Removable nanocoatings for siRNA polyplexes

Article abstract of DOI:10.1021/bc100197e

To assist in overcoming the in-herent instability of nucleic acid-containing polyplexes in physiological solutions, we have here set out to develop removable nanocoatings for modifying the surface of siRNA-based nanoparticles. Here, N-(2-hydroxypropyl)methacrylamide (HPMA) based copolymers containing carbonylthiazolidine-2-thione (TT) reactive groups in their side chains bound via disulfide spacers to the polymeric backbone were synthesized, and these copolymers were used to coat the surface of polyplexes formed by the self-assembly of anti-Luciferase siRNA with the polycations polyethylene imine (PEI) and poly(HPMA)-grafted poly(l-lysine) (GPL). The coating process was monitored by analyzing changes in the weight-average molecular weight (M _w), the hydrodynamic radius (R_h), and the zeta-potential (ζ) of the polyplexes, using both static (SLS) and dynamic (DLS) light scattering methods. The outlined methods resulted in the attachment of, on average, 28 polymer molecules to the surface of the polyplexes, forming a ～5-nm-thick hydrophilic stealth coating. Initial efforts to develop RGD-targeted coated polyplexes are also described. Atomic force microscopy (AFM) showed an angular polyplex structure and confirmed the narrow size distribution of the coated nanoparticles. The stability of the polymer-coated and uncoated polyplexes was evaluated by gel electrophoresis and by turbidity measurements, and it was found that modifying the surface of the siRNA-containing polyplexes substantially improved their stability in physiological solutions. Using polymer-coated GPL-based polyplexes containing anti-Luciferase siRNA, we finally also obtained some initial proof-of-principle for time- and concentration-dependent target-specific gene silencing, suggesting that these systems hold significant potential for further in vitro and in vivo evaluation.

Full text of DOI:10.1021/bc100197e

ARTICLE
pubs.acs.org/bc
Removable Nanocoatings for siRNA Polyplexes
,†
†
†
†
‡
‡
ꢀ
ꢀ
ꢀ
Libor Kostka,* Cestmír Koꢀnꢁak, Vladimír Subr, Milena Spírkovꢁa, Yoseph Addadi, Michal Neeman,
||
Twan Lammers,§, and Karel Ulbrich†
†Institute of Macromolecular Chemistry, v. v. i., Academy of Sciences of the Czech Republic, Heyrovskꢁy sq. 2, 162 06, Prague 6,
Czech Republic
‡Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel
§Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16,
3584 CA Utrecht, The Netherlands
Department of Experimental Molecular Imaging, RWTH - Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany
ABSTRACT: To assist in overcoming the in-
herent instability of nucleic acid-containing
polyplexes in physiological solutions, we have
here set out to develop removable nanocoat-
ings for modifying the surface of siRNA-based
nanoparticles. Here, N-(2-hydroxypropyl)-
methacrylamide (HPMA) based copolymers
containing carbonylthiazolidine-2-thione (TT)
reactive groups in their side chains bound via
disulfide spacers to the polymeric backbone
were synthesized, and these copolymers were
used to coat the surface of polyplexes formed
by the self-assembly of anti-Luciferase siRNA
with the polycations polyethylene imine (PEI)
and poly(HPMA)-grafted poly(L-lysine)
(GPL). The coating process was monitored
by analyzing changes in the weight-average
molecular weight (Mw), the hydrodynamic radius (Rh), and the zeta-potential (ζ) of the polyplexes, using both static (SLS) and
dynamic (DLS) light scattering methods. The outlined methods resulted in the attachment of, on average, 28 polymer molecules to
the surface of the polyplexes, forming a ∼5-nm-thick hydrophilic stealth coating. Initial efforts to develop RGD-targeted coated
polyplexes are also described. Atomic force microscopy (AFM) showed an angular polyplex structure and confirmed the narrow size
distribution of the coated nanoparticles. The stability of the polymer-coated and uncoated polyplexes was evaluated by gel
electrophoresis and by turbidity measurements, and it was found that modifying the surface of the siRNA-containing polyplexes
substantially improved their stability in physiological solutions. Using polymer-coated GPL-based polyplexes containing anti-
Luciferase siRNA, we finally also obtained some initial proof-of-principle for time- and concentration-dependent target-specific gene
silencing, suggesting that these systems hold significant potential for further in vitro and in vivo evaluation.
’ INTRODUCTION
that still restrict therapeutic use of nonviral vectors relate to their
instability in vivo and to nonspecific interactions of PEC with
cells of the immune system and blood plasma proteins during
their transport to target cells. Both instability and the uptake by
macrophages and the resulting rapid elimination of vectors from
the bloodstream by cells of the reticuloendothelial system can be
successfully suppressed by modifying the surface of polyplexes
with covalently linked hydrophilic polymers. Such stealth coat-
ings significantly improve stability, they prolong circulation times
Gene therapy provides a possibility for the treatment of
various human diseases in situations where conventional ap-
proaches are not feasible or ineffective.1 However, the expansion
of gene therapy is limited by the development of safe, sufficiently
stable, specific, and efficient gene delivery systems. The delivery
systems must be able to protect DNA or RNA from degradation
during transport, provide targeted delivery to the pathological
site, and promote target cell-specific uptake and intracellular
trafficking.2 The gene delivery vectors, such as polyelectrolyte
complexes (PEC) of DNA,1 RNA,3,4 or modified viruses, have
been developed to overcome these barriers and efficiently
mediate in vivo expression of genes. However, major limitations
Received: April 23, 2010
Revised:
December 6, 2010
Published: January 10, 2011
r
2011 American Chemical Society
169
dx.doi.org/10.1021/bc100197e Bioconjugate Chem. 2011, 22, 169–179
|

Bioconjugate Chemistry
ARTICLE
in plasma, and they suppress nonspecific cell entry.5,6 Moreover,
the wide tissue distribution of vectors can be restricted by binding
of ligands that are able to selectively interact with receptors ex-
pressed on the surface of target cells.7,8 However, although such
(targeted) polymeric coatings often sufficiently protect the vec-
tors from rapid dissociation and from undesirable interactions
with blood components, and although they generally enable
efficient and target cell-specific uptake, they often also prevent
the escape of DNA (or RNA) from the delivery system to the
cytosol, which results in dramatic decreases of transfection
efficiency.9
’ MATERIALS AND METHODS
Materials. Methacryloyl chloride, 1-aminopropan-2-ol, 3,30-
dithio-dipropionic acid (DTDP), 4,5-dihydrothiazole-2-thiol,
N,N0-dicyclohexylcarbodiimide (DCC), 4-(dimethylamino)pyr-
idine (DMAP), triethylamine (Et3N), 2,20-azobis(isobutyro-
nitrile) (AIBN), tetrahydrofurane (THF), N,N-dimethylfor-
mamide (DMF), dimethyl sulfoxide (DMSO) and branched
polyethylenimine (PEI, Mw = 25 000, dn/dc = 0.21) were pur-
chased from Sigma-Aldrich. N-(3-Aminopropyl)methacrylamide
hydrochloride was from Polysciences, Inc. FlucDsiRNA (siRNA,
Mw = 17 930, dn/dc = 0.185, mass/charge = 391 g/mol) was
obtained from Integrated DNA Technologies (Leuven, Belgium).
