Pyrrolidone Modification Prevents PAMAM Dendrimers from Activation

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Pyrrolidone Modification Prevents PAMAM Dendrimers from Activation of Pro-Inflammatory Signaling Pathways in Human Monocytes Anna Janaszewska,*,† Michał Gorzkiewicz,† Mario Ficker,‡ Johannes Fabritius Petersen,‡ Valentina Paolucci,‡ Jørn Bolstad Christensen,‡ and Barbara Klajnert-Maculewicz†,§ †

Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, 141/143 Pomorska St., 90-236 Lodz, Poland ‡ Department of Chemistry, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark § Leibniz-Institut für Polymerforschung Dresden e.V., Hohe Strasse 6, 01069 Dresden, Germany ABSTRACT: The biological features of dendrimers are affected by the character of highly reactive terminal moieties. In some polyamine dendrimer types the surface charge makes them bioincompatible and prevent their direct medical application. Moreover, foreign particles can induce the immune response which is undesirable due to the adverse side effects in vivo. The reduction of cytotoxicity of positively charged macromolecules is possible through chemical modifications of terminal groups. In our study, we have developed new derivatives of PAMAM dendrimers modified with 4-carbomethoxypyrrolidone and evaluated their immunomodulatory properties. The experiments were conducted on two human cancer myeloid cell lines: THP-1 and U937. To evaluate the cytotoxicity of dendrimers, the reasazurin assay was applied. The expression level of NF-κB targets (NFKBIA, BTG2) and cytokine genes (IL1B, TNF) was determined by quantitative real-time RT-PCR. The measurement of binding of NF-κB to a consensus DNA probe was determined by electrophoretic mobility shift assay. The ELISA cytokine assay was performed to measure protein concentration of IL-1β and TNFα. We have found that PAMAM-pyrrolidone dendrimers did not impact THP-1 and U937 viability even at high concentrations (up to 200 μM). The surface modification prevented PAMAM dendrimers from stimulating NF-κB-related signal transduction, which have been determined on the level of nuclear translocation, gene expression and protein secretion. Pyrrolidone modification efficiently prevents PAMAM dendrimers from stimulating pro-inflammatory response in human cancer myeloid cell lines, thus it can be used to improve the biocompatibility of positively charged dendrimers and to broaden the scope of their biological applications. KEYWORDS: PAMAM dendrimer, pyrrolidone, inflammation, NF-κB, biocompatibility

1. INTRODUCTION Since the emergence of nanoscience in the 1980s, a significant development of research in this field has been noticed. It is believed that application of nanotechnology in medicine will improve diagnosis and therapy of many diseases, and therefore several nanosystems are nowadays under investigation. The understanding of interactions between the nanostructures and cell cultures in vitro is particularly important in order to fully exploit their potential and define the mechanisms of action. To date, dendrimers are probably the best characterized nanoparticles. These monodisperse, polyvalent polymers with a highly branched sphere-shaped architecture possess a number of unique physicochemical properties, which make them exceptionally useful for biomedical applications.1 Due to the threedimensional structure with reactive terminal groups and several internal cavities, dendrimers may serve as efficient nanocarriers for different therapeutic or imaging agents, transporting them either physically entrapped inside the scaffold or attached to the surface moieties.1−4 The use of dendrimers as drug delivery © XXXX American Chemical Society

devices may help to overcome cellular resistance mechanisms and enable targeted transport, at the same time reducing the systemic toxicity.5 Both the scaffold and terminal groups of dendrimers are largely responsible for their biological and physicochemical features, e.g., high solubility and biopermeability.6 In most cases, surface moieties are highly reactive, and may be further modified in order to change the properties of macromolecules, or to generate the desired activity.7 However, the surface charge is responsible for the cytotoxicity of cationic dendrimers through their interactions with cell membranes.8 Therefore, it is important to find the right balance between toxic side effects and unique properties of dendrimers in order to enable their therapeutic and diagnostic utilization. In order to reduce the cytotoxicity Received: Revised: Accepted: Published: A

