INTERNATIONAL DOCTORAL
SCHOOL OF THE USC
Sand a
Díez Villa es
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Doc o al P og amme in Molecula Medicine
DOCTORAL THESIS
PRECLINICAL IMAGING AND
BIODISTRIBUTION STUDIES
OF LIPID NANOEMULSIONS
Sand a Díez Villa es
INTERNATIONAL PHD SCHOOL OF THE UNIVERSITY OF SANTIAGO DE COMPOSTELA
PHD PROGRAMME IN MOLECULAR MEDICINE
SANTIAGO DE COMPOSTELA
2022
DECLARACIÓN DE LA AUTORA DE LA TESIS
Dña. Sand a Díez Villa es
Tí ulo de la esis: P eclinical imaging and biodis ibu ion s udies o lipid
nanoemulsions
P esen o mi esis, siguiendo el p ocedimien o adecuado al Reglamen o y decla o
que:
1) La esis aba ca los esul ados de la elabo ación de mi abajo.
2) De se el caso, en la esis se hace e e encia a las colabo aciones que u o
es e abajo.
3) Con i mo que la esis 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.
4) La esis es la e sión de ini i a p esen ada pa a su de ensa y coincide la
e sión imp esa con la p esen ada en o ma o elec ónico.
Y me comp ome o a p esen a el 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, 22 de diciemb e de 2022.
AUTORIZACIÓN DEL DIRECTOR DE LA TESIS
P eclinical imaging and biodis ibu ion s udies o lipid
nanoemulsions
D a. Ma ía de la Fuen e
INFORMAN:
Que la p esen e esis, se co esponde con el abajo ealizado po Dª.
Sand a Díez Villa es, bajo mi di ección, y au o izo 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 de es a no incu e
en las causas de abs ención es ablecidas en la Ley 40/2015.
De acue do con lo indicado en el Reglamen o de Es udios de Doc o ado,
decla a ambién que la p esen e esis doc o al es idónea pa a se de endida
en base a la modalidad de Monog á ica con ep oducción de
publicaciones, en los que la pa icipación de la doc o anda ue decisi a
pa a su elabo ación y las publicaciones se ajus an al Plan de In es igación.
En San iago de Compos ela a 22 de diciemb e de 2022
AUTORIZACIÓN DEL TUTOR DE LA TESIS
P eclinical imaging and biodis ibu ion s udies o lipid
nanoemulsions
D . Ra ael López López
INFORMAN:
Que la p esen e esis, se co esponde con el abajo ealizado po Dª.
Sand a Díez Villa es, bajo mi u o ización, y au o izo 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 de es a no incu e
en las causas de abs ención es ablecidas en la Ley 40/2015.
De acue do con lo indicado en el Reglamen o de Es udios de Doc o ado,
decla a ambién que la p esen e esis doc o al es idónea pa a se de endida
en base a la modalidad de Monog á ica con ep oducción de
publicaciones, en los que la pa icipación de la doc o anda ue decisi a
pa a su elabo ación y las publicaciones se ajus an al Plan de In es igación.
En San iago de Compos ela a 22 de diciemb e de 2022
Table o con en s
TABLE OF CONTENTS
Abs ac ................................................................................................................................. 19
Resumen ................................................................................................................................ 23
Resumen in ex enso ............................................................................................................... 29
Resumo in ex enso ................................................................................................................. 45
In oduc ion .......................................................................................................................... 59
Hypo hesis ............................................................................................................................. 89
Objec i es .............................................................................................................................. 95
Chap e I ............................................................................................................................. 101
Manganese Fe i e Nanopa icles Encapsula ed in o Vi amin E / Sphingomyelin Nanoemulsions
as Con as Agen s o High-Sensi i e Magne ic Resonance Imaging 103
Chap e II ............................................................................................................................ 139
Biodis ibu ion o 68/67Ga- adiolabeled sphingomyelin nanoemulsions by PET and SPECT
imaging 141
Chap e III ........................................................................................................................... 175
Quan i a i e PET acking o in a-a icula ly adminis e ed 89Z -pep ide-deco a ed
sphingomyelin nanoemulsions 177
Chap e IV ........................................................................................................................... 209
89Z adiolabeling o sphingomyelin nanoemulsions o long- e m in i o PET acking 211
Chap e V ............................................................................................................................ 229
T acing he a e o sphingomyelin nanoemulsions in lung cance models o es hei po en ial o
each me as a ic cells 231
O e all discussion ............................................................................................................... 265
Conclusions ......................................................................................................................... 285
Abb e ia ions ...................................................................................................................... 291
Ag adecimien os ................................................................................................................. 301
E hical issues and annexes ................................................................................................... 309
Abs ac
21
ABSTRACT
O e he las decades, nanomedicine has undoub edly e olu ionized he pha maceu ical
ield since i o e s unique and a ac i e op ions o imp o e he ea men o se e al
diseases and mo e owa ds he implemen a ion o p ecision medicine. In ac , a as
numbe o p omising p eclinical s udies and clinical ials a e being p oduced e e y yea ,
bu un o una ely, he e is an impo an gap be ween hese s udies and hose ha ge he
clinical app o al. In his ega d, non-in asi e imaging is being s ablished as a p omising
a enue o be e unde s and he in i o beha io o nanopa icles and o e come one o
he g ea es challenges acing clinical ansla ion. Techniques such as magne ic esonance
imaging (MRI) o nuclea imaging modali ies o e a ac i e possibili ies o moni o he
nanopa icle pha macokine ic beha io , he in i o s abili y, he d ug deli e y p ocess,
he he apeu ic e ec , o he a ge si e accumula ion, all in a eal- ime manne .
In his ame, ou g oup has ecen ly epo ed he p epa a ion and cha ac e iza ion
o a bioinspi ed o mula ion consis ing o lipid nanoemulsions, using a one-s ep,
ex emely simple and cos -e ec i e me hodology. P e ious esul s highligh hei high
biocompa ibili y and po en ial in d ug deli e y, due o hei capaci y o ca y
hyd ophobic d ugs and hyd ophilic biomolecules such as pep ides and nucleic acids.
Bea ing his in o ma ion in mind, he main aim o he p esen hesis was o ailo
hese lipid nanoemulsions o hei associa ed ca go wi h imaging agen s o explo e hei
in i o beha io by non-in asi e imaging modali ies. Addi ionally, o he analysis
echniques based on luo escence and low cy ome y we e also explo ed o con i m cell
a ge speci ici y.
SANDRA DÍEZ VILLARES
22
In he i s chap e , supe pa amagne ic manganese e i e nanopa icles we e
encapsula ed in o he oil co e o sphingomyelin nanoemulsions, o ob ain magne ic
nanoemulsions able o p oduce a nega i e magne ic esonance con as signal. In i o
MRI esul s con i med he o mula ion biocompa ibili y, as well as a good con as signal
in he b ain o a s. Ex i o magne ic esonance s udies allowed o de e mine he o gan
dis ibu ion. In he second, hi d and ou h chap e s, we mo ed on o nuclea imaging
modali ies. Speci ically, in he second chap e we adiolabeled sphingomyelin
nanoemulsions wi h wo adionuclides, gallium-68, and gallium-67 o PET and SPECT
imaging. We e alua ed he in luence o he ype o chela o , as well as he inco po a ion
o hyd ophilic coa ings wi h espec o hei in i o beha io . In he hi d chap e , we
adiolabeled a lipid-pep ide de i a i e wi h zi conium-89 in o de o assess i s e en ion
in he knee a e in a-a icula injec ion. We also associa ed he pep ide o
sphingomyelin nanoemulsions o op imize deli e y s a egies o pep idomime ics. In
he ou h chap e , we s udied di e en chela ing s a egies o di ec ly adiolabel he
nanoemulsions wi h zi conium-89 and explo e long- e m in i o a e a e in a enous
adminis a ion by PET imaging. Finally, in he las chap e we explo ed he
biodis ibu ion o luo opho e-labeled sphingomyelin nanoemulsions a he sub-o gan
le el by low cy ome y and p o ed hei abili y o accumula e in speci ic cell ype subse s
in lung cance animal models ep esen a i e o ad anced s ages o he disease.
O e all, his hesis p o es he po en ial o sphingomyelin nanoemulsions o
associa e di e en ypes o imaging agen s, (e.g., MR con as agen s, PET and SPECT
adionuclides and luo opho es) and p o ides aluable in o ma ion abou he in i o a e
o hese no el nanopla o ms and po en ial use in di e en biomedical applica ions.
Resumen
31
RESUMEN IN EXTENSO
Se denomina nanomedicina a la aplicación de la nano ecnología en el campo de la
biomedicina, ya sea pa a p e ención, diagnós ico o a amien o de en e medades.
Aunque no exis e una de inición exac a, ac ualmen e se conside an nanopa ículas
aquellas cuyo amaño se encuen a den o de la escala nanomé ica, gene almen e en el
ango comp endido en e 1 y 1000 nm. Es e amaño nanomé ico les con ie e unas
p opiedades únicas, esponsables de la g an e olución que han causado en el ámbi o
a macéu ico. En e ellas, cabe des aca la mayo biodisponibilidad, e icacia y selec i idad
que p esen an en compa ación con los á macos con encionales. Es as p opiedades en
conjun o hacen de los nano á macos o mulaciones segu as capaces de causa menos
e ec os secunda ios en pacien es.
El po encial de la nanomedicina ha quedado demos ado hace años con la
ap obación po pa e de la Adminis ación de Alimen os y Medicamen os de los Es ados
Unidos (FDA, po sus siglas en inglés) de las p ime as o mulaciones liposomales,
Dip i an® y Doxil®, en 1989 y 1995, espec i amen e. Desde en onces, se han ap obado
más de 80 nanomedicamen os en e la FDA y la Asociación Eu opea del Medicamen o
(EMA), siendo la más ecien e la e oluciona ia ap obación de eme gencia de las acunas
de ARNm desa olladas po P ize -BioNTech y Mode na pa a hace en e a la pandemia
causada po la COVID-19. De en e odos los nanomedicamen os ap obados, la mayo ía
han sido diseñados pa a a amien os oncológicos, aunque ambién pa a el a amien o
de un amplio ango de en e medades como in ecciones, en e medades in lama o ias,
anemia o pa ologías neu ológicas. En cuan o a la composición, las nanopa ículas
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lipídicas y las polimé icas se ponen en cabeza con espec o a o os diseños dada su
conocida biocompa ibilidad.
Además de los nanomedicamen os ap obados, el núme o de o mulaciones en
ensayos clínicos es cada ez mayo . De hecho, el impac o de las acunas de ARNm ha
impulsado de nue o el campo de la nanomedicina y, según las es adís icas de me cado,
se espe a que en los p óximos años los nanomedicamen os ap obados aumen en
conside ablemen e. En pa icula , exis e una endencia hacia el desa ollo de
nanopa ículas más so is icadas, que sean capaces de combina di e sas unciones en una
sola pla a o ma pa a log a e apias di igidas y de p ecisión. Es o pone de mani ies o la
impo ancia que es á adqui iendo la medicina pe sonalizada en clínica, sob e odo en
en e medades como el cánce , donde la he e ogeneidad en e pacien es di icul a la
e icacia de a amien os gené icos.
Aunque el u u o de la nanomedicina puede se p ome edo , la ealidad es que la
di e encia en e la in es igación y aquellas nanopa ículas que llegan a la p ác ica clínica
es abismal y la asa de aslación se es ima que sea in e io a un 10%. Es a b echa puede
p o eni de di e en es ac o es, desde las impo an es di e encias expe imen ales que
exis en en e animales y humanos, la an e io men e mencionada he e ogeneidad en e
pacien es, has a un mal diseño de las nanopa ículas en é minos de es abilidad, acilidad
de escalado y cos es de p oducción. Sin emba go, uno de los mo i os más ele an es que
impide una aslación adecuada es la al a de conocimien o del compo amien o in i o
de los nanomedicamen os, lo que di icul a el diseño de o mulaciones op imizadas
ga an es de una mayo e icacia.
Cuando los nanomedicamen os son adminis ados en el cue po ienen que supe a
una se ie de ba e as biológicas, isiológicas y mic oambien ales pa a llega de mane a
e icaz al luga de acción y ealiza su ac i idad e apéu ica. Es as ba e as, que dependen
p incipalmen e de la ía de adminis ación y del ipo y g ado de en e medad, pueden
a ec a a la es abilidad y biodisponibilidad de los nanomedicamen os, po lo que es
indispensable conoce las en de alle pa a ealiza un diseño óp imo de la o mulación. Si
bien es cie o que hay nanomedicamen os ap obados pa a di e sas u as de
Resumen in ex enso
33
adminis ación, la que más in e és ha gene ado, y po lo an o la más es udiada, es la ía
in a enosa. En es e con ex o, el g ado de in e acción con las ba e as mencionadas, el
eco ido y el compo amien o de los nanomedicamen os una ez adminis ados en el
cue po depende esencialmen e de sus ca ac e ís icas, es deci , de su o ma, amaño, ca ga
supe icial y composición. Po ejemplo, la o ma geomé ica de las nanopa ículas juega
un papel impo an e en el p oceso de ex a asación del o en e sanguíneo, siendo
aquellas con o ma no es é ica las que ienen mejo capacidad de des ia se de la
ci culación pa a adhe i se al aso sanguíneo. En lo que se e ie e al amaño y ca ga
supe icial de las nanopa ículas, es as p opiedades pueden se decisi as pa a de e mina
di e sos pa áme os, como el iempo de ci culación en sang e, la cap ación en ó ganos
como hígado o bazo e incluso la capacidad de acumulación e in e nalización en el umo .
Asimismo, los ma e iales con los que son ab icadas las nanopa ículas pueden a ec a a
su compo amien o in i o, especialmen e en el iempo de ci culación, ya que se ha
obse ado que las nanopa ículas ígidas, como las nanopa ículas ino gánicas, son
eliminadas del o en e sanguíneo de mane a más emp ana que nanopa ículas
o gánicas.
Todas es as a iables ponen en alo que un de allado seguimien o de las
nanopa ículas, conociendo el eco ido desde que se adminis an has a su luga de
acción, sea uno de los desa íos p incipales pa a consegui una co ec a aslación a la
clínica. En es e con ex o, las écnicas de imagen no in asi a o ecen g andes
posibilidades, dado que una combinación de las nanopa ículas con agen es de imagen
pe mi i ía la e aluación a iempo eal de la a macociné ica de los nanomedicamen os,
de su acumulación en el luga de acción e incluso la e aluación del e ec o e apéu ico.
Tan o en la p ác ica clínica como en la in es igación p eclínica exis en di e sas écnicas
de imagen no in asi a, y cada una de ellas iene p opiedades especí icas ú iles pa a
di e en es aplicaciones. Po ejemplo, la imagen po Resonancia Magné ica (MRI, po sus
siglas en inglés) des aca po su al a esolución espacial capaz de p opo ciona
in o mación ana ómica p ecisa y ha sido ampliamen e u ilizada pa a moni o iza la
biodis ibución de nanomedicamen os, e alua la libe ación de las moléculas
SANDRA DÍEZ VILLARES
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e apéu icas y de e mina su acción. Po o o lado, las écnicas de imagen nuclea o ecen
g andes en ajas g acias a su ex ema sensibilidad y a su na u aleza cuan i a i a,
pe mi iendo ob ene imágenes de cue po en e o de la biodis ibución de
nanomedicamen os, e incluso, han sido u ilizadas pa a la es a i icación y selección de
pacien es suscep ibles a a amien o, abo dando así la nanomedicina de p ecisión.
Además de la moni o ización in i o, exis en écnicas ex i o que esul an de g an
u ilidad pa a la ca ac e ización a ni el celula e in acelula de los nanomedicamen os,
po ejemplo, pa a de e mina poblaciones celula es esponsables de la cap ación de
nanopa ículas o pa a es udia los mecanismos de in e nalización celula .
Sin duda alguna, la e aluación in i o es un pa áme o c ucial pa a la aslación de
nanomedicamen os, sin emba go, an es de llega a ello, las nanopa ículas han enido que
se diseñadas desde una pe spec i a aslacional, es deci , con el obje i o de log a llega
algún día la p ác ica clínica. Es o supone la u ilización de ma e iales biodeg adables,
biocompa ibles, no inmunogénicos y asequibles (en é minos de cos es) en el p oceso de
p epa ación. Además, es e p oceso ha de se sencillo y ácil de escala sin comp ome e
la es abilidad ni las p opiedades de las nanopa ículas. Con odo es o en men e, nues o
g upo de in es igación ha desa ollado ecien emen e una nue a ecnología que iene
g an po encial pa a se u ilizada en el campo de la nanomedicina. Se a a de
nanoemulsiones lipídicas, compues as p incipalmen e po dos componen es na u ales,
Vi amina E, un excipien e designado como GRAS (del inglés, Gene ally Recognized as
Sa e) y es ingomielina, uno de los componen es mayo i a ios de las memb anas celula es.
Es as o mulaciones, denominadas en é minos gene ales nanoemulsiones de
es ingomielina, han demos ado una g an e sa ilidad pa a la asociación de di e sas
moléculas e apéu icas y ienen g an po encial pa a el a amien o di e sos ipos de
cánce , como el cánce de mama, colo ec al y de pánc eas. Sin emba go, en lo que
espec a a su compo amien o in i o, aún es á po explo a .
En is a a los an eceden es desc i os, el p incipal obje i o de es a esis consis e en la
e aluación del compo amien o in i o de las nanoemulsiones de es ingomielina a a és
de di e en es écnicas y di e sas u as de adminis ación, con el in de apo a nue a
Resumen in ex enso
35
in o mación que ayude a de e mina su po encial en nanomedicina. Pa a ello, se p opone
la asociación de agen es de con as e pa a su moni o ización po MRI, de adioisó opos
pa a su seguimien o po imagen nuclea y de luo ó o os pa a la de e minación ex i o
de la biodis ibución a ni el celula en modelos de cánce de pulmón no mic ocí ico.
En p ime luga , se encapsula on nanopa ículas supe pa amagné icas de e i a de
manganeso en el núcleo de las nanoemulsiones de es ingomielina, c eando
nanoemulsiones magné icas capaces de ac ua como agen es de con as e nega i o y de
aumen a signi ica i amen e la sensibilidad de la esonancia magné ica (Figu a 1). Pa a
ello, p ime o se sin e iza on y ca ac e iza on las nanopa ículas de e i a de manganeso,
de e minando su composición, amaño, mo ología y sus ca ac e ís icas
supe pa amagné icas.
Figu a 1. Resumen g á ico del abajo ealizado en el Capí ulo I cuyo obje i o consis ió en la
p epa ación y ca ac e ización de nanoemulsiones magné icas pa a e alua su po encial como
agen es de con as e en esonancia magné ica.
MANGANESE FERRITE
NANOPARTICLES
BIOINSPIRED
NANOEMULSIONS
MAGNETIC
NANOEMULSIONS
One-s ep Rep oducible
S able Biocompa ible
HIGH-SENSITIVE
MAGNETIC
RESONANCE IMAGING
SANDRA DÍEZ VILLARES
36
A con inuación, es as pa ículas se ecub ie on con Vi amina E, el acei e u ilizado
pa a la o mulación de las nanoemulsiones, a o eciendo así su encapsulación. Las
nanoemulsiones magné icas se p epa a on po el mé odo de inyección de e anol, un
mé odo que equie e de un único paso y que pe mi ió ob ene o mulaciones
homogéneas, con un amaño meno de 200 nm y es ables, de una mane a al amen e
ep oducible. Asimismo, es as nanoemulsiones magné icas se ca ac e iza on a ni el
mo ológico y se con i mó la p esencia de las nanopa ículas magné icas en su in e io .
Finalmen e, se demos ó la habilidad de las nanoemulsiones magné icas pa a ac ua
como agen es de con as e nega i o en esonancia magné ica. En es e con ex o, cabe
des aca que las nanoemulsiones magné icas p esen a on una mayo elaxi idad
ans e sal en compa ación con las pa ículas magné icas lib es.
Además, se es udió la biocompa ibilidad de las nanoemulsiones magné icas in i o.
Pa a ello, se inyec a on po ía in a enosa en a as Sp ague-Dawley y se midie on
alo es de bioma cado es indica i os de daño hepá ico y enal en di e en es iempos, sin
obse ase ninguna di e encia con espec o a los animales con ol, y con i mándose así,
que es as nanoemulsiones no p esen an oxicidad a las dosis es udiadas y pueden se
u ilizadas pa a su e aluación en animales. Po úl imo, se con i mó que las
nanoemulsiones pueden de ec a se de mane a e icaz po esonancia magné ica an o in
i o como ex i o, obse ando una buena señal nega i a en el ce eb o as inyección
in apa enquimal y una acumulación p incipalmen e en hígado y iñones, as inyección
in a enosa. Todo ello nos pe mi ió conclui que las nanoemulsiones de es ingomielina
pueden op imiza se ácilmen e pa a ac ua como agen es de con as e, dando luga a
nanoemulsiones magné icas biocompa ibles, con una al a capacidad de gene a un
con as e nega i o y que, además, pueden moni o iza se po MRI u ilizando dosis muy
bajas, po lo que son capaces de aumen a la sensibilidad de la écnica, lo que se conside a
su p incipal limi ación.
En segundo luga , se p opuso op imiza las nanoemulsiones de es ingomielina pa a
pode moni o iza su biodis ibución u ilizando en es e caso las écnicas de imagen
nuclea , Tomog a ía po Emisión de Posi ones (PET) y Tomog a ía Compu a izada de
Resumen in ex enso
37
Emisión Mono o ónica (SPECT), que p esen an cie as en ajas con espec o a la imagen
po MR, como mejo sensibilidad y mayo acilidad pa a adqui i imágenes de cue po
en e o. Pa a ello, se escogió el pa de adioisó opos galio-68 y galio-67, dado que el
p ime o es un emiso de posi ones (PET) y el segundo es un isó opo emiso gamma
(SPECT). Además, ambos ienen pe íodos de semidesin eg ación muy dispa es, siendo
de 68 minu os pa a el galio-68 y 3,3 días pa a el galio-67, lo que p opo ciona una g an
e sa ilidad pa a e alua in i o las nanoemulsiones a di e en es iempos, dependiendo
de la aplicación pa a la que se diseñen (Figu a 2).
Figu a 2. Resumen g á ico del abajo ealizado en el Capí ulo II cuyo obje i o consis ió en el
adioma caje de las nanoemulsiones de es ingomielina con los adionúclidos galio-68 y galio-67
pa a e alua su biodis ibución po las écnicas de PET y SPECT.
Pa a consegui un adioma caje e icaz de las nanoemulsiones de es ingomielina, es
necesa io inco po a agen es quelan es en su supe icie, es deci , moléculas que o man
complejos quela o con el adionucleido. Con es e in, se selecciona on dos quelan es, el
DTPA y el NOTA, y con el obje i o de man ene un mé odo de p epa ación sencillo y en
un solo paso, se u iliza on de i ados lipídicos de es as moléculas. Es o pe mi ió su
inco po ación en la supe icie de las nanoemulsiones en el momen o de su p epa ación y
dio luga a nanoemulsiones homogéneas y es ables. Ambas o mulaciones se ma ca on
con galio-68 e icazmen e, si bien es cie o que el ma caje de las nanoemulsiones que
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con enían NOTA esul ó se más es able que el de DTPA. La biodis ibución de ambas
nanoemulsiones pudo no obs an e e alua se an o in i o, median e la adquisición de
imágenes PET, como ex i o, cuan i icando la adiac i idad en los ó ganos de los a ones.
Ambos esul ados, aunque con impo an es di e encias, mos a on la cap ación ípica de
la mayo ía de las nanopa ículas, es deci , hígado y bazo, aunque ambién se obse ó
acumulación en co azón, o en e sanguíneo, pulmones, iñones y ejiga. La p esencia de
nanoemulsiones en el o en e sanguíneo con i mó la necesidad de u iliza
adionucleidos con iempos de semidesin eg ación más la gos pa a es udia su
compo amien o in i o, po lo que se selecciona on las nanoemulsiones modi icadas
con NOTA pa a adioma ca las con galio-67 y se adqui ie on imágenes has a 48 ho as
as la inyección. Asimismo, u ilizando la écnica SPECT, se e aluó si la inco po ación de
políme os hid o ílicos en la supe icie de las nanoemulsiones, como el ácido hialu ónico
(HA) y el polie ilenglicol (PEG), pod ían inc emen a el iempo de ci culación en sang e,
ob eniéndose esul ados posi i os en el caso de las o mulaciones PEGiladas. En
esumen, la capacidad de pode modula an o el adioma caje, la écnica de imagen
como el iempo de ci culación de las nanoemulsiones, pone de mani ies o el po encial
que ienen es os sis emas de es ingomielina pa a adap a se ácilmen e a las necesidades
que puedan eque i di e sas aplicaciones biomédicas.
Aunque, como se ha mencionado an e io men e, la ía in a enosa es la más
u ilizada en el desa ollo de nanomedicamen os pa a el a amien o de en e medades
como el cánce , exis en o as ías de adminis ación que pueden se in e esan es pa a
a a pa ologías más locales, como la a i is euma oide o la os eoa i is. En conc e o,
pa a el a amien o de es e ipo de en e medades la ía in a-a icula pe mi e la
libe ación selec i a de á macos en su luga de acción, a la ez que educe la exposición
sis émica y minimiza los e ec os secunda ios ad e sos que pod ían causa al se
adminis ados po o as ías. Sin emba go, uno de los mayo es p oblemas a sol en a es
la b e e pe manencia en la a iculación debido a la cons an e eno ación del líquido
sino ial, aspec o en el que la nanomedicina puede apo a soluciones e ec i as. Teniendo
es o en men e, se plan ea on dos obje i os pa a la ealización del siguien e capí ulo. En
Resumen in ex enso
39
p ime luga , se p opuso u iliza las nanoemulsiones de es ingomielina pa a mejo a la
e ención en la a iculación de un pép ido senolí ico que iene po encial pa a el
a amien o de la os eoa i is. En segundo luga , pa a de e mina es a e ención, se
p opuso hace un seguimien o po PET, ap o echando su na u aleza cuan i a i a
(Figu a 3).
Figu a 3. Resumen g á ico del abajo ealizado en el Capí ulo III, en el que el p incipal obje i o
ue el seguimien o po PET pa a compa a la e ención en la odilla de un pép ido senolí ico
adioma cado con zi conio-89 con la de nanoemulsiones deco adas con es e pép ido as su
adminis ación in a-a icula .
Pa a abo da es os obje i os, en p ime luga , se modi icó el pép ido senolí ico de
in e és con una cadena lipo ílica, acili ando así su pos e io inco po ación a las
nanoemulsiones, y con de e oxamina (DFO), un quelan e que pe mi e el ma caje de es a
biomolécula con el adionucleido zi conio-89. An es de p ocede al adioma caje, se
p epa a on y ca ac e iza on las nanoemulsiones deco adas con el pép ido senolí ico,
ob eniendo o mulaciones ca iónicas, con un amaño meno de 200 nm y es ables en
medios biológicos. A con inuación, se ealizó el adioma caje del pép ido y la p epa ación
de nanoemulsiones ca gadas con es e. Tan o el pép ido ma cado como las
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40
nanoemulsiones asociando el mismo, p esen a on ele ada pu eza adioquímica y buena
es abilidad en líquido sino ial y plasma sanguíneo. Po úl imo, se p ocedió a inyec a en
la a iculación de la odilla de a as Sp ague-Dawley an o el pép ido adioma cado en su
o ma lib e como asociado a las nanoemulsiones, y se e aluó su biodis ibución
adqui iendo imágenes du an e 14 días y cuan i icando la señal de ac i idad p esen e en
la odilla inyec ada y en o os ó ganos. Los esul ados con i ma on que las
nanoemulsiones de es ingomielina son capaces de a o ece una mayo pe manencia del
pép ido en la a iculación con espec o al pép ido lib e, que mos ó una eliminación más
ápida, con una g an acumulación en hígado y iñones. En conjun o, es e capí ulo
po encia la e sa ilidad de las nanoemulsiones de es ingomielina y con i ma que pueden
ene po encial pa a a amien o de en e medades in lama o ias. Además, dado que se
a a de un pép ido e apéu ico, ac ua ían como sis emas e anós icos, donde se combina
imagen y e apia en una sola pla a o ma.
En is a de los esul ados ob enidos, y a la e icacia obse ada con el zi conio-89 pa a
ma caje y seguimien o in i o du an e pe íodos de iempo p olongados, en el cua o se
quiso es udia la biodis ibución de las nanoemulsiones de es ingomielina as
adminis ación in a enosa, una ez op imizado el ma caje con zi conio-89. Es deci , en
es e capí ulo se quiso abo da un seguimien o del compo amien o in i o de las
nanoemulsiones a iempos más la gos que los u ilizados en el capí ulo II y ma cándolas
con adionucleidos PET. Con es e obje i o en men e, se e aluó la conjugación del
quelan e DFO a las nanoemulsiones de es ingomielina a a és de es es a egias
di e en es. En p ime luga , se lle ó a cabo una eacción química pa a asocia la DFO en
la supe icie de las nanoemulsiones. Pa a ello, la DFO modi icada con un g upo
iso iociana o se hizo eacciona con las nanoemulsiones que con enían un PEG lipídico
uncionalizado con una amina (DSPE-PEG-NH2). T as una ho a de incubación, las
nanoemulsions se pu i ica on, se cuan i icó la e icacia de eacción y se ca ac e iza on sus
p opiedades isicoquímicas. Po o o lado, se lle ó a cabo la modi icación p e ia del
quelan e DFO con un lípido (DSPE) y pos e io men e se incluyó con el es o de los
lípidos que con o man la memb ana de las nanoemulsiones en el momen o de su
47
RESUMO IN EXTENSO
Denomínase nanomedicina á aplicación da nano ecnoloxía no campo da biomedicina,
xa sexa pa a p e ención, diagnós ico ou a amen o de en e midades. Aínda que non
exis e unha de inición exac a, ac ualmen e considé anse nanopa ículas aquelas cuxo
amaño se a opa den o da escala nanomé ica, xe almen e no ango comp endido en e
1 e 1000 nm. Es e amaño nanomé ico con í elles unhas p opiedades únicas,
esponsables da g an e olución que causa on no ámbi o a macéu ico. En e elas, cabe
des aca a maio biodispoñibilidade, e icacia e selec i idade que p esen an en
compa ación cos á macos con encionais. Es as p opiedades en conxun o an dos
nano á macos o mulacións segu as capaces de causa menos e ec os secunda ios en
pacien es.
