Dendrimeric amide- and carbamate-linked lysine-based efficient molecular transporters

Article abstract of DOI:10.1039/c7ob02552a

Amide- and carbamate-linked dendrimeric oligomers are reported as molecular transporters. They effectively complex with pDNA and transport it into cells at an efficiency superior to Lipofectamine, when complexation is carried out by incubation overnight.

Full text of DOI:10.1039/c7ob02552a

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
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Dendrimeric Amide- and Carbamate-linked Lysine-based Efficient
Molecular Transporters
Received 00th January 20xx,
Amit Kumar Yadav,a,b Namit Dey,c Sabyasachi Chattopadhyay,c Munia Gangulic,* and Moneesha
Accepted 00th January 20xx
Fernandesa,b,
*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Amide- and carbamate-linked dendrimeric oligomers are reported poly(ethoxyethylglycinamide) (PEE-G).11 However, in the
as molecular transporters. They effectively complex with pDNA clinical context, there are still issues related to toxicity,
and transport it into cells at an efficiency superior to bioavailability and biocompatibility that limit their
Lipofectamine, when complexation is carried out by incubation application.12 Therefore, there exists a need for development
overnight. The carbamate-linked K2C, is superior to amide-linked of new efficient molecular transporters that are non-toxic,
K2A; their pDNA complexes have very low associated cytotoxicity. freely water-soluble, are resistant to degradative enzymes, and
that can carry cargo molecules inside cells.
Cationic molecular transporters, including mainly peptides, are
growing in importance to solve the problem of efficiently
delivering cell-impermeable cargo molecules such as drugs,
nucleic acids or diagnostic agents into cells. Besides peptides,
oligoureas,1 oligocarbamates,2 peptoids3 and even cationic
polymers4 have been explored for cell penetration. Among the
cell-penetrating peptides (CPPs),5 the presence of cationic
arginine residues and their spatial presentation along the
peptide backbone have been shown to be instrumental in
deciding the ability to penetrate cells.6
We describe herein, the synthesis, cell penetration and cargo
delivery properties of new lysine-based dendrimers that
contain either amide- or carbamate- linkages at their branch
points. The synthesis of a generation-2 dendrimeric structure
is described (Figure 1), that results in the presentation of eight
terminal guanidinium functionalities, for cell penetration.
Carbamate linkages were chosen on the basis of our earlier
report,2b where we showed that (R-X-R)-motif oligocarbamates
displayed highly desirable cell penetration properties.
gx
Dendrimers have
a polyvalent structure that present a
gx
xg
k
concentration of several functionalities of the same type,
enabling amplification of interactions with complementary
groups. They can be used for drug encapsulation and
controlled delivery; the degree of encapsulation generally
increases with the dendrimer generation. Cationic dendrimers
are also highly water-soluble and dendrimeric peptides are
more resistant to proteolytic degradation than linear peptides,
thus increasing their half-life in biological systems. They can,
therefore, function as transporters for the delivery of cargo
molecules into cells.7 Some commonly known dendrimeric
polymers that are capable of cell penetration include the
poly(amido amine) (PAMAM),8 poly(propylene imine) (PPI),9
k
k
k
k
(FAc/cf)
K
gx
gx
k
βAla NH2
gx
xg
k
gx
Figure 1. Schematic representation of the designed amide- and carbamate-linked
dendrimers. The coloured circles represent the branch points, where the linkages were
either amide or carbamate in K2A or K2C respectively; green and blue circles represent
the two different dendrimer generations. βAla = β-alanine, K = lysine, FAc = N-acetyl
phenylalanine, cf
residues with either amide- or carbamate- linkages at the attachment points; k = lysine
or lysine-derived carbonate monomer and 6-aminohexanoic acid or 6-
= 5(6)-carboxyfluorescein, lower case letters k and x represent
x
=
aminohexanoic acid-derived carbonate monomer, g = guanidinium group.
poly(lysine)-based
dendrimers10
and
The dendrimers were designed to have terminal guanidinium
groups, known to promote cell penetration, appended through
6-aminohexanoic acid-based spacers.
