Polycationic adamantane-based dendrons of different generations display high cellular uptake without triggering cytotoxicity

Article abstract of DOI:10.1021/ja411987g

Dendrons used as synthetic carriers are promising nanostructures for biomedical applications. Some polycationic dendritic systems, such as the commercially available polyethylenimine (PEI), 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 synthesis of a novel type of polycationic dendrons that are able to penetrate into cells without triggering cytotoxic effects. We synthesized first- and second-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 of the dendrons penetrated through 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.

Full text of DOI:10.1021/ja411987g

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Article
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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Journal of the American Chemical Society
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Polycationic Adamantane-based Dendrons of Different Generations
Display High Cellular Uptake without Triggering Cytotoxicity
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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 eꢀmail: a.bianco@ibmcꢀcnrs.unistra.fr
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 adamantaneꢀbased 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 cytotoxꢀ
icity 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 peneꢀ
trated 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 wellꢀdefined particle size and shape are widely explored for biomedical appliꢀ
cations such as drug delivery, gene transfection, and imaging. Dendrons (wedgeꢀshaped dendrimer sections) have
been investigated as ideal nanoscale carrier molecules for the delivery of bioactive materials into the cells.1 Moꢀ
lecular engineering of these hyperbranched, monodisperse, wellꢀdefined structures can be easily performed using
simple organic synthesis.2 Multivalency constituted by the multiple surface groups at the periphery of a dendron
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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.
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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 problematꢀ
ic toxicity profiles8 strongly dependent on the concentration used.9 PEI (polyethylenimine), a commercially availꢀ
able polyammonium dendrimer,10 is one of the most efficient transfection agents. However, PEI has shown an
undesired doseꢀdependent cytotoxicity which prevents this system for clinical studies. The first burden to overꢀ
come 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 chemoꢀ
therapy.
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Polyamineꢀbased carriers present positive charges at the physiological pH and they can bind polyanionic molꢀ
ecules 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 carꢀ
boxylates, phosphates and sulfates present in the lipid cell membrane bilayers. The bidentate hydrogen bond netꢀ
work 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 internaliꢀ
zation of dendrimers into cells is mainly regulated by the functional groups at their surface.16 Primary amino
groups can be easily guanidinylated using 1Hꢀpyrazoleꢀ1ꢀcarboxamidine hydrochloride reagent in one simple
step.17
We have previously synthesized different generation adamantaneꢀbased dendrons as multivalent scaffolds that
we called HYDRAmers.18 The adamantane core with a wellꢀdefined 3D conformation serves as a building block
orienting four arms tetrahedrally into the space.19 This tripodal arrangement on a rigid molecule permits the introꢀ
duction of additional functionality into the fourth bridgehead position without disturbing the geometry of adaꢀ
mantaneꢀbased scaffolds. These features give to the arborescent structures less steric hindrances between the atꢀ
tached 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 polyguaniꢀ
dinium adamantaneꢀbased dendrons. Triꢀ 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 denꢀ
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drons via click chemistry.
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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 celluꢀ
lar substrate in exam,20 we decided to evaluate the behavior of our different HYDRAmers on both phagocytic
RAW 264.7 murine macrophages and nonꢀphagocytic HeLa epithelial human cells by flow cytometry and confoꢀ
cal 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.
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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 adamanꢀ
tane starting from the inexpensive compound 1ꢀbromoadamantane.18c Following this method, we prepared amiꢀ
noadamantaneꢀ1,3,5ꢀtricarboxylic 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 aminoadamantaneꢀ1,3,5ꢀtricarboxylate
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 5ꢀhexynoic acid, activated with Nꢀ(3ꢀdimethylaminopropyl)ꢀN’ꢀ
ethylcarbodiimide hydrochloride (EDC×HCl) and 1ꢀhydroxybenzotriazole (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
Nꢀ(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 generaꢀ
tions of dendrons. All functionalized adamantane molecules were fully characterized by NMR, FTꢀIR, HPLC and
mass spectrometry.
