Fighting cardiovascular disease with nanoparticles that mimic cholesterol carriers

A creative group of nanomedicine researchers at Rutgers University led by Kathryn Uhrich and Prabhas Moghe have spent the past several years fine-tuning “nanolipoblockers,” nanoparticles that block the uptake of cholesterol by macrophages, the inflammatory cells that make up atherosclerotic plaques. These nanoparticles resemble oxidized low density lipoprotein (LDL) particles, binding to macrophages’ receptors for the cholesterol carriers so that the oxidized LDL itself cannot. This innovation is an example of how nanotechnology expands the range of ways to treat disease: rather than designing a small molecule to inactivate the receptor, which has so far proven unsuccessful, the tools of nanomedicine allow those working to treat cardiovascular disease to create structures that mimic the scale, charge, and other physical properties of the natural ligand. Further, because nanolipoblockers are assemblies of many parts, they can incorporate small molecule drugs to target other steps in the process of plaque formation carried out by macrophages.

Though atherosclerosis, the accumulation of fatty plaques in the arteries that causes heart attack and stroke, is a massive public health challenge, its treatment has only advanced incrementally in the past twenty years with the introduction of new types of statins. While these drugs were the first to actually address the underlying cause of atherosclerosis rather than simply lower blood pressure or treat chest pain, they only slow its development. Nanolipoblockers could represent a long sought-after next-generation therapy that would effectively block the accumulation of fatty material in artery walls in combination with statins.

Uhrich and Moghe’s breakthrough stemmed from their recognition that the low efficacy of small molecule blockers of the oxidized LDL receptor likely resulted from the fact that the receptor is designed to bind nanoscale particles. Inhibitors designed to form nanoscale complexes could therefore bind more effectively to the receptor, reducing the necessary dose. After finding that micelles (spherical assemblies of detergent-like molecules) they had previously designed for drug delivery inhibited the uptake of oxidized LDL by macrophages, they have spent the past seven years optimizing their structure to maximize uptake inhibition. Uhrich and Moghe have recently altered the stereochemistry (the orientation of various parts relative to one another) of the molecule composing the micelle to yield a version that inhibits uptake by 89% at submicromolar concentrations (1) (100 times lower than the effective concentrations of inhibitors studied by other groups) (2). These investigators have already shown that using these oxidized LDL receptor (specifically, the type A scavenger receptor)-blocking micelles to deliver a drug that promotes cholesterol efflux does indeed further reduce cholesterol accumulation and macrophage recruitment in vivo, though they did not examine effects on atherosclerosis itself (they studied acute carotid injury) (3).

Nanolipoblockers prevent uptake of oxidized low density lipoprotein (oxLDL)
Nanolipoblockers prevent uptake of oxidized low density lipoprotein (oxLDL). oxLDL was labeled with a red dye prior to uptake experiments in primary macrophages. Top, without, and below, with nanolipoblockers. Image from Poree et al., Biomacromolecules 2013.

The Rutgers team’s approach is quite clever, but the impact of these investigations will only become clear upon in vivo tests of whether nanolipoblockers block or slow atherosclerosis development in animal models. Given the complexity of the atherogenic process, accumulation of oxidized LDL in the extracellular space of the vessel wall (rather than in macrophages) might have other harmful effects. However, the results of a study in which uptake of oxidized LDL was blocked in a mouse atherosclerosis model by transduction of a gene for a soluble form of the type A scavenger receptor suggests that this therapy would be beneficial (4), as the authors observed a noticeable decrease in atherosclerotic lesion area. If results in atherosclerosis models are as impressive as those in carotid injury, Uhrich and Moghe could become well-known in the cardiovascular field.  

 

1. D. E. Poree, K. Zablocki, A. Faig, P. V. Moghe, K. E. Uhrich, Biomacromolecules,  (2013).
2.  K. Yoshiizumi, F. Nakajima, R. Dobashi, N. Nishimura, S. Ikeda, Bioorganic & Medicinal Chemistry 10, 2445 (2002).
3. N. M. Iverson et al., Biomaterials 32, 8319 (2011).
4. J. Jalkanen et al., Mol Ther 8, 903 (2003).

Drug conjugates that self-assemble into their own delivery vehicles

This post is part of a series highlighting the research of those invited to speak at NanoDDS’13.

Most drug-carrying nanoparticles are created by mixing drugs with lipids or polymers in complex mixtures of organic and aqueous solutions to create nanostructures (vesicles or polymer networks) surrounding a small amount of drug. But what if the drug and the nanocarrier material were the same molecule? Chemically attaching the two components not only simplifies the formulation process, but ensures that the drug molecules will remain inside the structure until it’s induced to disassemble and allows precise control over the ratio of drug to surrounding material in each nanostructure. 

A young professor at Johns Hopkins, Honggang Cui, has recently introduced just such a technology. His background developing peptides and polymers that self-assemble into nanostructures led him to recognize the power of this approach to overcome the limitations of other nanoformulations. For example, attaching several drug molecules to each macromolecule composing the surrounding structure enables each nanocarrier to incorporate high proportions of drug. Increasing the amount of drug incorporated into each nanoparticle would increase effectiveness, as it would require fewer nanoparticles to reach the target tissue to have the same therapeutic effect. The control over drug loading provided by the drug self-assembly approach is also a major advantage over delivery in liposomes or polymeric nanoparticles, in which loading varies significantly from batch to batch, which could affect the reliability of dosing.

The first demonstration of this concept, recently published in JACS, yielded an impressively high 38% loading of the anticancer drug camptothecin when each assembling molecule contains four camptothecin units (1). This study attached the drug molecules to a peptide derived from Tau protein that was already known to aggregate into filaments; future studies could employ other peptides that assemble similarly (into β sheets).

Self-assembling drug amphiphiles. Left, schematic; right, transmission electron micrograph. Blue ribbons represent the Tau peptide; red ovals, drug; green, degradable linkers. Image from Cheetham et al., JACS 2013.
Self-assembling drug amphiphiles. Left, schematic; right, transmission electron micrograph. Blue ribbons represent the Tau peptide; red ovals, drug; green, degradable linkers. Image from Cheetham et al., JACS 2013.

