A nanoparticle drug delivery system based on bioorthogonal reaction and a TCO-modified bioprosthetic valve

Abstract

The present invention discloses a nanoparticle drug delivery system based on bioorthogonal reaction and a TCO-modified biological valve, belonging to the technical field of medical devices. In the present invention, the anti-calcification drug octyl itaconate is screened through in vitro and in vivo cell and animal experiments. This drug can regulate the metabolic state of macrophages infiltrating inside the valve, inhibit their apoptosis and oxidative stress, thereby reducing the calcification of biological valves. In the present invention, the biological valve is modified with a TCO group, and the PLGA-PEG nanoparticles loaded with octyl itaconate are modified with a Tz group. The drug-loaded nanoparticles target and bind to the position of the biological valve through the bioorthogonal reaction of the TCO-Tz group to release the drug and play a significant anti-calcification effect. The present invention provides a new idea for the anti-calcification of biological valves.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical devices, and particularly relates to a nanoparticle drug delivery system based on bioorthogonal reaction and a TCO-modified biological valve. Background Art

[0002] Cardiac valve disease seriously endangers human health. Valve replacement is the main treatment method, and the market demand for valve substitutes is huge. It is estimated that the global number of valve replacements will reach 850,000 per year in 2050. Existing valve substitutes include mechanical valves and biological valves. Mechanical valves have problems such as the need to take anticoagulant drugs for life, easy bleeding or embolism, and loud valve opening and closing sounds, which seriously affect the patient's quality of life and survival. Biological valves do not require anticoagulation. After decades of technological iteration and update, they have surpassed mechanical valves and become the mainstream of valve substitutes. And the rapid development and popularization of transcatheter valve replacement in the past 20 years have further expanded the scope of use of biological valves. However, biological valves have the problem of limited durability. The main reason is that as the implantation time of biological valves in the body increases, the valve leaflets gradually calcify, and the valve opening and closing are restricted, resulting in clinical symptoms such as biological valve stenosis or regurgitation. The calcification of biological valves seriously affects the treatment effect of valves and has become an international problem that has not been solved for more than half a century. Developing a new type of biological valve with anti-calcification performance and lifelong durability has great scientific value and clinical significance.

[0003] Currently, common anti-calcification methods for biological valves include blocking residual aldehydes of biological valves, glycerol preservation method, and decellularization method. The above methods only regard the calcification of biological valves as a passive process of simple calcium and phosphorus deposition, without considering the effects of factors such as cells and body fluids in the body on biological valves themselves. Therefore, they can only delay the calcification process of biological valves, and the anti-calcification effect is limited, and the problem of biological valve calcification has not been fundamentally solved. Summary of the Invention

[0004] In the present invention, the anti-calcification drug octyl itaconate is screened through in vitro and in vivo cell and animal experiments. This drug can regulate the metabolic state of macrophages infiltrating into the valve, inhibit their apoptosis and oxidative stress, thereby reducing the calcification of biological valves. Drug targeted delivery is completed through bioorthogonal reaction, which is a fast and selective labeling reaction applied to living systems and a system that does not interfere with biochemical processes in the body. In the present invention, the biological valve is modified with a TCO group, and the PLGA-PEG nanoparticles loaded with octyl itaconate are modified with a Tz group. The drug-loaded nanoparticles target and bind to the position of the biological valve through the bioorthogonal reaction of the TCO-Tz group to release the drug and play an anti-calcification role.

[0005] The present invention provides a TCO-modified biological valve based on bioorthogonal reaction. The preparation method of the TCO-modified biological valve includes the following steps:

[0006] (1) The arterial valve is treated with a glutaraldehyde solution to obtain valve BHV;

[0007] (2) Valve modification system configuration: Dissolve 0.25 - 0.3 g of EDC, 0.03 - 0.04 g of NHS, and 0.4 - 0.5 g of MES in 45 - 55 mL of ddH2O, adjust the pH to 5, and add TCO - NH2 to make its concentration 0.8 - 1.2 mmol / L;

[0008] (3) After washing the biological valve with PBS, place it into the valve modification system and react on a shaker to obtain BHV - TCO.

[0009] Preferably, the concentration of the glutaraldehyde solution in step (1) is 0.625%.

[0010] More preferably, in step (2), the usage amounts of EDC, NHS, and MES are 0.2876 g, 0.0345 g, and 0.4881 g respectively, and the volume of ddH2O is 50 ml.

[0011] More preferably, the concentration of TCO - NH2 in step (2) is 1 mmol / L.

[0012] More preferably, the reaction temperature in step (3) is 37°C.

