Zwitterionic surface coating of quantum dots reduces protein adsorption and cellular uptake.

We have studied the effect of the zwitterionic surface coating of quantum dots (QDs) on their interaction with a serum supplemented cell medium and their internalization by human cervical carcinoma (HeLa) cells. Zwitterionic QDs showed negligible adsorption of human serum albumin (HSA) selected as a model serum protein, in contrast to similar but negatively charged QDs. The incorporation of zwitterionic QDs by HeLa cells was found to be lower than for negatively charged QDs and for positively charged QDs, for which the uptake yield was largest. Our results suggest that the suppression of protein adsorption, here accomplished by zwitterionic QD surfaces, offers a strategy that allows for reducing the cellular uptake of nanoparticles.

The Nature of a Hard Protein Corona Forming on Quantum Dots Exposed to Human Blood Serum.

Biological responses of cells and organisms to nanoparticle exposure crucially depend on the properties of the protein adsorption layer ("protein corona") forming on nanoparticle surfaces and their characterization is a crucial step toward a deep, mechanistic understanding of their build-up. Previously, adsorption of one type of model protein on nanoparticles was systematically studied in situ by using fluorescence correlation spectroscopy. Here, the first such study of interactions is presented between water-solubilized CdSe/ZnS quantum dots (QDs) and a complex biofluid, human blood serum. Despite the large number of proteins in serum, a protein layer of well-defined (average) thickness forming on QD surfaces is observed. Both the thickness and the apparent binding affinity depend on the type of QD surface ligand. Kinetic experiments reveal that the protein corona formed from serum is irreversibly bound, whereas the one formed from human serum albumin was earlier observed to be reversible. By using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry, the most abundant serum proteins contributing to the formation of a hard corona on the QDs are identified.

Surface Functionalization of Nanoparticles with Polyethylene Glycol: Effects on Protein Adsorption and Cellular Uptake.

Here we have investigated the effect of enshrouding polymer-coated nanoparticles (NPs) with polyethylene glycol (PEG) on the adsorption of proteins and uptake by cultured cells. PEG was covalently linked to the polymer surface to the maximal grafting density achievable under our experimental conditions. Changes in the effective hydrodynamic radius of the NPs upon adsorption of human serum albumin (HSA) and fibrinogen (FIB) were measured in situ using fluorescence correlation spectroscopy. For NPs without a PEG shell, a thickness increase of around 3 nm, corresponding to HSA monolayer adsorption, was measured at high HSA concentration. Only 50% of this value was found for NPs with PEGylated surfaces. While the size increase clearly reveals formation of a protein corona also for PEGylated NPs, fluorescence lifetime measurements and quenching experiments suggest that the adsorbed HSA molecules are buried within the PEG shell. For FIB adsorption onto PEGylated NPs, even less change in NP diameter was observed. In vitro uptake of the NPs by 3T3 fibroblasts was reduced to around 10% upon PEGylation with PEG chains of 10 kDa. Thus, even though the PEG coatings did not completely prevent protein adsorption, the PEGylated NPs still displayed a pronounced reduction of cellular uptake with respect to bare NPs, which is to be expected if the adsorbed proteins are not exposed on the NP surface.

Low affinity binding of plasma proteins to lipid-coated quantum dots as observed by in situ fluorescence correlation spectroscopy.

Protein binding to lipid-coated nanoparticles has been pursued quantitatively by using fluorescence correlation spectroscopy. The binding of three important plasma proteins to lipid-enwrapped quantum dots (QDs) shows very low affinity, with an apparent dissociation coefficient in the range of several hundred micromolar. Thus, the tendency to adsorb is orders of magnitude weaker than for QDs coated with dihydrolipoic acid.

Impact of protein modification on the protein corona on nanoparticles and nanoparticle-cell interactions.

Recent studies have firmly established that cellular uptake of nanoparticles is strongly affected by the presence and the physicochemical properties of a protein adsorption layer around these nanoparticles. Here, we have modified human serum albumin (HSA), a serum protein often used in model studies of protein adsorption onto nanoparticles, to alter its surface charge distribution and investigated the consequences for protein corona formation around small (radius ∼5 nm), dihydrolipoic acid-coated quantum dots (DHLA-QDs) by using fluorescence correlation spectroscopy. HSA modified by succinic anhydride (HSAsuc) to generate additional carboxyl groups on the protein surface showed a 3-fold decreased binding affinity toward the nanoparticles. A 1000-fold enhanced affinity was observed for HSA modified by ethylenediamine (HSAam) to increase the number of amino functions on the protein surface. Remarkably, HSAsuc formed a much thicker protein adsorption layer (8.1 nm) than native HSA (3.3 nm), indicating that it binds in a distinctly different orientation on the nanoparticle, whereas the HSAam corona (4.6 nm) is only slightly thicker. Notably, protein binding to DHLA-QDs was found to be entirely reversible, independent of the modification. We have also measured the extent and kinetics of internalization of these nanoparticles without and with adsorbed native and modified HSA by HeLa cells. Pronounced variations were observed, indicating that even small physicochemical changes of the protein corona may affect biological responses.

Carbohydrate-lectin recognition of sequence-defined heteromultivalent glycooligomers.

