A water-soluble nucleolin aptamer-paclitaxel conjugate for tumor-specific targeting in ovarian cancer

Article abstract of DOI:10.1038/s41467-017-01565-6

Paclitaxel (PTX) is among the most commonly used first-line drugs for cancer chemotherapy. However, its poor water solubility and indiscriminate distribution in normal tissues remain clinical challenges. Here we design and synthesize a highly water-solubl

Full text of DOI:10.1038/s41467-017-01565-6

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DOI: 10.1038/s41467-017-01565-6
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P a c l i t a xe l ( P T X ) i s a m o n g t h e m o s t c o m m o n l y u s e d fir s t - l i n e d ru g s f or c a n c e r c h e m o th e ra py .
H o w ev e r, i t s p o o r w a te r s o l u b i l i t y a n d i n d i s c r i m i n a te d i s t r i b ut i o n i n n or m a l t i s s u e s r e m a i n
c l i ni c a l c h a ll e n g e s . H e r e w e d e s i g n a n d s y n th e s i z e a h i g h l y w a t e r- s ol u b l e n uc l e o l i n a p t a m e r-
p a c l i t a xe l c o n ju g a t e ( N u c A - P TX ) t h a t s e l e c ti v e l y d e l i v e rs P T X t o t h e t u m o r s i te . B y c o n -
n e c t i n g a t u m o r- t a rg e t i n g n u cl e o l i n a p ta m er ( N u c A ) t o t h e a c t i v e h y d ro x y l g r ou p a t 2 ′
p o si t i o n o f P T X v ia a c a t h e p s i n B s e n s i t i v e d i p e p ti d e b on d , N u c A - P T X r em a i n s s t a b l e a n d
i n a c t i v e i n t h e c i rc u l a ti o n . N u c A f a c i l i ta te s t h e u p ta ke o f t h e c o n j ug a t e d P T X s p e c i fic a l l y i n
t u m o r c e l ls . O n c e i ns i d e c e l l s , t h e d i p e p t i d e b o n d l i n k e r o f N u c A - P T X i s c l e a v e d b y c a t h e ps i n
B a n d t h e n t h e c o n ju g a t e d P T X i s r e l e a s e d f o r a c t i o n. T he N uc A m o d i fic a t i o n a s s i s ts t h e
s e le c t i ve a c c u m ul a ti o n o f t h e c o n j u g a t e d P T X i n o v a r i a n t u m o r t i s s u e r a t he r t h a n n or m a l
t i ss u e s , a nd s u b se qu e n t l y r e s u l t i n g i n n o ta b l y i m p ro v ed a n t i t um or a c t i v i t y a n d r ed u c ed
t o x i c i t y .
1 Institute of Precision Medicine and Innovative Drug Discovery (PMID), School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR 999077,
China. 2 Institute for Advancing Translational Medicine in Bone and Joint Diseases (TMBJ), School of Chinese Medicine, Hong Kong Baptist University, Hong
Kong SAR 999077, China. 3 Institute of Integrated Bioinfomedicine and Translational Science (IBTS), School of Chinese Medicine, Hong Kong Baptist
University, Hong Kong SAR 999077, China. 4 College of Science, Northwest Agriculture and Forestry University, Yangling, 712100 Shaanxi, P.R. China.
5 School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR 999077, China. 6 Department of Orthopaedics and
Traumatology, Nanfang Hospital, Guangzhou 510515, China. Fangfei Li, Jun Lu, Jin Liu, Chao Liang, Maolin Wang and Luyao Wang contributed equally to this
work. Correspondence and requests for materials should be addressed to B.-T.Z. (email: zhangbaoting@cuhk.edu.hk)
or to A.L. (email: aipinglu@hkbu.edu.hk) or to G.Z. (email: zhangge@hkbu.edu.hk)
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A R T I C L E
N A T U R E C O M MU N IC A T I ON S | D O I: 1 0 . 1 0 3 8 / s 414 6 7 - 0 1 7 - 0 1 56 5 - 6
aclitaxel (PTX) is one of the most commonly used first-line promising tumor-targeting element for developing paclitaxel
drug for treating a variety of cancers in clinical che- derivatives.
P
motherapy1,2. However, its poor water solubility restricts its
Cathepsin B is an intracellular lysosomal protease and its
direct clinical applications3. More importantly, PTX does not general substrates include valine-citrulline and phenylalanine-
explicitly discriminate between cancer cells and normal cells and arginine21. Cathepsin B mainly locates in lysosomes with a
frequently leads to serious undesirable side effects4,5. Attaching to favorable pH from 4.5 to 5.022, and plays an important role in
tumor recognition elements has been proven beneficial for invasion and metastasis in tumor cells23,24. The trace amount of
selective delivery of anti-cancer compounds to tumor cells; cathepsin B in blood circulation normally stays inactive due to
however, few tumor-targeting elements was proved to be suc- unfavorable pH conditions and the existence of protease inhibi-
cessful. Although some antibody-drug conjugates (ADCs) have tors. Therefore, via a cathepsin B-labile valine-citrulline dipeptide
been approved for clinical treatment, there are still challenges linker, the tumor-targeting NucA can be connected to PTX at the
when utilizing antibodies for drug delivery, especially the risk of C-2′ position as a nucleolin aptamer-paclitaxel conjugate (NucA-
immunogenicity and the raising incidents of resistance6–9. In PTX). The intact NucA-PTX is supposed to have no or low
addition, difficulties in synthesis and chemical modification, and activity as the substitution of 2′-hydroxyl group on PTX with
rigorous storage requirement are also the major concerns8. other groups resulted in the loss of antitumor activity for the PTX
Therefore, it is highly desirable to develop paclitaxel derivatives derivative25. The conjugate is postulated to remain stable in blood
with tumor-targeting property.
circulation, whereas once delivered to tumor cells via NucA
Nucleic acid aptamers are small single-stranded DNA or RNA recognition to the cell surface nucleolin and taken up by endo-
oligonucleotide segments, which bind to their targets, such as cytosis, the linker can be cleaved by lysosomal proteases in
proteins with high affinity and specificity by unique three- tumors, resulting in PTX release for action. To ensure the com-
dimensional (3D) structure10–13. Aptamers have been widely used plete release of PTX, p-aminobenzylcarbonyl (PABC) can be
as tumor recognition elements13. In contrast to antibodies, incorporated between PTX and the dipeptide linker, which
aptamers have limited immunogenicity and are unlikely to facilitates the conjugate being hydrolyzed and releasing PTX
develop resistance11,14,15. Besides, rapid targeted cell penetration through spontaneous 1,6-elimination reaction. On the other side,
and excellent stability of aptamers are also desired advan- succinic acid can be introduced to reduce the steric hindrance
tages16,17. As one of the most successful tumor-targeted apta- between NucA and the dipeptide linker, which facilitates not only
mers18,19, a nucleolin aptamer (AS1411, NucA) in phase II trial the enzymolysis of the linker by cathepsin B, and also the binding
(NCT00740441) for treating metastatic renal cell carcinoma has of NucA to nucleolin.
been confirmed with excellent tumor-targeting property and
In this study, we synthesize the designed NucA-PTX which
exceptional safety20. NucA interacts with nucleolin protein, which shows both good water solubility and tumor-targeting property.
normally locates in the nucleus; however, also found expressing By utilizing a Fluorescein amidate (FAM) and Rhodamine B (Rh)
on the surface of cancer cells19. Hence, NucA could be a dual-labeled conjugate (FAM-NucA-PTX-Rd) with fluorescence
O
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= NucA
= CRO
Fig. 1 S y n t h e s i s o f a p t a m e r- p a c li t a xe l c o n j u ga t es . N o t e f o r r e a g e n t s a n d c o n d i t i o n s : ( i ) P A B OH , E E DQ , C H C l / C H O H ; ( i i ) P N P c h l o r o f o r ma t e , p yr i d in e ,
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N i t r o p h en yl , D M A P = 4 - D i m et h yl a m i n o p y r i d in e , B z = B e n z o y l, A c = A c e t y l , D M F = D i m e t h y l f o rm a m i d e , D IP E A = N, N′- D i i s o p r o py l e th y l a m i n e , T H F =
T e t ra h y d r o f ur a n , S u l f o - N HS = N- h y d r o xy s u lf o su c c i n i m i d e s o d i u m s a l t , E D C I = 1 - Et h y l - 3 - ( 3 - d i me t h y l l a m in o p r o p y l ) c a r bo d i i m i d e h y d r o c hl o r i d e , N uc A =
N u cl eo l i n a p t a m e r , C R O = C y to s i n e - ri ch o l i go n u c l e o t i de
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Wavelength (nm)
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Fig. 2 T h e c a t h e p s i n B - d e p e n d en t r e le a s e o f P T X i n F A M- N uc A -P T X - R h. a S c h e m a t i c d i a gr a m o f t h e u t i l i z a t i o n o f F R ET i n F A M - Nu c A- P T X - R h f or t r a c k in g
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c o m p o u n d s w e re a t a c o n c e n t r a t io n o f 2 μM . c F l u o r e s c e n ce e m i s s i o n s p e c t r a o f F A M - Nuc A - P T X - R h ( λe x = 4 7 0 n m ) i n t h e p r es e n ce o f c a t h e p s i n B u po n
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s ta n da r d d ev i a t i o n . n = 3 . e T h e i n t r a c e ll u l a r r e le a s e o f P T X - R h f ro m F A M - Nuc A - P T X - R h i n S K OV 3 c e l l s m o n i t o r e d b y flo w c y t o m e t r y . T h e r e l a t i v e m e d i an
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E r ro r b a r s i n d i c at e m e a n s t a n d a rd d e v ia t i o n . n = 3 , *P < 0 . 0 5
resonance energy transfer (FRET) existing between two fluor- Results
ophores, the stability of NucA-PTX in human serum and the Synthesis of aptamer-paclitaxel conjugates. Synthesis of NucA-
release of the conjugated PTX in tumor are verified. Moreover, PTX (7a, Fig. 1) commenced with 1 and PABOH in the presence
the PTX conjugation does not considerably affect the binding of EEDQ to provide 2. Then 3 was produced by treating 2 with
affinity between NucA and its target protein nucleolin, which is PNP chloroformate in the presence of pyridine. Transesterifica-
supported by both molecular dynamic simulation and isothermal tion of p-nitrophenoxy on 3 with PTX under the catalytic con-
titration calorimetry (ITC). The NucA modification is shown to dition of DMAP afforded 4. The Fmoc protecting group in 4 was
facilitate the uptake of the conjugated PTX in ovarian cancer cells quickly removed by piperidine in DMF, giving rise to 5. Without
mainly by macropinocytosis in a nucleolin expression-dependent further purification, the amino group in 5 was reacted with
manner, leading to higher cytotoxicity of the conjugated PTX to succinic anhydride in the presence of DIPEA, providing 6 with a
ovarian cancer cells compared to normal cells. The in vivo data free carboxyl group. 6 was then conjugated with NucA in the
collected from a human xenograft model of ovarian cancer presence of Sulfo-NHS and EDCI to afford NucA-PTX (7a).
