Self-assembled nano-drug for enhancing the treatment of echinococcosis by combination of sonodynamic therapy and anti-echinococcosis drugs, and preparation method and application thereof

Abstract

The application discloses a kind of self-assembled nanomedicine for enhancing sound dynamic combined anti-cysticercosis drug treatment hydatid disease and its preparation method and application, belong to the field of biological medicine, and preparation method includes the following steps: with hollow TiO2 Nanoparticle as carrier, Au / TiO2 Nanoparticle is prepared using in-situ photodeposition method, then it is carried drug, and anti-cysticercosis drug@Au / TiO2 is obtained, finally based on electrostatic attraction method, self-assembled nanomedicine is prepared.The application also discloses the self-assembled nanomedicine prepared by the above preparation method and its application in the preparation of sound dynamic combined anti-cysticercosis drug treatment hydatid disease.The self-assembled nanomedicine obtained by the application can prevent the toxic effect of anti-cysticercosis drug in peripheral tissue, and the characteristics of reactive oxygen species are generated by using nanocarrier, to enhance the anti-cysticercosis effect of anti-cysticercosis drug, improve the efficiency of anti-cysticercosis drug treatment hydatid, effectively play the role of anti-cysticercosis drug treatment hydatid disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a self-assembled nano-drug for enhancing the treatment of echinococcosis by a combination of sonodynamic therapy and anti-echinococcosis drugs, and a preparation method and application thereof. BACKGROUND

[0002] Echinococcosis, also known as hydatid disease, is a parasitic disease of humans and animals, which is distributed worldwide. In China, it is mainly distributed in the western pastoral areas of Xinjiang, Qinghai, Tibet and other places, seriously endangering human health and hindering the development of animal husbandry.

[0003] At present, surgical treatment is the preferred treatment for echinococcosis, but research has found that surgical treatment is only suitable for patients with a small number of cysts and early-stage echinococcosis. Drug treatment is irreplaceable for improving the quality of life of patients with recurrent, multiple and late-stage echinococcosis. The only drug recommended by WHO for clinical treatment of echinococcosis is benzimidazole, which is still the main treatment. Albendazole (ABZ) is the best pharmacological choice for treating echinococcosis, but it is insoluble in water and most organic solvents, resulting in poor intestinal absorption, low blood concentration and low bioavailability of albendazole, which limits its efficacy and leads to a low cure rate (only about 30%). In recent years, some scholars have devoted themselves to the research of new dosage forms of albendazole, and have obtained albendazole emulsion, albendazole liposome, albendazole chitosan and other dosage forms, and have carried out pharmacodynamic studies, and have made certain progress, but all have the defect of insufficient response. In addition to the influence of the drug properties of each drug itself, the number, size, type and location of the cysts and the cyst wall also affect the drug, making it difficult for the drug to completely penetrate the cyst wall and exert the best efficacy, so it is difficult to achieve the ideal therapeutic effect. The use of benzimidazole drugs and other drugs in combination to treat echinococcosis has achieved certain results in the prior art, but most of them have little effect or are still in the laboratory stage. Moreover, echinococcosis usually parasitizes in the liver, which increases the burden on the liver and reduces the detoxification effect, and high-dose long-term continuous administration greatly increases the toxicity of the drug. Therefore, how to improve the bioavailability of anti-echinococcosis drugs, strengthen the ability of the drug to penetrate the cyst wall, and increase the effective concentration in the cyst is the primary problem to be solved in the treatment of echinococcosis.

[0004] The inventors of the present application have previously used photodynamic therapy combined with albendazole to treat hydatid disease, which also achieved significant therapeutic effect (a patent has been applied for and granted: Photodynamic composition, preparation method and application thereof, Patent No.: ZL 202010479494.1), but the laser used in photodynamic therapy cannot reach the deep part of the organ, and it is difficult to treat deep lesions with photodynamic therapy. Therefore, to improve the clinical efficacy of albendazole, not only the solubility and bioavailability of the drug should be increased, but also the drug's penetration and delivery capacity to the hydatid capsule wall and multiple insecticidal functions should be considered.

