RSC Advances PAPER Bioevaluation of magnetic mesoporous silica rods: Cite this: RSC Adv., 2022, 12, 31878 cytotoxicity, cell uptake and biodistribution in zebrafish and rodents† Jan Grzelak, a Mariana Teles,b Nerea Roher, b Alba Grayston,c Anna Rosell,c Martı́ Gich*a and Anna Roig *a Mesoporous silica nanoparticles (MSN) characterized by large surface area, pore volume, tunable chemistry, and biocompatibility have been widely studied in nanomedicine as imaging and therapeutic carriers. Most of these studies focused on spherical particles. In contrast, mesoporous silica rods (MSR) that are more challenging to prepare have been less investigated in terms of toxicity, cellular uptake, or biodistribution. Interestingly, previous studies showed that silica rods penetrate fibrous tissues or mucus layers more efficiently than their spherical counterparts. Recently, we reported the synthesis of MSR with distinct aspect ratios and validated their use in multiple imaging modalities by loading the pores with maghemite nanocrystals and functionalizing the silica surface with green and red fluorophores. Herein, based on an initial hypothesis of high liver accumulation of the MSR and a future vision that they could be used for early diagnosis or therapy in fibrotic liver diseases; the cytotoxicity and cellular uptake of MSR were assessed in zebrafish liver (ZFL) cells and the in vivo safety and biodistribution was investigated via fluorescence molecular imaging (FMI) and magnetic resonance imaging (MRI) employing zebrafish larvae and rodents. The selection of these animal models was prompted by the well-established fatty diet protocols inducing fibrotic liver in zebrafish or rodents that serve to investigate highly prevalent liver conditions such as non-alcoholic fatty liver disease (NAFLD). Our study demonstrated that magnetic MSR do not cause cytotoxicity in ZFL cells regardless of the rods' length and surface charge (for concentrations up to 50 mg ml−1, 6 h) and that MSR are taken up by the ZFL cells in large amounts despite their length of ∼1 mm. In zebrafish larvae, it was observed that they could be safely exposed to high MSR concentrations (up to 1 mg ml−1 for 96 h) and that the rods pass through the liver without Received 12th September 2022 Accepted 29th October 2022 causing toxicity. The high accumulation of MSR in rodents' livers at short post-injection times (20% of the administered dose) was confirmed by both FMI and MRI, highlighting the utility of the MSR for liver DOI: 10.1039/d2ra05750f imaging by both techniques. Our results could open new avenues for the use of rod-shaped silica rsc.li/rsc-advances particles in the diagnosis of pathological liver conditions. Since the rst report on mesoporous silica as a drug delivery Introduction system,6 MSN have been studied extensively as therapeutic Mesoporous silica nanoparticles (MSN) are solid materials that carriers.7 Although MSN have not yet been approved for clinical contain empty monodispersed channels in the mesoporous trials, considerable progress has been made in designing and range (between 2 and 50 nm, according to the IUPAC developing MSN-based agents for diagnostic8,9 and/or thera- classication).1–3 MSN biosafety, large surface area, large pore peutic2,10 purposes. volume, robustness, and the possibility of surface modica- Worth noting is that the vast majority of research on silica tion,2,4,5 have made this system very attractive in biomedicine. materials focuses on spherical particles,2,11–13 while not much attention has been given to anisotropic shapes, possibly due to the challenging fabrication of particles with well-controlled rod-like a Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Campus UAB, 08193 morphology. Although some studies on mesoporous silica nano- Bellaterra, Catalonia, Spain. E-mail: mgich@icmab.es; anna.roig@csic.es rods (MSR) have been reported, this is still an emerging eld. b Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, Available studies showed that the shape of silica particles could 08193 Barcelona, Spain c inuence their biodistribution, major lung accumulation,14,15 Neurovascular Research Laboratory, Vall d'Hebron Research Institute (VHIR), 08035, cellular uptake,16–20 toxicity,15,21 degradation,14,15 drug release22 or Barcelona, Catalonia, Spain † Electronic supplementary information (ESI) available. See DOI: mobility,23 but further knowledge is still necessary to evaluate how https://doi.org/10.1039/d2ra05750f the rod shape could be a determinant factor in the context of 31878 | RSC Adv., 2022, 12, 31878–31888 © 2022 The Author(s). Published by the Royal Society of Chemistry Paper RSC Advances diseases lacking early-stage diagnostic tests or therapeutic strate- glycol)-block-poly(ethylene glycol) (Pluronic P123), tetraethyl gies. A relevant example is non-alcoholic fatty liver disease orthosilicate (TEOS), (3-aminopropyl)triethoxysilane (APTES), (NAFLD), which is a major cause of liver conditions worldwide.24–26 uorescamine and triethylamine were purchased from Sigma- NAFLD progresses in approximately 10–20% of cases to non- Aldrich and used as received. Ethanol was purchased from alcoholic steatohepatitis (NASH), characterized by hepatocellular PanReac. Iron(III) nitrate nonahydrate was purchased from steatosis, inammation, and brosis, and can lead to more severe ACROS Organics and used as received. Cyanine5 NHS ester and irreversible liver damage with fatal consequences.27,28 (Cy5-NHS) was purchased from Lumiprobe and used as Pioneering studies reported that