Designing therapeutic and proof of principle clinical trials of nanoparticles for cancer

After initial preclinical development, novel agents require clinical development which generally implies a series of Phase I-II and possibly III studies. Careful attention to early phase drug development including adequate trial design, clinical pharmacology and obtaining pharmacodynamic data related to the drug under investigation can greatly improve this process. Some new agents are thought to have failed clinical; development not because of the agent itself but because of poor trial design.

xxx

NanoMedicine Start-ups: Commercializing Research – Keystone Nano & Nano Therapies

Keystone Nano (KN) is developing Calcium Phosphate Nanoparticle products (NanoJackets) based on technology developed at Penn State University and the Hershey Medical Center.  These NanoJackets are: small (30-50nm), non-toxic, colloidally stable, can be functionalized to selectively target delivery and display pH dependent dissolution..

While Keystone Nano’s lead product, NanoJacketed Doxorubicin, is being developed to more effectively treat breast cancer, the company is also developing NanoJacketed products to treat non-solid tumors through its JV company – NanoTherapies. In addition, Keystone Nano is developing a series of NanoJacket products for industrial applications with Nalco, a large and profitable specialty chemicals manufacturer.   Jeff Davidson will introduce the commercialization programs at Keystone Nano and the company’s focus on creating high value NanoJacketed compounds.

Cense Biosciences is an early stage biopharmaceutical company whose mission is to build a high value company through the use of its proprietary Agglomerated Vesicle Technology (AVT), which enables the sensing and reactive release of therapeutic drugs. Our initial focus it to develop a unique glucose-sensing insulin delivery compound called Briodurance™, which effectively manages blood glucose levels in patients with diabetes. Current insulin treatments require constant monitoring of blood glucose levels and several insulin injections a day. Presently, there is no insulin treatment that promotes compliance in diabetics, given that approximately 93% of diabetics do not attain satisfactory glycemic control. Failure to consistently monitor glycemic levels elevates the risk of other health problems, including heart disease, stroke, blindness, and loss of limbs. Cense Biosciences aims to replace the current insulin regimen through the use of Briodurance™, a compound that can automatically maintain stable and desirable glycemic levels in a lifestyle-friendly manner. An automated mechanism, injected once daily, senses glucose levels, adjusts the insulin release rate, and releases insulin accordingly.

Tursiop Technologies is developing novel MRI coil devices utilizing proprietary nanotechnology, which will produce higher image quality and faster scan times resulting in a significant boost of the functional capabilities of any installed MRI system. Tursiop-generated imaging enhancements will apply across the entire range of MRI system field strengths, from low to moderate to high-field systems. In addition to increasing diagnostic competency across the board, it is anticipated that the resulting gains will also lead to much more open imaging systems, enabling expanded procedural opportunities within the areas of interventional, intra-operative and functional MRI and molecular imaging.

The Workings of NCI Nanotechnology Alliance for Cancer
an Opportunity for a New Class of Diagnostic and Therapeutic Solutions Based on Nanotechnology

National Cancer Institute is engaged in efforts to harness the power of nanotechnology to radically change the way we diagnose and treat cancer. Novel and multi-functional nanodevices will be capable of detecting cancer at its earliest stages, pinpointing its location within the body, delivering anticancer drugs specifically to malignant cells, and determining if these drugs are effective. Functionalized nanoparticles would deliver multiple therapeutic agents to tumor sites in order to simultaneously attack multiple points in the pathways involved in cancer. Such nano-therapeutics are expected to increase the efficacy of drugs while dramatically reducing potential side effects. In vivo biosensors would have the capability of detecting tumors and metastatic lesions that are far smaller than those detectable using current, conventional technologies. Furthermore, they willprovide rapid information on whether a given therapy is working as expected.
In order to further these research goals, NCI Alliance for Nanotechnology in Cancer has been formed in 2004. The Alliance is investing $144.3 million over the next 5 years to pursue applied nanotechnologies for cancer detection, therapy, and prevention with an aim to achieve clinical translational stage of these technologies towards culmination of the program. The Alliance funds Centers of Cancer Nanotechnology Excellence, the development of nanotechnology platforms, and intramural Nanotechnology Characterization Laboratory (NCL). NCL provides a spectrum of data on the physical parameters and pharmacological and toxicological characteristics of clinically promising nanomaterials. 
The eight CCNEs – Centers of Cancer Nanotechnology Excellence represent prime research institutions in the US: Carolina Center of Cancer Nanotechnology Excellenceat the University of North Carolina, Center for Cancer Nanotechnology Excellence Focused on Therapy Response at Stanford University, Center of Nanotechnology for Treatment, Understanding, and Monitoring of Cancer at the University of California, San Diego, Emory-Georgia Tech Nanotechnology Center for Personalized and Predictive Oncology at Emory University and Georgia Institute of Technology, MIT-Harvard Center of Cancer Nanotechnology Excellence at MIT and Harvard University, Nanomaterials for Cancer Diagnostics and Therapeutics at Northwestern University, Nanosystems Biology Cancer Center at California Institute of Technology, and the Siteman Center of Cancer Nanotechnology Excellence at Washington University. This presentation will describe the details behind the organization and science and technology of the Alliance.

