Manganese-based multifunctional nanoparticles for improving tumor hypoxia and reversing doxorubicin drug resistance

Abstract

The invention discloses a manganese-based multifunctional nanoparticle for improving tumor hypoxia and reversing doxorubicin drug resistance and a preparation method of the manganese-based multifunctional nanoparticle, and belongs to the technical field of pharmaceutical preparations. According to the method, the HSA-MnO2 nanoparticles are firstly prepared by adopting an ultrasonic method, then DOX is loaded through ultrasonic, the DOX / HSA-MnO2 nanoparticles are obtained, and the encapsulation efficiency and the drug loading capacity of the manganese-based multifunctional nanoparticles can be remarkably improved by adopting the method; meanwhile, the manganese-based multifunctional nanoparticles prepared on the basis of the method not only are uniform in distribution and have good preparation stability, photo-thermal stability and biocompatibility, but also have good TME response drug release and oxygen production characteristics. When photothermal therapy is combined, the expression level of HIF-1alpha and P-gp in tumor cells can be remarkably reduced, breast cancer cell MDR is effectively reversed, and the treatment effect of drug-resistant breast cancer is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a manganese-based multifunctional nanoparticle capable of improving tumor hypoxia and reversing doxorubicin resistance and a preparation method thereof. Background Art

[0002] It is reported that in 2020, the number of new cancer cases in the world reached 19.3 million, and nearly 10 million people died of cancer, becoming the largest cause of human death after cardiovascular disease. Among many tumors, breast cancer has become the first in the global tumor incidence, accounting for 11.7% of all tumor cases, of which the mortality rate of female breast cancer has reached 15.5%. At present, the main treatments for breast cancer are surgery, chemotherapy, radiotherapy, endocrine therapy, targeted therapy and immunotherapy. For patients who cannot undergo surgical treatment or fail to undergo surgical treatment in time, chemotherapy is the most commonly used treatment. However, chemotherapy drugs have problems such as poor targeting and large toxic side effects, and long-term use of chemotherapy drugs is prone to multidrug resistance (MDR), which ultimately leads to chemotherapy failure. In addition, MDR is also prone to cause the metastasis of tumor lesions, leading to an increase in the mortality rate of breast cancer patients. Therefore, improving the targeting of chemotherapy drugs and reversing their multidrug resistance are important issues that need to be urgently addressed to improve the effect of breast cancer chemotherapy.

[0003] In recent years, researchers have designed a catalase (CAT) that can mimic catalase and convert H 2 O 2 Converted to H 2 O and O 2 Molecular nanoparticles, including metal and metal oxide nanoparticles, carbon-based nanomaterials and coordination polymers. Among them, metal oxides, especially manganese dioxide (MnO 2 ) nanoparticles have attracted increasing attention due to their adjustable surface functionalization, controllable synthetic shape, and effective multi-enzyme activity. Manganese-based nanoparticles can react with GSH to convert Mn 4+ Reduction to Mn 2+ , which can not only consume the overexpressed GSH in tumor cells, but also produce Mn 2+ Can be used with H 2 O 2 The Fenton reaction forms hydroxyl radicals (·OH), which have a chemodynamic therapy (CDT) effect, thereby killing tumor cells. At the same time, manganese is also one of the elements of human growth, development, metabolism and antioxidant system, and excess Mn 2+ It can also be rapidly metabolized by the kidneys. 2+It has been used clinically as a contrast agent for magnetic resonance imaging (MRI). This shows that manganese-based nanomaterials have greater safety advantages over non-biodegradable inorganic materials such as mesoporous silica and graphene. In addition, nanomaterial carriers can increase the accumulation of drugs in drug-resistant cells through cellular uptake mediated by endocytosis, while nanocarriers with a size of 10-100 nm can avoid rapid systemic clearance and passively target tumors through enhanced permeability and retention (EPR) effects, thereby improving the targeting of chemotherapeutic drugs and reducing the toxic side effects of chemotherapeutic drugs. In addition, the use of MnO 2 The nanosystem can efficiently and continuously produce oxygen in situ in the tumor microenvironment (TME), avoiding the need for exogenous O 2 The adverse reactions caused by the infusion and the problem of oxygen leakage during the oxygen carrying process can directly increase the oxygen content of the TME, which is more effective than the two methods of inhibiting oxygen consumption and normalizing the blood vessels in the tumor to overcome the problem of TME hypoxia. 2 It is a nano drug delivery carrier with GSH and H 2 O 2 Dual responsiveness, namely, under acidic conditions of the TME, the overexpressed GSH and H 2 O 2 response, producing O in situ at the tumor site 2 , and the corresponding equations are shown in equations (1) and (2).

