Posts Tagged ‘nanotechnology’

Ferritin Cage Enzyme Encapsulation as a New Platform for Nanotechnology

 Reporter: Irina Robu, PhD

In bionanotechnology, biological systems such as viruses, protein complexes, lipid vesicles and artificial cells, are being developed for applications in medicine and materials science.  However, the paper published by Stephan Tetter and Donald Hilvert in Angewandte Chemie International Edition show that it is possible to encapsulate proteins such as ferritin by manipulating electrostatic interactions with the negatively charged interior of the cage.The primary role of ferritin is to protect cells from the damage caused by the Fenton reaction; where, in oxidizing conditions, free Fe(II) produces harmful reactive oxygen species that can damage the cellular machinery.

The ferritin family proteins are protein nanocages that evolved to safely store iron in an oxidizing world. Since ferritin family proteins are able to mineralize and store metal ions, they have been the focus of much research for the production of metal nanoparticles and as prototypes for semiconductor production. The ferritin cage itself is highly symmetrical, and is made up of 24 subunits arranged in an octahedral symmetry. Ferritins are smaller than other protein used for protein   encapsulation.   Their  outer  diameter is only 12 nm, whereas engineered lumazine synthase variants form cages with diameters ranging from about 20 to 60 nm.The ferritin cage displays remarkable thermal and chemical stability it is likely to modify the surface of the ferritin cage through the addition of peptide and protein tags. These characteristics have made ferritins attractive vectors for the delivery of drug molecules and as scaffolds for vaccine design.

In summary, the paper published in Angewandte Chemie International Edition is the first example of protein incorporation by a ferritin.  Dr. Donald Hilvert and colleagues have shown that AfFtn not only complexes positively charged guest proteins within its naturally negatively charged luminal cavity, but that the in vitro mixing technique can be extended to the encapsulation and protection of other functional  fusion proteins.

Hence, the recent discovery of encapsulated ferritins has identified an exciting new platform for use in bio nanotechnology. The use of synthetic biology tools will allow their rapid implementation in materials science, bio-nanotechnology and medical applications.



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Novel Blood Substitute – ErythroMer

Reporter: Irina Robu, PhD

For years, scientists have tried ineffectively to create an artificial molecule that emulates the oxygen-carrying function of human red blood cell but the efforts failed because of oxygen delivery and safety issues. Now, a group of researchers led by Dr. Alan Doctor at Washington University in Saint Louis, are trying to resuscitate blood substitutes with a new nanotechnology-based, artificial red blood cell may overcome the problems that killed products designed by a team of companies such as BiopureAlliance PharmaceuticalsNorthfield Labs and even Baxter. Dr. Alan Doctor’s new company, Kalocyte is advancing the development of the

The donut-shaped artificial cells, ErythroMer are one-fiftieth the size of human red blood cells. ErythroMer is a novel blood substitute composed of a patented nanobialys nanoparticle. A special lining and control system tied to changes in blood Ph allows Erythromer to grab onto oxygen in the lungs and then dispense the oxygen in tissues where it is needed. The new artificial cells are intended to sidestep problems with vasoconstriction or narrowing of blood vessels.

Erythromer is stored freeze dried and reconstituted with water when needed but it can also be stored at room temperature which makes it for military and civilian trauma.

Trials have been successful in rats, mice, and rabbits, and human trials are planned. However, moving Erythromer into human clinical trials is still 8-10 years away.


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Walking DNA Nanorobot

Reporter: Irina Robu, PhD

New research from California Institute of Technology headed by Anupama Thubagere and Lulu Qian built robots from DNA and programmed them to sort and deliver molecules to a specified location. These robots can potentially transform the drug delivery field to how body fights infections to how microscopic measurements are made. The dominant premise of DNA robots is that rather than creating molecular devices from scratch, we can use the power of molecular machinery by building microscopic-size robots and send them to places that are then impossible to reach, such as a cell or a hard-to-reach cancerous tumor. These robots demonstrated the ability to perform simple tasks, however this latest effort ramped up a path by programming DNA robots to perform a cargo‐sorting task and possibly many other tasks.

Each robot was built from a single-stranded DNA molecule of just 53 nucleotides equipped with “legs” for walking and “arms” for picking up objects. The robot are 20 nanometers tall and their walking strides measures six nanometers long, where one nanometer is a billionth of a meter. For the cargo, the researchers used two types of molecules, each being a distinct single-stranded piece of DNA. For the tests, the researchers placed the cargo onto a random location along the surface of a two-dimensional origami DNA test platform. The walking DNA robots moved in parallel along this surface, hunting for their cargo.

To see if a robot successfully picked up and dropped off the right cargo at the right location, the researchers used two fluorescent dyes to differentiate the molecules.

The researchers guess that each DNA robot took around 300 steps to complete its task, or roughly ten times more than in previous efforts. Though, more work is needed to figure out how these DNA robots perform under different environmental conditions. This new study suggests a worthwhile methodology for scientists to continue pursuing.



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3D DNA Images  Nanoscale design of printed vascular tissue

Curator: Larry H. Bernstein, MD, FCAP


New Detailed 3D DNA Images Could Aid Nanoscale Designs

This video shows techniques that scientists used to produce 3-D reconstructions of shape fluctuations in double-helix DNA segments attached to gold nanoparticles [Lei Zhang, Dongsheng Lei, Jessica M. Smith, Meng Zhang, Huimin Tong, Xing Zhang, Zhuoyang Lu, Jiankang Liu, A. Paul Alivisatos, and Gang “Gary” Ren]


Flexible double-helix DNA segments connected to gold nanoparticles are revealed from the 3-D density maps (purple and yellow) reconstructed from individual samples using a Berkeley Lab-developed technique called individual-particle electron tomography or IPET. Projections of the structures are shown in the background grid.[Berkeley Lab]


The general chemical structure of the DNA helix was described by James Watson and Francis Crick in 1953. Over the years that followed, scientists intensely studied the molecular structure of DNA to understand its behaviorin vivo and to exploit its unique properties for nanotechnology purposes.

Now, an international team of scientists working at the Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) has captured the first high-resolution 3D images from individual double-helix DNA segments attached at either end to gold nanoparticles. The images detail the flexible structure of the DNA segments, which appear as nanoscale jump ropes.

Using a cutting-edge electron microscopy (EM) technique, called individual-particle electron tomography (IPET), the researchers were able to visualize the shapes of the coiled DNA strands, which were sandwiched between polygon-shaped gold nanoparticles, and reconstruct high-resolution 3D images. The EM technique was coupled with a protein-staining process and sophisticated software that provided structural details to the scale of approximately 2 nanometers (two billionths of a meter).

“We had no idea about what the double-strand DNA would look like between the nanogold particles,” noted senior study author Gang Ren, Ph.D., staff scientist in the Molecular Foundry at Berkeley Lab. “This is the first time for directly visualizing an individual double-strand DNA segment in 3-D.”

The findings from this study were published recently in Nature Communications in an article entitled “Three-Dimensional Structural Dynamics and Fluctuations of DNA-Nanogold Conjugates by Individual-Particle Electron Tomography.”

Dr. Ren and his colleagues hope their unique imaging technique will aid in the use of DNA segments as building blocks for molecular devices that function as nanoscale drug-delivery systems, markers for biological research, and components for computer memory and electronic devices. Additionally, the research team speculates that the new method could also lead to images of important disease-relevant proteins that have proven elusive for other imaging techniques and of the assembly process that forms DNA from separate, individual strands.

The Berkeley Lab scientists flash froze samples to preserve their structure for study with cryo-EM imaging. The distance between the two gold particles in individual samples varied from 20 to 30 nanometers based on different shapes observed in the DNA segments. They then collected a series of tilted images of the stained objects and reconstructed 14 electron-density maps that detailed the structure of individual samples using the IPET technique. They gathered a dozen confirmations for the samples and found the DNA shape variations were consistent with those measured in the flash-frozen cryo-EM samples.

While the 3D reconstructions show the basic nanoscale structure of the samples, the investigators are looking at the next steps, which will be to work on improving the resolution to the subnanometer scale.

“Even in this current state we begin to see 3D structures at 1- to 2-nanometer resolution,” Dr. Ren explained. “Through better instrumentation and improved computational algorithms, it would be promising to push the resolution to that visualizing a single DNA helix within an individual protein.”

In future studies, Dr. Ren noted that researchers could attempt to improve the imaging resolution for complex structures that incorporate more DNA segments as a sort of “DNA origami”—with the hope of building and better characterizing nanoscale molecular devices using DNA segments that can, for example, store and deliver drugs to targeted areas of the body.

“DNA is easy to program, synthesize, and replicate, so it can be used as a special material to quickly self-assemble into nanostructures and to guide the operation of molecular-scale devices,” Dr. Ren stated. “Our current study is just a proof of concept for imaging these kinds of molecular devices’ structures.”


Three-dimensional structural dynamics and fluctuations of DNA-nanogold conjugates by individual-particle electron tomography

Lei ZhangDongsheng LeiJessica M. Smith, …., Jiankang LiuA. Paul Alivisatos & Gang Ren
Nature Communications7,Article number:11083

DNA base pairing has been used for many years to direct the arrangement of inorganic nanocrystals into small groupings and arrays with tailored optical and electrical properties. The control of DNA-mediated assembly depends crucially on a better understanding of three-dimensional structure of DNA-nanocrystal-hybridized building blocks. Existing techniques do not allow for structural determination of these flexible and heterogeneous samples. Here we report cryo-electron microscopy and negative-staining electron tomography approaches to image, and three-dimensionally reconstruct a single DNA-nanogold conjugate, an 84-bp double-stranded DNA with two 5-nm nanogold particles for potential substrates in plasmon-coupling experiments. By individual-particle electron tomography reconstruction, we obtain 14 density maps at ~2-nm resolution. Using these maps as constraints, we derive 14 conformations of dsDNA by molecular dynamics simulations. The conformational variation is consistent with that from liquid solution, suggesting that individual-particle electron tomography could be an expected approach to study DNA-assembling and flexible protein structure and dynamics.

