A ros-responsive nanoparticle and its application in sonodynamic mediation of hydatidosis treatment

Abstract

The present application relates to a kind of ROS response type nanoparticles and its application in sound power mediated hydatidosis treatment, it is related to medical technology field.The ROS response type nanoparticles of the present application uses the beta-cyclodextrin modified with targeting substance as shell, uses the mesoporous titanium dioxide nanoparticles for drug inclusion as core, the shell is covered to the core by ROS sensitive linker.The targeting nano-preparation of the present application can realize drug co-delivery through the barrier of capsule wall, can enrich drug in lesion, avoid the contact of drug with peripheral tissue, prevent the degradation of drug in peripheral tissue, reduce the systemic toxicity of drug, better play the therapeutic effect on hydatidosis.The present application can especially better realize the effect of reducing the toxic side effect of dehydropeimine, significantly improve the bioavailability of dehydropeimine and tirapazamine, more effectively exert the synergistic anti-hydatid effect of dehydropeimine and tirapazamine cascade amplification.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a ROS-responsive nanoparticle and its application in sonodynamic therapy of hydatidosis. BACKGROUND

[0002] Hydatidosis is a zoonotic parasitic disease caused by Echinococcus granulosus (Eg) infection, which seriously endangers human health and economic development.

[0003] The preferred treatment for hydatidosis is surgical removal, but due to multiple factors, not all patients meet the surgical indications. Drug therapy has become a powerful adjunct to the treatment of hydatidosis and has become the main direction of research on the disease. Currently, the WHO recommends fewer drug options for the clinical treatment of hydatidosis, and since the discovery of albendazole (ABZ), no other new drugs have been marketed. Harmine (HM) is a tricyclic β-carboline alkaloid extracted from the seeds of the Xinjiang local specialty medicine, Peganum harmala. Years of research have shown that HM is a highly potential drug for killing hydatidosis, with lower liver toxicity and higher anti-parasitic activity than ABZ. However, due to its central nervous system toxicity, it has been limited to laboratory research.

[0004] Currently, the problems encountered by various research groups in the study of hydatidosis drugs are usually: drug solubility, low single drug efficacy, and the barrier of hydatid cyst wall to drug molecules, which makes the penetration of most drug molecules through the hydatid cyst wall barrier poor (the drug concentration in the hydatid lesion is only 1 / 100 of the blood drug concentration), resulting in a small number of candidate drugs for treating hydatidosis and poor treatment effect; long-term chemotherapy may lead to adverse complications; and the depth of hydatid parasitism in the organ varies, resulting in uneven drug absorption, poor treatment effect, and inability to cure the disease, which is prone to recurrence. Therefore, the difficulty of drug treatment of hydatidosis is that drug molecules are difficult to reach the inside of the cyst and the deep parasitic part, and domestic and foreign scholars have made a lot of research, but there is still a lack of effective measures. Therefore, it is urgent to find a new strategy to improve the penetration of drug molecules through the hydatid cyst wall barrier and to release the drug specifically in the microenvironment of the hydatid parasitic tissue, to increase the local concentration of drug molecules in the hydatid cyst, and to increase the concentration of drug molecules in the hydatid cyst, thereby improving the cure rate of hydatidosis.

[0005] In recent years, nanotechnology has brought new ideas and conceptual changes for tumor treatment. Nanomedicine has been rapidly developed due to its significant advantages, such as reducing the frequency of drug administration, reducing side effects, enhancing targeting ability, and even changing the physical and chemical properties of drugs. Compared with conventional drug preparations, the new nanomedicine delivery system has the following potential: ① increasing the apparent solubility of drugs and improving the oral absorption of poorly soluble drugs; ② improving the in vitro and in vivo stability of drugs by encapsulating or complexing drugs, adjusting drug dissolution and release; ③ improving the selectivity of drugs to tissues, organs or cells and drug efficacy, and reducing side effects; ④ achieving new drug delivery routes and optimizing drug combination therapy strategies.

[0006] At present, a variety of nanomedicines have been approved by the US Food and Drug Administration (FDA) for the treatment of tumors, hepatitis, kidney transplants, endocrine, dental repair, and other diseases. Nanomedicine has also been valued by many scholars in the treatment of hydatid disease. Nanomedicine systems can load at least two anticancer drugs with different physicochemical properties and pharmacological properties into one drug delivery system, achieving better therapeutic effect, and have become a promising strategy for combined anticancer therapy. The proposal and extensive research and application of multifunctional nanoscale drug delivery platforms have made the multi-drug combination scheme better.

[0007] Nanomedicine reaches the inflammatory lesion site by active or passive targeting, and slowly releases the internal drug, mainly through natural release and microenvironment-responsive release. Microenvironment-responsive drug release mainly releases the drug loaded by the nanomedicine according to the special microenvironment of the lesion site, which often has low pH, high enzyme concentration, high ROS or glutathione concentration, etc., so that the nanomedicine can achieve the purpose of site-specific drug release. Because the microenvironment-responsive drug release system can achieve specific drug release at the lesion site, it can reduce drug release in other organ sites, enhance drug efficacy, and reduce side effects. In particular, ROS-responsive drug carriers have high selectivity and specificity for drug release (high ROS in inflammatory, tumor and other lesions, and low ROS in other normal physiological conditions), which have attracted much attention from researchers in recent years.