xxx

Translational Nanomedicine: Basic Mechanisms to Applications
Vinod Labhasetwar, Ph.D.
Department of Biomedical Engineering, Lerner Research Institute, and Taussig Cancer Center, Cleveland Clinic, Cleveland, OH

Central to the applications of nanoparticles as an efficient carrier system for intracellular drug delivery is the need to understand their interactions with cell surface, their intracellular trafficking, and retention [1].  We have previously demonstrated that PLGA-nanoparticles, following cellular internalization via endocytic pathway, undergo surface charge reversal (anionic to cationic) in the acidic pH of endo-lysosomes, thus facilitating their escape into the cytosolic compartment.  However, the subsequent studies showed that a significant fraction of nanoparticles are recycled out (undergo exocytosis) and only 15% of the internalized nanoparticles escape into the cytosolic compartment, thus limiting their efficiency for intracellular delivery of encapsulated therapeutics [2-4].  We have established a model to study nanoparticle-cellular interactions and the dynamics of intracellular trafficking of nanoparticles using atomic force (AFM) and confocal microscopy.  This presentation will feature the effect of surface properties of nanoparticles on endosomal escape of nanoparticles, their intracellular retention, and cytoplasmic delivery of encapsulated therapeutics.  One of the objectives of the translational nanomedicine is to explore biomedical applications of nanoparticles to improve therapeutic outcomes.  In this regard, we have been exploring drug/gene delivery applications of biodegradable nanoparticles in cancer therapy and stroke.  The results of these studies will be discussed in the presentation.  
REFERENCES:

Circulating Tumor Cells:   Let Me Do More Than Count the Ways
G. Thomas Budd, MD

Circulating Tumor Cells have been described in patients with advanced cancer for over a century.  Until recently, however, the detection of such cells was possible in only a minority of patients in a pre-terminal state.  Newly developing methods, however, have made the detection of such cells possible in 50% or more of patients with metastatic malignancy.  With the ability to find these cells has come the ability to study their significance and biology.  Clinical studies have indicated that the number of circulating tumor cells is of prognostic significance in breast, prostate, and colorectal cancer, and the response of these circulating tumor cells to therapy may presage the clinical outcome of patients receiving systemic therapy.  The ability to detect these cells in early stage cancer has been variable, however, and technical advances will be needed to allow further research in patients who are clinically free of disease.  With the ability to detect circulating tumor cells has come the opportunity to study them.  Promising studies have indicated that therapeutic targets can be detected in circulating tumor cells, raising the possibility of real-time monitoring of targeted therapies.  Preliminary studies have also indicated that gene expression profiling can be performed in tumor cells found in the circulation, suggesting the possibility that the process of tumor progression can be monitored and used to optimize therapy. 

Properties of Magnetite-polymer Nanoparticles in Physiological Media

J. S. Riffle and R. M. Davis, Macromolecules and Interfaces Institute, Virginia Tech, Blacksburg, VA, in collaboration with T. G. St. Pierre and R. C. Woodward, School of Physics, University of Western Australia, Perth, AU and A. V. Kabanov and T. Bronich, Dept. of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE

      Magnetite nanoparticles coated with polymers are desirable for many applications in biotechnology including cell and other molecular separations, contrast-enhancing agents for MRI, field-induced tumor hyperthermia, target nanoparticles for magnetic biochip sensors, and in our case, hydrophobic magnetic fluids for treating retinal detachment.  The molecular parameters of the polymeric coatings control solution sizes, dispersion, charge characteristics, interactions with cell membranes, targeting, and also affect NMR relaxivities.  The binding characteristics of functional polymers to magnetite are vastly different in water versus media that contain phosphate salts.  This lecture will describe our current findings regarding the synthesis, dispersion and cluster formation of magnetite-polymer nanoparticles in water and phosphate buffered saline (to simulate physiological media).  Hydrophilic poly(ethylene oxide) and amphiphilic poly(ethylene oxide-b-propylene oxide) dispersants with one to three ammonium ions, carboxylates, phosphates or phosphonates on one end have been bound to the magnetite surfaces, and the properties of these materials have been compared.  Our approach to learning how to control magnetic nanoparticle dispersions is to combine experimental measurements with colloidal theories.  Early results regarding effects of amphiphilic parameters on interactions with the lipid regions in cancer cell membranes and on NMR (transverse) relaxivities will also be discussed. Properties of Magnetite-polymer Nanoparticles in Physiological Media, J. S. Riffle and R. M. Davis, Macromolecules and Interfaces Institute, Virginia Tech, Blacksburg, VA, in collaboration with T. G. St. Pierre and R. C. Woodward, School of Physics, University of Western Australia, Perth, AU and A. V. Kabanov and T. Bronich, Dept. of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE   Magnetite nanoparticles coated with polymers are desirable for many applications in biotechnology including cell and other molecular separations, contrast-enhancing agents for MRI, field-induced tumor hyperthermia, target nanoparticles for magnetic biochip sensors, and in our case, hydrophobic magnetic fluids for treating retinal detachment.  The molecular parameters of the polymeric coatings control solution sizes, dispersion, charge characteristics, interactions with cell membranes, targeting, and also affect NMR relaxivities.  The binding characteristics of functional polymers to magnetite are vastly different in water versus media that contain phosphate salts.  This lecture will describe our current findings regarding the synthesis, dispersion and cluster formation of magnetite-polymer nanoparticles in water and phosphate buffered saline (to simulate physiological media).  Hydrophilic poly(ethylene oxide) and amphiphilic poly(ethylene oxide-b-propylene oxide) dispersants with one to three ammonium ions, carboxylates, phosphates or phosphonates on one end have been bound to the magnetite surfaces, and the properties of these materials have been compared.  Our approach to learning how to control magnetic nanoparticle dispersions is to combine experimental measurements with colloidal theories.  Early results regarding effects of amphiphilic parameters on interactions with the lipid regions in cancer cell membranes and on NMR (transverse) relaxivities will also be discussed.  

no info yet

Electrospun Nanofibers for Controlled Drug Delivery
Horst von Recum, Ph.d.

One disadvantage of use nano-sized drug carriers is that due to small device diameters, the diffusion-based release of incorporated drug is nearly instantaneous.   Additionally due to the small volume, there is not much potential payload.  In an interest to circumvent these problems our research group is exploring the delivery of drug bound to the surface of nano devices (such as nanoparticles and electrospun nanofibers).  This takes advantage of the high surface to volume ratio allowing more drug to be bound to nano-sized devices than micro or macro sized devices.   Additionally this affinity-based binding and release mechanism exploits the chemical interaction between drug and surface, such that release can be slowed to much lower than that seen by diffusion.
While our laboratory has some research in micro and nanoparticles using the affinity-based release mechanism, the primary topic of this talk is release from electrospun nanofibers.  Electrospinning is a process by which small fibers (from microns to tens or hundreds of nanometers in diameter) can be generated from a polymer solution or melt.  This talk will address two of the directions we are taking in the use of electrospun fibers or mats.  The first application is the delivery of antibiotics from microfibers for the treatment of surgical site infections. Another application discussed will be the incorporation of oligonucleotides, such as DNA or siRNA into spun fibers for subsequent therapeutic release.

Nanoparticle Gene Delivery; Rational Design and Biological Testing
Kevin G. Rice

Nonviral gene delivery systems have evolved to mimic viral particles that infect human cells and express genes. Increasingly sophisticated carrier molecules are being developed to package DNA into nanoparticle polyplexes that circulate and target DNA and siRNA. DNA binding peptides have been modified with targeting ligands and polyethylene glycol (PEG) to direct their biodistribution. The asialoglycoprotein receptor on the surface of hepatocytes has been the target of numerous in vivo gene delivery studies since it binds and internalizes galactosylated polyplexes. Glycopeptides and PEG-peptides have been developed that form stabilized crosslinked polyplexes. Sulfhydryl crosslinked PEGylated glycoproteins form polyplexes that undergo a triggered release of DNA in the reducing environment of the cytosol of hepatocytes. Fusogenic peptides, such as melittin, have been incorporated into PEGylated glycoproteins to facilitate the endosomal escape of DNA in hepatocytes. Proteasome inhibitors have been built in to peptide carriers to block the premature metabolism of polyplexes and increase gene expression. Mimicking the nuclear targeting of viruses remains the most challenging step of nonviral delivery. Application of these strategies to improve the level of expression from a nanoparticle gene delivery system will be discussed.

