Enginee ing amphiphilic alkenyl
lipids o sel -assembly in
unc ional hyb id nanos uc u es
Raúl Gimeno-Fe e o1, Vic o ia Valdi ia1, Inmaculada Fe nández1,
Ma ía Luisa Ga cía-Ma ín2,3,4 & Manuel Pe nia Leal 1
The de elopmen o biocompa ible hyb id nanosys ems o ad anced unc ional applica ions p esen s
signi ican challenges o he esea ch communi y. Key obs acles include he poo solubili y o hese
nanosys ems in wa e and he di icul y o p ecisely con olling hei nanos uc u e dimensions
and composi ion. A p omising app oach o o e coming hese challenges is he sel -assembly o
su ac an -based building blocks in o well-o de ed hyb id nanos uc u es. In his s udy, we explo e he
ela ionship be ween s uc u e and sel -assembly in no el low molecula weigh amphiphilic molecules
o p oduce s able and biocompa ible hyb id nanos uc u es. We in es iga ed he sel -assembly
beha io o wo amilies o amphiphiles de i ed om alkenyl lipids wi h one o wo double bonds,
leading o dis inc hyb id sup amolecula s uc u es acili a ed by he inco po a ion o hyd ophobic
i on oxide nanopa icles (IONPs) as empla es. The p esence o double bonds in he lipid ail and he
mo phology o he amphiphile in luence he a angemen on he hyd ophobic NPs. Amphiphiles wi h
a single double bond in he lipid ail o m highly wa e -soluble, well-o de ed micella -like s uc u es
on he IONP su aces, while hose wi h wo double bonds c ea e diso de ed lipid nanopa icles.
Fu he mo e, hese amphiphilic molecules can sel -o ganize in o highe -o de hyb id sup amolecula
s uc u es, such as esicles, wi h po en ial applica ions in magne ic esonance imaging (MRI).
Sel -assembly o building blocks o he cons uc ion o unc ional ma e ials has ecei ed signi ican in e es
in many ields, including nano echnology, ma e ials sciences, and biomedicine, in ecen decades1–8. The
di e si y in he design and na u e o he building blocks allows esea che s o expand and enhance he lib a y
o esul ing ma e ials, o e ing excellen op ical, elec onic, and magne ic p ope ies9–12. Many o hese sel -
assembled ma e ials a e based on ino ganic nanopa icles (NPs), which ac as building blocks o o m highly
o de ed supe s uc u es anging om 0D o 3D h ough hyd ophobic alkane-based su ac an s in e pa icle
in e ac ions. Ne e heless, he poo solubili y in physiological media and he challenges in con olling he
dispe si y, size, geome ical dimensions, and homogenous composi ion o hese sel -o ganized nanos uc u es
complica e hei use in biological applica ions such as d ug deli e y sys ems, biosenso s, and he apeu ic
agen s13–18. To o e come hese issues, one o he mos elegan and cos -e ec i e me hods is he su ac an -
assis ed coope a i e sel -assembly o hyd ophobic ino ganic NPs using amphiphilic molecules19,20. The sel -
o ganiza ion o hese componen s leads o he o ma ion o well-de ined and o de ed nanos uc u es. These
hyb id complex a chi ec u es a e o med h ough non-co alen in e ac ions, such as an de Waals o ces, π-π
s acking, o hyd ogen bonding, be ween he hyd ophobic capped su ac an s om he ino ganic NPs and he
hyd ophobic coun e pa o he amphiphilic molecules21–24. Micelles and esicles a e among he mos s udied
su ac an -assis ed coope a i e sel -assemblies due o he e sa ili y and ease o syn hesis o he amphiphilic
molecules4,25–27. Typical amphiphilic building blocks used in he o ma ion o hese sup amolecula s uc u es
ange om low molecula weigh (MW) amphiphilic molecules o small su ac an s o polyme ic lipids28–30.
