Ferritin cage nanocarriers for targeting and co-loading hydrophilic / hydrophobic drugs and their applications

Abstract

The present invention uses genetic engineering technology to display functional motifs containing hydrophobic peptides on the outer surface or inner surface of the ferritin cage, and through the hydrophilic ion channels in the ferritin nanocage and the electrostatic adsorption of the hydrophobic peptides displayed on the surface of the protein cage, respectively, the hydrophilic and hydrophobic drugs are loaded into the inner cavity or outer surface of the protein cage nanocarrier, thereby constructing a new type of targeted and hydrophilic and hydrophobic synergistic dual-drug co-loaded protein cage nanocarrier. It is found that the carrier can not only prolong the half-life of small molecule drugs and reduce side effects, but also promote the affinity and permeability of drugs to tumor cells through targeting, and can cross the blood-brain barrier to effectively aggregate in brain tumors. The drug-loaded ferritin cage nanomaterial can improve the therapeutic effect on tumors, especially malignant and drug-resistant tumors, through a synergistic cascade drug release strategy, and can be used as a new ferritin cage nanocarrier platform for synergistic hydrophilic and hydrophobic drug co-loading and targeted combined chemotherapy.

Description

Technical Field

[0001] The invention belongs to the technical field of ferritin loaded drugs, and in particular relates to a ferritin cage nanocarrier for targeting and co-loading hydrophilic / hydrophobic drugs and applications thereof. Background Art

[0002] Ferritin is an iron storage protein that plays an important role in cellular iron homeostasis and anti-oxidation. In recent years, the application of human H-ferritin (HFn) as a drug carrier has greatly developed. As a natural cage protein, HFn has a hollow spherical structure with inner and outer diameters of 8 nm and 12 nm, respectively, and is self-assembled from 24 subunits to form a unique 24-mer protein cage structure. HFn nanocages have the following characteristics: (1) HFn has good biocompatibility, that is, it is non-toxic and non-immunogenic, and can be biodegraded into amino acids, the nutrients needed by the human body; (2) HFn can remain stable and biologically active at high temperatures (80°C); (3) HFn has CD71 (also known as transferrin receptor, TfR 1, which is highly expressed in most tumors and blood-brain barrier (BBB) ​​endothelial cells) targeting, that is, without any targeting modification, HFn can actively target different types of tumor cells including lung cancer and breast cancer by interacting with the receptor (ZL201110122433.0, PCT / CN2012 / 075291), and can cross the BBB. Currently, several CD71-targeted nanodrugs have entered the clinical trial stage for brain tumor treatment, indicating the potential use of HFn in tumor targeting applications; (4) It can disassemble in acidic environments and high-concentration urea environments, and reassemble into a cage-like structure in neutral environments. It can be applied to drug loading and pH-responsive tumor drug delivery; (5) In addition, HFn nanocages can be stably and mass-produced by biological systems (such as E. coli systems). The product HFn protein has a uniform size and a unique cage structure, and other stimulus-responsive functions (such as enzyme responsiveness, etc.) and targeting functions can be introduced through genetic engineering technology, so that the modified HFn has multiple functions and is more intelligent, making it a potential nano-drug delivery system for targeted delivery of anti-tumor drugs. Therefore, due to its advantages such as single controllable molecular weight, pH-responsive release, long half-life, good biorecognition, biocompatibility and biodegradability, it is currently used in tissue engineering or as a delivery carrier for small molecule drugs, biological agents, nucleic acids, etc. In 2014, doxorubicin molecules were first loaded into the inner cavity of human HFn using the gradient urea method, and it has been shown that HFn-Dox has a good anti-tumor effect on three different mouse transplant tumor models of human breast cancer, colorectal cancer, and liver cancer. In 2018, HFn-Dox was shown to effectively penetrate the BBB and target the treatment of brain glioma. Taking advantage of the characteristics of HFn protein cages that they depolymerize and repolymerize under pH control and concentration regulation of denaturants such as urea, anti-tumor chemotherapy drugs (such as platinum drugs, doxorubicin, curcumin, atropine, 5-fluorouracil, etc.), small interfering RNA, photodynamic / photothermal therapy drugs (such as IR820), etc. have been reported to be successfully loaded into the inner cavity of HFn protein cages and exhibited good anti-tumor activity.In summary, the use of HFn for the delivery of chemotherapy drugs in vivo has achieved good therapeutic effects. These preliminary studies show that HFn, as a new generation of natural protein nanocarriers, has broad application prospects in the efficient delivery of small molecule drugs and is expected to become a new growth point and application outlet for the development of biomaterials.

[0003] At present, the drug loading methods of HFn mainly include directly chemically coupling the drug to the HFn surface, and pH or urea-mediated HFn depolymerization / repolymerization method (ZL201410230829.0, PCT / CN2014 / 14015 9). However, these methods will affect the physical and chemical properties of HFn, and cause potential toxicity, making it difficult to achieve industrial transformation, and the drug loading efficiency is limited, which cannot meet clinical needs. Recent studies have found that there are hydrophilic ion channels on HFn nanocages, which can allow hydrophilic small molecule drugs to be loaded in the natural state of ferritin. Heating can further open the channel and promote the loading of hydrophilic drugs (CN202011496039.9), providing a mild, stable and convenient drug loading method for HFn. However, due to the hydrophilicity of soluble proteins, it is still difficult to effectively load hydrophobic drugs on ferritin-based nano drug carriers, and hydrophobic drugs are usually quickly cleared from the body and have extremely low bioavailability, which limits their clinical application. It is a huge challenge to load hydrophilic and hydrophobic drugs on a HFn protein nanocarrier at the same time. Due to the excellent synergistic therapeutic effect of combined chemotherapy, co-loading and controllable delivery of hydrophilic and hydrophobic drugs are also the application requirements of combined chemotherapy in clinical practice. Therefore, it is necessary to further consider new strategies for drug loading using HFn as a nanocarrier. Summary of the invention

[0004] To achieve the above purpose, the present invention prepares a bioengineered ferritin cage nanocarrier (amphiphilic multi-drug loading protein nanocage, Am-PNCage, hereinafter Am-PNCage is uniformly used to refer to ferritin cage nanocarrier), and applies a convenient, fast, efficient, and sufficient method of co-loading hydrophilic and hydrophobic drugs under non-denaturing conditions while maintaining the integrity of the Am-PNCage structure. The Am-PNCage hydrophilic and hydrophobic dual drug co-loading system of the present invention can greatly increase the drug loading amount of ferritin, and thus the clinical dosage will be greatly reduced under the same treatment premise.

[0005] In a first aspect, the present invention provides a ferritin cage nanocarrier for targeting and co-loading hydrophilic / hydrophobic drugs, wherein at least one ferritin subunit in the ferritin cage nanocarrier is modified with a functional amino acid motif containing a hydrophobic polypeptide.

[0006] In certain embodiments, the modification is replacement of the fifth alpha helix of the ferritin subunit with the functional amino acid motif containing the hydrophobic polypeptide.

[0007] In certain embodiments, the hydrophobic polypeptide-containing functional amino acid motif is directly linked to the C-terminus of the ferritin subunit.

[0008] In certain embodiments, the functional amino acid motif containing the hydrophobic polypeptide consists of 5-200 amino acids, preferably 5-15 amino acids.

[0009] In certain embodiments, the hydrophobic polypeptide includes but is not limited to any one of the hydrophobic amino acids A, F, I, L, M, P, V, W, and Y, or a combination of at least two thereof.

[0010] In certain embodiments, the hydrophobic polypeptide is any one of AAVVVFAFAFAFA (SEQ ID NO: 1), AAIMV (SEQ ID NO: 2), LLLVVVAAA (SEQ ID NO: 3), PPWWYYYLVVAA (SEQ ID NO: 4), matrix metalloproteinase MMP-2 responsive peptide PLGLWA (SEQ ID NO: 5), or a combination of at least two thereof.

[0011] In certain embodiments, the functional amino acid motif containing a hydrophobic polypeptide further includes any polypeptide connected to the hydrophobic polypeptide, wherein the any polypeptide is a hydrophilic peptide and / or a targeting peptide.

[0012] In certain embodiments, the hydrophilic peptide is any combination of hydrophilic amino acid sequences, including but not limited to any one or a combination of at least two of the hydrophilic amino acids D, E, H, K, Q, R, S, and T, preferably HHHHHHKK (SEQ ID NO: 6), DDDDEDDDDE (SEQ ID NO: 7), RRQQHHHHHH (SEQ ID NO: 8), DDDSSTT (SEQ ID NO: 9), DDEEKKQS (SEQ ID NO: 10), and HHEQSSTTT (SEQ ID NO: 11);

[0013] In certain embodiments, the targeting peptide is preferably any one of the tumor targeting peptide GRGDS (SEQ ID NO: 12), the tumor targeting peptide QHWSYGLRPG (SEQ ID NO: 13), the fibrin targeting peptide CREKA (SEQ ID NO: 14), the brain capillary endothelial cell targeting peptide HAIYPRH (SEQ ID NO: 15), the macrophage targeting peptide YEQDPWGVKWWY (SEQ ID NO: 16), the nerve cell targeting peptide CLEVSRKNC (SEQ ID NO: 17), and the cardiomyocyte targeting peptide CSTSMLKAC (SEQ ID NO: 18), or a combination of at least two of them.

