Light-Triggered Drug Release from 3D-Printed Magnetic Chitosan

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Light-Triggered Drug Release from 3D-Printed Magnetic Chitosan Microswimmers Ugur Bozuyuk,†,§ Oncay Yasa,‡,§ I. Ceren Yasa,‡ Hakan Ceylan,*,‡ Seda Kizilel,*,† and Metin Sitti*,‡ †

Chemical & Biological Engineering Department, Koç University, 34450 Istanbul, Turkey Physical Intelligence Department, Max Planck Institute for Intelligent Systems, 70569 Stuttgart, Germany

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ABSTRACT: Advances in design and fabrication of functional micro/nanomaterials have sparked growing interest in creating new mobile microswimmers for various healthcare applications, including local drug and other cargo (e.g., gene, stem cell, and imaging agent) delivery. Such microswimmerbased cargo delivery is typically passive by diffusion of the cargo material from the swimmer body; however, controlled active release of the cargo material is essential for ondemand, precise, and effective delivery. Here, we propose a magnetically powered, double-helical microswimmer of 6 μm diameter and 20 μm length that can on-demand actively release a chemotherapeutic drug, doxorubicin, using an external light stimulus. We fabricate the microswimmers by two-photon-based 3D printing of a natural polymer derivative of chitosan in the form of a magnetic polymer nanocomposite. Amino groups presented on the microswimmers are modified with doxorubicin by means of a photocleavable linker. Chitosan imparts the microswimmers with biocompatibility and biodegradability for use in a biological setting. Controlled steerability of the microswimmers is shown under a 10 mT rotating magnetic field. With light induction at 365 nm wavelength and 3.4 × 10−1 W/cm2 intensity, 60% of doxorubicin is released from the microswimmers within 5 min. Drug release is ceased by controlled patterns of light induction, so as to adjust the desired release doses in the temporal domain. Under physiologically relevant conditions, substantial degradation of the microswimmers is shown in 204 h to nontoxic degradation products. This study presents the combination of lighttriggered drug delivery with magnetically powered microswimmer mobility. This approach could be extended to similar systems where multiple control schemes are needed for on-demand medical tasks with high precision and efficiency. KEYWORDS: microswimmer, chitosan, light-triggered, drug delivery, two-photon polymerization, biodegradation

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icroscopic swimmers powered by external magnetic fields possess significant potential in medical applications due to their wireless actuation, active locomotion, and precise localization capabilities.1,2 Their small size and untethered control could allow deep tissue penetration and thus could revolutionize minimally invasive surgeries and therapies.3,4 So far, synthetic and biohybrid magnetic microswimmers, which are actuated using either an external power source or an integrated microorganism, have been used in different platforms for targeted cargo delivery, object/cell manipulation, and tissue engineering applications.5−13 Particularly, helical magnetic microswimmers have recently gained interest in drug and cargo delivery applications due to the efficiency of magnetic torque over magnetic gradient pulling for microscale actuation.14 Helical microswimmers, operated in low Reynolds number (Re) regime with an external rotating magnetic field, were previously designed in millimeter scale using a small magnet incorporated in the head of a spirally bent copper wire.15,16 © XXXX American Chemical Society

Then, different fabrication techniques, including self-scrolling and glancing angle deposition, were utilized to fabricate helical magnetic swimmers at the micrometer scale.17,18 Afterward, the advancements in the two-photon direct laser writing (TDLW) technique realized three-dimensional (3D) fabrication of more complex polymeric microstructures, eased their local 3D patterning using versatile chemical moieties, and provided the possibility to embed biocompatible superparamagnetic iron oxide nanoparticles (SPIONs) into the microswimmers.19−23 Until now, different photosensitive materials have been used with the TDLW technique to fabricate biocompatible helical microswimmers toward biomedical applications.24 Initially, helical microswimmers functionalized with drug-loaded liposomes were utilized to Received: August 6, 2018 Accepted: August 31, 2018

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DOI: 10.1021/acsnano.8b05997 ACS Nano XXXX, XXX, XXX−XXX

