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Man Wang, Huajian Chen, Rui Sun, [Tianjiao Zeng](https://orcid.org/0000-0002-1286-0337), Chengyu Lu, [Toru Yoshitomi](https://orcid.org/0000-0003-3847-1812), [Naoki Kawazoe](https://orcid.org/0000-0003-3916-0709), Yingnan Yang, [Guoping Chen](https://orcid.org/0000-0001-6753-3678)

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[Influence of microenvironmental viscosity on the cellular uptake of Fe                    <sub>3</sub>                    O                    <sub>4</sub>                    nanoparticles and their anticancer effect](https://mdr.nims.go.jp/datasets/df3d24c6-3d59-4b4a-9769-aa9ba0c35d94)

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Influence of microenvironmental viscosity on the cellular uptake of Fe3O4 nanoparticles and their anticancer effectNanoscalePAPERCite this: Nanoscale, 2026, 18, 10691Received 4th December 2025,Accepted 8th April 2026DOI: 10.1039/d5nr05112frsc.li/nanoscaleInfluence of microenvironmental viscosity on thecellular uptake of Fe3O4 nanoparticles and theiranticancer effectMan Wang, a,b Huajian Chen,a Rui Sun,a Tianjiao Zeng, a,b Chengyu Lu,a,bToru Yoshitomi, a Naoki Kawazoe,a Yingnan Yangc and Guoping Chen *a,bViscosity is a characteristic property of extracellular microenvironments, and it varies among cancertissues. Although internalized magnetic nanoparticles are crucial for magnetic hyperthermia, theinfluence of microenvironmental viscosity on their cellular uptake remains elusive. In this study, the effectof microenvironmental viscosity on the cellular uptake of magnetic nanoparticles and the efficiency ofmagnetic hyperthermia were investigated by culturing colorectal cancer cells in media of different visco-sities. Results showed that the cellular uptake of magnetic nanoparticles significantly decreased withincreasing microenvironmental viscosity, which further decreased the intracellular heating effect of mag-netic nanoparticles. Furthermore, the proportion of apoptotic cells induced by magnetic hyperthermiawas significantly reduced in high viscosity microenvironments, and the most viscous microenvironmentexhibited the lowest apoptosis rate. This study revealed the important role of microenvironmental vis-cosity in regulating the cellular uptake of magnetic nanoparticles and the efficiency of magnetichyperthermia, which provides a novel perspective for optimizing the application of magnetic hyperther-mia in anticancer therapy.1. IntroductionNanomaterials have been extensively investigated and used inbiomedical applications because of their unique propertiessuch as small size, large specific surface area and high pene-tration capacity.1–3 Accordingly, nanomaterial-based thera-peutic strategies have garnered significant attention.4–6 Forexample, magnetic nanoparticle (MNP)-mediated magnetichyperthermia,5,7–10 which utilizes the unique heating propertyof MNPs under an alternating magnetic field (AMF) to locallyheat tumor tissues,11 has been developed as a prospectivemodality for cancer treatment.12–21Magnetic hyperthermia can induce cancer cell apoptosisthrough intracellular magnetic hyperthermia by internalizedMNPs or through extracellular magnetic heating by MNP-loaded scaffolds.3,6,11 Emerging evidences suggest that intra-cellular magnetic hyperthermia is more efficacious than extra-cellular magnetic heating in inducing cancer cellapoptosis.22–25 Intracellular magnetic hyperthermia not onlydestroys the internal structure of cancer cells moreeffectively22,26,27 but also minimizes damage to surroundinghealthy tissues.28,29 However, intracellular magnetic hyperther-mia requires the uptake of MNPs by cancer cells. An acceler-ated cellular uptake of MNPs can increase the therapeuticeffect of magnetic hyperthermia.The cellular uptake of nanomaterials proceeds via somepathways such as endocytosis, phagocytosis, micropinocytosisand direct fusion with the cell membrane.30 Not only cells butalso extracellular environment can affect the interactionbetween cells and nanoparticles and their cellular uptakebecause cells are surrounded by their extracellular microenvir-onments.31 Extracellular microenvironments interact with cellsthrough their specific motifs that bind with their receptors onthe cell membranes or through their physical characteristicssuch as viscoelasticity. Many studies have revealed the influ-ence of the viscoelastic properties of extracellular microenvir-onments on the adhesion, proliferation, migration and differ-entiation of cells.32,33 Substrate stiffness affects the cellularuptake of nanoparticles.34,35Viscosity, as a characteristic property of extracellular micro-environments, varies depending on the type of tissues, healthcondition and age. The viscosity of the mucus, bone marrow,synovial fluid and