Robert S. Langer - US grants

Extracorporeal medical machines such as the pump-oxygenator and artificial kidney rely on systemic heparinization to improve blood compatibility. However, heparin can lead to serious complications such as bleeding. With the prospect of even longer perfusion times with machines such as the membrane oxygenator, the problems due to heparinization become more severe. Although many approaches have been explored to solve this problem, such as the use of neutralizing compounds or heparin bonded surfaces, there still remains no real alternative to systemic heparinization. We propose a new method to control heparin levels using a blood filter containing immobilized heparinase. Such a filter might be used in situations where it is desired to heparinize the extracorporeal circuit without simultaneous heparinization of the patient. Alternatively, it could eliminate the use of neutralizing compounds such as protamine. Because the amount of data on heparinase has, until now, been limited ad the methods of producing it are inadequate for large scale use, research has focused not only on the development and testing of the filter, but on enzyme production and purification. Thus far we have been able (1) to define critical requirements for nutrients for fermentation which greatly reduced the cost of these procedures by eliminating the need for heparin as the inducer; (2) to completely purify heparinase to homogeneity and to characterize its entire amino acid content; (3) to demonstrate that the heparin degradation products do not build up in the body and are cleared much more rapidly than the parent compound, heparin, as demonstrated by both radio-labeled and radioautographic studies; (4) to develop approaches for immobilizing heparinase and develop models that can predict the behavior of the immobilized enzyme; (5) to test this reactor in a sheep model using both the reactor by itself and the reactor in conjunction with a dialyzer at flow rates as high as 200 cc/min. in as many as 16 separate perfusions in a given animal, and (6) to demonstrate that heparinase, in contrast to protamine, can neutralize a variety of low molecular weight heparins. Having made these findings, our specific aims are (1) improving purification and production procedures using genetic engineering and other procedures, (2) developing improved reactors and modeling them in vivo and (3) testing these reactors in hemodialysis and cardiovascular applications in vivo.

DESCRIPTION (provided by applicant): This is a grant renewal for studies on formulations of high molecular weight agents, such as proteins and nucleic acids. For the past 29 years, this particular grant has been central for our drug delivery research. We believe the impact of our NIH funded research in this area has been significant and that our work has helped provide some of the fundamental concepts necessary for the clinical development of controlled release of macromolecules and for localized drug delivery. Examples of such delivery systems are Lupron Depot, Gliadel and Nutropin Depot. In writing our last competing renewal grant proposal we felt that much of the fundamentals of peptide and protein release had been established and that the greatest impact could come from extending our research nucleic acid delivery. Our studies in this area have led to a greater mechanistic understanding, and have resulted in 35 peer-reviewed papers, including in such journals such as Science and Nature. The major barrier to the success of gene therapy in the clinic is the lack of safe and efficient DNA delivery methods. Modified viruses, while effective at transferring DNA to cells, suffer from serious toxicity and production problems. In contrast, non-viral systems offer a number of significant potential advantages, including ease of production, stability, low toxicity, and reduced vector size limitations. From our studies we have discovered that optimal intracellular delivery to different tissues in the body requires different materials. Therefore, we propose to develop combinatorial libraries of new materials with the specific goal of generating clinically useful, non-viral methods for gene therapy. Lung cancer will be used as a model disease. We propose to explore two complementary approaches: 1) developing lung-tumor cell specific DNA delivery systems for the anti-tumor therapy and 2) developing anti-lung cancer DNA vaccines by targeting and activating native immune cells. Our specific aims are: 1. To develop next-generation biodegradable polymers for use as efficient and non-toxic vectors for lung cancer and DNA vaccines. 2. Development of cell specific DNA delivery systems for lung tumor and dendritic cell targeting. 3. To test the in vivo gene delivery efficiency of biodegradable vectors composed of PEI- and PBAE- based polymer and that of their electrostatic ligand coated ternary polyplexes in normal and lung cancer mouse models. 4. To examine potential of PEI- and PBAE- based vectors for their use as DNA vaccines.

The controlled release of macromolecules from a porous polymer carrier is an example of diffusion through a porous medium; a complete understanding of the behavior of these systems in not available. A microstructural approach to the problem will be taken with the goal of elucidating the underlying physical mechanisms which determine the rate of transport in the porous network. The research involves a system for acquiring detailed microstructural data on the porous polymer devices. A detailed understanding permits the formulation of physically realistic models of the porous network; it will also provide a method for testing preliminary hypotheses. Release studies are also conducted for examining the kinetics of macromolecular release from the polymer devices. The major thrust of the project is exploratory experimentation on the physical mechanisms.

The controlled release of marcomolecules from inert polymeric martix devices has been extensively researched by our group. Furthermore, a recently synthesized class of bioerodible polymers, made from the non-peptide linking of typrosine dipeptide derivatives, demonstrates adjuvanticity in both monomeric and polymeric form. The aim of the proposed research is to develop a controlled release immunization implant which will have a two-fold effect. First, the device will produce sustained, controlled release of antigen in vivo, thereby initiating production of antibody specific for that antigen and successfully immunizing the animal. Second, the products formed during polymer hydrolysis will have an adjuvant effect on the immune response, thus enhancing the level and duration of antibody production and improving protection against subsequent challenge. To achieve this goal: 1) Poly(iminocarbonates) composed of N- and C-blocked derivatives of N-(L-tyrosyl)-L-tyrosine will be synthesized and characterized with regard to molecular weight mechanical properties, surface properties, and degradation behavior; 2) The release of model antigens from devices composed of these polymers will be measured and mathematically modelled; 3) Adsorption-desorption studies of polymer and antigen will be conducted to investigate the "depot" effect mechanism of adjuvanticity; 4) Polymeric devices containing antigen and polymer-antigen adsorbates will be implanted or injected in mice, and the antigen- specific antibody levels will be measured by ELISA; and 5) Release patterns from devices will be altered with ultrasound or formulation parameters to determine the effect of dosage on adjuvanticity.

It is proposed to study experimentally both nonerodible and bioerodible polymer pore structure and morphology. Both polymers exhibit structurally complex systems, and the bioerodible components will show a dynamically changing pore structure. Although it may not be possible to perform a fully computerized image analysis of these systems within the constraints of the grant, scanning electron microscopy (SEM) and light microscopy will provide quantitative insight into the pore morphology of these systems. Photomicrographs of the polymer taken at various times during the erosion process will allow the changing structure to be followed in order to provide the basis for the system model.

This is a competing renewal proposal of our previous grant application. The objective of the previous grant application was to investigate the effects of the use of ultrasound to enhance polymeric drug delivery and drug permeation through skin. In this past grant period, we investigated the effects of polymer molecular weight, cavitation, temperature, solution gas content, and other factors on ultrasound induced polymeric drug delivery; we showed that ultrasound could enhance transdermal drug delivery, that it could act as a trigger to terminate the activity of controlled release implants, and that it could provide a new rapid method of achieving protein blotting from electrophoretic gels. We believe that these studies will have wide applicability in a number of areas for ultrasound-assisted transport and, indeed, many of these avenues of application are now being pursued in other laboratories. In the current grant application, we wish to focus our attention on one of these areas- ultrasound-assisted transdermal drug delivery with special emphasis on protein delivery in part due to the reviewers' previous comments-because of the great impact that can now be made in this area. The specific goals of this proposal include: 1. Understanding the mechanisms of transdermal flux enhancement due to ultrasound. 2. Understand the dependence of sonophoresis on ultrasound parameters. 3. Investigate safety issues of sonophoresis. 4. Modeling ultrasound-mediated enhancement and transdermal fluxes.

The goal of this research is to identify and synthesize a polymer system to serve as a scaffold for functioning mammalian cells. The optimal polymer system would maximize cellular function in vivo. The basic requirements for the scaffold include biodegradability, biocompatibility, suitable mechanical properties, and an adhesive moiety to facilitate cell attachment. In order to reach this goal, several steps need to be completed. The exact ordering of the polymer processing and adhesion moiety attachment steps will be determined during the project. Cell culture studies will be conducted on these polymers in vitro, and surgical implantation of these systems with an assessment of structure and function in vivo will be attempted.

DESCRIPTION: (Adapted from the applicant's abstract) Extracorporeal medical machines such as the pump oxygenator and artificial kidney rely on systemic heparinization to improve blood compatibility. However, heparin can lead to serious complications such as bleeding. The applicants propose a new method to control heparin levels using a blood filter containing immobilized heparinase. Such a filter might be used in situations where it is desired to heparinize the extracorporeal circuit without simultaneous heparinization of the patient. Alternatively, it could eliminate the use of neutralizing compounds such as protamine. Their research has focused not only on the development and testing of the filter, but on heparinase production and purification as well. The specific aims of the current proposal are: (1) to employ genetic engineering to produce high levels of heparinase, (2) to improve reactor design, (3) to develop comprehensive in vivo models to predict reactor performance, and (4) to conduct in vivo studies using hemodialysis and cardiovascular models in animals, to assess the safety and efficacy of the proposed approach.

