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Advanced Review
NanobiosystemsMary-Margaret Seale-Goldsmith1 and James F. Leary2∗
‘Nanobiosystems’ is a relatively new term describing objects in the size rangebelow 150 nm and having structures or functions that link to biological functions.Key features are that these nanosized objects typically self-assemble, are notcapable of self-replication, and have functions that take advantage of its size.Nanobiosystems can be made entirely of biological or organic molecules that areorganized into nanoparticles (e.g., liposomes, dendrimers) or be totally inorganic(with the exception of surface coatings used for biocompatibility) nanoparticles(e.g., gold, iron oxide, quantum dot nanocrystals). More complex nanobiosystemsare inorganic/biologic hybrid composites that may include complex multilayeredstructures with targeting molecules (e.g., peptides, antibodies, aptamers), cellentry-promoting molecules (e.g., HIV-tat peptide sequence), drugs (smallmolecules), genes (therapeutic genes, reporter genes), and core nanomaterials(e.g., gold, quantum dot, iron oxide) that give the nanobiosystems sometimesunique detection capabilities by a variety of optical and non-optical modalities(fluorescence, surface plasmon resonance, magnetic resonance imaging) . 2009John Wiley & Sons, Inc. WIREs Nanomed Nanobiotechnol 2009 1 553–567
Nanobiosystems are non-existing in nature, buttheir properties have great potential for new
advances in research and medicine. Their uniquenanomaterial structures can have toxicities andin vivo distribution properties that are somewhatunpredictable. This will necessitate new research in thefield of nanotoxicity, where size as well as compositioncan significantly alter the toxicity properties of thesesystems. Since these nanobiosystems are small enoughto cross cell membranes, and even the blood–brainbarrier, new methodologies need to be developed tosafely contain them.
Nanobiosystems are devices designed and con-structed to interact with biological systems atthe nanolevel. Numerous biological and biomedi-cal phenomena occur at the nanometer level, andthe current research focus of many fields is nan-otechnology. Nanobiosystems provide the ability toprobe the sub-optical, molecular level and are becom-ing powerful tools to study biomolecular processes.Moreover, many uses and specific applications for
∗Correspondence to: [email protected] School of Biomedical Engineering, Purdue University,W. Lafayette, IN 47907, USA2Department of Basic Medical Sciences, School of VeterinaryMedicine and Weldon School of Biomedical Engineering, BindleyBioscience Center and Birck Nanotechnology Center, PurdueUniversity, W. Lafayette, IN 47907, USA
DOI: 10.1002/wnan.049
nanobiosystems have been elucidated. For example,biosensors that can detect and capture molecules orpathogens beyond the limits of detection for currentdevices are being developed. Nanobiosystems alsohold great promise for the field of nanomedicine,where nanostructures are designed to diagnose andprovide therapy at the single-cell level.
Numerous examples of nanobiosystems andnanostructure components exist in the literatureto date. The development of liposomes and den-drimeric polymers has greatly influenced the fieldof nanobiosystems and has provided materials andconcepts for the development of nanobiosystems.Core nanoparticles have been a common platform forthe nanobiosystem construction, notably in exampleswhere the core nanoparticle displays properties thataid in detection or manipulation of the nanobiosystem.Advanced construction of nanomaterials has beena significant more recent research area, and scien-tists are constructing reproducible, highly controllednanostructures with elaborate geometries and func-tions. Combining these advanced nanostructures andnanomaterial building blocks, construction and appli-cation of multifunctional nanobiosystems are beingperformed today. Multifunctional nanobiosystemswill perform a variety of biological functions at themolecular level, and these systems will interact withbiological systems through natural pathways to probesub-optical phenomena and direct cellular processes.
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With the development of nanobiosystems, manychallenges and questions have arisen regardingcharacterization and assessment of their function.For example, the detection and visualization ofnanobiosystems remain a current challenge today.However, many tools are being utilized for thestudy of nanobiosystems. Nanobiosystems madeof metallic nanomaterials can often be imagedby electron microscopy and light-scattering effects.Also, surface science techniques, such as X-rayphotoelectron spectroscopy (XPS), have providedinsightful data regarding the chemical compositionof nanobiosystems. Nanobiosystem detection in vitroand in vivo has been greatly aided by the incorporationof fluorescent or radiolabels as well as sensitiveimaging techniques, such as magnetic resonanceimaging (MRI).
Another focus area in the study of nanobiosys-tems is the interaction with biological systems. Atthe cellular level, nanobiosystems and their materialsmay exhibit unique responses because of size, shape,and metabolism of materials. These phenomena cantranslate into perturbation of cellular function andundesirable effects. However, much research is beingperformed to understand what materials and dosesare biocompatible and well-tolerated by cells. Forin vivo applications, large-scale toxicity and biodis-tribution effects are major areas of development.Currently, researchers are focusing on the selectionof non-immunogenic materials and understandingdimensions needed for nanostructures in order toguide nanobiosystems to the desired site in a complexin vivo environment.
Overall, nanobiosystems have great potentialto impact numerous fields in science and medicine.Nanobiosystems are being developed for a varietyof applications, and their successful application willlikely exceed the efficiency and sensitivity of manycurrent processes and techniques. Numerous exam-ples of nanobiosystems and a general understandingof their use will be presented in this review ofnanobiosystems research.
TYPES OF NANOBIOSYSTEMS
Building BlocksNanobiosystems today are the result of buildingblocks that have been developed in the field ofnanotechnology and biomaterials. Although numer-ous examples exist, some of the most notable andextensively studied nanostructures are liposomesand dendrimeric polymers. While liposomes werediscovered through the study of cell membrane phos-pholipids in the early 1960s,1 dendrimeric polymers
development became prominent in the 1990s.2 Also,metallic nanoparticles have become well-known fortheir ease of synthesis and numerous advantageousproperties at the nanolevel. These examples areamong the first structures developed that crossed thethreshold from micro- to nanostructures for uniquebiological applications.