[0014] In a second aspect, the present invention provides a use of the ferritin cage nanocarrier described in the first aspect in loading and delivering hydrophilic and / or hydrophobic small molecule drugs, wherein the loading is performed on the ferritin cage nanocarrier under non-denaturing conditions.

[0015] In certain embodiments, the small molecule drugs include but are not limited to chemical small molecule drugs, nucleic acid drugs, and radioactive drugs.

[0016] In certain embodiments, the loading molar mass ratio of the ferritin cage nanocarrier to the hydrophilic small molecule drug is 1:100 to 1:180, preferably, 1:130 to 1:150.

[0017] In certain embodiments, the hydrophilic small molecule drugs are anthracyclines, platinums, pyrimidines, and alkaloids; anthracyclines are preferably epirubicin and daunorubicin; platinums are preferably cisplatin, carboplatin, and oxaliplatin; pyrimidines are preferably gemcitabine, capecitabine, and cytarabine; and alkaloids are preferably irinotecan.

[0018] In certain embodiments, the molar mass ratio of the ferritin cage nanocarrier to the hydrophobic small molecule drug is 1:20 to 1:70, preferably, 1:30 to 1:50.

[0019] In certain embodiments, the hydrophobic small molecule drugs are pyrimidines, taxanes, and nitrosoureas; the pyrimidines are 5-fluorouracil and gimeracil, the taxanes are paclitaxel and docetaxel, and the nitrosoureas are temozolomide and nimustine.

[0020] In certain embodiments, the buffer in the drug-loaded reaction system is any buffer with a pH of 6 to 8, preferably, Tris-HCl buffer; and / or, preferably, the solution concentration is 10 to 300 mM.

[0021] In certain embodiments, the small molecule nucleic acid drug is preferably siRNA or lncRNA.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] 1. Through genetic engineering modification, a functional motif P composed of hydrophobic peptide-hydrophilic peptide-RGD peptide (targeting integrin ɑvβ3 highly expressed on the surface of tumor cells) was used. out By replacing the fifth helix of the human HFn subunit, the functional peptide motif is displayed on the outer surface of the ferritin cage; or by directly connecting the hydrophobic peptide motif Pin to the C-terminus of human HFn, the functional peptide motif is displayed on the inner surface of the ferritin cage, and two types of Am-PNCage recombinant plasmids are constructed, and the corresponding proteins are expressed and purified by E. coli. Therefore, its structure and function are completely precisely controllable.

[0024] 2. The present invention ensures that the Am-PNCage structure remains unchanged and under non-denaturing conditions, through a simple and easy two-step method, the hydrophilic drug EPI and the hydrophobic drug CPT are spatially loaded into the inner cavity and outer shell of the Am-PNCage nanocarrier respectively to prepare EPI@Am-PNCage-P out / CPT nanoparticles. Or, through a simpler one-step method, the hydrophilic EPI and hydrophobic CPT drug molecules are simultaneously loaded into the inner cavity of the Am-PNCage nanocarrier to prepare EPI / CPT@Am-PNCage-P in Nanoparticles. For EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P in , the molar ratios of EPI and CPT loadings on one nanocarrier were 1:132:50 and 1:150:39, respectively, which are the maximum loadings obtained by HFn-based nanocarriers so far.

[0025] 3. The present invention explored the EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P in The co-loading of drugs at different spatial sites and the cascade drug release kinetics triggered by the amphiphilic structure. out / CPT and EPI / CPT@Am-PNCage-P in The anti-cell proliferation biological activity, synergistic coefficient, targeting tumor cell affinity, tumor penetration ability and in vitro anti-resistant tumor therapeutic efficacy of EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P inThe pharmacokinetics, biodistribution, maximum tolerated dose, tumor targeting, therapeutic effects on different tumor models and biosafety of Am-PNCage were systematically studied. The effects of the structure and function of Am-PNCage on the release kinetics, activity, stability, in vivo pharmacokinetics, biodistribution, efficacy and biosafety of loaded drugs were systematically studied, and the synergistic mechanism was clarified.

[0026] 4. The bioengineering modification method proposed in the present invention can be used to conveniently, quickly, efficiently and in sufficient quantities to load different hydrophilic / hydrophobic anticancer drug combinations commonly used in the clinical frontline, and exhibit excellent anti-tumor proliferation activity and anti-drug resistance function. As a broad-spectrum multi-drug delivery platform, it can be widely used in targeted combined chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific implementation manner of the present invention or the technical solution in the prior art, the drawings required for describing the specific implementation manner are briefly introduced below.

[0028] Figure 1 Synthesis and characterization of Am-PNCage. (A) SDS-PAGE characterization of purified HFn and Am-PNCage proteins; (B) Size exclusion analysis of HFn and Am-PNCage; (C) TEM image analysis; (D) Hydrodynamic size of nanocarriers measured by DLS.

[0029] Figure 2 For EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P in Synthesis and characterization of HFn and Am-PNCage. (A) Molecular exclusion analysis after drug loading; (B) TEM image analysis; (C) DLS particle size analysis.

[0030] Figure 3 For EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P in In vitro drug release kinetics. (A) Stability in PBS and mouse serum for 72 hours at 37°C; (B) Release of EPI and CPT in PBS at different pH values ​​(5.0 and 7.4); (C) CLS M analysis of the endocytosis of dual-drug co-loaded Am-PNCage nanoparticles by U87 cells and the sequential release of EPI and C PT at different times. Purple, lysosome; red, nucleus; green, EPI; blue, CPT.

[0031] Figure 4For EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P in (A) U87, HepG2 and MCF7 / MDR cell viability after 48 h of treatment with Am-PNCage nanocarriers loaded with dual drugs or single drugs; (B) EPI@Am-PNCage-P out / CP T and EPI / CPT@Am-PNCage-P in CI values ​​in different cell lines; (CD)EPI@Am-P NCage-P out / CPT and EPI / CPT@Am-PNCage-P in Inhibitory effect on cell colony formation.

[0032] Figure 5 Dual-targeted EPI@Am-PNCage-P out / CPT specifically targets tumor cells and promotes drug penetration. (A)EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P in Binding to tumor cells; (B) Flow cytometry to detect the affinity of U87 cells; (C) EPI@Am- PNCage-P out / CPT permeability in MCF7 / MDR 3D cell spheroids; (D) EPI fluorescence intensity distribution of cross-sectional tomography at 70 μm; (E) EPI@Am-PNCage-P out / CPT and EPI / CP T@Am-PNCage-P in Inhibitory effect on the growth of MCF7 / MDR resistant cell spheres; (F) Volume changes of MCF7 / MDR cell spheres within 6 days.

[0033] Figure 6 For EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P in Changes in blood drug concentration over time.

[0034] Figure 7 For EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P in In vivo biodistribution of . Near-infrared fluorescence images (A, B) and quantification (C, D) in tumors and major tissues at 1, 4, and 24 h after injection (n=3).

[0035] Figure 8For EPI@Am-PNCage-P out / CPT(A) and EPI / CPT@Am-PNCage-P in (B) Maximum tolerated dose.

[0036] Fig. 9 For EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P in Antitumor efficacy against U87 brain glioma (A), HepG2 liver cancer lung metastases (B), and MCF7 / MDR (C) resistant breast cancer.

[0037] Fig.10 For EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P in (A) H&E staining of the main tissues and organs of mice after injection of the maximum tolerated dose; (B) Hematological and biochemical analysis.

[0038] Fig.11 The broad spectrum of hydrophilic and hydrophobic drugs co-loaded on Am-PNCage. (A) Absorption spectra of various small molecule anticancer drugs; (BK) Am-PNCage-P out and Am-PNCage-P in Absorption spectra after loading different combinations of synergistic hydrophilic and hydrophobic drugs. The dashed line at 280 nm indicates the absorption peak of Am-PNCage protein.

[0039] Fig.12 Am-PNCage-P out (A) and Am-PNCage-P in (B) Cytotoxicity assay after co-loading of different hydrophilic and hydrophobic drug combinations.

[0040] Fig.13 IC50 values ​​of Am-PNCage nanocarriers after loading dual drugs or single drug. DETAILED DESCRIPTION

[0041] The following will be described in detail with reference to the accompanying drawings. The following examples are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. The reagents used in the examples are all commercially available conventional products unless otherwise specified.

[0042] definition

[0043] The ferritin of the present invention refers to any ferritin that can form a cage-like structure, which can be a ferritin of natural origin, or a recombinantly expressed ferritin, or a mutant thereof, which can be derived from prokaryotes, protists, fungi, plants or animals, such as bacteria, fungi, insects, reptiles, birds, amphibians, fish, mammals, such as rodents, ruminants, non-human primates or humans, such as mice, rats, guinea pigs, dogs, cats, cattle, horses, sheep, monkeys, gorillas, and humans. From bacteria to humans, although the amino acid sequences of ferritins of different organisms are very different, their structures are similar and can form a protein shell structure. In some embodiments, the ferritin of the present invention is human ferritin, and in some embodiments, the ferritin of the present invention is a genetically engineered full-human heavy chain ferritin, and its subunit nucleotide sequence is shown in SEQ ID NO: 3.

