Nanoparticles for self-triggering proteolysis and RNA interference for tumor targeting as well as preparation method and application of nanoparticles

Abstract

The invention discloses a nanoparticle for self-triggering proteolysis and RNA (Ribonucleic Acid) interference for tumor targeting as well as a preparation method and application of the nanoparticle, and belongs to the technical field of biological medicines. The nanoparticle takes ferritin as a carrier, siRNA capable of up-regulating mitochondrial iron transporter is wrapped in the nanoparticle, the surface of the nanoparticle is modified with an E3 ligase ligand and a tumor targeting peptide, and the preparation method of the nanoparticle comprises the following steps: modifying the E3 ligase ligand and the tumor targeting peptide on the surface of the ferritin through Click reaction; the method comprises the following steps: loading siRNA into a ferritin carrier by using the pH-dependent depolymerization / recombination characteristic of ferritin to obtain the self-triggering proteolysis and RNA interference nanoparticles for tumor targeting. The nanoparticles can effectively avoid clearing of the liver, are effectively enriched in tumor parts, activate strong anti-tumor immunity by destroying iron homeostasis in tumor cell mitochondria and inducing tumor cell death, and can be used for preparing anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a nanoparticle for tumor targeting self-triggered proteolysis and RNA interference, a preparation method thereof, and an application thereof. Background Art

[0002] In the past decade, medical progress has revolutionized the traditional treatment methods for various cancers. With the continuous deepening of the understanding of cell biology and immunology, the role of mitochondria in enhancing tumor immunogenicity has become increasingly prominent. Mitochondria play a crucial role in the metabolism and bioenergetics of tumor cells, adapting to the needs of tumor cell proliferation in terms of quantity, structure, and function. Given the important role of mitochondria, mitochondrial disruption is often fatal to cells, causing irreversible damage to cells. In addition, mitochondria contain a variety of immune-stimulating factors, such as ATP and mitochondrial DNA, which are exposed and initiate downstream immune-related pathways when mitochondrial function is impaired. These characteristics have inspired the development of mitochondrial-targeted immunotherapy, and mitochondrial-targeted treatment systems are under development. However, the lack of tumor specificity of related treatments is a prominent defect in practical applications. Even if the tumor-promoting mechanism mediated by mitochondria is downregulated, due to its non-selective damage to the mitochondria of immune cells, it is difficult to ensure sufficient tumor-killing effects. Therefore, alternative strategies for selectively destroying the mitochondria of tumor cells to effectively treat cancer are needed. In mitochondria, iron, as an important cofactor, participates in the synthesis process of various enzymes such as cytochromes and iron-sulfur proteins. These iron-related proteins use the reversible valence change of iron to transfer electrons, forming an electron transport chain (ETC) to generate ATP to support cell activity and the main source of intracellular reactive oxygen species (ROS). Generally speaking, tumor cells have a strong survival need for iron to meet the needs of malignant proliferation. Due to the important role of mitochondria in iron metabolism, directly interfering with mitochondrial iron homeostasis may more effectively damage mitochondria, thereby killing tumor cells. However, existing iron supply means often damage normal cells, thereby bringing serious toxic side effects. Therefore, a precise and safe mitochondrial iron homeostasis disruption strategy needs to be found. Ferritin is a commonly existing iron storage protein in organisms, which chelates abundant iron ions and is an ideal iron supply protein for disrupting mitochondrial iron homeostasis. However, ferritin exhibits very prominent acid-base tolerance and does not release iron in the physiological environment. In addition, tumor cells have developed complex and strict mechanisms to maintain intracellular iron balance, and ferritin will only release free iron through ferritinophagy when iron is depleted. Therefore, an effective method for destroying ferritin is needed to induce acute iron stress in tumor cells.

[0003] Due to the important role of mitochondrial iron homeostasis in tumor growth and proliferation, how to develop an anti-tumor nanomaterial with high precision and strong safety has become a technical problem to be solved urgently. Summary of the Invention

[0004] The object of the present invention is to provide a tumor-targeted self-triggered protein degradation and RNA interference nanoparticle and its preparation method and application, which can efficiently target tumor tissues, disrupt the mitochondrial iron homeostasis of tumor cells, and then produce a highly efficient and low-toxic anti-tumor effect.

[0005] In the first aspect of the present invention, there is provided a tumor-targeted self-triggered protein degradation and RNA interference nanoparticle, which uses ferritin as a carrier, and siRNA is encapsulated in the ferritin cavity, and the siRNA can up-regulate mitochondrial iron transporter; the surface of the ferritin is modified with an E3 ligase ligand and a tumor-targeting peptide.

