CN113633789B - Liquid metal nano probe integrating light acoustic imaging and drug inclusion and preparation method thereof - Google Patents

Abstract

The invention discloses a liquid metal nano probe integrating light-sound imaging and drug inclusion and a preparation method thereof. The preparation method comprises the steps of mixing liquid metal and a tannic acid aqueous solution, carrying out ultrasonic treatment, taking upper-layer liquid, and further carrying out mild centrifugal purification to remove relatively large particles; and then, mixing the medicinal preparation with the liquid metal nano-particles to obtain the medicament-carrying liquid metal nano-probe. The medicine-carrying liquid metal nano probe provided by the invention is used for carrying out in-situ injection on tumors, so that the medicine-carrying liquid metal nano probe is uniformly distributed on tumor positions, the medicine-carrying liquid metal nano probe can be subjected to swelling deformation under the irradiation of laser, the medicine is released from the surface of the nano probe, and the photothermal treatment and the chemotherapy are combined for treatment, so that the tumors are killed under the effect of double management.

Description

A liquid metal nanoprobe integrating photoacoustic imaging and drug encapsulation and preparation method thereof

technical field

The invention relates to the field of biomedical nanomaterials, in particular to a liquid metal nanoprobe integrating photoacoustic imaging and drug encapsulation and its application.

Background technique

Cancer is a major killer that threatens human life and health. At present, the diagnosis methods of tumors mainly include four methods: biopsy, laboratory examination, pathological examination and magnetic resonance imaging (MRI). Many recent studies have focused the two separate processes of diagnosis and treatment on the same nanoprobe, thus constructing an integrated nanoplatform for diagnosis and treatment. Nanoprobes are the product of the multidisciplinary cross-integration of nanoscience and biology, physics, chemistry and other technologies. They have good biocompatibility and stability, and can be multifunctionally modified and encapsulated with antitumor drugs. and cancer diagnosis and treatment has shown great potential.

Liquid metal is a new type of material that has both metallic properties and fluid flow. The liquid metal gallium indium tin alloy (galinstan, 68wt% Ga, 22wt% In, 10wt% Sn) at room temperature not only has the characteristics of traditional metals, including excellent It also has high flexibility, moldability, low toxicity and good biocompatibility. However, the surface tension and density of liquid metal are relatively large, and it is difficult to disperse into small nano-sized particles. Therefore, the current research difficulties of liquid metal mainly focus on using simple means to disperse it into stable nanoparticles and further surface functionalization. The functionalized liquid metal nanoparticles will be better applied in the field of biomedicine.

Photoacoustic imaging is to irradiate biological tissue with pulsed laser or modulated light, and the absorbed light energy is completely or partially converted into heat energy, resulting in thermoelastic expansion to generate pressure waves, which are transmitted in the form of ultrasound in biological tissue, and finally pass through The signal converter converts the image. The contrast agents required for photoacoustic imaging are divided into endogenous contrast agents and exogenous contrast agents. Endogenous contrast agents are inherent components in biological tissues, including hemoglobin, melanin, water, etc.; exogenous contrast agents mainly include organic small molecular materials, polymer nanomaterials, inorganic non-metallic nanomaterials, and metal nanomaterials. Contrast agents change the local optical and acoustic properties of biological tissues, improve contrast and resolution, and can be used in non-invasive real-time imaging of tumor tissue, blood vessels, brain tissue, etc. It is also applied to reactive oxygen species (ROS), pH value, metal ions and other sensing. Therefore, it is of great significance to develop photoacoustic imaging contrast agents with biocompatibility, high resolution, and high penetration depth.

Tannin is a green and safe natural polyphenolic substance whose chemical structure is composed of a glucose core covalently linked to a gallic acid group through a hydrolyzable ester bond. Under neutral conditions, tannins exhibit electronegativity due to the large number of galloyl groups, so they can bind to positively charged species through electrostatic interactions. At the same time, the large number of aromatic rings in the tannic acid structure can promote its interaction with hydrophobic molecules. According to the structural characteristics of tannic acid, it is believed to be able to extensively link to molecules of different groups, and has great potential to modify the surface of nanoparticles, thereby controlling the interaction of nanoparticles with specific cells or providing them with additional functions, such as Coated with small molecules, synthetic polymers, proteins, polysaccharides and various drugs.