Considerable research remains to develop this concept towards clinical applicability. At this preliminary stage, these drug assemblies’ efficacy at killing cancer cells is actually lower than that of free drug, which likely results from their nanofiber shape; long, thin particles are not readily taken up by most cells. Altering the molecular design to promote the formation of spherical assemblies would increase cellular uptake. Because the linker between the drug and the rest of the molecule is towards the interior of the nanofiber and degrades upon exposure to reducing conditions, the assemblies must dissociate and diffuse into the cytosol to release active drug. Therefore, another potential route to increasing the amount of active drug within cells would be to engineer the assemblies to be less stable at low pH so that assemblies would dissociate within the cellular recycling vesicle. 

1.  Cheetham AG, Zhang P, Lin Y-a, Lock LL, & Cui H (2013) Supramolecular Nanostructures Formed by Anticancer Drug Assembly. J Am Chem Soc 135(8):2907-2910.

Synthetic nanodetectors for specific molecules

A new type of molecular detector, which can be rapidly manufactured and screened, has recently been introduced by scientists at MIT. In a paper published Nov. 24 in Nature Nanotechnology, a large team led by Michael Strano show that specific polymers without inherent molecular recognition abilities selectively recognize small molecules such as the vitamin riboflavin and the hormone estradiol when adsorbed to carbon nanotubes. By single-molecule fluorescent imaging and molecular dynamics simulations, the authors show that adsorption alters the polymer’s structure, creating new potential binding sites. If future work allows cost-effective design of polymer-nanotube detectors for specific molecules, the researchers speculate that these complexes could become an alternative to antibodies for everything from biological research to diagnostic assays to drug targeting.

Whether this novel platform will have such wide-ranging uses will remain unknown for some time; the results so far provided are a proof-of-concept that polymer-nanotube complexes can be rapidly screened for recognition. Among ~30 random polymers adsorbed to carbon nanotubes for binding to 36 small molecules (indicated by changes in the fluorescence of the carbon nanotubes), the MIT group discovered 3 that selectively recognized just one. While binding specificity could not be predicted based on a polymer’s structure prior to this study, the imaging and simulation approach the team developed may enable such prediction in the future. Further, binding affinity between a specific analyte and polymer-nanotube complex can be improved by tuning polymer chemistry and nanotube diameter. The fact that numerous variables influence binding affinity suggests that a polymer- nanotube sensor could be designed for almost any analyte.

Molecular recognition principle. Adsorption of polymer to carbon nanotubes (i) creates binding sites for analytes, and binding (ii) changes the nanotube's fluorescence. Reprinted by permission from Macmillan Publishers Ltd: [Nature Nanotech] (8, 896-7), copyright 2013.

 

This work builds on prior use of nanotube complexes to detect specific molecules; the novelty of this study lies in its open-ended approach. Rather than using enzymes, complementary DNA, or other chemical groups known to bind specific molecules, these researchers used an arbitrary set of polymers—that these complexes would selectively bind any of the target molecules was not entirely expected.

 

While the Strano group aims to provide a synthetic means of molecular recognition, which they speculate would be more consistent and less expensive than antibodies, carbon nanotube expert Davide Bonifazi expects a more limited set of applications, such as binding-triggered drug release, in his review of the article. A system that combines sensing and response would require development of a means of translating the electronic change in the carbon to a mechanical change in the drug-containing component. He also cautions that the current variability in carbon nanotube structure would limit the consistency of these sensors. Even without these future applications, the fluorescent sensors themselves could prove useful for cell-based studies; the nanotube-polymer complexes allow real-time imaging of riboflavin diffusion within cells.

 

Breakthrough in oral delivery of nanoparticles

Nanomedicine would be much more useful to treat non-life-threatening diseases if nanoparticle therapeutics could be administered in pill form, but oral delivery of nanoparticles is not yet possible because the intestine does not naturally transport particles across the epithelium and into the circulation. This major challenge has been a focus of decades of research, but so far, even the most effective strategies to promote uptake of nanoparticles by intestinal epithelia allow only small percentages of drug to reach the circulation (see this review).

One way to overcome limited alimentary uptake of nanoparticles would be to identify natural exceptions to the rule—large particles that are actively transported across intestinal epithelia—and attach the portion of the molecule that mediates uptake to nanoparticles. Omid Farokhzad’s group at Harvard has recently implemented this strategy to develop the most efficient approach yet introduced to deliver insulin orally. Not only is insulin an ideal drug to study oral delivery because of its rapid effects on blood sugar, but oral insulin capable of alleviating spikes in blood sugar would also represent a major public health breakthrough. While several pharmaceutical companies, including Oramed and Novo Nordisk, are developing oral formulations of insulin without nanoparticles (instead attaching polymers or surfactants to promote passive absorption), very small amounts are absorbed, so they are only useful as a means of slowing the onset of type 2 diabetes or as an adjunct to injected insulin.

Realizing that maternal antibodies (IgG) are actively taken up by the neonatal intestine to transfer immunity, the Harvard team attached the component of IgG that mediates this uptake (called Fc) to nanoparticles carrying insulin in their interior. This component binds to the neonatal Fc receptor, FcRn; others have used the same target to allow oral delivery of follicle-stimulating hormone (FSH, used in fertility treatments) by fusing Fc to the protein. The Farokhzad group chose instead to formulate insulin within nanoparticles to maximize the number of drug molecules transported per uptake event and to avoid genetic engineering, which could alter a therapeutic protein’s activity.

In the paper, published in Science Translational Medicine in late November, graduate student Eric Pridgen and colleagues observed a 30-45% decrease in blood glucose following oral administration of a standard insulin dose in biodegradable poly(lactic acid)–b-poly(ethylene glycol) (PLA-PEG) nanoparticles decorated with Fc. This effect is twice that of insulin delivered in non-targeted nanoparticles, which is surprising since intestinal FcRn expression decreases in adult mice. However, the group does provide evidence that uptake is mediated by FcRn: the hypoglycemic response to insulin delivered in targeted particles is half as strong in mice lacking the receptor. Since intestinal expression of FcRn in humans remains stable through adulthood, uptake of FcRn-targeted nanoparticles could be even more efficient in patients.

