CN115919774A - Magnetic targeting nano albendazole liposome and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of parasitic disease prevention and treatment, in particular to a magnetic targeting nano albendazole liposome and a preparation method thereof. The specific technical scheme is as follows: the magnetic targeting nano albendazole liposome is characterized in that: mixing the magnetic particles and the albendazole liposome, and homogenizing to obtain magnetic nano albendazole liposome; the particle size range of the magnetic nano albendazole liposome is 200-300 nm. The invention solves the problems of larger particle size, lower entrapment rate, lower oral bioavailability and nonideal drug targeting of the albendazole liposome in the prior art.

Description

Magnetic targeting nano albendazole liposome and preparation method thereof

Technical Field

The invention relates to the technical field of parasitic disease prevention and treatment, in particular to a magnetic targeting nano albendazole liposome and a preparation method thereof.

Background

Echinococcosis is a common parasitic disease of both human and livestock seriously harming human health, and among echinococcosis in human body, echinococcosis accounts for 70%. Xinjiang and Qinghai are high-traffic areas, the threatened population reaches 6600 ten thousand, and the economic loss is over 30 million yuan each year. Albendazole (ABZ) is used as one of the first-choice drugs for chemotherapy of echinococcosis specified by WHO, the common dosage form of albendazole has poor intestinal absorption and low liver distribution concentration, and the drug concentration in the echinococcus bursa of infection focus only accounts for 1/10-1/100 of the blood, so the clinical cure rate is only 30.1%, and the clinical application of albendazole is severely restricted.

The liposome is a dosage form which is researched more in recent years, has passive targeting, can be quickly taken by human Reticuloendothelial (RES) system, achieves higher concentration in organs such as liver, spleen and the like, and can also increase the solubility of insoluble drugs, so the liposome is an ideal drug carrier for treating the liver echinococcosis. Chinese patent CN02152519.6 provides a prescription of albendazole liposome (L-ABZ), which contains 0.5-1.0% of albendazole powder, 1.0-3.0% of lecithin, 0-0.3% of sodium benzoate, 0-0.02% of antioxidant and 0.7-0.9% of sodium chloride solution. The liposome is prepared by a neutralization method, the obtained L-ABZ can increase the solubility of the ABZ and improve the bioavailability, and the patent is successfully converted into a hospital preparation by the first subsidiary hospital of Xinjiang medical university and is widely used for treating clinical echinococcosis. There are three disadvantages, however: (1) the liposome has larger and non-uniform particle size; the ABZ encapsulation rate is lower, and is about 60-70% on average; (2) oral bioavailability remains to be improved; (3) the common liposome mainly has certain liver passive targeting, but the drug targeting of the infected part is not ideal.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a magnetic targeting nano albendazole liposome and a preparation method thereof, and solves the problems of larger liposome particle size, lower ABZ entrapment rate, lower oral bioavailability and unsatisfactory drug targeting in the prior art.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention discloses a magnetic targeting nano albendazole liposome, which is prepared by mixing magnetic particles with the albendazole liposome, and homogenizing the mixture to obtain the magnetic nano albendazole liposome; the particle size range of the magnetic nano albendazole liposome is 200-300 nm.

Preferably, the magnetic particles are ferroferric oxide modified by citric acid.

Correspondingly, the preparation method of the magnetic targeting nano albendazole liposome comprises the steps of adding magnetic particles into the albendazole liposome, and carrying out high-pressure homogenization to obtain the magnetic nano albendazole liposome.

Preferably, the homogenization pressure is 5000-25000 psi, the homogenization temperature is 0-60 ℃, and the homogenization times are 2-14 times.

Preferably, the magnetic particles are ferroferric oxide modified by citric acid.

Preferably, the mass ratio of the magnetic particles to the albendazole in the albendazole liposome is 1.

Preferably, the citric acid modified ferroferric oxide process comprises the following steps: feCl is added ₃ ·6H ₂ O and FeCl ₂ ·4H ₂ Dissolving the mixture O in dilute hydrochloric acid, continuously introducing nitrogen, rapidly stirring in a water bath condition, rapidly adding concentrated ammonia water, keeping rapid stirring, adding citric acid solution, and continuously keeping rapid stirring; heating and curing are carried out, the stirring speed is reduced, the introduction of nitrogen is kept, and the introduction of nitrogen is stopped after the reaction is finished; stopping heating, adjusting pH to neutral with dilute hydrochloric acid, dialyzing to obtain citric acid modified ferroferric oxide, and freeze-drying for use.

Preferably, the FeCl ₃ ·6H ₂ O and FeCl ₂ ·4H ₂ The molar ratio of O is 1.5-2.5, the mixture is mixed and dissolved in 1moL/L of dilute hydrochloric acid, the volume ratio of the total mass of the mixture to the dilute hydrochloric acid is 1-1, the water bath temperature is 20-60 ℃, the stirring time is 20-40 min, 25% concentrated ammonia water in mass-volume ratio is rapidly added, and the added concentrated ammonia water is concentratedThe volume ratio of the ammonia water to the dilute hydrochloric acid is 7-8, the rapid stirring time is kept for 20-40 min, the molar ratio of the citric acid in the citric acid solution to the mixture is 3; then heating to 80-90 ℃ and curing for 1-3 h; adjusting the pH value with 1moL/L dilute hydrochloric acid, and the dialysis time is 48-120 h.