silica rods exhibit higher received. The purity of all reagents was 98% or higher. diffusivity than spheres in the intestinal mucus, leading to deeper ZFL were cultured at 28 °C (optimal temperature for penetration and longer retention time in the gastrointestinal culturing zebrash cells38), 5% CO2 in Dulbecco's modied tract.29,30 The superior diffusivity of silica rods has been explained Eagle's medium (DMEM) with 4.5 g l−1 glucose, supplemented by rotational dynamics facilitated by the brous structure of with 0.01 mg ml−1 insulin, 50 ng ml−1 EGF, 5% (v/v) antibiotic/ mucosal tissue and shear ow. Besides, it is well known that rod- antimycotic solution, 10% (v/v) heat-inactivated fetal bovine shaped pathogenic bacteria of the gastrointestinal tract are also serum (FBS) and 0.5% (v/v) heat-inactivated trout serum (TS) as highly mobile in mucus.31 We hypothesize that similar behavior described in literature.39 The MTT (3-(4,5-dimethylthiazol-2-yl)- could occur with MSR in brotic liver tissue, and they could offer 2,5-diphenyltetrazolium bromide) substrate and DMSO were potential advantages over spherical particles in an early-stage purchased from Sigma-Aldrich. diagnosis or therapeutic opportunities in the context of NAFLD and NASH. This work sets the basis for further studies in the Synthesis and functionalization of silica rods abovementioned eld by reporting on the in vitro cytotoxicity and Extensive details on the synthesis and functionalization of the cellular uptake of MSR in zebrash liver (ZFL) cells and the in vivo MSR used in this work have been previously reported.32 Briey, biodistribution of the MSR in zebrash larvae and rodents with Pluronic P123 was dissolved in 1.7 M hydrochloric acid solu- already existing models of brotic liver. tion. Aer obtaining a homogeneous solution, the temperature Recently, we synthesized MSR with two distinct aspect ratios was increased to 40 °C and the solution was stirred at 700 rpm. (∼2 and ∼5).32 We equipped the MSR with multiple imaging Aer 3 h, tetraethyl orthosilicate (TEOS) was added dropwise. modalities, loading the pores with magnetic nanoparticles and Stirring was stopped aer either 4 or 5 min, and the reaction functionalizing their surface with uorophores of two emission was kept in static conditions for either 3 h or 24 h. Aer that, the wavelengths. Here, by using magnetic measurements, uores- reaction was kept at 80 °C for 24 h. Silica was then ltered and cence molecular imaging (FMI), and magnetic resonance dried at 55 °C overnight. The surfactant was removed by imaging (MRI), we investigated the toxicity and biodistribution of washing the product in ethanol and the material was calcined in the MSR in two in vivo preclinical models of different complexity. air at 550 °C for 5 h. On the one hand, zebrash (Danio rerio) is one of the simplest The rods were loaded with iron oxide nanoparticles (NPs) organisms with a liver, and zebrash larvae can be used without using a wet impregnation method, with iron(III) nitrate non- ethical clearance. The larvae are transparent, and their liver is ahydrate as the precursor. Impregnation was performed by readily visible at low magnications. Besides, well-established heating the silica powder and the iron precursor at 60 °C, fol- animal models of NAFLD can be induced in both larvae and lowed by a thermal treatment at 425 °C under an argon ow adult zebrash by a specic feeding protocol.33–35 Uptake and with 5% of hydrogen (v/v). Amine groups were graed to the toxicity in vitro studies were undertaken using ZFL while we silica rods surface by APTES aminosilanization to enable the studied toxicity and biodistribution in zebrash larvae and subsequent attachment of uorophores. A stock solution of rodents. Higher complexity rodent models are relevant because uorescamine or Cyanine5 was added to an acetone dispersion previous studies of murine exposure to high aspect ratio meso- of amino-functionalized MSR, leaving the mixture to react porous silica reported accumulation in the lungs, with different overnight. The particles were puried by washing three times levels of toxicity.36 Still, other works concluded that rod-shaped with ethanol and centrifugation. Finally, the precipitate was mesoporous silica had good biocompatibility.37 Due to the scar- dispersed in a small amount of acetone and dried in vacuum. city of studies on MSR and the variability of rod dimensions, The product was stored at 4 °C in the dark. Table 1 summarizes animal models, doses and exposure times between the different the physico-chemical characteristics and the labeling to identify experiments, more studies are needed to understand the rela- the materials used in the present study. tionship between the silica particle shape and toxicity in rodents. Our study shows low cytotoxicity and high liver accumulation of MSR in zebrash larvae and rodents, setting the ground for Zebrash liver cell viability studies further studies involving brotic liver models. Cytotoxic of MSR on ZFL were assessed using the MTT assay. ZFL were cultured in 75 cm2 cell culture asks with vented caps Materials and methods (Falcon®). Conuent cells were lied using 2 ml TrypLE Express (ThermoFisher) and seeded in 24 well plates (cells from one Materials ask used for seeding in two plates) in complete medium, 500 ml MSR were synthesized using the following reagents. Hydro- per well. Aer 16 hours, the medium was changed to a minimal chloric acid (37%), poly(ethylene glycol)-block-poly(propylene medium (0–0.5% FBS; 2% antibiotic/antimycotic). Aer 