CN112007168A - Hyaluronidase-modified layered double-metal hydroxide hybrid nano platform and preparation and application thereof - Google Patents

Abstract

The invention relates to a hyaluronidase-modified layered double metal hydroxide hybrid nano-platform and its preparation and application. The layered double metal hydroxide nanoparticles are synthesized by a co-precipitation method, and the hydrogen bond and electrostatic interaction are used to synthesize the nanometer particles. The hyaluronidase is modified on its surface, and the antitumor drug doxorubicin is loaded. The hyaluronidase-modified LDH nanomaterial prepared by the invention has the characteristics of uniform particle size, good stability and biocompatibility, pH response and the like.

Description

A hyaluronidase-modified layered double metal hydroxide hybrid nanoplatform and its preparation and application

technical field

The invention belongs to the field of nanocomposite materials and their preparation and application, in particular to a layered double metal hydroxide hybrid nanoplatform modified by hyaluronidase and its preparation and application.

Background technique

Pancreatic cancer is a common lethal malignancy with strong invasiveness and intractability, often invading surrounding stromal components, including lymphatic, vascular, and nervous systems, and eventually metastasizing to distant organs. Despite recent advances in the clinical treatment of cancer, pancreatic cancer survival remains the lowest of all cancer types, with a 5-year survival rate of only 8% in the United States. The annual incidence of pancreatic cancer in my country is 5.1/100,000, a three-fold increase compared with 20 years ago, and the incidence in some cities is close to the level of western developed countries. Therefore, it is very necessary and urgent to improve the diagnosis and treatment of pancreatic cancer.

The traditional treatment methods for pancreatic cancer include surgical resection, radiotherapy and chemotherapy. Although clinical surgical treatment has made significant progress in recent years, only about 15% of patients are clinically able to perform surgical resection, and most patients actually benefit only for a few months. In addition, most patients are found at an advanced stage, and often have local spread or distant metastases that cannot be surgically removed. Chemotherapy still plays a dominant role in adjuvant and neoadjuvant treatment of pancreatic cancer based on high-quality data from randomized controlled trials over the past few decades. In recent decades, the emergence of nanotechnology has had a profound impact on the diagnosis and treatment of tumors. Compared with conventional chemotherapeutic drugs, nano-drug systems constructed with nano-materials loaded with drugs can make use of the enhanced permeability and retention (EPR) effects of solid tumors to make nano-drugs more inclined to aggregate in blood vessels rich, The tumor tissue with wide vascular wall gap and poor structural integrity can reduce the intake of normal tissue with dense microvascular endothelial gap and complete structure, so it can reduce the damage to normal cells and improve the effect of tumor treatment.

At present, the commonly used nanocarriers in nanomedicine include organic types such as dendrimers, nanohydrogels, liposomes, proteins, etc., and inorganic types include mesoporous silica, gold nanoparticles, and layered double metal hydroxides. (LDH) et al. Among them, LDH, as an inorganic nanomaterial, has the characteristics of low synthesis cost, simple method and high transfer efficiency. Applying it to the drug delivery system has the following advantages: 1) good biocompatibility, low toxicity and degradability, almost no adverse reactions after oral administration or injection; 2) high drug loading capacity, forming different types of The drug/biomolecule-LDH nanocomposite; 3) has the characteristics of acid response, combined with the physiological characteristics of acidic pH in the tumor, which is conducive to the release of drugs at the lesion site, thereby reducing the side effects of chemotherapy drugs.

Studies have found that pancreatic cancer cells interact with their surrounding fibroblasts, immune cells and endothelial cells to have a strong desmoplastic response, forming a dense and complex extracellular matrix (ECM), which makes pancreatic cancer form dense The tumor microenvironment has extremely high tumor interstitial fluid pressures (IFPs). Higher tumor interstitial fluid pressures (IFPs) resist the penetration of chemotherapeutic drugs, thereby exhibiting drug resistance and reducing the efficacy of chemotherapeutic drugs. The extracellular matrix (ECM) contains collagen, fibronectin, laminin, and hyaluronic acid. Among them, hyaluronic acid (HA) is a key component of the extracellular matrix (ECM), consisting of repeats of N-acetylglucosamine and glucuronic acid disaccharide units, providing a hydrogel-like matrix to enhance tumor interstitial fluid pressure and support Tumor growth, which resists drug penetration.

