CN116473928B - Preparation method and application of arginine/calcium phosphate nanoparticles - Google Patents

Abstract

The invention discloses a preparation method and application of arginine/calcium phosphate nano particles, comprising the following steps: 1) L-arginine, caCl ₂ and deionized water, and magnetically stirring until the solution is clear and transparent; 2) Slowly dropwise adding 30-60 mL of isopropanol into the solution obtained in the step 1) under magnetic stirring, and stirring at room temperature for 1-2 h. 3) Adding Na ₂HPO₄ solution into the solution obtained in the step 2) under magnetic stirring, and stirring and reacting for 12-24 hours under the condition of 25-30 and ^o ℃. 4) Centrifuging and washing the mixed solution obtained in the step 3), and freeze-drying the precipitate at-50 ^o ℃ to obtain the arginine/calcium phosphate (L-Arg-Cap) nano-particles. The product has uniform particle size and good biocompatibility, has pH response capability, can inhibit the discharge of calcium ions while providing a large amount of calcium ions, and can be used in the fields of calcium ion overload mediated cancer treatment and the like.

Description

Preparation method and application of arginine/calcium phosphate nanoparticles

Technical Field

The invention belongs to the technical field of nano composite materials and drugs, and specifically relates to a method for preparing arginine/calcium phosphate nanoparticles and application thereof in preparing drugs for treating tumors.

Background technique

Hepatocellular carcinoma (HCC) is one of the most common cancers and accounts for a high proportion of cancer-related deaths. Most HCCs are preceded by chronic liver disease or cirrhosis, which is mainly caused by hepatitis B virus (HBV), hepatitis C virus and excessive alcohol consumption. In addition, other diseases such as diabetes and obesity also increase the risk of HCC. Currently, there are many treatments for liver cancer, including surgery (liver resection or liver transplantation) and non-surgical methods (arterial chemoembolization, radiotherapy, local ablation, microwave ablation) and systemic chemotherapy. However, among these treatments, surgery is the most direct and effective, but most HCC patients are diagnosed in the late stage, and surgical treatment is very limited. Existing chemotherapeutic drugs such as doxorubicin (DOX), camptothecin (CPT), cisplatin (Cis-Pt), epirubicin (EPI), etc. have been used to treat HCC, but due to the random biodistribution of chemotherapeutic drugs and the inherent drug resistance characteristics of HCC, they have not shown satisfactory clinical efficacy.

With the development of nanoscience and nanotechnology, "nanomedicine" is no longer a new term in the 21st century. The combination of nanoscience and technology with biology and medicine in multiple application fields such as biosensors, medical tracing, early diagnosis of diseases, and cancer treatment has gradually developed "nanomedicine" into a new research direction of multidisciplinary intersection, ushering in a new era of medical engineering. Faced with the actual needs for disease prevention, diagnosis and treatment and the urgent problem of damage to normal tissues and organs of the human body caused by the non-specific distribution of traditional drugs, the development of nanotechnology has brought new hope and opened up new ways to obtain more advanced drug delivery systems and achieve early detection and diagnosis. Faced with the actual needs for disease prevention, diagnosis and treatment and the urgent problem of damage to normal tissues and organs of the human body caused by the non-specific distribution of traditional drugs, multifunctional nanomaterials provide an important research platform for the precise positioning and early diagnosis, targeted and combined treatment of tumors. With the continuous development and improvement of nanotechnology and nanomaterials, nanoparticles have made significant progress in the treatment of cancer due to their unique structure and physicochemical properties [L. Mei, D. Ma, Q. Gao, X. Zhang, W. Fu, X. Dong, G. Xing, W. Yin, Z. Gu, Y. Zhao, Mater.Horiz. 2020, 7, 1834-1844; Z. Tang, Y. Liu, M. He, W. Bu, Angew. Chem. Int.Ed. 2019, 58, 946-956; Y. Wang, L. Shi, Z. Ye, K. Guan, L. Teng, J. Wu, Y.Xia, G. Song, X. Zhang, Nano Lett. 2020, 20, 176-183; M. Wang, M. Y. Chang, C.X. Li, Q. Chen, Z. Y. Hou, B. G. Xing, J. Lin, Adv. Mater. 2022, 34, 21060.]. In recent years, calcium, as a second messenger, plays an important role in intracellular signal transduction and can regulate the physiological functions of cells, such as cell cycle, apoptosis and cell proliferation. Mitochondrial calcium overload is considered to be the cause of tumor cell toxicity and usually leads to cell necrosis and apoptosis. A large amount of calcium ions can cause calcium ion signal transduction to reshape, leading to a continuous increase in intracellular calcium ions. Calcium ions are deposited in the mitochondria of tumor cells, causing cytochrome c to be released from mitochondria into the cytoplasm, and the mitochondrial membrane potential (ΔΨ) is reduced, thereby inducing tumor cell apoptosis. However, due to the limitation of the calcium ion efflux of the cell itself, it is difficult to have a real killing effect on cancer cells. Therefore, how to develop new nanomaterials to inhibit the cellular calcium ion efflux ability while providing high concentrations of calcium ions is a challenging topic. So far, there are no literature and patent reports on the synthesis of high-biosafety arginine/calcium phosphate nanoparticles using the method in this article.

