Fine-tuning the pH trigger of self-assembly

Article abstract of DOI:10.1021/ja211113n

The creation of smart, self-assembling materials that undergo morphological transitions in response to specific physiological environments can allow for the enhanced accumulation of imaging or drug delivery agents based on differences in diffusion kinetics. Here, we have developed a series of self-assembling peptide amphiphile molecules that transform either isolated from molecules or spherical micelles into nanofibers when the pH is slightly reduced from 7.4 to 6.6, in isotonic salt solutions that simulate the acidic extracellular microenvironment of malignant tumor tissue. This transition is rapid and reversible, indicating the system is in thermodynamic equilibrium. The self-assembly phase diagrams show a single-molecule-to-nanofiber transition with a highly concentration-dependent transition pH. However, addition of a sterically bulky Gd(DO3A) imaging tag on the exterior periphery shifts this self-assembly to more acidic pH values and also induces a spherical micellar morphology at high pH and concentration ranges. By balancing the attractive hydrophobic and hydrogen-bonding forces, and the repulsive electrostatic and steric forces, the self-assembly morphology and the pH of transition can be systematically shifted by tenths a pH unit.

Full text of DOI:10.1021/ja211113n

Communication
pubs.acs.org/JACS
Fine-Tuning the pH Trigger of Self-Assembly
Arijit Ghosh,†,‡ Mark Haverick,†,‡ Keith Stump,† Xiangyu Yang,§ Michael F. Tweedle,§
and Joshua E. Goldberger*,†
§
†Department of Chemistry and Biochemistry and Department of Radiology, The Ohio State University, Columbus, Ohio 43210,
United States
S
* Supporting Information
particularly attractive stimulus is the slightly acidic extracellular
microenvironment of tumor tissue (pH 6.6−7.4)4 that arises
ABSTRACT: The creation of smart, self-assembling
due to the enhanced rate of glycolysis.5 There are numerous
materials that undergo morphological transitions in
response to specific physiological environments can allow
for the enhanced accumulation of imaging or drug delivery
agents based on differences in diffusion kinetics. Here, we
have developed a series of self-assembling peptide
amphiphile molecules that transform either isolated from
molecules or spherical micelles into nanofibers when the
pH is slightly reduced from 7.4 to 6.6, in isotonic salt
solutions that simulate the acidic extracellular micro-
environment of malignant tumor tissue. This transition is
rapid and reversible, indicating the system is in
thermodynamic equilibrium. The self-assembly phase
diagrams show a single-molecule-to-nanofiber transition
with a highly concentration-dependent transition pH.
However, addition of a sterically bulky Gd(DO3A)
imaging tag on the exterior periphery shifts this self-
assembly to more acidic pH values and also induces a
spherical micellar morphology at high pH and concen-
tration ranges. By balancing the attractive hydrophobic and
hydrogen-bonding forces, and the repulsive electrostatic
and steric forces, the self-assembly morphology and the
pH of transition can be systematically shifted by tenths a
pH unit.
examples of materials that incorporate acid-cleavable linkages
that degrade under the lysosomal (pH 5.0−5.5) or the slightly
acidic tumor environment to release cargo;3,6 however, there
are far fewer examples of materials that reversibly transform to
larger, more slowly diffusing morphologies in response to the
extracellular cancer pH. Creating a material that, upon reaching
the acidic extracellular tumor environment, transforms into a
bulky, more slowly diffusing object could serve as a novel
mechanism for achieving a higher relative concentration of
imaging, drug delivery, or radiotherapeutic agent at the tumor
site compared to the bloodstream. Although a multitude of self-
assembling materials have pH-dependent assembly behavior,
there are very few biologically compatible systems designed for
in vivo use, with assembly behavior that can be reversibly
triggered at neutral pH values (6.6−7.4) in an ionic
environment that resembles serum. Both the concentration
and the valency of the ionic environment play key roles in
mediating the self-assembly of charged systems.7 Thus,
developing systems that function under the stringent set of
conditions for in vivo use requires considerable insight and
optimization.