In the prior art, photoacoustic imaging and drug-loaded diagnosis and treatment are usually implemented independently, and there are few nanoprobes that can realize the integration of photoacoustic imaging and drug-loaded diagnosis and treatment. In order to realize the wider application of liquid metal nanoparticles in the field of biomedical nanomaterials, it is hoped to combine photoacoustic imaging and drug loading to construct a nanoprobe integrating diagnosis and treatment, which has certain application prospects for clinical detection and treatment of cancer.

Purpose of invention

The purpose of the present invention is to proceed from the deficiencies of the prior art, and aims to provide a liquid metal nanoprobe integrating photoacoustic imaging and drug encapsulation and a preparation method thereof.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, there is provided a method for preparing a liquid metal nanoprobe integrating photoacoustic imaging and drug-carrying, comprising the following steps:

Step 1. Ultrasonic treatment is performed after mixing the liquid metal (1) with the tannic acid aqueous solution (2), the upper layer liquid is taken, and then, the micron-sized particles are removed by mild centrifugal purification to obtain liquid metal nanoparticles;

In step 2, the drug preparation is mixed with the liquid metal nanoparticles described in step 1 to obtain a drug-loaded liquid metal nanoprobe (6).

Preferably, the liquid metal is a gallium-based alloy, and the gallium-based alloy contains 68wt% gallium, 22wt% indium and 10wt% tin; the liquid metal nanoparticles have a diameter of 15nm-150nm.

Preferably, the volume concentration ratio of the liquid metal and the tannic acid is 1:2, and a 6mmφ probe with a model of Scientz-IID is used to perform 15min ultrasonic treatment by a cell crusher (3), and the power is set to 160W , use an ice bath to ensure that the temperature of the sample is controlled at 0 °C during the sonication process.

Preferably, the pharmaceutical preparation described in step 2 is doxorubicin hydrochloride (4), which is mixed with liquid metal nanoparticles overnight at room temperature using a magnetic stirrer (5).

According to another aspect of the present invention, a liquid metal nanoprobe prepared according to the above preparation method is provided.

Preferably, the drug-loaded liquid metal nanoprobe is used for breast cancer photoacoustic imaging.

More preferably, the drug-loaded liquid metal nanoprobe performs photoacoustic imaging under the excitation of a laser with a wavelength of 808 nm.

Preferably, the drug-loaded liquid metal nanoprobe is used for the combined treatment of breast cancer with photothermal therapy and chemotherapy.

Preferably, the drug-loaded liquid metal nanoprobe is irradiated by a laser with a wavelength of 808 nm and a power of 1 W to generate thermal expansion deformation to promote drug release.

Description of drawings

Figure 1 is a flow chart of the preparation of drug-loaded liquid metal nanoprobes.

Figure 2 is a schematic diagram of the photothermal therapy-chemotherapy combined treatment process of drug-loaded liquid metal nanoprobes at the tumor site,

Figure 3 shows the experimental results of photothermal therapy-chemotherapy combined therapy for tumor treatment.

Reference number:

Among them, 1- liquid metal, 2- tannic acid, 3- cell crusher, 4- doxorubicin hydrochloride, 5- magnetic stirrer, 6- drug-loaded liquid metal nanoprobe, 7- laser.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, but are not used to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. The experimental reagents can be obtained through commercial channels unless otherwise specified, and the experimental methods are conventional experimental methods unless otherwise specified.

Example 1: Preparation of Liquid Metal Nanoparticles

The preparation process of liquid metal nanoparticles is shown in Figure 1, which specifically includes the following steps:

(1) Dissolve 200 mg of tannic acid powder in 20 mL of water, and uniformly disperse it into a 10 mg/mL TA solution. Put 100 mg of liquid metal (Ga ₆₈ In ₂₂ Sn ₁₀ ) into a 50 mL sample tube containing 20 mL of tannic acid solution, and use a 6 mmφ probe (Scientz-IID) for 15 min of ultrasonic treatment (power 160 W) by a cell crusher, An ice bath was used to keep the temperature of the sample at 0°C during sonication.

(2) After sonication, the largest particles settle quickly, then the supernatant is removed from the vial and further purified by gentle centrifugation (1,000 rpm) to remove micron-sized particles.

(3) Take the supernatant for transmission microscopy to characterize its morphological characteristics. It can be known from the experimental results that the liquid metal nanoparticles prepared in this example have a statistical average particle diameter of 82 nm (N=100).

