The potential of nanomedicine to tackle many diseases is immense, but we really don’t know much about how they get into cells and what potential damage – as well as good – they could do. Here we learn why a combinatorial approach is set to change that…

Giovanna Fragneto, Head of Large Scale Structures and Soft Matter Science and Support at Institut Laue-Langevin 
Marco Maccarini, research scientist at the Université Grenoble Alpes

The ability to manipulate and control a variety of nanoparticles has revolutionised science in the past three decades. From creating entirely new materials, to understanding the structures behind unusual scientific properties, the relatively new field of nanotechnology is being implemented into our daily lives and has massive potential for the future.   

Nanoparticles are particles between 1 and 100 nanometres in size, and can be found naturally (for example as part of biological systems such as viruses or bone matrix) or can be artificially produced. They can range from carbon-based nanomaterials like fullerenes, to metal-based, such as silver and gold nanoparticles, nano-polymers, and composites of different nanomaterials. These are being used to engineer new materials and nanotechnologies across a variety of sectors.  

The small size of these particles means they have a very high surface area to volume ratio, and thus their properties depend strongly on their size, shape and bound molecules. The options for variables that can be altered to define the properties offer engineers greater flexibility when designing materials that may make their way into our everyday lives. Nanomaterials are already used in everything from golf clubs to racing cars, and are also found in sun creams and cosmetics. Nanoparticles are also expected to have an impact inside our bodies, with potential as drug delivery vehicles and as contrast agents for pharmaceuticals. 

Unwanted effects 
While we have been able to fine-tune and engineer the properties of nanoparticles by changing their size, shape, and surface chemistry, the downside of such a variety of possibilities means that predicting and controlling exactly how the particles behave at such a minute scale is an enormous challenge. This is of particular concern when it comes to the potential use of nanoparticles within the human body and in systems where they will impact the environment around them. While nanotechnology has improved lives and enabled massive progress in many areas of society, it must be acknowledged that until we are able to fully characterise and engineer the precise behaviours of nanoparticles at every level, enhanced use of nanoengineered materials exposes humans, animals, and the environment to their potential risks. 

Going gold 
Gold nanoparticles (AuNPs) are a metal-based material proving to be a next-generation tool in nanoengineering. At such a small dimension, gold is an effective catalyst. Thanks to their particular range of electronic, optical, sensing, and biochemical properties, AuNPs have long-been investigated for potential biological and medical potential applications, such as medical imaging, disease treatment, and drug delivery processes. AuNPs can be visualised as a cluster of gold atoms at the centre, surrounded by negative reactive groups on the surface. This surface can be manipulated to produce particular functions for the nanoparticle as a whole, through the addition of specific ligands that may target a particular type of molecule.  

Gold in particular is thought to be suited to systems that can deliver drugs to specific tissues and provide controlled release therapy

By exploiting this property, gold nanoparticles can make good carriers of large and small molecules, hence the interest in employing them as drug transporters to human cells. The idea of nanoparticle-based drug formulations provides a potential opportunity to address and treat challenging diseases.  

Nanotechnology has proven beneficial in the treatment of cancer, AIDS and many other conditions, as well as providing advancement in diagnostic testing. The nanoparticles can be developed into smart systems, encasing therapeutic and imaging agents, but gold in particular is thought to be suited to systems that can deliver drugs to specific tissues and provide controlled release therapy. These systems enable researchers to resolve the main critical issues encountered with conventional pharmaceutical treatments such as the nonspecific distribution, rapid clearance, uncontrollable release of drugs, and low bioavailability. The sustained and targeted drug delivery which could be provided by AuNPs would minimise the toxicity associated with many drugs, and the lack of excess substance means that patients can rely on less frequent doses.  

However, predicting exactly how far they are then absorbed by the cells, which is central to their toxicity, is very difficult, as is understanding any associated risks to health using these nanomaterials. Even with the potential medical benefits, as interactions between gold nanoparticles and living matter are not fully understood, they might not perform as expected in the human body. 

Collaborating to characterise  
To examine this behaviour further, a European collaboration of researchers, including scientists from the Institut Laue-Langevin (ILL), Tampere University, University of Helsinki, Norwegian University of Science and Technology, and Université Grenoble?Alpes, investigated the physical and chemical influences when gold nanoparticles interact with a model biological membrane. 

Conducting this research to identify the behavioural mechanisms taking place is vital to the eventual application of gold nanoparticles in medicine. Enhancing our understanding of the factors that determine whether nanoparticles are attracted or repelled by the cell membrane, whether they are adsorbed or internalised, or whether they cause membrane destabilisation, will help us to ensure that nanoparticles interact with our cells in a controlled way. Such atomic-level intricacies could be the difference between a drug being effectively delivered to a site and carrying out the desired effect, or a drug being unintentionally absorbed into cells, causing systematic damage.  

