Erythrocyte based achiral micromotors for localized therapeutic delivery

Abstract

Bio-hybrid micromotors, active structures composed of both biological and synthetic components, are promising for use in several biomedical applications including targeted drug delivery, tissue engineering, and biosensing. Among biological candidates, erythrocytes are well suited for use as the biological component of bio-hybrid micromotors due to their biocompatibility, mechanical deformability, and long circulation time. However, their symmetric shape and small size make controlled actuation of these devices particularly challenging. Here, we present a novel strategy to overcome these limitations by fabricating achiral erythrocyte micromotors with enhanced propulsion efficiency. Inspired by recent work on synthetic achiral microswimmers, we report two and three-cell micromotors fabricated through biotin-streptavidin binding. These self-assembled red blood cell (RBC) structures are then interfaced with magnetic beads enabling them to swim and roll under the propulsion of a single homogenous rotating magnetic field at a much greater velocity compared to single cell micromotors in both Newtonian and viscoelastic fluids. Further, to demonstrate biomedical application of these self-assembled micromotors, the chemotherapeutic agent doxorubicin is loaded into RBC achiral micromotors, which are magnetically directed to cancer cells within a microfluidic chamber, successfully delivering their anticancer payload. The fabrication and propulsion method reported here will aid in the development of future erythrocyte-based micromotors for drug delivery and cancer therapy.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13036-025-00537-5.

Introduction

The field of micromotors and nanomachines has rapidly evolved over the past two decades, driven by the aspiration to create autonomous, miniaturized devices capable of navigating complex environments for biomedical, environmental, and industrial applications [1, 2]. In particular, the development of synthetic micromotors—microscale constructs propelled by chemical reactions, acoustic waves, magnetic fields, and light has enabled a wide range of innovations in drug delivery [3–7], medical imaging [8–10], and biosensing [11–13]. However, despite the tremendous progress in propulsion mechanisms and material design, one of the major bottlenecks facing synthetic micromotors remains their limited biocompatibility, potential toxicity, and immunogenicity, especially in in vivo biomedical applications [14]. To overcome these limitations, recent efforts have increasingly turned toward the development of bio-hybrid micromotors, which integrate functional biological components with synthetic materials. These systems leverage the inherent biocompatibility, biodegradability, and biological functionalities of living or derived cells, enabling improved performance in physiological environments. A wide range of biological organisms and materials has been explored, including the use of bacteria [15], spermatozoa [16], platelets [17], cancer cells [18] and cell membranes [11, 19], each offering unique features such as autonomous propulsion, immune evasion or carrying capability. Microalgae-based micromotors have recently gained traction due to their intrinsic mobility and responsiveness to external stimuli [20, 21], demonstrating potential in applications like targeted drug delivery. Among these diverse biohybrid strategies, erythrocytes (red blood cells, RBCs), due to their abundance, deformability, immune evasion, and long circulation time have garnered significant attention as ideal chassis for bio-compatible micromotor platforms.

In this work, we present an approach that builds upon the unique properties of RBCs to create magnetically actuated micromotors capable of navigating complex environments, carrying therapeutic payloads, and delivering them with precision. Erythrocytes possess a biconcave shape and a high surfacearea-to-volume ratio, enabling both mechanical flexibility and large internal volume—two critical attributes for developing efficient micromotor systems [22–25]. Once matured and released into circulation, RBCs remain functional for up to 120 days [24], providing a long operational window for in vivo applications. Their high deformability allows them to traverse capillaries narrower than their own diameter, granting access to otherwise hard-to-reach or delicate tissue regions [26]. Moreover, their nucleus-free structure, biodegradability, and natural clearance mechanisms (via macrophages and the reticuloendothelial system) make them ideal candidates for biomedical micromotor applications [27, 28]. Previous studies have demonstrated several ways to functionalize RBCs for therapeutic purposes, including encapsulating drugs via hypotonic treatment [29–31], anchoring drugs to membrane proteins [32–34], or camouflaging synthetic particles with RBC membranes [35, 36]. More recently, the integration of magnetic nanoparticles (MNPs) has enabled remote, non-invasive control of RBC-based micromotors. Magnetic fields are particularly suitable due to their large penetration depth, their ubiquity in medical settings, and are considered generally safe at low intensities. Wu et al. [37]. reported an innovative drug delivery system that encapsulated both citrate-stabilized MNPs within RBCs, thereby imparting controllable motion using synergistic acoustic and magnetic fields. However, due to the natural asymmetry of erythrocytes, acoustic fields also influenced unmodified cells, introducing potential off-target effects. In another approach, Hou et al. [38] developed a surface-functionalized RBC-mimetic micromotor, which demonstrated controlled actuation under a rotational magnetic field. While promising, these designs have often been limited by inconsistent motion in bulk fluid, which impairs their utility in biologically relevant environments.

