Nanoparticles capable of continuously supplying NO and treating tumors in combination with photothermal therapy

Abstract

The invention relates to the technical field of chemistry, in particular to nano particles capable of continuously supplying NO and treating tumors in combination with photo-thermal treatment, a preparation method and application of the nano particles. According to the invention, TA-Fe coated CaO2 modified LA-loaded mesoporous MPDA nanoparticles (MPDA at LA-CaO2-TA-Fe) are constructed, and the mesoporous MPDA nanoparticles (MPDA at LA-CaO2-TA-Fe) are used for NO enhanced photothermal immunotherapy of breast cancer. According to the invention, a new mode for enhancing tumor resistance through the NO-enhanced phototherapy nano platform is established, so that new inspiration is provided for tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of chemical technology, and in particular to a nanoparticle capable of sustainably supplying NO combined with photothermal therapy for tumors, a preparation method and an application thereof. Background Art

[0002] Tumors have become a major disease that endangers human health and has become a global hot issue of concern. There is an urgent need to develop effective and safe treatment methods. The main methods for treating cancer are: surgical treatment, radiotherapy and chemotherapy. Among them, chemotherapy is the most commonly used method for treating cancer. However, due to poor targeting and low drug utilization during chemotherapy, it is difficult to effectively distinguish tumor tissue from normal tissue, and there are many serious side effects on normal tissues. It is easy to cause bone marrow suppression, liver and kidney dysfunction, and it is easy for patients to develop drug resistance and other adverse reactions. In general, the comprehensive efficacy is not ideal. Due to the above shortcomings, the development of a multimodal tumor treatment platform to change the drug delivery method to reduce the toxicity of chemotherapy to normal tissues, and to combine multiple treatment modes (gas therapy and photothermal therapy, etc.) to improve the effect of tumor treatment is one of the current research hotspots in the field of drug delivery.

[0003] In gas therapy, nitric oxide (NO) has become a star molecule. Studies have found that NO has the function of inhibiting tumor growth and migration. It plays a key regulatory role in a series of biochemical and physiological processes such as neural activity, cardiovascular homeostasis and immune response. In recent years, new evidence has shown that NO can enhance the efficacy of chemotherapy, radiotherapy or phototherapy, and directly induce tumor cell apoptosis at relatively high concentrations (>1μM), which has been developed as an effective therapeutic agent for cancer prevention and treatment. Summary of the invention

[0004] The present invention constructs TA-Fe-wrapped CaO2-modified LA-loaded mesoporous MPDA nanoparticles (MPDA@LA-CaO2-TA-Fe) for NO-enhanced breast cancer photothermal immunotherapy. The TA-Fe nanocoating disintegrates in the weakly acidic microenvironment of the tumor, the CaO2 nanospheres hydrolyze to produce H2O2, and the TA converts Fe 3+ Rapid reduction to Fe 2+ , which solves the problem of limited endogenous H2O2 content and low efficiency of Fenton reaction catalysts leading to limited Fenton reaction. 3+ The ·OH produced by the reaction with H2O2 can cause oxidative stress in tumor cells, desensitize the calcium ion protein of the cells, lead to calcium overload, and accelerate the death of cancer cells. 3+ Mediating the Fenton reaction to produce OH and "Ca 2+The two methods "overload" work synergistically. MPDA, as a highly biocompatible carrier, not only acts as a nanocarrier, but also provides photothermal therapeutic effects. The H2O2 provided by CaO2 can promote the decomposition of LA to produce NO. The present invention establishes a new mode of enhancing anti-tumor through NO-enhanced phototherapy nanoplatform, which will provide new inspiration for tumor immunotherapy.

[0005] In one aspect, the present invention provides a method for preparing nanoparticles that can sustainably supply NO combined with photothermal therapy for tumors, comprising the following steps:

[0006] 1) Preparation of MPDA

[0007] First, F127 was added to ethanol, and deionized water and TMB were added after ultrasonic dissolution. Dopamine hydrochloride was added to dissolve and then ammonia was added. After stirring at room temperature for 24, the mixture was centrifuged at 12000 rpm for 10 minutes, and the supernatant was discarded. The obtained product was washed three times with acetone: ethanol (v:v) = 1:3, and the product was freeze-dried and stored to obtain MPDA.

