Image‐guided diagnosis and treatment of glioblastoma

Glioblastoma (GBM) is the most aggressive primary brain tumor with poor prognosis and high recurrence rate. The presence of the blood–brain barrier (BBB) prevents diagnostic and therapeutic drugs from penetrating and working in GBM. In traditional surgical treatment, it is difficult to completely distinguish the boundary between tumor and surrounding normal tissue, resulting in incomplete resection of tumor. Therefore, the diagnosis and treatment of GBM are very challenging. Several molecular probes and nanoprobes have been reported to successfully penetrate the BBB, selectively target and accumulate in GBM to achieve in situ imaging of brain tumors, thus achieving accurate diagnosis and treatment of orthotopic or non‐orthotopic GBM. This paper reviews the advances of molecular probes and nanoprobes in image‐guided diagnosis and treatment of GBM. The design principle, application, and advantages of each probe are enumerated in detail. Finally, the prospects and potential challenges of probes in the diagnosis and treatment of GBM are discussed with a view to further promote the study and application of novel imaging probes in GBM.

of patients. 3 Therefore, appropriate diagnosis and treatment methods that can accurately identify GBM lesions and effectively excise the lesions are the keys to improve the survival rate of patients.
Currently, postoperative adjuvant chemotherapy and radiotherapy are commonly used management means to remove the infiltrative growth and highly invasive residual lesions of GBM. 4 The commonly used therapeutic drugs for GBM are temozolomide and bevacizumab, which can dramatically suppress the growth and invasion of tumor cells. 5 Unfortunately, the blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB) greatly limit the effective delivery of therapeutic drugs to intracranial GBM, which greatly impairs their therapeutic effectiveness in the postoperative management of GBM. 6 In addition, drug dose escalation increases the risk of adverse reactions rather than improving therapeutic efficiency. At the same time, the complexity and heterogeneity of GBM are the major factors closely related to multidrug resistance (MDR) in clinical oncology, which hinder the effective tumor suppression of drugs entering intracranial tumors. 4a,7 The development of GBM remains the most severe challenge in terms of diagnosis and therapeutic management due to the deficient accumulation of drugs and drug resistance in intracranial tumor lesions as well as the incomplete resection of tumor tissue. Therefore, there is an urgent need to develop promising diagnostic and therapeutic approaches to overcome these challenges.
In the face of the above challenges, rapid and accurate diagnosis of GBM is crucial for understanding the pathological function of GBM and determining treatment options. Clinical imaging techniques will be extremely useful noninvasive tools to provide guidance for the diagnosis, staging, and monitoring of therapeutic efficacy of GBM. 8 Various imaging modalities, such as magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), and fluorescence imaging, have irreplaceable imaging advantages and have been utilized in clinical practice. 9 In order to achieve accurate diagnosis of GBM, the selection of appropriate imaging agents is a prerequisite for accurate diagnosis. 10 More importantly, effort should be devoted to develop methods that can cross the BBB to target accumulation at tumor sites. Among them, the receptor-mediated transcytosis mechanism not only provides a noninvasive delivery system to facilitate the crossing of BBB for targeted carriers into the brain, but also acts selectively on GBM cells within the brain parenchyma. 11 In addition, GBM cytomembrane carriers have also emerged as novel delivery systems for GBM agents delivery based on targeting native proteins on the membrane.
Based on the special advantages of precise diagnosis, image-guided therapy can provide noninvasive means of imaging disease and evaluating pharmacokinetics and drug efficacy. 11 The emerging BBB-crossing carriers, with a variety of targeted ligand modifications, integrating diagnosis and treatment agents, can achieve early accurate diagnosis, determine treatment plans, and track therapy effect. 12 At the same time, by leveraging carriers to deliver functional therapeutics to lesion sites, the multifunctional platforms allow for a variety of treatment options, including photodynamic therapy (PDT), photothermal therapy (PTT), chemotherapy, immunotherapy, or their synergistic treatments, which can contribute to significantly S C H E M E 1 Overview of emerging theranostic agents based on small molecular probes and nanoplatforms for glioblastoma (GBM) diagnosis, image-guided therapy, and image-guided surgery improving noninvasive therapeutic efficacy. 13 These efforts are expected to generate revolutionary consequences on troublesome GBM.
In this review, we comprehensively summarize and rigorously discuss the current advances in accurate and effective diagnosis and therapy management of GBM (Scheme 1), and intellectually analyze the design strategies for BBB-crossing imaging and image-guided therapeutic platforms. Moreover, the significant achievements of different imaging probes and various nanocarriers delivery systems in GBM diagnosis and treatment are highlighted. Finally, the challenges and prospects of GBM targeted platforms for future clinical directions are discussed. We expect that this review will further deepen our understanding of GBM and facilitate the development of emergent clinical diagnostic and therapeutic techniques for GBM.

Imaging of GBM by molecular probes
Molecular probes are easy to synthesize, can detect a variety of biological targets, and have high sensitivity, nondestructive rapid analysis, and real-time monitoring capabilities. Therefore, it is one of the powerful tools for disease imaging and monitoring in biological systems. 14 F I G U R E 1 Chemical structures of small molecular probes for glioblastoma (GBM) detection. (A) Activatable molecular probes. (B) Targetable molecular probes Imaging of GBM with molecular probes can be achieved by fluorophores modification of the recognition groups of GBM overexpressed biomarkers and the modification of targeted peptides. These probes demonstrated good GBM targeting ability, could clearly visualize the margins between GBM tumor and surrounding normal tissues. Here, activatable or targetable molecular probes for GBM imaging are summarized (chemical structures in Figure 1).

