Structurally Engineered Silica Shells on Gold Nanorods for Biomedical Applications

Owing to their unique optical and chemical properties, gold nanorods (AuNRs) are among the most frequently used nanomaterials for biomedical applications, including cancer therapy, imaging, and drug delivery. In particular, the longitudinal dipole plasmon wavelength of AuNRs can be verified from the visible to the near‐infrared (NIR) region, allowing AuNRs to be used as photodynamic/photothermal and imaging contrast agents. At the same time, the silica shell is important as it enhances stability and facilitates the functionalization and biocompatibility of AuNRs, offering numerous advantages in biomedical applications. In this review, silica‐coated AuNRs from a bioapplication perspective are focused. First, the importance of AuNRs for biomedical applications is explained and the purpose of silica coating on AuNRs is discussed. Then, recent studies on the development of silica‐coated AuNRs from a biomedical perspective are reviewed. Subsequently, various strategies for engineering silica coatings and their properties in the biomedical field are reviewed. This review is expected to promote further research on next‐generation silica‐coated AuNRs for biomedical applications.


Introduction
Nanomaterials play a crucial role in the fabrication of various devices and functional materials because of their unique chemical and physical properties. [1]mong these nanomaterials, gold nanoparticles (AuNPs) are of great interest owing to their high-quality synthesis methods and unique optical and chemical properties.For example, external light causes collective oscillations of free electrons in metal nanostructures to form resonance, known as localized surface plasmon resonance (LSPR). [2]This resonance significantly enhances the electromagnetic field near the metal surface. [3]It is important to control the LSPR wavelength within the "NIR biological window" for biomedical applications.NIR-I, ranging from 650 to 950 nm, is considered the first biological window, while NIR-II, ranging from 1000 to 1200 nm, is known as the second biological window.Within this wavelength region, [4] light can penetrate biological tissues, allowing AuNPs to be used as efficient photothermal/photodynamic agents and imaging agents in vivo. [5,6]Gold nanorods (AuNRs) are particularly attractive materials for biomedical applications. [7]hey exhibit two LSPR modes, namely, transverse and longitudinal modes.0] Traditionally, synthesized AuNRs contain cetyltrimethylammonium bromide (CTAB) as a cationic surfactant to prevent aggregation or agglomeration. [11,12]However, CTAB is a potentially toxic chemical that may cause adverse effects, such as inhibition of adenosine triphosphate (ATP) synthase and destabilization of cell membranes. [13,14]Therefore, encapsulating AuNRs is important for achieving biocompatibility and stability under biological conditions.By coating with silica, the AuNRs no longer require a minimum concentration of CTAB for insolution dispersion and improved biocompatibility.In addition, silica has a high degree of thermal stability, allowing the morphology of AuNRs to be maintained during photothermal treatment. [15]urthermore, silica shells are suitable for the functionalization of surfaces with various biomolecules owing to click chemistry, enhancing their targeting ability in biomedical applications. [16,17]n 2001, Murphy et al. first reported the preparation of silicacoated AuNRs. [18]Since then, silica coating has been used to synthesize AuNRs suitable for biomedical applications, and research on its use has significantly increased (Figure 1).To the best of our knowledge, despite this growing interest, only a few articles have thoroughly reviewed silica-coated AuNRs with a focus on the synthesis methods and properties of the silica shell coating and its effect on biomedical applications.This review presents several novel viewpoints on silica-coated AuNRs for biomedical applications, emphasizing the recent progress in this field.First, the advantages of AuNRs and the benefits of silica coating of AuNRs for use in biological environments are discussed.Then, we present an in-depth history of various silica coating technologies used for AuNRs, their physicochemical properties, and their effects in biomedical applications.Finally, we discuss the development of next-generation silica-coated AuNRs for this application.We believe that this review provides guidance for the development of novel silica-coated AuNRs in the biomedical field.

Advantages of using AuNRs in Biomedical Applications
AuNRs are among the most widely used nanomaterials for various biomedical applications, including biomedical imaging, drug delivery, and photothermal therapy owing to their unique optical properties.When metal nanoparticles (NPs) are exposed to light, conduction band electrons resonate with the frequency of the light, causing a collective coherent oscillation known as surface plasmon resonance (SPR). [19,20]This oscillation causes charge separation between free electrons and the ionic metal core.Subsequently, it exerts a restorative Coulomb force, causing electrons to oscillate back and forth on the surface of the metal NPs and causing dipole oscillation similar to that of spherical AuNPs (Figure 2a).
The SPR oscillation of the spherical AuNPs causes strong light absorption in the visible region, resulting in a clear red wine color.In contrast, because AuNRs have an anisotropic structure, electron oscillations can occur in two directions depending on the polarization of light along the short and long axes. [21,22]he excitation of surface plasmon oscillations along the short axis, known as the transverse band, induces a weak absorption peak in the visible region, while the excitation of surface plasmon oscillations along the long axis, known as the longitudinal band, induces a strong absorption peak in the longer-wavelength region (Figure 2b).In particular, as the aspect ratio (length/ width) increases, the longitudinal peak is redshifted from the visible region to the NIR region, whereas the transverse peak is insensitive to the AuNR size. [22]The tunable optical resonance in the NIR region allows light to penetrate biological tissues and interact with AuNRs.Therefore, AuNRs are particularly attractive for in vivo biomedical applications as drug delivery carriers, photothermal therapeutic agents, and optical contrast agents.For example, Cortie et al. reported that heat stress, namely, the photothermal effect induced by AuNRs, causes cell death. [23]El-Sayed reported that when AuNRs are combined with transmission NIR imaging, tumors can be identified due to high NIR adsorption. [24]Von Maltzahn et al. [25] and Taylor et al. [26] reported that AuNRs have a long blood circulation time when used as a drug carrier.Compared with metal nanospheres that have a blood half-life of 4 h, AuNRs have a blood half-life of over 17 h in mice.