2 h of © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12, 31878–31888 | 31879 RSC Advances Paper Table 1 Physico-chemical characteristics of the materials used in test. One-way analysis of variance (one-way ANOVA) was per- these studies formed to assess signicant differences among groups. When signicant differences were found, the one-way ANOVA was Mesoporous silica rods (MSR) Long rods (LR) Short rods (SR) followed by the post-hoc Tukey's test to signal signicant Length (mm) 1.4  0.3 0.9  0.1 differences between groups and to the control group. The Width (mm) 0.3  0.1 0.4  0.1 signicance of the results was ascertained at p < 0.05. Aspect ratio 4.7  1.3 2.2  0.5 Zeta potential (mV) −38  5 −34  4 ZFL cell uptake studies Rods loaded with Fe2O3 NPs Fe2O3@LR Fe2O3@SR Before the exposure, the cells in culture were counted in NP size (nm) 73×52 73×52 a Neubauer chamber and 125 000 cells per well were seeded in Crystallographic phase Maghemite Maghemite 96 well plates. The cultures were incubated with MSR similarly (100%) (100%) as described for the MTT assay protocol. Aer 6 h, the cells were Iron oxide content (wt%) 15  1 15.0  0.3 washed with PBS and centrifuged (200 × g, 10 min at 4 °C). The Saturation magnetization at 43  4 42  4 uptake of MSR by ZFL was assessed using magnetometry 300 K (emu g−1 Fe2O3) r2 relaxivity (s−1 mM−1) 143  14 108  11 measurements at 10 K. The cell pellets were collected from the wells and their magnetic moment measured by super- Rods loaded with Fe2O3 NPs conducting quantum interference magnetometer device and functionalized with amine (SQUID, Quantum Design Inc.) up to a maximum applied eld groups Fe2O3@LR-NH2 Fe2O3@SR-NH2 of 60 kOe. The values of magnetic moment at remanence (i.e. at Zeta potential (mV) 33  5 34  5 zero magnetic eld aer applying the maximum eld) were divided by the number of cells calculated before the experiment. Rods functionalized with From previous measurements of the magnetization (magnetic uorescamine LR-FL SR-FL moment per unit mass) of MSR samples recorded in the same Emission wavelength (nm) 525 525 conditions, it was possible to obtain the mass of MSR contained in each pellet. The number of uptaken particles per cell was Rods loaded with Fe2O3 NPs determined from the relative content of Fe2O3 in Fe2O3@SR (15 and functionalized with Cy5 Fe2O3@LR-Cy5 Fe2O3@SR-Cy5 wt% see Table 1), assessing the mass of a MSR of about 0.2 pg in Emission wavelength (nm) 733 733 the case of short rods. This mass, m, was calculated from the formula m = (rpr2L)/(1 + rVtotal), where r is the density of silica (2.2 g cm−3), r is the mean radius of the MSR, L is the mean length of the MSR, and Vtotal is the pore volume determined from nitrogen adsorption experiments.40 incubation, cell cultures were treated with MSR (Fe2O3@LR, Fe2O3@LR-NH2, Fe2O3@SR, Fe2O3@SR-NH2, each MSR type Evaluation of toxicity and biodistribution by uorescence and condition in triplicate). Ten percent of culture medium imaging in zebrash larvae volume was removed and replaced with the same volume of Wild-type zebrash (Danio rerio) were kept in a re-circulating either water or stock solution of MSR, to obtain MSR concen- aquarium with water temperature maintained between 26 and trations of 0, 5, 20, 50 and 200 mg ml−1. The cells were incubated 28 °C. The lighting conditions were 14 : 10 h (light : dark) and with MSR for 6 h at 28 °C. Cells were then washed with PBS and adult sh were fed twice a day at a rate of 2% body weight, the MTT substrate was added to 10% of the total volume and following a normal feeding protocol. Ammonia and nitrite further incubated at 28 °C for 30 min. The solution was levels were kept below the detection level at a pH between 6.8 removed, the cells were solubilized in DMSO and the lysate and 7.5. The nitrate levels were maintained at <100 mg l−1. For absorbance was read on a Victor 3 plate reader (PerkinElmer) at in-tank breeding, one female and three males were transferred 550 nm. The experiments compared two sets of materials on to a breeding tank in the late aernoon. The divider was each plate (Fe2O3@LR with Fe2O3@SR, Fe2O3@LR with removed the next morning aer the onset of light. Eggs were Fe2O3@LR-NH2, Fe2O3@SR with Fe2O3@SR-NH2, or Fe2O3@LR- collected aer 1–2 h and cultured in embryo medium (E3 NH with Fe2O3@SR-NH2) in duplicates, and each type of medium) in a Petri dish (Deltalab). Fertilized eggs were sepa- material was assessed in 6 different experiments. The mean of rated from unfertilized eggs using a plastic pipette (Deltalab). control wells was set as 100% in each experiment. The medium was changed every 24 h and, aer hatching (approximately 3 days post-fertilization), the larvae were placed Statistical analysis in a 96-well plate (ThermoFisher), one larva per well containing Results are expressed as mean ± standard deviation (SD). 