Lactoferrin nano-drug as well as preparation method and application thereof

Abstract

The invention discloses a lactoferrin nano-drug as well as a preparation method and application thereof, the lactoferrin nano-drug is prepared from lactoferrin, dihydroartemisinin and hemin, and the mass ratio of the lactoferrin to the dihydroartemisinin to the hemin is 1: 1: 1-30: 1: 1. The lactoferrin is used as a carrier, dihydroartemisinin and hemin enter an internal hydrophobic cavity of the lactoferrin through an anti-solvent method to form a protein nano-drug, and the two drugs cooperate to achieve a more remarkable anti-tumor effect. The dihydroartemisinin is an artemisinin derivative with an active hydroxyl group, the hemin is an in-vitro purified form of natural heme, the lactoferrin is an iron-binding glycoprotein in a transferrin family, and the three substances are non-toxic to normal cells and have a synergistic anti-tumor effect that 1 + 1 is greater than 2 on the action mechanism.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to a lactoferrin nano drug and a preparation method and application thereof. Background Art

[0002] At present, breast cancer has replaced lung cancer as the world's number one cancer. Breast cancer can be treated by surgery, radiotherapy, chemotherapy, endocrine therapy and targeted therapy, but most chemotherapy drugs cause serious damage to normal tissues while killing tumor cells, limiting their clinical application. Nano drug delivery systems can control the delivery and release of drugs at the molecular level, and achieve passive or active targeted release of drugs at the tumor site by improving drug solubility and pharmacokinetic characteristics, thereby enhancing drug efficacy and significantly reducing systemic toxic side effects of drugs; however, the latest clinical data analysis results show that nanoformulations only improve the safety of chemotherapy, and do not significantly improve the anti-tumor effect of chemotherapy drugs. The combined application of chemodynamic therapy with other therapies (such as radiotherapy, photothermal therapy, photodynamic therapy, immunotherapy, etc.) can usually achieve therapeutic effects that cannot be achieved by a single treatment method, and the combined application of different tumor killing mechanisms can overcome multidrug resistance and reduce the dosage of drugs.

[0003] Dihydroartemisinin (DHA) is an inducer of ferroptosis and immunogenic cell death (ICD). Studies have shown that it can induce ferroptosis and regulate immune function to fight tumors. Due to its dual effects of ferroptosis and immunotherapy, DHA has been considered an excellent anti-tumor agent. 2+ It is essential for DHA to exert its therapeutic effect due to the cleavage of the endoperoxide bridges required for DHA. 2+ Extreme consumption in cancer cells is not conducive to DHA destroying the endoperoxide bridge and exerting its therapeutic effect. Another obstacle is that DHA has poor water solubility and a short blood circulation half-life, which greatly limits its application in anti-tumor. Solving the problem of iron deficiency In tumor cells, the water solubility of DHA becomes a key indicator. Solving the problem of Fe 2+ The problem of deficiency in tumor cells and the water solubility of DHA become key issues.

[0004] The present invention is designed to combine hemin with dihydroartemisinin. Hemin is an iron-based porphyrin molecule and a purified form of endogenous heme in vitro. It is decomposed by heme oxygenase (HO-1) in the cytoplasm to release iron ions, providing sufficient Fe for ferroptosis based on the Fenton reaction. 2+ At the same time, dihydroartemisinin can increase the activity of HO-1 and promote Fe 2+ , which can achieve cascade ferroptosis tumor treatment and has potential prospects for clinical translation. Summary of the invention

[0005] One of the purposes of the present invention is to provide a lactoferrin nanomedicine, which is made of lactoferrin, dihydroartemisinin and hemin, wherein the mass ratio of lactoferrin, dihydroartemisinin and hemin is 1:1:1-30:1:1. Preferably, the mass ratio of lactoferrin, dihydroartemisinin and hemin is 6:1:1-14:1:1.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned lactoferrin nanomedicine, wherein dihydroartemisinin and hemin are dissolved in different organic solvents respectively, and then the two solutions are mixed, and the mixed solution is added to the lactoferrin aqueous solution under stirring conditions, and ultrasonicated to obtain the lactoferrin nanomedicine.

[0007] Furthermore, the organic solvent is selected from dimethyl sulfoxide, ethanol, methanol, acetone and tetrahydrofuran.

[0008] Furthermore, the dihydroartemisinin is soluble in ethanol, and the hemin is soluble in dimethyl sulfoxide.

[0009] Furthermore, the volume ratio of the organic solvent to water is 1:1:4-1:1:16.

[0010] Furthermore, the stirring speed is 200-800 r / min, and the ultrasonic conditions are 50-200 W, 3-10 min.

[0011] In one embodiment of the present invention, the mass ratio of dihydroartemisinin, hemin and lactoferrin is 1:1:10, and the volume ratio of anhydrous ethanol, dimethyl sulfoxide and water is 1:1:14.

[0012] The third object of the present invention is to provide the use of the above lactoferrin nanomedicine in the preparation of tumor treatment drugs. Preferably, the tumor is breast cancer.

[0013] The present invention uses lactoferrin as a carrier, and dihydroartemisinin and hemin chloride enter the internal hydrophobic cavity of lactoferrin through an anti-solvent method to form a protein nano-drug, and the synergistic anti-tumor effect of the two drugs is more significant. Dihydroartemisinin is an artemisinin derivative with active hydroxyl groups, hemin chloride is an in vitro purified form of natural heme, and lactoferrin is an iron-binding glycoprotein in the transferrin family. These three substances are non-toxic to normal cells and have a synergistic anti-tumor effect of "1+1 is greater than 2" in terms of the mechanism of action.

