Abstract

The use of micromotors for active drug delivery via oral administration has recently gained considerable interest. However, efficient motor-assisted delivery into the gastrointestinal (GI) tract remains challenging, owing to the short propulsion lifetime of currently used micromotor platforms. Here, we report on an efficient algae-based motor platform, which takes advantage of the fast and long-lasting swimming behavior of natural microalgae in intestinal fluid to prolong local retention within the GI tract. Fluorescent dye or cell membrane–coated nanoparticle functionalized algae motors were further embedded inside a pH-sensitive capsule to enhance delivery to the small intestines. In vitro, the algae motors displayed a constant motion behavior in simulated intestinal fluid after 12 hours of continuous operation. When orally administered in vivo into mice, the algae motors substantially improved GI distribution of the dye payload compared with traditional magnesium-based micromotors, which are limited by short propulsion lifetimes, and they also enhanced retention of a model chemotherapeutic payload in the GI tract compared with a passive nanoparticle formulation. Overall, combining the efficient motion and extended lifetime of natural algae–based motors with the protective capabilities of oral capsules results in a promising micromotor platform capable of achieving greatly improved cargo delivery in GI tissue for practical biomedical applications.

INTRODUCTION

Oral delivery to the gastrointestinal (GI) tract has been one of the most widely used approaches for drug administration due to its high patient compliance, noninvasiveness, simplicity, and low cost (1, 2). Despite their potential advantages, oral drug formulations face several barriers within the GI tract, including poor stability in gastric fluid, limited drug interaction with the intestinal lining, and low solubility (3). Several engineered systems, based on microinjectors and microneedles, have been described recently for improving the oral delivery of large biological macromolecules via mechanical penetration mechanisms (4, 5). Other platforms, including bioadhesive patches, responsive hydrogels, mucus-penetrating particles, ionic liquids, and microgrippers, have been reported for enhancing GI delivery via improved tissue adhesion, prolonged retention, and improved drug localization (6–10). The use of micromotors is another promising and effective approach for the active transport of therapeutic agents to specific sites of interest (1, 11–14). A variety of synthetic micromotors, capable of propelling to hard-to-reach locations within the body, have been developed for active drug delivery and for other biomedical applications, including biosensing and microsurgery (15–19). Early in vivo studies using micromotors focused primarily on the GI tract, which can provide a favorable environment for movement (19, 20). These pioneering studies used chemically powered micromotors based on magnesium (Mg) or zinc microengines for the enhanced delivery of antibiotics, vaccines, and micronutrients (19–22). These self-propelled micromotors hold particular promise for the local delivery of drug payloads toward the treatment of GI diseases and disorders (19). Although these active platforms substantially enhanced delivery efficacy compared with corresponding static microcarriers, they suffer from short lifetimes of operation up to about 15 min once in contact with GI fluid. This decreases the potential of these synthetic micromotors to interact with GI mucosa and limits their overall retention, thus leading to decreased drug bioavailability. To address this limitation, active micromotor-based GI drug delivery systems with improved characteristics need to be developed.

Here, we report an algae motor–loaded capsule system that combines the efficient long-lasting movement of natural algae in the small intestine with the protective capabilities of oral capsules, thus enabling prolonged retention within the intestinal mucosa toward greatly improved GI delivery (Movie 1). Microorganisms such as bacteria, sperm, and microalgae have evolved over millions of years to develop robust actuation systems that enable autonomous motion (23). These natural cellular systems have recently emerged as attractive cargo carriers that are capable of transporting therapeutics to hard-to-reach body locations (24, 25). Combining these motile microorganisms with synthetic materials results in biohybrid systems capable of performing multiple tasks (26–28). Whereas sperm and bacteria have been used for over a decade in diverse biomedical applications ranging from cancer therapy to assisted fertilization (29–31), microalgae swimmers have rarely been explored as robotic actuators (32). Microalgae are eukaryotic swimmers that are facile to culture, and they offer attractive properties for biomedical applications, including efficient propulsion (>100 μm/s), autofluorescence, and phototactic guidance capabilities.