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Figure 1. Overview of the synthesis and fabrication processes. (A) Synthesis of the photo-cross-linkable methacrylamide chitosan from natural chitosan. (B) 3D printing of the microswimmers using two-photon direct laser writing technique. (C) Optical microscopy image of 3D-printed 3 × 3 array of the microswimmers. (D) Energy-dispersive X-ray spectroscopy elemental mapping showing the presence of iron atoms (red color) in the microswimmers.

perform single-cell drug delivery in vitro. In this work, drug molecules were successfully delivered to individual cells which were in contact with the microswimmers.25 Then, single-cell transfection was realized in vitro using plasmid DNA functionalized helical microswimmers. In the study, fluorescent protein encoding plasmid DNA was incorporated in the microswimmers, and later individual cells were controllably transfected through cell−microswimmer interaction.26 Finally, a swarm of helical microswimmers functionalized with fluorescent probes were navigated in the peritoneal cavity of a mouse. The results demonstrated that it is possible to navigate the swarm of microswimmers in vivo in the direction of an applied rotating magnetic field.27 Despite the enormous recent developments in the field, helical magnetic microswimmers still need to be strengthened to have physiologically relevant biodegradation and controlled local cargo release capabilities, which are essential for their potential medical applications. Biodegradation of administered microswimmers inside the body within a desired period of time by forming nontoxic degradation products is a critical aspect of medical applications. Recently, degradation of helical microswimmers, composed of various ratios of PEG-DA/PE-TA and SPIONs, through sodium hydroxide based hydrolysis reaction was demonstrated.28 However, use of a 1 M NaOH solution for the degradation of the microswimmers would not be preferred in vivo, and hence integration of natural, physiologically relevant degradation mechanisms to the microswimmers is indispensable for future medical applications. In addition, controlled release of concentrated therapeutics at disease sites by active microswimmers could increase the overall treatment efficiency.29 Helical microswimmers overcome active delivery issues of therapeutics to the site of action using rotating magnetic fields. However, controlled release of the therapeutics is still an issue that should be addressed in microswimmerbased drug delivery systems. Remotely triggered systems have

always been attractive for facilitating release of therapeutics to desired sites at desired times. Concentration of therapeutics at the site of action can be controlled and increased; thus, overall injected dose can be decreased using remotely triggered systems. Light-triggered release systems are especially practical among the other trigger mechanisms, including pH, temperature, ultrasound, and magnetic field, due to their high spatiotemporal accuracies.30 In ultraviolet (UV) light-triggered release systems, poor tissue penetration depth of the UV light restricts the potential medical applications to certain locations inside the body. However, optical upconversion processes, in which low-energy photons (e.g., near-infrared light that has more penetration depth) are transformed to high-energy ones (e.g., UV light), could be utilized to realize real life medical applications of UV light-triggered release systems in different parts of the body.31 Here, we report a magnetically powered chitosan-based helical microswimmer system that has a light-triggered drug release capability. The microswimmers had a 6 μm diameter and 20 μm length and were composed of double helices to operate in the low Reynolds number regime, Re ≈ 10−5, with a low-amplitude rotating magnetic field. They were fabricated from photo-cross-linkable methacrylamide chitosan macromolecules in the presences of photoinitiator and SPIONs using the TDLW technique. The microswimmers were actuated and controlled in an aqueous environment with an average speed of 3.34 ± 0.71 μm·s−1 under a 10 mT and 4.5 Hz rotating magnetic field. Azide-modified doxorubicin (DOX) was used as a model therapeutic for light-triggered drug release and bound to alkyne moieties of the microswimmers using an onitrobenzyl linker through an NHS−amine coupling reaction. Biodegradability of the microswimmers was demonstrated at 37 °C with different lysozyme concentrations, and the biocompatibility of the whole microsystem was investigated using these degradation products. Overall, hereby we reveal a biocompatible and biodegradable magnetic microswimmer B


