IRONSperm: Sperm-templated soft magnetic microrobots

Fabrication and characterization of IRONSperms

IRONSperms are fabricated using a suspension of rice grain–shaped maghemite nanoparticles with a 100-nm diameter and bovine sperm cells (~60 μm long). The particles in the suspension are influenced by electrostatic forces (van der Waals and Coulomb forces). The van der Waals attraction is limited and decays asymptotically as r⁻⁷, where r is the distance between adjacent particles, and results in aggregation when particles collide. The formed aggregates and sperm cells are oppositely charged, thereby inducing long-range Coulomb attractive forces that decay as ri−2, where r_i is the distance between the ith aggregate and the nearest point along the sperm cell (Fig. 1F). The energy needed to bring the ith aggregate to this point must be smaller than the thermal energy (41)14πϵ0qniqs(s)ri<3 kBT*(1)where q_ni is the charge of the ith aggregate of nanoparticles and q_s(s) is the charge along the sperm body as a function of the arc length s. Further, ϵ₀ is the permittivity, k_B is the Boltzmann factor, and T^* is the characteristic temperature. Figure 1F shows two representative simulation results based on the electric charge balance (Eq. 1) between the sperm cell and randomly arranged nanoparticles. These particles display an average surface zeta potential of 12.9 mV in water, while sperm cells display an overall net negative surface potential (38) with nonuniform distribution of charges, also allowing areas with neutral or positive charges. The net charge of the sperm cell and the nearby aggregates determine the amount of particles that coat the sperm cell. The resulting IRONSperms are shown exemplarily in Fig. 1A in electron micrographs and in Fig. 2A in optical microscopic images. We observe a nonuniform distribution of nanoparticles along the head, midpiece, principal piece, and distal end, as displayed exemplarily by cases in Figs. 1 (B to D) and 2A. The net charge and the spatial distribution along the sperm cell are important factors in the determination of the magnetization profile and flexibility of IRONSperm. The amount of particles per cell is indicative of the magnetic response to the external magnetic field, so the magnetization is preferably high to enable actuation in the milliTesla range (2 mT) while maintaining the flexibility of the flagellum to enable flagellar wave propagation. Figure 2A shows the nonuniform coating of nanoparticles along the cell that results in distributed actuation under the influence of an external magnetic field. The particle distribution is also highly dependent on the surface charge of the sperm cells. The magnetization along the sperm cell is approximated by four magnetic dipole moments according to the four morphological distinct segments of spermatozoa m_h (head), m_m (midpiece), m_p (principal piece), and m_d (distal end), as shown in Fig. 1 (B to E).

The distributed actuation of IRONSperm is represented by approximating the magnetic dipole moment of each segment along the sperm cell. These magnetic dipole moments generate magnetic torque (m_i × B) to counterbalance the drag torque in the medium, where B is the external magnetic field and i = h, m, p, d, indicating the head, midpiece, principal piece, and distal end, respectively. The governing equation of IRONSperm balances all forces normal to the body as follows (42)κi∂4φi∂s4(s,t)+cni∂φi∂t(s,t)=0 for i=h,m,p,d(2)where κ_i is the bending stiffness of the ith segment along IRONSperm and φ_i(s, t) is its tangent angle. The tangent angle is enclosed between the long axis (e₁(t)) of the sperm and the local tangent at s. Further, c_ni is the normal drag coefficient of the ith segment. The force κi∂4φ∂s4 tends to restore the flagellum to its original natural shape, and cni∂φ∂t is the viscous drag force exerted by the fluid. IRONSperms are driven by a bending moment applied by the external magnetic field at the location of the nanoparticle aggregates (Fig. 2A). These bending moments induce a traveling wave that propagates from the head to the distal end, producing a net thrust force that propels IRONSperm forward, as shown in Fig. 2B. Therefore, flexibility of the flagellum is required to achieve wave propagation and is tested by allowing the sample to reside on a surface (Fig. 2C). Once adhered to the surface, a magnetic torque is exerted to observe the flexibility of IRONSperm. Figure 2C shows that the flagellum deforms with the position of the distal end fixed (see also movie S2). The blue dots indicate the trajectory of the head and represent an ellipse with minor and major diameters of 50 and 82 μm, respectively. Therefore, the flagellum of IRONSperm maintains its flexibility after the electrostatic-based self-assembly.

