Nano Research
Nano Res
DOI
10.1007/s12274-014-0509-9
Nano modification of living organisms by biomimetic
mineralization
Wei Chen1, Guangchuan Wang2, and Ruikang Tang1,2 ()
Nano Res., Just Accepted Manuscript • DOI 10.1007/s12274-014-0509-9
http://www.thenanoresearch.com on June 5, 2014
© Tsinghua University Press 2014
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1
TABLE OF CONTENTS (TOC)
Nano
modification
of
living
organisms
by
biomimetic mineralization.
Wei Chen, Guangchuan Wang and Ruikang Tang*
Zhejiang University, China
Nano modification of living organisms by biomimetic
mineralization can create novel living-mineral complexes with
external shells or internal scaffolds, promising great potentials
in biological storage, protection, functionalization and drug
delivery etc.
Nano Research
DOI (automatically inserted by the publisher)
Review Article
Nano modification of living organisms by biomimetic
mineralization
Wei Chen1, Guangchuan Wang2, and Ruikang Tang1,2 ()
Received: day month year
ABSTRACT
Revised: day month year
In nature, a few living organisms such as diatoms, magnetic bacteria, and eggs
have developed specific mineral structures, which can provide extensive
protection or unique functions. However, almost others do not have such
structured materials due to the lack of biomineralization ability. An artificial
introduction of biomimetic-constructive nano minerals tends to be a great
challenging and promising attempt. In this overview, we highlight two typical
mineral-living complex systems. One is the biological surface-induced nano
material, which produces the artificial living-mineral core-shell structures such
as the mineral-encapsulated yeast, cyanobacteria, bacteria and virus etc. The
other is the biology internal nano minerals that could endow organisms with
unique structures and properties. The applications of these biomimetic
generated nano minerals are further discussed, mainly in four potential areas:
storage, protection, “stealth” and delivery. Since biomineralization combines
chemical, nano and biological technologies, we suggest that nano biomimetic
mineralization may open up another window for interdisciplinary researches.
Specifically, this is a novel material-based biological regulation strategy and the
integration of living organisms with functional nanomaterial can create ‘‘super’’
or intelligent nano living complexes for biotechnological practices.
Accepted: day month year
(automatically inserted by
the publisher)
© Tsinghua University Press
and Springer-Verlag Berlin
Heidelberg 2014
KEYWORDS
Biomineralization, living
organism, bioinspiration,
nanomaterial,
material-based
bio-modification.
1. Introduction
In the evolution of natural systems, living organisms,
whether very basic or highly complex, have
developed various biominerals, such as teeth, bones,
shells, carapaces, spicules and so on [1, 2]. Those
composite biomaterials often exhibit unique
structures and possess important functions such as
mechanical support, protection, motility, and sensing
of signals [3]. Marine organisms, e.g. diatoms, use
Address correspondence to [email protected]
biominerals as their exterior coats to protect
themselves from external stresses or aggressions [4,
5]. Another example is that, the magnetotactic
bacteria have the internal nano compass that consists
of a chain of discrete crystals of magnetite (Fe 3O4 or
Fe3S4), which align the organism along with lines of
force of the geomagnetic field [6, 7]. Chicken eggs are
perhaps the most familiar example since the
eggshells provide mechanical support for the
embryo and are also extremely important for
Nano Res.
maintaining the egg’s viability. This shell forms a
shield to prevent the enclosed cell from
contamination so that an egg with an intact shell
structure can be stored for several weeks in ambient
conditions. In addition, shells also provide the
embryo with the minerals needed for the generation
of organs high in calcium, such as the skeleton,
muscles, and brain [8, 9].
efficiently regulate nucleation and crystallization
[26-31], which dominate the hierarchical structures
and unique properties of biominerals [32, 33].
It has been demonstrated that the characteristic
minerals can improve the living organisms'
survivability. However, most cells in nature cannot
make their own shells and thereby, have the
relatively poor stabilities or insufficient functions.
Different from eggs with mineral shells, they can
only be stored under refrigeration conditions [10].
The living organisms cannot generate mineral
structures due to the lack of biomineralization ability.
Biomineralization is the process by which living
organisms make use of organic matrices to control
the formation of inorganic mineral-based structures
[1-3, 11]. Different from synthesized materials, the
biologically
produced
biominerals
exhibit
hierarchical structures from nano- to macro-scales
[12], which are induced and controlled precisely by
intricate biology systems [13].
Interestingly, those regulatory biomacromolecules
always contain specific functional groups to facilitate
organic-inorganic interaction [34]. The biomolecules
active in calcium-based minerals share a common
feature of that they contain the active domain rich in
negatively charged residues including phosphate,
carboxylate and sulfate [23, 35-38]. With these
functional groups, the biomacromolecules can
sequester and chelate calcium ions from
supersaturated metastable solutions, favoring the
heterogeneous nucleation and growth of calcium
minerals. Meanwhile, the biosilication-relative
macromolecules always contain polyamines that
may either serve as “flocculating agents” or absorb
silicic acid species to form “liquid precipitate” by
protonated amino groups [39-43]. In addition, the
biomacromolecules like proteins on magnetosome
membrane inside magnetospirillum always contain
specific acid amino residues such as glutamic acid
and aspartic acid residues, which have crucial
functions in the accumulation of iron, nucleation of
mineral, redox and pH control[6, 7, 44, 45].
In order to reveal that how biology systems
control inorganic material formation, there are many
researches concentrated on various biomineralization
systems including calcium carbonates [14],
hydroxyapatite [15], silicon [5] and magnetic
minerals [16] etc. Addadi and Weiner showed that
porous organic matrix sheets allow the crystals to
grow and keep them aligned in mollusks [17, 18].
Christopher and Wilt reveal a number of matrix
molecules and their biochemical complexity during
biomineralization of echinoderm endoskeleton [19].
In human bodies, physiological biomineralization in
collagenous matrices as well as non-collagenous
matrix largely influence the formation of skeleton,
teeth and so on [20]. Clearly, organic matrices are the
key to biomineralization controls since various
organic biomolecules can induce precursor
formation of biominerals [21, 22] or construct
inorganic-organic
composites
[23].
Therefore
molecular recognition [24] and tectonics [25] can
The
molecular-level
regulation
of
biomineralization can also be developed to control
nanomaterial formation and assembly in specific
composition, size, morphology, orientation, and
locations. For example, the mineral composition of
the magnetosome is under strict biochemical control,
because of that even when hydrogen sulphide is
present in the growth medium, the cells of several
cultured magnetotactic bacteria continue to
synthesize nano Fe3O4 but not Fe3S4. Besides, the
magnetosome crystals have narrow size ranges
(35-120
nm)
and
species-specific
crystal
morphologies (cuboidal, elongated prismatic and
tooth-, bullet- or arrowhead-shaped), as well as
specific
arrangements
within
the
cells
(one-dimension chain) [6, 7]. Such a precise control
should
be
derived
from
the
specific
organic-inorganic interactions and the specific
organic phase is the key to mediate the inorganic
mineralization.
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Inspired by the nature, artificially introducing and
rational
utilization
of
mineralization-relative
macromolecules
can
help
us
to
modify
non-mineralized organisms. We suggest that the
regulatory molecules inside the organisms may
induce artificial internal minerals and analogously,
the functional molecules on the organisms’ surface
may generate artificial external minerals. In this way,
the living organisms and the non-living materials
can be combined as a novel functional integration
and such a biomimetic approach follows a novel
strategy for biological modification of living systems
base upon nanomaterials rather than traditional
biotechnologies.
Table 1 List of organisms that are employed for nano modification by biomineralization.
