Targeted mPDA / MSNs nano-drug delivery carrier for inducing ferroptosis as well as preparation method and application of targeted mPDA / MSNs nano-drug delivery carrier

Abstract

The invention discloses a ferroptosis-inducing targeted mPDA / MSNs nano-drug delivery carrier as well as a preparation method and application thereof, and relates to the technical field of preparation of targeted nano-delivery carriers. The preparation method comprises the following steps: firstly, connecting and modifying-NH2 to the surfaces of MSNs to obtain amino modified MSNs-NH2; the preparation method comprises the following steps: coating with mPDA to obtain mPDA / MSNs-NH2, and reacting with ferrous lactate to obtain Fe < 2 + >-mPDA / MSNs-NH2; the final carrier RGD-Fe2 +-mPDA / MSNs-NH2 is prepared by coupling an RGD targeting peptide, the particle size is uniform, the physicochemical property is stable, the ATRA and DOX encapsulation efficiency and the drug loading capacity are high, the drug can be responsively released in a simulated tumor microenvironment, a drug-loaded sample RFP / MN atD / A can deliver a drug into tumor cells in a targeting manner, ferroptosis can be rapidly induced, and the tumor cells can be effectively killed through synergistic chemotherapy.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of targeted nano-delivery carriers, and more specifically to a targeted mPDA / MSNs nano-drug delivery carrier that induces ferroptosis, and a preparation method and application thereof. Background Art

[0002] It has been found that the mechanisms of ferroptosis include iron metabolism, lipid metabolism, antioxidant barrier, etc. Iron metabolism refers to the fact that excess iron can promote the generation of free radicals and lipid peroxidation, thereby inducing cell death. Iron metabolism is the main mechanism of ferroptosis, and intervention in the iron metabolism process can effectively promote or inhibit the occurrence of ferroptosis. Cancer cells have a much higher demand for iron than normal cells or benign tumor cells, and excess iron usually quickly induces ferroptosis. Therefore, cancer cells are more susceptible to ferroptosis than benign tumor cells or normal cells.

[0003] In the field of tumor treatment, the use of single therapy, especially chemotherapy, is usually ineffective. The tumor microenvironment (TME) is closely related to the development of tumors, and is mainly composed of tumor cells, immune cells, stromal cells and extracellular matrix. Some studies have shown that the infiltration of immune effector cells in TME is usually not sufficient to induce an effective anti-tumor response, and the presence of a large number of immunosuppressive cells and factors seriously hinders the role of the immune system. It is difficult to achieve the expected therapeutic effect by simply relying on immunotherapy to fight the complex and changeable TME. Based on this, the use of new multi-strategy synergistic treatment methods to inhibit tumor growth and proliferation in different mechanisms and ways can achieve the complementary advantages of various therapies, such as combining ferroptosis with immunotherapy, and adding the assistance of chemotherapy drugs to achieve the regulation of TME through the synergistic effect of the three, thereby producing a stronger anti-tumor effect. It is very difficult to achieve the synergistic effect of ferroptosis, chemotherapy and immunotherapy, which requires the development of a practical functionalized collaborative platform. Nanoparticles, due to their series of significant advantages, can be used as a delivery carrier for chemotherapeutic drugs and other diagnostic and therapeutic agents, providing a platform for efficient application of multi-strategy synergistic treatment. For example, mesoporous silica nanoparticles (MSNs) have unique properties such as regular pore size, high specific surface area, large pore size, and biodegradability, and have been widely studied and applied in many biomedical fields such as catalysis, adsorption, and sensing. In addition, physical or chemical surface modification on the surface of MSNs can enhance its biocompatibility and inhibit specific adsorption, providing the necessary functional groups for further coupling of biomolecules or modifiers. Based on the above advantages, it is possible to explore loading chemotherapeutic drugs and immune inducers into MSNs to achieve simultaneous and efficient delivery; then Fe 2+ Chelated on the outer surface, it promotes ferroptosis and achieves a synergistic anti-tumor effect of multiple therapies.

[0004] Dopamine is a neurotransmitter secreted by the human brain. It is also widely present in the foot silk protein secreted by mussels. It can be oxidized and self-polymerized to generate polydopamine (mPDA) in a weakly alkaline and aerobic environment. mPDA has adhesion, strong hydrophilicity and high biocompatibility. It can be conformally coated on a material substrate of almost any material and size. In the field of drug delivery, mPDA coating is widely regarded as one of the effective tools to prevent the premature leakage and release of loaded "cargo" such as drugs in the biological environment. On the other hand, mPDA has good pH responsiveness. In a pH-neutral physiological environment (such as blood, tissue fluid, etc.), the mPDA modified film can remain stable, but it can be degraded in the acidic environment inside the tumor, thereby releasing drugs in a concentrated manner. There are a large number of pore structures on the surface of MSNs, which are prone to rapid leakage and degradation after internal drug loading. A layer of mPDA can be coated on the surface of drug-loaded MSNs by interfacial polymerization, and a barrier can be added to the outside to prevent drug leakage and improve the drug loading effect of the carrier.

[0005] The catechol structure of mPDA makes it negatively charged, and the strong electrostatic repulsion makes the nanoparticles coated with mPDA on the surface show extremely strong stability. The mPDA with catechol structure is also a strong ligand for multivalent metal ions, such as iron ions and copper ions. Ferrous lactate is a widely used food-grade iron fortifier with good biosafety and better absorption effect than inorganic iron. It can be used as a food and feed additive and is also a common drug for treating anemia. In addition, the chelated ferrous ion (Fe 2+ After passively targeting the tumor site, the nanoparticles can undergo a Fenton reaction with endogenous hydrogen peroxide (H2O2) to produce a large amount of ROS, which attack phospholipids to produce phospholipid free radicals, triggering tumor cell ferroptosis, thereby achieving a tumor-suppressing effect.

