Highly selective organ distribution and cellular uptake of inorganic-organic hybrid nanoparticles customized for the targeted delivery of glucocorticoids

Journal of Controlled Release 319 (2020) 360–370

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Highly selective organ distribution and cellular uptake of inorganic-organic hybrid nanoparticles customized for the targeted delivery of glucocorticoids

T

Tina K. Kaisera, Mikhail Khorenkob, Amir Moussavic, Michael Engelkea, Susann Boretiusc, ⁎ Claus Feldmannb, Holger M. Reichardta, a

Institute for Cellular and Molecular Immunology, University Medical Center Göttingen, 37073 Göttingen, Germany Institute of Inorganic Chemistry, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany c Functional Imaging Laboratory, German Primate Center, Leibniz Institute for Primate Research, 37077 Göttingen, Germany b

A R T I C LE I N FO

A B S T R A C T

Keywords: Inorganic-organic hybrid nanoparticles Glucocorticoids Drug delivery Macrophages Endocytosis

We previously reported that inorganic-organic hybrid nanoparticles (IOH-NPs) containing the synthetic glucocorticoid (GC) betamethasone show efficient anti-inflammatory activity in mice. Here, we employed IOH-NPs 2− (AMP: adenosine monophosphate) to determine their in vivo with the chemical composition Gd3+ 2 [AMP]3 distribution by magnetic resonance imaging after intraperitoneal injection. We show that IOH-NPs distribute throughout the peritoneal cavity from where they get rapidly cleared and then localize to abdominal organs. Our findings were confirmed by analyzing individual mouse organs ex vivo following injection of IOH-NPs with the chemical composition [ZrO]2+[(BMP)0.9(FMN)0.1]2− (BMP: betamethasone phosphate, FMN: flavin mononucleotide) or [ZrO]2+[(HPO4)0.9(FMN)0.1]2− using inductively coupled plasma mass spectrometry and flow cytometry. To characterize the mechanism of cellular uptake in vitro, we tested different cell lines for their ability to engulf IOH-NPs by flow cytometric analysis taking advantage of the incorporated fluorescent dye FMN. We found that IOH-NPs were efficiently taken up by macrophages, to a lesser extent by fibroblasts, epithelial cells, and myoblasts, and hardly at all by both T and B lymphocytes. Characterization of the endocytic pathway further suggested that IOH-NPs were internalized by macropinocytosis, and imaging flow cytometry revealed a strong colocalization of the engulfed IOH-NPs with the lysosomal compartment. Intracellular release of the functional anions from IOH-NPs was confirmed by the ability of the GC betamethasone to downregulate the expression of surface receptors on bone marrow-derived macrophages. Taken together, our findings unveil the mechanistic basis of an anti-inflammatory GC therapy with IOH-NPs, which may entail translational approaches in the future.

1. Introduction Glucocorticoids (GCs) are the most widely employed class of antiinflammatory drugs. They strongly suppress innate and acquired immunity by regulating the activity of macrophages, granulocytes and T cells, and show efficient therapeutic activity in autoimmune and allergic diseases [1–3]. However, GCs impact physiological processes also in many other organs, thereby leading to severe adverse effects such as muscle atrophy, osteoporosis, and diabetes [4–6]. Therefore targeted delivery of GCs to selective cell types would be desirable and could make therapy more tolerable. Application of free GCs leads to a largely uniform distribution of the drug in all tissue via the circulation. Hence, the site of action is almost impossible to control. One option to increase the specificity of GCs is to alter their tissue distribution by encapsulation in nanosized structures,

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a strategy that entailed the development of a variety of nanoformulations of different physico-chemical compositions [7]. For instance, GC delivery using PEGylated liposomes was found to result in a preferential targeting of macrophages and monocytes and improve the treatment of autoimmune diseases [8–10]. However, liposomes can also cause adverse effects such as the activation of the complement system, which complicates their application in patients [11]. Other carrier systems for GC delivery include polymer-drug conjugates and polymeric micelles, which were shown to be suitable for anti-inflammatory therapy with reduced adverse effects in mouse models [12–14]. As an alternative to the existing material concepts, we have developed multimodal inorganic-organic hybrid nanoparticles (IOH-NPs) composed of an inorganic cation and one or several organic anions functionalized with a phosphate group [15]. We could show that such GC-loaded IOH-NPs with the chemical composition [ZrO]2+[(BMP)0.9(FMN)0.1]2− have a

Corresponding author at: Institute for Cellular and Molecular Immunology, University Medical Center Göttingen, Humboldtallee 34, 37073 Göttingen, Germany. E-mail address: [email protected] (H.M. Reichardt).

https://doi.org/10.1016/j.jconrel.2020.01.010 Received 16 September 2019; Received in revised form 11 December 2019; Accepted 6 January 2020 Available online 07 January 2020 0168-3659/ © 2020 Elsevier B.V. All rights reserved.

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any agglomeration that may have occurred. IOH-NPs with the chemical composition [ZrO]2+[(BMP)0.9(FMN)0.1]2− (designated BMP-NPs) and [ZrO]2+[(HPO4)0.9(FMN)0.1]2− (designated EP-NPs) were prepared as previously described [16] and redispersed at a concentration of 2.6 mg/ ml to 3.2 mg/ml in H2O.

