A nano-carrier, a nano-diagnosis and treatment agent, and a preparation method and application thereof

Abstract

The application provides a kind of nanocarrier, nanodiagnosis and treatment agent and its preparation method and application.The preparation method of the nanodiagnosis and treatment agent includes the following steps: after MAX phase material is etched in situ, ultrasonic peeling, add potassium ferricyanide and ferric chloride mixed solution in situ reduction, synthesis prussian blue loaded MXene nanosheet, then carry out drug loading and encapsulation.The nanodiagnosis and treatment agent of the application targets brain glioma by intravenous injection, and shows higher photo-thermal conversion efficiency and photoacoustic signal than single component under laser irradiation, has drug release function of specific response to tumor and light response, and has peroxidase-like activity, can eliminate various ROS produced in the treatment process, so as to effectively inhibit inflammatory response and tumor recurrence.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanobiomedical materials, and relates to a nano-diagnostic and therapeutic agent capable of photoacoustic imaging and synergistic treatment of brain glioma, and in particular to a preparation method of Prussian blue in-situ reduction loaded MXene nanosheets and application thereof. BACKGROUND

[0002] Glioma is the most common primary malignant tumor of the central nervous system, with an incidence of about 3-6.4 / 100,000 people per year, accounting for 81.9% of all central nervous system malignancies. According to the tumor and molecular pathological characteristics, the World Health Organization (WHO) central nervous system classification guide has further divided diffuse glioma into grades II, III and IV, among which GBM is the highest grade of grade IV cancer, with the highest annual incidence of about 3.23 / 100,000 people, accounting for 49.1% of all primary malignant central nervous system tumors and 60.8% of gliomas. The current standard treatment regimen is to remove the tumor in the largest range while preserving the patient's neurological function, and to perform radiotherapy combined with oral temozolomide concurrent chemotherapy followed by temozolomide adjuvant chemotherapy within 3-5 weeks after surgery. The Stupp standard treatment regimen. Before the introduction of the Stupp regimen in 2005, the median survival of glioblastoma was about 12 months, and later due to the improvement of treatment in multiple aspects, including intraoperative imaging protection total resection and progress in radiotherapy technology, the median survival has increased to 16 months. However, there has been little new treatment method showing significant efficacy and breakthrough progress in reducing glioblastoma. Most clinical data show that the 5-year survival rate is less than 5%, and all GBM patients will eventually relapse, which is associated with a significant reduction in progression-free survival (PFS) associated with local recurrence. Human brain glioma is similar to most malignant tumors, with tumor microenvironment acidification and hypoxia due to high activity of glycolysis in tumor tissue and lack of sufficient blood supply. Human brain glioma cells have high expression of CD44 on the cell surface relative to normal cells, and the expression level of hyaluronidase in the cytoplasm is increased.

[0003] Nanomaterials have unique photothermal, photodynamic or sonodynamic properties, which endow them with new treatment and imaging functions. At the same time, drug delivery technology based on nanotechnology can significantly improve drug solubility, specifically respond to tumor microenvironment, and improve drug bioavailability, so the nano-diagnostic and therapeutic agent integrating disease diagnosis and treatment functions has shown great potential.

[0004] Photothermal therapy (PTT) is a method that uses materials with good photothermal conversion performance as therapeutic agents to kill cancer cells by the heat energy generated by photothermal agents (PTAs) under external light source irradiation. With the development of nanotechnology, PTAs nanomaterials with near-infrared absorption characteristics show greater potential in tumor treatment due to their deep penetration depth, excellent light stability and multifunction. However, there are still many aspects that need to be improved, such as insufficient photothermal conversion efficiency and inflammation caused by PTT process which can lead to tumor recurrence and migration, which puts higher requirements on clinical PTAs. MXenes is a transition metal carbide, nitride or carbonitride with the general formula M n+1 X n T x wherein M represents transition metal sites (Ti, V, Sc, Cr, Zr, Mo, Ta, Nb, etc.), X represents carbon and / or nitrogen, T x (wherein X is variable) represents the functional groups on the surface of the outer transition metal layer, such as -O, -OH, -F. So far, more than 70 different elements of MXenes have been prepared and applied in catalysis, energy storage and conversion due to their unique electronic, (electro)chemical and physical properties. It is worth noting that Ti3C2 has been widely used in biomedical fields such as biosensors, antibacterial activity and PTT due to its absorption of near-infrared light and special surface activity. However, for cancer treatment, Ti3C2 MXenes only exhibit single photothermal ability and relatively low efficiency in killing cancer cells, so it is necessary to endow Ti3C2 MXenes with additional properties to achieve effective cancer treatment. Prussian blue (PB) nanoparticles are antidotes approved by the US Food and Drug Administration (FDA) for the clinical treatment of thallium poisoning, which have attracted widespread attention due to their excellent biocompatibility and biosafety, and are also a kind of multifunctional nanoenzyme with various ROS scavenging enzyme-like activities. It is a kind of nanomaterial with various enzyme-like activities, which has a broad application prospect in the treatment of inflammation and tumors. In practical applications, the photothermal conversion efficiency of single PB at 808 nm is also relatively low (about 30%).