[0044] The hydrophobic polypeptide of the present invention refers to an amino acid sequence containing hydrophobicity, including but not limited to any one or a combination of at least two of the hydrophobic amino acids A (alanine), F (phenylalanine), I (isoleucine), L (leucine), M (methionine), P (proline), V (valine), W (tryptophan), and Y (tyrosine), as well as a hydrophobic polypeptide with biological function. The hydrophilic polypeptide of the present invention refers to any combination of hydrophilic amino acid sequences other than hydrophobic amino acids, including but not limited to hydrophilic amino acids D (aspartic acid), E (glutamic acid), H (histidine), K (lysine), Q (glutamine), R (arginine), S (serine), and T (threonine).

[0045] The drug or entrapped drug of the present invention refers to any drug that can be entrapped in ferritin, as long as the molecular size of the drug is less than 8 nanometers. In some embodiments, the drug or entrapped drug of the present invention is selected from antibiotic tumor drugs, natural source anti-tumor drugs, metal compounds, radioactive isotopes, alkylating agents, antimetabolite anti-tumor drugs, and hormone anti-tumor drugs. The antibiotic anti-tumor drugs are selected from doxorubicin (doxorubicin hydrochloride), daunorubicin hydrochloride, valrubicin, bleomycin sulfate, mitomycin, epirubicin hydrochloride, idarubicin hydrochloride, actinomycin D, mithramycin, daunorubicin, pirarubicin, epirubicin, idarubicin, aclarubicin, bleomycin A 5, chromomycin A3, bleomycin hydrochloride, chromycin, pingyangmycin hydrochloride, daunorubicin, aclarubicin hydrochloride, azotocin, pirarubicin, pirarubicin hydrochloride, actinomycin C; the natural source anti-tumor drugs are selected from topotecan hydrochloride, 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, 1 0-hydroxycamptothecin, rubicin, resveratrol, camptothecin, paclitaxel, colchicine, etoposide, docetaxel, vinblastine sulfate, cantharidin, irinotecan, sunitinib, podophyllotoxin, Topotecan, crizotinib, homoharringtonine, epothilone, teniposide, palbociclib, monocrotaline, vincristine, vinorelbine, vindesine sulfate, indirubin, vincristine sulfate, norcantharidin, norcantharidin, podophyllotoxin, docetaxel, colchicine, cephalotaxine, epothilone C, epothilone E, cinobufina, vindesine sulfate, norcantharidin sodium, maytansine, scutellaria baicalensis, podophyllotoxin, vinorelbine bitartrate, iris root, brucea javanica oil, mitozazide, cephalotaxine, colchicin, hydroxycamptothecin, methylcantharidin; the metal compound is selected from carboplatin, cisplatin, nedaplatin, oxaliplatin, etc.; the radioactive isotope is selected from polonium, radon, francium, radium, actinium, thorium, Protactinium, uranium, neptunium, plutonium, technetium, promethium, americium, curium, berkelium, californium, einsteinium, fermium, mendelevium, nobelium, lawrencium, elements 104, 105, 106, 107, 108 and 109;The alkylating agent is selected from bendamustine hydrochloride, mizolastine, bepotastine besilate, busulfan, enbicin, dacarbazine, lomustine, phenylbutyrate nitrogen, mustard carmustine, triethylenethiophosphoramide, nimustine hydrochloride, melphalan nimustine, bendamustine, estramustine, sodium 2,3-dibromo-1,4-butenediol phosphate, estramustine, hexamethylmelamine, fotemustine, nimustine, gammustine, prednimustine, estramustine, icomustine phosphate, tamustine, amomustine, estramustine, bendamustine hydrochloride impurity A, nemustine, Bofumustine, epinastine hydrochloride, bendamustine hydrochloride impurity C, lomustine capsules, betahistine hydrochloride, betahistine hydrochloride, dithiomustine, tauromustine, epimustine, spiromustine, bendamustine hydrochloride impurity B, prednimustine, semustine, ranimustine, carboquinone, methyluracil nitrogen mustard, nitrogen mustard, epinastine, uramustine base, dibromomannitol, oxychloride mustard hydrochloride, oxychloride mustard, epipiperidine, o-lipid mustard, dehydrodole, iminoquinone, methoxy mustard, enprobamate, glycophosphodiesterone mustard, nitrocarbone, isoaromatic mustard, nitrogen methyl, triiminoquinone, mannitol nitrogen mustard, 2, 4,6-triethyleneimine-1,3,5-triazine; antimetabolite antineoplastic drugs selected from methotrexate, 5-fluoro- 2'-deoxyuridine nucleoside, gemcitabine, doxifluridine, cytarabine, 6-thioguanine, gemcitabine hydrochloride, fludarabine phosphate, vinorelbine tartrate, fludarabine, temozolomide, pentoxifylline, clorabine, nelarabine, cyclocytidine hydrochloride, tegafur, cytarabine hydrochloride, 6-mercaptopurine, 4-hydroxy-5-fluoropyrimidine, 5-fluorouracil, aminopterin, miltefosine, raltitrexed, deoxycoformycin, carmofur, amsacrine, ethiprodoxil, tegafur, cyclosporine, methylisoindigo, fluorouracil, inosine dialdehyde, 1-vinyl-1-methyl-2,4-di(prop-1-en-2-yl)cyclohexane, sulfathiopurine sodium; Hormonal anti-tumor drugs selected from exemestane, raloxifene, fulvestrant, letrozole, anastrozole, Flutamide, tamoxifen citrate, droloxifene, idoxifene, nilutamide, aminoglutethimide, formestane, tamoxifen, toremifene, aminoglutethimide. ;

[0046] The hydrophilic small molecule drugs of the present invention are anthracyclines, platinums, pyrimidines, and alkaloids. Anthracyclines are more preferably epirubicin and daunorubicin; platinums are more preferably cisplatin, carboplatin, and oxaliplatin; pyrimidines are more preferably gemcitabine, capecitabine, and cytarabine; alkaloids are more preferably irinotecan.

[0047] The hydrophobic small molecule drugs of the present invention are pyrimidines, taxols, and nitrosoureas. Pyrimidines are more preferably 5-fluorouracil and gimeracil; taxols are more preferably paclitaxel and docetaxel; nitrosoureas are more preferably temozolomide and nimustine.

[0048] Without being limited to any theory, the drug-loaded ferritin of the present invention can target tumors, release the loaded drug after binding to the tumor, and the drug acts on the tumor to achieve the prevention and / or treatment of the tumor. For example, human ferritin may bind to its receptor transferrin receptor (Transferrin receptor) in Receptor 1, TfR1) specifically targets human solid tumors and blood malignant cells, such as lung cancer, breast cancer, prostate cancer, cervical cancer, colorectal cancer, ovarian cancer, esophageal cancer, gastric cancer, thymic cancer, T lymphocytic leukemia, erythrocytic leukemia, etc.

[0049] In some embodiments, the drug or encapsulated drug of the present invention is selected from drugs other than anti-tumor drugs, that is, non-anti-tumor drugs. For example, such drugs can be drugs that do not require targeting, such as drugs for systemic administration, such as poorly soluble, unstable and / or drugs that are prone to interactions and become ineffective. In some embodiments, such drugs are selected from amphotericin B, glatiramer acetate, complex sodium iron gluconate, rapamycin, sevelamer hydrochloride sulfate binder, verteporfin for injection, iron sucrose, pegylated interferon α-2a / 2b, fenofibrate, pegfilgrastim, riprepifgrastim, amikacin, fentanyl, cyclosporine, cetirizine, capsaicin, ceramide, etc. In some embodiments, the non-anti-tumor drug of the present invention is selected from radiopharmaceuticals, neurotransmitter drugs, dopamine receptor agonists, central nervous system anticholinergics, cholinergic receptor agonist drugs, γ-secretase inhibitors, antioxidants or anesthetics, and more preferably, the radiopharmaceutical is selected from 64 Cu, 235 U, neurotransmitter drugs are selected from carbachol, atropine, scopolamine, dopamine and its derivatives, dopamine receptor agonists are selected from ergot alkaloid derivatives and non-ergot alkaloid derivatives such as bromocriptine, pergolide, apomorphine, etc., central nervous system anticholinergics are selected from trihexyphenidyl, benztropine and procyclidine, cholinergic receptor agonists are selected from muscarine and pilocarpine, gamma secretase inhibitors are selected from diflunisal, antioxidants are selected from melatonin, and anesthetics are selected from anthramines.

