nihms676796.pdf

Turning Erythrocytes to Functional Micromotors

Zhiguang Wu

†,‡,§

Tianlong Li

†,§

Jinxing Li

†,§

Wei Gao

†

Tailin Xu

†

Caleb Christianson

†

Weiwei Gao

†

Michael Galarnyk

†

Qiang He

‡

Liangfang Zhang

†,*

, and

Joseph Wang

†,*

†

Department of Nanoengineering, University of California, San Diego, La Jolla, California 92093,

United States

‡

The Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology,

Harbin 150080, China

Abstract

Attempts to apply artificial nano/micromotors for diverse biomedical applications have inspired a

variety of strategies for designing new motors with unique propulsion mechanisms and functions.

However, existing artificial motors are made exclusively of synthetic materials, which are subject

to serious immune attack and clearance upon entering the bloodstream. Herein we report an

elegant approach that turns natural red blood cells (RBCs) into functional micromotors with the

aid of ultrasound propulsion and magnetic guidance. Iron oxide nanoparticles are loaded into the

RBCs, where their asymmetric distribution within the cells results in a net magnetization, thus

enabling magnetic alignment and guidance under acoustic propulsion. The RBC motors display

efficient guided and prolonged propulsion in various biological fluids, including undiluted whole

blood. The stability and functionality of the RBC motors, as well as the tolerability of regular

RBCs to the ultrasound operation, are carefully examined. Since the RBC motors preserve the

biological and structural features of regular RBCs, these motors possess a wide range of antigenic,

transport, and mechanical properties that common synthetic motors cannot achieve and thus hold

considerable promise for a number of practical biomedical uses.

Keywords

synthetic motor; red blood cells; magnetic guidance; ultrasound; whole blood; biocompatibility

The development of nano/micromotors is a research area of intense activity due to numerous

potential applications.

–

While considerable attention has been given to catalytic motors

that exhibit self-propulsion in the presence of a hydrogen peroxide fuel, many practical

applications would require elimination of the need of chemical fuel.

–

Several groups have

thus explored fuel-free propulsion mechanisms based on externally applied magnetic or

ultrasound fields.

–

The increased capabilities and sophistication of these tiny fuel-free

Corresponding Author: josephwang@ucsd.edu; zhang@ucsd.edu.

Author Contributions:

Z.W., T.L. and J.L. contributed equally to this work.

The authors declare no competing financial interest.

Supporting Information.

Videos of the propulsion of the RBC nanomotors. This material is available free of charge

via

the Internet at

http://pubs.acs.org

HHS Public Access

Author manuscript

ACS Nano

. Author manuscript; available in PMC 2015 December 23.

Published in final edited form as:

ACS Nano

. 2014 December 23; 8(12): 12041–12048. doi:10.1021/nn506200x.

Author Manuscript

motors hold considerable promise for directed drug delivery, biopsy, cleaning clogged

arteries, precision nanosurgery, or localized diagnosis in hard-to-reach places. To fulfill

these exciting potential applications, particular attention is drawn to the biocompatibility of

the motors in biological environments and to their performance in undiluted biological

media. The metallic or polymeric components of common artificial nano/micromotors are

facing destructive immune attack once entering into the bloodstream due to the foreign

nature of these materials.

Natural cells and their derivatives are highly optimized by nature for their unique in vivo

functions and possess attractive features desired for systemic cargo delivery.

–

As a

result, various types of cells, such as red blood cells (RBCs, also named erythrocytes), white

blood cells, macrophages, engineered stem cells and so on, have been employed to carry and

deliver therapeutic or imaging agents.

The intrinsic properties of these natural carriers

have opened the door to creative cargo delivery strategies and novel biomaterials

development. Among these cell-based carriers, RBCs are of particular interest owing to their

vast availability, unique mechanical attribute, surface immunosuppressive property, and

versatile cargo-carrying capability.

–

As such, numerous RBCs based or inspired delivery

systems have been recently developed for cargo delivery, relying on the prolonged transport

property of RBCs in the bloodstream.

–

However, there are no early reports on how to

bestow active propulsion force upon the passively moving RBCs and thus to utilize the cells

as a powerful autonomous micromotor.

Here we demonstrate the attractive behavior of an ultrasound-powered, magnetically-

switchable RBC-based micromotor (denoted RBC motor). Recent efforts have demonstrated

that ultrasound field can trigger the propulsion of microscale objects, and that such

movement is driven by the interaction between the objects and the distribution of acoustic

forces within the field.

The new RBC motors are prepared by loading iron-oxide

nanoparticles into RBCs. The propulsion of the RBC motor can be attributed to the

asymmetric distribution of iron oxide nanoparticles within the cell, which is critical for

ultrasound-powered motion.