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Scheme 1. Building blocks synthesis
1
2
O
O
HOOC
EDC/HOBt/DIEA
3
4
5
6
HN
HN
KOH
MeOH/H2O
dry DMF
MeOOC
MeOOC
COOMe
HOOC
HOOC
COOH
93%
65%
7
NH2
NH2
8
9
3
2
SOCl2/MeOH
MeOOC
MeOOC
COOMe
HOOC
HOOC
COOH
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79%
NHCbz
NHCbz
KOH
MeOH/H2O
Cbz-OSu/Et3N
CH2Cl2
1
HOOC
HOOC
COOH
MeOOC
MeOOC
COOMe
56%
90%
5
4
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 spacꢀ
ers were prepared in large quantity starting from Bocꢀmonoprotected diamines in one easy step.21 By coupling 3
activated with EDC×HCl and HOBt to {2ꢀ[2ꢀ(2ꢀaminoethoxy)ethoxy]ethyl}carbamic acid tertꢀbutyl ester we
obtained G1 dendronꢀ(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 dendronꢀ(NH3 )3 7 in a quantitative yield. The next step was the conversion of ammonium into
Bocꢀprotected guanidines. We used N,N’ꢀdiꢀBocꢀ1Hꢀpyrazoleꢀ1ꢀcarboxamidine and Nꢀdiisopropylethylamine
(DIEA) to produce the guanylation of three ammonium followed by purification via column chromatography on
silica gel. We obtained G1 dendronꢀ(NHC(NHBoc)NBoc)3 8 with a yield of 67% and then deprotection of Boc
+
groups with TFA afforded the G1 dendronꢀ(NHC(NH2)NH2 )3 9 quantitatively (Scheme 2). Triammonium (7) and
triguanidinium (9) dendrons were fully characterized by NMR, FTꢀIR, HPLC and mass spectrometry (see SI for
details).
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Scheme 2. First generation HYDRAmer synthesis
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9
O
O
O
HN
HN
HN
NBoc
NHBoc
1)
O
O
O
O
O
O
N
O
O
HN
O
N
HN
HN
HN
O
NHBoc
O
HN
H2N
O
6
7
DIEA
CH3CN/THF
9
HN
HN
HN
EDC/HOBt/DIEA
dry DMF
TFA
CH2Cl2
O
HN
O
O
67%
O
O
O
O
O
O
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O
87%
quantitative
O
O
O
O
2)
TFA
CH2Cl2
O
O
quantitative
NH3
HN
H2N
O
NH3
NH
NH2
NH2
NHBoc
O
H2N
NHBoc
NH
NH2
NH3
HN
NHBoc
H2N
+ 3 CF3COO
HOOC
HOOC
COOH
+ 3 CF3COO
O
O
3
HN
HN
O
O
O
O
H2N
O
COOtBu
O
O
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EDC/HOBt/DIEA
dry DMF
O
O
HN
HN
10
HN
HN
11
KOH
MeOH/H2O
HN
HN
86%
O
O
O
O
O
O
O
72%
O
O
O
O
O
O
O
O
O
O
BuOOC
HOOC
t
BuOOC
HOOC
t
BuOOC
HOOC
t
We then prepared the building blocks for the second generation dendrons (G2) starting again from compound
. In this case, in order to speedꢀup the synthesis and being a triethylene glycol linker with a terminal ester group
3
not commercially available, we used amino acid tertꢀbutyl 12ꢀaminoꢀ4,7,10ꢀtrioxadodecanoate. The amidation
reaction between the tetraethylene glycol chains and 3 yielded G1 dendronꢀ(COOtBu)3 10 (86%). Saponification
of tertꢀbutyl ester functions produced G1 dendronꢀ(COOH)3 11 which is highly soluble in water. To remove the
salts after the reaction, we used the desalting column SephadexTM Gꢀ10 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 Bocꢀmonoprotected diamine spacers on 5 by direct amidation to give G1
Cbz protected dendronꢀ(NHBoc)3 12 in 89% yield (Scheme 3).