[0008] Sonodynamic therapy (SDT) as a new strategy for treating diseases is a non-invasive targeted treatment based on ultrasound. Through local ultrasound irradiation, the sonosensitizer can produce highly toxic ROS to induce target cell apoptosis, and there is no need to worry about drug resistance. It shows unique potential in combating cancer and bacterial infection. In addition, due to the precise irradiation, non-invasiveness and high tissue penetration of ultrasound, and the low risk of inducing systemic toxicity, the economic, portable and easy-to-operate ultrasound device is easy to implement, and SDT is often used as a strategy for treating deep tissue diseases, which has very wide application prospects.

[0009] However, there are still some problems in the treatment of deep tumors by sonodynamic therapy. The rapid development of nanomedicine provides a safer and more reliable guarantee for SDT to treat deep tumors. The main mechanism of SDT to exert anti-tumor effect is ROS, so the development of nano sonosensitizers that can effectively regulate ROS is an important part of improving the efficacy of SDT. The size-adjustable nanoparticles can enhance the EPR effect of tumors to enrich the sonosensitizer in the tumor area, promote ROS generation, and improve the tumor inhibition rate. Due to the complexity of the disease, the inhibition efficiency of a single functional nanoplatform on tumors is limited, and the combination of multiple regulation pathways is needed to maximize ROS production and fully exert the anti-tumor effect of SDT.

[0010] The use of nanotechnology can improve the targeting of sonosensitizers, enhance their ROS production rate or ultrasound cavitation effect, and also promote the combination of SDT with other treatment methods, and promote the development of SDT in the field of biomedicine. Nanoparticles can be formed by conjugation between various functional groups, or self-assembled from copolymers, which can be easily controlled by adjusting the polymerization method or reactants to control the size of nanoparticles. Inorganic nano sonosensitizers have good stability, controllable physical and chemical properties, easy chemical modification and surface modification, and outstanding potential in tumor diagnosis and treatment. Inorganic SDT nanomaterials have unique physical and chemical properties, which are beneficial to realize tumor photothermal therapy, photodynamic therapy and chemical dynamic therapy, etc. In addition, they are widely developed as drug delivery carriers due to their good drug loading capacity, biocompatibility, chemical modification and physical and chemical stability. SUMMARY

[0011] The purpose of the present application is to provide a ROS-responsive nanoparticle and its application in sonodynamic therapy of hydatid disease, in order to solve the problems existing in the prior art and achieve effective treatment of hydatid disease.

[0012] To achieve the above purpose, the present application provides the following solutions:

[0013] One of the technical solutions of the present application: a ROS-responsive nanoparticle, a β-cyclodextrin modified as a targeting material as a shell, and a mesoporous titanium dioxide nanoparticle wrapping a drug as a core; the shell is coated on the core through a ROS-sensitive linker;

[0014] The drug includes a first drug and a second drug;

[0015] The first drug includes one or more of dehydropeimine, dehydropeimine derivative, peimine, albendazole, albendazole sulfoxide, mebendazole, flubendazole, and oxfendazole;

[0016] The second drug is tirapazamine.

[0017] The mass ratio of the first drug and the second drug is 1:1-5.

[0018] Further, when the first drug is dehydropeimine, the mass ratio of dehydropeimine and tirapazamine is 1:1; the mass ratio of dehydropeimine derivative and tirapazamine is 1:1-5.

[0019] More specifically, the mass ratio of albendazole: tirapazamine is 1:1-5; the mass ratio of albendazole sulfoxide: tirapazamine is 1:1-3.5; the mass ratio of mebendazole: tirapazamine is 1:1-5; the mass ratio of flubendazole: tirapazamine is 1:1-5; and the mass ratio of oxfendazole: tirapazamine is 1:1-5.

[0020] Further, the first drug and the second drug are connected through a ROS-sensitive linker.

[0021] Further, the ROS-sensitive linker is a ketone thioether.

[0022] The ketone thioether TK can be selected as a ketone thioether COOH-TK-COOH (COOH-TK-COOH is a ROS active oxygen-responsive nanoparticle drug carrier-ketone thioether (TK) compound, which contains two carboxyl groups (COOH) and a TK group in its chemical structure. Due to the presence of two carboxyl atoms, COOH-TK-COOH can be used as a bifunctional compound).

[0023] The ROS-sensitive linker can be selected as:

[0024]

[0025] CAS: 4265-58-1, 2,2'-[propane-2,2-diylbis(thio)]diacetic acid; or

[0026]

[0027] CAS: 4265-59-2, propane-2,2-diylbis(sulfanyl)diacetic acid;

[0028] The ROS-sensitive linker can be selected from:

[0029]

[0030] The ROS-sensitive linker can also be HOOC-S-CH2-S-COOH.

