APPLIED
SCIENCES
AND
ENGINEERING
Autonomous
metal-organic
framework nanorobots
for
active mitochondria-targeted
cancer
therapy
Xiqi Peng
1,2
, Songsong
Tang
2,3
*, Daitian
Tang
1,2
, Dewang Zhou
2
, Yangyang Li
2
, Qiwei Chen
1,2
,
Fangchen
Wan
2
, Heather Lukas
3
, Hong
Han
3
, Xueji
Zhang
4
, Wei Gao
3
*, Song
Wu
1,2,5
*
Nanorobotic
manipula
tion to access subcellular
organelles
remains
unmet
due to the challenge
in achieving
intracellular
controlled propulsion.
Intracellular
organelles,
such as mitochondria,
are an emerging
therapeutic
target
with selectiv
e targeting
and curative efficacy. We report an autonomous
nanorobot capable
of active
mitochondria-targeted
drug delivery, prepared by facilely encapsula
ting mitochondriotr
opic doxorubicin-tri-
phenylphosphonium
(DOX-TPP)
inside
zeolitic
imidazola
te framework-67
(ZIF-67)
nanoparticles.
The catalytic
ZIF-67
body can decompose
bioavailable
hydrogen peroxide overexpressed
inside
tumor
cells to generate ef-
fective intracellular
mitochondriotr
opic movement
in the presence
of TPP cation. This nanorobot-enhanced
tar-
geted
drugdeliveryinduces
mitochondria-media
tedapoptosis
andmitochondrial
dysregulationtoimprovethe
in vitro anticancer
effect and suppression
of cancer
cell metastasis, further
verified
by in vivo evalua
tions in the
subcutaneous
tumor
model
and orthotopic
breast tumor
model.
This nanorobot unlocks
a fresh field of nano-
robot operation with intracellular
organelle
access, thereby introducing
the next generation of robotic
medical
devices
with organelle-lev
el resolution
for precision
therapy.
Copyright
© 2023 The
Authors,
some
rights
reserved;
exclusive licensee
American
Associa
tion
for the Advancement
of Science.
No claim to
original
U.S. Government
Works. Distributed
under
a Creative
Commons
Attribution
License
4.0 (CC BY).
INTRODUCTION
Micro-/nanor
obots have offered remarkable
revolutions
for bio-
medical
applica
tions benefiting
from their autonomous
movement
(
1
,
2
). These tiny machines
can harness
local energy
[e.g., magnetic
(
3
,
4
), chemical
(
5
,
6
), acoustic (
7
–
9
), and light (
10
,
11
)] to generate
propulsiv
e force, enabling
effectiv
e movement
within
confined
spaces and successful
transport
to hard-to-r
each sites, such as
blood
vessels (
12
), the vitreous (
13
), and the lungs (
14
). Given
that cellular
dysfunction
directlyaltersthe homeos
tasis of living or-
ganisms
to give rise to diverse diseases,
recent efforts
have extended
the operation scope of medical
robots from the organ level down to
the cellular
level, allowing precise operations to therapeutically
reg-
ulate cellular
dynamics.
Pioneer
works have reported
various
intra-
cellular
applica
tions of nanorobots, such as motion
within
cells (
15
,
16
), rapid internaliza
tion for intracellular
delivery [e.g., small inter-
fering RNA (
17
), oxygen
(
18
), enzyme
(
19
)], intracellular
sensing
(
20
,
21
), and scavenging
of reactive oxygen
species
(ROS) (
22
). In
addition,
nanorobots may help regulate cellular
metabolism
by tar-
geting the subsystem of organelles
involved, such asthe nucleus,
ly-
sosome,
mitochondrion,
endoplasmic
reticulum,
and Golgi
apparatus(
23
). The activityandchemical
composition
ofthese sub-
cellular
organelles
alters cell metabolism,
directly determining
the
homeos
tasisoflivingsystems(
24
). Targeting
theseorganelles
shows
great therapeutic
potential
to enhance
drug delivery and treatment
efficacy of prevalent
pathologies
(
25
–
27
).
However, the subcellular
manipula
tion of nanorobots to access the specific
organelles
within
the cytoplasm
remains
a bottleneck.
The lack of directed mobility
and manipula
tion within
the cell has limited
the development
and translation of nanorobots for cellular
modula
tion.
Herein, we present a self-po
wered metal-organic
framework
(MOF)
–
based
nanorobot capable
of active and targeted
drug deliv-
ery to mitochondria
for cancer
eradication and metastasis inhibi-
tion (Fig. 1A). Mitochondria
are used as the therapeutic
target
due to their pivotal role in adenosine
triphospha
te (ATP) produc-
tion, calcium
regulation, cellular
metabolism,
and apoptosis
in eu-
karyotic cells (
28
).
Mitochondrial
dysfunction
has been
demons
trated to contribute
to various
common
pathologies,
such
as cancer
growth and metastasis, inflamma
tion, and neurodegener-
ation (
29
). Zeolitic
imidazola
te framework-67
(ZIF-67)
capable
of
hydrogen peroxide (H
2
O
2
) catalysis was selected
as the material
founda
tion of the nanorobot, serving
as the power engine
(
30
).
The chemother
apeutic
drug, doxorubicin
(DOX),
conjuga
ted with
mitochondriotr
opic
triphenylphosphonium
(TPP
+
) cation
(denoted
as DOX-TPP),
was chosen
to enhance
the binding
of
nanorobots with mitochondria
(
31
). The lipophilic
TPP cation le-
verages the high mitochondrial
membr
ane potential
to passively
target the mitochondria
(
26
,
32
).
Inthiswork,theself-propelled
nanorobotswerefacilelyfabricat-
ed by encapsula
ting DOX-TPP
inside ZIF-67
nanoparticles
(NPs)
(denoted
as ZIF-67@DOX-TPP)
(Fig. 1A). The catalytic decompo-
sition of ZIF-67
in the presence
of H
2
O
2
, which
is overproduced
inside tumor
cells, generates sufficient
force to propel internalized
ZIF-67@DOX-TPP
nanorobots in the cytosol.
Meanwhile,
the en-
capsula
ted mitochondriotr
opic TPP
+
leadsto mitochondria-target-
ed movement
of untether
ed nanorobots,
yielding
a targeted
accumula
tion of nanorobots and subsequently
higher
local drug
concentr
ation at the mitochondria.
