Nanomaterials have a great potential for biomedical applications. When considering their use in drug delivery, they must be engineered and tailored for targeted delivery and to avoid toxicity. Dr Yahia and his colleagues investigate the use of Superparamagnetic Iron Oxide Nanoparticles (SPIONs) for the delivery of Nitrous Oxide (NO) during surgeries that require general anesthesia. NO has many beneficial uses in medicine as it is a natural neurotransmitter. In this case it would act as a pulmonary vasodilator, of therapeutic use during cardiopulmonary bypass. However, NO has a very short half-life in vivo, which is why attaching the molecule to SPIONs is being explored. SPIONs allow for controlled and prolonged NO release under physiological conditions.
Background
Sabrina: My first question is more of a validation of the conclusions I came to while reading the article and from what I learned in your course on biomaterials. I specifically remember that you made it an important point for us to know that the first event that occurs when a foreign material is introduced in the body, is the adsorption of proteins.
I also learned from Michel Meunier’s course on biomedical nanotechnology, that there are many aspects to consider when evaluating the toxicity and biocompatibility of nanoparticles. Their composition, shape, size, functionalization and surface charge are all significant factors.
As I realized that this article focused on the surface charge of the nanoparticles, I immediately concluded that it was to evaluate which proteins would be adsorbed since they would modulate the cell response and the overall toxicity of the SPIONs.
Dr Yahia: Yes, exactly. When a foreign material is introduced in the human body, the almost instantaneous reaction is the adsorption of endogenous proteins at its surface. In fact, the rapidity of the adsorption is what makes the surface charge so important when evaluating toxicity. As shown in figure 1, the adsorption of proteins happens in less than a few minutes. This aggregation is biologically active, and it is called a protein corona. It is what the cells encounter.
Figure 1: Steps with time frame of what foreign materials encounter when introduced in the body from Dr Yahia’s course slides
The most effective way of deciding which proteins are most likely to be adsorbed to obtain a desired effect, is by modulating the surface chemistry of the foreign material. According to the functionalization, which entails covering of the nanomaterial, the type of proteins adsorbed will differ.
In this study SPIONs were covered with either COOH groups or NH2 groups. With a COOH group functionalization, it was noticed that fibrinogen was most adsorbed, while in the case of NH2 group functionalization, it was mostly albumin.
We found that the toxicity of a COOH group functionalization is reduced since fibrinogen is an opsonin. More specifically, it can promote particle recognition by scavenger cells that can further the excretion process. Albumin has the opposite effect; the NH2 group functionalization increased toxicity.
Therefore, to validate your conclusion, by studying the surface chemistry of the SPIONs specifically, we get an idea of which functionalization is better according to the proteins adsorbed at its surface. This gives us an idea about what is needed to reduce toxicity.
Sabrina: How did the idea of using nanoparticles to carry the NO come about?
Dr Yahia: The project was initiated due to a demand form an anesthesiologist from the CHUM. She mentioned that, during general anesthesia, there were secondary effects such as memory loss and administering NO could prevent this. She had done tests on rats and observed the benefits. However, the FDA doesn’t authorize inhalation of NO as a gas for humans since it is unstable and dangerous when in contact with oxygen.
We came up with the idea of using nanoparticles to deliver the NO. By attaching NO to the nanoparticles, it can be released once in the alveola in the lungs. This can be done by changing the temperature or the pH, avoiding the toxicity of the NO previously described.
The problem with this method became obvious when researching the approach. When inhaled, foreign particles are not tolerated by lungs, they are rejected by physiological mechanisms such as coughing. Therefore, there is a physiological barrier to overcome. This can be done by using magnetic nanoparticles. By applying a magnetic field, we can force the nanoparticles to cross that barrier. In particular, superparamagnetic nanoparticles are used since they are the most biocompatible.
Outcomes of the study
Sabrina: With the resulting types of protein adsorbed and their associated increase or decrease in toxicity, do you recommend a specific functionalization? Would you generally suggest using a functionalization that attracts proteins such as fibrinogen on the inhaled particles?
