PAPER
Magali Ga y-Bobo, José A. Pomposo, Fabienne Dumoulin e al .
Topology e ec s in pho odynamic he apy wi h
ph halocyanine nanoca ie s
Ma e ials
Ad ances
sc.li/ma e ials-ad ances
ISSN 2633-5409
Volume 6
Numbe 1
7 Janua y 2025
Pages 1–466
148 | Ma e . Ad ., 2025, 6, 148–156 © 2025 The Au ho (s). Published by he Royal Socie y o Chemis y
Ci e his: Ma e . Ad ., 2025,
6, 148
Topology effec s in pho odynamic he apy wi h
ph halocyanine nanoca ie s†
Da ide A ena,
a
U¨mi I˙s-ci,
b
Me
´lanie Ono e,
c
Ch is ophe Nguyen,
c
Zeynel S-ahin,
b
Es e Ve de-Ses o,
ad
Amaia I u ospe,
a
A an xa A be,
a
Magali Ga y-Bobo, *
c
Jose
´A. Pomposo *
ade
and Fabienne Dumoulin *
Two diffe en amphiphilic copolyme s, a andom copolyme (RCP) and a block copolyme (BCP), ha e
been used o encapsula e a a - ed pho osensi izing hyd ophobic zinc ph halocyanine while o ming
sel -assembled nanoca ie s o diffe en opology: sel - olded single-chain nanopa icles (SCNPs) and
s a -like agg ega es (SLAs), espec i ely. Diffe en copolyme /ph halocyanine a ios ha e been es ed o
assess hei effec on s uc u al p ope ies which we e de e mined by small-angle X- ay sca e ing
(SAXS) measu emen s. The ele ance o hese ma e ials as pho osensi ize nanoca ie s o
pho odynamic he apy (PDT) has been s udied agains human b eas cance cells (MCF-7). A be e PDT
effec was ound o he SCNP-Pc2
x
(100 mgmL
1
) exci ed a 800 nm wi h a pulsed lase han o he
SLA-Pc2
x
unde iden ical condi ions. Con e sely, i adia ion a sligh ly lowe wa eleng hs (740 nm) o
MCF-7 cells incuba ed wi h SLA-Pc2
x
esul ed in a no o ious PDT effec when compa ed o ha
obse ed o MCF-7 cells incuba ed wi h he SCNP-Pc2
x
. These sys ems ep esen new s a egies o
he encapsula ion o pho osensi ize s o pho odynamic he apy.
1. In oduc ion
Pho odynamic he apy (PDT) is now a well- ecognized ea men
o cance , e en hough i is s ill oo o en desc ibed as eme ging.
The i s pho osensi ize s we e app o ed decades ago,
1,2
bu had
se e al d awbacks in e ms o cha ac e iza ion, umo a ge ing
and efficiency. Second gene a ion-pho osensi ize s ha e since
been app o ed o a e in ad anced clinical ials.
3–5
Using exci a-
ion wa eleng hs belonging o he i s pho o he apeu ic window
(600–1000 nm) o PDT is ad an ageous o wo easons: i s , i
a oids exci ing endogenous ch omopho es,
6
and second, i
allows deepe ligh pene a ion in o biological issues han wi h
sho e wa eleng hs.
7
Ph halocyanines a e syn he ic e apy olic
molecules o he po phy inoid amily and show ypically an
a e age maximum abso p ion a 700 nm. In his espec , ph ha-
locyanines a e excellen pho osensi ize molecula bases o an i-
cance PDT.
8–11
Many wa e -soluble ph halocyanines wi h ca io-
nic, anionic o neu al subs i uen s ha e been used o PDT.
12
Howe e , hey a e equen ly agg ega ed, which impai s hei
e iciency. Besides, excessi e wa e -solubili y o pho osensi ize s
may be de imen al since i induces a apid clea ance om he
body wi hou ha ing he chance o accumula e in he a ge ed
issues, and/o p e en s cellula in e naliza ion by limi ing he
cellula memb ane c ossing. The use o hyd ophobic pho o-
sensi ize s o e comes hese issues, bu a signi ican d awback
is ha hey a e no biocompa ible as hey canno ci cula e in he
bloods eam. This p omo ed he use o a ious nanoca ie s,
ei he by encapsula ion o by co alen g a ing o he hyd opho-
bic pho osensi ize .
13
An addi ional ad an age is ha nano-
o mula ed pho osensi ize s can bene i om he enhanced
pe meabili y and e en ion (EPR) e ec , which may ha e some
limi a ions
14
bu isanywayagoodway odeli e pho osensi ize s.
A liposomal o mula ion o unsubs i u ed zinc ph halocyanine
has en e ed ad anced clinical ials,
15
and se e al micella o -
mula ions o ph halocyanine de i a i es o PDT applica ions
ha e been epo ed.
16,17
Conjuga ing a mono-hyd oxyla ed ph ha-
locyanine o poly-L-glu amic acid allowed o ob ain polyme ic
nanopa icles o high e iciency,
18,19
and ph halocyanine-based
o gano-silica nanopa icles exhibi ed ema kable pho odynamic
a
Cen o de Fı
´sica de Ma e iales (CSIC, UPV/EHU) and Ma e ials Physics Cen e
MPC, Paseo Manuel La dizabal 5, 20018 Donos ia, Spain.
E-mail: jose xo[email p o ec ed]
b
Ma ma a Uni e si y, Facul y o Technology, Depa men o Me allu gical and
Ma e ials Enginee ing, 34854 Mal epe, Is anbul, Tu
¨ kiye
c
Ins i u des Biomole
´cules Max Mousse on, CNRS, ENSCM, 34293 Mon pellie ,
F ance. E-mail: [email p o ec ed]
d
IKERBASQUE–Basque Founda ion o Science, Plaza de Euskadi 5, 48009 Bilbao,
Spain
e
Depa amen o de Polı
´me os y Ma e iales A anzados, Fı
´sica, Quı
´mica y Tecnologı
´a,
Uni e si y o he Basque Coun y (UPV/EHU), 20800 Donos ia, Spain
Acıbadem Mehme Ali Aydınla Uni e si y, Facul y o Enginee ing and Na u al
Sciences, Depa men o Biomedical Enginee ing, 34752 A as-ehi , Is anbul,
Tu
¨ kiye. E-mail: [email protected].