demonstrated that the NucA modification facilitates the selective CRO-PTX (7b) was prepared as described above by using a
accumulation of the conjugated PTX in ovarian tumor tissue, and control aptamer CRO (C-rich oligonucleotide) for conjugation at
subsequently resulting in notably improved antitumor activity the last step (Fig. 1). The structures of key intermediates were
1
1
and reduced toxicity.
confirmed by H-NMR, 13C-NMR, 2D [1H, H] COSY spectro-
scopy and high-resolution mass spectrometry (Supplementary
Figs. 1−4). NucA-PTX was characterized by HPLC, mass
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a
b
c
Time (min)
Time (min)
0
10
20
30
40
0
10
20
30
40
0.05
0.05
0.00
NucA-PTX-Rh
CRO-PTX-Rh
NucA-PTX-Rh
CRO-PTX-Rh
0.00
–0.05
–0.10
–0.15
–0.20
–0.25
3000
2000
1000
0
3000
2000
1000
0
–0.05
–0.10
–0.15
–0.20
0.00
*
*
*
*
*
*
–0.30
*
0.00
*
*
*
–5.00
–10.00
–15.00
–20.00
–5.00
–10.00
–15.00
–20.00
0
0
1
2
3
4
5
6
60
120 250 500
1000
Time (h)
Concentration (nM)
NucA
Kd = 151 24 nM
NucA-PTX
Kd = 267 43 nM
0.0 0.5 1.0 1.5 2.0
Molar ratio
0.0 0.5 1.0 1.5 2.0
Molar ratio
d
e
100
80
60
40
20
0
100
80
60
40
20
0
100
100
80
60
40
20
0
L02
L02
Unstained
NucA Pre
CRO Pre
No Pre
L02
OVCAR3
SKOV3
OVCAR3
SKOV3
OVCAR3
SKOV3
80
60
40
20
0
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
FITC-A
PE-A
PE-A
PE-A
Surface nucleolin
NucA-PTX-Rh
CRO-PTX-Rh
NucA-PTX-Rh
Fig. 3 T h e e f f ec t o f N u cA m o d ific a t i o n o n t h e u p t a k e o f t h e c o n j u g a t ed P T X- R h . a T h e i n t e r a c ti o n o f n u c l e o l i n w i t h N u c A a n d N u c A - P T X, r e s p e c t iv el y,
d e t e ct e d b y i s o th er m al t i t r a t i o n c a l o r i m et r y ( I T C ) . N u c A o r N u c A - P T X w a s i n j e ct e d i n t o n u c l e o l i n a t a 1 0 : 1 r a t i o i n 1 9 p o r t i o n s ( e a c h p o r t i o n 2 μL ) a t 2 5 ° C .
b T he t i m e - d ep e n de n t c e l lu l a r u p t a k e o f t h e c o n j u ga t ed P T X -R h i n S K OV 3 c e l l s e x a m i n e d b y flo w c y t o m e t r y . S K OV 3 c e l l s w e r e i n c u b a te d w it h e i t h e r
N u cA - P T X- R h o r C R O - P T X -R h a t a c o n c e n t ra t i o n o f 5 0 0 n M f o r 1 t o 6 h . E r r o r b a r s i n d i c a t e m e a n s t a n d a r d d ev i a t i o n . n = 5 , *P < 0 .0 5 . c T he
c o nc e n tra t i o n - d ep en d en t c e l l u l a r u p t a k e o f t h e c o n j u ga t ed P T X - R h i n S K OV 3 c e l l s e x a m i n e d b y flo w c y t o m e t r y . S K OV 3 c e l l s w e r e i n c u b a t e d w i t h e i t h e r
N u cA - P T X- R h o r C R O - P T X -R h a t a r a n ge o f c o n c e n t r a t io n s f o r 2 h . E r r o r b a r s i n d i c a t e m e a n s t a n d a r d d ev i a t i o n . n = 5 , *P < 0 .0 5 . d T h e n u c l e o li n
e x p r e s s i o n o n c e l l s u r f a c e , a n d t h e u p t a k e o f N u c A - P T X -R h o r C R O- P T X- R h i n S K OV 3, O V C A R 3 a n d L 0 2 e v al u a t e d b y flo w c y t o m e t r y . T h e c e l ls w e r e
t re a t e d w i th N u cA - P T X- R h o r C R O - P TX -Rh a t a c o n c e n t ra t i o n o f 1 0 0 n M f o r 2 h . T h e n u c l e o l i n e x p r e s si o n w a s d e t e c te d u s i n g A l e x a 4 8 8 - l a be l ed a nt i -
n u cl e o l i n a n t i b o d y s h o w n i n t h e F I TC c h a n n el , a n d t h e r h o d a m i n e l e v e l o f t h e c o n j u g a t e s w a s s h o w n i n t h e P E c h a n n e l . e T h e c e l l u l a r u p t a k e o f N uc A -P T X-
R h i n S KO V 3 p r e - i n c u b a ted w it h e i th er N u c A ( N u c A P r e ) o r C R O ( C R O P r e ) a n a l y z e d b y flo w c y t o m e t r y . T h e c e l l s w e r e p r et r e a t e d w i t h 2 5 0 n M N uc A o r
C R O f o r 2 h b e f o r e i n c u b a t e d w it h 1 00 n M N u c A - P T X- R h f o r 2 h . T h e r h o d a m i n e l ev e l o f N u c A - P TX - R h w a s s h o w n i n t h e P E c h a n n e l
spectrometry and 1H-NMR, 2D [1H, 1H] COSY, 2D [1H, 1H] FAM-NucA-PTX-Rh was designed according to the fluorescence
NOESY spectroscopy (Supplementary Figs. 5, 6). The structure of resonance energy transfer (FRET) principle (Fig. 2a). Fluorescein
CRO-PTX was confirmed by HPLC and mass spectrometry amidate (FAM) was linked to the 5′ end of NucA as the donor
(Supplementary Fig. 7). To synthesize PTX-Rh (Supplementary and rhodamine B (Rh) was linked to the 7-hydroxyl group of
Fig. 17), the 2′-hydroxyl group on PTX was protected by TBS in PTX as the acceptor. When the dipeptide linker stays intact, the
the presence of TBSCl and imidazole to afford 8 in good yield. fluorescence of both fluorophores in FAM-NucA-PTX-Rh
Treated with Rhodamine B, 8 was converted into 9 upon for- remained stable. Whereas when the linker is hydrolyzed in the
mation of ester group in the presence of EDCI and DMAP. The presence of the specific protease—cathepsin B, FRET between
free 2′-hydroxyl group of 10 was obtained after deprotection of FAM and rhodamine B is interrupted, appeared as the decrease of
TBS group using hydrogen fluoride-pyridine. Compounds 11, 12, Rh intensity and the increase of FAM intensity. With an excita-
13, FAM-NucA-PTX-Rh (14a), NucA-PTX-Rh (14b), and CRO- tion wavelength at 470 nm, FRET was noted in the fluorescence
PTX-Rh (14c) were prepared with the same method as described emission spectrum of FAM-NucA-PTX-Rh compared to the
above. The structures of key intermediates were confirmed by 1H- individual spectra of FAM-NucA and PTX-Rh (Fig. 2b). Whereas,
NMR, 13C-NMR spectroscopy and high-resolution mass spec- in the presence of cathepsin B, increased emission at 520 nm and
trometry, and the conjugates were confirmed by HPLC and mass decreased emission at 590 nm upon time were observed in the
spectrometry (Supplementary Figs. 8−15). The water solubility of spectra of FAM-NucA-PTX-Rh (Fig. 2c). Accordingly, the con-
NucA-PTX was significant improved compared to that of free centration of intact FAM-NucA-PTX-Rh could be presented by
PTX (Supplementary Fig. 18).