Advantages of Silica-Coated AuNRs for Biomedical Applications
The aggregation of NPs reduces their utility in many applications.For biomedical applications, a coating process is essential to stably disperse NPs in a biological environment and increase their biocompatibility.Although the coating process can be performed with biocompatible polymers, such as polyethylene glycol (PEG), AuNRs coated with such polymers can have serious stability issues when exposed to light and heat.Therefore, fabrication of a more rigid shell layer is required. [27,28]Encapsulation of silica on AuNRs is known to enhance their biocompatibility, stability, and functionalization capabilities.

Enhanced Biocompatibility of AuNRs from Silica Coating
CTAB is potentially toxic for biomedical applications.As CTAB is a positively charged surfactant, it is strongly attracted to the negatively charged cell membranes.Therefore, its interaction with the phospholipid bilayer destabilizes the cell membrane and reduces cell membrane integrity or porosity, leading to cell death. [29,30]In addition, the catalytic activity of CTAB can generate a CTA þ cation and trigger the quenching of the enzyme ATP synthase, which causes energy deprivation and cell death. [31,32]A CTAB bilayer is present on the AuNR surface by the electrostatic interaction of ammonium head groups and an anionic AuNR surface with bromide complexes (Figure 3a). [33]This results in AuNRs that can be potentially toxic for biomedical applications.In contrast, silica is generally recognized to be safe by the United States Food and Drug Administration (FDA). [34]Therefore, encapsulating AuNRs with silica can reduce the concentration of CTAB, thereby reducing toxicity.
Yi et al. investigated the cytotoxic properties of bare AuNRs and silica-coated AuNRs through the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. [35]Their results showed a higher toxicity level for the CTAB-coated AuNRs, with 10 < half-maximal inhibitory concentration (IC 50 ) <25 μg mL À1 , than the silica-coated AuNRs, with IC 50 > 400 μg/mL (Figure 3b-e).This suggests that the silica coating can significantly reduce the toxicity of AuNRs.Jokerst et al. also investigated the toxicity of CTAB-coated AuNRs and silica-coated AuNRs through in vitro and in vivo tests. [36]They developed silica-coated AuNRs as a contrast agent for imaging and quantification of mesenchymal stem cells.Importantly, they demonstrated that the silica coating increases the uptake of AuNRs into cells by a factor of more than fivefold due to their low cytotoxicity.They confirmed that no toxicity or proliferation changes occur in cells loaded with silica-coated AuNRs.9][40]

Enhanced Stability of AuNRs Due to Silica Coating
Silica coating significantly improves both the dispersibility and thermal stability of AuNRs.In general, AuNRs tend to agglomerate due to high surface energy caused by their high surface area to volume ratio.In many cases, the particles must be dispersed to maintain their properties.
of AuNRs when used in applications with pulsed lasers. [27]ypically, once AuNRs coated with PEG, CTAB, or other small polymers absorb energy from pulsed lasers, they melt.In contrast, owing to the thermal stability of silica, silica-coated AuNRs can retain their 1D shape and LSPR during photothermal heating. [44]hen et al. reported that silica-coated AuNRs have a much more stable photoacoustic (PA) signal for imaging and improved photothermal stability under irradiation compared to CTABcoated and PEGylated AuNRs.Transmission electron microscopy (TEM) images showed that the shape of the CTAB-coated and PEGylated AuNRs changed from rod to sphere or oval after irradiation.However, only a few morphological changes were observed in the silica-coated AuNRs. [27]

Facile Functionalization of AuNRs by Silica Coating
Because of the variety of silane reagents available for click chemistry, the silane linker is condensed on the silica shell surface through an alkoxysilane group, allowing the targeting ligand to be easily coupled to the particles. [45]In addition, the loading and release of drugs can be easily controlled through surface modification.Surface functionalization is usually required to load appropriate drug molecules onto NPs (hydrophobic/ hydrophilic or positive/negative charges).Smart and multifunctional drug release can be achieved through functionalization of molecules that respond to stimuli or emit light. [46]ang et al. reported silica-coated AuNRs functionalized with DNA as nanovehicles for drug delivery and photothermal therapy (Figure 4). [47]DNA functionalization was easily achieved by reaction between an amine-reactive silica layer and aminemodified single-stranded DNA molecules.The authors hybridized functionalized DNA on the silica surface with a complementary DNA aptamer, which could be used as both a coating agent and a targeting agent.Exposure of the particles to NIR light led to the dehybridization of the linkage DNA by the photothermal effect of AuNRs, enabling the release of trapped drugs.In vitro studies have shown the possibility of using this nanocarrier as a targeted and noninvasive remote controlled drug delivery system because of its high spatial/temperature resolution.