[0008] 2) Preparation of CaO2-HA nanoparticles

[0009] In the vigorously stirred methanol, add the hyaluronic acid aqueous solution and the CaCl2 aqueous solution, stir evenly, then add 30% H2O2 at a rate of 10 seconds / drop, then add ammonia water to activate the reaction until white particles are produced, centrifuge at 12000 rpm for 10 minutes to collect the product, wash twice with methanol to obtain CaO2-HA nanoparticles;

[0010] 3) Preparation of CaO2-TA-Fe nanoparticles

[0011] CaO2-HA nanoparticles were weighed and dissolved in deionized water by ultrasonication, followed by addition of TA solution and Fe 3+ , vortexed for 20 s, the CaO2-TA-Fe nanoparticles were collected by centrifugation at 10000 rpm for 10 min, washed several times with ultrapure water, and then freeze-dried to obtain CaO2-TA-Fe nanoparticles;

[0012] 4) Preparation of MPDA@LA

[0013] Weigh L-arginine, dissolve it in deionized water, add MPDA, stir at room temperature for 24 hours, centrifuge at 10000 rpm for 10 minutes, and wash twice with water to obtain the product MPDA@LA;

[0014] 5) Preparation of MPDA@LA-CaO2-TA-Fe

[0015] MPDA@LA and CaO2-TA-Fe nanoparticles were added to deionized water and reacted for 24 h. The product was collected by centrifugation at 12000 rpm for 10 min and washed twice with water to obtain MPDA@LA-CaO2-TA-Fe.

[0016] Preferably, the weight volume ratio of F127 to ethanol in step 1) is 50 mg:1 mL, the weight volume ratio of F127:deionized water:TMB in step 1) is 50 mg:1 mL:20 mL, and the weight volume ratio of F127:dopamine hydrochloride:ammonia water is 50 mg:15 mg:90 μL.

[0017] Preferably, the concentration of the hyaluronic acid aqueous solution in the step 2) is 0.5 mg / mL, the concentration of the CaCl2 aqueous solution is 2 mol / L, and the methanol: hyaluronic acid aqueous solution: CaCl2 aqueous solution: 30% H2O2: ammonia water in the step 2) is 15 mL: 1 mL: 250 μL: 700 μL: 700 μL.

[0018] Preferably, the concentration of the TA solution in step 3) is 40 mg / mL, and the Fe 3+ The concentration of the nanoparticles is 10 mg / mL. In step 3), CaO2-HA nanoparticles: deionized water: TA solution: Fe 3+ 2mg:2mL:40μL:80μL.

[0019] Preferably, in the preparation of MPDA@LA in step 4), the ratio of L-arginine: deionized water: MPDA is 12.5 mg: 10 mL: 30 mg.

[0020] Preferably, in step 5), the ratio of MPDA@LA to CaO2-TA-Fe nanoparticles to deionized water is 3 mg: 10 mg: 10 mL.

[0021] On the other hand, the present invention provides a nanoparticle prepared by the above method that can sustainably supply NO combined with photothermal therapy for tumors, and the nanoparticle can be used for NO-enhanced photothermal immunotherapy for breast cancer.

[0022] In another aspect, the present invention provides a nanoparticle prepared by the above method for sustainable supply of NO combined with photothermal therapy for tumors as an application in the preparation of a nano drug delivery system for treatment / photothermal synergistic therapy.

[0023] Compared with the prior art, the present invention combines photothermal therapy with gas therapy and introduces chemodynamic therapy to enhance the anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1These are the scanning electron microscopy of (a) MPDA in Example 1, (b) transmission electron microscopy of MPDA, (c) transmission electron microscopy of MPDA@LA-CaO2-TA-Fe, (d) scanning electron microscopy of MPDA@LA-CaO2-TA-Fe, (e) transmission electron microscopy of CaO2-HA nanoparticles, and (f) transmission electron microscopy of CaO2-TA-Fe nanoparticles.