Synthesis of cyclic form of the RGD peptide c(RGDfK) and its
scrambled analogue was described previously.14 All other chemi-
cals and solvents were of analytical grade.
Monomer Synthesis. N-(2-Hydroxypropyl)methacrylamide
(HPMA) was synthesized by a modified reaction of methacryloyl
chloride with 1-aminopropan-2-ol in dichloromethane in the
presence of sodium carbonate.15 Synthesis of 3-(N-methacry-
loylglycylglycyl)thiazolidine-2-thione monomer (Ma-GlyGly-TT)
was performed as described in ref 16.
N-3-[3-(Methacryloylamino)propyl]-7-(diethylamino)cou-
marine-3-carboxamide. This was prepared by reaction of
N-(3-aminopropyl)methacrylamide (2.5 mg, 13.9 μmol) and
7-(diethylamino)coumarine-3-carboxylic acid N-succinimidyl
ester (5 mg, 13.9 μmol) in 300 μL DMF for one hour at room
temperature. Crude product was purified on a Sephadex LH-20
column with methanol as the mobile phase and recrystallized
from a mixture of ethyl acetate and diethyl ether. HPLC analysis
(Chromolith column, gradient of water in acetonitrile of
5-100%) gave a single peak at 340 nm with a retention time of
13.6 min.
Synthesis of N-1-3-[(3-[3-Oxo-3-(2-thioxo-1,3-thiazolan-
3-yl)propyl]disulfanylpropanoyl)amino]propyl-2-methyla-
crylamide (Ma-SS-TT). 2-Methyl-N-[3-[3-[3-oxo-3-(2-thiox-
othiazolidin-3-yl)propyl]disulfanylpropanoylamino]propyl]
prop-2-enamide (DTDP-(TT)2, 1.77 g, 4.3 mmol; synthesis
described previously17) was dissolved in 20 mL of THF. To this
solution, 0.36 g (2 mmol) of N-(3-aminopropyl)methacrylamide
hydrochloride dissolved in a mixture of 5 mL THF and 5 mL
methanol containing 0.33 mL (2.4 mmol) Et3N was added
dropwise, and the reaction mixture was stirred for 30 min at
room temperature. Product Ma-SS-TT was purified on a silica gel
column in a mobile phase of dichloromethane and acetone (1:1),
and then recrystallized from a mixture of dichloromethane and
diethyl ether. Yield: 0.34 g (47%). HPLC analysis (Chromolith
column, gradient of water in acetonitrile of 5-100%) gave a
single peak at 305 nm with a retention time of 8.6 min. Elemental
analysis: calcd/found C = 44.11/44.35%; H = 5.78/5.71%;
N=9.65/9.45%; S=29.44/28.92%. 1H NMR 300 MHz (CDCl3,
296 K) ppm: 1.66-1.69 (m, 2H), 1.97 (s, 3H), 2.61 (t, 2H),
2.92-2.99 (m, 4H), 3.27-3.35 (m, 6H), 3.67 (t, 2H), 4.58 (t,
2H), 5.34 (s, 1H), 5.76 (s, 1H), 6.37 (s, 1H), 6.67 (s, 1H).
Synthesis of Copolymers. All copolymers were prepared by
radical solution copolymerization of HPMA and the respective
comonomers in DMSO using AIBN as the initiator. Polymeri-
zation was carried out under a nitrogen atmosphere in a sealed
ampule at 60 °C for 6 h.15 Structures of all copolymers are shown
in Scheme 1.
