10 1021acs nanolett 5b04221

Prévia do material em texto

Cellular Cargo Delivery: Toward Assisted Fertilization by Sperm-
Carrying Micromotors
Mariana Medina-Sańchez,*,† Lukas Schwarz,*,† Anne K. Meyer,† Franziska Hebenstreit,†
and Oliver G. Schmidt*,†,‡
†Institute for Integrative Nanosciences, IFW Dresden, Helmholtzstraße 20, 01069 Dresden, Germany
‡Material Systems for Nanoelectronics, Chemnitz University of Technology, Reichenhainer Straße 70, 09107 Chemnitz, Germany
*S Supporting Information
ABSTRACT: We present artificially motorized sperm cellsa novel type
of hybrid micromotor, where customized microhelices serve as motors for
transporting sperm cells with motion deficiencies to help them carry out
their natural function. Our results indicate that metal-coated polymer
microhelices are suitable for this task due to potent, controllable, and
nonharmful 3D motion behavior. We manage to capture, transport, and
release single immotile live sperm cells in fluidic channels that allow
mimicking physiological conditions. Important steps toward fertilization are
addressed by employing proper means of sperm selection and oocyte
culturing. Despite the fact that there still remain some challenges on the way
to achieve successful fertilization with artificially motorized sperms, we
believe that the potential of this novel approach toward assisted
reproduction can be already put into perspective with the present work.
KEYWORDS: Artificially motorized sperm cell, cellular cargo delivery, assisted reproduction, asthenozoospermia, micromotors,
microswimmers
The operation of miniaturized vehicles that perform tasksand interact with living cells inside the human body
appears to be one more 20th century dream that today’s
engineers finally become ready to tackle. In recent years,
numerous approaches have emerged from various laboratories
to employ such micromotors that can be powered and
controlled on a scale that allows them to assist or interfere
with cellular processes.1−3 Most of these micromotors are
directly inspired by their natural counterparts which are, for
example, flagella or cilia of living microorganisms.4,5 These
nature-approved propulsion strategies were mimicked success-
fully with the help of external power sources like electric or
magnetic fields, ultrasound, light, or chemical fuels.6−8
However, carrying out tasks in the complex surroundings of
living cells requires more than just miniaturized motion alone.
For example, the most prominent micromotors application up
to date, the loading with drugs for targeted transport and
release, is still far from being realized clinically, due to various
shortcomings of current microcarrier systems. The two main
challenges are precise, active transport in three dimensions and
biocompatibility. Active transport of microscopic cargo should
be reasonably fast, and complex microcarrier movements
should be directly controllable both spatially and chronolog-
ically. In addition to these microengineering aspects, the
operation in biologically active environments brings about a
whole new set of problems that involves interactions with living
matter that mostly happen on the nanoscale. Biocompatibility
in this case not only means that the synthetic microcarrier must
not be toxic to cells but also implies that the microcarrier has to
actively take part in cellular and biomolecular interactions in
order to fulfill its task as biosensor, drug distributor, or
microsurgeon.
For example, self-diffusiophoretic Janus motors still rely on
fuels that are cytotoxic, and their motion is only indirectly
controllable by ratchet mechanisms or chemical gradients.9
Acid-powered motors based on propulsion by bubble
generation are limited to the gastric environment and were
not shown to be controllable.10 Hybrid biomicromotors based
on red blood cells functionalized with magnetic nanoparticles
could be actuated by ultrasound and steered by a magnetic field
and were shown to be biocompatible.11 However, the
movement controllability was fairly limited by the fixed
directionality of the ultrasound transducer, and specific,
controllable cargo release was not shown. Other hybrid
microcarriers that rely on on-board bacterial propulsion have
also been shown to be magnetically controllable, but only in 2D
and with relatively low propulsion speeds of less than 5 μm/s.12
Limitation to 2D motion is also the main drawback of a
magnetically actuated stick−slip motion microrobot.13 Martel
et al. proposed an approach that relies entirely on magneto-
tactic bacteria as controllable motor units which showed
Received: October 16, 2015
Revised: December 16, 2015
Letter
pubs.acs.org/NanoLett
© XXXX American Chemical Society A DOI: 10.1021/acs.nanolett.5b04221
Nano Lett. XXXX, XXX, XXX−XXX
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promising behavior in terms of 3D motion and cargo loading
capacity, but suffered from a limited lifetime in physiological
conditions and the yet unsolved question of defined cargo
release.14,15 Nelson et al. employed synthetic microhelices that
were able to manipulate microobjects16 or act as functionalized
drug carriers.17 These motile devices proved to be biocompat-
ible and precisely steerable in 3D. However, it remains to prove
that these highly individual motors, that are able to target single
cells,18 can deliver significant drug doses for therapeutic
purposes within a reasonable time frame.