2.1.1
Activatable molecular probes Relevant biomarkers are usually abnormally expressed in the disease. Monitoring the expression levels of these biomarkers can effectively diagnose the occurrence of diseases. Designing activatable molecular probes based on abnormally expressed biomarkers of GBM is an important means for real-time monitoring of GBM. Several activatable probes that can penetrate the BBB have been developed ( Figure 1A). In the hypoxia environment of GBM, the elevated level of intercellular reactive oxygen species (ROS) leads to overexpression of cysteine (Cys). The detection of Cys upregulation behavior is regarded as a new diagnostic direction for GBM. 15 A quinoline-based Cys fluorescent probe ZS-C1 with high selectivity was recently reported to monitor endogenous Cys in living U87MG cells. 16 By observing the distribution of ZS-C1 in 3D solid cell spheres, it was found that the penetrating depth of ZS-C1 reached 60 μm in solid tumors. However, this probe has not yet been applied to the orthotopic GBM. NPO-B, another highly selective and sensitive Cys-activated probe, showed good targeting in U87MG and SNU4098 cells xenograft GBM in brain. 17 In the z-stacked images of both GBM samples with NPO-B, the strongest fluorescence signal can be seen at a distance of 30-70 μm from the surface, validating the high tissue permeability of NPO-B. Subsequently, NPO-B exhibited a stronger fluorescence signal in human clinical GBM samples than in normal tissues. These results strongly demonstrate its ability to distinguish GBM cells in clinical samples from individual patients. Homocysteine (Hcy) is also an important biomarker of GBM. 18 Kim et al. constructed a fluorescent diagnostic probe (NPO-Pyr) to identify Hcy activity in GBM. 19 On days 7 and 14 after the tumor implantation, the fluorescence intensity in the plasma of NPO-Pyr-treated GBM xenograft mice was significantly higher than that of normal mice. NPO-Pyr is the first probe to monitor Hcy activity in the plasma of GBMxenograft mice, predicting the development of GBM at a relatively early stage.
In 2018, Liu et al. proposed a spray-type γglutamyltranspeptidase (GGT)-activated fluorescent probe (NC-B-Cys-γ-Glu) to track the real-time activity of GGT 20 (Figure 2A). GGT is an enzyme that can be overexpressed in glioma cells, but normally expressed in normal brain cells. 21 Local spraying does not involve the possibility of crossing the BBB, so NC-B-Cys-γ-Glu can be quickly activated by GGT. The fluorescence at 610 nm is decreased with the increased fluorescence at 558 nm when probe is exposed to GGT. After spraying NC-B-Cys-γ-Glu, the fluorescence performance of tumor site was obviously distinguished from the surrounding F I G U R E 2 (A) Sensing mechanism of fluorescence probe (NC-B-Cys-g-Glu) for detection of GGT in orthotopic glioblastoma (GBM). (B) In vivo fluorescence imaging of U87 orthotopic GBM mouse modal with or without GGsTOP pretreatment. Reproduced with permission from Ref. 20 Copyright 2018, Elsevier. (C) Schematic of Hydro-Cy5-RWrNR for targeting imaging of ROS in GBM. (D) In vivo imaging of U87MG GBM-bearing mice with Hydro-Cy5-RWrNR, Cy5-RWrNR, and Hydro-Cy5 without targeting group. Mice treated subcutaneously with PBS (blue circle) and LPS (green circle) to induce inflammation as controls. Reproduced with permission from Ref. 34 Copyright 2019, American Chemical Society normal tissue with an average fluorescence signal ratio of 3.2 ± 0.3 ( Figure 2B). Similarly, Kitagawa et al. developed another spray-type fluorescent probe PR-HMRG based on hydroxymethyl rhodamine green (HMRG) to detect GBM in response to calpain-1 (CAPN1). 22 CAPN1 is involved in neurodegenerative neuronal cell death and is therefore thought to be overexpressed in GBM. 23 PR-HMRG can be specifically recognized and subsequently fluoresced by CAPN1. Significant fluorescence contrast between tumor and peritumoral tissue was found after spraying during surgery in U87 xenograft model mice.
Besides, aldehyde dehydrogenase 1A3 (ALDH1A3) is an enzyme that is overexpressed in glioblastoma stem cells (GSCs), and is related to the stemness and invasiveness of GBM. 24 Gelardi et al. recently presented a series of curcumin-based fluorescent probes capable of binding to ALDH1A3. 25 Results indicated that probe 10 showed a 12-fold fluorescence enhancement on the U87 ALDH1A3positive cells with more selectiveness than other probes, and could clearly delimit the cancerous masses.

2.1.2
Targetable molecular probes GBM cell surface receptors play a key role in invading surrounding tissues and are often upregulated in tumor cells. Therefore, these receptors can serve as targets for GBM, and the design of targetable molecular probes is an innovative approach to achieve early diagnosis of GBM ( Figure 1B). 26 Angiogenic integrin α v β 3 , highly expressed in activated endothelial cells and surrounding solid tumor cells, is an important target of GBM. 27 α v β 3 can effectively recognize the tripeptide sequences Arg-Gly-Asp (RGD) in extracellular matrix proteins, so a series of RGD-modified probes have been developed for the precise diagnosis of GBM. Owing to the α v β 3 integrin receptor-mediated transcytosis, these molecular probes can effectively cross the BBB and target tumor cells even at early stage of GBM. 28 Liu et al. developed a multivalent two-photon fluorescent probe TAB-3-cRGD, which binds to integrin α v β 3 through the multiple cyclic RGD (cRGD) motifs. 29 One hour after injection of TAB-3-cRGD in the U87MG non-orthotopic xenograft GBM model, fluorescence enhancement could be clearly observed at the tumor site and persisted for about 0.5 h. TAB-3-cRGD showed great targeting ability to bind integrin α v β 3 in both cancer cells imaging and in vivo imaging of U87MG tumor mice.
Near-infrared fluorescence (NIRF) imaging is a noninvasive method to detect and monitor early-stage tumors. 30 NIR fluorophore Cy5.5 containing RGD or Asn-Gly-Arg (NGR) labeling can be used for GBM imaging. 31 Cy5.5-RKL, Cy5.5-NKL, and Cy5.5-DKL were successfully synthesized as visual tools for image-guided diagnosis of GBM. 32 Cy5.5-RKL and Cy5.5-NKL exhibited favorable imaging potential in U87MG xenograft mice. The peak value of tumor/normal brain tissue in U87 orthotopic GBM mice was 4.82 ± 0.80 after 3 h of Cy5.5-RKL injection, displaying its great potential of image-guided GBM resection. Subsequently, Zhang et al. identified a linear peptide Arg-Trp-( D -Arg)-Asn-Arg (RWrNK) with an unnatural D -arginine that can target integrin α v β 3 , and synthesized a probe Cy5-RWrNK to pass through the BBB and selectively accumulate in orthotopic U87MG tumors. 33 Based on Cy5-RWrNK, an NIRF probe hydro-Cy5-RWrNR response to ROS with the ability to synergistically target integrin α v β 3 was proposed ( Figure 2C). 34 To prove its ability to detect GBM with high sensitivity, three different experimental groups were designed. Results showed that hydro-Cy5-RWrNR could rapidly accumulate in GBM and exhibit strong fluorescent signals, while the control hydro-Cy5 produced much higher fluorescent signals in LPS-induced inflammatory muscles ( Figure 2D). More importantly, hydro-Cy5-RWrNR exhibited a higher targetto-background ratio, indicating that it can serve as a powerful tool to highly selective detection of GBM and is promising to diagnose early tumors.
In addition to integrins, other potential targets for targeted surveillance of GBM cells and tissues are gradually being discovered. An et al. recently disclosed a new triple receptor-targeting fluorescent probe BSA-OXN-SIWV for visualizing GBM. 35 BSA-OXN-SIWV contains a two-photon oxazepine dye (OXN), a tetra-peptide (SIWV) to target caveolin-1 receptor, 36 and a bovine serum albumin (BSA) protein to target albondin (gp60) and secreted protein acidic and rich in cysteine receptor. 37 The imaging of human GBM biopsy tissue showed that the fluorescence signal observed in GBM tissue was significantly higher than normal tissue, and showed higher permeability (160 μm). The successful applications on GBM cell lines and human clinical GBM tissues demonstrated that BSA-OXN-SIWV has a broad application prospect in clinical surgery.

