Christine K. Payne

Nanoparticle–Cell Interactions: Molecular Structure of the Protein Corona and Cellular Outcomes

Conspectus The use of nanoparticles (NPs) in biology and medicine requires a molecular-level understanding of how NPs interact with cells in a physiological environment. A critical difference between well-controlled in vitro experiments and in vivo applications is the presence of a complex mixture of extracellular proteins. It has been established that extracellular serum proteins present in blood will adsorb onto the surface of NPs, forming a “protein corona”. Our goal was to understand how this protein layer affected cellular-level events, including NP binding, internalization, and transport. A combination of microscopy, which provides spatial resolution, and spectroscopy, which provides molecular information, is necessary to probe protein–NP–cell interactions. Initial experiments used a model system composed of polystyrene NPs functionalized with either amine or carboxylate groups to provide a cationic or anionic surface, respectively. Serum proteins adsorb onto the surface of both cationic and anionic NPs, forming a net anionic protein–NP complex. Although these protein–NP complexes have similar diameters and effective surface charges, they show the exact opposite behavior in terms of cellular binding. In the presence of bovine serum albumin (BSA), the cellular binding of BSA–NP complexes formed from cationic NPs is enhanced, whereas the cellular binding of BSA–NP complexes formed from anionic NPs is inhibited. These trends are independent of NP diameter or cell type. Similar results were obtained for anionic quantum dots and colloidal gold nanospheres. Using competition assays, we determined that BSA–NP complexes formed from anionic NPs bind to albumin receptors on the cell surface. BSA–NP complexes formed from cationic NPs are redirected to scavenger receptors. The observation that similar NPs with identical protein corona compositions bind to different cellular receptors suggested that a difference in the structure of the adsorbed protein may be responsible for the differences in cellular binding of the protein–NP complexes. Circular dichroism spectroscopy, isothermal titration calorimetry, and fluorescence spectroscopy show that the structure of BSA is altered following incubation with cationic NPs, but not anionic NPs. Single-particle-tracking fluorescence microscopy was used to follow the cellular internalization and transport of protein–NP complexes. The single particle-tracking experiments show that the protein corona remains bound to the NP throughout endocytic uptake and transport. The interaction of protein–NP complexes with cells is a challenging question, as the adsorbed protein corona controls the interaction of the NP with the cell; however, the NP itself alters the structure of the adsorbed protein. A combination of microscopy and spectroscopy is necessary to understand this complex interaction, enabling the rational design of NPs for biological and medical applications.

Internalization and Trafficking of Cell Surface Proteoglycans and Proteoglycan‐Binding Ligands

Using multicolor live cell imaging in combination with biochemical assays, we have investigated an endocytic pathway mediated by cell surface proteoglycans, primary receptors for many cationic ligands. We have characterized this pathway for a variety of proteoglycan‐binding ligands including cationic polymers, lipids and polypeptides. Following clathrin‐ and caveolin‐independent, but flotillin‐ and dynamin‐dependent internalization, proteoglycan‐bound ligands associate with flotillin‐1‐positive vesicles and are efficiently trafficked to late endosomes. The route to late endosomes differs considerably from that following clathrin‐mediated endocytosis. The proteoglycan‐dependent pathway to late endosomes does not require microtubule‐dependent transport or phosphatidyl‐inositol‐3‐OH kinase‐dependent sorting from early endosomes. The pathway taken by these ligands is identical to that taken by an antibody against heparan sulfate proteoglycans, suggesting that this mechanism may be used generally by cell surface proteoglycans and proteoglycan‐binding ligands that lack secondary receptors.

Nanoparticle surface charge mediates the cellular receptors used by protein-nanoparticle complexes.

