Harmine derivative-PLGA (poly (lactic-co-glycolic acid)) nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses harmine derivative-PLGA nanoparticles and a preparation method and application thereof.The preparation method comprises the following steps that 1, a harmine derivative-containing water phase and a PLGA-containing oil phase are mixed, ice bath ultrasonic treatment is conducted, and primary emulsion is obtained; (2) mixing the obtained primary emulsion with a sodium taurocholate solution, and carrying out ice-bath ultrasonic treatment to obtain a multiple emulsion; the preparation method provided by the invention is simple, the condition is mild, the reproducibility of a preparation process is relatively good, the obtained harmine derivative-PLGA nanoparticles are stable in physical property, small and uniform in particle size, low in polydispersity index, high in encapsulation efficiency and high in drug loading capacity, and compared with a harmine derivative serving as a raw material, the harmine derivative-PLGA nanoparticles can remarkably improve the hydatid resistance curative effect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to PLGA nanoparticles loaded with a dehydrogenated harmine derivative, and a preparation method and application thereof. Background Art

[0002] This section merely provides background information related to the present disclosure and is not necessarily prior art.

[0003] Echinococcosis, also known as cystic echinococcosis (CE), is a chronic zoonotic parasitic infectious disease caused by the tapeworm Echinococcus granulosus. This pathogen mainly affects the liver, but can also parasitize in organs such as the lungs and spleen, posing a serious threat to human health. At present, the main treatment for echinococcosis is surgery, but in actual clinical work, due to the easy dissemination of echinococcosis during surgery, the recurrence and abdominal and pelvic dissemination of echinococcosis after surgery account for a large proportion, and can lead to various complex complications, and surgical treatment is also extremely difficult. Therefore, drug treatment has become an indispensable and important auxiliary treatment method.

[0004] Peganum harmala L. is a perennial herb of the Zygophyllaceae family, and dehydrogenated harmaline is the main component of Peganum harmala. A large number of literature reports show that dehydrogenated harmaline has certain anti-hydatid effect and is a compound with development potential. However, in studies, it was found that dehydrogenated harmaline has a large toxic effect, which is mainly manifested as neurological symptoms, fever, and cardiovascular system disorders, etc., so it has not been further developed and applied to clinical practice. Therefore, the research team used dehydrogenated harmaline as the parent nucleus, modified its structure, and obtained more than a thousand derivatives. After a lot of research in the early stage of the project, the derivative H8 with better anti-hydatid effect and low toxicity was screened out (see patent 202011613055.1 for details, the structural formula is as shown in the present invention Figure 1 However, the pharmacokinetic results showed that the half-life of the derivative was very short and the bioavailability was very low, only 2.84%, which affected the efficacy of the derivative H8 to a certain extent. The present invention encapsulates the drug in nanoparticles to control the dissolution efficiency and thus prolong the circulation time and improve the efficacy of the drug, thereby providing a new option for the treatment and prevention of cystic echinococcosis. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a dehydrogenated harmine derivative-PLGA nanoparticles and a preparation method and application thereof in view of the deficiencies in the prior art.

[0006] In order to solve the above technical problems, the present invention discloses the following technical solutions:

[0007] In a first aspect, the present invention discloses a method for preparing dehydrogenated harmine derivative-PLGA nanoparticles.

[0008] In some embodiments, the preparation method comprises the following steps:

[0009] (1) mixing the aqueous phase containing the dehydrogenated echinopsine derivative and the oil phase containing PLGA, and ultrasonicating the mixture in an ice bath to form colostrum;

[0010] (2) pouring the obtained colostrum into the sodium taurocholate solution, mixing the two, and homogenizing them by ultrasonication in an ice bath to obtain a double emulsion;

[0011] (3) The obtained double emulsion is subjected to rotary evaporation, and a first centrifugation is performed at a speed of 4000-5000 r / min for 15-25 min. The first obtained supernatant is subjected to a second centrifugation at a speed of 12000-16000 r / min for 20-40 min. The precipitate obtained by the second centrifugation is the dehydrogenated harmine derivative-PLGA nanoparticles;

[0012] (4) The obtained precipitate is dispersed in water to obtain a dehydrogenated harmine derivative-PLGA nanoparticle suspension.

[0013] In step (1), the concentration of the dehydrogenated harmine derivative in the aqueous phase is 1-10 mg / mL, such as 6.5, 7, 7.5, 8, 8.5 mg / mL.

[0014] In step (1), the PLGA is poly(lactic acid-co-glycolic acid); in some embodiments, it is PLGA 75:25.

[0015] In step (1), the concentration of PLGA in the oil phase is 1-10 mg / mL, such as 3, 4, 5, 6, 7 mg / mL.

[0016] In step (1), the solvent of the oil phase includes chloroform.

