Biomimetic stem cell membrane-camouflaged iron oxide nanoparticles for theranostic applications

Abstract

Superparamagnetic iron oxide nanoparticles (SPIO NPs) have been used extensively for various biomedical applications, such as magnetic resonance imaging (MRI), drug delivery, cellular tracking and magnetic hyperthermia therapy. Surface modifications of SPIO NPs are usually required to improve their rapid in vivo clearance profiles. In this study, we prepare novel stem cell membrane (STM)-camouflaged SPIO NPs for biomedical applications. The water-soluble and highly dispersed STM-SPIO NPs were prepared using a simple and mild sonication method. The increased size of STM-SPIO NPs compared to unmodified SPIO NPs suggested successful STM coating onto the SPIO NP surfaces. The STM coating was further confirmed by Transmission Electron Microscopy (TEM) imaging and dye retention assay. High preservation of STM-associated proteins on the SPIO NPs was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Specifically, retention of stem cell-specific marker CD44 was confirmed using an antibody binding assay. As a potential MRI agent, STM-SPIO NPs exhibited good magnetization (65.9 emu g⁻¹) and dose-dependent T2-weighted imaging contrasts (R₂ = 653.3 s⁻¹ mM⁻¹) in vitro. In comparison to unmodified SPIO NPs, the macrophage uptake of STM-SPIO NPs was found to be significantly less by Prussian blue staining and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis. Under the application of an alternating magnetic field (AMF), STM-SPIO NPs successfully induced cancer cell death via a magnetic hyperthermia mechanism. The results demonstrated good potential for STM-SPIO NPs for future theranostic applications.

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1. Introduction

In recent decades, inorganic nanoparticles have emerged as a novel drug delivery platform for disease treatment and diagnosis. Nanoparticle surfaces can be tailored to carry various functionalities for loading drugs or genes^1,2 and enhancing the cargo transport to tumor tissues via passive or active targeting mechanisms. In addition, the inherent physical and chemical properties of nanoparticles offer additional theranostic functions. For instance, gold nanoparticles are well known for their capability to generate photothermal and photoacoustic effects in vivo.³ Silver nanoparticles are found to be useful anti-microbial agents.⁴ To design nanoparticle-based drug delivery carriers for in vivo applications, the colloidal stability and blood circulation lifetime of the nanoparticles should be taken into consideration. Taking advantage of a leaky vascular structure and impaired lymphatic drainage in tumors, long circulating nanoparticles can accumulate in tumors via enhanced permeability and retention (EPR) effects.⁵ PEGylation has been widely applied to prepare long-circulating drugs or nanoparticles to passively target tumor sites. PEG molecules can effectively reduce nanoparticle uptake by macrophages in the epithelial reticular system (RES) thus lowering the undesired distribution of nanoparticles in liver, spleen and lung.⁶

Despite their effectiveness for nanoparticle shielding, the elicited immunogenic responses by PEG molecules have raised concerns in recent years.^7,8 Surface modification using cell membranes has emerged as a possible better solution to prepare long-circulating nanoparticles. Cell membranes are mainly composed of a phospholipid bilayer with numerous associated proteins and polysaccharides responsible for various cellular functions such as cell–cell recognition and cell signaling. For example, CD47 on red blood cells (RBC) acts as a marker of self to avoid phagocytic uptake by the macrophages of the immune system.^9,10 Zhang L. et al. utilized a physical extrusion method to successfully prepare mouse RBC-camouflaged PLGA nanoparticles displaying a comparable pharmacokinetics profile to PEGylated PLGA nanoparticles.¹¹ Other research also found that porous silicon nanoparticles coated by a leukocyte membrane could effectively prolong in vivo circulation time, avoid clearance by the immune system and increase nanoparticle accumulation in tumor sites.¹²

SPIO NPs have been approved as a clinical MRI contrast agent. In addition to their molecular imaging application, under a high-frequency AMF, SPIO NPs can rapidly change their magnetic moments whereby the friction caused by this produces heat for hyperthermia therapy applications.¹³ Surface modifications are often required to afford SPIO NPs with long circulating ability for better theranostic efficacy.¹⁴ In this study, we proposed using low immunogenic stem cell membrane as a novel surface modification material to prepare biomimetic membrane-camouflaged SPIO NPs. Mesenchymal stem cells (MSCs) are a class of multipotent cells which are capable of differentiating into various cell lineages such as osteoblasts, chondrocytes, adipocytes and myoblasts.¹⁵ In addition, MSCs are slightly immunogenic and exhibit immunomodulatory activity in vivo.^16,17 As a potential source of cell membranes, MSCs can be harvested from various tissues and expended in large quantity in vitro. So far, using stem cell membrane (STM) for the surface modification of functional nanoparticles has not been reported.

