New York Academy of Sciences, the Mushett Family Foundation, and the Nanotechnology Center at Memorial Sloan-Kettering Cancer Center
Nanotechnologies in Cancer Diagnosis, Therapy, and Prevention
Posted September 04, 2013
On June 11–13, 2013, researchers converged at the Memorial Sloan-Kettering Cancer Center in New York City for a conference on Nanotechnologies in Cancer Diagnosis, Treatment, and Prevention, presented by the New York Academy of Sciences, the Mushett Family Foundation, and the Nanotechnology Center at Memorial Sloan-Kettering Cancer Center. Most of the presentations and posters focused on the promise of nanoparticles that home in on primary or metastatic tumors, either to highlight the tumors in images or to deliver drugs. A wide variety of nanoparticle systems are being explored, including gold and magnetic nanoparticles, biodegradable polymers, liposomes, and micelles.
The conference included much discussion of mechanisms for targeting particles to specific tissues. In some cases nanoparticles selectively enter cancer tissue because of its unusual leaky blood vessels. But this passive targeting may not be effective for all human tumors, and many researchers go further by attaching ligands to nanoparticles that bind to receptors that are overexpressed in tumor cells, which can also induce the cells to internalize the nanoparticle. However, this active targeting may not work as planned in real bodily fluids that cover the particles in a corona of proteins.
There is much to be learned about how nanoparticles behave in the body, including the risk (or, for vaccines, the promise) of stimulating immune responses. Nonetheless, many of the nanoparticle systems discussed are being evaluated in clinical trials for either diagnostics or therapy. The ability to separately optimize the delivery vehicle from the payload, or to deliver multiple payloads to the same place, shows great promise for new strategies for treating cancer.
Use the tabs above to find a meeting report and multimedia from this event.
Presentations available from:
Gregory I. Berk, MD (BIND Therapeutics)
Michelle S. Bradbury, MD, PhD (Memorial Sloan-Kettering Cancer Center)
Charles Patrick Case, PhD (University of Bristol, UK)
Kenneth Dawson, MSc, PhD (University of Dublin, Ireland)
Marina A. Dobrovolskaia, PhD (Nanotechnology Characterization Lab SAIC–Frederick Inc.)
James R. Heath, PhD (California Institute of Technology)
Alexander V. Kabanov, PhD, DrSc (University of North Carolina at Chapel Hill)
Sylvain Martel, PhD (Polytechnique Montreal, Canada)
Michael R. McDevitt, PhD (Memorial Sloan-Kettering Cancer Center)
Moein Moghimi, PhD (University of Copenhagen, Denmark)
Jay Nadeau, PhD (McGill University, Canada)
Shuming Nie, PhD (Emory University)
Aliasger Salem, PhD (The University of Iowa)
Yi Yan Yang, PhD (Institute of Bioengineering and Nanotechnology, Singapore)
- 00:011. Introduction: ultrasmall hybrid silica technologies
- 05:542. C dots: fluorescent silica nanoparticles for nanomedicine
- 10:203. Intracellular tracking of c dots
- 14:424. First-in-human clinical trials
- 22:365. SLN mapping
- 27:026. Optical examples
- 33:177. Conclusions: modular approach to multifunctional particles; Ongoing development
Journal Articles, Websites, and Trials
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Enhanced permeability and retention
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Chan JM, Rhee JW, Drum CL, et al. In vivo prevention of arterial restenosis with paclitaxel-encapsulated targeted lipid-polymeric nanoparticles. Proc Natl Acad Sci U S A. 2011;108(48):19347-52.
Gu M, Vegas AJ, Anderson DG, et al. Combinatorial synthesis with high throughput discovery of protein-resistant membrane surfaces. Biomaterials. 2013;34(26):6133-8.
von Hoff DD, Mita M, Eisenberg P, et al. A phase I study of BIND-014, a PSMA-targeted nanoparticle containing docetaxel, in patients with refractory solid tumors. Presented at the American Association for Cancer Research Annual Meeting. 2013.
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Mieszawska AJ, Gianella A, Cormode DP, et al. Engineering of lipid-coated PLGA nanoparticles with a tunable payload of diagnostically active nanocrystals for medical imaging. Chem Commun (Camb). 2012;48(47):5835-7.
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Valencia PM, Hanewich-Hollatz MH, Gao W, et al. Effects of ligands with different water solubilities on self-assembly and properties of targeted nanoparticles. Biomaterials. 2011;32(26):6226-33.
Xiao Z, Levy-Nissenbaum E, Alexis F, et al. Engineering of targeted nanoparticles for cancer therapy using internalizing aptamers isolated by cell-uptake selection. ACS Nano. 2012;6(1):696-704.
Alidori S, Asqiriba K, Londero P, et al. Deploying RNA and DNA with functionalized carbon nanotubes. J Phys Chem C Nanomater Interfaces. 2013;117(11):5982-5992.
Goldberg MS, Hook SS, Wang AZ, et al. Biotargeted nanomedicines for cancer: six tenets before you begin. Nanomedicine (Lond). 2013;8(2):299-308.
Ruggiero A, Villa CH, Bander E, et al. Paradoxical glomerular filtration of carbon nanotubes. Proc Natl Acad Sci U S A. 2010;107(27):12369-74.
Shen H, Rodriguez-Aguayo C, Xu R, et al. Enhancing chemotherapy response with sustained EphA2 silencing using multistage vector delivery. Clin Cancer Res. 2013;19(7):1806-15.
Shi J, Xiao Z, Votruba AR, et al. Differentially charged hollow core/shell lipid-polymer-lipid hybrid nanoparticles for small interfering RNA delivery. Angew Chem Int Ed Engl. 2011;50(31):7027-31.
Villa CH, McDevitt MR, Escorcia FE, et al. Synthesis and biodistribution of oligonucleotide-functionalized, tumor-targetable carbon nanotubes. Nano Lett. 2008;8(12):4221-8.
Dunn SS, Byrne JD, Perry JL, et al. Generating better medicines for cancer. ACS Macro Lett. 2013;2(5):393-397.
Hammond PT. Polyelectrolyte multilayered nanoparticles: using nanolayers for controlled and targeted systemic release. Nanomedicine (Lond). 2012;7(5):619-22.
Kabanov AV. Polymer genomics: an insight into pharmacology and toxicology of nanomedicines. Adv Drug Deliv Rev. 2006;58(15):1597-621.
Klyachko NL, Sokolsky-Papkov M, Pothayee N, et al. Changing the enzyme reaction rate in magnetic nanosuspensions by a non-heating magnetic field. Angew Chem Int Ed Engl. 2012;51(48):12016-9.
Luxenhofer R, Han Y, Schulz A, et al. Poly(2-oxazoline)s as polymer therapeutics. Macromol Rapid Commun. 2012;33(19):1613-31.
Luxenhofer R, Schulz A, Roques C, et al. Doubly amphiphilic poly(2-oxazoline)s as high-capacity delivery systems for hydrophobic drugs. Biomaterials. 2010;31(18):4972-9.
Poon Z, Chang D, Zhao X, Hammond PT. Layer-by-layer nanoparticles with a pH-sheddable layer for in vivo targeting of tumor hypoxia. ACS Nano. 2011;5(6):4284-92.
Stephen W. Morton, Kevin P, et al. Scalable manufacture of built-to-order nanomedicine: spray-assisted layer-by-layer functionalization of PRINT nanoparticles. Adv Materials. 2013[Epub ahead of print]
Zhang X, Chibli H, Mielke R, Nadeau J. Ultrasmall gold-doxorubicin conjugates rapidly kill apoptosis-resistant cancer cells. Bioconjug Chem. 2011;22(2):235-43.
Zhao Y, Alakhova DY, Kim JO, et al. A simple way to enhance Doxil® therapy: Drug release from liposomes at the tumor site by amphiphilic block copolymer. J Control Release. 2013;168(1):61-9.
Andersen AJ, Robinson JT, Dai H, et al. Single-walled carbon nanotube surface control of complement recognition and activation. ACS Nano. 2013;7(2):1108-19.