This is a competing renewal proposal in the area of the development of controlled release systems for immunocontraception. There are several reasons why this is a very important area. First, for active immunization with sperm antigens, the ability to release the antigens for long periods of time will enhance it's immunogenicity. Second, for passive immunization with monoclonal antibodies against sperm or zona pellucida protein, the ability to provide long-term release of an active antibody would be very desirable. In the past grant period, we proposed the synthesis of a class of novel degradable polymers involving L-tyrosine peptide derivatives. The major objective was to design devices that would provide controlled release of the antigen for prolonged periods of time. In addition, one of the desirable features of these polymers would be that the products formed from polymer hydrolysis could have an adjuvant effect on the immune response and thereby enhance the level or duration of antibody production. In the last grant period, we successfully synthesized polymers composed of, or containing, a variety of tyrosine and tyrosine dipeptide monomers and fully characterized them with respect to a variety of properties relevant to antigen delivery. We developed micron-sized antigen-loaded spheres (i.e., microspheres) from these polymers and developed approaches to provide prolonged release of model proteins from these systems. A number of papers in very good journals have resulted from these studies. The specific aims of the current proposal are to further develop this area by utilizing specific antigens (such as PH-20, a peptide from the active site of a fertilin beta, and a recombinant fertilin beta extracellular domain) now available from our collaborators, and to expand potential methods of achieving controlled release immunocontraception, including new routes of administration and approaches for achieving pulsatile release. The specific aims are as follows: 1) To incorporate antigens from the other Centers into polymer microspheres based on tyrosine derivatives. 2) To pursue the idea of vaccine delivery by using a new approach involving FDA approved lactic/glycolic acid polymers which can provide pulses of vaccine. In addition, we will pursue a novel idea of stabilizing proteins in these polymers systems using an oil-based microencapsulation. 3) To develop delivery systems for mucosal immunization, in particular systems which can be administered by the pulmonary route.

DESCRIPTION (Adapted from the applicant's abstract) The objective of this grant proposal is to develop a noninvasive method to implant polymers using photopolymerization. This allows solid hydrogels to be implanted without surgical intervention. Enough light is able to penetrate tissue, including skin, to trigger the photopolymerization of an injected liquid, polymer solution to a solid hydrogel. Further study of transdermal photopolymerization using alternative photoinitiating systems and wavelengths of light is proposed in order to increase depths at which implants can be photopolymerized. Photopolymerization will be applied to the tissue engineering of cartilage. Cartilage lacks the ability to regenerate and its loss by trauma, congenital abnormalities or tumors gives few options to the physician for replacement. Preliminary data has shown that chondrocytes survive encapsulation, injection and photopolymerization to form neocartilage. Important biological factors, including growth factors and adhesion peptides, will be incorporated into photopolymerizing hydrogels to determine if they can accelerate cartilage development and produce cartilage with biochemical and biomechanical properties similar to native cartilage. The tissue engineering of cartilage will be examined both in vitro and in vivo, in particular in an immune competent animal. The integration of photopolymerized tissue engineered cell/polymer implants with surrounding native cartilage is critical to the clinical application of tissue engineered cartilage and will be addressed. In conclusion, this proposal addresses methods for noninvasive polymer implantation, which applied to cartilage tissue engineering, would provide physicians with a significant alternative to cartilage replacement for craniofacial reconstruction and orthopedic surgery. The specific goals of this proposal include: (1) development of a highly efficient, biocompatible transdermal or transtissue photopolymerization system for noninvasive polymer implantation; (2) development of an injectable tissue engineered cartilage using transdermal photopolymerization; (3) increase cellular biocompatibility of the polymer hydrogel through the incorporation of bioactive peptides and proteins; and (4) integration of tissue engineered and native cartilage for the correction of cartilage defects.

DESCRIPTION: (Adapted from the applicant's abstract) The concept and practice of seeding cells onto degradable polymer scaffolds, such as poly(lactic acid) (PLA) or poly (glycolic acid) (PGA), in vitro followed by eventual in vivo implantation has become an important methodology in tissue engineering. These scaffolds provide structural support for the cells until they develop their own extracellular matrix, but scaffold mechanical properties have yet to be specifically designed to provide an environment that will match that of the native tissue. The specific interest of this project is the engineering of cardiovascular tissues. In the investigators' experiences with existing PLA or PGA tubular and valvular leaflet scaffolds, it has been difficult to sew them into the native tissue, bend them to fit a desired or required conformation within the animal, or replace all three pulmonary valve leaflets without leading to unacceptable valvular stenosis. The investigators' goals are to develop the next generation of tissue engineering polymer scaffolds with mechanical properties (e.g. elasticity) that closely model those of the native tissue (pulmonary valve leaflets, arteries) and to investigate the influence of such an environment on cell proliferation, tissue formation, and function of engineered tissues in vivo. Their hypothesis is that significant advances in tissue engineering may be realized if the biomaterials to be used as scaffolds can be specifically designed to resemble the mechanical properties of the native tissue in question. Accordingly, the Specific Aims are: 1) Synthesis and characterization of elastic, degradable polymers with appropriate mechanical properties for use as cardiovascular tissue engineering scaffolds; 2) Test the degradation profiles of the new polymers and monitor the changes in mechanical properties over time; 3) Test the polymer-cell compatibilities and interactions (attachment and spreading) on polymer films using appropriate cell types (e.g. endothelial cells, smooth muscle cells, fibroblasts). Cells will be provided by Dr. Mayer at Children's Hospital; 4) Select best candidates based on Aims 1-3, then design and prepare scaffolds in the shapes of tubes for blood vessels and leaflets or whole valves for heart valves; 5) Carry out in vitro cell seeding studies to determine the influence of polymer properties and bioreactor conditions on cell differentiation, proliferation, and tissue formation in conjunction with Drs. Vacanti, Bischoff, and Roth at Children's Hospital; 6) Implantation of cell-scaffold devices at the appropriate time frame based on the findings of Aim 5 in small animal models (to be carried out by Drs. Mayer and Vacanti at Children's Hospital).

The objective of this project is to establish whether electrical
stimulation can provide similar, or potentially greater, levels of
control over neural cell behavior as the cocktails of chemical
factors currently used. To achieve this objective, two specific
aims are put forth by the Principal Investigator (PI):

1. Compare different patterns of stimulation with chemical
factors in their abilities to alter cellular behavior; as a
model, the PI will examine rescuing neurons from programmed
cell death (PCD).

2. Compare different patterns of stimulation with chemical
factors in effects on the calcium-sensitive cascade network
by measuring the signaling activity in different linear
branches of the network.

If electrical and chemical stimuli can elicit similar effects,
then a significant improvement in control over cellular behavior
will be achieved.

It is well known that the method by which a drug is delivered can have a significant effect on the drug's therapeutic efficacy. Most drugs have a concentration range in which they have maximum efficacy. Conventional drug delivery regimens result in sharp changes in systemic drug levels that can be fatal. Controlled drug delivery can alleviate the problems associated with conventional therapy by providing stable drug bioavailability in a therapeutically meaningful range and in addition can be used to localize the therapy to the tissue site of interest. We recently showed that it is possible to fabricate a solid-state silicon microchip in which a number of chemicals or drugs can be stored and released on demand by an external trigger. One advantage of this novel controlled release system is that it allows for simultaneous release of multiple drugs in complex release profiles. One can potentially develop a device that can be pre- programmed to delivery combination drugs in a pre-determined fashion. We believe that this novel delivery technology has broad utility in the biomedical area such as local delivery of anesthetics for pain management, sub-dermal delivery of vaccines, periodontal delivery of antibiotic and anti-inflammatory agents, localized delivery of anti-tumor and neoplastic agents, gene delivery, delivery of antiarrhythmic agents to name a few. Based on the above mentioned rationale and our preliminary results we propose the following specific aims: (1) Development of an active, silicon based microchip for controlled release of drugs that can operate autonomously, (2) Development of a passive, polymeric chip for the controlled release of drugs, (3) Evaluate the biocompatibility of active and passive microchip delivery device and (4) Evaluate the drug release both in vitro and in vivo, specifically: (a) show that predictable drug release is possible from both active and passive microchips (b) study a pathology such as brain tumors that maybe be better treated by combination therapy (c) evaluate the efficacy of these devices in this tumor model.

DESCRIPTION (provided by the applicant): Not provided.

0507449
Langer

This proposed research delineates a number of novel approaches for grafting antifungal molecules to polymeric surfaces or incorporating those compounds within the polymeric matrix. The chemistry is challenging, as inappropriate modifications may abolish the fungicidal activity of the molecules, and their specific chemical properties make them hard to work with. The methodologies developed here will be applicable not only to the set of chemistries described here, but to surface modification in general. The potential uses are also broad, ranging from the prevention of fouling of industrial fermenters, sewage systems, and shipping hulls, to preventing fungal infections of catheters. The project follows a systematic approach whereby molecules are synthesized to predetermined specifications, tested in vitro against C. albicans, and then - if shown to be effective - tested in animals for efficacy and safety.

DESCRIPTION (provided by applicant): The overall objective of this proposal is to develop a fundamental understanding of the role of localized transport regions (LTRs) in low-frequency sonophoresis (LFS), with an emphasis on: 1. Understanding if the LTRs are Indeed Regions of Highly-Localized Transdermal Transport for both Hydrophilic and Hydrophobic Permeants: Specifically, demonstrating whether the LTRs are regions of high permeability compared to the surrounding regions of ultrasound-treated skin (the non-LTRs) for a broad class of permeants. 2. Understanding the Mechanisms that Govern the Formation of the LTRs in LFS: Specifically, understand: i) the role of the surfactant sodium lauryl sulfate (SLS) in LTR formation, and ii) the roles of acoustic cavitation, the ultrasound acoustic field, and the skin surface topography in LTR formation. 3. Understanding the Nature of the Permeation Pathways that Exist within the LTRs and the Non-LTRs: Specifically, identify the type of permeation pathway (transcellular, intercellular, or a combination of both) followed by permeant molecules traversing the skin within the LTRs and the non-LTRs. 4. Investigating the Safety of LTR Formation on Skin Treated with LFS: Specifically, determining: i) the reversibility of LTR formation in the skin, and ii) the biological effects of LTR formation. 5. Mathematical Modeling of Transdermal Transport in the Presence of LTRs: Specifically, i) developing mathematical models to more accurately evaluate experimental transdermal permeabilities across ultrasound-treated skin with LTRs, and ii) developing models to confirm the presence of transcellular and/or intercellular transdermal pathways within the LTRs and the non-LTRs in the stratum corneum resulting from the ultrasound treatment.