CN 110013559 A discloses a HA-targeted double metal hydroxide-ultra-small iron nanomaterial and its preparation and application. Directly modifying hyaluronidase on LDH and maintaining its enzymatic activity can simplify the treatment process and achieve the therapeutic effect while reducing the number of injections.

Searching domestic and foreign literatures, there is no research report on the preparation of hyaluronidase-modified LDH nanoplatforms and their loading with chemotherapeutic drugs to explore the effect of tumor chemotherapy. Based on the above background, the invention of hyaluronidase-modified layered double metal hydroxide hybrid nanoplatform is of great significance and potential for clinical translation.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a hyaluronidase-modified layered double-metal hydroxide hybrid nano-platform and its preparation and application, which can directly modify the hyaluronidase on LDH and maintain enzymatic activity.

A layered double metal hydroxide composite nanomaterial of the present invention, the nanomaterial is a layered double metal hydroxide with surface modified hyaluronidase.

Further, the hyaluronidase is modified by hydrogen bonding and electrostatic interaction on the surface of the layered double metal hydroxide.

The mass ratio of the hyaluronidase to the layered double metal hydroxide is 0.8-1.2:0.8-1.2.

A preparation method of a layered double metal hydroxide composite nanomaterial of the present invention comprises:

Mix the aqueous solution of hyaluronidase (HAase) and the aqueous solution of layered double hydroxides (LDH), stir in the dark for 3-6 h, collect, wash and freeze-dry to obtain layered double hydroxides (LDH). Metal hydroxide composite nanomaterials (LDH-HAase).

The preferred mode of above-mentioned preparation method is as follows:

Further, the hyaluronidase (HAase) was dissolved in water, added to the aqueous solution of the layered double metal hydroxide LDH, stirred for 6 h under dark conditions, collected, washed and freeze-dried to obtain the layered double metal hydroxide. Hydroxide composite nanomaterials, namely hyaluronidase-modified layered double metal hydroxide nanomaterials (LDH-HAase).

The layered double metal hydroxide is prepared by the following method: stirring the two metal salt solutions, adding alkali to the mixed metal salt solutions to adjust the pH of the solution to obtain a milky white solution, continuing to stir at room temperature, aging, and The obtained solution is collected by centrifugation, washed and freeze-dried to obtain a layered double metal hydroxide; wherein the freeze-drying time is 24-48h.

Further, two kinds of metal salt solutions are added to the three-necked flask and continuously stirred, and alkali is added to adjust the pH of the solution, and stirring is continued at room temperature. oxide (LDH).

The two metal salt solutions are mixed aqueous solutions of Mg(NO ₃ ) ₂ .6H ₂ O and Al(NO ₃ ) ₃ .9H ₂ O, Mg(NO ₃ ) ₂ .6H ₂ O and Al(NO ₃ ) ₃ ·The molar concentration ratio of 9H ₂ O is 0.07～0.11:0.043～0.047; the solvent of the solution is ultrapure water with CO ₂ removed.

Further, two metal salts, Mg(NO ₃ ) ₂ .6H ₂ O and Al(NO ₃ ) ₃ .9H ₂ O, were dissolved in the solvent, continuously stirred in a three-necked flask, and added dropwise to the mixed metal salt solution. The pH was adjusted with NaOH solution to obtain a milky white solution, and the whole process was carried out under the protection of nitrogen, centrifuged and washed to remove free metal ions, and freeze-dried to obtain LDH.

The pH of the solution adjusted by adding the alkali is as follows: the alkali added dropwise is a 1M NaOH solution, the dropping rate is 0.5 mL/min, and the final pH value of the adjusted solution is 9.3-9.5; the whole process of preparing the layered double metal hydroxide Carry out under nitrogen protection; the aging time is 24-48h.

The mass ratio of the layered double metal hydroxide to the hyaluronidase is 0.8-1.2:0.8-1.2; the solvent of the solution is ultrapure water from which CO ₂ is removed.

Further, the mass ratio of the layered double metal hydroxide LDH and the hyaluronidase HAase is 1:1.

The present invention provides a drug-loaded nanomaterial, which is the layered double metal hydroxide composite nanomaterial of claim 1 as a carrier to load a drug.