The invention has the following beneficial effects:

the invention discloses a magnetic targeting nano albendazole liposome, which is a nano-scale liposome formed by wrapping magnetic nanoparticles in the inner water phase of the albendazole nano-liposome or embedding the magnetic particles and the albendazole liposome in a lipid bilayer to form a combination, and has the dual characteristics of the magnetic nanoparticles and the nano-liposome. On one hand, the magnetic nanoparticles are wrapped in the liposome, so that the defects of easy metabolism and short half-life period in the magnetic nanoparticles can be overcome, and the active magnetic targeting effect of the magnetic nanoparticles can be retained; on the other hand, the albendazole magnetic nano liposome still has the characteristics of easy drug entrapment, high entrapment rate, small and uniform particle size, high drug bioavailability, passive targeting, reduction of drug toxicity and the like of the albendazole nano liposome.

Drawings

FIG. 1 is a graph of the distribution of the L-ABZ and ML-ABZ particle sizes;

FIG. 2 is a TEM image of ML-ABZ;

FIG. 3 is a graph showing the behavior of 4 different formulations;

FIG. 4 shows the distribution of 4 preparations in liver and lung tissues;

FIG. 5 is a graph of pathological changes in liver tissue (HE, 200X) in mice;

FIG. 6 is a graph of mouse vesicle histopathological changes (HE, 200 ×);

FIG. 7 is a graph showing the ultrastructural change (10000X) of mouse vesicle tissue.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1

The invention discloses a magnetic targeting nano albendazole liposome, which is prepared by mixing magnetic particles with the albendazole liposome and homogenizing. Wherein, the magnetic particles are ferroferric oxide modified by citric acid.

Example 2 preparation of magnetically targeted Nanobendazole liposomes

The method specifically comprises the following steps:

(1) Citric Acid (CA) -modified ferroferric oxide (Fe) ₃ O ₄ ) Preparation of (2)

Weighing FeCl ₃ ·6H ₂ O3.76 g and FeCl ₂ ·4H ₂ O1.60 g, placed in a 250mL three-necked round bottom flask, dissolved by adding 8mL of dilute hydrochloric acid (1 moL/L), and purged with nitrogen. Rapidly stirring for 30min by a magnetic stirrer in a water bath kettle at 40 ℃, rapidly adding 60mL of concentrated ammonia water (25%), and maintaining the rapid stirring of the magnetic stirrer for 30min; after the ammonia water is added dropwise, adding 80mL of 0.5moL/L citric acid solution, continuously keeping a magnetic stirrer to stir rapidly for 30min, simultaneously heating to 85 ℃, slowing down the stirring speed, curing for 2h, keeping introducing nitrogen in the experimental process, stopping introducing nitrogen after the reaction is finished, stopping heating, adjusting the pH value of the solution to about 7.0 by using dilute hydrochloric acid (1 moL/L), and dialyzing the obtained black substance for 72h to obtain citric acid modified ferroferric oxide (Fe) ₃ O ₄ @ CA), lyophilized, ready for use.

(2) Preparation of magnetic nano albendazole liposome (ML-ABZ)

The preparation method of the albendazole liposome comprises the following steps: albendazole: mixing soybean phospholipids according to a mass ratio of 1.

Mixing Fe ₃ O ₄ @ CA Add Albendazole Liposome (Fe) ₃ O ₄ @ CA, albendazole =1, 15, g/g), and adopting a micro-jet high-pressure homogenizer to obtain the magnetic nano albendazole liposome under the homogenizing pressure of 5000-25000 psi, the homogenizing circulation frequency of 2-14 times and the homogenizing temperature of 0-60 ℃.

EXAMPLE 3 Effect of homogenization conditions on ABZ encapsulation efficiency

1. Pressure of homogenization

Fixing homogenizing cycle for 10 times, homogenizing at 40 deg.C, preparing ML-ABZ under different homogenizing pressure conditions of 5000, 10000, 15000, 20000, and 25000psi, respectively, measuring particle size and ABZ encapsulation rate, and calculating comprehensive score. The composite score was evaluated comprehensively using the particle size and ABZ encapsulation ratio as indices (weight coefficients of 50% and 50%, respectively). Composite score% = (minimum average particle size/average particle size) × 50% + (ABZ envelope/maximum ABZ envelope) × 50%.

The results are shown in Table 1. The results show that the ML-ABZ particle size is continuously reduced and the ABZ encapsulation efficiency is continuously increased along with the continuous increase of the homogenizing pressure. The 5000psi composite score was lowest and the 25000psi composite score was highest. Considering that the higher the pressure, the more the loss of the high pressure homogenizer is, which is not good for the enlargement of the production, 10000psi, 15000psi and 20000psi were selected for further investigation.

Table 1 effect of homogenization pressure on liposomes (n = 3)

Pressure (psi)	Particle size (nm)	Encapsulation efficiency (%)	Composite score: (％)
				5000	316.25±9.64	79.87±5.15	74.82
10000	275.11±8.53	91.59±3.20	85.89
				15000	255.67±5.26	93.52±2.17	91.245
20000	213.13±3.08	95.62±1.57	99.30
				25000	209.39±3.56	95.73±2.16	100.00

2. Number of homogenization cycles

Fixing homogenizing pressure at 20000psi, homogenizing temperature at 40 deg.C, preparing ML-ABZ for 2, 4, 6, 8, 10, and 12 times respectively under different homogenizing cycle number, measuring particle diameter and ABZ encapsulation rate, and calculating comprehensive score.

