Bioma e ials Ad ances 151 (2023) 213456
A ailable online 9 May 2023
2772-9508/© 2023 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
E y h ocy e−cance hyb id memb ane-coa ed educ ion-sensi i e
nanopa icles o enhancing chemo he apy e icacy in b eas cance
Somayeh Rezaei
a
, Raimundo Fe nandes de A aújo Júnio
a
,
b
,
c
,
*
, Isado a Luisa Gomes da Sil a
c
,
Timo Schomann
a
,
d
, Ch is ina Eich
a
,
**
, Luis J. C uz
a
,
**
a
T ansla ional Nanobioma e ials and Imaging (TNI) G oup, Depa men o Radiology, Leiden Uni e si y Medical Cen e , 2333 ZA Leiden, he Ne he lands
b
Pos g adua e P og am in Heal h Science, Fede al Uni e si y o Rio G ande do No e (UFRN), Na al 59064-720, B azil
c
Cance and In lamma ion Resea ch Labo a o y (LAICI), Pos g adua e P og am in Func ional and S uc u al Biology, Depa men o Mo phology, Fede al Uni e si y o
Rio G ande do No e (UFRN), Na al 59064-720, B azil
d
Depa men o Vascula Su ge y, Leiden Uni e si y Medical Cen e , 2333 ZA Leiden, he Ne he lands
ARTICLE INFO
Keywo ds:
Reduc ion-sensi i e nanopa icles
Chi osan
Biomime ic nanopa icles
Cance cell memb ane-coa ed nanopa icles
Homo ypic a ge ing
Red blood cells
Hyb id memb ane
B eas cance
ABSTRACT
Cell-memb ane-coa ed biomime ic nanopa icles (NPs) ha e a ac ed g ea a en ion due o hei p olonged
ci cula ion ime, immune escape mechanisms and homo ypic a ge ing p ope ies. Biomime ic nanosys ems om
di e en ypes o cell -memb anes (CMs) can pe o m inc easingly complex asks in dynamic biological en i-
onmen s hanks o speci ic p o eins and o he p ope ies inhe i ed om he sou ce cells. He ein, we coa ed
doxo ubicin (DOX)-loaded educ ion-sensi i e chi osan (CS) NPs wi h 4T1 cance cell -memb anes (CCMs), ed
blood cell -memb anes (RBCMs) and hyb id e y h ocy e-cance memb anes (RBC-4T1CMs) o enhance he de-
li e y o DOX o b eas cance cells. The physicochemical p ope ies (size, ze a po en ial and mo phology) o he
esul ing RBC@DOX/CS-NPs, 4T1@DOX/CS-NPs and RBC-4T1@DOX/CS-NPs, as well as hei cy o oxic e ec
and cellula NP up ake in i o we e ho oughly cha ac e ized. The an i-cance he apeu ic e icacy o he NPs
was e alua ed using he o ho opic 4T1 b eas cance model in i o. The expe imen al esul s showed ha DOX/
CS-NPs had a DOX-loading capaci y o 71.76 ±0.87 %, and ha coa ing o DOX/CS-NPs wi h 4T1CM signi i-
can ly inc eased he NP up ake and cy o oxic e ec in b eas cance cells. In e es ingly, by op imizing he a io o
RBCMs:4T1CMs, i was possible o inc ease he homo ypic a ge ing p ope ies owa ds b eas cance cells.
Mo eo e , in i o umo s udies showed ha compa ed o con ol DOX/CS-NPs and ee DOX, bo h 4T1@DOX/
CS-NPs and RBC@DOX/CS-NPs signi ican ly inhibi ed umo g ow h and me as asis. Howe e , he e ec o
4T1@DOX/CS-NPs was mo e p ominen . Mo eo e , CM-coa ing educed he up ake o NPs by mac ophages and
led o apid clea ance om he li e and lungs in i o, compa ed o con ol NPs. Ou esul s sugges ha speci ic
sel - ecogni ion o sou ce cells esul ing in homo ypic a ge ing inc eased he up ake and he cy o oxic capaci y
o 4T1@DOX/CS-NPs by b eas cance cells in i o and in i o. In conclusion, umo -disguised CM-coa ed DOX/
CS-NPs exhibi ed umo homo ypic a ge ing and an i-cance p ope ies, and we e supe io o e a ge ing wi h
RBC-CM o RBC-4T1 hyb id memb anes, sugges ing ha he p esence o 4T1-CM is c i ical o ea men
ou come.
1. In oduc ion
B eas cance is one o he mos le hal cance s and accoun s o o e
90 % o cance - ela ed dea hs in women. A majo challenge o cance
he apy oday is cance me as asis, which occu s when cance cells
sepa a e om a p ima y umo and a el o ano he pa o he body by
blood o he lymph sys em [1,2]. Chemo he apy s ill emains he s an-
da d me hod o ea men o b eas cance pa ien s who canno unde go
esec ion su ge y [3,4], bu pa ien s o en ace he side e ec s o an i-
cance d ugs due o non-selec i e up ake o hose d ugs by no mal
cells in heal hy issues.
Nanopa icles (NPs) ha e gained much a en ion o cance he apy
* Co espondence o: R. F. de A aújo Júnio , Pos g adua e P og am in Heal h Science, Fede al Uni e si y o Rio G ande do No e (UFRN), Na al 59064-720, B azil.
** Co esponding au ho s.
E-mail add esses: [email p o ec ed] (S. Rezaei), [email p o ec ed] (R.F. de A aújo Júnio ), [email p o ec ed] (C. Eich), [email p o ec ed]
(L.J. C uz).
Con en s lis s a ailable a ScienceDi ec
Bioma e ials Ad ances
jou nal homepage: www.jou nals.else ie .com/ma e ials-science-and-enginee ing-c
h ps://doi.o g/10.1016/j.bioad .2023.213456
Recei ed 7 Decembe 2022; Recei ed in e ised o m 7 Ap il 2023; Accep ed 30 Ap il 2023
Bioma e ials Ad ances 151 (2023) 213456
2
and diagnosis o e he las decades [5]. NP-encapsula ed d ugs can
imp o e d ug solubili y, can c oss he blood essel wall, ha e well
adap ed elease kine ics, educed side e ec s and inc eased he apeu ic
e icacy [6]. Fo cance ea men , he use o NP pla o ms has inc eased
he po ency o he adi ional he apeu ics [7]. Depending on he NPs’
physicochemical p ope ies and su ace coa ing, NPs ha en e he
blood ci cula ion o en ge co e ed by biomolecules which o m a p o-
ein co ona. This can lead o changes in NP s uc u e, unc ion and dy-
namics, esul ing in al e ed cellula ecogni ion and up ake o he NPs
[8]. The binding s eng h o he NPs o he p o ein co ona depends on
he NPs’ physicochemical p ope ies, such as size, cha ge, shape and
hyd ophilici y [9]. A commonly used s a egy o p e en he o ma ion
o a p o ein co ona and o inc ease he blood ci cula ion ime is su ace
modi ica ion o he NP wi h polye hylenglycol (PEG) [9]. In i o and in
i o s udies suppo he heo y ha he conjuga ion o a ge ing mo i s o
he NP su ace, including p o ein/pep ides, olic acid, an ibodies,
ap ame s and polysaccha ides, can enhance he selec i i y o cellula
up ake by cance cells and/o cy o oxici y [10].