A preparation method of a drug-loaded nanomaterial of the present invention comprises: adding an anticancer drug such as an aqueous solution of doxorubicin DOX to the aqueous solution of the layered double metal hydroxide composite nanomaterial, and stirring for 12-24 hours in the dark; The drug-loaded nanomaterials (such as DOX@LDH-HAase) can be obtained by centrifugal collection, washing, and freeze-drying; wherein the solvent water is ultrapure water with CO ₂ removed.

Further, the mass ratio of the layered double metal hydroxide composite nanomaterial LDH-HAase and the anticancer drug DOX is 1:1.

In the steps of centrifugation collection and washing, centrifugation was carried out at 4°C and 8000 r/min for 10 minutes.

The invention provides an application of the drug-carrying nanomaterial in the preparation of tumor chemotherapy drugs.

The invention utilizes a co-precipitation method to synthesize LDH nanomaterials with uniform particle size, then modifies hyaluronidase (HAase) through hydrogen bonds and electrostatic interactions to form hybrid nanomaterials (LDH-HAase), and finally loads anticancer drug DOX, Drug-loaded nanomaterials (DOX@LDH-HAase) were formed.

The present invention firstly synthesizes LDH by co-precipitation method, and its surface contains a large number of hydroxyl groups and is negatively charged, and the amino and carboxyl groups contained in hyaluronidase can utilize hydrogen bond and electrostatic interaction to modify hyaluronidase, and finally load DOX. The present invention can hydrolyze the hyaluronic acid in the extra-tumor matrix to improve the tumor penetration effect of the material and thus improve its chemotherapy efficiency. The invention uses ultraviolet absorption spectrometer (double metal hydroxide UV-Vis), infrared absorption spectrometer (FT-IR), X-ray crystal diffraction analysis, field emission scanning electron microscope (SEM), transmission electron microscope (TEM) and other technologies to analyze The synthesized drug-loaded nanomaterial DOX@LDH-HAase was subjected to corresponding physicochemical characterization, and the cytotoxicity of nanocomposite LDH and LDH-HAase and the inhibitory effect of DOX@LDH-HAase at the two-dimensional cell level were evaluated by CCK-8 experiment. The phagocytosis of the drug-loaded nanomaterial DOX@LDH-HAase by cells was qualitatively detected by laser confocal microscopy, and then the penetration and anti-tumor effects of DOX@LDH-HAase were explored by 3D cell spheroids of SW1990 cells, and finally established in nude mice. A subcutaneous tumor model of pancreatic cancer to explore its antitumor effect in vivo.

beneficial effect

(1) The synthesis process of the present invention is simple, the preparation period is short, the raw materials are abundant and the price is low, and the invention has the prospect of industrialized production.

(2) The hybrid nanomaterial LDH-HAase prepared by the present invention has good biocompatibility, and has the characteristics of pH-responsive release, which can control the release of drugs by utilizing the acidity in tumor cells and reduce the side effects of drugs.

(3) The present invention modifies hyaluronidase on the surface of LDH and loads anticancer drugs for the first time, thereby improving its chemotherapy effect by changing the tumor microenvironment, and providing a new strategy for clinical treatment of tumors.

(4) The nanomaterial prepared by the present invention has the function of degrading the abundant hyaluronic acid in the tumor extracellular matrix, regulating the tumor microenvironment, thereby enhancing the therapeutic effect of chemotherapeutic drugs, and has great clinical transformation and application potential in tumor therapy.

Description of drawings

1 is a schematic diagram of a synthetic method of a hyaluronidase-modified LDH nanoplatform provided by the present invention;

Figure 2 (a), (b) are respectively the SEM image and TEM image of DOX@LDH-HAase prepared by the present invention;

In Fig. 3 (a) is the UV absorption spectrum of HAase alone and LDH and LDH-HAase prepared by the present invention at 240-800 nm; (b) is the UV absorption spectrum of DOX alone and LDH-HAase prepared by the present invention, DOX@LDH-HAase at 240 nm -800nm UV absorption spectrum;

Fig. 4 is the infrared absorption spectrum of LDH, LDH@HAase, DOX@LDH-HAase prepared by the present invention;

