Programmed transport and release of nanoscale cargo by immune cells

Transport and delivery of (nanoscale) materials are crucial for many applications in biomedicine. However, controlled uptake, transport and triggered release of such cargo remains challenging. In this study, we use human immune cells (neutrophilic granulocytes, neutrophils) and program them to perform these tasks in vitro. For this purpose, we let neutrophils phagocytose a nanoscale cargo. As an example, we used DNA-functionalized single-walled carbon nanotubes (SWCNT) that fluoresce in the near infrared (980 nm) and serve as sensors for small molecules. Cells still migrate, follow chemical gradients and respond to inflammatory signals after uptake of the cargo. To program release, we make use of neutrophil extracellular trap formation (NETosis), a novel cell death mechanism that leads to chromatin swelling and subsequent rupture of the cellular membrane and release of the cell’s whole content. By using the process of NETosis we can program the time point of cargo release via the initial concentration of stimuli such as phorbol 12-myristate-13-acetate (PMA) or lipopolysaccharide (LPS). At intermediate stimulation with LPS (100 μg/ml), cells continue to migrate, follow gradients and surface cues for around 30 minutes and up to several hundred micrometers until they stop and release their cargo. The transported and released SWCNT sensor cargo is still functional as shown by subsequent detection of the neurotransmitter dopamine and reactive oxygen species (H2O2). In summary, we hijack a biological process (NETosis) and demonstrate how neutrophils can be used for programmed transport and delivery of functional nanomaterials.