The Use of Magnetic Nanoparticles to Enrich for Rare, Circulating Tumor Cells in Head and Neck Cancer Patients
Jeffrey Chalmers
Chemical and Biomolecular Engineering
The Ohio State University

The genesis of overt metastases in a number of different types of cancer is based on the concept that a tumor cell, or a tumor microemboli, dissociate from the primary tumor, circulates through the body either in blood vessels or lymphatic channels, coming to reside at a site in which metastatic tumors develop. Thus, detection of such cells, commonly referred to as circulating tumor cells, CTC’s, in peripheral blood of cancer patients is an appealing strategy to provide further information for fundamental understanding as well as for potential prognosis and treatment. Unlike a majority of other published studies which attempt to isolate CTC by specifically targeting the CTC, we have develop a device and methodology which targets, and removes “normal cells”, thereby enriching for rare CTC’s without biasing for those cells. Our approach, which uses magnetic nanoparticles that bind to tetrameric antibodies that also bind to the CD45 cell surface marker, can achieve a 5.76 log10 enrichment of the CTC’s in as little as 20 minutes. We are not aware of any system that can achieve this level of performance and provide the significant flexibility with respect to analysis that a purely depletion of normal cells provides. This performance is achieved through the pairing of specifically design magnet and flow channels to magnetic nanoparticles of specific size and magnetic susceptibility. In addition to providing an overview of the technology, I will also present our latest data on actual cancer patients as well speculations on the clinical relevance.

Biocompatibility of Particles for Magnetic Drug Delivery
Urs O. Häfeli
Faculty of Pharmaceutical Sciences
University of British Columbia, Vancouver, Canada

Magnetic drug delivery by particulate carriers is a very efficient method of delivering a drug to a localized disease site. For biomedical applications, magnetic carriers must be water-based, biocompatible, non-toxic, and non-immunogenic. In their first uses in medical applications, magnetic particles consisted of iron powder. Improved biocompatibility, however, was attained by encapsulating the magnetic components into matrix materials such as chitosan, dextran, poly(lactic acid), or albumin. In this presentation, the biocompatibility testing results of magnetic microspheres and nanoparticles made from materials such as cobalt, magnetite, and iron will be discussed. The in vivo applications for the tested magnetic particles will also be presented and include cancer therapy, wound healing, stem cell therapy, cardiac investigations and macular degeneration.
For more information about magnetic carriers and an extensive bibliography of this field, please visit the “Magnetic Carrier Home Page” at www.magneticmicrosphere.com.

Commercialization of nanomaterials for immunomagnetic cell separation and cancer diagnostics

Columbus NanoWorks ongoing research and development is focused on the design and commercialization of magnetic reagent kits in novel and commercial magnetic sorting devices having applications involving cancer cell enrichment. Cell separation is becoming increasingly commonplace for researchers and clinicians who isolate rare cells from complex cell populations to understand how cells function in different environments or to diagnose disease occurrence, recurrence or progression. Complex mixtures and searches for small variations in cell samples require new techniques that provide exquisite sensitivity with high throughput; therefore, advances in cell separation technology must allow the researcher to find low percentage cells of interest, as well as purify them to allow for accurate assessment of biological relevance.  Optimal magnetic cell separation requires immunomagnetic particles to have high magnetic susceptibility, narrow particle size distribution, and high-density attachment sites for targeted antibodies. Commercial immunomagnetic beads are either too large, lack size uniformity, or have low magnetic susceptibility.  Columbus NanoWorks is able to manufacture magnetic nanoparticles having uniform size (200 to 300 nm), ligand density (2 to 5 ug/ml Fe particles) and elicit a magnetic susceptibility of approximately 70 emu/g. These criteria for magnetic particles allowed the enrichment of 1 ovarian tumor cell in 107 total nucleated blood cells with an average recovery of 74.1% of spiked tumor samples, as well as an average 3.21 log10 depletion of contaminating cells.  Columbus NanoWorks is focused on the specifications of the particles so that greater than 70% recovery of desired cells, as well as a 4 log10 depletion of contaminating cells is achieved using any magnetic separation device.  The goal is for CNW customized reagents to become an integral part of gold standard testing in the current pathology repertoire.