Organism
Location
Method
Reference
Calcium phosphate
External
LbLa
[47]
Cyanobacteria
Silica
External
LbL modification
[48]
Yeast
Silica
External
LbL modification
[49-51]
Zebrafish embryo
LnPO4
External
LbL modification
[52]
Str. Thermophilus
ZnS
External
Sonochemistry
[58, 59]
Calcium phosphate
External
Direct mineralization
[61]
External
Direct mineralization
[62]
External
Direct mineralization
[64]
Yeast
Japanese encephalitis vaccine
Mineral materials
(JEV)
Adenovirus serotype 5 (Ad 5)
Calcium phosphate
Tobacco mosaic virus (TMV)
Silica,
PbS
and
CdS
modification
nanocrystals, Iron Oxide
Human enterovirus type 71 (EV 71)
Calcium phosphate
External
Genetic engineering
[67]
M13 bacteriophatte
Co3O4, Au
External
Genetic engineering
[81]
Yeast
Calcium Carbonate
Internal
Ions internalization
[87]
MCF10 epithelial cell
Au
Internal
Metal reduction
[90, 91]
Mouse embryonic fibroblast cell (NIH 3T3)
Ag
Internal
Metal reduction
[92]
Cowpea chlorotic mottle virus (CCMV)
Paratungstate
Internal
Spatially constrain
[93]
Human lung epithelial cell (A549)
(H2W12O4210-)
a
LbL represents layer by layer technique.
Herein, we provide a perspective about some
artificially mineralized nano structures on living
organisms by using biomimetic mineralization. The
approaches to produce nano materials within or on
living organisms are summarized in section 2. In
section 3, the material-based functionalizations of the
biomineralized living organisms and their potential
applications are discussed. And in section 4, we
remark on some important questions from the
current studies of nanomaterials-based modification
of living organisms and propose challenges and
perspectives for the further development.
2. Biomimetic mineralization
Up to now, various living organisms including
yeast, algae, bacteria virus and even human cells etc,
have been successfully employed for nano
modification through biomimetic mineralization
with diverse methods (Table 1). In this way, amount
of
novel
living-nonliving
integrations
are
constructed, which exhibit quite specific structures
and unique properties. The following discussions are
mainly from two aspects: external and internal
mineralization, which helps us understand more
about the mechanism that living organisms create
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biominerals and presents some new approach to
modify natural livings.
2.1. External mineralization
Living organisms can create amazing ways to
produce high performance materials. A number of
them have outer-surface proteinaceous membranes
as the templates for biomineralization, which induce
mineral shell structures [46]. The resultant thin
mineral layers are the functional coverings. For
example, eggshells provide mechanical support and
meanwhile, maintain the balance of oxygen, carbon
dioxide, water and nutrients required for enclosed
cells [8, 9]. However, most cells lack of proteinaceous
membranes on their surfaces and do not have such
functional exteriors. Through rationally chemical or
biological modification, scientists have succeeded in
introducing the “nucleation sites” onto the surfaces
of cells (yeast, cyanobacteria, zabrafish embryo, etc)
[47-52] to produce an artificial mineral shell.
(a)
(b)
(c)
(d)
(e)
Figure 1 Introducing artificial mineral shell on cells. (a) Scheme
of artificially constructing CaP shell on yeasts. (b) Scheme for
silica encapsulation on individual cyanobacteria. TEM
micrographs of microtome-sliced native cyanobacteria (c),
PDADMAC/PSS-multilayer-coated cyanobacteria (d), and
cyanobacteria@SiO2 (e) at different magnifications. Reproduced
by permission of Royal Society of Chemistry from Ref. [48]
Yeast is frequently chosen as a study model in cell
biology. The chemical compositions of the cell wall
are
weakly
electronegative
polysaccharides,
mannose, and N-acetylglucosamine [53], which
contain less mineralization-relevant groups. Thereby,
yeast hardly induces mineralization and deposition
of calcium biominerals spontaneously. However, the
deposition of poly(acrylic sodium) (PAA), onto the
cell can turn yeast into the biomineralized one. PAA
has a high density of carboxylate groups, which can
mimic biomineralization proteins to provide active
heterogeneous nucleation sites for calcium minerals
[35, 54].
Actually, the negatively charged PAA cannot
deposit onto the negatively charged cell surfaces
directly. During the chemical modification, the
positively charged poly(diallyldimethyl ammonium
chloride) (PDADMAC) plays an important role to
bridge cell and PAA. Layer-by-layer (LbL) is a
general strategy which can fabricate desired
polyelectrolytes on solid supports, including living
organisms [55]. Based on the electrostatic properties
of cells, functional polyelectrolytes may be directly
grown or built onto cells for the fabrication of
multicomponent films by several alternating cycles
of absorption and deposition of opposite-charged
polyelectrolytes. By using such a method, polymers
or polypeptides with desired nucleating residues can
be present on the living organisms, and the intensity
of nucleation sites can also be regulated by
controlling the repeating times (Fig. 1(a)). If the
adsorbed PAA molecules are on the outermost layer,
the carboxylate groups migrate toward the
water-polymer interfere and bind Ca2+ ions. Upon
contact with calcium-rich solutions, the reorganized
surfaces trigger in situ deposition of calcium
phosphate (CaP) minerals [47, 56, 57].
Silica is another important biomaterial in nature.
Biosilica shell formation of diatom cell wall is
catalyzed by polycationic peptides containing
long-chain polyamine (LCPA) [39-43]. Accordingly,
the similar structured cationic polyelectrolyte,
PDADMAC, has been introduced onto the
cyanobacteria surface to promote silica deposition.
And the surface adsorption of this polycation onto
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cells can be assisted by poly(styrene sulfonate) (PSS),
another biocompatible polyelectrolyte with negative
charge, which can generate the multilayer structure
of (PDADMAC/PSS)6-PDADMAC on the modified
cells (Fig. 1(b)). The exposed PDADMAC acts as a
catalytic template to induce in situ silicification [48].
It should be noted that polyelectrolyte modification
does not obviously change the morphology and
surface texture of cells (Figs. 1(c) and 1(d)). However,
the new biomineralization ability is conferred on the
cells after the modification. Thus, a shell–like matter
(30 nm thick) can be spontaneously formed onto the
cyanobacteria (Fig. 1(e)). In addition, shells with a
thickness of 10 nm and 100 nm can be prepared by
adjusting the number of multilayers. In this way, a
thickness-tunable nano shell can be effectively
constructed onto the cell surface, which protects the
enclosed cells and mediates the metabolism by
affecting organisms’ responses against environment.
biocompatible conditions (Fig. 2). In this way, a
fluorescence or bioselective silica shell can be
constructed, which largely alters the inherent biology
properties of yeasts [51].
Besides
the
biomineralization-relevant
macromolecules modification, rationally utilizing
some potential surface groups might be another
available method for inducing mineralization. Take
bacteria for instance, most gram-positive bacteria
such
as
Lactobacillus
contain
primarily
peptidoglycan (PG) as the main constitute of cell
wall, which is a polymer of N-acetylglucosamine and
N-acetylmuramic acid. The two other important
constituents are teichoic acid, a polymer of
glycopyranosyl phosphate, and teichuronic acid,
which is similar to teichoic acid but replaces the
phosphate functional groups with carboxyl groups.
Therefore, the cell wall surfaces are covered
predominantly
with
carboxyls
(R-COOH),
phosphomonoesters (R-OPO3H2) and hydroxyls
(R--OH), which might exhibit large potential for
inducing mineralization. Zhang etc develops a novel
sonochemical
route
for
encapsulating
Str.
