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Original Multivalent Gold (III) and Dual Gold(III)-Copper(II) Conjugated Phosphorus Dendrimers as Potent Antitumoral and Antimicrobial Agents Serge M. Mignani, Nabil El Brahmi, Saïd El Kazzouli, Regis Laurent, Sonia Ladeira, Anne-Marie Caminade, Elzbieta Pedziwiatr-Werbicka, Eligia M. Szewcyk, Maria Bryszewska, Mosto M. Bousmina, Thierry Cresteil, and Jean Pierre Majoral Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00771 • Publication Date (Web): 29 Sep 2017 Downloaded from http://pubs.acs.org on October 1, 2017

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Molecular Pharmaceutics

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Original Multivalent Gold (III) and Dual Gold(III)-Copper(II) Conjugated Phosphorus Dendrimers as Potent Antitumoral and Antimicrobial Agents Serge M. Mignani†,* Nabil El Brahmi‡, Saïd El Kazzouli‡, Regis Laurent§,¶, Sonia Ladeira§,¶, Anne-Marie Caminade§,¶, Elzbieta Pedziwiatr-Werbicka^, Eligia M. Szewczykw, Maria Bryszewska^, Mosto M. Bousmina‡, Thierry Cresteil ≠,* and Jean-Pierre Majoral§,¶.* †

Université Paris Descartes, PRES Sorbonne Paris Cité, CNRS UMR 860, Laboratoire de Chimie et de Biochimie pharmacologiques et toxicologique, 45, rue des Saints Pères, 75006 Paris, France ‡ EuroMed Research Institute, Euro-Mediterranean University of Fes, Route de Meknes, 30000 Fès, Morocco. § Laboratoire de Chimie de Coordination du CNRS, 205 route de Narbonne, 31077 Toulouse Cedex 4. France ¶ Université de Toulouse, UPS, INPT, Toulouse, France ≠ IPSIT, Faculté de Pharmacie, Université Paris Sud, 92290 Chatenay-Malabry, France ^ Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, 141/143 Pomorska Street, 90-236 Lodz, Poland w Department of Pharmaceutical Microbiology and Microbiological Diagnostics, Medical University of Lodz, 137 Pomorska Street, 90-235 Lodz, Poland * Address correspondence to [email protected], [email protected], [email protected] Abstract. Original metallo phosphorus dendrimers (generation 3, 48 terminal groups) have been prepared via the complexation of phosphorus dendrimers bearing imino-pyridino end groups with Au(III) or both with Au(III) and Cu(II) The complexation of the dendrimer with Au(III), leading to 1G3-[Au48] [AuCl4]48, strongly increased the anti-proliferative activities against both KB and HL-60 tumoral cell lines, showing IC50’s in the low nanomolar range. It can be noticed also that this gold conjugated phosphorus dendrimer displayed low activity on the quiescent cell line EPC versus its potent antiproliferative activity against actively dividing cells. In order to evaluate the potential synergistic effect between Au(III) and Cu(II) and the influence of the number of Au(III) moieties on the surface of dendrimer against the proliferative activities, nine other original dendrimers with several surface modifications have been prepared. Whatever the number of Au(III) moieties introduced on the surface of dendrimers, all the dendrimers prepared displayed similar potency (nanomolar range) to 1G3-[Au48] [AuCl4]48 against KB and HL60. In marked contrast synergistic effect on the antimicrobial activity of some of these phosphorus dendrimers are observed when both Au (III) and Cu(II) are present on the dendritic structure.

Keywords Dendrimers, gold complexes, copper complexes, antitumoral agents, antimicrobial agents

