Download Sterically stabilized self-assembling reversibly cross

Bionanotechnology II: from Biomolecular Assembly to Applications
Sterically stabilized self-assembling reversibly
cross-linked polyelectrolyte complexes with
nucleic acids for environmental and medical
applications
Martin C. Garnett*1 , Paolo Ferruti†, Elisabetta Ranucci†, Marco A. Suardi†, Mieke Heyde‡ and Rob Sleat‡
*School of Pharmacy, University Park, University of Nottingham, Nottingham NG7 2RD, U.K., †Department of Organic and Industrial Chemistry, University of
Milan, Via Venezian 21, 20133 Milan, Italy, and ‡EnviroGene Limited, Tredomen Innovation & Technology Centre, Tredomen Business Park, Ystrad Mynach,
Hengoed CF82 7FQ, Wales, U.K.
Abstract
One of the principal problems facing nucleic acid delivery systems using polyplexes is the instability of the
complexes in the presence of proteins and high salt concentrations. We have used a cross-linking polymer
to overcome this problem. Pendant thiol moieties have been incorporated into a PAA (polyamidoamine)
homopolymer and a PEG [poly(ethylene glycol)]–PAA–PEG copolymer reported previously as a selfassembling system. When mixed with DNA, small monodisperse sterically stabilized particles are formed in
quantitative yields. Optimization of the formulation resulted in nanoparticles which are stable in seawater.
This cross-linked formulation has been successfully tested in both freshwater and estuarine field trials as
a water tracer. Future work will develop these particles as a groundwater tracer and also for therapeutic
applications of nucleic acid delivery.
Introduction
One of the most difficult problems in medical therapeutics is
how to deliver nucleic acids such as genes and siRNA (small
interfering RNA) to cells. The problem relates to condensing
and protecting a large biodegradable molecule from a hostile
environment, while at the same time overcoming barriers
of adsorption and penetration of various cell membranes to
reach the target. At the end of this process, the nucleic acid
needs to be released in an active form [1]. These problems have
largely been overcome by viruses, but the use of viruses brings
other unwanted consequences. It would be more useful to be
able to incorporate the functionality of viruses into a wholly
synthetic construct [2]. One path which has been followed to
achieve this objective is the use of polymer-based complexes
known as polyplexes. These complexes are based on
an interaction of the highly negatively charged nucleic acid
with a positively charged polymer to form polyelectrolyte
complexes. With therapeutically useful sizes of nucleic
acid, this is a spontaneous and complete reaction typically
resulting in nanometre-sized (20–200 nm diameter) discrete
particles.
The theory of polyelectrolyte complexes is well known
and has been reported extensively in the literature (e.g.
[3]). Most living organisms use a similar process for DNA
condensation. A variety of cationic polymers have been used
Key words: cross-linking, DNA delivery, polyplex, self assembly, steric stabilization, water tracing.
Abbreviations used: PAA, polyamidoamine; PEG, poly(ethylene glycol); CP, PEG–PAA–PEG
copolymer; qPCR, quantitative PCR; XLP, short cross-linking homopolymer.
1
To whom correspondence should be addressed (email martin.garnett@nottingham.
ac.uk).
Biochem. Soc. Trans. (2009) 37, 713–716; doi:10.1042/BST0370713
to investigate DNA delivery [4], but few of these polymers
are both biodegradable and produce useful well-defined
non-aggregating complexes. Preparing polyplexes from a
cationic homopolymer and DNA requires an excess of one
of the components (typically the cationic polymer), resulting
in a particle which is charge-stabilized by the presence of the
excess polymer. The presence of the charged surface means
that, for some polymers, these particles can exist in a nonaggregated form. However, the presence of the charge means
that the particles can readily adsorb to negatively charged
substances and surfaces, and that they are destabilized in the
presence of salt, resulting in aggregation. To overcome
the charge problem, the usual solution is to create a sterically
stabilized surface in which a hydrophilic polymer is densely
packed on the surface leaving a neutral charge and an
entropic barrier to interactions between the particle and
other surfaces. The most effective way of achieving this
is to chemically attach PEG [poly(ethylene glycol)] to the
cationic polymer before formation of complexes [5] or to
the surface of pre-formed complexes [6].
