glycodendrimers

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Terminal sugar moiety determines immunomodulatory properties of poly(propyleneimine) glycodendrimers Michał Gorzkiewicz, Krzysztof Sztandera, Izabela Jatczak-Pawlik, Robin Zinke, Dietmar Appelhans, Barbara Klajnert-Maculewicz, and #ukasz Pu#aski Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 23 Mar 2018 Downloaded from http://pubs.acs.org on March 25, 2018

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Biomacromolecules

Terminal

sugar

immunomodulatory

moiety

determines

properties

of

poly(propyleneimine) glycodendrimers Michał Gorzkiewicz1*, Krzysztof Sztandera1, Izabela Jatczak-Pawlik1, Robin Zinke2, Dietmar Appelhans2, Barbara Klajnert-Maculewicz1,2, Łukasz Pulaski3,4*

1

Department of General Biophysics, Faculty of Biology and Environmental Protection,

University of Lodz, 141/143 Pomorska St., 90-236 Lodz, Poland 2

Leibniz Institute of Polymer Research Dresden, Hohe Str. 6, 01069 Dresden, Germany

3

Department of Molecular Biophysics, Faculty of Biology and Environmental Protection,

University of Lodz, 141/143 Pomorska St., 90-236 Lodz, Poland 4

Laboratory of Transcriptional Regulation, Institute of Medical Biology PAS, 106 Lodowa

St., 93-232 Lodz, Poland

ACS Paragon Plus Environment

Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract Poly(propyleneimine) (PPI) dendrimers fully surface-modified with disaccharide moieties (maltose, cellobiose and lactose) designed to mimic natural lectin receptor ligands, were tested for their bioactivity in two myeloid cell lines: THP-1 and HL-60. Depending on the sugar modification, we observed variable activation of NF-κB, AP-1 and NF-AT signaling pathways: lactose-coated dendrimers had the strongest impact on marker gene expression and most signaling events, with a notable exception of NF-κB activation in THP-1 cells. The two cell lines showed an overall similar pattern of transcription factor and gene expression activation upon treatment with glycodendrimers, suggesting the involvement of galectin and C-type lectin receptor types. An important result of this action was the overexpression of CD40 and IL8 genes, potentially leading to an activated, pro-inflammatory phenotype in the monocyte/macrophage

cell

lineage.

These

pharmacodynamic

characteristics

of

glycodendrimers need to be taken into account during their pharmaceutical applications, both in drug delivery and direct immunomodulation.

Keywords: PPI dendrimers, glycodendrimers, monocytes, immunomodulation


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Biomacromolecules

Introduction Glycodendrimers, which first appeared in the scientific literature 25 years ago1, are one of the most promising classes of highly-branched dendritic polymers with numerous biomedical applications2. Surface modification with sugar moieties has been primarily aimed at improvement of the biocompatibility of positively-charged dendrimers (such as PAMAM or PPI macromolecules) by lowering their cytotoxic activity associated with non-specific interactions with cellular membranes3,4. Later it was found that carbohydrates may additionally modulate biodistribution patterns5 and enhance molecular recognition potential of glycodendrimers due to the specific interactions with lectin receptors overexpressed on various types of cells6,7, providing receptor-mediated endocytosis or activation of intracellular signaling pathways. Due to these findings, glycodendrimers quickly became the object of interest of several research groups. Their studies focused on the application of dendritic glycopolymers as drug carriers or therapeutics per se, bringing promising results2. However, the expected mechanism of action of novel nanoparticles is usually accompanied by collateral biological activities, which may shed new light on their clinical applications. Therefore, it is crucial to thoroughly characterize their biomedical potential. Specific carbohydrate-protein interactions are involved in numerous cellular processes, including bacterial and viral adhesion, cell differentiation and growth regulation, and most importantly the immune response8,9. The recognition of molecular mechanisms by which glycodendrimers influence the cellular environment may provide a valuable tool for strategies using these particles clinically, not only as vectors for targeted drug delivery, but also as immune modulators, e.g. to trigger the anti-tumor activity of innate immune system. The wide range of macromolecular scaffolds and sugar moieties gives the possibility to design various branched glycopolymers with unique bioactivities. While fully surface-modified PPI glycodendrimers (so-called dense shell – DS) are unsuitable as drug carriers for anionic molecules10, they may



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interact with signaling pathways important for cellular function and exert an immunomodulatory

effect.