O po encial da nanomedicina quedou demos ado ai anos coa ap obación po
pa e da Adminis ación de Alimen os e Medicamen os dos Es ados Unidos (FDA, polas
súas siglas en inglés) das p imei as o mulacións liposomais, Dip i an® e Doxil®, en 1989
e 1995, espec i amen e. Desde en ón, ap obá onse máis de 80 nanomedicamen os en e
a FDA e a Asociación Eu opea do Medicamen o (EMA), sendo a máis ecen e a
e oluciona ia ap obación de eme xencia das acinas de ARNm desen ol idas po
P ize -BioNTech e Mode na pa a ace on e á pandemia causada pola COVID-19. De
en e odos os nanomedicamen os ap obados, a maio ía o on deseñados pa a
a amen os oncolóxicos, aínda que amén pa a o a amen o dun amplo ango de
en e midades como in eccións, en e midades in lama o ias, anemia ou pa oloxías
neu olóxicas. En can o á composición, as nanopa ículas lipídicas e as polimé icas
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póñense na cabeza con espec o a ou os deseños dada a súa coñecida
biocompa ibilidade.
Ademais dos nanomedicamen os ap obados, o núme o de o mulacións en ensaios
clínicos é cada ez maio . De ei o, o impac o das acinas de ARNm impulsou de no o o
campo da nanomedicina e, segundo as es a ís icas de me cado, espé ase que nos
p óximos anos os nanomedicamen os ap obados aumen en conside ablemen e. En
pa icula , exis e unha endencia ca a ó desen ol emen o de nanopa ículas máis
so is icadas, que sexan capaces de combina di e sas uncións nunha soa pla a o ma pa a
log a e apias di ixidas e de p ecisión. Is o pon de mani es o a impo ancia que es á
adqui indo a medicina pe sonalizada en clínica, sob e odo en en e midades como o
canc o, onde a he e oxeneidade en e pacien es di icul a a e icacia de a amen os
xené icos.
Aínda que o u u o da nanomedicina pode se p ome edo , a ealidade é que a
di e enza en e a in es igación e aquelas nanopa ículas que chegan á p ác ica clínica é
abismal e a axa de anslación es ímase que sexa in e io a un 10%. Es a b echa pode
p o i de di e en es ac o es, desde as impo an es di e enzas expe imen ais que exis en
en e animais e humanos, a an e io men e mencionada he e oxeneidade en e pacien es,
a a un mal deseño das nanopa ículas en e mos de es abilidade, acilidade de escalado e
cus os de p odución. Con odo, un dos mo i os máis ele an es que impide unha
anslación adecuada é a al a de coñecemen o do compo amen o in i o dos
nanomedicamen os, o que di icul a o deseño de o mulacións op imizadas ga an es
dunha maio e icacia.
Cando os nanomedicamen os son adminis ados no co po eñen que supe a unha
se ie de ba ei as biolóxicas, isiolóxicas e mic oambien ais pa a chega de manei a e icaz
ao luga de acción e ealiza a súa ac i idade e apéu ica. Es as ba ei as, que dependen
p incipalmen e da ía de adminis ación e do ipo e g ao de en e midade, poden a ec a
á es abilidade e biodispoñibilidade dos nanomedicamen os, polo que é indispensable
coñecelas en de alle pa a ealiza un deseño óp imo da o mulación. Aínda que é ce o
que hai nanomedicamen os ap obados pa a di e sas u as de adminis ación, a que máis
Resumo in ex enso
49
in e ese xe ou, e polo an o a máis es udada, é a ía in a enosa. Nes e con ex o, o g ao
de in e acción coas ba ei as mencionadas, o pe co ido e o compo amen o dos
nanomedicamen os unha ez adminis ados no co po depende esencialmen e das
ca ac e ís icas des e, é dici , da súa o ma, amaño, ca ga supe icial e composición. Po
exemplo, a o ma xeomé ica das nanopa ículas xoga un papel impo an e no p oceso
de ex a asación do o en e sanguíneo, sendo aquelas con o ma non es é ica as que
eñen mello capacidade de des ia se da ci culación pa a adhe i se ó aso sanguíneo. No
que se e i e ó amaño e ca ga supe icial das nanopa ículas, es as p opiedades poden se
decisi as pa a de e mina di e sos pa áme os, como o empo de ci culación en sangue,
a cap ación en ó ganos como ígado ou bazo e mesmo a capacidade de acumulación no
umo . Así mesmo, os ma e iais cos que son ab icadas as nanopa ículas poden a ec a o
seu compo amen o in i o, especialmen e no empo de ci culación, xa que se obse ou
que as nanopa ículas íxidas, como as nanopa ículas ino gánicas, son eliminadas do
o en e sanguíneo de manei a máis empe á que nanopa ículas o gánicas.
Todas es as a iables poñen en alo que un de allado seguimen o das
nanopa ículas, coñecendo o pe co ido desde que se adminis an a a o seu luga de
acción, sexa un dos p incipais desa íos pa a consegui unha co ec a anslación á clínica.
Nes e con ex o, as écnicas de imaxe non in asi a o ecen g andes posibilidades, dado
que unha combinación das nanopa ículas con axen es de imaxe pe mi i ía a a aliación
a empo eal da a macociné ica dos nanomedicamen os, da súa acumulación no luga de
acción e mesmo a a aliación do e ec o e apéu ico. Tan o na p ác ica clínica como na
in es igación p eclínica exis en di e sas écnicas de imaxe non in asi a, e cada unha delas
en p opiedades especí icas ú iles pa a di e en es aplicacións. Po exemplo, a imaxe
Resonancia Magné ica (MRI, polas súas siglas en inglés) des aca pola súa al a esolución
espacial capaz de p opo ciona in o mación ana ómica p ecisa e oi amplamen e
u ilizada pa a segui a biodis ibución de nano á macos, a alia a libe ación das
moléculas e apéu icas e de e mina a súa acción. Dou a banda, as écnicas de imaxe
nuclea o ecen g andes an axes g azas á súa ex ema sensibilidade e á súa na u eza
cuan i a i a, pe mi indo ob e imaxes de co po en ei o da biodis ibución de
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nanomedicamen os, e mesmo, o on u ilizadas pa a a es a i icación e selección de
pacien es suscep ibles a a amen o, abo dando así a nanomedicina de p ecisión.
Ademais do seguimen o in i o, exis en écnicas ex i o que esul an de g an u ilidade
pa a a ca ac e ización a ni el celula e in acelula dos nanomedicamen os, po exemplo,
pa a de e mina poboacións celula es esponsables da cap ación de nanopa ículas ou
pa a es uda os mecanismos de in e nalización celula .
Sen dúbida algunha, a a aliación in i o é un pa áme o c ucial pa a a anslación
de nanomedicamen os, con odo, an es de chega a iso, as nanopa ículas i e on que se
deseñadas desde unha pe spec i a anslacional, é dici , co obxec i o de log a chega
algún día a p ác ica clínica. Is o supón a u ilización de ma e iais biodeg adables,
biocompa ibles, non inmunoxénicos e alcanzables (en e mos de cus os) no p oceso de
p epa ación. Ademais, es e p oceso en que se sinxelo e ácil de escala sen comp ome e
a es abilidade nin as p opiedades das nanopa ículas. Con odo is o en men e, o noso
g upo de in es igación desen ol eu ecen emen e unha no a ecnoloxía que en g an
po encial pa a se u ilizada no campo da nanomedicina. T á ase de nanoemulsións
lipídicas, compos as p incipalmen e po dous compoñen es na u ais, Vi amina E, un
excipien e designado como GRAS (do inglés, Gene ally Recollized as Sa e) e
es ingomielina, un dos compoñen es maio i a ios das memb anas celula es. Es as
o mulacións, denominadas en e mos xe ais nanoemulsións de es ingomielina,
demos a on unha g an e sa ilidade pa a a asociación de di e sas moléculas
e apéu icas e eñen g an po encial pa a o a amen o di e sos ipos de canc o, como o
canc o de mama, o canc o de colon e o de pánc eas. Con odo, no que espec a ao seu
compo amen o in i o, aínda es á po explo a .
En is a aos an eceden es desc i os, o p incipal obxec i o des a ese consis e na
a aliación do compo amen o in i o das nanoemulsións de es ingomielina a a és de
di e en es écnicas e di e sas u as de adminis ación, co in de achega no a in o mación
que axude a de e mina o seu po encial en nanomedicina. Pa a iso, p oponse a asociación
de axen es de con as e pa a o seu seguimen o po MRI, de adioisó opos pa a o seu
Resumo in ex enso
51
seguimen o po imaxe nuclea e de luo ó o os pa a a de e minación ex i o da
biodis ibución a ni el celula en modelos de canc o de pulmón non mic ocí ico.
En p imei o luga , encapsulá onse nanopa ículas supe pa amagné icas de e i a
de manganeso no núcleo das nanoemulsións de es ingomielina, c eando nanoemulsións
magné icas capaces de ac ua como axen es de con as e nega i o e de aumen a
signi ica i amen e a sensibilidade da esonancia magné ica. Pa a iso, p imei o
sin e izá onse e ca ac e izá onse as nanopa ículas de e i a de manganeso,
de e minando a súa composición, amaño, mo oloxía e as súas p opiedades
supe pa amagné icas. A con inuación, es as pa ículas ecub í onse con Vi amina E, o o
acei e u ilizado pa a a o mulación das nanoemulsións, a o ecendo así a súa
encapsulación.
As nanoemulsións magné icas p epa á onse polo mé odo de inxección de e anol,
un mé odo que equi e dun único paso e que pe mi iu ob e o mulacións homoxéneas,
cun amaño meno de 200 nm e es ables, dunha manei a al amen e ep oducible. Así
mesmo, es as nanoemulsións magné icas ca ac e izá onse a ni el mo olóxico e
con i mouse a p esenza das nanopa ículas magné icas no seu in e io . Finalmen e,
demos ouse a habilidade das nanoemulsións magné icas pa a ac ua como axen es de
con as e nega i o en esonancia magné ica. Nes e con ex o, cabe des aca que as
nanoemulsións magné icas p esen a on unha maio elaxi idade ans e sal en
compa ación coas pa ículas magné icas lib es.
Ademais, es udouse a biocompa ibilidade das nanoemulsións magné icas in i o.
Pa a iso, inxec á onse po ía in a enosa en a as Sp ague-Dawley e medí onse alo es
de bioma cado es indica i os de dano hepá ico e enal en di e en es empos, sen obse a
ningunha di e enza con espec o aos animais con ol, e con i mándose así, que es as
nanoemulsións non p esen an oxicidade ás doses es udadas e poden se u ilizadas pa a
a súa a aliación en animais. Po úl imo, con i mouse que as nanoemulsións se poden
de ec a de manei a e icaz po esonancia magné ica an o in i o como ex i o,
obse ando unha boa sinal nega i a no ce eb o as inxección in apa enquimal e unha
acumulación p incipalmen e en ígado e iles, as inxección in a enosa. Todo iso
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pe mi iunos concluí que as nanoemulsións de es ingomielina se poden op imiza
acilmen e pa a ac ua como axen es de con as e, dando luga a nanoemulsións
magné icas biocompa ibles, cunha al a capacidade de xe a un con as e nega i o e que,
ademais, poden moni o iza se po MRI u ilizando doses moi baixas, polo que son
capaces de aumen a a sensibilidade da écnica, o que se conside a a súa p incipal
limi ación.
En segundo luga , p opúxose op imiza as nanoemulsións de es ingomielina pa a
pode moni o iza a súa biodis ibución u ilizando nes e caso as écnicas de imaxe
nuclea , Tomog a ía po Emisión de Posi ones (PET, polas súas siglas en inglés) e
Tomog a ía Compu a izada de Emisión Mono o ónica (SPECT, polas súas siglas en
inglés), que p esen an ce as an axes con espec o á imaxe po MR, como mello
sensibilidade e maio acilidade pa a adqui i imaxes de co po en ei o. Pa a iso,
escolleuse o pa de adioisó opos galio-68 e galio-67, dado que o p imei o é un emiso de
posi óns (PET) e o segundo é un isó opo emiso gamma (SPECT). Ademais, ambos eñen
pe íodos de semidesin eg ación moi dispa es, sendo de 68 minu os pa a o galio-68 e 3,3
días pa a o galio-67, o que p opo ciona unha g an e sa ilidade pa a a alia in i o as
nanoemulsións a di e en es empos, dependendo da aplicación pa a a que se deseñen.
Pa a consegui un adioma caxe e icaz das nanoemulsións de es ingomielina,
cómp e inco po a axen es quelan es na súa supe icie, é dici , moléculas que o man
complexos quela o con o adionucleido. Con es e in, seleccioná onse dous quelan es, o
DTPA e o NOTA, e co obxec i o de man e un mé odo de p epa ación sinxelo e nun só
paso, u ilizá onse de i ados lipídicos des as moléculas. Is o pe mi iu a súa inco po ación
na supe icie das nanoemulsións no momen o da súa p epa ación e deu luga a
nanoemulsións homoxéneas e es ables. Ambas o mulacións ma cá onse con galio-68
e icazmen e, aínda que é ce o que a ma caxe das nanoemulsións que con iñan NOTA
esul ou se máis es able que o de DTPA. La biodis ibución de ambas as nanoemulsións
puido non obs an e a alia se an o in i o, median e a adquisición de imaxes PET, como
ex i o, cuan i icando a adiac i idade nos ó ganos dos a os. Ambos esul ados, aínda
que con impo an es di e enzas, mos a on a cap ación ípica da maio ía das
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53
nanopa ículas, é dici , ígado e bazo, aínda que amén se obse ou acumulación no
co azón, o en e sanguíneo, pulmóns, iles e exiga. A p esenza de nanoemulsións no
o en e sanguíneo con i mou a necesidade de u iliza adionucleidos con empos de
semidesin eg ación máis longos pa a es uda o seu compo amen o in i o, polo que se
selecciona on as nanoemulsións modi icadas con NOTA pa a adioma calas con galio-
67 e adqui í onse imaxes a a 48 ho as as a inxección. Así mesmo, u ilizando a écnica
SPECT, a aliouse se a inco po ación de políme os hid o ílicos na supe icie das
nanoemulsións, como o ácido hialu ónico (HA) e o polie ilenglicol (PEG), pode ían
inc emen a o empo de ci culación en sangue, ob éndose esul ados posi i os no caso
das o mulacións PEGiladas. En esumo, a capacidade de pode modula an o o
adioma caxe, a écnica de imaxe como o empo de ci culación das nanoemulsións, pon
de mani es o o po encial que eñen es es sis emas de es ingomielina pa a adap a se
acilmen e ás necesidades que poidan equi i di e sas aplicacións biomédicas.
Aínda que, como se mencionou an e io men e, a ía in a enosa é a máis u ilizada
no desen ol emen o de nanomedicamen os pa a o a amen o de en e midades como o
canc o, exis en ou as ías de adminis ación que poden se in e esan es pa a a a
pa oloxías máis locais, como a i e euma oide ou a os eoa i e. En conc e o, pa a o
a amen o des e ipo de en e midades a ía in a-a icula pe mi e a libe ación selec i a
de á macos no seu luga de acción, á ez que educe a exposición sis émica e minimiza
os e ec os secunda ios ad e sos que pode ían causa ao se adminis ados po ou as ías.
Sen emba go, un dos maio es p oblemas que se an sol en a é a b e e pe manencia na
a iculación debido á cons an e eno ación do líquido sinon ial, aspec o no que a
nanomedicina pode achega solucións e ec i as. Tendo is o en men e, expuxé onse dous
obxec i os pa a a ealización do seguin e capí ulo. En p imei o luga , p opúxose u iliza
as nanoemulsións de es ingomielina pa a mello a a e ención na a iculación dun
pép ido senolí ico que en po encial pa a o a amen o da os eoa i e. En segundo luga ,
pa a de e mina es a e ención, p opúxose ace un seguimen o po PET, ap o ei ando a
súa na u eza cuan i a i a.
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Pa a abo da es es obxec i os, en p imei o luga , modi icouse o pép ido senolí ico
de in e ese cunha cadea lipo ílica, acili andoa súa pos e io inco po ación ás
nanoemulsións, e con de e oxamina (DFO), un que pe mi e a ma caxe des a biomolécula
co adionucleído zi conio-89. An es de p ocede ao adioma caxe, p epa á onse e
ca ac e izá onse as nanoemulsións deco adas co pép ido senolí ico, ob endo
o mulacións ca iónicas, cun amaño meno de 200 nm e es ables en medios biolóxicos.
A con inuación, ealizouse a adioma caxe do pép ido e a p epa ación de nanoemulsións
ca gadas con es e. Tan o o pép ido ma cado como as nanoemulsións asociando es e,
p esen a on ele ada pu eza adioquímica e boa es abilidade en líquido sinon ial e plasma
sanguíneo. Po úl imo, p ocedeuse a inxec a na a iculación da odilla de a as Sp ague-
Dawley an o o pép ido adioma cado na súa o ma lib e como asociado ás
nanoemulsións e a aliouse a súa biodis ibución adqui indo imaxes du an e 14 días e
cuan i icando a sinal de ac i idade, an o do xeonllo inxec ado, como aquela p esen e
nou os ó ganos. Os esul ados con i ma on que as nanoemulsións de es ingomielina
son capaces de pe manece na a iculación un empo máis p ologado que o pép ido e,
que mos ou unha eliminación máis ápida, cunha g an acumulación en ígado e iles. En
conxun o, es e capí ulo po encia a e sa ilidade das nanoemulsións de es ingomielina e
con i ma que poden e po encial pa a a amen o de en e midades in lama o ias.
Ademais, dado que se a a dun pép ido e apéu ico, ac ua ían como sis emas
e anós icos, onde se combina imaxe e e apia nunha soa pla a o ma.
En is a dos esul ados ob idos, no cua o capí ulo quixemos es uda a
biodis ibución das nanoemulsiones de es ingomielina a longo p azo, pe o, nes e caso,
u ilizando de no o a ía in a enosa e ma cando as nanopa ículas, en luga das
biomoléculas, con zi conio-89. É dici , nes e capí ulo quíxose abo da un seguimen o do
compo amen o in i o das nanoemulsións a empos máis longos que os u ilizados no
capí ulo II e ma cándoas con adionucleidos PET en luga de SPECT. Con es e obxec i o
en men e, a aliouse a conxugación do quelan e de e oxamina ás nanoemulsións de
es ingomielina a a és de es es a exias di e en es. En p imei o luga , le ouse a cabo
unha eacción química pa a asocia a DFO na supe icie das nanoemulsións. Pa a iso, a
Resumo in ex enso
55
DFO modi icada cun g upo iso iociana o íxose eacciona coas nanoemulsións que
con iñan un PEG lipídico uncionalizado cunha amina (DSPE-PEG-NH2). T as unha
ho a de incubación, as nanoemulsions pu i icá onse, cuan i icouse a e icacia de eacción
e ca ac e izá onse as súas p opiedades isicoquímicas. Po ou a banda, le ouse a cabo a
modi icación p e ia do DFO cun lípido (DSPE) e pos e io men e incluíuse co es o dos
lípidos que con o man a memb ana das nanoemulsións no momen o da súa p epa ación.
Po úl imo, as comp oba que pa e da asociación obse ada na es a exia de eacción
non e a especí ica, alo ouse a encapsulación de DFO sen modi ica no núcleo ou na
memb ana das nanoemulsións de es ingomielina.
Todas as es a exias de on luga a nanoemulsións monodispe sas e con ca ga
supe icial nega i a, sen g andes di e enzas en e elas. Así mesmo, os adioma caxes con
zi conio-89 esul a on se e icien es, adioquímicamen e pu os e es ables. Po úl imo,
u ilizando as nanoemulsións esul an es da p imei a es a exia, ealizouse un seguimen o
po PET du an e 8 días, as os cales se obse ou que as nanoemulsións p ac icamen e se
elimina an. des aca que, ao compa a a biodis ibución con zi conio-89, pódese concluí
que a ma caxe ob ida é moi es able, dado que non se obse a cap ación debida á
libe ación do adionucleido in i o. Po odo is o, aínda que es es son esul ados
p elimina es e necesí ase máis in es igación nes e eido, demos un paso máis ca a a unha
mello comp ensión do compo amen o in i o das nanoemulsións de es ingomielina.
Po úl imo, e dado que as nanoemulsións de es ingomielina u ilizá onse
p incipalmen e en in es igación oncolóxica, quixo a alia a capacidade des as
nanoemulsións de acumula se en umo es de canc o de pulmón non mic ocí ico e a
posibilidade de chega a ipos celula es especí icos, en conc e o células umo ais
diseminadas posi i as pa a un bioma cado especí ico e , esponsables do p oceso de
o mación de me ás ase. Pa a iso, en luga de seguimen o po imaxe, le ouse a cabo unha
biodis ibución ex i o po ci ome ía de luxo as ma ca as nanoemulsións cun
luo ó o o e emp ega di e sos an ico pos pa a dis ingui cada poboación celula
Pa a op imiza a cap ación umo al íxose uso dunha écnica ecen emen e desc i a,
que consis e na coadminis ación dos nano á macos de in e ese con "po enciado es da
SANDRA DÍEZ VILLARES
56
acumulación", dando luga así a al as doses de nanopa ículas (po encima dun illón).
Es es po enciado es son nanopa ículas non óxicas e biodeg adables, xe almen e
liposomas, cuxa unción consis e en se cap adas polo sis ema e iculoendo elial e
acumuladas no ígado, de o ma que ó log a unha sa u ación pa cial se educe a
cap ación dos nanomedicamen os de in e ese e, po an o, se a o ece a súa acumulación
no umo . No noso caso, dada a biocompa ibilidade das nanoemulsións de
es ingomielina, quixemos u iliza es as o mulacións, polo que es udamos se al as doses
de nanoemulsións pode ían da luga a maio acumulación en umo es de xenoinxe o
de i ados de pacien es (PDX). Ademais, u ilizando nanoemulsións uncionalizadas cun
ap áme o selec i o pa a TAS1R3, un bioma cado elacionado coa p og esión de
me ás ase do canc o de pulmón non mic ocí ico descube o p e iamen e polo noso
labo a o io es udouse a biodis ibución e a capacidade de chega a células umo ais
diseminadas nos pulmóns en modelos animais con di e en e ca ga me as ásica ( modelos
de a ón de xenoinxe o de i ados de células umo ais ci culan es, CTCs, e de células
umo ais diseminadas, DTCs, CDX e DDX espec i amen e). Obse ouse que a
acumulación das nanoemulsións en umo p ima io, é modelo-dependen e, e que,
ademais, den o do umo , a dis ibución das nanoemulsións no mic oambien e umo al
amén é di e en e dependendo do modelo. Obse ouse amén que as nanoemulsións son
capaces de chega á poboación TAS1R3 posi i a e, mesmo, ás células umo ais
diseminadas, o que pon de mani es o o seu po encial aplicación pa a o desen ol emen o
de u u as e apias selec i as pa a o a amen o de canc o de pulmón non mic ocí ico en
e apas a anzadas.
En esumo, op imizá onse as nanoemulsións de es ingomielina pa a o seu
seguimen o po écnicas de imaxe como MRI, PET e SPECT. Ademais, amén se
in es igou a súa dis ibución a ni el celula , dando luga a un coñecemen o in eg al do
compo amen o in i o des as o mulacións. Cabe des aca a e sa ilidade e o po encial
que p esen an pa a di e en es aplicacións, desde imaxe neu olóxica, as se
adminis adas po ía in apa enquimal, en e midades in lama o ias, ala súa
adminis ación in a-a icula , a a e apias oncolóxicas, as adminis a se de manei a
In oduc ion
63
Among he di e se ypes o nanopa icles, lipid-based and polyme -based
nanomedicines a e he mos p edominan ca ego ies and comp ise he 29% and 32% o
he ma ke , espec i ely, ollowed by nanoc ys als and ino ganic nanopa icles (Figu e
3a). Rega ding he applica ion ield, ma ke ed nanomedicines a e mainly ocused on
oncology, accoun ing o nea ly he 24% o he o al (Figu e 3b).10 Howe e ,
nanopa icles ha e also shown hei he apeu ic e icacy in o he sec o s such as
hema ology, in ec ions, neu ological p oblems, and some ha e been also app o ed o in
i o imaging.10,12
Figu e 3. Globally app o ed nanomedicines ca ego ized by (a) pa icle ype and (b) clinical
applica ion. Da a om Halwani A.10
Besides he abo emen ioned nanomedicines, he e is a la ge numbe o
nanopa icles in clinical ials, and i is con inuously g owing e e y yea o a wide ange
o applica ions.11 In ac , based on ecen ly epo ed s a is ics, he global nanomedicine
ma ke size was es ima ed o wo h USD 159.53 billion in 2021, and by 2030, i is
p ojec ed o hi USD 427.18 billion, which e lec s he impo ance o nano echnology-
based pha maceu icals in he upcoming u u e.13 Al hough he majo i y o he
nanop oduc s in clinical p ocess a e new e sions o p e iously app o ed d ugs, since
(b)(a)
Oncology
24%
+Hema ology
15%
+An i-
in ec i e
9%
Neu ology 9%
Bone-subs i u e
6%
Me abolic 5%
Au oimmune 5%
Vaccines 5%
In lamma o y 4%
Oph halmic 4%
Diagnos ic 2% Miscellaneous
13%
Lipid-based
NPs
29%
Polyme NPs
32%
Nanoc ys als
25%
Ino ganic
NPs 10%
Dend ime s 1%
P o ein-based
NPs 3%
SANDRA DÍEZ VILLARES
64
2019 mo e han 35 new nano o mula ions en e ed in clinical ials.9 As an o e iew o
he pipeline, an icance nanomedicines a e s ill in he lead o clinical esea ch, ollowed
by an imic obial nanod ugs.14 In addi ion, he impac o COVID-19 accines has
p omp ed he design o no el mRNA accines based on lipid nanopa icles.15,16
In e es ingly, he e has been a ecen ise in clinical esea ch s udies o mo e sophis ica ed
nanosys ems, known as nex -gene a ion, which can in eg a e mul iple unc ions such as
ac i e a ge ing, d ug con olled elease, combina ion he apies o e en he anos ic
app oaches.17 In his sense, he e is a clea end in mo ing o wa d o he
implemen a ion o p ecision nanomedicine, whe e no only mul i unc ional
nano he apeu ics would be used, bu also imaging-guided nanopa icles o
nano he anos ics would play a c ucial ole o ecognize he ea ly s ages o a disease,
de e mine he bes he apeu ic d ug sui ed o a speci ic g oup o pa ien s and, he e o e,
allow pa ien s a i ica ion o pe sonalized ea men s.18
E en hough he use o nanopa icles in he clinical p ac ice is undeniable and he
u u e end can be s ill encou aging, he u h is ha he ansla ion a e o
nanomedicines is less han 10%, meaning ha he e is a huge dispa i y be ween he
numbe o p eclinical p oduc s and hose a ailable o pa ien s.19 This gap can come om
di e en ac o s such as he impo an ansla ional di e ences be ween animal models
and humans, he he e ogenei y amongs pa ien s o he design o low ansla ional
nanopa icles ega ding in i o ins abili y, cos -e ec i eness o di icul ies in he scaling-
up p ocess.20,21 Howe e , he mos impo an ac o ha hampe s nanomedicine
ansla ion a ises om a lack o unde s anding o he nanopa icles' in i o beha io .22
The e o e, he nex sec ions a e in ended o p o ide iew o he cu en s a e o he a
ega ding he in i o beha io o nanopa icles and o e iew he echniques a ailable o
e alua e hei beha io in o de o b idge he gap be ween bench op and bedside
nanomedicine.
In oduc ion
65
2. IN VIVO FATE OF NANOMEDICINES: A GENERAL OVERVIEW
When nanopa icles a e adminis e ed in o he body, hey encoun e some biological,
physical, and mic oen i onmen al ba ie s ha could limi hei s abili y, ci cula ion
ime, and si e-speci ic bioa ailabili y, causing a lack o p ope he apeu ic ou comes. The
ex en o in e ac ion wi h each ba ie is mainly dependen on he nanopa icle design
and he adminis a ion ou e, besides o, o cou se, he ype o disease and he disease
p og ession.20
Rega ding he adminis a ion ou e o nanopa icles, a g ea amoun o li e a u e
has been ocused on he s udy o se e al adminis a ion modes and some nanomedicines
ha e been clinically app o ed o o al, sys emic, opical, and local deli e y.7 Among all
he possibili ies, in a enous injec ion is s ill he one ha ecei es he mos a en ion,
since i is he pa h commonly used o an icance d ug deli e y.21 Howe e , depending
on he equi ed applica ion, o he ou es such as o al adminis a ion o in a-a icula
injec ion a e being also widely explo ed in he esea ch scena io. Fo ins ance, o al
deli e y is he leas in asi e ou e ha ensu es he mos pa ien compliance and has
gained much a en ion o he deli e y o biomolecules, wi h many e o s ocused on he
o al deli e y o insulin, among o he s.23 On he o he hand, in a-a icula injec ion is
highly ecommended o he local ea men o in lamma o y diseases including
heuma oid a h i is and os eoa h i is, since in his case, i p o ides a highe he apeu ic
e ec wi h lowe side e ec s compa ed o o al an i-in lamma o y d ug deli e y.24 In all,
nanomedicines should be c i ically and a ionally designed o i hei speci ic applica ion
and adminis a ion ou e, as his design (i.e., composi ion, shape, size, and su ace
cha ge) will go e n he in i o beha io .25
Focusing on in a enous injec ion, he ollowing pa ag aphs a e aimed o p o ide a
gene al o e iew o he ba ie s ha nanopa icles migh con on du ing all hei in i o
pa h, om he injec ion poin o he a ge si e. Mo eo e , his sec ion is ocused on how
he nanopa icle physicochemical p ope ies a ec hese in e ac ions and how hey can
be modula ed o enhance he ba ie c ossing e iciency.
