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SCIENTIFIC RePoR s | (2018) 8:8867 | DOI:10.1038/s41598-018-26772-z
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P os a e-Speci ic Memb ane
An igen-Ta ge ed Si e-Di ec ed
An ibody-Conjuga ed Apo e i in
Nano ehicle Fa o ably In luences
In Vi o Side E ec s o Doxo ubicin
Simona Dos alo a1,2, Hana Polanska3, Ma ke a S obodo a3, Jan Bal an3,4, Olga K ys o o a1,2,
Yazan Haddad
1,2, Sona K izko a1,2, Michal Masa ik3, Tomas Eckschlage 5, Ma ie S ibo o a6,
Zbynek Hege 1,2 & Voj ech Adam
1,2
He ein, we desc ibe he in i o e ec s o doxo ubicin (DOX) encapsula ed in ubiqui ous p o ein
apo e i in (APO) and i s e iciency and sa e y in an i- umo ea men . APODOX is bo h passi ely
( h ough Enhanced Pe meabili y and Re en ion e ec ) and ac i ely a ge ed o umo s h ough
p os a e-speci ic memb ane an igen (PSMA) ia mouse an ibodies conjuga ed o he su ace o ho se
spleen APO. To achie e si e-di ec ed conjuga ion o he an ibodies, a HWRGWVC hep apep ide
linke was used. The p os a e cance - a ge ed and non- a ge ed nanoca ie s we e es ed using
subcu aneously implan ed LNCaP cells in a hymic mice models, and compa ed o ee DOX. P os a e
cance - a ge ed APODOX e ained he high po ency o DOX in a enua ion o umo s (wi h 55%
dec ease in umo olume a e 3 weeks o ea men ). DOX and non- a ge ed APODOX ea men
caused damage o li e , kidney and hea issues. In con as , no ele a ion in li e o kidney enzymes
and negligible changes we e e ealed by his ological assessmen in p os a e cance - a ge ed APODOX-
ea ed mice. O e all, we show ha he APO nanoca ie p o ides an easy encapsula ion p o ocol,
eliable a ge ing, high he apeu ic e iciency and e y low o - a ge oxici y, and is hus a p omising
deli e y sys em o ansla ion in o clinical use.
Va ious po en chemo he apeu ic d ugs ha e been de eloping o e he decades. Despi e hei p o ound he a-
peu ic e icacy1, hey cause nume ous dose-limi ing side e ec s2. Doxo ubicin (DOX) is bu one example o his
phenomenon, whe e DOX adminis a ion leads o a hy hmia o ca diomyopa hy caused by he o ma ion o
eac i e oxygen species and cy och ome c elease om mi ochond ia3 in up o 26% o pa ien s4. To dec ease hese
e ec s, DOX is o en co-adminis a ed wi h he ca diop o ec i e agen dex azoxane. Howe e , i s ca diop o ec i e
abili ies a e con en ious and many pa ien s ea ed wi h dex azoxane ha e de eloped seconda y malignancies5.
To elimina e he challenges o con en ional cance chemo he apy, p e e en ial deli e y o an i-cance d ugs
o umo cells is being in es iga ed. This can be achie ed using nano-scaled d ug-con aining pa icles, which a e
called nanoca ie s6. The ideal nanoca ie needs o no only be non- oxic bu also biocompa ible and biodeg ad-
able7. These p ope ies a e impo an o bo h he subjec s in ol ed in he ea men and he gene al public since
he nanopa icles a e o en exc e ed in o was e wa e and can pose a h ea o he en i onmen 8. Many di e en
ma e ials ha e been s udied o he p epa a ion o d ug nanoca ie s, bo h o ganic and ino ganic9. Ino ganic
1Depa men o Chemis y and Biochemis y, Mendel Uni e si y in B no, Zemedelska 1, B no, CZ-613 00, Czech
Republic. 2Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, Pu kyno a 123, B no, CZ-612
00, Czech Republic. 3Depa men o Pa hological Physiology and Depa men o Physiology, Facul y o Medicine,
Masa yk Uni e si y, Kamenice 753/5, B no, CZ-625 00, Czech Republic. 4TESCAN ORSAY HOLDING a.s., Libusina
ida 863/21, B no, CZ-623 00, Czech Republic. 5Depa men o Pedia ic Hema ology and Oncology, 2nd Facul y
o Medicine, Cha les Uni e si y and Uni e si y Hospi al Mo ol, V U alu 84/1, P ague 5, CZ-150 06, Czech Republic.
6Depa men o Biochemis y, Facul y o Science, Cha les Uni e si y, Hla o a 2030/8, P ague 2, CZ-128 43, Czech
Republic. Co espondence and eques s o ma e ials should be add essed o V.A. (email: [email p o ec ed])
Recei ed: 12 Sep embe 2017
Accep ed: 11 May 2018
Published: xx xx xxxx
OPEN
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SCIENTIFIC RePoR s | (2018) 8:8867 | DOI:10.1038/s41598-018-26772-z
exogenous ma e ials a e usually no biodeg adable and can be accumula ed in an o ganism ollowing epea ed
adminis a ion o p ema u ely cap u ed in o gans o he e iculoendo helial sys em10. They can also cause in lam-
ma o y esponse o neu o oxic eac ions8. O ganic exogenous pa icles also ha e some d awbacks. Cu en ly,
he e a e wo comme cially a ailable nanopha maceu icals con aining DOX: Myoce © (DOX in ba e liposomes)
and Doxil© (DOX in polye hylenglycola ed (PEGyla ed) S eal h® liposomes)11. Ba e liposomes we e ound o be
ecognized by pa ien ’s cy o oxic T cells and emo ed om he body p io o eaching he umo si e12. Al hough
PEGyla ed liposomes a e able o e ade he immune cells, hei cellula up ake is hampe ed due o he PEGyla ion.