Bo h ypes o su ac an s p esen se e al c ucial poin s o be conside ed ideal building blocks o sel -assembly
wi h ino ganic NPs, such as he concen a ion o su ac an s abo e he c i ical micelle concen a ion (CMC), he
use o hyd ophobic ino ganic NPs, and he applica ion o ene gy, such as hea ing, o e apo a e he apola sol en s
om he hyd ophobic NP solu ion. The e o e, hese key ac o s, combined wi h he design o he amphiphilic
molecule, lead o hei sel -o ganiza ion in micelles o esicles, passi a ing he hyd ophobic ino ganic NPs o
1Depa amen o de Química O gánica y Fa macéu ica, Facul ad de Fa macia, Uni e sidad de Se illa, c/ P o eso
Ga cía González, 2, Se illa 41012, Spain. 2Biomedical Magne ic Resonance Labo a o y-BMRL, Andalusian Public
Founda ion P og ess and Heal h-FPS, Se ille, Spain. 3Ins i u o de In es igación Biomédica de Málaga y Pla a o ma
en Nanomedicina-IBIMA Pla a o ma BIONAND, C/ Se e o Ochoa, 35, Málaga 29590, Spain. 4Biomedical Resea ch
Ne wo king Cen e in Bioenginee ing, Bioma e ials &Nanomedicine (CIBER-BBN), Mad id, Spain. email:
[email p o ec ed]
OPEN
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he cons uc ion o hyb id building blocks. In addi ion, he s abili y o hese sel -assemblies in aqueous media
emains a challenge o bo h ypes o su ac an s. In he case o he low MW su ac an s, due o hei na u e,
hey sel -assemble dynamically a ound he ino ganic NPs, in e digi a ing hei hyd ophobic coun e pa s wi h
he hyd ophobic capping ligands o he ino ganic NPs. The esul ing nanos uc u es a e highly dependen on
he su ac an concen a ion due o he sho esidence ime o he amphiphilic su ac an s16. On he con a y,
polyme ic su ac an s, such as block copolyme s, exhibi a high numbe o hyd ophobic chains in he polyme
ha inc ease he esidence ime, enhancing he e icacy and capaci y o he micella a chi ec u es31–33. Howe e ,
amphiphilic polyme s p esen some challenges, such as mo e p ecise and e sa ile syn he ic me hods, and
also equi e a deepe unde s anding o he assembly o polyme su ac an s wi h ino ganic NPs o yield well-
o de ed hyb id building blocks capable o o ming hie a chical supe s uc u es34. In ac , con olling he laye
composi ion and assembly o su ac an s in sup amolecula s uc u es inco po a ing ino ganic NPs emains a
ques in ma e ials science esea ch. In his wo k, we epo he syn hesis o wo amilies o low MW su ac an s
based on lipid chains wi h one and wo double bonds. These su ac an s a e capable o sel -assembling in o
a ious wa e -soluble, s able and well-o de ed hyb id sup amolecula s uc u es d i en by he addi ion o
hyd ophobic i on oxide NPs as empla es. The p esence o double bonds in he lipid chain, and he mo phology
o he su ac an , induce he ype o a angemen o he amphiphilic molecules on he hyd ophobic NPs,
as well as he maximal capaci y o accep ance and embedding o hyd ophobic NPs. Mo eo e , hese hyb id
sup amolecula s uc u es exhibi ed good cha ac e is ics as MRI con as agen s depending on he a angemen
o he amphiphiles on o he IONPs.
Expe imen al sec ion
Ma e ials
All eagen s and sol en s we e ob ained om comme cial supplie s Me ck and Fishe Scien i ic and used
wi hou u he pu i ica ion. Thin laye ch oma og aphy (TLC) was ca ied ou on Me ck silica gel 60 F254
analy ical pla es. The chemicals syn hesis was moni o ed by a handheld UV lamp (254nm) and e ealed wi h
a solu ion o 5% phosphomolybdic acid in E OH. Silica gel 60 (0.04–0.063mm) om Me ck was used o lash
ch oma og aphy ca ied ou wi h echnical g ade sol en s.
Me hods
– Nuclea magne ic esonance (NMR) spec a we e eco ded on a BRUKER AMX-500 appa a us. Deu e a ed
chlo o o m and me hanol we e used and indica ed in pa en heses o each compound. The chemical shi
alues (δ) we e e e ed o e ame hylsilane used as an in e nal e e ence.
– High esolu ion mass spec ome y (HRMS) was eco ded on a Q Exac i e Hyb id Quad upole-O bi ap
appa a us om The moFishe Scien i ic.
– T ansmission elec on mic oscopy (TEM) images we e ob ained on an HR Fei Talos 200X mic oscope op-
e a ed a an accele a ing ol age o 100kV. TEM samples we e p epa ed by d opping a solu ion o he co e-
sponding nanosys em on o a ca bon-coa ed coppe g id wi hou s aining. The mean sizes we e calcula ed on
an a e age o 100 nanopa icles measu ed. S ained TEM g id was pe o med by adding a d op o a solu ion
o 2% u anyl ace a e p e iously il e ed h ough a 0.2μm po e size sy inge il e (memb ane ma e ial: ac ylic
copolyme housing cellulose ace a e/su ac an - ee memb ane) o a d op o he nanopa icle sample. The
d op was le 2min, and hen, he g id was d ied using pape il e o elimina e u anyl ace a e excess.
– The in ensi y dis ibu ion o he nanosys ems was measu ed on a Ze asize Nano ZS90 om Mal e n Pana-
ly ical. The dynamic ligh sca e ing (DLS) measu emen s we e pe o med on a cell ype: ZEN0040 disposable
cu e e, se ing a e ac i e index o 2.3 o i on oxide NPs and 1.40 o he micelles wi hou IONPs. The meas-
u emen du a ion was se as au oma ic, and he numbe o measu emen s was h ee. The gene al pu pose,
no mal esolu ion, was chosen as analysis model. The nanosys ems we e suspended in he co esponding
sol en a a concen a ion o 1mg/L o micelles and/o IONPs.