In our group, so-called spermbots have been introduced as a
novel type of hybrid micromotor. Specifically, a spermatozoon
was used as on-board power supply and coupled to a
ferromagnetic microtube to allow remote control by an external
magnetic field while the sperm tail provides propulsion.19,20
This approach has opened up novel applications for micro-
motors as new alternatives for assisted reproduction biology
and related medical and fundamental studies.21 In general,
sperm cells have been a model of interest for micromotor
research and inspired many innovative activities.22−24
In the present work, we employ the aforementioned
magnetic microhelices for a particularly sophisticated and
relevant case of cellular cargo delivery that suits their described
advantages and continues the previous efforts of our group: the
transportation of a sperm cell to the oocyte with the goal of
fertilization. We show the capture and transportation of
immotile, but otherwise functional sperms25−27 to the oocyte
by coupling them to artificial helical micromotors that can be
actuated by rotating magnetic fields (see Figure 1). Artificial
propulsion of immotile sperms is of major interest for potential
reproduction, because poor sperm motility is one of the major
causes for male infertility and, despite numerous innovations in
the field of Assisted Reproductive Technology, can still not be
countered in a satisfactory way.28,29
We have chosen magnetic helices as micromotors because of
their relatively simple mechanism of motion that is widely
understood and easy to control in 3D by a common setup of
axial pairs of Helmholtz coils that create a rotating magnetic
field which is also biocompatible30,31 and therefore crucial for
potential in vivo applications.
We report the fabrication of polymer microhelices by Direct
Laser Writing32 with soft-magnetic NiTi bilayer coatings, which
show controllable 3D motion with speeds comparable to fast
microorganisms like sperms (up to 70 μm/s), under the
influence of rotating magnetic fields that are generated by a
customized set of Helmholtz coils. These microhelices are
shown to be able to capture, transport, and release single live
sperm cells under physiological conditions (in sperm medium
with adapted viscosity and temperature). Successful delivery of
sperm cells to the oocyte cell wall, in order to fertilize, was
achieved. However, for successful in vitro fertilization, several
requirements have to be met that could not be sufficed due to
various reasons which will be discussed in detail below. Despite
further challenges, it should be stressed that the strength ofour
fertilization approach lies in its potential in vivo applicability,
the benefits of which, as well as further challenges, will be also
discussed.
Swimming Performance of Microhelices. Polymer
microhelices were fabricated by Direct Laser Writing and
coated with a NiTi soft-magnetic bilayer according to a
procedure that was established by Nelson et al.33 for similar
micromotors. They were actuated with a rotating magnetic field
inside fluidic channels made from glass and Parafilm (see Figure
S1a in Supporting Information). Highly reproducible shape and
design features were attained by choosing proper writing and
coating parameters. These can be found in the Supporting
Information, alongside further details and images (Figure S2a).
Reproducible geometries are important to obtain similar
motion behavior. In Figures 2a,b the velocity profiles of helices
with three and four windings, respectively, related to the
actuation frequency f, are depicted for different media
conditions. First of all, they show the well-known behavior of
linear increase of the average velocity v with f up to a so-called
step-out frequency f S, followed by a reciprocal decrease of v
with f > f S, which was reported similarly in literature for these
kinds of helical micromotors.4 We observed this characteristic
behavior for different geometrical variations of helices, mainly
concerning the helix pitch, length and the number of helix
windings. The results of these measurements can be found in
Figure 1. An immotile sperm is captured by a remotely controlled
magnetic helix and delivered to the oocyte for fertilization.
Figure 2. Frequency−velocity profiles of helices with (a) three
windings and (b) four windings in water at room temperature, and in
Sp-TALP at room temperature and 38 °C, respectively; color bars
mark respective step-out frequencies, and error bars depict average
velocity variations between individual helices (n ≥ 3); (c) viscosity
measurements of Sp-TALP, compared to water viscosity values in the
temperature range of 20−40 °C; (d) rectangular track representation
for video analysis and scheme of Helmholtz coil setup with sample
holder.
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the Supporting Information (Figure S2b). Exemplarily, we
show here the difference between helices with three and four
windings. Considering the most important parameters, f S and
the maximum velocity vM, it is apparent that helices with four
windings generally reach higher velocities than those with three
windings, for example ca. 55 μm/s compared to ca. 35 μm/s at
50 Hz, respectively, in sperm medium (Sp-TALP) at 38 °C.
The fact that both types of helices are fastest under these
specific conditions will be further discussed below; however, it
is important to mention that these conditions were chosen to
mimic physiological conditions. The successful swimming
performance of the microhelices in physiological medium at
38 °C inside confined fluidic channels marks an important step
toward real in vivo microswimmers (this temperature is
optimized for fertilization with sperms and oocytes from
bovine origin34,35). The average swimming velocity of both
types of helices slightly increased, compared to helices in water
at room temperature, and compares well to natural motile
sperm cells (vSperm ≈ 10−70 μm/s36,37) in the same
environment. Apart from geometrical parameters of the
microswimmer’s architecture, its maximum velocity is mainly
determined by the viscosity of the surrounding medium.38,39
We can clearly observe this dependency when we look at the
performance in water and in Sp-TALP, respectively (see Figure
2a,b). At room temperature, the helices are slower in Sp-TALP,
compared to water at the same actuation frequency, because the
viscosity of Sp-TALP is higher than the one of water. However,
when the temperature of the Sp-TALP medium is increased to
38 °C, the helices reach speeds similar or even higher than the
ones in water at room temperature, because the viscosities of
water40 at room temperature on the one hand, and Sp-TALP at
38 °C on the other, are similar as well (see Figure 2c). The
average velocity of an individual helix at a certain actuation
frequency is determined by recording its velocity over a number
of points of a rectangular track at a given frequency and
calculating the arithmetic mean of these tracked points (Figure
2d). An exemplary track of an individual helix can also be
observed in Video S1 of the Supporting Information.