Progress in Silica Coating Strategies for AuNRs
For decades, several methods have been reported for silica coating of AuNRs.In the 1960s, Stöber et al. first reported the catalyzed hydrolysis and condensation of tetraethylorthosilicate (TEOS), which provided the basis for silica coating on inorganic NPs. [48]Murphy et al. first reported the preparation of silicacoated AuNRs in 2001. [18]They coated AuNRs with a thin silica shell (ranging from 5 to 10 nm) through the modification of 3-mercaptopropyltrimethoxysilane (MPTMS).Importantly, silica has a low affinity for gold surfaces. [49,50]Therefore, it is very useful to coat silica on AuNRs using silane coupling agents, such as MPTMS, or polymer templates, such as polyvinylpyrrolidone (PVP) and PEG. [51,52]Gorelikov et al. adapted a single-step silica coating process on AuNRs using CTAB as a template. [53]urrently, this single-step silica coating method is commonly used for the preparation of silica-coated AuNRs.This method has been continuously improved to control the thickness, porosity, and anisotropy of silica coating. [15,54,55]Herein, the development process of the silica coating method on AuNR surfaces and its properties are described in detail (Figure 5).

Silica Coating using Silane Coupling Agents
In the 2000s, Obare et al. first reported the coating of AuNRs with a thin silica shell using the CTAB exchange method with MPTMS as a surface primer (Figure 6a,b). [18]The use of MPTMS, one of the most frequently used silane coupling agents, creates a silanol-rich surface on AuNRs, which can deposit a silica layer by condensation of sodium silicate (Na 2 SiO 3 ).Importantly, this silane primer was preferred because the strong affinity of sulfur for gold could displace the strongly adsorbed CTAB on the AuNR surface. [18]Subsequently, Li et al. also reported the formation of thin silica shells on MPTMSfunctionalized AuNRs (MPTMS-AuNRs). [56]They showed that varying the amount of Na 2 SiO 3 as a silica precursor could adjust the silica shell thickness from 0.5 to 3.5 nm.Pellas et al. also reported the coating of CTAB-AuNRs with silica by CTAB exchange using MPTMS as a surface primer (Figure 6c). [54]he authors adjusted the silica shell thickness on AuNRs from 2 to 6 nm by varying the concentration of TEOS.
However, when MPTMS molecules were functionalized on the gold surface, the maximum thickness of the silica shell formed on the MPTMS-AuNR surface was very thin (less than 6 nm) because of the relatively short maximum length of MPTMS molecules on the AuNR surface.Additional processes, such as solution transfer of AuNRs, are required to increase the thickness of the silica shell.This is because the Stöber method requires an overgrowth process after the initial silica shell is coated with the MPTMS layer. [54]However, a disadvantage of this method is that the initial shell growth with sodium silicate is strongly dependent on the pH value and is difficult to control.Notably, it took more than 24 h to coat the initial silica shell on the AuNRs.

Silica Coating using Polymer Templates
In 2003, Graf et al. proposed another method in which PVP can be adsorbed onto CTAB on the AuNP surface, leading to a silica coating on the AuNP surface. [57]This physisorption can form an intermediate layer on CTAB-AuNRs, allowing for the transfer of water-stable colloidal NPs to the alcohol/H 2 O/NH 3 solution, which is typically used for the Stöber process (Figure 7a,b).However, it was found that the PVP coating failed to coat thick silica on the CTAB-AuNR surface owing to the neutral charge of PVP. [58]Furthermore, it often causes the remaining CTAB to promote the formation of silica NPs with uncoated AuNRs. [59,60]n 2006, Pastoriza-Santos et al. developed a layer-by-layer (LBL) adsorption process of charged surface primers, such as polystyrene sulfonate (PSS) and polyallylamine chloride (PAC), to achieve a thick silica coating on CTAB-AuNR surfaces.Using this process, CTAB-AuNRs were successively coated with negatively charged PSS and positively charged PAC in an aqueous solution.Next, neutrally charged PVP was added to reduce the surface charge of the AuNRs.The addition of PVP prevents AuNR aggregation by maintaining its surface charge below 20 mV.Finally, the catalytic hydrolysis and condensation of TEOS caused the growth of silica shells on the AuNR surface (Figure 7c,d). [61]However, few researchers prefer this method because the multistep process can be laborious and timeconsuming.It is very difficult to finely control the reaction conditions, such as the concentration and molecular weight of the charged surface primer, pH, and ionic strength of the solution.Reproduced with permission. [47]Copyright 2021, Springer Nature.b) Schematic and TEM images of silica-coated AuNRs synthesized using PVP.c) TEM images of silica-coated AuNRs synthesized using the layer-by-layer method.d) TEM and SEM images of silica-coated AuNRs synthesized using the single-step method.e) TEM image of silica-coated AuNRs synthesized using methoxypolyethylene glycol thiol (SH-PEG).f ) TEM images of silica-coated AuNRs with increasing silica shell thickness from left to right of 11, 14, 17, 22, and 26 nm.g) Schematic and TEM images of large-scale silica overcoating of AuNRs with tunable shell thicknesses.h) Schematic of the mechanism of anisotropic silica-coated AuNRs and TEM images of anisotropic silica-coated AuNRs.i) Schematic and TEM images of silica-coated AuNRs having controlled thickness and oriented porosity.Panel (a) Reproduced with permission. [18]Copyright 2001, Panel (b) Reproduced with permission. [57]Copyright 2003, Panel (c) Reproduced with permission. [61]Copyright 2006, Panel (d) Reproduced with permission. [53]Copyright 2008, Panel (e) Reproduced with permission. [63]opyright 2009, Panel (f ) Reproduced with permission. [67]Copyright 2014, Panel (g) Reproduced with permission. [15]Copyright 2015, Panel (h) Reproduced with permission. [72]Copyright 2017, and Panel (i): Reproduced with permission. [54]Copyright 2021, American Chemical Society.