Organic–inorganic-hybridized nanocrystals are a valuable class of new materials that are suitable for addressing many emerging challenges in biological and material sciences1, 2. Nanogold and quantum dot conjugates have been used extensively as biomolecular markers3, 4, whereas DNA base pairing has directed the self-assembly of discrete groupings and arrays of organic and inorganic nanocrystals in the formation of a network solid for electronic devices and memory components5. Discretely hybridized gold nanoparticles conjugated to DNA were developed as a molecular ruler to detect sub-nanometre distance changes via plasmon-coupling-mediated variations in dark-field light scattering3, 6. For many of these applications, it is desirable to obtain nanocrystals functionalized with discrete numbers of DNA strands7, 8. In all of these circumstances, the soft components can fluctuate, and the range of these structural deviations have not previously been determined with a degree of rigour that could help influence the future design and use of these assemblies.

Conformational flexibility and dynamics of the DNA-nanogold conjugates limit the structural determination by X-ray crystallography, nuclear magnetic resonance spectroscopy and single-particle cryo-electron microscopic (cryo-EM) reconstruction because they do not crystallize, are not sufficiently small for nuclear magnetic resonance studies and cannot be classified into a limited number of classes for single-particle EM reconstruction. In addition, three-dimensional (3D) structure averaged from tens of thousands of different macromolecular particles obtained without prior knowledge of the macromolecular structural flexibility could result in an absence of flexible domains upon using the single-particle reconstruction method, for example, two ankyrin repeated regions of TRPV1 were absent in its atomic resolution 3D density map9.

A fundamental experimental solution to reveal the structure of a flexible macromolecule should be based on the determination of each individual macromolecule’s structure10. Electron tomography (ET) provides high-resolution images of a single object from a series of tilted viewing angles11. ET has been applied to reveal the 3D structure of a cell section and an individual bacterium at nanometre-scale resolution12. However, reconstruction from an individual macromolecule at an intermediate resolution (1–3nm) remains challenging due to small molecular weight and low image contrast. Although, the first 3D map of an individual macromolecule, a fatty acid synthetase molecule, was reconstructed from negative-staining (NS) ET by Hoppe et al.13, serious doubts have been raised regarding the validity of this structure14, as this molecule received a radiation dose hundreds of times greater than the reported damage threshold15. Recently, we investigated the possibility based on simulated and real experimental NS and cryo-ET images10. We showed that a single-protein 3D structure at an intermediate resolution (1–3nm) is potentially achieved using our proposed individual-particle ET (IPET) method10, 16, 17, 18. IPET, an iterative refinement process using automatically generated dynamic filters and soft masks, requires no pre-given initial model, class averaging or lattice, but can tolerate small tilt errors and large-scale image distortion via decreasing the reconstruction image size to reduce the negative effects on 3D reconstruction. IPET allows us to obtain a ‘snapshot’ single-molecule 3D structure of flexible proteins at an intermediate resolution, and can be even used to reveal the macromolecular dynamics and fluctuation17.

Here we use IPET, cryo-EM and our previously reported optimized NS (OpNS)19, 20 techniques to investigate the morphology and 3D structure of hybridized DNA-nanogold conjugates. These conjugates were self-assembled from a mixture of two monoconjugates, each consisting of 84-bp single-stranded DNA and a 5-nm nanogold particle. The dimers were separated by anion-exchange high-performance liquid chromatography (HPLC) and agarose gel electrophoresis as potential substrates in plasmon-coupling experiments. By OpNS-ET imaging and IPET 3D reconstruction, we reconstruct a total of 14 density maps at a resolution of ~2nm from 14 individual double-stranded DNA (dsDNA)-nanogold conjugates. Using these maps as constraints, we derive 14 conformations of dsDNA by projecting a standard flexible dsDNA model onto the observed maps using molecular dynamics (MD) simulations. The variation of the conformations was largely consistent with that from liquid solution, and suggests that the IPET approach provides a most complete experimental determination of flexibility and fluctuation range of these directed nanocrystal assemblies to date. The general features revealed by this experiment can be expected to occur in a broad range of DNA-assembled nanostructures and flexible proteins.


Although the direct imaging of dsDNA has been previously reported using heavy metal shadowing32, 33 and NS methods34, 35, 36, to the best of our knowledge, the 3D structure of an individual dsDNA strand has not previously been achieved. It has been thought that individual dsDNA would be destroyed under the high energy of the electron beam before a 3D reconstruction, or even a 2D image, is able to be achieved. Our NS tilt images showing fibre-shaped dsDNA bridging two conjugated nanogold particles demonstrated that the dsDNA can in fact be directly visualized using EM, which is consistent with the recently reported single-molecule DNA sequencing technique via TEM36. The resolutions of our density maps ranged from ~14 to ~23Å, demonstrating that an intermediate-resolution 3D structure can be obtained for each individual macromolecule. This capability is consistent with our earlier report of a ~20-Å resolution 3D reconstruction of an individual IgG1 antibody using the same approach16, 17.

Notably, a total dose of ~2,000eÅ−2 used in our ET data acquisition is significantly above the limitation conventionally used in cryo-EM (~80–100eÅ−2), which can be suspected to have certain artefact from radiation damage. In cryo-EM, the radiation damage could cause sample bubbling, deformation and knockout effects; in NS, only the knockout phenomena is often observed, in which the protein is surrounded by heavy atoms that were kicked out by electron beam. Since the sample was coated with heavy metal atoms and were dried in air, the bubbling and deformation phenomena were not usually observed. The heavy metal atoms that coat the surface of the biomolecule can provide a much higher electron scattering than from a biomolecule only inside lighter atoms. The scattering is sufficiently high to provide enough image contrast at our 120-kV high tension; thus, a further increase to the scattering ability by reducing the high tension to 80kV may not be necessary for this NS sample. In addition, the heavy atoms can provide more radiation resistance and allow the sample to be imaged under a higher dose condition. The exact dose limitation for NS is still unknown. The radiation damage related artefact in NS samples is knockout, which could reduce the image contrast and lower the tilt image alignment accuracy and 3D reconstruction resolution. In our study, a total dose of 2,000eÅ−2 did not cause any obvious knockout phenomena, but provides a sufficiently high contrast for the otherwise barely visible DNA conformations in each tilt series. The direct confirmation of visible DNA in each tilt image is essentially important to us to validate each 3D reconstruction, especially considering this relatively new approach.

Our 3D reconstruction algorithm used an ab initio real-space reference-projection match iterative algorithm to correct the centres of each tilt images, in which the equal tilt angle step for 3D reconstruction of a low contrast and asymmetric macromolecule was used. This method is different from recently reported Fourier-based iterative algorithm, termed equally sloped tomography, in which the pseudo-polar fast Fourier transform, the oversampling method and internal lattice of a targeted nanoparticle are used to achieve 3D reconstruction at atomic resolution37.

It is generally challenging to achieve visualization and 3D reconstruction on an individual, small and asymmetric macromolecule by other conventional methods; our method demonstrated its capability for 3D reconstruction of 52kDa 84-bp dsDNA through these studies: IgG1 antibody 3D structural fluctuation17, peptide-induced conformational changes on flexible IgG1 antibody5, floppy liposome surface binding with 53-kDa proteins38, all of which suggest that this method could be used to serve the community as a novel tool for studying flexible macromolecular structures, dynamics and fluctuations of proteins, and for catching the intermediate 3D structure of protein assembling.

DNA-based self-assembling materials have been developed for use in materials science and biomedical research, such as DNA origami designed for targeted drug delivery. The structure, design and control require feedback from the 3D structure, which could validate the design hypothesis, optimize the synthesis protocol and improve the reproducible capability, while even providing insight into the mechanism of DNA-mediated assembly.


Printing Vascular Tissue

from Wyss Institute for Biologically Inspired Engineering at Harvard University

Printing vessel vasculature is essential for sustaining functional living tissues. Until now, bioengineers have had difficulty building thick tissues, lacking a method to embed vascular networks. A 3D bioprinting method invented at the Wyss Institute and Harvard SEAS embeds a grid of vasculature into thick tissue laden with human stem cells and connective matrix. Printed within a custom-made housing, this method can be used to create tissue of any shape. Once printed, an inlet and outlet own opposite ends are perfused with fluids, nutrients, and cell growth factors, which control stem cell differentiation and sustain cell functions. By flowing growth factors through the vasculature, stem cells can be differentiated into a variety of tissue cell types. This vascularized 3D printing process could open new doors to tissue replacement and engineering. Footage credit: David KA.S. Gladman, E. Matsumoto, L.K. Sanders, and J.A. Lewis / Wyss Institute at Harvard University For more information, please visit:

Scaling up tissue engineering

In this video, the Wyss Institute and Harvard SEAS team uses a customizable 3D bioprinting method to build a thick vascularized tissue structure comprising human stem cells, collective matrix, and blood vessel endothelial cells. Their work sets the stage for advancement of tissue replacement and tissue engineering techniques. Credit: Lewis Lab, Wyss Institute at Harvard University


Bioprinting technique creates thick 3D tissues composed of human stem cells and embedded vasculature, with potential applications in drug testing and regenerative medicine

(CAMBRIDGE, Massachusetts)  — A team at the Wyss Institute for Biologically Inspired Engineering at Harvard University and the Harvard John A. Paulson School for Engineering and Applied Sciences (SEAS) has invented a method for 3D bioprinting thick vascularized tissue constructs composed of human stem cells, extracellular matrix, and circulatory channels lined with endothelial blood vessel cells. The resulting network of vasculature contained within these deep tissues enables fluids, nutrients and cell growth factors to be controllably perfused uniformly throughout the tissue. The advance is reported March 7 in the journal Proceedings of the National Academy of Sciences.