June 19, 2017 October 17, 2017 November 30, 2017 November 30, 2017 DOI: 10.1021/acs.molpharmaceut.7b00515 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Figure 1. Chemical structures of dendrimers. (A) PAMAM-G3 dendrimer; (B) PAMAM-G3 dendrimer modified with 4-carbomethoxypyrrolidone (PAMAM-pyrrolidone-G3 dendrimer); (C) PAMAM-G4 dendrimer; (D) PAMAM-G4 dendrimer modified with 4-carbomethoxypyrrolidone (PAMAM-pyrrolidone-G4 dendrimer).

and immunogenicity of dendrimers, PEGylation9 and glycosylation10−12 are most commonly used. Attachment of poly-(ethylene glycol) chains may further increase the loading capacity and extend a blood half-life of therapeutics carried by dendrimers.13,14 In addition, some modifications, e.g., conjugating folate, dextran,15 or specific antibodies16 on the surface of dendrimers may enhance their targeting potential and improve the biodistribution pattern. An important aspect in the development of innovative nanotherapeutics is the reaction of the organism’s immune system to nanosystems. Nanoparticle-induced inflammatory response is undesirable in case of several clinical applications.17 Certain types of dendrimers, especially those with positive surface charge (e.g., native PAMAM) have been reported to elicit an immune response through the generation of oxidative stress.18,19 Surprisingly, an unexpected anti-inflammatory activity in vivo has been reported for both surface-modified and unmodified PAMAM dendrimers,20 which indicate the need for a more indepth study. PAMAM dendrimers with various terminal groups showed different level of inhibitory activity on LPS-induced nitric oxide (NO) and cyclooxygenase 2 (COX-2) production in rat peritoneal macrophages.20 Qi et al. found that anionic PAMAM G4.5-COOH and neutral G5-OH dendrimer and also G5-NH-acetamide (Ac)-TOS (alpha-tocopheryl succinate conjugate with PAMAM dendrimer) could inhibit the nuclear translocation of NF-κB in macrophages.21,22 Similar observations were made for alkyne−azide dendrimers with acetylene and hydroxyl terminal groups.23 Glycosylation of dendrimers provided both pro-24 and anti-inflammatory properties,25,26 depending on the type of sugar moieties. Phosphorusbased dendrimers terminated with anionic azabisphophonate (ABPs) were found to activate the cells of the immune system. ABPs dendrimers generated changes in morphology and phenotype of monocytes, increasing their phagocytosis properties,27 inducing the expression of mannose receptor MRC1 and antiinflammatory cytokines (IL-4, IL-10 and IL-13), at the same time decreasing the expression of CD64 and CD13.28 Moreover, ABPs promoted the amplification of human NK cells in cultures of PBMCs29 via specific inhibition of the proliferation of CD4+ T lymphocytes,30 and inhibited the pro-inflammatory cytokines secretion and osteoclastogenesis process in arthritic mice models.31 Further, poly-(ethylene oxide) dendrimers with hydroxylated surface lactose moieties,32 as well as dendritic

polyglycerol sulfates33 showed anti-inflammatory properties by blocking P- and L-selectins both in vivo and in vitro. Our studies concentrated on the development and assessment of new derivatives of PAMAM-G3 and -G4 dendrimers, modified with 4-carbomethoxypyrrolidone groups. We demonstrated previously that PAMAM-pyrrolidone dendrimers are not toxic toward various murine cell line models in vitro,34−36 do not influence the mitochondrial membrane potential and ROS generation34 and do not induce hemolysis.36 They are highly soluble37 and can form stable noncovalent complexes with carboxylates.38 Furthermore, PAMAM-pyrrolidone dendrimers can efficiently pass the cellular membranes and localize within various compartments of immortalized cell lines of different species and tissue origin, hinting at existence of variable internalization and intracellular trafficking mechanisms involved depending on the type of cell culture.39,40 Talking all these aspects into consideration, such constructs may prove to be efficient delivery systems. Therefore, it is crucial to investigate their impact on the immune system. Due to the fact that the pyrrolidone modification is indicated to influence the inflammatory response,20 in the present study we focus on the in vitro evaluation of immunomodulatory properties of native PAMAM and PAMAM-pyrrolidone variants, in order to complement the biological characteristics of these compounds.