Hypoosmotic Swelling and Sperm Cell Viability Tests.
To use sperm−microhelix hybrid swimmers as a tool for
assisted fertilization with immotile sperms requires proper
means of sperm selection to distinguish immotile, but otherwise
healthy, from completely infertile sperm cells. We chose the
Hypoosmotic Swelling Test (HOS) for this purpose since it is a
well-established method to indicate viable sperm cells without
damaging them.41,42 Figure 3a(i) shows swelled sperms in HOS
medium and how the swelled tails correspond well to cell
viability (DNA integrity) (Figure 3a(ii)). Figure 3b(i) high-
lights that the hypoosmotic swelling behavior also corresponds
well to acrosome integrity (stained blue). The acrosome is
connected to the cell membrane of a sperm head and has to be
intact until its reaction in immediate vicinity of an oocyte in
order to promote the activation of proteolytic enzymes to
digest the zona pellucida, which is important to achieve the
fusion of sperm and oocyte membranes.43 Analysis of acrosome
integrity shows that sperms after the HOS test have intact
acrosomes, which is indicated in Figure 3b(ii) by the blue color.
Sperms with damaged acrosomes do not show tail swelling but
green fluorescence, which is in this case caused by Pisum
sativum agglutinin (PSA) bonded to the exposed lectins in the
damaged acrosomal membranes (Figure 3b(iii)). The red color,
again, marks dead sperms, which leads to many sperms being
red and green at the same time because of damaged acrosomes
and subsequent apoptosis.
It has been demonstrated that acrosome integrity is essential
to achieve successful fertilization.44 There are several reports in
literature that prove that immotile sperms are not necessarily
infertile.25−27 The discrimination between immotile and dead
or otherwise defective sperms is thereby a crucial point to
increase fertilization success. The HOS procedure, which we
explained above, is a well-known technique that is also
employed by laboratories that perform intracytoplasmic
sperm injection (ICSI), which is currently the method of
choice for assisted reproduction with sperms with motion
deficiencies.28 Intact sperms are able to respond to
hypoosmotic pressure by regulating the uptake of surrounding
medium via their cell membranes. This leads to a swelling of
the sperm cells which is expressed by a curling of their tales
(see Figure 3a(i)). By subsequent staining of the sperm cells,
we could verify that indeed only alive (Figure 3a(ii)) and intact
(Figure 3b(ii)) sperms feature a curled tail, whereas defect or
dead sperms are still indistinguishable from healthy sperms that
did not undergo hypoosmotic swelling or staining. It is
important to stress that stained sperms are DNA-damaged
and thus not suited for fertilization anymore, whereas sperm
cells that underwent only hypoosmotic swelling would still be
able to fertilize.28 The partial curling of swelled sperm tails also
facilitates sperm coupling with the helical micromotors due to
the coupling mechanism which will be described below. Swelled
sperm cells are not always as clearly distinguished as in Figure
3b(ii). There are at least three types of differently curled sperm
tails that indicate hypoosmotic swelling that we illustrate in
detail in the Supporting Information (Figure S3). We observed
in our experiments that, if a helix accidently pierces a sperm and
thus causes membrane rupture and subsequent cell death, the
sperm tail willimmediately turn back to its full length. This
Figure 3. Sperm cell viability after HOS test: (a) bright field
micrograph (i) of sperm cells in HOS medium and (ii) fluorescence
image of its live/dead staining to demonstrate the compliance of
sperm swelling and viability. Green represents alive sperm cells; red
indicates dead ones. Arrows point at swelled sperm cells. (b)
Acrosome staining of sperm cells. Reacted and unreacted acrosomes
were visualized using FITC-PSA and Lysotracker blue, respectively.
Intact acrosomes are thus blue (ii), and damaged acrosomes are green
(iii), whereas dead sperms are red.
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happens because the damaged sperm membrane cannot uphold
the osmotic pressure any longer and thus the swelling recedes
(Figure 4). Additionally, when sperm membrane rupture
occurs, the viscous intracellular microenvironment leaks out
and leads to a local increase of viscous drag forces, caused by
the high viscosity of cell plasma (150−250 mPa·s)45 comparted
to the surrounding medium (0.8−1.0 mPa·s), which leads to
the helix becoming stuck in the cell. Another explanation for
this sticking could be the rapid cellular reaction of resealing the
ruptured membrane as reported by Terasaki et al.,46 which was
also observed and reported by Srivastava et al.47 with needle-
shaped microcarriers that pierced HeLa cells in our laboratories.
The corresponding video to the images of Figure 4 can be
found in the Supporting Information (Video S2).
We therefore assume in our further experiments that, if we
manage to capture a sperm cell, the coupled sperm is alive and
healthy as long as its tail is still swelled. To verify that the
microhelices are indeed not harmful to the sperms, we also
conducted a conventional viability test.
