A new approach to create targeted nanovesicles for cancer treatment

Researchers have used autologous immune cells from the mouse to create nanovesicles to be used in the delivery of drugs to tumors. This technique helped them to create a sufficient number of nanovesicles inexpensively to be used as drug delivery system.

This image shows ligands-grafted extracellular vesicles as drug delivery vehicles.
Credit: Xin Zou 

Cells naturally release nanovesicles to carry chemical messages between cells. To create targeted nanovesicles, ligands (short pieces of protein) need to be attached to the nanovesicle wall so they can recognize tumor cells. This is done by incorporating DNA into cells and collecting extracellular nanovesicles from cell culture supernatant. However, the yield of nanovesicles is poor using above process. Researchers now developed a new approach by chemically grafting lipid tagged ligands to the cell membrane and then passing them through a seave to create large amounts of fillable and targeted nanovesicles.  Research findings are published in journal Cancer Research.

“Currently, natural nanovesicles can be harvested from cell culture supernatant (the fluid surrounding cultured cells) and they are fillable,” said Yuan Wan, a postdoctoral fellow in biomedical engineering, Penn State. “However, there are two problems using them for cancer treatment. There aren’t enough nanovesicles produced in short timescales and they do not have targeting effect.”

“Pushing the cells through a filter is the engineered way to produce lots of nanovesicles,” said Zheng. “This approach enables us to create nanovesicles with different ligands targeting different types of tumors in about 30 minutes to meet actual needs,” said Zheng. “With this approach, we also can put different types of ligands on a nanovesicle. We could have one ligand that targets while another ligand says, ‘don’t eat me.'”

Reference: Wan, Yuan, Lixue Wang, Chuandong Zhu, Qin Zheng, Guoxiang Wang, Jinlong Tong, Yuan Fang, Yiqiu Xia, Gong Cheng, Xia He, and Si-Yang Zheng. “Aptamer-Conjugated Extracellular Nanovesicles for Targeted Drug Delivery.” Cancer Research 78, no. 3 (2017): 798-808. doi:10.1158/0008-5472.can-17-2880.

Research funding: Nanjing Science and Technology Development Foundation, Jiangsu Provincial Medical Youth Talent Award, Natural Science Foundation of Jiangsu Province, U.S. National Institutes of Health.

Adapted from press release by Penn State.

New nanosensor technology to detect osteoarthritis biomarker

Researchers at Wake Forest Baptist Medical Center have been able to analyze hyaluronic acid using solid-state nanopore sensor. This technique allows them to study its role in osteoarthritis and other inflammatory joint disorders. This technique is first of a kind and is a significant improvement regarding relative ease to perform and high precision from other techniques like gel electrophoresis, mass spectroscopy or size exclusion chromatography.

The study was led by Hall and Elaheh Rahbar, Ph.D., of Wake Forest Baptist, and conducted in collaboration with scientists at Cornell University and the University of Oklahoma. The study is published in the journal of Nature Communications.

“Our results established a new, quantitative method for the assessment of a significant molecular biomarker that bridges a gap in the conventional technology,” said Adam R. Hall. “The sensitivity, speed and small sample requirements of this approach make it attractive as the basis for a powerful analytic tool with distinct advantages over current assessment technologies.”

In the study researchers first employed synthetic hyaluronic acid polymers to validate the measurement approach. They then used the platform to determine the size distribution of hyaluronic acid extracted from the synovial fluid of a horse model of osteoarthritis.

The measurement approach consists of a microchip with a single hole or pore in it that is a few nanometers wide that is small enough that only individual molecules can pass. And as they do, each can be detected and analyzed. By applying the approach to hyaluronic acid molecules, the researchers were able to determine their size one-by-one. Hyaluronic acid size distribution changes over time in osteoarthritis so this technology could help better assess disease progression, Hall said.

Researchers hope to conduct their next study in humans, and then extend the technology with other diseases where hyaluronic acid and similar molecules play a role, including traumatic injuries and cancer.

Citation: Rivas, Felipe, Osama K. Zahid, Heidi L. Reesink, Bridgette T. Peal, Alan J. Nixon, Paul L. Deangelis, Aleksander Skardal, Elaheh Rahbar, and Adam R. Hall. “Label-free analysis of physiological hyaluronan size distribution with a solid-state nanopore sensor.” Nature Communications 9, no. 1 (2018). doi:10.1038/s41467-018-03439-x.