[0017] In step (1), the mass ratio of the dehydrogenated harmine derivative to PLGA is 1:3-4, such as 1:5.

[0018] In step (1), the power of the ice bath ultrasound is 200-600 W, such as 300, 400, or 500 W; and the time of the ice bath ultrasound is 4-12 min, such as 3, 4, 5, 6, 7, 8, 9, 10, or 11 min.

[0019] In step (2), the concentration of sodium taurocholate in the sodium taurocholate solution is 1-8 mg / mL, such as 3, 4, 5, 6, or 7 mg / mL.

[0020] In step (2), the volume ratio of colostrum to sodium taurocholate solution is 1:(5-12.5), such as 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12.

[0021] In step (2), the power of the ice bath ultrasound is 200-600 W, such as 300, 400, 500 W; the time of the ice bath ultrasound is 4-12 min, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 min.

[0022] In step (3), the temperature of the rotary evaporation of the emulsion is 60-65°C, such as 50-62°C, and the rotary evaporation temperature is generally 25-35min, so as to evaporate the chloroform by rotary evaporation.

[0023] In step (3), the first centrifugation speed is 4000-5000r / min, such as 4200, 4400, 4500, 4600r / min; the first centrifugation time is 15-25min, such as 18, 20, 22min; the second centrifugation speed is 12000-16000rpm, such as 13000, 14000, 15000r / min; the second centrifugation time is 20-40min, such as 25, 27, 30, 33, 35min. This scheme adopts a unique centrifugation method, through two centrifugations, as well as the adjustment of the speed, the collection of the precipitate and the supernatant respectively, the final nanoparticle size is very uniform.

[0024] In a second aspect, the present invention discloses a dehydrogenated harmine derivative-PLGA nanoparticle.

[0025] Wherein, the dehydrogenated echinopsine derivative-PLGA nanoparticles are prepared by the method described in the first aspect above.

[0026] In a third aspect, the present invention discloses the use of the dehydrogenated harmine derivative-PLGA nanoparticles described in the second aspect in the preparation of a product for preventing and / or treating echinococcosis granulosus.

[0027] In summary, the present invention provides a PLGA nanoparticle loaded with a dehydrogenated harmine derivative to enhance the efficacy of the dehydrogenated harmine derivative and improve the anti-hydatid efficacy. The method has a simple preparation process, and the obtained nanoparticles have a small particle size and a good sustained release effect. At the same time, the PLGA nanoparticles provided by the present invention have a high encapsulation rate, which can further enhance the bioavailability of the drug; at the same time, the PLGA nanoparticles have a large drug-loading space, and can use fewer carriers to load the dehydrogenated harmine derivative, saving preparation costs, and using fewer carriers, which is also beneficial to human health.

[0028] Beneficial effects:

[0029] The preparation method provided by the present invention is simple, the conditions are mild, and the preparation process has good reproducibility. The obtained dehydrogenated harmine derivative-PLGA nanoparticles have stable physical properties, small and uniform particle size, low polydispersity index, high encapsulation rate, and high drug loading capacity. Compared with the raw material drug dehydrogenated harmine derivative, the anti-hydatid efficacy can be significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0031] Figure 1 This is the structural formula of H8.

[0032] Figure 2 This is a flow chart for the preparation of dehydrogenated echinopsine derivative H8-PLGA nanoparticles.

[0033] Figure 3 This is the standard curve for H8 content determination.

[0034] Figure 4 The effect surface curve is the effect of particle size and encapsulation efficiency.

[0035] Figure 5 This is the particle size distribution diagram of dehydrogenated echinopsine derivative H8-PLGA nanoparticles.

[0036] Figure 6 This is the Zeta potential diagram of dehydrogenated harmine derivative H8-PLGA nanoparticles.

[0037] Figure 7 This is a scanning electron micrograph of the dehydrogenated echinopsine derivative H8-PLGA nanoparticles (×10000).

[0038] Figure 8 It is the blood drug concentration-time curve.

[0039] Fig. 9 This is the result of the changes in liver lesions in mice infected with hepatic echinococcosis treated with dehydrogenated harmaline derivative H8-PLGA nanoparticles.

[0040] Fig.10 The results show the changes in body weight, liver weight and liver proportion of mice after infection.

[0041] Fig.11 HE results of the liver of mice infected with hepatic echinococcosis treated with H8-PLGA nanoparticles, a derivative of harmaline.

[0042] Fig.12 The results are for the detection of mouse serum cytokines. DETAILED DESCRIPTION

[0043] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0044] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0045] The dehydrogenated harmine derivative H8 involved in the embodiment of the present invention is 1-methyl-9-(3-pyridyl)methyl-β-carboline, which has been disclosed in a Chinese patent application (application number "CN202011613055.1" and application name "Application of dehydrogenated harmine derivatives in the preparation of drugs for the treatment or prevention of cystic echinococcosis"), and its structural formula is shown in Figure 1 .