As outlined in the journal Small, the researchers used a combination of neutron scattering techniques and computational methods to study the interaction between positively charged cationic gold nanoparticles and model lipid membranes, with the aim of revealing the possible nanotoxicity. Nanotoxicity studies are of great complexity, partly due to the fact that in vitro observations of toxicity are often not representative nor directly transferable to in vivo studies. The results obtained in this study could help in the future to lay the foundations for further levels of investigation into how gold nanoparticles behave in the body. 

Lipid membranes, found in a continuous bilayer around all cells, act as a key barrier that keeps out or allows in selected ions, proteins, and other molecules when needed, and prevents the organelles of the cell from diffusing out. The study showed how the temperature and the charge of lipids in the membrane are clear factors that modulate the presence of energy barriers affecting the interaction of the nanoparticle with the membrane.  

The lipid charge is highly relevant for biological systems as plasma membranes are inherently negatively charged – this is critical to the effectiveness of ion pumps that move charged atoms in and out of the cell, creating polarisation central to the primary function of many cells. Understanding how the molecular mechanisms are influenced by temperature can be valuable as they can indicate how the natural biological system may be affected by varying temperature fluctuations. This understanding can then be used to tune the system across the phase of the lipid bilayer in the experimental environment. The results demonstrate how the presence of charged lipids determine the fate of AuNP – whether it is adsorbed or internalised by the cell – and how the AuNP-interaction responds to temperature in the case of non-charged and negatively charged bilayers.  

It takes two  
The study also shows, using the computational technique of coarse?grained molecular dynamics, how the lipid charge can affect the cooperative behaviour, or aggregation, of AuNPs. It was found that negatively charged lipids can favour the aggregation of nanoparticles and this cooperative effect can be fatal for the membrane stability. Furthermore, different molecular mechanisms for nanoparticle-membrane interactions were revealed that explain how nanoparticles become internalised in the lipid membranes. 

The specific neutron technique implemented in the study was neutron reflectometry. This technique provides a wealth of information on the structure of thin films and solid surfaces, and is particularly well suited to the study of interfaces between solids and liquids. The technique is highly versatile and able to examine a wide variety of materials. It is the perfect tool for examining the molecular details of the lipid/nanoparticle interaction, providing unambiguous insight into the behaviours of the molecular components. The instrument uses a beam of neutrons, directed at a sample, to allow researchers to visualise molecular structures across a range of length scales. 

Institut Laue-Langevin (ILL) is the world’s flagship neutron science facility, and provides the tools for scientists from across the globe and all scientific fields to further investigate the structures at the centre of their research. The D17 instrument at ILL hss a horizontal scattering geometry designed for high flux and flexibility, and is one of the ILL’s two reflectometers. It is highly flexible in resolution and modes of operation, and is well suited to the study of solid-liquid interfaces and membranes. 

In addition to the neutron reflectometry data, the researchers implemented molecular dynamics (MD) – a computational simulation method for studying the movement of atoms – to demonstrate how gold nanoparticles interact within the system at the atomic level. It provides a complementary tool to interpret and explain the data obtained on real systems by neutron reflectometry. As well as indicating the potentially destructive effects of gold nanoparticles on the cell membrane, the study provides an additional bonus for the field of demonstrating convincingly that the combination of neutron scattering and computational methods provides a better understanding than just one of these methods on its own.  

There are differences between the two methodologies, and so particular care was taken when comparing the results. One of these differences pertains to the timescales of the data provided. While neutron reflectometry provides insight about systems under equilibrium conditions at time scales of minutes or hours, MD simulations explore the system of interest over time scales of microseconds or milliseconds. In addition, it is important to note that the lipid bilayers studied in the ILL neutron experiments differed slightly from those in the simulations. Despite these differences, the two techniques give complementary and coherent views of the membrane systems, and the results provide detailed information on the effect of AuNPs on the membrane. 

Unlocking the future 
Since the first contact between a nanoparticle and a living cell occurs through a biological membrane, it is important to understand the basic mechanisms governing the interaction with the plasma membrane. However, real membranes are complex in terms of their structure, composition, and properties (for example, presence of several lipid types, cholesterol, membrane proteins, glycocalyx) and hence it is currently difficult to establish models that can predict the fate of nanoparticles interacting with a real plasma membrane. Instead, simpler models can be used to represent some essential membrane characteristics. 

There are thousands of different nanoparticles of different sizes and compositions, all of which can impact on cells differently. The complementarity of computational and neutron techniques highlighted in this study has helped to provide a clearer indication of what influences the behaviour of nanoparticles, which can help us predict how cells will interact with nanoparticles in future applications. Research into the possible mechanisms for implementing gold nanoparticles in medical applications such as drug delivery must be accompanied by a wealth of studies into where the high-potential properties could also have extensive yet unwanted effects. While nanoparticles are proving to be an invaluable tool to help us address a number of social challenges, it is important that we develop the tools to better investigate nanomaterials, so we can harness them effectively and safely. This ability can be enhanced through developments in neutron science techniques and advances in sample environment and sample preparation, performed at world-class facilities such as ILL.  

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