Thinking small : DNA Nanoparticles for Gene Transfer
Pamela B. Davis, M.D, Ph.D.
Case Western Reserve University

The enormous promise of gene therapy has gone largely unfulfilled. Viral vectors have been effective in several critical diseases, but have the disadvantage for some of integration and subsequent chromosomal damage, and for others, an immune response that precludes repeat administration. Nonviral strategies might be favored, but they have been inefficient, fraught with surprising inflammatory toxicity, often poor at transfecting dividing cells and difficult to manufacture. With favorable chemical properties, simplicity of manufacture, and favorable biological profiles, DNA nanoparticles particles are promising for clinical therapeutics. However, the route by which they enter the cell and the nucleus of nondividing cells has been unclear and has given rise to skepticism about their results. Recent studies indicate that DNA nanoparticles bind at the cell surface to nucleolin, a protein that shuttles between cytoplasm and nucleus and thereby may offer passage into the nucleus as well. Nucleolin takes a nondegradative route into the nucleus, allowing for efficiency, and does not release its cargo in the cytoplasm, reducing toxicity. The size of the nuclear pore through which nucleolin and presumably its cargo passes exactly coincides with the apparent size limitations of the DNA nanoparticles. This observation predicts the tissue tropism of the DNA nanoparticles, and offers opportunities to increase particle uptake by manipulating the arrival of nucleolin at the cell surface.

Magnetic Nanoparticles for Biomedicine

Sara A. Majetich
Physics Department
Carnegie Mellon University

We will focus on the synthesis and characterization of magnetic nanoparticles targeted toward biomedical applications. The starting point is the synthesis of monodisperse iron oxide nanoparticles and nanorods, which are then coated so that they form stable dispersions in biological media. For in vitro applications such as studies of single cells, it is desirable to be able to track the location of individual particles, and to control their position magnetically. Because the particles are much smaller than the optical diffraction limit, this must be done either through fluorescence, or through surface plasmon scattering from gold or silver attached to the iron oxide. Here we show how to deposit Au or Au onto iron oxide. The evolving morphology of the particles as they are coated is correlated with changes in the plasmon resonance. Energy filtered transmission electron holography of individual Ag-coated iron oxide particles shows the spatial and energetic extent of the surface plasmon. Dynamic light scattering shows the average particle size in aqueous dispersion, and dark field optical spectroscopy is used to observe Brownian motion of the particles. Magnetophoretic and drag force calculations predict the particle velocity as a function of the magnetic field gradient and the particle shape and size. Experimental results show the ability to collect the Au-coated iron oxide particles with a permanent magnet, and preliminary tests with patterned elements. When dealing with small particles, nanorods have advantages over nanospheres due to reduced Brownian motion.

Healthcare Biomagnetics: in vivo and in vitro sensing, moving and heating of magnetic nanoparticles for diagnosis and therapy

Quentin A. Pankhurst

Davy-Faraday Research Laboratory, The Royal Institution of Great Britain,
21 Albemarle Street, London W1S 4BS, U.K.

The emerging field of ‘Healthcare Biomagnetics’ – the sensing, moving and heating of magnetic nanoparticles either in vitro or in the human body for diagnostic and therapeutic purposes - will be reviewed. Examples will be given for each of the modalities, including:

Sensing: A high-Tc SQUID based sensor system, with a room temperature hand-held probe, designed for use in a hospital operating theatre to detect breast cancer sentinel lymph nodes (Figure 1). The system is currently being evaluated in patients, and has been used successfully in twelve operations.

Moving: A high field-gradient magnetic actuator designed to capture magnetic nanoparticle loaded haematopoetic stem cells for the treatment of atherosclerosis. Bench-top (Figure 2) and animal trials are under way to establish the efficacy of such a therapy, with promising results.

Heating: Magnetic field hyperthermia treatment for superficial and, as a long term goal, metastatic cancer, using antibody-targeted magnetic nanoparticles. Work is progressing on several fronts: the synthesis of improved magnetic particles for heat transduction (Figure 3), the engineering of new high-frequency drive circuits to produce rf fields in controlled geometries, and cell and animal studies of antibody-nanoparticle conjugation and tumour targeting.

Therapeutic Applications of DNA Nanoparticles
Mark J. Cooper, M.D.
Copernicus Therapeutics, Inc.

