Alkyl sulfonyl derivatized PAMAM-G2 dendrimers as nonviral gene delivery vectors with improved transfection efficiencies

Article abstract of DOI:10.1039/c0ob00355g

Amphiphilic dendrimer-based gene delivery vectors bearing peripheral alkyl sulfonyl hydrophobic tails were constructed using low-generation PAMAM-G2 as the core and functionalized by means of the aza-Michael type addition of its primary amino groups to vinylsulfone derivatives as an efficient tool for surface engineering. While the unmodified PAMAM-G2 was unable to efficiently transfect eukaryotic cells, functionalized PAMAM-G2 dendrimers were able to bind DNA at low N/P ratios, protect DNA from digestion with DNase I and showed high transfection efficiencies and low cytotoxicity. Dendrimers with a C18 alkyl chain produced transfection efficiencies up to 3.1 fold higher than LipofectAMINE 2000 in CHO-k1 cells. The dendriplexes based in functionalized PAMAM-G2 also showed the ability to retain their transfection properties in the presence of serum and the ability to transfect different eukaryotic cell lines such as Neuro-2A and RAW 264.7. Taking advantage of the vinylsulfone chemistry, fluorescent PAMAM-G2 derivatives of these vectors were prepared as molecular probes to determine cellular uptake and internalization through a clathrin-independent mechanism.

Full text of DOI:10.1039/c0ob00355g

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Alkyl sulfonyl derivatized PAMAM-G2 dendrimers as nonviral gene delivery
vectors with improved transfection efficiencies†
Julia Morales-Sanfrutos,‡a Alicia Megia-Fernandez,‡a Fernando Hernandez-Mateo,a
Ma Dolores Giron-Gonzalez,b Rafael Salto-Gonzalezb and Francisco Santoyo-Gonzalez*a
Received 30th June 2010, Accepted 14th October 2010
DOI: 10.1039/c0ob00355g
Amphiphilic dendrimer-based gene delivery vectors bearing peripheral alkyl sulfonyl hydrophobic tails
were constructed using low-generation PAMAM-G2 as the core and functionalized by means of the
aza-Michael type addition of its primary amino groups to vinylsulfone derivatives as an efficient tool
for surface engineering. While the unmodified PAMAM-G2 was unable to efficiently transfect
eukaryotic cells, functionalized PAMAM-G2 dendrimers were able to bind DNA at low N/P ratios,
protect DNA from digestion with DNase I and showed high transfection efficiencies and low
cytotoxicity. Dendrimers with a C18 alkyl chain produced transfection efficiencies up to 3.1 fold higher
than LipofectAMINETM 2000 in CHO-k1 cells. The dendriplexes based in functionalized PAMAM-G2
also showed the ability to retain their transfection properties in the presence of serum and the ability to
transfect different eukaryotic cell lines such as Neuro-2A and RAW 264.7. Taking advantage of the
vinylsulfone chemistry, fluorescent PAMAM-G2 derivatives of these vectors were prepared as molecular
probes to determine cellular uptake and internalization through a clathrin-independent mechanism.
lar weights and sizes.5 In particular, poly(amidoamine) (PAMAM)
Introduction
dendrimers that have easily cationized primary amino termini and
The potential of gene therapy for treating genetic disorders has
tertiary internal amino groups have become a standard tool in the
laboratory due to their easy synthesis and commercial availability.6
The PAMAM–DNA complexes, termed dendriplexes and formed
by the interaction with the primary amine groups,7 are positively
charged supramolecular systems that have the ability to interact
with the negatively charged membrane of cells allowing their
entrance through endosomal uptake without inducing significant
cytotoxicity and immunogenicity.8 Once internalized the buffering
capacity of the unprotonated amino groups permits them to act
as a proton sponge in the acidic environment of endosomes,
enhancing in this way the release of DNA into the cytoplasm
through the inhibition of pH-dependent endosomal nucleases.9
The influence of the dendrimer generation on the efficiency of
transfection (also denoted as the dendritic effect) was identified
early and medium sized dendrimers (G5–G10) are generally
the most efficient ones.10 Three different strategies have been
developed to improve the performance of PAMAMs as nucleic
acid carriers: (a) enhancing the flexibility of their branches,
(b) rendering the dendrimers amphiphilic and (c) making them
biocompatible.
led to prolific research activity on the design of safe and efficient
gene carrier vectors that compact and protect oligonucleotides and
DNA against degradation by serum nucleases. Nonviral vector
systems1 are an attractive alternative to viral carriers,2 retroviruses
and adenoviruses, to overcome the fundamental problems associ-
ated with these systems despite the high delivering efficiency that
they exhibit: toxicity, immunogenicity and limitations related with
scale-up production.3 Although synthetic vectors have inherently
reduced transfection efficiencies due to the extra- and intracellular
barriers that they find for their cellular uptake,1f these nonviral
vectors can easily be structurally modified to allow systematic
study of the factors implicated in transfection efficiency.
The design of cationic compounds including lipids, polymers,
dendrimers and peptides that interact in a non-covalent and strong
manner with the negatively charged phosphate backbone of DNA
has been demonstrated to be an useful strategy for developing effi-
cient synthetic transfecting agents. In this context, hyperbranched
dendrimers4 have proved to have greater potential as gene delivery
vectors compared to traditional polymeric systems as a result of
their unique characteristics: multivalent-functionalized terminal
surface and well-defined three-dimensional architectures, molecu-
The thermal degradation of high generation PAMAM den-
drimers produces fractured PAMAM-based vectors with a greater
flexibility that are commercialized under the name SuperfectTM
.
aDepartamento de Q. Orga´nica, Facultad de Ciencia, Instituto de Biotecno-
log´ıa, Universidad de Granada, Granada, 18071, Spain. E-mail: fsantoyo@
ugr.es; Fax: (+34)-958243186; Tel: (+34)-958248087
In addition, surface engineering by chemical modification in-
volving functionalization at the dendrimer’s periphery has been
extensively investigated in an effort to improve not only the
complex formation, interaction with the plasma membrane and
endosomal release but also to reduce the cytotoxicity and to
incorporate cell-targeting capabilities into the PAMAM-based
dendriplexes to promote cell-specific uptake.6f,8b,11 Conjugation of
the primary amino groups with poly(ethylenglycol), amino-acids,
peptides, proteins, sugars, cyclodextrins, lipids and glucocorticoids
bDepartamento de Bioqu´ımica y Biolog´ıa Molecular II, Facultad de Farma-
cia, Universidad de Granada, E-18071-Granada, Spain
† Electronic supplementary information (ESI) available: 1H- and 13C-
NMR spectra for compounds 4a–d, 5a–d, 6a–d, 8a–b, 8d, 9a–d, 10d, 11d
and 13, gel electrophoresis shift assays, DNase I protection experiments
and hydrodynamic diameter and z potential values for PAMAM-G2
derivatives and their complexes with DNA. See DOI: 10.1039/c0ob00355g
‡ These authors contributed equally to this work.
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Scheme 1 Synthesis of vinylsulfone reagents for the functionalization of PAMAM-G2.
has been described in the literature with differing results on their
transfection properties.
the basis of (a) its non-toxicity, attributed to the limited surface
charges of this compound,14 and (b) its easy synthesis with respect
to high generation PAMAMs that usually requires tedious and
low yielding procedures. The incorporation of hydrophobic alkyl
chains of different length was anticipated to be a structural
factor that would positively contribute to improving synergistically
the cellular uptake and internalization of the corresponding
dendriplexes.
Several studies have demonstrated the benefits of incorporating
lipophilic motifs to render the dendrimer amphiphilic in synthetic
gene delivery systems.1f With this concern, lipid derivatized
PAMAMs dendritic vectors have been engineered with topologies
in which the dendritic hydrophilic moiety was present on one
side of the vector while the hydrophobic moiety, mainly alkyl
chains of different length, was installed on the opposite side.12
Alternatively, PAMAM dendrimers having a hydrophilic interior
and a hydrophobic corona prepared by randomly grafting alkyl
tails at the dendrimer periphery have also been reported.13 In both
cases, the influence of parameters such as dendritic generation,
length of the alkyl chain and extent of functionalization on
the transfection efficiency and cytotoxicity has been evaluated.
Michael addition of acrylate derivatives as well as acylation of
activated esters fatty derivatives have been the chemical tools used
for the construction of such hybrid dendritic–lipidic transfection
agents.11–13
Herein we report on the synthesis and transfection efficiency
properties of novel amphiphilic dendrimer-based gene delivery
vectors bearing peripheral hydrophobic tails that are constructed
using low-generation PAMAM-G2 as the core and functionalized
by means of the aza-Michael type addition of its primary amino
groups to vinylsulfone derivatives containing hydrophobic alkyl
chains as an efficient conjugation strategy. Despite the poor
transfection properties usually exhibited by native PAMAM-G2
dendriplexes, this cationic polymer was chosen over other higher
PAMAM generations for the preparation of lipid conjugates on
Results and discussion
Chemical synthesis of functionalized PAMAM-G2 dendrimers
With a view to obtain PAMAM-G2 amphiphilic derivatives to be
used as nonviral vectors with improved transfection efficiencies
and diminished cytotoxicity, we selected a set of compounds
containing hydrophobic chains for conjugation with the amino
terminal groups of that dendrimer (Scheme 1). To evaluate the
influence of the hydrophobic chain length on the process of
transfection, commercially available reagents containing linear
alkyl chains with four, twelve and eighteen carbon atoms were
selected, namely, the bromide derivatives 1a–c, oleic alcohol
((Z)-9-octadecen-1-ol) 2, butyric chloride 7a, and lauric acid
(docedanoic acid) 7b, stearic acid (octadecanoic acid) 7c and oleic
acid (cis-9-octadecenoic acid) 7d. The aza-Michael type addition
of vinylsulfone derivatives of such hydrophobic compounds was
thought to be an optimal ligation tool for their coupling with the
peripheral amine groups as this reaction usually proceeds with
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Scheme 2 Synthesis of amphiphilic PAMAM-G2 dendrimers as nonviral vectors.
high efficiency by simple mixing of the reagents in an adequate
solvent without the formation of any undesired by-product.15
gave the disulfide 10d in high yield. Reduction of this compound
with powdered Zn followed by Michael addition to DVS afforded
the thioether derivative 11d in a one-step procedure (Scheme 1).
