SCIENCE ROBOTICS | RESEARCH ARTICLE

MICROROBOTS Copyright © 2022

The Authors, some
Light-driven carbon nitride microswimmers rights reserved;
exclusive licensee
with propulsion in biological and ionic media American Association
for the Advancement
and responsive on-demand drug delivery of Science. No claim
to original U.S.
Government Works
Varun Sridhar1, Filip Podjaski2*, Yunus Alapan1, Julia Kröger2,3, Lars Grunenberg2,3,
Vimal Kishore1,4, Bettina V. Lotsch2,3,5*, Metin Sitti1,6,7*

We propose two-dimensional poly(heptazine imide) (PHI) carbon nitride microparticles as light-driven mi-
croswimmers in various ionic and biological media. Their high-speed (15 to 23 micrometer per second; 9.5 ± 5.4
body lengths per second) swimming in multicomponent ionic solutions with concentrations up to 5 M and without
dedicated fuels is demonstrated, overcoming one of the bottlenecks of previous light-driven microswimmers.
Such high ion tolerance is attributed to a favorable interplay between the particle’s textural and structural nano­
porosity and optoionic properties, facilitating ionic interactions in solutions with high salinity. Biocompatibil-
ity of these microswimmers is validated by cell viability tests with three different cell lines and primary cells. The
nanopores of the swimmers are loaded with a model cancer drug, doxorubicin (DOX), resulting in a high (185%)
loading efficiency without passive release. Controlled drug release is reported under different pH conditions and
can be triggered on-demand by illumination. Light-triggered, boosted release of DOX and its active degrada-
tion products are demonstrated under oxygen-poor conditions using the intrinsic, environmentally sensitive

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and light-induced charge storage properties of PHI, which could enable future theranostic applications in oxygen-­
deprived tumor regions. These organic PHI microswimmers simultaneously address the current light-driven
microswimmer challenges of high ion tolerance, fuel-free high-speed propulsion in biological media, biocom-
patibility, and controlled on-demand cargo release toward their biomedical, environmental, and other poten-
tial applications.

INTRODUCTION positive and negative phototaxis and gravitaxis (21), depending on

Microswimmers are cell-scale mobile machines that can be self-­ their surface charge (22), enabling their steering control and targeting
propelled by converting energy made available to them from their a desired location (23).
environment or remotely (1–3), such as chemical reagents, light, Despite their many advantages over other self-propulsion methods,
and magnetic or acoustic energy (4, 5). One of the main targeted photocatalytic and chemical propulsion suffer from fundamental
applications of microswimmers is local, active, and on-demand propulsion limitations in electrolyte solutions (18), especially when
delivery of theranostic cargos, such as drugs, imaging agents, and diffusiophoretic and electrophoretic propulsion modes are opera-
stem cells inside the human body and organ-on-a-chip devices (6–8). tive. This drawback is due to the presence of ions hindering the
So far, chemical propulsion—for example, by dissolution of metals build-up of concentration gradients of reactants and products in-
(e.g., magnesium under acidic conditions)—enzymatic, and mag- volved in self-electrophoresis and self-diffusiophoresis around the
netic propulsion are the most widely used methods in such in vitro swimmer (24–26). Although different semiconducting inorganic
and in vivo biomedical applications (9). Chemical propulsion is photocatalysts (27–32) have been studied as light-driven micro­
predominantly used for swimming in acidic body fluids, such as swimmers for potential biological applications (33), donor-free
inside the stomach and gastrointestinal tract (9–12), whereas enzy- light-driven swimming in ionic media has only been demonstrated
matic propulsion can be used in other body regions (13–15). Both with sophisticated geometries in Si for concentrations below 10 mM.
methods need sacrificial and usually toxic agents as fuels to enable Thus, light-driven swimming in ionic media—such as NaCl,
swimming, which is disadvantageous for their prolonged use under Dulbecco’s phosphate-buffered saline (dPBS), and Dulbecco’s
biological conditions (16). Light, however, is a viable energy source modified Eagle’s medium (DMEM)—with concentration levels of
for the propulsion of microswimmers (17, 18), both with and with- more than 200 mM present in various biological solutions and cell
out the use of additional fuels, enabling also propulsion control and environments still remains an unresolved bottleneck. The EI50
steering (19, 20). Moreover, photocatalytic swimmers can exhibit number has been introduced previously as a measure of the ionic
concentration at which the speed of the microswimmers is reduced
by 50% (34). EI50 is less than 0.1 mM for self-diffusiophoretic and
1
Physical Intelligence Department, Max Planck Institute for Intelligent Systems, self-electrophoretic swimmers (25), reaching up to ~4 mM for
70569 Stuttgart, Germany. 2Nanochemistry Department, Max Planck Institute for geometrically optimized systems addressing this problem (34).
Solid State Research, 70569 Stuttgart, Germany. 3Department of Chemistry, Ludwig-­
Maximilians-Universität München, 81377 Munich, Germany. 4Department of Physics,
For biomedial applications, an EI50 value of more than 100 mM
Banaras Hindu University, Varanasi 221005, India. 5Cluster of Excellence e-conversion, is required. This limitation is attributed to the presence of a solid
Lichtenbergstrasse 4, 85748 Garching, Germany. 6Institute for Biomedical Engi- surface in the microswimmer, which does not allow fluids or ions to
neering, ETH Zurich, 8092 Zurich, Switzerland. 7School of Medicine and College of migrate through the swimmer. The presence of salt ions reduces the
Engineering, Koç University, 34450 Istanbul, Turkey.
*Corresponding author. Email: [email protected] (M.S.); [email protected] (B.V.L.); Debye length of the electrochemical layer formed on the surface of
[email protected] (F.P.) the microswimmers in contact with the solution from hundreds of