[0050] In some embodiments, when encapsulating a drug into ferritin, an additive needs to be added to the incubation solution to promote the dissolution of the drug and / or the aggregation of ferritin, thereby achieving and / or improving the drug encapsulation effect. For example, in some embodiments, it is necessary to add DMA, DMF, DMSO or a mixture thereof to the incubation solution when encapsulating a platinum drug. Depending on the drug to be encapsulated, the additives that need to be added may also be different accordingly.

[0051] In some embodiments, the buffer system of the incubation solution is a Tris-HCl buffer, a phosphate buffered saline (PBS), a carbonate buffer, a glycine buffer or a citrate buffer, the pH of the buffer is between 6-8, for example, 6.5, 7, 7.5, and the concentration of the buffer is between 20 and 500 mM, more preferably, 25 to 100 mM, more preferably, 25 to 50 mM. Depending on the different buffers used, their buffering capacity, pH range, required concentration, etc. may need to be adjusted.

[0052] Example 1 Construction of bioengineered ferritin cage nanocarrier Am-PNCage

[0053] 1. Using genetic engineering technology, a functional amino acid motif P composed of hydrophobic peptide-hydrophilic peptide-RGD peptide was used. out (AAVVVFAFAFAFAHHHHHHKKGRGDS (SEQ ID NO: 19)) replaced the fifth helix of the human HFn subunit and displayed the polypeptide functional motif on the outer surface of the ferritin cage to construct Am-PNCage-P out (SEQ ID NO: 20). The functional amino acid motif P composed of a hydrophobic peptide in (AAVVVFAFAFAFA (SEQ ID NO: 1)) was directly linked to the C-terminus of HFn, and the polypeptide functional motif was displayed on the inner surface of the ferritin cage to construct Am-PNCage-P in (SEQ ID NO: 21). The two Am-PNCage recombinant plasmid sequences and the DNA sequence of the HFn subunit (SEQ ID NO: 22) were synthesized (Generay, Shanghai), digested with NdeI and Bam H1 restriction endonucleases, and cloned into the E. coli expression vector pET22b(+) plasmid (Novagen) with NdeI and BamHI restriction enzyme sites, and the sequences were confirmed to be correct by DNA sequencing.

[0054] 2. Expression and purification of Am-PNCage

[0055] Protein expression: The plasmid obtained above was transferred into the E. coli BL21 (TransGen) expression strain. The transformed E. coli was grown overnight in LB medium containing 100 mg / L ampicillin, and then 0.5 mM IPTG (Sigma-Aldrich) was used for induction of protein expression at 30°C for 8 h.

[0056] Protein purification: The cells were collected by centrifugation at 4000g for 15 min and resuspended in Tris buffer (20mM Tris, pH 8.0). The resuspended E. coli cells were homogenized and crushed by high pressure, and the supernatant was collected by centrifugation at 12000g for 30 min. The supernatant was heat treated at 80°C for 20 min to denature and precipitate most of the E. coli impurities, and the supernatant was collected by centrifugation at 12000g for 30 min again. Subsequently, HFn and Am-PNCage proteins were purified and separated using anion exchange column Q-Sepharose Fast Flow (GE Healthcare), and finally purified and separated using superdex200 (10 / 300GL, GE Healthcare) molecular sieve. The concentration of HFn and its mutant proteins was determined using BCA protein detection kit (Pierce) with bovine serum albumin as the standard, and repeated three times.

[0057] The protein purity was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the successful self-assembly of Am-PNCage was verified by size exclusion chromatography (SEC). The morphology of Am-PNCage was further characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS).

[0058] The results are as follows Figure 1 As shown, SDS-PAGE analysis shows that the peptide-modified Am-PNCage has a single band similar to HFn at a molecular weight of about 21kDa, which is consistent with the theoretical value. The elution volume of Am-PNCage on the SEC column is similar to that of the 24-mer HFn protein cage, proving that Am-PNCage successfully self-assembles into a cage structure. And under TEM, it shows a spherical morphology similar to HFn, indicating that peptide modification will not affect the overall nanocage structure of the protein. DLS results show that the average particle size of Am-PNCa ge is 14-16nm, which is consistent with the TEM results.

[0059] Example 2 Method for Co-loading Synergistic Hydrophilic and Hydrophobic Drugs into Bioengineered Ferritin Nanocages (Am-PNCage)

[0060] 1. For polypeptide functional motif P out Am-PNCage-P displayed on the outer surface of the ferritin cage out , Am-PNCage-P out The nanocarrier and the hydrophilic drug EPI were incubated at 60°C in the dark for 4 h. The free EPI drug molecules were removed using a dialysis bag (MWCO 3500Da, Thermofisher Scientific) to obtain the product EPI@Am-PNCage-P outThen, it was mixed with the hydrophobic drug CPT and stirred overnight at room temperature in the dark, and the free CPT drug molecules were removed by dialysis to obtain the final product EPI@Am-PNCage-P out / CPT. Through a simple two-step method, the hydrophilic drug EPI and the hydrophobic drug CPT were spatially loaded into Am-PNCage-P out The inner cavity and outer shell of the nanocarrier were prepared according to the above method. out and Am-PNCage-P out / CPT was used as control.

[0061] 2. For polypeptide functional motif P in Am-PNCage-P displayed on the inner surface of the ferritin cage in , Am-PNCage-P in The nanocarrier was mixed with the hydrophilic drug EPI and the hydrophobic drug CPT and incubated at 60°C in the dark for 4 hours. The free drug molecules were removed using a dialysis bag to obtain the product EPI / CPT@Am-PNCage-P in EPI@Am-PNCage-P was prepared according to the above method. in and CPT @Am-PNCage-P in As a control.

[0062] 3. The successful loading of two drugs, EPI and CPT, was verified by SEC and the drug loading amount was calculated. The morphology of Am-PNCage before and after drug loading was characterized by TEM, and the hydrated particle size was determined by DLS.

[0063] The results are as follows Figure 2 As shown:

[0064] For P out Am-PNCage-P displayed on the outer surface of the ferritin cage out Through a simple two-step reaction, the hydrophilic EPI is first encapsulated into Am-PNCage-P through the hydrophilic ion channel of HFn by heat treatment. out The complex is then incubated with CPT, which interacts with the hydrophobic P displayed on the outer surface of the protein cage through hydrophobic interactions. out Peptide binding. The hydrophilic and hydrophobic drugs can be loaded into Am-PNCage-P out EPI@Am-PNCa ge-P on the inner cavity and outer shell of the nanocarrier out / CPT. The results showed that 1 Am-PNCage-P outThe nanocarrier can stably load about 132 EPI molecules and about 50 CPT molecules.

[0065] For P in Am-PNCage-P displayed on the inner surface of the ferritin cage in Through a simpler one-step reaction, the hydrophilic drug EPI and the hydrophobic drug CPT can be co-encapsulated into Am-PNCage-P in The product EPI / CPT@Am-PNCage-P was obtained in the inner cavity of the nanocarrier. in CPT interacts with the hydrophobic P displayed on the inner surface of the protein cage through hydrophobic interactions. in The results showed that 1 Am-PNCa ge-P in The nanocarrier can stably load about 150 EPI molecules and about 39 CPT molecules.

[0066] By comparison, the drug loading of this method is the highest drug loading of HFn-based nanocarriers reported so far. SEC results show that HFn can only load the hydrophilic drug EPI but not the hydrophobic drug CPT. The modification of hydrophobic peptides overcomes the limitation of HFn hydrophilicity, thus achieving the purpose of effectively loading hydrophilic and hydrophobic drugs on a protein nanocarrier. The eluate of Am-PNCage protein nanocage has two specific absorption peaks at 480nm and 365nm, corresponding to EPI and C PT, respectively, proving that EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P in The EPI@Am-PNCage-P out / CPT and EPI / CPT@A m-PNCage-P in The co-loading of drugs failed to affect the monodisperse state and self-assembled cage-like properties of HFn nanocages, with an average particle size of 14-16 nm.

[0067] Example 3 EPI and CPT were loaded into different spatial structural sites of Am-PNCage nanocarriers to induce different drug release mechanisms, showing the kinetic characteristics of drug cascade release

[0068] 1. In vitro stability and drug cascade release kinetics

[0069] The stability of EPI@Am-PNCage / CPT in serum and PBS and the drug release properties under different pH conditions were characterized in vitro at 37°C by dialysis method. out / CPT and EPI / CPT@Am-PNCage-P in To investigate the drug release characteristics of the drug, 10 mg of the sample was dissolved in 2 mL of PBS solution and placed in a dialysis tube, which was immersed in 30 mL of buffer solution with pH values ​​of 7.4 and 5.0, respectively, and dialyzed at 37°C. The released drug concentration was calculated by detecting the fluorescence intensity of EPI and CPT (λex = 480 nm for EPI and λex = 365 nm for CPT). The stability of the Am-PNCage nanocarriers loaded with dual drugs was evaluated by incubating them with PBS (pH = 7.4) or normal mouse serum in the same manner.