2. EXPERIMENTAL SECTION 2.1. Cell Culture. The THP-1 (acute monocytic leukemia) and U937 (histiocytic lymphoma) human cancer cell lines were purchased from ATCC (USA) and were maintained under standard conditions in RPMI-1640 Medium (Gibco) containing 10% fetal bovine serum (Sigma-Aldrich) at 37 °C in an atmosphere of 5% CO2. Cells were subcultured three times per week. 2.2. Dendrimers. The synthesis of pyrrolidone terminated PAMAM dendrimer was performed as described earlier.36,41 Shortly, a PAMAM dendrimer with amine terminal groups was stirred under nitrogen atmosphere with dimethyl itaconate. Reaction resulted in obtaining a PAMAM-pyrrolidone dendrimer ([core: 1,4-diaminobutane]; (4 → 2); dendri-{poly-(-amidoamine)(4-carbomethoxy pyrrolidone)64}, G = 4 PAMAM), as presented on Figure 1. The dendrimers synthesized according to these procedures where of high purity and monodispersity. Moreover, the applied analytical techniques showed stability of pyrrolidone B

DOI: 10.1021/acs.molpharmaceut.7b00515 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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the cell pellet as dry as possible. Nuclear extracts were then prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher) with the Protease Inhibitor Cocktail (Sigma-Aldrich) according to the manufacturer’s recommendation. Protein concentration of extracts was determined using the Microplate BCA Protein AssayKit - Reducing Agent Compatible (ThermoFisher) and aliquots were frozen at −80 °C until use. Nuclear extracts were analyzed for the presence of active, DNA-binding NF-κB using double-stranded oligonucleotides probes with the NF-κB consensus binding sequence, labeled with IRDye 700 infrared fluorescence dye (5′-AGT TGA GGG GAC TTT CCC AGG C-3′, consensus site is underlined, custom-synthesized by Metabion International AG). Extracts were incubated for 30 min at 4 °C with 0.5 μg/mL salmon sperm DNA in binding buffer: 5% glycerol, 10 mM MgCl2, 1 mM DTT, 50 mM NaCl, 0.1% NP-40, 0.4 μM ZnCl2, and 10 mM Tris-HCI, pH 8 with or without the addition of 2 pmol/μL of the competing, unstained oligonucleotide probe. Labeled NF-κB probes were subsequently added to the mixture at the final concentration of 0.02 pmol/μL and further incubated 30 min at 4 °C. DNA−protein complexes were analyzed by electrophoresis in denaturing conditions on a 12% polyacrylamide gel at 4 °C. The probe-protein complexes were visualized on an Odyssey IR imager (Li-Cor). Band intensities were quantified digitally using ImageJ software. 2.6. Cytokine Assay. Aliquots of 1.5 × 106 of THP-1 and U937 cells were cultured for 3 h with the dendrimers at the final concentration of 50 μM. The selected samples were subsequently subjected to further stimulation with 10 μg/mL LPS (Sigma-Aldrich) for 15 min. Then the cells were harvested, washed once with PBS, suspended in fresh medium and cultured for 21 h. Subsequently, cells were removed by centrifugation (5 min, 5000 × g, RT) and protein concentration of IL-1β and TNFα was measured in the supernatant using Quantikine ELISA kits (R&D Systems), according to manufacturer’s protocol. The absorbance was read in an EnVision plate reader (PerkinElmer) at 450 nm. 2.7. Statistics. For statistical significance testing we used one-way ANOVA for concentration series followed by posthoc Tukey’s test for pairwise difference testing. In all tests, p values 200 >200 10.31 ± 0.98 200 >200

PAMAMpyrrolidon G3

72 h

PAM AM G3 PAMAM G4 PAMAM-pyrrolidone PAMAM-pyrrolidone PAMAM G3 PAMAM G4 PAMAM-pyrrolidone PAMAM-pyrrolidone

Table 3. Effect of PAMAM and PAMAM-Pyrrolidone on the NF-κB Pathway and Cytokine-Related Gene Expression at the mRNA Levela