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Introduction Cancers remains one of the major leading cause of mortality in both developed and developing countries with over 10 million new cases every year.1 Oncology is one of the fastest-growing therapeutic areas in the pharmaceutical industry.2 Intensive and sustained research efforts in oncology during more than fifty years have led to the development of various partially curative strategies and the hope is now based on the combined efforts of various branches of science including oncology, biology, chemistry, physics and material science and engineering. New types of functional materials have been developed and are currently used in several fields of pharmaceutics and curative medicine including the field of oncology. Forty-five new drugs were approved in 2015, and the Food Drug Administration (FDA) approved 14 (~31%) cancer drugs in 2015.3 The oncology field has accounted for over 30% of the approvals for 3 of the past 4 years. Nanotechnology has immensely progressed in the last 20 years to supply important advances in several different domains such as early diagnosis and prevention, bioimaging, therapies, and drug and gene delivery systems.4,5,6 Within the nanotechnology universe, the remarkable unique and tunable properties of dendrimers have made them promising tools for diverse biomedical applications such as drug delivery, gene therapy and diagnostic methods.7 Dendrimers are highly branched macromolecules with nanometer-scale dimensions. Three different components define dendrimers: a central core, an interior dendritic structure (named branches), and an external surface with the possibility to graft multiple functional type groups. Numerous varied combination of these three components yield nanomaterials of different sizes, shapes internal cores, and are ideal candidates for instance for biological applications. A large number of small molecules or polymer metal complexes as therapeutic agents in the oncology domain were reported. Several informative reviews highlighted the use of metallodrugs in the oncology domain (unexhaustive list).8,9,10,11,12,13 To date, very few examples have described the preparation of dendrimer conjugated-metallodrugs. In early studies, Malik et al. conjugated G3.5 PAMAM-CO2H dendrimers with cisplatin giving PAMAMplatinate complexes (mono-, bi- and crosslinked-dentates).14,15,16 In in vivo studies, dendrimer conjugated-Pt displayed equi-activity with cisplatin by i.p. route against intra-peritoneally grafted L1210 leukaemia tumour model in mice with a T/C > 191% versus 171%, which is the maximal cisplatin tolerated dose (MTD) of cisplatin. At the highest dose of 15 mg/kg i.p., dendrimer conjugated-Pt, showed very good activity against B16F10 melanoma model (T/C = 129%), whereas cisplatin, at its MTD dose, did not. Beside PAMAM dendrimers, the first generation of the poly(propyleneimine)dendrimer (DAB(PA-PtCl)4) substituted with four trans-diaminechloroplatinum moieties has been described.17 This fourarmed complex can be expected to form crosslinks with DNA that are different from adducts formed with cisplatin, and consequently, potentially overcome cisplatin resistance in cancer cells. This complex showed low cytotoxicity against the L1210/0 and L1210/2 mouse leukaemia cell lines and against seven human tumor cell lines (MCF7, EVSA-T, WIDR, IGROV, M19, A498 and H226 cells). An anionic biocompatible G2-citric acid-based dendrimer-Pt showed potent in vitro cytotoxic activities not only against Pt sensitive human fibrosarcoma (HT1080) and carcinoma (CT26) tumour cell lines (up to 8-fold versus cisplatin alone), but also against Pt resistant cell lines such as human


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ovarian carcinoma (SK-OV-3). An approximatively 2-fold increase in comparison with cisplatin alone was observed.18 Several Pt(II)-dendrimer conjugates have been also prepared, and displayed weak cytotoxicity against human breast adenocarcinoma cell line (MCF-7) in contrast to complexes A(PtCl2)2 and C(PtCl2)2 showing good potency against MCF-7.19 In another example, Jang et al. have also reported interesting conjugates. Thus, cisdichlorodiamineplatinum(II), a derivative of cisplatin, was loaded onto phthalocyanine dendrimer, which led to polymer-metal-complex micelles that are useful for sustained drug releasing and photodynamic therapy. To the best of our knowledge, no antitumor activities have been reported for these products.20 Following Chu and collaborators, several works were carried out on the synthesis and cytotoxicity of transition metal complexes with Schiff base ligands. The authors have reported the synthesis of Pt(II), Pd(II), Cu(II), and Hg(II) complexes with N-[2-(pyridine-2-ylmethylidene)amino)ethyl]acetamide showing moderate to low activities against human leukaemia (MOLT-4) and breast cancer (MCF-7), and no activity against non-cancerous Chang Liver cell lines.21,22 More recently the importance of ruthenium metallodendrimers was nicely pointed out using either arene ruthenium carbosilane dendrimers,23 arene ruthenium metallacages pyrenyldendrimers,24 and ruthenium-PTA phosphorus dendrimers.25 Another example was reported by Sadler’s group consisting of the synthesis of different organometallic osmium arene complexes with substituted phenylazopyridines as chelating ligands. The most potent complexes were cytotoxic at nanomolar concentrations toward ovarian, lung, breast colon, prostate and bladder human cancer cells, with an order of magnitude higher than cisplatin alone.26 Recently, within our goal to develop new anticancer agents in the dendrimer space27, we have shown that the multivalent Cu-conjugated analogue 1G3-Cu48 displayed a high efficiency against several human cancer cell lines (IC50≈ 0.5-0.6µM), and thus might constitute a new anti-tumour candidate for chemotherapy (Figure 1).28 Its non-complexed analogue 1G3 was found to be less active against the same tumor cell lines (IC50≈1.2-1.5µM). Basically, we have shown that 1G3 moderately activated caspase-3 activity whereas 1G3-Cu48 actively promoted the translocation of Bax to the mitochondria, the release of AIF to the nucleus and a severe fragmentation of DNA, hallmark of apoptosis. 29 The promising results of 1G3 and 1G3-Cu48 prompt us to investigate the effect of the replacement of all the 48 Cu(II) groups of 1G3-Cu48 by Au(III) named 1G3-[Au48] [AuCl4]48 (Figure 1) on the antiproliferative and antimicrobial activities of this original metallodendrimer.