Andersen AJ, Windschiegl B, Ilbasmis-Tamer S, et al. Complement activation by PEG-functionalized multi-walled carbon nanotubes is independent of PEG molecular mass and surface density. Nanomedicine. 2013;9(4):469-73.
Borchard G, Flühmann B, Mühlebach S. Nanoparticle iron medicinal products: Requirements for approval of intended copies of non-biological complex drugs (NBCD) and the importance of clinical comparative studies. Regul Toxicol Pharmacol. 2012;64(2):324-8.
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Dobrovolskaia MA, McNeil SE. Understanding the correlation between in vitro and in vivo immunotoxicity tests for nanomedicines. J Control Release. 2013. [Epub ahead of print]
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Hamad I, Al-Hanbali O, Hunter AC, et al. Distinct polymer architecture mediates switching of complement activation pathways at the nanosphere-serum interface: implications for stealth nanoparticle engineering. ACS Nano. 2010;4(11):6629-38.
Moghimi SM, Andersen AJ, Ahmadvand D, et al. Material properties in complement activation. Adv Drug Deliv Rev. 2011;63(12):1000-7.
Raghunathan VK, Devey M, Hawkins S, et al. Influence of particle size and reactive oxygen species on cobalt chrome nanoparticle-mediated genotoxicity. Biomaterials. 2013;34(14):3559-70.
Schellekens H, Klinger E, Mühlebach S, et al. The therapeutic equivalence of complex drugs. Regul Toxicol Pharmacol. 2011;59(1):176-83.
Sood A, Salih S, Roh D, et al. Signalling of DNA damage and cytokines across cell barriers exposed to nanoparticles depends on barrier thickness. Nat Nanotechnol. 2011;6(12):824-33.
Joshi VB, Geary SM, Salem AK. Biodegradable particles as vaccine delivery systems: size matters. AAPS J. 2013;15(1):85-94.
Krishnamachari Y, Geary SM, Lemke CD, Salem AK. Nanoparticle delivery systems in cancer vaccines. Pharm Res. 2011;28(2):215-36.
Lemke CD, Geary SM, Joshi VB, Salem AK. Antigen-coated poly α-hydroxy acid based microparticles for heterologous prime-boost adenovirus based vaccinations. Biomaterials. 2013;34(10):2524-9.
Salem AK, Weiner GJ. CpG oligonucleotides as immunotherapeutic adjuvants: innovative applications and delivery strategies. Adv Drug Deliv Rev. 2009;61(3):193-4.
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Ghassemi S, Meacci G, Liu S, et al. Cells test substrate rigidity by local contractions on submicrometer pillars. Proc Natl Acad Sci U S A. 2012;109(14):5328-33.
de la Rica R, Thompson S, Baldi A, et al. Label-free cancer cell detection with impedimetric transducers. Anal Chem. 2009;81(24):10167-71.
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Wang J, Tham D, Wei W, et al. Quantitating cell-cell interaction functions with applications to glioblastoma multiforme cancer cells. Nano Lett. 2012;12(12):6101-6.
An assay used for characterizing nanoparticles at the Nanotechnology Characterization Laboratory.
Mark E. Davis, PhD
Mark E. Davis is the Warren and Katharine Schlinger Professor of Chemical Engineering and a member of the City of Hope Comprehensive Cancer Center’s Experimental Therapeutics Program at the California Institute of Technology. He holds an MS and a PhD in chemical engineering from the University of Kentucky. His research focuses on the synthesis of zeolites and molecular sieves, the synthesis of catalytic materials and biomaterials, and gene therapy. His group is working on designs for inorganic and hybrid organic–inorganic materials for catalysis and on biocompatible materials for the delivery of macromolecular therapeutics.
Omid Farokhzad, MD
Omid Farokhzad is an associate professor at Harvard Medical School (HMS) and a physician-scientist in the Department of Anesthesiology at Brigham and Women’s Hospital (BWH). He completed postgraduate clinical and postdoctoral research trainings, respectively, at BWH/HMS and MIT in the laboratory of Professor Robert Langer. He holds MD and MA degrees from Boston University School of Medicine. At BWH, Farokhzad directs the Laboratory of Nanomedicine and Biomaterials, which he established in 2004, and is a faculty member of the Brigham Research Institute Cancer Research Center. He is also a member of the Dana-Farber/Harvard Cancer Center Programs in Prostate Cancer and Cancer Cell Biology. Farokhzad has extensive experience with the development of therapeutic nanoparticle technologies; most notably, he pioneered the high-throughput combinatorial development and screening of multifunctional nanoparticles for medical applications.
Roger Kornberg, PhD
Roger Kornberg is the Mrs. George A. Winzer Professor in Medicine at Stanford University School of Medicine. He is a professor of structural biology and a member of Bio-X, a Stanford initiative that supports, organizes, and facilitates interdisciplinary research connected to biology and medicine. Kornberg was awarded the 2006 Nobel Prize in Chemistry for his work in understanding how DNA is converted into RNA, a process known as transcription. His current research focuses on gene regulation and transcription, the first step in the pathway of gene expression, including discovery of the molecular machines involved in transcription; reconstitution of the process with purified components; determination of the structure of the transcription machinery; and determination of the structure–function relationships in chromatin, the natural DNA template for transcription. This work is directed toward elucidating the structure of the entire transcription apparatus at atomic resolution and the mechanism of transcription control in living cells.
Robert S. Langer, ScD
Robert S. Langer is the David H. Koch Institute Professor at Massachusetts Institute of Technology. He holds a ScD in chemical engineering from MIT and served as a member of the FDA’s SCIENCE Board from 1995–2002, and as its chairman from 1999–2002. His research focuses on nano-based drugs, cancer immunology, and personalized medicine at the interface of biotechnology and materials science. A major focus is the study and development of polymers to deliver drugs, particularly genetically engineered proteins and DNA, continuously at controlled rates for prolonged periods of time. Langer is the recipient of awards including the 2006 United States National Medal of Science, the 2008 Millennium Prize, the 2012 Priestley Medal, and the Gairdner Foundation International Award. Forbes (1999) and BioWorld (1990) named Langer as one of the 25 most important individuals in biotechnology in the world and Forbes (2002) selected him as one of the 15 innovators worldwide who will reinvent our future.
Konstantin Severinov, PhD, DSc
Konstantin V. Severinov is a professor in the Department of Molecular Biology and Biochemistry and the Waksman Institute of Microbiology at Rutgers University. His work focuses on genetic and biochemical analysis of RNA polymerases from E. coli and yeast, as well as site-directed modification of proteins.
Brooke Grindlinger, PhD
The New York Academy of Sciences
Melanie Brickman Stynes, PhD, MSc
The New York Academy of Sciences
Omid Farokhzad, MD
Ralph Weissleder, MD, PhD
Ralph Weissleder is a professor at Harvard Medical School (HMS), the director of the Center for Systems Biology and an attending clinician for interventional radiology at Massachusetts General Hospital (MGH), a senior faculty member in the HMS Department of Systems Biology, and a member of both the Harvard Cancer Center and the Harvard Stem Cell Institute. Weissleder is a graduate of the University of Heidelberg, Germany, and completed postdoctoral and residency training at MGH. He has been on staff at HMS since 1991. He is the recipient of awards including the J. Taylor International Prize in Medicine, the Millennium Pharmaceuticals Innovator Award, the Society for Molecular Imaging Lifetime Achievement Award, and the Academy of Molecular Imaging 2006 Distinguished Basic Scientist Award. His lab studies cancer biology and inflammatory diseases, exploring disease biology using in vivo imaging and working to translate research discoveries into new drugs.