[0031] The second technical solution of the present application provides a preparation method of the ROS-responsive nanoparticle, comprising the following steps:

[0032] Preparation of mesoporous titanium dioxide nanoparticles with ROS-sensitive linkers on the surface: mesoporous titanium dioxide and ROS-sensitive linkers are reacted under a protective atmosphere to obtain mesoporous titanium dioxide nanoparticles with ROS-sensitive linkers on the surface;

[0033] Drug loading: the drug is loaded by using the mesoporous titanium dioxide nanoparticles with ROS-sensitive linkers on the surface to obtain drug-loaded mesoporous titanium dioxide nanoparticles with ROS-sensitive linkers on the surface;

[0034] Shell coating: the drug-loaded mesoporous titanium dioxide nanoparticles with ROS-sensitive linkers on the surface are coated by using β-cyclodextrin modified with a targeting substance to obtain the ROS-responsive nanoparticle.

[0035] The third technical solution of the present application provides a preparation method of the ROS-responsive nanoparticle, comprising the following steps:

[0036] Preparation of mesoporous titanium dioxide nanoparticles with ROS-sensitive linkers on the surface: mesoporous titanium dioxide and ROS-sensitive linkers are reacted under a protective atmosphere to obtain mesoporous titanium dioxide nanoparticles with ROS-sensitive linkers on the surface;

[0037] Drug connection: the first drug and the second drug are connected by using the ROS-sensitive linker to obtain the connected drugs;

[0038] Drug loading: the connected drugs are loaded by using the mesoporous titanium dioxide nanoparticles with ROS-sensitive linkers on the surface to obtain drug-loaded mesoporous titanium dioxide nanoparticles with ROS-sensitive linkers on the surface;

[0039] Shell coating: the drug-loaded mesoporous titanium dioxide nanoparticles with ROS-sensitive linkers on the surface are coated by using β-cyclodextrin modified with a targeting substance to obtain the ROS-responsive nanoparticle. ​

[0040] The fourth aspect of the present application provides the use of the ROS-responsive nanoparticle in the preparation of a sonodynamic therapy drug for echinococcosis.

[0041] Previous studies have found that harmine (HM) has a significant effect on echinococcosis and has development potential, but the dosage is large (the effective concentration for treating echinococcosis in mice is 50 mg / kg), which can easily cause toxic side effects (central nervous system toxicity) of the drug, thereby limiting further development as a clinical application drug. At the same time, the study also found that the main mechanism of action of HM against echinococcosis may be that HM can consume oxygen in the worm during the anti-worm process, increasing the ROS content in the worm, thereby causing DNA damage in the worm, and ultimately leading to apoptosis of the echinococcus.

[0042] Tirapazamine (TPZ) is a hypoxia-activated chemotherapeutic prodrug that can selectively kill hypoxic cells. In cells with normal oxygen content, the parent compound mainly exists, and the toxicity is small, and there is no damage to normal cells. In a hypoxic state, TPZ is reduced to free radicals that cause DNA double-strand breaks, single-strand breaks, and base damage, destroy the DNA structure, and cause cell death.

[0043] Titanium dioxide (TiO2) as a classic inorganic sonosensitizer has high chemical stability and excellent sonodynamic performance. Titanium dioxide nanoparticles (TiO2 NPs) are non-toxic to mammalian cells because they are chemically inert and stable under physiological conditions. Due to their biocompatibility, TiO2 NPs have been studied as potential carriers for anticancer drugs and genes. Their high drug loading capacity, the possibility of achieving local or even combined therapy make them a promising alternative for the development of advanced nanotherapies. Mesoporous titanium dioxide nanoparticles have revolutionized the field of drug controlled release systems, with characteristics such as adjustable shape, robustness, and ease of surface modification, making them an ideal platform for designing multifunctional nanosystems. However, the lack of cell targeting of TiO2 severely limits its research and application.

[0044] Short rod-shaped or strip-shaped blood flow signals can be seen around the echinococcosis lesions, and the lesions show the characteristics of "poor blood supply". There is a large amount of inflammatory factor infiltration around the parasitic tissue, and there is a high ROS microenvironment, which provides a theoretical basis for the targeted treatment of echinococcosis and the slow release of drugs in the inflammatory site microenvironment.

[0045] Sonosensitizers can directly activate reactive oxygen species (ROS) through acoustic cavitation, including singlet oxygen ( 1 O2), hydroxyl radicals (·OH － ) and superoxide anion (·O 2-), effectively damaging intracellular DNA and protein, and promoting cell lipid peroxidation, inducing tumor cell apoptosis.

[0046] The application proposes the following technical concept: the nanomedicine can target hydatid cysts and deliver drugs through the hydatid cyst wall barrier into the vesicle cavity, and efficiently treat hydatid through the DNA damage combined effect.