Results and discussion Anticancer activities

The first goal of this study is to analyse the influence of metal-type on the antiproliferative profile. Two different tumour cell lines representative markers for anticancer activity have been chosen: a solid tumour (KB, epidermal carcinoma) and the leukaemia HL60 (promyelocytic) cells. In addition, two non-cancer cell lines, namely, MCR5 (proliferative human lung fibroblasts) and the quiescent EPC



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(endothelial progenitor cells, Cyprinus carpio) have also been selected to figure a “safety ratio” by the evaluation of “normal” dividing and non-dividing cells.

Figure 1. Chemical structure of 1G3, 1G3-Cu48 and 1G3-[Au48] [AuCl4]48 dendrimers X = nothing for 1G3; X = CuCl2 for 1G3-Cu48; X = [AuCl2] [AuCl4] for 1G3-[Au48] [AuCl4]48

We first evaluated the antiproliferative potency of the mononuclear compound 1 which represents the terminal function of 1G3-[Au48] [AuCl4]48 (Figure 2), as well as the potential synergistic effects between Au(III) and Cu(II) and the influence of the number of Au(III) on the dendrimer surface against the proliferative - and also - antimicrobial activities by preparing new dendrimers containing Au(III) moieties and variable numbers of N-(pyridin-2-ylmethylene) ethanamine (named NN) groups, Cu(II)Cl2 named Cux where x is the number of Cu(II) units complexed to the NN ligands and PEG chains.


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As shown in Table 1, the replacement of Cu(II) by Au(III) increased considerably the anti-proliferative activity: 1G3-[Au48] [AuCl4]48 (prepared from the reaction of 1G3 with 96 equivalents of AuCl3,see supplementary information) showed IC50s of 7.5 nM and 3.3 nM against KB and HL60, respectively, while 1G3-Cu48 displayed IC50’s of 470 nM and 580 nM against KB and HL60, respectively. No or very weak effect has been observed against the viability of quiescent cell line EPC with both 1G3-Cu48 and 1G3-[Au48] [AuCl4]48 (IC50s ≥ 1µM), whereas IC50s of 1G3-Cu48 and 1G3-[Au48] [AuCl4]48 are of ~800nM and ~12nM against the proliferating non-tumoral MRC5 human cell line. Such values have to be compared with the known antitumoral activity of cisplatin showing IC50s of 6.3 and 11.6 µM against KB and HL60, respectively). Similar safety ratios can be calculated in actively dividing cells (IC50 MRC5/IC50 KB or HL60) for both 1G3-Cu48 and 1G3-[Au48] [AuCl4]48 whereas the safety ratio raised considerably in quiescent cells compared to actively dividing cells (IC50 EPC/IC50 KB or HL60) with 1G3-[Au48] [AuCl4]48 but remained unchanged with 1G3-Cu48. Thus, unlike 1G3-Cu48, 1G3-[Au48] [AuCl4]48 might preferentially target cells in active division. IC50 (nM)

KB

HL60

EPC

MRC5

Safety ratio

1G3-Cu48

470±20

580±70

1380±20

800±180

2.9-2.4*

1.7-1.4**

1G3-[Au48] [AuCl4]48

7.5±7.5

3.3±0.6

>1000

12±5

133-300*

1.6-3.6**

* IC50 (EPC)/IC50 (KB or HL60) ** IC50 (MRC5)/IC50 (KB or HL60) Table 1. Anti-proliferative activities of 1G3-Cu48 and 1G3-[Au48] [AuCl4]48 To delineate the respective role of the plain dendrimer 1G3 and AuCl3, the mixture of the noncomplexed 1G3 plus AuCl3 and of the stable complex 1G3-[Au48] [AuCl4]48 we measured their growth inhibition potency. As expected from previous data obtained with CuCl2,28 AuCl3 retained a low intrinsic activity (% inhibition: 55±6% cell death at 33 nM) and the association of AuCl3 with the plain 1G3 (% inhibition: 45±5% cell death at 33 nM) did not potentiate the intrinsic antiproliferative effect of 1G3 (% inhibition: 44±6% cell death at 33 nM) compared to that of the complexed 1G3-[Au48] [AuCl4]48 (% inhibition: 89±5% cell death at 33 nM). Therefore 1G3-[Au48] [AuCl4]48 represents a very interesting new nanodrug against solid tumour and leukaemia cell lines. The exact chemical structure of 1G3-[Au48] [AuCl4]48 has been determined from the X-ray determination of the Au(III)-monomer complex 1 (Figure 2). Two different Au(III) types were observed: one cationic [AuCl2]+ moiety complexed with N-(pyridin-2-ylmethylene) ethanamine moiety and one anionic Au moiety under Au(III)Cl4- counterion form. The mononuclear complex 1 was also tested against KB, HL60 and EPC cell lines to evaluate the role of the corresponding mononuclear entity on the anti-proliferative activity and the safety ratio. 1 displayed IC50’s of ~150 nM and 65-75 nM against KB and HL60, respectively, and >10 µM against EPC.



