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pH-induced size changes in solutions of cholesteric liquid- crystal polymers studied by SANS
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2014 J. Phys.: Conf. Ser. 554 012011
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Dynamics of Molecules and Materials-II
Journal of Physics: Conference Series 554 (2014) 012011
IOP Publishing
doi:10.1088/1742-6596/554/1/012011
pH-induced size changes in solutions of cholesteric liquidcrystal polymers studied by SANS
Mercedes Pérez Méndez1, Daniel Rodriguez Martinez2, 3 and Stephen M King3.
1
Institute of Polymer Science and Technology, Group of Physical-Chemistry and
Modelization of Macromolecules (PCMM), C/ Juan de la Cierva, 3. 28006 Madrid,
Spain
2
Retinal degeneration: from genetics to therapy Laboratory, Centro Andaluz de
Biología Molecular y Medicina Regenerativa Edif. CABIMER Avda. Américo
Vespucio, s/n Parque Científico y Tecnológico Cartuja 93. 41092 Sevilla, Spain
3
ISIS Facility, STFC Rutherford Appleton Laboratory, Harwell Oxford, Didcot,
Oxfordshire OX11 OQX, United Kingdom
E-mail: perezmendez@ictp.csic.es
Abstract. Polycations possessing substantial buffering capacity below physiological pH, are
intrinsically efficient transfection agents. These vectors have been shown to deliver genes as
well as oligonucleotides, both in vitro and in vivo, by protecting DNA from inactivation by
blood components. Their efficiency relies on extensive endosome swelling and rupture that
provides an escape mechanism for the polycation/DNA complexes. Recently, biocompatible
cationic cholesteric liquid-crystal polymers (ChLCP) have proved able to condense and
successfully transfect DNA, acting as non-viral vectors. Here the radius of gyration of the new
ChLCPs is determined by SANS as a function of pH, the ultimate aim being to correlate
changes in polymer conformation with membrane activity. With increasing pH the polymers
apparent radii of gyration increased to a maximum, before subsequently decreasing. This
molecular expansion, on passing from acidic pH environment (cf., lysosome pH 3.5- 4, late
endosome pH 5- 6, early endosome pH 6- 6.5) to neutral pH (cytosol pH=7-7.4), matches the
endocytic route through the cell, where the pH change is used as a signal to release
biomacromolecules, such as DNA. It confirms that the new cationic ChLCPs could act as an
endosomolytic release system in gene therapy according to the hypothesis of "the proton
sponge".
1. Introduction
The introduction of exogenous genetic material in cells is a key stage in the development of basic
research in cellular biology. The term “transfection” is used to indicate the transfer of DNA into the
nuclei of cells of higher organisms [1]. The development of non-viral DNA delivery vectors make it
possible to model viral assembly and gene transfer by incorporating functional groups that enable
particular assembly and transfer steps.
Cationic polymers (at physiological pH) are already used to condense anionic nucleic acids into
nano-sized particle-like complexes called “polyplexes”, through self-assembly driven by electrostatic
interactions. By compressing DNA molecules to a relatively small size cellular internalization is
facilitated and, thus, transfection efficacy is improved [2]. Whilst the intravascular route of
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Published under licence by IOP Publishing Ltd
1
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Dynamics of Molecules and Materials-II
Journal of Physics: Conference Series 554 (2014) 012011
IOP Publishing
doi:10.1088/1742-6596/554/1/012011
administration is an attractive approach for wide spread delivery, it is however particularly plagued by
toxicity and biodistribution problems [3, 4]. New types of delivery vector are always welcome.
Several non-permanent polycations possessing substantial buffering capacity below physiological
pH, such as lipopolyamines and polyethylenimines, are intrinsically efficient transfection agents, i.e.
they do not need the addition of lysosomotropic bases, or cell targeting, or membrane disruption
agents. These vectors have been shown to deliver genes as well as oligonucleotides both in vitro and in
vivo. Their efficiency relies on extensive endosome swelling and rupture that provides an escape
mechanism for the polycation/DNA polyplexes [5, 6]. The challenge, therefore, is to see if materials
chemistry can provide more efficient, and ‘smart’, delivery vectors. One promising area of research
uses liquid crystals (LCs).