Since the hypoglycemic effect induced by FcRn-targeted particles is similar to that of the most widely used form of insulin, these findings seem very likely to lead to a new formulation of insulin, and possibly other protein drugs, for clinical use.

Imaging of any tumor type by pH-triggered fluorescence activation using nanoparticles

Current approaches to detection of tumors by imaging are generally tumor type or subtype-specific, so their applicability is limited. Towards the goal of an imaging agent capable of detecting any tumor, numerous research groups have created imaging agents whose signal is activated by the pH characteristic of the tumor extracellular space (6.5-6.8, slightly lower than the normal 7.4). This decreased pH is observed in all tumors as a result of numerous mechanisms, including hypoxia and enhanced H+ efflux by tumor cells. However, previous pH-responsive imaging agents haven’t been promising because their signal gradually increases at decreasing pH, the difference between “on” and “off” states is only a few fold, or they become
activated over many hours, all of which would blur the distinction between normal and tumor tissue.

Jinming Gao’s group at UT Southwestern recently reported a system that overcomes all of these issues,  displaying a 300-fold greater fluorescent signal within tumors than in the circulation. This impressive degree of tumor specificity results from a sharp pH response and tumor targeting using a peptide that recognizes newly formed blood vessels, another universal feature of tumors. Though the agent’s overall design, a micelle encapsulating hundreds of dye molecules so that their fluorescence is quenched by energy transfer among them, is not new, the huge difference in signal between the “off” and “on” states is a major advance that results from careful engineering. The agent by its creators, responds to slight changes in pH (<0.25 pH units) with huge changes in fluorescent signal because the material (a block copolymer) composing the micelle ionizes at a specific pH, completely disrupting the structure to release all of the dye molecules within. The ionizable micelle design had been previously reported by the same group; this study tuned pH  responsiveness for tumor detection and incorporated the targeting peptide to allow greater accumulation within tumors through cellular uptake.

pH-activated fluorescence. Ionization of the copolymer composing the micelle disrupts the structure, releasing and activating all dye molecules. UPSe, nanosystem activated by tumor extracellular pH (pHe); UPSi, nanosystem activated by endosomal pH (pHi). Reprinted by permission from Nature Publishing Group.

Most importantly, lead author Yiguang Wang and  his team demonstrate the applicability of this approach to numerous tumor types in mice, including a transgenic model of breast cancer and orthotopic (consisting of injected human cancer cells) lung, head and neck, prostate tumors. In all of these cases, the fluorescent signal is clearly restricted to tumors.

These ultra pH-sensitive nanoprobes are an exciting development in the area of smart biomedical imaging materials, and could become a tool to resolve tumor borders during resection. However, tools for that purpose already exist, including the cell-penetrating protease-cleavable peptides developed by Roger Tsien’s group, so the clinical relevance of pH-sensitive nanoprobes depends upon whether they offer brighter signal or allow imaging of a wider variety of tumors than existing technologies. If it could be modified to silence and activate paramagnetic metal particles or ions to enable tumor-specific MRI, this design could one day be relevant to cancer diagnosis and treatment monitoring.

New capabilities for a cutting-edge in vivo imaging method: activatable photoacoustic contrast agent

The recent invention of an imaging method that promises to bridge the gap between microscopy and current low-resolution imaging methods used in the clinic, such as X-ray tomography, has spurred the introduction of a host of new contrast agents to enable detection of specific molecules. This imaging method, called photoacoustic tomography (PAT), was established roughly ten years ago and detects the ultrasonic waves produced when colored molecules absorb light. All photoacoustic imaging agents so far introduced detect disease by binding to proteins present at higher-than-normal concentrations in disease sites. As even unbound contrast agents produce signal, however, considerable background signal may remain; to more clearly distinguish disease sites from healthy tissue, activatable contrast agents that only produce signal upon encountering disease characteristics could be adapted from those already in use in optical imaging.

A research group at Stanford led by Jianghong Rao recently saw an opportunity for the materials they have developed for use in solar cells to serve as photoacoustic imaging agents. Because these semiconducting pi-conjugated polymers are designed to absorb near infrared light efficiently, the investigators hypothesized that they would produce high photoacoustic signal. Further, by incorporating a dye that’s inactivated by certain reactive oxygen species (ROS) into nanoparticles composed of one of these polymers, they created an “activatable” agent. ROS are present at higher-than-normal levels in atherosclerotic plaques, tumors, and degenerating brain tissue, so many ROS-activated imaging agents have been developed towards the goals of early detection and examining the role of ROS in the progression of these diseases. In the present design, the ratio of the signal of the semiconducting polymer (which remains constant) to that of the ROS-responsive dye correlates with the concentration of ROS with which it reacts.

ROS sensing by polymeric photoacoustic contrast agent. ROS inactivate the dye (IR775S, blue) that produces photoacoustic (PA) signal in response to irradiation at 820 nm, while signal upon 700 nm irradiation remains constant. SP1, superconducting polymer 1; DPPC, surfactant. Reprinted by permission from Nature Publishing Group.

The team, led by first author Kanyi Pu, demonstrated in a Nature Nanotechnology paper that nanoparticles of one type of semiconducting polymer provides much greater photoacoustic signal on a per mass basis than gold nanorods or carbon nanotubes in vitro and in vivo. (Not all of these polymers work equally well for this purpose; the study began by comparing two, and the polymer used in later experiments has four-fold greater signal.) They also showed that the activatable version detects inflammation caused by treatment with an immune-activating agent (zymosan).

While this is a clever demonstration of the potential relevance of this class of materials to photoacoustic imaging, the usefulness of this advance remains far from clear. As this is the first use of these materials in animals and involved only one acute exposure, their safety must be examined much more thoroughly. Another caveat is that while induced inflammation is detectable with this approach, ROS levels characteristic of chronic diseases may be less easily distinguished. More broadly, while PAT is a hot topic, the method is only now being introduced to the clinic (so far in non-contrast-enhanced applications, such as monitoring breast cancer response to therapy by measuring blood volume and oxygenation).

