DEPARTMENT OF ENGINEERING AND ARCHITECTURE

PHD SCHOOL OF NANOTECHNOLOGY

Dendrimers: from their interactions with albumin and genetic material !

to a taste of the crossing of biological membranes.!
Domenico Marson, Sabrina Pricla, Erik Laurinib
aSupervisor, bTutor

DEA (Department of Engineering and Architecture),

MOSE (Molecular Simulation Engineering), University of Trieste
Viologen dendrimers!

Core (C)

In contrast to the toxicity exhibited by isolated viologen monomer,

their introduction as building blocks for the design of polycationic
dendrimers allowed De Clercq et al.1 to point out the activity of various
viologen dendrimers against human immunodeficiency virus.
Recently, Ciepluch et al.2 studied the biological properties of
seven new viologen-phosphorus dendrimers, based on the monomers
showed on the azure box, with general structure C-L-V-(B-V)n-T.

R
R
P
S
R

Branching point (B)

or core (C)

R
N

N
P
R

Me
N

Linker (L)
R

Viologen (V)

Terminal (T)

R
R
OEt
P
O

OEt

100
80
60
40
20

rdf_Den of D6_PFS and D4 and D2_PFS and

15

R = 0.81673

10

05

0
0

11

13

15

Radius of gyration [ ]
10

15

17
20

19
25

21
30

35

dist

rdf_BZA of D6_PFS and D4 and D2_PFS and

0.12
D6_PFS
D4
D2_PFS

0.1
0.08

g(r)

(r)

Simulating each dendrimer alone in explicit

solvent, and also in complex with human
serum albumin (HSA) we gave some insight in
the structure of this new family of dendrimers.

D6_PFS
D4
D2_PFS

g(r)

Hemolytic activity
[%]

0.06
0.04
0.02
0
0

- we found correlations between some

biological properties studied by Ciepluch
and their morphology (first plot).

10

15

r[]

20

25

30

35

dist

rdf_VIN of D6_PFS and D4 and D2_PFS and

0.4
D6_PFS
D4
D2_PFS

g(r)

0.3

cyan
blue
red
green

0.2
0.1

- we described the tendency to backfold of

the terminal residues of higher generation
dendrimers (second plot, the R(g) of the
terminal residues of two G1, in green and
red, and one G2 dendrimer, blue).

= G1 dendrimer
= G2 dendrimer
= dendrimers residues not involved
= proteins residues involved

Radius of gyration
[]

Asphericity

Fractal
dimension

SASA [ 2 ]

CS1

8.90 0.27

0.0107

2.095

1823 60

CS2

10.02 0.18

0.0072

2.115

2632 56

CS3

9.49 0.24

0.0132

2.126

2170 60

CS4

10.62 0.20

0.0108

2.093

2520 53

Carbosilane Dendrimers!
Water-soluble carbosilane dendrimers containing peripheral amine groups have recently been
described by Klajnerts group as biocompatible molecules with potential as non-viral carriers
(low toxicity profiles, no antigenicity).1 It was previously shown that such dendrimers form
complexes (dendriplexes) with oligonucleotides. e eciency of formation and the stability
of the dendriplexes depend on electrostatic interactions with the oligonucleotides. Dendriplex
formation significantly decreases the interactions between oligonucleotides and albumin,
therefore its important for their pharmaceutical role.
rough all atom simulations in explicit solvent we derived some morphological features of
these dendrimer, and then we studied their interaction with two oligodeoxynucleotides.
In this step we used a kind of Steered Molecular Dynamics to pull the dendrimer near the
genetic material (as showed in the picture for three dierent docking positions). In this way
we avoided to arbitrary put each dendrimer in some random conformation near the
oligodeoxynucleotide.

0
0

10

15

20

25

30

35

dist

rdf_WAT of D6_PFS and D4 and D2_PFS and

3
D6_PFS
D4
D2_PFS

2.5

g(r)

2
1.5
1
0.5
0
0

10

15

20

25

30

35

dist

rdf_Na of D6_PFS and D4 and D2_PFS and

0.015
D6_PFS
D4
D2_PFS

0.01
g(r)

- exploiting the eective-binding concept,

we realized that having an higher number
of branches (G2) doesnt mean a dendrimer
will bind better with HSA.

