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How Leucocyte Cell Membrane Modified Janus Microcapsules are Phagocytosed by Cancer Cells Wenping He, Johannes Christoph Frueh, Zhenwei Wu, and Qiang He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b10885 • Publication Date (Web): 29 Jan 2016 Downloaded from http://pubs.acs.org on February 4, 2016

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ACS Applied Materials & Interfaces

How Leucocyte Cell Membrane Modified Janus Microcapsules are Phagocytosed by Cancer Cells Wenping He, Johannes Frueh *, Zhenwei Wu, and Qiang He* State Key Laboratory of Advanced Welding and Joining (HIT), Harbin Institute of Technology, Yikuang Street 2 , Harbin 150080, China E-mail: [email protected]; [email protected] Keywords: Janus capsule, leukocyte cell, biocompatible, target recognition, phagocytosis

Abstract: Modern drug delivery systems are relying on either antibody based single surface recognition or on surface hydrophobicity based approaches. For a tumor showing various surface mutations, both approaches fail. This publication hereby presents Janus capsules based on polyelectrolyte multilayer microcapsules exhibiting human leucocyte (THP-1 cell line) cell membranes for discriminating HUVEC cells from three different cancer cell lines. Despite destroying the cellular integrity of leucocyte cells, the modified Janus capsules are able to adhere to cancer cells. Leucocyte cell membrane coated Janus capsules are phagocytosed with the cellular membrane part pointing to the cells.

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1. Introduction Drug delivery systems like modified capsules with pH or chemo-sensitive release,1,2 chemically labeled antibodies3 or externally triggered laser based drug release4–7 are novel and promising candidates for fighting cancer, which is a leading death cause8 in industrial countries. The main drawback of an antibody based system is that cancer stem cells can renew the cancer while they are not being affected by the antibodies targeting the daughter cells.3 This effect renders antibody based therapies less efficient.3 Also, frequent mutations in fast dividing cancer cells can change the cell surface structure. pH and chemo-sensitive systems rely on bulk environmental changes1,2 which are only present in big tumors. The resolution of externally triggered systems relies on the detection resolution, which is also limited.4 Guidable systems, like Janus capsules and core-shell systems are able to detect cancer cells and offer solutions to afore mentioned drawbacks.9–12 This is because Janus capsules, are comprised of different functional surface entities.13,14 Multi-functional Janus capsules can enable drug delivery systems to perform a controlled stepwise drug release.15,16 These systems were shown to perform multiplexed biomolecular detection, by using one side of a Janus capsule as the analyzing and the other side as the reporting unit (e.g. fluorescence tags).17,18 Janus capsules being coated on one half with magnetic capsules and the other half being fluorescent enable simultaneous imaging while enabling magnetic guidance of the drug carrier.18 These Janus capsules are phagocytosed based on their physical and chemical properties, including size, surface chemistry, and mechanical stiffness.19– 22

One of the main problems of capsules based drug delivery is to keep the capsules circulating long enough in vivo to enable them to find their target.9 This problem was solved elegantly by


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coating such entities with cellular membranes.23,24 For example, Zhang et al. reported that red blood cell (RBC) membranes can be used to encapsulate nanoparticles for detoxification treatment and antitoxin immunity.25–27 The Machluf group reported a targeted delivery platform, based on nanoghosts (NGs) (cellular membrane vesicles without a core) reconstructed from the whole cell membrane of mesenchymal stem cells (MSCs).28 Despite the success in camouflaging the particles from the immune system and clearing systems off the body, the role of cellular membrane in particles internalization remains unclear.29–32 In this study, we report Janus micro capsules coated on one side with gold (sputtered rough film) and on the other side with leucocyte cellular membrane to discriminate healthy HUVEC (human umbilical vein endothelial cells) from three different kinds of cancer cells in in-vitro experiments. The targeting function of these capsules is delivered by the leucocyte cellular membrane. The monocyte leukemia cell line THP-1 was used as a model system in this work. It displays commitment towards macrophage differentiation.33,34 This kind of cell line resembles human monocytes with respect to numerous criteria such as expression of membrane antigens and expression of genes involved in lipid metabolism. In contrast to native human monocytes, a cell line such as THP-1 offers the advantage of a homogeneous population.35,36 An additional advantage of using these cells as detectors is the fact that they are able to migrate to disease sites and play a role in immunological and inflammatory responses.37 The ability of THP-1 cells, Janus capsules and THP-1 cell membrane modified Janus capsules (HTMJC) are used to discriminate HUVEC cells from three different cancer cell lines. In addition the cell entry path of HTMJC capsules is studied.


