Upload
others
View
0
Download
0
Embed Size (px)
Citation preview
Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/264557153
FerritinNanocages:ANovelPlatformforBiomedicalApplications
ArticleinJournalofBiomedicalNanotechnology·October2014
DOI:10.1166/jbn.2014.1980
CITATIONS
17
READS
496
6authors,including:
Someoftheauthorsofthispublicationarealsoworkingontheserelatedprojects:
Frictionanddurabilityofvirginanddamagedskinwithandwithoutskincreamtreatmentusing
atomicforcemicroscopyViewproject
NanomedicineViewproject
BharatBhushan
TheOhioStateUniversity
1,135PUBLICATIONS40,575CITATIONS
SEEPROFILE
UdayKumar
IndianInstituteofTechnologyRoorkee
23PUBLICATIONS318CITATIONS
SEEPROFILE
IshitaMatai
IndianInstituteofTechnologyRoorkee
20PUBLICATIONS393CITATIONS
SEEPROFILE
AbhaySachdev
IndianInstituteofTechnologyRoorkee
23PUBLICATIONS428CITATIONS
SEEPROFILE
AllcontentfollowingthispagewasuploadedbyGopinathPackirisamyon27January2016.
Theuserhasrequestedenhancementofthedownloadedfile.
https://www.researchgate.net/publication/264557153_Ferritin_Nanocages_A_Novel_Platform_for_Biomedical_Applications?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_2&_esc=publicationCoverPdfhttps://www.researchgate.net/publication/264557153_Ferritin_Nanocages_A_Novel_Platform_for_Biomedical_Applications?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_3&_esc=publicationCoverPdfhttps://www.researchgate.net/project/Friction-and-durability-of-virgin-and-damaged-skin-with-and-without-skin-cream-treatment-using-atomic-force-microscopy?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_9&_esc=publicationCoverPdfhttps://www.researchgate.net/project/Nanomedicine-32?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_9&_esc=publicationCoverPdfhttps://www.researchgate.net/?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_1&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Bharat_Bhushan11?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_4&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Bharat_Bhushan11?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_5&_esc=publicationCoverPdfhttps://www.researchgate.net/institution/The_Ohio_State_University?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_6&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Bharat_Bhushan11?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_7&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Uday_Kumar72?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_4&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Uday_Kumar72?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_5&_esc=publicationCoverPdfhttps://www.researchgate.net/institution/Indian_Institute_of_Technology_Roorkee?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_6&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Uday_Kumar72?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_7&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Ishita_Matai?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_4&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Ishita_Matai?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_5&_esc=publicationCoverPdfhttps://www.researchgate.net/institution/Indian_Institute_of_Technology_Roorkee?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_6&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Ishita_Matai?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_7&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Abhay_Sachdev?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_4&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Abhay_Sachdev?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_5&_esc=publicationCoverPdfhttps://www.researchgate.net/institution/Indian_Institute_of_Technology_Roorkee?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_6&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Abhay_Sachdev?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_7&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Gopinath_Packirisamy2?enrichId=rgreq-9cc35a51e06257bdc088662ac301496a-XXX&enrichSource=Y292ZXJQYWdlOzI2NDU1NzE1MztBUzozMjI0NjM2ODUxMjAwMDJAMTQ1Mzg5Mjc0MTc1NA%3D%3D&el=1_x_10&_esc=publicationCoverPdf
Copyright © 2014 American Scientific PublishersAll rights reservedPrinted in the United States of America
ReviewJournal of
Biomedical NanotechnologyVol. 10, 2950–2976, 2014
www.aspbs.com/jbn
Ferritin Nanocages: A Novel Platform forBiomedical Applications
Bharat Bhushan1, S. Uday Kumar1, Ishita Matai1, Abhay Sachdev1,Poornima Dubey1, and P. Gopinath1�2�∗1Nanobiotechnology Laboratory, Centre for Nanotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India2Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Ferritin is a ubiquitous iron storage protein responsible for maintaining the iron homeostasis in living organism and therebyprotects the cell from oxidative damage. The ferritin protein cages have been used as a reaction vessel for the synthesisof various non-native metallic nanoparticles inside its core and also used as a nanocarrier for various applications. Lackof suitable non-viral carrier for targeted delivery of anticancer drugs and imaging agents is the major problem in cancertherapy and diagnosis. The pH dependent reversible assembling and disassembling property of ferritin renders it asa suitable candidate for encapsulating a variety of anticancer drugs and imaging probes. Ferritins external surface ischemically and genetically modifiable which can serve as attachment site for tumor specific targeting peptides or moieties.Recent studies, further establishes ferritin as a multifunctional nanocarrier for targeted cancer diagnosis and therapy.Moreover, the biological origin of these protein cages makes it a biocompatible nanocarrier that stabilizes and protectsthe enclosed particles from the external environment without provoking any toxic or immunogenic responses. This reviewmainly focuses on the application of ferritin nanocages as a novel non-viral nanocarrier for cancer therapy and it alsohighlights various biomedical applications of ferritin nanocages.
KEYWORDS: Apoferritin, Protein Cages, Nanoparticles Synthesis, Biomedical Applications, Cancer Therapy, Cancer Imaging.
CONTENTSIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2950Ferritin and Its Biological Role . . . . . . . . . . . . . . . . . . . . . . 2952Structure of Ferritin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2953Routes of Loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2955Biomineralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2955Ferritin as a Template for Nanoparticles (NPs) Synthesis . . . . . . 2956Applications of Ferritin Protein Cages . . . . . . . . . . . . . . . . . . 2960
Tumor Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2960Tumor Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2961Tumor Targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2963Cellular Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2964Bioassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2964Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2965Biocatalyst . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2966Enzyme Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . 2967Artificial Antioxidant . . . . . . . . . . . . . . . . . . . . . . . . . . . 2967Magnetic Resonance Imaging (MRI)Contrasting Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2967Biocompatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2969
∗Author to whom correspondence should be addressed.Emails: [email protected], [email protected]: 20 January 2014Accepted: 28 March 2014
Other Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2969Conclusion and Future Perspectives . . . . . . . . . . . . . . . . . . . . 2970
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2970References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2970
INTRODUCTIONNanoscale materials play a vital role during the course ofevolution of life in the form of nanosized biomoleculessuch as nucleic acids, lipids, carbohydrates and peptides.In the 20th century, nanotechnology and nanoscience hasemerged as a fascinating area of research where manynanosized structures have proven their role in the specificfield particularly in their biomedical aspects.1 Physical andchemical properties of nanoparticles such as size, shape,composition and surface chemistry determine the suitabil-ity of these particles for such applications.2�3
A variety of nanoscale materials, such as metalbased nanoparticles,4–7 polymeric nanoparticles,8�9 mag-netic nanoparticles,10�11 fluorescent nanoparticles,12–14 andnanocomposites,15�16 has been extensively synthesized andstudied for their diagnosis and therapeutic roles. With
2950 J. Biomed. Nanotechnol. 2014, Vol. 10, No. 10 1550-7033/2014/10/2950/027 doi:10.1166/jbn.2014.1980
Bhushan et al. Ferritin Nanocages: A Novel Platform for Biomedical Applications
increase in knowledge in this field, effective techniquesare emerging against dreadful human diseases, particularlycancer in which conventional methods are not efficient.17�18
These nanostructures have comes out as blessing with dis-guise for human being as certain nanoparticles itself gen-erate toxicity and become a major concern for humanhealth.19 This provokes the researchers to search for morebiocompatible nanostructured materials for therapeutic anddiagnostic procedures.20�21
Bharat Bhushan received his B.Sc. degree in Industrial Chemistry and M.Sc. degreein Biotechnology from Aligarh Muslim University, India. Currently he is pursuing hisPh.D. degree from Centre for Nanotechnology, Indian Institute of Technology Roorkee,India. His research work focus on development of protein based nanocarriers for variousbiomedical applications.
S. Uday Kumar is pursuing his doctoral degree from Centre for Nanotechnology, IITRoorkee. At present he is involved in developing a multifunctional nanoscale carriersystem for lung cancer theranostics. Apart from this, his work also includes fabricationof cell specific-tissue engineering scaffold systems wherein the theranostic systems canbe evaluated. His research interest also includes tissue engineering and nanomedicine.
Ishita Matai received her M.Tech. in Nanotechnology from IIT Roorkee, India. Sheis currently a Ph.D. student in the Centre for Nanotechnology at IIT Roorkee, India.Her current research interests include developing multifunctional nanocomposites fortargeted delivery of anticancer agents.
Abhay Sachdev received his M.Tech. in Nanotechnology from IIT Roorkee, India.Presently he is pursuing his Ph.D. in the Centre for Nanotechnology at IIT Roorkee,India. His research work focus on development of biocompatible imaging agents forbioimaging applications.
In this regard, biologically derived protein cage nano-structures emerge as potential nanoplatform in thedevelopment of theranostic (therapeutic and diagnostic)nanocarrier for the simultaneous delivery of anticancer andimaging agents. Protein cages get self-assembled from lim-ited number of subunits to form a spherical nanocage hav-ing an interior cavity that is utilized for the storage ofvarious therapeutic materials while exterior surface canbe functionalized with tumor specific targeting moieties.
J. Biomed. Nanotechnol. 10, 2950–2976, 2014 2951
Ferritin Nanocages: A Novel Platform for Biomedical Applications Bhushan et al.
Poornima Dubey received her M.Sc. degree in Biotechnology from University ofMysore, India. Currently she is a Ph.D. student in the Centre for Nanotechnology atIIT Roorkee, India. She is investigating the molecular mechanism of toxicity of variousnanoparticles and nanocomposites. Her research interests include nanotoxicology andcancer biology.