Daniel G. Anderson, PhD
Thomas Lars Andresen, PhD
Gregory I. Berk, MD
Sangeeta N. Bhatia, MD, PhD
Michelle S. Bradbury, MD, PhD
Charles Patrick Case, PhD
Kenneth Dawson, MSc, PhD
Marina A. Dobrovolskaia, PhD
Paula T. Hammond, PhD
James R. Heath, PhD
Alexander V. Kabanov, PhD, DrSc
Takashi Kei Kishimoto, PhD
Gabriel Lopez-Berestein, MD
Sylvain Martel, PhD
Michael R. McDevitt, PhD
Moein Moghimi, PhD
Jay Nadeau, PhD
Shuming Nie, PhD
Aliasger Salem, PhD
Chris Scott, PhD
Yi Yan Yang, PhD
Hot Topics Presenters and Early Career Investigators
François Fay, PhD
Joel M. Friedman, MD, PhD
Saba Ghassemi, PhD
University of Pennsylvania
Michael Goldberg, PhD
Daniel A. Heller, PhD
Hiroshi Matsui, PhD
Louise Rocks, PhD
Jinjun Shi, PhD
Andrea Z. Tuckett, PhD
Don Monroe is a science writer based in Murray Hill, New Jersey. After getting a PhD in physics from MIT, he spent more than fifteen years doing research in physics and electronics technology at Bell Labs. He writes on physics, technology, and biology.
The Nanotechnologies in Cancer Diagnosis, Treatment, and Prevention conference focused on innovative work to apply nanotechnologies to cancer research, highlighting new ideas as well as established concepts that are going through clinical trials. The June 11–13, 2013, conference was relocated from its original venue in Moscow, Russia, to the Memorial Sloan-Kettering Cancer Center in New York City.
Most presentations featured nanoparticles that move through the body to accumulate in tumors, where they can be used for imaging or to deliver drugs. Researchers are still evaluating many different types of nanoparticles, which range from 1–100 nanometers (nm), including metallic and magnetic particles, biodegradable polymers, liposomes, and micelles. Nanoparticles are often passive carriers, and the challenge is to promote long-lasting circulation; for example, by coating them with poly-ethylene glycol (PEGylation), as in traditional drug development.
A primary goal for nanoparticle therapies is to target drug delivery to areas where treatment is needed and limit drug exposure in the rest of the body. Cancer may be particularly amenable to this strategy, since tumors often have leaky blood vessels that some nanoparticles can pass through, a characteristic known as enhanced permeability and retention (EPR). The conference featured much discussion of passive targeting that takes advantage of the EPR effect.
An alternative strategy is to actively target tumors; for example, by decorating nanoparticles with ligands that bind tumor-specific receptors, often to initiate endocytosis. Such targeting may be particularly useful for metastatic disease. However, nanoparticles in the body accumulate a "corona" of proteins that complicate molecular targeting, and some experiments have found this strategy to be less effective than drug delivery through the EPR effect. Researchers are also exploring novel targeting strategies, such as magnetic guidance of particles to the vicinity of a tumor.
For applications in imaging and diagnostics, nanoparticles need only to accumulate in cancer tissue or in individual cancer cells, where their presence can, for instance, alert surgeons to missed tumor tissue or diseased lymph nodes. However, there is also a need for "theranostics" applications, combining therapeutic and diagnostic functions in a single particle.
There is concern about the potential health side effects of nanoparticles in the body, including immune responses. However, at least one application of nanoparticles described at the conference, cancer vaccines, relies on the uptake of unprotected nanoparticles by immune cells. Talks highlighting cancer vaccines pointed to the potential for using nanotechnology in cancer prevention.
Rational design of complex nanoparticles to optimize toxicity, biodistribution, targeting, uptake, and drug release is a challenge. Several speakers described combinatorial design techniques that use high-throughput screening to select desired pharmacokinetics or release properties.
The exciting possibility of exploiting RNA interference to manipulate gene expression in the body, not just in the laboratory, depends on delivering small interfering RNA to specific tissues and cells. Several talks included applications of nanoparticle platforms for nucleic acid delivery. Researchers also proposed novel delivery strategies for other drugs that would delay drug release from a nanoparticle until after it arrives at a tumor.
In addition to nanoparticles, several talks described other nanotechnological approaches to cancer, which manipulate materials on a fine scale to improve cancer diagnosis and treatment. There were numerous references to ongoing clinical trials of nanoparticle systems, and it is clear that nanotechnology is moving out of the laboratory and being translated into applications for cancer treatment and diagnosis.
Kenneth Dawson, University College Dublin, Ireland
Jay Nadeau, McGill University, Canada
Yi Yan Yang, Institute of Bioengineering and Nanotechnology, Singapore
Thomas Lars Andresen, Technical University of Denmark
Early Career Investigator:
Louise Rocks, University College Dublin, Ireland
- In an environment that contains the diverse components found in bodily fluids, a corona of proteins surrounds nanoparticles, which can obscure molecular-targeting specificity.
- The unusual vasculature of tumors (the enhanced permeability and retention, or EPR, effect) is sometimes more important than molecular recognition in targeting cancer.
- It is unclear how strong the EPR effect is in humans. It is evident in naturally occurring tumors in dogs, which are more similar to humans than are mice, but it varies between tumors and even within a single tumor.
Nanoparticle surfaces get messy
The ability to independently control different components of nanoparticle-based therapies makes it possible to modify the nanoparticle surface to target molecules that are uniquely expressed in specific tissue, such as a tumor. These therapies may therefore deliver high local concentrations of a drug to the tumor, while reducing side effects in other tissues.
The effectiveness of molecular targeting with nanoparticles was a recurring topic at the conference, which featured talks on both the successes and failures of the strategy. Even without molecular targeting, many tumors easily accumulate nanoparticles because of their unusually leaky vasculature, and this enhanced permeability and retention (EPR) effect can be used to selectively target the tumors.
"There is no such thing as an untargeted nanoparticle."
"There is no such thing as an untargeted nanoparticle," said Kenneth Dawson of University College Dublin, although it may not be targeted at the intended tissues. A particle immersed in bodily fluids invariably acquires a coating of proteins, known as the corona. Proteins that stay on the surface for a long time can be characterized in the lab, but it is often difficult to predict which species will attach to any particular nanoparticle and the same proteins can attach to a diverse set of core particles. Thus, "it is by no means trivial to design a particle that functions in a realistic environment to target anything," Dawson said.
Another complication is that cells can almost always take up particles by other means than the pathway they are designed for. Therefore, researchers need to verify that selective targeting to the intended tissue is taking place in a realistic environment that mimics the body. Dawson said that researchers can verify molecular targeting "reasonably well" before using the particles in vivo; "give us several years and it will be a science."
Dawson's University College Dublin colleague Louise Rocks presented examples of protein-functionalized silica nanoparticles. Although transferrin- and EGF-functionalized particles selectively targeted cells with the respective receptors in vitro, this specificity was lost when the media contained serum proteins. However, non-specific cellular uptake continued via another pathway that does not involve uptake by lysosomes.
Gold nanoparticles against melanoma
Jay Nadeau and her colleagues at McGill University are exploring the use of small (2.7 nm diameter) gold nanoparticles that are capped with tiopronin. They use these particles to deliver doxorubicin, hoping to avoid the heart and lung toxicity of this drug. Compared to the free drug, gold nanoparticles conjugated with doxorubicin are more rapidly taken up in vitro by B16 melanoma cells. Moreover, 60% of the conjugate enters the nucleus, and it largely avoids the efflux pumps that normally make melanoma insensitive to doxorubicin. As a result, even without active targeting, the nanoparticles are effective at a concentration 20 times lower than the drug alone. Early results show promising effects in mouse melanoma models.
"It is unknown whether targeting is superior to the EPR effect."
Active targeting could be especially useful in treating melanoma because it is highly prone to metastasis. In general, however, researchers do not know whether molecular targeting is superior to the EPR effect, especially for very small particles like the ones Nadeau studies. Her team found that bare particles do accumulate in the tumor, indicating a robust EPR effect, but were also found in the liver and kidney. Surprisingly, PEGylating the particles to increase their circulation time actually decreased their accumulation in the tumor.