the fluorescence intensity ratio at 590 nm and 520 nm (RFI λ590/
λ520), while the concentration of free PTX-Rh could be inversely
reflected by the fluorescence intensity at 590 nm (FI λ590). When
FAM-NucA-PTX-Rh was incubated in human serum at 37 °C for
48 h, both RFI λ590/λ520 and FI λ590 remained the same upon time
(Fig. 2d), suggesting FAM-NucA-PTX-Rh existed as the intact
The stability of NucA-PTX and the release of paclitaxel. To
verify the stability of NucA-PTX in circulation and the intracel-
lular release of PTX, a dual fluorophore-labeled conjugate—
4
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A R T I C L E
a
b
Alexa488-
labeled marker
*
Rhodamine
Hoechst 33342
Overlay
Light
Clathrin pathway
Caveolae pathway
Macropinocytosis
*
1.0
0.5
0.0
Transferrin
NS
Choleratoxin
c
d
150
NS
150
100
50
*
Dextran
100
50
0
*
NS
0
f
LysoTracker
green DND-26
Rhodamine
Hoechst 33342
Overlay
Light
EIPA (μM)
Filipin (μM)
g
e
150
100
50
1.0
0.5
0.0
NS
*
0
Chlorpromazine (μM)
Fig. 4 T h e e f f ec t o f N u cA m o d ific a t i o n o n c e l l u l a r i n t e r n a l i z a t i on a n d t r a ffic ki n g . a R e p r e s e n t a t iv e i m a ge s s h o w i n g t h e c o - l o ca l i z a t io n o f t h e c o nj u g at e d
P TX - R h ( r ed ) i n N u cA - P TX - R h w it h A l e xa F l u o r 4 8 8 - la b l e d e n d o c y t i c m a r ke r s ( t r a n s f e r r i n , c h o l e r a t o x i n a n d d e x t r a n ; g r e e n ) b y c o n f o ca l m i c r o s c o py. T he
n u cl e i w e re c o u n t er s t a in e d w it h H o e c h s t 3 3 , 3 4 2 ( b lu e ) . S c a le b a r , 1 0 μm . b P e a r s o n’s c o r r e l a t i o n c o e ffic i e n t a n a l y s i s o f t h e c o - l o ca l i z a t io n b e t w e e n N uc A -
P TX - R h a n d e n d o cy t o si s m a r k e r s i n S K O V 3 c e l l s b y I m a ge J C o l o c2 . E r r o r b a r s i n d i c a t e m e a n s t a n d a r d d ev i a t i o n . n = 5 p e r g r o u p . E a c h r e p l i c a t e i s f r o m
o n e b i o l o gi c a l e xp er i m en t , q u a n t ifie d w it h 1 0 i n d e p e n d e nt fie l d s o f v i e w . * P < 0 .0 5 ; N S : n o t s i gn ific a n t d i f f e r e n c e . c- e T h e c h e m i c a l i n h i b i t i on o f c e ll u la r
u p ta k e f o r t h e c o n j u g a ted P T X - R h i n C R O - P TX -Rh i n S K O V 3 c e l l s . T h e r e l a t i v e flu o r e s ce n ce o f r h o d a m i ne w a s q u a n t ifie d a f t e r t h e t r e a tm en t w i t h
i n hi b i t o r s o f t h r e e e n d o c yt i c p a t h wa y s b y flo w c y t o m e t ry : E I P A ( m a c r o p i n oc y t o s i s , c) , F i l i p i n ( c a v eo l a e p a t h w a y , d) , a n d C h l o r pr o m a z i n e ( c l a th r i n p at h w ay ,
e) . E r ro r b a r s i n d i c at e m e a n s t a n d a r d d e v ia t i o n . n = 3 . * P < 0 . 05 ; N S : n o t s i gn ific a n t d i f f e r e n ce . f R e p r e s e n t a t i ve i m a ge s s h o w i n g t h e c o - l o ca l i za t io n o f t h e
c o nj ug a ted P T X - R h ( r ed ) w it h a l y s o s o m a l m a r k e r ( L y s o T ra c ke r G r ee n D N D- 2 6 ; g r e e n ) b y c o n f o c a l m i c r o s c o p y. T h e n u c l e i w e r e c o u n t e r st a in e d w i t h
H o e c h s t 3 3 , 3 4 2 ( b l u e ). S c a le b a r , 1 0 μm . g P e a r s o n’s c o r re l a t io n c o e ffic i e n t a n a l y s i s o f t h e c o - l o ca l i z a t io n b e t w e e n t h e c o n j u g a t e s a n d l y s o t r a c k e r i n
S K O V 3 c e l l s b y I m a g e J C o l o c 2 . T h e d a t a w er e p r e s e n t ed a s t h e m e a n s s t a n d a r d d ev i a t i o n . n = 5 p e r g r o u p . E a c h r e p l i c a t e i s f r o m o n e b i ol og i ca l
e x p e r i m en t , q u a n t ifie d w i t h 1 0 i n d e p e n d e nt fie l d s o f v i ew . *P < 0 . 0 5
form. In contrast, in a buffered solution of cathepsin B (pH 5.0), property of NucA-PTX. To investigate whether the conjugated
both RFI λ590/λ520 and FI λ590 decreased upon time, while those in PTX affects the interaction between NucA and its target protein
buffer without cathepsin B remained unchanged (Fig. 2d), nucleolin, their binding affinity before and after PTX conjugation
implying the successful cleavage of the linker and the release of was studied by both molecular dynamic simulation and iso-
free PTX-Rh. The stability and dissociation of NucA-PTX were thermal titration calorimetry (ITC). Nucleolin of eukaryotic cell
confirmed by HPLC analysis with consistent results (Supple- comprises several domains, including an acidic N-terminal
mentary Fig. 19). To further probe the intracellular release of domain, four RNA binding domains (RBD) and a C-terminal
PTX-Rh from FAM-NucA-PTX-Rh in ovarian cancer cells, RGG-rich “tail” (Supplementary Fig. 20a). In the simulated
SKOV3 cells were treated with FAM-NucA-PTX-Rh and the model, we found that both NucA and NucA-PTX conjugate
fluorescence of FAM and Rh of the cells at 1, 3, and 6 h was interacted with RGG-rich domain of the C-terminal of nucleolin,
analyzed by flow cytometry. In consistence of the results obtained which was consistent with previously reported26. Structurally, the
extracellularly (Fig. 2d), the relative fluorescence intensity RFI conjugated PTX segment laid at the end of the chain in the
(Rh/FAM) kept decreasing upon time (Fig. 2e), implying the simulated model (Supplementary Fig. 20b). Quantitatively, no
successful intracellular release of PTX-Rh from NucA-PTX.
considerable difference was observed in the calculated binding
free energy between NucA and NucA-PTX conjugate when
docking to nucleolin. Moreover, the binding of NucA or NucA-
PTX with Nucleolin was examined using isothermal titration
The effect of PTX on NucA and nucleolin interaction.
Nucleolin binding is essential for achieving the tumor-targeting
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a
SKOV3
OVCAR3
L02
1.5
1.0
0.5
1.5
1.5
NucA
CRO
PTX
CRO-PTX
NucA-PTX
1.0
1.0
0.5
0.5
0
20 40
200 400
0
20 40
200 400
0
20 40
200 400
Concentration (nM)
Concentration (nM)
Concentration (nM)
b
Untreated
CRO-PTX
NucA-PTX
PTX
80
60
40
20
0
80
60
40
20
0
800
600
400
200
Dip G1
60
40
20
0
Dip G2
Dip S
0
0
50 100 150 200 250
Channels (PI-A)
0
50 100 150 200 250
Channels (PI-A)
0
50 100 150 200 250
Channels (PI-A)
0
50 100 150 200 250
Channels (PI-A)
G0/G1: 68.6 1.2
S: 17.5 0.6
G0/G1: 40.6 0.9
S: 19.8 0.3
G0/G1: 24.4 0.7
S: 21.5 0.6
G0/G1: 23.1 1.0
S: 28.8 0.5
G2/M: 13.9 0.4
G2/M: 39.6 0.7
G2/M: 54.1 1.3
G2/M: 48.1 0.8
Dip G1
Dip G2
Dip S
120
90
60
30
0
80
60
40
20
0
800
600
400
200
60
40
20
0
0
0
50 100 150 200 250
Channels (PI-A)
0
50 100 150 200 250
Channels (PI-A)
0
50 100 150 200 250
Channels (PI-A)
0
50 100 150 200 250
Channels (PI-A)
G0/G1: 66.4 1.1
S: 14.9 0.7
G0/G1: 45.7 1.2
S: 21.0 0.8
G0/G1: 20.8 0.6
S: 36.9 0.9
G0/G1: 14.0 0.2
S: 45.1 1.8
G2/M: 18.7 0.5
G2/M: 33.4 1.4
G2/M: 42.3 2.1
G2/M: 40.9 1.4
Fig. 5 T h e e f f ec t o f N u c A m o d ific a t i o n o n t h e c y t o t o x i c i ty o f t h e c o n j u g a t ed P T X. a T h e c e l l v i a b i l i t i es o f S K OV 3 , O V C A R 3 , a n d L 0 2 a f t e r 2 4 h t r e a t m e n t o f
N u cA , C R O , P T X , C R O - P T X , o r N u c A - P T X e v al u a t e d b y C C K 8 a s s a y . T h e c o n c e n t ra t i o n r a n g e o f e a c h c o m p o u n d w a s f r o m 1 5 . 6 t o 5 0 0 n M . T h e d at a w e r e
p re s e n t ed a s t h e m e a n s s t a n d a r d d e v ia t i o n . n = 3 . b T h e c e l l c y c l e d i s t r i b u t i on o f S K OV 3 a n d O V C A R 3 c e l l s a f t e r 4 8 h t r e a tm e n t o f C R O - P T X , N uc A -
P TX , o r P T X a n a l y ze d b y flo w c y t o m e t r y
calorimetry. By titrating NucA or NucA-PTX into the nucleolin uptake of NucA-PTX-Rh was observed in SKOV3 cells with the
protein solution with a 10:1 ratio of concentration, results top nucleolin expression, and the lowest uptake in L02 cells with
demonstrated that the PTX conjugation did not significantly the least nucleolin expression (Fig. 3d). In contrast, the uptake of
affect the interaction between NucA and nucleolin (Fig. 3a), with CRO-PTX-Rh did not vary significantly among the three cell lines
the Kd value 151 24 nM before conjugation and 267 43 nM (Fig. 3d), implying the specificity of NucA targeting. When the
after conjugation, respectively.
