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Interactions of Nitroxide-Conjugated and non-Conjugated Glycodendrimers with Normal and Cancer Cells and Biocompatibility Studies Elisa Andreozzi, Antonella Antonelli, Michela Cangiotti, Barbara Canonico, Carla Sfara, Anna Pianetti, Francesca Bruscolini, Karin Sahre, Dietmar Appelhans, Stefano Papa, and Maria Francesca Ottaviani Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00635 • Publication Date (Web): 09 Jan 2017 Downloaded from http://pubs.acs.org on January 11, 2017
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Bioconjugate Chemistry
Interactions of Nitroxide-Conjugated and non-Conjugated Glycodendrimers with Normal and Cancer Cells and Biocompatibility Studies Elisa Andreozzi,ξ†,∆ Antonella Antonelli, ξ† Michela Cangiotti,‡ Barbara Canonico,† Carla Sfara,† Anna Pianetti,† Francesca Bruscolini,† Karin Sahre,§ Dietmar Appelhans,§ Stefano Papa,† and Maria Francesca Ottaviani*‡ †
Department of Biomolecular Sciences, University of Urbino Carlo Bo, Via Saffi 2, 61029 Urbino, Italy. Department of Pure and Applied Sciences, University of Urbino Carlo Bo, Via Ca’ Le Suore 2/4, 61029 Urbino, Italy. § Leibniz Institute of Polymer Research Dresden, Department Bioactive and Responsive Polymers, Hohe Strasse 6, 01069 Dresden, Germany. ∆ Current Address: Eastern Regional Research Center, Agricoltural Research Service, USDA, 600 East Mermaid Lane, 19038 Wyndmoor, PA, USA. ‡
*corresponding author: Maria Francesca Ottaviani, Department of Pure and Applied Sciences (DiSPeA), University of Urbino Carlo Bo, Via Ca’ Le Suore 2/4, 61029 Urbino, Italy tel. +39 0722 304320; Fax +39 0722 304306 e-mail:
[email protected] ξThese
authors contributed equally to this work
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ABSTRACT Poly(propyleneimine) glycodendrimers fully modified with maltose units were administered to different cancer cell lines and their effect on cell viability was evaluated by using MTS assay and flow cytometry. The mechanism of dendrimer-cell interactions was investigated by the electron paramagnetic resonance (EPR) technique by using a new nitroxide-conjugated glycodendrimer. The nitroxide groups did not modify both the biological properties (cell viability and apoptosis degree) of the dendrimers in the presence of the cells and the dendrimer-cell interactions. Since this class of dendrimers is already known to be biocompatible for human healthy cells, non-cancer cells such as human peripheral blood mononuclear cells (PBMCs) and macrophages were also treated with the glycodendrimer and EPR spectra of the nitroxide-conjugated glycodendrimer were compared for cancer and non-cancer cells. It was found that this dendrimer selectively affects the cell viability of tumour cells, while, surprisingly, PBMC proliferation is induced. Moreover, H-bond-active glycodendrimer-cell interactions were different for the different cancer and non-cancer cell lines. The nitroxide-conjugated glycodendrimer was able to interact with the cell membrane and eventually cross it, getting in contact with cytosol antioxidants. This study helps to clarify the potential anti-cancer effect of this class of dendrimers opening to future applications of these macromolecules as new anti-tumour agents.
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Bioconjugate Chemistry
INTRODUCTION Nanomaterial systems have been receiving increasing attention over time as powerful strategies simultaneously to work as therapeutic agents and to deliver, for instance, drugs, genes, siRNA, recombinant proteins, vaccines, and aptamers. Among the nanomaterials, dendrimers have rapidly grown interest for their application in nanomedicine in the last years, as reported in a large number of publications including several major reviews.1-5 The increase in the interest on the biomedical use of dendrimers can be explained by their unique and tunable properties: i) a structure constituted by a polyfunctional core with covalently attached monomers which form subsequent layers (termed generations, Gn, where n is the generation number). The dendrimer has a globular shape where the several external surface groups can be functionalized with drugs, targets, etc., ii) monodispersity, which favours reproducible pharmacokinetics; mainly, their size, shape, surface properties, influence pharmacodynamic and pharmacokinetic behaviours, iii) their size which is generation dependent and may be selected on the basis of various biomedical applications, iv) their high stability and the lack of immunogenicity that render the dendrimers preferable if compared to synthesized peptide carriers and natural protein carriers, v) a high cellular uptake level due to high penetration abilities through the cell membranes. These two last items describe different but related properties of the dendrimers: on one side the stability and the fact that the dendrimers show a concentration-dependent immune response in preclinical studies, and, on the other side, their ability to enter the cell membrane and therefore to work as drug and gene carriers, vi) a preferential uptake of materials by cancers and inflamed tissues due to the enhanced penetration and retention effect, vii) the various administration routes, including non-classical ones as trans-dermal diffusion, trans-nasal diffusion and ocular delivery. The possibility to use dendrimers as active drugs, per se, was well documented and could significantly increase the chance to fight against many diseases as cancers, neurodegenerative diseases, viral and inflammatory diseases.6-13 However, the potential of dendrimers bearing partially charged surface groups like the amino ones is limited in biological systems due to associated toxicity issues.14 The problem of toxicity is conditioned by the interaction between the cationic charged surfaces of the dendrimers and negatively charged biological membranes in vivo. This dendrimer toxicity is generation dependent, and higher generations are associated with greater toxicity.14-16 Since the presence of terminal functional groups enables the binding of various compounds such as amino acid residues or sugars to the dendrimer, surface modification may be the key to avoid the toxicity issues.17-19 Previous studies have indeed shown that poly(propyleneimine) (PPI) glycodendrimers, decorated with maltose or maltotriose functions, 3 ACS Paragon Plus Environment