SANDRA DÍEZ VILLARES
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2.1. Bloods eam and clea ance
The de elopmen o nanopa icles wi h enhanced ci cula o y p ope ies and low blood
clea ance can aid in si e-speci ic deli e y. To ob ain desi able ou comes, he e a e some
gene al design ea u es ega ding nanopa icle physicochemical p ope ies ha ha e
been led a e decades o esea ch (Figu e 4). Fo ins ance, i is well-known ha
nanopa icles smalle han 10 nm a e p omp ly clea ed h ough he kidneys, while
nanopa icles bigge han 200 nm can ac i a e he complemen sys em. In e ms o shape,
non-sphe ical nanopa icles (e.g. nano ods, ilomicelles, o nanodisks) p esen longe
ci cula ion imes han he sphe ical coun e pa s, which esul s om he endency o
hese pa icles o align wi h blood low.26
Figu e 4. Nanopa icle size, shape and su ace cha ge dic a e biodis ibu ion among he di e en
o gans including he lungs, li e , spleen, and kidneys. Adap ed om Blanco e al.26
The main eason behind he ailu e o he nanopa icles o each he si e o ac ion
wi h a su icien he apeu ic dose is due o ea ly cap u e by a ne wo k o phagocy ic cells
(p ima ily mac ophages) ha o m he mononuclea phagocy e sys em (MPS). Fo
example, s i nanopa icles a e clea ed mo e apidly by he MPS han so e
nanopla o ms. Rega ding su ace cha ge, ca ionic nanopa icles easily in e ac wi h he
SIZE SHAPE SURFACE CHARGE
> 150 nm 20 -150 nm < 5 nm 20 -150 nm 20 -150 nm
Li e
Lungs
Spleen
Kidneys
--
-
-
-
-
-
--+
+
+
+++
+
+
In oduc ion
67
nega i e cha ge on he memb ane o mac ophages and a e he ea lies o be clea ed,
ollowed by anionic nanopa icles, while a sligh ly nega i e su ace cha ge seems o be he
op imal o a oid MPS clea ance.27 Once in bloods eam, plasma p o eins such as
complemen , and immunoglobulins adso b on o he su ace o nanopa icles, in a
p ocess called opsoniza ion, and o m a “p o ein co ona”, which can comp omise he
d ug and nanopa icle s abili y.28 The gene a ed co ona is highly dependen on he
nanopa icle physicochemical p ope ies and will de e mine he nanopa icle hal -li e
and i s cap u e by li e and spleen mac ophages.29 In e es ingly, i has been epo ed ha
he co ona can also be o med by dysopsonins, i.e., apolipop o eins (ins ead o opsonins),
and his composi ion can inc ease he nanopa icles ci cula ion ime and, in some
ins ances, leads o deli e y ac oss he blood–b ain ba ie (BBB).30 These di e ences in
biodis ibu ion ha e p omp ed he design o new s a egies ha ins ead o iewing i as
an obs acle, ha ness he p o ein co ona o modula e he in i o beha io o
nanopa icles.23,24
To minimize mac ophage up ake and ex end blood ci cula ion ime, nanopa icles
su ace modi ica ion wi h polyme s is also a s a egy commonly used.26 Al hough o da e
PEGyla ion emains he mos popula coa ing o p o ide s eal h p ope ies, he e a e
impo an conce ns ela ed o he de elopmen o an i-PEG an ibodies a e
adminis a ion, which esul in an immune esponse known as accele a ed blood
clea ance (ABC) phenomenon.33 To o e come hese sho comings, o he s a egies such
as biomime ic coa ings wi h cell memb anes (e.g. pla ele s, ed blood cells (RBC) o
leukocy es) a e being cu en ly esea ched wi h p omising ou comes.34
2.2. Si e-speci ic ex a asa ion and a ge si e accumula ion
A e a p ope ci cula ion ime, nanopa icles mus ex a asa e om he blood essels o
success ully accumula e in he si e o ac ion. To do so, nanopa icles mus la e ally d i
om he blood low owa ds he ascula wall in a p ocess known as ma gina ion.
Se e al aspec s in luence in he adhesion o he ascula walls, and hese a e ela ed o
SANDRA DÍEZ VILLARES
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he hemodynamic p ope ies such as low pa e n, heology, shea s ess and eloci y.25
In his sense, pa icle geome y is a key pa ame e ha go e ns ma gina ion dynamics.
Unlike sphe ical nanopa icles, he non-sphe ical coun e pa s can unde go signi ican
la e al d i due o hei o a ional mo ion unde low wi hou he aid o an ex e nal o ce
and a a a e p opo ional o he nanopa icle aspec a io (Figu e 5a).35 Ex a asa ion is
also dependen o nanopa icle size, whe eby small nanopa icles a e able o c oss
capilla y walls mo e easily compa e o la ge nanopa icles.36
Figu e 5. (a) Nanopa icle low, ma gina ion and adhesi e p ope ies in blood essels a e
dependen on pa icle size and geome y. Adap ed om Blanco e al.26 (b) The EPR e ec scheme
(le ). Recen epo s sugges ed anscy osis h ough in a-endo helial channels o esicles as he
o e a ching mechanism o umo en y ( igh ). Adap ed om de Láza o and Mooney.37
Al hough some non-speci ic o gan biodis ibu ion is gene ally expec ed a e
in a ascula adminis a ion (mainly in li e and spleen), an icance nanomedicines ake
ad an age o he i egula hype ascula i y and inc eased blood essel pe meabili y o
p e e en ially accumula e in umo s. This passi e a ge ing app oach cha ac e is ic o
nanopa icles was e med as enhanced pe mea ion and e en ion (EPR) e ec by Maeda
e al. and was exploi ed o se e al yea s in cance nanomedicine esea ch (Figu e 5b,
le ).38 Rega ding he impac o nanopa icle p ope ies in he EPR e ec , he p e e ed
pa icle size alls in o he ange o 10–150 nm, while he su ace cha ge o nanopa icles
does no appea o a ec passi e a ge ing based on his e ec bu o o he cellula
(b)(a)
Tigh
junc ion
Endo helial
cell
Tumo
cell
Nanopa icles
EPR e ec T anscy osis
In oduc ion
69
mechanisms.39 Al hough EPR e ec can enhance he umo accumula ion o
nanopa icles, some cha ac e is ics o he umo mic oen i onmen such as he ele a ed
in e s i ial luid p essu es may s ill p o e de imen al o hei accumula ion in o
umo s.40 These limi a ions ha e been widely explo ed and epo s e ealed ha only a
0.76% (median) o nanopa icles a e success ully deli e ed o solid umo s.41–43 Mo eo e ,
he ele ance in humans is ques ionable, and he EPR e ec has been ound o be pa ien
and umo ype dependen .44 Besides, ecen epo s suppo ha he EPR e ec was
o e es ima ed, and he nanopa icles en y in o solid umo s is go e ned by speci ic
endo helial cells mo e han passi e di usion (Figu e 5b, igh ).43,45 The e o e, g ea
amoun o li e a u e on his opic has aimed o imp o e nanomedicines umo up ake o
acili a e hei ansla ion and mos p omising s a egies include, bu a e no limi ed o
wo king abo e a dose h eshold ecen ly desc ibed, o he use o speci ic ligands o ac i e
a ge ing app oaches.46,47
The in acellula ba ie is he inal biological hu dle agains deli e y o
he apeu ics. As expec ed, nanopa icle cha ac e is ics can in luence in cellula up ake
and endosomal compa men aliza ion.21,25 Fo example, nega i ely cha ged
nanopa icles can su e om epulsi e elec os a ic o ces due o cell memb anes,
whe eas ca ionic nanopa icles seem o up ake be e by cells. Howe e , high ca ionic
cha ge densi ies a e ela ed wi h cy o oxic e ec s. In addi ion, size and shape
cha ac e is ics can a ec he ac i e up ake mechanisms o c oss he cell memb anes i.e.,
pinocy osis, ca eolin-media ed endocy osis, cla h in-media ed endocy osis, o
independen endocy osis, whe eby de e mining he in acellula compa men s.21
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3. TECHNIQUES TO EVALUATE THE IN VIVO BEHAVIOR OF
NANOMEDICINES
As p e iously de ailed, when nanomedicines a e adminis e ed in o he body, hey
need o o e come se e al ac o s and limi a ions o speci ically each he a ge si e and
p oduce he desi ed he apeu ic e ec . The e o e, unde s anding he in i o a e o
nanomedicines is one o he p incipal challenges hinde ing e ec i e clinical ansla ion.
In his sense, he combina ion o nanopla o ms wi h non-in asi e imaging modali ies
can signi ican ly imp o e he ansla ion a e, since hese echniques o e a ac i e
possibili ies o moni o he nanopa icle pha macokine ic beha io , he in i o s abili y,
he d ug deli e y p ocess, he he apeu ic e ec , o he a ge si e accumula ion, all o
hem in a eal- ime manne .48,49 Mo eo e , in clinical p ac ice, imaging-guided
nanomedicines would play a powe ul ole since hey can acili a e he iden i ica ion and
selec ion o he pa ien s who would bene i he mos om a nano he apeu ic plan,
enabling pe sonalized medicine.18,50
Medical imaging echniques include magne ic esonance imaging (MRI), X- ay
compu ed omog aphy (CT), op ical imaging (OI), ul asound imaging (US),
pho oacous ic imaging (PAI), and he nuclea imaging modali ies single pho on emission
compu ed omog aphy (SPECT) and posi on emission omog aphy (PET).51 Wi h
ad an ages and d awbacks, each modali y has i s in insic p ope ies and can be use ul
o e y di e en speci ic applica ions in e ms o imaging-guided nanomedicine
e alua ion (Figu e 6).52 Fo example, MRI and CT s and ou o hei high spa ial
esolu ion o p o ide accu a e ana omical in o ma ion, whe eas PET excels o i s
sensi i i y and quan i a i e na u e. Gene ally, o o e come some o he limi a ions ela ed
o a echnique, wo modali ies can be combined in o a single ins umen , a e m known
as mul imodal imaging, and he e o e complemen a y in o ma ion can be acqui ed o
a o d an exhaus i e cha ac e iza ion.53 Pa icula ly, PET/CT has become a s anda d
modali y ha allows syne gis ic accu a e ana omical in o ma ion o he PET ace
loca ion wi h high spa ial esolu ion.54 Howe e , o he combina ions such as PET/MRI,
PET/OI o MR/PAI ha e been widely s udied in p eclinical esea ch.55–57 In he ollowing
In oduc ion
71
pa ag aphs, he common imaging modali ies will be b ie ly desc ibed, wi h a special ocus
on how hey can be used o imp o e he unde s anding o nanopha maceu icals’ in i o
beha io .
Figu e 6. Summa y o he p ope ies o imaging modali ies discussed in his sec ion. Da a om
Pellico e al.69
3.1. Magne ic esonance imaging
MRI is a non-in asi e and non-ionizing echnique widely used in clinical p ac ice ha
o e s impo an ea u es, such as excep ional spa ial esolu ion, high pene a ion dep h,
and excellen in insic so - issue con as allowing he acquisi ion o h ee-dimensional
ana omical images.58 This echnique elies on he magne ic p ope ies and spin
cha ac e is ics o ac i e nuclei, mos commonly hyd ogen om wa e molecules, bu
o he nuclei such as 19F-MRI ha e also been s udied. Unde an ex e nal magne ic ield,
he spin p o ons can be aligned ei he pa allel o an i-pa allel o his ield. Then, a e
applica ion o a adio equency pulse, he spin p o ons o ien a ion is dis u bed, and i is
SANDRA DÍEZ VILLARES
72
hei elaxa ion ime o he equilib ium s a e wha is measu ed and allow image con as .
This p ocess can gene a e wo independen elaxa ion pa ame e s known as he
longi udinal (T1) and ans e sal (T2) elaxa ion imes. MRI con as agen s can sho en
hese elaxa ion imes and, depending on which one a e capable o modula e, hey can
p o ide T1-weigh ed (posi i e, make he image b igh e ) o T2-weigh ed (nega i e, make
he image da ke ) con as .51 Al hough he main limi a ion o s anda d MRI is i s low
sensi i i y, MR con as agen s can signi ican ly imp o e i and allow he use o his
modali y a he molecula le el.59 As ep esen a i e examples based on nanoma e ials, he
mos common T1-weigh ed con as agen s a e Gadolinium (Gd3+)-chela ed
nanopa icles o pa amagne ic nanopa icles con aining Manganese (Mn2+). Rega ding
T2-weigh ed con as agen s, i on-based compounds, especially supe pa amagne ic i on
oxide nanopa icles (SPIONs) s and ou as enhance s o he nega i e con as signal and
ha e been al eady FDA-app o ed o clinical MRI.60 Rema kably, no only pu e i on
oxide ma e ials ha e eno mous po en ial, bu also dopped spinel e i es as o example
manganese e i e nanopa icles (MnFe2O4) showed p ope cha ac e is ics o his aim.61
When i comes o nanomedicine in i o acking, MR has been p o ed o be a
sui able echnique o moni o ing he d ug elease in a empo ally and spa ially
ashion.62–64 In his sense, Wang e al. de eloped magne osomes loaded wi h doxo ubicin
o pho o he mal- and chemo he apy o b eas cance and, a e i. injec ion in xenog a
mice models, hey ollowed-up he doxo ubicin elease by co ela ing he d ug le els wi h
T2-con as enhancemen .63 In addi ion, in a clinical s udy, nanomedicine-based MRI
showed po en ial in imaging-guided su ge y by enabling he p ecise localiza ion o
sen inel lymph nodes, he i s eached si e o me as a ic cance cells, using SPIONs as
MR con as agen s.65 In o he in e es ing clinical pilo s udy, Fe umoxy ol® (FDA-
app o ed o anemia ea men ) was used as MR con as agen o p edic he
nano he apy esponse o Oni yde® in pa ien s wi h ad anced solid umo s.66,67 Al hough
MR whole-body imaging is mo e challenging han o he imaging echniques, i was also
used o analyze he biodis ibu ion and pha macokine ic p o ile o nanomedicines.68
In oduc ion
79
as pa o Ma qibo®, an an icance nanomedicine.14 I should be no iced ha al hough
Ma qibo® has been wi hd awn in 2022, he easons we e no ela ed o o mula ion o
oxici y p oblems, bu we e due o di icul ies in pa ien ec ui men o pos -app o al
clinical s udies.110 On he o he hand, Vi E is an an ioxidan widely used in biomedicine,
wi h a well-known sa e y eco d and ecognized as GRAS-lis ed excipien .111 Thus,
sphingomyelin nanoemulsions (SNs) a e sa e-by-design, biodeg adable, and hei
biocompa ibili y has been al eady p o ed in i o and in i o.112 In addi ion, hey a e
highly s able unde s o age condi ions o e long pe iods o ime and also in di e en
biological media such as human plasma o simula ed gas ic luid, which could indica e
ha he ca go would no be p ema u ely eleased in his luids. Rega ding he
manu ac u ing p ocess, SNs a e p epa ed by e hanol injec ion, a low ene gy, cos -
e ec i e, and simple me hod ha allows he spon aneous o ma ion o monodispe se
nanoemulsions in one-s ep. Impo an ly, his me hodology can be easily scaled-up by
mic o luidic app oaches as desc ibed o o he lipid-based nanosys ems.113
Figu e 9. Ra ional design o sphingomyelin nanoemulsions ollowing he p ope ies ha
nanomedicines mus hold o mee ansla ional pu poses.
Recen epo s demons a ed ha SNs ha e shown g ea e sa ili y o accommoda e
di e en payloads o he po en ial ea men o di e en ypes o cance , such as
colo ec al, b eas and panc ea ic cance .112,114–119 Rema kably, SNs no only e icien ly
NANOMEDICINE DESIGN SPHINGOMYELIN NANOEMULSIONS
Biocompa ible
Biodeg adable
Cos -e ec i e
S able
Scalable
Biocompa ible
Biodeg adable
Cos -e ec i e
S able
Scalable
SANDRA DÍEZ VILLARES
80
encapsula e an icance small d ugs such as e oposide o gemci abine,115,116 bu also a e
able o associa e pep ides o a ge ing pu poses,115,119 and nucleic acids (e.g. miRNA,
pDNA and lncRNA) o gene he apy.114,117,118 As ecommended o nanomedicine
de elopmen ,108 in all hese s udies whe e SNs inco po a e di e en he apeu ic moie ies,
hey we e deeply cha ac e ized in e ms o hei mo phology, physicochemical p ope ies
and colloidal s abili y. Finally, he i s app oach o SNs in i o acking was desc ibed
by Nagachin a e al. and in ol ed he adiolabeling o his lipid nanoemulsions wi h
luo ine-18.120 B ie ly, he adioiso ope 18F was inco po a ed in o a 4-
[18F] luo obenzamido-N-e hylmaleimide ([18F]FBEM) and chemically linked o hiol
su ace-modi ied SNs ob aining adiolabeled nanoemulsions wi h mode a e
adiochemical yields. A e nanopa icle cha ac e iza ion SNs-[18F]FBEM we e
in a enously injec ed in mice and PET s udies success ully e ealed he pha macokine ic
and clea ance pa hways o his lipid nanoemulsions, pa ing he pa h o u he esea ch
in his ield.
The e o e, in he p esen hesis, he main aim is o ailo hese lipid nanoemulsions
wi h imaging agen s o be e explo e hei in i o beha io by di e en imaging
modali ies, and o p opose hei u ili y o speci ic biomedical applica ions, namely
os eoa h i is and me as a ic lung cance . This hesis p o ides aluable in o ma ion
abou he SNs in i o a e and opens a ga e o u he p eclinical esea ch o demons a e
he po en ial o his lipid nanoemulsions as ansla ional nanomedicines.
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Objec i es
97
OBJECTIVES
Based on he backg ound and hypo hesis ou lined, he main goal o his hesis is he
op imiza ion o sphingomyelin nanoemulsions o associa e di e en imaging moie ies
o he e alua ion o hei p eclinical biodis ibu ion h ough di e en echniques, such
as non-in asi e imaging o low cy ome y, and o assess hei po en ial o speci ic
biomedical applica ions.
This goal has been pu sued a e se ing up he ollowing pa ial objec i es (O):
Chap e I
O1. To encapsula e hyd ophobic supe pa amagne ic nanopa icles in o he co e o
sphingomyelin nanoemulsions and o ully cha ac e ize he de eloped magne ic
nanoemulsions.
O2. To e alua e he po en ial o he magne ic sphingomyelin nanoemulsions o
high-sensi i e MRI by de e mining hei ans e se elaxi i y alues and he
abili y o enhance he MR signal.
O3. To de e mine he biodis ibu ion o magne ic sphingomyelin nanoemulsions by
in i o and ex i o MRI.
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98
Chap e II
O4. To de elop and cha ac e ize chela o -modi ied sphingomyelin nanoemulsions,
speci ically designed o nuclea imaging, h ough a simple me hodology using
lipid-de i a i e chela o s.
O5. To adiolabel sphingomyelin nanoemulsions wi h 68Ga and 67Ga unde mild
condi ions and o e alua e hei biodis ibu ion by PET and SPECT imaging.
O6. To modula e he biodis ibu ion o he adiolabeled sphingomyelin
nanoemulsions by coa ing hei su ace wi h hyd ophilic polyme s.
Chap e III
O7. To chemically modi y a senoly ic pep ide o media e he adiolabeling wi h 89Z
and i s u he associa ion wi h sphingomyelin nanoemulsions and hen, o ully
s udy he adiochemical p ope ies o bo h adio ace s.
O8. To p o ide e idence o he po en ial o sphingomyelin nanoemulsions as in a-
a icula pep ide deli e y sys ems o ea men o os eoa h i is by acking he
join e en ion ime and he whole-body biodis ibu ion using quan i a i e PET
imaging.
Chap e IV
O9. To e alua e di e en s a egies o he associa ion o bi unc ional chela o s o
sphingomyelin nanoemulsions o u he 89Z adiolabeling.
O10. To ack he long- e m biodis ibu ion o 89Z - adiolabeled sphingomyelin
nanoemulsions by PET imaging.
Objec i es
99
Chap e V
O11. To p o e in i o and in i o ha he accumula ion o sphingomyelin
nanoemulsions in umo s can be op imized by p iming he li e and blocking
mac ophages up ake.
O12. To s udy he umo up ake and he speci ic cell dis ibu ion o a ge ed
sphingomyelin nanoemulsions as a key s ep owa ds designing no el
he apeu ic app oaches o ea men o me as a ic lung cance .
Chap e I
Manganese Fe i e Nanopa icles Encapsula ed in o Vi amin E /
Sphingomyelin Nanoemulsions as Con as Agen s o
High - Sensi i e Magne ic Resonance Imaging
Adap ed om: Díez-Villa es S. e al. Ad . Heal hc. Ma e . 10, 1–12 (2021).1
ISSN: 2192-2640
Chap e I
103
Manganese Fe i e Nanopa icles Encapsula ed in o Vi amin E /
Sphingomyelin Nanoemulsions as Con as Agen s o High-Sensi i e
Magne ic Resonance Imaging
Sand a Díez-Villa es,†1,2,3, Miguel A. Ramos-Docampo,†4,5 And és da Sil a-Candal,6 Pablo
He ella,6 Abi J. Vázquez-Ríos,1,3Ana B. Dá ila-Ibáñez,7 Ra ael López-López,3,7 Ramón
Iglesias-Rey,6 Ve ónica Salguei iño,* 4,5 Ma ía de la Fuen e*1,3.
1Nano-Oncology and T ansla ional The apeu ics g oup, Heal h Resea ch Ins i u e o San iago de Compos ela
(IDIS), SERGAS, San iago de Compos ela 15706, Spain
2Uni e si y o San iago de Compos ela (USC), San iago de Compos ela 15706, Spain
3Biomedical Resea ch Ne wo king Cen e on Oncology (CIBERONC) Mad id 28029, Spain
4Depa amen o de Física Aplicada, Uni e sidade de Vigo, 36310 Vigo, Spain.
5CINBIO, Uni e sidade de Vigo, 36310 Vigo, Spain
6Clinical Neu osciences Resea ch Labo a o y, Heal h Resea ch Ins i u e o San iago de Compos ela (IDIS),
SERGAS, San iago de Compos ela 15706, Spain
7Roche-CHUS Join -Uni , T ansla ional Medical Oncology G oup, Heal h Resea ch Ins i u e o San iago de
Compos ela (IDIS), SERGAS, San iago de Compos ela 15706, Spain.
ABSTRACT
Magne ic esonance imaging (MRI) is one o he mos powe ul non-in asi e imaging
modali ies used in clinics due o i s g ea spa ial esolu ion and excellen so - issue
con as , hough s ill less sensi i e han o he echniques such as he nuclea imaging
modali ies. This lack o sensi i i y can be conside ably imp o ed wi h he use o con as
agen s based on nanoma e ials. In ecen yea s, esea che s ha e been ocused on he
de elopmen o magne ic nanopa icles, gi en hei ole as enhance s o he con as
signal based on he magne ic esonance. Manganese e i e nanopa icles s and ou , gi en
hei high magne ic suscep ibili y and magne ic so na u e. He ein, 10 nm MnFe2O4
nanopa icles, unc ionalized wi h he na u al an ioxidan i amin E (Vi E-MFO) a e
encapsula ed in o simple, biodeg adable, and non- oxic nanoemulsions (SNs), by a
ep oducible one-s ep me hod ob aining s able 150 nm-sized magne ic nanoemulsions
(Vi E-MFO-SNs). A e encapsula ion, he supe pa amagne ic p ope ies o Vi E-MFO
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a e main ained and MR imaging s udies e eal an ex emely high ans e se elaxi i y o
Vi E-MFO-SNs (652.9 mM−1s−1), 2- old highe han Vi E-MFO alue. Mo eo e , Vi E-
MFO-SNs show g ea in i o biocompa ibili y and good signal a a low dose by in i o
and ex i o MRI, which indica es hei g ea po en ial o biomedical imaging enhancing
he nega i e MR con as and signi ican ly imp o ing he sensi i i y o MRI.
KEYWORDS
Magne ic esonance imaging, supe pa amagne ic manganese e i e nanopa icles,
i amin E, sphingomyelin, nanoemulsions, ans e se elaxi i y.
Chap e I
111
g id. The excess was washed wi h Milli Q wa e and he g id was allowed o d y o e nigh
a oom empe a u e. FESEM images we e acqui ed using a ZEISS FESEM ULTRA Plus,
mic oscope wi h wo de ec o s, STEM and InLens. Cha ac e iza ion o Vi E-MFO-SNs
was done by he same me hod desc ibed abo e o ee MFO. AFM cha ac e iza ion was
pe o med in a Scanning P obe Mic oscopy Mul imode Mul imode 8/Nanoscope V
ope a ing in apping mode. The MFM mode was employed using a Tip Roc < 35 nm
( adius) wi h a CoC coa ing, wo king a a nominal equency o 75-100 kHz and a
nominal coe ci i y o 40 mT. Samples o AFM/MFM we e p epa ed in Millipo e wa e
and allowed o d y o e a mica subs a e o e nigh a oom empe a u e.
2.7. In i o MR imaging
MR imaging was pe o med by aga -based phan oms p epa ed ollowing a p ocedu e
desc ibed elsewhe e.28 Aga solu ion (1.6% w/ ) was used o p epa e a mold wi h se e al
wells. Di e en dilu ions o he nanoemulsions we e p epa ed in o de o ob ain se ial
concen a ions o manganese e i e nanopa icles anging om 0.006 o 0.471 mM Fe.
Then, 100 µL o magne ic nanoca ie s we e mixed wi h 100 µL o aga solu ion,
deposi ed in o aga wells and co e ed wi h he emaining aga solu ion. Fo Vi E-MFO
phan oms dilu ions we e made in e hanol and hen mixed wi h aga solu ion. MR images
we e acqui ed on a B uke Biospec 9.4 T small animal MR scanne (ho izon al bo e
magne wi h 12 cm wide B uke BioSpin) equipped wi h ac i ely shielded g adien s
(440 mT m−1). Fo he acquisi ion o da a, a adio equency esona o (B uke ) was used
as ansmi e - ecei e (quad a u e olume coil 7 cm in diame e ).
T2-weigh ed images we e acqui ed using he mul i-slice mul i-spin-echo (MSME)
sequence wi h 10.26 ms o echo ime, 3 s o epe i ion ime, 16 echoes wi h 10.26 ms echo
spacing, 50 KHz spec al bandwid h, lip angle o 90 °, 14 slices o 1 mm, 1 a e age. T2-
weigh ed images we e ob ained wi h a ield o iew (FOV) o 7.5 cm × 7.5 cm (wi h
sa u a ion bands o supp ess signal ou side his FOV), and a ma ix size o 300 × 300,
gi ing an in-plane esolu ion o 250 µm/pixel and implemen ed wi hou a supp ession.
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Images we e p ocessed, and hei T2-maps we e calcula ed using he so wa e FIJI: Image
J (Rasband W, NIH). The elaxi i y cons an ( 2) was calcula ed as he slope o he cu e
ob ained by i ing he T2−1 alues e sus he Fe concen a ion in mM.
2.8. In i o s udies
Animal expe imen s we e conduc ed in he Clinical Neu osciences Resea ch Labo a o y
o he Uni e si y Clinical Hospi al o San iago de Compos ela (REGA ES 15078 029
2801). All expe imen al animal p ocedu es we e conduc ed unde he p ocedu e
numbe : 15010/2019/004 app o ed by he Animal Ca e Commi ee, acco ding o
Eu opean Union Rules and he Spanish egula ion (86/609/CEE, 2003/65/CE,
2010/63/EU, RD1201/2005 and RD53/2013, RD 53/2013). Sp ague-Dawley a s wi h a
weigh be ween 250-300 g we e used o in i o s udies. In all he expe imen s animals
we e anes he ized placing hem in a s e eo axic ame (S oel ing Co., Wood Dale) unde
se o lu ane anes hesia.
2.9. In i o biocompa ibili y o Vi E-MFO-SNs
Vi E-MFO-SNs (n=4, 2.7 mg lipids mL−1, 471 µM Fe) we e injec ed h ough he ail ein
in anes he ized a s (1 mL, 10.9 mg lipids kg−1, 1.88 µmol Fe kg−1) and hepa ic and enal
oxici ies we e assessed measu ing Glu ama e Oxaloace a e T ansaminase enzyme
(GOT) and C ea inine le els a di e en ime poin s (p io injec ion and 2, 4, 24, 48 h
pos -injec ion). The same olume o PBS was in a enously injec ed as a con ol. A he
selec ed ime poin s, blood was ex ac ed om he ail ain and collec ed in ubes wi h
hepa in (BD Vacu aine ® Hepa in Blood Collec ion Tubes). Then, 32 µL o blood we e
placed in eac i e s ips o GOT ( e : 10745120202 Roche), and c ea inine ( e :
10886874202 Roche) and analyzed in a Re lo on® plus (Roche, Basel, Swi ze land). GOT
le els we e no malized o he baseline le els o each animal.
Chap e I
113
2.10. Ex i o MRI
A e 48 h om PBS/Vi E-MFO-SNs in a enous injec ion (n=4), animals we e
anes he ized and pe used ansca dially wi h 100 mL PBS 0.1 M pH 7.4 and 150 mL 4%
o maldehyde (VWR Chemicals, Leu en, Belgium). O gans (lungs, kidneys, li e , and
spleen) we e ca e ully emo ed and pos ixed by imme sion in 4% o maldehyde un il
MRI analysis. Aga -based phan oms we e p epa ed wi h hese o gans o de e mine T2
elaxa ion imes. MR images we e acqui ed ollowing he p o ocol p e iously desc ibed
o in i o MRI s udies and T2-maps we e calcula ed om T2-weigh ed images. Image
analysis was pe o med selec ing each con ol o gan as a egion o in e es (ROI) and
measu ing a mean o he minimum elaxa ion imes alues. These alues we e ixed as
h eshold and we e applied o he o gans ea ed wi h Vi E-MFO-SNs in o de o
quan i y he di e ences caused by hese pa icles in he elaxa ion imes.
2.11. In i o b ain MRI
Fo b ain MRI Vi E-MFO-SNs, SNs and PBS we e injec ed di ec ly in he pa enchyma o
he animals (n=3, 2.7 mg lipids mL−1, 471 µM Fe) ollowing a p o ocol p e iously
desc ibed.29 B ie ly, a Hamil on sy inge (Hamil on; 10 μL) was illed wi h he
co esponding nanopa icle suspension, SNs (0.109 mg lipids kg−1) o Vi E-MFO-SNs,
(0.109 mg lipids Kg−1, 0.018 µmol Fe kg−1) and a olume o 10 µL was injec ed in he igh
hemisphe e o he b ain a a low a e o 1 µL min−1 o e 10 minu es. Same p ocedu e was
pe o med in he le hemisphe e injec ing 10 µL o PBS. The a s we e placed in an
animal box a e su ge y o eco e ing in a wa m place wi h access o ood.
MRI s udies we e conduc ed on a 9.4-T ho izon al bo e magne (B uke BioSpin)
wi h 12-cm wide ac i ely shielded g adien coils (440 mT m−1), and a combina ion o a
linea bi dcage esona o (7 cm in diame e ) o signal ansmission and a 2 × 2 su ace
coil a ay o signal de ec ion, posi ioned o e he head o he animal, which was ixed
wi h a ee h ba , ea plugs, and adhesi e ape. T ansmission and ecep ion coils we e
ac i ely decoupled om each o he . G adien -echo pilo scans we e pe o med a he
SANDRA DÍEZ VILLARES
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beginning o each imaging session o accu a e posi ioning o he animal inside he
magne bo e. To assess nanopa icles in i o, animals we e scanned ollowing T2-
weigh ed and T2*-weigh ed sequences in MRI o e alua e he p esence and he
dis ibu ion 1 h a e he injec ion. T2*-weigh ed images we e acqui ed using a MGE
sequence wi h a 2.9 ms echo ime, 1.5 s epe i ion ime, 16 echoes wi h 3.28 ms echo
spacing, implemen ed wi h a FA o 30 °, 2 a e ages, 14 slices o 1 mm, and wi h a 19.2 ×
19.2 mm2 FOV, a 192 × 192 image ma ix (iso opic in-plane esolu ion o
100 µm pixel−1). T2-weigh ed images we e acqui ed using a MSME sequence wi h a 9 ms
echo ime, 3 s epe i ion ime, 16 echoes wi h 9 ms echo spacing, implemen ed wi h a FA
o 180 °, 2 a e ages, 14 slices o 1 mm and 19.2 × 19.2 mm2 FOV, a 192 × 192 image
ma ix (iso opic in-plane esolu ion o 100 μm pixel−1). MRI pos -p ocessing was
pe o med using ImageJ so wa e (W. Rasband, NIH, USA).
