Immunomodulatory Effects of Dendritic Poly(ethyleneimine

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Cite This: Biomacromolecules 2019, 20, 2713−2725

Immunomodulatory Effects of Dendritic Poly(ethyleneimine) Glycoarchitectures on Human Multiple Myeloma Cell Lines, Mesenchymal Stromal Cells, and in Vitro Differentiated Macrophages for an Ideal Drug Delivery System in the Local Treatment of Multiple Myeloma Felix Schulze,† Bettina Keperscha,‡ Dietmar Appelhans,*,‡ and Angela Rösen-Wolff*,† Downloaded via UNIV OF SOUTHERN INDIANA on July 19, 2019 at 08:33:02 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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Department of Pediatrics, University Hospital Carl Gustav Carus, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany Leibniz Institute of Polymer Research Dresden, Hohe Str. 6, 01069 Dresden, Germany

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S Supporting Information *

ABSTRACT: The use of a drug delivery system (DDS) represents a novel therapeutic approach in the treatment of multiple myeloma in bone lesion. We show the immunomodulatory effects of anionic and cationic dendritic poly(ethyleneimine) glycoarchitectures (PEI-DGAs) on human myeloma cell lines and cells in their microenvironment, in vitro differentiated macrophages, and mesenchymal stromal cells (MSCs). PEI-DGAs do not influence the secretion of IL-6, which is a major growth and survival factor in multiple myeloma. Cationic PEI-DGAs in turn have cytostatic properties on multiple myeloma cell lines. Anionic PEI-DGAs induce the secretion of proinflammatory cytokines IL-1β, TNFα, and IL-6 in macrophages and MSCs, whereas cationic PEI-DGAs do not. Macrophages and MSCs show remarkably high cell viability in the presence of high concentration of PEI-DGAs. RNA sequencing of MSCs exposed to cationic PEI-DGAs supports the hypothesis that smaller cationic PEI-DGAs are less toxic and could improve osteogenic differentiation in an ideal DDS.

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resistance.10 Activated proinflammatory macrophages secreting IL-6 and TNFα then activate myeloma cells.12,13 Contactmediated interactions between macrophages and myeloma cells also strengthen drug resistance of the latter because of the uptake of drug molecules by macrophages. Myeloma cells are also activated by released mediators and MSC-derived cytokines (IL-6, IL-8, and TNFα) from the extracellular environment to enhance cell growth and survival.14−17 One conclusion of this complex interaction relationship between myeloma cells, macrophages, and MSCs is that the immune response of macrophages is a pivotal in the homeostasis of the myeloma niche,10 in addition to the immune response of MSCs to their extracellular environment. Our aim has been to validate the proinflammatory response and biocompatibility of cationic and anionic dendritic glycoarchitectures (Figure 1) against myeloma cell lines and parts of their interacting cells, macrophages as a component of the innate immune system, and MSCs, as part of bone marrow cells. The results of the present study provide evidence of further optimizing the composition of dendritic glycoarchitectures in having negligible immunomodulatory properties

INTRODUCTION Multiple myeloma is a malignant disease of clonal plasma cells,1,2 accumulating in the bone marrow and causing symptoms related to the formation of bone lesions, replacement of normal hematopoiesis, and production of monoclonal protein. As normal plasma cells, myeloma cells depend for their survival on interaction with the bone marrow microenvironment. After systemic therapy with chemotherapeutics, some malignant cells can survive in bone lesions of multiple myeloma patients, leading to a relapse.3−8 For the assessment of innovative therapeutics for the local treatment of relapsed myeloma in bone lesions (Scheme 1A) to reconstitute the natural bone remodeling (Scheme 1B), interactions in the multiple myeloma environment in the presence of the drug delivery system (DDS) have to be identified before key characteristics of an adaptive and smart DDS for the local therapy9 in bone lesions can be postulated. Interaction between tumor cells and the local microenvironment (e.g., osteoclasts, osteoblasts, macrophages, and bone marrow human mesenchymal stromal cells (MSCs)) is the deciding issue in understanding the underlying interaction principles between healthy and unhealthy cells in multiple myeloma.10,11 An interaction between myeloma cells, macrophages, and MSCs has been postulated such that each cell type can orchestrate myeloma cell growth, viability, and drug © 2019 American Chemical Society

Received: April 5, 2019 Revised: May 20, 2019 Published: June 25, 2019 2713

DOI: 10.1021/acs.biomac.9b00475 Biomacromolecules 2019, 20, 2713−2725

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Scheme 1. Validating the Influence of Dendritic Glycoarchitectures as a Potential DDS for Local Therapy of Multiple Myeloma (MMC) and Interacting Cells (MΦ, MSC)a

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MΦ and MSC enhanced the survival and growth of MMC. It is necessary to develop DDSs that do not stimulate the secretion of IL-6 and other cytokines in future local therapy of MM. (A) Pathological bone in multiple myeloma, (B) healthy bone status, (C) bone substituent with anionic glycoarchitectures, and (D) bone substituent with cationic glycoarchitectures. Ideas for Scheme 1 adopted from Klein et al.59 and further modified for own purpose.

