‘Just add water’ to activate freeze-dried brain cancer fighting nanoparticles

A fluorescence micrograph showing brain cancer cells producing a green fluorescent protein. DNA encoded to produce the protein was delivered to the cancer cells by new freeze-dried nanoparticles produced by Johns Hopkins biomedical engineers. Image: Stephany Tzeng/JHU

Biomedical engineers and clinicians at Johns Hopkins University have developed freeze-dried nanoparticles made of a shelf-stable polymer that only need the addition of water to activate their cancer-fighting gene therapy capabilities.

Principal investigator Jordan Green, assistant professor in the department of Biomedical Engineering at the Johns Hopkins School of Medicine, led the team that fabricated the polymer-based particles measuring 80 to 150 nanometers in diameter. Each particle, which is about the size of a virus, has the ability to carry a genetic cocktail designed to produce brain cancer cell-destroying molecules. After manufacture, the nanoparticles can be stored for up to 90 days before use. In principle, cancer therapies based on this technology could lead to a convenient commercial product that clinicians simply activate with water before injection into brain cancer tumor sites.

Because this method avoids the common, unpleasant side effects of traditional chemotherapy, “nanoparticle-based gene therapy has the potential to be both safer and more effective than conventional chemical therapies for the treatment of cancer,” Green said. But, he added current gene therapy nanoparticle preparations are just not practical for clinical use.

“A challenge in the field is that most non-viral gene therapy methods have very low efficacy. Another challenge with biodegradable nanoparticles, like the ones used here is that particle preparation typically takes multiple time-sensitive steps.” Green said. “Delay with formulation results in polymer degradation, and there can be variability between batches. Although this is a simple procedure for lab experiments, a clinician who wishes to use these particles during neurosurgery will face factors that would make the results unpredictable.”

In contrast, the nanoparticles developed by the Green lab are a freeze-dried, or “lyophilized,” formulation. “A clinician would simply add water, and it is ready to inject,” Green said. Green thinks this freeze-dried gene-delivery nanoparticle could be easily manufactured on a large scale.

Co-investigator Alfredo Quinones-Hinojosa, a Johns Hopkins Hospital clinician-scientist and associate professor in the departments of Neurosurgery and Oncology at the Johns Hopkins School of Medicine, said he could imagine particles based on this technology being used in conjunction with, and even instead of, brain surgery. “I envision that one day, as we understand the etiology and progression of brain cancer, we will be able to use these nanoparticles even before doing surgery,” Quinones said. “How nice would that be? Imagine avoiding brain surgery all together!”

Currently, patients with glioblastoma, or brain cancer, only have a median survival of about 14 months, Green said. “Methods other than the traditional chemotherapy drugs and radiation—or in combination with them—may improve prognosis,” he said.

Gene therapy approaches could also be personalized, Green said. “Because gene therapy can take advantage of many naturally-existing pathways and can be targeted to the cancer type of choice through nanoparticle design and transcriptional control, several levels of treatment specificity could be provided,” Green said.

The nanoparticles self-assemble from a polymer structural unit, so fabrication is fairly simple, said Green. Finding the right polymer to use, however, proved to be a challenge. Lead author Stephany Tzeng, a PhD student in biomedical engineering in Green’s lab screened an assortment of formulations from a “polymer library” before hitting on a winning combination.

“One challenge with a polymer library approach is that there are many polymers to be synthesized and nanoparticle formulations to be tested. Another challenge is designing the experiments to find out why the lead formulation works so well compared to other similar polymers and to commercially available reagents,” Green said.

Tzeng settled on a particular formulation of poly(beta-amino ester)s specifically attracted to glioblastoma (GB) cells and to brain tumor stem cells (BTSC), the cells responsible for tumor growth and spread. “Poly(beta-amino ester) nanoparticles are generally able to transfect many types of cells, but some are more specific to GBs and BTSCs,” Tzeng said.