[0046] The PLGA described in the embodiment of the present invention is PLGA 75:25.

[0047] Example 1: Preparation method of dehydrogenated harmine derivative H8-PLGA nanoparticles

[0048] In this study, PLGA nanoparticles were prepared by emulsification method. Figure 2 ): Weigh 100 mg of H8 and dissolve it in 20 mL of ultrapure water as the aqueous phase. Weigh 500 mg of PLGA and dissolve it in 100 mL of chloroform, vortex to dissolve it as the oil phase. Inject the aqueous phase into the oil phase, ultrasonicate at 300W for 5 min under ice bath conditions to obtain colostrum, pour all the obtained colostrum into 500 mL of TCA aqueous solution with a mass concentration of 4 mg / mL, and ultrasonicate at 600W for 10 min under ice bath conditions to obtain a double emulsion. Remove the organic solvent by rotary evaporation, centrifuge at 4500r / min for 20 min, discard the precipitate, collect the supernatant, centrifuge at 15000r / min for 30 min, and disperse the precipitate in ultrapure water to obtain a dehydrogenated harmaline derivative H8-PLGA nanosuspension.

[0049] Example 2: Determination of H8 Content in Haremine Derivative H8-PLGA Nanosuspension

[0050] 2.1 Chromatographic conditions

[0051] Chromatographic column: Inertsil ODS-SP C18 column (4.6×250mm, 5μm)

[0052] Mobile phase: methanol-0.1%wt formic acid water (35:65v / v)

[0053] Detection wavelength: 302nm

[0054] Volume flow rate: 0.8mL / min

[0055] Column temperature: 25°C

[0056] Injection volume: 20 μL

[0057] 2.2 Preparation of test solution

[0058] Take 0.2 mL of the dehydrogenated harmine derivative-PLGA nanoparticle suspension prepared in Example 1 and place it in a 2 mL centrifuge tube, add acetonitrile and make the volume to 2 mL, place it in an ultrasonic instrument and ultrasonicate for 30 min at 80 Hz, vortex oscillate for 120 s, and then filter it with a 0.22 μm pore size filter membrane. The filtrate is the test solution.

[0059] 2.3 Investigation of linear relationship

[0060] Accurately weigh 5.00 mg of the dehydrogenated harmaline derivative H8 reference substance, place it in a 25 mL volumetric flask, dissolve it with methanol and make up to volume to prepare a stock solution with a mass concentration of 200.0 μg / mL. Accurately measure 0.0625, 0.125, 0.25, 0.5, 1.0, 2.0, and 3.0 of the stock solution and place it in a 5 mL volumetric flask, make up to volume with methanol to obtain a series of gradient concentration solutions. Linear regression is performed with concentration as the horizontal coordinate and the peak area of ​​the derivative H8 as the vertical coordinate, and the regression equation is: Y = 66894X + 12345 (r = 0.9999) ( Figure 3 ), indicating that the derivative H8 has a good linear relationship between 2.5 μg / mL and 120 μg / mL.

[0061] 2.4 Precision experiment

[0062] The test solution was taken and sampled 6 times continuously, the peak area was recorded, and the relative standard deviation (RSD) was calculated. The result showed that the RSD was less than 2%, which met the requirements of the content determination methodology and indicated that the instrument had good precision.

[0063] 2.5 Stability test

[0064] Take the test solution, place it at room temperature for 0, 2, 4, 8, 16, 24 hours, then inject and test, record the peak area, and calculate the RSD. The result of RSD is less than 2%, indicating that the test solution has good stability.

[0065] 2.6 Repeatability Experiment

[0066] Six samples were prepared in parallel, injected and measured, the peak area was recorded, and the H8 content in the sample was calculated. The results showed that the average content of H8 in the sample was 40.30 μg / mL, and the RSD was less than 2%, indicating that the established method had good repeatability.

[0067] 2.7 Recovery rate experiment

[0068] Take 9 portions of the dehydrogenated harmine derivative-PLGA nanosuspension with a known content of 40.30 μg / mL prepared in Example 1, 1 mL each, and divide them into 3 groups on average. Add 80%, 100% and 120% of the derivative reference solution respectively, and measure after treatment to calculate the recovery rate of the sample. The results are shown in Table 1.

[0069] The recovery rate calculation formula is: measured amount / theoretical amount, that is, measured amount / (added amount + known content 40.30).

[0070] Table 1 Recovery rate experimental results

[0071]

[0072] Example 3: Determination of encapsulation efficiency and drug loading of dehydrogenated echinopsine derivative-PLGA nanosuspension

[0073] High-speed centrifugation combined with HPLC was used to determine the content of H8 in PLGA nanoparticles, and its encapsulation efficiency (EE) and drug loading efficiency (LE) were calculated.