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SCIENCE ROBOTICS | RESEARCH ARTICLE

nanometers to ~1 nm (35), thereby collapsing also the ionic gra- resulting in an unusual blend of optoelectronic and optoionic prop-
dient around the particle under illumination, which is responsi- erties (47–49). PHI does not only show higher hydrogen evolution
ble for the propulsion force. Therefore, commonly, the collapse of activity than melon-type CNx (44, 45, 50), but it is also able to store
the Debye layer stops the swimming for hard spheres. Besides, most light-induced electrons for subsequent use through electron-ion
light-driven microswimmers require high concentrations of H2O2 interactions (47–49, 51). Because PHI micro­swimmers exhibit not
or alcohols as additional chemical fuels (36), which should be only structural but also textural porosity, enabling ion intercalation
avoided in biological applications because of their toxicity. The and permeability that can be driven by light (42, 46–48), they are
availability of potential biocompatible fuels that are present in large promising platforms for light-driven propulsion (51) and cargo de-
enough quantities has been a pressing bottleneck in implement- livery in various biological media, while not requiring sophisticated
ing light-driven microswimmers in such applications (18), which shape control. Hence, the development of biocompatible and highly
require concentrations as high as ~30 mM of biocompatible fuels ion-tolerant, nontoxic light-­driven microswimmers that can be pro-
like glucose for effective light-driven propulsion (37). Furthermore, pelled purely by visible light in naturally occurring biofuels while
taxis-based direction-controlled propulsion and controlled cargo being able to carry and release cargo in a controllable fashion may
uptake and release of active products are highly desired properties solve many fundamental challenges (48).
of medical microswimmers, which are usually studied and realized
separately (38).
Here, we aim to solve the above issues and bottlenecks by using RESULTS
highly (photocatalytically) active and porous (texturally and struc- PHI characterization
turally) carbon nitrides (CNx) as light-driven microswimmers. CNx The PHI microparticles were obtained by sonication and centrifuge-­
are a family of organic macromolecular photocatalysts that have assisted separation from the original suspension (see Materials and
gained attention because of their facile synthesis (39), chemical Methods for details). Figure 1A shows a scanning electron mi-

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robustness, absorption of visible light, and favorable band positions croscopy (SEM) image of the PHI microparticles with a diameter
that enable various photocatalytic redox reactions, such as water ranging from 1 to 5 m. Because they represent agglomerates of
splitting (40). Besides, CNx are widely used for environmental re- smaller primary crystallites, the microswimmers have an irregular
mediation (41), sensing, and ion pumping (41–43). Most commonly, spherical shape, and textural nanopores occur spontaneously. These
a one-dimensional (1D) CNx called “melon” or “g-C3N4” is reported. larger voids enable efficient fluid movement and entrapment.
However, in this study, we use a recently found 2D CNx species called Figure 1 (B) illustrates the underlying molecular structure of the PHI
poly(heptazine imide) (PHI) (44–46), which hosts hydrated alkali microswimmers. Structural nanopores in the PHI backbone con-
metal ions (typically K+) or protons in its structural nanopores, sisting of imide-bridged heptazine units are filled with hydrated

Fig. 1. Structure, morphology, and optical properties of PHI-based organic microswimmer particles. (A) SEM image of representative PHI microparticles (gray) with
a size distribution of 1 to 5 m (scale bar, 5 m) and close-up of one particle (scale bar, 400 nm), showing the porous morphology. (B) Schematics of the PHI microswimmer
and structure of the PHI macromolecules consisting of heptazine moieties comprising carbon (blue), nitrogen (gray), and hydrogen (white). Solvated potassium ions
reside in structural nanopores (purple). (C) Absorbance spectrum of PHI microswimmers, showing the onset of bandgap absorption at 450 nm determined on the basis
of a Tauc plot. a.u, arbitrary unit. (D) Band positions of PHI and melon with a bandgap of 2.7 eV along with hydrogen evolution reaction (HER), oxygen reduction reaction
(ORR), and oxygen evolution reaction (OER) potentials at pH of 7.

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ions that can be exchanged (46). The absorbance spectrum in Fig. 1C PHI microswimmers also moved in the diluted red blood cell (RBC)
shows light absorption below 450 nm (bandgap of 2.7 eV) (47, 49), solution. Autofluorescence of the PHI microswimmers, i.e., the
enabling photocatalysis and, hence, propulsion driven by visible intrinsic emission of PHI without any fluorescent markers, enabled
light. The strongly positive valence band position (+2.2 V versus their label-free imaging and detection even in the optically dense
normal hydrogen electrode) gives rise to an even stronger oxidative RBC solution. The mean speed of the PHI microswimmers in the
power of the light-generated holes than for melon (Fig. 1D) (47), dilute RBC solution, where viscosity remains the same in compari-
providing the thermodynamic driving force for various photocata- son with pure DMEM solution (54), was 20.2 ± 0.8 m/s, also
lytic oxidation reactions, including water oxidation, which are often comparable with their mean speed in DMEM. The RBCs in the
the bottleneck for light-generated charge extraction and photocata- solution were also observed to be moving in the same direction,
lytic propulsion (51). which appears to be caused by the fluidic flow arising from the
collective propulsion of PHI microswimmers (note S2). The PHI
Light-induced swimming in different biological media microswimmers were not able to swim in whole blood, which could
To demonstrate the mechanism of propulsion of the PHI micro­ be ascribed to the increased viscosity and heterogeneous non-­
swimmers, the light from a photodiode was focused with an inten- Newtonian nature making photocatalytically driven effective swim-
sity of 1.9 W/cm2 at 385 nm on the particle chamber. The mean ming impossible. A comparison of the available literature and the
speed of the illuminated microswimmers in deionized (DI) water current work on ionic swimming is shown in table S1. For better
was 24.2 ± 1.9 m/s (Fig. 2A and movie S1). In this case, their comparison, a discussion of the wavelength and intensity depen-
photocatalytically induced swimming is attributed to water oxida- dence of the propulsion can be found in note S2, figs. S1 and S2,
tion and reduction of dissolved oxygen on the illuminated PHI and tables S2 to S4. In DMEM, light-driven propulsion was effi-
hemisphere (51). To explore and determine the influence of differ- cient and proportional to the intensities in the UV and blue light,
ent ionic and biological constituents on the propulsion efficiency of as shown in Fig. 2B.