“This latest work extends the capabilities of our multi-material bioprinting platform to thick human tissues, bringing us one step closer to creating architectures for tissue repair and regeneration,” says Wyss Core Faculty member Jennifer A. Lewis, Sc.D., senior author on the study, who is also the Hansörg Wyss Professor of Biologically Inspired Engineering at SEAS.

Three-dimensional bioprinting of thick vascularized tissues

David B. Koleskya,1Kimberly A. Homana,1Mark A. Skylar-Scotta,1, and Jennifer A. Lewisa,2     aSchool of Engineering and Applied Sciences, Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA 02138
PNAS  2016; 113(12): 3179–3184


Current tissue manufacturing methods fail to recapitulate the geometry, complexity, and longevity of human tissues. We report a multimaterial 3D bioprinting method that enables the creation of thick human tissues (>1 cm) replete with an engineered extracellular matrix, embedded vasculature, and multiple cell types. These 3D vascularized tissues can be actively perfused with growth factors for long durations (>6 wk) to promote differentiation of human mesenchymal stem cells toward an osteogenic lineage in situ. The ability to construct and perfuse 3D tissues that integrate parenchyma, stroma, and endothelium is a foundational step toward creating human tissues for ex vivo and in vivo applications.

The advancement of tissue and, ultimately, organ engineering requires the ability to pattern human tissues composed of cells, extracellular matrix, and vasculature with controlled microenvironments that can be sustained over prolonged time periods. To date, bioprinting methods have yielded thin tissues that only survive for short durations. To improve their physiological relevance, we report a method for bioprinting 3D cell-laden, vascularized tissues that exceed 1 cm in thickness and can be perfused on chip for long time periods (>6 wk). Specifically, we integrate parenchyma, stroma, and endothelium into a single thick tissue by coprinting multiple inks composed of human mesenchymal stem cells (hMSCs) and human neonatal dermal fibroblasts (hNDFs) within a customized extracellular matrix alongside embedded vasculature, which is subsequently lined with human umbilical vein endothelial cells (HUVECs). These thick vascularized tissues are actively perfused with growth factors to differentiate hMSCs toward an osteogenic lineage in situ. This longitudinal study of emergent biological phenomena in complex microenvironments represents a foundational step in human tissue generation.

The ability to manufacture human tissues that replicate the essential spatial (1), mechanochemical (2, 3), and temporal aspects of biological tissues (4) would enable myriad applications, including 3D cell culture (5), drug screening (6, 7), disease modeling (8), and tissue repair and regeneration (9, 10). Three-dimensional bioprinting is an emerging approach for creating complex tissue architectures (10, 11), including those with embedded vasculature (1215), that may address the unmet needs of tissue manufacturing. Recently, Miller et al. (15) reported an elegant method for creating vascularized tissues, in which a sacrificial carbohydrate glass is printed at elevated temperature (>100 °C), protectively coated, and then removed, before introducing a homogeneous cell-laden matrix. Kolesky et al. (14) developed an alternate approach, in which multiple cell-laden, fugitive (vasculature), and extracellular matrix (ECM) inks are coprinted under ambient conditions. However, in both cases, the inability to directly perfuse these vascularized tissues limited their thickness (1–2 mm) and culture times (<14 d). Here, we report a route for creating thick vascularized tissues (≥1 cm) within 3D perfusion chips that provides unprecedented control over tissue composition, architecture, and microenvironment over several weeks (>6 wk). This longitudinal study of emergent biological phenomena in complex microenvironments represents a foundational step in human tissue generation.

Central to the fabrication of thick vascularized tissues is the design of biological, fugitive, and elastomeric inks for multimaterial 3D bioprinting. To satisfy the concomitant requirements of processability, heterogeneous integration, biocompatibility, and long-term stability, we first developed printable cell-laden inks and castable ECM based on a gelatin and fibrinogen blend (16). Specifically, these materials form a gelatin–fibrin matrix cross-linked by a dual-enzymatic, thrombin and transglutaminase (TG), strategy (Fig. 1and SI Appendix, Fig. S1). The cell-laden inks must facilitate printing of self-supporting filamentary features under ambient conditions as well as subsequent infilling of the printed tissue architectures by casting without dissolving or distorting the patterned construct (Fig. 1A). The thermally reversible gelation of the gelatin–fibrinogen network enables its use in both printing and casting, where gel and fluid states are required, respectively (SI Appendix, Fig. S2). Thrombin is used to rapidly polymerize fibrinogen (17), whereas TG is a slow-acting Ca2+-dependent enzymatic cross-linker that imparts the mechanical and thermal stability (18) needed for long-term perfusion. Notably, the cell-laden ink does not contain either enzyme to prevent polymerization during printing. However, the castable matrix contains both thrombin and TG, which diffuse into adjacent printed filaments, forming a continuous, interpenetrating polymer network, in which the native fibrillar structure of fibrin is preserved (SI Appendix, Fig. S3). Importantly, our approach allows arbitrarily thick tissues to be fabricated, because the matrix does not require UV curing (19), which has a low penetration depth in tissue (20) and can be readily expanded to other biomaterials, including fibrin and hyaluronic acid (SI Appendix, Fig. S4).


Fig. 1.

Fig. 1.

Three-dimensional vascularized tissue fabrication. (A) Schematic illustration of the tissue manufacturing process. (i) Fugitive (vascular) ink, which contains pluronic and thrombin, and cell-laden inks, which contain gelatin, fibrinogen, and cells, are printed within a 3D perfusion chip. (ii) ECM material, which contains gelatin, fibrinogen, cells, thrombin, and TG, is then cast over the printed inks. After casting, thrombin induces fibrinogen cleavage and rapid polymerization into fibrin in both the cast matrix, and through diffusion, in the printed cell ink. Similarly, TG diffuses from the molten casting matrix and slowly cross-links the gelatin and fibrin. (iii) Upon cooling, the fugitive ink liquefies and is evacuated, leaving behind a pervasive vascular network, which is (iv) endothelialized and perfused via an external pump. (B) HUVECs growing on top of the matrix in 2D, (C) HNDFs growing inside the matrix in 3D, and (D) hMSCs growing on top of the matrix in 2D. (Scale bar: 50 µm.) (E and F) Images of printed hMSC-laden ink prepared using gelatin preprocessed at 95 °C before ink formation (E) as printed and (F) after 3 d in the 3D printed filament where actin (green) and nuclei (blue) are stained. (G) Gelatin preprocessing temperature affects the plateau modulus and cell viability after printing. Higher temperatures lead to lower modulus and higher HNDF viability postprinting. (H) Photographs of interpenetrated sacrificial (red) and cell inks (green) as printed on chip. (Scale bar: 2 mm.) (I) Top-down bright-field image of sacrificial and cell inks. (Scale bar: 50 µm.). (J–L) Photograph of a printed tissue construct housed within a perfusion chamber (J) and corresponding cross-sections (K and L). (Scale bars: 5 mm.)

To construct thick, vascularized tissues within 3D perfusion chips, we coprinted cell-laden, fugitive, and silicone inks (Fig. 1 H and I). First, the silicone ink is printed on a glass substrate and cured to create customized perfusion chips (Movie S1 and SI Appendix, Fig. S1). Next, the cell-laden and fugitive inks are printed on chip, and then encapsulated with the castable ECM (Fig. 1 J–L and Movie S2). The fugitive ink, which defines the embedded vascular network, is composed of a triblock copolymer [i.e., polyethylene oxide (PEO)–polypropylene oxide (PPO)–PEO]. This ink can be removed from the fabricated tissue upon cooling to roughly 4 °C, where it undergoes a gel-to-fluid transition (14, 23). This process yields a pervasive network of interconnected channels, which are then lined with HUVECs. The resulting vascularized tissues are perfused via their embedded vasculature on chip over long time periods using an external pump (Movie S3) that generates smooth flow over a wide range of flow rates (24).

Movie S3.

Fluorescent microscopy video of different perfusion rates through the embedded vasculature within the printed 3D tissue microenvironments.

Fig. 2.

Three-dimensional vascularized tissues remain stable during long-term perfusion. (A) Schematic depicting a single HUVEC-lined vascular channel supporting a fibroblast cell-laden matrix and housed within a 3D perfusion chip. (B and C) Confocal microscopy image of the vascular network after 42 d, CD-31 (red), vWF (blue), and VE-Cadherin (magenta). (Scale bars: 100 µm.) (D) Long-term perfusion of HUVEC-lined (red) vascular network supporting HNDF-laden (green) matrix shown by top-down (Left) and cross-sectional confocal microscopy at 45 d (Right). (Scale bar: 100 µm.) (E) Quantification of barrier properties imparted by endothelial lining of channels, demonstrated by reduced diffusional permeability of FITC-dextran. (F) GFP-HNDF distribution within the 3D matrix shown by fluorescent intensity as a function of distance from vasculature.

Movie S4.

Confocal microscopy video of cross-section through vascularized tissue after 45 d of perfusion.

