Method for loading medicine by non-denatured human H ferritin
阅读说明：本技术 一种非变性人h铁蛋白装载药物的方法 (Method for loading medicine by non-denatured human H ferritin ) 是由 孙国明 尹雨芳 陈向茹 于 2020-12-17 设计创作，主要内容包括：本发明属于铁蛋白封装药物技术领域,具体公开了一种非变性人H铁蛋白装载药物的方法。本发明通过HFn-小分子药物晶体结构分析,鉴定发现HFn的蛋白壳上存在12条药物通道,决定该通道的关键氨基酸是Asp89-Cys90-Asp91-Asp92,同时,该小分子药物通道的氨基酸具有温度因子调控的性质。基于上述发现,本发明在保证铁蛋白结构不变,非变性条件下,通过探索不同孵育温度和/或不同的孵育条件,在保持铁蛋白结构完整前提下,实现了对HFn包载药物最优条件的确定。利用本发明所获得的非变性条件下的包载条件,可以方便、快捷、高效、足量的利用HFn包载不同的小分子药物。(The invention belongs to the technical field of ferritin encapsulated drugs, and particularly discloses a method for loading a drug by non-denatured human H ferritin. According to the invention, through the analysis of HFn-micromolecule drug crystal structure, 12 drug channels exist on the protein shell of HFn, the key amino acid determining the channel is Asp89-Cys90-Asp91-Asp92, and meanwhile, the amino acid of the micromolecule drug channel has the property of temperature factor regulation. Based on the findings, the invention realizes the determination of the optimal condition of the HFn entrapped drug by exploring different incubation temperatures and/or different incubation conditions under the condition of ensuring that the ferritin structure is unchanged and non-denatured and on the premise of keeping the ferritin structure complete. The entrapping condition under the non-denaturing condition obtained by the method can conveniently, quickly, efficiently and sufficiently utilize HFn to entrap different small molecule drugs.)
1. A human H ferritin drug loaded channel comprising amino acids Asp89-Cys90-Asp91-Asp92(SEQ ID NO: 1).
2. A method for loading a drug by non-denatured human H ferritin, wherein the human H ferritin is loaded by ion/hydrophobic channels in the native state.
3. The method of claim 2, wherein the ion/hydrophobic channel comprises the amino acids Asp89-Cys90-Asp91-Asp92(SEQ ID NO: 1).
4. The method according to claim 2, further comprising adding a non-denaturing agent comprising polyols, sugars, amino acids, polymers, and the like, preferably glycerol, mannitol, sorbitol, polyethylene glycol, glucose, sucrose, lactose, mannose, heparin, 2-hydroxypropyl-beta cyclodextrin, dimethyl sulfoxide, N-dimethylformamide, Tween-80, sodium citrate, sodium dodecyl sulfate, more preferably glycerol.
5. The method of claim 2, wherein the drug includes but is not limited to small molecule drugs, preferably small molecule chemotherapeutic drugs and small molecule nucleic acid drugs.
6. The method of claim 5, wherein the small molecule chemotherapeutic agent comprises hydrophilic and amphiphilic small molecule drugs, preferably antibiotic-type oncology drugs, naturally derived antineoplastic agents, metallic compounds, radioisotopes, alkylating agents, antimetabolite-type antineoplastic agents, hormonal-type antineoplastic agents, more preferably doxorubicin, Epirubicin (Epirubicin), Cisplatin (cisclin), Oxaliplatin (Oxaliplatin); the small molecule nucleic acid medicament is preferably siRNA or lncRNA.
7. The method according to claim 2, wherein the incubation temperature of the loading is 20-70 ℃, preferably 25-70 ℃, more preferably 30 ℃, 35 ℃, 40 ℃, 45 ℃,50 ℃, 55 ℃, 60 ℃, 65 ℃, more preferably 55-65 ℃, more preferably 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃.
8. The method according to claim 2, wherein the loading is performed for an incubation time of 0.1-24h, preferably 1-12h, more preferably 1-6 h, more preferably 1h, 2h, 3h, 4h, 5h, 6 h.
9. The method according to claim 2, wherein the concentration of the human ferritin H in the loading reaction system is 0.1-90 mg/mL, preferably 0.5-9 mg/mL, more preferably 1-5 mg/mL, more preferably 1.5mg/mL, 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL, 4mg/mL, 4.5mg/mL, and the loading reaction system further comprises a buffer solution, wherein the pH value of the buffer solution is 6-8, preferably 6.5, 7, 7.5, and the concentration of the buffer solution is 20-500 mM, more preferably 25-100 mM, and more preferably 25-50 mM.
10. The method according to claim 2, wherein the loading mass ratio of the human H ferritin to the small molecule drug in the loading reaction system is 1: 10-10: 1, preferably 1: 1-5: 1, and more preferably 1: 1-3: 1.
Technical Field
The invention belongs to the technical field of ferritin encapsulated drugs, and particularly relates to a method for loading a drug by non-denatured human H ferritin.
Background
Ferritin (ferritin) is an iron storage protein that plays an important role in cellular iron homeostasis and in oxidation resistance. Ferritin has become a drug delivery carrier with great transformation prospect due to its unique structure of protein shell formed by self-assembly of 24 subunits and capable of realizing additional functions through gene and chemical modification and hollow cavity capable of encapsulating drugs1. Recent studies have shown that the receptor for human Heavy chain ferritin (HFn) is Transferrin receptor 1(Transferrin receptor1, TfR1), i.e. ferritin can actively target different types of tumor cells including lung and breast cancer (ZL201110122433.0, PCT/CN2012/075291) by interacting with the receptor TfR1 without any targeting modification, suggesting a potential use of ferritin in tumor targeting applications2-6. It can be seen that ferritin has the function of being a chemotherapeutic drug carrier in the field of tumor treatmentGreat potential7,8。
Numerous chemotherapeutic agents have been successfully loaded into ferritin and used in tumor therapy. However, its drug loading efficiency is still relatively low. Currently, methods for loading ferritin drugs are mainly divided into three, including direct chemical coupling of the drug to the ferritin surface, pH mediated depolymerization/complexation of ferritin, and urea mediated depolymerization/complexation of ferritin (ZL201410230829.0, PCT/CN 2014/140159). The drug prepared by the chemical coupling method is exposed on the surface of ferritin, which may cause the influence on the physicochemical property of ferritin, and is easy to cause potential toxicity due to the breakage of linker. Ferritin depolymerization/repolymerization encapsulates the drug inside the protein shell, which can avoid the above problems. In a pH-mediated ferritin depolymerization/polymerization loading system, a ferritin-drug complex is unstable after drug loading because the structure of the protein is damaged by severe pH change, so that industrial transformation is difficult to realize9(ii) a The urea-mediated ferritin depolymerization/multimerization system has long incubation time, large protein gradient dialysis loss, and most importantly, the drug loading efficiency is limited, which cannot meet clinical requirements. Therefore, there is a need in the art to develop a novel method for carrying H Fn drug that is mild, stable, and convenient.
Reference documents:
1.He,J.；Fan,K.；Yan,X.,Ferritin drug carrier(FDC)for tumor targeting therapy.Journal of Controlled Release 2019.
2.Fan,K.；Cao,C.；Pan,Y.；Lu,D.；Yang,D.；Feng,J.；Song,L.；Liang,M.；Yan,X.,Magnetoferritin nanoparticles for targeting and visualizing tumour tissues.Nat Nanotechnol 2012,7(7),459-64.
3.Liang,M.；Fan,K.；Zhou,M.；Duan,D.；Zheng,J.；Yang,D.；Feng,J.；Yan,X.,H-ferritin-nanocaged doxorubicin nanoparticles specifically target and kill tumors with a single-dose injection.Proc Natl Acad Sci U S A 2014,111(41),14900-5.
4.Fan,K.；Jia,X.；Zhou,M.；Wang,K.；Conde,J.；He,J.；Tian,J.；Yan,X.,Ferritin Nanocarrier Traverses the Blood Brain Barrier and Kills Glioma.ACS Nano 2018,12(5),4105-4115.