In light of this, novel, highly hydrophilic and multivalent
copolymers based on N-(2-hydroxypropyl)methacrylamide
(HPMA) containing reactive carbonylthiazolidine-2-thione
(TT) side chain groups bound via reductively cleavable disulfide
linkages to the polymer backbone have been developed. These
polymers enable surface modification of suitable DNA or RNA
nanoparticle carriers with surface nanolayers that are removable
by the reductive environment of the cytosol of target cells. The
disulfide bridge can be cleaved by an interaction with reductive
agents forming two thiol groups, or may interchange their
substituents with other thiols or disulfides.10,11 This reductive
reaction represents an important function in biological systems,
and is especially interesting in the design of drug, protein, RNA,
and DNA delivery systems, since the natural reductive agent
L-glutathione (L-R-glutamyl-L-cysteinyl glycine, GSH) occurs in
concentrations of 0.1-10.0 mmol/L inside living cells, while the
extracellular level of GSH is 3 orders of magnitude lower.12 New
reactive copolymers containing reductively degradable spacers
can be used in the design of new polymer-coated gene delivery
vectors that are relatively stable during their transport in the
bloodstream, but disintegrate and release siRNA or DNA after
cellular internalization. This process increases gene expression
and the efficacy of RNA interference. The properties of new,
highly hydrophilic polymers bearing reactive TT groups attached
via biodegradable spacers and the efficiency of coating of poly-
plexes with the polymers were compared with those of previously
described polymers containing nondegradable spacers, which
were successfully used for the coating of both viral and nonviral
gene delivery vectors.13
In this paper, as a first step toward the development of poly-
plexes with improved in vivo stability and prolonged in vivo
circulation times, we describe the synthesis and physicochemical
characterization of HPMA-based multivalent copolymers con-
taining TT reactive groups bound to the polymer backbone via
spacer-containing disulfide bonds. In addition, we also studied
their use for the coating of PEC prepared by the self-assembly of
antiluciferase siRNA with polyethylene imine (PEI) or poly-
(HPMA)-grafted poly(L-lysine) (GPL) copolymer, and we ex-
tensively analyzed their physicochemical properties. The coating
process was monitored by changes in the weight-average molec-
ular weight (Mw), the hydrodynamic radius (Rh), and the zeta
potential (ζ) using either static (SLS) or dynamic (DLS) laser
light scattering methods, and the stability of the coated (siRNA/
GPL/PHPMA) or uncoated (siRNA/GPL) siRNA complexes
was examined by gel electrophoresis and by turbidity measure-
ments in physiological solutions. Finally, to provide some initial
experimental evidence for biological activity, we also evaluated
the ability of the coated and uncoated complexes to induce
target-specific gene silencing; using MDA-MB-231 human breast
cancer cells double-transfected with luciferase and red fluores-
cence protein (RFP).
Polycationic graft copolymer PLL-g-poly(HPMA) (GPL,
Mw =2 ꢀ105, dn/dc = 0.18, mass/charge = 344.5 g/mol) was
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Scheme 1. Structure of HPMA Copolymers Containing TT Reactive Groups in Side Chains
Table 1. Characteristics of Reactive HPMA Copolymers
NaCl) to the filtered solution of GPL (8 mL, 0.584 mg/mL,
0.01 M NaCl).
Mwcp (g/mol)
Mw/Mn
TT (mol %)
Surface Modification of PEC. A freshly prepared aqueous
solution of HPMA copolymer containing TT groups was added
to a stirred solution of PEC prepared with an excess of amino
groups (j = 1.5). Optimal conditions for aminolysis were
achieved by addition of 1 M HEPES buffer (pH 8.7, 40 μL
per mL) to reach the final pH of 8.2. The reaction was carried out
at room temperature, typically for 0.5 to 1.0 h. An excess of
polymer TT groups in comparison to free amino groups on the
PEC surface was used and the concentrations of the coating
polymers ranged from 0.5 to 2.0 mg/mL. After the coating
reaction, the excess of the unreacted polymer was removed by
centrifugal filtration, repeated 5 times (Millipore Amicon Ultra-4
with Ultracel-100 membrane).
PEC Characterization Using Light Scattering Methods.
Weight-average molecular weight was determined by static light
scattering (SLS) carried out with an ALV goniometer. The
instrument was equipped with an intensity-stabilized 22 mW
He-Ne laser as the light source and a PC for data recording. The
accuracy of the measurements was found to be approximately
1%. The scattering curves were measured 2-3 times after prepa-
ration of PEC and 2-3 times after their surface modification.
The refractive index increments, dn/dc, of the individual
components of the complexes were taken from the literature.
The complex concentrations (cpec) and refractive index incre-
ments (dn/dc)pec of the complexes were calculated as a function
of the molar mixing ratio, j (positive to negative charge ratio),
on the basis of the model of complex formation given in our
previous paper.4
POL 1
POL 2
POL 3
85 300
56 000
52 800
1.7
1.9
1.4
9.9
8.2
9.0
prepared by reaction of poly(HPMA) grafts (Mn = 4 300)
terminated with succinimidyloxycarbonyl end groups with high-
molecular-weight PLL (Mw = 130 000).3
Characterization of Reactive Polymers. The content of TT
groups in copolymers was determined spectrophotometrically
using UV absorption with a Specord 205 (Analytik Jena,
Germany) spectrophotometer (ε305 = 9 500 L/mol.cm, methanol).
Weight (Mw) and number (Mn) average molecular weights were
determined by size exclusion chromatography on a HPLC
system LC-10 (Shimadzu, Japan) equipped with a DAWN 8
multiangle light scattering detector, an Optilab rEX (Wyatt
Technology Corp., USA), a UV detector, and a Superose
6 column (300 ꢀ 10 mm, GE Healthcare, USA) using 0.3 M
sodium acetate buffer, pH 6.5, as a mobile phase at a flow rate of
0.5 mL/min.16 Characteristics of reactive polymers are summar-
ized in Table 1.
Preparation of siRNA/PEI Complexes. Formation of siRNA/
PEI complexes (w/w = 1) was achieved by flash addition of filtered
PEI solution (40 μg/mL) to siRNA solution (40 μg/mL) with
vigorous stirring. Formed complexes were filtered over 0.22 μm
PVDF filters (saturated with PEI).
Preparation of siRNA/GPL Complexes. The siRNA/GPL
PECs were prepared with various charge ratios (j) from 20 to 1.
We found that the complexes prepared with j = 1.5 are the most
suitable for subsequent coating reactions and their stability was
reasonable. Since low concentrations of salt may decrease the
level of aggregation by 2 orders of magnitude,18 all these com-
plexes were prepared in the presence of 0.01 M NaCl. These
PECs were prepared by slow addition from a linear dispenser
(rate of 15 mL/h) of siRNA solution (4 mL, 0.69 mg/mL, 0.01 M
The static light scattering data were analyzed by a Zimm plot
2
Kcpec
Rg q2
1
¼
pec þ
3Mw
ð1Þ
pec
RðqÞ
Mw
where R(q) is the Rayleigh ratio of the scattering intensity,
q = (4π/λ)sin θ/2, λ is wavelength in the medium, θ is the
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scattering angle between the incident and the scattered beam,
K is a contrast factor containing the optical parameters, cpec is the
complex concentration, Mw is the weight-average of the molar
mass of the complex particles, and Rg is their radius of gyration
(calculated from the z-average of the square). The concentration
dependence was neglected, which seemed to be justified because
of the low concentrations of the PEC solutions (∼10-5 g/mL).