Fig. 5 is the X-ray crystal diffraction pattern of LDH, LDH@HAase, DOX@LDH-HAase prepared by the present invention;

Fig. 6 is the cumulative release curve of DOX in phosphate buffer solution of pH=7.4 and 5.8 in DOX@LDH-HAase prepared by the present invention;

Fig. 7 is the cytotoxicity test result of LDH and LDH@HAase prepared by the present invention;

Figure 8 is the in vitro anti-tumor activity test results of DOX alone and DOX@LDH-HAase prepared by the present invention at 24h;

Fig. 9 is the qualitative analysis of the phagocytosis of DOX@LDH-HAase prepared by the present invention and DOX@LDH of comparative example respectively incubated with SW1990 cells for 4 hours;

Fig. 10 is the drug penetration situation of DOX@LDH-HAase prepared by the present invention and comparative example DOX@LDH incubated with 3D cell spheroids for 6 hours;

Figure 11 is a quantitative analysis of the penetration of DOX@LDH-HAase prepared by the present invention and comparative example DOX@LDH and 3D cell spheroids incubated for 6 hours;

Figure 12 shows the inhibition of SW1990 cells to form 3D spheroids by DOX@LDH-HAase, comparative example DOX@LDH, DOX alone and control group prepared by the present invention;

Figure 13 shows the intratumoral injection of normal saline (NS), LDH-HAase, DOX (5 mg/kg, 100 μL), DOX@LDH (5 mg/kg, 100 μL) and DOX@LDH-HAase (5 mg/kg, 100 μL) according to the present invention ), changes in the relative volume of mouse tumors within 14 days;

Figure 14 shows that after intratumoral injection of NS, LDH-HAase, DOX (5 mg/kg, 100 μL), DOX@LDH (5 mg/kg, 100 μL) and DOX@LDH-HAase (5 mg/kg, 100 μL), Body weight change chart of mice within 14 days;

FIG. 15 is the SEM image and the TEM image of the comparative example DOX@LDH.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the application.

Reagent name source Magnesium nitrate hexahydrate (Mg(NO₃)₂·6H₂O) Sinopharm Group Chemical Reagent Co., Ltd. Aluminum nitrate nonahydrate (Al(NO₃)₃·9H₂O) Sinopharm Group Chemical Reagent Co., Ltd. Sodium Hydroxide (NaOH) Sinopharm Group Chemical Reagent Co., Ltd. Hyaluronidase (HAase) Beijing Soleibo Technology Co., Ltd. Doxorubicin (DOX) Beijing Huafeng Technology Co., Ltd. RPMI-1640 Medium Hangzhou Jinuo Biomedical Technology Co., Ltd. penicillin-streptomycin Hangzhou Jinuo Biomedical Technology Co., Ltd. Trypsin 0.25% solution Hangzhou Jinuo Biomedical Technology Co., Ltd. Cell Counting Kit-8 (CCK-8) Shanghai Biyuntian Biotechnology Co., Ltd. nude mice Shanghai Slack Laboratory Animal Center

Example 1

(1) Weigh out Mg(NO ₃ ) ₂ ·6H ₂ O (0.09M) and Al(NO ₃ ) ₃ ·9H ₂ O (0.045M) and dissolve them in 50 mL of ultrapure water with decarbonated carbon dioxide. The pump slowly added 1M NaOH solution dropwise at a speed of 0.5mL/min until the pH value was 9.5±0.2. At this time, a milky white solution was seen. The white solution was continued to be stirred and aged at room temperature for 48h. The synthesis and precipitation process The samples were collected by centrifugation at 8000 r/min for 10 min, washed to remove the two free salt ions, and the final product was lyophilized to obtain LDH for relevant characterization and subsequent experiments.

(2) Synthesize LDH-HAase with hyaluronidase (HAase) and layered double metal hydroxide (LDH) in a mass ratio of 1:1, that is, dissolve 16.5 mg of hyaluronidase (HAase) in 5 mL of ultrapure Water, added dropwise to 10 mL of LDH (concentration 1.65 mg/mL) aqueous solution, stirred for 6 h in the dark, centrifuged at 8000 r/min for 10 min and washed 3 times, lyophilized to collect the product LDH-HAase.

