acs_nn_nn-2014-07097k 1..7

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December 30, 2014

C

2014 American Chemical Society

Arti

fi

cial Micromotors in the Mouse's

Stomach: A Step toward

in Vivo

Use of

Synthetic Motors

Wei Gao,

†

Renfeng Dong,

†

Soracha Thamphiwatana,

†

Jinxing Li, Weiwei Gao, Liangfang Zhang,

and

Joseph Wang

Department of Nanoengineering, University of California, San Diego, La Jolla, California 92093, United States.

†

W.G., R.D., and S.T. contributed equally.

he development of small-scale syn-

thetic motors that convert energy

into movement and forces has been

a fascinating research area.

Impressive

progress made over the past decade has led

to a variety of powerful microscale motors

based on di

erent propulsion mechanisms

and design principles. New functionalities

and capabilities have been added to these

micromotors, leading to advanced micro-

scale machines that o

er a wide range of

important applications. In particular, the

movement of man-made micromachines

in biological

uids can bene

t biomedical

elds such as directed drug delivery, diag-

nostics, nanosurgery, and biopsy.

For

example, functional micromotors have

shown considerable promise for isolating

circulating tumor cells and bacteria from

raw biological

uids.

14,15

Mallouk et al. have

recently reported the ultrasound-driven

propulsion of nanowire motors in living

cells,

while Nelson et al. have explored

targeted drug delivery based on magneti-

cally propelled arti

cial

agella.

In vitro

testing by Pumera's team found no appar-

ent toxicity e

ects of catalytic microengines

on cell viability.

Although tremendous

progress has been made toward such bio-

medical applications,

there are no re-

ports so far illustrating and examining the

in vivo

operation and behavior of these tiny

micromotors. Lacking the characterization

and evaluation of these synthetic motors in

whole living organisms greatly hinders their

further development toward practical and

routine biomedical applications.

Here we demonstrate the

rst study of

synthetic motors under

in vivo

conditions,

involving acid-powered zinc-based micro-

motors in a living organism. Among the

variety of recently developed arti

cial mi-

cromotors, our recently reported zinc-based

motors hold great promise for

in vivo

use,

particularly for gastric drug delivery, be-

cause of their unique features, includ-

ing acid-powered propulsion, high loading

capacity, autonomous release of payloads,

and nontoxic self-destruction.

19,20

Fabricated

through established membrane templating

* E-mail: josephwang@ucsd.edu.

* E-mail: zhang@ucsd.edu.

Received for review December 12, 2014

and accepted December 30, 2014.

Published online

10.1021/nn507097k

ABSTRACT

Arti

cial micromotors, operating on locally supplied fuels and performing

complex tasks, o

er great potential for diverse biomedical applications, including autonomous

delivery and release of therapeutic payloads and cell manipulation. Various types of synthetic

motors, utilizing di

erent propulsion mechanisms, have been fabricated to operate in biological

matrices. However, the performance of these man-made motors has been tested exclusively

under

in vitro

conditions (outside the body); their behavior and functionalities in an

in vivo

environment (inside the body) remain unknown. Herein, we report an

in vivo

study of arti

cial

micromotors in a living organism using a mouse model. Such

in vivo

evaluation examines the

distribution, retention, cargo delivery, and acute toxicity pro

le of synthetic motors in mouse stomach via oral administration. Using zinc-based

micromotors as a model, we demonstrate that the acid-driven propulsion in the stomach e

ectively enhances the binding and retention of the motors as

well as of cargo payloads on the stomach wall. The body of the motors gradually dissolves in the gastric acid, autonomously releasing their carried

payloads, leaving nothing toxic behind. This work is anticipated to signi

cantly advance the emerging

eld of nano/micromotors and to open the door to

in vivo

evaluation and clinical applications of these synthetic motors.

KEYWORDS:

nanomotors

zinc

in vivo

cargo delivery

toxicity

ARTICLE

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the

article or any adaptations for non-commercial purposes.

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processes, the zinc-based micromotors can display

ffi

cient propulsion in a harsh acidic environment

without additional fuel and transport fully loaded

cargoes at high speeds. As the zinc body is dissolved

by the acid fuel, the motors are self-destroyed, leaving

no harmful chemicals behind.

