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Polycationic Adamantane-based Dendrons of Different Generations Display High Cellular Uptake without Triggering Cytotoxicity Maxime Grillaud, Julie Russier, and Alberto Bianco J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 20 Dec 2013 Downloaded from http://pubs.acs.org on December 23, 2013
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Polycationic Adamantane-based Dendrons of Different Generations Display High Cellular Uptake without Triggering Cytotoxicity MAXIME GRILLAUD, JULIE RUSSIER, ALBERTO BIANCO* CNRS, INSTITUT DE BIOLOGIE MOLECULAIRE ET CELLULAIRE, IMMUNOPATHOLOGIE ET CHIMIE THERAPEUTIQUE, STRASBOURG, FRANCE ADAMANTANE, DENDRONS, DELIVERY, CELLULAR UPTAKE, CYTOTOXICITY Corresponding e-mail:
[email protected] ABSTRACT: Dendrons used as synthetic carriers are promising nanostructures for biomedical applications. Some polycationic dendritic systems, like the commercially available PEI (polyethylenimine), have the ability to deliver genetic material into cells. Nevertheless polycationic vectors are often associated with potential cellular toxicity which prevents their use in clinical development. In this context our research focused on the design and the synthesis of a novel type of polycationic dendrons which are able to penetrate into cells without triggering cytotoxic effects. We synthesized 1st and 2nd generation polycationic adamantane-based dendrons via a combined protection/deprotection strategy starting from different adamantane scaffolds. The linker between the adamantane cores is constituted of short ethylene glycol chains and the periphery consists of ammonium and guanidinium groups. None of these dendritic structures which we previously called HYDRAmers displayed significant cytotoxicity effects on two different cell lines (RAW 264.7 and HeLa). Conjugation of the fluorescent probe cyanine 5 at their focal point via click chemistry permitted the evaluation of their cellular internalization. All dendrons penetrated trough the membrane with efficient cellular uptake depending of the dendron generation and the nature of the peripheral groups. These results suggest that the polycationic HYDRAmers are potentially interesting as new vectors in biomedical applications including gene and drug delivery.
Introduction Molecular nanostructures with well-defined particle size and shape are widely explored for biomedical applications such as drug delivery, gene transfection, and imaging. Dendrons (wedge-shaped dendrimer sections) have been investigated as ideal nanoscale carrier molecules for the delivery of bioactive materials into the cells.1 Molecular engineering of these hyperbranched, monodisperse, well-defined structures can be easily performed using simple organic synthesis.2 Multivalency constituted by the multiple surface groups at the periphery of a dendron ACS Paragon Plus Environment
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promotes higher binding affinity for ligand/receptor interactions.3 By controlling their synthesis, it is possible to manipulate both the molecular weight and chemical composition of the dendrons4 in order to solve problems of biocompatibility, toxicity, pharmacokinetics and organ specific targeting.5 Each part of the dendron consisting of three distinct moieties (i.e. the focal point, the branching and the dendritic surface) can play a distinctive role that can be tuned by modifying the characteristics of the functional groups. Different dendritic architectures have been studied for their ability to deliver genetic material into cells6 such as PAMAM (polyamidoamine) globular dendrimers.7 Polycationic dendrons based on polyamines have been commonly employed for gene delivery, but, although their good transfection activity, they also exhibit problematic toxicity profiles8 strongly dependent on the concentration used.9 PEI (polyethylenimine), a commercially available polyammonium dendrimer,10 is one of the most efficient transfection agents. However, PEI has shown an undesired dose-dependent cytotoxicity which prevents this system for clinical studies. The first burden to overcome is therefore the design of polycationic dendrons which have no potential of toxicity effects. Polyethylene glycol (PEG) based nanocarriers with low hydrophobicity11 displayed for example low cytotoxicity as well as a reduction in systemic clearance with enhanced permeability and retention (EPR)12 for effective cancer chemotherapy. Polyamine-based carriers present positive charges at the physiological pH and they can bind polyanionic molecules such as nucleic acids through electrostatic interaction. In comparison to amines, guanidino groups are highly basic (pKa = 12.5) and they can be fully protonated under physiological pH. This feature renders polyguanidinium carriers13 highly positively charged and endows them with strong electrostatic interactions. This type of molecules can also form both ionic and hydrogen bonding interactions with the negatively charged carboxylates, phosphates and sulfates present in the lipid cell membrane bilayers. The bidentate hydrogen bond network formed by guanidinium groups is strong and it can facilitate cellular uptake of the carriers.14 For example, it has been shown that the capacity of a series of guanidinylated dendritic polymers to enter cells was dependent on the number of guanidinium groups at their surface.15 Furthermore, it has