The nanoparticles work like a virus, co-opting the cell’s own protein-making machinery, but in this case, to produce a reporter gene (used to delineate a tumor’s location) or new cancer fighting molecule. “It is possible that glioblastoma-derived cells, especially brain tumor stem cells, are more susceptible to our gene delivery approach because they divide much faster,” Tzeng added.

Not only are the particles convenient to use, the team discovered that dividing cells continued to make the new protein for as long as six weeks after application. “The gene expression peaked within a few days, which would correspond to a large initial dose of a therapeutic protein,” said Green. “The fact that gene expression can continue at a low level for a long time following injection could potentially cause a sustained, local delivery of the therapeutic protein without requiring subsequent injection or administration. The cells themselves would act as a ‘factory’ for the drug.”

Once the nanoparticles release their DNA cargo, Tzeng said the polymer quickly degrades in water, usually within days. “From there, we believe the degradation products are processed and excreted with other cellular waste products,” Tzeng said.

Members of the Green Lab are now working on identifying the intracellular mechanism responsible for facilitating cell-specific delivery. “We also plan to build additional levels of targeting into this system to make it even more specific. This includes modifying the nanoparticles with ligands to specifically bind to glioblastoma cells, making the DNA cargo able to be expressed only in GB cells, and using a DNA sequence whose product is only effective in GB cells.”

So far, the team has only successfully transfected brain tumor stem cells using these nanoparticles in a plastic dish. The next step is to test the particle in animal models.

“We hope to begin tests in vivo in the near future by implanting brain tumor stem cells into a mouse and injecting particles. We also hope to begin using functional genes that would kill cancer cells in addition to the fluorescent proteins that serve only as a marker,” Tzeng said.

Other authors who contributed to this work are Hugo Guerrero-Cázares, postdoctoral fellow in Neurosurgery and Oncology, and Joel Sunshine, an M.D.-Ph.D. candidate, and Elliott Martinez, an undergraduate leadership alliance summer student, both from Biomedical Engineering. Funding for this work came from the National Institutes of Health, Howard Hughes Medical Institute, the Robert Wood Johnson Foundation and a pilot-grant from Johns Hopkins Institute for NanoBioTechnology (INBT). Green is an affiliated faculty member of INBT. The research will be published in Issue #23 (August 2011) of the journal Biomaterials and is currently available online.

Freeze-dried gene therapy system avoids virus, complications

Story by Mary Spiro

 

Nanowires Deliver Biochemical Payloads to One Cell Among Many

Imagine being able to drop a toothpick on the head of one particular person standing among 100,000 people in a sports stadium. It sounds impossible, yet this degree of precision at the cellular level has been demonstrated by researchers affiliated with The Johns Hopkins University Institute for NanoBioTechnology. Their study was published online in June in Nature Nanotechnology.

Arrow points to nanowire placed on cell surface. (Image: Levchenko/Chien labs)

The team used precise electrical fields as “tweezers” to guide and place gold nanowires, each about one-two hundredth the size of a cell, on predetermined spots, each on a single cell. Molecules coating the surfaces of the nanowires then triggered a biochemical cascade of actions only in the cell where the wire touched, without affecting other cells nearby. The researchers say this technique could lead to better ways of studying individual cells or even cell parts, and eventually could produce novel methods of delivering medication.

Indeed, the techniques not relying on this new nanowire-based technology either are not very precise, leading to stimulation of multiple cells, or require complex biochemical alterations of the cells. With the new technique the researchers can, for instance, target cells that have cancer properties (higher cell division rate or abnormal morphology), while sparing their healthy neighbors.

“One of the biggest challenges in cell biology is the ability to manipulate the cell environment in as precise a way as possible,” said principal investigator Andre Levchenko, an associate professor of biomedical engineering in Johns Hopkins’ Whiting School of Engineering. In previous studies, Levchenko has used lab-on-a-chip or microfluidic devices to manipulate cell behavior. But, he said, lab-on-a-chip methods are not as precise as researchers would like them to be. “In microfluidic chips, if you alter the cell environment, it affects all the cells at the same time,” he said.