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Scheme 3. Second generation ammonium HYDRAmer synthesis
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9
NHCbz
HN
NHCbz
O
NHBoc
O
O
H2N
O
O
O
TFA
EDC/HOBt/DIEA
dry DMF
NHCbz
CH2Cl2
O
O
HN
HN
13
12
HN
HN
HN
HOOC
HOOC
COOH
quantitative
O
O
O
O
89%
O
O
O
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5
O
O
O
O
O
NHBoc
NH3
NHBoc
NH3
NHBoc
NH3
+ 3 CF3COO
H3N
O
O
O
O
O
HN
HN
O
HN
NH
O
O
O
H2
Pd/C
MeOH
O
O
H
O
O
N
quantitative
O
HN
N
H3N
H
HN
O
O
O
17
NH2
O
O
O
1) 11
EDC/HOBt/DIEA
dry DMF
O
O
O
O
O
O
H3N
O
O
HN
HN
15
O
O
NH
2) TFA
CH2Cl2
HN
O
NH
HN
O
O
O
O
O
O
51%
O
HN
O
O
HN
O
HN
O
O
O
HN
H3N
HN
O
O
NH3
O
O
NHBoc
NHBoc
O
O
O
+ 9 CF3COO
O
NHBoc
NH3
NH3
NH3
NH3
+
In parallel, Boc deprotection afforded G1 Cbz protected dendronꢀ(NH3 )3 13 quantitatively (Scheme 4).
Guanylation of ammonium groups gave G1 Cbz protected dendronꢀ(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 dendronꢀ(NHBoc)3 15 (Scheme 3) and G1 free amine
dendronꢀ(NHC(NHBoc)NBoc)3 16 (Scheme 4) in quantitative yield, respectively. Compound 15 was coupled to
11 by amidation and after the workꢀup the product was directly used for Boc deprotection with TFA giving G2
+
dendronꢀ(NH3 )9 17 easily purified by preparative HPLC (51% overall yield) (Scheme 3).
+
The same way was employed to synthesize G2 dendronꢀ(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, FTꢀIR, HPLC and mass spectrometry (see SI for details). All HYDRAmers 7, 9, 17 and 18 are highly soluꢀ
ble 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 foꢀ
1
cal point. Cyanine 5 dye22 activated with N,N,N′,N′ꢀtetramethylꢀOꢀ(Nꢀsuccinimidyl)uronium tetrafluoroborate
(TSTU) was initially coupled to 5ꢀazidopentanꢀ1ꢀamine20a to give the modified Cy5ꢀN3 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.
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3
4
5
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Scheme 4. Second generation guanidinium HYDRAmer synthesis
NBoc
NHCbz
NHCbz
HN
NH2
N
NHBoc
O
O
N
O
O
O
O
H2
O
O
Pd/C
MeOH
DIEA
CH3CN/THF
O
O
HN
HN
O
HN
HN
13
14
HN
16
HN
HN
HN
quantitative
69%
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
NH3
HN
NH3
NH
NBoc
NBoc
HN
NH
NBoc
NBoc
BocHN
BocHN
NH3
NH
BocHN
BocHN
NH
NBoc
BocHN
NBoc
BocHN
+ 3 CF3COO
NH2
H2N
N
O
H
O
O
HN
HN
O
NH
HN
O
O
O
O
11
1)
EDC/HOBt/DIEA
dry DMF
2) TFA
CH2Cl2
O
O
H
N
O
O
H2N
H2N
O
O
O
HN
N
H
N
H
53%
HN
O
O
O
O
18
O
O
O
O
HN
H2N
O
NH2
O
O
NH
HN
NH
O
O
O
O
O
O
HN
O
HN
O
HN
H2N
H2N
O
HN
NH
HN
O
O
NH2
N
O
O
O
H
NH2
O
O
O
+ 9 CF3COO
NH
NH
HN
H2N
NH2
NH
NH2
NH2
H2N
H2N
H2N
NH2
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1
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Cellular viability
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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 nonꢀphagocytic HeLa epithelial human cells.