The presence of a foreign material, especially microstructures
fabricated from polymeric photoresists and magnetic metal
layers (Ni) could potentially cause harmful effects on sperm
cells. We investigate this influence by viability assessment using
two dyes, one being membrane-permeant nucleic acid stain
(SYBR 14 dye), and the other one commonly used dead-cell
stain which binds to the DNA only when the cell membrane is
damaged (propidium iodide).48 We analyzed several different
samples under different conditions (see Supporting Informa-
tion, Figure S4), the most relevant ones of which are presented
in Figure 5c: The control sample (sperms in Sp-TALP medium
on a glass substrate without helices), and sperms under the
same conditions in the presence of NiTi-coated polymer helices
or NiTi-coated polymer helices that were treated with Pluronic
F-127 in order to prevent unspecific adhesion, which is an
important functionalization step for our application. Sperms
that underwent the hypoosmotic swelling test were used for
these three conditions to take into account the modification
that is necessary for successful fertilization experiments.
The cell counts of the control sample indicated roughly 30%
viable sperms (Figure 5a,c). This value decreased slightly for
the sample that was in contact with several hundred NiTi-
coated helices (Figure 5b,c) and increased slightly for the
sample that was in contact with NiTi-coated helices that had
been functionalized with Pluronic F-127 (Figure 5c). On the
one hand, a slight decrease of the fraction of live sperms might
be caused by the potentially harmful nickel coating which we
intended to shield by a titanium layer. On the other hand, the
increase of the fraction of live sperms for functionalized helices
could be caused by the Pluronic F-127 which is a biological
detergent that the cells are able to recognize and apparently
react to in a positive way which we did not predict beforehand.
Altogether, considering the inherent variations in biological
specimens, the small differences in cell viability of the three
conditions presented in Figure 5c are not striking enough and
serve to prove that the NiTi-coated microhelices, functionalized
or not, are not overly harmful to sperm cells. However, it is
apparent from the control sample that the sperm cells are in
general of limited quality, as indicated by the relatively low
fraction of live sperms of roughly 30%. Intriguingly, this fact
allows us to mimic physiological conditions effectively, since we
are indeed interested in working with deficient sperm
specimens, i.e., sperm samples of low quality, since we aim to
assist naturally immotile sperm cells. The important precondi-
tion is to reliably distinguish live from dead sperms, which was
shown in Figure 3 and can also be verified in Figure 5a,b by the
correlation of swelled tails and green staining. We are thus
confident that we can select an immotile, but viable sperm cell
even in a sample with suboptimal quality.
Capture, Transport, and Release of Live Sperm Cells.
A sperm cell is successfully captured when its tail is confined
inside the inner part of the microhelix, while its head sticks out
at the front end of the helix and is loosely bound by the front
ring that acts like a noose to prevent the sperm head from
slipping back through the helix (see Figure 1 and Figure 6a).
This coupling mechanism is considered to be the most efficient
since it avoids any sticking or piercing mechanisms that could
damage the sperm cell, while it also does not impair the helical
propulsion of the artificial microswimmer. A severe impairment
of the helix movement would result in a drastic speed decrease,
as well as a loss of directionality, e.g. the ability of the helix to
follow the directions given by the orientation of the rotating
magnetic field. For the presented sperm coupling, the
surroundings were set slightly out of focus to visualize the
end of the sperm tail in order to identify the small curling of the
tail that indicates a viable, swelled sperm, and to simplify
Figure 4. Sperm pierced by a helix. (i) Helix close to a sperm, (ii) helix
approaching and contacting sperm membrane, and (iii) sperm death
after being drilled and pierced by a helix. Yellow circle and arrow in (i)
and (ii) indicates tail curling due to hypoosmotic swelling and in (iii)
swelling recession. Time scale in seconds.
Figure 5. Fluorescence image of live/dead staining in the presence of
different materials: (a) glass and (b) helices coated with Ni (100 nm)
and Ti (5 nm). Green represents alive sperm cells; red indicates dead
ones. (c) Fraction of live sperms after incubation of the control glass
substrate, substrates with added NiTi-coated helices, and ones with
added NiTi-helices that were functionalized with Pluronic F-127.