Considering that cholesterol has often been exploited1f as a
hydrophobic motif in synthetic transfection agents including
PAMAM-based vectors16 to give them a lipidic character, the
preparation of a vinylsulfone derivative of this steroid was
also envisaged in order to further synthesize the corresponding
cholesterol–PAMAM-G2 conjugate and evaluate if this struc-
tural modification allows an optimization on gene delivery.
Simple treatment of commercial cholesterol 12 with DVS affords
the corresponding vinylsulfone derivative 13 in moderate yield
(Scheme 1).
With the vinylsulfone derivatives (6a–d, 9a–d, 11d and 13)
in hand, the synthesis of the novel gene delivery vectors was
performed by the simple addition of a THF solution of these
reagents to an aqueous solution of PAMAM-G2. To evaluate
the effect of the extent of functionalization, three different
vinylsulfone : PAMAM-G2 molar ratios (I = 0.5, II = 1.0 and
III = 2) were used in each case, giving rise to the corresponding
conjugates [6a–d-G2(I–III), 9a–d-G2(I–III), 11d-G2(I–III) and 13-
G2(I–III)] after elimination of the reaction solvent (Scheme 2).
In addition, to perform studies aimed at the observation of the
intracellular events in the transfection of the PAMAM-G2 based
dendrimers and at the elucidation of their cellular uptake and
internalization mechanisms, the synthesis of fluorescent PAMAM-
G2 probes was also designed to be used in confocal microscopy
studies. The vinylsulfone rhodamine derivative 14 (Scheme 2),
previously reported by us,17 allows the easy incorporation of this
The different functionality of compounds 1a–c,
2 and
7a–b (bromide, alcohol and carboxylic acid, respectively) allows
the design of simple chemical strategies for the preparation
of their corresponding vinylsulfone derivatives, and also the
introduction of a structural variability in the linker that will
join the hydrophobic alkyl chain and the dendrimer core whose
influence on the transfection properties will be also evaluated.
Thus, nucleophilic displacement on the bromide derivatives 1a–c
and the mesylate derivative 3 of oleic alcohol 2 by the sulfur atom
of 2-thioethanol gave access to the hydroxyethyl thioethers 4a–d
in high yields. Oxidation with peroxyacetic acid and subsequent
dehydration of the corresponding sulfone derivatives 5a–d via their
mesylate derivatives led easily to the vinylsulfones 6a–d in which
the original functionality has been replaced by the vinyl sulfonyl
group (Scheme 1). Alternatively, a strategy was envisaged starting
from the fatty acids 7a–d which enables the introduction of a longer
vinylsulfone linker for the hydrophobic chains. Thus, reaction with
2-amino ethanol of the fatty acid chloride derivatives, using the
commercial reagent 7a or obtained in situ by treatment with thionyl
chloride in the case of the acids 7b–d, gave the corresponding
2-hydroxylethyl amides 8a–d. Reaction of these compounds with
divinylsulfone (DVS) gave the ether vinylsulfones 9a–d in moderate
yields (Scheme 1). Additionally, in the case of oleic acid the
homologous thioether vinylsulfone 11d was also prepared in order
to evaluate a possible influence of the nature of the heteroatom
of the linker on the transfection properties. This compound was
accessible by reaction of oleic acid chloride with cystamine, which
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fluorescent probe in native PAMAM-G2 to give the PAMAM-G2-
Rho dendrimer that will be later functionalized with hydrophobic
motifs on the basis of the results obtained in transfection efficiency
studies (see next Sections).
1H NMR spectroscopic data of the new amphiphilic PAMAM-
G2 derivatives confirm successful grafting of the hydrophobic
or the rhodamine vinyl sulfone reagents at the periphery of the
PAMAM-G2 as evidenced by the absence of the characteristic
vinyl sulfone proton peaks and the presence of new signals
ascribed to the alkyl chains and the fluorescent residue (see ESI
Fig S1†).
In the next phase, cellular uptake of the novel dendriplexes
was studied in CHO-k1 cells, a common standard cell line used
for a variety of transfection agents. Plasmid pEGFP-N3 that
encodes for the green fluorescent protein allows us to evaluate
the uptake by measuring the fluorescence of the cells transfected
with the complexes that have been grown 48 h after transfection
to allow the expression of eGFP. Cellular uptake has been studied
in the absence of serum at N/P ratios of 2.5–50 (Fig. 3A). 100%
value has been assigned to the transfection level obtained when
LipofectAMINETM 2000 (LP2000), a commercial reagent that has
been described as a high efficiency vector for the transfection
of many cell types, is used. The results obtained in these assays
clearly indicated that unmodified PAMAM-G2 have a very poor
transfection capability (less than 5% of the LP2000 value) which
is in accordance with previous reported observations10 and with
our own results: (a) no retardation of pDNA in gel electrophoresis
(Fig. 1A) and (b) absence of protection towards DNase I degra-
dation (Fig. 2A). These facts corroborate the benefits obtained by
the incorporation of a hydrophobic moiety to promote binding
of the dendrimer to the hydrophobic DNA bases. In addition, it
is observed that the incorporation of the moderately hydrophobic
moiety of rhodamine is enough to promote DNA binding to the
PAMAM-G2 (Fig. 1C) although it fails to protect DNA from
DNase I degradation (Fig. 2C).
From the assembly of the results obtained some conclusions can
be extracted concerning the influence of the chemical structure
of the engineered PAMAM-G2 vectors on their transfection
efficiency. PAMAM-G2 derivatives functionalized with a butyl
chain, 6a-G2 and 9a-G2, are not able to transfect the CHO-k1 cells
(data not shown) despite the enhanced pDNA complexing abilities
and protection from DNase I digestion that they exhibited. Among
the assayed compounds, the minimum length of the hydrophobic
chain that enables the PAMAM-G2 derivatives to efficiently
transfect CHO-k1 cells is 12 carbons (compounds 6b-G2 and
9b-G2). PAMAM-G2 derivative 6b-G2(I) exhibited a remarkably
high transfection level (p <0.001 compared to the LP2000 value)
at N/P ratios between 5 and 20 while the efficiency of 9b-G2(I)
to deliver pDNA is lower but similar to LP2000 at a N/P
Gene delivery capabilities of amphiphilic PAMAM-G2 derivatives
The capabilities of the novel amphiphilic PAMAMs to neutralize,
bind and compact plasmid DNA (pDNA) were first studied due
to their importance for efficient gene delivery. Plasmid pEGFP-
N3 [coding for the enhanced Green Fluorescent Protein (eGFP)]
was selected for the preparation of the DNA complexes and
transfection assays. The PAMAMs conjugates were mixed with
pDNA at several N/P ratios (0.5 to 50) for the formation of
the corresponding dendriplexes. Gel electrophoresis shift assays
were performed as they reflect the capability of the PAMAM-G2
derivatives to compact pDNA.18 Formation of the corresponding
complexes results in pDNA lacking migration in the gel due to
polycation binding and charge-neutralization. The agarose gel
shift assays performed revealed that binding of the majority of
the PAMAM-G2 derivatives to pDNA occurs at N/P ratios of
0.5–1 for the functionalized compounds at the three molar ratios
(I = 0.5, II = 1.0 and III = 2). (Fig. 1 and ESI Fig. S2†).
The capability of protecting pDNA from endonucleases degra-
dation was next evaluated as this is a key point to achieve a success-
ful transfection since unprotected pDNA are rapidly degraded by
the nucleases presented in the culture media serum.1e The results
obtained with the PAMAM-G2 amphiphilic derivatives (Fig. 2
and ESI Fig. S3†) showed that these derivatives have the capability
to efficiently protect pDNA from endonuclease degradation while
unmodified PAMAM-G2 failed to protect pDNA from digestion.
Fig. 1 Gel Electrophoresis Shift Assay. A.- Gel shift assays showing PAMAM-G2 or selected PAMAM-G2 derivatives–pDNA binding at N/P ratios
between 0 (pEGFP-N3 alone) and 10. l/H lane corresponds to l/HindIII molecular weight marker. The absence of plasmid band in the wells correlates
with the inhibition of the plasmid DNA electrophoretic mobility. I, II and III correspond to the three different vinylsulfone : PAMAM-G2 molar ratios
(I = 0.5, II = 1.0 and III = 2) used B.- Minimum N/P ratio needed to completely inhibit electrophoretic mobility in the shift assay. C.- Rhodamine labeled
PAMAM-G2 or PAMAM-G2 derivatives gel shift assays.