The results are shown in Table 2. The results show that the particle size of the liposome is continuously reduced with the continuous increase of the homogenization times, and the ABZ entrapment rate is increased and then reduced. The comprehensive score is lowest when the homogenization is performed for 2 times, and is highest when the homogenization is performed for more than 8 times, so that 6 times, 8 times and 10 times are selected for further investigation.

Table 2 effect of number of homogeneous cycles on liposomes (n = 3)

Number of homogenisations	Particle size (nm)	Encapsulation efficiency (%)	Composite score (%)
				2	254.37±3.07	91.07±1.71	86.91
4	235.80±4.38	92.69±2.46	90.88
				6	222.62±3.06	93.75±1.84	93.96
8	217.03±4.62	94.46±1.56	95.50
				10	210.13±3.35	96.46±2.09	98.06
12	208.92±2.52	95.79±1.22	97.99

3. Homogenization temperature

Fixing homogenizing pressure at 20000psi, homogenizing for 10 times, respectively preparing ML-ABZ at different homogenizing temperatures of 0, 20, 40, and 60 deg.C, measuring particle size and ABZ encapsulation rate, and calculating comprehensive score.

The results are shown in Table 3. The results show that the liposome particle size decreases and then increases with increasing homogenization temperature, while the encapsulation shows a first increase and then decrease. The highest comprehensive score for homogeneity at 40 ℃ was obtained, so 20 ℃, 40 ℃ and 60 ℃ were selected for further investigation.

Table 3 effect of homogenization temperature on liposomes (n = 3)

Homogenization temperature (. Degree. C.)	Particle size (nm)	Encapsulation efficiency (%)	Composite score (%)
				0	238.28±4.10	91.41±1.49	92.32
20	227.68±3.18	93.32±2.02	95.40
				40	213.64±3.50	96.24±1.88	100.00
60	248.11±5.33	89.78±1.65	89.70

4. Further optimizing homogeneity parameters in the preparation of ML-ABZ

The factors are: homogenizing pressure, homogenizing cycle number and homogenizing temperature, selecting three levels for each factor, and designing L ₉ (3 ⁴ ) Orthogonal experiments, see table 4.

TABLE 4 orthogonal experimental design

The orthogonal test was performed according to table 4, and the particle size, ABZ encapsulation efficiency, comprehensive score value and visual analysis of the test results are shown in table 5, and the analysis of variance is shown in table 6.

From the visual analysis of Table 5, when ML-ABZ is prepared by microfluidization high-pressure homogenization, the factors that most affect the particle size and the encapsulation efficiency of ABZ are A (homogenization pressure), B (number of homogenization cycles), and C (homogenization temperature). Namely A is more than B and more than C, and the optimal homogenizing process is A ₃ B ₃ C ₂ 。

As shown in the analysis of variance in Table 6, the factors A and B have statistical significance, which indicates that the homogenization pressure and frequency have significant influence on the particle size and the encapsulation efficiency (P is less than 0.05), and C (homogenization temperature) has no significant influence on the particle size and the ABZ encapsulation efficiency (P is more than 0.05). Therefore, the optimized homogenizing process is determined to be A by comprehensively considering the large-scale production and the experimental controllability ₃ B ₃ C ₁ I.e., 20000psi at 20 deg.C for 10 cycles.

TABLE 5 results and analysis of orthogonal experiments

TABLE 6 ANOVA TABLE

Source of variation	SS	V	MS	F	P
						Pressure of	226.30	2	113.15	25.78	0.001
Number of cycles	135.42	2	67.71	15.42	0.001
						Temperature of	18.97	2	9.48	2.16	0.17
Error of the measurement	17.25	2	4.39

5. Verifying the parameters of step 4 above

5 batches of ML-ABZ were prepared according to the preferred parameters of the orthogonal test and the particle size and ABZ encapsulation were determined, the results are shown in Table 7. The result shows that the result of the verification test by optimizing the homogeneous parameters is consistent with the result of the orthogonal test, and the method has stability and feasibility. Homogeneity parameters in the preparation process of the magnetic nano albendazole liposome are as follows: homogenization pressure 20000psi, number of homogenization cycles 10, and homogenization temperature 20 ℃. The process is stable and reliable, has good reproducibility and feasibility.

Table 7 verification test results (n = 5)

Sample number	1	2	3	4	5	Mean number	Standard deviation of	RSD％
									Particle size (nm)	214.11	209.17	211.04	209.32	210.19	210.77	2.01	0.96
ABZ encapsulation Rate (%)	96.18	96.43	95.87	95.97	96.36	96.16	0.24	0.25

Example 4 magnetic Nanobendazole Liposome characterization

The magnetic nano-albendazole liposomes prepared according to the optimal homogeneity parameters given in example 3 were characterized below.

1. Measuring the particle size of the magnetic nano albendazole liposome by adopting a laser scattering method: according to the volume ratio of 10% polysorbate 80 ML-ABZ of 4. As a result, the ML-ABZ particles had an average particle diameter of 211.57. + -. 1.23nm and a PDI value of 0.34. + -. 0.04, and it is clear from FIG. 1 (b) that the particle diameter distribution was uniform and the particle diameter ranged from 200 to 300nm. FIG. 1 (a) shows that the average particle diameters of L-ABZ are 571.82. + -. 2.16nm and the PDI value is 0.34. + -. 0.03, respectively.