[0047] HM can consume oxygen to generate ROS, cause worm apoptosis through DNA damage, and at the same time, due to oxygen consumption, the oxygen content is reduced, causing the downstream drug TPZ to play a pharmacological effect in anoxic environment, and converting into a toxic metabolite to cause DNA damage cascade to further amplify the damage to Eg. Therefore, the combination of HM and TPZ can reduce the dosage of HM, reduce the neurotoxic side effects of HM, and play a cascade amplification synergistic anti-hydatid effect, therefore, the research found that the synergistic enhancement interaction between HM and TPZ treatment can produce higher therapeutic effect under low-dose treatment, thereby reducing the side effects induced by high-dose HM.

[0048] The application provides a targeted nanometer preparation of dehydropegoenine and tiazolinium combined administration for treating hydatidosis, which comprises a targeted nanometer carrier and loaded drugs.

[0049] Preferably, the shell is a modified folate (FA) β-cyclodextrin (β-CD), and the core is a mesoporous titanium dioxide nanoparticle wrapped with a chemotherapeutic drug HM:TPZ (1:1).

[0050] The present application provides a new type of coated mesoporous titanium dioxide nanoparticles (MTN), according to the optimal combination ratio (1:1) of dehydropeimine (HM) and tirapazamine against echinococcosis, the two compounds are connected by ketone thioether (the purpose of connection: HM and TPZ are connected through ROS sensitive groups, only when active oxygen species exist, the ROS sensitive groups can be rapidly oxidized and destroyed to release HM and TPZ, achieve drug controlled release, effectively improve the biological safety, ensure that HM and TPZ can still be in a 1:1 relationship in the body, then, β-cyclodextrin (β-CD) is connected to the outer surface of mesoporous titanium dioxide nanoparticles through a linker sensitive to active oxygen species (ROS) to block the pores (MTN@HM / TPZ@β-CD). MTN@HM / TPZ@β-CD can wrap HM / TPZ in the pores, under the irradiation of focused ultrasound (US), MTN produces a large amount of ROS, which causes the ROS sensitive linker to be cut, separating the gatekeeper (β-CD) from MTN@HM / TPZ, and HM / TPZ can be rapidly released, only the ROS sensitive groups can be rapidly oxidized and destroyed, separating HM and TPZ into monomeric compounds to play a cascade therapy role. By integrating SDT and chemotherapy into a system, MTN@HM / TPZ@β-CD shows good anti-echinococcosis effect. More importantly, this new type of DDS significantly reduces the side effects of HM. Among them, the β-cyclodextrin (β-CD) is modified by folic acid.

[0051] The ROS sensitive linker of the present application contains a methylene-substituted or unsubstituted -S-CH2-S- bond, which is cut under the irradiation of focused ultrasound (US).

[0052] The present application discloses the following technical effects:

[0053] The targeted nano-preparation of the present application can realize drug delivery through the capsule wall barrier, and then enrich in the lesion, avoid the contact of the drug with the peripheral tissue, prevent the degradation of the drug in the peripheral tissue, play a role through US ultrasound focusing in the lesion area, reduce the systemic toxicity of the drug, and better play the therapeutic effect on echinococcosis. The present application can especially better realize the effect of reducing the toxic side effects of dehydropeimine, significantly improve the bioavailability of dehydropeimine and tirapazamine, and more effectively play the synergistic anti-echinococcosis effect of dehydropeimine and tirapazamine. BRIEF DESCRIPTION OF DRAWINGS

[0054] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0055] Figure 1 Effect diagram of the combination of HM and TPZ in the present application for in vivo and in vitro intervention of Eg;

[0056] Figure 2 Stability test results of the nanoparticles of the present application;

[0057] Figure 3 Drug release effect of the nanoparticles of the present application;

[0058] Figure 4 External morphology of the oncosphere after US action of the present application;

[0059] Figure 5 ROS production after drug action and US action of the present application. DETAILED DESCRIPTION

[0060] A number of exemplary embodiments of the present application are now described in detail. The following description of certain examples of the application should not be considered limiting of the present application, but rather a description of certain aspects, features and embodiments of the application.

[0061] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. Each smaller range that falls within the integer ranges are also specifically included. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0062] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in further detail the methods and / or materials associated with the documents. In the case of conflict between the present specification and any document incorporated by reference, the present specification will control.

[0063] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0064] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended and do not limit the object to which the terms are applied to the listed items only.

[0065] The present application is first directed to the in vitro and in vivo pharmacodynamic investigation of HM, TPZ and HM-TPZ against Echinococcus:

[0066] 1. In vitro pharmacodynamic investigation of the effect of drugs on protoscoleces

[0067] Methylene blue staining experiment was used to collect protoscoleces after 1-5 days of drug intervention, which were placed in 1.5 mL centrifuge tubes, the culture medium was discarded, and the protoscoleces were washed with normal saline for 3 times, then methylene blue staining was added, after 3 minutes, the protoscoleces in the 1.5 mL centrifuge tubes were taken to the glass slide, and then the protoscoleces were observed under an inverted microscope, and the survival rate of protoscoleces under the intervention of different concentrations of drugs was calculated according to the total number of survival and death of protoscoleces.

