Active oxygen and active nitrogen cascade anti-tumor nano-drug as well as preparation method and application thereof

Abstract

The invention provides an active oxygen and active nitrogen cascade anti-tumor nano-drug aiming at tumor treatment obstacles of insufficient hydrogen peroxide and ultra-short service life of nitric oxide in tumors, and the combined effect of plant aldehyde and natural amino acid is taken as the core content of anti-tumor treatment. Nanoparticles are used as a carrier, the carrier is coated with a biocompatible material, plant aldehyde is loaded or grafted, and natural amino acid is packaged. In a tumor acidic environment, the plant aldehyde releases hydroxyl radicals to realize active oxygen therapy. When hydroxyl radicals are generated, hydrogen peroxide can be slowly released by the plant aldehyde, then natural amino acid is triggered to be converted into nitric oxide, and active nitrogen therapy is achieved. The controlled release of the plant aldehyde enables the release of the nitric oxide to be controllable, and the realization of space-time release of the active oxygen and the nitric oxide is realized, so that the complementarity of the active oxygen and the nitric oxide in pharmacokinetics is fully utilized, and a strong anti-tumor effect is generated. The preparation method is easy to operate, the raw materials are easy to obtain, and the prepared nano-particles loaded with the plant aldehyde and the natural amino acid have good particle dispersity, chemical stability and biocompatibility and are an ideal chemical-gas therapy-ferroptosis cascade cancer treatment reagent.

Description

Technical Field

[0001] The present invention relates to an active oxygen and active nitrogen cascade anti-tumor nanomedicine and a preparation method and application thereof, specifically a nanomedicine of plant aldehydes and natural amino acids released in response to acid in a tumor microenvironment guided by single red upconversion fluorescence imaging and its application as a chemo-gas therapy-ferroptosis cascade therapeutic reagent and an upconversion fluorescence imaging agent in tumor diagnosis and treatment, belonging to the field of biomedical technology. Background Art

[0002] Nitric oxide (NO) gas combines with intracellular superoxide anions to produce ·N or ·O, which are toxic to cancer, and plays an important role in tumor therapy. With the development of nanomedicine, strategies to embed NO donors into multifunctional nanocarriers by physical encapsulation or chemical coupling have been widely reported, but the blood circulation of NO donors is difficult to control and the half-life is less than 5 s, so how to deliver them has become a very challenging problem.

[0003] The effective combination strategy of NO generating agents and reactive oxygen species (ROS) releasers can fully utilize the pharmacokinetic complementarity between ROS and NO. ROS has a short diffusion distance and a high redox potential, which can destroy the dense extracellular matrix and promote the free diffusion of short-lived, highly toxic NO from the intercellular space into the cell, thereby killing cancer cells. However, the level of ROS in the tumor microenvironment (TME) is often not high enough and is unevenly distributed. Therefore, the targeted therapeutic strategy of ROS self-supply can solve the above problems by selectively amplifying the level of ROS in tumors.

[0004] The integration of diagnostic and therapeutic methods into a nano-platform is a new and highly specific diagnostic and therapeutic concept developed in recent years. So far, among the light-induced tumor imaging technologies, upconversion fluorescence imaging has excellent imaging effects, so it has attracted widespread attention from scientific researchers. In particular, single red upconversion fluorescence imaging has broad application prospects in reducing background noise and achieving deep tissue penetration due to its low scattering or absorption of tissues. Therefore, there is an urgent need to develop a single red upconversion fluorescence imaging agent. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide an active oxygen and active nitrogen cascade anti-tumor nanomedicine and its preparation method and application. The drug is a water-dispersible nanodiagnostic probe loaded with a single red up-conversion fluorescent imaging agent, a plant aldehyde and a natural amino acid, which can be used as an up-conversion fluorescent contrast agent in tumor diagnosis, and a chemo-gas therapy-ferroptosis cascade therapeutic reagent in tumor treatment.

[0006] The present invention provides an active oxygen and active nitrogen cascade anti-tumor nanomedicine, including single red upconversion luminescent mesoporous nanoparticles, the mesoporous nanoparticles are used as carriers, plant aldehydes are grafted and natural amino acids are loaded, and in order to prevent drug leakage and increase tumor targeting, biocompatible materials are wrapped outside the carrier. The present invention takes the synergistic effect of plant aldehydes and natural amino acids as the core content of anti-tumor treatment. It realizes the sequential activation response mechanism of active oxygen and active nitrogen under the condition of activation of overexpressed lactic acid in the tumor microenvironment. After the plant aldehydes are activated by overexpressed lactic acid in tumor cells, highly toxic hydroxyl free radicals are generated to achieve active oxygen therapy. At the same time, the plant aldehydes will slowly release hydrogen peroxide, thereby inducing the conversion of natural amino acids into nitric oxide, achieving active nitrogen therapy, and the two treatment methods fully utilize the pharmacokinetic complementarity of active oxygen and active nitrogen to produce a powerful anti-tumor effect.

