The nanoparticle-labeled MSCs biodistribution was assessed in the model of renal tuberculosis in rabbits (Figure 1). Analysis of the TB progression was performed using the delayed-type skin hypersensitivity (DTH) test and visually assessed during the laparotomy followed by the histological verification of the biopsy. One month following the injection of Mtb, SPIONs-labeled MSCs were intravenously administered. At the designated time points (2, 3, and 7 days), animals were euthanized and histological analyses (H&E, Ziehl-Nielsen stain) for the tissue samples (kidney, lung, liver, spleen, and paratracheal lymph nodes) were performed. The biodistribution as well as the kinetics of the cells accumulating in different organs and tissues were determined on the days 2, 3, 7 following the injection of the SPIONs-loaded MSCs. The following organs were studied: kidney, lungs, liver, spleen, paratracheal lymph nodes, heart, brain, pancreas, stomach, small intestine, colon, bladder, femur, ovaries, inguinal lymph nodes, and blood. Additionally, the pharmacokinetic modeling of the MSCs biodistribution was performed.

Following aspiration of bone marrow from the ilium, an equal volume of phosphate-buffered saline (PBS) was added. Obtained bone marrow was dispersed by pipetting, filtered through a sterile 70-μm nylon filter (Becton Dickinson Biosciences, Bedford, MA, USA) and centrifuged at 400 × g for 5 min. For the removal of erythrocytes the cell pellet was resuspended in Red Blood Cell Lysis Solution (Panreac, Barcelona, Spain) for 5 min and centrifuged at 400 × g for 5 min. Cells (1 × 10⁷) were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA, USA) and antibiotics (100 U/mL penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, USA) at 37°C in the humidified 5% CO₂/95% air atmosphere. Following four days of incubation, the non-adherent cells were removed by replacing the medium. The culture medium was exchanged twice a week. At 80–90% confluence, cells were harvested with 0.05% trypsin-EDTA (Life Technologies, Carlsbad, CA, USA) for 3 min at 37°C. Before the experiments, cells were harvested in the log phase of growth and their viability was determined by 0.4% Trypan blue exclusion. The MSCs were used between the passages 4 and 6 in the current study.

The phenotype of rabbit MSCs was assessed according to the minimal criteria for defining multipotent mesenchymal stromal cells [13]. MSCs are presented as a homogeneous population of elongated fibroblast-like cells which showed plastic adherence and were maintained in the standard culture conditions (5% CO₂, 37°C). The MSCs obtained from the passage four were tested for the differentiation potential as described previously with minor modifications [38]. The MSCs differentiated into osteoblasts, adipocytes and, chondroblasts in vitro (Supplementary Figure 1).

Superparamagnetic nanocarriers were prepared with an iron salt solution containing FeCl₃ and FeSO₄ by co-precipitation as described previously (Shevtsov et al., 2015, [36]). To prevent sedimentation of the nanoparticles, 10 kDa dextran (Sigma-Aldrich, St. Louis, USA) was added to the nanosuspension during the application of ultrasound. The separated fine fraction was produced from the dextran treated suspension by sedimentation on the NdFeB permanent magnet to remove large particles. The resultant suspension was dialyzed against water. The prepared fine fraction exhibited high colloidal stability. Optical density of the fraction measured by spectrophotometry at λ = 575 nm changed only by 10% from the initial value after 1 year of storage at +4°C. The colloidal stability in magnetic field at different effective Fe concentrations of the dextran-coated MNPs during the time interval 90 min was measured by NMR relaxometry (Supplementary Figure 2). From the 1st to the 90th min the relaxation time of protons changed only within 20% of its original value even in the largest magnetic field ~7 T, indicating insignificant aggregation of the MNPs. When MNPs were stored in a refrigerator in the absence of magnetic field, the relaxation time of protons in the water suspension was constant within the measurement errors, indicating a stable state of MNPs. After long-term storage, before use, the colloidal solution of nanoparticles was shaken to resuspend possible formed aggregates. The hydrodynamic size and the surface charge of nanoparticles were assessed using the Zetasizer Nano (Malvern Instruments, GB). The size of the iron oxide core and the shape of the particles were analyzed employing TEM (JEOL-2000 microscope, Jeol, Japan) and X-ray diffraction. From X-ray diffraction the iron-oxide nuclei of SPIONs exhibited the structure of nanocrystalline magnetite Fe₃O₄. Highly sensitive non-linear magnetic response measurements were employed for the analysis of superparamagnetic properties of SPIONs. From combined TEM and NLR-M2 data, the dextran shell thickness was estimated to be 1.4–1.7 nm [39]. The MR contrast of the particles was assessed (i.e., R₁, R₂, and R2* coefficients) in the 7.1 T magnetic field of the NMR spectrometer CXP-300 (Bruker, Germany).