[0005] Photoacoustic imaging (PAI) is a hybrid imaging method that acoustically detects optical absorption contrast through the photoacoustic effect, which can complement and be compatible with optical imaging and ultrasonic imaging. PAI has the following advantages: (1) Because the degree of transparency of biological tissues to sound is several orders of magnitude higher than the degree of transparency to light, in terms of scattering mean free path, PAI provides much greater penetration capability than optical microscopes in scalable spatial resolution; (2) PAI can be used for functional, metabolic and histological imaging through endogenous contrast agents, i.e. inherent components in biological tissues, and can also be used for molecular, cellular and tissue imaging through exogenous contrast agents. Exogenous contrast agents optimize the optical and acoustic properties of biological tissues locally, improve contrast and resolution, and can be applied to non-invasive real-time imaging of tumor tissues, blood vessels, brain tissues and the like. Therefore, it is of great significance to develop photoacoustic imaging contrast agents with biocompatibility, high resolution and high penetration depth.

[0006] There are problems such as low photothermal efficiency, severe inflammatory reaction caused by photothermal treatment and difficulty in precise positioning imaging of intracranial glioma in existing brain glioma photothermal nano diagnosis and treatment agents. In order to further develop the wider application of MXene in the field of nanobiomedical materials, it is hoped to construct a nano preparation with photoacoustic imaging and integrated drug delivery diagnosis and treatment, which plays a positive role in promoting clinical detection and treatment of cancer. SUMMARY

[0007] In order to solve the above problems, the purpose of the present application is to provide a kind of nanocarrier, nanodiagnosis and treatment agent and its preparation method and application, the nanodiagnosis and treatment agent under 808nm Etc. Laser irradiation shows high photothermal conversion efficiency and photoacoustic signal better than single component, and has peroxidase-like activity, which can eliminate various ROS generated during treatment, thereby effectively inhibiting inflammatory reaction and tumor recurrence.

[0008] In order to achieve the above purpose, the present application provides a kind of nanocarrier, its composition includes MXene nanomaterial, prussian blue, wherein the prussian blue is synthesized in situ on the MXene nanomaterial.

[0009] According to the specific embodiment of the present application, preferably, the MXene nanomaterial includes one or more than two combinations of Ti3C2T x nanosheet, Nb2CT x nanosheet and Mo2CT x nanosheet and the like.

[0010] According to a specific embodiment of the present application, preferably, the preparation method of the nanocarrier comprises the following steps: taking MXene nanomaterial, adding a mixed solution of K3[Fe(CN)6] and FeCl3 dropwise, stirring, centrifugal washing, drying, and obtaining the nanocarrier; wherein the mass ratio of the sum of the mass of K3[Fe(CN)6] and FeCl3·6H2O to the mass of the MXene nanomaterial is (1-5):1, and the amount-of-substance ratio of K3[Fe(CN)6] to FeCl3·6H2O is 1:(0.8-1.5), more preferably 1:1.

[0011] According to a specific embodiment of the present application, preferably, the MXene nanomaterial can be in any suitable shape, for example, MXene nanosheet.

[0012] According to a specific embodiment of the present application, preferably, the MXene nanosheet is prepared by the following method: taking a MAX phase solid, etching in situ with an etching solution, and ultrasonic dispersion to obtain the MXene nanosheet.

[0013] According to a specific embodiment of the present application, preferably, in the above etching process, the etching solution is an HCl / LiF mixed solution.

[0014] According to a specific embodiment of the present application, preferably, in the above etching process, the concentration of HCl in the HCl / LiF mixed solution is 5-20 mol·L -1 .

[0015] According to a specific embodiment of the present application, preferably, in the above etching process, the in-situ etching is adding the MAX phase solid to the HCl / LiF mixed solution and etching at room temperature for 24-48 h.

[0016] According to a specific embodiment of the present application, preferably, in the above etching process, after ultrasonic dispersion, a MXene nanosheet dispersion liquid is obtained, and then the MXene nanosheet is collected by differential centrifugation.

[0017] According to a specific embodiment of the present application, preferably, in the above etching process, the MXene nanosheet dispersion liquid is a water dispersion liquid of MXene nanosheet, and the concentration is 1-10 mg / mL, more preferably 2 mg / mL.

[0018] According to a specific embodiment of the present application, preferably, in the above etching process, the differential centrifugation collection is centrifugation at 5000-8000 rpm for 20-60 min to precipitate, so as to make the particle size distribution of the nanosheet as concentrated as possible.