The controlled accessto the mi-
tochondria
allows the regulation of mitochondrial
dynamics
for
cancer
therapy benefiting
from the key role of mitochondria
in
modula
ting cancer
growth and metastasis. The mitochondria-tar-
geted
propulsion
of nanorobots
could
induce
mitochondria-
1
Luohu
Clinical
InstituteofShantou
UniversityMedical
College,
Shantou
University
Medical
College,
Shantou
515000,
P. R. China.
2
Institute
of Urology, The Third
Affiliated Hospital
of Shenzhen
University, Shenzhen
518000,
P. R. China.
3
Andrew and Peggy Cherng
Department
of Medical
Engineering,
California
Insti-
tute of Technology
, Pasadena,
CA 91125,
USA.
4
School
of Biomedical
Engineering,
Health
Science
Centre, Shenzhen
University, Shenzhen
518060,
P. R. China.
5
Department
of Urology, South
China
Hospital,
Medical
School,
Shenzhen
Univer-
sity, Shenzhen
518116,
P. R. China.
*Corresponding
author.
Email:
sstang@caltech.edu
(S.T.); weigao@caltech.edu
(W.
G.); wusong@szu.edu.cn
(S.W.)
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,
Sci. Adv.
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, eadh1736
(2023)
9 June 2023
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mediated apoptosis
and mitochondrial
dysfunction,
leading
to im-
proved anticancer
effects
against various
types of cancer
cells and
inhibited
cancer
cell migration and invasion
for in vitro tests.
Two animal
models,
the subcutaneous
tumor
model
and the lung
metastasis model
(orthotopic
breast cancer
model),
were used to
validate the enhanced
antitumor
efficacy and suppression
of
cancer
metastasis by ZIF-67@DOX-TPP
nanorobots.
Overall, this
design
expands
the field of nanorobot operation to the organelle
level with nanorobots acting as miniaturized
intracellular
surgeons
to directly modula
te cellular
physiology
for the treatment of patho-
logical
disorders.
Developing
medical
robotic
tools with organelle-
targeted
capabilities
presents an advanced
generation for precision
Fig. 1. Overall concept
of active mitochondria-targeted
cancer
therapy and characteriza
tion of ZIF-67@DOX-TPP
nanor
obots.
(
A
) Schema
tic of the fabrica
tion of
ZIF-67@DOX-TPP
nanorobots
and their intracellular
mitochondriotr
opic propulsion,
enabling
mitochondrial
targeting
drug delivery to effectiv
ely inhibit
cancer
growth
and metastasis. (
B
) Transmission
electron microscopy
(TEM) and energy-dispersiv
e x-ray images
of ZIF-67@DOX-TPP
nanorobots.
Scale bars, 50 nm. (
C
) Size and zeta (
ζ
)
potential
(
n
= 3; means
± SD) and (
D
) P2p x-ray photoelectr
on spectroscopy
(XPS) spectra of zeolitic
imidazola
te framework-67
(ZIF-67)
nanoparticles
(NPs), ZIF-67@DOX,
and ZIF-67@DOX-TPP
nanorobots.
DOX, doxorubicin;
TPP, triphenylphosphonium;
a.u., arbitrary units.
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, eadh1736
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therapy to achieve maximized
therapeutic
outcomes
with reduced
drug dosage.
RESUL
TS
Preparation and characteriza
tion of ZIF-67@DOX-TPP
nanor
obots
ZIF-67@DOX-TPP
nanorobots were facilely fabrica
ted via an in
situ biominer
alization approach by mixing
DOX-TPP
with the pre-
cursors
of ZIF-67,
cobalt nitrate, and 2-methylimidazole
(
33
). ZIF-
67 and DOX-encapsula
ting ZIF-67
(ZIF-67@DOX)
NPs were also
synthesized
as counterparts.
The transmission
electron microscopy
(TEM)
images
of both ZIF-67@DOX-TPP
nanorobots (Fig. 1B and
fig. S1, C and D) and ZIF-67
NPs (fig. S1, A and B) illustrated
uniform
size distribution
with the typical
morphology
of rhombic
dodecahedr
on (
34
), revealing the negligible
impact of drug encap-
sulation on the structur
e of ZIF-67
NPs. The homogenous
distribu-
tion of Co, O, and P in elemental
mapping
images
of nanorobots
(Fig. 1B) indicates the successful
construction
of the ZIF-67
entity, DOX,
and TPP molecules,
respectiv
ely. The nanorobots
wereexamined
withanaveragesizeof140.0nmandazetapotential
of 27.3 mV (Fig. 1C). The size of the nanorobots was smaller
com-
pared to ZIF-67
NPs, which might be attributed
to the specific
in-
teraction between DOX-TPP
and metal
ions with enhanced
coordina
tion bonding
(
35
,
36
). The nanorobot size (175.6
nm) in
the TEM image (Fig. 1B) made up a high proportion
in the hydro-
dynamic
size distribution
of nanorobots (fig. S2), indicating a con-
sistency in size measur
ements
across different methods.
The x-ray
photoelectr
on spectroscopy
(XPS)
analysis confirmed
that the P
element
(fig. S3, A to D) and two distinct peaks in P 2p spectra
(Fig. 1D) were only observed in ZIF-67@DOX-TPP
nanorobots
and were absent
in ZIF-67
and ZIF-67@DOX
NPs. The x-ray dif-
fraction (XRD)
analysis showed declined
intensity
of diffraction
peaks of ZIF-67
after loading
DOX and DOX-TPP
due to the coor-
dination between Co
2+
and encapsula
ted compounds
(fig. S3E)
(
37
). The ultraviolet-visible
(UV-vis)
spectra of nanorobots illus-
trated broader absorptions
involving
the peaks of building
blocks
and encapsula
ted drugs
(fig. S3F). The construction
of ZIF-
67@DOX-TPP
nanorobotswasfurther
confirmed
usingfluorescent
characteriza
tion by leveraging
the fluorescence
of DOX-TPP
(fig. S4).
Propulsion
performance
ZIF-67,
the active material of the nanorobot shell, can decompose
H
2
O
2
into water and oxygen
due to the catalytic
cobalt ion center
(
30
). Geometric
irregularities
are inevitable
during
the fabrication
of the rhombic
dodecahedr
al ZIF-67,
generating an asymmetric
MOF structur
e. Thereby, the catalytic
reaction of H
2
O
2
around
ZIF-67@DOX-TPP
nanorobots induces
uneven distribution
of de-
composed
products
with directional
flow, propelling
the nanorobot
in self-diffusiophor
etic movement
(
38
–
40
).