Development of NanostructuresLiposomes are vesicles comprised of amphiphilicmolecules, such as phospholipids that form bilayersand enclose upon themselves to form sphere-shapedparticles. Although there is wide-range of sizedistributions, liposomes typically have diametersnearly 100–200 nm. One of the key advantages inliposome technology is that liposomes self-assemble,meaning that under controlled conditions and withthe necessary materials, these vesicles assemble andencapsulate molecules for cellular delivery. Much ofthe liposome research has focused on development ofliposomes with synthetic polyethylene glycol (PEG)phospholipids for the encapsulation of hydrophobicdrugs. Some studies have investigated the effects oftumor targeting and gene delivery with antibody- orpeptide-conjugated liposomes.3 Currently, doxil isan Food and Drug Administration(FDA)-approvedliposomal form of the anti-cancer drug doxorubicinwhere doxorubicin is encapsulated within PEG-based liposomes.4 While liposomes may be an idealplatform for hydrophobic drug delivery, some of themajor drawbacks to liposomes as a platform fornanobiosystems are their short half-life in blood anddifficulty monitoring in vivo. Further opportunitiesfor liposomal nanobiosystems are likely to arise inthe future as a result of the numerous hydrophobicpharmaceutical agents used clinically; however, thelarge size of liposomes and shorter circulation times invivo are being challenged by smaller, more biologicallystable nanoparticle devices.
Another important building block in the devel-opment of nanobiosystems is dendrimeric polymers.Highly branched polymers, such as polyamidoamine(PAMAM), have been studied extensively for theirbiocompatible and non-immunogenic properties.Specifically, these polymers have been investigatedfor their ability to transfect cells with genetic materialbecause of their ability to cross cell membranes withminimal perturbation.5,6 More recently, investigatorshave focused on the nanomaterial properties of den-drimeric polymers for radiolabeled tumor detectionand antibody-mediated tumor targeting.7,8 One of thekey strengths with dendrimeric polymers is the verylarge surface area and variety of biomolecules that canbe attached to the dendrimer. In addition, dendrimer
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materials have been well-characterized and appear tobe generally biocompatible and non-immunogenic.For example, PAMAM dendrimers were conjugatedto an fluorescein isothiocyanate(FITC) fluorescenttag and a prostate-specific monoclonal antibody forcellular targeting in vitro.8 Similar to liposomes,polymeric dendrimers will face similar challenges invivo because of break down of the polymers by bloodenzymes. On the other hand, it is highly likely thatdendrimeric polymers will be a common theme inthe development of nanobiosystems because of theirmultifaceted ability to incorporate drugs, targetingligands, genetic materials, and labeling compoundsall within one structure.9,10 Other opportunities fordendrimeric polymers exist as coating materials formetallic core nanoparticles, especially as a means toenhance water solubility and increase surface areafor functionalization. However, their use in othernanobiosystems will depend on the size constraintsof the nanostructure because dendrimeric polymersare likely to increase the size of a nanostructure byapproximately 10–100 nm in diameter, depending onthe number of dendrimeric layers used.
Solid Core NanoparticlesAnother approach common among nanobiosystemdevelopment is construction around a core nanoparti-cle, where the core material displays unique propertiesfor stability and/or detection of the system. Corenanoparticles typically range in diameter from sev-eral nanometers up to 100 nm, and nanoparticles ofmany different shapes and composite formulationshave been developed. In the design of the nanobiosys-tem, biomolecules are incorporated either within thecore nanoparticle or onto the nanoparticle via surfacecoatings.
One of the most extensively studied corenanoparticle material is metals. Gold, silver, iron,cobalt, nickel, platinum, and various metal compos-ite nanoparticles have been developed and studiedextensively in the development of nanobiosystems.Some of the most notable advancements have beenmade with gold, iron oxide, and composite metalnanoparticles. Gold nanoparticles have been utilizedextensively because of their ease of detection byelectron microscopy, plasmon resonance properties,and photothermal effects. In addition, conjugationof biomolecules to gold surfaces is readily per-formed with thiol-containing molecules in aqueousbuffers.11 For example, Thaxton et al.12 reported aDNA barcode detection method that relies on DNAprobes attached to gold nanoparticles. Target DNA isdetected by the absorbance shift of the nanoparticlesupon binding target DNA or the magnetic separation
by hybridizing the bound target DNA with magneticmicroparticles. In comparison, iron oxide nanoparti-cles provide similar density properties for detectionby electron microscopy, but their magnetic propertiesfor magnetic resonance contrast and cell separationare unique as compared to other nanoparticle mate-rials. Researchers to date have focused on magneticnanoparticles as a platform for drug/gene delivery,13,14
rare cell detection and manipulation,15 and MRIenhancement in vivo.16,17 Metallic nanoparticles pro-vide stability and enhanced detection capabilities ofthe nanobiosystem; however, some metals are toxic inelemental form. Nanotoxicology is an embryonic fieldand the dynamics and toxicity of these nanomaterialsin vivo are not well-understood at this time. Toxic-ity of nanomaterials may well vary with nanoparticlesize, and toxicity is difficult to evaluate with the mask-ing presence of hydrophilic biocoatings used to coatthese generally hydrophobic nanostructures. Toxicitymay increase if these biocoatings are removed in vivoor inside cells. Metallic nanoparticle toxicity remainsa largely unresolved issue in the field of nanoparti-cle research, and other biodegradable nanobiosystemsthreaten the development of metallic nanoparticlesas nanobiosystems, especially in vivo. Opportuni-ties exist for FDA-approved formulations of metallicnanoparticles, such as dextran-iron oxide nanoparti-cles, to be developed as a platform for nanobiosystems.