Imaging of GBM by nanoprobes
Molecular probes have been applied to GBM imaging due to their good biocompatibility and the tailored structure for specific application. But there is also impaired tumor targeting and in situ imaging contrast due to rapid metabolism and poor brain aggregation. With the advantages of prolonged circulation in vivo and easy modification to build theranostic nanoplatforms, nanoprobes are designed with the improved BBB penetrable for high-performance GBM imaging. There are three main types of the BBB penetrable nanoprobes that have been reported: active targeting, 38 passive targeting, 39 and physical means of focused ultrasound (FUS). 40 Here, the research progress of nanoprobes in diagnosis of orthotopic GBM is summarized, including inorganic nanoprobes, rare earth nanoprobes, and other nanoprobes.

Rare earth nanoprobes
Rare earth nanomaterials are typical multichannel luminescent materials, which emit narrow fluorescence under NIR excitation. 41 Rare earth nanoprobes generally exhibit anti-Stokes properties and exhibit relatively hypochromatic-shift fluorescence under suitable light irradiation, thus being called up-conversion nanoparticles (UCNPs). 42 UCNPs are less disturbed by light scattering and background signals of biological tissues, and are ideal imageological materials for deep-tissue imaging. 43 In 2014, Ni et al. constructed Gd-doped and Angiopep-2 (ANG, TFFYGGSRGKRNNFKTEEY) decorated UCNPs (ANG/PEG-UCNPs) for MR/NIR dual-modal diagnosis of GBM. 44 Through the specific ligand of ANG to the lowdensity lipoprotein receptor-associated protein (LRP) in both the BBB and GBM cytomembranes, 45 ANG/PEG-UCNPs can specifically accumulate into GBM tissues. Meanwhile, the integration of Gd ion into the core endows ANG/PEG-UCNPs with T1-weighted imaging properties, making it the first MR/NIR dual-modal UCNPs for GBM imaging. MR/NIR dual-modal imaging has high sensitivity of fluorescence signal and high resolution of MR imaging in deep tissue, and can capture more comprehensive pathological information, which holds the great potential in GBM lesion localization.
Compared with NIR-I (650-900 nm) window, second near-infrared (NIR-II, 1000-1700 nm) fluorescence imaging has the advantages of autofluorescence-free and lower tissue attenuation, and has stronger penetrability for high-resolution imaging of deep tissue. Therefore, luminescent materials with NIR-II fluorescence have emerged as promising tools for imaging orthotopic GBM. To construct NIR-II probes for the detection of orthotopic GBM, Liu et al. achieved a significant fluorescence enhancement at 1340 nm by coating NaLuF 4 on the surface of NaNdF 4 nanoparticles. The particles were then modified with NIR dye IR-808 and PEG-5000 to afford a nanoprobe NaNdF 4 @NaluF 4 /IR-808@DSPE-PEG5000 for detecting the orthotopic GBM ( Figure 3A). 46 Due to the Förster resonance energy transfer (FRET) between IR-808 and Nd 3+ , the nanoprobe exhibited strong NIR-II fluorescence at 1080 and 1340 nm under 808 nm laser excitation. Moreover, with the collaboration of FUS, the nanoprobe was permitted to cross the BBB and delivered to the brain. Notably, the longer channel at 1340 nm showed higher resolution F I G U R E 3 (A) Schematic of construction and energy-transfer mechanism of NaNdF 4 @NaluF 4 /IR-808@DSPE-PEG5000. (B) In vivo NIR-II fluorescence imaging of orthotopic glioblastoma (GBM) tumor-bearing mice with different treatments. Reproduced with permission from Ref. 46 . Copyright 2019, Elsevier. (C) Schematic illustration of T2-weighted MRI images for monitoring tumor growth and in vivo NIR-II imaging of M2-type TAMs for the assessment of GBM. (D) FUS-mediated in vivo NIR-II imaging of orthotopic GBM tumor-bearing mice and dissected brains after intravenous injection of EDB-8.4 NPs and EDBM-8.4 NPs. Reproduced with permission from Ref. 48 Copyright 2022, Wiley-VCH than the channel at 1060 nm when it was used for fluorescence imaging of orthotopic GBM ( Figure 3B). Therefore, this study not only provides a novel NIR-II nanoprobe for high-resolution imaging of deep tumor, but also demonstrates the great potential of FUS technology in delivering nanomedicine.
Infiltrative M2-type tumor-associated macrophages (TAMs) have been reported to predominate in GBM and to be associated with chemoresistance and poor prognosis. 47 To map M2 TAMs in orthotopic GBM, Zhu et al. constructed an Er-based NIR-II nanoprobe (EDBM-8.4NPs) decorated with M2pep peptides (YEQDPWGVKWWY). 48 EDBM-8.4NPs exhibited bright up-conversion emission at 540 nm and down-shifting emission at 1525 nm under 980 nm laser excitation at the lowest power density (40 mW cm −2 ), which was suitable for the identification of M2 TAMs in vitro and in vivo. More importantly, EDBM-8.4 NPs showed significant M2-targeting after the synergic FUS treatment, enabling accurate in situ mapping of M2 TAMs in GBM tissues ( Figure 3C,D). The rare earth nanoprobes with NIR-II optical properties provide an effective means for GBM imaging and show great potential in noninvasive diagnosis of small lesions and assessment of GBM prognosis.