We have shown that dendritic glycoarchitectures, based on oligosaccharide-modified hyperbranched poly(ethyleneimine) (PEI) and poly(propyleneimine) (PPI) dendrimers, are suitable for DDS,18−25 showing a required concentrationdependent biocompatibility in both in vitro and in vivo applications.18−21,26,27 Previously, the influence of cationic dendritic PEI glycoarchitectures, PEI-5-Mal B and PEI-25-Mal B, was also employed (Figure 1), and their effects on primary MSCs and their differentiated cells into osteoblasts were investigated. One key issue was identified, that is, that the smaller PEI-5-Mal B provides superior biocompatibility over 28 days in the presence of MSCs and osteoblasts compared to larger PEI-25-Mal B macromolecules. Only at high dose (1 mg/mL) is the larger PEI-25-Mal B toxic and initiates clearly visible mitochondrial damage. Moreover, dendritic PEI glycoarchitectures are also suited in DDS to induce prolonged release of bortezomib from bone cement.23 Bortezomib is a successfully used proteasome inhibitor in the systemic administration of multiple myeloma patients.28,29 In the present study, three different dendritic glycoarchitectures (PEI-DGP; Figure 1) have been used here: PEI macromolecules with two different molecular weights of 5000 (PEI-5) and 25 000 (PEI-25) g/mol were modified with maltose (Mal) units to establish open shell (B) dendritic PEI glycoarchitectures, PEI-5-Mal B and PEI-25-Mal B.30 The third PEI-DGP (PEI-PGlu-Mal) is composed of a PEI-5 core decorated with Mal units and anionic oligo/polyglutamic acid chains (PGlu). The terminal primary amino groups are mostly modified with Mal units.31 The smaller PEI-5-Mal B can be considered a core−shell architecture,32 with a dominating Mal shell, whereas the larger PEI-25-Mal B is attributed with a fluffy globular structure at which a dynamic embedding of Mal units within the dendritic PEI scaffold becomes possible, besides the

Figure 1. Simplified molecular architectures of dendritic PEI glycoarchitectures. Mal = maltose. PGlu = polyglutamic acid. PEI25 = PEI with molecular weight of 25 000 g/mol. PEI-5 = PEI with molecular weight of 5000 g/mol. B = defines an open shell architecture in the outer shell of dendritic PEI scaffold, decorated with maltose units.42

(e.g., avoiding/minimizing the secretion of proinflammatory cytokines) of our DDS in future drug-targeted cell studies in multiple myeloma. Development of the potential DDS in the presence of malignant cells would be to have an inert DDS regarding healthy cells without activating their immune response, whereas DDS could be cytotoxic to malignant cells in this manner, such that cancer cell growth and survival is impaired. 2714


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Pam3CSK4 5 ng/mL, Nigericin 1 μM (1 h), MSU 150 μg/mL, or PMA 1 μg/mL. For most experiments, cells were stimulated for 24 h; the cell-free supernatant and the washed cells were then analyzed. Cytokine Measurement. Cytokine concentrations in the supernatants were determined by a cytometric bead array (CBA assay) for IL-1β, IL-6, IL-8, and TNFα according to manufacturer’s instructions. Briefly, 24.5 μL of capture bead diluent was mixed with 0.5 μL of capture beads of the appropriate cytokines, and 50 μL of cell culture supernatant was added. The suspension was mixed and incubated for 1 h at room temperature in the dark. Thereafter, 24.5 μL of the detection reagent diluent and 0.5 μL of the PE detection reagent, for each cytokine tested, were mixed and added to the bead supernatant suspension. After mixing, the samples were incubated for 1 h at room temperature in the dark. The beads were washed once and analyzed in a LSRII flow cytometer. The concentrations of cytokines in the samples were calculated with FCAP Array software, using a standard curve. Cell Viability and Mitotracker Staining. Cell viability was measured indirectly by the AlamarBlue Assay. Cell media were freshly replaced after stimulation before 10× AlamarBlue was added. After 2 h incubation, fluorescence was measured using with a multiplate reader (Tecan Infinite M200, Ex. 570 nm and Em. 585 nm). Cell death of multiple myeloma cells was analyzed after 48 h of incubation with PEI-DGAs by AnnexinV/PI staining. The cells were washed twice in FACS buffer (PBS + 2% FCS) and stained with AnnexinV-APC and propidium iodide. They were analyzed in a LSRII flow cytometer by gating single cells, with the results expressed as percentage of AnnexinV-APC and PI positivity. For visualization of mitochondrial damage, MitoTracker Deep Red and Green were used. After stimulation, the medium was removed, and the cells were washed three times with PBS. Thereafter, the medium including MitoTracker Green and Deep Red (50 nM) was added and the culture plates were incubated for 30 min. The cells were washed three times with PBS and detached with PBS/EDTA. The cell suspension was immediately analyzed by LSRII flow cytometry. The data were analyzed using FlowJo software. Confocal Microscopy and FACS Analysis. THP-1 cells were differentiated, as described above, on collagen-coated culture slides overnight before being incubated for 24 h with FITC-labeled PEIDGAs. Thereafter, supernatants were removed, the cells washed three times with PBS and finally fixed in 4% paraformaldehyde for 10 min at room temperature. Coverslips were mounted on glass slides in a DAPI-containing Vectashield mounting medium (Vector Laboratories). Images of the cells were taken with an inverted Zeiss LSM 510 confocal microscope (Carl Zeiss) with a 40× or 63× 1.4NA objective lens. An argon laser was used for excitation of FITC at a wavelength of 488 nm, whereas a laser diode (405 nm) was used for the excitation of DAPI. For flow cytometric analysis of the interaction of cells and FITCconjugated PEI-DGAs, THP-1 cells were differentiated in 24-well plates and incubated with 1 mg/mL of FITC-PEI-DGAs. After 24 h, the supernatant was removed and the cells were washed three times with PBS. Cells were detached with PBS/EDTA for FACS analysis. First, the whole FITC signal was detected by LSRII flow cytometry. To measure internally localized PEI-DGA, cells were washed with PBS in FACS tubes, and the supernatants were then discarded. The cell pellet was resuspended in 0.4% trypan blue solution (SigmaAldrich) and incubated for 5 min on ice before the analysis started. RNA Sequencing and Data Analysis. For RNA sequencing, 2 × 105 MSCs were seeded in 6-well plates and stimulated for 24 h (as indicated above). After the incubation, supernatants were removed and cells were washed three times with PBS. Thereafter, 1 mL of Trizol reagent was added, and incubation continued for 10−20 min at room temperature. Cells were lysed by pipetting several times, and the solution was transferred to a 1.5 mL tube. Chloroform (200 μL) was added, and incubation continued for 15 min at room temperature. Phase separation involved centrifugation at 11 500g for 15 min at 4 °C. The aqueous phase was transferred into a new 2 mL tube, and the