P. Gopinath is an Assistant Professor in the Department of Biotechnology at IndianInstitute of Technology (IIT) Roorkee, India. He received his B.Sc. degree in Micro-biology and M.Sc. degree in Biotechnology from Bharathidasan University, India. Heearned his Ph.D. in Biotechnology at Indian Institute of Technology Guwahati, India.He did his postdoctoral research at University of Rochester Medical Center, New York,USA. Currently his research group in nanobiotechnology laboratory is working on thedevelopment of various protein and polymer based nanocarriers for the delivery of var-ious anticancer agents including anticancer drugs, siRNA, genes etc. This group is alsoexploring the possibilities of various biocompatible imaging agents for cancer diagno-sis. In order to realize the efficacy of such therapeutic and imaging agents, they arevalidating these systems in an artificial scaffold which mimics the in vivo condition to
the closest extent.
These nanoparticles overcome various limitations of con-ventional therapy such as non-specific distribution and tar-geting of drug, poor solubility of drug, poor bioavailabilityand therapeutic efficacy of drug.The most commonly used protein nanocarrier includes
ferritin, heat shock protein (Hsp), and viral nanoparticlessuch as cowpea chlorotic mottle virus (CCMV) and cow-pea mosaic virus (CPMV), as discussed in the Table I.These protein nanocarriers have more advantages overother micrometer and sub-micrometer size delivery sys-tems, such as liposome because the protein nanocarriershave high surface area to volume ratio which increasestheir drug holding capacity, enhance the solubility of drug,increases their bioavailability by controlled release of drug,biocompatible and do not produce any toxic effect due toits biological origin.41
Among the protein nanocarriers, viral nanoparticles arethe most extensively studied protein cages. Several draw-backs have been coupled with the use of viral deliveryvectors, which includes evoking immune response, prob-ability of integrating with the host chromosome to pro-duce a replication-competent infectious virus, rapid renalclearance from the body, difficulties in the modificationof viral capsids for tumor specific targeted delivery andhigh cost of production.42 Thus more attention is givento non-viral protein cages as they offer advantages, suchas less immunogenicity, larger drug/DNA holding capac-ity, not removed by the complement system, repeatedlyadministered without generating adverse effects, cheap,easily modifiable for targeted delivery and have negligible
safety issues due to the non-viral nature of the deliverysystem. However, one disadvantage coupled with thesenanocarriers is low transfection efficiency. So, most ofthe recent research has been focused on the developmentof novel non-viral nanoscale delivery system by utilizingthe biologically originated protein cages having geneticallycontrolled ordered structural symmetries and modifiablesurface chemistries. Thus, by inducing genetic alterationsvarious novel functionalities, such as multiple ligands, pep-tides and small chemical entities can be anchored to thesenanocaged structures to make them competent for cancertheranostics and other biomedical applications.In last few decades, the uses of biological nanopar-
ticles, as nanocarriers become an emerging approachfor the development of theranostic nanoparticles. Amongthese supramolecular assemblies of protein subunits, fer-ritins form a synthetic biomimetic platform for the size-constrained synthesis of nanomaterials. Thus this reviewsummarizes the role of ferritin nanocages in the nanopar-ticles synthesis and also highlights their potential biomed-ical applications.
FERRITIN AND ITS BIOLOGICAL ROLEIn 1937, ferritin was first isolated from horse spleen43
and later its crystal structure was elucidated in 1991.44
The ferritin superfamily has been divided into two maingroups depending on their size namely: maxi-ferritin andmini-ferritin as described in Table II. Ferritin performstwo major functions in the body. Firstly, they act as an
2952 J. Biomed. Nanotechnol. 10, 2950–2976, 2014
Bhushan et al. Ferritin Nanocages: A Novel Platform for Biomedical Applications
Table I. Various types of protein cages, their structure and applications.
Protein Interior Exteriorcages diameter diameter Structure Properties and function Applications Refs.
HSA 8 12 Spherical shell composed of24-subunits, giving them octahedral(4:3:2) symmetry.
Disassemble at low pHand reassemble athigh pH.
Easily modifiable structureused in cell targetingand MRI imaging.
[22]
Dps 4.5 9 Spherical shell composed of 12 subunitswith 23 point group symmetry, alongwith two type of 3 fold symmetrychannel having size 0.7–0.9 nm.
Protect cell from oxidativestress
Template for synthesis ofvariety of NPs.
[23, 24]
CCMV 24 28 Capsid is composed of 180 copies of 20kDa coat protein, which assemble intoa T = 3 capsid with three positivesense RNA molecules packagedinside making a 28 nm virus and 2 nmpores exist at the quasi 3-fold axis.
Capsids swell at pHgreater than 6.5.
Easily modified by geneticand chemicalmodification and used inMRI imaging, celltargeting and imaging.
[25–27]
Mj sHsp 6.5 12 Composed of 24 subunits, which forms acage with cubic (4:3:2) symmetry andwith eight 3 nm pores located at the 3fold axes. Six smaller (1.7 nm) poresalso exist at the 4-fold axis.
Extremely stable protein,function as molecularchaperones and overexpressed during stress.
Easily modified, used todeliver variety ofmolecules such as MRIcontrasting agents etc.
[28–30]
LS 8 15 Hollow icosahedral shell with negativelycharged protein cavity, composed of60 beta subunit and 3 alpha subunit.
Enzyme involved in thesynthesis of lumazine,a precursor of riboflavin
Biomimetic packing ofGFP and HIV protease.
[31–33]
TMV 4 18 Contain ssRNA surrounded by 300nm×18 nm hollow protein tube,composed of 2130 capsomer subunitshaving both positively and negativelycharged amino acids on both surfacesthat act as the nucleation centres.
Rod shaped and havingdistinct amino acidcomposition in interiorand exterior.
pH dependent synthesis ofNPs. Used in synthesisof nanotubes and othernanoelectronic devices.
[34, 35]
P22 54 60 The mature phage form composed of415 copies of 46.6 kDa coat proteinassemble into a spherical T = 7structure with as many as 300 of 33kDa scaffold protein.10 nm pores arepresent in the P22 capsid.
P22 naturally infectsSalmonella typhimurium
Easily modified byattaching functionalmoieties such as biotinto encapsulate variety ofparticles.
[36, 37]
MS2 23 27 Self assembled structure composed of180 subunit having 32 pores ofdiameter 1.8 nm.
Infect E.coli Easily modifiable, used todeliver variety ofmolecules, such asimaging agent for PETand MRI.
[38–40]
Notes: HSA-Horse spleen apoferritin; Dps-DNA-binding protein from starved cells; CCMV-Cowpea chlorotic mottle virus; Mj sHsp-Heat shock protein fromMethanococcus jannaschii; LS-Lumazine synthase from Bacillus subtilis; TMV-Tobacco mosaic virus; P22-P22-Bacteriophage; MS2–MS2 Bacteriophage.
iron storage component and thereby maintain the availabil-ity of iron during biological synthesis of various proteins,which comprise iron as co-factor (such as heme protein,iron sulfur protein (Fe–porphyrin, Fe–S, and Fe)). Theseiron-containing proteins constitute a crucial component invarious biological processes, such as respiration, photosyn-thesis and play an important role in hydroxylation reac-tions and oxygen sensing.57�58 Secondly, ferritins play avital role in the iron metabolism and protect the cells fromoxidative damage.22�59
STRUCTURE OF FERRITINThe primary amino acid sequences of the ferritins doesnot have any homological similarities however a clearstructural homology were found at the 2�, 3�, and 4� levels,
indicating that the structure of ferritins remain conservedduring the evolution. The structure of ferritin is shown inFigure 1 having 24 identical subunits with octahedral sym-metry. These subunits possess a four-helix bundle alongwith a fifth E helix which is found at 60� to the four-helix bundle axis.44�61�62 Ferritin is a spherical hollow pro-tein cage with internal and external diameter of about120 Å and 75 Å, respectively.63 It can accumulate andstore approximately 4500 iron atoms.The apoferritin protein cage is composed of 80–90%
of L-chain (light chain) and 10–20% of H-chain (heavychain) subunits with 55% sequence homology. The differ-ence between these two subunits lies in their outer surface,cavity, and hydrophobic channel sequences whereas thehydrophilic channel sequence found to be identical.64�65
The negatively charged L chain subunit found inside the
J. Biomed. Nanotechnol. 10, 2950–2976, 2014 2953
Ferritin Nanocages: A Novel Platform for Biomedical Applications Bhushan et al.
Table II. Difference between maxi-ferritin and mini-ferritin.
Characteristics Maxi-ferritin Mini-ferritin References
Size 8–12 nm 4.5–9 nm [23]
Structure • 24 subunits (∼ 20 kDa), four-helix bundle fold,with octahedral symmetry (432 point groupsymmetry) forming a larger spherical cavity thataccumulate 4500 Fe atoms.
• Monomer is made up of a four-helix bundle(A, B, C and D helices) with a short fifth helix(E helix) at the C-terminus.
• Each subunit interacts with six adjacentmonomers through three types ofsymmetry-related interfaces.
• There are twelve dimerization interactioninterfaces at the two-fold axes, eighttrimerization interaction interfaces at thethree-fold axes and six tetramerizationinterfaces at the four-fold axes.
• 12 subunits, four-helix bundle fold with 32(tetrahedral) point group symmetry forming asmaller cavity that accumulate 500 Fe atoms and isa structural analogue of the maxi-ferritins.
• Monomer folds into a four-helix bundle (A, B, C andD helices), with no E helix
• Each subunit interacts with five surroundingmonomers through two types of symmetry-relatedprotein-protein interfaces.
• Six dimer interactions are at two-fold symmetryaxes, and four trimerization interactions arecentered at the three-fold axes. Two types ofnonequivalent three-fold interfaces exist in themini-ferritin tetrahedral dodecamer.