Abstract
Transport and delivery of (nanoscale) materials are crucial for many applications in biomedicine. However, controlled uptake, transport and triggered release of such cargo remains challenging. In this study, we use human immune cells (neutrophilic granulocytes, neutrophils) and program them to perform these tasks in vitro. For this purpose, we let neutrophils phagocytose a nanoscale cargo. As an example, we used DNA-functionalized single-walled carbon nanotubes (SWCNT) that fluoresce in the near infrared (980 nm) and serve as sensors for small molecules. Cells still migrate, follow chemical gradients and respond to inflammatory signals after uptake of the cargo. To program release, we make use of neutrophil extracellular trap formation (NETosis), a novel cell death mechanism that leads to chromatin swelling and subsequent rupture of the cellular membrane and release of the cell's whole content. By using the process of NETosis we can program the time point of cargo release via the initial concentration of stimuli such as phorbol 12-myristate-13-acetate (PMA) or lipopolysaccharide (LPS). At intermediate stimulation with LPS (100 µg/ml), cells continue to migrate, follow gradients and surface cues for around 30 minutes and up to several hundred micrometers until they stop and release their cargo. The transported and released SWCNT sensor cargo is still functional as shown by subsequent detection of the neurotransmitter dopamine and reactive oxygen species (H 2 O 2 ). In summary, we hijack a biological process (NETosis) and demonstrate how neutrophils can be used for programmed transport and delivery of functional nanomaterials.
A large drawback of conventional drug delivery systems is their incapability to move autonomously. Most of the above mentioned approaches rely on an external flow (e.g. of the vascular system) to reach a target zone as they do not own a self-propelling mechanism.
Therefore, crossing biological barriers and actively reaching a site of interest remains difficult. [24,25] One way to overcome this issue is to equip the cargo transporter with additional capabilities. For example, magnetic nanoparticles have been manipulated through external magnetic fields. [26] Another approach is to use and reprogram cells. [27] Din et al. engineered bacteria for programmed lysis in vivo resulting in the delivery of cytotoxic agents and a potential way to tackle cancer. [28] Another example is binding and transport of cargo molecules by surface-modified red blood cells, which form long-living, biocompatible hybrid carriers. [29,30] Neutrophilic granulocytes (neutrophils) are the most abundant type of white blood cells. They are interesting candidates for cargo delivery because they are able to take up materials (phago-/endocytosis) [31] , sense and migrate along chemical gradients (chemotaxis) to inflammatory sites [32,33] or cross dense borders, such as the blood-brain barrier. [34,35] The abilities of neutrophils have been used to take up silica particles to follow E.coli gradients and paclitaxel containing liposomes for cancer treatment. [34,36] However, so far it was not yet shown how to control cargo release. Another function of neutrophils is neutrophil extracellular trap (NET) formation (NETosis), a defense strategy and novel type of cell death. [37,38] During NETosis, the cell's chromatin is chemically modified, which leads to its expansion and ultimately the rupture of the cellular membrane and the release of their cytosolic content. [39] Neubert et al. showed that this process consists of different phases, including a first active phase in which the cell remains fully functional. [39] In summary, neutrophils possess several functions that are highly interesting for cargo delivery of nanoscale materials.
Here, we make use of these functions and demonstrate uptake, transport and programmed release of a nanoscale cargo. We show that neutrophils take up carbon-nanotube-based near infrared fluorescent sensors as cargo, transport them and release them again via NETosis. Importantly, we quantify time and length scales of this process and showcase that the cargo is fully functional after delivery by detecting small molecules with the transported nanosensors. Neutrophils use phagocytosis to destroy foreign objects. [31,40] Therefore, neutrophils should also be able to take up nanomaterials. In this study, we chose single-walled carbon nanotubes (SWCNT) as a model cargo to make use of their unique near infrared fluorescent sensing properties. Neutrophils readily took up DNA functionalized (GT) 15 -(6,5)-SWCNTs ( Fig.2a,b). The nIR signal (red) of the uptaken SWCNTs is localized in a region of the neutrophil outside the nucleus (blue) and this compartment remained in the rear of the cells during migration (Fig. 2a, Suppl. Movie 1). It is most likely that this compartment is the phagosome, which is often found in the actomyosin-rich back of polarized neutrophils. [41] Similarly, streptavidin-functionalized SWCNTs in HL60 cells, a model cell line for primary neutrophils were found in a similar location. [42] The nuclei, on the other side, stayed rather at the middle/front of the cell during migration as previously described. [43] Uptake of SWCNTs increased with concentration and incubation time as evidenced by the nIR fluorescent signal inside the cells (Fig. 2b). After around 15 min, uptake reached a plateau (Fig. 2b). For low SWCNT concentrations (0.1 nM) cells showed a normal behavior while at higher concentrations (> 1nM) we observed sometimes cell agglomerates (Suppl. Fig. S1a). For this reason, we used 0.1 nM SWCNT for uptake and all following experiments. Cells with a SWCNT cargo were still able to perform NETosis after stimulation with 100 nM PMA and demonstrated the well-documented time course of chromatin decondensation and subsequent cell rupture (Fig. 2c). Interestingly, the distribution of the SWCNT cargo changed during NET-formation. The size of the intracellular SWCNT cargo did not change in early phases. In contrast, in later stages of NETosis the compartment with the SWCNTs were compressed, parallel to the expansion of the neutrophil's chromatin ( Fig. 2c/d, Suppl. Fig. S2, Suppl.

Uptake and release of carbon nanotubes by neutrophils
Movie 2), which could be explained by an increasing intracellular pressure by chromatin swelling. [39] This finding also explains why SWCNTs ended up in close proximity to the cell membrane. Additionally, the SWCNT fluorescence intensity decreased during the time course of NETosis, which could be explained by changes in the pH or quenching because SWCNTs get closer to each other. Degradation of SWCNTs by myeloperoxidase (MPO) is well known but unlikely because it would occur on other time scales (hours to days). [44,45]