Mo eo e , hey ha e been p o en o cause palma -plan a e y h odyses hesia13 and pulmona y ib osis14.
In ligh o hese ac s, endogenous pa icles seem mo e p omising; especially hose in ol ed in he cellula
up ake pa hways. They a e na u ally biocompa ible and biodeg adable and hey also p o ide easy passage h ough
he cell memb anes15. These much-needed p ope ies can be p o ided by e i ins o be e apo e i ins (APO,
demine alized e i ins), ubiqui ous p o eins wi h high in e species sequence homology esponsible o he s o -
age and ans e o i on ions16. Ou p e ious s udy17, as well as s udies o o he s18–21, ha e p o en ha si e-di ec ed
APO could enhance he in i o selec i i y o encapsula ed cy o oxic d ug o cance issue, while e aining i s
po ency.
In he p esen s udy, we e alua ed he p os a e cance - a ge ed ho se spleen APO-encapsula ed DOX o he
i s ime in e ms o i s mechanisms o in e naliza ion in o umo cells, p os a e umo a enua ion in mu ine
ec opic xenog a s and i s e ec s on he o - a ge o gans o he adminis e ed mice. The si e-di ec ed o ien a ion
o a ge ing an ibodies was achie ed h ough p o ein A-de i ed hep apep ide, which was a ached o 1.3 nm gold
nanopa icle-modi ied APO su ace ia cys eine on i s C- e minus. O e all, we show ha while he nanoca ie
was able o a enua e he umo s wi h sligh ly lowe po ency han ee DOX, i signi ican ly spa ed he o - a ge
o gans om he unwan ed oxic ac ion o ee DOX.
Resul s
Elucida ing he cellula up ake e iciency and mechanism o APODOX-an i-PSMA. To choose
sui able cells o mice xenog a s udies, wo p os a e cance cell lines, LNCaP and 22RV1, we e e alua ed based
on hei abili y o bind and in e nalize APODOX-an i-PSMA (p os a e-speci ic memb ane an igen). As cellula
up ake o APO h ough ei he ans e in ecep o 1 (T R)22, T cell immunoglobulin and mucin domain-con-
aining p o ein-2 (TIM-2)23 o sca enge ecep o class A membe 5 (SCARA5)22 is based on his hea y and
ligh chain subuni a io, hei exp ession was es ed. Figu e1a shows he exp ession p o iles o T R, SCARA5
and PSMA as he mos p obable p e e en ial a ge s o APODOX-an i-PSMA binding, as well as he esul s
om A i-assay, e alua ing he binding o APODOX-an i-PSMA o 84 kDa PSMA p o ein. Densi ome ic analysis
e ealed signi ican di e ences (p < 0.05) be ween he wo es ed cell lines, wi h LNCaP ha ing highe exp ession
o bo h T R (1.3× highe ) and PSMA (2.0× highe ), as well as highe binding o APODOX-an i-PSMA (2.8×
highe ). No ewo hy, only negligible exp ession o p e e en ial ecep o o L- ype e i ins SCARA5 was ound
on hese cell lines.
Hence, we ocused ou a en ion on in e naliza ion o APODOX-an i-PSMA in LNCaP cells. To de e mine
up ake kine ics, we ob ained mean in acellula DOX luo escence alues om con inuous quan i a i e phase
imaging (24 h) o ea ed cells (Fig.1bi and bii). The signi ican ly (p < 0.05) highes up ake was obse ed o ee
DOX (3× highe luo escence han o APODOX and 1.7× o APODOX-an i-PSMA a e 20 h o ea men ).
Fi s signs o he cell sh inkage we e obse ed a e 1 h ea men wi h DOX, wi h memb ane budding clea ly
isible a e 4 h o ea men (Supplemen 1). APODOX exhibi ed lowe cellula up ake compa ed o ee DOX;
howe e , si e-di ec ed su ace modi ica ion wi h an ibodies a o ably in luenced he up ake (1.8× highe in a-
cellula luo escence o APODOX-an i-PSMA han o APODOX, Fig.1bi and bii). Mo eo e , he i s sh inking
cells we e isible a e 10 h o ea men and cell budding was obse ed a e mo e han 20 h o ea men wi h
APODOX (Supplemen 2). Whe eas, he cells ea ed wi h APODOX-an i-PSMA also showed i s signs o he
cell sh inkage a e 10 h o ea men bu he budding was isible a e 14 h, and signi ican ly as e han in he case
o APODOX (Supplemen 3).