2.12. S a is ical analysis
All he expe imen s we e pe o med a leas in iplica e. Da a a e exp essed as mean ±
s anda d de ia ion (SD). S a is ical analyses we e calcula ed wi h G aphPad P ism®
so wa e ( e sion 8.0). S uden ’s - es was used o compa e signi ican di e ences
be ween wo g oups. * (p ≤ 0.05), ** (p ≤ 0.01), ***(p ≤ 0.001) was conside ed s a is ically
signi ican .
3. RESULTS AND DISCUSSION
Supe pa amagne ic nanopa icles, MFO, we e ob ained by a chemical
he modecomposi ion o he me al p ecu so s (i.e., manganese and i on
ace ylace ona es) a high empe a u e. Figu e 1 summa izes he mo phological and
s uc u al cha ac e iza ion o he nanopa icles p oduced. The TEM image included in
Figu e 1a shows hei well-de ined sphe ical shape, wi h an a e age diame e o 9.86*/
1.02 nm (log-no mal i ). The s uc u al cha ac e iza ion o he sample was pe o med
Chap e I
115
using XRD and Raman spec oscopy. The XRD pa e n o he nanoc ys als (Figu e 1b)
ma ches he expec ed Fd-3m spa ial g oup o he ace-cen e ed cubic (FCC) spinel
s uc u e o he me al e i e, wi h a Le Bail e inemen o e ing a 0.84472 nm la ice
pa ame e , which ag ees wi h he expec ed o bulk manganese e i e. Ne e heless, he
ICP-OES analysis e ealed ha he manganese e i e is non-s oichiome ic
(Mn0.7Fe2.3O4). Figu e 1c includes he Raman spec um o he sample, egis e ed using a
785 nm exci a ion wa eleng h, a oom empe a u e. The powe was ixed below 0.5 mW,
in o de o a oid any s uc u al ansi ion in he sample.30 G oup heo y p edic s i e
ac i e Raman modes o he spinel s uc u e, acco ding o he Fd-3m spa ial g oup,31
namely A1g, 3T2g and Eg, hough o nanopa icles only he A1g (spli ), he Eg and one o
he h ee T2g a e usually egis e ed, as in his case (Lo en zial i , in g een). In his speci ic
e en o a non-s oichiome ic manganese e i e, he spli ing o he A1g band can be
associa ed o he p esence o wo di e en ca ions in e ahed al posi ions (Mn2+ and Fe3+)
acco ding o he ob ained s oichiome y. Acco dingly, he h ee main bands obse ed in
he spec um, which can be ela ed o he Eg (312 cm−1), he T2g(2) (450 cm−1) and he spli
A1g (A1g(1) a 610 cm−1) and A1g(2) a 670 cm−1) ib a ional modes, con i ming he spinel
e i e c ys alline s uc u e. These bands also appea sligh ly shi ed, likely because o he
nanome ic size o he c ys als. These magne ic nanopa icles a e ini ially s abilized by an
o ganic laye o OA molecules a ached o hei su ace, which keeps hem s able in
chlo o o m.
In o de o pa e he way o encapsula e hese MFO nanopa icles in o he SNs, hese
OA molecules a ached on he su ace o he nanopa icles a e i s displaced/ eplaced by
Vi E, ollowing he ligand exchange p o ocol de ailed in he expe imen al sec ion. Figu e
1d includes FTIR spec a o he sample be o e and a e p omo ing his ligand exchange
a he su ace o he nanopa icles. The black spec um co esponds o he oleic acid
coa ed nanopa icles, whe e he ypical ib a ional bands o oleic acid can be isualized,
wi h he bands cen e ed a 3496 cm−1 s emming om he O-H ib a ional modes, and he
bands loca ed a 1113 and 1580 cm−1 om he C-O and C=O bonds espec i ely; all o
hem ela ed o he ca boxylic g oup o he oleic acid. In addi ion o hose, he bands
SANDRA DÍEZ VILLARES
116
anging om 2852 o 2962 cm−1 and he one cen e ed a 1444 cm−1 co espond o he C-
H s e ching, o he C-H bending modes, espec i ely, o he hyd oca bon chain o he
molecule.
Figu e 1. (a) TEM image o he manganese e i e nanopa icles, and he size his og am i ed o a
log-no mal cu e (inse ). (b) Expe imen al (black) and i ed ( ed) XRD pa e n wi h he di ac ion
peaks indexed o he spinel s uc u e. (c) Raman spec um egis e ed using a 785 nm exci a ion
wa eleng h wi h he decon olu ed ib a ional modes (g een). (d) In a ed spec a o he oleic acid
(black spec um) and i amin E ( ed spec um) coa ed manganese e i e nanopa icles.
Al e na i ely, he ed spec um o e s he ib a ional modes a e he ligand
exchange. The disappea ing o he s e ching modes a 3496 and 1580 cm−1, associa ed o
he ca boxylic g oups o he oleic acid jus con i ms he ligand exchange. Though bands
cen e ed a 1458 cm−1 and in he egion anging om 2854 o 2968 cm−1 a e s ill p esen ,
hey can be associa ed in his case o he C-H ib a ional modes p esen in he i amin
molecule. The band a 1115 cm−1, which s ems om he e he bond, can as well be
(a) (b)
(c) (d)
Chap e I
117
associa ed o he i amin, p esen in i s molecule. Finally, i is necessa y o highligh he
p esence o a b oad band ex ended om 526 o 900 cm−1, which e e s o he me al-
oxygen bond, ypical o he ino ganic co e. Please, no e ha o e ones in 2000 –
2200 cm−1 a e ela ed o he en i onmen al ca bon dioxide. The selec ion o Vi E o
MFO coa ing was ein o ced by he well-known an ioxidan ac i i y o Vi E which migh
help imp o e he biocompa ibili y o he magne ic nanopa icles by educing hei
oxidan abili ies, inhibi ing o educing he p oduc ion o eac i e oxygen species (ROS)
in i o and hus, o e coming one o he main limi a ions o he ansla ion o hese
nanopa icles o clinic.32 In addi ion, his p e ious coa ing o he nanopa icles wi h Vi E
ende s easie he encapsula ion in he Vi amin E- based nanoemulsions ( ide in a).
To include he i amin E-coa ed MFO nanopa icles in o he SNs, he o ganic phase
composed by oil and su ac an s (wi h o wi hou Vi E-MFO) was injec ed in ul apu e
wa e as shown in he scheme included in Figu e S1. A homogeneous suspension o SNs
and Vi E-MFO-SNs (PDI = 0.19 in bo h cases) wi h an a e age size o 141 ± 3 nm o
149 ± 5 nm was spon aneously ob ained a e he injec ion, espec i ely, as shown by he
DLS analysis (Figu e 2a). Figu e 2b shows he z-po en ial alues o bo h SNs and Vi E-
MFO-SNs (− 51 ± 3 mV and – 42 ± 3 mV, espec i ely), ensu ing he suspension s abili y
due o elec os a ic epulsions, analogously as p e iously epo ed o o he magne ic
emulsions.33–36 The SNs and Vi E-MFO-SNs we e also cha ac e ized by acking hem
using NTA, ob aining a mean size o 128 ± 39 nm and 136 ± 39 nm and concen a ions
o 4x1011 and 6x1011 SNs pe mL (Figu e 2c). These esul s a e consis en wi h he DLS
analysis, bu NTA o e s be e esolu ion han DLS allowing he obse a ion o peaks
e y closed in size ha canno be de ec ed by DLS.37 The esul s ob ained om DLS and
NTA show ha he encapsula ion o Vi E-MFO in o he nanoemulsions did no p oduce
signi ican a ia ions in hei physicochemical p ope ies, gi ing simila size, ze a
po en ial and concen a ion o bo h o mula ions. Thus, we ha e desc ibed he
p epa a ion o magne ic emulsions by an ex emely simple and as me hod. In ac , bo h
o mula ions can be p epa ed in less han 10 minu es and in a e y ep oducible way as
shown in Figu e 2d. Fu he mo e, once p epa ed, colloidal s abili y s udies o he Vi E-
SANDRA DÍEZ VILLARES
118
MFO-SNs we e assessed in h ee di e en ele an media (i.e., DMEM cul u e medium,
phospha e bu e pH 7.4 and saline solu ion o NaCl 0.9%) by measu ing he a e age size
o e ime.
Figu e 2. (a) Physicochemical cha ac e iza ion o SNs and Vi E-MFO-SNs by DLS, LDA (b) and
(c) NTA (n=10). (d) Rep oducibili y o he p epa a ion me hod measu ing he hyd odynamic size
by DLS o 30 independen ba ches, ho izon al ba s ep esen mean and s anda d de ia ion (SD).
(e) E olu ion o he a e age size o he Vi E-MFO-SNs a e incuba ion in DMEM cul u e medium,
PBS 10 mM pH 7.4 and saline se um (NaCl 0.9%), a 37 °C o 24 h (n=5 pe g oup).
SNs Vi E-MFO-SNs
50
100
150
200
250
Size (nm)
0.1 110 100 1000 10000
0
5
10
15
Size (nm)
In ensi y (%)
SNs
Vi E-MFO-SNs
SNs Vi E-MFO-SNs
-80
-60
-40
-20
0
Ze a Po en ial (mV)
10 100 1000 10000
0
2×109
4×109
6×109
8×109
1×1010
Size (nm)
Concen a ion (pa icle/mL)
Vi E-MFO-SNs
SNs
024624 024624 024624
0
50
100
150
200
Time (hou s)
Size (nm)
Cul u e medium PBS Saline se um
(a) (b)
(c) (d)
(e)
Chap e I
119
The e y sligh a ia ion in a e age size (wi hin he e o ba ) (Figu e 2e) in he
h ee cases indica es he nanoemulsions keep he ini ial mo phology o e his pe iod o
24 h a 37 °C in di e en media (and he e o e exposed o di e en ionic s eng h o
di e en biologically ele an con en ), demons a ing he e o e hei high s abili y,
which is key o u he in i o and in i o s udies.38 Mo eo e , he magne ic
nanopa icles encapsula ion e iciency was de e mined by ICP-OES a e isola ion and
quan i ica ion o he ee Vi E-MFO sepa a ed om he magne ic nanoemulsions, gi ing
an encapsula ion yield o 98% (Table S1). Gene ally, he physicochemical p ope ies o
nanoma e ials ha e been demons a ed o play a key ole in he in i o beha io o he
o mula ion.39 Fo example, he mean size o nanoma e ials is a key poin in cance
applica ions as i s ongly in luences umo accumula ion and pene a ion; i is widely
assumed ha small nanopa icles can ex a asa e in he leaky ascula u e o umo s due
o he enhanced pe meabili y and e en ion (EPR) e ec ,40 and ha nega i e su ace
cha ge nanopa icles ha e longe blood ci cula ion imes and can each he a ge o gans
mo e e icien ly han posi i e ones.41
S uc u al cha ac e iza ion o SNs and Vi E-MFO-SNs was pe o med by FESEM,
TEM, AFM and MFM, wi h ep esen a i e images included in Figu e 3. Images o plain
SNs we e acqui ed by TEM (Figu e 3a) and FESEM, using wo de ec o modes, STEM
(Figu e 3b) and InLens (Figu e 3c), e lec ing he esicula mo phology and he
homogeneous size dis ibu ion o he nanosys ems. Cha ac e iza ion o he Vi E-MFO-
SNs was also pe o med, conside ing TEM, FESEM and AFM, o co ela e he 2D images
wi h he h ee-dimensional p o ile ob ained by he a omic o ces. Figu e 3d, e and
include ep esen a i e images o his pa ially o ganic, pa ially ino ganic s uc u e o he
Vi E-MFO-SNs o mula ion on which besides sphe ici y, we can app ecia e he Vi E-
MFO nanopa icles apped inside SNs, wi hou obse ing ee magne ic co es. This
ag ees wi h he e icien en apmen al eady men ioned, such ha he Vi E-MFO
nanopa icles canno eely di use in he ou wa d di ec ion h ough he lipidic shell. The
AFM analysis, showed in Figu e 3g, pe mi s as well o app ecia e he well-de ined
sphe ical shape, p ese ed e en a e he SNs ha e been loaded wi h he Vi E-MFO
SANDRA DÍEZ VILLARES
120
nanopa icles (wi h a oundness ac o o 0.92). AFM images plain SNs a e shown in
Figu e S2. Addi ionally, AFM images using he magne ic o ce mic oscopy mode we e
ca ied ou , in iew o he magne ic esponse o he MFO nanopa icles encapsula ed in
he nanoemulsions. The MFM mode allows he s udy o magne ic o ces a he nanoscale,
by scanning he g adien o magne ic o ce on he sample su ace while simul aneously
ob aining he opog aphic map. Acco dingly, Figu e 3 includes a 10 × 10 µm2-gene al
o e iews o he Vi E-MFO-SNs in phase con as (Figu e 3h) and MFM (Figu e 3i)
modes. Bo h images e idence he co ela ion be ween he SNs and he magne ic con as
s emming om he Vi E-MFO nanopa icles.
Figu e 3. (a) TEM image and FESEM images wi h (b) STEM and (c) InLens de ec o s showing he
homogenous size dis ibu ion o SNs. (d) Rep esen a i e images showing he esicula mo phology
o Vi E-MFO-SNs acqui ed by TEM and (e, ) FESEM. (g) 3D-high magni ica ion image o he
Vi E-MFO measu ed by AFM and gene al 2D opog aphic images o Vi E-MFO-SNs in (h) phase
con as and (i) MFM modes, eco ded in he same egions.
e
2 µm
150 nm
-70 nm
1 mV
-1 mV
200 nm 200 nm200 nm
200 nm 200 nm
2 µm
(a) (b) (c)
(d) (e) ( )
(g) (h) (i)
Chap e I
127
nanopa icles in lungs, which could be an indica i e ha he Vi E-MFO-SNs a e s able
and do no agg ega e causing obs uc ion in he pulmona y capilla ies. The e o e, ou
esul s indica e ha Vi E-MFO-SNs a e biocompa ible and can be de ec ed by ex i o
MRI a ex emely low doses ela ed o hei high ans e se elaxi i y.54
Apa om whole-body imaging applica ions, MRI is one o he p e e ed
echniques in he diagnosis o neu ological pa hologies due o i s high spa io empo al
esolu ion and unlimi ed issue pene a ion dep h, allowing he use o mul iple sequences
o isualize and e alua e he unc ionali y and s uc u e o di e en b ain egions.55,56 Fo
his eason, we also ha e pe o med a p oo o concep in i o s udy o e alua e he
po en ial applica ion o he Vi E-MFO-NE as con as agen s in he b ain.
SNs and Vi E-MFO-SNs we e injec ed in he igh hemisphe e o he b ain (10 µL,
471 µM Fe, 0.018 µmol Fe kg−1, n = 3), and as a con ol, PBS was injec ed in he le
hemisphe e (n = 3). Figu e 7 shows he s ongly nega i e con as o he de eloped
magne ic nanoemulsions a e in ac anial injec ions. T2*-weigh ed in i o MR images o
a b ains (Figu e 7a) and g aphics o he ela i e MR signal measu ed (Figu e 7b) show
he e icien nega i e con as p oduced by Vi E-MFO-SNs compa ed wi h PBS and plain
SNs. Simila esul s we e ob ained wi h T2-weigh ed images and a e shown in Figu e S5.
O e all, we ha e p o ed ha Vi E-MFO-SNs can d ama ically enhance he T2*-weigh ed
and T2-weigh ed signals, o e ing he e o e e icien o mula ions o high-sensi i e MRI.
Fu he expe imen s will in ol e es ing o hese o mula ion in ele an animal models
so hey will allow us o de e mine hei ull po en ial as high-sensi i e issue MR imaging.
SANDRA DÍEZ VILLARES
128
Figu e 7. (a) T2*-weigh ed MR images o b ain a s injec ed in he le ce eb al hemisphe e wi h
PBS as a con ol and in he igh hemisphe e wi h he o mula ions SNs (image on he le ) and
Vi E-MFO-SNs (image on he igh ). (b) Compa ison o T2*-weigh ed MR signals be ween PBS
and SNs (le ) and PBS and Vi E-MFO-SNs ( igh ). MR signals o SNs and Vi E-MFO-SNs a e
no malized o PBS as con ol (100%). (n=3 pe g oup) * (p ≤ 0.05), ** (p ≤ 0.01), ***(p ≤ 0.001) was
conside ed s a is ically signi ican .
4. CONCLUSIONS
We ha e success ully p epa ed a e y simple, easy o manu ac u e and biocompa ible
o mula ion o high-sensi i e T2 MRI by a one-s ep me hod. Fi s , manganese e i e
nanopa icles we e success ully syn hesized by he modecomposi ion o he me al
p ecu so s and coa ed o he i s ime wi h he an ioxidan Vi amin E o ob ain
hyd ophobic nanopa icles. Then, Vi E-MFO we e encapsula ed in o biocompa ible SNs
p o iding good-sized, homogeneous, nega i e cha ged and highly s able o mula ions
sui able o biomedical applica ions. Mo eo e , mo phological cha ac e iza ion o Vi E-
NE
Con ol (PBS) NEs Con ol (PBS) Vi E-MFO-NEs
T2*-weigh ed images
PBS Vi E-MFO-NEs
0
50
100
150
MR signal (%)
**
PBS NEs
0
50
100
150
MR signal (%)
ns
(a)
(b)
Chap e I
129
MFO-SNs con i med he na ow dis ibu ion o he pa icles and he e icien
en apmen o Vi E-MFO in o hei inne co e. We ha e demons a ed he magne ic
p ope ies o Vi E-MFO-SNs by con i ming ha he supe pa amagne ic beha io o he
nanopa icles was no comp omised a e encapsula ion. In i o MR s udies o Vi E-
MFO and Vi E-MFO-SNs showed a g ea enhancemen o he T2-weigh ed MR con as ,
being signi ican ly highe o Vi E-MFO-SNs. Mo eo e , we ha e p o ed he in i o
biocompa ibili y o Vi E-MFO-SNs and con i med ha hey can be de ec ed by ex i o
MRI and in i o MR b ain imaging. In summa y, his wo k p o ides an exhaus i e
cha ac e iza ion o biocompa ible magne ic nanoemulsions ha possess an ul ahigh T2
con as abili y and can e icien ly inc ease he sensi i i y o MRI and hus, open a ga e
o possible applica ions in biomedical imaging.
SANDRA DÍEZ VILLARES
130
SUPPORTING INFORMATION
Magne ic nanoemulsions o mula ion
Figu e S1. (a) Scheme o one-s ep e hanol injec ion me hod o ob ain magne ic nanoemulsions
and (b) pho og aph showing he Vi E-MFO-NEs appea ance and he pelle o non-encapsula ed
Vi E-MFO a e cen i uga ion.
Aqueous phase
wa e
O ganic phase
Sphingomyelin
Vi amin E
Magne ic nanoemulsions
(Vi E-MFO-SNs)
Manganese e i e
nanopa icles (Vi E-MFO)
(a)
(b)
Chap e I
131
ICP measu emen s
Vi E-MFO-NEs we e p epa ed by e hanol injec ion and non-encapsula ed magne ic
nanopa icles we e p ecipi a ed a e cen i uga ion 10 min a 5.000 ela i e cen i ugal
o ces ( c ). Magne ic nanoemulsions we e emo ed and he pelle s we e mixed wi h a
dilu ion 1:2 ni ic acid 65%:hyd ogen pe oxide 35%, ollowing by mic owa e diges ion.
The esul an solu ion was dilu ed o 5 mL o inal olume and analyzed by ICP-OES.
Table S1. ICP-OES quan i ica ion o he non-encapsula ed magne ic nanopa icles.
Sample
F ee MFO mass
(µg)
Encapsula ion yield (%)
1
2.0315 ± 0.0175
99, 14
2
1.7026 ± 0.0043
99,29
3
2.0759 ± 0,0299
99,13
4
3.8928 ± 0.0246
98,37
A e age
2.4257 ± 0,8592
98,98
Figu e S2. (a) AFM images and (b) 3D-high magni ica ion image o he plain nanoemulsions
(NEs).
(a) (b)
SANDRA DÍEZ VILLARES
132
Figu e S3. (a-c) Phase con as and (d- ) MFM images o Vi E-MFO-NEs scanning he same
egion a di e en li heigh s (70, 88 and 100 nm) abo e he su ace o he sample. MFM mode was
ope a ing in he p esence o a 40-mT magne ic ield.
In i o MR imaging and longi udinal elaxi i y.
To measu e he longi udinal elaxi i y o Vi E-MFO-NEs, aga -based phan oms we e
p epa ed ollowing he same p ocedu e desc ibed in he manusc ip and loaded wi h
di e en concen a ions o Vi E-MFO-NEs ( om 0.003 o 0.10 mM Fe). T1-weigh ed
images we e acqui ed using RAREVTR sequence wi h 8.7 ms o echo ime, 17.48 ms o
e ec i e echo ime, 4 a e ac o , 6 T1 expe imen s (900, 2000, 3500, 5000, 7000,
11000 ms), 1 a e age, 1 epe i ion, lip angle o 180 °, 14 slices o 1 mm, wi h a ield o
iew (FOV) o 7.5 cm × 7.5 cm (wi h sa u a ion bands o supp ess signal ou side his
FOV), and a ma ix size o 300 × 300, gi ing an in-plane esolu ion o 250 μm/pixel and
implemen ed wi hou a supp ession. The elaxi i y cons an ( 1) was calcula ed as he
slope o he cu e ob ained by i ing he T1-1 alues e sus he Fe concen a ion in mM.
2 µm
200 nm
-145 nm
2 µm
2.8 mV
-2.2 mV
500 nm
100 nm
-100 nm
3.0 mV
-2.4 mV
500 nm
200 nm
70 nm
-50 nm
1.8 mV
-1.8 mV
200 nm
70 nm 88 nm 100 nm
(a) (b) (c)
(d) (e) ( )
Chap e I
133
Figu e S4. Longi udinal elaxi i y ( 1) o Vi E-MFO-NEs acqui ed wi h 9.4 T ho izon al MR
scanne .
Figu e S5. (a) T2-weigh ed MR images o b ain a s injec ed in he le ce eb al hemisphe e wi h
PBS as a con ol and in he igh hemisphe e wi h he o mula ions NEs (image on he le ) and
Vi E-MFO-NEs (image on he igh ). (b) Compa ison o T2-weigh ed MR signals be ween PBS and
NEs (le ) and PBS and Vi E-MFO-NEs ( igh ). MR signals o NEs and Vi E-MFO-NEs a e
no malized o PBS as con ol (100 %). (n=3 pe g oup). * (p ≤ 0.05), ** (p ≤ 0.01), ***(p ≤ 0.001) was
conside ed s a is ically signi ican .
0.00 0.02 0.04 0.06 0.08 0.10 0.12
0.35
0.40
0.45
0.50
Fe concen a ion (mM)
1/T1 (s-1)
1 = 0.6535 mM-1s-1
R2 = 0.95
Vi E-MFO-SNs
NE
Con ol (PBS) SNs Con ol (PBS) Vi E-MFO-SNs
T2-weigh ed images
(a)
(b)
PBS Vi E-MFO-SNs
0
50
100
150
MR signal (%)
**
PBS SNs
0
50
100
150
MR signal (%)
ns
SANDRA DÍEZ VILLARES
134
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15. Ch is odoulou, E. e al. Pacli axel magne ic co e–shell nanopa icles based on poly(Lac ic
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chemo he apy ea men o cance . Pha maceu ics 11, 213 (2019).
16. Ba ow, M., Taylo , A., Mu ay, P., Rosseinsky, M. J. & Adams, D. J. Design
conside a ions o he syn hesis o polyme coa ed i on oxide nanopa icles o s em cell
labelling and acking using MRI. Chem. Soc. Re . 44, 6733–6748 (2015).
Chap e I
135
17. Ka imi, Z. e al. Pegyla ed and amphiphilic Chi osan coa ed manganese e i e
nanopa icles o pH-sensi i e deli e y o me ho exa e: Syn hesis and cha ac e iza ion.
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Rep. 8, 1–11 (2018).
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nanomicelles. In . J. Nanomedicine 11, 4051–4063 (2016).
20. Shana as, A., Sasidha an, S., Bahadu , D. & S i as a a, R. Magne ic co e-shell hyb id
nanopa icles o ecep o a ge ed an i-cance he apy and magne ic esonance imaging.
J. Colloid In e ace Sci. 486, 112–120 (2017).
21. An on, N., Halloua d, F., A ia, M. F. & Vandamme, T. F. Nano-emulsions o D ug
Deli e y and Biomedical Imaging. in In acellula Deli e y III, Fundamen al Biomedical
Technologies 8 (2016).
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28. T ekke , J. e al. Sensi i e in i o cell de ec ion using size-op imized supe pa amagne ic
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30. Tes a-An a, M., Ramos-Docampo, M. A., Comesaña-He mo, M., Ri as-Mu ias, B. &
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32. Angulo-Molina, A. e al. Magne i e Nanopa icles Func ionalized wi h Vi amin E
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34. Ja zyna, P. A. e al. I on oxide co e oil-in-wa e emulsions as a mul i unc ional
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35. P é o , G. e al. I on oxide co e oil-in-wa e nanoemulsion as ace o a he oscle osis
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43. Co do a, G., A wood, S., Gaikwad, R., Gu, F. & Leonenko, Z. Magne ic o ce mic oscopy
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Chap e II
143
1. INTRODUCTION
O e he las decades, pe sonalized medicine has g ea ly e ol ed wi h he de elopmen
o imaging ools ha imp o e he managemen o se e al diseases, especially cance .2
Among all he non-in asi e imaging echniques, he nuclea imaging modali ies Single
Pho on Emission Compu ed Tomog aphy (SPECT) and Posi on Emission Tomog aphy
(PET) s and ou mainly due o hei high sensi i i y p ospec o ob ain quan i a i e
in o ma ion. In ac , PET and SPECT imaging can p o ide de ailed in o ma ion abou
he in i o beha io and pha macokine ics o se e al compounds, such as
nanomedicines, and can acili a e hei ansla ion o clinics.3
In his sense, nanomedicine has eme ged as a p omising s a egy o imp o e diagnosis
and ea men o p e alen diseases including cance .4,5 Addi ionally, one o he mos
p omising ad an ages o nanomedicine is he possibili y o combine he apeu ic
molecules wi h diagnos ic agen s in o single mul i unc ional nanopa icles, known as
nano he anos ics, opening an en i e new ield o de elopmen owa ds he
implemen a ion o pe sonalized medicine.6,7 In ecen yea s, he combina ion o
nanopa icles wi h adionuclides is apidly g owing and he e a e a g ea numbe o
submissions o he Food and D ug Adminis a ion app o al.8 Di e en ypes o
nanopa icles a e in es iga ed o nuclea medicine applica ions and mul imodal
imaging.9 Fo example, ino ganic nanopa icles ha e been widely s udied due o hei
in insic physical p ope ies, ha con e hem in ma e ials wi h a high po en ial o
mul imodal imaging.10,11 Ne e heless, o ganic nanopa icles a e s ill he mos demanded
o he de elopmen o imaging p obes by i ue o hei biodeg adable and
biocompa ible composi ion, p e en ing a long- e m accumula ion in he body and
undesi able oxic side e ec s.12 Indeed, liposomes a e he mos ex ended ype o o ganic
nanopa icles o nuclea imaging applica ions. Liposomes can be adiolabeled by
di e en me hodologies, which can be adap ed o di e en kinds o nanopa icles such as
micelles, solid lipid nanopa icles, and nanoemulsions.13 Chela o -based adiolabeling
s a egies o e a high e sa ili y o he inco po a ion o adionuclides wi h di e en
p ope ies, sui able o complemen a y imaging echniques and nano he anos ics.14
SANDRA DÍEZ VILLARES
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Nanoemulsions a e de ined as nanoscale d ople s in which wo immiscible liquids a e
mixed o o m a single phase. Thei biocompa ible composi ion, easy p oduc ion by so
and scalable me hodologies and imp o ed d ug loading capaci y compa ed o liposomes,
a e ele an ad an ages ha ha e p omp ed hei use in biomedicine.15,16
Nanoemulsions ha e been widely s udied o luo escence, MRI and ul asounds
imaging.17,18 Howe e , hei use in nuclea imaging is s ill ecen and he e a e only ew
epo s desc ibing adiolabeled nanoemulsions.19–21 Ou g oup has ecen ly epo ed he
de elopmen and cha ac e iza ion o sphingomyelin nanoemulsions (SNs) ha
inco po a e sphingomyelin, one o he mail lipids in cell memb anes, and claimed hei
po en ial in d ug deli e y.22 The p incipal ad an ages o SNs ela e o hei sa e and simple
composi ion, long- e m colloidal s abili y, and capaci y o accommoda ion di e en
ypes o unc ionali ies and he apeu ic payloads.22–24 P e ious a emp s by ou esea ch
g oup ha e p o ed ha SNs can be adiolabeled wi h Fluo ine-18 o PET imaging
ollowing a maleimide eac ion. Howe e , adiochemical yields (RCY) we e ound o be
highly dependen on he c osslinking e icacy.25 The aim o his wo k was o p o ide an
op imized composi ion and s aigh o wa d me hodology o he adiolabeling o SNs
wi h 68Ga and 67Ga adioiso opes by a chela o -based s a egy. Radiolabeled o mula ions
can be used o indis inc applica ion in PET and SPECT imaging, and he e o e
adap able o speci ic needs and biomedical applica ions.
2. MATERIALS AND METHODS
2.1. Syn hesis and cha ac e iza ion o NOTA-s ea ylamine de i a i e
Oc adecylamine (s ea ylamine, >99%, Me ck G oup, Da ms ad , Ge many) was
conjuga ed o 2-(4-iso hiocyana obenzyl)-1,4,7- iazacyclononane-1,4,7- iace ic acid
(p-SCN-Bn-NOTA, NOTA, >94%, Mac ocyclics, Dallas, TX, USA) o ob ain a lipid
de i a i e chela o o u he inclusion in o he nanoemulsions. De ails abou he
eac ion p o ocol and he p oduc cha ac e iza ion a e included in he Suppo ing
In o ma ion.