MSCs were co-incubated with SPIONs (at Fe concentration of 150 μg/ml) for 1, 3, 12, and 24 h at 37°C in a CO₂-incubator. The non-treated cells were used as a control. Following co-incubation, cells were washed with PBS and their viability was assessed by 0.4% Trypan blue exclusion. The Vybrant® MTT Cell Proliferation Assay Kit was applied according to the manufacturer's protocol (Life Technologies, Carlsbad, CA, USA) for evaluation of the cytotoxicity of the nanoparticles and cell proliferation. To demonstrate ferric iron incorporation into the cells, Prussian blue staining of cells in the culture (BioPAL, Worcester MA, USA) was used in accordance to the manufacturer's protocol. Additionally, the nanoparticles incorporation into MSCs was analyzed employing confocal microscopy. The cells were allowed to settle on the glass slides and were co-incubated in the culture medium with SPIONs (at Fe concentration of 150 μg/ml) for 1, 3, 6, 12, and 24 h at 37°C. Following the co-incubation the cells were washed, fixed in 0.5% paraformaldehyde (PFA), and mounted in DAKO fluorescent mounting medium (Dako, North America Inc., USA). Nuclei were stained with DAPI (Sigma-Aldrich, USA). The intracellular localization of SPIONs was evaluated using Leica TCS SP5 confocal system (Leica Microsystems, Heidelberg, Germany). Obtained images were processed employing Adobe Photoshop PS software (Adobe Software, Palo Alto, United States). To assess the SPIONs released from the labeled cells into the fresh culture medium, a spectrophotometric method was applied. For this the MSCs loaded with SPIONs (300 μg/ml) were cultured in the fresh medium for 1, 3, 5, and 7 days. At designated time points the medium was collected and quantitative analysis of the iron content was performed. DMEM/F12 medium with 10% heat-inactivated fetal bovine serum and mixture of antibiotics (penicillin/streptomycin) was used as a control.

The nanoparticle uptake by MSCs was analyzed employing an iron assay kit (Sigma-Aldrich, St. Louis, USA) according to the manufacturer's protocol. Briefly, cells were co-incubated with SPIONs at various Fe concentrations (50, 100, 150, 300 μg/ml) for 24 h. Following the co-incubation, cells were washed with PBS and the intracellular iron content as well as the content of iron adsorbed on the outer cell membrane were assessed.

Following incubation with SPIONs for 24 h, MSCs were fixed in 2.5% glutaraldehyde in 0.1M cacodylate buffer, pH 7.4, for 1 h at 4°C, postfixed in 1% aqueous OsO₄ for 1 h, dehydrated, and embedded in Epon and Araldit. Utrathin sections of the cells were collected on fine mesh nickel grids. For examination, the samples were stained with uranyl acetate and lead citrate and analyzed employing Zeiss Libra 120 electron microscope.