Functionality of cargo-loaded neutrophils
Uptake and release of the cargo is a necessary step for a delivery system. However, it remained elusive if DNA-SWCNT loaded cells activated to perform NETosis were still functional and able to migrate or for how long. Therefore, live-cell imaging of neutrophils  The time for the neutrophils to reach a stationary phase (stopping time) decreased with increasing concentration of the activator for both LPS and PMA as expected (Fig. 3b). The migration velocity stayed relatively constant for all LPS concentrations ( Fig. 3c) but drastically decreased with increasing PMA concentration (≥10 nM). NETosis (indicated by chromatin decondensation) was assessed 160 min after activation and showed concentration dependent probabilities (Fig. 3d).
The results showed that low activator concentrations (0.1 -10 µg/ml LPS and 0.1 -1 nM PMA) did not trigger high NETosis rates but maintained the neutrophil's motility. On the contrary, too high concentrations (10 --100 nM PMA) resulted in high decondensation rates but also inhibited cell migration completely. Only for medium concentrations (100 µg/ml LPS

Transport of nanoscale cargo via migration of neutrophils
To further investigate whether SWCNTs inside neutrophils affect their migration, a gradient migration assay (under-agarose migration) was performed with cargo loaded and unloaded cells. [46] This assay mimics an in vivo scenario in which cargo loaded neutrophils are supposed to follow inflammatory cues and finally release their cargo at the inflammatory site. behavior of PMA-induced NETosis pathways (Fig. 4c). This result is in agreement with a recent study that shows that PMA induced NETosis does not require adhesion or mechanical input at all. [47] Interestingly, cells migrated over longer distances at higher serum concentration (20 % vs. 0.5 %) conditions, which highlights that this approach could also work in vivo (Suppl.

Programmed release of functional nanosensors
In the final step, we performed functionality tests of the SWCNT cargo in inactivated and ruptured cells to investigate whether the specific abilities of the internalized material remain intact throughout NETosis. SWCNTs are useful near infrared fluorescent building blocks of nanosensors for novel applications and their selectivity depends on the specific surface functionalization. [13,48] We used (GT) 15 -(6,5) SWCNTs because they are able to report the presence of the neurotransmitter dopamine. [16,49,50] Such sensors have been used to image dopamine release from cells with high spatiotemporal resolution. [51] As a second cargo, hemin-aptamer functionalized SWCNTs that decrease their fluorescence in the presence of H 2 O 2 were used. [52,53] In both cases, SWCNTs were taken up by neutrophils and their responses were measured via consecutive nIR imaging either while they were carried by nonactivated cells or after NETotic membrane rupture. Here, the addition of 100 nM dopamine led to an instantaneous increase of the sensors' intensity in case of disrupted cells. In comparison, in non-damaged cells dopamine should not get into the cell and indeed such cells showed no sensor response to dopamine (Fig. 5a, Suppl. Movie 5/6). This result indicates a successful release and accessibility of the cargo after NETosis and full functionality of the dopamine nanosensors after release. In contrast, 100 µM H 2 O 2 addition decreased the nIR signaling both intact and NETotic cells (Fig. 5b, Suppl Movie 7/8), which can be explained by the diffusion of H 2 O 2 through the cellular membrane. [54,55] Interestingly, we were also able to locate differences in the sensors' uptake behavior depending on the associated surface functionalization. While (GT) 15 -(6,5) SWCNTs appeared most often in larger, intracellular structures, Aptamer/Hemin-SWCNTs were found closer to the cell membrane in smaller agglomerates ( Fig. 5a/b, Suppl. Fig. S7a). Nevertheless, both sensor types were functional after cargo transport and rupture, as evidenced by the same fluorescence response performance prior to cellular uptake (Suppl. Fig. S7b) and control experiments (Suppl. Fig.   S7c/d).
Finally, we also demonstrated the transport and release of the functionalized nanosensors to specific target sites. For this purpose, fibrinogen was patterned on glass surfaces and SWCNT-loaded, activated neutrophils were allowed to migrate over the coated area resulting in an alignment of the cells along the fibrinogen pattern (Fig. 5c). Again, the immobilized sensors were still functional after cell rupture on the pattern and showed an instant response to 100 nM dopamine (Fig. 5d, Suppl. Movie 9). Thus, neutrophils can be programmed to take up such a nanoscale cargo, transport it to specific sites and release it in a functional state.
Of course SWCNTs can be different in length, functionalization and chirality. It is known that those properties affect uptake and retention in cells [56] . Therefore, one could envision to tailor the nanoscale cargo for specific uptake/retention kinetics. In this study, functionalized SWCNTs have been used as sensors for two important signaling molecules (dopamine, H 2 O 2 ).
However, sensors for many other interesting molecules have been developed and could be transported by this approach [16,17,[20][21][22][23]57] . For example, recently a SWCNT-based sensor for another important neurotransmitter (serotonin) has been introduced [18] . Another application is to use them as mechanical sensors. In this context, SWCNTs have been used to study movements in cells and the extracellular space in brain tissue [58,59] . Therefore, the cargo transport approach presented in this work could also be extended to bring SWCNT sensors into specific in vivo locations and explore the local mechanical properties.