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Figure 2: Chemical structure and X-ray structure of the Au(III)-monomer complex 1 The next step was the analysis of the influence of the number and the nature of metal moieties distribution on the surface of the 1G3 dendrimer (Figure 3). We focused our attention on the preparation of nine original dendrimers based on 1G3 chemical structure with several surface modifications such as keeping free some of the N-(pyridin-2-ylmethylene) ethanamine (named NN) ligands, or complexing them partially with Cu(II)Cl2, and Au(III)Cl3 or introducing some PEG chains (n = 11) to evaluate the potential synergistic effect between Au(III) and Cu(II) and the influence of the number of Au(III) moieties against the proliferative activities. PEG chains have been selected to increase both the water solubility of corresponding dendrimers, and also potentially to increase their biocompatibility e.g. improvement of the circulating time in the blood The following dendrimers have been prepared: 44 N-(pyridin-2-ylmethylene) ethanamine (NN) groups and 4 PEG chains termed 1G3[NN44-PEG4], 40 N-(pyridin-2-ylmethylene) ethanamine (NN) groups and 8 PEG chains termed 1G3[NN40-PEG8], 30 N-(pyridin-2-ylmethylene) ethanamine (NN) groups and PEG chains termed 1G3[NN30-PEG18], 30 Au(III), 15 N-(pyridin-2-ylmethylene) ethanamine (NN) groups, and 18 PEG named 1G3-[Au15-NN15-PEG18][AuCl4]15, 40 Au(III), 20 N-(pyridin-2-ylmethylene) ethanamine moieties and 8 PEG chains named 1G3-[Au20-NN20-PEG8] [AuCl4]20, 44Au(III), 22 N-(pyridin-2-ylmethylene) ethanamine moieties and 4PEG 1G3-[Au22-NN22-PEG4] [AuCl4]22, 80Au[III),8PEG named 1G3-[Au40PEG8] [AuCl4]40, 40 Au(III) 20 Cu(II) and 8 PEG named 1G3-[Au20-Cu20-PEG8] [AuCl4]20 which represent also first-in-class dual Cu(II)-Au(III) dendrimers; 20 Au(III), 20 Cu(II),10 N-N and 8 PEG named 1G3[Au10-Cu20-NN10-PEG8] [AuCl4]10 (Figure 3). All of these dendrimers incorporate therefore stochastic number of functional group types on their respective surface. The Table 2 highlights the anti-proliferative activities of the new dendrimers against KB, HL60 tumour cell lines and the EPC quiescent cell line. Figure 4 shows the antiproliferative activities (against KB and HL60 cells) of the prepared dendrimers, and the Figure 5 summarizes the antiproliferative activities in relation with the number of Au(III) moieties on the surface of 1G3 phosphorus dendrimers versus Cu(II), PEG and N-N groups on the surface. Interestingly, we noted that 1G3-[Au20-NN20-PEG8][AuCl4]20, 1G3-[Au10-Cu20-NN10-PEG8] [AuCl4]10, 1G3-[Au20-Cu20-PEG8] [AuCl4]20, 1G3-[Au22-NN22-PEG4][AuCl4]22 and 1G3-[Au40-PEG8][AuCl4]40 displayed similar potency to 1G3-[Au48] [AuCl4]48 against KB and HL60 whatever the number of Au(III) moieties introduced. These phosphorus dendrimers displayed highly anti-proliferative activities


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against KB and HL-60 in comparison with both 1G3-Cu48 and 1G3-[NN30-PEG18,], 1G3-[NN40-PEG8], and 1G3-[NN44-PEG4]. No cumulative effect of Cu(II) was observed in the presence of Au(III). Equal to or above 10 [AuCl2]+ introduced on the surface of 1G3 no further increase was noticed on the antiproliferative activities against KB and HL-60 cell lines, as well as the association of Cu(II) to Au(III) had no additive effect, suggesting that a threshold might exist in the antiproliferative activity of Au complexed dendrimers. Of interest is the fact that the increase of the number of PEG on the surface of the dendrimer provokes partially the decrease of the antitumoral potency of the dendrimer. This might be tentatively explained by a hindered access of the terminal Au units to the tumor cells due to the presence of 18 PEG units on the surface of dendrimer. Such effect is more pronounced in the case of KB cell lines: for example, 1G3-[Au15-NN15-PEG18] [AuCl4]15 displayed IC50’ of ~80-100 nM versus 10-20 nM for 1G3-[Au20-NN20-PEG8] [AuCl4]20. However such a phenomenon is not so marked when the number of PEG units is decreased from 8 to 4 (see Table 2)



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Figure 3: 2D chemical structure of 1G3-[NN44-PEG4], 1G3-[NN40-PEG8], 1G3-[NN30-PEG18], 1G3-[Au15NN15-PEG18] [AuCl4]15, 1G3-[Au40-PEG8] [AuCl4]40, 1G3-[Au20-NN20-PEG8] [AuCl4]20, 1G3-[Au22-NN22PEG4] [AuCl4]22, 1G3-[Au20-Cu20-PEG8] [AuCl4]20, 1G3-[Au10-Cu20-NN10-PEG8] [AuCl4]10