[0005] Sonodynamic therapy (SDT) is a new non-invasive treatment method based on PDT, which uses ultrasound-induced cavitation and free radicals generated after the activation of a sonosensitizer to kill surrounding cancer cells. Under the ultrasound (US) irradiation of sonodynamic therapy, the depth of penetration of human tissues can reach 7-10 cm, and the active oxygen induced locally can be activated and released by ultrasound irradiation of the sonosensitizer, thereby causing great damage to deep tumors and attracting widespread attention. Sonodynamic therapy has the advantages of strong penetration, low cost, precise treatment and slight damage, etc. Compared with the laser or microwave used in photodynamic therapy, ultrasound as a mechanical wave can penetrate deep into tumor tissue and accurately focus on tumor cell sites, effectively selecting and activating ultrasound sensitizers accumulated in tumors, which is beneficial to avoid damage to normal tissues along the propagation path of the ultrasound wave. It has strong targeting and safety for treating tumors, especially deep tumors. Therefore, it can overcome the shortcomings of poor penetration of excitation light in photodynamic therapy and has an irreplaceable role in treating deep tumor lesions in the body. Moreover, the ultrasound technology required for sonodynamic therapy has been very mature, and the clinical equipment is relatively inexpensive.

[0006] Therefore, how to provide a nuclear-targeted drug delivery system based on nanotechnology to deliver anti-hydatid drugs into the nucleus with satisfactory therapeutic effect is a technical problem that those skilled in the art need to solve. SUMMARY

[0007] To solve the above technical problems, the present application provides a self-assembled nanodrug for enhancing sonodynamic therapy combined with anti-hydatid drugs to treat hydatid disease and a preparation method and application thereof.

[0008] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0009] A preparation method of a self-assembled nanodrug for enhancing sonodynamic therapy combined with anti-hydatid drugs to treat hydatid disease, comprising the following steps:

[0010] The Au / TiO2 nanoparticles are prepared by in-situ photodeposition method with hollow TiO2 nanoparticles as carrier, then the obtained Au / TiO2 nanoparticles are added into an anti-echinococcosis drug solution for drug loading to obtain anti-echinococcosis drug@Au / TiO2, and finally HA / PFOB@Au / TiO2@anti-echinococcosis drug NPs are prepared based on electrostatic attraction method, that is, the self-assembled nanodrug for enhancing the treatment of echinococcosis by the combination of sonodynamic therapy and anti-echinococcosis drug.

[0011] Beneficial effects: the self-assembled nanodrug provided by the application is composed of a targeted nanocarrier and a drug loaded by the targeted nanocarrier; the targeted nanocarrier is composed of a shell and a core coated by the shell, the shell is modified hyaluronic acid (HA) and PFOB (perfluorooctylbromide) encapsulated on the surface of the carrier nanoparticle; the core is a chemotherapeutic drug anti-echinococcosis drug wrapped by mesoporous titanium dioxide nanoparticles (TiO2) loaded with gold nanoparticles. In the obtained HA / PFOB@Au / TiO2@anti-echinococcosis drug NPs, PFOB acts as an oxygen carrier to enhance the effect of sonodynamic therapy, PFOB enables the nanodrug to have the ability to carry oxygen, improves the hypoxic microenvironment in the echinococcosis vesicle, and then reverses the drug resistance of the anti-echinococcosis drug, improves the anti-echinococcosis effect, the obtained self-assembled nanodrug can enrich the anti-echinococcosis drug in the lesion, avoid the contact between the anti-echinococcosis drug and the peripheral tissue, prevent the toxic effect of the anti-echinococcosis drug on the peripheral tissue, and utilize the characteristic of the nanocarrier to produce active oxygen to enhance the anti-echinococcosis effect of the anti-echinococcosis drug, improve the treatment efficiency of the anti-echinococcosis drug for echinococcosis, and more effectively play the role of the anti-echinococcosis drug in treating echinococcosis.

[0012] Preferably, the preparation method of the hollow TiO2 nanoparticles specifically comprises the following steps:

[0013] After the polyvinylpyrrolidone aqueous solution, hydrochloric acid and anhydrous ethanol are uniformly mixed, the titanium tetrafluoride solution is added dropwise and stirred uniformly, then hydrothermal reaction is performed, after the reaction is completed, centrifugation, washing, drying and calcination are performed to obtain the hollow TiO2 nanoparticles.

[0014] Preferably, the concentration of the polyvinylpyrrolidone aqueous solution is 10 mg / mL, and the polyvinylpyrrolidone is PVP-K30;

[0015] The concentration of the hydrochloric acid is 0.1 moL / L;

[0016] The concentration of the titanium tetrafluoride aqueous solution is 0.8-1.2 mL / mL;

[0017] The volume ratio of the polyvinylpyrrolidone aqueous solution, hydrochloric acid, anhydrous ethanol and titanium tetrafluoride aqueous solution is 5:0.15:75:0.2;

[0018] The temperature of the hydrothermal reaction is 180℃, and the time is 3h;

[0019] The temperature of the calcination is 350℃, and the time is 3h.