presence of a Mal unit in the outer shell itself.33 Anionic PEIPGlu-Mal is also characterized by a core-double-shell and binary architecture in which anionic PGlu chains are preferred to be randomly organized and to undergo ionic interactions with the cationic PEI-5 core.31 In general, surface modification of PEI macromolecules has always improved the biocompatibility of PEI macromolecules with different cells, as well as their potential use in biological, medical, and pharmaceutical fields.18,20,21,34−36

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EXPERIMENTAL SECTION

General and Materials. Poly(ethyleneimines) (PEI) is used as a general abbreviation; Lupasol G100 with Mw 5000 g/mol and Lupasol WF with Mw 25 000 g/mol were obtained from BASF SE (Ludwigshafen, Germany). Maltose monohydrate (Mal), sodium borate, and borane pyridine complex (BH3*Py complex, 8 M solution in tetrahydrofuran) were purchased from Fluka (Germany). Dendritic PEI glycoarchitectures (PEI-DGAs) (Figure 1) were synthesized and characterized as described in refs 26 and 30. Some parameters of PEIDGAs have been characterized and can be found in the Supporting Information (Table S1 and Figure S1). Chloroform, ethanol (70%), Lglutamine, paraformaldehyde, PBS, penicillin/streptomycin mixture, PMA (phorbol-12-myristal-13-acetate), propidium iodide, and trypan blue solution were bought from Sigma-Aldrich (Taufkirchen, Germany). Alpha medium, FCS, FiColl, and RPMI1640 medium were provided by Biochrom GmbH (Berlin, Germany). Antibodies against TLR4 and TLR2, MSU, Nigericin, Pam3CSK4, and upLPS were purchased from Invivogen (Toulouse, France). AnnexinV-APC was purchased from Biolegend (Koblenz, Germany), Vectashield mounting medium was purchased from Vector Laboratories (Burlingame, USA), and BioCoat Collagen Culture Slides were purchased from BD Bioscience (Heidelberg, Germany). The following kits were used: Cytometric Bead Array from BD Bioscience (Heidelberg, Germany), RNaesy Mini Kit from Qiagen (Hilden, Germany), SMARTer Ultra Low RNA Kit from Clontech Laboratories (Mountain View, USA), and NEBNext ChIP-Seq Library Perp Master Mix Set for Illumina from New England Biolabs (Ipswich, USA). Methods. Handling of Dendritic PEI Glycoarchitectures for Cell Culture. Freeze-dried PEI-DGAs were weighed and dissolved in PBS at 50 mg/mL. The solution was filtered through 0.2 μm sterile filters and stored at −80 °C until use. For stimulation in cell cultures, PEIDGA solution was diluted to the required concentration in phenol red-free IMDM with 10% FCS. Cell Culture. Human THP-1 cells purchased from ATCC (ATCC TIB-202) were cultured in RPMI1640 supplemented with 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were passaged every 3−4 days and cultured at a starting density of 0.2 × 106 cells/mL. For differentiation, 0.5 × 106 cells/mL were cultured in 24-well plate and incubated with 5 nM phorbol-12-myristate-13-acetate (PMA) overnight. Human bone marrow MCSs were provided by the Translational Biomedical Research Group, Center of Regenerative Therapies, Dresden. These MSCs were enriched by density gradient centrifugation in Ficoll and cultured in Alpha medium containing 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. The culture medium was replaced after 4−6 days, and the cells were subsequently subcultured until they reached 80−90% confluence. Multiple myeloma cell lines (RPMI-8226, EJM, NCI-H929, SKMM, and U266) were obtained from the laboratory for myeloma research, Department of Internal Medicine V, University of Heidelberg. The cells were cultured in complete RPMI1640 medium (see THP-1 medium) and passaged twice a week. Stimulation of Cells with Dendritic PEI Glycoarchitectures. For stimulation with PEI-DGAs, the cell lines were seeded in 24-well plates at 5 × 106 cells/well. MSCs were used at 5 × 104 cells/well. The concentrations of stimuli were, if not stated otherwise, PEI-PGluMal, PEI-25-Mal B, and PEI-5-Mal B 1 mg/mL, upLPS 50 ng/mL, 2715


