Cationic poly(ester amide) dendrimers: Alluring materials for biomedical applications

Article abstract of DOI:10.1039/c8tb00639c

Novel cationic poly(ester amide) dendrimers have been synthesized by copper(i) azide-alkyne cycloaddition (CuAAC) of a tripropargylamine core and azide-terminated dendrons, in turn prepared by iterative amide coupling of the new monomer 2,2′-bis(glycyloxymethyl)propionic acid (bis-GMPA). The alternation of ester and amide groups provided a dendritic scaffold that was totally biocompatible and degradable in aqueous media at physiological and acidic pH. The tripodal dendrimers naturally formed rounded aggregates with a drug that exhibited low water solubility, camptothecin, thus improving its cell viability and anti-Hepatitis C virus (anti-HCV) activity. The presence of numerous peripheral cationic groups enabled these dendrimers to form dendriplexes with both pDNA and siRNA and they showed effective in vitro siRNA transfection in tumoral and non-tumoral cell lines.

Full text of DOI:10.1039/c8tb00639c

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Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
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DOI: 10.1039/C8TB00639C
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ARTICLE
Cationic poly(ester amide) dendrimers: alluring materials for
biomedical applications
Alexandre Lancelot,a‡ Rebeca González-Pastor,b,c‡ Rafael Clavería-Gimeno,c,d Pilar Romero,e Olga Abian,b,c,d,f Pilar Martín-
Duque,b,c* José L. Serrano,a* Teresa Sierra.e*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Novel cationic poly(ester amide) dendrimers have been synthesized by copper(I) azide-alkyne cycloaddition (CuAAC) of a
tripropargyl amine core and azide-terminated dendrons, in turn prepared by iterative amide coupling of the new
monomer 2,2’-bis(glycyloxymethyl)propionic acid (bis-GMPA). The alternation of ester and amide groups provided a
dendritic scaffold that was totally biocompatible and degradable in aqueous media at physiological and acidic pH. The
tripodal dendrimers naturally formed rounded aggregates with a drug that had low water solubility, camptothecin, thus
improving its cell viability and anti-Hepatitis C virus (anti-HCV) activity. The presence of numerous peripheral cationic
groups enabled these dendrimers to form dendriplexes with both pDNA and siRNA and these showed effective in vitro
siRNA transfection in tumoral and non-tumoral cell lines.
www.rsc.org/
applications. Indeed, these monodisperse macromolecules
have a globular shape that favours the presence of numerous
and available functional groups at their surface. These groups
can be easily modified with biological moieties, targeting
molecules or dyes, amongst other units, in order to turn a
dendrimer into a multifunctional platform that is able to
transport drugs and/or genes, which is useful from
investigation to clinical applications.7-11
Introduction
Synthetic vectors applied to drug delivery and gene therapy
offer new opportunities to cure diseases in very specific and
efficient way. The conjugation of drugs with a vector may
modify the pharmacokinetic profiles and improve various key
parameters, such as their bioavailability, biodistribution and
activity, thus limiting drug doses and undesired side effects.1-3
The delivery of exogenous genetic material into cells upon
conjugation with a vector may favour the production of a
missing protein, silence the expression of a deleterious gene or
specific cell suicide to cure the diseases in a highly precise
way.4,5 In order to perform these activities, the
biocompatibility and degradability in physiological media of
the synthetic vector is a crucial issue that must be considered
in their design.6
Commercially available poly(amidoamine) (PAMAM) and
poly(propylene imine) (PPI) dendrimers are the most
commonly used systems for these two applications. The
numerous nitrogen functional groups that make up the
dendrimers favour their entry into the cell cytoplasm, where
they can release their pharmaceutical cargo. These two
systems have demonstrated impressive abilities to improve
drug activity and to enhance gene transfection.12,13,14
Unfortunately, the weak biocompatibility and degradability of
these functional groups induce a cytotoxic effect that must be
tempered, usually by the addition of polyethylene glycol on
the dendrimer surface.15,16,17 Dendrimers based on oxygen
functions, such as 2,2’-bis(hydroxymethyl)propionic acid (bis-
MPA) polyester dendrimers18 or polyglycerol polyether
dendrimers,19 have also been investigated for the purposes
outlined above as they present excellent biocompatibility and
can be rapidly degraded in physiological media. However,
these materials generally show less marked biological
improvement than PAMAM and PPI derivatives, especially in
gene transfection.20
Among synthetic vectors, dendrimers show interesting
features that have prompted their development for biomedical
a Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, Spain
b Aragon Institute for Health Research (IIS Aragon), Zaragoza, Spain.
c Instituto Aragonés de Ciencias de la Salud (IACS), Zaragoza, Spain.
d Institute of Biocomputation and Physics of Complex Systems (BIFI), Joint Unit
IQFR-CSIC-BIFI, Universidad de Zaragoza, Spain
Instituto de Ciencia de Materiales de Aragón (ICMA), Facultad de Ciencias,
Universidad de Zaragoza-CSIC, Spain
f Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades
Hepáticas y Digestivas (CIBERehd), Barcelona, Spain.
g Fundación ARAID, Government of Aragon, Zaragoza, Spain,
‡ These authors contributed equally to the work. *Email - tsierra@unizar.es
Electronic Supplementary Information (ESI) available: [Detailed protocols carried
out for the anti-HCV studies and for the pDNA transfection. Synthesis and
characterization of the bis-GMPA monomer. Dendrons and dendrimers.
Biocompatibility of the dendrons and dendrimers and degradability of the
dendrons. Characterization of complexes formed between bis-GMPA dendrimers
and pEGFP. SI7. siRNA transfection efficacy of commercial reagents.]. See
DOI: 10.1039/x0xx00000x
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DOI: 10.1039/C8TB00639C
poly(ester amide)
architecture
dendrons
dendrons
spacer
core
spacer
core
+
D[G4]-(NH3
)
48
+
D[G3]-(NH3
)
24
Figure 1. 2,2’-bis(glyciloxy)propionic acid (bis-GMPA) dendrimers of 3rd and 4th generation.
In order to combine the advantages of nitrogen and oxygen poly(ester amide) dendrons with terminal amino groups. We
functions in the chemical structure of dendrimers, hybrid have previously reported the potential of glycyloxy amino-
structures containing ether and imine groups, such as terminated dendrons and dendrimers with
a bis-MPA
poly(propyl ether imine) (PETIM) dendrimers, have been polyester skeleton that formed biocompatible nanocarriers for
studied in both gene21 and drug delivery22 applications along drug delivery26,27 and/or gene transfection.20,28 The amino
with structures with ether and amide groups such as gallic groups corresponded in all cases to glycine moieties and these
acid-triethylene glycol (GATG) dendrimers.23 Although linear provided a dendritic scaffold that releases non-toxic glycine
poly(ester amide) polymers have shown interesting results for and bis-MPA molecules upon degradation. As a continuation of
biomedical applications,24 poly(ester amide) dendrimers are our investigation of such amino-terminated carriers, we have
still sporadic. The combination of the hydrolytic degradability implemented a synthetic procedure that involves iterative
of ester linkages and the stability and H-bond-forming ability amidation coupling reactions that lead to poly(ester amide)
of amido groups makes these polymers attractive candidates dendrons containing glycine and bis-MPA. The 3rd and 4th
to form synthetic vectors for drug and gene delivery. generation dendrons with 8 and 16 terminal ammonium
Furthermore, the inclusion of amino acids as a source of amino groups, respectively, were thus prepared and grafted onto a
groups imparts poly(ester amide)s with additional advantages tripropargyl amine core in order to obtain flexible tripodal
in terms of their biological properties. To our knowledge, only poly(ester amide) dendrimers (Figure 1). The biocompatibility
a poly(ester amide) dendrimer can be found in the literature, and degradability in aqueous media of such structures were
and this is based on glycine and bis-MPA monomer. This confirmed as along with their potential to deliver an anti-
material was synthesized by iterative esterification couplings hepatitis C virus (anti-HCV) drug with low-water soluble as well
and it has terminal hydroxyl groups, which help to improve the as plasmid deoxyribonucleic acid (pDNA) and small interfering
water solubility of glymepiride, an antidiabetic drug low- ribonucleic acid (siRNA).
water.25
We present here a novel 2,2’-bis(glycyloxymethyl)propionic
acid (bis-GMPA) monomer that allows the synthesis of
Results and discussion
2 | J. Name., 2012, 00, 1-3
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Synthesis and chemical characterization
ARTICLE
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DOI: 10.1039/C8TB00639C
A- Synthesis of bis-GMPA monomer
1) BnBr, KOH
2) Glyboc(OH),
DCC, DPTS
H2, Pd/C
AcOEt
96 %
62 %
bis-GMPA monomer
B- Synthesis of bis-GMPA dendrons
1st step (general procedure i):
1)
DCC, HOBt, DMAP
dry DCM/dry DMF, Ar
74 – 88 %
N3-[G2]-(NHBoc)4
2nd step (general procedure ii):
HCl,
EtOAC
81 – 93 %
+
N3-[G2]-(NH3
)
4
+
+
N3-[G3]-(NH3
)
N3-[G4]-(NH3
)
8
16
C- Synthesis of bis-GMPA dendrimers
1)
CuSO4·5H2O, L-Asc, TBTA
DMF, 45°C, Ar
2) HCl, EtOAc
78 – 85 %
Scheme 1. Synthesis of: (A) bis-GMPA monomer (Bn = benzyl), (B) Dendrons. The two-step procedure followed to grow the dendron generation is represented for the synthesis of
the 2nd generation dendron, N3-[G2]-(NH3+)4. The chemical structures of 3th and 4th generation dendrons used to synthesize the final dendrimers are represented. (C) Dendrimers.