[0020] Preferably, the Au / TiO2 prepared by the in-situ photodeposition method specifically comprises the following steps:

[0021] After adjusting the pH of the HAuCl4 solution, the hollow TiO2 nanoparticles are added and stirred uniformly, then the photodeposition is performed by using a xenon lamp, after the reaction is completed, the centrifugation, washing of the precipitate, drying, and calcination are performed to obtain the Au / TiO2.

[0022] Preferably, the pH is adjusted to 9.0;

[0023] The concentration of the HAuCl4 solution is 0.1g / mL;

[0024] The ratio of the addition amount of the HAuCl4 solution to TiO2 is 30mg:0.8mg;

[0025] The deposition time is 50min;

[0026] The temperature of the calcination is 350℃, and the time is 3h.

[0027] Preferably, the drug loading specifically comprises the following steps:

[0028] After the Au / TiO2 is uniformly mixed with the anti-echinococcosis drug solution and stirred in the dark, the obtained precipitate is washed to complete the drug loading, and the anti-echinococcosis drug@Au / TiO2 is obtained.

[0029] Preferably, the anti-echinococcosis drug is selected from one or more of albendazole, albendazole sulfoxide, mebendazole, flubendazole, oxfendazole, artemether, peganum alkaloids, and tetrandrine.

[0030] The stirring time is 48h;

[0031] The ratio of the addition amount of the Au / TiO2 to the anti-echinococcosis drug solution is 10mg:5ml;

[0032] The solvent of the anti-echinococcosis drug solution is DMSO, and the concentration is 1mg / mL.

[0033] Preferably, the preparation of the HA / PFOB@Au / TiO2@anti-echinococcosis drug NPs based on the electrostatic attraction method specifically comprises the following steps:

[0034] The hyaluronic acid solution and the perfluorooctyl bromide solution are added into the ethanol dispersion solution of the anti-echinococcosis drug Au / TiO2 in sequence, and the obtained solution is added into water and stirred in dark for 48h, and then the obtained solution is dialyzed and freeze-dried to obtain the HA / PFOB@Au / TiO2@anti-echinococcosis drug NPs.

[0035] A preparation method of a self-assembled nanodrug for enhancing the treatment of echinococcosis by a combination of sonodynamic therapy and anti-echinococcosis drugs.

[0036] The self-assembled nanodrug for enhancing the treatment of echinococcosis by a combination of sonodynamic therapy and anti-echinococcosis drugs.

[0037] The self-assembled nanodrug for enhancing the treatment of echinococcosis by a combination of sonodynamic therapy and anti-echinococcosis drugs.

[0038] (1) The mesoporous hollow TiO2 is used as a drug carrier in the present application, the drug can be well loaded by controlling the particle size of the nanoparticles, the release of the anti-echinococcosis drug is accelerated under ultrasonic response, and under the ultrasonic activation, the TiO2 generates electron-hole separation, reacts with the surrounding water or oxygen molecules to produce active oxygen (compared with the traditional sonodynamic agent, the TiO2 nanoparticle-mediated SDT can generate more singlet oxygen). The AuNPs are loaded on the mesoporous hollow TiO2, the characteristics of the AuNPs enhanced sonodynamic therapy are utilized, the treatment effect is improved, and the application value of the TiO2 sonodynamic therapy technology is also improved. The TiO2 NPs are wrapped by HA, and the macrophage targeting property of the sonodynamic agent is obtained (there are a large number of macrophages infiltrating around the host tissues parasitized by the echinococcosis vesicle);

[0039] (2) The nanodrug obtained in the present application can exert the sonodynamic therapy effect through ultrasonic, the treatment mode can be in-depth to the echinococcosis vesicle parasitized in the deep tissue, accurately focus on the echinococcosis parasitized site, effectively and selectively activate the ultrasonic sensitizing agent combined with the chemotherapeutic drug accumulated in the echinococcosis, so that a small amount of chemotherapeutic drug can completely take effect, and then the drug bioavailability is increased, the drug dose and the drug side effects are reduced, and the patient compliance is improved;