Ano he o m o a ge ed NPs, known as biomime ic NPs, can be
ob ained by combining syn he ic NPs (as co e) wi h a biological mem-
b ane coa ing (as shell). Biologically ele an su ace moie ies including
adhesion p o eins, an igens and memb ane s uc u es o sou ce cells a e
ans e ed on o he su ace o a NP by ansloca ing en i e cell mem-
b anes [7,11,12]. Recen ly, biomime ic NPs gained a en ion o he a-
peu ic and imaging applica ions. In addi ion, cell memb ane-coa ed NPs
e ain he e sa ili y and complexi y o na u al cell -memb anes o
pe o m speci ic unc ions, in pa icula bioin e acing. Biomime ic NPs
ha e shown a ge -homing abili ies as well as p olonged ci cula ion
imes, immune escape abili ies, homologous binding p ope ies and
good biocompa ibili y, which a e consis en wi h he p ope ies o a cell
memb ane [13]. Cu en ly, mos CM-based biomime ic NPs a e p e-
pa ed in h ee s eps: i) memb ane ex ac ion om he sou ce cells, ii)
p epa a ion o co e NPs and iii) me ging o CMs and NPs o o m a
memb ane shell a ound he NP co e [14]. P e ious s udies ha e shown
ha biomime ic NPs p e en he o ma ion o a p o ein co ona and
ep esen a p omising app oach o he ab ica ing o no el nano-
medicines [15]. RBCMs a e one o mul iple ypes o na u al memb anes,
which can be coa ed on o NPs. Howe e , hey canno ac i ely a ge
cance cells due o a lack o a ge ing molecules wi h umo - opism.
Con e sely, CCM-de i ed esicles, wi h inhe i ed a ini y ligands
na i e o he sou ce cance cells, which can ac i ely a ge cance cells,
bu a e p one o ecogni ion by immune cells due o he p esence o
umo an igens, and hus may no be s eal hy enough o e ade immune
su eillance [14,16,17]. Biomime ic nanosys ems can pe o m inc eas-
ingly complex asks in dynamic biological en i onmen s when made
om di e en ypes o CMs ha inhe i ed p o eins and o he p ope ies
om di e en sou ce cells [18]. Compa ed o ba e NPs, hyb id
memb ane-coa ed NPs, such as RBC-pla ele (PLT)-CM NPs educe
in lamma ion and p olong blood e en ion. These indings sugges ha
he usion memb ane-coa ing s a egy is an e ec i e me hod o inc ease
he accumula ion o NPs in umo cells [19].
S imuli- esponsi e NPs, such as pH-sensi i e and glu a hione (GSH)-
esponsi e NPs, ha e ecei ed g ea a en ion due o hei abili y o
achie e on-demand d ug elease in he umo mic oen i onmen (TME)
[20]. Among he s imuli- esponsi e NPs, GSH- esponsi e NPs a e o
g ea in e es because hey slowly elease d ugs in blood ci cula ion,
while hey excellen ly elease d ugs in o he cy oplasm o cance cells
[21], whe e he concen a ion o GSH is highe han in no mal issue
[22]. Chi osan (CS) is a well-known aminopolysaccha ide wi h a o -
able p ope ies, such as biocompa ibili y, biodeg adabili y, low oxici y,
Scheme 1. Schema ic illus a ion o he a ge ing and coa ing o DOX/CS-NPs by RBCMs, 4T1CMs and RBC-4TCMs o he apeu ic pu pose on 4T1 b eas cance .
S. Rezaei e al.
Bioma e ials Ad ances 151 (2023) 213456
3
high ca ionic cha ge and pH sensi i i y, which made i widely used in
biomedical applica ions [22].
He ein, we epo he de elopmen o DOX/CS-NPs coa ed wi h 4T1,
RBC and a combina ion o bo h CMs speci ically o he in a umo al
deli e y o DOX o ea b eas cance . As shown in Scheme 1, he plasma
memb ane de i ed om 4T1 cells and RBCs, wi h i s mul i ude o p o-
eins, including homologous a ge ing p o eins and immune escape
p o eins, bes ows 4T1 and RBCMs wi h mimicking p ope ies o adhe e
o sou ce cells. I has been shown ha in amu al injec ion o PLT
memb ane-coa ed NPs exhibi ed p olonged e en ion a he umo si e
and enhanced cellula in e ac ion in he TME [23]. Ou in i o da a
showed ha , a e in a umo al adminis a ion in i o, due o he ho-
mologous a ge ing abili y o 4T1 umo CMs, 4T1@DOX/CS-NPs could
selec i ely accumula e in he 4T1 TME, leading o a high local concen-
a ion o DOX. In addi ion, RBC@DOX/CS-NPs could limi he NP up-
ake by mac ophages. Bo h 4T1@DOX/CS-NPs and RBC@DOX/CS-NPs
could e ec i ely supp ess he umo g ow h in si u compa ed o ee
DOX, while o DOX/CS-NPs we e less e ec i e.
2. Ma e ial and me hods
2.1. Ma e ials and eagen s
Fo he p epa a ion o NPs, chi osan oligosaccha ide (CSO), lipoic
acid (LA), e hanol, 1-E hyl-3-(3-dime hylaminop opyl) ca bodiimide
(EDC) and N-hyd oxysuccinimide (NHS) we e ob ained om Sigma-
Ald ich (Zwijnd ech , he Ne he lands). Doxo ubicin hyd ochlo ide
(DOX.HCl) was ob ained om he Leiden Uni e si y Medical Cen e
Pha macy, Leiden, he Ne he lands. Cyanine5 ca bocyclic acid (Cy5)
was pu chased om Lumip obe, Hanno e , Ge many. Fe al bo ine
se um (FBS) and cell cul u e media we e ob ained om Gibco Labo a-
o ies (The mo Scien i ic™, Wal ham, Massachuse s, USA). Fo cell
cul u e, 4T1 (ATCC® CRL2539™) mu ine b eas cance cells, mouse
mac ophages RAW 264.7 (ATCC® TIB-71™; mouse mononuclea mac-
ophages), MC38 (mu ine colon adenoca cinoma cell), CT-26 (mu ine
colo ec al ca cinoma cells) and 3 T3 (emb yonic mouse ib oblas ) cells
we e used in his s udy. They we e pu chased om he Ame ican Type
Cul u e Collec ion (ATCC; Manassas, Vi ginia, USA). 4T1 cells we e
cul u ed and main ained in he Roswell Pa k Memo ial Ins i u e (RPMI)
medium, supplemen ed wi h 10 % FBS and 100 U mL
−1
o penicillin/
s ep omycin (P/S). O he cell lines, i.e., RAW 264.7, MC38, CT-26 and
3 T3, we e cul u ed in comple e Dulbecco’s Modi ied Eagle Medium
(DMEM) con aining 10 % FBS and 100 U mL
−1
P/S. The cells we e
cul u ed in a humidi ied incuba o a 37 ◦C wi h 5 % CO
2
. To al RNA
isola ion sys em and CellTi e 96(R) AQueous MTS Reagen Powde
we e pu chased om P omega (Madison, WI, USA). SnakeSkin™ Dial-
ysis Tubing (3.5 kDa MWCO, 22 mm), high-capaci y RNA- o-cDNA™
Ki , Ka yoMAX™Giemsa S aining Solu ion, Powe Up™ SYBR™ G een
Mas e Mix, LysoT acke ™ Deep Red, ypsin, P/S (10,000 U mL
−1
), and
FBS we e pu chased om The mo Scien i ic™ (Wal ham, MA, USA).