A
generation-2
a.Organic Chemistry Division, CSIR-National Chemical Laboratory (CSIR-NCL), Dr.
Homi Bhabha Road, Pune 411008, India. E-mail: m.dcosta@ncl.res.in
b.Academy of Scientific and Innovative Research (AcSIR), CSIR-NCL Campus, Pune,
India
dendrimeric structure was selected as it would result in the
presentation of eight guanidinium groups, comparable to the
same number of arginine residues in our control oligomer, (R-
X-R)4 (X = 6-aminohexanoic acid), one of the best-known
synthetic cell-penetrating peptides for cargo delivery.13 The
dendrimers are shown to be able to penetrate cells, complex
c. CSIR-Institute of Genomics and Integrative Biology, Mathura Road, New Delhi
110020, India. E-mail: mganguli@igib.in
Electronic Supplementary Information (ESI) available: experimental procedures,
chemical structures, NMR
DOI: 10.1039/x0xx00000x
&
MALDI-TOF spectra, cell studies. See
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Journal Name
with plasmid DNA (pDNA) cargo and transport it efficiently into and diisopropylcarbodiimide (DIPCDI) as the coupling agents
cells, with negligible associated cytotoxicity. yielded the resin-linked fluoresceinylated dendrimers 11A and
The carbonate monomers 3a and 3b2b for the synthesis of the 11C respectively. In the absence of carboxyfluorescein, L-
carbamate-linked dendrimers were synthesized according to phenylalanine (Phe) was coupled to the Nε-amino group of
Scheme 1. The ester functions in Nα-Boc-L-Lys(N-Boc)-methyl lysine in 9A or 9C (Scheme 3) to facilitate the calculation of
ester 1a and N-Boc-6-aminohexanoic acid methyl ester 1b concentration by UV-absorbance. In this case, the Nα-amino
were reduced by in situ-generated LiBH4 to yield the group of phenylalanine was subsequently acetylated (12A and
corresponding alcohols 2a and 2b respectively. The hydroxyl 12C respectively) to rule out its contribution towards the
groups in 2a and 2b were each activated as their p-nitrophenyl cationic charges of the dendrimer. TFA-mediated cleavage of
carbonates by treating with p-nitrophenylchloroformate in dry the Boc-protecting groups in 12A and 12C, followed by
pyridine and CH2Cl2 to get the N-Boc-protected monomers 3a guanidinylation, yielded the resin-linked dendrimers 13A and
and 3b2b respectively, amenable for use in oligocarbamate 13C respectively. The synthesized dendrimers 11A
,
11C
and 13C were cleaved from the solid support using TFA-TFMSA
cleavage protocol to obtain K2A-cf K2C-cf K2A and K2C
, 13A
synthesis.
,
,
respectively. The chemical structures of the dendrimers are
illustrated in the ESI (Figure S1). They were purified by RP-
HPLC and characterised by MALDI-TOF mass spectroscopic
analysis (Table 1), after re-checking their purity by analytical
RP-HPLC on a C18 column.
Table 1. Lysine-based dendrimers of the study.
Scheme 1. Synthesis of carbonate monomers for carbamate-linked dendrimer
synthesis. Reagents and conditions: (i) NaBH4:LiCl (1:1) (2.5 equiv), THF:EtOH (3:4) (2a,
90%; 2b, 92%); (ii) p-nitrophenyl chloroformate (1.2 equiv), dry pyridine (2.5 equiv),
CH2Cl2 (3a, 80%; 3b, 85%).
Sr.
Na
Dendrimer
MALDI-TOF Mass
No.
me
Calcd.
Obsd.
The generation-2 dendrimers of this study were synthesized in
a divergent strategy by solid phase synthesis, described in
Schemes 2 and S1 (ESI). For the carbamate-linked dendrimers
(Scheme 2), the N-Boc-protected p-nitrophenyl activated
1.