[0025] Figure 2 The drug loading amount (LE%) and encapsulation efficiency (EE%) of the nanoparticles in Example 2.

[0026] Figure 3 This is the NO release curve of MPDA@LA-CaO2-TA-Fe at pH 5.4 and pH 7.4 in Example 3.

[0027] Figure 4 The cell survival rate of 4T1 cells after 24 hours of action of nanoparticles of different concentrations in Example 4.

[0028] Figure 5 These are the physical images of mice in each group on days 0, 7, and 14 in Example 5.

[0029] Figure 6 The following are actual pictures of tumors in each group of tumor-bearing mice in Example 5. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below with reference to the accompanying drawings.

[0031] Scanning electron microscope method used in the present invention: The surface morphology of nanoparticles was evaluated by SEM (S-4800) in the experiment. All nanoparticles were evenly blown onto the silicon wafer. Before analysis, the sample was vacuum coated with a thin gold film, and then the sample was observed at an appropriate magnification at an accelerating voltage of 20 kV. The TEM analysis of the sample was tested by Lorentz transmission electron microscope. An appropriate amount of nanoparticle solution was evenly dripped onto a copper mesh, and TEM scanning observation was performed respectively.

[0032] The abbreviations used in the present invention are as follows:

[0033] F127: Polyoxyethylene polyoxypropylene ether

[0034] TMB: 3,3',5,5'-Tetramethylbenzidine

[0035] TA: Tannic acid

[0036] Fe 3+ :Trivalent iron ion

[0037] LA: L-Arginine

[0038] HA: Hyaluronic acid

[0039] Example 1: Preparation of carrier

[0040] 1) Preparation of MPDA

[0041] First, 250 mg of F127 was added to 5 mL of ethanol, and 5 mL of deionized water and 100 mL of TMB were added after ultrasonic dissolution. After ultrasonic dissolution, 75 mg of dopamine hydrochloride was added and dissolved, and then 450 μL of ammonia water was added. After stirring at room temperature for 24 hours, the mixture was centrifuged at 12000 rpm for 10 minutes, and the supernatant was discarded. The obtained product was washed three times with acetone: ethanol (v:v) = 1:3, and the product was freeze-dried and stored to obtain MPDA.

[0042] 2) Preparation of CaO2-HA nanoparticles

[0043] In 15 mL of methanol that was vigorously stirred, 1 mL of hyaluronic acid aqueous solution (0.5 mg / mL) and 250 μL of CaCl2 aqueous solution (2 mol / L) were added. After stirring evenly, 700 μL of 30% H2O2 was added at a rate of 10 seconds / drop, and then 700 μL of ammonia water was added to activate the reaction until white particles were produced. The product was collected by centrifugation at 12000 rpm for 10 min, and washed twice with methanol to obtain CaO2-HA nanoparticles;

[0044] 3) Preparation of CaO2-TA-Fe particles

[0045] Weigh 2 mg of CaO2-HA nanoparticles and add them to 2 mL of water for ultrasonic dissolution. Then, add 40 μL of TA solution (40 mg / mL) and 80 μL of Fe 3+ (10 mg / mL), vortexed for 20 s, and the CaO2-TA-Fe nanoparticles were collected by centrifugation at 10000 rpm for 10 min, washed several times with ultrapure water, and then freeze-dried to obtain CaO2-TA-Fe particles;

[0046] 4) Preparation of MPDA@LA

[0047] Weigh 12.5 mg of L-arginine, dissolve it in 10 mL of deionized water, add 30 mg of MPDA, stir at room temperature for 24 h, centrifuge at 10,000 rpm for 10 min, and wash twice with water to obtain the product MPDA@LA;

[0048] 5) Preparation of MPDA@LA-CaO2-TA-Fe

[0049] 30 mg of MPDA@LA prepared in step 4) and 10 mg of CaO2-TA-Fe prepared in step 3) were reacted in 10 mL of deionized water for 24 h, the product was collected by centrifugation at 12000 rpm for 10 min, and washed twice with water to obtain MPDA@LA-CaO2-TA-Fe.

[0050] The prepared carrier was tested by electron microscopy, and the electron microscopy results were as follows: Figure 1 shown.