We chose Chlamydomonas reinhardtii as a model microalgal swimmer for active payload delivery in the GI tract because of its many attractive properties, including cytocompatibility, cost-effective scalable production, good adaptability and motility in diverse aqueous environments, abundance of reactive surface groups for functionalization, and autofluorescence for ease of tracking in vivo (27, 32–34). C. reinhardtii swims by beating its two flagella synchronously at a frequency of 50 Hz (35), reaching high speeds up to about 200 μm/s (32, 36). We demonstrate that C. reinhardtii display substantially longer propulsion in intestinal fluid compared with synthetic chemically powered Mg micromotors, which are the only type of self-propelled microrobot swimmers that have been reported for in vivo operation in the GI tract (19–22). To protect the algae motors from the harsh gastric environment, we embedded them inside a protective capsule (Fig. 1, A to C), which was prepared with an inner hydrophobic coating to entrap aqueous solution for maintaining algae viability along with an outer pH-responsive enteric polymer coating. Upon release from the capsule, the algae display constant motility in intestinal fluid (Fig. 1D). Compared with the short lifetime of commonly used Mg micromotors, the algae motors remain motile for more than 12 hours at body temperature. In vivo, this prolonged movement leads to notably improved intestinal distribution (Fig. 1E), resulting in enhanced retention of a model chemotherapeutic doxorubicin (Dox) conjugated to the algae motors. Overall, our findings indicate that natural algae–based active carriers hold great promise for oral drug delivery to enhance the treatment of GI diseases.

RESULTS

Movement of algae-based micromotors in simulated intestinal fluid

We first studied the motion properties of C. reinhardtii, which are commonly used as a model algal species (37), and compared them with those of Mg micromotors (Fig. 2A). Simulated intestinal fluid (SIF), mainly composed of potassium dihydrogen phosphate at pH 6.8, was used to test the movement of the algae and Mg micromotors. In SIF, the natural algae motors exhibited a stable speed profile of about 120 μm/s that lasted for a minimum of 12 hours (movie S1). This consistent motile behavior is ascribed to the coordinated, self-sustained beating of algae flagella (38) even under suboptimal survival conditions, such as SIF. In contrast, the speed of Mg micromotors in SIF markedly decreased from an initial 180 to 80 μm/s after 15 min before reaching 0 μm/s after another 15 min (movie S2). This sharp drop in speed reflects the rapid dissolution and depletion of the Mg engine during propulsion. The percentage of motile Mg motors also dropped to 20% after 15 min of propulsion, whereas 89% of the algae motors remained moving after 12 hours (Fig. 2B). In tracing their motion, the movement patterns of algae tracked over 2-s intervals appeared consistent over the course of 12 hours (Fig. 2C and movie S3), whereas no movement was observed for Mg motors after 30 min (Fig. 2D and movie S4). These data illustrated that the algae motors could self-propel efficiently in SIF and maintain consistently fast motility over long periods of time, thereby supporting their potential for active GI delivery applications.

To demonstrate their potential for drug delivery, we modified the algae motors with two different cargos: a fluorescent dye and polymeric nanoparticles (NPs). The green dye fluorescein (excitation/emission = 494 nm/518 nm) was first chemically conjugated to the surface of the algae (39). After dye conjugation, the algae could be fluorescently tracked (Fig. 2, G and H, and movie S5), and the median speed calculated from 100 individual algae was nearly identical to that of unmodified algae (Fig. 2, E and F, and movie S5) and consistent with previously reported values (32, 36). Similarly, red blood cell (RBC) membrane–coated poly(lactic-co-glycolic acid) (PLGA) NPs (40) were linked to the algae via click chemistry. To visualize the NPs on the algae, we encapsulated the fluorescent dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI; excitation/emission = 550 nm/567 nm) inside the PLGA core during the fabrication process. The NP-modified algae motors (denoted “algae-NP motors”) exhibited a similar swimming pattern and speed distribution profile in SIF compared with unmodified algae motors (Fig. 2, I and J, and movie S5). In addition, the intrinsic phototaxis of the algae was not compromised after the NP functionalization (movie S6). The data here confirmed that different payloads, ranging from small molecules to NPs, can be successfully loaded onto algae motors without affecting their propulsion characteristics, further highlighting the active GI delivery potential of algae-based motor systems.