[0040] (3) The HA / PFOB@Au / TiO2@ anti-echinococcosis drug NPs obtained by the application are dual-function nanoparticles, the use of PFOB@Au / TiO2 enhances the accumulation of ROS in the body to play an oxidative damage anti-echinococcosis effect, and the combination of traditional drugs anti-echinococcosis drugs (inhibiting the uptake and utilization of glucose by parasites and hindering ATP generation) treatment has a synergistic effect against echinococcosis, which can amplify the insecticidal capacity of drugs and reverse the drug resistance of anti-echinococcosis drugs, achieve the effect of "1+1>2", and the HA / PFOB@Au / TiO2@ anti-echinococcosis drug NPs can more easily leak into the interstitial space of the tissue where the echinococcus is located, prolong the residence time, have the ability of passive targeting to the echinococcus site, and the ultrasonic technology has been very mature, and the clinical equipment is relatively cheap. Therefore, the new anti-echinococcosis treatment system in the application provides a new strategy for the clinical treatment of echinococcosis. BRIEF DESCRIPTION OF DRAWINGS

[0041] The drawings constituting a part of the application are used to provide a further understanding of the application, the illustrative embodiments of the application and the description thereof are used to explain the application, and do not constitute an improper limitation on the application. In the drawings:

[0042] Figure 1 is a result graph of the stability of the oxygen-carrying ability of the nanoparticles;

[0043] Figure 2 is a schematic diagram of the stability results of the nanoparticles;

[0044] Figure 3 is a schematic diagram of the drug release of the nanoparticles;

[0045] Figure 4 is a result graph of the active oxygen production intensity of the nanoparticles;

[0046] Figure 5 is the effect of the nanoparticles on the survival rate level of protoscoleces. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application.

[0048] In order to make the above-mentioned purposes, features and advantages of the application more obvious and easy to understand, the application will be further described in detail below with reference to the drawings and specific embodiments.

[0049] The raw materials in the embodiments of the application are obtained by market purchase.

[0050] Example 1

[0051] A preparation method of a self-assembled nanodrug for enhancing the treatment of echinococcosis by a combination of a sonodynamic effect and an anti-echinococcosis drug, comprising the following steps:

[0052] (1) Preparation of hollow TiO2 nanoparticles: 5 mL of a polyvinylpyrrolidone PVP-K30 (10 mg / mL) aqueous solution and 0.15 mL of hydrochloric acid (0.1 mol / L) were added to 75 mL of anhydrous ethanol, and the mixture was uniformly stirred at room temperature under magnetic stirring. Then, 10 mL of a titanium tetrafluoride aqueous solution with a concentration of (1 mg / mL) was added dropwise to the obtained dispersion under magnetic stirring, and after the dropwise addition was completed, the mixture was magnetically stirred for 1 h. Then, the obtained suspension was transferred to a stainless steel autoclave, and a hydrothermal reaction was performed at 180°C for 3 h. After the reaction was completed, the mixture was cooled to room temperature. The white product was collected by centrifugation and washed with water and ethanol several times. After drying at 60°C for 24 h, the product was calcined in a muffle furnace at 350°C for 3 h to obtain hollow TiO2 nanoparticles;

[0053] (2) Preparation of Au / TiO2: 5 mL of a HAuCl4 solution (0.1 g / mL) was added to 200 mL of water, and the pH value was adjusted to 9.0 with a 1 mol·L - 1 NaOH solution. Then, 0.8 mg of the hollow TiO2 nanoparticles obtained in step (1) was added, and the mixture was stirred for 3 h to obtain a suspension. The obtained suspension was subjected to photodeposition using a xenon lamp for 50 min, and the light purple product was collected by centrifugation and washed with high-purity water several times. After drying at 60°C for 24 h, the product was calcined in a muffle furnace at 350°C for 3 h to obtain purple Au / TiO2;

[0054] (3) Preparation of ABZ@Au / TiO2 by loading drugs on Au / TiO2: 10 mg of the Au / TiO2 obtained in step (2) was added to 5 mL of a free ABZ solution (solvent: DMSO, 1 mg / mL), and the mixture was stirred at room temperature in the dark for 48 h. After the stirring was completed, the precipitate was collected and washed with high-purity water several times until the supernatant was colorless to obtain ABZ@Au / TiO2. The drug loading amount was determined by the characteristic light absorbance of ABZ at λ = 295 nm, and the detected drug loading amount was 29%;

[0055] (4) Preparation of HA / PFOB@Au / TiO2@ABZ NPs based on electrostatic attraction method: ABZ@Au / TiO2 obtained in step (3) was dispersed in ethanol to obtain ABZ@Au / TiO2 ethanol solution with a concentration of 2 mg / mL. Under vigorous stirring, 1 mL of 0.15% (w / v) hyaluronic acid solution and 2 mL of 0.25 mg / mL perfluorooctyl bromide solution were added to 10 mL of the ABZ@Au / TiO2 ethanol solution, and the mixture was magnetically stirred in the dark for 48 h. After stirring, the obtained solution was slowly added to 15 mL of high-purity water, and the mixture was again magnetically stirred in the dark at room temperature for 48 h. After the reaction was completed, the reaction solution was dialyzed in deionized water (dialysis bag with a molecular weight cut-off of 8000-14000 Da), and then freeze-dried to obtain HA / PFOB@Au / TiO2@ABZ NPs, which are self-assembled nanodrugs for enhanced sonodynamic and anti-echinococcosis drug treatment of echinococcosis.