19,20

Such attractive be-

havior makes these zinc-based micromotors useful for

movement and operation in the harsh stomach envi-

ronment, hence opening the door to the

rst

in vivo

operation of micro/nanomotors in living animals.

In this study, the zinc-based micromotors are ap-

plied to the stomach of living mice through gavage

administration. The autonomous movement of these

motors in gastric acid and the motion-induced biodis-

tribution and retention of the micromotors on the

stomach wall are carefully evaluated, along with their

in vivo

toxicity pro

le. Our results demonstrate that the

self-propulsion of the micromotors leads to a drama-

tically improved retention of their payloads in the

stomach lining compared to the common passive

usion and dispersion of orally administrated pay-

loads. These

ndings, along with the absence of toxic

ects in stomach, indicate that the movement of

micromotors in the stomach

uid o

ers potentially

distinct advantages for

in vivo

biomedical applications

and pave the way for their future clinical studies.

RESULTS AND DISCUSSION

Figure 1a illustrates the self-propulsion and tissue

penetration of acid-driven poly(3,4-ethylenedioxythio-

phene)(PEDOT)/zinc(Zn) micromotors in the stomach

environment. The PEDOT/Zn bilayer micromotors were

fabricated using Cyclopore polycarbonate membrane

templates containing microconical pores (Figure 1b).

Due to solvophobic and electrostatic e

ects, the

monomers initially polymerized on the inner wall of

the membrane pores, leading to a rapid formation of

the outer PEDOT layer.

A zinc layer was subsequently

deposited galvanostatically within the PEDOT micro-

tube. The resulting PEDOT/Zn bilayer microstructures

were then released by dissolving the membrane tem-

plates. Figure 1c (left) displays a side-view SEM image

of two typical PEDOT/Zn micromotors. Such biconical

micromotors have a length of 20

μ

m and a diameter

of 5

μ

m. Energy-dispersive X-ray spectroscopy (EDX)

mapping analysis, carried out to con

rm the motor

composition (Figure 1c, right), illustrates the presence

of zinc throughout the motor body. Immersion of the

PEDOT/Zn micromotors in the gastric acid resulted in a

spontaneous redox reaction involving the Zn oxidation

and the generation of hydrogen bubbles essential for

the propulsion thrust. Figure 1d displays time-lapse

images, taken from the Supporting Information video S1,

for the movement of the PEDOT/Zn micromotor in a

simulated gastric acid (pH 1.2) over a 3 s period at 1 s

intervals (I

IV). These images illustrate a de

ned tail of

hydrogen microbubbles generated on the inner Zn

surface and released from one side of the micromotors,

propelling the micromotors at a high speed of

∼

μ

m/s. Such e

ffi

cient propulsion of the zinc-based

micromotors at the gastric pH indicates considerable

promise for

in vivo

evaluation and operation, as envi-

sioned in Figure 1a.

In order to demonstrate that the zinc-based micro-

motors hold such distinct advantages for

in vivo

opera-

tion and potential for biomedical applications, we

rst

Figure 1. Preparation and characterization of PEDOT/Zn micromotors. (a) Schematic of the in vivo propulsion and tissue

penetration of the zinc-based micromotors in mouse stomach. (b) Preparation of PEDOT/Zn micromotors using polycarbo-

nate membrane templates: (I) deposition of the PEDOT microtube, (II) deposition of the inner zinc layer, and (III) dissolution of

the membrane and release of the micromotors. (c) Scanning electron microscopy (SEM) image (left) of the PEDOT/Zn

micromotors and the corresponding energy-dispersive X-ray spectroscopy (EDX) data (right) of elemental Zn in the

micromotors. Scale bar, 5

μ

m. (d) Time lapse images (1 s intervals, I

IV) of the propulsion of PEDOT/Zn micromotors in

gastric acid under physiological temperature (37

°

C). Scale bar, 20

μ

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examined their retention properties on the stomach

tissues using a mouse model. The micromotors were

administered to the mice orally after the mice were

fasted overnight (in order to avoid the in

uence of

food). To examine the e

ect of the micromotor propul-

sion upon the tissue penetration, we compared the

retention behavior of PEDOT/Zn micromotors with that

of PEDOT/Pt micromotors (which cannot move in the

stomach environment and thus serve as a control).