[0068] After 10, 20, 30, 40, 50 and 100 μg / mL of HM and 5, 10, 20, 30, 40 μg / mL of TPZ were used to intervene protoscoleces, methylene blue staining was used, and the survival rate was calculated under an inverted microscope (protoscolex survival rate (%) = (total number of protoscoleces - death number) / total number of protoscoleces x 100%). The survival rate of each concentration of HM group is shown in Table 1 and Table 2. The results showed that in the HM group, the activity of 10, 50, 100 μg / mL group decreased to 95.42%, 87.10%, 79.65% on the 3rd day, and 88.57%, 70.19%, 66.49% on the 5th day, and the high concentration group (100 μg / mL) of HM had a significant inhibitory effect on the activity of protoscoleces, and the survival rate of 10 μg / mL of HM on the 5th day was significantly lower than that on the 3rd day. The survival rates of 5, 10, 20, 30, 40 μg / mL of TPZ group on the 5th day were 94.57%, 93.31%, 83.08%, 81.78%, and 80.33%, respectively, and it can be seen that the survival rate of 20 μg / mL of protoscoleces began to decrease significantly, so the concentration of 10 μg / mL of TPZ was selected as the concentration of the combination of the two drugs, which had no obvious change in survival rate after administration in vitro.

[0069] Table 1 Survival rate of protoscoleces after HM intervention (%)

[0070]

[0071] Table 2 Survival rate of protoscoleces after TPZ intervention (%)

[0072]

[0073] Notes: * Compared with the blank control group, P<0.05; ** Compared with the blank control group, P<0.01;

[0074] a. TPZ: HM synergistic ratio determination: According to the in vitro pharmacodynamic results of single drug, the TPZ concentration was fixed at 10 μg / mL, and the variable was HM. Different mass ratios of HM and TPZ mixed drugs (TPZ: HM = 1:0.25, 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5) were used to treat protoscoleces, and their insecticidal ability was measured. Through the synergistic ratio detection results, the best ratio of TPZ: HM was 1:1, and the survival rate of protoscoleces intervened by the ratio of TPZ: HM 1:1 was verified, and the inhibitory effect of TPZ: HM on the viability of protoscoleces was stable.

[0075] Table 3 Survival rate (%) of protoscoleces after intervention of HM-TPZ with synergistic ratio

[0076]

[0077] b. In vivo synergistic anti-echinococcosis effect of dehydromelilotine and tirapazamine

[0078] By fixing the tirapazamine drug concentration (10 μg / mL) and changing the dehydromelilotine concentration, the killing effect of different proportions of drug concentration on in vitro echinococcosis granuloma was investigated. The results showed that when the ratio of HM and TPZ was 1:1, the double drugs had good synergistic anti-echinococcosis effect in vitro and in vivo. The mortality rate of Eg intervened by HM and TPZ combined for 72 h in vitro was 76.46±2.82, which was much higher than that of HM (50 μg / mL) alone, which was 12.90±1.76. The cystic vesicle in the in vivo experiment: the cystic vesicle surface of the model control group was smooth and round, and was transparent; the cystic vesicle of the combined drug group was shrunk and the volume was smaller, which was significantly smaller than that of HM alone, and the degree of calcification was more obvious than that of HM (Fig. 4) Figure 1 ), and the cyst inhibition rate of mice after treatment in the combined drug group was significantly higher than that of HM alone (Table 4). The in vivo drug efficacy was obvious (Fig. 5) Figure 1

[0079] Table 4 Wet weight value of cystic tissue and cyst inhibition rate of each group after intervention of each drug in vivo

[0080]

[0081] Note: ** Compared with the model group, P<0.01; ## Compared with the HM 50 mg / kg group, P<0.01.

[0082] Example 1 Preparation of ultrasound-responsive HM-TPZ@MTN-ROS-DSPE-PEG2K-NH2@FA-β-CD

[0083] ​The present application includes but is not limited to HM-TPZ@MTN-ROS-DSPE-PEG (2K-5K)-NH2@FA-β-CD, and the present embodiment is described by taking HM-TPZ@MTN-DSPE-PEG2K-NH2@FA-β-CD as an example.

[0084] 1. Preparation of MTN (using the classic hard film plate method)

[0085] a. Preparation of SiO2nanospheres: SiO2nanospheres were prepared using the classic Stöber method: 5.2 ml purified water, 27 ml anhydrous ethanol and 0.6 ml ammonia water were mixed and magnetically stirred (500 rpm) for 1 h, 1 ml of tetraethyl orthosilicate was added dropwise to the mixture, and after magnetic stirring at 25°C for 8 h, the anhydrous ethanol was centrifuged and washed 3-5 times, and the dispersed SiO2nanospheres were obtained by drying at 80°C.