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For this purpose, both cell types were exposed to increasing concentrations of 1st and 2nd generation ammoniꢀ
um and guanidinium HYDRAmers ranging from 0.8 to 25 ꢁM during 24 h. After incubation, the cellular viability
was evaluated by flow cytometry using the double staining AnnV/PI (Figure 1).
GUANIDINIUM
1st Generation – Dendron 9
AMMONIUM
1st Generation – Dendron 7
*** ***
*** ***
100
75
50
25
0
100
75
50
25
0
Control DMSO 25 ꢀM 12.5 ꢀM 6.3 ꢀM 3.2 ꢀM 1.6 ꢀM 0.8 ꢀM
Control DMSO 25 ꢀM 12.5 ꢀM 6.3 ꢀM 3.2 ꢀM 1.6 ꢀM 0.8 ꢀM
2nd Generation – Dendron 18
2nd Generation – Dendron 17
*** *** *°
*°
**°
**°
**°
**
*** ***
100
75
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0
100
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Control DMSO 25 ꢀM 12.5 ꢀM 6.3 ꢀM 3.2 ꢀM 1.6 ꢀM 0.8 ꢀM
Control DMSO 25 ꢀM 12.5 ꢀM 6.3 ꢀM 3.2 ꢀM 1.6 ꢀM 0.8 ꢀM
Early Apoptotic Cells
Late Apoptotic & Necrotic Cells
N
Live 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 ꢁM) of 1st and 2nd generation ammonium and guanidinium HY-
DRAmers. Twoꢀways ANOVA followed by Bonferroni’s postꢀtest was performed to determine the statistical difꢀ
ferences versus control cells (*p<0.05; **p<0.01; ***p<0.001) and to compare the four HYDRAmers to each
other (°p<0.05 versus G1 ammonium HYDRAmer).
First, we would like to notice that RAW 264.7 and HeLa had a different basal viability (82.6 % and 90.3 %,
respectively) (see SI Table S1) and were differently sensitive to our death positive control. Indeed, the remaining
viable cells following DMSO exposure were almost zero in RAW 264.7 (0.7%), while HeLa cells showed still
37.3% of living cells in the same conditions. This observation anticipates a major sensitivity of RAW 264.7 in
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comparison to HeLa cells, as further confirmed after incubation with 1st and 2nd generation HYDRAmers. In fact,
the first observation was that the cellular viability of murine macrophages was always below the cellular viability
of human cells in the same conditions (Figure 1; see SI Table S1).
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More in details, we could see that the RAW 264.7 cellular viability was affected neither by 1st and 2nd generaꢀ
tion ammonium (7 and 17) nor by 1st generation guanidinium (9) dendrons. Only the 2nd generation guanidinium
HYDRAmer (18) determined a slight but significant reduction of cellular viability of these cells (0.8 to 25 ꢁM).
Even though this decrease was not dramatic (~ 60 % of residual viable cells), this result leads to the consideration
that the 2nd generation guanidinium HYDRAmer affected macrophage viability more than their counter ammoniꢀ
um HYDRAmer (17) which displayed a remaining cellular viability of ~ 70%. This trend between ammonium and
guanidinium dendrons of the same generation was also present to a minor extent between dendron 7 (~ 80 % of
cellular viability) and dendron 9 (~ 75 % of cellular viability). Interestingly, another trend was also observed beꢀ
tween 1st and 2nd generations. Considering ammonium or guanidiunium compounds separately, we could observe
that 2nd generation HYDRAmers affected RAW 264.7 cellular viability to a major extent respect to their related 1st
generation.
