Dr Alessia Besford | Understanding the Crown of a Nanoparticle

Medical nanoparticles are an innovative method of delivering drugs to highly specific locations in the body, such as tumours or across the blood-brain barrier. Once a nanoparticle has entered the bloodstream, it forms a crown of surrounding biomolecules called a corona. The composition of this corona depends on its biological environment and the material of the nanoparticle itself and it has significant implications for how the nanoparticle interacts with the human body. Dr Alessia Besford from the Leibniz Institute of Polymer Research Dresden studies these interactions and how they can be refined to develop more effective medical nanoparticles.

Medical Nanoparticles

Nanotechnology is a fascinating and expanding field of science that can be investigated within a multitude of fields from electrical engineering to quantum physics. It is also increasingly being utilised for medical applications including innovative imaging methods, artificial implants and targeted drug delivery. The latter is particularly exciting because the ability to send drugs to treat a very specific part of the body opens up all sorts of new, more effective treatment options known as nanomedicine.

This method uses nanoparticles, which are tiny, often synthetic, vesicles usually between 1 and 100 nm across – about 1 billionth of a metre. Nanoparticles can be made from metals, ceramics or biodegradable polymers (a larger structure made up of lots of smaller units). Whilst these materials are man-made, they are designed to break down in the body so that they can release their contents. Each particle contains a drug that has been specially designed to treat a specific disease and which works especially well when delivered to its specific target.

For example, nanoparticles can be engineered to bind only to specific cancer cells. Once a nanoparticle has been internalised by the cancer cell, it is transported to the endosome, which is acidic and acts like the cell’s stomach. As a result, the nanoparticle is dissolved and the drug inside is released and the individual cell dies, fighting the cancer whilst leaving surrounding healthy cells intact. This has a number of benefits in comparison to conventional chemotherapy treatments, including a controlled release of the drug with a specific, more effective distribution. Consequently, toxic side effects and adverse reactions are minimised in the patient.

Nanoparticle with a protein corona. Credit Alessia Besford.

The Crown of Nanoparticles

Although medical nanoparticles show incredible potential, more research is needed to understand how they interact with the human body and whether these interactions could be detrimental or even toxic. This is the focus of Dr Alessia Besford’s research at the Leibniz Institute of Polymer Research Dresden in Germany, where she works together with Prof Carsten Werner at the Institute of Biofunctional Polymer Materials. She has conducted multiple studies investigating how nanoparticles interact with the human immune system. Specifically, she studies how immune cells and blood proteins interact with the nanoparticles and how the formation of the biomolecular corona, predominantly consisting of proteins, around synthetic nanoparticles affects their distribution in the body.

‘El corona’ is the Spanish word for crown and it is commonly used in science to describe crown-resembling entities. This can be a part of the body or the halo of a star. When a nanoparticle enters a biological environment (usually via the bloodstream), biomolecules like proteins and lipids adsorb onto its spherical surface, creating a corona. This crown has two layers: the ‘hard’ inner and the ‘soft’ outer biomolecular corona. The ‘soft’ layer contains more loosely bound biomolecules that can exchange with surrounding molecules, whereas the ‘hard’ layer is bound strongly and permanent.

Both the biological milieu (blood) and the surface chemistry of the nanoparticle significantly impact the biological composition of the corona. This means that how the nanoparticle is made and what it is made from are important considerations when developing a nanoparticle vehicle. Materials that reduce the ability of proteins to absorb onto the nanoparticle are called low-fouling; fouling is a term that describes the accumulation of unwanted matter on a surface. For example, poly(ethylene glycol) is often used to coat nanoparticle surfaces which results in a water layer that acts as a physical and energetic barrier against proteins.

Many studies have shown the role of the biological milieu and the surface chemistry of the nanoparticles in corona formation, although there was a lack of studies on the dynamic nature of the blood and its constant flow in the body. Therefore, Dr Besford set out to apply microfluidics to study the protein absorption of nanoparticles under flow with a number of different factors. To do this, she created an environment for her experiments that precisely mimicked the geometry of blood vessels. A variety of particles with different coatings allowed her to carry out her incubation experiments with high-, medium- and low-fouling nanoparticles. Dr Besford also tested with human blood and human plasma (blood with all cells removed) leaving a yellowy liquid instead of red).