†Elec onic supplemen a y in o ma ion (ESI) a ailable. See DOI: h ps://doi.o g/
10.1039/d4ma00524d
Recei ed 21s May 2024,
Accep ed 5 h No embe 2024
DOI: 10.1039/d4ma00524d
sc.li/ma e ials-ad ances
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Ad ances
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e iciency.
20,21
I has been shown ha he na u e o he polyme ic
nanoca ie can g ea ly a ec he pho odynamic e iciency o a
nano-pho osensi izing sys em.
22
Sel -assembled nanoca ie s such as single-chain nano-
pa icles (SCNPs) ha e p o en o be efficien ca ie s o he
deli e y o hyd ophobic d ugs.
23–25
These wo ks a e ela i ely
ecen . Indeed, only a hand ul o e apy olic de i a i es com-
bined wi h SCNPs ha e been epo ed: a po phy in has been
g a ed on o he skele on o indi idual SCNPs;
26
ph haloni iles
(which a e he mos common p ecu so o ph halocyanines)
ha e been in oduced on a polys y ene backbone, he subse-
quen in amolecula o ma ion o he ph halocyanine moie y
leading o he o ma ion o colo ed single-chain polyme ic
nanopa icles;
27
and he axial coo dina ion o a polyme ic chain
con aining py idine moie ies by a cobal ph halocyanine p o-
duced CO
2
- educing ma e ials.
28
In his wo k, a pho osensi izing a - ed abso bing hyd o-
phobic zinc ph halocyanine has been designed o be encapsula ed
in o wo diffe en polyme ic nanoca ie s p epa ed om he same
monome s. The andom copolyme sel -assembled in o sel -
assembled single-chain nanopa icles (SCNPs), and he block
copolyme o med s a -like agg ega es (SLAs). A ca e ul cha ac e -
iza ion o he esul ing ph halocyanine-loaded nanoca ie s
(SCNP-Pc2
x
and SLA-Pc2
x
) was ca ied ou by means o small-
angle X- ay sca e ing (SAXS) expe imen s. Finally, we epo how
he e y diffe en opology o hese wo nanoca ie s modula e he
PDT efficiency on MCF-7 b eas cance cells.
2. Expe imen al
2.1. Chemis y
2.1.1. Ma e ials and me hods. Oligo(e hyleneglycol)mono-
me hyle he me hac yla e (OEGMA
300
) (99%), (2-ace oace oxy)-
e hyl me hac yla e (AEMA) (95%), 2,20-azobis(2-me hylp opio-
ni ile) (AIBN) (Z98%), 1,4-dioxane, n-hexane, deu e a ed chlo o o m
(CDCl
3
) (99.96 a om% D, con aining 0.03% ( / ) e ame hylsi-
lane, TMS), sodium phospha e dibasic hep ahyd a e (Na
2
HPO
4
7H
2
O), sodium phospha e monobasic monohyd a e (NaH
2
PO
4
H
2
O) we e pu chased om Sigma-Ald ich and used, unless speci-
ied, as ecei ed. 4-Cyanopen anoic acid di hiobenzoa e (CPADB)
(Z97%) was pu chased om S em Chemicals. Me hanol and
e ahyd o u an (THF) (HPLC g ade) we e pu chased om Scha -
lab. AIBN was ec ys allized om me hanol. OEGMA
300
and AEMA
we e pu i ied by passing h ough basic alumina. 3-Ni oph halo-
ni ile, isobu yl hiol, hexadeu e a ed dime hylsul oxide
(DMSO-d
6
), dime hyl o mamide (DMF), po assium ca bona e
(K
2
CO
3
), dime hyl sul oxide (DMSO), zinc ace a e Zn(OAc)
2
,
dime hylaminoe hanol and dichlo ome hane we e used as
pu chased. Column ch oma og aphies we e done using Me ck
Silica Gel 60 (0.040–0.063 mm). NMR spec a we e eco ded a
oom empe a u e in CDCl
3
o DMSO-d
6
solu ions on a Va ian
spec ome e (500 MHz o
1
H, 125 MHz o
13
C spec a). FT-IR
spec a we e eco ded be ween 4000 and 650 cm
1
using a
Pe kinElme Spec um 100 FT-IR spec ome e . Mass spec a
we e measu ed on a MALDI (ma ix assis ed lase deso p ion
ioniza ion) BRUKER Mic o lex LT (B emen, Ge many) using
2,5-dihyd oxybenzoic acid as he ma ix.
2.1.2. Syn hesis o 3-isobu yl hioph haloni ile (1). 3-Ni o-
ph haloni ile (5 g, 28.8 mmol), isobu yl hiol (2.9 g, 33.2 mmol)
and d y po assium ca bona e (30 g, 217.5 mmol) we e s i ed in
anhyd ous DMF (50 mL) unde a gon a 40 1C o 24 h un il
comple e disappea ance o 3-ni oph haloni ile in TLC mon-
i o ing. Then he eac ion mix u e was pou ed in o wa e
(500 mL), and he esul ing solid was collec ed by il a ion
and ho oughly washed wi h wa e . The d ied c ude p oduc
was ec ys allized om e hanol. Yield: 80% (4.98 g).
1
H NMR
(500 MHz, CDCl
3
)d, ppm: 7.61–7.56 (m, 3 H), 2.96 (d, 2H), 1.96
(m, 1 H), 1.11 (d, 6 H).
13
C NMR (125 MHz, CDCl
3
)d, ppm:
146.3, 132.6, 131.1, 129.4, 117.2, 115.3, 114.7, 113.8, 41.9, 28.1,
21.9. FT-IR (n,cm
1
): 3090, 2973, 2956, 2934, 2230, 1566, 1454,
1421, 1385, 1319, 1259, 1198, 1153, 1083, 958, 792, 726.
2.1.3. Syn hesis o ph halocyanine Pc2. A mix u e o 1(1 g,
4.62 mmol) and Zn(OAc)
2
(424 mg, 2.31 mmol) was e luxed in
dime hylaminoe hanol (10 mL) unde a gon o 12 h. A e
cooling o oom empe a u e, he eac ion mix u e was pou ed
in o wa e (100 mL). The esul ing p ecipi a e was il e ed off
and washed se e al imes wi h e hanol. Pc2 was isola ed by
ch oma og aphy on silica gel using a mix u e o dichlo o-
me hane/e hanol (100/1) as he eluen . Yield: 28% (300 mg).