[0007] The present invention uses mesoporous nanoparticles as carriers, single red upconversion fluorescent materials as imaging agents, and simultaneously loads plant aldehydes and natural amino acids, and is coated by biocompatible materials to synthesize acid-sensitive nanomedicines. The present invention introduces cinnamaldehyde, which is a trans-structured acrolein derivative that exists in large quantities in plants such as cinnamon. When exposed to the lactic acid conditions of the tumor environment, a large number of highly toxic hydroxyl radicals (OH) are rapidly generated, effectively inducing tumor cell apoptosis and performing chemotherapy. The hydrogen peroxide (H2O2) released at the same time triggers the conversion of L-arginine to NO, achieving reactive nitrogen (RNS) treatment. In addition, glutathione (GSH) in the tumor microenvironment is depleted by ROS and RNS, and GSH is inactivated by peroxidase 4 (GPX4) protein to perform ferroptosis treatment. This sequential activation response mechanism of chemical-gas therapy-ferroptosis makes full use of the pharmacokinetic complementarity of ROS and RNS to produce a powerful anti-tumor effect. In short, the cinnamaldehyde and L-arginine of the present invention have outstanding chemical-gas therapy-ferroptosis cascade treatment effects in tumor treatment.

[0008] The technical solution further optimized as the present invention is as follows: Furthermore, the porous nanocarrier particles can be porous metal organic framework nanoparticles, polymer nanoparticles, liposome nanoparticles, etc.; the fluorescent material can use quantum dots, organic small molecules and conjugated polymers with fluorescent properties, etc. The present invention uses single red luminescent upconversion nanoparticles @ mesoporous silica for further explanation; the plant aldehyde is cinnamaldehyde; the natural amino acid is L-arginine; the biocompatible material is human serum albumin.

[0009] The present invention also provides a method for preparing an active oxygen and active nitrogen cascade anti-tumor nanomedicine, comprising the following steps: Step 1, synthesizing single red upconversion luminescent nanoparticles with suitable mesopores as carriers; Step 2, preparing nanoparticles coated with biocompatible materials: dispersing the mesoporous nanoparticles obtained in step 1 in deionized water, adding biocompatible materials, and dripping acetone under vigorous stirring to obtain nanoparticles coated with biocompatible materials; Step 3, preparing nanoparticles loaded or grafted with plant aldehydes: dispersing the nanoparticles obtained in step 2 in acetone, adding plant aldehydes, and stirring to obtain nanoparticles grafted with plant aldehydes; Step 4, preparing nanoparticles encapsulating natural amino acids: selecting suitable natural amino acids, encapsulating and diffusing them into the mesopores of the nanoparticles grafted with plant aldehydes obtained in step 3, and obtaining nanoparticles encapsulating natural amino acids.

[0010] In step 1 of the present invention, in order to prepare single red light upconversion nanoparticles, NaTmF4 can be selected as the matrix and doped with Er 3+ ions and adjust the ratio of F source and Na source, and encapsulate NaLuF4 inert shell to enhance the luminescence intensity. In order to prepare small pore size mSiO2, short chain surfactants can be used as pore formers. The molar ratio of fluorescent material to mesoporous material can be regulated according to the size of the final nanoparticles required. There are many biocompatible materials available in step 2, such as bovine serum albumin, which has been reported, washed with deionized water and centrifuged at a speed of 9000 to 12000 rpm.

[0011] In the step 2, 10 to 50 mg of the nanoparticles obtained in step 1 are ultrasonically dispersed in 1 to 5 mL of deionized water, and then 2 to 10 mL (5 mg / mL) of human serum albumin is added, and 5 to 30 mL of acetone is slowly pushed in with a syringe, and the mixture is vigorously stirred for 10 to 24 h. After high-speed centrifugation, the product is collected and washed with deionized water to obtain nanoparticles loaded with human serum albumin.

[0012] In step 3, the nanoparticles obtained in step 2 are ultrasonically dispersed in 1-5 mL of acetone, and then 5-15 mL of the solution (V) is slowly added under stirring at room temperature. 肉桂醛 :V 丙酮 = 1:9), and stirred in the dark at room temperature for 12 to 24 h to obtain nanoparticles loaded with cinnamaldehyde.

[0013] In the step 4, 10 to 50 mg of the nanoparticles obtained in step 3 are ultrasonically dispersed in 2 to 10 mL of deionized water, and then 5 to 25 mL (5 mg / mL) of an L-arginine aqueous solution is slowly dropped into the solution under stirring at room temperature. After stirring at room temperature in the dark for 10 to 24 h, nanoparticles encapsulating L-arginine are obtained.