[23, 44–46]
Occurrence Bacteria, archaea, and eukaryotes Bacteria and archaea
Examples Human ferritin, HSA, bacterioferritins Dps
Function Store excess iron and protect from oxidativestress by removing iron and oxygen,predominantly dioxygen.
Protecting bacteria from oxidative damage byremoving iron and oxygen, predominantly hydrogenperoxide.
[47]
Ferrioxidasesite/active site
• Located in the middle of the monomericfour-helix bundle.
• 24 active sites are saturated with 48 Fe(II).
• Situated at the interface between two-foldaxis-related monomers.
• 12 active sites are saturated with 12 Fe(II) atoms.Except in proteins that can use dioxygen as thesubstrate, where 24 Fe(II) bind/cage.
[23, 48, 49]
Stability Highly resistant to chemical denaturation, pHchanges and heat. Stable in dimer form insolution and assembly proceed from dimers totetramers and octamers.
The protein was found to be extremely pH stable,Dps dissociated reversibly into dimers at conditionsabove pH 7.5 and below 6.0. Furthermore, dimersdissociate to monomers at pH 4.0.
[50–53]
Self assembly 6 amino acids at the end of the C-terminal tip ofthe D helix are essential for self-assembly.
26 residues of the C-terminus are essential forself-assembly.
[54]
Iron entry The channel carboxylates in 24 subunit ferritinsselectively control Fe2+ entry.
The channel carboxylate groups control both Fe2+entry and Fe2+ exit.
[55, 56]
inner cavity of assembled protein cage has clusters ofacidic residues (Glu and Asp) which form the mineralnucleation site. This site mainly performs the function ofdelivery of iron and help in the nucleation of ferrihydritecore.61�66 So, these chains were found in the extra cellularferritin as they act as an iron carrier for different cells.67
The heavy chain which catalyzed the oxidation of Fe+2 is
Figure 1. (a)–(c) Ribbon diagrams of L-ferritin taken fromPDB entry 1DAT: (a) the 24-subunit assembled cage; (b) theinner cavity; (c) the 3-fold axis channel. Reprinted with per-mission from [60], S. Abe, et al., Polymerization of pheny-lacetylene by rhodium complexes within a discrete space ofapo-ferritin. J. Am. Chem. Soc. 131, 6958 (2009). © 2009,American Chemical Society.
responsible for the iron mineralization and the formationof iron crystal.65 The nucleation site of H chain subunitfound in close proximity with the ferroxidase site shar-ing one glutamate residue between them.61 Recent studieson different ferritin further strengthened that iron storingcapability of ferritin is related to the number of L sub-units. Moreover, presence of small number of H subunitin ferritin obtained from iron storage organ reveal theimportance of oxidative process in iron storage.68 The fer-roxidase activity of the apoferritin gets affected in thepresence of metal nanoparticles as it has been found toget increased in the presence of platinum, gold and silvernanoparticles.69�70
There are 14 channels having a diameter of 0.3–0.4 nmeach, which are present at the junction of these sub-units. Out of these 14 channels, eight channels arehydrophilic in nature and posses four-fold symmetry, whilethe remaining six are hydrophobic and possess three-foldsymmetry.71 The aperture size of these hydrophilic chan-nels are adjusted according to the particles as demonstratedthat in presence of urea, these eight hydrophilic channels
2954 J. Biomed. Nanotechnol. 10, 2950–2976, 2014
Bhushan et al. Ferritin Nanocages: A Novel Platform for Biomedical Applications
attain sufficient flexibility and allow larger size moleculesto penetrate inside the apoferritin cavity.72
The molecular species enter into the protein cavitythrough these channels by charge selective process. More-over, flow of ion through the pore is regulated by thelocal folding and unfolding of the ion channel pore.The four highly conserved residues, such as arginine 72,aspartate 122, leucine110 and leucine 134 are respon-sible for the stability of pore and form the pore gate.These pores are less stable compared to the overall sta-bility of ferritin nanocages, even at low temperature andlow concentration of denaturants, such as urea and guani-dine, pores show instability. It has been suggested thatbiological regulators are present in vivo, which recog-nize the pore gates and hold it in either open or closeconformation to maintain the iron homeostasis.73 Ferritincage without the ferrihydrite mineral core is called asapoferritin.
ROUTES OF LOADINGThere are two major ways of loading materials inside apo-ferritin as shown in Scheme 1: First, by directly incubatingthe materials with the apoferritin in which the smaller par-ticles comparable to the size of channels move directly andget accumulated inside the inner cavity. This process mim-ics the natural biomineralization process. Second way isapplicable for the larger particles which cannot efficientlypass through the channels. In this route, the apoferritinprotein cage undergoes pH dependent assembly at higherpH and disassembly at lower pH.
BIOMINERALIZATIONFerritin protein cages have been used as nanosized con-tainers for the controlled synthesis of a variety of nanopar-ticles by biomimetic process. So, in order to synthesizenanoparticles inside its cavity it becomes important tounderstand, how the process of biomineralization of ironnaturally occurs in ferritin (Scheme 2).Iron biomineralization in ferritin is a multistep process
that includes:1. Entry and binding of iron ions inside the ferritin cagecavity.
Scheme 1. Schematic representation of different routes ofloading in ferritin nanocage.
Scheme 2. Schematic representation of naturally occurringbiomineralization process inside ferritin nanocage.
2. Oxidation of iron ions followed by nucleation andgrowth of ferrihydrite core.3. Release of iron ions from ferritin.
Step 1. Entry and binding of iron ions inside the ferritincage cavity. The iron enters into the ferritin through the15 Å long channels which are gated by extensions of thefour-helix bundle subunits. These metal ions were guidedinside the cavity by charged gradient of the channel cre-ated by the presence of conserved carboxylate residues:Glu130, Asp127, Ala26, Val42, Thr149.47�56 Moreoverthere are two basic types of functional channels present inferritin:
(1) Iron ion entry channels formed by three subunitsaround the 3-fold cage axes which allows the passage ofFe2+ substrate to oxidoreductase sites (Asp127, Glu130).(2) Iron ion nucleation channels, which are present at
the other side of the 4-helix bundles subunit around the4-fold cage axes (Ala26, Val42, Thr 149).
The Fe(II) ions reacts with O2 after binding to the activesite and produce diferric oxo products in eukaryotes. Thediferric peroxo intermediate (DFP) is first detectable inter-mediate which forms and decays in seconds or less intothe di-Fe(III)O product, a mineral precursor which is laterreleased into protein nucleation channels.48
Step 2. Oxidation of iron ions followed by nucleation andgrowth of ferrihydrite core. Two Fe2+ ions get oxidizedto Fe3+ in the presence of oxygen after binding to theferroxidase center. The Fe3+ ions then migrate to the fer-ritin inner cavity and finally a mineral core formation takeplaces at the nucleation sites of the L-chain ferritin. Thisis the initial process when no iron is present in ferritin,but as soon as the iron mineral core is formed, the irongets oxidized directly on the mineral core surface after
J. Biomed. Nanotechnol. 10, 2950–2976, 2014 2955
Ferritin Nanocages: A Novel Platform for Biomedical Applications Bhushan et al.
passing through the 3-fold channels.74 This oxidation pro-cess on mineral core is found to be more rapid than thatat the ferroxidase center, which remains functional aftera core is formed and with no significant contribution inFe2+ oxidation.75
It has been shown by various in vitro studies using vari-ous mutant ferritin cage, which lack nucleation site or fer-roxidase activity or both, directly affect the encapsulationor mineralization process or both.76 This indicates the roleof nucleation site in aggregating the ions at the highly nega-tively charged protein interface and in facilitating oxidativemineralization and ferroxidase centre in converting the sol-uble Fe2+ to insoluble Fe3+, absence of which leads to theuncontrolled growth and precipitation.76�77 Thus suggeststhat the ferritin biomineralization is highly specific for iron.Step 3. Release of iron from ferritin. In vitro removal ofiron from ferritin is a two-step process, which includesreduction of Fe3+ mineral followed by the chelation ofFe2+ from the mineral core. Four iron release reductionand chelation model namely subunit displacement, diffu-sion of molecules through the 3-fold channels, gated poresand electron transfer through the protein shell, has beenbriefly discussed in a review by Watt et al.78 Consideringall the possible ways of iron release mechanism and theirtransportation through the protein cage, the 3-fold chan-nels are currently accepted route for the passage of iron toenter and exit the protein cage.78 Moreover, the redox reac-tions occur during the iron mineralization and release areaccompanied by the simultaneous release of ion in order tobalance the charge on both sides of the cage. For example,the entries of electron during the reduction of iron in themineral core are accompanied by concomitant release ofnegative charge from the core. Some of the important ionsinvolved during this process are chloride and hydroxideions, moving throughout the protein cage and phosphateion release during the reduction process.78
Iron releasing occurs on exposure of ferritin to UV lightor ionizing radiation, the iron mineral core acts as photore-ceptor and result in the reduction of Fe (III). In the absenceof oxygen, redox reaction results in the iron mobilizationfrom ferritin catalyzed by the hydrated electron, which actsas a reducing agent. In the presence of oxygen superox-ide radical anion (O•−2 ) is responsible for the iron releaseprocess. This suggests the requirement of an iron chela-tor for Fe(II) mobilization from ferritin, in the absence ofwhich ferritin act as a electron-storage molecule.79 More-over, the reversal of process of biomineralization is veryslow, as shown in the in vitro study by removing the excessiron in sickle cell disease and thalassemia with the help ofchelators.80�81
FERRITIN AS A TEMPLATE FORNANOPARTICLES (NPs) SYNTHESISMetal nanoparticles can be fabricated inside the apoferritincavity, which act as a reaction vessel. Protein cage like
structure of apoferritin can be used for the size dependentencapsulation of various materials by serving as templateto restrain the NPs growth and prevent aggregation. Theseself assembled protein shell form a reaction chamber forthe synthesis of non-native materials of controlled dimen-sions, while exterior surface can be easily modified withvarious functional moieties through genetic and chemicalmodification. A variety of different precursor ions havebeen formed by nucleation and subsequent mineral growthsuggesting that other non-native metals could also be min-eralized within the ferritin core. Due to the sharper densityof these NPs as compared to higher-dimensional struc-tures, these NPs offer superior quality that can be used inbiosensors, nanoelectronic devices, bioimaging and vari-ous other biomedical applications.Artificial synthesis of ferromagnetic iron oxide nanopar-
ticles inside the apoferritin cavity has been reported byMann and co workers.82�83 They mimicked the naturalbiomineralization process and opened the way to utilizeapoferritin for the synthesis of various inorganic nanoparti-cles. Similarly, mini-ferritin (Dps) used for the synthesis ofNPs includes Co(O)OH and Co3O4 (dia 4�34±0�55 nm),84�-Fe2O3,
85�86 CdS87 and Pt.88
After loading, these NPs were undergoing various inter-mediate stages before leading to the final mineralizednanoparticles. Metal ions were reduced inside the cavity byusing a reducing agent for example H2, NaBH4, or light,then the encapsulated NPs were separated from the unen-capsulated ones by the implications of additional purifi-cation steps.89 Moreover the ferrihydrite core undergoesin situ reactions and gets modified to other iron productssuch as FeO,90 iron sulfide91 and semiconducting hematite(�-Fe2O3).
