Dopamine-conjugated apoferritin protein nanocage for the dual-targeting delivery of epirubicin

Document Type: Research Paper


1 Department of Applied Chemistry, Faculty of Chemistry, Razi University, Kermanshah, Iran

2 Department of Sciences, College of Basic Education, Al-Muthanna University, Al-Muthanna, Iraq

3 Faculty of Chemistry, Sensor and Biosensor Research Center and Nanoscience and Nanotechnology Research Center, Razi University, Kermanshah, Iran

4 Nano Drug Delivery Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran

5 Department of Chemistry, Kermanshah Branch, Islamic Azad University, Kermanshah, Iran

6 Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran


Objective(s): Nanocarriers are drug delivery vehicles, which have attracted the attention of researchers in recent years, particularly in cancer treatment. The encapsulation of anticancer drugs using protein nanocages is considered to be an optimal approach to reducing drug side-effects and increasing the bioavailability of anticancer drugs. Epirubicin (EPR) is an active chemotherapeutic medication used in the treatment of breast cancer. However, the toxicity of this drug against normal cells is a considerable limitation in therapy. EPR toxicity could be reduced using nanocarriers and dual-targeted drug delivery. Dual-targeted drug delivery system was developed by the conjugation of dopamine (DA) with horse spleen apoferritin (HsAFr)-encapsulated EPR to overcome the limitations of chemotherapeutic EPR in breast cancer treatment. HsAFr-EPR-DA complexes could target the scavenger receptors, transferrin receptors 1, and DA receptors, which are overexpressed on breast cancer cells.
Materials and Methods: UV-Visible, fluorescence, and circular dichroism (CD) spectroscopic techniques and transmission electronic microscope (TEM) have been applied to characterize HsAFr-EPR-DA complexes. In the present study, we utilized human breast cancer cell line (MCF-7), aiming to compare the cytotoxicity of HsAFr-EPR-DA complexes to free EPR.
Results: The toxicity was measured using the MTT assay, which demonstrated that the dual-targeted nanocarrier (HsAFr-EPR-DA) enhanced cytotoxicity against MCF-7 more significantly compared to non-targeted nanocarriers.
Conclusion: The findings of the current research indicated that the synthesized HsAFr-DA complex was an optimal nanocarrier for the dual-targeted delivery of anticancer drugs.


