# Internalization of Phospholipid-Coated Gold Nanoparticles
## 1 Introduction  
Nanoparticles have shown promise as platforms for curative treatments and detection of cancer and disease [1; 2; 3]. Due to the leaky vasculature of solid tumors, nanoparticles can passively penetrate through these openings and reach the outside cells of solid tumors, a process known as the enhanced permeability and retention effect [4; 5]. Gold nanoparticles are particularly favorable as they have shown to be used in a broad range of bio-health applications, specifically in photothermal therapy, drug delivery, and photodynamic therapy [6; 7]. Gold nanoparticles are also favorable because they possess the unique ability to bind with thiol groups, thus biomolecules can be easily functionalized to gold for various applications [8; 9]. In addition, gold nanoparticles are not significantly toxic in cells, based on lactate dehydrogenase (LDH), metabolic cell activity assays (MTT) [10], and other cell-based toxicity assays [11; 12]. Thus, gold nanoparticles are useful for drug delivery platforms and there is large motivation for in-vitro studies [13; 14; 15; 16; 17; 18; 19]. Studies have shown that gold nanoparticles can be internalized into cells by a process called clathrin-mediated endocytosis [20; 21; 22]. When internalized, the nanoparticles are compartmentalized into vesicles that fuse with early endosomes, which in turn mature into late endosomes, and ultimately fuse with acidic lysosomes for possible degradation [23; 24; 25; 26; 27]. Additionally, receptors are recycled back to the cell membrane via recycling endosomes that \"pinch\" off from the early endosome [28]. Thus, through clathrin-mediated endocytosis, gold nanoparticle processing could serve as a useful platform for cleavage of drugs in the acidic environment of endosomes for cellular processing [29; 30]. Endocytosis of any material, like gold nanoparticles, is strongly dependent on membrane trafficking between compartments, and Rab and LAMP proteins are important for assisting with this role [31; 32; 33]. Specific Rab and LAMP-1 proteins are known to localize to domains on endocytic compartments; they are referred to as _markers_ for these endosomes [34; 35]. Rab5 is used as a marker for early endosomes [36; 37; 38; 39] and Rab7 is used as a marker for late endosomes [40; 41; 42; 43]. Rab11 is used as a marker for recycling endosomes [44; 45; 46]. Few, if any Rab proteins have been identified to localize specifically to the lysosome, however the lysosomal associated membrane protein (LAMP-1) is known as a marker for lysosomes [47; 48; 49]. In this work, we used antibodies against Rab5, Rab11, Rab7, and LAMP-1 to study their distribution and possible co-localization with gold nanoparticles within the cell. It should be noted that in recent work we have shown that these markers are not as specific for the indicated endosomes as thought in the past [50]. Specifically, we found that Rab5, Rab7, and Lamp1 can all co-exist on the same endosome or lysosomes to varying extents in both C2C12 and A549 cells. Nevertheless, these markers are still clear markers for the endocytic pathway. Our laboratory created a new type of phospholipid-coated gold nanoparticle (PCAuNP) that can be labeled with fluorescent lipids for easy monitoring with fluorescence microscopy [51]. Subsequently, [PERSON] and [PERSON] provided a comprehensive characterization of PCAuNPs, in which the structure, stability, aggregation, quenching, FRET, size, and shape of the PCAuNPs were determined [52]. The key properties are described more fully in the materials section below. [PERSON] and [PERSON] demonstrated that these PCAuNPs were taken up by A549 cells into acidic compartments [51] but did not characterize which acidic compartments were involved. They determined that they were neither in mitochondria nor in peroxis, but by electron microscopy, they were seen in intracellular vesicular compartments, which could be endosomes, and in some cases, they were found in lamellar bodies, structures unique to A549 cells.