Inorganic nanoprobes
With the advancement of nanotechnology, inorganic nanoparticles such as gold nanoparticles, 49 mesoporous silica nanoparticles, and superparamagnetic iron oxide NPs 50 with large specific surface area and good biocompatibility have been used for establishing bioimaging nanoplatforms. 51 By effectively combining inorganic nanoplatforms and BBB-penetrable groups, nanoprobes for detecting orthotopic GBM have been constructed. In 2019, Guevel et al. constructed ultrasmall Au clusters (AuZwMe 2 , ∼2.4 nm) stabilized by short zwitterionic ligands for NIR fluorescence imaging of subcutaneous and orthotopic GBM. 52 AuZwMe 2 showed a maximum emission at 800 nm, and its ultrasmall size ensures its rapid clearance through urine. Zwitterionic modification improve higher tumor uptake and biodistribution of AuZwMe 2 . Furthermore, zwitterionic-modified AuZwMe 2 exhibited better accumulation and imaging performance than the well-described Au 25 GSH 18 nanoclusters in subcutaneous and orthotopic GBM mice models ( Figure 4A). This work indicates that optimized surface modification of nanoclusters plays a key role in the design of cancer diagnostic and therapeutic tools.
In 2018, Lee et al. constructed SiO 2 -coated iron oxide nanoparticles (NF-SIONs) with good water solubility and stable NIR fluorescence to map TAMs for delineating the entire boundary of GBM. 53 From fluorescence imaging, NF-SIONs showed higher affinity to GBM cells and TAMs than normal parenchymal cells. In both subcutaneous and orthotopic GBM mice models, clear GBM tissues were delineated after intravenous injection of NF-SIONs. This study demonstrated that NF-SIONs can effectively penetrate the BBB due to their good hydrophilicity, which provides an imaging strategy to delineate tumor margins.
Translocation protein (TSPO) is a membrane protein overexpressed in GBM, which has potential as a target to indicate disease status and improve probe delivery. 54 In 2019, Denora et al. successfully constructed GBM-targeted nanoprobes by decorating ultra-small iron oxide nanoparticles (USPIONs) with Cy5.5 and an imidazopyridinebased TSPO ligand. 55 Imaging experiments confirmed the selectivity of the nanoparticles for TSPO-overexpressing cell lines and demonstrated their ability to recognize the receptor in vivo through competitive studies with TSPO ligand PK 11195. In vivo fluorescence images and X-ray multi-species optical imaging systems of U87-MG xenografted tumor models demonstrated their effective visualization of tumor sites.
It has been reported that the chemotherapeutic factors released by brain tumor cells allow microglia to selectively reside around GBM. 56 Based on this theory, Guo et al. coated iron oxide nanoparticles and NIR fluorescent dye DiD with BV2 microglia to obtain engineered microglia DiDBV2-Fe. 57 DiDBV2-Fe showed increased BBB penetration and GBM targeting, becoming an effective optical means to guide GBM diagnosis. In addition, this study indicated that DiDBV2-Fe has a strong tendency for monocyte chemoattractant protein-1 (CCL2) secreted by U87MG tumor cells, leading to a 90% detectable fluorescence signal within encephalic region. Moreover, in vivo NIR imaging of disembodied brains, DiDBV2-Fe demonstrated a more pronounced ability to delineate GBM boundary than the commercial dye 5-ALA.
Each imaging modality has its own advantages and can provide meaningful diagnostic information, but inadequate information sometimes hinders the accuracy of diagnosis. Due to the complementary advantages of multimodal imaging, it has higher precision imaging capabilities. In 2022, Huang et al. constructed an NIRF/MR/photoacoustic tri-modal nanoplatform for accurate GBM diagnosis. 58 Furthermore, in order to solve the poor performance in preoperative diagnosis and intraoperative localization caused by poor targeting of signal target group, the tri-modal nanoplatform was conjugated with EGF and SEC61G for highly contrast imaging of GBM. 59 It was prepared by loading the amphiphilic dye indocyanine green (ICG), EGF ligand, and anti-SEC61G antibody on the surface of hydrophobic MRI agent SPIOs. Due to the simultaneous expression of epidermal growth factor receptor (EGFR) and membrane protein SEC61 translocon subunit gamma (SEC61G), the dual-target probe showed bright fluorescent signals, with a long imaging window and a high tumor-to-background (TBR) ratio. More importantly, the nanoprobes displayed obvious contrast enhancement and delineated clear tumor boundary by MR/MSOT images of orthotopic GBM mice ( Figure 4B). It is envisioned that the tri-modal nanoprobe incorporating dual-targeting property could serve as comprehensive tool for GBM diagnosis in clinical oncology.

2.2.3
Other nanoprobes Other nanomaterials, such as amphiphilic copolymer nanoparticles, have the advantages of good stability, controllable release, and high load capacity. 60 Based on their structural flexibility, they have been extensively explored for GBM targeted imaging. Organic copolymers are modified with proper ligands to produce targeting vehicle, which are then loaded with available fluorescent dyes, drugs, and so on. 61 At present, DNA nanotechnology has emerged as crucial role in biomedical research. Mirkin et al. proved that spherical nucleic acid (SNA) possesses superior properties of higher stability and cellular uptake than free DNA, and the decoration of SNA allowed Au nanoparticles to successfully cross the BBB through cytomembrane-bound scavenger receptors (SRs)-mediated transcytosis. Through the mild click reaction, Xiao et al. constructed an amphipathic DNA block copolymer (PS-b-DNA) with polystyrene (PS) as the hydrophobic module, which is the monomer of SNA. 62 Then the NIR-II dye (IR-FE) was loaded into the hydrophobic core of SNA, followed by decoration of U87MG-specific aptamer through chain hybridization, and finally the nanoprobe FE@PS-b-DNA SNA/Apt (29.2 nm) was obtained. In vivo fluorescence imaging of orthotopic GBM models demonstrated that FE@PS-b-DNA SNA/Apt successfully crossed the BBB, and both the SNA structure and U87MG-specific aptamer modification were helpful for brain tumor imaging and GBM diagnosis ( Figure 4C).

Molecular probes for treatment of GBM
As mentioned earlier, molecular probes have the advantage of structural designability, so fluorescence-guided surgery (FGS) can be realized with molecular probes. In addition, molecule therapeutic probes can be obtained by integrating chemotherapeutic drugs or PDT/PTT agent on fluorophores.

3.1.1
Surgical resection of GBM guided by molecular probe Surgical resection is an important treatment approach in clinical practice. 63 However, due to the characteristics of tumor infiltration, it is a great challenge in clinical surgery to accurately identify and remove all tumor lesions. Complete resection of the GBM tissues with maximal preservation of surrounding normal tissue has a positive impact on patient survival. 64 Fluorophores-based FGS has become a useful tool for detecting tumor location and margins during surgery. 65 The excellent performance of molecular probes can be used to improve the accuracy of malignant GBM resection. Several molecular probes have been successfully applied to guide surgery in living animals.
Hettie et al. investigated the NIR fluorescence imaging probe based on cetuximab-IRDye800 targeting EGFR to obtain visualization between lesion tissues. 66 Recent preclinical data have shown that anti-EGFR antibody therapeutics such as cetuximab can bypass the BBB. 67 Patientderived GBM39 cells orthotopic tumor models showed significantly enhanced signals compared with controls as measured by NIR fluorescence in vivo. Miller et al. first applied this fluorescence-labeled cetuximab-IRDye800 to image human GBM during GBM resection. 68 The TBR of the human GBM tissue (4.0 ± 0.5) injected with this probe was significantly higher than that of the control group (1.2 ± 0.3). This probe proved that antibody-based intraoperative imaging strategy can be used for human GBM resection, while some limitations still need to be addressed, such as small sample size and uncertainty of EGFR expression.
CH1055-4Glu-AE105, a targeted NIR-II fluorescent probe, was synthesized by combining the NIR-II fluorophore CH1055 with the targeting peptide AE105 of urokinase plasminogen activator receptor (uPAR). 69 The AE105 peptide was able to target the overexpressed uPAR on GBM cells, 70 enabling active targeting of the probe to orthotopic GBM. CH1055-4Glu-AE105 can clearly visualize the tumor profile with a TBR as high as 2.7, and NIR-II fluorescence successfully guided almost complete resection of orthotopic GBM in mice. Folate receptor is upregulated in GBM, so it can be a promising target. 71 64 Cu-DOTA-FA-ICG was synthesized by integrating folic acid (FA)-modified ICG dye and 64 Cu-radiolabeled DOTA chelating agent. The probe 64 Cu-DOTA-FA-ICG was also used to guide GBM resection with NIR-II fluorescence. 72 More significantly, 64 Cu-DOTA-FA-ICG also introduces NIR-I fluorescence and PET imaging for co-monitoring and treatment of GBM. Incidentally, GBM-targeted PET and fluorescence imaging can be achieved simultaneously.
After 24 h injection of the probe in the U87 orthotopic xenograft GBM mice, the probe showed the largest tumornormal brain ratio (TNR) of 2.74 for NIR-II and 2.50 for NIR-I. The PET signal appeared 1 h after injection and lasted for at least 48 h. This probe was found to specifically accumulate in an orthotopic GBM model by using PET, NIR-II, and NIR-I fluorescence imaging. Tumor tissue is considered to be completely excised by repeated imaging and resection until no fluorescent tissue remains, demonstrating the feasibility for image-guided surgery of GBM. Overall, this approach has led to further development of strategies to enable image-guided surgery of GBM.

