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Associate Professor Zhang Tuo, China Agricultural University, et al.: Research progress on the application of ferritin nanocarriers in the field of nutrition and health

 Last Update: 2022-11-14
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Ferritin is an iron storage protein widely found in organisms, and its main biological functions are two: first, to convert ferric ions into a soluble, non-toxic and bioavailable form of ferric iron and store it in the cavity inside the protein to regulate the balance of iron metabolism; The second is to scavenge ferrous ion-mediated free radical reactions and protect cells from oxidative damage
.
Natural ferritin consists of a protein shell consisting of 24 identical or different subunits of a hollow cage-like protein molecule with a highly symmetrical structure, and an internal iron nucleus, which is composed
of a large amount of iron hydroxide and phosphate.
Under anaerobic conditions, the iron nucleus can be removed by reduction reaction to prepare a ferritin shell
with an internal cavity.
The cage-like shell of ferritin is very stable, with strong acid-base resistance (pH 2.
0~12.
0) and thermal stability (protein denaturation temperature of 70~80 °C).

The advanced structure of ferritin is mainly maintained by hydrogen bonding and hydrophobic interaction between subunits, so its subunits can be dissociated and reassembled by physicochemical means, and its reversible dissociation and recombination characteristics lay the foundation
for the application of ferritin nanocages as embedded shells and delivery carriers.

So far, ferritin has been widely used to encapsulate anthocyanins, curcumin, rutin and other food nutrients to improve their water solubility, stability and cell absorption, and the use of ferritin to load anticancer drugs, contrast agents and other components can be used to achieve targeted drug delivery, cell imaging and tumor treatment research
.
In addition, due to the single structure of natural proteins, ferritin is also widely used by scientists to artificially design and manufacture new cage-shaped proteins with different structural properties, which greatly expands the application range
of ferritin as a nanocarrier.
Liu Bo, Zhang Chenxi and Zhang Tuo* from the College of Food Science and Nutritional Engineering, China Agricultural University, systematically summarized the structural characteristics of ferritin molecules, and focused on their research progress
as nanocarriers in food science, nutrition and health and medical imaging.

1.
The structure of ferritin

Typical structural features of ferritin

The subunit structure consists of a α helix cluster (A, B and C, D helix) that are oppositely parallel and a shorter α helix (E helix), which is connected by a chain of amino acids called a BC-ring, and the E helix is located at the tail end of the α helix at an angle of 60° (Figure 1A).

The amino terminus of the ferritin subunit, BC-ring, A helix and C helix form the outer surface of ferritin, while the B helix and D helix are located on the inner surface
.
The inner surface of ferritin is rich in acidic amino acid residues such as glutamic acid and aspartic acid, so under the condition that the pH value is neutral, the inner surface of ferritin has a high negative charge density, while the net charge of the outer surface is close to zero or slightly positive
.
Ferritin molecules have three chemically different active interfaces: the inner surface, the outer surface, and the interunit interface (Figure 1B), and all three interfaces can be chemically and genetically modified to achieve the functional regulation
of ferritin.
Chemical modification refers to the chemical coupling of functional small molecules such as dye molecules and quencher with specific amino acids of ferritin, and genetic modification is a targeted mutation, addition or deletion of specific amino acids to change the gene sequence
of ferritin.
The coupling and modification of some functional groups on the outer surface of ferritin can give it characteristics such as luminescence imaging, the inner surface is mainly used as a nanoreactor to synthesize inorganic nanomaterials or for embedding active nutritional small molecules, and the interunit interface can be used to regulate the dissociation and recombination of ferritin molecules and design new cage-like proteins
.
Through the modification and modification of these three active interfaces, the application space
of ferritin molecules can be expanded.
As a typical cage-shaped protein, ferritin molecules self-assemble to form a highly symmetrical quaternary structure
by complex and precise forces between subunit interfaces.
The interunit interfaces involved in the assembly of ferritin cage structures include C2, C3, C4, and C3-C4 interfaces, of which C2 and C3-C4 interfaces are the main regions that maintain the interaction between subunits (Figure 1C).