[0014] In the step 1, the fluorescent material is selected as a single red up-conversion luminescent material, the small-pore mesoporous material is selected as mesoporous silica, and a coating method is selected. In the step 2, the biocompatible material is selected as a coating method.

[0015] The present invention develops a tumor treatment method guided by single red upconversion fluorescence imaging, which overcomes the limitations of high background noise and poor tissue penetration of traditional upconversion fluorescence imaging and improves biomedical diagnostic capabilities.

[0016] In the step 3, the optimal mass ratio of the nanoparticles to the cinnamaldehyde is 1:20; and the mixture is dialyzed and purified at room temperature.

[0017] In the step 4, the optimal mass ratio of the nanoparticles to L-arginine is 1:2; after the reaction is completed, the material is washed with deionized water and collected by centrifugation, and the centrifuge speed is 9000-12000 rpm.

[0018] The nano drug provided by the present invention is used in chemical-gas therapy-ferroptosis cascade anti-tumor treatment.

[0019] The present invention targets the treatment obstacles of insufficient hydrogen peroxide and ultra-short half-life RNS in tumors, and loads cinnamaldehyde and L-arginine in nanocarriers. In the tumor microenvironment, cinnamaldehyde reacts with lactic acid overexpressed in tumor cells to produce a large amount of highly toxic OH, thereby achieving chemotherapy; while producing OH, cinnamaldehyde also releases a large amount of hydrogen peroxide, thereby quickly activating the conversion of L-arginine to nitric oxide, thereby achieving reactive nitrogen therapy. In addition, GSH in the tumor microenvironment is depleted by ROS and RNS, GPX4 protein is inactivated, and ferroptosis treatment is performed. This cascade-action chemical-gas therapy-ferroptosis therapy, the controlled release of cinnamaldehyde makes the release of RNS controllable, and the spatiotemporal release of ROS and RNS is achieved, thereby making full use of the pharmacokinetic complementarity of reactive oxygen and reactive nitrogen. The controlled release of L-arginine by the mesoporous silica carrier and the consumption of H2O2 can maintain the trace amount of L-arginine in the tumor, while providing L-arginine to immune cells, limiting the availability of L-arginine to tumor cells in the microenvironment, and producing a strong anti-tumor effect. In summary, the present invention realizes the sequential activation response mechanism of chemo-gas therapy-ferroptosis under the conditions of overexpressed lactic acid activation in the tumor microenvironment, and provides a chemo-gas therapy-ferroptosis anti-tumor cascade treatment method by combining two natural compounds, cinnamaldehyde and L-arginine, with an effective combination strategy of reactive oxygen and reactive nitrogen, providing a new idea for the development of tumor drugs.

[0020] Furthermore, the nanomedicine releases cinnamaldehyde in response to the acid in the tumor microenvironment and controls the release of L-arginine through mesoporous silica to achieve combined chemo-gas therapy-ferroptosis anti-tumor treatment, as well as integrated diagnosis and treatment guided by monochromatic red light upconversion fluorescence imaging.

[0021] Compared with the prior art, the present invention adopts the above technical scheme and has the following technical effects: upconversion nanoparticles @ mesoporous silica nanoparticles with loading effect are selected as carriers, and cinnamaldehyde and L-arginine are loaded in the carriers. The raw materials of the present invention are easily available from natural products, and the preparation process is easy to operate. The prepared nanoparticles loaded with cinnamaldehyde and L-arginine have good particle dispersibility, chemical stability and biocompatibility, and are an ideal cancer treatment agent that effectively combines active oxygen and active nitrogen. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the anti-cancer mechanism of cinnamaldehyde and L-arginine in Example 3 of the present invention.

[0023] Figure 2 This is a high-resolution transmission electron microscope image of NaTmF4:4%Er@NaLuF4 (U) in Example 1 of the present invention.

[0024] Figure 3 The product (U) in Example 1 of the present invention is at 980 nm, 1 W / cm 2 Upconversion fluorescence image under near-infrared light irradiation; the inset shows the red fluorescence emission characteristics characterized in a cuvette.

[0025] Figure 4 The nitrogen adsorption / desorption curve (a) and pore size distribution (b) of the product (US) in Example 2 of the present invention are shown.

[0026] Figure 5 The UV-visible spectra of human serum albumin (HSA) in Example 3 of the present invention before and after loading in ethanol.

[0027] Figure 6 The UV-visible spectra of cinnamaldehyde (CA) in Example 3 of the present invention before and after grafting in acetone.

[0028] Figure 7 The following are Fourier transform infrared spectra of the intermediates and products of Examples 1 to 3 of the present invention.