IC50 (nM)

KB

HL60

EPC

1G3-[NN44-PEG4]

>1000

>1000

>1000

1G3-[NN40-PEG8]

>1000

>1000

>1000

1G3-[NN30-PEG18]

>1000

>1000

>1000

1G3-[Au15-NN15-PEG18] [AuCl4]15

87±7

>1000

>1000

1G3-[Au20-NN20-PEG8] [AuCl4]20

15±5

4.5±0.5

>1000

1G3-[Au22-NN22-PEG4] [AuCl4]22

6.7±4.6

3±0.5

>1000

1G3-[Au20-Cu20-PEG8] [AuCl4]20

8.5±0.7

2.5±0.7

>1000

1G3-[Au10-Cu20-NN10-PEG8] [AuCl4]10

10±3

4±3

>1000

1G3-[Au40-PEG 8 ] [AuCl4 ]40

5.5±0.5

1.7±0.5

>1000

Table 2. Antiproliferative activities of 1G3-[NN44-PEG4], 1G3-[NN40-PEG8], 1G3-[NN30-PEG18], 1G3[Au15-NN15-PEG18] [AuCl4]15, 1G3-[Au40-PEG8] [AuCl4]40, 1G3-[Au20-NN20-PEG8] [AuCl4]20, 1G3-[Au22NN22-PEG4] [AuCl4]22, 1G3-[Au20-Cu20-PEG8] [AuCl4]20, 1G3-[Au10-Cu20NN10-PEG8] [AuCl4]10



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Figure 4. Antiproliferative activities (KB and HL60) of 1G3-Cu48, 1G3-[Au10-Cu20-NN10-PEG8] [AuCl4]10, 1G3-[Au15-NN15-PEG18] [AuCl4]15, 1G3-[Au20-NN20-PEG8] [AuCl4]20,1G3-[Au20-Cu20-PEG8] [AuCl4]20, 1G3-[Au22-NN22-PEG4] [AuCl4]22, 1G3-[Au40-PEG8] [AuCl4]40 and 1G3-[Au48] [AuCl4]48

Figure 5. Antiproliferative activities related to the number of AuCl2+ moieties on the surface of 1G3 phosphorus dendrimers. The antiproliferative activity of 1G3-Cu48 was the result of the activation of the apoptotic cascade leading to DNA fragmentation. Therefore, we evaluate the capacity of 1G3-[Au48] [AuCl4]48 to promote DNA fragmentation, estimated by TUNEL analysis. Basically, nick-end DNA strands were tagged by BrdU and a FITC-labelled anti BrdU antibody. Cells were separated by flow cytometry technique (FACS: fluorescence-activated cell sorting) and the green fluorescence intensity was monitored in individual cells separated also by flow cytometry. Consequently, the green fluorescence intensity is the reflect of the DNA fragmentation in each cell. The amount of total cellular DNA in the same cells was detected with propidium iodide giving a red fluorescence (supplemental data S1). The number of HL-60 cells with fragmented DNA raised from 2±1% in cells treated with DMSO alone, to 12±2% in cells treated for 24h with 20 nM AuCl3 + DMSO and from 15±8 to 55±20% with increasing concentration of 1G3-[Au48] [AuCl4]48 ranged 5 to 20 nM. In the same conditions 1 µM 1G3 elicited a modest 10±10% augmentation of DNA fragmentation in HL-60 cells. This clearly suggested that 1G3[Au48] [AuCl4]48 like 1G3-Cu was capable to activate the apoptotic process in dividing cells. Through the above example it is clear that the antiproliferative activities against KB and HL60 cell lines necessitate only a small amount of Au complexed on the surface of the phosphorus dendrimer of generation 3.


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Next, we decided to test 1G3-[Au48] [AuCl4]48, 1G3-[Au20-Cu20-PEG8] [AuCl4]20 (dual Au-Cu phosphorus dendrimer) and 1G3-[Au40-PEG8] [AuCl4]40 against MCF7 human breast adenocarcinoma cell line, PC3 prostatic small cell carcinoma and MRC5 non-tumoral human lung cells (Table 3).