200 ml of aquarium water with uorescent MSR (functionalized Statistical analysis was done using GrahPad Prism (version 7.01 with uorescamine) at 0, 20, 50, 100, 200, 400 and 1000 mg ml−1. for Windows, GraphPad Soware, San Diego, California USA, For mortality studies, groups of larvae (n $ 5 per condition) www.graphpad.com). The assumptions of normality and were used and monitored for 96 h using an optical microscope homogeneity of data were veried using the Shapiro–Wilk (Olympus). For uptake studies, groups of 4 larvae per condition 31880 | RSC Adv., 2022, 12, 31878–31888 © 2022 The Author(s). Published by the Royal Society of Chemistry Paper RSC Advances were used. Aer 24 h and 48 h of exposure, the uptake was congured with 2 transmission channels and 4 reception conrmed by imaging the larvae using a uorescence stereo- channels. The experiments were carried out using a volumetric microscope (Nikon SMZ800) coupled with a camera (Nikon DS- antenna of 72 mm in diameter with the transmission and Fi2) and a mercury lamp (Nikon C-LHGFI HG lamp, 130 W, reception of the signal optimized for the body of a rat. The wavelength range: 380–600 nm). animals were kept under anesthesia during the measurements using isourane (1–1.5%). The temperature of the animal was Fluorescence imaging in mice kept at approximately 37.0 °C through a system of hot air (SA instruments, NY, USA) and its respiration and temperature Biodistribution studies by FMI were performed at the Preclin- during the MRI image acquisition were monitored through ical Imaging Platform from VHIR (Barcelona). Ethical approval a system SAII M1030 (SA instruments, NY, USA), which at the for the experiments was received by the Comité same time was used to synchronize the acquisition of images d'experimentació animal del Vall d'Hebron Institut de Recerca, with the animal respiration. The monitoring was done at very protocol 70.18/10860. short time intervals. The MRI session started with the acquisi- FMI was performed to track the biodistribution of tion of basal scans before the injection of MSR. For the injec- Fe2O3@SR-Cy5 in vivo and ex vivo using an IVIS® Lumina LT tion, the MRI bed where the animal was located was taken out Series III imaging system (PerkinElmer, Waltham, MA). All without changing the position of the animal and intravenous images were acquired at the following lex/lem ranges: 625–655 injection with 1.2 ml of dispersion of Fe2O3@SR (3 mg ml−1 in nm/695–770 nm, centered at 640 nm/732 nm, respectively. an aqueous solution of D-mannitol, 55 mg ml−1) via the tail vein To study the in vivo/ex vivo biodistribution, C57BL/6JRj adult was performed. Aer the injection, the bed was introduced male mice (8 weeks of age) were injected via the tail vein with 200 inside the scanner in the same position as before. The scans ml of 3 mg ml−1 Fe2O3@SR-Cy5 dispersion in a D-mannitol aqueous were repeated until acquiring the last scans at t = 60 min aer solution (55 mg D-mannitol/ml H2O). For the in vivo acquisitions, the injection. For T2 maps, MRI signal was measured at coronal mice were anesthetized with isourane via facemask (5% for planes using the sequence of Bruker MSME (multi slice multi- induction, 1.5% for maintenance in 95% O2) and in vivo images in echo) with the following parameters: 20 echo times (TEs) the dorsal and ventral views of the whole body were acquired at within the interval TE = 7.5–150 ms, respiration synchronized 30 min and 60 min post-injection. At the end of the scan, mice with the acquisition, field-of-view (FOV) = 60 × 60 mm, ACQ were euthanized by cervical dislocation under anesthesia and matrix = 256 × 256, RECO matrix = 256 × 256, slice thickness brain, heart, lungs, liver, spleen, and kidneys were dissected to = 1.5 mm, N slices = 8, averages = 2. For T*2 maps, MRI signal measure the uorescence of the principal organs ex vivo. A control was measured at axial planes using the sequence of Bruker MGE animal was used as a background measurement both in vivo and (Multi gradient echo) with the following parameters: 10 TEs ex vivo. Four out of ve mice were successfully injected. There were within the interval TE = 3–39 ms, respiration synchronized with problems with injecting one animal, therefore, this animal will not the acquisition, FOV = 60 × 60 mm, ACQ matrix = 256 × 192, be considered in the following discussion. RECO matrix = 256 × 256, slice thickness = 1.5 mm, N slices = For quantication, circular ROIs were manually drawn 8, averages = 2. surrounding the uorescence signal and total radiant efficiency Aer the MRI session, the animals were euthanized, (TRE; [photons per s]/[mW cm−2]) was measured using the perfused with a saline serum and their livers and lungs extrac- Living Image soware (PerkinElmer, Waltham, MA) and cor- ted. Organs of an untreated animal were also extracted and used rected by the TRE from the corresponding ROI in the back- as a control. The organs were frozen at −80 °C and freeze-dried ground control animal. For the in vivo analysis, abdominal ROIs in a LyoQuest-Telstar lyophilizer (0.05 mBar, 72 h). Then the were drawn on the ventral images due to the position of the organs were crushed and made into a homogeneous powder. organs of interest. For the ex vivo analysis, ROIs of each Magnetic hysteresis loops of the dried organ powders were dissected organ were drawn in both dorsal and ventral posi- collected at 10 K in a superconducting quantum interference tions, and the mean value was considered. magnetometer device (SQUID, Quantum Design Inc.) with a maximum applied eld of 50 kOe. MRI imaging in rats The chemical analysis of the dry organs was made by MRI in vivo experiments were performed in CIC BiomaGUNE inductively-coupled plasma and optical spectroscopies (ICP- (Donostia – San Sebastián) in the Singular Scientic and Tech- OES). For this analysis, approximately 0.50 g of liver sample in nical Infrastructure (ICTS) of ReDIB-Molecular and Functional duplicate or 0.05 g of lung sample in duplicate were digested Imaging Facility. The animal license of the ICTS is AE- with a mixture of concentrated HNO3 and HF in a microwave biomaGUNE-1116. Five female rats of RjHan:Sb type (9 weeks oven (Milestone, Ultrawave model). The resulting digestions