Blood-Brain Barrier Conquest in Glioblastoma Nanomedicine: Strategies, Clinical Advances, and Emerging Challenges

1. Introduction

Glioblastoma (GBM) is the most prevalent malignant primary brain tumor (accounting for 13% of all tumors and 50.1% of all malignant tumors) with a 5-year survival rate of less than 7% [1,2]. Despite progress in multimodal treatments encompassing surgery, radiotherapy, chemotherapy, and targeted therapy, the overall prognosis remains unfavorable, with a median survival duration of merely 8 months [1]. Figure 1 illustrates seminal milestones in GBM research and treatment. In 2021, GBM, classified as IDH-wildtype and defined as a WHO grade 4, specifically refers to diffusely infiltrative gliomas exhibiting classic histopathological features or molecular alterations [3]. Due to its heterogeneity, invasiveness, and difficulties in defining malignant tissue margins, compared to other solid tumors, along with the BBB, leading to rapid disease progression in up to 70% of patients within one year of diagnosis. Chemotherapy is one of the standardized steps in GBM treatment [4]. However, non-targeted administration often leads to low efficacy and systemic toxicity, while the presence of BBB demands higher dosages from chemotherapeutic drugs. Developing brain-targeting drugs or formulations that can cross this barrier are urgently needed for effective treatment while minimizing toxic side effects.

The blood-brain barrier (BBB) is a natural defense mechanism between the bloodstream and brain tissue. The intact BBB effectively segregates brain tissue from peripheral blood and plays a pivotal biological role in upholding the fundamental stability of the internal environment of brain tissue and the normal physiological state of the central nervous system (CNS) (Figure 2a). The BBB restricts nearly all large molecules and over 98% of small molecule candidate drugs from permeating into it [5,6,7]. Therefore, the key issue in drug therapy for GBM is to design and develop targeted nanomedicine delivery systems (NDDS) that can effectively cross the BBB and ensure the drugs reach their intended sites in the brain. Two main approaches to delivering chemotherapy drugs to GBM involve either crossing or bypassing the BBB (Figure 2b).

Biomaterials, encompassing both natural and synthetic sources, are materials that possess excellent biocompatibility and physicochemical properties, utilized in drug delivery. These materials can either be directly conjugated with drugs to enhance their pharmacokinetic properties or serve as excipients, leveraging nanotechnology to encapsulate drugs within nanocarriers (10-500nm) or attach/adsorb them onto the carrier surface, thereby forming NDDS that achieve sustained release, controlled release and targeted delivery of drugs [8]. The development of NDDS represents a promising avenue for GBM treatment, albeit not without its challenges. A significant bottleneck lies in the transition from preclinical research to clinical trials. The heterogeneity of GBM presents a challenge as it may lead to varied therapeutic responses. Moreover, the BBB further complicates drug delivery. Despite notable advancements made by NDDS in overcoming the BBB, translating these preclinical successes into clinical practice has been hindered by issues related to scalability, regulatory pathways, and clinical validation.

Recent reviews have delved into the application of Nanomedicine in brain tumor pharmacotherapy, highlighting the significance of nanotechnology in overcoming the BBB [9,10,11]. Nevertheless, amidst the heterogeneity of GBM and the constraints imposed by the BBB on drug delivery to tumor sites, the clinical advancements of nano-drug delivery systems remain largely uncharted territories. This review aims to systematically classify strategies for crossing and bypassing the BBB, highlight the key research accomplishments from the past 5 years using these systems, and discuss the strengths and weaknesses of different BBB-overcoming strategies. Finally, we offer our perspectives on the future developments and challenges in this field, aiming to provide more comprehensive and in-depth guidance for the practical application of nanomedicine in the treatment of GBM.

2. Strategies for NDDS to Cross the BBB

The BBB is a formidable obstacle for drug delivery to the brain due to its tightly regulated selective permeability. However, several strategies have been developed to enhance the delivery of nanomedicines across the BBB (Table 1).