In our previous work, we reported RBC biohybrid micromotors, formed though biotin-streptavidin bonding, exhibit dual motion modes under rotating magnetic fields. However, these micromotors were limited by their low translational speed. The achiral micromotors were first introduced by Cheang at al [39]., who demonstrated that geometric chirality is not a prerequisite for propulsion in low Reynolds number fluids. By orienting micromotor magnetic dipoles asymmetrically within a structurally symmetric assembly of beads, propulsion was enabled using a uniform rotating magnetic field. Inspired by this design and propulsion strategy, we designed RBC-based achiral micromotors. Although the assembled RBC micromotors are not strictly achiral, due to the distribution of beads and cell orientation, they follow the same design logic of achieving motion through magnetic actuation without geometric chirality. The term “achiral” here is used in conceptual alignment with previous non-helical propulsion strategies. We engineered RBCs to self-assemble into two- or three-cell constructs by attaching streptavidin-coated magnetic microbeads to biotinylated erythrocyte membranes. This modular assembly not only creates structural asymmetry necessary for generating propulsion under a rotational magnetic field, but also maintains the inherent biocompatibility and flexibility of RBCs. The resulting micromotors exhibit significantly improved propulsion efficiencies in various environments, including phosphate-buffered saline (PBS), bovine serum, and closely mimicking physiological conditions (methylcellulose). Beyond propulsion, a key feature of these RBC micromotors is their ability to encapsulate and deliver therapeutic agents in a controlled manner. To assess this, we used fluorescein isothiocyanate (FITC)-Dextran as a mock drug known for its biocompatibility and frequent used in enhancing the pharmacokinetic properties of cancer drugs, severing as a fluorescently labeled polymer widely employed in drug delivery investigations [40–42]. FITC-Dextran with various molecular weights (3,000, 10,000, 40,000 kDa) was used to demonstrate the loading and passive releasing efficiency of different drugs. Fluorescent markers enabled tracking the mock drug and analyzing it quantitively. Additionally, we demonstrated the ability of loaded RBC micromotors to move in a controlled manner in a microfluidic chamber. In our experiments, doxorubicin hydrochloride (DOX-HCl) - loaded RBC micromotors propelled by an external magnetic field were controllably navigated toward breast cancer cells through microfluidic channels and were shown to be effective at locally delivering the anticancer payload.

Experimental methods

Fabrication of RBC achiral micromotor

The fabrication of RBC achiral micromotor involved three main steps, as shown in Fig. 1(a). First, red blood cells (RBCs) were separated from whole bovine blood (Carolina Biological Supply Company, # 828574) by centrifugation at 3,000 ×g for three minutes and subsequently washed twice with 1× phosphate-buffered saline (PBS). The washed RBCs were then incubated with biotin (Thermo Scientific, # 21338) overnight at 4 °C. After incubation, the biotinylated RBCs were washed twice to remove excess biotin and byproducts and resuspended in 1× PBS. For single-cell micromotor assembly, 1 µL of biotinylated RBCs was mixed with 1 µL of streptavidin-coated magnetic beads (0.21 μm in diameter; Spherotech Inc., #SVM-025–5 H, 0.5%w/v) in 100 µL of 1× PBS.

Fig. 1.

(a) Schematic of fabrication process of erythrocyte (RBC)-based achiral micromotors. (b) Image of (i) natural RBC, (ii) single-cell RBC micromotor, (iii) Two-cell RBC achiral micromotor, (iv) Three-cell RBC achiral micromotor under differential interference contrast (DIC); 60 magnification, scale bar is 10 μm

To fabricate two-cell and three-cell achiral micromotors, the biotinylated RBCs were split into two equal portions. One portion was incubated with streptavidin at 4 °C for one hour and washed twice to eliminate unbound streptavidin. Then, biotinylated RBCs were first mixed with streptavidin-coated RBCs, followed by the addition of 1 µL of 0.21 μm streptavidin-coated magnetic beads. As shown in Fig. 1(b), bright-field images captured the geometry of the RBC micromotors and bead attachment. The size distribution of self- assembled RBC achiral micromotors are shown as Figure S1. Observing under the microscope and propulsion with the MFG, we found that approximately 40% of RBCs attached by beads and show active response to magnetic field, when using a 1:1 ratio of biotinylated RBCs and streptavidin-coated magnetic particles. The ratio between biotinylated and streptavidin-coated RBCs determined the final micromotor configuration: a 1 to 1.5 ratio promoted the formation of two-cell micromotors, while a higher ratio (1.5 to 2) favored three-cell assemblies. Additionally, aggregations of four or more cells were observed. However, they typically formed disordered, bulk-like structures rather than linear chains. These configurations do not offer a meaningful basis for comparing propulsion efficiency due to their irregular geometry and as a result they were excluded from analysis. To minimize four or more cells aggregation, we maintained a low cell concentration during the mixing of the two RBC batches and gently pipetted the suspension several times before preparing slides for experiments. These RBC achiral structures demonstrated strong biotin-streptavidin bonding and remained stable, showing no disassembly after 30 min of continuous exposure to the magnetic field. The final micromotor suspensions were diluted in the experimental fluid prior to use. To ensure optimal quality, fresh RBC achiral micromotors were prepared before each experiment. For short-term storage, micromotors can be stored at low concentration in PBS at 4 °C for at most 4–5 days. However, the RBC achiral micromotors will degrade overtime, which may influence the membrane mechanical properties or the magnetic response.

Preparation polymer fluids

A 2% (w/v) stock solution of methylcellulose (MC, Sigma, M0262, molecular weight 41 kDa) was prepared by slowly adding MC powder into half the volume of hot PBS (~ 80 °C) under vigorous stirring until fully dispersed. The remaining volume of chilled PBS was added to reach the final 2% concentration. The stock solution was stirred for 6 h at 4 °C to ensure complete dissolution and then filtered using a 5 μm filter to remove any undissolved particles. Working solutions of 0.5% and 0.25% MC were freshly prepared by diluting the stock solution with PBS prior to use in the experiments.