Formulation of algae motors into protective capsules

To effectively use algae motors for delivery to the GI tract in vivo, it is necessary to overcome the harsh acidic environment of the stomach, which can degrade the algae before they reach the small intestines. To address this, we modified a commercial capsule to encapsulate viable algae in an internal aqueous medium for safe passage through the stomach. First, an organosilicon solution, consisting of 4% octadecyltrimethoxysilane (OTMS) (41), was prepared to create a thin hydrophobic coating on the inside of the capsule via a thermal evaporation technique (fig. S1A). To test the stability of a capsule with this internal coating, we encapsulated an aqueous solution containing rhodamine dye. Visually, the capsule remained unchanged, whereas notable deformation was observed for a control uncoated capsule (fig. S1, B and C). By changing the number of coating layers, we could modulate the degradation of the capsules, which is reflected by the release of the dye, with 10 layers of coating offering the longest delay in release in SIF (fig. S1, D and E). Second, the exterior surface of the capsule was coated with Eudragit L100-55, a pH-responsive polymer commonly used for protecting oral medication from harsh gastric acid conditions (fig. S2A); previous studies have demonstrated the utility of Eudragit L100-55 as an enteric coating for enhancing the delivery of micromotors to the intestines (42). To imitate physiological conditions in the stomach, we used simulated gastric fluid (SGF) at pH 1.5, containing sodium chloride and hydrochloric acid. In our case, three layers of coating using a 7% (w/v) polymer solution offered full protection of the encapsulated cargo from SGF (fig. S2B). Upon changing from SGF to SIF, burst cargo release was observed within 10 min because of dissolution of the enteric coating at higher pH values (fig. S2C). These results confirmed that it was possible to load cargo in aqueous solutions using suitably coated capsules for the GI delivery of algae motors.

Next, we investigated the encapsulation of live algae into the modified capsules and their release in vitro. Algae were suspended in tris-acetate-phosphate (TAP) medium and loaded into capsules with 10 inner OTMS layers and 3 outer enteric coating layers. For visualization, the OTMS and enteric coating layers were labeled with 3′,3′-dioctadecyloxacarbocyanine (DiO; excitation/emission = 484 nm/501 nm) and Pacific Blue (excitation/emission = 410 nm/455 nm), respectively. Under fluorescence microscopy, a strong signal was observed for both coating layers along with the autofluorescence of the algae (excitation/emission = 647 nm/680 nm), confirming the successful encapsulation of algae motors into the fabricated capsule platform (Fig. 3A). In SGF, it was demonstrated that there was no release of algae from the capsules, whereas the algae motors could be released over time in SIF (Fig. 3B). The release in SIF was gradual for about 30 min, after which most of the algae was released by 45 min from the start of the experiment. The total number of released algae reached 9.15 × 10⁵ after 45 min (Fig. 3, C and D). Motion tracking of algae motors within the capsule and algae motors released from the capsule revealed similar patterns of movement (Fig. 3E and movie S7). The median speed calculated from 100 individual algae also remained unchanged throughout the fabrication process and after release from the capsules (Fig. 3, F and G, and fig. S3). These data indicated that the algae motors could be effectively encapsulated and then released from the modified capsule with negligible effect on their swimming performance.