Conclusion
Over the years much effort has been put forward into developing biocompatible transport and drug delivery systems. Here, we demonstrated a novel approach that makes use of particular and unique functions of neutrophils including phagocytosis, migration and NET formation. As we show, precise chemical activation of neutrophils determines how long the cells migrate and the time point of cargo release. In this process, the internalized nanoscale cargo remains functional at all times and is protected from most extracellular influences. This new type of transport-and-release mechanism might be of great benefit for various biomedical applications as it combines the biocompatibility and targeting capabilities of cells and a tool to program the time scale of release. For example, one could envision isolation of neutrophils from a patient, loading with functional nanoparticles and reinjection for programmed release. At the same time, this work also emphasizes the utility of SWCNTs as versatile chemical sensors that can be transported inside cells and are highly stable. In conclusion, we present a concept for nanoscale cargo transport and delivery by programming immune cells via NETosis.

Cell isolation of human neutrophils
Neutrophils were isolated from human venous blood of healthy donors. The study itself was approved by the ethics committee of the university medical center in Göttingen and all donors fully consent after being informed about possible consequences of the procedure. Isolation of human neutrophils was performed according to a standard protocol. [60] In brief, fresh blood of

SWCNT modification with ssDNA
Surface modification of single-walled carbon nanotubes (SWCNTs) was performed as described previously. [49,61] Briefly, 125 µl ssDNA solution (2 mg/ml stock in phosphate buffered saline (PBS))(Sigma Aldrich) and 125 µl (6,5)  Measuring the intensity of SWCNT inside the cells was then performed using ImageJ's thresholding system (v3.52i). nIR images were tuned to 8-bit depths, cropped into 20x20 µm areas containing only the respective SWCNTs and thresholded via common MinError thresholding algorithm to calculate a mean intensity. SWCNT spots that couldn't be correlated with a cell area in the corresponding phase contrast image, as well as sensors that were found in cell agglomerations, were excluded from the analysis. The remaining data was averaged (weighted mean of all experiments) and normalized over the corresponding background value (I br = 1) to calculate a normalized mean intensity for each condition. Subsequently, the sample was placed into a preheated incubation system (37 °C) (Cat. 11922, ibidi), on top of the custom build near-infrared microscope mentioned in the previous section.

Live cell imaging of SWCNTs during NETosis
Using a 100x oil objective (UPLSAPO 100XO, Olympus) consecutively, phase contrast as well as chromatin (DAPI) and nIR-images were taken manually from the chosen sample position every ten minutes for 150 minutes after addition of 100nM phorbol myristate acetate (PMA) (Sigma-Aldrich) to the cell sample. SWCNTs were excited by a common fluorescence lamp in combination with a built-in 561nm filter cube (F48-553, AHF Analysentechnik) and excitation powers and exposure times were kept constant to ensure data comparability Analysis of chromatin and SWCNT area as well as SWCNT intensity was similar to the uptake study described before.