[0029] Figure 8 This is the UV-visible spectrum of L-Arg before and after L-Arg encapsulation in Example 3 of the present invention.

[0030] Fig. 9 The powder diffraction patterns of the products in Examples 2 and 3 of the present invention.

[0031] Fig.10 is the Zeta potential of the intermediate and product of Example 3 of the present invention.

[0032] Fig.11is the hydrated particle size of the products in Examples 2 and 3 of the present invention.

[0033] Fig.12 This is a transmission electron micrograph of the product (USAHC) in Example 3 of the present invention.

[0034] Fig.13 This is a transmission electron micrograph of the product (USAHC) in Example 3 of the present invention after being immersed in a pH 5.5 buffer solution for 24 hours.

[0035] Fig.14 This is a diagram showing the release of cinnamaldehyde in Example 3 of the present invention under different pH conditions; Fig.15 This is a diagram showing the release of L-arginine in Example 3 of the present invention under different pH conditions.

[0036] Fig.16 This is a diagram showing the generation of hydroxyl radicals by the product in Example 3 of the present invention under different pH conditions in vitro; Fig.17 a is a fluorescence image of ROS release in SACC cells at different pH values ​​of the product in Example 3 of the present invention.

[0037] Fig.17 b is a fluorescence image of RNS release of SACC cells acted upon by the product in Example 3 of the present invention.

[0038] Fig.18 a is a graph showing the change in the release of reactive oxygen species (ROS) in tumor cells by the product in Example 3 of the present invention over time.

[0039] Fig.18 b is a graph showing the change in the release of reactive nitrogen (RNS) in tumor cells by the product in Example 3 of the present invention over time.

[0040] Fig.19 a is the CCK8 method for evaluating the apoptosis of SACC cells induced by the product in Example 3 of the present invention.

[0041] Fig.19 b is the use of Hoechst 33342 probe to characterize the apoptosis of SACC cells induced by the product in Example 3 of the present invention.

[0042] Fig.19 c is the use of the JC-1 kit to characterize the effect of the product in Example 3 of the present invention on the mitochondrial membrane potential in SACC cells.

[0043] Fig. 20 a is a diagram showing the decrease in glutathione (GSH) levels caused by the product in Example 3 of the present invention in tumor cells.

[0044] Fig. 20b is a diagram showing the increase in lipid peroxide (LPO) levels caused by the product in Example 3 of the present invention in tumor cells.

[0045] Fig. 20 c is a diagram showing that the product in Example 3 of the present invention causes a decrease in the level of GSH-dependent peroxidase 4 (GPX4) protein in tumor cells.

[0046] Fig.21 This is a diagram showing the in vivo tumor treatment effect of the product in Example 3 of the present invention.

[0047] Fig. 22 These are H&E, Ki67 and GPX4 staining fluorescence images of tumor sections after treatment with the product in Example 3 of the present invention.

[0048] Fig.23 This is a diagram showing the in vivo tumor targeting effect of the product in Example 3 of the present invention.

[0049] Fig.24 This is an analysis of the cytotoxicity of the product in Example 3 of the present invention to MOVAS cells in a neutral culture medium.

[0050] Fig.25 These are H&E staining images of major organs after tumor treatment with the product in Example 3 of the present invention.

[0051] Fig.26 The blood biochemistry and hematology data of mice treated with the product in Example 3 of the present invention are shown in Table 1. All data were collected 18 days after intravenous injection.

[0052] Fig. 27 Body weight curves of healthy Balb / c nude mice injected with different doses of USAHC (0 mg / kg, 10 mg / kg, and 20 mg / kg) via tail vein.

[0053] Fig.28 This is the body weight curve of tumor-bearing mice treated with the product in Example 3 of the present invention during an 18-day treatment period. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and specific operation process are given, but the protection authority of the present invention is not limited to the following embodiments.

[0055] The present invention uses small-pore mesoporous silica (SiO2) as carrier particles, upconversion nanoparticles (NaTmF4: 4% Er@NaLuF4) as monochromatic red light upconversion fluorescent reagents, human serum albumin as a biocompatible material for loading, and further grafting cinnamaldehyde and encapsulating arginine to prepare USAHC core-shell nanoparticles. Figure 1As shown, the following is the specific preparation method of the present invention: Example 1 Preparation of Monochromatic Red Light Upconversion NaTmF4:4%Er@NaLuF4 (U) Core-Shell Nanoparticles Using NaTmF4 as the matrix, doped with 4% Er 3+ ions and adjusted the ratio of F source and Na source (NH4F / NaOH: 1.6 / 1mmol), synthesized monochromatic red light upconversion nanoparticles by existing thermal decomposition method and epitaxial growth method, and further used hydrochloric acid / ethanol washing method to remove oleic acid on the surface of nanoparticles for later use. Figure 2 As shown, transmission electron microscopy was used to confirm its core-shell structure and element distribution.