To elucida e he mechanisms o cellula up ake, a compe i i e assay be ween APODOX-an i-PSMA and
an i-T R/an i-PSMA an ibodies was pe o med (Fig.1bi and biii). The compe i ion be ween SCARA5 and
APODOX-an i-PSMA was no s udied, due o he e y low SCARA5 exp ession. The compe i ion wi h an i-T R
an ibodies did no inhibi he up ake o APODOX-an i-PSMA and he signs o ea ly and la e apop osis we e
obse ed e en ea lie (cell sh inkage a 5 h o ea men and budding a 12 h– see Supplemen 4). Howe e , com-
pe i ion wi h an i-PSMA an ibodies showed signi ican ly lowe cellula up ake o APODOX-an i-PSMA (1.2×
lowe up ake a e 12 h o ea men ). The cell sh inkage was also obse ed a e 20 h o ea men wi h budding
a e 23 h o ea men (Supplemen 5). These esul s sugges ha APODOX-an i-PSMA does in e nalize in o cells
p edominan ly h ough he PSMA.
To ob ain insigh in o he s abili y o a ge ing an ibody (mouse an i-PSMA an ibody) binding o APODOX
su ace in he blood s eam, APODOX-an i-PSMA was incuba ed wi h human IgG a plasma concen a ion o
up o 48 h and mouse an ibodies we e de ec ed on APODOX su ace and in he solu ion a a ious ime poin s
(Fig.1c). The esul s showed ha he binding was s able o up o 48 h, whe e he compe i ion wi h human IgG
an ibodies did no cause any emo al o a ge ing mouse an ibodies om he su ace o APODOX.
To e alua e he p o ein p o ile o APO, APO a e pH-media ed disassembly and eassembly, APODOX and
APODOX-an i-PSMA, we esol ed he samples on SDS and na i e PAGE (Fig.1d). The SDS-PAGE showed he
con en o ~15 and ~12 kDa p o eins in he sample o APO as ecei ed om he manu ac u e , al hough hese
we e no obse ed on na i e PAGE. The esul s showed ha nei he he disassembly/ eassembly, no encapsula-
ion o DOX changed he p o ein p o ile o APO, whe eas, as expec ed, changes we e obse ed a e he modi i-
ca ion wi h PSMA- a ge ing an ibody.
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SCIENTIFIC RePoR s | (2018) 8:8867 | DOI:10.1038/s41598-018-26772-z
Size measu emen s (Fig.1e) e ealed he unchanged 12-nm size o APO a e DOX encapsula ion while he
a ge ing an ibody inc eased he size o he whole nanocons uc o 91 nm which was p esumably due o a pa ial
agg ega ion. These esul s co espond o esul s ob ained om TEM mic og aphs (Fig.1e inse s), showing an i-
bodies bound on APO su ace wi hou u he changes o he mo phology o assembled APO. The ζ-po en ial in
Figu e 1. In i o cellula up ake o APODOX-an i-PSMA in p os a e cance cell lines. (a) Exp ession p o iles
o GAPDH, T R, SCARA5 and PSMA and a ini y binding o APODOX-an i-PSMA o PSMA on LNCaP and
22RV1 p os a e cance cell lines. The indi idual blo s we e c opped om di e en pa s o he same memb ane
(as indica ed by di iding whi e spaces). Righ pa shows he densi ome ic analysis o exp ession o T R,
SCARA5 and PSMA and binding o PSMA ela i ely compa ed o he exp ession o he house-keeping p o ein
GAPDH. The densi ome ic analyses we e pe o med on unc opped images using Azu eSpo so wa e. The
alues a e exp essed as means ± s anda d de ia ions o independen iplica es. Ve ical ba s indica e s anda d
de ia ion. *Indica e signi ican di e ences (p < 0.05) be ween he wo cell lines. (b) DOX luo escence ob ained
om con inuous quan i a i e phase imaging o LNCaP cells ea ed wi h DOX/APODOX/APODOX-an i-
PSMA and he compe i i e assay be ween APODOX-an i-PSMA and an i-T R/an i-PSMA an ibodies. (i) DOX
luo escence mic og aphs ob ained a a ious ime poin s o ea men . (ii) DOX luo escence alues om cells
ea ed wi h DOX/APODOX/APODOX-an i-PSMA a a ious ime poin s exp essed as means ± s anda d
de ia ions o six independen measu emen s. Ve ical ba s indica e s anda d de ia ion. *Indica e signi ican
di e ences (p < 0.05) among he es ed g oups. (iii) DOX luo escence alues om compe i i e assay be ween
APODOX-an i-PSMA and an i-T R/an i-PSMA an ibodies exp essed as means ± s anda d de ia ions o
six independen measu emen s. Ve ical ba s indica e s anda d de ia ion. *Indica e signi ican di e ences
(p < 0.05) be ween cells p e- ea ed wi h an ibodies and APODOX-an i-PSMA cellula up ake wi hou
compe i ion. (c) S abili y o mouse an i-PSMA binding o APODOX e alua ed by incuba ion o APODOX-
an i-PSMA wi h 10 mg·mL−1 human IgG o 0, 0.5, 24 and 48 h wi h subsequen de ec ion o mouse an ibodies
conjuga ed on APODOX (i) and eleased in o solu ion (ii). (d) 12.5% SDS-PAGE (i) and na i e-PAGE (ii) gels
showing p o ein con en o DOX (1) APO (2), APO a e disassembly and eassembly (3), APODOX (4) and
APODOX-an i-PSMA (5). Ma ked a e he > 250 kDa APO, ~60 kDa an ibody, and ~19 kDa ligh subuni . (e)
Size, ζ-po en ial and TEM mic og aphs (inse ) o APO (i), APODOX (ii) and APODOX-an i-PSMA (iii).