5.Cheng,X.；Fan,K.；Wang,L.；Ying,X.；Sanders,A.J.；Guo,T.；Xing,X.；Zhou,M.；Du,H.；Hu,Y.；Ding,H.；Li,Z.；Wen,X.；Jiang,W.；Yan,X.；Ji,J.,TfR1 binding with H-ferritin nanocarrier achieves prognostic diagnosis and enhances the therapeutic efficacy in clinical gastric cancer.Cell Death Dis 2020,11(2),92.
6.Jia,X.；Fan,K.；Zhang,R.；Zhang,D.；Zhang,J.；Gao,Y.；Zhang,T.；Li,W.；Li,J.；Yan,X.；Tian,J.,Precise visual distinction of brain Glioma from Normal tissues via targeted Photoacoustic and fluorescence navigation.Nanomedicine:Nanotechnology,Biology and Medicine 2020,102204.
7.Fan,K.；Gao,L.；Yan,X.,Human ferritin for tumor detection and therapy.WIREs Nanomedicine and Nanobiotechnology 2013,5(4),287-98.
8.Jin,Y.；He,J.；Fan,K.；Yan,X.,Ferritin variants:inspirations for rationally designing protein nanocarriers.Nanoscale 2019,11(26),12449-12459.
9.Kim,M.；Rho,Y.；Jin,K.S.；Ahn,B.；Jung,S.；Kim,H.；Ree,M.,pH-dependent structures of ferritin and apoferritin in solution:disassembly and reassembly.Biomacromolecules 2011,12(5),1629-40.
disclosure of Invention
In order to achieve the purpose, the invention identifies the small molecule drug channel existing on the surface of the natural human heavy chain ferritin by a crystallography structure method, and finds that the small molecule drug channel is regulated and controlled by temperature and can realize effective drug loading on a ferritin cavity under a non-denaturing condition. The invention provides a mild, effective and convenient new thought and method for loading the ferritin drug carrier.
In a first aspect, the invention provides a human H ferritin drug-loaded channel comprising amino acids Asp89-Cys90-Asp91-Asp92(SEQ ID NO: 1).
In certain embodiments, the drug-loaded human H ferritin may be genetically engineered fully human heavy chain ferritin, with the nucleotide sequence shown in SEQ ID No. 2 and the encoded amino acid sequence shown in SEQ ID No. 3, or may be all variants of ferritin, including ferritin without limitation surface modifications, conjugated to any functional ligand or fluorescent molecule, and ferritin engineered to display functional peptide fragments, nanobodies, etc. on its surface or inside.
In certain embodiments, the drug is a small molecule drug, including but not limited to small molecule drugs, preferably small molecule chemotherapeutic drugs and small molecule nucleic acid drugs. The small-molecule chemotherapeutic drugs comprise hydrophilic and amphiphilic small-molecule drugs, preferably antibiotic tumor drugs, natural source antitumor drugs, metal compounds, radioactive isotopes, alkylating agents, antimetabolite antitumor drugs and hormone antitumor drugs, and more preferably adriamycin, Epirubicin (Epirubicin), Cisplatin (cissplatin) and Oxaliplatin (Oxaliplatin); the small molecule nucleic acid drug is preferably siRNA, lncRNA.
In a second aspect, the present invention provides a method for loading a drug with human H ferritin, which is loaded with the drug through ion/hydrophobic channels in the native state.
In certain embodiments, the drug-loaded human H ferritin may also be all variants of ferritin, including, without limitation, ferritin surface modified, conjugated to any functional ligand or fluorescent molecule, and ferritin genetically engineered to display functional peptide fragments, nanobodies, and the like on its surface or within.
In certain embodiments, the ion/hydrophobic channel comprises the amino acids Asp89-Cys90-Asp91-Asp92(SEQ ID NO: 1).
In certain embodiments, the method further comprises adding a non-denaturing agent comprising polyols, sugars, amino acids, polymers, and the like, preferably glycerol, mannitol, sorbitol, polyethylene glycol, glucose, sucrose, lactose, mannose, heparin, 2-hydroxypropyl-beta cyclodextrin, dimethyl sulfoxide, N-dimethylformamide, tween-80, sodium citrate, sodium dodecyl sulfate, more preferably glycerol. The addition amount of the non-denaturing agent is 15%, 20%, 5%, 10%, 15%, 20%, 25%, 30%, preferably 15%.
In certain embodiments, the drug includes, but is not limited to, small molecule drugs, preferably small molecule chemotherapeutic drugs and small molecule nucleic acid drugs.
In certain embodiments, the small molecule chemotherapeutic agent comprises hydrophilic and amphiphilic small molecule agents, preferably antibiotic-type antineoplastic agents, naturally-derived antineoplastic agents, metallic compounds, radioisotopes, alkylating agents, antimetabolite-type antineoplastic agents, hormonal antineoplastic agents, more preferably doxorubicin, Epirubicin (Epirubicin), Cisplatin (cissplatin), Oxaliplatin (Oxaliplatin); the small molecule nucleic acid drug is preferably siRNA, lncRNA.
In certain embodiments, the loading is incubated at a temperature of 20-70 deg.C, preferably 25-70 deg.C, more preferably 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C, more preferably 55-65 deg.C, more preferably 56 deg.C, 57 deg.C, 58 deg.C, 59 deg.C, 60 deg.C, 61 deg.C, 62 deg..
In certain embodiments, the loading is performed for an incubation time of 0.1 to 24h, preferably 1 to 12h, more preferably 1h to 6h, more preferably 1h, 2h, 3h, 4h, 5h, 6 h.
In some embodiments, the concentration of the human H ferritin in the loading reaction system is 0.1-90 mg/mL, preferably 0.5-9 mg/mL, more preferably 1-5 mg/mL, more preferably 1.5mg/mL, 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL, 4mg/mL, 4.5 mg/mL.
In certain embodiments, the loading reaction system comprises a buffer solution, wherein the buffer system is Tris-HCl buffer, Phosphate Buffer Solution (PBS), carbonate buffer solution, glycine buffer solution or citrate buffer solution, the pH value of the buffer solution is 6-8, 9, and the concentration of the buffer solution is 20-500 mM, more preferably 25-100 mM, and more preferably 25-50 mM.
In some embodiments, in the loading reaction system, the loading mass ratio of the human H ferritin to the small molecule drug is 1: 10-10: 1, preferably 1: 1-5: 1, and more preferably 1: 1-3: 1.
In a third aspect, the invention further provides drug-loaded human H ferritin prepared based on the drug loading method provided by the invention.
In a fourth aspect, the present invention also provides a pharmaceutical composition comprising the human H ferritin of the third aspect, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. These pharmaceutical compositions may be formulated with pharmaceutically acceptable carriers or diluents and any other known excipients according to conventional techniques such as those disclosed in: the Science and Practice of Pharmacy, 22 nd edition, eds Gennaro, Mack Publishing Co., 2013.
In a fifth aspect, the invention also provides an application of the pharmaceutical composition provided in the fourth aspect in preparing a drug for targeted therapy of tumors. The tumors include, but are not limited to, human solid tumors and hematologic malignant cancerous cells, such as lung cancer, breast cancer, prostate cancer, cervical cancer, colorectal cancer, ovarian cancer, esophageal cancer, gastric cancer, thymus cancer, T-lymphocyte leukemia, erythroleukemia, and the like.
Compared with the prior art, the invention has the following advantages:
1) according to the invention, through a crystallographic structure method, the existence of 12 drug channels on the HFn protein shell is identified and found, the channels are different from the classical hydrophilic triple symmetry axis and hydrophobic quadruple symmetry axis on the iron protein shell, and the key amino acid determining the channels is Asp89-Cys90-Asp91-Asp 92. The amino acid of the drug channel has the property of temperature factor regulation, and the swing of amino acid residues is increased along with the rise of temperature, so that the lateral shift is generated, and the drug channel is enlarged.
2) The invention finally realizes high-efficiency drug loading of human H ferritin on the premise of keeping intact ferritin structure by exploring different incubation temperatures and/or different incubation conditions, such as incubation time of 1H-6H, incubation buffer solution pH of 6-8 and incubation temperature of 30-70 ℃ under the condition of ensuring unchanged ferritin structure and non-denaturation. The entrapping condition under the non-denaturing condition obtained by the method can conveniently, quickly, efficiently and sufficiently utilize HFn to entrap different small molecule drugs.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.