[0006] Further, the siRNA is siRNA targeting and interfering with ENO1, and its sequence is preferably AGUAUGACCUGGACUUCAAGUTT (SEQ ID NO.1).

[0007] Further, the E3 ligase ligand includes one or more of VH032, VHL Ligand 8, VHL Ligand 14, Pomalidomide, etc.

[0008] Further, the tumor-targeting peptide includes one or more of cyclo-(RGDfK), GE11 peptide (YHWYGYTPQNVI), B18 peptide (CRTIGPSVC), etc.

[0009] In the second aspect of the present invention, there is provided a preparation method of the above-mentioned tumor-targeted self-triggered protein degradation and RNA interference nanoparticle, and the preparation method includes: modifying the E3 ligase ligand and the tumor-targeting peptide on the surface of ferritin through a Click reaction; using the pH-dependent depolymerization / reorganization property of ferritin to load siRNA into ferritin to obtain a nanoparticle for tumor-targeted self-triggered protein hydrolysis and RNA interference.

[0010] Further, the preparation method includes:

[0011] S1. Mix ferritin with dibenzocyclooctyne-N-hydroxysuccinimide ester (DBCO-NHS) for reaction to obtain a dibenzocyclooctyne-labeled ferritin carrier;

[0012] S2. Mix the azide-labeled E3 ligase ligand and the azide-labeled tumor-targeting peptide with the dibenzocyclooctyne-labeled ferritin carrier for reaction to obtain a tumor-targeted self-triggered protein degradation ferritin carrier;

[0013] S3. Load siRNA into the cavity of the ferritin carrier obtained in step S2 by pH-regulated depolymerization / reassembly to obtain nanoparticles for tumor-targeted self-triggered proteolysis and RNA interference.

[0014] In the above steps S1 and S2, the reaction is preferably carried out in an aqueous solution; in the above step S3, the reaction is preferably carried out in a sodium chloride solution, and the concentration of the sodium chloride solution is preferably 20 - 30 mM; the reaction temperature in the above steps S1, S2, and S3 is preferably 20 - 35 °C.

[0015] Further, step S1 specifically includes:

[0016] Mix the ferritin solution and dibenzocyclooctyne-N-hydroxysuccinimide ester for reaction, and then wash and ultrafilter to obtain a dibenzocyclooctyne-labeled ferritin carrier.

[0017] Among them, the mass ratio of the ferritin to the dibenzocyclooctyne-N-hydroxysuccinimide ester is 1:0.1 - 0.2.

[0018] Further, step S2 specifically includes:

[0019] Add the azide-labeled E3 ligase ligand and the azide-labeled tumor-targeting peptide to the solution of the dibenzocyclooctyne-labeled ferritin carrier for reaction, and then wash and ultrafilter to obtain a ferritin carrier for tumor-targeted self-triggered protein degradation.

[0020] Among them, the mass ratio of the azide-labeled E3 ligase ligand, the azide-labeled tumor-targeting peptide to the dibenzocyclooctyne-labeled ferritin carrier is 0.1 - 0.15:0.05 - 0.15:1.

[0021] Further, step S3 specifically includes:

[0022] Dropwise add an acid solution to the solution of the ferritin carrier for tumor-targeted self-triggered protein degradation to adjust the solution pH to 2 - 3; then add siRNA and mix well; dropwise add an alkali solution to the reaction system to adjust the solution pH to 7 - 7.5, and continue the reaction to load siRNA into the cavity of the ferritin carrier; then centrifuge and wash to obtain nanoparticles for tumor-targeted self-triggered protein degradation and RNA interference.

[0023] Among them, the acid solution is preferably a hydrogen chloride solution, and its concentration is preferably 0.1 - 1 M. The alkali solution is preferably a sodium hydroxide solution, and its concentration is preferably 0.1 - 1 M. The solution of the ferritin carrier for tumor-targeted self-triggered protein degradation has a solute of 20 - 30 mM sodium chloride solution. The concentration of the siRNA is preferably 0.2 - 0.4 μM.

[0024] In the third aspect of the present invention, there is provided the use of the tumor-targeted self-triggered protein degradation and RNA interference nanoparticles in the preparation of anti-tumor drugs.