been recently reported that the internalization of dendrimers into cells is mainly regulated by the functional groups at their surface.16 Primary amino groups can be easily guanidinylated using 1H-pyrazole-1-carboxamidine hydrochloride reagent in one simple step.17 We have previously synthesized different generation adamantane-based dendrons as multivalent scaffolds that we called HYDRAmers.18 The adamantane core with a well-defined 3D conformation serves as a building block orienting four arms tetrahedrally into the space.19 This tripodal arrangement on a rigid molecule permits the introduction of additional functionality into the fourth bridgehead position without disturbing the geometry of adamantane-based scaffolds. These features give to the arborescent structures less steric hindrances between the attached entities and we might expect an improvement of multivalent ligand/receptor interactions of this novel type of dendron.3,18a,19 Here, we report the synthesis and the characterization of different generation polyammonium and polyguanidinium adamantane-based dendrons. Tri- and tetraethylene glycol chains were used as flexible, water compatible
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branching units and an alkyne group at the focal point was introduced to attach a fluorescent probe on the dendrons via click chemistry. Cellular investigations were carried out to determine the influence of the nature of the peripheral moieties and their number on cytotoxicity and cellular internalization of the different 1st and 2nd generation ammonium and guanidinium HYDRAmers. As the cellular interactions of dendrons have been shown to be dependent on the cellular substrate in exam,20 we decided to evaluate the behavior of our different HYDRAmers on both phagocytic RAW 264.7 murine macrophages and non-phagocytic HeLa epithelial human cells by flow cytometry and confocal laser scanning microscopy. None of these dendrons displayed significant cytotoxicity effects and they all had an effective cellular uptake depending of the generation and the nature of the peripheral groups.
Results and Discussion
Building blocks and HYDRAmer syntheses The detailed syntheses and characterization data for all compounds are provided in the Supporting Information (SI). In a previous work we have described a synthetic route to obtain the unsymmetrical tetrasubstituted adamantane starting from the inexpensive compound 1-bromoadamantane.18c Following this method, we prepared aminoadamantane-1,3,5-tricarboxylic acid in five steps with high yields. We chose this intermediate to exploit his functional groups for the construction of the dendritic HYDRAmers via a protection/deprotection strategy of the amines and carboxylic acids. We first esterified the tricarboxylic acid intermediate to form trimethyl aminoadamantane-1,3,5-tricarboxylate 1 with a yield of 79% (Scheme 1). At this point two different paths were followed for the protection of the free amine. The first one was the coupling between 5-hexynoic acid, activated with N-(3-dimethylaminopropyl)-N’ethylcarbodiimide hydrochloride (EDC×HCl) and 1-hydroxybenzotriazole (HOBt), and 1 affording compound 2 (65%). The alkyne moiety is available for click chemistry to attach the desired azide modified molecules to the dendrons. The second way was the introduction of a Cbz (carbobenzyloxy) protecting group on the amine using N-(benzyloxycarbonyloxy)succinimide and triethylamine (Et3N) to give compound 4 (56%) (Scheme 1). Basic hydrolysis of both compounds 2 and 4 afforded the corresponding tricarboxylic acids 3 and 5 in 93% and in 90% yield, respectively. The latter served as building blocks for the construction of the different generations of dendrons. All functionalized adamantane molecules were fully characterized by NMR, FT-IR, HPLC and mass spectrometry.
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Scheme 1. Building blocks synthesis O
O
HOOC HN
HN KOH MeOH/H2O
EDC/HOBt/DIEA dry DMF 65%
COOMe
93%
MeOOC
NH2
NH2
MeOOC
HOOC
COOH
HOOC
3
2 SOCl2/MeOH
HOOC HOOC
COOH
MeOOC
COOMe
79% MeOOC
1
NHCbz
Cbz-OSu/Et3N CH2Cl2 56%
MeOOC
NHCbz
KOH MeOH/H2O COOMe
90%
HOOC
COOH
HOOC
MeOOC
4
5
To synthesize the first generation dendrons (G1) we started from compound 3 on which we introduced the TEG chains to improve flexibility, biocompatibility and solubility in water of the final HYDRAmers. These spacers were prepared in large quantity starting from Boc-monoprotected diamines in one easy step.21 By coupling 3 activated with EDC×HCl and HOBt to {2-[2-(2-aminoethoxy)ethoxy]ethyl}carbamic acid tert-butyl ester we obtained G1 dendron-(NHBoc)3 6 (Scheme 2). The three amide bonds were formed with a yield of 87%. Then the three Boc protecting groups at the periphery of the dendron were easily removed using TFA to yield the desired compound G1 dendron-(NH3+)3 7 in a quantitative yield. The next step was the conversion of ammonium into Boc-protected guanidines. We used N,N’-di-Boc-1H-pyrazole-1-carboxamidine and N-diisopropylethylamine (DIEA) to produce the guanylation of three ammonium followed by purification via column chromatography on silica gel. We obtained G1 dendron-(NHC(NHBoc)NBoc)3 8 with a yield of 67% and then deprotection of Boc groups with TFA afforded the G1 dendron-(NHC(NH2)NH2+)3 9 quantitatively (Scheme 2). Triammonium (7) and triguanidinium (9) dendrons were fully characterized by NMR, FT-IR, HPLC and mass spectrometry (see SI for details).