[0019] According to a specific embodiment of the present application, preferably, in the above etching process, the average diameter of the MXene nanosheet is 100-500 nm.

[0020] According to a specific embodiment of the present application, preferably, in the above preparation process, the K3[Fe(CN)6] and FeCl3·6H2O are mixed and dissolved in a pre-acidified KCl solution, and the mixed solution of K3[Fe(CN)6] and FeCl3 is obtained after ultrasonic dissolution.

[0021] According to a specific embodiment of the present application, preferably, in the above preparation process, the KCl concentration in the pre-acidified KCl solution is 0.1 M, and the pH is 1-3.

[0022] The present application also provides a nanodiagnosis and treatment agent prepared by loading the drug into the above nanocarrier.

[0023] According to a specific embodiment of the present application, preferably, the drug is a small molecule chemotherapeutic drug.

[0024] According to a specific embodiment of the present application, preferably, the small molecule chemotherapeutic drug comprises one or more than two combinations of doxorubicin hydrochloride, vincristine, temozolomide, lomustine, and procarbazine.

[0025] According to a specific embodiment of the present application, preferably, the mass ratio of the drug to the nanocarrier is (1-3):1.

[0026] According to a specific embodiment of the present application, preferably, the nanodiagnosis and treatment agent is further encapsulated by glycosaminoglycan.

[0027] According to a specific embodiment of the present application, preferably, the glycosaminoglycan comprises hyaluronic acid and / or chondroitin sulfate, and more preferably, hyaluronic acid; the encapsulation of hyaluronic acid improves the stability of the nanocarrier in the body fluid and prevents the aggregation for a long time, and on the other hand, hyaluronic acid and chondroitin sulfate can recognize the CD44 overexpressed on the surface of glioblastoma, thereby achieving the targeting effect; in addition, the hyaluronidase expressed in glioblastoma can decompose the hyaluronic acid encapsulated on the surface of the nanocarrier, thereby achieving the controllable slow release of the chemotherapeutic drug loaded in the nanocarrier in the tumor cells.

[0028] According to a specific embodiment of the present application, preferably, the relative molecular mass of the hyaluronic acid is 40-100 kDa.

[0029] According to a specific embodiment of the present application, preferably, when the encapsulation of hyaluronic acid is performed, the mass ratio of the drug-loaded nanodiagnosis and treatment agent to hyaluronic acid is (2-10):1.

[0030] The application also provides application of the nanocarrier or the nanodiagnostic and therapeutic agent as a photoacoustic imaging agent or in preparation of a photothermal-chemical synergistic therapeutic drug.

[0031] According to a specific embodiment of the application, preferably, the photothermal-chemical synergistic therapeutic drug is used for treating brain glioma.

[0032] According to a specific embodiment of the application, preferably, the nanodiagnostic and therapeutic agent is used for photoacoustic imaging and / or photothermal conversion under laser (wavelength, for example, 808 nm or 1064 nm) irradiation.

[0033] According to a specific embodiment of the application, the preparation method of the nanodiagnostic and therapeutic agent comprises the following steps:

[0034] (1) M n+1 X n T x is to selectively remove A elements (usually Al) from the layered structure of MAX phase ceramics. Take the MAX phase solid, etch in situ with the etching solution, ultrasonically disperse to obtain a dispersion solution of MXene nanosheets, and then collect by differential centrifugation to obtain MXene nanosheets;

[0035] (2) A certain amount of K3[Fe(CN)6] and FeCl3·6H2O is weighed and dissolved in a pre-acidified KCl solution, and after ultrasonic dissolution, it is slowly added to the MXene nanosheet solution obtained in step (1), stirred at room temperature, centrifuged and washed, and freeze-dried to obtain the nanocarrier PB@MXene;

[0036] (3) Mix the small molecule chemotherapeutic drug solution with the PB@MXene nanocarrier solution in a certain proportion, stir overnight at room temperature, and centrifuge to remove the unloaded small molecule chemotherapeutic drugs;

[0037] (4) Dissolve the PB@MXene nanocarrier loaded with small molecule chemotherapeutic drugs in a certain concentration of glycosaminoglycan solution (such as hyaluronic acid solution), stir overnight at room temperature, and centrifuge to collect the precipitate to obtain the nanodiagnostic and therapeutic agent.