2. Morphology of magnetic nano albendazole liposome

The ML-ABZ with the concentration of 0.1g/mL is completely dissolved by water, diluted by 10 times, dripped on a copper net coated with a carbon film, dyed by 2 percent phosphotungstic acid after being dried, absorbed by filter paper to absorb redundant liquid, dried and observed under a transmission electron microscope. The TEM results show (FIG. 2) that the prepared ML-ABZ is round or quasi-round particles and has uniform size.

3. The encapsulation rate of the prepared magnetic nano albendazole liposome is measured

(1) Encapsulation efficiency of ABZ in ML-ABZ

The determination is carried out by using Sephadex column chromatography, namely selecting Sephadex G-50 with the particle size of 50-150 um, and carrying out column packing after 0.9% of NaCl injection solution is swelled for 24 hours under the temperature. Gel column diameter height ratio of 1.2, loading amount of 0.5mL, elution with 0.9% NaCl injection, elution flow rate of 5mL min ^-1 Separation of liposomes and free drug was performed. Collecting the first 4-20 mL of primary eluent containing ML-ABZ (17 mL of A), then gently stirring the chromatographic column by using a glass rod, and collecting the second 26-35 mL of eluent containing free ABZ (10 mL of B); separately, the same amount of ML-ABZ suspension before column loading was diluted with 0.9% NaCl solution injection to the same volume as (A) to obtain an unseparated solution (C). Respectively placing 2.5mL of A, B and C in 10mL measuring flask, adding 2.5mL of glacial acetic acid for dissolving, adding anhydrous ethanol to constant volume to scale, shakingHomogenizing; precisely sucking 2.0mL of each of A, B and C from the solution into a 5mL measuring flask, fixing the volume of absolute ethyl alcohol to a scale, and shaking up to be measured; using absolute ethyl alcohol as a blank control, measuring the absorbance at the maximum absorption wavelength of 295nm, and calculating the ABZ content according to a standard curve, wherein the ABZ entrapment rate calculation formula of ML-ABZ is as follows:

the results are shown in Table 8 below. The results showed that the encapsulation efficiency of ABZ in ML-ABZ was 96.27. + -. 1.14%.

TABLE 8 ABZ encapsulation efficiency assay in ML-ABZ (n = 5)

Sample number	1	2	3	4	5	Mean number	Standard deviation of	RSD％
									Encapsulation efficiency (%)	96.03	97.32	95.54	97.55	94.89	96.27	1.14	1.19

(2) Encapsulation of iron in ML-ABZ

Centrifuging ML-ABZ 1mL at 10000r/min for 10min, sucking supernatant, adding 4.0mL of 5% nitric acid and 0.1% triton to lower-layer precipitate, demulsifying and reducing iron ions, and measuring the content of the iron ions by using an inductively coupled plasma emission spectrometer (ICP-OES) to obtain the content of the iron wrapped in the pure liposome; and (3) directly adding 4.0mL of 5% nitric acid and 0.1% triton demulsification reduced iron ions into 1.0mL of the prepared ML-ABZ by the same method, determining the content of the iron ions by adopting ICP-OES (inductively coupled plasma-optical emission spectrometry), taking the content of the iron ions as the total iron content in the magnetic nano-liposome, and calculating the encapsulation rate of the magnetic nano-particles by dividing the content of the iron wrapped in the pure liposome by the content of the iron in the total magnetic nano-liposome.

Plotting the concentration of iron (mu g/mL) as an abscissa (X) and the absorption intensity as an ordinate (Y) to obtain a linear regression equation, wherein the regression equation of ML-ABZ is as follows: y =33710x +1736.2 (r = 0.9999), the encapsulation efficiency of iron in the prepared ML-ABZ was calculated to be 73.25 ± 1.08% (n = 3).

Example 5 bioavailability of magnetic Nanobendazole liposomes

Wistar rats 32 with male and female halves and weight 220 + -20 g. Randomly divided into 4 groups, which are albendazole tablet (T-ABZ) group, albendazole liposome (L-ABZ) group, and albendazole nanoliposome suspension (NL-ABZ) group (the preparation method is the same as that of step (2) of example 2, but Fe is not added ₃ O ₄ @ CA suspension), the magnetic nano albendazole liposome (ML-ABZ) group of the invention. Each group comprises 8 female and male halves. The administration dose is 70 mg/kg ^-1 Before administration, fasting and water-forbidden for 12h, respectively performing intragastric administration according to the weight of the rat, wherein ML-ABZ, and applying a magnetic field with certain intensity to the abdominal and chest part. 0, 0.5h, 1h, 2h, 4h, 8h, 12h,1mL of 24h and 48h fundus venous plexus blood is collected and placed in an Ep tube with 1% heparin sodium anticoagulation. After slight shaking at 8000r min ^-1 Centrifuging for 10min, collecting 500 μ L of plasma, determining the concentration of albendazole sulfoxide (ABZSO) which is the main active metabolite of albendazole in blood by adopting ultra performance liquid chromatography-tandem mass spectrometry (UPLC-TQS), calculating pharmacokinetic parameters of blood concentration-time data of different dosage form groups by using a Winnolin 8.1 pharmacokinetic software non-compartmental model analysis method, carrying out statistical analysis on experimental data by using SPSS19.0 software, and carrying out model embedding by using a GraphPad Prism 6 software drawing practical pharmacokinetic calculation program. The concentration of albendazole sulfoxide (ABZSO), the major active metabolite of albendazole, in the blood is shown in table 9 (mean blood concentration-time data (n = 8) for ABZSO for each dosage form group) and fig. 3.

