DCCNE Developmental Activities
Developmental activities arising within the Dartmouth Center of Cancer Nanotechnology Excellence will be designed to:
- Foster research in cancer nanotechnology
- Facilitate interactions of oncologists at the Norris Cotton Cancer Center (NCCC) with engineers and materials scientists at Thayer School of Engineering
- Facilitate efforts by DCCNE investigators to propose studies and collaborate on projects that are made feasible by the composite capabilities of the institutions participating in the NCI Alliance for Nanotechnology in Cancer
- Alliance Challenge Projects
Dartmouth demonstrated its commitment to foster research in cancer nanotechnology by investing funds to support the initial work of the Cancer Nanotechnology Working Group in 2006. This support facilitated interactions of oncologists at NCCC with engineers and materials scientists at Thayer School of Engineering to initiate collaborations that would allow cancer researchers to apply various nanotechnologies to solve specific problems in cancer. In addition to funding symposia, retreats, and interest group meetings, NCCC initiated a pilot award program in cancer nanotechnology and awarded 11 peer-reviewed grants.
In 2009, 4 additional pilot projects were funded in response to an NCCC/Thayer School request for applications to support innovative cancer-related nanotechnology pilot projects. The purpose of these awards is to stimulate interactive projects that are likely to become self-sustaining through external funding mechanisms or contribute new insights to the Cancer Nanotechnology Working Group (CNWG) efforts.
The DCCNE continues to fund new pilot projects on an annual basis. A call for proposals is issued each Spring, with an application deadline of June 1st for annual projects proposed to start August 1st. All Dartmouth faculty are eligible to propose new work related to current DCCNE studies. Questions should be directed to the Administrative Program Manager, Robert Gerlach.
2012 Pilot Projects
Quantifying Surface Temperature of Activated Magnetic Nanoparticles: Probing Thermal Effects as a Prospective Therapeutic Mechanism
PI: Karl E. Griswold
Read abstract »In the absence of global heating, the exact mechanism by which remotely-activated magnetic nanoparticles kill target cells remains poorly understood. Neither intensified research efforts nor access to progressively more advanced technologies has clarified this issue. Possible modes of action include but are not limited to localized mechanical disruption of membranes via particle motion, shear-induced protein unfolding and/or DNA damage resulting from particle rotation, and most prominently, highly localized heating derived from particle mediated energy deposition. In some camps, the latter hyperthermia mechanism is now accepted as a given, whereas heat transfer modeling suggest this concept is a nonstarter. Here, we propose to use spatially confined reaction kinetics to quantify temperature at the nanoscale surface of activated particles. In our opinion, this key parameter has yet to be determined accurately. Well-behaved thermal initiators from the polymer industry will be leveraged as thermolabile linkers between fluorescent reporter molecules and magnetic nanoparticles. The modified particles will be exposed to alternating magnetic fields of varying strength, and the corresponding rate of fluorophore release will be determined spectroscopically. For each experiment, the temperature at the nanoparticle surface will be determined from Arrhenius plots of kinetic runs in which particles are subjected to controlled bulk phase heating. We anticipate that the proposed studies will yield accurate estimates of local temperatures at the nanoparticle surface, and will represent a key milestone in DCCNE efforts to confirm or disprove the hyperthermia mechanism for magnetic nanoparticle-based cancer therapies, a question of particular interest to both Dartmouth and the NCI.
High-Efficiency AMF Coil Designs with Reduced Power and Cooling Requirements for Magnetic Hyperthermia
PI: Charles R. Sullivan
Read abstract »Clinical applications of magnetic hyperthermia require applying a strong alternating magnetic field (AMF) at high frequency to the region to be treated. The equipment needed to create this field comprises a high-frequency inverter (sometimes called a generator or power supply), a coil, and cooling apparatus for the coil. Present designs, based on industrial induction heating equipment, can perform the necessary function, but are difficult to use and are unattractive for clinical applications. They require power levels as high as 25 kW or more, resulting in a large, heavy, expensive inverter constrained to a lab with special power service, and a large chiller for cooling. We propose to develop new coil technology that can produce the same field strength and distribution as the apparatus used now, but with an order of magnitude lower power loss. This will allow the use of a much smaller portable inverter and a correspondingly smaller cooling system, perhaps even allowing air cooling. To do this, we will take a new approach, taking advantage of the differences between this application and induction heating while also addressing the unique challenges that are not as difficult in industrial induction heating.