The team has also explored the effectiveness of decorating nanoparticles with peptides that target tumor-cell receptors. The peptides sped the uptake of the particles somewhat compared to the EPR effect alone but were not necessary for accumulation in the tumor.
Mixed polymer micelles
Yi Yan Yang of the Institute of Bioengineering and Nanotechnology in Singapore also reported disappointing results using molecular targeting. She and her colleagues attached galactose to nanoparticles, hoping that the particles would be preferentially taken up by the galectin-3 in hepatocellular carcinoma blood vessels. However, the functionalized nanoparticles actually accumulated more in healthy liver tissue, while unmodified nanoparticles went to the tumor. In this case, she said, "the EPR effect was more pronounced than galactose targeting."
Yang reported success in delivering doxorubicin with passively targeted polymer micelles, similar to those described by Alexander V. Kabanov. Her team significantly increased the drug loading of the nanoparticles by making "mixed micelles" that exploit hydrogen bonding with the doxorubicin payload to promote stability. Mouse models of breast cancer showed uptake of the micelles in tumors, but not in the heart or lungs, and significant tumor reduction with no body-weight loss.
Yang also described a strategy for targeting cancer stem cells, whose growth can actually be stimulated by doxorubicin treatment, leading to a recurrence of cancer. The team co-delivered micelles loaded with thioridazine and micelles loaded with doxorubicin. The combination was effective against both cancer stem cells and cancer non-stem cells.
Liposomes taken up by natural canine tumors
Although implanted tumor models, such as xenografts in mice, generally show significant nanoparticle uptake due to the EPR effect, it is unclear how strong this effect is in human tumors. Thomas Lars Andresen and his colleagues at the Technical University of Denmark studied this issue by examining a small number of naturally occurring tumors in dogs, which are more similar to humans than are mice. They used liposomes that can be kept ready on the shelf to carry the short-lived isotopes used in PET imaging, which provides high-quality images of particle accumulation.
Different tumor types showed different degrees of EPR, with carcinomas readily taking up the nanoparticles and sarcomas hardly at all. Even in different regions within a single tumor, there was significant heterogeneity in accumulation. Andresen and his colleagues saw little benefit of molecular targeting with various peptides for their liposomes, which are more than 20 nm in size. But he emphasized that passive uptake could differ for smaller particles, so active targeting might be more important. He said that many questions need to be answered, including the role of the corona.
Andresen noted that the ability to monitor uptake in particular patients, and in specific tumors, could be critical for selecting patients who are most likely to benefit from nanoparticle-based drug delivery.
Successes of active targeting
Other researchers had better success with molecular targeting. Shuming Nie described work aimed to target mouse tumors using metal nanoparticles with a peptide that targets the EGF receptor. But he noted that the effectiveness of such targeting is disputed, and the nanoparticle he is using for tumor visualization relies primarily on the EPR effect. Michelle S. Bradbury reported that although there is some nonspecific tumor uptake of 7 nm silica nanoparticles, those displaying the RGD (arginine-glycine-aspartate) peptide that targets integrin had increased uptake by melanoma, which went away when the receptor was blocked. Similarly, Gregory I. Berk showed that block-copolymer nanoparticles accumulate by EPR in a prostate tumor model but accumulate even more when the targeted antigen is expressed.
Sangeeta N. Bhatia, Massachusetts Institute of Technology
Chris Scott, Queen's University Belfast, UK
Sylvain Martel, Polytechnique Montreal, Canada
- Rather than bundling targeting and delivery in a single nanoparticle, an alternative is to combine simpler particles that together achieve these functions.
- Antibodies could be used to target nanoparticles, delivering many more drug molecules than antibody–drug conjugates.
- In large blood vessels magnetic fields can guide particles to the vicinity of a chosen target, while in small vessels magnetotropic bacteria can guide them to a specific location.
- Direct injection can focus drug exposure to target tissues.
Teams of nanoparticles
Directing nanoparticles to a chosen location is critical to both diagnosis and treatment of cancer. Researchers are exploring ways to do this that go beyond the passive targeting of leaky blood vessels and the molecular targeting of tumor-specific receptors.
"This nanoparticle just has to find the tumor and send a signal."
Sangeeta N. Bhatia of MIT suggested that systems that rely on communication between relatively simple nanoparticles can sometimes be both easier to design and more effective than complex multifunctional nanoparticles. For example, even state-of-the-art complex particles typically deliver no more than a percent or two of their drug load to a tumor, even when they are chemically targeted. Drawing an analogy with the immune system, Bhatia suggested an alternative strategy in which one type of particle simply targets a tumor while another carries the drug. "The [first] nanoparticle doesn't actually have to accumulate very efficiently, it just has to find the tumor and send a signal," she said. This signal—which could be amplified by biological processes—then attracts the drug-carrying particle. In a proof-of-concept study, targeted gold nanorods were heated by an electromagnetic field to stimulate coagulation, attracting particles containing doxorubicin, which deliver roughly 35 000 drug molecules per nanorod.
Bhatia and her colleagues are exploring the possibility of generating other collective behaviors among nanoparticles. In principle, "swarms" of particles could show coordinated activity like birds or fish, even without central control or knowledge of their position.
Chemically bonded combinations uniting antibodies with anticancer drugs have generated a lot of excitement recently, said Chris Scott of Queen's University Belfast. But these antibody–drug conjugates have significant drawbacks. For example, since each antibody delivers at most a few drug molecules, "the drug that you choose must be very, very toxic." The drug is also exposed to the environment, where it can cause damage or be degraded, and the chemical linkage can deactivate the drug.
These problems may be avoided by conjugating the antibody to a nanoparticle, which can carry a much larger drug payload that is not chemically modified and is shielded from the environment. Scott described the targeted delivery of the cytotoxic drug camptothecin (CPT), a water-insoluble molecule that is easily incorporated into the hydrophobic core of a PLGA nanoparticle. Scott's team conjugated such a nanoparticle to an antibody to death receptor 5 (DR5), whose binding stimulates apoptosis. Even with the antibody attached, a PEG-coated particle showed reduced uptake by macrophages, and although the antibody alone suppressed tumor growth in vivo, conjugating it to the CPT-containing nanoparticle suppressed growth even more.
Directing a drug to a particular location is best accomplished in multiple stages, said Sylvain Martel of Polytechnique Montreal. A catheter in a chosen artery carries drugs to a particular region, but this approach is limited to large vessels; thus, "it might help a bit, but you'll still have secondary toxicity," he said.
Steering in smaller vessels can be achieved with magnetic nanoparticles. To generate large forces, such particles should be as large and as magnetizable as possible and should be placed in a high field, such as that of an MRI magnet. (The magnitude of the force also depends on large spatial field gradients, such as those that occur in the fringe fields of MRI magnets.)
Martel and his colleagues have demonstrated targeting of composite polymer particles that include many magnetic particles and a cancer drug. They use an MRI machine to both image and direct the particles, quickly switching between measuring the particles' position and pushing them into a chosen vessel.
Unfortunately, particles that could fit into capillaries were too small to generate large magnetic forces. To overcome this problem, Martel and his colleagues are using self-propelled magnetotactic bacteria, loaded with drug-filled liposomes, to penetrate into a tumor. These bacteria naturally contain internal nanocompasses that guide them along magnetic fields. Martel envisions injecting large composite particles into the largest vessels, pushing them magnetically through medium-sized vessels, and then breaking them apart to allow bacteria to guide them through small vessels into tumors. This process might take about an hour.
Joel M. Friedman of Albert Einstein College of Medicine gave a hot topic presentation on the magnetic guiding of particles, this time based on gadolinium oxide. This technique could deliver nitric oxide, which might enhance the EPR effect and improve drug delivery to tumors.
Getting ahead of breast cancer
In a hot topic presentation, Michael Goldberg of the Dana-Farber Cancer Institute illustrated the utility of using targeting strategies for ductal carcinoma in situ (DCIS), a common premalignant form of breast cancer. Although some cases progress to malignancy, many do not, so there is a strong incentive to block DCIS progression without the side effects of more aggressive treatment. A systems-biology analysis identified the HoxA1 gene as a potential master regulator for breast cancer. Goldberg showed that nanoparticles injected into a duct spread throughout the duct but remain within it, and can deliver siRNA to knockdown this gene, reducing cancer penetrance in a disease model from 100% to about 20%.