[0025] The present invention has at least the following technical effects or advantages:

[0026] 1. The tumor-targeted self-triggered protein degradation and RNA interference nanoparticles provided by the present invention. The ferritin modified with tumor-targeting peptides has the ability to evade liver clearance and tumor targeting, can be effectively enriched in the tumor site, and improve the targeting tumor treatment effect.

[0027] 2. The tumor-targeted self-triggered protein degradation and RNA interference nanoparticles provided by the present invention. During the tumor treatment process, the nanoparticles can recruit E3 ligase to initiate the ubiquitination process, release the internally chelated ferrous ions and siRNA through proteolysis, up-regulate the mitochondrial iron transport channels, and then lead to mitochondrial iron overload and the enrichment of harmful mitochondrial ROS, thereby resulting in tumor cell death and the activation of subsequent immune responses. This treatment strategy combines two treatment methods of proteolysis and gene therapy, providing an efficient, precise and low-toxic anti-tumor effect.

[0028] 3. The tumor-targeted self-triggered protein degradation and RNA interference nanoparticles provided by the present invention release the internally chelated ferrous ions through proteolysis. At the same time, siRNA can up-regulate mitochondrial iron transporters, thereby further promoting the enrichment of mitochondrial iron. Under the synergistic effect of these two effects, the mitochondrial iron homeostasis of tumor cells is effectively disrupted, and the subsequent treatment effect and immune response are significantly enhanced.

[0029] 4. The tumor-targeted self-triggered protein degradation and RNA interference nanoparticles provided by the present invention, by disrupting the mitochondrial iron homeostasis of tumor cells, not only cause tumor cell death, but also can significantly enhance the tumor suppression effect mediated by immunotherapy.

[0030] 5. The tumor-targeted self-triggered protein degradation and RNA interference nanoparticles provided by the present invention have a simple preparation method, a simple process, and high operability. Brief Description of the Drawings

[0031] Figure 1 It is a flowchart for the preparation of nanoparticles (cRGD-VFs) for tumor-targeted self-triggered proteolysis and RNA interference.

[0032] Figure 2 It is the construction of nanoparticles (cRGD-VFs) for tumor-targeted self-triggered proteolysis and RNA interference and its anti-tumor mechanism.

[0033] Figure 3Characterization of the pH-responsive depolymerization / re-reassembly properties of ferritin (Fn) and a ferritin carrier with tumor-targeted self-triggered proteolysis (cRGD-VF).

[0034] Figure 4 TEM image of nanoparticles (cRGD-VFs) for tumor-targeted self-triggered proteolysis and RNA interference.

[0035] Figure 5 Cytotoxicity of nanoparticles (cRGD-VFs) for tumor-targeted self-triggered proteolysis and RNA interference; wherein, Figure 5 A shows the cytotoxic effects of different nanoparticles on Hepa1-6 tumor cells at different concentrations. The experimental groups are the blank control group (Control), the ferritin group (Fn), the self-triggerable degradation ferritin group (VF), the ferritin group with RNA interference function (Fs), the self-triggerable degradation RNA interference nanoparticle group (VFs), and the nanoparticles for tumor-targeted self-triggered proteolysis and RNA interference (cRGD-VFs); Figure 5 B shows the cytotoxic effects of different nanoparticles on 3T3 normal cells; Figure 5 C shows the changes in the level of mitochondrial ferrous ions in tumor cells caused by different nanoparticles; Figure 5 D shows the changes in the level of mitochondrial ROS in tumor cells caused by different nanoparticles.

[0036] Figure 6 In vivo antitumor effect of nanoparticles (cRGD-VFs) for tumor-targeted self-triggered proteolysis and RNA interference; wherein, Figure 6 A shows the results of changes in tumor size, Figure 6 B shows the results of changes in body weight; Figure 6 C shows the survival curve of tumor-bearing mice. Detailed implementation manners

[0037] The present invention will be specifically described below in combination with the detailed implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are used to illustrate the present invention, rather than to limit the present invention.

[0038] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In case of contradiction, this specification shall prevail.

[0039] Unless otherwise specified, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or by existing methods.

[0040] The present invention provides a nanoparticle (cRGD-VFs) for tumor targeting with self-triggered proteolysis and RNA interference. It uses ferritin as a carrier, and siRNA is encapsulated within the ferritin cavity. The siRNA targets and interferes with ENO1 and can upregulate mitochondrial iron transporters. The surface of the ferritin is modified with an E3 ligase ligand and a tumor-targeting peptide. The preferred E3 ligase ligand is VH032, and the preferred tumor-targeting peptide is cyclo-(RGDfK). The preparation and anti-tumor mechanism of the nanoparticle (cRGD-VFs) for tumor targeting with self-triggered proteolysis and RNA interference provided by the present invention are as Figure 1 、 2 shown.