[0004] MnO 2 + H 2 O 2 + 2H + = Mn 2+ + 2H 2 O + O 2 (1) MnO 2 + GSH + H + = Mn 2+ +GSSH+H 2 O (2) In addition, studies have reported that the combination of photothermal therapy (PTT) and drug-delivering nanoparticles can effectively reverse tumor MDR. PTT is a typical photon-triggered treatment method that kills tumor cells by generating a local temperature of 41-42°C or above by a photothermal agent (PTA) under visible or near-infrared light. Photothermal therapy can not only ablate drug-resistant cells, but also change the permeability of tumor blood vessels to increase blood flow in the tumor, indirectly increasing the oxygen content in the hypoxic tumor site, thereby inhibiting MDR-related proteins and ultimately reversing tumor cell resistance. It has been reported that MnO 2 It also has photothermal properties. At the same time, the research team has also verified that MnO 2 Nanocarriers have significant photothermal effect under 635 nm laser irradiation and can be used as PTA to achieve photothermal therapy of tumors. 2 As a carrier of chemotherapy drugs, it can improve the chemotherapy effect of breast cancer in multiple ways.

[0005] At present, the preparation of MnO 2 Nanoparticles are commonly used in the form of silicon dioxide (SiO 2 ) template method and polyallylated ammonium chloride (PAH) template method, but these methods have problems such as complex preparation process, low stability and poor biocompatibility, which are not conducive to their clinical application. Biomineralization is a method of combining biological macromolecules (such as proteins) and inorganic materials to prepare functionalized nanocarriers. Compared with traditional chemical nanotechnology, biomineralization-mediated nanostructures can usually be prepared under mild and environmentally friendly conditions with high controllability. Albumin has been successfully used as a nanocarrier for a variety of drugs due to its good biocompatibility and low immunogenicity. Albumin-based biomineralization has many advantages such as simple preparation process, no need for further modification, high stability and high biocompatibility. In addition, albumin can promote the delivery of nanoparticles to tumor cells and enhance cell uptake by interacting with the albumin receptor gp60 overexpressed on tumor cells. Therefore, the biomineralization method using albumin as a template scaffold was used to prepare MnO 2 Nanoparticles are an efficient, safe and stable method. Human serum albumin (HSA) is a protein abundant in human serum with high biocompatibility. It has been proven to be an ideal drug carrier for chemotherapeutic drugs, hypoglycemic drugs, photosensitizers and radioisotopes. Therefore, HSA is used as a biomineralization method to prepare MnO 2 The template scaffold of the nanocarrier not only has good biocompatibility, but also can significantly increase the targeting of drug-loaded nanoparticles. Summary of the invention

[0006] The purpose of the present invention is to provide a manganese-based multifunctional nanoparticle and a preparation method thereof for improving tumor hypoxia and reversing doxorubicin resistance. The prepared manganese-based multifunctional nanoparticles are not only uniformly distributed, have good formulation stability, photothermal stability and biocompatibility, but also have good TME-responsive drug release and oxygen production characteristics.