The propulsion
performance
of ZIF-67@DOX-TPP
nanorobots
wasfirstevaluatedinphospha
te-buffer
edsaline (PBS)solution
with
various
concentr
ations of H
2
O
2
(0, 0.1, 1, and 10 mM). The nano-
robots showed enhanced
movement
with elongated motion
trajec-
tories upon the rising concentr
ation of H
2
O
2
fuel (Fig. 2A and
movie S1). The calcula
ted mean
square displacement
(MSD)
raisedlinearly
with time andimprovedby the increasing H
2
O
2
con-
centrations (Fig. 2B). The diffusion
coefficient
(
D
eff
; Fig. 2C) and
speed
(Fig. 2D) of nanorobots also increased upon the raised
H
2
O
2
concentr
ation. The catalytic
propulsion
of ZIF-67
NPs was
also examined
in 100 μM H
2
O
2
and showed only slight difference
compar
ed to that of ZIF-67@DOX-TPP
nanorobots (fig. S5 and
movie S2), indicating the negligible
influence
of drug loading
on
the catalytic performance
of ZIF-67.
To expand
the realm of oper-
ation of ZIF-67@DOX-TPP
nanorobots to various
biological
envi-
ronments,
we explored their motion
behavior in 1640 cell culture
medium
(fig. S6 and movie S3) and extracted cytoplasm
(Fig. 2, E
to H, and movie S4). The movement
of nanorobots was enhanced
by increasing
H
2
O
2
concentr
ation from 0 to 10 mM in both media,
where the motion
parameters
of nanorobots were compar
able with
that in PBS solution.
Overall, these results demons
trate that ZIF-
67@DOX-TPP
nanorobots present effectiv
e propulsion
in various
biological
fluids with H
2
O
2
fuel, including
cytoplasmic
extraction,
which
is a prerequisite
for their subsequent
intracellular
applica
tions.
Drug
loading
and release
Next, we explored the loading
capacity and release
behaviors of
DOX-TPP
in ZIF-67@DOX-TPP
nanorobots. The loading
capacity
was explored upon various
fabrication durations and drug inputs.
At the DOX-TPP
input of 1 mg/ml,
the loading
amount
of DOX-
TPP first increased and then saturated upon the increasing
reaction
time (Fig. 2I). The maximum
loading
amount
(347.8
μg/mg)
and
efficiency
(36.2%)
were optimized
at2 hoursofincubation.Increas-
ing the concentr
ation of DOX-TPP
resulted
in an increased loading
amount
of DOX-TPP
until saturation, at which point excess DOX-
TPP input
resulted
in decreased
loading
efficiency
(Fig. 2J).
Maximal
loading
capacity of DOX-TPP
was optimally
achieved at
an input concentr
ation of 1 mg/ml.
The DOX-TPP
released
from ZIF-67@DOX-TPP
nanorobots
wasthenanalyzed
inPBSsolution
undervarious
pHandH
2
O
2
con-
ditions
over 36 hours.
Acceler
ated drug release was observed at the
acidic pH of 5.5 (Fig. 2K) and higher
H
2
O
2
concentr
ation (Fig. 2L).
The nanorobot degradation was also examined
to be accelerated in
the solution
with lower pH and higher
H
2
O
2
concentr
ation using
TEM characterization (fig. S7), contributing
to the enhanced
drug
release behavior of nanorobots.
The slow degradation of ZIF-67
body in neutral PBS was attributed
to the binding
affinity of phos-
phate species
with metal centers,
altering
the coordina
tion equilib-
rium of Co
2+
and the organic
ligand
(
41
). Such degradation was
accelerated in acidic conditions
due to the intensified
competition
between proton and metal ion to coordina
tewith the organic
linker
(
42
) and in the presence
of H
2
O
2
due to the oxidizing
breakage
of
the C
═
C and C
═
N bonds in the network
(
43
). The increased fluid
flow in the porous ZIF-67
structur
e during
the propulsion
of nano-
robots in H
2
O
2
solution
mayalso improve the drug release of nano-
robots.
These release behaviors may promote
the drug delivery of
ZIF-67@DOX-TPP
nanorobots in the tumor
microenvironment
with the acidic condition
and high content
of H
2
O
2
. The loading
and release
behaviors of ZIF-67@DOX
were also evaluated (fig.
S8) and were similar
to that of ZIF-67@DOX-TPP
nanorobots.
Intracellular
movement
The overexpressed H
2
O
2
in cancer
cells (up to 100 μM) offers bio-
available
fuelto propel ZIF-67
–
based
nanorobots(Fig.3A) (
44
–
46
).
The biocompa
tible endocytosis
of the nanorobots by tumor
cells
and subsequent
lysosome
release
are keys to the nanorobots
’
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transport
into the cytoplasm
and their intracellular
operation (
47
).
ZIF-67
NPs were first examined
with good biocompa
tibility
with
negligible
toxicity
to various
cell lines below the concentr
ation of
128 μg/ml (fig. S9), which wasthe operation range in our study. Re-
garding
lysosome
escape,
ZIF-67@DOX-TPP
nanorobots were in-
cubated with T24 bladder
tumor
cells for 8 hours.
The cellular
nucleiandlysosomes
werethenstainedwithHoechs
t33342andLy-
soTracker Green dye, respectiv
ely.Thecolocaliza
tionratio ofthely-
sosome
andDOXfluorescence
wascalcula
tedthroughthePearson
’
s
correlation coefficient
(PCC; the right columns
in fig. S10) (
32
,
48
).
The nanorobots spread to the cytosol
as demons
trated by the intra-
cellular
dispersion
of DOX fluorescence
(red) and decreased PCC
value from 0.67 to 0.35 over the incuba
tion period
(fig. S10).
These results suggest successful
cellular
uptake, lysosomal
release,
and cytosolic
dispersion
of the nanorobots.