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Fig. 2 DNase I protection experiments. A.- Representative agarose electrophoresis of samples corresponding to pEGFP-N3 incubated in the absence (-)
or presence (+) of DNase I as controls. pEGFP-N3 samples that have been complexed with the PAMAM-G2 derivatives before the DNase I treatment
(as described in the experimental section) have been run in parallel. B.- Quantitation of the relative intensity (untreated pEGFP-N3 value equal to 100)
of the sum of relaxed and supercoiled electrophoretic plasmid bands corresponding to the pEGFP-N3 samples complexed with PAMAM-G2 derivatives
and treated with DNase I. Results are expressed as means SEM (n = 4). I, II and III correspond to the three different vinylsulfone : PAMAM-G2 molar
ratios (I = 0.5, II = 1.0 and III = 2) used. C.- Samples of PAMAM-G2 derivatives labeled with Rhodamine have been processed as above and the DNase I
protection has been assayed by gel electrophoresis. (a) dendrimer–pDNA complexes, (b) labeled dendrimer alone. Since the dendrimers are labeled with
fluorescence, the gels have been photographed before (UV) and after ethidium bromide staining (EtBr). Notice that due to the interaction of the free
labeled dendrimer with the SDS used in the sample preparation, the free labeled dendrimers have a net negative charge in the electrophoresis.
ratio of 20 (Fig. 3). Both compounds showed enhanced pDNA
complexing abilities (ESI Fig. S2†) although, as expected from its
transfection efficiency, 9b-G2(I) partially failed to protect pDNA
from DNase I degradation (ESI Fig. S3†). Amphiphilic dendrimer
6c-G2(I) bearing a C18 hydrophobic chain produced the highest
levels of transfection in CHO-k1 (approximately between 3.1
fold (p <0.001) more efficiently than LP2000), a capacity that is
complemented with the formation of stable complexes warranting
full pDNA protection from the environment (Fig. 2). However,
compound 9c-G2 which has an aliphatic chain of the same length
presented very poor transfection levels that correlate well with the
observed susceptibility to endonucleases of the relaxed pDNA
band in the DNase I protection assay (ESI Fig. S3†). Oleic-
containing PAMAM-G2 derivatives 6d-G2(I), 9d-G2(III) and 11d-
G2(III), which have a double bond in the C18 aliphatic chain,
exhibit higher levels of transfection than LP2000 (1.6 to 2.7 fold
higher than LP2000, p <0.01) (Fig. 3). These dendrimers also
presented high complexing abilities (Fig. 1 and ESI Fig. S2†)
and fully protected pDNA from DNases (Fig. 2, and ESI Fig.
S3†). Overall, it can be stated that although the inclusion of a
hydrophobic moiety is enough to obtain a tight binding of the
PAMAM-G2 dendrimers to the pDNA, an alkyl chain length
of 18 carbons suits better for optimal DNase I protection and
transfection efficiency.
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Fig. 3 In vitro gene transfection efficiency of the PAMAM-G2 based dendrimer complexes in CHO-k1 cells. CHO-k1 cells were transfected with dendriplexes
using pEGFP-N3 plasmid as described in the Experimental section. For each condition, DNA was mixed with the PAMAM-G2 derivatives at N/P
ratios of 2.5 to 50. As a positive control (LP), a transfection was performed using LP2000. Fluorescence was assayed 48 h after transfection. The ratio
fluorescence/proteins for each compound is shown. The eGFP fluorescence value for the LP2000 transfection was normalized to 100% in each experiment.
Results are expressed as means SEM (n = 8).
Concerning the extension of the functionality by hydrophobic
chains, a comparison of dendrimers from series 6 and 9 indicates
that the optimal transfection is related to the number of alkyl
chains grafted per dendrimer. Compounds from 6 series are
very good transfection reagents with only 0.5 hydrophobic chain
grafted per PAMAM-G2 (I = 0.5) while the functionalized
dendrimers of series 9 needed two alkyl chains grafted (III = 2) to
be efficient at transfecting pDNA. The reasons for this different
behavior are not clear.
On the other hand, the nature of the heteroatom of the ether
linkage, oxygen for compound 9d and sulfur for compound
11d, seems to have no influence on the transfecting abilities
of these compounds since both exhibited similar efficiencies
(Fig. 3).
Chemical functionalization of PAMAM-G2 with cholesterol,
compounds 13-G2(I–III), does not improve the abilities of the
corresponding dendriplexes as for these compounds high pro-
tection of pDNA (Fig. 2B and ESI Figs. S2 and S3†) but very
low transfection efficiency (data not shown) are observed. This
behavior could be tentatively ascribed to a strong binding of the
pDNA to those PAMAM-G2 derivatives that makes difficult the
release of the pDNA free into the nucleus.
Taken together, the results obtained allows us to conclude that
from the set of the amphiphilic PAMAM-G2 derivatives prepared
the best transfecting agents are the dendrimers 6c-G2(I) and 9d-
G2(III).
In the next stage of our study, dynamic light scattering and z-
potential measurements were carried out for the more efficient
dendrimers at the best N/P ratios, to determine the size and
electrokinetic properties of the formed complexes (ESI Table S1†).
As expected, z-potential values decreased when the functionalized
dendrimers were complexed withpDNA. However, norelationship
between particle size and transfection efficiency can be deduced
since the best transfecting agents 6c-G2(I) and 9d-G2(III) have
hydrodynamic diameters of 524 18 and 226 3 nm, respectively.
Once the influence of the structure on the transfection effi-
ciencies had been studied for the amphiphilic PAMAM-G2-based
vectors, the cytotoxicity of their pDNA complexes was evaluated
by determining the percentage of cell viability with respect to
unexposed cells using a MTT assay (Fig. 4). Cell viability was
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Fig. 4 Cytotoxicity of the complexes. Cytotoxicity of the complexes
formed by the functionalized dendrimers and pDNA was evaluated in
CHO-k1 cells 24 h after transfection by determining the percentage of cell
viability using a MTT assay. The dendrimer derivatives to be assayed were
those that showed a higher transfection efficiency. The complexes were
assayed for cytotoxicity using the most efficient N/P ratio for transfection
(6b-G2(I): 10; 6c-G2(I): 2,5; 6d-G2(I): 10; 9b-G2(I): 20; 9d-G2(III): 10;
11d-G2(III): 10). Results are reported as % viability based on the untreated
control cells normalized to 100% viable.
studied in CHO-k1 cells as a function of the functionalized
PAMAM-G2 derivative and the N/P ratio at which the compound
showed its higher transfection levels. Unmodified PAMAM-G2
complexed with pDNA showed itself to be not toxic as expected
while lipoplexes with LP2000 are not exempt of toxicity as they
reduced by 50% the cell viability (p <0.001). For the PAMAM-G2
derivatives the most efficient transfecting compounds, 6c-G2(I)
and 9d-G2(III), displayed a high level of cell viability
The effect of serum in the media on the transfection efficiency
of the pDNA was evaluated on the PAMAM-G2 derivative 6c-
G2(I), which was chosen considering that it is one of the best at
protecting pDNA against DNases and for its high transfection
efficiency at very low N/P ratios (Fig. 5A). In the presence of
serum, the transfection levels of this vector were similar to those
obtained with LP2000 at 5–10 N/P ratios. The increase in N/P
ratio needed for transfection due to the presence of serum has been
reported previously for cationic liposomes.19
To assess the pDNA transfection capabilities in other cell lines,
the PAMAM-G2 derivative 6c-G2(I) has also been used in assays
using neuroblast cells (Neuro-2a) and a cell line derived from
macrophages (RAW 264.7) which usually exhibits low transfection
levels.20 A 100% value was assigned to the transfection levels
observed when LP2000 in CHO-k1 cells was used. Compound
6c-G2(I) has the capability to transfect these three cell lines in a
similar or better way than LP2000 (Fig. 5B).
Fig. 5 Factors that modulate the transfection efficiency of PAMAM-G2
derivatives complexes. A.- Effects of serum in the transfection media.
LP2000 (as a positive control) or 6c-G2(I) dendrimer (N/P ratios
2.5–50) have been complexed with pEGFP-N3 plasmid and then used
for transfection of CHO-k1 cells in the absence or presence of 10% fetal
bovine serum in the transfection media. Fluorescence was assayed 48 h
after transfection. The ratio fluorescence/proteins for each condition is
shown. The eGFP fluorescence value for the LP2000 transfection carried
out in the absence of fetal bovine serum was normalized to 100%. Results
are expressed as means SEM (n = 7). B.- Cell type. CHO-k1, Neuro-2a
or RAW 264.7 cells were transfected using 6c-G2(I) dendrimer complexed
with pEGFP-N3 plasmid as described in the Experimental section. For
each condition, DNA was mixed with the PAMAM-G2 derivative at N/P
ratios from 2.5 to 50. For each cell line a transfection was performed
using LP2000 as a positive control. Fluorescence was assayed 48 h after
transfection. The ratio fluorescence/protein for each condition is shown.
The eGFP fluorescence value for the LP2000 transfection in CHO-k1 cells
was normalized to 100%. Results are expressed as means SEM (n = 8).
in the uptake of DNA complexes, dependent on cell type, the
nature of the carrier and particle size. The clathrin-dependent
pathway is preferred for microspheres up to 200 nm in size.22
The clathrin independent route includes uptake from lipid rafts
in caveolae or via flotillin-dependent pathway.23 To determine the
pathway of the cellular uptake of the functionalized PAMAM-
G2 dendrimers two chemical inhibitors of endocytosis were used:
chlorpromazine and genistein as inhibitors of clathrin-dependent
and -independent endocytosis, respectively.21 Fig. 6 shows that
chlorpromazine significantly increased (p <0.001) the efficiency of
transfection using LP2000, 6c-G2(I) or 9d-G2(III) while it did not
modify the capability of transfection of compounds 6b-G2(I), 6d-
G2(I), 9d-G2(III) y 11d-G2(III). Conversely, preincubation with
genistein decreased the transfection levels of the majority of
compounds, including LP2000. Therefore, from these observations
Cellular uptake and internalization mechanism studies for
amphiphilic PAMAM-G2-based vectors
Nonviral gene complexes can enter into mammalian cells through
different endocytic pathways.21 Therefore, for a novel vector
it is important to characterize its cellular uptake because this
determines how the gene carrier is processed intracellularly and
therefore influences its transfection efficiency. Most of the reports
suggest that endocytosis is the preferred route of cellular uptake
mechanism for nonviral gene carriers. Two main endocytic path-
ways, clathrin-dependent and clathrin-independent, are implicated
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free pDNA released to the nucleus) is increased. These results,
together with the transfection efficiency values measured in the
presence of endocytosis inhibitors, confirm that the clathrin-
independent pathway is the productive endocytic pathway used
by these functionalized PAMAM-G2 derivatives.