FIG. 1 the natural small molecule drug channel existing on the iron protein shell
Wherein, panel A is molecular exclusion analysis of HFn-Dox, the 280nm absorption peak is HFn absorption peak, and the 485nm absorption peak is Dox absorption peak; FIG. B is a Native Page analysis of HFn and HFn-Dox; panel C shows the direct observation of Dox in HFn-Dox over Native Page, where the red color of the Dox molecule is visible to the naked eye.
FIG. 2HFn-Dox shows the crystallographic structure of the protein, which shows the presence of small molecule drug channels on the HFn protein shell, and the key amino acids are Asp89-Cys90-Asp91-Asp 92.
FIG. 3HFn shows the mutation verification of the Dox drug loading channel on the surface of protein. FIGS. A-D are schematic diagrams of HFn, Block-HFn (C90S), Enlarged-HFn-1(D92G) and Enlarged-HFn-2(R43G + R79G) mutants, respectively.
FIG. 4HFn shows mutation verification of the Dox drug loading channel on the surface of protein. Wherein, panel A is SEC analysis of HFn mutant and HFn, panel B is SDS-PAGE and Native Page analysis of HFn and HFn mutants, and panel C is comparison of doxorubicin-loading efficiency of HFn and HFn mutants.
FIG. 5 is a graph showing that the loading of small molecule drugs is facilitated by heating to control the opening and closing degree of a small molecule drug loading channel. Wherein, the graph A is a temperature factor graph of HFn small molecule drug channels. The structure shows that the channel can increase the switching degree of the channel along with the increase of the temperature. Panel B is the effect of incubation temperature on HFn loading Dox. Panel C is the effect of incubation temperature on HFn recovery. Panel D is the combined effect of incubation temperature on the Dox loading rate and recovery at HFn. Panel E is the effect of incubation time on HFn loading Dox. Panel F is the effect of incubation time on HFn recovery. Graph G is the combined effect of incubation time on HFn loading Dox loading rate and protein recovery. The asterisks indicate the optimal conditions.
The heating method of fig. 6 can stably load Dox in HFn lumen without affecting ferritin HFn structure. Wherein, the graph A shows that the secondary structure of HFn is not changed before and after loading the drug by different temperature treatment. FIG. B, C shows that the size exclusion position of HFn was not changed before and after loading Dox for different temperature treatments. Panel D shows the position of HFn at Native Page before and after loading the drug with different temperature treatments.
FIG. 7 shows that the loading efficiency of the non-denaturing temperature loading method is greatly improved as compared with the Urea, pH renaturation method. Temperature, urea, pH three methods load doxorubicin (a), HFn recovery (B), color after loading (C), round-color before and after loading (D), stability in serum (E) and drug release under acidic conditions (F) analysis were compared.
FIG. 8 the non-denaturing temperature loading method is also applicable to other small molecule drugs. Panel A shows HFn comparison of epirubicin, cisplatin and oxaliplatin loading under non-denaturing temperature-dependent conditions. Panel B is an analysis of HFn recovery after loading different drugs with non-denaturing temperature treatment.
The LFn surface of fig. 9 does not have similar drug loading channels. FIG. A shows that LFn does not have particularly strong loading ability in the same non-denaturing temperature loading method. Panel B shows HFn color comparison with LFn loaded doxorubicin after treatment with t the same method.
FIG. 10 compared to the urea based de/complexed drug loading method, the non-denaturing temperature drug loading method synthesized HFn-Dox showed excellent biosafety (A, C) and ability to kill tumors efficiently (B, D).
FIG. 11 non-denaturing temperature loading method can effectively load HFn with siRNA and shows very good antitumor effect in vivo brain tumor antitumor experiment. In each group of three mice with brain tumor carcinoma in situ, the fluorescence signal is the fluorescence signal of the collected tumor cell self Luciferase catalytic substrate.
FIG. 12 comparison of doxorubicin loading efficiency of 12HFn and HFn mutants.
FIG. 13 shows HPLC standard curve drug concentration detection of non-denaturing temperature loading of other anti-tumor drugs, wherein A-G are drugs sunitinib (sunitinib), crizotinib (crizotinib), Palbociclib (Palbociclib), irinotecan (irinotecan), maytansine (maytansine), 5-fluorouracil (5-fluorouracil), and pentoxifylline (Pentoxiphylline), respectively.
FIG. 14 comparison of HFn loading for other anti-tumor drugs under non-denaturing temperature method conditions.
FIG. 15 analysis of HFn recovery after loading different drugs with non-denaturing temperature treatment.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby. It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains. The reagents used in the examples are commercially available conventional products unless otherwise specified.
Definition of
Ferritin in the present invention refers to any ferritin that may form a cage structure, which may be a ferritin of natural origin, or may be recombinantly expressed ferritin, or a mutant thereof, which may be derived from a prokaryote, protist, fungus, plant or animal, e.g. from a bacterium, fungus, insect, reptile, avian species, amphibian, fish, mammal, e.g. from a rodent, ruminant, non-human primate or human, e.g. mouse, rat, guinea pig, canine, cat, bovine, equine, ovine, monkey, gorilla, human. From bacteria to humans, protein shell structures can be formed with similar structures, although ferritin amino acid sequences of different organisms vary greatly. In some embodiments, the ferritin of the present invention is human ferritin, and in some embodiments, the ferritin of the present invention is genetically engineered fully human heavy chain ferritin, having a nucleotide sequence set forth in SEQ ID NO:2, respectively.
The drug or drug-encapsulated drug of the present invention refers to any drug that can be encapsulated in ferritin, as long as the molecular size of the drug is less than 8 nanometers. In some embodiments, the drug or drug-containing agent of the present invention is selected from antibiotic-type antineoplastic agents, naturally derived antineoplastic agents, metallic compounds, radioisotopes, alkylating agents, antimetabolite-type antineoplastic agents, and hormonal-type antineoplastic agents. Wherein the antibiotic antineoplastic agent is selected from adriamycin (doxorubicin hydrochloride), zorubicin hydrochloride, valrubicin, bleomycin sulfate, mitomycin, epirubicin hydrochloride, idarubicin hydrochloride, actinomycin D, mithramycin, daunorubicin, pirarubicin, epirubicin, idarubicin, aclarubicin, bleomycin A5, tryptomycin A3, bleomycin hydrochloride, palmomycin, pingyangmycin hydrochloride, daunorubicin, doxorubicin hydrochloride, azomycin, pimaricin, pirarubicin hydrochloride, actinomycin C; the natural antineoplastic is selected from topotecan hydrochloride, 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, rubitecan, resveratrol, camptothecin, paclitaxel, colchicine, etoposide, docetaxel, vinblastine sulfate, cantharidin, irinotecan, sunitinib, podophyllotoxin, topotecan, crizotinib, homoharringtonine, epothilones, teniposide, palbociclib, monocrotaline, vincristine, vinorelbine, vindesine sulfate, indirubin, vincristine sulfate, norcantharidine, podophyllotoxin, docetaxel, colchicine, cephalotaxine, epothilone C, epothilone E, cinobufagin, vindesine sulfate, sodium cantharidinate, maytansine, sarcandra, podophyllum, pellitorin, vinorelbine bitartrate, irisquinone, oleum fructus Bruceae, mitohydrazide, harringtonine, autumn schizoamine, hydroxycamptothecin, and methylcantharidimine; the metal compound is selected from carboplatin, cisplatin, nedaplatin, oxaliplatin and the like; 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, 104, 105, 106, 107, 108, and element No. 109; the alkylating agent is selected from bendamustine hydrochloride, mizolastine, bepotastine besylate, busulfan, enbisine, dacarbazine, lomustine, benzene butyric acid nitrogen, mustard carmustine, triethylenethiophosphoramide, nimustine hydrochloride, melphalan nimustine, bendamustine, estramustine, sodium phosphate 2, 3-dibromo-1, 4-butenediol, estramustine, altretamine, fotemustine, nimustine, galamustine, splatemustine, estramustine, etomomustine phosphate, tamustine, amimustine, estramustine, bendamustine hydrochloride impurity A, nemamustine, brivustine, eputistine hydrochloride, bendamustine hydrochloride impurity C, lomustine capsule, betahistine hydrochloride, dithiomustine, oxtemustine, emetine, and spiromustine, bendamustine hydrochloride impurity B, prednimustine, semustine, ramustine, carboquinone, mechlorethamine, epinastine, uramustine, dibromomannitol, mechlorethamine hydrochloride, oxaziclomethine, epipipradine, melphalan, dianhydrogalactitol, iminoquinone, methamphetamine, entinostoc, mechlorethamine, nitracarmustine, isoerucin, mechlorethamine, triimiquinone, mechlorethamine, chlorambucil, 2, 4, 6-triethyleneimine-1, 3, 5-triazine; the antimetabolite antineoplastic agent is selected from methotrexate, 5-fluoro-2' -deoxyurea nucleoside, gemcitabine, deoxyfluorouridine, cytarabine, 6-thioguanine, gemcitabine hydrochloride, fludarabine phosphate, vinorelbine tartrate, fludarabine, temozolomide, pentoxifylline, clorfarabine, nelarabine, cyclocytidine hydrochloride, tegafur, cytarabine hydrochloride, 6-mercaptopurine, 4-hydroxy-5-fluoropyrimidine, 5-fluorouracil, aminopterin, miltefosine, raltitrexed, desoxyhelpicolin, carmofluorine, amsacrine, emithidine, tegafur, ciclocyridine, methylisoidine, fluorouracil, inosine dialdehyde, 1-vinyl-1-methyl-2, 4-bis (prop-1-en-2-yl) cyclohexane, sodium sulfydryl; the hormone antineoplastic agent is selected from exemestane, raloxifene, fulvestrant, letrozole, anastrozole, flutamide, tamoxifen citrate, droloxifene, idoxifene, nilutamide, aminoglutethimide, formestane, tamoxifen, toremifene, aminoglutethimide.
