While “top-down” approaches (starting with bulk gold and removing material until nanoparticles remain) exist for synthesis of gold nanoparticles, “bottom-up” approaches (building nanoparticles from reduced metal ions) have become sufficiently optimized to produce relatively monodisperse particles in bulk and will be the focus here. One common method for gold nanosphere production involves reduction of gold(III) derivatives, such as HAuCl₄, using a weak reducing agent such as citric acid [45, 46]. The ratio of the reducing agent to the salt and reducing time determine the size of the nanoparticles, which are citrate stabilized in the solution. Particles can then be surface capped through PEGylation for purification and used for in vivo applications. Synthesis of anisotropic nanoparticles, most notably nanorods, begins with a similar method but using stronger reducing agents, (such as sodium borohydride) to produce small gold nanospheres (~4 nm). These small spheres act as “seeds” onto which carefully controlled epitaxial gold ion deposition is continued through the use of selective growth restriction surfactants that bind to faces of the seed with preferential crystal structures and atom spacing. In the case of gold nanorods, cetyltrimethylammonium bromide (CTAB) is used in combination with silver nitrate to block gold deposition on the {111} or {100} side faces, leaving the {110} common axis faces free for anisotropic growth {denote atomic crystal lattice structures} [47]. Similar to spheres, nanorods, and other anisotropic shapes can be stabilized through PEGylation for in vivo applications. Many other shapes (see Fig. 2) can be synthesized by varying synthesis parameters including temperature, component ratios, and surfactants. Using these synthesis methods combined with bioconjugation of targeting moieties allows fine tuning of nanoparticle composition for specific applications of photoacoustic imaging of cancer .

The use of nanoparticles as contrast agents for photoacoustic imaging of cancer currently remains in the preclinical research stage, though several interesting studies have been explored. For example, gold nanorods have been used to image multiple types of cancers. Jokerst et al. [48] demonstrated the use of gold nanorods to image three types of ovarian cancer tumors: 2008, HEY, and SKOV3 subcutaneously grown in mice (n = 3). They reported a 2.3-fold photoacoustic signal increase with intratumoral injection and a 3.5-fold increase with intravenous injection of gold nanorods at excitation wavelength of 756 nm respectively. The work by Zhong et al. [49] further demonstrates the applicability of gold nanorods beyond imaging, as they reported photoacoustic therapy using gold nanorods conjugated with folic acid in mice bearing HeLa (human cervical cancer) tumors that overexpress folate receptor. Folic acid conjugation allows the nanoparticles to bind to folate receptor whose expression is minimal in healthy tissues and high in cancers such as lung, breast, kidney, brain, cervix, or ovary cancer [50, 51]. Photoacoustic therapy although similar in principle to photoacoustic imaging , destructs individual target cells with laser-induced shock waves. When a photoabsorber is irradiated with a pulsed laser, the optical energy is transformed into mechanical energy, resulting in a shock wave without the side effect of heating or cavitation [52]. In photoacoustic therapy, the generation of shock waves rely on the efficiency of photoabsorbers to convert light energy into acoustic energy, with suitable candidates reported as indocyanine green containing nanoparticles (ICG-PL-PEG), single-walled carbon nanotubes , and gold nanorods [49, 53, 54]. When compared with images of mice injected with folic acid conjugated single-walled carbon tubes, and unconjugated gold nanorods, the tumor growth rate in mice treated with folic acid-conjugated gold nanorods was observed to be significantly lower over a duration of 24 days after treatment.

