The goals of this project are: 1) to construct an innovative proteinbased nanometer-sized self-assembling delivery system ("adaptosome") containing metal particles that will be used for specific cell targeting and 2) to characterize and refine these structures by Raman spectroscopy. The Localized Surface Plasmon (LSP) and Surface enhanced Raman Scattering (SERS) properties of these metal-protein hybrid nanoparticles will be exploited for their inherent signal amplification and transduction properties. These properties have direct application for molecule and cell identification and for non-destructive sensing. The adaptosome will be a superior molecular probe capable of precise target cell identification in tissues. The adaptosome can be used for local inactivation and degradation of targeted molecules and cells. To achieve these goals a new protein shell based on in vitro self-assembling gene transfer agent (GTA) prohead system will be constructed and modified with the peptides designed to mediate specific attachment to, uptake into, and movement across cell membranes. Highly sensitive molecule identification will be accomplished by monitoring the vibrational signatures in the local tissue/cellular environment of the particles when bound to the target. Inactivation of target cells by visible and near IR light when it is needed will be possible.
Experimental analyses of the formation and propagation of local electromagnetic fields in specific biological environments will allow for rational construction of protein shells and precise selection of nanoparticle size and structure. Spectroscopic testing of the constructed adaptosomes will be performed both in vitro and in situ to optimize the structure of the protein shell in the generation of an LSP and, more specifically, to elucidate the effect of the internal structural organization and pattern of the protein on the metal nanoparticles. Information gathered from modeling and spectroscopic analysis will substantially accelerate the proposed research by a priori elimination of non-working structures and will be used to refine the design principles during construction of the particles.
Intellectual Merit: The members of this team bring to bear expertise in separate disciplines and will interact in a direct, cohesive way. This team has collaborated successfully for several years on the generation of bio-inspired devices for energy generation. The work described in this proposal will lead to the construction of an innovative biosensor and drug delivery system based on protein encapsulated metallic nanoparticles. The product of the work itself will have its most direct application in the development of chemical and biological sensors and cancer therapy, through cellspecific targeting. It will also impact areas of modeling of bio-inorganic interfaces, mathematical optimization theory, and in the physical chemistry of protein-nanoparticle assembly and interaction.
Broader Impact: This proposal is focused on a major unsolved problem in the current field of chemical and biological defense and in medicine, namely how to target biosensors or therapeutic agents to tissues and specific cells. Construction of this cell target-specific probe capable of delivery of a therapeutic agent will open the door to practical application of innovative principles of chemical and biological sensing and will facilitate exploration of new avenues in patient-addressed personalized medicine. The strategy of the utilization of metallic nanoparticles and their inherent SERS diagnostics that accompanies them will lead to the development of a sub-cellular labeling system with greatly increased sensitivity and versatility. The proposed system will allow for simultaneous identification of a non-predefined unlimited number of molecules (including previously unknown targets) based on their Raman signature. The knowledge that will be gained from developing the ideas presented in this project will contribute broadly to several areas in the emerging fields of nanomedicine, protein-metal interactions, nanoplasmonics, and nanobiology.
The blood brain barrier (BBB) is composed in large part of the micro-capillary system in the brain and the cells that constitute and closely surround it. Collectively, these structures act as a gate to keep brain material in, to keep out all but the essential nutrients found in the bloodstream, and to keep out unwanted toxins or microbes that could impair brain function. The BBB works very well, indeed perhaps too well from a medical point of view, since it is normally not possible to get drugs or therapeutic agents across the barrier to treat neurological diseases or cancers. There is therefore a great interest in being able to selectively cross the blood brain barrier and to target compounds or drugs to areas of the brain. Many approaches have been tried to accomplish this task and to date there is no single universal effective method. Additionally, there are side effects and limitations found with virtually all of them. In this work, we have explored the possibility of building a protein-based metallic nanoparticle complex to selectively cross the BBB and target the brain. The idea was to create a working artificial virus-like sphere, made out of protein and metal, that assembled by itself, and which would use a set of surface-bound molecules to act as a code to gain entry and deliver the contents of the sphere onto brain targets. A bottom-up approach was taken in this work. We tried to assemble the sphere from a minimum number of component parts into a well characterized structure. We analyzed the assembly properties of the sphere and examined and modify some of the relevant physical properties that the sphere should have if it was to be used as a drug delivery system. These include 1) stability, which is the ability to remain stable under certain conditions present in the blood and 2) reversibility, to disassemble under conditions expected to be present inside a cell. This property of reversibility means that once the loaded spheres enter the cell, they will dis-assemble to deliver their cargo. We initiated a series of temperature studies designed to probe the rate of growth of the spherical protein shell. This allows us to understand the limits of what can be encapsulated, and provides valuable information about assembly under conditions that might be needed with temperature-sensitive cargoes. It also addressed basic questions of particle self-assembly. We employed several physical biochemical methods to gain insight into the properties of the assembled spheres, and into the 3-dimensional structure of the single protein that assembles into the sphere. Such a structure lets you know where all the atoms in the sphere are, and makes modifications to the surface of the sphere predictable and much easier to accomplish. This requires that the protein be crystallized. We were able to make crystals, and we tried many conditions and were able to make significant improvement in the properties of the crystals, but we did not get the resolution we needed to obtain the 3 dimensional x-ray structure. What is the relevance of these studies in the broader sense? Right now, many neurological diseases and traumatic brain injuries can only be treated by modest "watch and wait" approaches, since there is no good means of intervention. The cost to society is enormous. For traumatic brain injury alone the NINDS estimates we spend $56 billion dollars per year (www.ninds.nih.gov/disorders/tbi/detail_tbi.htm). Many neurological diseases await 1) new drugs that effect a treatment and 2) the means to carry the drugs across the BBB. The focus of this proposal was to create and characterize one type of vehicle whose construction would, if and when successful, provide an efficient delivery system and be sufficiently flexible to permit multipurpose use. We have made inroads into this system. The PI is actively seeking funds to further the work. The National Science Foundation has, as one of its most important missions, the responsibility to train the next generation of scientists. Apart from the obvious scientific and potentially beneficial medical aspects of the funded work, the PI has trained, and continues to train a diverse group of 10 undergraduate students in his laboratory, most of whom have gone on to medical or scientific (graduate) school. Currently there are 3 undergraduate students of nanomedicine and biochemistry in the lab.