3.1.2
Noninvasive treatment of GBM guided by molecular probe Traditional approaches to GBM tumor resection have some drawbacks. First, it causes great trauma to the body and poor prognosis; second, it is very difficult to completely resect GBM and maintain the integrity of normal tissues, resulting in a high recurrence rate. 73 With the indepth study of GBM treatment, some noninvasive methods have been proposed.
PDT offers a promising, effective, and precise treatment for GBM. 74 RB-1 is a β-gal-activated PDT agent based on an iodinated resorufin core and has shown selective photocytotoxicity against U87 cancer cells. 75 RB-1 possesses a high singlet oxygen ( 1 O 2 ) quantum yield (54%), turns on fluorescence in β-gal overexpressing U87MG cells, and induces late apoptosis/necrosis in most cells upon light irradiation.
In 2020, Li et al. developed iRGD-ILD, another theranostic molecular probe comprising Ga chelated by DTPA as an MRI contrast agent capable of crossing the intact BBB. 76 An additional advantage of this probe is the introduction of ICG moiety, which can generate ROS and heat to exert the PDT/PTT effect. Specifically, 33.3% of ILD+NIRtreated mice survived more than 23 days, while 66.6% of iRGD-ILD+NIR-treated mice survived more than 28 days.
Taking advantages of tumor microenvironment induced self-immolation. Ge et al. utilized overexpressed H 2 S in GBM cells as a potential stimulator for prodrug activation. 77 This H 2 S responsive probe, SNF, consists of amonafide (ANF), a self-immolative linker and an H 2 Strigger group. After being activated by H 2 S in lysosomes, ANF with strong fluorescence is continuously released, and then escapes from lysosomes into the nucleus, causing DNA damage and blocking the cell cycle. In U87MG 3D multicellular tumor spheroids, ANF-treated spheroids became loose after 2 days, while cisplatin-treated spheroids showed no obvious morphological changes. This strategy provides some useful insights into the development of prodrugs for the treatment of GBM.

Nanoprobes for treatment of GBM
Nanoprobes, which have been utilized in the diagnosis of GBM by imaging, have the advantages of good targeting, prolonged retention time, and good accumulation at tumor sites. In addition, nanoprobes have the ability to clearly depict tumor margins through imaging guidance, which have great design potential for image-guided GBM therapy. Meanwhile, nanoprobes have been utilized as carriers to deliver nanomedicines to GBM sites, which show enhanced therapeutic efficacy. Here, imageguided nanoprobes for GBM therapy, including imageguided tumor resection, PDT, PTT, photoimmunotherapy, chemotherapy, multimodality synergistic therapy, and other treatment options, are summarized.

Surgical resection of GBM guided by nanoprobes
To accurately identify tumor margins during surgery, several imaging nanocarriers have been developed to improve the diagnostic accuracy and surgical outcome of GBM foci. In 2018, Yue et al. designed a multimodal GBM transmembrane receptor EGFRvIII-targeted nanoprobe (AuP-FAL) to guide glioma surgery. 78 The nanoprobe AuP-FAL not only successfully delineated the orthotopic GBM boundary via MR imaging, but also accurately guided GBM excisions in ex brain with a hand-held Raman scanner.  79 The nanoprobe used poly (β-L -malic acid) (PMLA) as a platform, and chlorotoxin for high targeting ability against U87MG glioma cells, and is combined with ICG and fluorescence enhancement agent tri-leucine peptide (LLL). NIA achieves deep BBB penetration and high-contrast imaging of GBM after tail vein injection, while tumor resection rate is more than 98% under the guidance of NIR imaging.
In addition, another NIR fluorescent nanomaterial is available for targeted GBM multiforme resection and chemotherapy. It is difficult to observe invasive tumors in general image-guided surgery, and no chemotherapeutic drugs are used to treat the remaining GBM tissue. 80 Reichel et al. reported an NIRF-based fluorescent nanoparticle platform (HMC-FMX), which can visualize tumor boundary to guide surgery and accumulate chemotherapeutic drugs in GBM. 81 These nanoparticles are based on a MRI-sensitive superparamagnetic iron oxide nanoparticle ferumoxytol (FMX), which binds to the NIRF ligand hepthamethine cyanine (HMC) and targets overexpressed organic anion transporter polypeptide (OATP) in GBM. HMC-FMX nanoparticles can penetrate the BBB and selectively gather in tumors in an orthotopic GBM mouse model to treat the GBM tumor and visualize tumor tissue infiltration ( Figure 5A). In this process, encapsulated chemotherapy drugs such as paclitaxel or cisplatin can be efficiently delivered to residual GBM tumors and inhibit tumor growth. These experimental results indicate that HMC-FMX nanoparticles are promising nanoprobes for GBM surgery and chemotherapy.
In 2021, Ren et al. demonstrated the ability of the Erbased lanthanide nanoparticles in NIR-IIb image-guided surgery resection of orthotopic GBM. 82 They proposed an energy cascade down-conversion strategy to dramatically increase the fluorescence intensity of Er-based DCNPs at 1525 nm, and modified the tumor-targeting peptide ANG to obtain optimized nanoparticles (Er-DCNPs-Dye-BP). The nanoparticles were efficiently delivered into the orthotopic GBM with a high TBR ratio (TBR = 12.5) by synergistic FUS processing. In vivo experiments demonstrated that the strong NIR-II fluorescence of nanoprobes could precisely portray the orthotopic GBM diameter (2 mm), and the tumor was successfully resected under the guidance of high-contrast NIR-IIb fluorescence imaging ( Figure 5B,C). To improve imaging contrast and surgical resection effect, Li et al. successfully designed biomimetic lanthanide-doped nanoprobes (CC-LnNPs) to enhance the BBB permeability. 83 CC-LnNPs were prepared by NaYbF 4 :Gd, Er, Ce@NaYF 4 LnNPs as core and U87 cell membrane as shell ( Figure 5D). The probe avoids immune clearance, enhances the accumulation at orthotopic GBM, and guides surgical resection with precise NIR-II imaging. In vivo imaging demonstrated that the brain tumor cell membrane-coated nanoparticles had a marked enhancement of the BBB permeability ( Figure 5E). More importantly, after intravenous injection of CC-LnNPs, the orthoptic GBM with a size of 2.3 mm was accurately identified by NIR-II fluorescence imaging and successfully guided surgical resection of the brain tumor ( Figure 5F).
Zeng et al. reasonably designed an uPAR-targeted phototheranostic nanoprobe (CH4T@MOF-PEG-AE) with MR/NIR-II bimodal imaging for GBM treatment and resection. 84 In vivo studies demonstrated efficient MR/NIR-II bimodal imaging of nanoprobes in subcutaneous GBM mode. Furthermore, after intravenous injection, it was found that the nanoprobes could effectively cross the BBTB, clearly delineate the boundary of orthotopic GBM through real-time intraoperative NIR-II imaging, and accurately guide the surgical resection of GBM.