24 h

830.4 ± 10.6*

IC50 ± SEM [μM] THP-1 37.77 ± 2.19 9.19 ± 0.46 >200 >200 14.07 ± 0.35 2.04 ± 0.03 >200 >200

NFKBIA

PAMAMpyrrolidon G4 + LPS

Table 2. Effect of PAMAM and PAMAM-Pyrrolidone on the Viability of THP-1 and U937 Cellsa

74.5 ± 2.9



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Figure 2. Effect of PAMAM and PAMAM-pyrrolidone on IL-1β secretion on THP-1 (A) and U937 (B) cell lines, determined by ELISA. Data presented as percentage of cytokine levels in supernatants from control (untreated) cells, average ± SEM of four experiments. *Statistically significant difference compared to untreated control at p < 0.05. †Statistically significant difference compared to the control stimulated with LPS at p < 0.05.

Figure 3. Effect of PAMAM and PAMAM-pyrrolidone on TNFα secretion on THP-1 (A) and U937 (B) cell lines, determined by ELISA. Data presented as percentage of cytokine levels in supernatants from control (untreated) cells, average ± SEM of four experiments. *Statistically significant difference toward untreated control at p < 0.05. †Statistically significant difference toward control stimulated with LPS at p < 0.05.

in 3.5-fold increase in TNFα secretion in THP-1. In U937 upon LPS stimulation the level of TNFα was lower than after the treatment with unmodified PAMAM dendrimers. The synergistic induction of TNFα secretion was observed in U937 cells after stimulation with PAMAM-G4 and LPS (Figure 3). Levels of secreted cytokines, measured by ELISA in cellular supernatants, were not influenced by PAMAM-pyrrolidone variants, neither in untreated nor LPS-stimulated cells (Figures 2 and 3). As the induction of marker gene expression is not a direct proof of signaling activation, we evaluated the involvement of the NF-κB pathway in cellular response to dendrimers. The function of NF-κB upon activation depends on its translocation into the nucleus and ability to bind specific regulatory DNA sequences in order to influence the transcription profile. To confirm that the changes seen on the mRNA and protein level for modified and unmodified PAMAM dendrimers were directly associated with NF-κB signal transduction pathway, we measured the amount of active factor in the nucleus using the electrophoretic mobility shift assay (EMSA). In THP-1 cells, PAMAM-G4 stimulation promoted stronger binding of a nuclear protein to an NF-κB-specific consensus oligonucleotide, which could be seen as increased intensity of a mobility-shifted band. An even stronger enhancement was obtained for the positive control stimulus LPS. As expected, the stimulation with PAMAM-pyrrolidone G4 did not result in enhancement of the band compared with the control (Figure 4, upper panel). Another shifted band which appeared below the band of interest had been found previously not to correspond to an active p65 binding. This may be due to the nonspecific interaction of oligonucleotide probes with nuclear proteins.43

Therefore, we quantified the upper specific band in biological replicate experiments using image analysis (Figure 4, lower panel). LPS and PAMAM-G4 were able to induce the DNA binding by an active nuclear NF-κB by 2-fold and 2.6-fold, respectively. In U937 cell line (Figure 5), LPS increased the NF-κB binding capacity by 1.3-fold, with even stronger effect seen for the unmodified PAMAM-G4 stimulation (2.1-fold enhancement). Similarly to THP-1, PAMAM-G4 modified with pyrrolidone did not exert any effect. In case of both cell lines, the pattern of obtained results was highly similar to those seen for marker gene expression.

4. DISCUSSION In order to elaborate an efficient nanocarrier that would provide an enhanced cellular uptake of therapeutic agents without triggering further cellular stress, various surface modifications of dendritic scaffold have been examined.44,45 Terminal moieties of positively charged dendrimers, such as PAMAM or PPI, are responsible for their high cytotoxicity even at low concentrations; therefore, it is crucial to moderate this detrimental effect. This may be achieved by the attachment of various molecules on the surface of the dendrimer.11 This surface groups can be inert or even have targeting properties.46,47 Foreign compounds, like nanoparticles, may be recognized by the immune system in vivo, which can lead to a multilevel inflammatory response, increasing the host’s susceptibility to the infections and decreasing the therapeutic efficacy of the drug. Thus, it is extremely important to ensure that the delivery system avoids unfavorable immune recognition.48 Features like E