Another commonly studied core nanoparticleis the ‘quantum dot’, named for its unique opticalproperties and high quantum yield. Quantum dotsare typically comprised of semiconductor materials,including cadmium–selenium, cadmium–tellurium, aswell as indium–phosphorous.18 The exceptionallybright and photostable properties, as well as theirdifferent emission spectra for quantum dots of thesame nanomaterials based on their size, of quantumdots are the reasons for their use as fluorescentnanobiosystems. Quantum dot research has revolvedaround sensitive optical molecular imaging andmonitoring, and quantum dot technology has greatpotential to improve the sensitivity of diagnosticsand molecular detection assays.19 The main drawbackto quantum dot based nanobiosystems is their toxicelemental materials, namely cadmium. Quantum dotshave the greatest potential for in vitro and smallanimal nanobiosystem development because of theirfluorescent properties. But the ability to detect thesefluorescent nanoparticles is limited to typically afew millimeters of depth through skin and tissuemaking their use limited to near the surface oftissue or if detected by endoscopic analysis. Overall,core nanoparticles are an attractive material for theconstruction of nanobiosystems because nanoparticle
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materials provide enhanced sensitivity for detectionand interaction with the biological environment bymeans of electron density, magnetic properties, lightscattering, or fluorescence.
Advanced ConstructionIn the past decade, many researchers have focusedon synthesizing reproducible nanostructures withcontrollable sizes and geometries. A unique conceptin the development of these nanobiosystems isthat size and geometry of the nanostructure maydictate very critical biological and material effects.Therefore, many research groups have investigatednanostructures with alternative geometric features.
Nanostructure GeometryOne of the most studied structures with a uniquegeometric configuration is carbon nanotubes. Carbonnanotubes are lattices of carbon atoms that wrap intoa tube with dimensions of 10–100 nm in diameterand up to several hundred microns in length,20 andthese lattices are typically single- or double-wallednanotubes. Although carbon nanotube research hasbeen predominantly in the field of materials scienceand chemistry, recent advances for biological applica-tions of carbon nanotubes have been made.21,22 Forexample, Shim et al.23 reported a coating method toimmobilize functional PEG molecules and streptavidinonto the carbon nanotube surfaces for future applica-tions of attaching biomimetic peptides and antibodiesto these functional PEGs. Nanowires have a simi-lar structure to nanotubes with diameters less than100 nm and lengths up to several microns. Nanowireshave been constructed for various applications, suchas electrical biosensors for molecular detection. Forexample, Hahm et al.24 developed a peptide nucleicacid coated Si nanowire for detection of DNA muta-tions as a model for detection of cystic fibrosis.Despite the meticulous construction of nanowires andnanotubes, these nanostructures have as yet few spe-cialized applications as nanobiosystems and are notas versatile as other nanobiosystems.
Gold nanoshells and gold nanorods are nanos-tructures where geometric modifications have beenstudied extensively because of the substantial effectthese modifications have on nanostructure properties.Gold nanoshells synthesized on silica core nanoparti-cles display high optical scattering and tunable absorp-tion of near infrared (NIR) light.25 These propertieshave been used for optical detection of tumor tar-geting and photothermal ablation of cancer cells.26,27
In another variation of gold nanostructure geometry,gold nanorods demonstrate detectable single-particle
plasmon wavelength shifts that have been utilizedto measure target binding to antibody-conjugatednanorods.28 In addition, nanorods hold great poten-tial for nanobiosystem development because someresearch suggests that particles with higher aspectratios will promote reduced phagocytic uptake29
and longer circulation times in vivo.30 Nanorodsand nanoshells, in particular those made with gold,are highly advantageous for the construction ofnanobiosystems because of their biocompatibility andoptical detection properties.
Biologic NanostructuresIn addition to varying size and shape, some researchershave developed advanced nanostructures with biolog-ical materials. Biomolecules, such as peptides, RNA,and DNA, are generally biocompatible and readilyintegrated with other molecules for drug or genedelivery. In one approach, Falciani and colleaguessynthesized branched peptides on a PEG backbone fortumor targeting to increase circulation time.31 Othergroups have designed DNA nanomotors for power-ing nanodevices,32 while RNA hairpin probes havedemonstrated mRNA detection in live cells.33
Nanomotors and nanotubes constructed ofnucleic acid molecules are a predominant area of bio-logic nanostructure research. phi29 packaging RNAhas been used as a nanomotor tool for targeted deliv-ery of siRNA to cancer cells by Guo and colleagues.34
This RNA nanomotor engineered from bacteriophageentry mechanisms is a versatile tool for deliveryof environmentally sensitive genetic material. DNAhas also been used for the construction of biolog-ical nanostructures, such as nanotubes.35 O’Neillet al.36reported synthesis of a more thermally sta-ble DNA nanotube because of T4 ligation. Theserecent advancements in the field of biologic nanos-tructures suggest that this biomolecular engineeringwill promote the use of DNA, RNA, and peptidesfor advanced applications in nanobiosystems. Despitethe unique applications for biologic nanostructures,their complex functions may limit their application asnanobiosystems. For example, protection of nucleicacids is required for in vivo applications in order toevade enzymatic degradation. In addition, fluorescentor other tags must be incorporated with the bio-logic molecule to provide means for detection. On theother hand, biologic nanostructures may present newopportunities if combined with other platforms fornanobiosystems, such as more stable gold nanoparti-cles or quantum dots.