nucleolin on SKOV3 cells were partially blocked by preincubation
with NucA, the uptake of NucA-PTX showed a significant
decrease (Fig. 3e). While the preincubation with CRO aptamer
resulted in a less blocking effect and consequently a smaller
decrease of NucA-PTX-Rh uptake (Fig. 3e), further suggesting the
uptake of NucA-PTX-Rh in cells was correlated with available
nucleolin on the cell surface. It is noteworthy that there was
considerable amount of uptake for CRO-PTX-Rh in SKOV3 cells;
however, its indiscriminate uptake in other cell lines with dif-
ferent nucleolin surface expressions suggested that the uptake of
CRO-PTX-Rh was non-specific and nucleolin-independent
(Fig. 3b–d).
The effect of NucA modification on cellular uptake. The uptake
of NucA-PTX-Rh or CRO-PTX-Rh in SKOV3 cells was examined
by detecting the rhodamine fluorescence intensities in SKOV3
cells using flow cytometry. The uptake of NucA-PTX-Rh in
SKOV3 cells was shown to increase upon time and concentration,
which was significantly higher than that of its counterpart CRO-
PTX-Rh (Fig. 3b, c). To examine whether the uptake of the
conjugated PTX-Rh was nucleolin-dependent, the uptake of
NucA-PTX-Rh in three cell lines: two human ovarian cancer cell
lines—SKOV3 and OVCAR3, and one human normal liver cell
line—L02 were examined in this study. Different expression levels
of nucleolin on the cell surface were observed for the three cell The effect of NucA modification on cellular internalization. To
lines, with L02 the lowest and SKOV3 the highest (Fig. 3d). The explore whether the conjugated PTX was taken up by SKOV3
uptake of NucA-PTX-Rh in the three cell lines was found cells through endocytosis resulted from the NucA modification,
increasing along with the nucleolin expression. The highest SKOV3 cells was incubated with NucA-PTX-Rh or CRO-PTX-Rh
6
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A R T I C L E
a
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Rhodamine
Nucleolin
Merged
with DAPI
H&E
Fluorescence
(× 107)
2 h after injection
4 h after injection
Heart
Liver
3.0
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2.6
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Lung
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Kidney
Tumor
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2 h after injection
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4 h after injection
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Heart
Tumor
Tumor
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Spleen
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Fig. 6 T h e e f f ec t o f N u c A m o d ific a t i o n o n t h e d i s t r i b u t i on o f t h e c o n j u g a t e d P T X- R h . a T h e d i s t r i b u t i on o f t h e c o n j u g a t ed P T X- R h i n t u m o r a nd m a jo r
v i s c e ra ( h e a r t, l i v er , s p l ee n , l u n g , a n d k i d n ey ) a t o r g a n l ev e l 2 h a n d 4 h a f t e r i n t r a v e n o u s i n j e ct i o n o f N u c A - P TX - R h o r C R O - P T X - R h v i s u a l i z e d b y
b i op h o t o ni c i m ag i n g. b P e r c e n ta ge o f i n j ec t e d d o s e i n t u m o r a n d m a j o r v i s c e r a 2 h a n d 4 h a f t e r i n t r a v e n o u s i n j e ct i o n o f N u c A - P TX - R h o r C R O -P T X - R h
e v al u a t e d b y m e a su r i n g t h e flu o r e s c e n t i n t e n s i t y o f r h o d a m i n e . T h e d a t a w e r e p r es e n t ed a s t h e m e a n s s t a n d a r d d e v ia t i o n . n = 6 , N S : n o t s i gn ific a n t , * P <
0 . 0 5 . c T h e r e p r e se nt a ti v e flu o r e s c e n t m i c r o g r a p h s o f t h e d i s t r i b u t i o n o f t h e c o n j u g a t ed P T X- R h i n t u m o r a t t i s s u e l ev e l 4 h a f t e r i n t r a v e n o u s i nj e c t io n o f
N u cA - P T X- R h o r C R O - P T X -R h e x a m i n e d b y c o n f o c a l m i c r o s c o p y. N u c A - P TX - R h a n d C R O - P T X -R h w e r e i n d i c a t e d b y r h o d a m i ne ( r e d ) .
I m m u n oflu o r es c e n ce s t a i n in g w a s p e r f o r m e d b y A l e xa 4 88 - l a b e l e d a n t i - n u c le o l i n a n t i b o d y ( g r e e n ) . M e r g e d i m a ge s w i t h D A P I s t a i n i n g s h o w c o -
l o c a li z a t i o n o f t h e c o n j u g a t es w it h n u c l eo l i n p o s i ti v e c e l l s . H &E s t a i n i n g i m a ge s w e r e f r o m t h e s a m e c r y o s e ct i o n s . S c a l e b a r s , 8 0 μm . d T h e P e a r s o n’s
c o e ffic i e n t a n a l ys i s f o r t h e d i s t r i b u t i on o f t h e c o n j u ga te d P T X -R h ( r e d ) w i t h n u c l e o l i n - e x p r e ss i n g t u m o r c e l l s ( g r e e n ) . e R e p r e s e n ta t i v e flu o r e s c e n t
m i c ro g r ap h s o f t h e d i s tr i b u t i on o f t h e c o n j u ga t ed P T X -R h i n k i d n e y a n d l i v e r a t t i s s u e l ev e l 4 h a f t e r i n t r a v e n o u s i n j e ct i o n o f N u c A - P T X- R h o r C R O - P T X - R h
e x a m i n e d b y c o n f o c a l m i c r o s c o p y . N u c A - P T X -R h a n d C R O - P TX - R h w e r e i n d i c a t e d b y r h o d a m i n e ( r e d ) . M e r g e d i m a ge s w i t h D A P I s t a i n i n g s h ow
r ho d a m i ne p o s i t i v e c e l l s . H &E s t a i n in g i m a g e s w er e f ro m t h e s a m e c r y o s e ct i o ns . S c a l e b a r s , 8 0 μm . f T h e p e r c e n t a g e o f r h o d a m i ne p o s i t i v e c e ll s p e r
m i c ro s co p ic v i e w . N o te : C R O- P T X - R h , t h e x e n o gr a f te d m i c e t r e a t e d w i t h C R O - P T X - Rh . N u c A - P T X-R h , t h e x e n o gr a f t e d m i c e t r e a te d w i t h N uc A -P T X - Rh .