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Figure 5. Impact of PAMAM and PAMAM-pyrrolidone on DNAbinding activity of nuclear NF-κB in U937 cells, determined by EMSA (nuclear extract incubated with IRDye700-labeled probe, imaging on Odyssey IR imager) after 2 h of treatment. Stimulation with LPS was used as positive control of NF-κB activation. Upper panel: representative EMSA image. (A,E) Untreated cells; (B,F) LPS [10 μg/mL]; (C,G) PAMAM G4 [50 μM]; (D,H) PAMAM-pyrrolidone-G4 [50 μM]; (A,B,C,D) No probe competition; (E,F,G,H) 100 × excess of unlabeled probe. Lower panel: the quantification of the specific NF-κB-bound band (computer image analysis using ImageJ software). Data presented as percentage of band intensity in control (untreated) cells, average ± SEM of three experiments. *Statistically significant difference toward untreated control at p < 0.05.

Figure 4. Impact of PAMAM and PAMAM-pyrrolidone on DNAbinding activity of nuclear NF-κB in THP-1 cells, determined by EMSA (nuclear extract incubated with IRDye700-labeled probe, imaging on Odyssey IR imager) after 2 h of treatment. Stimulation with LPS was used as positive control of NF-κB activation. Upper panel: representative EMSA image. (A,E) Untreated cells; (B,F) LPS [10 μg/mL]; (C,G) PAMAM G4 [50 μM]; (D,H) PAMAM-pyrrolidone-G4 [50 μM]; (A,B,C,D) No probe competition; (E,F,G,H) 100 × excess of unlabeled probe. Lower panel: the quantification of the specific NF-κB-bound band (computer image analysis using ImageJ software). Data presented as percentage of band intensity in control (untreated) cells, average ± SEM of three experiments. *Statistically significant difference toward untreated control at p < 0.05.

maturation state. These commonly used myeloid models have been well characterized in terms of pro-inflammatory signaling pathways, thus being a perfect tool for our studies.52 The toxicity of PAMAM dendrimers against tested cell lines was time- and generation-dependent, which is consistent with previous reports.53 As expected, pyrrolidone modification eliminated the cytotoxicity of PAMAM dendrimers toward human cells. At the concentrations up to 200 μM concentration no harmful effect on cellular growth and viability has been noticed even after prolonged incubation (72 h). Our results show that pyrrolidone modification prevents PAMAM dendrimers from stimulating the NF-κB signaling pathway, one of the most significant pathways for innate immunity.54 Using the electrophoretic mobility shift assay, we have demonstrated that unmodified PAMAM dendrimers are able to induce NF-κB nuclear translocation in vitro. This leads to stimulation of expression of NF-κB target genes (NFKBIA and BTG2) and those of related pro-inflammatory cytokines (IL1B and TNF). The increased expression of interleukin 1β and TNFα in both cell lines has been confirmed on the protein level. Since unmodified PAMAM dendrimers penetrate cell membranes via classical, clathrin-dependent mechanism,55 it is unlikely that these macromolecules activate NF-κB in receptormediated manner. The most probable explanation for observed

size, charge, and additional modifications have been shown to significantly affect the compatibility of nanoparticles with the immune system.49−51 Taking the above-mentioned aspects into consideration, we set out experiments to evaluate the induction of pro-inflammatory signaling by PAMAM dendrimers modified with 4-carbomethoxypyrrolidone for their future application in drug delivery. This surface-modification has been previously shown to significantly decrease the toxicity of PAMAM dendrimers against Chinese hamster fibroblasts (B14), rat liver derived cells (BRL-3A),34 mouse embryonic hippocampal cells (mHippoE-18),34,35 and mouse neuroblastoma cells (N2a).36 Moreover, PAMAMpyrrolidone variants did not influence the intracellular ROS production or mitochondrial membrane potential,34 and did not show the hemolytic activity.36 They were characterized by high solubility in aqueous solutions and organic solvents,37 the ability to form stable dendrimer-carboxylate complexes38 and efficient intracellular uptake,39,40 which make them promising host molecules for drug transport and release. In order to verify the potential immunomodulatory activity of both modified and unmodified PAMAM dendrimers of third and fourth generations, we chose two human cancer monocytic cell lines, THP-1 and U937, which differs in the origin and F