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capture of the tail by a nearby microhelix. The helix was then
controlled to swim with a slight downward tilt in z-direction
and the tail was successfully captured by the holding ring at the
helix front. Figure 6b shows the transport; e.g., artificial
propulsion of the sperm cell on a semirectangular track that was
recorded. The sperm cell release is shown in Figure 6c, where
the rotation axis of the magnetic field is inverted. Figure 6a, b,
and c all show the same helix and sperm cell. The
corresponding video to the images of Figure 6 can be found
in the Supporting Information (Video S3). In these experi-
ments sperms were diluted in Sp-TALP medium and
transferred to trisodium citrate solution for hypoosmotic
swelling. This medium was heated to 38 °C to mimic
physiological conditions. The tail of the sperm features a tiny
curled part at its end which is marked in Figure 6a ii, but can
also be seen in Figures 6b and c. This is due to swelling in
hypoosmotic medium and serves to confirm that the sperm cell
is alive, although immotile. As previously mentioned,
hypoosmotic swelling could not always be observed as clearly
as for example in Figure 3b, but was nonetheless present and
could beverified by comparing the length of the sperm tail to
an unswelled sperm. Finally, Figure 6d compares speeds before
and after coupling of six different cases and reveals an average
speed decrease of the hybrid microswimmers to ca. 39.4% of
the initial helix velocity, with a relative standard deviation of
23.2%. Such deviation is attributed to the variability of
differently swelled sperms and their influence on the lose
coupling between sperm tail and microhelix. There are different
types of tail curling, as previously mentioned and shown in
Figure S3 of the Supporting Information. Consequently, the
sperm cargo is variable in shape and influences the performance
of the hybrid micromotor substantially considering the
hydrodynamics that are dominated by drag in this low
Reynolds number regime. The remarkable differences between
sperm cells with individually curled tails might also lead to an
insufficient mechanical coupling between sperm and helix in
some cases. The speed and controllability of the microhelix
decreases drastically if the respective sperm cell does not couple
to the helix in the ideal way that was described above. For
example, the hybrid micromotor presented in Figure 6 (and
Video S3) showed fairly good coupling of sperm and helix and
reached velocities as high as 17.6 ± 3.53 μm/s. Another good
though slightly slower example is presented in Video S4 in the
Supporting Information. In that particular case, complete
release of the sperm cell after transportation for a certain
distance was also achieved (see Video S4).
Sperm Cell Delivery to the Oocyte Wall. Transportation
of an immotile sperm cell to the oocyte cell wall is also
demonstrated with the helical micromotors. In Figure 7, the
sperm delivery procedure is shown in different steps: (i)
coupling, (ii) transport, (iii) oocyte approach and contact, and
(iv) sperm release. The corresponding video can be found in
the Supporting Information (Video S5). Although the helix
velocity decreased due to the cell load and the disturbance
caused by the sperm tail, it was possible to transport the sperm
toward the oocyte and release it once it adhered to the oocyte
wall by inverting the helix rotation via reversal of the magnetic
field rotation. Another case of successful sperm delivery to an
oocyte is presented in Video S6 in the Supporting Information.
In that particular case, transportation over a relatively long
distance with a speed of 19.7 ± 0.67 μm/s was achieved while
clearly retaining the sperm tail curling that verified viability due
to hypoosmotic swelling until delivery to the oocyte membrane
(see also Figure S5 in the Supporting Information).
Unfortunately, unlike in the case presented in Figure 7,
complete release of the sperm cell after delivery was not
successful due to unspecific adhesion of the microhelix. The
irreversible adhesion of helices to sperms, to the oocyte, or to
the substrate is a general problem that we tried to counter by
proper molecular surface functionalization of the helices and
the fluidic channel substrate, which is described in detail in the
Experimental Section. Unfortunately, this functionalization did
not always succeed in avoiding this problem, and we will strive
to find more efficient approaches in future works.
Figure 6. Sperm capture and release. (a) Sperm capture, (b) transport,
(c) release; (d) relative velocity of microhelices before and after sperm
coupling; error bars depict average velocity variations of six individual
helices. Yellow arrow in a(ii) indicates tail curling due to hypoosmotic
swelling.
Figure 7. Sperm cell coupling (i), transport (ii), approach to the
oocyte membrane (iii), and release (iv).
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Generally, limitations in reliability and reproducibility are the
main reasons why fertilization remains a challenge. In current
clinical practice, the oocyte fertilization yield with one immotile
sperm by ICSI is around 40−50% under otherwise optimal
conditions.28 In our setup, we have to transfer sperms and
oocytes from proper culture dishes to the fluidic platform
causing unwanted time delays and temperature fluctuations.
Current statistical limitations arising from the complex
individual capture of single sperm cells require extensive and
focused experimental work to increase the probability to
achieve successful fertilization. Still, this work serves to
demonstrate a new approach to artificial reproduction that is,
in principal, also applicable in vivo and would thus allow to
avoid all complications that arise from oocyte culturing and
subsequent embryo transfer, i.e., reimplantation. To make that
happen, there is however much exciting work to be done
starting from the first steps reported here. For instance, a
problem that all current micromotor systems that aim for an in
vivo application share, is in vivo imaging and tracking in order
to freely control their motion inside the living body. There are
several imaging techniques that are applied for this purpose
based on different concepts like optical fluorescence,49 infrared
emission,50 X-ray analysis,51,10 magnetic resonance,52,53 or
ultrasound imaging.54 Now and in the future, scientists of
different fields are striving to achieve the trinity of real-time,
deep-tissue, and high-resolution imaging that would allow many
future micromotor applications inside the human body. For the
specific application that is presented in this work, in vivo
imaging is indispensable in order to successfully navigate
through the uterine cavity. Other factors that are hard to mimic
in vitro but will be addressed in future studies are immune
response and navigation through confined, elastic surroundings.