Activation of anticancer prodrug by palladium particles to prevent toxicity outside tumors

The major drawback of traditional chemotherapeutic drugs is that they don’t selectively kill or prevent proliferation of tumor cells; instead, they act on all dividing cells, causing the well-known, debilitating side effects of chemotherapy such as nausea and fatigue. Thus, a major goal in cancer drug development is to eliminate these off-target effects while preserving the drugs’ ability to block tumor growth. This challenge is one of the major motivations for the development of the field of nanomedicine, but targeting drug-carrying nanoparticles is only one solution. Another approach is to modify the drug so it’s activated specifically within the tumor; that is, to create a prodrug. However, no tumor-specific means of activation is active enough to ensure generation of sufficient drug; current prodrugs generally involve less specific mechanisms like acid-catalyzed hydrolysis.

A research group at the University of Edinburgh has recently introduced a potential solution to this challenge: creating a prodrug activated by a non-biological catalyst, and implanting that catalyst in the tumor. Ideally, the catalyst would not affect other biological processes; as an entire field has recently developed around this type of chemistry (termed “bioorthogonal”), the group, led by Asier Unciti-Broceta, had plenty of knowledge on which to build their work. Of the materials known to catalyze bioorthogonal reactions, the most biocompatible is palladium-zero (chemical symbol Pd0). First author Jason Weiss and the rest of the team chose to create a prodrug from 5-fluorouracil, a chemotherapeutic for which dosages are severely limited by its toxicity to bone marrow and the intestinal epithelium (causing low white blood cell counts and diarrhea), because the reaction by which it’s incorporated into nucleotides points to a modification that would block its activity. They made several modifications to 5-fluorouracil that would be stable against biological enzymes but susceptible to Pd0-mediated activation, then examined whether any could be activated by Pd0 at 37 °C in physiological buffer (Pd0 is generally used at much higher temperatures). Luckily, one of these modifications proved compatible with biological conditions.

Implanted catalyst to activate prodrug
Activation of 5-fluorouracil prodrug by Pd0. Upon activation, the drug diffuses into cells and is incorporated into a uracil nucleotide, which then inhibits production of an essential nucleoatide for DNA replication, or is incorporated into DNA or RNA, which prevents further synthesis. Both actions prevent tumor cell growth and proliferation.

The study’s key results are that the Pd0-activated prodrug kills cancer cells and that implantation of Pd0 does not affect development of zebrafish embryos, suggesting that it would be safe. However, Unciti-Broceta’s group did not assess tumor growth inhibition in vivo. Though the reason for this limitation is not discussed, it may relate to the number of variables involved in setting up such a study, since the distance the activated drug can diffuse and the amount of Pd0 required to convert sufficient prodrug to significantly affect tumor growth, among other parameters, remain unknown.

Given the requirement for implantation of the Pd0 catalyst, this strategy has better potential as a follow-up than a first-course treatment. The Pd0 particles could be implanted following resection to ensure eradication of any potentially remaining tumor cells.

Source: Weiss JT, Dawson JC, Macleod KG, Rybski W, Fraser C, Torres-Sánchez C, Patton EE, Bradley M, Carragher NO, Unciti-Broceta A. Extracellular palladium-catalysed dealkylation of 5-fluoro-1-propargyl-uracil as a bioorthogonally activated prodrug approach. Nat Commun 2014; 5.

Light activation of motor neurons to treat paralysis

Recent work by neuroscientists, geneticists, and bioengineers has developed a means by which to control the activity of individual neurons using light. Though this technology seems futuristic, it relies on bacterial genes for light-responsive ion channels (thus its name, optogenetics). Because it requires genetic manipulations, which despite many years of research towards gene therapy remains challenging in patients, the concept has long been considered simply a research tool, but recent work published in Science suggests that it may eventually be useful in the clinic.

The cell type-specific control of optogenetics promises relevance to a variety of neurological conditions, including neuropathic pain, paralysis, epilepsy, and Parkinson’s. However, the clinical relevance of previous studies has been limited by the difficulty of inducing stable expression of these ion channels, called opsins. In this new development, researchers at King’s College London and University College London solved this problem by expressing the channels in transplanted cells, in this case derived from embryonic stem cells (ESCs). This approach is especially appropriate to treating paralysis because it both replaces damaged neurons and allows selective activation of transplanted and not surviving endogenous neurons. No currently available method of transplanting ESC-derived neurons allows integration with inputs from the brain, so they must be externally activated; electrical stimulation not only triggers excessive, spasmodic muscle contractions but may also activate sensory neurons and thus cause pain.

The most likely clinical application would be to support breathing in patients with high-level spinal cord injury by transplanting optogenetically altered neurons into the phrenic nerve, as these contractions would not require coordination with other muscles. Given the challenges of modeling such a severe condition in animals, the team led by Linda Greensmith and Ivo Lieberam instead proved the principle in the sciatic nerve.  Transplantation of ESC-derived, channelrhodopsin 2-transfected motor neurons was shown to allow light-triggered contraction of the lower hindlimb muscles, illustrating that this approach leads to sufficient expression of the channel. As has been shown in a previous optogenetic study, light activation induces a contraction pattern that more closely matches that caused by endogenous motor signals than electrical stimulation (which minimizes muscle fatigue), suggesting that this combination of optogenetics with regenerative medicine has advantages over either approach alone.

Light control of motor neurons
Expression of a light-activated ion channel in ESC-derived transplanted neurons enables light-triggered muscle contraction in an anesthetized mouse with peripheral nerve injury in an anesthetized animal. Image by H. MacDonald, Science Magazine.

While these results are an exciting step forward, translation to patients is still a far-off goal. Most importantly, an implantable light-emitting device must be developed; in this work, the nerve had to be surgically exposed to be accessible to the LED. Further, the long-term effects of exogenous ion channel expression and stem cell-derived neuron transplantation must be characterized, and the work must be replicated (or built upon in the phrenic nerve setting) using induced pluripotent stem cells, so that a future therapy could use a patient’s own cells rather than foreign ESCs.