An i-mouse CD163-Pe CP-eFluo ™ 710 (Clone TNKUPJ), an i-mouse
CD68-FITC (clone FA-11) and an i-mouse CD86-FITC (clone GL1) we e
ob ained om eBioscience (San Diego, CA, USA). An i-NF-κB (clone F-6)
was pu chased om San a C uz Bio echnology (Dallas, TX, USA). TRIzol
was pu chased om In i ogen (The mo Scien i ic™, Wal ham, MA,
USA). Bio inyla ed pan-speci ic uni e sal seconda y an ibody and
s ep a idin/HRP-conjuga ed an ibody we e pu chased om Vec o
Labs (Vicbio Bio echnology Co., L d.，Beijing, China). Dia-
minobenzidine and hema oxylin we e pu chased om DAKO (San a
Cla a, CA, USA). Hoechs 33342 and 3,3′-dioc adecyloxaca bocyanine,
pe chlo a e (DiO) (Vyb an ™ DiO Cell-Labeling Solu ion) we e pu -
chased om The mo Fishe Scien i ic (Wal ham, MA, USA).
2.2. P epa a ion o educ ion-sensi i e DOX/CS-NPs
Chi osan Lipoic acid (CSLA) NPs we e p epa ed as p e iously
epo ed, bu wi h some modi ica ions [21]. B ie ly, 10 mg CSO was
dissol ed in deionized wa e and s i ed a oom empe a u e. Subse-
quen ly, he solu ion was sonica ed o 30 min (250 wa s; Soni ie 250;
B anson Ul asonics, Danbu y, CT, USA). A e wa ds, he CSO solu ion
was adjus ed o pH 5. LA was dissol ed in e hanol and added o he CSO
solu ion. The eac ion was kep a 45 ◦C o 12 h o syn hesize CS-LA
NPs. Nex , CS-LA NPs we e collec ed and pu i ied by means o dial-
ysis. The DOX and Cy5-loaded CS-LA NPs we e syn hesized using he
same p o ocol by adding DOX and Cy5 [22]. The encapsula ion e i-
ciency (EE) and loading capaci y (LC) o DOX-loaded CS-NPs we e
de e mined by Spec aMax® iD3 Mul i-Mode Mic opla e Reade s
(Ma shall Scien i ic, Hamp on, NH, USA).
2.3. Cha ac e iza ion o DOX/CS-NPs
The size and ze a po en ial o DOX/CS-NPs we e measu ed by dy-
namic ligh sca e ing (DLS; Ze asize , NANO-ZS, Mal e n L d., UK).
The ea e , he NPs we e cha ac e ized by p o on nuclea magne ic
esonance
1
H NMR (B uke , B emen, Ge many).
2.4. P epa a ion o RBCMs and 4T1CMs
To p epa e RBCMs, pe iphe al blood o BALB/c mice was washed
wi h 1×PBS and cen i uged a 800 ×g o emo e blood plasma. The
RBC ghos s we e ob ained by ea men o RBCs wi h a hypo onic bu e
(T is, MgCl2, KaCl, CaPO
4
wi h p o ease inhibi o ) a 4 ◦C o 4 h. The
RBCMs we e ob ained by cen i uga ion a 21,000 g o 30 min [24,25].
The RBCMs we e u he washed wi h deionized wa e , sonica ed and
inally s o ed a −80 ◦C un il u he use. In o de o p epa e he 4T1
CM-coa ed NPs, 4T1 cells we e cul u ed in RPMI medium in T-175
cul u es lasks. A e he cells we e g own o ull con luency, hey we e
washed wi h PBS and ha es ed using a cell sc ape . The 4T1 cells we e
suspended in cold hypo onic bu e and u he sonica ed. The sample
solu ion was cen i uged a 3200 ×g a 4 ◦C o 5 min. Nex , he su-
pe na an s we e cen i uged a 21,000 ×g o 45 min. Finally, 4T1 CMs
we e sonica ed, dissol ed in H
2
O and s o ed a −80 ◦C un il u he use
[1,26,27].
2.5. Gene a ion and cha ac e iza ion o RBC-4T1 hyb id memb anes,
RBC-4T1@DOX/CS-NPs, RBC@DOX/CS-NPs and 4T1@DOX/S-NPs
To ob ain he RBC-4T1 hyb id memb ane, RBCMs and 4T1CMs we e
used using he sonica ion me hod [28]. RBC-4T1 CMs we e cha ac e -
ized by DLS and nanopa icle acking analysis (NTA; NanoSigh ®
NS300, Mal e n). DLS was used o de e mine he size and ze a po en ial,
while NTA was used o quan i y he concen a ion o RBCMs and 4T1
CMs. RBCMs we e added o 4T1 CMs a he weigh a ios o 1:1,1:2 and
1:3. Then, hey we e sonica ed (130 W; 42 kHz) a 37 ◦C o 10 min o
ob ain RBC-4T1 CMs [5,29,30]. The DOX/CS-NP solu ion in H
2
O was
added o a RBC-4T1 solu ion in PBS a he weigh a ios o 1:2:1 and
sonica ed (130 W; 42 kHz) a 37 ◦C o 10 min o ob ain RBC-
4T1@DOX/CS-NPs. RBC@DOX/CS-NPs we e also ob ained and cha -
ac e ized by he same me hod a he weigh a io o 2:1 CMs o DOX/CS-
NPs.
2.6. Pa icle su ace and mo phology
The mo phology and shape o all NPs we e imaged and analyzed by
ansmission elec on mic oscopy (TEM) B ie ly, be o e s aining,ca bon-
coa ed g ids (Fo m a /Ca bon on 200 Mesh Coppe ; AGS162; Van
LoenenIns umen s; Zaandam, he Ne he lands) we e glow-discha ged
using he Emi ech K950X Tu bo E apo a o (Quo um Technologies;
Ash o d, UK) a 2 ×10
−1
mba and 20 mA o 1 min. Nex , 3
μ
L o sample
solu ion we e applied on he eshly glow-discha ged g id and allowed
o adhe e o 1 min. A e wa ds, excess liquid was disca ded by blo ing
on o a il e pape and 3
μ
L o 2 % u anyl ace a e in dis illed wa e we e
S. Rezaei e al.
Bioma e ials Ad ances 151 (2023) 213456
4
applied o he g id o nega i e s aining o he sample. A e 1 min,
excess u anyl ace a e was emo ed by blo ing and he sample was ai -
d ied o 10 min. G ids we e moun ed on a oom empe a u e holde and
examined using a Tecnai 12 Twin (FEI Company;Hillsbo o, O egon,
USA) equipped wi h an OneView Came a Model 1095 (Ga an; Pleas-
an on, Cali o nia, USA) a a ol age o 120 kV. Digi al images we e ac-
qui ed and s o ed using Digi al Mic og aph 3.4 (Ga an).