[0007] To achieve the above object, the present invention adopts the following technical solution: A manganese-based multifunctional nanoparticle for improving tumor hypoxia and reversing doxorubicin resistance, wherein human serum albumin (HSA) is used as a template scaffold to prepare HSA-MnO 2 The resulting HSA-MnO nanoparticles 2 Nanoparticles were used as carriers to load doxorubicin (DOX) by adsorption and chemical bonding. The DOX / HSA-MnO 2 Nanoparticles; the particle size is less than 100 nm, the encapsulation efficiency is 96.56±0.17%, and the drug loading is 31.91±0.86%. 2 The nanoparticles are not only evenly distributed, with good formulation stability, photothermal stability and biocompatibility, but also have good TME-responsive drug release and oxygen production characteristics. When combined with photothermal therapy, they can significantly reduce the expression levels of HIF-1α and P-gp in tumor cells, effectively reverse the MDR of breast cancer cells, and improve the treatment effect of drug-resistant breast cancer.

[0008] The preparation of the manganese-based multifunctional nanoparticles is to firstly use an ultrasonic method to make HSA and KMnO 4 Oxidation-reduction reaction occurs to synthesize HSA-MnO in one step 2 Nanoparticles were then loaded with DOX by ultrasound to obtain DOX / HSA-MnO 2 Nanoparticles; which specifically comprises the following steps: 1) HSA-MnO 2 Preparation of nanoparticles: HSA solution was added dropwise to KMnO 4 The solution was treated with ultrasound and then purified by ultrafiltration to obtain HSA-MnO 2 a solution of nanoparticles; 2) DOX / HSA-MnO 2 Preparation of nanoparticles: The HSA-MnO prepared in step 1) 2 The solution of nanoparticles was mixed in deionized water, and then DOX solution was added dropwise. After ultrasonic treatment, ultrafiltration was performed to obtain DOX / HSA-MnO 2 Nanoparticle solution.

[0009] Furthermore, the HSA used in step 1) and KMnO4 The mass ratio is 1.25:1~5:1; preferably, the mass ratio is 2.5:1.

[0010] Furthermore, the HSA used in step 1) and KMnO 4 The concentration ratio is 1:1~5:1; preferably, the concentration ratio is 2.5:1.

[0011] Furthermore, the power of the ultrasonic treatment in step 1) is 320 W to 800 W, and the time is 0.5 h to 1.5 h; preferably, the power is 320 W, and the time is 1 h.

[0012] Furthermore, the HSA-MnO used in step 2) 2 The mass ratio of the nanoparticles to DOX is 1:1 to 6:1; preferably, the mass ratio is 1.5:1.

[0013] Furthermore, the HSA-MnO used in step 2) 2 The concentration ratio of the nanoparticles to DOX is 1:0.75 to 1:3; preferably, the concentration ratio is 1:1.5.

[0014] Furthermore, the power of the ultrasonic treatment in step 2) is 320 W to 800 W, and the time is 1 h to 4 h; preferably, the power is 640 W, and the time is 3 h.

[0015] The manganese-based multifunctional nanoparticles have the performance of improving breast cancer hypoxia and reversing doxorubicin resistance, and can be used to prepare anticancer drugs, especially anti-breast cancer drugs.

[0016] The beneficial effects of the present invention are: (1) The present invention uses HSA to synthesize DOX-loaded HSA-MnO in a simple one-step biomineralization method. 2 Nanoparticles have the characteristics of simple preparation process, high drug loading capacity, high encapsulation rate, high stability and good biocompatibility.

[0017] (2) HSA-MnO constructed by the present invention 2 Nanoparticles themselves have photothermal properties and can be used not only as carriers but also as photothermal converters. When combined with chemotherapy drugs, they can achieve chemotherapy-photothermal combined therapy.

[0018] (3) DOX / HSA-MnO prepared by the present invention 2 Nanoparticles not only have TME responsiveness and can release drugs in a targeted manner to reduce the toxic side effects of chemotherapy drugs, but can also directly produce oxygen at the tumor site to increase the oxygen content at the tumor site. They can also increase vascular permeability through photothermal effects, further alleviate the tumor hypoxic microenvironment, improve hypoxia through multiple pathways, reverse MDR, and improve the effect of chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 HSA, KMnO used in the examples 4 , DOX and prepared HSA-MnO 2 and DOX / HSA-MnO 2 Comparison of nanoparticle aqueous solutions.