Extrapolation to the zero scattering angle was carried out by
linear or quadratic fits of the scattering curves. The experimen-
tal error of Mw determination for the complexes was typically
about 2%.
scattering amplitude from DLS. This procedure could be suc-
cessfully used when the scattered intensities of different particle
types were comparable and their contributions to the distribution
of particle sizes (e.g., Rh) were sufficiently distinct. Since both of
these requirements were fulfilled in the systems under investiga-
cc
tion, the weight-average molecular weight of coated PEC (Mw
)
could be estimated even in the presence of coating polymers and
small amount of high-molecular-weight PEC aggregates (nanogels
formed by the reaction of excessive PEI and multivalent poly-
mer) in the solution.
If the light scattering intensity is comparable to that of PEC,
the weight fraction of the aggregates must be on the order of
0.01 (1 wt %) and Mwpec can be calculated using the scattering
intensity generated from coated PEC alone at their original
concentration, cpec. The weight-average molecular weight of PEC
is then calculated using the regular Zimm plot procedure.
ζ-Potential. The ζ-potentials of uncoated and coated PEC
were measured using a Nano-ZS, model ZEN3600 (Malvern
Instruments, UK). At least 10 measurements of each sample were
carried out to check reproducibility. The measurements of the
electrophoretic mobility were converted to ζ-potentials (mV)
using the Smoluchowski approximation. A reference measure-
ment using the Malvern ζ-potential standard was run prior to
each sample analysis to check for correct instrument operation.
Atomic Force Microscopy (AFM). The nanoparticles (con-
centration 1 mg/mL) were deposited on fresh mica substrate,
and the surface morphology (height image) and the sum of
tip-sample interactions (phase image) were characterized by
AFM. All measurements were performed under ambient condi-
tions using a commercial atomic force microscope (NanoScope
Dimension IIIa, MultiMode Digital Instruments, Santa Barbara,
CA, USA) equipped with a SSS-NCL probe, Super Sharp Silicon -
SPM-Sensor (NanoSensors Switzerland; spring constant
35 Nm-1, resonant frequency 173 kHz). The tapping mode
AFM technique was used for collecting the images. This techni-
que allowed two- and/or three-dimensional information to be
obtained of both height and material heterogeneity contrast
with high resolution when recording height and phase shifts
simultaneously.
Kinetics of Complex Coagulation. The kinetics of coagula-
tion of the siRNA/polycation complexes was measured by
turbidity in 0.15 M NaCl solutions. The complexes were prepared
and coated with polymer, as described above, at standard
conditions in 0.01 M NaCl solutions. The turbidity of the com-
plexes was measured spectrophotometrically at λ = 500 nm and
started immediately after increasing the ionic strength of the
complex solutions up to 0.15 M NaCl by the fast addition of 4 M
NaCl stock solution.
Electrophoresis. The ability of coating copolymers to cova-
lently bind on the PEC surface to retard the release of siRNA was
determined by horizontal agarose gel electrophoresis. The coat-
ed complexes of siRNA/GPL with POL1 or POL3 (siRNA/
GPL/PHPMA) were formed as described above at j = 1.5. The
samples were loaded on E-Gel agarose gel (2%) with SYBR
Safe and run for 15 min in an E-Gel iBase from Invitrogen. The
gel images were made by a Canon EOS 400D digital camera at
280 nm.
The hydrodynamic radius of particles (Rh) was determined by
dynamic light scattering (DLS) carried out on the same ALV
instrument in the angular range 30-140°. An ALV 6000 multi-
bit, multitau autocorrelator covering approximately 12 decades in
delay time (τ) was used for measurements of time autocorrela-
tion functions. The autocorrelation functions were analyzed
using the method of cumulants (a quadratic fit). From the first
cumulant, Γ, the diffusion coefficient, D, was calculated from the
equation D = Γ(q)/q2, where q is the scattering vector q = 4πn
sin(θ/2)/λ, n is the refractive index of a solvent, θ is the
scattering angle, and λ is the wavelength of the incident light.
The polydispersity index (PDI), which reflects the size poly-
dispersity of the complexes, was obtained from the second
moment (cumulant), μ2, where PDI = μ2/Γ2. The inverse
h
Laplace transform using the REPES19 method of constrained
regularization, which is similar in many respects to the inversion
routine CONTIN,20 was used for an analysis of the time
autocorrelation functions obtained from multicomponent solu-
tions. REPES directly minimizes the sum of the squared differ-
ences between the experimental and calculated intensity time
correlation functions using nonlinear programming. This method
uses an equidistant logarithmic grid with fixed components
(here, a grid of 20 components per decade) and determines
their amplitudes. As a result, a scattered light intensity distribu-
tion function, A(τ), of delay times was obtained (τ = 1/Γ) that
could be easily transformed in a distribution function of hydro-
dynamic sizes.
The hydrodynamic radius (Rh) was calculated from the
diffusion coefficient (D) using the Stokes-Einstein equation
Rh ¼ kT=6πηD
ð2Þ
where k is the Boltzmann constant, T is the absolute temperature,
and η (0.894 cP) is the viscosity of water at 25 °C. The
experimental error of Rh determination for the complexes was
typically about 3%.