MALDI-TOF-MS (DHB) m/z: 930.301 [M] +; calcula ed o C
48
-
H
48
N
8
S
4
Zn: 930.588. FT-IR (n,cm
1
) 3059, 2953, 1592, 1565,
1462, 1430, 1365, 1330, 1311, 1222, 1163, 1103, 1041, 946, 900,
881, 792, 757, 735, 679.
1
H NMR (500 MHz, DMSO-d
6
)d, ppm:
8.97–8.91 (m, 3H), 8.66–8.60 (m, 2 H), 8.06–7.59 (m, 8 H), 3.38–
3.16 (m, 8 H), 2.23 (m, 4 H), 1.31 (m, 24 H). UV- is (THF): l
max
,
nm (loge): 710 (5.3), 641 (4.6), 340 (4.9).
2.1.4. Syn hesis o he andom copolyme poly(OEGMA
300
-
-AEMA) (RCP). 10.55 mg (0.038 mmol) o 4-cyanopen anoic
acid di hiobenzoa e (CPADB) and 1.29 mg (0.0077 mmol) o
AIBN, 1.83 mL (6.4 mmol) o OEGMA
300
, 0.31 mL (1.6 mmol) o
AEMAa and 3.37 mL o dioxane we e added in his o de . The
sys em was sealed wi h a ubbe sep um and he mix u e was
hen le o degas unde ine gas o 15 minu es. A e ha
ime, he eac ion mix u e was main ained a 70 1C, unde
ni ogen posi i e p essu e and unde con inuous magne ic
s i ing o 17 hou s. M
n
(kDa)= 99.6, PDI= 1.04, dn/dc=
0.185. AEMA con en (mol%) = 20.
1
H NMR (400 MHz, CDCl
3
)
d, ppm: 4.37 (m, CCO
2
C
H
2
CH
2
CO
2
CH
2
), 4.18–4.11 (m, 2H,
CCO
2
CH
2
C
H
2
CO
2
), 3.83–3.58 (m, CH
2
O
C
H
2
CH
2
), 3.41 (s,
O
C
H
3
), 2.33 (s, CH
2
CO
C
H
3
), 1.81 (m, CCH
2
), 1.04 (m, CCH
3
),
0.87 (m, CCH
3
).
2.1.5. Syn hesis o he block copolyme poly(OEGMA
300
)-b-
poly(AEMA) (BCP). 29.25 mg (0.105 mmol) o CPADB and
3.45 mg (0.021 mmol) o AIBN we e dissol ed in 1.5 mL o
anhyd ous 1,4-dioxane. Then, 0.6 mL (3.144 mmol) o AEMA
was added and he esul ing mix u e was le degassing pu ging
ni ogen o 20 minu es. A e his ime, he solu ion was le
s i ing unde ni ogen posi i e p essu e and a 70 1C o
5 hou s. Then, he eac ion was quenched by eezing he
mix u e a 78 1C. 1 mL (0.014 mmol o mac o-CTA) o he
c ude, 6 mL (21 mmol) o OEGMA
300
, 2.3 mg (0.014 mmol) o
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AIBN and 18 mL o 1,4-dioxane we e degassed pu ging ni ogen
o 20 minu es, hen le s i ing a 70 1C unde posi i e
ni ogen p essu e o 17 hou s. A e his ime, he eac ion
was quenched by cooling he mix u e a 78 1C. The polyme
was p ecipi a ed in a la ge excess o n-hexane (40) o h ee
consecu i e imes and d ied unde dynamic acuum. M
n
(kDa) =
71.8, PDI = 1.02, dn/dc = 0.185. AEMA con en (mol%) = 14.
1
H NMR (400 MHz, CDCl
3
)d, ppm: 4.37 (m, CCO
2
C
H
2
CH
2-
CO
2
CH
2
), 4.18–4.10 (m, 2H, CCO
2
CH
2
C
H
2
CO
2
), 3.77–3.57
(m, CH
2
O
C
H
2
CH
2
), 3.40 (s, O
C
H
3
), 2.32 (s, CH
2
CO
C
H
3
), 1.87
(m, CCH
2
), 1.04 (m, CCH
3
), 0.87 (m, CCH
3
).
2.1.6. Cha ac e iza ion o RCP and BCP. Molecula
s uc u e and copolyme composi ion we e in es iga ed ia
1
H NMR spec oscopy, using a B uke spec ome e ope a ing
a 400 MHz. AEMA con en , exp essed as %AEMA (mol%) was
calcula ed acco ding o eqn (1):
%AEMAðmol%Þ¼
SAEMA
COCH3
SAEMA
COCH3þSOEGMA
OCH3
100 (1)
whe e SAEMA
COCH3is he signal in ensi y o he me hylic p o ons o
AEMA unc ional g oups (d: 2.3 ppm) and SOEGMA
OCH3is he signal
in ensi y o me hoxylic p o ons o OEGMA moie ies (d: 3.4 ppm),
bo h in eg a ed aking SOEGMA
OCH3as in e nal s anda d.
Numbe -a e age molecula weigh (M
n
) and dispe si y (Ð)
we e de e mined ia size exclusion ch oma og aphy/mul i-
angle ligh sca e ing (SEC/MALS) measu emen s, which we e
pe o med a 30 1C on an Agilen 1200 sys em equipped wi h
PLgel 5 mm Gua d and PLgel 5 mm MIXED-C columns and iple
de ec ion: a diffe en ial e ac i e index (dRI) de ec o (Op ilab
Rex, Wya ), a mul i-angle lase ligh sca e ing (MALS) de ec o
(MiniDawn T eos, Wya ), and a iscosime ic (VIS) de ec o
(ViscoS a -II, Wya ). THF was used as eluen a a low a e o
1 mL min
1
.