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2. Experimental Section 2.1. Materials The used capsules consist out of polyelectrolyte multilayers formed from alternating deposition of polyelectrolytes onto colloidal templates.38 The Janus capsules contain biocompatible polyelectrolytes like poly (allylamine hydrochloride) (harmless in complexes39), poly (styrene sulfonate) (PSS) (already FDA approved)40 and natural THP-1 human monocytic leukemia cell membranes.40 The spherical silica particles with an average diameter of 5µm were obtained from Microparticles GmbH (Berlin, Germany). PSS Mw = 70000, PAH Mw = 70000, Methoxy-omega-mercapto-polyethylene glycol (HS-PEG), fluorescein isothiocyanate (FITC), DID labeling solution [1, 1-Dioctadecyl-3, 3, 3, 3-tetramethylindodicarbocyanineperchlorate], phosphate buffered saline (PBS, 10mM phosphate buffer, 2.7mM KCl, 137mM NaCl, pH 7.4) were purchased from Sigma-Aldrich (St. Louis, USA). Sodium chloride (NaCl) and hydrofluoric acid (HF) were obtained from Chemical Reagent Company (Harbin, China). FITC-modified PAH (FITC-PAH) was prepared through labeling PAH with FITC according to reference49. The THP-1 cell line was selected on logarithmic phase and purchased from Chinese Academy of Sciences Institute for Cell Resource Center, Shanghai, China. All commercial materials were used without further purification.

2.2 Preparation of Janus capsules The silica micro-particles were coated with a polyelectrolyte multilayer (PEM) following the LbL procedure described in reference 38. Briefly, the particles were immersed into PAH solution (2g/L, 0.5M NaCl) for 15 minutes under continued shaking, following three washing and centrifugation steps (30 seconds each). Each of the washing steps utilized 0.1M NaCl solution.


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Subsequently the particles were immersed into PSS solution (2g/L, 0.5M NaCl) following above mentioned method. This process was repeated 5 times forming 5 bilayers of PSS and PAH onto the particles. In order to make sure the outmost layer of capsules is positively charged, PEM coated silica particles were immersed in aforesaid PAH solution for 15 min again. Following polylelectrolyte deposition sequence was therefore used: (PAH/PSS)5 PAH. In order to observe the morphology of particles in fluorescence microscopy, one PAH layer was replaced with Fluorescein Isothiocyanate (FITC) labeled PAH (PAH-FITC). The Janus structure is prepared by depositing a monolayer of PEM coated silica microspheres on silicon substrates, and sputtering ~100 nm Au onto the surface of the PEM coated silica particles, followed by peeling off the particles from the substrate.41 The silica cores were then dissolved by treating the particles for several minutes with 0.3 M HF42 (Caution: HF is extremely toxic and can penetrate the skin!). The capsules were purified by three centrifugation/water washing steps. All obtained capsule solutions were stored at 4 oC in ultra-pure water (resistance >18.6 MΩ cm, Elga Labwater, Beijing, China).