The virulent Mtb strain H37Rv (TBC # 1/47, Institute of Hygiene and Epidemiology, Prague) was obtained from the Federal Scientific Center for Expertise of Medical Products (RF Ministry of Health Care). Mycobacteria were cultured on Middlebrook's 7H11 agar medium (Sigma-Aldrich, St. Louis, USA) supplemented with 10% Middlebrook OADC Enrichment (Sigma-Aldrich, St. Louis, USA). CFUs were determined after 2–3 weeks of culture at 37°C.

Male Chinchilla rabbits weighing 3–3.5 kg (in total 18 animals) were obtained from the Rappolovo Laboratory Animal Nursery of the Russian Academy of Medical Sciences (St. Petersburg, Russia). For sedation prior to anesthesia the mixture of fentanyl/droperidol (Moscow Endocrine Plant, Moscow, Russia; Dalkhimpharm, Vladivostok, Russia) 0.2 mg/kg was intramuscularly injected. Intravenous administration of ketamine (Moscow Endocrine Plant, Moscow, Russia) 10–50 mg/kg and xylazine (Alfasan International B.V., Woerden, Netherlands) 1–3 mg/kg mixture was employed for an anesthesia.

Following anesthesia, the kidney was exposed through a midline laparotomy incision of 4 cm. Inoculation of the Mtb H37Rv (10⁷ colony-forming units (CFU)/0.2 ml) by intraparenchymal injection was performed. In the postoperative period, all animals were given one intramuscular injection of 300,000 EU of the antibiotic bicillin-3 (benzathine benzylpenicillin; Sintez, Kurgan, Russia) every 3 days (a total of four injections). TB progression control was performed using delayed-type skin hypersensitivity (DTH) test with the allergen recombinant TB ≪Diaskintest≫ (Generium, Moscow, Russia) and was visually evaluated during diagnostic laparotomy as described previously [25]. The inflammation of the kidney was visually assessed during the laparotomy followed by the histological verification of the biopsy. On day 30 following inoculation of Mtb, the highly positive skin DTH probe was detected in all experimental animals with a diameter of 19.8 ± 1.4 mm (Supplementary Figure 3). The skin DTH probe was not detected in the control group and was regarded as a negative result. For the analysis of MSCs biodistribution in the model of renal TB in the non-treated (control) and treated with anti-TB drugs, the animals were randomly divided into two groups (nine animals in each group). The suspension of MSCs (5 × 10⁷ cells in 2 ml of PBS) labeled with SPIONs was intravenously administrated using a 2-ml syringe (23 G) via the marginal ear vein.

One month following intraparenchymal injection of Mtb, standard aTBT was performed for a period of 2 months according to the National clinical guidelines [40]. The treatment protocol included daily intramuscular injection of isoniazid (Biosintez, Penza, Russia) 10 mg/kg with the concomitant oral administration of rifampicin (Kraspharma, Krasnoyarsk, Russia) 10 mg/kg, ethambutol and pyrazinamide (Pharmasintez, Irkutsk, Russia) 20 mg/kg and 15 mg/kg, respectively. After intravenous administration of the MSCs the sustaining treatment with izoniazid and rifampicin was performed daily until termination of the experiment for a period of 7 days.

At the designated time points, animals were euthanised by CO₂ asphyxiation, and organs (i.e., kidney, lung, liver, spleen, and paratracheal lymph nodes) were extracted and fixed in 10% formalin. Paraffin sections of 3–5 μm were stained with haematoxylin and eosin (H&E). The presence of acid-resistant bacteria (Mtb) was identified using Ziehl-Nielsen stain. For the detection of iron-labeled MSCs, the extracted samples were embedded in Tissue-Tek® (Sakura Finetek Europe BV, Alphen an den Rijn, The Netherlands) and stored at −80°C. Sections (5–10 μm thick) were used for the detection of MSCs labeled with magnetic nanoparticles employing confocal microscopy (Olympus FV3000 confocal system (Olympus IX83 microscope, Olympus Corporation, Tokyo, Japan). Cover glasses were mounted in the fluorescent mounting medium supplemented with 4′,6-diamidino-2-phenylindole (DAPI) (Abcam, Cambridge, GB). For the analysis of SPIONs intracellular localization, reflected laser (504 nm) scanning was applied. For evaluation of nanoparticle localization, single Z-planes were analyzed with Olympus confocal software (FluoView FV31S-SW; Olympus Corporation) and ImageJ 1.51 (National Institutes of Health, Maryland, USA).