2.1. Receptor-Mediated Transport

This is a pivotal approach in the nanomedicine delivery system for crossing the BBB, as it exploits the natural transport mechanisms of the brain's protective interface. The existence of nutrient transport receptors on BBB endothelial cell membranes is crucial for sustaining normal brain function in humans, with some overexpressed on GBM cell membranes. A tailored nanomedicine delivery system enhances BBB permeability and GBM cell targeting by binding to these receptors[43] (Figure 3).

2.1.1. Transferrin Receptors and Lactoferrin Receptors

The overexpression of transferrin receptors (Tf) and lactoferrin receptors (Lf) on BBB endothelial cells and glioma cells presents an opportunity for targeted drug delivery. Tf-functionalized nanoparticles (Tf-NPs) crossing BBB and targeting GBM in intracranial orthotopic models, reducing tumor burden and extending survival [12]. Lf, an iron-binding glycoprotein, surpasses Tf and OX-26 in BBB permeation [44]. Dong et al. developed a glioma-targeted drug delivery system utilizing biodegradable periodic mesoporous organosilica nanoparticles modified with Lf ligands, enhancing drug delivery to brain gliomas [13]. Taskeen et al. introduced an Lf receptor-mediated nanomedicine, USLP-NH₂-PEG-TMZ-Lf, with enhanced BBB permeability and cytotoxicity for GBM treatment [14].

2.1.2. Acetylcholine Receptors

Nicotinic acetylcholine receptors are widely distributed on the surface of the BBB, serving as a crucial target for drug delivery. 2-methacryloyloxyethyl phosphorylcholine (MPC)-based protein nanocapsules enhance CNS protein transport. Chen et al. engineered an NDDS containing TMZ (TMZ@RVG-Zein NPs), penetrating BBB via receptor-mediated endocytosis. This NDDS exhibits exceptional biocompatibility and is capable of penetrating into GBM (U87) cell lines, thereby facilitating the release of TMZ for therapeutic effect [15].

2.1.3. Folate Receptors

BBB and glioma cells express folate receptors (FR), crucial for tumor DNA replication. Folate-modified NPs are internalized by tumor FR via receptor-mediated endocytosis [45]. Xiang Yang Zhong et al. engineered folate/iRGD-modified NPs, enhancing targeting and uptake, delivering TMZ to nuclei, inhibiting tumor proliferation. FR can integrate with other drugs for novel delivery systems, e.g., WGA/FA-MPEG-PCL NPs loaded with ETO, BCNU, DOX, targeting BBB to inhibit GBM growth [16].

2.1.4. Low-Density Lipoprotein Receptors

Members of the LDL receptor superfamily exhibit binding affinity towards a diverse range of ligands, including lipoproteins, proteases, and protease inhibitor complexes, facilitating their transport into the cell nucleus. Specifically, apolipoprotein E demonstrates selective binding to LDL receptors, while solid lipid nanoparticles functionalized with apolipoprotein E have been demonstrated to enhance BBB delivery by 1.5 times compared to non-functionalized nanoparticles [46]. Jiantang Liang and colleagues designed the versatile biomimetic nanoplatform L-D-I/NPs to selectively target GBM by binding to low density lipoprotein receptacle-associated protein-1 (LRP1) and crossing the BBB. Effectively inhibited the progression of in orthotopic GBM and significantly prolonged survival [47]. Angiopep-2 modified nanoparticles selectively target LRP1. Compared with the control group，intravenous injection of Ti@FeAu-Ang nanoparticles resulted in a 10-fold reduction in tumor volume [17]. The Au-DOX@PO-ANG NPs designed by Chen He and colleagues also significantly reduced the size of GBM in mice[18].

