Proteomic studies on intracellular nanocarrier trafficking and regenerative bone substitute materials
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Abstract
The design and medical performance of nano- and biomaterials remain impacted by the adsorption of biomolecules, specifically the adsorption of proteins. Protein adsorption is inevitably initiated upon contact with biological fluids or tissues, leading to a change in the surface properties of the materials. These changed surface properties evoke undesired biological effects, posing a major drawback for indented medical purposes. To overcome this drawback, or even exploit protein adsorption, it is necessary to analyze the composition of adsorbed proteins and study the interactions with cells, tissues, and organisms. This thesis addresses the challenges of protein adsorption on nanoparticles (NPs) in intracellular environments and on calcium phosphate (CaP) biomaterial surfaces for bone substitution.
The layers of adsorbed proteins on NPs are termed protein corona. The formation of the protein corona is influenced by the protein milieu and the physicochemical properties of the NPs. The protein corona has been studied with various biological fluids and NP systems, but the comparability of these studies remains challenging. To address the lacking comparability, the study presented in chapter A of this thesis analyzed the protein corona formation under different plasma protein concentrations, temperatures, and NP surface modifications. Here, we observed different outcomes in protein adsorption and cell uptake when varying surface charge and surfactant on polystyrene NPs at consistent concentration and temperature. Notably, decreasing protein concentration and temperature during the protein corona formation resulted in increased cellular uptake for all studied NP types. The results highlight the necessity to thoughtfully select experimental conditions for protein corona studies.
Despite numerous studies of the protein corona in extracellular environments, the cellular processing of NPs and the formation of the intracellular protein corona continue to be poorly studied. Considering that the protein corona influences the cellular uptake and intracellular NP cargo release, it is crucial to characterize the intracellular protein corona. The first study of chapter B investigated the intracellular separation and fate of a preabsorbed protein corona on polystyrene NPs. By utilizing correlative light and electron microscopy and flow cytometry, the endosomal separation of corona proteins and NPs into morphologically distinct endosomal compartments was demonstrated. Eventually, the NPs were exocytosed, and the protein corona was processed for lysosomal degradation. The second study of chapter B revealed the intracellular trafficking of two biocompatible NP types by implementing proteomic analysis. We demonstrated a gradual evolution of the protein corona for hydroxyethyl starch NPs with a slower uptake while demonstrating a stable protein corona for human serum albumin nanocapsules with an accelerated uptake. Additionally, by unraveling the intracellular protein corona, we reconstructed molecular details during the intracellular trafficking andomplemented the results by flow cytometry and microscopy. For the third study of chapter B, the analysis of the intracellular protein corona was applied to investigate the endocytosis of gold NPs for imaging applications in stromal cells. The exocytosis of gold NPs was dependent on the performed loading protocol. Especially higher loading with gold NPs resulted in lower exocytosis when compared to a lower loading. Here, the analysis of the intracellular protein corona revealed that a higher loading led to an enrichment of intracellular proteins, decreasing exocytosis. Overall, the studies in this chapter demonstrated that detailed characterizations of the intracellular protein corona will improve NP application in drug delivery and imaging.
Chapter C focuses on protein adsorption on bulk biomaterial with nanostructured surfaces. To provide regenerative properties to synthetic biomaterials for bone substitution, the biomaterials are combined with growth factor-rich hemoderivatives. However, the composition of the adsorbed proteins is rarely investigated. The study in chapter C investigated the protein adsorption from hemoderivative protein sources on CaP bone substitutes regarding their regenerative potential for angiogenesis. Using proteomic studies, we identified abundantly adsorbed non-angiogenic and anti-angiogenic proteins. Furthermore, we measured the depletion of pro-angiogenic growth factors. The pro-angiogenic effects were analyzed by tube- formation assays with endothelial cells. Here, we observed pro-angiogenic effects when the CaPs were kept in the hemoderivative protein source but not after washing with PBS. These results emphasize the importance of analyzing protein adsorption to improve the regenerative, e. g. pro-angiogenic capabilities of biomaterials.
In conclusion, the presented studies emphasize the benefits of protein adsorption analysis for the development of nano- and biomaterials with medical applications. The future design of these materials will tremendously profit from studying surface-adsorbed proteins and exploiting this knowledge to modulate desired biological effects.