Adapted from press release by Wake Forest Baptist Medical Center.

Gene therapy using lipid based nanoparticles

Lipid nanoparticles (SLNs and NLCs) are regarded as highly promising systems for delivering nucleic acids in gene therapy. Literature review by researchers at PharmaNanoGene describes these systems and their main advantages in gene therapy, such as their capacity to protect the gene material against degradation, to facilitate cell and nucleus internalization and to boost the transfection process.

View of lipid nanoparticles.
View of lipid nanoparticles. Credit: UPV/EHU

“At PharmaNanoGene we are working on the design and evaluation of SLNs for treating some of these diseases using gene therapy. We are studying the relationship between formulation factors and the processes involving the intracellular internalisation and disposition of the genetic material that condition the effectiveness of the vectors and which is essential in the optimisation process, and for the first time we have demonstrated the capacity of SLNs to induce the synthesis of a protein following their intravenous administration in mice,” they stressed.

The publication also includes other pieces of work by this University of the Basque Country research group on the application of SLNs in the treatment of rare diseases, such as chromosome-X-linked juvenile retinoschisis, a disorder in which the retina becomes destructured due to a deficiency in the protein retinoschisin.

 “One of the main achievements of our studies in this field has been to demonstrate, also for the first time, the capacity of a non-viral vector to transfect the retina of animals lacking the gene that encodes this protein and partially restore its structure, showing than non-viral gene therapy is a viable, promising therapeutic tool for treating degenerative disorders of the retina,” specified the researchers.
The application of SLNs for treating Fabry disease, a serious, multi-system metabolic disorder of a hereditary nature, has also been studied at PharmaNanoGene.

 “This is a monogenic disease linked to the X-chromosome which is caused by various gene mutations in the gene that encodes the galactosidase A enzyme. In cell models of this disease we have demonstrated the capacity of SLNs to induce the synthesis of galactosidase A enzyme”. They have also reviewed the application of lipid nanoparticles to the treatment of infectious diseases: “Our work in this field shows that SLNs with RNA interference are capable of inhibiting a replicon of the hepatitis C virus in vitro, which was used as proof-of-concept of the use of SLN-based vectors as a new therapeutic strategy for treating this infection and others related to it”.

Citation: Ana del Pozo-Rodríguez, María Ángeles Solinís, Alicia Rodríguez-Gascón, Applications of lipid nanoparticles in gene therapy, European Journal of Pharmaceutics and Biopharmaceutics, Volume 109, December 2016, Pages 184-193.
DOI: 10.1016/j.ejpb.2016.10.016.
Adapted from press release by University of the Basque Country.

Large synthetic nanoparticles mimic biomolecules

Chemists at Carnegie Mellon University have demonstrated that synthetic nanoparticles can achieve the same level of structural complexity, hierarchy and accuracy as their natural counterparts biomolecules. The study, published in Science, also reveals the atomic-level mechanisms behind nanoparticle self-assembly.

The structure of the largest gold nanoparticle to-date, Au246(SR)80, was resolved using x-ray crystallography.
Credit: Rongchao Jin Carnegie Mellon University.

The findings from the lab of Chemistry Professor Rongchao Jin provide researchers with an important window into how nanoparticles form, and will help guide the construction of nanoparticles, including those that can be used in the fabrication of computer chips, creation of new materials, and development of new drugs and drug delivery devices.

“Most people think that nanoparticles are simple things, because they are so small. But when we look at nanoparticles at the atomic level, we found that they are full of wonders,” said Jin.

Nanoparticles are typically between 1 and 100 nanometers in size. Particles on the larger end of the nanoscale are harder to create precisely. Jin has been at the forefront of creating precise gold nanoparticles for a decade, first establishing the structure of an ultra-small Au25 nanocluster and then working on larger and larger ones. In 2015, his lab used X-ray crystallography to establish the structure of an Au133 nanoparticle and found that it contained complex, self-organized patterns that mirrored patterns found in nature.

In the current study, they sought to find out the mechanisms that caused these patterns to form. The researchers, led by graduate student Chenjie Zeng, established the structure of Au246, one of the largest and most complex nanoparticles created by scientists to-date and the largest gold nanoparticle to have its structure determined by X-ray crystallography. Au246 turned out to be an ideal candidate for deciphering the complex rules of self- assembly because it contains an ideal number of atoms and surface ligands and is about the same size and weight as a protein molecule.