[0051] Example 2: Drug loading (LE%) and encapsulation efficiency (EE%)

[0052] 30 mg of MPDA was dispersed in different concentrations of LA solution (500 mg / L, 750 mg / L, 1000 mg / L, 1250 mg / L, 1500 mg / L), stirred for 24 hours, and centrifuged (12000 rpm, 10 min). The precipitate after centrifugation was freeze-dried and weighed, and the product was recorded as MPDA@LA. The absorbance of LA in the supernatant was measured at a wavelength of 540 nm, and the drug loading (LE%) and encapsulation efficiency (EE%) were calculated using the following formula to optimize the optimal carrier and LA concentration ratio, such as Figure 2 shown.

[0053]

[0054] Where W0 is the total content of LA added, W1 is the free LA content in the supernatant after sample centrifugation, and W2 is the mass of the nanocarrier after drug loading.

[0055] from Figure 2 It can be seen that as the LA concentration increases, the loading amount of MPDA on LA increases accordingly. This is because when the LA concentration increases, LA is more likely to enter the pores of MPDA. When the LA concentration increases from 1250 mg / L to 1500 mg / L, the MPDA encapsulation rate decreases from 59.73% to 50.37%. This is because the LA concentration is too high and exceeds the encapsulation capacity of MPDA. Considering the loading amount and encapsulation rate, the study chose to load the drug when the LA concentration was 1250 mg / L.

[0056] Example 3: NO release in vitro

[0057] There is a difference in pH between the tumor site and normal tissue. The pH of normal tissue is 7.4, while the pH of the tumor site is lower, which is a weakly acidic environment overall. Therefore, the experiment investigated the release effect of MPDA@LA-CaO2-TA-Fe nanoparticles under pH 5.4 and pH 7.4.

[0058] 50 mg of MPDA@LA-CaO2-TA-Fe was dispersed in 10 mL of PBS with different pH values ​​(7.4 and 5.4) to measure the release of NO. The solution was then placed in a shaker at 120 rpm and 37 ° C. 1 mL of supernatant was centrifuged at 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, and 48 h (an equal amount of buffer was added after sampling) for NO testing, and the amount of NO produced was determined by a NO assay kit.

[0059] like Figure 3 As shown in the figure, the NO generation at pH 5.4 is significantly higher than that at pH 7.4. When MPDA@LA-CaO2-TA-Fe is immersed in pH 5.4 PBS for 48 hours, the NO release can reach 12.08μM, which is enough to kill cancer cells. However, at pH 7.4, after immersion for 48 hours, the NO release can only reach 8.816μM. The above results show that the release of MPDA@LA-CaO2-TA-Fe nanoparticles is pH-targeted and can well deliver drugs to slightly acidic tumor sites.

[0060] Example 4: Cytotoxicity Experiment

[0061] The CCK-8 method was used to evaluate the cytotoxicity of the prepared nanoparticles to 4T1 tumor cells. 4T1 cells were cultured in Biosharp RPMI 1640 medium (containing 1% penicillin-streptomycin solution and 10% fetal bovine serum) at 37°C and 5% CO2 in a cell culture incubator until the logarithmic culture phase. 3 The cells were inoculated in a 96-well plate at a density of 180 μL per well. After 24 hours of culture, 20 μL of PBS buffer solution of nanoparticles at concentrations of 0, 2, 4, 6, 8, 12, and 16 μg / mL was added to each well. After 24 hours, the culture medium in each well was aspirated, and 100 μL of 10% CCK-8 culture medium solution was added to each well, and the culture was continued for 90 minutes. The absorbance of each well was measured at 450 nm using an ELISA reader, and the cell survival rate was calculated as follows.

[0062]

[0063] In the formula, the absorbance value of the cell wells treated with nanoparticles is recorded as OD 阳 The absorbance value of the blank-treated cell wells is recorded as OD 阴 OD 空 .