Conclusions
In summary, PAMAM-G2-based amphiphilic dendrimers are
prepared in a straightforward and efficient manner by using
the aza-Michael type addition of vinylsulfone derivatives as an
optimal tool for the grafting of hydrophobic alkyl chains to
the peripheral amino groups of those dendrimers. This chemical
strategy is general and allows the facile functionalization of
any amino-containing polymer for the preparation of synthetic
gene carriers. The transfection properties are dependent on
the chemical structure of the hydrophobic chain and on the
extension of the functionalization of the dendrimers surface.
Some of these engineered PAMAM-G2 dendrimers are useful
vectors with improved transfection properties when compared to
unmodified PAMAM-G2 and commercial transfection reagents
such as LipofectAMINETM: higher transfection efficiencies, lower
cytotoxicity, the capability to maintain their transfection efficiency
in the presence of serum and the ability to transfect different
eukaryotic cell lines. The flexibility of the vinylsulfone based
chemistry also allowed the fluorescent labeling of these PAMAM
derivatives for the preparation of probes to monitor the endocytic
pathway used for successful transfection by the dendrimers and to
gain insights in the cellular uptake and internalization mechanisms
of these vectors.
Fig. 6 Effects of inhibitors on cellular transfection of PAMAM-G2
derivatives complexes. CHO-k1 cells were pretreated with chlorpromazine
(14 mM) or genistein (200 mM) for 30 min before transfection with den-
drimer complexes at their most efficient N/P ratio (6b-G2(I): 10; 6c-G2(I):
2,5; 6d-G2(I): 10; 9b-G2(I): 20; 9d-G2(III): 10; 11d-G2(III): 10). As a
control (LP), a transfection was performed using LP2000. Fluorescence
was assayed 48 h after transfection. The ratio fluorescence/proteins for
each compound is shown. The eGFP fluorescence value for the LP2000
transfection was normalized to 100% in each experiment. Results are
expressed as means SEM (n = 6).
it can be concluded that amphiphilic PAMAM-G2 derivatives use
mainly a clathrin independent pathway.
Finally, the internalization process of the dendriplexes was
studied by confocal microscopy using Cy5-labeled pDNA and
rhodamine-labeled dendrimer 6c-G2(III)-Rho and 9d-G2(III)-
Rho. These compounds were prepared following the vinylsulfone-
based chemical strategy indicated in Scheme 2 that implies the
functionalization of PAMAM-G2-Rho with the stearic and oleic
vinylsulfone derivatives 6c and 9d, respectively. For compound 9d-
G2(III)-Rho confocal images are shown. The transfected cells have
been identified by the expression of eGFP, since the experiments
have been performed using pEGFP-N3 plasmid. Images were
collected 24 h post-transfection to allow eGFP expression. Fig. 7A
corresponding to visible differential interface contrast (Nomarki)
images and fluorescence images shows the distribution of fluo-
rescence in the transfected and non-transfected cells. In all cells,
red fluorescence corresponding to the labeled dendrimer can be lo-
cated in the cytosol of the cells, close to the nucleus. Almost all blue
fluorescence corresponding to the labeled-pDNA is associated
with the red labeled-dendrimers (purple particles in the overlayed
images). Only in the transfected cells, free blue fluorescence
corresponding to the pDNA released from the dendrimers can
be located inside the nucleus (Fig. 7B). No free blue fluorescence
is located in the nucleus of the non-transfected cells. Similar results
have been obtained for the rhodamine-labeled 6c-G2(III)-Rho
(data not shown). These results confirm that the rate limiting
step in hydrophobic PAMAM-G2-based vectors transfection is
the release of the pDNA from the endosomes to the nucleus, since
all the cells are able to uptake the dendrimer complex, but only in
a subset is the pDNA released from the internal reservoirs to the
nucleus. Fig. 7C shows CHO-k1 cells that have been pre-incubated
with chlorpromazine prior to transfection with rhodamine-labeled
9d-G2(III)-Rho complexed with Cy5-labeled pEGFP-N3 plasmid.
In these cells the uptake of dendrimers to the perinuclear space
is greatly reduced and shows an apical distribution. However, the
transfection efficiency (the blue-fluorescence associated with the
Experimental
General experimental procedures
Generation 2 PAMAM dendrimer (PAMAM-G2) was purchased
from Aldrich. Commercially available reagents 1a–c, 2, 7a–d, 12
and solvents were used as purchased without further purification.
Compound 3 was obtained from oleyl alcohol following a
procedure described in the literature.24 TLCs were performed on
Merck Silica Gel 60 F254 aluminium sheets. Reagent used for
developing plates include potassium permanganate (1% w/v),
ninhydrin (0.3% w/v) in ethanol and UV light when applicable.
Flash column chromatography was performed on Silica Gel Merck
(230–400 mesh, ASTM). Melting points were measured on a
Gallenkamp melting point apparatus and are uncorrected. Optical
rotations were recorded on a Perkin-Elmer 141 polarimeter
at room temperature. IR spectra were recorded on a Satellite
Mattson FTIR. 1H and 13C NMR spectra were recorded at room
temperature on a Varian Direct Drive (300, 400 and 500 MHz)
spectrometer. Chemical shifts are given in ppm and referenced to
internal CDCl3. J values are given in Hz. FAB mass spectra were
recorded on a Fissons VG Autospec-Q spectrometer, using m-
nitrobenzyl alcohol or thioglycerol as matrix. NALDI-TOF mass
spectra were recorded on an Autoflex Brucker spectrometer using
NaI as matrix. Electrospray Ionization (ESI) mass spectra were
recorded on a LCT Premier Spectrometer. LipofectAMINETM
2000 (LP2000) was purchased form Invitrogen (Carlsbad, CA,
USA). pEGFP-N3 plasmid (Genbank U57609) was obtained from
Clontech Laboratories (Palo Alto, CA). This 4729 bp plasmid
858 | Org. Biomol. Chem., 2011, 9, 851–864
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Fig. 7 Confocal microscopy. CHO-k1 cells were seeded onto coverslips and transfected with dendrimer complexes based on Rhodamine labeled-9d-G2
(III) PAMAM derivative and Cy5 labeled pEGFP-N3 plasmid DNA at an N/P ratio of 10. 24 h after transfection, cells were fixed and confocal
microscopy was performed to detect eGFP protein in the transfected cells (green), Rhodamine labeled-9d-G2 (III) (red) or Cy5 labeled pEGFP DNA
(blue). A merged image as well as images for each independent channel are shown. A and B.- CHO-k1 transfected cells at two magnifications. C.- CHO-k1
cells preincubated with chlorpromazine prior to transfection.
encodes for an enhanced red-shifted variant of wild-type GFP
(eGFP). Plasmid was purified from transformed bacteria using
standard methods.25 DNA concentration was measured by a
fluorimetric method using the Hoechst 33258 dye.26 For confocal
microscopy experiments, DNA was covalently labeled for 30 min
1.4 mmol) for 4a–c or Cs2CO3 (650 mg 2.0 mmol) for 4d. The
solution was magnetically stirred at room temperature until TLC
showed complete disappearance of starting materials (2 h for
4a,d, 16 h for 4b–c). After filtration, the solvent was evaporated
under reduced pressure and the crude was purified by column
chromatography. In the case of compound 4c, filtration was
followed by partial evaporation of the solvent under reduced
pressure, addition of H2O (30 mL) and extraction with CH2Cl2 (2 ¥
30 mL). The combined organic extracts were then dried (Na2SO4)
and the solvent removed under reduced pressure to give a crude
that was purified by column chromatography.
R
with Cy5 dye using Mirus labelITꢀ Tracker Intracellular Nucleic
Acid Labeling Kit CyTM5 (Madison, WI, USA) at a DNA : label
ratio 0.5 : 1 according to the manufacturer’s instructions. Unbound
label was removed by ethanol precipitation.
Synthesis of vinyl sulfone reagents for the functionalization of
PAMAM-G2
Compound 4a. Column chromatography (hexane–ether 1 : 1)
of the crude gave 4a as a liquid (127 mg, 95%), previously described
General procedure for the synthesis of thioether 4a–d. To a
solution of 2-thioethanol (70 mg, 0.9 mmol for 4a–c; 155 mg,
2.0 mmol for 4d) in deoxygenated acetonitrile (10 mL) (for 4a,b,d)
or DMSO-THF 1 : 1 (for 4c) was added the bromo derivative
1a–c or the mesyl derivative 3 (1.0 mmol) and K2CO3 (193 mg,
in the literature.27 max(film)/cm-1: 3372, 2958, 2873, 1465, 1275
n
and 1046; 1H-NMR (CDCl3, 300 MHz): d 3.73 (t, 2 H, J = 5.9 Hz),
2.74 (t, 2 H, J = 5.9 Hz), 2.54 (t, 2 H, J = 7.4 Hz), 2.32 (br s, 1 H),
1.59 (m, 2 H), 1.43 (m, 2 H), 0.93 (t, 3 H, J = 7.3 Hz); 13C-NMR
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(CDCl3, 75 MHz): d 60.3, 35.4, 31.9, 31.8, 22.0, 13.7; HRMS (m/z)
(NALDI-TOF) calcd. for C6H13S [M - OH]+: 117.0738; found:
117.0736.
3.07 (t, 2 H, J = 8.0 Hz), 1.85 (m, 2 H), 1.50–1.18 (m, 30 H),
0.88 (t, 3 H, J = 6.6 Hz); 13C-NMR (CDCl3, 100 MHz): d 56.5,
54.9, 54.7, 32.0, 29.8, 29.7, 29.6, 29.5, 29.4, 29.2, 28.6, 22.8, 22.0,
14.2. HRMS (m/z) (FAB+) calcd. for C20H42O3SNa [M + Na]+:
385.2752; found: 385.2755.