Magnetic actuation

A sealed chamber was constructed using polydimethylsiloxane (PDMS) and cover slides to create an observational cell minimizing external flows. The sample slide was placed on a Nikon Eclipse Ti-2 inverted microscope equipped with a 60× oil immersion objective or 40× air immersion objective. A homogenous rotating magnetic field was generated by a magnetic field generator (MagnetibotiX MFG-100-i). To ensure experimental consistency, a constant-strength magnetic field was maintained throughout the experiment. Magnetic field frequencies were systematically increased from 0 to 10 Hz in a gradual manner to evaluate the translational velocity of RBC micromotors at different applied frequencies. The modulation of magnetic frequency was precisely controlled using a MATLAB script, enabling accurate measurement and analysis. The displacements of RBC achiral micromotors were recorded by videos at 30 frames per second. We noted that the 3-cell micromotors selected for the experiment exhibited an arc angle range from 90° to 150°.

RBC achiral micromotor biocompatibility assay

Hemocompatibility assay

RBCs were initially separated from whole blood and washed at least five times with 1× PBS until the supernatant was clear. Subsequently, different concentrations (40, 80, 120 µL/mL) of RBC achiral micromotors were co-incubated with 50 µL RBCs in 1mL PBS separately at 37 °C for three hours. A positive control (RBCs in DI water) and a negative control (untreated, RBCs in 1×PBS) were also set up. Notably, to minimize the influence of different blood types or other variables that may affect the result, all RBCs used in this experiment were obtained from the same batch and same bottle of bovine blood. Following incubation, the absorbance of the supernatant, obtained by centrifugation at 5,000 ×g for three minutes, was measured using a wavelength of 405 nm. The hemolytic ratio of RBCs can be represented by [43]:

ODs are optical density values of individual solutions.

In vitro biocompatibility

To assess the potential toxicity to cells caused by RBC achiral micromotors, the human embryo kidney cells (HEK-293) were used. HEK-293 cells were cultured in 96-well plates, with a seeding density of 5 × 10³ cells per well and incubated for 72 h at 37 °C with 5% CO₂. Various concentrations of RBCs (1 × 10⁵, 2 × 10⁵ RBC/mL), RBC achiral micromotors (1 × 10⁴, 2 × 10⁴ RBCM/mL), and streptavidin beads (10 µL/mL) were added and co-cultured with HEK-293 cells for 24 h and 72 h. After incubation, the cell culture media and co-culture components were discarded, and the wells were washed once with PBS. A mixture of 90 µL serum-free media and 10µL MTT solution (5 mg MTT in 1 mL PBS) was added and incubated for 3 hours. Subsequently 100 µL of the SDS-HCL solution was added to each well and mixed thoroughly before absorbance was measured at 595 nm.

Dextran and DOX-HCl load and release

The mock drug dextran and chemotherapy drug were loaded using the same method: a hypotonic treatment method reported by Wu et al. [37]. RBC achiral micromotors, along with each loading component, were suspended in a low osmolarity solution and incubate at 4 °C for two hours, with gently vortexing to ensure uniform mixing and efficient loading. In the mock drug experiment, dextran of various molecular weight was used at a consist concentration of 10 mg/ml. In the therapeutic treatment experiment, DOX-HCl was added at concentration of 20 µM. Afterwards, RBCs were resuspended in an isotonic solution (37 °C) and incubated for 10 min to allow membrane resealing. The RBC achiral micromotors were then washed twice (2500 ×g, 3 min) to remove any unencapsulated drug. In the mock drug experiment, following the washing steps, all RBCs with dextran loaded were resuspended in 37 °C 1× PBS. The release of dextran from the RBCs occurs via passive diffusion. During the releasing process, the RBC suspension was places at room temperature on the microscope stage for continuous observation.

Results and discussion

RBC achiral micromotor swimming in fluid

Previously, we established a biotin-streptavidin based interaction strategy to fabricate erythrocyte-based biohybrid micromotors, where surface exposed amines on RBC membranes were selectively biotinylated and bound to streptavidin coated magnetic beads [44]. Unlike many previous approaches that use isolated RBC membrane [37] or coated RBC membrane on synthesis micromotors [38] to enhance the biocompatibility, our method uses intact RBCs as the foundation of micromotors. The preservation of the natural structure is important for maintaining biological properties. This method enables stable assemble while preserving membrane integrity and supports dual swimming and rolling behaviors under a single external driving field. This dual-mode actuation will be particularly advantageous in navigating complex in vivo environments, where challenges such as endothelial roughness and cell-free layers exist near boundaries. However, the translational speed of the single-cell RBC biohybrid micromotors was suboptimal. Inspired by synthetic achiral micromotors, a configuration of two or three beads achiral micromotors [45, 46], where two- or three-bead configurations demonstrate enhanced propulsion, particularly three-bead swimmers in viscoelastic fluids [47, 48]. We hypothesized that linking RBCs into two- or three-cell assemblies could increase propulsion efficiency by enhancing asymmetry. RBC achiral micromotors were self-assembled into single-cell, two-cell or three-cell achiral structures in PBS, as Fig. 1a. A sealed chamber was constructed using PDMS and cover slides to create an observational cell. Micromotors were actuated by a homogenous rotating magnetic field with a fixed field strength (10mT). Magnetic field frequencies were systematically increased from 0 to 10 Hz in a gradual manner to evaluate the translational velocity of RBC micromotors at different applied frequencies. The modulation of magnetic frequency was precisely controlled using a MATLAB script, enabling accurate measurement and analysis. The displacement of RBC achiral micromotors were recorded by a Nikon Eclipse Ti2 inverted microscope. We noted that the 3-cell micromotors selected for the experiment exhibited an arc angle range from 90° to 150°.