Thermophilus with ZnS nano minerals [58, 59]. They
demonstrate that the perfect combination of the two
factors (bacteria and sonochemistry) realizes the
one-step synthesis of nanoshell structures, which
maximally conserves the geometry of the original
biotemplate (Fig 3(a)).
(a)
(b)
Figure 2 Encapsulation of individual yeast cells in the
thiol-functionalized silica (SiO2SH) shells and the introduction of
fluorescein or streptavidin onto the SiO2SH shells. Reproduced by
permission of Wiley-VCH from Ref. [51]
In an analogue way, individual yeast cells are also
encapsulated with artificial silica shell to form
similar structures [49, 50]. Furthermore, Choi etc
develop the systems by introducing fluorescein or
streptavidin onto the thiol-functionalized silica
(SiO2SH) shells through the specific reactions of thiol
group
with
maleimide
derivatives
under
Figure 3 (a) Schematic illustration of the in situ one-step
formation of ZnS hollow spheres using Str. Thermophilus as
template and (b) the formation mechanism ())): represent the
ultrasonic treatment). Reproduced by permission of American
Chemistry Society Publications from Ref. [58]
The possible mechanism of formation of ZnS nano
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shell structure on the cell surface from Zn(Ac)2
solution with the help of ultrasound could be
understood from following equations (Fig 3(b)).
Equations 1-3 represent the main reactions leading to
the formation of ZnS nanoclusters after introducing
Zn complexes onto the cell surface. Equation 4
represents the formation of ·CH2COO radicals (·AC)
under sonochemical conditions, which has already
been well established [60]. As the sonochemical
treatment ensued, the surface of the bacteria have a
strong interaction with solute radicals based on the
reactivity of surface groups mentioned above and
solute radicals (sonochemically formed Zn(Ac)2),
producing better adhesion of ZnS nanoparticles.
Taking carboxyls as an example, the illustration of
reactivity of carboxyls and solute radicals was shown
in equation 5. In such a way, a general controlled
synthesis method was developed to construct
mineral on bacterial surface. Although the current
works are mainly focused on the metal sulfide, a
similar approach is applicable to other minerals. This
is not only a good way to biomineralization modified
living organisms like bacteria, but also an ideal way
to prepare hollow materials as the biotemplate can
be easily removed according to Zhang’s description.
(a)
(c)
(b)
(d)
Figure 4 Characterization of JEV and biomineralized JEV
(B-JEV). JEV and B-JEV solutions (in Dulbecco’s Modified
Eagle’s Medium (DMEM)) before (a) and after (b) centrifugation;
circle shows the precipitated biomineralized vaccine particles. (c)
Direct observation of B-JEV particles; insert is negatively
stained B-JEV. (d) EDX analysis of B-JEV (the Si signal was
attributed to the silica substrate). Reproduced by permission of
Wiley-VCH from Ref. [61]
After introducing nano shells onto cells, a new
challenge lies in the shellization on noncellular
organisms, viruses. Some viruses are suitable for
direct biomineralization modification due to the
abundant anionic peptides on their surfaces. For
instance, viruses such as Japanese encephalitis
vaccine (JEV) or adenovirus serotype 5 (Ad5) show
strong negative charge with the zeta potentials below
-25mV under physiological conditions [61, 62].
Under calcium-rich condition, these viruses can
absorb and concentrate cationic calcium ions
spontaneously around the particles to increase the
local
supersaturation,
triggering
the
biomineralization and resulting in an ultrathin
coating.
After biomineralization modification, the original
clear viral solution of JEV turns to a stable colloid
solution (Fig. 4(a)). Under a normal-speed centrifuge
at 16,000 g for 10 min, a few of white precipitate can
be separated from the modified JEV solutions while
no precipitate is observed for the bare JEV solution
(Fig. 4(b)). It should be mentioned that virus
separation and concentration with normal-speed
centrifugation are extremely difficult, mainly due to
the relatively small sizes, low gravity densities, and
weak surface charge. However, biomineralized
viruses overcome these difficulties through altering
the surface repulsive force of the virus [63]. More
interestingly, after the nano modification, the surface
electron density of viruses is enhanced by the
adsorption of artificial shells, which makes the
modified viruses without staining visible under
transmission electron microscopy (TEM) (Fig. 4(c)).
Using a negative stain treatment of phosphotungstic
acid for biomineralized particles, the enclosed
vaccine is identified, suggesting the eggshell-like
structures. Energy dispersive X-ray spectroscopy
(EDX) finds that the biomineralized virus surface is
composed of inorganic Ca and P rather than organic
C and N atoms (Fig. 4(d)). Collectively, a simple
separation, collection, and observation can be
achieved by using nano-mineralized viruses.
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(a)
(b)
(d)
(c)
(e)
Figure 5 Scheme for synthesis of nanotube deposits using TMV
templates (a) and computer-generated images of six TMV
subunits arranged as in the native virion and viewed parallel to
the helical axis. (b) Glutamic acid channel. (c) Aspartic acid. (d)
Arginine: note the high density in the RNA groove. (e) Lysine.
Reproduced by permission of Wiley-VCH from Ref. [64]
Besides that the surficial potential could trigger the
virus mineralization, amino acids residues exposed
on the outer surface might play another significant
role [64]. Take tobacco mosaic virus (TMV) for
example, it could serve as templates for fabrication of
a range of nano tubular inorganic-organic
composites
(sol-gel
condensation
(silica));
co-precipitation (PbS and CdS naocrystals); oxidative
hydrolysis (iron oxide)) (Fig. 5(a)). This should be
attributed to the surface chemistry that TMV coupled
with the unusual high stability of the protein
assembly
provide
a
structured
substrate
(simultaneously contain glutamic acid, aspartic acid,
Arginine and Lysine residues) for the site-specific
nucleation of a variety of inorganic minerals at a
range of pH values and reaction conditions (Figs.
5(b), 5(c), 5(d) and 5(e)). In the case of silica
mineralization from acidic solutions (below pH 3),
there would be strong interactions between the TMV
surface and anionic silicate species formed by
hydrolysis of tetraethoxysilane (TEOS), because the
protein surface is positively charged below the
isoelectric point, and there are also a significant
number of arginine and lysine groups. In
comparison, preferential deposition of CdS, PbS, and
Fe oxides on the TMV external surface can be
explained by specific metal-ion binding at the
numerous glutamate and aspartate surface groups.
These observations and discussions clearly reveal
that proper amino acid residues on the surface of
some viruses could induce deposition of various
minerals directly in the appropriate conditions, in
this way some characteristic viruses could develop
mineral shell when they are in suitable
mineralization environments.
Unfortunately, most of viruses cannot possess
strong negative charged surfaces or high density of
mineralization-relevant amino acid residues, thus
hardly induce the mineral shell formation
spontaneously. Therefore a chemical or biological
modification is additionally required to enhance
their biomineralization ability. We expect to endow
viruses with the capability of mineralization through
introducing some nucleation-relative functional
groups. LbL is a good way to introduce typical
chemical modification for the virus system. PSS, a
negative charged polyelectrolyte rich in sulfate for
example, may serve as potential nucleation groups
for calcium mineralization [54]. This molecule can be
introduced onto virus with the help of a polycation,
poly(allylamine hydrochloride) (PAH) (Fig. 6(a)).
Initially, the strongly charged cationic PAH can
assemble around the relative weakly negative
viruses by electrostatic interaction. After adsorption,
the unbound PAH molecules are removed by
centrifugation against a regenerated cellulose
ultrafiltration membrane. During the centrifugation
process, the adsorbed PAH roll up around the
viruses and the surface potential (ζ) of YF-17D/PAH
hybrid is around +61 mV. Thisζvalue returns to
-14.7 mV following the subsequent adsorption of a
negatively charged PSS [65]. Thus this two-step
operation offers a convenient method for the
preparation of sulfate-modified viruses, which
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provides a possibility to produce calcium shell onto
the weakly negative viruses.