92 Similarly, high temperature synthesis wascarried out using ferritin from thermophilic archaeon Pyro-cocus furious, which retains its cage-like structure evenat 120 �C.93 This can be further used as a template forsynthesis of magnetite94 and other metallic NPs such asgold, silver, lead, copper, nickel and semiconductors NPssuch as CdS. The outer surface of ferritin modified withPEG prevents the bulk precipitation and improves the yieldof NPs in ferritin cavity.95 The noble metals ions (Au3+,Ag+) bind to the exterior surface of the protein. In orderto facilitate internalization of these metal ions, reactivecysteine and histidine residues are removed from the exte-rior surface of ferritin and soft cysteine ligands are intro-duced in the interior surface.96 The metals ions bind tothe specific binding site present on the protein shell, bothinterior and exterior surfaces of the protein cage that pro-mote the growth of NPs both inside and outside the cage.Moreover, modification on the surface of ferritin leads tochange in their properties. For instance, the alkylation ofthe ferritin protein using a monoamine-terminated alkaneoligomer (dodecylamine) changes the charge of the proteinand type of interactions by converting the primary car-boxylic acid groups on the ferritin surface into hydropho-bic groups.97 Recently, reported recombinant apoferritin
2956 J. Biomed. Nanotechnol. 10, 2950–2976, 2014
Bhushan et al. Ferritin Nanocages: A Novel Platform for Biomedical Applications
Table
III.
Listofva
riousmaterials
synthes
ized
usingferritin
cages
andtheirap
plic
ations.
Type
ofType
sof
Load
ing
Prope
rtiesof
ferritin
material
Sou
rceof
material
effic
ienc
ypa
rticles
App
lications
Referen
ces
HSA,RA,CA,
PfFt
Pdan
dits
orga
nometallic
complexes
K2PdC
l 4,[PdII(allyl)C
l]296
–500
Catalytic
and
non-mag
netic
.•Use
din
high
lysp
ecificae
robicox
idationof
alco
hols
inwater
•Use
din
size
-selec
tiveolefi
nhy
drog
enation.
[99–
102]
CF,
CA,HSA
Cuan
dits
radioa
ctive
isotop
e
64CuC
l 2,CuS
O4·5H
2O
225–
2000
Catalytic.
•Tu
mor
spec
ifictargeting.
•Can
beus
edin
PETim
agingan
dna
noelec
tron
icde
vice
s.•Ferritin
actin
gas
photoc
atalys
tin
pres
ence
ofvisiblelig
htredu
cing
copp
er.
•Electrontran
sportatio
nstud
ies.
[103
–106
]
HSA
CuS
NS
NS
Sem
icon
ductor
•Can
beutilize
das
aco
mpo
nentsforna
no-electric
device
s,su
chas
solarba
tterie
san
dliq
uidcrys
tals
[107
]
HSA
CuF
e[Fe(CN) 6]3
−22
5Mag
netic
•Bas
icload
ingstud
ies.
[108
]
HSA,CA,
reco
mbina
ntFtLi,AvB
F
Coan
dits
oxides
/ox
yhyd
roxide
Co
2+,CoS
O4,
Co(NO
3) 2·6H
2O,
Co(OAc)
2·4H
2O
200–
2000
Mag
netic
andca
talytic
•Pos
siblyutiliza
blein
nano
elec
tron
ics.
•Electroca
talyst
inelec
troc
hemical
reac
tionforthe
detectionof
gluc
ose.
•Nov
elbios
enso
rca
nbe
used
inmed
ical
and
indu
stria
lfields
tode
tect
diffe
rent
analytes
•Electrontran
sportatio
nstud
ies.
[84,
105,
106,
109–
115]
RA,CA
CoP
t(N
H4) 2PtC
l 4,
(CH
3COO) 2Co·4H
2O
1000
Mag
netic
•Can
beutilize
din
theprep
arationof
high
-den
sity
reco
rdingmed
ia.
[116
,11
7]
HSA
CoN
iCoS
O4,NiSO
4NS
Mag
netic
prop
ertie
s•Can
beus
edin
MRI
[118
]
RA,HSA
Nia
ndits
hydrox
ide
Ni2+
8000
Mag
netic
.•Pos
siblyus
able
inna
noelec
tron
ics.
•Prepa
ratio
nof
zero
valent
NPs.
[105
,10
9,11
9]
Rec
ombina
ntPfFt,HuH
Ft,
FtLi,RA,
HSA,HuH
Ft,
CA,AfFtn
Iron
andits
compo
unds
(oxide
,su
lphide
and
radioa
ctiveisotop
e)
FeC
l 2,FeS
O4,
(NH
4)Fe(SO
4� 2
100–
7200
Mag
netic,ca
talytic,an
dpo
tentialM
RI
contrast
agen
t
•Use
din
tumor
spec
ifictargetingan
dim
aging
•Fa
cilitatece
llularup
take
•Act
asph
otoca
talyst
inpres
ence
ofUV/Visible.
•Use
din
prod
uctio
nof
SWCNTan
dco
ntrolle
dits
size
•Use
din
fluores
cent
andMR
imag
ing(in
vivo
/invitro).
•Use
din
ironab
sorptio
nan
delec
tron
tran
sfer
stud
ies.
•Can
beutilize
din
nano
device
.
[82,
83,85
,91
,10
4,10
6,11
4,12
0–13
8],
CA
FeC
o(N
H4) 2Fe(SO
4)·6
H2O,
(Co(NO
3) 2·6H
2O
1000
Mag
netic
.•Mag
netis
mfoun
dwith
ferritin.
[139
]
HSA
Iron
arse
nate,
phos
phate,
vana
date,molyb
date
particles.
NS
2000
Catalytic.
•Iron
load
ingstud
ies
[140
]
PfFt
FeP
t(N
H4) 2Fe(SO
4) 2,
K2PtC
l 4
500
Mag
netic
•Bas
icbiom
imetic
synthe
sis.
[141
]
J. Biomed. Nanotechnol. 10, 2950–2976, 2014 2957
Ferritin Nanocages: A Novel Platform for Biomedical Applications Bhushan et al.
Table
III.
Continued
.
Type
ofType
sof
Load
ing
Prope
rtiesof
ferritin
material
Sou
rceof
material
effic
ienc
ypa
rticles
App
lications
Referen
ces
RA
Inox
ide
In2(S
O4� 3
NS
Sem
icon
ductor
•Pos
siblyutiliza
blein
nano
elec
tron
ics
[136
]
HSA
Des
ferrioxa
mineB
Des
ferrioxa
mineB
3Catalytic
•Poten
tialtobe
utilize
din
ironch
elationtherap
y.[142
]
NS
AuP
tK
2PtC
l 4,HAuC
l 4NS
Antioxida
nt•To
stud
ytherece
ptor-m
ediatedce
llularup
take
ofNPs.
[143
]
RA,HSA,
HuH
Ft,
Auan
dits
compo
unds
AuC
l 3,HAuC
l 4,KAuC
l 425
0–40
00/Au,
clus
ter
16–5
0,/
Au 2S-300
0
Catalytic,
photolum
ines
cenc
e,se
micon
ducting
particles
•In
vivo
kidn
eytargetingan
dbiom
edical
Imag
ing
•Produ
ctionof
SWCNTan
dgo
ldna
nosh
ell.
•Enh
ance
catalytic
activ
ity.
•Pos
siblyutiliza
blein
nano
elec
tron
ics
[69,
70,96
,98
,14
4–15
0],
RA
AuP
dKAuC
l 4,K
2PdC
l 428
9–44
1Catalytic.
•Enh
ance
catalytic
activ
itydu
ringolefi
nhy
drog
enation
[151
]
HSA
Au–
AgNPsalloy
AgN
O3,HAuC
l 4NS
Catalytic
•Catalyzed
theredu
ctionof
4-nitrop
heno
linthe
pres
ence
ofNaB
H4.
[152
]
Apo
PPF
Au-5F
UNH
4AuC
l 4,5F
U1/45
Antican
cerdrug
•Can
beus
edin
chem
othe
rapy
ofca
ncerou
sce
lls[153
]
HSA
Pho
spha
tepa
rticles
from
Cd,
Zn,
Pb,
Cu
Cdc
l 2,Zn(NO
3) 2,
Pb(NO
3) 2,Cu(NO
3) 2,
NS
Red
oxmarke
rs•Partic
lesus
edas
labe
lsin
elec
troc
hemical
immun
oass
ayan
dus
edforde
tectionof
individu
alsing
lenu
cleo
tidepo
lymorph
isms(S
NPs)
[154
–156
]
CA,HuH
Ft
HuL
Ft,PfFt,
RA,HSA
Ag
AgN
O3
250–
5000
Antim
icrobial
activ
ityan
dca
talytic
activ
ity.
