Seed-Mediated Growth Method

Since the first approach of GNRs, the synthesis methods of GNRs have been greatly developed (Lohse and Murphy 2013). Seed-mediated synthesis is currently the most commonly used method, which is to add a certain amount of gold nanoparticle seeds into the growth solution followed by the growth of seeds into GNRs with the help of surfactants. Jana et al. were the first to synthesize GNRs using the seed-mediated growth method (Jana et al., 2001). The process can be divided into three steps: 1) citrate-capped gold nanospheres used as seeds are formed after the reduction of HAuCl₄ by NaBH₄, 2) the growth solution that contained HAuCl₄ and cetyltrimethylammonium bromide (CTAB) is prepared, and 3) GNRs are acquired by adding seeds to the growth solution and ascorbic acid (AA; Jana et al., 2001; Xia et al., 2015). However, the low yield and unsatisfied size of GNRs synthesized in this original way make it inappropriate for applications (Scarabelli et al., 2015; Allen et al., 2017), so there appears an endless stream of improvements in accordance with the requirements based on GNRs applications in PTT.

GNRs suitable for PTT require good monodispersity, small size (An et al., 2017; Cheng et al., 2019), anisotropy, no toxicity, and the need to be produced in high yields. To obtain ideal GNRs, previous studies have discussed the influence of various factors on the synthesis process (Scarabelli et al., 2015) and adopted different improvement methods, including silver-assisted seeded growth (Nikoobakht and El-Sayed 2003), using CTAB-capped seeds instead of citrate-capped ones, etc., which can help grow GNRs to the desired length (He et al., 2017). The improved methods are not limited to this. Various improved methods follow, aiming at obtaining GNRs with better biocompatibility to guarantee safety and better controlling the AR, size, and rod shape.

At present, CTAB is usually introduced as a surfactant in the seed synthesis method of GNRs and is an indispensable step in the seed synthesis method. The CTAB concentration is closely related to the yield, shape, and size of GNRs. Studies have shown that CTAB as a surfactant can prevent isotropic grain growth, thus preventing it from forming spherical by-products (Mbalaha et al., 2019). CTAB of different suppliers can lead to different synthesis results of GNRs (Scarabelli et al., 2015). However, it is worth noting that CTAB molecules remaining on both the suspension solution and the GNRs surface are identified as the source of cytotoxicity. CTAB can cause damage to mitochondria and induce apoptosis by entering cells with or without GNRs. Therefore, how to effectively control the toxicity of surfactant CTAB during the preparation of GNRs is an urgent problem to be solved (Qiu et al., 2010; Golubev et al., 2016). During the production process, the toxic effects of CTAB can be reduced by repeated cleaning and replacement of nontoxic modifiers. Several protocols have emerged to remove CTAB from the surface of GNRs in previous studies, but most of them require tedious steps and costly reagents. He et al. proposed a simple “one-pot method” to completely remove CTAB from the surface of GNRs. This procedure adds sodium borohydride to remove CTAB as efficiently as the commercially available GNRs sample (He et al., 2018). Studies have shown that replacing AA with dopamine can also reduce the concentration of CTAB (Requejo et al., 2017).

Besides removing CTAB as much as possible to reduce its concentration, switching to other nontoxic surfactants is also an excellent way to reduce the toxicity of GNRs. Xu, Blahove et al. led the synthesis of GNRs with less toxicity using a less toxic surfactant, dodecyl dimethyl ammonium bromide (C12edmab), as an alternative (Allen et al., 2017). Hollow GNRs with controllable AR were synthesized by Cai et al. with nontoxic modifiers, which also reduced toxicity (Cai et al., 2018). Although the above methods reduce the toxicity of residual CTAB to some extent, how to develop a more simple and convenient method to reduce toxicity is still worthy of further studies. Above all, how to choose a nontoxic synthesis method or material to replace CTAB, which can be produced on a large scale and pass clinical trials, is a key and challenging point. The method described here to reduce the toxicity of GNRs is only aimed at the synthesis process of GNRs themselves, and it will be mentioned later on how to conceal the residual CTAB by surface functionalization or substitutions on GNRs.

GNRs with distinct sizes, ARs, and end shapes have been designed for specific uses. First, studies on the factors affecting the ARs of GNRs are condemned to be valuable, as longitudinal SPR (LSPR), which influences the optical properties of GNRs dramatically, can be finely tuned by adjusting the ARs. Numerous studies have demonstrated that GNRs with high ARs could be obtained by increasing the concentration of silver nitrate in the process of silver-assisted seed-mediated synthesis (Su et al., 2015; Gallina et al., 2016). Tong et al. found that silver nitrate plays a vital role in the symmetry-breaking point, and it was the [HAuCl₄]/[AgNO₃] ratio in the growth solution that critically controls the final width of GNRs and thus the ARs (Tong et al., 2017). Other factors, through the synthesis, such as reaction time (Zhang J. et al., 2017), temperature (Liu et al., 2017c), pH (Zhang L. et al., 2014; Chang and Murphy 2018), the concentration of AA (Li P. et al., 2018), seeds (Su et al., 2015), CTAB (Hormozi-Nezhad et al., 2013), types of reductants (Wu Z. et al., 2019), and surfactants and additives (Wang Y. et al., 2016), also have a great influence. Recently, Requejo et al. have proven that the addition of bioadditives, such as glutathione (GSH) or small thiolated molecules, in nanomolar and micromolar concentrations during the growth stage facilitated the formation of GNRs with tunable ARs and LSPR (Requejo et al., 2020). Additionally, more techniques, such as thermal reshaping, have been gradually used in the production of AR-tuned GNRs (Huang et al., 2018).