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Overall, the cellular viability experiments allowed us to evidence a double trend in the impact of our HY-
DRAmers towards RAW 264.7 macrophages (see SI Table S1). First, ammonium dendrons (7 and 17) affected
less cellular viability than their counter guanidinium HYDRAmers (9 and 18, respectively). Second, considering
ammonium and guanidinium dendrons separately, the 2nd generation affected cellular viability more than the 1st
generation. These two considerations were fully represented by the significant difference of cellular viability beꢀ
tween RAW 264.7 treated with dendron 7 (ammonium 1st generation HYDRAmer) and those treated with dendron
18 (guanidinium 2nd generation HYDRAmer) at almost all the tested concentrations (1.6 to 25 ꢁM).
In contrast, none of the HYDRAmers had an impact on HeLa cell viability (Figure 1; see SI Table S1). Even at
the highest concentration (25 ꢁM), the viability was not affected neither by ammonium nor by guanidinium 1st
and 2nd generation dendrons.
In a more general perspective, our results showed that the 1st and 2nd generation ammonium and guanidinium
HYDRAmers had no remarkable effects on cellular viability of RAW 264.7 and HeLa cells. Indeed, even though
the 2nd generation guanidinium HYDRAmer showed a certain degree of toxicity on murine macrophages, the reꢀ
sidual living cells were still above 50 %. Moreover, it is important to note that, since other guanidinium denꢀ
drimers were shown to create reversible poration of the cellular membrane, this phenomenon could also be partly
responsible for the augmented percentage of AnnV and PI positive cells.20a
We would like to point out that the range of concentrations used herein was higher respect to the concentraꢀ
tions reported in the literature by others and evidencing cytotoxicity of polycationic dendrimers.8 Among polyꢀ
cationic compounds, PAMAM dendrimers are considered good candidates for gene delivery. Studies on the imꢀ
pact on cellular viability of different types of PAMAM dendrimers nevertheless demonstrated that these comꢀ
pounds triggered cellular death in a concentration and generation dependent way.8b Although some studies eviꢀ
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denced a good cytocompatibility of these polycationic systems at low concentration ranging from 100 nM to 1.5
ꢁM,16a other reports showed a significant cytotoxicity at concentrations higher than 500 nM.9d,20a,23,24
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Our results on cellular viability experiments on both RAW 264.7 and HeLa cells, together with the lack of proꢀ
inflammatory effect on macrophages (i.e. interleukin 6 production; data not shown), allow us to conclude that our
1st and 2nd generation ammonium and guanidinium HYDRAmers have a good biocompatibility compared to
PAMAM dendrimers, thus representing very promising molecules for their future development as drug or gene
delivery systems.
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Study of HYDRAmer cell uptake
Besides a good biocompatibility, another key characteristic for a promising drug/gene delivery system is its
capability to reach the intracellular compartments. Polycationic dendrimers have been shown to be differently
internalized based on the properties of their peripheral functions and on the type of cells under investigation.16a,b
In order to follow the cellular uptake of our 1st and 2nd generation ammonium and guanidinium HYDRAmers, we
used cyanine 5ꢀconjugated dendrons (see SI Scheme S2). RAW 264.7 and HeLa cells were exposed to 1st and 2nd
generation ammonium (20 and 22) and guanidinium (21 and 23) fluorescentlyꢀlabelled HYDRAmers (5 ꢁM) durꢀ
ing 2 h in the absence or in the presence of active cellular uptake inhibitors.
We initially assessed that our HYDRAmers were present inside both phagocytic RAW 264.7 and nonꢀ
phagocytic HeLa cells using flow cytometry (Figure S64 and Figure 2) and confocal microscopy (Figure 3).
Then, we evaluated the effect of two energyꢀdependent cellular uptake inhibitors, namely 4°C and NaN3, on the
internalization of our dendrons. The good cellular viability under inhibitory conditions was also ascertained (see
SI Figure S65).