Liquid crystals are self-organizing systems that form intermediate mesophases, combining the order
of perfect crystals with the mobility of fluids [7]. These mesophases may be formed either by the
effect of temperature (thermotropic) or of solvent concentration (lyotropic). Lyotropic liquid crystals,
in the form of lipids, are of course ubiquitous throughout the life sciences [8]. They are a prerequisite
for the development of life and the ability of cells to function.
In all LCs the longitudinal molecular axes are aligned parallel to each other, with the distribution of
the molecular centres determining the type of LC. The most common types are the nematic (centres
distributed isotropically), figure 1a, and smectic (where they are arranged in layers), figure 1b. Less
common are the cholesteric, figure 1c, where nematic ordering is stacked in a superhelical structure,
with a certain pitch, figure1d. It is these LCs that are the focus of this paper.
a
b
c
d
Figure 1. Liquid Crystal mesophases. (a) Nematic, (b) Smectic, (c, d) Cholesteric
Cholesteric liquid-crystal polymers (ChLCP), designated PTOBDME [C34H36O8]n with the
monomeric chemical formula shown in figure 2, and PTOBEE [C26H20O8]n having the same mesogenic
rigid group along the main chain but different side chain length, have been synthesised in our
laboratory, through a stereoselective polycondensation reaction [9, 10]. The amphiphilic nature of the
monomers makes them polymerize into helical chains.
These ChLCP have been extensively characterized [11]. They are both thermotropic and lyotropic,
and adopt different conformations when dispersed in solution, depending on the solvent nature and
concentration. They are also optically active and biocompatible against macrophages and fibroblast
cellular lines, making them truly multifunctional materials.
We have previously shown that due to their lyotropic behaviour, these ChLCPs can entrap smaller
molecules inside [12] and interact with liquid-crystalline biomacromolecules, such as nucleic acids
and lipids (both neutral and cationic). The structures of the complexes formed have been previously
studied by simultaneous synchrotron SAXS/WAXS [13, 14, 15].
Subsequently, formulations of the synthetic ChLCPs and cationic monomeric surfactants were
developed in our lab to entrap an anionic plasmid of DNA. These complexes were successfully tested
as non-viral vectors in gene therapy, both in vitro and in vivo [16, 17].
2
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Dynamics of Molecules and Materials-II
Journal of Physics: Conference Series 554 (2014) 012011
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doi:10.1088/1742-6596/554/1/012011
SPACER
O
R’ - O
O
19 C 18 C
O
O
17 C
20C
MESOGEN
Hc
O
O
14 C 13 C
12 C
O
*
Ha
φ
11 C
16 C 15 C
20H
16H
Hd
10 C
H 26C
H2
PTOBDME
Hf
9C
H 28C
4C
H 22C
Hg
7CH
5CH
2
2
3CH
1 CH
R’’
n
Hb
15H
He
α−end-group
R’= H (acid group)
O
ω−hydroxy
end-group
R’’ = H
2
3
SIDE CHAIN
Figure 2. Chemical formula of PTOBDME [C34H36O8]n subdivided into mesogen, spacer and side
chain. PTOBEE [C26H20O8]n shares the same mesogen and spacer but only has two carbon atoms in
the side chain.
In order to specifically interact with complex polynucleotides, new cationic ChLCPs were
synthesized, by functionalization of the precursors PTOBEE and PTOBDME with nitrogenated groups
(NH2, choline, etc) [18]. The molecular model of Poly[PTOBEE]10-NH2 is shown in figure 3.
Figure 3. Molecular model of Poly[PTOBEE]10-NH2.
In the lower right sectional perspective view the terminal N atom of
the amine end group is represented by a ball.
Polyplexes of the cationic ChLCP PTOBEE-NH2 with the commercial anionic polynucleotide
[Poly-C-Poly-G] have been studied by contrast variation small-angle neutron scattering (SANS),
exploiting the difference in neutron scattering length densities (NSLD) of the two components
(1.887E+10/cm2 for PTOBEE-NH2, 3.320E+10/cm2 for [Poly-C-Poly-G]) to separately distinguish
both component structures, and to highlight the whole polyplex, without any selective deuteration
[19]. This provided complementary information to that obtained by SAXS [20].