The RBC motor is propelled by the pressure gradient

generated by the ultrasound waves due to the inherent asymmetric geometry of the RBC as

well as the asymmetric distribution of magnetic particles inside the RBCs. The latter also

provides a net magnetization that enables magnetic alignment and guidance under acoustic

propulsion. The magnetic guidance (orientation) of these RBC motors can be switched ‘On’

and ‘Off’ by applying an external magnetic field. The resulting RBC motors possess highly

efficient, ultrasound-powered, magnetically-guided propulsion (Figure 1a). Of particular

interest is the efficient prolonged movement that the RBC motors display in the bloodstream

over an extended period of time with no apparent biofouling effects. The RBC membrane

serves as an intrinsic shield to protect the magnetic nanoparticles from etching by co-

existing ions (e.g., chlorides, phosphates) in the blood, hence obviating the need for

commonly used protective coating.

Moreover, one of the most important factors of

micromotors for biomedical applications is biocompatibility, or the ability of the motors to

prevent detection and uptake by immune cells such as macrophages. Due to their inherent

biomimetic properties, the new RBC motors are not susceptible to uptake by macrophages,

displaying remarkable biocompatibility essential for practical biomedical uses.

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RESULTS AND DISCUSSION

The new RBC motors were prepared using a hypotonic dilution/encapsulation method to

load iron oxide nanoparticles (20 nm) into RBCs (Figure 1b).

In the experiment, the RBCs

were briefly incubated with citrate-stabilized iron-oxide nanoparticles in hypoosmotic lysing

buffer to undergo a hypotonic hemolysis process at low temperature (4 °C). It has been

reported that the influx of fluid creates pores with a diameter of up to 100 nm in the RBC

membrane.

These nanopores allow for inward diffusion of the magnetic nanoparticles

from the surrounding medium into the cell and in parallel allow for outward diffusion of

intracellular hemoglobin protein. The cells were held at low temperature for one hour so that

the inner and outer particle concentrations reach equilibrium. Upon equilibrium, the solution

reached isotonicity, when the cell membrane resealed by restoration of osmolarity. The

temperature was then increased to 37 °C and the encapsulated magnetic nanoparticles were

trapped inside the RBCs. Such loading protocol resulted in efficient encapsulation of

magnetic nanoparticles into cells while minimizing damage to the cell membrane. The

optical microscope image in Figure 1c demonstrates that the RBC motors mostly retain the

characteristic erythrocyte shape with a diameter of 6–8 μm. The transmission electron

microscopy (TEM) image in Figure 1d shows the magnetic nanoparticles as black spots

within the RBC, located primarily inside the cell. During the hypotonic process, the

nanoparticles aggregate asymmetrically within the RBCs into large magnetic particles,

–

which reflects the interaction between the nanoparticles and the remaining hemoglobin

proteins to form an agglomerate. Such asymmetric distribution of the magnetic nanoparticle

aggregates provides a net magnetization to the cellular structure that subsequently allows

magnetic alignment under an external magnetic field.

The RBC motors are acoustically powered and magnetically guided by an applied, external

magnetic field. In order to prove that the RBC motors hold efficient guided motion under the

combination of ultrasound and magnetic fields, a set of control experiments was conducted

under different combinations of these external stimuli. Figure 2a and corresponding

supplementary movie S1 show that the position of the RBC motor remained static in the

absence of both ultrasound and magnetic fields. Application of a rotating magnetic field to

the RBC motor, in the absence of ultrasound field, resulted in corresponding rotation of the

motor, yet without its displacement (Figure 2b and Video S1), reflecting that the magnetic

field affects only the orientation of the magnetic nanoparticles inside the RBC motor. Such

magnetically-driven rotation was not observed for regular RBCs that do not contain internal

magnetic nanoparticles (Figure 2c and Video S1). Application of an ultrasound field alone to

the RBC motor led to directional motion of the motor, as displayed by the tracking line in

Figure 2d and Video S1. It has been well documented that blood cells and microrganisms

migrate toward pressure nodes under the ultrasound field.

However, such responses of

regular cells are different from the controlled propulsion of MNPs-loaded RBC motors. As

illustrated in Figure 2e and Video S1, the simultaneous application of both the ultrasound

and magnetic fields results in a guided motion of the RBC motor, reflecting the reversible

alignment of the magnetization vector (discussed below). In order to confirm that this

guided motion is selective to the RBC motors and not to other coexisting micro-objects,

additional control experiments were performed by using regular RBCs (non-magnetic) as a

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negative control. Figure 2f and corresponding Supplementary Video S1 illustrate that when

both ultrasound and magnetic fields were applied to the system containing the regular and

magnetic RBCs, only the RBC motors exhibited controlled motion (with a “Z” shape

trajectory).