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the PHI microswimmers, different pH neutral buffers and relevant
biological media were tested (see note S1 for the ingredients and Ionic tolerance for light-induced propulsion with NaCl
concentrations of all studied liquids). In brief, dPBS was used as the To better understand the ionic tolerance of PHI, the microswimmers
buffer solution for cell washing, which contains NaCl, KCl, Na2HPO4, were exposed to various NaCl concentrations and compared with
and KH2PO4 at ca. 10 g/liter (~150 mM) in total. DMEM, commonly the “sister material” melon under 385-nm illumination, as shown in
used to culture cells, contains the same salts (~160 mM), amino Fig. 2C. In comparison with DI water (24.2 ± 1.9 m/s), the PHI
acids (ca. 2 g/liter, ~10 mM), and trace amounts of vitamins and swimmers showed a 25% decreased speed (18.1 ± 3.4 m/s) at 1 mM
glucose (4.5 g/liter, ~25 mM), some of which can be used as reduc- NaCl, with no obvious further decrease in speed observed until
ing agents and, hence, hole extraction fuel to power light-driven 100 mM (18.6 ± 3.4 m/s). Changing the electrolyte cation to potassium
microswimmers (52). Under illumination, the mean speed of the at the same concentration (100 mM KCl) has negligible influence
microswimmers in dPBS was 19.0 ± 3.3 m/s (21% slower than in on the propulsion speed within the margin of error (20.7 ± 3.5 m/s).
DI water) and 23.7 ± 2.6 m/s in DMEM (comparable with DI At 1 M NaCl, they showed a further decrease (12%) in speed
water), as seen in Fig. 2A. The ions present in dPBS (~150 mM) (16.1 ± 3.9 m/s) (see movie S3). Hence, the speed was reduced only
hence only have a relatively small influence on the speed of PHI, by 33% with respect to DI water and the characteristic EI50 is not yet
whereas the additional components in DMEM do not hamper reached. Only at 5 M NaCl, the speed is reduced to 9.7 ± 2.4 m/s
the efficiency of photocatalytic reactions being responsible for the (60% less than the speed in DI water), surpassing the EI50.
propulsion (movie S2). To emulate a realistic cell environment, the Hence, PHI’s ion tolerance surpasses all reported light-driven micro­
growth-supplementing medium fetal bovine serum (FBS) contain- swimmers by two orders of magnitude, as summarized in table S1,
ing complex proteins and other components (53) was added to and maintains fast light-induced propulsion even at 5 M salt
DMEM. The illuminated PHI microswimmers still moved with a concentrations.
mean speed of 12.5 ± 3.1 m/s in DMEM medium with 10% FBS, We attribute the constant speed between 1 and 100 mM to a
hence 47% slower than in DMEM alone. This decrease in speed may constantly high ionic mobility between the PHI microswimmer and
be ascribed to the viscosity change and deposition of protein and ionic environment, which only becomes limiting at higher concen-
other components on PHI, thus blocking surface reactions partially trations. To further investigate the effect of textural porosity on the
(50). In addition, a high concentration (1 M) of sodium phosphate high ion tolerance, PHI and melon were tested under identical con-
buffer, an important component of DMEM, was also used to test ditions. Although both melon and PHI exhibit textural porosity
the propulsion of PHI microswimmers. We observed a mean speed [see Fig. 1A and figs. S7 and S8 for SEM and Brunauer-Emmett-Teller
of 9.4 ± 1.7 m/s, which is 54% lower than in the 160 mM salt (BET) measurements], melon has no structural porosity because it
containing DMEM. These findings show that PHI microswimmers consists of close-packed 1D heptazine imide chains, hence enabling
can be used efficiently in many realistic biological environments only surface ionic interactions. The mean speed of the melon micro­
and in salt solutions at concentrations even beyond those of bio- swimmers under UV illumination was 11.7 ± 2.9 m/s in DI water
logical fluids. (43% lower than PHI). At 10 mM NaCl, the propulsion speed decreased
Active motion in heterogeneous and complex biological fluids, weakly (9%), with EI50 being reached at ~100 mM (6 ± 1.3 m/s).
such as blood, is also crucial for future biomedical applications These findings also show that this 1D form of CNx has a high ion
inside the human body. To test the propulsion capability of the PHI tolerance (higher than any other material reported except PHI),
microswimmers in blood, they were mixed with blood cells with a which we tentatively attribute to its textural porosity, enabling ionic
25% hematocrit (in DMEM medium), which is in the physiological permeation by an internal flow of ions and liquid under illumina-
range. When illuminated with ultraviolet (UV) light (385 nm), the tion (42, 43). However, at 1 M NaCl, the melon microswimmers

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Fig. 2. PHI microswimmer propulsion in different ionic fluids. (A) Mean speeds (N = 50) of PHI microswimmers in various biological media (30 g/ml) swimming under
385-nm light illumination, and the error bars indicate the SD in all plots. (B) Mean speeds (N = 50) of the PHI microswimmers in DMEM medium under different illumination
wavelengths. The propulsion speed is constant under dilute (3 g/ml) and under normal conditions (30 g/ml). (C) Mean speed (N = 50) of the PHI and melon CNx swimmers
in increasing concentrations of NaCl under 385-nm illumination. (D) Sample 2D trajectories of the PHI microswimmers in DMEM medium swimming under 365-nm illumi-
nation from the side with S and E indicating the start and end of trajectories, showing a positive phototactic behavior. (E) Polar plot of the directed propulsion of the PHI
microswimmers under the same conditions as (D) after 15 s of illumination. Radial scale bar, 50 m. (F) Phototactic behavior of PHI microswimmers in DMEM medium
swimming under 385-nm illumination from the bottom to a point in the image. S and E indicate the start and end of the trajectories. (G) Schematics of the band structure
of PHI along with the reduction and oxidation reactions responsible for the photocatalytic propulsion of PHI microswimmers and the light-induced intercalation of alkali
metal ions (K+ or Na+, e.g., purple) into the particle, enabled by optoionic effects assisting photocharging in PHI (blue). (H) Proposed mechanism for the phototactic propul-
sion of porous CNx particles (nanopores not to the real scale and density; particle shape not perfectly spherical in reality) caused by asymmetric illumination and photocatalysis,
resulting in ion flow around and through the particle. The movement of cations (purple) into and through the particle’s pores counteracts the Debye layer collapse and
enables ionic tolerance. Pseudocapacitive photocharging effects (blue) present in PHI increase ionic tolerance with respect to melon. Here, A and D represent electron
donor and acceptor reactions, respectively. Also, h+ and e– represent the holes and electrons, respectively.

