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Liver Tumor Spheroid Reconstitution for Testing Mitochondrial Targeted Magnetic Hyperthermia Treatment Xuqi Peng, Bingquan Wang, Yu Yang, Yihan Zhang, Yonggang Liu, Yuan He, Ce Zhang, and Haiming Fan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01630 • Publication Date (Web): 30 Jan 2019 Downloaded from http://pubs.acs.org on February 2, 2019

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ACS Biomaterials Science & Engineering

Liver Tumor Spheroid Reconstitution for Testing Mitochondrial Targeted Magnetic Hyperthermia Treatment Xuqi Peng1,2¶, Bingquan Wang3¶, Yu Yang4, Yihan Zhang1, Yonggang Liu5, Yuan He1, Ce Zhang3* and Haiming Fan1,2*. 1

Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of the Ministry of

Education, College of Chemistry and Materials Science, Northwest University, Xuefu Street No. 1, Xi’an, 710127, China 2

School of Chemical Engineering, Northwest University, Xuefu Street No. 1, Xi’an, 710069,

China 3

State Key Laboratory of Cultivation Base for Photoelectric Technology and Functional Materials,

Laboratory of Optoelectronic Technology of Shaanxi Province, National Center for International Research of Photoelectric Technology & Nanofunctional Materials and Application, Institute of Photonics and Photon-Technology, Northwest University, Xuefu Street No. 1, Xi’an 710127, China 4

College of Life Science, Northwest University, Xuefu Street No. 1, Xi’an, 710069, China

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Laboratory of Stem Cells and Tissue Engineering, College of Basic Medicine, Chongqing

Medical University, Medical School Road NO. 1, Chongqing 400016, China Corresponding Author *E-mail: [email protected]（CZ） *E-mail: [email protected]（HMF） Author Contributions ¶

These authors contributed equally to this work.

ABSTRACT. Mitochondria-targeting nanotherapy receive great attention these days for its capacity in disrupting mitochondria function and inducing tumor cell apoptosis through external magnetic and optical stimulations. However, the effect is significantly diminished when applying to animal models. The key factors include environmental complexity in-vivo and intrinsic protective features of tumor tissues. To address these obstacles and reduce expenses on drug screening, we herein introduce a methodology for producing millimeter-sized spheroids with structural and functional characteristics of tumor tissues in-vivo. The necessity of spheroid as liver tumor model is demonstrated by comparing the effect of TPP-SPIONs (triphenylphosphonium cation-superparamagnetic iron oxide nanoparticles) on monolayer-cultured HepG2 cells and spheroids. Our study reveals that large scale spheroid, in contrast to monolayer cells, reflects more in-vivo tumor characters and is less responsive to TPP-SPIONs during magnetic hyperthermia treatment.


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KEYWORDS: Superparamagnetic iron oxide nanoparticles; Magnetic hyperthermia; 3D; Spheroid; Droplet; Pipette tip.

INTRODUCTION Magnetic hyperthermia distinguishes itself by its compatibility with Magnetic Resonance Imaging (MRI), application as heat mediator and controllability through magnetic field guidance.1, 2

For example, superparamagnetic iron oxide nanoparticles (SPIONs) have been used as a

ultrasensitive MR nanoprobe for in vivo high-resolution MRI,3 and demonstrate that multifunctional SPIONs as a theranostic nanoplatform for precise diagnostics guided cancer treatment.4-7 When exposed to alternative magnetic field (AMF), mitochondria-targeting SPIONs can generate heat through oscillation of their magnetic moment and induce mitochondria dysfunction effectively.8-10 As promising as it is, little has been reported regarding mitochondriatargeting magnetic hyperthermia treatment in animal models and clinical trials. A viable in-vitro tumor model for nanoparticle-mediator and therapy screening tests may be the missing key, linking in-vitro and in-vivo cellular conditions. In place for conventional 2D cell culture in-vitro, spheroids maintained in a 3D environment better simulates tissue-specific functions and behaviors in vivo (e.g. enzymatic actives).11-13 The established 3D organotypic human cancer models can thus contribute to pre-clinical research delineating the underlying mechanism of tumor progression, and drug screening tests for personalized therapies.14-18 Over the years, techniques focusing on 3D spheroid models establishment for biomedical researches has been developed.19-22 Either by providing biochemical cues which modulate signaling pathways (e.g. EGF, HGF and inhibitors), or providing suitable physical support like Matrigel,23 spheroids capable of self-renewal, self-organization and exhibit