(3) The LDH-HAase and doxorubicin hydrochloride (DOX) feeding mass ratio is 1:1 to synthesize DOX@LDH-HAase, that is, 18.6 mg doxorubicin (DOX) is dissolved in 5 mL ultrapure water, and added dropwise to In 10 mL of LDH-HAase (concentration 1.86 mg/mL) aqueous solution, stirred in the dark for 24 h, centrifuged at 8000 r/min for 10 min, collected and washed the product 3 times, and finally lyophilized to obtain solid DOX@LDH-HAase.

Example 2

Physical and chemical characterization of the materials prepared in Example 1: The samples of LDH, LDH-HAase and DOX@LDH-HAase prepared in Example 1 were prepared into 0.5 mg/mL aqueous solutions, and their ultraviolet absorption spectra were measured with an ultraviolet spectrophotometer; The DOX@LDH-HAase solution with a concentration of 0.1 mg/mL was dropped on tin foil and dried with a gun tip. The sample was attached to the SEM stage, sprayed with gold, and its morphology was observed with a Hitachi S400 scanning electron microscope. The DOX@LDH-HAase aqueous solution with a concentration of 0.1 mg/mL was dropped on the copper grid for transmission electron microscopy, and the morphology was observed using a Japanese JEOL electron microscope. Take 0.1 mg of the lyophilized powder, and measure its infrared absorption spectrum with a KBr tablet method using a Fourier transform infrared spectrometer (NEXUS-670). Take 5 mg of the lyophilized solid powder to examine its crystal structure by X-ray diffractometer. As shown in Fig. 2(a) SEM image and Fig. 2(b) TEM image, the synthesized DOX@LDH-HAase has a hexagonal shape and a particle size of about 90 nm. Figure 3(a) shows that HAase has an absorption peak at 280 nm. Compared with LDH alone, LDH-HAase has a UV absorption peak at 280 nm, which proves that hyaluronidase is successfully modified on LDH. As can be seen from Figure 3(b), DOX@LDH-HAase has an obvious UV absorption peak at 490 nm, which proves that DOX is successfully loaded onto LDH-HAase to form a nanocomposite DOX@LDH-HAase.

As shown in Figure 4, it is shown that LDH has infrared absorption at 400-800cm ^-1 , and the peak in this part is caused by the vibration of the bond formed between the metal and oxygen atoms; 1384cm ^-1 is the absorption peak caused by NO ₃ ^- vibration; 3454cm ^-1 and 1632cm ^-1 are absorption peaks caused by interlayer and surface water molecules and hydroxyl groups, these characteristic peaks indicate the successful synthesis of LDH. After modification of HAase, an absorption peak appeared at 3735cm ^-1 , which proved that HAase was successfully modified to LDH. After loading with antitumor drugs, the change of its absorption peaks at 1384 cm ^-1 and 1083 cm ^-1 proves the successful loading of DOX, which is consistent with other experimental results above. The X-ray crystal diffraction patterns of LDH, LDH@HAase and DOX@LDH-HAase in Figure 5 show that the peaks of the three nanomaterials 003, 006 and 009 are sharp and narrow, and the peak shapes are consistent with those reported in the literature, indicating that the synthesized LDH The crystal form is complete, and its crystal structure does not change after modifying HAase and loading DOX.

Example 3

The material DOX@LDH-HAase obtained in Example 1 was dissolved into a solution with a concentration of 1 mg/mL with a phosphate buffer of pH=7.4 and pH=5.8, respectively, and 1 mL was put into a dialysis bag for fixation, and placed in a 9mL containing solution. 50mL centrifuge tubes of different pH phosphate buffers were placed in a shaker at 37°C. Samples were taken at fixed time points. Take 1 mL of the liquid outside the dialysis bag each time, and then add 1 mL of the corresponding buffer solution to the outside of the dialysis bag to keep the total volume unchanged, and measure its absorbance at 490 nm. The DOX release was calculated from the absorbance value, and the DOX release curves of the nanocomposite DOX@LDH-HAase under different pH conditions in vitro were obtained by plotting. As shown in Figure 6, it can be seen that the nanocomposite DOX@LDH-HAase released 43% of the amount in 6 hours under acidic conditions (pH=5.8) in a tumor-like environment, while in neutral environment (pH=7.4) , only 9% was released in 6 hours, and the release was slow, which proved that DOX@LDH-HAase had pH-responsive drug release behavior. This property facilitates the release of chemotherapeutic drugs at the tumor site, thereby reducing the damage of antitumor drugs to normal tissues.