DESCRIPTION (provided by applicant): Inflammatory bowel disease (IBD) is one of the most prevalent gastrointestinal disease burdens in the US, with an overall health care cost of more than $1.7 billion and affecting an estimate of 1.4 million patients in the US. Crohn's disease (CD) and ulcerative colitis (UC) are the two major types of inflammatory bowel disease (IBD), both characterized by chronic and relapsing inflammation in the intestinal tract which commonly requires a lifetime of care. Meanwhile, infants with microvillus inclusion disease (MVID) have severe malabsorption caused by hypoplasia and/or atrophy of the intestinal epithelium. These infants require total parental nutrition to survive but quickly develop liver failure and potential death within 2 years. The only effective therapeutic approach is small intestine transplantation that requires a donor and is logistically complicated and impractical. One potential long term treatment for IBD and MVID would be therapeutic intestinal stem cell (ISC) transplantation. The goal of this renewal grant is to use our biodegradable and biocompatible elastomers as a multifunctional delivery platform for drug and cell delivery to treat diseases affecting intestinal mucosa. Although ISCs could be delivered and engraft to intestinal epithelium, initial cell retention with high efficiency is not easily achieved owing to the harsh intestinal environment and the lack of supportive stem cell niche prior to engraftment. Through combining biodegradable elastomers with our high efficiency ISC expansion protocols, we can deliver stem cells under the protection of a defined synthetic niche. By maximizing rapid adhesion and engraftment, we aim to achieve high efficiency delivery of stem cells to diseased epithelium. We propose to utilize the delivery platform either as small cell/drug carriers, which can disperse quickly into the colon via enema for Ulcerative Colitis, or as larger patches that will be delivere via endoscope for Crohn's disease or intestinal anastomotic failure. These delivery vehicles will achieve 1. Efficient and selective adhesion to damaged or diseased intestinal epithelium; 2. Survival of delivered stem cells before and during engraftment, and 3. local delivery of anti-inflammatory agents such as IL-10, IL-22, or TNF-? neutralizing antibodies to facilitate and maximize the engraftment of stem cells and maintain remission. We aim to 1. Develop biodegradable and biocompatible elastomer patch enabling efficient and selective mucosal adhesion via both endoscopic (Crohn's disease) and enema-based (ulcerative colitis) delivery in vivo. 2. Engineer elastomer patch to support intestinal stem cell growth while control- releasing anti-inflammatory agents in vitro. 3. Evaluate drug and cell therapeutic efficacy in vivo.

DESCRIPTION (provided by applicant): 1 of the major obstacles in tissue engineering of thick, complex tissues (such as the heart and the liver) is the need to vascularize the tissue in vitro. Vascularization in vitro could restore cells viability during growth of the tissue, induce structural organization and promote integration upon implantation. Embryonic stem (ES) cells have the capability to differentiate and form blood vessels de novo in a process called vasculogenesis. We have shown that human ES (hES) cells can differentiate into endothelial cells (ECs) forming vascular-like structures when formation of embryoid bodies is induced and that these cells can be isolated and grown in culture. We hypothesize that the vasculogenic potential of hES-derived endothelial progenitors can be used to induce vascularization in engineered human tissue. Our goal will be to induce vasculogenesis in engineered tissue constructs grown on three dimensional (3D) polymer scaffolds. We will use co culture systems of embryonic endothelial and cardiac cells cultured on biodegradable polymer scaffolds designed to meet cellular and mechanical properties needed for a cardiac patch. The vascularized constructs will be implanted and examined for integration with the host vasculature. We hypothesize that the vessel network created in vitro will promote the vascularization of the tissue in vivo.

DESCRIPTION (provided by applicant): We propose to use our established and well documented history in the development of biomaterials as scaffolding for tissue replacement as a platform for stem cell approaches in craniofacial tissue regeneration. The governing hypotheses of this work include: i) that human stem cells of embryonic origin can be differentiated into craniofacial tissue producing cells such as osteoblasts and chondrocytes, ii) that highthroughput techniques can be used for the screening of large numbers of media and polymer candidates for potential use with tissue engineering scaffolds, and iii) that the appropriate combination of stem cells with 3-dimensional polymer scaffolds can be used for the production of complex craniofacial tissues. To test these hypotheses, we propose the following: Aim 1: Examine the in vitro differentiation of human embryonic stem cells into precursors of craniofacial tissues. Our previous work investigating the in vitro differentiation of human embryonic stem cells into various cell types and tissues will be continued to assess conditions necessary for differentiation into cell types necessary for craniofacial tissue formation. Aim 2: Investigate the interaction of human stem cells with polymer surfaces using high-throughput screening technology. Recently, we have developed techniques for the rapid screening of cell/polymer interactions using nanoliter-scale microarrays of various monomers and polymers. Success will be assessed through cell proliferation and the presence of tissue specific markers for craniofacial tissues. Aim 3: Assess in vitro tissue production by differentiated and undifferentiated human embryonic stem cell seeded polymeric scaffolds incorporating various genes and growth factors. Polymeric materials that support the differentiation of human stem cells into craniofacial tissues will be fabricated into 3-dimensional scaffolds incorporating various genes and growth factors and analyzed by histological staining of various markers. Aim 4: Optimal candidates from the previous aims will be seeded with differentiated and undifferentiated cells and implanted either subcutaneously or in a critical sized defect model in rats. The subcutaneous model in athymic rats will be used to assess tissue production by stem cell seeded scaffolds in an in vivo environment, including scaffolds with cells differentiated into multiple phenotypes. The cranial defect model in athymic rats is a clinically relevant model that is commonly used as a measure of bone regeneration.

DESCRIPTION (provided by applicant): We propose to use our established and well documented history in the development of biomaterials as scaffolding for tissue replacement as a platform for stem cell approaches in craniofacial tissue regeneration. The governing hypotheses of this work include: i) that human stem cells of embryonic origin can be differentiated into craniofacial tissue producing cells such as osteoblasts and chondrocytes, ii) that highthroughput techniques can be used for the screening of large numbers of media and polymer candidates for potential use with tissue engineering scaffolds, and iii) that the appropriate combination of stem cells with 3-dimensional polymer scaffolds can be used for the production of complex craniofacial tissues. To test these hypotheses, we propose the following: Aim 1: Examine the in vitro differentiation of human embryonic stem cells into precursors of craniofacial tissues. Our previous work investigating the in vitro differentiation of human embryonic stem cells into various cell types and tissues will be continued to assess conditions necessary for differentiation into cell types necessary for craniofacial tissue formation. Aim 2: Investigate the interaction of human stem cells with polymer surfaces using high-throughput screening technology. Recently, we have developed techniques for the rapid screening of cell/polymer interactions using nanoliter-scale microarrays of various monomers and polymers. Success will be assessed through cell proliferation and the presence of tissue specific markers for craniofacial tissues. Aim 3: Assess in vitro tissue production by differentiated and undifferentiated human embryonic stem cell seeded polymeric scaffolds incorporating various genes and growth factors. Polymeric materials that support the differentiation of human stem cells into craniofacial tissues will be fabricated into 3-dimensional scaffolds incorporating various genes and growth factors and analyzed by histological staining of various markers. Aim 4: Optimal candidates from the previous aims will be seeded with differentiated and undifferentiated cells and implanted either subcutaneously or in a critical sized defect model in rats. The subcutaneous model in athymic rats will be used to assess tissue production by stem cell seeded scaffolds in an in vivo environment, including scaffolds with cells differentiated into multiple phenotypes. The cranial defect model in athymic rats is a clinically relevant model that is commonly used as a measure of bone regeneration.

0609182
Langer

This proposal was received in response to Nanoscale Science and Engineering initiative NSF 05-601 category NIRT A. The objective of this research is to nanofabricate biologically interfacing materials with well defined surface morphologies. Through controlling the surface features of biocompatible polymers on the nanoscale, and through systematically developing an understanding of how nanotopography influences adhesion to biological tissues, a new class of self-adhesive nano-structured biomaterials will be developed. Using nano-molding techniques, pillars resembling hairs or spatulaeon the palms of geckos will be created which will substantially increase the surface area and render these materials adhesive through increased van der Waals and capillary interactions. Through using soft flexible materials, the nanostructured surfaces should easily conform to irregularly shaped surfaces, such as those encountered in the body, thereby maximizing molecular interactions at the interface. It is anticipated that these materials will be readily transferable into a wide range of minimally invasive medical treatments.