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Nanobiosystems: Current Focus and FutureOutlookFrom the discovery of initial nanostructures toadvanced construction of tunable properties at thenanolevel, nanobiosystems have emerged as nanos-tructures designed to perform systematic functionsat the molecular level. Multilayered nanoparticles areone example of nanobiosystems that are being devel-oped with many different materials to perform a seriesof functions in the biological environment. Moreover,the advancement of nanobiosystems beyond theresearch setting will rely on the integration of thesemulti-functions and the application to the biologicalenvironment.
Many advanced functions are needed for theapplication of autonomous nanobiosystems in abiological environment. One aspect for advancednanobiosystem construction is self-assembly.Although complex molecules and materials willlikely be constructed for the nanobiosystem, naturalincorporation of these molecules will increase theability to produce a functional nanobiosystem.For example, the layer-by-layer method has beenused for self-assembly, where charged moleculeinteractions promote the spontaneous development ofcoatings and thin films on surfaces.37 Layer-by-layerfunctionalization of gold nanoparticles has alsobeen demonstrated as a means to develop a widelyapplicable coating process for gold nanodevices.38
These and other methods of self-assembly are likely tobe common routes in the development of multilayerednanodevices because they mimic and work togetherwith native chemical and biological processes, thus
dictating the function of these nanostructures inbiological environments.
In addition to self-assembly, multifunctionalnanobiosystems are needed for complex rolesin biological applications, such as regenerativenanomedicine at the single-cell level. Some key func-tions that a nanobiosystem must be able to performinclude: targeting, monitoring, external manipulation,error-checking, delivery, and degradation/stealth func-tion. A general schematic of such multifunctionalnanobiosystems as described in several publications39–43 is shown in Figure 1. For example, doxorubicin-loaded thermo-sensitive micelles combined with ultra-sound sonication were able to target and deliver drugto cancer cell xenografts in nude mice, and resultsindicated significant reduction in tumor volumes.44
This study demonstrated passive targeting of thenanobiosystem via leaky tumor vasculature, deliveryof drug to the tumor site via thermal release, andstealth function of the polymers micelle materials toenable sufficient circulation times for tumor uptake.Gross monitoring of micelle accumulation within thetumor site was also performed by ultrasound imag-ing. One specific challenge for passively targetednanobiosystems arises when intracellular activationand cytoplasmic delivery are needed for therapeu-tic efficacy. This is because of the limited amountof drug or therapeutic agent that can be deliverywith a nanobiosystem. Merely delivering nanopar-ticles in the vicinity of tumor cells through leakyvasculature does not guarantee that the nanoparticleswill have the desired therapeutic properties, espe-cially if they are taken up through endocytosis andhave not been designed to tolerate low intracellu-lar pH environments. Therefore, active targeting and
(1) Multilayered nanoparticle
(2) Multilayered nanoparticle targeting to cell membrane receptor and entering cell
(3) Intracellular targeting to specific organelle
(4) Delivery of therapeutic gene
Targeted cellular organelleTargeted cell
Cell membrane
Multilayered nanoparticles for multifunctional nanomedicine Designing ‘‘programmable’’ multifunctional nanomedical systems with feedback control of
gene/drug delivery within single cells
(a) (b)
Cell targeting and entry
Intracellular targeting
Therapeutic genes
Magnetic or Qdot core (for MRI or optical imaging)
Biomolecular sensors (for error-checking and/or gene switch)
Targeting molecules (e.g. an antibody, an DNA, RNA or peptide sequence, a ligand, an aptamer) in proper combinations for more precise nanoparticle delivery
FIGURE 1 | (a) Nanobiosystems undergo a multi-step process that conceptually, and sometimes physically, require a multilayered structure, (b)Multifunctional nanobiosystems can contain specific molecules for targeting, entry, biosensing, and therapy constructed to accomplish a complex setof tasks that through de-layering and chemistry, these nanobiosystems can be considered ‘‘programmable’’ by controlling an ordered sequence ofevents.
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receptor-mediated uptake, where the nanobiosystembinds and activates a cellular receptor for intracellulardelivery, is needed to selectively increase the amountof drug delivered inside of the diseased cell. This mayalso allow for less total drug to be delivered overall,leading to reduced side effects and nonspecific toxicity.
Additional studies are being performed tomaintain monitoring and external manipulation oftargeted cells with nanobiosystems. Bright, photo-stable quantum dots have been coupled with targetingpeptides and siRNA molecules to explore targetedgene knockdown in cancer cells and provide a meansfor visualizing localization of the nanobiosystem. Inone study, enhanced green fluorescent protein (EGFP)siRNA was delivered to EGFP-expressing HeLa cellsby peptide-targeted quantum dots. Interestingly, thisgroup noted that quantum dots were trapped inendosomal vesicles and cationic complex-inducedrelease of the quantum dot particles was needed toachieve EGFP knockdown.45 Nanobiosystems withexternal modulation of therapeutic properties are alsobeing developed for cancer cell removal. El-Sayedand colleagues synthesized antibody-targeted goldnanorods for photothermal removal of cancer cells.For this application, laser light in the visible rangeapplied to the nanorod-targeted tumor site resultedin heating sufficient to kill targeted cancer cells.46
Another way to deliver a therapeutic response onnanobiosystems is to deliver a therapeutic genesequence tethered to a nanoparticle and expressedunder the control of an upstream molecular biosensor
switch. This concept has been tested on in vitro sys-tems modeling the oxidative stress damage that occursin retinopathies47,48 using reporter gene constructsthat express in response to an oxidative stress biosen-sor as shown in Figure 2. These studies demonstrateadditional advantages of targeted nanobiosystems formonitoring of therapeutic delivery (siRNA) and selec-tive ablation of diseased cells. Construction of suchmultifunctional nanobiosystems is a current challengebecause of the difficulty of assessing whether the allof the biomolecules (e.g., siRNA, targeting peptides)remain functional after crosslinking or conjugationto the nanobiosystem. Current characterization tech-niques are rapidly advancing to address the need tostudy smaller structures at a more detailed level, andsome of these techniques are described in Section 2.0.