Chap e II
145
2.2. P epa a ion o sphingomyelin nanoemulsions
SNs we e p epa ed ollowing a me hod p e iously epo ed by ou g oup wi h mino
modi ica ions.22 B ie ly, oleic acid (5 mg, 65-88%, Me ck G oup,Da ms ad , Ge many),
egg sphingomyelin (0.5 mg, 98%, Lipoid GmbH, Ludwigsha en, Ge many) and he
su ac an C16/C18-COO-C9H9O3 (0.5 mg, 96%, GalChimia S.L, A Co uña, Spain) wi h
a lipid a io 1:0.1:0.1 w/w we e dissol ed in 100 µL o absolu e e hanol (99.7%, Cieny ech
S.L., A Co uña, Spain). All he addi ional lipid de i a i es used o unc ionalize SNs, such
us 1,2-dimy is oyl-sn-glyce o-3-phosphoe hanolamine-N-
die hylene iaminepen aace ic acid (DTPA, 0.05 mg, >99%, A an i Pola Lipids,
Alabama, Al, USA), NOTA (0.05 mg) o an oleic acid modi ied polye hylene glycol (PEG,
2 kDa, 0.125 mg, Nanocs, New Yo k, NY, USA) we e included in he o ganic phase. Then,
his o ganic phase was injec ed in 1 mL o MilliQ wa e (Millipo e Milli-Q sys em) unde
magne ic s i ing using an insulin sy inge (0.5 mL, 0.33 × 12 mm ICO.C.1) and
nanoemulsions (SNs, DTPA-SNs, NOTA-SNs o PEG-SNs) we e spon aneously o med.
To p epa e SNs coa ed wi h hyalu onic acid (HA-SNs), he o ganic phase con aining he
lipids and NOTA was injec ed unde s i ing in 1 mL o an aqueous solu ion o sodium
hyalu ona e (HA, 170 kDa, 2 mg mL−1, >95%, Bioibe ica, S.A.U, Ba celona, Spain).
2.3. Physicochemical and mo phological cha ac e iza ion
All he nanoemulsions we e physiochemically cha ac e ized using a Nanosize 2000®
(Mal e n Ins umen s, Mal e n, UK). The mean size and i s dis ibu ion, de ined by he
polydispe si y index (PDI), we e measu ed by Dynamic Ligh Sca e ing (DLS).
Measu emen s we e pe o med in disposable mic ocu e es (ZEN0040, Mal e n
Ins umen s) upon dilu ion o he SNs in MilliQ wa e , eaching a inal lipid
concen a ion o 0.5 mg mL−1. The ze a po en ial (ZP) was analyzed by Lase Dopple
Anemome y (LDA) dilu ing SNs in MilliQ wa e (lipid concen a ion 0.12 mg mL−1) in
Folded capilla y cu e es (DTS1070, Mal e n Ins umen s). The s abili y o SNs, DTPA-
SNs and NOTA-SNs was es ed unde s o age condi ions a 4 °C up o one mon h and
SANDRA DÍEZ VILLARES
146
also a e incuba ion wi h human se um a 37 °C o 72 h. The colloidal p ope ies we e
measu ed by DLS main aining he condi ions men ioned be o e. Pa ame e s such as he
medium (wa e ) and he empe a u e (25 °C) we e ixed o all he measu emen s.
The mo phology o SNs was obse ed by Field Emission Scanning Elec on
Mic oscopy (FESEM) using a ZEISS FESEM ULTRA Plus, mic oscope (Ca l Zeiss Mic o
Imaging, GmbH, Ge many). Be o e he measu emen , 20 µL o sphingomyelin
nanoemulsions (0.5 mg mL−1) we e s ained wi h 20 µL phospho ungs ic acid (2% w/ ).
Then, 20 µL o he mix u e was placed on a ca bon coa ed g id and le o 2 minu es. The
excess was emo ed using a il e pape and he g id was allowed o d y. The g id was
washed 5 imes wi h 100 µL o il e ed MilliQ wa e and i was d ied o e nigh .
2.4. Cell cul u e condi ions
A549 (ATCC® CCL-185), MDA-MB-231 (ATCC® HTB-26) we e cul u ed in Dulbecco’s
modi ied Eagle’s medium high glucose (DMEM, Me ck G oup, Da ms ad , Ge many)
and OMM-2.5 (kindly p o ided by Ma ine J. Jage om Leiden Uni e si y Medical
Cen e , Leiden, The Ne he lands) we e g own in RPMI (Gibco, The mo Scien i ic S.L.,
Wal ham, MA, Uni ed S a es). Bo h media we e supplemen ed wi h 10% e al bo ine
se um (FBS) and 1% penicillin:s ep omycin (Gibco, The mo Scien i ic S.L., Wal ham,
MA, Uni ed S a es). Cells we e main ained a 37 °C wi h 95% ela i e humidi y and 5%
CO2.
2.5. Cellula up ake o SNs
Cellula up ake o SNs was s udied by con ocal mic oscopy. 8×104 cells/well we e seeded
in an 8 wells µ-chambe (SPL Li e Sciences Co., L d., Gyeonggi-do, Ko ea). A e 24 h,
cells we e ea ed wi h SNs (0.13 mg mL−1 pe well) labeled wi h C11-TopFluo
sphingomyelin (>99%, A an i Pola Lipids, Alabama, Al, USA). A e 4 h o incuba ion
a 37 °C, cells we e washed wi h 1x Phospha e bu e saline (PBS) wice and ixed wi h 4%
Chap e II
147
pa a o maldehyde o 15 min. Cells we e hen washed wice wi h 1x PBS and he cellula
nuclei we e coun e s ained wi h Hoechs 33342 (The mo Scien i ic S.L., Wal ham, MA,
Uni ed S a es) o 5 min. A e washing, he slide was moun ed wi h Mowiol (Me ck
G oup, Da ms ad , Ge many) and a co e slip. The samples we e le o d y in da kness
o e nigh a RT, ollowing hei s o age a − 20 °C, un il aken o obse a ion unde he
con ocal mic oscope (Con ocal Lase Mic oscope Leica SP8®).
2.6. Radiolabeling o DTPA-SNs and NOTA-SNs wi h 68Ga
68Ga ( ½ = 68 min, β+ = 89% and EC = 11%) was ob ained om a 68Ge/68Ga gene a o
sys em (ITG Iso ope Technologies Ga ching GmbH, Ge many) in which 68Ge ( ½ = 270
d) was a ached o a column based on an o ganic ma ix gene a o . The 68Ga was elu ed
wi h 4 mL o 0.05 M hyd ochlo ic acid. Then, 500 µL o DTPA-SNs o NOTA-SNs
(13 mg mL−1) we e mixed wi h 500 µL o 4-(2-hyd oxye hyl)-1-pipe azinee hanesul onic
acid bu e (HEPES, 0.5 M, pH 5.05). The mix u e was incuba ed wi h 1.5 mL o 68Ga
(≈300 MBq) a 30 °C o 30 min and pu i ied by PD-10 columns. The inco po a ed
adioac i i y was measu ed in an ac i ime e (A omLabTM500, Biodex).
2.7. Radiolabeling o NOTA-SNs, NOTA-HA-SNs and NOTA-PEG-SNs wi h 67Ga
[67Ga]Ga-ci a e ( ½ = 78.3 h, 100% EC = 39% γ 93 keV, 21% γ 185 keV, 17% γ 300 keV)
was ob ained om CURIUM (F ance) as s e ile solu ion wi h a pH be ween 5 - 8 and a
adiochemical pu i y a leas equal o 95%. [67Ga]Ga-ci a e solu ion was con e ed o
[67Ga]GaCl3 wi h a me hod p e iously desc ibed.26 In b ie , 2 mL o [67Ga]Ga-ci a e
(37 MBq) dilu ed in dis illed wa e was il e ed wi h a SEP-PAK® Plus silica ca idge
(ABX, Ad anced Biochemical Compounds, Ge many) using a 5 mL plas ic sy inge.
A e wa ds, he silica ca idge was washed h ee imes wi h 5 mL o dis illed wa e o
emo e he ee ci a e ions. The [67Ga]Ga3+ ions we e elu ed wi h 3 mL o HCl 0.1 M,
ob aining a solu ion o [67Ga]GaCl3 which was concen a ed in a o a y acuum
e apo a o o ge a inal olume o 500 µL. The pH was adjus ed o 4-5 wi h NaOH 0.1 M
SANDRA DÍEZ VILLARES
148
and he solu ion was incuba ed wi h 500 µL o NOTA-SNs, NOTA-HA-SNs o NOTA-
PEG-SNs (10 mg mL−1) dilu ed in HEPES bu e (1.5 M, pH 5.05) o 1 h a 37 °C. The
labeled nanoemulsions we e e en ually pu i ied wi h PD-10 columns and he
adioac i i y was measu ed in he ac i ime e .
2.8. Radiochemical cha ac e iza ion
The adiochemical yield (RCY) was calcula ed as a pe cen age o decay co ec ed ac i i y
ound in he pos -pu i ica ion solu ion compa ed o he s a ing ac i i y. The
adiochemical s abili y (RCS) wi h 68Ga-labeled nanoemulsions was assessed by
incuba ing he emulsions wi h animal se um a 37 °C o 4 hou s. In case o 67Ga-labeled
nanoemulsions, he s abili y was measu ed a e incuba ion wi h animal se um o 0, 24,
48 and 72 h, acco ding o he acquisi ion ime poin s. In bo h cases, a e he incuba ion
ime he mix u e was pu i ied by a PD-10 column and he ac i i y o he elu ion was
measu ed and decay co ec ed. Radiochemical pu i y (RCP) was analyzed by ins an hin
laye ch oma og aphy (iTLC) and de ails ega ding expe imen al p o ocols a e included
in he Suppo ing In o ma ion.
2.9. In i o biodis ibu ion by PET/CT and SPECT imaging
In i o PET/CT imaging was pe o med in heal hy mice (C57BL/6) wi h a nanoPET/CT
small-animal imaging sys em (Mediso Medical Imaging Sys ems, Budapes , Hunga y).
Lis -mode PET da a acquisi ion commenced 2 hou s pos bolus injec ion o ~12 MBq o
[68Ga]Ga-DTPA-SNs o [68Ga]Ga-NOTA-SNs (12 MBq, n = 5) h ough he ail ein and
con inued o 30 minu es. A he end o PET, mic oCT was pe o med o a enua ion
co ec ion and ana omic e e ence. The dynamic PET images in a 105x105 ma ix ( ame
a es: 3 x 10 min, 1 x 30 min, 1 x 60 min) we e econs uc ed using a Te a-Tomo 3D
i e a i e algo i hm. Acquisi ion and econs uc ion we e pe o med wi h p op ie a y
Nucline so wa e (Mediso, Budapes , Hunga y). Quali a i e Image analysis in mice was
pe o med using Osi ix so wa e (Pixmeo, Swi ze land). Animal expe imen s we e
Chap e II
149
conduc ed acco ding o he e hical and animal wel a e commi ee a CNIC and he
Spanish and UE legisla ion. Expe imen al p o ocols ha e been app o ed by Mad id
egional go e nmen , (PROEX16/277).
SPECT s udies we e ca ied ou on male Sp ague-Dawley a s wi h an a e age
weigh o 299.5 ± 23.45 g supplied by he animal acili y a he Uni e si y o San iago de
Compos ela (Spain). Plana dynamic SPECT images we e acqui ed in a single-head
clinical Siemens O bi e gamma came a (Siemens Medical Solu ions, Inc., USA) using a
pa allel collima o speci ically designed o low-ene gy pho ons and high spa ial
esolu ion. Da a we e acqui ed in lis -mode o ma o apply ene gy and spa ial linea i y,
and uni o mi y co ec ions. In i o NOTA-SNs and unchela ed 67Ga biodis ibu ion
we e s udied a e he in a enous injec ion (17.70 ± 8.5 MBq, n = 5) in heal hy a s a
di e en ime-poin s: 24, 48 and 72 h. To compa e he di e ences in biodis ibu ion
be ween NOTA-SNs, HA-SNs and PEG-SNs, heal hy a s we e in a enously injec ed
(13.2 ± 0.3 MBq, n = 3) and he images we e acqui ed dynamically du ing he i s 60 min
a e injec ion (30 ames/2min). All images we e analyzed using AMIDE so wa e
(amide.sou ce o ge.ne ). Quan i a i e analysis was ca ied ou in he dynamic s udy by
using ci cula ly delinea ed Regions o In e es (ROIs) a hea and li e , wi h 9 mm in
diame e . The mean up ake was calcula ed o e ime in e e y egion a e aged each 3
ames (6 min) and he esul s we e epo ed as hea o li e a io.
2.10. Ex i o biodis ibu ion s udies
Ex i o biodis ibu ion o 68Ga-labeled nanoemulsions was conduc ed 4 h pos -injec ion.
In case o 67Ga-labeled nanoemulsions, biodis ibu ion s udies we e pe o med 72 h pos -
injec ion. Animals we e sac i iced in a CO2 chambe , o gans we e ex ac ed and coun ed
wi h a Wiza d 1470 gammacoun e (Pe kin Elme ) o 1 min each (n=5 pe expe imen ).
Radioac i i y decay was co ec ed and biodis ibu ion was p esen ed as he pe cen age o
injec ed dose pe g am (% ID/g).
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2.11. S a is ical analysis
All he expe imen s we e pe o med a leas in iplica e. Da a a e exp essed as mean ±
s anda d de ia ion (SD). S a is ical analyses we e calcula ed wi h G aphPad
P ism® so wa e ( e sion 8.0). S uden ’s - es was used o compa e signi ican
di e ences be ween wo g oups. * (p ≤ 0.05), ** (p ≤ 0.01), ***(p ≤ 0.001) was conside ed
s a is ically signi ican .
3. RESULTS AND DISCUSSION
3.1. P epa a ion and cha ac e iza ion o SNs, DTPA-SNs and NOTA-SNs
We desc ibe he e he adiolabeling o SNs wi h Gallium-68 and Gallium-67 o hei
applica ion in PET and SPECT imaging. SNs we e p epa ed by e hanol injec ion, a one-
s ep mild echnique ha allows ob aining colloidal nanoemulsions wi hin seconds
(Figu e 1a, le ). The ep oducibili y o he p epa a ion me hod (Figu e 1a, igh ) was
ob ained a e measu ing 24 independen ba ches by DLS ( aw da a a e showed in Table
S1). SNs showed sphe ical mo phology, as obse ed in FESEM images (Figu e 1b).
Addi ionally, SNs we e e icien ly in e nalized in cance cells (Figu e 1c), which is a
ele an ac o o conside in o de o de e mine he po en ial o a o mula ion o
biomedicine applica ions. S abili y de e mina ions in cell cul u e media we e also
pe o med and a e shown in Figu e S1, Suppo ing In o ma ion. To con e SNs in
sui able p obes o PET and SPECT imaging, we ollowed a chela o -media ed app oach,
which is one o he mos used me hods o adiolabel nanopa icles wi h adionuclides
such as 64Cu, 68Ga, 99mTc o 111In.14,27 Labeling o ganic nanopa icles, and specially lipid
nanopa icles, can be done using lipid-de i a i e chela o s. These conjuga es can be
inse ed in he memb ane o he lipid pa icles a he ime o hei p epa a ion.28–30 In his
s udy, we used wo di e en lipid-de i a i e chela o s o de e mine he bes candida e o
in i o imaging. Fi s , we selec ed he acyclic chela o die hylene iaminepen aace ic acid
modi ied wi h a dimy is oyl-sn-glyce o-3-phosphoe hanolamine chain (DTPA).
Chap e II
151
Figu e 1. (a) Scheme o he one-s ep me hod used o he p epa a ion o SNs (le ) and he me hod
ep oducibili y a e measu ing he hyd odynamic size o 24 independen ba ches by DLS ( igh );
ho izon al ba s ep esen size mean and s anda d de ia ion (127 ± 9 nm). (b) Rep esen a i e Field
Emission Scanning Elec on Mic oscopy (FESEM) images o SNs acqui ed wi h STEM ( op) and
InLens (bo om) de ec o s. (c) Con ocal mic oscopy images showing he in e naliza ion o SNs in
di e en cance cell lines. SNs a e labeled in g een (TopFluo -SM) and cell nuclei a e labeled in
blue (Hoechs ).
Second, we syn hesized an amphiphilic de i a i e o he mac ocyclic chela o 1,4,7-
iazacyclononane-1,4,7- iace ic acid (NOTA) as p e iously desc ibed.31–33 In b ie , a
s ea ylamine was eac ed wi h he iso hiocyana e mac ocycle p-SCN-Bn-NOTA (1,
OMM-2.5 MDA-MB-231 A549
Con ol
SNs
Hoechs , TopFluo -SNs
SNs
STEMInLens
200 nm
200 nm
O ganic phase
Sphingomyelin
Oleic acid
Aqueous phase
Wa e
Sphingomyelin nanoemulsions
SNs
ONE-STEP METHOD REPRODUCIBLE NANOEMULSIONS
25 µm 25 µm 25 µm
25 µm25 µm25 µm
(a)
SNs
0
50
100
150
200
Size (nm)
(b) (c)
SANDRA DÍEZ VILLARES
152
Figu e S2a, Suppo ing In o ma ion). The nucleophilic subs i u ion in in N,N-
dime hyl o mamide a o ded he co esponding hiou ea de i a i e (2, NOTA-
s ea ylamine, Figu e S2a, Suppo ing In o ma ion) a e ec ys alliza ion in mode a e
yield (23 %). The NOTA-s ea ylamine de i a i e 2 was cha ac e ized by high esolu ion
mass spec ome y (Figu e S2b Suppo ing In o ma ion) and NMR, con i ming i s
s uc u e (Figu e S3 and Figu e S4 Suppo ing In o ma ion). Bo h lipid-modi ied
chela o s we e spon aneously inco po a ed in he lipidic laye o SNs. Acco ding o
esul s shown in Table 1, a sligh inc ease in size was obse ed o DTPA-SNs and
NOTA-SNs wi h espec o he con ol SNs, which could be indica i e o he e icien
inco po a ion o he chela o s. In all cases we obse ed a na ow dis ibu ion o he
pa icles wi h a PDI ≤ 0.2.
Table 1. Physicochemical Cha ac e iza ion o SNs, DTPA-SNs and NOTA-SNs Measu ed by DLS
and LDA (Resul s a e Exp essed as Mean ± S anda d De ia ion, n = 3)
Fo mula ion
Size (nm)
PDIa
ZPb (mV)
SNs
125 ± 1
0.14 ± 0.05
− 49 ± 5
DTPA-SNs
136 ± 3
0.20 ± 0.02
− 51 ± 3
NOTA-SNs
151 ± 7
0.15 ± 0.01
− 50 ± 5
aPDI: Polydispe si y Index; bZP: Ze a Po en ial
S abili y s udies unde s o age condi ions a 4 °C showed ha all he o mula ions
we e highly s able du ing he es ed pe iod (Figu e 2a), indica ing ha he inco po a ion
o he lipid-de i a i e chela o s does no comp omise he colloidal p ope ies o SNs. In
addi ion, hey showed high s abili y in human se um o 72 h a 37 °C, as shown in Figu e
2b, demons a ing hei po en ial o in i o applica ions. Al hough o ganic
nanopa icles o e ele an ad an ages espec o ino ganic nanopa icles, gene ally,
hei p epa a ion is s ill complex, as dend ime s, liposomes o nanogels end o equi e
mul i-s ep p epa a ion me hods and/o ypically he use o high ene gy echniques. On
Chap e V
255
analyzed by IHC (Figu e 1).22 In his ame, we can conclude ha he o e all equency
o TAS1R3 cells is qui e low when is measu ed by low cy ome y, while posi i i y o IHC
appea s highe , indica ing ha mo e s udies a e needed o cla i y he po en ial ele ance
o in acellula TAS1R3 exp ession. No ewo hy, he p esence o TAS5F-SNs could be
obse ed in e e y pai o models (Figu e 8c), and he up ake pa e n ollowed by hese
nanoemulsions co ela es qui e well wi h he ma ke exp ession, which indica es and
con i m he selec i e a ge ing o he ap ame -deco a ed o mula ions.
As p e iously men ioned, TAS1R3 was ound o be highly exp essed by cance
s em-like cells, bu he insigh s o hese ela ion a e s ill being in es iga ed. Cance s em
cells (CSCs) ep esen a small p opo ion o cells wi hin he umo mass wi h unique
cha ac e is ics, such as unlimi ed sel - enewal capaci y, plu ipo ency and g ea plas ici y
ha allows hem o change om a "do man " s a e o a mo e agg essi e one depending
on he en i onmen in which hey a e ound. Fo his eason, his cell subpopula ion is
associa ed wi h esis ance o ea men , capaci y o ini ia e umo s, umo dissemina ion
p ocesses, me as asis o ma ion and ecu ence.35,36 Due o his ea u es, his popula ion
ep esen a challenging and impo an a ge and many e o s a e ocused on i o cance
ea men .37 As CSCs also play a ole on me as asis, we wan ed o u he in es iga e he
p esence o TAS5F-SNs in his subpopula ion, iden i ied by he exp ession o CD133
ma ke .23 In addi ion, Be olini e al epo ed ha co-exp ession o CD133 wi h CXCR4
has a be e abili y o seed and ini ia e me as ases (me as a ic ini ia ing cells MIC) in
dis an o gans han CD133+CXCR4- cells o bulk popula ion.38 The e o e, we also
in es iga ed he possible accumula ion o TAS5F-SNs in CD133+CXCR4+ MICs.
As shown in Figu es 9a and 9b, he pe cen age o CD133 and MIC we e
d ama ically highe in he pa ien -de i ed models, compa ed o he ones de i ed om
he immo alized cell lines. Besides, hese exp essions we e impo an ly en iched in he
me as a ic models DDX111, highligh ing hei ela ionship wi h he me as asis p ocess.
When we looked o TAS5F-SNs posi i i y inside hese wo subse s, we could obse e
ha nanoemulsions we e e icien ly in e nalized bu in a comple ely unspeci ic manne ,
wi hou impo an di e ences be ween g oups (Figu e 9c).
SANDRA DÍEZ VILLARES
256
Figu e 9. (a) CD133 and (b) CD133/CxCR4 (MIC) exp ession in he umo s o he di e en
models. (c) Pe cen age o TAS5F-SNs in he CD133 and CD133/CxCR4 (MIC) posi i e
subpopula ions. Ba s ep esen mean ± SD (n=4), *** (p ≤ 0.001) **** (p ≤ 0.0001).
A e dissemina ion om he p ima y umo , a small pe cen age o CTCs can
ex a asa e om he bloods eam and colonize a dis an o gan o e en ually become
DTCs.39 This subse o cells mus de elop an adap i e me as a ic pheno ype o p oli e a e
and ul ima ely o m mac ome as asis. This p ocess can ake mon hs o decades o occu
(i i does), which could open a he apeu ic window o clinical ea men .40 Howe e ,
a e seeding, DTCs can en e in o a "do man " s a e, which makes ex emely di icul o
emo e he me as asis po en ial om he body using cu en ea men s, and only a single
do man cell is needed o e en ually cause me as asis.41 The e o e, i is ex emely
impo an o de elop new he apeu ic s a egies o de ec and e adica e DTCs. He e, we
ha e iden i ied lung DTCs using FACS analysis, ollowing an app oach p e iously
desc ibed (ga ing s a egy desc ibed in Suppo ing in o ma ion).24,42 As shown in Figu e
X H460
CDXH460
X H1299
DDXH1299
PDX111
DDX111
0
2
4
30
60
90
Pe cen age o CD133+ cells
CD133 Me as a ic ini ia ing cells
X H460
CDXH460
X H1299
DDXH1299
PDX111
DDX111
0.0
0.2
20
30
40
Pe cen age o MIC+
(a) (b)
CD133 Me as a ic ini ia ing cells
PDX111 DDX111
0
5
10
15
TAS5F-SNs
in CD133+ cells (%)
PDX111 DDX111
0
5
10
15
TAS5F-SNs
in MIC+ cells (%)
(c)
**** ***
Chap e V
257
10a, he pe cen age o DTCs was model dependen , bu mo e impo an ly, we we e able
o de ec he p esence o TAS5F-SNs in he DTCs o all he models (Figu e10b). Fu he
s udies a e aimed o alida e hese indings and in es iga e i he nanoemulsions up ake
is ela ed o TAS1R3 exp ession.
Figu e 10. (a) Pe cen age o dissemina ed umo cells (DTCs) in he lungs om he p ima y
umo . (b) Pe cen age o TAS5F-SNs in DTCs. Ba s ep esen mean ± SD (n=4). * (p ≤ 0.05), ** (p
≤ 0.01) was conside ed s a is ically signi ican .
CONCLUSIONS
Fi s , we ha e demons a ed ha blocking SNs can be used o speci ically block
mac ophage up ake and inc ease nanopa icle accumula ion in lung cance cells, bo h in
i o and in i o. Then, we con i med he speci ici y o TAS5F SNs o a ge ing TAS1R3
in i o, a bioma ke associa ed wi h me as a ic p og ession in non-small cell lung cance .
No ably, he a ge ing abili y was e alua ed in mul iple models, ep esen a i e o di e en
s ages o NSCLC. Finally, we also con i med he p esence o TAS5F-SNs in o he speci ic
cell subse s associa ed wi h me as a ic o ma ion and p og ession, such as CD133 posi i e
subpopula ion, MIC and DTCs, opening new a enues o he de elopmen o speci ic
ea men s o me as a ic lung cance . All in all, gi en he e sa ili y o SNs o
accommoda e se e al he apeu ic agen s and biomolecules o a ge ing pu poses, his
echnique could p o ide impo an ad ances o he p eclinical e alua ion o hese
o mula ions in di e en ypes o cance .
X H460
CDXH460
X H1299
DDXH1299
PDX111
DDX111
0.0
0.5
1.0
1.5
2.0
% DTCs in lungs
(a) (b)
DTCs in he lungs
X H460
CDXH460
X H1299
DDXH1299
PDX111
DDX111
0
10
20
30
TAS5F-SNs in DTCs (%)
TAS5F-SNs in DTCs
**
ns
ns
*
*ns
SANDRA DÍEZ VILLARES
258
SUPPORTING INFORMATION
Table S1. Clinicopa hological pa ame e s o he NSCLC pa ien s o PDX es ablishmen .
Name
Sex
His ology
S age
Smoking habi s
258
male
SCC
IIIB
Smoke
111
emale
ADC
IIB
Non-smoke
Table S2. Physicochemical Cha ac e iza ion o block-SNs and luo-SNs measu ed by DLS and
LDA (Resul s a e Exp essed as Mean ± S anda d De ia ion, n = 3)
Fo mula ion
Size (nm)
PDIa
ZPb (mV)
Block-SNs
106 ± 1
0.11 ± 0.03
− 20 ± 3
luo-SNs
94 ± 7
0.15 ± 0.02
− 26 ± 2
aPDI: Polydispe si y Index; bZP: Ze a Po en ial
Table S3. Summa y o he NSCLC mice models used o in i o s udies.
Tumo model
O igin
Rep esen a i e o
Ob ained
PDX258
Pa ien
P ima y umo
X H460
Cell line
P ima y umo
CDXH460
Cell line
Me as a ic s age
om CTCs o
X H460
X H1299
Cell line
P ima y umo
DDXH1299
Cell line
Me as a ic s age
om DTCs o
X H1299
PDX111
Pa ien
P ima y umo
DDX111
Pa ien
Me as a ic s age
om DTCs o
PDX111
Chap e V
259
Figu e S1. Cellula iabili y o RAW264.7 mac ophages a e 2 h incuba ion wi h di e en doses
o block-SNs measu ed by low cy ome y a e 7AAD s aining. Ba s ep esen mean ± SD, n=3.
Figu e S2. (a) Schema ic ep esen a ion o he p o ocol ollowed o e alua e he a ge ing
e ec i ennes o TAS5F-SNs. (b) Cellula dis ibu ion o he umo s, whe e mos o he cells a e
mouse cells and he e o e co espond o he umo s oma. All ba s ep esen mean ± SD, n=4.
w/o
Block-SNs
0.5 1 2 5
0
20
40
60
80
100
Concen a ion block-SNs
(mg mL-1)
Pe cen age o li e cells
(7AAD nega i e)
Tumo s o Tumo s o
0
50
100
Celulla dis ibu ion
inside he umo (%)
Mouse cells Human cells
luo-SNs
g oup
TAS5F-SNs
g oup
High dose
TAS5F-SNs
luo-SNs
Tumo
Li e
Spleen
Lungs
Single cell suspension
FACS analysis
PDX258
Block-SNs
(a)
(b)
SANDRA DÍEZ VILLARES
260
Figu e S3. Ex i o biodis ibu ion o TAS5F-SNs in he pai s (a) X and CDX H460, (b) X and
DDX H1299 and (c) PDX and DDX111. (d) Nega i e co ela ion be ween he pe cen age o
TAS5F-SNs measu ed in he umo and he umo olume. (e) Cellula umo s dis ibu ion in he
di e en models, whe e he umo s oma is shown in blue and umo cells a e shown in beige. ( )
Cellula dis ibu ion o TAS5F-SNs inside he umo s, wi h a gene al majo i y accumula ed in he
umo s oma, excep o X and DDXH1299, and PDX111. Ba s ep esen mean ± SD, n= 4 pe
g oup.
Li e
Spleen
Lungs
Tumo
0
20
40
60
Pe cen age o
TAS5F-SNs+ cells
X H1299
DDXH1299
Li e
Spleen
Lungs
Tumo
0
20
40
60
Pe cen age o
TAS5F-SNs+ cells
PDX111
DDX111
Li e
Spleen
Lungs
Tumo
0
20
40
60
Pe cen age o
TAS5F-SNs+ cells
X H460
CDXH460
0500 1000 1500 2000 2500
0
10
20
30
40
50
Tumo olume (mm3)
Pe cen age o TAS5F-SNs
in he umo
R = -0.695
p alue 0.0002
(c)
(b)(a)
(d)
(e) ( )
X H460
CDXH460
X H1299
DDXH1299
PDX111
DDX111
0
40
80
120
Celulla dis ibu ion
inside he umo (%)
Mouse cells Human cells
X H460
CDXH460
X H1299
DDXH1299
PDX111
DDX111
0
5
10
20
30
40
50
Pe cen age o SNs+ cells
Mouse cells
Human cells
Chap e V
261
GATING STRATEGIES
Flow-cy ome y ga ing s a egy o de ec ion o nanopa icles in in i o cell lines
To es ima e by low-cy ome y analysis he nanopa icles up ake o RAW264.7 and lung
cance cells line, a mo phological ga e was se , and i al cells we e selec ed as 7-AAD-
cells. The ga e o nanopa icles-TopFluo -SM was hen se based on he
au o luo escence o nega i e con ol i al cells in he speci ic 488 channel and hen
e alua ing he pe cen age and he mean luo escen in ensi y (M.F.I.) o posi i e cells
ea ed wi h nanopa icles.