The magneto-switchable guidance of the RBC motor is demonstrated in Figure 3.

Application of the magnetic field (while the ultrasound power is on) provides a net

magnetization that enables magnetic alignment and reversible guidance under the acoustic

propulsion (Figure 3a). The time-lapse image in Figure 3b and 3c illustrates such reversible,

magneto-switchable, controlled acoustic propulsion. The on/off magnetic switching allowed

the motion of the RBC motor to be periodically re-oriented (Figure 3b, c). Compared with

the controlled movement of the RBC motor (Figure 3b), the natural RBC exhibited no

significant change in direction upon turning the magnetic field on or off (Figure 3c). The

switchable behavior of the RBC motor observed in Figure 3 demonstrates the crucial role of

the magnetic nanoparticles in controlling the direction of the ultrasound-powered RBC

motor. We hypothesize that the ultrasound propulsion of the RBC motor is caused by an

asymmetric distribution of the encapsulated magnetic nanoparticles inside the RBC motor

(that leads to asymmetric intracellular density gradient) as well as the inherent asymmetric

geometry of the RBC. Further, the asymmetry of the magnetic particles within the RBC

creates a net magnetization within the cell in the presence of the magnetic field. The latter

aligns the magnetization vector to become parallel with the field, altering the direction of the

asymmetry. The magnetic orientation of the RBC motors can thus be switched ‘On’ and

‘Off’ by applying an external magnetic field. This data clearly indicates that encapsulating

magnetic nanoparticles into the RBC motors, along with application of magnetic field, are

essential for creating guided motion under the ultrasound field.

For practical biomedical applications, it is critical to test the propulsion performance of the

RBC motor in relevant biological environments.

As illustrated in Figure 4a and

corresponding Video S3, the RBC motors can operate readily in diverse media ranging from

PBS buffer solution to undiluted whole blood. The ultrasound-powered RBC motors display

a linear movement under the magnetic alignment. The 3 second track lines of such

movement (Figure 4a) indicate that the speed of the RBC motor decreased from 16 μm/s in

the PBS solution to 13, 12, and 5 μm/s in the cell medium, serum, and whole blood,

respectively, reflecting the increased environmental viscosity of these biofluids. The average

speeds of the RBC motor in different biological media are measure and displayed in Figure

4b. While these media affect the motor speed, the RBC motor still moves efficiently in the

different environments, indicating the robustness of the motor for diverse biomedical

applications.

Of particular biomedical significance is the efficient propulsion and behavior of the RBC

motor in undiluted whole blood. Most of the previous micromotor studies in biological

fluids were focused on serum or highly diluted blood samples. Ghosh reported recently the

magnetically actuated movement of cytocompatible ferrite-coated helical nanohelices in

whole blood.

The RBC motor displayed magnetically-guided movement in undiluted

whole blood over both short and long periods, consistent with the long life span of natural

RBCs.

For example, the time-lapse images in Figure 4c and corresponding Video S4

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illustrate controlled movement of RBC motors through whole blood at 15 minute intervals

over a 30 minute period. During this prolonged operation the motor displayed not only

controllable movement with orthogonal turning, but also a negligible change of speed (14

μm/s at 0 minute, 13 μm/s at 15 minute, and 14 μm/s at 30 minute). Figure 4d illustrates such

propulsion of the RBC motor in whole blood along a predetermined Z-shaped trajectory. To

demonstrate their resistance to biofouling, the RBC motor was incubated in undiluted whole

blood for 24 hours followed by testing its performance. As shown in Figure 4d,e and Video

S5, the motor exhibits a similar magnetically-guided acoustic propulsion before and after the

incubation; the “Z” trajectory of the RBC motor and the migration of regular RBCs with a

speed of 5 μm/s can be observed. Note that such long immersion in whole blood has a

minimal effect upon the speed of the RBC motor (12

11 μm/s, before

after the

incubation), reflecting the absence of protein biofouling and salt-etching effects on the

motor behavior. Overall, the data of Figure 4 clearly indicates that RBC motor can operate

in diverse environments, confirming the protection of the magnetic nanoparticles by the

RBC membrane.