[0006] Generally, the effect of passive targeting of carriers to tumor sites through high permeability and long retention effect (EPR effect) is limited. In order to improve the targeting of drug delivery systems, some small targeting groups or ligands are used to modify the surface of nanocarriers, which can effectively promote the efficiency of targeted drug delivery to tumor sites and promote cell uptake. αγβ3 integrin receptors are highly expressed in a variety of tumor vascular endothelial cells. Arginine (R)-glycine (G)-aspartic acid (D) (RGD peptide) sequence is a known classic αγβ3 integrin targeting sequence. Therefore, targeting αγβ3 integrin receptors on the surface of tumor cells through RGD peptide sequence can significantly improve the affinity of RGD peptide-modified nanocarriers to tumor cells.

[0007] Therefore, how to provide a targeted mPDA / MSNs nanodrug delivery carrier that induces ferroptosis is an urgent problem that needs to be solved by those skilled in the art. Summary of the invention

[0008] In view of this, the present invention provides a targeted mPDA / MSNs nanodrug delivery carrier that induces ferroptosis, and a preparation method and application thereof.

[0009] In order to achieve the above object, the present invention adopts the following technical solution:

[0010] On the one hand, an embodiment of the present invention provides a targeted mPDA / MSNs nano drug delivery carrier that induces ferroptosis, wherein the nano drug delivery carrier is RGD-Fe 2+ -mPDA / MSNs-NH2; particle size range is 180-260nm.

[0011] The second aspect of the embodiment of the present invention provides a method for preparing the nano drug delivery carrier, the process comprising:

[0012] (1) Connecting and modifying -NH2 on the surface of MSNs to obtain amino-modified MSNs-NH2;

[0013] (2) coating the MSNs-NH2 obtained in step (1) with mPDA to obtain mPDA / MSNs-NH2;

[0014] (3) Mix ferrous lactate and mPDA / MSNs-NH2 in a Tris base solution, react, and freeze-dry to obtain Fe 2+ -mPDA / MSNs-NH2;

[0015] (4) Coupling the RGD targeting peptide to the Fe 2+ -mPDA / MSNs-NH2 surface to obtain the final carrier RGD-Fe 2+ -mPDA / MSNs-NH2.

[0016] Preferably, in step (1), the particle size of MSNs is 125-130 nm.

[0017] Preferably, the specific process of step (1) connecting and modifying -NH2 is: adding 3-aminopropyltriethoxysilane APTES to MSNs, heating and stirring, centrifuging, dispersing with anhydrous ethanol, collecting and washing the precipitate after centrifugation, and vacuum drying to obtain MSNs-NH2.

[0018] Preferably, in step (2), the specific process of coating is: dopamine hydrochloride and mPDA are mixed in a ratio of 1:2 in a Tris base solution, stirred for reaction at room temperature, centrifuged, dispersed in anhydrous ethanol, collected and washed the precipitate after centrifugation, and vacuum dried to obtain mPDA-coated MSNs composite nanoparticles mPDA / MSNs-NH2.

[0019] Preferably, in step (3), the mass ratio of ferrous lactate to mPDA / MSNs-NH2 is 1:2.

[0020] The third aspect of the embodiment of the present invention provides that the nano drug delivery carrier prepared by the method is RGD-Fe 2 + -Application of mPDA / MSNs-NH2 in the preparation of drugs for targeted induction of ferroptosis and treatment of tumors.

[0021] Preferably, RGD-Fe 2+ -mPDA / MSNs-NH2 increased the embedding efficiency and drug loading of DOX and ATRA; and, RGD-Fe 2+ -mPDA / MSNs-NH2 has pH and H2O2 responsive properties.

[0022] A fourth aspect of the present invention provides a targeted nanomedicine for inducing ferroptosis, wherein the nanomedicine comprises RGD-Fe 2+ -mPDA / MSNs-NH2@DOX / ATRA

[0023] It can be seen from the above technical solution that, compared with the prior art, the present invention constructs a composite nanocarrier RGD-Fe with a functional core / shell structure that promotes ferroptosis and mediates chemotherapy and immunotherapy. 2+ -mPDA / MSNs-NH2(RFP / MN). The preparation method is simple, and the obtained RFP / MN sample has uniform particle size and stable physical and chemical properties. It has high encapsulation rate and drug loading for ATRA and DOX, and can responsively release drugs in a simulated tumor microenvironment. The drug-loaded sample RFP / MN@D / A can deliver drugs into tumor cells in a targeted manner, rapidly induce ferroptosis, and effectively kill tumor cells in synergistic chemotherapy. In addition, ATRA has been reported to be able to differentiate MDSCs into mature dendritic cells, macrophages, and granulocytes, and enhance tumor-specific immune responses. Therefore, RFP / MN@D / A is an efficient drug delivery system with significant development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0025] Figure 1 The attached figures show the particle size and potential measurement results of different nano samples. A: particle size result diagram, B: potential result diagram.

[0026] Figure 2 The attached figures are transmission electron micrographs of different nano-samples. A: M, B: MN, C: P / MN, D: FP / MN, E: RFP / MN.

[0027] Figure 3 The attached figures are scanning electron micrographs of different nano-samples. A: M, B: MN, C: P / MN, D: FP / MN, E: RFP / MN.

[0028] Figure 4 The attached figure shows the infrared absorption spectra of different nano-samples. a: M, b: MN, c: P / MN, d: FP / MN, e: RFP / MN.

[0029] Figure 5 The attached figures are DSC curves of different nano samples: a: M, b: MN, c: P / MN, d: FP / MN, e: RFP / MN.

[0030] Figure 6 The attached figure shows the specific surface area analysis results of different nano samples. A: specific surface area, B: adsorption-desorption curve, C: pore size distribution.

[0031] Figure 7 The attached figures are XPS spectra of FP / MN. A: C1s spectrum, B: O1s spectrum, C: Si2p spectrum, D: Fe2p, E: XPS Survey spectrum.

[0032] Figure 8 The attached figure is the result of hydroxyl radical detection. 2+ The ability of carrier (FP / MN, 100 μg / mL) and ferrous lactate (10 μg / mL) to degrade methylene blue. A: pH = 7.4, B: pH = 5.0.

[0033] Fig. 9 The attached figures are the in vitro release curves of each drug-loaded sample under different pH conditions. A, B: in vitro release curves of DOX and ATRA under pH 7.4; C, D: in vitro release curves of DOX and ATRA under pH 5.0.