Ralph Weissleder, Massachusetts General Hospital
Michelle S. Bradbury, Memorial Sloan-Kettering Cancer Center
Shuming Nie, Emory University
- Positron emission tomography from nanoparticle probes can provide high-resolution molecularly targeted images of tumors.
- Real-time optical emission can help surgeons to identify cancerous tissue that might otherwise be left behind.
- Non-bleaching fluorescence from carbon nanotubes could allow quantitative local measurements of cellular processes.
- Magnetic nanoparticles can be used for sensitive NMR probing at the bedside.
Combining quantitative and optical measurements
The successful targeting of nanoparticles results in their accumulation in specific tissues. Even if the particles do not enter cells or deliver payloads on their own, this accumulation can be used for diagnosis and imaging.
Michelle S. Bradbury of Memorial Sloan-Kettering Cancer Center described a versatile platform that combines two visualization modes for imaging tumors, either before or during surgery. Before surgery, tumors can be imaged using optical emission because the system uses silica nanoparticles that contain fluorescent dye. During surgery, encapsulation enhances the dye's light emission by a factor of ten or more, and the fluorescence can identify cancerous tissue. The results can be viewed with relatively inexpensive portable equipment during either open or laparoscopic surgery and can be enhanced with preoperative mapping.
Bradbury and her colleagues decorated these fluorescent nanoparticles with 124I for positron emission tomography (PET) imaging. The hybrid particles combine real-time optical imaging with PET quantification of the delivery, uptake, and retention of particles, thus allowing for assessment of the actual drug dose delivered.
In a small clinical trial, the particle was well tolerated in humans. Surprisingly, Bradbury said, in the associated biodistribution study, "it looks and behaves like a macromolecule." Intriguingly, even without specific targeting the nanoparticle accumulated in a pituitary lesion in one patient.The versatile particles could assist in mapping the spread of cancer to sentinel lymph nodes, and thus in accurately staging cancers. In a miniswine (miniature pig) that is prone to melanoma, the optical imaging mode identified even very small cancer burdens in lymph nodes.
Helping surgeons to see tumor cells
Shuming Nie of Emory University elaborated on the potential importance of visualizing cancer during surgery. For example, delineating the margins of a tumor would help surgeons to avoid leaving behind tumor cells that could seed recurrence, and optical emission from malignant cells could flag lymph nodes that contain metastases. These features could help surgeons balance the risk of leaving cancer cells intact against that of removing too much healthy tissue.
Nie and his colleagues are exploiting the enormous amplification of Raman scattering intensity that occurs near colloidal gold nanoparticles. The largest increase, by as much as a factor of 1014, occurs where two particles meet and allows for detection of a single molecule at room temperature. Since the spectrum reflects detailed vibrational modes, it provides a unique fingerprint for each molecular species. Nie is working to commercialize a pen-like device for Raman measurements, as well as a system to combine the images with anatomic and other information in real time.
Nie showed that naturally occurring canine tumors (such as those studied by Thomas Lars Andresen) show significant uptake of fluorescently labeled nanoparticles as a result of the EPR effect. Such particles are currently being evaluated in a clinical trial as an adjunct to human surgery. Encouragingly, Nie reported that the team "found some unexpected metastatic deposits which would have been missed without the fluorescent images."
Daniel A. Heller of Memorial Sloan-Kettering Cancer Center gave a hot topic talk on fluorescent probes based on carbon nanotubes. Unlike traditional dyes, nanotubes do not bleach under continued illumination, and different tube geometries emit at different wavelengths, so they could allow a multiplexed, quantitatively reproducible reporter of local cellular processes.
Molecular diagnosis at the bedside
In his keynote talk, Ralph Weissleder of Massachusetts General Hospital described external use of nanoparticles for diagnosis, in which the particles enhance the sensitivity of chemical detection to allow bedside analysis of bodily fluids using a palm-sized nuclear magnetic resonance (NMR) machine.
"Biopsy could have been prevented with a simple blood test."
The high sensitivity of this tool arises from two technological advances. In the first, the researchers wait for antibodies to find their targets and only then attach nanoparticles using a type of click reaction. By relieving the antibodies of the need to drag along bulky nanoparticles while they seek their targets, this technique achieves a tenfold increase in sensitivity. In the second advance, developed by Weissleder and his colleagues, magnetic materials for nanoparticles increase the sensitivity of water-based NMR measurements by a hundredfold.
Weissleder described three studies employing this NMR-amplification technology. The first measured a suite of biomarkers in fine-needle biopsies. Although individual markers were disappointing, a combination of four markers gave a 96% diagnostic accuracy for cancer in a comparison with traditional techniques.
A second study used the combined tumor biomarkers to look for circulating tumor cells (CTCs) in patients' blood. The team detected CTCs in many (about three quarters) of the patients with a positive tumor biopsy, in contrast to the frequent assumption that they are rare. In these cases, Weissleder said, "the biopsy could have been prevented, because with a simple blood test we could have [determined] whether they have cancer or not." The detailed biomarker signature also correctly predicted the type of cancer in 80%–90% of patients.
The third study identified ovarian cancer cells in excess abdominal fluid. Because such samples contain a large amount of "debris," the researchers developed a microfluidic chip that sorts fluid-borne particles by size and removes benign mesothelial cells before analysis. Weissleder noted that this relatively noninvasive technique could allow practitioners to track the effectiveness of cancer treatment as the treatment progresses.
Sangeeta N. Bhatia described an intermediate diagnostic technique in which nanoparticles penetrate a tumor bearing distinct tags that can be cleaved by specific proteases that indicate the presence and type of cancer. A single protease molecule can thereby create many cleavage fragments that can be detected in the urine using fluorescence or mass spectrometry to a high degree of sensitivity.
Moein Moghimi, University of Copenhagen, Denmark
Charles Patrick Case, University of Bristol, UK
Marina A. Dobrovolskaia, Nanotechnology Characterization Lab SAIC–Frederick Inc.
- Many nanoparticles trigger an immune response by activating the pattern recognition of the complement system.
- DNA damage can occur even across a cellular barrier in response to oxidative stress, nanoparticles, and other insults.
- The National Characterization Lab provides extensive characterizations of novel nanoparticle systems with no fee.
- Many of the immune responses observed for nanoparticle samples arise because the particles are contaminated with endotoxin.
- The regulation of nanoparticles may require a new paradigm, differing from those for small molecules or biosimilar materials.
In addition to their intended activity at a tumor, nanoparticles may cause unintended side effects such as immune responses elsewhere in the body. Assessing the safety of nanoparticles is challenging because there are many different types of particles and their manufacture often produces a range of particles in one sample. Moreover, the flexibility to mix and match various components of a nanoparticle complicates the overall assessment.
The complement system, a key component of the innate immune response, responds to particular molecular patterns, explained Moein Moghimi of the University of Copenhagen. Unfortunately, many nanomaterials activate this pattern-recognition function. In some cases, the complement response could enhance the cancer-fighting capacity of a nanoparticle therapy, but in others it promotes tumor growth.
For Doxil, a liposome-based vehicle for delivering doxorubicin, complement activation was traced to the specific chemical linkage used to incorporate the drug into the particle. In this case the linkage was easy to fix, but the specific molecular configurations that activate the complement system can be difficult to anticipate. The response may depend on seemingly unimportant details, such as particular configurations of a molecule; for example, a configuration in which the molecule resembles mannose, triggering the lectin pathway. "The activation is a net effect of an entire set of properties," said Moghimi, who repeatedly cautioned the nanoparticle community that "we have to slow down" to avoid setbacks like those experienced for gene therapy.