2.7. In i o elease o DOX-loaded CSLA-NPs
The elease p o iles o DOX-loaded CS-NPs, RBC@DOX/CS-NPs,
4T1@DOX/CS-NPs and RBC-4T1@DOX/CS-NPs we e measu ed by he
dialysis bag di usion me hod a 37 ◦C in di e en elease media,
including Phospha e Bu e ed Saline (PBS; pH 7.4), GSH (10 mM), PBS
(pH 5.3), and GSH (10 mM) [21]. The dialysis ube (SERVAPOR®,
MWCO 12000–14,000, Heidelbe g, Ge many) was used o quan i y he
DOX elease. A e wa ds, he eleased DOX was e alua ed by Spec-
aMax® iD3 Mul i-Mode Mic opla e Reade s (Ma shall Scien i ic,
Hamp on, NH, USA).
2.8. Memb ane p o ein cha ac e iza ion
2.8.1. Sodium dodecyl sul a e polyac ylamide gel elec opho esis (SDS-
PAGE)
4T1 CMs, 4T1@DOX/CS-NPs, RBCMs, RBC@DOX/CS-NPs,RBC-
4T1CMs and RBC-4T1@DOX/CS-NPs wi h he loading bu e we e
hea ed a 95 ◦C o 15 min. A e wa ds, 30
μ
L o he samples we e loaded
in o each well o a 10 % SDS-PAGE gel and hen un a 100 V o 1 h.
Nex , he gel was s ained wi h Coomassie Blue o 10 min and hen
washed o isualize p o ein bands by ChemiDoc™ Touch Imaging Sys-
em (Bio-Rad, he Ne he lands) [31].
2.8.2. Wes e n blo ing (WB)
A e elec opho esis, he p o ein bands we e ans e ed on o PVDF
memb ane (150 V, 60 min). Then, he PVDF memb ane was s ained wi h
p ima y an ibodies ollowed by de ec ion wi h IRDyeR680CW-labeled
seconda y an ibody. Finally, he bands we e isualized wi h he Li-Co
Odyssey 9120 In a ed Imaging Sys em (Lincoln, NE, U.S.A) [32].
2.9. In i o cellula s udies
2.9.1. Cy o oxici y assays
3-(4,5-dime hyl hiazol-2-yl)-5-(3-ca boxyme hoxyphenyl)-2-(4-sul-
ophenyl)-2H- e azolium (MTS) assay was used o de e mine he
cellula cy o oxici y o ou o mula ions. B ie ly, 4T1 cells we e seeded
in 96-well pla es (5.0 ×10
3
cells/well) and cul u ed a 37 ◦C o 24 h.
Subsequen ly, 4T1 cells we e ea ed wi h ee DOX, DOX/CS-NPs,
4T1@DOX/CS-NPs, and RBC@DOX/CS-NPs o ano he 24 h. A e -
wa ds, he 4T1 cells we e incuba ed wi h 100
μ
L o esh medium con-
aining 20
μ
L MTS eagen in an incuba o o 2 h. The cell iabili y was
de e mined by measu ing he abso bance a a wa eleng h o 490 nm
using a Spec aMax M3 Mul i-Mode Mic opla e Reade (Molecula De-
ices, Silicon Valley, CA, USA).
2.9.2. In i o cellula up ake and dis ibu ion o DOX-loaded NPs by
con ocal lase scanning mic oscopy (CLSM)
5 ×10
3
cell 4T1 cells we e seeded on mic oscope slides in a 24-well
pla e and we e incuba ed o 24 h. Subsequen ly, he cul u e medium
was eplaced wi h medium con aining DOX, DOX/CS-NPs, 4T1@DOX/
CS-NPs and RBC@DOX/CS-NPs wi h he inal concen a ion o 10
μ
g/
mL DOX. A e incuba ion o 3 h, he cells we e washed wi h PBS and
ixed wi h 4 % pa a o maldehyde (PFA) o 10 min. A e s aining he
nuclei o cells wi h Hoechs 33342 o 15 min, he slides we e imaged by
a CLSM Leica TCS SP8 (Leica Mic osys ems, We zla , Ge many).
2.9.3. Immune e asion de ec ion
Cells o he mouse mac ophage cell line RAW 264.7 we e seeded in
24-well pla es (1 ×10
4
cells/well) and cul u ed o 24 h. A e incu-
ba ion, he cells we e cul u ed wi h DOX/CS-NPs,4T1@DOX/CS-NPs,
RBC@DOX/CS-NPs and RBC-4T1@DOX/CS-NPs (wi h he same con-
cen a ion o DOX: 10
μ
g/mL) o 3 h and 6 h. Then, he cells we e
washed wi h PBS and s ained wi h Hoechs 33342 o 10 min. A e -
wa ds, he cells we e imaged by CLSM and analyzed using LAS X. To
s udy he up ake o DOX/CS-NPs,4T1@DOX/CS-NPs, RBC@DOX/CS-
NPs and RBC-4T1@DOX/CS-NPs by RAW 264.7 cells (1 ×10
4
cells/
well) we e seeded and cul u ed o 24 h. The day a e , he cells we e
ea ed wi h NPs o 3 h and 6 h. Subsequen ly, he cells we e collec ed
o low cy ome y (BD Biosciences, F anklin Lakes, NJ, USA). expe i-
men . The esul s we e analyzed wi h he FlowJo™ 10.7.1 so wa e
(BD Biosciences).
2.9.4. Homo ypic a ge ing s udy o 4T1 cells by CLSM and low cy ome y
To p o e he in i o homo ypic a ge ing e ec s o 4T1@Cy5/CS-
NPs on 4T1 quali a i ely, CLSM was used o in es iga e he in acel-
lula up ake o 4T1@Cy5/CS-NPs by 4T1 cells. The e o e, 1 ×10
4
cells
(4T1, MC38, CT-26, RAW 264.7 and 3 T3) we e seeded on mic oscope
slides and incuba ed a 37 ◦C o 24 h. Then, he cells we e incuba ed
wi h 4T1@Cy5/CS-NPs (50
μ
g /ml) o 4 h. The cells we e washed wi h
PBS and ixed wi h 4 % PFA. This was ollowed by nuclea s aining wi h
Hoechs 33342 o 10 min. As a con ol, 4T1 cells we e incuba ed wi h
Cy5/CS-NPs a he same concen a ion o 4 h. Finally, he in acellula
up ake o 4T1@Cy5/CS-NPs and Cy5/CS-NPs we e analyzed by a CLSM
Leica TCS SP8. The images we e analyzed using LAS X (Leica Applica-
ion Sui e X) so wa e. To de ec he phagocy ic e iciency o 4T1@Cy5/
CS-NPs and DiD@PLGA-NPs (con ol NPs) in di e en cell ypes. B ie ly,
cells (4T1, MC38, CT-26, RAW 264.7 and 3 T3) we e seeded in o 96-well
pla es a a densi y o 1 ×10
4
cells/well o 24 h. Subsequen ly, he cells
we e incuba ed wi h 4T1@Cy5/CS-NPs and DiD@PLGA-NPs o 1 and 4
h. Un ea ed cells we e used as a nega i e con ol. The cells we e hen
washed wi h PBS and diges ed wi h ypsin. La e , he luo escence in-
ensi y o 4T1 cells was de ec ed by means o low cy ome y.
