Nano-drug integrating activatable probe and oxygen-dependent and oxygen-independent sound-sensitive agents as well as preparation and application of nano-drug

Abstract

The invention belongs to the technical field of medicine, and relates to a nano-drug integrating an activatable probe and oxygen-dependent and oxygen-independent sound-sensitive agents as well as preparation and application of the nano-drug, asymmetric Janus AuNRs-mSiO2 is taken as a skeleton, Ag / Ag2S and hyaluronic acid are modified on the surface of the skeleton, and AIPH is loaded in mesopores of the Janus AuNRs-mSiO2; aIPH is an oxygen-independent type sound-sensitive agent, and Ag / Ag2S is used as an activatable probe and is also used as a precursor of the oxygen-independent type sound-sensitive agent. The nano-drug integrates multiple functions, activatable imaging is achieved, and ultrasonic radiation starting time is guided through images; the oxygen-dependent ROS and the oxygen-independent ROS generated by the two sound-sensitive agents have a synergistic enhancement effect, and the sonodynamic treatment effect of atherosclerosis is effectively enhanced. The nano medicine is ingenious in design, the preparation method is simple, the nano medicine can be used for sonodynamic treatment of atherosclerosis, and the market prospect is wide.

Description

Technical field:

[0001] The present invention belongs to the field of medical technology, and relates to an integrated nanomedicine that integrates an activatable probe and an oxygen-dependent and oxygen-independent sonosensitizer, and its preparation and application in activatable imaging-guided sonodynamic therapy. The activatable near-infrared II (NIR-II) fluorescent probe Ag / Ag2S and the oxygen-independent sonosensitizer 2,2'-azabis(2-imidazoline) dihydrochloride (AIPH) are loaded into the Janus AuNRs-mSiO2 nanomaterial, and the Ag / Ag2S is converted into the oxygen-dependent sonosensitizer Ag2S in response to the overexpressed H2O2 in the pathological environment, thereby turning on the NIR-II fluorescent signal to guide the start time of ultrasonic radiation; under ultrasonic radiation, both sonosensitizers produce reactive oxygen, synergistically enhancing the sonodynamic therapeutic effect on atherosclerosis. Background technology:

[0002] Nanomaterial-based sonodynamic therapy has attracted much attention in the treatment of atherosclerosis due to its advantages such as non-invasiveness, deep penetration, high specificity and good safety. Sonodynamic therapy can produce reactive oxygen species (ROS) in the desired area through cavitation effect by using low-intensity ultrasound and sonosensitizers, induce cell death, and thus play a therapeutic role. Although studies have shown that sonodynamic therapy can quickly inhibit the development of atherosclerotic plaques by selectively removing inflammatory macrophages in plaques. However, problems such as insufficient intake of sonosensitizers in the plaque area and low ROS production have led to its low therapeutic effect. Therefore, increasing the intake of sonosensitizers and the production of ROS in the plaque area is the core technology to enhance the effect of sonodynamic therapy. Increasing the intake of sonosensitizers in the plaque area can be achieved by connecting targeting molecules that can target the plaque area to nanomedicines. Increasing the production of ROS in the plaque area, especially in deep tissues, is still a core challenge in the current field of sonodynamic therapy.

[0003] Another factor that affects the effect of sonodynamic therapy is the starting time of ultrasound radiation. After intravenous administration, the sonosensitizer reaches the lesion area through cell uptake. The time of ultrasound radiation directly affects the effect of sonodynamic therapy. At present, there is no mature technology to guide the starting time of sonodynamic therapy. Summary of the invention:

[0004] The purpose of the present invention is to provide an integrated nanomedicine with targeted drug delivery capability, pathological microenvironment responsive imaging capability, nanomotor-driven deep tissue penetration capability, and oxygen-dependent and oxygen-independent ROS generation capability, so as to solve the problem of low efficiency of existing sonodynamic therapy for atherosclerosis.

[0005] In order to achieve the above-mentioned purpose, the present invention provides a nano drug integrating an activatable probe and an oxygen-dependent and oxygen-independent sonosensitizer, wherein an asymmetric Janus AuNRs-mSiO2 is used as a nano drug skeleton, Ag / Ag2S and hyaluronic acid (HA) are loaded on the surface of Janus AuNRs-mSiO2, and AIPH is loaded in the mesopores of Janus AuNRs-mSiO2; the AIPH is an oxygen-independent sonosensitizer, the Ag / Ag2S serves as both an activatable probe and an oxygen-dependent sonosensitizer precursor; and the HA is a targeting molecule.

[0006] The present invention preferably uses AIPH as an oxygen-independent sonosensitizer and preferably uses Janus AuNRs-mSiO2 as a nanodrug skeleton. This is because under ultrasonic radiation, AIPH can generate nitrogen, driving Janus AuNRs-mSiO2 to penetrate into the deep area of ​​the diseased tissue; at the same time, it generates oxygen-independent ROS to induce apoptosis of inflammatory macrophages. The AIPH is loaded in the mesopores of Janus AuNRs-mSiO2, which improves its stability, prolongs its residence time in the body, and overcomes its shortcomings of chemical instability and easy degradation.

[0007] The present invention preferably uses Ag / Ag2S as an activatable probe and preferably uses HA as a targeting molecule. HA can target the glycoprotein CD44 on the surface of inflammatory macrophages, thereby improving the active targeting ability of nanomedicines. Ag / Ag2S is converted into Ag2S under the action of H2O2 overexpressed in the pathological microenvironment, illuminating the NIR-II fluorescence signal. Ag2S also acts as an oxygen-dependent sonosensitizer. Under ultrasonic irradiation, Ag2S produces oxygen-dependent ROS to induce apoptosis of inflammatory macrophages.