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organ functionality can be produced.24,25 Techniques based on droplets receive great attention because of its fluid-air interface and sphere shape.26-28 In 2014, a ‘body-on-a-chip’ microfluidic platform based on hanging-droplet technology is presented.29 Microtissue sized a few hundreds of micrometers are successfully cultured and stimulated. To date, spheroids have been reported for a range of tissues: retina, kidney, intestine, stomach, lung, brain, liver, to name just a few, and from primary tumors, either as models for tumor biology or as a more realistic system for in vitro drug screens.30-35 The tedium of the current 3D spheroid techniques is, however, mainly caused by the complex operation procedure, high cost and often the uncontrollable spheroid morphology. To address these limitations, we herein introduce a method for producing structurally and morphologically well-defined spheroids. Through coculturing 3T3 fibroblasts and HepG2 cells in droplets hanging from pipette tiptop, spheroids with controllable size (μm to mm), comparable morphological characteristics and self-organized construct, can be produced at ease. We then examined the effect of TPP-SPIONs (mitochondrial-guiding agent triarylphosphonium) on liver tumor HepG2 cell viability in the form of monolayer and spheroid. TPP is one major class of mitochondrial-targeting molecules. Hyeon et.al. demonstrated that FITC-TPP-iron oxide NPs with core size of 3 nm and 10 nm are localized to mitochondria while 18 nm TPP-iron oxide NPs and iron oxide NPs without TPP conjugation regardless of the core size remain mostly in cytoplasm in SH-SY5Y and HeLa cells.9 Association of TPP to SPIONs will help magnetic hyperthermia target at subcellular organelles. Studies reveal that drugs and therapies targeting at subcellular organelles (cytosol, nucleus and mitochondria etc.) are more effective in tumor treatment as compared to cell-specific treatment.3639

Mitochondrion is one of the most vital subcellular organelle modulating energy production via

oxidative phosphorylation. Responding to heat shock, numerous reactive oxygen species (ROS)


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are produced, which cause apoptotic cell death. Mitochondria targeted nanoparticles and small molecule fluorophores, which are responsive to optical stimulation (e.g. near-infrared) and AMF, are identified as efficient oncotherapy candidates.40-42 In this study, we demonstrate that TPPSPIONs magnetic hyperthermia is more efficient in reducing monolayer cultured tumor cells (HepG2) viability in contrast to cocultured stromal cells (fibroblasts). Furthermore, we employ liver tumor spheroid as disease model and find that even though TPP-SPIONs surpasses SPIONs in killing single tumor cells, their capacity is not significantly different when dealing with spheroids. Similar to tumor tissues in-vivo, efficiency of TPP-SPIONs in magnetic hyperthermia treatment is inhibited by ECM (extracellular matrix) as a barrier.43

MATERIALS AND METHODS Spheroid formation in pipette tips Size and shape of the droplet is regulated by adjusting loading solution volume, and diameter of the cutoff cross-section (Figure 1 and Figure S9). As demonstrated, solution containing 3T3 fibroblasts and HepG2 cells is loaded into the truncated tip. Hanging from the tiptop, droplet balances itself between gravitational force and water surface tension, and the contact angle maximizes at 30 o regardless of the truncated position. For life cell imaging, p65-/- 3T3 mouse fibroblasts containing H2B-GFP nuclear marker have been used.44 HepG2 cell with no fluorescence marker are cultured using protocols described before.3 Liver tumor spheroids are formed by mixing 3T3 fibroblasts and HepG2 cells, and maintained in droplets as described above. After ~ 3 days, spheroids are transferred to well plates for magnetic hyperthermia treatment (MHT) and cell viability assays. Seeding cell densities range


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from 1 × 104 to 1 × 105 cells/ml for 3T3 fibroblasts, and constant 1 ×106 cells/ml for HepG2 to obtain different spheroid structures

Liver tumor spheroid viability and Fe uptake assessment Liver tumor spheroid viability is assessed through continuous live cell imaging (morphology), and acridine orange/ethidium bromide (AO/EB) assay. AO/EB staining is used to visualize nuclear changes and apoptotic body formation that are characteristic of apoptosis. Viability of monolayer cells are assessed by mixing cells (plated at 0.5 × 106 to 2.0 × 106 cells/ml density) and AO/EB solution (1/25 of the seeding solution), followed by immediate fluorescence imaging. Differently, spheroids are firstly dissociated using trypsin, and then transferred to 96-well plate for cell counting, EB/AO staining and trypan blue cell viability test. To measure NPs (Fe3O4) uptake, spheroids are firstly dissociated using trypsin following standard protocol, and then placed into 96-well plate for manual cell counting. In the form of monolayer, HepG2 cells and 3T3 fibroblasts are digested. The supernatant is then collected for inductively coupled plasma−atomic emission spectroscopy (ICP-AES) measurement. Fe content is quantified and normalized to the overall cell number to obtain averaged uptake of NPs (Fe3O4) per cell.