[0056] The upconversion red fluorescence emission performance was evaluated. First, a stable aqueous dispersion of U nanoparticles was prepared by ultrasound and placed in a 4 cm cuvette. The dispersion was illuminated in a dark room under a 980 nm laser at 1 W / cm 2 Under the irradiation conditions, a CCD camera is used to take pictures. Figure 3 As shown in the inset, the U nanoparticle material has good upconversion red fluorescence emission performance. 2 The upconversion fluorescence spectra of U nanoparticles were recorded under irradiation conditions. Figure 3 As shown, the two main emission peaks are 673 nm and 815 nm, corresponding to Tm 3+ of 1 G4→ 3 F4 and 3 H4→ 3 The monochromatic red light upconversion nanoparticles reduce the tissue absorption problem of green light, making it capable of deep tissue imaging. In summary, these experiments show that U nanomaterials inherently have upconversion red fluorescence emission properties, providing a simple method for achieving deep tissue fluorescence imaging.

[0057] Example 2 Preparation of small pore size mesoporous NaTmF4:4%Er@NaLuF4@SiO2 (US) core-shell nanoparticles Dissolve 1 g of CTAC (hexadecyltriethylammonium chloride) in 40 mL of deionized water, stir slowly for 10 minutes, and add 18 μ After stirring for 1.5 h at room temperature, 15 mL of NaTmF4:4%Er@NaLuF4 deionized water dispersion was added and the stirring was continued for another 1.5 h. The whole system was then heated to 90 °C, 0.15 mL of TEOS (tetraethyl orthosilicate) was added, and 50 μL APTES (3-aminopropyltriethoxysilane) was reacted at a speed of 1800 r / min for 4 hours. After cooling to room temperature, the product was collected by high-speed centrifugation and washed repeatedly with ethanol 3 times (using a centrifuge for washing at a speed of 9000-12000rpm); to remove the CTAC template, NaCl-methanol solution (mass fraction of 1 wt%) was used, vigorously stirred at room temperature for 3 hours, separated by high-speed centrifugation, and washed alternately with deionized water and ethanol 3 times. This process was repeated 3 times to obtain small-pore mesoporous US core-shell nanoparticles US. Figure 4 As shown in the figure, the nitrogen adsorption / desorption curves and the pore size distribution of US confirm that US core-shell nanoparticles are mesoporous materials with a diameter of ~3 nm. US nanoparticles are loaded with small-pore mesoporous SiO2 on the surface, which enables them to encapsulate the small molecule natural compound L-arginine.

[0058] Example 3 Preparation of reactive oxygen and reactive nitrogen cascade anti-tumor nanomedicine Step 1, loading of human serum albumin: ultrasonically disperse the small mesoporous US (30 mg) obtained in Example 2 in 1 mL of deionized water to form a uniform and stable suspension, then stir at room temperature for 10 min, drop 5 mL (5 mg / mL) of human serum albumin (HSA) aqueous solution into the US solution, slowly push 10 mL of acetone with a syringe, and stir vigorously for 5 h. The collected product after high-speed centrifugation was washed with deionized water to remove unreacted HSA, and then the human serum albumin-modified mesoporous core-shell nanoparticles USH were dispersed in 1 mL of acetone for later use. Figure 5 As shown in the figure, the presence of HSA UV characteristic peaks at 220 nm and 280 nm indicates that human serum albumin loading is successful. USH nanoparticles are modified with human serum albumin on the surface to make them biocompatible and tumor-targeting.

[0059] Step 2: Cinnamaldehyde (CA) grafting: 10 mL of the mixed solution (V CA :V acetone :1:9) was slowly dripped into the USH NPs acetone solution under a stirrer. After stirring for 24 h, the mixture was dialyzed and purified to obtain CA-grafted NaTmF4:4%Er@NaLuF4@SiO2@HSA / CA(USHC) nanoparticles. Figure 6 and Figure 7 As shown, the UV and IR characteristic peaks of CA indicate that CA was successfully grafted.

[0060] Step 3, NaTmF4:4%Er@NaLuF4@SiO2-L-Arg@HSA / CA (USAHC NPs) nanoparticles encapsulating L-arginine: USHC (20 mg) was ultrasonically dispersed in 10 mL of L-arginine (L-Arg) aqueous solution (5 mg / mL), and then transferred to a 100 mL round-bottom flask. After stirring at room temperature for 24 hours, the mixture was centrifuged to obtain USAHC nanoparticles encapsulating L-Arg. Infrared spectroscopy characterization proved that L-Arg encapsulation was successful ( Figure 7 The absorbance value of L-Arg decreased significantly before and after encapsulation, and the encapsulation rate of L-Arg in USAHC was calculated to be 66% ( Figure 8 ). The powder diffraction analysis results show that the prepared USAHC nanoparticles have good purity and crystallinity ( Fig. 9 The Zeta potential of the USAHC nanoparticle surface is ‒11.6 mV ( Fig.10 ), the hydrated particle size is 232 nm ( Fig.11 ). Further application of transmission electron microscopy confirmed the core-shell structure of the prepared USAHC nanoparticles ( Fig.12 ) and the silicon shell of the nanoparticles collapses and dissociates after encountering acid ( Fig.13 ).

