Tailo ing ca bon nano ube su aces wi h glyconano ings:
new bionanoma e ials wi h specific lec in affini yw
Nou eddine Khia ,*
a
Manuel Pe nia Leal,
a
Rachid Baa i,*
b
Ch is ine Ruhlmann,
c
Cha les Mioskowski,z
b
Pa ick Schul z
c
and Inmaculada Fe na
´ndez
d
Recei ed (in Camb idge, UK) 9 h Ma ch 2009, Accep ed 1s May 2009
Fi s published as an Ad ance A icle on he web 2nd June 2009
DOI: 10.1039/b904717d
Rema kably s able, wa e -soluble glyconano ing-coa ed
SWCNTs we e p epa ed by sel o ganiza ion and pho o-
polyme iza ion o neu al diace ylene-based glycolipids on he
nano ube su ace; he nanocons uc s a e able o engage
in specific ligand–lec in in e ac ions in a simila way o
glycoconjuga es on cell memb anes.
Endowed wi h unique size, shape and physical p ope ies,
single wall ca bon nano ubes (SWCNTs) a e cu en ly being
ac i ely in es iga ed as a ehicle o he in i o sma deli e y
o biologically ele an molecules, as nanome ic senso s,
and po en ially, o cance ea men .
1
In e es ingly, ecen
in es iga ions, including an in i o pilo s udy, ha e concluded
ha con enien ly unc ionalized wa e -soluble SWCNTs a e
non- oxic.
2
The e o e, he de elopmen o efficien s a egies
o he cons uc ion o wa e -soluble, biocompa ible CNTs is a
challenge o pa amoun impo ance.
3,4
Owing o hei high
su ace a ea, bioenginee ed CNTs can be used o expose a
la ge numbe o biomolecules, in a manne ha is simila o
he species in ol ed in hose c i ical biological p ocesses
ha a ou mul i alen ligand– ecep o in e ac ions.
5
A
pa adigma ic example o such e en s a e hose media ed by
ca bohyd a e–p o ein in e ac ions,
4
which include cell
adhesion, inflamma ion, umou cell me as asis, and pa hogenic
in ec ions.
6
Building on ou in e es in he syn hesis o
CNT-based bionanoma e ials,
7
he ein, we epo a new
bo om-up app oach o he p epa a ion o ema kably s able
wa e -soluble SWCNT–ca bohyd a e nanohyb ids wi h a
nanoabacus opology and wi h specific lec in affini y.
Ou a ionally designed app oach o he desi ed glyconano ing-
coa ed SWCNTs, Fig. 1, is based on he p e iously epo ed
sel o ganiza ion a ound ca bon nano ube su aces o cha ged
anionic su ac an s,
7a,8
and on he known selec i e and
smoo h pho opolyme iza ion o diace ylene-based glycolipids.
9
Compa ed o o he amphiphiles, glycolipids a e cha ac e ized
by a complexi y o in e ac ions among he suga g oups, which
add fine de ails o he hyd ophilic–hyd ophobic balance
go e ning hei sel assembly.
10
Thus, o ailo he p ope ies
o he suga -based biosu ac an o he desi ed sel o ganiza ion
on he nano ube, a diace ylene-based neoglycolipid Iwas
designed, which exhibi s a 25 ca bon-based hyd ophobic ail
o an efficien an de Waals in e ac ion wi h he CNT,
wi h a a iable space and suga g oup o fine uning o he
hyd ophilic–hyd ophobic balance o he molecule. Neu al
de e gen s 7–11 used in his s udy (Scheme 1), we e ob ained
by a new, con e gen , and modula s a egy, s a ing om
(2-aminoe hyl)-pe -O-ace yla ed-1- hio-glycosides 1,2, and 3,
o which a one s ep app oach has been de eloped (see ESIw).
Sonica ion o SWCNTs in he p esence o a homogeneous
aqueous solu ion o Lac-7affo ded uns able agg ega es as
e idenced by apid sedimen a ion o he ca bon nano ubes in
he medium. This esul e eals ha lipid Lac-7is unable o
sel o ganize on he nano ube side-wall, mos p obably due o
i s in insically un a o able hyd ophilic–hyd ophobic balance.