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concentrations of nanoparticles from 0.1 to 10 mg·mL−1 resulted in inefficient 3D printing and poor structures above 5 mg·mL−1. However, this problem could be potentially circumvented with iron oxide nanoparticles that have better colloidal stability, and hence they have lesser tendency for aggregation at higher concentrations. In this regard, this threshold concentration (5 mg·mL−1) was used in the rest of the microswimmer preparations, as they were homogeneously dispersed in the solution without any particle aggregation or agglomeration. A single microswimmer had 20 μm length and 6 μm outer diameter (Figure 1C). The average printing rate was around 10 s for an individual microswimmer. Energydispersive X-ray spectroscopy (EDS) elemental mapping confirmed homogeneous dispersion of iron atoms in the microswimmers (Figure 1D). The 3D structure of the microswimmers was affected during the EDS elemental mapping due to the applied energy (15 keV) and accusation time (10 min) (Figure S3). Magnetic Actuation and Control of the Microswimmers. The microswimmers were actuated and steered using a five-coiled electromagnetic setup, which can be mounted on an inverted optical microscope. The experiments were performed using a 10 mT rotating magnetic field. Initially, the step-out frequency of the microswimmers was investigated by gradually increasing the frequency of the applied rotating magnetic field from 1 Hz to 6 Hz with 0.5 Hz steps. It was demonstrated that the fabricated microswimmers were actuated and steered optimally at 4.5 Hz under a 10 mT rotating magnetic field (Figure 2A). The average forward velocity of the microswimmers at optimum actuation frequency was measured to be 3.34 ± 0.71 μm·s−1. Finally, the microswimmers were steered in different paths to demonstrate the controllability of the microsystem, and it was shown that it is possible to steer the microswimmers at both 4.5 and 5 Hz under a 10 mT rotating magnetic field (Figure 2B, Figure S4, Supplementary Movie 1). Enzymatic Degradation of the Microswimmers. Biodegradable materials have gained increasing attention in medicine since they are able to naturally break down and disappear from the body after performing their functions.43 Chitosan, as a biodegradable material, is primarily degraded by lysozyme enzyme, which is present in various tissues and body fluids with a concentration range of approximately 1−15 μg· mL−1.44−46 Lysozyme enzyme cuts off the glycosidic bonds between monomers in the polymer backbone, and the resulting small chains are removed naturally.47,48 Here, three different lysozyme enzyme concentrations (1.5, 15, and 150 μg·mL−1) were chosen for the biodegradation of the microswimmers, where 150 μg·mL−1 represents an unrealistically high condition. Biodegradation of the microswimmers with 15 μg· mL−1 lysozyme concentration is presented in Figure 3A. Biodegradation of the microswimmers occurred by a surface erosion mechanism, in which water and enzymes could not penetrate inside the cross-linked structures and, thus, started to degrade initially the exterior surface of the structures.49 Namely, helices and sharp edges of the microswimmers were degraded first by the lysozyme enzyme. For all groups, the microswimmers were partially degraded after 204 h. As expected, the unrealistically high lysozyme enzyme concentration group (150 μg·mL−1) had the lowest diameter and length, whereas the 1.5 μg·mL−1 lysozyme concentration group had the highest after 204 h (Figure 3B and C). There was rapid diameter and length changes in all groups since helices and

design strategy, composed of natural chitosan macromolecules, and combine the active locomotion of the microswimmers with a controlled drug release mechanism to further decrease the side effects of the whole microsystem in minimally invasive therapies.