Without being limited by any theory, the ferritin carrying the drug can target tumors, and the carried drug is released after being combined with the tumors, so that the drug acts on the tumors, and the prevention and/or treatment of the tumors are realized. For example, human ferritin may specifically target human solid tumors and hematologic malignant neoplastic cells, such as lung cancer, breast cancer, prostate cancer, cervical cancer, colorectal cancer, ovarian cancer, esophageal cancer, gastric cancer, thymus cancer, T-lymphocyte leukemia, erythroleukemia, and the like, by binding to its receptor Transferrin receptor (TfR 1).
In some embodiments, the drug or drug-in-package of the present invention is selected from drugs other than antineoplastic drugs, i.e., non-antineoplastic drugs. For example, such a drug may be a drug that does not require targeting, e.g., a systemically administered drug, e.g., a drug that is poorly soluble, unstable, and/or susceptible to interaction and failure. In some embodiments, such a drug is selected from amphotericin B, glatiramer acetate, complex ferric sodium gluconate, rapamycin, sevelamer sulfate hydrochloride binding agents, verteporfin for injection, ferric sucrose, peginterferon alpha-2 a/2B, fenofibrate, pefilgrastim, risperidone, amikacin, fentanyl, cyclosporine, cetirizine, capsaicin, ceramide, and the like. In some embodiments, the non-antineoplastic agent of the present invention is selected from the group consisting of a radiopharmaceutical, a neurotransmitter-like agent, a dopamine receptor agonist, a central nervous anticholinergic agent, a cholinergic agonist-like agent, a gamma secretase inhibitor, an antioxidant, and an anesthetic, and more preferably, the radiopharmaceutical is selected from the group consisting of a radiopharmaceutical, a neurotransmitter-like agent, a cholinergic agonist-like agent, a gamma secretase inhibitor, an antioxidant, and an anesthetic64Cu、235U, the neurotransmitter is selected from carbachol, atropine, scopolamine, dopamine and derivatives thereof, the dopamine receptor agonist is selected from ergot derivatives such as bromocriptine, pergolide and apomorphine and non-ergot derivatives, the nerve center anticholinergic agent is selected from trihexyphenidyl, benzalkonium and propidin, the choline receptor agonist is selected from muscarinic and pilocarpine, the gamma secretase inhibitor is selected from bifluoride, the antioxidant is selected from melatonin, and the anesthetic is selected from anthryl amine.
In some embodiments, it may be desirable to add additives to the incubation solution to facilitate dissolution of the drug and/or aggregation of ferritin when encapsulating the drug into ferritin order to achieve and/or improve drug encapsulation. For example, in some embodiments involving entrapment of a platinum-based drug, it may be desirable to add DMA, DMF, DMSO, or mixtures thereof to the incubation solution. The additives to be added may be different according to the drug to be encapsulated.
In some embodiments, the buffer system of the incubation solution is Tris-HCl buffer, Phosphate Buffered Saline (PBS), carbonate buffer, glycine buffer or citrate buffer, the pH of the buffer is between 6 and 8, such as 6.5, 7, 7.5, and the concentration of the buffer is between 20 and 500mM, more preferably between 25 and 100mM, more preferably between 25 and 50 mM. Depending on the buffer used, its buffering capacity, pH range, desired concentration, etc. may need to be adjusted.
Example 1 structural analysis of the Small molecule drug doxorubicin (Dox) channel on the protein Shell 1HFn
The structure of the channel of the small molecule drug doxorubicin (Dox) on the HFn protein shell was analyzed by the following method steps:
step (1) construction of HFn expression plasmid: HFn (shown as SEQ ID No:2 and SEQ ID No: 3) was subjected to whole gene synthesis (general, Shanghai), then cut with NdeI and BamHI restriction enzymes, cloned into E.coli (E.coli) expression vector pET22b (+) plasmid (Novagen) with NdeI and BamHI restriction sites, and the sequence was confirmed to be correct by DNA sequencing.
Expression and purification of step (2) HFn: the plasmid obtained above was transferred into E.coli BL21(TransGen) expression strain, and the transformed E.coli was grown overnight in LB medium containing 100mg/L ampicillin, and then cultured with 0.8mM IPTG (Sigma-Aldrich) at 30 ℃ for 8h to induce protein expression.
And (3) protein purification: the cells were collected by centrifugation at 4000 Xg for 15min and resuspended in Tris buffer (20mM Tris, pH 8.0). After the resuspended E.coli cells were homogenized and disrupted at high pressure, the supernatant was collected by centrifugation at 12000 Xg for 30 min. The supernatant is heat treated at 80 deg.C for 20min to denature and precipitate most of Escherichia coli impurity protein, and centrifuged again at 12000 Xg for 30min to collect supernatant. The HFn and its mutant proteins were then purified by anion exchange column Q-Sepharose Fast Flow (GE Healthcare) and finally purified by superdex 200(10/300GL, GE Healthcare) molecular sieves. Using bovine serum albumin as standard, and adopting BCA protein assayThe concentration of HFn and its mutant protein was determined with a test kit (Pierce) in triplicate. The resulting protein was concentrated to 10mg/mL and crystal growth was performed by sitting-drop method in a 48-well sitting-drop plate. The crystal growth pool liquid is 100mM Bicine, pH 9.0, 1.6-2.0M MgCl2. Mixing the raw materials in a ratio of 1: 1mL of protein and 1mL of pool liquid are mixed in a proportion of 1, and the mixture is placed in a sitting drop hole and protected from light under 291K for crystal growth. Then, the crystal which can be used for data collection is transferred into pool liquid containing 0.5mM adriamycin, soaked for 2-5 days under 291K in dark place, treated by frozen stock solution and stored in a liquid nitrogen tank.
And (4) data collection and structure analysis: the frozen crystals were subjected to collection of diffraction data. After the collected data are processed by HKL-3000 software, HFn crystal structure (5N27) is used as a model, a molecular replacement method is used for breaking the phase, and the HFn crystal structure containing adriamycin in 1.6 angstroms is finally analyzed through multiple rounds of optimization.
The results show that: the 24-mer ferritin molecules can entrap Dox molecules in the natural state without subunit depolymerization (as shown in fig. 1), but the only channels at the triple or quadruple axis of the intact ferritin molecules are small and cannot pass through the Dox molecules, so the mechanism of action of the Dox molecules into the ferritin core is still unknown. Meanwhile, the HFn crystal structure of Dox which can be loaded in a small amount is almost the same as the original structure (root mean standard deviation (r.m.s) is 0.09 angstroms), in the newly analyzed structure, residues 89-92 in the ferritin loop region are found to have the same structure as the original HFn structure, the interaction with nearby residues or other molecules is less, the flexibility of the fragment is far higher than that of ferritin which iron ions are not loaded, as shown in a Fo-Fc density chart (shown in figure 2), and the main chain of the residue is in negative density, which implies that the fragment residue is likely to have local conformational change during the loading of Dox and participate in the channel formation of Dox.