2.10. Animals
Female BALB/c mice [RRID:IMSRCRL:028] app oxima ely 7–9
weeks old weighing be ween 21 and 28 g we e used o he expe imen al
o ho opic b eas cance model and oxici y s udies. Animals we e
pu chased om he Keizo Asami Immunology Labo a o y (LIKA; Reci e,
PE, B azil) and ea ed acco ding o he e hical p inciples o animal
expe imen a ion. All animal p ocedu es we e app o ed by he Labo a-
o y Animal Managemen Commi ee and E hics Commi ee o Fede al
Uni e si y o Rio G ande No e (No. 063/2016). Mice we e kep in an
ai -condi ioned oom a 21–22 ◦C, 50 % humidi y, speci ic pa hogen-
ee condi ions, and a 12-h ligh /da k cycle. A g oup o i e mice we e
housed in a cage. They had ee access o ood and wa e and we e
andomly g ouped (n =6) and kep in di e en cages. A e 1 week and
adap ion o he mice o he en i onmen , he animal expe imen was
commenced.
2.10.1. In i o an i umo ac i i y
In his expe imen , 4T1 cells (1 ×10
6
cells/100
μ
l FBS- ee DMEM)
we e inocula ed below he ou h le b eas in BALB/c mice anes-
he ized wi h xylazine and ke amine (2:8). Then, he mice we e an-
domized in o 6 g oups wi h n =6 animals each. Tumo g ow h was
moni o ed e e y wo days. When he umo s eached 3 mm in diame e ,
he mice we e ea ed pe i umo ally wi h s e ile saline (nega i e con ol
g oup), DOX (0.125 mg/Kg; posi i e con ol), DOX/CS-NPs (0.125 mg/
Kg), RBC@DOX/CS-NPs (0.125 mg/Kg), 4T1@DOX/CS-NPs (0.125 mg/
Kg) and RBC-4T1@DOX/CS-NPs (0.125 mg/Kg). E e y 5 days, ea -
men s we e epea ed o a o al o 3 imes o e 15 days. The animals
we e anes he ized and killed by ce ical disloca ion on day 19.
S. Rezaei e al.
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5
Subsequen ly, blood ( om he hea ca i y), umo , lungs and li e we e
collec ed o biochemical and his opa hological analysis.
2.10.2. Biochemical and hema ological analysis
A e s o age, s anda d hema ological echniques we e pe o med on
EDTA- ea ed blood. Thus, e y h ocy e and leukocy e coun as well as
hemoglobin quan i ica ion and hema oc i es we e ca ied ou . The
blood o all animals in each g oup (n =6) was analyzed in iplica e.
2.10.3. qRT-PCR
Pa ial o ho opic umo s om BALB/c mice we e analyzed o
asce ain he gene exp ession. The o al RNA o ho opic umo was
ob ained wi h In i ogen™ TRIzol™ eagen (Fishe Scien i ic, USA)
and pu i ied wi h SV To al RNA Isola ion Sys em (P omega, USA) ac-
co ding o he manu ac u e ’s ins uc ions. Nex , cDNA was syn hesized
using high-capaci y RNA- o-cDNA™ ki (Applied Biosys ems, USA).
Powe Up SYBR G een Mas e Mix (Applied Biosys ems, USA) was used
o eal- ime ampli ica ion. In he (Supplemen a y Table 1), he o wa d
and e e se p ime s sequences (The mo Fishe Scien i ic, USA) a e lis-
ed. The expe imen s we e pe o med in iplica e. Gene exp ession da a
we e no malized ela i e o he housekeeping gene β-ac in using 2-
ΔΔC . All animals in each g oup (n =6) we e analyzed in duplica e.
2.10.4. His ology and immunohis ochemis y
Fo immunohis ochemical analysis, he umo s and o gans o BALB/c
mice we e collec ed. B ie ly, a e depa a iniza ion, ehyd a ion and
an igen eco e y, he umo issue sec ions we e incuba ed wi h an i-NF-
κB, an i-CD163 and an i-CXCL12 a 4 ◦C o e nigh . As a seconda y
an ibody, bio inyla ed pan-speci ic uni e sal an ibody was used ol-
lowed by incuba ion wi h HRP-conjuga ed s ep a idin. Nex , dia-
minobenzidine (DAB; DAKO) was used o e eal he labeling. Sec ions
we e coun e s ained wi h hema oxylin and analyzed unde a Nikon
E200 LED ligh mic oscope (Mina o, Japan). Acco ding o Cha a e-
Jau e [33] o immuno eac i i y classi ica ion, he pe cen age o
posi i ely s ained cells and in ensi y o posi i e cells a e immuno-
s aining was calcula ed. Immunohis ochemical analyses o umo issues
we e pe o med independen ly by wo ained esea che s. Blind da a
analysis was applied o he sco ing o he umo issues. Fo each
an ibody, h ee his ological sec ions we e e alua ed.
2.11. S a is ical analysis
Analyses we e pe o med using G aphPad P ism 8.1.1 so wa e
(G aphPad So wa e, San Diego, CA, USA). All da a a e p esen ed as
mean ±s anda d de ia ion (SD). All da a we e s a is ically analyzed
using s uden ’s - es , analysis o a iance (ANOVA; nonpa ame ic)
wi h Bon e oni es , unpai ed and Mann-Whi ney U es . Signi icance
le els we e de ined as # (no signi ican , P ≥0.05), *P ≤0.05, **P ≤
0.01, ***P ≤0.001 and ****P ≤0.0001.