Considering potential applications in daily surgical practice, these materials may decrease surgical complications, such as infection, by reducing traumatic tissue handling, reducing excessive bleeding, and reducing operating times. Through providing new methods to quickly seal tissues and to bond tissues together, this work may impact society by reducing complications associated with many common medical procedures thereby improving the quality of life. In addition to surgical applications, the proposed technology is potentially useful in areas from tissue engineering of replacement organs, where adhesion between various tissues is essential for proper function. As a primary component or our outreach program, undergraduate students will assist in the development of a website to increase awareness of nanotechnology in medicine. To foster integration of research and education, key results from this work will be presented to rural and intercity high school students and to the general public at the Boston Museum of Science.

DESCRIPTION (provided by applicant): We propose to use our established and well documented history in the development of biomaterials as scaffolding for tissue replacement as a platform for stem cell approaches in craniofacial tissue regeneration. The governing hypotheses of this work include: i) that human stem cells of embryonic origin can be differentiated into craniofacial tissue producing cells such as osteoblasts and chondrocytes, ii) that highthroughput techniques can be used for the screening of large numbers of media and polymer candidates for potential use with tissue engineering scaffolds, and iii) that the appropriate combination of stem cells with 3-dimensional polymer scaffolds can be used for the production of complex craniofacial tissues. To test these hypotheses, we propose the following: Aim 1: Examine the in vitro differentiation of human embryonic stem cells into precursors of craniofacial tissues. Our previous work investigating the in vitro differentiation of human embryonic stem cells into various cell types and tissues will be continued to assess conditions necessary for differentiation into cell types necessary for craniofacial tissue formation. Aim 2: Investigate the interaction of human stem cells with polymer surfaces using high-throughput screening technology. Recently, we have developed techniques for the rapid screening of cell/polymer interactions using nanoliter-scale microarrays of various monomers and polymers. Success will be assessed through cell proliferation and the presence of tissue specific markers for craniofacial tissues. Aim 3: Assess in vitro tissue production by differentiated and undifferentiated human embryonic stem cell seeded polymeric scaffolds incorporating various genes and growth factors. Polymeric materials that support the differentiation of human stem cells into craniofacial tissues will be fabricated into 3-dimensional scaffolds incorporating various genes and growth factors and analyzed by histological staining of various markers. Aim 4: Optimal candidates from the previous aims will be seeded with differentiated and undifferentiated cells and implanted either subcutaneously or in a critical sized defect model in rats. The subcutaneous model in athymic rats will be used to assess tissue production by stem cell seeded scaffolds in an in vivo environment, including scaffolds with cells differentiated into multiple phenotypes. The cranial defect model in athymic rats is a clinically relevant model that is commonly used as a measure of bone regeneration.

As stated above, the Project Manager, Shannon Cozzo, will oversee day-to-day management ofthe program. She currently serves in this role as a conduit between the PI/PD, Project Leaders and the centralized Koch Institute staff. Ms. Cozzo is involved in coordinating activities among affiliated institutions ofthe CCNE. These different units have separate and somewhat distinct financial management structures that will affect management ofthe various child and facility account that comprises the CCNE. The Project Manager is also responsible for coordinating all CCNE Alliance-related activities, steering committees, advisory boards and joint group meetings, monthly seminar series and the Education, Training and Outreach units, which are under the umbrella ofthe Administrative Core. She is located in the Koch Institute. This arrangement will ensure efficient coordination of all operations including management of finances, regulatory compliance and human resource support.

With advances in nanotechnology, it is now possible to develop highly selective and effective cancer therapeutics by combining specialized biomaterials with currently available chemotherapeutic agents. The aim of project 1 of this U54 proposal is to develop technologies for in vivo targeted delivery of cytotoxic drug encapsulated controlled release polymer nanoparticles to cancer cells. This could allow for a large amount of drug to be delivered to cancer cells and make it possible to reach a steady state cytotoxic drug concentration at the tumor site over an extended period of time. The combination of targeted delivery and controlled release could also decrease the likelihood of significant systemic toxicity since the drug is encapsulated and biologically unavailable during transit in systemic circulation. We will build upon our previous research on long circulating nanoparticles as published in Science and engineer biocompatible and biodegradable nanoparticles and optimize these for targeted drug delivery to cancer cells. By using high throughput combinatorial methods, we will develop a library of more than 3000 targeted nanoparticle formulations and optimize multiple parameters including, size, chemical structure and charge of nanoparticles;and density of targeting molecules on the nanoparticle surface for targeted delivery. As a model targeting molecule, we will use nuclease resistant RNA ligands that bind to tumor-antigens on the surface of prostate cancer cells. By isolating a panel of RNA ligands that bind to prostate tumor epitopes, we propose to develop polyvalent targeted nanoparticles that may overcome tumor heterogeneity which in some cases has hindered the clinical translation of previous targeted approaches. We believe this project has broad significance, since the successful development of this technology may result in the development of the next generation of nanoscale cancer therapeutic modalities.

7. TRAINING, EDUCATION, KNOWLEDGE TRANSFER 7.1 Training This CCNE will function to train the next generation of scientists and engineers in the use of the tools of nanotechnology to understand and ameliorate diseases, including cancer. Specifically, through the research projects funded by the Center as well as the associated educational activities, we will train graduate students and postdoctoral fellows at the interface between nanotechnology and cancer biology. We believe strongly that a true integration of different disciplines is best achieved by the joint training and supervision of trainees so that they become expert in all relevant areas of study. Accordingly, we place significant emphasis on efforts to attract students with backgrounds in cancer biology and basic cell and molecular biology as well as those from chemistry, physics andengineering disciplines. The CCNE will offer an ideal environment for the cross-training of such individuals, and we will work with the graduate and other training programs at the represented institutions to ensure that the students and fellows are able to perform their work in this cross-disciplinary setting.. In fact, there is a large pool of excellent students and MIT/Harvard already have in place administrative and educational mechanisms for encouraging interdisciplinary activities among these students and the respective faculty members. The training component will include participation in a CCNE-specific lecture series, classroom training and workshops. At both institutions, there are multiple courses with "nano" in their titles and several more that are closely related to the fields of study in the CCNE. Similar courses are also offered at Harvard. The educational component of the CCNE will provide support for the interdisciplinary training of 2 graduate students and 2 postdoctoral fellows per year and 5 undergraduates. This is in addition to the individuals funded directly by the projects and pilot projects Overall, we estimate to train 15-20 students and an equal number of post-doctoral fellows per year in the CCNE. 7.2 Interface with other nanotechnology training programs Harvard's Nanoscale Science and Engineering center (NSEC;PI: Westervelt) has an extensive educational and outreach program that has been in existence for 5 years. The program (for details see http://www.nsec.harvard.edu/education.htm) has lecture series, webcasts, interactions with the Museum of Science, cablecasts via New England Cable News, fellowships for women and minorities, undergraduate programs, K-12 Teacher programs and knowledge transfer programs to the public. We will harness these existing mechanism and in close collaboration with Dr. Westervelt plan for joint programs in the future open to participants of both programs. A second existing training program is that of MIT's Institute of Soldier Nanotechnologies (ISN;PI: Ned Thomas) and which will be tightly integrated with this consortium. There are currently over 130 graduate students and nearly 40 postdoctoral fellows working on different projects of the ISN. All standard MIT research center communications are implemented: web site, newsletters, brochure, video, hosting of groups for overviews and tours (everyone from school kids to alumni to four-star generals). There is a monthly seminar series, a K-12 outreach program, and contributions to the MIT Museum's family program. 7.3 Interface with NIH training programs There also exist a number of training programs associated with members of the CCNE that focus on cancer biology, biology, biophysics, materials engineering, physical sciences. This includes training grants in the MIT Biology Department (5 T32 GM07287-30), Computational Systems Biology Initiative (1-T90- DK070114-01, 1-R90-DK071503-01), Division of Biological Engineering (5-T32-ES070-2O), Chemistry Department (T32-ES-07020, T32-CA-09112). Also included are a T32 training program at CMIR (T32 CA79443), a P50 associated Career Development program (P50 CA86355). 7.4 Lecture series The program will have a monthly lecture series with alternating topics covering the three focus areas: nanotechnology, in vivo targeting/testing and oncology. The lectures will be given by scientists and engineers involved in the CCNE. In addition postdoctoral fellows will periodically present progress of individual projects. We will also invite outside guest speakers as part of our lecture series. 7.5 Annual retreat We plan an annual off-campus retreat to further foster interactions among CCNE members. Our experience has been that such retreats provide an outstanding opportunity for cross fertilization of ideas and for establishing new, formal collaborations. Although many of the Departments and Centers at MIT and Harvard, including the he MIT CCR and the DFHCC, hold such retreats, we feel that it is important to have a specific retreat for the faculty and trainees of the CCNE. Retreats will include platform and poster presentations as well as special lectures and workshops. We anticipate inviting the members of our internal and external advisory boards to attend the retreat. 7.5 Exchange program A major aspect of our educational program is its multidisciplinary training of students, postdoctoral fellows and staff. Formal mechanisms are in place to assign students and fellows to interdisciplinary joint programs (e.g. projects 2, 4, 5) through a central mechanism. We also encourage and facilitate student exchanges between programs. Several of our consortium members also have official exchange programs (visiting professors, sabbaticals, student exchanges, visitor programs) with other Universities and Programs. These programs are set up to share facilities, carry out collaborative research and cross-train oncology, chemistry, physics and engineering students. 7.6 Outreach to elementary schools: NanoSleuths This is a new project targeted at Cambridge inner city elementary school students. Dr. Angela Belcher has initiated this program to get students in local schools excited about nanoscience. This program has three major goals 1) to get students think about the nanoworld 2) to help increase the science infrastructure in local elementary schools 3) engage MIT undergraduate and graduate students in outreach to local schools. The NanoSleuths program will prepare special modules and teach applications of nanotechnology to cancer. Specifically, the NanoSleuths program will focus on modules for students to work in teams to solve "cases" that involve nano mysteries. MIT students and Dr. Belcher will help facilitate the cases. The cases to be investigated will be 1) "The Case of the Racing Slimes" 2) "The Case of the Slippery Atom" 3) The Case of the Stolen Gene 4) The Case of the Disappearing Light and 5) Nanorobots in cancer vessels, among others. 8. INTERACTIONS, OUTREACH 8.1 Interactions with Cancer Centers This CCNE is focused exclusively on the application of nanotechnology to problems in cancer diagnosis, treatment and disease monitoring, as this is reinforced by the tight integration with two NCI-designated Cancer Centers. The CCNE will be organized and administered by the MIT CCR (Dr. Jacks, Director), and will interact closely with the DFHCC (see letter from Dr. Livingston, Deputy Director). In addition, there multitude of Specialized Program of Research Excellence (SPORE) which are involved in the Projects. Projects 1 and 3 interact closely with the Prostate Spore at DFHCC lead by Dr. Kantoff. Drs. Kantoff and Rubin have played a pivotal role in the design of the current projects. They have also collaborated with Drs. Langer, Farokhzad, Josephson and Weissleder over a number of years. Project 2 will interact with research groups at the MIT CCR in developing siRNA-based targeting strategies for lung and brain tumor models under development. Through Dr. Jacks, Project 2 will interact with the DF/HCC lung cancer SPORE. Project 4 will rely on the Mouse Models Core, MIT CCR collaborators in the Jacks and Housman laboratory and the Ovarian Cancer group at MGH (Seiden, Chabner). Drs Weissleder and Seiden are corecipients of a Doris Duke award on ovarian cancer and have collaborated on developing methods for early detection and relapse of ovarian cancer in patients. Project 5 also relies on the Mouse Models Core and interactions with the clinical gastrointestinal research groups at DFHCC of which Dr. Weissleder is a member. Drs. Weissleder, Bhatia, Farokhzad and Ramaswamy are practicing physicians with appointments at MGH and/or BWH. Dr. Phillip Kantoff (collaborator of project 1 and 3) is Director of GU Oncology at DF/HCC and director of a GU Spore. Dr. Bruce Chabner (collaborator of project 3) is the Clinical Director of the MGH Cancer Center and Director of the "Phase 1-group" at DFHCC. Dr. Michael Seiden serves as Chairman of the MGH Cancer Center Clinical Protocol Committee, as well as Chairman of the Dana Farber-Harvard Gynecologic Oncology Research Committee. Dr. Daniel Haber is the Director of the MGH Cancer Center and Dr. David Livingston is the Deputy Director of the DFHCC. 8.2 Industrial outreach program (IOP) The consortium will establish a semi-annual meeting with industry leaders with joint sponsorship from MIT and Harvard Departments. The IOP is aimed at a) facilitating the rapid translation of most promising nanotechnology developments, b) strengthening external collaborations by facilitating mutually beneficial relationships and c) reviewing internal projects. The program will invite leading scientists from industry and our network of local laboratories to participate. The workshop will including talks and poster sessions on research, educational programs, shared experimental facilities and outreach activities. Based on our collective experience from the past several years the following large companies (among others) will be invited to participate in this Program: Merck, Novartis, Lilly, Abbot, Aventis, Glaxo, BMS, Johnson and Johnson, Proctor and Gamble, Genentech, Biogen, Analog Devices, Nanosys, Quantum Dot Corporation, General Electric and Siemens (see attached letters of interest from invitations already extended). 8.3 Clinical translation Members of this consortium have considerable expertise in clinical medicine, drug development, phase 1 clinical trials and translational research. Collectively, the team has developed the following materials for clinical use and/or has participated in clinical trials: [unreadable] Gliadel wafer, SonoPrep and others (76, 109) [unreadable] First clinical trials of magnetic nanoparticles (106) [unreadable] Development (119) and first clinical trials of lymphotropic magnetic nanoparticles (43, 44) [unreadable] Development and clinical testing of novel synthetic graft copolymers (17).