[0034] Fig.10 The attached figure shows the in vitro release curves of each drug-loaded sample under the condition of excess H2O2 (100 μmol / L) simulating TME. A, B: in vitro release curves of DOX and ATRA under pH 7.4; C, D: in vitro release curves of DOX and ATRA under pH 5.0.

[0035] Fig.11The attached figures show the cytotoxicity test results of different nanocarriers. A, B: Cell survival rate of L-02 cells after 24h and 48h treatment with different concentrations of nanocarriers; C, D: Cell survival rate of MCF-7 cells after 24h and 48h treatment with different concentrations of nanocarriers; E, F: Cell survival rate of MCF-7 cells after 24h and 48h treatment with different concentrations of drug-loaded samples.

[0036] Fig.12 The attached figure shows the intracellular Fe 2+ Qualitative test result diagram.

[0037] Fig.13 The attached figure shows the intracellular Fe 2+ Qualitative test result diagram.

[0038] Fig.14 Figure 1 shows chelated Fe 2+ carrier (FP / MN, concentrations of 50 μg / mL, 75 μg / mL, 100 μg / mL) and Fe 2+ The changes in GSH content after cells were incubated with the donor - ferrous lactate (concentrations were 10μg / mL and 20μg / mL, respectively) for 6h, 12h, and 24h.

[0039] Fig.15 The attached figure shows the detection of various nano drug delivery systems in IC 50 The changes of intracellular GSH content after incubation of cells for 6h, 12h, and 24h at the maximum concentration.

[0040] Fig.16 Figure 1 shows chelated Fe 2+ carrier (FP / MN, concentrations of 50 μg / mL, 75 μg / mL, 100 μg / mL) and Fe 2+ After incubating the cells with ferrous lactate (10 μg / mL and 20 μg / mL, respectively) for 6 h, 12 h, and 24 h, the Fe 2+ Changes in content.

[0041] Fig.17 The attached figure shows the detection of various nano drug delivery systems in IC 50 At the lowest concentration, the intracellular Fe 2 + Changes in content.

[0042] Fig.18 The attached figure shows the fluorescence photos of MCF-7 cells after incubation with each nano-drug delivery system for 4 hours.

[0043] Fig.19The attached figure shows the fluorescence photos of MCF-7 cells after incubation with each nano-drug delivery system for 24 hours.

[0044] Fig. 20 The attached figure is a fluorescent photograph of ROS generated by each nanodrug delivery system after incubating MCF-7 cells for 4 hours.

[0045] Fig.21 The attached figure is a fluorescent photograph of ROS generated by each nanodrug delivery system after incubating MCF-7 cells for 24 hours. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Abbreviations in the examples: MSNs (M), MSNs-NH2 (MN), mPDA / MSNs-NH2 (P / MN), Fe 2+ -mPDA / MSNs-NH2(FP / MN), RGD-Fe 2+ -mPDA / MSNs-NH2(RFP / MN), RGD-Fe 2+ -mPDA / MSNs-NH2@DOX / ATRA(RFP / MN@D / A).

[0048] MCF-7 cells and L-02 cells were purchased from Shanghai Bogu Biotechnology Co., Ltd.

[0049] Example 1 Preparation of Nano-drug Delivery Carrier RGD-Fe 2+ -mPDA / MSNs-NH2(RFP / MN)

[0050] Step 1: Synthesis of MSNs (M)

[0051] The sol-gel method was used. The specific method was as follows: 1g of ammonium fluoride (NH4F) and 0.61g of hexadecyltrimethylammonium bromide (CTAB) were weighed and dispersed in a 250mL three-necked flask, 160mL of ultrapure water was added, a magnet was placed and a reflux device was installed, and magnetic stirring was performed in an 80℃ oil bath to completely dissolve the mixture. When the temperature rose to 80℃, 9mL of tetraethyl silicate (TEOS) was added dropwise to the mixture, and the mixture solution was vigorously stirred for 6h. The sample was taken out and naturally cooled to room temperature, centrifuged at 12000rpm for 20min, and the precipitate was collected and washed once with distilled water and three times with anhydrous ethanol. The product was added to a mixture of 400mL of ethanol and 8mL of hydrochloric acid, refluxed at 80℃ for 24h, and repeated twice to completely remove the excess CTAB, and vacuum dried at 40℃ for 24h, and the product was collected to obtain pure MSNs (denoted as M).

[0052] Step 2 Synthesis of MSNs-NH2(MN)

[0053] In order to load more drugs, MSNs were amino-modified: 20 mg MSNs were dispersed in 20 mL ethanol, added to 1.2 mL APTES, and refluxed at 88 °C for 12 h. The sample was taken out and naturally cooled to room temperature, centrifuged at 12000 rpm for 20 min, washed several times with ethanol and dried to obtain MSNs-NH2 (denoted as MN).

[0054] Step 3 Synthesis of mPDA / MSNs-NH2(P / MN)

[0055] Take 50 mg of MN and ultrasonically disperse it in 50 mL of Tris-HCl buffer (10 mM, pH = 8.5), add 25 mg of dopamine hydrochloride, stir for 24 h in the dark, centrifuge at 12000 rpm for 20 min, and wash with anhydrous ethanol several times to obtain the mPDA-coated carrier mPDA / MSNs-NH2 (denoted as P / MN).

[0056] Step 4 4Fe 2+ Preparation of -mPDA / MSNs-NH2(FP / MN)

[0057] Take 50 mg of the above P / MN and put it in a flask. According to the feed ratio of ferrous lactate: P / MN = 1:2, weigh 25 mg of ferrous lactate and add 10 mL of Tris-HCl buffer (10 mM, pH = 8.5). Stir at room temperature for 12 h, centrifuge at 12000 rpm for 20 min, wash with anhydrous ethanol several times, and freeze-dry to obtain Fe 2+ -mPDA / MSNs-NH2 (denoted as FP / MN).

[0058] Step 5 RGD-Fe 2+Preparation of -mPDA / MSNs-NH2(RFP / MN)

[0059] Take 30 mg FP / MN in a flask, add water to disperse, then add 4.79 mg EDC and 5.75 mg NHS, activate for 15 min, add 1.5 mg DSPE-PEG-RGD to the mixture solution, stir for 8 h, centrifuge at 12000 rpm for 20 min, freeze-dry and collect the product to obtain RGD-Fe 2+ -mPDA / MSNs-NH2 (denoted as RFP / MN).