Similar to single-cell RBC micromotors [44], all types of micromotors generally exist in two different positions: near (< 30 μm) the bottom substrate and in the bulk suspension (~ 100 μm from the substrate). As shown in Video S1, under a single rotating magnetic field, RBC micromotors have two modes of motion depending on their location away from a solid boundary. Notably, in this video, the focal plane was set for the micromotor in the upper focal plane and the one in a lower focal plane is out of focus. RBC micromotors in different focal planes display distinct propulsion behavior. The micromotor in focus swims in the direction along the rotating axis, while the micromotor near the bottom surface (out of focus) moves perpendicularly, consistent with rolling behavior. RBC achiral micromotors near the bottom substrate roll along the surface, propelled dominantly by interactions with the substrate. Those in the bulk suspension swim in the direction perpendicular to rolling. The biconcave shape of the red blood cell and its deformability allow terms in the coupling viscous mobility tensor to be nonzero, enabling forward translational speed [39]. Following the fabrication of the RBC achiral micromotors and subsequent characterization of their motion modes in the suspension, we now focus on evaluating their velocity and propulsion efficiency. To achieve this, the RBC achiral micromotor suspension was diluted (400×) to minimize the impact of fluid flow generated by neighboring micromotors before being sealed within a PDMS chamber. Micromotors were actuated by a magnetic field rotated along the y-z plane, with a range of magnetic frequencies (1–10 Hz) at a fixed strength (10mT) being used to propel the micromotors.

Under the influence of magnetic torque, RBC achiral micromotors in the bulk suspension rotate and translate in the direction along the rotating axis, which we refer to as the RBC swimmers, as shown in Video S2. Generally, as external field frequency increases, the velocities of swimmers also increase until reaching the step-out frequency, at which the hydrodynamic torque balanced with magnetic torque, leading to a maximum velocity. As the field frequency continues to increase, the swimmers are no longer able to maintain synchronous rotation with the external field. Displacement of swimmers in the x-coordinate was recorded to determine micromotor velocity (plotted in Figure S3(a)), which was then normalized by the average end-to-end body length of each type of micromotor to obtain the propulsion efficiencies. This represents the number of body lengths traveled per second, as shown in Fig. 2 (a). All the two-cell and three-cell micromotors reached their maximum efficiency at 4 Hz, while 6 out of 10 single-cell micromotors peaked at 5 Hz, and the remaining, at 4 Hz. Among all three types, three-cell micromotors exhibited the highest propulsion efficiencies at their respective step-out frequencies. Although two-cell RBC swimmers showed slightly higher velocities than single-cell RBC swimmer, their propulsion efficiency was not significantly statistical different from 1-cell swimmers (p = 0.4327, p > 0.05 indicating no significant difference; n = 10). The higher efficiency of three-cell swimmers over two-cell and single cell swimmers can be attributed to the increase of their higher structure complexity and asymmetries. The translational speed of swimmers can be defined as , where is the magnetic torque applied on the swimmer, C is the coupling viscous mobility tensor, a parameter that depends on swimmer’s geometry. Two-cell and single-cell motors rotate with relatively symmetric drag profiles, as similar fluid drag on both sides of the structure attenuate thrust. However, three-cell swimmers, with an arc angle of 90° to 150°, introduce structural asymmetry that can break the balance of fluid drag, and promote the propulsion efficiency [47]. Here, we also noticed that single-cell micromotors displayed higher step-out frequencies compared to two- and three-cell swimmers. This difference was due to their simpler geometry, which induced lower hydrodynamic drag applied on them, allowing them to maintain synchronization with higher-frequency magnetic fields.

Fig. 2.

Single-cell, two-cell and three-cell RBC achiral swimmer propulsion efficiency in (a) PBS, (b) bovine blood serum; with respect to magnetic field frequency under a 10mT rotating magnetic field. N = 10. (c) Single-cell, two-cell and three-cell RBC achiral swimmer propulsion efficiency in 0.25% and 0.5% methylcellulose, 10mT, 3 Hz rotating magnetic field, N = 10. (d) Displacement of a three-cell achiral swimmer in PBS propelled by 3 Hz, 5,10, 15,17 mT magnetic field. (e) Single-cell, two-cell and three-cell RBC achiral swimmer propulsion efficiency in PBS related to external field strength, propelled by 3 Hz, 5, 10, 15, 17 mT magnetic field, N = 10

After conducting experiments in PBS, we next aimed to assess their performance in more complex and physiologically relevant environments. Compared to PBS, blood serum has a higher viscosity (Figure S2(a)) and richer in biomolecules that can interact with micromotors. A similar velocity sweep experiment was conducted with fresh bovine serum (Figure S3(b)). Similar to the results of swimmers in PBS, all two-cell and three-cell micromotors reached their maximum efficiency at 4 Hz (Fig. 2(b)). Notably, only 3 out of 10 single-cell micromotors reached their peak efficiency at 5 Hz, representing a significantly smaller proportion compared to those operating in PBS. Three-cell swimmers have the highest propulsion efficiencies at step-out. In addition, we noticed that at low frequencies (1–2 Hz), the velocity of a swimmer in serum is not linearly correlated with the frequency of the external field, as in the case of PBS. A linear fit analysis was conducted for the frequencies below their step-out frequencies and indicate the same results (shown as Table S1).