In this paper SANS is used without contrast variation to investigate the pH-dependent
conformational behaviour of three cationic ChLCPs. The pH value is a critical environmental
parameter during transfection as it can be used as a signal to promote the efficient delivery of
biomacromolecules or DNA to the correct intracellular compartment. The pH of an endosome is lower
than that of the cytosol by up to two pH units, depending on the stage of endosomal development.
2. Experimental
Three cationic ChLCPs have been studied: PTOBDME-choline, PTOBDME-NH2, and PTOBEE-NH2.
All samples were dispersed in aliquots of D2O (to maximise the scattering contrast), pH-adjusted with
3
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doi:10.1088/1742-6596/554/1/012011
5M HCL or 5M NaOH (to match the conditions encountered during endocytic internalization; pH 3 to
7.4), at a fixed polymer concentration of 7 mg/ml. The pH’s of the solutions were re-checked after
dissolution of the polymers. In adjusting the pH, allowance was made for the small offset between pH
and pD: pD=pH+0.4.
Contrast variation (via adjusting D2O/H2O ratio) was not employed in this work, since the neutron
scattering length densities (NSLD) of all the ChLCPs studied (between 1.3 - 3.3 (x1010 cm-2)), are
significantly different to that of D2O (+6.3) to provide excellent contrast. As the SANS signal is
proportional to the square of the difference in NSLD between the solute and solvent, using solvent
mixtures with H2O (-0.5 on the same scale) would have merely reduced the contrast. Contrast variation
would, however, provide a means to elucidate individual contributions to the overall scattering in the
polyplexes in future work.
SANS data were obtained on the LOQ small-angle diffractometer at the ISIS Pulsed Neutron Source
(STFC Rutherford Appleton Laboratory, Didcot, U.K.) [21a, 21b]. This is a fixed-geometry “white
beam” time-of-flight instrument which utilizes neutrons with wavelengths, λ, between 2 and 10 Å.
Data are simultaneously recorded on two, two-dimensional, position-sensitive, neutron detectors, to
provide a simultaneous Q (=4π/λ.sin(θ/2)) range of 0.008−1.6 Å-1, where θ is the scattering angle.
Each polymer sample and corresponding solvent background was placed in 1 mm path length quartz
disc-shaped cuvettes (volume 0.25 ml) and was measured for a total of 2 h in order to gather data of
sufficient statistical precision. Each raw scattering data set was then corrected for the detector
efficiencies, sample transmission and background scattering and converted to scattering cross-section
data (denoted here as I vs Q) using the instrument-specific software [21c, 21d]. These data were
placed on an absolute scale (cm-1) using the scattering from a standard sample (a solid blend of
hydrogenous and perdeuterated polystyrene) in accordance with established procedures [21e]. The
data from the ChLCPs were analysed with a combination of partial Zimm plots (to extract the apparent
radius-of-gyration of the nanostructure) [22] and Log-Log plots (aka Swollen Coil plots, to determine
the fractal scaling of the nanostructure). The Zimm plots were fitted over the range 0.008≤Q≤0.02 Å-1,
whilst the Log-Log plots were fitted over a slightly extended range 0.008≤Q≤0.04 Å-1.
3. Results and Discussion.
Figure 4 shows the small-angle neutron scattering (SANS) patterns, in log I(Q) versus Q
representation, from cationic ChLCPs PTOBDME-Choline, PTOBDME-NH2 and PTOBEE-NH2
dispersed in D2O at 7 mg/ml, as a function of pH (= 1.3, 3.58, 5.58, 7.2). These pH values were chosen
to match the values in different cellular compartments (lysosome between pH 3.5 - 4, late endosome
pH 5 - 6, early endosome pH 6 - 6.5 to cytosol, pH=7 - 7.4). Though superficially similar, there are
some marked differences in both the intensity and Q-dependence of the scattering from the polymers
as the pH changes, indicating that the size and/or shape of the solvated species is changing.
A zeroth-order approach to quantifying these changes is simply to determine the radius-of-gyration,
Rg, of the ChLCPs from the SANS data. Traditionally this can be done through one of two low-angle
linearisations, the Guinier plot or the Zimm plot [22]. We have used a variation of the latter as it has a
slightly wider range of applicability (ie, is a little more robust to the presence of polydispersity).