[0070] 2. Intracellular drug delivery process

[0071] U87 glioma cells were incubated with EPI@Am-PNCage-P at 37°C. out / CPT or EPI / CP T@Am-PNCage-P in After incubation for different time (0.5, 2, 6, 24 and 48 h), the cells were washed with cold PBS and fixed with 4% formaldehyde for 20 min at room temperature. Lysosomes were further stained with lysosomal associated membrane protein 1 (LAMP1) and nuclei were stained with propidium iodide (PI). EPI@Am-PN Cage-P was observed under confocal laser scanning microscopy (CLSM, Carl Zeiss LSM 700). out / CPT or EPI / CPT@Am-PNCage-P in The process of endocytosis into tumor cells and intracellular localization, as well as the rate and order of drug release within cells.

[0072] The results are as follows Figure 3 As shown:

[0073] Using dialysis to study drug release performance, EPI@Am-PNCage-P out / CPT and EPI / CPT@ Am-PNCage-P in It was stable in mouse serum and PBS (pH=7.4), with no significant drug release within 72 hours. However, a faster release was observed when incubated at pH 5.0: nearly 68%-69% of EPI and 47% of CPT were released after only 6 hours, while 90% of EPI and CPT were released after 48 hours, indicating that low pH caused the decomposition of HFn nanocages and showed a sequential release of EPI and CPT.

[0074] CLSM was used to further study the release process of the drug in cells. out / CPT or EPI / CPT@Am-PNCage-P in After incubation of U87 cells for 2 hours, the nanocarriers were mainly located in the lysosomes. After 6 hours, although EPI (green fluorescence) was mainly located in the nucleus, CPT (blue fluorescence) was still mostly retained in the lysosomes. 24 hours after incubation, both EPI and CPT released from Am-P NCage nanocarriers accumulated in the nucleus, acting on DNA and activating apoptotic pathways, leading to nuclear lysis and death within 48 hours. Therefore, EPI and CPT are released from the nanocarriers in lysosomes in sequence and eventually enter the nucleus to exert their anticancer effects. This may be due to the different drug loading pathways and release mechanisms of EPI and CPT. EPI@Am-PNCage- P out / CPT or EPI / CPT@Am-PNCage-P in After the targeted binding receptor is internalized into the cell, it is absorbed by the lysosome pathway. In the acidic environment of the lysosome, the EPI is released as the nanocage slowly disintegrates. As the hydrophobic effect on the surface gradually weakens, CPT is further slowly released. The spatiotemporally programmed cascade drug release kinetics prolongs the retention time of the drug in the cell, which helps to enhance the anti-tumor activity.

[0075] Example 4 Am-PNCage-loaded hydrophilic and hydrophobic dual drugs improve cytotoxicity and anti-resistance efficacy through synergistic effects

[0076] 1. CCK8 cytotoxicity assay

[0077] U87 brain glioma cells, HepG2 liver cancer cells and MCF7 / MDR resistant breast cancer cells were selected and cell proliferation kit 8 (CCK8) was used to study the dual-drug co-loaded EPI@Am-PNCage-P through cell proliferation assay. out / CPT and EPI / CPT@Am-PNCage-P in The toxicity to tumor cells was compared with that of Am-PNCage nanocarriers loaded with one of the drugs and free small molecule drugs. The median lethal dose (IC50) was calculated using Origin 8.0 (OriginLab, Northampton, MA).

[0078] 2. Drug interaction studies

[0079] The interaction properties and synergistic parameters of the two drugs EPI and CPT co-loaded in Am-PNCage were calculated by the drug interaction equation CIx = Da / Dxa+Db / Dxb. The inhibitory concentration (ICx) value was determined using Origin 8.0 (OriginLab, Northampton, MA). Where Da and Db are the concentrations of drug a and drug b that work together. Dxa and Dxb are the concentrations of individual drugs that achieve the same effect. CI>1 indicates antagonism, CI=1 indicates additive reaction, and CI<1 indicates synergism.

[0080] 3. Tumor cell colony formation assay

[0081] 2×10 3 U87 cells were seeded into 6-well plates and incubated with the drug-loaded Am-PNCage nanocarriers at 37°C for 24 hours. The drug solution was then replaced with fresh culture medium and cultured for 10 days until macroscopic cell colonies were formed. The cells were stained with crystal violet and counted to study the EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P in Anti-tumor cell efficacy over a longer period of time.

[0082] The results are as follows Figure 4 As shown in Table 1:

[0083] Dual-drug co-loaded EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P in The inhibitory effect on U87 brain glioma cells, HepG2 liver cancer cells, and MCF7 / MDR resistant breast cancer cells was significantly greater than that of the single drug carrier (EPI@Am-PNCage-P out 、Am-PNCage-P out / CPT, EPI@Am-PNCage-P in and CPT@Am-PNCage-P in ). According to the IC50 value results, EPI@Am-PNCage-P out / CPT has higher toxicity to tumor cells than EPI / CPT@Am-PNCage-P in , and can more effectively overcome the drug resistance of tumor cells, which may be due to the fact that P out The peptide contains the RGD sequence that targets the tumor cell surface integrin ɑvβ3, so EPI@Am-PNCage- P out / CPT overcomes the drug efflux effect of transporters by enhancing drug entry mediated by dual targeting of CD71 and ɑvβ3 and increasing intracellular drug concentration.

[0084] In order to demonstrate the synergistic effect of EPI and CPT loaded on Am-PNCage nanocarriers, the combination index (CI) of drug interaction properties was calculated according to the equation CIx = Da / Dxa + Db / Dxb. out / CPT and EPI / CPT@Am-PNCage-P in The CI values ​​were all less than 1 within the effective drug level range, indicating that there was a significant synergistic effect between EPI and CPT.

[0085] The results of cell colony formation experiments showed that out / CPT and EPI / CP T@Am-PNCage-P in The colony formation was significantly inhibited in the treated tumor cells, which further proved that EPI and CPT loaded with Am-PNCage had excellent synergistic effects and could inhibit tumor cell proliferation for a long time.

[0086] Table 1: IC50 values ​​of Am-PNCage nanocarriers loaded with dual drugs or single drug

[0087]

[0088] Example 5 Dual targeting promotes specific binding, tumor penetration and in vitro therapeutic effects

[0089] 1. CLSM study of target specificity

[0090] In order to study EPI@Am-PNCage-P out / CPT interacts with CD71 and αvβ3, and E PI@Am-PNCage-P out U87 tumor cells treated with 10 μM / CPT were co-incubated with anti-CD71 and anti-α v β 3 monoclonal antibodies or one of them. The cells were then fixed with 4% pre-cooled formaldehyde and the nuclei were further stained with PI. The EPI@Am-PN Cage-P bound to the tumor cell membrane was observed by CLSM. out / CPT content to study the specificity of dual targeting. In order to study the interaction of EPI / CPT@Am-PNCage-P with CD71 receptor in Receptor-mediated targeting of EPI / CPT@Am- PNCage-P inU87 tumor cells treated with (10 μM) were incubated with anti-CD71 monoclonal antibodies, and CD71 targeting specificity was studied by CLSM as described above.

[0091] 2. Flow cytometry to study affinity

[0092] For binding capacity analysis, Cy5-labeled Am-PNCage and HFn were loaded with drugs and 100 μL of dispersed U87 cell suspension (2.5×10 6 cells / mL) were respectively treated with 10 μM EPI@ Am-PNCage-P out / CPT,EPI@Am-PNCage-P out , Am-PNCage-P out / CPT, Am-PNCage-P out or EPI / CPT@Am-PNCage-P in ,EPI@Am-PNCage-P in , Am-PNC age-P in / CPT,Am-PNCage-P in , and HFn and PBS were incubated at 4°C for 45 minutes. The treated tumor cells were analyzed for drug fluorescence intensity using a flow cytometer system to compare the dual-targeting function of Am-PNCage-P out Vector and single Am-PNCage-P targeting CD71 in The tumor cell affinity was compared with that of HFn.

[0093] 3. 3D cell spheroid experiment

[0094] (1) Dual targeting promotes drug tumor penetration

[0095] 2×10 3 MCF7 / MDR drug-resistant breast cancer cells were seeded into ultra-low adhesion round-bottom 96-well plates (Corning, USA) to form 3D cell spheres. out / CPT, EPI@Am-PNCage-P out , Am-PNCage-P out / CPT, EPI@HFn, free EPI, and free CPT were added to culture for 8 hours, and fluorescence images were obtained using laser confocal tomography. The dual-targeted Am-PNCage-P out The tumor penetration ability of the nanocarriers was compared with that of single targeted HFn.

[0096] In vitro efficacy against drug-resistant tumors

[0097] In order to study EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P in In vitro treatment effects were observed. MCF7 / MDR resistant cells were cultured to form 3D cell spheres according to the above method. EPI@Am-PNCage-P was added on day 0. out / CPT,EPI@Am-PNCage-P out ,Am-PNCa ge-P out / CPT, EPI@HFn, or EPI / CPT@Am-PNCage-P in ,EPI@Am-PNCage- P in , Am-PNCage-P in / CPT, as well as free EPI and CPT, recorded the volume changes of MCF7 / MDR 3D cell spheroids within 6 days. Evaluate the therapeutic efficacy of Am-PNCage co-loaded with hydrophilic and hydrophobic dual drugs against drug-resistant tumors.