In this study, the STM-camouflaged SPIO NPs were prepared by utilizing a simple sonication method. The prepared STM-SPIO NPs were characterized to find the particle sizes. The STM coating on the SPIO NPs was tested using TEM, fluorescence dye retention assay, total membrane protein analysis and antibody binding assay. The magnetic properties of STM-SPIO NPs were examined using a superconducting quantum interference device (SQUID) and T2 MRI imaging. The effects of STM coating on the prevention of macrophage uptake were examined by Prussian blue staining and ICP-MS methods. The magnetic hyperthermia properties of STM-SPIO NPs were measured and applied for cancer cell treatment.

2. Experimental

2.1 Materials

Magnesium chloride, sucrose, sodium phosphate dibasic, sodium phosphate monobasic, citric acid, Coomassie Brilliant Blue R250 and ammonium hydroxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tris-base and MTT were purchased from MDBio, Inc. (Taipei, Taiwan). Fluorescein isothiocyanate (FITC)-anti-CD44 antibody (103006) and PE-Rat IgG2b/κ Isotype Ctrl Antibody (400608) were purchased from Biolegend (San Diego, CA, USA). Anhydrous iron(II) chloride and iron(III) chloride were purchased from Alfa Aesar (Ward Hill, MA, USA). Collagenase I was purchased from Worthington (Freehold, NJ, USA). Dulbecco’s Modified Eagles Medium (DMEM) and Minimum Essential Medium-alpha (α-MEM) were purchased from Gibco (Carlsbad, CA, USA). Penicillin–streptomycin (P/S) and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT, USA).

2.2 Synthesis of citric acid-capped SPIO NPs (CA-SPIO NPs)

The citrate-coated SPIO NPs were synthesized by a co-precipitation method.¹⁸ Briefly, 324.4 mg of FeCl₃ and 127 mg of FeCl₂ (Fe³⁺ : Fe²⁺ = 2 : 1 of molar ratio) were dissolved in 15 mL of DI H₂O in a three-necked flask under argon and heated up to 75 °C. Then 5 mL of 28% NH₄OH was added into the mixture with vigorous stirring for 30 minutes. Afterwards, 200 mg of citric acid dissolved in 2 mL DI H₂O was slowly introduced into the reactant and kept stirring at 75 °C for another 30 minutes to obtain the SPIO NPs. The as-synthesized SPIO NPs were collected by magnetic attraction and washed with DI H₂O three times to remove NH₄OH and excess citric acid. The purified SPIO NPs were resuspended in 10 mL DI H₂O and the iron concentration was determined by potassium thiocyanate (KSCN) methods.¹⁹

2.3 Quantitation of total iron by KSCN methods

The SPIO NP samples (50 μL) were mixed with 50 μL 12 N HCl and 50 μL 30% ammonium persulfate and incubated at 65 °C for 1 hour to dissociate the SPIO NPs and oxidize ferrous ions to ferric ion. After cooling down to room temperature, the mixture was mixed with 100 μL 5% KSCN and the absorbance at 470 nm was determined. A series of ferric chloride diluted solutions prepared in 12 N HCl were used for standard calibration.

2.4 Isolation and characterization of adipose-derived MSCs

The adipose-derived MSCs were obtained from C57BL/J mice.²⁰ The mice were sacrificed by CO₂ and the adipose tissue was dissected from a subcutaneous site of the lower abdomen. The tissue was cut into pieces and washed by PBS several times until the color of PBS turned transparent. This was then digested with 0.1% collagenase I in PBS for 1 hour at 37 °C. During the digestion, the sample was shaken every 20 minutes. The released cells were centrifuged at 300g for 5 minutes to remove the floating fat. The cell pellet was resuspended with PBS and filtered through a 70 μm cell strainer (BD). The cells were collected by centrifugation at 300g for 5 minutes and then resuspended with α-MEM supplemented with 20% FBS and 1× penicillin and streptomycin. The whole cells were sorted and the CD44 and CD73 positive cells were collected using FACSAria™ III (BD). Animals used in this study were handled in accordance with protocol approved by the Institutional Animal Care and Use Committee at the National Tsing Hua University, Taiwan.​