Virus surface-engineering base on chemical
modification does provide virus with new surface,
but their efficiency is relatively low as it can only
provide a throwaway modification. It means that
progeny of the modified virus may lose the coating
materials and need a new modification again. In
order to develop inheritable modification, gene
engineering is thereby introduced. With the
advances of molecular biology techniques, genetic
engineering has emerged as a powerful tool to
manipulation organisms at genetic level, which
affects various protein expressions [66]. In this way,
biomineralization-relative polypeptides can be
genetically incorporated onto the virus surface as
nucleators [67].
(a)
(b)
(c)
Figure 6 Chemical and biological modification for viruses for
inducing mineralization. (a) Scheme for preparing of
virus/PAH-PSS hybrid complex. Reproduced by permission of
Wiley-VCH from Ref. [65] (b) EV71 genome and the insertion
site of the β-(BC)-loop of viral protein 1 (VP1). (c) Homology
modeling of the mutant viral protein. EV71 capsid proteins VP1,
VP2, VP3 are shown in cyan, yellow, and orange, respectively.
The inserted peptides are marked with blue, which may induce in
situ biomineralization to form a CaP mineral exterior (gray) for
the vaccine. Reproduced by permission of National Academy of
Science from Ref. [67]
Among various nucleating peptides, two types
of representative nucleatiors, phosphate chelating
agents (N6p) or calcium chelating agents (NWp and
W6p) have been selected. N6p is a reported
CaP-binding peptide identified through phage
display [68] which has emerged as a powerful
method to identify peptide motifs [69, 70] when they
possess selective and enhanced binding affinity to
both organic [71-73] and inorganic substrates [74-77].
In addition NWp and W6p are derived from the
fragment of salivary statherin, a CaP high-affinity
protein [78, 79], and they all have showed large
potential in biomimetic mineralization. The coding
nucleotides of the nucleating peptides are
successfully cloned into the infectious full-length
cDNA of the attenuated human enterovirus type 71
(EV71) strain A12 (Fig. 6(b)) using standard DNA
recombination technology [80]. Structural modeling
shows that the insert peptides can be uniformly
displayed on the surface of EV71 without affecting
the original structure of EV71 virion (Fig. 6(c)). It
should be noted that not all the nucleating peptides
show the same biomineralization efficacy. There is no
significant difference on shellization efficacy
between EV71 and EV71-N6, meaning that the
integrated N6p peptide fails to induce virus
biomineralization.
However,
the
engineered
EV71-NW and EV71-W6 viruses demonstrate the
enhanced biomineralization capacity, especially for
latter, over 90% of infectious virions can form
mineral shells spontaneously under physiological
conditions. More importantly, it should be
emphasized that the serial passage of modified virus,
EV71-W6, in cells does not cause any loss of the W6p
coding sequence. The coding sequence of W6p is still
genetically stable after passaging 10 times and the
progeny EV71-W6 virus is still efficient on
self-biomineralization
[67].
Thus,
selfbiomineralization becomes an inheritable trait of
EV71-W6, and future generations retain the
inheritable calcium phosphate nanoshell even under
physiological conditions, which is attributed to the
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combination
of
biomineralization.
genetic
engineering
and
(a)
(c)
(b)
(d)
(e)
(f)
Figure 7 (a) Schematic diagram of the virus-enabled synthesis
and of Co3O4 nanowires. (b) TEM image of virus-templated
Co3O4. (c) High-resolution TEM image of a Co3O4 nanowire.
Electron diffraction pattern (Inset, upper right) confirmed that the
crystal structure was Co3O4. Magnified image (inset, lower right)
shows the lattice fringe of Co3O4 nanowire along the major coat
p8 proteins of a virus. The measured lattice spacing corresponds
to the (311) and (400) planes of Co3O4. (d) Visualizations of the
genetically engineered M13 bacteriophage viruses. P8 proteins
containing a gold-biding motif (yellow) were doped by the
phagemid method in E4 clones, which can grow Co3O4. (e) TEM
images of the assembled gold nanoparticles on the virus Control
experiments showed that gold nanoparticles were bound by the
gold specific peptides. (f) TEM image of hybrid nanowires of Au
nanoparticles/ Co3O4. Reproduced by permission of Science
Publishing Group from Ref. [81]
The
genetic
engineering
biomineralization for viruses is a quite precise
process. Different specific metal-binding peptides
could be incorporated into the filament coat of M13
virus (A filamentous bacteriophage) to induce
distinct mineral disposition [81]. Belcher etc
engineered the virus to satisfy the purpose of induce
Co3O4 disposition on the virus surface to act as an
electrode materials. Tetraglutamate (EEEE-) was
fused to the N terminus of each copy of the major
coat proteins (P8), which helically wrap around the
single DNA of M13 virus, with 100% expression. This
clone named E4, might serve as a general template
for growing nanowires through the interaction of the
glutamate with various metal ions (Fig. 7(a)). After
incubation of the E4 virus templates in aqueous
cobalt chloride solution (1 mM) for 30 min at room
temperature to promote cobalt ion binding,
reduction with NaBH4 and spontaneous oxidation in
water, monodisperse crystalline Co3O4 nanowires
were produced. Figs 7(b) and (c) show TEM images
of the virus-templated Co3O4 nanocrystals, where
Co3O4 nanocrystals of around 2 to 3 nm in diameter
were uniformly mineralized along the length of the
virus. The high-resolution TEM electron diffraction
pattern and lattice spacing together with x-ray
diffraction, confirm that the crystal structure is
Co3O4.
Furthermore,
in order to improve the
electrochemical performance of the nanowires, they
designed a bifunctional virus template that
simultaneously expressed two different peptide
motifs. To accomplish the purpose, they isolated a
gold-binding peptide motif (LKAHLPPSRLPS) by
screening against a gold substrate with a phage
display library [69, 70]. After phagemid constructs
[82] were inserted into host bacterial cells encoding
the gold-binding peptide motif, the infarction of the
plasmid-incorporating host cells with the E4 virus
resulted that E4 p8 proteins also displayed the
gold-specific peptide (named AuE4). Therefore, two
types of p8 proteins were produced: intact p8
proteins of E4 viruses and engineered p8 proteins
containing the gold-binding peptide motif, randomly
packaged onto the virus progeny (Fig. 7(d)).
Incubation of the amplified AuE4 clones with a 5-nm
controlled
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colloid suspension resulted in 1D arrays of Au
nanoparticles bound to the gold-binding peptides
distributed among p8 proteins (Fig. 7(e)). In this
clone, Co3O4 was still nucleated and grown via the
tetraglutamate functionality, resulting in hybrid nano
structures of 5-nm Au nanoparticles spatially
interspersed within the Co3O4 wires (Fig. 7(f)).
Obviously, precise gene manipulation could
specifically control different mineral disposition by
affecting specific surface protein expression,
indicating that gene engineering may precisely
control biomineralization in an originally genetic
level.
Many examples have demonstrated that, with
the help of chemical or biological modification, and
rational utilization of biomineralization-relevant
molecules, non-mineralized organisms can overcome
the difficulties such as low surface potential or lack
of mineral-relative functional groups, to induce
templated mineralization. This biomimetic strategy
can help living organisms to protect themselves or
endow them with new properties. In such a way,
amount interesting biological phenomenon will be
observed from new perspectives, and applications
for some natural livings may be significantly
improved.
cannot play their roles well, as the intracellular
concentration of mineralization-relevant ions is
relative low due to the semi-permeability of cell
membrane [85], leading to low nucleation driving
forces [86]. Artificially breaking the ion balance
seems to be a possible mean to activate the
mineralization-relevant macromolecules and switch
on the intracellular mineralization.
