Cellular Cargo Delivery: Toward Assisted ... - ACS Publications

We manage to capture, transport, and release single immotile live sperm cells in fluidic channels ..... Small-Scale Machines Driven by External Power ...
0 downloads 0 Views 7MB Size
Download PDF
Letter pubs.acs.org/NanoLett

Cellular Cargo Delivery: Toward Assisted Fertilization by SpermCarrying Micromotors Mariana Medina-Sánchez,*,† 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

T

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 magnetotactic bacteria as controllable motor units which showed

he operation of miniaturized vehicles that perform tasks and 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 successfully 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 chronologically. 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 © XXXX American Chemical Society

Received: October 16, 2015 Revised: December 16, 2015

A

DOI: 10.1021/acs.nanolett.5b04221 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

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 of our 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

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 biocompatible 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 micromotors 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

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.

Figure 1. An immotile sperm is captured by a remotely controlled magnetic helix and delivered to the oocyte for fertilization.

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

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 B

DOI: 10.1021/acs.nanolett.5b04221 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters 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) highlights 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

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.

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 will immediately turn back to its full length. This C

DOI: 10.1021/acs.nanolett.5b04221 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters 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

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.

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 needleshaped 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 Information, 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 NiTicoated 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

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.

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 precondition 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 D

DOI: 10.1021/acs.nanolett.5b04221 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

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

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.

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 experiments 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 be verified 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

Figure 7. Sperm cell coupling (i), transport (ii), approach to the oocyte membrane (iii), and release (iv).

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. E

DOI: 10.1021/acs.nanolett.5b04221 Nano Lett. XXXX, XXX, XXX−XXX

Nano Letters

■

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 fertilization. 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.

Letter

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.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: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. 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 Nicolás Pérez 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 realtime 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.; Merkoçi, 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-Martı ́nez, C. C.; Merkoçi, A. Chem. Rev. 2014, 114 (12), 6285−6322.

F

DOI: 10.1021/acs.nanolett.5b04221 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters (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-Fernández de Á vila, 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.; ZenobiWong, 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-Sánchez, 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.; López, P.; CaballeroPeregrin, 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.; Işik, A. Z.; Vicdan, K.; Ö zgür, S.; Ö zgün, 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.; FrancoObregõn, 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-Sánchez, 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.

G

DOI: 10.1021/acs.nanolett.5b04221 Nano Lett. XXXX, XXX, XXX−XXX