The opology o RCP and BCP in wa e was de e mined om
small-angle X- ay sca e ing (SAXS) measu emen s on polyme
solu ions a a ixed polyme concen a ion o 1 mg mL
1
. SAXS
expe imen s we e conduc ed on a Rigaku 3-pinhole PSAXS-L
equipmen ope a ing a 45 kV and 0.88 mA. The Mic oMax-002+
X-Ray Gene a o Sys em is composed by a mic o ocus sealed
ube sou ce module and an in eg a ed X-Ray gene a o uni
which p oduces CuKa ansi ion pho ons o wa eleng h l=
1.54 Å. The adius o gy a ion (R
g
) and size scaling exponen
(n)o RCP we e de e mined h ough i s o he expe imen al da a
o a gene alized Gaussian coil
29
unc ion. Fo BCP, analysis o
he SAXS da a in e ms o he Dozie
30
s a model p o ided R
g
and he a e age numbe o a ms, . In bo h cases he SAS i
p og am was used.
31
UV- is measu emen s o RCP and BCP we e ca ied ou in
wa e a 25 1C in an Agilen 8453A appa a us wi h Pel ie
he mos a ic cell holde , T-con olle 89090A.
2.1.7. Gene al p ocedu e o he encapsula ion o Pc2 in o
RCP o BCP o gi e SCNP-Pc2
x
and SLA-Pc2
x
, espec i ely.
A s ock solu ion o Pc2 (0.25 mg mL
1
) was p epa ed by
dissol ing 1 mg (1.1 mmol) o Pc2 in 4 mL o THF. 10 mg o
RCP o BCP we e dissol ed in 1 mL o THF and Pc2 s ock
solu ion was added (186 mL o 447 mL). The esul ing solu ion
was le s i ing o 48 hou s un il comple e e apo a ion o he
THF and he o ma ion o a hin ilm. A e his ime, 10 mL o
deionized wa e was added, and he esul ing mix u e was le
s i ing a oom empe a u e o 3 days. Pc2 concen a ion is
he e o e ei he 5 o 12 mM, and he espec i e nanopa icles
a e deno ed as SCNP-Pc2
x
o SLA-Pc2
x
(x= 5 o 12).
2.1.8. Cha ac e iza ion o SCNP-Pc2
x
and SLA-Pc2
x
.SCNP-
Pc2
x
and SLA-Pc2
x
we e cha ac e ized by SAXS and UV- is, ollow-
ing he same p ocedu es used o RCP and BCP, espec i ely.
2.2. Op ical measu emen s
UV- isible elec onic abso p ion spec um was eco ded on a
Shimadzu 2001 UV spec opho ome e .
Fluo escence quan um yield o Pc2 was de e mined by he
compa a i e me hod acco ding o he equa ion:
FF¼FFðs dÞF:AðS dÞ:n2
FðS dÞ:A:n2
ðS dÞ
whe e Fand F
S d
a e he a eas unde he emission cu es o he
sample and s anda d, espec i ely. Aand A
S d
a e he abso -
bance a he exci a ion wa eleng h o he sample and s an-
da d, nis he e ac i e index o he sol en s o he sample and
e e ence. Unsubs i u ed ZnPc in DMSO was used as a s anda d
(FF
(S d)
= 0.20) and sample and s anda d we e exci a ed (a l=
640 nm) a he same wa eleng h in DMSO.
Single oxygen quan um yield o Pc2 was ob ained in DMSO
solu ion(inai ,nooxygenbubbled)byindi ec me hodwi hzinc
ph halocyanines (ZnPc) as a e e ence and 1,3-diphenylisobenzo-
u an (DPBF) as a chemical quenche o single oxygen, using he
allowing equa ion:
FD¼FS d
D
RIS d
abs
RS d Iabs
whe e FS d
Dis he single oxygen quan um yield o he s anda d
ZnPc (F
D
=0.67inDMSO).Rand R
S d
a e he DPBF pho obleach-
ing a es in he p esence o Pc2, and he s anda d, espec i ely.
I
abs
and I
S d
abs
a e he a es o ligh abso p ion by Pc2 and s anda d,
espec i ely. Mix u e o ph halocyanine (B6mM, abso p ion B1.2
a 717 nm) and DPBF (B30 mM, abso p ion B0.8 in 417 nm) was
i adia ed in he Q band egion in 10 cycles o 5 s each one. DPBF
deg ada ion was moni o ed a 417 nm and he F
D
alue was
de e mined using he se up desc ibed abo e.
32,33
2.3. Biology
2.3.1. Ma e ials. Human b eas cance cells (MCF-7) we e
pu chased om Ame ican Type Cul u e Collec ion (ATCC), Dul-
becco’s modi ied Eagle’s Medium (DMEM/F12), e al bo ine se um
(FBS), and penicillin/s ep omycin we e pu chased om Gibco Li e
Technology, 3-(4,5-dime hyl hiazol-2-yl)-2,5-diphenyl e azolium
b omide (MTT) was pu chased om Al a Aesa (The mo ishe ).
2.3.2. Cell cul u e. MCF-7 cells we e main ained in DMEM/
F12 supplemen ed wi h 10% FBS and 1% penicillin/s ep omycin.
Cells g ew a 37 1C, in humidi ied a mosphe e and unde
5% CO
2
.
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2.3.3. Cy o oxici y s udies. MCF-7 cells we e seeded in a
plas ic mul i-well (96) pla e. The day a e seeding, cells we e
incuba ed wi h inc easing doses o nanopa icles ( om 0
o 200 mgmL
1
) o 72 h. The iabili y o he cells incuba ed
wi h nanopa icles was e alua ed using MTT assay. Fo his,
cells we e incuba ed o 4 h wi h 0.5 mg mL
1
o MTT in media.
The MTT/media solu ion was hen emo ed, and he p ecipi a ed
o mazan c ys als we e dissol ed in 150 mLo anequal olume
solu ion o e hanol/DMSO. A e 30 min, he op ical densi y (OD)
o samples was ead a 540 nm using mic opla e eade . The OD
alues a e di ec ly co ela ed wi h he numbe o li ing cells in
well. Cell iabili y was calcula ed as % iabili y = OD o ea ed
cell/OD o ehicle con ol 100.