2.1.5. Epidermal Growth Factor Receptors

Epidermal growth factor receptors (EGFR), an ErbB family tyrosine kinase receptor, governs GBM angiogenesis and determines high proliferation and drug resistance. EGFR-targeted drugs like Cetuximab, panitumumab, nimotuzumab, and necitumumab have been clinically approved [48]. EGFR overexpression occurs in 40-70% GBM patients, with EGFRvIII, a cancer-specific deletion, present in 25-50% pleomorphic GBMs [48]. EGFRvIII's absence in normal tissues makes it a prime target for GBM-specific receptor therapies. The antitumor efficacy of Panitumumab-conjugated and TMZ-loaded poly (lactic-co-glycolic acid) nanoparticles (PmAb-TMZ-PLGA-NPs) in GBM cells with overexpressed EGFR is significantly enhanced through the inhibition of caspase-mediated autophagy by this novel NDDS, thereby promoting apoptotic cell death [19].

2.1.6. Human Insulin-like Growth Factor-1 Receptors

Insulin-like growth factor-1 receptors (IGF1R), a receptor tyrosine kinase, is prominently expressed in brain endothelial cells, brain microvessels, and specific neuronal regions. It mediates endocytosis upon high-affinity binding with IGF1, making it a suitable target for brain drug delivery [20,49]. High-affinity anti-IGF1R monoclonal antibodies penetrate the BBB after in situ brain perfusion (ISBP) administration [20]. Additionally, IGF1R5, effectively binds to IGF1R on the BBB, enhancing drug transport [49].

2.1.7. Integrins

The αvβ3/αvβ5 integrins are upregulated in angiogenic sites and GBM, underpinning the reliance on their BBB endothelial and GBM cell expression for GBM-targeted drug delivery systems [50]. Studies confirm that cyclic-Arg-Gly-Asp (cRGD)-installed micelles enhance the targeted delivery and therapeutic efficacy of epirubicin in orthotopic GBM models [51]. Additionally, α5β1 integrin receptor overexpression in GBM cells facilitates the use of RGDk-lipid nanoparticles for concurrent delivery of chemotherapeutics and siRNA, significantly inhibiting tumor growth in mouse models [21]. These discoveries underscore the efficacy of integrin-mediated carriers for drug delivery in aggressive GBM.

2.1.8. CD13

CD13 is widely overexpressed on glioma neovasculature endothelial cells and glioma cell surfaces [52]. Asn-Gly-Asp (NGR), a structural motif for endothelial cells and tumor neovasculature, functions as an effective tumor-targeting drug carrier. Circular iNGR in the blood can specifically and rapidly bind to CD13, demonstrating strong tumor vascular targeting ability [53]. Sai An et al. engineered PEGylated iNGR-modified RNAi nanoparticles that exhibit superior tumor accumulation and penetration, offering a novel avenue for GBM's targeted therapy [52].

2.1.9. Neuropilin-1

Neuropilin-1 (NRP-1), a transmembrane glycoprotein, is overexpressed in GBM cells and serves as a co-receptor for semaphorin3A and vascular endothelial growth factor, contributing significantly to tumor angiogenesis, growth, and metastasis [54]. The CPT-S-S-PEG-iRGD@IR780 nanoparticles were modified by iRGD peptide and photosensitizer IR780, which can be used as drug delivery system. Leveraging αvβ integrin and NRP-1-mediated transport, this system efficiently traverses the BBB to target GBM, enhancing anti-tumor efficacy in combined therapies [22].

2.1.10. Heat Shock Protein 70

Heat shock protein 70 (Hsp70) is selectively expressed on GBM cell membranes, serves as a precise target for GBM therapy, enhancing binding efficiency [23]. Acid-triggered gold nanoparticles (D-A-DA/TPP) selectively deliver DOX to glioma tissue by Hsp70 targeting, with TPP (a peptide that binds to Hsp70 on the membrane of glioma cells) facilitating cellular uptake. Under the weakly acidic tumor microenvironment, D-A-DA/TPP aggregation prolongs circulation, augments binding, and triggers pH-responsive DOX release [23]. Jianfen Zhou et al. devised pHA-AOHX-VAP-DOX, a nanodrug system traversing the BBB via dopamine and GRP78 receptors, a heat shock protein family member, to extend survival in nude mice with intracranial U87 glioma [24].