of age) were used in this study. Four rats treated with MSR and were introduced into an ICP-OES spectrometer (Agilent, model an untreated control animal were imaged for biodistribution 5900), and the content of Fe and Si was determined. studies. Magnetic resonance imaging (MRI) in vivo experiments were Results and discussion performed at 7 T on a Bruker Biospec 70/30 equipped with a system of gradients of 12 cm interior diameter (400 mT m−1) MSR of Santa Barbara Amorphous-15 (SBA-15) type were and connected to a console Bruker AVANCE III (300 MHz) synthesized using a sol–gel method described in our previous © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12, 31878–31888 | 31881 RSC Advances Paper work.32 Herein, we focus on the safety and biodistribution of rod-like silica particles of two distinct lengths (1.4 and 0.9 mm) and aspect ratios (AR = 4.7 and 2.2), denominated as long rods (LR) and short rods (SR) and with additional specic function- alizations. Namely, those rods were modied by growing superparamagnetic maghemite (g-Fe2O3) nanocrystals within the pores (Fe2O3@LR, Fe2O3@SR). In a subsequent step, the rods were functionalized with amine (NH2), rendering them with a positive surface charge (Fe2O3@LR-NH2, Fe2O3@SR- NH2), or further enabling the graing of uorophores (uo- rescamine and Cyanine5) with emissions at l = 525 and 730 nm respectively (Fe2O3@LR/SR-Cy5 and LR/SR-FL). Representative images of the two MSR systems are included in Fig. S1.† Cytotoxicity and cell uptake studies in ZFL cells Cell viability and cellular uptake assays were performed using Fig. 2 Number of the MSR uptaken by ZFL cells presented as the mass ZFL cells (Fig. 1a). ZFL cell viability was studied using the MTT of sample per cell (left axis) and number of MSR per cell (right axis). **P reduction assay (Fig. 1b) aer exposing the cells for 6 h to long # 0.01, ****P # 0.0001. and short rods with negative and positive surface charges (Fe2O3@LR, Fe2O3@LR-NH2, Fe2O3@SR, Fe2O3@SR-NH2). It can be observed that none of the four MSR types caused cyto- The uptake of MSR by the ZFL cells was then studied by toxicity in ZFL cells for concentrations up to 50 mg ml−1. At 200 SQUID magnetometry at 10 K. Concentrations of the short rods mg ml−1, viable cells decreased signicantly for negatively (Fe2O3@SR, Fe2O3@SR-NH2) up to 100 mg ml−1 were used for (Fe2O3@LR) and positively charged (Fe2O3@LR-NH2) long rods. this experiment which we have considered the safest ones. The The lowest toxicity in ZFL cells at 200 mg ml−1 was observed for high sensitivity of the SQUID technique allows detecting very positively charged short rods Fe2O3@SR-NH2. small magnetic moments. The remanent magnetic moment is A relation between the aspect ratio of MSR and cytotoxicity proportional to the amount of the magnetic material and it is has been reported in the literature.17,21 However, this effect is not affected by the diamagnetic signal from organic matter.43 complex and greatly depends on the cell type.41,42 The cell Fig. S2a and b† show a representative hysteresis loop of MSR viability assay results performed on ZFL cells indicate that the and a ZFL cell pellet. The values calculated for each type of MSR toxicity depends on the concentration and aspect ratio of MSR. are shown in Table S1† and depicted in Fig. 2. This method At the same time, the surface charge does not have a signicant allowed us to calculate the uptake of MSR by ZFL cells and effect on the studied conditions. Note that the MSR of AR = 2.2 express it as the mass of the sample and an estimation of the (Fe2O3@SR, Fe2O3@SR-NH2) induced less cytotoxicity in ZFL number of uptaken rods per cell (see Methods section). cells at 200 mg ml−1 than rods of AR = 4.7 (Fe2O3@LR, The uptake of MSR was concentration-dependent for both Fe2O3@LR-NH2). types of short rods. There were no signicant differences in the Fig. 1 (a) ZFL cells observed by an optical microscope, (b) ZFL cell viability after 6 h of exposure to the different types of MSR and four concentrations (n = 6 with technical replicates), plotted as mean + SD. The correspondence of p-values to the number of asterisks is the following: *P # 0.05, ***P # 0.001. The horizontal dotted line at 100% viable ZFL cells corresponds to the average number of viable cells of the control group, against which the viabilities were normalized. The asterisks above the bars represent statistically significant differences with respect to the control group. 31882 | RSC Adv., 2022, 12, 31878–31888 © 2022 The Author(s). Published by the Royal Society of Chemistry Paper RSC Advances uptake between the negatively and positively charged rods. MSR h), aer the subsequent 24 h exposure (i.e., aer 48 h), it were readily uptaken by the ZFL cells without inducing cyto- appeared that the uorescence signal moved to the intestine toxicity despite a large number of internalized rods (>500 per (Fig. 4c, f and i). cell) and their dimensions (average length 0.9 mm). Globally, the experiments on zebrash larvae suggest that MSR pass through the liver of the larvae without causing toxicity, even when exposed to concentrations as high as 1000 mg ml−1. Biodistribution in zebrash larvae and effects on survival This sets the ground for the future use of MSR in zebrash fed Zebrash larvae were used as an intermediate model between in with a high-fat diet as a fatty liver disease model.33,44 vitro studies and rodents in vivo experiments to study the uptake and toxicity of the MSR. The larvae were incubated with MSR functionalized with uorescamine