2.3 Coating Janus capsules and homogeneous capsules and particles with THP-1 human monocyte leukemia cell membrane The Janus capsules were pre-treated with a binary mixture of HS-PEG solution to modify the gold shell of Janus capsules before coating the capsules with cell membrane. This step is necessary to decrease the surface energy and prevent the adsorption of cell membrane onto the gold. The binary mixture enables an efficient modification of the gold shell, and it minimizes unspecific binding of the HTMJC. Removing of residual HS-PEG was achieved by washing the capsules with phosphate buffer saline 3 times. To assemble the cell membranes onto the PEM


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side of HS-PEG modified Janus capsules, the Janus capsules were co-incubated with fresh cell membrane fragments and kept shaking together for 3 h followed by 3 washing steps in PBS.43–45 Homogeneous particles and capsules were coated with THP-1 membrane similar to Janus capsules with the difference, that homogeneous particle and capsules did not contain a gold cap.

2.4 Characterization An Olympus BX53 fluorescence microscope (Olympus, Tokyo, Japan) was utilized to record the cell status and to observe the co-incubation between capsules and cells. Scanning electron microscopy (SEM) (Hitachi S-5200, Tokyo, Japan) and atomic force microscopy (AFM) (Agilent, Palo Alto, USA) were used to observe the capsules. For AFM tests, a drop of sample solution was dropped onto a silicon wafer with sequential drying at room temperature overnight. Transmission electron microscopy (TEM) was performed using a Tecnai G2 F30 microscope (Tokyo, Japan). Copper grids coated with carbon films were used as supports for the sample in TEM. For this a drop of solution with dispersed capsules was dropped onto the grid and dried at room temperature overnight. Fluorescence images were obtained using a Leica TCS SP5 II confocal laser scanning microscope (CLSM) (Heidelberg, Germany). The excitation light wavelengths were 488 and 633 nm respectively. Quartz Crystal Microbalance (QCM) (Q-Sense E4, (Biolin company, Sweden)) experiments were performed to follow the PEM and cell membrane assembly in real time. The used sensors were silicon dioxide coated sensors from Qsense (QSX 303). The quartz sensor was washed with ethanol and ultra-pure water and then coated with 5 polyelectrolyte layers which were alternately adsorbed from PSS and PAH polyelectrolyte solutions (same concentration and ionic strength as for the Janus capsules) pumped through the


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sample chamber at a flow rate of 50 µL min-1 at room temperature (25oC). After equilibration the solution was switched from ultra-pure water to PBS. The fresh ultra-sound treated cell membrane solution was pumped into the measurement chamber. The formation of the ultra-thin composite films was monitored in real time. A Zeta PALS Analyzer (Holtsville, NY, USA) was used to analyze the membrane surface charge changes caused by PEM and cell membrane assembly onto the capsules. UV-VIS-NIR absorption spectra were recorded using a HITACHI U-4100 (Tokyo, Japan).

2.5. Cell experiments Human cervical cancer cells (HeLa) were used for cytotoxicity studies and cultured in RPMI 1640 growth medium supplemented with 10% fetal bovine serum and 1% penicillinstreptomycin at 37oC in an atmosphere of 5% CO2 and 70% humidity. When the cell density reached 80 to 90 %, cells were diverted to 96-well plates using a standard trypsin-based technique46 with a final concentration of 5×104 cell·ml-1. When cells in 96-well plates were cultured at a concentration of about 90%, 20 µl of HTMJC microcapsule solution was added to a well. After 2 hours 20 µl of HTMJC microcapsule solution was added to another well, to determine the effect of a lower incubation time. This step was repeated until 24 hours passed to determine the effect of up to 24 hours incubation time. Cell survival was studied using standard MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-nyltetrazoliumbromide), Sigma, St. Louis, USA) assay.47,48 After incubation for 24h, the cultured medium was removed and replaced. MTT (20 µl, 5 mg·mL-1) in PBS was added and incubated at 37oC for 4 h. Then the remaining MTT was exchanged by DMSO (100 µl) to solubilize the purple formazan crystals. After 20 min, all wells were characterized at a wavelength of 490 nm by using a Wallace Victor 3 1420 multilabel