Again, these are problems that do not only occur in the uterine
cavity and oviduct, but are also relevant for micromotors that
are meant to navigate through blood vessels for biosensing or
drug delivery applications.55−60
We implemented magnetically actuated polymer−metal
composite helices as microcarriers that can actively capture,
transport, and release single live sperm cells that would
otherwise be immotile due to pathological defects. In order to
set up an environment that would allow these artificially
motorized sperm cells to fertilize an oocyte, we mimicked in
vivo conditions and applied hypoosmotic swelling as a method
for sperm selection in a microfluidic channel platform where we
managed to deliver a single sperm cell to an oocyte cell wall.
Unfortunately, similar to many promising applications in
biomedical engineering, it appears to be still a long way from
artificially motorized sperm delivery to actual oocyte fertiliza-
tion. There is a lot of future work to do, considering proper
oocyte culturing, functionalization of helices to create
important biochemical clues, and further improvement of
targeted sperm capture and delivery, in order to achieve a
critical rate of fertilization trials that would lead to successful in
vitro fertilization. It remains to stress that, ultimately, the
strength of this novel fertilization approach lies in its potential
in vivo applicability, since it will not be necessary to explant
(and reimplant) oocytes for artificial reproduction if we can
target and fertilize the oocyte in its natural environment.
■ ASSOCIATED CONTENT
*S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.nano-
lett.5b04221.
More details of the fabrication and characterization
methods and used materials (PDF)
Helix performance, Figure 2 (AVI)
Piercing a sperm cell, Figure 4 (AVI)
Coupling, Figure 6 (AVI)
Coupling and release (AVI)
Sperm delivery, Figure 7 (AVI)
Sperm delivery 2, Figure S5 (AVI)
■ AUTHOR INFORMATION
Corresponding Authors
*E-mail: m.medina.sanchez@ifw-dresden.de.
*E-mail: l.schwarz@ifw-dresden.de.
*E-mail: o.schmidt@ifw-dresden.de.
Author Contributions
M.M.-S. and L.S. contributed equally to this work.M.M.S., L.S.,
and O.G.S. conceived the project; M.M.S. and L.S. designed the
experiments with help from A.K.M and F.H. O.G.S. supervised
the study. M.M.S and L.S performed and analyzed all
experiments. M.M.S and L.S wrote the manuscript. All authors
commented on and/or edited the manuscript and figures. All
authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
The authors thank Masterrind GmbH for kind donation of
cryopreserved bovine semen, as well as Südost Fleisch GmbH
(Altenburg) for the donation of ovaries. We thank Martin
Bauer, Cindy Kupka, Cornelia Krien, Ronny Engelhard, and
Sandra Nestler for clean room support. We also value the
discussions with Nicolaś Peŕez and Lin Gungun during the
Helmholtz coil design. We really appreciate the great work
made by Dr. Hartmut Siegel, Dr. Torsten Seidemman, Holger
Günter, Uwe Biscop and Samuel Grasemann in the fabrication
and control implementation of the Helmholtz coil setup.
Thanks to Veronika Magdanz, Maria Guix, Sarvesh Kumar
Srivastava for further helpful discussions along our work and to
Britta Koch for her support in the fluorescence imaging. Finally,
we thank Sharath Tippur Narayana Iyengar for his help on real-
time imaging of micromotor experiments with sperm cells and
oocytes.
■ REFERENCES
(1) Peyer, K. E.; Zhang, L.; Nelson, B. J. Nanoscale 2013, 5 (4),
1259−1272.
(2) Man, Y.; Lauga, E. Phys. Fluids 2013, 25 (7), 071904−071901.
(3) García, M.; Orozco, J.; Guix, M.; Gao, W.; Sattayasamitsathit, S.;
Escarpa, A.; Merkoci̧, A.; Wang, J. Nanoscale 2013, 5 (4), 1325−1331.
(4) Mahoney, A. W.; Nelson, N. D.; Peyer, K. E.; Nelson, B. J.;
Abbott, J. J. Appl. Phys. Lett. 2014, 104 (14), 1−5.
(5) Morozov, K. I.; Leshansky, A. M. Nanoscale 2014, 6 (3), 1580−
1588.
(6) Guix, M.; Mayorga-Martıńez, C. C.; Merkoci̧, A. Chem. Rev. 2014,
114 (12), 6285−6322.
Nano Letters Letter
DOI: 10.1021/acs.nanolett.5b04221
Nano Lett. XXXX, XXX, XXX−XXX
F
http://pubs.acs.org
http://pubs.acs.org/doi/abs/10.1021/acs.nanolett.5b04221
http://pubs.acs.org/doi/abs/10.1021/acs.nanolett.5b04221
http://pubs.acs.org/doi/suppl/10.1021/acs.nanolett.5b04221/suppl_file/nl5b04221_si_001.pdf
http://pubs.acs.org/doi/suppl/10.1021/acs.nanolett.5b04221/suppl_file/nl5b04221_si_002.avi
http://pubs.acs.org/doi/suppl/10.1021/acs.nanolett.5b04221/suppl_file/nl5b04221_si_003.avi
http://pubs.acs.org/doi/suppl/10.1021/acs.nanolett.5b04221/suppl_file/nl5b04221_si_004.avi
http://pubs.acs.org/doi/suppl/10.1021/acs.nanolett.5b04221/suppl_file/nl5b04221_si_005.avi
http://pubs.acs.org/doi/suppl/10.1021/acs.nanolett.5b04221/suppl_file/nl5b04221_si_006.avi
http://pubs.acs.org/doi/suppl/10.1021/acs.nanolett.5b04221/suppl_file/nl5b04221_si_007.avi
mailto:m.medina.sanchez@ifw-dresden.de
mailto:l.schwarz@ifw-dresden.de
mailto:o.schmidt@ifw-dresden.de
http://dx.doi.org/10.1021/acs.nanolett.5b04221
(7) Zhang, L.; Abbott, J. J.; Dong, L.; Kratochvil, B. E.; Bell, D.;
Nelson, B. J. Appl. Phys. Lett. 2009, 94 (6), 064107 1−3..