3. Resul s
3.1. P epa a ion and cha ac e iza ion o DOX/CS-NPs, 4T1@DOX/CS-
NPs, RBC@DOX/CS-NPs and RBC-4T1@DOX/CS-NPs
DOX/CS-NPs we e syn hesized as educ ion-sensi i e NPs o he
ea men o 4T1 umo s. Va ious educ ion-sensi i e polyme ic NPs o
a ge ed he apy o b eas cance , including he iple-nega i e b eas
cance cell line 4T1, ha e been epo ed [34]. To p epa e he 4T1CM
and RBCM-coa ed DOX/CS-NPs, 4T1CMs and RBCMs we e isola ed
using hypo onic lysis bu e and di e en ial ul acen i uga ion. RBCMs
and 4T1CMs we e mixed o he gene a ion o usion memb anes a a
a io o 1:1, 1:2 and 1:3 and we e quan i ied by NTA (Fig. S1) [5]. To
u he con i m he success ul usion o 4T1CMs and RBCMs, we labeled
4T1CMs wi h DiD (pu ple; λex/λem =646/663 nm) and RBCMs wi h
DiO (g een; λex/λem =484/501 nm). A dilu ed sample o RBCMs and
4T1CMs was p epa ed and imaged by CLSM and con i med success ul
usion (Fig. 1A). Ou da a con i med he success ul usion o RBC-4T1
memb anes, and es ablished ha he a io o 1:2 was op imal. Nex ,
4T1CMs, RBCMs and RBC-4T1CMs we e coa ed on o DOX/CS-NPs a a
a io o 2:1, 2:1 and usion memb anes a a io o 1:1:1, 1:2:1 and 1:3:1
(Fig. 1B). The size o RBC-4T1 usion memb anes a a a io o 1:3 was
bigge han a a a io o 1:2 and 1:1. In addi ion, a a a io o 1:1, he
posi i e su ace cha ge o DOX/CS-NPs was no su icien ly dec eased
(Fig. S 2), sugges ing incomple e coa ing o DOX/CS-NPs. TEM images
e ealed ha CM-coa ed DOX/CS-NPs we e o sphe ical shape, which
we e composed o DOX/CS-NPs as inne co e and 4T1CMs, RBCMs and
RBC-4T1CMs as he shell (Fig. 1C). In addi ion, he NPs we e cha ac-
e ized by DLS measu emen s. The DOX/CS-NPs showed an a e age
hyd odynamic diame e o 278 ±1.62 nm, polydispe si y index (PDI) o
0.352 ±0.092 and a su ace cha ge o +44 ±0.15 mV (Fig. 1D,E).
Success ul su ace coa ing o DOX/CS-NPs wi h 4T1CMs and RBCMs was
addi ionally con i med by an inc ease in pa icle size and a educ ion in
NP su ace cha ge o 4T1CM-, RBCM- and 4T1-RBCM-coa ed DOX/CS-
NPs, as measu ed by DLS (Fig. 1D, E). To his end, o coa ing o
DOX/CS-NPs wi h RBC-4T1CMs op imized a io was ob ained a 1:2:1,
indica ing sui able size o NPs and su icien coa ing (Fig. 1B).
The success ul conjuga ion o LA o CSO was in es iga ed by
1
H NMR
(Fig. 2A). As epo ed in ou p e ious s udy, he de e mined p o on
peaks o pen a-he e ocyclic s uc u e o LA a 2.5–3.5 ppm in CS-LA NPs
a e a p oo o LA conjunc ion [21]. The EE% and LD% o DOX in CS-NPs
we e calcula ed o be 71.76 ±0.87 and 38.03 ±0.45, espec i ely. The
elease o DOX om DOX/CS-NPs, RBC@ DOX/CS-NPs,4T1@ DOX/CS-
NPs and RBC-4T1@ DOX/CS-NPs was s udied a 37 ◦C in pH 7.4 and pH
5.3, wi h and wi hou addi ion o 10 mM GSH (Fig. 2B-E). The esul s
showed ha he addi ion o 10 mM GSH signi ican ly induced (**** p
0.0001) he elease o DOX om DOX/CS-NPs (Fig. 2B-E) [21,22]. In
addi ion, an acidic milieu (pH 5.3) signi ican ly (**** p ≤0.0001)
inc eased he elease o DOX om DOX/CS-NPs (app oxima ely 82 %,)
a e 24 h, compa ed o pH 7.4 (44 %). Howe e , RBC@ DOX/CS-NPs
and 4T1@ DOX/CS-NPs as well as RBC-4T1@ DOX/CS-NPs sus ained
DOX elease p o ile in pH 7.4 and pH 5.3, wi h and wi hou 10 mM GSH
(Fig. 2C, E) indica ing he e iciency o exci ing coa ing and s abili y o
memb ane-coa ed NPs du ing he physiological s a es [3,35]. The suc-
cess ul ans e o memb ane p o eins is impo an o he exploi a ion o
CMs o umo a ge ing. In o de o de e mine he memb ane p o eins
o 4T1CM-coa ed DOX/CS-NPs, we employed SDS-PAGE and WB
(Fig. 2D, E). SDS-PAGE o cell lysa es o 4T1 cell, 4T1CM, 4T1@DOX/
CS-NPs, RBCMs, RBC@DOX/CS-NPs, RBC-4T1CMs and RBC-
4T1@DOX/CS-NPs demons a ed he success ul ans e o p o eins
om 4T1CMs, RBCMs and RBC-4T1CMs on o he ou e laye o DOX/CS-
NPs (Fig. 2D, E). Nex , we in es iga ed he success ul ans e o CM by
WB labeling o he memb ane p o ein CD47, which is exp essed by 4T1
umo cells and is ele an o homo ypic a ge ing and educ ion o
up ake by mac ophages [1,14,36]. Ou esul s showed ha CD47 was
p esen in he lysa es o 4T1 cells, 4T1CMs and 4T1@DOX/CS-NPs,
con i ming he success ul coa ing o DOX/CS-NPs wi h 4T1CMs.