(4) The precipitate was washed three times with water and dissolved in 1 mL of water to obtain a liquid metal nanoparticle solution for photoacoustic and photothermal testing. It can be seen from the experimental results that the liquid metal nanoparticles prepared in this example have a relative value of photoacoustic signal of 1365 (a.u.) and a photothermal conversion efficiency of 55%.

Example 2: Preparation of Liquid Metal Nanoparticles

The preparation process of liquid metal nanoparticles is shown in Figure 1, which specifically includes the following steps:

(1) The difference from Example 1 is that the concentration of the tannic acid solution involved in ultrasound is 5 mg/mL.

(2) The supernatant was taken for transmission microscopy to characterize its morphological characteristics. It can be known from the experimental results that the liquid metal nanoparticles prepared in this example have a statistical average particle diameter of 48 nm (N=100).

(3) The precipitate was washed three times with water and dissolved in 1 mL of water to obtain a liquid metal nanoparticle solution for photoacoustic and photothermal testing. It can be seen from the experimental results that the relative value of the photoacoustic signal of the liquid metal nanoparticles prepared in this example reaches 643 (a.u.), and the photothermal conversion efficiency is 48%.

Example 3: Preparation of Liquid Metal Nanoparticles

The preparation process of liquid metal nanoparticles is shown in Figure 1, which specifically includes the following steps:

(1) The difference from Example 1 is that the concentration of the tannic acid solution involved in ultrasound is 15 mg/mL.

(2) Take the supernatant for transmission microscopy to characterize its morphological characteristics. It can be seen from the experimental results that the liquid metal nanoparticles prepared in this example have a statistical average particle diameter of 53 nm (N=100).

(3) The precipitate was washed three times with water and dissolved in 1 mL of water to obtain a liquid metal nanoparticle solution for photoacoustic and photothermal testing. It can be seen from the experimental results that the relative value of the photoacoustic signal of the liquid metal nanoparticles prepared in this example reaches 718 (a.u.), and the photothermal conversion efficiency is 47%.

Example 4: Preparation of drug-loaded liquid metal nanoprobes

The preparation process of the drug-loaded liquid metal nanoprobe is shown in Figure 1, which specifically includes the following steps:

(1) Mix 1 mL of liquid metal nanoparticles of Example 1 with a concentration of 500 μg/mL and 1 mL of doxorubicin hydrochloride with a concentration of 10 μg/mL, and incubate at 37° C. for 1 hour.

(2) By centrifuging at 8000 r/min for 30 min, after removing the supernatant, 1 mL of water was added to obtain a drug-loaded liquid metal nanoprobe solution.

Example 5: Biocompatibility of Liquid Metal Nanoparticles

The cell verification experiment was conducted in accordance with the relevant provisions of the National Standard of the People's Republic of China GB/T16886.5 (Biological Evaluation of Medical Devices: In Vitro Cytotoxicity Test) and the International Standard for Biological Evaluation of Medical Devices ISO10993-5, using mouse breast cancer cells 4T1 as the research object , the in vitro biosafety evaluation of the drug-loaded liquid metal nanoprobe in Example 4 was carried out.

Take 1 mL of the liquid metal nanoparticle solution of Example 1 for UV sterilization for 4 hours, add RPMI-1640 basal medium (containing 10% fetal bovine serum), and prepare the experimental group culture with concentrations of 100, 200, 300, 400, and 500 μg/mL, respectively. base. Select vigorously growing 4T1 cells and inoculate about 50,000 cells/well in a 96-well plate, add 100 μL of the experimental group medium to each well of the experimental group, add 100 μL of complete medium to each well of the control group, and add only complete medium without cells in the blank group. culture medium. After being placed in a 37°C, 5% CO ₂ incubator for 6 hours, 10 μL of CK8 reagent was added, and after 4 hours of incubation, the absorbance of each well solution at 450 nm was measured with a microplate reader. And according to the calculation formula: cell survival rate=[(A experimental group-A blank group)/(A control group-A blank group)]×100% to calculate the cell survival rate. After calculation, using liquid metal nanoparticles co-cultured with 4T1 cells, the cell viability did not change significantly with the increase of concentration, which indicated that liquid metal nanoparticles had no obvious cytotoxicity and had good biological safety.