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SCIENTIFIC RePoR s | (2018) 8:8867 | DOI:10.1038/s41598-018-26772-z
plasma, anging om −19.8 mV o APO, o −26.5 mV o APODOX and −22.0 mV o APODOX-an i-PSMA,
highligh s p onounced s abili y in biological milieu.
E ec o ea men on umo a enua ion and plasma biochemis y. Nex , we p oceeded o e al-
ua e he in i o e ec s o APODOX-an i-PSMA u ilizing mu ine xenog a s, ocusing on i s e ec s on umo
g ow h and damage o he o - a ge o gans. In i o cy o oxici y es s on p os a e cance and heal hy cell lines
we e concluded in de ail and we e e o esul s in ou p e ious wo k17. Mu ine xenog a s we e induced by s. c.
inocula ion o LNCaP cells. Figu e2a shows he expe imen al wo k low o he in i o expe imen , showing he
ea men cou se, e mina ion and subsequen analyses. Al hough all mice gained weigh h oughou he cou se
o he expe imen , DOX- ea ed mice showed signi ican (p < 0.05) losses o weigh ela i e o he saline-injec ed
con ols (Fig.2b). No mice died o had o be eu hanized p io o he end o he expe imen .
The 3D econs uc ion o umo s (Fig.2c) e ealed hei signi ican (p < 0.05) a enua ion in all ea ed g oups.
The as es a enua ion was obse ed in DOX- ea ed mice wi h 42 and 78% dec ease in umo mass a e 1 and
3 weeks o ea men , espec i ely. Mice ea ed o 3 weeks wi h APODOX and APODOX-an i-PSMA showed
dec ease in umo mass by 62 and 55%, espec i ely.
Blood was collec ed a eu hanasia o assess enal and li e unc ion. Va ious biochemical pa ame e s we e
es ed, including c ea inine, alanine amino ans e ase (ALT), aspa a e amino ans e ase (AST), o alkaline phos-
pha ase (ALP) as he esul s a e shown in Fig.3. The li e unc ion ma ke s ALT and AST we e signi ican ly
ele a ed in mice ea ed wi h DOX (AST 6.23 µka ∙L−1 and ALT 0.89 µka ∙L−1) and APODOX (AST 6.25 µka ∙L−1,
ALT 0.97 µka ∙L−1), while mice ea ed wi h APODOX-an i-PSMA showed simila alues o ha o saline-injec ed
mice and wi hin he no mal ange (AST 2.19 µka ∙L−1, ALT 0.67 µka ∙L−1 in APODOX-an i-PSMA- ea ed mice,
AST 3.63 µka ∙L−1, ALT 0.47 µka ∙L−1 in saline-injec ed mice). No ele a ion in ALP le els was obse ed in any o
he ea ed g oups. C ea inine le els, as a ma ke o kidney unc ion, did no show signi ican ele a ion in any o
he ea ed g oups compa ed o saline-injec ed mice. F om he da a ob ained, i ollows ha no ob ious hepa ic o
enal oxici y was obse ed in ea ed mice. Glucose, lac a e, p o eins o albumin a io also showed no signi ican
ele a ion. T iacylglyce ides showed sligh ele a ion in all ea ed g oups compa ed o saline-injec ed mice.
His ologic assessmen o excised o gans. Hea , li e and kidney we e collec ed om each mouse o
es he dis ibu ion o he a ious o ms o DOX in o - a ge o gans and hei oxici y. To e alua e DOX dis-
ibu ion in he o gans, we employed i s speci ic luo escen p ope ies in he issue slices (Fig.4a), as well as in
he homogena es a e ex ac ion by acidi ied isop opanol (Fig.4b). The au o luo escence o all es ed o gans
and hei homogena es excised om saline-injec ed mice was used as a backg ound. DOX- ea ed mice showed
high DOX concen a ion in umo and hea , wi h low concen a ion in li e and unde ec able concen a ion
in kidney. APODOX- ea ed mice showed low DOX concen a ion in umo (2.2× lowe han in DOX- ea ed
mice), wi h s ill high concen a ion in hea (1.1× lowe han in DOX- ea ed mice) and connec i e issue o
li e and unde ec able concen a ion in kidney. Tumo s excised om APODOX-an i-PSMA- ea ed mice showed
he highes obse ed DOX concen a ion among all he mice g oups (3.6× highe han in DOX- ea ed mice).