Confocal
laser scanning
microscopy
(CLSM)
was used to
examine
the intracellular
motion
of ZIF-67@DOX-TPP
nanoro-
bots. The fluorescence
channel
of CLSM
was used to enhance
the
contrast of nanorobots inside the cell, enabling
the visualiza
tion of
DOX fluorescence
(red) of the nanorobots along with the observa-
tion of bright field. ZIF-67@DOX-TPP
nanorobots exhibited
effec-
tivemotion
trackingwithcoloredtrajectories
insidetheT24bladder
cancer
cell (Fig. 3B and movie S5) and 4T1 breast cancer
cell
(Fig. 3C and movie S5). When
switching
the tumor
cell to a
human
uroepithelial
cell line (SV-HUC-1),
only Brownian
motion
and negligible
displacement
of nanorobots were observed
in the normal
cell due to the ultralow intracellular
H
2
O
2
level
(Fig. 3D and movie S5) (
46
,
49
). To further
confirm
the essential
role of ZIF-67
in such motion
behavior, another
type of MOF,
ZIF-8,
lacking the catalytic property
to H
2
O
2
, was used to encapsu-
late DOX-TPP
(ZIF-8@DOX-TPP)
(
33
). ZIF-8@DOX-TPP
NPs
with the rhombic
dodecahedr
al morphology
also showed smaller
size (166.0
nm) compar
ed to ZIF-8 NPs (195.8
nm) (fig. S11, A
and B), which
is similar
to the size difference between ZIF-
67@DOX-TPP
nanorobots
and ZIF-67
NPs. As expected,
the
absence
of the catalytic
power only led to Brownian
motion
of
ZIF-8@DOX-TPP
in PBS at various
H
2
O
2
concentr
ations (fig.
S11, C to F, and movie S6). The Brownian
motion
was slightly
en-
hanced
by increasing H
2
O
2
concentr
ation, which may be attributed
to the lower kinema
tic viscosity
and enhanced
degradation with
smaller
particles
in higher
H
2
O
2
concentr
ation (
50
). ZIF-8 NPs ex-
hibited
similar
motion
behavior compar
ed to ZIF-8@DOX-TPP
NPsin100μMH
2
O
2
(fig.S11,GandH,andmovieS7).TheBrown-
ian motion
of ZIF-8@DOX-TPP
NPs was also observed within
the
Fig. 2. Motion
study, drug loading,
and drug release
profiles
of ZIF-67@DOX-TPP
nanor
obots.
(
A
) Typical
motion
trajectories
(over 20 s), (
B
) mean square displace-
ment (MSD),
(
C
) diffusion
coefficient
(
D
eff
),and (
D
) speed
of ZIF-67@DOX-TPP
nanorobots in phospha
te-buffer
ed saline(PBS) solution
with different H
2
O
2
concentr
ations
(
n
=15;means
±SEM).(
E
) Typicalmovement
trajectories
(over20s),(
F
) MSD,(
G
)
D
eff
,and(
H
) speed
ofZIF-67@DOX-TPP
nanorobotsinthesolution
ofcytoplasmic
extracts
with various
H
2
O
2
concentr
ations (
n
= 15; means
± SEM). Loading
capacity of ZIF-67@DOX-TPP
nanorobots
upon various
incuba
tion time (
I
) and DOX-TPP
input con-
centrations (
J
) (
n
= 3; means
± SD). The cumula
tive release
of DOX-TPP
from nanorobots upon different pH values
(
K
) and H
2
O
2
concentr
ations (
L
) (
n
= 3; means
± SEM).
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Peng
et al.
,
Sci. Adv.
9
, eadh1736
(2023)
9 June 2023
4 of 14
T24 tumor
cell (Fig. 3E and movie S5). These results demons
trate
the effectiv
e propulsion
of ZIF-67@DOX-TPP
nanorobots inside
tumor
cells, ascribed
to the catalytic
decomposition
between ZIF-
67 and overexpressed H
2
O
2
fuel in cancerous cells.
Active mitochondria-targeted
behavior
The mitochondrial
targeting
capability
was evaluated by incubating
T24 cells with various
solutions,
including
DOX, DOX-TPP
, and
ZIF-8 NPs that encapsula
ted with DOX (ZIF-8@DOX)
or DOX-
TPP (ZIF-8@DOX-TPP),
ZIF-67@DOX,
and ZIF-67@DOX-TPP
nanorobots.
The cell nuclei
and mitochondria
were labeled
with
Hoechs
t 33342 and MitoTracker Green dye, respectiv
ely, for fluo-
rescentcharacterization(Fig.3F).Thecolocaliza
tionratioofthemi-
tochondria
and DOX fluorescence
was calculated through the PCC,
shown in the right columns
of Fig. 3F. The PCC value of the DOX-
TPP group (0.39)
was higher
than the DOX group (0.11).
ZIF-
8@DOX
displayed low fluorescence
colocaliza
tion percentage
of
mitochondria
and DOX with a small PCC value of 0.24. Changing
the cargo to DOX-TPP
(ZIF-8@DOX-TPP)
increased the PCC
value to 0.38, affirming
the preferential targeting
of mitochondria
by lipophilic
and cationic
TPP
+
due to the high mitochondrial
membr
ane potential
(
31
). Comparable PCC was achieved in the
groups of ZIF-67@DOX
in contrast with that of the ZIF-8@DOX-
TPP group, revealing that the chemical
propulsion
of ZIF-67
–
based
NPs could enhance
their adhesion
with mitochondria
possibly
through increased collisions.
The colocaliza
tion ratio was greatly
enhanced
by ZIF-67@DOX-TPP
nanorobots with a much higher
PCC value of 0.79. The PCC value was also shown to increase
with extended
incubation period
of the nanorobots and T24 cells
from 2 to 12 hours (fig. S12). The catalytic-driv
en movement
thus
works
synergis
tically
with loaded
mitochondriotr
opic TPP
+
to
enhance
the binding
efficacy of ZIF-67@DOX-TPP
nanorobots
with mitochondria,
yielding
effectiv
e mitochondrial
targeting
be-
havior. Such active mitochondria-targeted
performance
of ZIF-
67@DOX-TPP
nanorobots was also validated in 4T1 breast cancer
cell line (fig. S13), which exhibited
similar
trend as in T24 cells.
Fig. 3. Intracellular
autonomous
propulsion
and mitochondrial
targeting
of ZIF-67@DOX-TPP
nanor
obots.
(
A
) Schema
ticofthemitochondria-targeted
movement
of nanorobots
inside
the tumor
cell. Confocal
laser scanning
microscopy
(CLSM)
images
of the merged
optical
and DOX channels
showing intracellular
motion
trajec-
tories of ZIF-67@DOX-TPP
nanorobots inside
the T24 bladder
tumor
cell (
B
), 4T1 breast tumor
cell (
C
), and the SV- HUC-1
human
uroepithelial
cell (
D
). DOX fluorescence
(red) represents
ZIF-67@DOX-TPP
nanorobots.
