TARGETED NANOPARTICLE DRUG DELIVERY

Z.B. Bilgiçer, H.-C. Chang, C. D’Souza-Schorey, B. Smith, and E. Y. Zhu

In the last decade, research in single-drug delivery by nanoparticles or nano-vesicles

has led to 24 commercial products with sales exceeding 5 billion US$. The next phase of
research is on nanotechnology based directed drug delivery and controlled release. Using the
fundamental understanding of the biology of the diseased tissue, our goal is to design
nanoparticles encapsulating multi-functional combinatoric drugs, and functionalize their surfaces
with recognition elements (such as RGD-peptidomimetics, antibodies, scFvs, etc.) that can
target specific diseased cells. (1-7)
Mammalian cells require oxygen and nutrients for their survival and are therefore located
within 100 to 200 µm of blood vessels (the diffusion limit for oxygen). Multicellular organisms
develop new blood vessels for growth and repair through a process called angiogenesis which
is regulated by a balance between pro- and antiangiogenic molecules. An imbalance in this
process can be initiated by numerous angiogenetic diseases such as malignant tumors,
inflammatory, ischaemic, infectious and immune disorders.(8, 9)(10) Angiogenic diseases cause
the blood vessels surrounding the tissue to grow larger and become more permeable (leaky)
than healthy vessels by increasing the porosity of the vasculature. Liposomes and nano-
particles (with sizes between 100 nm to 200 nm) get trapped in these larger than average
porous regions of the blood vessels.(2, 11) This discovery has been used as means for
passively targeting of angiogenic diseases with nanoparticles encapsulated drugs (Figure 1a),
and the first applications reached the clinical trials in the mid-1980s. Passive targeting with
nanoparticles, however, encounters multiple obstacles on the way to their target; these include
mucosal barriers, non-specific uptake of the particle and non-specific delivery of the drug (as a
result of uncontrolled release).(1, 2) Therefore, two most important aspects of nanoparticle drug
delivery must be: i) the specific targeting of the diseased tissue with nanoparticles (appropriate
size and functionalization with antibodies—or other means of selective binding—provides
means of enhanced delivery of drugs and reduced non-specific toxicity); and ii) the timed
release of the drug (to prevent non-specific toxicity the drug must not diffuse out of the particle
while it is still in the circulatory system, and must remain encapsulated until the particle binds to
the target).

a) b)

Figure 1. a) Passive tissue targeting by

nanoparticles in blood vessels. Particles get
tFFped in the target tissue as a result of
leaky vessels and ineffective lymphatic
drainage; b) active cellular targeting of
nanoparticles with conjugated antibodies.

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 One way to overcome the first issue is to functionalize the nanoparticles with recognition
elements on their surfaces towards receptors present on the particular diseased tissue (Figure
1b). The conjugated antibodies or short chain variable fragments (scFvs) will provide selective
binding to the specific cell’s surface, and their endocytosis will be enhanced with suitably
adjusted binding affinities. We will refer to Başar Bilgiçer’s expertise in the physical
understanding of biomolecular interactions, antibody and scFv targeting, conjugation and
liposome chemistry to design the surface functionalized nanoparticles for active cellular
targeting. The specificity can be fine-tuned by adjusting the surface density and the monovalent
affinity of the antibodies and/or scFvs for a particular disease. (12, 13)
To address the second issue, we will engineer multilayered nanoparticles, where each
layer will contain one drug from the cocktail, and their release will be sequenced in accordance
with the appropriate timing of the delivery of
each drug for combination therapy.
Currently a significant amount of research
shows that combination therapy is more
effective than traditional therapies.
Furthermore, progressive and sequential
delivery of multiple drugs from multi-layered
nanoparticles has been proven to be an
effective way of treating tumors. (14) The
kinetics of the release of the drug from the
nanoparticles depend on: i) the strength of
the interactions (usually the dominant Figure 2. Liposome nanoparticles produced by the
interaction is hydrophobic interactions) Zhu group.
between the polymer and the drug as well
as the permeability of the polymer, when the drug is noncovalently encapsulated inside the
nanoparticles; ii) the rate of hydrolysis of the covalent bond (usually an ester) between the drug
and the polymer, when the drug is covalently conjugated to the polymer or the liposome. We will
investigate the optimal chemistry for the polymer/drug combination for the desired timing of the
release of the drug from the nanoparticle.
There is already some preliminary effort in compound drug synthesis within the group.
Elaine Zhu has successfully used AC electric field to synthesize multi-layered silicated large
liposomes and PEGylated polymeric nanoparticles. She has also stabilized the liposomes with
macro-counterions—nanocolloids of opposite charge from the ionic surfactants. Depending on
the field frequency, spherical liposomes of different dimensions and different number of lamellae
layers can be selectively synthesized (Figure 2). These multi-lamellae liposomes can be
endowed with high mechanical stability when silicated or after condensation of oppositely
charged nanocolloid. The Russian-doll capsules allow encapsulation of different drugs within
each aqueous layer and perhaps hydrophobic drugs within the bilayers.
We also propose to develop a high-throughput microfluidic platform, with integrated
imaging components, for screening nanoparticles (and possibly synthesize future drug
candidates) (Figure 3). We shall explore several new biocompatible methods for encapsulation
of different nanovesicles with
different drugs, a larger delivery
capsule that is mechanically
stable; and can yet release its
load, perhaps sequentially, upon
docking. The PIs have experience
Figure 3. Microfluidic platform for screening the size and in synthesizing core-shell polymer
loading of nanoparticles. colloids and multi-layered
liposomes. The microfluidic