Analysis of Au246’s structure revealed that the particles had much more in common with biomolecules than size. They found that the ligands in the nanoparticles self-assembled into rotational and parallel patterns that are strikingly similar to the patterns found in proteins’ secondary structure. This could indicate that nanoparticles of this size could easily interact with biological systems, providing new avenues for drug discovery.

The researchers also found that Au246 particles form by following two rules. First, they maximize the interactions between atoms, a mechanism that had been theorized but not yet seen. Second the nanoparticles match symmetric surface patterns, a mechanism that had not been considered previously. The matching, which is similar to puzzle pieces coming together, shows that the components of the particle can recognize each other by their patterns and spontaneously assemble into the highly ordered structure of a nanoparticle.

“Self-assembly is an important way of construction in the nanoworld. Understanding the rules of self-assembly is critical to designing and building up complex nanoparticles with a wide-range of functionalities,” said Zeng, the study’s lead author.

In future studies, Jin hopes to push the crystallization limits of nanoparticles even farther to larger and larger particles. He also plans to explore the particles’ electronic and catalytic power.

Citation: Zeng, Chenjie, Yuxiang Chen, Kristin Kirschbaum, Kelly J. Lambright, and Rongchao Jin. “Emergence of hierarchical structural complexities in nanoparticles and their assembly.” Science 354, no. 6319 (2016): 1580-1584.
DOI: 10.1126/science.aak9750
Research funding: Air Force Office of Scientific Research, Camille Dreyfus Teacher-Scholar Awards Program.
Adapted from press release by Carnegie Mellon University.

Development of thin-film biomaterials using nanofibres to be used as scaffolding for bone regeneration

In future, it may be possible to use nanofibres to improve the attachment of bone implants, or the fibres may be used directly to scaffold bone regeneration. This would aid the healing of fractures and may enable the care of osteoporosis. This is detailed in a new dissertation.

Hydroxyapatite nanofibre film produced at the University of Helsinki Credit: Riitta-Leena Inki

In his doctoral research, Jani Holopainen of the Department of Chemistry at the University of Helsinki has developed processes for fibrous and thin-film biomaterials that can be used as scaffolding for bone regeneration and in other bone implants. He also studied the apparatus used for nanofibre production.

At best, bone-reforming scaffolds that regenerate at the same rate as bones could be used as implants. The scaffolds activate the bone cells to generate new bone that slowly replaces the disintegrating scaffold and the implant exits the body naturally without separate removal surgery, Jani Holopainen says.

Holopainen selected hydroxyapatite, the main component of the bone mineral, as the focus of his research. This is why the synthetic hydroxyapatite structures he has developed are very compatible with bone.

Holopainen developed the electrospinning apparatus for producing hydroxyapatite fibres and a new kind of needle less twisted wire electrospinning setup, which is more productive than the generally known electrospinning method. The prototypes for the equipment used in the research were manufactured at the Department of Chemistry at the University of Helsinki. The equipment will have to be developed further in order to enhance production to an industrial scale.

This promising method still has a long way to go before it will become a real medical application, though cellular tests have already been made, says Professor Mikko Ritala of the Department of Chemistry and the Atomic Layer Deposition centre of excellence at the University of Helsinki, who was the adviser of the doctoral research.

Citation: Holopainen, Jani. “Bioactive Coatings and Fibers for Bone Implants and Scaffolds by Atomic Layer Deposition, Electrospinning, Solution Blow Spinning and Electroblowing.”Doctoral dissertation, University of Helsinki (2017).
Adapted from press release by University of Helsinki.

Liquid biopsy chip based on carbon nano-tubes to detect circulating cancer cells

A chip developed by mechanical engineers at Worcester Polytechnic Institute (WPI) can trap and identify metastatic cancer cells in a small amount of blood drawn from a cancer patient. The breakthrough technology uses a simple mechanical method that has been shown to be more effective in trapping cancer cells than the microfluidic approach employed in many existing devices.

A close-up of a prototype liquid biopsy. The chip is able to capture circulating tumor cells in a very small sample of blood. Etched into it are 76 individual test units, each with a small well containing antibodies for specific cancer cell surface markers attached to carbon nanotubes. Credit: Worcester Polytechnic Institute (WPI).