This journal is © The Royal Society of Chemistry 20xx
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+
The 2nd, 3rd and 4th generation bis-GMPA dendrons were D[G4]-(NH3 )48, respectively, i.e., far below the value of the
synthesized starting from 6-azidohexyl bis(hydroxymethyl) recommended dietary allowance of copper.34
propionate, previously described by us.31 This compound was
DOI: 10.1039/C8TB00639C
functionalized with t-Boc-glycine moieties, as described for the
bis-GMPA monomer (Scheme 1A), and subsequent amino
group deprotection (see Scheme SI.3.2). The dendron
a)
generation was grown by employing repetitive amidation
coupling reactions using dicyclohexylcarbodiimide (DCC), 1-
hydroxybenzotriazole hydrate (HOBt) and 4-(dimethylamino)
pyridine (DMAP) with a mixture of dry dichloromethane and
dry dimethylformamide as solvent (Scheme 1B). Subsequent
deprotection of the terminal amino groups was carried out
under acidic conditions (3M HCl in ethyl acetate). Under these
conditions the terminal amines form the ammonium salt and
this precipitates from the reaction mixture. The precipitation
allows an easy purification process and protects the internal
amide and ester groups of the dendron from acidic hydrolysis.
This two-step growth procedure, i.e. amido coupling and
deprotection, gaved the products in yields between 62 and 78
%.
H-15
*
H-12
H-13
*
*
H-6,7
H-5,8
Finally, the tripodal dendrimers of 3rd and 4th generation,
+
+
D[G3]-(NH3 )24 and D[G4]-(NH3 )48
,
respectively, were
H-9
1H NMR dendron
obtained by coupling three dendrons on the tripropargyl
amine core by means of copper(I) azide-alkyne cycloaddition
(CuAAC) (Scheme 1C). Given the high conversion yields and the
low number of by-products generated, CuAAC “click-
chemistry” has been used for the synthesis of tripodal
dendrimers with tris(alkyne)-derived cores,32 but the
tripropargyl amino core has rarely been used.33 In order to
favour the complete functionalization of the core and impart
flexibility to the final dendritic structures, an hexamethylenic
chain was included as a spacer between the core and the bis-
GMPA dendron. This reaction was carried out with the t-Boc
protected dendrons in order to avoid undesired copper
complexation by the dendrons, which may lower the reaction
yields, and to simplify the purification of the dendrimers.
Copper(I) was prepared in situ by reduction of copper(II)
sulfate by (L)-sodium ascorbate and tris[(1-benzyl-1H-1,2,3-
triazol-4-yl)methyl]amine (TBTA), which was added to the
reaction mixture to increase its stability. Once again, the t-Boc
protective groups were removed under acidic conditions in
ethyl acetate to obtain defect-free bis-GMPA dendrimers of 3rd
and 4th generation, which contained 24 and 48 terminal
ammonium groups and 18 and 46 internal amide groups,
respectively, and these were obtained in yields of 85 and 78 %.
At the end of the synthesis, the amount of copper that might
have remained in the dendrimers, and which could provoke
cytotoxic bioaccumulation, was measured by inductively
coupled plasma atomic emission spectroscopy (ICP-AES). The
b)
bis-GMPA
dendron
*
H-1,4
1H NMR dendrimer
H-8
H-5
2.0
H,3, N-H
8.0
7.0
6.0
5.0
4.0
3.0
0
1.0
C-10,14
C-1,11
C-12
C-9,13
*
C-16[G0,1]
C-15
C-16[G2]
13C NMR
C-5,
6,7,8
dendrimer
C-4
C-3
C-2
100
180
160
140
120
100
80
0
60
40
20
c)
SEC
0,9
FTIR
d)
dendron
dendrimer
dendron
-N3 st
0,6
0,3
0,0
dendrimer
N-H+ st
C-H st
-O-C=O st
-N-C=O st
-N-H+
δ
13
18
retention time (min)
Figure 2. Chemical structures and characterization of the 3rd generation bis-GMPA
+
dendron, N3-[G3]-(NH3+)8, and dendrimer D[G3]-(NH3
)
24. a) 1H NMR of the bis-GMPA
dendron in CD3OD, b) 1H and 13C NMR of dendrimer D[G3]-(NH3
spectra of the dendron and the dendrimer and d) SEC chromatogram of the dendron
) in CD3OD, c) FTIR
24
+
amounts of copper that remained in the two final compounds
and dendrimer with terminal t-Boc groups. * asterisks indicate solvent signals.
+
were extremely low, 16 and 54
μ
g/g for D[G3]-(NH3 )24 and
4 | J. Name., 2012, 00, 1-3
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The 1H-NMR spectrum of the 3rd generation bis-GMPA
dendron, N3-[G3]-(NH3 )8, is depicted in Figure 2a as an
View Article Online
DOI: 10.1039/C8TB00639C
+
D[G3]
D[G4]
example to illustrate the correct synthesis of the dendron. The
signals corresponding to the glycine methylenic groups, H-15,
are observed as singlets between 3.6 and 4.0 ppm and the
oxymethylene protons, H-13, appear as ABq signals between
3.6 and 4.6 ppm. The signals corresponding to the 6-
azidohexyloxy chain are also observed at 1.43-1.69 ppm (H-5
to H-8) and at 4.16 ppm (H-9), except for the triplet
corresponding to the methylenic protons, H-4, which appears
overlapped with the signal of the solvent. Nevertheless, the
correlation of this triplet with the signal corresponding to its
vicinal methylenic protons H-5 could be observed in 1H-1H
COSY spectrum (see Figure SI.3.1). Additionally, the chemical
structure, monodispersity and purity of each dendron were
confirmed by Fourier transform infrared (FTIR) spectroscopy,
mass
spectrometry
(MALDI-TOF),
size
exclusion
chromatography (SEC) and elemental analysis (EA). Mass
spectrometry and SEC results for each dendron whose
terminal amino groups were protected by t-Boc are gathered
in the supporting information (see Figure SI.3.2).
Ctr D[G3] D[G4]
Ctr D[G3] D[G4]
+
+
Figure 3. Cytotoxicity of the dendrimers, D[G3]-(NH3
)
24
and D[G4]-(NH3
)
48,
on HeLa
and mMSCs. A. Cell viability after incubation with 1 mg/mL of bis-GMPA dendrimers
was analysed by the Alamar Blue® assay and the fluorescence was normalized to cells
receiving medium alone (** p < 0.01, ns not significant). B. Effect of 1 mg/mL of
exposure for 24 h on the phases of the cell cycle.
1
The H NMR, 13C NMR and FTIR spectra, as well as the SEC
chromatograms, of the 3rd generation tripodal dendrimer,
+
D[G3]-(NH3 )24
, are also depicted in Figure 2 as a
representative example. The correct synthesis of the
dendrimer was confirmed by the appearance of the signals
corresponding to the formation of the triazole ring between
the tripropargyl core and the dendrons, i.e., a peak at 8.42
ppm in the 1H NMR spectrum corresponding to the proton H-3
and two peaks at 128.7 and 137.9 ppm in the 13C NMR
spectrum corresponding to carbons C-3 and C-2, respectively.