After evaluating the release of algae motors from the capsules under in vitro conditions, we investigated the delivery capabilities of the platform within the GI tract. First, mimicking the physiological conditions of the intestines, the motion behavior of algae motors and Mg motors was evaluated in SIF at 37°C in vitro (Fig. 4, A and B). The speed of the algae motors was affected by the elevated temperature (movie S8), decreasing from 113 to 83 μm/s within 15 min of self-propulsion and further dropping to 40 μm/s after 12 hours. At the experimental endpoint, about 70% of the algae remained motile, indicating that they could survive for prolonged periods of time even under unfavorable conditions. In comparison, the conventional Mg motors rapidly lost their movement (movie S9), with only 33% still propelling at a high speed of 180 μm/s after 5 min. After another 10 min, only 8% remained motile, with an average speed of 80 μm/s. This fast drop in speed and the fraction of motors displaying active motion reflected the rapid depletion of the Mg engine. The algae motors also exhibited the ability to swim in viscous simulated mucus (43) over an extended period of time, whereas the Mg motors did not (fig. S4). These findings illustrated the greatly improved behavior of the algae motors compared with their Mg-based counterparts for potential GI applications requiring prolonged propulsion.

In vivo biodistribution of algae motors after oral delivery

After the in vitro tests, a study was carried out to assess the in vivo biodistribution and retention of the algae motors when delivered orally in capsule form. To facilitate imaging and quantification in biological tissue, we labeled the algae motors and Mg motors with the same fluorescent dye (fig. S5). The algae motors were directly conjugated with fluorescein, whereas the Mg motors were first coated with poly-l-lysine (44), then conjugated with the dye. As a result, both motors could be easily visualized under fluorescence microscopy and displayed near-identical signals that did not diminish after 6 hours in SIF at 37°C. Next, the algae motors and Mg motors were embedded in the protective capsules and administered by oral gavage to mice. At 5 hours after administration, the mice were euthanized, and their GI tracts were imaged ex vivo (Fig. 4C). Whereas a narrow distribution was observed from the fluorescent signal of the Mg motors, the signal for the algae motors was more broadly distributed through the intestines (fig. S6). Quantification of the total radiant efficiency within the small intestines corroborated the improved retention of the algae motors compared with the Mg motors (Fig. 4D). The observed differences in biodistribution suggested that algae, with their long-lasting movement properties, could be effective at delivering drug payloads locally within the GI tract.

In vivo delivery of therapeutic drugs using algae motor capsules

To better understand the mechanism behind the improved biodistribution and retention of algae motors within the small intestine, we compared our algae motor capsule formulation with different control groups, including TAP medium only (negative control), free algae without a capsule, and static algae in a capsule. To prepare the static algae control, we deflagellated live algae using acetic acid and resuspended them in phosphate-buffered saline (PBS) for encapsulation. Optical visualization and scanning electron microscopy (SEM) images confirmed successful deflagellation and that the resulting static algae lost their motion capabilities in SIF at 37°C (fig. S7 and movie S10). The intrinsic fluorescence of chlorophyll a in algal chloroplasts allows for noninvasive fluorescence imaging of algae without the need for chemical modification (45). We then proceeded to perform ex vivo fluorescence imaging on the GI tracts of mice receiving the various formulations with equivalent fluorescence to determine the influence of active movement and capsule protection on biodistribution (Fig. 5A and fig. S8). At 5 hours after oral administration, algae motors delivered inside capsules were more broadly distributed across the intestines compared with static algae that were also encapsulated. This result highlighted the importance of self-propulsion, which likely helped to increase the interaction of the algae with the intestinal wall, thus leading to enhanced retention. In addition, there was almost no signal observed in the intestines after administration of free algae, demonstrating the necessity of using the capsules to protect from the harsh stomach acid. Quantification of the fluorescent signal from each sample further supported the imaging results, as the fluorescence from the encapsulated algae motor group was 3.5-fold greater than that of the encapsulated static algae group (Fig. 5B). To control for the background signal from food contaminants within the stomach (42, 46), we delivered capsules containing algae motors labeled with Cyanine7 (Cy7; excitation/emission = 750 nm/773 nm) orally, and it was confirmed that most of the algae were distributed within the intestine (fig. S9).