[0061] Example 4 Application of reactive oxygen and reactive nitrogen cascade anti-tumor nanomedicine The reactive oxygen and reactive nitrogen effectively combined with USAHC nanoparticle diagnostic and therapeutic probe prepared in Example 3 can be used for monochromatic red light upconversion fluorescence imaging under near-infrared light irradiation on the one hand, and can be used for chemo-gas therapy-ferroptosis treatment of tumors on the other hand.

[0062] The following experiment was conducted to evaluate the therapeutic effect of the combined treatment of cinnamaldehyde / L-arginine.

[0063] 1. Acid-sensitive release of cinnamaldehyde and L-arginine Cinnamaldehyde and L-arginine were released very slowly in neutral medium, with release rates of ~17% and ~8% at 72 h, respectively, whereas in acidic medium, the release rates reached 93% and 95%, respectively. Fig.14 and Fig.15 As shown. In addition, the drug release was very fast in the first 4 h, and the release curves of the two were similar. Most importantly, in this design, CA was released faster than L-Arg. In the first few hours, a large amount of L-Arg was rapidly released and reacted with endogenous and CA-produced H2O2. L-Arg was consumed without being captured by tumor cells, and the in situ production of NO activated immune cells. Achieving controlled release of L-Arg and supplementation of L-Arg by immune cells may be an important strategy for cancer treatment.

[0064] 2. Acid-sensitive release of hydroxyl radicals (·OH) Electron spin resonance (ESR) spectroscopy was used to detect ·OH using 5,5-dimethyl-1-pyrrolidine-oxide (DMPO) as a free radical scavenger. Fig.16 As shown in the figure, compared with the counterpart at pH 7.4, the counterpart at pH 5.5 showed a characteristic peak of ·OH in a ratio of 1:2:2:1. The results revealed that CA in USHC nanoparticles is an acid-sensitive substance that releases ·OH in acidic medium.

[0065] 3. Release of ROS and RNS Using DCFH-DA as a probe, the total ROS generation efficiency in SACC cells was first evaluated (Figure 17a). Compared with the corresponding group with a pH value of 5.5, the group with a pH value of 7.4 showed a weaker green signal, indicating that the acidic environment is conducive to the generation of ROS such as ·OH and H2O2. Subsequently, 3-amino-4-aminomethyl-2',7'-fluorescein diacetate (DAFDA) was used to detect the generation of intracellular RNS (Figure 17b). Among them, strong green fluorescence was detected in SACC cells treated with USAHC, which is because the H2O2 released by CA triggered the effective release of NO. In contrast, the release level of RNS was significantly increased after H2O2 treatment, indicating that USHAC has a high encapsulation capacity of L-Arg.

[0066] The release kinetics of total ROS in tumor cells is shown in Figure 18a. After 3 hours of incubation, the amount of ROS generated reached a maximum, then gradually decreased, and a very small amount of ROS was still released after 24 hours, which was consistent with the CA release curve at pH 5.5. Compared with the PBS group, the USHC group showed a stronger green signal, revealing the ROS released by CA in tumor cells. ROS has a short diffusion distance and a large reduction potential, which is an important cancer treatment strategy.

[0067] Further tumor cell tumorigenesis dynamics, such as Fig.18 As shown in Figure 2b. Compared with the PBS group, the USAHC-treated group showed strong green fluorescence, indicating that L-Arg reacted with H2O2 to generate more NO. Moreover, the release rate of NO was fast at first and then slowed down, which was consistent with the release curve of L-Arg and CA. A considerable amount of NO was still generated within 24 hours, indicating that USAHC achieved the controlled release of L-Arg and in situ NO generation.

[0068] 4. In vitro chemo-gas therapy-ferroptosis cascade treatment Oxidative stress caused by intracellular ROS and RNS can induce cell apoptosis. The apoptosis of SACC cells was evaluated by CCK8 method (Figure 19a). At pH 5.5, the apoptotic rate increased significantly. These results were further confirmed by using Hoechst 33342 (blue fluorescence) as a probe to distinguish apoptotic SACC cells (Figure 19b). Fig.19 As shown in c, the mitochondrial membrane potential was further analyzed using the JC-1 kit. The results confirmed that the target of ROS and RNS-induced apoptosis is mitochondria.