Scale bars, 10
μ
m. (
E
) CLSM
image
showing intracellular
motion
trajectories
of ZIF-8@DOX-TPP
NPs inside
the T24 cell.
Scale bar, 10
μ
m. (
F
) Representa
tive fluorescence
images
showing mitochondrial
colocaliza
tion in T24 cells upon various
incuba
tions for 12 hours,
including
DOX, DOX-
TPP, ZIF-8@DOX,
ZIF-8@DOX-TPP
, ZIF-67@DOX,
and ZIF-67@DOX-TPP
nanorobots.
Nuclei
were stained
by Hoechs
t 33342
(blue).
Mitochondria
were labeled
with Mito-
Tracker Green FM (green). The red fluorescence
represents
the loaded
DOX or DOX-TPP
. The right columns
show the calcula
ted Pearson
’
s
correlation coefficients
of the
colocaliza
tion of mitochondria
and nanorobots
in T24 cells (
n
= 5; means
± SD). Scale bars, 10
μ
m.
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In vitro antitumor
and antimetas
tatic effect
The active mitochondrial
targeting
of ZIF-67@DOX-TPP
nanoro-
bots can enhance
the drug accumula
tion around mitochondria,
leading
to improved mitochondrial
damage
and dysfunction.
Because
of the central role of mitochondria
in controlling cell apo-
ptosis and metastasis, such an effect on mitochondria
is expected
to
induce
mitochondria-media
ted apoptosis
and inhibited
cell metas-
tasis (
51
–
53
).
This apoptotic
pathway was first evaluated using JC-1
dye, a type of membr
ane-permeant
probe for monitoring
the mito-
chondrial
membr
ane potential
(
54
). The aggregated dye in healthy
mitochondria
fluoresces red, whereas, in damaged
and depolarized
mitochondria
with low membr
ane potential,
the monomeric
dye
fluoresces green (
55
). The ratio of red to green fluorescence
can
be used as an indicator of mitochondrial
depolariza
tion and early
apoptosis.
Here, T24 cells were incuba
ted with nanorobots and
other constructs for 8 hours and then stained
with JC-1 dye. T24
cells that incubated with ZIF-67@DOX-TPP
nanorobots exhibited
the smalles
t red/green ratio of 0.85 compar
ed to controls (Fig. 4A
and fig. S14), suggesting the capability
of nanorobots to evoke a
stronger and earlier
apoptotic
response.
Then, we evaluated the viability
of T24 cells that were cultured
with nanorobots and other constructs for 48 hours.
The DOX con-
centration in the drug-loaded
constructs wastested at 2.5, 5, and 10
μg/ml.
The highest cell-killing
efficacy was achieved in the group of
ZIF-67@DOX-TPP
nanorobots in contrast with other constructs
(Fig. 4B). To examine
the influence
of oxygen
generated by H
2
O
2
decomposition
on the anticancer
efficacy of DOX-TPP
, the
mixtur
e of free ZIF-67
NPs and H
2
O
2
was used to generate
oxygen
and incubate with DOX-TPP
and T24 cells for 48 hours.
No apparent alteration occurred on the cell viability
when cells
were incubated with the mixtur
e of ZIF-67,
H
2
O
2
and DOX-TPP
,
and other control groups (fig. S15), suggesting the negligible
influ-
ence of H
2
O
2
decomposition
on the anticancer
effect of DOX-TPP
.
To further
elucida
te the apoptotic
mechanism,
a Western blot was
performed
to examine
the involved protein expression.
Mitochon-
drial outer membr
ane permeabiliza
tion is a signaling
pathway that
initiates cell death, which
is balanced
by the proapoptotic
protein
Bax and antiapoptotic
protein B cell lymphoma
2 (Bcl-2).
The mi-
tochondria-media
tedapoptotic
pathwayactivatesBaxbutsuppress-
es the expression
of Bcl-2, leading
to the increased permeability
of
themitochondrial
outermembr
ane.This allowsthereleaseofinter-
membr
ane space proteins (e.g., cytochr
ome c) to trigger
the caspase
cascade,
inducing
the cleavage of Caspase-9
and Caspase-3
(
56
).
Treatment
with ZIF-67@DOX-TPP
nanorobots resulted
in the
largest up-regulation of Bax, cleaved caspase-9,
and cleaved
caspase-3
and down-regulation of Bcl-2 in comparison
to other
constructs
(Fig. 4C). These
results verify the capability
of ZIF-
67@DOX-TPP
nanorobots in stimulating mitochondria-r
egulated
apoptosis
to achieve enhanced
anticancer
efficacy dueto theireffec-
tive mitochondria-targeted
drug delivery. Besides
T24 cells, ZIF-
67@DOX-TPP
also exhibited
a strong anticancer
response
against
other kinds of cancer
cells, including
4T1 breast cancer
cells and
DOX-r
esistant BIU-87/ADR
bladder
cancer
cells (fig. S16), reveal-
ing the merit of active mitochondriotr
opic delivery in killing
a
broad array of cancer
cells, even cell lines that have previously
ex-
hibited
chemother
apeutic
drug resistance.
The mitochondrial
metabolism
also plays a critical
role in regu-
lating cancer
metastasis. As the powerhouse
of the cell, mitochon-
driaproduceATPforcellular
activities,
including
cellmigrationand
invasion
(
52
). ROS generated by the mitochondrial
electron trans-
port chain have been shown to promote
cancer
migration by acti-
vating the phospha
tidylinositol
3-kinase
pathway (
57
,
58
). In
addition,
mitochondrial
DNA mutations and dynamic
morpholog-
ical changes
of mitochondria
are involved in the alteration of met-
astatic behavior (
59
,
60
). The pivotal role of mitochondria
in cancer
metastasisencour
agesustoexploretheimpactofnanorobotsonthe
metastatic competence
of cancer
cells. The in vitro wound
healing
assay was first conducted.
A gap acting as the
“
wound
”
was created
by linearly
scratching
the confluent
T24 cells. Then, T24 cells were
incubatedwithnanorobotsandothercontrolgroupsfor12hoursto
examine
the wound
closure percentage.
ZIF-67@DOX-TPP
nano-
robots induced
the smalles
t closure rate (26.3%)
compar
ed to other
groups (Fig. 4, D and E), representing
their significant
suppression
of cell migration. A transwell migration assay using a Matrigel
barrier
was further
performed
to evaluate cell invasion.