NLR-M2 was used to characterize the magnetic state of SPIONs (single- or multi-domain) and to determine their magnetic parameters outside and inside nanoparticle-labeled cells (MSC-SPIONs) as well as to assess the biodistribution of the SPION-loaded MSCs in organs and tissues of the animals under study [37]. The home-made setup was employed, registering the NLR response of magnetic nanoparticles (MNPs) to the weak ac magnetic field h(t) = h sinωt parallel to the steady field H (with the frequency f = ω/2π =15.7 MHz and h =13.8 Oe). The setup included two coaxial Helmholtz coils (providing the linear scanning of the steady field H) combined with the electromagnet to create the constant field H₀, if necessary, and the transmitter–receiver high frequency coil of the two-mode resonant system. The resonant system, on the one hand, creates the ac field on the sample under study and selects from the total NLR the second harmonic M₂ of the magnetization generated by the sample [41]. The M₂-signal phase components i.e., ReM₂(H) and ImM₂(H) are recorded simultaneously as functions of H. The frequency F_sc of the H scan can be changed within 8–0.02 Hz, which allows of testing the state (single- or multi-domain) of MNPs. Indeed, in the multi-domain state, the magnetization reversal is determined by fast motion of domain boundaries and the H hysteresis does not depend on F_sc, whereas in single-domain MNPs, the field hysteresis appears in the blocking regime below the blocking temperature T_b. This temperature can be found from the condition τ = τ_mes of the hysteresis appearance where τ is the longitudinal magnetization relaxation time and τ_mes is the measurement time given by F_sc. In the case of uniaxial anisotropy applicable to our MNPs, the relationship 1/τ = f ₀exp(–E_a/k_BT_b) is valid, where E_a is the anisotropy energy, k_B is the Boltzmann constant, and the frequency f ₀ is of the order 1 ns⁻¹ so that T_b = E_a/[k·ln(f ₀/F_sc)] [42]. The dependence of the H hysteresis on F_sc evidences the single-domain state of MNPs. Usually in the measurements, h is small enough to ensure the condition M₂ ∞ h². The main contribution to ReM₂(H) arises due to the non-linear behavior of M(H), so in the regime close to SPM it can be semiquantitatively described as ∂²L(H)/∂H² + k_PMH, where L(H) is the Langevin function, and the term k_PMH takes into account a possible small linear contribution from the low-molecular paramagnetic fraction [43]. The ReM₂(H) reaches an extreme value near the inflection point of M(H), whose position depends on the average moment of MNPs. This description is rather approximate since it does not take into account magnetization dynamics. This account becomes possible with the data processing formalism based on the numerical solution of the Fokker-Planck equation [39]. As shown previously, the cellular structure does not change the form of M(H) and ReM₂(H) dependencies [44]. This allows one to determine relative concentrations of SPIONs in different organs of experimental animals by comparing the signal amplitudes and thus to control the biodistribution of MNPs in the organism.

The NLR-M₂ data were processed with the formalism based on the numerical solution of the kinetic Fokker-Planck equation for SPM particles:

with

Here W(t) is the non-equilibrium probability-density function for the direction of the particle magnetic moment m, α is the damping constant, the time scale τ_N is the Néel relaxation time, i.e., the free-diffusion (zero-potential) correlation time, which in the Landau-Lifshitz form is proportional to α⁻¹, and β = 1/k_BT. The magnetic potential H is the sum of the uniaxial anisotropy energy and the energy of the magnetic moment in the total magnetic field H = H₀ + h:

where n is the anisotropy axis direction. The direction of m defined by the spherical angles ϑ and φ thermally fluctuates in the field H.