•Antibac
teria
laga
inst
S.aureu
s.•Increa
sein
ferrox
idas
eac
tivity
offerritin.
[69,
70,96
,14
8,15
7–15
9],
NS
pHindica
tormolec
ules
NS
NS
pHindica
tor
•Use
dforstud
ying
ferritin.
[160
]
CA,HSA
Gdan
dits
complexes
Gd-Me 2DO2A
/Gd-
DOTA
,GdH
PDO3A
,Gd(NO
3) 3·6H
2O,
LnCl 3.
Com
plexe8
-36
/oxide
-17
00
Mag
netic
particlesan
dMRIco
ntrast
agen
t•Use
din
MRIex
perim
ents
(invitroan
din
vivo
).•Visua
lizationof
tumor
angiog
enes
isby
mag
netic
reso
nanc
e
[161
–166
]
HSA
Curcu
min
C21H
20O
69�5±2
The
rape
utic
agen
t•Use
dforev
alua
tingdrug
deliveryeffic
ienc
yin
mice
[161
]
CA,HSA
Pban
dits
compo
unds
(pho
spha
te,su
lhide)
Pb(NO
3) 2,Pb(AcO
) 213
00Signa
lamplifica
tion
andqu
antum
dot
•Electroch
emical
immun
oass
ayforqu
antifi
catio
nof
phos
phorylated
acetylch
olines
terase
.•Can
beus
edas
bioc
ompa
tible
agen
tin
bioimag
ing.
•Act
asan
tican
cerag
entan
dindu
ceap
optosis.
[167
–171
]
HSA
YPO
4Y
(NO
3) 3
500
Rad
ionu
clideNPs
•Poten
tialtobe
utilize
din
radioimmun
othe
rapy
ofca
ncer
[172
]
FtLi,HSA,RA
Cdan
dits
compo
unds
(sulph
ide,
selenide
,ph
osph
ate)
CdC
l 2,Cd(CH
3CO
2) 2
55–1
350
Sem
icon
ductor
quan
tum
dots
and
marke
rmolec
ule
•Pos
siblyutiliza
blein
nano
elec
tron
ics.
•Use
das
fluores
cent
biom
arke
rin
bioa
ssay
s.•Use
din
sequ
ence
-spe
cific
DNA
detection.
[24,
87,
173–
177]
HSA
Methy
lene
blue
.C
16H
18N
3SCl
NS
Pho
tose
nsitize
r•Cou
ldbe
useful
inph
otod
ynam
ictherap
yof
canc
er.
[178
,17
9]
HSA
Fluores
cein
C20H
10Na 2O
5NS
Fluores
cent
particles.
•Partic
lesus
edas
labe
lsin
bioa
ssay.
[180
]
HSA
Hex
acya
noferrate
K3Fe(CN) 6
65–1
50Marke
rmolec
ule.
•Partic
lesus
edas
labe
lsin
elec
troc
hemical
immun
oass
ay.
[180
,18
1]
2958 J. Biomed. Nanotechnol. 10, 2950–2976, 2014
Bhushan et al. Ferritin Nanocages: A Novel Platform for Biomedical Applications
Table
III.
Continued
.
Type
ofType
sof
Load
ing
Prope
rtiesof
ferritin
material
Sou
rceof
material
effic
ienc
ypa
rticles
App
lications
Referen
ces
Apo
PPF,
CA,
HSA
Cisplatin,ca
rbop
latin
andox
aliplatin
PtC
l 2(N
H3) 2,
500
Antican
cerdrug
.•Deliver
drug
andindu
ceap
optosis
•Sho
wsincrea
sedce
llularup
take
[182
–184
]
HAS,RA
Znan
dits
compo
unds
(selen
ide,
oxide)
(Zn(NO
3) 2·6H
2O)
1500
Sem
icon
ductor
•Cou
ldbe
used
inna
noelec
tron
ics
[185
,18
6]
HSA,RA,AfFtn
Mnan
dits
oxide,
oxyh
ydroxide
MnC
l 212
00–4
000
Mag
netic
•MRIse
nsor
formelan
inform
ationin
meloa
nomace
ll•Use
das
MRIco
ntrast
agen
t.[106
,11
3,11
5,13
3,18
7–18
9]
HSA,CA
Pt
K2PtC
l 4,K
2PtC
l 6,
(NH
4) 2PtC
l 4
250–
4000
Catalytic
activ
ity.
•Increa
seferrox
idas
eac
tivity
offerritin.
•Use
das
artifi
cial
antio
xida
nt.
•Morebioc
ompa
tible
andinternalizevia
rece
ptor-m
ediateden
docy
tosis
[70,
190–
192]
RA
Cerium
oxide(C
eO2,
Ce 2O
3)
CeC
l 3·7H
2O
400
Catalys
t•Activeartifi
cial
redo
xen
zymewith
mim
etic
SOD
activ
ity[193
,19
4]
CA
Eva
nblue
ordirect
yellow
dye
NS
NS
Dye
•Use
dto
prep
arefree
stan
ding
mes
oporou
sprotein
thin
filmsan
dstud
yof
theirpH
depe
nden
treleas
e[195
]
CA
Dau
nomyc
inC
27H
29NO
10·H
Cl
NS
Antican
cerdrug
•Binding
stud
ieswith
DNA
[196
]
CA,RA
Dox
orub
icin
C27H
29NO
11·H
Cl
5–50
Antican
cerdrug
•Prelim
inarycy
totoxicity
hasbe
ende
mon
strated
[195
–199
]
CA
Gluco
seox
idas
e(G
Ox)
GOx
7.8
Catalytic
activ
ity•Catalyzed
oxidationof
D-gluco
seto
D-gluco
no-1,5-la
cton
ean
dhy
drog
enpe
roxide
.[200
]
HSA
PEI
NS
NS
Coreac
tant
•Biose
nsingan
dbioa
ssay
applications
[201
]
RA,CA
Euox
idean
dits
complexes
EuC
l 314
–100
0La
belm
oiety
•Bioaffin
ityas
sayan
dbio-im
aging
[134
,20
2,20
3]
CA
Tio
xide
Ti(IV)
1000
NS
•Bas
icload
ingstud
ies.
[134
]
RA
RhCom
plexes
[Rh(nb
d)Cl] 2
58Catalytic
•Catalyzethepo
lymerizationof
phen
ylac
etylen
e.[60]
RA
Ruco
mplexes
[Ru(pcy
men
e)Cl 2] 2
88±1
1Catalys
ts•Bas
icload
ingstud
ies.
[204
]
HSA
LuPO
4Lu
Cl 3
NS
NS
•Cou
ldbe
used
inca
ncer
therap
y.[205
]
RA
Caan
dits
compo
und
(carbo
nate)
Ca(HCO
3) 2,CaC
O3
NS
Che
mically
stab
le•New
mec
hanism
ofmineralizationus
ingco
ntrolle
delec
tros
tatic
potential
[206
]
HSA
CaC
O3,SrC
O3,BaC
O3
andCa 3(P
O4) 2
CaC
l 2,SrC
l 2or
BaC
l 214
60–1
600
NS
•Bas
icload
ingstud
ies.
[207
]
RA,HSA
Crhy
drox
ide
Cr3
+48
00NS
•Pos
siblyus
able
inna
noelec
tron
ics.
[119
]
CA,HSA
Uan
dits
oxide/ox
yhyd
roxide
UO
2+80
0–40
00Rad
ioac
tivepa
rticles
•Can
beus
edin
uran
ium
neutron-ca
pturetherap
y[133
,20
8]
HSA
Pruss
ianblue
K4Fe(CN) 6
NS
Catalytic
•Enz
ymemim
etic
activ
ityca
nbe
utilize
dfor
biolog
ical
detection.
[209
]
Notes
:NS—Not
spec
ify;HSA—Horse
spleen
ferritin;
Apo
PPF-Apo
-pig
panc
reas
ferritin;
CA—Com
mercial
apoferritin;RA—Rec
ombina
ntap
oferritin;CF—Chimeric
ferritin
(mixture
of2
heav
ych
ain
ferritins
);HuH
Ftan
dHuL
Ft—
Hea
vyan
dlight
human
ferritinch
ains
;PfFt—
Ferritin
from
hype
rthe
mop
hilic
bacterium
Pyroc
occu
sfurio
sus;
AfFtn—Mutan
tArcha
eoglob
usfulgidus
ferritin;
FtLi—
Ferritin
from
Listeria
inno
cua;
AaL
S—Lu
maz
inesyntha
sefrom
Aqu
ifexae
olicus
;AvB
F—Azo
toba
cter
Vinelan
diib
acterio
ferritin.
J. Biomed. Nanotechnol. 10, 2950–2976, 2014 2959
Ferritin Nanocages: A Novel Platform for Biomedical Applications Bhushan et al.
having gold-binding peptide and titanium-binding peptideat the C- and N-terminus, respectively that specificallycatch gold NPs and deliver them to the silicon dioxide sur-face under specific conditions.98 List of various moietiesencapsulated inside the ferritin protein cage is discussedin Table III.
APPLICATIONS OF FERRITINPROTEIN CAGESFerritin nanocages have been widely used in various bio-logical and biomedical applications as discussed below(Scheme 3).