[0056] It was detected that the particle size of the obtained self-assembled nanodrug was 80-400 nm.

[0057] Comparative Example 1

[0058] A preparation method of a self-assembled nanodrug for enhanced sonodynamic and anti-echinococcosis drug treatment of echinococcosis, which is different from Example 1, wherein step (5) comprises the following steps:

[0059] (5) Preparation of PFOB@Au / TiO2@ABZ NPs based on electrostatic attraction method: ABZ@Au / TiO2 obtained in step (3) was dispersed in ethanol to obtain ABZ@Au / TiO2 ethanol solution with a concentration of 2 mg / mL. Under vigorous stirring, 2 mL of 0.25 mg / mL perfluorooctyl bromide solution was added to 10 mL of the ABZ@Au / TiO2 ethanol solution, and the mixture was magnetically stirred in the dark for 48 h. After stirring, the obtained solution was slowly added to 15 mL of high-purity water, and the mixture was again magnetically stirred in the dark at room temperature for 48 h. After the reaction was completed, the reaction solution was dialyzed in deionized water (dialysis bag with a molecular weight cut-off of 8000-14000 Da), and then freeze-dried to obtain PFOB@Au / TiO2@ABZ NPs drug.

[0060] Application Example 1

[0061] 1. Detection of oxygen carrying capacity of HA / PFOB@Au / TiO2@ABZ nanoparticles

[0062] The UV-VIS (Ultraviolet-visible spectroscopy) is a UV-visible light absorption spectrum determination. KMnO4 is prepared into a series of concentrations of 0, 0.1, 0.2, 0.4, 0.8 mmol / L. The UV absorption values of KMnO4 of different concentrations are measured by UV-VIS, and the standard curve of KMnO4 is drawn. The standard curve equation is y = 0.97 + 0.019, R = 0.9983. 2

[0063] The same concentration and volume of PFOB@Au / TiO2@ABZ and HA / PFOB@Au / TiO2@ABZ nanoparticles are taken and reacted with excess Na2SO3, respectively. The unreacted Na2SO3 reacts with KMnO4. The oxygen content of PFOB@Au / TiO2@ABZ and HA / PFOB@Au / TiO2@ABZ nanoparticles is quantitatively measured according to the change of absorbance before and after the reaction of KMnO4 and the molar mass ratio of the redox reaction. The oxygen stability is measured according to the above redox reaction at the same time on 1, 2, 3, 4, 5 days. As shown in Figure 5, the oxygen content of PFOB in PFOB@Au / TiO2@ABZ is 0.381 ± 0.057 mg / mL, and the oxygen content of PFOB in HA / PFOB@Au / TiO2@ABZ nanoparticles is 0.367 ± 0.081 mg / mL. As shown in Figure 5, within 5 days, only a few nanoparticles have decreased oxygen content, and the oxygen carrying capacity is relatively stable. Figure 1

[0064] 2. Measurement of particle size and surface potential of nanoparticles:

[0065] The nanoparticles Au / TiO2@ABZ and HA / PFOB@Au / TiO2@ABZ prepared in Example 1 are diluted with ultrapure water to prepare a suspension with a mass concentration of 10%. Dynamic light scattering particle size analyzer (DLS) is used to measure the particle size distribution and surface Zeta potential of nanoparticles Au / TiO2@ABZ, PFOB@Au / TiO2@ABZ, and HA / PFOB@Au / TiO2@ABZ. The particle size distribution and potential results of each nanoparticle are shown in Table 1. Each nanoparticle shows negative electric properties.

