Lipid-Coated Biodegradable Particles as "Synthetic
Pathogens" for Vaccine Engineering
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Citation
Bershteyn, A. et al. “Lipid-coated biodegradable particles as
“synthetic pathogens” for vaccine engineering.” Bioengineering
Conference, 2009 IEEE 35th Annual Northeast. 2009. 1-2.
©2009 Institute of Electrical and Electronics Engineers.
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http://dx.doi.org/10.1109/NEBC.2009.4967679
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Lipid-Coated Biodegradable Particles as "Synthetic
Pathogens" for Vaccine Engineering
A. Bershteyn, J.P. Chaparro, E.B. Riley, R.S, Yao, R.S. Zachariah, and D.J. Irvine
Massachusetts Institute of Technology
77 Massachusetts Ave.
Cambridge, MA 02139
The physicochemical context in which molecules are presented
at the surfaces of microbes has tremendous implications for the
immune response to vaccination. The spacing and mobility of
molecules may control interactions of their receptors,
influencing immune cell activation, pathogen uptake, and
antigen processing. The chemical environment of antigens also
influences the specificity of the humoral immune response,
because antibodies recognize antigen in its three-dimensional
shape and context. Finally, physical properties of antigen, such
as diameter, impact immune response on both a cellular and
tissue level. We have constructed "synthetic pathogens"
consisting of a biodegradable core polymer coated by a lipid
shell to mimic a bilayer-enveloped pathogen. Synthesized in an
oil-in-water emulsion, these particles have an average diameter
on the order of either 100 nm, mimicking a lipid-enveloped viral
pathogen, or 1 micron, mimicking a bacterial pathogen.
CryoEM reveals self-assembled lipid layers at the particle
surface. With tunable chemical and physical properties, these
particles can be used to study the importance of specific
properties of biomaterials when used in vaccination. Because all
components are biodegradable, the particles may provide a
clinically applicable way of implementing structural features of
microbes in synthetic vaccines.
I. INTRODUCTION
Antigen-presenting cells (APes) such as dendritic cells
(Des) recognize pathogens by detecting pathogen-associated
molecular patterns (PAMPs) using Toll-like receptors (TLRs)
[1]. APes cross-present antigen to cytotoxic T lymphocytes
lOOO-fold more efficiently when the antigen is delivered on a
particle rather than in soluble form [2]. Particle size and
surface chemistry also playa role in transport of antigen from
the periphery to the lymph node, where T and B cells respond
to antigen. Particles under 20 nm in size can arrive at the
lymph node within hours, draining directly through the
lyphatics, whereas particles above 200 nm in size arrive
within days, and must be transported by cells [3]. Particles
"cloaked" by a hydrophilic shell of poly(ethylene glycol)
exhibit greater mobility in mucosal enviroments [4] and are
better able to leave the periphery to enter lymph nodes.
Although increasing PEG molecular weight improves the
ability of particulate antigen to leave the periphery, this
comes at a cost of poorer retention in the draining lymph
node [5].
We have developed a biodegradable particulate vaccine
carrier of a size that can mimic either viruses (order of 100
nm) or bacteria (order of 1 micron) [6]. The particles have a
poly(lactide-co-glycolide) (PLGA) core and a lipid-based
shell that includes phospholipids conjugated to 45-monomer
PEG chains. The lipid-coated, PEG-cloaked nanoparticles are
best suited for conveying lipid-like PAMPs and antigens,
though other molecules can be encapsulated into the particle
core or chemically coupled to function ali zed lipids at the
surface. The carriers can serve as models in studies of
immune response to pathogens, but are also based on
biocompatible materials for potential use as vaccines.