On he con a y, e y low o unde ec able concen a ion o DOX was ound ei he in hea (2.8× lowe han in
Figu e 2. The a enua ion o s.c. mu ine LNCaP xenog a s ea ed wi h DOX/APODOX/APODOX-an i-
PSMA. (a) Schema ic depic ion o expe imen al wo k low beginning wi h he LNCaP cells (5 × 106) s.c.
inocula ion. The schema ics o mouse and sy inge we e adop ed unde Gene al Public Licence om pixabay.
com. (b) A e age weigh o mice de e mined o e he cou se o expe imen wi h he weigh a he s a o
expe imen designa ed as 100%. (c) Changes in umo size du ing he expe imen . (i) 3D econs uc ion
o umo s. (ii) Changes in umo olumes compa ed o he olume a he s a o expe imen . The alues
a e exp essed as means ± s anda d de ia ions o independen iplica es. *Indica e s a is ically signi ican
di e ences (p < 0.05) when compa ing ea men s wi h con ol (saline).
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SCIENTIFIC RePoR s | (2018) 8:8867 | DOI:10.1038/s41598-018-26772-z
DOX- ea ed mice) o in kidney o hese mice. Low DOX concen a ion was obse ed in connec i e issue o
li e .
The mic os uc u e and i on con en we e assessed in hea , li e and kidney (Fig.5 o s uc u e and Fig.5
inse o i on con en ). The his ological examina ion o hea sec ions e ealed signi ican (10–25%) myoca di-
ocy e acuola ion in DOX- ea ed mice. Howe e , he APODOX and APODOX-an i-PSMA mice showed only
sligh acuoliza ion in he hea (5–10% o APODOX and 1–5% o APODOX-an i-PSMA). The e was no e i-
dence o changes in hea i on con en in any o he es ed g oups. His ological sec ions o he li e showed
diso ganiza ion o he no mal appea ance, inc eased li e single cell nec osis and cen ilobula hepa ocy e
binuclea ion and conges ed cen al eins in DOX- ea ed mice. Inc eased cen ilobula hepa ocy e binuclea ion
was also obse ed in APODOX- ea ed mice, bu only spo adically appea ed in APODOX-an i-PSMA ea ed
mice. A sligh dec ease in li e i on con en was also obse ed in bo h DOX-and APODOX- ea ed mice while
APODOX-an i-PSMA- ea ed mice showed simila i on con en o saline-injec ed ones.
His ological assessmen o kidney showed he p esence o la ge numbe o in aluminal p o ein cas s in
DOX- ea ed mice (in 25–50% o ubula p o iles), while in mice ea ed wi h APODOX and APODOX-an i-PSMA,
hese p o ein cas s we e o med only spo adically (in less han 5% o ubula p o iles). I on was only spo ad-
ically ound in capilla ies o all g oups, oo. The ob ained esul s show ha encapsula ion o DOX in p os a e
cance - a ge ed APO lowe s he in luence o DOX on o - a ge o gans.
Sequence homology o e i ins om di e en o ganisms. Since he APO used in his wo k was
isola ed om equine spleen, conce ns abou i s immunogenici y in mice and u u e human pa ien s can a ise.
Fo his eason, sequences o ligh - and hea y-chain APO om mouse, human and ho se we e downloaded om
Unip o da abase and subjec ed o mul iple sequence alignmen (Fig.6a). The L-chain and H-chain APO om
hese o ganisms showed e y high homology, wi h mos amino acid changes esul ing in amino acid wi h he
same cha ge and only e y mino changes o he s uc u e. The phylogene ic ee (Fig.6b) also shows he high
sequence homology.
Discussion
Ou o he 3.4 illion USD annually spen on heal h ca e in he USA only23, 30.1 billion dolla s is spen owa ds
he impac and managemen o ad e se d ug eac ions due o he inc eased need o hospi aliza ion and addi ional
clinical in es iga ions24. Wi h i s sha e o 125 billion dolla s spen 25, an i- umo ea men and i s many se e e
side e ec s a e he cause o a la ge pa o hese unneeded ex a expenses, no only dec easing he quali y o
pa ien s’ li es bu causing socioeconomic damage as well. The ca dio oxici y o one o he mos commonly used
Figu e 3. Plasma biochemis y analyses e ealing plasma le els o AST, ALT, ALP, c ea inine, glucose, lac a e,
o al p o ein, albumin and iacylglyce ides in mice ea ed wi h DOX/APODOX/APODOX-an i-PSMA and
saline-injec ed con ols. The alues a e exp essed as means ± s anda d de ia ions o independen iplica es.
Ve ical ba s indica e s anda d de ia ion. Dashed lines indica e uppe limi o physiological alues o selec ed
plasma ma ke s. *Indica e s a is ically signi ican inc ease (p < 0.05) abo e he physiological limi .

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SCIENTIFIC RePoR s | (2018) 8:8867 | DOI:10.1038/s41598-018-26772-z
chemo he apeu ic agen s, DOX, was obse ed in up o 26% o adul pa ien s4 and, mo eo e , in 65% o pa ien s
who ecei ed ea men in hei childhood26.
