Proteomics reveals time-dependent protein corona changes in the intracellular pathway.

The intracellular protein corona has not been fully investigated in the field of nanotechnology-biology (nano-bio) interactions. To effectively understand intracellular protein corona formation and dynamics, we established a workflow to isolate the intracellular protein corona at different uptake times of two nanoparticles - magnetic hydroxyethyl starch nanoparticles (HES-NPs) and magnetic human serum albumin nanocapsules (HSA-NCs). We performed label-free quantitative LC-MS proteomics to analyze the composition of the intracellular protein corona and correlated our findings with results from conventional methods for intracellular trafficking of nanocarriers, such as flow cytometry, transmission electron microscopy (TEM), and confocal microscopy (cLSM). We determined the evolution of the intracellular protein corona. At different time stages the protein corona of the HES-NPs with a slower uptake changed, but there were fewer changes in that of the HSA-NCs with a more rapid uptake. We identified proteins that are involved in macropinocytosis (RAC1, ASAP2) as well as caveolin. This was confirmed by blocking experiments and by TEM studies. The investigated nanocarrier predominantly trafficked from early endosomes as determined by RAB5 identification in proteomics and in cLSM to late endosomes/lysosomes (RAB7, LAMP1, cathepsin K and HSP 90-beta) We further demonstrated differences between nanoparticles with slower and faster uptake kinetics and determined the associated proteome at different time points. Analysis of the intracellular protein corona provides us with effective data to examine the intracellular trafficking of nanocarriers used in efficient drug delivery and intracellular applications. STATEMENT OF SIGNIFICANCE: Many research papers focus on the protein corona on nanoparticles formed in biological fluids, but there are hardly any articles dealing with proteins that come in contact with nanoparticles inside cells. The "intracellular protein corona" studied here is a far more complex and highly demanding field. Most nanocarriers are designed to be taken up into cells. Given this, we chose two different nanocarriers to reveal changes in the proteins in dendritic cells during contact at specific times. Further studies will allow us to examine molecular target proteins using these methods. Our research is a significant addition towards the goal of understanding and thus improving the efficacy of drug nanocarriers.

Systematic modulation of the lipid composition enables the tuning of liposome cellular uptake.

As liposomes have been widely explored as drug delivery carriers over the past decades, they are one of the most promising platforms due to their biocompatibility and versatility for surface functionalization. However, to improve the specific design of liposomes for future biomedical applications such as nanovaccines, it is necessary to understand how these systems interact with cell membranes, as most of their potential applications require them to be internalized by cells. Even though several investigations on the cellular uptake of liposomes were conducted, the effect of the liposome membrane properties on internalization in different cell lines remains unclear. Here, we demonstrate how the cellular uptake behavior of liposomes can be driven towards preferential interaction with dendritic cells (DC2.4) as compared to macrophages (RAW264.7) by tuning the lipid composition with varied molar ratios of the lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Cellular internalization efficiency was analyzed by flow cytometry, as well as liposome-cell membrane co-localization by confocal laser scanning microscopy. The corresponding proteomic analysis of the protein corona was performed in order to unravel the possible effect on the internalization. The obtained results of this work reveal that it is possible to modulate the cellular uptake towards enhanced internalization by dendritic cells just by modifying the applied lipids and, thus, mainly the physico-chemical properties of the liposomes. STATEMENT OF SIGNIFICANCE: In the field of nanomedicine, it is of key importance to develop new specific and efficient drug carriers. In this sense, liposomes are one of the most widely known carrier types and used in clinics with good results. However, the exact interaction mechanisms of liposomes with cells remain unclear, which is of great importance for the design of new drug delivery platforms. Therefore, in this work we demonstrate that cellular uptake depends on the lipid composition. We are able to enhance the uptake in a specific cell type just by tuning the content of a lipid in the liposome membrane. This finding could be a step towards the selective design of liposomes to be internalized by specific cells with promising applications in biomedicine.

Endosomal sorting results in a selective separation of the protein corona from nanoparticles.

The formation of the protein corona is a well-known effect when nanoparticles (NP) are exposed to biological environments. The protein corona is the most important factor, which determines the rate and route of endocytosis, and decisively impacts cellular processes and even the release of the active pharmaceutical ingredient from the nanoparticles. While many studies concentrate on the effect of the protein corona formation extracellularly or the uptake consequences, little is known about the fate of the protein corona inside of cells. Here, we reconstruct for the first time the separation of the protein corona from the NPs by the cell and their further fate. Ultimately, the NPs and protein corona are separated from each other and end up in morphologically different cellular compartments. The cell directs the NPs towards recycling endosomes, whereas the protein corona gathers in multivesicular bodies. From this, we conclude that the NPs are prepared for subsequent exocytosis, while the protein corona remains in the cell and is finally metabolized there.