Tumor TherapyApoferritin can encapsulate a variety of therapeutic agents,which can be utilized in different strategies for tumor treat-ment. Some of the strategies include:
Neutron Capture TherapyNeutron capture therapy is a promising methodology forthe treatment of cancer. Boron and uranium are the basicelements used in this technique. They are localized to thetargeted tumor cell and irradiated with slow neutron, whichleads to the disintegration of nuclei into smaller fragments
Scheme 3. Schematic representation of various applications of ferritin nanocages.
along with ionizing particles that kill the cell. In early90’s, Hainfeld first described a method to deliver 235U byencapsulating it in apoferritin cage which minimize theimmune response and heavy metal toxicity. Antibody Fab-fragments were chemically coupled to the protein cage fortumor specific targeting. The isotope was then fissioned byneutron beam that produced the required localized lethalradiation for the tumor therapy.208
Radioimmunoimaging and RadioimmunotherapyLutetium-177 is a radionuclide having a physical half-life of 6.7 days and other radiological properties suchas emission of low energy beta particles and gammaradiation have been utilized for targeting small tumorsfor radioimmunoimaging and radioimmunotherapy ofcancer.210 A radionuclide nanoparticles (NPs) have beensynthesized by conjugating apoferritin with lutetiumphosphate (LuPO4) or yttrium phosphate (YPO4) andfunctionalized them with biotin. The pretargeting capa-bilities of these nanoparticle conjugates were studiedusing biotin-modified LuPO4 or YPO4-apoferritin withstreptavidin-modified magnetic beads and in addition withthe aid of streptavidin-modified fluorescein isothiocyanate(FITC) tracer. This method can be further exploitedfor the preparation of radioactive LuPO4 or YPO4
2960 J. Biomed. Nanotechnol. 10, 2950–2976, 2014
Bhushan et al. Ferritin Nanocages: A Novel Platform for Biomedical Applications
conjugates that can be utilized in radioimmunotherapy ofcancer.172�205
Photodynamic TherapyPhotodynamic therapy (PDT) has emerged as an importanttool in the field of tumor therapy and has been utilizedfor the treatment of both oncological (e.g., tumors anddysplasias) and non-oncological (e.g., age-related maculardegeneration, localized infection and non-malignant skinconditions) applications.211 In this strategy, photosensitiz-ing agents, light, and oxygen take part in photochemi-cal reaction. A photosensitizing agent, methylene blue hasbeen successfully encapsulated inside apoferritin cage, thatcan be internalized by the tumor cells and on irradiationwith a light of suitable wavelength (i.e., 633 nm) generatesa cytotoxic agent, a singlet oxygen intracellularly for pho-todynamic therapy (PDT) which induced cytotoxic effectson the human breast adenocarcinoma cells (MCF-7).178�179
Recently, a RGD4C-modified ferritin encapsulated withZinc hexadecafluorophthalocyanine (ZnF16Pc), an effec-tive photosensitizer showed a high tumor accumulationrate, less toxicity toward major organs and effective tumorinhibition on light irradiation by inducing phototoxicity inU87MG subcutaneous tumor models.212
Anticancer Drug CarrierToxicity and drug resistance of platinum based anticancerdrug limited their use for cancer therapy. Apoferritin canbe exploited as a drug delivery system to these plat-inum based drugs (cisplatin, carboplatin and oxaliplatin)to overcome these drawbacks and to enhance the cellu-lar uptake of anticancer drugs.182 Cisplatin and carboplatinloaded apoferritin showed a primary toxicity against ratpheochromocytoma PC12 cells.183 Recently, a novel nano-sized construct of cisplatin core-apo pig pancreas ferritin(NCC-PPF) has been developed and its anticancer activityon gastric cancer cells BGC823 (GCC) were studied.184
Daunomycin (anticancer drug) used for the treatment ofacute myeloid leukemia and lymphocytic leukemia havebeen successfully encapsulated into apoferritin which ismodified by incorporating a negatively charged polypep-tide poly-L aspartic acid (PLAA) to improve their drugholding capacity.196 Similarly, apoferritin has been utilizedfor the encapsulation of anticancer drug doxorubicin.197�198
A simple and easy method for preparation of thin meso-porous protein films has been developed for efficient load-ing and releasing of dye or doxorubicin by controlling thepH. It loaded at lower pH and released the drug at higherpH than the isoelectric point of protein.195
The differential effect of near-infrared apoferritin-PbS(AFt-PbS) nanocomposites on cell cycle progression innormal and cancerous human cells has been recentlyreported. The nanocomposite did not alter the cell cycle innormal cell at concentration up to 1 mg mL−1 whereas inhuman breast cancer cell line it triggered apoptosis at con-centration > 0.2 mg mL−1. These nanocomposites entered
the cell through endocytosis and further could be used forthe in vivo imaging studies.170 Another anticancer com-pound Ru complex has also been successfully immobilizedto the ferritin cages by His residue present on the ferritinsurface.204
A bio-inspired nanoconstruct have recently been devel-oped using an apoferritin-gold nanoconstruct loadedwith anticancer drug 5-fluorouracil (5-FU) that exhib-ited a high selectivity towards cancerous cells and alsoincreased the cellular uptake of 5-FU via receptor-mediated endocytosis.153 Moreover, a genetically modifiedferritin (RFRTs) nanocages having a tumor targeting pep-tide Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys (RGD4C)attached on its surface has been used for the deliveryof anticancer drug doxorubicin as shown in Figure 2.Such nanoconstruct showed high drug loading efficiencyin presence of Cu(II) as a helper agent. This nanoconstructhave improved tumor suppression ability and reduced car-diotoxicity, when studied on U87MG subcutaneous tumormodels.199
Other Therapeutic ApplicationsIron is one of the essential elements for all the livingbeings, but if present in excess becomes toxic. Humanbody is incapable to remove this excess iron, which leadsto their accumulation in the liver and other organs lead-ing to serious health complications and eventually death.213
To remove this excess of iron, Desferrioxamine B (DFO)drug produced by Streptomyces pylosus is used for ironchelation therapy by encapsulating it inside the apoferritincage, which upon further reaction with Fe III gives rise toencapsulated [DFOFe] complex within the apoferritin.142
This nanocontainer can also be utilized for the treatmentof other infectious disease. As in a newly developed strat-egy in which silver (I) ions were loaded into apoferritinto function as an antimicrobial agent.157 Recently, a noveltheranostic agent has been constructed utilizing the apofer-ritin cage to simultaneously deliver the therapeutic agent(curcumin) and imaging agent (GdHPDO3A) to hepato-cyte in mice. This nanoconstruct can be used to preventhepatocellular damage in the thioacetamide-induced hep-atitis and can simultaneously evaluate the drug deliveryefficiency via Magnetic resonance imaging (MRI), as apo-ferritin cage is efficiently taken up by hepatocyte scav-enger receptor class A type 5 from blood via the ferritintransporting route.161
Tumor ImagingIn order to improve the quality and accuracy of dis-ease management, a fused technique has been developedby coupling the multiple imaging techniques as shownin Figure 3. Near-infrared fluorescence (NIRF) imagingand positron emission tomography (PET) are combinedin order to minimize the chances of misdiagnosis andused for in vivo imaging. A chimeric ferritin nanocagehas been developed by introducing RGD4C and Cy5.5 on
J. Biomed. Nanotechnol. 10, 2950–2976, 2014 2961
Ferritin Nanocages: A Novel Platform for Biomedical Applications Bhushan et al.
Figure 2. (a) Schematic illustration of D-RFRTs. Dox was precomplexed with Cu, and then encapsulated into RFRTs. (b) Gel-filtration chromatography analysis of RFRTs and D-RFRTs. The same peak at around 27.4 min was observed for both RFRTs andD-RFRTs. (c) Cumulative drug release curves of D-RFRTs in PBS (pH 7.4) and FBS. (d) Therapy studies performed on U87MGtumor-bearing nude mice (n = 5/group). On day 18, significant difference in tumor growth was found between D-RFRT treatedmice and those treated with PBS, RFRTs and free Dox (P < 0�05). Eighteen days after the onset of the treatment, a TGI rate of89.6% was observed for D-RFRTs, in comparison to that of 74.0% for free Dox. Reprinted with permission from [199], Z. Zhen,et al., RGD-modified apoferritin nanoparticles for efficient drug delivery to tumors. ACS Nano 7, 4830 (2013). © 2013, AmericanChemical Society.
the exterior surfaces of hybrid ferritin cage via geneticand chemical means. These nanocages loaded with 64Cuonto heavy chain of ferritins have a potential as mul-tifunctional loading and multimodality imaging probes.This hybrid nanoprobe has both PET and NIRF func-tionalities for tumor imaging in conjugation to integrinspecific targeting, when injected intravenously into tumor-bearing mice.103 Similarly, it has been demonstrated thatthe engineered human ferritin protein cages conjugatedwith either fluorescent Cy5.5 molecule or encapsulatingmagnetite nanoparticles, can serve as a nano-platform toimage vascular inflammation in vivo. They can be success-fully taken up by the macrophages in murine atheroscle-rotic carotid arteries and thus served as a novel platform asMR or Near-infrared (NIR) contrast agents for detecting
macrophage infiltration within atherosclerotic plaques todetect high-risk atherosclerotic plaques.130
In a recent study, a multifunctional ferritin cage-basednanostructure has been developed for the fluorescence andMR imaging and for detection of �v�3 integrin upregula-tion in tumor cells by attaching green fluorescent protein(GFP) and Arg–Gly–Asp (RGD) peptide on the exteriorsurface of the ferritin cages and ferrimagnetic iron oxidenanoparticles to the interior cavity.132 Paired gold clustershave been synthesized within the interior cavity of apofer-ritin cage with tunable fluorescent emissions, suggestingthe occurrence of fluorescence resonance energy transfer(FRET) effects between the clusters and use of these novelbiomolecule-metal complexes for in vivo kidney targetingand biomedical imaging.145
2962 J. Biomed. Nanotechnol. 10, 2950–2976, 2014
Bhushan et al. Ferritin Nanocages: A Novel Platform for Biomedical Applications
Figure 3. (a) Schematic illustration of the process of triple-loading. First, we introduced RGD4C and Cy5.5 onto the surfacesof two sets of ferritins, via genetic and chemical means. These two ferritins were then mixed and broken down into subunits atpH = 2 and incubated with 64CuCl2 to achieve radiolabeling. The pH was then adjusted back to 7.4 to facilitate the reformationof nanostructures. The reconstituted chimeric ferritin nanocages have both RGD4C and Cy5.5 on their surfaces and 64Cu loadedin their cavities. In vivo (b) PET and (c) NIRF images after the administration of ferritin probes. In the comparison group, ablocking dose of c (RGDyK) was injected 30 min prior to the ferritin probe administration. Reprinted with permission from [103],X. Lin, et al., Chimeric ferritin nanocages for multiple function loading and multimodal imaging. Nano Lett. 11, 814 (2011). © 2011,American Chemical Society.