– Small Angle X- ay sca e ing (SAXS) was measu ed using a B uke D8 DISCOVER di ac ome e . The sam-
ple is loca ed in a bo osilica e capilla y o 1mm o diame e wi h a concen a ion o 1mg/mL. The wo-di-
mensional a ea de ec o (Eige 2 500K) allows o as measu emen s wi h high angula esolu ion and a la ge
dynamic ange. Addi ionally, a Goebel mi o wi h a 0.3mm collima o is used o educe he di e gence o
he inciden beam. The mi o and he de ec o a e used in combina ion wi h addi ional acuum-enclosu es
o measu e he SAXS powde samples in ansmission geome y.
Resul s and discussion
Design and syn hesis o alkenyl-based amphiphilic molecules
To de e mine he in luence o he alkenyl-based lipid coun e pa on he sel -assembly o amphiphilic molecules,
wo amilies o low MW su ac an s we e designed. One amily was based on a hyd ophobic chain wi h a single
double bond (cis alkene, as oleic acid), while he o he amily had a hyd ophobic chain wi h wo double bonds
(cis-cis alkene, as linoleic acid). In bo h amilies, h ee ypes o amphiphiles we e syn hesized wi h di e en
hyd ophobic/hyd ophilic balance and s uc u al mo phology (I-VI). These ypes included a single hyd ophilic
pola head wi h a single hyd ophobic ail (I and IV), wo hyd ophilic pola heads wi h a single hyd ophobic ail
(II and V), and a single hyd ophilic pola head wi h wo hyd ophobic ails (III and VI). The chosen pola head
was a e ae hylene glycol (TEG) chain wi h an amino as e minal g oup (Fig.1).
Amphiphilic molecules I and IV, wi h a single hyd ophilic pola head and a mono alkenyl-based lipid,
we e syn hesized h ough a wo-s ep eac ion, as shown in Scheme 1. The alkyne-based lipids 1 and 2 we e
combined wi h he hyd ophilic chain, amino-TEG-azide, ia a Cu(I)-ca alyzed [3 + 2] cycloaddi ion (known
as click eac ion), yielding he amphiphilic molecules I and IV. The Y-shape su ac an s II, III, V and VI we e
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p epa ed using dopamine as he s a ing building block ma e ial (Scheme 1). The choice o dopamine was based
on i s g ea e sa ili y de i ed om he di e en eac ion si es i possesses, allowing o o hogonal eac ions o
cons uc a a ie y o complex molecula sca olds. The amida ion o he alkenyl-based lipid acids, oleic acid,
and linoleic acid, wi h dopamine, esul ed in he amide 3 and 6, espec i ely. Following he inco po a ion o
wo e minal alkynes in o he dopamine de i a i es and subsequen double click eac ions wi h wo molecules
o aminoBOC-TEG-azide, he co esponding iazoles 5 and 8 we e ob ained, wi h excellen yield o he oleyl
de i a i e and mode a e yield o he linoleyl de i a i e. Subsequen dep o ec ion o aminoBOC in he p esence
o i luo oace ic acid and dichlo ome hane ga e he desi ed Y-shaped amphiphiles II and V, wi h wo pola
heads and a mono alkenyl-based lipid. The o he Y-shaped amphiphiles III and VI, wi h a single hyd ophilic
pola head and wo alkenyl-based lipids, we e also syn hesized ia a ou -s ep eac ion, as shown in Scheme 1.
Un o una ely, he bis-linoleyl amphiphilic molecule VI could no be syn hesized. The inco po a ion o wo
linoleic acids in o he dopamine-de i ed compound esul ed in mainly aces o he bis-linoleyl p oduc , as
de e mined by HRMS. This poo eac i i y was likely due o he high s e ic hind ance o he i s a ached linoleyl
lipid, which hinde ed he inco po a ion o he second one. The e o e, u he s udy o he bis-linoleyl-de i ed
su ac an was no pu sued. This s e ic hind ance was also obse ed in all eac ions in ol ing he linoleyl-based
lipid, which exhibi ed low yields compa ed o hei oleyl analogues. All hese low molecula weigh alkenyl-
based amphiphilic molecules we e cha ac e ized using1H and13C NMR spec oscopies and high mass esolu ion
spec ome y (Figu es S1-S16).
Sel -assembly beha iou o alkenyl-based amphiphilic molecules
The capaci y o he syn hesized alkenyl-based amphiphilic de i a i es o sel -assemble in o micelles was
de e mined by measu ing he c i ical micelle concen a ion (CMC). The CMC alues we e ob ained using he
py ene me hod in wa e 35,36, esul ing excellen alues om 0.049 o 0.074 mM o he amphiphilic molecules
I, II and III (Fig.2 and Figu e S17). These alues ep esen an imp o emen o e he CMC alues o low MW
su ac an s and all wi hin he same ange as hose epo ed o amphiphilic polyme s in he li e a u e37,38. In
ac , he CMC alues o ou oleyl amphiphiles dec ease wi h an inc ease in he pola head o he su ac an ,
pa icula ly in he amphiphilic molecule II, which p esen s wo hyd ophilic chains. On he con a y, he p esence
o bis-oleyl lipid in he su ac an III esul ed in he highes CMC alue.