Encapsula ion o subop imal he apeu ics inside sui able nano-scaled ca ie s and hei p e e en ial deli e y
o umo cells can help o e come hese limi a ions. Nanoca ie s no only p o ec he d ug ca go om hos ile
en i onmen s in he body and inc ease i s ci cula ion ime bu , mo e impo an ly, all nanoca ie s o 20–200 nm
ha e he abili y o selec i ely accumula e in umo issue due o he Enhanced Pe meabili y and Re en ion (EPR)
e ec – hei abno mal ascula u e lacking smoo h-muscle laye and con aining la ge enes a ions, no cons an
Figu e 4. (a) Mic og aphs showing DOX dis ibu ion in his ological sec ions o excised umo , hea , li e and
kidney om mice ea ed wi h DOX/APODOX/APODOX-an i-PSMA and saline-injec ed con ols. (b) DOX
dis ibu ion in he o gan homogena es a e ex ac ion by acidi ied isop opanol.
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SCIENTIFIC RePoR s | (2018) 8:8867 | DOI:10.1038/s41598-018-26772-z
blood low and impai ed lympha ic d ainage27. Howe e , i has la ely been disco e ed ha he EPR e ec amoun s
o inc eased accumula ion o nanopa icles in umo issue by only 20–30%28 and e en less in umo s wi h poo
ascula iza ion, such as p os a e o me as a ic li e cance 29. Thus, nanoca ie s elying only on his passi e way
o a ge ing a e s ill no e ec i e enough. Mo eo e , e en i EPR did signi ican ly imp o e he accumula ion
in umo icini y, ca go deli e y inside he ac ual cance cells is no ensu ed. S ill, sui able size is an impo an
pa ame e in o de o a oid p ema u e emo al o he d ug om pa ien ’s body and inc ease, e en hough only
sligh ly, i s accumula ion in he umo 30.
To ully u ilize he p ope ies nano-scaled ca ie s p o ide, a mo e e ec i e ea men s a egy has la ely
been exploi ed, wi h ac i e a ge ing o one o he many memb ane-bound p o eins o e exp essed in mul iple
ypes o cance cells, mos ly due o hei inc eased me abolic needs31. These a ge s include sca enge ecep o s
ype B132, insulin ecep o s33 o ola e ecep o s34. Many cance cells also need inc eased up ake o i on, as i is
impo an o la ge numbe o cellula p ocesses ela ed o ene gy me abolism, espi a ion and DNA syn hesis.
Fo his pu pose, hose cance cells o en o e exp ess ecep o s o ans e in (i on- anspo ing p o ein), and
ans e in-conjuga ed nanoca ie s can be e ec i ely used o inc ease cellula up ake in o hese cells10,19,21,35. The
d awback is ha me abolically highly ac i e cells also o en o e exp ess hese ecep o s, so he deli e y is no as
Figu e 5. Uppe pa : H&E-s ained issue sec ion o o - a ge o gans (hea , li e and kidney) collec ed om
mice ea ed wi h DOX/APODOX/APODOX-an i-PSMA and saline-injec ed con ols. Black a ows in hea
show myoca diocy e acuola ion. Black a ows in li e show cen ilobula hepa ocy e binuclea ion. CV shows
he conges ed cen al ein. Black a ows in kidney show in aluminal p o ein cas s. Inse s showdis ibu ion
o i onde ec ed byPe ls’ P ussian blue s aining. Lowe pa : His opa hological indings and se e i y sco es in
hea , li e and kidney in mice ea ed wi h he a ious DOX o mula ions. (a) Ca diomyocy e acuola ion (%
a ec ed ca diomyocy es): 1 = 1–5%; 2 = 5–10%; 3 = 10–25%; 4 = g ea e han 25%. (b) Li e single cell nec osis:
1 = dead hepa ocy es we e a ely obse ed (1 pe 10 high powe ield/400×). (c) In aluminal p o ein cas s:
1 = de ec ed in less han 5% o he ubula p o iles; 2 = de ec ed in 5–25% o he ubula p o iles; 3 = de ec ed in
25–50% o he ubula p o iles; 4 = de ec ed in g ea e han 50% o he ubula p o iles.
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SCIENTIFIC RePoR s | (2018) 8:8867 | DOI:10.1038/s41598-018-26772-z
speci ic as equi ed36. The e o e, mo e speci ic a ge s a e equi ed. The nanoca ie in he p esen ed s udy was
a ge ed owa ds PSMA, a 100 kDa ype II memb ane glycop o ein, highly exp essed on almos all p os a e can-
ce cells37 o endo helial cells o umo ascula u e o many non-p os a ic solid malignancies bu no on heal hy
cells38. Since Liu and cowo ke s ound ha an i-PSMA an ibodies a e cons i u i ely endocy osed in LNCaP cells
ia cla h in-coa ed pi s39, we expec endocy osis o be he majo in e naliza ion ou e o APODOX-an i-PSMA.
Fe i ins a e i on s o age p o eins ha can be ubiqui ously ound in all li e o ms, excep yeas 40. Once emp ied
o hei i on con en , hey c ea e a hollow icosahed al p o ein cage – APO41. Thei qua ena y s uc u e is o med
by 24 subuni s wi h a ious a ios o hea y (H-, 21 kDa) and ligh (L-, 19 kDa) chain. Based on his H-/L-chain
a io, APO can en e cells ia T R ( o H-chain APO)22, TIM-2 ( o H-chain APO)42,43 o SCARA5 ( o L-chain
APO)22,44,45, al hough some wo ks ha e mis akenly iden i ied T R as he main ecep o o bo h H- and L-chain
APO46–48.