Summary of the invention

The purpose of the present invention is to provide a method for preparing arginine/calcium phosphate nanoparticles which have the characteristics of good biocompatibility, uniform particle size, good dispersibility, etc. and can be used for tumor treatment and its application.

The preparation method of arginine/calcium phosphate nanoparticles comprises the following steps:

1) In a 100 mL round-bottom flask, add 10 to 20 mg L-arginine, 3 to 5 mg _CaCl2 , and 2 to 5 mL deionized water. Stir magnetically for 5 to 10 min until the solution becomes clear.

2) Slowly add 30 to 60 mL of isopropanol to the solution obtained in step 1) under magnetic stirring and stir at room temperature for 1 to 2 h.

3) Add 100 ~ 200 μL Na ₂ HPO ₄ solution (30 mg mL ^-1 ) to the solution obtained in step (2) under magnetic stirring, and stir the reaction at 25 ~ 30 ^o C for 12 ~ 24 h.

4) The mixed solution obtained in step 3) was centrifuged (7000-9000 rpm, 6-8 min), washed with water 2-3 times, and the obtained precipitate was dried in a -50 ^° C freezer for 12-24 h to obtain arginine/calcium phosphate (L-Arg-CaP) nanoparticles.

The present invention provides a preparation method and application of arginine/calcium phosphate nanoparticles, which comprises: 1) L-arginine, _CaCl2 and deionized water are magnetically stirred until the solution is clear and transparent; 2) 30 to 60 mL of isopropanol is slowly added dropwise to the solution obtained in step 1) under magnetic stirring, and stirred at room temperature for 1 to 2 hours. ₃ ) _Na2HPO4 solution is added to the solution obtained in step 2) under magnetic stirring, and stirred for 12 to 24 hours at 25 to 30 ^° C. 4) The mixed solution obtained in step 3) is centrifuged and washed with water, and the precipitate is freeze-dried at -50 ^° C to obtain arginine/calcium phosphate (L-Arg-CaP) nanoparticles. The product has uniform particle size, good biocompatibility, pH responsiveness, inhibits calcium ion efflux while providing a large amount of calcium ions, and can be used in the fields of calcium ion overload-mediated cancer treatment.

The present invention has the following advantages:

1) The synthesis method of the present invention is simple and is the first to use a one-step method to synthesize highly biocompatible arginine/calcium phosphate nanoparticles.

2) The arginine/calcium phosphate nanoparticles obtained by the present invention can regulate the intracellular calcium ion concentration, stimulate tumor immune response through calcium ion overload, and achieve tumor killing. It is a new type of drug-free therapeutic nanomaterial.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a transmission electron microscope image of arginine/calcium phosphate nanoparticles obtained in the present invention;

Fig. 2 is a surface scanning image of arginine/calcium phosphate nanoparticles obtained by the present invention;

Figure 3. Confocal laser scanning microscopy images of HepG-2 cells cultured with arginine/calcium phosphate nanoparticles for different time periods;

Figure 4. CLSM images of changes in mitochondrial membrane potential in cells;

Figure 5. Activity of HepG2 cells with different concentrations of arginine, calcium phosphate and arginine/calcium phosphate nanoparticles;

Figure 6 shows the photos of mice and dissected tumors after 14 days of treatment with arginine/calcium phosphate nanoparticles, calcium phosphate nanoparticles, arginine, and blank control groups (from right to left).