To develop materials capable of reversible pH-triggered
morphological changes, we sought to design amphiphilic
molecules that would exist as either single molecules or
spherical micelles under normal physiological conditions (pH
7.4) and would self-assemble into nanofibers upon encounter-
ing the acidic environment (pH 6.6) of the tumor vasculature
(Figure 1). Peptide amphiphiles (PAs, Chart 1) are an attractive
here has been much interest in understanding the
T
influence of size, shape, and mechanical properties of
nanomaterials on their biodistribution, to design more effective
drug delivery and imaging agents. For example, the enhanced
permeation and retention of spherical materials with 20−200
nm diameters in the leaky, non-lymphatic vasculature of tumor
tissue has ultimately led to the development of FDA-approved
liposomal therapies.1 More recently, the size and shape of
nanomaterials has been found to play a significant role in the
distribution and circulation lifetimes of these objects when
delivered intraveneously.2 For example, cylindrical polymeric
micelles have been shown to have a 10 times longer circulation
time in the bloodstream compared to their spherical counter-
parts.2a Still, most of these materials tend to be either static
objects that do not transform in the cancer environment or
carriers that fragment into smaller objects to release cargo when
they get to the target.3
Figure 1. Schematic of the target reversible, pH-triggered morpho-
logical transition of self-assembling peptide amphiphiles.
class of molecules in this regard since they are biocompatible,
can spontaneously self-assemble into a variety of morphologies,
and have intermolecular forces that can be precisely tuned with
Designing nanomaterials that can spontaneously change
shape and size in response to specific physiological stimuli has
the potential to exploit the differential diffusion kinetics to
amplify the accumulation of these agents. For cancer, one
Received: November 27, 2011
Published: February 6, 2012
© 2012 American Chemical Society
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morphology at pH > 6.82. At more acidic pH, the peptides start
self-assembling into a structure with β-sheet character, which is
indicative of a nanofiber morphology.8d The transition pH from
random coil to β-sheet occurred at pH 6.6. We defined the
transition pH to be the value at which the ellipticity at 205 nm
rises to zero, followed by the appearance of a minimum at 218−
220 nm.
Chart 1. PA Structure and Design
The transition between random coil and β-sheet structure
was rapid and reversible. At pH 7.75, HCl was added until the
pH was 6.1, and the resulting β-sheet CD spectrum was
collected within 3 min. An appropriate amount of NaOH was
then added to reverse the pH back to 7.70, and random coil
behavior was observed again. This process was repeated three
times, and the CD spectra were found to be superimposable
with respect to pH (SI-3), indicating that this self-assembly
transition occurs under thermodynamic equilibrium and
requires 3 min or less to achieve the expected morphology.
Conventional transmission electron microscopy (TEM)
imaging was used to determine the morphology of 10 μM
PA1 at pH 6 and 8 (SI-4). The TEM grids were prepared
within 3 min of pH adjustment. At pH 6, both individual and
bundled fibers were present but dilute, and the isolated fibers
the peptide sequence.8 Our PA molecules consist of three main
segments: a hydrophobic alkyl tail, a β-sheet-forming peptide
sequence, and a charged amino acid sequence. Decreasing the
repulsive interaction of the charged region either via electro-
static screening or by lowering the degree of side-chain
ionization with pH induces assembly into nanofibers. For
instance, it was recently shown that upon addition of 5 mM
Ca2+, a specific peptide amphiphile (palmitoyl-VVAAEEEE-
GIKVAV) underwent a spherical-to-nanofiber transition at pH
7.4.8d From this observation, we reasoned that, by balancing the
relative attractive and repulsive forces via the peptide sequence,
it would be possible to enable the transition to occur at the
desired pH in physiological salt concentrations.
Herein, we have developed a PA design strategy for tuning
the pH at which the self-assembly transition into nanofibers
occur by tenths of a pH unit, in simulated serum salt solutions
(150 mM NaCl, 2.2 mM CaCl2) at 10 μM PA.9 As one of our
eventual goals is to develop Gd3+-based magnetic resonance
imaging agents, 10 μM is the minimum diagnostic concen-
tration of these agents in blood.10 The PAs in this study contain
a palmitic acid tail, an XAAA β-sheet-forming region, where X is
an amino acid with a nonpolar side chain, and four glutamic
acid residues (Table 1). A ratio of one strongly hydrophobic
had an average length of 590
200 nm and an average
diameter of 9.1 1.5 nm. This fiber diameter corresponds to
roughly twice the molecular length from MM+ molecular
simulations, corresponding approximately to the expected
diameter of cylindrical fibers consisting of hydrophobically
collapsed β-sheets. At pH 8, no fibers were present, confirming
that the β-sheet character corresponds to the existence of fibers.