The easily pe o med encapsula ion p o ocol o small molecules in APO is based on i s esponsi eness o he
su ounding pH49,50. The qua e na y s uc u e o APO e e sibly disassembles in pH be ween 2.0 and 3.4 and
eassembles once in a pH abo e 7.051. The APO-encapsula ed d ug has a e y good s abili y du ing long- e m
s o age52.
Di e en app oaches o in oducing ac i ely a ge ed moie ies on o APO su aces, ha e been deployed.
Some wo ks elied on na u al APO ecep o s47,48,53, o he s in oduced a ibodies o sho pep ides o a ge -
ing o umo cells6 o ascula u e54. We employed a ge ing ia si e-di ec ed an ibodies. APO su ace was unc-
ionalized by adso p ion o gold nanopa icles o mul i udes o posi i ely and nega i ely cha ged amino acid
esidues. These adso bed gold nanopa icles u he enabled binding o a hep apep ide de i ed om p o ein A
(by i s cys eine- ich C- e minus). We achie ed si e-di ec ed o ien a ion o an ibodies due o he high a ini y o
N- e minus o his pep ide owa ds Fc agmen o immunoglobulins (KD = 10 µM o IgG1)55,56. This enables a
s aigh acing o pa a opes owa ds an igens, while inc easing he immunoe iciency. In non-di ec ed sys ems,
immunope o mance is inhibi ed by andom in e ac ions be ween pa a opes and nanoca ie ’s su ace caus-
ing s e ical blockades17. In he p e iously pe o med in i o oxicological es s, we we e able o conclude ha
PSMA- a ge ed APO selec i ely deli e ed DOX in o PSMA-o e exp essing cance cells. This cons uc inhibi ed
he g ow h o cance cells wi h a simila po ency as ee DOX while signi ican ly spa ing non-malignan cells
om he nega i e e ec s o DOX17.
Nume ous nanoca ie s es ed o e he pas decades ha e had e y p omising in i o esul s, only o ail,
due o mul iple easons, once adminis e ed in o ganism. Some nanoca ie s unwan edly in e ac wi h biological
milieu and hei ou e su ace needs o be modi ied wi h polyme s o pep ides, dec easing hese in e ac ions19,57,58.
Figu e 6. (a) Mul iple sequence alignmen o mouse L-chain (Unip o da abase accession P29391), mouse
H-chain (Unip o da abase accession P09528), human L-chain (Unip o da abase accession P02792), human
H-chain (Unip o da abase accession P02794), ho se L-chain (Unip o da abase accession P02791) and ho se
H-chain (Unip o da abase accession Q8MIP0) e i in. (b) Phylogene ic ee showing he dis ance be ween
mouse L-, mouse H-, human L-, human H-, ho se L- and ho se H-chain e i ins.
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SCIENTIFIC RePoR s | (2018) 8:8867 | DOI:10.1038/s41598-018-26772-z
Some nanoca ie s ha e high p ema u e e lux o d ug molecules in blood, meaning ha nanoca ie s o en each
umo si e as d ug- ee30. Ou p e ious esul s p o ed ha DOX is no p ema u ely eleased om APO du ing i s
ci cula ion in bloods eam, bu is only emo ed once in in acellula en i onmen 17.
To e alua e he in i o e ec s o PSMA- a ge ed, APO-encapsula ed DOX, xenog a mice we e ea ed wi h
DOX/APODOX/APODOX-an i-PSMA. Fi s , we s udied he a e o cellula up ake o hese a ious DOX o ms
in PSMA-o e exp essing cells. Al hough he highes cellula up ake was obse ed o ee DOX, PSMA- a ge ing
was able o signi ican ly inc ease he up ake compa ed o non-modi ied APODOX. We pe o med a compe i i e
assay wi h an i-PSMA an ibodies o elucida e ha APODOX-an i-PSMA does indeed employ PSMA o he
in e naliza ion in a ge cells ins ead o ecep o s ha a e employed by ba e e i ins. As ho se spleen APO is
composed o 22/24 L-chain subuni s, i p e e en ially in e nalizes in o cells h ough SCARA522,44,45,59. Howe e ,
since he exp ession o L-chain e i in ecep o SCARA5 on LNCaP cells was negligible and 8% o ho se spleen
APO subuni s a e hea y, we es ed he in luence o compe i ion wi h H-chain T R an ibody and p o ed ha his
compe i ion had no signi ican e ec on APODOX-an i-PSMA in e naliza ion.
Since he p o ein A-de i ed hep apep ide used o bind a ge ing an i-PSMA an ibody has a ini y owa ds Fc
egion o an ibodies p oduced in a ious o ganisms56, we u he con i med he s abili y o mu ine an i-PSMA
binding e en a e p olonged compe i ion wi h human IgG an ibodies.
Nex , he a enua ion o PSMA-o e exp essing umo s in mice ea ed wi h hese a ious o ms o DOX was
e alua ed. The as es a enua ion was obse ed o ee DOX, which is in acco dance wi h in i o expe imen s.