The presence of biomolecules like proteins in bovine serum introduces a hydrodynamic drag exerted on the RBC micromotors, which is especially prominent when operating at low field strengths, as the magnetic torque applied to the RBC micromotor is relatively small. Across all configurations, RBC micromotors demonstrated lower propulsion efficiency in serum than in PBS. To more comprehensively evaluate micromotor performance, we considered physiologically relevant challenges that swimmers may encounter in vivo. As tumors grow, they can compress surrounding lymphatic vessels and impair fluid drainage, resulting in elevated viscosity near the tumor site [49, 50]. The viscosity of the interstitial fluid varies up to 3.5 mPaS, while it can exceed to 8 mPaS for pathological abnormalities. To simulate the abnormally high viscosity fluid surrounding the tumor, we employed 0.25% and 0.5% methylcellulose (MC) solutions and evaluated the performance of the three micromotor types in these viscoelastic environments. To characterize the local viscoelastic properties of 0.25% and 0.5% MC solutions, active microrheology was performed using optical tweezers force measurements. Multiple 2 μm beads at different locations within sample chambers were used as rheological probes. Particles were trapped and oscillated sinusoidally at various frequencies (). The loss modulus () was used to estimate the dynamic viscosity () using the relation: . Measured values for 0.25% and 0.5% MC solutions are shown in Figure S2.

Because we observed that for certain batches of swimmers, particularly the velocity of three-cell swimmers in PBS the increase from 3 Hz to 4 Hz is not linear, suggesting that the step-out frequency of three cell swimmer lies between 3 and 4 Hz. To ensure stable and consistent propulsion across different batches, we use 3 Hz, 10mT rotational magnetic field to analyze the propulsion of swimmers in two MC solutions. The swimmer velocity was measured from the displacement in x axis (Figure S3(d)). Then the propulsion efficiencies of each type of swimmers obtained (Fig. 2(c)) by normalized by the average body length. In both concentrations of MC solution, three-cell swimmers exhibited the highest propulsion efficiency, which were also greater than those found in PBS. We believe this is due to the asymmetric arc-like configuration of three-cell swimmers, which enables them to harness viscoelastic recoil more effectively from the surrounding fluid, thereby enhancing their propulsion efficiency. This finding highlights the contribution of swimmer geometry in optimizing performance in complex fluids, which indicates three-cell swimmers may be more promising that other configurations (i.e. single cells) for in vivo application.

In addition, a series of experiments was conducted to investigate whether higher propulsion efficiency could be achieved by increasing the strength of the applied magnetic field. For this purpose, single-cell, two-cell, and three-cell RBC micromotors were randomly selected and subjected to rotating magnetic fields of 5, 10, 15, and 17 mT at a constant frequency of 3 Hz (see Video S3). The x-direction displacement of each swimmer was recorded, and a representative displacement profile of a three-cell swimmer is shown in Fig. 2. Each experiment was repeated four times per swimmer type, and the resulting propulsion efficiencies in relation to different magnetic field strengths are summarized in Fig. 2(e). Overall, all three types of swimmers demonstrated enhanced propulsion efficiency under stronger magnetic fields. However, at the lowest field strength of 5 mT, the swimmers failed to rotate synchronously, suggesting that the step-out frequency was exceeded under this condition. This indicates that the magnetic torque generated at 5 mT was insufficient to overcome the opposing hydrodynamic torque. In contrast, synchronous rotation was observed at 10, 15, and 17 mT, allowing the swimmers to maintain efficient propulsion. These findings suggest that applying stronger magnetic fields could enable swimmers to maintain synchronized rotation at higher frequencies, thereby further improving their propulsion performance in future applications.

RBC achiral micromotor rolling in fluid

Under the influence of magnetic torque, RBC achiral micromotors near the bottom substrate rotate and translate along the direction perpendicular to the rotating axis. In this scenario we termed the micromotors as RBC rollers, as shown in Video S4. The same experimental and analysis method was employed to illustrate the profile of rollers, and the profile of propulsion efficiencies related to magnetic field frequencies (Fig. 3a). Compared to RBC swimmers, rollers generally exhibit higher translational speed and propulsion efficiencies, achieving approximately twice the swimming performance under similar actuation conditions. This enhanced performance is primarily attributed to wall-induced frictional asymmetry, which facilitates more effective conversion of rotational motion into forward propulsion. Two-cell and three-cell RBC rollers demonstrated higher translational speeds compared to single-cell rollers (as Figure S4). However, when normalized by end-to-end body length, single-cell rollers exhibited significantly greater propulsion efficiency. The reduced efficiency of multi-cell rollers can be attributed to increased interactions between the micromotor body and the substrate, which hinder smooth rolling motion. Additionally, three-cell rollers reached their step-out frequency at a lower actuation rate (3 Hz), achieving peak propulsion efficiency earlier than both single- and two-cell rollers, which peaked at 4 Hz. As the external field frequency increases above step-out, the rolling velocities of the three-cell rollers decrease dramatically. The difference in performance among the three rollers is directly related to micromotor geometry. We also examined roller propulsion in bovine serum to simulate a more complex biological environment. Single-cell rollers have the highest propulsion efficiencies among all types. At low frequencies, roller speed in serum slightly deviates from the expected linear correlation, akin to the swimmer (Table S1). Unlike swimmers, there is minimal difference in propulsion efficiency of rollers between PBS and serum. These results indicate that for future applications involving navigation along blood vessels or other endothelial surfaces, single-cell micromotors represent the optimal choice, as they exhibit the highest propulsion efficiency in both PBS and bovine serum.