[0060] Example 2 Detection of particle size, potential and electron microscope image of each carrier

[0061] Loading ATRA, DOX:

[0062] Weigh 50 mg of M into a flask, dissolve it in 10 mg / mL ATRA solution (solvent: DMSO:H2O=9:1, volume: 5 mL) by ultrasonication, incubate it at 37°C for 12 h, remove free ATRA by centrifugation and washing, and then redisperse the sample in DOX solution (10 mg / mL in ultrapure water). After incubation for 12 h, centrifuge and wash, and collect the product to obtain ATRA and DOX-loaded MSNs (MSNs@DOX / ATRA, denoted as M@D / A).

[0063] Weigh 50 mg of MN and place it in a flask, and operate in the same manner as in Example 1 to obtain ATAR and DOX-loaded MSNs-NH2 (MSNs-NH2@DOX / ATRA, denoted as MN@D / A).

[0064] Using the prepared MN@D / A as raw material, the modified MSNs samples loaded with ATRA and DOX were prepared according to the method of Example 1, namely mPDA / MSNs-NH2@DOX / ATRA (denoted as P / MN@D / A), Fe 2+ -mPDA / MSNs-NH2@DOX / ATRA (denoted as FP / MN@D / A), RGD-Fe 2+ -mPDA / MSNs-NH2@DOX / ATRA (denoted as RFP / MN@D / A). The drug-loaded samples were freeze-dried and stored at 4°C in the dark for future use.

[0065] The particle size and Zeta potential of the samples M, MN, P / MN, FP / MN, RFP / MN, and RFP / MN@D / A are shown in Tables 1 and Figure 1 .

[0066] Table 1 Particle size and Zeta potential measurement results of different nano samples

[0067] Sample name Particle size (nm) PDI Zata potential (mV) M 129.51±2.36 0.329 -20.8±1.91 MN 139.83±4.21 0.363 15.3±1.23 P / MN 146.75±1.52 0.060 -20.2±0.82 FP / MN 184.14±1.69 0.065 -12.1±1.36 RFP / MN 195.29±3.22 0.426 -26.8±0.80 RFP / MN@D / A 258.07±3.16 0.150 -11.3±1.74

[0068] From Table 1 and Figure 1 It can be seen that the potential of MSNs is negative, which is mainly due to the silanol groups on its surface; since -NH2 is positively charged, the surface potential of the modified MSNs-NH2 becomes positive; since the surface of mPDA contains negatively charged catechol groups, after the surface of MSNs is coated with mPDA, the surface potential of the nanoparticles becomes negative again, and the particle size also increases slightly; after Fe 2+ After chelating on the surface of mPDA, the electronegativity of the nanoparticle surface decreased; after connecting the targeting modifier RGD peptide, the aspartic acid in the peptide carried a certain negative charge, which led to the increase in the size of the nanoparticles and further reduction in the potential. The above results preliminarily indicate that the RFP / MN composite nanocarrier was successfully constructed, with a particle size of about 195.29nm and a Zata potential of about -26.8mV.

[0069] The prepared M, MN, P / MN, FP / MN, RFP / MN, and RFP / MN@D / A samples were analyzed by transmission electron microscopy and scanning electron microscopy. The results are shown in Figure 2 and Figure 3 .

[0070] Depend on Figure 2 As can be seen, the transmission electron micrographs of each nanocarrier are shown in Figure 1. A is MSNs. It can be seen from the figure that the particle size of MSNs is uniform and the surface is a mesoporous structure; B is -NH2-modified MN, which is dispersed spherical with uniform size and regularly arranged pores can be clearly seen on the surface; Figure C is P / MN, after mPDA modification and coating, the outer layer has obvious coating, the particle size is slightly increased, but the morphology has not changed significantly; D is FP / MN, compared with MSNs, -NH2 connection, coating, chelated Fe 2+ Modifications such as α and β did not significantly change their morphology; E is RFP / MN, and it can be seen that the morphology of MSNs did not change significantly after being connected to the targeting peptide.

[0071] Depend on Figure 3 It can be seen that Figure 3 is a scanning electron microscope image of each nanocarrier. Figure 2 The samples in the figure correspond to each other. From A and B, it can be seen that the particle size of MSNs and MNs is uniform and evenly dispersed, and the surface is mesoporous. From C, it can be seen that after the mP DA pore blocking modification, the outer layer has obvious coating, but the morphology has not changed significantly. From D and E, it can be seen that the chelated Fe 2+ There was no obvious change in the morphology of MSNs after connection with targeting peptides.

[0072] Example 3

[0073] The carriers provided in Example 1, M, MN, P / MN, FP / MN, and RFP / MN, were used for infrared spectroscopy analysis, specific surface area (BET) measurement, and other characterizations. Figure 4 , Figure 5 , Figure 6 , Figure 7 and Figure 8 .

[0074] in, Figure 4 This is the infrared absorption spectrum of each nanosample, with the scanning wave number range of 500-4000cm -1 , the Y axis represents the transmittance (%), and the X axis represents the wave number, the unit is cm -1 As can be seen from the figure, MSNs are at 802cm -1 、1087cm -1 There are obvious Si-O-Si stretching vibration peaks and bending vibration peaks at 3450cm -1 The absorption peak at 1507cm is the bending vibration peak of Si-OH and water molecules. -1 The NH stretching vibration peak of the -NH2 functional group at 1662 cm -1 The peak at 1225 cm is the stretching vibration peak caused by C=C in the special indole structure of mPDA. -1 The P=O absorption peak at 1540 cm -1 、1280cm -1 The peaks at are respectively assigned to the vibration peaks of -C=O and -CO-NH2 on the RGD peptide, proving the successful connection of the RGD targeting peptide.