[0074] Take 5 mL of the dehydrogenated harmaline derivative-PLGA nanoparticle suspension and centrifuge at high speed (4°C, 16000 rpm, 30 min). The supernatant contains free drugs. Take 1 mL of the supernatant, add methanol to 5 mL, vortex and filter with a 0.22 μm microporous membrane. The obtained filtrate is tested according to the chromatographic conditions under Example 2 to measure the concentration of free drugs, which is recorded as W. 游离 . Separately, 1 mL of the dehydrogenated harmaline derivative-PLGA nanoparticle suspension was precisely measured, methanol was added to make the volume 5 mL, ultrasonic demulsification was performed, and the total drug concentration was measured and recorded as W. 总 , and calculated the encapsulation efficiency and drug loading.

[0075] Encapsulation efficiency calculation formula: EE% = (W 总 -W 游离 ) / W 总 ×100%

[0076] The calculation formula of drug loading is: LE% = (W 总 -W 游离 ) / W m ×100%

[0077] (W m represents the mass of H8 in the nanoparticles + the mass of the carrier material).

[0078] Example 4: Prescription screening of dehydrogenated harmine derivative H8-PLGA nanoparticles

[0079] 4.1 Study on the concentration of dehydrogenated echinopsine derivatives

[0080] According to the prescription process, the PLGA mass concentration was fixed at 5 mg / mL, the colostrum ultrasound power was 300 W, the ultrasound time was 5 min, the TCA concentration in 500 mL TCA aqueous solution was 4 mg / mL, the double emulsion ultrasound power was 600 W, and the ultrasound time was 10 min. The effects of different mass concentrations of H8 (1 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, and 10 mg / mL) (the volume of the aqueous phase remained unchanged, only the mass of H8 was changed) on the nanoparticles were investigated, and the particle size, Zeta potential, polydispersity index (Polydispersion index, PDI), and EE were used as evaluation indicators. The experimental results are shown in Table 2. As the concentration of H8 increases, the particle size does not change much, the encapsulation efficiency gradually increases, and the encapsulation efficiency decreases when the concentration exceeds 7.5 mg / mL. Therefore, 7.5 mg / mL of H8 was selected for subsequent experiments.

[0081] Table 2 Effect of H8 concentration on dehydrogenated echinopsine derivative-PLGA nanoparticles

[0082]

[0083] 4.2 PLGA concentration

[0084] According to the prescription process, the H8 mass concentration was fixed at 7.5 mg / mL, the colostrum ultrasound power was 300 W, the ultrasound time was 5 min, the TCA concentration in 500 mL TCA aqueous solution was 4 mg / mL, the emulsion ultrasound power was 600 W, and the ultrasound time was 10 min. Keeping other parameters unchanged, the effects of PLGA concentrations of 1 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, and 10 mg / mL (the oil phase volume remained unchanged, only the mass of PLGA was changed) on nanoparticles were investigated, and the particle size, Zeta potential, PDI, and EE were used as evaluation indicators. The results are shown in Table 3. As the amount of PLGA increased, the particle size decreased and the encapsulation efficiency increased. However, when the concentration of PLGA exceeded 5 mg / mL, the particle size of the prepared nanoformulation increased and the encapsulation efficiency decreased. The reason may be that the high carrier concentration hindered the drug from forming a nanosystem, resulting in a decrease in the encapsulation efficiency. Therefore, 5 mg / mL of PLGA was selected for subsequent experiments.

[0085] Table 3 Effect of PLGA concentration on dehydrogenated echinopsine derivative-PLGA nanoparticles

[0086]

[0087] 4.3 Investigation of Ultrasonic Power of Colostrum Suspension

[0088] According to the prescription process, the H8 mass concentration was fixed at 7.5 mg / mL, the PLGA mass concentration was 5 mg / mL, the colostrum ultrasound time was 5 min, the TCA concentration in 500 mL TCA aqueous solution was 4 mg / mL, the emulsion ultrasound power was 600 W, and the ultrasound time was 10 min. The effects of colostrum ultrasound power of 200, 300, 400, 500, and 600 W on PLGA nanoparticles were investigated, and the particle size, Zeta potential, PDI, and EE were used as evaluation indicators. The experimental results are shown in Table 4. When the colostrum suspension ultrasound power was 500 W, the prepared nanoparticles had a smaller particle size and the highest encapsulation rate, the PDI value fluctuated within an appropriate range, and the potential was moderate. When the power increased to 600 W, the particle size increased, the encapsulation rate decreased, and the absolute value of the Zeta potential also decreased, which was not conducive to the stability of the nanosystem, indicating that excessive ultrasound power may destroy the nanoparticles. In summary, the ultrasound power of the colostrum suspension was selected to be 500 W.