Immunomodulation

is

a

very

important

goal

in

nanopharmacology, since specific and receptor-targeted activation or inhibition of immune cell proliferation, cytokine release or antibody secretion may be superior to currently available chemical immunomodulators which cause severe side effects11. Thus, immune systemtargeted sugar-modified dendrimers are an exciting area of research not only with regard to delivering drugs or antigens, but also in terms of their direct impact on immune system function and regulation. In our previous studies, we verified the immunomodulatory properties of dense shell maltosemodified poly(propylene imine) glycodendrimer of the 4th generation (PPI-Mal DS G4) in human monocytic cell line THP-112. Maltose-coated PPI dendrimers exhibited a very good combination of biocompatibility and bioactivity. We have shown that PPI-Mal DS G4 may activate the NF-κB signaling pathway, which is one of the most important pathways for innate immunity. PPI-Mal DS G4 enhanced the transcriptional activity of NF-κB, causing a moderate induction of gene expression but not affecting the secretion of pro-inflammatory cytokines by THP-1 cells. This significant finding generates a number of additional questions about the impact of glycodendrimers on the delicate signaling homeostasis in myeloid cells, influencing their potential application as adjuvants or drug delivery vehicles. In the light of our discoveries, we decided to further analyze pro-inflammatory mechanisms triggered by maltose-coated PPI dendrimer. Moreover, in order to fully understand the influence of the composition of surface sugar shell on cellular environment, we introduced two additional PPI glycodendrimers to our research – macromolecules modified with cellobiose (PPI-Cel DS G4) and lactose moieties (PPI-Lac DS G4), since these disaccharides are known to strongly and specifically interact with cell surface lectin receptors13,14. Our research focused on identification

and

evaluation

of

inflammatory

signaling


pathways

triggered

by

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Biomacromolecules

glycodendrimers due to their specific interactions with biological systems in two myeloid cell line models - THP-1 and HL-60.

Materials and methods 1. Dendrimers Poly(propylene imine) dendrimer of the 5th generation with 64 primary amino surface groups was obtained from Symo-Chem (Eindhoven; Netherlands) and modified with maltose (PPIMal DS G4, MW 46000 g/mol), cellobiose (PPI-Cel DS G4, MW 42500 g/mol) or lactose (PPI-Lac DS G4, MW 40000 g/mol) to 90% extent (maximal sterically possible surface coverage; “dense shell” glycodendrimers). Glycodendrimers (Fig. 1) were synthesized, purified and characterized as previously described15. The molecular weight of PPI glycodendrimers was determined by MALDI-TOF mass spectrometry. According to Tomalia and Rookmaker16, the nomenclature for Tomalia-type PAMAM dendrimers can be used for PPI dendrimers. Hence, we adopted the suggested classification, describing commercially available PPI dendrimer of the 5th generation (DAB-Am-64) as 4th generation.



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PPI-Mal DS G4, PPI-Cel DS G4 or PPI-Lac DS G4 R2N

NR2 R N 2 NR2

NR2 R2N NR2 R2N

NR2 R2N

NR2 NR2

R2N R2N R2N

N

N

N

N

R2N

NR2 NR2

N

N

N N

R2N

NR2 NR2

N

N

N

R2N

N

N

N

R2N N

N R2N

N

N

N

N

NR2

N N

R2N

N

N

NR2

N

R2N

NR2 NR2

N

N

N N

N

R2N N

N

NR2

N

R2N

N

R2N

N

N

N R2N

N

NR2

N

NR2 NR2

N N

N N

R2N

N

N

R2N N

N

N

NR2

N

N

R2N N R2N R2N

N

NR2 NR2

N

N

N N

R2N

NR2

N

N

N

N

N

NR2 NR2

N

R2N R2N

NR2

R2N NR2 R2N NR2 R2N NR2 R2N

NR2

NR2

NR2NR2

OH OH O

=R

HO

HO

=R

O

HO

OH

OH

=R OH

maltose = Mal

cellobiose = Cel

lactose = Lac

Figure 1. Simplified structures of dense shell (DS) 4th generation (G4) poly(propyleneimine) (PPI) dendrimers decorated with different disaccharide units.

2. MALDI-TOF-Mass Spectrometry. The MALDI-TOF experiments were performed on Autoflex Speed TOF/TOF system (Bruker Daltonics GmbH). The measurements were carried out in linear mode and positive polarity by pulsed smart beam laser (modified Nd:YAG laser). The ion acceleration voltage was set to 20 kV. For the sample preparation, the polymers were mixed with 2,5-dihydroxybenzoic acid (DHB) as matrix and dissolved in Millipore water. The preparation was done without the addition of salts.


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Biomacromolecules

3. Cell culture THP-1 (acute monocytic leukemia) and HL-60 (acute promyelocytic leukemia) human cell lines were purchased from ATCC (Manassas, VA, USA) and maintained under standard conditions in RPMI-1640 Medium (ThermoFisher) supplemented with 10% fetal bovine serum (Sigma-Aldrich) at 37°C in an atmosphere of 5% CO2. Cells were sub-cultured three times per week.