T24 cells
were injected
in the upper layer and incubated with various
solu-
tions for 24 hours (Fig. 4F). The addition
of ZIF-67@DOX-TPP
nanorobots resulted
in the lowest invasion
rate of T24 cells that mi-
grated through the barrier
(Fig. 4G), demons
trating much stronger
inhibition
on cancer
cell invasion.
This enhanced
suppression
of
nanorobots on cell migration and invasion
was also verified
in
4T1 cells (fig. S17). Together,
these results highlight
the improved
tumor
cell apoptosis
and the suppressed tumor
cell migration by
mitochondrial
targeting
nanorobots,leading
toaneffectiv
eantican-
cer and antimetas
tatic chemother
apeutic
paradigm.
In vivo anticancer
efficacy agains
t a subcutaneous
tumor
model
The in vivo antitumor
effect of nanorobots was first evaluated using
a subcutaneous
T24 tumor
model
(Fig. 5A). The T24 tumor-
bearing
mice were intratumorally injected
with ZIF-67@DOX-
TPP nanorobots and other control groups. The tumor
growth was
monitor
ed during
the treatment
course
(Fig. 5B). The smalles
t
tumor
volume
was achieved by the group of ZIF-67@DOX-TPP
nanorobots,
which
was at least 2.71-fold
lower than other groups.
Such effectiv
e tumor
inhibition
of nanorobots was further
verified
by the weight of resected
tumors
after finishing
the treatment
(Fig. 5C and fig. S18). The excised
tumors
were also stained
with
hematoxylin
and eosin (H&E),
terminal
deoxynucleotidyl
transfer-
ase
–
media
ted deoxyuridine
triphospha
te nick end labeling
(TUNEL),
and Ki67 (Fig. 5E). ZIF-67@DOX-TPP
nanorobots
induced
more noticeable
cell necrosis and apoptosis
with inhibited
proliferation rate compar
ed to other conditions.
The resected
liver
slices from the control and nanorobot groups and tumor
slice from
the nanorobot group were stained
with HLA-DRA
polyclonal
anti-
body, which could label the HLA-DRA
protein, one of the human
leukocyte antigen
(HLA)
class II alpha chain paralogs and specifi-
cally expressed on human-deriv
ed cells. The tumor
tissue showed
the positive signal of HLA-DRA
with brown color due to the inter-
action with human-deriv
ed T24 bladder
cancer
cells, whereas no
signal (blue) was observed in the mouse
liver slice (fig. S19). Such
results indicate that the subcutaneous
tumor
did not develop any
liver metastases during
the treatment course.
Regarding
the toxicity
profile of nanorobots,
no mice experi-
enced distinct weight loss during
the treatment
course
(Fig. 5D).
Main organs
of mice, including
the heart, liver, spleen,
lungs, and
kidneys were processed
with H&E staining
(fig. S20). ZIF-
67@DOX-TPP
nanorobots had negligible
influence
on the tissue
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Fig. 4. In vitro evalua
tion of ZIF-67@DOX-TPP
nanor
obots
for cancer
cell death and metas
tasis inhibition.
(
A
) Fluorescence
images
of T24 cells stained
with JC-1
dyes after incuba
ted with various
solutions
for 8 hours,
including
PBS, ZIF-8@DOX-TPP
, ZIF-67@DOX,
and ZIF-67@DOX-TPP
nanorobots.
The right columns
represent the
calcula
ted fluorescence
ratio of J-aggregate (red) and J-monomer
(green) (
n
= 5; means
± SD). Scale bars, 20
μ
m. (
B
) Viability
of T24 cells afterincuba
tion with nanorobots
and other control groups for 48 hours
(
n
= 3; means
± SD). (
C
) Western blots for characteristic proteins
involved in mitochondria-media
ted apoptosis
in T24 cells after
treatment
with nanorobots
and other control groups. Bcl-2, B cell lymphoma
2. (
D
) Optical
images
showing in vitro wound
healing
assay and (
E
) corresponding
wound
closure percentages.
The wound
(cell gap) was built by a straight scratch across T24 cancer
cells (0 hours).
The wound
closure rate was examined
after incuba
tion with
nanorobots
and other control groups for 12 hours (
n
= 5; means
± SD). Scale bar, 50
μ
m. (
F
) Schema
tic and images
of invaded
T24 cells across the Matrigel barrier
after
treatment
with nanorobots and othercontrol groups in the upper
chamber
of transwell assayand (
G
) corresponding
invasiv
e contents
(
n
= 5; means
± SD). Scale bar, 100
μ
m. **
P
< 0.01; ****
P
< 0.0001;
one-way analysis of variance
(ANOVA).
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structur
e and integrity
compar
ed with the negative control (PBS),
suggesting the favorable biosafety of ZIF-67@DOX-TPP
nanoro-
bots for in vivo applica
tions.
In vivo cancer
treatment
agains
t orthotopic
breast cancer
Next, we used an orthotopic
unilateral breast tumor
model
to eval-
uate the dual function
of ZIF-67@DOX-TPP
nanorobots in inhib-
iting tumor
growth and metastasis (Fig. 6A). The formed
4T1
tumors
can spontaneously
metastasize to distant organs,
especially
the lungs (
61
). The obtained
tumor-bearing
mice were treated with
nanorobots or other control groups. The tumor
growth and body
weight of mice were monitor
ed during
the treatment
course.
After the treatment,
the breast tumor
was resected
for imaging
and weighting.
Among
the groups, ZIF-67@DOX-TPP
nanorobots
displayed the most effectiv
e inhibition
of primary
breast tumor
growth with the smalles
t tumor
volume
(Fig. 6B) and weight
(Fig. 6, C and D). To evaluate the pulmonary
metastasis of breast
tumor,
thelungsofmiceweredissected
attheendpointofthetreat-
ment course
forimaging
andH&Estaining
(Fig. 6E)to quantify
the
metastaticnodule
(Fig.6F).Theaveragenumber
ofpulmonary
met-
astatic nodules
of ZIF-67@DOX-TPP
nanorobots was quantified
as
2.6, representing
at least a sevenfold
lower quantity
than the groups
of free DOX-TPP
(17.8),
ZIF-8@DOX-TPP
(17.4),
and ZIF-
67@DOX
(19.8).
Verifying
the biosafety of the nanorobots, no no-
ticeable
weight change
of mice was observed (Fig. 6G). In addition,
the results of H&E staining
showed that the overall structur
e and
integrity
in the tissues
of main organs
were not affected
by ZIF-
67@DOX-TPP
nanorobots and were compar
able to the healthy
control (PBS)
(Fig. 6H). These
results validate the function
of
ZIF-67@DOX-TPP
nanorobots
in effectiv
ely suppressing
the
growth and lung metastasis of orthotopic
breast tumor
model
due
to the active mitochondriotr
opic movement
of nanorobots inside
tumor
cells.