Natural ferritin characteristics from different sources

Animal ferritin is usually composed of
two subunits, H-type (heavy chain, molecular weight of about 21 kDa) and L-type (light chain, molecular weight of about 19.
5 kDa), which vary greatly in function.
The H-type subunit contains 1 ferrous oxidation center that oxidizes ferric ions to ferric ions
.
1 ferrous oxidation center can bind 2 ferric ions at the same time, the ferrous oxidation center contains 2 iron ion binding sites, A site is composed of Glu27, Glu61, Glu62, His65 and Glu107 5 amino acid residues, and B site is composed of
Tyr34 and Gln141 amino acid residues.
The L-type subunit does not have the function of ferrous oxide ions, but it has 1 nucleation center responsible for converting oxidized ferric ions into mineralized nuclei
.
The proportion of ferritin H and L subunits is different in different organs and tissues, and ferritin is rich in H subunit in organs with strong metabolism such as brain and heart, while the proportion of ferritin L subunit is higher
in organs such as spleen and liver.
H and L subunits can specifically recognize the surface receptors of human cells, one is transferrin receptor (TfR)1, which specifically recognizes ferritin H type subunits; The other is scavenger class A receptor protein 5, which recognizes the L-type subunit
of binding ferritin.

Structural modification of ferritin

Due to the relatively single size and shape of natural proteins, their application range
is limited.
So scientists built a new ferritin nanocage
through artificial design.
The interunit interface of four subunits of ferritin molecules is very important for the formation of ferritin cage-like structure, among which the surface area of the C3-C4 interface is the largest, followed by the C2, C3 and C4 interfaces
.
Zhang Shengli et al.
converted 24 mer ferritin to 16 mer and 48 mer caged ferritin by inserting or deleting amino acids at key positions at the C3-C4 interface (Figure 2A).

16 polymer ferritin is joined together by hydrophobic interaction of two similar 8 mers to form a flat spherical hollow structure
.
The outer diameter of 48 mer ferritin is expanded to 17 nm, and its internal cavity capacity is about 4.
3 times that of the original, but under solution conditions, 48 mer is unstable and will be converted to bowl-shaped 8 mer ferritin
with a diameter of about 10 nm 。 Zang Jiachen et al.
then used the bowl-shaped 8 meric ferritin as a template and successfully converted it into three novel cage-shaped proteins by designing the intra-chain disulfide bond and the interchain disulfide bond as force, respectively: cage-shaped 24 mer (outer diameter 12 nm), elliptic 16 mer (major axis 10 nm, minor axis 8 nm), and cage 48 mer (outer diameter 17 nm) (Figure 2B).

When the C3-C4 interface of ferritin is completely eliminated, that is, 49 residues at the carboxyl end of the ferritin subunit are deleted, the ferritin will be converted to an 8-mer nanocyclic structure (Figure 2C).

The inner wall of this nanoring consists only of a B helix, while its outer wall is composed of a A helix, a C helix and a BC-ring with a height of 5.
1 nm and an inner and outer diameter of 3.
2 nm and 7 nm
, respectively.

2.
Application of ferritin in the field of food nutrition

Mineral element carriers

Iron is one of the essential trace elements of the organism, and iron deficiency anemia (IDA) is one of the most common nutrient deficiencies in the world, seriously affecting human nutrition and health, mainly due to insufficient or excessive iron intake, so scientists are constantly exploring efficient and safe iron supplementation preparations
.
At present, the common iron supplements on the market are mainly represented by ferrous sulfate or ferrous gluconate, although the iron content is high and cheap, but the nature is unstable and easily affected by other components of food, and at the same time will have certain side effects on the human body, such as diarrhea, vomiting, growth retardation, etc
.
Natural plant ferritin, especially ferritin in legume seeds, has attracted the attention of scientists due to its wide source and rich iron content, and is considered to be a new natural dietary iron supplementation factor
.
Thanks to the protection of the protein shell, the iron of plant ferritin is not susceptible to dietary chelating agents such as phytic acid and tannic acid
.
In legume seeds, more than 90% of iron is stored in amyloids in the form of ferritin, and the researchers compared the iron absorption effect of ferrous sulfate and isolated soy ferritin in rats and found that there was no significant difference between the two groups, so the iron in soy ferritin can be well absorbed by the human body and has a similar iron supplementation effect
as ferrous sulfate.
Ferritin can inhibit the auto-oxidation of ferrous ions to generate reactive oxygen species with toxic side effects, and has a detoxifying effect, and protein can be used as a new iron supplement preparation
from plant sources.