Long-term quantitative imaging of oxygen levels via an injectable MRI agent

Quantitative measurement of oxygen levels would be highly useful in the oncology clinic, as hypoxia confers resistance to radiotherapy, impedes the action of chemotherapeutics, and promotes metastasis. However, no method of measuring oxygen in tissues has yet been introduced that meets clinical needs. Current approaches are either invasive, only useful in superficial tissues, or require specialized, expensive equipment not available in most medical centers.

A research group at MIT has recently introduced a creative solution to this challenge: an injectable MRI agent that forms a depot. This unusual strategy ensures that a sufficient concentration of the oxygen-responsive component, decamethyltetrasiloxane (DMTSO), is present within the region in which oxygen levels are to be imaged. The team, led by Michael Cima, a materials scientist, encapsulated this compound into polydimethylsiloxane (PDMS) microparticles at a 7:3 ratio so that it would be retained for long periods. Tissue oxygen concentrations can be calculated by correlation with a calibration curve generated by imaging DMTSO:PDMS exposed directly to varying concentrations of oxygen. In this study, the ability of the DMTSO:PDMS depot to detect oxygen level variation in vivo was determined by imaging rats whose calf muscles had been injected with the microparticles while they breathed air containing varying proportions of oxygen.

Injectable O2-sensing MRI agent
Oxygen tension (pseudocolored) extracted from the MR signal from a DMTSO/PDMS depot overlaid on anatomical images (coronal slices of the leg).

While the paper argues the advantages of depot MR agents, including a consistent concentration for repeated measurements, and demonstrates the biocompatibility of their material up to one month, an injected depot may not be as clinically translatable as an agent that could be cleared from the body, such as a nanoparticle. Whether DMTSO could be incorporated into a smaller agent is not discussed, perhaps because of the difficulty of inducing a sufficient concentration of nanoparticles to accumulate within the anatomical region of interest.

Liu et al., Solid MRI contrast agents for long-term, quantitative in vivo oxygen sensing, Proc Natl Acad Sci 2014; published online April 21.

Nanoparticles to prevent infiltration of immune cells into inflamed tissue

Many serious inflammatory conditions, including acute lung injury, sepsis and ischemia-reperfusion injury, result from excessive infiltration of neutrophils, and blocking this process has been shown effective in reducing inflammation. However, such inhibition has so far relied on antibodies against the adhesion molecules ß2 integrins, and these antibodies also reduce neutrophils’ ability to kill bacteria. This limitation motivated a team at the University of Illinois, Chicago to seek an alternative means of inhibiting neutrophil adherence to blood vessel walls. The team, led by vascular biologist Asrar Malik, demonstrated that albumin nanoparticles encapsulating an inhibitor of spleen tyrosine kinase (Syk) reduced neutrophil infiltration in a model of acute lung injury. While the concept of using nanoparticles to prevent neutrophil adherence to the endothelium could prove useful to the treatment of many severe diseases, whether the effect is indeed selective warrants further study.

Piceatannol-loaded albumin nanoparticles (NPs) block neutrophil adherence. Intravital microscopy in mice in a region adjacent to injection of TNF-α reveals neutrophils carrying either Alexa Fluor 488-conjugated anti-granulocyte receptor-1 (Gr1) antibodies (top) or Alexa Fluor 488-conjugated, drug-loaded NPs (lower panels). Antibody-carrying neutrophils remain in place for over 3 min, while those that have taken up the Syk inhibitor-loaded NPs move along the vessel. Used with permission from Nature Publishing Group.

 

The selectivity of both nanoparticle uptake and the drug’s effects are partially established in this early-phase investigation. While no uptake by macrophages is observed in inflamed regions, nanoparticle accumulation in other cell types and non-inflamed tissues remains to be examined. Similarly, nanoparticle uptake by neutrophils is shown to be blunted in mice lacking the FcγR, which is known to activate endocytosis upon binding of denatured proteins, but the potential contribution of nanoparticle attributes such as size and charge is not addressed. Study of the drug-carrying particles’ therapeutic effects focuses entirely on neutrophil adherence and infiltration, which are only one aspect of the inflammatory response. As the drug employed, piceatannol, also acts as an antioxidant and promotes apoptosis, the formulation could reduce inflammation via multiple mechanisms. 

Selective targeting of neutrophils would prove enormously useful for the delivery of many immune-modulating drugs. If albumin nanoparticles do possess such ability, the simplicity of their preparation and history of FDA approval in formulations such as Abraxane would facilitate development of future therapies.

Wang Z, Li J, Cho J, Malik A. Prevention of vascular inflammation by nanoparticle targeting of adherent neutrophils. Nature Nanotechnol 2014; 9: 204–210.

 

 

 

Hemostatic nanoparticles reduce mortality following blast trauma

Minimizing bleeding is essential to save lives following major injuries, but existing options to control internal bleeding have major limitations. The two currently available therapies include allogeneic platelets, which have a short shelf life and can cause immune reactions, and recombinant clotting factor 7 (NovoSeven), which is extremely expensive, can trigger thromboembolic events and has not been shown to reliably reduce mortality. These challenges have led several groups to design synthetic platelet substitutes consisting of polymeric nano- or microparticles or liposomes decorated with peptides that bind activated platelets, which accelerates clotting.

One of these groups, led by Erin Lavik at Case Western Reserve University, has recently shown that their synthetic platelets, which they term hemostatic nanoparticles, reduce mortality and lung injury in mice exposed to pressure waves mimicking a blast. This advance is especially impressive given that no animal model of blast trauma yet existed. Further, blast trauma causes multiple severe injuries and is likely one of the most challenging settings in which these nanoparticles might be used.

