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Research Topics > Self-Assembling Systems: Lipids and Peptides
Self-Assembling Systems: Lipids and Peptides
A significant research effort in our group over the past few years involves the
study of phospholipid and peptide self-assembly. Self-assembly of biological
molecules is being used to form hydrogel networks and nanoscale tubules, filaments,
and ribbons.
Lipid Self-Assembly
Biological phospholipid bilayer membranes are perhaps the most ubiquitous forms
of self-assembly, organizing living matter into compartments and acting as physical
barriers that partition the aqueous phase into several volumes (extracellular,
cellular, and subcellular) that are spatially, chemically, and functionally
distinct. The establishment and maintenance of compartment chemical composition,
mediated by the lipid bilayer membrane and molecules embedded within or attached to
it, is a critical aspect of biosynthesis, energy transduction, information storage,
and cellular recognition. Furthermore, many rapid biological events, such as muscle
fiber contraction, nerve impulse transmission, and blood coagulation, are regulated
by transient changes in compartmental ion concentration.
Over the last few decades, the use of liposomes for systemic (i.e intravenous)
delivery of drugs and diagnostic reagents has received widespread attention.
Historically, the major barrier to using liposomes for systemic drug delivery has
been the rapid clearance of liposomes from the bloodstream by the reticuloendothelial
system. Although there is renewed interest in systemic liposome administration due
to recent progress in the use of steric stabilization for increasing the blood
circulation lifetimes of liposomes, it is important to note that significant
challenges still exist for systemic delivery of liposomes, including the targeting
of liposomes to specific tissues or organs. In recent years, considerable effort
has been directed at constructing lipid vesicles that can be induced to release
entrapped materials in response to an applied stimulus. Typically, the goal of
these efforts has been to trigger site-directed delivery of therapeutic agents in
response to photo, pH, and other stimuli.
Most of our work has centered on the use of thermally triggered release of reagents
from lipid vesicles. Our approach exploits the release of entrapped substances from
liposomes at temperatures near the lipid chain melting transition (Tm), a temperature
at which bilayer permeability is known to be significantly enhanced. At temperatures
below Tm saturated phosphatidylcholine bilayers are in the gel phase (Lβ), which has
low permeability to encapsulated monovalent and divalent cations. However at Tm,
lipid bilayer permeability has been observed to increase by several orders of
magnitude, a phenomenon which has been attributed to the presence of highly permeable
interfacial regions between coexisting gel (Tm) bilayer domains.
This fundamental characteristic of phospholipid bilayers permits the design of
reagent-filled liposomes that are relatively impermeable to encapsulated molecules
at one temperature but highly permeable at another temperature. If the initial
concentration of an ion or low molecular weight molecule is high inside the liposome
but low outside, heating to Tm will result in diffusion of the entrapped species
down a large concentration gradient, effectively releasing the entrapped species
into the extravesicular medium. In general, we employ liposomes comprised of lipids
having Tm between ambient and body temperature (37°C), so that the thermal release
mechanism could be used to trigger biomaterial formation when a fluid precursor
suspension is injected into a warm tissue or body cavity.
In most cases we utilize saturated phosphatidylcholines, in which Tm is determined
primarily by the fatty acid chain length. By selecting miscible phospholipids with
appropriate fatty acid chain lengths, such as dimyristoyl phosphatidylcholine (DMPC)
and dipalmitoyl phosphatidylcholine (DPPC), we are able to tailor the Tm of the
bilayer. Although a 9:1 molar ratio of DPPC to DMPC is typically used because this
mixture has a Tm that coincides with the body’s temperature of 37°C, by adjusting
the DPPC/DMPC molar ratio we can tailor Tm to less than 37°C; such flexibility could
be useful in tailoring these systems to lower temperatures, such as might be
encountered in a limb or on the external surface of the body.
In collaboration with Prof. David Thompson at Purdue,
we are also employing light-responsive liposomes for in-situ formation of
biomaterials. For example, we have demonstrated the use of near infrared (NIR)
sensitive liposomes to trigger TG-mediated formation of fibrinogen hydrogels
(Bioconjugate Chemistry, vol. 13, 2002, pp. 640-46). Additionally, we demonstrated the use of both temperature and light
to trigger release of metal salts for gelation of a self-assembling peptide
(Journal of the American Chemical Society, vol. 123, 2001, pp. 9463-9464).
The use of NIR light to trigger biomaterial formation is
attractive because of the ability of 800nm light to penetrate soft tissue to a
much greater depth than either visible or UV light, in principle permitting
noninvasive phototriggering of sol-gel transformations and drug release.
Self-Assembly of Phospholipid Tubules/Ribbons.
In this research, we are studying the self-assembly of phospholipids into
nonvesicular structures for potential use as biomaterials and in materials
synthesis. Much of our work in this area has focused on understanding phospholipid
self-assembly in mixtures of polymerizable and nonpolymerizable lipids. The
diacetylenic lipid 1,2-bis(tricosadienoyl)-sn-glycero-3-phosphocholine (DC8,9PC)
has previously been shown to form hollow microtubules of length 50-200 microns and
average diameter of approximately 0.5 microns. A significant finding that we
published is the discovery of phospholipid nanotubules of diameter 50-60 nm and
length up to 100 microns, which transform into twisted nanoribbons
(Langmuir, vol. 15, 1999, pp. 4464-4471).