DISCUSSION
The capability
of micro/nanor
obots to move in a confined
space at
micro/nanoscale
has brought
distinct merits
to cellular-lev
el
Fig. 5. In vivo evalua
tion of the antitumor
effect
of ZIF-67@DOX-TPP
nanor
obots
using
a subcutaneous
tumor
model.
(
A
) Schema
tic of the mouse
model
that
bears subcutaneous
T24 bladder
tumor
and the following treatment
protocol.
(
B
) The tumor
growth kinetics
of tumor-bearing
mice that were treated with various
intratumoral injections,
including
PBS, ZIF-67,
DOX-TPP
, ZIF-8@DOX-TPP
, ZIF-67@DOX,
and ZIF-67@DOX-TPP
nanorobots,
over the treatment
process (
n
= 5; means
±
SEM). (
C
) Excised
tumor
weights
from mice at the end of treatment
with nanorobots
and other control groups (
n
= 5; means
± SD). (
D
) Body weight changes
of tumor-
bearing
mice thatweretreatedwithnanorobotsandothercontrol groupsover thetreatment
process(
n
= 5;means
±SEM).(
E
) Representa
tiveimages
ofhematoxylin
and
eosin(H&E),
terminal
deoxynucleotidyl
transferase
–
media
teddeoxyuridine
triphospha
tenickendlabeling
(TUNEL),
andKi67staining
ofresected
tumor
tissues
frommice
that were adminis
trated with nanorobots
and other control groups. Scale bars, 100
μ
m. *
P
< 0.05; **
P
< 0.01; ***
P
< 0.001;
****
P
< 0.0001;
one-way ANOVA.
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biomedical
applica
tions, such as intracellular
delivery, sensing,
and
detoxifica
tion (
62
). However, major efforts
have been devoted to
cargo transport
to the cell or cell vicinity
. The interaction of nano-
robots with subcellular
organelles
and their related impact on cellu-
lar dynamics
have not yet been explored due to the barrier
of
achieving
controlled propulsion
within
the restricted
intracellular
environment.
On the other hand,
organelles
are the essential
systemtocontrolcellular
andbodyhomeos
tasis.Treatingorganelles
asthe therapeutic
target holds great potential
to improve the target-
ing and therapeutic
efficacy for drug delivery (
27
), but the
development
of organelle-targeted
systems is impeded
by passive
nanopla
tforms
that lack the motility
to reach desired sites.
To overcome these limitations, we have demons
trated MOF-
based nanorobots with intracellular
autonomous
propulsion
to ac-
tively target mitochondria
inside tumor
cells. ZIF-67
–
based
nano-
robotscan catalyzethe decomposition
of thebioavailable
H
2
O
2
that
is overproduced
inside tumorcellsto generate effectiv
e intracellular
propulsion.
The loaded
mitochondriotr
opic TPP
+
facilitates self-
powered nanorobots to bind preferentially
to mitochondria,
over-
coming
the constraints of traditional
passive nanosystems with
limited
transport
efficacy to the specific
organelle
(
63
). This active
Fig. 6. In vivo cancer
treatment
of ZIF-67@DOX-TPP
nanor
obots
using
an orthotopic
breast tumor
model.
(
A
) Schema
tic of the mouse
model
bearing
orthotopic
4T1 breast cancer
and the following treatment
protocol.
(
B
) The tumor
growth kinetics
of tumor-bearing
mice that were intratumorally injected
with PBS, ZIF-67,
DOX-
TPP, ZIF-8@DOX-TPP
, ZIF-67@DOX,
and ZIF-67@DOX-TPP
nanorobots
over the treatment
process (
n
= 5; means
± SD). (
C
) Tumor images
and (
D
) weights
of excised
primary
tumors
from mice at the end of treatment
with nanorobots
and other control groups (
n
= 5; means
± SD). (
E
) Representa
tive images
and H&E staining
showing metastatic nodules
(labeled
with circles) in resected
lungs from tumor-bearing
mice at the end of treatment
with nanorobots
and other control groups and
(
F
) quantified
pulmonary
metastatic nodules
(
n
= 5; means
± SD). Scale bar, 1 mm. (
G
) Body weight changes
of tumor-bearing
mice treated with nanorobots
and other
control groups over the treatment
process (
n
= 5; means
± SD). (
H
) H&E staining
of histological
sections
from main organs,
including
the heart,
liver, spleen,
and kidney,
resected
fromtumor-bearing
micethatwereintratumorallyinjected
withPBSornanorobotsattheendofthetreatment
course.
Scalebar,100
μ
m. **
P
<0.01;***
P
<0.001;
****
P
< 0.0001;
one-way ANOVA.
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targeting
behavior leads to enhanced
accumula
tion of nanorobots
aroundmitochondria
withhigher
localdrugconcentr
ationsdamag-
ing and dysregulating the mitochondria.
Compared to ZIF-
67@DOX
and static ZIF-8@DOX-TPP
NPs, ZIF-67@DOX-TPP
nanorobots led to improved anticancer
efficacy against different
types of cancer
cells due to the induced
mitochondria-media
ted ap-
optosis.
Such improved chemother
apeutic
performance
of the
nanorobots was also verified
in animal
tests via the subcutaneous
tumor
model
and the orthotopic
breast tumor
model.
The capabil-
ity to effectiv
ely kill different kinds of cancer
cells demons
trates the
promising
potential
to generalize this active targeted
drug delivery
to treat a wide range of cancerous sites. Moreover, the impact of
ZIF-67@DOX-TPP
nanorobots on mitochondria
also greatly sup-
pressed lung metastasis of orthotopic
breast tumors.
Using bioavailable
fuel to propel nanorobots with chemota
ctic
guidance
introduces
a feasible
route for the micro/nanor
obotic
community
to create a robotic
platform capable
of effectiv
e and or-
ganelle-targeted
propulsion
inside cells (
1
), obviating the require-
ment for extra fuel and power sources. The catalytic
MOF body,
ZIF-67
showed enhanced
degradation in the acidic and H
2
O
2
con-
ditions.