Compound 4b. Column chromatography (hexane–ether 2 : 1)
of the crude gave 4b as a syrup (214 mg, 87%). nmax(film)/cm-1:
3315, 2918, 2849, 1461, 1044 and 997; 1H-NMR (CDCl3,
300 MHz): d 3.72 (t, 2 H, J = 6.0 Hz), 2.73 (t, 2 H, J = 5.8 Hz), 2.52
(t, 2 H, J = 7.4 Hz), 2.43 (s, 1 H), 1.66–1.52 (m, 2 H), 1.45–1.15
(m, 18 H), 0.89 (t, 3 H, J = 6.6 Hz); 13C-NMR (CDCl3, 75 MHz): d
60.5, 35.5, 32.2, 31.9, 30.0, 29.9, 29.9, 29.8, 29.8, 29.6, 29.5, 29.1,
22.9, 14.3. HRMS (m/z) (ESI): calcd. for C14H31OS [M + H]+:
247.2096; found: 247.2098.
Compound 5d. Column chromatography (hexane–EtOAc 1 : 1)
gave 5d as a syrup (245 mg, 68%). nmax(film)/cm-1: 3384, 2918,
2847, 1460, 1323, and 1121; 1H-NMR (CDCl3, 400 MHz): d 5.30–
5.26 (m, 2 H), 4.06 (t, 2 H, J = 5.2 Hz), 3.13 (t, 2 H, J = 5.2 Hz),
3.00 (t, 2 H, J = 8.1 Hz), 2.47 (br s, 1 H), 1.94 (dd, 4 H, J = 11.9
and 6.1 Hz), 1.83–1.73 (m, 2 H), 1.42–1.32 (m, 2 H), 1.30–1.12
(m, 20 H), 0.81 (t, 3 H, J = 6.8 Hz); 13C-NMR (CDCl3, 100 MHz):
d 130.3, 129.9, 56.6, 55.0, 54.8, 32.1, 30.0, 29.9, 29.7, 29.5, 29.5,
29.4, 29.3, 29.2, 28.7, 27.4, 27.3, 22.9, 22.0, 14.3; HRMS (m/z)
(ESI) calcd. for C20H41O3S [M + H]+: 361.2776; found: 361.2775.
Compound 4c. Column chromatography (hexane–ether 1 : 1)
of the crude gave 4c as a solid (304 mg, 92%). M.P. 60–61 ◦C;
1
n
max(film)/cm-1: 3213, 2918, 2849, 1460, 1063 and 719; H-NMR
General procedure for the synthesis of vinyl sulfones 6a–d.
A
(CDCl3, 400 MHz): d 3.71 (t, 2 H, J = 6.0 Hz), 2.72 (t, 2 H, J =
5.6 Hz), 2.51 (t, 2 H, J = 7.4 Hz), 2.11 (s, 1 H), 1.58 (m, 2 H),
1.41–1.20 (m, 30 H), 0.87 (t, 3 H, J = 6.7 Hz); 13C-NMR (CDCl3,
100 MHz): d 60.3, 35.5, 32.0, 31.8, 29.9, 29.8, 29.8, 29.7, 29.7,
29.6, 29.4, 29.3, 29.0, 22.8, 14.2.
solution of the corresponding sulfone 5a–d (1 mmol) in anhydrous
CH2Cl2 (20 mL) was cooled by means of an ice bath. Et3N
(0.41 mL, 3 mmol for 5a–c; 0.82 mL, 6 mmol for 5d) and mesyl
chloride (0.12 mL, 1.5 mmol for 5a–c; 0.24 mL, 3.0 mmol for
5d) were then added and the solution was left to warm to room
temperature until TLC showed complete disappearance of starting
materials (2 h for 4a–b, 24 h for 4c, 6 h for 4d). The solvent was
removed under reduced pressure and the resulting crude purified
by column chromatography.
Compound 4d. Column chromatography (hexane–ether 2 : 1)
of the crude gave 4d as a syrup (308 mg, 94%). nmax(film)/cm-1:
3371, 3003, 2923, 2852, 1461 and 1045; 1H-NMR (CDCl3,
400 MHz): d 5.36–5.28 (m, 2 H), 3.69 (t, 2 H, J = 6.0 Hz), 2.70
(t, 2 H, J = 5.6 Hz), 2.49 (t, 2 H, J = 7.4 Hz), 2.35 (s, 1 H), 1.99
(dd, 4 H, J = 12.4 and 6.5 Hz), 1.60–1.52 (m, 2 H), 1.41–1.25 (m,
22 H), 0.86 (t, 3 H, J = 6.8 Hz); 13C-NMR (CDCl3, 100 MHz):
d 130.1, 129.9, 60.4, 35.4, 32.1, 31.8, 29.9, 29.8, 29.7, 29.6, 29.5,
29.4, 29.3, 29.0, 27.4, 27.3, 22.8, 14.3; HRMS (m/z) (ESI) calcd.
for C20H41OS [M + H]+: 329.2878; found: 329.2865.
Compound 6a. Column chromatography (hexane–ether 1 : 1)
gave 6a as a syrup (136 mg, 92%). nmax(film)/cm-1: 2919, 1673,
1463, 1384, 1312, 1130, 978 and 808; 1H-NMR (CDCl3, 400 MHz):
d 6.64 (dd, 1 H, J = 16.6 and 10.1 Hz, CH =), 6.44 (d, 1 H, J =
16.4 Hz, = CH2trans), 6.17 (d, 1 H, J = 10.2 Hz, = CH2cis), 2.99 (t,
2 H, J = 8.2 Hz), 1.80–1.72 (m, 2 H), 1.46 (m, 2 H), 0.95 (t, 3 H,
J = 7.4 Hz); 13C-NMR (CDCl3, 100 MHz): d 136.1, 130.4, 54.0,
24.3, 21.6, 13.5. HRMS (m/z) (ESI) calcd. for C6H13O2S [M + H]+:
149.0636; found: 149.0640.
General procedure for the synthesis of sulfones 5a–d. To a
solution of the corresponding thioether derivative 4a–d (1 mmol)
in AcOH (5 mL) was added 33% H2O2 (2 mL). The solution was
kept in the dark for 16 h. Evaporation of the solvent under reduced
pressure gave a crude that was purified by column chromatography.
Compound 6b. Column chromatography (hexane–ether 1 : 1)
gave 6b as a syrup (218 mg, 84%). nmax(film)/cm-1: 2925, 1719,
1
Compound 5a. Column chromatography (ether) gave 5a as a
solid (144 mg, 87%). M.P. 42–43 ◦C; nmax(film)/cm-1: 3450, 2961,
2875, 1462, 1272, 1122 and 1065; 1H-NMR (CDCl3, 400 MHz): d
4.12 (t, 2 H, J = 4.8 Hz), 3.21 (t, 2 H, J = 5.0 Hz), 3.09 (t, 2 H,
J = 7.9 Hz), 2.62 (br s, 1 H), 1.84 (m, 2 H), 1.49 (m, 2 H), 0.97 (t,
3 H, J = 7.3 Hz); 13C-NMR (CDCl3, 75 MHz): d 56.4, 54.9, 54.4,
23.8, 21.7, 13.6. HRMS (m/z) (ESI) calcd. for C6H15O3S [M + H]+:
167.0742; found: 167.0743.
1647, 1459, 1307, 1131, 1069, 973 and 666; H-NMR (CDCl3,
400 MHz): d 6.60 (dd, 1 H, J = 16.6 and 9.9 Hz, CH =), 6.37
(d, 1 H, J = 16.6 Hz, = CH2trans), 6.11 (d, 1 H, J = 9.9 Hz, =
CH2cis), 2.92 (t, 2 H, J = 8.1 Hz), 1.75–1.67 (m, 2 H), 1.36–1.18
(m, 18 H), 0.83 (t, 3 H, J = 6.8 Hz); 13C-NMR (CDCl3, 100 MHz):
d 136.1, 130.4, 54.3, 31.9, 29.6, 29.5, 29.3, 29.2, 29.0, 28.3, 22.7,
22.3, 14.1. HRMS (m/z) (ESI) calcd. for C14H29O2S [M + H]+:
261.1888; found: 261.1879.
Compound 5b. Column chromatography (ether) gave 5b as a
solid (230 mg, 83%). M.P. 81–82 ◦C; nmax(film)/cm-1: 3392, 2914,
2844, 1259 and 1116; 1H-NMR (CDCl3, 400 MHz): d 4.14 (t, 2 H,
J = 5.1 Hz), 3.20 (t, 2 H, J = 5.1 Hz), 3.07 (t, 2 H, J = 8.0 Hz), 1.80–
1.70 (m, 2 H), 1.48–1.40 (m, 2 H), 1.38–1.20 (m, 16 H), 0.88 (t, 3 H,
J = 6.7 Hz); 13C-NMR (CDCl3, 100 MHz): d 56.4, 54.8, 54.6, 31.9,
29.6, 29.5, 29.3, 29.2, 29.0, 28.4, 22.7, 21.8, 14.1. HRMS (m/z)
(ESI) calcd. for C14H31O3S [M + H]+: 279.1994; found: 279.1989.
Compound 6c. Column chromatography (hexane–ether 1 : 1)
gave 6c as a solid (279 mg, 81%). M.P. 61–62 ◦C; nmax(film)/cm-1:
3062, 2915, 2847, 1462, 1284, 1124, 908 and 730; 1H-NMR (CDCl3,
400 MHz): d 6.62 (dd, 1 H, J = 16.6 and 9.8 Hz, CH =), 6.43 (d, 1
H, J = 16.6 Hz, = CH2trans), 6.15 (d, 1 H, J = 9.8 Hz, = CH2cis),
2.96 (t, 2 H, J = 8.0 Hz), 1.77 (m, 2 H), 1.46–1.20 (m, 30 H),
0.88 (t, 3 H, J = 6.7 Hz); 13C-NMR (CDCl3, 100 MHz): d 136.3,
130.4, 54.4, 32.0, 29.8, 29.7, 29.7, 29.7, 29.6, 29.4, 29.3, 29.1, 28.5,
22.8, 22.4, 14.2. HRMS (m/z) (FAB+) calcd. for C20H40O2SNa
[M + Na]+: 367.2647; found: 367.2644.