[0069] In addition, GSH was added to the NaTmF4:4%Er@NaLuF4@SiO2-L-Arg@HSA / CA prepared in Example 2 to detect the performance of GSH depletion. Fig. 20 As shown in a, it was found that the diagnostic probe consumed the overexpressed GSH / GSSG in the TME. The GSH level in the USAHC-treated group was only 31%, indicating that USAHC effectively released NO, leading to increased GSH consumption. Furthermore, the product caused an increase in lipid peroxide (LPO) levels in tumor cells, as shown in Fig. 20 As shown in b. Fig. 20 As shown in c, Western blot analysis showed that USHC slightly inhibited the activity of GPX4, which was attributed to the generation of ·OH-induced severe oxidative stress imbalance. When L-Arg was conjugated to USHC, GPX4 activity was significantly inhibited, only 29% of the control group, indicating that ROS / NO synergistically inhibited GPX4 activity.

[0070] 5. Chemotherapy-gas therapy-ferroptosis cascade tumor treatment effect like Fig.21 As shown in the figure, the USHC group also had a significant inhibitory effect on tumor growth. After USHC was delivered to the tumor site, CA was effectively released in the weakly acidic TME and produced a large amount of ·OH, which promoted the accumulation of ROS and effectively inhibited the tumor. The USAHC group had the highest efficacy after 18 days of treatment, and the chemo-gas therapy-ferroptosis cascade treatment produced a strong anti-tumor effect. Fig. 22 The results of tumor tissue section staining showed that the tumor treatment mechanism of USAHC nanoparticles involved the combined effects of apoptosis and ferroptosis.

[0071] The tumor targeting of USAHC nano anti-tumor drugs is evaluated by monochromatic red light upconversion fluorescence imaging: BALB / c tumor-bearing mice were intravenously injected with USAHC nanoparticles (10 mg / kg), and UCL images were collected under 980 nm laser excitation at 0 minutes, 15 minutes, 30 minutes, 40 minutes, etc., until they reached the tumor area. The tail vein injection and tumor site of the mice were recorded with a CCD camera. After the imaging, the mice were euthanized by cervical dislocation. As shown in Figure 23a. 60 minutes after injection, strong red fluorescence signals were observed in the tumor site and in vitro tumors (Figure 23b), indicating that USAHC nanoparticles have excellent retention effects in tumors and the nanoprobe has good upconversion fluorescence imaging effects.

[0072] The following experiments are used to evaluate the biosafety of USAHC nano anti-tumor drugs: 1. Evaluation of cytotoxicity to normal biological tissues As shown in Figure 24, using the MOVAS cell line as the research object, the cell survival rate remained at a high level (about 95%) at a higher concentration (150 μg / mL) at pH 5.5, which indicates that the toxicity of USAHC to normal biological tissue cells is negligible.

[0073] 2. Blood biochemical analysis After the evaluation period ended (the tumor was basically eliminated), the mouse eyeballs were collected for blood sampling, and the blood samples were used for blood biochemical analysis to evaluate the effects of USAHC on the liver and kidney functions of mice ( Fig.25 The blood cell levels, liver function indicators such as alanine transferase (ALT), aspartate transferase (AST) and albumin (ALB), and the renal function indicator mouse creatinine level did not change significantly within 18 days, indicating that USAHC did not cause obvious liver and kidney toxicity damage.

[0074] 3. Evaluation of side effects of tumor treatment H&E staining analysis was performed on the ex vivo organs of tumor-bearing mice treated with USAHC nanoparticles ( Fig.26 ), there was no obvious damage or inflammatory infiltration in major organs such as the heart, liver, spleen, kidney, and lung, indicating that the side effects of the treatment were negligible.

[0075] 4. Weight and health status assessment First, Balb / c healthy nude mice were used as research subjects, and the weight and health of the nude mice were observed every 3 days during the 24-day evaluation period (Figure 27). The weight of the nude mice in the drug group was not significantly suppressed, indicating that the toxicity of USAHC is very low.

[0076] Secondly, Balb / c tumor-bearing nude mice were used as research subjects, and the weight and health status of the nude mice were observed every 3 days during the 18-day treatment period (Figure 28). There was no significant suppression in the weight of nude mice in the medication group, indicating that USAHC has good safety.

[0077] The above description is only a specific implementation of the present invention, but the protection scope of the present invention is not limited thereto. Any person familiar with the technology can understand and think of any changes or substitutions within the technical scope disclosed by the present invention, which should be included in the scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. An active oxygen and active nitrogen cascade anti-tumor nanomedicine, characterized in that: The invention comprises inorganic nanoparticles, which are used as carriers, coated with biocompatible materials, loaded or grafted with plant aldehydes, and encapsulated with natural amino acids; the nanoparticles are upconversion nanoparticles@mesoporous silica; the biocompatible material is human serum albumin; the plant aldehyde is cinnamaldehyde; and the natural amino acid is L-arginine.