hydrodynamic diameter of 30–40 nm, suppress the activity of macrophages in vitro and in vivo and ameliorate inflammation in a mouse model of multiple sclerosis [16]. Furthermore, they can be detected in cells and mice based on incorporated fluorescent or infrared dyes [16,17]. Due to the versatility of this material concept, both components of the IOH-NPs can be exchanged for other organic molecules or inorganic metal cations, thus rendering them highly potent theranostic agents [18]. GC application in vivo can be achieved via different routes. Whereas intravenous (IV) infusion is the preferred method for drug administration in the clinic, intraperitoneal (IP) injection can be advantageous especially when drugs are delivered with the help of nanoformulations. IV administration often results in a rapid opsonization of NPs in the blood, which leads to their rapid clearance from the systemic circulation and a deposition in liver and spleen. This in turn reduces drug availability and may cause fibrosis and inflammation [19]. In contrast, IP injection generally results in a retarded release of NPs from the peritoneal cavity into the circulation via the lymphatics and the portal circulation [20,21], which results in an accumulation in a variety of abdominal organs. Therefore, NPs which are administered IP often have a longer biological half-life and show a more even distribution in the body than those ones injected IV [22]. GCs passively enter the cell by diffusion across the plasma membrane due to their lipophilic nature, while NPs need to be actively taken up via endocytic pathways [23,24]. Phagocytosis mainly takes place in cells of the immune system such as macrophages and involves the recognition of particular matter by specialized receptors. In contrast, macropinocytosis is a pathway for the unspecific uptake of external fluid, soluble molecules and small particles via membrane ruffles [25,26]. Clathrin- and caveloae-dependend endocytosis are initiated by recognition of target structures via cell surface receptors and classified as micropinocytic pathways [27]. Regardless of the employed endocytic mechanism, the vesicles containing the engulfed material can have different intracellular fates and travel to lysosomes, mitochondria or back to the plasma membrane. Generally, the mechanism by which NPs are taken up is determined by their size, surface hydrophobicity, charge and shape but also by individual features of each target cell. Hence the chemical composition of the nanoformulation and the cell type both influence the endocytic pathway and intracellular trafficking of NPs, and thus determine their suitability as drug delivery vehicles for biomedical applications. To further improve the features of IOH-NPs, detailed insights into their mechanism are needed. Therefore we set out to characterize their tissue distribution after IP injection into mice in vivo and ex vivo, and their uptake into different cell types in vitro. Based on these results, we would predict that an improved although not entirely specific activity profile can be achieved with this nanoformulation, thereby potentially providing a more tolerable form of GC therapy.

2.2. Animal experimentation Wildtype BALB/c mice were bred and kept in our animal facility at the University Medical Center Göttingen under specific-pathogen-free conditions in individually ventilated cages. Female mice were used at an age of 8–12 weeks. All experiments comply with the ARRIVE guidelines, were conducted accordingly to Lower Saxony state regulations for animal experimentation and approved by the responsible authority (LAVES, Oldenburg, Germany). 2.3. Magnetic resonance imaging (MRI) Wildtype BALB/c mice were anesthetized by subcutaneous injection with 0.6 ml/kg ketamine and 0.4 ml/kg medetomidine. Subsequently, the mice were intubated and ventilated with a mixture of oxygen and ambient air and with a constant respiratory frequency of 85 breaths/ min by using an Animal Respirator Advanced (Technical & Scientific Equipment, Bad Homburg, Germany). Anesthesia was maintained with 0.5 to 1% isoflurane during the entire MRI experiment. Initially, a baseline scan was recorded. Thereafter, the mice received 500 μl of a 1:8 dilution of GAP-NPs in H2O containing 0.1 mg gadolinium via IP injection, and MR data of the abdominal region were continuously acquired for a period of 6 h. MR images were obtained at 9.4 T (BioSpec 94/30, Bruker BioSpin GmbH, Ettlingen, Germany) using a spoiled radial FLASH sequence with the following parameters: 201 spokes, repetition time (TR) = 150 ms, echo time (TE) = 2 ms, flip angle = 60°, Field of View (FOV) = 19.2 × 19.2 mm2, acquisition bandwidth = 50 kHz, spatial resolution = 0.15 × 0.15 × 0.5 mm3, 3 averages and 36 slices in 3 concatenations resulting in a total acquisition time of 4.5 min per data set. Regions of interest (ROI) were defined for the intraperitoneal cavity and selected organs as illustrated in the SI (Fig. S6). Data were analyzed using the MATLAB software. 2.4. Estimation of the detection limit of GAP-NPs by MRI A contrast-to-noise-ratio (CNR) > 3 was considered to be sufficient to detect differences in MR signal intensity [28]. The CNR was calculated for each time point using the equation depicted below by subtracting the signal intensity of the native image taken before GAP-NP injection (S0) from the signal intensity at the respective time point after GAP-NPs injection (Sc). To estimate the GAP-NPs' concentration that led to a CNR of 3, R1 and R2 relaxation rates were determined in samples of different concentrations of GAP-NPs (range: 1.5–600 μM, dissolved in 100 mg/ml BSA) by varying the repetition time and echo time, respectively. Corresponding specific relaxivities (r1, r2) were then obtained from the slope of the linear relationship between the relaxation rate and the contrast agent concentration. Knowing the specific relaxivities (r1 and r2) of the contrast agent, the expected CNR was calculated for different contrast agent concentrations. In more detail:

2. Material and methods 2.1. Synthesis of IOH-NPs 2− To obtain Gd3+ IOH-NPs (designated GAP-NPs), 0.5 ml of 2 [AMP]3 an aqueous solution of GdCl3 × 6H2O (18.6 mg, 99%, Sigma-Aldrich, Taufkirchen, Germany) were injected into 50 ml of a vigorously stirred aqueous solution of Na2(AMP) (39.1 mg, > 99%, Sigma-Aldrich) at room temperature. After 2 min, IOH-NPs were separated via centrifugation (15,000 rpm, 15 min) and redispersed in 10 ml of H2O. Centrifugation and subsequent redispersion were repeated twice to remove all remaining salts and residual starting materials. The colourless 2− Gd3+ IOH-NPs were redispersed in H2O at a concentration of 2 [AMP]3 8 mg/ml and employed for analytical characterization. Alternatively, they were stabilized by addition of 100 mg/ml BSA and used for animal experimentation. Although GAP-NPs prepared as such remain stable for two weeks, they were careful mixed before injection into mice to revert

CNR =

SC − S0 ∂noise

with Sc being the signal intensity at a given contrast agent concentration (c), S0 being the signal intensity without contrast agent, and δnoise being the standard deviation of the noise. By using T1 and T2 relaxivities measured in vivo for different tissues of interest, Sc was simulated using the signal equation of the applied FLASH sequence:

Sc = M0 sin α 361

1 − e−TR / T 1c e−TE / T 2c 1 − cos αe−TR / T 1c

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1 1 = + ri [C ], i = 1, 2 Tic Ti

2.9. Cell culture experiments

Wildtype BALB/c mice received 500 μl BMP-NPs containing 0.2 mg zirconium via IP injection and were sacrificed 5 or 24 h later by CO2 inhalation and cervical dislocation. The lung, the left kidney, a central piece of 20 cm length of the small intestine, the left and caudate liver lobes, and the stomach were removed, washed with PBS, weighted and stored at −80 °C. The samples were sent for analysis to a commercial microanalytical laboratory (Labor Pascher, Remagen, Germany). In brief, 40 to 200 mg of the tissue samples were dissolved in a PFApressure system with HNO3/HF at 180 °C overnight, and after addition of rhodium/iridium as an internal standard, zirconium was detected using an iCAP™ Q ICP-MS instrument (Thermo Fisher Scientific, Waltham, MA).