The Lavik lab’s hemostatic nanoparticles consist of poly(lactic-co-glycolic) acid (PLGA) and poly(L-lysine) cores coated with polyethylene glycol chains conjugated to GRGDS (glycine-arginine-glycine-aspartate-serine) peptides, which bind to the activated platelet receptors glycoprotein IIb-IIIa and integrin αvβ3. These particles have been shown over the past five years to halve bleeding time following femoral artery injury and to increase survival following blunt trauma injury. In the most recent study, hemostatic nanoparticles reduced mortality following a 20 psi pressure wave from 40% (4/10 mice) to 5% (1/11 mice). While this effect was larger than that of NovoSeven or nanoparticles to which a control peptide (that would not bind activated platelet receptors) was conjugated, these differences were not significant because of the necessarily small sample size.

Hemostatic nanoparticles and platelets
Hemostatic nanoparticles (pseudocolored green) bind platelets (pseudocolored purple). Scanning electron micrograph.

As these nanoparticles remain effective even after two weeks of storage at room temperature, they represent a promising alternative to biologically-derived blood products. Nonetheless, studies in larger animals are required before clinical trials may begin.

Lashof-Sullivan MM, Shoffstall E, Atkins KT, Keane N, Bir C, VandeVord P, Lavik EB. Intravenously administered nanoparticles increase survival following blast trauma. Proc Nat Acad Sci USA 2014; published online June 30.

Standing waves to pattern tissue constructs

Pattern generation by sonic vibrations is a well-known phenomenon often used in physics demonstrations (see this entertaining video by UCSD’s own Physics Girl, recent winner of Alan Alda’s Flame Challenge). However, it has only recently been applied to the challenge of patterning engineered tissues, which is surprising given its simplicity and the tolerance of cells for these vibrations.

A team led by Utkan Demirci at Stanford University recently showed in Advanced Materials that standing waves in a liquid can cause cultured cells to assemble into sub-millimeter scale patterns, which can be immobilized using blood clotting proteins (including fibrinogen in the cell solution and adding thrombin after pattern generation). As this approach is bottom-up rather than top-down, it is far less time- and resource-intensive than many methods of cell patterning, such as bioprinting and micromolding. Further, it employs equipment available in most biology labs, and could thus be easily adopted by many researchers if it proves able to generate structures similar to those of native tissues. This method, termed liquid-based templated assembly (LBTA) by the researchers, can generate an enormous variety of patterns depending on the combination of frequency and geometry of the chamber.

Pattern generated by vibrating a solution of NIH 3T3 fibroblasts in OptiPrep/ PBS (also containing fibrinogen). Cells were later fixed with thrombin and stained with cell tracker CFSE. Image used with permission from Wiley and Sons Publishing, © 2014.


According to the New Scientist, the team is currently pursuing application of LBTA to engineering liver tissue.

 

Reprogramming of heart muscle cells into pacemaker cells in a large animal

Slow heartbeats caused by age or heart disease are treated by implantation of a pacemaker, a device that delivers precisely timed electrical impulses to the heart muscle. While these are widely used and generally effective, they can fail or cause complications such as infections or damage to the surrounding tissues caused by device migration. An alternative means of restoring normal heart rate that is no more invasive than pacemaker implantation (a minor surgery performed under local anesthesia) would avoid such complications. However, targeting such futuristic therapies, either stem cell-derived pacemaker cells or genes to convert heart muscle cells to pacemaker cells, to a small area of the heart (necessary to induce synchronous beating) represents a major challenge.

A research team at Cedars-Sinai Heart Institute led by Eugenio Cingolani has made a major advance towards this goal, demonstrating restoration of beating in pig hearts with experimentally induced heart block by a virally delivered gene that reprograms heart muscle cells. Most importantly, this induction of endogenous pacemaking was achieved via a minimally invasive procedure; the viral vector was introduced to the heart muscle via a steerable catheter.

Specifically, Yu-Feng Hu (the study’s first author) and co-workers delivered transcription factor T-box 18 (TBX18), previously demonstrated to convert heart muscle cells to pacemaker cells (also called sinoatrial node cells) in vitro and in guinea pigs. This intervention restored normal heart rate (75-80 bpm) in pigs with heart block induced by radio frequency ablation of the atrioventricular node (in which animals heart rate was 60-65 bpm) from days 5-8 following gene transfer, after which time heart rate decreased, suggesting that transduced cells had begun to be eliminated. (Virally transduced cells are known to be cleared by the immune system.) Restoration of heart rate supported increased physical activity.  

Heart muscle cells converted to pacemaker cells
Tbx18 adenovirus reprograms heart muscle cells into sinoatrial node cells. Reprogrammed cells then generate electrical impulses to restore normal heart rate. Image from Science, 345 (6194): 268-269.

While this study demonstrates the feasibility of genetic reprogramming as a therapeutic approach, and is a convincing preclinical basis for human trials of a therapy that would provide temporary pacemaker function, other gene delivery vehicles are needed to allow retention of reprogrammed cells. Current research in nanomedicine, either involving synthetic nanoparticles or engineered viruses or cells, could lead to such vehicles. Nonetheless, even a temporary effect would be clinically relevant, as it would allow normal heart function between removal of an electronic pacemaker following complications and implantation of another.

Synthetic myelination substrates for high-throughput screening of multiple sclerosis therapeutics

In multiple sclerosis (MS), neurons degenerate because the electrical insulation surrounding them, myelin, is gradually lost. Myelin is formed by oligodendrocytes, another cell type in the central nervous system, and damage to myelin can be repaired by their precursors, which are present in adults and are recruited to sites of damage. Remyelination prevents neuronal damage, but cannot keep pace with the damage in MS; drugs that promote it would be an obvious means of slowing progression. However, no such drugs have yet been identified.

A possible reason for this lack of disease-modifying drugs in MS is that screening candidates for promotion of such a complex process would be highly time-intensive, as quantifying myelination in neuronal cultures is laborious. However, the recent observation that oligodendrocytes can myelinate electrospun fibers led a team at UC San Francisco supervised by Jonah Chan to design a substrate for high-throughput screens. As an assay that yields a simple, countable output would be most efficient, postdoc Feng Mei and colleagues examined whether vertical silica micropillars could act as myelination substrates, as these would yield rings detectable by staining for a myelin surface marker.