interstitial fluid has different values. Theviscosity of colorectal cancer (CRC) tissue is significantlyhigher than that of the normal colorectal tissue due toaResearch Center for Macromolecules and Biomaterials, National Institute forMaterials Science, Ibaraki 305-0044, Japan. E-mail: Guoping.CHEN@nims.go.jpbGraduate School of Science and Technology, University of Tsukuba, Ibaraki 305-8577, JapancGraduate School of Life and Environmental Science, University of Tsukuba, Ibaraki305-8572, JapanThis journal is © The Royal Society of Chemistry 2026 Nanoscale, 2026, 18, 10691–10702 | 10691Published on 28 April 2026Licensed under CC-BY-NC 4.0http://rsc.li/nanoscalehttp://orcid.org/0000-0002-4009-0296http://orcid.org/0000-0002-1286-0337http://orcid.org/0000-0003-3847-1812http://orcid.org/0000-0001-6753-3678http://crossmark.crossref.org/dialog/?doi=10.1039/d5nr05112f&domain=pdf&date_stamp=2026-05-26https://creativecommons.org/licenses/by-nc/4.0/increased mucus production and extracellular matrix remodel-ing.36 Recently, some studies have disclosed that the viscosityof the microenvironment can affect stem cell differentiationand anticancer drug resistance of colon cancer cells.37–41Despite increasing interest in nanomaterial-based thera-pies, it remains unclear how the viscosity of the extracellularmicroenvironment affects the cellular uptake of MNPs.Elucidating the effect of microenvironmental viscosity on thecellular uptake of MNPs is crucial for the subsequent selectionof conditions for magnetic hyperthermia treatment becauseheat generation and temperature increase during magnetichyperthermia are correlated with the amount of magneticnanoparticles in cancer cells. Therefore, the objective of thisstudy is to investigate the influence of viscosity on the cellularuptake of Fe3O4 NPs in CRC (SW480) cells (Scheme 1). The cel-lular uptake of Fe3O4 NPs was analyzed and their magnetother-mal effect was investigated. Viscosity could inhibit the cellularuptake of NPs and reduce their therapeutic capacity.2. Materials and methods2.1. ReagentsSodium hydroxide (granular), FeCl3·6H2O, FeCl2·4H2O, sodiumcitrate, polyethylene glycol (PEG) 8M, Mw 8 000 000, PEG 35K,Mw 35 000, DMEM with high glucose and trypsin-EDTA solu-tion (0.25%) were purchased from Sigma-Aldrich, USA.Millipore syringe filter with a 0.45 μm mesh size was pur-chased from Merck Millipore. The human colorectal cancercell line SW480 was purchased from the American TypeCulture Collection (ATCC, Manassas, VA, USA). Prussian BlueStain Kit was bought from ScyTek Laboratories, USA. AnnexinV-FITC/PI Apoptosis Detection Kit was purchased fromMedChemExpress, USA. Ethyl acetate, diethylene glycol (DEG),N-methyldiethanolamine (NMDEA), ethanol, 4% Para-formaldehyde Phosphate Buffer Solution (PFA, 4%), bovine serumalbumin (BSA), hydrogen peroxide (H2O2, 30%) and nitric acid(HNO3, 67%) were bought from Wako Pure Industries, Ltd.2.2. Synthesis and characterization of Fe3O4 NPsFe3O4 NPs were synthesized via a solvothermal method, asdescribed previously.11,42 Initially, FeCl3 and FeCl2 were mixedin NMDEA and DEG polyols at a molar ratio of 2 : 1 and stirredunder a nitrogen atmosphere for 1 h. Subsequently, a sodiumhydroxide polyol solution was added dropwise to the ferricchloride mixture and agitated for an additional 3 h. The result-ing mixture was then heated to 220 °C, and after 12 h of reac-tion, a black precipitate was separated using a magnet andwashed with an ethanol-ethyl acetate mixture. The separatedblack precipitate was then dispersed in an aqueous sodiumcitrate solution and reacted at 60 °C for 24 h to facilitate chela-tion between NPs and citrate ions. The final citrate-NPs wereobtained through centrifugation (12 000 rpm, 15 min) andwashed three times with pure water. The morphology and sizeof the citrate-NPs were examined utilizing a JEOL JEM-ARM200Ftransmission electron microscope (TEM). Fourier-transforminfrared (FT-IR) spectra of the prepared citrate-NPs wererecorded. Dynamic light scattering (DLS, Beckman Coulter) wasemployed to analyze the hydrodynamic size and zeta potentialof citrate-NPs in pure water and cell culture media. In addition,in order to evaluate the protein corona formation, scanning elec-tron microscopy (SEM) coupled with energy-dispersive X-rayspectroscopy (EDS) was further utilized to characterize theelemental composition of citrate-NPs before and after incu-bation in a cell culture medium.2.3. Preparation of PEG solutions with different viscositiesTo regulate the viscosity of cell culture media, two types ofPEG were employed: a high-molecular-weight PEG (PEG 8M,Mw 8 000 000) and a low-molecular-weight PEG (PEG 35K, Mw35 000).37 The 1.5% (wt/v) PEG stock solution and a 5.75-foldconcentrated DMEM with high glucose were individually pre-pared and sterilized using a Millipore