Silica Coating through a Single-Step Process
Based on the Stöber process, Gorelikov et al. were the first to report a single-step silica coating process for AuNRs. [53]They successfully developed a simple protocol to obtain silica-coated AuNRs using CTAB as a template (Figure 8a-c).Briefly, AuNRs were redispersed in water at pH 10-11, and a TEOS solution in alcohol (methanol, ethanol, etc.) was added to this solution under stirring.The mixed solution was allowed to react for several hours before use.The authors confirmed the dependence of the silica shell thickness of AuNRs on the reaction time (Figure 8d).Importantly, when single-step silica coating was first performed on AuNRs, it led to the formation of core-free particles along with uncoated AuNRs.To solve this issue, a centrifugation process is usually performed between the AuNR synthesis and silica coating steps.Centrifugation can reduce the concentration of CTAB in the AuNR solution before adding a silica precursor, such as TEOS. [15,53,62]The single-step silica coating process is now commonly used for preparing silicacoated AuNRs because this method is not only simple but also forms mesoporous structures by CTAB micellar templates that are favorable for drug loading.Importantly, several studies have consistently reported controlled thickness, porosity, and anisotropy of silica based on this method. [15,54,55]4.Silica Coating using Thiol-Reactive Ligands Thiol-tagged PEG (thiol-PEG) is another widely used surface primer to perform the CTAB exchange process for silica coating of AuNRs.This primer can interact strongly with the AuNR surface via Au-thiol chemistry, while oxygen-rich PEG causes the nucleation of silica by the condensation of TEOS.[63,64] In 2009, Liz-Marzán was the first to develop a fast and simple strategy to obtain silica-coated AuNRs using thiol-PEG for CTAB exchange (Figure 9a,b).[63] The authors adjusted the silica shell thickness of the AuNRs from 3.7 to 30.7 nm by simply varying the amount of TEOS added.Importantly, the total length of the thiol-PEG molecule depends on the number of PEG spacers.For example, a thiol-PEG with a 12-unit PEG spacer has a total length of 4.8 nm, whereas a 4-unit PEG spacer makes its length nearly 1.6 nm.[65,66] This indicates that once thiol-PEG is functionalized on the gold surface, the maximum distance between them can be tuned within a wide range.In other words, long thiol-PEGs can form thick silica shells on the AuNR surface.Wang et al. also reported the formation of silica shells by CTAB exchange with thiol-PEG.[64] They showed that the use of sodium hydroxide (NaOH) as an etching agent could alter the porosity of the silica shell.However, this process requires multistep surface modification.