Evaluation of the Weight-Average Molecular Weight of
PEC in the Presence of Coating Polymer and Particle
Aggregates. In the presence of aggregates and coating polymer
molecules in solution, the weight-average molecular weight of
polyplexes (Mwpec) was estimated using a combination of
dynamic and static light scattering experiments.21 If several kinds
of particles were present in the solution under investigation, the
total scattered intensity (Ist) could be expressed as the sum of
their individual contributions. The analysis of dynamic light
scattering data provided, in addition to hydrodynamic sizes of
particles, corresponding relative scattering amplitudes of the
distinguished particle types present in the tested solution. Thus,
the scattering intensity generated by particles of special interest
(e.g., PEC) could be extracted from the total scattering intensity
of the mixed solution by multiplication of Ist by their relative
Luciferase Assay. MDA-MB-231 human epithelial breast
adenocarcinoma cells expressing both luciferase and red fluores-
cent protein (RFP) were kindly provided by Prof. Yoram
Salomon and were grown in RPMI media supplemented with
10% FCS L-glutamin and antibiotics. Cells were plated in 96
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ARTICLE
well plates (at a density of 55 000 cells per well), and 24 h after
plating, various amounts of siRNA-containing particles equiva-
lent to 1, 10, and 20 μg of siRNA were added to a final volume of
125 μL per well. In some experiments, siRNA/Lipofectamine
complex was included as a control, and it was used according to
the instructions of the manufacturer (i.e., Invitrogen). Plates
were analyzed after 48, 72, and 96 h of incubation, using a Victor
plate reader (PerkinElmer, USA), and both luminescence and
fluorescence were quantified. The luminescent signal was pro-
duced by means of the Bright-Glo Luciferase Assay System
(Promega, USA), and its reduction (as compared to untreated
controls) upon incubation with the complexes was used as a
measure for overall gene silencing. The reduction of RFP-
mediated fluorescence (as compared to untreated controls)
was used as a measure for nonspecific gene silencing/toxicity.
To determine target-specific gene silencing, the percentage of
reduction in fluorescence was subtracted from the percentage of
reduction in luminescence.
described in the literature.22 We then compared the stability of
the PEI-based vectors with the stability of poly(HPMA)-grafted
poly(L-lysine) (GPL) vectors, and we evaluated their suitability
for further improvement of vector properties by coating their
surfaces with hydrophilic N-(2-hydroxypropyl)methacrylamide
(HPMA) copolymers. It is known that PEI-siRNA complexes are
not suitable for in vivo application,23 especially because of their
improper stability,24 and coating of this complex does not result
in improvement of its stability (see Figure 1). This result rein-
forced our focus on further development of siRNA complexes
based on GPL polycations.
It is well-known, however, that PLL-a polycation routinely
used for DNA delivery-does not form compact complexes with
short oligonucleotides, such as siRNA.24 With the aim of im-
proving the ability of PLL to form stable complexes with siRNA,
we decided to use a new type of PLL-based polycation, grafted
with short chains of hydrophilic poly(HPMA) developed and
studied in our laboratory.3
In this paper, a comparison of the properties of uncoated
siRNA/PEI and HPMA copolymer-coated complexes with the
properties of siRNA/GPL complexes and their coated analogues
is presented and discussed.
’ RESULTS AND DISCUSSION
An important limitation of RNAi-based therapy is the diffi-
culty in achieving effective delivery of RNAi-bearing vectors to
target cells and tissues in vivo. Many laboratories developing
PEC-based delivery vectors for small oligonucleotides, like
siRNA, are designing oligonucleotide delivery vectors using
commercially available branched polyethylene imine (PEI). With
the aim of using these complexes as controls in our studies, we
prepared these PEI based PECs using standard methodologies
Properties of siRNA/PEI Polyplexes. Basic physical charac-
teristics of siRNA/PEI complexes prepared as reference PECs
and models for the study of the coating reaction of PECs with
multivalent reactive copolymers bearing carbonylthiazolidine-2-
thione (TT) reactive groups attached to the polymer backbone
via degradable and nondegradable spacers were determined by
pec
pec
light scattering methods (Table 2). The Rh and Mw of
complexes coated with various copolymers differed only slightly,
which demonstrated a good reproducibility in PEC preparation.
The size of the PEI-based PECs was found to be close to that
published in the literature.22 The ratio Rh/Rg = 0.8 found for
the PECs corresponded with that predicted for solid spheres
(0.78).26 The polydispersity of PEI PEC (PDI=0.14) was slightly
higher than that of DNA polyplexes.13 As the PECs were
prepared with an excess of polycation, their zeta potentials were
positive (Table 2).
These PEI-based complexes were then coated with HPMA
copolymers containing nondegradable (POL 1) and degradable
(POL 2) spacers terminating with TT reactive groups in buffer at
pH 8.2. We incorporated the fluorescent probe coumarin into the
copolymer structure of POL 2, which enabled better monitoring
of the coating polymer during the purification step. Particle Rh
distributions found in solutions of uncoated PECs and PECs
coated with POL 2 were measured at θ = 150°, and data are
shown in Figure 1.
While Rh distribution of PECs dominates before coating, as
shown in Figure 1, the distribution of particle size in solution
after the coating procedure was more complex. The size dis-
tribution showed, besides the coated particles (35 nm), new
Figure 1. Rh distributions for uncoated (---) and coated (___) siRNA/
PEI complexes with POL 2 copolymer (ccp = 1ꢀ 10-3 g/mL) after
incubation for 220 min; cpec = 2 ꢀ 10-5 g/mL, θ = 150°.
Table 2. Effect of Coating on the Characteristics of siRNA/PEI Polyelectrolyte Complexes Coated with the Reactive Copolymers
POL 1 and POL 2
reactive copolymer cpec ꢀ 105 (g/mL) ccp ꢀ 10-3 (g/mL) Mwa ꢀ 10-6 (g/mol) ΔMct ꢀ 10-6 (g/mol) Fh g/mL nct Rh (150°) (nm) ζ potential (mV)
-
2
-
4.4
0.065
30
>29
>29
35
þ12
POL 1
POL 1
POL 2
1.85
1.85
1.85
2
10.7a
10.0a
17.2a
3.1
2.9
5.1a
36
34
91
0.5
1
0.14
-5
a Contribution of the coating polymer and “new particles” to the SLS was subtracted.
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pec
Figure 3. Dependence of molecular weight Mw (a) and Rh (b) of
siRNA/GPL complexes on the mixing ratio j. The concentration of
starting polycation solution was 3.91 ꢀ 10-4 g/mL.