Synthesis of Superparamagnetic Iron Oxide Nanoparticles (SPIONs) Superparamagnetic iron oxide nanoparticles were synthesized following procedure described before.45 Typically, a mixture of 0.178 g FeO(OH) fine powder (2.00 mM), 1.69 g oleic acid (6.00 mM) and 5.00 g 1-octadecene was heated under stirring to 320 ºC and maintained for 30 min under a constant argon flow. Subsequently, cooling of the reaction mixture to room temperature naturally, and 20 ml of ethanol was added to the solution. Separation of the precipitated


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nanoparticles was carried out by centrifugation, and washed by repeated dispersion and precipitation using hexane and ethanol, respectively. The final product was dispersed completely in hexane or tetrahydrofuran (THF) for further use.

Phase transfer of hydrophobic SPIONs Monodispersed hydrophobic SPIONs were transfer to an aqueous phase via ligand exchange for biomedical applications.46 In detail, 50 mg of 3, 4-dihydroxyhydrocinnamic acid (DHCA) was dissolved in 6 ml of THF in a three-neck flask (25 ml). The resulting solution was heated to 50 ºC under argon flow. Then, 20 mg of hydrophobic SPIONs dispersed in 1 ml of THF were added dropwise. After 3 hours, the reaction was cooled to room temperature, and 500 μl NaOH (0.5 M) was introduced to the solution to precipitate the SPIONs. The precipitate was collected by centrifugation (3000 rpm) and re-dispersed in 2 ml water.

Synthesis of the TPP-SPIONs The TPP-SPIONs was obtained from SPIONs by reaction with (PPh3+(CH2)2NH2)Cl- in aqueous solution through amidation reaction (Figure S5). Briefly, SPIONs (100 μL, 0.1 mmol) and EDCꞏHCl (0.1 mmol) were dissolved in 1 mL PBS buffer (pH=7.4) and stirred under a nitrogen atmosphere at room temperature for 15 mins. Then, NHS (0.12 mmol) and (PPh3+(CH2)2NH2)Clwere added for further reaction of 2 h. The precipitate was collected by centrifugation (8000 rpm/min). Washed with deionized water for three times before the TPP-SPIONs were finally redispersed in water.

Characterization of SPIONs and TPP-SPIONs Transmitted electron microscopy (TEM) images of the SPIONs were taken on a JEOL 100CX transmission electron microscope operating at 200 kV (293 K) using an accelerating voltage of


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100 kV (Figure S4 and S5). The crystal phase of SPIONs was verified by X-ray diffraction (XRD) patterns which were recorded on a powder diffractometer (Bruker D8 Advanced Diffractometer System) with a Cu Kα(1.5418 Å) source. The Scherrer equation was utilized to determine the mean particle size of SPIONs. Magnetic properties were determined using a vibrating sample magnetometer (VSM) operating at room temperature. Fe concentrations of hyperthermia samples was determined by inductively coupled plasma (ICP) analysis using a Perkin-Elmer Dualview Optima 5300 DV ICP-OES system. The hydrodynamic diameter of SPIONs was determined using Malvern Zetasizer Nano-ZS. Samples were equilibrated at 37 ºC for 2 min before measurement and 5 sets of measurements were taken. Fourier-transformed infrared spectroscopy (FT-IR) of SPIONs were recorded on a Varian 3100 FT-IR (Excalibur series) instrument. About 2 mg of SPIONs were added to the FT-IR stage and scanned from 4000 cm−1 to 400 cm−1. X-ray photoelectron spectroscopy (XPS) measurements were performed on a VG ESCALAB 210 XPS system with Mg Kα (1253.6 eV) source. Heating capacities of TPP-SPIONs under AMF are characterized as well (Figure S6)

Magnetic hyperthermia treatment (MHT) To investigate MHT effect on monolayer cells, 200 l of HepG2 cells at a density 1×106 viable cells/ml were cultured and then stabilized in 6-well plates for 24 h. After 24 h incubation, the medium was then replaced by fresh culture medium containing SPIONs (50 g/ml) with designated Fe concentrations. Cells were incubated with SPIONs for 24 h. The excessive SPIONs were removed by repeated washing with PBS buffer and the cells were detached and collected into a plastic tube for MHT. The AMF is maintained at 300 kHz and 30 A for 10 min. The processed cells were again seeded in 96 well plates containing fresh medium for 24 h before live cell fluorescence imaging and cell viability assay. As for spheroid MHT, liver tumor spheroid was


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transferred from droplet to 35 mm culture dish with 1 ml DMEM medium containing SPIONs (50 μg/ml). After 24 h of co-incubation, excessive SPIONs were removed by repeated centrifuge (200 rpm) and washing with PBS buffer. Spheroid was then exposed to AMF at 300 kHz frequency before imaging and cell viability assay.

