Table 1.

Overview of the included studies.

Reference	Model	Route of adm.	Shape	Size (nm)	GNP coating and imaging tags	BBB-crossing enhancers	Imaging modality	Main conclusions
Gao et al. (21)	In vivo: U87 GBM orthotopic xenograft in nude mice	I.V.	GNS	20	PEG, Gd-DTPA, Raman tag (IR783B), Alkyne/azide group	LRP-1	MRI, SERS microscopy	–The acidic brain tumor environment triggers nanoclustering of alkyne-GNS with azide-GNS, preventing them from returning in the blood stream. –Xenograft edges are persistently enhanced by Gd-DTPA. –Tumor resection is guided by SERS signal
Huang et al. (20)	In vivo: RCAS-PDGF/N-tva transgenic mouse model of GBM (overexpression of integrin αvβ3)	I.V.	GNSt/GNS	60	PEG, Raman tag (N,N-dimethylformamide) RGDyK/RADyK	RGDyK	SERS microscopy	–RGDyK-GNSts penetrate the GBM significantly better than RADyK-GNSt (non-integrin targeted GNSt). –RGDyK-GNSts define the tumor edges, the local infiltration and satellite foci
Pohlmann et al. (22)	In vitro: GS9-6/NOTCH1 + GBM cells	Culture	GNR	50	PVP	No	TEM	–TEM allows visualizing the interaction between cells and GNPs at different GNP concentrations, and between GNPs within the tumor cells
Lai et al. (23)	In vivo: GNS-loaded U87 GBM and GBM8401 glioma orthotopic xenograft in mice	Culture	GNS	N/A	Fluorescent tag (MUA)	No	TXM, Fluo	–The GNPs allow tumor localization, visualization of anomalous tumor vasculature and detection of the BBB leakage typical of brain tumors
Kempen et al. (24)	In vivo: TS543 GBM xenograft in severe combined immunodeficiency mice	I.V.	GNS	60	Silica shell, Gd, Raman tag trans-1,2-bis(4-pyridyl)-ethylene	No	SEM, Optical microscopy	–By complementing the SEM imaging with optical imaging, the GNPs can be identified and localized within the tumor itself
Dixit et al. (25)	In vitro: U87 GBM cells, U227 GBM cells. In vivo: U87 GBM orthotopic xenograft in mice	Culture/I.V.	GNS	41	PEG, Fluorescent tag (Pc4)	Tf	Fluo	–Tf conjugation significantly enhanced the GNP uptake by GBM orthotopic xenograft with respect to the GNPs non-conjugated with Tf. –Regardless of the Tf conjugation, the GNPs were found to be highly specific for brain tumor tissue, with negligible accumulation in other organs
Dixit et al. (26)	In vitro: U87 GBM cells U227 GBM cells. In vivo: U87 GBM orthotopic graft in mice	Culture/I.V.	GNS	41	PEG, Fluorescent tag (Pc4)	Tf, FGF	Fluo	–Double-targeted GNPs cross the BBB more efficiently than untargeted GNP-Pc4, leading to higher accumulation levels and to a faster rate of accumulation. –Double-targeted GNPs accumulate in critical organs less than single-targeted GNPs
Cheng et al. (27)	In vitro: U87 GBM cells, U251 GBM cells, GBM43 cells, GL261 GBM cells In vivo: U87 GBM orthotopic xenograft implanted in athymic nude mice	Culture/I.V.	GNS	21	PEG, Gd, Dox	TAT	MRI	–When compared with Gd-chelates alone, the TAT-GNP-Gd conjugates cause more intense and more lasting enhancement of the brain tumor with signal still detectable after 24 h. –GNPs are washed out from the normal brain within 24 h. –TAT-GNPs conjugated with Dox cross the BBB and are selectively uptaken by tumor cells that are killed. –TAT-GNPs conjugated with Dox significantly increase mice survival with respect to Dox alone or TAT-GNPs alone. –TAT-GNPs conjugated with Dox or with Gd cause no adverse effects in vivo
Diaz et al. (28)	In vitro: gliosarcoma 9L cells GBM cells, C6 glioma cells, U87 GBM cells, A172 GBM cells, U251 GBM cells, U373 GBM cells, BT2012036 oligodendroglioma, GLINS1 GBM stem cells In vivo: GNP-loaded U87 orthotopic xenograft in nude mice; 9L gliosarcoma orthotopic xenograft in mice	Culture (U87 model), I.V or intra-arterial (9L model)	GNS	50/120	PEG, Silica shell, Fluorescent tag (Cyto647) Raman tag (trans-1,2-Bis(4-pyridyl)-ethylene)	MRgFUS, anti-EGFR Ab	MRI, TEM, Fluo, SERS microscopy	–Fluo allows monitoring the growth of GNPs-loaded xenograft. –GNPs cross the BBB in areas treated with MRgFUS. –Anti-EGFR functionalization promotes GNPs uptake by tumor cells. –SERS-active GNPs allow enhancement of the brain tumor edges after MRgFUS in vivo
Yuan et al. (29)	In vivo: D270 glioma xenograft in mice	I.V.	GNSt	80	PEG	Ultra-short pulsed laser	MPM	–MPM allows micro-angiographic visualization of GNPs in the tumor vasculature. –Low-potency image-guided pulsed laser irradiation allows selective GNSs uptake by the tumor
Schultke et al. (30)	In vivo: GNP-loaded C6 glioma xenograft in Wistar rats	Culture	GNS	50	No	No	SynCT	SynCT allows single-cell spatial resolution of GNP-loaded glioma xenograft ex vivo
Astolfo et al. (31)	In vivo: GNP-loaded F98 glioma xenograft in mice	Culture	GNS	50	No	No	SynCT	–SynCT allows 3D reconstruction and volumetric analysis of GNP-loaded tumor xenograft in vivo and ex vivo. –Tumor doubling time is assessed by SynCT
Nedosekin et al. (32)	In vitro: B16F10 Melanoma cells, MDA-MB-231 Breast cancer cells In vivo: MDA-MB-231 Breast cancer xenograft implanted in mice breast	Culture/intratumoral	GNR	<100	PEG	Folate, EpCam, CD 45	PAFC PTC	–Photothermal imaging allows to identify GNRs labeled cancer cells and to detect the intracellular clustering of GNRs. –By applying a laser over the cisterna magna of mice, the GNRs were used to label and detect breast cancer metastasis to the CNS before those became macroscopically evident
Cho et al. (16)	In vitro: GNP-loaded U87 glioma cells. In vivo: GNP-loaded U87 glioma cell suspension injected subcutaneously in mice	Culture	GNC	50	No	RGDyK (the experiment did not require BBB crossing)	MPM, PAM	–MPM estimates the intracellular uptake of GNCs by glioma cells in vitro. –PAM allowed estimating the total number of RADyK-GNCs within the tumor at each time point and quantifying the growth of the tumor
Kircher et al. (19)	In vitro: eGFP+U87MG cells. In vivo: eGFP+U87MG xenograft in mice	Culture/I.V.	GNS	60	Silica shell, Gd, Raman tag (trans-1,2-bis(4-pyridyl)-ethylene) Aka MPR NP	No	MRI, PAM, SERS microscopy	–MPR NP allows MRI, photoacoustic and Raman imaging. –MRI, photoacoustic, and Raman imaging co-localize in vivo between them and with histological analysis. –SERS microscopy allowed guiding GBM resection in mice
Noreen et al. (33)	In vivo: U87 GBM cells xenograft in mice injected with GPNs	I.V.	GNS	20	No	No	FTIR	–FTIR in vitro reveals microvascular architecture of brain tumors (enhanced by BaSO4 nanoparticles) and their micro-fenestration (revealed by GNP leak into the extravascular space)