In co-cul u e expe imen s, an i-MHC-I an ibody was added o disc imina e
RAW264.7 mac ophages om umo cells. The pe cen age o luo-SNs+ cells was hen
e alua ed wi hin MHC-I+ mu ine li e cells and MHC-I nega i e li e umo cells.
Flow-cy ome y ga ing s a egy o in i o models
E olu ion o nanopa icles up ake in he in i o xenog a s o PDX models, was
pe o med by se ing mo phological and i al ga es as desc ibed abo e and hen analyzing
he pe cen age and M.F.I o TopFluo luo escen cells wi hin he ga e o MHC-I+ mouse
cells and wi hin he ga e o human MHC-I nega i e cells.
In lung issue, nanopa icles up ake was e alua ed bo h in mu ine cells and wi hin
lung dissemina ed umo cells (DTC). DTCs we e iden i ied by he exclusion o dead cells
and MHC-I+ mouse cells, as p e iously desc ibed in Be olini e al Cance Res 2015.
Wi hin ga ed DTC, he posi i i y o luo-SNs was hen es ima ed.
Fo analysis o li e and spleen, only i al mu ine cells we e conside ed o analysis
since any o a negligible ac ion o umo cells can be de ec ed.
Human i al cells we e also analyzed o he exp ession o CD133-PE, CXCR4-
Pe CP and TAS1R3-Alexa 647, by se ing he ga e on he au o luo escence o un ea ed
and uns ained i al cells in he p ope channel. Wi hin he ga e CD133+ cells,
CD133+CXCR4+ and TAS1R3+ popula ion, he pe cen age o cells posi i e o
nanopa icles was de e mined by he posi i i y o TopFluo -SM.
SANDRA DÍEZ VILLARES
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O e all Discussion
271
in es iga e i s po en ial o a ge me as a ic lung cance and speci ically TAS1R3 posi i e
cells in p eclinical models. No ewo hy, in all cases, e icien labeling o SNs wi h imaging
agen s was achie ed unde mild condi ions, ollowing s aigh o wa d me hodologies.
Mo eo e , we used simple ye e ec i e pu i ica ion me hods. Indeed, SNs can be
o mula ed, labeled wi h imaging agen s, and pu i ied o concen a ed o in i o
adminis a ion in less han 3-4 hou s, o e ing imesa ing, eliable, and e icien
o mula ions o in i o acking.
Rega ding he physicochemical p ope ies, imaging SNs showed op imal and
ep oducible cha ac e is ics, as can be obse ed in Table 1, which summa izes he
cha ac e iza ion o ep esen a i e o mula ions de eloped in his hesis.
Table 1. Physicochemical cha ac e iza ion o ep esen a i e SNs, measu ed by DLS and LDA.
Fo mula ion
Size (nm)
PDIa
ZPb (mV)
Vi E-MFO-SNs
149 ± 5
0.19 ± 0.05
− 42 ± 3
DTPA-SNs
136 ± 3
0.20 ± 0.02
− 51 ± 3
NOTA-SNs
151 ± 7
0.15 ± 0.01
− 50 ± 5
PEP-D o-SNs
145 ± 5
0.12 ± 0.04
+ 19 ± 4
D o-SNs
116 ± 7
0.10 ± 0.03
− 43 ± 2
[89Z ]Z -D o-SNs
132 ± 1
0.15 ± 0.04
− 40 ± 6
TAS5F-SNs
191 ± 1
0.18 ± 0.01
− 37 ± 2
aPDI: Polydispe si y Index; bZP: Ze a Po en ial
Rema kably, he hyd odynamic diame e o all SNs was main ained below 200 nm,
an impo an ac o in i o applica ions since nanopa icles la ge han 200 nm isk
ac i a ing he complemen sys em.27 Addi ionally, nanopa icles in he ange o 30-200
nm seem o be op imal o passi e umo accumula ion.5 Rega ding su ace cha ge,
excep PEP-D o-SNs all he o mula ions esul ed in high nega i e ze a po en ials.
Ex ensi e s udies ha e p o en ha besides nanopa icle size, su ace cha ge has a c i ical
impac on he blood ci cula ion and biodis ibu ion o nanoca ie s. In his sense, a e
SANDRA DÍEZ VILLARES
272
in a enous adminis a ion ca ionic NPs a e mo e p one o be clea ed han he anionic
coun e pa s, due o elec os a ic in e ac ions wi h he nega i e memb ane o
mac ophages.6 Mo eo e , he su ace cha ge densi ies also play a c ucial ole in his
ma e , and sligh ly nega i e nanopa icles a e p e e ed o p olonged ci cula ion hal -
li es.7 O ele ance, he ca ionic na u e o PEP-SNs, can ep esen an ad an age o IA
adminis a ion as ca ionic compounds ha e been desc ibed o enhance he pene a ion
and e en ion in o he a icula ca ilage. This ac is mainly due o hei elec os a ic
in e ac ion wi h he highly nega i e p o eoglycans o ming he ca ilage ma ix and he
p esence o HA in he syno ial luid.28–30
O e all, SNs can be easily and con enien ly ailo ed wi h se e al imaging agen s o
allow in i o nanopa icle acking wi hou comp omising he physicochemical
p ope ies and ollowing mild and s aigh o wa d me hodologies, impo an ac o s o
conside o de eloping ansla ional nanomedicines.
SNs can be acked by imaging echniques and adap ed o mee speci ic biomedical
applica ions
As p e iously men ioned, unde s anding he in i o a e o nanomedicines can be
e ec i ely se led by hei isualiza ion h ough non-in asi e imaging echniques.3,8,31
When nanomedicines a e adminis e ed in o he body, hey need o o e come se e al
ac o s and limi a ions o speci ically each he a ge si e. The ex en o in e ac ion wi h
each ba ie is la gely dependen on he nanopa icle design and he adminis a ion
ou e.6 In gene al, imaging-guided nanopa icle e alua ion is pe o med ollowing
in a enous adminis a ion, since i is he mos common ou e in nanomedicine
esea ch, mainly aimed a wo speci ic applica ions: he deli e y o an icance
nano he apeu ics and gene he apy.32 None heless, local adminis a ion can gi e ise o
se e al ad an ages, such as o e coming sys emic oxic side e ec s o imp o ed
he apeu ic e ec s in he a ge si e.33
O e all Discussion
273
Wi h his in mind, we aimed o explo e he po en ial o imaging-guided SNs bo h
o sys emic and local adminis a ion. Rega ding he la e , we ocused on wo ele an
biological applica ions whe e nanomedicine can p o ide p omising al e na i es o
con en ional pha maceu ics, neu ological pa hologies, and in lamma o y diseases.34,35
In he con ex o neu ological diso de s, o e coming he blood b ain ba ie is one
o he majo challenges aced by d ugs, and hus local adminis a ion may some imes be
necessa y o achie e e ec i e pha macological ea men s.36 Imaging-guided
nanomedicines can assis in he diagnosis and ea men o se e al neu ological
condi ions, including b ain umo s, ce eb al ischemia, and s okes, among o he s.37,38 In
his sense, MRI is one o he p e e ed echniques in he diagnosis o neu ological
pa hologies due o i s high spa io empo al esolu ion and unlimi ed issue pene a ion
dep h, allowing he use o mul iple sequences o isualize and e alua e he unc ionali y
and s uc u e o di e en b ain egions.39,40 In he scope o his hesis, magne ic SNs
(Vi E-MFO-SNs) e ec i ely enhanced he MR con as in he b ain a low adminis a ion
doses, due o hei high ans e se elaxi i y. As shown in Figu e 3a, magne ic SNs
p oduced a good nega i e signal a e di ec injec ion in o he b ain, compa ed wi h plain
SNs and PBS. Al hough hese a e p elimina y esul s, we can se le ha Vi E-MFO-SNs
showed po en ial as nega i e con as agen s o u he neu oimaging applica ions.
On he o he hand, in lamma o y diseases such as os eoa h i is o heuma oid
a h i is a e one o he majo causes o disabili y in he elde ly, mainly cha ac e ized by
pain in he join .41 In a-a icula (IA) adminis a ion o d ugs is a common s a egy o
selec i ely deli e ac i e molecules a hei si e o ac ion. Howe e , he syno ial luid
eplacemen and clea ance limi he d ugs’ hal -li e and es ic s hei e ec i i y.35 In
esponse, se e al deli e y sys ems ha e been de eloped aimed o inc ease he join
e en ion ime o he apeu ic moie ies.42,43 Indeed, in he hi d chap e o he p esen
hesis we p o ed ha pep ide-deco a ed SNs we e able o signi ican ly enhance join
e en ion compa ed o he ee senoly ic pep ide (Figu e 3b). On he con a y, he pep ide
showed as e clea ance, wi h highe accumula ion in he li e and kidneys. In e es ingly,
we desc ibed a simple adiolabeling app oach wi h 89Z , ha allows us o p epa e eady-
SANDRA DÍEZ VILLARES
274
o-injec 89Z -pep ide-deco a ed SNs di ec ly a e he adiolabeling o he pep ide and
s udy he biodis ibu ion o bo h compounds wi h only one adiolabeling p ocess.
Mo eo e , we epo ed o he i s ime he quan i a i e PET acking o an in a-
a icula ly adminis e ed nanopa icle, a s a egy ha could be easily adap ed o a wide
ange o d ug deli e y sys ems.
Figu e 3. (a) In apa enchymal injec ion o Vi E-MFO-SNs. T2*-weigh ed MR b ain images o
a s injec ed wi h Vi E-MFO-SNs compa ed wi h PBS and he MR signal quan i ica ion. (b) In a-
a icula injec ion o 89Z -pep ide-deco a ed SNs. Whole-body PET images showing he join
e en ion o [89Z ]Z -PEP-SNs compa ed wi h he ee pep ide and he quan i ica ion o adioac i e
signal in he injec ed knee o e ime. * (p ≤ 0.05), ** (p ≤ 0.01), ***(p ≤ 0.001).
When i comes o sys emic adminis a ion, he e a e impo an ac o s ha need o
be conside ed in he e alua ion o nanomedicines, as hey a e key playe s ha de e mine
he in i o po en ial o he de eloped nanopa icles. These include bu a e no limi ed o
he cap u e by he mononuclea phagocy e sys em (MPS), he clea ance pa hways, he
blood ci cula ion ime, and he umo up ake (in he case o oncological applica ions).5
In his wo k, he in i o beha io o SNs in heal hy oden s was in es iga ed using MRI,
Con ol (PBS) Vi E-MFO-SNs
T2
*-weigh ed image
NE
[89Z ]Z -PEP [89Z ]Z -PEP-SNs
Knee Knee
PET 72 h pos injec ion
INTRA-ARTICULAR
INJECTION
INTRAPARENCHYMAL
INJECTION
O e all Discussion
275
PET, and SPECT. Rep esen a i e esul s a e shown in Figu e 4, which allow us o
gene ally elucida e all he a o emen ioned ac o s in he speci ic case o SNs.
Fi s , we can clea ly s a e ha SNs show high accumula ion in he MPS, being
mainly cap u ed by he li e and spleen. This p ope y can be obse ed bo h in sho - and
long- e m biodis ibu ion s udies (4 h, 24 h, 48 h, and 4 days). None heless, his is a
common end in lipid-based nanopa icles, whe e a la ge p opo ion o he injec ed dose
( ypically mo e han 30% ID) is abso bed by hese o gans, and ye hey emain leade s in
clinical p ac ice because o hei excep ional biocompa ibili y.22,44,45 Based on he PET
images and he biodis ibu ion g aphs we can also obse e accumula ion in kidneys,
bladde , and eces, sugges ing ha bo h hepa obilia y and enal pa hways a e in ol ed in
he exc e ion o SNs a e hei biodeg ada ion.45,46 Impo an ly, we ound ha SNs can
be comple ely clea ed om he body a e 8 days o adminis a ion, a oiding undesi able
side e ec s obse ed wi h some nanomedicines.47,48
Finally, we can also see om Figu e 4 ha SNs had a ela i ely long ci cula ion ime.
Indeed, a 4 hou s a e in a enous injec ion, 10% o he injec ed dose emained in
bloods eam, and his was u he op imized a e PEGyla ion o SNs o he s udies wi h
gallium-67 and zi conium-89. In his ega d, liposomes gene ally exhibi longe
ci cula ion imes, mainly due o di e ences in hyd ophobici y.5 Howe e , i should be
no ed ha longe blood ci cula ion imes do no gua an ee be e he apeu ic e icacy in
he clinic.49 A clea example o his ac was demons a ed in a phase III clinical ial
compa ing he liposomal o mula ions Doxil® and Myoce ®. In his s udy, Myoce ® had
only 10% emaining in he blood a e 24 hou s compa ed wi h 50% o Doxil®, e en a e
48-72 hou s o ci cula ion.50 Howe e , no di e ences in he apeu ic e icacy we e
obse ed, and mo e impo an ly, Myoce ® did no exhibi he skin side e ec s obse ed
wi h Doxil® due o i s long ci cula ion p ope ies.51
SANDRA DÍEZ VILLARES
276
Figu e 4. (a) MRI ex i o biodis ibu ion o Vi E-MFO-SNs compa ed wi h PBS as a con ol
analyzed 48 h pos -adminis a ion. (b) Whole-body PET/CT images o [68Ga]Ga-NOTA-SNs 2 h
a e i. . injec ion and hei sho - e m biodis ibu ion 4 h pos -adminis a ion. (c) PET/CT images
o [89Z ]Z -D o-SNs up o 8 days pos i. injec ion.
Biodis ibu ion and clea ance o nanopa icles can also be a ec ed by se e al human
ac o s, as o example he pa ien sex o he umo p esence.52,53 Thus, in he las chap e
O e all Discussion
277
we aimed o in es iga e he biodis ibu ion o SNs in umo bea ing NSCLC models and,
mo e speci ically, o demons a e ha ap ame -deco a ed SNs can e ec i ely a ge a
ecep o ela ed wi h me as a ic s ages o he disease. To his end, we i s demons a ed
ha ea ly mac ophage sca enge o SNs can be e icien ly educed by high doses o
nanopa icles, consis en wi h he ecen ly disco e ed dose h eshold ha leads o
enhanced umo deli e y54 No ably, his s a egy has ou lanked o he ecen ly desc ibed
app oaches, such as he MPS blockade.55–58 In his con ex , i has been shown ha p io
injec ion o p iming nanopa icles is no necessa y o o e whelm mac ophages up ake
a es and imp o e umo accumula ion, and we ha e con i med his ac in i o and in
i o (Figu e 5a and b).
Figu e 5. (a) Mac ophages up ake sa u a ion by co-incuba ing luo escen SNs wi h block-SNs,
eaching high doses o nanopa icles (le ). Enhanced deli e y lung cance cells due o mac ophages
blockade in cocul u e wi h A549 cells ( igh ). (b) Reduc ion in li e up ake and inc ease in umo
accumula ion a e in a enous injec ion o high doses o SNs in NSCLC models. G aphs
ep esen ed as old-change o low doses. ** (p ≤ 0.01) **** (p ≤ 0.0001).
30 min 1 h 2 h 30 min 1 h 2 h
0
50
100
Pe cen age o
luo escen cells
wi hou block-SNs coincuba ion block-SNs
** **** ****
(a)
High doses o SNs
TAS5F-SNs Block-SNs
wi hou block-SNs coincuba ion block-SNs
A549
RAW264.7
**
2h Block 2h
0
50
100
150
Celulla dis ibu ion (%)
(b) Tumo
Li e
X H460
CDXH460
PDX258
DDX111
0
1
2
3
4
Tumo up ake
( old change o low dose)
X H460
CDXH460
PDX258
DDX111
0.0
0.5
1.0
1.5
2.0
Li e up ake
( old change o low dose)
SANDRA DÍEZ VILLARES
278
Subsequen ly, he a ge ing e iciency was con i med in NSCLC models wi h
di e en disease s ages de i ed om bo h pa ien s and immo alized cell lines. Mo eo e ,
accumula ion o TAS5F SNs was de ec ed no only in he TAS1R3 subse , bu also in
o he ele an cell subpopula ions associa ed wi h me as asis p og ession, such as cance
s em cells, cells ini ia ing me as asis, and dissemina ed umo cells. This highligh s he
po en ial o SNs o he de elopmen o u u e a ge ed he apies.59,60 Wi hin his ame,
he a ional design o an icance nanomedicines may change in he upcoming u u e, due
o new indings ha challenge he cen al dogma o cance nanomedicine, he EPR
e ec .61 Speci ically, i has been disco e ed ha a iny ac ion o nanopa icles
accumula es in umo s by passi e di usion ac oss in e -endo helial gaps, as desc ibed by
he EPR e ec . In ac , hese gaps we e ound o be a less abundan in he umo
ascula u e han p e iously hough . Ins ead, 97% o nanopa icles each solid umo s by
ac i e anscy osis ac oss he endo helial ba ie s.62 These obse a ions ha e spa ked
deba e abou he mechanism o nanopa icle en y in o solid umo s and he dis ibu ion
o nanopa icles wi hin he umo . Fo example, ecen epo s sugges ha umo -
associa ed mac ophages play a pi o al ole in he s oma and in he in a- umo al
dis ibu ion o nanopa icles a e ex a asa ion.63 All in all, hese indings clea ly
highligh he impo ance o unde s anding nano-bioin e ac ions o achie e e icien
deli e y o nanomedicines.4
OVERVIEW AND FUTURE PERSPECTIVES
O e all, in his hesis, we ha e aken he i s s ep owa d unde s anding he in i o
beha io o SNs and demons a ed ha hese nanopla o ms can be ailo ed wi h imaging
agen s and acked using di e en imaging modali ies. O ele ance, we ha e
demons a ed e icien umo deli e y in ele an mouse models o NSCLC o u u e
a ge ed cance he apies, as well as g ea join e en ion o in a-a icula deli e y o
pep idomime ics. In addi ion, he o gan dis ibu ion, blood ci cula ion ime and
elimina ion pa hways ha e been elucida ed in heal hy oden s. Clea ly, u he s udies a e
needed o ul il he unde s anding o SNs in i o a e, such as oxicological s udies,
O e all Discussion
279
in e ac ions wi h he immune sys em, and de ailed pha macokine ic p o iles, among
o he s.64
Addi ionally, gi en he known e sa ili y and e iciency o SNs in inco po a ing
small d ugs and he apeu ic biomolecules, u u e p ospec s may ocus on combining
hese he apeu ic SNs wi h some o he imaging agen s included in his wo k, gi ing ise
o nano he anos ics. The ield o nano he anos ics is likely o lead o mo e e icien
p eclinical de elopmen and clinical implemen a ion, as well as be e he apeu ic
ou comes.65 In his sense, he abili y o isualize and quan i y he whole-body biological
dis ibu ion and pha macokine ic o nanomedicines p o ides aluable in o ma ion in
he ea ly and la e s ages o hei p eclinical de elopmen . Conside ing u he ha pa ien
and disease he e ogenei y is widesp ead in cance , imaging migh be a key playe in he
clinic by enabling he iden i ica ion and selec ion o pa ien s mos likely o espond o
nanomedicine ea men , e e ed o as "p ecision nanomedicine".64
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i o acking by PET. Colloids Su aces B Bioin e aces 188, 110793 (2020).
20. Angulo-Molina, A. e al. Magne i e Nanopa icles Func ionalized wi h Vi amin E
Analogues: An icance E ec s. in Ma e ials Today: P oceedings 3, 703–707 (Else ie L d.,
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Cance Nano he anos ics. Ma e ials (Basel). 13, 266 (2020).
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287
CONCLUSIONS
In his hesis, o gain a be e unde s anding o he in i o beha io o sphingomyelin
nanoemulsions, hese nanopla o ms ha e been ailo ed wi h speci ic imaging agen s and
hei p eclinical in i o and ex i o biodis ibu ion has been e alua ed by di e en
echniques such as MRI, PET, SPECT and FACS. The expe imen al esul s ob ained led
us o wi hd aw he ollowing conclusions:
Chap e I
1. Sphingomyelin nanoemulsions ha e demons a ed hei po en ial o he e icien
encapsula ion o nega i e MR con as agen s, speci ically i amin E-coa ed
supe pa amagne ic manganese e i e nanopa icles. The magne ic nanoemulsions
we e ob ained by a one-s ep me hodology and showed sui able physicochemical
and mo phological p ope ies, simila o he plain o mula ions.
2. The de eloped magne ic nanoemulsions we e able o success ully enhance he MR
signal in i o and showed 2- old highe ans e se elaxi i y alues compa ed o
he ee supe pa amagne ic nanopa icles, con i ming ha he magne ic beha io
was no comp omised a e encapsula ion.
3. Magne ic sphingomyelin nanoemulsions we e p o en o be biocompa ible gi en
ha hey did no cause in i o hepa ic o enal oxici y. Mos impo an ly, hey we e
e icien ly de ec ed by ex i o MRI and in i o MR b ain imaging a low doses,
demons a ing hei po en ial as high-sensi i e MR con as agen s.
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Chap e II
4. Sphingomyelin nanoemulsions ha e been ailo ed o nuclea imaging applica ions
by inco po a ing wo di e en lipid-de i a i e chela o s h ough a simple and
ep oducible me hodology.
5. Chela o -modi ied nanoemulsions we e success ully adiolabeled wi h 68Ga and
67Ga unde mild condi ions, showing op imal adiochemical p ope ies. Mo eo e ,
hey we e ollowed-up by PET and SPECT imaging a wo di e en acking
pe iods.
6. The in i o biodis ibu ion o adiolabeled sphingomyelin nanoemulsions has been
modula ed upon modi ica ion o hei su ace p ope ies by he addi ion o
hyd ophilic polyme s.
Chap e III
7. A no el senoly ic pep ide was success ully adiolabeled wi h 89Z and hen, 89Z -
adiolabeled pep ide-loaded nanoemulsions we e e icien ly o mula ed
h ough a one-s ep me hodology. The join e en ion and he whole-body
biodis ibu ion we e acked by quan i a i e PET imaging a e in a-a icula
adminis a ion o bo h adio ace s.
8. The po en ial o sphingomyelin nanoemulsions o he in a-a icula deli e y
o senoly ic pep ides has been demons a ed since hey ha e shown a
signi ican ly highe e en ion in he injec ed join compa ed wi h he ee
pep ide.
Chap e IV
9. The bi unc ional chela o , iso hiocyana obenzyl-modi ied de e oxamine, used
o 89Z adiolabeling, has been success ully associa ed o sphingomyelin
Conclusions
289
nanoemulsions h ough h ee di e en app oaches: i) by pe o ming a chemical
eac ion, ii) by using a lipid-de i a i e and iii) by encapsula ing i in o he oil
co e o he nanoemulsions.
10. All he de e oxamine-deco a ed sphingomyelin nanoemulsions we e e icien ly
adiolabeled wi h 89Z and, in case o he chemical eac ion, nanoemulsions we e
also acked up o 8 days a e in a enous injec ion by PET imaging.
Chap e V
11. I has been demons a ed ha i is possible o speci ically block mac ophages
up ake and inc ease he nanopa icle accumula ion in lung cance cells by
p iming he li e wi h high doses o sphingomyelin nanoemulsions.
12. The ex i o biodis ibu ion o luo opho e-labeled sphingomyelin
nanoemulsions has been success ully de e mined by low cy ome y, p o ing
ha i is possible o imp o e umo accumula ion and each speci ic cell subse s
ha a e ela ed o he o ma ion and p og ession o me as asis, opening new
enues o he de elopmen o speci ic ea men s o me as a ic lung cance .
Abb e ia ions
293
ABBREVIATIONS
%ID/g
Pe cen age o injec ed dose pe g am
[18F]FBEM
[18F] luo obenzamido-N-e hylmaleimide
[89Z ]Z -PEP
89Z - adiolabeled senoly ic pep ide
[89Z ]Z -PEP-SNs
89Z - adiolabeled pep ide-modi ied sphingomyelin
nanoemulsions
%ID/o gan
Pe cen age o injec ed dose pe o gan
13C-NMR
Ca bon-13 nuclea magne ic esonance
1H-NMR
P o on nuclea magne ic esonance
67GaCl3
Gallium-67 chlo ide
ABC
Accele a ed blood clea ance
ACN
Ace oni ile
AFM
A omic o ces mic oscopy
ATCC
Ame ican Type Cul u e Collec ion
Block-SNs
Blocking sphingomyelin nanoemulsions
CDXs
CTC-de i ed xenog a
Cn l-SNs
Sphingomyelin nanoemulsions wi hou he eac i e g oup
CO2
Ca bon dioxide
COVID-19
Co ona i us disease
CT
Compu ed omog aphy
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CTCs
Ci cula ing umo cells
DDS
D ug deli e y sys ems
DDXs
DTC-de i ed xenog a
DFO
De e oxamine
D o-SNs
Chemically linked de e oxamine sphingomyelin
nanoemulsions
DIPA
Diisop opylamine
DIPEA
Diisop opyle hylamine
DLS
Dynamic ligh sca e ing
DMEM
Dulbecco’s modi ied Eagle’s cul u e medium
DMF
N,N-dime hyl o mamide
DMSO
Dime hyl sul oxide
DMSO-d6
Deu e a ed dime hyl sul oxide
DNA
Deoxy ibonucleic acid
DSPE-D o
1,2-Dis ea oyl-sn-glyce o-3-phospho yle hanolamine modi ied
de e oxamine
DSPE-PEG
Dis ea oyl-sn-glyce o-3-phosphoe hanolamine-poly(e hylene
glycol)
DSPE-PEG-2k-
NH2
1,2-dis ea oyl-sn-glyce o-3-phosphoe hanolamine-N-
[amino(polye hylene glycol)-2000]
DTCs
Dissemina ed umo cells
DTPA
1,2-dimy is oyl-sn-glyce o-3-phosphoe hanolamine-N-
die hylene iaminepen aace ic acid
DTPA-SNs
Sphingomyelin nanoemulsions including he DTPA chela o
EMA
Eu opean Medicines Agency
en.D o-SNs
sphingomyelin nanoemulsions encapsula ing de e oxamine
EPR
Enhanced pe meabili y and e en ion e ec
Abb e ia ions
295
ESI-MS
Elec osp ay ioniza ion mass spec ome y
FA
Folic acid
FACS
Fluo escence-ac i a ed cell so ing
FBS
Fe al bo ine se um
FC
Field-cooling
FCC
Face-cen e ed cubic
FDA
Food and D ug Adminis a ion
Fe(acac)3
I on(III) ace ylace ona e
FESEM
Field emission scanning elec on mic oscopy
luo-SNs
luo opho e-labeled sphingomyelin nanoemulsions
FOV
Field o iew
GEE
Gene alized es ima ed equa ions
GOT
Glu ama e oxaloace a e ansaminase
GRAS
Gene ally ecognized as sa e
H
Hypo heses
HA
Hyalu onic acid
HCl
Hyd ochlo ic acid
HEPES
4-(2-hyd oxye hyl)-1-pipe azinee hanesul onic acid
HPLC
High pe o mance liquid ch oma og aphy
i.
In a enous injec ion
IA
In a-a icula
IC
In o ma ion coe icien
ICP-OES
Induc i ely coupled plasma op ical emission spec ome y
iTLC
Ins an hin laye ch oma og aphy
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KCl
Po assium chlo ide
KH2PO4
Po assium phospha e monobasic
LDA
Lase Dopple anemome y
lip.D o-SNs
sphingomyelin nanoemulsions con aining lipid-de i a i e
de e oxamine
lncRNA
Long non-coding RNA
mAb
Monoclonal an ibody
MAR
Mo ional a e age egime
MFM
Magne ic o ces mic oscopy
MFO
Manganese e i e nanopa icles
MICs
Me as a ic ini ia ing cells
miRNA
Mic o-RNA
MLEM
Maximum likelihood expec a ion maximiza ion
Mn(acac)2
Manganese (II) ace ylace ona e
MPS
Mononuclea phagocy e sys em
MRI
Magne ic esonance imaging
mRNA
Messenge RNA
MSME
Mul i-slice mul i-spin-echo
MW
Molecula weigh
Na2CO3
Sodium ca bona e
Na2HPO4
Sodium phospha e dibasic
NaCl
Sodium chlo ide
NaOH
Sodium hyd oxide
303
Ag adecimien os
A San iago ine, lige i a, caminando, y con mi pa agüi as; p epa ada pa a odo lo que
llega a, e ilusionada po deja la química pu a a un lado y aden a me en el mundo de
a macia a a és de un más e de in es igación y desa ollo de medicamen os.
In es igación e a el oco del más e y, aun así, ilusa de mí, lo que menos eía eni e a que
acaba ía haciendo una esis doc o al. Aho a, seis años después, dejo San iago con más de
20 pa aguas pe didos y me oy de odo, menos lige i a. Y es que, Compos ela ha jugado
con los lími es de mis emociones más de mil eces y ha conseguido que aho a me aya,
no solo con un lib o en la mano, que de po sí ya pesa (sob e odo psicológicamen e), sino
ambién ca gada de con ianza, buenísimos ecue dos, muchísimas ca cajadas, algún que
o o disgus ín y, an e odo, llena a e en a de un inmenso ca iño y apoyo. Y es que, a
pesa de las incon ables ho as de abajo y de odo el es ue zo que he dedicado pa a da
lo mejo de mí, no me cabe la meno duda de que si no hubie a sido po ese ca iño y
apoyo de odas las pe sonas que menciono a con inuación, me hubie a ido hace años, con
las manos acías.
En p ime luga , me gus a ía ag adece a mi di ec o a de esis, Ma ía de la Fuen e,
y a mi u o , Ra a López, po habe me acogido en el g upo, guiado du an e la elabo ación
de es a esis y, sob e odo, po habe me pe mi ido ap ende a ues o lado. Ma ía, g acias
po habe con iado en mi desde el p ime minu o, po que uis e ú la que un día me
con enció pa a comenza es e camino. G acias po se ejemplo, po de ende que un
expedien e académico no a ligado al alen o, po hace nos espabila pa a saca nos las
cas añas del uego soli as y po u in ini o in e és en e nos c ece p o esionalmen e. Ha
sido odo un place o ma pa e de u g upo y e lo e oluciona a lo la go de es os años.
SANDRA DÍEZ VILLARES
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C eo que hay pocas cosas más en iquecedo as en la in es igación que pode
dis u a de la colabo ación que hay en ella. He enido la inmensa sue e de colabo a con
eno mes p o esionales que han apo ado muchísimo alo a cada capí ulo de es a esis,
con sus ideas, su apoyo incondicional y, sin duda, haciendo un abajo excepcional.
G acias de co azón, po que es a esis no se ía ni la mi ad sin oso os.
G acias a Ve ónica Salguei iño y Miguel A. Ramos, po que con oso os empecé
es e camino y aho a enemos una boni a publicación, e lejo de odo el cu o que nos
pegamos. G acias al g upo de neu o, en especial a Ramón Iglesias, And és da Sil a y Pablo
He ella. G acias po aguan a me cada ez que bajaba a queja me desespe ada con
nue as dudas, po ayuda me en odo lo que es aba en ues a mano y, sob e odo, po
hace lo siemp e con una son isa. G acias, no sabéis el ca iño que os engo. Pablo, g acias
a i en especial, po que u ayuda ha sido in ini a, siemp e man eniendo u oque de
humo , acile y ganas de co illeo. A la hogue a con las ino gánicas.