Treatment of GBM by PDT
PDT as an effective cancer treatment tool is based on photosensitizers upon external irradiation-induced tumor cell death, 85 wherein nanocarrier-based photosensitizers showed negligible toxicity in the absence of external irradiation. In 2020, Teng et al. reported a nanoprobe-coated ICG and Chlorin-e6 (Ce6) on superparamagnetic ferric oxide nanoparticles (SPIONs) to generate nanoclusters ICG-Ce6-SPION (ICS) for PDT of GBM. 86 NIR imaging results demonstrated that the nanoclusters could clearly detect abnormal and intracranial GBM lesions in tumor mice. Importantly, postoperative PDT results showed that at day 23, the volume of recurrent neoplasm was significantly smaller than that of the surgically resected group without PDT, suggesting that nanoclusters can be developed for clinical integration. Fluorescence imaging confirmed that PDT was highly effective in killing GL261 cells and suppressing recurrence in the flanking tumor model. PDT exhibited promising prospects for the therapy of GBM, but the existence of BBB and hypoxic environment in deep tumors impeded its overall therapeutic effect. In 2022, Lv et al. designed diagnostic and theranostic nanoplatforms, which consisted of YOF:Nd 3+ with NIR-II emission as the core, MnO 2 as shell, photosensitizer ICG, glucose oxidase (GOx) and lactoferrin with BBB penetration and targeted capabilities (YOF:Nd 3+ @MnO2-ICG-GOx-LF, YMIGL) for NIR II-guided synergistic starvation, and PDT therapy of orthotopic GBM. 87 GOx can consume glucose to produce H 2 O 2 and gluconic acid to achieve starvation treatment at the tumor sites, 88 and the O 2 concentration in situ was enhanced through the cascade reaction between GOx and MnO 2 to improve therapeutic efficiency of PDT. NIR-II fluorescence imaging of orthotopic GBM revealed that YMIGL can effectively penetrate the BBB and accumulate in the tumor region compared to YMIG without targeted peptide. Furthermore, the inhibitory effect of orthotopic glioma suggests that the generation of O 2 in situ can effectively enhance the therapeutic effect of PDT.

Treatment of GBM by PTT
PTT is a focal therapy of cancer by generating heat to kill cancer cells, which relies on materials with high photothermal conversion efficiency to convert light energy into heat energy under external light source. 89 PTT has been extensively used for GBM therapy due to its noninvasive and effective treatment. 90 In 2018, Wang et al. innovatively constructed a folic acid-targeted and caspase-3activated gold nanostar-based nanoagent (AuNS@probe) for PTT of U87-MG tumor cells. 91 However, theranostics for GBM has only been applied at the cellular level through image-guided apoptosis assay, and further exploration of in vivo therapeutic applications is required. In 2020, Gao et al. designed a novel in situ assembled nanoplatform (R/Au-ICG) based on RGD peptide-modified Bis(DPA-Zn)-RGD and ultrasmall Au-ICG nanoparticles. 92 Bis(DPA-Zn)-RGD can selectively target GBM tumor sites and in situ assembles into nanoclusters R/Au-ICG with subsequently administrated Au-ICG for enhancing imaging contrast and improving inhibition effect. R/Au-ICG-treated orthotopic GBM mice showed enhanced fluorescence/photoacoustic signals compared to single Au-ICG-treated mice. Moreover, with the aid of image-guided PTT treatment, a 93.9% tumor growth inhibition efficiency was observed in the R/Au-ICG-treated group. Furthermore, Wang et al. engineered brain-targeted apolipoprotein E peptide (ApoE)modified AIE nanoparticle (ApoE-Ph NPs) showed excellent PTT therapeutic effect with 57 days of survival for orthotopic GBM guided by NIR-II imaging. 93 To further enhance BBB penetration and improve therapeutic efficacy, it is feasible to modify multiple targeting blocks on nanocarriers. Cai et al. designed and synthesized dual-targeted nanoprobes (FA-cRGD-TNSP NPs) to penetrate BBB and achieve precise NIR image-guided PTT of In addition to receptor-mediated endocytosis mechanism, the camouflage of biocompatible cell membranes can ingeniously escape the immune system into the brain parenchyma. Currently, biomimetic delivery systems coated with different cell membranes have been widely used for imaging and treatment of GBM. In 2019, Lai et al. designed biomimetic nanoprobes (MDINPs) for NIR-Ib image-guided diagnosis and therapy of GBM ( Figure 6A). 95 DSPE-PEG loaded with NIR-Ib fluorophore IR-792 is coated by the macrophage plasma membrane, endowing with the ability to penetrate the BBB. In vitro studies found that U87 cells were decimated through photothermal effects of MDINPs. Moreover, orthoptic GBM in vivo could be clearly observed by NIR-Ib fluorescence imaging ( Figure 6B). The growth of GBM tumors in the MDINP-treated group was effectively inhibited, and the survival time (22 days) was prolonged ( Figure 6C,D). In addition, recent studies have shown that various cancer cell membranes are able to cross the BBB and can be used to construct biomimetic delivery systems. In 2020, Wang et al. made the first attempt to design nanoparticles disguised with the cell membrane of brain metastasis (BMS) for PTT in early brain tumors ( Figure 6E). 96 Using different metastatic tumor cells coated with ICG-loaded polymer nanoparticles, two nanocarriers B16-PCL-ICG and 4T1-PCL-ICG were finally synthesized. Compared with naked nanoparticles and nanoparticles covered with tumor cell membranes, nanoparticles disguised with BMS cell membranes significantly enhanced BBB crossing ( Figure 6F). Moreover, B16-PCL-ICG-treated orthoptic GBM mice with PTT showed significant tumor suppression efficiency.

Treatment of GBM by chemotherapy
Many traditional chemotherapeutics are ineffective in the treatment of glioma due to the low permeability of BBB. 97 Fortunately, the development of nanoprodrugs can overcome these obstacles by taking advantage of the nontoxic and tumor-specific targeting properties of nanoprodrug systems. 98 In 2013, Lee et al. showed a camptothecin (CPT) nanoprodrug that can selectively cross the BBB, prepared from the antioxidant CPT prodrug and α-tocopherol. 99 By imaging U87MG cells, this CPT nanoprodrug penetrates the cell and continuously accumulates the cellular drug in the cell through the enhanced permeability and retention (EPR) effect, which makes the accumulation more efficient than endocytosis, making the tumor and normal tissue more pronounced. As predicted, in U87 MG subcutaneous xenograft tumor mice, the CPT nanoprodrug crosses the BBB and accumulates exclusively in brain tumor tissue to prevent cancer cell proliferation for optimal therapeutic efficacy. Compared with the control group, the CPT nanoprodrug inhibited more than 80% of subcutaneous tumor growth and significantly prolonged the survival time of mice. Doxorubicin (DOX), as a traditional chemotherapeutics, is also limited by the presence of the BBB in the treatment of GBM. 100 To address this issue, Shen et al. prepared nanoprobes SPIO@DSPE-PEG/DOX/ICG NPs, including hydrophobic SPIONPs, DSPE-PEG2000, DOX, and ICG, to achieve BBB penetration and accumulation of probe in C6 glioma-bearing rats. 101 In vivo fluorescence and MR imaging demonstrated that the nanoprobes could not only efficiently cross the BBB but also selectively accumulate at tumor sites. More importantly, it can realize the sustained release of DOX, thereby realizing the theranostics of glioma. It was found that the C6 glioma-bearing rats in the nanoprobe group had the least weight loss, the smallest tumor volume, and the longest survival time, demonstrating the powerful advantages of this probe in treating GBM in vivo.