[0008] The structure of the Janus AuNRs-mSiO2 nano drug skeleton of the present invention is that one end of the gold nanorod is coated with SiO2 having a mesoporous structure.

[0009] The present invention also provides a method for preparing a nano drug integrating an activatable probe and an oxygen-dependent and oxygen-independent sonosensitizer, characterized in that the specific steps are as follows:

[0010] (1) preparing gold nanorods;

[0011] (2) Synthesis of Janus AuNRs-mSiO2 NPs

[0012] The gold nanorod aqueous solution is slowly added to isopropanol and stirred, and then 4-mercaptophenylacetic acid and polyacrylic acid are added to react, and then tetraethyl orthosilicate, 3-aminopropyltriethoxysilane and ammonia water are added and stirred for reaction; then centrifuged and washed to obtain a Janus AuNR-SiO2 NPs sample; subsequently, a polyvinyl pyrrolidone solution is added to the Janus AuNRs-SiO2 sample, heated, and then cooled to room temperature, centrifuged and washed to obtain Janus AuNRs-mSiO2 NPs;

[0013] (3) Synthesis of Janus AuNRs-mSiO2@AIPH NPs

[0014] Mix AIPH and Janus AuNRs-mSiO2 prepared in step (2) in water and stir to load AIPH; finally, centrifuge and wash to obtain Janus AuNR-mSiO2@AIPH NPs;

[0015] (4) Preparation of Janus Ag / Ag2S NPs;

[0016] (5) Preparation of Janus AuNRs-mSiO2-Ag / Ag2S@AIPH nanodrugs

[0017] First, HS-PEG 5000 -COOH was added dropwise to the Janus Ag / Ag2S NPs aqueous solution and stirred; free PEG was then removed by ultrafiltration centrifugal filter to obtain PEG 5000 -COOH-Ag / Ag2S NPs; PEG 5000 -COOH-Ag / Ag2S NPs were redispersed with ultrapure water, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added; the above solution was adjusted to pH = 7.2 and dropped into the Janus AuNR-mSiO2@AIPH solution with continuous stirring; finally, Janus AuNR-mSiO2-Ag / Ag2S@AIPH was purified by ultracentrifugation;

[0018] (6) Hyaluronic acid (HA) modification

[0019] HS-PEG 5000-HA was added dropwise to the Janus AuNR-mSiO2-Ag / Ag2S@AIPH aqueous solution and stirred, and then the free HS-PEG5000-HA was further removed by an ultrafiltration centrifugal filter to obtain hyaluronic acid-modified Janus AuNR-mSiO2-Ag / Ag2S@AIPH (HA-JASAA), which is a nanodrug integrating an activatable probe and oxygen-dependent and oxygen-independent sonosensitizers.

[0020] In step (2) of the present invention, the reaction time after adding 4-mercaptophenylacetic acid and polyacrylic acid is 30-60 minutes; the reaction time after adding tetraethyl orthosilicate, 3-aminopropyltriethoxysilane and ammonia water is 4-9 hours; the heating temperature after adding polyvinyl pyrrolidone solution is 80-120° C. and maintained for 1-5 hours.

[0021] The specific preparation method of step (4) of the present invention is: add ultrapure water, hexadecyltrimethylammonium bromide aqueous solution, AgNO3 aqueous solution, and ammonia water in a round-bottom flask in sequence, stir, and then quickly inject acetamide aqueous solution to form a brown complex liquid; magnetically stir in the dark, collect with an ultrafiltration centrifugal filter, wash, and obtain Ag / Ag2S NPs; the volume ratio of the ultrapure water, hexadecyltrimethylammonium bromide aqueous solution, AgNO3 aqueous solution, and NH3·H2O solution is 50:6:2.705:5.41, the concentration of the hexadecyltrimethylammonium bromide aqueous solution is 10mM; the concentration of the AgNO3 aqueous solution is 10mM; the concentration of the NH3·H2O solution is 4M; the concentration of the acetamide aqueous solution is 4mM.

[0022] The specific steps of step (5) of the present invention are: adding 1 ml of 5 mg / mL (HS-PEG 5000 5 mL of -COOH aqueous solution with a concentration of 0.5 mg mL -1 The Ag / Ag2S NPs aqueous solution was stirred; then, the free PEG was removed by ultrafiltration centrifugal filter to obtain PEG modified with PEG 5000 -COOH-Ag / Ag2S NPs; in 1mL of PEG with a concentration of 10mg / mL 50002 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2 mg N-hydroxysuccinimide were added to the Ag / Ag2S NPs solution, and the solution was stirred continuously for 10-50 min at pH = 6 to activate the carboxyl groups on the surface of Ag / Ag2S NPs. Then, the solution was adjusted to pH = 7.2 and dropped into 1 mL of 10 mg / mL Janus AuNR-mSiO2@AIPH solution, and the mixture was stirred continuously at 15-25 ° C for 1-5 hours. Finally, the Janus AuNR-mSiO2-Ag / Ag2S@AIPH nanodrug was obtained by three rounds of centrifugation purification.

[0023] The present invention also provides the use of the nano drug integrating the activatable probe and the oxygen-dependent and oxygen-independent sonosensitizers in fluorescence imaging, which remains inactive before being triggered by H2O2 and turns on fluorescence after being activated by H2O2, thereby realizing activatable fluorescence imaging.

[0024] The present invention also provides the use of the nano drug integrating the activatable probe and the oxygen-dependent and oxygen-independent sonosensitizers in sonodynamic therapy. The nano drug can guide the start time of ultrasound radiation through activatable fluorescence imaging. Under the action of H2O2 overexpressed in the lesion area, the nano drug lights up the NIR-II fluorescence signal. The time point when the signal intensity reaches a peak value is determined as the starting point of ultrasound radiation, thereby realizing image-guided sonodynamic therapy.