As can be seen from Table 9, C of ML-ABZ _max Compared with NL-ABZ, the compound has no significant difference but is improved to a certain extent, and is significantly improved compared with T-ABZ and L-ABZ (P is less than 0.01); indicating that the uptake of ML-ABZ of the present invention is significantly increased.

The AUC of ML-ABZ is obviously improved (P is less than 0.01) compared with T-ABZ and L-ABZ, and is improved to a certain extent although no obvious difference is generated compared with NL-ABZ; formula of relative bioavailability: f _rel ＝AUC _t /AUC _r X 100% (footnote t and r represent test and reference formulations, respectively). F of L-ABZ with T-ABZ as reference formulation _rel 119.84% F of NL-ABZ _rel F of ML-ABZ at 376.21% _rel It was 396.44%.

ML-ABZ has a significant decrease in clearance (Cl) compared to T-ABZ and L-ABZ (P < 0.01), and an increase in Mean Residence Time (MRT), indicating that ML-ABZ has a relatively faster rate of decrease in plasma drug concentration, but a slightly slower rate of clearance, and an extended in vivo residence time, suggesting that ML-ABZ is more rapidly and more readily accessible to tissue and metabolism than T-ABZ, and has an extended in vivo residence time.

Compared with T-ABZ and L-ABZ suspensions, ML-ABZ significantly increases the drug absorption amount, is easier to enter tissues and metabolize, prolongs the retention time in vivo, and significantly improves the relative bioavailability of oral administration of rats.

TABLE 9 pharmacokinetic parameters for the different formulations

Note: compared with the T-ABZ group, ^a p＜0.05， ^aa p is less than 0.01; compared with the L-ABZ group, ^b p＜0.05， ^bb p is less than 0.01; in contrast to the NL-ABZ group, ^c p＜0.05， ^cc P＜0.01。

example 6 rat targeting

256 Wistar rats, randomly divided into 4 groups of 8 rats each, were: the preparation method comprises the following steps of (1) preparing albendazole tablets (T-ABZ), albendazole liposome (L-ABZ), albendazole nanoliposome suspension (NL-ABZ-suspension) and magnetic nano albendazole liposome (ML-ABZ) according to the invention, wherein the medicines are administrated in an oral intragastric administration mode, ML-ABZ is adopted, and a magnetic field with certain intensity is applied to the abdominal and chest positions. Respectively taking liver and lung tissues at 0.5h, 2h, 4h, 8h, 12h, 24h, 36h and 48h before and after administration, measuring the concentration of albendazole sulfoxide (ABZSO) in the liver and lung tissues by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-TQS), analyzing and processing the data by Winnolin 8.1 pharmacokinetic software and SPSS19.0, and taking the drug peak concentration (C) in the liver and lung tissues after the rats take four preparations orally _max ) The area under the curve (AUC), mean Retention Time (MRT), and relative uptake (Re) are shown in tables 10 to 12 and FIG. 4. When Re is more than 1, the tissue has certain uptake capacity to the medicine, the medicine is prompted to have certain targeting, and when Re is less than or equal to 1, the tissue does not have the uptake capacity to the medicine, and the calculation formula is as follows: re = AUC _{Targeted formulations} /AUC _{Reference preparation (T-ABZ)} 。

The results show that ML-ABZ can be widely distributed in liver and lung tissues. The same dose is givenAfter administration, NL-ABZ and ML-ABZ have drug C in liver and lung tissues _max AUC and MRT are both obviously larger than T-ABZ (P is less than 0.01), cl is smaller than T-ABZ (P is less than 0.01), and the distribution and the average residence time of the two preparations in liver and lung tissues are obviously increased; c of ML-ABZ compared to NL-ABZ _max AUC, MRT and Cl are also obviously different (P is less than 0.01), which shows that the preparation can obviously improve the distribution of the medicament in tissues; the Re values of the L-ABZ, the NL-ABZ and the ML-ABZ are all larger than 1, which indicates that the 3 preparations have liver and lung targeting; and the Re value of the ML-ABZ is respectively greater than that of the L-ABZ and that of the NA-ABZ, which shows that the ML-ABZ targeting effect is superior to that of the L-ABZ and the NL-ABZ. The ML-ABZ is suggested to greatly improve the medicine intake of the liver and the lung, so that the medicine can be accumulated in the liver and the lung tissues to play a therapeutic role.