MH-S cells were cultured in RPMI 1640 medium with 10% FCS, 1% Penicillin/Streptomycin (P/S) and 0.05 mM 2-mercaptoethanol. WEHI 7.1, WEHI 231, L929 and C2C12 cells were cultured in DMEM medium with 10% FCS and 1% P/S. LA-4 cells were cultured in F12 NutMix medium with 15% FCS and 1% P/S. All media and supplements were purchased from Thermo Fischer Scientific. To analyze IOH-NP uptake, the cell lines were seeded at 1 × 105 cells/ml in 6-well plates and cultured in 3 ml of medium at 37 °C and 5% CO2 for 48 h followed by the addition of BMP-NPs or EP-NPs for another 30 min to 6 h. Alternatively, cells were first cultured for 24 h in the absence of IOHNPs and then for another 24 h in the presence of BMP-NPs or EP-NPs. MH-S, L929, C2C12 and LA-4 cells were detached from the wells by incubation with 0.25% Trypsin-EDTA (Thermo Fischer Scientific) for 5 min whereas WEHI 7.1 and WEHI 231 cells were collected by centrifugation. To study endocytosis, MH-S cells were seeded at 5 × 105 cells/ml in 24-well plates and cultured in 1 ml of medium for 24 h followed by incubation with BMP-NPs for another 24 h. Alternatively, cells were first cultured for 48 h and then BMP-NPs were added for 30 min to 6 h. 1 μg/ml cytochalasin D (cytoD), 1 mM amiloride hydrochloride hydrate (amiloride), or 100 μM monodansyl-cadaverine (MDC) were added to MH-S cells concomitantly with BMP-NPs. All inhibitors were purchased from Sigma-Aldrich.

2.6. Cell isolation from mouse organs

2.10. Bone marrow-derived macrophages (BMDMs)

Wildtype BALB/c mice received 1 ml EP-NPs via IP injection and were sacrificed 5 h later by CO2 inhalation and cervical dislocation. Single cell preparations were obtained from the spleen by passing it through a 40 μm cell strainer followed by the removal of erythrocytes using Tris-buffered amonium chlorid buffer according to standard procedures [29]. To obtain single cell preparations from the small intestine, we first removed the epithelial cells by incubation in PBS/ 60 mM EDTA/ 3 mM DTT followed by repeated vigorous shaking, and then digested the remaining tissue with a mixture of collagenase type II, collagenase type 1A and DNaseI (Sigma-Aldrich) as described previously [30]. Finally, the cells were resuspended in PBS and analyzed by flow cytometry.

Tibia and femur from wildtype BALB/c mice were flushed with PBS/ 0.1% BSA and single-cell suspensions from the bone marrow were cultured in the presence of L929 cell-conditioned medium (LCCM) as a source of M-CSF for seven days as previously described [38]. When differentiation was completed, BMDMs were harvested, seeded at 1 × 106 cells/ml in DMEM medium with 10% FCS and 1% P/S, and cultured in 1 ml of medium in 24-well plates. To study regulation of cell surface molecules, BMDMs were cultured with IOH-NPs for 24 h, harvested by incubation with PBS/ 2 mM EDTA for 30 min at 37 °C and 5% CO2, and analyzed by flow cytometry.

(TR – repetition time, TE – echo time, M0 – amplitude of gradient echo at TE = 0 and TR = ∞, α – flip angle, Ti – relaxation times, ri – specific relaxivities, [C] concentration of GAP-NPs). From that, together with the standard deviation of noise and the signal intensity obtained from the native image in vivo, concentrations leading to CNR of 3 were calculated. 2.5. Inductive coupled plasma mass spectrometry (ICP-MS)

2.11. Cell viability test 2.7. Flow cytometry (FACS)

Cell lines were seeded at a density of 1 × 106 cells/ml and cultured in 100 μl in 96-well plates in the presence or absence of IOH-NPs for 3 to 24 h. Cell viability was assessed by using a colorimetric MTS Assay Kit according to the manufacturer's instructions (Promega, Mannheim, Germany) employing a Power Wave 340 plate reader (BioTek, Bad Friedrichshall, Germany). In some experiments, cells were cultured in the presence of 1 μg/ml CytoD, 1 mM Amiloride, or 100 μM MDC.

Cellular uptake of IOH-NPs was analyzed by flow cytometry detecting fluorescence emission by FMN contained in IOH-NPs. To this end, live cells were gated based on their forward and sideward scatter, followed by measuring the percentage of cells showing green fluorescence. Leukocyte surface molecules were stained with monoclonal antibodies directly conjugated to fluorophores (BioLegend, Uithoorn, The Netherlands) as previously described [31]: anti-mouseCD3 (17A2), anti-mouseCD11b (M1/70), anti-mouseCD86 (GL-1), anti-mouseMHC II (I-Ad haplotype, AMS-32.1). To prevent unspecific antibody binding to Fc-receptors, cells were incubated with anti-mouseCD16/32 for 20 min. All analyses were carried out with a FACS Canto II device (BD Biosciences, Heidelberg, Germany), the data were processed with FlowJo software (Treestar, Ashland, OR).