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counter (PerkinElmer Life Sciences, Waltham, MI, USA). It is noted, that A-549, HUVEC and HepG2 cells were processed in similar manner like HeLa cells. Membrane of the Human Monocytes cell line (THP-1) was used for modifying the Janus capsules. Briefly, THP-1 cells were suspended and cultured in RPMI 1640 growth medium (Jiayu biological reagent company, Harbin, China) supplemented with 10% fetal calf serum and 1% penicillin and streptomycin (complete RPMI) at 37oC in an atmosphere of 5% CO2 and 70% humidity for 48h. When the cell density reached 70%, the cells were washed with PBS (pH 7.4) solution, then incubated with 2 µg/ml DID labeling solution for 15min to trace the cell membrane. PBS solution was used to wash the cells 3 times. Subsequently ultrasound was used to dissemble these cells in an ice bath (Sonics Vibra-Cell (Branson, USA)) to get the fresh aseptic cell membranes in PBS solution.

3. Results and Discussion 3.1. Janus Structure and Characterization UV-Vis-NIR absorption spectra of gold coated Janus capsules have a maximum absorption wavelength in the near-infrared region (650-900 nm, see SI Figure S1). This is beneficial for NIR laser based treatment, since NIR irradiation can penetrate deep into the tissue and minimize damage to healthy skin and tissue.39 The functionalized HTMJC are therefore able to function as cargo carrier as well as photothermal agents in future applications. Janus capsules can be driven into desired directions via controlled short laser pulses, which is known as “nudging”.49 Other authors used a more manual approach for controlling thermophoretic movement of drug filled Janus capsules to guide them to cancer cells.12,50 In our study thermophoretic motion away from laser irradiation was found, as supporting information Video S1 shows. Homogeneous non-gold


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cap containing capsules showed no thermophoretic motion and no absorption peak in UV-VISNIR spectra (results not shown). HTMJCs can be successfully prepared using THP-1 cellular membranes. SEM images in Figure 1A show that the gold coating covers one half of the Janus capsules, the hollow Janus morphology obtained after dissolving the silica core which is displayed in the inset in Figure 1A. The THP-1 cell membrane covers the other half of the Janus capsules, as TEM images illustrate in SI Figure 2A. The diameter of single membrane fragments, on the outer wall of capsules, is found to be in the range of 10 to 50 nm. The resulting HTMJC membrane is obviously more rough and massy compared to pristine PEM capsules as can be seen by comparing the inset SI Figure S2A with SI Figure S2B. The Zeta potential value of the THP-1 cells is in average -21.9 ± 2.51 mV. For this reason self-assembly via electrostatic forces is possible.51 Prior to coating the capsules with cellular membranes the zeta potential is +33.9 ± 1.4 mV on the PEM part of the Janus capsule. After coating the capsule with THP-1 membrane the value decreases to -26.4 ± 2.14 mV which is close to the value of original THP-1 cell membrane charge. These results prove a successful cell membrane transfer onto the Janus capsules. For QCM measurements,52 the polyelectrolyte wall of the Janus capsules was prepared onto a Quartz sensor by using 5 layers of PSS/PAH, followed by flushing cell membrane emulsion into the measurement chamber. The process of membrane coating on top of the polyelectrolyte multilayer was followed in real time. The shift in resonance frequency of the quartz crystal was studied since it is a measurement of the mass deposited onto the sensor.53 When the negatively charged cell membrane emulsion is transported into the reaction chamber, it starts coating the positive charge terminated PAH top layer. At the same time, the line of the resonance frequency