After showing that oligodendrocytes do indeed extend myelin around micropillars, they then modified the assay to identify compounds that promote myelination by oligodendrocyte precursors, since these are the true drug target. Staining for both a precursor marker and a mature oligodendrocyte marker identified compounds that promoted differentiation and myelination (indicated by a high ratio of mature marker-stained to precursor marker-stained rings). Demonstration that one of the active compounds promotes remyelination in mice treated with an agent that causes demyelination indicates that this rapid assay yields valid candidates.
 


Micropillar assay provides easily analyzed results: green rings (positive for PDGFRα) indicate wrapping by oligodendrocyte precursors, while red rings (positive for myelin basic protein, MBP) indicate wrapping by mature oligodendrocytes. Ideal drug candidates would promote both myelination and differentiation; clemastine was the most effective compound among those screened in promoting both outcomes. (Thyroid hormone T3 is known to promote myelination.) Used with permission from Nature Publishing Group.

 

While the family of compounds that promote remyelination in this assay are not likely drug candidates given their sedative and psychoactive properties, they do indicate a direction for future drug screening, as all effective compounds inhibit a particular class of acetylcholine receptor. However, an important caveat to these results is that drugs were screened in healthy oligodendrocyte precursors; whether they would have the same effect in MS-affected cells is unknown. Employing patient-derived cells (perhaps via induction of pluripotency in skin or other accessible cells, followed by differentiation into oligodendrocyte precursors) in future studies would provide more directly relevant candidates.

This work suggests that micropatterned surfaces could be an important tool in drug screening. While micropillars are ideal substrates for myelination assays, other patterns may simplify image analysis for other cell fates or outcomes; for example, wavy or ridged surfaces could be used in assays related to skeletal muscle differentiation.

 

Mei F et al., Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis, Nature Medicine 2014; published online July 6.

Porous scaffolds for in vitro modeling of traumatic brain injury

Aiming to create an in vitro model of the brain, a group at Tufts University led by David Kaplan has reported layered structures formed by piecing together sponge-like scaffolds seeded with neurons from rat brains and filled with collagen gel. While claims of its similarity to brain structure and function may be overblown, the work represents an important advance in the development of materials for 3D neuron culture that support long-term viability.

Much of the excitement regarding this paper in the media (and even from NIH) appears to rest on the title’s assertion of similarity to the brain, but that similarity is limited. The structures have six layers like the human cortex, but the layers in these constructs are far thicker than the thinnest in the human brain (1-2 mm vs. <100 µm), and these layers are all seeded with the same mix of cortical cells, while neurons in each layer of mammalian cortex have particular shapes, sizes, and densities. Mimicking such complexity in vitro will likely require a far more sophisticated approach, perhaps involving a combination of top-down engineering and development from stem or progenitor cells.

More deserving of attention (though less interesting to the general public) is the long-term viability enabled by the scaffold’s porous structure, created either by foaming gas through a solution of solubilized silk or by directional rapid freezing (to align pores), followed by drying or lyophilization, respectively. Previous attempts at neuron culture in 3D systems did not support neurons for more than a couple of weeks, as most gel materials collapse over time, lowering permeability to oxygen and nutrients. In contrast, the authors show their cultures maintain viability for five weeks. Such viability is surprising given the mechanical properties the Kaplan group has previously reported for silk-based scaffolds, which are far stiffer than those of the brain. Though this paper demonstrates similar compressive moduli for their spongy constructs following seeding of cells and addition of collagen to those of whole rat brains, they do not report smaller scale mechanical measures.

'Sponge' scaffold for neurons
Neurons cultured in silk fibroin (blue)/ collagen system. Neurons were immunostained for β3 tubulin (green) to mark axons and microtubule-associated protein-2 (red) to mark dendrites. Neurons establish connections across scaffold pores.

Similar mechanical properties are the rationale for examining the response of neurons cultured within this scaffold to mechanical trauma. Min Tang-Schomer et al. show that weight-drop impact transiently increases electrophysiological activity, which mimics findings in animal models. However, the extended impact-induced release of glutamate, the excitatory neurotransmitter, compared to that in animals (lasting over ten minutes vs. less than one) suggests that incorporation of astrocytes (brain cells that recycle glutamate) is necessary to make the model reflect the in vivo response.

This is the first system enabling study of neuronal responses to direct impact in vitro; previous cellular models of traumatic brain injury (TBI) involve other mechanical deformations, which may not cause the same effects. While a layered, brain-like structure is likely not essential to model TBI in vitro, the ability of the Kaplan group’s material to allow real-time recordings during impact is a distinct advantage over other materials for 3D culture such as hydrogels.

Tang-Schomer M et al., Bioengineered functional brain-like cortical tissue, Proc Nat Acad Sci USA 2014; published online Aug 11.

Nanoparticle-based transcription factor mimics

Biologists have been enhancing expression of specific genes with plasmids and viruses for decades, which has been essential to uncovering the function of numerous genes and the relationships among the proteins they encode. However, tools that allow enhancement of expression of endogenous genes at the transcriptional level could be a powerful complement to these strategies. Many chemical biologists have made enormous progress developing molecular tools for this purpose; recent work by a group at Rutgers suggests how nanotechnology might allow application of this strategy in living organisms, and perhaps one day in patients.

In a paper published in ACS Nano, researchers led by KiBum Lee synthesized gold nanoparticles bearing synthetic or shortened versions of the three essential components of transcription factors (TFs), the proteins that “turn on” expression of specific genes in cells. Specifically, polyamides previously designed to bind to a specific promoter sequence, transactivation peptides, and nuclear localization peptides were conjugated to the nanoparticle surface. These nanoparticles enhanced expression of both a reporter plasmid (by ~15-fold) and several endogenous genes (by up to 65%). This enhancement is much greater than that possible using previous constructs lacking nuclear localization sequences; the team incorporated a high proportion of those peptides to ensure efficient delivery to the nucleus.