[0098] The results are as follows Figure 5 As shown:

[0099] CLSM results demonstrate that EPI@Am-PNCage-P out / CPT can bind to the U87 glioma cell membrane by binding to CD71 and αvβ3. in It only binds to the U87 cell membrane by binding to CD71. Flow cytometry (FACS) results showed that the fluorescence intensity of HFn binding to cells targeting CD71 was about 50, and Am-PNCage-P in The fluorescence curve of Am-PN Cage-P overlaps with that of HFn, indicating that peptide modification did not change the affinity of ferritin cage to cells, while the dual-targeted Am-PN Cage-P out The fluorescence intensity of the nanocarrier was approximately 100, which indicated that the dual-targeted Am-PNCage-P out The ability of nanocarriers to bind to tumor cells is enhanced.

[0100] On this basis, we further evaluated the dual-targeted EPI@Am-PNCage-P using MCF7 / MDR resistant breast cancer 3D cell spheres. out / CPT's ability to penetrate into tumor tissues, and found that the cell spheres incubated with free EPI and CPT were relatively dim, and the fluorescence mostly appeared in the edge area. outAfter incubation with / CPT, EPI and CPT fluorescence were present in the edge and core of the spheres, indicating that the dual-targeted nanocarriers further enhanced the drug penetration ability. The EPI fluorescence intensity spectrum of the cross-sectional tomography at 70 μm showed that the spheres were not as good as those without the targeting peptide. out Compared with the EPI@HFn with only ferritin cage CD71 single targeting, the dual targeting EPI@Am-PNCage-P out Significantly more drugs aggregated into 3D cell spheroids.

[0101] With EPI / CPT@Am-PNCage-P in Compared with EPI@Am-PNCage-P out / CPT was more effective in inhibiting the growth of MCF 7 / MDR drug-resistant cell spheres in vitro. out The nanocarrier has a better affinity for tumor cells than the single-targeted Am-PNCage-P in , and significantly enhanced tumor penetration ability, effectively overcoming the drug resistance of 3D tumor cell spheres. The reasons may be the effective endocytosis mediated by CD71 and ɑvβ3 receptors, the high drug concentration in intracellular lysosomes, and the cascade release and synergistic effect of EPI and CPT, which mediated the increased sensitivity to drugs.

[0102] Example 6 Pharmacokinetic Properties

[0103] EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P in 10mg / mL and an equivalent dose of EPI@Am-PNCage-P out , Am-PNCage-P out / CPT,EPI@Am-PNC age-P in , CPT@Am-PNCage-P in, EPI and CPT were intravenously injected into BALB / c nude mice. At different time points after injection, blood was collected from the tail vein and plasma was separated. The obtained plasma samples (10 μL) were incubated with 490 μL of acidified isopropanol at -20 °C overnight in the dark to extract free drugs. The mixture was then centrifuged at 15,000 × g for 20 min, and the supernatant was loaded onto a 96-well plate (Corning). The concentration of EPI or CPT was determined by measuring fluorescence at 480 nm or 365 nm using a Varioskan Flash spectral scanning multi-mode plate reader (ThermoFisher Scientific). The fluorescence of blood samples from untreated mice was used as the background. After deducting non-specific background fluorescence, the drug concentration of the blood sample was quantified and the changes in drug concentration in the blood over time were analyzed by comparison with the fluorescence standard curve.

[0104] The results are as follows Figure 6 As shown in Table 2:

[0105] We studied EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P in The pharmacokinetic properties of nanocarriers and free drugs EPI and CPT were analyzed. out The drug half-life in the plasma of mice administered with EPI@Am-PNCage-P (6.79±0.07h) was 8.5 times longer than that of free EPI (0.80±0.12h) and 12.6 times longer than that of CPT (0.54±0.26h). out The area under the curve (AUC) of EPI / CPT@Am-PNCage-P was 76-fold (2.10±0.42%ID / mL*h) and 111-fold (1.44±0.35%ID / mL*h) greater than that of free EPI and CPT. in The pharmacokinetic properties of Am-PNCage were similar to those of Am-PNCage, and there was no significant difference in the pharmacokinetics of Am-PNCage in mice loaded with dual drugs and single drugs, indicating that loading the drugs into Am-PNCage nanocarriers is the reason for prolonging the drug circulation time.

[0106] Table 2: Pharmacokinetic parameters of Am-PNCage nanocarriers loaded with dual drugs or single drug (n=3)

[0107]

[0108] Example 7 Am-PNCage nanocarrier can promote the targeted enrichment of loaded drugs in tumors and prolong the retention time of drugs in tumors

[0109] In order to study the biodistribution of the drug, a U87-luc glioma mouse model was established and EPI@Am-PNCage-P was injected into the tail vein at a dose of 3 mg / kg EPI equivalent. out / CPT,EPI@Am-PNCage-P out ,EPI / CPT@Am-PNCage-P in ,EPI@Am-PNCage-P in and free EPI. 1, 4 and 24 hours after injection, the tumors and major organs (heart, liver, spleen, lung, kidney) of the mice were collected and near-infrared imaging of in vitro tissues was performed using IVIS spectral imaging system (Xenogen). The fluorescence intensity of EPI drugs in each tissue and organ was analyzed using Living Image software.

[0110] The results are as follows Figure 7 As shown, EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNC age-P in After intravenous injection of U87 glioma-bearing mice for 1, 4, and 24 hours, tissues, organs, and brain tissues were collected for ex vivo imaging. out / CPT and EPI / CPT@ Am-PNCage-P in The enrichment in tumors within 24 hours was much better than that of free drug. Compared with free EPI, EPI@Am-PNCage-P out / CPT,EPI@Am-PNCage-P out ,EPI / CPT@Am-PNCage-P in ,EPI@Am-PNCage-P in The EPI signal was significantly enhanced at the tumor site. out / CPT and EPI / CPT@A m-PNCage-P in The tumor fluorescence intensity of the mice in the control group at 1 h after injection was 15.2 and 10.4 times that of the mice treated with free EPI, respectively. Strong fluorescence signals were detected in the tumors at 4 and 24 h after injection, but almost no signal was found in the tumors of mice given free EPI due to rapid clearance by the blood.

[0111] Example 8 Am-PNCage nanocarrier greatly increases the body's maximum tolerance dose of the loaded drug

[0112] Six-week-old healthy female SPF BALB / c mice were used to study the changes in the maximum tolerated dose of Am-PNCage after co-loading hydrophilic and hydrophobic drugs. out / CPT, EPI / CPT@Am-PNCage-P at 30, 35, 40 mg / kg body weight in Mice were injected with 5, 10, 15 mg / kg of free EPI or CPT by tail vein injection (n=3). Changes in mouse body weight and survival rate were monitored daily for two weeks. Mice that lost more than 15% of their pre-treatment body weight were euthanized. The highest dose with no animal death and no more than 15% body weight loss was defined as the maximum tolerated dose.

[0113] The results are as follows Figure 8 To evaluate the maximum tolerated dose, mice were injected with different doses of E PI@Am-PNCage-P out / CPT or EPI / CPT@Am-PNCage-P in For EPI@Am-PN Cage-P out In the / CPT group, mice had almost no visible functional impairment and no significant changes in body weight when given 25 and 30 mg / kg, but mice given 35 mg / kg EPI@Am-PNCage-P out / CPT mice gradually lost weight and dropped to less than 85% of their initial weight within 6 days. out The maximum tolerated dose of / CPT is about 30mg / kg. Similarly, EPI / CPT@Am-PNCage-P in The maximum tolerated dose of EPI and CPT is about 35 mg / kg. In contrast, the maximum tolerated dose of a single injection of free EPI and CPT is only 10 mg / kg. Therefore, Am-PNCage nanocarriers co-loaded with hydrophilic and hydrophobic drugs can significantly improve the maximum tolerated dose of EPI and CPT, allowing higher doses of EPI and CPT to be used for cancer treatment.

[0114] Example 9 Dual-targeted Am-PNCage nanocages co-loaded with hydrophilic and hydrophobic drugs exhibited high tumor killing ability

[0115] All mice and their corresponding studies in this study were approved by the Animal Care and Use Committee of the Chinese Academy of Sciences (approval number: SYXK2019021).

[0116] Evaluation of therapeutic effects on U87 glioma

[0117] By intracerebral injection of 1 × 10 6 The mouse brain glioma model was established by using U87MG cells expressing luciferase (U87-luc). The mice were weighed and randomly divided into 6 groups, and each group was intravenously injected with 3 mg / kg of EPI@Am-PNCage-P out / CPT,EPI / CPT@Am-PNCage-P in EPI@Am-PNCage-P out ,EPI@Am-PNCage-P in , EPI and equivalent CPT doses of Am-PNCage-P out / CPT, CPT@Am-PNCage-P in , CPT. The drug was administered once every 3 days, for a total of 3 times. The IVIS Spectrum Imaging System (Xenogen) was used for in vivo imaging every 3 days to compare the changes in tumor volume. The survival rate and body weight of the mice were measured during the experiment. The fixed tumor sections were stained using the TUNEL cell death detection kit (Roche). Tumor tissue sections were observed using a fluorescence microscope (IX7 1, Olympus).