Anti-EGFR Ab, anti-epidermal growth factor receptor antibody; BBB, blood-brain barrier; Cyto647, cytochrome 647; FGF, fibroblast growth factor; Fluo, fluorescent imaging; FTIR, Fourier-transform infrared imaging; I.V., intravenous; Gd, gadolinium; GNR, gold nanorod; GNS, gold nanosphere; GNSt, gold nanostar; LRP-1, low-density lipoprotein-receptor-related protein-1; MPM, multiphoton microscopy; MPR, magnetic resonance imaging–photoacoustic imaging–Raman imaging; MRgFUS, MR-guided focused ultrasound; MRI, magnetic resonance imaging; MUA, mercapto-urodecanoic acid; PAFC, photoacoustic flow cytometry; PAM, photoacoustic microscopy; PEG, polyethylene glycol; Pc4, phthalocyanine 4; PTC, photothermal cytometry; PVP, poly(vinylpyrrolidone); RADyK-GNC, gold nanocage conjugated with protein RADyK; RADyK-GNSt, gold nanostar conjugated with protein RADyK; RGDyK-GNSt, gold nanostar conjugated with integrin RGDyK; Route of adm., route of administration; SEM, scanning electron microscopy; SERS, surface-enhanced Raman scattering; SynCT, synchrotron-based CT; TAT, transactivator of transcription; TEM, transmission electron microscopy; Tf, transferrin; TXM, tridimensional X-ray microscopy.