Dynamic ligh sca e ing (DLS) analysis and ansmission elec on mic oscopy (TEM) analysis o he sel -
assemblies I, II and III (he ea e e e ed o as Mc1, Mc2, and Mc3, espec i ely) con i med he o ma ion
o micelles wi hou he p esence o la ge agg ega es, PDI alues anging om 0.24 o 0.37 (Fig.2A-C). In
ac , hese analyses exhibi ed a simila end o he CMC alues. The su ac an wi h he highe hyd ophilic/
hyd ophobic balance, amphiphile II, which possesses a double TEG chain, o med micelles Mc2 wi h he
smalles hyd odynamic size in wa e , 5.9 ± 0.4nm. A sligh inc ease in he HD size o 7.8 ± 0.5nm was obse ed
o Mc1 de i ed om amphiphile I wi h a single TEG chain, while a mo e han 2- old inc ease o 13.7 ± 0.8nm
was no ed o Mc3 de i ed om he bis-oleyl lipid-based su ac an III. Indeed, a change in he hyd ophilic/
hyd ophobic balance o he amphiphilic molecule, achie ed by he in oduc ion o wo oleyl lipid chains
(Y-shaped amphiphile), led o an expansion o he hyd ophobic inne co e o he sel -assemblies, esul ing in an
inc ease in size and he numbe o su ac an s molecules equi ed o sel -assemble in micelles (CMC alue). As
shown in he TEM images in Fig.2D and E, he inco po a ion o wo double bonds (cis, cis) in he lipid chain
o he low MW su ac an s esul ed in di e en sel -assemblies, named as lipid nanopa icles (LNP), compa ed
o hose obse ed o de i a i es wi h a single double bond (cis) in he alkenyl chain. LNPs de i ed om mono-
TEG linoleyl IV (LNP1) and di-TEG Y-shaped V (LNP2) exhibi ed much la ge HD sizes han hei oleyl
analogues, measu ing 461 ± 10nm and 410 ± 29nm, espec i ely (Fig.2D-E). These esul s clea ly indica ed a
di e en mode o sel -o ganiza ion compa ed o micelles o he cis, cis linoleyl lipid amphiphilic molecules. The
p esence o he second double bond in he lipid chain es ic s he mo emen o he amphiphilic molecule o
Fig. 1. Mono- and bis-alkenyl based amphiphilic molecules s udied in his wo k.
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adop he mos ene ge ically a ou able a angemen , esul ing in inc eased igidi y and olume. This induces
he o ma ion o non-o de ed s uc u es such as LNPs.
Explo ing he NP-d i en assembly o low MW su ac an s
As men ioned abo e, he syn hesis o well-o de ed hyb id building blocks is c ucial o de eloping new ma e ials
o a wide ange o applica ions, pa icula ly in biomedical ields. The e o e, i u ges he necessi y o de elop
wa e -soluble hyb id building blocks ha can be used o his pu pose. In his sense, we ocused ou a en ion on
i on oxide nanopa icles (IONPs) as empla es o p epa ing hyb id building blocks. The p epa ed IONPs we e
s abilized by oleic acid molecules, which could in e ac wi h he lipid ails o he low MW su ac an s desc ibed
abo e, leading o s able and well-o de ed nanos uc u es. The ini ial expe imen s we e pe o med simila ly o
he o ma ion o micelles Mc1 by adding he amphiphilic molecule I a a concen a ion abo e hei CMC in
he p esence o a ixed concen a ion o IONPs e-suspended in oluene. These expe imen s we e ca ied ou a
Scheme 1. Syn hesis o mono- and bis-alkenyl based amphiphilic molecules.
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di e en sonica ion imes anging om 1 o 30min o op imize he esul ing ensembles Mc1-IONP. As shown
in Fig.3A, he sys em equi ed a minimum o 10min o each equilib ium in e ms o size, measu ing 125.1nm,
likely due o he e apo a ion ime o he oluene om he IONPs. Hyb id sys ems o med wi h less han 10min o
sonica ion exhibi ed signi ican a ia ions in size, wi h he appea ance o some agg ega es a e se e al mon hs.
Thus, he sel -assembly p ocess o he amphiphiles on o he IONPs is d i en by he e apo a ion o he o ganic
sol en , esul ing in he embedding o he lipid ails in he oleic acid laye o he NP, leading o highly s able
colloidal s uc u es. The e o e, he speed o sol en e apo a ion plays an impo an ole in he kine ics o he
o ma ion o well-o de ed sup amolecula s uc u es4,13,20. To demons a e his e ec , oluene was subs i u ed
o a low boiling poin apola sol en such as hexane in he sel -assembly p ocess. Consequen ly, he 30min
Fig. 3. (A) Size s. sonica ion ime g aph o he Mc1-IONPs in wa e ob ained om oluene as apola sol en .
(B) Summa y able o he Mc-IONPs, ob ained om oluene and hexane as s a ing apola sol en s, con aining
he sizes and PDI alues de e mined by DLS in wa e . P oposed sel -assembly models o Mc1-IONPs om (C)
oluene and (D) hexane as apola sol en s. Rep esen a i e TEM images o (E) Mc1-IONPs, (F) Mc2-IONPs
and (G) Mc3-IONPs ob ained om hexane as s a ing apola sol en . Scale ba co esponds o 100nm o
low magni ica ion images and 25nm o he inse s. (H) TEM image o Mc2-IONPs, ob ained om hexane as
s a ing apola sol en , s ained wi h u anyl ace a e showing he o ganic coa ing and ino ganic nucleus. Scale
ba co esponds o 200nm.