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Figure 2. Effects of PEI-DGAs on cytokine release from human multiple myeloma cells. Human multiple myeloma cell lines (RPMI-8226, EJM, NCI-H929, SKMM, and U266) were stimulated for 24 h with 1 mg/mL PEI-DGAs, 1 μg/mL upLPS, 1 μg/mL Pam3CSK4, or 50 ng/mL PMA. Cytokine concentration was determined by CBA. Data shown as mean ± S.E.M., n = 5. (A) IL-6. (B) IL-8. (C) TNFα. same volume of 70% ethanol was added before transferring to RNaesy spin columns. The RNaesy protocol was followed along with instructions that included DNAseI digestion. RNA concentration was measured using the Nanodrop system, and RNA quality control used an Agilent 2100 Bioanalyzer (Agilent). RNA quality was controlled by comparing the 28S to 18S RNA peak ratio (2:1 in non-degraded RNA-samples) or the so-called RNAintegration number. cDNA was synthesized using a SMARTer Ultra-Low RNA Kit. Briefly, 1 μL of total RNA (5 ng) was added to 1 μL of 12 μM SMART CDS Primer IIA and 2.5 μL of Dilution Buffer before the samples were incubated at 72 °C for 3 min, followed by 42 °C for 2 min. Samples were supplemented with 2 μL of first-strand buffer, 0.25 μL of 100 mM DTT, 1 μL of 10 mM dNTPs, 1 μL of SMARTer IIA oligonucleotide, 0.25 μL of RNAse inhibitor, and 1 μL of SMARTscribe reverse transcriptase, followed by incubation at 42 °C for 1 h. After clean-up with AMPure XP Beads, cDNA was preamplified using the SMARTer Ultra-Low RNA Kit protocol. The amplified cDNA was fragmented using ultrasound, and the cDNA library was prepared using NEBNext ChIP-Seq Library Perp Master Mix Set for Illumina. The libraries were sequenced on a NextSeq 500 sequencer (Illumina). RNA-Sequencing DATA were analyzed as previously reported.37 Next-generation-sequencing RNA fastq-Files were aligned and reads were counted with STAR,38 with default options. As a reference, GRCh38 genome and annotation information of Ensembl databases release 82 were used. Normalization and differential expression analysis used R39 (version 3.3; http://www.r-project.org/) and edgeR.40 Genes with at least 1 read per million in at least three samples were selected. Normalization factors, library size, and counts per million were computed. Differential expression analysis was estimated using a quasilikelihood F-test. Genes were counted as differentially expressed with an adjusted p-value ≤0.05 and absolute fold change of ≥2.

The goana function of edgeR was used to carry out over representation analysis for gene ontology (GO) terms of the differentially expressed genes. GO terms with p-value ≤0.01 were selected. Statistical Analysis. The data are given as mean ± S.E.M., and asterisks mark statistically significant differences to the corresponding control. The differences between groups were analyzed by one-way ANOVA with Dunn’s multiple comparison test. For calculation of significance, GraphPad Prism 6 software was used. P ≤ 0.05 was taken as being significant, with p ≤ 0.005 being highly significant.

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RESULTS AND DISCUSSION

Effects of Dendritic PEI Glycoarchitectures on Cytokine Release and Viability of Myeloma Cell Lines. The proinflammatory cytokine IL-6 is important as a survival and proliferation factor in myeloma cells.1 This factor should not be induced by all three PEI-DGAs when applying them as potential DDS for local treatment of bone lesions in relapsed myeloma cells. IL-6, IL-8, and TNFα release were determined after stimulation of five different multiple myeloma cell lines with different stimulants (1 μg/mL upLPS, 1 μg/mL Pam3CSK4, 50 ng/mL PMA, or 1 mg/mL PEI-DGAs) (Figure 2). All three cytokines were unaffected by either anionic PEI-PGlu-Mal or cationic PEI-DGAs, PEI-25-Mal B, and PEI-5-Mal B because of below (e.g., PEI-25-Mal B in Figure 2C) or marginal above (e.g., PEI-PGlu-Mal in Figure 2B) values for positive controls. In the case of positive controls, (upLPS, Pam3CSK4, and PMA), we could induce secretion of cytokines from these cells. No positive control was suitable for inducing cytokine secretion in all of the cell lines, but one of the positive control stimulators induced the release of 2716


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Figure 3. Influence of PEI-DGAs on cell death in human multiple myeloma cells. Human multiple myeloma cell lines (RPMI-8226, EJM, NCIH929, SKMM, and U266) were stimulated for 48 h with 1 mg/mL PEI-DGAs. Cell viability was analyzed by AlamarBlue Assay (A) and AnnexinV/ PI staining (B). Data shown as mean ± S.E.M., n = 2 × 5; asterisks mark statistically significant differences from respective control *p ≤ 0.05 **p ≤ 0.005.

Figure 4. Effects of PEI-DGAs on proinflammatory cytokine release from THP-1 cells. THP-1 cells were differentiated with PMA overnight and stimulated for 24 h with 150 μg/mL MSU or 1 mg/mL PEI-DGAs. Cytokine concentration was determined by CBA. Data shown as mean ± S.E.M., n = 3−6; asterisks mark statistically significant difference from respective control *p ≤ 0.05 **p ≤ 0.005. (A) IL-1β. (B) IL-6. (C) TNFα.

These results indicate that both cationic PEI-DGAs were suitable for application as DDS because of their own potential to induce cell death in myeloma cell lines without affecting cytokine secretion. Effect of Dendritic PEI Glycoarchitectures on Cytokine Release from Macrophages. To imitate the effect of maltose-modified PEI-DGAs (Figure 1) interacting with multiple myeloma cells, differentiated THP-1 cells were used as a model for innate immune cells of macrophages being stimulated with all three PEI-DGAs (1 mg/mL) for 24 h. Thereafter, proinflammatory cytokines were measured, with the results being summarized in Figure 4. Secretion of IL-1β as a major proinflammatory cytokine was significantly increased by anionic PEI-PGlu-Mal, whereas cationic PEI-DGAs, PEI25-Mal B, and PEI-5-Mal B, did not increase the secretion of

measureable amounts of one cytokine for at least every cell line. These results show that all of the tested PEI-DGAs had no effect on cytokine secretion of myeloma cell lines, especially because IL-6, as a growth and survival factor, was not released.41−43 On the other hand, cationic PEI-DGAs, PEI-5Mal B, and PEI-25-Mal B did affect apoptosis of several human myeloma cell lines, as determined by AlamarBlue and AnnexinV/PI staining after 48 h of incubation of PEI-DGAs (Figure 3). Cell apoptosis was also observed by light microscopy after 24 h of incubation of PEI-DGAs (Figure S2). The AlamarBlue assay indicated only a slight reduction in energy metabolism (Figure 3A), but AnnexinV/PI staining clearly showed late apoptotic cells with a damaged cell membrane after cationic PEI-DGA treatment (Figure 3B). 2717