Additionally, the signals corresponding to the methylenic
protons adjacent to the triazole ring, H-4 and H-5, were shifted
downfield, moving from 3.23 to 4.52 ppm and from 1.62 to
1.99 ppm, respectively. The protons of the tripropargyl amino
core appeared at 4.52 ppm in a merged signal. The correct
assignment of the proton and carbon signals was confirmed by
The biocompatibility of dendrons and dendrimers was studied
in tumoral cells (HeLa) and mesenchymal stem cells of mouse
origin (mMSCs) (Figure 3 and SI.5.1.1-2). Cell viability remained
constant for normal mMSCs (> 95%), whereas a decrease was
observed on tumoral HeLa cells between 24 h and 72 h. In any
case, all of the dendrons and dendrimers showed cell viability
values above 70% for the two cell lines for concentrations up
to 1 mg/mL, even after 72 hours (Figure 3A), thus confirming
their biocompatibility. Moreover, significant changes were not
found in cell cycle distribution (Figure 3B).
+
The degradability of the bis-GMPA dendrons, N3-[G2]-(NH3 )4,
+
+
N3-[G3]-(NH3 )8 and N3-[G4]-(NH3 )8 was studied by 1H NMR
experiments in aqueous buffer at pH = 7.4, i.e., physiological
pH, and at pH = 5.0, which corresponds to the pH observed in
the late endosomes. The most revealing zones of the proton
spectra corresponding to the degradation study of N3-[G2]-
1
1H-1H COSY and H-13C HSQC experiments (Figures SI.4.1. and
SI.4.2.). In the FTIR spectra, the band corresponding to the
azide group dissappeared after the CuAAC reaction.
Characteristic bands of ester, amide and protonated amino
groups were observed, respectively, at 1749, 1655 and as a
broad signal between 3600 and 2600 cm-1 (Figure 2C). The
monodispersity was confirmed by SEC, which provided
chromatograms in which the retention time decreased after
the CuAAC reaction and a residual peak corresponding to
uncoupled free dendrons could not be observed (Figure 2D
and Figure SI.4.3.).
+
1
(NH3 )4 at different times are shown in Figure 4. The H NMR
+
spectra for the degradation study of N3-[G3]-(NH3 )8 and N3-
[G4]-(NH3 )16 are gathered in Figures SI5.2.
+
At pH = 7.4, new signals near 3.6 ppm appeared over time.
These signals correspond to hydroxymethylenic protons, i.e. –
CH2OH, as described previously,31 and arise from the slow
hydrolysis of the external ester functions, which release
glycine units into the medium. This interpretation was
corroborated by the modifications observed near 3.84 ppm,
which are consistent with the appearance of methylenic
protons of free glycine. Additionally, the proton signals
corresponding to the CH2-OC(O) and NHC(O) groups at 4.20-
4.40 and 8.30 ppm, respectively, are modified during the
hydrolysis process. All of the changes outlined above occur
slowly since the signals become clearly visible between 2 and 7
days.
Biocompatibility and Degradability
As a first criterion to assess the potential of these cationic
poly(ester amide) dendrons and dendrimers for biomedical
applications, their biocompatibility and degradability were
assessed.
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tested in anti-hepatitis C virus (anti-HCV) experiments for the
dendrimer D[G3]-(NH3 )24 using camptothecin (CPT). Indeed,
CPT was previously identified as a potential inhibitor of the
virus replication during in vitro assays.35 Although CPT is highly
toxic, it has previously been reported that its encapsulation
within organic nanostructures can improve its anti-HCV activity
while preserving cell viability.27,36,37
View Article Online
DOI: 10.1039/C8TB00639C
+
hydrolysis
The procedure based on the solvent diffusion technique for
drug loading was employed to prepare dendrimer-drug
complexes with a ratio of 58 µmol CPT/mol dendrimer.
Although this drug content is not high, we found that in the
presence of dendrimer the CC50 referred to CPT was higher
+
pH = 7.5
than 1
M, compared to 0.5
M for free CPT. At the same
time, viral inhibition at a 0.01
M concentration of CPT
increased from 50% using free CPT to 80% for
+
D[G3](NH3 )24/CPT (Figure 5). These data showed that CPT was
more active and less cytotoxic when offered to infected cells
associated with the dendrimer.
The dendrimer-drug system was studied by TEM (Figure 5) and
this showed rounded objects with a mean size of 21 ± 5 nm,
which is twice the size of the nanoobject formed by the empty
dendrimer (10 ± 3 nm). Furthermore, Isothermal Titration
Calorimetry (ITC) measurements showed that CPT-dendrimer
association is energetically favoured with an association
CH2-OC(O)
CH2-NHC(O)
CH2-OH
NHC(O)
pH = 5
constant of Ka = 4.1 ± 0.6
moderate to high Gibbs energy of interaction (
kcal
mol-1) dominated by the entropic contribution (–T
9.9 ± 0.9 kcal
mol-1), with an unfavourable interaction enthalpy
H= 0.9 ± 0.1 kcal
mol-1). This could mean that drug and

106 M-1. This value corresponds to a
G= -8.9 ± 0.8
S= –




(

dendrimer binding is led by non-specific interactions (mainly
hydrophobic forces), which is consistent with the drug being
encapsulated within the dendritic nano-objects observed by
TEM, and this allows its cell viability and antiviral activity to be
enhanced.
CH2-OC(O)
CH2-NHC(O)
CH2-OH
NHC(O)
Figure 4. Degradation of the bis-GMPA dendron N3-[G2]-(NH3+)4 in aqueous buffers at
pH = 7.4 and 5.
At acidic pH (i.e. 5.0), the degradation of the dendrons by
hydrolysis was clearly accelerated and, after the first day,
signals at around 3.6 ppm, corresponding to CH2OH protons,
were visible. All of the peripheral glycine moieties were then
totally released after one week. After this first step, the
dendron degradation continued, as evidenced by the
appearance of peaks with lower intensity close to 3.78 ppm
and by the modifications of the remaining signals close to 4.25
ppm (CH2-OC(O) area) and 8.15 ppm (NHC(O) area).
Figure 5. Evaluation of viral load reduction (lines) of HCV replicon system in human
hepatoma cells (Huh5-2) with cell growth (bars) while increasing compound
Drug delivery
+
+
concentration and TEM images of D[G3](NH3
free CPT (grey) is represented as a control in D[G3](NH3
)
24/CPT (red) and D[G3](NH3
)
(green);
24
+
)24/CPT graph. *UTC:
The possibility of using these poly(ester amide) dendrimers as
carriers to improve the activity of small drugs was initially
untreated controls.
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pDNA and siRNA dendriplexes formation and transfection
and siRNA complexation was not observed for VtiehweArstimcleaOllnelinset
dendrimer. Accordingly, the number ofDpOeI:r1i0p.h10e3r9a/lCa8TmBi0n0e6s39iCn
the dendrimer seems to be crucial to enhance good
complexation abilities at low ratios.42 For these reasons,
The potential of the novel cationic bis-GMPA poly(ester amide)
dendrimers to be used as non-viral gene vectors was
investigated with pDNA (pEGFP) and siRNA (siGFP and siLuc).
GFP and firefly luciferase are widely used as reporters to study
gene expression. The GFP system is based on the production of
a green fluorescence protein that can be detected after its
transfer, and the luciferase system is based on
bioluminescence, where the enzyme luciferase catalyses a
two-step reaction in which oxidation of D-luciferin yields light.
Although it is more common to use the transfer of pDNA
encoding a protein that is missing or defective into a patient’s
cells, in the last few years there has been considerable interest
in the use of short double-stranded RNAs in gene therapy, as
there is evidence for the inhibition of proliferation of tumour
cells by sequence-specific silencing of factors that support
tumour growth or facilitate the generation of chemotherapy-
resistant cells.38-41
+
D[G4]-(NH3 )48 was chosen to explore further the transfection
efficiency of its corresponding dendriplexes with pEFGP and
siGFP.
+
The correct formation of the dendriplexes with D[G4]-(NH3 )48
and pDNA was confirmed by AFM (Figure 6B). At low ratios
(w/w 1/1), free pDNA appears as fibres with a low height
(around 0.7 nm), whereas at higher ratios (w/w 10/1) these
fibres are no longer observed and only rounded dendriplexes
are visible. This is consistent with the results obtained during
gel electrophoresis experiments (Figure 6A).