[0075] Figure 5 It is the DSC curve of each nano sample. As can be seen from the figure, MSNs has a characteristic peak at about 75℃, indicating that MSNs transforms to a crystalline state. MN has an endothermic peak at about 95℃, indicating that its stability is higher than that of the original MSNs. P / MN has a strong endothermic peak at about 90℃, indicating that it transforms from a solid state to a gel state. There are two endothermic peaks in the heating curve of FP / MN. The right side is the endothermic peak of alloy melting, and the corresponding temperature point is the melting point. The peak on the left may be caused by the dissolution of the strengthening phase, or other phase changes have occurred. The specific phase change type can be determined by metallographic method. In the curve of RFP / MN, there is a strong exothermic peak at about 75℃ and an endothermic peak at about 230℃, and the stability has increased.

[0076] Figure 6Figure 1 is the specific surface area and pore size distribution curve of each nano sample. As shown in Figure A, MSNs has the largest specific surface area, about 881.6 m 2 / g, which is due to the clear and abundant mesoporous structure of MSNs. The specific surface area of ​​MN is 394.1m 2 / g, which is lower than that of MSNs. After mPDA coating, the specific surface area of ​​P / MN is 24.1m 2 / g, indicating that the mPDA coating was successfully modified and effectively blocked the holes on the surface of MSNs. With the continuous improvement of the modification degree, the specific surface area of ​​the nanocarrier further decreased, and the specific surface area of ​​RFP / MN almost dropped to zero. Figure B is the pore size distribution curve. The pore size of MSNs is mainly distributed around 2.35nm, and the pore size of MSNs-NH2 is mainly distributed around 1.55nm. 2+ After chelation and connection of RGD peptide, the pore size distribution curve of RFP / MN is basically a straight line, which again shows that the functional modification of the carrier is successful. Figure C is the adsorption-desorption curve. As the degree of modification continues to increase, the closed area of ​​the curve gradually decreases to disappear. The smaller the area under the curve, the fewer nitrogen molecules enter the pore and the smaller the space in the pore.

[0077] Figure 7 Is chelated Fe 2+ XPS spectrum of the post-support (FP / MN). XPS can accurately detect the elemental composition and content of nano-samples. Figure A is the C1s peak diagram. It can be seen from the figure that the sample has a C1s peak at 285eV, and the peak shape is good, indicating that the prepared nano-support has good stability. Figure B is the O1s peak diagram. The sample has a large O1s peak at 532eV. This is because there are a large number of oxygen-containing groups in MSNs. Figure C is the Si2p peak diagram. Silicon (Si) is one of the main elements that make up MSNs. MSNs have a large Si2p peak (103eV). Figure D is the Fe2P peak diagram, which has a peak at 710eV, indicating that Fe in the material 2+ Figure E is the XPS Survey spectrum, the sample has obvious Si2p peaks and Fe2P peaks, indicating that Fe 2+ The chelating and core / shell structured MSNs carriers were successfully constructed. 2+ The presence of ferroptosis can promote ferroptosis after its internalization into cancer cells and enhance the anti-tumor effect.

[0078] Figure 8 This is the test result of the generation of hydroxyl radicals (·OH) in vitro. Fenton's reagent has a very strong oxidizing ability. In the Fenton system, H2O2 2+ / Fe 3+Under catalytic action, a large amount of ·OH can be produced. Figures A and B show the conditions of pH 7.4 and pH 5, respectively, containing Fe 2+ The results of the degradation of methylene blue (MB) by carrier (FP / MN, 100 μg / ml) and ferrous lactate (10 μg / mL). 2+ In the Fenton reaction system, the Fe 2+ The presence of can promote H2O2 to decompose ·OH, and ·OH can quickly degrade MB and make it fade. As can be seen from the figure, under pH 7.4, the degradation rate of MB by ferrous lactate reaches about 20% with the extension of time. After adding H2O2, the degradation rate can reach 30%. The degradation rate of FP / MN is 50%, and after adding H2O2, the degradation rate reaches 60%. Under pH 5, the degradation rate in each group further increases. The above results show that the degradation rate of ferrous lactate on MB is about 20% with the extension of time. After adding H2O2, the degradation rate can reach 30%. 2+ The carrier (FP / MN) can effectively degrade MB in a pH- and H2O2-dependent manner. In the TME with low pH and high H2O2 concentration, it can induce the production of a large amount of ·OH, causing lipid peroxidation and triggering ferroptosis.

[0079] Based on the above characterization results, it can be seen that the present invention successfully prepared RFP / MN nanocarriers, which have good physical stability, good dispersibility, and successful modification of various functional groups. It can be used as an excellent delivery carrier for loading anti-tumor drugs and immune inducers and enhancing ferroptosis.

[0080] Example 4

[0081] The drug loading and encapsulation efficiency were determined using the nano-samples M@D / A, MN@D / A, P / MN@D / A, FP / MN@D / A, and RFP / MN@D / A (i.e., each nano-preparation) loaded with ATRA and DOX provided in Examples 1 and 2. Each nano-preparation was taken, the volume of the supernatant was measured after centrifugation, and the two drugs were determined by HPLC and UV methods respectively. The measured peak area values ​​and OD values ​​were substituted into the standard curve to obtain the concentrations of ATRA and DOX in the supernatant, and the encapsulation efficiency and drug loading of each carrier for ATRA and DOX were calculated according to the formula.

[0082] Determination of ATRA by HPLC:

[0083] 355 nm was selected as the detection wavelength of ATRA, and an HPLC method for quantitative determination of ATRA was established.

[0084] Establish a standard curve: Take 50μg / mL ATRA (solvent is DMSO:H2O=9:1) solution, use it as the mother solution, and dilute it with a mixed solvent (DMSO:H2O=9:1) to a series of ATRA solutions with a concentration gradient, namely 1.0, 3.0, 5.0, 8.0, 10.0, 15.0, 20.0μg / ml. The chromatographic column is Agilent column (ZORBAX SB-C18, 5μm, 4.6×250mM), the mobile phase is a methanol-2% glacial acetic acid mixed solution (volume ratio 90:10), the injection volume is 10μL, the flow rate and column temperature are set to 1.0mL / min and 25±5℃. Use ATRA concentration as the horizontal axis and peak area as the vertical axis to establish a standard curve. Prepare for the determination of ATRA content, encapsulation efficiency and drug loading in the in vitro release experiment, see Table 2.