A safe and effective non-viral nanoparticle technology has been developed by Copernicus Therapeutics that compacts single molecules of nucleic acids with polycationic carriers, such as polyethylene glycol (PEG)-substituted lysine 30-mer peptides.  These nanoparticles are non-immunogenic, non-inflammatory, and non-toxic, and transfect various non-dividing tissues in vivo with efficiencies comparable to viral vectors.  DNA nanoparticles achieve transfection efficiencies of 30% to 80% of surface epithelial cells in the lung, are equivalent in efficiency to viral vectors in the brain, and transfect up to 99% of retinal photoreceptors after local eye delivery.  Nanoparticles can be repetitively administered to the lungs of mice without any reduction of transgene activity.  The gene transfer efficiency is probably related to the unimolecular nature of the DNA nanoparticle, resulting in an effective diameter of < 12 nm, and to its association with cell surface nucleolin, which facilitates efficient cellular internalization, non-degradative (non-lysosomal) intracellular trafficking, and nuclear uptake.  Based on its safety profile and gene transfer activity, Copernicus initiated a program for CFTR replacement therapy to the lungs of patients with cystic fibrosis (CF).  In an intranasal single dose escalation phase I/IIa human clinical trial in CF subjects, there were no adverse events attributed to the nanoparticles and 8/12 subjects had partial to complete functional correction of the CFTR chloride channel.  In preparation for a lung aerosol trial in CF subjects, DNA nanoparticle aerosols have been shown to be stable and to effectively transfect the lungs of intubated rabbits.  Following a single dose to the lungs of mice, hCFTR mRNA expression is >100% of endogenous mCFTR and expression persists for at least 2 months.  In other studies that provide further proof of concept for the use of DNA nanoparticles, subretinal delivery of compacted DNA prevented blindness in a mouse model of retinitis pigmentosa.  In a rat model of Parkinson’s disease, compacted DNA encoding rat glial cell line-derived neurotrophic factor prevented development of motor symptoms.  In summary, these findings suggest that compacted DNA nanoparticles may provide safe and effective therapies for cystic fibrosis, retinitis pigmentosa, and Parkinson’s disease, and have potential for treating various other disorders of the lung, eye, and brain.

Immunolipopolyplex Nanoparticals of ODN and siRNA for Targeted Cancer Therapy
By
L. James Lee1,2,3 , Bo Yu2, Chee Guan Koh2, Yan Jin1, Xulang Zhang1, Xiaoguan Yang3, Yuan Yuan1, Xiofang Yue2, Michael Paulaitis2, , Raj Muthusamy4, Guido Marcucci4, John Byrd4 and Robert Lee1,3

1. Center for Affordable Nanoengineering of Polymeric Biomedical Devices,
2. Department of Chemical and Biomolecular Engineering,

(3) College of Pharmacy
(4) College of Medicine
The Ohio State University

The problem of efficient delivery of oligonucleotide compounds remains unsolved. Multi-functional nanoparticles, defined as nanoscale particulate entities designed for overcoming various barriers in drug delivery, can be regarded as a possible solution to this problem. We formulate both anti-sense oligonucleotide and siRNA into tumor-targeted lipopolyplexes with antibody and transferrin targeting. Both conventional mixing and a novel microfluidic hydrodynamic focusing methods are used for particle formation. Characterization and transfection efficiency are evaluated. Particle size and zeta-potential are also measured. Using both cell line and patient cells as a model, cell viability and gene transfection efficiency as well as protein down regulation are examined. In addition to in vitro study, cancer cells are planted into nude mice to grow solid tumor and lipoplexes are injected. Measurements of tumor size and mice survival rate, analysis of the protein down regulation and gene expression in tumor tissue by cell and molecular level, and pharmacokenatices study for nanoparticles are also carried out. We compare the efficiency and cytotoxicity of free oligonucleotide and its nanoparticles both in vitro and in vivo. It is found that properly formulated immunolipopolyplex nanoparticles can provide good specific targeting and transfection efficiency. Microfluidic focusing is able to produce more uniform nanoparticles and consequently better protein down-regulation.

Near-infrared Fluorescence of Single-Walled Carbon Nanotubes:
  a Tool for Developing Medical Applications

