2007 Progress Report – Executive Summary
The NIH-Nanomedicine Center at Purdue University was established
in October, 2006. Currently, the NDC center involves 25 faculties
distributed in seven universities, including Duke, Northwestern,
U of California- Davis, U of Illinois-Urbana-Champaign, Purdue,
U of Southern Mississippi, and U of Cincinnati. The role
of different faculties in the center is classified into the
following categories: PI (1), co-PI (3), Key personnel (16),
and collaborators (5). Our long-term goal is to develop a
delivery device that can recognize specific targets for the
active pumping of therapeutics into cells in a controllable
fashion. To create a new generation of embedded motor for
passive diffusion and active pumping of DNA, RNA, drugs or
other therapeutic molecule to specific cancer, viral infected
or ailing cells we are initially focusing on covering the
gap in knowledge at the bio- and nanomaterials interface
by constructing matrix- or lipid/polymer membrane-adapted
motors based on the wellstudied bacteriophage Phi29 DNA packaging
motor. Our goal is a good match to the Vision of the
NIH Roadmap for Medicine RFA, including “a system of
molecular motors”, a model for the study of “energy transduction”,
and a tool for “transport of materials across membranes”.
The engineering principles underlying the construction and
operation of the embedded motor will be elucidated by an
interdisciplinary team including physicists, chemists, engineers,
mathematicians, physicians, computation scientists and molecular
biologists. Although extensive studies on the native bacteriophage
Phi29 DNA packaging motor have been carried out for years,
adaptation to a matrix substrate for therapeutic applications
is unique and distinct. Our primary aim is to apply the knowledge
of the Phi29 motor to a membrane-adapted form, embedded in
a polymer or lipid membrane sheet, liposome, polymersome
or template-directed foreign environment.
THEME: Phi29 DNA-Packaging Motor for Nanomedicine
Nature has achieved tremendous design optimization and efficiency
to perform work in biological systems. Deep and broad fundamental
research over the past 20 years has led to a good understanding
of the molecular mechanisms of Phi29 DNA-packaging motor
activity. For example, the Phi29 connector is a self-assembled
12- fold symmetrical ring of gp10 proteins. The resulting
cylindrical structure contains a 3.6 nm central channel,
which makes it an ideal candidate as a building block for
nanotechnology and nanomedicine applications. The goal of
this Nanomedicine Development Center (NDC) is to create biologically
compatible lipid or polymer membranes and metal or silicon
arrays with embedded active Phi29 DNA-packaging motors for
applications in medicine. To achieve this goal, three important
research challenges are elaborated:
- Re-Engineering the Phi29 Motor for Incorporation into
Lipid Bilayers
- Mechanistic Studies of the Re-Engineered Membrane Adapted
Motor
- Active Nanomotor/metal and silicon Arrays for Drug Delivery
and Diagnostics
Challenge 1: Re-Engineering the Phi29 Motor for Incorporation
into Lipid Bilayers
A lipid bilayer membrane is an appropriate vehicle to host
and direct the nanomotor activity for diagnostic or therapeutic
activity in humans. Activities in the first challenge are
directed at co-opting the simplicity and efficiency of design
of the Phi29 motor to provide effective machinery for movement
and transport of cargo in human cells and tissue systems.
We explore the feasibility of incorporating the native Phi
29 connector into the membrane of liposomes, polymersomes,
and sheets composed of lipid or polymers, which will enable
both passive and active delivery of drugs and/or DNA to the
infected cell. For passive membrane-bound motors, the connector
will be incorporated into the membrane and serve only as
a pore. The main concern is that the alternating hydrophobicity
and hydrophilicity of the three-layered motor connector structure
is unfavorable to its insertion into the membrane of liposomes.
Initial studies indicate several approaches to incorporate
the Phi29 nanomotor into liposomes that might be successful.
The emerging promise is that liposomes of various types can
be generated for different applications such as to deliver
specific therapeutic cargo or for targeting to specific cells
and organs. Activities in Challenge 1 are already underway
to establish the best strategies to reengineer the connector,
to attach or insert the connector to liposomes or lipid sheets.
Activity in this challenge includes development of strategies
to incorporate active and functional Phi29 motors into liposomal
membranes. Gaining control over the orientation of motor
assembly and incorporation will be a major goal. The stabilization
of both protein and nucleic acid motor components in the
heterologous lipid or polymer environment will also be required.