Figure 8 Formation mechanism of the functionalized cell with
endogenous
production
of CaCO3
nanoparticle
scaffold.
Reproduced by permission of Wiley-VCH from Ref. [87]
2.2. Internal mineralization
Highly efficient natural biosystems, such as bacteria
and fungi have potential for the fabrication of
intracellular minerals [83, 84], which may serve as
kinds of special ‘’inorganic organelles’’ to help the
organisms response correctly to the external
stimulation. For example, magnetic bacteria have an
ability to synthesize an internal “compass needle”
that consists of a chain of discrete magnetite (Fe3O4 or
Fe3S4) [6, 7]. Together the crystals align the organism
along the lines of force of the geomagnetic field.
These intracellular structured minerals can efficiently
functionalize the organisms by means of their unique
physicochemical characteristics. In order to form the
nano biominerals, intracellular macromolecules like
some reductive proteins play a decisive role on
nucleation and crystal growth, as well as the
orientation [6, 44]. However, many of the molecules
S.cerevisiae cells hardly produce any intracellular
calcium nanomaterials as they always keep the
concentration balance of calcium ions inside the cells.
An excess introduction of free calcium ions in culture
environment can break the balance and lead to
intracellular Ca2CO3 mineral scaffold construction.
Despite some complex factors that can affect the
mineralized process, a simplified model can be used
to explain the possible generation mechanism
according to the basic mineralization principles (Fig
8). Firstly, S.cerevisiae is reactivated in maltose
solution and carbon dioxide is produced by
respiration. Secondly, addition of a saturation
solution of Ca(OH)2 leads to a large amount of Ca2+
and OH- ions entering the yeast cell due to the
concentration gradient. In the basic environment, the
carbon dioxide inside the yeast cells can form CO32-
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ions. Meanwhile, Ca2+ ions interact with the
biomolecules derived from the cell such as acidic
proteins and polysaccharides, which contain the
“nucleating sites”. Finally, the CaCO3 nanocrysals are
crystallized and they are stabilized by the
biomolecules.
Interestingly,
these
inside
nanominerals exhibit nearly no effects on the cells
metabolic status but act as new “inorganic
organelles” to alter the membrane permeability to
other substances [87]. In this way scientists endow
the cells with the new capability of drug loading and
heavy metal bio-sorption, leading them as novel
biological species that can be potentially applied in
the fields of nano medicine and environmental
science.
In addition to the calcium minerals, analogous
strategy can be introduced to artificially construct
some intracellular metallic mineral nanoparticles,
which may exhibit more diverse functions. It has
been known for a long time that certain
microorganisms have the capacity to reduce metallic
ions into elemental metals. Nanoparticles of various
shapes, sizes and compositions can be synthesized
using different microorganisms (bacteria, fungus,
yeast, actinomycetes) [84, 88, 89]. Although the exact
mechanism of the reduction process has not been
elucidated quite clearly, this reducing capacity has
been largely observed in various systems even
including human cell lines. Those intracellularly
produced metallic nanoparticles exhibit super
biological stability and can not only provide detail
information about the intracellular metabolism
process, but also functionalize the cells with novel
properties. For example, intracellularly grown Au
nanoparticles serve as potential surface-enhanced
Raman scattering (SERS) substrates for confocal
Raman spectrometry [90, 91], which help a single cell
to present characteristic SERS spectra. Intracellularly
produced silver nanoclusters create fluorescent cells
due to their stable intracellular states and unique
optical property [92]. Different from the other
synthetic materials, the intracellularly generated
nanoparticles do no harm to the livings, indicating
super biocompatibility. Thus the introduction of
intracellular metallic minerals provides organisms
with new functions and preserves their biological
property, which is another pathway to modify living
organisms.
(a)
(b)
(d)
(c)
(e)
Figure 9 (a) Schematic illustration of the synthetic approach for
mineralization within the virus particle. Step Ⅰ involves the
removal of viral RNA and purification of the empty virus particle
by ultracentrifugation of sucrose gradients. StepⅡinvolves the
selective mineralization of an inorganic paraungstate species
within the confines of the virus particle. Cryo electron
microscopy and image reconstruction of the cowpea chlorotic
mottle virus (CCMV) (b) in an unswollen condition induced by
low pH; (c) in a swollen condition induced by high pH. TEM
images of paratungstate-mineralized viruses show discrete
electron dense cores in (d) an unstained sample and show the
mineral core surrounded by the intact virus protein cage in (e) a
negatively stained sample. Reproduced by permission of Nature
Publishing Group from Ref. [93]
Besides the intracellular mineralization, virus
internal mineralization was another useful method to
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construct unique mineral structures and materials.
Douglas etc used well defined cowpea chlorotic
mottle virus (CCMV) as a model system for
reversibly gated entrapment of inorganic minerals
[93]. Purified viral coat protein subunits can be easily
assembled in vitro into empty virion particles [94].
The dimensions of the virion cavity define the upper
limit for crystal growth of the entrapped mineral (Fig.
9(a)). In addition, each coat protein subunit presents
at least 9 basic residues (arginine and lysine) to the
interior of the cavity [95]. This crates a positively
charged interior cavity surface, which provides an
interface for inorganic crystal nucleation and growth.
However, inorganic precursor ions could not
freely enter into the interior cavity as there is no
proper channel or pores on the outer surface when
the virion particles are in unswelling conditions.
Fortunately CCMV undergoes a reversible
pH-dependent swelling which results in a 10%
increase in virus dimension and formation of 60
separate pores (20 Å ) [96]. Under swollen conditions
(pH ＞ 6.5) these opening allow free molecular
exchange between the virus cavity and the bulk
medium (Fig. 9(b)). In contrast, in the nonswollen
form (pH ＜ 6.5) there is no apparent exchange of
large molecules between the cavity and bulk
medium (Fig. 9(c)). This dynamic structural
transition [96, 97] allows the virus particle to
selectively entrap and release materials from within
the central cavity.
As the oligomerization of aqueous molecular
tungstate (WO42-) species to form paratungstate
(H2W12O4210-) polyanions is induced by lowering the
pH, the pH-dependent gating mechanism of virion
could be utilized to facilitate the reaction. The empty
virions were incubated with the inorganic precursor
ions under conditions (pH ＞ 6.5) where the virus
exists in its open (swollen) form and allows all ions
access to the central cavity. After incubation, the pH
was lowed to a point where the virion undergoes a
structure transition and the pores in the protein shell
closed. The virion gating, coupled to the tungstate
oligomerization, results in the spatially selective
crystallization and entrapment of paratungstate ion
within the virion. TEM observation for the produced
minerals reveals that electron-dense mineral cores
were commensurate in size and shape with the
internal diameter of the virus particle (average 150 Å )
(Fig. 8(d)). Negative stain of these samples shows the
presence of the intact capsid protein shell
surrounding these cores. This result indicates the
spatial selectively of the mineralization inside the
virus cavity, showing a special mineralization way
for virus, forming an exquisite organic-inorganic
complex structure, which largely differed from
conventional viruses or nanocrystals.
It can be seen that an artificially introduction of
mineralization-relevant ions into intracellular
environment (rich in biomacromolecules) and
control the permeability of the external structure of
organisms can induce and control mineralization,
which is an alternative way to produce
nanomaterials as well as a powerful tool for
biological modification. Unlike the exogenously
introduced nanomaterials, these in vivo produced
ones within cells and viruses are featured by their
excellent biocompatibility and functions, exhibiting
great potentials in nano science and technology.