Dendri
mer

0.005

References:
0
0
5
10
15
20
25
30
35
1.
Asaftei, S.; De Clercq, E. "Viologen Dendrimers as Antiviral Agents: e Eect of Charge Number and
Distance.
J. Med. Chem.
2010, 53,
34803488.
dist
2.
Ciepluch, K.; Katir, N.; El Kadib, A.; Felczak, A.; Zawadzka, K.; Weber, M.; Klajnert, B.; Lisowska, K.; Caminade, A.; Bousmina,
M.; Bryszewska, M.; Majoral, J. P. Biological Properties of

rdf_Cl of D6_PFS and D4 and D2_PFS and

New Viologen-Phosphorus Dendrimers Mol. Pharmaceutics, 2012, 9 (3), 448457.
0.02
0.015
g(r)

Impact of siRNA Overhangs for Dendrimer-Mediated siRNA

Delivery!

D6_PFS
D4
D2_PFS

References:
1.
Ortega P, Bermejo JF, Chonco L, de Jesus E, Javier de la Mata F, Fernndez G et al (2006) Novel water-soluble carbosilane dendrimers: synthesis and biocompatibility. Eur J Inorg Chem
7:13881396. doi:10.1002/ejic.200500782

Membrane-crossing!

0.01

0.005

10

15

20

25

30

35

dist

Dendrimers are emerging as

appealing small interfering RNA
(siRNA) delivery vectors; siRNA
molecule have attracted considerable
attention as compelling therapeutics
recently.
Using siRNA with complementary
overhangs oer the best action in
term of gene silencing
rdf_PFS of D6_PFS and D4 and D2_PFS and

0.1

D6_PFS
D4

g(r)

0.08
0.06
0.04
0.02
0
0

10

15

20

25

30

35

dist

MD snapshot of the complex between

dendrimer G5 and the sticky siRNA
dimer [siRNA(A7/T7)]. "

Another key issue in the behavior of dendrimers as nanocarriers is their ability to cross the cell
membrane to ultimately release their cargo inside the cell. us, we are currently studying the
crossing of a bilayer membrane by dendrimers using a combined umbrella sampling/SMD
approach. We are currently trying to use all-atom simulation, with the recently released
Amber Lipid11 forcefield.

Here are shown two snapshots from our

simulations (water is represented as VMDquicksurf, while the lipid heads and the
dendrimer are represented as solventaccesible surfaces). e membrane in the
pictures is made of POPE, POPC and
cholesterol in 2:2:1 proportion; the
dendrimer is a G0 viologen dendrimer.

While siRNA bearing noncomplementary long overhangs show considerably higher gene
silencing potency than normal siRNA, they are nevertheless still less eective than sticky siRNA
bearing complementary overhangs, most probably through the formation of concatemers in this
last case. For siRNA bearing noncomplementary overhangs, the sum of eects of overhang
length, nature, and flexibility plays a major role in determining their ultimate performance in
siRNA delivery and the resulting gene silencing.

We studied the interactions between the structurally flexible

fifth-generation TEA-core PAMAM dendrimer (G5) and
siRNA molecules with dierent overhangs.1
Our results, from computer modeling, show that among the
siRNA with noncomplementary overhangs considered, those
characterized by (dT)n/(dT)n overhangs appear to oer the
best compromise in terms of dendrimer binding and
unbinding characteristics. ese conclusions greatly correlate
with experimental findings according to which siRNA with
(dT)n/(dT)n overhangs are endowed with the best G5mediated siRNA delivery and gene silencing,

MD snapshot of the complex between

dendrimer G5 and the siRNA with 7nucleotide long (dT)7/(dT)7 overhangs."

References:
1.
Paola Posocco, Xiaoxuan Liu, Erik Laurini, Domenico Marson, Chao Chen, Cheng Liu, Maurizio Fermeglia, Palma Rocchi, Sabrina Pricl, and Ling Peng, "Impact of siRNA Overhangs for
Dendrimer-Mediated siRNA Delivery and Gene Silencing, Mol. Pharm. 2013, 10, 32623273, doi: 10.1021/mp400329g.

e picture above is a snapshots from the

equilibration phase, with the dendrimer
right near the membrane. e second
picture is a cross-section of the dendrimer
while its starting to penetrate the
membrane.