Table 10 liver tissue distribution results for the 4 formulations

Note: compared with the T-ABZ group, ^a p＜0.05， ^aa p is less than 0.01; compared with the L-ABZ group, ^b p＜0.05， ^bb p is less than 0.01; in comparison with the NL-ABZ group, ^c p＜0.05， ^cc P＜0.01。

table 11 lung tissue distribution results for the 4 formulations

Note: compared with the T-ABZ group, ^a p<0.05， ^aa P<0.01; compared with the L-ABZ group, ^b p<0.05， ^bb P<0.01; in comparison with the NL-ABZ group, ^c p<0.05， ^cc P<0.01

TABLE 12 relative uptake (Re) in tissue for the formulations

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Example 7 quantitative analysis of active metabolite ABZSO in cyst fluid

ABZ enters the human body and produces its main active metabolite ABZSO through the action of liver granule enzymes, and this metabolite is further metabolized into ABZSO ₂ . The insecticidal activity of ABZ is mainly derived from ABZSO ₂ Does not have any insecticidal activity. The successfully modeled hydatid mice were randomly divided into 8 groups, namely a blank control group, a model group, a positive drug 1 group (ABZ tablet, T-ABZ), a positive drug 2 group (ABZ liposome, L-ABZ), a positive drug 3 group (nano ABZ liposome, NL-ABZ) and a drug group (ML-ABZ high dose group, ML-ABZ medium dose group and ML-ABZ low dose group), wherein each group contains 20 mice. Continuously orally administering ML-ABZ for 15d and 30d, applying a magnetic field with certain intensity to abdomen and chest, killing animals after 4h of the last administration, dissecting out mouse vesicles, sucking out the vesicle liquid by a syringe, and storing at-80 ℃.

And (3) quantitatively analyzing ABZSO which is a main metabolite of the albendazole in the cyst fluid of the mouse by adopting a UPLC-TQS method. The results are shown in table 13, where ABZSO concentration could not be determined at lower vesicle levels for T-ABZ. The ABZSO concentration in ML-ABZ is higher than that in L-ABZ and NL-ABZ groups under the same dosage, the significant difference is realized (P is less than 0.01), and the ABZSO concentration in each ML-ABZ dosage group is increased along with the increase of the drug concentration and time, so that the obvious dose-effect relationship is realized, and the active metabolite ABZSO of ML-ABZ can enter the hydatid cyst of the mouse more than that of L-ABZ and NL-ABZ, and the target property is good, so that the high efficiency of ML-ABZ in resisting echinococcosis is further proved.

TABLE 13 measurement of capsule fluid albendazole sulfoxide concentration in mice (X. + -. S, n = 10)

Note: ND, not detected; compared with the L-ABZ group, ^a p＜0.05， ^aa p is less than 0.01; in comparison with the NL-ABZ group, ^b p＜0.05， ^bb P＜0.01。

example 8 magnetic Nanodiazole Liposome drug Effect

1. Preparation of echinococcus granulosus

Collecting sheep liver naturally infected with echinococcus granulosus from Uflow slaughterhouse in Xinjiang, performing surface sterilization with 75% alcohol, lightly inserting into liver capsule with disposable syringe, extracting cyst fluid, transferring into 50mL centrifuge tube, and standing to allow natural precipitation of original metacercaria. Washing the metacercaria with sterile PBS containing 1% double antibody (streptomycin) under aseptic condition for 3-5 times, adding 1% (by weight) pepsin (pH 2.0), digesting at 37 deg.C for 30min, washing with sterile PBS for 20 times, adding RPMI1640 culture medium, and standing at 37 deg.C and 5% CO ₂ Culturing in a constant temperature incubator, and replacing the culture solution every 4-5 days for later use.

2. Establishment of cystic echinococcosis mouse animal model

Selecting echinococcus granulosus vesicles with uniform size of 2-3 mm under aseptic condition, carrying out intraperitoneal injection and inoculation on Kunming mice with age of 6-8 weeks and weight of 20-25 g, carrying out B-mode ultrasonic detection about 3-4 months after infection, and successfully molding the Kunming mice with the echinococcus granulosus vesicles with diameter of more than 0.5 cm.

3. Experiment grouping

The successfully molded hydatid mice are randomly divided into 8 groups, namely a blank control group, a model group, a positive drug 1 group (ABZ tablet, T-ABZ), a positive drug 2 group (ABZ liposome, L-ABZ), a positive drug 3 group (nano ABZ liposome, NL-ABZ) and a drug intervention group (ML-ABZ high dose group, ML-ABZ medium dose group and ML-ABZ low dose group), and each group comprises 20 mice.

4. Cystic dampness and its cyst inhibiting rate

As shown in table 14, after the infection of the abdominal cavity with KM mice, the treatment was performed with drug intervention for 15 and 30 days, respectively, wherein ML-ABZ was applied, and then a magnetic field of a certain intensity was applied to the abdominal and thoracic region. After the treatment is finished, the mice are subjected to autopsy to measure the wet weight of the bursa, and the reduction amount of the wet weight of the bursa is calculated. The average bursa wet weight of each administration group is reduced to different degrees, and the difference has statistical significance (P is less than 0.01). The wet weight from the sac results show: the NL-ABZ and ML-ABZ dose groups show better anti-hydatid effect after 15 days of intervention and have better treatment effect after 30 days of continuous administration intervention, but the ML-ABZ group has statistical difference (P is less than 0.01) in the bursa wet weight compared with the NL-ABZ at the same dose; ML-ABZ presents typical dose-effect relationship and aging relationship, and the capsule inhibiting effect is stronger and stronger along with the increase of the dose.

The results show that the T-ABZ group has a cyst inhibition rate of 3.6 percent, an L-ABZ cyst inhibition rate of 42.83 percent, an NL-ABZ cyst inhibition rate of 67.93 percent and an ML-ABZ cyst inhibition rate of 84.14 percent under the same dosage along with the prolonging of the administration period; meanwhile, the research finds that the effect of ML-ABZ in the (25 mg/kg) dose for interfering 15 days on the hydatid resistance is equivalent to the effect of ML-ABZ in the NL-ABZ (50 mg/kg) dose for interfering 30 days on the hydatid resistance, and is superior to other positive control groups. The ML-ABZ is prompted to reduce the course of medicine taking if the dosage is unchanged on the basis of improving the anti-hydatid effect; or the treatment course is unchanged, and the dosage of the medicine is reduced, thereby being beneficial to improving the compliance of patients and reducing the occurrence of adverse reactions.

