Trafficking of Gold Nanorods in Breast Cancer Cells: Uptake, Lysosome Maturation, and Elimination

Gold nanorods (AuNRs) have been largely investigated driven by their promising potentials in drug delivery, imaging, and photodynamic therapy because of their distinctive physicochemical properties. It is widely known that AuNRs can be taken up by different cells, however, the trafficking of the nanorods in cells are less known. In this work, the behaviors and fate of AuNRs in the human breast cancer cell line MDA-MB-231 were intensively probed by transmission electron microscopy (TEM) with detailed time resolution, together with induced couple plasmon mass spectroscopy (ICP-MS), confocal microscopy, Western blot, and cell viability assay. We reveal that AuNRs enter the classic lysosome maturation through endocytosis and are sequestered in the vesicular system even during cell division. AuNRs can escape from the lysosomes occasionally and the escaped AuNRs are recycled back into the lysosomal system through cytoprotective autophagy. The dilution of AuNRs in cells is mainly attributed to the cell division rather than exocytosis, because expelled AuNRs can be re-endocytosed by the cells. The feature of vesicular restriction guarantees other organelles such as mitochondria and nucleus are exempted from the direct exposure to AuNRs

Trafficking of Gold Nanorods in Breast Cancer Cells: Uptake, Lysosome Maturation, and Elimination

Gold nanorods (AuNRs) have been largely investigated driven by their promising potentials in drug delivery, imaging, and photodynamic therapy because of their distinctive physicochemical properties. It is widely known that AuNRs can be taken up by different cells, however, the trafficking of the nanorods in cells are less known. In this work, the behaviors and fate of AuNRs in the human breast cancer cell line MDA-MB-231 were intensively probed by transmission electron microscopy (TEM) with detailed time resolution, together with induced couple plasmon mass spectroscopy (ICP-MS), confocal microscopy, Western blot, and cell viability assay. We reveal that AuNRs enter the classic lysosome maturation through endocytosis and are sequestered in the vesicular system even during cell division. AuNRs can escape from the lysosomes occasionally and the escaped AuNRs are recycled back into the lysosomal system through cytoprotective autophagy. The dilution of AuNRs in cells is mainly attributed to the cell division rather than exocytosis, because expelled AuNRs can be re-endocytosed by the cells. The feature of vesicular restriction guarantees other organelles such as mitochondria and nucleus are exempted from the direct exposure to AuNRs

Distribution of AuNRs in MDA-MB-231 cells evaluated by TEM observation with detailed time-resolution.

<p>(A, C, E, G, I, K, M, O) The representative images of AuNRs in cells with different incubation time. The area rectangle labeled is further magnified and shown in the middle column. (B) AuNRs are shown adsorbed on the membrane and retained in the endocytic vesicle (ev). (D) AuNRs are observed in an early endosome (ee). (F) AuNRs are located in a late lysosome (ll) which is featured with multilayer structure and near Golgi apparatus. (H) AuNRs are in a late endosome (le) appeared as multivesicular body (MVB) in perinuclear region. (J, I, N, P) AuNRs-contained lysosomes located in perinuclear region after 6, 12, 24 and 48 h of incubation with AuNRs. The black arrows point lysosome, white arrows point mitochondria, and black and white arrow heads mark the Golgi apparatus and rough endoplasmic reticulum (rER), respectively. (n: nucleus, m: membrane).</p

Schematic illustration of the itinerary of AuNRs in MDA-MB-231 cells.

<p>AuNRs are first taken up by endocytosis and immediately retained in endocytic vesicles (ev). The AuNRs then experience lysosome maturation process including early endosome (ee), late endosome (le) and late lysosome (ll) location. In the cell, individual AuNRs gradually aggregated one another and located towards perinuclear region. The rER and Golgi represents rough endoplasmic reticulum and Golgi apparatus, respectively.</p

Analysis of AuNRs internalized by MDA-MB-231 cells vs AuNRs concentration.

<p>(A) Typical absorption curves of AuNRs in cells cultured with different AuNRs dosages for 6 hours. The curves are offset for clarity. (B–C) A comparison between the quantification methods of ICP-MS and TSPR peak area calculation. (D) Digital photographs of AuNRs-contained cell pellets co-cultured with different amount of AuNRs in media for 6 hours. (E) Dark field microscopic observations of cells incubated with different concentrations of AuNRs in the media. The red scale bar is 50 µm.</p

Analysis of AuNRs internalized by MDA-MB-231 cells vs incubation time.

<p>(A) Pellets of the cells incubated with AuNRs for 0.25, 0.75, 1.5, 3, 6, 12, 24, and 48 hours. Cells with no treatment are control. (B) The representative UV-vis-NIR absorption spectra of the cells incubated with AuNRs for different hours. The curves are offset for clarity. (C) The absorption spectra of AuNRs in cells. The absorption from the control cells is subtracted as background and the spectra are offset for clarity. (D) Comparison between results obtained from ICP-MS and TSPR peak area value. (E) The data acquired from the ICP-MS and TSPR peak area is converted to number of AuNRs contained in each cell. (F) Bright and dark field microscopic observation of the cells incubated with AuNRs for different hours (TSP/AuNRs is 250). The first line is the bright field microscopic observation. The second and third line is the dark field microscopic images with and without false color. The scale bar represents 50 µm.</p

Characterization of AuNRs dispersed in water and culture media.

