Supramolecular strategies for intracellular delivery and design of new thermoresponsive materials

TESIS DOCTORAL
SUPRAMOLECULAR STRATEGIES
FOR INTRACELLULAR DELIVERY
AND DESIGN OF NEW
THERMORESPONSIVE
MATERIALS
Héc o Fe nández Ca o
ESCUELA DE DOCTORADO INTERNACIONAL
PROGRAMA DE DOCTORADO EN CIENCIA Y TECNOLOGÍA QUÍMICA
SANTIAGO DE COMPOSTELA
AÑO 2020
DECLARACIÓN
DEL AUTOR DE LA TESIS
SUPRAMOLECULAR STRATEGIES FOR INTRACELLULAR DELIVERY
AND DESIGN OF NEW THERMORESPONSIVE MATERIALS
Pa a de ensas elemá icas
D. Héc o Fe nández Ca o
P esen o mi Tesis, siguiendo el p ocedimien o adecuado al Reglamen o, y decla o que:
1) La Tesis aba ca los esul ados de la elabo ación de mi abajo.
2) De se el caso, en la Tesis se ha á e e encia a las colabo aciones que u o es e abajo.
3) La Tesis es la e sión de ini i a p esen ada pa a su de ensa y coincide con la e sión en iada en
o ma o elec ónico.
4) Con i mo que la Tesis no incu e en ningún ipo de plagio de o os au o es ni de abajos
p esen ados po mí pa a la ob ención de o os í ulos.
Y me comp ome o a p esen a el ejempla imp eso de la Tesis en el plazo de un mes desde que la EDIUS
me lo equie a, así como del Comp omiso Documen al de Supe isión en el caso que el o iginal no es é
deposi ado en la Escuela.
En San iago de Compos ela, ... de ..... de 2020
Fdo. Héc o Fe nández Ca o
AUTORIZACIÓN
DEL
DIRECTOR
/
TUTOR DE LA
TESIS
SUPRAMOLECULAR STRATEGIES FOR INTRACELLULAR
DELIVERY AND DESIGN OF NEW THERMORESPONSIVE
MATERIALS
D. Juan Ramón G anja Guillán
D. Ja ie Mon eneg o Ga cía
INFORMAN:
Que la p esen e Tesis se co esponde con el abajo ealizado po D. Héc o Fe nández Ca o, bajo nues a di ección,
y au o izamos su p esen ación, conside ando que eúne los equisi os exigidos en el Reglamen o de Es udios de
Doc o ado de la USC, y que como di ec o es de es a no incu e en las causas de abs ención es ablecidas en la Ley
40/2015.
En San iago de Compos ela, ... de ..... de 2020
Fdo. Juan Ramón G anja Guillán
Fdo. Ja ie Mon eneg o Ga cía

Ag adecimien os
En p ime luga , me gus a ía exp esa mi eno me ag adecimien o a los di ec o es de es a
Tesis doc o al, Ja ie Mon eneg o y Juan R. G anja. G acias po da me es a opo unidad y
pe mi i que odo es o haya sido posible.
A Ja ie Mon eneg o, po da me la opo unidad de o ma pa e de es e p oyec o. G acias
po asmi i nos día as día u pasión po la in es igación, po us mul iples enseñanzas pa a
pode desa olla in es igación de al a calidad y po u apoyo a ni el pe sonal en los momen os
di íciles. Toda ía ecue do mis inicios cuando me enseñabas a hace mis p ime as columnas
c oma og á icas. Sin duda, odo lo conseguido ha sido u o de innume ables ho as de abajo,
es ue zo y ap endizaje. G acias po con ia en mí.
A Juan R. G anja, po us innume ables enseñanzas, ideas pa a mejo a el desa ollo de los
p oyec os y discusiones cien í icas en los semina ios. Po ansmi i nos u eno me igo
cien í ico y po enseña nos y demos a nos los alo es que debe cumpli un excelen e
in es igado y p o eso . Po enseña nos que de los e o es ambién se ap ende. Muchas g acias.
Ag adece a la Xun a de Galicia la concesión de mi beca p edoc o al (ED481A-2017/047)
que me ha pe mi ido desa olla es a Tesis doc o al y po habe me dado la opo unidad de
ealiza una es ancia p edoc o al en No hwes e n Uni e si y. Además, ambién me gus a ía da
las g acias a odas las ins i uciones que han inanciado la labo in es igado a del g upo du an e
es os años.
A Alejand o Méndez, po odo lo que me has enseñado. Po posee esa capacidad pa a
econduci los p oyec os y pa a hace que lo di ícil pa ezca ácil. A I ene Los alé, po enseña me
a hace los p ime os expe imen os en células y explica me odo lo necesa io pa a pode en ende
la pa e biológica de los p oyec os. A Julián Be guei o, po con ia en mí y pe mi i me ap ende
de í, día as día, en los dis in os p oyec os que compa imos. Muchas g acias.
A los doc o es Manuel Amo ín, Rebeca Ga cía-Fandiño y al p o . Luis Cas edo. Po las
in e esan es discusiones cien í icas du an e los semina ios de g upo. A Pa icia Lago, po u po
u ayuda y amabilidad a la ho a de esol e cualquie ipo de ges ión.
Al p o . Na han Gianneschi po habe me b indado la opo unidad de ealiza una es ancia
en su g upo de in es igación en No hwes e n Uni e si y, E as on, Illinois. A And ea S. Ca lini,
Mollie A. Tou e, Ma hew P. Thompson, Wei Cao, Ma y F. Cassidy, Spence Bu on, Claudia
Ba is ella, Hao Sun, Ch is Fo man, Wonmin Choi y al es o del g upo po habe me ayudado a
c ece como in es igado y acompaña me en es a g an expe iencia.
Al p o . Jose Luis Masca eñas po sus ideas, consejos y ayuda en el desa ollo del p oyec o.
A Miguel Ma ínez-Cal o po su ayuda en la p epa ación de los anspo ado es y, ambién, a
Jesús Mosque a. Al p o . Jona han R. Ni schke y su g upo de in es igación po la p epa ación
de la caja sup amolecula .
A Rebeca Menaya-Va gas po su ayuda con los cul i os celula es y su eno me in e és y
dedicación pa a que odo salga siemp e a la pe ección.
A la RIAIDT de la Uni e sidad de San iago de Compos ela po pone a mi disposición sus
medios écnicos pa a el co ec o desa ollo de es a Tesis doc o al. En especial me gus a ía
des aca a Es eban Gui ián po odos los análisis de espec ome ía de masas ealizados.
Además, me gus a ía ag adece a odo el pe sonal écnico del CIQUS, en especial a A cadio,
Lau a, Pablo y Noela.
Me gus a ía ag adece le eno memen e a odas/os y cada una/o de mis compañe as/os de
labo a o io, que después de odo es e iempo, engo el place de, a muchas/os, pode llama las/os
amigas/os.
A Ale, c eo que ninguna palab a puede ag adece odo lo que has hecho po mí du an e
es os años, ya no solo odo lo que me has enseñado en el labo a o io, sino como amigo, me has
hecho ei , has sabido comp ende me y anima me en odo momen o. E es una pe sona y un
amigo imp esionan e y e deseo odo lo mejo po que e lo me eces.
A Al onso, g acias po eco e es e du o camino conmigo. Pienso en el pasado y ecue do
millones de buenos momen os, chis es, isas, iajes, ies as, es i ales, con e saciones en el
desie o. Pe o, sin duda, lo que nos ha hecho se g andes amigos es el man ene nos unidos y
anima nos en los malos momen os. Muchas g acias po odo, mago.
A Ma isa, po enseña me y cuida me desde que llegué al labo a o io y ayuda me a con ia
en el pép ido. Ma isa, con ía en el pép ido. A Juanillo, po enseña me en los inicios y que e
siemp e lo mejo pa a mí. A I án, po odos esos ab azos y animos econ o an es, po las
gominolas a úl imas ho as de la a de.
A Jose Juan ¨Jose e¨, po ayuda me cuando las cosas no salían, po hace me ei día a día,
po aconseja me como a un hijo, po u o ma de aplica ma emá icas. A Ma a, po
acompaña me en es e camino y pe mi i me obse a en p ime a pe sona la g an in es igado a
en la que e has con e ido. A I ene, po ayuda nos siemp e en odo lo necesa io, po us b omas.
A Albe o, po us millones de consejos, po ayuda me con el p oyec o inicial y con muchas
o as cosas, po con a nos miles de eces la misma his o ia, haciéndonos c ee , a eces, que u
e as el p o agonis a, po los pa idos de padel mañane os. A Ál a o, po se pa e del g upo, po
nues as comidi as y ca eci os, po p opo ciona nos siemp e cualquie eac i o.
A Julián, po c ea en menos de un segundo millones de ideas pa a millones de nue os
p oyec os, po con ia en mí pa a los nue os p oyec os, po ene siemp e un chis e malo pa a
compa i . A Gee , po llega al labo a o io odos los días con una son isa. También a Ma ie e,
g acias po u eno me gene osidad. A Rebe, po us ganas de ap ende y po suminis a me
chocola e en iempos de esc i u a. También, a Jose Ma ía.
A Lamas, po odos los g andes momen os compa idos, po demos a nos como se hace
un buen e és en enis. A Nacho, po las clases p ác icas, po las isas y b omas compa idas,
po us inespe ados lanzamien os de pipas. A Giulia, po odas las con e saciones, po
enseña nos i aliano, po o ganiza las mejo es cenas y ies as. A Ma ín Cal elo, po odas las
anécdo as g aciosas que i imos, po o ma pa e del equipo ámi e.
A E i a, po odas las con e saciones de camino al abajo, aunque, a eces, e quedases
do mida, po u apoyo y us enseñanzas. A Ángel, po u bondaz, u es ue zo y dedicación que
me han enseñado que odo es ue zo, al inal, iene su ecompensa. A Vicky, po i adia esa
aleg ía ca ac e ís ica. A Fede, po u gene osidad, espe o que odo aya inc eíble en u nue a
e apa. A Alicia, po se una magni ica ecina de i ina. A I ia, po ayuda me en los inicios. A
los más jó enes, Sand a, Ma cos, Ezequiel, An ía, Cha lene e c. es oy segu o que os i á muy
bien. A Richa d, Ghibom y a Sahnawaz, po odos los momen os compa idos.
Además, ambién, a odos los compañe os de o os g upos de in es igación, en especial a
Jaime, po anima me día as día, po espe a me mien as acababa los expe imen os an es de i
a en ena , po se el g an capi án. También a Felipe, po ecibi me siemp e con una son isa y
algo nue o que con a , po compa i u sabidu ía. A Tomás Pose, po anima me siemp e a
supe a mis limi es, po los ma a ones noc u nos a la ciudad de la cul u a y uel a. A Jacobo, a
Da id y a Ma c, po odas las cha las siemp e di e idas. A Bea, po a a me siemp e an bien.
A odas las demás pe sonas que han con ibuído en mayo o meno medida al desa ollo de
es e pe íodo de mi ida y no los haya ci ado an e io men e. Todas es as pe sonas han apo ado
su g ani o de a ena, an o den o como ue a del labo a o io, en es a Tesis. Muchísimas g acias
a odos.
En ando en un ámbi o más pe sonal, me gus a ía ag adece le odos los animos ecibidos y
expe iencias i idas a odos mis amigos/as du an e es os años.
A mis amigos que conocí hace ya muchos años en la uni e sidad y oda ía o man pa e
de mi ida. En especial oy a des aca a los pila es básicos en mi ida:
A And ea, po acompaña me en mi desa ollo como in es igado y, a la ez, pe sonal, po
hace e apo a se odos los p oblemas con un pa de palab as, po u empuje y mo i ación
du an e odos es os años.
A Bus elo, po se el cla o ejemplo de op imismo y ansmi í selo a oda la gen e que e
odea. Tengo sue e de ene e ce ca.
A Ma ín, po acudi al esca e siemp e que lo necesi o y hace me ei has a más no pode .
A Reims, po comp ende me y aconseja me, a Rocío, a Ne ea y a And ea López po su
apoyo con inuado.
A mis amigos de oda la ida, en especial a Rica. Siemp e dispues os a aconseja me,
ayuda me y i i nue as expe iencias. Po muchos años que pasen conse amos nues a
amis ad y eso es un au én ico eso o. A mis compañe os/amigos del equipo de ú bol, que me
pe mi is eis libe a odas las ensiones del doc o ado y llega a o ma pa e de ues a ¨ amilia¨.
A Ca liños, po a a me an bien y enseña me an o inglés.
A Jo ge, po con e i e en una pieza muy impo an e en mi ida, po eco e el mundo
conmigo, po que e siemp e lo mejo pa a mí, po ega la me siemp e los mejo es consejos.
A Sa a, g acias po ansmi i me odos los días us ganas de i i y u aleg ía, po
con e i e en la pe ec a compañe a de a en u as, expe iencias y iajes, po i a de mi cuando
las cosas no salen bien, po hace que es e baile alga la pena. G acias po odo lo i ido, ha
sido apasionan e. Me aleg a pensa odo lo que nos queda po i i . Ag adece , ambién, a Ana,
Césa y Ani a odo el apoyo que me habéis b indado y po a a me siemp e an bien.
Po úl imo, me gus a ía dedica le unas líneas a mi amilia po apoya me y anima me en
odo momen o. A mis abuelos, en especial a mi abuela Lola, que me demues a día a día que la
edad no impo a sino las ganas de i i . A mi sob ino Ál a o, que desde que llegó a la amilia,
nos ha p opo cionado un eno me soplo de ai e esco. A Luis. A mi he mana, Jessica, siemp e
dispues a a ayuda me y, sin duda, uno de mis g andes apoyos en la ida. A mis pad es, po
a anza en iempos en los que sólo es udiaban algunos p i ilegiados, po p ome e se, has a
consegui lo, que sus hijos, al e és que ellos, pod ían i a la uni e sidad y po o ma es a
amilia de la cual es oy an o gulloso. G acias po an o.

Abb e ia ions
17
Abb e ia ions
δ
ζ
λem
λex
Aa
Abs
Ac
Ad
ACPPs
ACN
AFM
Ahx
Ala
an i-EGFR
A g
ATRP
AuNPs
BCECF
BCECF-AM
Boc
b s
13C-NMR
CA
calcd
CB
β-CD
CD
CDs
β-CD@AuNPs
CD@AuNPs
chemical shi (ppm)
ze a
emission wa eleng h
exci a ion wa eleng h
amino acid
abso bance
ace yl
adaman ane
ac i able cell-pene a ing pep ides
ace oni ile
a omic o ce mic oscope
6-aminohexanoic acid
alanine
an i-epide mal g ow h ac o ecep o
a ginine
a om ans e adical polyme iza ion
gold nanopa icles
2′,7′-Bis(2-ca boxye hyl)-5(6)-ca boxy luo escein
2′,7′-Bis(2-ca boxye hyl)-5(6)-ca boxy luo escein
ace oxyme hyl es e
e -Bu oxyca bonyl
b oad single
13C nuclea magne ic esonance
calixa ene
calcula ed
cucu bi u il
β-cyclodex in
ci cula dich oism
cyclodex ins
β-cyclodex in-modi ied AuNPs
cyclodex in-modi ied AuNPs
Héc o Fe nández Ca o
18
CDP
CF
CP
CPPs
CPT
CRISPR
CTC
d
DCM
DLS
DNA
DMEM
DMSO
DIEA
DMF
DP
DYN
E9
EDC
EDTA
EBPs
EBPPA
ELPs
EPR
EYPG
Fab′
FBS
FDA
FITC
Fmoc
FRET
FSC
Gln
cyclodex in-con aining polyme
ca boxy luo escein
cyclic pep ide
cell-pene a ing pep ides
camp o hecin
clus e ed egula ly in e space sho palind omic epea s
2- chlo o i yl chlo ide
double
dichlo ome hane
dynamic ligh sca e ing
deoxy ibonucleic acid
Dulbecco's Modi ied Eagle Medium
dime hylsul oxide
N, N-Diisop opyle hylamine
N, N-Dime hyl o mamide
deg ee o polyme iza ion
dynaso e
nona-L-glu amic acid
1-e hyl-3-(3-dime hylaminop opyl)ca bodiimide hyd ochlo ide
e hylenediamine e aace ic acid
elas in-based polyme s
elas in-based side-chain helical poly(phenylace ylene)
elas in-like polypep ides
enhanced pe meabili y and e en ion
egg yolk phospha idylglyce ol
an igen-binding agmen
e al bo ine se um
ood and d ug adminis a ion
luo escein iso hiocyana e
9- Fluo enylme hoxyca bonyl
luo escence esonance ene gy ans e
o wa d sca e channel
glu amine
Abb e ia ions
19
Gly
Glu
GSH
1H-NMR
HEPES
HFIP
His
HKR
HPLC-MS
HPTS
HR-MS
ICP-MS
J
Ka
Kd
LCST
Leu
Lys
m
MeOH
MES
mRNA
MS
MTT
M
N-HATU
N-HBTU
NHS
glycine
glu amic acid
glu a hione
p o on nuclea magne ic esonance
4-(2-hyd oxye hyl)-1-pipe azinee hanesul onic acid
1,1,1,3,3,3-hexa luo o-2-p opanol
his idine
HEPES-K ebs-Ringe bu e
high-pe o mance liquid ch oma og aphy coupled wi h mass
spec ome y
8-hyd oxypy ene-1,3,6- isul onic acid (py anine)
high- esolu ion mass spec ome y
induc i ely coupled plasma mass spec ome y
coupling cons an
a ini y cons an
dissocia ion cons an
lowe c i ical solu ion empe a u e
leucine
lysine
mul iple
me hanol
2-(N-mo pholino)e hanesul onic acid
messenge RNA
mass spec ome y
(3-(4,5- dime hyl hiazol-2-yl)-2,5-diphenyl e azolium b omide)
e azolium;
4-me hyl i yl
N-[(Dime hylamino)-1H1,2,3- iazolo[4,5-b]py idine
1ylme hylene]-N-me hylme hanaminium-hexa luo ophospha e
N-oxide;
N-[(1HBenzo iazol-1-yl)-(dime hylamino)me hylene]-N-
me hylme hanaminium hexa luo ophospha e N-oxide
N-hyd oxysuccinimide
Héc o Fe nández Ca o
20
NMR
NPs
O2Oc
PAMAM
Pb
PBS
Phe
pDNA
PEG
PEI
PNAs
PNA-AuNPs
PNIPAM
POEG
Ppm
P o
PyAOP
PVCL
RAFT
R4
R8
RGD
RP-HPLC
R
s
SD
Se
SCID
SCPNs
siRNA
sgRNA
shRNA
nuclea magne ic esonance
nanopa icles
8-amino-3,6-dioxaoc anoic acid
polyamidoamine dend ime s
2,2,4,6,7-Pen ame hyldihyd obenzo u an-5-sul onyl
phospha e-bu e ed saline
phenylalanine
plasmid DNA
polye hylene glycol
polye hyleneimine
pep ide nucleic acids
poly alen nucleic acid capped AuNPs
poly(N-isop opylac ylamide)
poly(oligoe hyleneglycol)
pa s pe million
p oline
(7-Azabenzo iazol-1-yloxy) ipy olidinophosphonium
hexa luo ophospha e;
poly(N- inlycap olac am)
e e sible addi ion− agmen a ion chain- ans e
e aa ginine
oc aa ginine
a ginine-glycine-aspa ic acid pep ide
e e sed-phase high-pe o mance liquid ch oma og aphy
e en ion ime
single
s anda d de ia ion
se ine
se e e combined immunode iciency
sel -assembling cyclic pep ide nano ubes
small in e e ing RNA
single guide RNA
small hai pin RNA
Abb e ia ions
21
SPNs
SPPS
TAMRA
TCP
TEM
TF
TFA
TFE
TFRC
TGA
TIS
TNF
TRAIL
UCST
uHPLC
UV- is
Val
sup amolecula nanopa icles
solid-phase pep ide syn hesis
iple
5-ca boxy e ame hyl hodamine
cloud poin empe a u e
ansmission elec on mic oscopy
ansc ip ion ac o
i luo oace ic acid
i luo oe hanol
ans e in ecep o
he mog a ime ic analysis
iisop opylsilane
umo nec osis ac o
umo nec osis ac o - ela ed apop osis-inducing ligand
uppe c i ical solu ion empe a u e
ul a-high pe o mance liquid ch oma og aphy
ul a iole - isible
aline