Engineering Thickness, Porosity, and Anisotropy of Silica Capping on AuNRs
As mentioned above, single-step silica coating is the most popular method for the preparation of silica-coated AuNRs.Consequently, many researchers have investigated parameters, such as CTAB or TEOS concentration, pH, and the amount of alcohol used during this process, to control the silica shell thickness and porosity.
Murphy et al. first demonstrated that the silica shell thickness can be adjusted by controlling the concentration of CTAB. [67]As the concentration of CTAB increases, more CTAB becomes free in solution and the thickness of the silica shell decreases.Wu et al. developed a large-scale silica overcoating method to achieve tunable shell thickness by varying the amount of injected silica precursor or by adding PEG-silane to terminate shell growth. [15]hey demonstrated that the silica shell thickness was adjusted from 3.3 to 17.3 nm by varying the amount of added TEOS.In addition, when PEG-silane was added prior to the completion of the silica shell growth, a thin shell of approximately 2 nm was obtained.[70] Based on the modified Lamer Theory, they adjusted the amount of silica precursor to suppress homogeneous nucleation and injected silica precursor to induce only heterogeneous nucleation.The authors were able to control the thickness of the silica shell with a high degree of precision, close to 1 nm; therefore, the rotational diffusion mode of NPs could be observed by simply controlling the silica precursor injection times (Figure 10a-c).
Meanwhile, Pellas et al. reported producing AuNRs with controlled silica shell thickness and porosity suitable for biosensing. [54]They showed that the orientation of silica porosity was tailored to be either perpendicular to or more unexpectedly parallel to the AuNR surface with a change in pH value.Xu et al. reported producing bacteria-like silica-coated AuNRs by adapting an oil-water biphase reaction system. [71]They successfully controlled the pore size of silica from 4 to 8 nm by controlling the  c) TEM image of silica-coated AuNRs synthesized using the MPTMS primer and pH monitoring at a 1/1.8 Au/TEOS ratio with AuNRs dimensions: 94 nm Â 39 nm.Panel (b): Reproduced with permission. [18]opyright 2001, American Chemical Society.Panel (c): Reproduced with permission. [54]Copyright 2003, American Chemical Society.Panel (d): Reproduced with permission. [61]Copyright 2011, Royal Society of Chemistry.(a-c): Reproduced with permission. [68]opyright 2018, American Chemical Society.Panel (d): Reproduced with permission. [72]Copyright 2017, American Chemical Society.The anisotropic coating of AuNRs has also been reported in which the oxidation state of poly(ethylene glycol) methyl ether thiol (PEG-SH) plays an essential role. [72]The authors found that oxidation of PEG-SH leads to the formation of PEG-disulfide, leading to selective coating on the ends of the AuNR surface (Figure 10d).They reported that the terminal thiols of PEG-SH could be attached to the ends and sides of AuNRs, whereas the SS bonds of PEG-disulfide embedded deep in the polymer could prevent adsorption to the AuNR sides.Therefore, PEG-SH prefers to attach to the ends of AuNRs with a low density of CTAB.In this regard, the silica shell also prefers to be deposited at the ends because of the loosely packed CTAB layer.Therefore, dumbbell-shaped silica-shell AuNRs can be easily synthesized by kinetic control.Using this principle, Wang et al. synthesized tip-selective and side-selective silicacoated AuNRs.Silica is deposited selectively on both ends of the nanorods because of its high curvature or on the sides when the ends are blocked with PEG-SH. [73]Huang et al. synthesized dumbbell-like and lollipop-like nanostructures using dimethyl formamide (DMF). [74]These structures are synthesized when DMF penetrates the CTAB layer and further loosens the packing on the tip of the AuNRs.Tracy et al. reported that the concentration of TEOS in the TEOS/MeOH mixture determines whether the SiO 2 shell uniformly coats the entire AuNR or forms lobes at the ends of the AuNR. [75]The authors demonstrated that the formation of a dumbbell-shaped silica coating could be observed as the TEOS fraction in the TEOS/methanol solution was in the range of 3-7 vol%.

Biomedical Applications of Silica-Coated AuNRs
Silica-coated AuNRs are suitable nanocarriers owing to their biocompatibility, tunable optical properties, and photothermal effects. [76]Therefore, several biomedical applications, such as photodynamic therapy (PDT), photothermal therapy (PTT), imaging, and drug delivery, have been widely reported.[79] Therefore, many researchers have attempted to prove that silica-coated AuNRs have good biocompatibility in terms of chronic exposure. [80]

PDT
PDT is a crucial treatment that adapts photosensitizers (PSs) and lasers for oncological diseases, infectious diseases, and skin diseases. [81]In particular, cell apoptosis or necrosis is caused by reactive oxygen species (ROS), such as singlet oxygen ( 1 O 2 ), hydroxyl radical (•OH), and hydrogen peroxide (H 2 O 2 ), generated via light irradiating PSs. [81,82][85] Indeed, this ultrashort pulsed laser can generate nanoplasma via multiphoton emission of electrons from AuNRs, resulting in the production of ROS, namely, •OH and H 2 O 2 . [84,86]However, it should be considered that ROS may block the progression of DNA/RNA polymerase, resulting in cell death or necrosis. [87,88]In addition, ROS can interact with DNA and cause mutagenesis. [89]Therefore, it is critical to prevent undesired side effects while generating ROS.
Palpant et al. investigated the effect of silica coating on AuNRs in terms of the correlation between ROS generation and safety in biomedicine and biology. [85]They demonstrated that the silica shell could prevent the generation of 1 O 2 and •OH by AuNRs under irradiation.The silica shell separates the dissolved triplet oxygen ( 3 O 2 ) from the AuNR surface, and as a result, hinders the production of 1 O 2 between 3 O 2 and "hot" electrons generated by the plasmon excitation.In addition, they showed that the silica shell blocked the generation of •OH caused by electron emission around the AuNRs.This indicates that the silica coating can minimize ROS production, rendering AuNRs safe for several biomedical applications (Figure 11a).