2.5 Isolation of STM

The STM was isolated according to a published procedure.²¹ In brief, MSCs (1 × 10⁷ cells) were harvested, washed with PBS and resuspended in cold Tris–Magnesium buffer (TM-buffer pH = 7.4). The cells were homogenized at 22 000 rpm for 1 minute before adding TM-buffer/60% sucrose to a final concentration of 5% sucrose. The homogenized cells were collected by centrifugation at 6000g for 15 minutes and then washed twice with 0.15 M sucrose/TM-buffer (pH 7.4). To completely remove the cell organelles and obtain smaller fragments of STM, the collected pellet fraction was subjected to further homogenization by Sonics VCX 750 W probe sonication (Sonics, Newtown, CT, USA; maximum power: 750 W; frequency: 20 kHz; probe diameter: 13 mm) at 27% amplitude of the maximum power for 5 seconds and washed twice as described above. The STM was collected by centrifugation at 6000g for 15 minutes and resuspended in DI H₂O for STM-SPIO NP preparation.

2.6 STM-SPIO NP preparation by sonication

To assemble the STM onto the SPIO NPs, a fixed amount (1 mg) of STM was mixed with the SPIO NPs at various weight ratios (STM : SPIO NPs = 1 : 1, 3 : 1, 5 : 1, 20 : 1) in 4 mL of 10 mM Na₂HPO₄/NaH₂PO₄ buffer. The mixtures were incubated on ice for 30 minutes and then subjected to probe sonication at 27% amplitude for 40 seconds (5 seconds/5 seconds; on/off, 8 times). The as-synthesized STM-SPIO NPs were collected by magnetic attraction, washed twice with DI H₂O and resuspended in DI H₂O for further usage.

2.7 Physicochemical characterizations of the SPIO NPs and STM-SPIO NPs

The particle sizes of the as-synthesized STM-SPIO NPs in DI H₂O were measured using a ZetaSizer Nano Series (Malvern, UK). For the colloidal stability test, STM-SPIO NPs were added to DI H₂O, 25% FBS/DMEM or 50% FBS/DMEM for particle size measurement. The magnetism of SPIO NPs or STM-SPIO NPs were measured using a SQUID at 300 K (Quantum Design MPMS-XL, USA). For TEM imaging (JEOL JEM-1200EX II, Japan), samples were prepared by dropping SPIO NP or STM-SPIO NP solution onto copper TEM grids and were then negatively stained with 1% tungstophosphoric acid before the images were acquired.

2.8 Coverage of STM on SPIO NPs by dye retention assay

To verify the presence of STM on the SPIO NPs, 1 μL or 10 μL of DiO (0.5 mg mL⁻¹ in DMSO) was mixed with 1 mg of STM-SPIO NPs or unmodified SPIO NPs in 4 mL of 10 mM Na₂HPO₄/NaH₂PO₄ buffer and then sonicated for 20 seconds (5 seconds/5 seconds; on/off). The SPIO NP-containing samples were collected by magnetic attraction and washed with DI H₂O twice to remove the unbound DiO. The STM-SPIO NPs or SPIO NPs were resuspended in DI H₂O. The STM-SPIO NPs or SPIO NPs containing 80 μg Fe were analyzed for DiO fluorescence using a fluorescence plate reader (ex: 460 nm/em: 495–600 nm).

2.9 Protein retention by SDS-PAGE

The protein retention of STM on the STM-SPIO NPs was confirmed by SDS-PAGE. STM extracted from 7 × 10⁶ MSCs or STM-SPIO NPs prepared with the same amount of STM was mixed with 6× loading buffer to a total volume of 70 μL and then subjected to a protein denaturation process (heating at 85 °C for 10 minutes). The samples were analyzed by SDS-PAGE (12% polyacrylamide for protein separation). The gel was stained by Coomassie Brilliant Blue for 20 minutes and then destained in 30% methanol/10% acetic acid before gel images were acquired using an optical scanner (TX220, EPSON).

