2008 Progress Report – Executive Summary
The overall vision of our Nanomedicine Center is to advance the technology, science, and biomedical application of membranes supported on nanoporous substrates. Our philosophy is explicitly biomimetic. Because of all the things that biological membranes can do (energy transduction and production, electrical signaling, chemical signaling, sequestering, etc.) we have a proof of concept that if we can capture the essence of the biological membrane we should be able to adapt all those functions for nano-engineered therapies. Our engineered membranes are constructed by self-assembly (as are biological membranes). As with biological membranes, the self-assembly of our engineered membranes relies on the balance of polar and non-polar regions in membrane molecules to induce the membrane formation and the embedding of proteins in the membrane that provide its active character. In our most common construct, the supports that our membranes sit on are made of amorphous silica, which is generally recognized as safe in the human body (for example, it is a common food additive).
In order to fulfill our vision, our Center has a wide variety of scientific disciplines represented among our principal investigators, including materials science, mechanical engineering, electrical engineering, chemistry, and physics, as well as the more traditional biomedical disciplines of physiology and biochemistry. Early in the project we organized our Center along the lines of particular technologies (computational, experimental, and design) to reflect our initial developing fundamental technologies. More recently we have reorganized our NDC into five subprojects that reflect the evolution of our project as we have accomplished initial technology development and move to greater emphasis on particular clinical targets and classes of biomedical problems. The subprojects are 1) Functional Protocells, 2) Direct Protein Therapy for Cystic Fibrosis, 3) Oxidative Damage to Membranes, 4) Design of Channels and Transporters, 5) Biobattery
The functional protocell subproject is built around our team’s ability to make synthetic cells by loading a nanoporous silica sphere with internal electrolyte as below:
and then depositing a membrane with a defined set proteins on the surface as below:
The functional protocell architecture is particularly powerful because of the ability to independently engineer the interior contents and the external surface, and for the ability of the surface to communicate with, and modify, the interior contents. Our overall goal for the functional protocell construct is to selectively engineer into it capabilities of complete cells that will be useful for such purposes as targeted delivery of therapeutics and absorption and/or inactivation of toxins or viruses.
The most important milestones that we have passed in functional protocell technology in the past year are:
- Verification that functional protocells can readily achieve entry into the cytoplasm of both mammalian and bacterial cells.
- In vitro demonstration that protocells coated with human receptor protein (“honeypot cells) can specifically and drastically reduce infection with “pseudotyped” viruses that faithfully represent Nipah and Hendra viruses, emerging lethal human pathogens.
Major broad goals for this subproject are specific antimicrobial therapy that can circumvent drug resistance, using the functional protocell as the delivery agent, and specific antiviral therapy using the functional protocell in the “honeypot” mode. Bacterial targets of particular interest include methicillin-resistant Staphylococcus aureus (MRSA) and the array of infections that arise in the airway mucus of cystic fibrosis patients. (We have identified experimental and bioinformatics collaborators for both of these targets, as well as being already embarked on collaboration for the Nipah/Hendra viruses (henipavirus) studies that is being supported by our initial Pathways to Medicine supplement.) The work in the coming year will be directed towards those goals.
Direct Protein Therapy for Cystic Fibrosis
Cystic fibrosis is caused by a defect in a gene coding for an ion channel that selects for the negative ions chloride and bicarbonate over other ions. The particular gene is the Cystic Fibrosis Transport Regulator (CFTR) and is expressed in a variety of tissues, including the linings of the lung and the gut, cardiac muscle, and bone. Researchers at our center have developed a synthetic anion selective channel that enters membranes readily. The scaffold for the synthetic channel is the bacterial toxin alpha-hemolysin (structurally a beta-barrel channel) that is made anion selective by one of two ways: 1) Mutation of residues lining the lumen of the channel, and 2) Covalent linkage of a cyclic sugar molecule (beta-cyclodextrin) in the lumen of the channel, forming a synthetic selectivity filter. The ongoing work is to test the hypothesis that inserting the synthetic channel into epithelia whose function has been impaired by cystic fibrosis will cause a recovery of function—especially the ability to transport water. The ongoing work has the following components:
- Experimental work in which the existing construct is inserted into CF epithelial preparations (airway and gut) and function measured.
- Further channel modification to adjust selectivity and to introduce the possibility of channel regulation by intracellular messengers.