In a similar way, gadolinium 1,4,7-tris(carboxymethyl)-10-(2′-hydroxypropyl)-1,4,7,10-tetraazacyclododecane (Gd-HP-DO3A) loaded apoferritin probe has been used for MRvisualization of tumor blood vessels (tumor angiogenesis)in a mouse model by utilizing biotin-streptavidin affinityand targeting neural cell adhesion molecules.165 Recently, aartificial luminescent protein has been developed by encap-sulating a strongly luminescent Eu3+ complex, N ,N ,N1,N1-[40-(1-naphthyl)-2,20:60,200-terpyridine-6,60 0-diyl]bis(methylenenitrilo) tetrakis(acetic acid) (NTTA–Eu3+�into cavity of apoferritin which act as a bioprobe fortime-gated luminescence bioimaging. This bioprobe canbe used to understand the distribution and function ofapoferritin inside complex living systems.203
Tumor TargetingProtein based NP systems are the promising tool for thetargeted delivery of imaging and therapeutic agents. Theadvantage of these NPs over other conventional systemslies in their ease to undergo cage modification and extendsto the wide possibility for loading a variety of moieties
for diagnostic and therapeutic purposes. These functionalmoieties include targeting agents that can effectively rec-ognize the receptor, over expressed by specific cells andtissues.Magnetic nanoparticles loaded apoferritin conjugated
with fluorescently labeled RGD-4C peptide can be takenup by macrophages more efficiently due to their spe-cific affinity with amelanotic melanoma cells and THP-1monocyte cells, which are known to overexpress integrin�v�3.
214 Similarly, a multifunctional NPs have been for-mulated for cell specific targeting by encapsulating ironoxide (magnetite) NPs within the interior cavity of genet-ically engineered human H-chain ferritin (HFn) and a flu-orescent dye, Fluorescein- 5-maleimide along with cellspecific targeting peptide, RGD-4C as shown in Figure 4.RGD-4C were attached on its exterior surface whichenabled specific binding to �v�3 integrins upregulated ontumor vasculature and C32 melanoma cells in vitro.122
Recently, multifunctional nanoparticles have been devel-oped by genetically and chemically modifying the heavychain of the human protein ferritin (HFt), stabilizing andmasking them with polyethylene glycol (PEG) molecules,
J. Biomed. Nanotechnol. 10, 2950–2976, 2014 2963
Ferritin Nanocages: A Novel Platform for Biomedical Applications Bhushan et al.
Figure 4. TEM images (left) and DLS analysis (right; insets are the corresponding correlation functions) of empty HFn andRGD4C-Fn. Both HFn and RGD4C-Fn show 12–14 nm in diameter. Reprinted with permission from [122], M. Uchida, et al., Tar-geting of cancer cells with ferrimagnetic ferritin cage nanoparticles. J. Am. Chem. Soc. 128, 16629 (2006). © 2006, AmericanChemical Society.
rhodamine fluorophores and magnetic resonance imag-ing contrasting agents for selective melanoma-targetingboth in vitro and in vivo. These constructs were specifi-cally targeted to the melanoma cell by attaching selectivetargeting moiety, such as �-melanocyte stimulating hor-mone (�-MSH) peptide on the surface of protein, whichbinds to the receptors expressed only by melanoma cellsand to some extend by melanocytes. In this study, therewas considerable reduction in non-specific recognitionand uptake by the reticuloendothelial and mononuclearphagocytic systems as HFt-MSH-PEG were easily recog-nized and taken by the melanoma cells and not by otherhuman cancer cells or mouse tissues (expect by dedicatedphagocytes).128
Cellular UptakeFerritin in natural conditions enters into cell though recep-tor mediated endocytosis due to the presence of endoge-nous ferritin receptors and for site specific targeting offerritin their exterior surface could be modified. The recep-tor for ferritin varies with the type of cell and tissue andon their developmental stages. The ferritin receptors werefound on different types of cells including lymphocytes,215
placental microvilli,216 and erythroid precursors.217 Theseare also found on various cell lines, such as giant HeLacells,218 K562 cells,219 and human intestinal carcinoma
Caco-2 cells, which can even internalize plant ferritin.220
In absence of transferrin receptors, ferritin L-chain recep-tors (scara 5) have been found on developing kidney forthe iron uptake.221
It has been previously reported that ferritin bind to themembrane of HeLa cells and is internalized through fer-ritin receptors via endocytosis.218 For example, in embryoTim2 is reported as the receptor for H-ferritin,222 whereasin many other cell lines such as HeLa cells and immuno-genic cells such as mitogen-activated T- and B-cells, cel-lular uptake is facilitated via human transferrin receptor-1(TfR1).223
Apoferritin nanocage can act as a natural and bio-compatible carrier for the cellular delivery of bioac-tive molecules through receptor-mediated endocytosis andprovide a non-destructive (to the cell membrane) andswitchable control of their cellular uptake by inhibitionof endocytosis which make them a highly flexible andpractical nanocarrier for drug delivery. The control of thedelivery system was tested on human intestinal epithelialCaco-2 cells, as they exhibit ferritin receptors.143
BioassaysIn the modern era of nanotechnology, use of nanoparticleshas emerged as an important tool in the field of biomedicalapplications because of their simplicity, high surface area
2964 J. Biomed. Nanotechnol. 10, 2950–2976, 2014
Bhushan et al. Ferritin Nanocages: A Novel Platform for Biomedical Applications
and unique physiochemical properties at the nanoscale.They have been widely utilized in development of highlysensitive bioassays for biomolecular diagnosis. Apoferritinin combination with other metal NPs have been extensivelystudied for molecular diagnosis, bioimaging, targeted drugdelivery and therapeutics.A fluorescence marker (fluorescein anion) and a redox
marker [hexacyanoferrate (III)] loaded apoferritin hasbeen synthesized that can be used as bioassay labels for
Figure 5. (A) Magnetic beads and electrochemical sandwichimmunoassay protocol based on biotin-functionalized hexa-cyanoferrate MLAN labels. (B) Typical square wave voltam-mograms of electrochemical immunoassay with increasingconcentration of the IgG (from a to e, 0.1, 0.5, 2, 10, and20 ng mL−1 IgG, respectively). A baseline correction of theresulting voltammogram was performed using the “linearbaseline correction” mode of the CHI 660 (CH Instruments)software. Also shown (insets), (top) the resulting calibrationplot and (bottom) the square wave voltammograms (with-out baseline correction) of 0.1 and 0 ng mL−1 (control) IgG.After the sandwich hybridization assay, the magnetic bead-hexacyanoferrate loaded apoferritin hybrid was dispersed in50 �L of 0.1 M HCl/KCl to release the captured hexacyano-ferrate. Following a magnetic separation, the solution wastransferred to a SPE surface for SWV scanning. Reprintedwith permission from [180], G. Liu, et al., Versatile apoferritinnanoparticle labels for assay of protein. Anal. Chem. 78, 7423(2006). © 2006, American Chemical Society.
microscopic fluorescence immunoassay and electrochem-ical immunoassay, respectively as shown in Figure 5. Itsdetection limits were estimated to be of 0.06 (0.39 pM)and 0.08 ng mL−1 (0.52 pM) IgG with fluorescein andhexacyanoferrate, respectively.180�181 The biologically pro-duced functionalized NPs were also used as labelingagents in bioaffinity assay. In this study, Eu3+ ions wereused as labeling agent and were loaded inside the fer-ritin, while a binding moiety i.e., single chain Fv fragment(scFv) of an antibody was attached on its surface in orderto aid their specific binding to the thyroid stimulating hor-mone (TSH).202
A new highly sensitive and selective magnetic parti-cle (MP)-based electrochemical immunoassay has beendemonstrated, having a detection limit of 0.01 ng/mLusing carbon nanospheres (NS) and lead phosphate loadedprotein cage nanoparticles (PCN) for signal amplification.This system has been used to analyze the phosphorylatedprotein human phospho-p5315, a potential biomarker ofgamma-radiation exposure.167
In a similar way, a co-reactant based highly sensitiveelectro chemiluminescence (ECL) immunoassay approachhas been devised based on PEI loaded apoferritin NPs,probes for the specific quantification of human chorionicgonadotrophin (HCG) by enhancing the ECL of ruthe-nium (II) tris(2,2′-bipyridyl) (Ru(bpy)+32 ).
201 Moreover, forrapid, sensitive, selective and inexpensive quantification oforganophosphorylated acetylcholinesterase (OP-AChE), anexposure biomarker of organophosphate based pesticides.A new sandwich type electrochemical immunoassay hasbeen developed using apoferritin templated lead phosphatelabel for quantification of OP-AChE, having a detectionlimit of 0.02 nM.168 These new apoferritin based nanopar-ticle labels hold great promise in the field of biomoleculedetection and in enhancing the sensitivity of various otherbioassays.