[0025] The present invention also provides the use of the nano drug integrating the activatable probe and the oxygen-dependent and oxygen-independent sonosensitizers in the sonodynamic therapy of atherosclerosis, wherein the nano drug is used at a concentration of 100 μg / mL; when the nano drug is used for 24 hours, US irradiation is performed, and the US intensity is 2.0 W cm -2 , time is 10 minutes.

[0026] In order to increase the production of ROS in the plaque area, especially in the deep tissue, the present invention loads oxygen-dependent and oxygen-independent sonosensitizers and targeting molecules on the Janus AuNRs-mSiO2 nanomotor, thereby realizing the targeted delivery of nanomedicines. Under ultrasonic radiation, the motor generates nitrogen to drive the sonosensitizer to the deep area of ​​the plaque, realizing the generation of oxygen-dependent and oxygen-independent ROS, thereby improving the therapeutic effect.

[0027] In order to determine the starting point of ultrasound radiation during sonodynamic therapy, the present invention introduces a pathological microenvironment-activatable probe Ag / Ag2S during the preparation of nanomedicine. After intravenous administration, the probe and the sonosensitizer enter the lesion area at the same time. Under the action of H2O2 overexpressed in the plaque, Ag / Ag2S is converted into Ag2S, thereby lighting up the NIR-II fluorescence signal. The time point when the signal intensity reaches the peak value is determined as the starting point of ultrasound radiation (at this time, the sonosensitizer is most enriched in the plaque area), thereby realizing image-guided sonodynamic therapy. When the nanomedicine integrating an activatable probe and oxygen-dependent and oxygen-independent sonosensitizers is applied to atherosclerosis, after intravenous injection of HA-JASAA, the hyaluronic acid modification can specifically target pro-inflammatory macrophages in atherosclerotic plaques, react with H2O2 in the atherosclerotic microenvironment, and turn on the NIR-II fluorescence signal; when the intensity of the NIR-II fluorescence signal reaches a peak value (at this time, the sonosensitizer is enriched in the plaque area to the maximum), US irradiation is performed; AIPH loaded in HA-JASAA is converted into oxygen-independent alkyl free radicals (R·, a type of ROS) and nitrogen, which promotes HA-JASAA to penetrate deeply into the plaque tissue. In addition, under US activation, two ultrasound sensitizers AIPH and Ag2S respectively generate oxygen-independent and oxygen-dependent ROS, inducing apoptosis of diseased macrophages, thereby significantly inhibiting the progression of atherosclerotic plaques.

[0028] Compared with the prior art, the nano drug of the present invention integrates the ability of targeted drug delivery, imaging ability of pathological microenvironment response, deep tissue penetration ability driven by nano motors, and oxygen-dependent and oxygen-independent ROS generation ability. Ag / Ag2S is used as an activatable probe, which is converted into oxygen-dependent sonosensitizer Ag2S under the action of H2O2 overexpressed in the pathological microenvironment, lighting up the NIR-II fluorescence signal, thereby realizing specific fluorescence imaging of atherosclerosis; the time point when the signal intensity reaches the peak is determined as the starting point of ultrasound radiation, thereby realizing the guidance of sonodynamic therapy by imaging; under ultrasound radiation, AIPH can produce nitrogen, driving the nano drug to penetrate into the deep area of ​​the diseased tissue, and at the same time, the oxygen-independent and oxygen-dependent ROS generated by the two sonosensitizers AIPH and Ag2S respectively have a synergistic enhancement effect, inducing apoptosis of inflammatory macrophages. The nano drug of the present invention cooperates with sonodynamic therapy to realize the non-invasive treatment of atherosclerosis and effectively enhances the sonodynamic therapy effect of atherosclerosis. The nano drug of the present invention integrates multiple functions, has an ingenious design, and has a simple preparation method. It can be used for sonodynamic therapy of atherosclerosis and has broad market prospects. Description of the drawings:

[0029] Figure 1The present invention relates to a nanomedicine (HA-JASAA) that integrates an activatable probe and oxygen-dependent and oxygen-independent sonosensitizers, wherein A is a transmission electron microscope image; B is a HAADF-STEM image and corresponding elemental mapping; C is a UV-Vis-NIR spectrum; and D is a Fourier transform infrared spectroscopy (FT-IR) analysis result diagram.

[0030] Figure 2 These are NIR-II fluorescence images of the HA-JASAA nanomedicine involved in the present invention after being treated with or without H2O2 (1 mM) for different periods of time.

[0031] Figure 3 The present invention relates to the ability of HA-JASAA to generate oxygen-independent ROS after being treated with ultrasound at different intensities.

[0032] Figure 4 The present invention relates to the ability of HA-JASAA to generate oxygen-dependent ROS after being treated with ultrasound at different intensities.

[0033] Figure 5 This is a fluorescent image of HA-JASAA involved in the present invention producing ROS at the cellular level.

[0034] Figure 6 The present invention relates to the ability of HA-JASAA to induce apoptosis of inflammatory macrophages.

[0035] Figure 7 This is a fluorescent image of HA-JASAA in atherosclerosis in mice.

[0036] Figure 8 The present invention relates to the in vivo treatment of atherosclerotic plaques by HA-JASAA-mediated sonodynamic therapy, wherein A represents the plaque size and B represents the lesion area.

