Interaction of Positively Charged Gold Nanoparticles with Cancer Cells Monitored by an in Situ Label-Free Optical Biosensor and Transmission Electron Microscopy

Abstract

Functionalized nanoparticles (NPs) can penetrate into living cells and vesicles, opening up an extensive range of novel directions. For example, NPs are intensively employed in targeted drug delivery and biomedical imaging. However, the real-time kinetics and dynamics of NP-living cell interactions remained uncovered. In this study, we in situ monitored the cellular uptake of gold NPs-functionalized with positively charged alkaline thiol-into surface-adhered cancer cells, by using a high-throughput label-free optical biosensor employing resonant waveguide gratings. The characteristic kinetic curves upon NP exposure of cell-coated biosensor surfaces were recorded and compared to the kinetics of NP adsorption onto bare sensor surfaces. We demonstrated that from the above kinetic information, one can conclude about the interactions between the living cells and the NPs. Real-time biosensor data suggested the cellular uptake of the functionalized NPs by an active process. It was found that positively charged particles penetrate into the cells more effectively than negatively charged control particles, and the optimal size for the cellular uptake of the positively charged particles is around 5 nm. These conclusions were obtained in a cost-effective, fast, and high-throughput manner. The fate of the NPs was further revealed by electron microscopy on NP-exposed and subsequently fixed cells, well confirming the results obtained by the biosensor. Moreover, an ultrastructural study demonstrated the involvement of the endosomal-lysosomal system in the uptake of functionalized NPs and suggested the type of the internalization pathway.

Strategies for the Development of Metallic‐Nanoparticle‐Based Label‐Free Biosensors and Their Biomedical Applications

“…AuNP colorimetric detection of up to single base pair mismatched DNA difficultyi nthe adsorption of longer length DNA by AuNPs [43] AuNP colorimetric NP size effect in the detection of mismatched DNA and importance of target-to-particle ratio for aggregation of NPs interference from other biomolecules;atmospheric effects on the detection processwere not tested [47] AuNP colorimetric detection of mismatchedD NA in longer length ssDNA and dsDNA, as well as the effect of chemical modification of DNA on the detection process lower sensitivity and specificity in longer length DNA [49] AgNP fluorescence use of NC in the fluorescence detection of mutant genes LOD was little bit highert han other reported mechanism [59] AgNP fluorescence fluorescence turn-on sensor for the detection of AChEa ctivity impact of different lengths of DNA on fluorescence intensityo fDNA-Ag NCs was not studied [65] CuNP fluorescence methodt od etect sulfur-containing drugse ffect of the presenceo fa tmospheric thiol-containing compounds was not illustrated [80] CuNP fluorescence recognition of ALP activityP ie xtraction methodw as not presented because Pi inhibits ALP activity and can interfere with real data [82] AuNP SPR examined the entrance mechanism of AuNPs into HeLa cancer cell no band was observed if the concentration of AuNPs was very low and only 50 %o ft he area of an evanescent field sensor was coveredb ycells [94] AuNP SPR developed an on-chipL SPR-based biosensor for multiplex detection of breast cancer biomarkers LOD of CA125 and CEA protein biomarkers for breast cancer are higher than the clinically relevant amount [100] AgNP SPR used triangularAgNPs to sense serum p53 protein effect of other proteinsa nd concentrations on LSPR band shifting was not studied [104] AuNP SERS demonstrated away to prepare uniform AuNPs up to alarge area SERS shift near 518 cm À1 was not defined [117] AuNP SERS studied lipidconstitution and modificationinc ompositions after addition of CH groups by the attachment of AuNPs effect of various chemically stabilizedA uNPs was not represented [118] AgNP SERS studied site-specific drug delivery and intercellular molecular determination with Ag@NGO cell viability for normal cells was much lower than that of cancer cells [126] AgNP SERSr epresented the SERS-based cascade amplificationb ioassay methodf or the determination of miRNA involved multiple washinga nd ultracentrifugation steps, which could affectthe sensing performance [132] CuNP SERSd emonstrated the preparation of CuNPsb yP LAL method involved the oxidation of CuNPs, whichc ould affect the band position [137] AlNP MEFutilized AlNPs for increasing fluorescence of adenine spacing effect of NPs and analytea nd interference from foreign molecules werenot explored [152] AgNP MEFrevealedt he effect of spacing between fluorophorea nd AgNPs, density and interparticle distance, and spectral overlap between AgNPs and fluorophores on fluorescenceemission impact of d...…”

“…Gold NPs are known for targeted drug delivery and for destroying cancer cells. Trimethylamine (TMA)‐functionalized AuNPs of 4.6 nm in size were utilized to examine their entry into adhered HeLa (cervical cancer) cells by employing evanescent field biosensors . As shown in Figure , there is an evanescent field above the waveguide grating layer that responds by changing the refractive index through any means of perturbation.…”

“…Trimethylamine (TMA)-functionalized AuNPs of 4.6 nm in size were utilized to examinet heir entry into adhered HeLa (cervical cancer) cells by employing evanescent field biosensors. [94] As shown in Figure 6, there is an evanescent field above the waveguide grating layer that responds by changing the refractive index through anym eans of perturbation. Modification of the refractive index was measured by the shifto ft he signal by aw aveguide biosensor composed of microplates.…”

Surface chemistry governs the sub-organ transfer, clearance and toxicity of functional gold nanoparticles in the liver and kidney

“…The shapes of NMs have been shown to dictate endocytosis by either normal or cancer cells, as well as clearance by the MPS [10,11]. Surface chemistry has been shown to particularly influence NM cellular uptake, protein "corona" formation, and subsequent immune system activation, as well as biodistribution [12,13]. Importantly, to effectively use NMs in biomedicine, the appropriate surface modifications are necessary in order for them to resist rapid clearance, pass-through biological barriers, and rapidly distribute to target organs and tissues [14].…”

Human primary endothelial label-free biochip assay reveals unpredicted functions of plasma serine proteases

“…The detection is typically limited to a probing depth of ~150 nm due to the exponentially decaying electromagnetic evanescent field of the sensing waveguide mode 3,4 . The technology is commercially available and was successfully employed previously to monitor the kinetics of cell-surface and cell membrane receptor-ligand interactions 3,[5][6][7] , binding affinity 8 , cellular signaling 9,10 , cytotoxicity 11 , nanoparticles 12 and the functional state of surface-adhered cells down to the single cell level 13 . The output of the sensor is an integrated signal of dynamic mass redistribution (DMR) taking place in the sensing depth above the bottom of each well 14 .…”