Research will be undertaken to develop methods for encapsulation
of therapeutic and diagnostic cargo into the motor studded
liposomes. These procedures must eventually meet clinical
standards and therefore must be robust, reproducible and
efficient. Ongoing work will focus on methods for tagging
motor-studded and loaded liposomes to track these materials
in cell cultures and ultimately in vivo as well.
A key issue for our approach to incorporate this nanomotor
into liposomes is the potential toxicity and immune response
associated with these constructions as therapeutics. We expect
the materials to be very well tolerated based on the excellent
track record for liposomes in the clinical trials. However,
this must be rigorously tested in cells and animal systems.
Similarly, pharmacokinetic studies will be carried out to
evaluate adsorption, distribution, metabolism and excretion
of these materials as required for characterization of therapeutics.
Challenge 2: Mechanistic Studies of the Re-Engineered
Membrane Adapted Motor
To effectively employ the Phi29 DNA packaging motor for therapeutic
and diagnostic purposes as proposed in Challenge 1, we must
better understand the engineering principles underlying the
design and function of the membrane-adapted motor. Substantial
molecular characterization of the native motor system has
been obtained in the laboratory of the PI and many others.
However, a deeper and system-based understanding of the reengineered
and membrane adapted motor will be required to co-opt and
even to improve upon the motor for nanomedicine applications.
The engineers, physicists and other NDC team investigators
are currently working to obtain this deep understanding of
motor reengineering and the resulted artificial motor. This
team has the resources and capabilities to re-engineer motor
components and its activity for specific applications.
Methodologies that will strengthen our efforts to gain control
on the Phi29 motor function include ultra-high resolution
imaging and ultra-fast photography. Systems with single molecule
sensitivity using Total Internal Reflection Fluorescence
(TIRF) microscopy, Fluorescence Correlation Spectroscopy
(FCS), cryo and variable vacuum scanning electron microscopy
are all in use to achieve more detailed structural characterization
of the modified motor components and the altered motor to
elucidate relationships between structure and function in
membrane adaptation.
Physical methods to measure the interaction forces of motor
components with the lipid or polymer are beeing developed
and applied by the NDC investigators. Various bead and cantilever
based approaches already hold promise for accurate and facile
monitoring of motor activity in membrane crossing and transport.
Lessons learned from those approaches will be key stepping
stones for gaining control over the assembly of functional
motors incorporated into the membrane or embedded in a foreign
environment. The first step will be to apply these findings
to control the motor orientation upon reengineering or incorporation,
especially in heterologous systems such as the liposome.
The empirical measurements will be complemented by molecular
modeling approaches. This will generate new hypotheses concerning
the nanomotor function which can be further tested in the
laboratory.
Importantly, it is anticipated that this mechanistic understanding
of motor function will directly suggest alternative approaches
to DNA nanoparticle assembly. With component parts of the
motor understood at the molecular and atomic scale, it will
be crucial to formulate chimeric motors with ‘bullet proof’
components to provide in vivo durability and therapeutic
impact. The developed re-engineered motors will, of course,
require characterization with respect to activity, biocompatibility,
durability, toxicity and biodistribution as described above
for challenge 1.
Challenge 3: Active Nanomotor/Metal, DNA or Silicon
Arrays that Enable Drug Delivery and Diagnostics
a) The short-term objective in this challenge is to construct
connector arrays that can serve as templates for further
construction of superlattices. A key component to meet the
goals of this challenge is to immobilize stable and functional
nanomotors in metal, DNA or silicon arrays. The concept for
the ability to ‘array’ motor components has been demonstrated
in the first year of the NDC award, as revealed by the self-assembly
of motor components as well as the capability of vectoral
localization of motor components in a planar lipid bilayer.
Risks for the proposed Center
- Although it is very possible that a membrane/motor chimera
could be constructed and the passive transportation of
drugs or ion could occur, there is a risk that the motor
might not be able to carry out its active transportation
role by at first. It is expected that this is a low risk,
since one of the key motor components is an RNA and the
other one is a DNA. Both RNA and DNA are easier to manipulate
than other macromolecules such as proteins.
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