(8) Abbott, J. J.; Peyer, K. E.; Lagomarsino, M. C.; Zhang, L.; Dong,
L.; Kaliakatsos, I. K.; Nelson, B. J. Int. J. Rob. Res. 2009, 28 (11−12),
1434−1447.
(9) Ma, X.; Hahn, K.; Sanchez, S. J. Am. Chem. Soc. 2015, 137, 4976−
4979.
(10) Gao, W.; Dong, R.; Thamphiwatana, S.; Li, J.; Gao, W.; Zhang,
L.; Wang, J. ACS Nano 2015, 9 (1), 117−123.
(11) Wu, Z.; Esteban-Fernańdez de Ávila, B.; Martín, A.;
Christianson, C.; Gao, W.; Thamphiwatana, S. K.; Escarpa, A.; He,
Q.; Zhang, L.; Wang, J. Nanoscale 2015, 7 (32), 13680−13686.
(12) Carlsen, R. W.; Edwards, M. R.; Zhuang, J.; Pacoret, C.; Sitti, M.
Lab Chip 2014, 14, 3850−3859.
(13) Steager, E. B.; Sakar, M. S.; Magee, C.; Kennedy, M.; Cowley,
A.; Kumar, V. Int. J. Robot. Res. 2013, 32 (3), 346−359.
(14) Taherkhani, S.; Mohammadi, M.; Daoud, J.; Martel, S.;
Tabrizian, M.; et al. ACS Nano 2014, 8 (5), 5049−5060.
(15) De Lanauze, D.; Felfoul, O.; Turcot, J.-P.; Mohammadi, M.;
Martel, S. Int. J. Robot. Res. 2014, 33 (3), 359−374.
(16) Huang, T.-Y.; Qiu, F.; Tung, H.-W.; Peyer, K. E.; Shamsudhin,
N.; Pokki, J.; Zhang, L.; Chen, X.-B.; Nelson, B. J.; Sakar, M. S. RSC
Adv. 2014, 4 (51), 26771−26776.
(17) Qiu, F.; Fujita, S.; Mhanna, R.; Zhang, L.; Simona, B. R.;
Nelson, B. J. Adv. Funct. Mater. 2015, 25 (11), 1666−1671.
(18) Mhanna, R.; Qiu, F.; Zhang, L.; Ding, Y.; Sugihara, K.; Zenobi-
Wong, M.; Nelson, B. J. Small 2014, 10 (10), 1953−1957.
(19) Magdanz, V.; Sanchez, S.; Schmidt, O. G. Adv. Mater. 2013, 25
(45), 6581−6588.
(20) Magdanz, V.; Medina-Sańchez, M.; Chen, Y.; Guix, M.; Schmidt,
O. G. Adv. Funct. Mater. 2015, 25 (18), 2763−2770.
(21) Magdanz, V.; Koch, B.; Sanchez, S.; Schmidt, O. G. Small 2015,
11 (7), 781−785.
(22) Magdanz, V.; Guix, M.; Schmidt, O. G. Robot. Biomimetics 2014,
1 (1), 1−11.
(23) Magdanz, V.; Schmidt, O. G. Expert Opin. Drug Delivery 2014,
11 (8), 1−5.
(24) Nadal, F.; Lauga, E. Phys. Fluids 2014, 26, 082001 1−28..
(25) Nuñez-Calonge, R.; Cortes, S.; Gago, M.; Loṕez, P.; Caballero-
Peregrin, P. ISRN Urol. 2012, 2012, 1−6.
(26) Nijs, M.; Vanderzwalmen, P.; Vandamme, B.; Segal-Bertin, G.;
Lejeune, B.; Segal, L.; van Roosendaal, E.; Schoysman, R. Hum. Reprod.
1996, 11 (10), 2180−2185.
(27) Kahraman, S.; Isi̧k, A. Z.; Vicdan, K.; Özgür, S.; Özgün, O. D.
Hum. Reprod. 1997, 12 (2), 292−293.
(28) Ortega, C.; Verheyen, G.; Raick, D.; Camus, M.; Devroey, P.;
Tournaye, H. Hum. Reprod. Update 2011, 17 (5), 684−692.
(29) Nagy, Z. P.; Liu, J.; Joris, H.; Verheyen, G.; Tournaye, H.;
Camus, M.; Derde, M. C.; Devroey, P.; Van Steirteghem, A. C. Hum.
Reprod. 1995, 10 (5), 1123−1129.