The WPI device uses antibodies attached to an array of carbon nanotubes at the bottom of a tiny well. Cancer cells settle to the bottom of the well, where they selectively bind to the antibodies based on their surface markers (unlike other devices, the chip can also trap tiny structures called exosomes produced by cancers cells). This “liquid biopsy,” described in journal Nanotechnology, could become the basis of a simple lab test that could quickly detect early signs of metastasis and help physicians select treatments targeted at the specific cancer cells identified.

Metastasis is the process by which a cancer can spread from one organ to other parts of the body, typically by entering the bloodstream. Different types of tumors show a preference for specific organs and tissues; circulating breast cancer cells, for example, are likely to take root in bones, lungs, and the brain. The prognosis for metastatic cancer (also called stage IV cancer) is generally poor, so a technique that could detect these circulating tumor cells before they have a chance to form new colonies of tumors at distant sites could greatly increase a patient’s survival odds.

The focus on capturing circulating tumor cells is quite new,” said Balaji Panchapakesan, associate professor of mechanical engineering at WPI and director of the Small Systems Laboratory. “It is a very difficult challenge, not unlike looking for a needle in a haystack. There are billions of red blood cells, tens of thousands of white blood cells, and, perhaps, only a small number of tumor cells floating among them. We’ve shown how those cells can be captured with high precision.”

The device developed by Panchapakesan’s team includes an array of tiny elements, each about a tenth of an inch (3 millimeters) across. Each element has a well, at the bottom of which are antibodies attached to carbon nanotubes. Each well holds a specific antibody that will bind selectively to one type of cancer cell type, based on genetic markers on its surface. By seeding elements with an assortment of antibodies, the device could be set up to capture several different cancer cells types using a single blood sample. In the lab, the researchers were able to fill a total of 170 wells using just under 0.3 fluid ounces (0.85 milliliter) of blood. Even with that small sample, they captured between one and a thousand cells per device, with a capture efficiency of between 62 and 100 percent.

The carbon nanotubes used in the device act as semiconductors. When a cancer cell binds to one of the attached antibodies, it creates an electrical signature that can be detected. These signals can be used to identify which of the elements in the array have captured cancer cells. Those individual arrays can then be removed and taken to a lab, where the captured cells can be stained and identified under a microscope. In the lab, the binding and electrical signature generation process took just a few minutes, suggesting the possibility of getting same-day results from a blood test using the chip, Panchapakesan says.

In a paper published in the journal Nanotechnology, Panchapakesan’s team, which includes graduate students Farhad Khosravi, the paper’s lead author, and researchers at the University of Louisville and Thomas Jefferson University, describe a study in which antibodies specific for two markers of metastatic breast cancer, EpCam and Her2, were attached to the carbon nanotubes in the chip. When a blood sample that had been “spiked” with cells expressing those markers was placed on the chip, the device was shown to reliably capture only the marked cells.

In addition to capturing tumor cells, Panchapakesan says the chip will also latch on to tiny structures called exosomes, which are produced by cancers cells and carry the same markers. “These highly elusive 3-nanometer structures are too small to be captured with other types of liquid biopsy devices, such as microfluidics, due to shear forces that can potentially destroy them,” he noted. “Our chip is currently the only device that can potentially capture circulating tumor cells and exosomes directly on the chip, which should increase its ability to detect metastasis. This can be important because emerging evidence suggests that tiny proteins excreted with exosomes can drive reactions that may become major barriers to effective cancer drug delivery and treatment.”

Panchapakesan said the chip developed by his team has additional advantages over other liquid biopsy devices, most of which use microfluidics to capture cancer cells. In addition to being able to capture circulating tumor cells far more efficiently than microfluidic chips (in which cells must latch onto anchored antibodies as they pass by in a stream of moving liquid), the WPI device is also highly effective in separating cancer cells from the other cells and material in the blood through differential settling.

“White blood cells, in particular, are a problem, because they are quite numerous in blood and they can be mistaken for cancer cells,” he said. “Our device uses what is called a passive leukocyte depletion strategy. Because of density differences, the cancer cells tend to settle to the bottom of the wells (and this only happens in a narrow window), where they encounter the antibodies. The remainder of the blood contents stays at the top of the wells and can simply be washed away.”