Following 2 h of the micromotors administration, the

mice were sacri

ced and their entire stomach was

excised and opened. Subsequently, the luminal lining

was rinsed with PBS and

attened for imaging and

micromotors counting. The gastric tissue obtained

from the mice treated with PEDOT/Zn micromotors

displayed a large amount of micromotors retained on

the stomach lining 2 h post-administration (Figure 2a).

In contrast, a signi

cantly smaller amount of the con-

trol PEDOT/Pt micromotors (which are very stable in

the stomach environment and do not exhibit autono-

mous propulsion) was retained on the stomach wall

using the same experimental conditions (Figure 2d).

Apparently, the propulsion of the Zn-based micromo-

tors in the acidic stomach environment greatly im-

proved their tissue penetration and retention. It is well

documented that the inner surface of the stomach is

covered by a 170

μ

m thick mucus layer, which is

composed primarily of cross-linked and entangled

mucin

bers.

When the conical motors are actively

propelled in the stomach, they have a great chance to

penetrate into the porous, gel-like mucus layer and be

trapped within the mucus. The retention of PEDOT/Zn

micromotors in the gastric tissue was further examined

at 6 and 12 h after their administration (Figure 2b,c).

The number of micromotors retained in the stomach

tissues decreased gradually from 285 per mm

70 per mm

and to 25 per mm

at 2, 6, and 12 h

post-oral administration, respectively (Figure 2e). Such

a time-dependent decrease of motor retention in the

mouse stomach is likely due to further degradation of

the anchored motors under gastric conditions as well

as their transfer to the subsequent digestive systems

such as the small intestine. Nevertheless, the results

clearly demonstrate that even after 12 h micromotors

are still observed in the stomach tissue, indicating the

ffi

cient tissue penetration of the motors.

Next, we tested the

in vivo

functionalities of such

PEDOT/Zn micromotors for possible applications, par-

ticularly for cargo delivery in living organisms. In our

previous study,

we reported the

in vitro

capabilities

of Zn-based micromotors for combinational cargo

delivery and multifunctional operation, including

autonomous release of cargo and self-destruction of

the motors during the acid-driven movement of the

zinc motors. Taking advantage of these attractive

capabilities, we demonstrated here that PEDOT/Zn

micromotors could e

ectively deliver the cargoes

in vivo

. Compared to the previously reported fully

loaded zinc micromotors that could operate only in

an extremely strong acid and had very short lifetimes

(<1 min),

the presence of the outer PEDOT polymeric

layer here greatly enhances the propulsion perfor-

mance and lifetime of the zinc micromotors under a

broad spectrum of acidic conditions characteristic of

the stomach environment. Such PEDOT based micro-

motors can be self-propelled for

∼

10 min in the

stomach environment under physiological tempera-

ture (pH up to 2). In this study, gold nanoparticles

(AuNPs) were employed as a model cargo because of

their widespread use as imaging agents and drug

carriers.

Figure 3a (left) shows an SEM image of a

PEDOT/Zn micromotors loaded with AuNPs (

∼

50 nm

diameter) through vacuum in

ltration before the zinc

deposition. The corresponding EDX mapping analysis

indicates a uniform distribution of elemental Zn and Au

over the entire micromotor body (Figure 3a, center and

right panel, respectively), which con

rms the success-

ful encapsulation of the AuNP cargoes. Note that the

AuNPs are hardly observed in the SEM image because

of their extremely small size and con

nement within

the Zn body. Supporting Information video S2 dis-

plays the autonomous propulsion of two AuNPs loaded

Figure 2. Tissue retention of PEDOT/Zn micromotors. (a

Microscopic images illustrate the retained micromotors on

the stomach tissues collected at (a) 2 h, (b) 6 h, and (c) 12 h

post-oral administration of PEDOT/Zn micromotors and

(d) 2 h post-oral administration of PEDOT/Pt micromotors

(serving as a negative control). Scale bars, 100

μ

m. (e)

Enumeration of the density of PEDOT/Zn and PEDOT/Pt

micromotors retained on the stomach tissues at the di

er-

ent times after the administration.