R. Bruce Weisman, Department of Chemistry
Rice University Houston, Texas

Single-walled carbon nanotubes (SWCNTs) are a family of artificial nanomaterials composed entirely of carbon atoms covalently bonded into highly ordered tubular structures. They typically have diameters near 1 nm and lengths of hundreds of nanometers. SWCNTs exhibit unique physical and chemical properties that hold promise for applications in medical diagnosis, drug delivery, and thermal ablation therapy. Although pristine SWCNTs are highly hydrophobic, they can be stably suspended in aqueous media when noncovalently coated by artificial surfactants, DNA, RNA, or proteins. Because of their well defined p-electron states, most SWCNT structures emit intrinsic fluorescence at specific near-IR wavelengths between 900 and 1600 nm when excited by visible light. The minimal autofluorescence in this emission region allows SWCNTs in biological environments to be detected and imaged with very high sensitivity and selectivity. Moreover, as fluorophores SWCNTs offer very large Stokes shifts, unsurpassed photostability, and an absence of blinking. Results will be described in which near-IR fluorescence methods have been used to monitor and track SWCNTs in cells, tissues, and organisms. In one project, macrophage cells were grown in the presence of SWCNTs. The resulting uptake of nanotubes was quantified by bulk fluorimetry and imaged by near-IR fluorescence microscopy. In an in vivo study, SWCNTs fed to Drosophila larvae were imaged in the living animals and in dissected tissues. In these tissue specimens it was possible to observe the location, orientation, and structural identities of individual nanotubes. Evidence for successful targeting of injected SWCNT/antibody conjugates to specific tissue structures in Drosophila larvae will also be presented. A mammalian in vivo study explored the pharmacokinetics of SWCNTs after intravenous injection into rabbits, using near-IR fluorescence to selectively monitor nanotube concentrations. A circulation half-life of 1.0 hours was determined, and examination of tissue specimens taken 24 h after exposure revealed significant SWCNT concentrations only in the liver. In a current project involving both in vitro and in vivo studies, SWCNTs are shown to be effective transfection agents for delivering siRNA into cancer cells and causing selective inhibition of gene expression. Future prospects for the use of SWCNT optical properties in biomedical application development will also be discussed.

Use of Nanoparticles for Biomedical Applications
Clemens Burda, Ph.D.

In this presentation, the interactions between inorganic nanoparticles and organic drug loads are discussed based on our work over the past few years. The drug loading, coupling, and transport on inorganic nanoparticles can be useful for in vitro and in vivo explorations. We have conducted a thorough study on these properties and most recent results will be presented.

The uniform magnetic field effect enables  targeting magnetic nanoparticles to stents--local delivery and cell therapy studies

Robert J. Levy, M.D., The Children’s Hospital of Philadelphia

ntravascular expandable metallic stents have now been used to treat tens of millions of patients with vaso-occlusive disease with generally beneficial results However, this novel and new therapy needs to evolve, since it is clear that current stenting strategies are not beneficial for all, and reobstruction of stented arteries, a process know as restenosis, occurs commonly and can even occur with drug eluting stents, especially in high risk subjects such as diabetics.  Furthermore, following implantation of a drug-eluting stent reendothelialization is impaired thereby leading to an increased risk of myocardial infarction and death.       We investigated the hypothesis that uniform magnetic fields, such as those that are required for MRI studies, are homogeneous and therefore can interact with ferromagnetic materials within the uniform field, such as a steel stent, by creating very high, localized gradients along the boundaries of the magnetizable object within the field.  In the case of a stent this involves very high gradients between each set of wire struts that can attract and bind magnetic nanoparticles (MNP), that are composed of biodegradable polylactic acid and mixed iron oxides.  Furthermore, cells, such as endothelial cells, can be  preloaded with MNP ex vivo by applying MNP with magnetic exposure in culture.   These MNP-loaded cells, if injected in proximity to a deployed stent that is in a uniform field, also become magnetized in the field and are also strongly attracted to stent wires, due to the high magnetic gradients generated by  the uniform field.  Thus, we have demonstrated magnetic targeting to stents of either MNP or endothelial cells loaded with MNP both in vitro and in vivo .  This talk will present the results of these studies, and will examine the physical and biologic mechanisms involved in this novel magnetic targeting approach investigating nanoparticles for local delivery

Nanoscale MRI Contrast Agents for Molecular Imaging
Marty Pagel

Molecular imaging may diagnose pathologies at molecular levels. Magnetic Resonance Imaging (MRI) can use a contrast agent to detect a molecular composition of interest, while also providing exquisite images of soft tissue anatomies. However, MR image contrast is often changed by other physiological characteristics in addition to the molecular composition of interest, such as tissue pharmacokinetics, pH, temperature, metal ions, and other proteins and metabolites. Therefore, the absolute change in image contrast produced by a single agent can be difficult to interpret. In order to translate molecular imaging with MRI to pre-clinical and clinical studies, a relative change in image contrasts caused by two MRI contrast agents must be measured, in which two agents are equally responsive to all other physiological characteristics, but only one agent is responsive to the molecular composition of interest. Yet to translate this approach to pre-clinical and clinical studies, these two MRI contrast agents must be linked, and must be present at high concentrations for adequate detection. Nanoparticles that carry a high payload of both MRI contrast agents provide excellent solutions to these challenges. In particular, nanoscale dendrimers possess excellent characteristics for the delivery of MRI contrast agents for molecular imaging. Dendrimers have also been used as drug delivery nanocarriers, which provide opportunities to combine therapy with diagnostics. Examples of our research with nanoscale MRI contrast agents for molecular imaging will be used to highlight these new opportunities in nanomedicine.