[0037] Fig. 9 This is a graph of the in vivo biosafety assessment experiment results of the HA-JASAA nanomotor involved in the present invention, wherein A is the plasma concentration-time curve of the HA-JASAA nanomotor; B is the hemolysis test of HA-JASAA at different concentrations, and the illustrations are images of solutions at different concentrations; C is a routine blood test of mice treated with different methods; D is the liver function of mice treated with different methods; E is the effect of different treatments on the renal function of mice; F is a blood lipid test of mice treated with different methods; G is an H&E staining image of the main organs (heart, liver, spleen, lungs and kidneys) of different groups, scale bar: 200μm. Specific implementation method:

[0038] The technical solution of the present invention is further described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0039] Embodiment 1:

[0040] This embodiment relates to a method for preparing a nanomedicine that integrates an activatable probe and an oxygen-dependent and oxygen-independent sonosensitizer. The specific steps are as follows:

[0041] (1) Synthesis of gold nanorods (Au NRs)

[0042] Au NRs were synthesized by seed-mediated growth method. First, 100 μL of 50.8 mM chloroauric acid trihydrate solution (HAuCl4·3H2O) was added to 10 mL of 0.1 M hexadecyltrimethylammonium bromide (CTAB) aqueous solution and stirred for 5 min to obtain a mixed solution. Subsequently, 460 μL of a mixed solution of NaBH4 and NaOH was quickly added to the mixed solution under vigorous stirring to obtain a gold seed solution. The mixed solution of NaBH4 and NaOH was prepared by dissolving 4 mg of sodium borohydride (NaBH4) and 4 mg of NaOH powder in 10 mL of ice water. Then, 600 μL of the gold seed solution was quickly added to the growth solution, reacted at 30 °C for 12 h, and centrifuged (8 000 r min). -1 , 10min) to collect Au NRs samples, and wash them twice with ultrapure water, and dry them to obtain AuNRs; finally, dilute Au NRs with ultrapure water to 50mL, store them at 4°C, and obtain AuNRs aqueous solution for standby use; the gold seed solution should be aged in the dark for 30 minutes before use; the growth solution is prepared by mixing CTAB aqueous solution (50mL, 0.1M), HAuCl4·3H2O aqueous solution (500μL, 50.8mM), AgNO3 aqueous solution (350μL, 0.1M) and hydroquinone aqueous solution (2.5mL, 0.1M);

[0043] (2) Synthesis of Janus AuNRs-mSiO2 NPs

[0044] 6 mL of 10 mg / ml AuNRs aqueous solution was slowly added to 30 mL of isopropanol while stirring vigorously (800 r min -1 , 10min), then add 200μL of 5mM 4-mercaptophenylacetic acid (4-MPAA) ethanol solution and 720μL of 0.645mM polyacrylic acid (PAA) aqueous solution, react for 45min, then add 9mL of 8.9mM tetraethyl orthosilicate (TEOS) aqueous solution, 32μL 3-aminopropyltriethoxysilane (APTES) and ammonia water (1mL), stir and react at room temperature for 6h; then centrifuge (7000r min -1, 8min) collect the precipitate, wash with water, and obtain the Janus AuNR-SiO2 NPs sample; then, add 1mL of 4wt% polyvinyl pyrrolidone (PVP) aqueous solution to the prepared Janus AuNRs-SiO2 sample to obtain a Janus AuNRs-SiO2 suspension, heat the suspension to 100°C and keep it for 3h; then cool to room temperature, centrifuge, wash with ultrapure water and ethanol several times to remove excess alkali, and dry to obtain Janus AuNRs-mSiO2 NPs (abbreviated as AuNRs-mSiO2);

[0045] (3) Synthesis of Janus AuNRs-mSiO2@AIPH NPs

[0046] The concentration was 5 mg mL -1 The AIPH (2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride) aqueous solution was mixed with the Janus AuNRs-mSiO2 prepared in step (2) in water, and then gently stirred for 24 hours to achieve the loading of AIPH; finally, the precipitate was collected by centrifugation, washed several times with ultrapure water to remove excess free AIPH, and dried to obtain JanusAuNR-mSiO2@AIPH NPs (abbreviated as AuNR-mSiO2@AIPH);

[0047] (4) Preparation of Janus Ag / Ag2S NPs

[0048] In a 100 mL round-bottom flask, 50 mL of ultrapure water, 6 mL of 10 mM CTAB aqueous solution, 2.705 mL of 10 mM AgNO3 aqueous solution, and 5.41 mL of 4 M NH3·H2O solution were added in sequence, stirred for 5 min, and then 3 mL of 4 mM acetamide (TAA) aqueous solution was quickly injected to form a brown complex solution; after magnetic stirring for 24 h in the dark, the solution was collected with an ultrafiltration centrifugal filter (cutoff molecular weight of 10 kDa), washed 3 times with ultrapure water to remove excess CTAB, and dried to obtain JanusAg / Ag2S NPs (abbreviated as Ag / Ag2S);

[0049] (5) Preparation of Janus AuNRs-mSiO2-Ag / Ag2S@AIPH nanodrugs

[0050] First, 1 ml of 5 mg / mL thiolated polyethylene glycol acid (HS-PEG 5000 -COOH,) aqueous solution was added dropwise with 5 mL of 0.5 mg mL -1The Ag / Ag2S NPs aqueous solution was stirred for 6 h; then, the free PEG was removed by ultrafiltration centrifugal filter, and the 5 kDa MW was cut off at 5000 rpm to obtain PEG modified with PEG 5000 -COOH-Ag / Ag2S NPs, PEG 5000 -COOH-Ag / Ag2S NPs were redispersed with ultrapure water; then, 1 mL of PEG with a concentration of 10 mg / mL was added. 5000 2 mg EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 2 mg NHS (N-hydroxysuccinimide) were added to the aqueous solution of -COOH-Ag / Ag2SNPs and stirred continuously for 30 minutes at pH = 6 to activate PEG 5000 -COOH-Ag / Ag2S NPs-Ag / Ag2S NPs surface carboxyl groups; then, the above solution was adjusted to pH = 7.2, and it was dropped into 1 mL of Janus AuNR-mSiO2@AIPH aqueous solution with a concentration of 10 mg / mL obtained in step (3), and the mixture was continuously stirred at a constant temperature of 25°C for 2 hours; finally, Janus AuNR-mSiO2-Ag / Ag2S@AIPH nanodrug (abbreviated as JASAA) was obtained by three rounds of ultracentrifugation purification; the obtained JASAA was prepared with water to a concentration of 0.5 mg mL -1 JASAA aqueous solution, set aside;