These examples underscore the versatility and potential of RMT in nanomedicine, particularly for GBM therapy. By binding to specific receptors overexpressed on the BBB and tumor cells, nanoparticles can be guided to their target, enhancing drug delivery efficiency while minimizing systemic side effects. The continued exploration and refinement of RMT strategies hold great promise for improving the prognosis and treatment of brain-related diseases.

2.2. Transporter-Mediated Transport

Membrane transport proteins in neurovascular units, comprising 10-15% of their structure [55], are essential for transmembrane movement of diverse substrates, including small water-soluble molecules. These proteins, categorized into four types based on their mechanisms, undergo conformational changes to facilitate transport and can be targeted by nanoparticles for drug delivery [56]. Their strategic distribution and unique mechanisms on brain capillary endothelial cells (BCECs) enhance the targeting and uptake of nanomedicines in brain tissue (Figure 4).

2.2.1. Glucose Transporters

Glucose is the brain's primary energy source, with glucose transporter-1 (GLUT1) being the predominant transporter on BCEC membranes, facilitating its rapid translocation into brain tissue [57]. Leveraging post-fasting hyperglycemia, nanocarriers can enhance drug delivery across the BBB via GLUT1 [58]. Glycosylated derivatives of heptapeptide ATWLPPR (A7R) have shown improved serum stability and enhanced BCEC uptake via GLUT1-mediated transcytosis, thereby improving BBB penetration and glioma cell absorption [59]. Zhang et al. developed a 2-DG nanocapsule system that exploits GLUT1 overexpression for targeted delivery to the GBM tumor microenvironment [25].

2.2.2. Choline Transporters

The choline transporters (ChTs) protein, prominently expressed on cerebral capillary endothelial cell lumens, efficiently traverses the BBB to transport acetylcholine and choline analogs into the CNS [60]. Li's team engineered a cholinergic-derivative-modified delivery system that significantly increased glioma drug accumulation, inducing apoptosis and enhancing therapeutic outcomes [61]. MPC-n (IVIg) utilizes high-affinity ChT1 overexpression for targeted brain tissue drug delivery, reducing therapeutic doses [62]. Hairong Wang's team devised a universal BBB-permeable smart polymer, pMPC-co-(anti-PD-L1-pPEGMA), that crosses the BBB via choline transporters to effectively treat malignant gliomas [26].

2.2.3. Amino Acid Transporters

L-type amino acid transporter 1 (LAT1) is overexpressed in both BBB and GBM cells [63], facilitating targeted brain drug delivery and minimizing peripheral exposure [64]. MeHg-L-cys harnesses LAT1 to enhance malignant glioma cell targeting and mitigate normal brain tissue toxicity [65]. Amphi-DOPA-loaded wp1066 nano-carriers significantly improved survival in orthotopic mouse GBM models [27].

2.2.4. Vitamin Transporters

The sodium-dependent vitamin C transporter (SVCT2), expressed in choroid plexus epithelial cells and brain tumor cell lines, emerges as a promising nanomedicine target [66]. Ascorbic acid's (AA) CNS penetration, facilitated by its reversible oxidation to dehydroascorbic acid (DHAA), leverages the SVCT2-mediated transport mechanism, enhancing DHAA CNS levels and offering a strategy for drug delivery systems with potential in Alzheimer's therapy [66,67]. Yao Peng's research introduced a glucose-vitamin C modified liposome for paclitaxel delivery to the brain, demonstrating superior targeting efficacy over unmodified or singly modified formulations [28].