RESULT AND DISCUSSION Hanging droplet formation from pipette tip Different from hanging droplet technologies relying on micro-machinery, the strategy we proposed utilizes only commonplace pipette tips (Figure 1).29 Hydrophobic properties of pipette tip prevent liquid from spreading. When solution is applied through the tip opening, it is drawn to the other end by gravitational force. The shape and size of the droplets are determined by two factors: the diameter of the tip end opening (D); and contact angle () by varying loading solution volume (Figure 1B). Droplets of various morphologies were produced in this study. Our results reveal that spheroid formation is optimized at a contact angel of ~30°. At liquid–air interface, undesired cell adhesion is prevented. 3D spheroid conformation can thus be preserved. The open culture system allows for optimal gas exchange (for example, oxygen and CO2), and nutrient delivery. After culture and stimulation, spheroids and supernatant can be harvested through simple pipetting.


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Figure 1. An in vitro strategy for constructing 3D liver spheroid. (A) Schematic for the work flow of constructing hepatic spheroid in a droplet hanging from the top of a pipette tip. The steps include, 1. Cut a 200 l tip at positions 5 mm to 11 mm away from the tiptop; 2. Sectional cutoff surface is polished for better droplet formation; 3. Solution containing 3T3 fibroblasts and HepG2 cells is loaded into the tip; 4. Incubation of aggregates composed by 3T3 and HepG2 cells within the droplet, which leads to 3D liver spheroid formation in-vitro. (B) Optical image of a droplet hanging from a pipette tiptop. The contact angle () depends on the loaded solution volume: (1) 80 l; and (2) 50 l, and diameter of the cutoff region 𝐷′ . (C) Contact angle () of the hanging droplet as a function of solution volume, which is loaded into the pipette. Different color coding denotes different cutoff positions along the tip.

Spheroid formation in droplet The initial cell loading is performed through pipetting, which is no more difficult than operating in a 200 μl centrifugal tube. A defined solution volume with designated cell density determines the droplet shape, and thus the spheroid morphology (Figures S1 and S2). We observe that shape of the spheroid depends mostly on the shape of the hanging droplet (contact angle ) (Figure S1A), which is close to sphere when the contact angle is ~30° or larger, and irregular when the liquid– air interface is flat. The self-organization process of spheroids has been extensively reported before.47,

48

In a

Transwell system, Mingxing Lei, etc. demonstrated that cells aggregate and form multiple ‘islands’


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on a flat surface, which may later progress into individual spheroid. While, in commercialized plate utilizing hanging drop technique, single spheroid with comparable characteristics in terms of size, longevity, and functional tissue characteristics can be produced separately in each well. Even though the underlying mechanism of spheroid formation remain unclear, it is reasonable to conclude that surface geometry plays important role in determining spheroid morphology. It is worth noting that single spheroids are produced in each hanging droplet regardless of the bottom surface geometry (Figure S1). Possibly, the lack of ECM molecules (e.g. fibronectin) at liquid-air interface inhibits cell attachment, together with gravitational force promotes cell aggregation into single entities. Contribution of gravitational force is determined by the shape of the hanging droplet (contact angle: ). At larger angles, cells are ‘pulling’ more towards the center. As a result, round-shaped spheroids can be formed (Figure S1A, Figure 3 and 4). When the air-liquid interface is relatively flat, shape of the produced spheroids becomes increasingly irregular, but carrying the same structural characteristics. Our hypothesis is confirmed by experimenting on a super-hydrophobic surface. 3T3 fibroblasts and HepG2 cells start to aggregate after 2 h (Figure S3). Hint of cell spreading after 20 h suggests secretion of cytokines (e.g. fibronectin), which promote cell adhesion.49, 50 No spheroid is observed after incubation for up to 28 h. The rod-like shape of spheroids formed on surface resembles those formed in droplets with small contact angles. The lack of driven force for cell accumulation and aggregation at one single site may be the main cause. Mature spheroid is transferred to well-plate for continuous culture, simulation and imaging through a simple ’push’ using pipette. 3T3 fibroblasts employed in this study carry H2B-GFP marker, which allows them to be easily distinguished from non-fluorescent HepG2 cells. 3T3 fibroblasts are observable in both bright field and fluorescence imaging, and HepG2 only with