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AMMONIUM
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Without Inhibitors
4 C
NaN3
9
1st Generation – Dendron 20
2nd Generation – Dendron 22
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GUANIDINIUM
100
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*
***
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***
Without Inhibitors
4 C
NaN3
1st Generation – Dendron 21
2nd Generation – Dendron 23
Figure 2. Quantification of the cellular internalization of ammonium and guanidinium Cy5ꢀlabeled HY-
DRAmers in RAW 264.7 (colored empty bars) and HeLa cells (colored hatched bars) by flow cytometry. The
cellular uptake of 1st and 2nd generation HYDRAmers was evaluated in the absence and in the presence of the inꢀ
hibitors of energyꢀdependent pathways of internalization (4 °C and NaN3). Oneꢀway ANOVA followed by Dunꢀ
nett’s postꢀtest was performed to determine the statistical differences between the data obtained with inhibitors
versus normal conditions (*p<0.05; **p<0.01; ***p<0.001).
As expected, under nonꢀinhibitory conditions phagocytic macrophages internalized a higher amount of HY-
DRAmers in comparison to nonꢀphagocytic cells. The mean fluorescence intensity (MFI) in RAW 264.7 was 1.7
to 2.6 times higher than that observed in HeLa cells (see SI Figure S64). Figure 2 shows flow cytometry analysis
of cellular uptake of dendrons 20ꢀ23 under inhibitory conditions, allowing us to further observe some differences
of internalization between the two types of cells and the different types of dendrons. In the case of macrophages,
we measured a significant reduction of the cellular internalization of the 1st and 2nd generation ammonium HY-
DRAmers (20 and 22) both at 4 °C and in the presence of NaN3, resulting in about 90 % and 70 % of internalizaꢀ
tion inhibition, respectively. Similarly, the cellular uptake of 1st and 2nd generation guanidinium (21 and 23) was
also significantly reduced by the inhibitory conditions. Only 12.9 % to 17.8 % of dendrons 21 and 23 were found
inside the cells at 4°C, while NaN3 was less effective in blocking cellular uptake (20.6 % for 21 and 39.3 % for
23).
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These results suggest that our HYDRAmers were mainly internalized via endocytosis. Nevertheless, up to 40
% of fluorescence was still registered under inhibitory conditions, evidencing that both active and passive pathꢀ
ways of internalization likely contributed to the cellular uptake of our dendrons. In addition, it seems that the
inhibitory conditions were slightly less effective in blocking the cellular uptake of guanidinium compounds reꢀ
spect to the ammonium HYDRAmers, especially considering the 2nd generation dendrons (23).
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5
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7
8
9
A similar behavior of our dendrons was confirmed by studies on HeLa cells. The incubation at 4 °C or with
NaN3 determined a significant reduction of the uptake of dendrons 20 and 22, but the decrease was smaller than
in the case of RAW 264.7 (see SI Table S1). In fact, cellular uptake of dendron 20 was reduced to 38.6 % while
cellular internalization of dendron 22 was 22.2 % of the nonꢀinhibitory condition during 4 °C incubation (respect
to ~ 10 % in RAW 264.7 for both dendrons). In the presence of NaN3, the residual internalization of dendron 20
and dendron 22 by HeLa was even greater: 67.8 % and 42.3 % of the normal condition, respectively.
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The inhibitory conditions did not significantly influence the cellular uptake of the 1st and 2nd generation guaniꢀ
dinium HYDRAmers. Only internalization of dendron 21 was affected at 4 °C, still allowing a detection of 55.1 %
of the initial fluorescence inside HeLa cells. A residual uptake of ~ 60 to ~ 80 % respect to the nonꢀinhibited conꢀ
dition was instead registered for dendron 21 in the presence of NaN3 and for dendron 23 both at 4°C and with
NaN3.