[0020] Figure 2 HSA-MnO prepared in the example 2 Nanoparticles (A) and DOX / HSA-MnO 2 TEM image of nanoparticles (B).

[0021] Figure 3 is the photothermal effect of nanoparticle solution, where (A) is different MnO 2 Temperature rise after 10 min of irradiation at different laser powers; (B) 100 μg / mL MnO 2 Temperature rise of concentration at different laser powers.

[0022] Figure 4 The photothermal stability test of the nanoparticle solution, where (A) is HSA-MnO 2 Nanoparticle solution; (B) DOX / HSA-MnO 2 Nanoparticle solution.

[0023] Figure 5 is the photothermal conversion efficiency of the nanoparticle solution, where (A) is HSA-MnO 2 Temperature rise-fall curve of nanoparticle solution; (B) is HSA-MnO 2 The linear relationship between the cooling time of the nanoparticle solution and -lnθ; (C) is DOX / HSA-MnO 2 Temperature rise-fall curve of nanoparticle solution; (D) is DOX / HSA-MnO 2 Linear relationship between the cooling time of nanoparticle solution and -lnθ.

[0024] Figure 6 DOX / HSA-MnO 2 The inhibitory effect of nanoparticles on MCF-7 / ADR cells under normoxic and hypoxic conditions, where (AB) was incubated for 24 h; (CD) was incubated for 48 h.

[0025] Figure 7 DOX / HSA-MnO 2 The inhibitory effect of nanoparticles on MCF-7 cells under normoxic and hypoxic conditions, where (AB) was incubated for 24 h; (CD) was incubated for 48 h.

[0026] Figure 8 This is the change curve of relative tumor volume of nude mice in different treatment groups in the pharmacodynamic experiment.

[0027] Fig. 9 These are the anatomical images and weights of the tumors in each treatment group after 21 days of treatment in the pharmacodynamic experiment. DETAILED DESCRIPTION

[0028] In order to make the contents of the present invention easier to understand, the technical scheme of the present invention is further described below in conjunction with the specific implementation methods, but the present invention is not limited to this. The present invention uses a single-factor experiment to screen out the optimal ratio and then uses dynamic scattering analysis (DLS), Zeta potential, transmission electron microscopy (TEM), ultraviolet-visible spectroscopy (UV-vis) and other means to characterize; then the MTT method is used to evaluate the cytotoxicity of the nanoparticles, and the fluorescence microscope and flow cytometer are used to examine the cellular uptake of DOX and nanoparticles; the protein immunoblotting method (WB) is used to examine the expression levels of HIF-1α and P-gp in tumor cells after chemotherapy, photothermal therapy, and chemotherapy combined with photothermal therapy; a mouse in situ tumor model is established for anti-tumor experiments. The specific contents are as follows: Example

[0029] DOX / HSA-MnO 2 The preparation of nanoparticles includes the following steps: 1) HSA-MnO 2 Preparation of nanoparticles: HSA solution was added dropwise to KMnO 4 The solution was treated with ultrasound at 320 W to 800 W for 0.5 h to 1.5 h and then placed in an ultrafiltration tube (MWCO = 10 kDa) for centrifugation and purification to obtain HSA-MnO 2 a solution of nanoparticles; 2) DOX / HSA-MnO 2 Preparation of nanoparticles: The HSA-MnO prepared in step 1) 2 The nanoparticle solution was mixed in deionized water, and then the DOX solution was added dropwise. After ultrasonic treatment at 320 W to 800 W for 1 h to 4 h, the solution was placed in an ultrafiltration tube (MWCO = 10 kDa) for centrifugation and purification to obtain the DOX / HSA-MnO 2 Nanoparticle solution.

[0030] Optimal ratio screening test: 1. HSA-MnO 2 Investigation of Ultrasonic Power in Nanoparticle Preparation Table 1 Effect of the first ultrasonic power on the preparation of DOX / HSA-MnO 2 Effects of Nanoparticles

[0031] From the results in Table 1, it can be seen that when the ultrasonic power is smaller, the DOX / HSA-MnO 2 The smaller the particle size and PDI of the nanoparticles, the smaller the difference in potential, encapsulation efficiency and drug loading. 2 The ultrasonic power of the nanoparticles was 320 W.