Figure 2. Zimm plot of scattering curves of the siRNA/GPL complexes
in relation to the mixing ratio, j (top, j = µ; bottom, j = 1).
particles with Rh of about 120 nm. These particles (nanogel)
were probably formed by chemical reaction between amine
groups of free PEI polymers (used in excess) and TT reactive
groups of the coating copolymers. The formation of similar
nanogels was also observed in PEC solutions after coating with
reactive copolymer POL 1 containing a nondegradable spacer.
The small peak at Rh ≈ 8 nm corresponds with the peak of free
coating copolymers, which were used in excess. Unfortunately,
purification of complexes from excessive polycations turned out
to be impossible, because of low stability of the generated
complexes, and disintegration of the PEI-based polyplexes was
observed after dialysis, column filtration, and centrifugation
(results not shown). In the case of such complicated particle
mixtures as those obtained after the coating procedure, the
cc
weight-average molecular weights of coated polyplexes (Mw
)
could be estimated using a combination of dynamic and static
light scattering experiments.21 The increase in the molecular
weight of PEC due to coating with reactive copolymer POL 1 and
POL 2, ΔMct, is shown in Table 2.
In order to characterize the compactness of complexes, the
structural density (Fh) of PEC was calculated from the hydro-
dynamic volume of particles (VH, in cm3) and from their
Figure 4. Zimm plot for uncoated (9) and coated siRNA/GPL
complexes with copolymers POL 1, POL 3, POL 3 þ c(RGDfK), and
POL 3 þ c(RDGfK) (j = 1.5, ccp = 1 ꢀ 10-3 g/mL) after an incubation
time of 24 h; cpec = 5.5 ꢀ 10-4 g/mL.
corresponding molecular weight, Mw (Mw þ ΔMct). These
results are shown in Table 2. The value of Fhcc = 0.14 obtained for
coated siRNA/PEI complexes (Table 2) was comparable with
those found for micelles in organic solvents,27 for DNA/PLL
complexes coated with HPMA copolymer13 and oligonucleotide/
(PLL-g-PHPMA) complexes.3,4 Values for Fhpec of uncoated
siRNA/PEI complexes were approximately 2-fold smaller (Table 2).
This result meant that the coating procedure increased the
observed density of PEC.
cc
cp
On the basis of these findings, it can be concluded that the
purification of polymer-coated siRNA/PEI complexes is impossible
due to their poor stability and the formation of large particles by
chemical reaction of an excess of PEI with reactive coating polymers.
This represents a serious problem for the proper characterization of
coated siRNA/PEI complexes for possible future medical applica-
tions. Therefore, we have designed and tested another polyelec-
trolyte system (i.e., siRNA/GPL) more suitable for medicinal in
vivo applications.
Properties of siRNA/GPL Polyplexes. First, we studied the
formation of the complexes as a function of the molar mixing
ratio (j). Typical results are shown in Figure 2, where the Zimm
plot of the siRNA/GPL complexes is shown for a variety of
mixing ratios.
The coating efficiency was characterized by the number of
molecules attached to the PEC surface, nct, as calculated from
ΔMct, using the formula nct = ΔM/Mwcp, where Mw is the
cp
weight-average molecular weight of used coating polymer. The
results obtained for this parameter for POL 1 and POL 2 are
shown in Table 2. The nct was found to be higher for the coating
copolymer POL 2 due to its lower molecular weight.
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The behavior of Mwpec and Rhpec as a function of j is shown in
Figure 3a,b. The Mwpec was found to increase with increasing j,
with the increase accelerated in the vicinity of the charge
compensation point, j ≈ 1. Since the size of PEC was too small
for reliable determination of the radius of gyration by static light
scattering, the hydrodynamic radius (Rh) of the complexes was
estimated by DLS. The j-dependence of Rhpec was more com-
plex than that of Mwpec, while Rhpec of complexes was practically
independent of j at j > 2.5. The size of the complexes was
strongly increased by enhanced hydrophobic aggregation at j ≈
1. No flocculation was observed in the investigated region of j =
data used for Figure 2, with nagg calculated to be 6.7 and
the number of siRNA molecules per PEC was found to be
nrn ≈ 10.
Static and dynamic light scattering methods were also used to
monitor the efficiency of the coating reaction of siRNA/GPL
complexes. Changes in the weight-average molecular weight
(Mw) and hydrodynamic radius (Rh) of the PEC were examined.
The sensitivity of the methods is demonstrated in Figures 4
and 5, where Zimm plots and the Rh-distribution functions for
uncoated and coated siRNA/GPL (siRNA/GPL/PHPMA)
complexes are shown.
The complexes were prepared at j = 1.5 and were coated
with copolymer POL 1, which was attached to the complex
surface via nondegradable spacers, and polymer POL 3, which
was connected with degradable linkages (siRNA/GPL/PHPMA)
(Scheme 1). To provide some initial proof-of-principle for the
feasibility of developing polymer-coated PECs for targeted
delivery to certain receptor-overexpressing cells, we also incor-
porated the standard targeting moiety c(RGDfK) and a control
oligopeptide (i.e., c(RDGfK)) into the copolymer structure, by
means of a polymer analogous reaction (i.e., POL3þc(RGDFK)
and POL3þc(RDGfK)), and these copolymers were used for
coating of complexes. Zimm plots of uncoated and coated siRNA/
GPL complexes measured after 24 h are shown in Figure 4. The
concentration of coating copolymer was again 1 mg/mL in these
experiments. Results of light scattering measurements obtained
for all copolymer-coated complexes are shown in Table 3. The
efficiency of coating with POL 1 was slightly higher than that
obtained with POL 3 due to its higher molecular weight
(Table 1). The number of siRNA molecules incorporated in a
complex was between 3 and 3.5. It was calculated using data on
molecular weight of the complexes, their density, known molec-
ular weight of used polycation, and the N/P (j) ratio.
pec
pec
10-1.05. The obtained Rh and Mw of siRNA/GPL com-
plexes corresponded well with results obtained earlier for other
oligonucleotides.3,4 For all the following experiments, we
decided to use complexes prepared at j = 1.5, which was in the
region of suitable Rh and Mw values of the complexes. The
presence of a free polycation in the PEC solution was also not
observed at this j; in contrast to PEI, the chemical coating
reaction was not contaminated by the reaction of excessive
coating copolymers with free polycations resulting in formation
of nanogel.
pec
From the Mw values of the complexes, their aggregation
s
number (nagg = Mwpec/Mw ) could be estimated, where Mws was
the molecular weight of the unimolecular siRNA/GPL complex.