Confocal microscopy studies were performed to further investigate the cellular internalization of ammonium
and guanidinium 1st and 2nd generation HYDRAmers (Figure 3). The confocal microscopy observations were in
good agreement with the previous flow cytometry analysis under either normal or inhibitory conditions. But conꢀ
focal microscopy allowed also to assess other crucial endpoints such as the intracellular fluorescence intensity
and subcellular localization of the different HYDRAmers.
Similarly to the observations by flow cytometry under nonꢀinhibited conditions (see SI Figure S64), it was
possible to observe that 2nd generation ammonium and guanidinium HYDRAmers (22 and 23) were more internalꢀ
ized than their relative 1st generation dendron (20 and 21) in both types of cells. The other trend showing that
guanidinium compounds were more internalized respect to their counter ammonium HYDRAmers was also obꢀ
served in RAW 264.7 cells and, even better, in HeLa cells (Figure 3).
Considering confocal microscopy analysis under inhibitory conditions, we could draw two main conclusions.
First, it was possible to detect that a more intense fluorescence was present close to the cellular membrane, eviꢀ
dencing that our dendrons were somehow blocked to the surface of the cells without being able to be internalized.
Second, confocal microscopy allowed examining in higher details the subcellular localization of the HYDRAmers.
Independently of the conditions, our HYDRAmers were present into the cytoplasm of both RAW 264.7 and
HeLa cells but were never localized inside the nucleus. Under normal cellular uptake conditions, we could notice
that HYDRAmers were present in the cytoplasm, mainly as wellꢀdefined dotted red areas. This subcellular organiꢀ
zation of the internalized HYDRAmers supports an active mechanism of internalization leading to a preferential
phagosomal/endosomal localization of the different dendrons in both types of cells (Figure 3, Panel A and Panel
B, left column).25 The endosomes could be better distinguished in HeLa cells likely because, being nonꢀ
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phagocytic, they have lower endocytic rate and therefore a lower amount of endosomes/phagosomes is formed in
comparison to RAW 264.7. In the second case, the extremely high concentration of the phagosomal vesicles reꢀ
sulted in a dense fluorescence instead of wellꢀdefined vesicle localized signals. When inhibitory conditions were
applied, the cellular uptake of HYDRAmers was reduced but it was still possible to observe a diffused fluoresꢀ
cence inside the cells, confirming the previous flow cytometry results and demonstrating the contribution of a
passive pathway in the internalization of our dendrons.26
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Beyond the interest of understanding which mechanisms account for the cellular uptake of the different HY-
DRAmers, it is important to remember that their way of internalization can have a direct impact on the fate of the
loaded drugs and/or nucleic acids. In particular, if we consider the potential risk of inactivation of a therapeutic
agent by acid hydrolases from the endoꢀlysosomal compartments, adamantaneꢀbased drug/gene delivery systems
that can escape the endocytotic/phagocytic pathways would be of great interest. On the other hand, if active upꢀ
take is predominant, it will be necessary for the HYDRAmers, as suggested for other nanovectors, to evade from
the endosomes for their cargoes to exert their therapeutic activity.13b,21
Comparing the results on cellular viability and uptake between ammonium and guanidinium HYDRAmers and
between 1st and 2nd generation dendrons, their different behaviors are consistent with previous studies regarding
other polycationic dendrons and polymers.16a,b,26
As guanidinium is more basic than ammonium, its degree of protonation is higher. This property is hypotheꢀ
sized to be responsible for its capacity to form strong and stable bidentate hydrogen bonds with the negatively
charged cellular membrane components leading to an enhanced cellular internalization, a phenomenon called
adaptive translocation.14c,27 Once anchored to negatively charged cellular membrane components (e.g. phosphates,
sulfates, carboxylated), the actual polarity of the dendron changes allowing its penetration in the nonꢀpolar memꢀ
branes through adaptive nonꢀcovalent association with membrane constituents, finally sliding into the cytosol
under the influence of a membrane potential.14c The passive internalization of polycationic dendrons is then not