[0089] Table 4 Effect of ultrasonic power of colostrum suspension on dehydrogenated echinopsine derivative-PLGA nanoparticles

[0090]

[0091]

[0092] 4.4 Ultrasonication time of colostrum suspension

[0093] According to the prescription process, the H8 mass concentration was fixed at 7.5 mg / mL, the PLGA mass concentration was 5 mg / mL, the colostrum ultrasound power was 500 W, the TCA concentration in 500 mL TCA aqueous solution was 4 mg / mL, the emulsion ultrasound power was 600 W, and the ultrasound time was 10 min. The effects of colostrum ultrasound time of 4, 6, 8, 10, and 12 min on PLGA nanoparticles were investigated, and the particle size, Zeta potential, PDI, and EE were used as evaluation indicators. The experimental results are shown in Table 5. It can be seen from the results that when the ultrasound time is in the range of 8-12 min, the encapsulation rate does not change much. When the ultrasound time reaches 12 min, although the particle size is small, the absolute value of the Zeta potential decreases, the nanoparticles are unstable, and the encapsulation rate is slightly reduced at this time. When the ultrasound time is 10 min, each index is relatively good, so the colostrum suspension time of 10 min is selected for subsequent experiments.

[0094] Table 5 Effect of ultrasonic power of colostrum suspension on dehydrogenated echinopsine derivative-PLGA nanoparticles

[0095]

[0096] 4.5TCA concentration

[0097] According to the prescription process, the H8 mass concentration was fixed at 7.5 mg / mL, the PLGA mass concentration was 5 mg / mL, the colostrum ultrasound power was 500 W, the ultrasound time was 10 min, the emulsion ultrasound power was 600 W, and the ultrasound time was 10 min. The effect of TCA concentrations of 1, 2, 4, 6, and 8 mg / mL (the volume of the TCA solution remained unchanged, only the mass of TCA was changed) on PLGA nanoparticles was investigated, and the particle size, Zeta potential, PDI, and EE were used as evaluation indicators. The results are shown in Table 6. When the concentration of sodium taurocholate was 4 mg / mL, the nanoparticles prepared had a smaller particle size and a higher encapsulation rate, the PDI value fluctuated within an appropriate range, and the potential was moderate. Therefore, a 4 mg / mL sodium taurocholate solution was selected for subsequent experiments.

[0098] Table 6 Effect of sodium taurocholate concentration on dehydrogenated echinopsine derivative-PLGA nanoparticles

[0099]

[0100]

[0101] 4.6 Ultrasonic power of emulsion suspension

[0102] According to the prescription process, the H8 mass concentration was fixed at 7.5 mg / mL, the PLGA mass concentration was 5 mg / mL, the colostrum ultrasound power was 500 W, the ultrasound time was 10 min, the concentration of sodium taurocholate in 500 mL TCA aqueous solution was 4 mg / mL, and the emulsion ultrasound time was 10 min. The effects of emulsion ultrasound power of 200, 300, 400, 500, and 600 W on nanoparticles were investigated, and the particle size, Zeta potential, PDI, and EE were used as evaluation indicators. The experimental results are shown in Table 7. The ultrasonic power of the emulsion suspension had little effect on the encapsulation rate, but as the power increased, the particle size decreased. However, when the power exceeded 500 W, the particle size increased and the absolute value of the Zeta potential decreased. Therefore, the 500 W ultrasonic power of the emulsion suspension was selected for subsequent experiments.

[0103] Table 7 Effect of ultrasonic power of multiple emulsion suspension on dehydrogenated echinopsine derivative-PLGA nanoparticles

[0104]

[0105] 4.7 Investigation of ultrasonic time of emulsion suspension

[0106] According to the prescription process, the H8 mass concentration was fixed at 7.5 mg / mL, the PLGA mass concentration was 5 mg / mL, the colostrum ultrasound power was 500 W, the ultrasound time was 10 min, the concentration of sodium taurocholate in 500 mL TCA aqueous solution was 4 mg / mL, and the emulsion ultrasound power was 500 W. The effect of emulsion ultrasound time of 4, 6, 8, 10, and 12 min on PLGA nanoparticles was investigated, and the particle size, Zeta potential, PDI, and EE were used as evaluation indicators. The results are shown in Table 8. The ultrasound time has little effect on the encapsulation rate, but the changes are obvious. When the ultrasound time of the emulsion suspension is 10 min, the particle size of the prepared nanoparticle is the smallest and the PDI value fluctuates within an appropriate range, and the absolute value of the potential is the largest. Therefore, the 10-min ultrasound time of the emulsion suspension was selected for subsequent experiments.