Nanoscript, a synthetic transciption factor
Diagram of the synthetic TF mimic (termed NanoScript). Decorated particles are ~35 nm in diameter. Letters are amino acid sequences; Py-Im, N-methylpyrrole-N-methylimidazole.

These nanoparticles offer an alternative to delivering protein TFs, which remains extremely challenging despite considerable effort towards the development of delivery systems that transport cargo into cells. Among other barriers to the use of native TFs, incorporating them into polymeric or lipid-based carriers often alters their shape, which would likely reduce their function.

While the group suggests future generations of these nanoparticles might one day be used to treat diseases caused by defects in TF genes, many questions remain. First, the duration of gene expression enhancement is not known; the study only assesses effects at 48 h post-administration. Further, whether gold is the best material for the core remains unclear, as its non-biodegradability means the particles would likely accumulate in the liver over time; synthetic TFs with biodegradable cores might also be considered.

Patel S et al., NanoScript: a nanoparticle-based artificial transcription factor for effective gene regulation, ACS Nano 2014; published online Sep 3.

Spleen-inspired device to clear blood of sepsis-causing pathogens

Microfluidics experts at Harvard’s Wyss Institute have made another big splash, expanding their reputation for creating useful devices that mimic organ function. Recent research funded by DARPA has yielded an impressively effective portable dialysis-like technology to treat sepsis. The device clears blood of pathogens by adding magnetic beads coated with a human protein that recognizes many bacterial (and some fungal) carbohydrates, which are then removed from the blood (in complexes with pathogens) by magnets that pull them across narrow channels into an adjacent stream of saline.

Magnetic beads in artificial spleen
Magnetic beads bound to S. aureus (left) and E. coli (right). Pseudocolored scanning electron micrographs. From Nature News, “Artificial spleen cleans up blood.”

Sepsis is a major public health challenge; its mortality rate is 30–50% even in leading treatment centers. The first line of treatment is usually intravenous broad-spectrum antibiotics, but these are only effective if the pathogen is susceptible. As antibiotic-resistant strains become more prevalent, the need for alternative therapies is increasingly urgent.

The Wyss team’s solution seems simple—bind pathogens and filter them out—but the device’s success relies on some smart tweaks. For example, the protein on the beads’ surface (mannose-binding lectin, MBL) was engineered to only bind pathogens; its pro-inflammatory and pro-clotting functions were removed. Further, thoroughly removing the magnetic beads from blood while maintaining a clinically feasible filtration rate requires a precise ratio of flow rates, for which the researchers took inspiration from the spleen. Analysis of filtered blood in initial experiments revealed not-insignificant quantities of magnetic beads remaining, so lead author Joo Kang and colleagues ingeniously added large, uncoated magnetic beads to collect the smaller ones that do not bind pathogens.

Microfluidic spleen
Cartoon schematic; in the device, blood and saline flow through many channels in parallel. Opsonin refers to a pathogen-binding bead. From Kang J et al., Nature Med 2014.

The device clears ~90% of live S. aureus and E. coli from septic rats’ blood within 1 h and reduces lethality from >80% to ~10%. Removal of pathogens by this process also improves respiratory rate and partially restores leukocyte levels, suggesting that it would speed patient recovery.

While the MBL-coated beads are effective in clearing some fungi and a large proportion of microbes in complex cecal mixtures, the device likely will not be effective against all sepsis pathogens. For example, MBL does not bind Pseudomonas aeruginosa or Haemophilus influenzae, both of which can cause sepsis. Perhaps additional beads bearing other pathogen-binding proteins such as toll-like receptors (or fragments thereof) could be added to broaden the range of pathogens that could be cleared. Another challenge to the development of this device is the large difference in scale between the rats employed in the study and humans.

Nanoparticles allow targeting of inflammation progression in cardiovascular disease

Cardiovascular disease, the leading cause of death in the industrialized world, is currently treated with statins, which lower cholesterol levels in the blood. While this does reduce risk of heart attacks by slowing the formation of atherosclerotic plaque (accumulations of fat and white blood cells), it doesn’t prevent them, so the search for better therapies continues. Because the attribute that makes plaques deadly is their ability to break free and block arteries at narrower points downstream, one promising strategy is to prevent the transition to this necrotic, breakage-prone state. This transition results from chronic inflammation, so drugs that promote the resolution of inflammation would keep plaques from becoming necrotic.

The challenge in developing such a therapy is that allowing it to distribute throughout the body would have massive immune side effects and likely reduce its ability to fight infection. To overcome this, a team at Columbia University led by Ira Tabas collaborated with nanoengineers at MIT, led by Omid Farokhzad. The Farokhzad group specializes in targeted drug delivery, and had previously shown that incorporating a collagen IV-binding peptide increases accumulation of nanoparticles at sites of inflammation, where this component of the vascular wall becomes exposed. As this also occurs surrounding atherosclerotic plaques, the team used the same nanoparticles to deliver an inflammation-resolving peptide, and found that it decreases plaque necrotic area in mice by almost two-thirds (Fredman et al., Sci Transl Med 2015).

These impressive results could still be improved, as the strategy for enhancing accumulation in plaques was not based on a screen of many targeting moieties or surface coatings. The collagen IV-binding peptide they use may indeed be among the most efficient strategies available, as it doubles accumulation in plaque and reduces that in the liver and spleen (all nanoparticles accumulate in those organs, which function to clear exogenous particles from the bloodstream).

The potential for nanoparticles to carry drugs to atherosclerotic plaques has led others to work on this problem as well, but the pro-resolving peptide results are at least as promising as anything else yet published. Other notable work in this area includes delivery of statins to plaques by loading in high-density lipoprotein (HDL), a natural cholesterol carrier, which reduces plaque area and collagen-degrading enzyme activity (Duivenvoorden et al., Nat Comm 2014). A nanoparticle system shown to block uptake of oxidized low-density lipoprotein (LDL) in vitro (previously covered on this site) had similar effects in a mouse model of atherosclerosis (Lewis et al., Proc Nat Acad Sci 2015). The clinical potential of all of these systems remains unknown, however, since heart attacks do not result from atherosclerosis in mice and none have yet been tested in larger animals.