Summa y
23
Summa y
Syn he ic o ganic chemis y has played a key ole in he de elopmen o no el s a egies
o ob aining chemically unc ional compounds. Despi e he g ea ad ance ha o ganic
syn hesis has unde gone in he las decades, co alen s a egies p esen in insic limi s o
achie ing s uc u es wi h la ge dimensions such as a chi ec u es commonly ound in Na u e,
such as hose o med by he assembling o p o eins. The p epa a ion o hese complex
molecules by only using s a egies based on he o ma ion o co alen bonds is almos
unapp oachable om he poin o iew o ime and human e o equi ed oge he wi h he lack
o app op ia ed syn he ic s a egies. Addi ionally, ce ain collec i e phenomena such as sel -
o ganiza ion equi e he coo dina ed in e ac ion be ween di e en chemical en i ies, which is
ou side he scope o adi ional co alen chemis y. Acco dingly, a no el discipline coined
sup amolecula chemis y eme ged in he 70-80’s. This ield was i s de ined by Jean-Ma ie
Lehn as ¨ he chemis y beyond he molecule¨. This discipline ocuses on he s udy o
in e ac ions be ween di e en chemical en i ies h ough non-co alen in e ac ions, which
include Van de Waals o ces, hyd ogen o halogen bonding, ion-ion/dipole, dipole-dipole,
ca ion o anion π in e ac ions, o π-π in e ac ions. This s a egy allows he cons uc ion o
ex emely complex h ee-dimensional s uc u es om syn he ically simple p ecu so s.
Fu he mo e, he use o sup amolecula chemis y con e s o hese s uc u es many di e en
ad an ages such as e e sibili y, sel -healing, o adap i e esponse o di e en s imuli. Fo he
p epa a ion o no el sup amolecula sys ems mainly wo p ocesses ha e been explo ed he
molecula ecogni ion and sel -assembly. Molecula ecogni ion has been de ined as he
speci ic binding o a gues en i y o a complemen a y hos molecule o gene a e a hos -gues
complex by using non-co alen in e ac ions. Impo an ly, in some examples, hos -gues binding
cons an s ha e eached alues almos compa able o co alen bonds. Howe e , hese hos -gues
complexes s ill main ain he ad an ages ha o e he use o sup amolecula chemis y. A
ele an example in his sense is he molecula ecogni ion o bio in by he p o ein, s ep a idin.
On he o he hand, sel -assembly has been de ined as a p ocess by which a non-o ganized
sys em o molecula componen s, o a pa o a molecule, as a consequence o non-co alen
in e ac ions, spon aneously gene a es an o ganized s uc u e. Na u e has also used molecula
sel -assembly o achie e complex and unc ional s uc u es such as he double helix s uc u e
o DNA, he assembly o he obacco mosaic i us, o he cons uc ion o lipid memb anes.
Since he day o i s incep ion o nowadays, sup amolecula sys ems ha e gained momen um in
he de elopmen o unc ional ma e ials, such as di e se deli e y and he mo esponsi e
sys ems.
In his Ph.D. disse a ion, we ha e applied sup amolecula s a egies o p omo e
in acellula deli e y o di e se molecules and design po en ial he mo esponsi e sys ems.
Hos -gues chemis y has been exploi ed o encapsula e and deli e di e en anionic p obes
such as py anine, as well as anionic pep ides wi h low sel -deli e y e iciency. Finally, we ha e
designed po en ial he mo esponsi e sup amolecula ma e ials based on sel -assembling cyclic
pep ide nano ubes (SCPNs).
Du ing he las decades, new p omising he apeu ic and diagnos ic molecules ha e been
syn hesized. Howe e , some o hese molecules p esen ed limi a ions o c oss he cellula
memb ane. To o e come his p oblem, a wide ange o cell deli e y sys ems ha e been
desc ibed o p omo e he in acellula in e naliza ion o di e en en i ies such as he apeu ic
nucleic acids, p o eins, o small molecules such as d ugs o luo escen p obes. Di e en
examples o deli e y sys ems commonly used o in e nalize biologically impo an
Héc o Fe nández Ca o
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mac omolecules and small molecules a e liposomes, polyme s, o me al nanopa icles. A
ele an example o p omising deli e y ehicles is cell-pene a ing pep ides (CPPs). These
pep ides a e small-sized pep ides ha showed imp essi e capabili ies o in e nalizing a wide
a ie y o memb ane-impe meable he apeu ic and diagnos ic mac omolecules and also small
molecules in o he cell cy osol. CPPs wi h di e en physicochemical p ope ies ha e been
desc ibed such as ca ionic, hyd ophobic, o amphipa hic. Some ele an examples o widely
s udied CPPs a e pene a in, TAT, oc aa ginine, MPG, o PEP-1. Du ing he las decades,
di e en designs and s uc u al modi ica ions ha e been explo ed o imp o e he ansloca ion
capabili ies o hese molecules. An inno a i e s a egy used o p epa e new CPPs has been he
o ma ion o dynamic bonds such as oxime, hyd azone, o disul ide bonds. This s a egy
allowed o p epa e a lib a y o CPPs in a sho pe iod o ime, la ge quan i ies, and high yields.
Fo ins ance, in ou g oup, we ha e used he alcoxyamine g oup o a aching di e en
elec ophilic aldehydes and o achie e he easy modula ion o he p ope ies o he model CPP.
Mo eo e , ou g oup has also explo ed he hyd azide g oup o he inco po a ion o di e en
hyd ophobic aldehydes such as oleic acid in o he CPP sca old. I was ound ha his
modi ica ion impo an ly imp o ed he ansloca ion o a pseudohelical model pep ide. The
inco po a ion o he ca go in o he deli e y sys em has been achie ed mainly by applying
co alen o non-co alen sup amolecula s a egies. Co alen a achmen o he ca go has been
adi ionally used o he p epa a ion o he d ug-deli e y sys em en i y. On he o he hand,
sup amolecula me hodologies ha e shown impo an ad an ages such as imp o emen o
ca go elease and educ ion o he syn he ic e o . Fo ins ance, cell-pene a ing pep ides ha e
been non-co alen ly a ached o he apeu ic p o eins such as Cas9 ibonucleop o ein, nucleic
acids such as siRNA o plasmid DNA, and small molecules such as d ugs o p obes.
In he i s chap e o his hesis, we ha e de eloped a no el gene a ion o highly e icien
and biocompa ible sup amolecula ehicles o he anspo o molecula p obes inside li ing
cells. Nega i ely cha ged molecula p obes such as py anine o Alexa dyes a e o in e es o
p obing he in acellula space. Howe e , he ine icien anspo o hese p omising molecules
ac oss he plasma memb ane, due o he epulsi e in e ac ions be ween molecula p obes and
he cellula memb ane, cu en ly emains a undamen al limi a ion. To o e come his p oblem,
we ha e demons a ed ha a hyb id sys em composed o a memb ane-impe meable
sup amolecula cage and a co alen ly ancho ed e aa ginine pep ide can be exploi ed o he
cy osolic deli e y o nega i ely cha ged luo opho es o di e en cell lines (HeLa and Ve o
cells). The sup amolecula cage C used o he p epa a ion o hese ca ie s shows a
isbipy idyl s uc u e and, in a p e ious epo by p o . Ni schke g oup, demons a ed o
encapsula e wi h high-a ini y di e en anions o la ge sizes, such as py anine, in an aqueous
en i onmen . Based on hese p e ious esul s, we p epa ed ace yla ed and luo escen ly labeled
(TAMRA) R4 pep ides by using solid-phase pep ide syn hesis, and hen he ca ionic
sup amolecula cage C was coupled by s anda d condi ions o amide bond o ma ion o
gene a e deli e y agen s AcR4C and TmR4C. Fluo escence expe imen s showed sup amolecula
complexa ion be ween hese hyb id ec o s and py anine p obe, wi h calcula ed dissocia ion
cons an s o Kd = 189 nM and Kd = 12.6 µM, espec i ely. In p e ious epo s by he g oup o
p o . Ma ile, in i o U- ube expe imen s showed he abili y o di e en molecules o ac as
ca ie s. In his expe imen , chlo o o m was placed a he bo om o a U- ube, and wo di e en
aqueous bu e s we e added a bo h sides o he o ganic phase (cis and ans bu e s). Then, he
ans e ence om cis o ans bu e was moni o ed. To assess anspo ac oss he model apola
o ganic sol en -chlo o o m-, we ca ied ou in i o U- ube expe imen s in he p esence o
na u ally occu ing phospholipids. Time-dependen luo escence measu emen s indica ed ha
he pep ide-cage ca ie AcR4C a o ed he anspo o py anine p obe om cis o ans aqueous
Summa y
25
phase. A e a pe iod o 12 h, he ans aqueous phase con ained 4 imes mo e py anine dye
when he pep ide ca ie was used in compa ison wi h con ol expe imen s. This indica ed ha
he pep ide-cage ca ie AcR4C ac s as a ca ie o he py anine p obe ac oss he bulk apola
laye . Deli e y expe imen s wi h py anine-complexed o AcR4C and TmR4C showed cy osolic
elease in HeLa and Ve o cells. Compe i ion assays in he p esence o a compe ing molecula
p obe (TAMRA) showed ha TmR4C was able o selec i ely in e nalize py anine in o he
cy osol by endocy osis. This mechanism o en y was con i med by expe imen s in he p esence
o endocy osis inhibi o s and by obse ing py anine-ca ie colocaliza ion by con ocal
mic oscopy expe imen s. We con i med he in eg i y o he cellula memb ane in he py anine
anspo by nuclea s aining. In hese expe imen s, a e he anspo expe imen s wi h he
p obe, we incuba ed he cells wi h DAPI and also wi h p opidium iodide, p obes ha would
cause nuclea s aining when he cellula memb ane is damaged. Mo eo e , compe i ion
expe imen s also ensu ed non-memb ane pe meabiliza ion. Finally, we ex apola ed his
s a egy o he deli e y o mul iple anionic p obes, namely ca boxy luo escein and Alexa Fluo s
488, 546, and 568, and we also con i med hei elease in he cy osolic medium by con ocal
mic oscopy expe imen s. On he o he hand, he modi ica ion o he physiological in acellula
pH has been desc ibed o be ela ed o he eme gence o di e en pa hologies such as cance .
Fo ins ance, cance cells p esen a sligh ly acidic pH compa ed o no mal cells. The e o e, he
me iculous s udy o he in acellula pH would allow a be e unde s anding o impo an
cellula p ocesses and diseases. A wide ange o molecules can be used o ca y ou in acellula
pH measu emen s. Fo ins ance, py anine p esen s wonde ul p ope ies and can be used as a
pH indica o . Howe e , agg essi e s a egies, such as elec opo a ion, ha e o be used o he
in acellula deli e y o his molecule. The e o e, in his chap e , we inally decided o use he
p e iously syn hesized ca ie s o e icien ly in e nalize py anine and o ack he in acellula
pH. Using con ocal mic oscopy expe imen s, we con i med he e iciency o he de eloped
me hodology o ca y ou he a iome ic in acellula pH acking using he py anine p obe.
In e es ingly, we we e able o disc imina e he pH o di e en cellula compa men s such as
he neu al pH o cy osol (pH: 7.5), he mode a ely acidic pH o he ea ly endosomes (pH: 7.0),
and he mo e acidic pH o la e endosomes (pH: 6.5).
In he second chap e , we ha e explo ed sup amolecula nanopa icles o he deli e y o
hyd ophilic pep ides. The e m sup amolecula nanopa icles (SNPs) has no mally been used o
de ine nanopa icles cons uc ed mainly by non-co alen in e ac ions. Sup amolecula
in e ac ions ha e been used o he unc ionaliza ion wi h di e en moie ies which p o ide
di e en unc ionali ies o he sys em. The sca olds used o cons uc hese ma e ials can be
so o ha d, which ha e ele an consequences no only in hei p ope ies bu also in hei
he apeu ic e ec . Sup amolecula nanopa icles based on so ma e ials ha e been used o he
in acellula deli e y o no only he apeu ic nucleic acids bu also small molecules such as
d ugs. In e es ingly, examples o SNPs ha ha e al eady eached clinical ials ha e been
epo ed. Mo eo e , co-deli e y using SPNs o di e en ca gos, such as small d ugs and nucleic
acids, has been also explo ed. On he o he hand, me allic nanopa icles ha e been widely used
and s udied in Nano echnology. Fo ins ance, hese pa icles ha e been used in he de elopmen
o no el d ug deli e y ehicles. Du ing he las decades, gold nanopa icles (AuNPs) ha e
shown applica ions in ca alysis, as senso s, in biomedicine, e c. Fo he de elopmen o e icien
and non- oxic he apeu ics, mul icomponen unc ionaliza ion o he nanopa icle su ace is
undamen al. Fu he mo e, he inco po a ion o e e sibili y by sup amolecula in e ac ions has
eme ged as an inno a i e app oach o he design o no el mul icomponen nanopa icles. A
ele an example is he combina ion o AuNPs and sup amolecula mac ocycles. The use o
sup amolecula in e ac ions allowed he p epa a ion o complex sys ems by easy mixing o he
Héc o Fe nández Ca o
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uncionalización con di e en es en idades que p opo cionan di e en es uncionalidades al
sis ema. Los ma e iales u ilizados pa a cons ui es os sis emas pueden se blandos o du os, lo
que iene consecuencias ele an es no solo en sus p opiedades sino ambién en su e ec o
e apéu ico. Las nanopa ículas sup amolecula es basadas en ma e iales blandos se han
u ilizado pa a el anspo e in acelula no solo de ácidos nucleicos e apéu icos sino ambién
de moléculas pequeñas como los á macos. Cu iosamen e, se han publicado ejemplos de SNPs
que han alcanzado ensayos clínicos. Además, ambién se han p epa ado SPNs que pe mi en el
anspo e in acelula simul áneo de di e en es ca gos, como á macos y ácidos nucleicos. Po
o o lado, las nanopa ículas me álicas han sido ampliamen e u ilizadas y es udiadas en
nano ecnología. Po ejemplo, es as pa ículas se han u ilizado en el desa ollo de nue os
ehículos de anspo e de á macos. Du an e las úl imas décadas, las nanopa ículas de o o
(AuNPs) han mos ado aplicaciones en ca álisis, como senso es, en biomedicina, e c. Pa a el
desa ollo de e apias al amen e e icien es y con baja oxicidad, la inco po ación de a ios
componen es en la supe icie de las nanopa ículas es undamen al. Además, el empleo de
in e acciones sup amolecula es se ha con e ido en un en oque inno ado pa a el diseño de
nue as nanopa ículas mul icomponen e. Un ejemplo ele an e es la combinación de AuNPs y
mac ociclos sup amolecula es. El uso de in e acciones sup amolecula es pe mi e la p epa ación
de sis emas complejos simplemen e mezclando los di e en es componen es. Además, es a
me odología e i a pasos sin é icos di íciles y pu i icaciones complicadas de los ma e iales
inales. Du an e los úl imos años, se han p epa ado AuNPs uncionalizadas con an i iones
mac ocíclicos como cucu bi u ilos o ciclodex inas (CDs). Po ejemplo, las CDs con ienen una
ca idad in e na al amen e hid o óbica, lo que hace que es as en idades sean excelen es
an i iones pa a un amplio ango de moléculas hid o óbicas, como los á macos hid o óbicos.
Du an e las úl imas décadas, el uso de nanopa ículas me álicas, basadas en química an i ión-
huésped, con aplicaciones e apéu icas ha susci ado un g an in e és en la comunidad cien í ica.
La o mación de complejos an i ión-huésped se ha u ilizado adicionalmen e en la
solubilización e in eg ación en nanopa ículas de di e en es á macos hid o óbicos como los
p o á macos de cispla ino, doxo ubicina, pacli axel o me o exa o. Sin emba go, la mayo ía de
es as es a egias han explo ado la libe ación e in e cambio de ca gos hid o óbicos. Has a donde
sabemos, aún no se ha in es igado la libe ación y el in e cambio dinámico en e pép idos
huésped hid o ílicos y nanopa ículas deco adas con an i iones. En es e p oyec o, uno de los
p incipales desa íos es la complejidad química de los pép idos, que puede conduci a
in e acciones inespecí icas como, po ejemplo, in e acciones elec os á icas o de an de Waals.
Po o o lado, adicionalmen e pa a la uncionalización de la supe icie de las nanopa ículas
con pép idos hid o ílicos se han empleado enlaces co alen e e in e acciones elec os á icas. Sin
emba go, ejemplos que u ilizan el econocimien o sup amolecula an i ión-huésped siguen
siendo limi ados.
El segundo capí ulo de es a Tesis, se desa olló una es a egia sup amolecula pa a la
inco po ación de pép idos y políme os hid o ílicos en β-CD@AuNPs median e el empleo de
p ocesos sup amolecula es an i ión-huésped. P epa amos nanopa ículas de o o ancladas a β-
ciclodex inas (β-CD@AuNPs) y las ca ac e izamos median e expe imen os de TEM, DLS,
UV-Visible y TGA. Los expe imen os de TGA indica on que es as poseen un p omedio de 20
β-CD po nanopa ícula, mien as que expe imen os de TEM y DLS demos a on la p epa ación
de nanopa ículas de un amaño de 2-3 nm y un diáme o hid odinámico de ap oximadamen e
8 nm. Se sin e izó una quimio eca de pép idos pene an es de células hid ó ilicos ( e a y oc a-
a ginina) modi icados con adaman anos, mono (AdR4 y AdR8) y di alen es (Ad2R4 y Ad2R8)
empleando la condensación alcoxiamina-aldehído en e el g upo alcoxiamina del pép ido y el
g upo aldehído del huésped. En es e abajo, elegimos el adaman ano (Ad) como huésped

Resumen
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debido a su ele ada cons an e de a inidad po el an i ión, β-CD. Po o o lado, los huéspedes
mono y di alen es se u iliza on pa a explo a el in e cambio dinámico de pép idos en la
supe icie de las β-CD@AuNPs median e e ec os es adís icos o mul i alen es. Expe imen os
de po encial ζ con i ma on la inco po ación de es os pép idos mono y di alen es en las β-
CD@AuNPs median e in e acciones especí icas an i ión-huésped. Además, los pép idos
con ol sin huésped no ecub ie on de o ma ele an e las β-CD@AuNPs. Los esul ados de
los expe imen os de po encial ζ se u iliza on pa a p opo ciona una idea es imada de las
cons an es de asociación an i ión-huésped, que se encon a on en un ango adecuado pa a la
in e acción indi idual Ad-CD. Además, la in e p e ación de las cons an es de asociación
apa en es calculadas indica que el p oceso p edominan e es una unión es adís ica o
in e acciones en e pa ículas. Además, los expe imen os de compe ición en p esencia de β-CD
lib e mos a on un descenso en el po encial ζ de las nanopa ículas que indica e e sibilidad
e modinámica. Po o o lado, los políme os biocompa ibles como el polie ilenglicol (PEG) se
han u ilizado pa a p e eni el ecub imien o de la supe icie de nanopa ículas con di e en es
p o eínas del medio biológico. Aquí, decidimos sin e iza y ca ac e iza cadenas de PEG
hid o ílicos mono (AdPEG) y di alen es (Ad2PEG), u ilizando la me odología sin é ica
desc i a an e io men e, pa a inco po a o a uncionalidad en el nanosis ema. Cons uimos
nanopa ículas mul icomponen e compues as po pép idos de polia ginina y cadenas de PEG
uncionalizados con el huésped adaman ano. Los expe imen os de amaño y po encial ζ en
p esencia de una mezcla de polia ginina y PEG mono alen es/di alen es sugi ie on que la
es abilización elec os á ica es la causan e de la es abilización del sis ema, mien as que el PEG
pod ía ene un e ec o nega i o al obs aculiza el alcance de alo es ele ados de po encial.
Exclusi amen e en zonas de es abilidad elec os á ica, la adición de PEG dio como esul ado
una modulación a o able del amaño. Po úl imo, plan eamos la hipó esis de que el in e cambio
de pép idos sob e las β-CD@AuNPs, median e in e acciones an i ión-huésped, pod ía lle a se
a cabo empleando pép idos que p esen asen una alencia supe io . Demos amos que la
o mación de complejos en e pép idos poliglu ámicos impe meables a la memb ana (AdE9) y
β-CD@AuNPs p omue e el anspo e in acelula de es os pép idos. Además, los
expe imen os de mic oscopía con ocal y los ensayos de ci ome ía celula con i ma on el
in e cambio de es os pép idos aniónicos AdE9 po pép idos di alen es como Ad2R8 en el
in e io de células i as.
El úl imo capí ulo de es a Tesis se ha cen ado en el desa ollo de nue os sis emas
nano ubula es sup amolecula es e mo esponsi os. Du an e las úl imas décadas, los sis emas
que esponden a los es ímulos, ambién llamados "ma e iales in eligen es", han a aído un g an
in e és debido a su capacidad pa a esponde a una amplia gama de es ímulos, como el pH, la
ue za iónica, la luz, el campo magné ico o eléc ico o la empe a u a. En e ellos, los ma e iales
e mo esponsi os, que poseen la capacidad de adap a se a los cambios de la empe a u a
ambien al, han demos ado nume osas aplicaciones biomédicas, como en e apia génica o en la
adminis ación de á macos. Se ha demos ado que la elas ina, que es una p o eína es uc u al,
p esen a p opiedades e mo esponsi as. La opoelas ina se de inió como el p ecu so de la
elas ina y su es uc u a p ima ia es á compues a po secuencias epe idas de pen apép idos. La
secuencia pep ídica más comúnmen e encon ada es Val1P o2Gly3Val4Gly5 o VPGVG. Du an e
las úl imas décadas, se han sin e izado polipép idos ipo elas ina (ELP) y políme os basados en
elas ina (EBP). Al in es iga es e ipo de ma e iales, se ha encon adon compo amien os LCST
( empe a u a de solución c í ica in e io ) o compo amien os UCST ( empe a u a de solución
c í ica supe io ) y múl iples aplicaciones, como po ejemplo como ehículos e mo esponsi os
pa a la in e nalización celula de á macos. Po o o lado, los nano ubos cons i uidos po
pép idos cíclicos (SCPNs) ep esen an uno de los ejemplos más ele an es de en idades
Héc o Fe nández Ca o
34
sup amolecula es. Po ejemplo, SCPNs se han p epa ado u ilizando D,L-α-ciclopép idos (D,L-
α-CP) que consis en en un núme o pa de aminoácidos con qui alidad al e nan e. El
au oensamblaje de pép idos cíclicos se basa en el apilamien o de múl iples subunidades
median e la o mación de una ed de enlaces de hid ógeno. Desde el descub imien o de los
SCPNs, se han lle ado a cabo di e en es modi icaciones es uc u ales y ambién se han desc i o
una amplia a iedad de aplicaciones. Los SCPNs han demos ado capacidades como análogos
de canales de iones de memb ana o como senso es de iones. Además, es as en idades
sup amolecula es se han u ilizado no solo como ehículos de adminis ación de á macos sino
ambién como agen es an ibac e ianos o an i i ales. Du an e las úl imas décadas, nues o g upo
de in es igación ha diseñado y sin e izado un amplio ango de es uc u as de pép idos cíclicos
de di e en es amaños capaces de au oensambla se en nano ubos adqui iendo una al a
expe iencia en es e campo. Recien emen e, en nues o g upo, se ha log ado demos a el
au oensamblaje de pép idos cíclicos den o de esículas. Más ecien emen e, la o mación de
nanohojas se ha demos ado en condiciones isiológicas y se ca ac e iza on como bicapas de
SCPNs. Pa a p opo ciona le a es os sis emas sup amolecula es la capacidad pa a esponde a
dis in os es ímulos, pép idos cíclicos se han uncionalizado con políme os que conducen a la
cons ucción de nano ubos basados en híb idos de pép idos cíclicos y políme os. Es os
ma e iales híb idos han demos ado esponde a es ímulos ambien ales como la empe a u a o
el pH. Sin emba go, la unión de los políme os a i iciales e mo esponsi os a pép idos cíclicos
ca ece de una es a egia sin é ica ácil que pe mi a la modi icación con olada de las
empe a u as de ansición y un es udio de allado del au oensamblaje de los SCPNs con la
a iación de la empe a u a. Además, cuando se usan en aplicaciones biomédicas, es os híb idos
cíclicos pép ido-políme o no malmen e p oducen oxicidad celula . Po lo an o, la unión de
cadenas pep ídicas e mo esponsi as, como la elas ina, a pép idos cíclicos pe mi i ía cons ui
no solo ma e iales sup amolecula es e mo esponsi os de una o ma más sencilla sino ambién
sis emas plenamen e biocompa ibles.
Po lo an o, en el e ce capí ulo de es a Tesis doc o al, hemos es udiado las di e en es
posibilidades pa a diseña pép idos cíclicos modi icados con elas ina con po enciales
capacidades pa a au oensambla se en nano ubos de pép idos cíclicos median e a iaciones en
el pH del medio. Es os pép idos cíclicos es án cons i uidos po ocho aminoácidos con qui alidad
al e nan e (D/L-α-CP) deco ados con una cadena de elas ina, que con ie e capacidades
e mo esponsi as al sis ema. Diseñamos es pép idos cíclicos base di e en es que poseen
di e en es aminoácidos sensibles al pH pa a p omo e el au oensamblaje a pH básico (CP1.1 y
CP1.2), a pH neu o (CP2.1 y CP2.2) y a pH ácido (CP3.1 y CP3.2). En es e diseño, decidimos
inco po a una cadena de elas ina a los pép idos cíclicos en dos o ien aciones di e en es: el
segmen o de elas ina na u al [H-(V-P-G-V-G-)OH] y la es uc u a in e sa, ambién
e mo esponsi a (H-(G-V-G-P-V-)OH). Además, ambién diseñamos los pép idos de con ol
sin la cadena de elas ina CP1, CP2 y CP3. En es a Tesis, sin e izamos dos pép idos cíclicos
di e en es modi icados con elas ina CP1.1 y CP3.1, en ambos pép idos inco po amos el
segmen o na u al de elas ina [H-(V-P-G-V-G-)OH]. Aquí plan eamos la hipó esis de que CP1.1
pod ía au oensambla se en es uc u as nano ubula es a pH básico, mien as que CP3.1 a pH
ácido. En CP1.1, inco po amos a la secuencia de pép ido cíclico base (CP1) es esiduos
sensibles al pH (2 His y 1 Lys) pa a log a el au oensamblaje en nano ubos de pép idos cíclicos
a pH básico. En es e pep ido cíclico, la basi icación del medio acuoso p omo e ía la
desp o onación de las cadenas la e ales de his idina (pKa ≈ 6.0) que conduci ía a la apa ición
de enlaces de hid ógeno his idina-his idina. Además, las cadenas la e ales de Lys (pKa ≈ 10.5)
a pH básico es a ían desp o onadas, po lo an o, las epulsiones elec os á icas ca iónicas se
minimiza ían. En CP3.1, pa a log a la o mación de nano ubos a pH ácido, inco po amos es
Resumen
35
esiduos de Glu a la secuencia base del pép ido cíclico (CP3). En es e pép ido, la acidi icación
del medio acuoso p oduci ía la p o onación de las cadenas la e ales de Glu (pKa ≈ 4.2) que
conduci ía a la o mación de enlaces de hid ógeno. Pa a la p epa ación de es os dos pép idos
cíclicos di e en es modi icados con elas ina (CP1.1 y CP3.1), u ilizamos exclusi amen e la
sín esis de pép idos en ase sólida. Usando es a es a egia sin é ica, el c ecimien o del agmen o
de elas ina y la sín esis y ciclación del agmen o D, L se lle a on a cabo en la esina clo o i ilo.
Además, hemos es udiado el au oensamblaje y la capacidad de espues a é mica de es os
pép idos cíclicos modi icados con elas ina. Es udiamos el au oensamblaje de CP1.1 a pH básico
median e expe imen os de dic oísmo ci cula (CD). Sin emba go, no pudimos co elaciona la
señal de CD ob enida con el au oensamblaje de pép idos cíclicos a pH básico. También
in es igamos, median e expe imen os de u bidime ía, la capacidad de espues a é mica de
CP1.1 y, desa o unadamen e, no encon amos compo amien o e mo esponsi o a di e en es
empe a u as y/o pH. Po o o lado, ambién lle amos a cabo la explo ación de las capacidades
de au oensamblaje de CP3.1 en condiciones de pu i icación p e iamen e desc i as.
Gene al In oduc ion