3. Applications
mineralization
of
nano
biomimetic
3.1. Storage
Most of the living organisms and biological
molecules are sensitive to heat as their structure
cannot preserve stability at high temperature
conditions. Thus many biological agents, such as
protein drugs, blood, vaccination and so on, are
stored under refrigeration conditions (cold chain)
[98-100]. Keeping them at low temperatures is
difficult and expensive. For example, maintaining
the cold chain accounts for 80 % of the financial cost
of vaccination programs [101, 102], while
cryopreservation will induce permanent damage to
the stored cells, such as physical cell rupture caused
by the detrimental effects of cellular volumetric
fluctuations and intracellular ice crystal formation[10,
103]. However, nano shellization can provide
alternative
strategy
to
improve
biological
thermostability.
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According to our attempts, nanoshell does show
excellent stabilizing effect on different biological
systems at relatively high temperatures [49]. One
example is the silica-coated yeast. When uncoated
yeast cells are exposed at 50C for 10 min, the
excessive evaporation inside cells enhances osmotic
stress and results in an outflow of water, which
directly cause cell deformation, especially the wall
defect formation (Figs. 10(a) and 10(b)). Theoretically
and experimentally, silica coating increases the water
absorption/retention capability of cells through
hydrogen bonds formed between water and
hydroxyl groups on silica surfaces. A uniform silica
nanoshell can suppress overall water loss, allowing
the cell to be maintained in a fluid, water-rich
microenvironment. Silica nanoshell serves as an
extracellular inorganic exoskeleton that provides
structural rigidity, thus maintaining cell wall
integrity and even ensuring the preservation of
cytoplasm and organelles exposed to thermal stress
(Figs. 10(c) and 10(d)).
(a)
(c)
is another useful system. Currently, the poor efficacy
and instability of vaccine products lead to
incomplete immunization and rapid loss of potency
during storage and delivery [104], which severely
limit the coverage of vaccination programs.
Interestingly, biomineralization modification can
improve the thermal stability of the vaccine
significantly [67]. When biomineralized virus
(EV71-W6-CaP),
genetic
engineering
virus
(EV71-W6), and parental EV71 are incubated at room
temperature (26C), physiological temperature (37C)
and tropical zone temperature (42C). Their
infectivities are titrated by plaque assays and the
data are represented in a logarithmic scale as a
function of the storage time.
(a)
(d)
(b)
(e)
(c)
(f)
(b)
(d)
Figure 10 Effect of heat exposure on structures of yeast cells.
Uncoated cells ((a) and (b)) exhibit severe cell wall damage,
whereas the organelles and walls of silica-coated cells ((c) and
(d)) remain almost unchanged. Shown by the arrows that the
silica shell can prevent hemorrhaging of the cell (leaking of
cytosol) better compared with the uncoated cells. Reproduced by
permission of Wiley-VCH from Ref. [49]
Figure 11 In vitro and in vivo tests of virus thermostability.
Thermal-inactivation kinetics are determined at 26 ℃ (a), 37℃
(b), 42 ℃ (c). The remaining percentage of infectivity is
represented in a logarithmic scale as a function of incubation
time. In animal tests, EV71-specific lgG titers (d) and
neutralizing antibody responses by EV71, EV71-W6, and
EV71-W6-CaP after 5 days of storage at 37 ℃ (e). (f)
Frequencies of EV71-specific INF-γ-secreting splenocytes in
the immunized mice before and after storage (*, p＜0.05; **, p
＜0.01). Reproduced by permission of National Academy of
Science from Ref. [67]
Besides the heat-resistant cells, thermostable virus
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The results show that EV71-W6 and parental EV71
share similar thermal inactivation kinetics at ambient
temperature (Fig. 11(a)), exhibiting 〜 1-log10
plaque-forming unit (PFU; 90%), 2-log10 PFU (99%),
and 4-log10 PFU (99.99%) losses after 3, 6, and 14 days
of storage, respectively. According to indices of the
World Health Organization requirement for vaccine
efficacy, no more than 1-log10 reduction of the initial
titer is permitted [105]. Thus the room temperature
storage for EV71 or EV71-W6 should be less than 3
days. It follows that genetic engineering alone does
not alter the viral thermal stability. After
biomineralization modification, EV71-W6-CaP shows
an obviously slower inactivation rate and its storage
period prolongs to more than 9 days at room
temperature. Similarly, when the vaccine storage
period is estimated at high temperature ＞30C (Fig.
11(b)) or even ＞ 40C (Fig. 11(c)), the CaP
mineralization can still reduce inactivation kinetics
for the engineered vaccine EV71-W6. The
immunogenicity of heat-treated vaccines is verified
by using mice. After incubation at 37C for 5 days,
the EV71-W6-CaP induces a similar amount of
EV71-specific IgG titers and neutralization
antibodies in comparison with the fresh one, where a
significant decreasing in the level of IgG titers and
neutralization antibody is observed for the native or
only genetic engineered viruses (Figs. 11(d) and
11(e)). Additionally, the stored EV71-W6-CaP can
induce high frequencies of EV71-specific IFN- γ
-secreting cells (representing T-cell response [106])
with the same level of fresh ones, while the same
storage results in a significant decreasing in the
frequencies of these cells induced by EV71-W6 and
parental EV71 (Fig. 11(f)). Collectively, the
genetically induced biomineral shell significantly
improves the thermostability of the vaccine to the
point that it can be viable for use in a vaccine
program even after a week’s storage at ambient
temperatures without any refrigeration. This new
and important feature indicates that the
biomineralized vaccines can be less dependent upon
cold chain, which may be used to improve the
immunization effect in poor and tropical zones such
as Africa.
The thermostablity improvement by nanoshell can
be understood by the isolation of enclosed vaccine
from destructive external adverse factors by the
biomineralized CaP nano exterior, which protects
viral proteins from thermal inactivation by insulating
the direct interactions between water solutions and
vaccines [107]. Furthermore, an attachment of the
mineral phase can stabilize the protein and subunit
structures on vaccine surfaces by the direct binding
effect between CaP and biomineralization-relative
peptide. In this way, biomineralized vaccines keep
their activities after storage at relatively high
temperature conditions, simplifying the applications.
Furthermore, the nanoshell-based storage strategy
tends to be applied for more biomedical systems, like
some protein drug preservation, which is also
limited by the temperature [108]. Once introducing
appropriate nucleation sites on the protein molecules,
a rational biomineralized protein can be designed to
improve their thermal stability.
3.2. Protection
Living organisms in nature are always in face of
foreign aggression and hostile threaten, which
largely reduce their life activities. For example, a
lytic enzyme mixture can digest the cell wall that
protects S. cereveisiae from rupture by osmotic stress,
resulting in about 80% cells die within only 3 h.
However, most of the encapsulated S.cerevisiae cells
by CaP mineral are still alive in such a hostile
solution (Fig. 12).
Figure 12 Survival of bare and shellized S.cerevisiae cells in the
presence of zymolyase. The two inserts are the corresponding
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optical photos of the cells at the end of the tests. Reproduced by
permission of Wiley-VCH from Ref. [47]
Clearly, the artificial mineral shell shows extra
protection, and the encapsulated cells even resist this
natural toxin [47]. The reason may be that the
containment of a cell within a shell can prevent most
extracellular items from passing through the shell to
contact with inside cells. Thus the nanoshells ensure
the cells’ viability by isolation of the enclosed ones
from destructive external lytic enzyme mixture. It
should be noted that the artificially produced
nanoshells are porous, which permit only molecules
smaller than the shell pore channel dimension to
pass through the coating structure. Thus the
size-selective characteristics can provide the enclosed
cells with sufficient small nutrient transport to
ensure their life activities. Therefore, we suggest that
various protective strategies can be specifically
designed for different aggressions by adjusting the
porous structure.