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Figure 5. Influence of PEI-DGAs on mitochondrial status and effects on cell viability due to PEI-DGAs internalization. (A) Cells were treated for 24 h with the indicated stimuli before their viability was determined by alamarBlue Assay. (B) Differentiated THP-1 cells were stimulated with 1 mg/mL of PEI-DGAs for 24 h or 5 μM Nigericin for 1 h. The cells were stained with Mitotracker Deep-Red and Green and were analyzed by flow cytometry to detect damaged mitochondria. (C) Cells were incubated with 1 mg/mL FITC-labeled PEI-DGAs for 24 h and the distribution of PEIDGAs was determined by confocal microscopy. (D) Flow cytometry analysis of FITC-PEI-DGAs distribution, total (without trypan blue), and internal fluorescence (after trypan blue quenching) were analyzed. (E) Quenching of external fluorescence on a percentage basis. Data presented as mean ± S.E.M., n = 3.

cytokines. Processing of IL-1β needs two signals; first, pro-IL1β is synthesized by NF-κB signaling, and second, IL-1β is cleaved by active caspase-1 to its mature form. As a positive control, MSU crystals were also used because these crystals can induce inflammation by triggering IL-1β production by activating the NLRP3 inflammasome, which in turn activates caspase-1.47,48 MSU crystals can indeed induce secretion of IL1β, but not other NF-κB depending cytokines (Figure 4), which indicates that no other proinflammatory cytokine secretion is induced by the autocrine effects of IL-1β.49 This accepted matter is directed by PMA priming of THP-1 cells to induce translation of pro-IL-1β during differentiation of THP1 cells. A concentration of PMA (5 ng/mL) is sufficient to induce the production of pro-IL-1β, but only small amounts of other cytokines.50 At this concentration, it is possible to respond well to secondary weak stimuli without being overwhelmed by unwanted gene upregulation induced by PMA.50 Moreover, increased secretion of IL-6 and TNFα after stimulation with PEI-PGlu-Mal (Figure 4) undoubtedly indicates activation of NF-κB pathways by anionic PEI-PGluMal. Effect of Dendritic PEI Glycoarchitectures on Cell Viability and Mitochondrial Status of Macrophages. The next experimental series were directed at determining cell

IL-1β compared to the controls. Secretion of IL-6 and TNFα was also significantly increased in cells that had been stimulated with anionic PEI-PGlu-Mal. On the other hand, cationic PEI-DGAs, PEI-25-Mal B, and PEI-5-Mal B did not increase the secretion of both cytokines compared to unstimulated cells (Figure 4). Cationic lipid nanocarriers and amino-functionalized polystyrene nanoparticles are able to induce several proinflammatory markers.44,45 Similar biological behavior of cationic PEI-DGAs was not found in relation to any increased secretion of proinflammatory cytokines (e.g., IL1β, IL-6, or TNFα) from THP-1 cells. One possible explanation is that maltose units in the outer sphere of cationic PEI-DGAs prevent any significant interaction with receptors outside and inside of THP-1 cells that would induce proinflammatory signaling. Similar results were seen when PEIcoated poly(lactide-co-glycolide) particles were given to macrophages.46 This also indicates that even more neutralized charges of cationic PEI in any particle composition can minimize biological interaction with immune receptors of cells. In contrast, anionic PEI-DGA PEI-PGlu-Mal has another function in the secretion of proinflammatory markers in the presence of THP-1 cells, as found with cationic PEI-DGAs. This dendritic PEI glycoarchitecture, with a double and binary shell (Figure 1), smoothly induced secretion of all three 2718


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Figure 6. Influence of NF-kB signaling inhibition on cytokine release during PEI-DGAs stimulation. Differentiated THP-1 cells were preincubated for 1 h with selective inhibitor of IκB kinase (10 μM) and stimulated with 50 ng/mL upLPS, 5 ng/mL Pam3CSK4 or 1 mg/mL PEI-DGAs for 24 h. Cytokine concentration was measured by CBA. Data presented as mean ± S.E.M., n = 3. (A) IL-1β. (B) IL-6. (C) TNFα.

suggested that 68% of PEI-PGlu-Mal, 60% of PEI-25-Mal B, and 80% of PEI-5-Mal B were located intracellularly. This distribution is partially explained by the different diameters of PEI-DGAs (Figure 1) in combination with its pH-dependent surface charge (Figure S1). Almost 20% more of the smallest PEI-DGA, PEI-5-Mal B, was located within the cells compared to the larger PEI-25-Mal B (Figure 5D). By contrast, anionic PEI-PGlu-Mal showed overall the least cellular uptake (Figure 5D) because of the presence of anionic surface charge (Figure S1). Validating the Participation of NF-κB Signaling for the Release of Proinflammatory Cytokines from Macrophages. It is postulated that the secretion of all cytokines followed (see Figure 4) are dependent on NF-κB activation and signaling. To determine if NF-κB signaling was involved and caused cytokine release from anionic PEI-DGA-stimulated cells, NF-κB signaling was inhibited with IKK16 that is a selective inhibitor of IκB kinase that blocks further NF-κB signaling.51 After 1 h of preincubation of THP-1 cells with IKK16, cells were stimulated with the three PEI-DGAs for 24 h. As positive controls for NF-κB activation, upLPS and Pam3CSK4 were included. Both stimulants induced, as expected, increased cytokine release (IL-1β, IL-6, and TNFα), whereas inhibition of NF-κB reduced the amount of cytokines secreted (Figure 6). Cytokine release from cells stimulated with PEI-PGlu-Mal was clearly reduced (IL-1β, IL6, and TNFα). By contrast, no secretion of IL-6 and TNFα occurred in the presence and absence of IKK16 for cationic PEI-DGA-stimulated cells. Thus, NF-κB signaling-dependent release of cytokines is mainly responsible for PEI-PGlu-Malstimulated THP-1 cells. This also implies that the anionic dendritic PEI glycoarchitecture PEI-PGlu-Mal can activate NFκB pathways. Induction of NF-κB in THP-1 cells by other dendritic glycoarchitectures has recently been shown. Maltosemodified PPI glycodendrimer was unable to induce a strong secretion of cytokines 52 because of the use of low glycodendrimer concentration (≤100 μM) that was given.57