syringe filter (0.45 μm).The complete DMEM with varying viscosities was formulated bycombining the PEG solution (1.5% (wt/v)) and concentratedDMEM solution (5.75-fold) at a volume ratio of 4 : 1. This mixturewas subsequently supplemented with 1% (v/v) fetal bovine serum(FBS), L-glutamine (584 mg L−1), and antibiotics (100 U mL−1penicillin and 100 μg mL−1 streptomycin) to achieve a final con-centration of 1-fold DMEM and 1.0% (wt/v) PEG.The culture media with varying viscosities were prepared byincorporating PEG 35K, PEG 8M, and their combinations atdifferent weight ratios. These groups were named according totheir relative viscosity levels as follows: normal viscosity (NV,no PEG); low viscosity (LV, PEG 8M : PEG 35K = 1 : 1); middleviscosity (MV, PEG 8M : PEG 35K = 2 : 1); high viscosity (HV,PEG 8M : PEG 35K = 4 : 1) and very high viscosity (VHV, PEG8M : PEG 35K = 1 : 0). To prepare media containing Fe3O4 NPs,an NP stock solution (10 mg mL−1) was added to the PEG-con-Scheme 1 Influence of viscosity on the cellular uptake of Fe3O4 NPs inCRC (SW480) cells and their intracellular magnetic hyperthermiaefficiency. The illustration was generated using BioRender (https://app.biorender.com/).Paper Nanoscale10692 | Nanoscale, 2026, 18, 10691–10702 This journal is © The Royal Society of Chemistry 2026https://app.biorender.com/https://app.biorender.com/https://app.biorender.com/taining culture media to achieve a final Fe3O4 NP concen-tration of 120 μg mL−1. The mixtures were homogenized bystirring overnight before use.2.4. Measurement of viscosityAn MCR 302 rheometer (Anton Paar, Germany) was used todetermine the viscosity of the culture medium in the rotationalshear mode.37,38 The procedure involved placing 2 mL of thesample on a preheated plate (PP-50) and compressing it withanother parallel plate, maintaining a 1.0 mm gap. To preventevaporation during the measurement, low-viscosity silicone oilwas applied to the periphery of the sample. Measurementswere conducted at 37 °C with shear rates ranging from 0.1 to100 s−1. The zero-shear viscosity was defined as the viscosity ata shear rate of 0.1 s−1. For each measurement, three sampleswere used to calculate the mean and standard deviation.2.5. Cellular uptake of Fe3O4 NPsSW480 colorectal cancer cells were cultured in DMEM serumsupplemented with 1% FBS, L-glutamine, and antibiotics.37The cells were maintained in an incubator with controlledhumidity at 37 °C and 5% CO2. Upon reaching approximately75% confluence, the cells were dissociated and seeded in a24-well plate at a density of 2.5 × 105 cells per well. After 24 h,a final concentration of 120 μg mL−1 of Fe3O4 NPs was intro-duced into the media with different viscosities to investigatethe effect of viscosity on the cellular uptake of NPs. Followingincubation for different durations (0, 6, 12, 24, 36, and 48 h),the cells were washed five times with PBS to remove non-inter-nalized NPs. The cellular uptake of Fe3O4 NPs was monitoredvia Prussian blue staining and quantified by ICP-OES. ForPrussian blue staining, the cells were fixed using 4% PFA, andthen, the fixed cells were treated with an iron stain solutionfor 3 min. To provide contrast, cell nuclei were stained with anuclear fast red solution for 5 min. The stained cells were thenexamined using an optical microscope in the bright-fieldmode.ICP-OES was used to quantify the cellular uptake amount ofFe3O4 NPs. SW480 cells were seeded in 24-well plates and incu-bated for 24 h. Subsequently, the cells were exposed to NPs ata concentration of 120 µg ml−1 in various viscous media.Following different incubation periods, the cells were washedfive times with PBS to remove non-internalized NPs. The cellswere then detached using trypsin/collagenase treatment andcollected by centrifugation (1100 rpm, 5 min). The harvestedcells were enumerated and subjected to digestion using amixture of HNO3 : H2O2 (1 mL, v/v = 2 : 1). This digested cellsolution was subsequently diluted with pure water, filteredand analyzed for iron content using ICP-OES.2.6. Intracellular pH measurementThe intracellular pH was measured using the pHrodo™ AMvariety pack following the manufacturer’s protocol. Briefly, thecells were seeded in glass-bottom confocal dishes and allowedto adhere overnight. The culture medium was then exposed toNPs (120 µg ml−1) in various viscous media. After pre-incu-bation for 24 h under the respective viscosity conditions, cellswere incubated with pHrodo™ AM ester diluted in a serum-free medium and incubated for 30 min at 37 °C. After incu-bation, cells were washed three times with PBS and imagedimmediately using a confocal laser scanning microscope.2.7. Lysosomal trafficking of Fe3O4 NPsTo gain deeper mechanistic insights into the intracellulartrafficking route of the internalized NPs, rhodamine-labeledFe3O4 