To explore emergent phenomena in complex microenvironments, we created a heterogeneous tissue architecture (>1 cm thick and 10 cm3 in volume) by printing a hMSC-laden ink into a 3D lattice geometry along with intervening in- and out-of-plane (vertical) features composed of fugitive ink, which ultimately transform into a branched vascular network lined with HUVECs. After printing, the remaining interstitial space is infilled with an HNDF-laden ECM (Fig. 3A) to form a connective tissue that both supports and binds to the printed stem cell-laden and vascular features. In this example, fibroblasts serve as model cells that surround the heterogeneously patterned stem cells and vascular network. These model cells could be replaced with either support cells (e.g., immune cells or pericytes) or tissue-specific cells (e.g., hepatocytes, neurons, or islets) in future embodiments. The embedded vascular network is designed with a single inlet and outlet that provides an interface between the printed tissue and the perfusion chip. This network is symmetrically branched to ensure uniform perfusion throughout the tissue, including deep within its core. In addition to providing transport of nutrients, oxygen, and waste materials, the perfused vasculature is used to deliver specific differentiation factors to the tissue in a more uniform manner than bulk delivery methods, in which cells at the core of the tissue are starved of factors (25). This versatile platform (Fig. 3A) is used to precisely control growth and differentiation of the printed hMSCs. Moreover, both the printed cellular architecture and embedded vascular network are visible macroscopically with this thick tissue (Fig. 3B).

Fig. 3.

Fig. 3.

Osteogenic differentiation of thick vascularized tissue. (A) Schematic depicting the geometry of the printed heterogeneous tissue within the customized perfusion chip, wherein the branched vascular architecture pervades hMSCs that are printed into a 3D lattice architecture, and HNDFs are cast within an ECM that fills the interstitial space. (B) Photographs of a printed tissue construct within and removed from the customized perfusion chip. (C) Comparative cross-sections of avascular tissue (Left) and vascularized tissue (Right) after 30 d of osteogenic media perfusion with alizarin red stain showing location of calcium phosphate. (Scale bar: 5 mm.) (D) Confocal microscopy image through a cross-section of 1-cm-thick vascularized osteogenic tissue construct after 30 d of active perfusion and in situ differentiation. (Scale bar: 1.5 mm.) (E) Osteocalcin intensity across the thick tissue sample inside the red lines shown in C. (F) High-resolution image showing osteocalcin (purple) localized within hMSCs, and they appear to take on symmetric osteoblast-like morphologies. (Scale bar: 100 µm.) After 30 d (Gand H), thick tissue constructs are stained for collagen-I (yellow), which appears to be localized near hMSCs. (Scale bars: 200 µm.) (I) Alizarin red is used to stain calcium phosphate deposition, and fast blue is used to stain AP, indicating tissue maturation and differentiation over time. (Scale bar: 200 µm.)

In summary, thick, vascularized human tissues with programmable cellular heterogeneity that are capable of long-term (>6-wk) perfusion on chip have been fabricated by multimaterial 3D bioprinting. The ability to recapitulate physiologically relevant, 3D tissue microenvironments enables the exploration of emergent biological phenomena, as demonstrated by observations of in situ development of hMSCs within tissues containing a pervasive, perfusable, endothelialized vascular network. Our 3D tissue manufacturing platform opens new avenues for fabricating and investigating human tissues for both ex vivo and in vivo applications.


Steve Dufourny Hello Mr Bernstein, it is relevant.I have a model with quantum sphères and spherical volumes correlated with my theory of spherisation in theoretical physics(in a very simple resume quant sphères…..spherisation encodings…….Cosmol sphères…….Universal sphères with a central BH)more two équations about matter and energy and sphères.The quantizations and properties can be computed.The adn, amino acids ,protiens INE ASE are the keys.I search the main gravitationalcodes.It is more far than our standard model in logic.Regards


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Nanoparticle Delivery to Cancer Drug Targets

Curator: Larry H. Bernstein, MD, FCAP



Image for unlabelled figure

Lipid-based drug delivery (LBDD) systems have gained much importance in the recent years due to their ability to improve the solubility and bioavailability of drugs with poor water solubility9. The absorption of drug from lipid based formulation depends on numerous factors, including particle size, degree of emulsification, rate of dispersion and precipitation of drug upon dispersion4 and 10.
Diagram of liposome showing a phospholipid bilayer surrounding an aqueous interior
This diagram shows several ways in which transport across the BBB works. For nanoparticle delivery across the BBB, the most common mechanisms are receptor-mediated transcytosis and adsorptive transcytosis
Targeted Polymeric Nanotherapeutics
Author: Jeff Hrkach

New drug-delivery strategies will lead to safer, more effective treatments for previously intractable diseases.

This paper provides an overview of steps being taken by BIND Biosciences Inc. to translate innovative research conducted at the Massachusetts Institute of Technology (MIT) and Harvard Medical School into novel, targeted, polymeric nanotherapeutics.

Figure 1 Schematic diagram of a BIND targeted polymeric nanoparticle.
Schematic diagram of a BIND targeted polymeric nanoparticle.

Drugs delivered by nanoparticles hold promise for targeted treatment of many diseases, including cancer. However, the particles have to be injected into patients, which has limited their usefulness so far.

Now, researchers from MIT and Brigham and Women’s Hospital (BWH) have developed a new type of nanoparticle that can be delivered orally and absorbed through the digestive tract, allowing patients to simply take a pill instead of receiving injections.

The BIND Targeted Nanoparticle
BIND Biosciences Inc. (BIND), a biopharmaceutical company that was founded upon the research of two pioneers in nanoparticle drug delivery, Professor Robert Langer of MIT and Professor Omid Farokhzad of Brigham and Women’s Hospital of the Harvard Medical School, has developed methods of engineering targeted nanoparticles composed of biodegradable and biocompatible polymers with precise biophysicochemical properties optimized to deliver drugs for specific therapeutic applications (Gu et al., 2008).

The foundational research by Langer and Farokhzad put BIND in a position to pursue the development of targeted polymeric nanotherapeutics for treating several diseases. BIND’s lead program is focused on translating their innovative academic findings into improved treatments for patients with cancer. The BIND technology offers a unique combination of long-circulating nanoparticles with the capability of targeting diseased cells specifically and releasing drugs from nanoparticles in a programmable, controlled way.

Figure 1 is a schematic diagram of a BIND targeted nanoparticle. The targeting ligand enables the nano-particle to recognize specific proteins or receptors on the surface of cells involved in disease, or in the surrounding extracellular matrix, and bind, with high specificity and avidity, to its intended cellular target site. Many types of cancer have been shown to have cell-surface receptors that are highly expressed on the cancer cells (e.g., prostate cancer [prostate-specific membrane antigen, PSMA], breast cancer [human epidermal growth factor receptor 2, HER-2], and lung cancer [epidermal growth factor receptor, EGFR]), and many drugs are being evaluated that might improve treatment outcomes.

Surface Functionalization
Surface functionalization imparted by a PEG component shields the targeted nanoparticles from MPS immune clearance, while providing an attachment site for the targeting ligand on the particle surface at precise, controlled levels through proprietary linkage strategies. A key to the successful development of BIND targeted nanoparticles is the optimization of the nanoparticle surface, which requires a precise balance between the targeting ligand and PEG coverage so the nanoparticle surface is masked enough to provide circulation times long enough to reach the disease site and enough targeting ligand on the surface to effectively bind to the target cell surface receptors. This delicate balance requires precise control over the nanoparticle production process. It also requires the discovery and selection of ligands that are potent and specific enough to bind selectively to the targeted disease cells while remaining bound to the nanoparticle surface.

The polymer matrix, the bulk of the nanoparticle composition, encapsulates the drug in a matrix of clinically safe, validated biodegradable and biocompatible polymers that can be designed to provide appropriate particle size, drug-loading level, drug-release profile, and other critical properties. A variety of drugs or therapeutic payloads can be incorporated into the targeted nanoparticles, including small molecules, peptides, proteins, and nucleic acids, such as siRNA.

Composite magnetic nanoparticle drug delivery system
US 20120265001 A1

A composite magnetic nanoparticle drug delivery system provides targeted controlled release chemotherapies for cancerous tumors and inflammatory diseases. The magnetic nanoparticle includes a biocompatible and biodegradable polymer, a magnetic nanoparticle, the biological targeting agent human serum albumin, and a therapeutic pharmaceutical composition. The composite nanoparticles are prepared by oil-in-oil emulsion/solvent evaporation and high shear mixing. An externally applied magnetic field draws the magnetic nanoparticles to affected areas. The biological targeting agent draws the nanoparticles into the affected tissues. Polymer degradation provides controlled time release delivery of the pharmaceutical agent.

Patent Drawing
Patent Drawing
Nanoparticle delivery systems for cancer therapy: advances in clinical and preclinical research.
Nanoparticle drug delivery systems exploit the abnormal characteristics of tumour tissues to selectively target their payloads to cancer cells, either by passive, active or triggered targeting.
Drug delivery and nanoparticles: Applications and hazards
The use of nanotechnology in medicine and more specifically drug delivery is set to spread rapidly. Currently many substances are under investigation for drug delivery and more specifically for cancer therapy. Interestingly pharmaceutical sciences are using nanoparticles to reduce toxicity and side effects of drugs and up to recently did not realize that carrier systems themselves may impose risks to the patient. The kind of hazards that are introduced by using nanoparticles for drug delivery are beyond that posed by conventional hazards imposed by chemicals in classical delivery matrices. For nanoparticles the knowledge on particle toxicity as obtained in inhalation toxicity shows the way how to investigate the potential hazards of nanoparticles. The toxicology of particulate matter differs from toxicology of substances as the composing chemical(s) may or may not be soluble in biological matrices, thus influencing greatly the potential exposure of various internal organs. This may vary from a rather high local exposure in the lungs and a low or neglectable exposure for other organ systems after inhalation. However, absorbed species may also influence the potential toxicity of the inhaled particles. For nanoparticles the situation is different as their size opens the potential for crossing the various biological barriers within the body. From a positive viewpoint, especially the potential to cross the blood brain barrier may open new ways for drug delivery into the brain. In addition, the nanosize also allows for access into the cell and various cellular compartments including the nucleus. A multitude of substances are currently under investigation for the preparation of nanoparticles for drug delivery, varying from biological substances like albumin, gelatine and phospholipids for liposomes, and more substances of a chemical nature like various polymers and solid metal containing nanoparticles. It is obvious that the potential interaction with tissues and cells, and the potential toxicity, greatly depends on the actual composition of the nanoparticle formulation. This paper provides an overview on some of the currently used systems for drug delivery. Besides the potential beneficial use also attention is drawn to the questions how we should proceed with the safety evaluation of the nanoparticle formulations for drug delivery. For such testing the lessons learned from particle toxicity as applied in inhalation toxicology may be of use. Although for pharmaceutical use the current requirements seem to be adequate to detect most of the adverse effects of nanoparticle formulations, it can not be expected that all aspects of nanoparticle toxicology will be detected. So, probably additional more specific testing would be needed.