There are abundant examples and formulationsof nanobiosystems (Table 1). The development ofnanobiosystems today is the result of nanomaterialbuilding blocks and advanced construction of nanos-tructures. Current research is focused on the incorpo-ration of multiple functions within nanobiosystems inorder to apply these systems as biosensors, diagnostic,and therapeutic agents.
HOW DO WE SEE AND MEASURENANOBIOSYSTEMS?
Nanobiosystems are Sub-opticalSince nanobiosystems are by definition very small,typically 150 nm or lower (National Institutes of
Tathered gene expression on magnetic nanoparticles for nanomedicine
(a)
(b)
(d)
(e)
(c)CMV DsRed
EGFP
polyA
polyAARE
MW 1 2
+
+
Lipid Lipid coated nanoparticle clusters
Lipid
DNA
Streptavidin
Dextran
Iron oxide
DNA tethered nanoparticle
Streptavin coated magnetic nanoparticle
Biotin labeled TAPS
FIGURE 2 | (a) Tethered genes onnanoparticles can express genes and highefficiency, (b) Multiple layers can be built oniron oxide nanoparticle cores, (c) Reportergenes (DsRed and eGFP) can have theirexpression driven by a cytomegalovirus(CMV) promoter or can even be controlledby upstream antioxidant response element(ARE) molecular biosensor switches, (d) toproduce eGFP reporter gene products inresponse to oxidative stress under control ofa stress biosensor, or (e) simply expressDsRed reporter gene product freely under aCMV promoter.
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TAB
LE1
Exam
ples
ofN
anob
iosy
stem
san
dth
eir
Des
igne
dA
pplic
atio
ns
Type
ofna
nobi
osys
tem
Dim
ensi
ons
Mat
eria
ls(n
otin
clud
ing
coat
ing)
Appl
icat
ions
Refe
renc
e
Lipo
som
esSp
heric
alw
ithdi
amet
ers:
200
nm49
95nm
Doxi
l50
110
nmm
Ab-D
oxil
50
136,
165,
209,
275,
and
318
nm51
Phos
phat
idyl
chol
ine,
chol
este
rol,
PEG
-pho
spha
tidyl
etha
nola
min
e49
Dist
earo
ylph
osph
atid
ylch
olin
e,ch
oles
tero
l,PE
G-
dist
earo
ylph
osph
atid
ylet
hano
lam
ine
α-t
ocop
hero
l51
¬TAT
-pep
tide
lipos
omal
tran
sfec
tion49
¬In
vest
igat
ion
ofan
tibod
y-ta
rget
eddo
xoru
bin
lipos
omes
50
¬Bi
odis
trib
utio
nof
lipos
omes
with
vary
ing
diam
eter
s51
¬Li
poso
mal
plas
mid
deliv
ery
toin
hibi
tbre
astc
ance
rin
mic
e3
Torc
hilin
49
Luky
anov
50
Awas
thi51
Chen
3
Dend
rimer
icpo
lym
ers
Cylin
dric
al:
3.6
nm(G
3)an
d5.
4nm
(G5)
indi
amet
er9
G2
and
G3
poly
plex
dend
rimer
s20
0–25
0nm
indi
amet
er52
Poly
amid
oam
ine
(PAM
AM)9
Poly
prop
ylen
imin
e52
Also
poly
ethe
rs,p
olye
ster
s,po
ly(e
ther
amid
es),
etc.
53
¬An
tibod
y-ta
rget
edde
ndrim
ers
toca
ncer
cells
8
Synt
hetic
vect
orfo
rgen
ede
liver
y52
Esum
9
Inou
e53
Russ
52
Thom
as8
Met
alna
nopa
rticl
esSp
heric
alw
ithdi
amet
ers:
40nm
46
29nm
14
4–10
nm16
Gol
d,46
silv
er,54
iron
oxid
e14
Com
posi
tem
etal
s:Iro
nco
balt16
¬G
ene
deliv
ery
with
mag
netic
field
assis
tanc
e14
¬Du
alm
agne
ticre
sona
nce
and
near
infra
red
(NIR
)im
agin
gag
ents
16
Seo16
Mor
ishi
ta14
Mor
ones
54
El-S
ayed
46
Qua
ntum
dots
(QDs
)N
ears
pher
ical
with
diam
eter
s:2.
4–4.
5nm
55
10–1
5nm
with
coat
ings
19
Cadm
ium
sele
nium
(CdS
e)55
Core
-she
llCd
Se-z
inc
sulfi
de19
¬In
vest
igat
eth
ecy
toto
xici
tyof
quan
tum
dots
with
diffe
rent
surfa
ceco
atin
gs55
¬Pe
ptid
e-ta
rget
edde
liver
yof
siRN
Aw
ithN
IRQ
Ds45
¬An
tibod
y-ta
rget
edqu
antu
mdo
tsfo
rin
vivo
tum
orta
rget
ing19
Kirc
hner
55
Derfu
s45
Gao
19
Carb
onna
notu
bes
(CN
Ts)
Cylin
dric
al:
1nm
indi
amet
erby
400
nmle
ngth
56
50nm
indi
amet
er20
Unm
odifi
edca
rbon
nano
tube
s¬
Invi
trobi
osen
sorf
orce
llula
rrel
ease
ofni
tric
oxid
e22
¬St
udy
ofin
vitro
toxi
city
from
seve
ralf
orm
sof
func
tiona
lized
CNTs
57
¬PE
Gfu
nctio
naliz
edCN
Tin
vivo
biod
istr
ibut
ion20
Du22
Saye
s56
Liu57
Li20
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Advanced Review www.wiley.com/wires/nanomed
TAB
LE1
cont
inue
d
Type
ofna
nobi
osys
tem
Dim
ensi
ons
Mat
eria
ls(n
otin
clud
ing
coat
ing)
Appl
icat
ions
Refe
renc
e
Nan
orod
sRo
dsh
aped
:1.