Compound 5c. Column chromatography (EtOAc) gave 5c as
a solid (308 mg, 85%). M.P. 93–94 ◦C; nmax(film)/cm-1: 3368,
1
2913, 2847, 1594, 1470, 1256, 1133 and 1059; H-NMR (CDCl3,
Compound 6d. Column chromatography (hexane–EtOAc 5 : 1)
gave 6d as a liquid (274 mg, 80%). nmax(film)/cm-1: 3003,
400 MHz): d 4.13 (t, 2 H, J = 5.0 Hz), 3.19 (t, 2 H, J = 5.0 Hz),
860 | Org. Biomol. Chem., 2011, 9, 851–864
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1
2922, 2852, 1613, 1463, 1314, 1132 and 973; H-NMR (CDCl3,
in THF (50 mL) was added DVS (0.21 mL, 2 mmol) and t-
BuOK (11 mg, 0.10 mmol). The reaction mixture was magnetically
stirred (20 min) at room temperature. Evaporation of the solvent
under reduced pressure gave a crude that was purified by column
chromatography.
500 MHz): d 6.56 (dd, 1 H, J = 16.7 and 9.7 Hz), 6.37 (d, 1 H, J =
16.7 Hz), 6.09 (d, 1 H, J = 9.7 Hz), 5.32–5.23 (m, 2 H), 2.90 (t, 2
H, J = 8.1 Hz), 1.96–1.92 (m, 4 H), 1.74–1.67 (m, 2 H), 1.38–1.16
(m, 22 H), 0.81 (t, 3 H, J = 6.7 Hz); 13C-NMR (CDCl3, 125 MHz):
d 136.1, 130.3, 130.0, 129.6, 54.3, 31.9, 29.7, 29.6, 29.5, 29.3, 29.3,
29.1, 29.0, 28.3, 27.2, 27.1, 22.7, 22.3, 14.1; HRMS (m/z) (ESI)
calcd. for C20H39O2S [M + H]+: 343.2671; found: 343.2664.
Compound 9a. Column chromatography (EtOAc–hexane 2 : 1
→ EtOAc) gave 9a as a syrup (150 mg, 60%). nmax(film)/cm-1: 3380,
1
2963, 2874, 1649, 1544, 1313, 1126 and 756; H-NMR (CDCl3,
Synthesis of 2-hydroxylethyl amide 8a. A solution of butyric
acid chloride (10 mmol) in anhydrous Cl2CH2 (30 mL) was added
dropwise to a solution of 2-amino ethanol (9 mL, 15 mmol) and
Et3N (2.8 mL, 20 mmol) in anhydrous Cl2CH2 (30 mL) The
reaction mixture was kept at room temperature (15 min) and
then evaporated under reduced pressure to give a crude that
was purified by column chromatography (EtOAc) yielding 8a,
400 MHz): d 6.67 (dd, 1 H, J = 16.6 and 9.9 Hz), 6.35 (d, 1 H, J =
16.6 Hz), 6.29 (br s, 1 H), 6.07 (d, 1 H, J = 9.9 Hz), 3.82 (t, 2 H,
J = 5.5 Hz), 3.49 (t, 2 H, J = 5.1 Hz), 3.37 (q, 2 H, J = 5.2 Hz),
3.20 (t, 2 H, J = 5.5 Hz), 2.09 (t, 2 H, J = 7.5 Hz), 1.58 (m, 2 H),
0.86 (t, 3 H, J = 7.4 Hz); 13C-NMR (CDCl3, 100 MHz): d 173.3,
137.5, 129.4, 69.8, 63.8, 54.5, 38.6, 38.4, 19.0, 13.7. HRMS (m/z)
(NALDI-TOF) calcd. for C10H19NO4SNa [M + Na]+: 272.0932;
found: 272.0934.
previously described in the literature,28 as a syrup (1.18 g, 90%).
1
n
max(film)/cm-1: 3294, 2940, 2873, 1649, 1558 and 535; H-NMR
Compound 9b. Column chromatography (EtOAc–hexane 2 : 1
→ EtOAc) gave 9b as a solid (191 mg, 53%). M.P. 64–66 ◦C;
(CDCl3, 400 MHz): d 4.41 (br s, 1 H, NH), 3.56 (t, 2 H, J = 4.7 Hz),
3.26 (q, 2 H, J = 5.3 Hz), 2.08 (t, 2 H, J = 7.5 Hz), 1.53 (m, 2 H),
0.83 (t, 3 H, J = 7.4 Hz); 13C-NMR (CDCl3, 100 MHz): d 174.5,
61.4, 42.2, 38.3, 19.1, 13.6.
n
max(KBr)/cm-1: 3306, 3061, 2914, 2848, 1636, 1551, 1460, 1380,
1
1295, 1248, and 1123; H-NMR (CDCl3, 500 MHz): d 6.70 (dd,
1 H, J = 16.6 and 9.9 Hz), 6.43 (d, 1 H, J = 16.6), 6.12 (d, 1 H,
J = 9.9 Hz), 3.89 (t, 2 H, J = 5.5 Hz), 3.55 (t, 2 H, J = 5.1 Hz),
3.44 (q, 2 H, J = 5.2 Hz), 3.24 (t, 2 H, J = 5.5 Hz), 2.17 (t, 2 H,
J = 7.6 Hz), 1.61 (m, 2 H), 1.24 (br s, 16 H), 0.87 (t, 3 H, J =
6.8 Hz); 13C-NMR (CDCl3, 125 MHz): d 173.4, 137.6, 129.4, 70.0,
63.8, 54.6, 38.7, 36.7, 29.6, 29.6, 29.5, 29.3, 29.3, 25.7, 22.6, 14.1.
HRMS (m/z) (NALDI-TOF) calcd. for C18H35O4SNa [M + Na]+:
384.2184; found: 384.2189.
General procedure for the synthesis of 2-hydroxylethyl amides
8b–d. A solution of the corresponding acid 7b–d (10 mmol) in
Cl2SO (11 mL for 8b–c; 22 mL for 8d) was magnetically stirred
for 1 h at room temperature. Evaporation and coevaporation
with anhydrous toluene (3 ¥ 15 mL) under reduced pressure gave
a crude that was dissolved in anhydrous Cl2CH2 (30 mL) for
8b,d or anhydrous THF (50 ml) for 8c. This solution was added
dropwise to a solution of 2-amino ethanol (0.9 mL,15 mmol)
and Et3N (2.8 mL, 20 mmol) in anhydrous Cl2CH2 (30 mL) for
8b,d or anhydrous THF (50 ml) for 8c. The reaction mixture was
kept at room temperature (15 min) and then evaporated under
reduced pressure to give a crude that was purified by column
chromatography.
Compound 9c. Column chromatography (EtOAc) gave 9c as a
solid (249 mg, 56%). M.P. 89–91 ◦C; nmax(KBr)/cm-1: 3309, 2917,
1
2847, 1636, 1552, 1460, 1380, 1294, 1254, 1123, and 1089; H-
NMR (CDCl3, 400 MHz): d 6.70 (dd, 1 H, J = 16.6 and 9.9 Hz),
6.44 (d, 1 H, J = 16.6 Hz), 6.13 (d, 1 H, J = 9.9 Hz), 6.1 (br s,
1 H), 3.89 (t, 2 H, J = 5.5 Hz), 3.56 (t, 2 H, J = 5.0 Hz), 3.45
(q, 2 H, J = 5.2 Hz), 3.25 (t, 2 H, J = 5.5 Hz), 2.17 (t, 2 H, J =
7.7 Hz), 1.62 (m, 2 H), 1.24 (m, 28 H), 0.87 (t, 3 H, J = 6.7 Hz); 13C-
NMR (CDCl3, 100 MHz): d 173.4, 137.6, 129.4, 70.0, 63.8, 54.6,
38.7, 36.7, 31.9, 29.7, 29.6, 29.6, 29.6, 29.5, 29.4, 29.3, 29.3, 25.7,
22.7, 14.1. HRMS (m/z) (NALDI-TOF) calcd. for C24H47NO4SNa
[M + Na]+: 468.3123; found: 468.3124.
Compound 8b. Column chromatography (EtOAc) gave 8b
◦
as a solid (2.25 g, 93%). M.P. 89–91 C (lit.29 90–92 ◦C);
n
max(film)/cm-1: 3292, 2984, 2917, 1740, 1640, 1373, 1240 and
1046;1-NMR (CDCl3, 400 MHz): d 6.18 (br s, 1 H), 3.69 (t, 2
H, J = 4.9 Hz), 3.39 (q, 2 H, J = 5.1 Hz), 2.20 (t, 2 H, J = 7.6 Hz),
1.60 (m, 2 H), 1.24 (m, 16 H), 0.86 (t, 3 H, J = 6.7 Hz); 13C-NMR
(CDCl3, 100 MHz): d 174.6, 62.3, 42.4, 36.7, 31.9, 29.6, 29.6, 29.5,
29.3, 29.3, 29.3, 25.7, 22.6, 14.1.