[0041] The preparation method of the nanoparticle for tumor targeting with self-triggered proteolysis and RNA interference provided by the present invention includes:

[0042] S1. Mix ferritin and dibenzocyclooctyne-N-hydroxysuccinimide ester for reaction to obtain a dibenzocyclooctyne-labeled ferritin carrier.

[0043] The step S1 specifically includes:

[0044] Mix the ferritin solution and dibenzocyclooctyne-N-hydroxysuccinimide ester for reaction, then wash and ultrafilter to obtain a dibenzocyclooctyne-labeled ferritin carrier;

[0045] Among them,

[0046] The concentration of the ferritin solution is 0.5 - 2 mg / mL;

[0047] The mass ratio of ferritin to dibenzocyclooctyne-N-hydroxysuccinimide ester is 1:0.1 - 0.2.

[0048] S2. React the dibenzocyclooctyne-labeled ferritin carrier with an azide-labeled E3 ligase ligand VH032 and an azide-labeled tumor-targeting peptide cyclo-(RGDfk) to obtain a tumor-targeting ferritin carrier with self-triggered protein degradation.

[0049] The step S2 specifically includes:

[0050] Add the azide-labeled E3 ligase ligand VH032 and the azide-labeled tumor-targeting peptide cyclo-(RGDfk) to the dibenzocyclooctyne-labeled ferritin carrier solution for reaction, then wash and ultrafilter to obtain a tumor-targeting ferritin carrier with self-triggered protein degradation;

[0051] Among them,

[0052] The mass ratio of the azide-labeled E3 ligase ligand VH032, the azide-labeled tumor-targeting peptide cyclo-(RGDfk) to the dibenzocyclooctyne-labeled ferritin carrier is 0.1-0.15:0.05-0.15:1.

[0053] S3. Adjust the pH of the ferritin carrier solution for tumor-targeted self-triggered protein degradation to acidic, add siRNA and mix well, and then adjust the pH to neutral for reaction to obtain nanoparticles for tumor-targeted self-triggered protein degradation and RNA interference.

[0054] The step S3 specifically includes:

[0055] Dropwise add hydrochloric acid solution to the ferritin carrier solution for tumor-targeted self-triggered protein degradation to adjust the solution pH to 2-3; then add siRNA and mix well; dropwise add sodium hydroxide solution to the reaction system to adjust the solution pH to 7-7.5, and continue the reaction for 2 hours; then centrifuge and wash to obtain nanoparticles for tumor-targeted self-triggered protein degradation and RNA interference.

[0056] Among them,

[0057] The concentration of the hydrochloric acid solution is 0.1-1M;

[0058] The concentration of the sodium hydroxide solution is 0.1-1M;

[0059] The solute of the ferritin carrier solution for tumor-targeted self-triggered protein degradation is a 20-30 mM sodium chloride solution;

[0060] The dosage of the siRNA is 1 / 10 to 1 / 5 of the molar amount of the ferritin carrier.

[0061] The reactions in the above steps can all be carried out at room temperature.

[0062] The present invention provides the application of the nanoparticles for tumor-targeted self-triggered proteolysis and RNA interference in the preparation of anti-tumor drugs.

[0063] The nanoparticles for tumor-targeted self-triggered proteolysis and RNA interference target and regulate mitochondrial iron homeostasis, initiating liver cancer immunotherapy. The nanoparticles for tumor-targeted self-triggered proteolysis and RNA interference have a structure similar to proteolysis-targeting chimeras. They innovatively utilize the natural ubiquitin-proteasome system to degrade ferritin, releasing iron ions and siRNA targeting and interfering with ENO1. By interfering with its transcription, the expression of ENO1 is downregulated, thereby regulating the ENO1-IRP1-Mfrn1 pathway. Eventually, the expression of Mfrn1 is upregulated, increasing the mitochondrial iron transport channels. In particular, the influx of mitochondrial iron is significantly enhanced, promoting the accumulation of reactive iron in mitochondria, leading to the overproduction of toxic ROS, and inducing mitochondrial dysfunction. Thus, the collapse of mitochondria disrupts the redox homeostasis of the whole cell and ultimately triggers tumor cell death. As Figure 2 shown, mitochondrial iron overload mediated by the nanoparticles for tumor-targeted self-triggered proteolysis and RNA interference can activate a powerful anti-tumor immunity by upregulating immune-related pathways, thereby eliminating tumors, which represents a potential strategy for enhancing anti-tumor immunotherapy by interfering with mitochondrial iron homeostasis.