RESULTS AND DISCUSSION Synthesis of Methacrylamide Chitosan. Chitosan is a linear and cationic polymer that is obtained from chitin, the second most abundant natural polymer in the world.32 Its inherent properties, such as biocompatibility, biodegradability, bioadhesivity, and antimicrobial, antitumor, and antioxidant activities, make chitosan an ideal polymer for medical applications.32−37 Polymers without photosensitive characteristics like chitosan can be chemically modified while their polysaccharide backbones remain unchanged. In this work, we initially synthesized a photosensitive form of chitosan, methacrylamide chitosan (ChMA), as previously described, by reacting amino groups of the polymer with methacrylic anhydride.38 The amino groups of the chitosan transformed into photosensitive methacrylamide groups according to the methacrylic anhydride/chitosan ratio at constant reaction time (Figure 1A).39,40 The newly formed polymer chains had the capability to cross-link with each other, in the presence of a photoinitiator, around 350 nm wavelength UV light. After the synthesis, the methacrylation degree of ChMA macromolecules was determined using the 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay. TNBS is a photospectroscopic reagent used to determine free amino groups.41 The methacrylic anhydride/ chitosan ratio was changed at fixed reaction times, and the assay demonstrated that amino groups were consumed with increasing methacrylic anhydride/chitosan ratio (Figure S1). Methacrylamide chitosan macromolecules with 70% methacrylation degree were selected for 3D printing, which means that we had ChMA macromolecules with a backbone composed of 70% photosensitive methacrylamide groups, and therefore we accordingly optimized our fabrication procedure. 3D Printing of the Microswimmers. Chitosan-based microswimmers were fabricated in double-helical geometry using the TDLW technique. Prepolymer solution was prepared in 8% (v/v) acetic acid containing ddH2O and composed of 30 mg·mL−1 ChMA, 20 mg·mL−1 phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator, and 5 mg·mL−1 PEG/ amine-functionalized 50 nm SPIONs (Figure 1B). Photopolymerization of the prepolymer solution in predefined structures was performed in a closed channel, which had two sites to place permanent magnets for the alignment of SPIONs (Figure S2). Embedded SPIONs enabled control and steering of the microswimmers in 3D aqueous environments using rotating magnetic fields. In addition, use of SPIONs in microswimmer designs has two main advantages: (1) they are often considered to be biocompatible and have no severe side effects in vivo,42 and (2) they dramatically increase the availability of drug and cargo release sites compared to cobaltor nickel-based surface coatings. The total amount of magnetic nanoparticles loaded in the mesh network of the microswimmer body is critical for the magnitude of magnetization, and hence it is the limiting factor for the maximum swimming speed. Decreasing the amount will result in lower step-out frequencies, thereby reducing the maximum swimming speeds. Therefore, we aimed to maximize the volume fraction of the magnetic nanoparticles in the precursor suspension. Increased C


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biocompatibility of the degradation products was investigated using the SKBR3 breast cancer cell line. The SKBR3 cells were treated with the degradation products for 1 day and then stained with a live−dead assay for toxicity analysis. The results showed that the degradation product of the microswimmers did not have a toxic effect on SKBR3 cells and the live/dead cell ratios for both control and treated groups were similar and approximately 90% of the whole cell populations (Figure 3D and E). Photocleavable Linker and Drug Binding to the Microswimmers. Photocleavage-based light-triggered delivery systems have been studied to control the release of different drug molecules. In these systems, drug molecules are chemically bound to photocleavable linker molecules, and the prerequisite is that there should be available chemical sites allowing for chemical modification without impairing the therapeutic efficacy of the modified drug in the released form. The photocleavable linker molecules split into two parts upon light exposure, and drug molecules are released from the attached structures.31 o-Nitrobenzyl is a photocleavable group, and functional o-nitrobenzyl derivatives have been used for the delivery of various biomolecules.54,55 An o-Nitrobenzyl derivative that has N-hydroxysuccinimide ester (NHS) and alkyne can be quite effective for the release of molecules due to its chemical functionality.55 NHS groups selectively react with amino groups (known as NHS−amine coupling), and alkyne groups react with azide groups (known as the copper(I)catalyzed Click reaction). In this part of the study, the NHS ends of photocleavable linker molecules were conjugated to free amino groups of the microswimmers. Then, azidemodified DOX, which was utilized as a model drug, was linked to the alkyne ends of the attached photocleavable linker molecules. Two different chemical reactions were performed to obtain DOX-functionalized microswimmers (Figure 4A). In the first step, the microswimmers were treated with the onitrobenzyl photocleavable linker molecules containing solution. Alkyne-ended microswimmers were obtained after this, the so-called NHS−amine coupling reaction. As a second step, alkyne-ended microswimmers were treated with azideDOX-containing reaction mixture. To confirm azide-DOX was bound to the microswimmers by the Click reaction, only the second step was performed with another group of microswimmers as a negative control group. In the negative control group, azide-modified DOX could not be bound to the microswimmers since there was no reaction between amino and azide groups. DOX-modified and negative control groups were compared using fluorescence microscopy. The DOXmodified group had significantly higher and homogeneous fluorescence emission than the negative group at same light intensity and time (Figure S5). Meanwhile, low-fluorescence emission from the negative group was due to diffusion of the drug molecules into the microswimmers. These results confirmed the chemical conjugation of o-nitrobenzyl linker and azide-modified DOX to the microswimmers. Bleaching Tests and Controlled Drug Release from the Microswimmers. o-Nitrobenzyl linker molecules between the microswimmers and DOX experienced selective bond cleavage with light irradiation at 365 nm wavelength. For drug release experiments, our main assumption was that the initial fluorescence intensity of the microswimmers corresponds to 100% drug loading to the microswimmers. The drug release from the microswimmers was characterized based on the fluorescence intensity decrease over time. Initially,