[0014] The present invention uses lactoferrin as a carrier of the preparation, which can enhance the solubility and bioavailability of the drug, enhance the biostability and retention, and improve the ability to reach the expected site through active targeted delivery. At the same time, lactoferrin also has certain anti-tumor activity and immunomodulatory function.

[0015] Lactoferrin Inspired by the success of albumin-paclitaxel nanoparticles, recent advances in protein-based nanomedicine have generated new therapeutics for the treatment of challenging diseases such as cancer and viral infections. However, lack of specific drug targeting, poor pharmacokinetics, premature drug release, and off-target toxicity remain formidable challenges in the clinic. Therefore, alternative protein-based nanomedicines have been developed to overcome these challenges. In this regard, lactoferrin (Lf), a glycoprotein of the transferrin family that can specifically recognize overexpressed transferrin receptors on tumor cell membranes, provides a promising biodegradable, well-tolerated material for use as active therapeutics and drug nanocarriers. The main pharmacological effects of Lf include anticancer, antiviral, and immunomodulatory effects.

[0016] Dihydroartemisinin (DHA) and hemin are loaded on lactoferrin. When the nanodrug reaches the tumor tissue, it enters the lysosome in the cancer cell and is degraded, releasing DHA and hemin. Hemin reacts with intracellular GSH (highly expressed in tumor cells) to produce Fe 2+ It can induce Fenton / Fenton-like reactions, which enhance ferroptosis by promoting ROS generation. At the same time, due to GSH depletion, GPX4 activity is inhibited, which further leads to ferroptosis. Afterwards, the endoperoxide bridge of DHA in Fe 2+ There are breaks, which produce toxic carbon-centered free radicals ROS, further inducing ferroptosis. The massive production of ROS also leads to cell apoptosis. Importantly, the two-pronged approach of Hemin / DHA stimulates oxidative stress, leading to high immune activation of ICD. It can promote the maturation of dendritic cells (DCs), enhance the infiltration of CD4 and CD8 T cells, and promote macrophage polarization. In addition, DHA is used as an immunomodulator to suppress Treg cells and stimulate beneficial aspects of the host immune response. DHA can then induce the cancer cell ferroptosis pathway by inhibiting GPX4, while heme, an important component for transporting oxygen in the blood, can be broken down by heme oxygenase-1 (HO-1), thereby increasing the intracellular Fe2+ concentration to further induce ferroptosis of cancer cells. At the same time, DHA can enhance the activity of HO-1, further promoting Fe from Hemin. 2+ Therefore, the present invention provides a cascade ferroptosis tumor treatment strategy for potential clinical application.

[0017] Beneficial Effects

[0018] (1) The nanoparticles of the present invention are a completely new concept, namely, the lactoferrin carrier nanoparticles provided by the present invention. The combined use of dihydroartemisinin and hemin has the advantage of not only being able to actively transport to tumor tissues by utilizing the targeting effect of lactoferrin, but also being able to achieve responsive drug release in tumor tissues, thereby achieving multi-mechanism tumor killing.

[0019] (2) The self-assembled nanoparticles have a uniform particle size of 90-100 nm and can exist stably in the blood.

[0020] (3) It has tumor targeting, specifically recognizes the transferrin receptor overexpressed on the tumor cell membrane, and then enters the lysosome of the tumor cell through endocytosis. Lactoferrin nanoparticles are further hydrolyzed in the lysosome to release hemin and dihydroartemisinin. At pH = 7.4, only 4% dihydroartemisinin and 8% hemin are released after 24 hours of lactoferrin nanoparticles. At pH = 4.5, 85% dihydroartemisinin and 90% hemin are released after 24 hours.

[0021] (4) The dihydroartemisinin and hemin contained in the lactoferrin nanoparticles can achieve a synergistic anti-tumor effect of chemodynamic therapy and immunotherapy. The tumor volume and weight of mice administered with lactoferrin nanoparticles were significantly smaller than those of mice administered with dihydroartemisinin and hemin alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Graph showing the particle size and potential of lactoferrin nanoparticles in Example 2 of the present invention.

[0023] Figure 2 This is a transmission electron micrograph of the lactoferrin nanoparticles in Example 2 of the present invention.

[0024] Figure 3 This is the circular dichroism spectrum of lactoferrin nanoparticles in Example 3 of the present invention.

[0025] Figure 4 Graphs a and b are graphs of the particle size-time variation of the nanoparticles in Example 4 of the present invention in physiological solution, wherein graphs a and b are graphs of the particle size-time variation of the nanoparticles in Example 1 of the present invention in physiological saline and PBS solution containing 10% serum, respectively.

[0026] Figure 5 1 is the drug release diagram of the nanoparticles in Example 5 of the present invention under different pH conditions, wherein a and b are the drug release diagrams of dihydroartemisinin and hemin under different pH conditions, respectively.