Recent years have witnessed unprecedented growth of research and applications in the area of nanoscience and nanotechnology. There is increasing optimism that nanotechnology, as applied to medicine, will bring significant advances in the diagnosis and treatment of disease. Anticipated applications in medicine include drug delivery, both in vitro and in vivo diagnostics, nutraceuticals and production of improved biocompatible materials (Duncan 2003; De Jong et al 2005; ESF 2005; European Technology Platform on Nanomedicine 2005; Ferrari 2005). Engineered nanoparticles are an important tool to realize a number of these applications. It has to be recognized that not all particles used for medical purposes comply to the recently proposed and now generally accepted definition of a size ≤100 nm (The Royal Society and Royal Academy of Engineering 2004). However, this does not necessarily has an impact on their functionality in medical applications. The reason why these nanoparticles (NPs) are attractive for medical purposes is based on their important and unique features, such as their surface to mass ratio that is much larger than that of other particles, their quantum properties and their ability to adsorb and carry other compounds. NPs have a relatively large (functional) surface which is able to bind, adsorb and carry other compounds such as drugs, probes and proteins. However, many challenges must be overcome if the application of nanotechnology is to realize the anticipated improved understanding of the patho-physiological basis of disease, bring more sophisticated diagnostic opportunities, and yield improved therapies. Although the definition identifies nanoparticles as having dimensions below 0.1 μm or 100 nm, especially in the area of drug delivery relatively large (size >100 nm) nanoparticles may be needed for loading a sufficient amount of drug onto the particles. In addition, for drug delivery not only engineered particles may be used as carrier, but also the drug itself may be formulated at a nanoscale, and then function as its own “carrier” (Cascone et al 2002; Baran et al 2002; Duncan 2003; Kipp 2004). The composition of the engineered nanoparticles may vary. Source materials may be of biological origin like phospholipids, lipids, lactic acid, dextran, chitosan, or have more “chemical” characteristics like various polymers, carbon, silica, and metals. The interaction with cells for some of the biological components like phospholipids will be quite different compared to the non biological components such as metals like iron or cadmium. Especially in the area of engineered nanoparticles of polymer origin there is a vast area of possibilities for the chemical composition.

Although solid NPs may be used for drug targeting, when reaching the intended diseased site in the body the drug carried needs to be released. So, for drug delivery biodegradable nanoparticle formulations are needed as it is the intention to transport and release the drug in order to be effective. However, model studies to the behavior of nanoparticles have largely been conducted with non-degradable particles. Most data concerning the biological behavior and toxicity of particles comes from studies on inhaled nanoparticles as part of the unintended release of ultrafine or nanoparticles by combustion derived processes such as diesel exhaust particles (reviewed by Oberdörster 1996; Donaldson et al 2001, 2004; Borm 2002;Donaldson and Stone 2003; Dreher 2004; Kreyling et al 2004; Oberdörster, Oberdörster et al 2005). Research has demonstrated that exposure to these combustion derived ultrafine particles/nanoparticles is associated with a wide variety of effects (Donaldson et al 2005) including pulmonary inflammation, immune adjuvant effects (Granum and Lovik 2002) and systemic effects including blood coagulation and cardiovascular effects (Borm and Kreyling 2004;Oberdorster, Oberdörster et al 2005). Since the cut-off size for both ultrafine and nanoparticles (100 nm) is the same, now both terms are used as equivalent. Based on the adverse effects of ultrafine particles as part of environmental pollution, engineered nanoparticles may be suspected of having similar adverse effects. It is the purpose of this review to use this database on combustion derived nanpoarticles (CDNP) obtained by inhalation toxicology and epidemiology and bridge the gap to engineered nanoparticles.

Nanoparticles and drug delivery

Drug delivery and related pharmaceutical development in the context of nanomedicine should be viewed as science and technology of nanometer scale complex systems (10–1000 nm), consisting of at least two components, one of which is a pharmaceutically active ingredient (Duncan 2003; Ferrari 2005), although nanoparticle formulations of the drug itself are also possible (Baran et al 2002; Cascone et al 2002; Duncan 2003; Kipp 2004). The whole system leads to a special function related to treating, preventing or diagnosing diseases sometimes called smart-drugs or theragnostics (LaVan et al 2003). The primary goals for research of nano-bio-technologies in drug delivery include:

  • More specific drug targeting and delivery,
  • Reduction in toxicity while maintaining therapeutic effects,
  • Greater safety and biocompatibility, and
  • Faster development of new safe medicines.

The main issues in the search for appropriate carriers as drug delivery systems pertain to the following topics that are basic prerequisites for design of new materials. They comprise knowledge on (i) drug incorporation and release, (ii) formulation stability and shelf life (iii) biocompatibility, (iv) biodistribution and targeting and (v) functionality. In addition, when used solely as carrier the possible adverse effects of residual material after the drug delivery should be considered as well. In this respect biodegradable nanoparticles with a limited life span as long as therapeutically needed would be optimal.

Table 1  presents some of the types of chemical structures and possibilities for the preparation of nanoscale materials used as pharmaceutical carrier system (reviewed in Borm and Muller-Schulte 2006). Certainly none of the so far developed carriers fulfill all the parameters mentioned above to the full extent; the progress made in nanotechnology inter alia emerging from the progress in the polymer-chemistry, however, can provide an intriguing basis to tackle this issue in a promising way.

Table 1

Overview of nanoparticles and their applications in Life Sciences

Particle class Materials Application
Natural materials or derivatives Chitosan
Drug/Gene delivery
Dendrimers Branched polymers Drug delivery
Fullerenes Carbon based carriers Photodynamics
Drug delivery
Polymer carriers Polylactic acid
Block copolymers
Drug/gene delivery
Ferrofluids SPIONS
Imaging (MRI)
Quantum dots Cd/Zn-selenides Imaging
In vitro diagnostics
Various Silica-nanoparticles
Mixtures of above
Gene delivery

Nanoparticle delivery system to tackle cancer

Directing drug treatment to tumors is a hit-or-miss activity. Considerable research efforts are going into improving targeted drug delivery. A new approach centers on nanotechnology
Nanoparticle delivery system to tackle cancer 

Cancer drugs are injected into the bloodstream and move through the body seeking out fast-growing cancer cells. One consequence of chemotherapy is the unintended effect on different parts of the body, including messing up the digestive system. Such side effects can be minimized if the drug is better targeted.

Another consequence of the poor targeting of some chemo drugs is that they miss cancer cells entirely.

For these reasons, different research groups are focusing on drug delivery: finding smart ways to direct the anti-cancer drug to the required target. One such research team is led by Professor Warren Chan of the University of Texas.

Professor Chan thinks the answer to more effective targeting is the use of nanoparticles. In trials, the research group has used nanoparticles attached to strands of DNA that can, remarkably, change shape to gain improved access to cancerous tissue.

Interviewed by Pharmaceutical Processing, Professor Chan explains: “Your body is basically a series of compartments.” He added: “Think of it as a giant house with rooms inside. We’re trying to figure out how to get something that’s outside, into one specific room.”

The complication with the approach is based on different cancers. Because different types of cancer differ in morphology, and cancers at different stages equally vary, selecting the appropriate nanoparticle is important. Here the research group have been looking at nanoparticles of varying sizes and shapes, as well as different coatings.

The solution is to create nanoparticles that can change shape to meet different types of tumors. This structural alteration makes the technology more versatile and means treatments could be delivered more quickly, rather than waiting for test results to assess the size and shape of the tumor.

The shape-shifting has been achieved by constructing the nanoparticles from tiny fragments of metal and then attaching DNA to them. The DNA acts as a means for marking the cancer, and then allowing the chemotherapy drug to attack the tumor.

The research is published in the journal Proceedings of the National Academy of Sciences. The paper is titled “Tailoring nanoparticle designs to target cancer based on tumor pathophysiology.”

Tailoring nanoparticle designs to target cancer based on tumor pathophysiology


Nanotechnology is a promising approach for improving cancer diagnosis and treatment with reduced side effects. A key question that has emerged is: What is the ideal nanoparticle size, shape, or surface chemistry for targeting tumors? Here, we show that tumor pathophysiology and volume can significantly impact nanoparticle targeting. This finding presents a paradigm shift in nanomedicine away from identifying and using a universal nanoparticle design for cancer detection and treatment. Rather, our results suggest that future clinicians will be capable of tailoring nanoparticle designs according to the patient’s tumor characteristics. This concept of “personalized nanomedicine” was tested for detection of prostate tumors and was successfully demonstrated to improve nanoparticle targeting by over 50%.