only correlated to the nature of the moieties they are bearing on their surface (with guanidinium better than amꢀ
monium) but also on the properties of the cellular membrane itself such as its composition and its potential.16a,28
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Figure 3. Intracellular localization of of 1st and 2nd generation ammonium and guanidinium HYDRAmers by
RAW 264.7 (A) and HeLa cells (B) using confocal microcopy. Cells were incubated with dendrons alone (Panel A
and Panel B, left column) or under conditions that inhibited the energyꢀdependent internalization, namely incubaꢀ
tion at 4°C (Panel A and Panel B, middle column) and NaN3 (Panel A and Panel B, right column). Dendron localꢀ
ization is evidenced in red, cellular membrane (CD11b labeling in RAW 264.7 or CD95 labeling in Hela cells) in
green and nuclei (DAPI staining) in blue. The left insets display both the dendron and the cellular membrane asꢀ
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sociated signals while the right insets show the overlay of the dendron, the cellular membrane and the nuclei asꢀ
sociated signals. The images represent one of at least five experiments with similar results.
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Moreover, it has also been demonstrated that the architecture of the dendrimer scaffold bearing the positively
charged moieties plays an important role in their interaction with cellular membrane, thus influencing the cellular
internalization.14a,b,29 As two negatively charged components of the cellular membrane are unlikely to be close to
each other due to the electrostatic repulsion, it has been demonstrated that the generation of the dendron also inꢀ
fluences its interaction with the cellular membrane.13b It has been evidenced that the optimal number of guanidiꢀ
um exposed on the periphery of the dendrons or polymers is 8 to 9,15b,30 corresponding to our 2nd generation guanꢀ
idinium HYDRAmer (23). Consistently with these earlier observations, this dendron was in fact the one that was
the most internalized by the cells suggesting that these HYDRAmers are able to strongly interact with cellular
membrane, likely supporting the subsequently enhanced active and passive cellular uptake.
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Conclusions
In this study, we described the synthesis and full characterization of a novel type of polycationic dendrons.
They are built from different adamantane scaffolds using a straightforward protection/deprotection strategy. Cyaꢀ
nine 5 dye was covalently linked to the target dendrons via click chemistry to obtain fluorescently labeled moleꢀ
cules for cell imaging. The cellular investigations allowed us to determine the influence of the nature of the peꢀ
ripheral moieties and their number on the cytotoxicity and the cellular internalization of 1st and 2nd generation
ammonium and guanidinium dendrons. Our studies revealed that the soꢀcalled HYDRAmers had an improved
biocompatibility respect to other polycationic dendrons proposed as potential drug/gene delivery systems. Furꢀ
thermore, our HYDRAmers were well internalized by both phagocytic and nonꢀphagocytic cells. The cellular upꢀ
take analyses permitted to evidence a combination of active and passive mechanisms of uptake, dependent on
cellular type, peripheral groups and dendron generation.
In comparison to other polycationic carriers, HYDRAmers do not show cytotoxicity despite the presence of
positive charges and their high cellular uptake. This feature provides to these adamantaneꢀbased dendrons a
strong potential for biomedical applications. Additional studies to assess the capacity of the HYDRAmers to comꢀ
plex nucleic acids and intracellularly deliver this type of therapeutic molecules are currently underway.
Acknowledgements
The authors acknowledge the CNRS (Centre National de la Recherche Scientifique). M. G. wishes to thank the
icFRC (International Center for Frontier Research in Chemistry) and Alsace Region for financing his PhD.
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Supporting Information Available
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Experimental Section including material and methods, details on the synthesis and characterization of all derivaꢀ
tives, precursors and final dendrons, NMR and MS spectra, cell culture protocols and flow cytometry analysis.
This information is available free of charge via the internet at http://pubs.acs.org.
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