Example 6: Use of drug-loaded liquid metal nanoprobes for photoacoustic imaging diagnosis of tumors

0.1 ml of mouse breast cancer 4T1 cells with a density of 10 ⁷ were subcutaneously injected into the right groin of Balb/c nude mice. When the tumor volume reached about 100 mm ³ , it could be used for in vivo experiments. The drug-loaded liquid metal nanoprobes of Example 4 were injected into the tumor in situ, and the photoacoustic images of the tumor at 808 nm were measured at 0, 6, 12, and 24 hours after injection, respectively.

Example 7: Drug-loaded liquid metal nanoprobes for photothermal therapy-chemotherapy combined therapy of tumors

The drug-loaded liquid metal nanoprobe of Example 4 was used for the tumor treatment, as shown in FIG. 2 . In situ injection of drug-loaded liquid metal nanoprobes into tumors. 24 hours after injection, the tumor site was irradiated with an 808 nm laser for 10 minutes. After laser irradiation, the drug-loaded liquid metal nanoprobes would swell and deform, and tannic acid and doxorubicin would be released from the surface of the nanoprobe, and tannic acid would enhance doxorubicin. The killing effect of breast cancer cells, photothermal therapy and chemotherapy combined treatment, two-pronged approach to eliminate tumors.

The above tumor sites were injected with drug-loaded liquid metal nanoprobes for photothermal therapy-chemotherapy combined treatment group, while the untreated tumor was used as a blank control group, and the tumor site was injected with liquid metal nanoparticles of Example 1 as a hyperthermia group. The experimental results are shown in Figure 3. The joint effect of hyperthermia and chemotherapy can kill tumor cells more effectively, and realize the combination of chemotherapy and hyperthermia to treat tumors.

To sum up, the present invention has the following beneficial effects:

1. Tannic acid-modified liquid metal nanoprobes were synthesized by one-step ultrasonic method using tannic acid.

2. The liquid metal nanometer is realized and the surface of the liquid metal is modified. The drug-loaded liquid metal nanoprobe can be prepared by simply mixing doxorubicin hydrochloride and the above-mentioned liquid metal nanoparticles.

3. The synthesis process is simple, the cost is low, the efficiency is high, and it has good dispersibility and stability in water, and has good biocompatibility.

4. It can not only realize the diagnosis of tumor by photoacoustic imaging, but also perform the combined treatment of photothermal therapy and chemotherapy for the tumor.

Claims (6)

1. A preparation method of a liquid metal nanoprobe integrating light-sound imaging and drug inclusion is characterized by comprising the following steps:

mixing liquid metal (1) with a tannin (2) aqueous solution, performing ultrasonic treatment, taking upper-layer liquid, and then removing micron-sized particles through mild centrifugal purification to obtain liquid metal nanoparticles; the liquid metal (1) is a gallium-based alloy containing 68 wt% of gallium, 22 wt% of indium and 10 wt% of tin; the diameter of the liquid metal nanoparticles is 15nm-150 nm; the concentration ratio of the aqueous solution of the liquid metal (1) to the aqueous solution of the tannic acid (2) is 1:2, a 6mm phi probe with the model of Scientz-IID is used, ultrasonic treatment is carried out for 15min by a cell disruptor (3), the power is set to be 160W, and an ice bath is used in the ultrasonic treatment process to ensure that the temperature of a sample is controlled at 0 ℃;

step two, mixing the medicinal preparation with the liquid metal nanoparticles in the step one to obtain a medicine-carrying liquid metal nanoprobe (6); the pharmaceutical preparation is doxorubicin hydrochloride (4), and the doxorubicin hydrochloride and liquid metal nanoparticles are mixed overnight at normal temperature by a magnetic stirrer (5).

2. A drug-loaded liquid metal nanoprobe, which is prepared by the preparation method of claim 1.

3. The drug-loaded liquid metal nanoprobe of claim 2, which is used for photoacoustic imaging of breast cancer.

4. The drug-loaded liquid metal nanoprobe of claim 3, wherein photoacoustic imaging is performed under laser excitation at a wavelength of 808 nm.

5. The drug-loaded liquid metal nanoprobe of claim 2, which is used for combined photothermal therapy-chemotherapy treatment of breast cancer.

6. The drug-loaded liquid metal nanoprobe of claim 5, which generates thermal expansion deformation under the laser irradiation with wavelength of 808nm and power of 1W to promote drug release.

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