[0085] UV method for determination of DOX:

[0086] Select 485nm as the detection wavelength of DOX, and establish a UV method for quantitative determination of DOX. Establish a standard curve as above, with DOX concentration as the horizontal axis and the measured absorbance OD value as the vertical axis to establish a standard curve. Prepare for the determination of DOX content, encapsulation efficiency and drug loading in the in vitro release experiment, see Table 2.

[0087]

[0088] Table 2 Encapsulation efficiency and drug loading of different nanoformulations

[0089]

[0090] Table 2 shows that due to its large specific surface area, MSNs can effectively load ATRA and DOX, with encapsulation efficiency of 61.35% and 20.45%, respectively. The encapsulation efficiency of MN@D / A is about 70.54% and 23.51%, respectively. The encapsulation efficiency of RFP / MN@D / A for ATRA and DOX is 72.98% and 24.33%, respectively. The more modified components are connected to the carrier surface, the more likely it is that it is not conducive to the drug loading of the carrier. The encapsulation efficiency and drug loading of the final drug-loaded sample RFP / MN@D / A are almost the same as those of other samples, indicating that the nanocarrier of the present invention is more effective in connecting various modifiers (such as Fe 2+ After addition of RGD peptide, the drug loading performance was not affected. This showed that it had good loading effect on ATRA and DOX and excellent drug loading performance.

[0091] Example 5

[0092] The nano-samples loaded with ATRA and DOX provided in Example 2 of the present invention, M@D / A, MN@D / A, P / MN@D / A, FP / MN@D / A, and RFP / MN@D / A, were used. The membrane dialysis method was used to determine the drug release results of different drug-loaded nano-samples in an environment of pH 7.4 and pH 5, and the in vitro release in an environment of pH 7.4 and pH 5 was determined after adding H2O2 at a concentration of 100 μmol / L to the release medium. Among them, a mixed solution of anhydrous ethanol: PBS = 4:1 was selected as the release medium. Take 5 ml of each nano-preparation with a concentration of 1 mg / ml, place it in a dialysis bag, add 50 mL of the release medium, shake at 37°C, and adjust the speed to 100 rpm. Take 2 mL of samples at 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, 96h, and 120h, and replenish fresh medium at the same time. The HPLC and UV methods were used to detect and calculate the contents of released ATRA and DOX.

[0093] The in vitro release calculation formula is:

[0094]

[0095] Results Fig. 9 and Fig.10 .

[0096] Fig. 9 Figure 1 is the result of pH-responsive drug release. A and B are the in vitro release curves of DOX and ATRA in each drug-loaded sample at pH 7.4, and C and D are the in vitro release curves of DOX and ATRA in each drug-loaded sample at pH 5. As can be seen from the figure, the release of each nanocarrier under acidic conditions is higher than that under neutral conditions. Since the mPDA coating is pH-responsive, the release of P / MN@D / A under pH 5 is significantly higher than that under pH 7.4, and compared with MN@D / A, the release of P / MN@D / A is significantly reduced, indicating that the mPDA coating can control drug release. Due to the Fe 2+ Can promote the Fe nton reaction to generate free Fe 2+ , which cleaves the mPDA coating and exposes the pores on the surface of MSNs, releasing more ATRA and DOX. Therefore, the release rates of FP / MN@D / A and RFP / MN@D / A are higher than those of P / MN@D / A. The above results prove that each modified carrier has good pH-responsive drug release properties.

[0097] Fig.10The results of H2O2-responsive drug release were shown. H2O2 at a concentration of 100 μmol / L was added to the release medium. A and B are the in vitro release curves of DOX and ATRA of each drug-loaded sample at pH 7.4; C and D are the in vitro release curves of DOX and ATRA of each drug-loaded sample at pH 5. Fig. 9 By comparison, it can be seen that in the presence of excess H2O2, the release rate of each carrier increases, proving that it has H2O2-responsive drug release characteristics. In particular, FP / MN@D / A and RFP / MN@D / A have a more significant increase in release rate than other samples. Therefore, the functionalized nanocarrier constructed by the present invention has excellent pH and H2O2 dual-responsive drug release capabilities.

[0098] Combination Fig. 9 and Fig.10 Come and see, Fig. 9 It shows that each drug-loaded sample has pH responsiveness to the release of the two drugs. The release of each drug-loaded sample under pH 5 is higher than that under pH 7.4. In addition, the release of DOX from FP / MN@D / A and RFP / MN@D / A under pH 5 is higher than that from other samples. Fig.10 After adding H2O2 to the release medium, under the same pH conditions, the release of both drugs by each drug-loaded sample increased, indicating that the release of each sample was responsive to H2O2. Fig.10 In the two CD graphs, under pH 5, the release of DOX by FP / MN@D / A and RFP / MN@D / A is only slightly lower than that of MN@D / A, while the release of ATRA by RFP / MN@D / A is significantly higher than that of other samples. It is generally recognized that the tumor microenvironment has a low pH and high H2O2 concentration. Therefore, in general, under the actual conditions of the tumor microenvironment, RFP / MN@D / A has obvious advantages in drug release performance. In other words, the advantage of RFP / MN@D / A in drug release is not unconditional, and its advantage in drug release can only be clearly manifested under conditions close to the tumor microenvironment.

[0099] Example 6

[0100] The cytotoxicity of the nanosamples provided in Examples 1 and 2 of the present invention - M, MN, P / MN, FP / MN, RFP / MN and the nanosamples loaded with ATRA and DOX - M@D / A, MN@D / A, P / MN@D / A, FP / MN@D / A, RFP / MN@D / A (i.e., various nanoformulations) were investigated.

[0101] Cell culture: MCF-7 cells and L-02 cells were cultured in RP MI1640 medium containing 10% fetal bovine serum (FBS). The cells were cultured in an incubator at 37°C and 5% CO2. The culture medium was replaced every two days.