By expanding W(t) in the series on spherical harmonics:

and in the Fourier series, Equation (1) is reduced to a linear set of equations which, in turn, can be expressed as a continuous-fraction matrix equation

where the matrices Q_n, Q_n⁺ and Q_n+1 (n = 1, 2,…) are composed of the spherical harmonics indices, direction cosines of the magnetic fields, as well as of the parameters entering Equations (1–3) and the magnetic potential H. The normalized kth Fourier harmonic of the magnetic moment in the field direction is expressed as

where cijk(ω) are Fourier transforms of c_lm(t) in Equation (4) and γ_i are the direction cosines of the magnetic fields relative to the anisotropy axis n. The quantities cijk(ω) are the elements of the column vector S₁, the solution of Equation (5). To obtain the fit function for the M₂ response, m_k(ω) for k = 2 should be convoluted with the distribution over magnetic moments:

where M~ is the saturation magnetization of the SPM system and f_M corresponds to the distribution over volumes if all the particles are magnetically homogeneous with the same, size independent, magnetization.

Computation of the fitting function (7) at each experimental point implies the multiple solving of the recursion Equation (5). The solution accuracy is determined by the number of equations in the set, i.e., the retained terms in the Fourier and spherical harmonics expansions. By the simultaneous fit of the real and imaginary components of the measured signal with Equation (7), the following quantities can be directly obtained: (i) the saturation magnetization of the sample M~, (ii) the mean magnetic moment M_C and the width of the lognormal distribution σ, (iii) the number of magnetically responsive centers in the sample, (iv) the mean anisotropy energy E_a and field H_a, (v) the mean angle Ψ between n and H, and (vi) the magnetization dynamics parameters such as the damping constant α and the free-diffusion relaxation time τ_N. With the knowledge on the iron concentration, some more information can be extracted, viz., the mean number of Fe ions in the magnetic centers, the mean size of the centers and some others.

The data processing has been performed at the PIK Data Center of PNPI.

To describe the biodistribution of MSCs, a physiologically based pharmacokinetic (PBPK) model was set up. The rabbit body was divided into separate compartments representing blood and organs, while the transportation rates of MSCs were described by differential equations.

The model was based on the previously published PBK model by Wang et al. for MSC distribution in mice [45]. The body in this model is reduced to the most important organs, while the rest is summarized in one compartment. As the main transportation route, blood is divided into venous and arterial. Inside organs MSCs can be trapped in microvessels, which is described by a separate respective compartment for each organ. Arrested cells can either remain arrested, be released back into the corresponding organ or be depleted. Figure 2 represents a sketch of the model compartments and their transition pathways. Each transition rate is described by one kinetical constant. Both, parameter estimation and simulations were performed in MATLAB. Since the data were in the form of concentrations, all data points were first multiplied by their respective organ weights, which were taken from literature. The results were then used to compute a set of optimal parameters using a previously described approximation strategy ([46–54]).

For analysis of two continuous variables, the parametric Student's t-test was used. The significance level was equal to 0.05 for all tests with the confidence intervals at the 95% level. For comparison of multiple groups which had few observations, a non-parametric analog to the one-way ANOVA test, the Kruskal–Wallis test, was used. Statistica Version 9.2 software was employed for all the tests.

