Nature has been utilizing nanoscale machines since the emergence of prokaryotic organisms. These devices provide myriad examples from which biomedical researchers may harness opportunities for new nanoscale devices and approaches in medicine. By developing artificial nanomachines, we will be able to interact more effectively with biological entities and to influence their behavior for desired outcomes. Bacteriophage phi29, a virus that infects bacteria, provides unique and sophisticated examples of novel nanomachines. phi29 uses nanomotors to package its genome into a nanometer dimension protein capsid, creates nanoplugs that can resist high internal pressures of the packaged genome, has nano-tweezers that can hold onto the surface of host cells, and assembles nanochannels and nanopores for transporting its genome during replication. Further, viral capsids are protein self-assemblies that display unique and outstanding mechanical properties. The genome of phi29 is packaged into a preformed protein shell during replication. This energetically unfavorable DNA translocation process is accomplished by a DNA packaging motor geared by a hexameric ring composed of six pRNAs (packaging RNA). The pRNA assembles into dimers, trimers, and hexamers via hand-in-hand interactions between two right and left interlocking loops. The unique feature of the two interlocking loops makes phi29 pRNA a promising nano-tool for nanomachine fabrication, pathogen detection, and co-delivery of multiple therapeutic reagents with ligands for specific cell targeting. The core component of the phi29 motor is a dodecameric connector composed of 12 copies of protein gp10, which forms a channel with a cross-section area of 10 nm2 acting as a path for dsDNA translocation. Until now, our fundamental understanding of nature’s nanomotors has not yet effectively translated into new biomedical applications. This intricately articulated ATPdriven motor has inspired its application to problems in nanotechnology and nanomedicine. Incorporation of this motor or connector into a membrane will potentiate its applications for single molecule sensing, microreactors, gene delivery, drug loading, and sequencing of dsDNA. A key challenge in this regard is to develop our molecular-level understanding of how the nanomotor components transform chemical energy to mechanical work, ultimately so that the nanomachine may be actuated in artificial, non-viral environments. For example, currently there is no nanodevice available for actively pumping drugs, DNA/RNA and other therapeutic molecules into specifically targeted cells. This is also an extremely well-characterized nanomachine, providing an excellent opportunity for conducting the experiments necessary to reveal the biophysical properties of the motor and manipulating these for medical applications.

The goal of the Phi29 DNA-Packaging Motor for Nanomedicine team is to bridge the knowledge gap at the bio- and nanomaterials interface by employing the well-studied bacterial phage phi29 DNA packaging motor as a model for development of nanomedicine applications with a highly interdisciplinary team. The team currently includes key investigators from University of Cincinnati, Purdue University, University of Illinois, and from the City of Hope Research Center, as well as collaborative investigators from other six universities, that address three key challenges; 1) creation of multivalent pRNA nanoparticles for therapeutic and diagnostic uses, 2) employ the phi29 connector protein for translocation of therapeutic cargo from liposomes, and 3) deploy active engineered nanomotors on solid state membranes and in liposomes for therapeutic and diagnostic applications.

Several advantages of this system over other nanoparticle-based nanomedicine vehicles are summarized below.

• Controlled synthesis. Homogeneous nanoparticles derived from the dodecameric phi29 connector and the hexameric pRNA that can be reproduced with known stoichiometry is an important advantage to our system. A current serious limitation for competing nanoparticle approaches is that the materials are difficult to consistently reproduce with respect to the copy number within each nanoparticle. Our Phi29 motor and its components, on the other hand, can be “manufactured” with high reproducibility and known stoichiometry.
  
  
• Multi-valence. The Phi29 motor includes a connector core that is composed of 12 copies of a protein subunit and the pRNA ring that is composed of six copies of the pRNA. Each subunit may be separately functionalized to carry therapeutic, reporter, or targeting payloads (i.e., a total of 12 or 6 equivalents of drug and/or reporter, etc., at once).
  
  
• Targeted delivery and detection. Since each element can be individually programmed to provide a unique function, the Phi29 motor is ideally suited toward multiple target selection, reporter function and therapeutic response properties within a single Phi29-based agent. This degree of multifunctionality is extremely difficult to achieve with conventional nanoparticle-based vehicles.
  
  
• Advantageous size of the nanoparticle. Depending on configuration, phi29 nanoparticles can be produced in the 20 to 40nm size range. This multimeric particle is not so small that it will be rapidly excreted and not so large that it is unavailable for active uptake by cells. Since many studies suggest that particles in the 10- 100 nm range are the optimal size for a nonviral vector because they are large enough to avoid renal filtration, yet small enough to enter the cell via receptor-mediated endocytosis. This delivery strategy has the potential to improve the pharmacokinetics, pharmacodynamics, biodistribution, and toxicology of the newly emerging therapeutic nanoparticle modality.
  
  
• Expected minimal induction of antibody response by Phi29 pRNA nanoparticles will enable repeated treatments. Protein-free pRNA nanoparticles with RNA aptamers as anti-receptors will yield superior specificity and the lower antibody-inducing activity compared to protein anti-receptors, thus providing an opportunity for repeated administration and treatment of chronic diseases. Concerning the entire motor that includes the connector, which is a non-endogenous protein to humans, antibody induction may be a concern. Since the motor is covered by pRNA, this may mask the accessibility of the connector to immune recognition in a manner similar to the reduced optimization rates that occur for PEG-coated surfaces and proteins.
  
  
• Motor activity. The Phi29 motor exists to actively package viral DNA and is one of the most powerful nanomotors thus far studied and reported. An ultimate goal of our NDC is to harness that activity for loading and unloading of vesicle-based (liposome) and solid-phase (arrays) systems in therapeutic and diagnostic settings. Motor function may be critical to improve therapeutic efficacy since the active delivery of drug payload could provide an alternative to avoid the receptor-mediated endocytosis uptake pathway that ultimately leads to lysosomal degradation (i.e., for carriers that are not designed to escape the endosome).
  
  

The Phi29 NDC is focused on understanding the engineering principles of the unique Phi29 DNA packaging nanomotor. Our team will then exploit this knowledge of the motor and re-engineer the components of this intriguing nanomachine for medical applications. We anticipate that it will also be possible to extend these principles to other motors for eventual clinical use.

The specific achievements of the Phi29 NDC team to date have added to our understanding of this unique DNA packaging motor system in the following areas:

1. Re-engineering the phi29 motor for incorporation into lipid, diblock polymer films or cell membranes.
   
   
   • N- and C-terminal histidine-tagged variants of gp10 were prepared for oriented insertion into membranes;
     
     
   • stable incorporation of the gp10 connector in bilayer lipid membranes has been achieved and single channel conductivity of the connector determined under a wide variety of conditions;
     
     
   • oriented incorporation of the gp10 connector in bolalipid membranes has been achieved using methods that are amenable to large scale processing for animal testing;
     
     
2. Mechanistic studies of the re-engineered membrane adapted motor.
   
   
   • the mechanism of motor and component assembly was studied in Years 1–2 of the project;
     
     
   • the structure and function of the resulting motor intermediates and nanoparticle assemblies was elucidated;
     
     
3. Active nanomotor/metal and silicon arrays for that enable drug delivery and diagnostics.
   
   
   • preparation of solid nanopore structures for docking gp10 connectors was achieved in Years 1-2 of the project;
     
     
   • various strategies for aligning pre-assembled gp10 connectors with nanoporous substrates have been pursued.
     
     

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