T h e d a t a w e re p r e s e n t e d a s t h e m e a n s s t a n d a r d d ev i a t i o n . n = 4 , * P < 0 .0 5 v s . C R O - P T X - R h g r o u p
in the presence of Alexa Fluor 488-labeled endocytic markers and was also confirmed by the reduced uptake of NucA-PTX-Rh
analyzed by confocal microscopy. The quantification of the co- when cells were pretreated with EIPA (an inhibitor of macro-
localization of the conjugated PTX and endocytic markers was pinocytosis) at various concentrations (Fig. 4c), while the uptake
carried out using Pearson’s correlation coefficient analysis by was not substantially affected by the inhibition of the other two
ImageJ. A remarkable co-localization of NucA-PTX-Rh with pathways (Fig. 4d, e). The distribution of CRO-PTX-Rh in
Dextran (a marker for macropinocytosis) in SKOV3 cells was SKOV3 cells was more dispersed compared to that of NucA-
observed (Fig. 4a), with Pearson’s correlation coefficient ~0.8 PTX-Rh, and no significant co-localization of CRO-PTX-Rh with
(Fig. 4b). In contrast, a significant lower co-localization of NucA- any of the three markers was observed (Supplementary Fig. 22a,
PTX-Rh with transferrin (a marker for clathrin-mediated endo- b). The inhibition of three endocytic pathways did not dramati-
cytosis) or choleratoxin (a marker for caveolae-mediated endo- cally affect the uptake of CRO-PTX-Rh (Supplementary Fig. 22c),
cytosis) were detected (Fig. 4a, b), indicating that NucA-PTX-Rh implying the non-specific uptake of CRO-PTX-Rh in SKOV3
was mainly taken up by SKOV3 cells via macropinocytosis. This cells. The cellular trafficking of NucA-PTX-Rh in SKOV3 cells
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12 16 20 24 28
Date (days)
c
PBS
PTX
CRO
NucA
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NucA-PTX
d
PBS
PTX
CRO
NucA
CRO-PTX
NucA-PTX
Fig. 7 T h e e f f ec t o f N u cA m o d ific a t i o n o n t h e a n t it u mo r a c t i vi t y o f t h e c o n j u g a t ed P T X. a M a c r o s co pi c v i e w s o f t h e x e n o gr a f t e d t u m o r a f t e r 4 w e e k s o f
d i f f e re n t t r ea t m e n t s f r o m t h e g ro u p s i n d i c a t e d. S c a l e b a r , 1 c m . b A n a l y s i s o f t u m o r v o l u m e a f t e r 4 w e e k s o f d i f f e r e n t t r e a tm e n ts i n d i c a t e d a t a d os i ng
f r e q u e n cy o f t w i c e a w e ek v i a i n t r a v e n o u s i n j ec t i o n . T h e d o s i n g p o i n t s w e r e i n d i c a t e d w i t h b l u e a r r o w s . T h e d a t a w e r e p r es e n t ed a s t h e m e a n s s t a nd ar d
d ev i a t i o n . n = 6 , *P < 0 . 0 5 . c K i - 6 7 i m m u n o h is t o ch e m i c a l s t a in i n g a n a l y s i s o f o v a r i a n t u m o r s e c t i o n s f r o m t h e g r o u p s i n d i c a t e d . K i - 6 7 - p o si t i v e s t a in i ng w a s
i n di c a t e d b y b r o w n a n d n u c l ei w er e s t a in e d b y b l u e . S c a l e b a r s , 1 0 0 μm . d H & E s t a i n i n g a n a l y s i s o f o v a r i a n t u m o r s e c t i o n s f r o m e a c h g r o u p . A r r o w s
i n di c a t e c e l l a p o p t o s i s a n d n e c ro s i s i n t h e t u m o r s e c t io n s . S c a l e b a r s , 1 0 0 μm . N o t e : P B S , t h e x e n o gr a ft e d m i c e t r e a te d w i t h P B S . P T X, t h e x e n o g r af t e d m i c e
t re a t e d w i th p a c l i t a xe l . C R O , t h e x e n o gr a f te d m i c e t r e a t e d w it h C R O a p t a m e r . N u c A , t h e x e n o gr a f t e d m i c e t r e a te d w i t h n u c l e o l i n a p t a m er . C R O - P T X, t h e
x e n o g r a f t ed m i ce t r ea t e d w it h C R O - P TX . N u c A - P T X, t h e x e n o g r a f t e d m i c e t r e a te d w i t h N u c A - P T X
after macropinocytosis was then presented by the predominant cathepsin B and efficiently release PTX. In addition, the co-
co-localization of NucA-PTX-Rh with lysotrackers in contrast to localization of NucA-PTX-Rh with endocytic markers in L02 cells
its CRO counterpart (Fig. 4f, g). The lysosome transportation for were also investigated. The lack of co-localization with any of the
NucA-PTX-Rh could facilitate the rupture of the cathepsin B- endocytic markers and the unaltered uptake by all endocytic
labile linker in the conjugate in the presence of lysosomal pathway inhibitors were also noted (Supplementary Fig. 23).
8
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D O I : 1 0 . 1 0 3 8 / s 4 1 4 6 7 - 0 1 7 - 0 1 5 6 5 - 6 w ww .n a t u r e . c o m / n a t u re c o m m u n i c a t i o n s
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Date (days)
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Fig. 8 T h e e f f ec t o f N u cA m o d ific a t i o n o n t h e t o x i c i t y o f t h e c o n j u g a t e d P T X. a B o d y w e i gh t c h a n g e s a f t e r 4 w e e k s o f d i f f e r e n t t r e a tm en t s i nd ic at e d i n
x e n o g r a f t ed m i ce . B o d y w ei g h t w a s n o r m a l iz ed t o e a c h b o d y w e i g h t b e f o r e t r e a tm e n t. T h e d o s i n g p o i n t s w e r e i n d i c a t e d w i t h b l u e a r r o w s . b T he w h it e
b l oo d c e l l c o u n t d e t e rm in e d b y a u t o m a t ed h e m a t o lo gy a n a l y z e r b e f o r e a n d a f t e r d i f f e r e n t t r e a tm en t s i n x e n o gr a f t e d m i c e . c N e u r o p h y si o l o g i c al a na ly s i s o f
c a ud a l N C V a n d d i g i t a l N C V i n t h e x e n o g ra f t e d m i c e 4 d a y s b e f o r e t h e e n d o f t h e d i f f e r e n t t r e a tm en t s i n d i c a t e d. d H i s t o l o g i c al a s s e s s m en t s o f t i s s ue s
u si n g H & E s t a i n i n g i n d i f f e r e n t t r e a t m e n t g ro u p s . A r r o w s i n H ea r t i m a ge s i n d i c a t e t h e m y o fib r i l l a r l o s s a n d a t r o p h y o f m y o c a r di a l c e l l s . A r r o w s i n L i v e r
i m a g e s i n d i ca te t h e h e p at i c c o r d s l o s s , s t e a to s i s a n d d i l a t a ti o n o f b l o o d s i n u s . A r r o w s i n S p l e e n i m a ge s i n d i c a t e t h e w h i t e p u l p a t r o p h y . A r r o w s i n L u n g
i m a g e s i n d i ca te t h e b r o ke n l u n g fib e r s . S c a l e b a r s , 1 00 μm . e S e m i - q u a n ti t a ti v e h i s t o l o g i c a l a n a l y s i s o f t h e H & E - s t a i n e d t i s s u e s e c t i o n s i n d i f f e r e n t
t re a t m e n t g r o u p s . N o te : P B S , t h e x e n o g raf t e d m i c e t r e a t e d w it h P B S . P T X, t h e x e n o gr a f t e d m i c e t r e a te d w i t h p a c l i t a x e l . C R O , t h e x e n o gr a ft e d m ic e t r e a t e d
w i t h C R O a p t a m e r . N u cA , t h e x e n o gr a f te d m i c e t r e a t e d w it h n u c l e o l i n a p t a m er . C R O - P T X , t h e x e n o gr a f t e d m i c e t r e a te d w i t h C R O - P T X . N u c A -P T X, t h e
x e n o g r a f t ed m i ce t r ea t e d w it h N u c A - P T X . T h e d a t a w er e p r e s e n t e d a s t h e m e a n s s t a n d ar d d ev i a t i o n . n = 6 , *P < 0 .0 5 v s . N u c A - P T X g r o u p
Hence, endocytosis of NucA-PTX-Rh in normal cells was unlikely that to cancer cell lines. NucA and CRO exhibited no consider-
involved and the uptake could be much less efficient than that in able cytotoxicity in all cell lines within the concentration range
cancerous cells.
examined (Fig. 5a, Supplementary Fig. 24). Furthermore, the
effect of NucA-PTX on cell cycle was also investigated. NucA-
PTX increased the fraction of cells in G2/M phase in the cell cycle
of SKOV3 and OVCAR3 as PTX did after 48 h treatment; how-
ever, for CRO-PTX the majority of cells were still in G0/G1 phase
(Fig. 5b), which is consistent with the cytotoxicity results.
The effect of NucA modification on the cytotoxicity. To
examine whether the NucA modification affects the antitumor
activity of the conjugated PTX in ovarian cancer cells, the 24 h
and 72 h cytotoxicity of NucA-PTX, CRO-PTX, PTX, NucA, and
CRO on two human ovarian cancer cell lines—SKOV3 and
OVCAR3, and a normal liver cell line—L02, was examined using The effect of NucA modification on in vivo distribution. To
Cell Counting Kit-8 (CCK8). The IC50 values at 72 h is shown in examine the tumor-targeting property of the NucA conjugated
Supplementary Table 1. For two ovarian cancer cell lines, NucA- PTX-Rh, the tissue distribution of PTX-Rh was evaluated in a
PTX exhibited comparable cytotoxicity as PTX but higher cyto- xenografted mice model of human ovarian cancer by biophotonic
toxicity than CRO-PTX (Fig. 5a, Supplementary Fig. 24, Sup- imaging. It was shown that the Rh fluorescence intensities in
plementary Table 1). When it comes to the normal liver cell line ovarian tumor tissues collected from the NucA-PTX-Rh treated
L02, though NucA-PTX had a slightly inhibitory effect on cell mice were significantly higher than those from the CRO-PTX-Rh
proliferation, it was still significantly less cytotoxic compared to treated mice at 2 h and 4 h after intravenous injection of NucA-
CRO-PTX or PTX (Fig. 5a, Supplementary Fig. 24, Supplemen- PTX-Rh and CRO-PTX-Rh, respectively (Fig. 6a). Moreover, the
tary Table 1). The cytotoxicity of NucA-PTX to L02 was less than Rh fluorescence intensities in livers and kidneys from the NucA-
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PTX-Rh treated mice were remarkably lower when compared to active cell proliferation. In contrast, the NucA-PTX treated group
those from the CRO-PTX-Rh treated mice at 4 h after injection showed a lower Ki-67-positive cells than PTX treated groups and
(Fig. 6a). Interestingly, the fluorescence signal was hardly detected the other control groups (Fig. 7c), suggesting a higher inhibition
in hearts, spleens and lungs in both groups (Fig. 6a). The per- effect on tumor growth. For H&E staining, the nuclei were
centage of injected dose of NucA-PTX-Rh and CRO-PTX-Rh in stained with hematoxylin in blue and the cytoplasm was stained
tumor and other major viscera at 2 h and 4 h were also analyzed by eosin in pink. The PBS-treated and both aptamer-treated
to quantitatively examine the effect of NucA on the distribution groups showed rhombic or polygonal cells with large spherical or
of the conjugated PTX-Rh. In consistency, the accumulation of spindle-shaped nuclei, which indicating the normal cells without
NucA-PTX-Rh in tumor was significantly higher than that CRO- apoptosis and necrosis (Fig. 7d).
