Nano Res

1

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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Nano Research

DOI 10.1007/s12274-014-0509-9

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 modification of living organisms by biomimetic

mineralization

Wei Chen1, Guangchuan Wang2, and Ruikang Tang1,2 ()

Received: day month year

Revised: day month year

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.

ABSTRACT

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.

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

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 (Fe3O4 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 Research

DOI (automatically inserted by the publisher)

Address correspondence to rtang@zju.edu.cn

Review Article

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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].

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].

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

efficiently regulate nucleation and crystallization

[26-31], which dominate the hierarchical structures

and unique properties of biominerals [32, 33].

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].

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 Mineral materials Location Method Reference

Yeast Calcium phosphate External LbLa modification [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]

Japanese encephalitis vaccine (JEV) Calcium phosphate External Direct mineralization [61]

Adenovirus serotype 5 (Ad 5) Calcium phosphate External Direct mineralization [62]

Tobacco mosaic virus (TMV) Silica, PbS and CdS

nanocrystals, Iron Oxide

External Direct mineralization [64]

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

Human lung epithelial cell (A549)

Au Internal Metal reduction [90, 91]

Mouse embryonic fibroblast cell (NIH 3T3) Ag Internal Metal reduction [92]

Cowpea chlorotic mottle virus (CCMV) Paratungstate

(H2W12O4210-)

Internal Spatially constrain [93]

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.

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

(a)

(b)

(c) (d) (e)

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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.

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

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)).

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

(a)

(b)

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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.

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.

(a) (b)

(c) (d)

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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

(a)

(b) (c)

(d) (e)

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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].

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, self-

biomineralization 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

(a)

(b)

(c)

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combination of genetic engineering and

biomineralization.

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 controlled

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

(a)

(b) (c)

(d)

(f) (e)

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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.

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

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]

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.

Figure 9 (a) Schematic illustration of the synthetic approach for

mineralization within the virus particle. StepⅠ 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

(a)

(b) (c)

(d) (e)

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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 of nano biomimetic

mineralization

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 50C 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)).

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]

Besides the heat-resistant cells, thermostable virus

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 (26C), physiological temperature (37C)

and tropical zone temperature (42C). Their

infectivities are titrated by plaque assays and the

data are represented in a logarithmic scale as a

function of the storage time.

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]

(a) (b)

(c) (d)

(a)

(c)

(d)

(b) (e)

(f)

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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 ＞30C (Fig.

11(b)) or even ＞ 40C (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 37C 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.

Figure 14 Scheme of biological stealth though

biomineralization-based virus shell engineering (BVSE).

Reproduced by permission of Wiley-VCH from Ref. [62]

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

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 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]

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.

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

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) (b)

(c) (d)

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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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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.

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). References

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