[0027] Figure 6Graphs showing in vitro ROS generation of different preparations in Example 6 of the present invention, wherein a is a graph showing the degradation of methylene blue when the preparations are incubated under different pH conditions for 24 h; b is a graph showing the degradation of methylene blue when the preparations are incubated with different concentrations of H2O2 for 24 h; and c is a graph showing the degradation of methylene blue when the preparations of different concentrations are incubated with the same concentration of H2O2 for 0, 4, 8, 12, and 24 h.

[0028] Figure 7 Graphs showing in vitro GSH depletion of different preparations in Example 7 of the present invention, a showing GSH depletion when the preparations were incubated under different pH conditions for 24 h; b showing GSH depletion when the preparations were co-incubated with the same concentration of GSH at 0, 2, 4, 8, 12, 24, and 48 h.

[0029] Figure 8 : This is a graph showing the effects of different preparations on transferrin receptor expression in Example 8 of the present invention, a is a graph showing the effects of different preparations on transferrin receptor expression, b is a graph showing the effects of preparations on transferrin receptor expression in different cells, c is a graph showing the effects of preparations of different concentrations on transferrin receptor expression, and d is a graph showing the effects of transferrin receptor expression at different times.

[0030] Fig. 9 These are the cytotoxicity graphs of different preparations in Example 9 of the present invention, wherein a is the cytotoxicity graph of each preparation on 4T1 cells; b is the cytotoxicity graph of each preparation on 3T3 cells; and c is the cytotoxicity graph of each preparation on MCF-7 cells.

[0031] Fig.10 These are the cytotoxicity graphs in Example 10 of the present invention, a is the cytotoxicity graph of the preparation on 4T1 cells after adding the Z-VAD-FMK inhibitor; b is the cytotoxicity graph of the preparation on 4T1 cells after adding the DFO inhibitor; c is the cytotoxicity graph of the preparation on 4T1 cells after adding the Fer-1 inhibitor; d is the cytotoxicity graph of the preparation on 4T1 cells after adding the Lip-1 inhibitor.

[0032] Fig.11 Graph showing the effects of different preparations on ATP levels in 4T1 cells in Example 11 of the present invention, a is a graph showing intracellular ATP content, and b is a graph showing extracellular ATP content.

[0033] Fig.12 This is a graph showing the effects of different preparations in Example 12 of the present invention on the HMGB1 level in 4T1 cells.

[0034] Fig.13 This is a time chart of the uptake of the preparation by 4T1 cells in Example 13 of the present invention.

[0035] Fig.14 This is a graph showing the effects of different preparations in Example 14 of the present invention on the lipid peroxidation level of 4T1 cells.

[0036] Fig.15 This is a graph showing the effects of different preparations in Example 15 of the present invention on the expression of different proteins in 4T1 cells.

[0037] Fig.16 These are in vivo anti-tumor experimental diagrams of different preparations in Example 16 of the present invention, wherein a, b, c, and d are mouse body weight, mouse survival curve, tumor volume diagram, and tumor actual photograph, respectively. DETAILED DESCRIPTION

[0038] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0040] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0041] The reagents (kits) used for performance testing of nanomedicines included: TFR1 detection kit, CCK-8 kit, ATP detection kit, MDA kit, HMGB1 detection kit, and BCA protein detection kit, which were purchased from Beyotime (Shanghai, China). C11-BODIPY581 / 591 was purchased from Thermo Fisher Scientific. 3T3, 4T1, MCF-7, and HT-29 cells were purchased from Wuhan Pronocell Life Science Co., Ltd. Female BALB / c mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. Unless otherwise specified, all reagents were of analytical grade and used without further purification.

[0042] Material characterization: Transmission electron microscopy (TEM) imaging was performed on a JEM-2100 field emission transmission electron microscope (Hitachi, Japan). The particle size and potential test of nanomedicine were performed on a nanoparticle size potentiometer (Zetasizer Nano ZS90, Malvern Panalytical). UV-visible absorption spectra were recorded by a UV-visible spectrophotometer (Cary 60, Agilent Technologies). The absorbance in the CCK-8 assay was measured on an ELISA reader (EP0CH2, China). Flow cytometry analysis was performed on a flow cytometer (Cytoflex, A00-1-1102). Cell images were measured by a FV3000 laser confocal microscope.

[0043] Example 1

[0044] Optimal formulation of lactoferrin nanoparticles

[0045] Preparation of lactoferrin nanoparticles: Weigh 30 mg lactoferrin (LF) and dissolve it in 7 mL ultrapure water, place it in a refrigerator overnight to fully dissolve it, which is the antisolvent phase; ultrasonically dissolve 3 mg dihydroartemisinin (DHA) in 0.5 mL anhydrous ethanol, and ultrasonically dissolve 3 mg hemin (Hemin) in 0.5 mL dimethyl sulfoxide, and mix the two to form the solvent phase. Use a syringe to slowly drip the solvent phase into the lactoferrin solution in a magnetic stirrer (400 r / min) at a rate of 1 mL / min, and then homogenize the obtained nanoparticles on a probe sonicator (power 100 W, amplitude 10%, open 2s, close 3s, cycle 3min). Finally, centrifuge at 4000 r / min for 10 min to separate free dihydroartemisinin and hemin, and the supernatant passes through a 0.45 μm microporous filter to obtain a lactoferrin nanoparticle solution.

[0046] Referring to the above preparation method, the volume ratio of solvent to anti-solvent, stirring speed, ultrasonic power, ultrasonic time, and drug concentration ratio were respectively optimized, and the screening conditions were the encapsulation rate of the two drugs and the particle size of the preparation.