3.2. E ec i e in e naliza ion and enhanced cy o oxici y o DOX/CS-NPs
coa ed wi h 4T1CMs by 4T1 b eas cance cells
The cellula up ake o ee DOX, DOX/CS-NPs, 4T1@DOX/CS-NPs,
RBC@DOX/CS-NPs and RBC-4T1@DOX/CS-NPs was de e mined by
CLSM and low cy ome y. 4T1 cells we e incuba ed wi h ou NP o -
mula ions o 4 h. Cellula NP up ake depends on many ac o s including
NP size, PDI, su ace cha ge and su ace modi ica ion [9]. CLSM da a
showed ha he posi i ely cha ged RBC@DOX/CS-NPs and 4T1@DOX/
CS-NPs, as well as he nega i ely cha ged RBC-4T1@DOX/CS-NPs
exhibi ed highe up ake by 4T1 cells compa ed o DOX/CS-NPs and
ee DOX (*p ≤0.05, ***p ≤0.0001), and he e o e imp o ed cellula
up ake o DOX by NPs (Fig. 3A, B). P e ious epo s showed ha ee
DOX di used o he nuclei 2 o 8 h a e up ake [37]. CLSM analysis
S. Rezaei e al.
Bioma e ials Ad ances 151 (2023) 213456
6
e ealed ha , while ee DOX localized o he nucleus 4 h a e incu-
ba ion, he majo i y o NP-encapsula ed DOX localized o he cy osol, in
line wi h ou elease kine ics da a showing ha only a ac ion o DOX
was eleased a his imepoin (Fig. 2B). Bo h CLSM and low cy ome y
da a showed ha up ake o NP-encapsula ed DOX was o e all highe
han up ake o ee DOX (Fig. 3A,B). Flow cy ome ic analysis u he
e ealed ha be ween he a ge ed NPs, up ake o 4T1@DOX/CS-NPs
was highes (Fig. 3B). This inc eased up ake o 4T1CMs can be a ib-
u ed o he ans e o a ge ing p o eins p esen on 4T1CMs, which
media e homo ypic a ge ing owa ds sou ce cells. Cell iabili y assays
o 4T1 cells incuba ed wi h ee DOX and DOX/CS-NPs e ealed an IC
50
o 0.85 and 0.91
μ
g mL
−1
, espec i ely (Fig. 3C). Howe e , when DOX/
CS-NPs we e coa ed wi h 4T1CMs (4T1@DOX/CS-NPs), hey displayed
an IC
50
o 0.59
μ
g mL
−1
, which is 1.5- old lowe han ha o non-
a ge ed NPs (con ol). Thus, 4T1CM-coa ing led o highe cy o oxici y
likely as a esul o inc eased cellula in e naliza ion o 4T1@DOX/CS-
NPs by 4T1 cells, compa ed o DOX/CS-NPs (Fig. 3C). Compa ed o
4T1@DOX/CS-NPs and DOX/CS-NPs, RBC@DOX/CS-NPs did no
signi ican ly inc ease cellula up ake o educed he IC
50
(Fig. 3C, D)
[38]. The cy o oxici y o NPs wi hou DOX, CS-NPs, 4T1@CS-NPs,
RBC@CS-NPs and RBC-4T1@CS-NPs, was s udied in 4T1 and 3 T3
cells. None o he con ol NPs exhibi ed any signi ican cy o oxici y
(Fig. 3E, F), showing ha CS-NPs possess good biocompa ibili y.
3.3. Inhibi ion o mac ophage-media ed NP up ake by CM-coa ing
Nex , we s udied he immune escape mechanism o DOX/CS-NPs,
4T1@DOX/CS-NPs, RBC@DOX/CS-NPs and RBC-4T1@DOX/CS-NPs.
Mac ophages a e esponsible o he in i o immune clea ance o NPs
[39]. To in es iga e whe he ou NP o mula ions could escape
mac ophage-based clea ance, we cul u ed he mac ophage RAW 264.7
cell line wi h DOX-loaded CS-NPs. Then, he cells we e imaged by CLSM
a e 3 and 6 h o incuba ion. The luo escence images showed ha RAW
264.7 cells incuba ed wi h DOX/CS-NPs had a he in ensi y o he ed
luo escence was signi ican ly highe han ha o RAW 264.7 cells
incuba ed wi h CM-coa ed DOX/CS-NPs a 3 h and 6 h (Fig. 4A, B). The
in ensi y o 4T1CM, RBCM and RBC-4T1CM-coa ed DOX/CS-NPs was
lowe , he eby sugges ing ha 4T1CM, RBC and RBC-4T1CM ep e-
sen ing he shell o DOX/CS-NPs educed he cellula up ake o DOX/CS-
NPs. To quan i y he up ake da a, low cy ome ic analysis was pe -
o med (Fig. 4B). The in ensi y o he ed luo escence o DOX/CS-NPs
was abou 1.3-, 1.3- and 1.2- old highe han ha o 4T1@DOX/CS-
NPs, RBC@DOX/CS-NPs and RBC-4T1@DOX/CS-NPs g oups, espec-
i ely (** p ≤0.01, *** p ≤0.001, **** p ≤0.0001). The da a indica e
ha he p esence o CD47 on CM-coa ed DOX/CS-NPs could supp ess
he up ake o NPs by mac ophages, which is in line wi h he li e a u e
[4,40,41].
3.4. 4T1 cance cell sel - ecogni ion by homo ypic a ge ing
To in es iga e he homo ypic a ge ing capaci y o CM-coa ed
Cy5@DOX/CS-NPs (Table S2), DiD/PLGA-NPs (con ol NPs, Table S2),
we incuba ed NP o mula ions wi h b eas cance cell line (4T1 cells)
and he colo ec al umo cell lines (MC38 and CT-26 cells), as well as
wi h non- umo igenic cell lines (3 T3 and RAW 264.7 cells) (Fig. 5A-C
and Fig. S3), and analyzed he cellula up ake by CLSM and low
cy ome y. To his end, we used NPs encapsula ed wi h he luo escen
dye Cy5. Bo h MC38 and 4T1 cells we e ea ed wi h 4T1@Cy5/CS-NPs,
MC38@Cy5/CS-NPs, CT26@Cy5/CS-NPs, RAW@Cy5/CS-NPs and 3
T3@Cy5/CS-NPs. Flow cy ome ic analysis showed a highe Cy5 signal
in 4T1 cells ea ed wi h 4T1@Cy5/CS-NPs compa ed o he CM-coa ed-
Cy5/CS-NPs de i ed om MC38, CT-26, RAW and 3 T3 cells. The
luo escence in ensi y in he 4T1@Cy5/CS-NPs g oup was abou 2.7-,
1.3-, 2.1, 1.6- and 1.8- old highe han in 4T1 cells ea ed wi h
Cy5@CS-NPs, MC38@Cy5/CS-NPs, CT26@Cy5/CS-NPs, RAW@Cy5/
CS-NPs and 3 T3@Cy5/CS-NPs, espec i ely. To con i m ou low
cy ome y da a, we incuba ed bo h MC38 and 4T1 cells wi h
MC38@Cy5/CS-NPs, CT26@Cy5/CS-NPs, RAW@Cy5/CS-NPs and 3
Fig. 1. Cha ac e iza ion o di e en NP o mula ions. A) CLSM images o DiD-labeled 4T1CMs (pu ple), RBCMs-labeled DiO (g een), and he me ged images o
4T1CMs and RBCMs (scale ba =5
μ
m). B) Schema ic illus a ion showing he o e iew o he combina ions o he 4T1CMs, RBCMs and DOX/CS-NPs used o
designing DOX/CS-NPs, 4T1@DOX/CS-NPs, RBC@DOX/CS-NPs and RBC-4T1@DOX/CS-NPs. C) TEM images o I) DOX/CS-NPs, II) 4T1CMs, III) 4T1@DOX/CS-NPs,
IV) RBCMs, V) RBC@DOX/CS-NP, VI) RBC-4T1 CMs and VII) RBC-4T1@DOX/CS-NPs. D, E) Pa icle sizeand ze a po en ial o DOX/CS-NPs, 4T1@DOX/CS-NPs,
RBC@DOX/CS-NPs and RBC-4T1@DOX/CS-NPs by DLS.
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T3@Cy5/CS-NPs o 4 h o analyze he up ake using CLSM. The images
showed ha he luo escence signal o 4T1@Cy5/CS-NPs (pu ple) was
highe han in he o he samples (Fig. 5 A, B; # =no signi ican , P >
0.05, **P <0.01, ***P <0.001, and ****P <0.0001).In addi ion, up ake
da a o con ol NPs (DiD@PLGA-NPs)(Fig. S3) demons a ed he abili y
o DiD@PLGA-NPs o homo ypic a ge ing. Toge he , hese esul s
showed ha he sel - ecogni ion capabili y o memb ane-coa ed NPs o
homo ypic a ge ing [29,42].