1.Torre LA, Bray F, Siegel RL, Ferlay J, Lortet‐Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015; 65(2): 87-108.
2.Pais-Silva C, de Melo-Diogo D, Correia IJ. IR780-loaded TPGS-TOS micelles for breast cancer photodynamic therapy. Eur J Pharm Biopharm. 2017; 113: 108-117.
3.Miller KD, Siegel RL, Lin CC, Mariotto AB, Kramer JL, Rowland JH. Cancer treatment and survivorship statistics, 2016. CA Cancer J Clin. 2016; 66(4): 271-289.
4.Zagar TM, Cardinale DM, Marks LB. Breast cancer therapy-associated cardiovascular disease. Nat Rev Clin Oncol. 2016; 13(3): 172.
5.Mordente A, Meucci E, EttoreMartorana G, Tavian D, Silvestrini A. Topoisomerases and anthracyclines: recent advances and perspectives in anticancer therapy and prevention of cardiotoxicity. Curr Med Chem. 2017; 24(15): 1607-1626.
6.Perrino C, G Schiattarella G, Magliulo F, Ilardi F, Carotenuto G, Gargiulo G, et al. Cardiac side effects of chemotherapy: state of art and strategies for a correct management. Curr Vasc Pharmacol. 201412(1):106-16..
7.Netíková IRŠ, Slušná M, Tolasz J, Št’astný M, Popelka Š, Štengl V. A new possible way of anthracycline cytostatics decontamination. New J Chem. 2017; 41(10): 3975-3985.
8.Karimi F, Shojaei AF, Tabatabaeian K, Shakeri S. CoFe2O4 nanoparticle/ionic liquid modified carbon paste electrode as an amplified sensor for epirubicin analysis as an anticancer drug. J Mol Liq. 2017; 242: 685-689.
9.Rother M, Nussbaumer MG, Renggli K, Bruns N. Protein cages and synthetic polymers: a fruitful symbiosis for drug delivery applications, bionanotechnology and materials science. Chem Soc Rev. 2016; 45(22): 6213-6249.
10.Lee EJ, Lee NK, Kim I-S. Bioengineered protein-based nanocage for drug delivery. Adv Drug Deliv Rev. 2016; 106: 157-171.
11.Zangabad PS, Karimi M, Mehdizadeh F, Malekzad H, Ghasemi A, Bahrami S, et al. Nanocaged platforms: modification, drug delivery and nanotoxicity. Opening synthetic cages to release the tiger. Nanoscale. 2017; 9(4): 1356-1392.
12.Zang J, Chen H, Zhao G, Wang F, Ren F. Ferritin cage for encapsulation and delivery of bioactive nutrients: From structure, property to applications. Crit Rev Food Sci Nutr. 2017; 57(17): 3673-3683.
13.Zhen Z, Tang W, Chen H, Lin X, Todd T, Wang G, et al. RGD-modified apoferritin nanoparticles for efficient drug delivery to tumors. ACS nano. 2013; 7(6): 4830-4837.
14.Mosca L, Falvo E, Ceci P, Poser E, Genovese I, Guarguaglini G. Use of Ferritin-Based Metal-Encapsulated Nanocarriers as Anticancer Agents. Applied Sciences. 2017; 7(1): 101.
15.Wei J, Li YL, Gao PC, Lu Q, Wang ZF, Zhou JJ, et al. Assembling gold nanoparticles into flower-like structures by complementary base pairing of DNA molecules with mediation by apoferritins. Chem Commun (Camb). 2017; 53(33): 4581-4584.
16.Jang JS, Choi SJ, Kim SJ, Hakim M, Kim ID. Rational Design of highly porous SnO2 nanotubes functionalized with biomimetic nanocatalysts for direct observation of simulated diabetes. Adv Funct Mater. 2016; 26(26): 4740-4748.
17.Zhang S, Zang J, Chen H, Li M, Xu C, Zhao G. The size flexibility of ferritin nanocage opens a new way to prepare nanomaterials. Small. 2017; 13(37): 1701045.
18.Pontillo N, Ferraro G, Helliwell JR, Amoresano A, Merlino A. X-ray Structure of the Carboplatin-Loaded Apo-Ferritin Nanocage. ACS Med Chem Lett. 2017; 8(4): 433-437.
19.Gomhor JAH, Kashanian S, Rafipour R, Mahdavian E, Mansouri K. Development and characterization of folic acid-functionalized apoferritin as a delivery vehicle for epirubicin against MCF-7 breast cancer cells. Artif Cells Nanomed Biotechnol. 2018; 46(sup3): S847-s854.
20.Dostalova S, Heger Z, Kudr J, Vaculovicova M, Adam V, Stiborova M, et al. Apoferritin: protein nanocarrier for targeted delivery. Nano Based Drug Delivery Zagerb: IAPC Publishing. 2015: 217-33.