Figure 2. Actuation and steering of the microswimmers using a rotating magnetic field. (A) Forward velocity of the microswimmers as a function of magnetic excitation frequency demonstrating a step-out frequency of 4.5 Hz. (B) Controlled swimming trajectory snapshots (red lines) of the microswimmers (pseudocolored in green) under a 10 mT rotating magnetic field at 4.5 Hz.

edges were degraded first due to the surface erosion. After biodegradation of the helices and the edges, which had a smaller volume compared to whole body of the microswimmers, the rate of diameter and length changes dramatically decreased as expected. This did not necessarily mean a decrease in the biodegradation rate, because lysozyme enzyme then tried to degrade the cylindrical microswimmer body, which had a lower surface area to volume ratio compared to the helices. Because of the surface erosion phenomenon, it became harder to observe biodegradation, length, and diameter changes after a certain point in time. Partial biodegradation for the microswimmers in 204 h was consistent with the literature, where full degradation was not observed after several weeks for most of the studies.50−53 In addition to biodegradation, in vitro D


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Figure 3. Enzymatic degradation of the microswimmers using lysozyme. (A) Optical microscopy images of the microswimmers treated with 15 μg·mL−1 lysozyme, (i) t = 0 h and (ii) t = 204 h, revealing a surface-corrosion-based degradation mechanism. (B) Changes in length of the microswimmers in time with different lysozyme concentrations. (C) Changes in diameter of the microswimmers in time with different lysozyme concentrations. (D) Live−dead staining of SKBR3 breast cancer cells (i) nontreated or (ii) treated with the degradation products of the microswimmers for 1 day. Green represents live cells and red represents dead cells. (E) Quantification of viability of SKBR3 breast cancer cells treated with the degradation products. The viability did not alter in the cells treated with the degradation products (Student’s t test, p > 0.05). Error bars represent the standard deviation (n.s.: not significant).

DOX was released within 5 min for 3.4 × 10−1 W/cm2 light intensity (Figure 4C). The release rate was dramatically decreased after 5 min. The incomplete release is due to low photochemical conversion observed for nitrobenzyl groups.56,57 Slow release after 5 min was observed probably due to slower diffusion of DOX molecules which were cleavedoff from the center of the microswimmers. Slower drug release was observed in the case of 6.7 × 10−2 W/cm2 compared to 3.4 × 10−1 W/cm2 light intensity. The cumulative drug release rate decreased and converged approximately to 40% (Figure 4C). The lower drug release could be explained as slower reaction kinetics due to the lower light intensity.56,57 Provided the correct photon absorption wavelength is matched for the photo-cross-linkable linker, the photocleavage reaction rate dictates the drug release kinetics. To extend the sustained release, the light intensity should be tailored to lower intensities accordingly. Smart dosing of therapeutics is another important consideration of various delivery systems since many drugs have serious off-target side effects.58 Therefore, it was presented that drug release from the microswimmers could

bleaching tests were performed to confirm that there were no photobleaching- and diffusion-related fluorescence intensity changes in the microswimmers. Accordingly, the negative group was exposed to light at 365 nm, which was used for cleavage of the linker molecules, and 470 nm, which was used for DOX excitation and fluorescence intensity change analysis, wavelengths (Figure S6). No decrease in the fluorescence intensity was observed for excitation both at 470 nm (Figure S6A) and at 365 nm wavelengths (Figure S6B and C), which means that the drug molecules did not lose their fluorescence upon light exposure, but the fluorescence change in the whole microsystems was due to the controlled drug release. Two different light intensities at 365 nm wavelength, 6.7 × 10−2 and 3.4 × 10−1 W/cm2, were selected to demonstrate on-demand light-triggered drug release. For 3.4 × 10−1 W/cm2, there was significant reduction in the fluorescence intensity after 30 min, which means that DOX was released from the microswimmers. In addition, functionalization of the microswimmers with drug molecules using chemical reactions did not affect the magnetic actuation capabilities of the microswimmers (Figure 4B, Supplementary Movie 2). Approximately 60% of the bound E