PTX-Rh at both time points, while the clearance of NucA-PTX-
Rh in liver and kidney at 4 h was considerably faster (Fig. 6b).
The serum half-life of NucA-PTX-Rh was evaluated to be ~2.17 h The effect of NucA modification on in vivo toxicity. Con-
(Supplementary Fig. 25). To further confirm whether NucA could sidering the toxicities of paclitaxel (e.g., hematological toxicity
selectively deliver the conjugated PTX-Rh into tumor cells on and neurotoxicity) reported in clinical application, we further
which nucleolin was highly expressed, we examined the co- evaluate the toxicity of NucA-PTX in the xenografted mouse
localization of PTX-Rh with the nucleolin-expressing cells in the model. First, we measured the body weight of the treated mice
tumor cryosections from the xenografted mice injected with every 4 days during the treatments. PTX-treated group showed
NucA-PTX-Rh or CRO-PTX-Rh by immunofluorescent analysis obvious weight loss (about 85%) throughout the experiment,
with confocal imaging. Although there were numerous instances indicating the potential of systematic toxicity. In contrast, no
of nucleolin-expressing cells in the tumor cryosections of both remarkably body weight loss was observed in PBS-, CRO-, NucA-,
groups, more co-localization of Rh fluorescent signal with and NucA-PTX-treated groups, revealing a slightly low potential
nucleolin-expressing cells were found in the NucA-PTX-Rh- of systemic toxicity (Fig. 8a). Next, we investigated the myelo-
treated mice as compared to the CRO-PTX-Rh treated mice at 4 h suppression of NucA-PTX through white blood cell (WBC) count
after injection (Fig. 6c, d). In addition, there were lower percen- test. No obvious change of WBC count was observed in xeno-
tages of nephrocytes and hepatocytes with Rh fluorescent signal grafted mice after treatment with PBS and CRO, whereas PTX
(Rh+ cells) per microscopic view in the NucA-PTX-Rh-treated and CRO-PTX induced a significant decrease in the value of
mice as compared to the CRO-PTX-Rh treated mice at 4 h after WBC count. The value of WBC count in the NucA-PTX-treated
injection (Fig. 6e, f). Furthermore, we wonder whether the linker mice was also decreased compared with the value before
of NucA-PTX-Rh remained intact when the conjugate reached administration but to a much lesser extent than that in PTX-
the tumor site, which is crucial for the tumor-specific accumu- treated group (Fig. 8b). Furthermore, we evaluated the potential
lation of the conjugated PTX. After a single dose of NucA-PTX- neurotoxicity of NucA-PTX using an electromyography appara-
Rh and FAM-NucA-PTX-Rh, the fluorescence of rhoamine in tus. PTX and CRO-PTX induced a marked and significant
NucA-PTX-Rh treated mouse tumors (representing the con- reduction in caudal and digital nerve conduction velocities
centration of PTX-Rh in the conjugate) and the fluorescence of (NCVs) when compared to the PBS- and CRO-treated groups,
rhodamine and FAM in FAM-NucA-PTX-Rh treated mouse hinting the potential neurotoxicity of PTX. NucA-PTX also
tumors (FAM/Rh representing the intact conjugate illustrated in induced caudal and digital NCVs reduction compared to PBS
Fig. 2) were analyzed. The PTX-Rh gradually accumulated in control group, but the value of NCVs was higher than that in
tumor tissue in the first 2 h after injection, and started being CRO-PTX-treated group (Fig. 8c). Finally, The PTX-related
cleared from the tissue after 2 h (Supplementary Fig. 26a). toxicities to major organs were evaluated by histology analysis
Interestingly, the relative fluorescence intensity (RFI) Rh/FAM with H&E staining and serum biochemical assay, respectively. As
exhibited a plateau at the beginning, and then gradually expected, serious damages were observed in the H&E-stained
decreased after 2 h (Supplementary Fig. 26b). The initial plateau sections of heart, liver, spleen, and lung, respectively, in the
of RFI Rh/FAM could be explained by the continuous accumu- xenografted mice with PTX- and CRO-PTX treatment (Fig. 8d).
lation of intact conjugate at the tumor site. The subsequent For heart damage, myofibrillar loss and atrophy of myocardial
reduction of RFI Rh/FAM could possibly because when there was cells were found. For liver damage, hepatic cords loss, mild
no accumulation of new intact conjugate after 2 h, the release of steatosis, and dilatayion of blood sinus were observed. For lung
PTX-Rh from the conjugate in the tumor tissue could be injury, broken lung fibers were found. For spleen damage, the
observed.
atrophy of the white pulp was detected. However, the above
damages to these tissues was greatly reduced or absent in NucA-
PTX treated group when compared to those in PTX and CRO-
The effect of NucA modification on in vivo efficacy. The in vivo PTX treated groups. Consistently, the histological scores of liver,
antitumor effect of NucA-PTX was evaluated in a xenografted kidney, lung, heart, and spleen were all significantly lower in the
mice model of human ovarian cancer. Compared with PBS- NucA-PTX-treated mice as compared to the mice treated with
treated group (control group) and CRO-treated group (aptamer PTX or CRO-PTX (Fig. 8d). Interestingly, there is no visible
control group), the others groups exhibited significantly lower damage in kidneys from all the groups (Fig. 8d, e). Moreover,
tumor volumes, wherein the NucA-PTX treated group showed a serum biochemical assays showed that the levels of liver function
better inhibitory effect against tumor growth that PTX-treated enzymes, i.e., aspartate aminotransferase (AST) and alanine
group or nucleolin aptamer-treated group (Fig. 7a). Especially, aminotransferase (ALT), and cardiac function enzymes, i.e.,
the average tumor volume of the NucA-PTX treated group was creatine phosphokinase (CPK) and creatine kinase myocardial
significantly smaller than that treated with PTX and other groups bound (CK-MB) were all significantly lower in the NucA-PTX-
(P < 0.05) on day 28 (Fig. 7b). Then, Ki-67 antigen staining, a treated mice as compared to the mice treated with PTX or CRO-
commonly used cell proliferation marker, together with H&E PTX (Supplementary Table 2). Moreover, the serum cytokine
staining, were performed to further investigate the tumor sup- levels (TNF-α, INF-γ, IL-1β, IL-6, IL-10) of both nude mice
pression efficiency of NucA-PTX. For Ki-67 staining, we found (immune-incompetent) and Balb/c mice (immune-competent)
numerous instances of Ki-67-positive cells in tumor tissue from after NucA-PTX treatment was assessed and no notable change in
PBS-treated and aptamer-treated xenografted mice, indicating levels was observed in both species (Supplementary Fig. 27).
10
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Discussion
reduced toxicity of NucA-PTX in vivo was most likely attributed
To connect PTX with the tumor-targeting NucA, the active 2′- to the stability of the linker in circulation and the selective
hydroxyl group on PTX was the optimal site for substitution. accumulation in tumor tissues with high nucleolin expression led
However, it was reported that the introduction of other functional by NucA.
groups on this site resulted in the loss of antitumor activity for the
AS1411, the same sequence as NucA, has been reported with
obtained PTX derivatives25. Thus, the cathepsin B-labile dipep- tumor growth inhibition and utilized in phase II clinical trials20.
tide linker was utilized to facilitate the intracellular release of PTX However unexpectedly, no activity was observed for NucA either
from the conjugate21,24. In addition, spacers (PABC on PTX side in vitro or in vivo in our studies. This could be explained by the
and succinic acid on NucA side) were incorporated to the linker following possible reasons. For in vitro cytotoxicity study, the
for reducing the steric hindrance for cathepsin B. Previously concentration range that we examined was set based upon the
reported antibody-drug conjugate with the similar dipeptide activity of PTX, which was within nanomolar range34. While
linker was shown to release drug efficiently in tumor tissues27. By AS1411 was reported to be cytostasis and only inhibit the growth
monitoring the FRET change of the dual fluorophore-labeled of cancer cells in micromolar range35,36. In addition, the induc-
conjugate FAM-NucA-PTX-Rh, our in vitro results demonstrated tion of cell death by AS1411 occurs only after prolonged exposure
that the dipeptide linker exhibited remarkable stability in human to AS1411 and varies from different cell lines35. For in vivo anti-
serum, and the release of PTX was observed either upon addition cancer efficacy studies, normally a continuous infusion of AS1411
of cathepsin B or inside tumor cells (Fig. 2). In vivo fluorescence at a relative high dose for 4 or 7 days was chosen as the route of
analysis of the fluorophore-labeled conjugates in tumor tissue also administration for clinical studies35. However, the in vivo dose
suggested that the linker still remained intact when initially used in our study was also set according to PTX37–40, which was
reaching the tumor site, and then gradually ruptured and released lower and less frequent—twice a week. Besides, ovarian cancer
PTX once inside tumor cells (Supplementary Fig. 26).
