Synthesis And Characterization Of Functionalized Gold Nanoparticles And Their Interactions With Proteins And Cells

Nanomaterials have been proposed as ideal carriers for drugs, genes and proteins to treat diseases. Nanomaterials have also been developed as contrast agent for disease diagnosis. Nanomaterials itself have some abilities to kill cells, such as photothermal effects. Besides medical applications, nanomaterials are also widely used in electronics, photonics, and catalysis. However, it also raises societal concerns on the toxicity of nanomaterials. It is urgent to understand their in vivo distribution, metabolism, excretion and body damage because of organ accumulation. This is a new research discipline, named nanotoxicology. In nanomedicine or in nanotoxicology, there is a pressing need to gain a thorough understanding of the fundamental interactions between nanoparticles and proteins or cells. All physiological responses caused by nanomaterials are on the basis of interactions between nanoparticles and proteins or cells, including protein adsorption, change protein conformation, specific cell uptake, killing cells, organ accumulation and so on. Understanding these interactions will guide us design safe and effective vectors, and help us to control and decrease the damage of nanomaterials to human body.Usually, undesired cytotoxicity of nanomaterials, such as cell dysfunction, cell cycle arrest, and cell apoptosis, are resulted from non-specific interactions between nanomaterials and cells, including electrostatic, hydrophobic, hydrogen bonds, steric, π-π staking and so on. Electrostatic interaction between nanoparticles and cells is one of the most important interactions. Typically, three types of charged nanoparticles, positively, neutral, negatively charged, were employed to investigate the electrostatic interaction, without considering the effect of surface charge density and the existing of other non-specific forces. To systematically investigate the electrostatic attraction between nanoparticles and cells and avoid other forces, we synthesized a gold nanoparticle array with continuous change in surface charge density by adjusting the ratio of structure similar charged ligands and neutral ligands. Nanoparticles in the array are approximately5nm sphere particles. Ligands associated on nanoparticles were quantified by elemental analysis and LC/MS/MS after iodine cleavage. According to the loaded ligand number, surface charge density of the array is calculated. Surface charge density of nanoparticles with100%covered by ligands with one charged group was assumed as1.0. The range of surface charge density of the array is from+2.87to-4.18. After12hours incubation with cells at25and50μg/mL, the amount of the array cell uptake didn’t parallel change with surface charge density. The amount of internalization of negatively charged and neutral nanoparticles are negligible. There was a sharp increase in the cell uptaken of the GNP array at GNP05(surface charge density is+0.52), reaching a level equal to that of the highly positively charged GNP01-GNP04(surface charge density is from+2.87to+1.0). Due to mutual shielding effect, only outmost layer of surface charge provide the electrostatic attraction. Although some nanoparticles in the array were overloaded with charged ligands, their internalization didn’t increase. The outmost layer of surface charge density was termed as effective surface charge density.So the effective surface charge density determines the electrostatic attraction between nanoparticles and cells. The cell uptake of the array was confirmed by time-dependent cell uptake, TEM images of uptaken nanoparticles cells and dark-field microscopy images of uptaken nanoparticles cells. The electrostatic properties for nanoparticles are commonly characterized by zeta potential. In water, zeta potential values of the array has transformed from positive to negative at GNP09(neutral). The trend is similar the cell uptake of the array. In cell culture medium, protein corona was created on particle surface, zeta potential values of these protein-bound nanoparticles were all negative and nearly leveled off The bound proteins were separated by SDS-PAGE-and didn’t show amount difference except GNP01. LC/MS/MS analysis of the nanoparticle-associated proteins showed that each nanoparticle bound to～60proteins, of which～30were common proteins that bound to all nanoparticles. Binding proteins to nanoparticles is a dynamic process and maintains an equilibrium. Therefore, bound proteins didn’t determine the electrostatic interactions between nanoparticles and cells. From all these results, we conclude that huge amount of positively charged nanoparticles were uptaken, only when the positive surface charge density was big enough to provide strong enough electrostatic attraction between nanoparticles and cells. Although protein coating altered the zeta potential of positive charged nanoparticles from positive to negative, the positive charged nanoparticle were still able to bind to the cell surface.To specific and effectively delivery drugs, nanoparticles have been conjugated with targeting molecules, such as antibodies, peptides, organic molecules and so on. These targeting molecules will only mediate nanoparticles into disease cells with specific receptors or over-expressed receptors. So targeting nanoparticles can specific enter and kill disease tissues or cells, and didn’t show side effect. However, when nanoparticles enter physiological environment, such as blood, proteins will be adsorbed to the surface of nanoparticles and create protein corona. So far, there are very few reports about the effect of protein adsorption on nanoparticle targeting. Targeting nanoparticles were designed and constructed without considering the effect of protein adsorption. To investigate the effects of protein adsorption on nanoparticle targeting, targeting nanoparticles with three sizes were synthesized. On the basis of TEM images of nanoparticles, statistics sizes are4.5±2.5nm,14.2±1.6nm,38.8±4.9nm respectively. In cell culture medium with serum, proteins bound to the surface of targeting nanoparticles analyzed by SDS-PAGE. Compared with nanoparticles themselves, amount of cell uptake and uptaken pathway were different with protein-bound targeting nanoparticles. The differences were related to their sizes, doses and the density of targeting receptors. Small nanoparticles with big curvature cannot multivalent bind to cell membrane receptors Adsorbed proteins prevent the ligand-receptor recognition and nanoparticles cannot bind to cells, so the amounts of cell uptake of small nanoparticles decreased. Big nanoparticles with small curvature can multivalent bind to receptors. Adsorbed proteins cannot completely prevent the ligands binding to targeting receptors. In addition, adsorbed proteins can multivalent bind to cell membrane and provide extra forces, so the amounts of cell uptake of big nanoparticles slightly increased. However, effect of protein binding on ligand-receptor recognition in medium size nanoparticle was dose dependent. At a high dose, cell uptake was prevented; at a low dose, cell uptake was enhanced. Because at a low dose, receptors were not saturated bound by targeting ligands, there is high opportunity for multivalent interactions. At a high dose, receptors were saturated bound by targeting ligands, there is low opportunity for multivalent interactions. When available receptors density decreased, the ratio of cell uptake deceasing after protein binding in small nanoparticles was also decreased. This is resulted from all nanoparticles were difficult to bind to receptors at low density of receptors.In big nanoparticles, the amounts of cell uptake of protein-bound nanoparticles were always a bit more than nanoparticles themselves. This is resulted from the multivalent interactions between the adsorbed proteins and cell membrane. In summary, adsorbed proteins have some effects on cell targeting nanoparticles. When designing targeting nano-vectors, several factors need to be considered, including size, dose in clinical use, and receptor density.