[0064] The results are as follows Figure 4As shown. The MPDA group had no obvious killing effect on cells (cell survival rate> 80%), which is due to the good biocompatibility of MPDA. Figure 4 M-CaO2-TA-Fe) and MPDA@LA-CaO2-TA-Fe( Figure 4 Compared with the MPDA@LA-CaO2-TA-Fe group (abbreviated as ML-CaO2-TA-Fe), there is a significant difference, which shows that NO has a significant killing effect on tumor cells. The survival rate of the group with HA added to the culture medium is 30.34% higher than that of the cells without special treatment. This shows that the addition of HA to the culture medium occupies the CD44 receptor on the surface of 4T1 cells, reducing the uptake of nanoparticles. This proves the targeting effect of the nanoparticles. Figure 4 The cell survival rate of the MPDA@LA-CaO2-TA-Fe+Laser group (abbreviated as ML-CaO2-TA-Fe+Laser) (only 19.59%) was significantly different from that of the MPDA@LA-CaO2-TA-Fe non-light-irradiated group, which was due to the good photothermal effect of MPDA@LA-CaO2-TA-Fe nanoparticles and the synergistic treatment of gas. The results showed that MPDA@LA-CaO2-TA-Fe nanoparticles had good photothermal responsiveness and pH targeting.

[0065] Example 5: In vivo anti-tumor experiment

[0066] To study the effects of nanoparticles on the vital signs of tumor-bearing mice, the tumor-bearing mice were randomly divided into five groups (5 mice in each group), namely PBS group, MPDA group, MPDA-CaO2-TA-Fe group ( Figure 5 abbreviated as M-CaO2-TA-Fe), MPDA@LA-CaO2-TA-Fe group ( Figure 5 abbreviated as ML-CaO2-TA-Fe), MPDA@LA-CaO2-TA-Fe+Laser( Figure 5 The MPDA@LA-CaO2-TA-Fe+Laser group was abbreviated as ML-CaO2-TA-Fe+Laser. 200 μL of the corresponding drug (20 mg / kg) was injected through the tail vein on days 0, 2, 4, 6, 8, 10, and 12, and the MPDA@LA-CaO2-TA-Fe+Laser group was irradiated with an 808 nm light source at 12 h after administration, with a light power of 1.0 W / cm 2 , time for 5 minutes, and use an infrared thermal imager to record the temperature changes in the tumor site of the mouse. The weight and tumor size of the mouse were recorded every two days, and the tumor volume was calculated according to the following formula. The tumor volume of the same group of mice was photographed and recorded on days 0, 7, and 14. On the 14th day, the mice were killed by cervical dislocation, and the solid tumor tissues and major organs were collected. The tumors were photographed and weighed for comparison.

[0067]

[0068] Where V is the tumor volume, a is the tumor length, and b is the tumor width.

[0069] like Figure 5 As shown in the figure, on the 0th, 7th and 14th days of tumor treatment, one mouse in each group was injected with anesthetic intraperitoneally and photographed. The results showed that on the 0th day, the initial tumor volume of mice in each group was consistent. On the 7th day, the tumor volume in the PBS group was the largest, which is the natural growth state of the tumor. The tumor volume of the MPDA group was larger than that of the MPDA-CaO2-TA-Fe( Figure 6 This is because CaO2-TA-Fe undergoes a Fenton reaction to produce ·OH. MPDA@LA-CaO2-TA-Fe( Figure 6 The tumor size of the MPDA@LA-CaO2-TA-Fe+Laser( Figure 6 The tumor volume in the PBS group was the smallest, which was the result of the synergistic treatment of gas therapy and photothermal therapy. On the 14th day of treatment, the tumor volume in the PBS group continued to increase, and the comparison trend in other groups was more obvious, indicating that the synergistic treatment of gas therapy and photothermal therapy has a significant effect on anti-tumor.

[0070] After 14 days of treatment, the tumors were dissected, photographed and weighed. Figure 6 As shown, the results show that MPDA@LA-CaO2-TA-Fe( Figure 6 ML-CaO2-TA-Fe) nanoparticles can significantly inhibit tumor growth and show good anti-tumor effects.