[0064] The preparation and application of the nanoparticles for tumor-targeted self-triggered proteolysis and RNA interference of the present invention will be described in detail below in conjunction with examples and experimental data.

[0065] Example 1 Preparation of dibenzocyclooctyne-labeled ferritin carrier

[0066] Ferritin was purchased from Sigma-Aldrich, with the catalog number F4503, and extracted from horse spleen. Ferritin was dispersed in ultrapure water to obtain a ferritin solution. Dibenzocyclooctyne-N-hydroxysuccinimide ester was added to the ferritin solution. The mass ratio of ferritin to dibenzocyclooctyne-N-hydroxysuccinimide ester was 1:0.15, and the concentration of the ferritin solution was 0.5 mg / mL. The mixture was stirred and reacted for 8 hours, and after washing and ultrafiltration, a dibenzocyclooctyne-labeled ferritin carrier was obtained.

[0067] Example 2 Preparation of dibenzocyclooctyne-labeled ferritin carrier

[0068] Ferritin was dispersed in ultrapure water to obtain a ferritin solution. Dibenzocyclooctyne-N-hydroxysuccinimide ester was added to the ferritin solution. The mass ratio of ferritin to dibenzocyclooctyne-N-hydroxysuccinimide ester was 1:0.15, and the concentration of the ferritin solution could be 1 mg / mL. The mixture was stirred and reacted for 8 hours, and after washing and ultrafiltration, a dibenzocyclooctyne-labeled ferritin carrier was obtained.

[0069] Example 3 Preparation of dibenzocyclooctyne-labeled ferritin carrier

[0070] Disperse ferritin in ultrapure water to obtain a ferritin solution; add dibenzocyclooctyne-N-hydroxysuccinimide ester to the ferritin solution. The mass ratio of ferritin to dibenzocyclooctyne-N-hydroxysuccinimide ester is 1:0.15, and the concentration of the ferritin solution is 2 mg / mL. Stir and react for 8 hours, and after washing and ultrafiltration, obtain a dibenzocyclooctyne-labeled ferritin carrier.

[0071] Example 4 Preparation of a Ferritin Carrier for Tumor-Targeted Self-Triggered Protein Degradation

[0072] Dissolve azide-labeled E3 ligase ligand VH032 (purchased from MCE) in ultrapure water to obtain an E3 ligase ligand VH032 solution with a concentration of 1 mg / mL; dissolve azide-labeled tumor-targeting peptide cyclo-(RGDfk) (purchased from Haode Biology) in ultrapure water to obtain a tumor-targeting peptide cyclo-(RGDfk) solution with a solution concentration of 0.5 mg / mL; add the azide-labeled E3 ligase ligand VH032 solution and the azide-labeled tumor-targeting peptide cyclo-(RGDfk) solution to the dibenzocyclooctyne-labeled ferritin carrier solution for reaction. The mass ratio of azide-labeled E3 ligase ligand VH032, azide-labeled tumor-targeting peptide cyclo-(RGDfk) to dibenzocyclooctyne-labeled ferritin carrier in the reaction system is 0.12:0.1:1; stir and react for 4 hours, and then wash and ultrafilter to obtain a tumor-targeted self-triggered protein degradation ferritin carrier.

[0073] Example 5 Preparation of a Ferritin Carrier for Tumor-Targeted Self-Triggered Protein Degradation

[0074] Dissolve azide-labeled E3 ligase ligand VH032 in ultrapure water to obtain an E3 ligase ligand VH032 solution with a concentration of 1 mg / mL; dissolve azide-labeled tumor-targeting peptide cyclo-(RGDfk) in ultrapure water to obtain a tumor-targeting peptide cyclo-(RGDfk) solution with a solution concentration of 1 mg / mL; add the azide-labeled E3 ligase ligand VH032 solution and the azide-labeled tumor-targeting peptide cyclo-(RGDfk) solution to the dibenzocyclooctyne-labeled ferritin carrier solution for reaction. The mass ratio of azide-labeled E3 ligase ligand VH032, azide-labeled tumor-targeting peptide cyclo-(RGDfk) to dibenzocyclooctyne-labeled ferritin carrier in the reaction system is 0.12:0.1:1; stir and react for 4 hours, and then wash and ultrafilter to obtain a tumor-targeted self-triggered protein degradation ferritin carrier.