Calcium is the most abundant inorganic element in the human body, which participates in a variety of physiological processes in the body and plays an important role
in the growth and development of the human body and nutritional health.
At present, the phenomenon of calcium deficiency in China is still common, calcium supplement preparations on the market are mainly divided into inorganic calcium, bioactive calcium, organic calcium and amino acid chelated calcium, etc.
, these calcium preparations have certain application defects, and then calcium preparations with peptides or proteins as the main ligand have developed rapidly, and studies have found that calcium or other mineral elements can be absorbed
by small intestinal cells through peptide transport mechanisms or protein-specific receptors.
Although dairy products are a good source of bioavailable calcium, they are not suitable for people such as vegetarians, so it is particularly important
to develop calcium supplementation preparations with plant-derived proteins as carriers.
In order to solve the above problems, the researchers loaded calcium ions into the cavity of plant ferritin to prepare a ferritin calcium complex
.
Cell absorption experiments have found that plant ferritin can protect calcium ions from the interference
of dietary inhibitors such as oxalic acid and tannic acid.
In addition, unlike the known divalent metal ion transporter 1 (DMT1)-mediated divalent metal ion absorption pathway, cells can absorb the ferritin-calcium complex through the new TfR1 participation pathway, so ferritin-coated calcium ions do not interfere with the absorption
of other divalent metal ions.
Subsequently, zinc, copper, selenium and other mineral elements are loaded into the ferritin cavity has been experimentally verified, and its special hollow structure and cage-shaped protein shell can overcome the shortcomings of low solubility of metal ions and easy interference from the external environment, and significantly improve the bioavailability
of mineral elements.
In summary, ferritin has broad application prospects
as a mineral nutrient carrier.

Embedding of active nutrients

There are many highly bioactive small molecules in food, such as β-carotene, curcumin, rutin, anthocyanins, lutein, proanthocyanidins, etc.
, which are considered to have antioxidant, anti-cancer and anti-inflammatory biological activities, and have great promoting functions
for human health.
However, most of these compounds are sensitive to environmental conditions, such as water insolubility or light and thermal instability, which greatly limits their application in nutrition and human health, so it is important
to overcome these constraints.
In recent years, animal and plant ferritin has received extensive attention in the fields of food science and nutritional health, and scientists have used the reversible dissociative assembly properties of ferritin controlled by pH value to encapsulate various water-soluble or fat-soluble active nutrients
.
This method is simple and environmentally friendly, and it has been found that the water solubility, photothermal stability and cell uptake activity of small nutritional molecules after embedding are significantly better than those in the free state, and ferritin molecules can also prevent the embedded substances from being interfered
with by other components in the food.

Heavy metal detection

Mercury is a highly toxic heavy metal that bioaccumulates through the food chain and poses a serious threat to human health
.
Mercury compounds can be divided into inorganic mercury and organic mercury
.
Inorganic mercury mainly exists in the environment in the form of divalent mercury ions (Hg²⁺), ingestion or inhalation will cause serious harm to human nerves, gastrointestinal system and internal organs, so the establishment of rapid and accurate detection of mercury ions is of great significance
for food safety, human health and environmental testing.
At present, atomic absorption spectroscopy, atomogenesis spectroscopy, inductively coupled plasma mass spectrometry, electrochemical method and other instrumental methods can be used to detect heavy metal ions such as Hg²⁺, but these traditional methods have application defects
such as high cost and cumbersome processing process.
In recent years, fluorescent probe detection technology has gradually attracted extensive attention
from researchers due to its advantages of high sensitivity, good selectivity and convenient application.
Wang Yingjie et al.
successfully prepared a protein nanocage (HFn-MBP)
with high binding ability to^Hg2+ by attaching a segment of MBP to the outer surface of the HFn nanocage.
The fluorescence of HFn-MBP labeled with fluorescein isothiocyanate (FITC) can be quenched by GO, while the addition of Hg²⁺ to the above system can restore the quenched fluorescence in a dose-dependent manner (Figure 4B).

The sensor consists of FITC-labeled HFn-MBP and GO and has high sensitivity and selectivity for Hg²⁺ with a detection limit of up to 1.
0 nmol/L
.
This study has opened up a new way
for the application of ferritin nanocages in the detection of heavy metal ions.

3.
Application of ferritin in the field of biomedicine

Anti-cancer drug delivery

The increasing cancer mortality rate indicates that there are still great challenges in tumor treatment, and in addition to the research and development of various anti-cancer drugs, the research on nanomedicine vectors has also attracted widespread attention
.
Since human heavy chain ferritin can specifically recognize TfR1, the structure of the complex between TfR1 and human heavy chain ferritin was analyzed by cryo-electron microscopy and found that the amino terminus, BC-ring and carboxyl end of the H-type subunit A helix were the corresponding regions
where ferritin binds to TfR1.
TfR1 is overexpressed in tumor cells, and with its inherent tumor targeting, human heavy chain ferritin has been widely used in loading platinum compounds such as cisplatin, carboplatin, oxaliplatin, Dox, gold-based compounds (Au2phen and Auoxo4), ruthenium complex (DiRu-1) and other anticancer drugs
.
Studies have found that ferritin as a drug carrier can cross the blood-brain barrier and achieve targeted therapy for malignant gliomas in situ (Figure 4C).