Figure 13 Parallel development stages of the bare (up) and the
LnPO4 coated (down) zebrafish embryo under UV radiation at
different time. Reproduced by permission of Public Library of
Science from Ref. [52]
The changing environment is another threat to
living organisms, such as strong ultraviolet radiation.
Environmental relevant levels of mid-ultraviolet (UV)
radiation negatively impact various natural
populations of marine organisms, which is attributed
to the suppressed embryonic development by the
increased radiation [109]. Before the natural
evolution to generate UV-protective structure,
artificial UV-blocking functional shells can be
developed by the biomimetic mineralization. Taking
rare earth materials lanthanide phosphate (LnPO4) as
the components of cell coat, the UV penetration can
be greatly reduced. We have demonstrated
experimentally that the Zebrafish embryos enclosed
with UV-absorbable shells can develop normally
even exposed to radiation (Fig. 13). However, all the
native embryos without the nano mineral coating die
under the same condition [52]. At the end of
development, the Larvae can grow property and
break the ultra-thin mineral nanoshells. In this way,
vulnerable lives can be protected effectively by the
shell structures.
Another example is the photoinhibition in
cyanobacteria
system.
Photosynthesis
in
cyanobacteria is an inherently inefficient process in
nature because their photoautotrophic growth is
always limited by numerous environmental stresses
[110, 111]. Excess light can significantly inhibit their
photosynthesis and even lead to photooxidative
destruction of the photosynthetic apparatus [112,
113]. Therefore, alleviate the high light stresses
invariably plays an important role in protecting the
cyanobacteria and improving the photosynthesis.
Bio-inspired by the diatom, a nanostructured silica
shell is introduced to the surface of cyanobacteria
[48]. This biomimetic shell as an absorbing material
can reduce light transmission to alleviate the
photoinhibitory effects. It follows that the
biomineralization treatment keeps the viability of the
enclosed cyanobacteria under high light conditions
and enhances its biomass synthesis.
Apparently, the nanostructured shells provide
more survival opportunities for the biological items.
To some extents, artificial shellization can be
considered as a way for manual selection. After the
external modification, lots of natural organisms are
no longer vulnerable and inefficient, which is of
importance to expand their applications in the
biology, medicine and environment areas.
3.3 "Bio-stealth”
Molecular recognition [114] plays important role in
biological systems and is observed between
receptor-ligand, antigen-antibody, DNA-protein,
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suger-lectin, RNA-ribosome, etc. It can help
biological systems to delivery signals and defend
against exogenous detrimental factors [115, 116]. But
unfortunately, some undesired recognition may limit
efficiently therapeutic measures base on biomaterials.
For example, viral vectors exhibit large potential in
the gene therapy as they can transfer genes into a
wide variety of human cells and featured by high
transfer rates [117, 118]. But during in vivo tests, they
are always recognized as exogenous organisms by
human bodies, followed by neutralization and quick
clearance [119]. This unexpected recognition and
removal restrict the applications of viral
nanoparticles in biomedical fields. The recognition
depends upon the interaction of virus surface and
specific biomolecules in plasma. Thus viral surface
engineering is an idea approach to tailor the viral
surface
to
hidden
recognition
sites
and
biomineralization is a helpful strategy.
protein cannot be determined under the same
condition, indicating that the virus become
biologically stealthy. Meanwhile, their original
infection is independent upon the recognition
elimination. Native Ad5 adopts a coxsackievirus and
adenovirus receptor (CAR) dependent pathway to
enter cells so that this viral nanoparticle fails to
establish infection in CAR deficient cell lines [120].
However, the biomineralized Ad5 can still result in
effective infection in these deficient cells, implying
that an alternative CAR-independent uptake
pathway. Interestingly, the biomineralized Ad5 virus
acts like Trojan horse and the CaP coat can be
degraded spontaneously within lysosomes after the
internalization. The stealthy internalization tends to
avoid neutralization and expand the tropism for the
viral vectors (Fig. 14), which can improve their
biomedical applications especially in gene therapy.
Figure
14 Scheme
of
biological
stealth
though
biomineralization-based virus shell engineering (BVSE).
Reproduced by permission of Wiley-VCH from Ref. [62]
Figure 15 Biomineralization provides a Trojan pathway for
Pt-drug uptake through endocytosis, avoiding inhibited Ctr1
pathway. Reproduced by permission of Royal Society of
Chemistry from Ref. [123]
Biomineralization-based virus shell engineering
can construct a core-shell structure, which shows a
CaP covered surface and a biological core [62]. Due
to the shielding effect by the coating shells, the
enclosed viral particles can prevent the recognition
from specific antibodies under the biological
conditions. Compared to the control experiment that
anti-hexon antibody can easily detect the virus
capsid, the biomineralization shielded virus capsid
The analogous biological hidden technologies can
be further applied in other recognition-relevant
fields, even in some non-living systems. Cisplatin is
one of classic anti-cancer drugs for various
malignancies. However, it encounters the same fate
as many other medicines used in therapy – namely,
drug resistance [121], which is a major complication
in chemotherapy and accounts for the failure in
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curing a majority of cancer patients. Among various
possible factors for resistance, the copper transport
protein (Ctr1) deletion is considered as a vital one,
which reduces the cisplatin intracellular uptake
because the protein is the main carrier to transfer
cisplatin [122]. Facing the problem of that “how to
bypass
the
Ctr1-dependent
pathway”,
biomineralization-based stealth is a suitable choice.
After a substitution of carbonate and phosphate
under physiological conditions, the Pt drugs can
readily be mineralized in calcium-rich environment
to form spherical particles at nano scale [123]. In this
way, the resistant tumor cells adopt a new uptake
pathway, endocytosis, for the biomineralized
particles, without the involvement of Ctr1. More
importantly, this kind of mineral complexes can be
also dissolved with lysosomes after the cellular
internalization. Thereby, the enclosed Pt drugs can be
released from the “stealth” ones and then react with
nucleus to kill the tumor cells (Fig. 15).
Biomineralization modification is a recently
developed method for biological stealth. By means of
shielding the recognition-relative site of biological or
chemical materials, living organisms will recognize
them as endogenic materials rather than some
ectogenic ones. Therefore, the utilization of many
biomedical agents could be largely improved.
intracellular ultra-small nano CaCO3 minerals with
acceptable
loading
efficiency.
And
this
drug-mineral-cell system possesses the potential for
application in the development of efficient drug
carriers due to its ideal biocompatibility. Notably,
only mineral modified S.cerevisiae cells exhibit strong
red autofluorescence of DOX while the normal cells
show nothing under the same drug treatment (Figs.
16(c) and 16(d)). This phenomenon indicates that the
intracellular scaffold induces DOX to enter into cells.
More importantly, this living and non-living
combinative system acts as a pH sensitive carrier
with the spontaneous DOX release at tumor tissues
(pH 6) or lysosomes inside cancer cells (pH 4.5)
conditions [124, 125]. This should be attributed to the
crystals dissolution and organisms’ smart response
[87, 126, 127]. Cytotoxic test has confirmed the
effective drug delivery. The result shows that the
death rate of tumor cells is greatly increased when
treated with the drug-mineral-cell systems compared
to DOX alone, while the modified cells without drug
show almost no cytotoxicity. Therefore a
mineral-cell–based delivery system is developed
with a number of advantages, including increased
bioavailability, improved pharmacokinetics, and
reduced toxicities. It follows a new research field by
combining living and nonliving systems.