Example 2 mutation verification of Dox drug loading channel on surface of 2HFn protein
To verify the presence of Dox drug loading channels on the surface of HFn protein, we synthesized HFn series of mutants. Comprises HFn C90S/C102S/C130S (SEQ ID No: 4) mutant for proving the existence of the channel, and Enlarged-HFn-1(D92G) (SEQ ID No: 5), Enlarged-HFn-2(R43G + R79G) (SEQ ID No: 6) for expanding the channel and Block-HFn (C90S) (SEQ ID No: 7) for blocking the channel. HFn the residues in the 89-91 part of the protein shell can shift laterally, with little interaction between Cys90 and Asp91 and surrounding residues or water molecules, and the flexibility is particularly high. The region between C90S and C102S of the C90S/C102S/C130S mutant forms a hydrogen bond network formed by adjacent residues and bound water molecules, the hydrogen bond network relatively fixes the conformation of residues 89-91, so that the residues 89-91 are stable, side shift caused by flexibility in wild type HFn is inhibited, Dox is difficult to encapsulate in a drug encapsulation experiment under a non-denaturing condition by the mutant, and the fact that a channel does exist can be proved, and opening of the channel needs to keep certain flexibility at the residues 89-91. By mutating aspartic acid D at position 92 of the Dox channel into glycine G and removing the interaction between the amino acid side chain on the aspartic acid D and other residues, the mutation of D92G can further increase the flexibility of residues 89-91, so that the Dox channel is easier to open, and thus a mutant Enlarged-HFn-1(D92G) is designed. Through mutating R43 and R79 on the other side of the small molecule drug channel into glycine G, the amino acid side chain on arginine R is removed, and the drug channel is expanded, so that the mutant Enlarged-HFn-2(R43G + R79G) is designed. The hydrophilicity of the region is enhanced by mutating cysteine C at the position 90 of the Dox channel into serine S, a complete hydrogen bond ring is formed by the combined water molecules and surrounding residues, and the residue is stabilized in a Dox channel closed state, so that a mutant Block-HFn (C90S) is designed.
The specific method comprises the following steps: the DNA sequences of HFn C90S/C102S/C130S, Enlarged-HFn-1(D92G), Enla ged-HFn-2(R43G + R79G) and Block-HFn (C90S) mutants were synthesized for the whole gene (general, Shanghai), cut with NdeI and BamHI restriction enzymes, cloned into E.coli (E.coli) expression vector pET22b (+) plasmid (Novagen) with NdeI and BamHI restriction sites, and the sequences were verified to be correct by DNA sequencing. The subsequent experimental procedures were carried out as in steps (2) to (4) of example 1.
The results show that: WT-HFn, Enlarged-HFn-1, Enlarged-HFn-2, and Block-HFn proteins were obtained by prokaryotic expression purification as shown by SDS-PAGE (FIG. 2B). The results of transmission electron microscopy, Native-PAGE and size exclusion SEC show that Enlarged-HFn-1, Enlarged-HFn-2, Block-HFn and WT-HFn have consistent molecular weights in their Native state, and form 24-mer spherical shell structures by self-assembly (as shown in FIG. 3). Enlarged-HFn-1, Enlarged-HFn-2, Block-HFn and WT-HFn had hydrated particle sizes of 13.50nm, 13.77nm, 13.74nm and 12.38nm, respectively.
We verified that Dox could enter the ferritin lumen through the drug loading channel present on the surface of HFn protein by incubating HFn and Dox directly mixed. We found that ferritin HFn was able to load around 25.8 molecules of Dox after incubation of HFn and Dox by direct mixing for 4 hours at 37 degrees celsius, whereas Block-HFn had a Dox loading of only 3.4, significantly less than HFn, 12.67% of HFn under the same conditions; the Dox loading of Enlarged-HFn-1 was 58.2, significantly higher than HFn, and 225.7% of HFn; the Dox loading for Enlarged-HFn-2 was 53.1, significantly higher than HFn, and 205.7% of HFn (as shown in FIG. 4C).
Example 3 facilitating Loading of Small molecule drugs by controlling temperature
Based on the analysis result of the ferritin structure, we speculate that the ferritin drug channel can be further opened by a heating method, so that drug loading is facilitated.
The method comprises the following steps:
1. temperature gradient experiment
15% glycerol, WT-HFn 2mg, doxorubicin Dox0.75mg, and 50mM Tris buffer were added to 1mL of the reaction mixture, respectively, to make up to 1mL, pH 8.0. Incubate at 4 deg.C, 25 deg.C, 37 deg.C, 42 deg.C, 50 deg.C, 60 deg.C, 65 deg.C, and 72 deg.C for 4 h. Centrifuging at 12000rpm and 4 deg.C for 10min, collecting supernatant, desalting with Hiprep columnTM26/10 Desainting treatment removed free doxorubicin, a HFn-Dox ferritin doxorubicin sample was obtained. The concentrations of HFn and Dox in the HFn-Dox sample were measured to calculate the Dox loading and HFn protein yield, respectively.
2. Time gradient experiment
15% glycerol, WT-HFn 2mg, doxorubicin Dox0.75mg, and 50mM Tris buffer were added to 1mL of the reaction mixture, respectively, to make up to 1mL, pH 8.0. Respectively at 60 deg.CAnd (5) incubating for 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 16h, 20h and 24 h. Centrifuging at 12000rpm and 4 deg.C for 10min, collecting supernatant, desalting with Hiprep columnTM26/10 Desainting treatment removed free doxorubicin, a HFn-Dox ferritin doxorubicin sample was obtained. The concentrations of HFn and Dox in the HFn-Dox sample were measured to calculate the Dox loading and HFn protein yield, respectively.
3. Glycerol concentration gradient experiment in reaction System
Different glycerol concentration gradients (0%, 5%, 10%, 15%, 20%, 25%, 30%), WT-HFn 2mg, doxorubicin Dox0.75mg, 50mM Tris buffer were added to 1mL of the reaction system to make up to 1mL, pH 8.0. Incubate at 60 ℃ for 4 h. Centrifuging at 12000rpm and 4 deg.C for 10min, collecting supernatant, desalting with Hiprep columnTM26/10 Desainting treatment removed free doxorubicin, a HFn-Dox ferritin doxorubicin sample was obtained. The concentrations of HFn and Dox in the HFn-Dox sample were measured to calculate the Dox loading and HFn protein yield, respectively.
The results are shown in FIG. 5:
1. at low or room temperature (25 ℃ C., 4 ℃), Dox adsorbs on HFn surface, resulting in HFn total denaturing precipitates. As the temperature increased, Dox began to enter the HFn lumen from the channel, the Dox loading of HFn increased gradually and reached a peak loading (about 100) at 65 ℃, but after the temperature exceeded 60 ℃, the stability of HFn-Dox began to decrease, HFn began to precipitate and the protein yield began to decrease. After comprehensively comparing the loading capacity and the loading stability, the inventor finds that about 90 adriamycin molecules can be stably loaded by HFn under the incubation condition of 60 ℃, and the recovery rate of HFn protein reaches about 90%.
2. Under the condition of 60 ℃, the Dox loading reaches a peak value (90-100) after 4-6h of incubation, particularly HFn keeps about 90% of recovery rate when 4h of incubation is carried out, the time is increased, and the recovery rate of HFn is reduced at first.
3. The glycerol as a dissolving promoter is added into an HFn-Dox loading system in a certain proportion, so that the stability of HFn can be further improved, the recovery rate of HFn is improved, and the loading effect is optimal under the condition of 15% glycerol.
The above results all show that, in addition to the mutated Dox loading channel, the Dox channel can be further opened by heating, and more Dox molecules can be rapidly loaded in the HFn inner cavity.