Although Fig. 2C indicates relatively large deformation of IRONSperm when its distal end is fixed, applying rotating magnetic field is not likely to yield the same deformation during propulsion. One valid argument that then arises is whether the motion demonstrated in Fig. 2B is due to (i) rigid body rotation of IRONSperm under the action of the rotating magnetic field or (ii) helical traveling waves along a flexible flagellum under the influence of the same actuating field. In swimming motion using wave propagation along a flexible filament, the amplitude of the wave pattern is proportional to the characteristic penetration length (43), ℓw=(κωcn)1/4, where ω is the angular velocity of the filament. This length depends only on the bending stiffness of the flagellum, the frequency of oscillation, and the viscosity of the medium. Variation of these parameters only affects the wave pattern in the case of propulsion with traveling waves (44). Therefore, we apply rotating magnetic fields at a frequency range of 3 ≤ f ≤11 Hz. The time-dependent deformation and tangent angle of the flagellum are measured for successive intervals of motion. At each frequency, the maximum amplitude of the overall wave pattern is measured as the vertical distance between the envelopes of motion, indicated by the dashed curves in the left column of graphs in Fig. 3. At f = 3 Hz, the maximum amplitude is measured as 37 μm (Fig. 3A). The amplitude decreases to approximately 30 μm at actuation frequencies of 5 and 7 Hz (Fig. 3, C and E). As the frequency increases, we observe that the overall wave pattern scales down and decreases to 23 (Fig. 3G) and 18 μm (Fig. 3I) at 9 and 11 Hz, respectively. Therefore, we attribute the propulsion of IRONSperm to the traveling wave along its flexible flagellum. Figure 3 (B, D, F, H, and J) also shows the calculated tangent angle along the flagellum based on the measured wave patterns for an actuation frequency range of 3 ≤ f ≤11 Hz.

We measure the area covered by rice grain–shaped 100-nm nanoparticles at each segment of IRONSperm to obtain the ratio of magnetic material per cell segment and its net magnetization (see Materials and Methods and fig. S1). The average magnetic dipole moment of IRONSperm is 5.9 × 10⁻¹¹Am² at a magnetic field of 5 mT, whereas the average remanent magnetization is 5.2 × 10⁻¹¹ Am². The magnetization vector changes its direction when the field is applied and is enclosed between the local tangent of each segment and the magnetic field at this segment (45). The ratio of magnetic material per segment over the total magnetic material per cell for the head, midpiece, principal piece, and distal tip are 31, 8, 51, and 10%, respectively (see Materials and Methods and fig. S1 in supporting information). The samples are likely to have a different distribution of nanoparticles owing to the cell-to-cell variability in surface charge (38). These ratios are used in the determination of the magnetic dipole moments of each segment, and Eq. 2 is numerically solved to predict the response of IRONSperm. The contribution of the magnetic torque in Eq. 2 is included by specifying the boundary conditions between each segment (see Materials and Methods). Equation 2 can also be used to predict the response of IRONSperm in close proximity to a solid boundary by specifying the drag coefficient (c_ni) as a function of the distance to this boundary (46).

Magnetization distribution and magnetic actuation

Similar to sperm cells, IRONSperms display either a planar two-dimensional or a helical three-dimensional flagellar wave (47). In the case of a planar flagellar wave, periodic magnetic fields constrained to a plane can be used to propel IRONSperm. In the case of a helical flagellar wave, periodic out-of-plane fields with a cone angle (α_m) enable the head to follow a finite steradian. The axis of the steradian concedes with the long axis of IRONSperm, as shown in Fig. 4A. On the basis of the magnetization distribution experiments and the measured magnetic properties, we enter the measured magnetization of each segment into the hydrodynamic model (Eq. 2). The time-dependent flagellum deformation is calculated for actuation frequency of 1 ≤ f ≤ 20 Hz and a magnetic field cone angle of 10° ≤ α_m ≤ 90°.

Figure 4A shows the time-dependent flagellum deformation for f = 1 Hz and α = 15°. The maximum amplitude (vertical distance between the propulsion axis e₁ and the envelope) of the propagating wave is 2 μm, producing negligible net thrust. This amplitude is determined on the basis of the transverse deviation from the propulsion axis to the flagellum, as a function of the arc length. For f = 1 Hz and α =30°, we also observe a similar response and zero net displacement is achieved for a propagating wave with an amplitude of 4 μm (Fig. 4B). For f = 1 Hz and α = 45°, the amplitude of the propagating wave increases to 6 μm (Fig. 4C), producing a net thrust force that propels IRONSperms forward at a speed of 2 μm/s. Therefore, a relatively large cone angle would maximize the amplitude of the propagating wave and enhance the propulsion. The actuation frequency also has a substantial influence on the swimming velocity. Figure 4 (D to F) shows the time-dependent flagellum deformation for actuation frequencies of 1, 5, and 10 Hz and α = 45°, respectively. For f = 1 Hz and α = 45°, IRONSperm propels at a speed of 2 μm/s by a propagating wave with an amplitude of 6 μm. For f = 5 Hz and α = 45°, the speed increases to 7 μm/s for a wave with an amplitude of 3 μm, as shown in Fig. 4E. For f = 10 Hz and α = 45°, a maximum amplitude of 4 μm along the wave is generated and enables IRONSperms to propel at a speed of 9 μm/s (Fig. 4F). The dependence of IRONSperm velocity on cone angle and frequency as calculated by our model is depicted in Fig. 4G. Similar to the cone angle of the actuating magnetic field, the actuation frequency linearly increases the swimming speed and decreases slowly above a step-out frequency of approximately 10 Hz, as shown in Fig. 4G.