IC50 (nM) 1G3-[Au48] [AuCl4]48 1G3-[Au40-PEG8 ] [AuCl4 ]40 1G3-[Au20-Cu20-PEG8] [AuCl4]20

MCF7 6±2 2.5±0.7 60±15

PC3 1.5±0.7 0.5±0.1 13±2

MRC5 6±2 2.7±0.3 32±8

Table 3. Anti-proliferative activities of 1G3-[Au48] [AuCl4]48, 1G3-[Au20-Cu20-PEG8] [AuCl4]20 and 1G3[Au40-PEG8] [AuCl4]40 These three metallo-dendrimers displayed moderate to good anti-proliferative activities against all three cell lines. 1G3-[Au48] [AuCl4]48 showed good anti-proliferative activities with IC50s =1.5 to 6nM, 1G3-[Au40-PEG8] [AuCl4]40 is the most potent dendrimer against the three cell lines (IC50s= 0.5 to 2.7nM) whereas (1G3-[Au20-Cu20-PEG8] [AuCl4]20 is the less potent one (IC50s= 13 to 60 nM). These data fully confirm the potential antitumoral profile of phosphorus dendrimers bearing gold metal, or dual gold-copper metals. The next step was to check if a modification of the backbone of the dendrimer can modify the potency of the dendrimer. For such a purpose a green chromophore having outstanding two photon absorption properties was introduced at the level of the dendrimer of generation 1, then the construction of the generation 2 from the twelve chromophoric branches was carried on leading to a dendrimer bearing 24 terminal P(S)Cl2 Simultaneous addition of 12 equivalents of phenol PEG and 12 equivalents of the phenol bearing iminopyridino ligands affords the dendrimer of generation 2 which was submitted to complexation with Au(III) to give the phosphorus dendrimer 2G2-[Fluo12-Au16PEG8][Au Cl4]16 It is interesting to note that the size of this dendrimer is roughly the same to that of 1G3-[Au20-PEG8] [AuCl4]20 due to the length of the internal chromophoric linkages (Scheme 1). Remarkably such a fluorescent dendrimer keeps in a high level the antiproliferative activities against KB and HL60: IC50s of 60-70 nM and 40-50 nM against KB and HL60 respectively. The presence of PEG units maintains the water solubility, thus allowing us to consider such a dendrimer as a good candidate for further in vitro and in vivo imaging studies in tumor cell lines.



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Scheme 1. Synthesis of the dendrimer bearing 12 internal fluorophore units 2G2-[Fluo12-Au16] [PEG]8 [AuCl4 ]16

Antimicrobial activities Some of these metallophosphorus dendrimers were evaluated for an antibacterial and antifungal activity against the strains: Gram positive bacterium (Staphylococcus aureus ATCC 6538), Gram negative bacteria (Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 27853) and yeasts (Candida albicans ATCC 10231), but also against drug-resistant clinical strains: Gram positive bacteria (Staphylococcus aureus ZMF KSK, Enterococcus faecalis ZMF BD 156) and yeasts Candida glabrata ZMF SZP 4. The MIC (minimal inhibitory concentration) values were the lowest concentrations of the compound with no measurable optical density increase. The MBC (minimal bactericidal concentration) values were the lowest concentrations of the compound that killed all cells and as a result there was no growth of a subculture on the surface of a Petri dish. The obtained results (Figures 6, 7 and Table 4) indicate that all dendrimers have a wide spectrum of bacteriostatic/fungistatic activity. Also in some cases they have bactericidal and fungicidal activity (MIC=MBC) in a concentration range of 3.5-500 mg/L. 1G3-[Au48] [AuCl4]48 dendrimer has the biggest number of monomers 1 in the structure, resulting in the biggest number of Au atoms (96) and the highest antimicrobial activity. In turn the monomer 1 having two Au atoms is the second one in antimicrobial activity ranking. There are many reports on Au(I) and Au(III) complexes studied for antimicrobial activity against a wide variety of microorganisms.30,31,32,33 Among studies on antimicrobial potential of different nanoparticles, the gold nanoparticles represent a broad group of compounds.34,35,36,37 Mechanism of antibacterial activity of AuNP, proposed by Cui et al. suggests that the death of bacterial cells is due to attachment of these nanoparticles to the bacterial membrane followed by membrane potential modification and ATP level decrease and secondly inhibition of tRNA binding to the ribosome.38 The most sensitive bacterium towards all tested compounds is Gram