EXAMPLE 4 non-denaturing temperature Loading method
1. Circular Dichroism (CD):
CD spectra were obtained from HFn-Dox samples loaded at HFn and different temperature conditions at 25 ℃ by Chirasca n-Plus circular dichroism spectroscopy (Applied Photophysics). The samples were resuspended in PBS at a concentration of 0.2 mg/mL. Spectra from 260nm to 200nm at 0.1nm resolution were measured using a quartz cuvette with a 1cm path length.
2. High performance liquid chromatography Size Exclusion (SEC):
HFn samples with the same protein concentration (0.5mg/mL) and HFn-Dox samples loaded under different temperature conditions were taken, respectively, and TSKgel G4000SW was usedXLColumn was subjected to size exclusion analysis by high performance liquid chromatography with mobile phase 50mM Tris-HCl, pH 7.2, and ultraviolet absorbance at 280nm and 485nm was measured. The loading amount was 100. mu.L.
3. Native-PAGE:
HFn samples of the same protein amount (50. mu.g) and HFn-Do x samples loaded under different temperature conditions were taken for Native-PAGE analysis.
4. Dynamic Light Scattering (DLS)
HFn-Dox samples (100. mu.L, 0.25mg/mL), PBS buffer were prepared. DLS analysis was performed using DynaPro Titan (Wyatt Technology) set at 25 ℃.
The results are shown in FIG. 6:
1. after HFn-Dox was obtained by incubation at 60 ℃ for 4h, we confirmed that Dox was stably loaded in the HFn lumen by size exclusion and Native-PAGE. The SEC data results showed that the HFn protein a280nm absorption peak overlaps with the Dox a485nm absorption peak in peak position, indicating that HFn forms a stable complex with Dox. Data from Native-PAGE show that in unstained condition, the position of the Dox indicated by the red band is the same as that of the HFn protein band in Coomassie blue stained condition, and that the HFn-Dox and HFn band positions are substantially the same, indicating that Dox is stably loaded in the HFn lumen.
2. Through the analysis of molecular exclusion SEC, we find that Dox itself can not be degraded and can be kept stable under the condition of incubation for 4h at 60 ℃, and the feasibility of loading Dox by a warming method is further proved.
3. Through Circular Dichroism (CD) analysis, the HFn-Dox sample loaded under HFn and different temperature conditions shows obvious alpha helix structure characteristics, and the secondary structure of HFn is not changed before and after Dox loading.
The detection results of SEC showed that the molecular weight and size of HFn did not change before and after Dox loading, and the HFn-Dox sample loaded under different temperature conditions detected a specific absorption peak of Dox at 485nm, while no Dox signal was detected in the control HFn.
The results of Native-PAGE showed no change in protein molecular weight and assembly status of HFn-Dox samples loaded under different temperature conditions compared to HFn control.
Example 5 non-denaturing temperature Loading method the Loading efficiency was greatly improved compared to Urea, pH renaturation method
1. Loading of doxorubicin by urea renaturation
HFn was dissolved in 8M urea solution (Amredco) at a concentration of 1mg/mL and gently stirred at room temperature for 30min to ensure HFn was completely denatured and dissolved. Dox (Sangon Biotech) was added at a concentration of 1 mg/mL. After incubation for 30min at room temperature in the dark, the mixture was transferred to dialysis bags (molecular weight cut-off 3KDa, thermolfisher Scientific) and dialyzed sequentially against a gradient urea solution containing 1mg/mL Dox (6, 5, 4, 3, 2, 1 and 0M) to renature the protein. Then, dialysis was performed in PBS buffer (pH7.4) to remove free Dox. Finally, in Supe rdexTMHFn-Dox protein was purified by molecular sieves on a 200pg gel filtration column (GE Healthcare). The concentration of HFn-Dox was determined by BCA protein assay kit using bovine serum albumin as standard, and three groups were determined in parallel. At 485nm (ε ═ 1.00X 104·M-1·cm-1) The molar extinction coefficient of Dox in HFn-Dox was determined and used to determine the concentration of Dox in HFn-Dox nanoparticles.
Loading of Adriamycin by pH renaturation
The loading of doxorubicin into the lumen of ferritin by the pH renaturation method was carried out according to the method described in the literature (Kilic, M.A.; Ozlu, E.; Calis, S., A novel protein-based anticancer drug encapsulation nanosphere: aporitin-doxorubicin complex. journal of biological nanotechnology 2012,8(3),508-14.), and the procedure was briefly as follows: 20mg of HFn protein was added to 40mL of 10mM glycine Acetate buffer Gly-Acetate buffer (pH 2.5) and incubated at a final concentration of 3mM Dox for 15min with mixing at 4 ℃ to allow ferritin to be sufficiently disintegrated. Then, about 50mL of 0.1M Tris buffer at pH 9.2 was slowly added to adjust the pH of the reaction system to about 4.0, so that the ferritin began to gradually recombine. The reaction was then dialyzed overnight against 20mM Tris buffer and PBS buffer, respectively, allowing the ferritin to self-assemble back into a native fullerene-like structure and doxorubicin to load into its lumen. The HFn-Dox sample can be obtained by centrifuging at 12000rpm for 30min at 4 ℃ and taking the supernatant.
pH5.0 in vitro drug Release assay and stability analysis assay for HFn-Dox
HFn-Dox nanoparticles in PBS buffer (600. mu.M Dox equivalent, 800. mu.L) were transferred to a D-Tube dialysis Tube (cut-off molecular weight 6-8kDa, Novagen), gently stirred at 37 ℃ in the absence of light, and then incubated in sodium acetate buffer (pH 5.0), PBS buffer (pH7.4), Fetal bovine serum (Fetal bovine serum, 1/1000NaN plus, respectively3) Performing dialysis. The release of free Dox in the dialysis buffer was determined at different time points according to the peak absorbance at 485nm using a NanoDrop 2000(Thermo Scientific).
4. Circular Dichroism (CD):
CD spectra were obtained from HFn and HFn-Dox samples loaded under different conditions at 25 ℃ by Chirascan-Plus circular dichroism spectroscopy (Applied Photophysics). The samples were resuspended in PBS at a concentration of 0.2 mg/mL. Spectra from 260nm to 200nm at 0.1nm resolution were measured using a quartz cuvette with a 1cm path length.
5. High performance liquid chromatography Size Exclusion (SEC):
HFn samples with the same protein concentration (0.5mg/mL) and HFn-Dox samples loaded under different conditions were taken, respectively, using TSKgel G4000SWXLColumn was subjected to size exclusion analysis by high performance liquid chromatography with mobile phase 50mM Tris-HCl, pH 7.2, and ultraviolet absorbance at 280nm and 485nm was measured. The loading amount was 100. mu.L.
The results are shown in FIG. 7:
comparing and analyzing a method for loading Dox by heating and a method for loading Dox by urea and pH variation, the method finds that the loading efficiency of Dox by the heating method is greatly improved. The Dox loading (90) for the warming method was significantly increased compared to the urea (33), pH (30) renaturation loading Dox. Moreover, the heated load Dox method was more stable, with ferritin recovery (90%) significantly higher than the urea renaturation (35%) and pH renaturation (15%) (see fig. 7A-B).
The results of Circular Dichroism (CD) and SEC detection show that the protein secondary structure is not changed after Dox loading by a heating method and the uniformity of ferritin is kept good after Dox loading compared with the prior Urea and pH renaturation method.
We further analyzed and compared the stability and drug release capacity of HFn-Dox produced by the three Dox loading methods. We found that HFn-Dox was significantly more stable in PBS and fetal calf serum than the pH renaturation loading Dox when Dox was loaded by the warm loading method and the urea renaturation method. Particularly, the drug is basically prevented from leaking out after the heating loading Dox is dialyzed for 7 days in PBS and fetal calf serum at 37 ℃. Also, no significant precipitation occurred with HFn-Dox in the warmed-loaded Dox when left at room temperature for 1 month, indicating that the HFn-Dox sample in the warmed-loaded Dox exhibited excellent stability.
Meanwhile, the HFn-Dox samples synthesized by the three Dox loading modes can realize drug release under the acidic condition of pH 5.0.
The results show that the Dox loading efficiency of the heating method is obviously higher than that of the existing Urea and pH renaturation method, and HFn-Dox synthesized by the heating method has better stability and drug release capacity under acidic conditions.