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Figure 4. Light-triggered drug release from the microswimmers. (A) Schematic showing the reaction pathway to obtain DOX-modified microswimmers. Amino groups on the microswimmers react with the NHS group of o-nitrobenzyl photocleavable linker molecules. Then, azide-modified DOX reacts with alkyne ends of the microswimmers. (B) DOX release from the microswimmers exposed to 3.4 × 10−1 W/ cm2 light intensity for 30 min. Decrease in the fluorescence intensity indicates the cleavage of DOX from the microswimmers and its release. (C) Cumulative DOX release from the microswimmers for 6.7 × 10−2 and 3.4 × 10−1 W/cm2 light intensity. (D) Smart dosing of DOX from the microswimmers. A 365 nm wavelength light is on in the red regions. Approximately 15% of DOX was released per minute from the microswimmers.

be controlled on-demand by switching the light on and off. Light at 365 nm wavelength was turned on for 1 min and then turned off for an additional 5 min, and this was repeated two times. A sharp drug release from the microswimmers was observed when light was on (3.4 × 10−1 W/cm2 light intensity) for 1 min, and afterward, there was no or slight drug release from the microswimmers when light was off for 5 min (Figure 4D). Approximately, 15% of the total drug was released per dose. This showed that the user can control the on-demand drug release profile from the microswimmers. Also, the amount of drug that is dosed can be tuned by changing either light intensity or exposure time. It was also demonstrated that the drug release can be localized by focusing light on a specific group of microswimmers. Drug was released from a specific group of microswimmers while others retained the drug inside (Figure S7A). Moreover, it was even possible to release the drug more precisely from a half-body of the microswimmers (Figure S7B). Overall, we showed that the o-nitrobenzyl group is an efficient photocleavable linker molecule for the precise

release of therapeutics from the microswimmers. Drug dose can be also thoroughly controlled by changing the light intensity or switching the light on/off. A general disadvantage of UV-based drug release systems is that UV photons have very limited penetration depth through human skin or water based biological systems, while NIR photons, which have a higher penetration depth, have insufficient energy to break the covalent bonds of the photocleavable linker.31 Upconversion processes assisted by endoscopic techniques could enable implementation of the described system in a biomedical setting. Available low-power continuous wave lasers are able to provide upconversion through certain nanoparticles.31 In this scheme, NIR provides high penetration depth, while the UV upconversion provides the energy required to split the bonds of the linker. Incorporation of UV photon upconversion strategy to the microrobotic swimmer could provide opportunities for lightbased microrobotic drug release. Along this line, previous F