2011 Pilot Projects
Magnetic Nanoparticle Induced Hyperthermia for Enzyme-Prodrug Therapy
PI: Barjor Gimi
Read abstract »
Cells can be engineered to express specific enzymes for enzyme-prodrug therapy, and then grafted for therapy. Such enzyme-prodrug therapy facilitates the localized synthesis of chemotherapeutic drugs and can therefore overcome systemic toxicity. The encapsulation of these bioengineered cells in immunoprotective matrices enhances their survival post grafting. Initiating enzyme expression in the graft by external factors such as thermal stress or irradiation can help temporally coordinate prodrug therapy with other therapy such as radiation, thereby amplifying therapeutic efficacy over monotherapy. We propose to engineer E. coli designed to express the enzyme cytosine deaminase under the influence of a heat induced promoter and encapsulate these cells in magnetic nanoparticle (MNP) embedded alginate microbeads through surface modification of nanoparticles that will be covalently bound to alginate. We will irradiate the microbeads with radio frequency to induce hyperthermia and trigger enzyme expression, and then administer the non-toxic prodrug 5-FC. Enzyme activity of the encapsulated E. coli will be characterized by monitoring the conversion of 5-FC to the toxic drug 5-FU.
As a first step towards the development of engineered E. coli cells capable of expressing the enzyme cytosine deaminase (CD) under the transcriptional control of a heat induced promoter, we have designed a thermally inducible expression vector. The cloned plasmid was then transformed into BL21 competent cells to yield the desired bacterial cell line capable of heat induced CD expression. Transplantation of these encapsulated engineered E. coli at the tumor location should reduce systemic toxicity and overcome the limitation of the drug's short half-life; the encapsulated cells can be resident at the tumor location to serve as a watchdog against recurrence. A successful outcome of this project will provide a cell based strategy to treat cancer using hyperthermia, particularly as a temporally coordinated adjuvant to radiation therapy.
Modulation Of Hypoxia To Enhance Nanoparticle Uptake And Tumor pO2-Guided Radiotherapy With Magnetic Hyperthermia
Eunice Y. Chen and Nadeem Khan
Read abstract »
- Aim 1: Determine the effect of oxygen environment (hypoxia and hyperoxia) on nanoparticle uptake
- Aim 2: Assess tumor pO2 variation with nanoparticle hyperthermia treatment and determine therapeutic outcome when the temporal changes in tumor pO2 are used to schedule radiotherapy
Study Design/Methods: Breast and head and neck cancer cell lines were used in vitro and in vivo as xenograft tumors in nude mice.
Results: Decreased uptake of iron oxide nanoparticles by breast and head and neck cancer cells was found under hypoxic and hyperoxic in vitro conditions. Under hypoxia, nanoparticle uptake, as quantified by inductively coupled plasma mass spectrometry, was decreased by a factor of 11 in Fadu cells and 3.6 in SCC cells. In an orthotopic xenograft mouse model, MDA-MB 231 tumors were hypoxic (< 5 mm Hg) when they reached a volume of 200 mm3. Nanoparticle hyperthermia treatment resulted in an immediate increase in tumor pO2, which was sustained for greater than 1 week. Tumors treated with nanoparticle hyperthermia grew at a slower rate than the untreated tumors.
Summary: Hypoxia decreased nanoparticle uptake in breast and head and neck cells in vitro. The mechanism is unknown. In hypoxic tumors in vivo, nanoparticle hyperthermia effectively increased tumor oxygenation immediately and for a sustained period of time, which may be used to improve the effectiveness of adjuvant radiotherapy.
2010 Pilot Projects
SQUID Susceptometry Imaging of Magnetic Nanoparticles
PI: Solomon G. Diamond
Project update (PDF)
Read abstract »Novel techniques are needed to noninvasively image magnetic nanoparticles in support of research on magnetic hyperthermia treatment of tumors. Superconducting quantum interference devices (SQUIDs) offer extremely high sensitivity to magnetic signals. SQUIDs have been used previously to measure the bulk magnetic susceptibility of superparamagnetic nanoparticles. We propose to develop a novel nanoparticle imaging system using SQUID susceptometry together with tomographic image reconstruction. For these experiments, an existing SQUID system that was originally designed for magnetoencephalography (MEG) imaging will be converted to perform susceptometry imaging. The MEG system has an array of 37 SQUID gradiometers that are arranged in concentric rings covering a 27 cm diameter downward-facing concave dish with 5 cm depth. The MEG system was designed for positioning over any desired region of the human head but could be easily positioned over other body parts such as the abdomen, breast, or neck. Three orthogonal Helmholtz coils will be designed and installed to supply an external magnetic field in the region of interest. Energizing each of the 6 coils in turn maximizes the rank of the forward model at 6 x 37 = 222 effective measurements for tomographic reconstruction. Based on simulations of the field distortions due to the presence of superparamagnetic nanoparticles and 3D tomography calculations, we anticipate spatial resolution of approximately 1 cm up to a depth of 5 cm. Quantification of the nanoparticle concentration within the field of view is possible within about 10 percent error. These imaging performance measures will be experimentally tested in this project.