to visualize the rods inside Fluorescence imaging studies in mice the larvae using uorescence microscopy. The biodistribution of MSR in rodents aer intravenous injec- Fig. S3a–d† shows the adult zebrash and the stages between tion was studied by FMI and MRI. For these experiments, we zebrash eggs and larvae. The zebrash eggs were collected and selected the short magnetic rods, which had the lowest toxicity placed in E3 medium. Fertilized eggs were separated from in the cell studies. Cyanine5-labeled short rods with iron oxide unfertilized ones. Aer hatching, 3 days post-fertilization larvae NPs (Fe2O3@SR-Cy5) were used in FMI studies performed on were placed in a 96 well plate (1 larva per well) and incubated C57BL/6JRj mice. A clear uorescence signal was observed at with a dispersion of uorescamine-functionalized long and 30 min in the abdominal area of animals 1, 2 and 4 (Fig. S4a†) short rods (LR-FL and SR-FL). The toxicity of MSR on the larvae and remained relatively unchanged at 60 min (Fig. S4b†). At was monitored by observing the larvae in an optical microscope 60 min, uorescence was also observed in the abdomen of up to 96 h aer exposure to various concentrations (20, 50, 100, animal 3. The quantitative analysis of uorescence intensity 200, 400, and 1000 mg ml−1; n $ 5), 100% survival was observed (table included as Fig. S4c†) in the abdomen has shown similar at 96 h for each studied concentration (Fig. S3e†). uorescence values in the rst three animals at 30 min, and The uptake of MSR by zebrash larvae was then studied in a slight decrease of these values was observed at 60 min. a uorescence stereomicroscope. At 96 h of exposure, a strong Aer imaging at 60 min, the mice were sacriced and the green uorescence of uorescamine-functionalized rods was major organs (brain, lungs, liver, spleen, and kidneys) were easily distinguishable from the larva autouorescence, showing extracted. The uorescence images of the organs of treated the nanoparticles' location in the liver and intestine (Fig. 3). animals were compared with control (Fig. 5a) and uorescence The evolution of MSR in the zebrash larvae was tracked intensity was quantied (Fig. 5b). As expected, high uores- using the highest exposure concentration of 1000 mg ml−1. Aer cence was observed in the livers and similar intensity in the 24 h exposure, the larvae were observed in the uorescence spleen and kidneys. No uorescence was observed in the brains. microscope and transferred to clean aquarium water. Aer an In contrast to the results of biodistribution studies of MRI additional 24 h, the same larvae were observed. While at 24 h of imaged rats (see next section), the uorescence studies also exposure, MSR seemed to accumulate in the liver (Fig. 4b, e and indicate accumulation of MSR in the lungs. Fig. 3 MSR uptake by zebrafish larvae. Bright-field microscopy images (top) and corresponding fluorescence images (bottom) of an untreated larva (a) and (b) and two distinct representative larvae incubated with LR-FL (1 mg ml−1) for 96 h (c)–(f). Red arrows mark the fluorescence from MSR. Scale bars: 1 mm. © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12, 31878–31888 | 31883 RSC Advances Paper Fig. 4 Evolution of MSR uptake in zebrafish larvae. Bright-field optical microscopy images of an untreated larva (a) and a larva treated with LR-FL at 1000 mg ml−1 (b) and (c). Corresponding fluorescence images after 24 h (d) and (e) and 48 h (f). Corresponding high magnification images showing regions of interest (g), (h) and (i). Scale bars: 1 mm. Fluorescence from MSR is indicated with arrows. MRI studies in rats time values (Fig. S5g†). Due to the characteristics of the lungs, The magnetic MSR were further used as MRI T2 contrast agents changes in MRI T2-contrast in this organ cannot be easily seen. Therefore, the potential pulmonary involvement was assessed in experiments on rats. Four rats were injected with a dispersion indirectly by monitoring the animal respiration rate. During the of Fe2O3@SR (3 mg ml−1 in D-mannitol, 1.2 ml) and the MRI T2 acquisition of MRI scans, no critical changes in animal respi- contrast was observed until 60 min post-injection. MRI scans ration or body temperature have been observed (Fig. S5h†). All were performed in coronal and axial planes. T2 relaxation time four animals survived the experiment. maps recorded in coronal planes for two studied rats are shown T*2 relaxation time maps recorded in axial planes are shown in Fig. S5a–f.† Slices in which the liver covers the most extensive area were selected and compared. The coronal MRI T2 images in Fig. 6a–f. Similar to the T2 maps, slices with the largest area did not show a signicant contrast change throughout the occupied by the liver were analyzed. It can be seen that aer the injection of MSR, the contrast in the liver decreases. The maps experiment. These ndings are conrmed by the T2 relaxation Fig. 5 (a) Ex vivo fluorescent images of organs extracted from mice treated with Fe2O3@SR-Cy5, (b) fluorescence values measured in the major organs. 