[0051] (6) HA modification

[0052] HS-PEG 5000 -HA (1 mL, 5 mg / mL) (provided by Xi'an Ruixi Biological Company) was added dropwise to 5 mL of the JASAA aqueous solution (0.5 mg mL -1 ) and stirred for 6 h. Free PEG was then further removed by an ultrafiltration centrifugal filter to obtain Janus HA-PEG-AuNR-mSiO2-Ag / Ag2S@AIPH (abbreviated as HA-JASAA), which is a nanodrug integrating an activatable probe and oxygen-dependent and oxygen-independent sonosensitizers. It was redispersed with ultrapure water to obtain a solution with a concentration of 100 μg / ml for subsequent experiments.

[0053] The prepared HA-JASAA was characterized by TEM, HAADF-STEM images and corresponding elemental mapping, UV-visible-near infrared spectroscopy and Fourier transform infrared spectroscopy (FT-IR). Figure 1 shown.

[0054] from Figure 1 A combination Figure 1B It can be seen that according to the distribution of Au, Si, O and Ag, SiO2 is selectively coated on one end of AuNRs, and SiO2 has a mesoporous structure; Ag / Ag2S is attached to the surface of SiO2, and this attachment occurs because SiO2 provides sufficient amino groups to covalently crosslink with the functionalized carboxyl groups on Ag / Ag2S.

[0055] from Figure 1 The UV-visible-near infrared absorption spectra of AuNRs, Janus AuNRs-mSiO2, Janus AuNR-mSiO2@AIPH and HA-JASAA of C show that AuNRs have an absorption peak at about 1050nm, while a red shift is observed in the absorption peak of AuNRs-mSiO2, which is mainly attributed to the change of the refractive index around the AuNR surface. In addition, the absorption band around 360nm indicates that AIPH is successfully loaded into AuNRs-mSiO2.

[0056] from Figure 1 The Fourier transform infrared spectroscopy (FT-IR) results of D show that there are several characteristic peaks related to HA: 1097 cm -1 A peak at 1600 cm indicated the presence of CO groups; -1 There is a peak at 3350 cm-1, corresponding to the presence of the amide II group; -1 There is a peak at 2900 cm-1, confirming the stretching vibration of OH; the stretching vibration of CH is at 2900 cm-1. -1 A peak appears near the HA, confirming that HA loading is successful.

[0057] Embodiment 2:

[0058] This example relates to the performance test of HA-JASAA prepared in Example 1.

[0059] 1. The NIR-II fluorescence signal of the HA-JSASS nanomotor has a H2O2 response turn-on performance

[0060] A 1 mM H2O2 solution was added or not added to the HA-JASAA aqueous solution, and the NIR-II fluorescence images of HA-JASAA were taken at different times. The results are shown in Figure 2 As shown. Figure 2 It can be seen that the addition of H2O2 increases the fluorescence of the HA-JASAA solution over time compared with the absence of H2O2, indicating that HA-JASAA has good NIR-II fluorescence signal opening characteristics for H2O2.

[0061] 2. Detection of the ability of HA-JASAA to produce oxygen-independent ROS under US irradiation

[0062] 2,2'-Amino-bis(3-ethylbenzothiazole-6-sulfonic acid) free radical (ABTS*) was used as an indicator. The mixed solution of ABTS* and HA-JASAA was irradiated with US of different intensities, and the UV-visible-near infrared spectrum of the irradiated solution was detected. The results are as follows Figure 3 As the US irradiation intensity increased, the characteristic absorbance in the wavelength range of 550-900 nm gradually increased, indicating that the non-oxygen-dependent ROS produced by HA-JASAA gradually increased.

[0063] 3. Detection of the ability of HA-JASAA to produce oxygen-dependent ROS under US irradiation

[0064] Singlet oxygen ( 1 O2) is a typical oxygen-dependent ROS, and its production can be monitored by monitoring 1,3-diphenylisobenzofuran (DPBF) in the presence of 1 O2 decomposes into 1,2-dibenzoylbenzene, which leads to a decrease in UV absorbance at 410nm. The HA-JASAA solution containing DPBF was irradiated with ultrasound, and the absorbance of the solution after treatment with different US intensities was recorded. The results are shown in Figure 4 As shown in the figure, the DPBF absorbance decreased significantly with the increase of ultrasound intensity. The results showed that HA-JASAA had a good oxygen-dependent ROS generation ability under ultrasound irradiation.

[0065] Before ultrasonic irradiation, H2O2 was used to turn on the fluorescence signal of HA-JASAA solution, that is, Ag / Ag2S in HA-JASAA was converted into Ag2S.