NPs (Rho-Fe3O4 NPs) were employed in combinationwith LysoTracker and Hoechst staining for fluorescenceimaging, thereby enabling visualization and co-localization oftheir subcellular distribution within endo/lysosomal compart-ments. SW480 cells were seeded in plates and then exposed toRho-Fe3O4 NPs (120 µg ml−1) in various viscous media for24 h. After washing cells with PBS for 5 times, the cells werestained with LysoTracker and Hoechst. The stained cells werethen visualized using a fluorescence microscope.2.8. Magnetic heating performance of intracellular Fe3O4NPsTo investigate the influence of various viscous culture mediaon the cellular uptake of Fe3O4 NPs and the subsequentimpact on the magnetic heating effect of intracellular NPs,SW480 cancer cells (5 × 106 cells) were cultured in Petri dishes(P-100, Falcon) using diverse viscous media containing 120 µgmL−1 Fe3O4 NPs for 24 h. The cells were subsequently washedfive times with PBS to remove the non-internalized NPs, result-ing in cells containing varying quantities of NPs. These NP-loaded cells were then detached, collected (1100 rpm, 5 min)and resuspended in 100 µL of PBS before being transferredinto 0.5 mL Eppendorf tubes. The samples were positioned inthe central area of AMF Double H CoilSets and subjected to10 min irradiation (frequency: 373.6 kHz; field intensity: 130Oe). During the AMF exposure, the temperature of the cell sus-pension was recorded utilizing a fiber optic thermometer(Rugged Monitoring, Canada).To ensure thermal consistency, all heating experimentswere conducted in a temperature-controlled environment,using preheated insulation to maintain the ambient tempera-ture at 37 °C ± 0.5 °C, simulating physiological conditions andminimizing the external thermal interference. The baselinetemperature before AMF exposure was 37 °C. Duringirradiation, the temperature of the cell suspension was con-tinuously monitored using a fiber optic thermometer (RuggedMonitoring, Canada), with the probe placed directly in thetube to allow accurate, real-time temperature measurement.Temperature increment was normalized to the cellular uptakeamount of Fe3O4 NPs by dividing the temperature incrementwith the respective amount of uptaken Fe3O4 NPs.2.9. Magnetic hyperthermia effect of intracellular Fe3O4 NPsTo assess the magnetic hyperthermia effect of differentamounts of intracellular Fe3O4 NPs, cell apoptosis of SW480cancer cells was examined following AMF irradiation. Controlcells were cultured in DMEM without NPs and PEG. Cells fromNanoscale PaperThis journal is © The Royal Society of Chemistry 2026 Nanoscale, 2026, 18, 10691–10702 | 10693the control group and those loaded with various amounts ofNPs (as described in section 2.6) were subjected to AMFexposure. Prior to and following AMF irradiation, the cellswere suspended in a 1× binding buffer and incubated withAnnexin V-FITC conjugate and PI for 10 min in darkness. Thestained cell suspensions were filtered through a 35 μm coverand transferred to 12 × 75 mm plastic test tubes. The cell fluo-rescence was immediately quantified using a flow cytometer(BD Accuri C6 Plus). The viable cells remained unstained byboth PI and Annexin V-FITC conjugate. Early apoptotic cellswere identified by Annexin V-FITC conjugate staining alone,whereas late apoptotic cells exhibited staining with bothAnnexin V-FITC conjugate and PI. The apoptosis rate was cal-culated using both early and late apoptotic cells.2.10 Statistical analysisAll quantitative experiments were conducted in triplicate (n =3), and the results are presented as mean ± standard deviation(S.D.). Statistical analysis was performed using one-way ana-lysis of variance (ANOVA). A p value of 0.05 was established asthe threshold for statistical significance, with the data categor-ized as follows: *p < 0.05, **p < 0.01 and ***p < 0.001. All ana-lyses were performed using GraphPad Prism 9.0 (GraphPadSoftware, CA, USA).3. Results3.1. Synthesis and characterization of Fe3O4 NPsThe Fe3O4 NPs were synthesized and modified with citrate toimprove the stability of NPs. The morphology of the preparedcitrate-NPs was characterized by TEM. As shown in Fig. 1A, thesynthesized citrate-Fe3O4 NPs displayed a flower-like shapewith an average size of 31.4 ± 4.8 nm. FT-IR spectroscopy wasemployed to verify the citrate modification of NPs. The spectraof bare Fe3O4 NPs, citrate-Fe3O4 NPs and sodium citrate areillustrated in Fig. 1B. The characteristic absorption peaks at1384.9 and 1577.8 cm−1 are attributed to the symmetric andFig. 