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Scheme 2. First generation HYDRAmer synthesis O
O O
HN
HN
HN
O
H2N
NHBoc
O
HN O
6
HN
N
O
O
87%
O
quantitative
HN
O
O
O
TFA CH2Cl 2
O
quantitative
NH3
O
H2N
NH3
NH
NH2 H2N
COOH
NH2
H2N
NH2
+ 3 CF3COO
HOOC
3
O
O
HN O
HN
NH
+ 3 CF3COO
H2N
9 O
O 2)
NHBoc
HOOC
HN HN
67%
O
NH3
NHBoc
NHBoc
DIEA CH3CN/THF
O
O
O O
7
O
HN O
O
O O
O
NHBoc
N
HN HN
TFA CH2Cl2
HN
O
O
O
HN O
EDC/HOBt/DIEA dry DMF
NBoc
1)
O O
O
O
O
COOtBu
EDC/HOBt/DIEA dry DMF
HN O
O
HN O
10
HN HN
86%
O
KOH MeOH/H2O O
O O
72% O
O O
O O
O
O
O
O
O t BuOOC
11
HN HN
O O
O
O
HN O
O t BuOOC
HOOC
t BuOOC
HOOC HOOC
We then prepared the building blocks for the second generation dendrons (G2) starting again from compound 3. In this case, in order to speed-up the synthesis and being a triethylene glycol linker with a terminal ester group not commercially available, we used amino acid tert-butyl 12-amino-4,7,10-trioxadodecanoate. The amidation reaction between the tetraethylene glycol chains and 3 yielded G1 dendron-(COOtBu)3 10 (86%). Saponification of tert-butyl ester functions produced G1 dendron-(COOH)3 11 which is highly soluble in water. To remove the salts after the reaction, we used the desalting column SephadexTM G-10 and we obtained the tricarboxylic acids dendron 11 in 72% yield as shown in Scheme 2. Compound 5 with the Cbz protected amine served as the scaffold for the building of the “second layer” of G2 dendrons. We initially introduced the Boc-monoprotected diamine spacers on 5 by direct amidation to give G1 Cbz protected dendron-(NHBoc)3 12 in 89% yield (Scheme 3).
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Scheme 3. Second generation ammonium HYDRAmer synthesis NHCbz O
H2N
O
NHCbz
O
EDC/HOBt/DIEA dry DMF
NHCbz HOOC
NHBoc
O
O
HN O
TFA CH 2Cl2
12
HN
O
HN O
COOH
HN
O
89%
HOOC
quantitative
O
O
O
O
5
O
O
O
O
O
O
O
NHBoc
NHBoc
NH3
NH3
NHBoc
H 3N
O
O
O
H3N
O
O
HN
NH
H N
O
O
O
O
O
O
O
HN
N H HN
HN
O
O
O
O
O
NH 3
+ 3 CF3COO
O
H2 Pd/C MeOH
O HN
17 O
NH O
O
HN
O HN O
O O + 9 CF3COO NH3
O
O
NH3
O
2) TFA CH2Cl2
HN
51%
O HN O
H 3N O
O
O
HN O O
NH
HN O
NH2
1) 11 EDC/HOBt/DIEA dry DMF
O
O O
quantitative
O
O
H 3N
13
HN
HN
NH 3
O HN
O
O O
O O NH3
O
15
HN HN
O
O O
NHBoc
NHBoc NHBoc
NH3
In parallel, Boc deprotection afforded G1 Cbz protected dendron-(NH3+)3 13 quantitatively (Scheme 4). Guanylation of ammonium groups gave G1 Cbz protected dendron-(NHC(NHBoc)NBoc)3 14 (69%). Cbz group at the focal point of 12 and 14 (Boc protected dendrons) was selectively removed to liberate the amine available to react with carboxylic acids of the “first layer” 11. Cbz deprotection of 12 and 14 by hydrogenolysis using H2 and a catalytic amount of Pd/C produced G1 free amine dendron-(NHBoc)3 15 (Scheme 3) and G1 free amine dendron-(NHC(NHBoc)NBoc)3 16 (Scheme 4) in quantitative yield, respectively. Compound 15 was coupled to 11 by amidation and after the work-up the product was directly used for Boc deprotection with TFA giving G2 dendron-(NH3+)9 17 easily purified by preparative HPLC (51% overall yield) (Scheme 3). The same way was employed to synthesize G2 dendron-(NHC(NH2)NH2+)9 18 by coupling compound 16 to 11 followed by Boc deprotection and HPLC purification (53% overall yield) (Scheme 4). The second generation nonaammonium (17) and nonaguanidinium (18) dendrons were fully characterized by NMR, FT-IR, HPLC and mass spectrometry (see SI for details). All HYDRAmers 7, 9, 17 and 18 are highly soluble in water and their potential cytotoxic effects were subsequently evaluated (vide infra).
