ACS Nano. 2017 Mar 28;11(3):3262-3273.

Unveiling the in Vivo Protein Corona of Circulating Leukocyte-like Carriers.

Claudia Corbo, Roberto Molinaro, Francesca Taraballi, Naama E Toledano Furman, Kelly A Hartman, Michael B Sherman, Enrica De Rosa, Dickson K Kirui, Francesco Salvatore, Ennio Tasciotti

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Last year we published a groundbreaking study on the development of leukocyte-like liposomal carriers. Such vesicles, referred to as leukosomes, mimic immune cells through the integration of leukocyte membrane proteins onto their surface. This unique biomimetic composition allowed delayed clearance by filtering organs (liver, spleen), and targeting of the inflamed endothelia.1

Unraveling the interactions between nanoparticles and biological systems is imperative to further map the mechanisms that govern nanoparticles’ function in the body. Upon systemic injection, protein components from the blood stream bind to nanoparticles’ surface and form an exterior coating called “protein corona” which is responsible for the modification of the nanoparticles’ expected properties.2 Previous studies have suggested that protein corona composition is affected not only by the structure and composition of the nanomaterial, but also by the makeup of the microenvironment in which the nanoparticle is in, and by the patient’s specific disease.3-5 As a result, the very same nanoparticle ends up forming protein coronas with different compositions according to their location in the body and to the type of disease affecting the patient, a concept described as personalized protein corona.6

The formation of a protein corona can decrease the ability of functionalized nanoparticles to selectively adhere to target cells and tissues. This happens through the masking effect of the receptor-ligand interactions that govern targeted delivery.7 Conversely, by controlling the adsorption of specific plasma proteins that can function as targeting moieties it is possible to take advantage of the protein corona to increase nanoparticles accumulation in target tissues. We hypothesized that by grafting leukocyte proteins in the lipid bilayer of our biomimetic leukocyte-like carriers (leukosomes) we could control the formation and composition of the protein corona.8 We were able to follow and characterize the formation of the protein corona in a living organism, and provided a unique insight into how biomimetic particles interact with the host’s immune system after injection.8 Figure 1 describes the experimental workflow of our study.

As the leukosome’s surface mimics that of an immune cell, the host’s defense system recognizes the particles as self and their clearance is much lower than that of non-biomimetic nanoparticles (i.e. regular liposomes). By analyzing the biodistribution of leukosomes using intravital microscopy, we showed decreased accumulation in the liver and spleen (the most efficient filtering organs) and enhanced circulation time. From a therapeutic standpoint, the ability of nanoparticles to flow through blood vessels undisturbed results in an increased probability of encountering the specific target site.

To determine if the ability of leukosomes to avoid immune clearance was due to their protein coronas, we investigated in vitro the uptake of bare and protein corona-coated nanoparticles by J774 macrophages. Our results revealed that the presence of protein corona induced an increased macrophage uptake of liposomes and a reduced uptake of leukosomes.

The protein corona of leukosomes, like liposomes and most nanoparticles, is characterized by the presence of IgGs, complement and coagulation factors, recognized by immune cells as “eat-me” signals.

These proteins are, in general, adsorbed on nanoparticles’ surface through electrostatic and hydrophobic interactions. However, the presence of receptors on the leukosome’s surface directs the adsorption of specific ligand proteins in the corona through receptor-to-ligand binding. This causes their recognition domains to be oriented inward – toward the leukosomes – and, therefore, hidden from macrophages’ recognition. On the other hand, proteins adsorbed on liposomes are randomly oriented, making liposomes more subject to macrophage recognition and consequent uptake. Our work revealed that the neutralizing effect that the leukosome’s surface receptors have on protein corona-mediated uptake by immune cells contributes to the superior efficacy of these nanoparticles.8

This work is the result of a multidisciplinary study combining nanotechnology, proteomics and biology, and provides a deeper insight into the properties of nanoparticles in circulation. Considering the impact of the protein corona on nanoparticles properties and biological fate, we envision that future works will need to consider not only the changes that the protein corona induces on the chemical physical properties of nanoparticles, but also the nanoparticle uptake from cells of interest. To have a complete understanding of the nanoparticles’ potential therapeutic efficacy, there is an urgent need to revise all these questions in the context of the protein corona formation.

 

 

Figure 1. Workflow of the study. Lipid vesicles were injected in mice, recovered and their protein coronas were analyzed by SDS-PAGE gels, LC-MS and bioinformatic analysis. Nanoparticles have been characterized before and after protein corona formation. The effects of the protein corona formation on nanoparticles biodistribution and cellular uptake have been also evaluated.

 

 

Figure 2. First and last author: Claudia Corbo and Ennio Tasciotti. This work has been performed at the Center for Biomimetic Medicine of the Houston Methodist Research Institute.

 

References

1. Molinaro, R., Corbo, C., Martinez, J., et al. Biomimetic Proteolipid Vesicles for Targeting Inflamed Tissues. Nature materials 2016, 15, 1037-1046.
2. Corbo, C., Molinaro, R., Parodi, A., Furman, N. E. T., Salvatore, F., Tasciotti, E. The Impact of Nanoparticle Protein Corona on Cytotoxicity, Immunotoxicity and Target Drug Delivery. Nanomedicine 2016, 11, 81-100.
3. Corbo, C., Molinaro, R., Taraballi, F., et al. Effects of the Protein Corona on Liposome–Liposome and Liposome–Cell Interactions. International Journal of Nanomedicine 2016, 11, 3049.
4. Hajipour, M. J., Raheb, J., Akhavan, O., et al. Personalized Disease-Specific Protein Corona Influences the Therapeutic Impact of Graphene Oxide. Nanoscale 2015, 7, 8978-8994.
5. Lundqvist, M., Stigler, J., Elia, G., Lynch, I., Cedervall, T., Dawson, K. A. Nanoparticle Size and Surface Properties Determine the Protein Corona with Possible Implications for Biological Impacts. Proceedings of the National Academy of Sciences 2008, 105, 14265-14270.
6. Corbo, C., Molinaro, R., Tabatabaei, M., Farokhzad, O. C., Mahmoudi, M. Personalized Protein Corona on Nanoparticles and Its Clinical Implications. Biomaterials Science 2017, 5, 378-387.
7. Salvati, A., Pitek, A. S., Monopoli, M. P., et al. Transferrin-Functionalized Nanoparticles Lose Their Targeting Capabilities When a Biomolecule Corona Adsorbs on the Surface. Nature nanotechnology 2013, 8, 137-143.
8. Corbo, C., Molinaro, R., Taraballi, F., et al. Unveiling the in Vivo Protein Corona of Circulating Leukocyte-Like Carriers. ACS nano 2017, 11, 3262-3273.