1 Characterization of citrate-Fe3O4 NPs. (A) TEM images of citrate-Fe3O4 NPs (scale bar: 20 nm). (B) FT-IR spectra of bare Fe3O4 NPs, citrate-Fe3O4 NPs and sodium citrate. (C ) Photograph of citrate-Fe3O4 NPs in DMEM. (D) Hydrodynamic size distribution of citrate-Fe3O4 NPs in purewater. (E) Hydrodynamic size distribution of citrate-Fe3O4 NPs in DMEM. (F) Zeta potential of bare-Fe3O4 NPs, citrate-Fe3O4 NPs and citrate-Fe3O4NPs in DMEM. (G) SEM image with the corresponding elemental mapping of the citrate-Fe3O4 NPs. (H) SEM image with the corresponding elementalmapping of the citrate-Fe3O4 NPs after incubation in a cell culture medium.Paper Nanoscale10694 | Nanoscale, 2026, 18, 10691–10702 This journal is © The Royal Society of Chemistry 2026asymmetric stretching vibrations of the COO− group,43,44which are observed in the spectra of sodium citrate andcitrate-Fe3O4 NPs, but not in the bare Fe3O4 NPs. This resultrevealed the surface modification of Fe3O4 NPs with citrateions, suggesting the successful synthesis of citrate-Fe3O4 NPs.The citrate-Fe3O4 NPs demonstrated superior dispersioncharacteristics in the cell culture medium (DMEM), maintain-ing stability without precipitation (Fig. 1C). The hydrodynamicsize and size distribution of citrate-Fe3O4 NPs dispersed inwater (Fig. 1D) or in DMEM (Fig. 1E) were measured via DLS.The hydrodynamic diameters of citrate-Fe3O4 NPs were 95.1 ±12.9 nm in water and 132.4 ± 34.2 nm in DMEM, respectively.The PDI value of the hydrodynamic size of citrate-Fe3O4 NPs inpure water and DMEM was 0.148 and 0.174, respectively. Inboth media, citrate-Fe3O4 NPs exhibited a unimodal size distri-bution, and the relatively small increase in particle size inDMEM indicates that the prepared citrate-Fe3O4 NPs main-tained good colloidal stability under physiological conditions.Zeta potential analysis revealed a surface charge of +36.5 ±3.8 mV for bare Fe3O4 NPs, which shifted to −31.6 ± 4.2 mVfollowing citrate modification, and further to −9.8 ± 2.1 mVafter co-incubation in DMEM (Fig. 1F). The slight enlargementof hydrodynamic size together with the reduction in surfacecharge in DMEM can be attributed to the adsorption of serumproteins and the consequent formation of a protein corona,which effectively screens the negative surface charge.Moreover, the SEM image and relative elemental mapping ofcitrate-Fe3O4 NPs (Fig. 1G) revealed the co-localization of Fe, O,and C, confirming the presence of a citrate coating. Inaddition, the appearance of N after the incubation of citrate-Fe3O4 NPs in a cell culture medium (Fig. 1H) demonstratedthe formation of protein corona. These results suggested thatwhile the formation of a protein corona alters the interfacialproperties of the NPs, it does not compromise their colloidalstability, thereby ensuring reliable performance under biologi-cally relevant conditions. The citrate-Fe3O4 NPs were used forcell culture to investigate the influence of viscosity of culturemedia on their uptake.3.2. Preparation and characterization of cell culture media ofdifferent viscositiesThe cell culture media with various viscosities were preparedby adding the same amount but different ratios of low-mole-cular-weight PEG 35K and high-molecular-weight PEG 8M inthe culture media. A rheometer was utilized to assess the vis-cosity of cell culture media at 37 °C, employing shear ratesranging from 0.1 to 100 s−1. The results (Fig. 2A) demonstratedthat the medium’s viscosity decreased with the increasingshear rate. Furthermore, the viscosity value at a shear rate of0.1 s−1 was determined as the zero-shear viscosity39 and thezero-shear viscosity (Fig. 2B) of the culture media exhibited anincrease as the proportion of PEG 8M increased. The viscosityof the culture medium was modulated within a range of 32.6 ±1.86 to 619.8 ± 70.4 mPa s (Table 1) by altering the weight ratioof PEG 8M and PEG 35K, encompassing a viscosity rangecharacteristic of the mucus layer.453.3. Prussian blue staining and quantification of internalizedFe3O4 NPsThe internalization of Fe3O4 NPs by cells during the cultivationprocess was visualized via Prussian blue staining. SW480colon cancer cells were cultured in culture media of variousviscosities containing 120 µg mL−1 NPs for a duration of0–48 h. The results of Prussian blue staining (Fig. 3) demon-strated that although the cells were capable of internalizingNPs in media of different viscosities, large differences amongthe cells cultured in media of different viscosities duringuptake were observed. In the control group, medium withoutPEG (PEG 8M : 35K = 0 : 0), the uptake of Fe3O4 NPs by thecells increased gradually during the initial 12 h of culture.However, as the incubation time was extended to 48 h, a slightdecrease in Fe3O4 NPs uptake was observed, potentially due tocell division and metabolic processes. In contrast, the uptakeof NPs by cells was reduced in the PEG-containing media.This phenomenon might be attributed to the effect ofincreased medium viscosity on the diffusion behavior of NPs.46Fig. 2 Characterization of the viscosity of cell culture media containing different