[0066] 4. Sonodynamic therapeutic effect of HA-JASAA at the cellular level

[0067] To verify the synergistic enhancement of the therapeutic effect achieved by HA-JASAA, oxygen-independent and oxygen-dependent sonosensitizers were included. The ROS probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) was used to observe the intracellular ROS level. DCFH-DA reacts with ROS to produce a green fluorescent signal. US (2.0 W cm -2 The HA-JASAA aqueous solution (100 μg / mL concentration) was irradiated with US for 10 min, and the fluorescence signal was observed, which was recorded as the HA-JASAA+US group. In the same way, the AuNR-mSiO2-Ag / Ag2S (HA-JAS-A) solution and the AuNR-mSiO2@AIPH (HA-JAS@A) solution were irradiated with US, respectively, and they were recorded as the HA-JAS-A+US group and the HA-JAS@A+US group. A separate US group, HA-JASAA group and blank control group were set up to observe the fluorescence signal. The results are shown in the figure. Figure 5 As shown. Figure 5It can be seen that the fluorescence signals of the control group, US group and HA-JASAA group are negligible; the HA-JAS-A+US group or HA-JAS@A+US group shows a strong green fluorescence signal due to the generation of ROS activated by US; the green fluorescence signal of the HA-JASAA+US group is the strongest among all groups, which further proves that HA-JASAA contains oxygen-independent and oxygen-dependent sonosensitizers that can enhance the generation of ROS under US stimulation.

[0068] Flow cytometry was used to study the enhanced therapeutic effect of HA-JASAA on RAW264.7 macrophages under US irradiation. Figure 6 As shown. Figure 6 It can be seen that neither HA-JASAA alone nor US alone induced significant cell death. Only 2.6% and 3.4% of cells in the HA-JASAA alone group and the US alone group were damaged, respectively. However, the HA-JAS-A+US group caused a significant increase in cell damage, with a cell death rate of 33.2%. The HA-JAS@A+US combination also showed a significant increase in cell damage, with a corresponding cell death rate of 54.4%. These results indicate that ROS can induce cell death independently of oxygen availability. Figure 6 It can be seen that the cell death rate in the HA-JASAA+US group reached the highest level (78%), which indicates that there is a synergistic enhancement effect between oxygen-dependent and oxygen-independent ROS.

[0069] The AuNR-mSiO2-Ag / Ag2S (HA-JAS-A) differs from HA-JASAA in that AIPH is not loaded.

[0070] The difference between the AuNR-mSiO2@AIPH (HA-JAS@A) and HA-JASAA is that Ag / Ag2S is not loaded.

[0071] Before irradiating the HA-JASAA and HA-JAS-A solutions with US, H2O2 was used to turn on the fluorescence signal of the solutions, that is, to convert the Ag / Ag2S in HA-JASAA and HA-JAS-A into Ag2S.

[0072] Embodiment 3:

[0073] This example relates to the application test of Janus HA-PEG-AuNR-mSiO2-Ag / Ag2S@AIPH (abbreviated as HA-JASAA) prepared in Example 1.

[0074] 1. Establishing an animal model of atherosclerotic plaque

[0075] ApoE - / -Mice were used as experimental subjects to establish an animal model of atherosclerosis. - / - Mice were purchased from Huafukang Biological Company and were adaptively fed in the first week to adapt to the culture environment. They were fed a high-fat diet from the second to the 13th week, and mice were randomly selected for dissection and oil red staining to confirm the establishment of the arterial model. In vivo imaging and sonodynamic therapy were performed from the 14th to the 15th week.

[0076] 2. Verification of the fluorescence imaging function of HA-JASAA

[0077] HA-JASAA was dissolved in PBS solution (concentration of 100 μg / mL) and intravenously injected into atherosclerotic mice. Normal mice injected with HA-JASAA were used as the control group. The in vivo fluorescence imaging of the two groups of mice was observed by the in vivo imaging system, and the corresponding changes in the average fluorescence intensity were recorded. The results are shown in Figure 2. Figure 7 shown.

[0078] from Figure 7 It can be seen that 6 h after the injection of HA-JASAA in the model group, an obvious NIR-II fluorescence signal was observed in the plaque area, and the signal intensity continued to increase until 24 h, but in the following 12 h, the NIR-II fluorescence signal gradually weakened. Figure 7 The corresponding changes in the recorded mean fluorescence intensity at 1250 nm in the plaque area are consistent with the changes in the NIR-II fluorescence signal observed from the plaque area image. The control group, that is, the NIR-II fluorescence signal of normal mice injected with HA-JASAA, did not show significant changes. This shows that the activation of the NIR-II fluorescence signal in the plaque area is attributed to the conversion of Ag / Ag2S to Ag2S induced by the atherosclerotic microenvironment. This is because Ag / Ag2S has no fluorescence, and H2O2 in the atherosclerotic environment can etch away Ag, turning on the fluorescence of Ag2S.

[0079] The HA-JASAA of the present invention is able to respond to H2O2 and, as an activatable probe, remains inactive until triggered by overexpressed H2O2 in the atherosclerotic microenvironment. This feature provides an improved signal-to-background ratio, as well as enhanced sensitivity and specificity.

[0080] 3. Verification of the efficacy of HA-JASAA-mediated sonodynamic therapy against atherosclerosis