PILOT PROJECTS During the preparation of this application, there has been a tremendous interest by research groups to join the consortium through pilot projects. We view these projects as an important adjunct of the CCNE's mission and from which full projects can emerge. The pilot projects also provide a mechanism by which to attract new researchers and to rapidly fund the most promising ideas. New pilot projects beyond year 02 will be chosen by the internal steering committee in consultation with the NCI program officer. The following section lists some of the current examples of pilot projects: Pilot project 1: Nanoparticle labels for high-sensitivity mass detection of cancer biomarkers Project Leader: Scott Manalis Ph.D, Associate Professor, MIT Department: Biological Engineering, MIT Background Despite progress in the development of new therapeutic agents for the treatment of cancer, there has been very little progress in the development of molecular markers for the early detection of cancer. With the exception of the Prostate Specific Antigen (PSA) which is currently used to screen men for the presence of prostate cancer, most cancers have no molecular marker in clinical use. One possible reason is that early tumors are quite small, often under 10 mm in diameter. It is clear that the amount of protein secreted by such tumors will be also be small, requiring sensitive assays able to detect proteins in biological fluids at concentrations of 0.1 -1.0 ng/ml or less. Pilot project 2: Microfluidic Sorting of Circulating Tumor Cells Project Leader: Mehmet Toner, Ph.D., Professor HMS;Daniel Haber M.D, Professor, HMS Departments: NIGH Center for Engineering in Medicine and MGH Cancer Center Background Human cancers generate small numbers of cells that circulate in the vasculature. Some of these may be destined to seed sites of cancer metastasis, while the majority may not be viable but simply reflecting microvascular invasion at local sites of disease. The ability to identify, recover and study these cells offers a potentially accurate, affordable, reliable, and noninvasive screening and surveillance tool for early diagnosis and treatment monitoring. There are a growing number of reports on the isolation and characterization of CTC in cancer patients before the primary tumor is detected (1). There is also evidence that CTC are originated from the primary tumor. Thus, CTC may ultimately provide to be a very valuable source in providing diagnostic, prognostic, monitoring, as well as genetic and immunophenotypic information about the primary tumor. Equally important is the ability to use CTC for targeted therapies in cancer, such as non-small-cell lung cancer. Unfortunately, only about ten percent of patients with non-small-cell lung carcinoma have a robust clinical response to the tyrosine kinase inhibitor gefitinib. We and others have recently demonstrated that this subgroup of patients has specific mutations in the epidermal growth factor receptor (EGFR) gene (2-3). Thus, screening for EGFR mutations in lung cancers may identify patients who will have a response to a specific treatment. To this end, it is important to develop a noninvasive blood test such as the noninvasive isolation of CTC from blood of patients already diagnosed with lung cancer. Furthermore, the monitoring of the number of CTC of those patients with the EGFR mutation may provide invaluable information about the efficacy of the treatment with the tyrosine kinase inhibitor. The ability to use CTC to screen populations, to monitor therapies, to predict recurrence, and to identify patient subpopulations for targeted therapies, in combination with new molecular techniques, will likely result in significant progress toward improving survival rates in cancer. Pilot project 3: Nanomodels of Metastatic Cancer Project Leader: Sridhar Ramaswamy, Ph.D., Assistant Professor, HMS Department: MGH Cancer Center, Broad Institute Background Metastasis is the major cause of cancer-related deaths, but its molecular basis is poorly understood. As a result, current approaches to cancer drug development have not led to increased survival for most patients with advanced solid tumors (1). Metastasis mostly results from the interplay of acquired mutation, epigenetic regulation, and inheritance (2). Highly complex cellular and molecular interactions in cis- and trans- likely cause the clinical features of metastatic cancer that make it particularly difficult to treat;namely, tumor growth at distant sites and resistance to chemotherapy. These interactions, however, are difficult to functionally examine in a comprehensive way using traditional approaches. This hinders the development of effective chemotherapy for advanced cancer (3). Animal models of metastasis (autograft, allograft, xenograft, or genetically-engineered), for example, are limited in many ways including low-throughput, high cost, low-genetic complexity, and unclear relation to human disease. In vitro modeling of metastasis, usually limited to cancer cell invasion and migration assays, while relatively inexpensive and high-throughput, do not adequately recapitulate the cellular and molecular complexity of human tumors in vivo. Our aim is to develop next-generation in vitro cancer models using new developments in nanotechnology to more faithfully mimic the complexity of metastatic human tumors. Our long-term goal is to use these systems to screen for small-molecule compounds that inhibit the rate-limiting step in cancer metastasis: survival and growth of metastatic cancer cells at distant sites. Cancer cell behavior is highly dependent on micro-environmental cues and context (4). We hypothesize that successful end-organ colonization results from interactions between cancer cells (with particular mutations) and host cells (with specific genetic and epigenetic features) in target tissues. We are experimentally exploring a wide spectrum of such interactions through the systematic co-culture of different human cancer cell lines (mutations) with panels of normal fibroblasts from different patients (genetics) and organs (epigenetics). These 2-D co-cultures, albeit crude, preliminarily demonstrate that interactions of a cancer cell with different fibroblast populations can result in inhibitory, enhancing, or null effects on in vitro cancer proliferation (Figure 1). These results suggest that in vitro cancer models that mimic multi-cellular interactions will yield very different views of human cancer cell behavior compared with unicellular models, and that such experimental systems might more accurately model tumor biology in vitro. Pilot project 4: Hybrid Integrated Circuit / Microfluidic chips for the manipulation of cells Project Leaders: Robert Westervelt Ph.D, Professor Harvard University;Donhee Ham, Ph.D. Assistant Professor, Harvard Department: Division of Engineering and Applied Sciences, Harvard University Background The manipulation of biological systems using spatially patterned magnetic and electric fields is an important tool. Conventional approaches use relatively simple methods to create the electromagnetic fields, limiting the range of their applications. Pilot project 5: Ultrasensitive chemical probing at the single molecule level using surface enhanced Raman scattering in local optical fields of gold nanoparticles Project Leaders: Katrin Kneipp Ph.D, Associate Professor, Harvard Department: Wellman Center for Photomedicine, MGH Background Cancer is currently being missed at its earliest stages. With regard to this situation , the objective of this project is to explore and to develop a novel method based on ultrasensitive molecular structural probing and imaging inside living cells for the discovery of cellular changes during the development of cancer. The method has also the potential capability to monitor the "chemical" response of ceils to therapy and interventions. The applied approach exploits the phenomenon of surface enhanced Raman scattering (SERS), where Raman scattering takes place in the local optical fields of silver and gold nanostructures resulting in the increase of Raman signals up to 14 orders of magnitude. This allows molecular structural information from single molecules and from nanometer scaled volumes. Pilot project 6: Functionalized linear-Dendritic Diblock Copolymers for Targeted, Tumor-Selective Nucleic Acid Delivery Project Leaders: Paula Hammond Ph.D, M. Hyman Associate Professor MIT;Dane Wittrup, PhD, J. Mares Professor MIT Department: Chemical Engineering and Bioengineering, MIT Background The application of nucleotide-based therapeutics in clinical medicine has the potential to revolutionize the treatment of human disease. The success of gene therapy is dependent upon the ability to deliver genes that express key proteins when and where they are needed. To address this challenge, a spectrum of viral and non-viral delivery systems has been developed. One of the most promising delivery approaches involves the use of cationic polymers, and a range of linear, branched, and dendritic polymers have been explored, including poly (b-amino esters), poly (ethylenimines), and poly (amidoamines), respectively. Unlike viral delivery systems, which are often highly immunogenic, prone to insertional mutagenesis, and refractory to repeated administrations, non-viral (polymeric) delivery systems can be synthesized with low immunogenicity and toxicity, though they frequently suffer from cytotoxicity, poor tissue targeting, rapid clearance from circulation, and low expression efficiency (1-2). Pilot project 7: Targeted Nanoparticles for siRNA Delivery in Cancer Project Leader: Clark Cotton Ph.D, Professor, MIT Department: Chemical Engineering, MIT Background We have developed novel nanoparticles that have promise for siRNA delivery to tumor cells. The nanoparticles are composed of a unique alternating copolymer backbone consisting of hydrophilic polyethylene glycol (PEG) segments and hydrophobic trifunctional linkers to which are bound hydrophobic side chains terminated with hydrophobic, hydrophilic, or charged moieties. When placed into water above its critical micelle concentration, 8 to 12 of these amphiphilic polymer chains self assemble into a micelle structure with the linker forming the surface of a sphere, the PEG chains externalized as loops and the hydrophobic side chains internalized. Typically the micelles have a molecular weight about 200 and a hydrodynamic diameter about 5 nm. When mixed with contrast agents or drugs that are encapsulated as cargo, nanosphere size increases to as much as 50 nm. In addition to encapsulation of cargo, the side chain or terminal group can be replaced with a covalently bound agent. These micelle nanoparticles have advantages over other approaches because 1) their small size enhances access to cells within a tumor, 2) their chemical structure can be easily modified, and they are synthesized by a straight forward chemo-enzymatic method that is more practical and economical than the complex protection-deprotection schemes needed for purely chemical synthesis of such structures, and 3) a single platform can accommodate a wide variety of bound or encapsulated agents useful for improved imaging of tumor cells and drug delivery to tumor cells. We are currently investigating different tumor targeting peptides (e.g. those developed by Ruoslahti (Project 2) or the Weissleder group (Project 5)). The peptides are bound to the free hydroxyl end groups of the PEG. As a consequence, large numbers of the nanoparticles are rapidly taken up selectively by tumor cells. Pilot project 8: Nanowire, nanolaser as optical probe for high resolution cellular imaging and manipulation Project Leaders: Yu Huang, PhD Department: MIT Material Science and Engineering/LLNL Backqround Nanotechnology can enable many unique tools to probe/image biosystems at an unprecedented molecular level and reveal new phenomena. For example, scanning near-field optical microscopy (SNOM) is an interesting technique in biophysics for the visualization of biological objects, e.g. cellular membrane, with high spatial resolution. This technique represents a powerful approach for high resolution imaging of bio-species by combining topographic information with optical fluorescence or light transmission imaging. However, the metallic coated probe is limited in several ways. First, only a tiny fraction (<0.01% for 100 nm tip) of the light coupled into the fiber is emitted by the aperture because of the cutoff of propagation of the waveguide modes. The low light throughput and the finite skin depth of the metal are the limiting factors for resolution. Many applications require spatial resolutions that are not obtainable with the aperture technique. Moreover, the aperture technique has other practical complications: (1) it is difficult to obtain a smooth metal coating on nano scale which introduces irreproducibility in probe fabrication, as well as measurements;(2) the absorption of light in the metal coating causes significant heating and poses a problem for biological applications. To address these issues, significant efforts have been devoted to searching for alternative probes such as aperture-less probe including metallic probes or fluorescence active probes. They represent exciting new directions, but often suffer from low signal-to-noise ratio due to low light intensity.