[0107] Table 8 Effect of ultrasonic time of double emulsion suspension on dehydrogenated echinopsine derivative-PLGA nanoparticles

[0108]

[0109] 4.8 Volume ratio of colostrum to TCA solution

[0110] According to the prescription process, the H8 mass concentration was fixed at 7.5 mg / mL, the PLGA mass concentration was 5 mg / mL, the colostrum ultrasound power was 500 W, the ultrasound time was 10 min, the TCA concentration was 4 mg / mL, the emulsion ultrasound power was 500 W, the ultrasound time was 10 min, and the volume of TCA solution was changed to investigate the effect of the volume ratio of colostrum to TCA solution of 1:1, 1:2.5, 1:5, 1:75, and 1:10 on PLGA nanoparticles. The particle size, Zeta potential, PDI, and EE were used as evaluation indicators. The experimental results are shown in Table 9. When the volume ratio of colostrum to TCA solution was 1:10, the prepared nanoparticle had the smallest particle size and the highest encapsulation rate, the PDI value fluctuated within an appropriate range, and the absolute value of potential was the largest. Therefore, the volume ratio of colostrum to TCA was selected as 1:10.

[0111] Table 9 Effect of the volume ratio of colostrum to sodium taurocholate on the effect of harmine derivative-PLGA nanoparticles

[0112]

[0113] 4.9 Response surface methodology for optimizing prescriptions

[0114] For nanoformulations, particle size and encapsulation efficiency are important indicators. The single factor results showed that H8 mass concentration, PLGA mass concentration and colostrum to TCA volume ratio were the main factors affecting the encapsulation efficiency and particle size of nanoparticles. Therefore, according to the response surface design principle, the H8 mass concentration (A), PLGA mass concentration (B) and colostrum to TCA volume ratio (C) were used as factors, and the encapsulation efficiency (R1) and particle size (R2) were used as response values ​​for experimental design. The factor level table was randomly given by the Design-expert software, and the results are shown in Table 11. The software was used to analyze and fit the factors and indicators, and the resulting model was: Encapsulation efficiency = 89.55 + 3.04A + 1.24B + 3.20C - 0.1072AB + 0.9577AC - 2.97BC - 8.11A 2 -3.07B 2 -8.10C 2 (R 2 =0.9677); particle size = 216.78-14.32A+0.1488B-0.0512C+1.58AB+5.29BC-1.43BC+43.67A 2 +0.4090B 2 +36.86C 2 (R 2 =0.9421). The results are shown in Tables 10-11. The effect surface curves of the effects of two factors on the encapsulation efficiency are shown in Figure 4 The best preparation process conditions calculated by software analysis are: when the mass concentration of H8 is 6.8 mg / mL, the mass concentration of PLGA is 4.2 mg / mL, and the volume ratio of colostrum to TCA is 1:10, the theoretical particle size can reach 205.5 nm, and the encapsulation rate can reach 89.76%. The smaller the particle size, the higher the encapsulation rate.

[0115] Table 10 Factor level table

[0116]

[0117] 4.10 Process Validation

[0118] Three batches of dehydrogenated harmaline derivative H8-PLGA nanoparticles were prepared according to the optimal prescription and process obtained by optimization. The particle size and encapsulation efficiency were measured, compared with the predicted value, and the deviation between the actual value and the predicted value was calculated [deviation = (predicted value - actual value) / predicted value]. The results showed that the relative deviation between the actual value and the predicted value was less than ±5%.

[0119] Comparative Example

[0120] The best recipe process obtained by optimization is different in the centrifugation method.

[0121] After two centrifugations, the first centrifugation was at 3000 r / min for 10 min, and the second centrifugation was at 10000 r / min for 15 min. The obtained nanoparticles had a non-uniform size (450.23±100.20) nm, and the encapsulation efficiency was 65.3%.

[0122] The organic solvent was removed by centrifugation and rotary evaporation, and the mixture was centrifuged at 12000 r / min for 30 min. The particle size of the obtained nanoparticles was (285.43±50.20) nm, and the encapsulation efficiency was 56.8%.

[0123] Example 5 Characterization of Haremine Derivative-PLGA Nanoparticles

[0124] 5.1 Particle size and potential

[0125] The dehydrogenated harmaline derivative H8-PLGA nanoparticles prepared by the optimal process were placed in a Malvern laser particle size analyzer to measure the particle size distribution and Zeta potential of the nanoparticles. The results showed that the average particle size of the prepared nanoparticles was (123.42±12.25)nm and the average potential was (-34.54±2.58)mV. The results are shown in Figure 5 and Figure 6 .

[0126] 5.2 Morphological observation

[0127] Take an appropriate amount of drug-loaded nanoparticles on the sample stage, treat them with gold spray, and observe the surface morphology of the microspheres under a scanning electron microscope. Figure 7 The dehydrogenated harmaline derivative H8-PLGA nanoparticles are spherical, uniform in size, and have smooth and round spherical walls.