Proteomics reveals differential adsorption of angiogenic platelet lysate proteins on calcium phosphate bone substitute materials.

Protein adsorption on biomaterials for bone substitution, such as calcium phosphates (CaP), evokes biological responses and shapes the interactions of biomaterials with the surrounding biological environment. Proteins adsorb when CaP materials are combined with growth factor-rich hemoderivatives prior to implantation to achieve enhanced angiogenesis and stimulate new bone formation. However, the identification of the adsorbed proteins and their angiogenic effect on bone homeostasis remain incompletely investigated. In this study, we analyzed the adsorbed complex protein composition on CaP surfaces when using the hemoderivatives plasma, platelet lysate in plasma (PL), and washed platelet lysate proteins (wPL). We detected highly abundant, non-regenerative proteins and anti-angiogenic proteins adsorbed on CaP surfaces after incubation with PL and wPL by liquid chromatography and mass spectrometry (LC-MS) proteomics. Additionally, we measured a decreased amount of adsorbed pro-angiogenic growth factors. Tube formation assays with human umbilical endothelial cells demonstrated that the CaP surfaces only stimulate an angiogenic response when kept in the hemoderivative medium but not after washing with PBS. Our results highlight the necessity to correlate biomaterial surfaces with complex adsorbed protein compositions to tailor the biomaterial surface toward an enrichment of pro-angiogenic factors.

Higher Loading of Gold Nanoparticles in PAD Mesenchymal-like Stromal Cells Leads to a Decreased Exocytosis.

Cell therapy is an important new method in medicine and is being used for the treatment of an increasing number of diseases. The challenge here is the precise tracking of cells in the body and their visualization. One method to visualize cells more easily with current methods is their labeling with nanoparticles before injection. However, for a safe and sufficient cell labeling, the nanoparticles need to remain in the cell and not be exocytosed. Here, we test a glucose-PEG-coated gold nanoparticle for the use of such a cell labeling. To this end, we investigated the nanoparticle exocytosis behavior from PLX-PAD cells, a cell type currently in clinical trials as a potential therapeutic agent. We showed that the amount of exocytosed gold from the cells was influenced by the uptake time and loading amount. This observation will facilitate the safe labeling of cells with nanoparticles in the future and contribute to stem cell therapy research.

PEG Spacer Length Substantially Affects Antibody-Based Nanocarrier Targeting of Dendritic Cell Subsets.

Successful cell targeting depends on the controlled positioning of cell-type-specific antibodies on the nanocarrier's (NC) surface. Uncontrolled antibody immobilization results in unintended cell uptake due to Fc-mediated cell interaction. Consequently, precise immobilization of the Fc region towards the nanocarrier surface is needed with the Fab regions staying freely accessible for antigen binding. Moreover, the antibody needs to be a certain distance from the nanocarrier surface, influencing the targeting performance after formation of the biomolecular corona. This can be achieved by using PEG linker molecules. Here we demonstrate cell type-specific targeting for dendritic cells (DC) as cellular key regulators of immune responses. However, to date, dendritic cell targeting experiments using different linker lengths still need to be conducted. Consequently, we focused on the surface modification of nanocarriers with different molecular weight PEG linkers (0.65, 2, and 5 kDa), and their ability to reduce undesired cell uptake, while achieving efficient DC targeting via covalently immobilized antibodies (stealth targeting). Our findings demonstrate that the PEG linker length significantly affects active dendritic cell targeting from cell lines (DC2.4) to primary cells (BMDCs, splenocytic conventional DCs type 1 (cDC1)). While antibody-functionalized nanocarriers with a shorter PEG length (0.65 kDa) showed the best targeting in DC2.4, a longer PEG length (5 kDa) was required to specifically accumulate in BMDCs and splenocytic cDC1. Our study highlights that these crucial aspects must be considered when targeting dendritic cell subsets, which are of great importance in the fields of cancer immunotherapy and vaccine development.

Polysaccharide-Based pH-Responsive Nanocapsules Prepared with Bio-Orthogonal Chemistry and Their Use as Responsive Delivery Systems.

Bio-orthogonal reactions have become an essential tool to prepare biomaterials; for example, in the synthesis of nanocarriers, bio-orthogonal chemistry allows circumventing common obstacles related to the encapsulation of delicate payloads or the occurrence of uncontrolled side reactions, which significantly limit the range of potential payloads to encapsulate. Here, we report a new approach to prepare pH-responsive nanocarriers using dynamic bio-orthogonal chemistry. The reaction between a poly(hydrazide) crosslinker and functionalized polysaccharides was used to form a pH-responsive hydrazone network. The network formation occurred at the interface of aqueous nanodroplets in miniemulsion and led to the production of nanocapsules that were able to encapsulate payloads of different molecular weights. The resulting nanocapsules displayed low cytotoxicity and were able to release the encapsulated payload, in a controlled manner, under mildly acidic conditions.