Damage across barriers
Charles Patrick Case of the University of Bristol is investigating DNA damage caused by nanoparticles. To explore whether DNA damage crosses natural biological barriers, his team used a model placenta consisting of placental cells on a perforated plastic membrane. To their surprise, they found that the barrier sometimes enhanced the damage to distant cells from nanoparticles on the other side, as in the radiation "bystander effect." The damage appears to require two layers of cells, with the first layer signaling through direct gap junctions to the second layer. The cells that receive this signal then send a damage-promoting signal through the solution to distant cells.
In addition to its implications for nanoparticles, this phenomenon may contribute to the teratogenic effects of the thousands of compounds that create free radicals. But Case speculated that it might also be possible to exploit it to enhance nanoparticle therapies.
Marina A. Dobrovolskaia represented the Nanotechnology Characterization Laboratory, a collaboration of the National Cancer Institute (NCI), the Food and Drug Administration (FDA), and the National Institute of Standards and Technology (NIST) that aims to "accelerate transition of the basic and technology concepts into the field." To further this goal, she said, "everyone is entitled to a free evaluation of materials" for potential negative health effects.
The assessment of particles includes physical and chemical characterization, in vitro and in vivo toxicology, and immunology. For characterization and toxicology, particle stability is the primary challenge; for immunology, it is that more than 30% of submitted samples are contaminated during or after manufacture by endotoxin, the lipopolysacharide that inhabits the membrane of gram-negative bacteria. Such contamination could derail the further exploration of nanomaterials that are actually safe.
It is difficult to anticipate immune responses to new types of nanoparticles because "all nanoparticles are different," Dobrovolskaia said. The protein corona that invariably decorates them has important effects on uptake by macrophages and on other immune responses. But "the presence of complement proteins on the particle surface does not necessarily mean that the particle activated complement [immune responses]," she noted. In spite of these challenges, Dobrovolskaia emphasized that there are success stories in which reformulation of drugs into nanotechnology platforms has reduced toxicity compared to the traditional drug.
Are current regulations adequate?
Stefan Mühlebach, chairman of the Working Group on “non-biological complex drugs” (NBCD) and a professor at the University of Basel, explained in his hot topic talk that current regulation of nanoparticular NBCDs and their first follow-on versions (similars) is modeled on established procedures for either well-characterized small molecules (generics) or large molecules that are "biosimilar." But in these new drugs, "the entire complex formulation with the active pharmaceutical ingredient" is the drug product, he said, and "the elaborate manufacturing process is fundamental to create the product and difficult to control." Mühlebach and his colleagues argue a new regulatory approach, recognizing these complexities, will therefore be necessary for these drug products and their follow-on versions when it comes to comparability assessments with reference products for market authorization.
Aliasger Salem, The University of Iowa
Takashi Kei Kishimoto, Selecta Biosciences
- Simultaneous delivery of an adjuvant with an antigen in a nanoparticle generates more robust humoral and cellular immune responses than when either is in solution.
- CpG DNA sequences act through the toll-like receptor system as an adjuvant of vaccine action.
- Nanoparticles are roughly the same size as viruses, and they can be engineered with many components to optimize the immune responses that they induce.
The power of co-delivery
The immune responses induced by nanoparticles are often a serious drawback, but for vaccines, including cancer vaccines, the ability to induce a specific immune response is the desired goal. Robust immune responses generally require the simultaneous presence of an adjuvant with an antigen, so delivering both in the same time and place, near a target such as a tumor, is likely to be a powerful advantage for engineered nanoparticle vaccines.
One potent class of adjuvant, said Aliasger Salem of the University of Iowa, is the CpG oligonucleotides, which include a cytosine followed in sequence by a guanine. These sequences are agonists for toll-like receptor (TLR) 9, and probably contributed to Coley's historic success in recruiting the immune system to fight cancer.
Salem and his colleagues explored the encapsulation of CpG into biodegradable polymers, which are relatively benign biologically. Nanoparticles made of these materials are naturally taken up by macrophages and dendritic cells, where they degrade and stimulate an immune response. There is no need to coat them with PEG or other materials.
Salem showed that adding CpG to a desired antigen in one particle dramatically improves its adjuvant effect. "There is a 20-fold increase in the interferon-gamma secretion when you package the CpG and the antigen into the particle, versus having higher concentrations of the antigen and CpG in solution," he said. Moreover, TLR9 receptors within the phagosomes of dendritic cells respond to the CpG and generate a cellular immune response from CD8+ T cells. The effects are largest for particles of approximately 300 nm and are greater than those seen when CpG and the antigen are only chemically conjugated.
Salem also described a novel "in situ immunization" process in which CpG is co-delivered in a nanoparticle with a cytotoxic drug such as doxorubicin. The presence of an adjuvant can stimulate an immune response to the molecular fragments that emerge from the killed cells.
Synthetic vaccines particles
Takashi Kei Kishimoto of Selecta Biosciences also showed that co-delivery of adjuvant and antigen using a nanoparticle generates a more robust immune response, especially the T-cell response. He and his colleagues are designing what they call "targeted Synthetic Vaccine Particles" for use against cancer and other conditions. Their lead drug for developing this platform, now in phase I trials, is a vaccine against nicotine to aid smoking cessation.
"By co-delivering antigen plus TLR agonists in the particle, we're getting the right instructions to the right cells and minimizing systemic side effects."
The nanoparticle platform for this drug is based on the sorts of block copolymers described by Omid Farokhzad. It can present B-cell antigens on the surface while encapsulating a T-cell peptide and a TLR agonist to act as an adjuvant. Kishimoto contrasted the small scale required for vaccine production, which only needs to deliver the instructions for making many molecules, with the larger scale required for manufacturing other drugs.
When the adjuvant was included in the nanoparticle rather than free in solution, subcutaneous injection in mice showed a large increase in the cytokines that promote T-cell responses in the local draining lymph node. At the same time, the systemic release of pro-inflammatory cytokines, often associated with flu-like side effects, was dramatically lower with the nanoparticle. "By co-delivering antigen plus TLR agonists in the particle, we're getting the right instructions to the right cells and minimizing systemic side effects," Kishimoto said.
The flexible copolymer system also allowed the team to optimize the release kinetics for specific immune responses. One formulation, for example, gave rise to a sustained response to a single injection, which promoted effector memory T cells. This optimized release increased survival in both an in vivo tumor model and in a metastasis model, and it generated a robust immune response against a fresh tumor.
Omid Farokhzad, Brigham and Women's Hospital, Harvard Medical School
Gregory I. Berk, BIND Therapeutics
Daniel G. Anderson, Massachusetts Institute of Technology
- Nanoparticle pharmacokinetics, targeting, and delivery are all sensitive to the many design choices in particle construction.
- Rather than attempting rational design, an alternative is experimental exploration of the many combinations by high-throughput screening using animal models.
- This exploration platform is being commercially developed to achieve targeted delivery of cancer drugs, as well as to improve synthetic vaccines.
- A similar technique uncovered new materials for delivering short interfering RNA.
To be used successfully in cancer diagnosis, therapy, or vaccination, a nanoparticle must navigate an intricate series of biological processes. The complex nanostructure that is required is difficult to design rationally. As an alternative, several groups are using combinatorial techniques that create and assess many different variants.
Keynote speaker Omid Farokhzad of the Brigham and Women's Hospital and Harvard Medical School has been exploring nanoparticle drug delivery for more than a decade. Early work on intratumorally injected polymer nanoparticles that delivered docetaxel to prostate cancer by targeting a prostate-specific membrane antigen (PSMA) were well received by the research community. But his industry colleague Steve Zale warned that the surface chemistry involved in making them would not be reproducible enough for commercial pharmaceuticals.
To address this challenge, Farokhzad and his colleagues developed nanoparticles that self-assemble from block copolymers in which one block is the drug. They soon realized that this system, which works by physically mixing block copolymers with different constituents, is ideal for combinatorial experiments. New particles can be made, using a single process, simply by varying the composition to optimize their circulation and delivery properties.