Gene al In oduc ion
39
1. Sup amolecula chemis y
Jean-Ma ie Lehn de ined sup amolecula chemis y as he “chemis y beyond he
molecule”.
1
Due o hei impo an disco e ies in his a ea, Lehn1, C am
2
, and Pede sen
3
ecei ed he Nobel p ize in 1987. Sup amolecula chemis y ocuses on he design and
p epa a ion o complex sys ems ha held oge he chemical componen s by non-co alen
in e ac ions.
4
,
5
,
6
Ins ead o ne wo ks o co alen bonds, he d i ing o ce o he assembly o
sup amolecula sys ems elies on a gamu o non-co alen o ces, such as ion-ion/dipole,
dipole-dipole, hyd ogen and halogen bonding, ca ion o anion-π in e ac ions, an de Waals
o ces o π-π in e ac ions.6,
7
The eme gence o sup amolecula chemis y allowed o ob ain complex s uc u es ha
p esen unc ional g oups o ien ed in a p ecise di ec ion o he h ee-dimensional space. The
p epa a ion o hese complex ensembles by he o ma ion o co alen bonds esul s ex emely
challenging om he poin o iew o ime and human esou ces.
8
Mo eo e , sup amolecula
chemis y p o ided a possibili y o manipula ing sup amolecula building blocks a a molecula
le el, allowing he “bo om-up” syn hesis o complex molecula a chi ec u es wi h con olled
size and shape.
9
Due o he use o non-co alen in e ac ions, sup amolecula ma e ials ha e
shown e e sibili y, allowing con enien complex dissocia ion and associa ion a a low ene gy
cos .
10
In e es ingly, his ype o sys ems has also shown adap i e beha io in esponse o
ex e nal s imuli ha can igge he s uc u e a ia ion o sup amolecula ma e ials.
11
2. Molecula ecogni ion and sel -assembly
Sup amolecula sys ems a e mainly assembled by molecula ecogni ion o sel -assembly.
In bo h cases, he in e ac ion be ween he molecules occu s based on hei s uc u al
in o ma ion. This is cons i u ed mainly o wo elemen s: chemical complemen a i y, which
implies he co ec dis ibu ion o he ma ching in shape and chemical g oups in he molecule,
and he p eo ganiza ion, which e e s o he adequa e con o ma ional disposi ion o he
in e ac ing uni s. The main di e ence be ween bo h p ocesses is ela ed o he complexi y o
he ob ained sup amolecula sys ems. Molecula ecogni ion is ocused on simple p ocesses in
which only a ew molecules a e in e ac ing, while sel -assembly deals wi h mo e complex
sys ems, gene ally in ol ing an in ini e numbe o molecules. Molecula ecogni ion is de ined
as he speci ic binding o a gues molecule o a complemen a y hos en i y o gene a e a hos -
1
J.-M. Lehn, Angew. Chem. In . Ed. 1988, 27, 89-112.
2
D. J. C am, Angew. Chem. In . Ed. 1988, 27, 1009-1020.
3
C. J. Pede sen, Angew. Chem. In . Ed. 1988, 27, 1021-1027.
4
J.-M. Lehn, Chem. Soc. Re . 2017, 46, 2378-2379.
5
J.-M. Lehn, Eu . Re . 2009, 17, 263-280.
6
J. W. S eed, D. R. Tu ne , K. Wallace, Co e Concep s in Sup amolecula Chemis y and Nano echnology, Jon
Wiley & Sons, Chiches e , 2007.
7
J. D. Badjić, A. Nelson, S. J. Can ill, W. B. Tu nbull, J. F. S odda , Acc. Chem. Res. 2005, 38, 723-732.
8
D. N. Reinhoud , Science 2002, 295, 2403-2407.
9
V. Balzani, A. C edi, M. Ven u i, Chem. Eu . J. 2002, 8, 5524-5532.
10
K. Liu, Y. Kang, Z. Wang, X. Zhang, Ad . Ma e . 2013, 25, 5530-5548.
11
B. Ryb chinski, ACS Nano 2011, 5, 6791-6818.
Héc o Fe nández Ca o
40
gues complex by he use o non-co alen in e ac ions. On he o he hand, molecula sel -
assembly is de ined as he cons uc ion o sys ems wi hou guidance om an ou side sou ce.6,
12
3. Molecula sel -assembly
Sel -assembly is a p ocess by which a non-o ganized sys em o molecula componen s, o
a pa o a molecule, spon aneously gene a es an o ganized s uc u e as a consequence o non-
co alen in e ac ions.
13
The molecules in ol ed in his ype o p ocess a e p og ammed
chemical en i ies. The e o e, hese molecules include unc ional complemen a y g oups ha
di ec he o ganiza ion o ma e owa ds he gene a ion o a chi ec u es wi h a p e-de ined
opology and p ope ies.
14
Molecula sel -assembly has been also he s a egy selec ed by na u e
o cons uc complex and unc ional a chi ec u es.
15
In ac , he mos sophis ica ed examples o
sel -assembly can be ound in na u al sys ems, such as he double helix s uc u e o DNA, he
assembly o he obacco mosaic i us, he olding o p o eins o ob ain hei h ee-dimensional
s uc u e ha de e mines i s biological unc ion o he o ma ion o lipid memb anes.
16
The
implemen a ion o sel -assembly in syn he ic chemis y allowed o cons uc a wide ange o
complex s uc u es wi h di e en opologies such as sphe es,
17
helica es,
18
ba els,
19
nanowi es,
20
cubes
21
, o nano ubes.
22
4. Hos -gues chemis y
4.1. Gene al p inciples
An impo an phenomenon s udied by sup amolecula chemis y is he o ma ion o
inclusion complexes. He e, a hos molecule can in e ac wi h a gues en i y in o de o o m a
hos -gues complex, as shown in Figu e 1.
23
The d i ing o ce is he o ma ion o non-co alen
sup amolecula in e ac ions such as an de Waals o ces.
24
The geome ical i ing o he gues
o he hos pocke is he e o e an essen ial ac o in maximizing hese s abilizing in e ac ions
while minimizing s e ical epulsion. The s abili y o he hos -gues complex a a gi en
empe a u e and sol en is ypically measu ed by he he modynamic equilib ium o ma ion
cons an (Ka), o he dissocia ion cons an (Kd, being Kd = Ka−1). Ka is exp essed as he
concen a ion o o med hos -gues complex di ided by concen a ions o ee hos and gues
(Figu e 1, igh ). In some cases, hos -gues binding cons an s can each magni udes almos
6 J. W. S eed, D. R. Tu ne , K. Wallace, Co e Concep s in Sup amolecula Chemis y and Nano echnology, Jon
Wiley & Sons, Chiches e , 2007.
12
D. Rasale, A. Das, In . J. Mol. Sci. 2015, 16, 10797-10820.
13
Whi esides, G. M.; G zybowski, B. Science 2002, 295, 2418-2421.
14
Hosseini, M. W. Chem. Commun. 2005, 5825-5829.
15
Lehn, J.-M. P oc. Na l. Acad. Sci. U. S. A. 2002, 99, 4763-4768.
16
Whi esides, G. M.; Ma hias, J. P.; Se o, C. T. Science 1991, 254, 1312-1319.
17
A. R. S e ankiewicz, J. K. M. Sande s, Science 2010, 328, 1115-1116.
18
M. Boiocchi, L. Fabb izzi, Chem. Soc. Re . 2014, 43, 1835-1847.
19
Y. Lim, M. Lee, J. Ma e . Chem. 2011, 21, 11680-11685.
20
C. Qian, F. Kim, L. Ma, F. Tsui, P. Yang, J. Liu, J. Am. Chem. Soc. 2004, 126, 1195-1198.
21
A. S ephenson, M. D. Wa d, Dal on T ans. 2011, 40, 10360-10369.
22
Chapman, R.; Danial, M.; Koh, M. L.; Jolli e, K. A.; Pe ie , S. Chem. Soc. Re . 2012, 41, 6023-6041.
23
S. M. Man oo h, B. G. Munoz-Robles, M. J. Webbe , Mac omol. Biosci. 2019, 19, 1800281.
24
C. B. Rodell, J. E. Mealy, J. A. Bu dick, Bioconjug. Chem. 2015, 26, 2279-2289.
Gene al In oduc ion
41
compa able o co alen bonds. A clea example in his sense is he molecula ecogni ion o
bio in by he binding pocke o s ep a idin p o ein,
25
wi h a Kd ~ 4 × 10-14 M-1.
Figu e 1. In e ac ion be ween a ca i and hos ( ed) and a i ing gues (blue) o gi e a hos -gues complex,
whe e kon and ko a e he o ma ion and dissocia ion a e cons an s espec i ely. The binding cons an
(Ka) de e mines he specia ion o ee hos , ee gues , and hos -gues complex.23
4.2. Mul i alency
In sup amolecula chemis y, mul i alency ypically desc ibes mul iple non-co alen
in e ac ions ha occu be ween a mul i alen hos and a mul i alen gues . This esul s,
gene ally, in enhanced binding a ini ies and speci ici ies,
26
which plays a pi o al ole in
in e cellula adhesion
27
o p o ein-ca bohyd a e ecogni ion.
28
Depending on he spacing and
lexibili y o he mul i alen mo i es, a mul i alen gues can bind o a mul i alen hos in an
in amolecula o in e molecula ashion. In amolecula binding ypically leads o ela i ely
high associa ion cons an s wi h espec o mono alen binding and he o ma ion o well-
de ined complexes. In con as , in e molecula binding migh lead o he o ma ion o la ge
agg ega es ha o en p ecipi a e om solu ion. Binding shows associa ion cons an s
compa able o hose o he co esponding mono alen in e ac ions, while i e e sible
p ecipi a ion and agg ega ion lead o dec eased dissocia ion a es and consequen ly o an
appa en binding enhancemen (Figu e 2A).
29
In addi ion o i s impo ance in ele an biological p ocesses, mul i alen in e ac ions a e
he ounda ion o se e al echnologies in nanomedicine and ma e ial sciences. Fo example,
i al in ec ions ypically p oceed by an ini ial binding o memb ane ecep o s which
subsequen ly igge s cell endocy osis and s a s he in ec ion. Vi al inhibi o s can be designed
by including mul iple copies o he an igen in o a mul i alen pla o m, which shows highe
a ini ies owa ds he hos cell and he e o e p e en s i us binding by mul i alen compe i ion.
As example, Naza io Ma in and col. demons a ed ha gian mul i alen glyco ulle enes a e
po en inhibi o s o Ebola i us in ec ion showing subnanomola hal -maximum inhibi o y
concen a ions. These mul i alen compounds demons a ed inhibi ion o lec in-media ed i al
in ec ions in cellula expe imen s. In e es ingly, he hal -maximum inhibi o y concen a ions
o e passed by h ee o de s o magni ude ( wo i he numbe o mannoses is conside ed) hose
exhibi ed by hexakis adduc s showing 12 mannoses (Figu e 2B).
30
On he o he hand, ma e ials
wi h enhanced adhesion can be designed by co e ing la ge su aces wi h hos -gues pai s,
yielding unde wa e “ elc os”. Fo example, mul i alen in e ac ions a ising be ween a hos
25
N. M. G een, Me hods Enzymol. 1990, 184, 51-67.
26
M. J. W. Ludden, D. N. Reinhoud , J. Huskens, Chem. Soc. Re . 2006, 35, 1122-1134.
27
Y. Zhang, S. Si asanka , W. J. Nelson, S. Chu, P oc. Na l. Acad. Sci. 2009, 106, 109-114.
28
T. K. Dam, R. Roy, S. K. Das, S. Osca son, C. F. B ewe , J. Biol. Chem. 2000, 275, 14223-14230.
29
A. Mulde , J. Huskens, D. N. Reinhoud , O g. Biomol. Chem. 2004, 2, 3409-3424.
30
A. Muñoz, D. Sigwal , B. M. Illescas, J. Luczkowiak, L. Rod íguez-Pé ez, I. Nie enga en, M. Holle , J. Remy,
K. Bu e , S. P. Vincen , e al., Na . Chem. 2016, 8, 50-57.

In oduc ion
Chap e I: In oduc ion
51
1. The cellula memb ane as a selec i e na u al ba ie
The cellula memb ane is p ima ily esponsible o he p o ec ion and he s ic con ol o
he exchange o molecules be ween he cellula in e io and he ex acellula en i onmen .
73
This selec i e na u al ba ie is mainly o med by wo componen s: lipids and p o eins.
Memb ane lipids ha e a hyd ophobic ail and a hyd ophilic head ha a e o ien ed o o m a
p o ec i e ba ie , he lipid bilaye . In addi ion o lipids, he cellula memb ane con ains
di e en ypes o p o eins. These memb ane p o eins can ac , o example, as anspo e s
(con olling he exchange o di e en molecules be ween he cellula in e io and i s
en i onmen ) o as ecep o s (being esponsible o cell signaling).
74
The anspo o di e en subs ances inside o he cells can be pe o med by using a a ie y
o mechanisms ha can be mainly classi ied in passi e di usion, acili a ed di usion, and
ac i e anspo . T anspo by passi e di usion allows he cells o anspo di e en subs ances
ac oss he cell memb ane in a o o an elec ochemical o a concen a ion g adien . The e o e,
his mechanism does no equi e ene gy om he cell. An impo an poin is ha his mechanism
only allows he anspo o hyd ophobic small molecules ac oss he cell memb ane. To imp o e
he anspo o biologically impo an hyd ophilic molecules o he cell such as ions o suga s,
cells use acili a ed di usion. This mechanism occu s helped by di e en memb ane p o eins
and i does no equi e ene gy om he cell.
75
Figu e 5. Schema ic ep esen a ion o he di e en endocy ic pa hways. Rep in ed wi h pe mission om
e . 77. Copy igh 2007 Sp inge Na u e.
Ac i e anspo allows cells o anspo ex acellula molecules agains an elec ochemical
o a concen a ion g adien , a mechanism ha demands ene gy consump ion by he cell. An
impo an ype o ac i e anspo is endocy osis, which allows he cell o anspo luids and
solu es om i s su oundings o he cell in e io . Endocy osis is a phenomenon whe e he e is a
con o ma ional change o he cell memb ane o in oduce di e en ex acellula subs ances
73
J. Lomba d, Biol. Di ec 2014, 9, 32.
74
A. Uzman, B. Albe s, A. Johnson, J. Lewis, M. Ra , K. Robe s, P. Wal e , Molecula Biology o he Cell, 4 h
edi ion, Ga land Science, New Yo k, 2003.
75
N. J. Yang, M. J. Hinne , Me hods Mol. Biol. 2015, 1266, 29-53.
Héc o Fe nández Ca o
52
h ough he o ma ion o small anspo esicles. Endocy osis can be classi ied in o wo majo
classes: phagocy osis and pinocy osis. Phagocy osis is an impo an p ocess ha allows he cell
o in e nalize la ge pa icles and occu s exclusi ely in specialized cells such as neu ophils and
mac ophages. Howe e , pinocy osis is a mechanism ha allows he non-specialized cells o
anspo a a ie y o ex acellula molecules and luids h ough he cell memb ane.
Fu he mo e, pinocy osis can be also classi ied in di e en in e naliza ion pa hways such as
mac opinocy osis, ca eolin-dependen endocy osis, cla h in-dependen endocy osis, and
cla h in and ca eolin-independen endocy osis (Figu e 5).
76
,
77
This me iculous con ol o he anspo h ough he cell memb ane impo an ly in luences
he deli e y o he apeu ics as he e ec i eness o d ugs and o he bioac i e compounds
depends on hei e ec i e deli e y. The main eason is ha he si e o ac ion o many molecules
is placed in he cell in e io . Thus, he d ug needs o c oss he plasma memb ane o accomplish
i s pha maceu ical e ec . Fo his eason, in he las yea s, many inno a i e cell deli e y
sys ems ha e eme ged o imp o e he anspo ac oss he cell memb ane o la ge hyd ophilic
molecules. O e he las wen y yea s, cell-pene a ing pep ides ha e eme ged as an inno a i e
app oach o imp o e he in acellula deli e y o di e en he apeu ic and diagnos ic ca goes
such as d ugs, imaging agen s, nucleic acids, p o eins, e c.
78
2. Cell-pene a ing pep ides
In 1988, i was disco e ed ha he TAT p o ein o he human immunode iciency i us
(HIV-1), consis ing o 86 amino acids, had he abili y o c oss he cell memb ane.
79
,
80
A e
s udying his phenomenon, i was disco e ed ha he sequence ha p o ided hese anspo
capabili ies was a small pep ide (RKKRRQRRR) called TAT.
81
In 1994, ansloca ion
capabili ies we e also ound in he homeodomain o An ennapedia.
82
I was disco e ed ha
hese anspo p ope ies we e p o ided also by a small pep ide which was called Pene a in
(RQIKIWFQNRRMKWKK).
83
Following hese p elimina y indings, he scien i ic communi y
was ascina ed by he anspo capabili ies o hese pep ide sequences and began o de elop
modi ica ions o hese sequences designing new s uc u es wi h pene a ing capabili ies, hese
pep ides we e named as cell-pene a ing pep ides (CPPs).
84
Cell-pene a ing pep ides (CPPs) a e small-sized pep ides (usually less han 30 amino
acids) capable o anspo ing a wide ange o memb ane-impe meable he apeu ic agen s in o
he cell in e io . An impo an cha ac e is ic o hese pep ides is ha hey ha e he abili y o
anspo molecules wi h low cellula oxici y a low mic omola concen a ions. Di e en
classi ica ions ha e eme ged o he wide a ie y o CPPs, o example, CPPs can be easily
classi ied acco ding o hei physical-chemical p ope ies in di e en ypes:
76
G. J. Dohe y, H. T. McMahon, Annu. Re . Biochem. 2009, 78, 857-902.
77
S. Mayo , R. E. Pagano, Na . Re . Mol. Cell Biol. 2007, 8, 603-612.
78
F. Hei z, M. C. Mo is, G. Di i a, B . J. Pha macol. 2009, 157, 195-206.
79
A. D. F ankel, C. O. Pabo, Cell 1988, 55, 1189-1193.
80
M. G een, P. M. Loewens ein, Cell 1988, 55, 1179-1188.
81
E. Vi ès, P. B odin, B. Lebleu, J. Biol. Chem. 1997, 272, 16010-16017.
82
D. De ossi, A. H. Jolio , G. Chassaing, A. P ochian z, J. Biol. Chem. 1994, 269, 10444-10450.
83
D. De ossi, G. Chassaing, A. P ochian z, T ends Cell Biol. 1998, 8, 84-87.
84
J. D. Ramsey, N. H. Flynn, Pha macol. The . 2015, 154, 78-86.
Chap e I: In oduc ion
53
Ca ionic CPPs: This ype o CPPs is cha ac e ized by hei con en o mainly ca ionic
esidues such as a ginine and lysine, which con e a high ne posi i e cha ge o he CPP. Some
examples in his ca ego y a e TAT, pene a in, o polya ginines.
Hyd ophobic CPPs: These pep ides mainly consis o hyd ophobic amino acids. An
example o a hyd ophobic pep ide is K-FGF (AAVLLPVVLLAAP).
Amphipa hic CPPs: hese pep ides con ain wo di e en egions, a hyd ophobic domain,
and hyd ophilic and ca ionic domain.
85
This class o CPPs no mally can o m s able non-
co alen complexes wi h di e en ca gos inducing he in e naliza ion in he cell. An
amphipa hic pep ide, Pep-1 (KETWWETWWTEWSQPKKKRKV) has been applied o
deli e ing a wide numbe o ca gos. Fo ins ance, his pep ide can o m non-co alen
in e ac ions wi h o he pep ides and p o ein ca gos o allow hei memb ane ansloca ion.
Ano he example o his ca ego y is he MPG (GALFLGFLGAAGSTMGAWSQPKKKRKV),
a pep ide ha has been disco e ed able o o m s able complexes wi h di e en nucleic acids.
86
Mo eo e , amphipa hic p oline- ich pep ides (VXLPPP)n ha e been p epa ed and also showed
good memb ane ansloca ion capabili ies.
87
,
88
Many s udies ha e been published ega ding he mechanism o up ake o he CPPs. This
opic has gene a ed g ea con o e sy. No mally, hese pep ides a e ansloca ed inside o he
cell ollowing di e en endocy ic pa hways. I is known ha he e is a high in luence o he
concen a ion o he pep ide and he cha ac e is ics o he ca go deli e ed and he ype o CPP.
89
A powe ul s a egy o he syn hesis o new cell-pene a ing pep ides is he inco po a ion
o dynamic bonds, such as oximes, hyd azones, o disul ides. Dynamic bonds a e esponsi e o
di e en ex e nal s imuli and, du ing hei in e naliza ion pa hway, hese bonds a e sensi i e o
he en i onmen . F om he syn he ic poin o iew, dynamic bonds a e also in e es ing as hey
lead o he inal p oduc s wi h high yields, wi h he gene a ion o a wa e molecule as he only
side p oduc , and in sho pe iods o ime.
90
Recen ly, in ou esea ch g oup, we ha e designed
new pene a ing pep ides ha con ain dynamic bonds.91 Fi s , we syn hesized ca ionic pep ides
con aining amino acids modi ied wi h an alcoxyamine g oup. These pep ides we e hen eac ed
wi h di e en aldehydes esul ing in new oxime amphiphilic pep ides. These amphiphilic
ca ionic pep ides we e able o anspo a a ie y o anionic DNA molecules in esicle anspo
expe imen s.
91
Subsequen ly, we applied he same s a egy o syn hesize an amphiphilic
pseudo-helical pa en pep ide, which was easily eac ed wi h di e en hyd ophobic and
hyd ophilic elec ophiles (Figu e 6).92 In his wo k, we explo ed he capaci y o DNA anspo
in esicles o his ype o pep ides and he di e ence ha exis s depending on he hyd ophilici y
o he elec ophiles used. Mo eo e , we s udied he modi ica ion o he memb ane ansloca ion
capabili ies o he pep ides in cells wi h he modula ion o hei amphiphilici y.
92
In ou esea ch
g oup, dynamic linkages, such as hyd azone bonds, ha e also been applied o syn hesize
85
D. Rauche , J. S. Ryu, T ends Mol. Med. 2015, 21, 560-570.
86
M. C. Mo is, S. Deshayes, F. Hei z, G. Di i a, Biol. Cell 2008, 100, 201-217.
87
A. D. F ankel, C. O. Pabo, Cell 1988, 55, 1189-1193.
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Y. A. Fillon, J. P. Ande son, J. Chmielewski, J. Am. Chem. Soc. 2005, 127, 11798-11803.
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A. Rioboo, I. Gallego, J. Mon eneg o, An. Química 2019, 115, 9-21.
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C. Gehin, J. Mon eneg o, E. K. Bang, A. Caja a ille, S. Takayama, H. Hi ose, S. Fu aki, S. Ma ile, H. Riezman,
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91
J. M. P iegue, J. Mon eneg o, J. R. G anja, Small 2014, 10, 3613-3618.
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M. Pazo, H. Fe nández-Ca o, J. P iegue, I. Los alé-Seijo, J. Mon eneg o, Synle 2017, 28, 924-928.