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descends significantly, proving that the cell membrane readily adsorbs on the PEM. It is worthy to note that the adsorbed membrane mass is several times larger than in the case of polyelectrolyte self-assembly (see Figure 1C). When PBS solution is substituted for cell membrane emulsion, no significant change of the frequency is observed, proving an irreversible adsorption of cellular membrane onto the PEM. CLSM images (Figure 1D) reveal the successful preparation of cell membrane functionalized Janus capsules. The bright field channel in Figure 1D (bright) shows the distinct Janus structure of the capsule because of the gold shell. FITC labeled PAH within the polyelectrolyte film, shows for this reason a green semi-circle shape in FITC channel. The cytomembrane was labeled with DID which has an emission band at 633 nm, and appears therefore red in DID channel. The overlay images show that the labeled cellular membrane covers exclusively the polyelectrolyte side of the Janus capsules. This effect is caused by HS-PEG (polyethyleneglycolemercaptoalcohole) which was used to protected the gold shell.54 As SI Figure S3 shows, the HSPEG does not adsorb on PEM and does also not shield the THP-1 adsorption on PEM, proving the in afore mentioned assumption. The multi-functionality of these capsules like gold for NIR laser absorption and cancer targeting via cellular membranes allows theoretically the application of photothermal cancer therapies as UV-VIS-NIR absorption spectra in SI Figure S1 shows.39,55 The cytomembrane should theoretically allow for HUVEC versus cancer cell discrimination.56 The diameter of HTMJC is in average 5 ± 0.5µm, which is in accord with the diameter of the silica particle template. The HTMJC are stable for more than 24 hours in PBS or cellular media, as MTT tests and microscopic observations showed. Such findings are in agreement with Neutron reflectometric studies performed in reference 57. As pointed out in reference 57 the stability of a lipid bilayer on


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top of PEM films is strongly pH dependent and slight deviations can cause the membrane to leave the PEM or not to assemble from the start. It is additionally pointed out, that the PEM can dissemble upon larger changes of pH or ionic strength due to shielding of PAH or PSS charges as stated in reference 51. The capsules were found to be stable over time frames of more than one day, whereby the PEM capsules kept their shape up to a month. On these larger time scales (>2 days) no cell targeting tests were performed. A necessary prerequisite for being phagocytized by cancer cells is that used materials of Janus capsules are biocompatible. Figure S4 displays that the HTMJC show no adverse effects on HeLa cells when being co-incubated. For the whole observed time frame of 24 h (lifetime development was investigated with a resolution of 2 hours) no significant changes in cell viability were detected. The cell survival rates at different intervals were similar to the control group which proves the biosafety and biocompatibility of HTMJCs.

Figure 1. (A) SEM image of gold sputtered Janus micro-particles (for display purposes the core was not removed in this image) (Scale bar = 2µm), inset: Janus capsule without particle template (scale bar = 1µm); (B) Zeta potential of THP-1 cells (black), Janus microcapsules (before coating,


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red) and HTMJC (blue); (C) QCM measurements following THP-1 cell membrane self-assembly onto PAH terminated PEM film, demonstrating the successful adsorption of cell membrane fragments onto PEM film; (D) CLSM images of the HTMJC; Bright-field channel (gray); FITC channel (green); DID channel (red), THP-1 cell membrane labeled with DID dye; overlay of FITC and DID channel (orange), merged image demonstrates the relationship of cell membranes and polymer side of Janus microcapsules. Scale bar = 8µm.

3.2 Autonomous Recognition of Cancer Cells The molecular recognition ability of their cell membranes, allows THP-1 cells and HTMJC to detect antigens expressed by tumor cells.58,59 This should allow them to discriminate between healthy and cancer cells. To prove this assumption, the recognition ability of THP-1 cells was tested by co-incubating THP-1 cells with three different cancer cell lines as well as the healthy HUVEC cell line. An increased binding probability of THP-1 cells to cancer cells was found, as shown in supporting information (SI) Figure S5. After co-incubation for 5 hours, the THP-1 monocyte leukemia cells stay with a significant higher probability adherent to the three kinds of cancer cell lines compared to the HUVEC cell line. After three washing steps, the THP-1 cells were still sticking with a higher probability to the cancer cell lines, proving the strong cancer cell to THP-1 cell membrane interaction. The efficiency of washing can be seen by comparing before and after washing images in SI Figure S5 (E) and (F). On the basis of these findings, the ability of this cellular membrane to not only camouflage capsules from the immune system, as found in similar systems10, also autonomous targeting of cancer cells seems feasible. The capsule coating and cancer cell targeting properties of these membranes was therefore tested. To demonstrate the cancer cell targeting capability of HTMJCs