stopped ballistic swimming (only active Brownian motion was observed), and Na+ ions (hydrodynamic radius of 1.25 and 1.84 Å, respectively),
which may be attributed to the absence of optoionic properties and making the molecular backbone of PHI permeable to ions. We have
a smaller thermodynamic driving force for oxidation (Fig. 1D). demonstrated in earlier work that the light-induced hole extraction
In contrast to melon, PHI has not only textural but also structural and photo-charging ability of PHI is intimately linked to the presence
nanopores, with a radius on the order of 3.84 Å (44, 45, 47). The of ions in the pores of PHI and the surrounding electrolyte, which
pores are large enough to host and allow the passage of hydrated K+ may lead to light-induced ion transport throughout the structural

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and textural nanopores of PHI and thus the movement of ions explained by the illumination from one side only, shadowing the
toward the (photogenerated) electrons on PHI (42, 45, 46, 48), as other half of the particle, resulting in the creation of an artificial
illustrated in Fig. 2 (G and H). We believe it is these intrinsic Janus-type structure that breaks the microswimmer’s symmetry,
optoionic properties of PHI, which are absent in melon, paired with independent of the irregular size and shape of the swimmers. The
porosity, that lead to sustained motion in high ionic strength media negative zeta potential of PHI (−35 mV as measured in 10 mM NaCl)
on the time scale of the experiments. The presence of both textural (44) led to positive phototaxis in this situation (22, 56–58).
and structural pores invalidates the assumption of a hard sphere When PHI microswimmers were illuminated under the optical
(the latter excluding liquid or ion flow through the volume of microscope with light focused onto the image plane, the particles
the particle), which is commonly used to describe phoresis. Hence, the moved toward the middle of the light beam (Fig. 2F and movie S1),
Helmholtz-Smoluchowski equation, U = eE0, where e is the enabling collective assembly in one direction. Further effects and
electrophoretic mobility and E0 is the magnitude of the electric possible thermal contributions in the case of dense suspensions are
field, cannot be used to sufficiently describe the motion. Therefore, discussed in note S3, and figs. S4 to S6 show control experiments
different theoretical models are needed to be developed in the with nonpropelled, passive polystyrene particles. The propulsion
future to fully capture the reason for the high ion tolerance of this under these conditions was shown to last for 2 hours with minimal
organic semiconductor. speed reduction (−18%, from 23.5 ± 3.5 to 19.8 ± 3.2 m/s), attributed
Despite the presence of ions and no dedicated fuel, the speed of to crowding and shading (fig. S7). The phototactic properties of
light-driven PHI microswimmers (9 to 23 m/s) is comparable the PHI microswimmers, which originate predominantly from
with, and even higher, than the speed of other light-driven swim- their light-induced photocatalytic propulsion (Fig. 2, G and H, and
mers in absence of ions and in presence of dedicated fuels (5 to movie S4), as evidenced most directly by the propulsion at low par-
25 m/s) (22, 55). The limitations on phoresis arising from the ticle concentrations and the decreasing speed with increasing ionic
accumulation of ions around the particle are shown to be efficiently concentration, can be used to control the direction of their motion