Example 4

Hyaluronidase can degrade hyaluronic acid into disaccharides, monosaccharides and smaller hyaluronic acid fragments, so the enzymatic activity of DOX@LDH-HAase can be measured by turbidimetry. The reagents required for the experiment were prepared according to the protocol provided by Sigma Aldrich (https://www.sigmaaldrich.com/china-mainland/technical-documents/protocols/biology/enzymatic-assay-of-hyaluronidase.html). The specific experimental steps are as follows:

(1) Add the reagent to the test tube according to the amount in the following table;

(2) Add 1 mL of hyaluronic acid solution with a concentration of 0.3 mg/mL to the upper test tube respectively;

(3) Shake and mix, incubate at 37°C for 45 minutes;

(4) 0.5mL of each test substance and blank group were transferred to a centrifuge tube containing 2.5mL of acidic albumin solution and mixed, transferred to a cuvette and left to stand for 10 minutes;

(5) measure the % transmittance at 600nm;

(6) Calculate according to the following algorithm;

in:

df = dilution factor of enzyme

14.84 = extinction coefficient determined by Sigma-Aldrich

mL of enzyme solution = volume of enzyme solution used in the reaction

The calculated enzymatic activity of DOX@LDH-HAase is shown in Table 1. Compared with HAase alone, DOX@LDH-HAase maintained 86% of the enzymatic activity.

Table 1. Viability comparison of DOX@LDH-HAase and HAase.

Example 5

Using SW1990 cells as a model, the cytocompatibility of nanomaterials LDH and LDH-HAase was evaluated by CCK-8 method. SW1990 cells at 1×10 ⁴ /well were seeded in a 96-well plate and cultured in a 37°C, 5% CO ₂ incubator for 24 h. Pour off the old medium and re-add 100μL of medium solution containing different concentrations of LDH, LDH-HAase (0, 1, 2.5, 5, 10, 25, 50, 100, 100, 250, 500μg/mL) to the 96-well plate In each group, six parallel wells were set up and cultured for 24 h. The medium was poured out, washed three times with PBS, and then 100 μL of fresh medium containing 10% CCK-8 was added to the well plate, and incubated for 4 h in the incubator. Finally, the absorbance value of each well at 450 nm was measured by a microplate reader, and the average cell viability was calculated based on the measured absorbance value with the PBS group as the blank group.

The in vitro antitumor effect of nanomaterial DOX@LDH-HAase was evaluated by CCK-8 method, and the steps were the same as above. The medium containing LDH, LDH-HAase was replaced with 100 μL medium containing DOX@LDH-HAase at different concentrations (0, 0.5, 1, 2.5, 5, 10, 25, 50 μg/mL) only during the incubation step. In a 96-well plate, the subsequent steps are the same as above.

As can be seen from the results in Figure 7, when the LDH concentration reached 500 μg/mL, LDH and LDH-HAase were incubated with SW1900 cells for 24 hours, and their cell viability remained above 90%, which proved that the synthesized LDH and LDH-HAase were less toxic. Has good cytocompatibility. The 24-h in vitro anti-tumor experiments are shown in Figure 8. With the increase of DOX concentration, the anti-tumor effects of DOX@LDH-HAase and the comparative example DOX@LDH gradually increased, and DOX@LDH-HAase was more effective than DOX@LDH. Strong tumor suppressor effect.

Example 6

The phagocytic effect of SW1990 on DOX@LDH-HAase and DOX@LDH was observed by laser confocal microscopy, and the cells were seeded in confocal culture dishes at a density of 1 × 10 ⁵ SW1990 cells per well at 37 °C, 5% CO ₂ . Incubate overnight in an incubator, then pour out the original medium, and re-add 1 mL of medium containing DOX@LDH-HAase and DOX@LDH nanocomplexes, with a DOX concentration of 2 μg/mL, for 4 h. Washed 3 times with PBS solution, fixed with 2.5% glutaraldehyde for 15 min, washed 3 times with PBS solution, and stained with DAPI for 10 min. Finally, the phagocytosis of nanocomplexes by cells was observed by laser confocal microscope. As shown in Figure 9, the fluorescence intensity of DOX@LDH-HAase group was stronger than that of DOX@LDH group, indicating that HAase hydrolyzed the hyaluronic acid around tumor cells, which was beneficial for tumor cells to phagocytose nanomaterials.