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compared to pristine Janus capsules, the two kinds of capsules were incubated with HUVEC and three cancer cell lines in vitro. The added Janus microcapsules and HTMJCs were respective 40µl at a concentration of 2×105 THP-1 or capsules ml-1. The samples were washed with PBS solution 3 times after co-incubating them for 3h and subsequently fixing the cells for CLSM (confocal laser scanning microscopy) imaging. Figure 2 displays a typical case of capsule attachment to HeLa cells. The amount of HTMJC attaching to the cancer cells was in all samples and repetitions (3 repetitions) significantly larger than that of Janus capsules without THP-1 cellular membrane. This proves the targeting ability of the HTMJCs. Most of the HTMJCs adhered to cancer cells with the THP-1 modified side while the gold shell of capsules faced upwards or sidewards away from the cells, proving that the cyto-membrane is fulfilling the targeting function.

Figure 2. CLSM images showing in vitro targeting ability. Same quantities of Janus capsules and HTMJC were incubated with HeLa cells for 3 hours, the samples were fixed for CLSM imaging after three washing steps. Pictures were obtained in bright field, FITC and DID channel.


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(A) Janus capsules; (B) HTMJC, scale bar = 50µm. Note that not all Janus capsules or HTMJC appear in green and red channel, due to shielding of the capsules fluorescence by the gold cap, as well as DID dye based quenching of the FITC fluorescence, see SI Scheme S1.

Evaluating the coverage of cells with pristine Janus capsules, HTMJC or THP-1 cells, one can not only see a selectivity towards cancer cells for the THP-1 cells. Even the PEM coated capsules show a significant selectivity towards HeLa and A-549 but not towards HUVEC and HepG2 cells, as shown in Figure 3A-D. Such an effect is probably due to hydrophilichydrophobic interactions.60 The strong increase in cancer cell coverage from HTMJC compared to native THP-1 cells is not fully understood. It could be attributed to a combined effect between uncoated PEM and THP-1 cellular membrane parts or due to substrate based structural receptor changes like in reference 61. Dividing the cell coverage rate of the two capsule types by the THP-1 cells coverage rate, one is able to compare the target recognition behavior to THP-1 cells using the same concentrations.61 The performed experiments show that the pristine Janus capsules are less sensitive towards cancer cells than THP-1 cells, but more towards healthy HUVEC cell lines (see Figure 3D). This is most likely due to hydrophobic, as well as electrostatic interactions. The HTMJC show increased target recognition of cancer cells compared to Janus capsules but are also more likely to bind to HUVEC cells than THP-1 cells. This effect can be explained with an imperfect PEM surface coverage by the THP-1 cell membrane as shown in SI Figure S2. The fact that the coverage of HUVEC cells by HTMJC is close to the binding level of Janus capsules (see Figure 3B and C) supports this assumption. The authors would like to point out that pristine Janus capsules do adhere to HUVEC cells with a similar probability like they adhere to HepG2


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cells. This proves, that the adhering probability of the capsules is responsible for internalization and not the cancer cells food uptake level. It is noted, that homogeneous particles and capsules also offer such cell targeting and detection properties with similar efficiency like HTMJCs. Due to presentation reasons such results are not shown in this study. Experiments in a cytometer showed a similar tendency like Figure 3, as can be seen in SI Figure S6. The necessity to trypsinate the cells prior to cell counting caused however to a large decrease of Janus and HTMJCs from cell surfaces. This causes an underestimation of the detected particles, since only internalized particles could be detected.

Figure 3. Statistical evaluation of four kinds of the cell lines coverage for equimolar amounts of THP-1 cells or capsules; coverage with (A) THP-1 cells; (B) HTMJCs (C) normal Janus capsules and; (D) coverage of different cells by HTMJC and Janus capsules relative to THP-1 cell


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coverage (∆S). Evaluations for all error bars are done by 3 independent samples and 4 images per samples at 10x zoom (~5×104 cells, 2×105 THP-1 or capsules ml-1). The changes in cell coverage rate (selectivity towards cell type) of HTMJC versus Janus capsules were normalized by THP-1 cell coverage rate. For A-C % is (number of capsules per cell*100). Adsorption images were acquired in bright field and fluorescence as can be seen in SI Figure S 7.