An important feature of the RBC motor is its anti-phagocytosis capability against

macrophages which is crucial for evading the immune attack for prolong lifetime in the

bloodstream. Given that the RBC motor retains intact membrane structure and antigens of

natural RBCs including CD47 that prevents phagocytosis by macrophages through its

interaction with inhibitory receptor SIRP

Therefore, the RBC motor is expected to

share the functionality of natural RBCs. To investigate the biocompatibility of the RBC

motor, a macrophage uptake study was carried out by cultivating the J774 murine

macrophage cells with RBC motors or unencapsulated magnetic nanoparticles for 1 hour. To

establish samples with equal amounts of iron, the magnetic nanoparticles were obtained

from same amount of RBC motors which are completely lysed by the addition of Triton

X-100. The macrophages with natural RBCs were cultivated as a background control, which

showed negligible uptake of RBCs (Figure 5a). Similar to natural RBCs, the RBC motors

showed inhibited macrophage uptake as well (Figure 5b). In contrast, the incubation of

macrophages with unencapsulated magnetic nanoparticles resulted in a significant number

of dark spots in the intracellular and perinuclear regions of the cells, indicating that the

magnetic nanoparticles were actively taken up by the cells (Figure 5c). Inductively-coupled

plasma/mass spectrometry (ICP-MS) analysis was conducted to further quantify the iron

uptake by the macrophage cells. As shown in Figure 5d, an uptake of 22.88 ng iron per 1000

cells was observed from the magnetic nanoparticles, while the RBC motors had an uptake of

2.38 ng per 1000 macrophage cells. The near 10-fold reduction in the amount of iron clearly

demonstrates that the RBC motor can effectively inhibit the uptake by the macrophage cells.

The inhibition is largely due to the immunosuppressive antigens of the RBC membrane

present on the RBC motors; the encapsulation of magnetic particles exhibits a negligible

effect on the stealthy properties of the RBC.

To test the tolerability of regular RBCs to the long period of ultrasound treatment, we next

examined the properties of natural RBCs propelled by ultrasound at different transducer

voltages (1–6 V) for a period of 1 hour. The images of Figure 6a,b show a 1% suspension of

regular RBCs before and after the ultrasound treatment, respectively. The geometry of RBCs

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exhibited negligible change after the treatment, indicating that the ultrasound field did not

cause adverse effect on the RBCs. Moreover, the absorption spectrum of regular RBCs, over

the 300–800 nm wavelength range, showed no detectable change at various ultrasound

powers (Figure 6c). The ultrasound-treated regular RBCs were next subject to hemolytic

lysis to quantify the remaining hemoglobin within these cells by measuring the hemoglobin

absorbance at 540 nm. We found that all hemoglobin was retained inside the cells after the

ultrasound treatment, corresponding to near 100% hemolysis after the hemolytic treatment

(Figure 6d). Such negligible change in the degree of hemolysis further confirms the stability

of regular RBCs under the ultrasound field.

CONCLUSIONS

We reported an RBC-derived approach for developing a new generation of cell-based

micromotor that is powered by ultrasound and activated by a magnetic field. The RBC

motor was fabricated by loading magnetic nanoparticles into natural RBCs. Switchable

guided propulsion of RBC motors can be achieved by using a combination of the ultrasound

and magnetic fields. The RBC motors can perform controlled propulsion in undiluted whole

blood over extended periods with no apparent biofouling. The inhibited macrophage uptake

confirms the biocompatibility of the RBC motors. The ability to load natural RBCs with a

variety of functional components,

together with the efficient propulsion in a broad

spectrum of biological fluids, holds great promise for developing multifunctional cell-based

micromotors for a variety of

in vitro

and

in vivo

biomedical applications, and for bridging

the gap between synthetic motors and the biological world.

EXPERIMENTAL SECTION

Synthesis of Citrate-Stabilized Magnetic Nanoparticles

Citrate-stabilized Fe

nanoparticles were synthesized using the previously reported

protocol.

Briefly, a mixture of 0.43 g of FeCl

and 0.70 g of FeCl

was mixed in 40 mL of

water, which was degassed with nitrogen before mixing under the protection of nitrogen.

Subsequently, 2 mL of NH

OH were added to the mixture solution under vigorous stirring

and heated at 80 °C for additional 30 minutes. The supernatant was discarded while the

nanoparticles were obtained in the reaction flask using a magnet, and then fresh degassed

water was added. Citric acid solution (2 mL, 0.5 g/mL) was added, and the reaction mixture

was maintained at 95 °C for 90 minutes. The reaction mixture was allowed to cool to room

temperature under nitrogen. The nanoparticle suspension was washed three times with

deionized water and then collected for the subsequent use.