2.3.4. PDT expe imen s. Fo con inuous exci a ion (633,
650 o 740 nm), MCF-7 cells we e seeded in a plas ic mul i-well
(96) pla e. The day a e , cells we e incuba ed wi h 100 mgmL
1
o
SCNP-Pc2
x
o SLA-Pc2
x
o 24 h. A e he incuba ion ime, cells
we e exposed o no o ligh sou ce a 633, 650 o 740 nm. The
exci a ion a 633 nm (4.96 J cm
2
)waspe o medwi hanEVOS
5000 mic oscope using Cy5 EVOSLEDligh cube du ing 1 min,
64,9 mW a magni ica ion 4.Theexci a iona 650nm
(39 J.cm
2
) was pe o med wi h a po able lase du ing 20 min.
The exci a ion a 740 nm (11.25 J m
2
)waspe o medwi haLED
spo ligh om E oluChem o 10 min. Fo pulsed lase exci a-
ion (800 nm), MCF-7 cells we e incuba ed in a mul i-well
(384) pla e wi h glass bo om. The day a e , cells we e incuba ed
wi h 100 mgmL
1
o SCNP-Pc2
x
o SLA-Pc2
x
o 24 h, and hen
submi ed o no o lase i adia ion wi h a con ocal Ca l Zeiss
wo-pho on mic oscope a 800 nm and maximum lase powe
(3 W inpu , 1100 mW ou pu a lens). The lase beam was ocused
by a mic oscope objec i e lens (Ca lZeiss10- oldmagni ica ion/
objec i e 0.3 EC Plan-Neo lua ) and i adia ions we e pe o med
by 3 scans o 1.5 s each. Two days a e i adia ion, he pho o-
oxici y effec o nanopa icles was assessed using MTT assay as
p e iously desc ibed. Cells ea ed wi h he ehicle blank nano-
ca ie s (RCP and BCP) we e conside ed as a con ol.
2.3.5. S a is ical analysis. S uden ’s - es was pe o med o
compa e pai ed g oups o da a. A p alue o0.05 was consid-
e ed as s a is ically signi ican .
3. Resul s and discussion
3.1. Molecula design and syn hesis o iso-bu yl hio non-
pe iphe ally e asubs i u ed Zn ph halocyanine (Pc2)
The pho o-p ope ies o ph halocyanines can be modula ed by
hei subs i u ion pa e n.
34
The p esence o S-alkyl subs i uen s
shi s hei maximum abso p ion owa ds he ed, as well as
non-pe iphe al subs i u ion. Bulky subs i uen s a e known o
limi agg ega ion. Isobu yl moie ies we e selec ed o be in o-
duced ia hioe he unc ions in one non-pe iphe al posi ion
o each o he ou isoindole subuni s o a Zn ph halocyanine.
As shown in Scheme 1, he syn hesis was eadily achie ed in wo
s eps. Fi s , ph haloni ile 1was p epa ed by he eac ion
be ween isobu yl hiol and 3-ni oph haloni ile, in 80% yield.
The co esponding ph halocyanine Pc2 was ob ained by eac ing
ph haloni ile 1in p esence o Zn(OAc)
2
, yielding he desi ed
ph halocyanine in 28% yield and in a he la ge scale (300 mg).
All analyses con i med he p oposed s uc u e.
3.2. Pho o-p ope ies o Pc2
UV- is spec a o Pc2 ha e been eco ded in THF, DMSO and
chlo o o m a 10 mic omola , condi ions o which Pc2 is no
agg ega ed. DMSO has been selec ed as he sol en o he nex
measu emen s o use he unsubs i u ed ZnPc as he s anda d,
as all necessa y e e ence da a a e a ailable.
33
The maximum o
he Q band is loca ed a 717 nm, e lec ing he ba hoch omic
shi effec expec ed om bo h he non-pe iphe al and he
alkyl hio subs i u ion (Fig. 1A), compa ed o unsubs i u ed
ph halocyanine ha abso bs a 675 nm in DMSO
33
co es-
ponding o a subs an ial ba hoch omic shi o mo e han
40 nm. The luo escence spec um eco ded in DMSO showed
he expec ed shape and small S okes shi . The luo escence
quan um yield o Pc2 in DMSO was de e mined o be 0.07, in
line wi h i s high oxygen gene a ion quan um yield (F
D
= 0.78).
All he pho o-p ope ies o Pc2 in DMSO a e summa ized in
Table 1. All hese collec ed da a con i m he ele ance o using Pc2
o an i-cance PDT, bo h o i s s ong abso p ion a a - ed
wa eleng h and i s high abili y o gene a e single oxygen. The
luo escence quan um yield o Pc2 is lowe han hose o ZnPc,
while i s SO quan um yield is highe . This is due o he p esence o
he sul u a om on he ph halocyanine mac ocycle, which is known
o exe a hea y a om effec inc easing he in e sys em c ossing.
35
3.3. Syn hesis o RCP and BCP nanoca ie s and
encapsula ion o Pc2.
3.3.1 Syn hesis o RCP and BCP. Two kind o nanoca ie s
comp ising hyd ophilic oligo(e hylene glycol) monome hyl e he
me hac yla e (OEGMA
300
) and hyd ophobic (2-ace oace oxy)e hyl
me hac yla e (AEMA) epea uni s we e in es iga ed: a andom
copolyme (RCP) and a block copolyme (BCP)(Scheme2).Due
o hei in insic amphiphilic na u e, bo h can sel -assemble in
aqueous media in ul a-dilu e condi ions, yielding sel -assembled
s uc u es whose opology can a y wi h he a chi ec u e o he
s a ing copolyme . Bo h nanoca ie s we e syn hesized ia RAFT
copolyme iza ion o he comme cially a ailable monome s, using
4-cyanopen anoic acid di hiobenzoa e (CPADB) as chain ans e
agen (Scheme 2). A e pu i ica ion ia p ecipi a ion in n-hexane
and d ying unde dynamic acuum, he isola ed RCP and BCP we e
cha ac e ized by means o SEC/MALS and quan i a i e
1
H NMR
Scheme 1 Syn hesis o ph halocyanine Pc2 (only one o he exis ing
egioisome s is shown).
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spec oscopy o he de e mina ion o M
n
,Ðand copolyme
composi ion (see Table 2).