[0118] Evaluation of therapeutic effects on lung metastases of HepG2 liver cancer

[0119] 2 × 10 were injected via tail vein 6 The mouse model of liver cancer lung metastasis was established by using HepG2 cells expressing luciferase (HepG2-luc). The mice were weighed and randomly divided into 6 groups. The maximum tolerated dose of EPI@Am-PNCage-P was intravenously injected into each group. out / CPT(30mg / kg), EPI / CPT@Am-PNCag eP in (35 mg / kg) and the equivalent EPI dose of EPI@Am-PNCage-P out ,EPI@Am-PNC age-P in , EPI and equivalent CPT doses of Am-PNCage-P out / CPT, CPT@Am-PNCage- P in, CPT. In vivo imaging was performed every 3 days using the IVIS Spectrum Imaging System (Xenogen) to compare changes in tumor volume. The survival rate and body weight of mice were measured during the experiment. The lungs were obtained after the mice were killed on day 30, washed three times with PBS, fixed with 10% neutral formalin, stained with hematoxylin and eosin (H&E), and imaged by optical microscopy (DM5500B, Leica).

[0120] Evaluation of therapeutic effects in MCF7 / MDR-resistant breast cancer

[0121] In situ injection of 1×10 7 The drug-resistant MCF7 / MDR cells were used to establish a drug-resistant breast cancer mouse model. 3 At 14:00, the mice were weighed and randomly divided into 6 groups, and each group was intravenously injected with the maximum tolerated dose of EPI@Am-PNCage-P out / CPT(30mg / kg), EPI / CPT@Am-PNCage-P in (35 mg / kg) and the equivalent EPI dose of EPI@Am-PNCage-P out ,EPI@Am-PNCage-P in , EPI and equivalent CPT doses of Am-PNCage-P out / CPT, C PT@Am-PNCage-P in During the experiment, the tumor size and body weight of the mice were measured every 3 days. 2 / 2 to calculate the tumor volume, where L represents the maximum diameter of the tumor and W represents the minimum diameter of the tumor.

[0122] The results are as follows Fig. 9 As shown:

[0123] After the mice were injected with U87-luc tumor cells intracerebrally, they were given intravenous treatment on days 7, 10, and 13. The results showed that the tumors in the PBS group continued to grow, while the EPI@Am-PNCage-P out The treatment with EPI / CPT@Am-PNCage-P resulted in significant tumor regression, and the dual-targeting enhanced the affinity and permeability of Am-PNCage to tumor cells, which was superior to the single-targeting EPI / CPT@Am-PNCage-P in The TUNEL method was further used to characterize the apoptosis of tumor cells. out / CPT and EPI / CPT@Am-PNCage-P inThe treatment group was significantly higher than that of single-drug loaded EPI@Am-PNCa ge, EPI@Am-PNCage-P in and free EPI induced more tumor cell apoptosis. The results showed that EPI@Am-PNCage-P out / CPT and EPI / CPT@Am-PNCage-P in The superior ability of the drug to kill gliomas in vivo may be due to its BBB penetration ability, selective targeting of tumor cells, and the synergistic effect of the drugs.

[0124] The therapeutic effects of various drugs on HepG2-Luc tumors were evaluated by a single injection of the maximum tolerated dose on day 15. out / CPT (30 mg / kg) and EPI / CPT @Am-PNCage-P in In mice treated with 1% paraformaldehyde (35 mg / kg), HepG2-Luc tumors in the lungs were significantly inhibited. In contrast, mice injected with EPI, CPT, or PBS had increased tumor sizes and died before day 33.

[0125] After a single administration at the maximum tolerated dose, EPI@Am-PNCage-P out / CPT and EPI / CPT@ Am-PNCage-P in The growth of MCF7 / MDR-resistant tumors was effectively blocked, while Am-PNCage loaded with a single drug only partially inhibited tumor growth. out / CPT and EPI / CPT@Am-PNCage-P in All mice in the EPI@Am-PNCage group survived, significantly prolonging their survival time (less than 60 days in the Am-PNCage group alone, while the average survival time of mice in the EPI, CPT or PBS group was less than 36 days). out / CPT and EPI / CPT@Am-PNCage-P in It has effective targeting and killing effects on mouse resistant MCF7 / MDR cells.

[0126] Example 10 Am-PNCage nanocages loaded with hydrophilic and hydrophobic drugs exhibit excellent biosafety

[0127] 1. Histological examination

[0128] Inject EPI@Am-PNCage-P at the maximum tolerated dose out / CPT or EPI / CPT@Am-PNC age-P in Afterwards, the main tissues and organs were fixed with formalin, paraffin sections were made and stained with H&E, and the tumor tissue sections were subjected to histological examination and morphological observation using a Nikon Eclipse 90i microscope.

[0129] 2. Hematological examination

[0130] For hematological examination, blood samples were collected retro-orbitally and analyzed by an automatic biochemical analyzer to obtain clinical hematological parameters, mainly including: alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as markers of liver function, creatinine (C REA) and blood urea nitrogen (UREA) as markers of renal function, and creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH) as markers of cardiac function.

[0131] The results are as follows Fig.10 As shown, in order to evaluate the biosafety after the maximum tolerated dose injection, tissue sections, blood cells and biochemical parameters were examined histologically. The main organs of mice, including heart, liver, lung, spleen and kidney, were collected for histological examination. out / CPT and EPI / CPT@Am-PNCage-P in H&E stained sections of the treatment group showed no obvious organ damage. The blood biochemical indicators of the mice showed that their heart, liver and kidney functions were normal, with no significant difference from the free drug or PBS treatment groups. out / CPT and E PI / CPT@Am-PNCage-P in It has no significant organ and tissue toxicity and is biocompatible.

[0132] Example 11 Am-PNCage nanocages exhibit broad spectrum of co-loading hydrophilic and hydrophobic drugs

[0133] According to the same two-step or one-step reaction method as in Example 2, Am-PNCage nanocages were used to co-load other hydrophilic and hydrophobic drug combinations with clinical synergistic effects to prepare dual-drug co-loaded Oxaliplatin@Am-PNCage / 5-Fluorouracil, Gemcitabine@Am-PNCage / 5-Fluorouracil, Gemcitabine@Am-PNCage / Docetaxel, Epirubicin@Am-PNCage / Docetaxel, and Irinotecan@Am-PNCage / Temozolomide, and the successful loading of the two drugs was verified by SEC.

[0134] The results are as follows Fig.11 As shown in Figure 2, other hydrophilic / hydrophobic anticancer drug combinations were loaded on Am-PNCage nanocarriers by two-step or one-step incubation methods. SEC results demonstrated that the absorption spectra of Oxaliplatin@Am-PN Cage / 5-Fluorouracil, Gemcitabine@Am-PNCage / 5-Fluorouracil, Gemcitabine @Am-PNCage / Docetaxel, Epirubicin@Am-PNCage / Docetaxel, and Irinotecan @Am-PNCage / Temozolomide all matched the corresponding free drug spectra except for the protein absorption peak at 280 nm, indicating the successful loading of the two drugs. Therefore, in addition to EPI / CPT, Am-PNCage also has the ability to effectively load a variety of synergistic hydrophilic / hydrophobic anticancer drugs used in the clinic and may have a wide range of clinical applications in the future.

[0135] Example 12 Am-PNCage nanocages co-loaded with hydrophilic and hydrophobic drug combinations have a broad spectrum of synergistic anti-tumor proliferation abilities

[0136] U87 brain glioma cells, HepG2 liver cancer cells and MCF7 / MDR resistant breast cancer cells were selected for clinical application of drugs. Cell proliferation kit 8 (CCK8) was used to study the toxicity of Oxaliplatin@Am-PNCage / 5-Fluorouracil, Gemcitabine@Am-PNCage / 5-Fluorouracil, Gemcitabine@Am-PNCage / Docetaxel, Epirubicin@Am-PNCage / Docetaxel, and Irinotecan@Am-PNCage / Temozolomide co-loaded with dual drugs on tumor cells through cell proliferation assays, and compared with Am-PNCage nanocarriers loaded with one of the drugs and free small molecule drugs. Using Orig in 8.0(Orig in The median lethal dose (IC50) was calculated by ELISA.

[0137] The results are as follows Figure 12-13 As shown, on the basis of effectively loading a variety of synergistic hydrophilic / hydrophobic anti-tumor drugs, the anti-tumor cell proliferation ability of the dual-targeted Am-PNCage after co-loading a variety of hydrophilic and hydrophobic drug combinations was further evaluated through cytotoxicity experiments. It was found that Am-PNCage co-loaded with paired hydrophilic and hydrophobic synergistic anti-cancer drugs showed significantly enhanced cytotoxic effects than when hydrophilic drugs and hydrophobic drugs were loaded separately, indicating that Am-PNCage has a broad-spectrum synergistic anti-tumor cell proliferation ability after co-loading hydrophilic and hydrophobic drug combinations, and has broad clinical application prospects.