2.10 Antibody binding assay

To further confirm the preservation of stem cell marker CD44 on the STM-SPIO NPs, an antibody binding assay was performed. 5 μg CD44-FITC antibody was mixed with 10 μg or 100 μg STM-SPIO NPs as w/w ratios (CD44-Ab : STM-SPIO NPs) of 1 : 2 or 1 : 20 respectively. 2 μg of isotype-PE control antibody was mixed with 100 μg STM-SPIO NPs and used as a negative control. The mixtures were incubated on ice for 2 hours. The STM-SPIO NPs were collected by magnetic attraction and the supernatant which contained the unbound antibody-FITC was analyzed for fluorescence intensity using a plate reader. (FITC: ex: 490 nm/em: 520 nm; PE: ex: 540 nm/em: 580 nm).

2.11 T2-weighted imaging of STM-SPIO NPs

T2-weighted images and transverse relaxivity (R₂) of STM-SPIO NPs were determined using a 7T MR imaging system (Bruker biospec 70/30 MRI, USA). Different ferric iron concentrations of the STM-SPIO NPs (1, 0.5, 0.25, 0.125 and 0.0625 mM Fe) in DI H₂O were transferred into 250 μL PCR tubes for image scanning. A multislice multiecho (MSME)-T2 map pulse sequence²² with fixed TR (6000 ms) and 32 echoes in 11 ms intervals was used to measure the spin–spin relaxation times (T2) of the STM-SPIO NPs. A T2-weighted spin-echo sequence (TR/TE = 6000 ms/11 ms) with the following parameters: TR/TE = 6000 ms/11 ms, matrix size = 256 × 256, FOV = 6 × 6 mm and NEX = 3 was used for MR imaging.

2.12 Cell culture

TRAMP-C1 and RAW246.7 cells were cultured in DMEM supplemented with 10% FBS and 1× P/S maintained at 37 °C and 5% CO₂ atmosphere. MSCs were cultured in α-MEM supplemented with 20% FBS and 1× P/S at 37 °C and 5% CO₂ atmosphere. The cells were subcultured at a split ratio of 4 : 1 while cell confluency reached 90%.

2.13 Macrophage uptake of the STM-SPIO NPs

1 × 10⁵ RAW246.7 cells were seeded in 24-well plates and cultured at 37 °C overnight. After the cell confluency reached 60%, the cells were rinsed with PBS and the medium was replaced with 25% FBS/DMEM. The Fe content of the SPIO NPs or STM-SPIO NPs was determined using KSCN methods as previously described. SPIO NPs or STM-SPIO NPs were added to the cells at final Fe concentrations of 10, 20 or 30 μg mL⁻¹ and were then incubated for 4 hours. After that, the cells were washed twice with PBS and then fixed by 4% paraformaldehyde/PBS at room temperature for 10 minutes. To visualize the intracellular SPIO NPs, the cells were treated with Prussian blue staining (10% hexacyanoferrate in 20% hydroxyl chloride solution) for 20 minutes and washed twice with DI H₂O before the cell images were taken using a Zeiss Axio Observer D1 (Carl Zeiss, Oberkochen, Germany). On the other hand, the cells were detached by trypsinization then subjected to fixation to determine the amount of uptake of SPIO NPs using ICP-MS. The results were normalized to the amount of total cell proteins measured by BCA assay (Pierce).

2.14 Magnetically-assisted cellular uptake of the STM-SPIO NPs

1 × 10⁵ TRAMP-C1 cells were seeded in 24-well plates and cultured at 37 °C overnight. After the cell confluency reached 60%, the cells were rinsed with PBS and the medium was replaced with 10% FBS/DMEM. STM-SPIO NPs were added into the wells to a final concentration of 20 μg mL⁻¹ or 150 μg mL⁻¹ and then incubated for 4 hours with or without magnetic attraction. After that, the cells were washed twice with PBS and then fixed by 4% paraformaldehyde/PBS before Prussian blue staining as previously described.

2.15 Magnetic hyperthermia treatment

The magnetic hyperthermia effect of the SPIO NPs or STM-SPIO NPs was first evaluated by placing SPIO NP or STM-SPIO NP solutions (200 μg mL⁻¹) under AMF treatment for 20 minutes (Power-cube 32 High Frequency Induction System, President Honor Industries Co., New Taipei City, Taiwan) with field frequency 1.024 MHz, average applied power on coil 32 kvar and maximum absorbed power 2.8 kW. To further evaluate the magnetic hyperthermia effect of the STM-SPIO NPs on TRAMP-C1 cells, 4 × 10⁵ cells were seeded in 6-well plates and cultured overnight. When the cell confluency reached 70%, the TRAMP-C1 cells were washed with PBS and the medium was replaced with fresh 10% FBS/DMEM. STM-SPIO NPs were added into the medium at 100 μg mL⁻¹ or 150 μg mL⁻¹ which was then incubated with or without magnetic attraction for 4 hours. Next, the cells were washed with PBS twice and trypsinized before being resuspended in 250 μL medium at a cell concentration of 1.5 × 10⁶ cells per mL to receive AMF treatment (same parameters as mentioned above). 5, 10, 15, and 20 minutes after the beginning of the AMF treatment, the solution temperature was recorded using an infrared thermal camera (AVIO F30S, NEC Avio Infrared Technologies, Tokyo, Japan). After that, the cells were reseeded into 24-well plates and cultured for an additional 20 hours before determining the cell viability using an MTT assay.

