Nanomedicine Development Centers
Cell Propulsion LabCenter for Cell ControlCenter for Protein Folding MachineryNanomedicine Center for Nucleoprotein MachinesNanotechnology Center for Mechanics in Regenerative MedicineNational Center for Design of Biomimetic NanoconductorsOptical Control of Biological FunctionPhi29 DNA-Packaging Motor for Nanomedicine

National Center for the Design of Biomimetic Nanoconductors

Executive Summary

While many research teams study biological membranes, including their medical applications, our center fills a unique niche in that we study membranes as systems to be engineered. We aim to acquire a full understanding of membranes, including the structure and function of ion channels and transporters and the regulation of membrane function. This knowledge will allow us to directly modify abnormalities in channel or membrane-bound regulatory proteins, or possibly construct new membrane components to treat and cure diseases. One of our distinctive technical innovations is a "functional protocell," which consists of a nanoporous solid containing reactants enveloped by a protein-containing membrane. We have also developed technologies to modify biological membranes in living systems. We are eager and ready to engage in collaborations with translational and clinical researchers in the fields of cystic fibrosis, hemodialysis, infectious disease, and oxidative membrane damage.

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Introduction

Creating structures at the nanoscale can be pursued by two methods. Top-down nanofabrication shrinks larger scale processes down to the nanoscale, and an example is nanolithography. In contrast, bottom-up nanofabrication is the formation of desired structures due to interactions of molecular components in close proximity. A key concept underlying bottom-up approaches in biology is self-assembly, the ordering of molecules into well-defined structures by noncovalent bonding. We believe that bottom-up nanofabrication technology has enormous future utility because of its potential for a very high degree of molecular-level specificity of interactions, and also because of the generally smaller dimensions that can be readily achieved with this approach. We know from biology that useful structures can be created at the nanoscale through self-assembly. For example, proteins, which are a few nanometers in size, acquire their three dimensional structures via protein folding, a self-assembly process. The surfaces of biological cells and organelles are formed by lipid bilayer membranes, themselves created through self-assembly, as is the process of inserting proteins into cell and organelle membranes. However, spontaneous self-assembly of a particular structure does not always produce the desired result (e.g., misfolded proteins) in natural systems, experimental systems, or in engineered nanoscale devices. We believe that directed self-assembly, under conditions that will produce a useful and desired structure, is the key to achieving optimal utility at the nanoscale.

Assembling the NDC team

Realizing the potential of supported membranes in particular, and directed self-assembly in general, is far beyond the scope of any individual laboratory and any particular scientific or technical discipline. Because the most powerful and compelling examples of useful self-assembled structures are biological, the project needs to include a strong molecular biophysics component. Manipulation of materials at the nanoscale requires a strong nanotechnology component. Engineering proteins requires a strong biochemistry and synthetic chemistry component. Understanding the structure of materials requires a strong materials science component. Because the theoretical and computational aspects of self assembled systems are still in a developmental stage, a strong theory and computation component is required for the team, focusing on the areas of transport through channels and membrane organization.

NDC Goals

Membrane transport accomplishes several basic functions in biological systems including: a) Electrical and electrochemical signaling, b) generation of osmotic pressures and flows, c) generation of electrical power, and d) energy transduction, and e) molecular recognition and chemical signal transduction. Our specific aims are two fold. We wish to:

  1. Engineer nanoscale systems with synthetic membranes that accomplish the functions of natural membranes in the service of therapy.
  2. Combine a systems understanding of membranes with an ability to manipulate the molecular components of membranes to repair defective natural membranes.

Highlights of Recent Accomplishments

We have invented and created the first prototype of the Functional Protocell

We define the "functional protocol" as a nanoporous solid surrounded by a membrane. The cavities in the solid can be filled with any desired electrolyte up to the limit of solubility. The surrounding membrane can contain any combination of membrane proteins. Thus the functional protocell can be imbued with any array of intracellular and membrane processes that are desired. It can be considered analogous to either a biological cell or an organelle such as a mitochondrion or a chloroplast (which started life as bacteria the order of 1018 nanoyears ago).

The phrase "functional protocell" as we are using it should be distinguished from the term "minimal protocell" that is commonly applied to a (so far hypothetical) minimal assembly of molecules that would have all the essential properties of cellular life, including self-replication. The "functional protocell" does not have all the essential properties of life, but would have specific designed properties that would make it technologically or biomedically useful. In some ways, the functional protocells would have the basic components of cells---a membrane that could do molecular recognition and transport, and an intracellular network of reactions that could sustain cell-like functioning. But it would have no properties that are not specifically built into it; especially the "functional protocell" would not self-replicate. The rationale for creating the functional protocell stems from the observation that cells have enormous flexibility and specificity of behavior compared to human-made entities on the same size scale. The key is the combination of a complex surface coating for specific molecular recognition and transport tasks, and complex contents comprised of networks of molecules organized to do specific tasks. Biological cells and viruses are often questionable therapeutic agents because of unpredictable side effects. Living cells and viruses may be modified by engineering to give some desirable properties, but because their functioning and their effects on human function are not completely understood, undesirable side effects can not be fully predicted and eliminated. Completely synthetic functional protocells, on the other hand, would only have components that are put into them, and could be guaranteed not to replicate.