The sel -assembled blank nanopa icles (wi hou ph halo-
cyanine) SCNP-Pc2
0
( o med by RCP) and SLA-Pc2
0
( o med by
BCP) a high dilu ion ([polyme ] = 1 mg mL
1
) in wa e we e
in es iga ed by SAXS measu emen s. As illus a ed in Fig. 2a,
RCP in wa e a high dilu ion was ound o o m SCNP-Pc2
0
as
de e mined by SAXS expe imen s. Due o he impossibili y o
pe o m SAXS expe imen s in he condi ions ha mimic he cell
cul u e, SAXS expe imen s we e pe o med in wa e in dilu e
condi ions. P e ious wo ks by ou g oup ha e con i med he
sel -assembly o hese amphiphilic andom copolyme s in o
globula like co e–shell single-chain nanopa icles a a concen-
a ion well abo e he o e lap concen a ion.
36,37
Conce ning
he use o he gene alized Gaussian coil model o i he SAXS
da a o RCP i is well-es ablished in he li e a u e.
25
The a e age
adius o gy a ion (R
g
) o he SCNPs was R
g,wa e
= 10.4 nm
(a alue smalle han ha ound in THF sol en , R
g,THF
=
14.4 nm). The SCNPs we e ound o ha e a spa se con o ma ion
as e ealed by he high alue o he size scaling exponen (n=
0.59). We a ibu e he open con o ma ion o he SCNPs o he
ela i ely small amoun o hyd ophobic AEMA uni s in RCP.On
he o he hand, BCP a high dilu ion in wa e sel -assembled
in o s a -like assemblies (SLA-Pc2
0
)o R
g,wa e
= 10.0 nm ha ing
(on a e age) 2 a ms each one (i.e., o ming dime s). As we will
see below, he numbe o a ms pe s a inc eases upon encap-
sula ion o Pc2. Conce ning he SAXS da a o BCP, he alidi y
o he Dozie S a model o i he SAXS da a o s a -like
polyme s is suppo ed bo h by simula ions and expe imen al
Fig. 1 (A) Supe imposed UV- is spec a o Pc2 in chlo o o m, DMSO and
THF (10 mM). (B) UV- is abso p ion spec a o Pc2 in DMSO (2–12 mM).
Inse : Abso p ion s. concen a ion. (C) Abso bance, emission (i adia ion:
640 nm), and exci a ion spec a o Pc2 in DMSO. (D) De e mina ion o
single oxygen quan um yield o Pc2 in DMSO (6 mM). Inse : Plo o DPBF
abso bance a 417 nm s. ime.
Table 1 Pho o-p ope ies o Pc2 in DMSO
l
max
(nm) logel
Em
(nm) l
Exc
(nm) Dl
S okes
(nm) F
F
F
D
717 5.29 736 720 16 0.07 0.78
Scheme 2 (A) Pic o ial ep esen a ion o he copolyme iza ion eac ion
o he hyd ophobic monome AEMA (depic ed in g ey) and he hyd ophilic
monome OEGMA
300
(depic ed in blue) o gi e RCP. (B) Reac ion scheme
o he syn hesis o he amphiphilic block copolyme BCP.
Table 2 Numbe a e age molecula weigh (M
n
), dispe si y (Ð)and
copolyme composi ion o RCP and BCP
Copolyme M
n
/kDa ÐAEMA/mol% OEGMA
300
/mol%
RCP 99.6 1.04 20 80
BCP 71.8 1.02 14 86
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esul s.
38
I is wo h no ing ha he UV- is spec a o SCNP-Pc2
0
( o med by RCP) and SLA-Pc2
0
( o med by BCP) showed no
abso p ion bands in he 600–700 nm wa eleng h egion
(Fig. S8, ESI†).
3.3.2. Encapsula ion o Pc2. Bo h Pc2-con aining SCNP-
Pc2
x
and SLA-Pc2
x
we e p epa ed using he same encapsula ion
p ocedu e by s a ing wi h RCP o BCP, espec i ely. The expe i-
men al condi ions o he encapsula ion o Pc2 we e i s op imized
explo ing diffe en polyme concen a ions in solu ion. As explained
abo e, we obse ed ha he op imal wo king ange o sel - olding
in o SCNPs is om 0.1 mg mL
1
o 5 mg mL
1
o RCP and ha
BCP dime s s a o o m om a concen a ion o 1 mg mL
1
.
Rega ding he concen a ion o loadable Pc2, we looked o he
highes achie able quan i y o Pc2 o be compa ible wi h he
o ma ion o SCNPs when using he less possible amoun o RCP
p ecu so , ensu ing he highes possiblePc2/polyme a io. This
la e is a less lexible sys em, when compa ed o mul i-chain
agg ega es, in e ms o hyd ophilic/hyd ophobic in e ac ions
changes in he media, and cons i u ed indeed ou limi ing ac o
o he op imiza ion o he encapsula ion p ocedu e. We a emp ed
he encapsula ion ixing he pho osensi ize concen a ion a
20 mM, which esul ed in i e e sible agg ega ion when using
RCP as nanoca ie . Fo his eason, we p og essi ely lowe ed he
pho osensi ize loading up o 12 mM, which inally esul ed o be he
maximum wo king Pc2 concen a ion. In addi ion, we selec ed he
polyme concen a ion o 1 mg mL
1
as i is he lowes nanoca ie
amoun needed in solu ion o efficien ly encapsula e he pho o-
sensi ize a i s maximum achie able loading. To explo e he
effec o he loading amoun , wo diffe en concen a ions o Pc2
(x=5mMo 12mM) we e used o bo h copolyme s, yielding o
bo h he nanopa icles a chi ec u es (SCNPs o SLAs) he i le
ph halocyanine pho osensi ize -encapsula ed nanoca ie s a he
loading egimes o 4.7 mg pe mg o polyme o SCNP-Pc2
5
and SLA-Pc2
5
and 11.2 mg pe mg o polyme o SCNP-Pc2
12
and
SLA-Pc2
12
.