Whilst light scatterers will be familiar with the Zimm plot as a complex double extrapolation to
infinite dilution and Q=0 (to correct for the strong angular dependence of the scattering), this is not
actually necessary in order to estimate an apparent Rg as the slope of the Zimm plot, 1/I(Q) vs Q2, is
just (Rg2/3) x intercept as Q < 0, and SANS is always considered point scattering because the neutron
wavelength is much larger than the object from which it is being scattered (the nucleus). The SANS
data in Figure 4 are replotted in this partial Zimm representation (to denote that extrapolation to zero
concentration was not performed) in Figure 5. Linearity is expected when Q.Rg < π (note: this is a
slightly broader limit [23] than Zimm’s original definition, which allows for the presence of some
polydispersity). For Rg’s up to ~200 Å this would imply 0 < Q2 < 0.00025 Å-2. In some instances
linearity would seem to extend beyond this range, in others a slight curve to the Zimm plot is evident
4
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Dynamics of Molecules and Materials-II
Journal of Physics: Conference Series 554 (2014) 012011
IOP Publishing
doi:10.1088/1742-6596/554/1/012011
(a)
PTOBDME,NH2$
Log$I$(Q)$/cm$,1)$
1,E+00'
pH'1,3'
pH'3,58'
pH'5,58'
pH'7,2'
1,E$01'
(b)
1,E$02'
1,E$03'
1,E$04'
0,000'
0,050'
0,100'
0,150'
Q$$=$4π$/λ .$sin$(θ$/2)$Å,1$$
(c)
Figure 4. Small-angle neutron scattering patterns (SANS) of cationic
ChLCPs, dispersed in D2O at 7 mg/ml, as a function of pH:
(a) PTOBDME-Choline; (b) PTOBDME-NH2; (c) PTOBEE-NH2.
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Journal of Physics: Conference Series 554 (2014) 012011
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doi:10.1088/1742-6596/554/1/012011
(a)
(b)
(c)
Figure 5. Partial Zimm plots of the cationic ChLCPs, dispersed in D2O
at 7 mg/ml, as a function of pH: (a) PTOBDME-Choline;
(b) PTOBDME-NH2; (c) PTOBEE-NH2. The slope is proportional to
Rg2/3 at small Q.
suggesting that a more limited range might be more appropriate. But for consistency we report Rg’s
determined over the same range of Q2.
In Figure 6 the apparent z-average radii of gyration, Rg, of the polymers extracted from the partial
Zimm plots are displayed as a function of pH. With increasing pH, the apparent Rg of all the polymers
increases up to a maximum value, before subsequently decreasing.
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Dynamics of Molecules and Materials-II
Journal of Physics: Conference Series 554 (2014) 012011
IOP Publishing
doi:10.1088/1742-6596/554/1/012011
Figure 6. Variation of the apparent radius of gyration (Å) of the cationic
polymers dispersed in D2O at [7 mg/ml] derived from the SANS data as
a function of pH: (triangles) PTOBDME-Choline (squares) PTOBDMENH2 and (circles) PTOBEE-NH2.
The cholesteric liquid crystalline nature of these new cationic polymers means they might
reasonably be expected to have ‘less random coil’ solution conformations than other synthetic
polymers, and there would seem to be evidence of this. In figure 7, the swollen coil fits to the SANS
data from PTOBDME-NH2 at pH 1.3 and pH 7.2 are shown. The gradients of the two linear regions
are both significantly larger than what would be expected for an extended (filamentous) nanostructure,
at -1.00, a ‘self-avoiding chain’ (ie, in a good solvent environment), at -1.66, or a ‘Gaussian chain’ (ie,
in a theta solvent environment), at -2.00. Indeed the scaling displayed is more typical of some sort of
network structure. The change observed in fractal scaling is also modest despite a large change in the
apparent Rg (almost a factor of 3 for this polymer).
Figure 7. Swollen coil fits to the SANS from PTOBDME-NH2 at pH
1.3 and pH 7.2. The gradients of the straight lines are -2.55 and -3.04,
respectively.