Morphological studies of the dendriplexes were carried out by
cryoTEM at different w/w ratios (Figure 7 and Table 1), and
their hydrodynamic size and superficial charge were measured
+
by DLS and
ζ
potential measurements (Table 1). D[G4]-(NH3 )48
forms spherical objects when mixed with pDNA. the addition
of further quantities of dendrimer (ratio 100/1) allows the
formation of smaller and less polydisperse dendriplexes. When
mixed with siRNA, spherical dendriplexes were observed at the
lowest ratio (50/1). The addition of a larger quantity of
dendrimer (ratio 100/1) triggered the formation of bigger and
more elongated objects. All of the dendriplexes showed a
positive superficial charge that increased on the addition of
dendrimer.
A
+
D[G3]-(NH3
)
24
+
D[G4]-(NH3 )48
pDNA 10/1
200 nm
pDNA 100/1
200 nm
+
D[G3]-(NH3 )24
+
D[G4]-(NH3 )48
siRNA 50/1
100 nm
siRNA 100/1
200 nm
Figure 6. A. Electrophoretic mobility of free and complexed pDNA and siRNA in SFM.
Gel retardation of pDNA (up) and siRNA (down) in the presence of increasing
+
Figure 7. CryoTEM images of the dendriplexes formed by D[G4]-(NH3
with pDNA or siRNA.
) when mixed
48
+
+
+
concentrations of D[G3]-(NH3
)
and D[G4]-(NH3
)
48
(D[G3]-(NH3 )24, 6256 g/mol, 24
24
N+/molecule; D[G4]-(NH3
)
48, 12671 g/mol, 48 N+/molecule; pEGFP, 3.12·106 g/mol,
+
Table 1. Diameter (DH) and ζ potential values of the dendriplexes.
9400 P-/molecule; siGFP 14520 g/mol, 44 P-/molecule). B. AFM images of dendriplexes
+
formed between D[G4]-(NH3
)
and pDNA at 1/1 and 10/1 w/w ratios. Free pDNA
48
DH (nm)
cryoTEM
ζ potential
(mV)
chains are highlighted with green arrows.
Dendriplexes
DLS
+
D[G4]-(NH3 )48/pDNA
57 ± 4
30 - 70
Gel electrophoresis results (Figure 6A) showed differences in
+ 12
+ 23
+ 19
+21
10/1
244 ± 21
120 - 200
the complexation capacity of the two dendrimers, with D[G4]-
+
D[G4]-(NH3 )48/pDNA
+
85 ± 22
63 ± 9
30 - 120
35 - 60
50 -120
(NH3 )48 being the most efficient to spontaneously form
100/1
dendriplexes with either pDNA or siRNA. As for pDNA
complexation, a w/w ratio of 5/1 for pDNA (N/P ratio 6.3) was
+
D[G4]-(NH3 )48/siRNA
50/1
+
observed whereas the ratio was 50/1 for D[G3]-(NH3 )24. As for
+
D[G4]-(NH3 )48/siRNA
179 ± 29
siRNA complexation, a w/w ratio of 80 for pDNA (N/P ratio
100/1
+
100/1) was deduced for the largest dendrimer, D[G4]-(NH3 )48
,
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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Journal of Materials Chemistry B
Page 8 of 14
ARTICLE
Journal Name
reduction of Luciferase on SKOV3-Luc), although not as
View Article Online
DOI: 10.1039/C8TB00639C
significant as the reduction achieved with Lipofectamine (79%
GFP reduction on MDCK-GFP and 89% reduction of Luciferase
on SKOV3-Luc).
+
D[G4]-(NH3
)
48
Figure 8. A. Confocal z-stack projections and corresponding lateral views of the
internalization with pEGFP-Cy5 after 4 h incubation in SFM. Arrows indicate the
positions of dendriplexes. Nuclei were labelled with DAPI (light blue), actin filaments
with AlexaFluor 488-Phalloidin (green) and pEGFP with Cy5 (dark blue). Scale bars: 20
μm. B. EGFP expression after 48h transfection with D[G4]-(NH3+)48 (scale = 100 µm).
Figure 9. A. Reduction of GFP expression on MDCK-GFP cells after 72h of transfection
+
+
with D[G4]-(NH3
) (compared to control cells with 100% expression of GFP). Scale =
48
The efficacy of pEGFP transfection using D[G4]-(NH3 )48 as a
500 µm. B. Relative quantification of the reduction of Luciferase expression in SKOV3-
vector was explored on HeLa cells. The internalization of the
dendriplexes was first observed by confocal microscopy after 4
h incubation (Figure 8A). The majority of the complexes
(identified with an arrow) were found either at, or passing
through the membrane and few were observed on the
cytoplasm, indicating a limited internalization. Consequently,
+
Luc after 72h of transfection with D[G4]-(NH3
) .
48
Experimental
Materials
+
transfection levels with D[G4]-(NH3 )48 remained low (Figure
Unless otherwise stated, the reagents used for the synthesis of
the bis-GMPA dendrimers were purchased from Sigma-
Aldrich® or Acros OrganicsTM and were used without further
purification. Solvents were purchased from Sigma-Aldrich® or
Scharlab, S.L.; dichloromethane was distilled prior to use as a
reaction solvent. Camptothecin (CPT) was purchased from
Sigma-Aldrich®. Enhanced green fluorescent protein DNA
plasmid (pEGFP 4733bp) was obtained from BD Biosciences
Clontech®. siRNA duplex targeting green fluorescent protein
(siGFP) sequence (GFP Duplex I, 5'-GCA AGC UGA CCC UGA
AGU UC-3') and siRNA against luciferase (siLuc) (anti Luciferase
I, 5'-GAU UAU GUC CGG UUA UGU AUU-3') were obtained
from Thermo Scientific Dharmacon.
8B), with no significant differences on increasing the D[G4]-
+
(NH3 )48/pEGFP ratios. Nevertheless, although
a smaller
proportion of cells was transfected at a ratio of 100/1, the
level of EGFP expression per cell was higher than that obtained
at a ratio of 10/1. This result could be related to the rather
large size distribution of the complexes.
More interestingly, the efficacy of dendriplexes formed
+
between D[G4]-(NH3 )48 and siRNA to reduce specific protein
expression was assessed in two different cell lines stably
transformed to express an exogenous protein, i.e., a non-
tumoral cell line expressing GFP (MDCK-GFP) and a tumoral
cell line expressing luciferase (SKOV3-Luc) (Figure 9). siRNA
transfection experiments gave positive results on both cell
+
lines, with D[G4]-(NH3 )48/siGFP at a 50/1 ratio (w/w) showing
Synthesis
the highest efficacy with a 12% reduction on GFP expression
on MDCK-GFP and a 22% reduction on luciferase expression on
SKOV3-Luc. In both cases, cell viability after transfection was
high, thus proving that the reduction of the protein expression
was due to siRNA transfection and not to cell death. The values
obtained for dendriplexes at a 50/1 ratio were higher than
those found for dendriplexes at a 100/1 ratio, even though not
Fully details of the synthesis and complete characterization of
all the materials are provided in the supplementary
information (SI.3 and SI.4). The general procedures carried out
for the synthesis of the bis-GMPA dendrons and dendrimers
and their chemical characterization are described below.
General procedure i): generation growth amidation reaction.
The ammonium salt of the corresponding starting dendron
+
all of the siRNA was complexed with D[G4]-(NH3 )48 at the
(1.00 mol) was dissolved in
a
mixture of dry
50/1 ratio. It can be deduced that spherical complexes with a
smaller size, close to 60 nm, could be better internalized,
which in turn favours a more efficient gene transfection.
Compared with commercial transfection agents, these
transfection efficacies were similar to those obtained with
FuGene (10% GFP expression reduction on MDCK-GFP and 13%
dimethylformamide and dry dichloromethane and the bis-
GMPA monomer (1.20, 1.50 and 1.75 mol per amino group
according to the dendron generation), HOBt (1.20, 1.50 and
1.75 mol per amino group according to the dendron
generation) and DMAP (1.4 mol per amino group) were added.