3.3.3. Cha ac e iza ion o SCNP-Pc2
x
.SAXS expe imen s
con i med he success ul o ma ion o SCNP-Pc2
5
and SCNPs-
Pc2
12
, as illus a ed in Fig. 3. Hence, a small educ ion in size
and in scaling exponen was obse ed upon inc easing he
amoun o encapsula ed Pc2. This may be because Pc2 is
hyd ophobic, so o encapsula e i he polyme ic chain w aps
he Pc2 complex, and his gene a es a compac ion o he chain
accompanied by a educ ion in size. Table 3 shows he alues o
he i ing pa ame e s o SAXS cu es o SCNP-Pc2
0
(blank
nanopa icles wi hou ph halocyanine), SCNP-Pc2
5
and SCNP-
Pc2
12
in wa e , applying a gene alized Gaussian coil unc ion.
UV- is spec a o SCNPs-Pc2
5
and SCNPs-Pc2
12
in wa e
e ealed an in ense abso p ion in he 600–700 nm wa eleng h
egion (Fig. 4). The spli ed shape o he Q band indica es ha
Pc2 is agg ega ed inside he nanoca ie .
3.3.4. Cha ac e iza ion o SLA-Pc2
x
.SAXS esul s o SLA-
Pc2
5
and SLA-Pc2
12
a e illus a ed in Fig. 5. Bo h nanoca ie s
in wa e a high dilu ion o m s a -like assemblies (SLAs) wi h a
la ge numbe o a ms and sligh ly la ge size han BCP ( =2
a ms,R
g,wa e
= 10.0 nm). Analysis o he SAXS o m ac o o
SLA-Pc2
5
in e ms o he Dozie s a model p o ided = 4 a ms
wi h R
g,wa e
= 10.1 nm, whe eas SLA-Pc2
12
displayed = 8 a ms
and R
g,wa e
= 10.2 nm. Table 4 shows he alues o he i ing
pa ame e s o SAXS cu es o BCP,SLA-Pc2
5
and SLA-Pc2
12
in
wa e , applying he Dozie s a model.
The cha ac e is ic UV- is abso p ion bands o Pc2 encapsu-
la ed in o SLA-Pc2
5
and SLA-Pc2
12
a e epo ed in Fig. 6 and
show he agg ega ed s a e o he ph halocyanine inside he
nanopa icles.
3.4. In i o pho odynamic efficiency o SCNP-Pc2
x
and SLA-Pc2
x
The biological ac i i y o he SCNP-Pc2
x
and SLA-Pc2
x
was
s udied on MCF-7 human b eas cance cells. Blank nano-
ca ie s wi hou Pc2 (RCP and BCP) we e also es ed o assess
hei own biocompa ibili y.
Fig. 2 (A) SAXS esul s e ealing he o m ac o o SCNP-Pc2
0
in wa e .
Line is a i o a gene alized Gaussian coil unc ion, yielding he alues
o he R
g
and npa ame e s. (B) SAXS esul s e ealing he o m ac o o
SLA-Pc2
0
in wa e . Line is a i o he Dozie s a model, yielding he alues
o he R
g
and pa ame e s.
Fig. 3 SAXS esul s and espec i e depic ions o he mo phologies
e ealed, wi h o m ac o in wa e o SCNP-Pc2
5
(A) and SCNP-Pc2
12
(B),Values o R
g
and nwe e ob ained h ough i s o a gene alized Gaussian
coil unc ion (con inuous lines) o he expe imen al da a.
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3.4.1. Cy o oxici y. Fi s , he cy o oxic effec in he da k was
analyzed by incuba ing he cells du ing 72 h wi h inc easing
concen a ions o SCNP-Pc2
x
o SLA-Pc2
x
( om 1 o
200 mgmL
1
). The co esponding concen a ions o Pc2 o
each nanoca ie we e calcula ed (in mgmL
1
and in mM) and
epo ed in he Table S1 (ESI†). The cells we e submi ed o a
MTT colo ime ic assay, and he quan i ica ion o li ing cells
was pe o med (Fig. 7A). All he nanoca ie s, ei he emp y o
ph halocyanine-loaded, induced no mo e han 50% cell dea h,
e en a he highes concen a ions (200 mgmL
1
). This obse a-
ion le us o ix a concen a ion o 100 mgmL
1
o PDT
expe imen s.
3.4.2. Pho odynamic he apy. Nex , o e alua e he pho o-
dynamic po en ial o he Pc2-loaded nanoca ie s, MCF-7 cells
we e incuba ed wi h 100 mgmL
1
o dis inc nanoca ie s
loaded wi h 5 o 12 mM o ph halocyanine (i.e.,SCNP-Pc2
5
,
SCNP-Pc2
12
,SLA-Pc2
5
and SLA-Pc2
12
) o 24 h and exposed o
no o lase i adia ion a a ious wa eleng hs (633, 650 and
740 nm). These wa eleng hs co espond o he abso p ion
ange o Pc2 when encapsula ed in o he nanoca ie s, as i s
Q band is enla ged due o hei agg ega ed s a e inside he
nanoca ie . Li ing cell pe cen age quan i ica ion was pe -
o med 2 days a e i adia ion (Fig. 7B). SCNP-Pc2
5
and
SCNP-Pc2
12
did no exhibi signi ican pho o oxici y, while
SLA-Pc2
5
and SLA-Pc2
12
esul ed in signi ican efficiency. SLA-
Pc2
12
exci ed a 650 nm and SLA-Pc2
5
exci ed a 740 nm ga e
he bes esul s wi h 47% and 73% cell dea h, espec i ely.
When compa ing he abso p ion spec a o SCNP-Pc2
5
wi h
SCNP-Pc2
12
(as well as o SLA-Pc2
5
s.SLA-Pc2
12
), no signi i-
can diffe ence was obse ed. Fo his eason, we asc ibed he
augmen ed PDT-effec in SLA-Pc2
x
o he mo e lexible s uc-
u e and peculia opology o hese BCP-based nanoca ie s
ha accommoda e Pc2 in a manne a o ing likely be e ROS
gene a ion. In pa allel, we ha e e i ied he cy o oxici y and
PDT po en ial o ee Pc2. Fo his, MCF-7 cells we e incuba ed
Table 3 Fi ing pa ame e s alues o SAXS cu es o SCNP-Pc2
0
,SCNP-
Pc2
5
and SCNP-Pc2
12
in wa e , applying a gene alized Gaussian coil
unc ion
SCNP-Pc2
0
SCNP-Pc2
5
SCNP-Pc2
12
R
g
(nm) 10.4 10.1 9.4
n0.60 0.56 0.53
I
0
0.048 0.053 0.053
Fig. 4 UV- is spec a o SCNP-Pc2
5
and SCNP-Pc2
12
in wa e (1 mg mL
1
).