[0032] 2. HSA-MnO 2 Ultrasonic time observation of nanoparticle preparation Table 2 Effect of the first ultrasonic time on the preparation of DOX / HSA-MnO 2 Effects of Nanoparticles

[0033] It can be seen from the results in Table 2 that when the ultrasound time is short, DOX / HSA-MnO 2 The PDI of the nanoparticles was too large. When the time was extended appropriately, the particle size and PDI of the nanoparticles became smaller. When the ultrasonic time was extended, the particle size and PDI tended to increase, while the potential, encapsulation efficiency and drug loading were not much different. Therefore, HSA-MnO was finally selected. 2 The ultrasonic time was 1 h.

[0034] 3. Investigation of ultrasonic power of DOX loading Table 3 Effect of the second ultrasonic power on the preparation of DOX / HSA-MnO 2 Effects of Nanoparticles

[0035] It can be seen from the results in Table 3 that when the ultrasonic power is high, DOX / HSA-MnO 2 The particle size and PDI of the nanoparticles are both small, but when the power is lower than 640 W, the particle size tends to increase, while the potential, encapsulation efficiency and drug loading are not much different. Therefore, the drug loading ultrasound power was finally selected as 640 W.

[0036] 4. Ultrasonication time study of DOX loading Table 4 Effect of the second ultrasonic time on the preparation of DOX / HSA-MnO 2 Effects of Nanoparticles

[0037] It can be seen from the results in Table 4 that when the ultrasonic time is short, DOX / HSA-MnO 2 The PDI of nanoparticles was too large. After prolonging the ultrasonic time, the PDI of nanoparticles became smaller, while the particle size, potential, encapsulation efficiency and drug loading were not much different. Therefore, the ultrasonic time for drug loading was mainly screened based on PDI, and the final ultrasonic time for drug loading was 3 h.

[0038] 5. HSA and KMnO 4 Quality ratio of Table 5 HSA and KMnO 4 Preparation of DOX / HSA-MnO by mass comparison 2 Effects of Nanoparticles

[0039] From the results in Table 5, it can be seen that in KMnO 4 Under the condition of constant quality, with the increase of HSA dosage, DOX / HSA-MnO 2 The drug loading of the nanoparticles decreased, and the PDI was relatively small. When the mass ratio was 2.5:1, the PDI of the nanoparticles was the smallest and the encapsulation efficiency was the highest. Therefore, HSA and KMnO were finally selected. 4 The mass ratio is 2.5:1.

[0040] 6. HSA and KMnO 4 The concentration ratio of Table 6 HSA and KMnO 4 Preparation of DOX / HSA-MnO by comparison of concentrations 2 Effects of Nanoparticles

[0041] From the results in Table 6, it can be seen that with the increase of HSA concentration, DOX / HSA-MnO 2 The particle size of the nanoparticles gradually increased, while the PDI showed a trend of first decreasing and then increasing. Therefore, HSA and KMnO 4 The concentration ratio is 2.5:1.

[0042] 7. DOX and HSA-MnO 2 The quality ratio Table 7 DOX and HSA-MnO 2 Preparation of DOX / HSA-MnO by mass comparison 2 Effects of Nanoparticles

[0043] From the results in Table 7, it can be seen that with the increase of DOX dosage, DOX / HSA-MnO 2 The particle size and drug loading of the nanoparticles also increased, while the PDI showed a trend of first decreasing and then increasing. Therefore, DOX and HSA-MnO 2 The mass ratio is 1:1.5.

[0044] 8. DOX and HSA-MnO 2 The concentration ratio Table 8 DOX and HSA-MnO2 Preparation of DOX / HSA-MnO by comparison of concentrations 2 Effects of Nanoparticles

[0045] It can be seen from the results in Table 8 that with the increase of DOX concentration, PDI shows a trend of first decreasing and then increasing. When the concentration ratio is 1:1.5, DOX / HSA-MnO 2 Nanoparticles have the smallest PDI, so DOX and HSA-MnO were finally selected. 2 The concentration ratio is 1:1.5.