For example, Farokhzad's team found an optimum density of ligands for PSMA on the nanoparticle surface. Increasing the density improves the targeting in vitro, as expected, but in vivo it increasingly attracts the attention of the excretion apparatus, which removes the particles from circulation. Different formulations of the particles allow the researchers to vary the particle pharmacokinetics independently of the pharmacokinetics of the drug.
Farokhzad also described high-throughput screening for peptides that are taken up by cancer cells but do not bind to ordinary tissue. In addition to applications in targeting therapy or tumor visualization, the selected ligands can be attached to magnetic beads to purify and identify the antigens that they bind to.
Finally, Farokhzad showed that targeting a nanoparticle to a known translocation pathway could transfer particles across the gut or lung membrane and may allow for oral delivery of nanoparticle drugs. "These are, to the best of my knowledge, the first example of nanoparticles with very high bioavailability for oral administration," he said.
Nanoparticle delivery in the clinic
Gregory I. Berk is the chief medical officer of BIND Therapeutics, which is exploring the commercial use of the PSMA-targeted docetaxel nanoparticles describe by Farokhzad. "BIND has decided to start with known targets and known drugs" like these, "but plans to address novel targets and drugs in the future," he said. As one example, the platform could be useful for delivering modern chemotherapy drugs that target specific molecular pathways, such as kinase inhibitors.
Even non-targeted nanoparticles are taken up in tumors, presumably as a result of the EPR effect. But he showed that targeting PSMA clearly improves both drug concentration and efficacy in animal models. Phase I trials of the company's lead drug, BIND-014, showed promising pharmacokinetics and predictable side effects, primarily neutropenia, in terminally ill patients. One subject died of the side effects, but another showed complete remission of cervical cancer. The drug is currently entering phase II trials.
"We need to prove this in head-to-head trials versus conventional docetaxel," Berk said. He added that the drug may be useful for cancers that are ordinarily not sensitive to docetaxel at acceptable doses, and thus may improve that therapeutic index.
Takashi Kei Kishimoto described the use of similar nanoparticles as vaccines, specifically the potential to tailor delivery kinetics by high-throughput screening to optimize particular immune responses.
Exploring thousands of molecules
For delivery of small interfering RNA (siRNA) in therapeutic applications, delivery to the proper cells is the major challenge. "One of the strategies we've focused on is developing systems for accelerated discovery of nanoparticulate delivery materials," said Daniel G. Anderson of MIT.
"We've focused on developing systems for accelerated discovery."
Anderson and his colleagues found a flexible synthetic pathway that allowed them to explore thousands of materials without re-optimizing synthesis for each one and found a family of nanoparticle-forming materials they call "lipidoids." They used an in vivo assay to optimize siRNA delivery to hepatic cells, achieving gene knockdown at doses of hundredths of milligrams per kilogram of body weight. This efficient delivery means that the platform could be used to simultaneously deliver multiple siRNAs.
Another formulation showed successful gene knockdown in various endothelial cells, despite relatively poor silencing in hepatocytes. Delivery of an endogenous microRNA using this model significantly extended life in a mouse model of lung cancer.
Sangeeta N. Bhatia drew on screening results from her collaborators, led by Erkki Ruoslahti at the Sanford-Burnham Medical Research Institute, who have developed a technique to inject and track an entire library of bacteriophages into test animals. This technique identified peptides that target nanoparticles to specific parts of the body, as well as peptides that allow nanoparticles to penetrate deep into a tumor to deliver siRNA there.
Gabriel Lopez-Berestein, MD Anderson Cancer Center
- Delivery is the major hurdle to realizing the therapeutic promise of RNA interference.
- Challenges include avoiding immune responses to free nucleic acids and delivering siRNA into target cells.
- Nanoparticles provide many options for the tunable control of delivery of these potentially powerful molecules.
Turning genes off
RNA interference, in which gene activity is post-translationally inhibited by siRNA, has become a versatile and popular tool in the laboratory. Its use for therapeutic manipulation of gene expression is also promising for applications including cancer treatment. However, as for its predecessor anti-sense DNA, the usefulness of siRNA depends on the ability to deliver it to cells. Nanoparticles hold the promise of protecting nucleic acids from degradation and of avoiding immune responses, while also shuttling the siRNA to, and into, specific cells.
As noted by Daniel G. Anderson, foreign substances are often removed by the liver, so hepatic cells have been a somewhat easier target for early siRNA exploration (although he and his colleagues have also found formulations that target endothelial cells). Similarly, Michael R. McDevitt described the delivery of siRNA to the kidneys, taking advantage of the unusual biodistribution of carbon nanotubes to that excretion system. Michael Goldberg showed that directly injecting nanoparticles into the breast ducts of mice delivers siRNA specifically to ductal cells as a potential prophylactic intervention for ductal carcinoma in situ.
Targeting ovarian cancer
Delivery to tumors can potentially take advantage of the EPR effect. Gabriel Lopez-Berestein of MD Anderson Cancer Center described a phospholipid-based nanoliposome that can entrap large amounts of siRNA. Specifically, his work aims to knock down the ephrin EphA2, whose expression correlates strongly with decreased survival.
Lopez-Berestein and his colleagues developed a formulation that evades macrophage uptake and silences the target gene for more than a week in an orthotopic model of ovarian cancer. Based on these results, they have embarked on a phase I clinical trial of these nanoparticles in EphA2-positive ovarian cancer patients.
Many ways to deliver RNA
Jinjun Shi of Brigham and Women's Hospital and Harvard Medical School gave a hot topic talk on another approach to delivering siRNA, intended to improve its delivery. He and his colleagues used a double-emulsion technique to create hollow nanoparticles that resemble a liposome, but in which the hydrophobic interior of the shell wall is filled with biodegradable polymer. The thickness and properties of this layer can be varied to improve the release kinetics of the siRNA.
Alexander V. Kabanov, Paula T. Hammond, and Sangeeta N. Bhatia all described how their respective nanoparticle systems could be used to deliver siRNAs. Bhatia suggested that such systems could be useful not only in therapy but also in research to validate the importance of genes as inferred from genomic studies. Aliasger Salem and Takashi Kei Kishimoto also both described nanoparticles systems for delivering nucleic acids, in their case oligonucleotides to stimulate immune responses for vaccines.
Alexander V. Kabanov, University of North Carolina at Chapel Hill
Paula T. Hammond, Massachusetts Institute of Technology
Michael R. McDevitt, Memorial Sloan-Kettering Cancer Center
Early Career Investigator:
François Fay, Mount Sinai School of Medicine
- Polymeric micelles are a versatile system for delivering insoluble drugs, and the block copolymers that comprise them have biological activity on their own.
- Drug payloads can be activated by low-frequency magnetic fields, not because of heating but because of their mechanical effect.
- Building particles layer by layer allows for the optimization of pharmacokinetics as well as of the chemistry that releases drugs in the acidic tumor environment.
- A hybrid nanoparticle can present different surface properties upon exposure to proteases in a tumor.
- Carbon nanotubes can deliver siRNA to the kidney by passing lengthwise through narrow pores that normally block large molecules.
Amphiphilic block copolymers
A nanoparticle that survives its circuit through the bloodstream to arrive in the target tissue still must deliver its drug payload inside the cells. Nanoparticles are sometimes non-selectively taken up by cells, but targeting of cell-surface receptors often triggers endocytosis as well as recognition. Researchers are also exploring other ways to improve or tailor the delivery.
Because of the interaction of block copolymers with biological structures, "it's not just putting stuff in a container."
Alexander V. Kabanov of the University of North Carolina at Chapel Hill is a longtime champion of polymeric micelles, block copolymers that self-assemble into a hollow shell. By properly choosing the internal surface of the micelle, these structures can carry a variety of drug payloads, including insoluble ones. This scheme avoids the need to covalently attach the drug to the carrier, allowing for easier release.
Polymeric micelles have been examined in several clinical trials, all using PEG as the hydrophilic segment of the copolymers. PEG is used with many other drugs to improve blood circulation and is relatively nontoxic, but its ubiquity in pharmaceuticals and personal-care products means that about a quarter of people have antibodies to it, complicating its use in drugs.