Héc o Fe nández Ca o
54
dynamic pene a ing pep ides wi h hyd ophobic ails which o med complexes by non-co alen
in e ac ions and, hen, igge ed he anspo o plasmid DNA
93
and Cas9
94
in cells.
Figu e 6. A) Chemical s uc u e o he amphiphilic pep ide showing wo alcoxyamine eac i e g oups
whe e he di e en elec ophilic aldehydes we e a ached. B) Top- iew helical diag am and ci cula
dich oism spec a o he pa en pep ide. C) Schema ic ep esen a ion o pep ide unc ionaliza ion wi h
he di e en aldehydes o a o d oxime-modula ed pep ides o memb ane anspo applica ions.
Rep in ed om e . 92 unde he e ms o he C ea i e Commons CC-BY-NC-ND license.
3. Cell deli e y o biologically ac i e mac omolecules
P o eins and nucleic acids ha e eme ged as excellen he apeu ics o he ea men o a
a ie y o diseases.
95
,
96
Mac omolecula he apeu ics, such as nucleic acids o ecombinan
p o eins, ha e o e icien ly c oss he cellula memb ane due o hei si e o ac ion is loca ed in
he cy osol o he cell nucleus. Un o una ely, he cy osolic deli e y o nucleic acids and
p o eins is ine icien mainly because o endosomal en apmen and enzyma ic diges ion.
97
,
98
The e o e, he deli e y o mac omolecules in o cells and issues emains a majo challenge, and
new deli e y sys ems o o e come hese limi a ions a e needed. Du ing he las yea s, di e en
me hods can be di e en ia ed o deli e he apeu ic mac omolecules in o he cell in e io
including physical and mechanical me hods and chemical co alen and non-co alen me hods.
93
I. Louzao, R. Ga cía-Fandiño, J. Mon eneg o, J. Ma e . Chem. B 2017, 5, 4426-4434.
94
I. Los alé-Seijo, I. Louzao, M. Juanes, J. Mon eneg o, Chem. Sci. 2017, 8, 7923-7931.
95
J. Nguyen, F. C. Szoka, Acc. Chem. Res. 2012, 45, 1153-1162.
96
Y. Zhang, J. J. Røise, K. Lee, J. Li, N. Mu hy, Cu . Opin. Bio echnol. 2018, 52, 25-31.
97
V. To chilin, D ug Disco . Today Technol. 2008, 5, 95-103.
98
M. L. Read, A. Logan, L. W. Seymou , Ad . Gene . 2005, 53, 19-46.
A)
B)
C)
Chap e I: In oduc ion
55
3.1. Physical and mechanical me hods
Se e al physical and mechanical s a egies ha e been de eloped o achie e di ec cy osolic
deli e y o mac omolecules such as DNA o p o eins.
99
,
100
Mechanical me hods, such as mic oinjec ion, a e a ypical powe ul s a egy.
Mic oinjec ion is one o he mos di ec and simples me hods o inco po a e DNA in o he
nucleus o he cy osol o cells. In his echnique, he elease o he biomolecule is ca ied ou
using a needle on a single cell. This s a egy is ca ied ou when a p ecise and accu a e deli e y
o he mac omolecule is necessa y. Howe e , his me hodology does no show good esul s
when a high numbe o cell ans ec ion is equi ed.
101
Physical me hods, such as elec opo a ion ha e been also widely applied. This echnique
induces sho high ol age elec ic cycles o he cell suspensions. This ol age p oduces
ansien po es in he cell memb ane and he soluble desi ed p o eins can c oss he cell
memb ane by simple di usion.
102
The main ad an age o his echnique is ha i can be applied
o di e en cell lines and a la ge numbe o cells can be ans ec ed. Howe e , he main
limi a ion o elec opo a ion me hods is he impo an cy o oxici y induced by he high ol age
cycles and he damage p oduced o he cell memb ane. Mo eo e , his echnique is limi ed o
in i o usage.
103
3.2. Chemical co alen me hods
Du ing he las yea s, mac omolecules, such as DNA o p o eins, ha e been co alen ly
linked o di e en deli e y sys ems including cell-pene a ing pep ides,
104
gold
nanopa icles,
105
polyme s,
106
and s ain-p omo ed hiols.
107
3.2.1. Cell-pene a ing pep ides
The co alen a achmen o mac omolecules o cell-pene a ing pep ides has been applied
o deli e di e en he apeu ic mac omolecules such as p o eins o nucleic acids.104
Deli e y o p o eins. Di e en CPPs such as TAT pep ide, Pene a in, T anspo an, e c.
ha e demons a ed, in di e en in i o examples, hei abili ies o e icien ly deli e
p o eins.
108
,
109
Fo ins ance, i has been demons a ed ha TAT pep ide has he abili y o deli e
la ge p o eins such as β-galac osidase. The in i o adminis a ion o he co alen conjuga e
99
M. S. Al-Dosa i, X. Gao, AAPS J. 2009, 11, 671.
100
S. Du, S. S. Liew, L. Li, S. Q. Yao, J. Am. Chem. Soc. 2018, 140, 15986-15996.
101
S. Mehie -Humbe , R. H. Guy, Ad . D ug Deli . Re . 2005, 57, 733-753.
102
M. Chipe , K. Niede ei he , G. Zube , Ad . Heal hc. Ma e . 2018, 7, e1701040.
103
A. Fu, R. Tang, J. Ha die, M. E. Fa kas, V. M. Ro ello, Bioconjug. Chem. 2014, 25, 1602-1608.
104
D. Kala a o ic, E. Gi al , Molecules 2017, 22, 1929.
105
D. A. Giljohann, D. S. Se e os, W. L. Daniel, M. D. Massich, P. C. Pa el, C. A. Mi kin, Angew. Chem. In . Ed.
2010, 49, 3280-3294.
106
R. Duncan, Na . Re . D ug Disco . 2003, 2, 347-360.
107
N. Chua d, G. Gaspa ini, D. Mo eau, S. Lö che , C. Pali an, W. Meie , N. Sakai, S. Ma ile, Angew. Chem. In .
Ed. 2017, 56, 2947-2950.
108
S. G. Pa el, E. J. Saye s, L. He, R. Na ayan, T. L. Williams, E. M. Mills, R. K. Allemann, L. Y. P. Luk, A. T.
Jones, Y. H. Tsai, Sci. Rep. 2019, 9, 6298.
109
M. Mäe, Ü. Langel, Cu . Opin. Pha macol. 2006, 6, 509-514.
Héc o Fe nández Ca o
56
be ween β-galac osidase and TAT has shown an e icien deli e y no only in he di e en
issues o he mice bu also in he b ain.
110
To success ully each i s in acellula a ge and
p oduce i s he apeu ic e ec he p o ein should no emain apped inside o he endosome.
111
To p omo e p o ein deli e y, he use o co alen ly a ached endosomoly ic pep ides has also
been epo ed.
112
The co alen conjuga ion be ween p o eins and CPPs can be achie ed by
exp essing CPP- usion p o eins.
113
In addi ion, he CPP can be co alen ly linked o he side
chain o a lysine o he p o ein o o a hiol o a cys eine o achie e speci ic modi ica ions.
114
,
115
Deli e y o nucleic acids. CPPs ha e also demons a ed hei abili ies o e icien ly deli e
bioac i e nucleic acids. 5´- hiol unc ionalized siRNA has been co alen ly a ached o
T anspo an and Pene a in pep ides modi ied wi h e minal cys eines. This complex CCP-
siRNA showed an e icien knockdown o mammalian cells.
116
Mo eo e , pep ide nucleic acids
(PNAs) ha e been also co alen ly a ached o di e en CPPs o imp o e hei ansloca ion
capabili ies.
117
,
118
The use o co alen bonds wi h CPPs has been success ully applied o he
cellula deli e y o a a ie y o he apeu ic mac omolecules. Howe e , his s a egy has also
d awbacks such as modi ica ion o he chemical p ope ies o he ca go, which can lead o
changes in i s biological ac i i y.
119
In addi ion, his s a egy is no as compa ible wi h he
unc ional deli e y o la ge biomolecules such as pDNA and he syn hesis and pu i ica ion o
he co esponding conjuga es esul s edious as i no mally equi es mul iple s eps.
120
3.2.2. Me al nanopa icle conjuga es
Gold nanopa icles (AuNPs) ha e also been applied o deli e mac omolecules by
co alen s a egies. The g oup o p o . Chad Mi kin p epa ed a ype o poly alen nucleic acid
capped AuNPs (pNA-AuNPs) by co alen ly unc ionalizing AuNPs wi h hiol-modi ied
oligonucleo ides o siRNA-based gene silencing.
121
The inco po a ion o he oligonucleo ides
on he su ace o hese AuNPs showed p o ec ion o he ca go and inhibi ion o i s deg ada ion
by nucleases. In e es ingly, hese pNA-AuNPs we e e icien ly in e nalized in mo e han i y
di e en cell lines.105,
122
Mo e ecen ly, o a ge speci ic cells, his g oup modi ied he pNA-
110
S. R. Schwa ze, A. Ho, A. Voce o-Akbani, S. F. Dowdy, Science 1999, 285, 1569-1572.
111
P. Lönn, A. D. Kacsin a, X. S. Cui, A. S. Hamil, M. Kaulich, K. Gogoi, S. F. Dowdy, Sci. Rep. 2016, 6, 32301.
112
M. Li, Y. Tao, Y. Shu, J. R. LaRochelle, A. S einaue , D. Thompson, A. Schepa z, Z. Y. Chen, D. R. Liu, J.
Am. Chem. Soc. 2015, 137, 14084-14093.
113
J. Jo, S. Hong, W. Y. Choi, D. R. Lee, Sci. Rep. 2014, 4, 4378.
114
S. B. Gunnoo, A. Iye , W. Vannecke, K. W. Decoene, T. Hebb ech , J. Ge emans, M. Laga, S. Lo e ix, I.
Las e s, A. Madde , Chem. Commun. 2018, 54, 11929-11932.
115
D. A. Shannon, E. Wee apana, Cu . Opin. Chem. Biol. 2015, 24, 18-26.
116
A. Mu a o ska, M. R. Eccles, FEBS Le . 2004, 558, 63-68.
117
Y. Wol , S. P i z, S. Abes, M. Biene , B. Lebleu, J. Oehlke, Biochemis y 2006, 45, 14944-14954.
118
S. Abes, J. J. Tu ne , G. D. I ano a, D. Owen, D. Williams, A. A zumano , P. Clai , M. J. Gai , B. Lebleu,
Nucleic Acids Res. 2007, 35, 4495-4502.
119
G. Guido i, L. B ambilla, D. Rossi, T ends Pha macol. Sci. 2017, 38, 406-424.
120
T. Leh o, K. Ku iko , Ü. Langel, Expe Opin. D ug Deli . 2012, 9, 823-836.
121
D. S. Se e os, A. E. P igodich, D. A. Giljohann, P. C. Pa el, C. A. Mi kin, Nano Le . 2009, 9, 308-311.
105 D. A. Giljohann, D. S. Se e os, W. L. Daniel, M. D. Massich, P. C. Pa el, C. A. Mi kin, Angew. Chem. In . Ed.
2010, 49, 3280-3294.
122
P. C. Pa el, D. A. Giljohann, W. L. Daniel, D. Zheng, A. E. P igodich, C. A. Mi kin, Bioconjug. Chem. 2010,
21, 2250-2256.
Chap e I: In oduc ion
57
AuNPs wi h monoclonal an ibody-DNA conjuga es (Figu e 7, le ). Impo an ly, highe cell
selec i i y and gene knockdown we e achie ed in cells ha o e exp ess he a ge an igen.
123
Figu e 7. Le : Hyb idiza ion o pNA-AuNPs using monoclonal an ibody-DNA conjuga es. Rep in ed
wi h pe mission om e . 123. Copy igh 2012 Ame ican Chemical Socie y. Righ : S ain-p omo ed
hiols s a egy applied o deli e liposomes and polyme somes inside li ing cells. Rep in ed om e .
107 wi h pe mission om John Wiley and Sons. Copy igh 2017 Wiley‐VCH Ve lag GmbH & Co.
KGaA, Weinheim.
3.2.3. S ain-p omo ed hiols
S ain-p omo ed hiols a e a ecen ly de eloped s a egy om he g oup o he p o .
Ma ile.107 The applica ion o his s a egy allowed he deli e y o polyme somes and liposomes
in o HeLa Kyo o cells.107 Based on p e ious wo k om his g oup on s ained cyclic disul ides,
he ans e in ecep o (TFRC) was ound o be ela ed o he cellula in e naliza ion o he
co esponding pene a ing pep ides.
124
Recen ly, ca ionic amphiphiles we e modi ied wi h
cyclic disul ides o ob ain liposomes and polyme somes and, a e disul ide exchange wi h
cellula exo acial hiols, he highly s ained disul ides lead o an e icien cy osolic elease o
hese gian subs a es (Figu e 7, igh ).107
3.2.4. Polyme -d ug conjuga es
Polyme -d ug conjuga es a e a ac i e molecules o deli e d ugs inside cells. The
inco po a ion o di e en mac omolecules, such as p o eins, in o polyme ic sca olds, has
eme ged as an inno a i e app oach o o e come endosomal en apmen . Fo ins ance, he
limi a ions o p o ein he apeu ics such as as clea ance, low s abili y, and high
immunogenici y can be sol ed by PEGyla ion.
125
Conjuga ion o PEG wi h p o ein he apeu ics
educe immunogenici y, by shielding i s an igenic epi opes, and imp o ing i s he apeu ic
sa e y. Mo eo e , p o ein PEGyla ion demons a ed o a oid opsoniza ion and diges ion by
p o eoly ic enzymes. Fu he mo e, molecula mass inc ease helps o educe plasma clea ance
and he e o e he dosage equency.
126
Du ing he las yea s, se e al PEGyla ed p o eins such
123
K. Zhang, L. Hao, S. J. Hu s , C. A. Mi kin, J. Am. Chem. Soc. 2012, 134, 16488-16491.
107 N. Chua d, G. Gaspa ini, D. Mo eau, S. Lö che , C. Pali an, W. Meie , N. Sakai, S. Ma ile, Angew. Chem. In .
Ed. 2017, 56, 2947-2950.
124
D. Abegg, G. Gaspa ini, D. G. Hoch, A. Shus e , E. Ba olami, S. Ma ile, A. Adibekian, J. Am. Chem. Soc.
2017, 139, 231-238.
125
P. Calice i, F. M. Ve onese, Ad . D ug Deli . Re . 2003, 55, 1261-1277.
126
I. Ekladious, Y. L. Colson, M. W. G ins a , Na . Re . D ug Disco . 2019, 18, 273-294.
Héc o Fe nández Ca o
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4. Cell deli e y o small molecules
Small molecules ha e a s ong po en ial as in acellula p obes and d ugs o he ea men
o a wide ange o diseases.
157
,
158
,
159
Howe e , many small molecules, such as d ugs, ha e hei
si e o ac ion loca ed in he cy osol, in he cell nucleus, o in acellula o ganelles. As examples,
e e se ansc ip ase inhibi o s o s a ins can be highligh ed.
160
,
161
Mo eo e , some d ugs su e
om cell e lux media ed by P-glycop o eins (P-gp) o mul id ug esis ance p o eins (MRP).
162
The e o e, o achie e he desi ed e ec , small molecules ha e o be able o c oss he cell
memb ane, o be e icien ly deli e ed in o he cell cy osol, and o a oid he cellula e lux.
Hyd ophilic small d ugs wi h in acellula a ge s, such as doxo ubicin, ha e eme ged as
aluable an icance d ugs. Doxo ubicin is a widely adminis e an icance d ug whose
mechanism o cell damage is ela ed o inhibi ion o he DNA- opoisome ase II complex.
163
Un o una ely, i s hyd ophilic cha ac e makes i s cy osolic deli e y e y ine icien and he
e lux pumping, by mul id ug esis ance p o eins (MRPs) and P-glycop o eins (P-gp), makes
manda o y o adminis a e a high dose o doxo ubicin o each he p ope an icance e ec .
164
The e o e, he e icien deli e y o small hyd ophilic d ugs in o cells and issues emains a
majo challenge, and new deli e y me hods a e needed o o e come hese p oblems.
Hyd ophilic anionic small luo escen molecules a e ele an in acellula p obes such as
sul ona ed Alexas, py anine, o ca boxy luo escein (Figu e 13). Mo eo e , py anine has shown
excellen p ope ies as a pH indica o . Un o una ely, he nega i e ne cha ge a physiological
pH o hese hyd ophilic anionic small molecules makes hei cy osolic deli e y e y
ine icien .
165
The e o e, he deli e y o small hyd ophilic luo escen molecules in o cells and
issues emains a majo challenge, and new deli e y sys ems o o e come hese limi a ions a e
needed.
Figu e 13. Examples o hyd ophilic and anionic luo escen p obes which do no pass h ough he
cellula memb ane a low mic omola concen a ion.
157
S. Weggen, M. Roge s, J. E iksen, T ends Pha macol. Sci. 2007, 28, 536-543.
158
M. Vogle , D. Dinsdale, M. J. S. Dye , G. M. Cohen, Cell Dea h Di e . 2009, 16, 360-367.
159
N. K. De a aj, S. Hilde b and, R. Upadhyay, R. Mazi schek, R. Weisslede , Angew. Chem. In . Ed. 2010, 49,
2869-2872.
160
W. Lewis, B. J. Day, W. C. Copeland, Na . Re . D ug Disco . 2003, 2, 812-822.
161
C. S ancu, A. Sima, J. Cell. Mol. Med. 2001, 5, 378-387.
162
M. M. Go esman, Annu. Re . Med. 2002, 53, 615-627.
163
S. Da wish, S. Moza a i, K. Pa ang, R. Tiwa i, Te ahed on Le . 2017, 58, 4617-4622.
164
A. Nas olahi Shi azi, R. Tiwa i, B. S. Chhika a, D. Mandal, K. Pa ang, Mol. Pha m. 2013, 10, 488-499.
165
G. Y. Wiede schain, Biochem. 2011, 76, 1276-1276.

Chap e I: In oduc ion
65
Du ing he las yea s, di e en me hods ha e been explo ed o he deli e y o small
hyd ophilic molecules o he cell in e io including physical and mechanical me hods, cell-
pene a ing pep ides (CPPs), sup amolecula me hods, o by ansi o y educ ion o cha ge.
4.1. Physical and mechanical me hods
Physical and mechanical me hods ha e been applied o cy osolic deli e small molecules
such as anionic p obes. Fo example, mic oinjec ion has been used when he numbe o cells o
ans ec is small. Mo eo e , he use o s ong hypo onic s ess o he cells has been also
applied.
166
Fu he mo e, small molecules, such as an icance d ugs o luo opho es, ha e been
inco po a ed inside he cell by elec ope meabiliza ion. This echnique uses sho high- ol age
cycles o p oduce ansi o y pe meabiliza ion o he cell memb ane o allow small molecules o
c oss he cell memb ane by simple di usion.
167
,
168
4.2. Co alen me hods using cell-pene a ing pep ides
The co alen a achmen o a CPP cons i u es an al e na i e s a egy o he cy osolic
deli e y o small molecules. Di e en app oaches ha e been de eloped whe e he CPP is
co alen ly a ached o small an icance d ugs such as doxo ubicin o me ho exa e
169
and also
o luo opho es.
170
T adi ionally, he co alen bond o ma ion was one o he mos used
me hods. To inco po a e he ca go in o he pep ide by co alen bonding, one o he possible
s a egies is based on he ancho ing o he ca go in o he cys eine o he lysine side chain o one
o he pep ide esidues.
171
Among he di e en ypes o co alen bonding be ween he ca go
and he CPP, i is necessa y o highligh he disul ide bond, as his bond has he ad an age o
being sensi i e o he cy osolic glu a hione, which allows he dynamic disconnec ion o he
ca go inside he cells. An excep ional example was desc ibed by he g oup o p o . Paul
Wende , in which a CPP was connec ed o luci e in by a disul ide bond. The ca go-luci e in
complex can c oss he plasma memb ane and once inside he cell, glu a hione educes he
disul ide bond causing he elease o he luci e in ha igge s luci e ase ac i i y and ligh
emission (Figu e 14).
172
166
B. S. Gan, E. K ump, L. D. Sh ode, S. G ins ein, Am. J. Physiol. 1998, 275, C1158-C1166.
167
M.-P. Rols, Biochim. Biophys. Ac a 2006, 1758, 423-428.
168
A. Pena, J. Rami ez, G. Rosas, M. Calaho a, J. Bac e iol. 1995, 177, 1017-1022.
169
K. M. S ewa , K. L. Ho on, S. O. Kelley, O g. Biomol. Chem. 2008, 6, 2242-2255.
170
Z. Qian, P. G. Doughe y, D. Pei, Chem. Commun. 2015, 51, 2162-2165.
171
M. Zo ko, U. Langel, Ad . D ug Deli . Re . 2005, 57, 529-545.
172
L. R. Jones, E. A. Goun, R. Shinde, J. B. Ro hba d, C. H. Con ag, P. A. Wende , J. Am. Chem. Soc. 2006, 128,
6526-6527.
Héc o Fe nández Ca o
66
Figu e 14. Schema ic illus a ion showing in acellula anspo o luci e in co alen ly a ached o a
cell-pene a ing pep ide h ough a eleasable linke and, he subsequen , in acellula elease o he ca go
media ed by glu a hione. Rep in ed wi h pe mission om 172. Copy igh 2006 Ame ican Chemical
Socie y.
CPPs ha e been used o imp o e he in e naliza ion o a wide ange o d ugs (Figu e 15).
Fo example, he p od ug 5-aminole ulinic acid was a ached o pene a in o cell deli e y.
173
Fu he mo e, cyclospo ine A-CPP conjuga es we e also syn hesized and i was ound ha his
conjuga e is able o pass h ough he s a um co neum and o ca y ou i s he apeu ic e ec .
174
Some o hese co alen conjuga es we e also applied in i o, showing good p ope ies o c oss
he b ain memb ane. Fo example, he a achmen o benzylpenicillin o a CPP impo an ly
inc eased he d ug deli e ed in o he b ain in compa ison wi h he d ug alone.
175
In e es ingly, he co alen a achmen o CPPs o an icance d ugs also showed o o e come
an icance -d ug esis ance in cells.
176
These conjuga es showed he abili y o a oid he e lux
pump p o eins, such as in he case o he co alen conjuga ion o doxo ubicin o TAT which
has shown high cy o oxici y agains doxo ubicin- esis an cell lines.
177
A me ho exa e-CPP
conjuga e has also shown in e naliza ion and oxici y agains cell lines esis an o his
an icance d ug.
178
A p ominen s a egy based on ac i able cell-pene a ing pep ides (ACPPs) was de eloped
by he g oup o p o . Roge Tsien (Figu e 15, igh ).179 A usion pep ide was p epa ed by a
combina ion o a ca ionic CPP and an anionic amino acid sequence ha ac s as an inhibi o o
he pene a ing ac i i y. A consensus pep ide sequence ecognized by p o eases ha a e
o e exp essed in umo s cells was inco po a ed be ween he ca ionic and he anionic sequences.
The e o e, p o eases clea age o he inhibi o sequence igge s selec i e pene a ion o he
ca go such as a luo opho e o selec i ely label umo cells.
179
Imaging agen s ha e been
173
L. Bou é, F. Giun ini, I. M. Eggles on, M. Wilson, A. J. MacRobe , B . J. Cance 2009, 100, 723-731.
174
J. B. Ro hba d, S. Ga ling on, Q. Lin, T. Ki schbe g, E. K eide , P. L. McG ane, P. A. Wende , P. A. Kha a i,
Na . Med. 2000, 6, 1253-1257.
175
C. Rousselle, P. Clai , J. Temsamani, J. M. Sche mann, J. D ug Ta ge . 2002, 10, 309-315.
176
S. B. Fonseca, M. P. Pe ei a, S. O. Kelley, Ad . D ug Deli . Re . 2009, 61, 953-964.
177
J. F. Liang, V. C. Yang, Bioo ganic Med. Chem. Le . 2005, 15, 5071-5075.
178
M. Lindg en, K. Rosen hal-Aizman, K. Saa , E. Ei íksdó i , Y. Jiang, M. Sassian, P. Ös lund, M. Hällb ink,
Ü. Langel, Biochem. Pha macol. 2006, 71, 416-425.
179
T. Jiang, E. S. Olson, Q. T. Nguyen, M. Roy, P. A. Jennings, R. Y. Tsien, P oc. Na l. Acad. Sci. 2004, 101,
17867-17872.
Chap e I: In oduc ion
67
inco po a ed in o hese pep ides o hei use as umo issues labelling p obes in i o
180
,
181
and
o guided su ge y.
182
,
183
Figu e 15. Le : Examples o d ugs deli e ed in o cells by applying co alen a achmen o CPPs.
Rep oduced om Re . 169 wi h pe mission om The Royal Socie y o Chemis y. Righ : Schema ic
illus a ion o he mechanism o in e naliza ion o ac i able cell-pene a ing pep ides (ACPPs).
Rep in ed om e . 179 Copy igh 2004 Na ional Academy o Sciences.
Mo eo e , his s a egy has been ex ended o he ield o chemo he apy by he inco po a ion
o an an icance d ug such as doxo ubicin. This idea allowed o inc ease he speci ici y in
umo s ha o e exp ess enzymes such as MMPs (ma ix me allop o einases).
184
Al hough he e
a e many examples o CPP-molecula conjuga es by co alen bonds, his s a egy has also some
limi a ions such as al e a ions o he biological e ec o he ca go and some imes edious
syn he ic s eps. Fo his eason, du ing he las decades, di e en sup amolecula s a egies ha e
been de eloped o o e come some o he limi a ions o he co alen a achmen .
4.3. Non-co alen sup amolecula me hods
Sup amolecula s a egies can use di e en non-co alen in e ac ions, such as hos -gues
encapsula ion, elec os a ic in e ac ions, o hyd ophobic o ces, o inco po a e small molecules,
such as d ugs o p obes, in o e icien deli e y ehicles. Du ing he las yea s, mul iple
sup amolecula s a egies ha e eme ged o he cy osolic deli e y o a wide ange o small
molecules including pep ides,
185
ionopho es,
186
liposomes,
187
mac ocyclic capsules,188
sup amolecula nanopa icles
188
o s imuli- esponsi e esicles.
189
180
T. A. Aguile a, E. S. Olson, M. M. Timme s, T. Jiang, R. Y. Tsien, In eg . Biol. 2009, 1, 371-381.
181
E. S. Olson, T. A. Aguile a, T. Jiang, L. G. Ellies, Q. T. Nguyen, E. H. Wong, L. A. G oss, R. Y. Tsien, In eg .
Biol. 2009, 1, 382-393.
182
Q. T. Nguyen, R. Y. Tsien, Na . Re . Cance 2013, 13, 653-662.
183
Q. T. Nguyen, E. S. Olson, T. A. Aguile a, T. Jiang, M. Scadeng, L. G. Ellies, R. Y. Tsien, P oc. Na l. Acad.
Sci. U. S. A. 2010, 107, 4317-4322.
184
N. Q. Shi, W. Gao, B. Xiang, X. R. Qi, In . J. Nanomedicine 2012, 7, 1613-1621.
185
S. J. Song, S. Lee, K. S. Ryu, J. S. Choi, Bioconjug. Chem. 2017, 28, 2266-2276.
186
P. R. B o he hood, A. P. Da is, Chem. Soc. Re . 2010, 39, 3633-3647.
187
U. Bulbake, S. Doppalapudi, N. Kommineni, W. Khan, Pha maceu ics 2017, 9, 12.
188
M. J. Webbe , R. Lange , Chem. Soc. Re . 2017, 46, 6600-6620.
189
W. C. de V ies, D. G ill, M. Tesch, A. Ricke , H. Nüsse, J. Klingau , A. S ude , V. Ge ke, B. J. Ra oo, Angew.
Chem. In . Ed. 2017, 56, 9603-9607.
Héc o Fe nández Ca o
68
4.3.1. Cell-pene a ing pep ides
Non-co alen conjuga es be ween cell-pene a ing pep ides and small molecules cons i u e
an excellen me hod o accomplish he cy osolic deli e y o small molecules. Despi e co alen
conjuga ion has been ex ensi ely used, he p epa a ion o he co esponding pep ide conjuga es
some imes equi es mul iple s eps. In con as , he p ocedu e o cellula deli e y by non-
co alen in e ac ions using pene a ing pep ides ep esen s a much mo e simple and
s aigh o wa d idea.
An elegan example o non-co alen deli e y o luo opho es has been de eloped in he
g oup o p o . Tampé (Figu e 16, le ).190 A pep ide consis ing o six N- e minal his idines was
connec ed by a linke (GGGS) o he ypical pene a ing pep ide TAT (RKKRRQRRR). On he
o he hand, a luo escen mul i alen chela o made o a mul i alen de i a i e o he N-
ni ilo iace ic acid ( isNTA) was modi ied wi h di e en luo opho es, such as Alexa Fluo
647. The isNTA was able o coo dina e wi h he his idine amino acids and when mixed
oge he he isNTA and he pep ide His6-TAT in he p esence o Ni (II), a complex was
o med. This complex was able o c oss he cell memb ane and he luo escen mul i alen
chela o was eleased inside o he cell.
190
Ano he example o non-co alen deli e y o
luo opho es wi h amphiphilic cell-pene a ing pep ides was shown wi h a non-co alen
complex including he mally ac i a ed delayed luo escence (TADF) moie ies.191 In his wo k,
he au ho s designed an amphiphilic cell-pene a ing pep ide, F6G6( R)3R2, ha was able o o m
sup amolecula complexes wi h TADF compounds such as 4CzIPN, NAI-DPAC, and BTZ-
DMAC (Figu e 16, igh ). These luo escen molecules and he pep ide sel -assembled in o
luo escen nanopa icles, hese NPs we e able o easily c oss he cell memb ane and o allow
he acquisi ion o ime- esol ed luminescence images.
191
Fu he mo e, amphipa hic pep ides
o ming nano ibbons coa ed wi h a second laye o cell-pene a ing pep ides we e also able o
deli e luo opho es inside o he cell.
192
Figu e 16. Le : Chemical s uc u e o isNTA luo opho e and His6- agged TAT cell-pene a ing
pep ide. Rep in ed wi h pe mission om e . 190. Copy igh 2018 Ame ican Chemical Socie y. Righ :
Chemical s uc u e o amphiphilic CPP and he di e en TADF luo opho es. Rep in ed and adap ed
wi h pe mission om e . 191. Copy igh 2018 Ame ican Chemical Socie y.
190
R. Wieneke, N. Labò ia, M. Rajan, A. Kollmannspe ge , F. Na ale, M. C. Ca doso, R. Tampé, J. Am. Chem.
Soc. 2014, 136, 13975-13978.
191
Z. Zhu, D. Tian, P. Gao, K. Wang, Y. Li, X. Shu, J. Zhu, Q. Zhao, J. Am. Chem. Soc. 2018, 140, 17484-17491.
192
Y. Lim, E. Lee, M. Lee, Angew. Chem. In . Ed. 2007, 46, 3475-3478.
Chap e I: In oduc ion
69
On he o he hand, amphiphilic pep ides, using non-co alen in e ac ions, we e shown o
anspo d ugs such as cu cumin o doxo ubicin.185,193 Fo example, amphiphilic pep ide
nano ods we e ecen ly used o encapsula e cu cumin and subsequen ly o deli e his molecule
in cance cell lines and zeb a ish models.185 Mo eo e , a new amphiphilic pep ide was ecen ly
shown o sel -assemble o o m pH- esponsi e micelles ha we e able o en ap and deli e
doxo ubicin in cells.
193
4.3.2. Ionopho es
Ano he sup amolecula me hod o he anspo o small molecules is he ionopho es.
This amily o syn he ic molecules ge s inspi a ion om a a ie y o na u ally occu ing
p oduc s ha a e able o complex ions in a e y igh and e icien manne and p omo e hei
ansloca ion ac oss lipid bilaye s.
194
To ca y ou such a challenging ask, na u al ionopho es
p esen a s uc u e ha is gene ally cyclic o ben -shaped, in which he ex e nal su ace o he
molecules p esen s a s ong hyd ophobic cha ac e , which g an s a good pa i ioning in o he
a y-acid ich memb ane lumen. The hyd ophilic egion o ionopho es is ich in coo dina ing
he e oa oms esponsible o he in e ac ion wi h he ions, no mally being able o mimic he
hyd a ion sphe e o he ion ha is ansloca ed.
195
,
196
Va ious esea ch g oups ha e modi ied
na u al molecules o design new anspo e s. In his g oup, p odigiosins a e a class o
molecules based on 4-me hoxy-bipy ole wi h di e en subs i uen s (Figu e 17).
197
In ecen
yea s, i has been ound ha his ype o molecules can unc ion as H+/Cl-
198
and Cl-/HCO3-
199
exchange an ipo e s.
To achie e seg ega ion be ween an ex e nal lipophilic su ace and a coo dina ion- ich inne
ca i y, esea che s ha e employed mul ipodal sca olds o ob ain a p ea anged idimensional
disposi ion o key unc ional g oups.
200
In his ega d, he cholapods, which a e a amily o
s e oids de i ed om cholic acid, ha e been designed (Figu e 17B). In hese molecules, one o
he hyd ophobic aces is p ese ed and g oups capable o complexing anions such as
squa amides, hiou eas, o u eas a e inco po a ed in o a ious posi ions o he polycyclic
ing.186 In e es ingly, cholapods wi h high e iciency o he anspo o Cl- in model esicles
ha e been disco e ed.
201
Also applying his s a egy, he unc ionaliza ion wi h h ee u eas o
185 S. J. Song, S. Lee, K. S. Ryu, J. S. Choi, Bioconjug. Chem. 2017, 28, 2266-2276.
193
J. Liang, W. Wu, X. Xu, R. Zhuo, X. Zhang, Colloids Su aces B Bioin e aces 2014, 114, 398-403.
194
A. Roy, D. Saha, A. Mukhe jee, P. Talukda , O g. Le . 2016, 18, 5864-5867.
195
J. Ru kowski, B. B zezinski, Biomed Res. In . 2013, 2013, 162513.
196
P. A. Gale, Acc. Chem. Res. 2011, 44, 216-226.
197
J. L. Sessle , L. R. Elle , W. S. Cho, S. Nicolaou, A. Aguila , J. T. Lee, V. M. Lynch, D. J. Magda, Angew.
Chem. In . Ed. 2005, 44, 5989-5992.
198
K. Tanigaki, T. Sa o, Y. Tanaka, T. Ochi, A. Nishikawa, K. Nagai, H. Kawashima, S. Ohkuma, FEBS Le .
2002, 524, 37-42.
199
J. T. Da is, P. A. Gale, O. A. Okunola, P. P ados, J. C. Iglesias-Sánchez, T. To oba, R. Quesada, Na . Chem.
2009, 1, 138-144.
200
N. Busschae , M. Wenzel, M. E. Ligh , P. Iglesias-He nndez, R. P ez-Toms, P. A. Gale, J. Am. Chem. Soc.
2011, 133, 14136-14148.
186 P. R. B o he hood, A. P. Da is, Chem. Soc. Re . 2010, 39, 3633-3647.
201
S. J. Edwa ds, H. Valkenie , N. Busschae , P. A. Gale, A. P. Da is, Angew. Chem. In . Ed. 2015, 54, 4592-
4596.