Multivalency as a key principle in nature has been successfully adopted for the design and synthesis of artificial glycoligands by attaching multiple copies of monosaccharides to a synthetic scaffold. Besides their potential in various applied areas, e.g. as antiviral drugs, for the vaccine development and as novel biosensors, such glycomimetics also allow for a deeper understanding of the fundamental aspects of multivalent binding of both artificial and natural ligands. However, most glycomimetics so far neglect the purposeful arranged heterogeneity of their natural counterparts, thus limiting more detailed insights into the design and synthesis of novel glycomimetics. Therefore, this work presents the synthesis of monodisperse glycooligomers carrying different sugar ligands at well-defined positions along the backbone using for the first time sequential click chemistry and stepwise assembly of functional building blocks on solid support. This approach allows for straightforward access to sequence-defined, multivalent glycooligomers with full control over number, spacing, position, and type of sugar ligand. We demonstrate the synthesis of a set of heteromultivalent oligomers presenting mannose, galactose, and glucose residues. All heteromultivalent structures show surprisingly high affinities toward Concanavalin A lectin receptor in comparison to their homomultivalent analogues presenting the same number of binding ligands. Detailed studies of the ligand/receptor interaction using STD-NMR and 2fFCS indeed indicate a change in binding mechanism for trivalent glycooligomers presenting mannose or combinations of mannose and galactose residues. We find that galactose residues do not participate in the binding to the receptor, but they promote steric shielding of the heteromultivalent glycoligands and thus result in an overall increase in affinity. Furthermore, the introduction of nonbinding ligands seems to suppress receptor clustering of multivalent ligands. Overall these results support the importance of heteromultivalency specifically for the design of novel glycoligands and help to promote a fundamental understanding of multivalent binding modes.

Effects of surface functionalization on the adsorption of human serum albumin onto nanoparticles - a fluorescence correlation spectroscopy study.

By using fluorescence correlation spectroscopy (FCS), we have studied the adsorption of human serum albumin (HSA) onto Fe-Pt nanoparticles (NPs, 6 nm radius), CdSe/ZnS quantum dots (QDs, 5 nm radius) and Au and Ag nanoclusters (1-4 nm radius), which are enshrouded by various water-solubilizing surface layers exposing different chemical functional groups (carboxyl, amino and both), thereby endowing the NPs with different surface charges. We have also measured the effects of modified surface functionalizations on the protein via succinylation and amination. A step-wise increase in hydrodynamic radius with protein concentration was always observed, revealing formation of protein monolayers coating the NPs, independent of their surface charge. The differences in the thickness of the protein corona were rationalized in terms of the different orientations in which HSA adsorbs onto the NPs. The midpoints of the binding transition, which quantifies the affinity of HSA toward the NP, were observed to differ by almost four orders of magnitude. These variations can be understood in terms of specific Coulombic interactions between the proteins and the NP surfaces.

Temperature: the "ignored" factor at the NanoBio interface.

Upon incorporation of nanoparticles (NPs) into the body, they are exposed to biological fluids, and their interaction with the dissolved biomolecules leads to the formation of the so-called protein corona on the surface of the NPs. The composition of the corona plays a crucial role in the biological fate of the NPs. While the effects of various physicochemical parameters on the composition of the corona have been explored in depth, the role of temperature upon its formation has received much less attention. In this work, we have probed the effect of temperature on the protein composition on the surface of a set of NPs with various surface chemistries and electric charges. Our results indicate that the degree of protein coverage and the composition of the adsorbed proteins on the NPs' surface depend on the temperature at which the protein corona is formed. Also, the uptake of NPs is affected by the temperature. Temperature is, thus, an important parameter that needs to be carefully controlled in quantitative studies of bionano interactions.

Studying the protein corona on nanoparticles by FCS.

Engineered nanoparticles (NPs) have found widespread application in technology and medicine. Whenever they come in contact with a living organism, interactions take place between the surfaces of the NPs and biomatter, in particular proteins, which are currently not well understood. We have introduced fluorescence correlation spectroscopy (FCS) and dual-focus FCS (2fFCS) to measure protein adsorption onto small NPs (~10-30 nm diameter). FCS allows us to measure, with subnanometer precision and as a function of protein concentration, the increase in hydrodynamic radius of the NPs due to protein adsorption. Investigations of the adsorption of a number of important serum proteins onto negatively charged, carboxyl-functionalized NPs revealed a stepwise increase of the NP size due to protein binding, clearly indicating that a protein monolayer enshrouds the NP. Structure-based calculations of the protein surface potentials reveal positively charged patches through which the proteins interact electrostatically with the negatively charged NP surfaces; the observed protein layer thickness is correlated with the molecular dimensions of the proteins binding in suitable orientations.

Characterization of protein adsorption onto FePt nanoparticles using dual-focus fluorescence correlation spectroscopy.

Using dual-focus fluorescence correlation spectroscopy, we have analyzed the adsorption of three human blood serum proteins, namely serum albumin, apolipoprotein A-I and apolipoprotein E4, onto polymer-coated, fluorescently labeled FePt nanoparticles (~12 nm diameter) carrying negatively charged carboxyl groups on their surface. For all three proteins, a step-wise increase in hydrodynamic radius with protein concentration was observed, strongly suggesting the formation of protein monolayers that enclose the nanoparticles. Consistent with this interpretation, the absolute increase in hydrodynamic radius can be correlated with the molecular shapes of the proteins known from X-ray crystallography and solution experiments, indicating that the proteins bind on the nanoparticles in specific orientations. The equilibrium dissociation coefficients, measuring the affinity of the proteins to the nanoparticles, were observed to differ by almost four orders of magnitude. These variations can be understood in terms of the electrostatic properties of the proteins. From structure-based calculations of the surface potentials, positively charged patches of different extents can be revealed, through which the proteins interact electrostatically with the negatively charged nanoparticle surfaces.