ratios of PEG 35K and PEG 8M. (A) Steady shear rate measurementof apparent viscosity of cell culture media ranging from 0.1 to 100 s−1 at 37 °C. (B) Zero-shear viscosity of cell culture media measured at a shearrate of 0.1 s−1.Nanoscale PaperThis journal is © The Royal Society of Chemistry 2026 Nanoscale, 2026, 18, 10691–10702 | 10695Higher viscosity restricts the ability of NPs to diffuse in themedium, thereby reducing their probability of reaching the cellsurface and consequently inhibiting the internalizationefficiency.35 Furthermore, the high viscous microenvironmentmay affect the fluidity and deformability of cell membranes,47–49further inhibiting the uptake of NPs. Notably, the uptake ofFe3O4 NPs by cells exhibited a decreasing trend with the increas-ing medium viscosity, further suggesting that the rheologicalproperty of the extracellular microenvironment plays a crucialrole in regulating the cellular uptake of NPs.The cellular uptake of Fe3O4 NPs was quantified by ICP-OESafter the cells were cultured in media of different viscosities inthe presence of 120 μg mL−1 Fe3O4 NPs for 6, 12, 24, 36 and48 h. During cell culture, the cells proliferated and cellnumber changed. To calculate the average amount of interna-lized Fe3O4 NPs per cell, the cell number was first measuredTable 1 Zero-shear viscosity of cell culture media with varying PEG weight ratios measured at a shear rate of 0.1 s−1Sample NV LV MV HV VHVViscosity (mPa s) 32.6 ± 1.86 112.7 ± 9.0 205.4 ± 46.8 362.0 ± 35.5 619.8 ± 70.4Fig. 3 Internalization and quantification of Fe3O4 NPs in SW480 cells cultured in media of different viscosities. (A) Prussian blue staining of theinternalized Fe3O4 NPs in SW480 cells cultured in media of different viscosities in the presence of 120 μg mL−1 Fe3O4 NPs for 0, 6, 12, 24, 36 and48 h. Change in the (B) cell number and (C) cellular uptake amount of Fe3O4 NPs during culture in different viscosity media in the presence of120 μg mL−1 Fe3O4 NPs for 0, 6, 12, 24, 36 and 48 h. Data are expressed as mean ± S.D. (n = 3). Significant difference: *p < 0.05. N.S.: no significantdifference.Paper Nanoscale10696 | Nanoscale, 2026, 18, 10691–10702 This journal is © The Royal Society of Chemistry 2026(Fig. 3B). The cell number increased with culture time. Thecell number increased more slowly in the high-viscositymedium than did in the low viscosity medium.The ICP-OES quantification results (Fig. 3C) revealed thatthe cellular uptake of NPs in all groups initially increased andsubsequently decreased with culture time. Additionally, theculture medium viscosity significantly influenced the cellularuptake of NPs, with the increased viscosity resulting indecreased cellular uptake of NPs. In a culture medium withoutPEG (PEG 8M : PEG 35K = 0 : 0), the intracellular amounts ofNPs reached the maximum at 12 h. In a culture medium withPEG 8M : PEG 35K ratios of 1 : 1, 2 : 1 and 3 : 1, the cellularuptake amounts of NPs peaked at 24 h. In the PEG 8M : PEG35K = 4 : 1 group, the peak of NP uptake was delayed until 36 h.The results indicated that the viscosity of the medium couldslow down and suppress the cellular uptake of Fe3O4 NPs.3.4. Intracellular pH measurementsTo determine whether the changes in extracellular viscosityalter intracellular acidification, the intracellular pH underdifferent viscosity conditions using the pHrodo™ AM indicatorwas measured. As shown in Fig. 4, no significant difference influorescence intensity was observed among the tested groups,indicating that the intracellular pH remained stable despitevariations in environmental viscosity. These results demon-strate that the viscosity modulation does not disrupt intracellu-lar pH homeostasis, and therefore, is unlikely to interfere withFe3O4 NP degradation and magnetothermal activity.3.5. Lysosomal trafficking of Fe3O4 NPsThe above Prussian blue staining and ICP quantification(Fig. 3) results demonstrated that the medium viscositystrongly influenced the overall uptake efficiency, and theseobservations alone could not reveal the subsequent intracellularfate of the NPs. To further elucidate the intracellular traffickingpathway of the internalized NPs, Rho-Fe3O4 NPs were employedin combination with LysoTracker and Hoechst staining to visual-ize their subcellular localization. Therefore, fluorescence co-localization analysis was used to investigate whether the interna-lized NPs were delivered to lysosomes, the major destination ofendocytic vesicles. The merged fluorescence images confirmedthe co-localization of Rho-Fe3O4 NPs with LysoTracker-stainedlysosomes (Fig. 5), indicating that the cellular internalization ofFe3O4 NPs predominantly occurs through the endo-lysosomalpathway. Importantly, this lysosomal trafficking patternremained unchanged across media of different viscosities,suggesting that