[0086] b. Preparation of SiO2 / TiO2nanospheres: SiO2 / TiO2nanospheres were prepared using an oil bath thermal deposition method, 0.5 g of silica nanospheres were dispersed in 200 ml of anhydrous ethanol and ultrasonically treated, 0.8 ml of tetrabutyl titanate ethanol solution (tetrabutyl titanate to anhydrous ethanol at a volume ratio of 1:5) was added dropwise and stirred for 30 min, and then mixed with 0.42 ml of ammonia water. Then the mixed solution was heated in an 85°C oil bath and stirred for 1 h. After centrifugation with water and ethanol, it was dried at 60°C. Finally, SiO2 / TiO2nanospheres were prepared by calcining at 600°C with a heating rate of 5°C / min for 2 h.

[0087] c. Preparation of porous TiO2hollow spheres: SiO2 / TiO2nanospheres were etched with an appropriate amount of 1 mol / L sodium hydroxide aqueous solution for 6 h. After stirring and centrifugation, the hollow TiO2was obtained by washing with high-purity water and ethanol three times, and then filtering after cooling to room temperature, and drying the filter cake at 60°C to obtain porous TiO2hollow spheres, i.e. MTN.

[0088] 2. Synthesis of MTN-NH2: 100 mg of MTN was uniformly suspended in 400 mL of toluene by ultrasonic treatment. (3-Aminopropyl)triethoxysilane (APTES, 4 mL) was added to the suspension, which was then stirred at 80°C under nitrogen for 48 h. The precipitate was centrifuged at 13,000 rpm and washed with ethanol three times to obtain the MTN-NH2product, which was then dried at 60°C under vacuum for 24 h.

[0089] 3. MTN-ROS sensitive linker: MTN-NH2(100 mg) was sonicated in 10 mL of high purity water, then EDC-HCl (96 mg) and NHS (60 mg) were added to the suspension. The ROS sensitive linker (keto-thioacetal: HOOC-S-CH2-S-COOH 50 mg) was added dropwise to the mixture, then stirred at 60 °C under nitrogen for 72 h. The resulting MTN-ROS was washed with absolute ethanol 3 times, then with water 3 times to remove unreacted materials and other reagents, then dried at 60 °C under vacuum for 24 h.

[0090] 4. Synthesis of MTN-ROS-DSPE-PEG2K-NH2: MTN-ROS 100 mg was sonicated in ethylenediamine tetra chloromethane: water (2 ml: 20 mg) solution, 100 mg DSPE-PEG2K-NH2 was dissolved in appropriate amount of DMSO, the mixture of 100 mg DSPE-PEG2K-NH2 was stirred at 60 °C under nitrogen for 72 h. The product (MTN-ROS-DSPE-PEG2K-NH2) was washed with water 10 times to remove unreacted DSPE-PEG2K-NH2 and other reagents, then dried at 50 °C under vacuum for 24 h. The ROS-responsive nanoparticle dispersion was obtained by ultrasonic emulsification (200 W, 3 min) ice bath treatment, centrifugation to take the supernatant, and dialysis (MWCO = 3.5 kDa) with deionized water for 3 days.

[0091] 5. FA-β-CD preparation: β-CD (500 mg) was dispersed in 40 mL of toluene, refluxed for 8 h, then 500 μL of APTES (3-aminopropyl)triethoxysilane) was added. The mixed solution was stirred at 50 °C for 8 h, then β-CD-NH2 was dried in vacuum after centrifugation and ethanol washing. Then, FA (130 Mg), DEC (68 Mg) and NHS (76 Mg) were dispersed in 30 mL of dimethyl sulfoxide, stirred at 50 °C for 6 h, and finally β-CD-NH2 (500 mg) was added, and the reaction was continued at the same temperature for 24 h. The product FA-β-CD was dried at 40 °C under vacuum after centrifugation, dimethyl sulfoxide and deionized water washing.

[0092] 6. HM-TPZ linking

[0093] HM-TPZ was linked using keto-thioacetal:

[0094] (1) HM-TPZ linking reaction 1:

[0095]

[0096] Substrate a (Tirapazamine) (1 mmol) and ketosulfide b (1 mmol) were taken in a reaction flask along with dry DCM (5-10 mL) solution, N, N'-dicyclohexyl carbodiimide DCC (2 mmol) and 4-dimethylamino pyridine DMAP (1.2 mmol) were added. After stirring for 48 h, the reaction mixture was diluted with DCM and filtered with celite. The filtrate was evaporated and purified by column chromatography using ethyl acetate-pet ether to get c.