Self-assembling PAA (polyamidoamine)
polyplexes
In our work, we have extensively investigated the linear PAAs
as cationic polymers for DNA delivery [7–11]. This is a
family of biodegradable polymers produced from a pair of
co-monomers, giving a range of possible charge and structural
characteristics [12]. These polymers generally have low cytotoxicity [7,13] and are also easily modified to produce A-B-A
C The
C 2009 Biochemical Society
Authors Journal compilation 713
714
Biochemical Society Transactions (2009) Volume 37, part 4
Figure 1 Cartoon of polyion micelles showing postulated
structure of non-cross-linked self-assembling DNA nanoparticles
DNA is thought to be circumferentially wrapped in a condensed form
and stabilized by a combination of cationic homopolymer (XLP) and the
PEG–PAA–PEG copolymer (CP).
block copolymers with terminal PEG groups. Early work
identified a particular PAA produced from MBA (methylene
bisacrylamide) and DMEDA (dimethylethylene diamine)
which produced well-structured non-aggregating particles
and had good biological activity for transfecting DNA [8].
Initial attempts by us to use the PEGylated block copolymer
to produce complexes were largely unsuccessful, as these
complexes, also known as polyion micelles, did not condense
DNA well. Similar findings were made by other groups with
other PEG–polycation copolymers. We demonstrated that
this was due to an incorrect PEG/cationic polymer ratio,
which could be overcome by using appropriate mixtures of
homopolymer and PEG copolymer [10]. This methodology
resulted in small discrete non-aggregating particles with a
neutral surface, indicating that steric stabilization had been
achieved (the proposed structure of particles is shown in
cartoon form in Figure 1). These nanoparticles formed
instantaneously and resulted in an essentially quantitative
incorporation of both DNA and cationic polymer.
Despite this success in self-assembly, polyelectrolyte
complexes suffer from two major shortcomings. First that
they are equilibrium systems like micelles, and secondly
that they are unstable (disintegrate) in the presence of high
salt concentrations. How far the equilibrium is in favour
of particle formation and particle stability are difficult
to measure. However, the instability is an acknowledged
problem in developing polyplex DNA-delivery systems [14].
We (M.C.G. and P.F.) were approached by EnviroGene
Ltd who wanted to use DNA as a tracer in water courses and
also required a protected DNA nanoparticle system. Their
requirements for this application were very similar to ours,
in that they needed to protect the DNA, condense it into a
very large number of small particles, have particles which did
not adsorb to materials in the environment and to be readily
released to be able to determine the sequence and amount
of DNA present in a water sample by qPCR (quantitative
PCR). In addition, they needed a biocompatible system
which was relatively cheap and easy to manufacture in large
C The
C 2009 Biochemical Society
Authors Journal compilation quantities. Working together, we have devised and produced
an appropriate reversibly cross-linked formulation.
Development of self-assembling
cross-linked polyplexes
We wanted to incorporate a disulfide-based cross-linker to
allow easy release of the DNA after sampling. Our twocomponent self-assembling system was an ideal starting
point, as disulfide bonds can be formed readily by reacting
a reduced thiol group with a thiol group activated by 2thiopyridine. The introduction of thiol groups was readily
accomplished by a new PAA polymer synthesis where some
of the aminic component was replaced by a mono-Boc (tbutoxycarbonyl)-protected cystamine [15]. This disulfidecontaining polymer can then be readily converted into either
the reduced thiol- or thiopyridyl-substituted polymer. To
give the two-component system required, PEG–PAA–PEG
copolymer (CP) with reduced thiol groups and a short crosslinking homopolymer (XLP) with thiopyridyl activated
groups were produced (the polymer structures are presented
in Figure 2). Mixing the copolymers with DNA resulted in
the spontaneous formation of cross-linked nanoparticles. A
cartoon depicting the cross-linked structure is shown in Figure 3. One of the important features of the chemistry used in
the production of these cross-linked particles is that the only
side product is the release of a small quantity of thiopyridyl
group on disulfide bond formation, so no clean-up of product
is required. Initial experiments showed that >99% of added
oligonucleotide was incorporated into the complexes.
The main work on developing this system was the
relationship between formulation and nanoparticle function
and properties. The non-cross-linked system already has a
number of variables such as polymer molecular mass, ratio
of CP to XLP, cationic polymer segment/DNA ratio, which
affect nanoparticle size, structure and surface properties.
For the cross-linked polymer, this is made more complex by
other factors such as amount and positioning of cross-linking
groups in each polymer component. We have examined a
number of different combinations of CPs and XLPs varying
in these different polymer specifications over a range of
XLP/CP and PAA/DNA ratios. For the environmental
system, a 60–80-mer oligonucleotide is required, so initial
work was carried out with a degraded salmon sperm DNA
preparation and later work with defined oligonucleotides.