although extracellular rheological conditionsregulate the efficiency of NP uptake, they do not alter the funda-mental intracellular internalization route.3.6. Magnetic heating effect of intracellular Fe3O4 NPsBased on the results shown in Fig. 3B and C, the cells culturedin different viscosity media containing 120 μg mL−1 Fe3O4 NPsfor 24 h were used for investigating magnetic heating effect.The results in Fig. 6A and B revealed that cells without NPs(control) did not induce temperature increase, while differentamounts of intracellular NPs resulted in varying degrees oftemperature changes. The cells cultured without PEG exhibitedthe highest uptake of NPs (Fig. 6B) and consequently causedthe most significant temperature change, with a temperaturerise of 7.3 ± 0.5 °C. This was followed by the group cultured inthe viscous medium prepared by adding PEG 8M and PEG 35Kat a ratio of 1 : 1, in which the intracellular NPs led to atemperature increase of 5.4 ± 0.7 °C. When PEG 8M : PEG35K = 1 : 0 were added in the culture medium, the cells wereexposed to the highest viscosity and internalized the leastamounts of Fe3O4 NPs, resulting in an increase in tempera-ture of only 2.3 ± 0.3 °C. When the temperature incrementwas normalized to the cellular uptake amount of Fe3O4 NPs,the temperature increment per amount of uptaken Fe3O4NPs was not significantly different among the 5 samples(Fig. 6C). The results indicated that the temperature incre-ment was predominantly due to the uptake amount of Fe3O4NPs.3.7. Magnetic hyperthermia effect of internalized Fe3O4 NPsWhen the temperature slightly exceeds the normal physiologi-cal limit, it can cause proteins to unfold, become entangledand form non-specific clumps.50 This can trigger the heatshock response and lead to the onset of programmed celldeath.51,52 The various temperature increments generated bydifferent intracellular amounts of NPs might induce differentdegrees of cell apoptosis. Therefore, cell apoptosis was ana-lyzed before and after AMF irradiation, and the results areshown in Fig. 7A and B.Cells cultured in a normal medium without Fe3O4 NPs andPEG were used as a control. Before AMF irradiation, there wasno significant difference in cell apoptosis compared to cells inthe control group, indicating that PEG and NPs could notinduce cell apoptosis. However, after AMF irradiation, theapoptotic rate of cells cultured in the normal medium withoutPEG significantly increased to 40.2% ± 4.8% compared to theFig. 4 Intracellular pH measurements under different viscosity media using the pHrodo™ AM assay.Nanoscale PaperThis journal is © The Royal Society of Chemistry 2026 Nanoscale, 2026, 18, 10691–10702 | 10697control group. Moreover, with the increase in the viscosity ofcell culture medium by adjusting the weight ratio of PEG 8Mand PEG 35K from 1 : 1 (LV) to 1 : 0 (VHV), the cell apoptoticrate gradually decreased from 22.8% ± 2.2% to 9.45% ± 0.7%because the various viscosities of culture media influenced theNPs internalization and further influenced the magnetichyperthermia effect.4. DiscussionIn this study, the influences of microenvironmental viscosityon the uptake of Fe3O4 NPs by CRC cells and the intracellularmagnetic hyperthermia were investigated. The results demon-strated that both the uptake rate and the uptake amount ofFe3O4 NPs decreased significantly with the increasing microen-vironmental viscosity, which led to the limitation of intracellu-lar magnetic hyperthermia and ultimately reduced the apopto-sis rate. These findings suggest that the viscosity of microenvi-ronment could regulate Fe3O4 NP uptake and the efficacy ofmagnetothermal therapy.The influence of viscosity on the cellular uptake of Fe3O4NPs may be explained through its dual effect on Fe3O4 NPdiffusion and cell membrane dynamics. The diffusion of NPsin the cellular microenvironment can be influenced by avariety of physicochemical factors, among which the solutionviscosity is an important regulatory parameter. According tothe Stokes–Einstein equation,53 the diffusion coefficient isinversely related to the solution viscosity, and thus, thediffusion of Fe3O4 NPs is limited in a high viscosity microenvi-ronment, resulting in a reduced chance of them to reach thecell membrane. In addition, the high viscosity may reduce thefluidity of the cell membrane, thereby affecting the distri-bution and function of endocytosis-associated receptors andfurther inhibiting receptor-mediated endocytosis and other NPuptake pathways.47–49Fig. 5 Fluorescence imaging of SW480 cells incubated with Rho-Fe3O4 NPs (red) under media of different viscosities, co-stained with Lyso Tracker(green) to visualize lysosomes and Hoechst 33258 (blue) for nuclear staining.Paper Nanoscale10698 | Nanoscale, 2026, 18, 10691–10702 This journal is © The Royal Society of Chemistry 