2.2.5. Organic Cation Transporters

Organic cation/carnitine transporter 2 (OCTN2, SLC22A5), a member of the OCTN family, is implicated in drug BBB permeation [68] and is upregulated in GBM [69]. Kou's team engineered L-Carnitine-conjugated nanoparticles (LC-PLGA NPs) that exploit OCTN2 overexpression on brain endothelial and glioma cells for enhanced BBB permeability and targeted glioma cell internalization [29].

2.2.6. Organic Anion Transporters

Organic anion-transporting polypeptides (OATPs), a family of multi-specific transporters, include OATP1A2, which facilitates CNS drug uptake at the human BBB [70]. Statins illustrate this by crossing the BBB via the Oatp1a4 transporter, underscoring OATPs' utility in targeted CNS drug delivery. Brain-specific anion transporter 1 (BSAT1), exclusively present in brain microvascular endothelial cells, is pivotal in targeted drug delivery within tumors and their surrounding areas. Research demonstrates platinum-nanogel conjugates with Cx43 and BSAT1 antibodies effectively shrink tumors [30]. Given OATP substrate selectivity, current research prioritizes stroke therapy, with glioma treatment often considered a secondary or dual-targeted approach.

2.2.7. Monocarboxylate Transporters

Monocarboxylate transporter (MCT) family members mediate the cellular translocation of monocarboxylic acids like lactate and pyruvate across various tissues. MCT1-4, integral to the plasma membrane, facilitate the bidirectional exchange of short-chain monocarboxylic acids and protons in mammalian cells [71]. Glioma cells uptake β-Hydroxy-β-methylbutyrate through H+-coupled MCTs [71], and the overexpression of MCT1 and MCT4 on their surfaces correlates with poor prognosis, indicating their diagnostic and therapeutic potential [72]. Huber's research demonstrated a link between MCTs and the brain penetration of cyclic C5-curcuminoids, emphasizing their role in BBB traversal [73].

2.3. Adsorptive-Mediated Transport

Nanocarrier size and surface charge are pivotal for BBB endocytosis, with cationic systems like PEG enhancing drug solubility and cellular uptake through electrostatic interactions with the endothelial cell membrane [74]. Tumor blood vessels' and cells' upregulation of negatively charged glycoproteins increases nanocarrier accumulation in brain tumors [75] (Figure 5). While common transport receptors like albumin and cell-penetrating peptides (CPPs) improve brain penetration [76], the lack of selectivity in electrostatic targeting limits its systemic drug administration use, necessitating combination with specific ligands for enhanced targeting.

2.3.1. Cationic Albumin

Electrostatic interactions between cationic and anionic microdomains on BBB endothelial membranes initiate AMT, facilitating brain drug delivery, particularly for macromolecules [77]. Cationized albumin (pI>8) accumulates in tumor cells via AMT [75], can be integrated with anti-glioma drugs into a nano-drug delivery system to induce tumor cell death and retard growth [78]. Cationized immunoglobulin, monoclonal antibodies, and histones possess brain-targeting properties through similar mechanisms. Given AMT's non-selectivity, it is often combined with other approaches to enhance BBB permeation of nanoborne drug systems [79].

2.3.2. Cell-Penetrating Peptides

CPPs encompass natural proteins (e.g., TAT, penetration peptide, Syn-B carrier) and synthetic cationic, highly hydrophilic proteins like polyarginine peptides [80]. Coupling CPPs with nanobodies enhances brain penetration [76]. TAT exhibits BBB-targeting potential, with brain targeting efficacy correlating positively with its bound ligand's positive charge [81]. TAT-modified gold NPs outperform free doxorubicin in BBB traversal, GBM targeting, and circulation time [31]. However, CPPs' non-specific electrostatic membrane interactions lack cell selectivity, elevating side effects. Researchers have devised dual/multi-ligand systems by conjugating CPPs with specific targeting ligands to optimize BBB traversal, impart cell selectivity, and enhance drug delivery efficiency [31,82].