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bright field. Taking advantage of the distinction, we reveal that spheroids formed in hanging droplets share a layered structure with 3T3 fibroblasts forming ECM shell (green fluorescence) and HepG2 (dark) at core (Figure 2). The hypothesis is further verified using Col-F Collagen Binding Reagent to verify the existence of ECM elements. Our results demonstrate that surface of HepG2/3T3 spheroids is covered with ring-like structures with diameter ranging from 10 to 20 m after 1 hour staining (Figure S14). These ‘rings’ colocalize with fibroblasts occupying the spheroid marginal region, suggesting the existence of collagen in between fibroblasts forming ECM (Figure S15).

Moreover,

HepG2

cells

stained

with

CMAC

live

cell

tracer

(7-amino-4-

chloromethylcoumarin) locate in the center (blue), and 3T3 fibroblasts with H2B-GFP located in the outer layer (green) (Figure 3).51 Bright field and fluorescence imaging show that spheroid gradually disassembles during 7-days’ culture in well plate (Figures 2D and E). Clear separation boundary between different participating cell types is observed, which coincide with our proposed layered spheroid structure.


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Figure 2. The structural character of the liver spheroid formed through 3T3 fibroblasts and HepG2 seeding in the droplet. (A) Bright-field images showing 3D conformation of liver spheroids after 3 days incubation in droplet. (B) Schematic drawing demonstrates that 3T3 fibroblasts and HepG2 cells self-organize into layered structures with HepG2 cells concentrating at the core region. The finding is revealed by automated Z-stack imaging on an inverted microscope, from layer 1 to 4 corresponding to different z-axis position from 0 to 120 m. (C) Fluorescence images of liver spheroid at different layers (z-axis position) show that the outer layer of the 3D liver spheroid is composed mostly by 3T3 fibroblasts with H2B-GFP fluorescence marker. (D) Bright-field, and (E) fluorescence image of mature liver spheroid, which was cultured in droplet for 3 days and then transported to well plate for 7-days’ continuous culture. In Figure 2D, images in the right panel are magnified view of Area 1 and Area 2, showing regional distribution of 3T3 fibroblasts and HepG2 cells. White arrows in Figure 2E identify 3T3 fibroblasts with H2B-GFP fluorescence marker. Scale bars denote 500 μm in all images.


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Viability of the spheroid is assessed using Acridine Orange/Ethidium Bromide (AO/EB) assay. Spheroid with a diameter of ~ 1 mm is obtained by co-culture HepG2 cell (1 x106 cell/ml) and 3T3 fibroblasts (1 x105 cell/ml) in a hanging droplet for 3 days. The pipette tip housing liver tumor spheroid is refilled with fresh cell culture medium daily to compensate for the solution volume loss due to evaporation. With contact angel of ~30° , droplet is stable during operation. The spheroid is then transferred to 96-well plate for EB staining and imaging (Figure 4). To exclude disturbance of 3T3-H2B-GFP signal, AO staining targeting at live cells is not used. Consistently, a densely packed cell ring (ECM) is observed at different depth of the spheroid in GFP channel. In contrast, signal from EB is weak at all layers possibly due to high cell viability and limited EB penetration into ECM layer (Figure S11). The presence of ECM is further verified by 3T3 fibroblast migration when the spheroid is transferred from droplet to well plate. After 8 h culture in plate, spheroid started to disintegrate with 3T3 fibroblast migrating hundreds of micrometers (Figure S10). Without the pre-existence of secretases like fibronectin in the spheroid shell, it will take long time for 3T3 fibroblasts to adhere, spread and migrate. It is worth noting that variations in well-plate incubation time before further process alter neither the round-shaped morphology, nor the compact edge conformation.


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Figure 3. The structural character of the liver spheroid formed by coincubation 3T3 fibroblasts carrying H2B-GFP markers and HepG2 cells stained with CMAC (7-amino-4chloromethylcoumarin). (A) Bright-field and fluorescent images of monolayer cultured HepG2 cells stained with CMAC. (B) Fluorescence image of mature liver spheroid, which was formed by seeding H2B-GFP 3T3 and CMAC HepG2 cells in droplet for 3 days. Consistent with the layered spheroid structure shown in Figure 2, HepG2 cells stained with CMAC stay in the center. While, 3T3 cells with H2B-GFP marker are observed mostly in the peripherical region. Scale bars denote 200 μm in all images.