Assuming full charge compensation of GPL macromolecules by
siRNA at j = 1, Mws was calculated using mass/charge data and
In principle, the coating reaction with a multivalent polymer
can result in aggregation of complexes. Inadequate increases in
Rh of complexes after coating can be used as evidence of such
aggregation. Additional evidence of particle aggregation can be
represented by increased polydispersity (broadening of Rh dis-
tribution) of the coated particles, which should be close to that of
uncoated particles. It can be seen in Figure 5 that the width of the
Rh distribution of the coated complexes was slightly smaller than
that of the original complexes. Therefore, particle aggregation
was practically negligible. The maximum of the distribution curve
of coated PECs was found to be shifted by 5 nm to higher Rh
values. Thus, the mean thickness of the coating layer (ΔRhct) was
found to be 5 nm. The number of molecules attached to the PEC
surface (nct) was 28 after 24 h of coating. The results above also
show that attachment of targeting oligopeptide does not sig-
nificantly influence the coating reaction and such copolymers are
suitable for preparation of coated and oligopeptide-targeted
delivery vectors.
Figure 5. Rh distributions for siRNA/GPL complexes (j = 1.5) and
coated PEC after 24 h; coating copolymer POL 1, ccp = 1 ꢀ 10-3 g/mL
and cpec = 4.2 ꢀ 10-4 g/mL.
Table 3. Characteristics of siRNA/GPL PECs Modified by Reactive Copolymers at j = 1.5, Obtained after 24 h
Mwa ꢀ 10-6
ΔMw ꢀ 10-6
Fpec,cc
(g mL-1
Rh (θ = 0°)
ζ potential
ct
coating polymer
(g mol-1
)
(g mol-1
)
nct
Nrn
)
nm
(mV)
-
0.80
7.92
5.32
6.69
6.65
3
0.007
0.042
0.032
0.031
0.029
37
42
40
44
45
þ7
-3
POL 1
2.04
1.45
1.66
1.65
28
27
POL 3
POL 3 þ c(RGDfK)
POL 3 þ c(RDGfK)
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Figure 6. AFM amplitude images of siRNA/GPL complexes (a,b) (j = 1.5) and their POL 3-coated analogues (c,d) (ccp = 1 ꢀ 10-3 g/mL): (a,c) field
of view 2 μm; (b,d) field of view 500 nm.
Figure 7. (a) Time dependence of Mwpec and Rhpec (θ = 0) of PEC prepared at j = 1.5. (b) Time dependence of Mwcc and Rhcc (θ = 0) of PEC prepared
at j = 1.5 and coated with POL 3 (ccp = 1 ꢀ 10-3 g/mL).
Figure 8. Time dissociation of siRNA/GPL complexes from horizontal
electrophoresis.
Atomic Force Microscopy (AFM). AFM was used as a direct
method for characterization of PEC particle morphology, which
consisted of size, shape, and size polydispersity. The AFM
amplitude images of siRNA/GPL complexes (j = 1.5) obtained
in the tapping mode are shown in Figure 6a,b. The sizes of dried
polyplexes obtained by the analysis of AFM images were only
slightly higher (the radius was in the range 60-70 nm) than
the Rh values presented in Table 3. Surprisingly, the uncoated
polyplexes exhibited a regular angular structure (Figure 6b).
Time Stability of PEC. Despite the slow speed of preparation
of siRNA/GPL complexes (15 min), the final complexes
were still in a nonequilibrium state. Therefore, we tested the
Figure 9. Time dependence of the turbidity of uncoated and coated
PEC as investigated in 0.15 M NaCl solvent.
medium-term stability of their parameters (Rh, Mw). The results
of stability for uncoated PECs (siRNA/GPL) and PECs coated
with copolymer POL 3 (containing a degradable spacer) ob-
tained within three weeks are shown in Figure 7a,b.
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Figure 10. Concentration (A) and time (B) dependence of target-specific gene silencing in vitro in MDA-MB-231 human epithelial breast
adenocarcinoma cells expressing both luciferase (to determine siRNA-specific gene silencing) and RFP (to determine unspecific gene silencing/toxicity)
induced by uncoated and POL3-coated antiluciferase siRNA/GPL complexes. Values were compared to those of untreated controls and to those
induced by Lipofectamine/siRNA complexes. In B, lipofectamine was used at an optimal dose of 0.5 μg of siRNA (according the manufacturer’s
instructions), while the coated and uncoated polyplexes were used at doses corresponding to 10 μg of siRNA.
As can be seen, the Mw of both types of complexes decreased in
the first four days of observation. After that, coated complexes
approached equilibrium, but the Mw of uncoated PEC continued
physiological environments, we measured the time dependence
of turbidity in saline solution (0.15 M NaCl). These measure-
ments are related to the kinetics of PEC aggregation followed by
phase separation. Results are shown in Figure 9 for uncoated and
polymer-coated complexes. Complexes prepared from PEI poly-
cations showed the fastest coagulation rate, while the rate of
coagulation was lower for PECs prepared by self-assembly of
siRNA with GPL. Furthermore, both types of polymer-coated
PEC were stable in saline solution; the turbidity did not change
with time. Thus, it can be concluded that coating the PEC with
HPMA copolymers substantially increased the colloidal stability
of the polyplexes, with stability of GPL complexes being superior
to those of PEI, and it seems reasonable to assume that also, in
vivo, the stability of the coated PEC might be higher than that of
uncoated PEC.
cc
to decrease slightly until the experiment ended. The Rh of
coated PEC decreased more rapidly than their Mwcc, probably
due to their internal restructuring.