[0047] 1. Volume ratio of solvent to antisolvent

[0048] When the volume ratios of ethanol, dimethyl sulfoxide and ultrapure water were 1:1:4, 1:1:8, 1:1:10, 1:1:12, 1:1:14 and 1:1:16 respectively, the lactoferrin nanoparticles were prepared under the conditions of a stirring speed of 200 r / min, an ultrasonic power of 50 w and an ultrasonic time of 10 minutes.

[0049] When the volume ratio of ethanol, dimethyl sulfoxide and ultrapure water is 1:1:4, 1:1:8, 1:1:10, 1:1:12, 1:1:14 and 1:1:16, lactoferrin nanoparticles can be formed, and their encapsulation efficiency and particle size distribution are shown in Table 1.

[0050] Table 1 Investigation of antisolvent / solvent volume ratio

[0051]

[0052] 2. Stirring speed

[0053] According to the screening, the volume ratio of the solvent to the anti-solvent was fixed at 1:1:14, and under the conditions of an ultrasonic power of 50w and an ultrasonic time of 10 minutes, different stirring speeds (200r / min, 400r / min, 600r / min, 800r / min) were examined to prepare the lactoferrin nanoparticles.

[0054] When the magnetic stirring speed is 200, 400, 600, and 800 r / min, lactoferrin nanoparticles can be formed, and their encapsulation efficiency and particle size distribution are shown in Table 2.

[0055] Table 2 Stirring speed investigation

[0056]

[0057] 3. Ultrasonic power

[0058] According to the screening, the volume ratio of the solvent to the anti-solvent was fixed at 1:1:14, and under the conditions of a stirring speed of 400 r / min and an ultrasonic time of 10 minutes, different ultrasonic powers (50 W, 100 W, 150 W, 200 W) were examined to prepare the lactoferrin nanoparticles.

[0059] When the ultrasonic power is 50W, 100W, 150W, and 200W, lactoferrin nanoparticles can be formed, and their encapsulation efficiency and particle size distribution are shown in Table 3.

[0060] Table 3 Ultrasonic power investigation

[0061]

[0062] 4. Ultrasound time

[0063] According to the screening, the volume ratio of the solvent to the anti-solvent was fixed at 1:1:14, and under the conditions of a stirring speed of 400 r / min and an ultrasonic power of 100 W, different ultrasonic times (0 minutes, 3 minutes, 5 minutes, and 10 minutes) were investigated to prepare the lactoferrin nanoparticles.

[0064] When the ultrasonic time was 0, 3, 5, and 10 minutes, lactoferrin nanoparticles were formed, and their encapsulation efficiency and particle size distribution were shown in Table 4.

[0065] Table 4 Ultrasound time investigation

[0066]

[0067] 5. Drug concentration ratio

[0068] According to the screening, the volume ratio of solvent to antisolvent was fixed at 1:1:14, and under the conditions of stirring speed of 400 r / min, ultrasonic power of 100 W, and ultrasonic time of 3 minutes, different protein to drug ratios (6:1:1, 8:1:1, 10:1:1, 12:1:1, 14:1:1) were investigated to prepare the lactoferrin nanoparticles.

[0069] When the ratio of protein to drug was 6:1:1, 8:1:1, 10:1:1, 12:1:1, and 14:1:1, lactoferrin nanoparticles were formed, and their encapsulation efficiency and particle size distribution were shown in Table 5.

[0070] Table 5 Investigation of protein drug ratio

[0071]

[0072] Example 2

[0073] Preparation of lactoferrin nanoparticles

[0074] Weigh 30 mg of lactoferrin (LF) and ultrasonically dissolve it in 7 mL of ultrapure water. Place it in a refrigerator overnight to fully dissolve it, which is the antisolvent phase; ultrasonically dissolve 3 mg of dihydroartemisinin (DHA) in 0.5 mL of anhydrous ethanol, and ultrasonically dissolve 3 mg of hemin in 0.5 mL of dimethyl sulfoxide, and mix the two to form the solvent phase. Use a syringe to slowly drip the mixed solution into the lactoferrin solution in a magnetic stirrer (400 r / min) at a rate of 1 mL / min, and then homogenize the prepared nanoparticles on a probe sonicator (power 100 W, amplitude 10%, on 2 s, off 3 s, cycle 3 min). Finally, centrifuge at 4000 r / min for 10 min to separate free dihydroartemisinin and hemin, and pass the supernatant through a 0.45 μm microporous filter to obtain a DHA / Hemin@LFNPs solution.

[0075] Blank LFNPs were prepared in the same manner except that dihydroartemisinin and hemin were not added.

[0076] DHA@LFNPs and Hemin@LFNPs can be prepared by using only dihydroartemisinin solution or hemin solution as the solvent phase.

[0077] like Figure 1 As shown, the prepared nanoparticles have a particle size of about 100 nm and a negative charge, which can effectively avoid being cleared by the human body and have stronger penetrability.

[0078] The appearance of DHA / Hemin@LFNPs was observed by transmission electron microscopy. The DHA / Hemin@LFNPs nanoparticles were uniform in shape and spherical in appearance. Figure 2 shown.