31884 | RSC Adv., 2022, 12, 31878–31888 © 2022 The Author(s). Published by the Royal Society of Chemistry Paper RSC Advances Fig. 6T*2 relaxation time maps of the abdominal area of (a)–(c) animal 1 and (d)–(f) animal 2 recorded at various times: t = 0 (pre-injection), t = 30 min and t = 50 min post-injection of Fe2O3@SR, (g) T*2 values in the liver of the two studied rats, before and 50 min after MSR injection. have been used to determine the T*2 values in the liver (Fig. 6g). quantitative biodistribution of magnetic rods was obtained by The results indicate a two-fold decrease of the MRI T*2 contrast SQUID magnetometry and ICP-OES. Magnetization vs. magnetic in the liver for both animals. We thus conclude that T*2 eld measurements of liver and lung samples of control and sequences of axial planes were more effective in imaging the treated animals were measured at 10 K. Fig. 7e–f shows the data liver with the Fe2O3@SR. Furthermore, considering the better corresponding to half loop measurements (sufficient for our relaxivity value for the Fe2O3@LR,32 this system should also be purposes) aer correcting the diamagnetic contribution. The investigated in the future now that it is proved that the rats did organs of both treated animals displayed similar magnetization not show adverse effects under the administration of SR. curves. In the case of livers, a small magnetic remanence was observed for the control animal, and this value was subtracted Ex vivo MSR quantication from the Mr measured for the livers of treated animals. Following the scans, the animals were sacriced and the major The remanence values and the MSR content in the organs organs of interest were extracted to conrm the high accumu- calculated from the remanent magnetization of Fe2O3@SR (Mr lation of MSR in the liver and assess the involvement in the = 3.3 emu g−1) are listed in Table 2. lungs that could not be observed by MRI. Livers and lungs were The results were compared with data obtained from ICP- lyophilized and made into a homogeneous powder (Fig. 7a–d). A OES, where the amount of iron and silicon in liver and lung Fig. 7 Lyophilized organs of rats: (a) lungs and (b) liver. Homogenized samples of (c) lungs and (d) liver. Scale bars: 1 cm. Magnetic hysteresis loops at 10 K were recorded for (e) livers and (f) lungs. Table 2 Fe2O3@SR quantification by SQUID in livers and lungs of rats, 60 min after administration Total organ MSR mass in the Organ Mr (10−3 emu g−1 sample) mass (g) Total mr of the organ (10−3 emu) organ (mg) Liver 1 1.00 2.18 2.18 0.66 Liver 2 1.15 2.15 2.47 0.75 Lungs 1 2.86 0.23 0.66 0.20 Lungs 2 2.14 0.24 0.51 0.16 © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12, 31878–31888 | 31885 RSC Advances Paper Table 3 Fe2O3@SR quantification by ICP-OES analysis of Fe and Si in the organs calculated from Fe and Si analysis by ICP-OES was livers and lungs of control (CT) and treated (1, 2) rats then compared with data derived from SQUID measurements (Fig. 8). The total mass of MSR injected in each animal was Total organ Fe content Si content Total Fe Total Si Organ mass (mg) (mg g−1) (mg g−1) (mg) (mg) 3.6 mg. The results of MSR quantication by SQUID are in good Liver CT 1927 0.57 <0.01 1.10 0.02 agreement with those derived from Si elemental analysis. Both Liver 1 2182 0.50 0.16 1.10 0.35 techniques indicate circa ve-fold higher uptake by the liver Liver 2 2152 0.58 0.13 1.25 0.28 Lungs CT 232 0.09 <0.05 0.02 0.01 compared to the lungs. Moreover, approximately 20% of the Lungs 1 230 0.29 0.30 0.07 0.07 injected sample accumulated in the liver 60 min aer admin- Lungs 2 244 0.26 0.12 0.06 0.03 istration. Accumulation in these organs was compared with other results reported in the literature for different MSN (Table 4). For comparison, the accumulation was expressed in % of samples of control and treated animals were measured (Table injected dose per gram of tissue (% ID per g tissue). 3). The quantication of iron using this method was not Our liver accumulation values are comparable to those of ref. possible for liver samples due to the endogenous iron present in 45 and 46, while longer times are related to higher liver accu- the liver, as seen from the control organ. Endogenous iron was mulation. Regarding lungs, the results seem to indicate that for also found in the lungs, although in smaller quantities. the smallest particle size or the shortest post-injection times, The total mass of MSR sample in the organs was then ref. 37 and 45, the highest lung accumulation occurs. However, calculated from ICP-OES, assuming 10.5 wt% iron in Fe2O3@SR the results can not be easily compared due to the differences in and similarly, 39.8 wt% of silicon. The total amount of rods in time post-injection, the animal model used, and the size of the particles. More experiments at longer exposure times and a systematic study for different MSR sizes and aspect ratios are needed to establish optimal features for an increased liver accumulation. Conclusions We have studied the in vitro cytotoxicity and in vivo safety and biodistribution of MSR with 1.4 and 0.9 mm in length, distinct aspect ratios (LR, AR = 4.7 and SR, AR = 2.2) and different surface charges, functionalized with magnetic nanoparticles and uorophores (uorescamine and Cyanine5). Studies carried out on ZFL cells showed no toxicity at 6 h for concen- trations up to 50 mg ml−1 of both long and short rods, negatively and positively charged. At 200 mg ml−1, the toxicity was signif- icantly higher for long rods and still very low for both types of short rods. We observed that the rod's surface charge does not signicantly affect cytotoxicity for this cell type. The uptake of MSR by ZFL cells was found to be concentration-dependent and Fig. 8Biodistribution in livers and lungs calculated from SQUID and similar for negatively and positively charged rods. The results ICP-OES data. indicate that despite its dimensions, a large number of rods Table 4 Comparison of the liver and lung accumulation of MSR. The accumulation of MSR expressed