21.Kim M, Rho Y, Jin KS, Ahn B, Jung S, Kim H. pH-dependent structures of ferritin and apoferritin in solution: disassembly and reassembly. Biomacromolecules. 2011; 12(5): 1629-1640.
22.Dostalova S, Polanska H, Svobodova M, Balvan J, Krystofova O, Haddad Y. Prostate-Specific Membrane Antigen-Targeted Site-Directed Antibody-Conjugated Apoferritin Nanovehicle Favorably Influences In Vivo Side Effects of Doxorubicin. Sci Rep. 2018; 8(1): 8867.
23.Roy K, Patel YS, Kanwar RK, Rajkhowa R, Wang X, Kanwar JR. Biodegradable Eri silk nanoparticles as a delivery vehicle for bovine lactoferrin against MDA-MB-231 and MCF-7 breast cancer cells. Int J Nanomedicine. 2016; 11:25.
24.Danilo C, Gutierrez-Pajares JL, Mainieri MA, Mercier I, Lisanti MP, Frank PG. Scavenger receptor class B type I regulates cellular cholesterol metabolism and cell signaling associated with breast cancer development. Breast Cancer Res. 2013; 15(5): R87.
25.Dostalova S, Cerna T, Hynek D, Koudelkova Z, Vaculovic T, Kopel P. Site-directed conjugation of antibodies to apoferritin nanocarrier for targeted drug delivery to prostate cancer cells. ACS Appl Mater Interfaces. 2016; 8(23): 14430-14441.
26.Kitagawa T, Kosuge H, Uchida M, Iida Y, Dalman RL, Douglas T. RGD targeting of human ferritin iron oxide nanoparticles enhances in vivo MRI of vascular inflammation and angiogenesis in experimental carotid disease and abdominal aortic aneurysm. J Magn Reson Imaging. 2017; 45(4): 1144-1153.
27.Zhao J, Liu M, Zhang Y, Li H, Lin Y, Yao S. Apoferritin protein nanoparticles dually labeled with aptamer and horseradish peroxidase as a sensing probe for thrombin detection. Anal Chim Acta. 2013; 759: 53-60.
28.Le TH, Kim JH, Park SJ. Fabrication of CdTe quantum dots–apoferritin arrays for detection of dopamine. J Cryst Growth. 2017; 468: 788-791.
29.Borcherding DC, Tong W, Hugo ER, Barnard DF, Fox S, LaSance K. Expression and therapeutic targeting of dopamine receptor-1 (D1R) in breast cancer. Oncogene. 2016; 35(24): 3103.
30.Yin T, He S, Shen G, Ye T, Guo F, Wang Y. Dopamine receptor antagonist thioridazine inhibits tumor growth in a murine breast cancer model. Mol Med Rep. 2015; 12(3): 4103-4108.
31.Wu M, Zhang D, Zeng Y, Wu L, Liu X, Liu J. Nanocluster of superparamagnetic iron oxide nanoparticles coated with poly (dopamine) for magnetic field-targeting, highly sensitive MRI and photothermal cancer therapy. Nanotechnology. 2015; 26(11): 115102.
32.Kim S, Jang Y, Jang LK, Sunwoo SH, Kim T-i, Cho S-W. Electrochemical deposition of dopamine–hyaluronic acid conjugates for anti-biofouling bioelectrodes. J Mater Chem B2017;5(23):4507-. 13.
33.Lee D-W, Yun Y-P, Park K, Kim SE. Gentamicin and bone morphogenic protein-2 (BMP-2)-delivering heparinized-titanium implant with enhanced antibacterial activity and osteointegration. Bone. 2012; 50(4): 974-982.
34.Motiei M, Kashanian S. Preparation of amphiphilic chitosan nanoparticles for controlled release of hydrophobic drugs. J Nanosci Nanotechnol. 2017; 17(8): 5226-5232.
35.Vantangoli MM, Madnick SJ, Huse SM, Weston P, Boekelheide K. MCF-7 Human Breast Cancer Cells Form Differentiated Microtissues in Scaffold-Free Hydrogels. PloS one. 2015; 10(8): e0135426-e.
36.Shadkam M, Mansouri K. DNA binding and cytotoxicity studies of magnetic nanofluid containing antiviral drug oseltamivir AU - Shahabadi, Nahid. J Biomol Struct Dyn. 2018: 1-9.
37.Kilic MA, Ozlu E, Calis S. A novel protein-based anticancer drug encapsulating nanosphere: Apoferritin-doxorubicin complex. J Biomed Nanotechnol. 2012; 8(3): 508-514.
38.Luo Y, Wang X, Du D, Lin Y. Hyaluronic acid-conjugated apoferritin nanocages for lung cancer targeted drug delivery. Biomater Sci. 2015; 3(10): 1386-1394.
39.Zalewski P, Firlej A, Medenecka B, Jankowska J, Mielcarek J, Oszczapowicz I. The use of UV-VIS spectroscopy for determining the photostability of epirubicin solutions2009. 43-8 p.