[0102] MCF-7 and L-02 cells were seeded in 96-well plates at a density of 5 × 10 3 Each well was added with 10, 20, 50, 100, and 200 μg / ml of each nanocarrier and each nanopreparation after adherence. After culturing for 24 or 48 hours, the culture medium was removed, and 20 μL of 5 mg / mL MTT solution and 80 μL of culture medium were added to each well. After incubation at 37°C for 4 hours, the liquid in the well was aspirated, and 100 μL of dimethyl sulfoxide was added to each well to dissolve the formazan crystals. The 96-well plate was shaken on a circular shaker at 100 rpm for 10 minutes to completely dissolve the formazan crystals. The OD value was measured at a wavelength of 490 nm using an enzyme marker to calculate the relative content of live cells. Results are shown in Fig.11 It can be seen that the pure materials have low toxicity to L-02 cells, and the cell survival rate is higher than 80% after 24h or 48h. 2+ After the sample was treated with , the cell survival rate decreased to a certain extent, indicating that the Fenton reaction occurred in the cells, lipid peroxides accumulated, and cell death occurred, proving that the constructed nanocarrier has good biocompatibility and low toxicity. Figures C and D show the toxicity test results of MCF-7 cells treated with pure materials for 24h or 48h. It can be seen that the cell survival rate is similar to the toxic effect of the blank carrier on L-02 cells, which once again proves that the nanocarrier has good biocompatibility and low toxicity. Figures E and F show the toxicity test results of MCF-7 cells treated with drug-loaded samples for 24h or 48h. As the modification degree of the carrier increases, the cell survival rate decreases. Because the drug loading of the -NH2-modified sample MN@D / A is significantly increased, after it is treated with cells, the cell survival rate drops to about 35%. Overall, the biosafety of pure materials is relatively stable, and the drug-loaded samples have a strong killing ability. FP / MN@D / A and RFP / MN@D / A have Fe in the carrier. 2+ , which can induce ferroptosis, and combined with the effects of the loaded DOX and ATRA, it has a stronger cytotoxic effect.

[0103] Further Fig.11For analysis, A and B are the survival results of L-02 cells (normal liver cells) treated with each nanocarrier (i.e., blank carrier, without DOX and ATRA). Usually, it is not desirable for the blank carrier to be too toxic to normal cells, as too high toxicity indicates poor biosafety. Therefore, the cytotoxicity of RFP / MN@D / A is lower than that of FP / MN@D / A, which is not a bad result. Figures E and F are the cell survival results of each drug-loaded sample. MN@D / A does have the strongest toxic effect on MCF-7 cells, but RFP / MN@D / A is second. At 48 hours, the toxicity results of the two samples are basically equal. The reason why MN@D / A has high cytotoxicity is that it is not coated with polydopamine (mPDA) on its surface, and the drug release is faster in a short time (48h), but the degree of modification of this sample is low. In terms of performance verified by other cell experiments, such as cell uptake, induction of ROS production, and ferroptosis, the effect of MN@D / A is obviously inferior to that of several other mPDA-coated samples, especially RFP / MN@D / A, which has the best performance. These performances are important prerequisites for the drug-loaded samples to finally exert their anti-tumor effects. Therefore, overall, RFP / MN@D / A, as a highly modified drug-loaded sample, has significantly better performance than MN@D / A.

[0104] Example 7: Using FerroOrange as a fluorescent probe to detect Fe 2+ Perform fluorescence imaging

[0105] MCF-7 cells were inoculated in a fluorescent culture dish and cultured overnight. The supernatant was discarded on the next day, and the cells were washed three times with serum-free medium. The medium containing drugs was replaced and cultured for 12 hours or 24 hours. After the end, the medium was removed, and the cells were washed three times with serum-free medium. FerroOrange solution with a concentration of 1μmol / l was added. After culturing for 30 minutes, the cells were observed under a fluorescence microscope. Fig.12 , is the fluorescence image after 12 h of culture, where red fluorescence represents DOX and yellow fluorescence represents Fe 2+ In the control group, the yellow fluorescence was very weak. In the first few groups of materials, the change in the yellow fluorescence intensity was not obvious. 2+ After the samples were treated with iron chelator 2,2'-Bipyridyl (Bpy), the yellow fluorescence was significantly enhanced. When the cells were co-incubated with RFP / MN@D / A, the yellow fluorescence basically disappeared, proving that FerroOrange binds to intracellular Fe 2+ Efficient response. Fig.13 The fluorescence image is shown after 24 h of culture. Both red and yellow fluorescence are enhanced, indicating that RFP / MN@D / A can effectively increase the intracellular Fe 2+ content, achieving the effect of promoting ferroptosis.

[0106] Ferroptosis usually causes a decrease in glutathione (GSH) content. The present invention investigates the effect of chelated Fe 2+ carrier (FP / MN, 50 μg / mL, 75 μg / mL, 100 μg / mL) and Fe 2+ The changes in GSH content after incubating cells with ferrous lactate (10 μg / mL, 20 μg / mL) for 6 h, 12 h, and 24 h, respectively. Fig.14 As shown, compared with the control group, each concentration of FP / MN could reduce the intracellular GSH content, among which 100 μg / mL FP / MN reduced the GSH content by more than 50% after incubation for 24 hours, indicating that the vector can effectively induce ferroptosis.

[0107] In addition, the IC of each drug delivery system was calculated using the MTT results. 50 The changes in intracellular GSH content caused by each drug delivery system were detected. Fig.15 As shown, each drug delivery system can reduce the GSH content and chelate Fe 2+ The effect is more significant for drug delivery systems, especially for chelated Fe 2+ The RFP / MN@D / A modified with RGD peptide can reduce the GSH content by about 58% after 24 hours. Compared with the blank vector, this drug delivery system can more effectively reduce the intracellular GSH content.

[0108] Iron plays a key role in ferroptosis. Excessive accumulation of iron can lead to oxidative damage and cell death. 2+ It can react with H2O2 to produce excessive ROS, enhance oxidative damage, and lead to cell ferroptosis. 2+ carrier (FP / MN, 50 μg / mL, 75 μg / mL, 100 μg / mL) and Fe 2+ After incubating cells with ferrous lactate (10 μg / mL, 20 μg / mL) for 6 h, 12 h, and 24 h, the intracellular Fe 2+ The content of Fig.16 As shown in Figure 2, compared with the control group, FP / MN increased the intracellular Fe 2+ The content of Fe 2+ The content was significantly higher. This result once again shows that the carrier has the ability to promote ferroptosis.