[0066] Table 1 Particle size and potential of different nanoparticles

[0067] Sample Average particle size (nm) Average potential (mV) Au / TiO2@ABZ 152.01±10.12 -13.23±2.17 [PFOB@Au / TiO2@ABZ] 160.35±14.73 -15.22±1.31 HA / PFOB@Au / TiO2@ABZ 166.93±10.89 -14.08±3.55

[0068] 3. Evaluation of nanoparticle stability

[0069] ​​The prepared sonodynamic nanoparticles were placed at 4°C and 37°C, respectively, sampling was performed, laser particle size analyzer was used to determine the particle size of the nanoparticles, and the stability of the nanoparticles was reflected by the change of the particle size, the results showed that the particle size of the nanoparticles did not change obviously within 10 days under different storage conditions, indicating that the prepared HA / PFOB@Au / TiO2@ABZ nanoparticles had good stability (see Figure 2 ).

[0070] 4. Drug release rate determination

[0071] Evaluation of drug release behavior of HA / PFOB@Au / TiO2@ABZ nanoparticles under ultrasound

[0072] The nanoparticles prepared in Example 1 were placed at the focal point of focused ultrasound, the main parameters of the ultrasound device used in the experiment were: frequency 1.0 MHz, different ultrasound intensities (US0-0W / cm 2 , US1-0.2W / cm 2 , US2-0.3W / cm 2 , US3-0.4W / cm 2 , US4-0.5W / cm 2 , US5-0.6W / cm 2 ) were used, and the ultrasound treatment lasted for 5 min, then dialysis method was used to determine the drug release rate, the results showed that under the action of ultrasound, the drug ABZ in HA / PFOB@Au / TiO2@ABZ NPs could be released quickly, and the drug release was obviously accelerated with the increase of ultrasound intensity (see Figure 3 ), while in the absence of Au / TiO2 loading (ABZ NPs: nanoparticles encapsulated by PLGA-PEG) combined with ultrasound treatment, the drug release rate was significantly reduced, wherein ABZ NPs refers to nanoparticles without MTN loading, and the preparation method is as follows:

[0073] 100 mg of high molecular material PLGA-PEG and 10 mg of albendazole were accurately weighed and dissolved in 1 mL of DMSO, after ultrasonic dissolution, the solution was slowly added into 5 times the volume of stirring phosphate buffer (PBS, 10 mM, pH = 7.4) using a pipette, after the addition was completed, it was stirred at room temperature for 20 min, then centrifuged at 3500 r / min for 5 min, and the unencapsulated drug was removed, thereby obtaining ABZ NPs.

[0074] 5. Evaluation of ROS production of nanoparticles under ultrasound

[0075] The nanoparticles prepared in Example 1 were placed in the focus of focused ultrasound after adding ROS probe DCFH-DA and treated with ultrasound, the ultrasound device was the same as in Example 4, US3 was selected as the ultrasound intensity, and then the intensity of reactive oxygen species was observed by fluorescence inverted microscope. The results showed that the intensity of HA / PFOB@Au / TiO2@ABZ+SDT was significantly higher than that of Au / TiO2@ABZ+SDT.

[0076] 6. Investigation of the effect of nanoparticles on the level of Eg ATP

[0077] The protoscoleces were cultured in 24-well plates at a concentration of 2000 per well, each nanoparticle was administered, and US3 was selected as the ultrasound intensity. The US intervention was 5 min, 100 μL of ATP lysis solution was added to the culture plate well, and then the lysis was performed and transferred to an EP tube. Centrifugation was performed at 4°C, 12700xg, 10 min, and the supernatant was aspirated. Protein quantification was performed by BCA method, and the final concentration of protein in each group was adjusted to the same. According to the instructions of the ATP detection kit, the luminescence values of ATP standard solution and sample were detected, and the ATP concentration of the sample was obtained according to the ATP concentration standard curve. The results are shown in Table 2. Compared with the Eg control group, the ATP levels in the Au / TiO2@ABZ, PFOB@Au / TiO2@ABZ, and HA / PFOB@Au / TiO2@ABZ groups were significantly reduced (all P<0.01); compared with the ABZ NPs group, the ATP level was significantly reduced (all P<0.01) compared with the photodynamic result.

[0078] Table 2 Effect of SDT nanoparticles on the level of Eg ATP

[0079]

[0080]

[0081] Note: *P<0.05 compared with the blank control group; **P<0.01 compared with the blank control group; ## P<0.01 compared with the blank control group.