These phospholipid nanostructures were formed from a mixture of DC8,9PC and
the short chain saturated lipid 1,2-bis(dinonanoyl)-sn-glycero-3-phosphocholine
(DNPC). The three-dimensional lipid assemblies being investigated in this project
are significant in that the diameters of the DC8,9PC/DNPC nanotubules are
approximately an order of magnitude less than previously reported lipid tubules, and
therefore may open up new applications of lipid tubules as drug delivery vehicles,
or even as nanowires or channels for conducting electrical or chemical signals.
More detailed studies of the mechanism and properties of nanotubules may lead to a
better understanding of the relationship between phospholipid chemistry, molecular
shape and aggregate morphology. Toward this end, a collaboration with scientists at
the Naval Research Laboratory revealed that the nanotubule/twisted nanoribbon
transformation is accompanied by an inversion of the circular dichroism signal,
which suggests that the nanotubule/twisted ribbon transformation involves significant
molecular reorganization(Nano Letters, vol. 1, 2001, pp. 375-378).
From a practical perspective, both the nanotubule and network gel phase can be
considered as candidate biomaterials that could be useful for hard and soft tissue
reconstruction. The rapid physical gelation of the three-dimensional network gel
is a potentially useful property that can be exploited for biomedical use. For
example, such a gel could be used as an injectable drug delivery matrix which
undergoes in-situ gelation. Macromolecular drugs and other therapeutic agents could
be entrapped within the porous lipid network and diffuse into the surrounding tissue,
at a rate which would be influenced by the lipid network structure and density.
Self-Assembling Peptides.
We are also investigating the design and self-assembly of peptides and
peptide-modified polymers. Such systems greatly expand the potential for tailored
biomaterials since specific functional groups can be incorporated for enhanced
physical and biological properties. For example, we are studying self-assembling
peptides that form hydrogels in response to increases in ionic strength. This work
is based on peptides such as n-FEFEFKFKFEFEFKFK-c (FEK16), a peptide consisting of
alternating hydrophobic and hydrophilic residues and a complimentary pattern of
charged residues that allow it to self-assemble into β-sheet hydrogels. In solutions
of pure water, FEK16 is highly soluble, but in the presence of millimolar
concentrations of salts (NaCl, CaCl2, etc.) it forms biocompatible, non-immunogenic
hydrogels which are able to support the growth of a wide variety of cell types. In
order to trigger the formation of the hydrogel in response to these materials being
placed in the body, we designed formulations that included either temperature or
light sensitive liposomes (Journal of the American Chemical Society, vol. 123, 2001, pp. 9463-9464). By utilizing liposomes that
contain gelation-inducing salts, we were able to form peptide hydrogels at a specific
temperature or by exposure to NIR light. Freeze-fracture TEM images revealed that
the self-assembled gel was composed of a highly crosslinked network of fibrils
containing embedded liposomes. FTIR and CD spectroscopy indicated that the fibrils
contained a high degree of anti-parallel beta-sheet secondary structure.
Subsequently, a de novo peptide that self-assembles into fibrillar structures and
serves as a substrate for the cross-linking enzyme tissue transglutaminase was
developed (CH3COO-QQKFQFQFEQQ-NH2) (Bioconjugate Chemistry, vol. 14, 2003, pp. 748-755). Congo
red staining, circular dichroism, and FTIR spectroscopy showed that this 11-amino
acid peptide produced predominantly β-sheet structures. TEM with negative staining
and quick-freeze deep etch (QFDE) TEM showed that the peptide structures were
composed of a highly entangled fibrillar network. These β-sheet fibrillar
nanostructures were then covalently coupled to pendant amine-containing biomolecules
via tissue transglutaminase, using the glutamine (Q) and lysine (K) residues
included for this purpose. MALDI-TOF mass spectrometry and HPLC were utilized to
monitor the extent of the transglutaminase modification of the peptide, showing
that as many as five glutamines in the peptide were reactive via transglutaminase
for covalent conjugation.
Recently, we have also reported the self-assembly of peptide-polymer conjugates
consisting of a water soluble polymer (PEG) and short peptides
(Advanced Materials, vol. 16, 2004, pp. 907-10).
We designed and investigated the solution behavior of a
series of peptide-polymer conjugates that self-assemble via β-sheet fibril formation,
identifying a poly(ethylene glycol) (PEG)-conjugated peptide that self-assembles
into very uniform fibrils with little to no lateral aggregation. Quick-freeze
deep-etch (QFDE) TEM showed that this peptide-polymer formed aligned and uniformly
spaced fibrils of unprecedented regularity for β-sheet fibril-based assembly. In
summary, these self-assembling peptide systems have potential applications for drug
delivery, tissue engineering, or other biomedical applications.
of pure water, FEK16 is highly soluble, but in the presence of millimolar
concentrations of salts (NaCl, CaCl2, etc.) it forms biocompatible, non-immunogenic
hydrogels which are able to support the growth of a wide variety of cell types. In
order to trigger the formation of the hydrogel in response to these materials being
placed in the body, we designed formulations that included either temperature or
light sensitive liposomes (