Her results revealed that protein adsorption onto the nanoparticles is increased when they are incubated with blood compared to serum and that a dynamic flow results in a more complex corona compared to a static incubation. They also showed that as the fouling degree of the surface increases, so does the amount of different absorbed proteins. Interestingly, she found that an initial hard corona layer on the particle surface is formed 2 seconds from incubation commencement and in a second kinetic phase, a more loosely bound hard layer forms.

Dr Besford’s work here showed how design parameters and the biological environment of a nanoparticle influence the composition of its corona. Understanding corona formation can help to create more accurate drug delivery nanoparticles as predicting how they will interact in the body will affect how effective they are in vivo.

The studies of Dr Besford evolve around nanoengineered materials in human blood and how the composition of the corona affects the association of nanoparticles with human immune cells. Her aim is to predict these associations (i.e., the reactions of the human immune system) based on the composition of the corona. Credit Alessia Besford.

Investigating Nanoparticle Corona to Improve Its Medical Properties

Further work from Dr Besford identified that there are three phases of protein binding. Using an innovative technique, she mimicked the human average blood flow and utilised confocal laser scanning microscopy to observe the proteins attaching to the nanoparticles over time. In phase one, proteins irreversibly attach directly to the nanoparticle surface. In phase two, these proteins irreversibly interacted with other preabsorbed proteins. The ‘soft’ corona was formed in phase three, where proteins were bound reversibly.

This study also investigated low-fouling zwitterionic nanoparticles – particles with equal positive and negative charges to give a net zero charge. In these cases, only a soft corona formed as no proteins bound irreversibly. Through these novel approaches, Dr Besford provided a pathway for a state-of-the-art procedure for kinetics and protein fouling experiments to improve medical nanoparticle production.

Another important consideration to make when developing medical nanoparticles is how the immune system will respond to them. Lots of research has been conducted to construct low-fouling materials that also have minimal interaction with human immune cells, giving them the name stealth materials. As the nanoparticles are a foreign substance, if they are detected too readily by the immune system, they may be destroyed before they can deliver their drug load. The previous assumption was that being a low-fouling material goes hand-in-hand with being a stealth material – but Dr Besford wanted to investigate this. Her in-depth study utilising different levels of fouling materials and immune components proved that this theory is correct.

Credit Alessia Besford.

Finding the Material for Nanoparticles

Dr Besford has also dedicated research into the best materials for medical nanoparticles. One popular material is glycogen, which is a natural polysaccharide (sugar) and is therefore biodegradable and does not provoke the immune system (it has low immunogenicity). However, because it has to be synthesised to create an appropriate nanoparticle, some of these beneficial properties can be lost in the process. Therefore, along with a team led by Dr Quinn Besford (Group Leader at IPF), she developed a unique nanoparticle from oyster glycogen (OG) and polymers called PNIPAM. Excitingly, the OG-PNIPAM nanoparticles both retained the biodegradability of native glycogen and did not induce an inflammatory response from the immune system.

In one of her latest papers, Dr Besford explored spider silk-based materials due to their similarly positive elements – they are biocompatible, processable and biodegradable. The study revealed that when the silk materials are positively charged, the corona that consequently formed were made up largely of fibrinogen-base proteins. These are proteins that are necessary for blood clotting and the tests showed an increase in clotting with these positively charged particles. Comparatively, negatively-charged silk materials prevented blood clotting. The properties of the spider silk can be refined through genetic engineering, which means that a scientist could pick and choose the surface properties of the nanoparticles synthesised from it.

With a strong portfolio of nanoparticle research behind her, Dr Besford is continuing her work with more studies planned in the near future. In a significant stride forward from previous research, the PhD project of her student Vaidehi Londhe combines aspects of nanotechnology, nanoimmunotherapy and immunology to develop immune evasive nanoparticles.

As such, Dr Besford is continuing to build on her experience and expertise to delve deeper into the properties of nanoparticle materials, how they interact with the human body and its immune system and how collating her research can help to develop low-fouling, stealthy nanoparticles for effective medical solutions.