Héc o Fe nández Ca o
70
hiou eas has been ca ied ou using is(2-aminoe hyl)amine, and he esul ing compounds
showed good e iciency as bica bona e anspo e s.
202
Figu e 17. Chemical s uc u es o di e en examples o ionopho es: A) P odigiosin.197 B) Example o
cholapod.186 C) Rep esen a i e example o ans-decalin de i a i e.204
O he anspo e s o high in e es a e di unc ionalized ans-decalins in he 4,8-diaxial
posi ions. In his ype o molecules, he hyd ophobic moie y o he co e molecule is main ained
while in he o he pa is modi ied wi h wo hiou ea o u ea g oups wi h a oma ic
subs i uen s.
203
In e es ingly, one o he ans-decalin de i a i es was he mos e ec i e
anspo e o he Cl- anion in model esicles (Figu e 17).204 This anspo e consis s o a
modi ied ans-decalin wi h wo a yl- hiou ea g oups in which he a oma ic ings a e
unc ionalized wi h wo CF3 g oups in posi ions 3 and 5.
204
Mo e ecen ly, we should highligh
ha hese anspo sys ems a e s a ing o be conside ed in i o o he ea men o cys ic
ib osis, in which hese a i icial molecules could ha e he po en ial o mimic Cl- anspo ing
channels (CFTR p o eins) absen o mal unc ioning in he pa ien s ha su e om his
disease.
205
,
206
4.3.3. Liposomes
Liposomes a e one o he mos used d ug deli e y sys ems. Classical liposomes consis o
sphe ical esicles wi h an in e nal aqueous co e ha is su ounded by one o mo e lipid bilaye s
in he ex e nal laye . F om he pha maceu ical poin o iew, hese en i ies a e capable o
inco po a ing bo h hyd ophobic (in he bilaye ) and hyd ophilic molecules (in he aqueous
space).
207
When classical liposomes we e adminis e ed ollowing he in a enous pa hway, i
was ound ha hey su e om a as clea ance, ha ing sho ci cula ion imes. Howe e ,
clea ance was educed ca ying ou he PEGyla ion o he liposome. A ew yea s ago, he
PEGyla ed liposomal doxo ubicin (Doxil®) was FDA app o ed and showed an inc ease in he
d ug le els and educ ion o ca diomyocy e damage in compa ison wi h doxo ubicin alone.
Ano he example o using a PEGyla ed liposomal sys em, in his case, o i ino ecan deli e y
is Oni ydeTM.187 Recen ly, mul i unc ional liposomes (Figu e 18, le ) ha e eme ged o imp o e
202
N. Busschae , P. A. Gale, C. J. E. Haynes, M. E. Ligh , S. J. Moo e, C. C. Tong, J. T. Da is, W. A. Ha ell,
Chem. Commun. 2010, 46, 6252-6254.
203
S. Hussain, P. R. B o he hood, L. W. Judd, A. P. Da is, J. Am. Chem. Soc. 2011, 133, 1614-1617.
204
H. Valkenie , L. W. Judd, H. Li, S. Hussain, D. N. Sheppa d, A. P. Da is, J. Am. Chem. Soc. 2014, 136, 12507-
12512.
205
K. A. Mu aglia, R. S. Cho ghade, B. R. Kim, X. X. Tang, V. S. Shah, A. S. G illo, P. N. Daniels, A. G. Cio i,
P. H. Ka p, L. Zhu, e al., Na u e 2019, 567, 405-408.
206
P. A. Gale, J. T. Da is, R. Quesada, Chem. Soc. Re . 2017, 46, 2497-2519.
207
T. O. B. Olusanya, R. R. H. Ahmad, D. M. Ibegbu, J. R. Smi h, A. A. Elko dy, Molecules 2018, 23, 907.
187 U. Bulbake, S. Doppalapudi, N. Kommineni, W. Khan, Pha maceu ics 2017, 9, 12.
A)
B)
C)
Chap e I: In oduc ion
71
he pha maceu ical p ope ies o liposomes. This kind o liposomes can inco po a e di e en
unc ionali ies in he same en i y such as a ge ing ligands (an ibody o ca bohyd a es), imaging
agen s, hyd ophilic o hyd ophobic d ugs, he apeu ic nucleic acids, e c.
208
Fo ins ance, an
inno a i e app oach has been ecen ly de eloped in he g oup o p o . Alexande K os.209 In
his wo k, a pai o complemen a y coiled-coil lipopep ides we e designed and inco po a ed
in o liposomes. The usion o he coiled-coil pep ides allowed he elease o he an icance d ug,
doxo ubicin, encapsula ed inside he liposomes in cells, and zeb a ish models.
209
4.3.4. Hos -gues deli e y sys ems
Hos -gues p ocesses a e a powe ul ool o de elop new ca ie s o he encapsula ion and
he deli e y o small molecules. As we ha e p e iously commen ed, classical hos molecules
such as cyclodex ins, cucu bi u ils, o calixa enes ha e been applied o deli e a a ie y o
he apeu ic molecules.188 Cyclodex ins (CDs) ha e been shown o be sui able deli e y ca ie s
o di e en ca gos due o hei high bioa ailabili y and low cy o oxici y. Fo ins ance, β-CDs
ha e been demons a ed no only o ecognize a a ie y o subs a es bu also o p omo e he
deli e y o di e en an icance d ugs such as doxo ubicin
210
o pacli axel.
211
Fo example, hos -
gues molecula ecogni ion be ween β-CDs and pacli axel has been ecen ly used o deli e
his an i umo al d ug in li ing cells and also in i o (Figu e 18, igh ).212 Kim and co-wo ke s
ha e designed a sys em cons i u ed by poly-pacli axel and poly-CDs conjuga ed by es e bonds
o a polyme chain. Mo eo e , a pep idic a ge ing ligand was also inco po a ed in o he sys em.
Once inside he cell, in acellula enzymes clea e he es e bonds and he
cyclodex in/pacli axel complexes a e eleased om he polyme s. Finally, hese complexes
dissocia e and he ee pacli axel p oduces i s umo cy o oxic e ec .
212
208
M. Riaz, M. Riaz, X. Zhang, C. Lin, K. Wong, X. Chen, G. Zhang, A. Lu, Z. Yang, In . J. Mol. Sci. 2018, 19,
195.
209
J. Yang, A. Bah eman, G. Daudey, J. Bussmann, R. C. L. Ols hoo n, A. K os, ACS Cen . Sci. 2016, 2, 621-630.
188 M. J. Webbe , R. Lange , Chem. Soc. Re . 2017, 46, 6600-6620.
210
H. Hyun, S. Lee, W. Lim, D. Jo, J. S. Jung, G. Jo, S. Y. Kim, D. Lee, S. Um, D. H. Yang, e al., J. Ind. Eng.
Chem. 2019, 70, 145-151.
211
J. Jing, A. Sza pak-Jankowska, R. Guillo , I. Pigno -Pain and, C. Pica , R. Auzély-Vel y, Chem. Ma e . 2013,
25, 3867-3873.
212
R. Namgung, Y. Mi Lee, J. Kim, Y. Jang, B. H. Lee, I. S. Kim, P. Sokka , Y. M. Rhee, A. S. Ho man, W. J.
Kim, Na . Commun. 2014, 5, 3702.
Héc o Fe nández Ca o
72
Figu e 18. Le . Schema ic ep esen a ion showing he di e en ypes o liposomes: A) Con en ional
liposomes, B) PEGyla ed liposomes, C) Ligand a ge ed liposomes and D) Mul i unc ional liposomes
such as he anos ic liposomes. Rep in ed om e . 208 unde he e ms o he C ea i e Commons CC
BY license. Righ . Schema ic illus a ion o he mechanism o in e naliza ion and pacli axel elease om
he sup amolecula hos -gues nano-assembly. Rep in ed wi h pe mission om e . 212. Copy igh 2014
Sp inge Na u e.
4.3.5. Redox and pH- esponsi e encapsula ing esicles
Redox- esponsi e nanocon aine s ha e been shown o deli e hyd ophilic ca gos such as
py anine o phalloidin. These nanocon aine s consis o a shell o med by a edox- esponsi e
polyme a ached by hos -gues p ocesses o a cyclodex in co e h ough an adaman ane moie y
(Figu e 19, le ). The encapsula ion o di e en ca gos was achie ed by disul ide bonds ha
we e inco po a ed in o he polyme ic shell as edox clea able unc ions. In his wo k, he
au ho s showed ha hese nanocon aine s we e able o elease di e en molecula payloads in
he cell in e io and d i en by he edox igge .189
On he o he hand, pH- esponsi e co e-shell nanopa icles ha e also been desc ibed o
deli e luo escen p obes and small molecula d ugs simul aneously (Figu e 19, igh ).213
These nanopa icles consis o a shell o med by he nega i ely cha ged py anine wi h a co e o
posi i ely cha ged ca boxyme hylhexanoyl chi osan. The elec os a ic in e ac ions be ween he
shell and co e polyme s p oduce he assembly in o pH- esponsi e nanopa icles. In e es ingly,
he encapsula ion o he an i umo al d ug camp o hecin in o hese NPs o pH-con olled d ug
elease was achie ed. The au ho s we e able o de elop a he anos ic sys em wi h po en ial
he apeu ic and diagnosis capabili ies.
213
189 W. C. de V ies, D. G ill, M. Tesch, A. Ricke , H. Nüsse, J. Klingau , A. S ude , V. Ge ke, B. J. Ra oo, Angew.
Chem. In . Ed. 2017, 56, 9603-9607.
213
H. S. Chou, M. H. Hsiao, W. Y. Hung, T. Y. Yen, H. Y. Lin, D. M. Liu, J. Ma e . Chem. B 2014, 20, 6580-
6589.
Chap e I: In oduc ion
73
Figu e 19. Le . P epa a ion o edox- esponsi e nanocon aine and edox- igge ed payload elease.
Rep in ed e . 189 wi h pe mission om John Wiley and Sons. Copy igh 2017 Wiley‐VCH Ve lag
GmbH & Co. KGaA, Weinheim. Righ : Schema ic ep esen a ion o pH- esponsi e co e-shell
nanopa icles. Rep oduced om Re . 213 wi h pe mission om The Royal Socie y o Chemis y.
4.4. T ansi o y educ ion o cha ge
T ansi o y educ ion o cha ge is one o he mos widely applied me hods o igge he
in acellula elease o small hyd ophilic molecules. This s a egy is based on inc easing he
hyd ophobici y o he molecule by o ming hyd ophobic es e s a phospho ic o ca boxylic
acids, among o he s. The esul ing molecule, wi h highe hyd ophobic p ope ies, can c oss he
cell memb ane and, a e he ac ion o in acellula enzymes, he hyd ophilic molecule is
eleased.
214
,
215
The main limi a ions o his s a egy a e he equi ed syn he ic modi ica ion o
he ac i e molecule and he need o e icien in acellula hyd olysis o he co esponding es e .
A ew decades ago, he p od ug s a egy has been de eloped.
216
This s a egy is based on
he modi ica ion o ac i e d ugs ha a e su e ing an in acellula clea age, by enzymes o
chemicals, will lead o he elease o he ac i e d ug. This me hodology has been applied o
inc ease he e iciency o a wide ange o d ugs.
217
Fo ins ance, cido o i is a highly
hyd ophilic d ug, used as he apy o he pes i uses, which ha e s ong limi a ions o c oss he
plasma memb ane. Howe e , when he phospha e g oup o his molecule is modi ied by
es e i ica ion wi h a hyd ophobic molecule, he esul ing molecule can be in e nalized by simple
di usion, and subsequen ly, he es e enzyma ic hyd olysis o he es e inside he cell allows
he eco e y o he ac i e p inciple. Impo an ly, he adminis a ion o he p od ug o cido o i
causes a ou - old ac i i y enhancemen compa ed o he unmodi ied d ug (Figu e 20).
218
In
addi ion o bioac i e molecules and d ugs, his s a egy has also been applied o di e en
luo escen p obes.
219
In ecen yea s, luo escein de i a i es ha e been used o ca y ou
in acellula pH measu emen s by luo escence me hods. One o he mos used compounds is
2´,7´-Bis(2-ca boxye hyl)-5(6)-ca boxy luo escein (BCECF) (Figu e 20).
220
This small highly
214
P. Ozkan, R. Mu ha asan, Biochim. Biophys. Ac a 2002, 1572, 143-148.
215
J. Rau io, H. Kumpulainen, T. Heimbach, R. Oliyai, D. Oh, T. Jä inen, J. Sa olainen, Na . Re . D ug Disco .
2008, 7, 255-270.
216
J. Rau io, N. A. Meanwell, L. Di, M. J. Hageman, Na . Re . D ug Disco . 2018, 17, 559-587.
217
K. M. Hu unen, H. Raunio, J. Rau io, Pha macol. Re . 2011, 63, 750-771.
218
E. De Cle cq, H. J. Field, B . J. Pha macol. 2006, 147, 1-11.
219
L. D. La is, T.-Y. Chao, R. T. Raines, Chem. Sci. 2011, 2, 521-530.
220
N. Boens, W. Qin, N. Basa ić, A. O e, E. M. Tala e a, J. M. Al a ez-Pez, J. Phys. Chem. A 2006, 110, 9334-
9343.

Chap e I: Objec i es
81
Based on p e ious esul s on cage complexa ion o plana anionic molecules,69 we decided
o explo e he po en ial caging s a egy o he ansi o y masking o he hyd ophilici y o plana
p obes and o achie e hei deli e y in he cy osol. The sup amolecula na u e o he app oach
will allow us o deli e hese p obes in o he cell cy osol d i en by he di e en concen a ions
o he p obe be ween he ex acellula and he in acellula en i onmen and he high
concen a ion o di e en anions p esen in he cell in e io (Figu e 24).
The speci ic objec i es o his i s chap e would be:
 Conjuga ion o a memb ane-impe meable sup amolecula ca ionic cage wi h di e en
cell-pene a ing pep ides o di e en leng hs, which would dona e cell memb ane
anspo capabili ies o he sup amolecula cage and e alua ion o hei cy o oxici y.
 S udy o he in e ac ion be ween he sup amolecula cage and he py anine p obe by
i a ion expe imen s, o unde s and he anspo mechanism o he p obe by U- ube
expe imen s.
 In acellula cy osolic deli e y o py anine in di e en cell lines using he p e iously
syn hesized pep ide-cage ca ie s.
 S udy o he mechanism o anspo o hese sup amolecula complexes inside li ing
cells by ensu ing he non-pe meabiliza ion o he cell memb ane and by using
endocy osis inhibi o s.
 E alua ion o he speci ici y o he app oach by compe i ion assays wi h di e en
ca gos.
 Ex ension o he app oach o s udy in e ac ion wi h he cage and cellula deli e y
e iciency o di e en anionic p obes (CF and Alexa Fluo ´s).
 Cell pH acking by using pep ide-cage ca ie and py anine as a pH-sensi i e p obe.
Figu e 24. A) Objec i e: de elopmen o a pep ide-cage ca ie ha consis s o a pep ide (blue sphe es)
and a posi i ely cha ged sup amolecula cage ha is capable o deli e ing memb ane-impe meable
plana anionic p obes in o li ing cells and elease hem om endosomes. B) Examples o highly aluable
luo escen p obes which a e no able o c oss cell memb anes a low mic omola concen a ions.
69 J. Mosque a, S. Za a, J. R. Ni schke, Angew. Chem. In . Ed. 2014, 53, 1556-1559.
A)
B)
Resul s and discussions
The esul s o his chap e ha e been published in:
H. Fe nández-Ca o, I. Los alé-Seijo, M. Ma ínez-Cal o, J. Mosque a, J. L. Masca eñas, J.
Mon eneg o, Chem. Sci. 2019, 10, 8930-8938.
Chap e I: Resul s and discussions
85
1. An eceden s
The g oup o p o . Ni schke has ecen ly de eloped a ca ionic sup amolecula cage (Figu e
25) ha showed he abili y o in e ac by hos -gues molecula ecogni ion wi h he py anine
p obe in aqueous en i onmen wi h a Kd o 1.2 nM.69 La e , in collabo a ion wi h P o .
Masca eñas g oup, a py anine-pep ide gues was designed by connec ing a ypical cell-
pene a ing pep ide (oc aa ginine) o a polyanionic oligoglu amic e mina ed wi h a py anine
moie y. This pep ide was no able o c oss he cell memb ane due o he anionic cha ac e o
he pendan . Howe e , when he sup amolecula cage was inco po a ed, he ca ionic cage
o med a hos -gues complex wi h he anionic py anine, masking i s nega i e cha ge and
p omo ing he cellula in e naliza ion o he memb ane-impe meable py anine-pep ide (Figu e
25).
241
Mo e ecen ly, in a ui ul collabo a ion be ween he g oup o p o . Masca eñas and he
g oup o p o . Liz-Ma zán, he sup amolecula in e ac ion py anine-cage was u he explo ed
o swi ch he cellula up ake o gold nanopa icles (AuNPs) (Figu e 25).242 In his wo k, AuNPs
we e unc ionalized wi h py anine moie ies. Due o hei high nega i e cha ge, hese AuNPs
did no c oss h ough he cell memb ane. Howe e , a e he o ma ion o he hos -gues
complex be ween he cage and he py anine, hei nega i e cha ge was masked and he cellula
in e naliza ion o he py anine-modi ied AuNPs was achie ed. In e es ingly, i was ound ha
he cellula up ake o he py anine-modi ied AuNPs could be e e sibly u ned on and o wi h
he addi ion o he sup amolecula cage.
242
Figu e 25. Schema ic ep esen a ions showing he in acellula deli e y o memb ane-impe meable A)
py anine- unc ionalized pep ide and B) py anine-modi ied AuNPs igge ed by he addi ion o he
ca ionic sup amolecula cage. A) Rep in ed wi h pe mission om e . 241. Copy igh 2017 Ame ican
Chemical Socie y. B) Rep in ed wi h pe mission om e . 242. Copy igh 2018 Ame ican Chemical
Socie y.
69 J. Mosque a, S. Za a, J. R. Ni schke, Angew. Chem. In . Ed. 2014, 53, 1556-1559.
241
J. Rod íguez, J. Mosque a, J. R. Coucei o, J. R. Ni schke, M. E. Vázquez, J. L. Masca eñas, J. Am. Chem. Soc.
2017, 139, 55-58.
242
J. Mosque a, M. Hen iksen-Lacey, I. Ga cía, M. Ma ínez-Cal o, J. Rod íguez, J. L. Masca eñas, L. M. Liz-
Ma zán, J. Am. Chem. Soc. 2018, 140, 4469-4472.
A)
B)