[0128] Example 6: In vivo bioavailability experiment

[0129] Twelve SD rats, half male and half female, were randomly divided into two groups, H8 group and H8-PLGA nanoparticle group (H8-PLGA nanosuspension of dehydrogenated harmine derivative prepared by the best process, H8-PLGA-NPs), with 6 rats in each group. The dosage was 50 mg / kg (based on H8 content). 0.4 mL of blood was collected from the fundus venous plexus at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h after administration, and placed in an Ep tube with 1% sodium heparin anticoagulation, centrifuged at 3500 r / min for 15 min, 0.1 mL of supernatant was aspirated, 10 μL of tinidazole internal standard solution with a mass concentration of 8.16 μg / mL was added, vortexed, 290 μL of chromatographic acetonitrile was added, vortexed for 120 s, and centrifuged at 12000 r / min for 15 min (4 ° C). 200 μL of supernatant was injected for analysis, blood drug concentration was determined, and blood drug concentration-time curve was drawn. Figure 8The pharmacokinetic parameters are shown in Table 12. The results showed that the peak time of blood drug concentration in H8 group was very fast, t max 0.5h, C max The blood concentration of the drug decreased rapidly after that. The biological half-life (t 1 / 2 ) was 1.08h, and the drug concentration was basically 0 at 8h. max Extended to 1.0h, C max (232.70±37.13) ng / mL, t 1 / 2 The MRT was significantly longer than that of the H8 group at 3.42 h, and the drug could still be detected after 24 h. The results showed that H8-PLGA nanoparticles had a certain sustained release effect. According to the blood drug concentration-time curve area (AUC), the bioavailability of the nanodrug was increased to 2.9 times the original. The results showed that H8-PLGA nanoparticles could slow down the elimination rate of drugs, increase the absorption of drugs, and effectively improve the bioavailability of H8.

[0130] Table 12 Pharmacokinetic parameter results (n=6)

[0131] parameter H8 H8-PLGA nanoparticles <![CDATA[C max (ng / mL)]]> 197±47.66 232.70±37.13 <![CDATA[t max ]]> 0.5±0.00 1.00±0.00 <![CDATA[t 1 / 2 (h)]]> 1.08±0.32 3.42±1.36 AUC (ng / mL*h) 338.75±58.20 992.79±262.16 MRT(h) 1.96±0.21 4.35±0.88

[0132] Embodiment 7:

[0133] Evaluation of PLGA nanoparticles derived from harmine against hepatic echinococcosis in mice

[0134] 7.1 Establishment of animal model of echinococcosis

[0135] Anesthesia was induced in 6-8 week old SPF grade Kunming mice, and the abdomen was opened after fixation to expose the portal vein. 0.2 mL of Echinococcus suspension (containing about 3000 Echinococcus granulosus, with vitality > 98%) was inoculated into the mice through the portal vein via a scalp needle. The wound was sutured and the mice were placed on an electric blanket for resuscitation. After waking up, they were transferred to a normal environment for feeding. Six months after infection, the model formation of the mice was observed by B-ultrasound.

[0136] 7.2 Pharmacodynamics of dehydrogenated harmaline derivative-PLGA nanoparticles against hepatic echinococcosis

[0137] 7.2.1 Grouping

[0138] In order to verify the effect of dehydrogenated harmaline derivative-PLGA nanoparticles against hepatic Echinococcus granulosus, an in vivo pharmacodynamic experiment was performed. The model mice were randomly divided into 5 groups, namely, negative control group (CON), model group, positive drug group and 2 drug intervention groups, with 6 mice in each group. The negative control group and the model group were given normal saline; the positive drug group was given albendazole (Albenzole, ABZ) at a dose of 50 mg / kg; the drug intervention groups were the derivative group (H8) and the derivative nano group (dehydrogenated harmaline derivative H8-PLGA nanosuspension prepared by the best process, H8-PLGA-NPs), and the dose was 50 mg / kg (based on H8 content). The drug was administered by gavage once a day for 30 consecutive days.

[0139] 7.2.2 Pharmacodynamic test results

[0140] After the drug intervention, the mice were weighed, and the free liver was observed and weighed. Results The liver lesions of the mice in the model group were obvious, and the lesion area occupied a large volume of the liver. The ABZ group, H8 group, and H8-PLGA-NPs group all had a certain improvement effect on the lesions. Among them, the H8-PLGA-NPs group had a more significant improvement effect than the ABZ group and H8 group, with a significant reduction in the number of lesions, a significant reduction in the volume of lesions, and the ability to calcify the lesions. Representative pictures are shown in Fig. 9 As shown. After intervention with different drugs, the liver weight of mice decreased to varying degrees, but there was no significant difference in liver weight between the ABZ and H8 groups and the model group (p>0.05). Compared with the model group, the liver weight and liver proportion of the H8-PLGA-NPs group were significantly reduced (p<0.01), and the liver weight was also significantly different from that of ABZ and H8 (p<0.01). Representative pictures are shown in Fig.10 The results suggest that H8-PLGA-NPs have a more obvious effect in improving liver lesions and have a more advantageous anti-hydatid effect.