2.
3.
4.
K2A
{[(C(=NH2+)NH2-X)2-K]2-K}2-K-
K(Phe-Ac)-βAla-NH2(amide)
2542.74
2711.71
2992.90
3163.81
2547.29
2717.16
2998.39
3170.11
K2A {[(C(=NH2+)NH2-X)2-K]2-K}2-K-K(cf)-
carbonate monomers 3a
& 3b were incorporated at
appropriate positions during synthesis on the solid support,
while for the amide-linked dendrimers (ESI, Scheme S1), Nα-
Boc-Lys(Nε-Boc)-COOH and N-Boc-6-aminohexanoic acid were
employed. MBHA resin was used as the solid support and Boc-
chemistry protocols were used to assemble the dendrimers by
following repetitive cycles of deprotection, neutralization and
-cf
βAla-NH2(amide)
K2C
{[(C(=NH2+)NH2-x)2-k]2-k}2-k-
K(Phe-Ac)-βAla-NH2(carbamate)
K2C
{[(C(=NH2+)NH2-x)2-k]2-k}2-k-K(cf)-
βAla-NH2(carbamate)
-cf
coupling.
spacer, to give
residue, yielding
β
-Alanine was first coupled to the solid support as a
4
, followed by an orthogonally-protected lysine
K = lysine, X = 6-aminohexanoic acid, k = carbamate-linked lysine derivative from
5
, to enable differential attachment of 3a, x = carbamate-linked 6-aminohexanoic acid derivative from 3b, C(=NH2+)NH2-
= guanidinylation at N-terminus of dendrimer, Phe-Ac = Nα-acetyl phenylalanine,
cf = 5(6)-carboxyfluorescein, βAla = β-alanine at the C-terminus of dendrimer.
fluorescein or phenylalanine prior to cleavage from the
support. The deprotection and coupling steps were monitored
by the Kaiser test. For the coupling step, pre-activated
monomers in the form of p-nitrophenyl carbonates were used
for generation of carbamate linkages (Scheme 2), while
appropriate coupling agents were used with the designated N-
Boc-protected amino acids for the generation of amide
linkages (ESI, Scheme S1). Accordingly, resin-linked compounds
6C through 9C were obtained bearing carbamate linkages,
while compounds 6A through 9A contained amide linkages.
For the synthesis of fluorescently-labeled dendrimers (Scheme
3), 9A or 9C was first treated with TFA to cleave the Boc-
protecting groups, followed by guanidinylation of the resulting
amine using 1H-pyrazole-1-carboxamidine hydrochloride in the
presence of Hünig's base, to obtain 10A and 10C respectively.
Removal of the Fmoc-protecting group of the lysine-Nε-amino
group was achieved by treatment with piperidine, and
subsequent coupling with 5(6)-carboxyfluorescein using HOBt
CD spectroscopic studies were carried out to study the
secondary structural features of the dendrimers. Spectra were
recorded in water and in the presence of TFE (Figure 2). The
CD spectrum of the carbamate-linked dendrimer, K2C, was
almost featureless, probably
a result of the flexibility
associated with the introduction of the carbamate linkage, as
observed earlier for (r-x-r)-motif carbamates.2b On the other
hand, the CD spectrum of the amide-linked dendrimer, K2A,
that showed a maximum at 216 nm and a minimum at 201 nm
in water, changed significantly in the presence of TFE, when it
displayed minima at 206 nm and 228 nm. Thus, the less
structured and flexible K2C, with its cationic guanidinium
groups, might be more amenable to adopt conformations
favouring interactions with cellular membranes resulting in
better cellular entry.