3.3 Cancer Cell Targeting and Cell Entering Properties A single Janus capsule may approach a cell from various orientations (membrane coated, gold coated or both sides at the same time). No obvious capsule uptake was observed when the metal-coated side faced the cell first, which is in line with the observation of others.59 On the contrary when HTMJCs faced the cancer cells with their cell-membrane modified parts they were taken up via endocytosis readily. This is the more surprising, since macrophages (final stage of THP-1 cells) usually engulf cells and not the other way round. A representative endocytosis event is illustrated in Figure 4 by a series of SEM images. There are 3 different statuses after the initial HTMJCs-cell contact during the first 5 hours. Step 1 (Figure 4A), the side coated with cellular membrane attaches to the cancer cell and stays there. The interaction between the cancer cell and THP-1 cell membrane, is therefore considered as a kind of ligand-receptor binding. Such binding strength and persistence was not observed for the PEG modified gold shell. Step 2 (Figure 4B and 4C), the HTMJC is swallowed nearly half by the cancer cell. The cancer cell membrane extends over the side of the cellular membrane to completely engulf it. Step 3 (Figure 4D) complete engulfing of the capsule, Figure 4E is its zoomed-in image. EDX mapping (Figure 4F) confirms the gold shell of the Janus capsule is also embedded in the cancer cell and that the cancer cell finished phagocytosis


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completely. In our study the total time of a complete HTMJC engulfment is not clearly determinable, since all stages are found in same SEM investigation. Comparing the uptake rate of THP-1 cells or HTMJC of different temperatures, one can see a decrease in uptake rate of the cells or HTMJC (see SI Figure S8). This is most likely due to a decreased enzymatic activity and receptor interaction, significantly decreasing eating speed of the HeLa cells. It is pointed out, that temperatures of 4°C for an extended period of time decreased viability of HeLa cells significantly. For this reasons, the experiments were performed at 10°C. Experiments at this temperature showed for 3 hours incubation time a decreased amount of phygoytosed capsules compared to 37°C. After 5 hours at 10°C a significant portion of HeLa cells rounded up and showed decreased viability (see SI Figure S 8). For this reason particle and capsule uptake dynamics proves to be difficult to estimate.

Figure 4. SEM micrographs of HTMJCs phagocytosed by HeLa cells. (A) THP-1 cell membrane modified polyelectrolyte side attached to HeLa cell; (B-C) intermediate state; (D) complete


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phagocytosis, scale bar = 10 µm; (E) is the zoomed-in image of (D); (F) EDX mapping analysis the gold shell of HTMJC (at position (E)) which is engulfed in HeLa cell, scale bar = 2 µm.

CLSM images were used additionally to illustrate phagocytosis stages of bio-functionalized HTMJCs. DID was chosen in this investigation as a type of lipophilic fluorescent dye for labeling membranes and other hydrophobic structures. This dye is usually used in vital cell staining. Once applied to cells, this dye diffuses laterally within the cellular plasma membranes. To confirm that the asymmetric membrane extension formation is induced by the Janus geometry, Janus capsules were prepared utilizing cell membrane without DID stain. The HeLa cells were


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Figure5. 3D CLSM images of HTMJCs being swallowed by a HeLa cell. (A) FITC channel; (B) DID channel; (C) overlay of FITC and DID channel; (D) cross section scans with HTMJC attached with membrane part on top of cell being swallowed (green pixels are the PEM part, the yellow line is due to overlay of PEM and THP-1 labeled membrane, the gold cap is not visible and partly blocking the cell and PEM fluorescence); (E) HTMJC completely phagocytized completely by HeLa cell, the direction of the green PEM and cellular membrane part which points to the Petri dish side and shows the cell entry direction from top to bottom, starting with THP-1 coated side. The inner part of the capsule looks red, due to cell membrane above the gold cap (which can therefore not be shielded) scattering into the detector. Scale bar for A-D= 50µm, for E= 25µm.