3.2.5
Treatment of GBM by synergistic therapy PDT, PTT, chemotherapy, surgery, and other therapeutic methods provide strong technical support for the treatment of GBM. In order to achieve a better therapeutic effect, some researchers combine the above two or more methods to achieve the synergistic treatment of GBM, so that multiple treatments methods play a more efficient role to overcome the limitations of conventional therapies.
In recent years, PDT/PTT combined chemotherapy has received considerable attention as a therapeutic approach to treat GBM multiforme by precisely stimulating the release of reactive drugs. In 2019, Kaundal et al. prepared a casein nanoformulation ICG-Gen@CasNPs tagged with ICG and encapsulated with genistein (Gen). 102 Gen can inhibit tumor cell oxidative stress, abrogate angiogenesis, and induce cell apoptosis, while ICG is used in antitumor PDT with NIR emitting. 103,104 The results showed that the nanoprobes can rapidly accumulate in the brain after crossing the BBB, and demonstrated superior antitumor effects on a 3D glioma spheroid model.
In the same year, DOX-loaded ultra-small Cu 2-x Se nanoparticles (CS-D NPs) were synthesized for the treatment of orthotopic malignant GBM under NIR-II imageguided PDT and chemotherapy. 105 In this system, CS NPS produced a large amount of ROS in response to NIR-II irradiation to achieve PDT, while DOX was used for chemotherapy. In orthotopic malignant GBM mice, it was also found that CS-D NPs could be effectively delivered into malignant GBM, inhibit the growth of tumor, and prolong the survival time without tissue damage or inflammatory lesions.
Subsequently, Lu et al. developed another nanoprobe CPT-S-S-PEG-iRGD@IR780 (CPD@IR780) for combination chemotherapy and PDT based on CPT and photosensitizer IR780. 106 IR780 was loaded to a disulfide bondconjugated prodrug polymer composed of CPT and PEG, and iRGD peptide was modified to obtain the nanoprobe CPD@IR780. The drug-loading ratio of CPT in this micelle was up to 11.07%. More notably, after the release of CPT in cells, the photosensitizer IR780 was encapsulated into micelles and subsequently released, realizing the combination of chemotherapy and PDT. In U87 orthotopic glioma-bearing mice, micelles showed more selective aggregation at the glioma site at different times, showing the highest glioma-targeting effect. CPD@IR780-treated mice survived longer than the control group, demonstrating the feasibility of this chemotherapy-PDT synergistic treatment.
Same as PDT, PTT can also be used in combination with chemotherapy drugs for the synergistic treatment of glioma. Lu et al. developed a new multicomponent self-assembled nanocomplex Ang-PEG-g-PLL@CPT-RT@IR783 (APCI) in 2021 ( Figure 7A). 107 Amino acidmodified clinical antitumor drug CPT could self-assemble with IR783, and then angiopep-2-modified PEGylated poly-L -lysine was coated on the two-component nanoparticles through electrostatic interaction to improve the BBB penetration. It was worth noting that APCI exhibits an ultrasensitive pH-responsive behavior ex vivo due to the presence of PEG-g-PLL. In addition, APCI was able to successfully cross the BBB and was enriched at glioma sites with good photothermal capacity ( Figure 7B). APCItreated glioma mouse models had the least weight loss and significantly improved survival rate ( Figure 7C,D). These results suggested that APCI-mediated PTT combined with chemotherapy can significantly inhibit the growth of glioma.
Meanwhile, targeted chemotherapy/PDT/PTT synergistic therapy has shown great potential in the treatment of GBM. Zhang et al. designed a multifunctional phototheranostic agent DTRGD NP, which were composed of octadecane-modified chemotherapy drug temozolomide (TMZ-C18), dicysteamine-modified photosensitizer hypocrellin derivative (DCHB), and GBM-targeting unit cRGD. 108 Benefiting from EPR effect, 109  same design idea and has also achieved good chemotherapy/PDT/PTT therapeutic effect. 110 The platform was composed of inherently fluorescent poly(levodopamine) nanoparticles (FLs) loaded with DOX and ICG. The killing rates of 2D C6 glioma cells and 3D spheres by FLDIP under the NIR laser irradiation were 94% and 87%, respectively, indicating that the effect of chemotherapy combined with PDT/PTT was significantly improved.
In addition to the BBB, blood-tumor barrier (BTB) proposed by Yang et al. is also an important factor in the treatment of aggressive GBM multiforme. A multifunctional liposome system called siRNA and DTX combination delivery system has been proposed to anchor two receptor-specific and penetrable peptides. 111 RNA interference (RNAi)-mediated VEGF silencing is a promising tumor therapy technique that can selectively inhibit the expression of target genes, thereby delaying tumor growth. 112 The combination of siRNA and DTX delivery system realizes the combination of gene therapy and chemotherapy for GBM by inhibiting angiogenesis and synergistic killing of tumor cells. In this system, NIR imaging of U87MG GBM-bearing mice demonstrated that DiD-labeled dual peptides-modified liposomes (At-Lp) could enter GBM through the BBB and BTB. This combination therapeutic system has significant advantages in prolonging survival time, improving anti-angiogenesis and apoptosis effects, and even regulating the GBM multiforme microenvironment.
Zhang et al. also synthesized nanoparticles (BK@AIE NPs) that selectively penetrate the BTB. 113 [des-Arg[9]]bradykinin was used to modify AIE-active luminescent gens (AIEgens), which act as NIR-II photothermal therapeutic agents, 114 to obtain BK@AIE NPs, which can carry out PTT and local immune response activation in the synergistic treatment of brain tumors ( Figure 7D). The BK ligand actively targeted GBM to achieve the aggregation of BK@AIE NPs in GBM. After spatiotemporal PTT, tumor growth was effectively inhibited and the immune response was activated. PTT stimulates the activation of T lymphocytes and natural killer cells (NK), and promotes the differentiation of M0 macrophages into M1 macrophages (MΦ), thereby killing tumor cells and achieving PTT and local immune therapy. As expected, mean tumor signal intensity increased almost three-fold after 24 h in the BK@AIE NPs-treated group ( Figure 7E,F), and tumor growth could be remarkably inhibited in U87-MG-glioma-bearing mice for 18 days.
Li et al. has also proposed a dual-targeted nanoradiotherapy for GBM photoimmunotherapy. 115 The NIR-II fluorophore MRP, self-assembled from drug precursor JQ1 and tumor-targeting T7 ligand-modified PEG5k-DSPE, can trigger the in situ release of tumor antigens under 808 nm laser, generates antitumor immunogenicity, and recruits tumor-infiltrating cytotoxic T lymphocytes. Multifunctional TNP@JQ1/MRP nanoparticles successfully crossed the BBB and accumulated at brain, showing a stronger NIR-II fluorescence signal at the tumor site of G422-Luc tumor-bearing mice. After 36 h injection at TNP@JQ1/MRP nanoparticles group, the tumor growth rate was delayed, and the survival time of mice was significantly prolonged. This method provided a new idea for the precise photoimmunotherapy of GBM.