2 | J. Name., 2012, 00, 1-3
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NH-Fmoc
O
NH-Fmoc
O
NH-Boc
O
O
O
O
O
(ii), (iv)
(ii), (iv)
(ii), (iii)
(i)
N
Boc-HN
N
H
Boc-HN
N
H
H
N
H2N
N
Boc-HN
N
H
H
H
6C
4
5
O
O
6
NH-Boc
Boc-HN
HN
O
O
6
O
O
NH
O
HN
HN
NH-Boc
O
O
N
NH-Boc
O
O
H
6
NH-Boc
NH
O
HN
NH-Boc
O
O
Boc-HN
NH-Fmoc
O
O
Boc-HN
O
Boc-HN
O
6
N
O
N
O
H
NH-Boc
H
O
O
O
O
NH-Fmoc
NH-Fmoc
O
N
(ii), (v)
(ii), (iv)
O
O
N
H
NH
O
N
H
H
N
N
O
O
H
H
O
O
O
O
O
O
N
N
NH
O
N
N
NH
NH
N
H
N
H
H
O
H
H
H
O
O
NH
O
O
O
O
O
NH
O
NH-Boc
O
NH
O
6
O
NH
NH-Boc
7C
6
O
O
NH-Boc
NH
O
NH
Boc-HN
HN
Boc-HN
O
O
O
Boc-HN
O
6
NH-Boc
Boc-HN
6
NH-Boc
Boc-HN
9C
8C
Bocx
xBoc
Bocx
k
k
k
H
Bocx
KFmoc Ala
=
k
β
N
Bocx
k
k
Bocx
xBoc
k
Bocx
carbamate-linked
Scheme 2. Solid phase synthesis of carbamate-linked lysine-based dendrimer precursor 9C. Reagents and conditions: (i) Boc-βAla-OH (3.0 equiv), HOBt (3.0 equiv), TBTU (3.0
equiv), DIPEA (3.0 equiv), DMF; (ii) 50 % TFA/CH2Cl2; (iii) Boc-Lys(Fmoc)-OH (3.0 equiv), HOBt (3.0 equiv), TBTU (3.0 equiv), DIPEA (3.0 equiv), DMF; (iv) 3a (3.0 equiv), DIPEA (3.0
equiv), DMF; (v) 3b (3.0 equiv), DIPEA, DMF.
xg
k
xg
xg
xg
xg
k
k
xg
k
k
xg
H
N
(iii), (iv)
k
k
k
xg
xg
xg
H
Kcf
xg
βAla N
k
KFmoc βAla
xg
k
xg
(v)
k
k
xg
k
k
xg
k
xg
xg
xg
xg
k
(i), (ii)
g = guanidine
cf = 5(6)-carboxyfluorescein
k
xg
xg
xg
k
Kcf
xg
βAla NH2
10A/10C
11A/11C
k
k
K2A-cf
K2C-cf
Bocx
xBoc
k
xg
Bocx
k
k
k
Bocx
Bocx
Bocx
H
k
KFmoc βAla
xBoc
N
xg
k
xg
k
k
xg
k
k
k
k
Bocx
k
k
xg
xg
xg
KFAc βAla NH2
9A/9C
k
K2A
K2C
xg
(iii), (vi), (iii), (vii)
xg
xg
Bocx
xg
k
xBoc
xg
Bocx
k
(v)
k
k
k
k
k
H
xg
xg
xg
(i), (ii)
Bocx
Bocx
Bocx
H
N
KFAc βAla
k
KFAc βAla
N
k
k
k
k
xg
xBoc
k
k
xg
Bocx
KFAc
= N-acetylphenylalanine
g = guanidine
12A/12C
13A/13C
Scheme 3. Synthesis of guanidinylated and fluorescein-labeled dendrimers. Reagents and conditions: (i) 50 % TFA/CH2Cl2; (ii) 1H-pyrazole carboxamidine hydrochloride (10.0 equiv
per amine), DIPEA (10.0 equiv per amine), DMF; (iii) 20 % piperidine/DMF; (iv) 5(6)-carboxyfluorescein (10.0 equiv), HOBt (10.0 equiv), DIPCDI (10.0 equiv), DMF; (v) TFA, TFMSA, 1,
2-ethane dithiol, thioanisole; (vi) Fmoc-Phe-OH (3.0 equiv), HOBt (3.0 equiv), TBTU (3.0 equiv), DIPEA (3.0 equiv), DMF; (vii) Ac2O (10.0 equiv), pyridine.