Novel NK Cell Receptor Targeting of Nanoparticles Against Tumor Cells
Ming-Ru Wu, Tong Zhang, Charles L. Sentman
Read abstract »
NKG2D is a NK cell receptor that recognizes ligands expressed on the majority of human tumors and local immunosuppressive cells, but these ligands are not expressed on normal cells under most conditions. Thus, NKG2D is a potential means to target tumor cells and the tumor microenvironment. The aim of this pilot project was to produce NKG2D-coated nanoparticles and test the ability of these to specifically recognize ligand expressing tumor cells in vitro.
We have produced specific constructs of extracellular NKG2D to attach the protein in proper orientation on FeOxide nanoparticles, but it has been challenging to produce sufficient amounts of protein for large scale production of NKG2D-coated nanoparticles that are required for in vivo testing. In order to more easily test the specificity of such nanoparticles to target ligand+ tumor cells and not nearby ligand- cells, we used a soluble NKG2D-Fc reagent to attach NKG2D to nanoparticles consisting of a FeOx core surrounded by a coat of BNF-starch and Protein A. Control nanoparticles included IgG and uncoated nanoparticles in all experiments. Using mixtures of ligand+ tumor cells with ligand- tumor cells or normal mouse spleen cells, we have been able to demonstrate specific enrichment of ligand+ tumor cells that is dependent upon the amount of NKG2D coated on the particles and the expression of ligand on the tumor cells. Enrichment of ligand+ tumor cells was effective even when the ligand+ cells were only a small percentage of the total cells. A small number of normal spleen cells were also isolated (<2%) with these nanoparticles. This was not specific for NKG2D as similar numbers and cell types were bound to the control IgG-nanoparticles. We are currently examining in vivo localization and potential efficacy against tumor cells.
Alliance Challenge Projects
The DCCNE looks forward to the opportunity to propose studies and collaborate on projects that are made feasible by the composite capabilities of the institutions participating in The Alliance. We anticipate that the DCCNE can contribute to The Alliance's unique strengths in biomedical engineering and physical sciences (from Thayer School faculty) and in biological and medical sciences (from The Geisel School of Medicine at Dartmouth faculty). Our experience in fostering pilot projects supports our ability to pursue preliminary results at this site that establish the rationale for, and feasibility of, larger multi-institutional studies built upon the outcomes of such initial local studies.
Our approach to the opportunity for Alliance Challenge Projects is three-fold:
- The DCCNE Steering Committee will intentionally include in the discussion of each active and each pilot project whether significant ancillary and/or correlative studies would become feasible with participation of other Alliance Centers. The most promising concepts would be developed for presentation at Alliance forums for further consideration.
- In anticipation that other members of The Alliance may perceive Alliance-based opportunities in Dartmouth-based projects, we will circulate widely reports of work underway at the DCCNE.
- The DCCNE Director will present to the DCCNE Steering Committee the opportunities for Dartmouth faculty to join as a performance site on Alliance Challenge Projects proposed by other Alliance Centers.
The DCCNE continues to fund new Alliance Challenge Projects every 18 months. The next call for proposals is anticipated in Spring 2013 for projects proposed to start August 1, 2013. All current DCCNE participants are eligible to propose new work related to current DCCNE studies. Questions should be directed to the Administrative Program Manager, Robert Gerlach.
