Nanomedicine

Phi29 DNA-Packaging Motor for Nanomedicine

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:

1. Re-Engineering the Phi29 Motor for Incorporation into Lipid Bilayers
2. Mechanistic Studies of the Re-Engineered Membrane Adapted Motor
3. 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

1. 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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