We next explored the feasibility of using algae motors for delivering therapeutic drugs to the GI tract. Dox, a commonly used frontline chemotherapeutic agent (47), was selected as a model drug payload. First, RBC membrane–coated NPs were loaded with Dox [denoted “NP(Dox)”] via a double emulsion solvent evaporation technique (48, 49). Transmission electron microscopy (TEM) imaging confirmed the core-shell structure of the NPs (fig. S10). Fluorescence imaging showed colocalization of the Dox-loaded PLGA cores and the DiO-labeled RBC membrane coating, verifying successful drug loading (fig. S11). Next, NP(Dox) was linked to the algae [denoted “algae-NP(Dox)”] by click chemistry (Fig. 6A). Fluorescence and SEM imaging confirmed the effective binding of NP(Dox) to the algae (Fig. 6, B and C). To test the Dox loading onto the algae, we incubated 1 × 10⁶ algae with different concentrations of NP(Dox). The Dox loading yield onto the algae was measured at different NP(Dox) inputs, and the maximum loading amount (15 μg) was obtained with a 50-μg input of Dox, corresponding to a 30% loading efficiency per 10⁶ cells (Fig. 6D). It was also confirmed that Dox, either in free form or nanoparticulate form, did not influence the viability of the algae (fig. S12). The mean and median speed of algae-NP(Dox) measured from 100 individual algae motors were 108.69 and 108.78 μm/s, respectively, and these values were comparable with those of the bare algae (Fig. 6, E and F). Furthermore, it was demonstrated that the drug release profile of NP(Dox) was not affected by binding to the algae motors (Fig. 6G). After loading onto algae motors, the NP(Dox) payload retained its cytotoxic activity, as it was demonstrated that algae-NP(Dox) could inhibit the growth of B16F10 melanoma cell lines in vitro (Fig. 6H). Algae-NP(Dox) and NP(Dox), both loaded into protective capsules, were then administered at the same drug dosage, followed by extraction of the intestines to quantify Dox concentration (Fig. 6I). Compared with the tissue homogenates of mice administered with NP(Dox), the samples from mice receiving algae-NP(Dox) exhibited significantly higher drug levels at all of the time points (3, 6, and 9 hours) that were tested. These data further supported the benefits of using algae motors with prolonged active self-propulsion to enhance the delivery and retention of therapeutic payloads in the small intestinal tissues. Future studies will focus on evaluating the potential of algae motors for drug delivery to treat diseases in suitable animal models, such as for inflammatory bowel disease and bacterial gastroenteritis.

In vivo toxicity evaluation of algae motor capsules

Last, we evaluated the in vivo safety profile of the algae motor capsule platform after oral administration. A comprehensive blood chemistry panel and blood cell count were conducted 24 hours after administration (Fig. 7, A and B). Compared with untreated control mice, the levels of all serum biochemistry markers and numbers of blood cells (RBCs, white blood cells, and platelets) in the mice receiving algae motor capsule treatment remained at normal levels. A longer-term safety study in which mice were administered with one algae motor capsule on days 0, 2, 4, and 6 also yielded the same result, whereby negligible toxicity, indicated by the minor changes in metabolic biomarkers and blood cell counts, to the mice was observed. Histological analysis of GI tract tissue sections from algae motor-treated mice stained with hematoxylin and eosin (H&E) revealed that structural integrity was preserved and that there was no immune cell infiltration into the mucosa or submucosa, indicating the lack of an inflammatory response (Fig. 7C). There was also no observable inflammation or pathological changes on H&E-stained sections from other major organs such as the heart, lungs, liver, kidneys, and spleen (Fig. 7D). Overall, these results suggested that the algae motor capsule platform is safe to use for oral drug administration.