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bypassed by suitable porosity, and in addition, it is hypothesized in various future biomedical applications, such as targeted drug
that optoionic effects enabling ion transport increase the ionic delivery and cargo transport. Besides, thermal effects can assist the
tolerance and speed of the swimmers. In the presence of buffers and swarming behavior under crowded conditions. Phototactic swim-
biological fluidic media, the additional species partially negate the ming of microswimmers in curvilinear paths and complex paths
speed reduction, presumably by offering additional redox reaction can be demonstrated by moving the light source dynamically with
pathways with respect to water and NaCl, hence increasing the respect to the swimmers, thereby changing the illumination angle.
efficiency of the photocatalytic propulsion process. The swimming in curvilinear paths is essential for applications such
as cargo transport, where the swimmer would need to navigate
Phototaxis of the PHI microswimmers against obstacles. Such phototactic swimming is shown in fig. S8
Light-induced propulsion itself lacks directional control. However, and movie S6.
phototactic properties of the microswimmers and light illumination
direction control enable their steering to a desired location, which is Cytotoxicity tests
very beneficial for their biomedical and other practical applications. A low cytotoxicity profile of the microswimmers is an essential
To test possible phototactic properties of the PHI particles, they requirement for their future biomedical applications. Therefore, we
were illuminated with 365-nm UV light at a 45° angle from one tested the cytotoxicity of the PHI microswimmers with a normal
side, with an intensity of 115 ± 15 mW/cm2, in DMEM medium. cell line (NIH 3T3 fibroblast cells) and two cancer cell lines (HT-29
This created a light gradient along the x axis as shown in Fig. 2D, colorectal cancer cells and SKBR3 breast cancer cells). Live/dead
which the particles followed. The mean speed of the microswimmers staining of the tested cell lines incubated with varying concentrations
was 12.5 ± 0.4 m/s in this case, which is substantially higher (78%) of PHI microswimmers (0 to 30 g/ml) for 24 hours showed no
compared with the lateral motion measurement with the perpendic- decrease in cell viability for all cell lines (Fig. 3, A and B). Other than
ular illumination through the microscope objective at 400 mW/cm2 particle cytotoxicity, we also tested the effect of illumination and
(6.5 ± 1.2 m/s), and hence as fast as with 10× stronger illumination catalytic activity of PHI microswimmers on cellular viability.
from the bottom (1.2 W/cm2). This apparent increase in speed is Illumination at 415 nm (440 mW/cm2) and 385 nm (1.1 W/cm2) of
attributed to a change in directional component of the measured a high density of PHI microswimmers (30 g/ml) on the fibroblast
speed. In the case of illumination from the side, there was a larger cells for varying durations (0 to 30 min) showed no negative effects
parallel component resulting in a higher lateral speed; when illumi- on their viability immediately after testing (Fig. 3, C and D).
nating from the bottom, the parallel component was slower, whereas Coupled to the efficient propulsion of the PHI microswimmers in
the z component (parallel to the illumination) could not be ana- biological media, their operation in cellular environments is highly
lyzed, resulting in a lower effective speed. Besides, tumbling and promising. When illuminated at 415 nm for 30 min, the cells re-
rotational motion may cause a sidewise motion with the perpendicu- tained viability up to 24 hours. With 385-nm UV light, 24-hour
lar illumination (21). As can be seen from the trajectories in Fig. 2D, viability is retained up to 10-min illumination. Because strong UV
the polar plot in Fig. 2E, and movie S4, the PHI particles followed illumination for long durations is harmful for the cells (59), they are
the direction of illumination very precisely; also, the speed was not not viable if illumination lasts for 30 min. Studies with primary
affected by decreasing the particle concentration to very dilute cases cells from mouse splenocytes further confirmed that there was no
(from 30 to 3 g/ml). At 415-nm side illumination, the same photo- detectable level of interleukin-12 (IL-12; proinflammatory marker)
tactic behavior resulted, with a speed of 5.3 ± 1.3 g/ml. When the in either of the untreated samples in concentrations used above in
direction of illumination was changed, the direction of swimming the dark. As shown here, long-duration exposure to short wave-
also changed, as shown in movie S4. This behavior could be length light can cause damage to the cells; therefore, the swimmer

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Fig. 3. Cytotoxicity of the PHI

microswimmers. (A) Cell viability
of fibroblasts and cancer cells incu-
bated with the PHI microswimmers at
varying concentrations after 24 hours.
Data represent the means and the error
bars represent the SD of ~300 cells.
(B) Live/dead staining of healthy BJ
human fibroblast cells after 24 hours
of incubation with the PHI micro­
swimmers. Green and red indicates
the live cells and dead cells, respec-
tively, along with the bright-field
images in the same conditions indi-
cating the cells and PHI (black dots).
Scale bars, 100 m. (C) Cell viability
of BJ fibroblast cells in presence of
PHI microswimmers (30 g/ml) right
after and 24 hours after illumination
for varying durations at 385 nm and
415 nm. Data represent means ± SD.
(D) Live/dead staining of BJ fibroblast
cells after swimming of PHI swim-

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mers, not in the picture (30 g/ml),
via light (415 nm) after 30 min, along
with the bright-field images under
the same conditions indicating the
cells and PHI (black dots). Scale bars,
100 m.

exposure times need to be limited to 10 min only for 385-nm wave- efficiency of 110% (110 g) resulted under the same conditions.
length light, whereas it can last 30 min or more for 415-nm wave- Astonishingly, no passive release was observed for PHI in the absence
length light. Such durations are typically long enough for the of illumination for more than 30 days, even at room temperature.
desired in vitro or in vivo biomedical functionalities. In vivo studies The stronger attachment and higher loading of DOX to the surface of
are required as future work for validating the complete biocompat- PHI is likely due to enhanced interactions between the oxygen-rich
ibility of the PHI microswimmers; however, the cytotoxicity and DOX backbone and PHI, due to its more negative zeta potential in
immunogenicity experiments are important steps in this direction. comparison to melon (44, 45, 61). Moreover, the DOX molecules
loaded on PHI particles did not show a strong negative effect on
Drug loading and hypoxically, pH-, and light-triggered the propulsion speed of the loaded PHI microswimmers in DMEM
drug release (18.5 ± 0.9 m/s, 22% less than the speed in DI water). This can be
Biocompatible PHI microswimmers are capable of actively carrying rationalized by the fact that the large DOX molecules are predomi-
and releasing drugs and other cargos attached to their porous body nantly physisorbed in the particle volume, rather than in the
structure at a target location. The concept of their targeted drug structural pores or on the outer particle surface, which does not
delivery is illustrated in Fig. 4A with an anticancer model drug, significantly block the ion flow through the inner structure or affect
doxorubicin (DOX). The presence of textural nanopores in both the outer surface photocatalytic reactions of PHI with the surround-
PHI and melon microswimmers is beneficial for enhanced drug ing medium that give rise to a field gradient around the particle and,
loading efficiency (BET surface area is ~13 and 26 m2/g and pore hence, swimming propulsion. On the other hand, the speed reduc-
size distribution is from 5 to 20 nm and 5 to 40 nm for the PHI and tion with loaded DOX again underlines that porosity and permea-
melon microswimmers, respectively; see fig. S9 and note S4), bility for ions seem to be important parameters to enable propulsion
enabling the adsorption of drugs within the microparticle pores in ionic media.
(44, 60). We further anticipate that drug uptake is assisted by the Illuminating the PHI and melon particles triggers the release of
amine surface groups of both CN x, enabling hydrogen bonding DOX, thus enabling a fully controlled, on-demand release of the
interactions with the drug, and by the negative zeta potential of drug. We used a 415-nm blue light at an intensity of 170 mW/cm2
melon and especially PHI, which intrinsically attracts the posi- to study the release of DOX and related products from the mi-
tively charged DOX molecules at pH 7 (44, 60, 61). To test this hy- croswimmers in both ambient and O2-free environments (Fig. 4C
pothesis, 200 g of DOX was added to a suspension of 100 g of and fig. S11A). Bandgap illumination of PHI microswimmers in
PHI microswimmers dispersed in 1 ml of DMEM, resulting in oxygen-deficient suspensions triggers (oxidative) photocharging of
185 g of DOX encapsulated with a DOX loading efficiency of 185% the material, which is accompanied by a change of optoelectronic
on PHI after 24 hours, which is far higher than previously reported properties and a color change from yellow to blue (47, 49). Hence,
values of 20 to 70% (62–64). Figure 4B shows the fluorescence this charging effect is expected to influence the release, too (Fig. 4A).
image of DOX loaded on the PHI particles. For melon, a loading The charging effect was also observed for denser PHI suspensions