- Theory of three sorts, a) dynamic modeling of the epithelia to define “engineering specifications” for the synthetic channel, b) molecular modeling to go hand-in-hand with chemical modification of the channel to tune the channel to match design specifications, and c) informatics to augment experimental work on the control mechanisms governing ion transport in the epithelia, and the connection between CFTR and osteoporosis manifest in bone.
Oxidative Damage to Membranes
This work builds on the abilities of members of our Center to experimentally measure membrane organization under different conditions and with different membrane molecules, and to do corresponding theory. The theory is enabled by the ability of Center researchers to use Mean Field Langevin Dynamics to extend the time and spatial scales achievable by molecular dynamics, enabling simulation of membrane organization.
The biomedical issue is that oxidative damage disrupts membrane organization. To a first approximation, this may be due to a change in the ratio between ceramide and more complex sphingolipids. We are doing coordinated experimental and simulation work to understand at a basic level what is happening in multicomponent membranes subjected to this type of disruption. This may lead to a possible intervention by adding a “shim” membrane molecule. This is by analogy to macroscopic shims used in engineering to compensate for parts that do not fit together precisely. At the membrane level, it seems that membrane molecules fit together by complementary charge and shape distribution, with the basic shape being a “wedge”, the taper of which is defined by the relative cross-section of the hydrophilic (water-loving) head groups and the hydrophobic (wateraverse) tails. We will test the hypothesis that the understanding we gain of detailed membrane structure by experiment and simulation can lead to the design of molecules that, when inserted into damaged membranes, will Program Director/Principal Investigator (Last, First, Middle): Jakobsson, Eric restore correct domain structure. Delivery of such molecules can be by liposomes that will fuse with the membrane.
Design of Channels and Transporters
In addition to the work specifically targeted at cystic fibrosis direct protein therapy (mentioned above), we are working toward the design of a “toolkit” of robust channels with various properties useful for a variety of investigations and therapies. These channels would mimic the properties of, but be more robust than, native helix-bundle channel proteins. A major motivation for this activity is to have tools for specific engineering of functional protocell membranes. Beyond that, we note that cystic fibrosis is only one (albeit a particularly important one) of a large number of known channelopathies that in principle could respond to direct protein channel therapy, if proteins of the right properties can be designed and delivered. A major theoretical milestone was achieved by researchers in our Center in the past year, with the elucidation of the most subtle ion selectivity in biology, that between sodium and potassium. This work was done using both channels and transporters, and gives us confidence that we will be able to design desired selectivities into a toolkit of ion transport and permeation proteins for therapy and devices.
Our vision for the biobattery is for an implantable battery for prosthetic devices that will be made of biomimetic materials and will be rechargeable by the body’s own metabolism. The biological prototype of the biobattery is the organ of the electric eel, which consists of many flat excitable cells in series. When the cells are simultaneously stimulated, the organ generates for a short time a very large current and a large voltage.
We have attacked the biobattery problem at both the theoretical/engineering design level and the experimental device level. At the theoretical level we have made a dynamical model of the electric eel organ and done parameter optimization for various design criteria. The eel optimizes the design for maximizing a short burst of current, but the same overall design can be optimized by other criteria, for example efficiency in converting metabolic energy to electrical power.
On the experimental side, workers associated with the Nanomedicine Center have constructed a prototype biobattery, capable of producing a voltage across a network of cells. However the cellular architecture that has proved feasible for the experimental prototype is quite different from the electric eel organ. Whereas the electric organ is a stack of cells, each with a bilayer membrane, the prototype experimental biobattery is a network of vesicles, the surface of each of which is a monolayer of lipid. The vesicles are formed in a bath of oil, with electrolyte inside facing the lipid headgroups and the lipid tails facing the oil bath. The vesicles can be “snapped” together in a fashion like Lego blocks. The snapping together is done via an ion channel in the surface of one of the vesicles, so that once the vesicles are snapped together the interiors of the two vesicles are connected through the lumen of the ion channel, much as cells in syncytial tissue are connected by gap junction channels. By creating ion concentration gradients across the channel connecting the cells, it was found to be possible to create measurable voltage gradients across a network of cells.
In the next stage of the work the design calculations will switch from the cellular to the syncytial architecture, to consider how to make a device in this architecture---what should be the properties of the transporters, how can the energy gradients be restored, etc.
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