[0046] 9. Determination of the final process According to the results of single factor investigation, HSA-MnO 2 The ultrasonic power of the prepared samples was 320 W and the ultrasonic time was 1 h. The ultrasonic power of the drug-loaded samples was 640 W and the ultrasonic time was 3 h. 4 The mass ratio of HSA to KMnO is 2.5:1. 4 The concentration ratio of DOX is 2.5:1, the mass ratio of DOX to carrier is 1:1.5, and the concentration ratio of DOX to carrier is 1:1.5.

[0047] The final process was as follows: 1 mL of HSA solution (10 mg / mL) was added dropwise to 1 mL of KMnO 4 The HSA-MnO 2 Then take 1 mL of the obtained HSA-MnO 2 After the nanoparticle solution was mixed with 1 mL of deionized water, 2 mL of DOX solution (1 mg / mL) was added dropwise at an ultrasonic power of 640 W. After ultrasonication for 3 h, ultrafiltration was performed to obtain DOX / HSA-MnO. 2 Nanoparticle solution. After testing, its encapsulation efficiency was 96.56±0.17% and the drug loading was 31.91±0.86%.

[0048] HSA-MnO prepared by the final process 2 Nanoparticle solution and DOX / HSA-MnO 2 The nanoparticle solutions were subjected to the following performance tests.

[0049] Performance Testing (1) TEM test Take an appropriate amount of HSA-MnO diluted to a certain concentration 2 and DOX / HSA-MnO 2The nanoparticle solution was added dropwise onto a 200-mesh copper grid coated with a carbon film and allowed to stand for 10 min. The excess nanoparticle solution at the edge of the copper grid was removed with filter paper. The copper grid was then air-dried at room temperature and the morphology of the nanoparticles was observed using TEM. Figure 2 .

[0050] Depend on Figure 2 It can be seen that the obtained nanoparticles are evenly distributed spherical and the morphologies of the two are different, indicating that the carrier-loaded DOX changes the morphology of the nanoparticles.

[0051] (2) Photothermal effect test of nanoparticle solution Take 100 μL of different concentrations of MnO 2 (0, 50, 100, 150, 200 μg / mL) HSA-MnO 2 The nanoparticle solution was placed in a 96-well cell culture plate (human breast cancer DOX-resistant cell line) and laser light with a wavelength of 635 nm was used at different power densities (0.5, 1, 1.5, and 2 W / cm 2 ) conditions, and the temperature change of the nanoparticle solution was recorded by an infrared thermal imager (the temperature was recorded every 30 s, for 10 min, and each group was repeated three times). 2 The temperature of the nanoparticle solution changes with time under laser irradiation with different power densities. The results are shown in Figure 3 .

[0052] Depend on Figure 3 It can be seen that under the same laser power density, MnO 2 The higher the concentration of MnO, the higher the temperature of the nanoparticle solution. 2 The higher the laser power density, the higher the temperature of the nanoparticle solution. The temperature of the nanoparticle solution is related to the concentration of MnO 2 The concentration of MnO is positively correlated with the laser power density. Considering that the photothermal treatment temperature needs to be above 42 °C, and a smaller amount of MnO should be used as much as possible, 2 It can meet the photothermal requirements, so 100 μg / mL of MnO 2 The temperature rise under different laser power densities was investigated by measuring the concentration. Considering that too high a temperature may cause burns to the skin surface, and in order to avoid damage to the operator's eyes during the test, the power density of the laser was finally selected to be 1.5 W / cm 2 .

[0053] (3) Photothermal stability test of nanoparticle solution The laser was used with a wavelength of 635 nm and a power of 1.5 W / cm 2The power density of HSA-MnO was irradiated 2 Nanoparticle solution and DOX / HSA-MnO 2 The nanoparticle solution was heated for 10 min, and then the laser was turned off. The cycle was repeated three times to investigate the photothermal stability of the nanoparticles. The results are shown in Figure 4 .