6µm
×17
0nm
indi
amet
er58
65nm
leng
thby
11nm
wid
th59
Aspe
ctra
tios
2.8,
4.528
Sepa
rate
Auan
dN
isec
tions
onAl
2O
3na
noro
d58
Gol
d28,5
9
¬In
vitro
asse
ssm
ento
fT-c
ell
resp
onse
forn
anor
odva
ccin
eap
plic
atio
n58
¬PE
G-m
odifi
edna
noro
dsfo
rto
impr
ove
invi
voci
rcul
atio
ntim
e59
¬M
ultip
lex
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Health (NIH) has defined nanomedicine as therealm from 100 nm diameter and lower), they arebelow the normal optical limit and cannot bevisualized as individual entities by light or fluorescencemicroscopy. They can be visualized as groups ofnanobiosystems, typically by fluorescence microscopy.Many of the quantum dot nanoparticles shown inpublications as apparent single fluorescent particlesare actually groups of hundreds or even thousands ofagglomerated particles that cannot be distinguishedindividually but appear as larger, optically visibleobjects. This includes quantum dots shown clusteredinside cells, as described in Figure 4 in this review.
Physicists for several generations have been ableto ‘see’ sub-atomic particles by their indirect effects.Other than through the use of transmission electronmicroscopy (TEM) and, more recently atomic forcemicroscopy (AFM), biologists are not accustomedto dealing with ‘entities’ that cannot be directlymeasured, but inferred by indirect means, e.g., throughtheir effects on cells or through measurements that areunique to nanobiosystems and do not exist in thenatural biological world.
Nanobiosystem Characterization
Direct Visualization of Nanobiosystems byElectron MicroscopyTEM and scanning electron microscopy (SEM)allow direct visualization of nanobiosystems providedthese systems are electron dense. Typically, thisproperty is exhibited by most metals; therefore,numerous metallic nanoparticle formulations arevisible by electron microscopy. The main differencefor nanobiosystem characterization between TEMand SEM is that TEM provides a two-dimensionalslice of the sample being analyzed, while SEM imagesshow the surface configuration of the sample. Whencombined with standard image analysis techniques,TEM and SEM analyses of nanobiosystems can givesize distribution analyses of those systems (at least theelectron-dense portions).
Indirect Measures of Size and Charge by‘Zeta-sizing’ TechnologiesWhile individual nanobiosystems are below the opti-cal limit and cannot be individually imaged, it does notmean that they do not still interact with light waves.Dynamic light scattering (DLS) is a standard techniqueused to provide size distributions of nanobiosystems.Interactions of nanobiosystems with cells and tissuesare largely governed by the zeta potential of both thecell and the nanobiosystems. Zeta potential is the netelectrical charge of the nanobiosystems or cell seen at a
distance. Biological cells usually have a net zeta poten-tial of approximately -30 mV because of the presenceof many negatively charged molecules (e.g., sialic acidmolecules) on the cell surface.61 For a nanobiosystemsto get close enough to the cell surface to interact withthe cell through cell surface receptors, it must have azeta potential in the range of -5 to -20 mV. This zetapotential can, and frequently does, change as it goesfrom one pH or ionic strength fluid compartment.
Topographical Analysis of Nanobiosystems byAFMAFM attempts to use nanoengineered cantilevers tointeract with cell surfaces. This is not difficult todo if cells are fixed and the measurements can beperformed in an air environment. But these measure-ments become much more challenging if performedin a natural ‘water’ (or more precisely an isotonicbuffer) environment for living cells. The so-calledbio-AFM remains challenging but has progressedconsiderable in recent years. If the nanobiosystemshave a magnetic component, such as the presence ofa core nanoparticle made of ferric oxide, then a morerecent form of this technology known as magneticforce microscopy (MFM) can be used. MFM has theadvantage that it uses the magnetic properties of thenanobiosystems that are not present in most naturalbiological materials.62 Hence it gives a distinct signalfree from background in a way analogous to theadvantages of fluorescence microscopy over standardlight microscopy.
What is Actually on the Surface ofNanobiosystems?One of the most important things to know whendesigning a nanobiosystems is whether or not amolecule added to the system, such as a targetingmolecule or a stealth layer (e.g., PEG) is present.In multilayered nanosystems, this technology can beused to see if a new layer is actually where it shouldbe. A relatively recent nanometrology tool knownas XPS is used to bombard the surface layers ofnanobiosystems to release inner core electrons thathave a distinct pattern of energies. XPS analysis candetermine whether specific molecules are attached tothe nanoparticle as shown in Figure 3.42
Detection of Nanobiosystems in BiologicalEnvironments
Taking Advantage of Nanobiosystems withMetallic PropertiesAnother application of TEM is imaging of thinsections of cells and tissues to investigate nanobiosys-tem interactions. Recently, new forms of TEM can
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x104(a) Iron oxide nanoparticles (uncoated)Si 2s Si 2p1/2 Si 3s
Si 3pSi 3p1/2Si 3p3/2
Si 2pSi 2p3/214
12
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S
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x104(b) APTMS coated iron oxide nanoparticlesSi 2s Si 2p1/2 Si 3s
Si 3pSi 3p1/2Si 3p3/2
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FIGURE 3 | (a) X-ray photoelectron spectroscopy (XPS) analysis of uncoated iron oxide nanoparticles, (b) XPS analysis of APTMS(3-aminopropyltrimethoxysilane) polymer silica coated iron oxide nanoparticles shows presence of silicon atoms in this coating layer.
produce images similar to 3D confocal microscopyimages but at the nanolevel.63 One challenge associ-ated with TEM is that living system must traditionallybe chemically fixed prior to TEM analysis to renderthem capable of surviving the near vacuum conditionsunder which most TEM instruments must operate.Some attempts at visualizing ‘frozen’ or even ‘live’cells or tissue under high-speed conditions thatallow them to visualize in a state nearer to living(non-fixed), but those conditions themselves arehardly equivalent to studying the natural interactionsof nanobiosystems and living cells.