and mitochondrial viability of macrophages (THP-1 cells) in the presence of PEI-DGAs (Figure 1). Therefore, THP-1 macrophages were incubated for 24 h with PEI-DGAs and cell viability measured indirectly by AlamarBlue assay (Figure 5A). All three PEI-DGAs failed to induce an increase in cell death compared to unstimulated cells. Light microscopy of THP-1 cells stimulated with PEI-DGAs also showed no changes in their physical appearance (Figure S3), which further supports the viability of THP-1 cells in the presence of all three PEIDGAs. To support the cell behavior of THP-1 cells in the presence of PEI-DGAs, mitochondrial functioning was examined. Mitochondria damage in MSCs occurred when 1 mg/mL PEI-25-Mal B was added to the cells.26 Finally, the same concentration of all PEI-DGAs was used for the mitotracker assay (Figure 5B), based on a flow cytometry analysis to determine a possible mitochondrial damage induced by PEIDGAs. As positive control, the pore-forming toxin nigericin was used. This toxin reduces mitochondrial membrane potential shown by decreased MitoTracker-Deep-Red staining after 1 h of incubation. In contrast, all PEI-DGAs tested failed to reduce the mitochondrial membrane potential after 24 h of stimulation, a finding that confirms all three PEI-DGAs to be biocompatible in differentiated THP-1 cells. To gain better insights into the molecular mechanisms of PEI-PGlu-Malinduced proinflammatory immune response of THP-1 cells (presented below), the cellular uptake and intracellular localization of the 3 PEI-DGAs were examined by confocal microscopy and FACS analysis. For this purpose, FITCconjugated PEI-DGAs are being used. The three dendritic scaffolds were located in and around the cells, but not in the nucleus (Figure 5C), which indicates a similar cellular distribution behavior of PEI-DGAs. To distinguish between extra- and intra-cellular distribution, flow cytometry with quenching of the extracellular fluorescence by trypan blue showed that all cells took up FITC-conjugated PEI-DGAs (Figure 5D). The quenching of the extracellular fluorescence 2719


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Figure 7. Effects of TLR2 and TLR4 signaling inhibition on cytokine release induced by PEI-PGlu-Mal. Differentiated THP-1 cells were preincubated for 1 h with neutralizing antibodies directed against TLR2 und TLR4 (aTLR, 10 μg/mL) followed by stimulation with 50 ng/mL upLPS, 5 ng/mL Pam3CSK4, or 1 mg/mL PEI-DGA for 24 h. Cytokine concentration was measured by CBA. Data presented as mean ± S.E.M., n = 3. (A) IL-1β. (B) IL-6. (C) TNFα.

Figure 8. Effects of PEI-DGAs on proinflammatory cytokine release from human MSCs. Human MSCs were stimulated for 24 h with 1 mg/mL PEI-DGAs or 50 ng/mL upLPS before cytokine concentration was determined by CBA. Data shown as mean ± S.E.M., n = 5−6; asterisks mark statistically significant differences from respective mock control *p ≤ 0.05 **p ≤ 0.05. (A) IL-6. (B) IL-8.

A kinetic experiment comparing cytokine release from upLPS- and PEI-PGlu-Mal-stimulated THP-1 cells showed that the induction of cytokine release mainly occurs between 2 and 8 h after stimulation (Figure S4). In combination with the identified localization of PEI-DGAs and PEI-PGlu-Mal (Figure 5C,D), we assumed that extracellular receptors are involved to activate NF-κB signaling. Toll-like receptors TLR2 and TLR4 are both major extracellular immune receptors that induce NFκB signaling. To establish whether inhibition of TLR2 and TLR4 results in the reduction of cytokine release, blocking antibodies (termed TLR) for both TLRs were used (Figure 7). IL-1β, IL-6, and TNFα release was sensitive to TLR4 blockade after upLPS stimulation and to TLR2 blockade after Pam3CSK4 stimulation, indicating that blocking of TLR with

neutralizing antibodies is suitable and specific. Release of proinflammatory cytokines after stimulation of the cells with PEI-PGlu-Mal was also inhibited. IL-1β release was reduced by both antiTLR4 and antiTLR2 ∼30%, which increased to 50% reduction when antiTLR4 and antiTLR2 were used in combination (Figure 7A). IL-6 release was reduced 90− 100% by the antiTLR4 antibody and 75% by antiTRL2 treatment. The combination of antiTLR4/2 completely inhibited IL-6 secretion (Figure 7B). In addition, TNFα release was also clearly reduced by antiTLR4 (50%) and antiTLR2 (60%). However, in combination, they did not increase this effect as did IL-1β or IL-6 (Figure 7C). These results show that PEI-PGlu-Mal induces both TLR2 and TLR4 signaling. This was not anticipated because TLR2 preferentially 2720