[0054] Depend on Figure 4 The results show that both nanoparticle solutions have good temperature rise-fall repeatability, indicating that MnO 2 The nanoparticle solution has excellent photothermal stability, and even when loaded with the chemotherapy drug DOX, the photothermal stability of the nanoparticles remains good.

[0055] (4) In vitro photothermal conversion efficiency test According to the recorded temperature rise-fall result data, the light-to-heat conversion efficiency η was calculated using the following formula. The results are shown in Table 9 and Figure 5 .

[0056] η(%)= ×100%, Where h is the heat transfer coefficient, s is the container surface area, T max refers to the equilibrium temperature, T surr Refers to the ambient temperature, Q Dis Refers to the amount of heat dissipated by the solvent and container when absorbing light, that is, the amount of heat absorbed by the blank aqueous solution or container under the same conditions, Q Dis =hs(T max，water -T surr，water ), I refers to the laser power (the laser power density selected in this experiment is 1.5 W / cm 2 , spot area is 0.5 cm 2 , so I is 0.75 W), A 635 Represents the absorbance of the sample at 635 nm.

[0057] Table 9

[0058] The calculated HSA-MnO 2 Nanoparticle solution and DOX / HSA-MnO 2 The photothermal conversion efficiencies of the nanoparticle solutions were 23.97% and 21.05%, respectively.

[0059] (5) Cellular pharmacodynamics test MTT method was used to investigate the 2 The inhibitory effect of nanoparticles on MCF-7 / ADR and MCF-7 cells. Figure 6 , Figure 7 .

[0060] Depend on Figure 6 , 7 The results showed that for MCF-7 / ADR cells, whether incubated under normoxic or hypoxic conditions for 24 h, DOX / HSA-MnO 2 The nanoparticles showed better inhibitory effects than the free chemotherapeutic drug DOX, and the inhibitory effects became more obvious as the incubation time was extended to 48 h. However, for MCF-7 cells, after 24 h of incubation under normoxic conditions, the inhibitory effect of free DOX on cells was stronger than that of DOX / HSA-MnO 2 When the incubation time was extended to 48 h, the inhibitory effect of free DOX at low concentrations was still stronger, while the inhibitory effect of nanoparticles at medium and high concentrations was gradually stronger than that of free DOX. 2 The inhibitory effect of nanoparticles on MCF-7 cells increased with the extension of incubation time and the increase of concentration. After 24 h of incubation under hypoxic conditions, the inhibitory effect of free DOX at high concentrations was still stronger than that of DOX / HSA-MnO 2 nanoparticles, while at low concentrations, DOX / HSA-MnO 2 The inhibitory effect of nanoparticles was stronger than that of free DOX, and when the incubation time was extended to 48 h, the inhibitory effect of nanoparticles on MCF-7 cells was stronger than that of free DOX. 2 The inhibitory effect of nanoparticles on MCF-7 cells was enhanced.

[0061] (6) In vivo pharmacodynamics testing of manganese-based multifunctional nanoparticles The tumor volume was 70-100 mm. 3 The in vivo pharmacodynamics study was conducted in nude mice of different sizes. The specific operation was as follows: the tumor-bearing nude mice were randomly divided into a saline group, a free DOX group, and a HSA-MnO group. 2 Group, DOX / HSA-MnO 2 Group, HSA-MnO 2 +Laser group and DOX / HSA-MnO 2 + Laser group, 4 mice in each group. Then, the drugs were injected into the tail vein according to the above groups (normal saline was used as the solvent, and the dosage was DOX 2 mg / kg, MnO 2 The mice in the Laser group were injected via tail vein at 1.5 W / cm2 1 h after the injection. 2 The tumor site was irradiated with a power density of 100 μg / cm2 for 10 min, and the tumor volume and weight changes of nude mice were measured and recorded every three days for 21 consecutive days. The results are shown in the table. Figure 8 .