From TEM the iron oxide core of SPIONs was found to be 9.46 (5) nm in diameter with the lognormal size distribution width σ_d = 0.383 (4) [39]. From dynamic light scattering of the colloidal PBS solution of dextran-coated SPIONs, the mean hydrodynamic diameter 100.4 (1.7) nm and Z-potential −43.2 (4.5) mV were obtained (Figure 3A). Subsequent measurements of the hydrodynamic diameter and Z-potential over the period of 3 months did not show the aggregation of the nanoparticles. After 12 weeks of storage at +4°C the mean diameter constituted 107.2 (3.1) nm and Z-potential −33.11 (5.7) mV. Relaxometry NMR measurements of spin-spin and spin-lattice relaxations demonstrated increased R₂ and R2* relaxation rates that indicated the strong negative MR contrast enhancement due to SPIONs (data not presented).

NLR-M₂ measurements of the aqueous suspension revealed disappearance of the field hysteresis in the ReM₂(H) signal from SPIONs at increasing temperature and decreasing the frequency of H scans thus indicating the dynamical character of the hysteresis [39]. Such a behavior is typical of the single-domain state of magnetic nanoparticles inherent to the superparamagnetic regime (Figure 3B). Fitting the registered M₂(H) signals with the formalism described above (Figures 3Bc,d) indicated formation of SPIONs aggregates in agreement with the TEM observation. The following parameters were determined: (i) the mean magnetic moment of aggregates M_C = 61,200 (100) μ_B close to the double mean magnetic moment of SPIONs; (ii) the corresponding mean “magnetic” diameter D_C = 41.7(1) nm; (iii) the mean amount of SPIONs in the aggregates n_SP = 55 (3); (iv) the mean anisotropy energy E_a = 254 (22) K. The magnetic and geometric characteristics did not change with increasing the SPIONs concentration from 0.02 mM(Fe)/L to 2 mM(Fe)/L that indicated the stable chemical nature of the binding of SPIONs dextran shells in water and the absence of noticeable magnetic interaction between the aggregates. Furthermore, subsequent NLR-M₂ measurements of the nanosuspension over the period of 3 years (storage at +4°C) demonstrated the single-domain state of the SPIONs without a change of their magnetic properties.

Following co-incubation of MSCs with SPIONs (at a Fe concentration of 150 μg/ml) for 24 h, Prussian blue staining (Figure 4A) and subsequent reflective laser scanning by confocal microscopy demonstrated internalization of nanoparticles in the cytoplasm (Figure 4B). SPIONs are visualized as red dots present in the cytosol but not in the nucleus. MSCs without SPIONs were used as an internal negative control. Additionally, incorporation of nanoparticles by cells at the ultrastructural level was analyzed by TEM. The cytoplasm contained prominent heteromorphous endosomes with a diameter of 0.5 μm filled with the aggregates of numerous small electron-dense particles (Figure 4C). Apart from endo-lysosomes nanoparticles were also found in the cytoplasm as free aggregates. MTT analysis did not reveal any significant toxic effect of the nanoparticles on the proliferation of MSCs at concentrations ranging from 50 to 150 μg/ml. However, an increase in cytotoxicity (up to 20%) was detected when SPIONs were employed at a concentration above 300 μg/ml (Figure 4D). A linear regression analysis confirmed that an increase in the concentration of SPIONs above a range of 0–300 μg/ml was accompanied by a significant decrease in the number of surviving cells after a co-incubation time of 24 h (Figure 4D). The iron assay kit (Sigma-Aldrich, St. Louis, USA) was employed for an assessment of the Fe concentration in a sample of 5 × 10⁶ cells. An increase in the concentration of SPIONs led to an increase of the iron concentration in MSCs, viz., 19, 32, 34, 62 pg/MSC for the Fe concentrations 50, 100, 150, and 300 μg/ml, respectively (Figure 4E). When comparing the number of lysosomes in nanoparticle-labeled cells and control cells (Figure 4G), no significant difference was found (Wilcoxon test p = 0.91, n = 10). Intriguingly, the subsequent co-localization analysis demonstrated that SPIONs were predominantly present in the cytoplasm with a minor pool of nanoparticles present in the endo-lysosomal compartment. Subsequent spectrophotometric experiments that were performed to assess the release of MNPs from the MSCs demonstrated that over a period of 7 days only a tiny fraction of SPIONs (~2 μg/ml) was excreted into the culture medium (Figure 4F).