[0082] Table 3 ATP content of Eg in each drug intervention group of PDT (n=3, x±SD)

[0083] Group ATP content (nmoL / mg) Control group 301.10±5.28 ABZ NPs group 292.54±6.556 Ce6 group 222.06 ± 8.91 ** ]] ABZ NPs+PDT 38.59 ± 5.94 ** ]]

[0084] Note: *P<0.05 compared with the blank control group; **P<0.01 compared with the blank control group;

[0085] 7. Investigation of the dual effect of nanoparticles against hydatid in vitro

[0086] The protoscoleces were inoculated in 96-well plates at 200 per well, and the protoscoleces were incubated with nanoparticles containing drugs (ABZ NPs, Au / Ti02@ABZ, HA / PFOB@Au / Ti02@ABZ) for 12 h, then the medium was replaced, and the protoscoleces were treated with ultrasound (parameters and devices consistent with 4) for 5 min, and then the protoscoleces were incubated at 37°C for 24 h, and the mortality was observed by methylene blue staining. The results showed that before the ultrasound, the nanoparticles had no obvious effect on the mortality of the protoscoleces, but after the ultrasound, the mortality of the protoscoleces increased significantly, and increased with the increase of the ultrasound intensity (see Table 4).

[0087] Table 4 Survival rate of protoscoleces after nanoparticle intervention (%)

[0088]

[0089] Note: * compared with the blank control group, P<0.05; ** compared with the blank control group, P<0.01.

[0090] 8. Comparison of Eg survival rate of in vitro PDT and SDT

[0091] The protoscoleces were inoculated in 96-well plates at 200 per well, ① the protoscoleces were incubated with nanoparticles containing 25 μg / mL albendazole (HA / PFOB@Au / Ti02@ABZ NPs) for 12 h, then the medium was replaced, and the protoscoleces were treated with ultrasound (parameters and devices consistent with 4) at US3 intensity for 5 min, and then the protoscoleces were incubated at 37°C for 24 h, and the mortality was observed by methylene blue staining; ② the protoscoleces were co-cultured with 10 μg / mL Ce6 culture solution for 2 h, the culture solution was removed, the protoscoleces were washed with PBS three times, and then new culture solution was used, and the protoscoleces were irradiated with 635 nm visible light at a light dose of 5 mW / cm2for 5 min. Then the protoscoleces after photodynamic therapy were co-cultured with nanoparticles containing 50 μg / mL albendazole for 24 h. 10 μL of protoscoleces were taken three times from each well for H&E staining to count the survival rate. 2 The protoscoleces were inoculated in 96-well plates at 200 per well, ① the protoscoleces were incubated with nanoparticles containing 25 μg / mL albendazole (HA / PFOB@Au / Ti02@ABZ NPs) for 12 h, then the medium was replaced, and the protoscoleces were treated with ultrasound (parameters and devices consistent with 4) at US3 intensity for 5 min, and then the protoscoleces were incubated at 37°C for 24 h, and the mortality was observed by methylene blue staining; ② the protoscoleces were co-cultured with 10 μg / mL Ce6 culture solution for 2 h, the culture solution was removed, the protoscoleces were washed with PBS three times, and then new culture solution was used, and the protoscoleces were irradiated with 635 nm visible light at a light dose of 5 mW / cm2for 5 min. Then the protoscoleces after photodynamic therapy were co-cultured with nanoparticles containing 50 μg / mL albendazole for 24 h. 10 μL of protoscoleces were taken three times from each well for H&E staining to count the survival rate.

[0092] The mortality of protoscoleces by the two methods was compared, and the results showed that the mortality of protoscoleces after ultrasound was significantly higher than that of photodynamic therapy, see Table 5, Figure 5 .

[0093] Table 5 Effect of each nanoparticle on the survival rate of protoscoleces

[0094] Group Survival rate Eg control 98.57±5.53 ABZ NPs 97.23±2.36 ABZ NPs+PDT 75.58±6.17** HA / PFOB@Au / TiO2@ABZ + SDT 27.43 ± 5.24** ## ]]

[0095] Note: * compared with the blank control group, P<0.05; ** compared with the blank control group, P<0.01; ##P < 0.01 compared with blank ABZ NPs group; ABZ NPs.

[0096] The above merely is the preferred specific implementation of the present application, but the protection scope of the present application is not limited to this, any person skilled in the art can easily think of the changes or replacements within the technical range disclosed by the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for preparing self-assembled nanodrugs for enhancing the treatment of echinococcosis by the combination of sonodynamic therapy and anti-echinococcosis drugs, characterized in that, The method comprises the following steps: The Au / TiO2 nanoparticles are prepared by in-situ photodeposition method using hollow TiO2 nanoparticles as carrier, and then the obtained Au / TiO2 nanoparticles are added into an anti-echinococcosis drug solution for drug loading to obtain anti-echinococcosis drug@Au / TiO2, and finally HA / PFOB@Au / TiO2@anti-echinococcosis drug NPs, i.e. the self-assembled nanodrug for enhancing the treatment of echinococcosis by combining the sonodynamic therapy and the anti-echinococcosis drug therapy, are prepared based on electrostatic attraction method.