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Fig. 4. Drug loading and hypoxically, light-, and pH-triggered drug release with the PHI microswimmers. (A) Schematic drug loading (DOX) and triggered release
from the PHI microswimmers under ambient and hypoxic conditions. (B) Map of the DOX-loaded PHI microswimmers [185 g/ml of DOX in PHI (100 g/ml)] showing DOX
fluorescence emission at 595 nm. Scale bar, 10 m. (C) Photo of the DOX-loaded PHI microswimmers immediately after 30 min of illumination under oxygen-deficient
conditions showing the charged blue state of PHI and the released DOX and by-products in red color. (D) Light-triggered cumulative release signal of DOX and by-products
in DMEM medium at different time points under 415-nm illumination, with the supernatant being removed at each interval in ambient (black) and Ar-purged (red). The
noncumulative release data are shown in fig. S12, and the error bars indicate the SD of N = 3 samples. (E) HPLC analysis of the main products found after photocata-
lytic DOX release from the PHI microswimmer in ambient and Ar-purged conditions at different illumination times, normalized to the highest signal of the DOX that
was observed from the HPLC (see note S6 for the details). (F) pH-triggered release of DOX in PBS medium at pH 3.5 (green) with 54% of DOX being released after 60 min
versus control (pH 6.7, black) showing negligible (~2%) release. (G) Optical microscopy (bright-field image) and fluorescence overlaid images of SKBR cancer cells (indi-
cated within the circle) with preloaded PHI microswimmers under 415-nm illumination for 20 min showing released DOX uptake by the cells under emission at 595-nm fluo-
rescence in red. (H) PHI autofluorescence image at 470 nm showing the PHI microswimmers adhering to the cancer cells after some amount of DOX release. The two
fluorescent images are taken from the same frame. Scale bars in (G) and (H), 10 m.

in DMEM even under ambient conditions (i.e., in the presence of conditions, an optical equivalent of about 26 g (14%) was released
oxygen), which appears to originate from photocatalytic oxygen every 10 min, with 64 g (35%) in 30 min cumulatively. When the
consumption near the PHI surface (fig. S11B and note S5). The amount of oxygen is decreased by purging the suspension with
cumulative release signal of DOX from PHI in DMEM is shown in argon (Ar) 5 min before and during the illumination, enabling
Fig. 4D for 3-, 10-, and 30-min intervals of illumination in both photocharging of PHI, an increase (almost doubling) in stepwise and
ambient and O2-free environments. Results of the DOX release with cumulative release was observed, resulting in 114.8 ± 5.2 g (62%)
continuous illumination are shown in fig. S12. Under ambient of DOX equivalently being released after 30 min (Fig. 4, C and D).

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Tumor cells have oxygen-deficient, i.e., hypoxic, regions. Hence, a cell media, such as dPBS, DMEM, DMEM/FBS, and diluted blood,
microswimmer that releases drugs faster in oxygen-deficient condi- without any additional fuel. Such particles exhibit positive photo-
tions is not only beneficial but could also be used in a theranostic taxis, which can be used to steer and locate the microswimmers into
sense for hypoxically triggered drug delivery in tumor regions. target locations (18) and can be photocharged even under oxygen-­
For melon, a light-triggered and rather constant DOX release rich conditions. Their high ion tolerance for phototaxis (>5 M) is
was also observed after 3 and 10 min of illumination, although at attributed to a favorable interplay between PHI’s textural and struc-
lower overall amounts than for PHI, although at similar propor- tural porosity, as well as possible optoionic effects enabling ion
tions (~17 mg of DOX equivalent, 15% of loading and hence signifi- motion into and through the particle, in the presence of high photo-
cantly less than 67% release possible with PHI despite higher initial catalytic activity (47, 48). As such, light-induced and controlled
loading), and with no significant differences under oxygen-depleted propulsion is realized without the need of dedicated particle mor-
conditions for these short time scales (fig. S13). phology selection. Next, the biocompatibility of the PHI swimmers
Upon longer illumination however, beyond 60-min illumination was verified with three different cell lines without and with visible
time intervals, the DOX amount released from PHI was reduced light illumination and primary cells. Besides, because of their
under ambient (O2-rich) conditions, suggesting a light-triggered textural porosity, PHI microswimmers are shown to have a very
degradation process, similar to observations made on DOX-loaded high capacity for drug uptake (~185% of their own mass using the
TiO2 microparticles (65). Because degraded products of DOX show example of the anticancer drug DOX), which stays bound stably to
similar fluorescence and absorption bands, a further examination of the particle over a month, until a fast release is triggered by a pH
the release products was only possible qualitatively by separation change or bandgap illumination. Intriguingly, PHI shows stimuli-­
and mass spectroscopy (MS), which was realized by high-performance responsive drug release, because the release can be enhanced or
liquid chromatography–MS (HPLC-MS) analysis of the supernatants modified by the intrinsic memristive photocharging ability under
after release. Besides pristine DOX, modified DOX by-products oxygen-deficient conditions, which are prevalent in hypoxic tumor

