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The ageing population, the high expectations for better quality of life and the changing lifestyle of European society call for improved, more efficient and affordable health care.
Nanotechnology can offer impressive resolutions, when applied to medical challenges like cancer, diabetes, Parkinson's or Alzheimer's disease, cardiovascular problems, inflammatory or infectious diseases.
Experts of the highest level from industry, research centers and academia convened to prepare the present vision regarding future research priorities in NanoMedicine. A key conclusion was the recommendation to set up a European Technology Platform on NanoMedicine designed to strengthen Europe's competitive position and improve the quality of life and health care of its citizens. This article has been extracted from the vision paper “European Technology Platform on NanoMedicine - Nanotechnology for Health” produced by the European Commission.
Nanotechnology Based Diagnostics Including Imaging
Introduction
The application of micro- and nanobiotechnology in medical diagnostics can be grouped into two areas, invitro (biosensors and integrated devices) and in-vivo (implantable devices, medical imaging) applications.
The basis of modern medicine was laid already in the middle of the 19th century by the recognition that the cell is the source of health and disease. It followed that basic research to provide a better understanding of the highly complex working of cells is mandatory for medicine. Improvement and combination of methods to characterize cells or cell compartments in-vitro (like novel optical microscopy, scanning probe microscopy, electron microscopy and imaging mass spectrometry) will be of importance for NanoMedicine.
In-vitro diagnosis for medical applications has traditionally been a laborious task; blood and other body fluids or tissue samples are sent to a laboratory for an analysis, which could take hours, days or weeks, depending on the technique used, and be highly labour intensive. The many disadvantages include sample deterioration, cost, lengthy waiting times (even for urgent cases), inaccurate results for small sample quantities, difficulties in integrating parameters obtained by a wide variety of methods and poor standardisation of sample collection. Steadily, miniaturisation, parallelisation and integration of different functions on a single device, based on techniques derived from the electronics industry, have led to the development of a new generation of devices that are smaller, faster and cheaper, do not require special skills, and provide accurate readings. These analytical devices require much smaller samples and will deliver more complete (and more accurate) biological data from a single measurement.
The requirement for smaller samples also means less invasive and less traumatic methods of extraction. Nanotechnology enables further refinement of diagnostic techniques, leading to high throughput screening (to test one sample for numerous diseases, or screen large numbers of samples for one disease) and ultimately point-of-care diagnostics.
These technological advancements pave the way towards major changes in the way drugs can be prescribed in future, by enabling the goal of personalised medicine that is tailored to individual needs. It is interesting to note that many new in-vitro techniques developed for medical testing are also finding important diverse applications, such as in environmental monitoring and security.
Medical imaging has advanced from a marginal role in healthcare to become an essential tool of diagnostics over the last 25 years. Molecular imaging and image guided therapy is now a basic tool for monitoring disease and in developing almost all the applications of in-vivo NanoMedicine. Originally, imaging techniques could only detect changes in the appearance of tissues when the symptoms were relatively advanced.
Later, contrast agents were introduced to more easily identify and map the locus of disease. Today, through the application of nanotechnology, both imaging tools and marker/contrast agents are being dramatically refined towards the end goals of detecting disease as early as possible, eventually at the level of a single cell, and monitoring the effectiveness of therapy.
The convergence of nanotechnology and medical imaging opens the doors to a revolution in molecular imaging (also called nano-imaging) in the foreseeable future, leading to the detection of a single molecule or a single cell in a complex biological environment.
One of the challenges has been to define research partnerships between the imaging industry and the contrast agent industry, which bring different, complementing competencies to the table. The proposed European Technology Platform in NanoMedicine would be timely to accelerate their integration.