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4
3
K2A in water
K2C in water
K2A in 90% TFE/water
K2C in 90% TFE/water
observed for K2C at charge ratio charge ratio 5, while at a
charge ratio of 10, no uncondensed pDNA was observed. In
comparison, with K2A, a small amount of uncondensed pDNA
was visible even at the charge ratio of 10. On the whole, the
charge ratio 10 was considered suitable for pDNA
complexation and used in all further cargo delivery studies.
The release of complexed pDNA from its complexes with
dendrimers was studied by exposure to anionic challenge,
achieved by addition of increasing heparin concentrations
(Figure 5). Complete release of pDNA was observed from the
complexes formed by K2A at a ratio of 1:5 (dendrimer:heparin,
w/w), while no release was observed from the K2C complexes
even at a ratio of 1:10 (dendrimer:heparin, w/w). This could
account for the lower transfection efficiency observed for this
dendrimer with complexes prepared by 1 h incubation as
described in subsequent sections. However, when the
complexes were prepared by incubation for 24 h, very high
release of pDNA was observed from the K2C complexes even
at lower heparin concentrations. This indicates a better
stability-release balance in case of the K2C complexes in
comparison to K2A complexes.
2
1
0
-1
-2
-3
-4
200
210
220
230
240
250
260
Wavelength (nm)
Figure 2. CD spectra of the K2A and K2C oligomers (500 µM) in water and 90%
TFE/water.
The ability of the dendrimers to penetrate cells was evaluated
in CHO-K1 and HaCaT cells. As seen in Figure 3, the dendrimers
were able to penetrate cells efficiently, with almost 100 % cells
showing the presence of internalized dendrimers with high
mean fluorescence intensity. The percentage of fluorescent
cells was similar to the corresponding non-dendrimeric
oligomer (R-X-R)4 in CHO-K1 and HaCaT cells; and the mean
intensities were either slightly lower or slightly higher than the
non-dendrimeric counterpart indicating the efficiency of the
designed dendrimers.
Figure 4. DNA condensation profile for complexes of (A) K2A and (B) K2C dendrimers
with pDNA at increasing charge ratios.
Figure 3. Uptake of the dendrimers in (A) CHO-K1 and (B) HaCaT cells. The cells were
treated with the dendrimers (5 µM) for 4 h at 37 °C.
The ability of the dendrimers to carry pDNA cargo into cells
was next evaluated. Each dendrimer in the present study bears
eight guanidinium groups that are positively-charged at
physiological pH. The cationic nature of the dendrimers
conveniently allows their complexation with negatively-
charged cargo such as DNA through charge interactions.
Further, the efficacy of the cargo is significantly dependent on
its release from the molecular transporters, once inside the
cells. We therefore evaluated the ability of the dendrimers to
form complexes with pDNA; subsequent release of the pDNA
was studied through exposure to anionic challenge by the
addition of heparin. Thus, K2A and K2C were separately pre-
mixed with pDNA at different charge ratios (Z+/-, N:P;
Figure 5. Heparin-induced pDNA release study from the complexes with K2A (left
panels) and K2C (right panels). The complexes were prepared by incubating the
dendrimers with pDNA for 1 h (upper panels) or overnight (lower panels) respectively.
U/T = untreated.