2.16 Statistical analysis

All statistical evaluations were carried out using unpaired two-tailed Student’s t-test. A p-value of less than 0.05 was considered significant (p < 0.05, *; p < 0.01, **; p < 0.001, ***).

3. Results and discussion

3.1 Preparation and characterization of the STM-SPIO NPs

Adipose-derived MSCs were collected and treated with hypotonic (0.25 × PBS) solution followed by mild homogenization to break up cells into cytosol, nucleus and cell membranes. The cell membranes were washed by repeated centrifugation steps to remove nucleic acids and cytosolic components. The aforementioned procedure for STM isolation was performed in an ice bath to minimize the denaturation of cell membrane-associated proteins. SPIO NPs were synthesized by a co-precipitation method. The as-synthesized SPIO NPs were water-soluble and exhibited good superparamagnetic properties. To fabricate STM-SPIO NPs, STM was added into SPIO NP solution which was then subjected to a mild sonication in an ice bath. Sonication provided the energy to force the cell membrane to physically disassemble and reassemble with the SPIO NPs into STM-SPIO NPs in the aqueous solution (Scheme 1). To optimize the preparation of the STM-SPIO NPs, different weight ratios of STM and SPIO NPs (STM : SPIO NPs = 1 : 1, 3 : 1, 5 : 1, 20 : 1) were investigated (Fig. 1A). The hydrodynamic size of the prepared STM-SPIO NPs was measured using a ZetaSizer. At all the tested weight ratios, the formed STM-SPIO NPs exhibited increased size compared to the SPIO NPs. The increased size might be due to the coverage of STM on the surface of the SPIO NPs. However, further increase in the STM amount contributed to the formation of large aggregated nanoparticles. Based on these results, STM : SPIO NPs at a weight ratio of 1 : 1 were used to fabricate nano-sized STM-SPIO NPs for the subsequent studies. In addition, the Fe content in the STM-SPIO NPs (1 : 1) was 85.17 ± 5.94% as measured using KSCN methods. To visualize the presence of the membrane structure on the STM-SPIO NPs, the particles were negatively stained with tungstophosphoric acid and observed using TEM (Fig. 1B). The results suggest SPIO NP clusters were covered with membrane structures in the STM-SPIO NP material. The STM coating on the surface of SPIO NPs was further characterized by a dye retention assay where the hydrophobic fluorescent dioctadecyloxacarbocyanine perchlorate (DiO) was employed. DiO was mixed with the SPIO NPs or STM-SPIO NPs in an aqueous solution which were then sonicated followed by repeated magnetic attraction to remove the unbound DiO. The fluorescence intensity of DiO (ex: 460 nm/em: 495–600 nm) was detected using fluorescence spectroscopy. As the negative control, the STM and SPIO NPs were verified to be non-fluorescent. The results (Fig. 2) show that higher fluorescence intensity was observed from the STM-SPIO NPs compared to the SPIO NPs, which can be explained by the presence of a lipid bilayer structure in STM providing a hydrophobic reservoir for the hydrophobic DiO molecules. The intensity of DiO fluorescence was increased by loading a higher amount of DiO to the STM-SPIO NPs. Next, SDS-PAGE analysis was utilized to analyze the retention of STM-associated proteins on the STM-SPIO NPs. Similar overall protein profiles were observed from the fresh STM and extracts of the STM-SPIO NPs (Fig. 3A) suggesting that the optimized fabrication procedure did not cause a significant decline in the total proteins from the STM.

Scheme 1 Schematic representation of STM-SPIO NP preparation procedure.

Fig. 1 Characterization of the STM-SPIO NPs. (A) The effect of STM coating on the particle size of STM-SPIO NPs. (B) TEM images of the SPIO NPs (i/ii) and STM-SPIO NPs (iii/iv).