[0141] H&E staining was performed on liver sample sections. Vesicles and cyst wall structures were clearly observed in liver sections of mice in the model group. The liver structure was obviously damaged, liver cells were arranged in disorder, and inflammatory cells infiltrated in the liver lobules. The liver structures of the ABZ group, H8 group, and H8-PLGA-NPs group were alleviated to varying degrees compared with the model group. The damage to the liver lobules was improved, and the infiltration of inflammatory cells was reduced. The improvement effect of the H8-PLGA-NPs group on the liver was more significant. Representative pictures are shown below. Fig.11 shown.

[0142] 7.2.3 Immunoreactivity Detection of Haremine Derivative-PLGA Nanoparticles

[0143] In the study, Luminex liquid phase chip technology was used to quantitatively detect the changes in inflammatory factors in mice in different groups. This technology detects cytokines by quantifying the protein in mouse serum samples, and is particularly used to detect the effects of dehydrogenated echinococcus derivative-PLGA nanoparticles on mice infected with Echinococcus granulosus. Mouse serum samples were transported to Shanghai Unionway Company for testing using dry ice to ensure the stability of the samples and the accuracy of the test results. The experimental results are as follows Fig.12 As shown in the figure, in the H8-PLGA-NPs group, compared with the model group, inflammatory factors IL-2, IL-6, TNF-α, MIP-1α, IL-13, KC, GM-CSF, G-CSF and other cytokines showed a significant decrease trend. These cytokines play a key role in the inflammatory response, among which IL-1, IL-6 and TNF-α are key proinflammatory cytokines. They regulate cell-mediated immune responses and play an important role in regulating the immune system. These inflammatory factors usually regulate the growth, activation, differentiation and homing of immune cells to the site of infection, ultimately controlling and eradicating intracellular pathogens.

[0144] The anti-inflammatory effect of harmine PLGA nanoparticles suggests that they may have potential application value in the treatment of echinococcosis by reducing the levels of proinflammatory cytokines and alleviating inflammatory responses. This discovery provides an important experimental basis for further research on the pharmacological effects and clinical applications of harmine derivative-PLGA nanoparticles.

[0145] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing dehydrogenated echinopsine derivative-PLGA nanoparticles, characterized in that: The following steps are involved: (1) mixing the aqueous phase containing the dehydrogenated harmaline derivative and the oil phase containing PLGA, and ultrasonicating them in an ice bath to obtain colostrum; (2) The obtained colostrum is mixed with sodium taurocholate solution and ultrasonicated in an ice bath to obtain a double emulsion.

2. The preparation method according to claim 1, characterized in that: In step (1), the concentration of the dehydrogenated harmine derivative in the aqueous phase is 1-10 mg / mL.

3. The preparation method according to claim 1, characterized in that: In step (1), the concentration of PLGA in the oil phase is 1-10 mg / mL; and the solvent of the oil phase includes chloroform.

4. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio of the dehydrogenated echinopsine derivative to PLGA is 1:3-4.

5. The preparation method according to claim 1, characterized in that: In step (1), the power of the ice bath ultrasound is 200-600 W, and the time of the ice bath ultrasound is 4-12 min.

6. The preparation method according to claim 1, characterized in that: In step (2), the concentration of sodium taurocholate in the sodium taurocholate solution is 1-8 mg / mL; and the volume ratio of the colostrum to the sodium taurocholate solution is 1:(5-12.5).

7. The preparation method according to claim 1, characterized in that: In step (2), the power of the ice bath ultrasound is 200-600 W, and the time of the ice bath ultrasound is 4-12 min.

8. The preparation method according to claim 1, characterized in that: Also includes: The obtained double emulsion is subjected to rotary evaporation and a first centrifugation, and the supernatant obtained by the first centrifugation is subjected to a second centrifugation, and the precipitate obtained by the second centrifugation is the dehydrogenated harmine derivative-PLGA nanoparticles; preferably, the obtained precipitate is dispersed in water to obtain a dehydrogenated harmine derivative-PLGA nanoparticle suspension; preferably, the speed of the first centrifugation is 4000-5000rpm, and the time of the first centrifugation is 15-25min; preferably, the speed of the second centrifugation is 12000-16000rpm, and the time of the second centrifugation is 20-40min.

9. A dehydrogenated harmine derivative-PLGA nanoparticle, characterized in that: Made by the method described in any one of claims 1 to 8.

10. Use of the dehydrogenated harmine derivative-PLGA nanoparticles according to claim 9 in the preparation of a product for preventing and / or treating echinococcosis granulosus.

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