Possible changes in the state of SPIONs during their incorporation into MSCs were controlled by subsequent NLR-M₂ measurements. The signals were registered from the suspensions of the SPION-labeled MSCs. The M₂ responses of SPION-labeled MSCs co-incubated with 150 μg/ml Fe in PBS (further used for injection to the experimental animals) and after fixation in formalin are presented in Figure 5 while the fit parameters are presented in Table 1. The amount of Fe (and SPIONs, respectively), absorbed by cells was determined as the difference in the Fe concentration in the culture medium before and after co-incubation, which depended on the effective concentration of Fe during the incubation, and subtracting also the concentration of SPIONs adsorbed on the cell membranes and removed by flushing the SPIONs off the cell surfaces. As a result, the average amount of Fe (and SPIONs, correspondingly) absorbed by MSCs in the sample containing 1.5 × 10⁵ cells was found to be 11.9 (1.3) μg for C(Fe) = 150 μg/ml. The change of the M₂(H) response amplitudes was observed from the suspensions of MSCs co-incubated with different concentrations of Fe while the extrema positions changed only slightly. A weak field hysteresis disappeared at decreasing F_sc down to 0.25 Hz indicating transfer to the superparamagnetic regime thus validating the NLR-M₂ data processing formalism. The best-fit magnetic parameters, viz., the saturation magnetization, the mean magnetic moment, the log-normal distribution width, the anisotropy field and energy, the damping constant and the Néel relaxation time, appeared to be nearly the same for all the samples regardless of the solvent, PBS or formalin. The small anisotropy energy, E_a = 7–11 K, and the mean “magnetic” diameter [i.e., relating to the magnetic component) D_C = 59 (1) nm (corresponding to the mean magnetic moment M_C (Table 1)] much exceeding the size of the SPION magnetic core indicate the M₂ response to come from the aggregates of SPIONs. The distribution width σ_V = 0.73 turned out to be even smaller than that of the SPIONs ensemble, σ_V = 3σ_d = 1.15. This quantity was underestimated due to the assumption on the homogeneous distribution of magnetization in the aggregates. The dipole-dipole coupling causes magnetic correlations with the finite correlation radius. Its account could yield somewhat larger, more realistic, distribution width and mean magnetic moment [39], unfortunately, not available in the present study. The ratio M~/MC (Table 1) yields the amount of magnetic centers in the sample N_C = 1.24 × 10¹⁰ or 8.27 × 10⁴ aggregates per MSC. With the known concentrations of absorbed iron in the measured co-incubated samples, the mean number of SPIONs per cell, N_SP = 1.3 × 10⁷, and n_SP = 160 SPIONs per aggregate were found, considerably larger than in the aqueous suspension. The diameter D_C determined from NMR-M₂ includes only the magnetic component of the aggregates. To obtain the real mean diameter, the “magnetic” diameter D_C should be multiplied by the factor ξ = 1.52 which corrects the aggregate size for the dextran shell and the interparticle space [39], giving the true mean diameter value 90 (2) nm. This value well-agrees with the size of the aggregates in the TEM image of Figure 4C and is close to the hydrodynamic diameter 100 nm obtained by dynamic light scattering. The sensitivity of the NLR-M₂ setup characterized by the minimal measurable amount of SPION-labeled MSCs in the sample corresponds to the signal-to-noise ratio equal to 1 and was found to be 1,300 cells.

The mean aggregate magnetic moment should obey the scaling law M_C = nspm, where m ~ (2.2–3.1) × 10⁴ μ_B is the SPION magnetic moment, and for n_SP = 160 was expected to be M_C = (2.8–3.9) × 10⁵ μ_B. However, the dipole-dipole coupling of SPIONs inside the aggregate tends to minimize the magnetostatic energy and, thus, strongly suppresses the aggregate magnetic moment down to 3.14 (6) × 10⁴ μ_B comparable with that of one SPION.