Nanoparticles can provide significant improvements in the diagnosis and treatment of cancer. How nanoparticle size, shape, and surface chemistry can affect their accumulation, retention, and penetration in tumors remains heavily investigated, because such findings provide guiding principles for engineering optimal nanosystems for tumor targeting. Currently, the experimental focus has been on particle design and not the biological system. Here, we varied tumor volume to determine whether cancer pathophysiology can influence tumor accumulation and penetration of different sized nanoparticles. Monte Carlo simulations were also used to model the process of nanoparticle accumulation. We discovered that changes in pathophysiology associated with tumor volume can selectively change tumor uptake of nanoparticles of varying size. We further determine that nanoparticle retention within tumors depends on the frequency of interaction of particles with the perivascular extracellular matrix for smaller nanoparticles, whereas transport of larger nanomaterials is dominated by Brownian motion. These results reveal that nanoparticles can potentially be personalized according to a patient’s disease state to achieve optimal diagnostic and therapeutic outcomes.



Curr Pharm Des. 2013;19(37):6560-74.
Mechanisms for targeted delivery of nanoparticles in cancer.
With the evolution of the “omics” era, our molecular understanding of cancer has exponentially increased, leading to the development of the concept of personalized medicine. Nanoparticle technology has emerged as a way to combine cancer specific targeting with multifunctionality, such as imaging and therapy, leading to advantages over conventional small molecule based approaches. In this review, we discuss the targeting mechanisms of nanoparticles, which can be passive or active. The latter utilizes small molecules, aptamers, peptides, and antibodies as targeting moieties incorporated into the nanoparticle surface to deliver personalized therapy to patients.
PMID: 23621529



Nanoparticle-based targeted drug delivery

Rajesh Singh1 and James W. Lillard Jr.1
Exp Mol Pathol. 2009 June ; 86(3): 215–223.

Nanotechnology could be defined as the technology that has allowed for the control, manipulation, study, and manufacture of structures and devices in the “nanometer” size range. These nano-sized objects, e.g., “nanoparticles”, take on novel properties and functions that differ markedly from those seen from items made of identical materials. The small size, customized surface, improved solubility, and multi-functionality of nanoparticles will continue to open many doors and create new biomedical applications. Indeed, the novel properties of nanoparticles offer the ability to interact with complex cellular functions in new ways. This rapidly growing field requires crossdisciplinary research and provides opportunities to design and develop multifunctional devices that can target, diagnose, and treat devastating diseases such as cancer. This article presents an overview of nanotechnology for the biologist and discusses the attributes of our novel XPclad© nanoparticle formulation that has shown efficacy in treating solid tumors, for single dose vaccination, and oral delivery of therapeutic proteins.

The development of a wide spectrum of nanoscale technologies is beginning to change the scientific landscape in terms of disease diagnosis, treatment, and prevention. These technological innovations, referred to as nanomedicines by the National Institutes of Health, have the potential to turn molecular discoveries arising from genomics and proteomics into widespread benefit for patients. Nanoparticles can mimic or alter biological processes (e.g., infection, tissue engineering, de novo synthesis, etc.). These devices include, but are not limited to, functionalized carbon nanotubes, nanomachines (e.g., constructed from interchangeable DNA parts and DNA scaffolds), nanofibers, self-assembling polymeric nanoconstructs, nanomembranes, and nano-sized silicon chips for drug, protein, nucleic acid, or peptide delivery and release, and biosensors and laboratory diagnostics.

Nanotechnology-based Drug Delivery in Cancer

Drug delivery in cancer is important for optimizing the effect of drugs and reducing toxic side effects. Several nanotechnologies, mostly based on nanoparticles, can facilitate drug delivery to tumors.


Hydrogel-nanoparticles are based on proprietary technology that uses hydrophobic polysaccharides for encapsulation and delivery of drug, therapeutic protein, or vaccine antigen. A novel system using cholesterol pullulan shows great promise. In this regard, four cholesterol molecules gather to form a self-aggregating hydrophobic core with pullulan outside. The resulting cholesterol nanoparticles stabilize entrapped proteins by forming this hybrid complex. These particles stimulate the immune system and are readily taken up by dendritic cells. Alternatively, larger hydrogels can encapsulate and release monoclonal antibodies.

Curcumin, a substance found in the cooking spice turmeric, has long been known to have anti-cancer properties. Nevertheless, widespread clinical application of this relatively efficacious agent has been limited due to its poor solubility and minimal systemic bioavailability. This problem has been resolved by encapsulating curcumin in a polymeric nanoparticle, creating “nanocurcumin” (Bisht et al., 2007). Further, the mechanism of action of nanocurcumin on pancreatic cancer cells mirrors that of free curcumin, including induction of apoptosis, blockade of nuclear factor kappa B (NFκB) activation, and downregulation of pro-inflammatory cytokines (i.e., IL-6, IL-8 and TNF-α). Nanocurcumin provides an opportunity to expand the clinical repertoire of this efficacious agent by enabling soluble dispersion. Future studies utilizing nanocurcumin are warranted in preclinical in vivo models of cancer and other diseases that might benefit from the effects of curcumin.

Micelles and liposomes

Block-copolymer micelles are spherical super-molecular assemblies of amphiphilic copolymer. The core of micelles can accommodate hydrophobic drugs, and the shell is a hydrophilic brush-like corona that makes the micelle water soluble, thereby allowing delivery of the poorly soluble contents. Camptothecin (CPT) is a topoisomerase I inhibitor that is effective against cancer, but clinical application of CPT is limited by its poor solubility, instability, and toxicity. Biocompatible, targeted sterically stabilized micelles (SSM) have been used as nanocarriers for CPT (CPT-SSM). CPT solubilization in SSM is expensive yet reproducible and is attributed to avoidance of drug aggregate formation. Furthermore, SSM composed of PEGylated phospholipids are attractive nanocarriers for CPT delivery because of their size (14 nm) and ability to extravasate through the leaky microvasculature of tumors and inflamed tissues. This passive targeting results in high drug concentration in tumors and reduced drug toxicity to the normal tissues (Koo et al., 2006).

Stealth micelle formulations have stabilizing PEG coronas to minimize opsonization of the micelles and maximize serum half-life. Currently, SP1049C, NK911, and Genexol-PM have been approved for clinical use (Sutton et al., 2007). SP1049C is formulated as doxorubicin (DOX)-encapsulated pluronic micelles. NK911 is DOX-encapsulated micelles from a copolymer of PEG-DOX-conjugated poly(aspartic acid), and Genexol-PM is a paclitaxelencapsulated PEG-PLA micelle formulation. Polymer micelles have several advantages over other drug delivery systems, including increased drug solubility, prolonged circulation halflife, selective accumulation at tumor sites, and lower toxicity. However, at the present time this technology lacks tumor specificity and the ability to control the release of the entrapped agents. Indeed, the focus of nano-therapy has gradually shifted from passive targeting systems (e.g., micelles) to active targeting.

Super paramagnetic iron oxide particles can be used in conjunction with magnetic resonance imaging (MRI) to localize the tumor as well as for subsequent thermal ablation. This has been used, for example, to target glioblastoma multiforme (GBM), a primary malignant tumor of the brain with few effective therapeutic options. The primary difficulty in treating GBM lies in the difficulty of delivering drugs across the BBB. However, nanoscale liposomal iron oxide preparations were recently shown to improve passage across the BBB (Jain, 2007).


Nanomaterial formulation

Nanomaterials have been successfully manipulated to create a new drug-delivery system that can solve the problem of poor water solubility of most promising currently available anticancer drugs and, thereby, increase their effectiveness. The poorly soluble anticancer drugs require the addition of solvents in order for them to be easily absorbed into cancer cells. Unfortunately, these solvents not only dilute the potency of the drugs but create toxicity. Researchers from the University of California Los Angeles California Nanosystem Institute have devised a novel approach using silica-based nanoparticles to deliver the anticancer drug CPT and other water insoluble drugs to cancer cells (Lu et al., 2007). The method incorporates the hydrophobic anticancer drug CPT into the pores of fluorescent mesoporous silica nanoparticles and delivers the particles into a variety of human cancer cells to induce cell death. The results suggest that the mesoporous silica nanoparticles might be used as a vehicle to overcome the insolubility of many anticancer drugs.


Novel nanosystems can be pre-programmed to alter their structure and properties during the drug delivery process, allowing for more effective extra- and intra-cellular delivery of encapsulated drug (Wagner, 2007). This is achieved by the incorporation of molecular sensors that respond to physical or biological stimuli, including changes in pH, redox potential, or enzymes. Tumor-targeting principles include systemic passive targeting and active receptor targeting. Physical forces (e.g., electric or magnetic fields, ultrasound, hyperthermia, or light) may contribute to focusing and triggering activation of nano systems. Biological drugs delivered with programmed nanosystems also include plasmid DNA, siRNA, and other therapeutic nucleic acids.

Using a degradable, polyamine ester polymer, polybutanediol diacrylate co amino pentanol (C32), a diptheria toxin suicide gene (DT-A) driven by a prostate-specific promoter was directly injected into normal prostate and prostate tumors in mice (Peng et al., 2007). This C32/DT-A system resulted in significant size reduction, apoptosis in 50% of normal prostate. However, a single injection of C32/DT-A triggered apoptosis in 80% of tumor cells present in the tissue. It is expected that multiple nanoparticle injection would trigger a great percentage of prostate tumor cells to undergo apoptosis. These results suggest that local delivery of polymer/DT-A nanoparticles may have application in the treatment of benign prostatic hypertrophy and prostate cancer.