Compound 9d. Column chromatography (EtOAc–hexane 2 : 1
→ EtOAc) gave 9d as a syrup (226 mg, 51%). nmax(film)/cm-1: 3303,
1646, 1541, 1460, 1379, 1312, 1249, and 1124; 1H-NMR (CDCl3,
400 MHz): d 6.71(dd, 1 H, J = 16.4 and 9.7 Hz), 6.45 (d, 1 H, J =
16.4 Hz), 6.14 (d, 1 H, J = 9.6 Hz), 6.12 (br s, 1 H), 5.35 (m, 2 H),
3.91 (t, 2 H, J = 5.4 Hz), 3.57 (t, 2 H, J = 5.0 Hz), 3.46 (q, 2 H, J =
5.2 Hz), 3.26 (t, 2 H, J = 5.4 Hz), 2.18 (t, 2 H, J = 7.7 Hz), 2.01 (m, 4
H), 1.62 (m, 2 H), 1.28 (several m, 20 H), 0.88 (m, 3 H); 13C-NMR
(CDCl3, 75 MHz): d 173.5, 137.6, 130.0, 129.8, 129.5, 70.0, 63.9,
54.6, 38.8, 36.7, 32.0, 29.8, 29.8, 29.6, 29.4, 29.4, 29.2, 27.3, 27.3,
25.8, 22.8, 14.2; HRMS (m/z) (FAB+) calcd. for C24H45NO4SNa
[M + Na]+: 466.2967; found: 466.2968.
Compound 8c. Column chromatography (Cl2CH2–MeOH
15 : 1 → 5 : 1) gave 8c as a solid (3.0 g, 91%). M.P. 106–108
◦C. nmax(film)/cm-1: 3294, 2917, 2848,1639, 1549, 1460 and 1214.
Spectroscopic data identical to those previously reported in
literature.30
Compound 8d. Column chromatography (EtOAc–hexane 2 : 1
→ EtOAc) gave 8d as a solid (2.88 g, 88%). M.P. 59–61 ◦C (lit.31 62–
63 ◦C); nmax(KBr)/cm-1: 3301, 1642, 1561, 1464, 1265, 1211, 1057,
and 1035; 1H-NMR data identical to those reported in literature;29
13C-NMR (CDCl3, 75 MHz): d 174.7, 130.1, 129.8, 62.4, 42.5, 36.7,
32.0, 29.8, 29.8, 29.6, 29.4, 29.4, 29.3, 29.3, 29.2, 27.3, 27.2, 25.8,
22.7, 14.2.
Compound 10d. A solution of oleic acid 7d (850 mg, 3 mmol)
in Cl2SO (10 mL) was magnetically stirred for 1 h at room
temperature. Evaporation and coevaporation with anhydrous
toluene (3 ¥ 15 mL) under reduced pressure gave a crude that was
General procedure for the synthesis of vinyl sulfones 9a–d. To a
solution of the corresponding 2-hydroxyethyl amide 8a–d (1 mmol)
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dissolved in anhydrous Cl2CH2 (30 mL). This solution was added
dropwise to a solution of cystamine dihydrochloride (260 mg,
1.15 mmol) and Et3N (0.98 mL, 6.9 mmol) in anhydrous Cl2CH2
(30 mL). The reaction mixture was kept at room temperature (15
min) and then evaporated under reduced pressure to give a crude
that was purified by column chromatography (ether–hexane 2 : 1
(several m, 21 H), 0.99 (s, 3 H), 0.92 (d, 3 H, J = 6.4 Hz), 0.86 (d,
6 H, J = 6.6 Hz), 0.67 (s, 3 H); 13C-NMR (CDCl3, 125 MHz): d
140.2, 138.0, 128.5, 122.1, 79.8, 61.5, 56.7, 56.1, 55.4, 50.1, 42.3,
39.7, 39.5, 38.8, 37.0, 36.8, 36.2, 35.8, 31.9, 31.8, 28.2, 28.1, 28.0,
24.3, 23.8, 22.9, 22.5, 21.0, 19.3, 18.7, 11.8; HRMS (m/z) (FAB+)
calcd. for C31H52O3SNa [M + Na]+: 527.3535; found: 527.3535.
◦
→ ether) yielding 10d as a solid (720 mg, 92%). M.P. 83–85 C;
n
max(KBr)/cm-1: 3320, 1632, 1536, 1464, 1419, 1261, and 1192; 1H-
Synthesis of amphiphilic PAMAM-G2 dendrimers
NMR (CDCl3, 300 MHz): d 6.28 (br s, 2 H), 5.34 (m, 4 H), 3.57 (q,
4 H, J = 6.3 Hz), 2.82 (t, 4 H, J = 6.4 Hz), 2.20 (t, 4 H, J = 7.5 Hz),
2.00 (m, 8 H), 1.63 (m, 4 H),1.28 (several m, 40 H), 0.88 (m, 6
H); 13C-NMR (CDCl3, 75 MHz): d 173.9, 130.2,129.9, 38.6, 38.1,
36.8, 32.1, 30.0, 29.9, 29.8, 29.7, 29.6, 29.6, 29.5, 29.5, 29.4, 27.4,
25.9, 22.9, 14.3; HRMS (m/z) (FAB+) calcd. for C40H76N2O2S2Na
[M + Na]+: 703.5246; found: 703.5250.
General procedure. To a solution of PAMAM-G2 (0.3 mM,
1 mg mL-1) in milliQ-water (15 mL) was added the vinyl sulfone
reagents 6a–d, 9a–d, 11d and 13 (0.15 mM for I, 0.3 mM for II and
0.6 mM for III) in THF (15 mL). The solution was magnetically
stirred at room temperature for 1 day. Then the solvent (THF) was
evaporated under reduced pressure and the water was freeze-dried.
The products were directly used in the transfection assays.
Compound 11d. To a solution of 10d (960 mg, 1.4 mmol) in
AcOH (20 mL) was added powdered Zn (1.1 g, 17 mmol). The
reaction mixture was magnetically stirred at 50 ◦C for 40 min.
After cooling and filtration over zeolite, Cl2CH2 (70 mL) was
added and the resulting organic solution was washed with water
(2 ¥ 30 mL), saturated NaHCO3 (2 ¥ 30 mL) and additional water
(30 mL). The organic phase was dried (Na2SO4) and evaporated
under reduced pressure to give a crude that was dissolved in
previously deoxygenated THF : isopropanol (2 : 1, 25 mL) by
bubbling Ar for 5 min. DVS (809 mL, 5.65 mmol) and Et3N (40
mL, 0.28 mmol) were added and the new reaction mixture was
magnetically stirred at room temperature under an Ar atmosphere
for 1.5 h. Evaporation of the solvent gave a crude that was purified
by column chromatography (ether) yielding 11d as a solid (890 mg,
69%). M.P. 85–87 ◦C; nmax(KBr)/cm-1: 3319, 1632, 1536, 1463,
Synthesis of fluorescent PAMAM-G2. To a solution of
PAMAM-G2 (0.6 mM, 2 mg mL-1) in milliQ-water (7.5 mL) was
17
added the vinylsulfone rhodamine B 14 (0.3 mM) in milliQ-
water (7.5 mL). The solution was magnetically stirred at room
temperature for 1 day and then water was freeze-dried. The
product (G2-Rho) was directly used in the transfection assays.
Synthesis of fluorescent amphiphilic PAMAM-G2 derivatives.
To a solution of PAMAM-G2 (0.6 mM, 2 mg mL-1) in milliQ-
water (7.5 mL) was added the vinylsulfone rhodamine B 14 (0.3
mM) in milliQ-water (7.5 mL). The solution was magnetically
stirred at room temperature for 1 day. Then compound 6c (0.15
mM) or 9d (0.6 mM) were added in THF (15 mL). The reaction
mixture was again magnetically stirred at room temperature for 1
day. The solvent (THF) was evaporated under reduced pressure
and the water was freeze-dried. The products (6c-G2(I)-Rho and
9d-G2(III)-Rho) were directly used in the transfection assays.
1
1418, 1261, and 1191; H-NMR (CDCl3, 300 MHz): d 6.70 (dd,
1H, J = 16.6 and 9.7 Hz), 6.48 (d, 1 H, J = 16.6 Hz), 6.23 (d, 1 H,
J = 9.7 Hz), 5.85 (br s, 1 H), 5.36 (m, 2H), 3.45 (q, 2 H, J = 6.4 Hz),
3.26 (m, 2 H), 2.91 (m, 2 H), 2.71 (t, 2H, J = 6.5 Hz), 2.18 (t, 2 H,
J = 7.6 Hz), 2.00 (m, 4 H), 1.62 (m, 2 H), 1.28 (several m, 20 H),
0.88 (m, 3 H); 13C-NMR (CDCl3, 75 MHz): d 173.5, 136.3, 131.5,
130.2, 129.9, 54.5, 38.5, 36.9, 32.8, 32.7, 32.2, 32.1, 30.0, 29.9,
29.7, 29.5, 29.5, 29.4, 27.4, 27.4, 25.9, 23.9, 22.9, 14.3; HRMS
(m/z) (FAB+) calcd. for C24H45NO3S2Na [M + Na]+: 482.2738;
found: 482.2734.
Preparation of amphiphilic PAMAM-G2/Plasmid pEGFP-N3
complexes
Amphiphilic PAMAM-G2/pDNA complexes were prepared at
several N/P ratios where N = number of primary amines in the
conjugate and P = number of phosphate groups in the pDNA
backbone. Plasmid pEGFP-N3 was used for the preparation of
the DNA complexes and for transfection assays. The quantities
of functionalized PAMAM-G2 dendrimers used were calculated
according to the desired DNA concentration of 0.1 mg mL-1,
the N/P ratio, the molecular weight and the number of positive
charges in the selected PAMAM derivative. Experiments were
performed for N/P 0.5 to 50. The desired amount of functionalized
PAMAM-G2 dendrimer was added from a 20 mg mL-1 stock
solution (1 : 2 DMSO–H2O). The solution was previously diluted
so that the desired N/P ratio was reached by mixing with an equal
volume of the plasmid solution. In this way, the DMSO proportion
was identical in all experiments (16.7% v/v). The preparation was
vortexed for a few seconds and incubated for 20 min.