Fig. 5 SAXS esul s and espec i e depic ions o he mo phologies
e ealed, wi h o m ac o in wa e o SLA-Pc2
5
(A) and SLA-Pc2
12
(B).
Values o R
g
and a m numbe ( ) we e ob ained h ough i s o he Dozie
s a model o he expe imen al da a (con inuous lines).
Table 4 Fi ing pa ame e s alues o SAXS cu es o SLA-Pc2
0
,SLA-Pc2
5
and SLA-Pc2
12
in wa e , applying Dozie s a model
SLA-Pc2
0
SLA-Pc2
5
SLA-Pc2
12
R
g
(nm) 10.0 10.1 10.2
248
n0.60 0.60 0.60
I
0
0.040 0.052 0.068
Fig. 6 UV- is spec a o SLA-Pc2
5
and SLA-Pc2
12
in wa e (1 mg mL
1
).
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wi h inc easing concen a ions o Pc2 and didn’ show any
oxici y o hese concen a ions (Fig. S11A, ESI†). Cells we e
hen incuba ed wi h 0.5 o 1.2 mgmL
1
o Pc2 (co esponding
o 100 mgmL
1
o SCNP-Pc2
5
,SCNP-Pc2
12
,SLA-Pc2
5
,SLA-Pc2
12
)
and submi ed o 633 nm, 650 nm and 740 nm exci a ion. Da a
demons a ed an absence o PDT po en ial wi hou encapsula-
ion (Fig. S11B, ESI†). This esul highligh s he necessi y o Pc2
encapsula ion o a biological applica ion. In addi ion, eac i e
oxygen species (ROS) p oduc ion was de ec ed unde ligh
exci a ion a 650 nm when cells we e incuba ed wi h SLA-
Pc2
12
bu no wi h ee Pc2 (1.2 mgmL
1
) (Fig. S12, ESI†).
I is well-known ha agg ega ed pho osensi ize s - including
ph halocyanines - ha e a sel -quenched in e -sys em c ossing
pa hway, nega i ely affec ing hei pho odynamic efficiency
when exci ed classically by monopho onic ligh sou ce. On he
o he hand, agg ega ed ph halocyanines, as in he case o
ph halocyanine-based o ganosilica nanopa icles p e iously
epo ed by some o us,
20,21
may exhibi good pho o-killing effec
when exci ed by pulsed lase a NIR wa eleng hs. The pho o-
dynamic efficiency o SCNP-Pc2
5
,SCNP-Pc2
12
,SLA-Pc2
5
and SLA-
Pc2
12
, s ill a 100 mgmL
1
, was he e o e s udied wi h a pulsed
lase i adia ing a 800 nm (Fig. 7C) in he i s NIR pho o he -
apeu ic window. Unde such condi ions, SCNP-Pc2
5
esul ed o be
he only nanopa icle o exhibi signi ican pho o-killing abili y.
Indeed, we can obse e 36% cell dea h when cells, p e iously
incuba ed wi h SCNP-Pc2
5
, we e submi ed o 800 nm exci a ion
wa eleng h using a pulsed lase , om Chameleon lamp o a
con ocal mic oscope (3 successi e scans o 1.5 s). The compa ison
be ween SCNP-Pc2
5
(4.7 mg pe mg o polyme ) and i s highe -Pc2-
loaded analogue SCNP-Pc2
12
(11.2 mg pe mg o polyme ) shows
ha he e also he loading is impo an , as oo much ph halocya-
nine in he SCNP ca ie p e en s a good efficency. O e all, he
diffe en beha io o hese ph halocyanine-loaded nanoca ie s
can be a ibu ed o hei diffe en nanoca ie opology: sel -
olded single-chain nanopa icles (SCNPs) s. s a -like agg ega es
(SLAs), espec i ely.
4. Conclusions
Two diffe en amphiphilic copolyme s encapsula ing Pc2
wi hin sel - olded single-chain nanopa icles (SCNPs) and
s a -like agg ega es (SLAs), ha e demons a ed hei ele ance
as pho osensi ize nanoca ie s o PDT o cance cells, unde
exci a ion in he in a ed ange wi h pulsed lase o in he
isible ange wi h con inuous lase , espec i ely. In addi ion,
ee Pc2 is no biologically ac i e in hese condi ions. All
oge he , hese esul s demons a e he in e es and he no el y
o hese nanoca ie sys ems o he encapsula ion and deli e y
o pho osensi ize s o PDT.
Au ho con ibu ions
DA, MO, CN, ZS-: da a cu a ion, in es iga ion, o mal analysis
and manusc ip w i ing. U
¨I
˙, EVS, AI, AA: da a cu a ion, supe -
ision, o mal analysis, me hodology and manusc ip w i ing.
FD, JAP and MGB: supe ision, concep ualiza ion, esou ces,
p ojec adminis a ion and manusc ip w i ing.
Da a a ailabili y
The da a suppo ing his a icle ha e been included as pa o
he ESI.†
Con lic s o in e es
The e a e no con lic s o decla e.
Fig. 7 (A) Cell iabili y s udy o MCF-7 cells incuba ed 72 h wi h inc easing
concen a ions ( om 1 o 200 mgmL
1
)o SCNP-Pc2
5
,SCNP-Pc2
12
,SLA-
Pc2
5
and SLA-Pc2
12
. (B) PDT effec o SCNP-Pc2
5
,SCNP-Pc2
12
,SLA-Pc2
5
and SLA-Pc2
12
(100 mgmL
1
) incuba ed 24 h wi h MCF-7 cells and exci ed
a 633, 650 and 740 nm. (C) PDT effec o SCNP-Pc2
5
,SCNP-Pc2
12
,SLA-
Pc2
5
and SLA-Pc2
12
(100 mgmL
1
) incuba ed 24 h wi h MCF-7 cells and
exci ed a 800 nm, wi h a pulsed lase . Da a a e means SEM; n=3.*po
0.05, signi ican ly diffe en om ‘‘No lase ’’.
Pape Ma e ials Ad ances
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