[0038] The application has the following beneficial effects:

[0039] (1) The MXene nanosheet in situ reduction loaded Prussian blue has a simple preparation method, low cost, mild conditions, uniform component distribution, small particle size, and can be mass produced or industrialized;

[0040] (2) It has good dispersibility and stability in water, no obvious hemolysis and cytotoxicity, and good biological safety (biocompatibility);

[0041] (3) The MXene nanosheet and the integrity of the in-situ reduced Prussian blue nanoparticle structure are reserved, and the excellent performance of MXene and Prussian blue is combined;

[0042] (4) The photothermal conversion efficiency of the brain glioma nanodiagnostic and therapeutic agent prepared in the application is increased to 47.3%, which is improved compared with the performance of pure Prussian blue and MXene, and the brain glioma nanodiagnostic and therapeutic agent can be used as a photoacoustic contrast agent to realize photoacoustic imaging diagnosis of tumors and can also perform photothermal-chemical synergistic treatment on tumors; under 1W / cm 2 , 808nm or 1064nm laser irradiation, the brain glioma nanodiagnostic and therapeutic agent has a selective killing effect on brain glioma cells, and has a significant anti-tumor effect; under 808nm or 1064nm laser excitation, the brain glioma nanodiagnostic and therapeutic agent can perform photoacoustic imaging;

[0043] (5) The brain glioma nanodiagnostic and therapeutic agent prepared in the application utilizes the wide ROS scavenging activity of the Prussian blue nanoparticles to relieve oxidative stress generated in the photothermal treatment process, thereby avoiding inflammation and tumor recurrence; the Prussian blue nanoparticles have good mimic enzyme properties such as catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD), and can effectively scavenge various ROS, inhibit the recruitment of neutrophils and various pro-inflammatory cytokines in vivo, and have good application in various inflammatory diseases;

[0044] (6) The coating of hyaluronic acid and chondroitin sulfate improves the stability and targeting of the nanodiagnostic and therapeutic agent, and can also optimize the realization of controlled drug release. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 It is a preparation flow chart of the brain glioma nanodiagnostic and therapeutic agent;

[0046] Figure 2 It is a scanning electron microscope image of the nanocarrier prepared in Example 2;

[0047] Figure 3 It is an element distribution graph of the nanocarrier prepared in Example 2;

[0048] Figure 4 It is a Zeta potential graph;

[0049] Figure 5 It is a photothermal stability result graph of the nanodiagnostic and therapeutic agent;

[0050] Figure 6 It is a result graph of the brain glioma nanodiagnostic and therapeutic agent prepared in Example 2 in the treatment of U87-MG cells;

[0051] Figure 7 It is an in-vivo photoacoustic image of the nanodiagnostic and therapeutic agent;

[0052] Figure 8Figure for in vivo photothermal-chemical synergistic therapy result of nanodiagnostic and therapeutic agent;

[0053] Figure 9 Figure for in vivo anti-inflammatory investigation result of nanodiagnostic and therapeutic agent. DETAILED DESCRIPTION

[0054] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application are described in detail as follows, but cannot be understood as limiting the implementable scope of the present application. The experimental reagents can be obtained through commercial channels if no special description is made, and the experimental methods are conventional experimental methods if no special description is made.

[0055] Example 1: Ti3C2T x Preparation of nanosheet

[0056] The present embodiment provides a Ti3C2T x nanosheet, the preparation process of which is shown in Figure 1 , and specifically comprises the following steps:

[0057] (1) 1.5 g of LiF is dissolved in 20 mL of 12M HCl, and after sufficient dissolution, 1.0 g of Ti3AlC2 powder is slowly added, and etching is carried out at room temperature under 500 rpm stirring for 40 h;

[0058] (2) The acidic mixture is washed by centrifugation with deionized water, and the cycle is repeated multiple times until the pH value of the supernatant is 4-5;

[0059] (3) The dispersed clay-like solid suspension (aqueous solution) is exfoliated by ultrasonic exfoliation for 1 hour, and after centrifugation at 3500 rpm for 20 min, the supernatant is centrifuged at 5000 rpm for 30 min, and the precipitate is resuspended in water to obtain a water dispersion (colloidal dispersion) of Ti3C2T x nanosheet.

[0060] Example 2: Preparation of Prussian blue loaded Ti3C2T x (PB@Ti3C2T x )

[0061] The present embodiment provides a Prussian blue loaded Ti3C2T x (PB@Ti3C2T x ), the preparation process of which is shown in Figure 1 , and specifically comprises the following steps:

[0062] (1) A 0.1 mM KCl solution is prepared, and the pH value is adjusted to 2 with hydrochloric acid;

[0063] (2) Take 16.5 mg K3[Fe(CN)6] and 13.5 mg FeCl3·6H2O and dissolve them in 10 mL of the above KCl solution, and ultrasonic oscillation to make it fully dissolved to obtain a mixed solution;

[0064] (3) Slowly add 5 mL of the mixed solution prepared in step (2) to 5 mL of the Ti3C2T -1 nanosheet aqueous dispersion obtained in Example 1 with a concentration of 2 mg·mL x , and stir slowly, and react at room temperature for 40 min;

[0065] (4) Centrifugal wash the reaction solution with deionized water, cycle three times, and finally centrifugal at 5000 rpm for 30 min to obtain Prussian blue loaded Ti3C2T x (PB@Ti3C2T x ), and the Prussian blue is in-situ grown on the Ti3C2T x .