PTT
PTT, which involves localized hyperthermia, is a popularly adopted method in cancer therapy through noninvasive treatment. [90]nterestingly, the fundamental mechanism of PTT is closely related to PDT, and most researchers have recently considered the effects of both PTT and PDT when developing novel cancer treatment methods. [91,92][95][96] Therefore, AuNRs have been regularly used for PTT on cancer cells for a few decades.
Yi et al. investigated the effect of silica-coated AuNRs on PTT in cancer cells. [97]They demonstrated that the cell viability of silica-coated AuNRs was over 70% even at 50 μg mL À1 , while that of CTAB-AuNRs was less than 20% at the same concentration (Figure 11b).They explained that silica coating is essential for improving the biocompatibility of CTAB-AuNR during PTT.Using NIR laser irradiation, they demonstrated that the silica-coated AuNRs are effective in killing cancer cells.Chen et al. reported that the combination of chemo-PTT using silicacoated AuNRs could maximize therapeutic efficacy in cancer cells. [98]They found that silica-coated AuNRs irradiated with NIR lasers alone showed a negligible decrease in cell viability, whereas the viability of silica-coated AuNRs containing doxorubicin (DOX) as a model drug was significantly reduced by NIR laser irradiation (Figure 11c).Since then, Monem et al. have also reported the efficacy of combined chemo-PTT treatment using silica-coated AuNRs with DOX through in vitro and in vivo tests. [99]Importantly, they first found that silica coating helps to increase the photothermal conversion efficiency of AuNRs for multiple iterations of NIR irradiation.
site.This was attributed to the high number of Nanocom-ICGs that can aggregate at the tumor site, leading to dual heat production by AuNRs and ICG.
In a study by Cheng et al., the efficacy of combinatorial chemo-PDT-PTT treatment using silica-coated AuNRs was reported. [101]The researchers coloaded the chemotherapy DOX and photosensitizer (IR820) onto the particles.Notably, NIR laser triggered the generation of reactive oxygen species and also resulted in remarkable photothermal efficacy from the AuNRs, leading to a highly efficient antitumor outcome in vitro and in vivo.
Surface modification of silica-coated AuNRs interface can improve chemo-PTT treatment.Li et al. covalently grafted the cell-penetrating TAT peptide (YGRKKRRQRRR) onto the silica surface of silica-coated AuNRs, utilizing an in situ graftingcleavage strategy. [102]TAT-functionalized silica-coated AuNRs showed a significant enhancement in intracellular uptake.The researchers loaded the drug onto the particles and demonstrated synergistic effects on photothermal-controlled drug release and inhibition of tumor growth in vivo by NIR laser irradiation.

Imaging
Imaging is a fundamental method used to improve the efficacy of several therapies, such as stem cell therapy and cancer cell therapy.It can also aid in the proper delivery of cells and monitor the long-term and short-term viability and death of the delivered cells. [103,104]Several nanomaterials, including AuNRs, have been employed as imaging agents to enhance the sensitivity of images. [105]In particular, AuNRs absorb pulsed light, which leads to the production of acoustic waves. [52]Consequently, AuNRs can be used as imaging agents for PA imaging.
In general, PA imaging is a noninvasive technique that can reach much deeper into turbid materials than other optical imaging tools. [106,107]This is attributed to the acoustic waves being less scattered in turbid materials, such as tissue.Chen et al. first reported the use of silica-coated AuNRs as imaging contrasts for PA imaging. [52]They showed that silica-coated AuNRs produce a much stronger PA signals than PEG-coated AuNRs (Figure 12a).In addition, they demonstrated that silica coating increases the PA signal of three types of AuNRs, CTAB, PEG, and polyelectrolyte-coated AuNRs but at different levels because of their different interfaces (Figure 12b).The signal enhancement of the silica-coated AuNRs was attributed to the interfacial heat conduction changing from gold to water because of the silica shell.Jokerst et al. showed that silica-coated AuNRs can be used as a PA contrast agent to monitor stem cells in living mice. [36]hey also demonstrated that silica coating could increase the PA signal of AuNRs in an in vivo system.
In addition to PA imaging, fluorescence imaging is one of the most important methods for in vivo imaging.Duan et al. demonstrated that silica-coated AuNRs could enhance fluorescence intensity, enabling the detection of cancerous cells in the form of red dots (Figure 12c). [40]X-ray imaging and terahertz imaging using silica-coated AuNRs have also been reported. [108,109]Copyright 2011, American Chemical Society.Panel (c): Reproduced with permission. [40]Copyright 2021, Elsevier.

Drug Delivery
Drug delivery refers to the process of administering a pharmaceutical compound to achieve a therapeutic effect. [110]Numerous nanomaterials are currently used for targeted drug delivery to enhance the uptake of poorly soluble drugs. [111,112]Among them, silica-coated AuNRs are highly likely to be used as a reservoir for chemotherapeutic agents with great potential.In particular, mesoporous silica is widely used as an AuNR-coated shell for drug delivery because of its large surface area and large pore volume, which enables efficient drug loading. [113]iu et al. proposed a nanoscale drug delivery system using AuNRs coated with mesoporous silica shells (MSNP-coated AuNRs). [114]They demonstrated that MSNP-coated AuNRs exhibit excellent release of DOX as a model drug by NIR light irradiation, resulting in effective killing of cancer cells.Since then, several researchers have reported the potential of MSNP-coated AuNRs as nanocarriers for drug delivery. [99,115,116]While most studies have only reported the drug delivery effects of MSNP-coated AuNRs using in vitro tests, there are a few relevant experimental results from in vivo tests.For example, Monem et al. reported the effect of MSNP-coated AuNRs as a drug carrier through an in vivo test. [99]Through histopathological examination, they showed that MSNP-coated AuNRs loaded with DOX exert a great therapeutic effect on cancer cells.However, they did not investigate whether MSNP-coated AuNRs containing DOX had a negative effect on other organs.It should be noted that this study is the first to report the therapeutic efficacy of drug-loaded MSNP-coated AuNRs via in vivo testing.However, further research on the biological toxicity of MSNP-coated AuNRs in vivo is required.