Fig. 2. Rep esen a i e TEM images, CMC alues and DLS da a o (A) Mc1, (B) Mc2 and (C) Mc3. Scale ba in
A co esponds o 20nm, in B and C co esponds o 100nm. Rep esen a i e TEM images and DLS da a o (D)
LNP1 and (E) LNP2. Scale ba s co espond o 1μm o low magni ica ion images and 200nm o he inse s.
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sonica ion o amphiphile I in he p esence o he IONPs e-suspended in hexane esul ed in la ge s uc u es wi h
a HD size o 173.6nm (Fig.3B). This inc ease o ca. 50nm in size o he sup amolecula s uc u es demons a es
he e ec o he sol en e apo a ion. The as e apo a ion o he o ganic sol en inc eases he en opy o he
hyb id sys em, esul ing in he o ma ion o kine ic sup amolecula assembly, meaning a mo e diso de ed
sys em wi h poo in e linking o he hyd ophobic chains in he oleic acid laye on he IONPs. On he con a y,
slow e apo a ion o he o ganic sol en leads o he o ma ion o he modynamic sup amolecula assemblies
h ough he inse ion o he hyd ophobic chains o he amphiphiles in o he hyd ophobic laye o he IONPs.
This e ec induces a comp ession and a mo e s able sys em a ising om mul iple an de Waals in e ac ions a
he NP su ace, esul ing in he o ma ion o smalle sup amolecula assemblies (Fig.3C-D). The Y-shaped oleyl
amphiphiles II and III showed dispa a e esul s compa ed o amphiphilic molecule I. The bis-oleyl amphiphile
III exhibi ed he same end as he mono oleyl amphiphile I. The use o oluene as he apola sol en in he sel -
assembly p ocess led o a well-o de ed hyb id sys em, Mc3-IONP, wi h a size o 148nm. In he case o hexane,
he hyb id sys em eached a size o 280nm (Fig.3B). As men ioned abo e, apid sol en e apo a ion esul ed
in inc eased en opy, leading o a mo e diso de ed sup amolecula s uc u e wi h la ge sizes. In ac , wi h he
Y-shaped amphiphilic molecule III, he size o he sys em inc eased ca. 100% due o he p esence o he bis-oleyl
chains in he amphiphile, which con e mo e igidi y and olume o he sys em Mc3-IONP. Howe e , he o he
Y-shaped amphiphilic molecule II, wi h he double TEG pola head, exhibi ed a dec ease in he size o he hyb id
sys em Mc2-IONP om 122nm in oluene o 94nm in hexane (Fig.3B). A possible explana ion is ha in he
assembly p ocess wi h oluene, he bis-TEG pola head o he Y-shaped amphiphiles is comple ely s e ched
owa ds he aqueous phase since he oleyl chains a e embedded wi hin he oleic acid co e o he NP. In hexane,
he oleyl chains om he Y-shaped amphiphiles mainly in e ac wi hou embedding in o he hyd ophobic laye
o he NPs, meaning ha he lipid ails a e deposi ed pa allel o he su ace o he NP wi h he bis-TEG pola
head posi ioned pe pendicula , educing he size o he sup amolecula s uc u e (Figu e S18). TEM analysis o
he h ee di e en Mc-IONPs showed he p esence o monodispe sed hyb id NPs as mesua ed by DLS (Fig.3E-
G). In ac , he comple e obse a ion o he hyb id sup amolecula s uc u e, showing he o ganic coa ing and
ino ganic nucleus, was ob ained by s aining wi h u anyl ace a e (Fig.3H).
Small-angle X- ay sca e ing (SAXS) s udies we e ca ied ou o con i m and de e mine he s uc u al
o ganiza ion o he di e en assemblies (Fig.4 and Figu e S19). The pai dis ibu ion unc ion plo agains
dis ance ( ) ypically showed wo main popula ions. In he smalle egion, he main popula ion co esponds
o he sca e ing o he ino ganic IONP co e, simila o he sca e ing o he oleic acid-capped IONPs (Figu e
S20). In he la ge egion, he maximal alue co esponds o he size o he hyb id sup amolecula assembly,
which is sligh ly smalle han he HD size de e mined by DLS. This di e ence a ises due o he supp ession o he
con ibu ion o he hyd a ion laye a he su ace o he sys em in he SAXS analysis (Fig.3B)39. Mo eo e , he
o he main popula ion a la ge indica es he hickness o he o ganic shell o he nanosys ems. This dis ibu ion
displays he adius o he o ganic shell and he ino ganic NP co e (Figu e S21). The e o e, he size o he o ganic
laye o Mc1-IONPs, Mc2-IONPs and Mc3-IONPs in oluene was de e mined o be 54nm, 54nm and 72nm,
Fig. 4. No malized pai dis ibu ion o he SAXS measu emen s s. dis ance g aphs o Mc1-IONP, Mc2-
IONP and Mc3-IONP ob ained om oluene ((A), (C) and (E), espec i ely) and hexane ((B), (D) and (F),
espec i ely) as s a ing apola sol en s.