2. A method for preparing self-assembled nano-drugs for enhancing the treatment of echinococcosis by the combination of sound and the anti-echinococcosis drugs according to claim 1, characterized in that, The preparation method of the hollow TiO2 nanoparticles specifically comprises the following steps: The polyvinylpyrrolidone aqueous solution, hydrochloric acid and anhydrous ethanol are uniformly mixed, then the titanium tetrafluoride solution is added dropwise and stirred uniformly, then hydrothermal reaction is performed, after the reaction is completed, centrifugation, washing and drying are performed, and then calcination is performed to obtain the hollow TiO2 nanoparticles.

3. The method for preparing the self-assembled nanodrug for enhancing the treatment of echinococcosis by combining the sonodynamic therapy and the anti-echinococcosis drug therapy according to claim 2, characterized in that: The concentration of the polyvinylpyrrolidone aqueous solution is 10 mg / mL, and the polyvinylpyrrolidone is PVP-K30; The concentration of the hydrochloric acid is 0.1 moL / L; The concentration of the titanium tetrafluoride aqueous solution is 0.8-1.2 mL / mL; The volume ratio of the polyvinylpyrrolidone aqueous solution, hydrochloric acid, anhydrous ethanol and titanium tetrafluoride aqueous solution is 5:0.15:75:0.2; The temperature of the hydrothermal reaction is 180℃, and the time is 3 h; The temperature of the calcination is 350℃, and the time is 3 h.

4. A method of preparing self-assembled nano-drugs for enhancing the treatment of echinococcosis by the combination of sound and the anti-echinococcosis drugs according to claim 1, characterized in that, The in-situ photodeposition method for preparing Au / TiO2 specifically comprises the following steps: After the pH of the HAuCl4 solution is adjusted, the hollow TiO2 nanoparticles are added and stirred uniformly, then photodeposition is performed by using a xenon lamp, after the reaction is completed, centrifugation, washing and drying are performed, and then calcination is performed to obtain the Au / TiO2.

5. A method of preparing self-assembled nano-drug for enhancing the treatment of echinococcosis by the combination of sound and the anti-echinococcosis drug according to claim 1, characterized in that, The pH is adjusted to 9.0; The addition amount ratio of the HAuCl4 solution to TiO2 is 20 mg:0.8 mg; The deposition time is 50 min; The temperature of the calcination is 350℃, and the time is 3 h.

6. A method of preparing self-assembled nano-drugs for enhancing the treatment of echinococcosis by the combination of sound and the anti-echinococcosis drugs according to claim 1, characterized in that, The drug loading specifically comprises the following steps: After the Au / TiO2 is uniformly mixed with an anti-echinococcosis drug solution, stirring is performed in the dark, after the stirring is completed, the obtained precipitate is washed, and the drug loading is completed to obtain anti-echinococcosis drug@Au / TiO2.

7. A method of preparing self-assembled nano-drugs for enhancing the treatment of echinococcosis by the combination of sound and the anti-echinococcosis drugs according to claim 1, characterized in that, The stirring time is 48 h; The addition amount ratio of the Au / TiO2 to the anti-echinococcosis drug solution is 10 mg:5 ml; The solvent of the anti-echinococcosis drug solution is DMSO, and the concentration is 1 mg / mL; The anti-echinococcosis drug is selected from one or more of albendazole, albendazole sulfoxide, mebendazole, flubendazole, oxfendazole, artemether, peganum alkaloids and tetrandrine.

8. A method of preparing self-assembled nano-drugs for enhancing the treatment of echinococcosis by the combination of sound and the anti-echinococcosis drugs according to claim 1, characterized in that, The preparation of HA / PFOB@Au / TiO2@anti-echinococcosis drug NPs based on electrostatic attraction method specifically comprises the following steps: The hyaluronic acid solution and the perfluorooctyl bromide solution were added into the ethanol dispersion of the anti-echinococcosis drug Au / TiO2 successively, and the obtained solution was stirred in dark for 48 h, then was added into water and stirred in dark for another 48 h, and then was dialyzed and freeze-dried to obtain the HA / PFOB@Au / TiO2@anti-echinococcosis drug NPs.

9. The self-assembled nanodrug prepared by the method of any one of claims 1-8.

10. The self-assembled nanodrug of claim 9 for use in the preparation of a medicament for enhancing the treatment of echinococcosis by the combination of sonodynamic therapy and anti-echinococcosis drug.

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