[0075] Example 6 Preparation of a Ferritin Carrier for Tumor-Targeted Self-Triggered Protein Degradation

[0076] Dissolve the azide-labeled E3 ligase ligand VH032 in ultrapure water to obtain an E3 ligase ligand VH032 solution with a concentration of 1 mg / mL; dissolve the azide-labeled tumor-targeting peptide cyclo-(RGDfk) in ultrapure water to obtain a tumor-targeting peptide cyclo-(RGDfk) solution with a solution concentration of 2 mg / mL; add the azide-labeled E3 ligase ligand VH032 solution and the azide-labeled tumor-targeting peptide cyclo-(RGDfk) solution to the dibenzocyclooctyne-labeled ferritin carrier solution for reaction. The mass ratio of the azide-labeled E3 ligase ligand VH032, the azide-labeled tumor-targeting peptide cyclo-(RGDfk) to the dibenzocyclooctyne-labeled ferritin carrier in the reaction system is 0.12:0.1:1; stir the reaction for 4 hours, and then wash and ultrafilter to obtain a tumor-targeting self-triggered protein degradation ferritin carrier.

[0077] Example 7 Preparation of Nanoparticles for Tumor-Targeted Self-Triggered Protein Degradation and RNA Interference

[0078] Disperse the tumor-targeting self-triggered protein degradation ferritin carrier in 25 mM sodium chloride solution with a concentration of 1 mg / mL (concentration about 2 μM); add hydrochloric acid solution to the tumor-targeting self-triggered protein degradation ferritin carrier solution. The concentration of the hydrochloric acid solution is 1 M, adjust the solution pH to 2, and stir for 5 minutes; then add siRNA targeting ENO1 (sequence: AGUAUGACCUGGACUUCAAGUTT) to make the siRNA concentration in the solution 0.2 μM, and stir for 15 minutes; then add sodium hydroxide solution to the reaction system. The concentration of the sodium hydroxide solution is 1 M, adjust the solution pH to 7, and stir the reaction for 2 hours; then centrifuge and wash to obtain nanoparticles for tumor-targeted self-triggered protein degradation and RNA interference.

[0079] Experimental Example 1 Characterization of pH-Responsive Depolymerization / Reassembly Characteristics of Ferritin Carrier for Tumor-Targeted Self-Triggered Protein Degradation

[0080] Use transmission electron microscopy (TEM) to characterize the pH-responsive depolymerization / reassembly characteristics of ferritin and the ferritin carrier for tumor-targeted self-triggered protein degradation. As Figure 3 shown, under the condition of pH 2, both ferritin (Fn) and the ferritin carrier for tumor-targeted self-triggered protein degradation (cRGD-VF) depolymerize into ferritin subunits, showing irregular fragments; after readjusting the pH to 7, both ferritin (Fn) and the ferritin carrier for tumor-targeted self-triggered protein degradation (cRGD-VF) reassemble into a cage-like structure with a uniform spherical morphology and a size of about 10 nm.

[0081] Structural Characterization of Nanoparticles for Tumor-Targeted Self-Triggered Protein Degradation and RNA Interference in Experimental Example 2

[0082] The morphology and size of ferritin and nanoparticles for tumor-targeted self-triggered protein degradation and RNA interference were characterized using transmission electron microscopy (TEM); as Figure 4 shown, ferritin (Fn) presented a uniform spherical shape with a cage-like structure and a size of approximately 10 nm; the nanoparticles for tumor-targeted self-triggered protein degradation and RNA interference (cRGD-VFs) presented a uniform spherical shape with the cavity structure filled and a size of approximately 10 nm.

[0083] Application Example 1 In Vitro Experiment

[0084] An in vitro co-culture experiment was carried out using Hepa1-6 tumor cells and nanoparticles for tumor-targeted self-triggered protein degradation and RNA interference (cRGD-VFs), and the in vitro anti-tumor performance of this material was studied by detecting cell viability. Different experimental groups were set up: blank control group (PBS, Control), ferritin group (Fn), self-triggerable degradable ferritin group (VF), ferritin group with RNA interference function (Fs), self-triggerable degradable RNA interference nanoparticles group (VFs), and nanoparticles for tumor-targeted self-triggered proteolysis and RNA interference (cRGD-VFs). Among them, VF is ferritin modified only with the E3 ligase ligand VH032, Fs is ferritin encapsulated only with siRNA, and VFs is VF encapsulated with siRNA.