Effects of Engineered Silica Shells on AuNRs for Biomedical Applications
Controlling the thickness, porosity, shell anisotropy, and complex structure of the silica shell coating on the surface of AuNRs is crucial for their successful use in biomedical applications.For example, the amplification and reduction of the signal intensity from a fluorescent molecule or Raman reporter used as an imaging probe is dependent on the distance from the NPs. [117]herefore, the use of silica shells as tunable gap spacers allows fine-tuning of the signal intensity. [118,119]Herein, research on how the silica shell coating on AuNRs is engineered for use in biomedical applications is introduced.During the process of engineering the silica shell, it is possible to fine-tune the optimal conditions for maximizing the efficiency of AuNRs for biomedical applications.

Shell Thickness Engineering of Silica-Coated AuNRs
By engineering the silica shell thickness on the AuNRs, the degree of scattering or the distance between the imaging molecule and the AuNRs can be controlled.Copyright 2022, Royal Society of Chemistry.
Murphy et al. proposed that silica-coated AuNRs can be applied as fluorescence imaging agents. [67]They demonstrated wavelength-and distance-dependent fluorescence emission with approximately tenfold maximum fluorescence intensity enhancement observed in the fluorescence hotspot.
Yang et al. reported that silica-coated AuNRs can significantly improve the photothermal effect and PA intensity (Figure 13a-c).They showed that the photothermal efficiency of silica-coated AuNRs depended on the shell thickness.The photothermal effect was maximized at a thickness of 20 nm but decreased thereafter, which is explained by the increased contribution of thermal conduction and scattering of silica.Furthermore, it was confirmed that AuNRs coated with a 20 nm-thick silica shell had the maximum effect on cellular uptake.

Shell Porosity Engineering of Silica-Coated AuNRs
In the biomedical field, controlling the silica shell porosity of AuNR coatings can be used to increase drug-loading capacity by increasing the specific surface area.Shell porosity can also affect the thermal conduction coefficient and optical properties.
Wang et al. synthesized silica-coated AuNRs with a significantly enlarged pore size (4-8 nm) and surface area (470 m 2 g À1 ), resulting in highly efficient parameters for loading DOX. [79]u et al. monitored the radiative relaxation of silica-coated AuNRs with different porosities. [120]As water has a larger thermal conduction coefficient than silica, entrapped water molecules deactivate the population of the vibrationally excited states of the silica layer, reducing the temperature of the entire AuNR in an efficient and nonradiative way.
Additionally, Mercadal et al. demonstrated that the surfaceenhanced Raman spectroscopy enhancement factor of silicacoated AuNRs with a molecular probe can be used to determine shell porosity under an NIR laser. [121]

Shell Anisotropy or Complex Structure in Silica-Coated AuNRs
Engineering silica shells with unique structures creates the possibility of amplifying their efficiency or synthesizing them into multifunctional particles for use in biomedical applications.
NPs, these particles not only exhibit a smaller shift of the LSPR wavelength and a higher absorption efficiency of light but also have a higher loading capacity and lower cytotoxicity (Figure 14b-d).Furthermore, the Janus particles effectively enter cells targeted for cellular imaging and demonstrate a significant photothermal effect.
Yoon et al. synthesized AuNRs embedded in silica (Figure 14e). [123]This organosilica-based clustering platform can control the distance and orientation between the AuNRs.In addition, various NPs with different surface polarities, such as spherical AuNPs, AuNRs, superparamagnetic NPs, and quantum dots, can be used as building blocks in this method, enabling their use as multimodal imaging agents with tunable enhancement and quenching (Figure 14f ).

Conclusions and Prospect
Silica-coated AuNRs have been used in biomedical applications since the early 2000s.Silica is an FDA-approved, nontoxic material that enhances the biocompatibility of AuNRs.Importantly, it helps improve the stability and dispersibility of AuNRs in biological environments.Therefore, several silica coating strategies have been proposed for the use of AuNRs in biomedical applications.The silica coating strategies of AuNRs adapted for each biomedical application are presented in Table 1.However, several challenges remain in the use of silica-coated AuNRs in biomedical applications.Therefore, it is expected that future extensive research efforts will lead to widespread use of silica-coated AuNRs for use in biomedical applications.
Herein, we discuss the remaining issues and research directions of silica-coated AuNRs as next-generation nanomaterials for biomedical applications.1) There is still controversy over whether silica-coated AuNRs are toxic or nontoxic in biomedical applications.This is because only a few studies have investigated the biocompatibility of silica-coated AuNRs using in vivo tests.The effect of silica-coated AuNR as an in vivo drug carrier system has recently been reported in a previous study. [99]However, this study only showed the therapeutic efficacy in cancer cells using silica-coated AuNRs via histopathological examination, without any results on their toxicity evaluation.To date, the adverse effects of silica-coated AuNRs have not been investigated.Therefore, more fundamental studies on the biological toxicity of silica-coated AuNRs must be conducted in vivo to expand their use for biomedical applications.2) Most silica coating procedures only produce a limited yield of silica-coated AuNRs, usually below 10 mg. [15,124]However, the typical dosage of NPs used in drug delivery is approximately 30-40 mg for in vivo systems, [125] indicating that it is difficult to obtain an adequate amount of silica-coated AuNRs for biomedical applications, such as drug delivery, using current silica coating methods.3) Further studies on engineering silica shells are needed to fine-tune the optical behavior of silica-coated AuNRs for biomedical applications.Many researchers agree that owing to their optical properties, silica-coated AuNRs are powerful nanomaterials for biomedical applications, such as PDT, PTT, imaging, and drug delivery.However, it is very challenging to efficiently treat cancer cells by PTT using only silica-coated AuNRs. [98]Recently, studies that maximize the effectiveness of biomedical applications by precisely engineering silica shells have been reported, and some of them have been introduced in this review.For the practical use of silica-coated AuNRs in biomedical applications, such silica shell engineering requires further study.Photothermal therapy (PTT) Silica coating using silane coupling agents MPTMS 3-5 nm [97]   Silica coating by single step TEOS %30 nm [98]   Silica coating using thiol-reactive probes Thiol-PEG 10-13 nm [99]   Silica coating using polymer templates PVP 15 nm [100]   Imaging Silica coating using thiol-reactive probes Thiol-PEG 6-75 nm [52]   Silica coating by single step TEOS 20 nm [36]   Silica coating by single step TEOS 11-26 nm [67]   Silica coating by single step TEOS 20 nm [40]   Silica coating by single step TEOS %18 nm [108]   Silica-coating by single step TEOS 6-25 nm [109]   Drug delivery Silica coating by single step, and Silica coating using silane coupling agents TEOS, and APTES %35 nm [114]   Silica coating by single step TEOS 10-13 nm [99]   Silica coating by single step TEOS %20 nm [116]