Fig. 2 Verification of the STM coating on SPIO NPs using a dye retention assay. Fluorescence spectra were taken (from top to bottom): (i) STM-SPIO NPs + DiO (high). (ii) STM-SPIO NPs + DiO (low). (iii) Unmodified SPIO NPs + DiO. (iv) STM. (v) Unmodified SPIO NPs. (Ex: 460 nm/em: 495–600 nm).

Fig. 3 (A) Membrane protein retention analysis by SDS-PAGE. The total protein profiles of STM and STM-SPIO NPs were comparable. (B) Retention assay of stem cell-specific makers (CD44) on the STM-SPIO NPs. FITC-labelled anti-CD44 antibody (CD44-FITC) was incubated with STM-SPIO NPs at w/w 1 : 20 or 1 : 2. A centrifugation step was performed to spin down the STM-SPIO NPs along with the bound CD44-FITC. The fluorescence spectrum of the free CD44-FITC in the supernatant fraction was analyzed. An isotype antibody was used as the negative control.

CD44-positive MSCs were used as the source of STM in this study. The retention of stem cell marker CD44 in the membrane structure of the prepared STM-SPIO NPs was further characterized using an antibody binding assay (Fig. 3B). FITC-labelled anti-CD44 antibody (CD44-FITC) was incubated with STM-SPIO NPs at the weight ratios of 1 : 2 or 1 : 20 for 2 hours and then subjected to centrifugation to pull down the CD44-FITC/STM-SPIO NP complexes. The unbound CD44-FITC in the supernatant was measured for fluorescence intensity using a fluorescence plate reader. A decrease of fluorescence intensity was observed from the higher binding ratio of the STM-SPIO NPs to CD44-FITC. In contrast, unchanged fluorescence was observed from the isotype control antibody/STM-SPIO NP binding group. The result suggests stem cell marker CD44 was well preserved after the mild sonication procedure used for STM-SPIO NP preparation.

3.2 Colloidal stability of the STM-SPIO NPs

Taking advantage of their nano-size and magnetic properties, SPIO NPs could be directed to cancer sites via passive (EPR effect) or active targeting (magnetic targeting) for potential theranostic applications on cancers. Without appropriate surface modifications, SPIO NPs are prone to aggregation in serum-containing conditions.²³ Poor in vivo colloidal stability of unmodified SPIO NPs is responsible for their rapid clearance by the RES organs such as liver and lung thus limiting their theranostic applications. To achieve efficient drug delivery, it is crucial to develop carriers with good colloidal stability under physiological conditions. In this study, the size evolution of STM-SPIO NPs under various conditions including DI H₂O, 25% serum and 50% serum-containing DMEM was studied using DLS techniques (Fig. 4). The initial size (10 minutes incubation) of the STM-SPIO NPs was measured as: 141.1, 391.1 and 282.9 nm in DI H₂O, 25% or 50% serum respectively. These particle sizes still fall within the range for EPR effects.²⁴ After an extended incubation time (4 and 24 hours), the size of the STM-SPIO NPs in DI H₂O was not significantly changed suggesting they have a stable colloidal structure. Similar size evolution trends were also observed from STM-SPIO NPs incubated under serum-containing environments. It was noticed that the size of the STM-SPIO NPs was larger in the serum-containing medium compared to H₂O. This phenomenon might be attributed to the salt-mediated charge neutralization on the surface of STM-SPIO NPs, which could promote particle aggregation. Overall, the results suggest that STM-SPIO NPs possess good colloidal stability and are worth further evaluation for potential in vivo theranostic applications.

Fig. 4 Colloidal stability of STM-SPIO NPs in DI H₂O, 25% FBS/DMEM or 50% FBS/DMEM. The particle size was measured after incubating the nanoparticles in the designated environments for 10 minutes, 4 hours and 24 hours.

3.3 Magnetic properties of STM-SPIO NPs

The magnetization and hysteresis loop of STM-SPIO NPs were examined using SQUID (Quantum Design MPMS-XL, USA). The magnetization of the SPIO NPs and STM-SPIO NPs was measured to be 72.7 and 65.9 emu g⁻¹ respectively (Fig. 5A). Both samples showed typical superparamagnetic curves, and the lower magnetization of STM-SPIO NPs might be attributed to the weight contribution of STM. To evaluate the potential of using STM-SPIO NPs as a novel MRI contrast agent, various concentrations of SPIO NPs or STM-SPIO NPs were fixed in agarose phantoms and imaged by a 7T MR imaging system. The transverse relaxivities (R₂) for STM-SPIO NPs and SPIO NPs were 653.3 and 786.0 s⁻¹ mM⁻¹ respectively measured by a multislice multiecho (MSME)-T2 map pulse sequence (Fig. 5B). A concentration dependent T2-weighted MRI imaging contrast was observed from the STM-SPIO NP solutions (Fig. 5C).