Although we have control over the actuation frequency and the cone angle of the magnetic fields, there is intrinsic variation in the distribution of the nanoparticles along the four segments of the sperm cell. The charge distribution of sperm is not uniform and also changes depending on the sperm’s developmental state. It is therefore expected that many different configurations of sperm-particle constructs are obtained by self-assembly of nanoparticles with a bull semen sample that naturally contains sperm of different quality. The number of nanoparticles, and also their attachment location on the sperm’s membrane, vary from case to case. The large experimental deviation of IRONsperm velocities over the range of applied frequency can thus be explained by the various configurations of IRONsperms.

This variation is evident in the characterized frequency response of IRONSperm, as shown in Fig. 4H. In this experiment, IRONSperms are allowed to swim under the influence of a rotating magnetic field at a frequency range of 0 to 20 Hz. Each data point in Fig. 4H represents the average speed of an IRONSperm sample at the respective actuation frequency. The error bar depicts the SD of the measurements for 15 different samples. At an actuation frequency of 1 Hz, an IRONSperm swims at an average speed of 2.3 ± 1.7 μm/s. The swimming velocity increases linearly with the actuation frequency up to f = 8 Hz. At this actuation frequency, the swimming velocity is 6.8 ± 4.1 μm/s, and we observe a step-out behavior above an actuation frequency of 10 Hz. Figure 4 (G and H) shows quantitative agreement between our measurements and theoretical predictions. Our theoretical predictions and measurements show that as the angular frequency increases from zero, the swimming velocity increases, reaches a maximum at approximately 8 to 10 Hz, and decreases slowly. In the case that the actuation frequency is below 8 Hz and the cone angle is between 30° to 50°, our measurements are in quantitative agreement with the numerical results. At f = 2 Hz, IRONSperms swim at 3.3 ± 1.8 μm/s, whereas numerical results indicate speeds of 1.7, 2.8, and 3.9 μm/s for cone angles of 30°, 40°, and 50°, respectively. The numerical model predicts also that, as the cone angle increases to 90°, the swimming speed increases. However, this cone angle is not achieved experimentally owing to the limited flexibility of IRONSperm. With a flexible flagellum, the capability of the magnetic head to align with the rotating magnetic field is governed by the magnetoelastic coupling between the flagellum and the magnetic torque. Therefore, the numerical model suggests that, by increasing the cone angle of the head, the swimming velocity increases. In experiments, this enhancement is possible by increasing the applied magnetic field or by increasing the magnetic moment of the head. Increasing the magnetic field is limited by the projection distance from the source, so the magnitude is preferably in the milliTesla range. This range enables the actuating field to span a few centimeters at most, and we limit the magnetic field to 2 mT in all our experiments. In the case of increasing the magnetic moment of the head, the nanoparticle aggregates that coat the sperm cell govern the magneto-elastic coupling. One can increase the induced magnetic torque exerted on the head by increasing the concentration of nanoparticle aggregates per cell. Increasing the concentration generates a stronger torque, but there is an upper limit on the amount of nanoparticles that covers the cell based on the net surface charge between the sperm cell and the particles (Fig. 1F). In addition, our measurements show that the head and principal piece of the cell are, on average, coated with 31 and 51% of the total amount of nanoparticle aggregates, and it is essential to maintain good magnetization and minimal adverse effect on the flexibility of the flagellum.