Live cell imaging of activated neutrophil migration
To record the migration behavior of activated neutrophils, around 200 µl of RPMI 1640 medium containing 75000 untreated cells and 1µg/ml Hoechst 33342 stain were incubated in 8 Well µ-slides (ibidi) for 20 minutes Subsequently, the µ-slides were incorporated in pre-

Migration analysis of preactivated neutrophils
Cell tracking was performed by ImageJ's TrackMate plugin. [62] Briefly, all chromatin images gained by the image process were combined to form a z-stack and implemented into the plugin first. Segmentation was then performed using the Laplacian of Gaussian (LoG) detector module with an estimated blob diameter of 20 pixels (6.5 µm) and a 5 pixel (1.6 µm) threshold. As a result, all cell trajectories within a stack could be traced back by using a subsequent simple LAP tracker model with a maximal linking and gap-closing distance of 50 pixels (16 µm) and a maximal frame gap of two frames. All trajectories were then further analyzed by a custom-build MATLAB code (v. Matlab 2014a) which was able to calculate the traveled distance d for every cell and frame i according to the formula with N defining the number of frames until the stopping time and τ the frame time (2 minutes) between two images.

Decondensation rate analysis
Counting decondensed and intact/lobular shaped nuclei was performed according to existing protocols. [39,63] Briefly, chromatin images of the recorded positions were taken after 180 minutes and analyzed via ImageJ's Cell Counter plugin. Nuclei that appeared in its known, compressed shape were counted and defined as intact/condensed whereas nuclei that showed increased, roundish chromatin distributions defined a basis for the decondensed/NETotic state. Cells were counted and the number of decondensed cells was divided by the total number of cells to generate a relative decondensation value.

Migration in a gradient
Under-agarose assays were performed to measure cell migration of SWCNT-loaded neutrophils in a gradient. Gels were manufactured according to the protocol of B. Heit et al.. [46] A HBSS/RPMI 1640 solution was prepared by mixing 5 ml HBSS (w/o Ca 2+ , Mg 2+ , Thermo Fisher Scientific) and 10 ml RPMI containing 0.75 % FCS (Merck) in a common 50ml Eppendorf tube and heated up to 68°C using a common water bath. Meanwhile, 0.24 g ultra-pure agarose (Roth) was added to a vial containing 5 ml of milliQ and the solution was vortexed extensively in order to suspend the agarose homogeneously. The latter was subsequently heated up until boiling by the use of a common Bunsen burner and quickly vortexed for three times. The HBSS/RPMI solution was added to the agarose and 3 ml each of the mixture was evenly distributed on a plastic-bottom petri dish (Cat. 81156, ibidi). Agarose gels were then allowed to solidify at room temperature and samples were stored overnight at 4 °C with the dish lid covered in dust-free, milliQ saturated cloths to avoid gel draining. Shortly before the cell experiment, two wells with a diameter of 3 mm and a distance of 2.2 mm were punched in each gel using a dermal biopsy punch (Cat. KBP-48101, kai medical) and remaining agarose within each of the wells was extensively removed by vacuum aspiration.
Lastly, gels were equilibrated with RPMI 1640 medium for one hour (37 °C, 5% CO 2 ) and the supernatant medium was again removed by vacuum aspiration shortly before cell loading.
For the latter, around 100000 neutrophils ((GT) 15 -(6,5) SWCNTs loaded or without any pretreatment) were poured in one of the prepared agarose wells using a 5 µl of medium plus 1.6 µM Hoechst 33342 solution and were allowed to equilibrate for 20 minutes at 37 °C, 5% CO 2 .

Cell patterning
Cell patterns on substrates shown in Fig, 5c were achieved by light-induced fibrinogen printing controlled by a PRIMO micropatterning machine (alvéole). Briefly, 18 x 18 mm glass coverslips (Fisher Scientific) were washed with 75% ethanol twice and plasma treated for five minutes to clean and improve the hydrophilicity of the sample. Subsequently, PDMS stencils with a circular well (r = 2 mm) in the center were pressed onto the glass and filled with 0.1 mg/ml PLL-g-PEG (Sigma) in PBS for one hour to guarantee a homogeneous passivation layer. Next, the so prepared sample was fixed on the respective PRIMO setup, calibrated according to the manufacturers' instructions (microscope specs: Olympus IX83 with an