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doi:10.1088/1742-6596/554/1/012011
The next best level of analysis would be to try and fit the SANS data to analytical descriptions of
different shaped objects. We have tried this, using the well-known model-fitting program, FISH [28].
However we were unable to obtain a fit in which we had both confidence and belief that it represented
physical reality. As an example, in Figure 8 fits of the SANS from PTOBDME-NH2 at pH= 1,3 and
7,2 against a Debye Gaussian coil model are shown [29]. Neither calculation is a good description of
the data; a not altogether surprising fact given the fractal scaling seen in Figure 7 but it illustrates the
difficulties. The solution architecture of the ChLCPs is evidently quite challenging to determine. The
analysis most likely to elucidate the structure is perhaps a MD simulation constrained against the
experimental SANS data. We note that software packages to achieve this, explicitly accounting for
solvent, are starting to become available [30].
The apparently large pH-induced change in apparent Rg (almost a factor of 3) for these ChLCP, on
passing from acidic pH environment (cf. lysosome, endosome), towards neutral pH (cf. cytosol) and
the posterior decrease in the more basic extreme, could be explained by the hypothesis of a molecular
expansion up to a maximum, similar to the coil expansion described for other polymers at the same pH
values [24], [25], [26]. The effect of extreme protonation/ deprotonation should be considered on
intramolecular H-bridges (N-H…O) and on possible cleavage through ester groups, due to charge
change.
The cleavage mechanism of a polyplex carrier, for delivery of a plasmid DNA, had been reported
through S-S bonds when passing from pH 7 to 5.5 [27].
We have previously reported a decrease in the Rg of the precursor PTOBEE in both D2O and in
ethanol-d6 when terminal amine groups are attached to form cationic PTOBEE-NH2. This was ascribed
to the presence of the nitrogen atoms affecting auto-aggregation, that is, a compaction of one molecule
by the introduction of positive charges or by formation of H-bonds with the neighbouring oxygen
atoms, or by a combination of the two [19]. The apparent Rg of PTOBEE-NH2 calculated then (also
from SANS data) at pH= 7, was 81 Å, assuming a rod-like nanostructure and 110 Å assuming a
globular nanostructure. The latter figure is consistent with the value in Figure 5.
Work to better understand the origin of the pH-induced structural changes in these ChLCPs is in
progress. However the results so far would seem to confirm the potential application of these new
cationic ChLCP as endosomolytic polymers.
(b)
(a)
Figure 8. Debye Gaussian coil model fits to the SANS data from PTOBDME-NH2 at
pH 1,3 (a) and pH 7,2 (b). The red circles represent the experimental data, the blue
line is the model calculation. The green line is the difference between the two,
superimposed on a yellow abcissa. The fitting was conducted with the FISH program
(see text).
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4. Conclusions
The SANS patterns of synthetic cationic cholesteric liquid-crystal polymers PTOBDME-Choline,
PTOBDME-NH2 and PTOBEE-NH2, dispersed in D2O at 7 mg/ml, have been obtained at different pH
values (= 1.3, 3.58, 5.58, 7.2), chosen to match the values in different cellular compartments
(lysosome between pH 3.5 - 4, late endosome pH 5 - 6, early endosome pH 6 - 6.5 to cytosol, pH=7 7.4).
The apparent radius of gyration of the polymers, derived from the scattering data, proved to be
highly sensitive to pH changes, initially increasing in size with increasing pH up to a maximum value,
before subsequently decreasing once more at higher pH. The ChLCPs adopt rather different solution
conformations to more conventional synthetic polymers, but the exact nature of the solution
nanostructure and the explanation for the pH-sensitivity is, as yet, not fully understood.
The results shown here help indicate the potential of these new cationic ChLCPs as an endosomolytic
release system in gene therapy, according to the hypothesis of "the proton sponge" where an endocytic
pH change could be used as a signal to release a ‘captive’ biomacromolecule, such as DNA, in the
correct intracellular compartment.
5. Acknowledgements
DR-M wishes to thank the MEC for a Scholarship of Specialization in International Organisms
conducted at the ISIS Facility. STFC and ISIS are thanked for the provision of neutron beamtime.
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