Example 7

Using 3D spheroids to simulate solid tumors to explore the penetration and anti-tumor conditions of DOX@LDH-HAase and DOX@LDH. First, wash the Microtissue template (purchased from Sigma-Aldrich) with sterile water, and sterilize the template; weigh 1 g of agarose powder, sterilize it with high temperature and high pressure in an autoclave, and add 50 mL of Bacteria physiological saline, heat to dissolve the agarose completely; after the agarose is cooled to 60-70 °C, use a pipette to suck 550 μL of agarose solution, and slowly hit the template, if bubbles are generated, use a pipette to remove the bubbles Scrape off slowly; after the agarose is cooled and solidified in the template, slowly remove the solidified 3D Petri Dish from the template and place it in a 12-well plate; take 2.5 mL of medium and add it to the 12-well plate with 3D Petri Dish; Soak for 15min, suck out the medium and repeat the operation again. Aspirate the medium in and around the wells of 3D Petri Dish, and slowly drop 190 μL of the cell suspension containing ⁵ x 105 SW1900 cells into the inner wells of the 3D Petri Dish agar block. After standing for 10 min, 2.5 mL of medium was slowly added to each well, and then the 12-well plate was placed in a 37° C., 5% CO ₂ incubator for 3 days to form 3D cell spheres.

In the permeation experiments exploring DOX@LDH-HAase and DOX@LDH, the medium inside and around the 3D Petri Dish groove was poured out, and fresh medium containing DOX@LDH-HAase and DOX@LDH was added, respectively, at the concentration of DOX. Both were 2 μg/mL, incubated for 6 hours, washed three times with PBS, pipetted the cell pellets into the Confocal dish, and detected and analyzed the penetration of DOX by Z-stack scanning with a confocal laser microscope. As shown in Fig. 10, it can be seen that the fluorescence of DOX@LDH group becomes weaker and weaker with the increase of scanning depth. At the depth of 75 μm, there is almost no fluorescence, while DOX@LDH-HAase is still at the depth of 100 μm. There is fluorescence of DOX. The quantitative analysis in Figure 11 is consistent with the above results. The experimental results prove that DOX@LDH-HAase penetrates into 3D cell spheroids more easily, and the reason may be that HAase decomposes hyaluronic acid inside the cell spheroids, which is beneficial to the penetration of DOX@LDH-HAase.

In the experiment to explore the inhibition of DOX@LDH-HAase, DOX@LDH and DOX alone on cell spheroids, the operation steps were the same as above, but the concentration of DOX in each group was 10 μg/mL, and the incubation time was 24 hours and 48 hours, respectively. The state of the 3D spheroids was then observed and analyzed using a phase contrast microscope. The results are shown in Figure 12. With the prolongation of incubation time, except for the control group, the diameters of the DOX, DOX@LDH and DOX@LDH-HAase groups alone gradually became smaller, and the diameter of the 3D cell spheres in the DOX@LDH-HAase group decreased. The smallest is the most, indicating that this group has the best inhibition effect.

Example 8

2×10 ⁶ SW1990 cells were inoculated into the right side of the back of nude mice, and after 2 weeks of feeding, when the tumor volume reached about 100 mm ³ , the pancreatic cancer tumor model was constructed, which could be used for chemotherapy studies in nude mice. Nude mice bearing subcutaneous tumors were randomly divided into 5 groups, 6 mice in each group, recorded as day 0. The specific groups are as follows: NS, LDH-HAase, DOX (5 mg/kg, 100 μL), DOX@LDH (5 mg/kg, 100 μL) and DOX@LDH-HAase (5 mg/kg, 100 μL). The body weight and tumor volume of nude mice were recorded every 2 days, and the drug was injected intratumorally every 2 days for 4 times, and the observation was carried out for 14 days. Plot the relative tumor volume change curve and body weight change curve. The relevant calculation formulas (1) and (2) are as follows:

Tumor volume (V) = a×b ² /2 (1)

Relative tumor volume = V/V ₀ (2)

where a and b represent the maximum and minimum tumor diameters, respectively, and V ₀ is the size of the tumor at the start of treatment. The results of relative tumor volume changes are shown in Figure 13. It can be seen that DOX@LDH-HAase has the slowest growth in tumor volume compared with other groups, and there is a significant difference compared with other groups, indicating that in vivo solid tumor experiments in nude mice DOX@LDH-HAase has a significant inhibitory effect on pancreatic cancer. The body weight changes of mice in each group are shown in Figure 14. Except for the single DOX group, the body weight of other groups increased slightly, while the body weight of the single DOX group decreased slightly, which may be caused by the side effects of the single drug, indicating that DOX@LDH-HAase has a strong effect. The tumor-inhibiting effect also has less drug side effects.

Comparative Example 1

The preparation method of DOX@LDH in the control group includes:

Synthesize DOX@LDH with DOX and LDH at a mass ratio of 1:1, that is, weigh 18.6 mg of doxorubicin (DOX), dissolve it in 5 mL of ultrapure water, and add dropwise to 10 mL of LDH (concentration of 1.86 mg/mL). ) aqueous solution, stirred in the dark for 24 h, centrifuged at 8000 r/min for 10 min, collected and washed the product, and finally lyophilized to obtain solid DOX@LDH. Its characterization method is the same as that of the material prepared in Example 1. The particle size of DOX@LDH is similar to that of DOX@LDH-HAase, which is 90-120 nm as shown in Fig. 15 SEM images (a) and TEM images (b).

Claims (10)

1. A layered double metal hydroxide composite nanomaterial, characterized in that, the nanomaterial is a layered double metal hydroxide of surface modified hyaluronidase. 2 . The nanomaterial according to claim 1 , wherein the mass ratio of the hyaluronidase to the layered double metal hydroxide is 0.8-1.2:0.8-1.2. 3 . 3. A preparation method of a layered double metal hydroxide composite nanomaterial, comprising: The aqueous solution of the hyaluronidase and the aqueous solution of the layered double metal hydroxide are mixed, stirred for 3-6 hours in the dark, collected, washed and freeze-dried to obtain the layered double metal hydroxide composite nanomaterial. 4. preparation method according to claim 3 is characterized in that, described layered double metal hydroxide is prepared by following method: two kinds of metal salt solutions are stirred, add alkali to adjust pH of solution, continue stirring at room temperature, After aging, the obtained solution is collected by centrifugation, washed, and freeze-dried to obtain a layered double metal hydroxide. 5 . The preparation method according to claim 4 , wherein the two metal salt solutions are mixed aqueous solutions of Mg(NO ₃ ) ₂ .6H ₂ O and Al(NO ₃ ) ₃ .9H ₂ O, and Mg( The molar concentration ratio of NO ₃ ) ₂ ·6H ₂ O and Al(NO ₃ ) ₃ ·9H ₂ O is 0.07-0.11:0.043-0.047; the solvent of the solution is ultrapure water with CO ₂ removed. 6. The preparation method according to claim 4, characterized in that, said adding alkali to adjust the pH of the solution is: the alkali added dropwise is a 1M NaOH solution, and the final pH value of the adjusted solution is 9.3 to 9.5; the layered bimetallic The whole process of the preparation of hydroxide is carried out under nitrogen protection; the aging time is 24-48h. 7. The preparation method according to claim 3, wherein the mass ratio of the layered double metal hydroxide to the hyaluronidase is 0.8-1.2:0.8-1.2; the solvent of the solution is removed _CO2 ultrapure water. 8 . A drug-loaded nanomaterial, characterized in that, the material is a drug-loaded drug-loaded layered double metal hydroxide composite nanomaterial according to claim 1 . 9. A preparation method of a drug-loaded nanomaterial, comprising: adding an anticancer drug aqueous solution to the aqueous solution of the layered double metal hydroxide composite nanomaterial according to claim 1, stirring for 12-24h in the dark, centrifugally collecting, After washing and lyophilization, the drug-loaded nanomaterials can be obtained. 10. An application of the drug-loaded nanomaterial according to claim 8 in the preparation of tumor chemotherapy drugs.

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