was probably not among the most sensitive cancer types to
The cellular uptake of NucA-PTX-Rh was shown in a AS1411 compared to acute myeloid leukemia, or metastatic renal
nucleolin-dependent manner and significantly higher in cancer cell carcinoma, which were chosen as indications of AS1411 for
cells, while the uptake of CRO-PTX-Rh was moderate and did not phase II studies20,35. Thus, it is reasonable that we did not observe
exhibited notable difference in cell lines with various surface anti-cancer activities for NucA in our studies.
nucleolin expression levels, suggesting that the interaction
Other investigators have reported utilizing the nucleolin
between NucA and nucleolin played a crucial role in the entrance aptamer as a targeting ligand on nanoparticles for PTX
of the conjugated PTX into cancer cells. NucA-PTX-Rh was delivery37–40. Guo et al.37 developed AS1411-functionalized PEG-
mainly taken up by cancer cells through macropinocytosis, which PLGA nanoparticles and enhanced the accumulation of PTX in
is consistent with the findings previously reported that macro- glioma. AS1411 modified pH-sensitive micelles38 and AS1411
pinocytosis was the endocytic pathway of AS141128. In contrast, modified HSA protein nanoparticles39 were also investigated for
the endocytosis of CRO-PTX-Rh in cancer cells and NucA-PTX- tumor targeted delivery of PTX. However the inhomogeneity,
Rh in normal cells were relatively non-specific and no clear instability, immunogenicity, and potential toxicity of those
pathways was noted. As macropinosomes would finally fused into nanoparticles or their lipid/polymer materials remain con-
lysosomes29, the co-localization of NucA-PTX-Rh and lysotracker cerns41,42. By contrast, our NucA-PTX as a single macromolecule
was also observed in this study. Lysosomes are the intracellular with direct connection of the nucleolin aptamer with paclitaxel,
sites where cathepsin B is most abundant and active at its optimal exhibits great improvements in terms of these properties. In
pH (4.5−5.0)22. The localization of NucA-PTX-Rh in lysosomes addition to excellent stability and homogeneity, our studies also
suggests that the intracellular environment would be suitable for revealed the lack of immunogenicity and toxicity for NucA-PTX,
the sufficient cleavage of the cathepsin B-labile linker in cancer which represents a step further toward the clinical translation for
cells.
aptamers. As alternatives of ADCs, this work establishes an
Compared to its CRO counterpart, NucA-PTX exhibited innovative technology platform for ApDC development. More-
higher cytotoxicity in cancer cells but lower cytotoxicity in nor- over, by altering aptamers that target different cancer cells (e.g.,
mal cells, which was consistent with the uptake results. It was Her2 positive breast cancer cells, triple negative breast cancer
notable that CRO-PTX showed a certain amount of uptake and a cells, non-small cell lung cancer cells, osteosarcoma cells, and so
moderate cytotoxicity in cancer cells in vitro, in the meanwhile on), the personalized paclitaxel derivatives could be developed for
NucA-PTX had a low level of uptake and a slight cytotoxicity in the patients with the according cancer types. Importantly, it
normal cells at high concentrations in vitro. This could possibly provides an efficient approach to the next-generation of smart
because during the in vitro incubation, there were non-specific antitumor drug discovery toward precision medicine, i.e., perso-
interactions between aptamers and cell surfaces regardless of nalized aptamer-drug conjugates.
aptamer sequence or nucleolin expression level, and the selectivity
of NucA to cancer cells may stand out in vivo when both tumor
Methods
and normal cells were presented. Accordingly, in the human
ovarian cancer xenografted mouse model, the NucA modification
facilitated the accumulation of the conjugated PTX in tumor
tissue rather than major organs, subsequently resulting in
improved antitumor efficacy—smaller tumor size and less active
proliferation in tumor tissue than CRO-PTX treated mice.
Emerging evidence showed that PTX chemotherapy induces
severe hematologic toxicities (neutropenia and leukopenia) and
organ damages30,31. Neurotoxicity of PTX is also a clinical
challenge, and cancer patients receiving PTX treatment may
develop peripheral neuropathies32,33. In our studies, no sig-
nificant WBC toxicity, neurotoxicity and major organ damage
were observed in mice treated with NucA-PTX compared to that
Cell culture. Human ovarian cancer cell lines SKOV3 (ATCC HTB-77) and
OVCAR3 (ATCC HTB-161) were purchased from American Type Culture Col-
lection (ATCC, USA). Human normal liver cell line L02 was purchased from China
Center for Type Culture Collection (CCTCC, China). The cells were maintained in
ATCC-formulated DMEM (for SKOV3 and OVCAR3) or RPMI-1640 (for L02)
medium, supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin,
and incubated at 37 °C with 5% CO2 and 95% humidity. The cell lines were
negative for mycoplasma.
Paclitaxel intracellular release. SKOV3 cells were seeded in a 24-wells plate at a
density of 2 × 105 cells each well and incubated overnight. FAM-NucA-PTX-Rh at
a concentration of 200 nM was added to the media of cells for 1 h incubation. The
cells were then washed and re-suspended in culture medium, analyzed immedi-
ately, or at 3 h and 6 h by a FACScan cytometer (BD 831 Immunocytometry
treated with PTX or CRO-PTX. The improved efficacy and Systems).
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Cellular uptake of aptamer-PTX conjugates in vitro. SKOV3 cells were seeded in
a 24-well plate at a density of 2 × 105 cells per well and incubate overnight. For
time-dependent uptake experiment, NucA-PTX-Rh or CRO-PTX-Rh were added
to a final concentration of 500 nM. After 1, 2, 3, 4, 5, and 6 h of incubation, the cells
were washed three times with PBS, trypsinized and re-suspended in 400 μL PBS
after centrifugation (1000 r.p.m., 5 min). The fluorescence intensity of rhodamine
was determined with a FACScan cytometer (BD 831 Immunocytometry Systems)
by counting 10,000 events. SKOV3 cells without any drug incubation were per-
formed as a blank control to measure background signals which were subtracted
from the final calculations. For concentration-dependent uptake assay, the final
concentration of NucA-PTX-Rh and CRO-PTX-Rh was 60 nM, 120 nM, 250 nM,
500 nM, and 1 μM, respectively. After 2 h of incubation, the same procedures were
conducted as the time-dependent uptake experiment. The experiments were per-
formed with triplicates and were repeated for three times. For nucleolin-dependent
uptake assay, SKOV3, OVCAR3, and L02 cells were incubated with Alexa 488-
labeled-nucleolin antibody, NucA-PTX-Rh or CRO-PTX-Rh for 2 h. The cells were
then centrifuged, washed and re-suspended for analysis by flow cytometry. For
aptamer blocking experiment, the SKOV3 cells were pre-incubated with 250 nM
either NucA or CRO for 2 h, and then incubated with 100 nM NucA-PTX-Rh for
2 h. The cells were then centrifuged, washed and re-suspended for analysis by flow
cytometry. Gating strategy of flow cytometry analysis is shown in Supplementary
Fig. 21.
Mice modeling. Eight-week-old female BALB/c nude mice were inoculated sub-
cutaneously with 2 × 106 SKOV3 cells in the left armpit. Tumors were observed
2 weeks after inoculation. The tumor-bearing nude mice were randomly divided
into groups with six mice in each group for further studies.
Tissue distribution by biophotonic imaging analysis. NucA-PTX-Rh and CRO-
PTX-Rh at a single dose of 5 mg kg−1 were given to two groups of the tumor-
bearing nude mice, respectively, by intravenous route (i.v.) via the tail vein. The
mice were killed 2 h and 4 h after injection. Tumors and major organs (hearts,
lungs, livers, spleens, and kidneys) were collected for biophotonic imaging. Bio-
photonic imaging was performed using an IVIS Lumina XR imaging system. All
images were acquired with an exposure time of 5 s. The distribution of fluorescence
within tissues was represented as a pseudo-color image. The photographic and
fluorescent images were individually acquired and then overlaid.
Ex vivo localization of NucA-PTX-Rh in liver, kidney, and tumor sections. The
liver, kidney, and tumor tissues were collected and placed in 4% paraformaldehyde
leaving for overnight. The tissues were then incubated in 30% sucrose for 6 h,
before embedding in optimal cutting temperature medium (OCT). The embedded
tissues were then cryosectioned, washed, and permeabilized by 0.1% Triton X-100.
Finally, the sections were stained with Alexa Fluor 488 Phalloidin (Invitrogen) and
mounted with ProLong Antifade Reagent with DAPI (Molecular Probes). A total of
six sections of the liver and kidney from each mouse were randomly selected for
immunostaining and image acquisition by a confocal microscopy, respectively. To
determine the co-localization of PTX-Rh with nucleolin-expressing tumor cells, the
Pearson’s coefficient analysis were performed on the fluorescent micrographs of
tumor cryosection by ImageJ software (National Institutes of Health, USA). To
determine the distribution of PTX-Rh in liver and kidney, the percentage of rho-
damine positive nephrocytes and hepatocytes per microscopic view were calculated
from the confocal images of six different views per section with the ×20 microscope
objective.