In-vitro Diagnostics
An in-vitro diagnostic tool can be a single biosensor, or an integrated device containing many biosensors. A biosensor is a sensor that contains a biological element, such as an enzyme, capable of recognising and ‘signalling’ (through some biochemical change) the presence, activity or concentration of a specific biological molecule in solution. A transducer is used to convert the biochemical signal into a quantifiable signal. Key attributes of biosensors are their specificity and sensitivity. Nanoanalytical tools like scanning probe microscopy or imaging mass spectrometry offer new opportunities for in-vitro diagnostics, like molecular pathology or reading out highly integrated ultra-sensitive biochips.
Techniques derived from the electronics industry have enabled the miniaturisation of biosensors, allowing for smaller samples and highly integrated sensor arrays, which take different measurements in parallel from a single sample. Higher specificity reduces the invasiveness of the diagnostic tools and simultaneously increases their effectiveness significantly in terms of providing biological information such as phenotypes, genotypes or proteomes.
Several complex preparation and analytical steps can be incorporated into ‘lab-on-a-chip’ devices, which can mix, process and separate fluids, realising sample analysis and identification. Integrated devices can measure tens to thousands of signals from one sample, thus providing the general practitioner or the surgeon with much more complementary data from his patient’s sample. Some nanobiodevices for diagnostics have been developed to measure parts of the genome or proteome using DNA fragments or antibodies as sensing elements and are thus called gene or protein chips. ‘Cells–on-chips’ use cells as their sensing elements, employed in many cases for pathogen or toxicology screening.
Integrated devices can be used in the early diagnosis of disease and for monitoring the progress of therapy.
New advancements in microfluidic technologies show great promise towards the realisation of a fully integrated device that directly delivers full data for a medical diagnosis from a single sample. Recent developments aim at developing in-vitro diagnostic tools to be used in a standard clinical environment or e.g. as ‘point-of-care’ devices.
In-vivo Nano-Imaging
In-vivo diagnostics refers in general to imaging techniques, but also covers implantable devices. Nanoimaging includes several approaches using techniques for the study of molecular events in-vivo and for manipulation of molecules. Imaging techniques cover advanced optical imaging and spectroscopy, nuclear imaging with radioactive tracers, magnetic resonance imaging, ultrasound, optical and X-ray imaging, all of which depend on identifying tracers or contrast agents that have been introduced into the body to mark the disease site
Targeted molecular imaging is important for a wide range of diagnostic purposes, such as the identification of the locus of inflammation, the visualisation of vascular structures or specific disease states and the examination of anatomy. It is also important for research on controlled drug release, in assessing the distribution of a drug, and for the early detection of unexpected and potentially dangerous drug accumulations.
The ability to trace the distribution of a drug leads to the possibility of activating it only where needed, thus reducing the potential for toxicity.
A wide range of particles or molecules is currently used for medical imaging. Some recent developments focus on using nanoparticles as tracers or contrast agents. Fluorescent nanocrystals such as quantum dots are nanoparticles which, depending on their coating and their physical and chemical properties, can target a specific tissue or cell and be made to fluoresce for imaging purposes. They offer a more intense fluorescent light emission, longer fluorescence lifetimes and increased multiplexing capabilities compared to conventional materials. Quantum dots are expected to be particularly useful for imaging in living tissues, where signals can be obscured by scattering. Toxicological studies are being undertaken to precisely study their impact on humans, animals and the environment. New developments are focusing on the nanoparticle coating, to improve its efficiency of targeting and biocompatibility.
The main benefits of nano-imaging for in-vivo diagnostics are the early detection of disease, the monitoring of disease stages (e.g. in cancer metastasis), in patient selection leading to individualised medicine and in the real-time assessment of therapeutic and surgical efficacy.