[0081] In view of the above verification that the NIR-II fluorescence signal reaches a peak 24 hours after the injection of HA-JASAA in model mice, this experiment chose to start sonodynamic therapy when the fluorescence signal reaches a peak. In order to verify the effect of HA-JASAA-mediated sonodynamic therapy, 4 comparative examples were designed. The specific steps were as follows: 25 ApoE mice carrying atherosclerotic plaques were injected with - / -The model mice were randomly divided into 5 groups for different treatments: (1) control group, no treatment; (2) US group, intravenous injection of PBS, once every other day, 24 hours after each injection, ultrasonic irradiation for 10 minutes, a total of 7 injections and 7 ultrasonic irradiations; (3) HA-JAS-A+US group, intravenous injection of HA-JAS-A, the difference between HA-JAS-A and HA-JASAA is that it does not contain the sonosensitizer AIPH; injection once every other day, 24 hours after each injection, ultrasonic irradiation for 10 minutes, a total of 7 injections and 7 ultrasonic irradiations. times, and ultrasonic irradiation 7 times; (4) HA-JAS@A+US group, intravenous injection of HA-JAS@A, the difference between HA-JAS@A and HA-JASAA is that there is no Ag / Ag2S; injection once every other day, ultrasonic irradiation for 10 minutes 24 hours after each injection, a total of 7 injections, ultrasonic irradiation 7 times; (5) HA-JASAA+US group, intravenous injection of HA-JASAA, injection once every other day, ultrasonic irradiation for 10 minutes 24 hours after each injection, a total of 7 injections, ultrasonic irradiation 7 times. The ultrasonic irradiation intensity is 2.0W cm -2 After the treatment of each group, the mice were euthanized and the aorta was removed one week later for Oil Red O (ORO) staining and quantification of the ratio of plaque area to the whole aorta. Figure 8 shown.

[0082] from Figure 8 As can be seen in A, Oil Red O staining shows aortic atherosclerotic plaques, and the red area indicates the presence of plaques. The ratio of plaque area to the entire aorta was then quantified. In all treatments, HA-JASAA+

[0083] The US group had the smallest lesion area, indicating that it has a better therapeutic effect on aortic atherosclerosis. HA-JASAA+US treatment can significantly reduce the aortic root plaque area to 0.082mm 2 , respectively, compared with the HA-JAS-A+US group and the HA-JAS@A

[0084] The +US group decreased by approximately 2.95-fold and 1.74-fold.

[0085] The cross-sectional aortic root samples were then stained with hematoxylin and eosin (HE) to assess aortic pathological morphology ( Figure 8 B) From Figure 8 B shows that the ratio of necrotic core area to lesion area in the HA-JASAA+US group was significantly reduced, indicating that HA-JASAA+US treatment can significantly inhibit plaque progression. Compared with other groups, mice treated with HA-JASAA+US showed the least atherosclerotic pathology.

[0086] The above results indicate that HA-JASAA can effectively prevent the progression of atherosclerotic plaques in vivo through the synergistic sonodynamic therapy of nanomedicines that integrate activatable probes and oxygen-dependent and oxygen-independent sonosensitizers.

[0087] Embodiment 4:

[0088] This example relates to an in vivo biosafety test of HA-JASAA.

[0089] The metabolic behavior of HA-JASAA in ApoE- / - mice carrying plaques was studied. The half-life of HA-JASAA in the blood circulation was 1.878 h as determined by ICP-MS at different time points after injection, indicating that HA-JASAA has good biocompatibility and fast blood clearance ( Fig. 9 A). In addition to metabolic behavior, the in vivo biosafety of HA-JASAA was evaluated by multiple parameters. Hemocompatibility was evaluated by incubating erythrocytes with HA-JASAA at concentrations ranging from 0 to 100 μg / mL. At concentrations up to 100 μg / mL, the hemolysis rate remained below 5%, indicating that HA-JASAA has good hemocompatibility in the blood circulation ( Fig. 9 B).

[0090] In addition, there was no statistical difference in the weight curves of mice in the control group and the HA-JASAA+US group within 14 days after treatment. Peripheral blood analysis 14 days after treatment showed no significant changes in blood routine parameters such as red blood cell, white blood cell, platelet and hemoglobin levels ( Fig. 9 C), indicating that HA-JASAA+US did not cause a decrease in systemic monocytes / macrophages. Similarly, no significant changes were observed in liver and kidney function indicators ( Fig. 9 D and 9E). In addition, the treatment did not affect the lipid profile of mice fed a high-fat cholesterol diet ( Fig. 9 F), which suggests that the anti-atherosclerotic effect of sonodynamic therapy is not due to changes in blood lipid levels.

[0091] Finally, the mice were killed and their main organs (heart, liver, spleen, lung, and kidney) were taken for pathological examination. Compared with the control group, no obvious tissue damage and inflammatory lesions were found in the H&E stained sections of the main organs of the HA-JASAA and other treatment groups ( Fig. 9 G). Taken together, these findings confirm that HA-JASAA-mediated sonodynamic therapy has a good biosafety profile and highlight its potential for future clinical applications.

Claims

1. A nanomedicine integrating an activatable probe and an oxygen-dependent and oxygen-independent sonosensitizer, characterized in that: Asymmetric Janus AuNRs-mSiO2 is used as a nanodrug skeleton, the surface of Janus AuNRs-mSiO2 is modified with Ag / Ag2S and hyaluronic acid (HA), and AIPH is loaded in the mesopores of Janus AuNRs-mSiO2; the AIPH is an oxygen-independent sonosensitizer, the Ag / Ag2S serves as both an activatable probe and an oxygen-dependent sonosensitizer precursor; the HA is a targeting molecule.

2. The nanomedicine integrating an activatable probe and an oxygen-dependent and oxygen-independent sonosensitizer according to claim 1, characterized in that: The structure of the Janus AuNRs-mSiO2 nano drug skeleton is that one end of the gold nanorod is coated with SiO2 having a mesoporous structure.