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 9 of 14
Journal of Materials Chemistry B
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Journal Name
ARTICLE
The reaction mixture was stirred under argon and cooled down CH2–, –NHCO2C(CH3)3), 1.61 (m, 2H, N3–CH2–CHV2i–ew),A1rti.c6le6On(lmine,
DOI: 10.1039/C8TB00639C
to 0 °C. DCC (1.20, 1.50 and 1.75 mol per amino groups 2H, –CH2–CH2–OCO–), 3.27 (t, 2H, J = 6.8 Hz, N3–CH2–), 3.88
according to the dendron generation) was dissolved in dry (m, 32H, –CH2N–[G4]), 3.98 (m, 28H, –CH2N–[G1,2,3]), 4.11 (t, J
dichloromethane and was added drop wise to the reaction = 6.4 Hz, 2H, –(CH2)5–CH2–OCO–), [4.20–4.45] (m, 60H, –CH2O–
mixture. The mixture was stirred at room temperature under [G1,2,3,4]), 5.55 (bs, –NHBoc), 7.19 (bs, –NHCO2–). 13C NMR
argon for 24 h. The white precipitate, N,N’-dicyclohexylurea (100 MHz, CDCl3)
δ (ppm): [17.7–18.0], 25.3, 26.3, 28.3, 28.6,
(DCU), was filtered off and the solvent was evaporated under 41.3, 42.3, [46.0–46.1], 51.3, 65.3, [66.3–66.5], 80.0, 156.1,
vacuum to give an orange oil. The remaining DCU and HOBt 169.6, [170.2–170.3], 172.6, [172.9–173.2]. MS (MALDI+, DCTB)
were precipitated in a mixture of hexane and ethyl acetate and m/z (%): Found: 5220.2 (100); Calcd. for [C221H351N33O108,Na]+:
filtered off. The organic phase was washed 3 times with brine, 5218.3. FTIR (νmax/cm–1, ATR): 3362 (N–H st), 2978–2930–2852
dried over anhydrous MgSO4 and the solvent was evaporated. (C–H st), 2098 (N3 st), 1749 (C=O ester st), 1695 (C=O
The remaining HOBt was precipitated into cold ethyl acetate (- carbamate st), 1668 (C=O amide st), 1520 (N–H δ), 1456 (CH2,
16ºC) and filtered off. The solvent was evaporated under CH3 δ), 1367 (C–N st), 1284 (CO–O st), 1155 (N–CO–O st). EA
reduce pressure. The crude product was purified on silica gel (%): Found: C, 52.2; H, 7.1; N, 8.8; Calcd. for C221H351N33O108: C,
(eluting solvent is detailed with the chemical characterization 51.1; H, 6.8; N, 8.9. SEC (ref PMMA): Mw 4463 g.mol–1; Ð: 1.04.
of each product) to obtain a white product. In the case of the General procedure ii): deprotection of the terminal amino
3rd generation, the crude product was precipitated in a mixture groups. t-Boc protected dendron (1 mol) was dissolved in ethyl
of hexane and ethyl acetate before purification by flash acetate. A saturated solution of HCl in ethyl acetate was
chromatography on silica gel (yields: 74 – 88%).
carefully added to it. The reaction mixture was stirred at room
(ppm): 1.27 (s, temperature during 1 hour and a white precipitate formed.
1
N3-[G2]-(NHBoc)4. H NMR (400 MHz, CDCl3)
δ
3H,
-CH3[G1]),
1.29
(s,
6H, The reaction mixture was diluted in ethyl acetate and was
–CH3[G2]), 1.41 (m, 4H, –CH2–CH2–CH2–CH2–), 1.45 (s, 36H, – stirred for an additional 30 min. The mixture was stirred under
NHCO2C(CH3)3), 1.62 (m, 2H, N3–CH2–CH2–), 1.67 (m, 2H, –CH2– vacuum to remove the hydrochloric acid vapours. The white
CH2–OCO–), 3.29 (t, 2H, J = 6.8 Hz, N3–CH2–), 3.91 (d, J = 5.6 residue was recovered by centrifugation and was washed
Hz, 8H, –CH2N–[G2]), 3.98 (d, J = 5.2 Hz, 4H, –CH2N–[G1]), 4.15 twice with pure ethyl acetate (yields: 81 – 93%).
+
1
(t, J = 6.4 Hz, 2H, –(CH2)5–CH2–OCO–), [4.17–4.38] (m, 10 H, – N3-[G2]-(NH3 )4. H NMR (400 MHz, CD3OD)
CH2O–[G1,2]), 5.29 (bs, –NHBoc), 7.08 (bs, –NHCO2–). 13C NMR 3H, –CH3[G1]), 1.37 (s, 6H, –CH3[G2]), 1.43 (m, 4H, –CH2–CH2–
(100 MHz, CDCl3) (ppm): 17.8, 18.1, 25.4, 26.3, 28.3, 28.7, CH2–CH2–), 1.62 (m, 2H, N3–CH2–CH2–), 1.69 (m, 2H,
δ (ppm): 1.27 (s,
δ
41.0, 42.4, 46.1, 46.3, 51.3, 64.9, 65.3, 66.5, 80.1, 155.9, 169.6, –CH2–CH2–OCO), 3.93 (m, 8H, –CH2N–[G2]), 3.96 (m, 4H, –
170.3, 172.5, 172.8. MS (MALDI+, DIT) m/z (%): Found: 1256.8 CH2N–[G1]), 4.16 (t, J = 6.4 Hz, 2H, –(CH2)5–CH2–OCO–), 4.32
(100); Calcd. for [C53H87N9O24,Na]+: 1256.6. FTIR (νmax/cm–1, (ABq, J = 11.6 Hz, ΔνAB = 9.4 Hz, 4H, –CH2O–[G1]), 4.45 (ABq, J
ATR): 3379 (N–H st), 2980–2943–2864 (C–H st), 2098 (N3 st), = 11.2 Hz, 8H, –CH2O–[G2]); note: N3–CH2– signal (~3.2 ppm) is
1742 (C=O ester st), 1699 (C=O carbamate st), 1676 (C=O overlapped with the signal of CH3OD, nevertheless the
amide st), 1520 (N–H δ), 1458 (CH2, CH3 δ), 1367 (C–N st), 1246 correlation between N3–CH2–CH2– and N3–CH2–CH2– is
(CO–O st), 1155 (N–CO–O st). EA (%): Found: C, 52.2; H, 7.5; N, observed in 1H-1H COSY as well as the correlation between N3–
1
10.1; Calcd. for C53H87N9O24: C, 51.6; H, 7.1; N, 10.2. SEC (ref CH2– and N3–CH2– is observed in H-13C HSQC. 13C NMR (100
PMMA): Mw 1581 g.mol–1; Ð: 1.02.
N3-[G3]-(NHBoc)8. H NMR (400 MHz, CDCl3)
MHz, CD3OD)
δ (ppm): 17.8, 18.1, 26.5, 27.3, 29.4, 29.7, 41.1,
1
δ (ppm): 1.25 (s, 42.1, 47.3, 47.6, 52.3, 66.6, 67.2, 68.5, 168.2, 170.9, 174.0,
6H, –CH3[G2]), 1.26 (s, 3H, –CH3[G1]), 1.28 (s, 12H, –CH3[G3]), 174.9. MS (MALDI+, DIT) m/z (%): 834.7 (100); Calcd. for
1.44 (m, 76H, –CH2–CH2–CH2–CH2–, –NHCO2C(CH3)3), 1.61 (m, [C33H55N9O16,Na]+: :834.4. FTIR (νmax/cm–1, ATR): 3600–2600 (bs
2H, N3–CH2–CH2–), 1.66 (m, 2H, –CH2–CH2–OCO–), 3.27 (t, 2H, N–H+ st), 3222 (N–H st), 2941–2861 (C–H st), 2098 (N3 st), 1747
J = 6.8 Hz, N3–CH2–), 3.90 (d, J = 5.2 Hz, 16H, –CH2N–[G3]), 3.95 (C=O st ester), 1662 (C=O st amide and N–H+ δ), 1541 (N–H
δ),
(d, J = 5.2 Hz, 4H, –CH2N–[G1]), 4.00 (d, J = 5.2 Hz, 8H, –CH2N– 1478 (CH2, CH3 δ), 1407 (C–N st), 1246 (νas CO–O), 1138 (O–C–C
[G2]), 4.13 (t, J = 6.4 Hz, 2H, –(CH2)5–CH2–OCO–), [4.20–4.38] st). EA (%): Found: C, 39.9; H, 6.5; N, 12.9. Calc. for
(m, 28H, –CH2O–[G1,2,3]), 5.42 (bs, –NHBoc), 7.19 (bs, – C33H59Cl4N9O16: C, 40.5; H, 6.1; N, 12.9.