Detailed ways

Example 1: Preparation of Arginine/Calcium Phosphate Nanoparticles

10 mg L-arginine, 3 mg CaCl ₂ and 2 mL deionized water were added to a 100 mL round-bottom flask in sequence and magnetically stirred for 5 min until the solution was clear and transparent. 30 mL isopropanol was slowly added dropwise to the above solution under magnetic stirring and stirred at room temperature for 1 h. 100 μL Na ₂ HPO ₄ solution (30 mg mL ^-1 ) was added to the solution under magnetic stirring and stirred at 25 ^o C for 12 h. The mixed solution was centrifuged (7000 rpm, 7 min), washed with water 3 times, and the resulting precipitate was dried in a -50 ^o C freezer for 12 h to obtain arginine/calcium phosphate (L-Arg-CaP) nanoparticles.

Example 2: Preparation of Arginine/Calcium Phosphate Nanoparticles

20 mg L-arginine, 5 mg CaCl ₂ and 5 mL deionized water were added to a 100 mL round-bottom flask in sequence and magnetically stirred for 10 min until the solution was clear and transparent. 60 mL isopropanol was slowly added dropwise to the above solution under magnetic stirring and stirred at room temperature for 1 h. 200 μL Na ₂ HPO ₄ solution (30 mg mL ^-1 ) was added to the solution under magnetic stirring and stirred at 30 ^o C for 24 h. The mixed solution was centrifuged (8000 rpm, 6 min), washed with water 3 times, and the resulting precipitate was dried in a -50 ^o C freezer for 24 h to obtain arginine/calcium phosphate (L-Arg-CaP) nanoparticles.

Example 3: Preparation of Arginine/Calcium Phosphate Nanoparticles

15 mg L-arginine, 4 mg CaCl ₂ and 7 mL deionized water were added to a 100 mL round-bottom flask in sequence and magnetically stirred for 8 min until the solution was clear and transparent. 50 mL isopropanol was slowly added dropwise to the above solution under magnetic stirring and stirred at room temperature for 1 h. 150 μL Na ₂ HPO ₄ solution (30 mg mL ^-1 ) was added to the solution under magnetic stirring and stirred at 29 ^o C for 16 h. The mixed solution was centrifuged (9000 rpm, 6 min), washed twice with water, and the resulting precipitate was dried in a -50 ^o C freezer for 20 h to obtain arginine/calcium phosphate (L-Arg-CaP) nanoparticles.

Experimental Example 1 Cellular uptake behavior of L-Arg-CaP in HepG-2 cells

HepG-2 cells were seeded in 20 mm glass-bottomed cell culture dishes at a density of 10 ⁵ cells per well and cultured at 37 °C in a 5% CO ₂ environment for 24 h. Then, L-Arg-CaP NPs (10 ug/mL) labeled with rhodamine B were incubated with the cells for 1, 3, and 6 hours, respectively. The cell supernatant was then discarded and the cell monolayer was rinsed three times with PBS. Hoechst 33342 was then added to the cells to stain the nuclei for 20 minutes. Finally, CLSM was used for observation. Figure 3 shows confocal laser scanning microscopy photos of L-Arg-CaP and HepG-2 cells cultured for 1 h, 3 h, and 6 h, respectively. The red color is rhodamine B-labeled nanoparticles, and the blue color is Hoechst 33342-stained cell nuclei. It can be seen from the confocal that the red fluorescence intensity gradually increases over time. After incubation for 6 h, the red fluorescence in the cytoplasm was significantly enhanced, indicating that L-Arg-CaP NPs can be effectively internalized by cancer cells.