2.4. Cell-Mediated Transport

The unique structures, mechanical properties, and surface ligands of human cells dictate their diverse physiological functions, fostering the development of cell-based targeted drug delivery systems [83,84]. Cells serve as drug delivery vectors, encapsulating or attaching drugs and utilizing their homing mechanisms for efficient targeted delivery [85]. Promising candidates include erythrocytes, leukocytes, and stem cells. Furthermore, synthetic nano-drug delivery systems utilize native cell membranes to enhance BBB penetration and active targeting, broadening the range of cells for drug delivery applications [83].

2.4.1. Erythrocytes

Erythrocytes, owing to their abundance, distinctive biconcave morphology, and prolonged circulation lifespan of 110-120 days in circulation, have garnered attention as promising nanodrug carriers [86]. Their structural characteristics, notably the biconcave shape and absence of a nucleus, enhance drug encapsulation efficiency by up to 67% [87]. However, direct drug integration into or onto erythrocyte membranes can inflict irreparable damage, hastening clearance by the reticuloendothelial system and diminishing circulatory persistence [86]. To circumvent this, CPPs are utilized for drug loading, safeguarding membrane integrity and function [88]. Despite these merits, erythrocytes as drug carriers encounter limitations such as uncontrolled drug release and absence of specific targeting receptors. To address these, red blood cell (RBC)-NPs, nanoparticles coated with RBC membranes (RBCms), combine membrane functionalities with nanoparticle physicochemical properties, enhancing drug-loading capacity, stability, biocompatibility, and prolonged retention [89]. Furthermore, RBC-NPs exhibit refined BBB penetration and tumor-targeting abilities, facilitated by surface- modified ligands [90]. The novel nano-drug Ang-RBCm@NM-(Dox/Lex), functionalized with the surface of Angiopep-2-modified RBCms, exhibits prolonged circulation time, superior BBB penetration, and enhanced tumor accumulation, effectively suppressing tumor growth and significantly extending the median survival time of orthotopic U87MG human GBM tumor-bearing nude mice [33]. Dong Luo and colleagues developed a NDDS using the tumor-penetrating peptide iRGD (CRGDK/RGPD/EC) derived from the RBC membrane as a carrier to overcome the BBB, enhance drug targeting, and increase the 30-day survival rate from 0% to 100% [34]. Mingming Song et al. devised LMP RFA NPs, a biomimetic nanodrug delivery system for targeted GBM therapy. This system encapsulates lomitapide (LMP)-loaded tetrahedral DNA nanocages within a folate-modified erythrocyte-cancer cell-macrophage hybrid membrane (FRUR) shell, demonstrating high BBB permeability, precise tumor targeting, low side effects, and extended survival in tumor-bearing mice [32].

2.4.2. Leukocytes

Leukocytes migrate to disease sites, traverse the BBB, and penetrate hypoxic tumor regions, facilitating targeted drug delivery to challenging areas [84]. These cells, including T cells, neutrophils, monocytes/macrophages, and dendritic cells, exhibit innate affinity towards inflammation and are recruited to lesion sites by inflammatory factors[91].

Chimeric antigen receptor (CAR)-T cells are genetically modified autologous T cells that can be employed for GBM-targeted drug delivery [92,93]. Despite genetic modification of T cells, limited transport across the BBB remains one of the primary challenges CAR-T cell therapy encounters. Focused ultrasound (FUS) has been utilized to open the BBB and enhanced survival rates by 129% compared to CAR-T cell therapy alone [94]. Gloria B. Kim and her team have developed a combined selective NDDS that utilizes high-affinity TQM-13 CAR T cells as drug carriers, integrating with nanoparticles and DOX to enhance drug bioavailability while mitigating systemic toxicity [35]. Overall, T cells remain a very promising delivery vehicle for GBM treatment.