Fig. 1 O e iew o he glyconano ing s a egy.
Scheme 1 S uc u es o (2-aminoe hyl)-pe -O-ace yla ed-1- hio-lac oside 1,
cellobioside 2, and mannoside 3, he bi unc ional space s 5–6, and he
diace ylenic acid 4used o he syn hesis o diace ylene-based Lac-7,
Lac-8, Cellob-9, Man-10, and Man-11.
a
Ins i u o de In es igaciones Quı´micas, C.S.I.C-Uni e sidad de
Se illa, c/. Ame
´ ico Vespucio, 49, Isla de la Ca uja, 41092 Se illa,
Spain. E-mail: [email p o ec ed]; Fax: +34 95 44600565;
Tel: +34 95 448 9559
b
Uni e si e
´de S asbou g, Facul e
´de Pha macie CNRS UMR 7199
Labo a oi e de Chimie des Sys e
`mes Fonc ionnels, 74, ou e du Rhin,
67401 Illki ch-G affens aden, F ance
E-mail: [email p o ec ed]
c
Ins i u de Ge
´ne
´ ique e de Biologie Mole
´culai e e Cellulai e,
CNRS/INSERM/ULP, 1 ue Lau en F ies, BP163, F-67404 Illki ch
Cedex, F ance
d
Depa amen o de Quı´mica O ga
´nica y Fa mace
´u ica, Facul ad de
Fa macia, Uni e sidad de Se illa, 41012 Se illa, Spain
wElec onic Supplemen a y In o ma ion (ESI) a ailable: Expe imen al
p ocedu es, lec in binding assays, NMR, IR and Raman spec oscopies
and addi ional TEM images. See DOI: 10.1039/b904717d
zDeceased. Dedica ed o he memo y o D Cha les Mioskowski.
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/ Table o Con en s o his issue
In o de o alida e his hypo hesis, he mo e hyd ophilic
lac ose based su ac an Lac-8was syn hesized. This has been
achie ed by he inco po a ion o he bi unc ional e hylene
glycol de i ed space 5, ob ained om e ae hylene glycol
in h ee s eps (see ESIw) ia amide bond linkage, o he
(2-aminoe hyl)-2,20,3,30,40,6,60-hep a-O-ace yl-1- hio-b-lac oside 1.
A e S audinge azide educ ion, a syn he ic ou e simila o
ha used o Lac-7, affo ded glycolipid Lac-8in good yield.
Consequen ly, by simply mixing he neu al lipid Lac-8wi h
CNT in wa e wi hou any addi i e, ollowed by sonica ion o
30 min, a black solu ion which emained s able o mon hs
was ob ained (Fig. 2A, ial 2). A simila s able black
suspension was ob ained when glycolipid Cellob-9de i ed
om cellobiose disaccha ide was used wi h he same SWCNTs
(Fig. 2A, ial 3). These esul s show ha an inc ease in he
hyd ophilic–hyd ophobic balance o he glycolipids, by
adding a poly(e hylene glycol) (PEG) chain, is c ucial o he
unc ionaliza ion o SWCNTs and he o ma ion o s able
nanocons uc s in pu e aqueous condi ions. This obse a ion
is co obo a ed by he ac ha : (i) a dec ease o he hyd ophilic–
hyd ophobic balance o he su ac an by using a
monosaccha ide such as in Man-10, ga e exclusi ely uns able
agg ega es ha p ecipi a ed quickly in wa e , and (ii) he use
o neoglycolipid Man-11 wi h he same suga epi ope
bu a longe hyd ophilic space 6allows he ema kable
solubiliza ion and unc ionaliza ion o ca bon nano ubes in
pu e wa e . The mac oscopic obse a ion o a s able black
suspension is a esul o he change in he na i e hyd ophobici y
o he nano ube side wall, becoming mo e hyd ophilic, due o
he unc ionaliza ion o he SWCNT’s su ace wi h glycolipids
and he exposu e o he suga epi opes o he pola
wa e phase. The cha ac e iza ion o SWCNT–neoglycolipid
nanocons uc s was ca ied ou by ansmission elec on
mic oscopy (TEM), and nuclea magne ic esonance (NMR),
in a- ed (IR) and Raman spec oscopies (see ESIw).