Fig. 3.

Single-cell, two-cell, and three-cell RBC achiral roller propulsion efficiency in (a) PBS, (b) bovine blood serum; with respect to magnetic field frequency under a 10mT rotating magnetic field, N = 10

Biocompatibility of RBC achiral micromotors

The utilization of naturally occurring cells in the body for fabricating micromotors can aid in mitigating the risk of foreign body immune response. However, surface treatment of RBCs with biotin during micromotor fabrication necessitates assessing biocompatibility. A hemolysis experiment was conducted by incubating a mixed population of RBC achiral micromotors at various concentrations with the same number of pure RBCs in PBS. The hemolytic rates of RBC samples at various micromotor concentrations are shown in Fig. 4. For all experimental concentrations, the hemolytic rates remained below 2%, which can be categorized as a nonhemolytic biomaterial [51], indicating no harm to the blood cell membrane. We also investigated the in vitro biocompatibility of various concentrations of a mixed population of assembled RBC micromotors on human embryonic kidney cells (HEK-293). An MTT assay was performed after 24 h and 72 h of incubation (Fig. 4), confirming that the self-assembled RBC achiral micromotors maintain the biocompatibility property of RBCs.

Fig. 4.

(a) Hemolysis analysis of the washed RBCs with RBC achiral micromotors of different concentrations: 0, 40, 80, 120 µL RBC achiral micromotor /mL RBC. Experiment repeats for three times. (b) Histograms represent the percentage, with respect to control cells (100%), of viable cells after co-culture with: 110⁴ RBC, 210⁴ RBC, 110⁴ RBCM, 210⁴ RBCM, 10 µL 0.5%w/v (50 µg) magnetic beads (experiment repeat three times, for each experiment, n = 6). Statistically significant analysis based on one-way ANOVA, compared with the control group; * represents p < 0.05, ns represents p > 0.05

Loading and releasing of a mock drug

To qualitatively evaluate the drug-loading and release capabilities of RBCs, we employed fluorescein isothiocyanate-labeled dextran (FITC-dextran) with molecular weights of 3,000, 10,000, and 40,000 Da as model drug. FITC-dextran was chosen due to its relatively large molecular size and slower diffusion rate, making it suitable for mimicking the loading and release behavior of therapeutic macromolecules. The mock drug was loaded using a hypotonic treatment, placing RBCs in a low osmolarity suspension leading to an enlargement of the RBC membrane pore, permitting mock drug diffusion into RBCs. Afterwards, RBCs were resuspended in an isotonic solution, and washed twice to eliminate any excess dextran. To indirectly verify the encapsulation of dextran, we performed a laser-induced lysis experiment (Video S6). Upon localized irradiation, the RBC membranes were ruptured, and a sudden release of fluorescent dextran was observed, which indirectly support the prove the internalized of dextran. Micrographs of dextran-loaded RBCs were taken using bright-field and florescence microscopy (Figure S5). Image analysis was performed in ImageJ by counting the total number of RBCs in the bright field image and the number of RBCs with fluorescent signal, in the florescent channel. As shown in Fig. 5, after two washing steps, a similar percentage of dextran-loaded was observed across all three molecular weights (initial time point shown in each figure).

Fig. 5.

Percentage of RBCs loaded with FITC-dextran (mock drug) of different molecular weights: (a) 3,000 Da, (b) 10,000 Da, and (c) 40,000 Da. Dextran loading was performed in two hypotonic solutions: 186 mOsmol/kg (67% PBS, 33% DI water) and 161 mOsmol/kg (60% PBS, 40% DI water). “0 min” represents the initial percentage of RBCs loaded with dextran immediately after loading and washing. Time points at 30, 60, 90, and 120 min (or 10, 20, 30, and 40 min, respectively, depending on panel) indicate the percentage of RBCs retaining the mock drug after passive release over time. Experiments were independently repeated twice, with N = 5 for each group