+
1
NHCO2–). 13C NMR (100 MHz, CDCl3)
δ (ppm): 17.7, 18.0, 18.3, N3-[G3]-(NH3 )8. H NMR (400 MHz, CD3OD) δ (ppm): 1.28 (s,
25.3, 26.2, 28.3, 28.6, 41.3, 42.3, [46.0–46.1], 51.2, 65.2, 66.4, 3H, –CH3[G1]), 1.33 (s, 6H, –CH3[G2]), 1.38 (s, 12H, –CH3[G3]),
80.0, 156.0, 170.6, 172.5, 172.8. MS (MALDI+) m/z (%): Found: 1.43 (m, 4H, –CH2–CH2–CH2–CH2–), 1.62 (m, 2H, N3–CH2–CH2–),
2578.7 (100); Calcd. for [C109H175N17O52,Na]+: 2578.7. FTIR 1.69 (m, 2H, –CH2–CH2–OCO), 3.93 (s, 16H, –CH2N–[G3]), 3.97
(
νmax/cm–1, ATR): 3356 (N–H st), 2978–2928–2854 (C–H st), (s, 4H, –CH2N–[G1]), 4.01 (s, 8H, –CH2N–[G2]), 4.16 (t, J = 6.4
2106 (N3 st), 1742 (C=O ester st), 1699 (C=O carbamate st), Hz, 2H, –(CH2)5–CH2–OCO–), 4.32 (m, 14H, –CH2O–[G1,2]), 4.45
1664 (C=O amide st), 1528 (N–H (ppm):
), 1458 (CH2, CH3 δ), 1367 (C– (ABq, 16H, –CH2O–[G3]). 13C NMR (100 MHz, CD3OD)
δ
δ
N st), 1250 (CO–O st), 1157 (N–CO–O st). EA (%): Found: C, 17.8, 18.1, 26.6, 27.4, 29.4, 29.8, 41.2, 42.2, 47.4, 47.7, 52.4,
51.3; H, 7.3; N, 9.6; Calcd. for C109H175N17O52: C, 51.2; H, 6.9; N, 66.5, 67.2, 67.8, 68.5, 168.3, 170.8, 171.1, 174.1, 175.0, 175.2.
9.3. SEC (ref PMMA): Mw 2966 g.mol–1; Ð: 1.02.
FTIR (νmax/cm–1, ATR): 3600–2600 (bs N–H+ st), 3342 (N–H st),
(ppm): [1.24– 2984–2928 (C–H st), 2091 (N3 st), 1755 (C=O st ester), 1659
1
N3-[G4]-(NHBoc)16. H NMR (400 MHz, CDCl3)
δ
1.28] (m, 45H, –CH3[G1,2,3,4]), 1.45 (m, 148H, –CH2–CH2–CH2– (C=O st amide and N–H+
δ), 1541 (N–H δ), 1475 (CH2, CH3 δ),
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
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Journal of Materials Chemistry B
Page 10 of 14
ARTICLE
Journal Name
1406 (C–N st), 1236 (νas CO–O), 1171 (O–C–C st). EA (%): 42.2, [47.4–47.5], 47.7, 51.6, 66.4, [67.2–68.6], 128.7, 137.9,
View Article Online
–1
DOI: 10.1039/C8TB00639C
Found: C, 39.7; H, 6.5; N, 11.5. Calc. for C69H119Cl8N17O36: C, 168.3, [170.8–175.2]. FTIR (νmax/cm , ATR): 3600–2600 (bs N–
40.5; H, 5.9; N, 11.6.
H+ st), 2947–2633 (C–H st), 1749 (C=O st ester), 1655 (C=O st
(ppm): 1.23 (s, amide and N–H+ δ), 1545 (N–H
), 1473 (CH2, CH3 δ), 1379 (C–N
N3-[G4]-(NH3 )16. 1H NMR (400 MHz, CD3OD)
δ
δ
+
3H, –CH3[G1]), 1.28 (s, 6H, –CH3[G2]), 1.33 (s, 12H, –CH3[G3]), st), 1221 (CO–O st).
1.38 (s, 24H, –CH3[G4]), 1.43 (m, 4H, –CH2–CH2–CH2–CH2–), D[G4]-(NH3 )48. 1H NMR (500 MHz, CD3OD)
δ (ppm): 1.23 (s,
+
1.62 (m, 2H, N3–CH2–CH2–), 1.69 (m, 2H, –CH2–CH2–OCO), 3.95 9H, –CH3[G1]), 1.28 (s, 18H, –CH3[G2]), 1.33 (s, 36H, –CH3[G3]),
(m, 32H, –CH2N–[G4]), 4.01 (m, 4H, –CH2N–[G1,2,3]), 4.16 (t, J 1.38 (s, 72H, –CH3[G4]), 1.45 (m, 12H, –CH2–CH2–CH2–CH2–),
= 6.4 Hz, 2H, –(CH2)5–CH2–OCO–), 4.33 (m, 28H, –CH2O– 1.68 (m, 6H, –CH2–CH2–OCO–), 1.99 (m, 6H, –C2H1N3–CH2–
[G1,2,3]), 4.46 (m, 32H, –CH2O–[G4]), 8.35 (bs, –NHCO2–). 13C CH2–), 3.96 (s, 96H, –CH2N–[G4]), 4.01 (m, 84H, –CH2N–
NMR (100 MHz, CD3OD)
δ (ppm): [17.9–18.2], 26.6, 27.4, 29.4, [G1,2,3]), 4.15 (m, 6H, –(CH2)5–CH2–OCO), 4.33 (m, 84H, –
29.8, 41.2, 42.2, [47.3–47.7], 52.4, 66.4, [67.2–67.9], 68.5, CH2O–[G1,2,3]), 4.46 (m, 96H, –CH2O–[G4]), 4.56 (m, 12H, N–
168.3, [170.8–171.3], 174.2, [174.9–175.3]. FTIR (νmax/cm–1, CH2–C2H1N3–CH2–), 8.30 (bs, –NHCO2–), 8.44 (s, 3H, –C2H1N3–).
ATR): 3600–2600 (bs N–H+ st), 3318 (N–H st), 2986–2883 (C–H 13C NMR (125 MHz, CD3OD)
δ
(ppm): [17.9–18.2], 26.4, 27.1,
st), 2102 (N3 st), 1744 (C=O st ester), 1655 (C=O st amide and 29.4, 31.2, 41.2, 42.3, [47.4–47.5], 47.7, 51.6, 65.7, [66.4–
N–H+ ), 1479 (CH2, CH3 δ), 1410 (C–N st), 1230 68.6], 128.9, 137.7, 168.3, [170.9–177.0]. FTIR (νmax/cm–1,
), 1539 (N–H
νas CO–O), 1176 (O–C–C st). EA (%): Found: C, 39.4; H, 6.5; N, ATR): 3600–2600 (bs N–H+ st), 3391 (N–H st), 2935–2615 (C–H
10.7. Calc. for C141H239Cl16N33O76: C, 40.5; H, 5.8; N, 11.1.
st), 1749 (C=O st ester), 1655 (C=O st amide and N–H+ δ), 1545
), 1477 (CH2, CH3 δ), 1385 (C–N st), 1211 (CO–O st).
δ
δ
(
General procedure iii): dendrimer synthesis by means of (N–H
CuAAC. Tripropargylamine (1.00 mol) and t-Boc protected bis-
GMPA dendron (1.10 mol per alkyne group) were dissolved in
δ
Chemical Characterization.
dimethylformamide (7 mL) in a Schlenk flask. Three vacuum- 1H NMR and 13C NMR experiments were performed on Bruker
argon cycles were carried out to flush the flask from air. The AMX-300 (1H: 300 MHZ, 13C: 75 MHz), Bruker AV-400 (1H: 400
reaction mixture was stirred under argon at 45 °C. CuSO4, MHz, 13C: 100 MHz) or Bruker AV-500 (1H: 500 MHz, 13C: 125
5·H2O (0.5 mol per alkyne group), L-ascorbate (1.0 mol per MHz) spectrometers employing deuterated chloroform
alkyne group) and TBTA (0.1 mol per alkyne group) were (CDCl3), deuterated methanol (CD3OD) or deuterated water
dissolved into dimethylformamide (3 mL) in a second Schlenk (D2O) as solvents. The chemical shifts are given in ppm relative
flask. 3 cycles vacuum-Argon were made in order to flush the to TMS and the coupling constants in Hz; the solvent residual
flask from air. The copper solution was stirred at 45 °C during peak was used as internal standard. Mass spectrometry (MS)
15 min and was then added through a cannula to the azide- was performed with a Bruker Microflex employing the MALDI-
alkyne reaction mixture. The reaction mixture was stirred at 45 TOF technique with nitrogen laser (337 nm) and dithranol
°C for 2 days. Brine (100 mL) was added and the product was (DIT), 2,5-dihydroxybenzoic acid (DHB) or trans-2-[3-(4-tert-
extracted twice with ethyl acetate (2 x 70 mL). The organic butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB)
phase was washed three times with brine (3 x 100 mL), once as matrix, and sodium trifluoroacetic acid as cationic agent or
with aqueous KCN solution (15 mg in 100 mL of water) and with an ESI Bruker Esquire 3000 plus spectrometer employing
twice with brine (2 x 100 mL), dried over anhydrous MgSO4 the electrospray technique. Infrared spectra were obtained on
and the solvent was evaporated under reduced pressure. The a Bruker Vertex 70 spectrophotometer in ATR mode and
crude product was precipitated in a mixture of hexane and recorded between 4000 and 800 cm–1 or using an FTIR ATI-
ethyl acetate (4:6 for D[G3]-(NHBoc)24 and 5:5 for D[G4]- Mattson Genesis series II system while the sample was
(NHBoc)48, 100 mL); the crude product was first dissolved in suspended in nujol in NaCl cells and recorded between 4000
ethyl acetate and hexane was slowly added to the solution. cm-1 and 600 cm-1. Size exclusion chromatography was
The product was recovered by centrifugation and the solid was performed on a Waters e2695 Alliance system employing two
washed once with the same mixture of hexane and ethyl in series Styragel columns HR4 and HR1 (500 and 104 Å of pore
acetate. Finally, the product was dialyzed against methanol size) and a Waters 2424 evaporation light scattering detector
(cellulose acetate, MW 1000 Da) to give a white or pale yellow with a sample concentration of 1.00 mg/mL. The solvent was
powder. The terminal t-Boc protective groups were removed THF (HPLC grade) with a flow rate of 1.00 mL/min at 35 °C;
under acidic conditions, as explained in general procedure ii) PMMA was used as the standard for calibration. Elemental
(yields: 75 – 88%).