TEM images o na i e SWCNTs and SWCNTs-coa ed wi h
neoglycolipid Lac-8a e gi en in Fig. 2B and 2C, espec i ely.
As a consequence o he hyd ophobic in e ac ions, an de
Waals o ces, and p-s acking among indi idual nano ubes, as
p oduced SWCNTs usually exis as dense bundles o opes
ha a e deeply in e connec ed, Fig. 2B.
11
Qui e ema kably, a
simple unc ionaliza ion wi h neoglycolipids Lac-8, Cellob-9
o Man-11 in pu e wa e and s ic ly neu al condi ions,
allowed he CNTs packages o ex olia e p oducing small
mainly indi idual suga -coa ed nano ubes, as e idenced by
TEM analysis (Fig. 2C, D and E). In e es ingly, he TEM
images show ha he su ace o he nano ube is comple ely
co e ed by s ia ions o hemi-micelles, ha can slide on he
nano ube in a simila way o beads on a single wi e in an
an ique abacus. Simila associa ions we e p e iously epo ed
o cha ged and zwi e ionic amphiphilic lipids,
7,8
bu as a as
we know his is he fi s ime whe e such o ganiza ion is
epo ed o a neu al, non-ionic su ac an in pu e wa e .
12
While up o h ee mechanisms we e p oposed o explain he
dispe sion o CNTs wi h su ac an s,
13
he obse ed pe iodic
s ia ions sugges ha he mic oscopic binding mode is he hal
cylinde mode.
7a
In e es ingly, unde such sup amolecula
o ganiza ion i is well documen ed ha diace ylene lipids
unde go efficien and clean polyme iza ion ia a 1,4-addi ion
eac ion o o m al e na ing ene-yne polyme chains upon ligh
i adia ion a 254 nm,
9
affo ding s ong polyme ized glyconano-
ings a ound he su ace nano ubes as shown in Fig. 3.
14
A e sonica ion o SWCNTs in he p esence o aqueous
solu ion o Lac-8as be o e, he mix u e was hen i adia ed by
a labo a o y UV lamp (254 nm) o 12 h in o de o p omo e he
polyme iza ion o he diyne unc ional g oups a ound he
nano ube. The high s abili y o he polyme ized glyconano ings
was asce ained by compa ison wi h ha o he non-polyme ized
coun e pa nanohyb ids. While he la e p ecipi a ed a e h ee
mon hs he o me ones emained s able o a leas six mon hs.
Addi ionally, he s able suspension o CNTs unc ionalized wi h
polyme ized glyconano ings emained unchanged a e washing
wi h me hanol, hea ing in wa e a 80 1C o one week, and when
dilu ed in Hepes buffe (20 mM a pH 7.5)—in con as wi h he
non-polyme ized CNT–glycolipid nanocons uc s and o mos o
he su ac an -based CNT-solubiliza ion sys ems de eloped so
a (see ESIw). These esul s indica e ha he SWCNTs a e
coa ed wi h polyme ized polydiace ylene-based glyconano ings
(PDA-GNR), o ming s able, wa e -soluble mul icomposi es
namely SWCNT-PDA-GNR-Lac-8. I is wo h men ioning ha
his ea u e ex ends he u ili y o ou bionanoma e ials, since
polydiace ylene- e he ed biomolecules ha e ecen ly ecei ed
a g ea deal o a en ion as biosenso s, due o hei unique
p ope ies upon ex e nal s imula ion.
9b
A cen al issue o his s udy was o demons a e he abili y
o such suga epi ope-bea ing bionanoma e ials o engage in
specific in e ac ions wi h p o ein ecep o s. Theo e ically, each
polyme ized nano ing on he nano ube su ace is su ounded
by a la ge numbe o suga epi opes, much like he glycocalyx
Fig. 2 (A) Pho og aphs o ials con aining aqueous solu ion o : 1.