Such is not the case with the gold nanowires, which are metallic cylinders a few hundred nanometers or smaller in diameter. Just as the unsuspecting sports spectator would feel only a light touch from a toothpick being dropped on the head, the cell reacts only to the molecules released from the nanowire in one very precise place where the wire touches the cell’s surface.

With contributions from Chia-Ling Chien, a professor of physics and astronomy in the Krieger School of Arts and Sciences, and Robert Cammarata, a professor of materials science and engineering in the Whiting School, the team developed nanowires coated with a molecule called tumor necrosis factor-alpha (TNF?), a substance released by pathogen-gobbling macrophages, commonly called white blood cells. Under certain cellular conditions, the presence of TNF? triggers cells to switch on genes that help fight infection, but TNF? also is capable of blocking tumor growth and halting viral replication.

Exposure to too much TNF?, however, causes an organism to go into a potentially lethal state called septic shock, Levchenko said. Fortunately, TNF? stays put once it is released from the wire to the cell surface, and because the effect of TNF? is localized, the tiny bit delivered by the wire is enough to trigger the desired cellular response. Much the same thing happens when TNF? is excreted by a white blood cell.

Additionally, the coating of TNF? gives the nanowire a negative charge, making the wire easier to maneuver via the two perpendicular electrical fields of the “tweezer” device, a technique developed by Donglei Fan as part of her Johns Hopkins doctoral research in materials science and engineering. “The electric tweezers were initially developed to assemble, transport and rotate nanowires in solution,” Cammarata said. “Donglei then showed how to use the tweezers to produce patterned nanowire arrays as well as construct nanomotors and nano-oscillators. This new work with Dr. Levchenko’s group demonstrates just how extremely versatile a technique it is.”

To test the system, the team cultured cervical cancer cells in a dish. Then, using electrical fields perpendicular to one another, they were able to zap the nanowires into a pre-set spot and plop them down in a precise location. “In this way, we can predetermine the path that the wires will travel and deliver a molecular payload to a single cell among many, and even to a specific part of the cell,” Levchenko said.

During the course of this study, the team also established that the desired effect generated by the nanowire-delivered TNF? was similar to that experienced by a cell in a living organism.

The team members envision many possibilities for this method of subcellular molecule delivery. “For example, there are many other ways to trigger the release of the molecule from the wires: photo release, chemical release, temperature release. Furthermore, one could attach many molecules to the nanowires at the same time,” Levchenko said. He added that the nanowires can be made much smaller, but said that for this study the wires were made large enough to see with optical microscopy.

Ultimately, Levchenko sees the nanowires becoming a useful tool for basic research. “With these wires, we are trying to mimic the way that cells talk to each other,” he said. “They could be a wonderful tool that could be used in fundamental or applied research.” Drug delivery applications could be much further off. However, Levchenko said, “If the wires retain their negative charge, electrical fields could be used to manipulate and maneuver their position in the living tissue.”

The lead authors for this Nature Nanotechnology article were Fan, a former postdoctoral fellow in the departments of materials science and engineering and in physics and astronomy; and Zhizhong Yin, a former postdoctoral fellow in the Department of Biomedical Engineering. The co-authors included Raymond Cheong, a doctoral student in the Department of Biomedical Engineering; and Frank Q. Zhu, a former doctoral student in the Department of Physics and Astronomy.

Regarding the faculty members’ participation, Chien led the group that developed the electric tweezers technique and collaborated with Levchenko on its biological applications.

The research was funded by the National Science Foundation and the National Institutes of Health.

Johns Hopkins Institute for NanoBioTechnology

Beyond academia and industry

Penelope Lewis, acquisitions editor at the American Chemical Society, spoke at the summer’s second Professional Development Seminar hosted by The Johns Hopkins Institute for NanoBioTechnology (INBT) on June 30 at 11 a.m. in Maryland Hall 110.