[0062] Depend on Figure 8 The results showed that compared with the saline group, the DOX group and HSA-MnO 2 The tumor volume of the DOX / HSA-MnO group changed little, indicating that the therapeutic effect on drug-resistant breast cancer was not obvious (p>0.05), while the DOX / HSA-MnO 2 The tumor growth in the group was slower, that is, it showed an inhibitory effect on tumor growth (p<0.01), indicating that the nanoparticles can improve the therapeutic effect of chemotherapy drugs on drug-resistant breast cancer. 2 Compared with the DOX group, the DOX / HSA-MnO group also had a better tumor inhibition effect. The tumor volume treated with laser irradiation showed a continuous downward trend. Compared with the non-laser irradiation group, it had a significant therapeutic effect on the tumor (p<0.01), indicating that photothermal therapy can effectively inhibit tumor growth. Compared with the Saline group and the DOX group, the DOX / HSA-MnO 2 The + Laser group showed a very significant tumor inhibition effect (p<0.0001). During the treatment period, it not only inhibited the continued growth of the tumor, but also ablated the original tumor. Some tumor-bearing nude mice even saw the tumor disappear after 21 days of treatment. This shows that nanoparticle-mediated chemotherapy combined with photothermal therapy can significantly reverse breast cancer MDR and improve the treatment effect of drug-resistant breast cancer.

[0063] After 21 days of treatment, the nude mice in each group were dissected, and the tumor tissues were completely removed, weighed, photographed, and recorded. Fig. 9 shown.

[0064] Depend on Fig. 9 It shows that DOX / HSA-MnO 2 In the +Laser group, only one nude mouse still had tumor tissue, which was consistent with the results of tumor volume measurement. 2 Nanoparticle-mediated chemotherapy combined with photothermal therapy has excellent therapeutic effect on drug-resistant breast cancer.

[0065] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A manganese-based multifunctional nanoparticle for improving tumor hypoxia and reversing doxorubicin resistance, characterized in that: The manganese-based multifunctional nanoparticles are DOX / HSA-MnO2 nanoparticles prepared by using HSA-MnO2 nanoparticles as carriers and loading DOX; the particle size is less than 100 nm, the encapsulation efficiency is 96.56±0.17%, and the drug loading is 31.91±0.86%.

2. A method for preparing manganese-based multifunctional nanoparticles according to claim 1, characterized in that: HSA-MnO2 nanoparticles were first prepared by ultrasonic method, and then DOX was loaded by ultrasonic method to obtain DOX / HSA-MnO2 nanoparticles.

3. The method for preparing the HSA-MnO2 nanoparticle solution according to claim 2, characterized in that: The steps include: 1) Preparation of HSA-MnO2 nanoparticles: HSA solution was added dropwise to KMnO4 solution, and then ultrafiltration was performed for purification after ultrasonic treatment to obtain a solution containing HSA-MnO2 nanoparticles; 2) Preparation of DOX / HSA-MnO2 nanoparticles: The solution containing HSA-MnO2 nanoparticles prepared in step 1) was mixed in deionized water, and then the DOX solution was added dropwise. After ultrasonic treatment, the solution was purified by ultrafiltration to obtain a solution containing DOX / HSA-MnO2 nanoparticles.

4. The method for preparing the HSA-MnO2 nanoparticle solution according to claim, characterized in that: The mass ratio of HSA to KMnO4 used in step 1) is 1.25:1~5:

1.

5. The method for preparing the HSA-MnO2 nanoparticle solution according to claim, characterized in that: The power of the ultrasonic treatment in step 1) is 320 W to 800 W, and the time is 0.5 h to 1.5 h.

6. The method for preparing the HSA-MnO2 nanoparticle solution according to claim, characterized in that: The mass ratio of HSA-MnO2 nanoparticles to DOX used in step 2) is 1:1~6:

1.

7. The method for preparing the HSA-MnO2 nanoparticle solution according to claim, characterized in that: The power of the ultrasonic treatment in step 2) is 320 W to 800 W, and the time is 1 h to 4 h.

8. Use of the manganese-based multifunctional nanoparticles according to claim 1 in the preparation of anticancer drugs, characterized in that: Specifically, anti-breast cancer drugs.

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