The flagellar propulsion of IRONSperms and motile sperm cells show good resemblance, although 51% of the nanoparticle aggregates coat its principal piece. A representative flagellar waveform of IRONSperms is illustrated in Fig. 5. In this experiment, a periodic magnetic field is applied at an actuation frequency of 2 Hz using triaxial Helmholtz electromagnetic coils, as shown in Fig. 5A. Figure 5 (B and C) demonstrates the flexibility of the flagellum. First, the time-dependent tail deformation is measured over consecutive beat cycles (Fig. 5B), and the amplitudes of the head and the distal end are measured (Fig. 5C). In contrast to the head, the amplitude of the distal end decreases from 21.2 μm at t = 0 s to 7.9 μm at t = 13 s. We attribute this decrease to the flexibility of the flagellum, which is also influenced by the properties of the medium (consisting of an IRONSperm and nanoparticle aggregates). In this trial, the average swimming speed is measured as 10.8 μm/s. The amplitude of the waveform near the distal end is nearly twice the amplitude near the head, as shown in Fig. 5C. This proves that an asymmetrical shape deformation occurs, which is an important requirement to break Purcell’s scallop theorem (48).

Riedel-Kruse et al. (49) and Friedrich et al. (50) have shown that the flagellar shape forms (Figs. 2 and 5D) can be used to determine the zeroth (Ψ₀) and first (Ψ₁) Fourier modes of the Fourier representation of the tangent angle φ(s, t). They have implemented this Fourier analysis of the waveforms on two-dimensional projections of the flagellum during circular swimming near a surface. In our case, the cell is not alive. Its flagellum is passive and does not undergo similar anisotropic and asymmetric beating as live cells (51), which is originated by the active bending moment of the flagellum. Therefore, the flagellar waveform of IRONSperm is preserved during rotation and two-dimensional projections can be used to estimate the time-averaged shape, bending amplitude, and the wave propagation speed. The zeroth mode provides a measure for the asymmetry of the flagella, whereas the first mode is a measure for the magnitude of the bending amplitude along the flagellum and the wavelength. In Fig. 5D, the angle enclosed between the propulsion axis of IRONSperm and the local tangent along the flagellum is calculated from the measured waveform of the flagellum over a complete beat cycle. The zeroth and first Fourier modes are calculated at all arc length positions s, as shown in Fig. 5E. The zeroth mode Ψ₀ indicates that the curvature along the flagellum is approximately 8.8 rad/mm. The curvature is calculated by fitting a line to the zeroth mode data points. Similar to the curvature, the amplitude of the first Fourier mode Ψ₁ indicates that the amplitude rise of the flagellum is 13.5 rad/mm. The amplitude rise is calculated by fitting a line to the amplitude of Ψ₁. Last, the wavelength is calculated by fitting a line to the phase angle of the first Fourier mode of the tangent angle. The calculated wavelength is 150 μm.

Riedel-Kruse et al. (49) and Friedrich et al. (50) have shown that the flagellar waveform of sperm cells is characterized by average curvature, amplitude rise, and wavelength of 13.4 ± 4.8, 14.6 ± 1.2 rad/mm, and 66 ± 8 μm, in water at 36°C. IRONSperms are characterized by a curvature of 8.1 rad/mm, an amplitude rise of 13.5 rad/mm, and a wavelength of 150 μm. Therefore, there is a resemblance between the flagellar waveform of the passive flagellum of IRONSperms and the active flagellum of live sperm cells (52).

Magnetic control and localization

IRONSperms have the capability to swim controllably by a rotating magnetic field produced around an arbitrary axis in space. These fields are generated using a triaxial Helmholtz electromagnetic coil setup, as shown in Fig. 5A. The IRONsperms are actuated in the bulk of the fluid, while unbound nanoparticle aggregates remain at the bottom of the reservoir. A representative open-loop control experiment is demonstrated in Fig. 6A. Rotating magnetic fields are generated around the propulsion axis (e₁) of the flagellum at a frequency of 4 Hz. The propulsion axis (e₁) is oriented toward the bottom-left corner of a square reference trajectory with an edge length of 260 μm. Second, the field is rotated around this axis to generate the flagellar propulsion. At t = 0 s, the IRONSperm is located at the bottom left corner of the square, and its propulsion axis is aligned along the x axis while simultaneously rotating the field. The IRONSperm swims at an average speed of 7.2 μm/s along the square reference trajectory. At t = 47 s, the propulsion axis is rotated along the y axis while simultaneously rotating the field. To keep the IRONSperm in the same plane, the pitch angle of its propulsion axis is set to 15° to compensate for gravitational effects (53). In another open-loop experiment, an IRONSperm is allowed to reverse its swimming direction by rotating the propulsion axis gradually with respect to the z axis, as shown in Fig. 6B. This trial indicates that the IRONSperm undergoes a U-turn trajectory while swimming at an average speed of 12.5 μm/s, under the influence of a rotating magnetic field at 6 Hz. These experiments demonstrate that IRONSperms have the capacity to be actuated using relatively weak magnetic fields (2 mT). The magnetic constituent of IRONSperm not only offers magnetic remote control and actuation of single sperm cells but also enables localization of groups of IRONSperm using ultrasound images. One of the key challenges of using IRONSperms in targeted in vivo drug delivery applications is the localization and control using a medical imaging modality. Therefore, we investigate the localization of IRONSperms by ultrasound imaging, which is a high-resolution, real-time, and noninvasive medical imaging method (1).