3. A method for preparing a nanomedicine integrating an activatable probe and an oxygen-dependent and oxygen-independent sonosensitizer, characterized in that: The specific steps are as follows: (1) preparing gold nanorods; (2) Synthesis of Janus AuNRs-mSiO2 NPs The gold nanorod aqueous solution is slowly added to isopropanol and stirred, and then 4-mercaptophenylacetic acid and polyacrylic acid are added to react, and then tetraethyl orthosilicate, 3-aminopropyltriethoxysilane and ammonia water are added and stirred for reaction; then centrifuged and washed to obtain a Janus AuNR-SiO2 NPs sample; subsequently, a polyvinyl pyrrolidone solution is added to the Janus AuNRs-SiO2 sample, heated, then cooled to room temperature, centrifuged and washed to obtain Janus AuNRs-mSiO2 NPs; (3) Synthesis of Janus AuNRs-mSiO2@AIPH NPs Mix AIPH and Janus AuNRs-mSiO2 prepared in step (2) in water and stir to load AIPH; finally, centrifuge and wash to obtain Janus AuNR-mSiO2@AIPH NPs; (4) Preparation of Janus Ag / Ag2S NPs; (5) Preparation of Janus AuNRs-mSiO2-Ag / Ag2S@AIPH First, HS-PEG 5000 -COOH was added dropwise to the Janus Ag / Ag2S NPs aqueous solution and stirred; free PEG was then removed by ultrafiltration centrifugal filter to obtain PEG 5000 -COOH-Ag / Ag2S NPs; PEG 5000 -COOH-Ag / Ag2S NPs were redispersed with ultrapure water, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added; the above solution was adjusted to pH = 7.2 and dropped into the Janus AuNR-mSiO2@AIPH solution with continuous stirring; finally, Janus AuNR-mSiO2-Ag / Ag2S@AIPH was purified by ultracentrifugation; (6) Hyaluronic acid modification HS-PEG 5000 -HA was added dropwise to the Janus AuNR-mSiO2-Ag / Ag2S@AIPH aqueous solution and stirred, and then the free HS-PEG5000-HA was further removed by an ultrafiltration centrifugal filter to obtain a nanodrug integrating an activatable probe and oxygen-dependent and oxygen-independent sonosensitizers.

4. The method for preparing the nanomedicine integrating the activatable probe and the oxygen-dependent and oxygen-independent sonosensitizer according to claim 3, characterized in that: In step (2), the reaction time after adding 4-mercaptophenylacetic acid and polyacrylic acid is 30-60 minutes; the reaction time after adding tetraethyl orthosilicate, 3-aminopropyltriethoxysilane and ammonia water is 4-9 hours; the heating temperature after adding polyvinyl pyrrolidone solution is 80-120° C. and maintained for 1-5 hours.

5. The method for preparing the nanomedicine integrating the activatable probe and the oxygen-dependent and oxygen-independent sonosensitizer according to claim 3, characterized in that: The specific preparation method of step (4) is as follows: ultrapure water, hexadecyltrimethylammonium bromide aqueous solution, AgNO3 aqueous solution, and ammonia water are added to a round-bottom flask in sequence, stirred, and then acetamide aqueous solution is quickly injected to form a brown complex liquid; magnetic stirring is performed in the dark, and the mixture is collected with an ultrafiltration centrifugal filter and washed to obtain Ag / Ag2S NPs.

6. The method for preparing the nanomedicine integrating the activatable probe and the oxygen-dependent and oxygen-independent sonosensitizer according to claim 5, characterized in that: The volume ratio of ultrapure water, hexadecyltrimethylammonium bromide aqueous solution, AgNO3 aqueous solution, and NH3·H2O solution is 50:6:2.705:5.41, the concentration of hexadecyltrimethylammonium bromide aqueous solution is 10mM; the concentration of AgNO3 aqueous solution is 10mM; the concentration of NH3·H2O solution is 4M; and the concentration of acetamide aqueous solution is 4mM.

7. The method for preparing the nanomedicine integrating the activatable probe and the oxygen-dependent and oxygen-independent sonosensitizer according to claim 3, characterized in that: Step (5) The specific steps are: add 1 ml of 5 mg / mL (HS-PEG 5000 5 mL of -COOH aqueous solution with a concentration of 0.5 mg mL -1 The Ag / Ag2S NPs aqueous solution was stirred; then, the free PEG was removed by ultrafiltration centrifugal filter to obtain PEG modified with PEG 5000 -COOH-Ag / Ag2S NPs; in 1mL of PEG with a concentration of 10mg / mL 5000 2 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2 mg N-hydroxysuccinimide were added to the Ag / Ag2S NPs solution, and the solution was stirred continuously for 10-50 min at pH = 6 to activate the carboxyl groups on the surface of Ag / Ag2S NPs. Then, the solution was adjusted to pH = 7.2 and dropped into 1 mL of 10 mg / mL Janus AuNR-mSiO2@AIPH solution, and the mixture was stirred continuously at 15-25 ° C for 1-5 hours. Finally, the Janus AuNR-mSiO2-Ag / Ag2S@AIPH sonosensitizer was obtained by three rounds of centrifugation purification.

8. Use of the nanomedicine integrating an activatable probe and an oxygen-dependent and oxygen-independent sonosensitizer as claimed in any one of claims 1 to 2 in fluorescence imaging, characterized in that: It remains inactive until triggered by H2O2, and turns on fluorescence after being activated by H2O2, achieving fluorescence imaging.

9. Use of the nanomedicine integrating an activatable probe and an oxygen-dependent and oxygen-independent sonosensitizer as claimed in any one of claims 1 to 2 in sonodynamic therapy, characterized in that: The nanomedicine can guide the start time of ultrasound irradiation through activatable fluorescence imaging.

10. The use of the nanomedicine integrating the activatable probe and the oxygen-dependent and oxygen-independent sonosensitizers in sonodynamic therapy according to claim 9, characterized in that: The nanomedicine lights up the NIR-II fluorescence signal under the action of H2O2 overexpressed in the lesion area, and the time point when the signal intensity reaches the peak is determined as the starting point of ultrasound radiation, thereby realizing image-guided sonodynamic therapy.

Images