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To study the cell internalization capacity of our dendrons we linked the fluorescent probe cyanine 5 at their focal point. Cyanine 5 dye22 activated with N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) was initially coupled to 5-azidopentan-1-amine20a to give the modified Cy5-N3 19 purified by preparative HPLC (47%). This azide derivative was then conjugated to the HYDRAmers 7, 9, 17 and 18 bearing an alkyne group using “click” chemistry. Compound purifications by preparative HPLC yielded the fluorescent dendrons with cyanine 5 connected at the focal point via a triazole moiety (see SI for the molecular structures of the four dendrimers). Relatively good yields were obtained (38 to 41%) after HPLC purification.
Scheme 4. Second generation guanidinium HYDRAmer synthesis
NBoc NHCbz O
N N
O
HN O
NH2
O
DIEA CH3CN/THF
13
HN
NHCbz
NHBoc
O
HN O
14
HN HN
HN O
69%
O
O
O
H2 Pd/C MeOH
O
HN O
quantitative
O
O
O
O
O O
O
O
O
O
O
O
O
O NH3
NH3
O HN
NH BocHN
NH3
NH
NBoc
16
HN HN
BocHN
NBoc
HN
NH BocHN
BocHN
NBoc
NBoc
BocHN
NBoc
BocHN
NH NBoc
+ 3 CF 3COO NH2 H 2N N H
O O
H2N
N H2N H
O
O
H N
O
NH O O
HN O
O
O
1) 11 EDC/HOBt/DIEA dry DMF
O O
O HN
N H HN
O
HN
O
O
O
HN
53% O
O
18
O
O O
HN H2N NH
2) TFA CH2 Cl2
O
O
2
O
O
NH
HN O H2N
NH O
O
HN
O
HN
HN
HN O
NH
H2N
O
O
O HN
O O
O
H2N
NH NH2
HN H2N
O O
NH2 N H NH2
O
O
+ 9 CF3COO
O
O
NH
NH2
NH H2N
NH2
H2N
NH2
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Cellular viability The assessment of cellular toxicity represents an important issue towards the development of new types of vectors for drug delivery. The first step in our biological investigations was to determine the impact of 1st and 2nd generation ammonium and guanidinium HYDRAmers towards two cell lines in vitro: the phagocytic RAW 264.7 murine macrophages and the non-phagocytic HeLa epithelial human cells. For this purpose, both cell types were exposed to increasing concentrations of 1st and 2nd generation ammonium and guanidinium HYDRAmers ranging from 0.8 to 25 µM during 24 h. After incubation, the cellular viability was evaluated by flow cytometry using the double staining AnnV/PI (Figure 1). GUANIDINIUM
AMMONIUM 1st Generation – Dendron 7
1st Generation – Dendron 9
*** ***
100
% of Total
% of Total
100 75 50 25 0
*** ***
75 50 25 0
Control DMSO 25 µM 12.5 µM 6.3 µM 3.2 µM 1.6 µM 0.8 µM
Control DMSO 25 µM 12.5 µM 6.3 µM 3.2 µM 1.6 µM 0.8 µM
2nd Generation – Dendron 17
2nd Generation – Dendron 18
*** ***
100
% of Total
100
% of Total
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75 50 25
*** ***
*°
*°
**°
**°
**°
**
75 50 25 0
0 Control DMSO 25 µM 12.5 µM 6.3 µM 3.2 µM 1.6 µM 0.8 µM
Live Cells
Early Apoptotic Cells
Control DMSO 25 µM 12.5 µM 6.3 µM 3.2 µM 1.6 µM 0.8 µM
Late Apoptotic & Necrotic Cells Necrotic & Late Apoptotic Cells
Figure 1. Flow cytometry analysis of cellular viability in RAW 264.7 (empty bars) and HeLa (hatched bars) exposed to different concentrations (0.8 to 25 µM) of 1st and 2nd generation ammonium and guanidinium HYDRAmers. Two-ways ANOVA followed by Bonferroni’s post-test was performed to determine the statistical differences versus control cells (*p