2.12. Imaging flow cytometry A total of 5 × 106 MH-S cells were cultured in 30 ml medium in 175 cm2 flasks for 24 h with BMP-NPs. Alternatively, BMP-NPs were added only during the last 6 h. To label intracellular compartments, cells were washed and incubated for another 30 min with 100 nM LysoTracker® Deep Red (Thermo Fischer Scientific) or 100 nM MitoTracker® Deep Red FM (Cell Signaling, Danvers, MA). Subsequently, the cells were washed, detached by incubation with 0.25% Trypsin-EDTA, and analyzed using an ImageStreamX Mk II Imaging Flow Cytometry (Amnis; Luminex, Austin, Tx). For each sample a total of 2000 focused events were acquired and analyzed with the software IDEAS (v6.2) using the co-localization wizard. Bright Detail Similarity was assessed after gating on FMN and Deep Red double-positive events.

2.8. Cell lines MH-S is a murine alveolar macrophage cell line, WEHI 7.1 are derived from a mouse thymoma and WEHI 231 were generated from an immature mouse B cell lymphoma [32–34]. L929 are murine fibroblasts of subcutanous tissue origin, LA-4 are derivatives of mouse alveolar type II cells, and C2C12 are reminiscent of murine myoblasts [35–37]. All cell lines were from ATCC (Manassas, VA). 362

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Fig. 1. Chemical composition of the three IOH-NPs employed in the study. BMP-NPs, EP-NPs and GAP-NPs contain [BMP]2−, [FMN]2−, [HPO4]2− or [AMP]2− as organic anions and [ZrO]2+ or Gd3+ as inorganic cations in the indicated stoichiometry.

3. Results

SI: Table S1, Fig. S4). Due to their individual advantages, IOH-NPs containing either Gd3+ or [ZrO]2+ were employed in our study. GAP-NPs were exclusively used for MRI experiments due to their paramagnetic features. BMP-NPs or EP-NPs were used in all other approaches since [ZrO]2+ is less toxic for cells than Gd3+ and more easily detectable by chemical methods such as ICP-MS. Importantly, previous studies and the one at hand indicated that the chemical properties of different IOH-NPs are largely independent of which specific cations and anions they are composed of 2− [39]. For instance, the three IOH-NPs Gd3+ 2 [AMP]3 , [ZrO]2+[(BMP)0.9(FMN)0.1]2− and [GdO]+[ICG]− (ICG: indocyanine green) all have a hydrodynamic diameter between 50 and 80 nm, a negative zeta potential at neutral pH and show a low solubility in water [15,16,18]. Thus, we considered the availability of different IOH-NPs of varying chemical composition as a good opportunity to demonstrate the flexibility of our material concept.

3.1. Material synthesis and characterization Three types of IOH-NPs were employed in this study (Fig. 1). In most cases, the previously described BMP-NPs with the chemical composition [ZrO]2+[(BMP)0.9(FMN)0.1]2− were used, which contain the GC betamethasone (BMP) and the fluorescent dye flavin mononucleotide (FMN). EP-NPs with the chemical composition [ZrO]2+[(HPO4)0.9(FMN)0.1]2− containing [HPO4]2− instead of [BMP]2− served as a control [16]. Both IOH-NPs have a hydrodynamic diameter of 60–80 nm and are highly stable for at least 24 h when redispersed in water [16]. To enable detection by MRI in living animals, IOH-NPs with the 2− composition Gd3+ were prepared by injecting a concentrated 2 [AMP]3 aqueous solution of GdCl3 × 6H2O into an aqueous solution of Na2(AMP) at room temperature (Fig. 1). The synthesis protocol of GAPNPs is comparable to the one of BMP-NPs [15,18,39]. The organic anion adenosine is functionalized by a phosphate group and forms insoluble saline nanoparticles after the addition of Gd3+ as inorganic cation, which are well dispersable in water (Fig. 2A). According to SEM analysis, GAP-NPs exhibit a spherical shape and a mean diameter of 51 ± 10 nm (Fig. 2B,C). Dynamic light scattering (DLS) confirmed this size in aqueous suspension with a mean hydrodynamic diameter of 52 ± 13 nm (SI: Fig. S1). Zeta potential measurements show a surface charge of −10 mV at neutral pH (SI: Fig. S2), for which reason BSA was added for steric stabilization. GAP-NPs exhibit magnetic properties as expected for Gd3+-containing NPs (Fig. 2D). The chemical composition of GAP-NPs was further characterized by Fourier-transform infrared (FT-IR) spectroscopy, energy-dispersive Xray spectroscopy (EDXS), thermogravimetry (TG) and elemental analysis (EA). The presence of gadolinium and phosphorus were validated by EDXS, whereas the presence of [AMP]2− was qualitatively proven by FT-IR (SI: Fig. S3). The composition was quantified by total organic decomposition via TG and by EA showing a weight loss of 59% and C/ H/N contents of 24.1 wt-% C, 3.1 wt-% H and 14.0 wt-% N. These values are in agreement with the calculated data based on the composition of the GAP-NPs (total organic content: 59%; carbon content: 26.7 wt-%, hydrogen content: 2.7 wt-%, nitrogen content: 15.6 wt-%;

3.2. MRI analysis of IOH-NPs in living mice after IP injection A major determinant of the therapeutic activity of drugs delivered by nanomaterials in vivo is their abundance in different target organs. Here we used MRI to visualize the distribution of IOH-NPs in living mice after IP injection of GAP-NPs containing the paramagnetic Gd3+ cation and [AMP]2− as an example of a functional organic anion, which is representative for a large range of potentially interesting organic compounds including pharmaceutical agents (Figs. 1,2). Initially, the specific relaxivities r1 (8.1 l mmol−1 s−1) and r2 (14.4 l mmol−1 s−1) of GAD-NPs were determined (SI: Fig. S5). For comparison, r1 (5.3 l mmol−1 s−1) and r2 (5.5 l mmol−1 s−1) of Gadovist®, a common contrast agent in clinical routine, was measured. With these results, the estimated minimal detectable concentration of GAD-NPs was found to be in the range of 60 to 125 μM depending on the relaxation rate of the targeted tissue (SI: Fig. S5). Subsequently, images of abdominal crosssections were recorded before and after IP injection of GAP-NPs into anesthetized mice. The presence of gadolinium was continuously visualized in the peritoneum and abdominal organs for six hours after administration (Fig. 3). A strong signal originating from GAP-NPs was detected throughout the entire peritoneal cavity briefly after IP injection, in particular between hepatic lobes, in intestinal loops, and around 363

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2− Fig. 2. Characterization of Gd3+ IOH-NPs. (A) Aqueous GAP-NP suspension with Tyndall cone. (B) Particle size distribution (statistical evaluation of 100 2 [AMP]3 particles on SEM images). (C) SEM detail and overview images. Size bar: 200 nm (D) Magnetic separation of GAP-NPs.