3.5. An i- umo e ec o NPs in i o
The 4T1 o ho opic mamma y umo me as asis model was used o
e i y he in i o an i- umo e ec s o all ou NP o mula ions. To his
end, mice we e inocula ed wi h 1 ×10
6
4T1 cells on day 1. When he
umo s eached a size o 3 mm in diame e , he mice we e injec ed
in a umo ally wi h saline, ee DOX, DOX/CS-NPs, 4T1@DOX/CS-NPs,
RBC@DOX/CS-NPs o RBC-4T1@DOX/CS-NPs a a dose o 0.125 mg/
kg
−1
(n =6; Fig. 6A). The umo g ow h was moni o ed o e he cou se
o 19 days (Fig. 6B, C). The umo s o he saline g oup showed a apid
g ow h (Fig. 6B-C). All DOX-loaded CS-NPs coa ed wi h ei he 4T1CMs
o RBCMs, as well as ee DOX and DOX/CS-NPs, signi ican ly inhibi ed
he umo g ow h, compa ed o he saline g oup (***P <0.001; Fig. 6B).
In e es ingly, we obse ed a comple e umo eg ession in one mouse o
he g oup ea ed wi h 4T1@DOX/CS-NPs (Fig. 6A). A e coa ing he
DOX/CS-NPs wi h 4TCMs and RBCMs, he umo g ow h was d ama i-
cally educed by 95 % and 90 %, espec i ely, compa ed o he saline
g oup (***P <0.001; Fig. 6C). As shown in Fig. 6D, umo weigh was
signi ican ly educed in he g oups ea ed wi h ee DOX, DOX/CS-NPs,
4T1@DOX/CS-NPs, RBC@DOX/CS-NPs and RBC-4T1@DOX/CS-NPs,
compa ed o he saline con ol g oup. The umo weigh cu es
Fig. 2. A)
1
H NMR spec um o DOX/CSLA-NPs in D
2
O. B, C, D,E) In i o DOX elease p o ile o DOX/CS-NPs, RBC@ DOX/CS-NPs,4T1@ DOX/CS-NPs and RBC-
4T1@DOX/CS-NPs a pH 7.4 and pH 5.3, in he p esence and absence o 10 mM GSH. F) SDS-PAGE and WB analysis o I) 4T1cell lysa es, II) 4T1CMs and III)
4T1@DOX/CS-NPs. G) SDS-PAGE o I) RBCM, II) RBC@DOX/CS-NPs, III) RBC-4T1CM and IIII) RBC-4T1@DOX/CS-NPs. H) Wes e n blo analysis o 4T1 cell, 4T1CM,
and 4T1@DOX/CS-NPs o cha ac e is ic 4T1CM ma ke s CD47. Da a a e ep esen ed as mean ±SD; ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001.
S. Rezaei e al.
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Fig. 3. In e naliza ion and cy o oxici y o he NPs o mula ions. A, B) Cellula up ake o 4T1 cells wi h di e en samples a e 4 h o incuba ion. DOX ( ed) and nuclei
(blue) by means o CLSM and low cy ome y (scale ba =10
μ
m). C, D) In i o cy o oxici y o DOX, DOX/CS-NPs, 4T1@DOX/CS-NPs and RBC@DOX/CS-NPs on 4T1
cells a e 24 h. Da a a e ep esen ed as mean ±SD (n =3). E, F) In i o cell iabili y o 4T1 cells and 3 T3 cells, espec i ely, a e 24 h o incuba ion o 4T1CMs,
RBCMs, wi hou DOX CS-NPs, 4T1@ CS-NPs, RBC@ CS NPs and RBC-4T1@ CS-NPs. Da a a e ep esen ed as mean ±SD; ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001.
S. Rezaei e al.
Bioma e ials Ad ances 151 (2023) 213456
9
illus a ed ha DOX/CS-NPs coa ed wi h RBC and 4T1CMs had a simila
e ec on educing umo weigh . F ee DOX educed he umo weigh o
120 mg, while umo weigh o he g oups ea ed wi h saline, DOX/CS-
NPs and RBC-4T1@DOX/CS-NPs we e 305.5 mg, 130.75 mg and 127.1
mg, espec i ely. In he g oups ea ed wi h RBC@DOX/CS-NPs and
4T1@DOX/CS-NPs, he umo weigh was educed o 41.16 mg and
16.9 mg, espec i ely. The da a o he umo olume a endpoin simi-
la ly illus a ed a signi ican educ ion in umo olume in he g oups
ea ed wi h DOX, DOX/CS-NPs, 4T1@DOX/CS-NPs, RBC@DOX/CS-
NPs and RBC-4T1@DOX/CS-NPs, compa ed o he saline g oup
(Fig. 6C). The esul s sugges ha 4T1CM-coa ed NPs induced homo-
ypic a ge ing, which enhanced he up ake e iciency o 4T1@DOX/CS-
NPs in 4T1 sou ce cells in i o and imp o ed DOX accumula ion [14]. In
addi ion, due o he RBCM coa ing, RBC@DOX/CS-NPs we e less likely
o be clea ed by he immune sys em han uncoa ed NPs [43]. The blood
leukocy e coun o he mice a e ea men wi h NPs was measu ed o
show whe he he ea men had any dele e ious e ec on he immune
sys em (Fig. 6E). The da a illus a ed ha he leukocy e coun s in he
g oups ea ed wi h DOX we e signi ican ly lowe han hose ea ed
wi h he saline. Howe e , he educ ion in leukocy e coun s in he
4T1@DOX/CS-NP- ea ed g oup was less compa ed o DOX ea men .
3.6. Inhibi ion o b eas cance me as asis by NPs
4T1 cells ha e been shown o apidly di ide and spon aneously
me as asize [44]; hey can sp ead in o he li e , lungs and lymph nodes
[45]. In his s udy, a e ea men o mice wi h DOX-loaded CS-NPs, he
p esence o in il a ing umo cells was his ologically analyzed in he
li e and lungs o 4T1 umo -bea ing mice (Fig. 7A, B, a owheads).
Sco ing o in il a ing umo cells e ealed ha in all expe imen al
g oups, umo cells in il a ed he li e , excep o g oups ea ed wi h
CS-NPs- and RBC-4T1@DOX/CS-NPs. Howe e , compa ed o he saline
g oup, he numbe o me as a ic niches was educed in mice ea ed wi h
DOX, 4T1@DOX/CS-NPs and RBC@DOX/CS-NPs (*P <0.05, **P <
Fig. 4. Cellula up ake o di e en NP o mula ions. A) In acellula up ake o DOX/CS-NPs, 4T1@DOX/CS-NPs, RBC@DOX/CS-NPs and RBC-4T@ DOX/CS-NPs in
RAW 264.7 cells a e 3 and 6 h o incuba ion (scale ba =20
μ
m). The nucleus was s ained wi h Hoechs 33342 (blue) and he NPs we e labeled wi h DOX ( ed). B)
In acellula luo escence in ensi y o DOX measu ed by means o low cy ome y a e 3 and 6 h o incuba ion. Da a a e ep esen ed as mean ±SD (n =3); ** p ≤
0.01, *** p ≤0.001, **** p ≤0.0001.
S. Rezaei e al.