[0097] (2) HM-TPZ ligation reaction 2:

[0098]

[0099] A mixture of trifluoroacetic anhydride TFAA (3.5 mmol) and intermediate c (1 mmol) was stirred for 20 min until the solid dissolved. After stirring for another 20 min, substrate d (dehydroregulating) (1 mmol) was added. To this mixture, 85% phosphoric acid (0.1 mmol) was added for 20 min. The mixture was then stirred for 2 h (monitored by thin layer chromatography) and excess TFA / TFAA was distilled off at atmospheric pressure. The remaining liquid was extracted with ethyl acetate (30 mL) and water (15 mL). The organic layer was separated, washed with 5% sodium hydroxide (7 mL) and then with brine (8 mL). The mixture was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. Purification by column chromatography using ethyl acetate-pet ether gave the desired product e (HM-TPZ).

[0100] 7. Loading of HM-TPZ

[0101] MTN-ROS-DSPE-PEG2K-NH2(20 mg) was added to the ethanol solution (10 mL) containing HM-TPZ (20 mg) and sonicated for 6 h at room temperature with 400 W ultrasonic tissue disruption system for 10 times. The nanosuspension was centrifuged to remove free HM-TPZ to obtain HM-TPZ@MTN-ROS-DSPE-PEG2K-NH2.

[0102] 8. FA-β-CD capping: HM-TPZ@MTN-ROS-DSPE-PEG2K-NH2(10 mg) and FA-β-CD (10 mg) were added to appropriate amount of water respectively, stirred for 5 h in dark and the prepared HM-TPZ@MTN-ROS-DSPE-PEG2K-NH2@FA-β-CD was collected by centrifugation.

[0103] Example 2 Preparation of MTN@FA-β-CD

[0104] MTN@FA-β-CD capping: HM-TPZ@MTN-ROS-DSPE-PEG2K-NH2(10 mg) and FA-β-CD (10 mg) prepared in Example 1 were added to appropriate amount of water, respectively, and stirred in the dark for 5 h. The prepared MTN@FA-β-CD was collected by centrifugation.

[0105] Example 3 Preparation of HM-TPZ-NPs

[0106] Accurately weigh 100 mg of high molecular material PLGA-PEG and 10 mg of HM-TPZ, dissolve in 1 mL of DMSO, and ultrasonically dissolve. Slowly add the solution to 5 times the volume of phosphate buffer (PBS, 10 mM, pH = 7.4) under stirring. After the addition is complete, stir at room temperature for 30 min. Then centrifuge at 3000 r / min for 5 min to remove unencapsulated drugs. Use an ultrafiltration tube (molecular weight cutoff 10 KDa) to remove organic phase in PBS, and concentrate the HM-TPZ nanoparticles.

[0107] Effect verification example

[0108] 1. Measurement of nanoparticle size and surface potential:

[0109] The prepared nanoparticles MTN@FA-β-CD, MTN-ROS-DSPE-PEG2K-NH2, and HM-TPZ@MTN-ROS-DSPE-PEG2K-NH2@FA-β-CD were diluted with ultrapure water, and the above substances were prepared into suspensions of a certain concentration. Dynamic light scattering particle size analyzer (DLS) was used to measure the particle size distribution and surface Zeta potential of nanoparticles MTN@FA-β-CD, HM-TPZ@MTN-ROS-DSPE-PEG2K-NH2, and HM-TPZ@MTN-ROS-DSPE-PEG2K-NH2@FA-β-CD. The particle size distribution and potential results of each nanoparticle are shown in Table 5, and each nanoparticle showed negative charge.

[0110] Table 5 Particle size and potential of different nanoparticles

[0111]

[0112] 2. Evaluation of nanoparticle stability

[0113] The prepared ROS-responsive sonodynamic nanoparticles (prepared in Example 1) were placed at 4°C and 37°C, respectively, sampled, and the particle size of the nanoparticles was determined by a laser particle size analyzer. The stability of the nanoparticles was reflected by the change in particle size. The results showed that the particle size of the nanoparticles did not change significantly within 10 days under different storage conditions, indicating that the prepared ROS-responsive nanoparticles had good stability. The results are shown in Figure 2 .

[0114] 3. Drug release rate determination

[0115] The drug release behavior of the ROS-responsive nanoparticles under the action of ultrasound was evaluated.

[0116] Freshly prepared ROS-responsive 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, duty cycle 50%. Different ultrasound intensities (US0-0 W / cm 2 , US1-0.1 W / cm 2 , US2-0.2 W / cm 2 , US3-0.4 W / cm 2 , US4-0.6 W / cm 2 ) were used, and the ultrasound was treated for 8 min. Subsequently, the dialysis method was used to determine the drug release rate. The results showed that under the action of ultrasound, the drug HM-TPZ in the HM-TPZ@MTN-ROS-DSPE-PEG2K-NH2@FA-β-CD NPs could be quickly released, and the drug release was significantly accelerated with the increase of ultrasound intensity (see Figure 3 ). However, in the absence of MTN loading (HM-TPZ NPs: nanoparticles encapsulated by PLGA-PEG) combined with ultrasound treatment, the drug release rate was significantly reduced, indicating that the ROS generated by ultrasound stimulation of MTN could quickly cleave the ROS-sensitive group (TL residue), resulting in the rapid release of the drug MTN from the ROS-responsive nanoparticles.