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Figure 4. 3T3 fibroblasts and HepG2 cells viability assessed by Ethidium Bromide assay in spheroid. After 3 days incubation in droplet, spheroid was collected and transferred to 96-well plate for Z-stack imaging and viability assessment. To avoid confusion with H2B-GFP signal, Acridine Orange is not used. In such way, all cell types in spheroid can be distinguished: live HepG2 cells (no fluorescence), dead HepG2 cells (EB fluorescence), live 3T3 fibroblasts (GFP fluorescence) and dead 3T3 (overlapping EB and GFP fluorescence). Scale bars in all images denote 200 m.

Magnetic Hyperthermia Treatment on Monolayer Cells The effect of TPP-SPIONs is firstly examined on monolayer HepG2 cells (detailed description of TPP-SPIONs can be found in supplementary information). With no observable cytotoxicity, the


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uptake of SPIONs with and without TPP coating increase significantly after incubation for 4 h, and saturate at ~ 17 pg/cell after 12 h (Figure 5B). No further increase is observed with prolonged incubation for up to 24 h, suggesting TPP coating bring no effect on trafficking SPIONs through cell membrane. With MHT, TPP-SPIONs induces approximately 76% HepG2 cell death, and ~ 26% without TPP coating (Figure 5C). The enhanced cytotoxicity is consistent with previous reports on photothermal therapy (PTT).52 The internalization and distribution of SPIONs with and without TPP coating within the cell membrane are examined using TEM (Figures 4D and E). After 12h incubation, SPIONs without TPP coating are found mostly in endosomes or lysosome. While, TPPSPIONs enter mitochondria, and maintain a spherical shape.

Figure 5. (A) Cell viability of SPIONs and TPP-SPIONs co-incubated with HepG2 cells using standard MTS colorimetric assays. (B) Time dependent uptake amount of SPIONs and TPPSPIONs. (C) Dose dependent of cell viability for HepG2 cells subjected to magnetic hyperthermia with SPIONs and TPP-SPIONs. Bio-TEM images of HepG2 cells incubated with SPIONs and TPP-SPIONs at the concentration of 50 μg/mL for 12 h. The arrows (D) in the images indicate that nanoparticles accumulated in the endo/lysosome, whereas the ones (E) show the distribution of mito-targeted nanoparticles in the mitochondria.


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The capacity of TPP-SPIONs in inducing HepG2 cell apoptosis is further demonstrated using Acridine Orange/Ethidium Bromide (AO/EB) assay (Figure 6). The combination of AMF and TPP-SPIONs induces the most significant tumor cell death. Lack of EB signal in conditions excluding either AMF or TPP coating shows the potential of TPP-SPIONs as drug free therapy for on-site tumor treatment.

Figure 6. Fluorescence images of HepG2 cells treat with (A) culture medium as control; (B) SPIONs; (C) TPP-SPIONs; (D) Alternating magnetic field (AMF); (E) SPIONs under AMF; (F) TPP-SPIONs under AMF. The concentration of SPIONs and TPP-SPIONs were 100 μg/ml. Untreated group is the blank control. The cell were stained with acridine orange/ethidium bromide for 5 min, the green color and the red color refer to live and apoptotic cells, respectively.

Magnetic Hyperthermia Treatment on Liver tumor spheroid In the next step, we examined MHT effect of TPP-SPIONs on liver tumor spheroid produced in hanging droplet to validate the strategy’s suitability for pre-clinical drug and therapy test (Figure 7 and Figure S12). We find that the efficiency of SPIONs with and without TPP coating depends on liver tumor spheroid structure, and shows no clear difference in penetrating spheroid ECM. To