Basis For A Strategic Research Agenda
In-vitro Diagnostics
The ultimate goal is the fast, reliable, specific and cost-effective detection of a few molecules (or even a single molecule) in a complex, non amplified and unlabelled biological sample. The improvement of invitro diagnostics towards this goal requires:
• nanoanalytical instruments of the highest spatial resolution, sensitivity and range of information and integrated, combined instruments
• better sensitivity of screening methods, enabling the sample size to be decreased or for the early detection of low concentrations of disease markers
• higher specificity, for quantitative detection of markers in complex samples
• stronger reliability, simplicity of use and robustness
• faster analysis
• integration of different technologies to provide data for complementary multi-parameter analyses
The commercialization of low cost, user-friendly lab-on-a-chip devices for point-of-care and disease prevention and control at home is driving research into areas of:
• user-friendly sample preparation techniques, enabling the detection of minute amounts of disease marker in a fairly concentrated blood drop and also minute quantities diluted in a relatively huge sample volume, e.g. 5 to 10 cancer cells in 100 ml of urine
• ultra-sensitive and label-free detection techniques aiming at a faster and more compact direct detection, using for example, cantilevers or conducting polymers
• synthetic recognition elements as sensors, in order to increase the sensitivity, specificity and ruggedness of recognition. This requires advancement in deposition techniques and surface chemistry including self assembly of biomolecules, hybrid conjugates of biomaterials with nanoparticles or Molecularly Imprinted Polymers
• integrated, complex devices based on advanced micro and nanofluidics, using for example active functionalised channel walls, and complex analysis protocols
• biomimetic sensors using molecules as sensors
In-vivo Nano-Imaging
The goal of in-vivo diagnostics research is to create highly sensitive, highly reliable detection agents that can also deliver and monitor therapy. This is the ‘find, fight and follow’ concept of early diagnosis, therapy and therapy control, that is encompassed in the concept of theranostics. With this strategy, the tissue of interest can firstly be imaged, using target specific contrast nanostructures. Then, combined with a pharmacologically active agent, the same targeting strategy can be used for applying therapy. Finally, monitoring of treatment effects is possible by sequential imaging.
Improved Detection
Research is needed to improve the efficiency and reliability of detection systems. A major objective for the coming years is to develop efficient, reasonably priced clinical cameras capable of acquiring whole-body images in one step and undertaking multi-isotope studies. The benefit would be a drastic increase in the throughput of whole body imaging, particularly important for cancer screening. Developments in nuclear imaging (which remains an expensive technology) require new detector architectures and new crystal growing methods to reduce manufacturing costs.
Combining different imaging modalities is a promising approach; for example, positron emission tomography with magnetic resonance imaging, magnetic resonance imaging with ultrasound or with electroencephalogram-based brain mapping, ultrasound with optical technologies and will lead to the possibility of benefiting from the advantages of each system. The fusion of magnetic resonance imaging and optical imaging modalities remains a challenge. In principle, this will require use of fluorescent nanoparticles as signal emitters, which function in both paramagnetic and infrared modes.
Once this is achieved, nanotechnology may lead to the miniaturisation of detection devices or the remote transduction of signals. In high-resolution measurements of electromagnetic fields, the development of new interfaces with nanostructured and/or biological functionalised surfaces, would improve continuous monitoring of biological parameters dramatically. Research is also required to improve methods of image analysis and visualisation, such as 3-D optical reconstruction, real-time intracellular tomography, stereo-imaging, virtual and augmented reality, holography, in-vivo imaging from optical catheters, and better endoscopic tools.
Furthermore, the ability to measure small local variations in temperature using radio frequency detectors has applications in identifying the onset and locus of many diseases, especially cancer.
Several other research issues are to be taken into consideration if molecular imaging is to become a reality. When dealing with the identification of tiny changes, achieving gains in the signal-to-noise ratio is critical, so also is the validation of the delicate measurements that images appear to provide for drug and therapy developments, as well as improvements to measurement techniques and data processing software. Lastly, effort has to be dedicated to the management of large amounts of data in order to fully gain advantage of nano-imaging results.