DESCRIPTION (provided by applicant): This application addresses broad challenge area (14) Stem Cells and the specific challenge topic 14-EB-101 Synthetic Delivery Systems for Generating Pluripotent Stem Cells The recent advent of cell reprogramming as a means of producing induced pluripotent stem (iPS) cells from somatic cells has produced great excitement in the biological and medical communities due to its potential to circumvent the immunological and ethical issues surrounding traditional embryonic stem cells. For this technology to prove useful as a therapeutic and/or experimental tool one must first develop a technique that can reprogram cells in a rapid, efficient and repeatable manner. Most importantly, one must find alternatives to the potentially cancer-causing retroviruses that are currently used for this process. In this proposal, we request funding to further develop a current, proof of concept, cell injection prototype and to utilize it as an enabling tool to conduct reprogramming studies. The device is capable of delivering pico liters of materials across the cell membrane in a high throughput, efficient manner. The microfluidic system utilizes a micron-scale nozzle to inject a jet of liquid into cells passing through a channel. This jet pierces the cell membrane, without causing cell lysis, and is capable of delivering the factors necessary to reprogram somatic cells. By further developing this device, we hope to use the system to produce iPS cells at a rate and efficiency comparable to viral transfection, while avoiding the issues of mutagenesis and toxicity. The efficacy of micro-injector based cell reprogramming will be thoroughly tested using published methods of inspecting iPS cells. Tissue cultures produced by the system will also be used in animal studies for verification of pluripotency in reprogrammed cells. Eventually we will use the device in tandem with our novel polymeric delivery mechanism as a means of further enhancing performance. In addition, the quantitative, high throughput nature of the device will allow us to conduct studies on the biological aspects of the reprogramming process itself. More specifically, we can determine what optimal combination of genes and factors will result in maximum reprogramming efficiency as well as the specific role of individual genes in the overall process. Due to the physical nature of the delivery system, our device can also explore the use of proteins as a means of enhancing reprogramming (or even replacing DNA) in the production of iPS cells. Over the past years, embryonic stem cells have revolutionized the field of regenerative medicine;their potential for producing entire organs and tackling genetic disorders, such as Multiple Sclerosis (MS), has made them a favorite among researchers. The cell injection device described herein will have the capability of producing stem cells in a rapid, safe and efficient manner by reprogramming a patient's existing adult cells. The system can thus not only circumvent the ethical and technical issues that surround embryonic stem cells, but also avoids many of the toxicity and cancer risks associated with existing virus-based reprogramming techniques.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (14) Stem Cells and specific Challenge Topic, 14-DE-103: Enhancing Human Embryonic Stem (ES) Cell Culture Systems. The overall objective of this proposal is to develop and optimize a feeder-free in vitro culture system with a controlled culture microenvironment for human pluripotent stem cell growth and directed differentiation. In particular, high-throughput approaches will be used to develop culture materials with optimized surface properties incorporating defined extracellular matrix (ECM) components that allow for accurate control of oxygen partial pressure of the cell surface (pO2cell) to provide more physiological conditions and allow strategies to better mimic normal development processes. The developing embryo is exposed in vivo to very low oxygen levels, and there is growing evidence that oxygen concentration, together with cell-protein interactions, are important factors in differentiation and proliferation. In typical culture systems, pO2cell is unknown because of gradients in the culture medium. Currently, there are no commercially available methods or equipment for culturing cells under known, hypoxic conditions. Membranes of silicone rubber, which have very high oxygen permeability, will be used as a culture substrate so that cells are exposed to the same oxygen level as in the gas phase. Silicone rubber surfaces will be investigated in various forms: (1) unmodified, (2) chemically modified and functionalized with synthetic polymers, and (3) chemically modified with synthetic polymers to which ECM components are attached by physical adsorption or covalent linkage, including defined proteins and mixtures thereof. Cell compatible, ECM protein microarrays on functionalized silicone rubber will be created with a high-throughput microarray platform that we previously developed for culture of cells in order to screen a large number of cell-ECM-biomaterial interactions. The various cell substrates will be examined for cellular attachment, proliferation, and gene expression patterns at various levels of pO2cell using combinatorial techniques. Confirmatory experiments in macro-scale culture using culture vessels will be carried out with the most promising combinations. Similar experiments with the high-throughput microarray platform and culture vessels will be carried out for differentiation of human pluripotent stem cells to cardiomyocytes, which has been shown to benefit from hypoxic culture, as a model system. Accomplishment of the goal of the proposed studies will lead to elimination of conventional feeder layers and undefined xenogenic proteins and to the development and validation of a more well-defined and physiological culture platform for simultaneous variation and study of soluble factors, ECM-cell interactions, and pO2cell, thereby enabling enhancement of our understanding of the role of these factors in maintenance and differentiation of human pluripotent stem cells. This novel, generally applicable platform, will upgrade the capability and quantity of research in this field. Pluripotent stem cells hold enormous promise for drug screening, in vitro modeling of genetic disorders, and cell therapies and the proposed research will have wide impact on human health. At its most basic level, this research will provide information about the interaction of biomaterials, ECM components, and undifferentiated or differentiating human pluripotent stem cells at different known values of pO2cell that mimic physiological conditions, data for which has not previously been available except under normoxic culture. This information will advance our knowledge in the fields of developmental and stem cell biology as well as human pluripotent stem cell technology. In particular, we expect to learn how the choice of ECM proteins and pO2cell levels interact to aid directed differentiation. From a broader standpoint, the proposed high-throughput platform and macro-scale culture vessels that derive therefrom are tools that constitute enabling technology to culture human pluripotent stem cells under defined physiological conditions. They will improve the research infrastructure in terms of the capability for culturing human pluripotent stem cells. The knowledge acquired and tools developed may have profound effects on a wide variety of applications in many fields of human health. This will accelerate developments in applications such as drug screening, in vitro models of genetic disease, therapeutic replacement of diseased cells in major diseases such as heart disease, diabetes, and neural diseases such as Parkinson's, which annually affect millions of people in the United States. Indeed, differentiation of human pluripotent stem cells to cardiomyocytes, the proposed model system, is itself of high interest for generating cells and tissues for repair of cardiac tissues following ischemic heart disease. Furthermore, the culture vessels to be tested will be useful in culturing cardiac tissue. This collaborative study will integrate recent progress in the fields of bioengineering, biomaterials, and developmental biology to design a new culture platform and protocols that will facilitate cell-based therapies for treatment of a multitude of human diseases and other ES cell applications. PUBLIC HEALTH RELEVANCE: This project concerns improved methods to work with human stem cells so that they can develop into medically useful cells and tissues. For example, this can lead to ways to grow functioning heart muscle cells that can be used to treat heart disease.