[0066] The above proves that the brain glioma nanodiagnosis and treatment agent PB@MXene nanocarrier is successfully synthesized by transmission electron microscopy (TEM), energy dispersive spectrometer (EDS), X-ray polychromatic diffraction (XRD), and X-ray photoelectron spectroscopy (XPS), and the transmission electron microscope image and element distribution of the nanocarrier are shown in Figure 2 and Figure 3 . The average diameter of the nanocarrier is 481 nm.

[0067] Example 3: Preparation of drug-loaded nanodiagnosis and treatment agent (PB@Ti3C2T x / DOX-HA)

[0068] This embodiment provides a drug-loaded nanodiagnosis and treatment agent (PB@Ti3C2T x / DOX-HA), and the preparation process thereof is shown in Figure 1 , and specifically comprises the following steps:

[0069] (1) Mix 2.0 mL of PB@Ti3C2T x aqueous solution (2.0 mg·mL -1 ) with 4.0 mL of DOX aqueous solution (2.0 mg·mL -1 ), and stir in the dark overnight;

[0070] (2) Centrifugal the mixture in step (1) at 5000 rpm for 30 min, and collect the supernatant;

[0071] (3) Resuspend the precipitate with 4.0 mL of 40-100 kDa hyaluronic acid aqueous solution (4.0 mg·mL -1 ), and centrifugal collect the precipitate after stirring for 6 hours to obtain PB@Ti3C2Tx / DOX-HA;

[0072] (4) The obtained PB@Ti3C2T x / DOX-HA was redispersed in PBS and stored at 4°C in the dark for later use.

[0073] The above proves the success of drug loading and encapsulation of the brain glioma nano-diagnostic and therapeutic agent by the change of Zeta potential, as shown in Figure 4 This embodiment uses the anti-tumor chemotherapy drug doxorubicin hydrochloride (DOX) as a model drug to construct a PB@MXene / DOX-HA nano-diagnostic and therapeutic agent (PB@MXene / DOX-HA for short). The drug loading of PB@MXene / DOX-HA was 0.72±0.09 μg DOX / μg MXene, and the encapsulation efficiency was 36.11±4.59% as detected by UV-visible absorption spectrum, calculated by the mass of MXene.

[0074] Test Example 1: Evaluation of the photothermal properties of the drug-loaded nano-diagnostic and therapeutic agent

[0075] The test process is as follows:

[0076] (1) 100 ppm (0.1 mg / mL) of the drug-loaded nano-diagnostic and therapeutic agent PB@Ti3C2T -2 / DOX-HA obtained in Example 3 was irradiated with 1 W·cm x , 808 nm laser at room temperature for 480 s, and the temperature change and thermal image every 15 s were recorded by an infrared thermal imager. PBS solution was irradiated under the same conditions as a blank control;

[0077] (2) When the temperature basically remained stable, the laser irradiation was stopped, and the temperature change every 15 s during the cooling process was recorded by the infrared thermal imager until the temperature returned to room temperature;

[0078] (3) As a comparison, 100 ppm of Ti3C2T -2 nanosheets and pure Prussian blue nanoparticles obtained in Example 1 were irradiated with 1 W·cm x , 808 nm laser at room temperature, and the temperature change curves of 0-480 s and 480-960 s were recorded by an infrared thermal imager at the same concentration;

[0079] (4) The photothermal conversion efficiency was calculated according to the formula:

[0080]

[0081] where T max is the equilibrium temperature of the sample solution, T surr corresponds to the experimental environment temperature; I represents the power density of the 808 nm laser (1 W·cm-2 ), A λ is the absorbance of the sample solution at 808 nm; Q dis represents the heat loss caused by the light absorption of the container itself, according to Q dis = (5.4 x 10 -4 ) x I. The value of hS can be calculated by another formula:

[0082]

[0083] wherein m is the mass of the sample solution, C water is the specific heat capacity of water 4.2 J·g -1 ·K -1 , and τ s is the time constant of the system.

[0084] Based on the above values, the photothermal conversion efficiency of the PB@Ti3C2T x / DOX-HA nanodiagnostic and theranostic agent was calculated to be 47.3%, the photothermal conversion efficiency of pure Ti3C2T x nanosheets was 25.2%, and the photothermal conversion efficiency of pure PB particles was 32.5%; in addition, steps (1) and (2) were repeatedly alternately performed for five times to verify the photothermal stability of the nanodiagnostic and theranostic agent, and the results are shown in Figure 5 , which proved that the nanodiagnostic and theranostic agent had good photothermal stability.

[0085] Test Example 2: Evaluation of the biocompatibility of the nanocarrier

[0086] The cell verification experiment was performed according to the national standard GB / T 16886.5 (Biological evaluation of medical devices: In vitro cytotoxicity test) and the international standard for biological evaluation of medical devices ISO 10993-5, with human glioma cells U87-MG as the research object, to evaluate the in vitro biological safety of the PB@Ti3C2T x obtained in Example 2.