Fig. S6). Taken together, our findings suggest that IP injection results in a slow exit of IOH-NPs from the peritoneal cavity, probably mediated by both lymphatic drainage and direct absorption via the portal circulation, followed by an accumulation in different abdominal organs, and finally the excretion via the intestinal tract.

kidneys, spleen and stomach (Fig. 3). The signal intensity in the peritoneal cavity then started to decline again, while concomitantly an increasing signal was observed in abdominal organs such as stomach and liver (Fig. 3). In some tissue folds in the peritoneal cavity, the signal intensity even dropped below the baseline, pointing to a local accumulation of GAP-NPs that lead to a predominantly R2 effect (Figs. 3 and 4A). A region of interest (ROI) analysis of the contrast-to-noise-ratio (CNR) confirmed a maximal signal intensity in the intraperitoneal cavity during the first 30 min after injection, which gradually declined during the next two hours and eventually became undetectable (Fig. 4A, SI: Fig. S6). In parallel, signal intensities in liver and stomach started to increase one hour after IP injection (Fig. 4B,C, SI: Fig. S6). GAP-NPs were detected with some delay also in the small intestine but not in the kidney, at least during the observation period (Fig. 4B,C, SI:

3.3. ICP-MS analysis of IOH-NPs in mouse organs after IP injection To independently confirm our MRI results, we analyzed the zirconium concentration in different organs after IP injection of BMP-NPs into mice using ICP-MS. Mice were sacrificed 5 and 24 h after injection, the organs were dissected and subjected to chemical trace analysis. The highest levels of zirconium at the 5 h time point were detected in liver and stomach, an intermediate level in the small intestine and only low amounts in kidney and lung (Fig. 4D). Analyses of liver and small

Fig. 3. In vivo MRI of the abdomen of a living mouse after IP injection of GAP-NPs. A BALB/c mouse was anesthetized and T1–weighted images were recorded before (−30 min) and after (+30 min, +3 h, +5 h) IP injection of GAP-NPs. Representative images of coronal sections at two different levels of the abdomen are depicted for each time point. Abbreviations: ip – intraperitoneal cavity; si – small intestine; li – liver; st – stomach, ki – kidney. 364

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Fig. 4. Quantification of IOH-NPs in different organs of mice by MRI and ICP-MS after IP injection. (A-C) Representative ROI analyses of the contrast-to-noise-ratio (CNR) in MR images at 30 min time intervals before and after IP injection of GAP-NPs into a wildtype mouse. Detection of IOH-NPs was performed in the peritoneal cavity as well as in kidney, liver, small intestine and stomach. The grey area indicates a CNR level of ≤3, which was assumed to be the detection limit. (D) Different organs were collected 5 h after IP injection of BMPNPs into mice and the zirconium concentration was determined by ICP-MS. The total zirconium (Zr) content in each tissue was then calculated by multiplying its concentration with the total weight of the respective organ (N = 4).

intestine 24 h after IP injection of BMP-NPs showed that the zirconium content in both organs had moderately declined again (SI: Fig. S7). In summary, there is a very good correlation between MRI and ICP-MS results. 3.4. FACS analysis of IOH-NPs in mouse organs after IP injection Next, we aimed to visualize the cellular uptake of IOH-NPs in different organs after IP injection into mice in vivo. As examples we chose the spleen, since it allows to evaluate the presence of IOH-NPs in the circulation, and the small intestine because it is one of the organs in which we demonstrated an accumulation of IOH-NPs by MRI and ICPMS. EP-NPs were injected IP into mice and after 5 h, the animals were sacrificed and the spleen and small intestine removed. Single cell preparations were stained with monoclonal antibodies directed against CD3 and CD11b to distinguish between T cells and myeloid cells. The efficacy with which the two cell types took up EP-NPs was then determined by flow cytometric analysis based on the green fluorescence of FMN contained in EP-NPs. Importantly, engulfment of EP-NPs could be demonstrated in the spleen and small intestine in vivo although to a different degree (Fig. 5, SI: Fig. S8). The percentage of cells that incorporated EP-NPs in the small intestine was higher than in the spleen, and in general, myeloid cells took up EP-NPs much more efficiently than T cells (Fig. 5, SI: Fig. S8). Taken together, our results confirm that IOH-NPs reach abdominal organs after IP injection and are selectively taken up by different cell types in vivo.

Fig. 5. Flow cytometric analysis of IOH-NP uptake by T cells and myeloid cells in spleen and small intestine following IP injection. Organs were collected 5 h after EP-NP administration and single cell preparations were stained with monoclonal antibodies recognizing CD3 or CD11b in order to distinguish between T cells and myeloid cells. Cell type-specific EP-NP uptake was determined by flow cytometric analysis of the percentage of FMN+ cells amongst the two immune cell subpopulations (N = 3–4).

observed any internalization into WEHI 7.1 and WEHI 231 cells (Fig. 6B). L929 cells rapidly incorporated EP-NPs as well, but LA-4 and C2C12 cells showed only a moderate uptake efficacy (Fig. 6B). In a second step, we incubated each cell line for 24 h either with EP-NPs or BMP-NPs. The uptake further increased after longer incubation as indicated by the higher percentages of FMN+ cells but the order of efficacy with which each cell line took up the IOH-NPs remained the same. Nevertheless, on the long run macrophages had a clear lead over the other cell lines (Fig. 6C). Finally, we tested the metabolic activity with an MTS assay, which confirmed that IOH-NPs did not affect cellular viability (Fig. 6D). Collectively, our results unveil marked differences in the ability to internalize IOH-NPs, which suggests that preferential targeting of selective cell types and tissues should be possible.