Confocal imaging for endocytosis pathways. SKOV3 or L02 cells were seeded in
glass bottom confocal dishes at a density of 5 × 104 per well and incubated over-
night. Cells were then incubated with 250 nM of NucA-PTX-Rh or CRO-PTX-Rh
and Alexa Fluor 488-labeled endocytic markers (25 μg mL−1 dextran, 25 μg mL−1
transferrin and 5 μg mL−1 CTX-B) at 37 °C for 2 h. Volume of 2 μg mL−1 Hoechst
33,342 then was added during the final 15 min of the incubation. After 2 h of
incubation, the cells were washed and visualized by confocal microscopy. To
investigate the sub-cellular trafficking, the cells were incubated with 250 nM of
NucA-PTX-Rh or CRO-PTX-Rh at 37 °C for 1 h. 75 nM LysoTracker Green DND-
26 (Invitrogen) was added to the cells for another 1 h. Volume of 2 μg mL−1
Hoechst 33,342 then was added during the final 15 min of the incubation. After 2 h
of incubation, the cells were washed and visualized by confocal microscopy.
Percentage of injected doses. NucA-PTX-Rh and CRO-PTX-Rh at a single dose
of 5 mg kg−1 were given to two groups of the tumor-bearing nude mice, respec-
tively, by intravenous route (i.v.) via the tail vein. The mice were killed 2 h and 4 h
after injection. Tumors and major organs (hearts, lungs, livers, spleens, and kid-
neys) were collected, weighed, chopped, and homogenized. The rhodamine fluor-
escence of the tissue homogenates were analyzed by a fluorescence
spectrophotometer. Tumor and organ homogenates from untreated mice were used
as blank controls. The fluorescence intensity of NucA-PTX-Rh and CRO-PTX-Rh
at injected dose was measured as the 100% standard. Percentage of injected doses
of tumor and each organ were calculated based upon the relative fluorescence
intensities.
Chemical inhibition of endocytosis pathways. SKOV3 or L02 cells were seeded
in 6-well plates at a density of 5 × 105 cells per well and incubated overnight. The
cells were then pre-incubated with the inhibitor of macropinocytosis (EIPA), the
inhibitor of clathrin pathway (Chlorpromazine) or the inhibitor of caveolae
pathway (Filipin) at various concentrations for 30 min prior to the addition of
NucA-PTX-Rh or CRO-PTX-Rh at a concentration of 500 nM. After 2 h, the cells
were trypsinized, centrifuged (1000 r.p.m., 5 min) and the supernatants were
removed. After washed twice with PBS, the cells were re-suspended in 400 μL of
PBS. Fluorescence was determined with a FACScan cytometer (BD 831 Immu-
nocytometry Systems) by counting 10,000 events. SKOV3 or L02 cells with DMSO
were performed as a blank control to measure background signals which were
subtracted from the final calculations.
Intactness of the conjugate at tumor site in vivo. NucA-PTX-Rh and FAM-
NucA-PTX-Rh at a single dose of 5 mg kg−1 were given to the tumor-bearing nude
mice, respectively, by intravenous route (i.v.) via the tail vein. The mice were killed
at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 h. The tumors were then collected, chopped and
homogenized. The fluorescence of rhoamine in NucA-PTX-Rh treated mouse
tumor homogenates and the fluorescence of rhodamine and FAM in FAM-NucA-
PTX-Rh treated mouse tumor homogenates were analyzed by a fluorescence plate
reader.
In vitro cell viability assay. The cell viability assay was conducted using a Cell
Counting Kit-8 (CCK8, Dojindo). According to the provided protocol, SKOV3,
OVCAR3, and L02 cells were seeded in 96-well plates with ~5000 cells in each well
and incubated overnight for adherence. Solutions of 500 nM NucA, CRO, PTX,
NucA-PTX, and CRO-PTX were made up in medium and a serial twofold dilution
of the solution were performed to achieve a range of concentrations from 15.6 to
500 nM. The media of cells were removed and the solutions were added for 24 h or
72 h incubation at 37 °C. At the end of the incubation, 10 µL of CCK8 solution was
added to each well. The plates were read after additional 3 h incubation by a
spectrometer at 450 nm. IC50 values of 72 h were calculated using Graph Pad based
on the viability curve data.
Plasma half-life. NucA-PTX-Rh at a single dose of 5 mg kg−1 was given to the
tumor-bearing nude mice by intravenous route (i.v.) via the tail vein. The rho-
damine fluorescence in plasma at various time points were measured. The fluor-
escence at 0 h was estimated by adding given dose of NucA-PTX-Rh to full volume
plasma from a mouse. The plasma clearance curve was presented by the relative
fluorescence intensities of the conjugate over time and the plasma half-life (t1/2
)
was calculated by performing a logarithm plot.
Cell cycle assay. Cell cycle analysis was performed by following the instructions of
the Propidium Iodide Flow Cytometry Kit (abcam). Briefly, SKOV3 and OVCAR3
cells (5 × 105 per well) were seeded in 6-well plates and incubated overnight. After
washing with PBS, the cells were incubated with 200 nM PTX, NucA-PTX or CRO-
PTX at 37 °C for 48 h. At the end of incubation, the cells were trypsinized, washed,
and fixed in 66% ethanol on ice. After storage at 4 °C overnight, the cells were
washed, re-suspended in 200 μL. 1 × Propidium Iodide + RNase Staining Solution
and incubated at 37 °C in the dark for 30 min. Finally, DNA content was measured
by Flow Cytometry (BD 831 Immunocytometry Systems), the percentage of cells in
each phase of the cell cycle was calculated using the ModFit software.
In vivo antitumor efficacy evaluation. CRO, NucA, NucA-PTX, CRO-PTX, or
PTX at a dosage of 2.4 μmol kg−1 (with the equivalent PTX concentration of 2 mg
kg−1) were given to mice of five groups by i.v. twice a week for 4 weeks. Another
group were given vehicle solution PBS as the control group. The tumor size and
body weight were monitored every 4 days. At the end of the treatment, the mice
were killed, blood was collected for WBC count analysis and biochemical analysis.
The tumors and major organs (hearts, lungs, livers, kidneys, and spleens) were
collected for immunohistochemistry and/or histological examinations.
Immunohistochemistry. Paraffin sections of tumors were deparaffinized using
xylene, and then rehydrated by immersing in alcohol at series of concentrations.
Endogenous peroxidase was quenched by 10 min incubation with 3% hydrogen
peroxide. Blocking serum of 1:10 was used for blocking the non-specific bindings of
epitopes. The sections were incubated with mouse Ki-67 antibody (Abcam) of
1:100 at 4 °C overnight. The slides were washed three times, incubated with a
biotinylated secondary antibody (sc-7207; Santa Cruz Biotechnology) for 30 min
and then with peroxidase substrate for 10 min. The sections were finally washed,
Animal handing. The mice were housed in Laboratory Animal House of Hong
Kong Baptist University. The animal house is temperature-controlled with a 12 h
light/dark cycle. Food and water were available ad libitum. At least a week’s
adaptation was given to mice before starting any experiments. The procedures of all
in vivo studies have gained ethics approval by the Animal Experimentation Ethics
Committee of the Hong Kong Baptist University.
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made, mounted on glass slides, deparaffinized, and subjected to hematoxylin and
eosin (H&E) staining using standard protocols. After mounting with coverslips, the
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the sections of liver, kidney, lung, heart, and spleen, respectively. Briefly, the liver
sections were scored ranging from 0 to 2, or 3 depending on the changes in
hepatocytes and hepatocyte rows as well as the presence of steatosis, fibrosis,
necrosis, macrophages, and lymphocytes46. The kidney sections were scored ran-
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epithelial cells, and medulla, as well as the presence of fibrin deposition, vascular
dilation, inflammatory cells, fibrosis, and necrosis46. The lung sections were graded
by a 4-point scoring method regarding the inflammatory changes of the lung tissue,
hemorrhage in the alveolar cavity, and edema and thickening of the alveolar wall47
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.
morphological changes in myocardial cell48. The spleen sections were graded with a
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positioned closed to the fourth toe on the same leg. The distances between sti-
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Statistical analysis. All the variables were expressed as mean standard devia-
tion. Student’s t-test or one-way analyses of variance (ANOVA) were performed in
statistical evaluation. A p-value <0.05 was considered to be significant.
Data availability. All data are available within the Article and Supplementary Files,
or available from the authors upon reasonable request.
Received: 24 January 2017 Accepted: 27 September 2017
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Author contributions
G.Z., A.L., B.-T.Z., S.S., F.J. and F.L. co-supervised the whole project. F.L., J. Lu, J. Liu, C.
L., M.W. and L.W. performed the major research and wrote the manuscript in equal
contribution. D.L., H.Y., Q.Z., J.W., Z.-K.Z., J. Li, Q.L., X.H., B.G., D.G., Y.Y., L.D., X.W.,
Y.L. and G.C. provided their professional expertise.
Additional information
Supplementary Information accompanies this paper at doi:10.1038/s41467-017-01565-6.
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Competing interests: The authors declare no competing financial interests.
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