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Biomacromolecules interacts with lipopeptides and TLR4 mainly interacts with lipopolysaccharide.53 Both lipid structures known to interact with TLRs are clearly different from applied dendritic PEI glycoarchitecture, PEI-PGlu-Mal, with a binary and double shell (Figure 1). A possible explanation of initiation of both signaling pathways by PEI-PGlu-Mal is its well-known receptor clustering on the membrane surface.18 The individual activation of each receptor can be mainly excluded, whereas the clustering of both TLR receptors amplifies cytokine inhibition, as noted in this study. This clustering effect (=avidity) leads to a close localization of both receptors and amplified induction of the signal transduction.18 In summary, anionic PEI-PGlu-Mal treatment of human THP-1 macrophages led to the release of proinflammatory cytokines, including IL-1β, IL-6, and TNFα, after incubation with its anionic dendritic scaffold. Induction of secretion was dependent on NF-κB activation induced by TLR2 and TLR4 signaling. However, no damage to the cells or mitochondria occurred. Cationic PEI-DGAs, PEI-25-Mal B, and PEI-5-Mal B, on the other hand, had no significant effect on the secretion of cytokines or cell morphology of THP-1 cells after 24 h stimulation. Effects of PEI-DGAs on Human MSCs. Following the concept of imitating the influence of PEI-DGAs on myeloma cells and exemplary interacting cells (Scheme 1), the effects of PEI-DGAs on bone-related cells were investigated. Human bone marrow MSCs were stimulated with PEI-DGAs (1 mg/ mL; Figure 1) for 24 h. Without stimulation, MSCs released IL-6 and IL-8 (Figure 8). Note that interaction of myeloma cells with MSCs also upregulates IL-6.54,55 Stimulation of these cells with anionic PEI-PGlu-Mal and upLPS as positive controls increased secretion of IL-6 and IL-8 compared to the unstimulated controls (Figure 8). By contrast, cationic PEIDGAs, PEI-25-Mal B, and PEI-5-Mal B reduced the release of both cytokines IL-6 and IL-8 to less than that of unstimulated cells (Figure 8). These results confirm our observation on the THP-1 cell model that anionic PEI-PGlu-Mal smoothly induced the secretion of IL-6 in both cell types, that is, THP-1 and MSCs (Figure 4). Hence, PEI-PGlu-Mal should not be used as DDS in local therapy for treating relapsed myeloma cells in bone lesions because of the self-amplification properties between myeloma cells, macrophages, and bone marrow MSCs in the presence of excess IL-6 secretion (Scheme 1C).54,55 On the other hand, PEI-25-Mal B and PEI5-Mal B had an anti-inflammatory effect. In particular, reduced secretion of IL-6 makes it a good tool as DDS for local therapy in multiple myeloma treatment (Scheme 1). Furthermore, the whole transcriptome of MSCs was analyzed by RNA sequencing to determine which genes can be upregulated and downregulated by PEI-DGAs. MSCs were incubated with all three PEI-DGAs (1 mg/mL). No genes were significantly affected by PEI-PGlu-Mal and PEI-5-Mal B compared to upLPS or PEI-25-Mal B (Table 1). PEI-25-Mal B affected gene expression most severely, with 602 genes being upregulated or downregulated compared to the medium control (Table 1). The following main findings were validated by analysis of the transcriptome(s) (Figure 9): the expression of NF-κBdependent cytokines and chemokines was significantly upregulated by upLPS compared to the medium control. IL6 and IL-8 gene expression were slightly upregulated by PEIPGlu-Mal (Figure 9A,B), which is comparable to the results on secreted IL-6 and IL-8 in the cell culture supernatant measured

Table 1. Number of Significantly Regulated Genes under Stimulation with PEI GPa

upLPS PEI-PGlu-Mal PEI-25-Mal B PEI-5-Mal B

upregulated genes to control

downregulated genes to control

11 0 254 0

0 0 348 0

a

Human MSCs were stimulated for 24 h with 1 mg/mL PEIPGlu346-Mal, PEI-25-Mal B, and PEI-5-Mal B or 50 ng/mL upLPS. Afterward transcriptome was analyzed through RNA sequencing. pvalue ≤0.05 and absolute fold change ≥2

by CBA (Figure 8). Both cationic PEI-DGAs slightly downregulated the expression of IL-6 and IL-8 genes compared to the mock control (Figure 9A,B). This underlines the possibility that cationic PEI-DGAs may have a more antiinflammatory effect on MSCs. Analysis of genes upregulated by the cationic PEI-25-Mal B glycopolymer showed that osteogenesis-related genes were also affected. ATF4 gene was significantly upregulated by PEI-25Mal B and slightly by PEI-5-Mal B compared to the mock control (Figure 9C). Upstream of ATF4 gene signaling pathway, TMEM119 gene (Figure 9D) has also a trend to be expressed at higher levels after incubation with both cationic PEI-DGAs. These results indicated that the interaction of human MSCs with cationic PEI-DGA may lead to an improved osteogenic differentiation by upregulation of TMEM119 and ATF456 genes. However, Lautenschläger et al. investigated this influence and did not detect significant differences in the ability of MSCs to differentiate into osteoblasts. It should be mentioned that in this study the influence of PEI-DGAs was determined during the chemical induction of osteogenic differentiation. On the other hand, it could be possible that under certain conditions (e.g., under in vivo conditions) these cationic PEI-DGAs per se are able to induce or improve osteogenic differentiation through TMEM119 and ATF4 genes. The high number of genes downregulated by PEI-25-Mal B may indicate that the cells react to the adverse effects of high doses of PEI-25-Mal B (1 mg/mL). Lautenschläger et al. also observed more apoptotic cells after long-term treatment with PEI-25-Mal B.26 Analysis of DNA damage response genes reveals that, depending on PEI-25-Mal B treatment, RAD51 gene (Figure 9E) was strongly downregulated. Combination of upregulated CDKN1a gene and downregulated LMNB1 gene expression (Figure 9F,G) resulted in an expression pattern of cells seen in senescence.57,58 This thoroughly indicates that these cells might first go into senescence and later into apoptosis. Stimulation with upLPS, PEI-PGlu-Mal, and PEI-5Mal B had little or no effects on these genes.