Nanoparticles are increasingly important for biological applications ranging from drug delivery to cellular imaging. In the course of these applications, nanoparticles are exposed to a complex environment of extracellular proteins that can be adsorbed onto the surface of the nanoparticle, altering nanoparticle-cell interactions. We have investigated how proteins found in blood serum affect the binding of nanoparticles to the surface of cells. Using fluorescence microscopy, we find that the cellular binding of cationic nanoparticles is enhanced by the presence of serum proteins, while the binding of anionic nanoparticles is inhibited. We have determined that this difference in cellular binding is due to the use of distinct cellular receptors. Competition assays, quantified with flow cytometry, show that the protein-nanoparticle complex formed from the cationic nanoparticles binds to scavenger receptors on the cell surface. Interestingly, the protein-nanoparticle complex formed from anionic nanoparticles binds to native protein receptors. As nanoparticles become increasingly important for in vivo applications, we expect these results will inform the design of nanoparticles with improved cellular binding.

Secondary structure of corona proteins determines the cell surface receptors used by nanoparticles.

Nanoparticles used for biological and biomedical applications encounter a host of extracellular proteins. These proteins rapidly adsorb onto the nanoparticle surface, creating a protein corona. Poly(ethylene glycol) can reduce, but not eliminate, the nonspecific adsorption of proteins. As a result, the adsorbed proteins, rather than the nanoparticle itself, determine the cellular receptors used for binding, the internalization mechanism, the intracellular transport pathway, and the subsequent immune response. Using fluorescence microscopy and flow cytometry, we first characterize a set of polystyrene nanoparticles in which the same adsorbed protein, bovine serum albumin, leads to binding to two different cell surface receptors: native albumin receptors and scavenger receptors. Using a combination of circular dichroism spectroscopy, isothermal titration calorimetry, and fluorescence spectroscopy, we demonstrate that the secondary structure of the adsorbed bovine serum albumin protein controls the cellular receptors used by the protein–nanoparticle complexes. These results show that protein secondary structure is a key parameter in determining the cell surface receptor used by a protein–nanoparticle complex. We expect this link between protein structure and cellular outcomes will provide a molecular basis for the design of nanoparticles for use in biological and biomedical applications.

Endo-lysosomal vesicles positive for Rab7 and LAMP1 are terminal vesicles for the transport of dextran.

The endo-lysosomal pathway is essential for intracellular transport and the degradation of extracellular cargo. The relationship between three populations of endo-lysosomal vesicles—Rab7-positive, LAMP1-positive, and both Rab7- and LAMP1-postive—was probed with fluorescence microscopy and single particle tracking. Of specific interest was determining if these vesicles were intermediate or terminal vesicles in the transport of extracellular cargo. We find that the major organelle in the endo-lysosomal pathway, both in terms of population and cargo transport, is positive for Rab7 and LAMP1. Dextran, a fluid phase cargo, shifts from localization within all three populations of vesicles at 30 minutes and 1 hour to primarily LAMP1- and Rab7/LAMP1-vesicles at longer times. This demonstrates that LAMP1- and Rab7/LAMP1-vesicles are terminal vesicles in the endo-lysosomal pathway. We tested two possible mechanisms for this distribution of cargo, delivery to mannose 6-phosphate receptor (M6PR)-negative vesicles and the fusion dynamics of individual vesicles. We find no correlation with M6PR but do find that Rab7-vesicles undergo significantly fewer fusion events than LAMP1- or Rab7/LAMP1-vesicles suggesting that the distribution of fluid phase cargo is driven by vesicle dynamics.

Fluorescent coumarin thiols measure biological redox couples.

In this report we present a new chemical probe, 3-HTC, that can reversibly and ratiometrically measure the thiol-disulfide equilibrium of biological systems. 3-HTC is composed of a coumarin that has a thiolate directly conjugated to its extended aromatic π system while formation of a disulfide attenuates this conjugation. The fluorescence and absorption properties of 3-HTC are therefore very sensitive to the redox state of its thiol. 3-HTC reacts reversibly with thiols and disulfides enabling its use to measure dynamic GSH/GSSH ratios in vitro as well as to monitor the reversible redox status of whole cell lysates.