in % of injected dose per g of tissue is the mean of the values calculated by the SQUID and ICP-OES techniques Time post-injection Liver accumulation Lung accumulation Source Material Dimensions (nm) (h) (% ID per g tissue) (% ID per g tissue) This work MSR loaded with Fe2O3 NPs 400 × 900 1 10  1 18  5 Ref. 45 MS spheres 120 0.5 17  3 147  37 2 11  3 140  52 MSR 136 × 1028 0.5 19.0  5.0 132  23 2 21.4  7.7 62 Ref. 37 MSR 150 × 250 3 45.0  5.0 18  3 MSR 150 × 450 3 41.0  5.0 18  4 Ref. 46 MS, almost spherical 900 1 18  3 12  0.1 Ref. 47 Porous silicon discoidal particles 400 × 1000 4 26  2 17  3 31886 | RSC Adv., 2022, 12, 31878–31888 © 2022 The Author(s). Published by the Royal Society of Chemistry Paper RSC Advances (>500) and up to 100 pg per cell were uptaken by the ZFL cells. In investigation, formal analysis, writing-review and editing. Anna vivo studies on zebrash larvae showed no toxicity at concen- Rosell: resources, investigation, formal analysis, writing-review trations up to 1 mg ml−1 for 96 h exposure time. Interestingly, and editing. Martı́ Gich: conceptualization, formal analysis, uorescence in the larvae liver was observed at 24 h, while at resources, funding acquisition, supervision, writing-original 48 h the uorescence came from the intestinal area. These dra, writing-review and editing. Anna Roig: conceptualiza- results open the door to evaluate further the diagnostic and tion, formal analysis, resources, funding acquisition, supervi- therapeutic potential of MSR for brotic liver using a zebrash sion, writing-original dra, writing-review and editing. model fed with a high-fat diet. Fluorescence imaging was per- formed in vivo in mice aer intravenous injection of Cyanine5- functionalized magnetic short rods (Fe2O3@SR-Cy5). Bright Conflicts of interest uorescence was observed in the abdomen at 30 min and There are no conicts to declare. 60 min post-injection. Importantly, ex vivo imaging of the major organs showed a substantial accumulation in the liver, con- rming our initial hypothesis. However, the accumulation in Acknowledgements mice's lungs and particularly kidneys, not observed in rats, deserves further studies. MRI studies were performed on rats The authors acknowledge nancial support from the Spanish aer intravenous injection with Fe2O3@SR. A decrease of MRI Ministry of Science and Innovation through the PID2021- T*2 signal in the axial plane was seen at 30 min and 60 min in the 122645OB-100 project, the ‘Severo Ochoa’ Programme for liver, corroborating a high accumulation in the liver. The Centers of Excellence in R&D (CEX2019-000917-S). The Gen- injection did not cause any abnormal respiration in the eralitat de Catalunya, projects 2017SGR765 and 2017SGR1427, animals. Biodistribution studies of extracted organs also are also acknowledged. The authors participate in the Aerogels conrmed liver accumulation. The MSR content in the liver and COST ACTION (CA 18125). J. G. has received nancial support lungs was quantied by SQUID magnetometry and ICP-OES through the “la Caixa” INPhINIT Fellowship Grant for Doctoral elemental analysis leading to similar estimates. Data show Studies at Spanish Research Centers of Excellence (grant code: that 20% of MSR were found in the liver, approximately ve LCF/BQ/DI17/11620041), “la Caixa” Banking Foundation times more than in the lungs. The observed low toxicity, cellular (ID100010434), Barcelona, Spain. J. G. was enrolled in the uptake, and signicant liver accumulation of the multifunc- doctoral program in Materials Science at the UAB. M. T. (ref. tional MSR highlight their potential use for their future evalu- RYC2019-026841-I) has a post-doctoral fellowship “Ramón y ation in some liver diseases, such as NAFLD. Studies using fatty Cajal” supported by the “Ministerio de Ciencia e Innovación”, liver animal models should be performed to determine whether Spanish Government. A. G. has been supported by the fellow- rods can accumulate more efficiently in fatty or brotic liver ship from Instituto de Salud Carlos III with FEDER funds (FI17/ tissue than their spherical counterparts. 00073). A. Rosell takes part of the RICORS-STROKE network from Instituto de Salud Carlos III with FEDER funds (RD21/ 0006/0007). This research work was performed in the frame- Ethical statement work of the Nanomedicine CSIC HUB (ref. 202180E048). Dr Fluorescence imaging in mice were performed at the Preclinical Daniel Padro is acknowledged for the supervision of MRI in vivo Imaging Platform from VHIR (Barcelona). All mice procedures experiments in ReDIB-Molecular and Functional Imaging were performed in accordance with the Guidelines for Care and Facility at CIC BiomaGUNE which were accessible through the Use of Laboratory Animals of VHIR. Ethical approval for the Spanish network of Singular Scientic and Technical Infra- experiments was received by the Comité d'experimentació structure (ICTS). Authors thanks Dr Fernando Herranz (IQM- animal del Vall d'Hebron Institut de Recerca, protocol 70.18/ CSIC) for fruitful discussions. 10860. MRI experiments in rats were performed in CIC Bio- maGUNE (Donostia – San Sebastián) within the Singular Notes and references Scientic and Technical Infrastructure (ICTS) of ReDIB- Molecular and Functional Imaging Facility. Animal proce- 1 J. L. Vivero-Escoto, I. I. Slowing, V. S.-Y. Lin and B. G. Trewyn, dures were performed in accordance with the Guidelines for Small, 2010, 6, 1952–1967. Care and Use of Laboratory Animals of ReDIB-Molecular and 2 M. 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