nanocomposite material is to be template with CTAB, in situ synthesis on graphene oxide Mesoporous silicon, monodisperse, with a diameter of 100-300 nm. 4. the preparation method of a kind of nanoparticle that kills cancer cells according to claim 2, is characterized in that, described acetylshikonin mixes with graphene/mesoporous silicon nanocomposite material by mass ratio 1:1, stirs 24 hr, loaded into the pores of mesoporous silica. 5. the preparation method of a kind of nanoparticle that kills cancer cells according to claim 2, is characterized in that, described hyaluronic acid is covalently modified on the surface of graphene/mesoporous silicon nanocomposite material as plugging agent and targeting molecules.