Analysis (EA) was performed with a Perkin-Elmer 2400 series II
δ (ppm): 1.28 (s, microanalyser.
+
D[G3]-(NH3 )24. 1H NMR (500 MHz, CD3OD)
9H, –CH3[G1]), 1.33 (s, 18H, –CH3[G2]), 1.38 (s, 36H, –CH3[G3]),
1.43 (m, 12H, –CH2–CH2–CH2–CH2–), 1.68 (m, 6H, –CH2–CH2–
Microscopy
OCO), 1.99 (m, 6H, –C2H1N3–CH2–CH2–), 3.95 (s, 48H, –CH2N- Transmission electron microscopy (TEM) and cryogenic
[G3]), 3.97 (s, 12H, –CH2N-[G1]), 4.01 (s, 24H, –CH2N–[G2]), transmission electron microscopy (cryoTEM) images were
4.15 (m, 6H, –(CH2)5–CH2–OCO), 4.33 (m, 36H, –CH2O–[G1,2]), obtained with a FEI TECNAI T20 with a beam power of 200 kV.
4.46 (m, 48H, –CH2O–[G3]), 4.52 (m, 12H, N–CH2–C2H1N3–CH2– For TEM, a droplet of the sample was deposited on a holey
), 8.30 (bs, –NHCO2–), 8.42 (s, 3H, –C2H1N3–). 13C NMR (125 carbon film 300 mesh coppered grid (Agar Scientific Ltd) and
MHz, CD3OD) δ (ppm): [17.9–18.2], 26.4, 27.1, 29.4, 31.2, 41.2, was allowed to penetrate for 30 seconds; the excess was
10 | J. Name., 2012, 00, 1-3
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Journal of Materials Chemistry B
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removed using a filter paper. A phosphotungstic acid aqueous after each injection was obtained from the integral of the
View Article Online
solution (3%) was used as negative stain; a droplet of the stain calorimetric signal. The heat due to theDbOiIn: 1d0in.10g3r9e/Ca8cTtiBo0n06w39aCs
solution was deposited on the sample grid, allowed to obtained as the difference between the reaction heat and the
penetrate during 10 seconds and the excess was removed corresponding heat of dilution, the latter estimated as a
using a filter paper. The grid was dried for at least 24 hours at constant heat throughout the experiment, and included as an
atmospheric pressure. The average size of the different adjustable parameter in the analysis. The association constant
aggregates measured by TEM was obtained by comparing at (Ka) and the enthalpy change (ΔH) were obtained through non-
least 100 aggregates in at least 5 photos.
linear regression of experimental data to a model considering
For CryoTEM, a droplet of the sample was deposited on Lacey one class of ligand binding sites. Data were analysed using
carbon film 300 mesh coppered grids (Agar Scientific Ltd) software developed in our laboratory and implemented in
previously ionized by glow discharge. Sample vitrification was Origin 7 (OriginLab, USA).
automatically processed using a vitrobot (FEI) and performed
in liquid ethane. A specific holder, Gatan for cold samples, was
Cells and replicon system.
used to stock the grids in liquid nitrogen between sample The highly permissive cell clone Huh /-Lunet, as well as HuH 7
vitrification and microscope observation.
cells containing subgenomic (HCV) replicons I389luc-ubi-
Atomic force microscopy (AFM) images were obtained in liquid neo/NS3-3’/5.1 (Huh 5-2), I377NS3-3’/wt (Huh 9-13), or
medium on a Veeco MultiMode 8 with a cantilever with a I389/hygro-ubi-NS3-3’/5.1 (a kind gift from Dr. V. Lohmann
force constant of 0.7 Nm-1 (Bruker) at 1 Hz, in tapping mode and Dr. R. Bartenschaler) has been described.29,30 Briefly, this
(150 kHz). Samples (20 μL) were deposited onto a previously system allowed the efficient propagation of genetically
exfoliated mica substrate. Before adding the samples, MgCl2 modified HCV RNAs (replicons) in a human hepatoma cell line
(10 mM) was added to the mica substrate to favour adsorption (Huh). The amount of RNA that has been transcribed and
for 1 minute and then the substrates were rinsed three times translated is determined through the quantification of a
with milliQ water.
reporter contained the replicon system (luciferase). The
amount of luminescence detected (after adding the substrate
specific for this enzyme) is proportional to the virus replication
Dendron degradation
Bis-GMPA dendrons (10 mg) were dissolved in a mixture of 4.5 rate (references). Cells were grown in a DMEM supplemented
mL of the corresponding aqueous buffer solution (phosphate with 10% heat-inactivated fetal bovine serum 1 x non-essential
buffered saline, pH =7.4, or citric acid–sodium citrate buffer, amino acids, 100 IU/mL penicillin, 100 μg/mL streptomycin and
pH = 5.0) and 0.5 mL of deuterated water. The different 250
samples were heated at 37 °C and 1H NMR spectra were
recorded at different times in an AV 500 spectrometer using
μg/mL geneticin (G418).
Antiviral and cytostatic assays with Huh 5-2 cells
the Excitation Sculpting pulse sequence for water suppression. Antiviral and cytostatic assays were performed according to
the protocol already described by us in reference 27. Full
Dendrimer/drug association.
details are included in the SI (SI.1.). The 50% effective
The solvent diffusion technique was carried out to associate concentration (EC50) was defined as the concentration of
+
camptothecin (CPT) and D[G3]-(NH3 )24. First, 200
μ
g/mL of compound that reduced the luciferase signal by 50 % and the
CPT in DMSO were added to the aqueous dendrimer solution 50% cytotoxic concentration (CC50) was determined
(1 mg/mL in distilled water) according to a feeding ratio of 100 employing the dose response equation (i.e., Hill equation).
g of CPT per mg of dendrimer. The mixture was stirred at 4 °C
Dendriplexes formation, size and ζ potential of the dendriplexes.
–
μ
for 16 hours to allow the formation of dendrimer/drug
aggregates. DMSO was removed by dialysis against distilled Dendriplexes were freshly prepared before used. Briefly, pDNA
water at 4 °C (cellulose acetate, 1000 Da cut-off). The non- or siRNA was added to a series of dendrimer solutions and
associated drug precipitated during the dialysis and was were mixed by vigorous pipetting and incubated for 20 min at
removed by filtration (cellulose acetate, 0.2
obtain the dendrimer/drug conjugate. The concentration of Gel Retardation Assay. 15
associated CPT was directly measured by fluorescence (λmax ratios, from 1/1 to 200/1 (w/w), containing 250 ng of pDNA or
μm) in order to room temperature.