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Degradation of Microswimmers and Cytotoxicity Investigation of the Degradation Products. 3D-printed microswimmers were treated with different concentrations of lysozyme solution (1.5, 15, and 150 μg·mL−1), prepared in 1× phosphatebuffered saline, at 37 °C. The length and diameter of the microswimmers were measured using a Nikon Eclipse Ti-E inverted microscope with 20× magnification in DIC mode with increasing time intervals (3, 6, 12, 24, 48 h). Enzyme solutions were refreshed every 12 h to prevent inactivation of the enzyme. Degradation products were used to investigate biocompatibility and cytotoxicity of the microswimmers. Briefly, SKBR3 breast cancer cells (passage #8) were seeded into a 96-well plate at 5000 cells/well. Then, they were treated either with the degradation products of 1000 3D-printed microswimmers or growth medium (control group) for 1 d until ∼80% confluence was reached. Finally, the cells were stained with live−dead imaging solution (Life Technologies) for 20 min at RT, imaged using a fluorescence inverted microscope, and counted using ImageJ for quantitative analysis. Integration of Photocleavable Linker and Drug Molecules to the Microswimmers. Initially, photocleavable o-nitrobenzyl linker (1-(5-methoxy-2-nitro-4-prop-2-ynyloxyphenyl)ethyl N-succinimidyl carbonate from LifeTein LLC) was bound to the surface of the microswimmers through an NHS−amine coupling reaction. Briefly, 500 μM of the linker was dissolved in anhydrous dimethyl sulfoxide and the microswimmers were treated with the linker solution for 4 h at RT. After that, for coupling azide-modified DOX (LifeTein LLC) to the alkyne ends of the linker molecules, bound to the microswimmers, a previously described protocol was adapted with some modifications.60 The microswimmers were treated with a solution containing 50 μM azide-modified DOX, 100 μM CuSO4, 5 mM sodium ascorbate, and 500 μM tris(3-hydroxypropyltriazolylmethyl)amine for 3 h at RT. Finally, the microswimmers were washed several times with ddH2O to remove unbound drug molecules and kept in the dark for further use. Bleaching Test and Controlled Drug Release from the Microswimmers. Drug-integrated microswimmers were equilibrated to RT, washed several times with ddH2O, and kept overnight in ddH2O. Controlled drug release from the microswimmers upon light exposure at 365 nm was investigated using a flourescence inverted microscope (DMi8, Leica Microsystems). Time-lapse fluorescent images were acquired every 10 s for a period of 30 min. Output light power was adjusted to either 3 mW (6.7 × 10−2 W/cm2) or 16.5 mW (3.4 × 10−1 W/cm2), and the exposure time was set to 1 s. Light intensities were measured using optical power and an energy meter (PM200, Thorlabs, Inc.) Fluorescence intensities of the microswimmers were analyzed using the LASX analysis toolbox (Leica Microsystems). The on-demand controlled drug release experiment was performed by 1 min of light exposure followed by 5 min of refractory period. In both cases, background fluorescence was subtracted from the measured values. Fluorescence bleaching of the microswimmers loaded with the drug molecules through passive diffusion was tested by exposure to light at 365 or 470 nm as in the controlled release experiments, and image acquisition. Bleaching tests for both 6.7 × 10−2 and 3.4 × 10−1 W/cm2 light intensities at 365 nm and light power at 470 nm were done for 30 min, and fluorescent images were acquired every 10 s. Similar to release experiments, fluorescent intensities of the individual microswimmers were measured through the LASX analysis toolbox (Leica Microsystems), and background was subtracted from the measured values. Statistical Analysis. All the quantitative values are presented as means ± standard deviation. All the experiments were performed with at least three replicates for each group. Student’s t test was used for statistical analysis, and a p-value of less than 0.05 was considered statistically significant.

studies demonstrated the cleavage of o-nitrobenzyl with pulsed NIR for drug release.59

CONCLUSION In summary, we developed a magnetically actuated biocompatible and biodegradable chitosan-based microswimmer, which has the capability of on-demand light-triggered drug release. The microswimmers are optimized to swim most efficiently inside water, and their optimization in non-Newtonian body fluids will be investigated in depth in a future work. We synthesized photosensitive methacrylamide chitosan macromolecules, then embedded SPIONs, and fabricated the microswimmers from this material using the TDLW technique in the dimensions of 20 μm length and 6 μm outer diameter. We demonstrated the actuation and steerability of the microswimmers at different frequencies under a 10 mT rotating magnetic field. Also, we presented biodegradation of the microswimmers, without generating any in vitro cytotoxic degradation products, using a natural enzyme found in the human body. Finally, we showed the combination of ondemand light-triggered drug release within the synthetic microswimmers, which makes the microsystem promising for the challenges associated with the active and controlled delivery of therapeutics for the treatment of various diseases. MATERIALS AND METHODS All materials were purchased from Sigma-Aldrich unless otherwise specified. Synthesis of Methacrylamide Chitosan. ChMA was synthesized according to a previously described protocol with some modifications.38 Initially, 3% (w/v) low molecular weight chitosan powder was dissolved in 3% (v/v) acetic acid solution at room temperature (RT) for 24 h. Methacrylic anhydride was added to the chitosan solution at 3.5:1 w/w ratio to obtain ∼70% methacrylation degree, and the reaction was performed for 3 h with a vortex mixer at RT. After performing the reaction, the reaction mixture was diluted with water and dialyzed (14 kDa cutoff) against water for 4 d. The resulting mixture was lyophilized and stored at −20 °C for further use. Degree of Methacrylation Characterization. Degree of methacrylation of ChMA macromolecules was analyzed with the TNBS assay that is based on the quantification of unmodified free amino groups. Briefly, unmodified chitosan, as a control group, and 0.05% (w/v) ChMA macromolecules were dissolved in 0.2% (v/v) acetic acid solution. An 80 μL amount of the solutions was incubated with 40 μL of 2% (w/v) NaHCO3 and 60 μL of 0.1% (v/v) TNBS reagent (Thermo Fisher Scientific) at 37 °C for 2 h. After the incubation period, 60 μL of 1 N HCl was added into the solutions, and then absorbance of the samples was measured at 345 nm using a plate reader (BioTek Gen5 Synergy 2, Bad Friedrichshall, Germany). Degree of methacrylation was calculated according to the following equation: Degree of Methacrylation% Absorbance of Sample = 100 − × 100 Absorbance of Unmodified Chitosan