While the ζ-potential of PEC was positive after the coating
reaction, similar to uncoated complexes, the polarity of the
ζ-potential of coated complexes switched overnight to become
negative (see Table 3). The drop in ζ-potential was evidently a
result of hydrolysis of the remaining reactive TT groups not
participating in the coating reaction (formation of -COOH
groups). If the excessive unreacted coating polymer was not
removed from the PEC solution, the drop in ζ-potential con-
tinued to a value of -22 mV after 22 days.
Initial in Vitro Evaluation of Gene Silencing Activity. To
provide some initial experimental evidence for the retention of
biological activity upon coating the siRNA-containing complexes,
their silencing efficacy was analyzed using MDA-MB-231 human
epithelial breast adenocarcinoma cells stably expressing lucifer-
ase and red fluorescent protein (RFP). To this end, both coated
and uncoated GPL-based antiluciferase PECs were prepared, and
their ability to specifically reduce the expression of luciferase was
compared to that of the standard transfection agent Lipofect-
amine. Here, luciferase knockdown upon exposure to different
concentrations (i.e., 1, 10, and 20 μg siRNA) and different
incubations times (i.e., 48, 72, and 96 h) was correlated with
loss of RFP signal (i.e., nonspecific silencing/toxicity), findings
were normalized to those of untreated controls, and they were
compared to those obtained for Lipofectamine (which according
the instructions of the manufacturer was always applied at an
optimal siRNA concentration of 0.5 μg). As shown in Figure 10,
it was found in these initial proof-of-principle analyses that, in the
majority of cases, luciferase-specific gene silencing induced by the
polymer-coated PEC was more or less comparable to that in-
duced by Lipofectamine, and that it in some cases even turned
out to be significantly better. Also, as compared to uncoated
PEC, the silencing efficacy of the polymer-coated PEC turned
out to be superior in the majority of cases (likely mostly because
of a reduction in nonspecific gene silencing/toxicity, rather than
because of an increase in specific gene silencing). These findings
are by no means conclusive, and additional in vitro efficacy, in
vitro uptake, and in vitro toxicity analyses are necessary before
SiRNA molecules were fixed inside the complexes by electro-
static interactions, therefore, they could be undesirably displaced
from complexes by an exchange reaction with polyanions.3 The
remaining coating copolymers were hydrolyzed, and their reac-
tive TT groups, which had not been aminolyzed in the coating
reaction, were changed into carboxylic acid groups. SiRNA could
then be released from the complexes during longer storage times
in their solutions by interaction with hydrolyzed coating copoly-
mers. Indeed, the release of siRNA from the unpurified com-
plexes was proven by agarose gel electrophoresis, and the
results of the measurements of 22-day-old samples are shown
in Figure 8.
In order to avoid this problem, we have developed a cleaning
procedure for freshly prepared coated PECs. To purify the PECs
from excessive coating polymer, the PEC solution was filtered
through the Millipore Amicon Ultra-4 centrifugation device
5 times with an Ultracel-100 membrane from regenerated
cellulose (cutoff 100 kDa) and diluted with 0.01 M NaCl buffer
to the original concentration. The concentration and size of the
complexes was verified by light scattering. The weight losses of
the PEC were less than 10% after this cleaning procedure. The Rh
of complexes was conserved during the cleaning procedure,
and results obtained after 22 days of storage of the purified
complexes are shown in Figure 8. Only the purified complexes,
therefore, were used for all subsequent experiments and for
biological testing.
Stability of Complexes in Saline Solution. To prove the
stability of the studied PECs under conditions mimicking
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Bioconjugate Chemistry
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transferring these systems to the in vivo situation. Nonetheless,
these initial in vitro insights already demonstrate quite clearly
that polymer-coated siRNA-containing PEC are able to specifi-
cally induce target-specific gene silencing in vitro, and they
thereby exemplify that even systems (physico-) chemically
optimized to eventually perform well in vivo compare well to
unstable and nonstealth systems primarily designed for gene
silencing in vitro.
dct - surface density of coating polymer (g/nm2 mol)
nct - number of coating molecules fixed on PEC surface
tct - characteristic reaction time of the slow coating process (min)
’ MEANING OF SUBSCRIPTS AND SUPERSCRIPTS
cp - coating polymer
pec - uncoated polyelectrolyte complexes
cc - coated PEC
ct - coating layer of PEC
’ CONCLUSIONS
’ DEFINITIONS
The synthesis and physicochemical properties of reactive
copolymers suitable for the coating of PEC and the physical
properties of coated and uncoated siRNA-containing PECs are
described. Results of stability measurements showed that often-
studied PEI-based PECs are not suitable for coating reactions
and consequently are likely suboptimal vectors for in vivo siRNA
delivery. We therefore set out to develop siRNA-containing
PECs based on GPL, as well as GPL PECs coated with HPMA-
based reactive hydrophilic copolymers. The synthesis and physico-
chemical characterization of HPMA-based copolymers contain-
ing TT reactive groups bound to the polymer chain via spacers
containing biodegradable disulfide bonds was described. The use
of the HPMA copolymers for siRNA/GPL complex coating
reactions resulted in attachment of (on average) 28 polymer
molecules to the nanoparticle surface, forming a 5-nm-thick
hydrophilic polymer protective layer that was selectively remo-
vable in reducing environments. Coating the siRNA/GPL vec-
tors with the stealthy hydrophilic polymer substantially enhanced
the stability of the vectors in aqueous media, and it might thereby
improve their potential for passive targeting to solid tumors by
means of the EPR effect. Preliminary results on the in vitro gene
silencing efficacy of the coated complexes furthermore showed
that they were able to specifically reduce target gene expression in
human epithelial breast adenocarcinoma cells, thereby suggest-
ing that these systems hold significant potential for further in
vitro and in vivo evaluation.
pec
ΔMwct = Mwcc - Mw
ΔRhct = Rhcc - Rh
pec
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