Modulation of Immune Cell Functions by Nanoparticles

Nanoparticles of various sizes and materials carrying different surface modifications have many industrial and medical applications. Macrophages act as a first line of defense of the body internalizing particulate matter including nanoparticles and retaining them eventually for extended periods of time. Surprisingly, however, the effects of long-term interaction of nanoparticles with cells of the immune system remain poorly investigated. Our goal is i) to explore in greater detail how distinct surface configurations of nanoparticles interact with cellular membranes, ii) which cellular consequences might be induced by endocytosed nanosized materials, and iii) how nanotechnology could be exploited for therapeutic purposes. Subsequently, some examples are given.

Effects of Superparamagnetic Carboxydextran-Coated Iron Oxide Nanoparticles on the Fate of Macrophages

Carboxydextran-coated superparamagnetic iron oxide nanoparticles (SPIO) are frequently used as contrast agents for magnetic resonance imaging (MRI). These particles are rapidly taken up by macrophages and have previously been considered as pharmacologically safe. However, we discovered that they induce delayed apoptosis in macrophages through the induction of ROS and subsequent JNK signaling. Indeed, we could demonstrate that lysosomal α-hydrolase time-dependently degrades the carboxydextran coating of these nanoparticles in a very effective manner. This process warrants liberation of molecular iron that subsequently catalyzes ROS generation in a Fenton type reaction. Accordingly, the cytotoxicity of those nanoparticles depends on the carboxydextran shell thickness. In line with these molecular mechanisms, we devised a pharmacological strategy employing ROS scavengers that indeed prevented the cytotoxicity of the nanoparticles under in vitroconditions.

Our data also demonstrated for the first time that carboxydextran-coated nanoparticles of 20-60 nm are internalized by macrophages via a scavenger receptor A-dependent, clathrin-mediated endocytotic process. Hence, the uptake of carboxydextran-coated nanoparticles occurs in a specific, receptor-mediated manner and depends on the coordination of multiple nanoparticle-receptor interactions, the polymerization of clathrin units, and membrane deformation-dependent signaling events. We developed a mathematic model that allows prediction of the uptake characteristics of engineered nanoparticles by living cells and provides a tool for a rational approach to direct and control cellular particle uptake.

As macrophages are equipped with all necessary tools to sense, internalize and digest particulate matter, it is not surprising that hepatic macrophages, the Kupffer cells, take up a significant portion of intravenously injected nanosized particles. Accordingly, we showed that the aforementioned cytotoxicity takes not only place in human macrophages in vitro but also in mice in vivo. Thus, 5 days after intravenous injection of a diagnostically relevant dose of the carboxydextran-coated nanoparticles, tissue levels of iron remained significantly elevated in murine liver and kidneys as compared to control animals. The observed increases in hepatic iron load appeared to be not very high. Hepatic tissue retained the largest amount (about 90 %) of the administered iron nanoparticles after 5 days, the remaining 10 % of SPIO were deposited in renal tissue. We could demonstrate that the iron from injected SPIO was exclusively absorbed by the Kupffer cells, which constitute only ~2% of all liver cells. The iron-loaded Kupffer cells exhibited caspase 3 in its activated form, which reflects a point of no return in apoptotic cell death. Consistent with these findings, three days after injection, the number of Kupffer cells in the liver was depleted by more than 66%. Consistent with our in vitro studies, we could demonstrate that the SPIO-mediated Kupffer cell toxicityin vivo can be abolished by the therapeutic ROS scavenger edaravone that has been registered for the treatment of stroke. Thus, scavenging ROS in the vulnerable phase of transition of molecular iron into the ferritin stores provides a pharmacological strategy to circumvent the adverse effects of such MRI contrast agents on macrophages. Therefore, the cytotoxicity of SPIO-based contrast agents specifically for professional phagocytes should be considered, particularly in patients with liver diseases.These results imply that the cytotoxic effects of iron oxide nanoparticles require more intensive study, specifically on a long-term basis and that such effects should be considered in biomedical applications.

Differential Uptake of Functionalized Polystyrene Nanoparticles by Human Macrophages and a Monocytic Cell Line

Tumor cell lines are often almost routinely used as models for the study of nanoparticle-cell interactions. Here we demonstrate that carboxy (PS-COOH) and amino functionalized (PS-NH2) polystyrene nanoparticles of ~100 nm in diameter are internalized by human macrophages, by undifferentiated and by PMA-differentiated monocytic THP-1 cells via diverse mechanisms. The uptake mechanisms also differed for all cell types and particles when analyzed either in buffer or in medium containing human serum. Macrophages internalized ~4 times more PS-COOH than THP-1 cells, when analyzed in serum-containing medium. By contrast, in either medium, THP-1 cells internalized PS-NH2 more rapidly than macrophages. Using pharmacological and antisense in vitro knockdown approaches, we showed that, in the presence of serum, the specific interaction between the CD64 receptor and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization in THP-1 cells occurred via dynamin II-dependent endocytosis. PMA-differentiated THP-1 cells differed in their uptake mechanism from macrophages and undifferentiated THP-1 cells by internalizing the particles via macropinocytosis. In line with our in vitro data, more intravenously applied PS-COOH particles accumulated in the liver, where macrophages of the reticuloendothelial system reside. By contrast, PS-NH2 particles were preferentially targeted to tumor xenografts grown on the chorioallantoic membrane of fertilized chicken eggs. Our data show that the amount of internalized nanoparticles, the uptake kinetics and its mechanism may differ considerably between primary cells and a related tumor cell line, whether differentiated or not, and that particle uptake by these cells is critically dependent on particle opsonization by serum proteins.

Methods

  • Protein expression
  • Western blotting with immunostaining
  • Co-immunoprecipitation
  • Peptide cross-linking
  • Flow cytometric analysis of the cell surface proteins
  • ELISA
  • Laser scanning microscopy
  • Spinning disc confocal laser microscopy
  • Fluorescence microscopy
  • Surface plasmon resonance
  • Electrophoretic mobility shift assay (EMSA)
  • RT-PCR
  • In vitro knockdown by antisense oligodeoxynucleotides
  • Isolation of primary cells by density gradient centrifugation
  • Macrophage differentiation
  • Chemical synthesis of polymeric and iron oxide nanoparticles
  • Chemical surface modifications of nanoparticles

 

Publications (selected)

Lunov O, Syrovets T, Loos C, Beil J, Delacher M, Tron K, Nienhaus GU, Musyanovych A, Mailänder V, Landfester K, Simmet Th. Differential uptake of functionalized polystyrene nanoparticles by human macrophages and a monocytic cell line. ACS Nano. 2011; 5:1657-69

Lunov O, Zablotskii V, Syrovets T, Röcker C, Tron K, Nienhaus GU, Simmet Th. Modeling receptor-mediated endocytosis of polymer-functionalized iron oxide nanoparticles by human macrophages. Biomaterials. 2011; 32:547-55

Lunov O, Syrovets T, Büchele B, Röcker C, Tron K, Nienhaus GU, Rasche V, Mailänder V, Landfester K, Simmet Th. Lysosomal degradation of the carboxydextran shell of coated superparamagnetic iron oxide nanoparticles determines the fate of professional phagocytes Biomaterials. 2010; 31:9015-22

Lunov O, Syrovets T, Büchele B, Jiang X, Röcker C, Tron K, Nienhaus GU, Walther P, Mailänder V, Landfester K, Simmet Th. The effect of carboxydextran-coated iron oxide nanoparticles on c-Jun N-terminal kinase-mediated apoptosis in human macrophages. Biomaterials. 2010; 31:5063-71