SWCNTs, 2. SWCNT-Lac-8, 3. SWCNT-Cellob-9, 4. SWCNTs-
Man-11. TEM images o he nanocons uc s nega i ely cha ged wi h
u anyl ace a e: (B) as p oduced SWCNTs, (C) SWCNT–Lac-8, (D)
SWCNT–Cellob-9, (E) SWCNT–Man-11.
Fig. 3 Fo ma ion o SWCNTs coa ed wi h polydiace ylene-based
glyconano ings in pu e wa e .
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a he cell memb ane su ace, and can ac as ligands o specific
ecep o s. The e o e he SWCNT-PDA-GNR-Lac-8exhibi ing
lac ose esidues on he nano ube’s su ace could be ecognized
by a lac ose-specific ecep o such as Peanu agglu inin (PNA)
om A achis hypogaea.
15
In o de o confi m his hypo hesis,
we incuba ed SWCNT-PDA-GNR-Lac-8, wi h PNA in Hepes
buffe a pH 7.4 o 1 hou , and he esul o he in e ac ion was
cha ac e ized by TEM. As can be seen om Fig. 4A, he
abacus-like geome y was los and he cha ac e is ic s ia ions
could no be obse ed. The lec ins co e ed ex ensi ely he
unc ionalized nano ubes whose su ace becomes uzzy and
om which indi idual lec in molecules can be ound o s ick
ou (Fig. 4A, whi e a ows). Whe e hey could be clea ly
delinea ed a he edges o he ubes, he sizes o he pa icles
a ached o he Lac-8-coa ed SWCNT we e measu ed. A alue
o 70 A
˚was ob ained (s=8A
˚,n= 13), which is consis en
wi h he size o he 110 kDa PNA homo e ame o
which a hyd odynamic adius o 39 A
˚was de e mined by
ul acen i uga ion analysis and dynamic ligh sca e ing
measu emen s.
16
Addi ionally, he size o he a ached pa icles,
Fig. 4A is iden ical o he size o pu ified lec in mac omolecules
obse ed by elec on mic oscopy in he absence o nano ubes
(see Fig. 4C). As a con ol expe imen o demons a e ha he
absence o he s ia ions o med by he glyconano ings is a
consequence o he specific in e ac ion be ween he lec in and
he nanohyb id, we pe o med he same eac ion be ween he
lec in and he glyconanohyb id SWCNT-PDA-GNR-Cellob-9,
exposing he cellobiose glycoligand which is no ecognized by
PNA. In his case he cha ac e is ic abacus-like geome y
can s ill be obse ed (Fig. 4B), as a consequence o he
absence o specific in e ac ions be ween PNA lec in and
SWCNT-PDA-GNR-Cellob-9. These esul s demons a e ha
SWCNTs coa ed wi h glyconano ings can engage in specific
molecula ecogni ion wi h a p o ein ecep o and p eclude
non-specific p o ein binding.
The specific in e ac ions wi h o he biologically ele an
species such as cells, bac e ia, and oxins, as well as s udies
de o ed o he emo al o hese ascina ing polyme ized
glyconano ings (GNR) om he nano ubes su ace, hei eco e y
and u he use, a e unde in es iga ion in ou labo a o ies.
This wo k was suppo ed by he DGICyT (g an No.
CTQ2006-15515-CO2-01 and CTQ2007-61185), he Jun a de
Andalucı´a (g an P06-FQM-01852 and P07-FQM-2774), he
CNRS (F ance) and CSIC (Egide Picasso 09543XA and PICS
P og am 2008).
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Fig. 4 TEM cha ac e iza ion o he specifici y acqui ed by he
agg ega es owa d specific ecep o s. (A) In e ac ion o PNA wi h
SWCNT-Lac-8. The abacus like geome y is los as he s ia ions a e
co e ed by lec in molecules (whi e a ows). (B) In e ac ion o PNA wi h
SWCNT-Cellob-9. The e is no specific in e ac ion and he cha ac e is ic
s ia ions a e s ill obse ed. (C) TEM image o pu ified PNA lec in alone.
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