Cancer
cells are known to have dysregulated pH with a
higher
intracellular
pH (>7.2)
than the extracellular
pH (~6.7 to
7.1) (
64
). The overexpression
of intracellular
H
2
O
2
is another
hall-
mark of tumor
cells compar
ed to normal
cells (
44
,
45
). The nano-
robot degradation in a practical intracellular
environment
thus
would
be mainly
accelerated by the increased concentr
ation of
H
2
O
2
, with the potential
for complete
degradation within
1 week
followingthedegradationtrendshowninfig.S7.Suchabiocompa
t-
ible platform could fulfill the therapeutic
mission
without
residual
unwanted side effects
and potentially
meet rigorous safety require-
ments
for clinical
trial. Alterna
tive catalytic
MOFs
with favorable
biocompa
tibility
and biodegr
adation could be readily
used for
future studies,
such as Zr-based
MOFs
(e.g., UIO-66)
and Fe-
based MOFs
(e.g., MIL-101)
(
65
).
Overall, this work pioneers
the subcellular
manipula
tion of
nanorobots with organelle-lev
el resolution
to modula
te cellular
functions,
introducing
a profound
dimension
for precision
therapy with maximized
therapeutic
outcomes
and reduced
drug
dosage.
The active organelle-targeted
system can be readilyexpand-
ed to other types of catalytic
entities
(e.g., catalase),
chemota
ctic
molecules
(e.g., dequalinium
chloride),
and organelles
(e.g.,
nucleus,
lysosome,
and endoplasmic
reticulum)
toward the treat-
ment of broader pathologies,
such as neurodegener
ative diseases
(
66
) and atherosclerosis (
67
).
MATERIALS
AND METHODS
Fabrica
tion of ZIF-67@DOX-TPP
nanor
obots
Atotalof9.7mgofcobaltous
nitratehexahydr
ate[Co(NO
3
)
2
·6H
2
O;
Aladdin]
was first dissolv
ed in 1 ml of deionized
(DI) water and
then mixed
with 2 ml of DOX-TPP
(4 mg) aqueous
solution
(Macklin) for 1 min. One milliliter
of 2-methylimidazole
aqueous
solution
(HmIm;
64.8 mg/ml;
Sigma-Aldrich)
was dropwise
added
tothemixtur
e upon magnetic
stirring
[1300 revolutions
per minute
(rpm)]
and reacted for 2 hours.
The resulting
ZIF-67@DOX-TPP
nanorobots were collected
by centrifuga
tion (13,800
g
,
10 min)
and washed twice with DI water. The counterparts,
ZIF-67@DOX
andZIF-67
NPs,werepreparedbychanging
theDOX-TPP
solution
to DOX or omitting
the drug solution,
respectiv
ely, in the afore-
mentioned
method.
Characteriza
tion of ZIF-67@DOX-TPP
nanor
obots
The morphology
and elements
of fabrica
ted ZIF-67@DOX-TPP
nanorobots and ZIF-67
NPs were examined
by TEM using a FEI
Talos F200X
instrument.
The hydrodynamic
size and zeta potential
weremeasur
edusingMalvernPanalytical
Zetasizer
NanoZS90.The
XRD analysis was conducted
using a D8 ADVANCE
diffractometer
(Bruker)viaCoKαradiation(λ=0.179026
nm).UV-vis
absorbance
spectra were assessed
by a Spark
multimode
microplate reader
(Tecan).XPSwastestedusingESCALAB250Xi(Thermo
FisherSci-
entific).
The fluorescent
images
of ZIF-67@DOX-TPP
were cap-
tured using CLSM
(Zeiss LSM800).
Drug
loading
and release
During
the fabrication process, various
DOX-TPP
or DOX inputs
(0.5, 1, 2, and 4 mg/ml)
were applied
upon 2-hour
incubation to
assess the influence
of drug inputs
on the loading
capacity of ZIF-
67
–
based
NPs at room temper
ature. The parameter
of reaction time
was evaluated with different durations (0.5, 1, 2, and 4 hours)
upon
the drug input of 1 mg/ml.
The DOX amount
was determined
by
the UV absorbance
at 485 nm in accordance
with the calibration
curve of DOX concentr
ations.
The drug loading
amount
was calculated by Eq. 1
Loading
amount
ð
μg
=
mg
Þ ¼
Mass of loaded
drug
Mass of drug-loaded
particles
ð
1
Þ
The loading
efficiency
was calculated by Eq. 2
Loading
efficiency
ð
%
Þ ¼
Mass of loaded
drug
Drug input
�
100
%
ð
2
Þ
To evaluate the drug release behavior, 1 mg of ZIF-67@DOX-
TPP nanorobots or ZIF-67@DOX
NPs were suspended
in 2 ml of
PBS with different pH values
(5.5, 6.5, and 7.4) or various
H
2
O
2
concentr
ations (0, 50, and 100 μM) and incubated for various
du-
rations (1, 2, 3, 4, 8, 12, 16, 20, 24, and 36 hours).
The superna
tants
werecollected
bycentrifuga
tionforUVabsorbance
detection
at485
nm.Thenanorobotsafterincubationfor12,24,and48hoursunder
each condition
were collected
for TEM characterization (FEI Talos
F200X)
to examine
their degradations.
Motion
study
ZIF-67@DOX-TPP
nanorobots were directly mixed
with PBS or
1640 cell culture medium
upon various
concentr
ations of H
2
O
2
(0, 0.1, 1, and 10 mM) on glass substrate, which
was covered by a
coverslip to prevent drift. The motion
videos
were recorded
by an
inverted optical
microscope
(Eclipse
Ti-U, Nikon Instruments
Inc.)
with a 40× objectiv
e using the dark-field
condenser.
The recorded
movies were analyzed
using NIS-Elements
Advanced
Research
5.21.02
software. The diffusion
coefficient
(
D
eff
) was measur
ed by
fitting
MSD data to the equation, MSD (Δ
t
) = 4
D
eff
Δ
t
, where Δ
t
represents
the time interval.
The motion
analysis of ZIF-67
NPs
was also conducted
in 0.1 mM H
2
O
2
.
Regarding
the motion
in cytoplasm
extract, T24 bladder
tumor
cells (1 × 10
7
cells) were suspended
in PBS solution
(0.2 ml) and
treated with three freeze-tha
w cycles to rupture cell membr
anes
(
68
,
69
). Cytoplasm
solution
was obtained
by centrifuging
the
SCIENCE
ADVANCES
|
RESEARCH
ARTICLE
Peng
et al.
,
Sci. Adv.
9
, eadh1736
(2023)
9 June 2023
10 of 14