Prior to the initiation of the NDC, the laboratory of Jeff Brinker (one of our NDC investigators) had developed a technique for creation of nanoporous silica objects, either as films or discreet spheres, with a size ranging from a few hundred nanometers to a micron (roughly the span from a virus to a small cell. The laboratory then acquired the ability to load the nanoporous spheres with any desired set of reactants in aqueous medium, by immersing the nanoporous spheres in a solution of the reactants, and then causing the reactants to be concentrated in the interior of the nanospheres by drying, as shown below:

Figure 1
Figure 1.

Within the past year, the Brinker lab succeeded in coating the nanoporous sphere in a protein-containing membrane by deposition of liposomes on the surface of the nanoporous sphere as shown below:

Figure 2
Figure 2.

The applications we envisage for the functional protocell will be given in a later section.

We have developed a computational method for simulating and predicting domain formation in heterogeneous membranes.

All biological membranes are made up of multiple types of molecules. Membranes made of more than one type of molecule will tend to form membrane domains, meaning that different parts of the membrane will have somewhat different properties. Membrane domain formation has high biological significance. The functioning of membrane proteins is modified by protein-lipid interactions that depend on the local lipid environment around the protein. Up to now, it has not been possible to rigorously simulate domain formation, because of how much computing time is necessary for atomically detailed molecular dynamics simulations of such a large system. Larry Scott of the NDC has developed a novel statistical mechanical method in which the results from atomically detailed molecular dynamics simulations of membranes are used to compute a mean interaction field for the different membrane components. The mean interaction field is utilized in a Langevin dynamics program. Scott and his co-workers have shown that for a phospholipid-cholesterol mixture (one of the most important classes of mixtures in membranes) this method successfully replicates the thermodynamic and structural properties of the mixed membranes. This prototypical result opens the way to predict and describe on an atomic level of detail domain formation in synthetic and natural membrane systems.

We have developed a covalently linked alpha-hemolysin and cyclodextrin anion-selective channel.

Alpha-hemolysin is a protein ion channel with a very large pore that is secreted by a bacterium and functions as a toxin, by inserting itself into cell membranes and permitting indiscriminate passage water and ions, essentially short-circuiting the cell membrane's role in regulating traffic between the cell interior and the exterior. Cyclodextrins are cyclic sugar molecules; informally one might describe them as shaped somewhat like donuts. Previously the Hagan Bayley lab of our NDC had shown that a cyclodextrin molecule would spontaneously insert into the lumen of an alpha-hemolysin channel and, while there, cause a reduced conductance moderately selective to anions. Now the Bayley lab has acquired the ability to covalently link the cyclodextrin into the channel lumen, creating a permanent anion-selective structure. This provides a platform for engineering channels of custom selectivity, by attaching various groups to the cyclodextrin. The first target will be a strongly chloride-selective channel, by addition of amine groups.

Other Advances

Applications

"Smart" hemodialysis utilizing functional protocells

In this application blood would be passed through a filter containing functional protocells. The protocell membranes would be equipped with proteins that would:

Combating Infection utilizing functional protocells

In this mode of functioning the protocell would have a surface coating that would recognize and bind to viruses or other pathogens. Once bound, a reaction would be triggered that would neutralize or kill the pathogen. One can reasonably envisage several modes of functioning, as follows:

Building a Biocompatible Battery that would be recharged by biological metabolism.

This was our founding mission, and we have made progress in the last year on both experimental and theoretical fronts. On the experimental front we have been able to create a network of synthetic cells that produces a voltage from one side of the network to the other, based on linking the network of cells with selective ion channels engineered on the framework of the bacterial toxin channel alpha-hemolysin. On the theoretical side we have made a dynamical model of the electric organ of the electric eel (the biological proof of concept for the biobattery) that can serve as an engineering design tool for the synthetic biobattery. Our next step is to adapt the analysis tools to the linked synthetic cell architecture and write engineering specifications for a battery built of linked cells that could be nutritionally sustained (i.e, take up glucose from its environment and produce electricity), and apply our knowledge of the linked cell fabrication technology to producing such a device. We envisage this to used as a power source for permanent implants in the body, such as the artificial retina or other neural prostheses.

Direct protein therapy for cystic fibrosis.

The common genetic defect in cystic fibrosis is in a gene that codes for the chloride-selective channel CFTR. While the gene is expressed in several epithelia (tissues composed of a layer of cells), the lethal symptoms are in the airway. The airway mucus becomes drier and more viscous, and thus cannot be moved by the cilia in the airway towards the mouth to clear inhaled foreign materials. The mucus becomes susceptible to opportunistic infection. Researchers in NCDBN have acquired the ability to convert a bacterial toxin, alpha-hemolysin, into an anion selective ion channel. The cystic fibrosis initiative within NCDBN is to explore the question of whether this finding by NCDBN can be the basis of direct protein therapy, in which a designed synthetic protein channel would play the role that the defective CFTR can not.

Understanding oxidative damage to membranes.

In another medical-directed initiative, NCDBN is utilizing an experimental technique developed by us (individually addressable arrays of membrane patches on a single nanoporous surface) and a computational technique newly applied by us to membranes (Mean Field Langevin Dynamics) to study in more detail than has previously been possible the changes in biological membrane organization that accompany oxidative stress. Changes in membrane properties under oxidative stress are implicated in many diseases, so we believe that the basic knowledge that we generate will ultimately be medically applied. Our initial focus is on oxidative membrane damage in macular degeneration and atherosclerosis.

References

Background

Current NDC Supported Papers

Contact Information

Center Website

Director

Key Investigators