Current Alliance Challenge Projects
Rationally Designed Theranostic Nanoconstructs For MR Imaging And Triggered Drug Release"
Paolo Decuzzi, The Methodist Hospital Research Institute; Brian Rutt, Stanford School of Medicine; Jack Hoopes, DVM, Dartmouth College; Andrew Dannenberg, Weill Cornell Medical College
Read abstract »
Mesoporous silicon particles can be designed rationally to lodge preferentially within the tumor microvasculature and can carry simultaneously multiple agents, providing both an imaging and a therapeutic capability. Here, we propose to develop theranostic nanoconstructs (TNCs) obtained by loading Gd-chelates (Gd-DOTA), Super Paramagnetic Iron Oxide nanoparticles (SPIOs), and nanogel-loaded drugs (NG) into mesoporous silicon particles. Discoidal particles, with a diameter of 1000 nm and thickness of 400 nm, have been shown to accumulate preferentially in two different orthotopic tumor models (melanoma and breast tumor) as compared to other particle sizes and shapes. The Gd-DOTA molecules will be conjugated on the surface of the mesoporous silicon particles to provide T1 MRI contrast enhancement; ~10 nm SPIOs will provide a T2 MRI contrast enhancement and, most importantly, will heat up under exogenous, non-invasive alternating magnetic field; ~30 nm NGs will carry curcumin molecules for tumor therapy. Both the SPIOs and the NGs will be loaded simultaneously , via capillary suction, into the ~40 nm pores of the silicon particles. Alternating magnetic fields, in the hundreds of kHz regime, will be used to heat up the SPIOs and induce the release of the NGs from the mesoporous silicon particles. The resulting nanoconstructs will be tested in vitro and in vivo using mouse models for breast tumor. Dr. Decuzzi (at TCCN) will be developing the nanoconstructs and run preliminary in vitro tests; Dr. Rutt (at CCNE-T) will characterize in vitro and in vivo the MRI properties of the nanoconstructs; and Dr. Hoopes (at D-CCNE) will test the therapeutic efficacy in mice. Dr. Dannenberg will provide his experience on breast tumor models and treatment with curcumin.
SWIFT MR Imaging for High Concentration Magnetic Nanoparticles
P. Jack Hoopes, DVM and Barjor Gimi, Dartmouth College; John Bischof, Michael Garwood, and Ryan Chamberlain, University of Minnesota
Read abstract »
The ability to image accurately the location and distribution of magnetic nanoparticles (mNP) is an essential step towards clinical application of mNP hyperthermia (mNPHT). However, the standard MR imaging techniques are unable to visualize concentrations greater than 1mg Fe/g tissue. Preliminary data suggest that a new technique from the Center for Magnetic Resonance Research (CMRR) at the University of Minnesota, entitled "Sweep Imaging with Fourier Transformation" (SWIFT), can supply this critical technology. Here we propose to examine the effects of mNP concentration, aggregation, and cellular and tissue uptake with SWIFT imaging for clinical translation.
Development Of A High HER2-Expressing Human Breast Cancer Xenograft Mouse Model For Dartmouth CCNE Projects 1 and 3
Lionel Lewis and P. Jack Hoopes, Dartmouth College; Dr. T. Ward, University of Miami Miller School of Medicine
Read abstract »
The ability to target cancer cell surface receptors using antibody-conjugated nanoparticles remains central to the success of nanoparticle-based cancer treatment. Projects 1 and 3 of the Dartmouth CCNE proposed to test such constructs in a mouse model to determine if targeting iron oxide nanoparticles to antigen-positive tumors results in increased particle deposition in tumor tissue. A HER2+ human breast cancer xenograft model is vital to achieve these goals as put forth by CCNE Projects 1 (PI: Gerngross) and 3 (PI: Hoopes). However, development and implementation of the BT-474 xenograft model deemed most appropriate revealed unforeseen complications due to poor take rate in the mice and serious estrogen supplementation-associated health problems [1, 2]. Project 3 has spent significant time and resources attempting to establish the published model with little success due to aforementioned problems. Dr. Toby Ward's team at the University of Miami successfully grows HER2+ luciferase-transfected BT-474 tumors without estrogen supplementation in NOD SCID IL2-/- mice using a modified implantation protocol. Dr. Ward's group reports a 100% take rate in challenged mice and measurable tumors in all mice at 30 days. We propose to establish this model at our facility with Dr. Ward's expertise, as well as co-develop with Dr. Ward's group an additional estrogen-free luciferase-transfected MCF7 xenograft (which would also otherwise require estrogen supplementation) for use as a control model in the experiments proposed by Projects 1 and 3. Without Dr. Ward's knowledge and participation, we would not be able to support successfully development of these critically important models.
- Justine A. Levin-Allerhand, Karen Sokol, and Jonathan D. Smith; Contemporary Topics by the American Association for Laboratory Animal Science; November 2003 42:6.
- Gail Pearse, Jeremy Frith, Kevin John Randall and Teresa Klinowska; Toxicol. Pathol. 2009 37:227.
Action of Magnetic Particles on Cells in Oscillating Magnetic Field
Eugene Demidenko, Fridon Shubitidze, and P. Jack Hoopes, Dartmouth College; Igor Sokolov, Clarkson University
Read abstract »
The use of super paramagnetic nanoparticles has shown its success for treatment of cancers in animal models. Oscillating magnetic field transfers its energy to the particles located inside tumors. This results in the tumor distraction. The mechanism of distraction of cancer cells by the action of super paramagnetic nanoparticles in the oscillating magnetic field is not clear. There are a number of open questions: Is this a pure mechanical action, heating, or both? How are the cells destroyed? To address these questions related to understanding the cell distraction mechanism, we propose:
- To make the fluorescent particles capable of measuring the temperature around superparamagnetic nanoparticles.