While the initial tests with the chip have focused on breast cancer, Panchapakesan says the device could be set up to detect a wide range of tumor types, and plans are already in the works for development of an advanced device as well as testing for other cancer types, including lung and pancreas cancer. He says he envisions a day when a device like his could be employed not only for regular follow ups for patients who have had cancer, but in routine cancer screening.

“Imagine going to the doctor for your yearly physical,” he said. “You have blood drawn and that one blood sample can be tested for a comprehensive array of cancer cell markers. Cancers would be caught at their earliest stage and other stages of development, and doctors would have the necessary protein or genetic information from these captured cells to customize your treatment based on the specific markers for your cancer. This would really be a way to put your health in your own hands.”

Citation: Khosravi, Farhad, Patrick J. Trainor, Christopher Lambert, Goetz Kloecker, Eric Wickstrom, Shesh N. Rai, and Balaji Panchapakesan. “Static micro-array isolation, dynamic time series classification, capture and enumeration of spiked breast cancer cells in blood: the nanotube–CTC chip.” Nanotechnology 27, no. 44 (2016): 44LT03.
DOI: 10.1088/0957-4484/27/44/44LT03
Adapted from press release by Worcester Polytechnic Institute.

MESO-BRAIN stem cell research project to develop 3D nanoprinting techniques to replicate neural networks

Aston University heading up major €3.3m stem cell research project to develop 3D nanoprinting techniques that could replicate brain’s neural networks.

Aston University has launched MESO-BRAIN, a major stem cell research project which it hopes will develop three-dimensional (3D) nanoprinting techniques that can be used to replicate the brain’s neural networks.

The cornerstone of the MESO-BRAIN project will be its use of pluripotent stem cells generated from adult human cells that have been turned into brain cells, which will form neural networks with specific biological architectures. Advanced imaging and detection technologies developed in the project will be used to report on the activity of these networks in real time.

Credit: Ashton University
Such technology would mark a new era of medical and neuroscience research which would see screening and testing conducted using physiologically relevant 3D living human neural networks. In the future, this could potentially be used to generate networks capable of replacing damaged areas in the brains of those suffering from Parkinson’s disease, dementia or other brain trauma.
The MESO-BRAIN initiative, which will span three years, received €3.3million of funding from the European Commission as part of its prestigious Future and Emerging Technology (FET) scheme. Aston University is leading the project, with partners from industry and higher education across Europe: Axol Bioscience Ltd, Laser Zentrum Hannover, The Institute of Photonic Sciences, University of Barcelona and Kite Innovations. This unique partnership brings together stem cell biologists, neuroscientists, photonics experts and physicists.

Head of the MESO-BRAIN project, Professor Edik Rafailov, said: “What we’re hoping to achieve with this project has, until recently, been the stuff of science-fiction.

“If we can use 3D nanoprinting to improve the connection of neurons in an area of the brain which has been damaged, we will be in a position to develop much more effective ways to treat those with dementia or brain injuries.

“To date, attempts to replicate and reproduce cells in this way have only ever delivered 2D tissues or poorly defined 3D tissues that do not resemble structures found within the human body. The new form of printing we are aiming to develop promises to change this. The MESO-BRAIN project could improve hundreds of thousands of lives.”

Dr Eric Hill, Programme Director for MSc Stem cells and Regenerative Medicine at Aston University, commented: “This research carries the potential to enable us to recreate brain structures in a dish. This will allow us to understand how brain networks form during development and provide tools that will help us understand how these networks are affected in diseases such as Alzheimer’s disease.”

Adapted from press release by Ashton University.

Nanomedicine: Nanotechnology and Nanoscience for better health

Nanoscience research involves molecules that are tiny compared to the size of cancer cells and that have the potential to profoundly improve the quality of our health and our lives. Now nine prominent Nano scientists look ahead to what we can expect in the coming decade, and conclude that nanoscience is poised to make important contributions in many areas, including health care, electronics, energy, food and water. The researchers discuss the need to safely implement new nanomaterials and present ideas for doing so. They also call for researchers to communicate their research with the public.