(30) Feychting, M. Prog. Biophys. Mol. Biol. 2005, 87 (2−3), 241−
246.
(31) Shellock, F. G.; Crues, J. V. Radiology 2004, 232 (3), 635−652.
(32) http://www.nanoscribe.de, Nanoscribe GmbH, 2007−2015.
(33) Tottori, S.; Zhang, L.; Qiu, F.; Krawczyk, K. K.; Franco-
Obregõn, A.; Nelson, B. J. Adv. Mater. 2012, 24 (6), 811−816.
(34) Lenz, R. W.; Ball, G. D.; Leibfried, M. L.; Ax, R. L.; First, N. L.
Biol. Reprod. 1983, 29, 173−179.
(35) Katska, L.; Smorag, Z. Anim. Reprod. Sci. 1985, 9 (3), 205−212.
(36) Van den Bergh, M.; Emiliani, S.; Biramane, J.; Vannin, a S.;
Englert, Y. Hum. Reprod. 1998, 13 (11), 3103−3107.
(37) Harvey, C. Reproduction 1960, 1, 84−95.
(38) Lauga, E.; Powers, T. R. Rep. Prog. Phys. 2009, 72 (9), 096601
1−36.
(39) Morozov, K. I.; Leshansky, A. M. Nanoscale 2014, 6 (3), 1580−
1588.
(40) Kestin, J.; Sokolov, M.; Wakeham, W. A. J. Phys. Chem. Ref. Data
1978, 7 (3), 941−948.
(41) Jeyendran, R. S.; Van der Ven, H. H.; Perez-Pelaez, M.; Crabo,
B. G.; Zaneveld, L. J. Reproduction 1984, 70 (1), 219−228.
(42) Smikle, C. B.; Turek, P. J. Mol. Reprod. Dev. 1997, 47 (2), 200−
203.
(43) Menkveld, R.; El-garem, Y.; Schill, W.; Henkel, R. J. Assist.
Reprod. Genet. 2003, 20 (10), 432−438.
(44) De Wagenaar, B.; Berendsen, J. T. W.; Bomer, J. G.; Olthuis,
W.; van den Berg, A.; Segerink, L. I. Lab Chip 2015, 15, 1294−1301.
(45) Kuimova, M. K. Phys. Chem. Chem. Phys. 2012, 14, 12671−
12686.
(46) Terasaki, M.; Miyake, K.; McNeil, P. L. J. Cell Biol. 1997, 139
(1), 63−74.
(47) Srivastava, S. K.; Medina-Sańchez, M.; Koch, B.; Schmidt, O. G.
Adv. Mater. 2015, DOI: 10.1002/adma.201504327.
(48) Graham, J. K.; Kunze, E.; Hammerstedt, R. H. Biol. Reprod.
1990, 43 (1), 55−64.
(49) Servant, A.; Qiu, F.; Mazza, M.; Kostarelos, K.; Nelson, B. J. Adv.
Mater. 2015, 27, 2981−2988.
(50) Filonov, G. S.; Piatkevich, K. D.; Ting, L. M.; Zhang, J.; Kim, K.;
Verkhusha, V. V. Nat. Biotechnol. 2011, 29, 757−761.
(51) Moosmann, J.; Ershov, A.; Weinhardt, V.; Baumbach, T.; Prasad,
M. S.; LaBonne, C.; Xiao, X.; Kashef, J.; Hofmann, R. Nat. Protoc.
2014, 9, 294−304.
(52) Pouponneau, P.; Bringout, G.; Martel, S. Ann. Biomed. Eng.
2014, 42 (5), 929−939.
(53) Ahrens, E. T.; Bulte, J. W. M. Nat. Rev. Immunol. 2013, 13, 755−
763.
(54) Olson, E. S.; Orozco, J.; Wu, Z.; Malone, C. D.; Yi, B.; Gao, W.;
et al. Biomaterials 2013, 34 (35), 8918−8924.
(55) Schamel, D.; Mark, A. G.; Gibbs, J.G.; Miksch, C.; Morozov, K.
I.; Leshansky, A. M.; Fischer, P. ACS Nano 2014, 8 (9), 8794−8801.
(56) Nelson, B. J.; Peyer, K. E. ACS Nano 2014, 8 (9), 8718−8724.
(57) Khalil, I. S. M.; Magdanz, V.; Sanchez, S.; Schmidt, O. G.; Misra,
S. IEEE Trans. Robot. 2014, 30 (1), 49−58.
(58) Zhu, L.; Lauga, E.; Brandt, L. J. Fluid Mech. 2013, 726, 285−311.
(59) Martel, S. IEEE Cont. Syst. 2013, 33 (6), 119−134.
(60) Martel, S.; Mohammadi, M.; Felfoul, O.; Lu, Z.; Pouponneau, P.
Int. J. Rob. Res. 2009, 28 (4), 571−582.
Nano Letters Letter
DOI: 10.1021/acs.nanolett.5b04221
Nano Lett. XXXX, XXX, XXX−XXX
G
http://www.nanoscribe.de
http://dx.doi.org/10.1002/adma.201504327
http://dx.doi.org/10.1021/acs.nanolett.5b04221