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demonstrate lower levels of cyto-, geno-, and hemato-toxicity on primary cells from healthy donors in vitro, when compared to their unmodified cationic PPI dendrimer counterpart.13,14,20 In addition, FraniakPietryga et al.13 proved that dense shell PPI glycodendrimers fully modified with maltotriose show a higher cytotoxicity against blood mononuclear cells derived from chronic lymphocytic leukemia patients than against cells derived from healthy donors. Surface modification based on coating of PPI dendrimers with maltose is an efficient method to reduce their genotoxicity and permits their use as drug carriers or therapeutic agents. Furthermore, a recent paper also suggested the possibility to use these dendrimers in the area of drug delivery because of their low in vitro haemolytic properties and low cytotoxicity on red blood cells.21,22 All these promising key characteristics of PPI glycodendrimers prompted us to try to characterize the biological and interacting behaviour of 3rd generation PPI glycodendrimers with dense maltose shell (Scheme 1: PPI-G3-DS-Mal) in the presence of cancer cells, compared to normal cells. Generation 3 (G3) was selected to ensure a high number of surface groups for undergoing desired Hbond interactions22 by biological environment.
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A
R
R R R R
R
N
N
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O N
R R N
N
O
R
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N R R R
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N R R
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HO O
PPI-G3-DS-Mal
R=
HO HO HO
O HO OH
R R N
N
S
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R H
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OH OH
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G3T10
B PPI-G3-DS-Mal
(N (
NH2
)x
(i)
(R
N
H
R R
)y
)32-y
(R
N
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)z
) R 32-z
(R
(ii)
+ R
(N
major component
y = 1-5
(N
G3T10
minor component
N
R
)y
)32-y
y = 1-5
+ z-w(
R N H
(N
) N
z = 6-15
H
R
(RHN
R R
S
)32-z )w
N
w = 1-10
O
Scheme 1. (A) Simplified structure of PPI glycodendrimer with dense maltose shell (PPI-G3-DS-Mal) and TEMPOconjugated PPI glycodendrimer with dense maltose shell (G3T10). (B) Reaction sequence for PPI-G3-DS-Mal and G3T10. Major and minor component are also simplified here. Secondary amino groups are not completely converted with SCN-TEMPO when y is ≤ 5 (further details in Supporting Information). (i) = reductive amination of PPI dendrimer with maltose units using borane*pyridine complex at 50°C. (ii) = conversion with SCN-TEMPO at room temperature.
To get information about the interactions occurring at a molecular level, with an in-situ technique, we selected the electron paramagnetic resonance (EPR) technique already demonstrated to be very suitable to obtain information about the site-site interactions occurring between dendrimers and several biocomponents.23-33 To pursue this goal, nitroxide-conjugation of the PPI-G3-DS-Mal dendrimer (Scheme 1) was performed by covalently attaching a nitroxide radical (tetramethylpiperidine-N-oxyl 5 ACS Paragon Plus Environment
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radical, termed TEMPO and indicated as T) to the external secondary amino groups of the dendrimer surface. The number of nitroxide groups, theoretically selected at 10 equivalents for the conversion with PPI-G3-DS-Mal, provides a quite high sensitivity of the EPR measurements, and it was found to be informative on the interactions without being perturbative. Therefore, the nitroxide-conjugated dendrimer, simply called G3T10, is the PPI-G3-DS-Mal glycodendrimer, partially modified with up to 10 TEMPO radicals at the external dendrimer surface (Scheme 1). The modification of PPI-G3-DS-Mal is limited due to the dense maltose shell as indicated in Scheme 1. The main part PPI-G3-DS-Mal of G3T10 was not modified with the TEMPO radicals. This corresponds to the expected assumption prior starting our biological study that the modification of PPI-G3-DS-Mal with TEMPO will not significantly influence the original biological properties of PPI-G3-DS-Mal (further explanation in Supporting Information). The in-vitro effect of PPI-G3-DS-Mal against tumour cells was studied by using tumour cell lines such as HEp-2 (laryngeal carcinoma cell line), HeLa (cervical adenocarcinoma cell line), Caco-2 (colorectal adenocarcinoma cell line) and Jurkat (acute T cell leukemia cell line), and, as a matter of comparison, primary cells such as human peripheral blood mononuclear cells (PBMCs) and macrophages. Biological interactions were evaluated in terms of cell viability and cell proliferation by both
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
(MTS) assay and flow cytometry after dendrimer treatment compared to the control in the absence of dendrimer. In parallel, EPR analyses were performed on the same cell types by using G3T10 in order to get information about cell-dendrimer interactions at a molecular level (H-bonds, ionic interactions, vande-Waals force and/or dipole-dipole interactions etc.).