In theory, higher molecular weight dextran, with larger hydrodynamic size, should have more difficulty diffusing through membrane pores. However, the hypotonic stress, governed by the osmolarity of the loading solution, also plays a critical role. Lower osmolarity can lead to more extensive membrane swelling and larger or longer-lived pores, which may partially compensate for the size related limitations of high molecular weight dextran. This could explain the comparable loading percentages observed between different molecular size dextran, or even more loading of macromolecules in the effect of solutions with lower osmolarity. Additionally, the mechanical stress from centrifugation during the washing process may further compromise membrane integrity in partial resealed cells. Some FITC-dextran may have been released, contributing to the overall similarity in unloading rates. Additionally, variation in fluorescence intensity between cells suggests potential differences in the amount of dextran loaded per cell. However, a quantitative assessment of intracellular loading amounts was not performed in this study. Figure 5 suggests that the osmolarity of a hypotonic suspension may influence the loading efficiency of RBCs, with the lower osmolarity condition used in our study resulting in a higher percentage of loaded RBCs compared to the higher one. Conversely, the molecular weight of dextran appears to have a more pronounced effect on the release rate, with lower molecular weight mock drugs releasing more rapidly than larger ones. As a result, in the group with 3,000 Da dextran, 70% of the erythrocytes lost their cargo after only 40 min. In comparison, in the 10,000 Da dextran group, only 60% of the erythrocytes were no longer loaded after 2 h. In the 40,000 Da group, only 20% of the erythrocytes were no longer loaded after two hours.

In vitro drug delivery and therapeutic treatment

To demonstrate the capability of RBC achiral micromotors loaded with actual drugs for targeted drug delivery and therapeutic treatment, we employed an open-loop control system to manipulate the orientation of the magnetic field, guiding the micromotors towards predetermined locations. Human breast cancer cells (MDA-MB-231) were utilized to assess therapeutic treatment efficiency. Doxorubicin (DOX-HCl), a potent chemotherapeutic agent, known to intercalate DNA strands and inhibit molecular biosynthesis, was chosen for its cytotoxic effects on cancer cells. DOX-HCl, with partial lipophilic properties, can interact with the lipid bilayer of RBC membranes to a limited extent. Biagiotti et al. [52] has shown that after intravenous administration, approximately 50% of DOX-HCl was found within the RBCs. However, in physiological conditions, the ionized form of DOX-HCl limits its ability to cross lip membranes through passive diffusion. Hypotonic treatment method is employed to enhance the loading efficiency by temporarily creating pores on RBC membrane [37, 53], allowing DOX-HCl diffuse. Later RBCs are resuspended in isotonic solutions enabling the membrane to reseal and entrap the drug. A confocal image of RBC loaded with DOX-HCl (as Figure S6) was taken to confirm that therapeutic agents can be successfully loaded into the micromotors. Lucas et al. [54] demonstrated that RBC-DOX is more stable and remains longer than free DOX. Although some leakage may occur, the RBC-DOX leads to significantly fewer side effects in the healthy tissues compare to free DOX. Once it reaches the target site, DOX-HCl is passively released from RBCs over time. The acidic extracellular pH of tumor microenvironments will help to enhance the drug releasing rate [55]. After efficiency loading, we demonstrate the maneuverability of RBC achiral micromotors with loading.

First, we demonstrate the locally targeted delivery capabilities of RBC achiral micromotor through rolling and swimming motions (Video S5). The trajectory of a representative micromotor is plotted in Fig. 6. Here, MDA-MB-231 cells were cultured in a confined area on one side of a microfluidic chamber, and RBC achiral micromotors were then introduced to the other side of the chamber. Under the manipulation of a rotating magnetic field, the micromotor was effectively propel toward the cancer cells. Once the magnetic field is deactivated, the micromotor can remain in the final position as the Brownian effects are minimal. Notably, in localized delivery experiments, we observed that more micromotors remained stationary than in mobility experiments. This phenomenon can be attributed to the culture plate we use. Unlike in mobility experiments which the glass slides were passivated using BSA to minimize the surface adhesion, these experiments were conducted on tissue culture treated plates, which are hydrophilic and promote stronger attachment of MDA-MB-231 cells. As a result, some RBC achiral micromotors seem to adhere to the substrate, resulting in hindered motion. Only slight wobbling can be observed under the influence of magnetic field.

Fig. 6.

Figure represents the trajectory of DOX-HCl. loaded RBC micromotor (a) roll (85s) (b) swim (50s) to target tumor cell with single 10 mT rotational magnetic field actuation. Scale bar = 20 μm

Given that interstitial pressure near tumor sites is typically higher than in surrounding tissues, our aim was to demonstrate that RBC micromotors can effectively navigate the final few micrometers and deliver therapeutic agents directly to the tumor sites, overcoming diffusion limitations and improving delivery precision. To evaluate the potential of RBC-based achiral micromotors for targeted drug delivery and localized cancer therapy, we employed a microfluidic chamber (as Fig. 7a, Figure S7) that mimics the tumor microenvironment. This chamber consists of three interconnected regions: a left reservoir, a narrow central channel, and a right reservoir. The right reservoir simulates the tumor site, where breast cancer cells were cultured to assess therapeutic efficacy. The central channel represents the blood vessel surrounding the tumor, while the left reservoir serves as the injection site, simulating the area surrounding the tumor where either free drug or RBC micromotors are introduced. Before the experiment, breast cancer cells (MDA-MB-231) were seeded and cultured in the right reservoir, reaching 80% confluency. Three groups of experiments were performed at the same time, RBC micromotors, RBC micromotors with DOX-HCl loaded, and DOX-HCl alone were added to the left side of the microfluidic chamber. After subjecting each group to a rotating magnetic field for 30 min and incubating the system for 48 h. After incubation, we observed RBC achiral micromotors retained their structure, without noticeable aggregation or disassembly. Subsequently, a live-dead assay was conducted to assess cancer cell viability. To compare the therapeutic efficacy of passive drug diffusion versus active, micromotor-based delivery, we conducted a series of parallel experiments using the microfluidic chamber. Three experimental groups were established: (1) free DOX-HCl (Doxorubicin hydrochloride, Sigma #D1515), representing passive diffusion; (2) RBC micromotors loaded with DOX-HCl, representing active, targeted delivery; and (3) unloaded RBC micromotors, included to control for any potential effects from the micromotors themselves. At the start of the experiment, each formulation was introduced into the left reservoir of separate microfluidic channels. All three channels were then placed in a rotating magnetic field for 30 min to allow RBC micromotors to move to the right side of the channel. Following, the devices were placed in an incubator at 37 °C with 5% CO₂ for 48 h to allow for drug interaction with the cancer cells cultured in the right reservoir. After incubation, the remaining culture media, containing either free drug or micromotors, was carefully removed, and the chambers were rinsed twice with PBS. A live/dead cell viability assay was then performed to assess the therapeutic outcome on the cancer cells.