When the CD spectra show a random coil morphology, the
PA molecules could either be self-assembled into spherical
micelles or exist as isolated molecules in solution.8d Because it
is difficult to distinguish between staining artifacts and sample
with TEM imaging at such a low concentration of sample, to
determine the morphology under basic pH values, the critical
aggregation concentration (CAC) was measured for PA1 at pH
6.6 using the pyrene 1:3 method (SI-3).11 The CAC was found
to be 6.0 μM, which is slightly below the 10 μM concentration
at which the CD spectrum was obtained. These two values are
in relative agreement considering the arbitrary nature of
defining the transition pH from the CD spectrum. Thus, the
random coil behavior corresponds to isolated molecules in
solution, as opposed to a spherical micellar morphology.
To determine the overall influence of concentration and pH
on the nature of this self-assembly transition, CAC measure-
ments were performed at pH 4.0−10.0 (SI-5), and CD spectra
were collected at 10−30 μM concentrations (SI-6). The
transition points determined from both techniques were
plotted to generate a concentration−pH self-assembly phase
diagram (Figure 2c). PA1 exhibited a strong dependence on
both concentration and pH in the self-assembly transition. This
concentration dependence was further confirmed via conven-
tional TEM imaging. At pH 6.0 and 10.0, both isolated and
bundled nanofibers were observed in samples prepared at 0.5
mM concentration (Figure 2a,b). At pH 6.0 and 10.0, the
isolated nanofibers had average diameters of 9.4 1.1 and 9.5
1.2 nm, respectively.
Table 1. Synthesized PA Molecules
molecule
sequence
PA1
PA2
PA3
PA4
PA5
PA6
palmitoyl-IAAAEEEE-NH2
palmitoyl-FAAAEEEE-NH2
palmitoyl-VAAAEEEE-NH2
palmitoyl-YAAAEEEE-NH2
palmitoyl-IAAAEEEEK(DO3A:Gd)-NH2
palmitoyl-VAAAEEEEK(DO3A:Gd)-NH2
amino acid (tyrosine (Y), valine (V), phenylalanine (F), or
isoleucine (I)) to four glutamic acids was essential to enable
this transition in the desired pH range of 6.0−6.6. PAs were
synthesized by solid-phase Fmoc synthesis and purified by
reverse-phase high-performance liquid chromatography
(HPLC) (SI-1). Their purity was assessed using analytical
HPLC, electrospray ionization mass spectrometry (ESI-MS),
and peptide content analysis (SI-1, SI-2).
Our target PA concentration (10 μM) is below the detectable
limit of conventional techniques to determine the morphology
such as cryoTEM and small-angle X-ray scattering. Con-
sequently, circular dichroism (CD) spectroscopy was initially
used to characterize the morphology of these PAs at various pH
values. PA1 was the first molecule synthesized that underwent a
self-assembly transition in our desired pH range of 6.6−7.4 at
10 μM PA in 150 mM NaCl and 2.2 mM CaCl2 (SI-3).8d The
secondary structure exhibited a superimposable random coil
By varying the β-sheet propensity of the amino acids in the
β-sheet-forming region, the transition pH can be systematically
tuned. In PA2−PA4, the isoleucine of PA1 was substituted
with the hydrophobic amino acids phenylalanine, valine, and
tyrosine. pH-dependent CD spectra of PA2−PA4 at 10 μM
also show a β-sheet-to-random coil transition at pH 6.0−6.6
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We then incorporated an MRI imaging moiety on the C-
terminus of the PA. An additional lysine, conjugated to a 1,4,7-
tris(carboxymethylaza)cyclododecane-10-azaacetylamide
(DO3A) tag was linked to the C-terminus of PA1 and P3 to
produce PA5 and PA6. The molecule-to-nanofiber transition
was still observed at 10 μM PA; however, the transition pH of
PA5 was shifted to 5.7 (SI-10). Since this imaging moiety does
not add excess charge, this shift toward more acidic pH is likely
due to the greater steric hindrance and additional hydrophilicity
of DO3A restricting the formation of the self-assembled state.
The concentration−pH self-assembly phase diagram was
mapped out for PA5 (Figure 3c). Under basic conditions and
Figure 2. TEM images of 0.5 mM of PA1, measured pH (a) 6.0 and
(b) 10.0. (c) Concentration−pH self-assembly phase diagram of PA1
as determined via CAC (blue diamonds) and CD (red squares)
measurements. All samples were prepared in 150 mM NaCl and 2.2
mM CaCl2. The concentration and pH values at which the TEM
images were obtained are labeled in (c). The white area corresponds to
a region where the self-assembled morphology is uncertain due to the
lack of suitable experimental techniques.