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espec i ely. La ge sizes we e ob ained in hexane, in acco dance wi h he DLS esul s, 77nm and 114nm o
Mc1-IONPs and Mc3-IONPs, espec i ely. In he case o Mc3-IONPs, he pai dis ibu ion plo exhibi s a hi d
popula ion ha di ec ly e lec s he size o he o ganic laye wi hou he p esence o he NPs. Fo Mc2-IONPs,
he hickness o he o ganic laye could no be de e mined due o he o e lapping o he wo popula ions.
Op imiza ion o he sup amolecula assembly o he oleyl-based amphiphilic molecules on
he NPs
So a , we ha e analyzed he s uc u e-sel -assembly ela ionship o wo amilies o amphiphilic molecules wi h
alkenyl-based lipids. Addi ionally, we ha e s udied he empla e e ec in he sel -assembly p ocess wi h he
p esence o IONPs and oleyl-based su ac an s. Howe e , i emains a challenge o demons a e he limi s and
capaci y o he sup amolecula sys ems based on hese new amphiphilic molecules d i en by he p esence o
NPs. The e o e, we ca ied ou mo e de ailed expe imen s o comp ehensi ely unde s and he beha iou o he
sup amolecula assemblies by inc easing he numbe o NPs. The addi ion o an inc eased numbe o IONPs in
he sel -assembly p ocess o oleyl-based amphiphiles I, II and III exhibi ed h ee di e en a eas in he size s.
concen a ion o NPs ep esen a ions (Fig.5A-C). The i s a ea co esponds o he p esence o sup amolecula
assemblies wi h and wi hou IONPs. The obse ed sizes ep esen he a e age be ween hese wo o med
sys ems. The addi ion o mo e NPs gene a ed he eme gence o a second a ea ha exhibi s simila sizes o he
o med assemblies (colou ed a ea in Fig.5A-C). This a ea ep esen s he sec ion o he comple e assembly o he
amphiphiles wi h he IONPs (Mc1-IONPs, Mc2-IONPs, and Mc3-IONPs). I means ha he e a e p ac ically no
sup amolecula s uc u es wi hou IONPs. The size o he assemblies in his a ea emains p ac ically unal e ed
upon he addi ion o mo e NPs. Once he amphiphiles a e occupied o ming he hyb id assemblies Mc-IONPs,
he ex a added IONPs p ecipi a ed du ing he p ocess wi hou a ec ing he nanos uc u e (Figu e S22). This a ea
is named as s abili y a ea, which co esponds o he p esence o homogeneous hyb id sys ems independen ly o
he numbe o IONPs. The sup amolecula assembly Mc1-IONPs om amphiphilic molecule I exhibi ed a wide
s abili y a ea compa ed o he assemblies Mc2-IONPs and Mc3-IONPs o Y-shape amphiphilic molecules II and
III, espec i ely. I is also impo an o highligh he assembly based on he Y-shape amphiphile II wi h a single
lipid ail, Mc2-IONPs, which showed he maximum inco po a ion o NP in ha a ea. Howe e , he addi ion o
mo e IONPs o he sys ems d i es he o ma ion o agg ega es. The e-o ganiza ion o he assemblies ha embed
mo e IONPs leads o he o ma ion o uns able sys ems ha p ecipi a e (inse s Fig.5B and Figu e S23).
Fig. 5. Size s. [IONPs]hexane g aphs o (A) Mc1-IONPs, (B) Mc2-IONPs and (C) Mc3-IONPs. S able a eas
in colou . PDI alues s. [IONPs]hexane g aphs o (D) Mc1-IONPs, (E) Mc2-IONPs and (F) Mc3-IONPs. The
e ical line shows he maximal accep ance (MA) poin . Embedded IONPs plo s o (G) Mc1-IONPs, (H) Mc2-
IONPs and (I) Mc3-IONPs.
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An exhaus i e analysis o he s abili y a ea o di e en amphiphile-based assemblies also p o ides key
in o ma ion abou he concen a ion o NPs equi ed o build he maximum numbe o well-o de ed hyb id
sup amolecula s uc u es. The polydispe si y index (PDI) o he Mc-IONPs s. concen a ion o NPs g aphs
show a minimum PDI alue, e e ed o as he maximal accep ance NP (MA) poin , co esponding o he
comple e embedding o he added IONPs in he medium (Fig.5D-F). These hyb id sup amolecula s uc u es
wi h he minimum PDI alue could be conside ed op imal building blocks o cons uc ing ad anced unc ional
ma e ials. The e o e, wi hin he s abili y a ea, adding ewe NPs han he MA poin esul s in highe PDI alues
(le side o he MA poin ) due o he p esence o sup amolecula assemblies wi hou IONPs. Con e sely, adding
a highe numbe o NPs also leads o highe PDI alues ( igh side), indica ing he onse o agg ega ion, o ming
a hi d a ea in he assembly p ocess consis ing o agg ega es and/o p ecipi a es. The h ee oleyl-based assemblies
exhibi ed simila MA poin s, 27.2 × 1012 IONPs pe mL, 24.4 × 1012 IONPs pe mL, and 26.1 × 1012 IONPs pe
mL o he assemblies Mc1-IONPs, Mc2-IONPs, and Mc3-IONPs, espec i ely (Fig.5D-F).