Lysosome transport as a function of lysosome diameter.

Lysosomes are membrane-bound organelles responsible for the transport and degradation of intracellular and extracellular cargo. The intracellular motion of lysosomes is both diffusive and active, mediated by motor proteins moving lysosomes along microtubules. We sought to determine how lysosome diameter influences lysosome transport. We used osmotic swelling to double the diameter of lysosomes, creating a population of enlarged lysosomes. This allowed us to directly examine the intracellular transport of the same organelle as a function of diameter. Lysosome transport was measured using live cell fluorescence microscopy and single particle tracking. We find, as expected, the diffusive component of intracellular transport is decreased proportional to the increased lysosome diameter. Active transport of the enlarged lysosomes is not affected by the increased lysosome diameter.

Membrane potential mediates the cellular binding of nanoparticles

The use of nanoparticles for cellular therapeutic or sensing applications requires nanoparticles to bind, or adhere, to the cell surface. While nanoparticle parameters such as size, shape, charge, and composition are important factors in cellular binding, the cell itself must also be considered. All cells have an electrical potential across the plasma membrane driven by an ion gradient. Under standard conditions the ion gradient will result in a -10 to -100 mV potential across the membrane with a net negative charge on the cytosolic face. Using a combination of flow cytometry and fluorescence microscopy experiments and dissipative particle dynamics simulations, we have found that a decrease in membrane potential leads to decreased cellular binding of anionic nanoparticles. The decreased cellular binding of anionic nanoparticles is a general phenomenon, independent of depolarization method, nanoparticle composition, and cell type. Increased membrane potential reverses this trend resulting in increased binding of anionic nanoparticles. The cellular binding of cationic nanoparticles is minimally affected by membrane potential due to the interaction of cationic nanoparticles with cell surface proteins. The influence of membrane potential on the cellular binding of nanoparticles is especially important when considering the use of nanoparticles in the treatment or detection of diseases, such as cancer, in which the membrane potential is decreased.

Imaging gene delivery with fluorescence microscopy

Gene delivery offers the promise of treatment for a range of human diseases. Although carried out initially with modified viruses, the use of synthetic molecules, including polymers, lipids and peptides, has extended the possibilities greatly for rationally designed vectors tailored to individual gene-delivery applications. Underlying the rational design of gene-delivery vectors is the need to understand the individual steps of the gene-delivery pathway. Using new methods in fluorescence microscopy, it is now possible to isolate individual steps along the gene-delivery pathway to characterize the mechanisms of cellular binding, cellular internalization and nuclear entry. This review describes the advances made in the gene-delivery field with the assistance of fluorescence microscopy. The focus of this review is the use of synthetic gene-delivery vectors, especially polyethylenimine, and the live-cell imaging and single-particle tracking techniques that reveal the intracellular dynamics of the gene-delivery process.

Femtosecond infrared studies of ligand rearrangement reactions:silyl hydride products from Group 6 carbonyls

The ultrafast dynamics of the SiH bond activation reaction by the Group 6 d6 organometallic compounds M(CO)5 (M=Cr, Mo, and W) have been studied in neat tri-substituted silanes under ambient conditions. The ultrafast spectral evolutions of the CO stretching bands were monitored following UV photolysis using femtosecond pump–probe spectroscopic methods. It was found that the coordinatively unsaturated species, which is formed following CO photolysis from the parent molecule, is quickly solvated (<2 ps) via the CH bonds of the solvent. These species then rearranged to the silyl hydride product on a timescale of a few nanoseconds. These results were augmented by rearrangement studies in neat ethanol, propanol and hexanol solutions in which the initially formed metal CH complex rearranged to the metal hydroxyl complex. The mechanism of this rearrangement was discussed by comparison of the data with various models in the literature. It was found that a mechanism that is primarily dissociative in nature provided the best description of the experimental data.