[0138] Unless otherwise specifically stated, the numerical value set forth in these embodiments does not limit the scope of the present invention. In all examples shown and described here, unless otherwise specified, any specific value should be interpreted as merely exemplary, rather than as restriction, and therefore, other examples of exemplary embodiments may have different values. Sequence Listing <110> Nanjing Namomei Technology Co., Ltd. <120> Ferritin cage nanocarriers for targeting and co-loading hydrophilic / hydrophobic drugs and their applications <130> 2021 <160> twenty two <170> SIPOSequenceListing 1.0 <210> 1 <211> 13 <212> PRT <213> Artificial sequence <400> 1 Ala Ala Val Val Val Phe Ala Phe Ala Phe Ala Phe Ala 1 5 10 <210> 2 <211> 5 <212> PRT <213> Artificial sequence <400> 2 Ala Ala Ile Met Val 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial sequence <400> 3 Leu Leu Leu Val Val Val Ala Ala Ala 1 5 <210> 4 <211> 12 <212> PRT <213> Artificial sequence <400> 4 Pro Pro Trp Trp Tyr Tyr Tyr Leu Val Val Ala Ala 1 5 10 <210> 5 <211> 6 <212> PRT <213> Artificial sequence <400> 5 Pro Leu Gly Leu Trp Ala 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial sequence <400> 6 His His His His His Lys Lys 1 5 <210> 7 <211> 10 <212> PRT <213> Artificial sequence <400> 7 Asp Asp Asp Asp Glu Asp Asp Asp Asp Glu 1 5 10 <210> 8 <211> 10 <212> PRT <213> Artificial sequence <400> 8 Arg Arg Gln Gln His His His His His 1 5 10 <210> 9 <211> 7 <212> PRT <213> Artificial sequence <400> 9 Asp Asp Asp Ser Ser Thr Thr 1 5 <210> 10 <211> 8 <212> PRT <213> Artificial sequence <400> 10 Asp Asp Glu Glu Lys Lys Gln Ser 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial sequence <400> 11 His His Glu Gln Ser Ser Thr Thr Thr 1 5 <210> 12 <211> 5 <212> PRT <213> Artificial sequence <400> 12 Gly Arg Gly Asp Ser 1 5 <210> 13 <211> 10 <212> PRT <213> Artificial sequence <400> 13 Gln His Trp Ser Tyr Gly Leu Arg Pro Gly 1 5 10 <210> 14 <211> 5 <212> PRT <213> Artificial sequence <400> 14 Cys Arg Glu Lys Ala 1 5 <210> 15 <211> 7 <212> PRT <213> Artificial sequence <400> 15 His Ala Ile Tyr Pro Arg His 1 5 <210> 16 <211> 12 <212> PRT <213> Artificial sequence <400> 16 Tyr Glu Gln Asp Pro Trp Gly Val Lys Trp Trp Tyr 1 5 10 <210> 17 <211> 9 <212> PRT <213> Artificial sequence <400> 17 Cys Leu Glu Val Ser Arg Lys Asn Cys 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial sequence <400> 18 Cys Ser Thr Ser Met Leu Lys Ala Cys 1 5 <210> 19 <211> 26 <212> PRT <213> Artificial sequence <400> 19 Ala Ala Val Val Val Phe Ala Phe Ala Phe Ala Phe Ala His His His 1 5 10 15 His His Lys Lys Gly Arg Gly Asp Ser 20 25 <210> 20 <211> 582 <212> DNA <213> Artificial sequence <400> 20 atgaccaccg cgagcaccag ccaggttcgt cagaactacc accaggatag cgaagcggcg 60 atcaaccgtc agatcaacct ggaactgtac gcgagctacg tttacctgag catgagctac 120 180. tacttcgatc gtgatgatgt tgcgctgaaa aacttcgcga aatacttcct gcaccagagc 240. gtgaacacgc ggaaaaactg atgaaactgc agaaccagcg tggtggtcgt atcttcctgc aggatatcaa aaaaccggat tgcgatgatt gggaaagcgg cctgaacgcg atggaatgcg cgctgcacct ggaaaagaac gttaaccaga gcctgctgga actgcacaaa 420. ctggcgaccg ataaaaacga tccgcacctg tgcgatttca tcgaaaccca ctacctgaac gaacaggtta aagcgatcaa agaactgggt gatcacgtta ccaacctgcg taaaatgggc gcgccggaaa gcggtggcgg tagcgcggcg gttgttgttt tcgcgttcgc gttcgctttc 540 gcgcaccacc accaccacca taaaaaaggc cgtggtgata gc <210> 21 <211> 588 <212> DNA <213> singular sequence (artificial sequence) <400> 21 atgaccaccg cgagcaccag ccaggttcgt cagaactacc accaggatag cgaagcggcg atcaaccgtc agatcaacct ggaactgtac gcgagctacg tttacctgag catgagctac 180. tacttcgatc gtgatgatgt tgcgctgaaa aacttcgcga aatacttcct gcaccagagc 240. gtgaacacgc ggaaaaactg atgaaactgc agaaccagcg tggcggtcgt atcttcctgc aggatatcaa aaaaccggat tgcgatgatt gggaaagcgg cctgaacgcg atggaatgcg cgctgcacct ggaaaagaac gttaaccaga gcctgctgga actgcacaaa 420. ctggcgaccg ataaaaacga tccgcacctg tgcgatttca tcgaaaccca ctacctgaac gaacaggtta aagcgatcaa agaactgggt gatcacgtta ccaacctgcg taaaatgggt gcgccggaa gcggcctggc ggaatacctg ttcgataaac acaccctggg tgatagcgat aacgaaagcg cggcggttgt tgttttcgct ttcgcgttcg cgttcgcg 588 <210> 22 <211> 549 <212> DNA <213> singular sequence (artificial sequence) <400> 22 atgaccaccg cgagcaccag ccaggttcgt cagaactacc accaggatag cgaagcggcg atcaaccgtc agatcaacct ggaactgtac gcgagctacg tttacctgag catgagctac 180. tacttcgatc gtgatgatgt tgcgctgaaa aacttcgcga aatacttcct gcaccagagc 240. gtgaacacgc ggaaaaactg atgaaactgc agaaccagcg tggcggtcgt atcttcctgc aggatatcaa aaaaccggat tgcgatgatt gggaaagcgg cctgaacgcg atggaatgcg cgctgcacct ggaaaagaac gttaaccaga gcctgctgga actgcacaaa 420. ctggcgaccg ataaaaacga tccgcacctg tgcgatttca tcgaaaccca ctacctgaac gaacaggtta aagcgatcaa agaactgggt gatcacgtta ccaacctgcg taaaatgggt gcgccggaa gcggcctggc ggaatacctg ttcgataaac acaccctggg tgatagcgat aacgaagc 549

Claims

1. A ferritin cage nanocarrier targeting and co-loading hydrophilic / hydrophobic drugs, characterized in that: The ferritin cage nanocarrier is Am-PNCage-Pout shown in SEQ ID NO.20, or Am-PNCage-Pin shown in SEQ ID NO.

21.

2. Use of the ferritin cage nanocarrier according to claim 1 in the preparation of a drug loaded with hydrophilic and / or hydrophobic small molecule drugs, characterized in that: The loading is to load the drug into the ferritin cage nanocarrier under non-denaturing conditions.

3. The use according to claim 2, characterized in that: The small molecule drug is a chemical small molecule drug.

4. The use according to claim 2, characterized in that: The loading molar mass ratio of the ferritin cage nanocarrier to the hydrophilic small molecule drug is 1:100 to 1:

180.

5. The use according to claim 4, characterized in that: The loading molar mass ratio of the ferritin cage nanocarrier to the hydrophilic small molecule drug is 1:130 to 1:

150.

6. The use according to claim 5, characterized in that: The hydrophilic small molecule drug is one or more of anthracyclines, platinums, pyrimidines and alkaloids.

7. The use according to claim 6, characterized in that: The anthracyclines are epirubicin and daunorubicin, the platinums are cisplatin, carboplatin and oxaliplatin, the pyrimidines are gemcitabine, capecitabine and cytarabine, and the alkaloids are irinotecan.

8. The use according to claim 2, characterized in that: The loading molar mass ratio of the ferritin cage nanocarrier to the hydrophobic small molecule drug is 1:20 to 1:

70.

9. The use according to claim 8, characterized in that: The loading molar mass ratio of the ferritin cage nanocarrier to the hydrophobic small molecule drug is 1:30 to 1:

50.

10. The use according to claim 9, characterized in that: The hydrophobic small molecule drug is one or more of pyrimidines, taxanes and nitrosoureas.

11. The use according to claim 10, characterized in that: The pyrimidines are 5-fluorouracil and gimeracil, the taxanes are paclitaxel and docetaxel, and the nitrosoureas are temozolomide and nimustine.

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