[0087] PB@Ti3C2T xUltraviolet sterilization for 4h, and then 1 mL of PBS solution was added to the U87-MG special culture medium (containing 10% fetal bovine serum) to prepare experimental group culture medium with concentrations of 10, 50, 100, 200, and 500 ppm, respectively. The U87-MG cells in vigorous growth were selected and inoculated in a 96-well plate at about 10,000 cells per well. The experimental group was added with 100 μL of the experimental group culture medium with different concentrations per well, the control group was added with 100 μL of complete culture medium per well, and the blank group was not added with cells but only with complete culture medium. After being placed in a 37°C, 5% CO2 incubator for 24 h, the culture medium was aspirated and washed with PBS for 3 times. Then, 100 μL of complete culture medium was added to each well, followed by the addition of 10 μL of CCK8 reagent. After being cultured for 4 h, the absorbance of the solution in each well at 450 nm was measured by an enzyme marker instrument. According to the calculation formula:

[0088]

[0089] The survival rate of the cells was calculated. After calculation, PB@Ti3C2T x nanocarriers had good biocompatibility. When the concentration reached 200 ppm, the survival rate of the cells was still greater than 95%, indicating that the PB@Ti3C2T x nanocarriers had good biocompatibility.

[0090] Test Example 3: Evaluation of the in-vitro treatment effect of the drug-loaded nanodiagnostic and therapeutic agent

[0091] The synergistic effect of PTT and chemotherapy of the drug-loaded nanodiagnostic and therapeutic agent prepared in Example 3 in vitro was evaluated.

[0092] The U87-MG cells in vigorous growth were selected and inoculated in a 96-well plate at about 10,000 cells per well. Six parallel groups were set. The control group and the laser irradiation group were added with 100 μL of complete culture medium per well. The chemotherapy group and the laser irradiation plus chemotherapy group were added with 100 μL of complete culture medium containing 50 μg / mL of DOX per well. The photothermal therapy group was added with 100 μL of PB@Ti3C2T x with a concentration of 100 ppm per well. The synergistic therapy group was added with 100 μL of PB@Ti3C2T x / DOX-HA with a concentration of 100 ppm per well. After being incubated in a 37°C, 5% CO2 incubator for 6 h, the culture medium was aspirated and washed with PBS for 3 times. Then, 100 μL of complete culture medium was added to each well, followed by the irradiation of 1 W·cm -2 808 nm laser on the laser irradiation group, the laser irradiation plus chemotherapy group, the photothermal therapy group, and the synergistic therapy group for 10 min per well. After being placed in a 37°C, 5% CO2 incubator for 6 h, 10 μL of CCK8 reagent was added to each well. After being cultured for 4 h, the absorbance of the solution in each well at 450 nm was measured by an enzyme marker instrument. The survival rate of the cells was calculated according to the calculation formula in Test Example 2. The experimental results are as follows:Figure 6 As shown, the results show that PB@Ti3C2T x / DOX-HA drug-loaded nanodiagnostic and therapeutic agent has a significant chemical-photothermal synergistic treatment effect on U87-MG cells.

[0093] Test Example 4: In vitro nanoscale enzyme activity determination of drug-loaded nanodiagnostic and therapeutic agent

[0094] The three kinds of nanoscale enzyme activities of the nanodiagnostic and therapeutic agent were detected by using the ammonium molybdate colorimetric hydrogen peroxidase activity detection kit, the guaiacol colorimetric peroxidase activity detection kit, and the nitrogen blue tetrazolium colorimetric superoxide dismutase activity detection kit, respectively. All kits were purchased from Boxbio Technology Co., Ltd. in Beijing, and the experimental operations were strictly performed according to the kit instructions. The results are shown in Table 1.

[0095] Table 1

[0096] Enzyme class CAT POD SOD Enzyme activity (U / mL) 24.87±0.82 43.29±6.85 30.80±6068

[0097] Test Example 5: Determination of POD-like nanoscale enzyme reaction kinetics of drug-loaded nanodiagnostic and therapeutic agent

[0098] The test process is as follows: in the presence of H2O2, 3,3',5,5'-tetramethylbenzidine (TMB) is used as a substrate in a PBS buffer solution at pH 7.4 at 25°C, and the PB@Ti3C2T x / DOX-HA drug-loaded nanodiagnostic and therapeutic agent obtained in Example 3 is subjected to enzyme reaction kinetics research. The absorbance change of the reaction system at a wavelength of 625 nm over time is determined using a UV-visible spectrophotometer. In order to obtain the steady-state kinetic parameters, the reaction kinetic curves under the concentrations of 5, 15, 30, 60 and 90 mM of hydrogen peroxide are determined. According to the Lambert Beer law, the concentration change rate of oxTMB is calculated:

[0099] A = εlc (Lambert Beer law);

[0100] The hydrogen peroxide concentration and initial reaction rate change graph is drawn, and the fitting curve follows the Michaelis equation:

[0101]

[0102] wherein V0 is the initial reaction rate, V max is the maximum reaction rate, [S] is the substrate concentration, and K m is the Michaelis constant; according to the fitted Michaelis-Menten saturation curve, the Michaelis constant is calculated. The equation can be transformed as follows:

[0103]

[0104] Based on the above values, the following parameters were calculated for PB@Ti3C2T under room temperature and pH 7.4 conditions. x / DOX-HA nanotherapeutic agents bind to substrate H2O2 with K m The value is 19.7 mM, V max =9.4×10 -8 M·s -1 .

[0105] Test Example 6: Drug-loaded nanotherapeutic agents for photoacoustic imaging diagnosis of tumors

[0106] Animal experiments were conducted in accordance with relevant national and Beijing laws, regulations, and technical specifications for laboratory animals. The in vivo photoacoustic imaging of the drug-loaded nanotherapeutic agent obtained in Example 3 was evaluated.

[0107] Male Balb / c nude mice were subcutaneously injected with 0.1 ml of solution at a density of 10 in the right groin. 7 Human glioma U87-MG cells, when the tumor volume reaches approximately 100 mm. 3 It can then be used in in vivo experiments. In the case of in situ injection of the drug-loaded nanotherapeutic agent from Example 3 into tumors, photoacoustic images of the tumor at 808 nm were measured at 0, 6, 12, and 24 hours after injection. Figure 7 As shown ( Figure 7 In the middle: Window Width (WW) and Window Level (WL).

[0108] Test Example 7: Drug-loaded nanotherapeutic agents for in vivo photothermal-chemosynergistic therapy of tumors

[0109] This test case evaluates the tumor-killing effect of the drug-loaded nanotherapeutic agent obtained in Example 3 on tumor killing in animals.

[0110] The drug-loaded nanotherapeutic agent obtained in Example 3 was used for the treatment of gliomas. The procedure was the same as in Test Example 6. Twenty-four hours after tail vein injection of the drug-loaded nanotherapeutic agent, the tumor site was irradiated with an 808nm laser for 10 minutes. The above-mentioned tail vein injection of the drug-loaded nanotherapeutic agent from Example 3 combined with radiation served as the photothermal-chemosynergistic therapy group. Simultaneously, an untreated tumor served as the blank control group. The tumor site was injected with Ti3C2T obtained in Example 1. x The nanosheets subjected to irradiation were used as the thermotherapy group, while the drug-loaded nanotherapeutic agent from Example 3, injected into the tumor site without irradiation, was used as the chemotherapy group. Experimental results show that the photothermal-chemical synergistic therapy can more effectively kill tumor cells, achieving synergistic treatment of tumors with chemotherapy and thermotherapy. Figure 8 As shown.

[0111] Test Example 8: Evaluation of the in vivo anti-inflammatory effect of drug-loaded nanotherapeutic agents

[0112] After the mice in Test Example 7 were treated with the nanodiagnostic and therapeutic agent and laser, the experimental animals were euthanized, the tumor tissues were removed, the samples were treated according to a ratio of 1:10 of tissue mass (g) to extraction volume (mL), homogenized in an ice bath, centrifuged at 8000g at 4°C for 10 min, and the supernatant was taken and placed on ice for testing. The contents of cyclooxygenase-2 (COX-2) and myeloperoxidase (MOP) in the samples were analyzed by ELISA technology. The results are shown in Table 1. Figure 9 x The levels of COX-2 and MOP in the tumor tissues of the hyperthermia group in which the MXene nanosheets were applied and irradiation was applied were significantly higher than those in the other groups. In contrast, the levels of COX-2 and MOP in the tumor tissues of the PB@Ti3C2T x / DOX-HA synergistic treatment group were lower than those in the other groups, indicating that the loading of PB can significantly alleviate the inflammation induced by PTT.

[0113] Test Example 9: Drug release evaluation of drug-loaded nanodiagnostic and therapeutic agent

[0114] This test example investigates the cumulative release of the chemotherapeutic drug in the nanodiagnostic and therapeutic agent under different pH, near-infrared light irradiation or not, and presence or absence of hyaluronidase. At 37°C, in a buffer solution at pH 7.4 or pH 6.5, with or without 1.0 W cm -2 , 808 nm laser irradiation (the temperature of the irradiated solution is heated to about 50°C), the drug-loaded nanodiagnostic and therapeutic agent solution obtained in Example 3 was centrifuged at different time intervals (0, 0.5, 1, 2, 3, 6, 9, 12, 24 and 48 h), 0.15 mL of supernatant was collected, and the release amount of doxorubicin was determined by UV-visible method. For hyaluronidase-reactive drug release, PB@Ti3C2T x / DOX-HA was incubated with a pH 7.4 buffer solution containing 0.5 mg / mL hyaluronidase, and the release amount of doxorubicin was determined according to the above operation.