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platform will also provide means for
selecting the nanoparticles that will have
the desired size and loading for providing
monodispersity to provide reliable
reproducibility of the experiments.
Elaine Zhu is currently working
with Chia Chang to synthesize such
compound capsules with microfluidic
devices such as the one shown in Figure
3. The electrokinetic chip can impart the
properly tuned AC field on the liposomes
at various stages on the chip to allow
sequential encapsulation and wrapping at
high throughput.
Once the particles are synthesized
and selected, the biological assays that
we will carry out to screen and study
these nanoparticles are described in the
schematics, and include the drug release
assays as well as the binding and uptake
studies. Our ultimate goal is to show the
efficacy of these targeted-controlled
release nanoparticles in the appropriate
preclinical disease models. More
specifically, we plan to use tumor
xenograft or syngeneic mouse tumor
models to study the efficacy of these
drugs in inhibiting tumor growth,
angiogenesis and metastasis. Furthermore, the pharmacokinetic studies will be accomplished in
rats to asses the in vivo drug release rates from the nanoparticles. Finally bio-distribution
studies will be accomplished to determine the degree of specificity and selectivity in targeting of
the diseased tissue by referring to the whole animal imaging expertise of Brad Smith’s group.
The biodistribution and pharmacodynamic properties of the drugs that are released from the
liposomes after reaching the target tumor is very different from that of free drug. It has been
shown that while free drug accumulates in all tissues because of its small size, liposomes
accumulate preferably in the tumors because of EPR effect. We plan to use free drug as a
negative control and compare tissue distribution properties of free drug and liposomal drug. If
the drug is released before it reaches the tumors, its biodistribution and pharmacodynamic
profile would resemble that of the free drug.
Further in vitro and in vivo experiments will identify whether the drug carrying
nanoparticles function via either or both of the two pathways: i) after the particle is positioned in
the proximity of the diseased tissue, passive diffusion carries the drug from inside the particle to
inside the cells; ii) the particle gets endocytosed as a whole (or sometimes smaller endosomes),
which is followed by the leaking of the drug inside the cell.
Endocytosis of larger endosomes or even the entire liposome delivery vehicle (> 10 nm)
remains a mystery. (15) C. D’Souza-Schorey’s expertise in endocytosis of cells will allow us to
fabricate microfluidic and imaging platforms for single cell assays. It may be quite possible that
endocytosis only occurs with some protein machinery rather than a physical phenomenon such
as rupture or fusion. After these preliminary studies, we shall develop a synthetic lipid bilayer
platform or, if necessary, a single-cell platform for rapid drug screening.

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 As bilayer rupture for such large nanocolloids requires an energy input in excess of 10 3
kT due to exposure of the hydrophobic surface, large nanocolloid internalization obviously
involves a restructuring of the bilayer, such as protrusion, inversion or fusion. However, the
exact nature of such topological rearrangement and how they are affected by dehydration
effects of the endosome and the membrane receptor (or receptor aggregates) or an internal
change in the lipid composition due to complexification after docking remains unknown. It is
quite possible that the large endosome form complexes with the membrane proteins or with the
bilayer in both mechanisms. Alternatively, there may be molecular transfer from the delivery
vehicle to the cell membrane. For example, if the nanocolloids Zhu uses to stabilize her vesicles
dissociate and condense onto charged protein sites on the membrane instead, the docked
vesicle would rupture spontaneously to release smaller endosomes or refuse with the cell to
allow internalization of the entire vesicle. A large nanocolloid or a polyvalent counter-ion may
also bridge the two surfaces, without dissociation from one, to produce a complex that favors
fusion.
It is quite possible that all the endocytosis routes can occur and specific drug design
requires careful screening of the pertinent internalization mechanism. (15) Such hypotheses on
colloid/liposome interaction will be first examined with synthetic lipid bilayers with the AFM,
FCS, TIRF and Surface Force Apparatus in Zhu’s lab. We shall support the lipid bilayers on
nanopillar supports fabricated to allow the study of internalization before testing the platform
against real cell membranes. Release of specific chemicals from nanofluidic channels will be
used to examine the effects of certain stimuli. Obviously, a synthetic bilayer platform would be
easier to run than one with real cells. The science of incorporating membrane proteins into the
artificial bilayers while retaining their bioactivity is well-developed and we shall include specific
receptors for our antibodies.
Ultimately our studies will establish the most efficient nanoparticles system with the right
qualifications, and the chosen system will be carried into clinical trials with the intention of
eventually improving patient outcome. Meanwhile, in the process, we will also be addressing
several unknown biophysical issues: how an antibody functionalized nanoparticle interacts with
a lipid bilayer membrane, how endosomes released by the vehicle are internalized via
endocytosis, how membrane protein aggregate patterns change due to docking with antibodies
functionalized to delivery vehicles and whether the protein aggregation can contribute to the
drug release and internalization.
The Faculty Fellow (FF) we seek is someone with extensive research experience in
nanoparticle drug delivery field who can choose and design the critical biological study plan to
evaluate and select the targeted drug-bearing nanoparticles with the highest physiological
relevance. The FF will complement the liposome and polymeric drug (antibody) synthesis
knowledge in Bilgicer’s group, imaging expertise in Zhu’s and Smith’s labs, the biological
strength of D’Souza-Schorey’s group and the microfluidic/nanofluidic fabrication know-how in
Chang/Bohn’s labs. The FF will have responsibilities of a project leader and project manager,
and will be expected to reach out to set up the right collaborations to make progress in all
aspects of this project to bridge biology with engineering. As such drug synthesis and fabrication
platforms should be of interest to the pharmaceutical industry and can lead to new start-ups,
some one with start-up experience would also be desirable.

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