4. Cytotoxicity assay To estimate the potential cytotoxic activity of glycodendrimers, resazurin assay was performed17. Cells were seeded into 96-well black plates at a density of 1.5×104 cells per well and treated with increasing concentrations of dendrimers (0.78−100 µM) for 24 hours. Following the incubation, resazurin was added to the culture medium to a final concentration of 10 µg/ml and the plates were incubated at 37°C in darkness to allow conversion of resazurin to resorufin. Fluorescence of metabolized resazurin in the cell suspension was measured after 30 and 90 minutes at 530 nm excitation and 590 nm emission using EnVision plate reader (PerkinElmer, Waltham, USA). Cell viability was calculated as the increase in resorufin fluorescence between 30 and 90 minutes and was presented as percentage of untreated control.

5. Gene expression assay The gene expression level was determined by quantitative real-time RT-PCR. Aliquots of 1×106 of HL-60 and THP-1 cells were cultured for 6 hours with glycodendrimers at final concentrations of 5 and 25 µM. Following incubation, cells were harvested and washed once with PBS. Total cellular RNA was isolated using TRI Reagent (Sigma-Aldrich) according to manufacturer’s protocol. Complementary DNA (cDNA) was transcribed from mRNA using



High Capacity cDNA Reverse Transcription Kit (ThermoFisher) and used for real-time PCR amplification with the GoTaq® qPCR Master Mix (Promega) according to manufacturer’s protocol. Each 16 µl reaction volume contained 1 µl of cDNA and 0.25 µM of forward and reverse intron-spanning primers (for primer sequences, see Table 1). The reference genes (HPRT1 and TBP) were selected according to the GeNorm procedure18. PCR reactions were performed in 96-well microplates using the CFX96 Real Time PCR DetectionSystem (BioRad). The expression level of assayed genes calculated by the ∆Ct method and expressed as number of cognate mRNA copies per 1000 copies of geometric-averaged mRNA for reference genes.

Table 1. Primer sequences Gene HPRT1 TBP NFKBIA TNFAIP3 MMP1 ACTG2 IFIT1 MCL1 F3 JAG1 CDKN1A CD40 IL8

Forward and reverse sequences (5’-3’) Fw: TGACACTGGCAAAACAATGCA Rv: GGTCCTTTTCACCAGCAAGCT Fw: CACGAACCACGGCACTGATT Rv: TTTTCTTGCTGCCAGTCTGGAC Fw: TGAAGGCTACCAACTACAATGGC Rv: TGACATCAGCACCCAAGGACAC Fw: CTTGACCAGGACTTGGGACTTTGC Rv: AGCCATTGTGCTCTCCAACACCTC Fw: GGGAGATCATCGGGACAACTC Rv: GGGCCTGGTTGAAAAGCAT Fw: GAAGATCTGGCACCACTCCT Rv: CAGAGGCATAGAGGGAGAGC Fw: CCTGGCTAAGCAAAACCCTG Rv: CCAGCAGTGCAGAAAGTGAG Fw: GGACATCAAAAACGAAGACG Rv: GCAGCTTTCTTGGTTTATGG Fw: CCGAACAGTTAACCGGAAGA Rv: TCAGTGGGGAGTTCTCCTTC Fw: CTTGCAAACTCCCAGGTGAC Rv: TTGAGACACGGCTGATGAGT Fw: TGGAGACTCTCAGGGTCGAAA Rv: GGCGTTTGGAGTGGTAGAAATC Fw: TCAGTGCTGTTCTTTGTGCC Rv: TACAGTGCCAGCCTTCTTCA Fw: CCTTGGCAAAACTGCACCTT Rv: CTGGCCGTGGCTCTCTTG


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Biomacromolecules

6. NF-κB translocation assay HL-60 and THP-1 cells were stimulated for 2.5 h with glycodendrimers at final concentration of 25 µM. Aliquots of 5×104 cells were then withdrawn from the culture and transferred to a thin-bottom 96-well plate coated previously with poly-L-lysine. After 10 minutes of sedimentation at 37°C, cells were centrifuged (5 min, 100×g, RT) to enhance cell adhesion to the plate. Following gentle aspiration of culture medium and a single wash with PBS, cells were immediately fixed for 20 min at room temperature with PBS-buffered 2% formaldehyde solution freshly prepared from paraformaldehyde. Subsequently, cells were washed once with blocking buffer (PBS, 1% BSA, 0.1% Triton X-100) and further incubated with the blocking buffer overnight at 4°C. NF-κB was then probed for one hour with DyLight488-conjugated antibody diluted in the blocking buffer (anti-NF-κB p65 DyLight488 rabbit monoclonal antibody, EP2161Y, Abcam, 1:100). Finally, after three wash cycles with the blocking buffer, nuclei were stained with 5µM Hoechst 33342 dye for 5 minutes and cells were stored protected from light in PBS/0,02% NaN3 at 4ºC until imaging. Measurement of NF-κB p65 translocation from cytoplasm to nucleus was conducted with a high-content screening platform (ArrayScanVTi from ThermoFisher) using manufacturer’s proprietary software and protocol for measuring the ratio of nuclear to cytoplasmic staining19. Measurements were made for >2500 cells per single well, ratios were averaged within the well and taken as single biological replicates. Data is presented as percentage of control (untreated) translocation ratio.