TABLE 14 vesicle wet weight and vesicle inhibition ratio of vesicles in mice of each group

Note: in comparison to the set of models, ^a p＜0.05， ^aa p is less than 0.01; compared with the T-ABZ group, ^b p＜0.05， ^bb p is less than 0.01; compared with the L-ABZ group, ^c p＜0.05， ^cc p is less than 0.01; in comparison with the NL-ABZ group, ^d p＜0.05， ^dd P＜0.01。

5. pathological change of liver

There were different degrees of improvement in the infiltration of inflammatory cells in the model group, positive control group and drug intervention group, with the ML-ABZ middle and high dose groups being the most significant (as shown in fig. 5). The pathological grading and results of the liver tissues are shown in tables 15 and 16.

TABLE 15 mouse liver histopathological section injury grading

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TABLE 16 pathological section observation results of mouse liver tissue of each experimental group

Group of	Inflammation (+, ++, +++)	Edema (+, ++++)	Necrosis (+, ++++)
				Model set	+++	+++	+++
T-ABZ	++	++	++
				L-ABZ	++	+	+
NL-ABZ	+	+	+
				In ML-ABZ	+		+
ML-ABZ Low	+	+	+
				ML-ABZ height	+		+

6. Pathological changes of vesicles

The hair growth layer structure of the model group vesicle is continuous and complete, the horny cortex is in a regular striation structure, the outer membrane is formed by host fiber cells, and the boundary with the horny cortex is clear; the hair growth layer structures of the positive control group and the medicament intervention group are damaged in different degrees; the outer stratum corneum cells were reduced to varying degrees, and the capsular lamellar structure was altered more significantly in the ML-ABZ, high dose group (see fig. 6). The classification of pathological damage of vesicle tissue and the results are shown in tables 17 and 18.

TABLE 17 pathological grading of vesicles in mice of each experimental group

TABLE 18 pathological section results of mouse vesicles from each experimental group

7. Transmission electron microscope observation of vesicles

The morphology results of each experimental group observed by TEM show that: the ultrastructure of the vesicles obtained from the model group showed an intact germinal layer, with the microprotrusions protruding into the lamina, an intact membrane of the undifferentiated nucleus, and an intact cytoplasm of these cells, rich in glycogen particles. The stratum corneum, biochemical layer and lamellar structure of the positive control group and the drug intervention group are damaged to different degrees; the micro-hair is reduced in different degrees; the cortical nucleoli disappeared to varying degrees (see fig. 7). The results of pathological grading of the vesicle tissue under transmission electron microscope are shown in tables 19 and 20.

TABLE 19 pathological grading of mouse vesicles by transmission electron microscopy

TABLE 20 results of transmission electron microscopy of vesicles from mice in each experimental group

8. Evaluation of in vivo safety

And (3) detecting pathological histological indexes, performing autopsy on the mice after the administration is finished, taking heart, liver, spleen, lung, kidney, brain and other organs of each group of mice, soaking and fixing the organs in 10% formaldehyde solution for 24 hours, washing, dehydrating, embedding paraffin, slicing, HE (high intensity staining) and performing pathological histological examination.

The histopathological changes of the mouse vital internal organs are described below:

heart: the myocardial fibers of mice in a blank control group, a T-ABZ group, an L-ABZ group, an NL-ABZ group and an ML-ABZ high-dose group are arranged in parallel, cytoplasm is uniformly dyed, and the nucleus is clear and elliptic; no clear myolytic foci or coagulative necrotic foci were found.

Liver: the liver lobules of mice in blank control group, T-ABZ, L-ABZ, NL-ABZ and ML-ABZ high-dose groups have clear structures, are radially arranged around the central vein, and have rich cytoplasm; inflammatory cells in the sink area do not infiltrate into fibrous tissues to proliferate and steatosis is not seen.

Spleen: the white marrow artery surrounding lymph sheath and the splenomesome in the spleen of the blank control group, T-ABZ, L-ABZ, NL-ABZ and ML-ABZ high dose groups have clear boundaries, and the spleen blood sinus and the splenomelia in the red marrow have clear structures and are not abnormal.

Lung: the blank control group, the T-ABZ, the L-ABZ, the NL-ABZ and the ML-ABZ high-dose groups have clear alveolar tissue form and structure, no collapse or rupture of alveoli, moderate alveolar filling and complete alveolar wall structure, and no abnormality is seen.

Kidney: in the kidneys of the blank control group, T-ABZ, L-ABZ, NL-ABZ and ML-ABZ high dose groups, the structure of the renal corpuscle is complete, and the glomerulus is located in the center of the corpuscle and is complete in structure, so that no abnormality is found.

Brain: the brain pyramidal cells of the blank control group, the T-ABZ, the L-ABZ, the NL-ABZ and the ML-ABZ high-dose groups are arranged regularly, the cell edges are clear, and the cells are polygonal and have no abnormality.