[0117] 4. Evaluation of ROS production amount of ROS-responsive nanoparticles under the action of ultrasound

[0118] Freshly prepared ROS-responsive nanoparticles (prepared in Example 1) were placed at the focal point of focused ultrasound after adding the ROS probe DCFH-DA. The ultrasound device was the same as in "3. Drug release rate determination". US3 and US4 were selected as the ultrasound intensity. Subsequently, the fluorescence inverted microscope was used to observe the intensity of reactive oxygen species. The results showed that under the action of ultrasound, a large number of small bubbles appeared on the body surface of the protoscolex, and the cyst membrane was damaged. The ROS production amount of US3 and US4 was significantly higher.

[0119] Figure 4To investigate the effect of US on the external morphology of P. nana after drug treatment; Figure 5 To investigate the production of ROS after drug treatment and US treatment.

[0120] 5. Investigation of the cascade amplification synergy of ROS-responsive nanoparticles in the sonodynamic precise treatment of echinococcosis

[0121] The P. nana was inoculated in 96-well plates at 200 per well, and after the cells were fully stretched, the nanoparticles (HM-TPZNPs, MTN@FA-β-CD and HM-TPZ@MTN-ROS-DSPE-PEG2K-NH2@FA-β-CD) were used to incubate the P. nana for 12 h, then the medium was replaced, and the P. nana was treated with ultrasound (the parameters and devices were consistent with those in "3. Drug release rate determination"), and then incubated for 24 h. After the incubation was completed, the mortality was observed by methylene blue staining, and the results showed that before the ultrasound treatment, the ROS-responsive nanoparticles had no obvious effect on the mortality of P. nana, but after the ultrasound treatment, the mortality of P. nana increased significantly, and increased with the increase of ultrasound intensity (see Table 6).

[0122] Table 6 Survival rate of P. nana after nanoparticle intervention (%)

[0123]

[0124] The above-described embodiments are only to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A ROS-responsive nanoparticle, characterized in that, The mesoporous titanium dioxide nanoparticles for wrapping drugs are taken as the inner core, and the β-cyclodextrin modified by a targeting substance is taken as the shell; the shell is obtained by coating the inner core with ketone thioacetal with double carboxyl groups; The drugs include dehydropegerine and tirapazamine. The mass ratio of dehydropegerine to tirapazamine is 1:1-5.

2. The ROS-responsive nanoparticle of claim 1, wherein, The mass ratio of dehydropegerine to tirapazamine is 1:

1.

3. The ROS-responsive nanoparticle of claim 1, wherein, The dehydropegerine and tirapazamine are connected by ketone thioacetal with double carboxyl groups.

4. The method for preparing ROS-responsive nanoparticles as described in claim 1, characterized in that, The method comprises the following steps: Preparation of mesoporous titanium dioxide nanoparticles with ketone thioacetal with double carboxyl groups connected on the surface: mesoporous titanium dioxide is reacted with ketone thioacetal with double carboxyl groups under a protective atmosphere to obtain mesoporous titanium dioxide nanoparticles with ketone thioacetal with double carboxyl groups connected on the surface; Drug loading: the drugs are loaded by using the mesoporous titanium dioxide nanoparticles with ketone thioacetal with double carboxyl groups connected on the surface to obtain drug-loaded mesoporous titanium dioxide nanoparticles with ketone thioacetal with double carboxyl groups connected on the surface; Shell coating: the drug-loaded mesoporous titanium dioxide nanoparticles with ketone thioacetal with double carboxyl groups connected on the surface are coated by using β-cyclodextrin modified by a targeting substance to obtain the ROS-responsive nanoparticles.

5. The method for preparing ROS-responsive nanoparticles as described in claim 4, characterized in that, The method comprises the following steps: Preparation of mesoporous titanium dioxide nanoparticles with ketone thioacetal with double carboxyl groups connected on the surface: mesoporous titanium dioxide is reacted with ketone thioacetal with double carboxyl groups under a protective atmosphere to obtain mesoporous titanium dioxide nanoparticles with ketone thioacetal with double carboxyl groups connected on the surface; Drug connection: dehydropegerine and tirapazamine are connected by using the ketone thioacetal with double carboxyl groups to obtain connected drugs; Drug loading: the connected drugs are loaded by using the mesoporous titanium dioxide nanoparticles with ketone thioacetal with double carboxyl groups connected on the surface to obtain drug-loaded mesoporous titanium dioxide nanoparticles with ketone thioacetal with double carboxyl groups connected on the surface; Shell coating: the drug-loaded mesoporous titanium dioxide nanoparticles with ketone thioacetal with double carboxyl groups connected on the surface are coated by using β-cyclodextrin modified by a targeting substance to obtain the ROS-responsive nanoparticles.

6. The ROS-responsive nanoparticles according to any one of claims 1-3 are used for preparing a sonodynamic therapy drug for treating hydatidosis.

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