Another important technique that takes advan-tages of difference between natural biological systemsand at least partially synthetic nanobiosystems is theinteraction of light waves with these metallic surfaces.Nanobiosystems with gold nanoparticle cores havemade particular advantage of these properties. If lighthits these metallic surfaces at the proper wavelengthand angle, energy is transferred to metallic electronswhich flow in the surface layers, a phenomenonknown as surface plasmon resonance (SPR). Thisleads to a decrease in the reflected light at given anglesfrom objects containing gold core nanobiosystemsduring SPR conditions.
Visualizing Nanobiosystems in vivo: NIR andMRIVisualization of nanobiosystems in vivo represents anumber of challenges even to visualize fairly largegroups of nanobiosystems. Peptide-guided quantumdot nanobiosystems targeting human cells in nudemice can be visualized in tissue sections64 as shown inFigure 4. However, even these visualizations showingexcellent homing of these nanobiosystems to tumorsin vivo involve thousands of agglomerated peptide-guided quantum dots per cell.
Visualizing nanobiosystems within living ani-mals or humans is much more challenging. Thescattering of light by surrounding tissue can con-found the visualization of nanobiosystems from thisvery large background. One way to get around thisproblem is to wavelength shift the nanobiosystemssignal through the use of an attached fluorescencemolecule that has good penetration through the tissueboth of its exciting wavelength and its fluorescenceemission spectrum. The best region of the spectrumfor accomplishing this is at the NIR. NIR probeshave been used in a variety of systems to visualizenanobiosystems (e.g., targeted to tumors beneath theskin in nude mice) in vivo. The limits of depth of theseNIR systems are still typically only a few millimeters.Intra-body cavities can also be explored by NIR-labeled nanobiosystems using endoscopy techniquescapable of capturing NIR fluorescence.65
For more penetrating in vivo visualizations atechnique already used in human patients is the useof nanobiosystems containing MRI contrast agents.Particularly as concerns about potential toxicityof gadolinium contrast agents grow, there will beincreased use of biodegradable iron oxide corenanobiosystems which can serve as targeted MRIcontrast agents.
INTERACTIONS BETWEEN NANO-AND BIOLOGICAL SYSTEMS
BiocompatibilityA critical issue for the application of nanobiosystemsis biocompatibility. Moreover, this concept is relevantfor nanobiosystems for in vitro, ex vivo, and in vivoapplications for accurate interpretation of nanobiosys-tem function. Biocompatibility, as described in the
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(a) (b)50 µµm 50 µm 50 µm
(c)
FIGURE 4 | The single bright spots in tumor cells within this histological section of SKBr3 human breast cancer cells excised from a xenograft froma nude mouse after necropsy, are thousands of agglomerated peptide-guided quantum dots nanoparticles that were injected into the tail vein of theanimals and targeted to the tumor site through its vasculature.
field of tissue engineering, refers to the ability ofa material to perform its designed function withoutdetrimental to the host environment.66,67 Comparingthis concept to nanobiosystem development, severalparadigms will be considered. First, materials mayelicit toxicity in the biological environment not onlybecause of their chemical formulation but also becauseof their nanostructure properties. Also, nanobiosys-tems are being developed for inducing cytotoxicresponses in diseased cells and pathogens;46,68 how-ever, nonspecific cytotoxic effects of the nanobiosys-tem or its components are not desired. Exampleswill be provided to highlight recent advancementsrelated to nanomaterial toxicity (nanotoxicity) as wellas nanobiosystem interaction with specific cellularpathways and functions. ‘Biocoatings’ essentially coatnanomaterials, which are generally hydrophobic, withhydrophilic molecules that allow these nanomaterialsto disperse in an aqueous environment since virtuallyall biological cells exist in water environments. Thesebiocoatings can also produce ‘biocompatibility’ withcells and tissues. But these biocoatings can detach orbe degraded from the nanomaterials exposing nanos-tructures than might be non-biocompatible or evennanotoxic.
NanotoxicityNanotoxicity, or cytotoxicity of nanostructures, is aprevalent topic and much research is being performedin this area. Two common themes in nanotoxicity are:(1) discovering mechanisms of known nanostructuretoxicity and (2) determining the level of nanotoxicityfor novel nanostructures. For example, carbon nan-otubes and CdSe quantum dots have been the subjectof toxicological scrutiny because of their materialformulations. However, several groups have reportedthat with biocompatible surface coatings, such asphenyl-SO3H for carbon nanotubes56 and PEG-silicafor quantum dots,55,69 these nanostructures are well
tolerated by cells in vitro. But these biocoatingscan also come off the quantum dots and exposemore cytotoxic nanomaterials. On the other hand,nanostructures that are generally thought to be bio-compatible, such as magnetic nanoparticles,70 havebeen shown to elicit cytotoxic response in certain celltypes under controlled environments.71 Overall, theimportance of nanotoxicity research is that these stud-ies must be performed for nanobiosystems and haveextreme importance in higher-risk applications, suchas stem cell labeling and in vivo applications.72,73 Nan-otoxicity is a new and emerging field and mechanismsof nanotoxicity are currently not well-understood.