II. PARTICLE SYNlHESIS
Particles are synthesized using an oil-in-water single
emulsion or a water-in-oil-in-water double emulsion
technique. The hydrophobic phase can be either
dichloromethane or chloroform. These solvents are wellsuited for particle synthesis because they dissolve lipid and
PLGA, and because they are volatile, and can thus be
removed from particles by solvent evaporation. An interior
aqueous phase may be dispersed into this organic solution of
lipid and polymer. The organic phase, in turn, is dispersed
into a larger bath of water, with the method of dispersion
controlling the particle size distribution. Sonication using a
vibrating probe tip outputting 12 Watts yields nanoparticles
with an average diameter of 116 nm, whereas
homogenization at 12,500 RPM yields microparticles 1-5 urn
in diameter, which can be further selected for size by
centrifugation. Figure 2 shows the sizes of microparticles and
nanoparticles visualized by scanning electron microscopy.
Figure 1. Scanning electron micrographs of microparticies (left)
made by homogenization of lipid/polymer solution in water, or
nanoparticies (right) made by sonication of the solution.
b
Figure 2. Cryo-TEM of lipid-coated silica nanoparticles (left) and
biodegradable lipid-coated PLGA nanoparticles (right). Arrows
show lipid leaflets directly visualized with this technique.
III. LIPID SEGREGATION TO P ARTICLE SURFACES
During dispersion and subsequent evaporation of the
organic solvent, the lipid component acts as a surfactant that
stabilizes the oil-water interface, finally forming a coating
around the solid PLGA microparticles or nanoparticles.
Figure 3 shows a cryo-transmission electron micrograph
(Cryo-TEM), comparing lipid-coated PLGA with a wellstudied lipid-coated silica control [7] to show the similarity in
lipid coating. This "lipid surfactant" strategy provides great
versaility because of the ease with which the surface
chemistry can be modified [8]. The lipid also exhibits twodimensional fluidity along the particle surface, as detected by
fluorescence recovery after photobleaching (FRAP), which
shows diffusion of fluorescent lipids into a region within
seconds of bleaching with a high-powered laser (Figure 3).
Figure 4. Cryo-TEM
showing the dynamic
conformations of liposomes
lacking a rigid core. Liposomes
formed by rehydration of dried
lipid, followed by 6 freeze-thaw
cycles and 11 passes through a
400 nm track-etch membrane.
This treatment yields a size
distribution similar to polymersupported nanoparticles.
In constast to the rigid spheres shown in Figure 3,
liposomes can adopt a variety of conformations, such as rodlike or bowl-like shapes shown in the Cryo-TEM "snapshot"
in Figure 5. Our analysis of the effects of particle rigidity in
vaccination may be instructive for vaccine design.
These biodegradable lipid-coated particles can be used to
implement structural features of pathogens, as a versatile
approach to designing vaccine carriers.
ACKNOWLEDGEMENT
We thank Dr. Kazuyoshi Murata for technical assistance with
Cryo-TEM. This research was supported by the Human
Frontier Science Program, the NSF, the NIH, and DARPA.
A.B. was supported by the Fannie and John Hertz
Foundation, the Paul and Daisy Soros Foundation, and the
NSF Graduate Research Fellowship Program. J.P.c., E.B.R.,
R.S.Y., and R.S.Z. were supported by the MIT Undergraduate
Research Opportunities Program.
IV. IMPLICATIONS FOR VACCINE DESIGN
As vaccine carriers, these particles mimic the structure and
chemistry of pathogen surfaces. PAMPs such as TLR2
agonists and TLR4 agonists are themselves lipid-like
molecules, and therefore can be incorporated into the lipid
coating by the same self-assembly process as with the other
lipid components. This gives the PAMPs a more physiologic
lipid context, and a two-dimensional mobility helpful for
bringing TLRs in close proximity and induce signaling.
The system may also offer insights into aspects of vaccine
design that are not well understood. For example, mechanics
of particute antigen may playa role in antigen transport and
processing. Viruses can change their physical properties
drastically during the cycle of viral replication - in the case of
HIV, by a factor of fourteen [9]. We are investigating this
parameter by the juxtaposing our lipid-coated particles with
more traditional liposome delivery vehicles.
100
50
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Figure 3. Fluorescence recovery after photobleaching of a region on
a lipid-coated micro particle (red) as compared to the entire particle
(blue) and background (green). The lipid coating contains a
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