As is known to all, most of the Ca ²⁺ in cells is stored in mitochondria and endoplasmic reticulum, and its abnormal retention can induce intracellular oxidative stress. Therefore, we first evaluated the effect of L-Arg-CaP on mitochondrial homeostasis. First, HepG-2 cells were cultured in 20 mm glass-bottomed cell culture dishes at a density of 10 ⁵ cells per well for 24 h to allow the cells to adhere to the wall. The culture medium was discarded, and fresh culture medium containing different treatment factors was added to the corresponding culture dishes for another 6 h. After washing with PBS three times, JC-1 solution was added and incubated at 37 °C for another 30 min. The fluorescence image of the mitochondrial membrane potential in the cells was observed under a fluorescence microscope (as shown in Figure 4). Under normal circumstances, the mitochondria in the cells are in a higher mitochondrial membrane potential, which makes JC-1 show red fluorescence due to the production of J aggregates. However, at low mitochondrial membrane potential, it shows green fluorescence due to the maintenance of JC-1 monomers. The green fluorescence of the cells in the L-Arg-CaP group was the strongest, indicating that the mitochondrial membrane potential was the lowest and the mitochondria were severely damaged, confirming that the synthesized nanomaterials have a significant mitochondrial killing effect.

Experimental Example 2 In vitro therapeutic effect of L-Arg-CaP NPs

We used the MTT method to determine the effect of different particles on the activity of HepG-2 cells. HepG-2 cells were seeded into 96-well plates at a density of 2.5×10 ⁴ cells per well and incubated in DMEM containing fetal bovine serum (FBS) for 24 h. Then, the culture medium was replaced with serum-free DMEM containing different concentrations of L-Arg, CaP, and L-Arg-CaP NPs. After incubation for 24 h, 10 μL of MTT (5 mg mL ^-1 ) was added to each well, and incubated for another 4 h. The culture medium was replaced with DMSO (150 μL), and the absorbance was measured at a wavelength of 490 nm using a microplate reader. The results showed that with the increase in the concentration of L-Arg-CaP, the survival rate of tumor cells decreased significantly, indicating that the L-Arg-CaP composite material has excellent anti-tumor effects (as shown in Figure 5).

In order to further verify the in vivo therapeutic effect of tumors, BALB/c mice with a tumor size of about ¹⁰⁰ mm3 were selected as the research subjects. The tumor-bearing mice were randomly divided into 4 groups (5 mice in each group): PBS group, L-Arg group, CaP group and L-Arg-CaP NPs group. The injection was performed once through the tail vein every 2 days, for a total of 7 injections. After the experiment, the tumors of BALB/c mice were peeled off and weighed. As shown in Figure 6, compared with the control group, the tumor in the L-Arg-CaP group was the smallest, and the tumor growth was significantly controlled. This was mainly due to the fact that NO promoted the increase of calcium ions in cells, thereby causing calcium death.

Claims (3)

1. A method for preparing arginine/calcium phosphate nanoparticles, characterized in that it comprises: 1) Add 10-20 mg L-arginine, 3-5 mg _CaCl2 and 2-5 mL deionized water in sequence and stir magnetically for 5-10 min until the solution becomes clear. 2) Slowly add 30-60 mL of isopropanol to the solution obtained in step (1) under magnetic stirring and stir at room temperature for 1-2 h; 3) Add 100 to 200 μL of 30 mg mL-1 Na ₂ HPO ₄ solution to the solution obtained in step (2) under magnetic stirring, and stir the reaction at 25 to 30°C for 12 to 24 h; 4) The mixed solution obtained in step 3) is centrifuged and washed with water for 2 to 3 times. The obtained precipitate is dried in a -50°C freezer for 12 to 24 h to obtain arginine/calcium phosphate L-Arg-CaP nanoparticles. 2. The method for preparing arginine/calcium phosphate nanoparticles according to claim 1, wherein the centrifugation is performed at 7000 to 9000 rpm for 6 to 8 min. 3. Application of arginine/calcium phosphate nanoparticles in the preparation of drugs for the treatment of liver tumors; The arginine/calcium phosphate nanoparticles are prepared by the method of claim 1.