3.5. Analysis of cell type-specific uptake of IOH-NPs in vitro The activity profile of nanoformulations is determined by the differential ability of individual cell types to take up particular matter via endocytic pathways [23,25]. We therefore selected six representative cell lines derived from the immune system and solid tissues (Fig. 6A) and studied their uptake of IOH-NPs by flow cytometric analysis of FMN contained in BMP-NPs and EP-NPs (SI: Fig. S9A). These data are well in agreement with a representative analysis of MH-S cells performed by confocal microscopy (SI: Fig. S9B). Initially, we incubated each cell line with EP-NPs and compared the percentages of cells that had incorporated them during the first 6 h. The uptake of EP-NPs by MH-S cells was very efficient, whereas we hardly

3.6. Characterization of IOH-NP uptake into macrophages Eukaryotic cells use different endocytic pathways to incorporate fluids, soluble molecules and particular matter [23,25]. Since our previous experiment identified macrophages as a major target cell of IOH365

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Fig. 6. Cell type-selectivity of IOH-NP uptake in vitro. (A) Schematic representation of the origin of the employed cell lines. (B) The cell lines were cultured in 6-well plates and incubated with 2.5 μg/ml EP-NPs containing FMN for up to 6 h. Cells analyzed before the addition of IOH-NPs served as a control (con). The efficacy of IOH-NP uptake was determined by flow cytometry based on the percentage of FMN+ cells (N = 3). (C) The cell lines were either incubated with 2.5 μg/ml EP-NPs or BMP-NPs for 24 h and analyzed for the percentage of FMN+ cells by flow cytometry (N = 3). (D) The cell lines were incubated with or without 2.5 μg/ml EP-NPs or BMP-NPs for 6 h and an MTS assay was performed to determine their viability based on the metabolic activity (N = 4). Cells cultured without (w/o) IOH-NPs served as a reference.

NPs, we characterized the uptake mechanism in MH-S cells (Fig. 7A). To this end, three pharmacological inhibitors were employed: cytochalasin D depolymerizes actin filaments and thereby suppresses both phagocytosis and macropinocytosis, amiloride inhibits Na+/H+ exchangers and mainly interferes with macropinocytosis, and monodansyl-cadaverine (MDC) blocks clathrin-dependent pinocytosis by inhibiting transglutaminase 2 [24,27]. Initially, we determined the concentration of each inhibitor at which a good efficacy but low toxicity were achieved (data not shown). Subsequently, we measured the uptake of BMP-NPs for 6 h by flow cytometry. Cytochalasin D as well as amiloride inhibited IOH-NP uptake by MH-S cells whereas MDC had no effect, suggesting that macrophages predominantly use the macropinocytosis pathway to take up IOH-NPs in vitro (Fig. 7B). When we investigated the dose-response of BMP-NP internalization after 24 h, a similar picture was obtained. The efficacy with which MH-S cells engulfed IOH-NPs increased with escalating concentrations, and both cytochalasin D and amiloride but not MDC interfered with their uptake (Fig. 7C). Importantly, the inhibitors were overall well tolerated at the selected concentrations for up to 24 h, although amiloride reduced the metabolic activity to some extent (Fig. 7D).

fates. Endosomes can fuse with lysosomes, but targeting to the mitochondria or the nucleus as well as a recycling back to the cell membrane have been reported, too [27]. Thus, we set out to determine the intracellular fate of the engulfed IOH-NPs in MH-S cells using imaging flow cytometry, a technique which combines the advantage of highthroughput analysis with the spatial information of microscopy [40]. MH-S cells were incubated with BMP-NPs containing the green fluorescent dye FMN for 6 or 24 h and subsequently loaded with a red fluorescent LysoTracker to visualize lysosomes or as a control with the mitochondrial marker MitoTracker (Fig. 7A). An extensive overlay of both signals resulting in yellow fluorescence was observed in cells labelled with LysoTracker. In contrast, an incubation of MH-S cells with BMP-NPs and MitoTracker resulted in distinct green and red spots (Fig. 8A). Analysis of the Bright Detail Similarity, which defines how well punctate stainings in two corresponding images correlate, revealed high scores for cells loaded with LysoTracker but not MitoTracker (Fig. 8B). Quantification of > 500 individual cells for each condition in three separate experiments confirmed that BMP-NPs selectively colocalized with the lysosomal compartment following engulfment by MH-S cells (Fig. 8C). In summary, our results indicate that microvesicles containing IOH-NPs fuse with lysosomes for subsequent degradation.

3.7. Intracellular fate of incorporated IOH-NPs

3.8. Biological activity of IOH-NPs Lysosomes contain enzymes that are able to release the biologically

Particular matter taken up by endocytosis may experience different 366

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Fig. 7. Analysis of the endocytic pathway of IOH-NP uptake into MH-S cells in vitro. (A) Scheme illustrating endocytic pathways, the activity of inhibitors, and the intracellular organelles that can be visualized with fluorescent trackers. (B) Cells were cultured in 24-well plates and incubated with 50 μg/ml BMP-NPs containing FMN for up to 6 h. Cells analyzed before the addition of IOH-NPs served as a control (con). Endocytosis inhibitors were added to the cells during the entire culture: cytochalasin D (cytoD, 1 μg/ml), amiloride (1 mM), or monodansyl-cadaverine (MDC, 100 μM). Some cells were left untreated as a control (w/o). The efficacy of IOH-NP uptake was determined by flow cytometric analysis of the percentage of FMN+ cells (N = 3). (C) Cells were incubated with ascending concentrations of BMP-NPs for 24 h with or without inhibitors and analyzed for the percentage of FMN+ cells by flow cytometry (N = 4). (D) Cells were incubated for up to 24 h with 6 μg/ml BMP-NPs with or without inhibitors. An MTS assay was performed to determine the metabolic activity as a measure of viability (N = 5). Cell cultured without NPs served as a reference.