The blood-brain barrier is a natural barrier to maintain central nervous system homeostasis and protect brain tissue from metabolite damage, almost all large molecule drugs and most small molecule drugs cannot cross the blood-brain barrier to reach brain tissue, so central nervous system diseases such as malignant brain tumors cannot be effectively treated
.
Since TfR1 can be highly expressed in brain endothelial cells and malignant tumor cells, the experimental results show that ferritin can cross the blood-brain barrier through the transcytosis of brain endothelial cells without blocking in lysosomes, and can be specifically enriched into lysosomes of malignant brain tumor cells through TfR1 for degradation
.
Ferritin encapsulates and targets the release drug Dox to target cancer cell
killing.
The specific targeted binding of ferritin to TfR1 and the ideal nanosize effect are important reasons why
it can cross the blood-brain barrier and has high selectivity in tumor tissue.

Diagnosis and photothermal therapy

The biomineralization capacity of ferritin cavities and the easy modification of surfaces make ferritin a good biological imaging device, and it is widely used
in magnetic resonance imaging (MRI) and PAI.
MRI is a powerful diagnostic technique that is widely used in tumor imaging
due to its high sensitivity and accuracy.
However, the lack of specificity of currently used gadolinium-based contrast agents for cancer cells leads to false positives, while MRI may not detect occult tumor microdeposition
due to insufficient spatial resolution.
Cao Changqian et al.
synthesized magnetite nuclei in ferritin nanocages as MRI contrast agents for in vivo detection of cancer, which has extremely high contrast performance and TfR1-dependent MRI signals
.

Conclusion

Due to the advantages of ferritin such as monodispersity, high solubility, high biosafety and high stability, it is of great significance
to use the characteristics of ferritin nanocage-like structure, dissociative self-assembly and easy modification to embed and target delivery of active substances or anticancer drugs.
After ferritin embedding, the water solubility, thermal stability, photostability and cell uptake rate of the active nutrients are significantly improved, and the drug molecules can accurately target and kill tumor cells and cross the blood-brain barrier to reach brain tissue
.
Many studies have shown ferritin as a new nanocarrier
for embedding bioactives.
In addition, ferritin nanocarriers also have broad application prospects
in food heavy metal detection and in vivo imaging diagnosis.
Although great progress has been made in the preparation and application of ferritin-embedded substances, there are still some problems that need to be solved
.
First of all, the embedding efficiency and loading capacity of ferritin still need to be improved, and it is possible to use the space of the assembly gap by artificially designing more new ferritin nanocages of different shapes and properties or preparing protein assemblies, but the specific application system needs to be deeply explored
.
Secondly, most of the current research focuses on the preparation methods and pharmaceutical applications of ferritin-embedded substances, and the application of ferritin nanocarriers in food nutrition and detection should receive more attention
.
Finally, the stability of ferritin in the stomach and the cell uptake efficiency of guest molecules need to be further improved, so as to ensure that the contribution of ferritin delivery system to human nutrition and health can be put into practical clinical application
.

·Corresponding Author Profile·

Zhang Tuo is an associate professor and distinguished researcher
at the College of Food Science and Nutritional Engineering, China Agricultural University.
He graduated from China Agricultural University with a bachelor's degree and a doctorate degree in engineering, and is a postdoctoral
fellow at Michigan State University.
His research interests include protein chemistry and nutrition, including mineral nutrition and health, trace element receptors and transmembrane transporter activity mechanisms
.
He has published more than 30 papers in related fields, and representative papers have been published in well-known journals in Science Advances
, Nature Communications, Science, Chemical Society Review, Critical Review in Food Science and Nutrition.

This article "Research Progress on the Application of Ferritin Nanocarriers in the Field of Nutrition and Health" is from Food Science, Vol.
43, No.
15, pp.
302-311, 2022, authors: Liu Bo, Zhang Chenxi, Zang Jiachen, Lv Chenyan, Zhang Tuo, Zhao Guanghua
.
DOI:10.
7506/spkx1002-6630-20210730-359
。 Click to view information about
the article.

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