[0079] Example 3

[0080] Investigation of targeting structure of lactoferrin nanoparticles

[0081] This example is a method for characterizing protein structure by utilizing the different absorption of left and right plane circular polarized light by optically active chromophores in protein molecules. The lactoferrin nanoparticles prepared in Example 2 were dispersed in 10 mL PBS (pH = 7.4), and commercial lactoferrin was dissolved in 10 mL PBS (pH = 7.4). The results were scanned by magnetic circular dichroism spectrometer. Figure 3As shown, there is no obvious change in the secondary structure of lactoferrin formed into nanoformulation and normal lactoferrin, indicating that the biological activity of lactoferrin nanoparticles is normal and can play a complete role.

[0082] Example 4

[0083] Stability of lactoferrin nanoparticles

[0084] 2 mL of lactoferrin nanoparticles prepared in Example 2 were dispersed in 4 mL of physiological saline (pH=7.4) and PBS (pH=7.4) containing 10% serum, and the particle size and PDI of the nanoparticles were monitored by a Malvern laser particle size analyzer. Figure 4 Figures a and b show the particle size changes of lactoferrin nanoparticles in physiological saline and PBS containing 10% serum at different times, respectively, confirming the stability of lactoferrin nanoparticles in a simulated physiological environment.

[0085] Example 5

[0086] In vitro drug release of lactoferrin nanoparticles under different conditions

[0087] This example uses dynamic dialysis to investigate the release behavior of drug-loaded nanoparticles. 5 mL of nanoparticle preparation was accurately measured and placed in a dialysis bag (molecular weight cutoff: 3500 Da), the mouth was tightened, and 25 mL of PBS containing 0.5% (w / m) Tween 80 with pH values ​​of 7.4, 6.5, and 5.0 was put into the bag, and the bag was shaken at 37°C ± 0.5°C and 100 r / min. 1 mL was sampled at 0, 1, 2, 4, 6, 8, 12, and 24 h, and fresh release medium of the same volume and temperature was added at the same time. The sample was filtered through a 0.45 μm microporous membrane, the primary filtrate was discarded, and 20 μL of the subsequent filtrate was taken. The DHA / Hemin content was determined by HPLC, and the cumulative release percentage was calculated.

[0088] like Figure 5 As shown, a is the cumulative release of dihydroartemisinin at pH = 7.4 after 24 hours, which is less than 20%, while at pH = 5.0 after 24 hours, the cumulative release is 80%; b is the cumulative release of hemin at pH = 7.4 after 24 hours, which is less than 40%, while at pH = 5.0 after 24 hours, the cumulative release is 90%. This confirms that lactoferrin nanoparticles are pH responsive.

[0089] Example 6

[0090] Investigation of ROS generation in vitro by lactoferrin nanoparticles

[0091] In this example, methylene blue (MB) was used to detect the generation of ROS in vitro. Under acidic conditions, ·OH produced by H2O2 under the catalysis of ferrous ions can degrade methylene blue. Buffer solutions containing 100 mM H2O2 at pH = 7.4, 6.5, and 5.0 were prepared and incubated with 2 mL of the preparation for 24 h, respectively. The supernatant was collected by centrifugation, and the ·OH-induced degradation of methylene blue was measured by scanning the ultraviolet absorption of the supernatant at 590 nm-730 nm with an ultraviolet spectrophotometer. The degradation results of methylene blue are shown in FIG. Figure 6 As shown in a. 100, 200, 300, 400, 500 mM H2O2 buffer solutions at pH = 5.0 were prepared and incubated with 2 mL of the preparation for 24 h, respectively. The supernatant was collected by centrifugation, and the ·OH-induced degradation of methylene blue was measured by scanning the ultraviolet absorption of the supernatant at 590 nm-730 nm using an ultraviolet spectrophotometer. The degradation results of methylene blue are shown in Figure 6 As shown in b. A buffer solution containing 100 mM H2O2 at pH = 5.0 was prepared and incubated with 7 mL of DHA preparations at 50, 100, and 200 μg / mL concentrations, respectively. 1 mL of the supernatant was collected by centrifugation at 0, 4, 8, 12, and 24 h, and the supernatant was scanned by ultraviolet spectrophotometer at 590 nm-730 nm to measure the degradation of methylene blue induced by ·OH. The degradation results of methylene blue are shown in Figure 6 As shown in c. Under acidic conditions, H2O2 basically does not degrade MB. Under acidic conditions, it reacts with Fe in hemin. 3+ Reactive oxygen species are produced under catalysis to degrade MB; different concentrations of H2O2 produce different concentrations of reactive oxygen species to degrade MB, and the concentration of dihydroartemisinin affects the rate of MB degradation.