The final results show that:

(1) The ML-ABZ prepared by homogenizing at the pressure of 20000psi, the cycle times of 10 times and the homogenizing temperature of 40 ℃ has the average grain diameter of 207.52 +/-1.5 nm, the PDI value of 0.21 +/-0.06, the ABZ encapsulation rate of 96.38 +/-2.07 and the Fe encapsulation rate of 73.25 +/-1.08 percent, and the preparation method has the advantages of stability, reliability, good reproducibility and feasibility.

(2) The research result of the relative bioavailability in a rat body shows that the ML-ABZ preparation can obviously increase the peak reaching concentration and the area under a drug-time curve, and the relative bioavailability is obviously improved (P is less than 0.01) compared with T-ABZ and L-ABZ dosage forms and is improved to a certain extent compared with NL-ABZ.

(3) The tissue distribution result shows that the content of the metabolite ABZSO of the ML-ABZ in the liver and the lung is higher, which indicates that the organ distribution participating in the treatment of the liver and lung echinococcosis is better; compared with T-ABZ, L-ABZ and NL-ABZ, the intake of ABZSO in the liver and the lung of ML-ABZ is relatively increased, and the ML-ABZ can obviously improve the distribution of infected tissues and improve the targeting property in the liver and the lung.

(4) The pharmacodynamic research result of ML-ABZ on echinococcosis mice shows that statistical analysis shows that the wet weight of the bursa of each period and each dosage group of ML-ABZ are smaller than that of T-ABZ, L-ABZ and NL-ABZ groups, and the mouse has dose-effect and time-effect relationship and statistical difference (P is less than 0.01); under the same dosage, the cyst inhibiting rate of ML-ABZ in each administration period and the concentration of albendazole sulfoxide in cyst fluid of the hydatid cyst are both obviously greater than those of T-ABZ, L-ABZ and NL-ABZ (P is less than 0.01), and the liver tissue, the pathology of the cyst and the ultrastructure of the cyst wall also present good hydatid resisting effect of ML-ABZ.

(5) Throughout the in vivo treatment period, we focused on the condition of animals, and in the experiment, with the increase of the dosage, although the anti-echinococcosis effect was enhanced, no damage to the heart, liver, spleen, lung, kidney, brain tissues, which are important organs of the animals, was found, and in the experiment, no diarrhea or death was found in the animals of each dose group of ML-ABZ, indicating that the administration regimen was well-tolerated in infected mice, and none showed side effects or toxic reactions.

Therefore, ML-ABZ can obviously improve the bioavailability of ABZ and the targeting property of liver and lung, and can obviously improve the effect of resisting echinococcosis after a certain period of treatment, and is expected to become a novel and effective medicament for treating echinococcosis.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (9)

1. A magnetic targeting nano albendazole liposome is characterized in that: mixing the magnetic particles and the albendazole liposome, and homogenizing to obtain magnetic nano albendazole liposome; the particle size range of the magnetic nano albendazole liposome is 200-300 nm.

2. The magnetically targeted nano albendazole liposome according to claim 1, characterized in that: the magnetic particles are ferroferric oxide modified by citric acid.

3. The method for preparing the magnetic targeting nano albendazole liposome according to claim 1, characterized by comprising the following steps: and adding the magnetic particles into the albendazole liposome, and homogenizing under high pressure to obtain the magnetic nano albendazole liposome.

4. The method for preparing the magnetic targeting nano albendazole liposome according to claim 3, characterized in that: the homogenizing pressure is 5000-25000 psi, the homogenizing temperature is 0-60 ℃, and the homogenizing times are 2-14 times.

5. The method for preparing the magnetic targeting nano albendazole liposome according to claim 3, characterized in that: the magnetic particles are ferroferric oxide modified by citric acid.

6. The method for preparing the magnetic targeting nano albendazole liposome according to claim 3, characterized in that: the mass ratio of the magnetic particles to the albendazole in the albendazole liposome is 1.

7. The method for preparing the magnetic targeting nano albendazole liposome according to claim 5, wherein the method comprises the following steps: the citric acid modified ferroferric oxide process comprises the following steps: feCl ₃ ·6H ₂ O and FeCl ₂ ·4H ₂ Dissolving the mixture O in dilute hydrochloric acid, continuously introducing nitrogen, rapidly stirring in a water bath condition, rapidly adding concentrated ammonia water, keeping rapid stirring, adding citric acid solution, and continuously keeping rapid stirring; heating and curing are carried out, the stirring speed is reduced, the introduction of nitrogen is kept, and the introduction of nitrogen is stopped after the reaction is finished; stop addingAnd (4) heating, adjusting the pH value to be neutral by using dilute hydrochloric acid, dialyzing to obtain citric acid modified ferroferric oxide, and freeze-drying for later use.

8. The method for preparing the magnetic targeting nano albendazole liposome according to claim 7, wherein the method comprises the following steps: the FeCl ₃ ·6H ₂ O and FeCl ₂ ·4H ₂ The molar ratio of O is 1.5 to 1.2.5, the mixture is mixed and dissolved in 1moL/L of dilute hydrochloric acid, the volume ratio of the total mass of the mixture to the dilute hydrochloric acid is 1; then heating to 80-90 ℃ and curing for 1-3 h; adjusting the pH value with 1moL/L dilute hydrochloric acid, wherein the dialysis time is 48-120 h.

9. The application of the magnetic targeting nano albendazole liposome prepared by the method of claim 3 in the field of echinococcosis diagnosis and treatment.

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