[0109] In the same way, each drug delivery system was used to detect the intracellular Fe 2+ The results are as follows: Fig.17 As shown, unchelated Fe 2+ Each drug delivery system induces intracellular Fe 2+ The effect of increasing the content is not obvious, and the chelated Fe2+ The drug delivery system can effectively induce Fe 2+ The content increased, and after different culture time, it caused Fe 2+ The contents of α-glucose and β-glutamic acid increased greatly, which was consistent with the GSH test results.

[0110] Example 8

[0111] Fluorescence localization method was used to investigate the uptake of each nanosample by MCF-7 cells. FITC-labeled nanosamples were used to act on MCF-7 cells, washed three times with PBS, and each FITC-labeled sample was added and incubated for 4h or 24h. The old culture medium was aspirated, washed three times with PBS, and Hoechst33342 staining solution was added and incubated for 30min, then washed with PBS and observed under an inverted fluorescence microscope.

[0112] Fig.18 The fluorescence images of each sample after incubation with MCF-7 cells for 4 hours. As can be seen from the figure, the cell nucleus of the control group showed blue fluorescence after 4 hours, and the free DOX showed weak red fluorescence and overlapped with the blue fluorescence of the cell nucleus, indicating that the small molecule DOX can passively diffuse through the cell membrane to reach the cell nucleus. After treatment with each nanoformulation, the fluorescence signals of DOX and FITC were significantly enhanced, indicating that each nanoparticle can be effectively taken up by cells. 2+ The intracellular fluorescence intensity of the nanoformulation was further enhanced, indicating that the chelated Fe 2+ The post-drug loading system is more easily taken up by cells. Fig.19 The fluorescence images of each nanoformulation after incubation with MCF-7 cells for 24 hours show that the fluorescence signal is significantly enhanced compared with 4 hours. The above results confirm that RFP / MN@D / A as a delivery carrier can greatly increase the drug uptake by MCF-7 cells and improve its bioavailability and pharmacodynamics.

[0113] When programmed cell death is triggered, a large number of ROS clusters are generated in the mitochondria. The indicator DCFH-DA is used to detect the production of intracellular ROS, which can be oxidized by ROS to green fluorescent DCF. The same method is used to incubate cells with each nanosample for 4h or 24h. After the incubation, the cells are treated with a ROS detection kit-fluorescent probe to detect the effect of each sample on the level of intracellular ROS generation, and the samples are placed under an inverted fluorescence microscope for observation.

[0114] Fig. 20 , Fig.21The ROS fluorescence images of each sample after 4h and 24h treatment, green fluorescence indicates ROS. Comparing the results of each treatment group, it can be found that the intracellular ROS fluorescence intensity after nanoformulation treatment is much greater than that of free DOX and the control group. Compared with 4h, the ROS fluorescence intensity of each group after 24h treatment is significantly enhanced, indicating that the nanoformulation induces ROS production in a time-dependent manner. 2+ After treatment with the drug delivery system, intracellular Fe 2+ Excessive accumulation and Fenton reaction with H2O2 produce excessive ROS, leading to cellular oxidative damage and inducing ferroptosis; after 4 hours of treatment, the drug-loaded nanosystem had no obvious effect on the production of ROS, but after 24 hours of treatment, it was clearly enhanced. In summary, all nano-drug delivery systems can stimulate the generation of ROS in cells, thereby promoting tumor cell death.

[0115] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0116] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A targeted mPDA / MSNs nanodrug delivery carrier that induces ferroptosis, characterized in that: The nano drug delivery carrier is RGD-Fe 2+ -mPDA / MSNs-NH2; particle size range is 180-260nm.

2. The method for preparing the nano drug delivery carrier according to claim 1, characterized in that the process include: (1) Connecting and modifying -NH2 on the surface of MSNs to obtain amino-modified MSNs-NH2; (2) coating the MSNs-NH2 obtained in step (1) with mPDA to obtain mPDA / MSNs-NH2; (3) Mix ferrous lactate and mPDA / MSNs-NH2 in a Tris base solution, react, and freeze-dry to obtain Fe 2+ -mPDA / MSNs-NH2; (4) Coupling the RGD targeting peptide to the Fe 2+ -mPDA / MSNs-NH2 surface to obtain the final carrier RGD-Fe 2+ -mPDA / MSNs-NH2.

3. The method according to claim 2, characterized in that In step (1), the particle size of MSNs is 125-130 nm.

4. The method according to claim 3, characterized in that: The specific process of step (1) connecting and modifying -NH2 is as follows: adding 3-aminopropyltriethoxysilane APTES to MSNs, heating and stirring, centrifuging, dispersing with anhydrous ethanol, collecting and washing the precipitate after centrifugation, and vacuum drying to obtain MSNs-NH2.

5. The method according to claim 4, characterized in that In step (2), the specific process of coating is as follows: dopamine hydrochloride and mPDA are mixed in a ratio of 1:2 in a Tris base solution, stirred for reaction at room temperature, centrifuged, dispersed in anhydrous ethanol, collected and washed the precipitate after centrifugation, and vacuum dried to obtain mPDA-coated MSNs composite nanoparticles mPDA / MSNs-NH2.

6. The method according to claim 5, characterized in that In step (3), the mass ratio of ferrous lactate to mPDA / MSNs-NH2 is 1:

2.

7. The nano drug delivery carrier prepared by any of the methods described in claims 2-6 is RGD-Fe 2+ -Application of mPDA / MSNs-NH2 in the preparation of drugs for targeted induction of ferroptosis and treatment of tumors.

8. The use according to claim 7, characterized in that: RGD-Fe 2+ -mPDA / MSNs-NH2 increased the embedding efficiency and drug loading of DOX and ATRA; and, RGD-Fe 2+ -mPDA / MSNs-NH2 has pH and H2O2 responsive properties.

9. A targeted nanomedicine that induces ferroptosis, characterized in that: The nano drug includes RGD-Fe 2+ -mPDA / MSNs-NH2@DOX / ATRA.

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