Multidrug resistance (MDR) of tumor cells is known to develop through a variety of molecular mechanisms. Glucosylceramide synthase (GCS) is responsible for the activation of the pro-apoptotic mediator, ceramide, to a nonfunctional moiety, glucosylceramide. This molecule is over-expressed by many MDR tumor types and has been implicated in cell survival in the presence of chemotherapy. A study has investigated the therapeutic strategy of co-administering ceramide with paclitaxel in an attempt to restore apoptotic signaling and overcome MDR in a human ovarian cancer cell line using modified poly(epsiloncaprolactone) (PEO-PCL) nanoparticles to encapsulate and deliver the therapeutic agents for enhanced efficacy (van Vlerken and Amiji, 2006). Results show that MDR cancer cells can be completely eradicated by this approach. Using this approach, MDR cells can be resensitized to a dose of paclitaxel near the IC50 of non-MDR cells. Molecular analysis of activity verified the hypothesis that the efficacy of this therapeutic approach is due to a restoration in apoptotic signaling, showing the promising potential for clinical use of this therapeutic strategy to overcome MDR.


Indiscriminate drug distribution and severe toxicity of systemic administration of chemotherapeutic agents can be overcome through encapsulation and cancer cell targeting of chemotherapeutics in 400 nm nanocells, which can be packaged with significant concentrations of chemotherapeutics of different charge, hydrophobicity, and solubility (MacDiarmid et al., 2007). Targeting of nanocells via bispecific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release. Doses of drugs delivered via nanocells are ∼1,000 times less than the dose of the free drug required for equivalent tumor regression. It produces significant tumor growth inhibition and regression in mouse xenografts and lymphoma in dogs, despite administration of minute amounts of drug and antibody. Indeed, reduced dosage is a critical factor for limiting systemic toxicity. Clinical trials are planned for testing this method of drug delivery.


In early studies, dendrimer-based drug delivery systems focused on encapsulating drugs. However, it was difficult to control the release of drugs associated with dendrimers. Recent developments in polymer and dendrimer chemistry have provided a new class of molecules called dendronized polymers, which are linear polymers that bear dendrons at each repeat unit. Their behavior differs from that of linear polymers and provides drug delivery advantages because of their enhanced circulation time. Another approach is to synthesize or conjugate the drug to the dendrimers so that incorporating a degradable link can be further used to control the release of the drug.

DOX was conjugated to a biodegradable dendrimer with optimized blood circulation time through the careful design of size and molecular architecture (Lee et al., 2006). Specifically, the DOX-dendrimer controlled drug-loading through multiple attachment sites, solubility through PEGylation, and drug release through the use of pH-sensitive hydrazone dendrimer linkages. In culture, DOX-dendrimers were >10 times less toxic than free DOX toward colon carcinoma cells. Upon intravenous administration to tumor bearing mice, tumor uptake of DOX-dendrimers were nine-fold higher than intravenous free DOX and caused complete tumor regression and 100% survival of the mice after 60 days.

Nanotubes Even though it was previously possible to attach drug molecules directly to antibodies, attaching more than a handful of drug molecules to an antibody significantly limits its targeting ability because the chemical bonds that are used tend to impede antibody activity. A number of nanoparticles have been investigated to overcome this limitation. Tumor targeting single-walled carbon nano-tube (SWCNT) have been synthesized by covalently attaching multiple copies of tumor-specific monoclonal antibodies (MAbs), radiation ion chelates and fluorescent probes (McDevitt et al., 2007). A new class of anticancer compound was created that contains both tumor-targeting antibodies and nanoparticles called fullerenes (C60). This delivery system can be loaded with several molecules of an anticancer drug, e.g., Taxol® (Ashcroft et al., 2006). It is possible to load as many as 40 fullerenes onto a single skin cancer antibody called ZME-108, which can be used to deliver drugs directly into melanomas. Certain binding sites on the antibody are hydrophobic (water repelling) and attract the hydrophobic fullerenes in large numbers so multiple drugs can be loaded into a single antibody in a spontaneous manner. No covalent bonds are required, so the increased payload does not significantly change the targeting ability of the antibody. The real advantage of fullerene-based therapies vs. other targeted therapeutic agents is likely to be fullerene’s potential to carry multiple drug payloads, such as taxol plus other chemotherapeutic drugs. Cancer cells can become drug resistant, and one can cut down on the possibility of their escaping treatment by attacking them with more than one kind of drug at a time. The first fullerene immuno-conjugates have been prepared and characterized as an initial step toward the development of fullerene immunotherapy.


Polymersomes, hollow shell nanoparticles, have unique properties that allow delivery of distinct drugs. Loading, delivery and cytosolic uptake of drug mixtures from degradable polymersomes were shown to exploit the thick membrane of these block copolymer vesicles, their aqueous lumen, and pH-triggered release within endolysosomes. Polymersomes break down in the acidic environments for targeted release of these drugs within tumor cell endosomes. While cell membranes and liposomes are created from a double layer of phospholipids, a polymersome is comprised of two layers of synthetic polymers. The individual polymers are considerably larger than individual phospholipids but have many of the same chemical features.

Polymersomes have been used to encapsulate paclitaxel and DOX for passive delivery to tumor-bearing mice (Ahmed et al., 2006). The large polymers making up the polymersome allows paclitaxel, which is water insoluble, to embed within the shell. DOX is water-soluble and stays within the interior of the polymersome until it degrades. The polymersome and drug combination spontaneously self-assembles when mixed together. Recently, studies have shown that cocktails of paclitaxel and DOX lead to better tumor regression that either drug alone, but previously there was no carrier system that could carry both drugs as efficiently to a tumor. Hence, this approach shows great promise.

Quantum dots

Single-particle quantum dots conjugated to tumor-targeting anti-human epidermal growth factor receptor 2 (HER2) MAb have been used to locate tumors using high-speed confocal microscopy (Tada et al., 2007). Following injection of quantum dot-MAb conjugate, six distinct stop-and-go steps were identified in the process as the particles traveled from the injection site to the tumor where they bound HER2. These blood-borne conjugates extravasated into the tumor, bound HER2 on cell membranes, entered the tumor cells and migrated to the perinuclear region. The image analysis of the delivery processes of single particles in vivo provided valuable information on MAb-conjugated therapeutic particles, which will be useful in increasing their anticancer therapeutic efficacy. However, the therapeutic utility of quantum dots remains undetermined.

XPclad® nanoparticles

The poor aqueous solubility of many drug candidates presents a significant problem in drug delivery and related requirements such as bioavailability and absorption. Recently, our laboratory has developed XPclad® nanoparticles that represent a novel formulation method that uses planetary ball milling to generate particles of uniform size (Figure 1), 100% loading efficiency of hydrophobic or hydrophilic drugs, subsequent coating for targeted delivery, and control of LogP for systemic, cutaneous, or oral administration of cancer drugs, vaccines, or therapeutic proteins (Figure 2).

The method for making XPclad® nanoparticles uses a novel and relatively inexpensive preparation technique (i.e., planetary ball milling), which allows for controlling the size of the particles (100 nm to 50 μm; ± 10% of mean size) with >99% loading efficiency, polymer- or ligand-coating for controlled-, protected-, and targeted-release and delivery of their contents. The nanoparticles produced thereby contain the desired biologically active agent(s) in a biopolymer excipient such as alginate, cellulose, starch or collagen and biologically active agents. Generally, there are two types of mills that have been employed for making particles: vibratory or planetary ball mills. The vibratory ball milling grinds powders by high velocity impact while planetary ball milling employs a grinding motion. Typically, planetary ball milling has been used only to generate micron-sized particles, while vibratory milling can yield nano-particles. However, the high impact resulting from the vibratory milling technique makes incorporating biologicals difficult. Planetary ball mills pulverize and mix materials ranging from soft and medium to extremely hard, brittle and fibrous materials. Both wet and dry grinding can be carried out. Minerals, ores, alloys, chemicals, glass, ceramics, plant materials, soil samples, sewage sludge, household and industrial waste and many other substances can be reduced in size simply, quickly and without loss. Planetary ball mills have been successfully used in many industrial and research sectors, particularly wherever there is high demand for purity, speed, fineness and reproducibility. The planetary ball mills produce extremely high centrifugal forces with very high pulverization energies and short grinding times. Because of the extreme forces exerted, the use of vibratory and planetary ball mills to formulate therapeutics has not been practiced until now. In general, XPclad® particle size can be engineered to range from 5 to 30 nm up to 10 to 60 μm by controlling the size and number of planetary balls, grinding speed, milling cycles, and centrifugal force by varying the revolutions per second and planetary jar velocity.


Nano delivery systems hold great potential to overcome some of the obstacles to efficiently target a number of diverse cell types. This represents an exciting possibility to overcome problems of drug resistance in target cells and to facilitate the movement of drugs across barriers (e.g., BBB). The challenge, however, remains the precise characterization of molecular targets and ensuring that these molecules only affect targeted organs. Furthermore, it is important to understand the fate of the drugs once delivered to the nucleus and other sensitive cells organelles.



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Dialysis alternative

Curators: Larry Bernstein, MD and Jennifer Schwartz, Ursulin College and Cleveland Clinic



VU Inside: Dr. William Fissell’s Artificial Kidney

by | Feb. 12, 2016 

Vanderbilt is using a microchip to build a first-ever artificial kidney

Vanderbilt University Medical Center nephrologist and Associate Professor of Medicine Dr. William H. Fissell IV, is making major progress on a first-of-its kind device to free kidney patients from dialysis. He is building an implantable artificial kidney with microchip filters and living kidney cells that will be powered by a patient’s own heart. The bio-hybrid device will mimic a kidney to remove enough waste products, salt and water to keep a patient off dialysis,” said Fissell.