3.2.6
Treatment of GBM by other therapy methods In addition to the most used PTT, PDT, and chemotherapy, some other effective treatments have also been reported, such as RNA interference (RNAi), sonodynamic therapy (SDT), gas therapy, and molecular inhibitor.
Researchers have expanded the design of biomimetic nanoprobes to treat GBM. RNAi therapy, a means of specifically inhibiting tumor growth by silencing oncogene expression, has the advantages of high efficacy and minimal toxicity. 116,117 Su et al. constructed a core-shell biomimetic nanoprobe (TMPsM) for RNAi therapy of GBM, which was composed of hollow MnO 2 carrying Pep-TPE and siRNA as the core, and TfR-aptamer-modified B16F10 cell membrane as the shell. 118 They proved that TMPsM exhibited the ability of TG2 gene silencing to induce apoptosis in vitro, 119 TG2 stimulated self-assembled AIE imaging to accurately diagnose GBM in vivo, and the released siRNA effectively silenced TG2 expression to induce apoptosis, inhibiting the development of orthotopic U87MG-luciferase tumor. For the first time, TMPsM achieved accurate diagnosis and effective treatment of GBM through TG2-triggered self-assembly imaging and RNAi therapy.
He et al. designed pH-sensitive red blood cell membrane-coated nanomaterials AM@NP(ABT/A12) modified with ApoE peptide to penetrate the BBB and deliver ABT and A12 to the brain for GBM therapy. 120 Small molecule inhibitor ABT promotes cell apoptosis, 121 and drug-resistant molecule Mcl-1 inhibitor A12 synergistically enhances the therapeutic effect of ABT. In addition, they demonstrated that the combination of ABT and A12 not only inhibited the activity of U87MG cells, drug-resistant U251-TR cells and patient-derived GBM stem cells (CSC-2) in vitro, but also effectively inhibited the growth of orthoptic glioma in vivo.
SDT is a promising method for tumor treatment. 122 Based on the production of ROS by sonosensitizer under ultrasound stimulation, SDT has the characteristics of deep tissue penetration, noninvasive, and nonradiation. 123 In 2022, Lv et al. established Nd 3+ -doped nanoparticles (YVO4:Nd 3+ -HMME@MnO 2 -LF, YHM), which were surface-modified by the targeting peptide lactoferrin (LF) and sonosensitizer hematoporphyrinmonomethyl ether (HMME) for NIR-II/MR imaging and sonodynamic therapy of orthotopic GBM. 124 The MnO 2 shell catalyzed the production of O 2 in the tumor environment and enhanced the therapeutic effect of SDT. The results demonstrated that YHM could effectively inhibit the growth of orthotopic glioma with no obvious weight change in vivo.
SO 2 is a messenger molecule of physiological process, 125 which is expected to treat tumors by inducing oxidative stress and inhibiting protein expression. 126 In 2022, Liu et al. prepared dye-sensitized up/down-conversion luminescence NaYf4:Yb/Tm@NaYF4:Nd nanoparticles for NIR-II imaging and SO 2 gas therapy of orthotopic GBM. 127 The ultraviolet up-conversion emission of nanoparticles was utilized to promote the release of SO 2 gas from the prodrug for gas therapy. By downregulating the expression of p62, the LC3-II/LC3-I ratio was upregulated, finally inducing cell apoptosis, promoting tumor cell death, and inhibiting tumor growth.

DISCUSSIONS AND PERSPECTIVES
Image-guided diagnostic and therapeutic probes for GBM have made remarkable achievements in accurate imaging detection, effective treatment, and tracking of therapeutic effects. Different types of imaging probes have become potential tools to promote the early diagnosis of GBM and provide important functional information. Due to its unique advantages, the superior BBB cross-integrated platform for diagnosis and therapy have exhibited encouraging results in preclinical animal trials, especially in the integrated development of noninvasive diagnosis and treatment for GBM and in situ monitoring curative progress.
Although the image-guided diagnosis and treatment of GBM have made good experimental progress, it is still necessary to further improve the efficacy of imaging diagnostic reagents in the following aspects in order to achieve practical clinical applications: 1. Development of imaging probes for clinical application. Although optical methods have been used for the accurate diagnosis of GBM, MR, a commonly used clinical detection method, has high accuracy and excellent performance in identifying small lesions. For example, MR image-guided GBM diagnosis has recently been reported, with MR agents based on superparamagnetic iron oxide particles 128 and paramagnetic organic PROXYL radicals 129 staying longer at the tumor site.
In addition, PET, as an advanced clinical examination imaging technology in the field of nuclear medicine, is also of great significance for the development of effective GBM imaging platforms. The diagnosis of GBM under the guidance of MR or PET imaging has practical significance for the identification of early GBM and the discovery of small lesions, and also has more clinical transformation value. 2. Development of multimodal imaging platform capable of accurate diagnosis and guidance. Some monomodal imaging carriers have the advantages of high sensitivity, noninvasive and real-time imaging. They provide a great opportunity for the diagnosis of GBM, and facilitate intraoperative real-time guidance to differentiation of malignant cells from healthy tissue. However, the dimension and depth of bioinformation of GBM nidus obtained from a single imaging is still limited. Therefore, it is essential to obtain comprehensive pathophysiological information in combination with other complementary imaging technologies, such as PA with the merits of optical imaging and ultrasound imaging, MRI with no penetration limitation, NIR II imaging with low background interference, and so forth. Advanced multimodal imaging probes can obtain comprehensive lesion information through integrated analysis of different signals, which contributes to further strengthening the research and indepth exploration of the early diagnosis of ultrasmall GBM tumors. 3. Construction of highly targeted and biosafety diagnosis and therapy platforms. Although existing approaches have enabled the effective delivery of imaging agents and therapeutic drugs in GBM, the untargeted accumulation of theranostic agents in other major organs remains a challenging problem. In addition, some metal-based nanocarriers, such as iron oxide, copper oxide, and carbon nanomaterials, may also cause toxicological reactions to some extent. More attempts can be made to camouflage the carrier with biocompatible cell membrane, such as red blood cells, macrophages, and diverse GBM cell membranes. Moreover, the multifunctional platforms modified by multiple brain tumor target ligands may contribute to highly selective accumulation of tumor lesions while minimizing off-target healthy organs and cranial nerve.
In summary, although imaging and therapeutic platforms have made tremendous progress in understanding of GBM malignancies, the application of molecular probes and nanotechnology to diagnose and treat brain cancer is still at the stage of basic research. There are still many obstacles that need to be addressed in practical clinical translation by academics and clinicians, such as toxicity assessment and biodistribution analysis. With the rapid development in the design and technologies of diagnostic and therapeutic approaches, multiple image-guided diagnosis and treatment probes are expected to promote further study on the biological behavior of GBM at the early stage, which has significant impact on improving patients' quality of life.