[0071] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A method for preparing nanoparticles that can sustainably supply NO combined with photothermal therapy for tumors, characterized in that: The following steps are involved: 1) Preparation of MPDA First, F127 was added to ethanol, and deionized water and TMB were added after ultrasonic dissolution. Dopamine hydrochloride was added to dissolve and then ammonia was added. After stirring at room temperature for 24, the mixture was centrifuged at 12000 rpm for 10 minutes, and the supernatant was discarded. The obtained product was washed three times with acetone: ethanol (v:v) = 1:3, and the product was freeze-dried and stored to obtain MPDA. 2) Preparation of CaO2-HA nanoparticles In the vigorously stirred methanol, add the hyaluronic acid aqueous solution and the CaCl2 aqueous solution, stir evenly, then add 30% H2O2 at a rate of 10 seconds / drop, then add ammonia water to activate the reaction until white particles are produced, centrifuge at 12000 rpm for 10 minutes to collect the product, wash twice with methanol to obtain CaO2-HA nanoparticles; 3) Preparation of CaO2-TA-Fe nanoparticles CaO2-HA nanoparticles were weighed and dissolved in deionized water by ultrasonication, followed by addition of TA solution and Fe 3+ , vortexed for 20 s, the CaO2-TA-Fe nanoparticles were collected by centrifugation at 10000 rpm for 10 min, washed several times with ultrapure water, and then freeze-dried to obtain CaO2-TA-Fe nanoparticles; 4) Preparation of MPDA@LA Weigh L-arginine, dissolve it in deionized water, add MPDA, stir at room temperature for 24 hours, centrifuge at 10000 rpm for 10 minutes, and wash twice with water to obtain the product MPDA@LA; 5) Preparation of MPDA@LA-CaO2-TA-Fe MPDA@LA and CaO2-TA-Fe nanoparticles were added to deionized water and reacted for 24 h. The product was collected by centrifugation at 12000 rpm for 10 min and washed twice with water to obtain MPDA@LA-CaO2-TA-Fe.

2. The method for preparing nanoparticles for sustainable supply of NO combined with photothermal therapy of tumors according to claim 1, characterized in that: The weight volume ratio of F127 to ethanol in step 1) is 50 mg:1 mL; the weight volume ratio of F127:deionized water:TMB in step 1) is 50 mg:1 mL:20 mL, and the weight volume ratio of F127:dopamine hydrochloride:ammonia water is 50 mg:15 mg:90 μL.

3. The method for preparing nanoparticles for sustainable supply of NO combined with photothermal therapy of tumors according to claim 1, characterized in that: The concentration of the hyaluronic acid aqueous solution in the step 2) is 0.5 mg / mL, and the concentration of the CaCl2 aqueous solution is 2 mol / L; in the step 2), the methanol: hyaluronic acid aqueous solution: CaCl2 aqueous solution: 30% H2O2: ammonia water is 15 mL: 1 mL: 250 μL: 700 μL: 700 μL.

4. The method for preparing nanoparticles for sustainable supply of NO combined with photothermal therapy of tumors according to claim 1, characterized in that: The concentration of the TA solution in step 3) is 40 mg / mL, and the Fe 3+ The concentration of the nanoparticles is 10 mg / mL. In step 3), CaO2-HA nanoparticles: deionized water: TA solution: Fe 3+ 2mg:2mL:40μL:80μL.

5. The method for preparing nanoparticles for sustainable supply of NO combined with photothermal therapy of tumors according to claim 1, characterized in that: In the preparation of MPDA@LA in step 4), the ratio of L-arginine: deionized water: MPDA is 12.5 mg: 10 mL: 30 mg.

6. The method for preparing nanoparticles for sustainable supply of NO combined with photothermal therapy of tumors according to claim 1, characterized in that: In the step 5), the ratio of MPDA@LA:CaO2-TA-Fe nanoparticles:deionized water is 3 mg:10 mg:10 mL.

7. A nanoparticle for continuously supplying NO combined with photothermal therapy for tumors prepared by the preparation method according to any one of claims 1 to 6, wherein the nanoparticle can be used for NO-enhanced photothermal immunotherapy for breast cancer.

8. Use of the nanoparticles as claimed in claim 7 in preparing a nano drug delivery system for therapeutic / photothermal synergistic therapy.

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