DISCUSSION

The development of microorganism-based micromotors for addressing key health care issues is still in its infancy (25). Although various in vitro studies have illustrated the use of bacteria-based drug delivery systems, there are major risks associated with their in vivo use when considering factors such as pathogenicity or immunogenicity (24). As an alternative cell type, microalgae are nonpathogenic and have been explored in the development of biohybrid systems (36, 50). Microalgal swimmers recently have been used as biomedical robotic actuators, primarily under in vitro settings (32). Early in vivo studies have mainly focused on the therapeutic delivery of algae-derived compounds (51). More recently, a few studies have reported the use of algae as drug carriers for in vivo operation (45, 52). For example, the spiral structure of Spirulina platensis was leveraged to passively prolong retention of curcumin in the intestines for the improved treatment of colon cancer and colitis (45). In addition, a magnetic NP-loaded microalgae biohybrid imaging system was reported recently, and a swarm of these microswimmers could be externally controlled by a magnetic field and tracked using magnetic resonance imaging (50). Despite these advances, there have been no studies exploiting the intrinsic self-propulsion of algae for enhanced GI delivery.

In the present study, several key points have been considered to tackle the challenges facing algae-based active GI delivery: protection from the harsh acidic environment of the stomach en route to the intestines, selection of a suitable algal strain with long-lasting self-propulsion in intestinal fluid, and cargo/drug loading capability. By addressing these issues, we demonstrated here an algae motor capsule system for effective intestinal targeting and prolonged tissue retention. Compared with the short lifetime of current Mg-based micromotors, the algae motors displayed prolonged propulsion in GI fluid toward enhanced local delivery for the treatment of potential GI diseases and disorders. To achieve intestinal delivery, we fabricated an enteric-coated capsule modified with an inner hydrophobic organosilicon layer to effectively protect encapsulated algae from the harsh gastric fluid while maintaining their viability. The capsule fabrication, encapsulation, and release processes had a negligible effect on the viability of the algae motors. Modifying capsules in the manner described here provides an effective approach for delivering motile living organisms in aqueous media to the GI tract. To demonstrate the distinct advantages of the platform for in vivo intestinal delivery, we administered algae motor capsules orally, and their biodistribution and retention properties were compared with various controls. The algae motors displayed broader distribution and stronger retention in the GI tract compared with synthetic Mg motors. This was likely mediated by the prolonged motion of the live algae, because it was shown that static algae that were incapable of propulsion exhibited considerably less retention in the intestines. We also demonstrated that the ability of the algae motor capsule system could also be used for the delivery of a model anticancer drug to the GI tract. Moreover, the platform displayed a favorable biosafety profile after oral administration.

The characteristics of the algae motor in a capsule formulation, particularly its long-lasting self-propulsion, can be leveraged for microrobotic biomedical applications beyond the treatment of GI diseases and disorders, ranging from GI detoxication to imaging and sensing. There are several approaches in which algae motors could be improved to enhance their utility for GI delivery applications. For example, the incorporation of imaging agents could enable direct visualization of algae movement as they operate within the intestines. A recent report described a photoacoustic computed tomography–guided microrobotic system for realizing real-time navigation and monitoring of synthetic Mg motors in the intestines in vivo (53). In another example, positron emission tomography combined with computed tomography was used to evaluate the swarm behavior of enzyme-powered nanomotors in the bladder (17). By conjugating magnetic microparticles to the algae surface (32), an external magnetic navigation system could be used to precisely guide and track algae motors to target sites. Whereas the current work focused on demonstrating the potential of algae motor capsules for enhancing intestinal delivery, future work will be required to confirm the therapeutic advantages of the platform in clinically relevant disease models, such as those for bacterial GI infection, irritable bowel disease, or colon cancer. Microalgae can also be engineered to express biologic payloads that can be produced in situ after oral administration (54, 55). The properties of the capsules can be tuned to more precisely target specific regions of the GI tract (42). Overall, functionalized algae motors, loaded within a protective capsule, represent an attractive biohybrid motor system that can be applied across a wide range of biomedical applications.