[0092] Example 7

[0093] Study on GSH depletion of different preparations in vitro

[0094] This embodiment uses 2-nitrobenzoic acid (DTNB) to detect GSSG generated by GSH reaction. The detection principle is that the reduced glutathione (GSH) in the sample to be tested reacts with the chromogenic substrate DTNB to produce stable yellow 2-nitro-5-mercaptobenzoic acid (TNB) and glutathione disulfide (GSSG), 2-nitro-5-mercaptobenzoic acid is a yellow product with maximum light absorption at a wavelength of 412nm. The absorbance at 412nm is measured by spectrophotometry (spectrophotometer) and compared with the corresponding treated GSH standard to obtain the GSH content of the sample. 0.01mol / L DTNB is dissolved in 0.05mol / L phosphate buffer (pH7.4) to prepare a DTNB standard solution, which is placed away from light and used immediately after preparation. Accurately weigh an appropriate amount of glutathione standard, dissolve it in a volumetric flask with deionized water, mix well, and make a standard product by fixed volume. Prepare buffer solutions with pH=7.4, 6.5, and 5.0 containing 100 mM GSH, and incubate with 2 mL of the preparation of Example 2 for 24 h, respectively. Collect the supernatant by centrifugation, add the same volume of DTNB standard solution, react for 30 min, and measure the absorbance by scanning the ultraviolet absorption of the reaction solution at 412 nm with an ultraviolet spectrophotometer. The GSH degradation results are shown in the figure. Figure 7 As shown in a. Under acidic conditions, Fe 3+ The reaction with GSH consumes GSH to generate GSSG. Similarly, the same GSH standard solution was prepared and incubated with 7 mL of the preparation. 1 mL of the sample was collected by centrifugation at 0, 2, 4, 8, 12, 24, and 48 h, and the supernatant was added with the same volume of DTNB standard solution. The reaction was continued for 30 min, and the absorbance was measured by scanning the ultraviolet absorption of the reaction solution at 412 nm with an ultraviolet spectrophotometer. The results are shown in Figure 7 As shown, the preparation depleted GSH in a time-dependent manner.

[0095] Example 8

[0096] Effects of different preparations on transferrin receptor expression

[0097] 4T1, 3T3, MCF-7, and HT-29 cells in the logarithmic growth phase were seeded in a 96-well plate at a density of 3000 cells / well and cultured until the cell confluence reached 80%. The free DHA, DHA nanoformulation, and final nanoparticle solution prepared in Example 2 were added to each well. The DHA content after dilution was used as the standard, and the DHA concentrations in Figure d were 37.5 μg / mL, respectively. Three replicate wells were set up, with 200 μL in each well. After 24 hours of incubation, proteins were extracted from the cells using RIPA cell lysis buffer (Sevier, China). After lysis, centrifuge at 4°C and 12000g for 10 minutes, and take the supernatant. In order to eliminate the error caused by the difference in protein amount during sample preparation, the protein concentration was determined according to the instructions of the BCA protein detection kit, and the protein concentration was determined according to the instructions of the TRF1 detection kit. The results are as follows Figure 8 As shown in a, the preparation can increase the expression of transferrin receptor on the surface of cancer cells by about 1 times, verifying whether the preparation has the same effect on different cancer cells. The results of administering different preparations to different cells are shown in Figure 8 As shown in b, different preparations have no effect on normal cells, but have the same effect on different cancer cells, indicating that it is mainly caused by DHA. In order to verify whether different time has an effect on lactoferrin receptor expression, the expression of transferrin receptor was measured after 12, 24, and 48 hours of administration. The results are shown in Figure 8 As shown in Figure c, there is no significant difference between different time periods, indicating that transferrin receptor expression will not change over time after reaching a certain level. At the same time, in order to verify the effect of the concentration of the preparation, different concentrations of preparations were given for 24 hours, and the test results are shown in Figure 5. Figure 8 As shown in middle d, the expression of transferrin receptor was concentration-dependent and stabilized after reaching a certain concentration.

[0098] Example 9

[0099] Cytotoxicity of different agents on cancer cells

[0100] 4T1, 3T3, MCF-7 cells in logarithmic growth phase are seeded in 96-well plates with a density of 3000 / well, and cultured to a cell confluence of 80%. Nanoparticles and free drugs prepared by embodiment 2, free drug mixture and free lactoferrin solution are added in each hole, with DHA content after dilution as standard, respectively 3.125, 6.25, 12.5, 25, 50, 100 μ g / mL, three multiple holes are set, 200 μ L in every hole, after 20h hatching, MTT solution (5mg / mL) 20 μ L is added in every hole, and 37 ℃ continue to hatch 4h. Discard supernatant subsequently, add 200 μ L DMSO to dissolve, measure absorbance at 492nm with microplate reader, measure the toxicity of nanoparticles to 4T1, 3T3, MCF7 cells.

[0101] The results are as follows Fig. 9 As shown, the toxicity results show that the cytotoxicity of the free drug mixture to 4T1 and MCF-7 cells is significantly stronger than that of the free drug alone, and the cytotoxicity of the nanoformulation is stronger than that of the free drug mixture, confirming the strong tumor killing effect of the nanoformulation. At the same time, it can be seen that the cytotoxicity is concentration-dependent, and each preparation is basically non-toxic to 3T3 cells.

[0102] Example 10

[0103] Effect of adding different inhibitors on the cytotoxicity of the preparation

[0104] 4T1 cells in the logarithmic growth phase were inoculated in 96-well plates at a density of 3000 cells / well and cultured until the cell confluence reached 80%. The nanoparticles prepared in Example 2 were added to each well, and three duplicate wells were set, 200 μL per well. After 4 hours of incubation, 20 μL of free inhibitors of different concentrations were added to each well, including pan-caspase inhibitors (Z-VAD-FMK), iron chelators (DFO), ferroptosis inhibitors (Fer-1), and lipstatin-1 (Lip-1). After 20 hours of incubation, 20 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated for 4 hours at 37°C. The supernatant was then discarded, 200 μL of DMSO was added to dissolve, and the absorbance was measured at 492 nm with a microplate reader to determine the toxicity of the nanoparticles to 4T1 cells.