The difference in acoustic impedance between sperm cells and their background fluids and soft tissue is relatively low. Therefore, localization of sperm cells using ultrasound feedback is not possible. The magnetic constituent of IRONSperms increases the acoustic impedance and thereby allows the detection of swarms of IRONSperms with ultrasound waves. The diameter and length of the elongated particles that coat IRONSperms are 10 and 100 nm, respectively, while the wavelength of a 10-MHz wave is approximately 1540/(10 × 10⁻⁶) m = 154 μm. This wavelength dictates the limit of the smallest objects that can be detected. It is possible to coat the whole cell (length of 60 to70 μm) with particles. Even then, only a 20-MHz wave could enable the localization of a single IRONSperm. However, clusters of IRONSperms can be detected, but a minimum size of the cluster has to be comparable to the wavelength of the wave.

To test the echogenicity of clusters of IRONSperms, we insert samples inside polyethylene tubes with an inner diameter of 380 μm (Fig. 7A). The samples are detected by a 16-MHz wave and visual feedback, as shown in Fig. 7 (B and C), respectively. In this trial, the tube is contained inside a transparent water reservoir to achieve air-free coupling between the tube and the ultrasound transducer. The water reservoir is mounted on an inverted microscope to compare ultrasound scans to microscopic images (Fig. 7A). Figure 7B shows the response of several clusters to an external magnetic field. The length of the clusters is approximately 200 μm and contains several IRONSperm samples. The green arrows indicate the location of these clusters in the ultrasound scans in Fig. 6C. This experiment shows that the magnetic constituent provides a relatively high contrast-to-noise ratio for the detection and localization of IRONSperms.

Loading of a molecular cargo for further therapeutic tasks

Doxorubicin hydrochloride (DOX-HCl), a widely applied anticancer drug, is used as a model drug to evaluate the loading capability of the here-presented IRONSperms (nonviable sperm decorated with magnetic nanoparticles). Thus, both the drug and IRONSperms were coincubated at room temperature and under mild agitation for about 1 hour, adapting an already reported protocol (29) (see Materials and Methods). After washing twice with water, drug-loaded IRONsperms were suspended in sperm medium (modified Tyrode’s albumin lactate pyruvate medium) to evaluate their drug-loading capability by using fluorescence microscopy and spectrophotometry. As DOX-HCl is autofluorescent at 485-nm excitation wavelength, fluorescent microscopy was sufficient to confirm the presence of the drug in IRONsperms (Fig. 8, A and C). As it can be seen in Fig. 8 (A and B), IRONSperms were successfully loaded with DOX-HCl. In contrast to the previously reported work, where the drug was mainly found in the nucleus of living sperm because of the high affinity of DOX-HCl to DNA (29), in the IRONsperms, the drug was found in the whole body, including the midpiece and tail. A possible reason might be the rupture of the sperm membrane during its interaction with the magnetic nanoparticles (in water), which facilitates the drug adsorption on the remaining proteins and lipid layers. However, further investigations using high-resolution microscopy are required to confirm their precise localization. Furthermore, DOX-HCl loading efficiency and loading amount per sperm were determined by optical spectroscopy. After 1 hour of incubation, 11.3 ± 0.5% of DOX-HCl was taken up by sperms when 1.3 × 10⁶ sperms/ml was immersed in a DOX-HCl solution at a final concentration of 50 μg/ml (see details in the Materials and Methods). A single IRONsperm was able to load 4.3 ± 0.2 pg of DOX-HCl. This amount was calculated after performing the linear regression of the amount found in the supernatant of the IRONsperm sample (supernatant was diluted 500 times to be in the linear range; see details in the Materials and Methods) (Fig. 8D). The cumulative release test shows the loading stability of the IRONsperm. After 72 hours of incubation, 21.4 ± 0.5% of the drug was kept inside the IRONsperm (Fig. 8E), although a burst release was observed in the first 8 hours. IRONsperms can be thus envisioned as potential drug carrier, thanks to their high encapsulation capacity and stability.