(1) −1

3D Printing of the Microswimmers. ChMA (30 mg·mL ), LAP initiator (20 mg·mL−1) (Tokyo Chemical Industry Co. Ltd.), and superparamagnetic iron oxide nanoparticles (5 mg·mL−1) (50 nm fluid MAG-PEG/amine from Chemicell GmbH) were dissolved in 8% (v/v) acetic acid solution. The resulting prepolymer solution was dropped on a trichloro(1H,1H,2H,2H-perfluorooctyl)silane-treated glass slide, and printing was performed with a commercially available direct laser writing system (Photonic Professional, Nanoscribe GmbH). After the fabrication, glass slides were thoroughly washed with ddH2O, and then the samples were kept at 4 °C for further use. G


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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.8b05997. Supporting figures for degree of methacrylation, microchannel setup used in 3D printing, elemental mapping of microswimmers with energy-dispersive X-ray spectroscopy, controlled swimming of chitosan-based microswimmers, chemical integration of drug molecules, bleaching tests, and localized drug release experiments (PDF) Video of magnetic actuation and steering of chitosanbased microswimmers (MOV) Video of light-triggered drug release from chitosan-based microswimmers (MOV)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Oncay Yasa: 0000-0002-0282-6127 Seda Kizilel: 0000-0001-9092-2698 Metin Sitti: 0000-0001-8249-3854 Author Contributions §

U. Bozuyuk and O. Yasa contributed equally to this work.

Notes

The authors declare the following competing financial interest(s): Max Planck Innovation and Koc University will file a joint patent application for the innovations in this paper.

ACKNOWLEDGMENTS This study is funded by the Max Planck Society and Koç University Visiting Scholar Program. S.K. would like to acknowledge support from Scientific and Technological Research Council of Turkey (TUBITAK) under International Support Program (COST Action - European Cooperation in Science and Technology - CA15216, project number: 116M995) and Koç University Seed Fund (SF.00074). REFERENCES (1) Sitti, M.; Ceylan, H.; Hu, W.; Giltinan, J.; Turan, M.; Yim, S.; Diller, E. Biomedical Applications of Untethered Mobile Milli/ Microrobots. Proc. IEEE 2015, 103, 205−224. (2) Sitti, M. Mobile Microrobotics; MIT Press, 2017. (3) Ceylan, H.; Giltinan, J.; Kozielski, K.; Sitti, M. Mobile Microrobots for Bioengineering Applications. Lab Chip 2017, 17, 1705−1724. (4) Nazli, C.; Demirer, G. S.; Yar, Y.; Acar, H. Y.; Kizilel, S. Targeted Delivery of Doxorubicin into Tumor Cells via MMP-Sensitive PEG Hydrogel-Coated Magnetic Iron Oxide Nanoparticles (MIONPs). Colloids Surf., B 2014, 122, 674−683. (5) Nelson, B. J.; Kaliakatsos, I. K.; Abbott, J. J. Microrobots for Minimally Invasive Medicine. Annu. Rev. Biomed. Eng. 2010, 12, 55− 85. (6) Xu, H.; Medina-Sánchez, M.; Magdanz, V.; Schwarz, L.; Hebenstreit, F.; Schmidt, O. G. Sperm-Hybrid Micromotor for Targeted Drug Delivery. ACS Nano 2018, 12, 327−337. (7) Park, B.-W.; Zhuang, J.; Yasa, O.; Sitti, M. Multifunctional Bacteria-Driven Microswimmers for Targeted Active Drug Delivery. ACS Nano 2017, 11, 8910−8923. H


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Light-Triggered Drug Release from 3D-Printed Magnetic Chitosan

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