Figure 1 .
Figure 1.The number of research articles per annum related to silica-coated AuNRs in terms of biomedical applications.All research articles were obtained using a Scopus search from 2001 to 2022.

Figure 4 .
Figure 4. a) Schematic images for NIR light-triggered release of drugs from the pore of aptamer-conjugated nanovesicles.b) TEM images of synthesized AuNRs and silica-coated AuNRs.c) NIR-induced heat generation of silica-coated AuNRs depending on power density (left) and concentration of silica AuNRs (right).Reproduced with permission.[47]Copyright 2021, Springer Nature.

Figure 8 .
Figure 8. a) Schematic of silica coating of AuNRs using the single-step process.b) TEM images of silica-coated AuNRs.c) SEM images of silica-coated AuNRs.d) TEM images of silica-coated AuNRs depending on reaction time.Panel (b-d): Reproduced with permission.[53]Copyright 2008, American Chemical Society.

Figure 7 .
Figure 7. a) Schematic of silica-coated AuNPs synthesized using PVP.b) TEM images of silica-coated AuNRs synthesized using PVP.c) Schematic of the LBL absorption process using surface primers, such as PSS, PAC, and PVP for thick silica coating of CTAB-AuNRs.d) TEM images of silica-coated AuNRs via the LBL absorption process.Panel (a,b): Reproduced with permission.[57]Copyright 2003, American Chemical Society.Panel (d): Reproduced with permission.[61]Copyright 2006, American Chemical Society.

Figure 10 .
Figure 10.a) TEM images of the silica-coated AuNRs with varied precursor injection times (S0 = 0 injection, S1 = 1 injection -S16 = 16 injections, scale bar = 50 nm).b) Silica shell thickness as a function of injection times.c) LSPR peaks of silica-coated AuNRs depending on the shell thickness of silica.d) Schematic and TEM images of synthetic conditions to produce anisotropic silica growth on AuNRs.Panel(a-c): Reproduced with permission.[68]Copyright 2018, American Chemical Society.Panel (d): Reproduced with permission.[72]Copyright 2017, American Chemical Society.
silica coating time.Furthermore, Wang et al. used NaOH as an etching agent and were able to alter the porosity of the silica shell in the AuNR.

Figure 11 .
Figure 11.a) Degree of generated ROS by ultrashort pulsed light between the bare AuNRs and silica-coated AuNRs.b) Cell viability of HeLa cells as model cancer cells after incubation with the bare AuNRs and silica-coated AuNRs.c) Cell viability of A549 cells as model cancer cells irradiated by an NIR laser with silica-coated AuNRs, doxorubin (DOX), and silica-coated AuNRs with DOX for 3, 4, and 8 min.d) Fluorescence images of A549 cells as model cancer cells with phosphate buffer solution (PBS), free indocyanine green (ICG), silica-coated AuNRs, and silica-coated AuNRs incorporating ICG after irradiation with an 808 nm laser (green: live cells, red: dead cells).Infrared microscopic images of the A549 tumor-bearing mice after 808 nm laser irradiation with PBS, free ICG, silica-coated AuNRs, and silica-coated AuNRs incorporating ICG.Panel (a): Reproduced with permission.[85]Copyright 2022, Royal Society of Chemistry.Panel (b): Reproduced with permission.[97]Copyright 2013, American Scientific Publishers.Panel (c): Reproduced with permission.[98]Copyright 2012, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.Panel (d): Reproduced with permission.[100]Copyright 2021, Springer Nature.

Figure 13 .
Figure 13.a) TEM images of the silica-coated AuNRs with different silica shell thicknesses, scale bar: 50 nm.b) The temperature changes of distilled water, CTAB-AuNRs, and silica-coated AuNRs when irradiated with an 808 nm laser for 5 min.c) Cell viability of the silica-coated AuNR with different thicknesses and CTAB-AuNRs after AuNR uptake.Panel (a-c): Reproduced with permission.[44]Copyright 2022, Royal Society of Chemistry.

Table 1 .
Silica coating strategies of AuNRs for several biomedical applications.