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PEDOT/Zn micromotors in gastric acid at physiological

temperature (37

°

C). No obvious di

erence in the

propulsion behavior was observed between regular

and AuNPs-loaded micromotors. Oral administration of

these AuNPs-loaded PEDOT/Zn micromotors into the

mouse stomach led to their movement in the gastric

uid and binding to the mucus layer on the stomach

wall. The zinc dissolution under the gastric acidic

conditions is accompanied by autonomous release

and delivery of the encapsulated AuNPs cargo to

the stomach tissue during this

in vivo

operation. The

motor-based active delivery strategy resulted in dis-

tinct improvement in the delivery e

ffi

ciency compared

to the common passive di

usion of orally admini-

strated AuNPs. The amount of the released AuNPs

retained on the mouse stomach was quanti

ed by in-

ductively coupled plasma/mass spectrometry (ICP-MS).

As shown in Figure 3b, this spectroscopic metal anal-

ysis demonstrates that under the same experimen-

tal conditions the mice treated orally with AuNPs

alone retained 53.6 ng Au per gram tissue compared

to 168 ng Au per gram tissue for the mice treated with

PEDOT/Zn micromotors loaded with an equivalent

amount of AuNPs. Regular PEDOT/Zn micromotors,

without AuNPs loading, were used as the control.

Apparently, loading the AuNPs cargo onto the self-

propelled

“

mother ship

”

micromotor leads to a sig-

cantly (>3-fold) larger retention of AuNPs on the

stomach tissue compared to the orally administrated

NPs. The high retention of AuNPs can be attributed

to the e

ective penetration and retention of the

micromotors that will concentrate and localize the

AuNP payloads on the stomach wall. Moreover, the

subsequent self-dissolution of the Zn-based motors

will release the AuNPs to the mucus layer, which will

further trap and retain the released AuNPs. These

ndings clearly illustrate the importance of the micro-

motor propulsion for enhancing the delivery e

ffi

ciency

compared to the common passive di

usion. While the

concept of

in vivo

cargo delivery of PEDOT/Zn micro-

motors was illustrated through the loading of model

AuNPs, it could be readily expanded to the simulta-

neous encapsulation and rapid delivery of a wide

variety of payloads possessing di

erent functions such

as therapy, diagnostics, and imaging. Unlike most

existing micromotors, Zn-based micromotors destroy

themselves upon completing their cargo delivery

mission.

It is also possible to add additional function-

alities to these micromotors through bulk or surface

modi

cations

14,15,19,20,24

toward diverse biomedi-

cal applications.

Finally, we evaluated the acute toxicity of the biode-

gradable micromotors on healthy mice. Toxicity is a

primary issue in any live animal experiment and hence

in practical real-world

in vivo

applications of micromo-

tors. The main degradation product of the present

micromotors is Zn

þ

, which is an essential multipur-

pose nutrient involved in many aspects of metabolism

and found in all body tissues.

In the study, mice were

orally administered with PEDOT/Zn micromotors,

AuNPs-loaded PEDOT/Zn micromotors, free AuNPs, or

PBS. Mice were fasted overnight before administrating

these samples to avoid the in

uence of food in the

digestive tracts. All the mice were sacri

ced 6 h after

administrating the micromotors or the AuNPs. The

longitudinal sections of gastric tissues obtained from

mice were collected and rinsed three times with PBS.

The tissue sections were stained with hematoxylin

and eosin (H&E). The gastric tissue treated with

PEDOT/Zn micromotors maintained an intact structure

with a clear layer of epithelial cells (Figure 4c), which

was similar to the gastric samples treated with PBS

(Figure 4a). The gastric tissue treated with free AuNPs

(Figure 4e) and with AuNPs-loaded PEDOT/Zn micro-

motors (Figure 4g) showed no apparent toxicity as well.

The potential toxicity of the PEDOT/Zn micromotors

was further evaluated using gastric tissue sections by a

terminal deoxynucleotidyl transferase-mediated deoxy-

uridine triphosphate nick-end labeling (TUNEL) assay

to examine the level of gastric epithelial apoptosis

as an indicator of gastric mucosal homeostasis.

apparent increase in gastric epithelial apoptosis was

observed for treatment groups involving PEDOT/Zn

micromotors (Figure 4d), free AuNPs (Figure 4f), and

AuNPs-loaded PEDOT/Zn micromotors (Figure 4h),

compared to the PBS control group (Figure 4b). The ab-

sence of any detectable gastric histopathologic change

and toxicity indicates that the orally administrated

Figure 3.