stained and incubated with HTMJC for 5 hours. The two stages of HTMJC in HeLa cells can be seen in Figure 5 D-E. FITC channel in Figure 5A is used to trace the Janus capsules. A sectional axonometric display of HeLa cells (Figure 5D) displays the phagocytosis intermediate state of a half swallowed HTMJC. A completely swallowed HTMJC located in the endosome or lysosome of a cancer cell is shown in Figure 5E. The red color stems from the HeLa cell membrane that extends over the membrane coated side of the HTMJC, which is in agreement with previous reports59 on the endocytosis pathway of Janus micro-capsules. In contrast to HTMJCs, homogeneous particles, capsules and cells cannot be utilized to determine the entrance direction as Figure 6 shows. It was however found, that the final capsule storage position within cells as well as their entry efficiency is similar to HTMJCs.


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Figure 6. Cell entry properties of homogeneous leucocyte cell membrane coated capsules. (A) FITC and DID overlay image, (B) bright field image, (C) axiometric image of HeLA cell phygocytosing a homogeneous capsule. (D) axiometric image of a HeLa cell with a completely phagocytosed capsule. (A)-(C) show same cell, while (D) displays a different cell. Scale bar= 25µm.

4. Conclusion Leucocyte cell membranes like those of THP-1 monocyte leukemia cell lines can be selfassembled onto PEM films based on electrostatic forces. Bio-functionalized Janus capsules are able to discriminate three cancer cell lines from a non-cancerous cell lines like HUVEC. This is


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in contrast to pristine Janus capsules which are not as sensitive as biofunctionalized ones. Unlike capsules that are uniformly functionalized, micrometer-sized Janus capsules functionalized with THP-1 cell membrane can enter HeLa cells in different steps: first: they adhere to the surface of cancer cells with the membrane coated hemisphere. Followed by a second step, which is described by rapid penetration into the cell, and third, the membrane absent hemisphere is phagocytozed, completing the capsule engulfment. The presented biofunctionalized hollow Janus capsules open interesting possibilities for target recognition based drug delivery vehicles. Using cell membrane based receptors for detecting cancer cells, allows for enhanced drug delivery efficiency. This is the more important since cancer cells make up only ~10 % of tumor tissue62. Sparing the non-cancerous ~90 % of the tumor can therefore enhance recovery speed compared to surgery or large area treatments.

ASSOCIATED CONTENT The associated content contains absorption spectra of the gold cap, a video of thermophoretic motion of HTMJCs, example micrographs of THP-1 cells attached to different cell lines, TEM images of the capsules, QCM data of PEM-HS-PEG interaction and a comparison between Janus gold caps exposed to THP-1 cellular membrane with and without HS-PEG exposure, MTT test data, a scheme of the HTMJC buildup, as well as the relationship of Janus capsules to cells and example images of cells exposed to HTMJCs at 10°C are displayed in the supporting information.


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AUTHOR INFORMATION Corresponding Author *Johannes Frueh, Qiang He. Email: [email protected]; [email protected]. Present Addresses † Micro/Nanotechnology Research Center, Harbin Institute of Technology, Yikuang Street 2 B1, Harbin 150080, China E-mail: [email protected]; [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT The authors thank the National Nature Science Foundation of China (21573053, 21503058) as well as HIT for the startup grant of J.F.

ABBREVIATIONS PEI= polyethylenimine, HTMJ= Human THP-1 membrane modified Janus capsule, PAH= polyallylamine hydrochloride, PSS= polystyrene sulfonate, FITC= fluorescein isothiocyanate, HS-PEG= Methoxy-omega-mercapto-polyethylene glycol, DID= [1, 1-Dioctadecyl-3,3, 3, 3tetramethylindodicarbocyanineperchlorate], PBS= phosphate buffered saline, CSLM= confocal scanning laser microscopy.


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