Fig. 5 Magnetic properties and MR imaging of STM-SPIO NPs. (A) Magnetism and hysteresis loop of SPIO NPs and STM-SPIO NPs measured by SQUID within 1 tesla. (B) Transverse relaxivities (R₂) and (C) T2-weighted images of STM-SPIO NPs at different concentration, measured by a 7T MR imaging system (TR/TE = 6000 ms/11 ms).

3.4 Macrophage uptake of STM-SPIO NPs

Foreign materials such as theranostic nanoparticles, after they are injected systemically, can elicit various host defense responses including opsonization and phagocytosis by macrophages residing in the reticuloendothelial system (RES) of liver and lungs. As a result, rapid clearance of nanoparticles could lead to ineffective theranostic efficacy and side effects in normal organs. It has been previously shown that surface modifications with red blood cell- or leukocyte-derived biomembranes represent an effective approach to prepare long-circulating nanoparticles. The underlying mechanism is attributed to the presentation of a marker of self on the biomembrane-camouflaged nanoparticles. For example, CD47 on a red blood cell membrane could inhibit macrophage uptake via interaction with the SIRPα (signal-regulatory protein alpha) on the macrophage surface.⁹ In this study, the effect of STM coating on preventing SPIO NP uptake by macrophages was examined. STM-SPIO NPs or SPIO NPs were incubated with a mouse macrophage cell line (RAW 264.7), which shows active phagocytic activity. The Fe content of the added SPIO NPs or STM-SPIO NPs was quantitated using KSCN methods. SPIO NPs or STM-SPIO NPs were added to the cells to make final Fe concentrations of 10, 20 or 30 μg mL⁻¹ and then incubated for 4 hours. After the incubation and washing steps, the intracellular SPIO NPs were visualized as blue spots using Prussian blue staining. The cell images (Fig. 6A) show that within the same incubation conditions (time length and SPIO NP concentration), the macrophage uptake of STM-SPIO NPs was less than that of the SPIO NPs. Furthermore, ICP-MS was used to accurately measure the intracellular Fe content. Macrophages incubated with SPIO NPs or STM-SPIO NPs were harvested and subjected to nitric acid digestion for ICP-MS analysis. The measured values of Fe for each group were 0.08 ppm (control), 16 ppm (SPIO NPs) and 6.55 ppm (STM-SPIO NPs) in the cell digest. After normalizing by cellular total protein amount, the results (Fig. 6B) show that the intracellular Fe content was significantly higher (p < 0.05) for SPIO NPs (0.02257 mg Fe per mg protein) than STM-SPIO NPs (0.00836 mg Fe per mg protein). Taken together, it is suggested STM coating could significantly decrease SPIO NP uptake by macrophages.

Fig. 6 (A) Macrophage uptake of SPIO NPs and STM-SPIO NPs. Macrophages were incubated with various concentrations (10, 20 and 30 μg mL⁻¹) of SPIO NPs (top) or STM-SPIO NPs (bottom), then observed using Prussian blue staining. (B) Quantitative measurement of intracellular Fe levels in the macrophages using ICP-MS. Data represents the mean ± S.E.; n = 3.