The biocompatibility of IRONsperm was tested by coculturing IRONsperm with Ishikawa cells, a human endometrial adenocarcinoma cell line. IRONSperms were cocultured with Ishikawa cells for 3 days. After 24 hours and after 3 days, we performed a trypan blue assay, a standard cytotoxicity test that stains any dead cells. No increased number of dead cells was observed after 24 hours or 3 days, respectively, as displayed in fig. S2. This proves the biocompatibility of IRONsperm and thus prospects for biomedical applications.

Synthesis of elongated Fe₂O₃ particles

Rice grain–shaped particles were synthesized following a procedure by Ohmori and Matijević (54). A solution of FeCl₃·6H₂O (0.02 M) was aged with a NaH₂PO₄ solution (0.0004 M) during 72 hours in a Pyrex bottle at 100°C. The obtained product was centrifuged and washed with acetone. The nanoparticles were repeatedly washed and centrifuged in distilled water.

Fabrication of IRONSperms

Cryo-preserved bovine sperm straws (Masterrind GmbH) were thawed in a 38°C water bath for 2 min and resuspended in sperm medium SP-TALP (Caisson Laboratories, Inc., modified Tyrode’s albumin lactate pyruvate medium; one straw per 1 ml of SP-TALP). The sperm suspension was centrifuged for 5 min at 300g in a soft mode. The supernatant was discarded, and the pellet was resuspended in distilled water. The sample was centrifuged again with the same settings and lastly resuspended in 500 μl of water.

Particle suspensions were centrifuged and resuspended in distilled water, vortexed, and well suspended by sonicating for 10 min before adding 50 μl of this particle suspension to 50 μl of sperm suspension. The mixture was incubated at room temperature for at least 24 hours before use. Because of the incubation of the bull sperm with particles in deionized water for 24 hours, the spermatozoa were not motile anymore. For storage, the IRONSperm samples were kept at 4°C for several weeks.

Structural characterization and attachment distribution

The average head length of the bovine sperm cells is 8 to 10 μm. The average values for midpiece, principal piece, and distal end are 13, 40, and 7 μm in length, respectively (55–57). The electrostatic self-assembly process causes the nanoparticles to nonuniformly adhere to the four segments of the sperm body. High-resolution microscope images of IRONsperm were obtained with a Leica DMi8 inverted microscope with a 100× oil immersion objective. ImageJ was used to subtract the background and calculate the particle-covered area for each segment (see fig. S1). The ratio between the adhered nanoparticles and each segment is measured. The average surface area of the head, midpiece, principal piece, and distal end are 16.5, 10.7, 56.9, and 11.4 μm², respectively. Optical microscopic images (×1000 total magnification, obtained with the Leica DMi8 inverted microscope, 100× oil immersion objective, Leica DFC 450 C camera) of IRONSperms are analyzed, and the ratio of area of adhered nanoparticles of each segment per the total nanoparticle area on the sperm cell is measured as 31, 8, 51, and 10% for the head, midpiece, principal piece, and distal end, respectively.

Cryo–scanning electron microscopy

The cryo-(FE-)SEM SUPRA 40VP-31-79 (Carl Zeiss SMT Ltd., Oberkochen, Germany) equipped with an EMITECH K250X cryo-preparation unit (Quorum Technologies Ltd., Ashford, Kent, UK) was used for imaging of the sperm-particle constructs. Two microliters of suspension was dropped onto a glass slide fixed to the cryo–scanning electron microscope (cryo-SEM) holder and immediately shock-frozen in liquid nitrogen in the slushing chamber. From there, it was transferred to the cryo-preparation chamber at −140°C, sublimed for 15 min at −70°C, and sputter-coated with platinum (layer thickness ca. 6 nm). Subsequently, it was transferred to the SEM and then examined in a frozen state at 5-kV accelerating voltage and −100°C temperature using the secondary electron detector.