Characterization of cross-linked polyplexes
It was found that the PAA/DNA ratio needed to be close
to 1. The XLP/CP ratio needs to vary to accommodate the
differing chain lengths of PEG and PAA, but the usable
range of ratios is limited because of the need to have
a reduced thiol/activated thiol ratio of more than 1 to
ensure full cross-linking. To achieve appropriate stability
of the nanoparticles, approx. 25% of the PAA monomers
needed to have pendant thiol groups. Varying the XLP/CP
Bionanotechnology II: from Biomolecular Assembly to Applications
Figure 2 Structures of homopolymer (XLP) and PEG–PAA–PEG copolymer (CP) containing thiol (sulphydryl) blocks prepared for
cross-linked nanoparticles
Note that only the right-hand end of the copolymer molecule is shown, the thiol and PEG blocks at the left-hand end of the
triblock copolymer have been omitted for clarity.
Figure 3 Cartoon depicting the cross-linking between the
homopolymer (XLP) and copolymer (CP) designed to occur at the
surface of the particle through placement of the thiol (sulphydryl)
blocks near the terminal PEG moieties
the DNA to pass through the membrane. Some release of
oligonucleotide from the cross-linked nanoparticles was
seen, particularly in the presence of high salt concentrations.
In these experiments, we could still detect particles by DLS
(dynamic light scattering), but when viewed by transmission
electron microscopy, the particles resembled empty cages.
Further small modifications to the formulation resulted
in particles which now gave an acceptable retention of
DNA even in 600 mM salt, equivalent in concentration to
seawater.
Manufacture of self-assembling polyplexes
and PAA/DNA ratios resulted in a variety of particle
morphologies ranging from toroids to spheres.
We determined the stability of the particles against
aggregation by measuring the turbidity of the particles in
the presence of sodium sulfate, but it was difficult to find
an objective assay for the stability of the particles to
disintegration in the present of salt. We eventually found
that filtering the particles through a centrifugal ultrafiltration
membrane and measuring the release of DNA by qPCR
proved to be a good indicator of stability. However, a
large-pore-size membrane (>100 kDa) is required to allow
The self-assembly properties of these particles were
important in providing an easy manufacturing process.
In small-scale preparation of these nanoparticles using a
batch method, factors such as the order of addition of
reagents are very important in determining size and quality
of nanoparticles. Polymer concentration is also important,
with high concentrations resulting in larger particles and
aggregated particles. We determined that using a continuous
flow mixing method actually resulted in a better particle
production with a smaller and more consistent particle size.
Because of the relatively low polymer concentration, the two
polymer components can be mixed together before assembly
without reacting substantially with each other until addition
of the DNA, when well-condensed particles are formed. We
have currently made nanoparticle batch sizes of up to 210 mg
of DNA using this procedure. We believe that this process
should be infinitely scalable for larger batch sizes.
Cross-linked polyplexes as water-borne
tracers
Using these cross-linked nanoparticles, field trials have
been carried out at both a freshwater and an estuarine site
C The
C 2009 Biochemical Society
Authors Journal compilation 715
716
Biochemical Society Transactions (2009) Volume 37, part 4
to compare the performance of the small DNA tracers
with the existing Rhodamine WT dye used in water-tracing
applications. Some details of the River Fruin trial are given
here. The River Fruin is well channelled, and the trial took
place over a 6.1 km stretch of the river. After depositing the
dye [2 litres of 20% (w/v) dye] and DNA tracer [1 litre, 1014
particles (2.5 mg of DNA)] in the river, samples were taken
at 10 or 20 min intervals and continued for 22 h. The dye was
detected fluorimetrically, and the DNA tracers were detected
by qPCR using a 20 μl sample of river water without further
treatment. For analysis of DNA, the heating cycle of the PCR
process is sufficient to break down the disulfide linkages and
release the DNA for enzyme action in the assay. Comparison
with the dye showed a similar time to peak for appearance
of the DNA tracer at the sampling site at 610 min. The dye
became undetectable after the main peak had passed, but
the presence of the DNA tracer was detectable well above
background for a further 5 h after the main flow peak had
passed.
These tests have demonstrated that the new tracers are
comparable with the previous dyes in terms of tracing water
flow, but have greater sensitivity, and can be used repeatedly
and with an infinite number of tracers at the same site,
which is not possible with the current dye technology. In
addition, DNA does not colour the river visibly as happens
with the large amount of dye necessary at present. Further
development of the water-tracing technology is in progress
to enable its use as a groundwater tracer.
The new technology developed for the water-tracing
application has many of the characteristics that are necessary
for use as a delivery system for therapeutic nucleic acids.