Another type of gold nanoparticles, nanoshells, have been used to study colon cancers. Li et al. [55] reported the intravenous use of PEGylated gold nanoshells in mice bearing CT26.wt colon cancer tumors grown subcutaneously. When imaged for 6 h using photoacoustic microscopy, the gold nanoshells were seen to be progressively taken up by the tumor foci, while those in the vessels were cleared out, indicating an enhancement in photoacoustic signals with a contrast ratio of 6.5 between the tumor foci to the vessels. Li et al. [56] reported the use of gold nanocages for photoacoustic imaging of cerebral cortex, sentinel lymph nodes, and melanoma in small animals. When gold nanocages were injected in rats to study their brain cortex [57], 81 % enhancement in blood absorption was reported over the intrinsic contrast. In a similar study conducted in rats to image sentinel lymph nodes [58], a gradual accumulation of gold nanocages was observed leading to a gradual increase in photoacoustic signal 28 min after injection. Likewise, gold nanocages functionalized with [Nle⁴, D-Phe⁷]-α-melanocyte-stimulating hormone were used for active targeting and resulted in a 300 % higher photoacoustic signal enhancement because of ligand-receptor binding on melanoma cells in mice compared to PEGylated gold nanocages 6 h after injection [59]. In a recent study by Shujing et al. [60], molecular imaging and photoacoustic therapy of gastric cancer stem cells using bioconjugated gold nanostars was reported. Gold nanostars conjugated with CD44v6 monoclonal antibody were intravenously injected in orthotopic and subcutaneous mice tumor models of human gastric cancer. Their results suggest that the bioconjugated gold nanostars successfully targeted the gastric vascular system at 4 h after injection resulting in 250 % increase in photoacoustic signal compared to the signal acquired from mice injected with unconjugated PEGylated gold nanostars. Moreover, tumor growth inhibition was also reported with an extension of survivability of tumor bearing mice.

Furthermore, introduction of gold nanospheres into a biological environment leads to complex interactions, such as aggregation of nanoparticles within cells, which can be visualized due to the resulting change in optical absorption spectra. Mallidi et al. [61] reported the synthesis and application of bioconjugated gold nanospheres (20 nm dia.) that can specifically bind to the cancer biomarker endothelial growth factor receptor (EGFR) for improved differentiation of cancer from background healthy tissue. EGFR-expressing human epithelial carcinoma cells (A431, n = 6) and EGFR-negative human breast cancer cells (MDA-MB-435, n = 3) were subcutaneously inoculated in mice followed by intravenous injection of EGFR-targeted or PEGylated gold nanospheres . Results of EGFR-targeted gold nanoparticles in A431 tumors are shown in Fig. 3a, b. A wavelength shift in the absorption of targeted gold nanospheres in EGFR expressing tumors was observed from 520 nm (green) to 720 nm (near infrared) due to plasmon resonance coupling. Effectively, cellular internalization and aggregation of nanoparticles into lysosomal compartments brings nanoparticle , and their individual free electron clouds, into close proximity. This grouping of nanoparticles then acts as a larger nanoparticle of a different shape with one associative electron cloud that has a distinct resonance frequency and associated optical absorption, changing the overall absorption spectrum from that of individual nanoparticles. Accumulation was confirmed by silver staining (which detects the presence of noble metals by depositing ionic silver until the nanoparticle can be visualized with light microscopy) of histological sections, along with an increase in photoacoustic signal over time (4 h after injection) shown in Fig. 3c, d. In contrast, images of mice with PEGylated gold nanospheres showed no significant change in photoacoustic signals, indicating that using targeted nanoparticles increased specificity in imaging EGFR -expressing cancer cells.

Overall, gold nanoparticles have been extensively used as contrast agents for photoacoustic imaging due to their excellent optical absorption and finely tunable absorption characteristic that can be optimized for specific applications. However, the bioaccumulation of gold nanoparticles remains a limiting factor in their use clinically.

Silver nanoparticles are synthesized very similarly to gold nanoparticles, predominantly through seed-mediated growth procedures. Reducing agents appropriate for silver particles include hydrazine, ascorbic acid, temperature increases, and light exposure. Silver seeds approximately 3 nm in diameter can be synthesized by the reduction of silver nitrate (AgNO₃) using sodium citrate and adding sodium borohydride (NaBH₄) in a dropwise fashion, followed by stabilization by bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium (BSPP) [65]. The further growth on anisotropic shapes is accomplished similarly to that of gold nanoparticles through the addition of silver salts and reducing agents [66].