[0115] The results show that the release of DOX in PB@MXene / DOX-HA is pH and temperature sensitive, and both the decrease in pH and near-infrared light irradiation can increase the release of DOX: the cumulative release amount of DOX in the pH 6.5 near-infrared light irradiation group (20.61%) is nearly 3 times that in the pH 7.4 non-irradiation group (7.48%), indicating that near-infrared light irradiation can significantly increase the release of DOX in the nanodiagnostic and therapeutic agent, thereby increasing the sensitivity of chemotherapy. Hyaluronidase can significantly increase the cumulative release amount of DOX in PB@MXene / DOX-HA under the same conditions, and the cumulative release amount of DOX in the hyaluronidase-containing group under the condition of pH 7.4 near-infrared light irradiation is as high as 60.65%.

[0116] ​The above merely describes preferred embodiments of the present application and is not used to limit the present application, and any modification, equivalent replacement, and improvement within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A nanotherapeutic agent for treating glioma, which is prepared by loading a drug onto a nanocarrier; The nanocarrier composition includes MXene nanosheets and Prussian blue; wherein... The Prussian blue was synthesized in situ on the MXene nanosheets, which were Ti3C2T. x Nanosheets; The preparation method of the nanocarrier includes the following steps: Take MXene nanosheets, add a mixed solution of K3[Fe(CN)6] and FeCl3, stir, centrifuge, wash, and dry to obtain the nanocarrier; wherein, the ratio of the sum of the masses of K3[Fe(CN)6] and FeCl3·6H2O to the mass of the MXene nanosheets is (1-5):1, and the molar ratio of K3[Fe(CN)6] and FeCl3·6H2O is 1:(0.8-1.5); The drug in question is doxorubicin hydrochloride; The nano-therapeutic agent is further encapsulated in hyaluronic acid; The MXene nanosheets are prepared by taking MAX phase solid, etching it in situ with an etching solution, and then ultrasonically dispersing it to obtain the MXene nanosheets. The etching solution is a mixture of HCl and LiF.

2. The nanotherapeutic agent according to claim 1, wherein, The mass ratio of the drug to the nanocarrier is (1-3):

1.

3. The nanotherapeutic agent according to claim 1, wherein, The relative molecular mass of the hyaluronic acid is 40-100 kDa.

4. The nanotherapeutic agent according to claim 1, wherein, When encapsulating hyaluronic acid, the mass ratio of the drug-loaded nano-therapeutic agent to hyaluronic acid is (2-10):

1.

5. The nanotherapeutic agent according to claim 1, wherein, The molar ratio of K3[Fe(CN)6] to FeCl3·6H2O is 1:

1.

6. The nanotherapeutic agent according to claim 1, wherein, In the HCl / LiF mixed solution, the HCl concentration is 5-20 mol·L⁻¹. -1 .

7. The nanotherapeutic agent according to claim 1, wherein, The in-situ etching involves adding the MAX phase solid to an HCl / LiF mixed solution and etching at room temperature for 24-48 h.

8. The nanotherapeutic agent according to claim 1, wherein, After ultrasonic dispersion, an MXene nanosheet dispersion was obtained, which was then collected by differential centrifugation to obtain the MXene nanosheets.

9. The nanotherapeutic agent according to claim 8, wherein, The MXene nanosheet dispersion is an aqueous dispersion of MXene nanosheets with a concentration of 1-10 mg / mL.

10. The nanotherapeutic agent according to claim 9, wherein, The concentration of the MXene nanosheet dispersion was 2 mg / mL.

11. The nanotherapeutic agent according to claim 8, wherein, The differential centrifugation was used to collect the precipitate by centrifugation at 5000-8000 rpm for 20-60 min.

12. The nanotherapeutic agent according to claim 1, wherein, The average diameter of the MXene nanosheets is 100 nm-500 nm.

13. The nanotherapeutic agent according to claim 1, wherein, K3[Fe(CN)6] and FeCl3·6H2O were mixed and dissolved in a pre-acidified KCl solution, and the K3[Fe(CN)6] and FeCl3 mixed solution were obtained after ultrasonic dissolution.

14. The use of the nanotherapeutic agent according to any one of claims 1-13 in the preparation of photoacoustic imaging agents or photothermal-chemical synergistic therapeutic drugs; The photothermal-chemical synergistic therapeutic drug is used to treat glioma.

15. The application according to claim 14, wherein, The nanotherapeutic agents undergo photoacoustic imaging and / or photothermal conversion under laser irradiation.

16. The application according to claim 15, wherein, The laser wavelength is 808 nm or 1064 nm.

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