Paul Weiss (UC presidential chair and distinguished professor of chemistry and biochemistry at UCLA) and Dr. Andre Nel, (Chief of nanomedicine at the David Geffen School of Medicine at UCLA) who written a paper about Nanoscience and Nanotechnology in Journal ACS Nano, think that significant progress is already made. Some important points made by authors in the publication include the following with respect to medicine and health.

  • Nanoparticles can be designed to target infectious disease. Nanomaterials may target the lungs to deliver potent antibiotics and anti-inflammatory drugs could fight bacterial and viral infection.
  • Nanoparticles may lead to more effective treatments of neurological disorders such as Parkinson’s disease and Alzheimer’s disease, as well as arthritis.
  • The emerging field of immuno-oncology is likely to produce advances that will activate the body’s immune system to attack tumor cells. Important advantages of nanoparticles are that they can bind selectively to receptors over-expressed on tumors and may be delivered to the same cell at a predetermined dose and timing, although significant scientific challenges remain.
Illustration of ultra thin materials.
Credit: Patrick Han, ACS Nano

Authors also feel that Nanotechnology will be a major part of other aspects of day today life including following areas.
  • The microelectronics industry has been manufacturing products with nanoscale structures for decades — a market currently valued at approximately $500 billion annually. The researchers say there is still plenty of room for major improvements, including many opportunities in creative design of devices for data processing and information storage.
  • Nanotechnology is likely to capture, convert and store energy with greater efficiency, and will help to safely produce sustainable and efficient large-scale energy production to meet the increasing worldwide demand for energy.
  • Nanotechnology principles are being used in water desalination and purification, and nanotechnology is poised to make major contributions to supplying clean water globally.
  • Technology is likely to become increasingly widespread, with the proliferation of “nano-enabled smart devices” in such areas as telecommunications, consumer staples and information technology.
  • Nanoscience advances may lead to advances in food safety and reductions in food contamination. Sensor technologies may be designed that exploit changes at the surface of nanostructures so they can detect disease-causing pathogens before they spread. Nanoscale sensor technologies also may lead to improvements in agrochemicals.

The researchers advocate strong federal support for nanoscience, and predict significant progress toward major scientific goals will be achieved by the end of this decade. They also advocate basic research to produce currently unforeseen discoveries.

Citation: Nano Day: Celebrating the Next Decade of Nanoscience and Nanotechnology
Authors: Cherie R Kagan, et., al.
DOI: http://dx.doi.org/10.1021/acsnano.6b06655
Journal: ACS Nano
Adapted from press release by UCLA

Researchers focus on using Nanoparticles to improve drug delivery in HIV patients

New research led by the University of Liverpool aims to improve the administration and availability of drug therapies to HIV patients through the use of nanotechnology.  The research, conducted by the collaborative nanomedicine research programme led by Pharmacologist Professor Andrew Owen and Materials Chemist Professor Steve Rannard, examined the use of nanotechnology to improve the delivery of drugs to HIV patients.

Currently, the treatment of HIV requires daily oral dosing of HIV drugs, and chronic oral dosing has significant complications that arise from the high pill burden experienced by many patients across populations with varying conditions leading to non-adherence to therapies.

Recent evaluation of HIV patient groups have shown a willingness to switch to nanomedicine alternatives if benefits can be shown. Research efforts by the Liverpool team have focused on the development of new oral therapies, using Solid Drug Nanoparticle (SDN) technology which can improve drug absorption into the body, reducing both the dose and the cost per dose and enabling existing healthcare budgets to treat more patients.

Presently, no clinically available oral nanotherapies exist for HIV populations and conventional paediatric HIV medicines are poorly available. The programme examined one current paediatric formulation that utilizes high ethanol concentrations to solubilize lopinavir, a poorly soluble antiretroviral.

Through the use of a rapid small-scale nanomedicine screening approach developed at Liverpool, the researchers were able to generate a novel water dispersible nanotherapy, hence removing the need to use alcohol in the paediatric medicine.

The research, funded by the UK Engineering and Physical Sciences Research Council is in ongoing human trials, and the preclinical development has been published in Nature Communications today (Friday, 21 October 2016).

Publication: Accelerated oral nanomedicine discovery from miniaturized screening to clinical production exemplified by paediatric HIV nanotherapies.
DOI: http://dx.doi.org/10.1038/ncomms13184
Authors:Marco Giardiello et.al,
Journal: Nature Communications – News
Adapted from press release by University of Liverpool