In vivo

cargo delivery. (a) SEM image of a AuNP-

loaded PEDOT/Zn micromotor (left) and EDX analysis illus-

trating the presence of Zn (middle) and Au (right) within the

motor. Scale bar, 5

μ

m. (b) Inductively coupled plasma-mass

spectrometry (ICP-MS) analysis of the amount of gold

retained on the stomach tissues. The AuNP-loaded PED-

OT/Zn micromotors or AuNPs were administered orally to

the mice, and the stomach tissues were collected 2 h post

the administration.

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PEDOT/Zn micromotors and AuNPs-loaded PEDOT/Zn

micromotors are safe for the model mouse. While these

Zn-based micromotors can penetrate the mucus layer

on the stomach wall and thus improve the retention of

the motors in the mouse's stomach, such penetration

and retention do not induce a destructive e

ect on the

gastric epithelial cells. The PEDOT polymer is a known

noncytotoxic material whic

h shows no apparent immu-

nological response.

Similarly, zinc is a biocompatible

“

green

”

nutrient trace element, vital for numerous body

functions and processes. Accordingly, the PEDOT/Zn

micromotors leave no harmful products following their

movement, cargo delivery, and self-destruction in the

stomach

uid, making them attractive nanoshuttles in

living organisms. The absence of toxic e

ects in the

stomachisinagreementwiththe

in vitro

study of the

uence of micromotors on cell viability.

It should be

pointed out that changing the micromotor composition

would require reassessment of the toxic response.

CONCLUSION

We reported the

rst

in vivo

study of arti

cial micro-

motors using a live mouse model and characterized

their distribution, retention, cargo delivery, and toxicity

pro

le in mouse stomach. These acid-powered micro-

motors were applied to the stomach of living mice

through gavage administration, and their retention on

the gastric tissue was investigated, along with a related

toxicity pro

le. The self-propulsion in the local stomach

environment led to greatly improved tissue penetra-

tion and retention. Autonomous and e

ffi

cient

in vivo

release and delivery of cargo payloads upon the self-

destruction of the motors were also demonstrated.

Such an active motor-based delivery strategy o

ers

dramatic improvement in the e

ffi

ciency compared to

the common passive di

usion of orally administrated

cargoes. While additional

in vivo

characterizations are

warranted to further evaluate the performance and

functionalities of various man-made micromotors in

living organisms, this study represents the very

rst

step toward such a goal. Our new

ndings and insights

are thus expected to advance the

eld of synthetic

nano/micromotors and to promote interdisciplinary

collaborations toward expanding the horizon of man-

made nanomachines in medicine.

METHODS

Synthesis of Zn-Based Micromotors.

The PEDOT/Zn micromotors

were prepared using a template-directed electrodeposition

protocol.

19,21

The Cyclopore polycarbonate membranes, con-

taining a 5

μ

m diameter (Catalog No. 7060-2513; Whatman,

Maidstone, U.K.) conical-shaped micropores, were employed as

the templates. A 75 nm thick gold film was first sputtered on one

side of the porous membrane to serve as the working electrode

using the Denton Discovery 18. Sputtering was performed at

room temperature for 90 s under a vacuum of 5

Torr, DC

power 200 W, Ar flow of 3.1 mT, and rotation speed of 65 rpm.

A Pt wire and a Ag/AgCl electrode (with 3 M KCl) were used as

counter and reference electrodes, respectively. The membrane

was then assembled in a plating cell with an aluminum foil

serving as a contact. First, the outer PEDOT layer of the micro-

tubes was prepared by electropolymerization at

þ

0.80 V using a

charge of 0.06 C from a plating solution containing 15 mM

EDOT, 7.5 mM KNO

, and 100 mM sodium dodecyl sulfate (SDS).