40.Hayat A, Andreescu D, Bulbul G, Andreescu S. Redox reactivity of cerium oxide nanoparticles against dopamine. J Colloid Interface Sci. 2014; 418: 240-245.
41.Barreto W, Ponzoni S, Sassi P. A Raman and UV-Vis study of catecholamines oxidized with Mn (III). Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 1998; 55(1): 65-72.
42.Fonseca BM, Rodrigues M, Cristóvão AC, Gonçalves D, Fortuna A, Bernardino L, et al. Determination of catecholamines and endogenous related compounds in rat brain tissue exploring their native fluorescence and liquid chromatography. J Chromatogr B. 2017; 1049 :51-59.
43.Chang SK, Zhang Y. Protein analysis. Food analysis: Springer; 2017. p. 315-31.
44.Satish L, Millan S, Bera K, Mohapatra S, Sahoo H. A spectroscopic and molecular dynamics simulation approach towards the stabilizing effect of ammonium-based ionic liquids on bovine serum albumin. New J Chem. 2017; 41(19): 10712-10722.
45.Matsumiya K, Suzuki YA, Hirata Y, Nambu Y, Matsumura Y. Protein–surfactant interactions between bovine lactoferrin and sophorolipids under neutral and acidic conditions. Biochem Cell Biol. 2017; 95(1): 126-132.
46.Jafari M, Karunaratne DN, Sweeting CM, Chen P. Modification of a designed amphipathic cell-penetrating peptide and its effect on solubility, secondary structure, and uptake efficiency. Biochemistry. 2013; 52(20): 3428-3435.
47.Kashanian S, Tarighat FA, Rafipour R, Abbasi-Tarighat M. Biomimetic synthesis and characterization of cobalt nanoparticles using apoferritin, and investigation of direct electron transfer of Co (NPs)–ferritin at modified glassy carbon electrode to design a novel nanobiosensor. Molecular biology reports. 2012; 39(9): 8793-8802.
48.Ito D, Itagaki H. Clarification of the inner microenvironments in poly (N-isopropylacrylamide) hydrogels in macrogel and microgel forms using a fluorescent probe technique. Eur Polym J. 2018; 99: 277-283.
49.Ji X-T, Huang L, Huang H-Q. Construction of nanometer cisplatin core-ferritin (NCC-F) and proteomic analysis of gastric cancer cell apoptosis induced with cisplatin released from the NCC-F. J Proteom. 2012; 75(11): 3145-3157.
50.Liu X, Wei W, Wang C, Yue H, Ma D, Zhu C, et al. Apoferritin-camouflaged Pt nanoparticles: surface effects on cellular uptake and cytotoxicity. J Mater Chem A. 2011; 21(20): 7105-7110.
51.Jiang Y, Pang X, Wang X, Leung AW, Luan Y, Zhao G, et al. Preparation of hypocrellin B nanocages in self-assembled apoferritin for enhanced intracellular uptake and photodynamic activity. J Mater Chem B. 2017; 5(10): 1980-1987.
52.Lin C-Y, Yang S-J, Peng C-L, Shieh M-J. Panitumumab-Conjugated and Platinum-cored pH-sensitive Apoferritin Nanocages for Colorectal Cancer-targeted Therapy. ACS Appl Mater Interfaces. 2018; 10(7): 6096-6106.
53.Zhang S, Zang J, Chen H, Li M, Xu C, Zhao G. The Size Flexibility of Ferritin Nanocage Opens a New Way to Prepare Nanomaterials. Small. 2017; 13(37).
54.Belletti D, Pederzoli F, Forni F, Vandelli MA. Protein cage nanostructure as drug delivery system: magnifying glass on apoferritin. 2017; 14(7): 825-840.
55.Conti L, Lanzardo S, Ruiu R, Cadenazzi M, Cavallo F, Aime Sl. L-Ferritin targets breast cancer stem cells and delivers therapeutic and imaging agents. Oncotarget. 2016; 7(41): 66713-66727.
56.Aleksandrowicz R, Taciak B, Krol M. Drug delivery systems improving chemical and physical properties of anticancer drugs currently investigated for treatment of solid tumors. J Physiol Pharmacol. 2017; 68(2): 165-174.
57.Li J, Yao Q-y, Xue J-s, Wang L-j, Yuan Y, Tian X-y, et al. Dopamine D2 receptor antagonist sulpiride enhances dexamethasone responses in the treatment of drug-resistant and metastatic breast cancer. Acta Pharmacol Sin. 2017; 38(9): 1282.