iron NPs and rare-earth metal nanocrystals each with a size of around 20 nm revealed significant differences in organ distribution [19,41] as well as the concentration in the circulation [42] dependent on the administration route. IV injection immediately resulted in high NP levels in the blood as expected, which declined within 30 min due to their sequestration in liver, spleen and lung. Following IP application, however, the concentration of the NPs slowly increased in the blood during the first 3 h due to their gradual clearance from the peritoneal cavity [19]. Concomitantly, the NPs accumulated in pancreas and mesentery due to lymphatic drainage, direct absorption and homing of macrophages. Subsequently, they were also found in liver, spleen, intestine, stomach, lung and kidney but not in heart and brain [19,41]. Hence, IP injection appears to result in a slow distribution of NPs to abdominal organs rather than their rapid and predominant sequestration in liver and spleen. Our new MRI and ICP-MS data are in good agreement with these earlier findings. IOH-NPs were drained from the peritoneum after IP injection and then distributed to abdominal organs such as stomach, liver and small intestine. Here, they were taken up by myeloid cells but only to a much lesser extent by T cells. In kidney and lung, however, IOH-NPs were essentially undetectable. IOH-NP concentrations in liver and small intestine slowly decreased over the first 24 h, and excretion appeared to mainly proceed via the intestinal tract rather than the kidneys. Altogether, our findings speak in favor of a distribution of IOHNPs by lymphatic drainage and direct absorption via the portal circulation when they are administered by IP injection [20,21]. It is

active GC betamethasone from BMP-NPs by hydrolysis of the phosphate ester bond. To test this feature of IOH-NPs, we incubated BMDMs for 24 h with ascending concentrations of BMP-NPs or as a control with EPNPs lacking GCs. Subsequently, surface levels of MHC II and CD86, two receptors known to be regulated by GCs, were analyzed by flow cytometry (Fig. 9). EP-NPs had only a minor effect on the percentages of MHC II+ and CD86+ BMDMs whereas treatment with BMP-NPs strongly reduced them in a dose-dependent manner (Fig. 9A,B). We conclude that the drug contained in the IOH-NPs, namely betamethasone, becomes released after engulfment and shows biological activity. 4. Discussion Inflammatory disorders are widely treated with GCs, which reduce clinical symptoms but often cause adverse effect, too [1,2]. One possibility to make GC therapy more tolerable would be to target them to selective cell types such as macrophages by using nanoformulations [7]. We recently reported on the synthesis of IOH-NPs and their successful application in the treatment of a mouse model of multiple sclerosis [15,16]. Here we investigate the organ distribution of IOH-NPs in mice after IP injection, their specificity and mechanism of cellular uptake, as well as their intracellular trafficking and drug release. IV injection is the major route of drug application in humans although several investigations indicated that IP application might be preferable in the case of nanosized materials. Analyses of copolymeric 367

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Fig. 8. Detection of internalized IOH-NPs in MH-S cells by imaging flow cytometry. (A) MH-S cells were incubated for 6 h with 6 μg/ml BMP-NPs and stained with 100 nM LysoTracker Deep Red (left panel) or 100 nM MitoTracker Deep Red (right panel). Exemplary brightfield and fluorescent images (each channel separately and as an overlay) are depicted. (B) Cells that are double-positive for the fluorescent signal of FMN and either the LysoTracker or MitoTracker after 6 or 24 h were analyzed for a colocalization of both signals using the Bright Detail Similarity feature. For each time point one representative experiment is depicted. (C) Quantification of BMP-NP colocalization with lysosomes or mitochondria after 6 or 24 h of culture, based on the Bright Detail Similarity, determined in three separate experiments, each of them including > 500 individual cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 9. Regulation of surface expression of macrophage receptors by betamethasone released from IOH-NPs. BMDMs were incubated with or without (w/o) ascending concentrations of BMP-NPs or EPNPs for 24 h. Subsequently, surface expression of MHC II (A) or CD86 (B) were analyzed by flow cytometry. For each condition the percentage of MHC II+ or CD86+ cells is depicted (N = 4/5).

efficacy, whereas T and B cells almost took up none of them. This finding is supported by our observation that macrophages present in mouse organs in vivo were also more efficient in taking up IOH-NPs than T cells. Furthermore, our data are in line with other reports that NP uptake by macrophages is generally much greater than by T and B cells [43]. Interestingly, when we tested cell types present in solid tissues, we found that fibroblasts also took up IOH-NPs very well whereas myoblasts and epithelial cells did so much less efficiently. Consequently, our data indicate that GC therapy with IOH-NPs will not be entirely specific for macrophages and that the suitability of such an approach will depend on the individual features of the disease that shall be treated. For instance, previous studies have shown that T cells are

noteworthy that a previous study analyzing the biodistribution of IOHNPs revealed a predominant accumulation in liver, gall bladder, kidney and lung after administration by IV injection [18]. Therefore, IP injection of IOH-NPs appears to result in a more favorable pharmacokinetics than IV injection in vivo. GCs are lipophilic hormones that enter cells by passive diffusion across the plasma membrane and subsequently bind to the ubiquitously expressed cytosolic GC receptor. In contrast, GCs encapsulated in IOHNPs need to be actively internalized by their target cells, which confers them a certain degree of specificity contingent upon the endocytic capacity of each cell type [43]. Our analysis of six different cell lines revealed that macrophages internalized IOH-NPs with the highest

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organ distribution of IOH-NPs in combination with their differential uptake by individual cell types is likely to confer a high degree of specificity to GC treatment when using this delivery vehicle, which thus holds great promise to improve anti-inflammatory therapy in the future.