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cellular model (human cancer myeloid cell lines). Therefore, the main conclusion from our research on pyrrolidone-modified PAMAM dendrimers is that they are ideal candidates for clinical applications such as drug delivery.

phenomenon is the activation of this particular signaling pathway by stimulation of reactive oxygen species (ROS) production.56 Because monocytes constitute an essential part of innate immune system, their response for environmental stress is rapid and involves so-called “oxidative burst”, which is meant to eliminate the invading pathogen and trigger additional proinflammatory pathways.57 This includes activation of MAPK signaling pathway which triggers numerous transcription factors, including NF-κB.58 The induction of inflammation via intracellularROS production has been reported for numerous nanoparticles,59 including PAMAM dendrimers.18,19 As a positive control for pro-inflammatory stimulation, we applied E. coli lipopolysaccharide (LPS), a characteristic component of Gram-negative bacteria cell wall, commonly used for triggering NF-κB-related immune response in monocyte/macrophage in vitro models by activation of TLR4.60 LPS stimulation was previously used for evaluation of immunomodulatory activity of both modified and unmodified PAMAM dendrimers in monocyte-derived macrophages (MDMs), immature monocytederived dendritic cells (DCs)25 and rat peritoneal macrophages.20 Interestingly, the level of immune response significantly differed in both cell lines. In THP-1 LPS stimulation resulted in stronger enhancement of pro-inflammatory signaling compared to PAMAM treatment, while in U937 the opposite effect was observed. This is not unexpected, since the different cytokine expression pattern for both cell lines has been reported,61 as well for other monocytic cell lines of various origin.62 This may be also explained by the activation of various signaling pathways triggered either by LPS or PAMAM-induced ROS production,63,64 as well as the susceptibility of both cell lines to different types of stimuli. The outcome of cytotoxicity assay indicates that U937 cells are more susceptible to unmodified PAMAM dendrimers than THP-1, which results in enhanced response to the environmental stress. The preincubation of cells with unmodified PAMAM dendrimers did not decrease the pro-inflammatory effects of subsequent LPS stimulation. On the contrary, the synergistic induction of gene expression was observed at the mRNA level in both cell lines, and at the protein level in U937. This outcome indicates that unmodified PAMAM dendrimers might not exert anti-inflammatory properties, as is was postulated previously.20 Moreover, these nanoparticles induce cellular stress, which may increase upon additional stimulation with LPS. This observation further confirms the limited applications of unmodified PAMAM dendrimers in medical sciences. The effects observed for unmodified dendrimers, including enhanced binding of active NF-κB to the consensus oligonucleotide probes, increased expression of NF-κB marker genes and related cytokines both on the mRNA and protein level were suppressed by the surface pyrrolidone modification. PAMAMpyrrolidone dendrimers exerted only mild anti-inflammatory activity in LPS-stimulated cells (manifesting in decreased expression of NF-κB marker genes in U937 cell line), indicating that they cannot be used as therapeutics per se. Nevertheless, pyrrolidone variants did not cause a generalized cellular stress leading to the enhancement of NF-κB activity, characteristic for unmodified variants.

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Anna Janaszewska: 0000-0002-8872-8092 Michał Gorzkiewicz: 0000-0001-9258-3626 Barbara Klajnert-Maculewicz: 0000-0003-3459-8947 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was sponsored by the National Science Centre, Poland (Project: “Intrinsically fluorescent dendrimers - spectrofluorimetric and cell biology studies” UMO-2014/14/M/NZ3/00498).

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REFERENCES

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5. CONCLUSIONS In summary, our study confirms that the pyrrolidone modification prevents PAMAM dendrimers from activating pro inflammatory signaling pathways. This demonstrates the overall high biocompatibility of PAMAM-pyrrolidone dendrimers in a novel G


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Pyrrolidone Modification Prevents PAMAM Dendrimers from Activation

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