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exclude the effect of 3T3 fibroblasts secretases, HepG2 cells and 3T3 fibroblasts are cultured as monolayer and exposed to MHT (Figure S7). It is revealed that HepG2 cells incubated with 3T3 fibroblasts take approximately the same amount of Fe as HepG2 monolayer cells alone. With no ECM, monolayer HepG2 cells are not protected against MHT at all fibroblast concentrations (Figure S7B and S7C). In contrast, TPP-SPIONs at concentration of 50 μg/ml does not induce significant 3T3 fibroblasts cell death after MHT at 30 A for 10 min (Figure S7A). Clearly, structural characters (i.e. ECM) of spheroid play important role in drug screening experiments. Employing a HepG2/3T3 ratio of 100/1, cell aggregates formed after 3 days in hanging droplet are loose in morphology, irregular in shape, and the height is as low as 1 to 2 cell layers, suggesting HegG2 cells are not capable of self-organizing into spheroid (Figure 7A). Consistently, no hint of 3T3 fibroblasts forming shell is observed. After MHT and AO/EB assay, green fluorescent signal (H2B-GFP and AO) dominates and uniformly distributes within the cell aggregates, suggesting majority of 3T3 fibroblasts are live. EB fluorescent signals (red), which decorates the cell aggregates, reflects that TPP-SPIONs can kill cells regardless of their positions. Our hypothesis is that spheroid can not be formed with inadequate supportive ECM material (e.g. fibroblasts, fibronectin and laminin), and thus HepG2 cells are not protected against small molecules and particles. While, with high 3T3 fibroblast content, spheroids are formed and share a round-shaped, more compact morphology (Figure 7B). Height of the spheroids are considerably higher (a few hundreds of micrometers) as compared to those with low 3T3 density, suggesting self-assembly into 3D structures. Lack of fluorescence in the central region suggests small molecules like AO and EB can not penetrate into the spheroid core. Applying TPP-SPIONs with MHT leads to reduced cell viability (~80%), but not as significant as monolayer cultured cells. As cell viability is assessed by


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firstly dissociating spheroids into monolayer cells after being exposed to 300 kHz AMF for 10 min, we are able to identify most dead cells as HepG2 cells, which lack of H2B-GFP signal. Even though EB staining on spheroid does not represent all dead cells, its distribution in peripherical region beneath the fibroblasts does show that SPIONs’ diffusion and penetration driven by high frequency AMF is inhibited by spheroid ECM shell. Considering the ~80% of HepG2 cells survives after MHT as compared to ~94% survivable rate without MHT, it is reasonable to conclude that most HepG2 cells in the core region are protected against TPP-SPIONs (Figure 7D and S13). While, spheroids formed by HL-7702 human hepatocytes and 3T3 fibroblasts share similar layered structures as the liver tumor spheroids originating from HepG2 cells (Figure S16). Even though the number of 3T3 fibroblasts is considerably less, no significant cell death is observed after treatment with TPP-SPIONs (Figure S13). One of the plausible explanations is that the effect exerted by TPP-SPIONs is more pronounced in tumor cells (HepG2) than normal tissue (HL-7702). 53,54 The uptake of TPP-SPIONs is assessed by digesting liver tumor spheroid and measure the Fe content. Consistent with our hypothesis that the invasion of TPP-SPIONs is inhibited by spheroid shell, Fe uptake amount is the highest in monolayer HepG2 cell (~ 17 pg/cell), and lowest in the form of spheroid (Figure 7C). The similarity between spheroid -100 (low 3T3 ratio, 1:100) and monolayer culture cells (3T3 ratio, 1:10) suggests that NPs can be in contact with most cells in both cases. In contrast, cells localized at the center of spheroid (3T3 ratio, 1:10) are prevented from contact with NPs by the ‘shell’, which is responsible for the significantly dropped Fe uptake amount (yellow bar in Figure 5C). Prolonged incubation in well-plate up to 24 h does not affect overall cell viability (Figure S13). Assuming each cell take the same amount of NPs, the number of cells in contact with NPs in a mature spheroid can be approximately 40% of monolayer cultured


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cells. Overall cell viability within the liver tumor spheroid after 10 min MHT is inversely related with the Fe uptake amount (Figure 7D). The efficiency of TPP-SPIONs during MHT is assessed by normalizing cell viability values to Fe uptake amount (Figure 7E). We find that TPP-SPIONs induced cell apoptosis is most significant when HepG2 cells are maintained as monolayer, and least effective when dealing with liver tumor spheroids.

Figure 7. Viability of 3T3 fibroblasts and HepG2 cells assessed by Acridine Orange/Ethidium Bromide (AO/EB) assay, and its correlation with liver spheroid composition. (A, B) Bright-field and fluorescence images of liver spheroid after AO/EB staining. Density of HepG2 cells are maintained at 1 10 in all experiments, and the ratio between HepG2 cells and 3T3 fibroblasts are 100 (Spheroid-100) (A), 10 (Spheroid-10) (B), respectively. (C) Uptake of TPP-SPIONs by HepG2 cells incubated with 3T3 fibroblasts as monolayer (3T3:HepG2 = 1: 10) or spheroid. Cells are incubated with TPP-SPIONs for 12 h before digestion for ICP-AES measurements. (D) HepG2 cells and 3T3 fibroblasts viability after AMF treatment (300 KHz, 30 A) for 10 min. In this experiment, all spheroids are incubated with TPP-SPIONs for 24 h and treated with AMF (300 kHz, 30 A) for 10 min. Spheroids were then collected and dissociated using trypsin. Dissociated HepG2 and 3T3 were transferred to 96-well plate for trypan blue cell viability test. (E) Cell


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viability normalized to TPP-SPIONs uptake quantity. Scale bars in all images are 200 μm.