target cells of GCs in the treatment of rheumatoid arthritis [44], macrophages and neutrophils are important in contact dermatitis and acute lung injury [45,46], and airway epithelial cells are crucial for the suppression of allergic asthma [47]. Similarly, adverse effects of GCs may either concern muscle cells, osteoblasts or hepatocytes, and thus the uptake of IOH-NPs by these cell types will determine whether detrimental activities of GCs can be circumvented. Similar to our findings presented here, two previous publications demonstrated that a Dex-copolymer was taken up efficiently by macrophage-like and fibroblast-like synoviocytes. Consequently, inflammation was well repressed in a mouse model of rheumatoid arthritis [48], whereas bone loss was less severe in comparison to the free drug [49]. While the concomitant targeting of macrophages and fibroblasts was beneficial in this model, it could also be disadvantageous under other circumstances. For instance, GCs were shown to regulate extracellular matrix remodeling in mesenchymal fibroblasts [50], for which reason it is conceivable that targeting BMP-NPs to this cell type may lead to an increased risk of fibrosis. Altogether, our data suggest that IOH-NPs represent a promising concept for the targeted delivery of GCs in inflammatory diseases although their specificity has its limitations and needs to be individually evaluated for each application. Nanosized material is taken up by endocytosis via different pathways that can be studied by using a panel of selective inhibitors [23,24]. Our data suggest that IOH-NP are internalized by macropinocytosis, at least in macrophages, although it is fair to assume that this pathway will probably be used by other cell types such as fibroblasts as well. The specificity of inhibitors is always a matter of debate [27]. We found that cytochalasin D prevented IOH-NP uptake while having minimal toxicity, which indicates that phagocytosis or macropinocytosis were involved. However, one should keep in mind that some authors believe that actin depolymerization leads to a general block of all internalization pathways [24]. In contrast, MDC had no effect, arguing against a role of clathrin-dependent micropinocytosis. Amilioride also prevented the uptake of IOH-NPs but at the cost of a partially compromised cellular viability. However, it appears unlikely that the reduced metabolic activity can be made responsible for the inhibition of IOH-NP uptake. Since amiloride mainly inhibits macropinocytosis, this pathway is presumably the most relevant one for IOHNPs. Macropinocytosis takes place in a variety of cell types including dendritic cells and macrophages but also fibroblasts [26]. The formation of membrane ruffles leads to a largely unspecific internalization of soluble molecules such as antibodies and particular matter like bacteria and cell fragments. The fact that target recognition by specific receptor is dispensable for macropinocytosis also explains why fibroblasts, which are unable to execute phagocytosis, still engulf IOH-NPs. It is noteworthy that all these experiments have been performed in vitro, and that NPs are often opsonized in vivo by antibodies, complement and serum proteins, which are recognized by their respective receptors possibly resulting in phagocytosis as well. Whether and to which degree opsonization of IOH-NPs occurs in the blood is currently unknown and needs to be determined. Taken together, our data suggest that IOH-NPs are mainly taken up by macropinocytosis, at least in vitro. The intracellular trafficking and fate of nanosized material is also important for determining the biological activity. We could show that IOH-NPs colocalize with the lysosomal compartment, which contains enzymes that are able to catalyze the release of the drug by hydrolytic cleavage of the phosphate ester bond. Furthermore, we observed that the GCs contained in BMP-NPs unfold their anti-inflammatory activity in macrophages as revealed by the down-regulation of crucial effector molecules of these cells. Hence, drugs contained in IOH-NPs reach the correct intracellular location necessary to exert their biological activity. In conclusion, IOH-NPs administered into the peritoneum show a delayed release from the site of injection and reach multiple abdominal organs. They are mainly engulfed by macrophages and fibroblasts via macropinocytosis, followed by their trafficking to the lysosomes where the drug gets released and exerts its biological activity. The selective

Author contribution TKK: performed most experiments and analyzed data; MK: synthesized nanoparticles; AM: performed MRI experiments; ME: was involved in imaging flow cytometry experiments; SB: supervised MRI experiments; CF: designed and supervised nanoparticle synthesis; HR: designed the project, supervised most experiments, analyzed data and wrote the manuscript. Funding This work was supported by the Deutsche Forschungsgemeinschaft (Re 1631/17-1) and the Elsbeth Bonhoff Stiftung (Grant number 203). Data availability The raw and processed data required to reproduce these findings are available upon request from the authors. Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgement We would like to thank Amina Bassibas, Nicole Klaassen, Tor Memhave, and Kristin Kötz for expert technical assistance. Appendix A. Supplementary data Supporting information (SI) for this article can be found online at https://doi.org/10.1016/j.jconrel.2020.01.010. References [1] U. Baschant, J. Tuckermann, The role of the glucocorticoid receptor in inflammation and immunity, J. Steroid Biochem. Mol. Biol. 120 (2010) 69–75. [2] N. Schweingruber, S.D. Reichardt, F. Lühder, H.M. Reichardt, Mechanisms of glucocorticoids in the control of neuroinflammation, J. Neuroendocrinol. 24 (2012) 174–182. [3] S. Whirledge, D.B. DeFranco, Glucocorticoid signaling in health and disease: insights from tissue-specific GR knockout mice, Endocrinol 159 (2018) 46–64. [4] A. Rauch, S. Seitz, U. Baschant, A.F. Schilling, A. Illing, B. Stride, M. Kirilov, V. Mandic, A. Takacz, R. Schmidt-Ullrich, S. Ostermay, T. Schinke, R. Spanbroek, M.M. Zaiss, P.E. Angel, U.H. Lerner, J.P. David, H.M. Reichardt, M. Amling, G. Schütz, J.P. Tuckermann, Glucocorticoids suppress bone formation by attenuating osteoblast differentiation via the monomeric glucocorticoid receptor, Cell Metab. 11 (2010) 517–531. [5] M.L. Watson, L.M. Baehr, H.M. Reichardt, J.P. Tuckermann, S.C. Bodine, J.D. Furlow, A cell-autonomous role for the glucocorticoid receptor in skeletal muscle atrophy induced by systemic glucocorticoid exposure, Am. J. Phys. 302 (2012) E1210–E1220. [6] D.H. van Raalte, D.M. Ouwens, M. Diamant, Novel insights into glucocorticoidmediated diabetogenic effects: towards expansion of therapeutic options? Eur. J. Clin. Investig. 39 (2009) 81–93. [7] F. Lühder, H.M. Reichardt, Novel drug delivery systems tailored for improved Administration of Glucocorticoids, Int. J. Mol. Sci. 18 (2017). [8] M.L. Immordino, F. Dosio, L. Cattel, Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential, Int. J. Nanomedicine 1 (2006) 297–315. [9] N. Schweingruber, A. Haine, K. Tiede, A. Karabinskaya, J. van den Brandt, S. Wüst, J.M. Metselaar, R. Gold, J.P. Tuckermann, H.M. Reichardt, F. Lühder, Liposomal encapsulation of glucocorticoids alters their mode of action in the treatment of experimental autoimmune encephalomyelitis, J. Immunol. 187 (2011) 4310–4318.

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