Penelope Lewis, acquisitions editor at the American Chemical Society (Photo: Mary Spiro)

Lewis discussed her experience as a scientist making the transition to non-profit, scholarly publishing.

As a PhD candidate, she felt she had only two options: academia or industry. She cautioned against having “too much of a single-minded focus,” as students can get “wrapped up in studying or getting stuck in the lab.” Lewis stressed the importance of having a broad outlook and being involved in a variety of activities to know where one’s true skills and interests lie.

Penelope Lewis advocated for an interactive and investigative approach to understanding career development: “My main piece of advice is to keep your eyes and ears open when considering different careers.” Academic publishing allowed Lewis to combine her interest in writing (she minored in English) with her love of science.

“Being able to communicate your research findings and their significance is such a critical skill. It is necessary not only for securing grants and publishing papers, but also as part of a responsibility that scientists and engineers have to act as good ambassadors for science, and to transfer their excitement and understanding to the public. This is especially important in newer fields like nanotechnology,” she said.

Penelope Lewis has a BS in Chemistry (English Minor) from Indiana University, a Chemistry PhD from Pennsylvania State University, and was a Postdoctoral Research Scientist at Columbia University.

For more information about INBT’s professional development seminars, click here.

Story by Sarah Gubara, Senior, Psychology, Krieger School of Arts and Sciences

Hopkins biomedical engineering doctoral student wins Weintraub Award

Deok-Ho Kim

Deok-Ho Kim, currently a postdoctoral fellow in the department of Biomedical Engineering, was among 13 graduate students from North America chosen to receive the 2010 Harold M. Weintraub Graduate Student Award, sponsored by the Basic Sciences Division of Fred Hutchinson Cancer Research Center in Seattle, Wash. Nominations were solicited internationally and winners were selected on the basis of the quality, originality and significance of their work.

The award, established in 2000, honors the late Harold M. Weintraub, Ph.D., a founding member of the FHC’s Basic Sciences Division, who in 1995 died from brain cancer at age 49. According to a press release from FHC, “Weintraub was an international leader in the field of molecular biology; among his many contributions, he identified genes responsible for instructing cells to differentiate, or develop, into specific tissues such as muscle and bone.”

Kim will receive a certificate, travel expenses and an honorarium from the Weintraub and Groudine Fund, established to foster intellectual exchange through the promotion of programs for graduate students, fellows and visiting scholars. Kim works in the laboratory of Andre Levchenko, associate professor of biomedical engineering at Johns Hopkins University’s Whiting School of Engineering and an affiliated faculty member of the Institute for NanoBioTechnology.

Read more about Kim’s research with Levchenko here.

APL scientist to explain self-assembled artificial cilia from cobalt nanoparticles

Jason Benkoski

Jason Benkoski

Can nanoparticles be used to engineer structures that could be as flexible and useful as the cilia that help bacteria move around?

Jason Benkoski, a senior scientist at Johns Hopkins Applied Physics Laboratory and an affiliated faculty member of Johns Hopkins Institute for NanoBioTechnology, will discuss his current research in this endeavor on March 1  at 1:30 p.m. in the Rome Room, Clark 110 at the Johns Hopkins University Homewood campus. Hosted by the Department of Biomedical Engineering, this talk also will be teleconferenced to the Talbot Library in Traylor 709 at the School of Medicine.

Abstract: Taking inspiration from eukaryotic cilia, we report a method for growing dense arrays of magnetically actuated microscopic filaments. Fabricated from the bottom-up assembly of polymer-coated cobalt nanoparticles, each segmented filament measures approximately 5–15 microns in length and 23.5 nanometers in diameter, which was commensurate with the width of a single nanoparticle. Boasting the flexibility of biological cilia, we envision applications for this technology that include micropumps, micro-flow sensors, microphones with hardware-based voice detection, surfaces with enhanced thermal transfer, switchable, tunable filters, and microscopic locomotion.

Additional Links:

Jason Benkoski’s INBT profile

Johns Hopkins Applied Physics Lab