Fluidic and structural effects

IRONSperm consists of a bovine sperm cell with nonuniform iron oxide nanoparticle coating. The prolate spheroidal head of length 2a and radius b has an average magnetic moment m_h. The head is connected to a midpiece of length l_m and radius r_m and average magnetic moment m_m. Flagellar beat is achieved by the principal piece and the distal end of the flagellum (radius r_f and r_d) with length of l_p and l_d and magnetic moment of m_p and m_d, respectively. Flagellar wave propagation along the flagellum (with bending stiffness κ) is achieved by exerting a dynamic magnetic torque on the magnetic moments of IRONSperm. This wave propagation enables IRONSperm to swim in a medium with viscosity μ, characterized by low–Reynolds number hydrodynamics (Re = ρv_x(L + 2a)/μ) where ρ, μ, and L are the density and viscosity of the medium and the length of the flagellum, respectively. Further, v_x is the swimming speed of IRONSperm. At actuation frequency range of 1 to 20 Hz, the average swimming speed varies between 0.007 to 0.5 body length/s, and Re is on the order of 𝒪(10⁻⁴). In this regime, the deformation of the flagellum is governed by elastic forces and viscous forces as followsκi∂4φi∂s4(si,t)+cni∂φi∂t(s,t)=0 for i=h,m,p,d(3)where κ_i is the bending stiffness of the ith segment along IRONSperm and φ_i(s, t) is its tangent angle. Further, c_ni is the normal drag coefficient of the ith segment and given bycni=4πμ/(ln(liri)+0.193)(4)

The influence of a nearby surface can be incorporated into the normal drag coefficient to study the response of IRONSperm near a surface (46). In (Eq. 3), φ_i(s, t) represents the deformation of the ith segment of IRONSperm, relative to a fixed frame of reference (e₁(t), e₂(t)), where e₁(t) and e₂(t) are orthonormal vectors such that e₁(t) is oriented along the long axis of the head. The position coordinates are calculated usingxi(s,t)=xi(0,t)+∑s=0sΔs cos(φi(s,t))yi(s,t)=yi(0,t)+∑s=0sΔs sin(φi(s,t))zi(s,t)=yi(s,t−T/4)(5)

The period time T is related to angular frequency by T = 2π/ω (Eq. 2), and ∆s is the discretization length ∆s=(lm+lp+ld)N with N = 100. The contribution of the magnetic torque (m_i × B) is included by specifying the boundary conditions between each segment, where B is the external magnetic field with the following componentsB=B(cos(αh)sin(αh)cos(ωt)sin(αh)sin(ωt))(6)

The head follows the magnetic field. At the right boundary of the head and the left boundary of the midpiece, s = 0,the midpiece orientation is φ_m(0, t) = α_m sin (ωt). At the right boundary of the midpiece, s = l_m, and the left boundary of the principal piece, s = l_m, the boundary condition is φ_m(l_m, t) = φ_p(l_m, t) = α_p sin (ωt). At the right boundary of the principal piece, s = l_m + l_p, and the left boundary of the distal end, the orientation is φ_p(l_p + l_m, t) = φ_d(l_m + l_p, t) = α_d sin (ωt). At the right boundary of the distal end, s = L, the flagellum is free from external forces and torques. Therefore, ∂2φd(L,t)∂s2=0 and ∂3φd(L,t)∂s3=0. The magnetic variables α_m, α_p, and α_d are determined by minimizing the magnetic energy U=−∑imi⋅B+0.5∫s=0s=Lκi(dφids)2ds, for i = h, m, p, d. The magnetization vectors are given bymi=mi((xi(s+Δs,t)−xi(s,t))(yi(s+Δs,t)−yi(s,t))(zi(s+Δs,t)−zi(s,t)))/|((xi(s+Δs,t)−xi(s,t))(yi(s+Δs,t)−yi(s,t))(zi(s+Δs,t)−zi(s,t)))|(7)

The angle of the magnetization of each segment is enclosed between the local tangent and the magnetic field for each segment along IRONSperm (45). Stokeslet points are distributed randomly on the surface of an ellipsoid representing the head with distance ∆s between the points (58, 59). Along the tail (x_i, y_i, and z_i), Stokeslet points are created in circles of radius r_i perpendicular to the tail tangent. The average distance between Stokeslet points is ∆s. The speed of the Stokeslet points (x_N, y_N, and z_N), is defined as u_N = (V_x, − ωz_i, ωy_i). With the Stokeslet relation relating force F= [F₁, …F_N] with velocities via the matrix A (60). The total force the propulsion axis (x axis) is determined for each V_x till the force in x direction is zero. This force balance provides the force free speed V_x.

Magnetic characterization and magnetic actuation

The magnetic moment of IRONSperm samples was measured using a vibrating sample magnetometer (VSM), Oxford Instruments, equipped with a variable temperature insert. For this purpose, the IRONSperm suspension in distilled water containing a concentration of 1.7 × 10⁵ cells/ml was placed in a plastic container and then attached to a sample holder made of polyether ether ketone material. The subsequent measurements were carried out at a temperature T = 300 K, a measurement frequency f = 55 Hz, and an amplitude x = 1 mm after calibration (determination of the geometry factor) with a nickel sample. The results show a hysteresis in the range of the external magnetic field μ₀H = ±2 T, characteristic for ferro- and ferrimagnetic materials. The curve and the low coercive field strength μ₀H < 0.1 T show the soft magnetic character of the magnetic sample fraction. The remanent magnetic moment of m_r = 5.2 × 10⁻¹¹ Am² is considerably smaller than the specific value of the magnetic starting material. Because of the known cell concentration in the suspension, the measured magnetic moment was related to the amount of IRONSperms in the sample. Samples with concentration of 1.7 × 10⁵ cells/ml provide a maximum magnetic dipole moment of 4 × 10⁻¹⁰ Am² at magnetic field of 2 T for each IRONsperm.