<p>(A) The UV-vis-NIR absorption spectrum of AuNRs dispersed in water. The inserted SEM image shows the morphology of AuNRs deposited from the aqueous solution. (B) Absorption spectra of AuNRs dispersed in serum containing media (SCM) with 0%, 2.5%, 5%, 10%, 20% and 30% of fetal bovine serum (FBS) as a function of incubation time. Concentration of AuNRs in the media was 120 pM. The corresponding ratios of total serum proteins (TSP) to AuNRs (TSP/AuNRs) were 0, 31.25, 62.5, 125, 250, and 375. (C) The absorption spectra of AuNRs dispersed in basic media containing different content of only bovine serum albumin (BSA) for 30 minutes. The ratios of BSA to AuNRs (BSA/AuNRs) were 1250, 125, 12.5, 1.25 and 0.125.</p

MOESM1 of Highly sensitive and robust peroxidase-like activity of Au–Pt core/shell nanorod-antigen conjugates for measles virus diagnosis

Additional file 1: Fig. S1. Typical photographs of TMB–H2O2 solution (left) TMB–H2O2-Au NRs (middle) and TMB–H2O2-Au@Pt NRs (right). Reaction conditions: 0.5 mM TMB, 20 mM H2O2 and 0.125 nM Au NRs/Au@Pt NRs. Fig. S2. Effects of substrates concentration (TMB), substrates concentration (H2O2), conjugate concentration (Au@Pt NR-antigen conjugates), temperature, reaction time and pH on catalytic activity of the Au@Pt NR-antigen conjugates. Reaction conditions: (A) 0.125 nM Au@Pt NRs, 20 mM H2O2, (B) 0.125 nM Au@Pt NRs and 0.5 mM TMB, (C) 0.5 mM TMB and 20 mM H2O2, (D-F) 0.125 nM Au@Pt NRs, 0.5 mM TMB and 20 mM H2O2

Near Infrared Laser-Induced Targeted Cancer Therapy Using Thermoresponsive Polymer Encapsulated Gold Nanorods

External stimuli, such as ultrasound, magnetic field, and light, can be applied to activate in vivo tumor targeting. Herein, we fabricated polymer encapsulated gold nanorods to couple the photothermal properties of gold nanorods and the thermo- and pH-responsive properties of polymers in a single nanocomposite. The activation mechamism was thus transformed from heat to near-infrared (NIR) laser, which can be more easily controlled. Doxorubicin, a clinical anticancer drug, can be loaded into the nanocomposite through electrostatic interactions with high loading content up to 24%. The nanocomposite’s accumulation in tumor post systematic administration can be significantly enhanced by NIR laser irradiation, providing a prerequisite for their therapeutic application which almost completely inhibited tumor growth and lung metastasis. Since laser can be manipulated very precisely and flexibly, the nanocomposite provides an ideally versatile platform to simultaneously deliver heat and anticancer drugs in a laser-activation mechanism with facile control of the area, time, and dosage. The NIR laser-induced targeted cancer thermo-chemotherapy without using targeting ligands represents a novel targeted anticancer strategy with facile control and practical efficacy

Activation of Oxygen-Mediating Pathway Using Copper Ions: Fine-Tuning of Growth Kinetics in Gold Nanorod Overgrowth

Growth kinetics plays an important role in the shape control of nanocrystals (NCs). Herein, we presented a unique way to fine-tune the growth kinetics via oxidative etching activated by copper ions. For the overgrowth of gold nanorods (Au NRs), competitive adsorption of dissolved oxygen on rod surface was found to slow down the overgrowth rate. Copper ions were able to remove the adsorbed oxygen species from the Au surface via oxidative etching, thus exposing more reaction sites for Au deposition. In this way, copper ions facilitated the overgrowth process. Furthermore, Cu²⁺ rather than Cu⁺ acted as the catalyst for the oxidative etching. Comparative study with Ag⁺ indicated that Cu²⁺ cannot regulate NC shapes via an underpotential deposition mechanism. In contrast, Ag⁺ led to the formation of Au tetrahexahedra (THH) and a slight decrease of the growth rate at similar growth conditions. Combining the distinct roles of the two ions enabled elongated THH to be produced. Copper ions activating the O₂ pathway suggested that dissolved oxygen has a strong affinity for the Au surface. Moreover, the results of NC-sensitized singlet oxygen (¹O₂) indicated that the absorbed oxygen species on the surface of Au NCs bounded with low-index facets mainly existed in the form of molecular O₂