As an alternative to PEG, Kabanov and his colleagues have explored poly(2-oxazoline)s, whose synthesis can also be modified to produce different degrees of polarity. This is particularly useful for delivering paclitaxel (currently formulated with an excipient as Taxol and bound to albumin as Abraxane), which has both polar and nonpolar regions and forms hydrogen bonds. A properly designed polymer formed micelles with a high drug-loading capacity for paclitaxel, 100 times that of Taxol and ten times that of Abraxane. The relative reduction in inactive ingredient for each drug molecule reduced complement activation and increased tolerable dose.
But the copolymers are also biologically active, so "it's not just putting stuff in a container," Kabanov said. The same hydrophobic/hydrophilic juxtaposition that leads to micelles also promotes their insertion into cellular membranes, including those of mitochondria. This latter effect may act synergistically with the cytotoxic doxorubicin in SP1049C, which is currently in clinical trials against multi-drug-resistant tumors.
With respect to delivery, after drug-containing liposomes have accumulated in a tumor, later administration of a block copolymer can disrupt the liposomes' membranes and release their payload. Another novel delivery mechanism uses magnetic beads attached to a nanoparticle. An oscillating magnetic field can release the drug, not through heating, but by direct mechanical jostling.
Layer-by-layer nanoparticle growth
Paula T. Hammond of MIT described a versatile layer-by-layer process that allows for the tailoring of many nanoparticle properties, including delivery characteristics. The initial seed particle can be a luminescent nanodot, a metallic or magnetic nanoparticle, or a drug-delivering lysosome, micelle, or biodegradable polymer. Alternating the self-limiting deposition of positively and negatively charged polyelectrolyte layers on a nanoparticle creates highly uniform coatings that can have a variety of compositions, including long circulation times.
An outermost layer can be chosen that will degrade in the more-acidic hypoxic environment of a tumor to locally deliver a payload. One strategy attaches an outer PEG layer by a degradable iminobiotin linkage. Another strategy uses hyaluronic acid as a polyelectrolyte in conjunction with poly(L-lysine). The degradation of this layer exposes a positive charge that induces cellular uptake. The hyaluronic acid also targets the CD44 receptor, which is overexpressed in many solid tumors.
Using alendronate, conjugated to poly-acrylic acid, as a polyelectrolyte resulted in accumulation in a metastatic osteosarcoma xenograft model as well as in bone tissue. The accumulation persisted over many days, in contrast to the day or so that is typical for the EPR effect. "Targeting makes a huge difference in these systems," Hammond said.
François Fay from Mount Sinai School of Medicine described an outer layer that changes in the tumor microenvironment. The particle uses the RGD (arginine-glycine-aspartate) peptide sequence to target the αVβ3 integrin, which is often overexpressed in tumors but is also expressed on a variety of endothelial cells. In what he calls a "surface-switching particle," this recognition sequence is protected by PEG, but this coating is attached by means of a linkage that is cleaved by proteases in the tumor environment.
Special delivery for the kidney
Michael R. McDevitt and his colleagues at Memorial Sloan-Kettering Cancer Center explored the pharmacokinetics of functionalized single-wall carbon nanotubes. Surprisingly, in view of their large size, these molecules were rapidly excreted intact in the urine. McDevitt suggested that the long, thin rods can slide lengthwise through the narrow pores in the slit barrier that ordinarily blocks passage of proteins and other macromolecules into the kidneys. "The slit diaphragm is seeing it as if it's a small molecule," he said.
This unusual biodistribution suggested that it would be possible to use nanotubes to deliver drugs specifically to the kidneys. In particular, siRNA wraps tightly around a functionalized nanotube, mostly through electrostatic and hydrogen bonding, and is protected from the usual degradation mechanisms in the bloodstream. Once within a kidney cell, however, the siRNA is liberated, as shown by in vitro silencing of a fluorescence gene.
McDevitt showed that radio-labeled siRNA on a nanotube carrier indeed goes preferentially to the kidneys of test animals, unlike bare siRNA. However, there was no indication of accumulation of the siRNA–nanotube complex in tumors.
James R. Heath, California Institute for Technology
- A compact nanotechnology-based tool for probing the associations between proteins down to the single-cell level provides unique insight into disease biology.
- Correct identification of distinct functional pathways at the cellular level allows for the more rational design of combination therapies that target several modes of cancer activity to avoid resistance.
- A microfluidic device allows for the detection and classification of cancer cells based on their elasticity differences with normal cells.
- An artificially nanostructured surface provides a thymus-like environment that supports the expansion of T-cell populations.
- A nanopatterned substrate can be made with the rigidity needed to promote the activation of human T cells.
Systems biology of individual cells
Although the majority of the conference was devoted to nanoparticle diagnostics and therapeutics, several talks described other nanotechnology approaches to cancer research.
"If you want to understand signaling pathways, you need to do it at the single-cell level."
To better reveal the molecular underpinnings of cancer, for example, James R. Heath and his colleagues at the California Institute for Technology have built microchips that can be used for ELISA analysis of very small samples. "If you want to understand signaling pathways at a high resolution of detail," he said, "you can't really do it from bulk tissue analysis. You need to do it at the single-cell level." Correlations between the quantities of various proteins reveal that cancer cells can be classified into distinct functional phenotypes with different signaling networks, both from each other and from the collective behavior of many cells.
Heath suggested that this cellular heterogeneity may be critical to the stability of tumors. In fact, isolated cells of a particular phenotype, whether aggressive or not, recapitulate the entire spectrum of behaviors when they multiply. For this reason, it may be counterproductive to try to isolate specific subpopulations of tumor cells for separate treatments.
These cell-level insights will be critical for choosing combination therapies that address drug resistance in cancers. Heath showed an analysis that identified distinct "modes" of action in a mouse model for glioblastoma multiforme (GBM). Using this information to choose pairs of drugs that simultaneously target both modes successfully inhibited the cancer in a mouse model. "The existing pharmacy for GBM is probably [effective] for at least a reasonable fraction of the patients," Heath said. "We just don't know how to use them."
Nanoscale cell manipulation
Hiroshi Matsui of Hunter College gave a hot topic talk on a chip-scale device, which distinguishes and even sorts cancer cells on the basis of their greater swelling in response to a sudden change of osmotic pressure.
Andrea Z. Tuckett of Memorial Sloan-Kettering Cancer Center and Saba Ghassemi of the University of Pennsylvania gave hot topic presentations describing new ways to encourage the growth and activation of T cells in artificial media. Tuckett developed a nanofabricated hydrogel structure with separate layers incorporating factors to encourage blood-vessel growth, fetal cells to provide growth signals, and T-cell precursors. When implanted in test animals, this scaffold supported both vascularization and sustained growth of T cells that could help patients deficient in these cells. Ghassemi described a growth substrate for activating T cells in the laboratory for potential use in adoptive cancer therapy. The mechanical rigidity of the substrate can be altered to manipulate the response of the cells.
In addition to her work on nanoparticle drug delivery, Yi Yan Yang described a hydrogel-based material that persistently delivered trastuzumab (Herceptin) into a tumor. This work shows that the impact of materials development for drug delivery, although critical for nanoparticles, has implications beyond this field.
Which nanoparticle systems will prove most useful for imaging and diagnosis, and which delivery vehicles will be best for therapies?
Is there a universal strategy for ensuring the uptake of drug payloads into cells, and their subsequent release?
Which human tumors show the enhanced permeability and retention (EPR) effect that can help localize therapy even without molecular targeting?
How effective are various strategies for molecular targeting of nanoparticles to tumors?
Is it possible to predict which proteins and other molecules will adsorb onto a particular core nanoparticle in a realistic environment in order to predict its biological activity?
How can regulatory strategies be improved to speed approval of complex combination therapies using nanoparticles while ensuring adequate safety?
In what ways do nanoparticles stimulate immune responses, and can those responses be managed?