Héc o Fe nández Ca o
86
2. Design and syn hesis
2.1. Syn hesis o he cage C and ini ial cellula in e naliza ion expe imen s
Fi s , in collabo a ion wi h he g oup o p o . Ni schke, we ollowed he p ocedu e epo ed
in he li e a u e o he syn hesis and he pu i ica ion o he sup amolecula cage C (Figu e
4A).69 Following he desc ibed p ocedu e, he 3,3’-bipy idine-6,6’-dica boxaldehyde was
eac ed wi h is(2-aminoe hyl)amine and cadmium(II) i luo ome hanesul ona e (Cd(OT )2)
in ace oni ile solu ion. The esul ing CdII- empla ed cage was hen educed using sodium
bo ohyd ide (NaBH4), leading o he deme alla ed cage C. Once, we ha e p epa ed he ca ionic
cage C, we wonde ed i he cage C alone would ha e he abili y o p omo e he cellula
in e naliza ion o py anine and, also, i py anine alone would be able o c oss he cell memb ane
a di e en concen a ions (Figu e 26). To s udy his phenomenon, we decided o ca y ou
p elimina y con ol expe imen s using con ocal mic oscopy.
Figu e 26. A) Schema ic ep esen a ion o he con ol expe imen s showing nei he in e naliza ion o
he complex py anine and cage C no py anine alone inside cells. B and C) Con ol anspo
expe imen s in HeLa cells. Con ocal mic og aphs o HeLa cells incuba ed wi h: B) Py anine (5 µM) C)
Py anine (5 µM) and only cage C (5µM). Nuclei s ained wi h Hoechs (blue). DIC on he le and me ge
o luo escence channels [Py anine emission (g een) + Hoechs 33342 emission (blue)] on he igh .
Fi s , we decided o e alua e he po en ial o he cage C o in e nalize py anine. Fo his,
we co-incuba ed di e en concen a ions o he cage C and py anine wi h HeLa cells. To ca y
ou his in e naliza ion expe imen , we ea ed HeLa cells wi h Hoechs 33342 o 30 min ( o
nucleus s aining), we washed (HKR bu e , 1x), and hen cells we e incuba ed wi h he di e en
concen a ions o cage and py anine, o ins ance, 5 μM o py anine and 5 μM o cage C (Figu e
26), in HKR bu e o 30 min a 37 ⁰C. Cells we e inally washed (HKR bu e , 3x) and
69 J. Mosque a, S. Za a, J. R. Ni schke, Angew. Chem. In . Ed. 2014, 53, 1556-1559.
A)
B)
C)
Chap e I: Resul s and discussions
87
obse ed unde he con ocal mic oscope. In he con ocal mic og aphs (Figu e 26), we did no
obse e any py anine (g een) luo escence inside he cells. This esul demons a ed ha he
sup amolecula cage C does no p omo e he in acellula deli e y o py anine. On he o he
hand, we also e alua ed he abili y o py anine alone o c oss he cell memb ane a di e en
concen a ions. Following he same p ocedu e, HeLa cells incuba ed wi h 5 μM o py anine
alone did no show any g een luo escence inside he cells (Figu e 26).
2.2. Ini ial design and syn hesis o he pep ide-cage hyb ids TmAC and
TmR8C
A e e i ying by con ocal expe imen s ha he sup amolecula cage C alone is no able
o in e nalize py anine a di e en concen a ions, we hen decided o co alen ly a ach o one
o he e ices o he cage di e en ca ionic pep ides wi h excellen in e naliza ion abili ies and
high aqueous solubili y. We hypo hesized ha hese pep ide pendan s would p omo e he
cellula in e naliza ion and also would inc ease he solubili y o he cage in he aqueous
en i onmen .
Wi h his pu pose, i s , we decided o co alen ly a ach o he cage a ypical cell-
pene a ing pep ide oc aa ginine (R8). This pep ide (Figu e 27) has been in ensi ely s udied and
demons a ed o be able o imp o e he solubili y and o p omo e he cellula ansloca ion o
di e en he apeu ically ac i e molecules.169 We also decided o inco po a e he amphiphilic
pep ide A (Figu e 27) o he cage.109 Fu he mo e, we inco po a ed he TAMRA luo opho e a
he N- e minus o bo h pep ides (Figu e 27) o ollow he dis ibu ion o he pep ide in cellula
expe imen s by con ocal mic oscopy wi hou causing any in e e ence wi h he py anine
emission.
Figu e 27. Chemical s uc u e o he pep ide-cage hyb ids TmR8C and TmAC.
To p omo e he in acellula deli e y o py anine, we syn hesized wo di e en pep ide-
cage hyb ids TmR8C and TmAC in which he Tm supe index deno es he inco po a ion o
TAMRA luo opho e a he N- e minus o he pep ides (Figu e 27). The syn he ic s a egy was
di ided in o wo s eps, i s , we syn hesized TmA and TmR8 wi h a ee ca boxylic acid g oup a
hei e minal ca boxylic ends and, in he second s ep, in collabo a ion wi h he g oup o he
p o . Masca eñas, we a ached he sup amolecula cage C (Figu e 28). To ca y ou he i s
s ep, SPPS by manual Fmoc solid-phase was applied by using a 2-chlo o i yl chlo ide esin.
243
This esin was employed o ob ain he co esponding ee C- e minal g oup o he subsequen
169 K. M. S ewa , K. L. Ho on, S. O. Kelley, O g. Biomol. Chem. 2008, 6, 2242-2255.
109 M. Mäe, Ü. Langel, Cu . Opin. Pha macol. 2006, 6, 509-514.
243
M. Alhassan, O. Al Musaimi, J. M. Collins, F. Albe icio, B. G. de la To e, G een Chem. 2020, 22, 2840-2845.
Héc o Fe nández Ca o
88
a achmen o he cage C. Impo an ly, we inco po a ed wo di e en linke s, i s , 8-amino-
3,6-dioxaoc anoic acid (O2Oc) and hen, 6-aminohexanoic acid (Ahx), be o e bo h pep ide
sequences. We hypo hesized ha he inco po a ion o bo h linke s would dec ease he s e ic
hind ance and he elec os a ic in e ac ions and would inc ease he yield o he eac ion wi h
he ca ionic cage in he las s ep. The i s coupling was ca ied ou in CH2Cl2 using DIEA as
he base. Fo he ollowing couplings, we used N-HBTU as ac i a o , DIEA as base, and DMF
as sol en . The dep o ec ion o he empo al Fmoc p o ec ing g oup was pe o med by ea ing
he esin wi h 20% pipe idine in DMF. To couple he luo opho e a he N- e minus we used
h ee equi alen s o 5(6)-Ca boxy e ame hyl hodamine (TAMRA) wi h 3 equi alen s o N-
HATU and 5 equi alen s o DIEA in DMF (0.2 M) o 60 min. The clea age/dep o ec ion s ep
was pe o med by ea men o he esin-bound pep ide o 2 h wi h he ollowing clea age
cock ail: 900 mL TFA, 50 mL CH2Cl2, 25 mL H2O, and 25 mL TIS. Pep ides we e p ecipi a ed
wi h E 2O and pu i ied by RP-HPLC. The pep ides we e cha ac e ized by HPLC-MS.
Once we ha e syn hesized and cha ac e ized TmA and TmR8 wi h he ee e minal
ca boxylic g oups, we p oceed wi h he second syn he ic s ep, he coupling o he
sup amolecula cage. To syn hesize TmAC and TmR8C, we i s dissol ed TmA and TmR8
pep ides in DMF (0.1 mM, 100 mL) and hen N-HATU (1 equi .) and DIEA (10 equi .) we e
added o he solu ion. This mix u e was added o a 0.1 mM solu ion o he cage C (1 equi .)
and he eac ion was le s i ing o e nigh . The esul ing p oduc was pu i ied by RP-HPLC
and he pep ide-cage pep ides we e cha ac e ized by mass spec ome y.
Figu e 28. Scheme o he syn hesis o TmAC and TmR8C.
2.3. P elimina y in e naliza ion expe imen s using TmAC and TmR8C
Once we had syn hesized and cha ac e ized he pep ides TmAC and TmR8C, we add essed
i hese pep ide-cage hyb ids would be able o in e nalize py anine inside cells. To s udy he
in e naliza ion o his p obe, we decided o ca y ou p elimina y anspo expe imen s using
low mic omola concen a ions o bo h py anine and he di e en pep ides. Fi s , we ca ied
ou py anine anspo expe imen s using TmAC. We incuba ed HeLa cells wi h Hoechs 33342
o 30 min, washed (HKR bu e , 1x), and hen we co-incuba ed he cells wi h 5 µM o TmAC
and 5 µM Py anine in HKR bu e o 30 min a 37 ⁰C. The cells we e inally washed (HKR
bu e , 3x) and hen obse ed unde he con ocal mic oscope. Unexpec edly, we ound high
cellula oxici y o he TmAC pep ide which can be app ecia ed in he con ocal images (Figu e
29) by he mo phological cellula al e a ions such as nuclea p o iling. Mo eo e , we also ound
ha his pep ide o ms agg ega es in he p esence o py anine in he cellula en i onmen .
Chap e I: Resul s and discussions
89
Then, we ca ied ou py anine anspo expe imen s wi h TmR8C ollowing he same
p ocedu e as be o e bu , in his case, using 5 µM o TmR8C and 5 µM Py anine. Howe e , we
ound again high cy o oxici y o TmR8C which was e iden by he cellula mo phological
al e a ions and he lowe numbe o cells obse ed (Figu e 29). Mo eo e , as he p e ious
hyb id, his pep ide is also o ming agg ega es in he p esence o py anine in he cellula
medium.
Figu e 29. T anspo expe imen s in HeLa cells wi h TmAC and TmR8C. Con ocal mic og aphs o HeLa
cells incuba ed wi h: A) 5 µM o TmAC and 5 µM Py anine B) 5 µM o TmR8C and 5 µM Py anine. DIC
on he le and me ge channels [Py anine emission (g een) + TAMRA emission ( ed) + Hoechs 33342
emission (blue)] on he igh .
2.4. Re-design and syn hesis o he pep ide cage-hyb ids AcR4C/TmR4C
Taking in o accoun he high cellula oxici y and high abili y o o m agg ega es when
incuba ed wi h py anine o p e iously designed pep ides TmAC and TmR8C. We e-designed ou
pep ide-cage hyb id and we decided o co alen ly a ach a sho e e aa ginine pep ide o one
o he e ices o he cage ins ead o he oc apep ide (Figu e 30). We hypo hesized ha his
sho e ca ionic pep ide would a oid cy o oxici y and agg ega ion p oblems.
The e o e, we syn hesized wo R4-cage hyb ids, AcR4C and TmR4C (supe indexes: Ac o
he ace yla ed hyb id and Tm o he TAMRA labeled de i a i e). Again, we p epa ed he
labeled e sion wi h he aim o ollowing he dis ibu ion o he pep ide in cellula expe imen s
using con ocal mic oscopy. On he o he hand, he ace yla ed e sion AcR4C (Figu e 30) was
p epa ed o e alua e he in luence o he luo opho e in he in e naliza ion.
The syn hesis o bo h AcR4C and TmR4C was ca ied ou ollowing he s a egy p e iously
desc ibed o TmAC and TmR8C. Fo he p epa a ion o AcR4, he ace yla ion o he N- e minal
g oup was pe o med unde s anda d Fmoc emo al condi ions (20% pipe idine in DMF)
ollowed by ea men wi h a solu ion o ace ic anhyd ide and 2,6-lu idine (1: 1, 1 mL) o 30
min. Mo eo e , hese pep ide-cage ec o s we e pu i ied and cha ac e ized by HPLC-MS
showing ha only one pep ide was a ached o a single cage.
A)
B)
Héc o Fe nández Ca o
96
To achie e an e icien py anine deli e y in cells, i s , we pe o med dose- esponse
anspo expe imen s wi h inc easing concen a ions o py anine (Figu e 37) in o de o ind
he bes concen a ion o ca ie /py anine o he bes anspo e iciency. To ca y ou his
expe imen , we decided o use he same concen a ion o TmR4C (5 μM) han in he p e ious
expe imen because i was shown o ha e good ansloca ion e iciency a his concen a ion.
Then, we used inc easing concen a ions o he py anine (2,5 μM, 5 μM, and 10 μM). In his
expe imen , HeLa cells we e incuba ed wi h he di e en concen a ions in HKR bu e o 30
min a 37 ⁰C. The cells we e inally washed (HKR bu e , 3x) and hen obse ed unde he
con ocal mic oscope.
In his dose- esponse expe imen , we ound ha TmR4C (5 µM) was able o anspo
py anine o he cell cy osol a 2.5 µM and 5 µM concen a ion. The bes anspo e iciency
was showed a TmR4C (5 µM) and py anine (5 µM). Mo eo e , we ound ha when we
inc eased he py anine concen a ion o 10 μM, he anspo o py anine was inhibi ed and
ex acellula agg ega ion was obse ed (Figu e 37).
Figu e 37. Dose- esponse anspo expe imen s o py anine in HeLa cells wi h TmR4C. Con ocal
mic og aphs o HeLa cells incuba ed wi h: A) TmR4C (5 µM), B) TmR4C (5 µM) and Py anine (2.5 µM),
C) TmR4C (5 µM) and Py anine (5 µM), D) TmR4C (5 µM) and Py anine (10 µM). DIC on he le and
me ge channels [Py anine emission (g een) + Tm emission ( ed)] on he igh .
Since he esul s o he dose- esponse expe imen sugges ed ha he bes anspo
e iciency was eached a equimola concen a ions o TmR4C (5 µM) and py anine (5 µM) in
HeLa cells (Figu e 37C), we decided o ca y ou u he cell in e naliza ion expe imen s in a
di e en cell line such as Ve o cells, achie ing also e icien cy osolic deli e y o py anine wi h
TmR4C (Figu e 38).

Chap e I: Resul s and discussions
97
Figu e 38. Py anine anspo expe imen s in HeLa (A) and Ve o (B) cells. A) Top: con ocal
mic og aphs o HeLa cells co-incuba ed wi h py anine (5 μM, g een) and TmR4C (5 μM, ed) o 30 min
in HKR bu e . Nuclei we e s ained wi h Hoechs (blue). The whi e a ow shows he nucleola
accumula ion o he pep ide-cage ca ie . Bo om: con ocal mic og aphs and he o hogonal p ojec ion
o he same HeLa cells showing py anine (g een) and TmR4C ( ed) cy osolic dis ibu ion and pa ial
endosomal co-localiza ion. B) In e naliza ion o py anine (15 μM, g een) in he p esence o TmR4C (15
μM) in Ve o cells, a e incuba ion o 30 min in HKR.
Mo eo e , we ca ied ou con ol anspo expe imen s in HeLa cells using he e a-
a ginine pep ide wi hou he sup amolecula cage C (TmR4). These expe imen s would allow us
o con i m he key ole o he sup amolecula cage in he in acellula anspo o py anine. In
his expe imen , we ollowed he same p ocedu e as be o e bu using TmR4 ins ead o TmR4C.
In e es ingly, we ound ha TmR4 does no p omo e any py anine in e naliza ion (Figu e 39A).
The e o e, we showed ha he sup amolecula cage has a key ole o p omo e he py anine
anspo inside o he cell. In addi ion, as we ha e p e iously men ioned, we also demons a ed
ha nei he he cage C alone co-incuba ed wi h py anine no py anine alone is able o en e he
cellula cy osol (Figu e 26B and C).
Once we showed he abili y o TmR4C o deli e py anine, we s udied he in acellula
anspo o py anine using he ace yla ed e sion o he pep ide-cage hyb id (AcR4C).
In e es ingly, we ound ha AcR4C is also able o deli e py anine p obe in o he cy osol o bo h
HeLa (Figu e 39B) and Ve o cells (Figu e 39D). Mo eo e , as expec ed, we also showed ha
he ace yla ed pep ide wi hou he cage C (AcR4) does no p omo e in acellula deli e y o
py anine inside cells (Figu e 39C).
A)
B)
Héc o Fe nández Ca o
98
A e disco e ing he abili y o bo h TmR4C and AcR4C o p omo e in acellula deli e y o
py anine. We hypo hesized i AcR4C would be able o cap u e ee py anine p e iously
dissol ed in he cellula medium and in e nalize his p obe inside o he cells. Wi h his pu pose,
we decided o ca y ou he sequen ial addi ion o bo h componen s, he py anine, and hen he
pep ide. In his expe imen , we i s incuba ed Ve o cells o en minu es only wi h py anine,
and hen we added d opwise he pep ide-cage hyb id AcR4C o Ve o cells in a inal concen a ion
o 10 µM ollowed by an incuba ion o 30 minu es a 37 ⁰C. In e es ingly, we ound ha AcR4C
is able o cap u e and o in e nalize py anine dissol ed in he ex acellula medium (Figu e
39D).
Figu e 39. Expe imen s in HeLa (A and B) and Ve o (C and D) cells. A) Con ocal mic og aphs o HeLa
cells incuba ed wi h TmR4 (5 µM) and Py anine (5 µM). Nuclei we e s ained wi h Hoechs . DIC on he
le and me ge o luo escence channels [Py anine emission (g een) + TAMRA emission ( ed) + Hoechs
33342 emission (blue)] on he igh . B) Con ocal mic og aphs o HeLa cells incuba ed wi h AcR4C (5
µM) and Py anine (5 µM; g een) o 30 min in HKR. C) Con ocal mic og aphs o Ve o cells incuba ed
wi h Py anine (10 µM, g een) and AcR4 (10 µM) in HKR o 30 min. D) Sequen ial addi ion o py anine
and AcR4C o Ve o cells. B, C, and D) DIC on he le and Py anine emission (g een) on he igh .
6. Demons a ion o cellula memb ane in eg i y cy osolic deli e y o
py anine
Once we showed ha bo h AcR4C and TmR4C can p omo e he in acellula anspo o
py anine wi h high e iciency and ep oducibili y, we decided o s udy he mechanism o
anspo o he sup amolecula complex inside li ing cells. To his aim, we ca ied ou wo
di e en expe imen s using nuclea s aining agen s o demons a e memb ane in eg i y du ing
py anine in e naliza ion and also compe i ion expe imen s.
A)
B)
C)
D)
Chap e I: Resul s and discussions
99
6.1. Memb ane in eg i y by nuclea s aining
Fi s , we decided o demons a e ha he in e naliza ion o py anine is no media ed by
memb ane pe meabiliza ion o by any cellula memb ane damage. Wi h his pu pose, we
ca ied ou py anine anspo expe imen s bu subsequen ly, we incuba ed he cells wi h wo
di e en dyes, DAPI o p opidium iodide. Bo h dyes would p oduce nuclea s aining in he case
o memb ane pe meabiliza ion. In he i s expe imen , we used DAPI (4',6-diamidino-2-
phenylindole) which is a luo escen p obe wi h he abili y o bind DNA. This dye will p oduce
a blue (λex = 405 nm and λem = 450/50 nm) nuclea luo escence in he case o memb ane
pe meabiliza ion (Figu e 40A). Fi s , we ca ied ou an ini ial expe imen o ensu e he abili y
o DAPI o de ec cell memb ane damage. In his expe imen , we ea ed he cells wi h a
memb ane de e gen (T i on X) o cause memb ane damage. Then, we incuba ed hese cells o
30 min wi h 2 μM DAPI o check he dye in ensi y in he cellula nucleus. As expec ed, in ense
blue nuclea luo escence was de ec ed in all he damaged cells (Figu e 40B).
Figu e 40. A) Chemical s uc u e o DAPI, his dye p oduces nuclea s aining in case o memb ane
damage. B) Con ocal mic og aphs o memb ane-damaged HeLa cells showing blue nuclea
luo escence o DAPI (2μM).
Once we demons a ed he abili y o DAPI o de ec cell memb ane damage. We ca ied
ou he py anine anspo expe imen whe e we incuba ed py anine (5 μM) wi h TmR4C (5 μM)
o 30 min wi h HeLa cells. Cells we e washed wi h HKR bu e se e al imes, and
subsequen ly, we incuba ed he cells o 30 min wi h 2 μM DAPI o check memb ane in eg i y.
We e alua ed he cells unde con ocal mic oscopy and no DAPI nuclea luo escence in he
cells loaded wi h py anine was ound (Figu e 41). The e o e, his expe imen indica ed ha
he e is no pe meabiliza ion o he cell memb ane du ing py anine anspo .
Figu e 41. Demons a ion o memb ane in eg i y by DAPI in py anine-loaded HeLa cells. Con ocal
mic og aphs o HeLa cells incuba ed wi h TmR4 (5 µM) and Py anine (5 µM), ollowed by washes (3x)
wi h HKR and, hen, incuba ed wi h 2 μM DAPI. Le panel: BF; second panel: py anine (g een); hi d
panel: DAPI (blue); igh panel: me ge o luo escence channels [Py anine emission (g een) + TAMRA
emission ( ed) + DAPI emission (blue)].
A)
B)
Héc o Fe nández Ca o
100
We hen con i med by p opidium iodide he memb ane in eg i y. This p obe p oduces ed
(λex = 493 nm and λem = 636 nm) nuclea s aining in he case o memb ane damage. In his
expe imen , we ca ied ou a sequen ial addi ion o py anine and AcR4C o Ve o cells. Fi s ,
cells we e incuba ed o en minu es wi h py anine be o e he d opwise addi ion o AcR4C o a
inal concen a ion o 10 µM o 30 minu es a 37 ⁰C. Then, cells we e washed wi h HKR
se e al imes and we e incuba ed wi h p opidium iodide o 10 minu es. This expe imen also
showed non-memb ane pe meabiliza ion o py anine-loaded cells. In e es ingly, in Figu e 42,
we can compa e a dea h cell wi h memb ane damage which shows a ed nucleus, wi h many
heal hy cells loaded wi h py anine wi hou luo escence de ec ed in he nucleus o cells.
Figu e 42. Demons a ion o memb ane in eg i y by p opidium iodide in py anine-loaded Ve o cells.
Con ocal mic og aphs o Ve o cells incuba ed wi h py anine be o e he addi ion o AcR4C o a inal
concen a ion o 10 µM. Then, cells we e washed wi h HKR and incuba ed wi h p opidium iodide. Le
panel: BF; second panel: py anine (g een); hi d panel: p opidium iodide ( ed); igh panel: me ge o
luo escence channels [Py anine emission (g een) + P opidium iodide emission ( ed)].
6.2. Memb ane in eg i y by compe i ion assays
Subsequen ly, we wonde ed i he pep ide-ca ie sys em would be capable o selec i ely
in e nalizing py anine when co-incuba ed wi h o he s uc u ally-simila p obes while
main aining he memb ane in eg i y. Wi h his pu pose, we ca ied ou compe i ion expe imen s
be ween py anine and TAMRA mixed in he ex acellula medium. Fi s , we ca ied ou wo
di e en in e naliza ion expe imen s wi h TAMRA alone and wi h AcR4C and TAMRA a low
concen a ion (Figu e 43). In hese expe imen s, we demons a ed ha TAMRA alone and in
he p esence o AcR4C is no able o c oss he cell memb ane (Figu e 43). Mo eo e , we ca ied
ou a i a ion expe imen by luo escence o s udy he in e ac ion be ween AcR4C and TAMRA
and we ound ha his p obe does no in e ac wi h AcR4C (Figu e 43). Once we demons a ed
ha TAMRA alone and in he p esence o AcR4C is no able o c oss he plasma memb ane and
nei he o in e ac wi h AcR4C, we decided o ca y ou compe i ion expe imen s using TAMRA
and py anine. The e o e, i he ca ie would ha e a high p e e ence o py anine, i would
selec i ely cap u e and would in oduce only his p obe inside cells (Figu e 43).
Chap e I: Resul s and discussions
101
Figu e 43. A and B) Con ocal mic og aphs o Ve o cells incuba ed in HKR bu e o 30 min a 37 ⁰C
wi h: A) only TAMRA (20 µM, ed channel). B) AcR4C (10 µM) in he p esence o TAMRA (20 µM,
ed channel). Le panels co espond o DIC images. C) Fluo escence spec a o he i a ion o 30 nM
TAMRA luo opho e wi h inc easing equi alen s (50, 100, and 200) o AcR4C. No e idence o
in e ac ion could be ound. D) Schema ic ep esen a ion o he compe i ion expe imen showing
py anine, TAMRA, and he pep ide-cage ca ie (AcR4C).
To ca y ou his expe imen , we co-incuba ed Ve o cells wi h AcR4C (10 μM) and wi h
py anine and TAMRA (20 μM each) o 30 min (Figu e 44). Impo an ly, we ound he
in e naliza ion o py anine (g een luo escence) bu no TAMRA up ake ( ed luo escence).
This expe imen demons a ed he ca ie selec i i y o py anine when co-incuba ed wi h o he
s uc u ally-simila p obe and he absence o memb ane pe meabiliza ion.
Figu e 44. TAMRA compe i ion expe imen s in Ve o cells. Con ocal mic og aphs o Ve o cells
incuba ed in HKR o 30 min a 37 ⁰C wi h AcR4C (10 µM) mixed wi h py anine (20 µM, g een) in he
p esence o TAMRA (20 µM, ed). Le panel: BF; second panel: TAMRA ( ed); igh panel: Py anine
emission (g een).
A)
B)
D)
C)