RESULTS AND DISCUSSION Cell Viability Assay. MTS results (Figure 1) evidenced a significant decrease in cell viability of HEp-2, Caco-2, HeLa and Jurkat cancer cells after PPI-G3-DS-Mal dendrimer treatment compared to control cell viability and this decrease appeared to be concentration dependent.
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Bioconjugate Chemistry
Figure 1. Evaluation of cell viability by MTS assay of different tumor cell lines at 24 h (A) and 48 h (B) post 1, 5 and 10 mM PPI-G3-DS-Mal dendrimer treatment. All values are expressed in percentage with respect to control cells as mean of three independent experiments; the error bars show the standard deviations (mean +SD). Asterisks indicate statistical significance of differences between treated cells compared to control cells; (*) p≤ 0.05, (**) p≤ 0.01 and (***) p≤ 0.001.
Specifically, when tumor cells were treated with 5 and 10 mM of PPI-G3-DS-Mal dendrimer, a cytotoxic effect was evident both at 24 and 48 h (Figures 1A and B), while the lower PPI-G3-DS-Mal dendrimer concentration (1 mM) led to a significant decrease (p≤0.05) of HEp-2 and HeLa cell viability only after 48 from treatment (Figure 1B). In detail, after 24h from dendrimer administration at 5 and 10 mM concentrations, respectively, all cell lines show a significant decrease of cell viability (p≤ 0.05 at least) versus control samples, while 1 mM concentration does not affect significantly the viability of all cell lines. In fact, with 1 mM dendrimer treatment, the percentages of cell viability for HEp-2 and HeLa cells correspond to 84.5% ± 7.5 and 88.7 ± 6, respectively, and are not significantly different (p> 0.05) from cell viability percentages of respective control cells (100 ± 4 and 100 ± 5.8). Instead, after 48h, all dendrimer concentrations (1mM, 5mM, 10mM) lead to a significant decrease of HEp-2 (84.9% ± 6.4, 77.7 ± 8.8, 77.5 ± 5.2 vs 100 ± 3.3 of control cells, respectively using 1 , 5 and 10 mM dendrimer) and HeLa cells viability (84.87% ± 7.7, 68.8 ± 10.8, 67 ± 9.7 vs 100 ± 4 of control cells, respectively using 1, 5 and 10 mM dendrimer) whereas the decrease of Caco-2 and Jurkat cell viability induced from 1mM dendrimer concentration still appears not significant. In general, it is useful to note that all cancer cell lines treated with 10 mM dendrimer showed a mean percentage of cell viability ranging from 65% to 80%. Our results agree with Ziemba et al.20 who have shown that similar dendrimers, using open shell glycodendrimers with maltose or maltotriose units, cause a significant decrease in cell viability of CEMSS and U87 cancer cells when administered at similar concentrations. However, like other types of non7 ACS Paragon Plus Environment
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charged dendrimers PPI-G3-DS-Mal showed a cytotoxic effect on cell viability lower than charged dendrimers, as reported by Cheng et al.34 On the other hand, charged dendrimers also exhibit cytotoxicity towards non-cancer cells. On the contrary, a time dependent increase of PBMC absorbance was observed after administration of PPI-G3-DS-Mal dendrimer at all concentrations (1, 5 and 10 mM) (Figure 2). In fact,
Figure 2. Evaluation of cell viability by MTS assay of peripheral blood mononuclear cells (PBMCs) at 24, 48 and 72 h post 1, 5 and 10 mM PPI-G3-DS-Mal dendrimer treatment. All values are expressed as mean of absorbance obtained from three independent experiments; the error bars show the standard deviations (mean +SD). Asterisks indicate statistical significance of differences between treated cells compared to control cells; (*) p≤ 0.05, (**) p≤ 0.01 and (***) p≤ 0.001.
at 24, 48 and 72 h, PBMC absorbance was significantly higher than that of control cells (p