7. Electrophoretic mobility shift assay (EMSA) Aliquots of 1×106 of THP-1 and HL-60 cells were cultured for 2.5 hours with glycodendrimers at final concentration of 25 µM. Following the incubation, cells were washed once with PBS and centrifuged at 500×g for 3 minutes. Supernatant was carefully removed, leaving the cell pellet as dry as possible. Nuclear extracts were then prepared using



the NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher) with the Halt Protease and Phosphatase Inhibitor Cocktail (ThermoFisher) according to the manufacturer’s recommendation. Protein concentration of extracts was determined using the Microplate BCA Protein Assay Kit – Reducing Agent Compatibile (ThermoFisher) and aliquots were frozen at -80°C until use. Nuclear extracts were analyzed for the presence of active (DNA-binding) AP-1, NF-AT and NF-κB using double-stranded oligonucleotide probes with the consensus binding sequences, labeled with IRDye 700 infrared fluorescence dye (AP1: 5'-GTG TGA TGA CTC AGG TTT G-3', NF-AT: 5'-AGA CTG TGT GGA AAA TGT AGA GT-3', NF-κB: 5'-AGT TGA GGG GAC TTT CCC AGG C-3', consensus sites are underlined), custom-synthesized by Metabion International AG. Extracts were incubated for 30 minutes at 4°C with 0.5µg/ml salmon sperm DNA in binding buffer: 5% glycerol, 10mM MgC12, 1mM DTT, 50 mM NaCl, 0.1% NP-40, 0.4µM ZnCl2 and 10 mM Tris-HCI, pH 8 with or without the addition of 2 pmol/µl of the competing, unstained oligonucleotide probe. After this time, labelled AP-1, NF-AT or NF-κB probes were added to the mixture at the final concentration of 0.02 pmol/µl and further incubated for 30 minutes at 4oC. DNA-protein complexes were analyzed by electrophoresis in denaturing conditions on a 12% polyacrylamide gel at 4oC. The probe-protein complexes were visualized on an Odyssey IR imager (Li-Cor). Band intensities were quantified digitally using ImageJ software.

8. In-cell Western Blot THP-1 and HL-60 cells were stimulated for 2.5 h with glycodendrimers at final concentration of 25 µM. Aliquots of 1×105 cells were then withdrawn from culture and transferred to a thinbottom 96-well plate coated previously with poly-L-lysine. After 10 minutes of sedimentation at 37°C, cells were centrifuged (5 min, 100×g, RT) to enhance cell adhesion to the plate.


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Biomacromolecules

Following gentle aspiration of culture medium and a single wash with PBS, cells were immediately fixed for 20 minutes at RT with PBS-buffered 2% formaldehyde solution (pH 7.2) freshly prepared from paraformaldehyde, and incubated with blocking buffer (PBS, 3% BSA) for 30 minutes at RT. Subsequently, cells were incubated with primary antibodies overnight at RT: Anti-c-Jun antibody [E254] (ab32137) or Anti-JunD (phospho S100) + c-Jun (phospho S73) antibody [EPR16586] (ab178858), Abcam, 1:300 in PBS with 1% BSA and 0.1% Tween 20. Following the incubation, cells were washed three times with PBS with 0.1% Tween 20 and incubated with secondary antibody (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773), Abcam, 1:500 in PBS with 1% BSA and 0.1% Tween 20) and CellTag 700 Stain (final concentration: 0.2 µM) for 1 hour at RT. Subsequently, cells were washed three times with PBS with 0.1% Tween 20 and 100 µl of PBS was added to each well. The antibody-protein complexes were visualized on an Odyssey IR imager (Li-Cor). Ratios were averaged within the well and normalized for the number of cells per well. Data is presented as percentage of control (untreated) phosphorylation ratio.

9. Statistics For statistical significance testing we used one-way ANOVA for concentration series followed by post-hoc Tukey’s test for pairwise difference testing. In all tests, p values

glycodendrimers

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