Further development of this self-assembling system for
medical applications is now being pursued.
Funding
The work on cross-linked formulations was supported by a CTC
fellowship for M.H. from an Engineering and Physical Sciences
Research Council Collaborative Training Account held by the
University of Nottingham [reference number GR/T11326/01] and
by funding from Envirogene Ltd.
C The
C 2009 Biochemical Society
Authors Journal compilation References
1 Garnett, M.C. (1999) Gene delivery systems using cationic polymers.
Crit. Rev. Ther. Drug Carrier Syst. 16, 147–208
2 Lehn, P., Fabrega, S., Oudrhiri, N. and Navarro, J. (1998) Gene delivery
systems: bridging the gap between recombinant viruses and artificial
vectors. Adv. Drug Delivery Rev. 30, 5–11
3 Kabanov, A.V. and Kabanov, V.A. (1998) Interpolyelectrolyte and block
ionomer complexes for gene delivery: physicochemical aspects.
Adv. Drug Delivery Rev. 30, 49–60
4 Gebhart, C.L. and Kabanov, A.V. (2001) Evaluation of polyplexes as gene
transfer agents. J. Controlled Release 73, 401–416
5 Seymour, L.W., Kataoka, K. and Kabanov, A.V. (1998) Cationic block
copolymers as self-assembling vectors for gene delivery. In
Self-Assembling Complexes for Gene Delivery: from Laboratory to
Clinical Trial (Kabanov, A.V, Felgner, P.L. and Seymour, L.W., eds),
pp. 219–239, Wiley, Chichester
6 Dash, P.R., Read, M.L., Fisher, K.D., Howard, K.A., Wolfert, M., Oupicky, D.,
Subr, V., Strohalm, J., Ulbrich, K. and Seymour, L.W. (2000) Decreased
binding to proteins and cells of polymeric gene delivery vectors surface
modified with a multivalent hydrophilic polymer and retargeting through
attachment of transferrin. J. Biol. Chem. 275, 3793–3802
7 Hill, I.R.C., Garnett, M.C., Bignotti, F. and Davis, S.S. (1999) In vitro
cytotoxicity of poly(amidoamine)s: relevance to DNA delivery. Biochim.
Biophys. Acta 1427, 161–174
8 Jones, N.A., Hill, I.R.C., Stolnik, S., Bignotti, F., Davis, S.S. and Garnett,
M.C. (2001) Polymer chemical structure is a key determinant of
physicochemical and colloidal properties of polymer–DNA complexes for
gene delivery. Biochim. Biophys. Acta 1517, 1–8
9 Rackstraw, B.J., Martin, A.L. Stolnik, S. Roberts, C.J., Garnett, M.C., Davies,
M.C. and Tendler, S.J.B. (2001) Microscopic investigations into PEGcationic polymer-induced DNA condensation. Langmuir 17, 3185–3193
10 Rackstraw, B.J., Stolnik, S., Davis, S.S., Bignotti, F. and Garnett, M.C.
(2002) Development of multicomponent DNA delivery systems based
upon poly(amidoamine)–PEG co-polymers. Biochim. Biophys. Acta 1576,
269–286
11 Heyde, M., Partridge, K.A., Howdle, S.M., Oreffo, R.O.C., Garnett, M.C. and
Shakesheff, K.M. (2007) Development of a slow non-viral DNA release
system from PDLLA scaffolds fabricated using a supercritical CO2
technique. Biotechnol. Bioeng. 98, 679–693
12 Ferruti, P. (1996) Ion-chelating polymer (medical applications). In
Polymeric Materials Encyclopedia (Salamonem, J.C., ed.), pp. 3334–3359,
CRC Press, Boca Raton
13 Ranucci, E., Spagnoli, G., Ferruti, P, Sgouras, D. and Duncan, R. (1991)
Poly(amidoamine)s with potential as drug carriers: degradation and
cellular toxicity. J. Biomater. Sci. Polym. Ed. 2, 303–315
14 Neu, M., Germershaus, O., Behe, M. and Kissel, T. (2007) Bioreversibly
crosslinked polyplexes of PEI and high molecular weight PEG show
extended circulation times in vivo. J. Controlled Release 124, 69–80
15 Ranucci, E., Ferruti, P., Suardi, M.A. and Manfredi, A. (2007)
Polyamidoamines with 2-dithiopyridine side substituents as
intermediates to peptide polymer conjugates. Macromol. Rapid
Commun. 28, 1243–1250
Received 5 January 2009
doi:10.1042/BST0370713