CONCLUSIONS In this study, we demonstrate the capacity of TPP-SPIONs in inducing tumor cell apoptosis under AMF, and its limitation when being applied to HepG2 spheroid. TPP modification facilitates SPIONs uptake by trafficking them from subcellular organelle endosomes and lysosomes to mitochondria, which enhances the effect of MHT as well. However, SPIONs with and without TPP coating both have trouble penetrating spheroid composed by HepG2 cells and 3T3 fibroblasts. HepG2 cell apoptosis distributes mostly at the outer layer, suggesting a protective mechanism. Our hypothesis is that within 24 h incubation in droplets hanging from pipette tip, participating cells self-organize into a layered structure with 3T3 fibroblasts forming an outer shell and HepG2 cell at core. When the quantity of fibroblasts is high enough to hold the spheroid structure, small molecules (e.g. AO and EB for cell viability staining) and SPIONs (7 nm) both have trouble penetrating through the outer layer, which could be composed by densely packed 3T3 fibroblasts and cellular secretases. Furthermore, to lower the expenses for drug testing before clinical trial, we propose a strategy for producing liver tumor spheroid with controllable size, morphology, and open to medium exchange. First, spheroids morphology can scale from micrometer to millimeter by adjusting the seeding cell density and droplet size (Figure S2). Second, medium exchange is possible by pipetting from the top without affecting spheroid positioned at droplet bottom. Third, and most importantly, spheroid production can now be performed at ease using regular benchtop equipment and standard cell culture protocols. In summary, we demonstrated that previously complex 3D spheroid protocols can be simplified and executed in a droplet hanging from a pipette tip. The


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controllability over spheroid structure (density, organization) and morphology (shape, size) features high usability and multifunctional applications. Our proposed strategy can play an important role in gaining new opportunities for interdisciplinary studies performed in labs, whose lack of both hardware and software for performing complex biological experiments stands in the way.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Figure S1: The sphericity of liver spheroids in association with the curvature of the pendant droplet. Figure S2: Liver spheroid formation in droplet hanging from pipette tip. Figure S3: Liver spheroid formation on top of a super-hydrophobic surface. Figure S4: Characterization of SPIONs. Figure S5: Characterization of TPP-SPIONs. Figure S6: Heating profiles of TPP-SPIONs under AMF. Figure S7: Monolayer culture 3T3 fibroblasts and HepG2 after magnetic hyperthermia treatment (MHT) with TPP-SPIONs and EB staining. Figure S8: Spheroid formed by seeding 3T3 fibroblasts and HepG2 cells in hanging droplet. Figure S9: Detailed operation procedure of producing a hanging droplet. Figure S10: Bright field images of spheroid. Figure S11: Fluorescent images of liver tumor spheroid. Figure S12: Bright-field and fluorescence images of liver spheroid after AO/EB staining. The following files are available free of charge. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]（CZ） *E-mail: [email protected]（HMF） ORCID Ce Zhang: 0000-0003-1284-7279 Haiming Fan: 0000-0002-0091-772X

Author Contributions


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Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Science Foundation of China (81771981, 81571809, 21376192 and 31400663). ABBREVIATIONS SPIONs, Superparamagnetic iron oxide nanoparticles; TPP, Triphenylphosphonium; AMF, Alternating magnetic field; MHT, Magnetic hyperthermia treatment; ROS, Reactive oxygen species; MRI, Magnetic resonance imaging; EGF, Epidermal growth factor; HGF, Hepatocyte growth factor; GFP, Green fluorescent protein; AO, acridine orange; EB, ethidium bromide; Calcein AM, Calcein acetoxymethyl; PI, Propidium iodide; THF, Tetrahydrofuran; DHCA, 3,4dihydroxyhydrocinnamic acid; ECM , Extracellular matrix ;

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Liver Tumor Spheroid Reconstitution for Testing Mitochondrial Targeted Magnetic Hyperthermia Treatment Xuqi Peng, Bingquan Wang, Yu Yang, Yihan Zhang, Yonggang Liu, Yuan He, Ce Zhang and Haiming Fan

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