[0085] First, Hepa1-6 cells were seeded in 96-well plates (1×10 5 cells / well) and cultured in complete DMEM medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) for 24 h. To verify the anti-tumor effect of cRGD-VFs, different concentrations of different nanoparticles were co-cultured with Hepa1-6 tumor cells for 24 h, then 10% CCK8 solution was added, incubated for 1 hour, and the absorbance of each well at a wavelength of 450 nm was measured using a microplate reader. As Figure 5 shown in A, Fn and VF showed negligible cytotoxicity within the set concentration range; in contrast, the cell viability decreased after treatment with VFs, while cRGD-VFs showed stronger tumor-killing ability at the same concentration, which was due to the enhanced uptake of cRGD-VFs by tumor cells due to the modification of the tumor-targeting peptide. Further, the cell viability was measured after co-culturing 80 μg / mL of different nanoparticles with 3T3 normal cells for 24 hours. As Figure 5As shown in Figure B, the nanoparticles (cRGD-VFs) for self-triggered protein degradation and RNA interference for tumor targeting did not show obvious toxic effects on normal cells, indicating the specific killing effect of cRGD-VFs on tumors and good biosafety.

[0086] Application Example 2 Evaluation of the Antitumor Mechanism of Nanoparticles for Self-Triggered Protein Degradation and RNA Interference for Tumor Targeting An in vitro co-culture experiment was carried out using Hepa1-6 tumor cells and nanoparticles (cRGD-VFs) for self-triggered protein degradation and RNA interference for tumor targeting. The in vitro antitumor mechanism of this material was studied by detecting the mitochondrial ferrous level and the mitochondrial reactive oxygen species level. Different experimental groups were set up: blank control group (PBS, Control), ferritin group (Fn), self-triggered degradable ferritin group (VF), ferritin group with RNA interference function (Fs), self-triggered degradable RNA interference nanoparticle group (VFs), and nanoparticles (cRGD-VFs) for self-triggered proteolysis and RNA interference for tumor targeting.

[0087] First, Hepa1-6 cells were seeded in 6-well plates (5×10 5 cells / well) and cultured in complete DMEM medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) for 24 h. Different nanoparticles at 80 μg / mL were co-cultured with Hepa1-6 tumor cells for 24 h, and then the mitochondrial ferrous ion probe MitoFerro-Green and the mitochondrial reactive oxygen species probe MitoSOX-Red were added for staining. As Figure 5 shown in Figure C, in Hepa1-6 tumor cells treated with cRGD-VFs, the green fluorescence of mitochondrial Fe 2+ was significantly enhanced, significantly stronger than that of other groups. Excessive mitochondrial iron can mediate the production of mitochondrial ROS. As Figure 5 shown in Figure D, the strongest red fluorescence signal of mitochondrial ROS was captured in tumor cells treated with cRGD-VFs.

[0088] Application Example 3 In Vivo Antitumor Performance Study of Nanoparticles for Self-Triggered Protein Degradation and RNA Interference for Tumor Targeting

[0089] Using 6-8-week-old C57BL / 6 mice as model animals, a Hepa1-6 tumor-bearing mouse model was constructed by subcutaneous injection of Hepa1-6 tumor cells. The effect of nanoparticles for self-triggered protein degradation and RNA interference for tumor targeting on tumor treatment after tail vein injection was systematically studied. When the tumors of the tumor-bearing mice grew to ~100 mm 3When the tumor-bearing mice were randomly divided into 5 groups: blank control group (PBS, Control), self-triggered degradable ferritin group (VF), ferritin group with RNA interference function (Fs), self-triggered degradable RNA interference nanoparticle group (VFs), and nanoparticle for tumor-targeted self-triggered proteolysis and RNA interference (cRGD-VFs). The drugs were injected into the mice via the tail vein on the 7th, 10th, and 13th days respectively (injection concentration was 2 mg / mL, and 100 μL was injected into each mouse). Every other day, the changes in body weight and tumor size of the mice were recorded and the tumor volume was calculated (Still formula: V = W 2 ×L / 2, where W is the short diameter length of the tumor and L is the long diameter length of the tumor).