Frequency response experiments were performed at a maximum magnetic field of 5 mT, and the average magnetic dipole moment of IRONSperms is 5.9 × 10⁻¹¹ Am² (at 5mT). The propulsion of IRONSperms is conducted on a triaxial Helmholtz coil system. The samples are contained in the common center of three orthogonal nested solenoids. The outer diameter of the outer, middle, and inner solenoids is 16, 12, and 7 cm, respectively. The system generates a maximum magnetic field of 5 mT using a current input of 1 A. The samples are actuated by homogeneous magnetic fields with a periodically varying component to enable the head to follow a finite steradian and induce a helical traveling wave along the flagellum.

Drug loading into IRONsperms

Bovine sperm cells with the immobilized magnetic nanoparticles at a concentration of 1.3 × 10⁶ sperms/ml were suspended in a final concentration of DOX-HCl of 50 μg/ml and coincubated at room temperature for ca. 1 hour. After the sample was washed twice with water by centrifugation at 300g for 5 min, the pellet of drug-loaded IRONSperm was redispersed in SP-TALP [modified Tyrode’s albumin lactate pyruvate medium (Caisson Laboratories, Inc.)] and stored under dark conditions before fluorescence imaging analysis. Fluorescence images were taken at an excitation wavelength of 470 nm (DOX-HCl: excitation, 470 nm; emission, 580 nm; Cell Observer, Carl Zeiss Microscopy GmbH).

To determine the drug loading efficiency, samples of DOX-HCl solution were prepared in a series of concentrations (1.25, 0.62, 0.31, 0.16, 0.08, and 0.04 μg/ml) to obtain the calibration curve in the linear range. After incubation of sperms in drug solution (50 μg/ml), the respective supernatant was collected after centrifugation and filtered through a 2-μm–pore size membrane. The concentration of DOX-HCl was determined with a fluorescence spectrometer in a fluorescence intensity mode (SpectraMax M2, Molecular Devices LLC), and the total concentration was calculated considering that the sample supernatant had to be diluted 500 times to be in the linear range of the drug calibration curve (Fig. 7D). After doing the linear regression and by multiplying the resulting value 500 times, the total amount of drug found in the supernatant was about 44.4 μg/ml. If the initial drug concentration was 50 μg/ml, the expected amount of drug loaded on the IRONsperm and surrounding particles is then the difference between the initial drug concentration and the measured drug concentration in the supernatant resulting in ca. 5.7 μg/ml DOX-HCl. Then, by dividing 5.67 μg/ml by 1.3 × 10⁶ sperms/ml, an amount of 4.26 ± 0.2 pg per sperm was obtained.

The accumulative release test was performed as reported elsewhere (29). DOX-HCl–loaded IRONsperms were incubated under dark conditions. At each time point (Fig. 7E), the samples were centrifuged, and 0.2 ml of supernatant was collected and replaced with 0.2 ml of water. The accumulative release ration was calculated as the ratio of the accumulated amount of released drug to the total loaded drug. SDs correspond to four technical replicates.

Cytotoxicity tests

Ishikawa cells were cultured in Dulbecco’s modified Eagle’s medium/F12 medium containing 10% fetal calf serum, 1% insulin, and 1% penicillin/streptomycin at 37°C and 5% CO₂. IRONsperms were prepared as described above from two cryo-preserved semen straws by incubating the thawed and washed sperm with iron oxide particles, resulting in 10⁷ IRONsperms. These IRONsperms were centrifuged and resuspended in 300 μl of phosphate-buffered saline (PBS) and distributed equally on three cell flasks, each containing 5 ml of Ishikawa cells in culture. After 24 hours of coculture at 37°C, the culture medium was removed, and 100 μl of PBS was added directly to the culture flasks. Then, 100 μl of 0.4% sterile-filtered Trypan Blue solution (Sigma-Aldrich) was added to each cell flask to obtain 0.2% trypan blue solution. The cells in each flask were immediately imaged with a Leica DMi8 inverted microscope.