(SI-7). Similar to PA1, this transition was observed to be
reversible (SI-8). Previous studies have shown that the
propensity for β-sheet formation of these amino acids follows
the trend I > F > V > Y.12 In PA1−PA4, the transition pH
shifts to lower values with decreasing β-sheet propensity of the
substituted hydrophobic amino acid (Table 2). The average
Figure 3. TEM images of 0.5 mM of PA5, measured at pH (a) 4.0 and
(b) 10.0. (c) Concentration−pH self-assembly phase diagram of PA5
as determined via CAC (blue diamonds) and CD (red square)
measurements. All samples were prepared in 150 mM NaCl and 2.2
mM CaCl2. The concentration and pH values at which the TEM
images were obtained are labeled in (c).
a
Table 2. CD Transition pH and pKa for PA1−PA4
molecule
CD transition pH
average pKa
PA1
PA2
PA3
PA4
6.6
6.6
6.2
6.0
4.66 0.10
4.94 0.09
4.86 0.11
4.70 0.10
above the CAC, a random coil secondary structure was
observed in the CD spectra, which is indicative of self-assembly
into a spherical micelle phase. The transition from nanofibers to
spherical micelles was confirmed via TEM imaging at 0.5 mM
PA at pH 4 and 10, respectively (Figure 3a,b). The nanofibers
and spherical micelles had diameters of 11.9 1.6 and 10.0
1.2 nm, respectively. In contrast to the nanofiber-to-molecule
transition, the pH for the nanofiber-to-micelle transition
showed relatively little concentration dependence. The nano-
fiber-to-micelle transitions at 0.5 mM and 20 μM PA occurred
at pH 6.0 and 5.7, respectively (SI-11). The steric bulk of the
DO3A moiety increases the headgroup size of PA5 relative to
PA1, thus inducing the spherical self-assembly morphology.13
The shift in transition pH due to the change in β-sheet
propensity still occurs when the Gd(DO3A) moiety is present.
For each concentration, PA6 had a nanofiber-to-micelle
transition that occurred 0.4 unit lower than for PA5 (SI-12).
Because the same trend occurs in these PAs irrespective of the
presence of a Gd(DO3A) moiety, this strategy of altering the β-
a
For 10 μM PA1−PA4, measured in 150 mM NaCl and 2.2 mM
CaCl2.
pKa values for the glutamic acid residues in each molecule were
determined from pH titration curves (Table 2) to be in the
range 4.66−4.94, with no specific correlation with the
hydrophobicity of the peptide (SI-9). Therefore, the difference
in self-assembly pH is not due to changes in the pKa of the
glutamic acid side chains. Rather, the transition is determined
by the balance between the relative attractive forces of the β-
sheet-forming and hydrophobic regions, and the repulsive
forces of the deprotonated glutamic acids in the peptide. With a
stronger β-sheet-forming segment, the transition shifts to more
basic pH. For PA1 and PA4, the transition at 10 μM occurred
when 98.8% and 91%, respectively, of the glutamic acids were
deprotonated.
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the transition pH.
Relaxivity values of water protons in the presence of PA5 at
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the tumor environment compared to the bloodstream, but the
improved relaxivity of fibers compared to spheres could serve as
a secondary mechanism for enhanced tumor detection.
In summary, we have shown that, through judicious design, it
is possible to use the power of self-assembly to develop
dynamic materials that change shape and size in response to
slight changes in pH, in solutions that have monovalent and
divalent ion concentrations similar to those of serum. This
morphological change is rapid and reversible and occurs under
thermodynamic equilibrium, which is ideal for in vivo imaging
and drug delivery applications. Although further optimization of
the spherical-to-nanofiber transition pH is required for in vivo
MRI applications, the molecules presented here outline a
design strategy for precisely tuning self-assembly behavior.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental methods, materials, and synthesis schemes;
HPLC chromatagrams, ESI-MS spectra, CD spectra, pH
titration curves, and CAC determination for relevant PAs.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
goldberger@chemistry.ohio-state.edu
Author Contributions
‡These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank N. Raju and K. Kumar for helpful discussions. TEM
images presented in this report were generated using the
instruments and services at the Campus Microscopy and
Imaging Facility at The Ohio State University. J.G. thanks The
Ohio State University Research Foundation, and M.T. thanks
the Stefanie Spielman Foundation for financial support.
REFERENCES
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