Fissell says the goal is to make it small enough, roughly the size of a soda can, to be implanted inside a patient’s body.


The key to the device is a microchip.

“It’s called silicon nanotechnology. It uses the same processes that were developed by the microelectronics industry for computers,” said Fissell.

The chips are affordable, precise and make ideal filters. Fissell and his team are designing each pore in the filter one by one based on what they want that pore to do. Each device will hold roughly fifteen microchips layered on top of each other.

But the microchips have another essential role beyond filtering.

“They’re also the scaffold in which living kidney cells will rest,” said Fissell.


close-up of microchip held by tweezers×299.jpg

An example of the microchip filter being used inside Fissell’s artificial kidney. (Vanderbilt University)


Living kidney cells

Fissell and his team use live kidney cells that will grow on and around the microchip filters. The goal is for these cells to mimic the natural actions of the kidney.

“We can leverage Mother Nature’s 60 million years of research and development and use kidney cells that fortunately for us grow well in the lab dish, and grow them into a bioreactor of living cells that will be the only ‘Santa Claus’ membrane in the world: the only membrane that will know which chemicals have been naughty and which have been nice. Then they can reabsorb the nutrients your body needs and discard the wastes your body desperately wants to get rid of,” said Fissell.

Avoiding organ rejection

Because this bio-hybrid device sits out of reach from the body’s immune response, it is protected from rejection.

“The issue is not one of immune compliance, of matching, like it is with an organ transplant,” said Fissell.

How it works

The device operates naturally with a patient’s blood flow.

The device operates naturally with a patient’s blood flow.

“Our challenge is to take blood in a blood vessel and push it through the device. We must transform that unsteady pulsating blood flow in the arteries and move it through an artificial device without clotting or damage.”

Fluid dynamics

And that’s where Vanderbilt biomedical engineer Amanda Buck comes in. Buck is using fluid dynamics to see if there are certain regions in the device that might cause clotting.

“It’s fun to go in and work in a field that I love, fluid mechanics, and get to see it help somebody,” said Buck.

She uses computer models to refine the shape of the channels for the smoothest blood flow. Then they rapidly prototype the new design using 3-D printing and test it to make the blood flow as smoothly as possible.


Amanda sitting at her desk looking at fluid dynamics models on a computer screen×299.jpg

Vanderbilt biomedical engineer Amanda Buck is using fluid dynamics to see if there are certain regions in the device that might cause clotting.


Future human trials

Fissell says he has a long list of dialysis patients eager to join a future human trial. Pilot studies of the silicon filters could start in patients by the end of 2017.

“My patients are absolutely my heroes,” said Fissell. “They come back again and again and they accept a crushing burden of illness because they want to live. And they’re willing to put all of that at risk for the sake of another patient.”

Federal investment

The National Institutes of Health awarded a four-year, $6 million grant to Fissell and his research partner Shuvo Roy from the University of California at San Francisco. The two investigators are longtime collaborators on this research. In 2003, the kidney project attracted its first NIH funding, and in 2012 the Food and Drug Administration selected the project for a fast-track approval program. The work is supported by NIH grant 1U01EB021214-01.

The National Kidney Foundation reports that in 2012, Federal Medicare dollars paid more than $87 billion caring for kidney disease patients (not including prescription medications).

Desperate need

Transplant of a human kidney is the best treatment for kidney failure, but donor kidneys are in short supply. According to the U.S. Organ Procurement and Transplantation Network, although more than 100,000 patients in the United States are on the waiting list for a kidney transplant, last year only 17,108 received one.

In all, the National Kidney Foundation says more than 460,000 Americans have end-stage renal disease and every day, 13 people die waiting for a kidney.

Read more about the NIH grant here.

By Amy Wolf

Media Inquiries: Craig Boerner, (615) 322-4747

Media Inquiries:
Amy Wolf, (615) 322-NEWS

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Advanced Nanospectroscopy

Larry H. Bernstein, MD, FCAP, Curator



Graphene Enables Nanoelectromechanical Systems Integration

BARCELONA, Spain, Jan. 21, 2016 — Combining nanoelectromechanical (NEMS) systems with on-chip optics holds promise as a method to actively control light at the nanoscale, and now a hybrid system has overcome the challenges of integrating such nanoscale devices with optical fields thanks to the material graphene.

Researchers from the Institute of Photonic Sciences (ICFO) have demonstrated an on-chip graphene NEMS suspended a few tens of nanometers above nitrogen-vacancy centres (NVCs), which are stable single-photon emitters embedded in nanodiamonds. The work confirms that graphene is an ideal platform for both nanophotonics and nanomechanics, the researchers said.

Due to its electromechanical properties, graphene NEMS can be actuated and deflected electrostatically over a few tens of nanometers with modest voltages applied to a gate electrode, the researchers found. The graphene motion can thus be used to modulate the light emission by the NVC, while the emitted field can be used as a universal probe of the graphene position. The optomechanical coupling between the graphene displacement and the NVC emission is based on near-field, dipole-dipole interaction.


False color scanning electronic micrograph of a hybrid graphene-nitrogen-vacancy nearfield nano-optomechanical system. Courtesy of ICFO.

The researchers observed that the coupling strength increased strongly for shorter distances and was enhanced because of graphene’s 2D character and linear dispersion. These achievements hold promise for selective control of emitter arrays on-chip, optical spectroscopy of individual nano-objects, and integrated optomechanical information processing. The ICFO team also said the hybrid device could advance quantum optomechanics.

The research was published in Nature Communications (doi: 10.1038/ncomms10218).

Electromechanical control of nitrogen-vacancy defect emission using graphene NEMS

Antoine Reserbat-PlanteyKevin G. SchädlerLouis GaudreauGabriele NavickaiteJohannes Güttinger, et al.

Nature Communications 2016; 7(10218)

Despite recent progress in nano-optomechanics, active control of optical fields at the nanoscale has not been achieved with an on-chip nano-electromechanical system (NEMS) thus far. Here we present a new type of hybrid system, consisting of an on-chip graphene NEMS suspended a few tens of nanometres above nitrogen-vacancy centres (NVCs), which are stable single-photon emitters embedded in nanodiamonds. Electromechanical control of the photons emitted by the NVC is provided by electrostatic tuning of the graphene NEMS position, which is transduced to a modulation of NVC emission intensity. The optomechanical coupling between the graphene displacement and the NVC emission is based on near-field dipole–dipole interaction. This class of optomechanical coupling increases strongly for smaller distances, making it suitable for nanoscale devices. These achievements hold promise for selective control of emitter arrays on-chip, optical spectroscopy of individual nano-objects, integrated optomechanical information processing and open new avenues towards quantum optomechanics.


Graphene is ideal substrate for brain electrodes, researchers find

February 1, 2016

This illustration portrays neurons interfaced with a sheet of graphene molecules in the background (credit: Graphene Flagship)

An international study headed by the European Graphene Flagship research consortium has found that graphene is a promising material for use in electrodes that interface with neurons, based on its excellent conductivity, flexibility for molding into complex shapes, biocompatibility, and stability within the body.

The graphene-based substrates they studied* promise to overcome problems with “glial scar” tissue formation (caused by electrode-based brain trauma and long-term inflammation). To avoid that, current electrodes based on tungsten or silicon use a protective coating on electrodes, which reduces charge transfer. Current electrodes are also rigid (resulting in tissue detachment and preventing neurons from moving) and generate electrical noise, with partial or complete loss of signal over time, the researchers note in a paper published recently in the journal ACS Nano.

Electrodes are used as neural biosensors and for prosthetic applications — such as deep-brain intracranial electrodes used to control motor disorders (mainly epilepsy or Parkinson’s) and for brain-computer interfaces (BCIs), used to recover sensory functions or control robotic arms for paralyzed patients. These applications require an interface with long-term, minimal interference.

Interfacing graphene to neurons directly

Scanning electron microscope image of rat hippocampal neurons grown in the lab on a graphene-based substrate, showing normal morphology characterized by well-defined round neural soma, extended neurite arborization (branching), and cell density similar to control substrates (credit: A. Fabbro et al./ACS Nano)

“For the first time, we interfaced graphene to neurons directly, without any peptide-coating,” explained lead neuroscientist Prof. Laura Ballerini of the International School for Advanced Studies (SISSA/ISAS) and the University of Trieste.

Using electron microscopy and immunofluorescence, the researchers found that the neurons remained healthy, transmitting normal electric impulses and, importantly, no adverse glial reaction, which leads to damaging scar tissue, was seen.

As a next step, Ballerini says the team plans to investigate how different forms of graphene, from multiple layers to monolayers, are able to affect neurons,  and “whether tuning the graphene material properties might alter the synapses and neuronal excitability in new and unique ways.”

Prof. Andrea C. Ferrari, Director of the Cambridge Graphene Centre and Chair of the Graphene Flagship Executive Board, said the Flagship will “support biomedical research and development based on graphene technology with a new work package and a significant cash investment from 2016.”

The interdisciplinary collaboration also included the University Castilla-La Mancha and the Cambridge Graphene Centre.

* The study used two methods of creating graphene-based substrates (GBSs).  Liquid phase exfoliation (LPE) — peeling off graphene from graphite — can be performed without the potentially hazardous chemical treatments involved in graphene oxide production, is scalable, and operates at room temperature, with high yield. LPE dispersions can also be easily deposited on target substrates by drop-casting, filtration, or printing. Ball milling (BM), with the help of melamine (which forms large hydrogen-bond domains, unlike LPE), can be performed in a solid environment. “Our data indicate that both GBSs are promising for next-generation bioelectronic systems, to be used as brain interfaces,” the paper concludes.




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