G acias ambién a Fe nando He anz, Juan Pellico, Ramón E i ja y San iago
G ijal o, po que a pesa de la dis ancia siemp e he sen ido ues o apoyo muy de ce ca.
Juan, a i e engo que ag adece especialmen e, po se mi p ime men o en el mundo
adioac i o y po ene siemp e palab as boni as hacia mi abajo. Espe o since amen e
que ol amos a coincidi algún día.
G acias al g upo de medicina nuclea , p ác icamen e mi segundo labo a o io. Pablo
Aguia , Noemí y La a, g acias de co azón, po que pod ía esc ibi o o lib o con ando
odo lo que he ap endido de oso os. Noemí, g acias po u dulzu a y amabilidad, las
a inas adiac i as es án en buenísimas manos bajo u cuidado. Pablo, has sido
p ác icamen e mi di ec o de esis en la somb a, g acias de e dad, po que más de la
mi ad de es e abajo ue bajo u supe isión y lo he dis u ado eno memen e. G acias po
u ce canía, po es a siemp e ahí pa a discu i ideas y echa una mano. La a, ú has sido
mi g an descub imien o de es os dos úl imos años. Tu ocación es admi able, me
con agias y de uel es la pasión po la in es igación cuando es oy a u lado, aunque sea
po un a i o. C eo que no he discu ido con nadie en el abajo an o como con igo, a cada
cual más cabezona con su campo, y, aun así, de odas las ho as que nos echamos jun as
Ag adecimien os
305
siemp e han salido g andes ideas. G acias, he ap endido muchísimo de i, la ciencia iene
sue e de ene e.
G azie mille al g uppo di Genomica Tumo ale dell'Is i u o Nazionale dei Tumo i.
Luca Roz e Giulia Be olini, g azie pe a e mi accol o e a o sen i e come una del g uppo,
ho impa a o mol o con oi. Max, C is ina, Fede, Mi iam, g azie pe esse e semp e s a i
dispos i ad aiu a mi, insegna mi e p ende si cu a di me in modo che mi sen issi semp e
a casa. G azie sop a u o ai miei e mosche ie i, Giuli, El i e Fabio, non sa e e quan o
i ing azio pe u o l'amo e che mi a e e da o in dal p imo gio no. G azie pe i ca è
quo idiani di sollie o, il p anzo insieme, le isa e den o e uo i il labo a o io e pe a e
condi iso un po' della os a I alia con me, i ado o e mi manca e mol issimo.
Millones de g acias ambién a mi g upo, nano se ha hecho eno me con oso as y
yo he enido la sue e de i i lo muy de ce ca. Ma a, Belén, Su asa, Fa imah, Lucía,
And ea, Alba, Miguel, Abi, Au o a, Ma ce, Rebeca, So ía, C is, Olaia, Raneem, Alex, Inés,
Lau a T, Bá ba a, Ne ea, Gab iela, g acias po habe compa ido conmigo es e g upazo.
Y aho a iene lo bueno, po que compañe as ya se queda muy co o pa a desc ibi el
g upo que hemos o mado y del que me sien o muy a o unada de o ma pa e. Qué
boni o ha sido compa i jo nadas in e minables, ca és de media mañana, comidas que
se ala gan ho as, cenas, cañas y ies as ue a del labo a o io, e incluso iajes jun as. Los
momen os du os se han hecho mucho más lle ade os a ues o lado. Jen, po u ac i ud
siemp e posi i a, u dulzu a y sabe es a , po apo a siemp e ese pun o de co du a y
sabidu ía, g acias collacia, la ep esen ación de la ie ina en el lab queda de u mano.
Ma ía, e como has e olucionado desde que en as e al lab siendo un ma de dudas ha
sido odo un lujazo. Oes, g acias po odos los ca és y cañas de desahogo y desconexión,
po es a siemp e a pie del cañón cuando alguien lo necesi a y po que es imposible no
que e e incluso cuando ese TOC de esc úpulos y o ganización choca de mo os con mi
yo desas e. Fue za, ojalá e ie as con mis ojos de o gullo, ya queda menos peixiño. A
mis nanobebes, So i y Lau, g acias in ini as po habe os quedado en el lab, desoyendo
odo consejo que os daban unas doc o andas e e anas exhaus as y has iadas. Sin duda
habéis aído luz, escu a y una inmensa aleg ía al g upo y yo es oy encan ada de ene os.
SANDRA DÍEZ VILLARES
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So i, mi bebe, po u na u alidad, u pe sonalidad y po esa bondad y e nu a que
desp endes, g acias. Lau, g acias po ese humo an uyo e inago able, po que he ganado
años de ida con odo lo que me eído a u lado, no conosco a nadie con esa sol u a y
g acia que ú ienes. A las dos, os desea ía sue e, pe o es que no la necesi áis, sois unas
cien í icas inc eíbles. Emma, hemos coincidido poquísimo y ya e gua do emendo
ca iño, g acias po o ece u ayuda desin e esadamen e y po apoya me casi sin
conoce me. Dejo en buenas manos el ele o del eam adioma caxe. Y, po encima de
odo, g acias de aquí a León pasando po Pon e ed a a mis dos pila es undamen ales,
mis pun os de apoyo y mo i ación incondicional, Sand i y Sai. Habe empezado y
compa ido nano-onco con oso as ha sido sin duda de lo mejo de es os años. Sand i,
po se mi gambe a a o i a y la e dade a p ime a o icial del labo a o io, po que los
ecue dos más di e idos de las noi es de Compos ela ienen g abado u nomb e, y los de
los días ambién; po p eocupa e po odos y da odo de i de la mane a más since a que
conozco, po celeb a jun as odos los log os y ahoga los d amas en muchas ce ezas,
po nues a complicidad y po u ue za, g acias pichi, de co azón, has sido ejemplo pa a
mi an o p o esional como pe sonalmen e. Sai, D a Lo es, a i in ini as g acias, po que
ienes mucha culpa de que haya conseguido llega has a aquí. No se me ocu e nadie
mejo que u pa a habe compa ido una esis en e a desde los inicios, con odos los
momen azos, los iajes a cong esos, las escapadas de ines de semana y e ano, y, ambién,
odos los momen os de ago amien o y desespe ación en los que a eces nos imos
en uel as. Tu no pudis e desc ibi lo mejo y yo e lo de uel o aquí, g acias po u ayuda
since a, po u emenda empa ía, y po es a siemp e dándome ideas pa a que
consiguie a hace las cosas un poqui o mejo . Y es que aho a ecue do con mo iña
nues os la gos días deba iendo sob e los p óximos expe imen os, p esen aciones o
publicaciones, aconsejándonos siemp e con el único in de hace nos b illa la una a la
o a, y aya que si b illas e. Y segui ás b illando Sai, y yo lo e é de ce ca.
Po supues o, ambién quie o da las g acias al club de Onco al comple o, po que
odo lo que dis u amos jun os se queda en mi ecue do pa a siemp e, po que hemos
con e ido en humo odos nues os d amas de labo a o io y las b omas son ya
Ag adecimien os
307
incon ables, po que los días de cañas, oncocenas y casas u ales siemp e ue on chu e de
ene gía pa a ol e con ganas al hospi al, po que las medias jo nadas acompañados se
lle an mejo y po que xun os a anzamos, siemp e. Ca ol, Nu ia, Pablico, Inés, Ai o ín,
Osca ín, Ana, Rachel, Manu y Ramón, a odos, g acias con odas mis ue zas. G acias de
mane a muy especial a Pa i, Ca los y Aida, po que engo la inmensa sue e de ene los
siemp e a mi lado. Pa i, g acias po se i amina, po la acilidad que ienes pa a aleg a le
el día a odo el que se c uza con igo con esa son isa imbo able, y po habe compa ido
los mejo es momen os de desconexión de es a e apa conmigo. Po más su y más iajes
jun as eco iendo islas, nos espe a uno de los g andes al e mina . Ca los, mi che
a o i o, mi ey del d ama y compañe o de inos, g acias po es a ahí a oque de llamada
pa a odo lo que hicie a al a, po las cenas en casa, los e mu s de abas os al escaso sol de
San iago y odas las ca cajadas y planazos que compa imos jun os, ya casi lo ienes,
ue za pichi. Aida, g acias po acompaña me du an e oda es a época, sob e odo du an e
la esc i u a de es e lib o, i a la pa con igo y pode compa i nues o agobio me ha dado
la ida. G acias po u emendo humo , los momen os de es alle y odos us consejos,
hemos llegado solo medio cucus, mil g acias chuli.
Me gus a ía ag adece ambién al es o de pe sonas del IDIS que me han
ayudado de una mane a u o a du an e es a época, en pa icula a odo el g upo de
ONCOMET, al labo a o io 13, al labo a o io 18, y al labo a o io de nanomedicina del
CIMUS, g acias po odo.
G acias de co azón a Albe , Alba M, Albi y Ne e, que hicie on que San iago ue a
mi segunda casa du an e es os años. Ne e, a i millones de g acias, po que i i con igo
ha sido un lujo, po las pelis y planes de umán icas, po que siemp e eco da é San iaguín
jun as, po eco e nos odas las e benas y concie os de Galicia pa a bailo ea al son
No edades Ca minha y O iga. Po nues os planes de in de semana en busca de sol,
como dos laga ijas, y po nues os iajes a O iedín en u cobe e, g acias cuca.
G acias a odos mis amigos de O iedín, po que los ines de semana de uel a a casa
siemp e ue on un soplo de ai e esco y las uel as a San iago solían hace se e e nas
después de habe dis u ado dos días a su lado, Ba e a, Méndez, San, Hila, Gonzolo y
SANDRA DÍEZ VILLARES
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demás peñi, g acias in ini as. G acias en pa icula a mis einas Luci, I e y Lau po
apoya me de mane a incondicional es os años. Lau, g acias po odos los ideoca és, po
da me con ianza y segu idad, po odo el iempo jun as, po us bo e adas de ealidad
cuando lo necesi aba y, an e odo, po es a siemp e a mi lado. I e, g acias po las
excu siones a pe de nos cuando necesi aba desconec a , po eno gullece e de mí más
que yo misma y po lui conmigo a donde ue a. G acias ambién a oda mi xen e de
Sanci, los de la plaza do Luga , en especial a Paloma, Ge mán, Kiko y Lucía, po que las
acaciones son a ues o lado desde pequeñi os. Luchi, g acias po se amiga pa a odo,
nos oca ese iajazo mochile o que an o lle amos espe ando. G azie mille o my
Lomba día People, Ma us, Ma io, Ioanna, Ma ía, y Diego, because i was such a su p ise
o mee you and because you made my s ay in Milan some o he bes mon hs o my li e.
Non edo l'o a di o na e la Da sena, con bi e e pa a ine, pe gua da e insieme il
amon o e cena e con gli a os icini alla ine ia. Lu u aga, Yamass.
Po úl imo, y po encima de odo, quie o da las g acias a mi amilia, po que me lo
han dado odo y han hecho lo posible pa a que hoy es é aquí. G acias a mis abuelos,
po que a odos les hubie a encan ado e he donde he llegado. Pa ón es o a po i.
Menchu, uis e mi maes a desde pequeña, no es el lib o li e a io que espe abas, pe o es
un lib o igualmen e y sé que es a ías o gullosa. G acias eno mes a mi abuela Pepa, po
acoge me y cuida me mien as esc ibía es o, po que da nos las buenas noches ue nues o
momen o especial del día du an e meses. Sul, g acias po se un g an apoyo en la
dis ancia, po que lle as mi esis en u piel y eso no se me ol ida. Y mis mayo es GRACIAS
es pa a mis pad es, po que odo es o es g acias a oso os y aunque muchas eces no lo
demues e lo su icien e, os quie o con locu a. G acias a los dos po sopo a me cuando
ni yo misma me aguan aba. Mamá, g acias po es a ahí siemp e, po hace que odo ue a
más sencillo pa a mí y po u amo in ini o. Papá, has sido y se ás mi ejemplo siemp e,
admi o u bondad, u empa ía, us ganas, la o ma que ienes de cuida nos y de que e me-
Siemp e se é niña de Papá.
A odos y cada uno, de e dad, in ini as g acias; es e lib o al comple o y pa e de quien
soy hoy os lo debo a oso os.
E hical issues and
annexes
311
ETHICAL ISSUES
1. ANIMAL STUDIES
All animal p ocedu es we e conduc ed in acco dance wi h he ins i u ional and na ional
guidelines o he animal ca e and wel a e, he ARRIVE guidelines and acco ding o he
Spanish and Eu opean Union ules (86/609/CEE, 2003/65/CE, 2010/63/EU and
RD53/2013). All expe imen al p o ocols we e app o ed by he co esponden Animal
Ca e Commi ee and he ins i u ional o ganiza ion. P ocedu e numbe s used o each
chap e a e de ailed below:
Chap e I – 15010/2019/004 app o ed by Xun a de Galicia
Chap e II – PROEX16/277, app o ed by Mad id egional go e nmen .
Chap e III and chap e IV – 15012/2022/011, app o ed by Xun a de Galicia
Chap e V – 50/2022-PR (P o . 74312.50), app o ed by he I alian Minis y o Heal h.
2. HUMAN CELL CULTURE
All cance lines used in his wo k we e acqui ed om comme cially a ailable esou ces
(Ame ican Tissue Cul u e Collec ion, ATCC) and cul u ed in he condi ions
ecommended by he manu ac u e s and only used o he esea ch pu poses speci ically
desc ibed in he p esen hesis.
SANDRA DÍEZ VILLARES
312
3. PATIENT’S SAMPLES
Blood samples we e collec ed in acco dance wi h he guidelines and p o ocols app o ed
by he Ins i u ional E hical Commi ees, Galician Clinical Resea ch E hics Commi ee,
SERGAS: code app o al: 2017/538. All indi iduals signed in o med consen o ms and
could wi hd aw hei consen a any ime. The s udy was pe o med in acco dance wi h
he Decla a ion o Helsinki.
4. IMAGES USE
Unless cla i ied, I ha e p oduced all he images p esen ed in his hesis. In he case o
images eused o adap ed om o he manusc ip s, pe mission has been asked o he
publishe s, and he mode o legal use has been cla i ied a he bo om o he
co esponding igu es. In addi ion, he ee esou ce Fla icon.com was used o he
igu e’s p epa a ion.
Annex II
319
PEGyla ion E ec s on he In e ac ion o Sphingomyelin
Nanoemulsions wi h Se um Albumin: A The modynamic
In es iga ion
Daniela Gheo ghe1, Sand a Díez-Villa es2,3,4, Romica Sandu1, Ana Neacsu1, Dana-And eea
Neacsu1, Ad iana Se ban1, Alina Bo ea-Pe cu, Jeni e Ga cia-Fe nandez2, Ra ael López2,4, Ma ia de
la Fuen e F ei e2,4,5*, Flo ina Teodo escu1* and Spe an a Tanasescu1*
1Labo a o y o Chemical The modynamics, “Ilie Mu gulescu” Ins i u e o Physical Chemis y, Splaiul
Independen ei 202, 060021, Bucha es , Romania
2Nano-Oncology and T ansla ional The apeu ics Uni , Heal h Resea ch Ins i u e o San iago de Compos ela
(IDIS), SERGAS, San iago de Compos ela 15706, Spain.
3Uni e si y o San iago de Compos ela (USC), San iago de Compos ela 15706, Spain.
4 Biomedical Resea ch Ne wo king Cen e on Oncology (CIBERONC), Mad id 28029, Spain.
5DIVERSA Technologies SL, Edi icio Emp endia, Uni e sidade de San iago de Compos ela, 15782 San iago de
Compos ela, Spain !
ABSTRACT
The ecen ocus in he de elopmen o no el nanosys ems o biomedical applica ions
lays i mly on hei in e ac ions wi h biomolecules. The modynamic pa ame e s d i ing
he in e ac ion be ween nanopa icles and p o eins p o ide insigh s in o complex
p ocesses a bio/nanoin e ace. The p esen wo k aims o in es iga e he binding
mechanisms and he dominan con ibu ions ha de e mine he adso p ion p ocesses
du ing he in e ac ions o a model p o ein, i.e., bo ine se um albumin, wi h a new ype
o d ug deli e y sys ems, Vi amin E / sphingomyelin nanoemulsions, plain and coa ed
wi h polye hylene glycol and D-ɑ- ocophe yl polye hylene glycol succina e. The binding
pa ame e s (binding cons an , binding s oichiome y, en halpy, Gibbs ene gy, and
en opy changes o binding) a e e alua ed by he iso he mal i a ion calo ime y wi h a
Mic oCaliTC200 equipmen . The e ec o nanoemulsions on he p o ein s abili y is
examined by measu ing he he modynamic pa ame e s o he p o eins un olding (hea
SANDRA DÍEZ VILLARES
320
capaci y; en halpy, en opy and ee ene gy changes) wi h a NanoDSC (TA Ins umen )
appa a us. The he modynamic p o ile shows o all composi ions an en opy-d i en
in e ac ion domina ed by hyd ophobic o ces due o he ea angemen s/displacemen
o he su ounding wa e molecules, while main aining he na i e con o ma ion o he
p o ein. All he in o ma ion acqui ed by he modynamic app oach may signi ican ly
enhance he knowledge wi h special ocus on PEGyla ed nanoemulsions used o
biomedical applica ions.
KEYWORDS
Sphingomyelin nanoemulsions, nanoemulsion - p o ein in e ac ions, he modynamic
pa ame e s, PEGyla ion, su ace coa ings, bo ine se um albumin.
!
!
!
!
!
In e ac ion a bio/nano in e ace
SNs
SNs-PEG50
SNs-PEG100
SNs-PEG50-TPGS
SNs-PEG100-TPGS
SNs-TPGS50
SNs-TPGS100
-20
-10
0
10
20
30
40
50
DH
T
DS
DG
kJ mol-1
The mal s abili y o he p o ein
ollowing he in e ac ion
TiO2(NM-101) >SiO2(NM-203)
En opic cha ac e o he in e ac ion
ΔG=ΔH-TΔS
Wa e – media ed
binding
in e ac ion
Annex II
321
The modynamic p o ile o he in e ac ion be ween ap ame -
unc ionalized sphingomyelin nanoemulsions and plasma p o eins
Ad iana Se ban1, Jeni e Ga cia-Fe nandez2, Daniela Gheo ghe1, Romica Sandu1, Ana Neacsu1,
Dana-And eea Neacsu1, Alina Bo ea-Pe cu, Sand a Díez Villa es2,3, Flo ina Teodo escu1, Ma ia
de la Fuen e F ei e2,3,4*, and Spe an a Tanasescu1*
1Labo a o y o Chemical The modynamics, “Ilie Mu gulescu” Ins i u e o Physical Chemis y, Splaiul
Independen ei 202, 060021, Bucha es , Romania
2Nano-Oncology and T ansla ional The apeu ics Uni , Heal h Resea ch Ins i u e o San iago de Compos ela
(IDIS), SERGAS, San iago de Compos ela 15706, Spain.
3Uni e si y o San iago de Compos ela (USC), San iago de Compos ela 15706, Spain.
4DIVERSA Technologies SL, Edi icio Emp endia, Uni e sidade de San iago de Compos ela, 15782 San iago de
Compos ela, Spain !
ABSTRACT
The s udy o he in e ac ions be ween nanopa icles and biomolecules is a undamen al
challenge o nanomedicine esea ch. The he modynamic pa ame e s d i ing hese
in e ac ions o e inno a i e insigh s in o he p o ein adso p ion p ocesses ha may
impac he biological esponses. In he p esen wo k he modynamic signa u e associa ed
o he in e ac ion o some Vi aminE-sphingomyelin nanoemulsions (SNs) conjuga ed o
ap ame s ( wo di e en sequences, APT1 and APT2) wi h h ee model p o eins (bo ine
se um albumin, immunoglobulin G and bo ine plasma ib inogen) we e in es iga ed.
SNs ha e good p ope ies in e ms o biodeg adabili y, s abili y in biological media,
biocompa ibili y, and signi ican e sa ili y, being able o associa e wi h a wide a ie y o
molecules in cance diagnosis and he apy. Ap ame s a e single-s anded
oligonucleo ides ha can bind o a ge molecules wi h excellen speci ici y and a ini y
due o hei 3D con o ma ional s uc u e. Thei ela i ely sho hal -li e and suscep ibili y
o nuclease deg ada ion made ap ame -nanoma e ials hyb ids become mo e compe i i e
candida es o clinical applica ions. The binding pa ame e s o he SNs and SNs-APT
SANDRA DÍEZ VILLARES
322
in e ac ion wi h p o eins (binding cons an , binding s oichiome y, binding en halpy,
en opy and Gibbs ee ene gy) we e e alua ed by iso he mal i a ion calo ime y. The
p o eins we e also analyzed ee in solu ion and adso bed on o he nanosys ems o
examine he changes in hei he mal s abili y. The he modynamic inge p in shows
ha he binding has an en opic cha ac e domina ed by he en opy gain associa ed wi h
sol en elease and eo ganiza ion. The en opic cha ac e inc easing depends on bo h
p o ein and ap ame ype. The he modynamic app oach o such binding e en s may aid
he u u e de elopmen o e icien o mula ions wi h po en ial o speci ic applica ions
in nanomedicine.
KEYWORDS
Sphingomyelin nanoemulsions; ap ame ; plasma p o eins, he modynamics in e ac ion,
p o ein co ona.
Annex II
323
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Cance is one o he majo heal h p oblems wo ldwide due o i s high a e o mo bidi y and mo ali y. Among
he di e en ypes o cance , b eas cance is he second mos p e alen , wi h an es ima ion o wo million new
cases in 2018, and is he leading cause o dea h in women wo ldwide1. I ep esen s a he e ogeneous g oup o
umo s ha is cu en ly classi ied in i e di e en sub ypes acco ding o hei his ological and molecula pa e ns2.
Among hese sub ypes, TNBC accoun s o 15–20% o he cases. TNBC lacks o he exp ession o es ogen and
p oges e one ecep o s and human epide mal g ow h ac o 2 ecep o (HER2). This sub ype is known o i s
he e ogenei y, immunogenici y and agg essi e biological beha io , high me as a ic a e, and complex me as a ic
pa e ns, al oge he leading o a high equency o elapse and low su i al a es2,3.
Conside ing he lack o e icien a ge ed he apies, chemo he apy is s ill he mos commonly used ea -
men o TNBC2,3. Howe e , chemo he apeu ic d ugs a e well known o being esponsible o se e al ad e se
e ec s in pa ien s due o hei poo accumula ion in he umo si e and me aboliza ion be o e eaching i , which
augmen hei in insic high oxic p o ile4. O he he apeu ics as DNA damage agen s, angiogenesis inhibi o s,
an i-and ogens and immune checkpoin inhibi o s a e unde clinical e alua ion5,6.
Edel osine (1ᒧOᒧoc adecylᒧ2ᒧOᒧme hylᒧsnᒧglyce oᒧ3ᒧphosphocholine o ET) is a syn he ic lipid membe
o alkyl-lysophospholipids amily ha unlike o he chemo he apeu ic d ugs does no ac on he le el o DNA.
I s high apop o ic ac ion on umo cells is in pa ela ed o i s accumula ion in hei plasma memb ane and
ac i a ion o lipid a s7,8. Edel osine was es ed in phase I9 and phase II10 ials in pa ien s wi h acu e leukemia,
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by in a enous and o al adminis a ion, showing p omising esul s in pu ging bone ma ow o ansplan a ion.
Edel osine was also es ed in a phase II s udy in pa ien s su e ing om b ain umo s (inope able o p e iously
ea ed wi h o he he apies showing no posi i e esul s), by o al adminis a ion. The esul s showed ha ET has
he capaci y o s op b ain umo g ow h and imp o e pa ien ’s quali y o li e11. S ill, when adminis e ed o al o
in a enously, his d ug leads o gas oin es inal and hemoly ic oxici y, espec i ely12–14.
Nano echnology has al eady demons a ed i s capaci y o imp o e he access o chemo he apeu ic d ugs o
he si e o ac ion while dec easing hei seconda y side e ec s. In his sense, di e en au ho s ha e p oposed
ET encapsula ion in lipidic nanosys ems in o de o dec ease i s ela ed oxici y issues while imp o ing i s bio-
a ailabili y, and he e o e i s e ec i eness on he ea men o di e en cance s, such as lymphoma15, leukemia16,
os eosa coma17, b eas cance 18, glioma19 as well as ela ed me as asis20, among o he s.
Rega ding b eas cance applica ion, Azna e al. obse ed a s ong inhibi ion o MCF7 cells (human b eas
cance cell line wi h es ogen and p oges e one ecep o s, i. e. no TNBC-de i ed) p oli e a ion and a no ably
dec ease in he cell iabili y upon he ea men wi h ET lipid nanopa icles in compa ison o he ee d ug.
Despi e he encou aging esul s, he pe o mance o his o mula ion was no s udied in i o. In he case o
TNBC, conside ing i s high me as a ic a e in b ain a ea lie s ages, Ren e al. showed ha mice injec ed wi h
b ain me as a ic cells de i ed om TNBC pa ien s, p esen ed a signi ican inhibi ion o b ain me as a ic umo
g ow h as well as o he o ma ion o mac o-me as ases upon he ea men wi h ee ET21.
Taking his in o conside a ion, in his wo k we p opose he p epa a ion o nanome ic emulsions comp ising
edel osine (ET-NEs) o he managemen o TNBC. Unlike he p e iously men ioned s udies ha used me hods
as high shea homogeniza ion and ul asonica ion o he p epa a ion o he lipidic nanopa icles, he ein we used
he simple and mild me hodology o e hanol injec ion, and na u al cell componen s as phospha idylcholine (PC)
and iglyce ides as excipien s, he eby a oiding he use o su ac an s.
ET-NEs’ oxici y was es ed bo h in i o, using a highly agg essi e and in asi e TNBC cell line (MDA-
MB-231) and in i o using zeb a ish (Danio e io) as animal model. Zeb a ish model was selec ed since i p o-
ides he complexi y o in i o condi ions ha he in i o cell assays canno and p esen s a ele an s uc u al and
unc ional homology o humans wi h mo e han 70% o o hologue human genes22. Fu he mo e, in compa ison
o commonly used oden animal models i is a mo e cos -e ec i e and less ime-consuming model due o i s
as e de elopmen and small physical size. Fo ins ance, basic de elopmen is nea ly comple ed wi hin 24h and
sexual ma u a ion is eached in 3–5mon hs. None heless, one o i s main a ac i e ea u es is he anspa en
body o zeb a ish emb yos and la ae, which allows a eal- ime acking o he adminis e ed o injec ed luo es-
cen ly labeled d ugs/nanoca ie s and cells o in e es 23.
Rega ding he cance ield, di e en zeb a ish cance models ha e been de eloped by ansgene exp ession
and xeno ansplan a ion o human umo cell lines o p ima y pa ien -de i ed cells, among o he me hods. The e
a e some unique ea u es ha make zeb a ish an ideal cance model, being especially ele an i s anspa ency,
which as men ioned abo e allows o moni o umo de elopmen and me as ases o ma ion and sp eading, as
well as hei esponse o ea men s24–26. Addi ionally, he delay in he adap i e immune sys em de elopmen
(10–14days) poses a clea ad an age o e oden models, as he immunosupp ession o animals is no equi ed27.
I is also wo h no ing ha neo ascula iza ion can be also s udied due o he simila i y be ween zeb a ish and
humans’ ascula u e28. Jus as impo an ly, acco ding o he Eu opean Food Sa e y Adminis a ion, zeb a ish
up o 5days pos - e iliza ion (dp ) a e less p one o expe ience any pain he eby complying wi h e hical con-
side a ions on animal expe imen a ion (3R p inciple)29. Fo hese easons, zeb a ish cance models a e gaining
ele ance and di e en esea ch g oups a e using hem o he e alua ion o a numbe o d ugs30–32 as well as
nanomedicines33–35, o en as complemen a y models o he mu ine ones. On he o he hand, Nada e al., used
zeb a ish MDA-MB-231 xenog a model o es ing hei pla inum/hyd oxyapa i e nanopa icles-based he apy
agains bone cance , conside ing he high a e o his ype o b eas o me as asize in o bone36.
Following his line, in he p esen wo k we decided o de e mine he e icacy o he de eloped nanosys em
in i o and in i o in a zeb a ish TNBC model o s udy he an i umo al po en ial o he p oposed edel osine
nanosys ems.
Ǥ ET-NEs, and hei con ol o mula ion (C-NEs) we e
p epa ed by adap ing he e hanol injec ion me hod p e iously op imized by ou g oup o he p epa a ion o
nanome ic emulsions on a single s ep37,38, allowing he s aigh o wa d o ma ion o he nanosys ems, as ep-
esen ed in Fig.1. Edel osine is composed by a long ca bon chain, and phospha e and qua e na y amine g oups.
Conside ing i s lipophilici y, i is expec ed ha i can o m emulsions upon combina ion wi h an oil (Miglyol)
Figu e1. Schema ic ep esen a ion o con ol (C-NEs) and edel osine (ET-NEs) nanosys ems.
Annex II
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and an addi ional phospholipid(PC). While he ca bon chain can be inco po a ed in o he oily co e o he s uc-
u e, he phospha e and qua e na y amine g oups can ge exposed on i s su ace, simila ly o PC. Indeed, ET
was e icien ly o mula ed ollowing his p ocess. ET-NEs composed o 85, 10.7 and 4.3% o Miglyol, ET and PC,
espec i ely, p esen ed a small pa icle size, a monodispe se popula ion, and a neu al ze a po en ial (Table1).
The con ol o mula ion (C-NEs) was also p epa ed by eplacing ET o PC, p esen ing a inal composi ion o
85 and 15% o he excipien s Miglyol and PC, espec i ely, which esul ed in a simila neu al ze a po en ial in
compa ison o ET-NEs and a sligh ly smalle a e age size o 100nm (Table1).