Transfection was studied in CHO-K1 and HaCaT cells that were
treated with complexes pre-formed by mixing K2A or K2C
dendrimers with pMIR-Report luciferase plasmid DNA (Figure 6
and 7 respectively). For complex formation, a charge ratio of
10 was used. LipofectamineTM 2000 was used as a standard
transfection agent for comparison. K2C-pDNA complexes were
found to be more efficient at transfection in comparison to
positively-charged
N in dendrimer to negatively-charged
phosphate in DNA). The resulting complexes were analysed by
their mobility in agarose gels in an electrophoretic mobility
shift assay (Figure 4). The carbamate-linked K2C was found to
be more efficient at condensing DNA, than the corresponding
amide-linked K2A. Almost complete condensation was
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K2A-pDNA complexes in both cell lines studied. When the HaCaT cells were treated with the fluorescein-labeled
complexes were prepared by overnight incubation, the dendrimers, K2A-cf and K2C-cf, following which, the cells were
transfection efficiency was even higher than that of imaged to show the intracellular localization of the
LipofectamineTM 2000. Given the fact that the transfection dendrimers. As seen in Figure S3 (ESI), K2C-cf was taken up to
efficiency of LipofectamineTM 2000 in vivo is limited by its high a larger extent than K2A-cf. Further, the fluorescence appears
associated cytotoxicity, this study illustrates the potential of to be distributed in the cytoplasm as well as in the nucleus and
the K2C dendrimer as a molecular transporter that can be used nucleoli, illustrating their cell penetration efficiency.
at a lower concentration, with minimal cytotoxic effects.
Conclusions
In summary, we have designed and synthesized efficient
lysine-based generation-2 dendrimers bearing amide- or
carbamate-linkages at the branch points. The dendrimers
possess good cell-penetration properties, and are capable of
delivering cargo molecules such as pDNA into cells at an
Figure 6. Transfection of CHO-K1 cells by pMIR-Report luciferase complexes with K2A
efficiency that is comparable or even better than the standard
and K2C dendrimers. (A) Dendrimer-pDNA complexes were prepared by incubation for
transfection agent, Lipofectamine, when their complexes with
1 h. (B) Comparison of transfection when K2C-pDNA complexes were prepared by
pDNA were prepared by incubation overnight. Taken together
incubation for 1 h or overnight.
with their lower cytotoxicity, this makes them worthy
candidates for further development as molecular transporters.
Conflicts of interest
There are no conflicts to declare.
Figure 7. Transfection of HaCaT cells by pMIR-Report luciferase complexes with K2A
Notes and references
and K2C dendrimers. (R-X-R)4/Dendrimer-pDNA complexes were prepared by
‡ MG and MF gratefully acknowledge research funding (BSC0302) from CSIR, New
Delhi. AKY thanks CSIR, New Delhi, for a research fellowship.
incubation (A) for 1 h or (B) overnight.
The cytotoxicity of the K2A and K2C dendrimers alone and in
the form of complexes with pDNA was assessed by the
standard MTT cell viability assay (Figures 8 and S2 (ESI)). While
K2A by itself showed a slight toxic effect in CHO-K1 cells
(Figure 8C), the cytotoxicity of the dendrimer-pDNA complexes
was minimal even after 24h. This data is consistent with that
obtained for the (R-X-R)4-amide and -carbamate oligomers,2b
making K2A and K2C advantageous over commercial
transfection agents such as Lipofectamine, which displayed
considerable cytotoxicity in comparison.2b
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Table of Contents entry
Graphical abstract
Carbamate- and amide-linked generation-2 dendrimeric oligomers transport pDNA into cells very
efficiently when complexed by incubation overnight.
H2N
NH2
NH
H2N
NH2
NH2
N
O
H2
HN
O
O
HN
HN
HN
O
O
O
O
NH
O
N
H
O
NHAc
O
N
O
NH2
HN
H
NH
O
H2N
O
N
O
H
N
O
O
H
O
O
O
N
NH
O
NH2
N
H
H
O
NH
O
O
O
O
O
NH
O
NH
O
O
NH
N
NH
NH
O
2NH
HN
NH
H2
Ctrl
Lipo
K2C
(R-X-R)4
K2A
H2
N
O
NH2
O
O
NH2
NH2
N
NH
H
H2
N
K2C
NH2