[0013] The present invention also provides a 4 - octyl itaconate - loaded PLGA - PEG - Tz nanoparticle based on bio - orthogonal reaction. The preparation method of the 4 - octyl itaconate - loaded PLGA - PEG - Tz nanoparticle includes the following steps:

[0014] (1) Dissolve 8 - 12 mg of PLGA - PEG and 0.3 - 0.7 mg of 4 - octyl itaconate in 0.4 - 0.6 mL of dichloromethane and mix well by ultrasound;

[0015] (2) Add 4 - 6 mL of 1% polyvinyl alcohol solution and perform ultrasonic emulsification;

[0016] (3) Stir for 4 - 8 h;

[0017] (4) Centrifuge, discard the supernatant, and the precipitate is the 4 - octyl itaconate - loaded PLGA - PEG nanoparticle;

[0018] (5) Dissolve 2 - 3 mg of Tz - PEG in 15 - 25 μL of dimethyl sulfoxide, then add 8 - 12 mL of PBS buffer solution with a pH of 7.4, adjust the pH to 8, and add it to the precipitate in step (4) and react for 2 - 6 h;

[0019] (6) Centrifuge, discard the supernatant, and the precipitate is the 4 - octyl itaconate - loaded PLGA - PEG - Tz nanoparticle.

[0020] Preferably, the amounts of PLGA-PEG, octyl itaconate, and dichloromethane used in step (1) are 10 mg, 0.5 mg, and 0.5 ml, respectively.

[0021] More preferably, the amount of 1% polyvinyl alcohol used in step (2) is 5 ml, and the stirring time in step (3) is 6 h.

[0022] More preferably, the amounts of Tz-PEG, dimethyl sulfoxide, and PBS buffer with a pH of 7.4 used in step (5) are 2.5 mg, 20 μL, and 10 mL, respectively, the reaction temperature is 37 °C, and the reaction time is 4 h.

[0023] The present invention also provides a product for reducing bioprosthetic valve calcification, which includes the above-mentioned TCO-modified bioprosthetic valve and the above-mentioned PLGA-PEG-Tz nanoparticles loaded with octyl itaconate.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) Significantly anti-calcification effect: Von Kossa staining of calcium salts in animal experiments shows that there is no obvious calcium salt deposition in the bioprosthetic valve after loading the nanoparticles with drugs; the calcium quantification results show that the degree of bioprosthetic valve calcification is significantly reduced after loading the nanoparticles with drugs;

[0026] (2) Good drug targeting and reduced drug side effects: Bioorthogonal reactions are chemical reactions that can occur in living cells or tissues without interfering with the biochemical reactions of the organism itself. They can occur at room temperature, do not require a catalyst or the catalyst is non-toxic and has low toxicity, and do not affect various substances in the biological system. Using a bioorthogonal reaction system can maximize the drug delivery efficiency and reduce the toxicity and side effects of the drug and the drug delivery system;

[0027] (3) There is no report on the treatment of bioprosthetic valve calcification with octyl itaconate. Octyl itaconate can inhibit bioprosthetic valve calcification by regulating the metabolic state of macrophages, and the treatment target is more specific. Description of the Drawings

[0028] Figure 1 Results of 1HNMR detection of BHV and BHV-TCO in Example 1.

[0029] Figure 2 Scanning electron microscope images of BHV and BHV-TCO in Example 1.

[0030] Figure 3 Scanning electron microscope image of PLGA-PEG-Tz nanoparticles in Example 1.

[0031] Figure 4It is the Zeta potential and particle size diagram of PLGA-PEG-Tz nanoparticles in Example 1.

[0032] Figure 5 It is the scanning electron microscope image of the binding of nanoparticles to the valve in vitro in Example 2.

[0033] Figure 6 It is the Von Kosaa calcium salt staining result after 30 days of subcutaneous embedding of the valve in Example 3.

[0034] Figure 7 It is the calcium quantitative detection result after 30 days of subcutaneous embedding of the valve in Example 3. Detailed implementation mode

[0035] Example 1

[0036] (1) Modification of biological valve with TCO:

[0037] ① The porcine aortic valve was crosslinked in 0.625% glutaraldehyde solution at room temperature in the dark for 48 hours to obtain the unmodified valve BHV.

[0038] ② Preparation of valve modification system: 0.2876 g of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), 0.0345 g of NHS (N-hydroxysuccinimide), and 0.4881 g of MES (2-morpholinoethanesulfonic acid) were dissolved in 50 mL of ddH2O, and the pH was adjusted to 5 using 10 mol / L NaOH solution, and TCO-NH2 was added to make its concentration 1 mmol / L.

[0039] ③ The biological valve was washed with PBS and then placed in the valve modification system, and reacted on a shaker at 37 °C for 24 h to obtain BHV-TCO.

[0040] (2) Preparation of PLGA-PEG-Tz nanoparticles loaded with octyl itaconate:

[0041] ① 10 mg of PLGA-PEG (Xi'an Haoran Biotechnology Co., Ltd., product number: R-PL005-4KD) and 0.5 mg of octyl itaconate (purchasing company and product number: selleck, product number: S5929) were dissolved in 0.5 mL of dichloromethane and mixed evenly by ultrasound;

[0042] ② 5 mL of 1% polyvinyl alcohol (Sigma, product number: 363170) was added and dissolved, and ultrasonic emulsification was performed;

[0043] ③ Stir at 1500 rpm on a magnetic stirrer for 6 h;

[0044] ④ The solution was centrifuged at 4500 g for 5 min, and the supernatant was discarded. The precipitate was the octyl itaconate-loaded nanoparticles;

[0045] ⑤ After dissolving 2.5 mg of Tz-PEG (Methyltetrazine-PEG5-NHS ester, purchased from Leyan (1185789)) in 20 μL of dimethyl sulfoxide, 10 mL of PBS buffer solution with a pH of 7.4 was added, the pH was adjusted to 8, and it was added to the precipitate obtained in the previous step, and the reaction was carried out at 37 °C for 4 h;

[0046] ⑥ Centrifuge at 4500 g for 5 min, discard the supernatant, and the precipitate is the nanoparticles loaded with octyl itaconate modified with Tz groups (for the detection results, see Figure 3 、 4 ).