Subsequently, the inner zinc tube was deposited galvanostati-

cally at

6 mA for 1 h from a commercial zinc plating solution

containing 80 g L

ZnSO

and 20 g L

(buffered to pH =

2.5 with sulfuric acid). For the PEDOT/Pt control micromotors,

the inner Pt tube was deposited galvanostatically at

2 mA for

10 min from a commercial platinum plating solution (Platinum

RTP; Technic Inc., Anaheim, CA). The sputtered gold layer was

removed by hand polishing with a 3

μ

m alumina slurry. The

membrane was then dissolved in methylene chloride for 10 min

to completely release the microtubes. The microtubes were

collected by centrifugation at 6000 rpm for 3 min and washed

repeatedly with methylene chloride, followed by ethanol and

ultrapure water (18.2 M

Ω

3

cm), three times each. Finally, the

microtubes from the whole piece of membrane were dispersed

into 1.2 mL of ultrapure water. The simulated gastric acid

(pH 1.2) was prepared by adding 2.0 g NaCl and 7 mL of HCl

(12 M) in 1.0 L of ultrapure water (18.2 M

Ω

3

cm).

Stomach Retention of PEDOT/Zn Micromotors.

To measure the

retention of the PEDOT/Zn micromotors, ICR male mice at 6

weeks of age were randomly assigned to 4 groups (

= 3) and

orally administered with 0.3 mL of the PEDOT/Zn micromotor

or PEDOT/Pt micromotor solution by oral-gavage. Mice were

sacrificed at 2, 6, or 12 h post-administration of PEDOT/Zn

micromotors and 2 h post-administration of PEDOT/Pt micro-

motors, and their stomachs were removed from the abdo-

minal cavity. The stomachs were cut open along the greater

curvature, the gastric content was removed, and the gastric

Figure 4. Toxicity evaluation of PEDOT/Zn micromotors.

The mouse stomach was treated with PBS bu

er (a, b),

PEDOT/Zn micromotor (c, d), AuNPs (e, f), and AuNP-loaded

PEDOT/Zn micromotors (g, h). At 6 h post-treatment, the

mice were sacri

ced and sections of the mouse stomach

were processed as described in the Methods section and

stained with H&E assay (a, c, e, and g) or TUNEL assay (b, d, f,

and h). Scale bars, 250

μ

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fluid containing excess micromotors was washed away. The

micromotors retained on the stomach lining of the mice from

each group were counted under an optical microscope.

Cargo Delivery by PEDOT/Zn Micromotors.

To study the capability

of PEDOT/Zn micromotors as a carrier for cargo delivery, AuNPs

were chosen as a model payload. AuNPs were prepared as pre-

viously described.

Briefly, a sodium citrate solution (2.2 mM)

was dissolved into DI water (150 mL) in a three-neck round-

bottom flask and heated to 100

°

C, followed by addition of a

HAuCl

aqueous solution (1.0 mL, 25 mM). The reaction mixture

was maintained at the boiling temperature for an additional

3.5 min followed by cooling to room temperature. The resulting

AuNPs (20 mL) were poured into HAuCl

(8 mL, 2.5 mM, 0.008g)

under magnetic stirring. Then, a freshly prepared NH

OH solu-

tion (40 mg in 4 mL) was added dropwise and stirred for 30 min

to allow the reduction of HAuCl

to form AuNPs with diameters

of 50 nm. The particle size (diameter, nm) was measured by

DLS on a Zetasizer Nano ZS (model ZEN3600 from Malvern

Instruments). To prepare the AuNPs-loaded micromotors, the

PEDOT outer layer was electropolymerized first in the 5

μ

m di-

ameter membrane template at

þ

0.80 V using a charge of 0.06 C

from a plating solution containing 15 mM EDOT, 7.5 mM KNO

and 100 mM sodium dodecyl sulfate (SDS). Then, AuNPs were

loaded into the membranes by the encapsulation method.

Solutions of AuNPs were passed through membrane pores by

vacuum infiltration before the zinc deposition. A polycarbonate

membrane with pore sizes of 15 nm was placed below the

sputtered Au film of the membrane containing PEDOT micro-

tubes to retain the AuNPs within the upper 5

μ

m membrane

pores. The membrane was then assembled in a plating cell for

electrodeposition of zinc.