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were indeed observed after release (Fig. 4E and fig. S14). The regions, thus enabling potential future neuromorphic or theranostic
dominant decomposition products have masses of m/z = 413, applications (48, 67–70). In addition to the multiple-stimuli respon-
399 and 148 (see scheme S1 for visualization of the reaction; more sive drug-release capability of the PHI microswimmers, we demon-
information on the drug release behavior is discussed in note S6). strate their swimming and ease of tracking in crowded heterogeneous
This gives evidence that not only the release of DOX itself but also biological fluids, such as diluted blood, by monitoring PHI’s inherent
of its oxidation products, which can act even more effectively on autofluorescence. These capabilities make the PHI microswimmers
cells (65), can be intentionally tuned by the illumination time while promising candidates for future in vitro and in vivo biomedical
being responsive to the presence of oxygen. applications in hypoxic tumor regions inside the human body and
Besides, the PHI microswimmers are sensitive to acidic pH organ-on-a-chip devices. Light penetration into tissues is a chal-
levels. A low pH triggers protonation of the PHI backbone and an lenge for all previous and our light-driven microswimmers for
increase in zeta potential, hence repelling the DOX (45, 61). Within in vivo deep-tissue medical applications because short wavelength
60 min at pH 3.5, induced by adding HCl to PBS, the microswim- light cannot penetrate the tissue. To overcome this challenge, long
mers released the loaded DOX (116.2 ± 3.3 g, 63%) without any wavelength near-infrared light-driven microswimmers need to be
light illumination (control) as shown in Fig. 4F. In comparison, the developed in the future, which could penetrate several centimeters
control (pH 6.7) showed negligible release of ~2%, within the deep into the tissues. Moreover, in specific potential medical appli-
margins of instrumental error. cations, a light source might be provided in deep regions by a
Next, the ability of PHI microswimmers to deliver DOX or its catheter that could also deploy the microswimmers in a target
active modifications to cancer cells under illumination was studied location. Because of PHI’s organic nature, we envision that the
to provide a proof of concept. The bright-field image of PHI microswimmers can be further optimized for tailored surface
microswimmers in a dense environment of cancer cells after 20-min functionalities and catalytic, morphological, and optical properties
illumination (415 nm) is shown in Fig. 4G. DOX or related products (45, 50), opening up different avenues for smart responsive micro-
(red color) were released from the PHI microswimmers and taken machines and theranostics in the future.
up by the cancer cells after 24 hours of incubation; some amounts
also diffused through the medium. Subsequently, the incubated cells
died. The fluorescence image of the PHI microswimmers shows MATERIALS AND METHODS
that the microswimmers stayed in the vicinity of the cancer cells PHI synthesis
(Fig. 4H) (66). This demonstrates that PHI microswimmers serve as PHI was synthesized according to a procedure described in the
smart light-driven cargo delivering agents under biological condi- literature (44). Shortly, melamine (5.0 g) was heated in a tube
tions. Their stimuli-responsive behavior triggers a theranostic furnace in a quartz glass boat to 550°C for 12 hours with a heating
function, making use of an intrinsic sensing property (i.e., charge rate of 5°C/min under Ar flow. After cooling to ambient tempera-
accumulation enabled in oxygen-poor environments) that triggers ture, a yellow powder (2.0 to 2.5 g) was obtained. A total of 1.5 g of
an enhanced release of the therapeutic agents. this product (melon) were thoroughly ground with KSCN (3.0 g),
which was heated overnight to 140°C in vacuum to evaporate water.
The mixture was heated in a tube furnace in an Alox boat to 400°C
CONCLUSION for 1 hour and 500°C for 30 min with a heating rate of 30°C/min
We have developed highly efficient carbon-based PHI microswimmers under Ar flow. The Alox boat with the CNx was sonicated two times
that can be propelled phototactically with light in aqueous salt for 15 min in 80 ml of water to disperse the yellow product. This
media with high molarity (up to 5 M NaCl) and realistic biological suspension was washed six times with DI water by centrifugation

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(20,000 rpm). The insoluble product was dried in vacuum at 60°C Cell isolation
overnight. Mouse spleen was provided by the Facility for Animal Welfare,
SEM images of the microswimmers were captured by a Zeiss Veterinary Service and Laboratory Animal Science at Eberhard Karl’s
Merlin SEM. To capture the swimming of the microswimmers, a University Tubingen. After mice were euthanized, the spleen was
Zeiss Axio A1 inverted optical microscope was used. A Thorlabs removed and kept on ice in PBS without Ca2+/Mg2+ for transport to
M365L2 (Germany) UV lamp, Zeiss Colibri fluorescence source, Max Planck Institute at Stuttgart. The spleen was forced through a
and Thorlabs M415 were used for illumination through the inverted cell strainer at 70 m, also containing DMEM media. After filtering,
microscope. The videos were recorded from the microscope using a the supernatant was centrifuged at 800g for 5 min. The upper layer
LD Plan-NeoFluar 40× objective lens and Axiocam 503 charge-­ of supernatant was removed, and the cells were washed with lysing
coupled device camera at 62 frames/s. The swimming behavior of buffer for RBCs removal. This occurred for 8 min at room tempera-
the microswimmers was systematically investigated by 2D mean ture. Then, the cells were centrifuged and resuspended in fresh
square displacement (MSD) analysis on the captured videos for 15 s medium before being placed into six-well plates at 1 million cells per
using a custom MATLAB and Python code. well. The chosen concentrations of the tested material were added
The absorptance spectra of these samples were measured with a to the wells for 24 hours before the medium was removed and frozen
double monochromator spectrophotometer (Edinburgh Instruments, before enzyme-linked immunosorbent assay (ELISA) analysis.
FLS-980). The measurements were performed locating the sample
in the center of an integrating sphere attached to FLS-980 working ELISA protocol (IL-12)
in synchronous mode to discern any photoluminescence signal A high affinity binding plate (Greiner) was coated with 100 l of
from the PHI particle. The sample suspension was measured in diluted, purified anticytokine capture antibody. The plate was sealed
degassed, aqueous solution while simultaneously being stirred to and incubated at 4°C overnight. The next day, the capture antibody
prevent sedimentation. was removed by decanting, and a blocking solution was added for