μ
L of dendriplexes at different
=
436 nm and λext = 368 nm, Perkin-Elmer-LS 55 system) in a ratios w/w 1-80 and 0.3 µg siRNA, were mixed with
mixture of DMSO/H2O (9/1). This process was carried out in appropriate amounts of 6X loading buffer (Takara, UK) and
duplicate.
then electrophoresed on a 0.8% (w/v) agarose gel in 1X TAE
Isothermal titration calorimetry (ITC) assay. The interaction of (40 mM TRIS/HCl, 1% acetic acid, 1 mM EDTA, pH 7.4)
+
CPT with D[G3]-(NH3 )24 was studied by ITC using a high containing SyberSafe for 30 min at 95V. The location of pDNA
sensitivity isothermal titration VP-ITC microcalorimeter and siRNA in the gel was analysed on
(MicroCal, USA). Experiments were performed at 25 °C in transilluminator (Syngene, UK).
a G:Box UV
aqueous media. A 10
the calorimetric cell and titrated with sequential injections of a performed with Brookhaven instruments Corporation 90 plus
100 M solution of CPT. Control experiments were performed particle size analyser. Samples for DLS measurements were
under the same experimental conditions. The heat evolved freshly prepared in distilled water at a concentration of 1.00
μM solution of dendrimer was placed in Dynamic light scattering (DLS). Measurements were
μ
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ARTICLE
Journal Name
mg/mL at two dendrimers:pDNA (w/w) ratios (10/1 and 100/1) evaluated by fluorescence microscopy (Olympus IX81
View Article Online
DOI: 10.1039/C8TB00639C
and at one dendrimer:siRNA (w/w) ratio 100/1. A series of 3 Olympus, Spain) at 72 h. For quantification of EGFP by flow
measurements were performed for each sample. Results are cytometry, cells were seeded at a density of 5·105 cells per
given in intensity.
ζ potential titration. Measurements were performed with a with 200 nM siGFP for 4h. For quantification of Luciferase with
Malvern Zetasizer Nano ZS in a NaCl-HEPES buffer containing 6 Bright-Glo™ Assay Reagent (Promega), after 72 of
well in 6-well plates and incubated with 750 µL of dendriplexes
h
mM of HEPES buffer pH 7.4 and 144 mM of NaCl. The transfection with siLuc, the medium was changed for 100 µL
dendriplexes were freshly prepared at a concentration of 10 fresh DMEM, 100 µL of Bright-Glo™ Assay Reagent (lysis buffer
µg·mL-1 of pDNA or siRNA and various dendrimer and luciferin) were added and cells were lysed by pipetting.
concentrations. Three measurements with a minimum of 10 After 5 min all the contents were transferred to an opaque 96
scans were carried out for each sample.
well plate (Nunc) in the dark. Luminescence was read on a
Synergy HT system (Biotek).
Cell lines and cell culture
Statistical analysis
mMSCS were grown in DMEM/F12 (Biowest) supplemented
with
10%
FBS
(Gibco), Results are reported as mean ± SD and experiments were
1%Penicillin/Streptomycin/Amphotericin (Biowest) and 2 mM performed at least in triplicate. Normally distributed data were
L-glutamine (Biowest) and maintained under hypoxic analysed by two-sample t-test and one-way ANOVA using
conditions (3% O2). HeLa, MDCK-GFP (Madin-Darby Canine Minitab 17 software (State College, PA). p < 0.01 (**) and p <
Kidney stably expressing GFP) and SKOV3-Luc derived from 0.05 (*) were considered to be statistically significant.
human ovarian carcinoma and stably expressing Luciferase
(kindly obtained from Cancer Research-UK Cell services) were
maintained in DMEM High Glucose (w/Glutamine, Biowest)
Conclusions
supplemented with 10% FBS and 1% Pencillin/
Streptomycin/Amphotericin at 37 °C and 5% CO2 in
humidified atmosphere. Cells were treated and analysed as
indicated for each experiment.
A two-step iterative synthetic procedure that consists of amide
coupling and t-Boc cleavage reactions involving a novel 2,2´-
bis(glycyloxy)propionic acid (bis-GMPA) monomer allowed the
synthesis of cationic poly(ester amide) dendrons. Following a
convergent strategy based on CuAAC click-chemistry, these
a
In vitro transfection and biocompatibility.
rd
th
dendrons provide cationic tripodal dendrimers of 3 and 4
For cytotoxicity evaluation, cells were seeded at a density of 5
× 103 cells per well in 96 multiwell culture plates. After 24 h
incubation, the culture medium was removed and 100 μL of
DMEM with increasing concentrations of the corresponding
dendrimer were added (0.1–1 mg/mL). After 24–72 h the
generation, with 24 and 48 terminal amino groups
respectively, in good yields. These dendrimers were found to
be fully biocompatible and degradable in water by hydrolysis.
They have demonstrated their potential for use as drug
delivery systems as they could enhance the cell viability and
the anti-HCV activity of camptothecin. On the other hand,
these materials can spontaneously form dendriplexes with
both pDNA and siRNA. Effective in vitro siRNA transfection was
observed into tumoral and non-tumoral cells. These positive
results reveal that ammonium-terminated poly(ester amide)
dendrimers are an interesting option for the design of carriers
for a broad range of therapeutic applications (drug delivery
and gene therapy).
solutions were replaced by 100 μL of fresh DMEM and 10 μL of
Alamar Blue® dye solution (Thermo Fisher Scientific). After
incubation for 3 h at 37 ºC, fluorescence was read at 530/590
(excitation/emission) on a Synergy HT (BioTek, USA) plate
reader. Untreated cells incubated with medium without
dendrimer were used as control and cytotoxicity is expressed
as the relative viability of the cells compared to control cells
(considered as 100% viability). Four replicates per
concentration were assayed for each of the three independent
experiments performed. Also, DNA cell cycle analysis was
measured on propidium iodide (PI)-stained nuclei of 106 cells
using a FACS Array system (Becton Dickinson). For all the
experiments, the control and sample cells were harvested by
trypsinisation, washed twice with PBS, fixed in ice-cold 70%
EtOH and maintained at 4 °C for at least 1 h prior to analysis.
For pEGFP transfection, cellular uptake and internalization,
experiments were carried out on mMSCs and HeLa cells as
previously described by us.28 Full details are included in the SI
(SI.2.)
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
This work was financially supported by the MINECO-FEDER
funds (under the projects CTQ2015-70174-P and MAT2015-
66208-C3-1-P), Miguel Servet Program from the Instituto de la
Salud Carlos III (CPII13/00017 to O.A.), Instituto de Salud
Carlos III and European Union (ERDF/ESF, “Investing in your
future”) (PI15/00663) and the Gobierno de Aragón-FSE (E04
and B01 research groups). Centro de Investigacion Biomédica
en Red en Enfermedades Hepaticas y Digestivas (CIBERehd);
Dendriplexes formed with 100-400 nM siGFP or siLuc in serum
free media (SFM) were incubated for 4 h with MDCK-GFP or
SKOV3-Luc cells seeded at a density of 1·104 cells per well in
96-well plates. For the positive controls, cells were incubated
with Fugene at ratio of 3/1 (w/w). EGFP expression was first
12 | J. Name., 2012, 00, 1-3
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22 A. Lakshminarayanan, V.K. Ravi, R. Tatineni, Y.B. Rajesh, V.
Maingi, K.S. Vasu, N. Madhusudhan, P.K. Maiti,ViAew.KA.rtSicoleoOdn,linSe.
and Asociación Española de Gastroenterologia (AEG). A.L. and
R.C-G thank the MECD for their FPU grants (FPU12/05210 and
FPU13/3870). R.G.-P. and R.C-G thank the DGA for their EPIF
grants. The authors are grateful to Dr. A. Gonzalez-Orive and
Dr. H.M. Osorio for their help with AFM studies, and M.
Savirón for her help with MS spectrometry studies. The
authors would like to acknowledge the use of Servicios
Científico-Técnicos of CEQMA (Universidad de Zaragoza-CSIC),
Servicio General de Apoyo a la Investigación-SAI (Universidad
de Zaragoza), microscopy, cell culture and cytometry
departments at CIBA (IACS-Universidad de Zaragoza) and the
LMA (INA-Universidad de Zaragoza).
Das, N. Jayaraman, Bioconjug. Chem. 2013, 24
DOI: 10.1039/C(89T)B, 010661392C–
1623.
23 A. Sousa-Herves, R. Novoa-Carballal, R. Riguera, E.
Fernandez-Megia, AAPS J. 2014, 16 (5), 948–961.
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26 J. Movellan, P. Urbán, E. Moles, J.M. de la Fuente, T. Sierra,
J.L. Serrano, X. Fernàndez-Busquets, Biomaterials 2014, 35
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27 A. Lancelot, R. Clavería-Gimeno, A. Velázquez-Campoy, O.
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DOI: 10.1039/C8TB00639C
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