For the

in vivo

cargo delivery experiments, ICR male mice at

6 weeks of age were randomly assigned to three groups (

=4)

to receive free AuNPs, AuNPs-loaded PEDOT/Zn micromotors,

and regular PEDOT/Zn micromotors as the control. Each mouse

in the

rst two groups was administered orally (by oral-gavage)

0.3 mL of the free AuNPs solution, and of the AuNPs-loaded

PEDOT/Zn micromotors containing the same amount of AuNPs.

After 2 h of administration, the mice were sacri

ced and the

stomach was removed from the abdominal cavity. The stomach

was cut along the greater curvature and rinsed with PBS. Gastric

tissue of the mouse from each group was weighted. The tissue

was added to 3 mL of aqua regia consisting of concentrated

nitric acid and hydrochloric acid (Sigma-Aldrich, trace element

analysis grade) in a volume ratio of 1:3. The mixture was left at

room temperature for 12 h, followed by annealing at 80

°

C for

6 h to remove the acids. The sample was then resuspended with

3 mL of DI water. Inductively-Coupled Plasma Mass-Spectro-

metry (ICP-MS) was used to quantify the amount of AuNPs

delivered and retained in the stomach tissue.

In Vivo

Toxicity Study.

To evaluate the toxicity of PEDOT/Zn

micromotors

in vivo

, ICR male mice at 6 weeks of age were orally

administered with 0.3 mL of the PEDOT/Zn micromotors, as well

as with free AuNPs or AuNPs-loaded PEDOT/Zn micromotors.

Mice administered with PBS were used as a negative control. At

6 h after the administration, the mice were sacrificed and the

stomachs were collected for histological analysis. The long-

itudinal sections of the gastric tissue were fixed in neutral-

buffered 10% formalin and then embedded in paraffin. The

tissue sections were stained with hematoxylin and eosin (H&E).

Epithelial cell apoptosis was evaluated by the terminal deoxy-

nucleotidyl transferase-mediate

d deoxyuridine triphosphate nick-

end labeling (TUNEL) assay (Boehringer Mannheim, Indianapolis,

IN). Sections were visualized by the Hamamatsu NanoZoomer

2.0HT, and the images were processed using NDP viewing soft-

ware. All animal experiments were in compliance with institutional

animal use and care regulations.

Equipment.

Template electrochemical deposition of micro-

motors was carried out with a CHI 661D potentiostat (CH

Instruments, Austin, TX). SEM images were obtained with a

Phillips XL30 ESEM instrument, using an acceleration potential

of 20 kV. The SEM images were taken using fresh micromotor

samples. Mapping elemental analysis was carried out using an

Oxford EDX attached to the SEM instrument and operated by

Inca software. Microscope images and videos were captured by

an inverted optical microscope (Nikon Instrument Inc. Ti-S/

L100), coupled with a 10

objective, using Hamamatsu digital

camera C11440 along with the NIS-Elements AR 3.2 software.

The propulsion experiments under physiological temperature

were carried out using a Peltier - thermoelectric cooler module

(CH-109-1.4-1.5) coupled with a K type thermocouple and a

dual digital display PID temperature controller SSR. The size of

the gold particle (diameter, nm) was measured by DLS on a

Zetasizer Nano ZS (model ZEN3600 from Malvern Instruments).

The amount of the released AuNPs retained on the mouse

stomach was quantified using an ICP-MS analyzer (PerkinElmer

Optima 3000 DV).

Conflict of Interest:

The authors declare no competing

nancial interest.

Supporting Information Available:

Supporting methods and

videos. This material is available free of charge via the Internet at

http://pubs.acs.org.

Acknowledgment.

This project received support from the

Defense Threat Reduction Agency-Joint Science and Technol-

ogy O

ffi

ce for Chemical and Biological Defense (Grant Nos.

HDTRA1-14-1-0064 and HDTRA1-13-1-0002) and from the Na-

tional Institute of Diabetes and Digestive and Kidney Diseases of

the National Institutes of Health (Award Number R01DK095168).

W.G.isaHHMIInternationalStudentResearchfellow.R.D.acknowl-

edges the China Scholarship Council (CSC) for the

nancial sup-

port. We thank Michael Galarnyk and Zhiguang Wu for their

assistance.

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