The plasmonic properties of GNRs also depend on specific sizes. Larger GNRs represent the higher scattering/absorption ratio, allowing scattering-based applications, such as imaging, whereas smaller GNRs exhibit great potential in PTT toward tumors due to their comparably larger absorption cross-sections. However, traditional synthesis methods of ultrasmall GNRs, by increasing the concentration of seeds added in the growth solution, show a lot of inevitable problems, such as the decreased growth yield and the more by-products, such as nanospheres (Tatini et al., 2014; Chang and Murphy 2018; Cheng et al., 2019; Mbalaha et al., 2019). Therefore, new synthesis strategies of small-sized GNRs should be put forward as soon as possible. Some researchers have attempted to explore the relationship between different end shapes of GNRs and their SPR effects. Wang et al. demonstrated that the addition of hydrochloric acid (HCl) successfully slowed down the growth rate of GNRs, creating longer cylindrical GNRs, whereas short, dogbone-shaped GNRs were fabricated in the group without HCl (Wang Y. et al., 2016). Interestingly, in another research, an inconsistent phenomenon was observed that the end shape of GNRs changed from an arrowhead shape to a dumbbell-like and a dog bone-like shape as the concentration of HCl increased through the second growth of GNRs. The subsequent results showed it was the number of anions rather than the pH altered by HCl that chiefly worked in this process (Kim et al., 2016).

Apart from the specific size, AR, and end shape, the ideal synthetic product of GNRs should guarantee the superior monodispersity and reproducibility of the synthesis. Many studies have synthesized GNRs with quite narrow size distribution and high shape purity by replacing the AA with weaker reductants, such as hydroquinone (Ghosh et al., 2017), 3-aminophenol (Vigderman and Zubarev 2013), pyrogallol (Huang et al., 2015), dopamine (Su et al., 2015), etc. However, good reproducibility remains an unattainable goal for the lack of understanding of the fabrication mechanism at a molecular level (Gallina et al., 2016). Improved reproducibility was achieved through continuous agitation and at a constant temperature of 30°C, which was believed to guarantee the complete solubilization of CTAB, by Gallina et al. (2016). Poor reproducibility is believed to be associated with the stochastic nature of the symmetry-breaking event, which plays a critical role in the subsequent anisotropic growth process during the synthesis of GNRs (Walsh et al., 2017b). Thus, Gonzalez-Rubio et al. attempted to separate the symmetry-breaking step from the seeded growth process. n-Decanol was addicted to the surfactant CTAB to generate a micellar aggregate. They first prepared the intermediate anisotropic seeds (small GNRs) with smaller dispersions in size and shape and subsequently induced GNRs based on them with specific AR and size by controlling the pH, temperature, and Ag⁺ concentration. GNRs with LSPR bands ranging from 600 to 1270 nm are accessible by this method without significantly affecting their dispersion in size and shape, which simultaneously optimize the symmetry breaking and the seeded growth process (Gonzalez-Rubio et al., 2019). Besides, postsynthesis modification, including the secondary growth (Ratto et al., 2010; Khlebtsov et al., 2014a; 2014b) and controlled etching (Szychowski et al., 2018) of GNRs, serves as an effective strategy to improve the reproducibility and precisely control the shape of GNRs.

Although research on the synthesis of various shapes of GNRs has made progress, there are still some problems that have not been solved, such as the limitations of the existing synthesis method of small-sized GNRs, the lack of understanding of the underlying molecular mechanisms in the growth of GNRs, the unattainable reproducibility of the synthesis, etc.

The conversion of Au salt precursor into GNRs contains two main parts. One is the reduction of Au [3] into Au [0] and the other is the formation of nanorods (Park et al., 2017). Various methods have been used to improve the yield of GNRs, involving increasing the total amount of Au0 in suspension, improving the ratio of GNRs to by-products, and enhancing the monodispersity of the GNRs and for the ultimate goal of expanding the production scale.

To raise the yield of reduced Au, Kozek et al., and Ratto et al. reported a secondary growth process in which AA was continuously added to deposit Au precursor remaining in the solution on GNRs (Ratto et al., 2010; Kozek et al., 2013). Although AA is critical for the growth of GNRs, a high concentration of it is accompanied by increasing by-products. Therefore, different reactants are added to decrease the side products. Some of them are weaker reducing agents, such as hydroquinone (Zhang L. et al., 2014; Chang and Murphy 2018; Requejo et al., 2018) and 3-aminophenol (Wu Z. et al., 2019) instead of AA. The addition of silver ions helps cut down the percentage of spherical nanoparticles in the product and form stable GNRs (Hormozi-Nezhad et al., 2013; Jessl et al., 2018), but Ag⁺ needs to be freshly prepared like AA (Burrows et al., 2017). Seeds, Au [3], AA, and CTAB in the correct proportion are necessary for producing high-yield GNRs (Sau and Murphy 2004). The purity of CTAB, mainly affected by bromide ion, also influences the yield of GNRs (Rayavarapu et al., 2010; Si et al., 2012). The reaction conditions and separation methods are the prime factors that affect the yield, too. Adding sodium hydroxide raises the pH value, resulting in an increase in the yield of rod-shaped nanoparticles (Xia et al., 2015), whereas adding HCl retards the growth of GNRs (Wang Y. et al., 2016). Surfactants could be used to preliminarily assist the precipitation of gold nanoparticles of different shapes in a concentrated dispersion (Xia et al., 2015). To extract pure nanorods, Nguyen et al. separated seeds and by-products by asymmetric-flow field flow fractionation (A4F) and finally increased the output of GNRs (Nguyen et al., 2016). Besides, it is necessary to improve resource utilization efficacy and simplify purification steps (Park et al., 2017).