The central aim of this CCNE project is to develop nanotechnologies for targeted combination pharmacotherapy using existing compounds with suboptimal pharmaceutical properties. The genomic revolution has resulted in the identification of approximately ~320 molecular targets and attempts to therapeutically utilize many of these have faced considerable development challenges (1, 2). More recently, advances in systems biology have aided in identifying synergistic pathways among these newly identified targets that may be concurrently utilized for more effective treatment of cancers. The development of nanotechnologies for effective delivery of multiple drugs or drug candidates in a temporally regulated manner to cancer cells can potentially overcome the development challenges faced to date, and result in harnessing the maximal benefits of cancer genomics and systems biology (3, 4). Our early work supported by the MIT-Harvard CCNE focused on engineering targeted nanoparticles for delivery of a single chemotherapeutic agent (docetaxel) for prostate cancer (PCa) therapy. Using a combinatorial process for engineering libraries of targeted nanoparticles by selfassembly, which is reproducible, we screened and Identified particles with optimal biophysicochemical properties. Particles with optimal properties are now in clinical development and approaching an IND in 2010./n the context of this proposal we hypothesize that 1) by engineering and blending distinct drugfunctionalized and ligand-functionalized polymers, with or without encapsulation of additional free drug molecules, we will be able to reproducibly engineer and characterize nanoparticles capable of delivering 2 or more drugs; and 2) by targeting these drug loaded nanoparticles to cancer cells we can achieve synergistic drug effects which may translate to better efficacy and tolerability making them suitable for potential clinical development. Herein we propose to develop technologies for co-delivery of up to 3 distinct anticancer agents for targeted combination chemotherapy. As a model cancer, building on our previous efforts, we propose to develop long circulating drug-conjugated targeted nanoparticles for differential uptake by PCa cells. We will aim to develop targeted nanoparticles with up to 3 distinct anticancer agents and place one candidate formulation on a development path toward an IND submission in 2014, setting the stage for clinical validation of our TempoSpatially-controlled Combination Chemotherapy (TSCC) platform in patients with hormone refractory prostate cancer (HRPC).

OVERALL VISION FOR DEVELOPMENTAL ACTIVITIES Over the last 5 years of our CCNE, there has been a tremendous interest by researchers to join the consortium through pilot projects and/or collaborative research. We view such projects as an important adjunct of the CCNE's mission and from which full projects and important science can emerge. For example, in the past our consortium has funded the selected following projects: ? Paula Hammond and Dane Wittrup (pilot project) on functionalized linear-dendritic diblock copolymers for tumor-selective nucleic acid delivery. One ofthe postdocs (Greg Thurber) now works in the Weissleder lab and confinues interactions (85, 86) ? Stephen Lippard (pilot project) on functionalizing gold nanoparticles with DNA duplexes containing tethered, releasable plafinum conjugates at their termini in collaboration with Chad Mirkin at Northwestern CCNE (87) ? Robert Westen/elt (pilot project) on hybrid integrated circuit/microfluidic chips forthe manipulation of cells (88, 89) ? Sridhar Ramaswamy (pilot project) on nanomodels of metastatic cancer (90) ? Mehmet Toner and Daniel Haber (pilot project) on microfluidic sorting of circulating tumor cells. This original pilot project has resulted in several high impact publications (91-93) and funding outside the consortium, including a recent SU2C grant involving several CCNE members (Bhatia, Weissleder). Pilot, Alliance Challenge and other exploratory projects provide a mechanism by which to attract new researchers and to rapidly fund the most promising ideas. The following section lists some of our general plans on continuing developmental activities.

MIT and Harvard have excepfionally strong research and education programs in the biological/medical sciences on the one hand and in the physical/engineering sciences on the other, as well as strong programs in nanotechnology. There is a large pool of excellent students and MIT already has administrative and educational mechanisms in place for encouraging interdisciplinary activities among these students and the respective faculty members. We have structured our program to leverage this advantageous situation.

DESCRIPTION (provided by applicant): Based on our work during past grant periods, low-frequency sonophoresis (LFS) is now clinically used, but its application is limited to only certain low-molecular weight drugs. To broaden the application of LFS, we aim to improve the treatment regimen of skin using a combination of LFS and the surfactant sodium lauryl sulfate (SLS), as well as to design nano-scale delivery vehicles to be used after LFS/SLS treatment to increase the transdermal delivery of high-molecular weight permeants, such as proteins and vaccines, to therapeutic levels. The proposed studies involve: 1) Understanding the mechanisms of heterogenous skin perturbation with LFS and the observed synergism between LFS/SLS on skin permeability enhancement. These mechanisms will be investigated by studying the cavitation field that develops in the coupling medium between the ultrasound horn and the skin. The synergistic mechanism of LFS/SLS-enhanced transdermal penetration may be identified by correlating the extent of enhanced skin permeability with the partitioning of novel fluorescent surfactants (which allow for molecular-level imaging of surfactant behavior using two-photon fluorescence microscopy) between major skin structural components. Upon determination of these mechanisms, the LFS application and topical formulation may be improved to more effectively deliver drugs through a treated skin area; 2) Promoting the formation of localized transport regions (LTRs, highly permeabilized regions that form on the surface of the skin as a result of LFS application) by controlling the nucleation of LFS-induced cavitation events, in order to reduce skin treatment areas, minimize variability between skin treatment sites, and decrease ultrasonic power input requirements; 3) Designing polymeric micelle delivery vehicles to enhance the transdermal delivery of permeants through skin pre-treated with LFS/SLS by utilizing functionalized nanoparticles (quantum dots and biomimetic gold nanoparticles) to identify optimal permeant properties for passive transdermal delivery of permeants through skin pre-treated with LFS/SLS; 4) Transport modeling of the permeant diffusion pathways through the skin, which will aid in proper clinical drug dosing by predicting the lag time to reach steady state and the steady state permeability of a drug; and 5) Investigating the safety of the LFS/SLS skin treatment by determining the reversibility of the LTRs and their biological effects. Overall, the studies proposed here aim to expand the clinical utility and efficacy of LFS/SLS as a painless and non-invasive method of transdermal drug delivery for a broad class of drugs.