[0105] The results are as follows Fig.10 As shown, the toxicity results showed that there was no significant change in the toxicity to 4T1 cells after the addition of the apoptosis inhibitor Z-VAD-FMK. The addition of different ferroptosis inhibitors showed that the cytotoxicity of the nanoformulation was significantly reduced and showed concentration dependence. The nanoformulation killed tumor cells mainly through ferroptosis rather than apoptosis.

[0106] Embodiment 11

[0107] Effects of different preparations on ATP levels in tumor cells

[0108] 4T1 cells in the logarithmic growth phase were cultured at 5×10 6The density of each well was inoculated in a 6-well plate and cultured until the cell confluence reached 80%. The nanoparticles prepared in Example 2 and the free drug, free drug mixture and free lactoferrin solution were added to each well, and the DHA content after dilution was 37.5 μg / mL as the standard, and the final nanoparticle solution was added to each well, and the DHA content after dilution was 37.5 μg / mL as the standard, and added to each well, three duplicate wells were set, 200 μL per well, and after 24h incubation, the cells were collected and centrifuged, and the supernatant was detected and RIPA cell lysis solution (Saiweier, China) was used to lyse the cells. After lysis, 4 ° C 12000g was centrifuged for 10 minutes, and the supernatant was taken. In order to eliminate the error caused by the difference in protein amount during sample preparation, the protein concentration was determined by the BCA protein detection kit specification, and the ATP concentration was determined by the ATP detection kit specification.

[0109] The results are as follows Fig.11 As shown, the release of ATP from dying cells into the extracellular space constitutes one of the main signs of ICD. It can be seen that free dihydroartemisinin can cause ATP to be released extracellularly. The effect of the preparation group is significantly better than that of the free dihydroartemisinin group, proving that the preparation has a more significant effect on ICD, and has obvious advantages in inducing the anti-tumor pathway of ICD through ferroptosis.

[0110] Example 12

[0111] Effects of different preparations on HMGB1 levels in tumor cells

[0112] 4T1 cells in the logarithmic growth phase were cultured at 5×10 6 The cells were inoculated in a 6-well plate at a density of 1000 μg / well and cultured until the cell confluence reached 80%. The nanoparticles prepared in Example 2, free drugs, free drug mixtures and free lactoferrin solutions were added to each well, and the DHA content after dilution was 37.5 μg / mL as the standard, and added to each well. Three duplicate wells were set, and each well was 200 μL. After 24 hours of incubation, the cell samples were centrifuged and the supernatant was taken (500 × g, 5 minutes). At the same time, RIPA cell lysis buffer (Sevier, China) was used to lyse the cells. After lysis, 4 ° C 12000g was centrifuged for 10 minutes, and the supernatant was taken. In order to eliminate the error caused by the difference in protein amount during sample preparation, the protein concentration was determined according to the instructions of the BCA protein detection kit, and the HMGB1 concentration of the cell sample supernatant was determined according to the instructions of the HMGB1 detection kit.

[0113] The results are as follows Fig.12As shown in the figure, the release of high mobility group protein 1 (HMBG1) is also one of the main signs of ICD. It can be seen that free dihydroartemisinin can cause the release of HMBG1 into the extracellular space, while there is no significant difference between the free hemin group and the control group. The effect of the preparation group is significantly better than that of the free dihydroartemisinin group, indicating that the combined use of the two drugs significantly enhanced the anti-tumor effect.

[0114] Embodiment 13

[0115] Uptake of nanomedicine by 4T1 cells

[0116] The therapeutic effect of nanomedicine depends on rapid uptake and effective accumulation in cells. Therefore, the cellular uptake of C6-labeled NPs (the preparation method of C6-labeled NPs is the same as that of Example 2, except that DHA is replaced by Cy5.5-labeled DHA) was evaluated by laser scanning confocal microscopy (LSCM). As time goes by, the green fluorescence intensity gradually increases, indicating that cellular uptake is time-dependent. Due to the acidity of the tumor microenvironment, pH-sensitive NPs will disintegrate after entering tumor cells, and the subsequent release of DHA and Hemin can cascade amplify LPO, leading to tumor cell apoptosis.

[0117] The results are as follows Fig.13 As shown, Figure a shows different preparations at the same time, and the uptake of the preparations is significantly better than that of the free drug. Figure b shows the uptake of the preparations at different times, and the preparations show time dependence.

[0118] Embodiment 14

[0119] Effects of different preparations on lipid peroxidation levels in 4T1 cells

[0120] 4T1 cells in the logarithmic growth phase were inoculated in a 12-well plate at a density of 10,000 cells / well and cultured until the cell confluence reached 80%. The nanoparticles prepared in Example 2, free drugs, free drug mixtures and free lactoferrin solutions were added to each well, and the DHA content after dilution was 37.5 μg / mL as the standard. Three replicate wells were set, 1 mL per well, and incubated for 24 hours. The culture medium was removed, and the cells were washed twice with PBS, incubated in a serum-free medium containing 2 μM C11 BODIPY 581 / 591 in dark at 37°C for 30 minutes, and the cells were washed twice with PBS, incubated in a medium containing 2 μM DAPI in dark at 37°C for 10 minutes, and the cells were washed twice with PBS. Finally, the fluorescence imaging results were obtained using a confocal laser scanning microscope.