Fig. 7.

(a) Schematic of microfluid chamber designed for therapeutic treatment experiment. (b) Fluorescence microscopy images of MDA-MB-231 cells treated with (i) free DOX-HCl. (20 µM), (ii) RBCMs with DOX-HCl loaded (2 µL/mL cell culture media), and (iii) RBCMs (2 µL/mL cell culture media). The living cells with green fluorescence were stained with calcein AM, and dead cells with red fluorescence were stained with BOBO-3 Iodide. (c) The statistics result from figure b, n = 5. Statistically significant analysis based on one-way ANOVA, compared with the control group; *** represents P ≤ 0.001, ns represents p > 0.05

Micrographs of stained cells were taken under bright-field, FITC, and TRIC channels (Fig. 7b). The area of fluorescent signal in each image was measured to quantify the relative amount of live or dead cells. Five random regions were selected for each group, the average cell viabilities are plotted in Fig. 7c. Notably, the control group, RBCMs without drug loading, showed approximately 97.5% cell viability after 72 h of incubation, indicating the biocompatibility of RBC achiral micromotors and their lack of cytotoxicity toward MDA-MB-231 cells, consistent with the results observed in HEK-293 cells. The group treated with DOX-HCl loaded RBC micromotors exhibited a significantly higher proportion of dead cancer cells compared to both the unloaded RBC micromotor group and the group receiving free DOX-HCl. This comparative experiment demonstrates the potential of RBC achiral micromotors as carriers for therapeutic payloads, enhancing the efficacy and precision of cancer treatment. Furthermore, the results highlighted the advantage of localized, magnetically controlled delivery, showing that RBC micromotors can be accurately guided and retained at the tumor site, enabling concentrated local drug release. Additionally, based on the reported IC50 values for DOX-HCl against MDA-MB-231 cells [56, 57], we estimated the local DOX-HCl concentration achieved in our experiment to be approximately 0.11µM or higher. While the estimate is indirect, it provides a useful approximation of the potential drug concentration delivered locally by the micromotors.

Conclusion

Here, we improved on previous RBC micromotors designs by assembling erythrocytes into two- and three- RBC micromotor configurations using efficient biotin-streptavidin specific binding. With the use of a single external field, RBC achiral micromotors can exhibit two modes of motion- swimming in bulk fluid and surface rolling. We systematically investigated the propulsion efficiency of the three types of micromotors with two different modes of motion in various environments. We observed increasing the complexity and asymmetry of RBC micromotors enhances swimming performance, where three-cell RBC swimmers exhibited a significant higher propulsion efficiency in PBS, bovine blood serum, and viscoelastic fluids. The presented assembly method remains simple to carry out for producing erythrocyte micromotors with enhanced propulsion efficiency. Additionally, we also confirmed that the RBC achiral micromotors are hemocompatible and have low cell cytotoxicity. Using a mock drug of different molecular weights, we demonstrated that therapeutics can be successfully loaded into RBCs though hypotonic treatment. Finally, we demonstrated the therapeutic potential of DOX-HCl -loaded RBC micromotors which can be actuated and navigated to cancer sites by rolling or swimming under the open-loop control of the magnetic field. Following incubation, the DOX-HCl -loaded micromotors exhibited significantly higher cancer cell death compared to both the unloaded micromotor and free drug groups. In summary, our results highlight the effectiveness of RBC achiral micromotors in loading and delivering therapeutics to targeted sites via magnetic field manipulation in vitro, and thus may have promise for future in vivo therapeutic applications.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

RBC

Red blood cell

MC

methylcellulose

DOX-HCl

Doxorubicin

PDMS

Polydimethylsiloxane

MNP

Magnetic nanoparticle

PBS

Phosphate-buffered saline

Author contributions

QW performed all experiments and analyzed all the data in the manuscript and led the writing of the original draft. JK led the review and editing of the manuscript. ND contribute to review and edit. JA led the conceptualization, funding acquisition and supervision. All authors read and approved the final manuscript.

Funding

This work was funded by the National Science Foundation (EES-2000202, EES-2306449, CMMI-2000330, and EES-2219558) and support by the NSF FAMU CREST Center award (EES-1735968). All the work was performed at the National High Magnetic Field Laboratory, which is supported by National Science Foundation Cooperative Agreement No. DMR-2128556 and the State of Florida.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Ethics approval not required.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.