The overall goal of this U54 application is to support a highly multidisciplinary team of expert chemists, engineers, biologists, material scientists and clinicians to develop and rapidly translate new nanotechnologies to better diagnose and treat cancer in the clinic. This team formed a Center for Cancer Nanotechnology Excellence (CCNE) in 2004 and over the past 5 years has shown extraordinary productivity through its integrated research programs and shared resources. The primary team includes investigators from Massachusetts Institute of Technology (MIT), Harvard Medical School (HMS), Massachusetts General Hospital (MGH) and Brigham and Women's Hospital (BWH) and Harvard Faculty of Arts and Sciences (FAS). The CCNE thus effectively bridges programs in basic sciences at two Universities (MIT and Harvard) and clinical programs at leading Hospitals (MGH and BWH). The program will continue to be headed up by Dr. Robert Langer and Dr. Ralph Weissleder as Co-Pls. Dr. Langer is Institute Professor at MIT and member of the Koch Institute for Integrative Cancer Research at MIT (Kl). Dr. Weissleder is a Professor at Harvard Medical School, the Director of the Center for Systems Biology at MGH, a practicing clinician at MGH and a member of the Dana Farber Harvard Cancer Center.

DESCRIPTION (provided by applicant): High throughout technologies has already significantly revolutionized fields such as genomics, proteomics, and drug discovery and formulation. This technology can similarly revolutionize the development of biomaterials for tissue engineering applications. A fundamental component of this proposal is to apply and advance our fully automated, high throughput discovery methods to push hESC tissue engineering closer towards clinical applications. Two key remaining limitations of existing hESC and iPSC methods are 1) the low efficiency and long time associated with stem cell differentiation into functional cell types such as chondrocytes and osteoblasts, and 2) suboptimal performance (e.g. mechanical properties, biocompatibility) of existing degradable materials used for tissue engineering. As such, we propose to develop both high throughput strategies to rapidly optimize the production of homogenous populations of key cell populations and degradable biomaterials that provide for improved cellular performance and low inflammation. Accordingly our specific aims are: Aim1. Develop efficient, rapid methods to differentiate human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into homogenous populations of craniofacial cells. We will use high-throughput approaches on hESC and iPSC cells to identify the optimal combinations of soluble (growth factor and small molecules), and insoluble factors (synthetic polymer surfaces) capable of committing ES cells to craniofacial precursor cells and fully committed osteogenic and chondrogenic cells. Aim 2: Develop biodegradable, non-inflammatory and mechanically appropriate 3D scaffold systems that can effectively deliver craniofacial cells to the site of injury. High-throughput libraries of hyaluronic acid (cartilage) and poly(2-amino ester) (bone) polymers will be synthesized and assessed for ability to form gels or solid porous scaffolds, respectively. Favorable materials will then be evaluated for either chondrocyte or bone cell compatibility. Aim 3: Assess performance of tissue engineered constructs developed in Aim 1 and 2 to generate cartilage and bone in vivo. Small animal model will be used to test osteogenesis in a cranial critical sized defect, while chondrogenesis will be evaluated subcutaneously. PUBLIC HEALTH RELEVANCE: We believe that this technology can similarly revolutionize the development of biomaterials for tissue engineering applications. A fundamental component of this proposal is to apply and advance our fully automated, high throughput discovery methods to push hESC and iPSC tissue engineering closer towards craniofacial clinical applications.

DESCRIPTION (provided by applicant): The purpose of this study is for a team of chemical engineers, materials engineers and neuroscientists at MIT to develop a combined micro-cannula and deep brain electrical stimulation device for the treatment of anxiety and mood disorders. Anxiety and mood disorders are common and debilitating disorders that afflict millions of Americans. The emerging thought is that these disorders are actually rooted in disruptions in activity across neural circuits, as opposed to defects in any one region. Current treatments comprising oral and i.v. administration of therapeutics are too coarse, both spatially and temporally, to appropriately attenuate the dynamic activity across neural circuits. Our goal is to develop an implantable micro-cannula device that is capable of simultaneous infusion of multiple therapeutics, as well as electrical stimulation and real time chemical sensing. This device will be micro-fabricated in such a way as to be minimally invasive, yet durable enough to be scaled to non-human primate use. The combination of precise anatomical targeting and diverse stimulatory (electrical & chemical) capabilities should improve our ability to modulate activity across specific neural circuits with the appropriate kinetics. This proposal, and the assembled research team, combines the varied fields of micro-fabrication, materials engineering, chemistry, biology and neuroscience. Our specific goals are summarized as follows: 1) Design for failsafe delivery of neuro-modulatory therapeutics. This aim will ensure that the desired doses are delivered accurately and reproducibly. 2) Evaluate methods of improving the structural integrity and biocompatibility of the device via metal deposition and chemical functionalization. Neuro-stimulatory electrodes have been shown to lose function over prolonged periods of implantation due to gliosis. Our proposed studies will develop a method for prolonging device function by retarding gliosis. 3) Refine the peripheral components (reservoir, pump and tubing) to be a stand-alone unit suitable for chronic implantation. This will increase the utility of the proposed device, as well as represent an important step towards clinical usage. 4) Demonstrate behavior change in animal (non-human primate) models of anxiety and mood disorders by delivering stimulation (electrical & chemical) via the proposed device. 5) Demonstrate that the behavioral change is a result of modulating neural circuit activity by the proposed device. Our aim is to, demonstrate the failsafe function of the device in vivo and investigate its capability to ameliorate anxiety and mood disorder based behaviors via precise spatiotemporal control. In a final step we plan to develop feedback based activation of the device by real time sensing of pathological activity. This represents an important step towards clinical usage where anxiogenic stimuli are frequently unknown and un-predictable.

Project Summary/Abstract Our long-term goal is the development of systems providing controlled drug delivery of a broad set of therapeutics including those that are limited to parenteral routes. In this proposal, we build on our prior work to focus on the gastrointestinal (GI) barrier and specifically propose a platform enabling the high-throughput GI transport evaluation of novel formulations rapidly. Developing therapies which are compatible with oral administration, requires significant formulation and in vitro and in vivo evaluation for maximal oral bioavailability in humans. Current in vitro models of GI absorption are limited by their throughput and approximation of the physiologic state. Consequently, we propose: 1) the development of systems enabling prolonged culturing of intact mammalian GI tissue coupled to 2) high-throughput robotics to transform formulation development and study of the GI tract. These investigations are supported by strong preliminary data demonstrating: 1) culture conditions which maintain the presence of a broad set of cellular markers and drug transporters ex vivo in porcine GI tissue, 2) fabrication of prototype systems enabling high-throughput interrogation, 3) demonstration of predictive capacity of drug absorption for a large panel of drugs, 4) demonstration of near order of magnitude enhancement of uptake of a model molecule following a large-scale excipient screen. Currently, promising therapeutics which are poorly absorbed through the oral route can manifest in drug development delays on the order of years and many times no formulation solution is identified. The proposed work will target a critical unmet clinical need by providing tools to rapidly identify formulations that enable: maximal drug solubility and absorption and minimal local toxicity. Moreover, the proposed system will enable interrogation and study of the GI tract entero-endocrine system enabling the discovery of new therapies for metabolic disorders. Through this proposal we will develop a novel set of formulations and novel material-drug combinations enabling the oral delivery of drugs previously restricted to parenteral routes. Moreover, we will develop novel modulators of the enteroendocrine system providing novel solutions to the metabolic disease epidemic. In sum, this proposal aims to provide a platform akin to an ?intestine-on-a-chip? with the capacity to transform formulation science and the study of the GI tract with the potential to transform treatments for metabolic disease and other diseases of the GI tract.

Project Summary The purpose of this study is for a team of materials scientists, biomedical engineers, analytical chemists, and neuroscientists at MIT to develop a micro-invasive implantable device for monitoring the biochemical composition of distinct brain regions. This analytical tool for sampling neurochemicals in brain interstitial fluid (ISF) promises to provide valuable insight into the dynamics of neural circuits in physiological and pathological states. We will apply this tool to study the role of neuropeptides in substance use disorder (SUD). The dynorphin family of neuropeptides has long been implicated in addiction, but no current analysis tool has been able to investigate the long-term spatiotemporal dynamics of these neurochemicals in vivo. Our goal is to demonstrate the efficacy of our sampling platform in measuring neuropeptide expression dynamically in a rodent model of SUD. This will lend greater insight into the biochemical basis of addiction and withdrawal, but perhaps more importantly establish our technology as an effective technique for understanding the onset and progression of neural diseases. Our specific goals are summarized as follows: 1) Design a minimally invasive and implantable device for sampling ISF chronically in vivo. The device will consist of a nanofluidic pump (nanopump) coupled to micro- scale probes (microprobes), with fluid flow characteristics optimized in vitro prior to translation to a stand-alone in vivo device. 2) Optimize the storage and processing of small volumes of sampled ISF, withdrawn via nanopump, for analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS). 3) Determine the detection limits for the dynorphin neuropeptide family in ISF in vitro prior to detection of these neurochemicals in in vivo samples at physiological and pathological concentrations. 4) Perform short-term monitoring of dynorphin at baseline and in acute stress to demonstrate the efficacy of this tool in tracking these large neuropeptides in real-time. 5) Track the dynorphin family of neuropeptides in a rodent model of cocaine SUD, lending greater insight into the biochemical basis of substance withdrawal and relapse. Our aim is to demonstrate the failsafe function of this sampling platform in vivo and establish its ability to monitor neuropeptide dynamics with precise spatiotemporal control. We aim to provide neuroscientists with a new tool for investigating the biochemical basis of neural pathology in well-established animal models, enabling more accurate diagnosis and treatment of neural disorders in humans in the future.