A mo e de ailed analysis o he s abili y a ea da a also showed he maximal concen a ion (MC) o IONP
o he di e en sup amolecula assemblies (Fig.5G-I). This alue, named as MC, indica es he maximal
capaci y o he amphiphilic molecules o embed and wa e -s abilize IONPs in he assemblies be o e eaching
he agg ega ion a ea. The sys em Mc2-IONPs exhibi ed he highes maximal capaci y o embedding IONPs,
18.1 × 1012 IONPs pe mL, ollowed by he Mc1-IONPs and Mc3-IONPs wi h 13 × 1012 IONPs pe mL and
7.5 × 1012 IONPs pe mL, espec i ely. These esul s a e consis en wi h he CMC alues ob ained o he oleyl-
based amphiphilic molecules, indica ing ha he bis-TEG pola head amphiphile II exhibi s he bes capaci y o
sel -o ganiza ion. Addi ionally, quan i ying he embedded IONPs in he assemblies a he MA poin allows us o
calcula e he e iciency o he amphiphilic molecules in he sel -assembling well-o de ed hyb id nanos uc u es.
Amphiphile II showed he highes e iciency a 70%, ollowed by amphiphilic molecules I and III wi h 53% and
31%, espec i ely. The e iciency was calcula ed as shown in he Suppo ing In o ma ion.
F om he da a abo e, we calcula ed he numbe o amphiphilic molecules pe NP and pe nm2 o NP. The
sup amolecula assemblies a maximal NP concen a ion exhibi ed 10 and 5 amphiphiles pe nm2 o he Y-shape
amphiphiles based Mc3-IONPs and Mc2-IONPs, espec i ely, and 10 amphiphiles pe nm2 o he Mc1-IONPs.
The s abili y o hese hyb id sup amolecula s uc u es Mc-IONPs, ob ained h ough he sel -assembly
me hods discussed abo e, was measu ed o e ime a Camphiphiles < CMC. I was ound ha he HD sizes o he
hyb id assemblies I and II emained unal e ed o wo mon hs (Figu e S24). Howe e , he nanosys em Mc3-
IONPs assembled by amphiphile III exhibi ed la ge sizes a e ha ime, o ming small agg ega es due o a
poo hyd ophilic/hyd ophobic balance o he amphiphilic molecule (Fig.6). In e es ingly, hese esul s clea ly
demons a e he o ma ion o s able hyb id nanos uc u es h ough s ong in e digi a ion o he amphiphiles
in o he IONP su aces.
Al hough he linoleyl-de i ed amphiphilic molecules IV and V a e unable o sel -o ganize in o micelles, hey
o m lipid nanopa icles. S udies we e also pe o med on he NP-d i en assembly o his amily wi h a double
alkene (cis, cis) in he alkenyl-based lipid ail (Fig.7). The hyb id sys ems LNP1-IONPs and LNP2-IONPs, based
on he amphiphilic molecules IV and V, espec i ely, exhibi ed a s abili y a ea simila o ha obse ed in he
s udies wi h hei mono alkene amphiphilic analogues I and II. In e es ingly, he HD sizes o 150nm and 220nm
o LNP1-IONPs and LNP2-IONPs, espec i ely, we e smalle han he esul ing LNPs wi hou he p esence
o IONPs. This indica es ha he p esence o IONPs du ing he sel -assembly p ocess se es as a empla e o
he a angemen o hese amphiphiles o e he IONPs in small assemblies. On he o he hand, he maximal
accep ance NP poin , e e ed o as he MA poin , was de e mined by obse ing he in e naliza ion o IONPs
in he assemblies wi hin he s abili y a ea (highligh ed in he colou ed egion in Fig.7A-B). As commen ed
abo e, in he amily o mono-alkene-based amphiphilic molecules, hese MA poin s we e de e mined by he
minimum PDI alue exhibi ed by he hyb id assemblies Mc-IONPs in he s abili y a ea. Since he double alkene
(cis, cis) lipid-based amphiphilic molecules a e no able o sel -o ganize in o well-o de ed s uc u es bu o m
small agg ega es as LNPs, he PDI alues a e no use ul as an agg ega ion pa ame e . The e o e, he MA poin s
o he hyb id LNPs we e de e mined by he maximal embedding o IONPs in he s abili y a ea (Fig.7C-D). In
bo h LNPs-IONPs, he amphiphilic molecules IV and V exhibi ed MA poin s o 48 × 1012 IONPs pe mL and
34 × 1012 IONPs pe mL o he assemblies LNP1-IONPs and LNP2-IONPs espec i ely, sligh ly highe alues
han hei mono-alkene based amphiphilic analogues. Rega ding he MC o NP, he hyb id nanosys ems Mc-
IONPs p esen ed highe alues, 175- old g ea e han hei linoleyl analogues, 7.4 × 1010 IONPs pe mL and
4.3 × 1011 IONPs pe mL o he LNP1-IONPs and LNP2-IONPs espec i ely. This signi ican di e ence is due
o he g ea e igidi y o he double alkene (cis, cis) lipid ail, which limi s and complica es hei a angemen
in o mo e o de ed and s able sup amolecula s uc u es. Mo eo e , TEM analysis o he assemblies LNP-IONPs
wi hin he s abili y a ea demons a ed he p esence o small agg ega es o IONPs, which indica es he o ma ion
o hese s uc u es (Fig.7E-F).