3.5 Magnetic hyperthermia by STM-SPIO NPs

When SPIO NPs are placed under an external AMF, heat will be generated from magnetic moment relaxation through a Neel or Brown mechanism. Using an infrared thermal camera (Fig. 7), it was observed that STM-SPIO NPs and SPIO NPs possessed similar magnetic induced hyperthermia effects. Next, the cellular uptake of STM-SPIO NPs by cancer cells was examined. Mouse prostate cancer cells (Tramp-C1) were incubated with STM-SPIO NPs (20 or 150 μg mL⁻¹) for 4 hours followed by washing out the un-internalized STM-SPIO NPs. Using Prussian blue staining (Fig. 8), it was observed that the uptake of STM-SPIO NPs by Tramp-C1 cells was dose-dependent. Under attraction by an external magnet, darker blue staining was observed inside the cells indicating the enhanced intracellular internalization of STM-SPIO NPs. The enhanced cellular uptake of STM-SPIO NPs by an external magnetic field could be explained by the accelerated sedimentation of nanoparticles to a cell surface which in turn promotes endocytosis.²⁵ The temperature raising effect of the STM-SPIO NPs in Tramp-C1 cells was investigated next. The cells were incubated with 2 different STM-SPIO NP concentrations with or without the attraction of an external magnet. The trend of the temperature raising effect is summarized as follows: 150 μg mL⁻¹ (Magnet+) > 150 μg mL⁻¹ (Magnet−) > 100 μg mL⁻¹ (Magnet+) > 100 μg mL⁻¹ (Magnet−) (Fig. 9A). For the group of 100 μg mL⁻¹ (Magnet+), the temperature was raised to 47 °C after receiving 20 minutes of AMF application. Under such magnet-induced hyperthermia circumstances, the viability of Tramp-C1 cells was measured using an MTT assay (Fig. 9B). A significant cancer cell viability decrease (∼87.3%) was observed from cells that received magnet-enhanced STM-SPIO NP internalization followed by 20 minutes of AMF application. In addition, it was noticed that nearly 100% viability was observed from cells that received STM-SPIO NPs without AMF treatment indicating the negligible cytotoxicity of these biomimetic nanomaterials.

Fig. 7 Magnetohyperthermia effect of STM-SPIO NPs. The temperature of SPIO NP or STM-SPIO NP solution under AMF treatment for 20 minutes was recorded using an infrared thermal camera. Data represents the mean ± S.E.; n = 3.

Fig. 8 Magnetically-assisted cellular uptake of STM-SPIO NPs by TRAMP-C1 cells. Cancer cells were incubated with (A) no treatment. (B) STM-SPIO NPs (20 μg mL⁻¹) w/o magnet attraction. (C) STM-SPIO NPs (20 μg mL⁻¹) w/magnet attraction. (D) STM-SPIO NPs (150 μg mL⁻¹) w/o magnet attraction. (E) STM-SPIO NPs (150 μg mL⁻¹) w/magnet attraction. Cellular uptake of SPIO NPs was observed by Prussian blue staining.

Fig. 9 STM-SPIO NP-mediated magnetic hyperthermia treatment on TRAMP-C1 cells. (A) Effect of AMF treatment on the temperature of cell solutions. Green line: cells incubated with 100 μg mL⁻¹ STM-SPIO NPs without magnetic attraction (Magnet−). Blue line: cells incubated with 100 μg mL⁻¹ STM-SPIO NPs with magnetic attraction (Magnet+). Cyan line: cells incubated with 150 μg mL⁻¹ STM-SPIO NPs without magnetic attraction (Magnet−). Pink line: cells incubated with 150 μg mL⁻¹ STM-SPIO NPs with magnetic attraction (Magnet+). (B) Viability assay of TRAMP-C1 cells incubated with 100 μg mL⁻¹ STM-SPIO NPs with or without magnetic attraction then received AMF treatment. The cell viability was measured using an MTT assay. Data represents the mean ± S.E.; n = 3.

4. Conclusions

In this study, we reported the fabrication, characterization and applications of a novel biomimetic STM-SPIO NP system. Water-soluble STM-SPIO NPs were successfully prepared via a simple sonication method. A coating of STM on the SPIO NPs significantly decreased the macrophage uptake. Magnetic hyperthermia-mediated cell death was observed from cells internalized with STM-SPIO NPs followed by AMF exposure in vitro. These results demonstrate the great potential of STM-SPIO NPs as a novel biomimetic nanoparticulate system for future theranostic applications.

Acknowledgements

This research was financially supported by Ministry of Science and Technology of Taiwan (102-2113-M-007-006-MY2), National Health Research Institutes (NHRI) of Taiwan (NHRI-EX103-10221EC) and National Tsing Hua University (104N2046E1/104N2732E1). We thank Ms C.-Y. Chien of Ministry of Science and Technology (National Taiwan University) for the assistance in TEM experiments and 7T animal MRI Core Lab of the Neurobiology and Cognitive Science Center, National Taiwan University for technical and facility support. The magnetization was measured using a SQUID magnetometer (MPMS XL-7) at the National Chiao Tung University.

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Footnote

† These authors contributed equally to the work.

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