The nanoca ie s p ope ies a e in luenced by he nanoca ie composi ion, encapsula ed d ug, as well as he
o mula ion echnique and used sol en s. Fo ins ance, using a di e en p opo ion o leci hin (a ype o PC)/
Miglyol o indome hacin encapsula ion, ace one as sol en , as well as he pou ing me hod, led o he o ma-
ion o pa icles o abou 220 nm39. Eskanda e al. also p epa ed leci hin/Miglyol pa icles o abou 213nm
( o ans- e inol deli e y) by using high p essu e homogeniza ion wi hou any o ganic sol en 40. On he o he
hand, he addi ion o ca ionic su ac an s as hexadecyl ime hylammonium b omide (CTAB) o Miglyol-leci hin
by Teijei o e al. led o he o ma ion o nanoca ie s wi h simila size and polydispe si y o ou nanosys em,
bu wi h a posi i e ne cha ge ins ead o neu al/nega i e one, which in ha wo k was equi ed o he coa ing
wi h hyalu onic acid34. The esul ing physicochemical cha ac e is ics ob ained o he ET o mula ion he ein
de eloped a e o pa icula in e es when in a enous adminis a ion is en isioned, since pa icles o his size
(< 200nm) a e able o passi ely en e in o he umo by he enhanced pe meabili y and e en ion (EPR) e ec 41
and a e less p one o accumula e in he li e and spleen, imp o ing hei accumula ion in he umo 42. On he
o he hand, he neu al ze a po en ial makes hese nanoca ie s less p one o opsoniza ion43 main aining hei
physicochemical p ope ies while ci cula ing in he blood and ex ending hei ci cula ion ime in he body. In
addi ion, i makes he nanoca ie s mo e biocompa ible in compa ison o posi i ely cha ged ones which in e ac
indisc imina ely wi h cells by elec os a ic in e ac ions44,45.
Aside om he physicochemical p ope ies o he de eloped nanoca ie , i s simple and sa e composi ion,
based on GRAS (Gene ally Recognized as Sa e by he Uni ed S a es FDA) ma e ials (Miglyol) and cell compo-
nen s (PC) makes i sui able o he in ended applica ion. In addi ion, he simple and s aigh o wa d me hodol-
ogy used o i s p epa a ion makes i mo e ad an ageous in e ms o a pha maceu ical indus y poin o iew,
han o he lipid nanoca ie s epo ed on li e a u e o ET deli e y, which we e p epa ed by ho and high shea
homogeniza ion ollowed by ul asonica ion17–19. Wi h espec o he concen a ion o ET in he suspension, we
epo ed abou 119µg ET/mg o mula ion ( heo e ical concen a ion o ET, calcula ed as µg ET/(mg Miglyol + mg
PC)) while o he wo ks ha e epo ed concen a ions o 13 o 33µg ET/mg o mula ion15,16,18,19.
In o de o de e mine he easibili y o hese o mula ions o hei es ing in i o, ET-NEs as well as he
con ol o mula ion o e e ence (C-NEs), we e incuba ed in cell cul u e media (DMEM wi h 1% FBS) a 37
ºC, and hei pa icle size was de e mined o a pe iod o 4h(Fig.2). As o mula ions showed a good s abili y
and main ained hei size, we p oceeded wi h hei cy o oxic p o ile e alua ion in i o in MDA-MB-231 cells.
Ǥ Taking in o accoun ET ela ed ad e se e ec s and i s lipophilici y, di -
e en au ho s ha e p oposed he use o lipid-based nanopa icles o ET deli e y15–17,19. Blanco-P ie o g oup has
showed ha lipid nanosys ems composed o Comp i ol o P eci ol and polyso ba e 80, p epa ed by ho and high
shea homogeniza ion combined wi h ul asonica ion, a e able o dec ease ET hema opoie ic oxici y, and a e
also esponsible o he imp o ed d ug an i umo al e icacy ei he in i o and in i o in glioma, lymphoma and
leukemia mice umo models15,16,19. Mo e ecen ly his g oup has combined ET wi h doxo ubicin17,20, and wi h
gemci abine-squalenic acid conjuga es46 o os eosa coma and ela ed me as asis. This g oup has also es ed he
e ec o ET lipid pa icles in i o in he b eas cance cell line MCF718. None heless, o he bes o ou knowl-
edge, ET has no been es ed o TNBC. Conside ing he agg essi eness o his ype o umo and he an i umo al
e icacy o ET in o he cance s, we decided o s udy i s e ec on TNBC by combining i wi h ou nanosys em
p epa ed by using he simple injec ion me hod, as p e iously men ioned.
In he p esen s udy, ET-NEs and C-NEs we e incuba ed wi h MDA-MB-231 cells o 24h, a inc easing
concen a ions o ET (1.3 o 210µg/mL co esponding o 12.5 o 2000µg/mL o NEs). As i possible o obse e
in Fig.3, and as expec ed, he con ol o mula ion C-NEs do no show cy o oxic e ec s a he es ed ange. This is
due o he ca e ul selec ion o he excipien s, Miglyol, a GRAS medium chain iglyce ide commonly used in sel -
emulsi ying sys ems, and PC, a majo cons i uen o cell memb anes ha is p esen in di e en nano o mula ion
composi ions. Wi h espec o ou o mula ion ET-NEs, i s an i umo al e icacy was dose-dependen , p esen ing
a hal maximal inhibi o y concen a ion (IC50) o 6.9µg/mL (13.2µM) a e 24h o incuba ion, while he IC50
o ee ET was an o de o magni ude highe (13.9µg/mL, 26.5µM).
As p esen ed in Table2, we obse ed he same endency in a lung adenoca cinoma (H460) cell line as well
as in panc ea ic adenoca cinoma cell line (MIA PaCa-2) and panc ea ic cells ep esen ing li e me as asis (L3.
pl6). These cance cell lines we e selec ed o being ep esen a i e o p e alen and di icul o ea umo s. As
Table 1. Physicochemical p ope ies o edel osine nanoemulsions (ET-NEs) and he con ol o mula ions
(C-NEs).
Nanosys em Size (nm) PDI Ze a po en ial (mV)
C-NEs 131 ± 3 0.1 − 2 ± 0
ET-NEs 123 ± 13 0.1 − 1 ± 0
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expec ed, he one p esen ing a highe esis ance o ea men , equi ing a highe ET-NEs dose, was ou main
a ge cell line MDA-MB-231 (Fig.3).
A dose- and ime-dependen e ec was obse ed by o he au ho s in human b eas cance (MCF7) and leu-
kemia cell lines wi h P eci ol-polyso ba e 80 lipid nanopa icles16,18. Ou esul s showed a highe po en ial o
ET o killing a esis an b eas cance cell line in ela ion o he esul s epo ed by Azna e al. in non- esis an
MCF7 b eas cance cells (IC50 12.9µg/mL a e a longe incuba ion ime o 72h)18. Resul s ela ed o leukemia
cell lines epo ed IC50 o 20µM in he esis an cell line K562, a e 72h ea men , and IC50 o 3.4µM o he
sensi i e cell line MOLM-1316. Al oge he , and conside ing he a iabili y due o cell lines es ed, we can conclude
ET can be e icien ly deli e ed o cance cells upon o mula ion as ET-NEs, p o iding highe e iciencies han
he d ug in solu ion (Table2), and a supe io beha io in ela ion o o he nanosys ems ha ha e been es ed
o longe pe iods o ime16,18.
Con ocal expe imen s addi ionally con i med ha ET-NEs we e e icien ly and highly in e nalized by he
MDA-MB-231, when compa ed o he C-NEs. Figu e4 p esen s a galle y showing se e al sec ions o show ha
di e ences a e e iden all h ough he sample (Fig.4a) as well as a ep esen a i e single plan o all he samples
(Fig.4b). The g een s aining ha is obse ed in cells ea ed wi h ET-NEs co esponds o he labelled o mula-
ion (ET-NEs include TopFluo -PC in a simila amoun as in he con ol o mula ion). The highe capaci y o
ET-NEs (p < 0.0001) o be in e nalized (Fig.4c) migh be explained by he well-desc ibed cha ac e is ic o ET o
accumula e in cells’ plasma memb ane7,8, which he ein p o ided ET-NEs he capaci y o be highly in e nalized.
ƤǤ Being awa e ha he in i o scena io does no e lec he com-
plexi y ound in i o, nex s ep in ou esea ch aimed o de e mine he oxici y, biodis ibu ion, and an i umo al
e icacy, o he ET-NEs in i o, using o ha pu pose zeb a ish emb yos. This animal model ep esen s a s ep
Figu e2. S abili y o con ol NEs (C-NEs) and edel osine NEs (ET-NEs) upon incuba ion wi h zeb a ish
medium (ZFM, s e ile dechlo ina ed ap wa e ) and cell cul u e medium (CCM, DMEM supplemen ed wi h 1%
FBS) o 0, 1 and 4h, a 37°C.
Figu e3. MDA-MB-231 cell iabili y upon he ea men wi h inc easing concen a ions o ee edel osine (ET)
edel osine nanoemulsions (ET-NEs) and con ol nanoemulsions (C-NEs) du ing 24h a 37°C.
Table 2. Hal maximal inhibi o y concen a ion (IC50) o ET-NEs and ee ET upon incuba ion wi h di e en
cance cell lines (lung (H460), panc ea ic (MIA PaCa-2), panc ea ic li e me as asis (L3.pl6)) o 24h a 37 °C.
Nanosys em
(µg/ml) MDA-MB-231 H460 MIA PaCa-2 L3.pl6
ET-NEs 6.9 2.2 1.2 2.5
F ee ET 13.9 6.1 3.0 5.9
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u he o unde s and umo he e ogenei y, umo cells beha io and di e en mechanis ic aspec s o cance .
Due o i s unique cha ac e is ics, i is conside ed a p omising ool in he de elopmen o no el he apeu ic
s a egies30,47.
P io o es hese o mula ions in i o, ET-NEs and C-NEs we e incuba ed in SDT wa e a 28 ºC, and
hei pa icle size de e mined o a pe iod o 4h, in o de o ensu e ha he e we e no changes on NEs’ phys-
icochemical p ope ies ha could comp omise he assay and lead o a misin e p e a ion o he esul s. Bo h
o mula ions we e able o main ain hei size (Fig.2), he e o e we p oceeded wi h hei in i o e alua ion in
zeb a ish emb yos.
ͶȂͺͽƤǤ Zeb a ish emb yos
we e incuba ed wi h inc easing concen a ions (up o 1000µg/mL) o NEs wi h and wi hou edel osine. Abo e
his concen a ion he media became whi e and he emb yos could no be clea ly obse ed h ough he mic o-
scope, he e o e we did no es highe concen a ions. In acco dance o wha was p e iously obse ed in i o,
a he es ed concen a ions, C-NEs did no lead o he dea h o 0 and 72 hp zeb a ish emb yos, 96h pos -
ea men , i espec i ely o he incuba ion empe a u e.
In he case o he ET-NEs, he LC50 o he zeb a ish main ained a 28 ºC was 12.89µg/mL (0 hp ) and
8.6µg/m (72 hp ), while he ones main ained a 34 ºC p esen ed a LC50 o 11.4µg/mL (0 hp ) and 3.2µg/mL
(72 hp ). The a ia ions in oxici y da a seem o be due o he incuba ion empe a u es used in he expe imen ,
as empe a u e is a ele an and highly a iable abio ic ac o in na u e ha plays an impo an ole du ing ish
emb yonic de elopmen 48. Fu he mo e, as empe a u e inc eases, i can become a physical s esso and aise
he aqua ic o ganisms’ ene gy me abolism and in u n, he bioa ailabili y o oxican s49.
Aside om LC50, o he oxici y indexes as LC10, LOEC and NOEC a e p esen ed in Table3. All he equi e-
men s o FET es we e accomplished: he mo ali y in nega i e con ol emb yos was ≤ 10%, he ha ching a e
was ≥ 80% o he nega i e con ol, whe eas he posi i e con ol was 100% dea hs (minimum equi ed is 30%).
The e we e no signi ican mo phological changes o ha ching delay in compa ison wi h he con ol g oup,
and ollowing he OECD guidelines, abno mal o non-ha ched emb yos a 96 hp we e excluded om he assay.
In gene al, he esul s show ha ET-NEs a e clea ly mo e oxic in con as o he con ol o mula ion (C-NEs),
a ac ha was expec ed due o he ca e ul selec ion o he con ol o mula ion composi ion (Miglyol and PC).
In speci ic, Miglyol, a GRAS medium chain iglyce ide (used in sel -emulsi ying sys ems), and PC, a majo
cons i uen o cell memb anes (also p esen in di e en nano o mula ions). These esul s a e in he same line
as he in i o assays, showing a high compa ibili y o he C-NEs and a dose-dependen oxici y o he ET-NEs.
Tak ing i n o con si de a ion he e su l s o he ox ic i y a ss ay ( Tabl e3), ET-NEs a 1.5µg/mL (LC10) was selec ed
o u he s udying he an i umo al e icacy o his nanosys em.
Figu e4. In e naliza ion o NEs in MDA-MB 231 cells. (a) Galle y o con ocal mic oscopy images o MDA-MB
231 cells incuba ed wi h 150µg/mL o C-NEs and ET-NEs labeled wi h TopFluo -PC (g een channel) o
4h a 37 ºC. (b) Rep esen a i e con ocal mic oscopy single-plan image o con ol s C-NEs and ET-NEs
in e naliza ion by MDA-MB 231 cells. (c) Fluo escence in ensi y (AU, a bi a y uni s) o wo maximum
p ojec ion images ( esul an om he combina ion o all he sec ions shown in (a)) was de e mined using
ImageJ so wa e. S a is ical analysis was pe o med using es . P alue ****p < 0.0001. Cell nuclei was s ained
wi h Hoechs (blue channel). Scale ba s co espond o 25µm.
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ͺ; ƤǤ Zeb a ish emb yos o 48 hp we e
injec ed wi h 5μg/ml o C-NEs and ET-NEs in he yolk sac o caudal ein and incuba ed a 28 ºC. As expec ed,
no dea h was obse ed 96h a e ea men wi h any o he es ed o mula ions. These esul s assu e ha he e
would be no dea hs as a esul o he injec ion i sel combined wi h he es ed nanosys ems concen a ion.
Ǥ One o he g ea es ad an ages o zeb a ish model o ganism is ha i o e s he
possibili y o s udying and as acking he dis ibu ion o nanopa icles h oughou o he o ganism due o i s
anspa ency. In addi ion, i allows a di ec obse a ion o nanopa icles ci cula ion and hei in e ac ion wi h
cells33–35. In his kind o s udy, choosing he igh dye and he app op ia e con ols is o ex eme impo ance in
o de o be su e ha we a e acking he nanosys ems indeed and a oid he misin e p e a ion o biodis ibu ion
da a. On one hand we selec ed DiR, which is commonly used in biodis ibu ion assays and is easily encapsula ed
in he nanosys em’s oil co e. None heless, i can be eleased om he oil co e o nanosys ems causing an appa -
en bu alse cellula up ake. The e o e, we also used TopFluo , co alen ly linked o he PC, and he ein ancho ed
o he memb ane o he NEs, o help us ensu ing ha he ob ained signal was due o he p esence o he NEs by
DiR/TopFluo -PC co-localiza ion, and no due o he p esence o ee luo opho es.
Emb yos o 72 hp we e incuba ed wi h 500µg/mL o DiR and TopFluo -PC labeled C-NEs o s udy i s
in e naliza ion and dis ibu ion. This high concen a ion was necessa y o be able o obse e he NEs wi h
enough amoun o luo escen labeling, unde he con ocal mic oscope. As men ioned be o e, C-NEs we e highly
compa ible e en a he maximum es ed concen a ion o 1000µg/mL (LC50 could no be assessed), whe eas
he de e mined LC50 o ET-NEs was 8.6µg/mL. Hence, a he necessa y concen a ion (500µg/mL) o he
biodis ibu ion assay, ET-NEs would be le hal o he emb yos, which lead us o pe o m he s udy only wi h he
con ol blank o mula ion (C-NEs).
Con ocal mic oscope analysis showed ha NEs we e e icien ly in e nalized by he exposed emb yos, espe-
cially in o he yolk sac (Fig.5), which demons a es he capaci y o he C-NEs o c oss biological ba ie s upon
di ec con ac wi h he skin o zeb a ish emb yos (wi hou cho ion). F ee DiR and TopFluo -PC (Fig.5a con ol)
we e jus included o e e ence and no o compa a i e pu poses. Zeb a ish o 48 hp a e p o ec ed by he cho-
ion. A e his pe iod o ime, he zeb a ish loses his laye (ha ching) and he skin becomes he main biological
ba ie p o ec ing he emb yo om he ex e nal en i onmen . As indica ed, in his s udy we incuba ed he NEs
wi h 72 hp emb yo meaning ha he NEs we e in di ec con ac wi h he emb yo skin. Teijei o-Valiño e al.
ound impo an di e ences be ween he zeb a ish biological ba ie s (cho ion s skin) in e ms o he pe me-
abili y and oxici y o posi i ely cha ged NEs and nega i ely cha ged nanocapsules (NCs)34. The NCs con aining
an ex e nal shell o hyalu onic acid/p o amine had he capaci y o pe mea e h ough he cho ion and skin laye s,
unlike he NCs con aining only a hyalu onic shell ha emained associa ed o he ex e nal laye o hese ba ie s
being unable o pene a e. Acco ding o he au ho s he pene a ion abili y o NCs con aining p o amine could
be due o p o amine, which aside om i s ne posi i e cha ge is known as a cell pene a ing pep ide, bu could
be also due o he p esence o PEG-s ea a e.
He ein he biodis ibu ion o he ET-NEs, unde he es ablished condi ions i. e. he equi ed concen a ion
o con ocal mic oscopy analysis (500µg/mL), was no s udied conside ing hei highe oxici y han he C-NEs.
Unlike nega i ely cha ged nanoca ie s de eloped by Teijei o-Valiño e al., ou nanosys ems p esen a neu al
su ace cha ge and he e o e we hypo hesized ha C-NEs capaci y o pene a e h ough he zeb a ish skin migh
be due o he p esence o PC, which is a majo cons i uen o cell memb anes. In addi ion, aking in o conside a-
ion he in i o in e naliza ion esul s in MDA-MB 321 cells, a simila and e en g ea e pene a ion capaci y
would be expec ed o ET-NEs.
ƥǦƤǤ Aside
om zeb a ish anspa ency, he possibili y o ansplan human cance cells in o his animal model is also use-
ul o de e mining he e icacy o an an i-cance he apy. In his sense, se e al models ha e been de eloped,
leading o a be e unde s anding o di e en c i ical aspec s o cance such as p oli e a ion and in asion, umo
o ma ion, angiogenesis, me as asis o immune cell esponse50–55. Fu he mo e, he umo mic oen i onmen
is mo e accu a ely ep esen ed han in in i o models, and he in e ac ion o umo cells wi h he hos can be
in e ed, o ins ance, om he immune cells’ beha io , due o he conse ed cell in e communica ion be ween
humans and zeb a ish56. These ea u es, oge he wi h hose p e iously men ioned, make he zeb a ish a highly
aluable pla o m no only o un a el umo beha io , bu also o es di e en po en ial an i-cance d ugs and
o pe o m high- h oughpu sc eenings o no el he apeu ic compounds. O e all, his b ings esea che s close
o unde s anding pa ien s’ esponse o ea men and, in u n, o pe sonalized medicine57.
Table 3. Toxici y o 0 and 72 hp emb yos exposed o ET-NEs o 96h. hp : hou pos - e iliza ion; LC10,
LC50, NOEC and LOEC a e ep esen ed in µg/mL.
hp T (ºC) LC10 LC50 NOEC LOEC
028 9.8 12.89 10 5
34 8.6 11.4 10 5
72 28 4.4 8.6 5 1
34 1.5 3.2 5 1
Annex III
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ANNEX III. ETHICAL APPROVALS FOR ANIMAL
EXPERIMENTATION
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RESOLUCIÓN DE AUTORIZACIÓN DE PROXECTO DE EXPERIM ENTACIÓN ANIM AL
Expedien e núm .: 150 12/20 22/0 11
Da a de inicio do expedien e: 29.3.2022
Pe soa solici an e: Anxo Vidal Figue oa
P ocedem en o: esolu ción d e au o izació n
Fo m a de inicio: solici ude da pe soa in e esada
ANTECEDENTES
A pe soa solici an e, com o ep esen an e do Cen o de Biomedicina Expe imen al
CEBEGA, p esen ou con da a 29.3.2022 unha solici ude pa a a ealización do
p oxec o de expe im en ación anim al (en ada no Rexis o elec ónico da Xun a de
Galicia 2022/854934), cuxos da os se de allan a con inuación:
Denom inación do p oxec o: Es udos de im axe m olecula e es u u al en a a
(R a u s n o e g ic u s ) e a o (M u s m u sc u lu s) con adminis ación de di e en es
con as es
Nom e do cen o usua io: Cen o de Biom edicina Expe im en al CEBEGA
Pe soa esponsable do p oxec o: P a b lo A g u ia F e n á n d e z
Es ablecem en o onde se ealiza án os p ocedem en os do p oxec o (ou luga
xeog áic o n o c a s o d e a b a llo s d e c a m p o ): Cen o de Biom edicina Expe im en al
da USC (CEBEGA)
Clasiic a c ió n d o p o x e c o : Tipo I T ip o II Tipo III
CONSIDERACIÓNS LEGAIS E TÉCNICAS
1 O Real dec e o 53/2013, de 1 de eb ei o (BO E 34, do 8 de eb ei o), polo que se
es ablecen as no m as básicas aplicables pa a a p o ección dos anim ais u ilizados
en expe im en ación e ou os =in s c ie n í=ic o s , in c lu ín d o a d o c e n c ia , e s a b le c e n o s e u
a igo 33 as condicións de au o izacións dos p oxec os con anim ais de
expe im en ación.
2 O a igo 88 da Lei 39/2015, de 1 de ou ub o, do p ocedem en o adm inis a i o
com ún das adm inis acións públicas (BO E 236, do 2 de ou ub o de 2015) es ablece
que a esolución que poña =in o p o c e d e m e n o d e c id i á o d a s a s c u e s ió n s e x p o s a s
CONSELLERÍA DO M EDIO RURAL
XEFATURA TERRITORIAL
SERVIZO D E G A N D A RÍA
Edi=ic io A d m in is a i o M o n e lo s
R/ Vicen e Fe e , 2, 4º anda
150 71 A C o uña
T: 981 184 565
se izo.ganda ia.a.co una@ xun a.gal
medio u al.xun a.gal
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Annex III
341
polos in e esados e aquelas ou as de i adas des e.
3 O Se izo de G anda ía da Co uña e isou a docum en ación achegada na solici ude
e o esul ado a o able da a aliación do p oxec o ealizada polo ó gano habili ado,
o Com i é de É ica de Expe im en ación Anim al da Uni e sidade de San iago de
Com pos ela.
Es a xe a u a e i o ial é com pe en e pa a di a unha esolución, de con o m idade
co D ec e o 149/2018, do 5 de decem b o, polo que se es ablece a es u u a o gánica
da Conselle ía do M edio Ru al e se m odi=ic a p a c ia lm e n e o D e c e o 17 7 /2 0 16 , d o 15
de decem b o, polo que se =ix a a e s u u a o g á n ic a d a V ic e p e s id e n c ia e d a s
conselle ías da Xun a de G alicia (D O G 235, do 11 de no em b o).
De aco do con odo o indicado, RESOLVO:
1 A u o iza o p oxec o solici ado.
2 O p oxec o non p ecisa som e e se a unha a aliación e ospec i a.
3 A au o ización des e p oxec o e á unha du ación de cinco anos e unha ez
ansco ido es e em po, es a au o ización debe á se eno ada.
A ci ada au o ización é unicamen e álida nas condicións que =ig u a n n o
expedien e. An e calque a cam bio signi=ic a i o n o p o x e c o q u e p o id a e e e c o s
nega i os sob e o benes a dos anim ais, debe á solici a a con=i m a c ió n d a
au o ización ao Se izo P o incial de G anda ía.
Es a au o ización pode á se suspendida no caso de que o p oxec o non se le e a
cabo de aco do coas condicións de au o ización e e i ala, p e io expedien e
am i ado ao que se lle da á audiencia.
Con a a p esen e esolución, que non pon =in á ía a d m in is a i a , p o d e á
in e p o ñ e u n e c u s o d e a lz a d a a n e o c o n s e lle i o d e M e d io R u a l. O p a z o
com eza á a con a dende o día seguin e ao da ecepción des a esolución. Todo
is o , s e g u n d o o d is p o s o n o s a ig o s 12 1 e 12 2 d a c i a d a L e i 3 9 /2 0 15 .
Median e es e esc i o no i=íc a s e lle a A n x o V id a l F ig u e o a e s a e s o lu c ió n s e g u n d o o
esixido no a igo 40.1 da an edi a Lei 39/2015.
A Co uña, 1 de ab il de 2022
O xe e e i o ial
Jose M anuel San os M anei o
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SANTUCCI VINCENZO UGO
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CN=SANTUCCI VINCENZO UGO
C=IT
2.5.4.4=SANTUCCI
2.5.4.42=VINCENZO UGO
RSA/2048 bi s
Annex IV
343
ANNEX IV. ETHICAL APPROVAL FOR PATIENT’S SAMPLES
Sec e a ia Técnica
Comi é Au onómico de É ica da In es igación de Galicia
Sec e a ia Xe al. Conselle ía de Sanidade
Edi icio Adminis a i o San Láza o
15703 SANTIAGO DE COMPOSTELA
Tel: 881546425. Co eo-e: ceic@se gas.es
Sec e a ia Técnica
Comi é Au onómico de É ica da In es igación de Galicia
Sec e a ia Xe al. Conselle ía de Sanidade
Edi icio Adminis a i o San Láza o
15703 SANTIAGO DE COMPOSTELA
Tel: 881546425. Co eo-e: ceic@se gas.es
DICTAMEN DEL COMITÉ DE ÉTICA DE LA INVESTIGACIÓN DE SANTIAGO-LUGO
Guille mo José P ada Ramallal, Sec e a io del Comi é de É ica de la In es igación de San iago-Lugo,
CERTIFICA:
Que es e Comi é e aluó en su eunión del día 21 de diciemb e de 2017 el es udio:
Tí ulo: Iden i icación de ma cado es de diagnós ico, p onós ico y seguimien o median e el
análisis de Biopsia Líquida en pacien es con cánce
P omo o : Ra ael López López
Tipo de es udio: Ou os
Ve sión: Ve sión 1 de 31 de Oc ub e de 2017
Código del P omo o :
Código de Regis o: 2017/538
Y, omando en conside ación las siguien es cues iones:
- La pe inencia del es udio, eniendo en cuen a el conocimien o disponible, así como los
equisi os legales aplicables, y en pa icula la Ley 14/2007, de in es igación biomédica, el
Real Dec e o 1716/2011, de 18 de no iemb e, po el que se es ablecen los equisi os básicos
de au o ización y uncionamien o de los biobancos con ines de in es igación biomédica y del
a amien o de las mues as biológicas de o igen humana, y se egula el uncionamien o y
o ganización del Regis o Nacional de Biobancos pa a in es igación biomédica, la ORDEN
SAS/3470/2009, de 16 de diciemb e, po la que se publican las Di ec ices sob e es udios
Pos au o ización de Tipo Obse acional pa a medicamen os de uso humano, y la Ci cula nº
07/2004, de in es igaciones clínicas con p oduc os sani a ios.
- La idoneidad del p o ocolo en elación con los obje i os del es udio, jus i icación de los
iesgos y moles ias p e isibles pa a el suje o, así como los bene icios espe ados.
- Los p incipios é icos da Decla ación de Helsinki igen e.
- Los P ocedimien os No malizados de T abajo del Comi é.
Emi e un dic amen FAVORABLE pa a la ealización del es udio po el/la in es igado /a del
cen o:
Cen os
In es igado es P incipales
C.H. Uni e si a io de San iago
Ra ael López López, Lau a Muinelo Romay,
Robe o Díaz Peña
En San iago de Compos ela, a 28 de diciemb e 2017.
El Sec e a io del Comi é Te i o ial de É ica de la In es igación de San iago Lugo,
Guille mo José P ada Ramallal
SANDRA DÍEZ VILLARES
344
Sec e a ia Técnica
Comi é Au onómico de É ica da In es igación de Galicia
Sec e a ia Xe al. Conselle ía de Sanidade
Edi icio Adminis a i o San Láza o
15703 SANTIAGO DE COMPOSTELA
Tel: 881546425. Co eo-e: ceic@se gas.es
Sec e a ia Técnica
Comi é Au onómico de É ica da In es igación de Galicia
Sec e a ia Xe al. Conselle ía de Sanidade
Edi icio Adminis a i o San Láza o
15703 SANTIAGO DE COMPOSTELA
Tel: 881546425. Co eo-e: ceic@se gas.es
Guille mo José P ada Ramallal, Sec e a io del Comi é de É ica de la In es igación de San iago-Lugo,
HACE CONSTAR QUE:
1.- El Comi é Te i o ial de É ica de la In es igación de San iago-Lugo cumple an o en su
composición como en sus PNTs los equisi os legales igen es (RD 1090/2015 de ensayos clínicos, y
la Ley 14/2007 de In es igación Biomédica).
2.- La composición ac ual del Comi é Te i o ial de É ica de la In es igación de San iago-Lugo es:
x Juan Manuel Vázquez Lago (P esiden e). Médico especialis a en Medicina P e en i a y
Salud Pública. Á ea de Ges ión In eg ada de San iago.
x Pila Rod íguez Ledo (Vicep esiden a). Médico especialis a en Medicina Familia y
Comuni a ia. Á ea de Ges ión In eg ada de Lugo.
x Guille mo José P ada Ramallal (Sec e a io). Médico especialis a en Fa macología Clínica.
Á ea de Ges ión In eg ada de San iago. Fundación Ramón Domínguez.
x Lo enzo A men e os del Olmo (Vicesec e a io). Médico especialis a en Medicina Familia
y Comuni a ia. Á ea de Ges ión In eg ada de Lugo.
x F ancisco Campos Pé ez. Biólogo. Ins i u o de In es igación Sani a ia de San iago de
Compos ela.
x Rosana Cas elo Domínguez. Fa macéu ica de A ención P ima ia. Á ea de Ges ión In eg ada
de San iago.
x Rica do Ga cía Ma ínez. Licenciado en De echo. Á ea de Ges ión In eg ada de Lugo.
x Jaime Gulín Dá ila. Fa macéu ico especialis a en Fa macia Hospi ala ia. Á ea de Ges ión
In eg ada de Lugo.
x Vic o He án Ca ei a. Pacien e. ADIL-Asociación de Diabé icos Lucense.
x Ma ía Jesús Lamas Díaz. Fa macéu ica especialis a en Fa macia Hospi ala ia. Á ea de
Ges ión In eg ada de San iago.
x Ca los Rod íguez Mo eno. Médico especialis a en Fa macología Clínica. Á ea de Ges ión
In eg ada de San iago.
x Ra ael Ca los Vidal Pé ez. Médico especialis a en Ca diología. Á ea de Ges ión In eg ada de
Lugo.
x Ma ía Jesús Wandosell Pica os e. En e me a. Á ea de Ges ión In eg ada de San iago.
Pa a que cons e donde p oceda, y a pe ición del p omo o /in es igado , en San iago de Compos ela,
a 28 de diciemb e de 2017.
El Sec e a io del Comi é Te i o ial de É ica de la In es igación de San iago Lugo,
Guille mo José P ada Ramallal
Annex VI
351
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