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negative E. coli. The difference in peptidoglycan layer’s thickness in Gram-positive and Gramnegative bacteria membrane (50% higher in Gram positive) may explain the fact, that in many studies larger doses of nanoparticles are required for Gram-positive bacteria. However, in case of metallophosphorus dendrimers high sensitivity was also observed for Gram positive S. aureus (type strains). Umadevi et al.39 showed that AgNPs were most effective against E. coli, which membrane exhibits a high permeability, leaving the bacterial cells incapable of properly regulating transport through the plasma membrane and causing cell death. It was shown that metal particles formed irregular-shaped pits in the outer membrane of E. coli and changed membrane permeability.40,41,42 Comparing the sensitivity of bacteria, the lower toxicity of tested compounds toward clinical resistant strains was observed (S. aureus ATCC 6538 versus S. aureus ZMF KSK). The mechanisms of bacterial resistance may be intrinsic to the species or acquired through mutation of intrinsic genes or horizontal exchange of genetic material encoding resistance determinants. P. aeruginosa and E. faecalis are the examples of highly intrinsic resistant bacteria. Low susceptibility of P. aeruginosa to antibiotics is mainly the consequence of the loss of porin43 and the presence of several genes encoding multidrug resistance (MDR) efflux pumps in its genome.44 Hollenback and Rice45 reviewed the mechanisms underlying antibiotic resistance in enterococci to virtually all clinically useful antibiotics (aminoglycosides, β-lactams, cephalosporins, lincosamides, glycopeptides and streptogramins). We have noticed that the lowest concentration 62.5 mg/L of 1G3-[Au48][AuCl4]48 killed all P. aeruginosa cells while the monomer 1 in a concentration of 125 mg/L has bacteriostatic activity against the same strain. E. faecalis ZMF BD 156 sensitivity toward monomer 1 is the same as P. aeruginosa sensitivity (MIC 125 mg/L, MBC 500 mg/L) and a little bit lower towards 1G3-[Au48][AuCl4]48 (bactericidal activity at 125 mg/L). Those results are very promising. However, some compounds (for example trimethoprimsulfamethoxazole) despite in vitro enterococcal susceptibility are ineffective in treating serious enterococcus infections.46 Among the studied molecules, 1G3-[Au10-Cu20-NN10-PEG8] [AuCl4 ]10 (with copper and gold in the structure) exhibited the greatest antifungal activity against Candida albicans ATCC 10231 and Candida glabrata ZMF SZP4 (MIC=MBC 62,5 mg/L). The main components of Candida membrane are β−1,3-glucan, β−1,6-glucan, chitin and two classes of proteins (they may constitute even 40%): GPI - linked to β−1,6-glucan through a glycophosphatidylinositol and Pir (proteins with internal repeats) - linked directly to the β-1,3-glucan.47 A large number of studies have used copper in various forms, concentration, and environment to test the antimicrobial properties, which are mostly based on “contact killing”. Metallic copper can alter the tertiary structure of proteins, which leads to damage of the bacterial envelope and might constitute a primary killing mechanism.48,49 Copper is also present in the structure of 1G3 [Cu44-PEG4], where the fungicidal effect is observed in a concentration of 250 mg/L. So, we can assume the synergistic effect between Au and Cu in case of 1G3-[Au10-Cu20-NN10-PEG8] [AuCl4]10. 1G3-[Au10-Cu20-NN10-PEG8] [AuCl4]10 presented also bacteriostatic activity to E. coli. Domek et al.50 claimed that copper considerably influences metabolism of E. coli. According to their results cooper impairs respiratory activity causing a decrease of oxygen uptake and in consequence the rate of utilisation of glucose and succinate which are the main substrates of their aerobic metabolism



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Figure 6. The MIC (minimal inhibitory concentration) values of best compounds against bacteria and yeasts.

Figure 7. The MBC (minimal bactericidal concentration) values of best compounds against bacteria and yeasts. 500* means >500.

Table 3. The MIC (minimal inhibitory concentration) and MBC (minimal bactericidal concentration) values of tested compounds against bacteria and yeasts. Monomer 1

1G3-[Au48]

1G3-[Au20-

1G3 [Cu44-


1G3-[Au10-Cu20-

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[AuCl4]48

NN20-PEG8] PEG4] [AuCl4 ]20 MIC MBC MIC MBC mg/L mg/L mg/L mg/L

NN10-PEG8] [AuCl4 ]10 MIC MBC mg/L mg/L

MIC mg/L

MBC mg/L

MIC mg/L

MBC mg/L

62.5

62.5

3.9

15.6

250

250

250

>500

500

500

7.81

15.6

7.8

15.6

62.5

62.5

500

500

62.5

500

125

500

62.5

62.5

250

250

>500

>500

250

>500

125

250

250

>500

>500

>500

250

250

62.5

62.5

125

>500

62.5

125

250

500

>500

>500

250

500

125

500

125

125

500

500

250

>500

250

>500

125

250

250

>500

125

>500

250

250

62.5

62.5

Type strains S. aureus ATCC 6538 E. coli ATCC 8739 P. aeruginosa ATCC 27853 C. albicans ATCC 10231