Nano Probes
The development of probes is an extremely active field where the miniaturization of complex reporting devices adapted to in-vivo imaging would be extremely beneficial. The major issues are specificity and the ability to penetrate the cell. The very earliest manifestations of disease in the body are indicated by changes in living cells, including defective cell adhesion, cell mis-signalling, mitotic errors, intercellular communication errors, and abnormal cytoplasmic changes. Major benefits are envisaged from being able to image and identify these changed states. Different imaging techniques require different reporting devices; for example, quantum dots may ‘report back’ by fluorescing on contact with diseased cells. It is not difficult to make the leap from a reporter device to a device that not only indicates the locus of the disease, but also delivers a cure; for example nanomagnetic particles ‘report back’ by providing increased contrast and also take part in the therapeutic process (in addition, this concept underpins the need for research into gene and cellular therapy and drug delivery).
To improve reporting, research is required into the design and composition of nanoparticles, enabling them to better target diseased cells, including those situated behind barriers such as epithelial tissues. Further research is required into the creation of an ‘all-purpose nanoparticle’ that can be imaged by the variety of existing instruments (e.g. optical, acoustic, magnetic, etc.).
Apart from identifying disease and delivering therapy, the non-toxic labelling of specific cell types is important for the imaging of intracellular trafficking.
Work is also urgently required to improve the biocompatibility of reporter devices and for minimising any potential toxicity, allergic or inflammatory response, taking into account natural elimination to prevent possible long lasting effects. Research is also required on encapsulating contrast agents to facilitate their delivery to the target site, or on attaching specific linkers to provide new properties.
Essentially, the development of new probes for molecular imaging requires a multidisciplinary approach, with the transfer of discoveries in material science (new nanostructures such as particles, tubes, capsules, fullerenes, dendrimers, new polymer structures, etc.).
Combined Techniques
Nanobiotechnology offers significant inputs to the improvement of detection devices and in the tagging of disease indicators administered in-vivo, which will lead to advancement in imaging. Potent driving forces include synergies, such as those between in-vitro diagnostics (probes and markers) and in-vivo imaging; and between contrast / probe development (in drug delivery and/or toxicology studies) and imaging technology (medical instrumentation). The combination of in-vitro diagnostics and in-vivo nano-imaging could lead to targeted tumor disruption or removal: Tagging tumor cells with functionalized nanoparticles, which react to external stimuli, allows for in-situ, localized ‘surgery’ (breaking up or heating of particles by laser, magnetic fields, microwaves, etc.) without invasiveness within the human body.
Surgery
In the clinic, the use of nanoparticles for diagnosis and manipulation may lead to an improvement of surgical techniques. This may be achieved, for example, through cancer distribution mapping using near-infrared quantum dots, and applying thermotherapy or heat treatment, the characterisation and non-destructive removal of cells or tissue in a specific area, the tracking of specific cell types used in therapy, as well as the visualisation of bio-therapeutic agents.
Nanotechnology has further application in transfection devices for therapeutic uses. An example would be the development of devices that can cross biological barriers (like the blood-brain barrier) to deliver multiple therapeutic agents at high local concentrations directly to cancer cells and the neighbouring tissues that play a critical role in the spread of the disease.
Implantable Devices For In-vivo Diagnostics
Nanotechnology also has many implications for invivo diagnostic devices such as the swallowable imaging ‘pill’ and new endoscopic instruments.
Monitoring of circulating molecules is of great interest for some chronic diseases such as diabetes or AIDS. Continuous, smart measurement of glucose or blood markers of infection constitutes a real market for implantable devices. Miniaturisation for lower invasiveness, combined with surface functionalisation and the ‘biologicalisation’ of instruments will help increase their acceptance in the body. Autonomous power, selfdiagnosis, remote control and external transmission of data are other considerations in the development of these devices.
Nanosensors, for example used in catheters, will also provide data to surgeons. Nanoscale entities could identify pathology/defects; and the subsequent removal or correction of lesions by nanomanipulation could also set a future vision.
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