Sel -assembly in liposomes o amphiphilic molecules wi h mono alkenyl-based amphiphilic
molecules
The oleyl-de i ed amphiphilic molecules I, II and III also demons a ed he abili y o sel -o ganize in o la ge
sup amolecula s uc u es, namely Lipo1, Lipo2 and Lipo3, espec i ely, compa ed o hose commen ed abo e.
In all cases, hey exhibi ed simila HD sizes, be ween 336 and 368nm by DLS, which is likely due o he sligh
a ia ion in composi ion used in he liposome o ma ion p ocess, speci ically a low p opo ion o he amphiphile
compa ed o oleic acid (a a a io 5:95) (Fig.8). The p esence o IONPs in he sel -assembly p ocess a ec ed poo ly
o he size and mo phology o he esul ing liposomes Lipo-IONPs (Figu e S25). The small size o he IONPs
allowed hem o be inco po a ed in o he hyd ophobic laye o he liposomes wi hou signi ican ly al e ing hei
size and shape. TEM analysis o he sup amolecula s uc u es, based on he amphiphile II, demons a ed he
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o ma ion o liposomes wi hou and wi h he p esence o he IONPs (Fig.8A-C). These hyb id s uc u es Lipo-
IONPs exhibi ed high s abili y o e ime, wi h no obse a ion o agg ega es.
Applica ion o hese hyb id sup amolecula s uc u es as MRI con as agen s
Al hough he p ima y goal o his wo k is he cons uc ion o o de ed hyb id sup amolecula s uc u es, we
analyzed he capaci y o hese sys ems as MRI con as agen s. The e ec i eness o an MRI con as agen in
dec easing he T2 elaxa ion ime o su ounding wa e p o ons is quan i ied by a pa ame e known as ans e se
elaxi i y, 2, exp essed in mM− 1 s− 1. The hyb id sys ems Mc-IONPs and Lipo-IONPs, based on mono-alkene
lipids, we e chosen due o hei be e size a angemen and con olled mo phology. Thus, he ans e se
elaxi i y, 2, o he sup amolecula s uc u es o med h ough a micella p ocess was measu ed. The 2 alues
o he oleyl amily o sup amolecula s uc u es Mc-IONPs exhibi ed a end dependen on he hickness o he
assemblies o he amphiphilic molecules o e he IONPs. These esul s showed ha he hyb id assembly Mc2-
IONPs exhibi ed he highes 2 alue o his ype o s uc u es, 6.2 mM− 1·s− 1 (Table1). This assembly showed
he smalles size wi hin he amily, wi h he hinnes hickness o amphiphiles o e he IONPs. Howe e , he
hyb id nanos uc u es Mc1-IONPs and Mc3-IONPs esul ed in 2 alues o 4.6 and 3.8 mM− 1·s− 1, espec i ely,
exhibi ing lowe 2 alues due o he hicke hyd ophobic laye 40.
On he o he hand, he hyb id liposomes Lipo-IONPs exhibi ed highe 2 alues compa ed o he o de ed
hyb id s uc u es Mc-IONPs commen ed abo e (Table1). This e ec is likely a ibu ed o he hinne hickness
o he hyd ophobic laye in which he IONPs a e embedded. In ac , he Lipo1-IONPs and Lipo2-IONPs, based
on mono-lipid amphiphiles, exhibi ed 1.5- old and 2.6- old highe 2 alues, espec i ely. Howe e , he Lipo3-
IONPs, based on he bis-lipid amphiphile, collapsed du ing he measu emen s.
Conclusions
We ha e demons a ed ha he p esence o double bonds in he lipid ail o low MW amphiphilic molecules,
wi h di e en mo phologies in hei design, can lead o highly wa e -soluble, s able and well-o de ed hyb id
Fig. 6. S abili y s udies a e wo mon hs o he hyb id nanos uc u es Mc-IONPs. (A) Size s. sonica ion
ime g aph o he Mc1-IONPs in wa e ob ained om oluene as apola sol en . Size s. [IONP]hexane g aphs
o (B) Mc1-IONPs, (C) Mc2-IONPs and (D) Mc3-IONPs. Ini ial measu emen s, = 0 (in black) and a e
wo mon hs (in ed). (E) Summa y able o he Mc-IONPs, ob ained om oluene as s a ing apola sol en ,
con aining he sizes de e mined by DLS in wa e .
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