Compound 13. To a solution of cholesterol 12 (300 mg,
0.77 mmol) in THF (20 mL) was added DVS (0.12 mL, 1.16 mmol)
and t-BuOK (9 mg, 0.077 mmol). The reaction mixture was
magnetically stirred at room temperature for 1 h. Amberlita IR
120H was then added and the magnetic stirring continued for
additional 30 min. After filtration, the solvent was evaporated
under reduced pressure. TLC of the crude showed the presence
of cholesterol. Ac2O (8 mL) and pyridine (4 mL) were added to
the resulting crude and the new reaction mixture was kept at room
temperature for 16 h. Acetylation of the crude reaction allowed the
separation of compound 13. Evaporation under reduced pressure
gave a crude that was purified by column chromatography (ether–
hexane 1 : 2) yielding 13 as a solid (204 mg, 52%). M.P. 133–135
◦C; [a]D -19 (c 1, chloroform); nmax(KBr)/cm-1: 3409, 1461, 1373,
1319, 1115, and 1052; 1H-NMR (CDCl3, 400 MHz): d 6.75 (dd, 1
H, J = 16.7 and 9.9 Hz), 6.40 (d, 1 H, J = 16.7 Hz), 6.07 (d, 1 H,
J = 9.9 Hz), 5.35 (br s, 1 H), 3.88 (t, 2 H, J = 5.6 Hz), 3.23 (t, 2 H,
J = 5.6 Hz), 3.19 (m, 1 H), 2.35–1.84 (several m, 7 H), 1.56–0.95
Gel electrophoresis shift assay
The binding capability of PAMAM derivatives for DNA was
analyzed by gel electrophoresis. PAMAM-G2 derivatives were
prepared in a DMSO–H2O (1 : 2) solution while DNA was
dissolved in NaCl 150 mM. pEGFP-N3 DNA (10 mL at 0.1
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mg mL-1) was mixed with an equal volume of PAMAM-G2
derivatives using N/P ratios 0–10 and incubated for 30 min at
room temperature before the addition of loading buffer (2 mL).
An aliquot (5 mL) of each sample was subjected to agarose gel
electrophoresis (0.8% w/v) in TAE buffer (40 mM Tris-acetate, 1
mM EDTA). Electrophoresis was carried out at 7 V cm-1 and gels
were stained after electrophoresis with ethidium bromide.
each data point was taken as an average over five independent
sample measurements. Solutions of dendriplexes were prepared
with a pDNA concentration of 0.1 mg mL-1 at different N/P
ratios (the optimum for transfection in each case) in the same
way as commented before. Measurements were taken on these
solutions before and after addition of DNA.
Cytotoxicity of the complexes
DNase protection assays
Cytotoxicity of the complexes formed by the functionalized den-
drimers and pDNA was evaluated 24 h after transfection. Cyto-
toxicity was assayed by determining the percentage of cell viability
(with respect to unexposed cells) using the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method,33 that
correlate the cellular metabolic activity and the number of viable
cells in culture. Results are reported as % viability based on the
untreated control cells normalized to 100% viable.
pEGFP-N3 DNA (10 mL at 0.1 mg ml-1) was mixed with 5 ml
of PAMAM-G2 derivatives to give a final N/P ratio of 5 and
incubated for 30 min at room temperature. To the mixture, 10 mL
of a solution of DNase I (0.05 mg mL-1 in Tris HCl 50 mM pH 8,
2000 U/mg) were added and incubated for 1 h at 37 ◦C. After the
digestion, 2 mL of a 10% SDS solution were added and the samples
incubated for 15 min at 65 ◦C before the addition of loading
buffer (4 mL). Finally, an aliquot (20 mL) of each sample was
subjected to agarose gel electrophoresis (0.8% w/v) in TAE buffer.
Quantification of the band intensity was performed with the NIH
Image Software.32 A value of 100 has been assigned to the intensity
of the band corresponding to the control undigested DNA.
Fluorescence and protein assay
Transfected cells were washed three times with PBS and 600 ml
of 0.5% Triton X-100 in PBS was added. Plates were shaken
for 10 min at room temperature and the lysate was recovered
and the eGFP fluorescence was quantified in a Shimadzu RF-
5301PC fluorimeter using an excitation of 480 nm (5 nm) and
510 nm (10 nm) emission wavelengths. Protein concentration
was measured using the Bio-Rad Protein Assay (Hercules, CA,
USA). In the transfection experiments, fluorescence in the positive
control using LP2000 polyplexes was assigned a value of 100
and the fluorescence results obtained in the transfections with
the PAMAM dendrimers were normalized to this value.
Cell culture and DNA transfection assays
Wild type Chinese Hamster Ovary (CHO-k1; ATCC no. CCL-
61), RAW 264.7 (ECACC No. 91062702) and Neuro-2a (ATCC
No. CCL-131) cells were grown in Dulbecco’s Modified Eagle’s
medium (DMEM) supplemented with 10% (v/v) fetal bovine
serum, 2 mM glutamine plus 100 U/mL penicillin, and 0.1
◦
mg mL-1 streptomycin. All cell lines were maintained at 37 C in
a humidified incubator containing CO2 (5%) and air (95%). Prior
to transfection, cells were seeded in 24 well plates at a density
of 2.25 ¥ 104 cells cm-2 and incubated for 24 h to reach a cell
confluence of 80–90%. For transfection experiments, pEGFP-
N3 plasmid (0.5 mg cm-2) was mixed with the corresponding
PAMAM-G2 derivative at N/P ratios of 2.5, 5, 10, 20 and 50
at room temperature for 20 min in a final volume of 20 mL. The
mixture was diluted to 0.5 mL with DMEM without serum or
supplemented with 10% serum and added to each well. Cells were
incubated with the DNA–PAMAM derivatives mixtures for 5 h,
then the transfection media was removed and cells were further
grown in DMEM media plus 10% FBS for an additional period
of 48 h. Untransfected cells and naked pEGFP-N3 were used as
negative controls. Transfections using unmodified PAMAM-G2
were always included as a control. LP2000 was also used as a
positive control in gene delivery experiments. LP2000 polyplexes
were prepared using 2 ml of LP2000 and 1.0 mg of DNA, according
to the manufacturer’s instructions. For the assay of the effects of
dendrimers internalizations, cells were preincubated for 30 min
with chlorpromazine (Sigma), 14 mM or genistein (Sigma), 200 mM
prior to the adition of the dendriplexes.
Confocal microscopy
CHO-k1 cells were seeded onto 12-mm coverslips in 24-well plates
at a density of 11250 cells cm-2 24 h before transfection. Poly-
plexes were prepared as described before, but using Cy5 labeled
pEGFP-N3 as plasmid DNA and rhodamine-labeled PAMAM
derivatives. Control experiments were carried out using either
unlabeled pEGFP-N3 plasmid or unlabeled PAMAM derivatives.
Transfection was carried out using 1 mg Cy5-labeled pEGFP-
N3 and the rhodamine labeled compound 9d-G2(III)-Rho at a
N/P ratio of 10. 5 h after transfection, the medium was changed
to complete medium and cells were grown for an additional
period of 24 h to allow expression of eGFP in the transfected
cells. Cells were fixed with 2% paraformaldehyde in PBS for 15
min at room temperature and coverslips were mounted on glass
slides using Vectashield mounting media (Vector Laboratories,
Inc., Burlingame, CA). Confocal microscopy was performed on
a Leica DMI6000 confocal microscope. To prevent cross-talk of
eGFP, rhodamine or Cy5 chromophores, distinct excitation laser
lanes and non-overlapping detection channels were selected for
their detection: eGFP was excited using the 488 nm line of a
krypton/argon laser and the emitted fluorescence was detected
with a 504–562 nm channel. Rhodamine was detected using the
He/Ne 543 nm excitation line and a 574–626 nm channel and
Cy5 was excited using a He/Ne 633 nm laser and a 651–688 nm
chanel. Under these conditions, no signal overspill among the
individual fluorescence channels was observed. For color analysis,
images were collected separately in single channel mode using a
Particle size and f potential measurements
The particle diameter was measured at 633 nm on a dynamic light
scattering instrument (MALVERN 4700C) at room temperature
with a detection angle of 60◦. Zeta potential measurements were
performed using a ZetaPALS instrument (Brookhaven, USA).
Zeta potentials were calculated using the Smoluchowsky model,
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sequential acquisition mode. All samples were exposed to laser
for a time interval not >5 min to avoid photobleaching. The laser
was set to the lowest power able to produce a fluorescent signal.
Maximum voltage of photomultipliers was used to decrease the
required laser power as much as possible. A pinhole of 1 Airy unit
was used. Images were acquired at a resolution of 1024 ¥ 1024.
Series were acquired in the xyz mode. Data was processed using
Leica AF software package.
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Statistical analysis
Results are expressed as mean
SEM for the number of
experiment indicated. The statistical significance of variations
was evaluated using ONE-WAY Analysis of Variance (ANOVA).
When significant effect was found, post hoc comparisons of the
means were done using the t adjusted Tukey’s test. A p value <0.05
was considered significant.
Acknowledgements
Financial Support was provided by Direccio´n General de Inves-
tigacio´n Cient´ıfica y Te´cnica (DGICYT) (CTQ2008-01754) Junta
de Andaluc´ıa (P07-FQM-02899) and Fundacio´n Marcelino Bot´ın.
J. M.-S. thanks the University of Granada for a research contract
(Programa Puente). A. M.-F. thanks the Spanish Ministerio de
Educacion for a research fellowship (FPU). Alberto Martin-
Molina and Cesar Rodriguez-Beas are greatly acknowledged for
their collaboration in the dynamic light scattering and z-potential
measurements.
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Megia-Fernandez, F. Perez-Balderas, F. Hernandez-Mateo and F.
Santoyo-Gonzalez, Org. Biomol. Chem., 2010, 8, 667.
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