[0090] As Figure 6 shown, compared with other control materials (the control materials were PBS, VF, Fs, and VFs), after the treatment with the nanoparticle cRGD-VFs for tumor-targeted self-triggered protein degradation and RNA interference, the tumor growth of the tumor-bearing mice was significantly inhibited, and the tumor volume was significantly smaller than that of other materials, indicating that cRGD-VFs had a good inhibitory effect on tumor growth. During the observation period, there were no abnormal fluctuations in the body weight of the mice, indicating that the materials had good biosafety. Moreover, the survival period of the tumor-bearing mice treated with cRGD-VFs was significantly prolonged, further confirming the outstanding anti-tumor effect of the nanoparticle cRGD-VFs for tumor-targeted self-triggered protein degradation and RNA interference.

[0091] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0092] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0093] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A tumor-targeted self-triggered protein degradation and RNA interference nanoparticle, characterized in that: The nanoparticles use ferritin as a carrier, with siRNA encapsulated in the cavity of ferritin. The siRNA can upregulate mitochondrial iron transporters. The surface of ferritin is modified with an E3 ligase ligand and a tumor-targeting peptide.

2. The tumor-targeted self-triggered protein degradation and RNA interference nanoparticles according to claim 1, wherein: The siRNA is siRNA targeting and interfering with ENO1.

3. The tumor-targeted self-triggered protein degradation and RNA interference nanoparticles according to claim 1, wherein: The E3 ligase ligand includes one or more of VH032, VHL Ligand 8, VHL Ligand 14, and Pomalidomide.

4. The tumor-targeted self-triggered protein degradation and RNA interference nanoparticles according to claim 1, characterized in that: The tumor-targeting peptide includes one or more of cyclo-(RGDfK), GE11 peptide, and B18 peptide.

5. The preparation method of the tumor-targeted self-triggered protein degradation and RNA interference nanoparticles according to any one of claims 1-4, characterized in that, Comprising: Modifying the E3 ligase ligand and the tumor-targeting peptide on the surface of ferritin through a Click reaction; using the pH-dependent depolymerization / reassembly property of ferritin to load siRNA into ferritin to obtain nanoparticles for tumor-targeted self-triggered proteolysis and RNA interference.

6. The preparation method of the tumor-targeted self-triggered protein degradation and RNA interference nanoparticles according to claim 5, wherein, Comprising: S1. Mix ferritin with dibenzocyclooctyne-N-hydroxysuccinimide ester for reaction to obtain a dibenzocyclooctyne-labeled ferritin carrier. S2. Mix the azide-labeled E3 ligase ligand and the azide-labeled tumor-targeting peptide with the dibenzocyclooctyne-labeled ferritin carrier for reaction to obtain a ferritin carrier for tumor-targeted self-triggered protein degradation. S3. Use a pH-adjusting depolymerization / reassembly method to load siRNA into the cavity of the ferritin carrier obtained in step S2 to obtain nanoparticles for tumor-targeted self-triggered proteolysis and RNA interference.

7. The preparation method of the tumor-targeted self-triggered protein degradation and RNA interference nanoparticles according to claim 6, characterized in that, Step S1 includes: Mix the ferritin solution and dibenzocyclooctyne-N-hydroxysuccinimide ester for reaction, then wash and ultrafilter to obtain a dibenzocyclooctyne-labeled ferritin carrier.

8. The preparation method of the tumor-targeted self-triggered protein degradation and RNA interference nanoparticles according to claim 6, wherein Step S2 includes: Add the azide-labeled E3 ligase ligand and the azide-labeled tumor-targeting peptide to the dibenzocyclooctyne-labeled ferritin carrier solution for reaction, then wash and ultrafilter to obtain a ferritin carrier for tumor-targeted self-triggered protein degradation.

9. The preparation method of the tumor-targeted self-triggered protein degradation and RNA interference nanoparticles according to claim 6, characterized in that, Step S3 includes: Dropwise add an acid solution to the ferritin carrier solution for tumor-targeted self-triggered protein degradation to adjust the solution pH to 2 - 3; add siRNA and mix well; dropwise add an alkali solution to the reaction system to adjust the solution pH to 7 - 7.5, and continue the reaction to load siRNA into the cavity of the ferritin carrier; then centrifuge and wash to obtain nanoparticles for tumor-targeted self-triggered protein degradation and RNA interference.

10. Use of the nanoparticles for tumor-targeted self-triggered protein degradation and RNA interference according to any one of claims 1 - 4 in the preparation of an anti-tumor drug.

Images