Encapsulation of Magnetic Particles in RBCs

Fresh RBCs were collected from six-week-old male ICR mice and anti-coagulated with

ethylenediamine tetraacetate. The cells were rinsed three times with PBS (300 mOsm, pH

8). For encapsulating magnetic nanoparticles into the RBCs, 300 μL suspension of RBC and

300 μL suspension of citrate-stabilized iron-oxide nanoparticles were mixed, which led to a

hypotonic condition (final osmotic pressure in RBC suspension, 100–160 mOsm). The

RBCs were incubated under stirring at 4 °C for 1 h. The loaded RBCs were washed three

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times with PBS (300 mOsm, pH = 8) at room temperature to remove the free hemoglobin

and excess Fe

nanoparticles. The resulting RBCs were resealed by incubation in 100 mL

PBS at 37 °C for 1h.

Ultrasound Equipment

The ultrasound experiments were carried out in a cell, as was reported previously.

The

cell was made in a covered glass slide (75 x 25 x 1 mm). A piezoelectric transducer (PZT),

consisting of a 0.5 mm thick ring with a 10 mm outside diameter and 5 mm inner diameter

was attached to the bottom center of the glass slide to create the ultrasonic field. The

continuous ultrasound sine wave was applied through the PZT,

via

an Agilent 15MHz

arbitrary waveform generator, which was connected to a power amplifier. The continuous

sine waveform had a frequency of 2.93 MHz and a voltage amplitude varied between 0 and

10.0 V, as needed for controlling the intensity of the ultrasonic wave. The electric signal was

monitored using a 20 MHz Tektronix 434 storage oscilloscope.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

This project received support from the Defense Threat Reduction Agency Joint Science and Technology Office for

Chemical and Biological Defense (Grants no. HDTRA1-13-1-0002 and HDTRA1-14-1-0064), National Institute of

Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (Award no. R01DK095168).

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Figure 1.

Red blood cells (RBC) motors. (a) Schematic illustration of magnetically-guided,

ultrasound-propelled RBC micromotors in whole blood. (b) The preparation of the RBC

motors: magnetic nanoparticles are loaded into regular RBCs by using a hypotonic dilution

encapsulation method. (c) Optical and (d) transmission electron microscopy images of the

RBC motors.

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Figure 2.

The propulsion performance of RBC motors. The RBC motor, suspended in PBS solution,

was subjected to various external stimulus conditions, including (a) without any stimulus;

(b) under magnetic field alone; (c) under magnetic field in the presence of regular (non-

magnetic) RBCs; (d) under ultrasound field alone; (e) under both magnetic and ultrasound

fields; and (f) under both magnetic and ultrasound fields in the presence of regular RBCs

(non-magnetic). Scale bars, 20 μm. Corresponding videos are provided in the supplementary

information (Video S1).

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Figure 3.

Magneto-switchable guidance of ultrasound-powered RBC motor. (a) Schematic illustration

showing the projected motion trajectory of the RBC motor under ultrasound field with On-

Off switchable magnetic field. (b) Actual time-lapse image, from corresponding Video S2,

illustrating the movement of the RBC motor under ultrasound field upon turning the

magnetic field On and Off; (c) as in (b) but in the presence of a regular natural RBC as a

control. Scale bars, 20 μm.

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Figure 4.

Movement of RBC motors in various media. (a) Images illustrating the propulsion of the

RBC motor in PBS, cell culture, serum, and whole blood, taken from corresponding Video

S3. (b) The quantitative velocity of the RBC motor in different media at ultrasound voltage

of 3 V and a frequency of 2.93 MHz. (c) Swimming behavior of the RBC motor in undiluted

whole blood over 30 minutes, taken from corresponding Video S4. Scale bars, 10 μm. (d, e)

Images showing the propulsion of the RBC motor in whole blood before (d) and after (e) a

24 hour incubation in the whole blood, respectively. Scale bars, 10 μm.

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Figure 5.

A macrophage uptake study to illustrate the biocompatibility of RBC motors. (a–c) Bright

field microscopic images of J774 murine macrophage cells incubated for 30 minutes, with

regular RBCs, RBC motors, and iron-oxide nanoparticles (Fe

NPs, with equal amounts

of iron to that of the RBC motors), respectively. (d) Quantitative analysis of macrophage

uptake of RBC motors and iron oxide NPs determined by ICP-MS measurements.

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Figure 6.

Tolerability of regular RBCs under ultrasound operations. (a and b) Optical images of

regular RBCs before and after the ultrasonic treatment, respectively. Scale bars, 2 μm. (c)

Absorption spectra of regular RBCs under ultrasound field with an applied frequency of

2.93 MHz and at different transducer voltages (0–6 V). (d) The relative hemolysis of regular

RBCs under various ultrasound transducer voltages.

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