Clinical strains S. aureus ZMF KSK E. faecalis ZMF BD 156 C. glabrata ZMF SZP 4

Conclusion In conclusion, new metallodendrimers have been prepared by the replacement of Cu(II) on the phosphorus dendrimer surface by Au(III). The complexation of the dendrimer with Au(III) strongly increased (~30-fold) the anti-proliferative activities against both KB and HL-60. 1G3-[Au48] [AuCl4]48 showed IC50’s in the low nanomolar range (IC50’s of 7.5 nM and 3 3 nM against KB and HL60, respectively). It can be noticed also that 1G3-[Au48] [AuCl4]48 displayed low activity on the quiescent cell line EPC versus its potent antiproliferative activity against actively dividing cells. This is in line with the activation of DNA fragmentation reported herein. Interestingly, equal to or above 10 Au(III) introduced on the surface of 1G3 no further increase of potency was noticed on the anti-proliferative activities against KB and HL-60 cell lines, as well as the association of Cu(II) to Au(III) had no additive effect, suggesting that a threshold might exist in the antiproliferative activity of Au complexed dendrimers. Taken together, these results strongly demonstrate that 1G3-[Au48] [AuCl4]48 and 1G3-[Aux]-[NN][PEG)] [AuCl4]x in which x is equal to or above 10 displayed similar impressive anti-proliferative activities (nanomolar range) and represent new nanodevices in the oncology domain. The oncology field remains one of the most challenging therapeutic area despite the considerable progress made with the new molecularly targeted therapies. For many patients the therapeutic options are still limited, and the process of bringing a new drug to patients is still frustratingly slow with high failure rates. Oncology is one of the poorest record for IND (Investigational NEW Drugs) in clinical development (~5%), low and decreasing response rates in Phase I oncology trials (~2-3%), and treatment-related to death rate (500 mg/L. Another two strains Enterobacter faecalis ZMF BD156 (Gram-positive bacteria) and Candida glabrata ZMF40 (yeasts) were isolated from clinical specimens from humans. Analysed compounds were suspended in dimethylsulfoxide (DMSO – analytical grade) and then in Mueller Hinton Broth (BioMaxime) for bacteria and in RPMI-1640 Medium (Sigma) for yeasts. Consecutive two-fold dilutions were prepared in a microtiter tray (concentrations from 500 to 3.9 mg/L). Tested strains were inoculated into each well of microtiter plate at concentration of 106 CFU per 1 mL. After 24-hour incubation at 37°C for bacteria and 48 hours at 25oC for yeasts an increase of turbidity was measured at 630 nm by means of microplate reader (MR 680 Bio-Rad). The MIC (minimal inhibitory concentration) values was the lowest concentration of the investigated compounds with no measurable optical density increase. The MBC (minimal bactericidal concentration) values was the lowest concentrations of the compound that killed all cells and as a result there was no growth of a subculture on the surface of Petri dish containing proper for each organism agar medium and after 24 or 48-hours incubation at an appropriate temperature.

Acknowledgment Thanks are due to CNRS (France) and the University of Lodz (Poland) for financial support Supporting informations Syntheses and characterization of dendrimers and gold (III) and Gold(III)Cu(II)dendrimer complexes. Figure and data on TUNEL assays.

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Figure 1. Chemical structure of 1G3, 1G3-Cu48 and 1G3-[Au48] [AuCl4]48 dendrimers X = nothing for 1G3; X = CuCl2 for 1G3-Cu48; X = [AuCl2] [AuCl4] for 1G3-[Au48] [AuCl4]48 164x163mm (300 x 300 DPI)



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Figure 2. Chemical structure and X-ray structure of the Au(III)-monomer complex 1 178x79mm (300 x 300 DPI)


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Figure 3. 2D chemical structure of 1G3-[NN44-PEG4], 1G3-[NN40-PEG8], 1G3-[NN30-PEG18], 1G3[Au15-NN15-PEG18] [AuCl4]15, 1G3-[Au40-PEG8] [AuCl4]40, 1G3-[Au20-NN20-PEG8] [AuCl4]20, 1G3[Au22-NN22-PEG4] [AuCl4]22, 1G3-[Au20-Cu20-PEG8] [AuCl4]20, 1G3-[Au10-Cu20-NN10-PEG8] [AuCl4]10 155x271mm (300 x 300 DPI)



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Figure 4. Antiproliferative activities (KB and HL60) of 1G3-Cu48, 1G3-[Au10-Cu20-NN10-PEG8] [AuCl4]10, 1G3-[Au15-NN15-PEG18] [AuCl4]15, 1G3-[Au20-NN20-PEG8] [AuCl4]20,1G3-[Au20-Cu20-PEG8] [AuCl4]20, 1G3-[Au22-NN22-PEG4] [AuCl4]22, 1G3-[Au40-PEG8] [AuCl4]40 and 1G3-[Au48] [AuCl4]48 289x179mm (96 x 96 DPI)


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Figure 5. Antiproliferative activities related to the number of AuCl2+ moieties on the surface of 1G3 phosphorus dendrimers 210x150mm (96 x 96 DPI)



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Figure 6. The MIC (minimal inhibitory concentration) values of best compounds against bacteria and yeasts. 146x88mm (120 x 120 DPI)


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Figure 7. The MBC (minimal bactericidal concentration) values of best compounds against bacteria and yeasts. 500* means >500. 146x88mm (120 x 120 DPI)



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Scheme 1. Synthesis of the dendrimer bearing 12 internal fluorophore units 2G2-[Fluo12-Au16] [PEG]8 [AuCl4 ]16 172x101mm (300 x 300 DPI)


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Conjugated Phosphorus Dendrimers as Potent ... - ACS Publications

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