2026Fig. 6 Magnetic heating effect of internalized Fe3O4 NPs. (A) Temperature–time curve of internalized Fe3O4 NPs during AMF exposure. (B)Temperature increment after 10 min of AMF exposure. (C) Temperature increment normalized to uptake amount of Fe3O4 NPs. Data are expressedas mean ± S.D. (n = 3). N.S.: no significant difference.Fig. 7 Apoptosis of SW480 cells induced by intracellular magnetic hyperthermia of internalized Fe3O4 NPs. (A) Representative flow cytometryimage and (B) apoptosis rate of SW480 cells with different intracellular amounts of NPs after AMF irradiation. Data are expressed as mean ± S.D. (n =3). Significant difference: **p < 0.01, ***p < 0.001. N.S.: no significant difference.Nanoscale PaperThis journal is © The Royal Society of Chemistry 2026 Nanoscale, 2026, 18, 10691–10702 | 10699The observed differences in magnetic hyperthermia per-formance are attributed to variations in the internalized Fe3O4NPs when the cells were cultured in different viscosity media.A larger amount of internalized Fe3O4 NPs could generatemore heat to reach a higher temperature and induce a highratio of apoptosis because the magnetothermal property ofFe3O4 NPs is correlated with the amounts of NPs. In a high-vis-cosity microenvironment, the magnetic heating effect was wea-kened due to the decreased intracellular uptake amount ofFe3O4 NPs, resulting in a decreased apoptosis rate. Thisphenomenon suggests that the microenvironmental viscositycould affect the magnetic hyperthermia efficiency through itsinfluence on the cellular uptake of Fe3O4 NPs. Therefore, themicroenvironmental viscosity of tumor tissues should be con-sidered when designing MNP-based magnetothermal therapyprotocols to optimize the therapeutic effects.In addition to viscosity, various physicochemical and bio-logical factors are known to affect the cellular nanoparticleuptake, such as particle size, surface charge, protein coronaformation, nanoparticle surface chemistry, cell type, and incu-bation conditions. In this study, the Fe3O4 NPs were coatedwith citrate, which provides good colloidal stability and a nega-tively charged surface, facilitating reproducible cellular inter-actions under different medium conditions. Although theintracellular pH may also influence nanoparticle trafficking,the present study focused primarily on extracellular viscosity;under standard culture conditions, pH variations are expectedto be limited and not anticipated to significantly affect theFe3O4 NP uptake.In summary, the present study demonstrated that microen-vironmental viscosity is an important factor in affecting Fe3O4NP uptake and intracellular magnetothermal efficacy. A highlyviscous microenvironment could limit the diffusion of Fe3O4NPs and inhibit cellular uptake, thus reducing the heatingeffect and apoptosis rate of magnetic hyperthermia. Thesefindings provide some useful information for the optimizationof CRC magnetothermal therapy and help to guide the designof nanomedicine-based strategies to improve the therapeuticefficacy of magnetic hyperthermia.5. ConclusionIn this study, the viscosity of cell culture media was modulatedby PEG of different molecular weights to simulate the viscousmicroenvironments of colorectal cancer for the investigationof microenvironmental viscosity effect on the cellular uptakeof Fe3O4 NPs and intracellular magnetic hyperthermia. Theresults showed that the uptake speed and amount of Fe3O4NPs by CRC cells decreased with the increase in microenviron-mental viscosity, which led to a decrease in intracellular mag-netic hyperthermia. The killing effect of magnetic hyperther-mia was reduced and the apoptosis rate was significantlydecreased in a high-viscosity microenvironment due to thedecreased amounts of internalized NPs. The results of thisstudy provide some new insights into the role of viscosity ofthe CRC tissue microenvironment in the regulation of NPuptake and magnetic hyperthermia.Author contributionsConceptualization: G. C., M. W., Y. Y.; funding acquisition:G. C., N. K.; project administration: G. C.; resources: G. C.;supervision: G. C.; date curation: G. C., M. W., N. K.; formalanalysis: G. C., M. W., H. C., T. Y.; investigation: G. C., M. W.,T. Z., C. L., R. S., H. C., T. Y.; methodology: G. C., M. W., T. Z.,C. L., H. C., R. S., T. Y., N. K., Y. Y.; validation: G. C., M. W.,N. K.; software: M. W., N. K.; visualization: M. W.; writing –original draft: all authors; writing – review and editing: allauthors.Conflicts of interestThe authors declare no competing interests.Data availabilityThe data that support the findings of this study are availablefrom the corresponding author on request.AcknowledgementsThis research was supported by JSPS KAKENHI GrantNumbers 22K19926 and 24K03289.References1 Z. 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