Dr Yahia: Unfortunately, it isn’t that easy. Attracting specific proteins is a challenge because there is a great amount of proteins to consider in a physiological setting, from 1000 to 3000. Additionally, there is the Vroman effect, a complex process where proteins are adsorbed by affinity to the surface, however this is done dynamically. Initially, certain proteins can be found at the surface, only to be competitively removed and replaced by other proteins as shown in figure 2. Determining which proteins win at what moment and remain at the surface is challenging.
Figure 2: Schematic representation of the Vroman effet
When evaluating this phenomenon in vivo, we only get a snapshot of the protein corona at one specific instant. There is no way of knowing if the protein corona will be composed of different proteins at a different time and how the immune systems adapts to this variable surface.
In this study, we consider a closed system, where the environment is controlled and predictable. The human body is an open system, which makes predicting the outcome difficult. Many studies follow this reductionist approach, where most variables are simplified or ignored to understand the basics. However, we are left with a model that isn’t representative of reality. This reductionist approach was also used for evaluating the human genome. Even though we have access to the entire human genome today, we can’t predict human behavior and outcomes due to the overbearing gene interactions.
Sabrina: In the discussion you mention administering a concentration of nanoparticles ten times higher than what is normally used in literature. What was the motivation behind this?
Dr Yahia: We wanted to deliver the maximum amount of NO until an undesired effect could be observed. This was a way of evaluating the dosage and the effects of administering high doses.
Sabrina: While evaluating the toxicity of the SPIONs, results are often validated by comparing with what exists in the literature. Were any of the results unexpected?
Dr Yahia: Unlike other groups researching SPION toxicity, we were able to make correlations between the physiochemical properties of the nanomaterial and the physiological response. This was possible because here at Polytechnique, we have access to equipment that allows us to characterise the nanomaterial and the adsorption of the proteins. Most of what is found in the literature is merely observation on the behaviour of nanoparticles regarding their toxic effects. They don’t have the necessary equipment to infer a correlation between the surface properties and the toxicity.
Most importantly, we showed that the characterization of nanoparticles needs to be integrated in norms of their fabrication. Their resulting properties after synthesis are not reproducible. There exist about 3-4 methods to synthesize SPIONs, and even when the same method is used in the same lab, the resulting properties differ. This presents a huge challenge especially in the medical field since application with variability is to be avoided for safety issues.
This project contributed the idea of the necessity of controlling the synthesis and how to avoid contamination from the environment during the procedure. There needs to be a great amount of normalization of manipulations while synthesizing nanoparticles. This can be done by characterization the nanoparticles after procedures.
Future directions
Sabrina: Has there been any progress in SPION use for delivery of NO to patients during surgery since the article was published?
Dr Yahia: As mentioned, there is too much inconsistency in the synthesis of SPIONs. Even the culture medium used across studies is not reproducible from one lab to another. As well, the existing norms for biocompatibility such as cytotoxicity need to be revaluated since they do not apply to nanomaterials. Indeed, they do not account for the protein corona formation and the cellular response to protein adsorption. The uncertainty about the safety of nanomaterials poses a challenge for further investigation of its use in medicine for obvious reasons.
There are many other applications for NO, particularly its ability to destroy bacteria. Following lack of funding, we explored the possibility of NO for antibacterial use, seeing as bacterial resistance is of huge concern in medicine today.
We also explored the use of SPIONs within an ex vivo system. They could potentially trap oxidised LDL, a known cause of atherosclerosis plaque formation. By magnetic retention through the lymphatic system, the patient could lower the count of oxidised LDL in their blood and avoid cardiovascular disease.
Reference:
Mbeh, D. and Yahia, L. (2015). Human Alveolar Epithelial Cell Responses to Core-Shell Superparamagnetic Iron Oxide Nanoparticles (SPIONs). Langmuir, 31, 3829-3839. doi: 10.1021/la5040646