Example 6 non-denaturing temperature Loading method is also applicable to other Small molecule drugs
Using the same loading method, we tried at 60 degrees CelsiusAnd (3) under the condition of incubation for 4h, loading other small molecule drugs, such as Epirubicin (Epirubicin), Cisplatin (Cisplatin) and Oxaliplatin (Oxaliplatin), so as to prove the universality of HFn surface small molecule drug loading channels. In the case of Dox loading, 15% glycerol, WT-HFn 2mg, doxorubicin Dox0.75mg, and 50mM Tris buffer were added to 1mL of the reaction system to make up to 1mL, pH8.0, respectively. Incubate at 60 ℃ for 4 h. Centrifuging at 12000rpm and 4 deg.C for 10min, collecting supernatant, desalting with Hiprep columnTM26/10 Desainting treatment removed free doxorubicin, a HFn-Dox ferritin doxorubicin sample was obtained.
Experiments show that the heating method can be used for loading Epirubicin (Epirubicin), Cisplatin (Cisplatin) and Oxaliplatin (Oxaliplatin) as well, the loading amounts respectively reach 45, 130 and 117, and the protein recovery rates respectively reach 83%, 63% and 60%, as shown in FIG. 8. The above results further confirm the presence of HFn surface drug loading channels.
Example 7LFn surfaces do not have similar drug loading channels
From the results of the structural analysis, we found that the LFn protein surface did not have the same drug loading channel as HFn, and to confirm the results of the structural analysis, we tried to load Dox into the LFn lumen by heating at 60 ℃ for 4 h. 15% glycerol, LFn 2mg, doxorubicin Dox0.75mg, 50mM Tris buffer were added to 1mL of the reaction system, pH8.0, respectively. Incubate at 60 ℃ for 4 h. Centrifuging at 12000rpm and 4 deg.C for 10min, collecting supernatant, desalting with Hiprep columnTM26/10 Desainting treatment removed free doxorubicin, and LFn-Dox ferritin doxorubicin samples were obtained.
The results are shown in FIG. 9: under the same loading conditions, the Dox loading of LFn is only about 25, which is significantly lower than HFn. The above results indicate that LFn surfaces do not have similar drug loading channels and thus do not allow for large loading of Dox by warming.
EXAMPLE 8 HFn-Dox synthesized by the non-denaturing temperature Loading method exhibits excellent biosafety and ability to kill tumors efficiently
Because HFn-Dox loaded by a pH renaturation method has poor stability, a large amount of HFn-Dox cannot be synthesized for animal experiments, and therefore the effects of HFn-Dox synthesized by a heating method and a urea renaturation method in-vivo application are analyzed and compared.
The method comprises the following steps:
1. maximum tolerance test
BALB/c wild type mice (Wintolite) were injected tail vein with different doses of Heat-HFn-Dox (40, 35, 30, 20, 10mg/kg, Dox equivalent/body weight), Urea-HFn-Dox (20, 10mg/kg, Dox equivalent/body weight), HFn (400mg/kg, protein/body weight) respectively, and the body weight of each group was measured daily after injection of the drug to 3 mice per group.
2. Experiment on tumor therapy
All mice and their corresponding studies in this study were approved by the animal protection and use committee of the chinese academy of sciences. Treatment evaluation of subcutaneously implanted HepG2 tumor model 1X 10 subcutaneous implantation of 6 weeks old female BALB/c nude mice (Wittingle)6HepG2 tumor cells. When the tumor volume is about 80mm3Then, the mice were randomly divided into 5 groups (n ═ 6/group), and Heat-HFn-Dox (10mg/kg, Dox equivalent/body weight), Urea-HFn-Dox (10mg/kg, Dox equivalent/body weight), free Dox (5mg/kg, Dox equivalent/body weight), HFn (200mg/kg, protein/body weight), PBS (200 μ L/group) were intravenously injected. During the experiment, the body weight and tumor volume of each group of mice were measured every other day. Tumor volume is expressed as L.times.W2The/2 calculation, where L represents the maximum diameter of the tumor and W represents the minimum diameter of the tumor.
The results are shown in FIG. 10:
from the experimental results, we found that when 10mg/kg Dox equivalent of Heat-HFn-Dox and Urea-HFn-Dox were injected into the tail vein, no significant weight loss occurred in the mice; when 20mg/kg Dox equivalent of Heat-HFn-Dox and Urea-HFn-Dox were injected into the tail vein, the mice both experienced weight loss, and the Heat-HFn-Dox group achieved weight recovery on day 3, while the Urea-HFn-Dox group achieved weight recovery on day 6; when the dose was increased to 25mg/kg Dox equivalent of Urea-HFn-Dox, mice in the Urea-HFn-Dox group died, indicating that the maximum tolerated dose of Urea-HFn-Dox was 20mg/kg Dox equivalent; while the maximum tolerated dose in the Heat-HFn-Dox group was increased to 35mg/kg Dox equivalent. The above results indicate that Heat-HFn-Dox shows better biosafety in vivo than Urea-HFn-Dox. In the following animal treatment experiments, we chose a safe dose of 10mg/kg Dox equivalent.
To confirm the superiority of Heat-HFn-Dox in tumor therapy experiments, we first constructed a subcutaneous tumor-bearing model of human hepatoma mice. Up to about 80mm in tumor growth3In size, mice were divided into 5 groups on average according to tumor volume, and injected intravenously with Heat-HFn-Dox (10mg/kg, Dox equivalent/body weight), Urea-HFn-Dox (10mg/kg, Dox equivalent/body weight), free Dox (5mg/kg, Dox equivalent/body weight), HFn (200mg/kg, protein/body weight), PBS (200. mu.L/mouse), respectively. Twice weekly dosing, the Dox group showed a sustained weight loss after twice dosing, thus the dosing was stopped; the Urea-HFn-Dox group showed a sustained weight loss after 3 administrations, and thus administration was discontinued; while the Heat-HFn-Dox group was administered for 6 times until the tumors of the control group (Dox group, HFn group, PBS group) were all over 1000mm3The body weight of the mice remained steady. Heat-HFn-Dox has obvious effect of inhibiting tumor growth, and is better than Urea-HFn-Dox.
Example 9 non-denaturing temperature Loading method HFn-siRNA synthesized by loading siRNA into HFn showed excellent anti-brain tumor activity
Using the same loading method, we tried loading nucleic acid drugs under incubation at 60 degrees Celsius for 4 h. In the case of loading siRNA targeting EGFR, 15% glycerol, WT-HFn 2mg, and siRNA targeting EGFR (Sense strand 5'-GGAGCUGCCCAUGAGAAAUtt-3'; Antisense strand 5'-AUUUCUCAUGGGCAGCUCCtt-3', Invitrogen)20mg, and 50mM Tris buffer were added to 1mL of the reaction system, respectively, to make up 1mL, pH 8.0. Incubate at 60 ℃ for 4 h. 12000rpm, 4 ℃ for 10min, supernatant, adding RNase (1mg/mL) in 37 degrees water bath for 0.5 h. Desalting column HiprepTM26/10 desaling replaced the protein sample with saline to obtain HFn-siRNA sample.
Culturing Luciferase-labeled human glioma cell U87-MG-Luc (purchased from cell bank of Basil medical college of Chinese academy of medical sciences) to 1 × 105About (culture)The culture conditions are as follows: DMEM medium (Sigma-Aldrich), 10% fetal bovine serum (Sigma-Aldrich), penicillin (100U/mL, Sigma-Aldrich) and streptomycin (100. mu.g/mL, Sigma-Aldrich), 37 ℃, 5% CO2Cultured under conditions).
Brain orientation instrument (Mouse) was performed by using Balb/c nude mice (purchased from Weitonghua Beijing)TMThe inventor successfully constructs a mouse glioma in situ cancer model by the technical means of Ster eotaxic Instrument, Stoelting30 Co.) positioning, microinjector (10 mu L, Hamilton) microinjection (10 mu L) of glioma U87MG cells and surgical suture. The tail vein was injected with HFn-siRNA and its control (HFn) at equal doses of 10mg/Kg ferritin three times, once every other day. Live small animal imaging was then performed using the small animal imaging system ivis (perkinelmer) (as shown in fig. 11).
HFn-siRNA loaded with siRNA can specifically target brain tumors and significantly inhibit the growth of brain tumors compared to controls. This example demonstrates that a non-denaturing temperature loading method, suitable for nucleic acid drugs, including, but not limited to, siRNA, can effectively target brain tumors, inhibiting the growth of brain tumors.