2. A method for preparing the active oxygen and active nitrogen cascade anti-tumor nanomedicine according to claim 1, characterized in that: The following steps are involved: Step 1, synthesizing single red upconversion luminescent nanoparticles with suitable mesopores as carriers; Step 2, preparing nanoparticles coated with biocompatible materials: dispersing the mesoporous nanoparticles obtained in step 1 in deionized water, adding biocompatible materials, and dripping acetone under vigorous stirring to obtain nanoparticles coated with biocompatible materials; Step 3, preparing nanoparticles loaded or grafted with plant aldehydes: dispersing the nanoparticles obtained in step 2 in acetone, adding plant aldehydes, and stirring to obtain nanoparticles grafted with plant aldehydes; Step 4, preparing nanoparticles encapsulating natural amino acids: selecting suitable natural amino acids, encapsulating and diffusing them into the mesopores of the nanoparticles grafted with plant aldehydes obtained in step 3, and obtaining nanoparticles encapsulating natural amino acids.

3. The method for preparing an active oxygen and active nitrogen cascade anti-tumor nanomedicine according to claim 2, characterized in that: In the step 1, a relatively small amount of NH4F / NaOH (1.6 / 1 mmol) is used to prepare single red upconversion luminescent nanoparticles through an epitaxial growth strategy, and after removing the oleic acid on the surface, suitable mesoporous silica is coated to obtain single red upconversion luminescent mesoporous nanoparticles with small pore size.

4. The method for preparing an active oxygen and active nitrogen cascade anti-tumor nanomedicine according to claim 2, characterized in that: In the step 2, 10 to 50 mg of the nanoparticles obtained in the step 1 are ultrasonically dispersed in 1 to 5 mL of deionized water, and then 2 to 10 mL of biocompatible material (5 mg / mL) is added, and 5 to 30 mL of acetone is slowly pushed in with a syringe, and the mixture is vigorously stirred for 10 to 24 hours. The product is collected after high-speed centrifugation and washed with deionized water to obtain nanoparticles coated with biocompatible materials. The biocompatible material is a human biocompatible material, and when the biocompatible material is a protein, a coating method is selected.

5. The method for preparing an active oxygen and active nitrogen cascade anti-tumor nanomedicine according to claim 2, characterized in that: In step 3, the nanoparticles obtained in step 2 are ultrasonically dispersed in 1-5 mL of acetone, and then 5-15 mL of the solution is slowly dripped into the mixture under stirring at room temperature. 植物醛 :V 丙酮 = 1:9, and after stirring in the dark at room temperature for 12 to 24 h, nanoparticles loaded with plant aldehydes were obtained, and the optimal mass ratio of nanoparticles to plant aldehydes was 1:20; the mixture was purified by dialyzing.

6. The method for preparing an active oxygen and active nitrogen cascade anti-tumor nanomedicine according to claim 2, characterized in that: In the step 4, 10 to 50 mg of the nanoparticles obtained in step 3 are ultrasonically dispersed in 2 to 10 mL of deionized water, and then 5 to 25 mL of a natural amino acid aqueous solution is slowly dropped into the solution under stirring at room temperature. After stirring at room temperature in the dark for 10 to 24 h, nanoparticles encapsulating natural amino acids are obtained.

7. The method for preparing an active oxygen and active nitrogen cascade anti-tumor nanomedicine according to claim 3, characterized in that: In the step 1, the fluorescent material is selected to be an up-conversion single red luminescent material, and the coated silicon dioxide is a mesoporous material with a small pore size.

8. The method for preparing an active oxygen and active nitrogen cascade anti-tumor nanomedicine according to claim 6, characterized in that: In step 4, the optimal mass ratio of the nanoparticles to the natural amino acids is 1:2; after the reaction is completed, the material is washed with deionized water and collected by centrifugation, and the centrifuge speed is 9000-12000 rpm.

9. Use of the reactive oxygen and reactive nitrogen cascade anti-tumor nanomedicine as claimed in any one of claims 1 to 8 in the preparation of gas therapy, chemotherapy and ferroptosis anti-tumor combined therapeutic drugs.

10. The medical use of an active oxygen and active nitrogen cascade anti-tumor nanomedicine according to claim 9, characterized in that: The nanomedicine releases plant aldehydes and natural amino acids in response to the acidity of the tumor microenvironment to achieve combined anti-tumor therapy, as well as chemotherapy-gas therapy-ferroptosis application guided by upconversion single red fluorescence imaging.

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