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The intensity of the illumination in the microscope was mea- 1 hour at room temperature. A series of washes was performed
sured at the place of the sample chamber with a calibrated Ocean between each step. The samples and standards were added and
Optics OCEAN-FX-XR1-ES spectrophotometer after attenuation incubated for 2 hours at room temperature. After rinsing, the working
by a neutral density filter and integration of the spectral irradiance. detector was added and incubated for 1 hour. The final rinse was
The results have been normalized to the filter attenuation and to the performed, the substrate solution was added, and the plate was read
spot size of the light beam in the microscope, which was measured after adding stop solution at 30 min.
to be 2.0 ± 0.5 mm in diameter, resulting in a relative experimental
error of 50% after the error propagation calculation. For side illumi- Swimming in biofluids
nation with the 365-nm diode, the light intensity was directly DMEM, dPBS, and DMEM + FBS (Sigma-Aldrich) were used as pur-
measured by a Thorlabs S310C/PM100D power meter. chased. The PHI swimmers were dispersed in the medium and illu-
minated inside a microfluidic chamber to measure their swimming.
Cytotoxicity tests
Human colorectal cancer cells, human breast cancer cells, and murine Cancer drug DOX loading
fibroblasts (American Type Culture Collection, Manassas, VA) were The efficiency of loading was measured by centrifuging the DOX-­
grown in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) loaded microswimmers and comparing the optical density (OD) of
penicillin/streptomycin (Gibco, Grand Island, NY, USA) at 37°C the supernatant with the precalibrated OD of DOX (200 g/ml) at
in a 5% CO2, 95% air-humidified atmosphere. Cells were reseeded 480 nm. PHI (100 g/ml) was dispersed with DOX (200 mg/ml),
after growing to confluence into -Slide eight-well plates (Ibidi GmbH, and this solution was stirred in dark for 24 hours to allow the DOX
Gräfelfing, Germany) at a cell density of 25 × 103 cells per well and to be adsorbed. After 24 hours, the suspension was centrifuged, and
incubated for 2 days. For cytotoxicity testing, all three cell lines the supernatant was used for measuring the DOX loading. The
were incubated with PHI microswimmers at varying concentra- DOX-loaded PHI was washed three times with water and stored at 4°C
tions (0 to 30 g/ml). Then, viability of difference cell lines was inves- for further delivery experiments. The DOX-loaded microswimmers
tigated using a LIVE/DEAD assay (Thermo Fisher Scientific, Waltham, were illuminated with 415-nm light at an intensity of 170 mW/cm2
MA) incorporating calcein-AM (green) and ethidium homodimer-1 from the bottom in a cylindrical quartz glass stirring simultaneously
(red) dyes. After 24 hours of incubation with the PHI microswimmers, (fig. S11A). To remove dissolved oxygen, the suspension was bubbled
live/dead cell viability was calculated from fluorescence microscopy with Ar through a needle for 5 min before the illumination and
images. Furthermore, cytotoxicity of microswimmers during light during the respective illumination time. For the pH release, the pH
actuation (385 nm, 415 mW/cm2) was tested by live/dead staining of the resulting HCl-diluted PBS solution was checked using a pH
of fibroblast cells right after and 24 hours after actuation of PHI meter to confirm the stability of the pH during the release experiments.
microswimmers for varying durations (0 to 10 min).
Sorption measurements
Cell culture Sorption measurements were performed on a Quantachrome In-
All cell culturing was performed under sterile conditions within a struments Autosorb iQ 3 with a coupled Cryosync for cooling and
biosafety cabinet. The cell culture used for the isolated cells was Ar as analysis gas at 87 K. The pore size distribution was determined
composed of DMEM from Gibco. This was supplemented with 10% from Ar adsorption isotherms using the NLDFT (cylindrical
heat-inactivated FBS and 1% penicillin and streptomycin (Gibco). nanopores, adsorption branch) kernel in carbon for Ar at 87 K im-
The storage condition for cells during incubation was an incubator plemented in the ASiQwin software version 3.01. Samples were ac-
set at 5% CO2, ~90% humidity, and 37°C. tivated in high vacuum at 120°C for 12 hours before measurement.

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SCIENCE ROBOTICS | RESEARCH ARTICLE

Deutsche Forschungsgemeinschaft (DFG) via the cluster of excellence “e-conversion” (project availability: All data needed to evaluate the conclusions in the paper are present in the paper
number EXC2089/1–390776260), and by the Center for NanoScience (CENS). Author or the Supplementary Materials.
contributions: V.S., F.P., M.S., J.K., and B.V.L. conceived and planned the research. V.S., F.P.,
Y.A., J.K., L.G., and V.K. performed the experiments or assisted in their realization. All authors Submitted 28 August 2021
contributed to the analysis and discussion of the data. M.S., F.P., and B.V.L. supervised the Accepted 17 December 2021
research. V.S. and F.P. wrote the manuscript with assistance from all other authors. Competing Published 19 January 2022
interests: The authors declare that they have no competing interests. Data and materials 10.1126/scirobotics.abm1421

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Sridhar et al., Sci. Robot. 7, eabm1421 (2022) 19 January 2022 12 of 12

 Light-driven carbon nitride microswimmers with propulsion in biological and ionic
media and responsive on-demand drug delivery
Varun Sridhar, Filip Podjaski, Yunus Alapan, Julia Krger, Lars Grunenberg, Vimal Kishore, Bettina V. Lotsch, and Metin
Sitti

Sci. Robot., 7 (62), eabm1421.

DOI: 10.1126/scirobotics.abm1421

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