Héc o Fe nández Ca o
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7. S udy o he mechanism o in e naliza ion using endocy osis
inhibi o s
P e ious con ocal mic oscopy expe imen s (Figu e 38) showed ha beyond he gene al
cy osolic dis ibu ion o bo h py anine and ca ie , a ac ion o he pep ide-cage ca ie was
able o colocalize wi h he p obe in he endosomes. The e o e, we hypo hesized ha he pep ide-
cage hyb id/py anine complex was being in e nalized inside he cells ollowing he endocy ic
pa hway.
Figu e 45. Quan i ica ion o he up ake o TmR4C (15 µM) and py anine (15 µM) in Ve o cells unde
di e en condi ions: 37 ⁰C (con ol), DYN (dynaso e, 80 µM), and 4 ⁰C. A) and C) median luo escence
in ensi y (MFI) co esponding o py anine luo escence (A) o TmR4C (C). B) and D) up ake no malized
o he con ol o TmR4C wi h py anine incuba ed a 37 ⁰C, co esponding o py anine (B) o TmR4C (D).
The las wo ba s in all cha s indica e he up ake o py anine (A, B) o TmR4C (C, D) when incuba ed
alone. E o ba s indica e he SD o h ee eplica es.
To in es iga e he mechanism o en y o he complex, we decided o pe o m cell anspo
expe imen s using endocy osis inhibi o s. Fo his, we quan i ied he in e naliza ion o TmR4C
and py anine by cell cy ome y expe imen s in he p esence o dynaso e (DYN) endocy ic
inhibi o and a low empe a u e (Figu e 45). The low- empe a u e expe imen s comple ely
inhibi he ene gy-dependen up ake and dynaso e is an inhibi o o mac opinocy osis and all
in e naliza ion pa hways which depend on dynamin. The e o e, i he complex is en e ing inside
he cells ollowing he endocy ic pa hway, a low empe a u e and in he p esence o dynaso e,
a educ ion o he up ake o TmR4C would be shown. To ca y ou his expe imen , we incuba ed
Ve o cells wi h TmR4C (15 µM) and py anine (15 µM) and TmR4C (15 µM) alone a low
empe a u es, wi h he dynaso e inhibi o , and a 37⁰C. In e es ingly, we ound a educ ion o
he up ake o TmR4C, wi h o wi hou py anine, a low empe a u e (4⁰C) and in he p esence o
dynaso e (Figu e 45). These esul s sugges ha he in e naliza ion o he TmR4C and he
TmR4C/py anine complex is media ed by an ene gy-dependen endocy osis mechanism.
Based on all p e ious esul s, we p opose ha he complex is en e ing inside he cell by an
endocy osis mechanism, ollowed by an escape om he endosomes and, hen, each he cell
A)
B)
C)
D)
Chap e I: Resul s and discussions
103
cy osol. The nucleola dis ibu ion o he pep ide also con i med ha he pep ide is capable o
escape om he endosome (Figu e 38). A simila in e naliza ion mechanism o he ace yla ed
pep ide can also be p oposed based on he analogous esul s showed by his ca ie .
8. In e ac ion and in acellula deli e y o di e en anionic
luo opho es
Once we deli e ed py anine inside di e en cell lines using he designed pep ide-cage
ca ie s. We wonde ed i we would be able o in e nalize new luo opho es wi h simila
s uc u es o py anine. We i s s udied he in e ac ion be ween pep ide-cage ca ie s
AcR4C/TmR4C and a ious anionic luo opho es wi h po en ial biological in e es . Fi s , we
decided o s udy he abili y o he pep ide-cage ca ie (AcR4C) o in e ac wi h
ca boxy luo escein (CF). Wi h his pu pose, we ca ied ou i a ion expe imen s o his
luo opho e wi h inc easing concen a ions o AcR4C in phospha e bu e a pH 7 (Figu e 46).
These expe imen s allowed he calcula ion o he dissocia ion cons an Kd = 16.2 µM o AcR4C.
In e es ingly, when labeled, TmR4C also educed i s a ini y wi h py anine Kd = 51.5 µM (Figu e
54).
Figu e 46. A) Chemical s uc u e o ca boxy luo escein (CF). B) Fluo escence spec a o he i a ion o
CF wi h AcR4C in phospha e bu e (pH 7). A ow indica es he dec ease o CF luo escence wi h
inc easing amoun s o he pep ide. C) 516 nm luo escence emission and cu e i o he i a ion o CF
wi h AcR4C in phospha e bu e (pH 7). The bes i o he da a using nonlinea analysis wi h O igin 8.5
o he Hill 1 equa ion.
Encou aged by he in e ac ion be ween CF and AcR4C, we decided o s udy he po en ial
ecogni ion o he pep ide cage hyb id wi h di e en Alexa luo opho es. We ca ied ou
di e en i a ion s udies wi h AcR4C and he di e en Alexa p obes (Figu e 47) and we ound
he in e ac ion o AcR4C wi h all he p obes wi h he ollowing binding cons an s: Kd = 6 µM
o Alexa Fluo 488, Kd = 1.37 µM o Alexa Fluo 546, and Kd = 9.5 µM o Alexa Fluo 568.
A)
B)
C)
Héc o Fe nández Ca o
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Figu e 47. A), D) and G): Chemical s uc u e o Alexa Fluo 488, 546 and 568, espec i ely. B, E, and
H: Fluo escence spec a o he i a ion o Alexa Fluo dyes wi h AcR4C. A ows indica e he dec ease
o luo opho e emission wi h inc easing amoun s o AcR4C. C), F) and I) Fluo escence emission a he
maximum o each luo opho e (513 nm o Alexa Fluo 488, 570 nm o Alexa Fluo 546 and 603 nm
o Alexa Fluo 568), and cu e i o he i a ion wi h AcR4C in phospha e bu e (pH 7). B), C): Alexa
Fluo 488; E), F): Alexa Fluo 546; H), I): Alexa Fluo 568. The bes i o he da a using nonlinea
analysis wi h O igin 8.5 o he Hill 1 equa ion.
Once we disco e ed ha ou pep ide-cage ca ie could encapsula e di e en
anionic/plana luo escen p obes, we decided o use ou inno a i e sys em o anspo hese
memb ane-impe meable luo opho es o he cell in e io . Fi s , we demons a ed ha AcR4C can
anspo ca boxy luo escein in wo di e en cell lines such as Ve o cells (Figu e 48) and HeLa
cells (Figu e 54). To con i m he po en ial deli e y o he Alexa p obes, we incuba ed Ve o
cells wi h he di e en condi ions showed in Figu e 48. In hese expe imen s, we ha e shown
ha he cage-pep ide ca ie was able o e icien ly deli e in o he cy osol o he Ve o cells he
di e en Alexa Fluo dyes (Figu e 48).
E)
F)
G)
H)
I)
B)
C)
A)
D)
Chap e I: Resul s and discussions
105
Figu e 48. T anspo expe imen s o di e en anionic luo opho es in o Ve o cells. A) Emission
wa eleng hs o he di e en luo opho es. B) o F) Chemical s uc u es and pseudocolo ed mic og aphs
o Ve o cells incuba ed wi h he di e en compounds showed o 30 min and dilu ed in HKR bu e . B)
Py anine (10 µM) and TmR4C (10 µM). C) CF (10 µM) and TmR4C (10 µM). D) Alexa Fluo 488 (20
µM) and TmR4C (10 µM). E) Alexa Fluo 546 (35 µM) and AcR4C (15 µM). F) Alexa Fluo 568 (25 µM)
and AcR4C (15 µM). In all cases, con ol panels indica e he incuba ion o he cells wi h he same amoun
o dye in he absence o he di e en pep ide-cage hyb id. Inse s show DIC images. Exci a ion and
emission wa eleng hs: py anine, CF, Alexa Fluo 488: ex = 488 nm, em = 525/50 nm; Alexa Fluo 546,
Alexa Fluo 568: ex = 561 nm, em = 620/20 nm.
9. pH acking in li ing cells
A e con i ming he abili y o pep ide/cage hyb ids o anspo a ious anionic p obes, we
in es iga ed he po en ial o py anine as a pH-sensi i e p obe o in acellula a iome ic pH
acking. Py anine has wo a iome ic pH-dependen exci a ion maxima a 405 nm (p o ona ed)
and 450 nm (dep o ona ed).240 In e es ingly, his p ope y will allow o dis inguish he pH o
he di e en cellula o ganelles.
As p e iously men ioned in he in oduc ion, he cu en s a egies o e icien ly deli e
py anine inside cells such as physical me hods ha e some limi a ions including cellula oxici y.
The e o e, we decided o apply ou me hodology o ca y ou cellula pH acking. Fo his, we
incuba ed TmR4C (10 μM) and py anine (10 μM) o 30 min wi h Ve o cells. In e es ingly, by
jus checking he acidic (exc = 405 nm) and he basic (exc = 488 nm) exci a ion wa eleng hs,
240 C. C. O e ly, K. D. Lee, E. Be hiaume, P. J. Hollenbeck, P oc. Na l. Acad. Sci. 1995, 92, 3156-3160.
A)
B)
E)
D)
C)
F)

Chap e I: Expe imen al sec ion
113
1. Chemical s uc u es
A) TmA
B) TmR8
C) TmR4
D) AcR4
Figu e 50. Chemical s uc u es o he pep ides A) TmA, B) TmR8, C) TmR4, D) AcR4.
O
O
H
N
O
N
H
O
H
N
NH
H2NNH2
O
N
H
O
H
N
NH
H2NNH2
O
N
H
O
H
N
NH
H2NNH2
O
N
H
O
H
N
NH
H2NNH2
O
N
H
O
O
N
N
O
O
O
H
NOH
O
NH
NH2
H2N
NH
NH2
H2N
NH
NH2
H2N
NH
NH2
H2N
O
O
H
N
O
N
H
O
N
H
O
H
N
NH
H2NNH2
O
N
H
O
H
N
NH
H2NNH2
O
O
N
N
O
O
O
H
NOH
O
NH
NH2
H2N
NH
NH2
H2N
O
O
H
N
O
N
H
O
N
H
O
H
N
NH
H2NNH2
O
N
H
O
H
N
NH
H2NNH2
O
OH
O
NH
NHHN
NH
NHH2N
Héc o Fe nández Ca o
114
A) TmAC
B) TmR8C
C) TmR4C
D) AcR4C
O
O
H
N
O
N
H
O
N
H
O
H
N
NH
H2NNH2
O
N
H
O
H
N
NH
H2NNH2
O
O
N
N
O
O
O
H
N
N
N
N
HN
N
N
O
NH
NH2
H2N
NH
NH2
H2N
Figu e 51. Chemical s uc u es o he pep ide-cage hyb ids A) TmAC, B) TmR8C, C) TmR4C, D) AcR4C.
Chap e I: Expe imen al sec ion
115
2. Figu es
Figu e 52. Viabili y assay in Ve o cells wi h py anine co-incuba ed in HKR wi h TmR4C (A, D) o AcR4C
(B, E) and only py anine (C, F) a di e en concen a ions a e 1 hou incuba ion ime (A, B, C) and
a e 24 hou s incuba ion ime (D, E, F) a 37 ºC. E o ba s ep esen he s anda d de ia ion o ou
eplica es.
Figu e 53. A) Fluo escence i a ion o py anine wi h he pep ide/cage AcR4C in phospha e bu e (pH
7). A ow indica es he dec ease o py anine luo escence wi h inc easing amoun s o he pep ide. B)
510 nm luo escence emission and cu e i o he i a ion o py anine wi h AcR4C in phospha e bu e
pH 7. The bes i o he da a using nonlinea analysis wi h O igin 8.5 o he Hill 1 equa ion. In B, i ing
was done wi h a ixed alue o n = 1, and Kd o AcR4C unde hese condi ions was 149 nM. The small
peak obse ed in A) a abou 480 nm a he highe concen a ions o pep ide co esponds o he Raman
sca e ing o wa e o an exci a ion wa eleng h o 415 nm.
A)
B)
A)
D)
B)
C)
E)
F)
Héc o Fe nández Ca o
116
Figu e 54. T anspo expe imen s o Ca boxy luo escein (CF) in HeLa cells. Con ocal mic og aphs o
HeLa cells incuba ed wi h: A) TmR4C (5 µM) and Ca boxy luo escein (5 µM). B) TmR4 (5 µM) and
Ca boxy luo escein (5 µM). DIC on he le and me ge channels [Ca boxy luo escein emission (g een)
+ Tm emission ( ed)] on he igh . HeLa cells we e incuba ed wi h he di e en componen s in he HKR
bu e o 30 min a 37 ºC. The cells we e inally washed (HKR bu e , 3x) and hen obse ed unde he
con ocal mic oscope.
Figu e 55. A) Fluo escence spec a o he i a ion o CF wi h TmR4C in phospha e bu e (pH 7). A ow
indica es he dec ease o CF luo escence wi h inc easing amoun s o he pep ide. The igh peak in C
co esponds o he TAMRA emission om TmR4C. B) 516 nm luo escence emission and cu e i o
he i a ion o CF wi h TmR4C in phospha e bu e (pH 7). The bes i o he da a using nonlinea
analysis wi h O igin 8.5 o he Hill 1 equa ion.
B)
A)
A)
B)
Chap e I: Expe imen al sec ion
117
Figu e 56. A) Abso p ion spec a o py anine in bu e s o di e en pH. B) The a io o he abso bance
a 450 and 405 nm plo ed as a unc ion o pH. C) Ra iome ic me hod o de e mining he pKa. pKa =
7.01.
A)
B)
C)

Héc o Fe nández Ca o
118
Figu e 57. pH s udies in Ve o cells. A) Ra iome ic imaging a e pH clamping wi h nige icin o Ve o
cells incuba ed wi h 10 µM py anine and 10 µM TmR4C. The op ow (BF) shows b igh - ield images.
The second (Py H) and hi d (Py -) ows show he emission o py anine a e exci a ion a 405 nm o
488 nm, espec i ely. Finally, he bo om ow shows he p ocessed images. B) Mean a io alues we e
ob ained o each ield and plo ed agains pH. Box and whiske plo o each da ase , g een poin s
ep esen indi idual obse a ions. C) Loga i hmic con e sion o he p e ious da a and linea i . E o
ba s indica e SD.
A)
B)
C)
Chap e I: Expe imen al sec ion
119
3. Ma e ials and me hods
Chemicals we e pu chased om Ca bosyn h, I is Bio ech, Sigma Ald ich, Al a Aesa ,
S eam Chemicals, and No abiochem and used wi hou u he pu i ica ion.
Comme cially a ailable 2-Chlo o i yl chlo ide esin, Fmoc-Ahx-OH, Fmoc-O2Oc-OH,
Fmoc-L-Ala-OH, Fmoc-L-Leu-OH, Fmoc-L-A g(pb )-OH, iisop opylsilane (TIS),
Disop opyle hyl amine (DIEA), Nige icin and 8-Hyd oxypy ene-1,3,6- isul onic acid
isodium sal (py anine) we e ob ained om Sigma-Ald ich. MTT and MES (2-(N-
mo pholino)e hanesul onic acid) we e pu chased om Al a Aesa ; 5(6)-Ca boxy luo escein
(CF) and 5-Ca boxy e ame hyl hodamine (TAMRA) we e a ailable om Ca bosyn h. L-α-
phospha idylglyce ol (Egg, Chicken) (sodium sal ) was pu chased om A an i Pola Lipids
and N-HATU om Glen ham li e sciences. N-HBTU was ob ained om I is. Hoechs 33342
T ihyd ochlo ide T ihyd a e, Alexa Fluo dyes (488, 546 and 594), and Dulbecco’s Modi ied
Eagle’s Medium (4500 mg/L glucose, L-glu amine, sodium py u a e, and sodium bica bona e)
we e pu chased in The moFishe . HEPES [4-(2-hyd oxye hyl)-1-pipe azinee hanesul onic
acid)] was pu chased om TCI Chemicals.
The sol en s o o ganic syn hesis we e o eagen g ade. D y sol en s we e bough om
Sigma-Ald ich. N, N-dime hyl o mamide, and i luo oace ic acid we e pu chased om
Scha lau, dichlo ome hane om Pan eac, and ace oni ile om Me ck. Wa e was deionized
and pu i ied on a Millipo e Milli-Q In eg al sys em.
The emo al o sol en s unde educed p essu e was ca ied ou on a o a y e apo a o
Büchi R-210 equipped wi h a he mos a ed ba h B-491, a acuum egula o V-850, and a
acuum pump V-700.
Pu i ica ion o p oduc s was accomplished using e e sed-phase high-pe o mance liquid
ch oma og aphy (RP-HPLC) using an Agilen Technologies 1160 In ini y using H2O (+ 0.1%
TFA) and CH3CN (+ 0.1% TFA) as eluen s and a Luna (C18)-Phenomenex column and on
Jasco LC-4000 wi h an Agilen Eclipse XDB-C18 column.
High-pe o mance liquid ch oma og aphy coupled wi h mass spec ome y (HPLC-MS)
analyses we e ca ied ou on Agilen Technologies 1260 In ini y II associa ed wi h a 6120
Quad upole LC-MS using an Agilen SB-C18 column o on DIONEX Ul ima e 3000 U-HPLC+
(The mo Scien i ic) wi h an Acclaim RSLC 120-C18 column wi h Sol en A: Sol en B
g adien s be ween 5:95 (Sol en A: H2O wi h 0.1% TFA; Sol en B: CH3CN wi h 0.1% TFA).
Fluo escence measu emen s we e pe o med using a Va ian Ca y Eclipse luo ome e and
UV-Vis spec a we e measu ed in an Agilen 8453 UV-Vis diode-a ay spec opho ome e o a
Bioch om Lib a S60 UV- is spec opho ome e .
Fo aking con ocal mic oscopy images a D agon ly con ocal spinning-disk sys em
moun ed on a Nikon Eclipse Ti-E equipped wi h an Ando Zyla 4.2 PLUS sCMOS digi al
came a was used. The 3D econs uc ions we e ob ained om he di e en indi idual con ocal
planes wi h Ima is bi plane 9.0.0 so wa e.
A Tecan In ini e F200P o mic opla e eade was used o measu e di ec ly in Cos a cell
cul u e 96-well pla es UV-Vis abso bance o he MTT iabili y assays.
Flow cy ome y was pe o med on a Gua a easyCy eTM cy ome e . Da a analysis was
pe o med wi h InCy e so wa e included in Gua aSo 3.2 (Millipo e).
The “U- ubes” we e house-made.
Héc o Fe nández Ca o
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4. Gene al p o ocol o syn he ic p ocedu es
Pep ides TmA, TmR8, TmR4, and AcR4 we e syn hesized acco ding o classic SPPS o pep ides
and u he de ails a e gi en below. The cage C was p epa ed and pu i ied ollowing he
p ocedu e epo ed in li e a u e.69,241
4.1. Syn hesis and cha ac e iza ion o he pep ides TmA, TmR8, TmR4 and
AcR4
All pep ides we e syn hesized by manual Fmoc solid-phase pep ide syn hesis on a 2-
Chlo o i yl chlo ide esin (1.14 mmol/g). The i s coupling was pe o med in CH2Cl2 using
DIEA as base whe eas o he ollowing couplings N-HBTU as he ac i a o , DIEA as he base,
and DMF as sol en we e used. The dep o ec ion o he empo al Fmoc p o ec ing g oup was
pe o med by ea ing he esin wi h 20% pipe idine in DMF. 5(6)-
ca boxy e ame hyl hodamine (TAMRA) was coupled using 3 equi alen s (0.15 mmol, 64.5
mg), 3 equi alen s o N-HATU, and 5 equi alen s o DIEA 0.2 M in DMF o 60 min.
Ace yla ion o he N- e minal g oup o AcR4 was pe o med by s anda d Fmoc emo al
condi ions (20% pipe idine in DMF) ollowed by ea men wi h a solu ion o ace ic anhyd ide
and 2,6- lu idine (1:1, 2 mL) o 30 min.
The clea age/dep o ec ion s ep was pe o med by ea men o he esin-bound pep ide o
2 h wi h he ollowing clea age cock ail: 900 μL TFA, 50 μL CH2Cl2, 25 μL H2O and 25 μL
TIS (1 mL o cock ail/40 mg esin) and pep ides we e p ecipi a ed in E 2O.
4.1.1. Syn hesis and cha ac e iza ion o pep ide TmA
The syn hesis o he pep ide TmA (TAMRA-Ahx-R-R-L-R-R-L-L-R-R-L-L-R-A-Ahx-
O2Oc-OH) was pe o med ollowing he abo e me hodology. TmA was ob ained a e RP-
HPLC pu i ica ion [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1%
TFA) 95:5→5:95 (5→35 min)] wi h an o e all yield o 12%. R 8.0 min [RP-HPLC Agilen
SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→12 min)] (Figu e 58).
MS (ESI, H2O) m/z: calcd o C118H204N39O23 [M+5H]5+: 507.3, ound: 507.5; calcd o
C118H203N39O23 [M+4H]4+: 633.9, ound 634.0; calcd o C120H203N39O25F3 [M+3H+TFA]3+:
882.9, ound: 883.0; calcd o C122H204N39O27F6 [M+3H+2TFA]3+: 920.9, ound: 920.9; calcd
o C122H203N39O27F6 [M+2H+2TFA]2+ : 1380.8, ound: 1380.7.
69 J. Mosque a, S. Za a, J. R. Ni schke, Angew. Chem. In . Ed. 2014, 53, 1556-1559.
241 J. Rod íguez, J. Mosque a, J. R. Coucei o, J. R. Ni schke, M. E. Vázquez, J. L. Masca eñas, J. Am. Chem. Soc.
2017, 139, 55-58.
Chap e I: Expe imen al sec ion
121
Figu e 58. Rep esen a ion o he HPLC-MS o TmA. A) HPLC ch oma og am (λabs = 550 nm) o TmA
and B) ESI-MS eco ded a 8.0 min o TmA.
4.1.2. Syn hesis and cha ac e iza ion o pep ide TmR8
The syn hesis o he pep ide TmR8 (TAMRA-Ahx-R-R-R-R-R-R-R-R-Ahx-O2Oc-OH) was
pe o med ollowing he abo e me hodology. TmR8 was ob ained a e RP-HPLC pu i ica ion
[Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95
(5→35 min)] wi h an o e all yield o 12%. R 10.9 min [RP-HPLC Agilen SB-C18 column,
H2O (0.1% TFA)/ CH3CN (0.1% TFA) 75:5→5:75 (0→21 min)] (Figu e 59). MS (ESI, H2O)
m/z: calcd o C91H156N37O18 [M+5H]5+: 411.0, ound: 411.3; calcd o C93H156N37O20F3
[M+4H+TFA]4+: 542.1, ound: 542.3; calcd o C93H155N37O20F3 [M+3H+TFA]3+: 722.4,
ound: 722.9; calcd o C95H156N37O22F6 [M+3H+2TFA]3+: 760.4, ound: 760.6; calcd o
C97H156N37O24F9 [M+2H+3TFA]2+: 1197.1, ound: 1197.6.
Figu e 59. Rep esen a ion o he HPLC-MS o TmR8. A) HPLC ch oma og am (λabs = 550 nm) o TmR8
and B) ESI-MS eco ded a 10.9 min o TmR8.
4.1.3. Syn hesis and cha ac e iza ion o pep ide TmR4
The syn hesis o he pep ide TmR4 (TAMRA-Ahx-R-R-R-R-Ahx-O2Oc-OH) was
pe o med ollowing he abo e me hodology. TmR4 was ob ained a e RP-HPLC pu i ica ion
[Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95
(5→35 min)] wi h an o e all yield o 12%. R 11.1 min [RP-HPLC Agilen SB-C18 column,
H2O (0.1% TFA)/ CH3CN (0.1% TFA) 75:5→5:75 (0→21 min)] (Figu e 60). MS (ESI, H2O)
A)
B)
A)
B)
Héc o Fe nández Ca o
128
8. Cell anspo expe imen s
HeLa o Ve o cells seeded he day be o e on glass-bo om dishes we e washed wi h
HEPES-K ebs-Ringe (HKR) bu e (5 mM HEPES, 137 mM NaCl, 2.68 mM KCl, 2.05 mM
MgCl2, 1.8 mM CaCl2, pH 7.4) and incuba ed o 30 min wi h 1 µM Hoechs 33342
(The moFishe ) in HKR o s ain he nucleus. This solu ion was emo ed and cells we e
incuba ed o ano he 30 min wi h py anine (o o he luo escen p obes such as TAMRA, CF,
o Alexa Fluo dyes) in combina ion wi h he pep ide, cage, o pep ide-cage hyb ids a he
concen a ions indica ed in he legend o he igu es. A e his incuba ion ime, cells we e
washed wice wi h HKR and examined on he con ocal mic oscope. In some cases, he s ep o
nuclea s aining wi h Hoechs was omi ed. To check memb ane in eg i y, a e incuba ion wi h
he complexes, cells we e washed wi h HKR and u he incuba ed wi h 2 µM o DAPI dilu ed
in HKR bu e o 30 min o 0.5 µg/mL o p opidium iodide o 10 min, be o e washing wi h
HKR and imaging. To in es iga e he possibili y o he in si u cap u e o he py anine in o he
cage o he ca ie , Ve o cells we e incuba ed wi h 12.5 µM py anine in HKR o 10 min be o e
adding, d opwise, a solu ion o AcR4C in HKR o a inal concen a ion o bo h pep ide and
py anine o 10 µM and incuba ed o 30 min a 37 °C be o e imaging.
9. In i o pH measu emen s
To con i m pH sensi i i y o he dye in he calib a ion bu e s used o in si u calib a ions
(10 mM MES, 10 mM HEPES, 20 mM glucose, 1 mM CaCl2, 1 mM MgCl2, 135 mM KCl, 20
mM NaCl; pH adjus ed wi h KOH), abso p ion spec a o py anine in he di e en bu e s we e
ob ained. Py anine was dilu ed a 70 µM in each bu e and abso bance be ween 330-510 nm
was measu ed in a Lib a S60 spec opho ome e . The a io be ween abso bances a 450 and 405
nm we e calcula ed, and hen ans o med using he ollowing equa ion:
𝑙𝑜𝑔10((𝑅𝑚𝑎𝑥−𝑅
𝑅−𝑅𝑚𝑖𝑛)·(𝐴405 𝑛𝑚
𝑏𝑎𝑠𝑖𝑐
𝐴405 𝑛𝑚
𝑎𝑐𝑖𝑑𝑖𝑐 ))
in which R ep esen s he a io A450/A405, Rmax and Rmin he maximal and minimal a io alues,
and Abasic405 and Aacidic405 he abso bance o he py anine a he highes and lowes pH es ed.
Da a we e i ed o a linea model. In e cep (7.01) co esponds o he pKa unde hese
condi ions.
10. pH s udies in cells
Fo pH a iome ic measu emen s, Ve o cells we e incuba ed wi h 10 µM py anine and 10
µM TmR4C pep ide o 30 min in HKR bu e . Cells we e hen washed wice wi h HKR and
imaged. A con ol wi h no py anine was also p epa ed o quan i y he backg ound. To p epa e
a calib a ion cu e, cells we e i s incuba ed wi h py anine and TmR4C unde s anda d
condi ions (30 min, pH = 7.4). Subsequen ly, he cells we e washed wice wi h high-po assium
pH calib a ion bu e s (10 mM MES, 10 mM HEPES, 20 mM glucose, 1 mM CaCl2, 1 mM
MgCl2, 135 mM KCl, 20 mM NaCl; pH adjus ed wi h KOH) a each indica ed pH. The cells
we e hen incuba ed a he co esponding pH o 20 min wi h he calib a ion bu e s con aining
10 µg/mL o he ionopho e nige icin o pH clamping be o e imaging. Images we e acqui ed

Chap e I: Expe imen al sec ion
129
wi h a Zyla 4.2 PLUS came a moun ed on a D agon ly spinning disk con ocal mic oscope
(Ando ), by exci a ion wi h 405 nm (p o ona ed o m, Py H) and 488 nm (dep o ona ed o m,
Py -) lase s, and de ec ing he luo escence a 500-550 nm. Images om he py anine channels
we e p ocessed wi h FIJI,
248
as ollows: he backg ound was sub ac ed om bo h channels and
hen image 488 was di ided by image 405. Mean alues pe ield we e ob ained (Figu e 57B)
and da a was linea ized by loga i hmic con e sion and adjus ed o a linea model (Figu e 57C).
The linea i was hen used o assign es ima ed alues o pH o he images.
11. Flow cy ome y
To u he in es iga e he up ake mechanisms o he sup amolecula complex o med by
py anine and TmR4C, Ve o cells seeded he day be o e a 10.000 cells/well o a 96-well pla e,
we e ea ed o 30 min wi h dynaso e (80 µM) dilu ed in DMEM wi hou se um o an ibio ics.
Cells we e hen washed wi h HKR and incuba ed wi h 15 µM TmR4C and 15 µM py anine o
30 min a 37 °C wi h he same concen a ion o he inhibi o in HKR bu e . Fo he incuba ion
a low empe a u e (4 °C), cells we e placed on ice be o e he incuba ion, and ice-cold solu ions
we e used o he washes and incuba ions. Con ols wi h 15 µM TmR4C alone and 15 µM
py anine alone we e also pe o med. A e 30 min o incuba ion, cells we e washed wi h HKR
and ypsinized. T ypsin was neu alized wi h 2 % FBS in PBS wi h 5 mM EDTA and cell
luo escence was measu ed on a Gua a EasyCy eTM cy ome e using wo lase s: a blue lase
(488 nm) wi h emission collec ed a 512/18 nm (py anine) and a g een lase (532 nm) collec ing
he emission a 575/25 nm (TAMRA). Cells wi h ypical FSC and SSC pa ame e s we e
selec ed and he median luo escence in ensi y (MFI) was calcula ed o each sample. Each
condi ion was done in iplica e. Fluo escence alues we e no malized o he up ake o each
un ea ed con ol (100%) a e blank sub ac ion. In all cases, da a analysis was pe o med wi h
InCy e so wa e included in Gua aSo 3.2 (Millipo e).
12. Cell iabili y
Cell iabili y was measu ed by MTT assay. To e alua e he oxici y o he ea men o
cells wi h TmR4C, AcR4C, and py anine, Ve o and HeLa cells we e submi ed o an MTT assay
1 hou and 24 h a e he incuba ion. One day be o e he assay, a suspension o HeLa and Ve o
cells we e pla ed in 96-well issue cul u e pla es by adding 100 µL (∼10.000 cells) pe well.
The nex day, he medium was emo ed and cells we e incuba ed in HKR in he p esence o
TmR4C, AcR4C, and py anine a di e en concen a ions (50 µL/well) du ing 1 hou o
incuba ion a 37 °C. A e he incuba ion, HKR wi h he compounds was emo ed and p e-
wa med DMEM con aining 10 % FBS was added o he wells. To e alua e he oxici y a 1
hou , MTT (5 mg/mL in PBS, 10 µL/well) was added and he cells we e u he incuba ed o
4 h. To e alua e he oxici y a 24 h, cells we e incuba ed o 24 h be o e adding he MTT. The
cells we e u he incuba ed o 4 h in he p esence o MTT. The supe na an was emo ed and
he wa e -insoluble o mazan sal was dissol ed in DMSO (100 µL/well). The abso bance a
570 nm was measu ed. Da a poin s we e collec ed in iplica e and exp essed as no malized
alues o un ea ed as con ol cells (100%) a e blank sub ac ion.
248
J. Schindelin, I. A ganda-Ca e as, E. F ise, V. Kaynig, M. Longai , T. Pie zsch, S. P eibisch, C. Rueden, S.
Saal eld, B. Schmid, e al., Na . Me hods 2012, 9, 676-682.
Chap e II:
Pep ide in eg a ion and exchange in o AuNPs
media ed by hos -gues molecula ecogni ion
In oduc ion