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CONCLUSIONS We demonstrated differential release of proinflammatory cytokine from multiple myeloma cell lines, macrophages, and MSCs for treatment with different PEI glycoarchitectures. Only high doses (1 mg/mL) of PEI glycoarchitectures have been used to provoke cellular responses. Three different PEI glycoarchitectures (Figure 1) were used to provide further important characteristics for the future development of a DDS for local therapy of relapsed myeloma cells in bone lesions. Anionic PEI-PGlu-Mal (Figure 1) induces a proinflammatory response in macrophage-like cells and human MSCs. This 2721


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Figure 9. Expression of stress response and apoptosis-related genes after 24 h stimulation. Human MSCs were stimulated with 50 ng/mL upLPS or 1 mg/mL of PEI-DGAs for 24 h. Expression was analyzed by RNA sequencing. Scatter blots of the log 2 expression of indicated genes presented as mean ± S.E.M. of 3 independent experiments. Asterisks mark statistically significant difference from respective control calculated by EdgeR (p ≤ 0.05). (A) IL-6. (B) IL-8 [CXCL8]. (C) ATF4. (D) TMEM119. (E) RAD51. (F) CDKN1A. (G) LMNB1.

Table 2. Properties of DDS for Development of Locally Applicable Bone Substitute Materials for Multiple Myeloma Treatment immunologically inert reduced IL-6 release of MSCs impact on cellular viability (MSCs/macrophages) ability to induce cell death in multiple myeloma cell lines osteogenic potential (gene regulation in MSCs) no apoptotic or senescence profile (gene regulation in MSCs) foreseen for further preclinical development

PEI-PGlu-Mal

PEI-25-Mal B

PEI-5-Mal B

− − + − − + −

+ + + + + − ∓

+ + + + + + +++

and IL-8 genes, whereas expression of osteogenesis-related genes was increased. Finally, PEI-25-Mal B led to the expression of a profile of DNA damage and senescence. The following key issues here can be extracted for the improvement and possible application of PEI glycostructures as a DDS: PEI-PGlu-Mal, as anionic glycopolymer, cannot be recommended in local multiple myeloma treatment because it induces IL-6 secretion, a survival factor for cancer cells. However, its proinflammatory effects could be used as a modest immunomodulatory adjuvant. It may be advantageous in other clinical applications or vaccines. Both cationic PEI glycoarchitectures are suitable in the future for local treatment of multiple myeloma lesions as DDS in bone substituents, but

response was due to activation of NF-κB signaling through recognition of TLR2 and TLR4. By contrast, cationic PEI-25Mal B and PEI-5-Mal B (Figure 1) had no proinflammatory effects on the macrophage cell model. All PEI glycoarchitectures that we tested failed to induce a significant cell death and mitochondrial damage after 24 h incubation and also had no effect on cytokine secretion of multiple myeloma cell lines (Scheme 1C,D). As demonstrated by transcriptomic analysis, PEI-PGlu-Mal induced the expression of proinflammatory cytokines, chemokines, and IL-6 and IL-8 genes, slightly, without any increased stress response genes or changes in the expression of osteogenesis-related genes. On the other hand, PEI-25-Mal B and PEI-5-Mal B reduced the expression of IL-6 2722


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the off-target effects of PEI-25-Mal B on interacting cells of the multiple myeloma cells are more drastic compared to PEI-5Mal B. Therefore, we suggest that PEI-5-Mal B has the best properties for potential DDS application (summarized in Table 2) in bone substituents for the local treatment of multiple myeloma lesions. This also implies that even the more nearly neutral or slightly cationic DDS with low content on their dendritic polyamine scaffold are favored to minimize any innate immune response from different cells in the local environment of multiple myeloma.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.9b00475.

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Article

Additional information about the characterization of dendritic glycoarchitectures and images and graphs of cell interaction study with dendritic glycoarchitecture (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (D.A.). *E-mail: angela.roesen-wolff@uniklinikum-dresden.de (A.R.W.). ORCID

Dietmar Appelhans: 0000-0003-4611-8963 Author Contributions

F.S. conceived and designed all experiments, analyzed data, and wrote the manuscript; B.K. synthesized and characterized the glycopolymers; D.A. and A.R.-W. supervised the study, interpreted data, and wrote the manuscript; the manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was solely funded by DFG, German Research Foundation (SFB/TRR79, subprojects M7 and B12), including PhD positions for F.S. and B.K. and expendables. The funding organization did not influence the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. We thank Dirk Hose, Anja Seckinger, and Martina Emde for helping by RNA sequencing and subsequent analysis as well as providing the used human myeloma cell lines within the research initiative SFB/TRR 79. We thank Hartmut Komber and the department of Analytics to support the characterization of dendritic glycopolymers.

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ABBREVIATIONS CBA, cytometric bead array; DDS, drug delivery system; IL, interleukin; ISGs, interferon stimulated genes; MSCs, mesenchymal stromal cells; MSU, monosodium urate crystals; PEI, poly(ethyleneimine); PGlu, oligo/polyglutamic acid chains; PI, propidium iodide; PMA, phorbol 12-myristate 13-acetate; PPI, poly(propyleneimine); TLR, toll-like receptor; TNFα, tumor necrosis factor alpha; upLPS, ultrapure lipopolysaccharide 2723


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Immunomodulatory Effects of Dendritic Poly(ethyleneimine

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