Example 10 the R63, E67, Y39, L35, M70, F81 amino acid residues near the ferritin drug-loading channel had no significant effect on ferritin drug loading
In order to verify that a complete drug-carrying channel exists on ferritin from outside to inside, we find that 6 amino acid residues of R63, E67, Y39, L35, M70 and F81 possibly respectively form the inner channel region, the middle channel region and the outer channel region of the drug-carrying channel through structural analysis. Wherein R63, E67, Y39 constitute the inner region of the channel, L35, M70 constitute the middle region of the channel, and F81 is located at the outer region of the channel. Therefore, we mutated the above 6 amino acid residues into serine (S) and alanine (a), respectively, by mutant design, and removed the side chains of these amino acid residues, thereby allowing the channel region to be completely opened. This mutation can result in an increase in drug loading if these 6 amino acid residues are in the drug loading channel. Therefore, we designed ferritin mutants directed to the inside, middle and outside of the channel, respectively: HFn-Inner1(R63S), HFn-Inner2(E67S), HFn-Inner3(Y39A), HFn-Innerrgroup (R63S + E67S + Y39A), HFn-Midggroup (L35A + M70A), HFn-outer4 (F81A).
The method comprises the following steps:
construction of mutant expression plasmids of HFn-Inner1(R63S), HFn-Inner2(E67S), HFn-Inner3(Y39A), HFn-Innergorroup (R63S + E67S + Y39A), HFn-Midgroup (L35A + M70A), HFn-outer4(F81A)
HFn-Inner1(R63S) (SEQ ID NO:8), HFn-Inner2(E67S) (SEQ ID NO:9), HFn-Inner3(Y39A) (SEQ ID NO:10), HFn-Innerrgroup (R63S + E67S + Y39A) (SEQ ID NO:11), HFn-Midgroup (L35A + M70A) (SEQ ID NO:12), HFn-outer4(F81A) (SEQ ID NO:13) mutants were cloned into E.coli (E.coli) expression vector pET30 (+) 30a plasmid (Novagen) with NdeI and BamHI restriction sites after whole gene synthesis (Genray, Shanghai) using NdeI and BamH1 restriction enzymes and DNA sequencing to identify the correct sequences.
Expression and purification of mutants of HFn-Inner1(R63S), HFn-Inner2(E67S), HFn-Inner3(Y39A), HFn-Innergorroup (R63S + E67S + Y39A), HFn-Midgroup (L35A + M70A), HFn-outer4(F81A)
The plasmid obtained above was transferred into E.coli BL21(TransGen) expression strain, and the transformed E.coli was grown overnight in LB medium containing 50mg/L kanamycin, followed by culture with 0.8mM IPTG (Sigma-Aldrich) at 30 ℃ for 8h to induce protein expression.
3. Protein purification: the cells were collected by centrifugation at 4000g for 15min and resuspended in Tris buffer (20mM Tris, pH 8.0). After the resuspended E.coli cells were homogenized and disrupted at high pressure, 12000g were centrifuged for 30min to collect the supernatant. The supernatant is heat treated at 80 deg.C for 20min to denature most of the Escherichia coli heteroprotein, and centrifuged again at 12000g for 30min to collect the supernatant. The HFn and its mutant proteins were then purified and separated by anion exchange column Q-Sepharose Fast Flow (GE Healthcare) and finally purified and separated by superdex 20010/300GL molecular sieves (GE Healthcare). The concentration of HFn mutant protein was determined in triplicate using a BCA protein assay kit (Pierce) using bovine serum albumin as standard.
Preparation of HFn-Dox: purified HFn mutant protein was mixed with 0.5mg/mlDox mix in 20mM Tris, pH8.0, 0.15M NaCl, 10% glycerol, 37 ℃ for 4 hours, after which unbound Dox is removed by desalting column and the buffer is replaced with 20mM Tris, pH8.0, 0.15M NaCl and concentrated to 10 mg/ml. Detection of A by Nanodrop280nmAnd A485nmThe absorption value.
As a result:
4.1, HFn-Inner1(R63S), HFn-Inner2(E67S), HFn-Inner3(Y39A), HFn-Innergorup (R63S + E67S + Y39A), HFn-Midgroup (L35A + M70A), HFn-outer4(F81A) proteins can be obtained by prokaryotic expression and purification methods. The results of the molecular exclusion SEC show that molecular weights of HFn-Inner1(R63S), HFn-Inner2(E67S), HFn-Inner3(Y39A), HFn-Inner group (R63S + E67S + Y39A), HFn-Midgroup (L35A + M70A), HFn-outer4(F81A) in the natural state are consistent, and 24-polymer spherical shell-like structures are formed through self-assembly. The hydrated particle sizes of HFn-Inner1(R63S), HFn-Inner2(E67S), HFn-Inner3(Y39A), HFn-Innerrgroup (R63S + E67S + Y39A), HFn-Midggroup (L35A + M70A) and HFn-outer4(F81A) are respectively 13.70nm, 13.57nm, 13.24nm, 12.88nm, 13.84nm and 12.68 nm.
2. We verified whether the 6 amino acid residues R63, E67, Y39, L35, M70 and F81 are related to a drug loading channel existing on the surface of HFn protein by a method of directly mixing and incubating HFn mutant and Dox. We found that ferritin HFn was able to load around 25.8 Dox molecules after incubation of HFn and Dox by direct mixing for 4 hours at 37 degrees celsius, whereas under the same conditions HFn-Inner1(R63S), HFn-Inner2(E67S), HFn-Inner3(Y39A), HFn-lnergroup (R63S + E67S + Y39A), HFn-Midgroup (L35A + M70A), HFn-outer4(F81A) had Dox loading levels of only 10.2, 8.4, 8.1, 9.7, 7.2, 10.5, significantly lower than HFn, respectively.
EXAMPLE 11 non-denaturing temperature Loading with other anti-tumor drugs
Using the same loading method, we tried loading of other antitumor drugs under incubation at 60 degrees for 4h, including: sunitinib (sunitinib), crizotinib (crizotinib), Palbociclib (Palbociclib), irinotecan (irinotecan), maytansine (maytansine), 5-fluorouracil (5-fluorouraci), pentoxifylline (pentaxiphyline). Wherein pentoxifylline is hydrophilic drug, and the rest drugs are hydrophobic drugs. Taking sunitinib loading as an example, 15% glycerol, WT-HFn 2mg, sunitinib 0.75mg, and 50mM Tris buffer were added to 1ml of the reaction system to make up to 1ml, pH8.0, respectively. Incubate at 60 ℃ for 4 h. Centrifuging at 12000rpm and 4 ℃ for 10min, taking supernatant, and removing free sunitinib by HiprepTM 26/10 desaling treatment of Desalting column to obtain HFn-sunitinib ferritin sunitinib sample. HFn were enzymatically cleaved using pepsin to allow leakage of the loaded drug and the drug concentration was measured by HPLC calibration (see FIG. 13). The concentration of HFn mutant protein was determined in triplicate using a BCA protein assay kit (Pierce) using bovine serum albumin as standard.
We found through experiments that the warming method can also be used to load natural (plant) anticancer drugs: sunitinib (sunitinib), crizotinib (crizotinib), Palbociclib (Palbociclib), irinotecan (irinotecan), maytansine (maytansine), antimetabolites: 5-fluorouracil (5-fluorouracil), pentoxifylline (Pentoxiphylline). As a result, as shown in fig. 14, the loading amounts were 14.9, 17.6, 7.5, 32.8, 3.0, 25.6, and 2.3, respectively. As shown in fig. 15, the ferritin recovery rates reached 86%, 79%, 84%, 81%, 86%, 80%, and 82%, respectively. The above results further confirm the presence of HFn surface drug loading channels.
In conclusion, the entrapping condition under the non-denaturing condition obtained by the method can conveniently, quickly, efficiently and sufficiently entrap different small molecule drugs by HFn.
Unless specifically stated otherwise, the numerical values set forth in these examples do not limit the scope of the invention. In all examples shown and described herein, unless otherwise specified, any particular value should be construed as merely illustrative, and not restrictive, and thus other examples of example embodiments may have different values.