(a)
3.4 Delivery
Nano biomimetic mineralization emerges as a
platform to construct various novel mineralized
structures, including nano shells, scaffold, particles
and so on. With excellent biocompatibility, they tend
to be combined with some biomedical molecules to
expand their applications. Intracellular mineral
scaffold inside S.cerevisiae is a novel artificial
Ca-based nano mineral material that has attracted
more and more attentions, which can be clearly
observed after being stained by tetracycline (Figs.
16(a) and 16(b)). Interestingly, without any
separation, these inside nanocrystals, incorporated
with the living organisms, yeast as integration, can
be applied directly in the fields of medicine,
environmental and material science. A clinical-used
antitumor drug, doxorubicin (DOX), is loaded into
(c)
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(b)
(d)
Nano Res.
Figure 16 Biomineralization modified yeast cells exhibit large
potential in the biological delivery. Optical photos of control (a)
and mineralization modified (b) S.ceresvisiae cells stained by
tetracycline. Confocal laser scanning images for the control
S.ceresvisiae cells (c) and mineralization modified cells (d) after
treatment with DOX. Reproduced by permission of Wiley-VCH
from Ref [87]
To endow this kind of living-mineral complex
system with target property, introducing manually
operable materials may be an ideal strategy.
Bio-inspired by the nature that magnetic bacteria
move in certain orientation under external magnetic
field with the help of magnetic particles [6, 7], thus
magnetic Fe3O4 can be used to control the movement
of organisms when incorporated into organisms. It
has been proven that Fe3O4 nanocrystals can be
co-precipitated with calcium phosphate and readily
added into calcium shells [47, 128]. This approach
has been used as an effective method to separate and
concentrate yeast cells (Fig. 17). Since the functional
mineral shells are moved under external magnetic
field control, the magnetic mineralized cells are
considered as available carriers for in vivo target
delivery. Clearly, the intracellular minerals help
living organisms load various biomedical agents
while the surface minerals provide them with target
properties. The combination of intracellular and
surface mineralization seems to be a desired strategy
to improve drug delivery and therapy.
Figure 17 Magnetic mineral shells help yeast cells aggregate
under external magnetic field. Red dots represent the magnetic
particles and blue circles represent the yeast cell. Reproduced by
permission of Wiley-VCH from Ref. [128]
Different from traditional drug delivery system,
the nano mineral-living system possesses both the
properties of inorganic materials and biological
livings. The mineral phases offer the capacities of
drug loading and easy manipulation, while the
living cells provide super biocompatibilities and
smart response. We believe that a combination of the
advantages of living and nonliving properties will be
a promising direction in future development of
biological and medical fields.
4. Perspectives and challenges
Great progresses have been made toward developing
novel nano biomimetic mineralization systems based
on living organisms, including the ones with
artificial nano shells and nano scaffolds. These
functionalized
organisms
exhibit
potential
applications in biomedical fields, such as biological
storage, protection, “stealth” and delivery. However,
there are several challenges that still need to be
addressed.
A major challenge in the future is to ensure the
biological safety [129] of the living-mineral systems.
With the protection of minerals like artificial shells,
enclosed
organisms
always
possess
super
survivability over natural ones. If the artificial
modified organisms, such as biomineralized viruses,
return to nature, they may act like new species that
never emerge in the world. This may break the
natural
balance,
leading
to
unimaginable
consequences. Thus manually controllable shell
removing technology needs to be fully developed to
restore them to the natural form. Besides the
frequently used acid-induced mineral dissolving,
many complex agents such as citrate and ethylene
diamine tetraacetic acid (EDTA), can be introduced.
These molecules are always used to suppress the
precipitation of calcium minerals in the solution by
means of binding to calcium ions [130-132], but their
shell-removing effects for the living organism
systems under precise control have not been well
understood.
Another challenge that should be addressed is to
extend the biomineralization systems. It should be
noted that not all the species are readily to be
modified through nano biomimetic mineralization,
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Research
Nano Res.
which is due to the unfavorable structure and
property. Mammalian cell is a typical example that
hardly induces organized shell mineral structures
without losing any biological activities even after
some modifications; otherwise it should be an ideal
model to develop cell ambition storage strategies due
to protective and isolative effects of artificial shells. A
main reason for the failure of mammalian cell
shellization remains on the lack of cell walls. Without
templates that can induce surface mineralization, the
nano building blocks that constituted the nano shell
are easily internalized into the cells through
endocytosis and then dissolved by lysosomes. The
unexpected uptake and dissolution inhibit the
mineral formation, and even reduce cell viabilities
[133]. Inspired by yeast structure, an extra
introduction of cell-wall-like structure may be a
resolution. Like the natural cell wall, hydrogel and
sol-gel shells have been developed to provide
mechanical
support,
chemical
stability,
immunological protection, and maintenance of cell
viability with sustained cellular functions [134-136].
These “organic shells” seem to be ideal operable
templates for subsequent modification and
biomineralization. In this way, we believe that
challengeable biomineralization for mammalian cells
will be ultimately achieved in the future.
The third challenge lies in the proportional use of
biological property. After nano biomimetic
mineralization
modification,
the
constructed
mineral-living complex systems possess unique
functions provided by inorganic minerals such as
drug loading and easy manipulation. But more
importantly, they are still living lives with various
biological properties. For example, once certain
chemical signals in their environment altered,
functionalized livings may response quickly and
direct their movements according to the alternation.
This characteristic is called chemotaxis [137], which
is important for bacteria to find food (e.g., glucose)
by swimming toward the highest concentration of
food molecules or to flee from poisons (e.g., phenol).
Obviously chemotaxis endows the organisms with
inherent targeting in biomedical environment,
suggesting potential applications in drug targeting
delivery. However reasonable use of chemotaxis is a
complicated project in biomedical fields as clinical
symptoms and syndromes will present amount of
signals that may confuse the organisms. Mineral
shell modification has been proven to provide
selective permeation for various biological molecules
[128], thus it could help the mineralized organisms
receive signals selectively. Thus rational design of
mineralized organisms is an ideal way for effective
improvement of artificially controlled chemotaxis. In
this way, more available mineral-living nano systems
should be developed with precise signal-receiving
selectivity, which may serve as potential candidate
for target therapy. The new system should load
various biomedical molecules, exhibit smart
response to the environment and give intelligent
biological targeting.
The combination of inorganic nano materials with
biological systems is an interesting research topic but
it remains largely unexplored. Actually, nature is the
best inspiration sources for the technical
developments in laboratories. The design of
nanomaterials from either natural constituents or
synthetic materials is of critical importance to
provide living organisms cells with new and unique
properties by the material incorporation. Since
structure determines function, the incorporated
materials can mimic natural features, such as
semi-permeability or can be well custom-designed.
Progress in material-based biological engineering
would find more and more applications throughout
the fields of biotechnology
Nano
biomimetic
mineralization
can
be
understood as a technology to produce various nano
inorganic solids based on living systems, which has
been considered as a good way to provide novel
biocompatible nano materials and reform exist living
organisms with new functions. As an important
branch of bioinorganic fields, it creates novel
living-mineral complex structures, which blur the
boundary between living and non-living systems.
The nano minerals from either natural constituents
or biomimetic synthetic materials can provide
organisms with new and unique property to
improve their applications. It is the main trend for
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Nano Res.
biological development to combining chemical,
material and nano technologies as integration in one
system. In this regard, nano biomimetic
mineralization would explore a promising field in
current scientific studies.
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Acknowledgements
The authors greatly thank Xiaoyu Wang, Ben Wang
and Wei Xiong for providing editable graphic
materials. This study was supported by the
Fundamental Research Funds for the Central
Universities of China and the Natural Science
Foundation of China (91127003).
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