Howe e , APODOX and APODOX-an i-PSMA showed simila an i-cance po ency, showing ha bo h o hese
o ms o DOX we e able o each he su ace o umo , likely due o EPR e ec . Howe e , a e he ea men
cou se ended, he in es iga ion o DOX dis ibu ion in umo slices clea ly showed ha he DOX was able o each
he inne pa o umo only in mice ea ed wi h APODOX-an i-PSMA while in mice ea ed wi h APODOX, i
only eached he su ace o he umo . This phenomenon can e y o en be obse ed o a ious nanopha maceu-
icals whe e hei high concen a ion on he su ace o umo and i s high in e s i ial p essu e can ac ually educe
umo pe usion and hus hinde he dose o d ug ha eaches he en i e umo mass9.
Besides easy encapsula ion p o ocol wi h a high encapsula ion e icacy, eliable a ge ing and con enien size,
sui able nanoca ie needs o be comple ely biocompa ible, a oxic and biodeg adable30. Many nanoca ie s ha
a e no na u al o pa ien ’s body can cause ad e se eac ions, accumula e in he body o p olonged ime a e
hei ca go is deli e ed o , upon en e ing he bloods eam, bind blood p o eins, be opsonized and aken up by
mononuclea phagocy es, ne e deli e ing hei ca go a all60. S a egies o a oid he immune esponse o en
include modi ica ion o he nanoca ie su ace by s eal h molecules, such as PEG. Howe e , hese modi ica ions
ac ually hampe he en y o cance cells and he deli e y o he ca go7. APO, as a ubiqui ously ound p o ein
among all li e o ms wi h a high sequence homology, is na u al o he body. Al hough e i in i sel is a oxic
o he o ganism, some s udies ha e shown ha apo e i in-based nano o mula ions can nega i ely in luence
i on me abolism, i. e. emo e i on om o - a ge issues61, due o i s highe na u al a ini y o i on han o
he encapsula ed d ug molecules15. In his wo k, we p o ed ha he i on le els in o - a ge o gans emained
in ac in APODOX-an i-PSMA- ea ed mice, al hough some changes we e obse ed in mice ea ed wi h ei he
DOX o APODOX. This p o ed ha DOX-loaded and si e-di ec ed APODOX does no nega i ely in luence i on
me abolism.
To in es iga e he non-speci ic oxici y o a ious ypes o DOX, he e ec o he ea men on o - a ge issues
was also s udied, namely hea , li e and kidney, which a e mos p ominen ly damaged by DOX. While DOX
ea men showed signi ican in luence on he body weigh o ea ed mice, no such end was obse ed o ei he
o he APO-encapsula ed DOX o mula ions.
On he o gan le el, biochemical analyses and his ological assessmen clea ly showed damage o li e o mice
ea ed wi h DOX and non- a ge ed APODOX, as e idenced by he ele a ion in le els o li e enzymes, conges ed
cen al eins and hepa ocy e binuclea ions. Al hough small numbe o hepa ocy e binuclea ions (as well as DOX
p esence in bounda ies o he lobules) was also obse ed in mice ea ed wi h APODOX-an i-PSMA, he le els o
li e enzymes showed no damage o he li e as a whole.
Fu he mo e, no signi ican inc ease in he le els o kidney enzymes and no DOX dis ibu ion in kidney we e
obse ed in any o he ea ed g oups, showing unc ional glome ula il a ion. Howe e , la ge numbe o in a-
luminal p o ein cas s was ound in DOX- ea ed mice. APODOX- and APODOX-an i-PSMA- ea ed mice also
showed he o ma ion o hese p o ein cas s, al hough hei numbe was much lowe , p o ing lowe damage o
kidney compa ed o ha caused by ee DOX.
In hea , he o gan which is clinically damaged by DOX ea men he mos , highe DOX concen a ion was
ound in he myoca dium o mice ea ed wi h DOX and APODOX, as well as acuola ion o he myoca diocy es.
The obse ed oxici y o non- a ge ed APODOX could be explained by i s a ini y o SCARA5, which is also
exp essed by hea muscle cells62. APODOX-an i-PSMA- ea ed mice had negligible amoun s o myoca diocy e
acuola ion and e y low DOX concen a ion was obse ed in hei myoca dium.
Ma e ial and Me hods
Chemicals. All chemicals we e ob ained in ACS pu i y om Sigma-Ald ich (S . Louis, MO, USA), unless
o he wise s a ed. The pH was measu ed using pH me e WTW inoLab (Weilheim, Ge many). Solu ion o 0.2
µm- il e ed ho se spleen APO in 0.135 M sodium chlo ide (ca . no. A3641), as well as doxo ubicin hyd ochlo ide
(ca . no. 44583) we e pu chased om Sigma-Ald ich.
Syn hesis o componen s and assembly o APODOX and APODOX-an i-PSMA. APODOX was
p epa ed ollowing he p o ocol published in ou p e ious s udy17. B ie ly, 200 µL o 1 mg∙mL−1 DOX (in wa e )
was added o 20 µL o 50 mg∙mL−1 ho se spleen APO and 100 µL o wa e . Hyd ochlo ic acid (2.5 µL, 1 M) was
added o dec ease he pH o he solu ion o 2.7 and disassocia e he APO. The solu ion was s i ed o 15 min