- To use the particles developed in the previous task to do proof of concept experiments investigating the most probable mechanism of destruction of individual cells.
Iron/Iron Oxide Nanoparticles for Magnetoresistive Microarray Labeling
Ian Baker, Dartmouth College and Michael Granger, University of Utah
Read abstract »
This project will utilize Dartmouth's Fe/Fe3O4 core/shell magnetic nanoparticles (MNPs) as immunometric assay labels for detection of low-level cancer markers using the University of Utah's magnetoresistive microarray (MRmArray) platform. The potential utility of MRmArray platforms for detection and discovery in the clinical arena hinges on the use of colloidally stable, monodisperse nanoparticle labels with high magnetic moment and low magnetic remanence. We believe using MNPs with a moderate mass magnetic moment, s, will provide an opportunity to reach the instrumental figures of merit (i.e., limit of detection, dynamic range, and analytical sensitivity) required of an MRmArray to function effectively as an in vitro diagnostic (IVD) platform for cancer biomarker detection and monitoring. The work will consist of:
- Producing a range of MNPs (Dartmouth College)
- Through magnetoresistive readout of MNPs, ascertain optimal synthesis parameters from the MNPs and compare these data to that of the superparamagnetic iron oxidenanoparticles (SPIOs) available from MicroMod GmbH, Ocean NanoTech, and Miltenyi Biotec. (University of Utah); determine magnetic, colloidal, and biological temporal stability of the bio-functionalized optimal MNPs (Dartmouth College and University of Utah)
- Synthesize 1 g of monodisperse, streptavidin-functionalized MNPs following any required optimization of magnetic character and surface chemistry (Dartmouth College)
- Using MMP7-spiked sera, compare and contrast the MNPs with the SPIOs available from MicroMod GmbH, Ocean NanoTech, and Miltenyi Biotec. (University of Utah)
Specific Drug Release from Mesoporous Silica Supported Lipid Bilayer Nanoparticles via Alternating Magnetic Fields
John B. Weaver, Ph.D., Dartmouth College; C. Jeffrey Brinker, Ph.D. and C. Willman, M.D., University of New Mexico
Read abstract »The University of New Mexico CNPP has developed a unique, promising drug delivery nanoparticle termed a "protocell". The protocell, a porous silica supported lipid bilayer NP, possesses a number of advantages over traditional lipid bilayer nanoparticles (i.e., liposomes) and free silica nanoparticles in vitro: increased cargo capacity, stability, target binding, and specificity. However, in vivo biodistribution and target specificity are currently unknown. In order to quantify biodistribution and develop a novel method of improving specificity, collaboration with the Dartmouth CCNE is proposed. In vitro, protocell specificity results primarily from peptides, conjugated to the lipid bilayer that are responsible for target cell binding, and internalization of the particle via receptor-mediated endocytosis. In vivo, protocells will be distributed passively to multiple organs following intravenous injection. The in vivo distribution will be studied. The structure of the protocell may provide an opportunity for increased specificity when combined with magnetic nanoparticle excitation via an externally applied alternating magnetic field (AMF). It is expected that the energy supplied to the magnetic protocell nanoparticle via the AMF will rupture the supported lipid bilayer, promoting release of the adsorbed drug cargo. Further, the energy deposited is a function of frequency and bound state, so we will find frequencies where bound protocells rupture and free protocells remain intact. The structure of the protocell will be modified to optimize AMF-induced rupture. The ability to rupture specifically the lipid bilayer of only particles bound to their cellular target will be investigated.
Vision for Other Trans-Alliance Activities
We anticipate that The Alliance could undertake consortia projects, such as development of:
- A directory of cancer nanotechnology expertise
- A database of active cancer nanotechnology projects and/or specialized resources
- A website for communication of developments in the cancer nanotechnology field
- National symposia to provide continuing education in cancer nanotechnology
Interface with NCI Nanotechnology Characterization Laboratory (NCL) and caNanoLab
Dartmouth's history of participating with NCI-wide initiatives, such as reliance on regional and national biorepositories, illustrates recognition of the justification for utilization of national reference laboratories that can assure a range and standardization of assays that exceed the capabilities of a single center. NCCC's well established Office of Clinical Research coordinates for investigators the preparation and shipping of specimens to national reference laboratories. NCCC's experience in offering this referral service confirms the feasibility of the DCCNE maintaining the interface necessary to rely upon collaborations with national resources such as the NCI Nanotechnology Characterization Laboratory and caNanoLab.