[0121] like Fig.14As shown. The weaker the fluorescence intensity of the reduced dye in C11 BODIPY 581 / 591, the stronger the degree of cellular lipid peroxidation. Free hemin has little effect on lipid peroxidation in 4T1 cells. When DHA is used in combination with Hemin, it can significantly increase the degree of lipid peroxidation in 4T1 cells. This is because it produces ROS with stronger oxidative activity under the catalysis of ferrous ions. ROS further oxidizes lipids in cells, and the free radicals generated by DHA and ferrous ions can also cause lipid peroxidation. The stronger uptake of nanoparticles by 4T1 cells leads to a stronger degree of lipid peroxidation in the preparation than a simple free drug mixture.

[0122] Embodiment 15

[0123] Effects of different preparations on the expression of different proteins in 4T1 cells

[0124] Protein blot analysis: Western blot analysis was used to determine the effects of different preparations on protein expression in the GPX4, PERK, IRP1, and Nrf2 pathways. Proteins were extracted from cells using RIPA cell lysis buffer (Solebo, China). The protein concentration was determined according to the instructions of the BCA protein detection kit. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to electrophoresed the relevant equal amounts of proteins, and the proteins were transferred to a PVDF membrane after the electrophoresis. A blocking solution containing skimmed milk powder was applied to the membrane, and then incubated with diluted anti-GPX4 primary antibody overnight at 4°C. The membrane was washed quickly and repeatedly, and incubated with the diluted corresponding secondary antibody (1:5000) at room temperature for 30 minutes. The ECL chemiluminescence detection kit was used for color development. The rest of the protein blot analysis was the same as above, and the results are shown in the figure. Fig.15As shown in the results, when the antioxidant systems such as GPX4, FTH, and xCT in the cell are inhibited, ferroptosis occurs, and the preparation causes a significant decrease in the expression of FTH, XCT, and GPX4 proteins. The production of intracellular ROS destroys the structure of the mitochondrial-associated endoplasmic reticulum membrane (MAM), leading to the upregulation of the PERK-mediated eukaryotic translation initiation factor 2α (eIF2α) phosphorylation pathway (p-eIF2α), and the expression of downstream proteins further aggravates the endoplasmic reticulum damage, thereby releasing a large amount of DAMPs (related damage molecules) such as ATP, which ultimately leads to cell immunogenic death. Inducing ICD, the preparation causes upregulation of eIF2α, p-eIF2α and ATF4. The preparation produces ROS, which leads to increased expression of IRP1, further stimulating downstream proteins, FPN and TFR1, so that TFR1 increases on the surface of cancer cells, creating a positive cycle for the uptake of the preparation. Nrf2 and keap1, as a key transcription factor for regulating antioxidant stress, play an important role in inducing the body's antioxidant response. Increased expression of Nrf2 and keap1 enhances antioxidant activity, thereby reducing intracellular ROS. The preparation leads to decreased expression of Nrf2 and keap1, destroying the antioxidant capacity of cancer cells and killing them.

[0125] Example 16

[0126] In vivo antitumor experiments of different preparations

[0127] 4T1 cell suspension (1×10 6 cells / 100 μL) were inoculated under the fourth mammary fat pad of female Balb / C mice. 3 At the same time, tumor-bearing mice were randomly divided into five groups each, and physiological saline, hemin, dihydroartemisinin, a physical mixture of hemin and dihydroartemisinin, and lactoferrin nanoparticles prepared in Example 2 were administered respectively. The administration was once every other day for 6 consecutive times. The dosage was 10 mg / kg based on dihydroartemisinin. The tumor inhibition effect was shown in FIG. Fig.16 Compared with the control group, free DHA and Hemin failed to significantly inhibit tumor growth; the combination of free Hemin and DHA had a stronger inhibitory effect on tumor growth by inducing cell ferroptosis in tumor cells; the nanoformulation had the strongest inhibitory effect on tumor growth by combining chemodynamic therapy and immunotherapy.

Claims

1. A lactoferrin nanomedicine, characterized in that: It is made of lactoferrin, dihydroartemisinin and hemin, and the mass ratio of lactoferrin, dihydroartemisinin and hemin is 1:1:1-30:1:

1.

2. The lactoferrin nanomedicine according to claim 1, characterized in that The mass ratio of lactoferrin, dihydroartemisinin and hemin is 6:1:1-14:1:

1.

3. The method for preparing the lactoferrin nano drug according to claim 1 or 2, characterized in that: Dihydroartemisinin and hemin are dissolved in different organic solvents respectively, and then the two solutions are mixed. The mixed solution is added to the lactoferrin aqueous solution under stirring conditions, and ultrasonicated to obtain the lactoferrin nanomedicine.

4. The preparation method according to claim 3, characterized in that: The organic solvent is selected from dimethyl sulfoxide, ethanol, methanol, acetone and tetrahydrofuran.

5. The preparation method according to claim 4, characterized in that: The dihydroartemisinin is soluble in ethanol, and the hemin is soluble in dimethyl sulfoxide.

6. The preparation method according to claim 3, characterized in that: The volume ratio of the organic solvent to water is 1:1:4-1:1:

16.

7. The preparation method according to claim 3, characterized in that: The stirring speed is 200-800 r / min, and the ultrasonic conditions are 50-200 W, 3-10 min.

8. Use of the lactoferrin nanomedicine according to claim 1 or 2 in the preparation of tumor therapeutic drugs.

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