Evading Clearance to enhance Nano-Delivery:
Surprises in Nanoscale Shape and Exploitation of a 'Marker of Self' ligand

Dennis Discher
University of Pennsylvania

Shape effects of drug delivery vehicles are largely unexplored, especially in vivo, but we have recently shown that flexible cylinders circulate in vivo longer than spheres of identical composition (1). Flexible filomicelles increase both dosage and tumor-selective effects in vivo relative to spheres and thus appear promising as anticancer drug delivery systems. However, any particle injected or surface implanted in us or any other mammal must contend with Macrophages that have – for eons – swept up invading bacteria, yeast, viruses, and other pathogens. At the same time, Macrophages leave our own ‘Self’ cells alone. The Foreign vs Self difference certainly does NOT reside in steric repulsion by the glycocalyx, which some have argued is well-mimicked by the modern polymer PEG. We have sought to address how Macrophages specifically recognize Self and to define its physicochemical limits, and we have focused on the cell-surface protein CD47 found on all of our own cells. We demonstrate a two-step procedure for recognizing intruders that helps avoid misdirected attacks. In the first step, which is well known, Macrophages adhere and begin engulfing objects studded with antibodies or plasma complement proteins that bind interlopers and will also bind to the body’s own cells. But before a macrophage engulfs its target, it also checks for the second form of identification on all self cells, CD47. The ~100 aa extracellular domain of CD47 proves sufficient to induce a macrophage to disengage a cell from the same species or even a synthetic particle decorated with this domain (2). We detail the divergence in Foreign vs Self adhesive signaling mechanisms, the dependence on protein densities, and the particle size dependence for this fundamental facet of immunocompatability. REFERENCES: (1) Y. Geng, P. Dalhaimer, S. Cai, R. Tsai, M. Tewari, T. Minko, and D.E. Discher. 2007. Shape effects of filaments versus spherical particles in flow and drug delivery. Nature Nanotechnology 2: 249-255. (2) R. Tsai and D.E. Discher. 2008. Inhibition of ‘Self’ Engulfment through deactivation of Myosin-II at the Phagocytic Synapse between Human Cells Journal of Cell Biology 180: 989-1003.

Nanoemulsions for molecular imaging and targeted therapeutics

Molecularly targeted perfluorocarbon nanoparticles exhibit unique interactions with cells as therapeutic agents as compared with other types of nanosystems. Upon ligation of specific receptors, hemifusion complexes are formed with the cellular membrane and drugs are delivered through lipid flow into the lipid bilayer and carried into the cell on lipid rafts, followed by trafficking to cytoplasmic compartments while avoiding endosomal sequestration. Examples of the unique carrier capacity of these agents will be discussed, such as their role in transporting the class of cytolytic “host defense” peptides that otherwise would destroy conventional carriers such as liposomes by pore formation.. Use of these short amphiphilic peptides to induce apoptosis in cancer will be presented.

Nanobubble-Nanoparticle Complexes for Imaging and Cancer Treatment

Agata Exner
Departments of Radiology and Biomedical Engineering
Case Western Reserve University

Of the many approaches to the treatment of cancer, only a minority can begin to address the complex nature of the disease. Targeted multifunctional nanoparticles have great potential for maximizing treatment directly at the site of the tumor and minimizing systemic side effects. Design criteria for an effective nanocarrier are: 1) prolonged circulation time, 2) imaging contrast, 3) targeting, and 4) responsiveness to physical or chemical stimuli to modulate drug release. This talk will discuss a new platform nanoparticle delivery system that meets these design criteria and is currently under development at Case Western Reserve University. The technology consists of a multifunctional nanobubble-nanoparticle composite (NNC) vehicle where a removable bubble ‘cloak’ is used to conceal a targeted, drug-loaded nanoparticle. The NNC is both a drug carrier and a contrast agent and possesses on-demand functionality modulated by ultrasound. This stimulus permits on-demand reduction of particle size to that suitable for extravasation, uncovering of concealed targeting ligands at the site of action, and sonoporation to improve cell uptake of the drug. The design, formulation and implementation of the NNCs will be discussed in relation to two applications: differentiation of inflammation and residual tumor in imaging follow-up of tumor ablation and specific targeting of drug-loaded particles for treatment of colorectal cancer.