[0047] Example 2

[0048] Dissolve the octyl itaconate-loaded PLGA-PEG-Tz nanoparticles prepared in Example 1 in 5 ml of PBS solution (pH = 7.2) with a final concentration of 10 mg / ml to simulate the in-vivo body fluid environment state of the nanoparticles. Immerse the BHV-TCO prepared in Example 1 in the octyl itaconate-loaded PLGA-PEG-Tz nanoparticle solution, and place it in a shaker at 37 °C and shake. After 2 hours, take out the valve, wash it three times with PBS to remove the unbound or loosely bound nanoparticles. After the washing is completed, fix it with an electron microscopy fixative for 8 hours and perform an electron microscopy scanning experiment. The results are shown in Figure 5 。

[0049] Example 3

[0050] Animal experiment: Male C57BL / 6 mice at 6-8 weeks old were subcutaneously implanted with the BHV and BHV-TCO valve materials prepared in Example 1 on the back. On the 7th, 14th, 21st, and 28th days after implantation in the experimental group, the octyl itaconate-loaded PLGA-PEG-Tz nanoparticles prepared in Example 1 were injected via the tail vein once a week, and the injection dose was 10 mg of nanoparticles per mouse per injection. The control group was not treated with tail vein injection of nanoparticles. The mice were sacrificed on the 30th day after implantation, and the subcutaneous valve tissues on the back were taken out for section staining and calcium quantification detection. The results are shown in Figure 6 、 7 。

[0051] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A product for reducing calcification of biological valves, characterized in that: The product includes trans-cyclooctene TCO modified bioprosthesis and 4-itaconate octyl PLGA-PEG-Tz tetrazine group nanoparticles; The method for preparing the trans-cyclooctene TCO-modified biological valve comprises the following steps: (1) The aortic valve is treated with glutaraldehyde solution to obtain the valve BHV; (2) Valve modification system preparation: Dissolve 0.25-0.3 g EDC, 0.03-0.04 g NHS, and 0.4-0.5 g MES in 45-55 mL ddH2O, adjust pH to 5, and add TCO-NH2 to a concentration of 0.8-1.2 mmol / L; (3) The bioprosthesis was washed with PBS and placed in the valve modification system, and the BHV-TCO was prepared by shaking the reaction. The preparation method of the 4-itaconate-carrying PLGA-PEG-Tz tetrazine group nanoparticles comprises the following steps: ① Dissolve 8-12 mg PLGA-PEG and 0.3-0.7 mg 4-itaconate in 0.4-0.6 mL dichloromethane and mix by ultrasonication; ② Add 4-6mL 1% polyvinyl alcohol to dissolve and perform ultrasonic emulsification; ③Stir for 4-8h; ④ Centrifuge and discard the supernatant. The precipitate is PLGA-PEG nanoparticles loaded with 4-itaconate; ⑤ Dissolve 2-3 mg Tz-PEG in 15-25 μL dimethyl sulfoxide, then add 8-12 mL PBS buffer with a pH of 7.4, adjust the pH to 8, and add to the precipitate in step ④, and react for 2-6 hours; ⑥ Centrifuge and discard the supernatant. The precipitate is the 4-octyl itaconate-loaded PLGA-PEG-Tz nanoparticles.

2. The product according to claim 1, characterized in that The concentration of the glutaraldehyde solution in step (1) is 0.625%.

3. The product according to claim 2, characterized in that In the step (2), the amounts of EDC, NHS and MES used are 0.2876 g, 0.0345 g and 0.4881 g respectively, and the volume of ddH2O is 50 ml.

4. The product according to claim 3, characterized in that The concentration of TCO-NH2 in step (2) is 1 mmol / L.

5. The product according to claim 4, characterized in that The reaction temperature in step (3) is 37°C.

6. The product according to claim 5, characterized in that In the step ①, the usage amounts of PLGA-PEG, 4-octyl itaconate and dichloromethane are 10 mg, 0.5 mg and 0.5 ml respectively.

7. The product according to claim 6, characterized in that The amount of 1% polyvinyl alcohol used in step ② is 5 ml, and the stirring time in step ③ is 6 hours.

8. The product according to claim 7, characterized in that In step ⑤, the amounts of Tz-PEG, dimethyl sulfoxide, and PBS buffer with a pH of 7.4 are 2.5 mg, 20 μL, and 10 mL, respectively. The reaction temperature is 37° C., and the reaction time is 4 h.

Images