BiosensorsThe metal encapsulated apoferritin NPs can be used invariety of nanodevices, such as single electron transistor,catalysis and floating gate memory. In similar way, semi-conductor NPs such as CdSe, ZnSe, and CdS encapsulatedapoferritin can be used as quantum dots and photofluores-cence markers. ZnSe is a n-type semiconductor that couldbe used as fluorescent labels for biological applications astheir fluorescent light does not quench easily.Ferritin molecules have redox property which remains
unchangeable until their electrochemical surrounding isfixed.224 In recent years, protein electrochemistry hasemerged as an interesting area in the development ofbiosensors and bioreactors. Various electron transfer reac-tion studies of ferritin have been conducted, such aselectron transfer of ferritin on bare gold electrode.225
The electrochemical behavior of ferritin adsorbed onindium–tin oxide (ITO) glass and single wall nano-tubes (SWNT)/ferritin composite on glassy carbon (GC)
J. Biomed. Nanotechnol. 10, 2950–2976, 2014 2965
Ferritin Nanocages: A Novel Platform for Biomedical Applications Bhushan et al.
disk electrode had been studied for nanoelectronicapplications.226�227 Moreover, electrochemical studies onferritin immobilized onto a self-assembled monolayer-modified gold electrode have been already reported.228 Thedirect electron transfer of ferritin in Dihexadecyl phos-phate (DHP) on Au film electrode was also evaluated.229
A ferritin/DNA complex was successfully constructedby chemically attaching maleimide modified DNA(M-DNA) to the exterior surface of a ferritin mutant pro-tein, which can be sterically attached to the complementaryDNA-functionalized GNPs. This complex can be utilizedin photo electrochemical biosensor fabrication as it canserve as a mediator between the DNA/RNA responsiblefor disease and dye-labeled photo reporter probe.137
A highly sensitive electrochemical approach have beenreported having a linear range from 2�0× 10−16 to 1�0×10−14 M and the detection limit was 5�1×10−17 M underoptimum condition, based on signal dual-amplificationwith Au NPs and marker-loaded apoferritin NPs for thesequence-specific DNA detection. The concentration oftarget DNA is quantified by electrochemical stripping anal-ysis of the electroactive cadmium markers released fromapoferritin NPs in acidic buffers. This proposed DNAbiosensor has high sensitivity, good reproducibility andselectivity even against two-base mismatched DNA.176
Recently, a direct electron transfer has been investigatedbetween cobalt NPs loaded apoferritin and a glassy carbonelectrode in thin film of dihexadecyl phosphate (DHP) bycyclic voltammetry (CV) in order to design a biosensingdevice that can be used in detection of various chemicaland biological analytes.112
In addition to this, an electrochemical approach has beendeveloped using metal phosphate nanoparticles loadedmonobase-conjugated apoferritin probe for the detection ofindividual single nucleotide polymorphisms (SNPs). Thebiotinylated DNA probes get hybridized with mutant andcomplementary DNA and the duplex DNA helix formwere captured on the surface magnetic beads by biotin-streptavidin based affinity binding. Signals were gener-ated and detected by electrochemical stripping analyses,when the probes get coupled to the mutant sites of formedduplex DNA by DNA polymerase, as each mutation cap-tures different nucleotide-conjugated apoferritin probe andgenerates distinct potential voltammogram peaks relativeto mismatch.156
An array of charged storage nodes in floating gate mem-ory had been developed using ferritin encapsulated NPs.230
Apoferritin loaded with Ni atoms catalyzed the fabricationof high quality polycrystalline silicon (Si) thin film froman amorphous Si thin film.231
In general, electrochemical biosensors utilize thepotentiometric and amperometric transducers that con-vert the biosensing information into the measurablesignal. Recently, apoferritin encapsulated gold nanoparti-cles have been utilized to perform electrochemical DNAbiosensing having a sensitivity up to 51 aM.176 Apoferritin
bionanomaterial also enhances electron transfer reactionsof hemoglobin in a wide pH range. Since, the Hb exhibitcatalytic activity toward H2O2, the construct can be usedfor the development of H2O2 biosensor.
232
BiocatalystApoferritin loaded nanoparticles also found their rolein catalyzing various chemical reactions. As shown inFigure 6, polymerization of phenylacetylene has been cat-alyzed by the Rhodium (Rh(nbd)) complexes immobilizedwithin the discrete space of apoferritin that can be usefulin investigating the behavior of a single polymer chain iso-lated within a nano-sized space.60 Similarly, they have alsodemonstrated that the ferrocenes and Pd(allyl) complexeswere immobilized on the interior surface of apoferritin.The Pd(allyl) complexes immobilized by forming a thiol-bridged dinuclear complexes and catalyzed the redox andSuzuki coupling reactions.101�233
Figure 6. Polymerization of phenylacetylene catalyzedby Rh(nbd) · apo-Fr. (a) Solution of Rh(nbd) · apo-Fr priorto addition of phenylacetylene (b) Reaction mixtureof Rh(nbd) ·apo-Fr and phenylacetylene after stirring for 3 h at25 �C. (c) Reaction mixture of [Rh(nbd)Cl]2 and phenylacety-lene under the same conditions. (d)–(f) Elution profiles fromsize-exclusion chromatography of (d) Rh(nbd) · apo-Fr afterthe reaction, (e) Rh(nbd) · apo-Fr, and (f) apo-Fr. Elutionwas monitored at both 280 nm (black line) and 383 nm (redline). Reprinted with permission from [60], S. Abe, et al.,Polymerization of phenylacetylene by rhodium complexeswithin a discrete space of apo-ferritin. J. Am. Chem. Soc. 131,6959 (2009). © 2009, American Chemical Society.
2966 J. Biomed. Nanotechnol. 10, 2950–2976, 2014
Bhushan et al. Ferritin Nanocages: A Novel Platform for Biomedical Applications
In a similar way, Pd has been encapsulated within thecore of a hyperthermophilic ferritin cages (from pyro-coccus furiosus) to form a hybrid catalysts that can beused for highly specific aerobic oxidation of alcohols inwater.99 In addition to this, it has been demonstrated thatthe apoferritin encapsulated Pd nanocluster catalyzes thesize-selective olefin hydrogenation.100
The Fe(O)OH-mineralized iron storage protein fer-ritin was used to catalyze the photoreduction of aque-ous Cr-(VI) species to Cr(III), Cu(II) to form a stable,air sensitive, colloidal dispersion of Cu(0) and reductionof cytochrome c and viologens as well as the oxidationof carboxylic acids, thiol compounds, and sulfite. Fer-ritin act as photocatalyst in presence of UV/visible lightand can be utilized for future photocatalytic applications,such as in environmental remediation chemistry.125�126�104
These NPs also acts as a catalyst for the growth of single-walled carbon nanotubes.124 Similarly, it has been reportedthat 1–2 nm and 3–5 nm diameter range discrete cat-alytic nanoparticles synthesized in apoferritin cavity canbe used for the growth of SWNTs on substrate by chem-ical vapor deposition (CVD) and diameter of nanotubeswas controlled by getting hold on the structure of catalyticNPs in core.127 Moreover, apoferritin encapsulated AuNPs exhibited catalytic synthesis of single-walled carbonnanotubes (SWCNTs) on various substrates by chemicalvapor deposition.146 In another similar attempt, ferritincage loaded with catalytic Au NPs were immobilized toa silicon substrate for the growth of silicon nanowire(SiNW) by CVD.147
The apoferritin encapsulated homogeneous gold-silveralloy NPs aid in the catalytic reduction of 4-nitrophenolinto 4-aminophenol in the presence of NaBH4.
152
A bimetallic nanoreactor is prepared by loading Au–PdNPs in apoferritin core that shows 2.5-fold higher catalyticreactivity of olefin hydrogenation as compared to Pd0 NPsin the cage.151
Enzyme ImmobilizationNow a days, researchers are interested in stabilizingenzymes and retaining their activity as they are promisingtools for wide range of applications including biocatalysis,bioassay, bioenergy conversion and environmental reme-diation. A large number of techniques are available forthe enzyme immobilization but most of them have certainlimitations, which include loss of enzymatic activity dur-ing immobilization, stability and low efficiency. Therefore,there is a need for development of new novel immobi-lization technique. A large number of inorganic materi-als are used for enzyme immobilization but they are notbiocompatible.Apoferritin provide a biocompatible nanosized con-
tainer for the synthesis of biomaterials. It has beenrecently shown that the apoferritin can also be usedin stabilizing enzymes and also to enhance their activ-ity. Immobilization of glucose oxidase (GOx) has been
reported on the surface of apoferritin by green syntheticapproach. A glucose oxidase–biotin/streptavidin/biotin–apoferritin conjugate (Apo–GOx) was formed by bridgingwith streptavidin. The Apo-GOx formed shows enhancedthermal and chemical stabilities.200
Artificial AntioxidantA naturally occurring antioxidant enzyme includes theendogenous superoxide dismutase (SOD), but it is foundto be incapable in protecting the cells from sudden oxida-tive damage. Therefore, current research is now focus-ing on the development of artificial antioxidant havinga high ROS-scavenging capability and low cytotoxicity.Nanoceria (nano-CeO2) is now being studied because oftheir SOD mimetic activity and other properties, such asreversibility and auto regeneration.234�235
Recently, a nano-CeO2 has been constructed withinthe cavity of apoferritin protein cage, which improvesbiocompatibility and manipulate electron localization onthe surface of nanoparticles thereby improving the ROS-scavenging activity of this nanocomposite. It was sug-gested that the increase in redox activity of CeO2 is dueto change in the surface morphology/surface defect orvacancies due to the charge transfer process that changethe electron localization on the surface of nano-CeO2,which e