The intravenous administration (via an ear marginal vein) of SPIONs-labeled MSCs was performed following the treatment of animals with a standard anti-TB protocol [40]. Then rabbits were evaluated on the days 2, 3, and 7 after MSCs administration. The untreated animals after injection of nanoparticle-labeled MSCs were used as an internal control. The specific TB inflammation of the kidney and lung was detected in animals from the experimental group. Acid-resistant bacteria (Mtb) were detected by the staining protocol according to Ziehl-Neelsen (Figure 6A). Giant multinucleated cells of Langhans (Figure 6B), small areas of caseous necrosis, and epithelioid cells were detected in the granulomas (Figure 6C). The cells with incorporated magnetic nanoparticles were visualized in various organs and tissues of the treated animals (i.e., kidney, lung, liver, and spleen) (Figure 7).

We performed NLR-M₂ measurements on the 10% formalin-fixed organ samples of TB. The biodistribution as well as the kinetics of cells accumulated in different organs and tissues were determined in the healthy rabbits on the days 2, 3, 7 after injection of the MSCs loaded with SPIONs. The following organs were studied: (i) kidneys; (ii) lungs; (iii) liver; (iv) spleen; (v) paratracheal lymph nodes; (vi) heart; (vii) brain; (viii) pancreas; (ix) stomach; (x) small intestine; (xi) colon; (xii) bladder; (xiii) femur; (xiv) ovaries; (xv) inguinal lymph nodes; (xvi) blood. The magnetically responsive samples were measured at two field scan frequencies, F_sc = 8 and 0.25 Hz to elucidate their magnetic state (single- or multi-domain). The M₂(H) dependences of organs giving the large signals, similar to those from SPION-labeled MSCs in PBS (kidneys, lungs, liver, spleen, paratracheal lymph nodes) together with the hysteretic response from kidneys at different time points after injection are presented in Figure 8, Table 2.

The signals from lungs, liver, spleen, paratracheal lymph nodes registered 2 days after injection together with their fitting curves are presented in Figure 9. The 2- and 3-day parameters for these organs are presented in Table 1. The probes of other organs (Supplementary Figures 4, 5, Table 2) reveal the similar hysteretic M₂ signals, except of pancreas and femur where the H hystereses decrease at reducing the H-scan frequency down to 0.25 Hz. The remaining organs (ovaries and inguinal lymph nodes) as well as blood do not show the presence of MNPs at the sensitivity level of the setup. In Table 2 the amplitudes of the real part of the M₂ response and the corresponding “coercive forces” H_C2 [that is analog of the coercive force of M(H)] are presented for all magnetically responsive organs. This allows of semi-quantitative comparison and assessment of the retention of SPION-labeled MSC in all organs including those exhibiting hysteretic response where the data processing formalism is inapplicable.

The M₂(H) responses from the healthy rabbits (injected by SPIONs-labeled MSC in the same manner as TB rabbits) are presented in Supplementary Figures 6–8. They demonstrated the predominant accumulation of cells in kidneys,colon, brain, bladder, femur, paratracheal lymph nodes and, to a lesser extent, in lungs, liver, and spleen after 72 h of administration with the subsequent significant decrease of the signal over the period of 7 days accompanied in some organs (brain, femur, paratracheal lymph nodes) by essential change of its dependence on the field H.

After estimation, the parameters were used for simulations to determine the fitting of the data. As shown in Figure 10, the simulations fit the data very well, suggesting the validity of the model for the MSC biodistribution in rabbits. According to the results, the venous blood releases the MSCs very quickly, resulting in an immediate peak in arterial blood and a peak in all investigated organs after some delay. Due to depletion mechanisms, all concentrations decrease after all MSCs left the system.