Perturbation of Cellular FunctionNumerous examples of nanobiosystems are beingdeveloped to interact with specific cellular pathwaysand functions. However, in some cases, undesirableperturbations to cellular function occur in the hostbiological environment. In the cellular environment,these perturbations may occur on many levels, whichcan be broadly divided into three levels of oxidativestress: enzymatic response (low), inflammationresponse (medium), and cytotoxicity (high).74 Forexample, Unfried et al.75 demonstrated that carbonnanoparticles activated Akt signaling pathway andgenerate in pro-inflammatory response in lung cells.More pronounced signs of cytotoxicity, such asintracellular reactive oxygen species generation andphosphatidyl serine expression, are readily detectedby cellular staining and apoptosis assays.76,77 Forexample, Lovric et al.77 reported that a breast cancercell line (MCF-7) generated nuclear reactive oxygenspecies, shown by intense nuclear fluorescence afterstaining with dihydroethidium, after exposure toquantum dots. Further, Berry and colleagues per-formed an assay for the early-stage apoptosis marker,annexin-V, in addition to clathrin immunofluores-cence to determine if dextran-iron oxide nanoparticles
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induced apoptosis upon intracellular uptake.78 Inconclusion, several biomarkers are used to detectperturbations in cellular function induced by nanos-tructures. These cellular assays are an importantscreening method to predict large-scale toxicity priorto in vivo animal studies. Measuring cellular perturba-tion in response to nanobiosystems is a critical link toin vivo studies because the nanobiosystem will inter-act with many different cell types in the body. Oneof the main challenges for cellular assays is that thenumerous methods to detect cytotoxicity or cellularstress lead to discrepancies in results. These conflictswill likely be resolved by performing a series of testson numerous formulations of the nanobiosystem ornanomaterials of interest.
Nanobiosystem Interactions in vivoOne of the ultimate goals for nanobiosystems is theirapplication in vivo. Whether the purpose is for enhanc-ing diagnostic sensitivity, targeted drug delivery, oranother aim, the nanobiosystem must overcome sev-eral unique challenges. First, evasion of inflamma-tion and immune response is critical; otherwise, thenanobiosystem will be removed from circulation priorto performing its task. Current research focuses onminimizing these adverse effects through controllingsize, generally between 10 and 100 nm, and selectionof stealth materials to improve circulation time.57,59
Another unique challenge for nanobiosystems isunderstanding biodistribution and metabolic effects.Nanobiosystems, which are one the order of one-billionth the volume of a single cell, must be followedand detected over the entire body. Nanobiosystem sizeand detection sensitivity are the two main challenges;7
however, metabolic pathways unique to nanomateri-als are another focus area being investigated.16 Thelast major concern for in vivo application is target-ing nanobiosystems to cells and tissues of interest.Even after the nanobiosystem has shown efficacy invitro, the final challenge is getting the nanobiosystemto the targeted cell or tissue.19 Many studies todayare focusing on combined physical and moleculartargeting, such as applying a magnetic field near atumor site for magnetic nanoparticle accumulation.79
Overall, the interaction between nano- and biologicalsystems culminates with the in vivo application ofnanobiosystems because this environment presentsunique challenges emphasizing the need for investi-gation in animal models and human clinical trials.
CONCLUSION
The general direction of nanobiosystems is to pro-duce multifunctional systems containing targeting(both cellular and intracellular), cell-entry, biosens-ing, and drug/gene delivery molecules in an integratedpackage. This strategy permits the use of multiplespecific-purpose molecules to be brought together ina coordinated way and in a controlled sequence offunctions. The power of in vivo imaging can also beincluded in this package through the use of taggingmolecules or core nanomaterials. These additionalfactors can be distinctly non-biological and haveproperties that highly distinguish themselves from bio-logical tissue to provide for greatly improved contrast.An example is the use of iron oxide superparamag-netic materials which serve not only as MRI contrastagents but also vehicles for manipulation by magneticfields to improve drug delivery or to allow for localizedheating. Composite nanomaterials and modified shapedesigns (e.g., nanorods) may improve circulation timein vivo and improve uptake by targeted cells.
The ability to simultaneously provide in vivodiagnostics and therapeutics (‘theragnostics’) in asingle nanobiosystem will allow for more sophisti-cated medical therapies and permit more sophisticatedappraisals of therapeutic efficacy, such as in vivoMRI measures of tumor shrinkage. The future ofnanobiosystems research will be to engineer systemssuch that they provide a means for swapping in or outtargeting and therapeutic molecules for treatment ofa wide variety of diseases. Such sophisticated nanode-livery systems should allow for not only improved tar-geting but also the use of far less overall drug, therebyreducing damage to bystander normal cells and tis-sue. Overall, nanobiosystems are being developed toperform efficient, multifunctional tasks in the biologi-cal environment, and their successful application willgreatly impact the fields of biology and medicine.
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FURTHER READING
A popular source of information on nanomedical systems is offered as a series of 21 online lectures presented aseither Breeze (PowerPoint) visual presentations with linked oral presentations, or as Podcasts. These are foundat the National Science Foundation’s NanoHUB website at http://www.nanohub.org/courses/nanomedicineA description of a synthesis process for production of water-soluble nanoparticle for nanomedicine is given at:http://docs.lib.purdue.edu/cgi/viewcontent.cgi?article=1021&context=nanoposterA good review of multifunctional magnetic nanoparticles is given by:McCarthy JR, Weissleder R. Multifunctional magnetic nanoparticles for targeted imaging and therapy.Adv Drug Deliv Rev 60:1241–1251, 2008.
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