Chap e II: In oduc ion
135
1. Nanopa icles as a ool o nanomedicine
Since he ad en o Nano echnology, nanosys ems wi h diagnos ic and/o he apeu ic
capabili ies ha e been ega ded as a new p omising pa adigm ha could imp o e in a signi ican
manne he apeu ical p oblems such as cance .
249
This has led o a as esea ch e o in o de
o unde s and how undamen al physicochemical p ope ies, such as size, cha ge, and chemical
composi ion, can be add essed in o de o imp o e he pha macokine ic capabili ies and
biodis ibu ion.
250
In ac , many limi a ions, such as limi ed deli e y o a ge issues a e s ill
majo hu dles o he de elopmen o sa e and e icien he apies.
251
Rega dless o hese
di icul ies, se e al nanomedicines a e cu en ly on di e en s ages o clinical ials. Up o
2016, he U.S. Food and D ug Adminis a ion had app o ed 51 nanomedicines, wi h 77
p oduc s in clinical ials.
252
An analysis o he nanoca ie ca ego y e ealed ha so
nanopa icles (e.g. micelles, polyme s) had mo e impo ance in compa ison wi h ha d ca ie s
(e.g. me allic nanopa icles). A i s sigh , his da a sugges some ine ia owa ds he success ul
use o nanopa icle sys ems in eal he apeu ic applica ions, bu also i migh e lec a plu alis ic
app oach owa ds he deli e y p oblem, i.e. a single ehicle migh no be well sui ed o
di e en deli e y he apeu ic p oblems. The e o e, i seems plausible ha di e en s a egies
owa ds he p epa a ion o nanosys ems could expand he possible applica ions in
nanomedicine.
In his con ex , he di e si y o s uc u e and unc ion accessible h ough sup amolecula
assembly holds p omise as a e sa ile ool o he p epa a ion o nanomedicine ools, including
micella , nano ubula , o nanopa icle-like sys ems.188 He e we will ocus on he use o hos -
gues ecogni ion and i s in eg a ion on nanosys ems in ended o he apeu ic applica ions.
2. Nanopa icles buil up by using hos -gues molecula ecogni ion
The use o hos -gues molecula ecogni ion o e s he possibili y o unc ionalize o e en
ully assemble nanopa icles by s able, ye e e sible in e ac ions. In he la e case, he e m
sup amolecula nanopa icles (SNPs) has o en been coined o indica e he o ma ion o
pa icles o nanome ic size buil undamen ally by non-co alen in e ac ions. One o he
simples examples o he p epa a ion o SNPs is he condensa ion, d i en by elec os a ic
in e ac ions, be ween polyanions (e.g. DNA) and posi i ely cha ged lipids o polyme s, which
ha e been adi ionally used o DNA deli e y.
253
On he o he hand, sup amolecula
in e ac ions can be used o he e e sible unc ionaliza ion wi h mo i s ha impa ce ain
unc ionali y. The sca olds used o build hese sys ems can be ei he so o ha d, which ha e
impo an consequences in hei physicochemical p ope ies and he he apeu ic unc ion. He e
we will discuss examples o nanopa icles ha in eg a e hos -gues elemen s acco ding o his
classi ica ion.
249
R. an de Meel, E. Sulheim, Y. Shi, F. Kiessling, W. J. M. Mulde , T. Lamme s, Na . Nano echnol. 2019, 14,
1007-1017.
250
B. Li, L. A. Lane, Wiley In e discip. Re . Nanomed. Nanobio echnol. 2019, 11, e1542.
251
S. Wilhelm, A. J. Ta a es, Q. Dai, S. Oh a, J. Aude , H. F. D o ak, W. C. W. Chan, Na . Re . Ma e . 2016, 1,
16014.
252
D. Bobo, K. J. Robinson, J. Islam, K. J. Thu ech , S. R. Co ie, Pha m. Res. 2016, 33, 2373-2387.
188 M. J. Webbe , R. Lange , Chem. Soc. Re . 2017, 46, 6600-6620.
253
O. Boussi , F. Lezoualc’h, M. A. Zan a, M. D. Me gny, D. Sche man, B. Demeneix, J. P. Beh , P oc. Na l.
Acad. Sci. 1995, 92, 7297-7301.
Héc o Fe nández Ca o
136
2.1. So sup amolecula nanopa icles o deli e y o he apeu ics based
on hos -gues molecula ecogni ion
2.1.1. Deli e y o he apeu ic nucleic acids
A pa icula ly success ul example o SNPs ha has mo ed om undamen al esea ch o
clinical ials is he wo k o Ma k E. Da is g oup. Since he end o he 90´s, his g oup has
de eloped a ge ed SNPs ha can deli e small in e e ing RNA (siRNA) o inhibi umo
g ow h.254 These SNPs consis o h ee di e en building blocks (Figu e 66A): a linea
cyclodex in-con aining polyme (CDP), an adaman ane-polye hylene glycol conjuga e (Ad-
PEG), and he a ge ing moie y ans e in linked o an adaman ane moie y (Ad-PEG-T ). The
sel -assembly o hese h ee componen s in o a hyb id polyme is d i en by hos -gues
p ocesses. The esul ing conjuga e is mixed wi h siRNA, which is subsequen ly cap u ed by
elec os a ic in e ac ions be ween he posi i ely cha ged polyme and he nega i ely cha ged
nucleic acid (Figu e 66B). This ou -componen cock ail inally yields so nanopa icles o
a ound 70 nm size. The ehicle emained s able in o he bloods eam and was able o en e in o
umo issues due o he EPR e ec . Enhanced deli e y e iciency in umo cells was achie ed
due o he ecogni ion be ween ancho ed ans e in and i s ecep o (T R) which is
o e exp essed in ce ain ypes o umo cells (Figu e 66C).
254
,
255
This he apeu ic p oduc
named CALAA-01 was e alua ed in phase I clinical ials in human pa ien s wi h solid umo s,
adminis a ed by in a enous sys emic dosing. Al hough some ad e se e ec s we e obse ed
( a igue, chill), hese s udies indica ed ha his nanosys em could be sa ely used in humans.
256
This illus a i e example was he inspi a ion o in ensi e esea ch in he a ea o gene deli e y
by using hos -gues sys ems. While Da is g oup wo k elied on he inco po a ion o he hos
in o ca ionic polyme s, many g oups ha e exploi ed he chemical unc ionaliza ion o he hos
as a way o designing homogeneous gene deli e y ec o s, which a e in consequence
“p eo ganized” owa ds in e ac ions wi h nucleic acids.
257
In his con ex , ema kable examples
include calixa enes
258
and cyclodex ins
259
hos s.
In he examples abo e, he size o he so nanopa icles was a consequence o he
in e ac ion be ween he nucleic acid and he hos polyme /molecules, and no size- uning was
epo ed. Howe e , con ol o e nanopa icle size is desi able, since many cell-nanopa icle
in e ac ions a e s ongly in luenced by hei con ac su ace, (e.g. de e mining he numbe o
ecep o -an igen in e ac ions).
260
Rema kably, he g oup o p o . Tseng de eloped size-
con ollable SNPs based on he sel -assembly o h ee di e en molecula building blocks
(Figu e 66D): i) mul i alen hos poly(e hylene imine) unc ionalized wi h β-CD (CD-PEI), ii)
mul i alen gues , Ad-poly(amidoamine) dend ime (Ad-PAMAM) and iii) mono alen gues
linked o a s e ic s abilize , Ad-PEG. The beau y o his wo k is ha uning o he nanopa icle
size is achie ed simply by changing he mixing a io o he di e en building blocks.
Impo an ly, i was shown ha his s a egy can be a p omising s a egy o he p epa a ion o
254
M. E. Da is, Mol. Pha m. 2009, 6, 659-668.
255
M. E. Da is, J. E. Zucke man, C. H. J. Choi, D. Seligson, A. Tolche , C. A. Alabi, Y. Yen, J. D. Heidel, A.
Ribas, Na u e 2010, 464, 1067-1070.
256
J. E. Zucke man, M. E. Da is, Na . Re . D ug Disco . 2015, 14, 843-856.
257
C. O iz Melle , J. M. Beni o, J. M. Ga cía Fe nández, Chem. Eu . J. 2010, 16, 6728-6742.
258
F. Sansone, M. Dudi, G. Dono io, C. Ri e i, L. Baldini, A. Casna i, S. Cellai, R. Unga o, J. Am. Chem. Soc.
2006, 128, 14528-14536.
259
L. Gallego-Ye ga, J. M. Beni o, L. Blanco-Fe nández, M. Ma ínez-Neg o, I. Vélaz, E. Aica , E. Junque a, C.
O iz Melle , C. T os de Ila duya, J. M. Ga cía Fe nández, Chem. Eu . J. 2018, 24, 3825-3835.
260
N. Hoshya , S. G ay, H. Han, G. Bao, Nanomedicine 2016, 11, 673-692.
Chap e II: In oduc ion
137
NPs wi h sizes be ween 30 nm and 100 nm and wi h a good polydispe si y. In e es ingly, he
size o he SNPs was easily uned by inc easing he quan i y o mul i alen gues , while keeping
cons an he concen a ion o he hos and he s abilize .
261
The con ol o e he assembly and
subsequen size dis ibu ion we e u he uned by modula ion o he mixing egimes in
mic o luidic chips.
262
,
263
These so SPNs we e used o he in acellula deli e y o DNA. SPNs
combined wi h DNA and he a ge ing ligand RGD (Figu e 66E) we e used o e icien ly deli e
DNA inside li ing cells.
264
Mo eo e , using his s a egy, an in ac a i icial ansc ip ion ac o
(TF) (GAL4-VP16) was encapsula ed in o SPNs leading o he a ge ed deli e y o an in ac
TF. In his wo k, a DNA plasmid (pG5E4T-Fluc) equipped wi h i e andem copies o GAL4-
VP16 ma ching ecogni ion sequences (Kd ≈ 10 nM) was used o imp o e he inco po a ion o
he TF in o he SPN by he o ma ion o an anionic TF/DNA complex.
265
Fu he examples o
he use o hese pla o ms in deli e y include he con olled elease o he an icance d ug
camp o hecin
266
o in ade mal elease o ke oconazole -an an i ungal d ug- in mice models.
267
261
H. Wang, S. Wang, H. Su, K.-J. Chen, A. L. A mijo, W.-Y. Lin, Y. Wang, J. Sun, K. Kamei, J. Cze nin, e al.,
Angew. Chem. In . Ed. 2009, 48, 4344-4348.
262
K. Liu, H. Wang, K.-J. Chen, F. Guo, W.-Y. Lin, Y.-C. Chen, D. L. Phung, H.-R. Tseng, C. K. F. Shen,
Nano echnology 2010, 21, 445603.
263
K. Liu, Y.-C. Chen, H.-R. Tseng, C. K.-F. Shen, R. M. an Dam, Mic o luid. Nano luidics 2010, 9, 933-943.
264
H. Wang, K.-J. Chen, S. Wang, M. Ohashi, K. Kamei, J. Sun, J. H. Ha, K. Liu, H.-R. Tseng, Chem. Commun.
2010, 46, 1851-1853.
265
Y. Liu, H. Wang, K. Kamei, M. Yan, K.-J. Chen, Q. Yuan, L. Shi, Y. Lu, H.-R. Tseng, Angew. Chem. In . Ed.
2011, 50, 3058-3062.
266
K.-J. Chen, L. Tang, M. A. Ga cia, H. Wang, H. Lu, W.-Y. Lin, S. Hou, Q. Yin, C. K. F. Shen, J. Cheng, e al.,
Bioma e ials 2012, 33, 1162-1169.
267
F. Wang, P. Yang, J. Choi, P. An o ski, Y. Zhu, X. Xu, T.-H. Kuo, L.-E. Lin, D. N. H. Kim, P.-C. Huang, e
al., ACS Nano 2018, 12, 6851-6859.
Héc o Fe nández Ca o
144
wo k (Figu e 68C), mul i unc ional CD@AuNPs o a ge ed cance he apy we e cons uc ed
by he inco po a ion o bo h adaman ane-hyd azone-doxo ubicin (Ad-Hyd-DOX) and
adaman ane-modi ied-PEG8-GRGDS (Ad-PEG8-GRGDS), playing he oles o an icance d ug
and a ge ing moie y espec i ely.
302
The GRGDS pep ide sequence has shown he abili y o
a ge cance cells ha o e exp ess α β3 in eg in.264 On he o he hand, he inclusion o he
hyd azone bond enabled he p o on-media e hyd olysis o he linke , esul ing in he elease o
he d ug a acidic pH alues. The au ho s showed ha hese mul i unc ional nanopa icles we e
able o be selec i ely in e nalized by cance cells ia ecep o -media ed endocy osis. Mo eo e ,
his nanosys em showed cance cell apop osis due o he elease o doxo ubicin a lysosomal
pH alues. O e all, one o he mos ema kable ea u es o his wo k is he exclusi e use o
hos -gues molecula ecogni ion and he use o a pH- esponsi e he apeu ic agen .302
302
W.-H. Chen, Q. Lei, G.-F. Luo, H.-Z. Jia, S. Hong, Y.-X. Liu, Y.-J. Cheng, X.-Z. Zhang, ACS Appl. Ma e .
In e aces 2015, 7, 17171-17180.
264 H. Wang, K.-J. Chen, S. Wang, M. Ohashi, K. Kamei, J. Sun, J. H. Ha, K. Liu, H.-R. Tseng, Chem. Commun.
2010, 46, 1851-1853.

Chap e II: In oduc ion
145
Figu e 68. A) Le . Schema ic and s uc u al ep esen a ion o he e ocene dime and he pe hiola ed
cyclodex in a ached o he su ace o he AuNPs. Righ . Fo ma ion o he sup amolecula ne wo k
be ween e ocene dime and cyclodex in- unc ionalized AuNPs.290 B) Schema ic ep esen a ion o he
p epa a ion o non- a ge ed and a ge ed CD@AuNPs o β-Lapachone in acellula deli e y.
Inco po a ion o building blocks in o AuNPs i) SH-CD and PEG-SH ii) β-Lapachone, iii) SH-CD, PEG-
SH, and NHS-PEG-SH, i ) an i-EGFR and ) β-Lapachone.298 C) Schema ic ep esen a ion o building
blocks and sel -assembly o mul i unc ional hos -gues CD@AuNPs o a ge ed an icance he apy,
in e naliza ion mechanism, and pH-media ed in acellula elease o doxo ubicin.302
A)
B)
C)
Héc o Fe nández Ca o
146
2.2.2. Achie ing he apeu ic esponse by changes in physical/ca aly ic
p ope ies
In addi ion o he inco po a ion o bioac i e d ugs, he use o ha d nanopa icles deco a ed
wi h hos ca i ies can addi ionally exploi sup amolecula p ocesses in o de o egula e a
he apeu ic esponse based on he physical o ca aly ic p ope ies o he nanopa icles. A
pionee ing epo o Ro ello’s g oup showed how dynamic exchange o gues s in li ing cells
could be used o igge ing a cy o oxic esponse.303 In pa icula , diaminohexane-
unc ionalized gold nanopa icles we e capped wi h CB[7] gues s. These pa icles we e eadily
in e nalized in MFC-7 cells. Addi ion o 1-adaman ylamine -a gues wi h a highe binding
a ini y owa ds CB[7] in compa ison o diaminohexane- o he cell media igge ed adaman ane
up ake and gues exchange in cells. Uncapped gold nanopa icles we e able o p omo e
endosomal escape and induce cell dea h.
303
Ske ching a simila concep , he use o edox-
esponsi e gues s ha e been exploi ed o igge in acellula nanopa icle agg ega ion.304 In
his epo , CD-coa ed gold nanopa icles and oxidized di alen e ocene c osslinke s we e co-
deli e ed o HepG2 cells. Flow cell cy ome y e ealed inc eased cell oxici y when
nanopa icles and c osslinke s we e deli e ed a he same ime in compa ison wi h he
indi idual componen s. As a mechanis ic p oposal, he au ho s claimed ha in acellula Fc+
was educed in he p esence o in acellula glu a hione, ini ia ing nanopa icle agg ega ion by
c osslinking. Quan i a i e ICP-MS e ealed ha he p esence o he c osslinke esul ed in
highe Au e en ion in cells. This accumula ion could induce apop osis in cells.
304
On he o he hand, Ro ello’s g oup has shown he egula ion o he enzyma ic ac i i y o
nanopa icle based-nano eac o s.305 The design is composed o AuNPs coa ed wi h N-Benzyl-
N,N-dime hyloligoe hylenglycol ligands. These can encapsula e ansi ion me al ca alys s and
hold hem by sup amolecula capping be ween CB[7] hos s and he ligands. Ca alys s can be
exposed o he media by eleasing he hos cap ia compe i ion wi h endocy osed
adaman ylamine. Once libe a ed, he ca aly ic ac i i y is ac i a ed, which was demons a ed by
he N-deacyla ion o an N-alkyla ed 5- luo ou acil p od ug. This igge ed he oxic esponse
o he d ug.
305
303
C. Kim, S. S. Agas i, Z. Zhu, L. Isaacs, V. M. Ro ello, Na . Chem. 2010, 2, 962-966.
304
Y. Wang, H. Li, Q. Jin, J. Ji, Chem. Commun. 2016, 52, 582-585.
305
G. Y. Tonga, Y. Jeong, B. Duncan, T. Mizuha a, R. Mou , R. Das, S. T. Kim, Y.-C. Yeh, B. Yan, S. Hou, e
al., Na . Chem. 2015, 7, 597-603.
Objec i es
Chap e II: Objec i es
149
The use o me allic hos -gues based-nanopa icles as mo i s o nano he apeu ic
applica ions has aised a g ea deal o in e es . In his sense, he e e sibili y o he hos -gues
complexes has been adi ionally used in he solubiliza ion and in eg a ion o di e en
hyd ophobic d ugs in o nanopa icle sys ems. Howe e , mos o hese s a egies ha e been
ocused on he elease and exchange o hyd ophobic hos s ca gos and, o he bes o ou
knowledge, he elease and dynamic exchange be ween hos deco a ed nanopa icles and
hyd ophilic pep ide gues s has no been explo ed. He e, a po en ial challenge is he chemical
complexi y o pep ides, which migh p omo e unspeci ic in e ac ions by elec os a ic o an de
Waals o ces wi h NPs o any o he componen . On he o he hand, he coa ing o nanopa icle
su aces wi h hyd ophilic (ca ionic o anionic) pep ides has been mos ly app oached by using
co alen and elec os a ic coa ing p o ocols, while examples using sup amolecula ecogni ion
a e sca ce, wi h li le in o ma ion epo ing he e ec o p og essi e coa ing o con ol
nanopa icle size and su ace po en ial. This is essen ial o unde s and he c i ical pa ame e s
ha de e mine nanopa icle s abili y. The e o e, in his second chap e , we will explo e he
inco po a ion o di e en gues -bea ing hyd ophilic pep ides, such as polya ginine o
oligoglu amic pep ides, in o β-cyclodex in- unc ionalized gold nanopa icles (β-CD@AuNPs)
d i en by hos -gues molecula ecogni ion (Figu e 69). Mo eo e , we will cons uc mul i-
componen nanopa icles wi h he inco po a ion o hyd ophilic s abilize s such as polye hylene
glycol chains. Finally, we will show he acili a ed up ake o memb ane-impe meable
hyd ophilic pep ides d i en by he o ma ion o sup amolecula pep ide/β-CD@AuNP hyb ids.
We hypo hesized ha he unc ional dynamic gues exchange o hyd ophilic biomolecules
would be possible inside li ing cells based on he highe a ini y o a mul i alen gues ,
exploi ing ei he s a is ical ebinding o mul i alen binding p ocesses.
The speci ic objec i es o his chap e would be:
 The syn hesis o β-CD@AuNPs by inco po a ion o pe - hiola ed-β-CD a he AuNP
su ace and hei cha ac e iza ion by TEM, DLS, UV-Visible, and TGA expe imen s.
 The syn hesis o a small lib a y o oligoa ginine pep ides bea ing mono- (AdR4 and
AdR8) o di alen (Ad2R4 and Ad2R8) adaman ane gues s a ached. We will exploi
he alcoxyamine-aldehyde condensa ion be ween pep ide and gues espec i ely.
Fu he mo e, mono- (AdPEG) and di alen (Ad2PEG) PEG moie ies will be also
p epa ed ollowing a simila s a egy.
 The in eg a ion o hese elemen s in o mul icomponen nanopa icles and hei
cha ac e iza ion by size and su ace po en ial measu emen s. The la e will be used o
quan i y he binding cons an s be ween unc ionalized pep ides and β-CD@AuNPs.
 The explo a ion o he in cellulo gues -bea ing pep ide exchange due o a ini y
di e ences based on gues alency. This will be ca ied ou by i s assessing he
pep ide exchange in i o using luo escence spec oscopy and la e inside li ing cells
by imaging (con ocal mic oscopy expe imen s) and cell cy ome y assays.

Héc o Fe nández Ca o
150
Figu e 69. A) Re osyn he ic s a egy o he p epa a ion o AdPEG exploi ing he alcoxyamine-
aldehyde condensa ion be ween PEG and gues espec i ely. B) Gues pool composed o
mono alen /di alen CPPs ( e a and oc a ginine based), mono alen /di alen PEG moie ies, and
mono alen model ca go oligoglu amic pep ide (AdE9). C) Schema ic ep esen a ion o he
inco po a ion o a mono alen pep ide ca go AdE9 in o β-CD@AuNP leading o he o ma ion o he
pep ide/β-CD@AuNP sup amolecula complex and dynamic exchange be ween gues s acco ding o
hei alence.
B)
C)
A)
Resul s and discussions
Chap e II: Resul s and discussions
153
1. Syn hesis and cha ac e iza ion o β-cyclodex in- unc ionalized gold
nanopa icles
We s a ed his p ojec by op imizing he condi ions o he p epa a ion o he NPs. The β-
CD@AuNPs we e p epa ed ollowing a sligh ly modi ied me hodology desc ibed in he
li e a u e.
306
In ou p ocedu e, we added a solu ion o NaBH4 ( educing agen ) and pe -
hiola ed-β-cyclodex in
307
in d y DMSO o a solu ion o HAuCl4 in d y DMSO (Figu e 70).
Du ing his pe iod, he Au(III) ca ions we e educed o elemen a y, coalescing Au(0). Then,
nanopa icles we e p ecipi a ed by adding ace oni ile and collec ed a e cen i uga ion.
Finally, he pa icles we e suspended in wa e and eeze-d ied.
Figu e 70. Syn he ic pa hway o he p epa a ion o β-CD@AuNPs.
To cha ac e ize he β-CD@AuNPs, we ca ied ou a ange o measu emen s in ended o
p o ide in o ma ion abou he physical p ope ies o he pa icles, as well as he deg ee o
unc ionaliza ion o he hos molecule on he su ace. Fi s , we s udied he size o he β-
CD@AuNPs by TEM and DLS. TEM mic og aphs showed he sphe ical shape and low
polydispe si y o β-CD@AuNPs (Figu e 71). Image analysis o he pa icle size dis ibu ion
e idenced an a e age nanopa icle size o 2-3 nm (inse Figu e 71). DLS expe imen s o β-
CD@AuNPs suspended in Milli-Q wa e showed a hyd odynamic diame e o a ound 8 nm
(Figu e 71). This disc epancy is based on he ac ha DLS measu emen s a e in luenced by
di usion ac o s, such as sol a ion laye s. The UV spec a o β-CD@AuNPs dispe sed in Milli-
Q wa e showed he ypical plasmon abso p ion band o well-dispe sed β[email protected]
Finally, we used he mog a ime ic analysis (TGA) o quan i y he amoun o o ganic ma e ial
a he su ace o he p epa ed β-CD@AuNPs. The mass loss up o 100°C was a ibu ed o
wa e e apo a ion, while he weigh loss a ibu ed o o ganic componen s we e obse ed
be ween 280-320°C. This indica es abou 13 % o ganic ma e ial, which would co espond o
app oxima ely 20 β-cyclodex ins co e ing each gold nanopa icle (Figu e 71). This is in
ag eemen wi h p e ious epo s.306
306
R. Mejia-A iza, J. Huskens, J. Ma e . Chem. B 2014, 2, 210-216.
307
M. T. Rojas, R. Koenige , J. F. S odda , A. E. Kai e , J. Am. Chem. Soc. 1995, 117, 336-343.