Neutrophils (NEs), the predominant immune cells with rapid responsiveness to inflammatory stimuli, are recruited to lesion sites upon activation and exhibit the ability to traverse the BBB, accessing inflamed brain tumor tissues [36,89]. A recent study harnessed this property by formulating a NEs-exosomes (NEs-Exos) system for GBM therapy, enabling the effective loading and intravenous delivery of DOX, significantly suppressed tumor growth and prolonged survival [36]. Inspired by CAR-T cell therapy, researchers have engineered CAR-neutrophils to specifically deliver tumor microenvironment-responsive nanodrugs to GBM, combining chemotherapy with immunotherapy to minimize off-target effects and extend the lifespan of tumor-bearing mice [37]. Utilizing neutrophil membrane as a biomimetic drug delivery system interacts with adherent proteins to target endothelial cell membranes within the BBB for crossing [95].

Monocytes/macrophages, recruited to lesion sites by chemotactic factors [89], exhibit robust migratory capacity towards GBM, rendering them ideal carriers for cell-mediated brain drug delivery [96]. Macrophage-derived delivery systems are tumor-targeted, releasing therapeutics for efficacy [89]. Inflammatory macrophage membranes enhance targeting, and their integration with nanoparticles augments BBB traversal and brain-targeting [97]. Xiao et al. achieved prolonged circulation, improved BBB penetration, and enhanced chemo/chemodynamic therapeutic efficacy for orthotopic C6 glioma in a mouse model through coating hybrid nanogels with macrophage membranes [38] (Figure 6).

Dendritic cells, specialized antigen-presenting cells, stimulate and regulate innate and adaptive immune responses, crucial for cytotoxic T cell activation and proliferation. Dendritic cell membrane proteins facilitate BBB traversal. Xiaoyue Ma et al. devised aDCM@PLGA/RAPA, a nano-platform coated with dendritic cell membranes, to effectively traverse the BBB, promoting tumor immune response and synergistically augmenting GBM eradication in conjunction with rapamycin (RAPA) [39].

Despite their potent anti-tumor and immune regulatory capabilities, natural killer (NK) cells have been underutilized as carriers in research. NK cells inherently possess direct cytotoxic effects, acting as effective "anti-tumor agents" [84]. They can induce target cell apoptosis by releasing perforin and granzymes, which may cause explosive release of tumor drugs and hinder the penetration of tumor drugs.

2.4.3. Stem Cells

Stem cells, such as mesenchymal stem cells (MSCs), neural stem cells (NSCs) and adipose-derived stem cells (ADSCs), are ideal carriers for glioma drug delivery due to their self-renewal, low immunogenicity and easy differentiation. These stem cells can penetrate the BBB, migrate to tumors, and are used to treat hypoxia, necrosis, inflammatory tissue and glioma, and even CNS diseases and other tumors [84,89]. MSCs and ADSCs, with their immunosuppressive properties, can be engineered to deliver therapeutic agents to tumors. Viral transfection of stem cells has been shown to induce immunoactive cytokine expression, thereby enhancing mouse survival rates [98]. ADSCs deliver apoptosation-inducing ligands (TRAIL) to brain tumors via viral transfection [99], and non-viral nanoparticles are also used to transfect stem cells targeting GBM in vitro [40]. The cancer homing ability of ADSCs and the therapeutic potential of TRAIL can induce apoptosis of primary tumor and microsatellite cells and prolong the survival time of GBM xenografts [40]. Transfection of ADSCs with bone morphogenetic protein 4 induces differentiation of brain cancer stem cells, preventing tumor recurrence, and their intranasal or intravenous administration has been shown to improve survival rates in F98 rats [100]. Furthermore, ADSCs can be engineered to express suicide genes like HSV-tk, and Malik et al. utilized PLL-PEI in conjunction with TRAIL and suicide gene strategies to extend survival in C6 glioma rats [41]. Integrating NSCs with nanoparticles in brain GBM models hastens tumor targeting and reduces nanoparticle clearance [101]. MSCs cross the BBB of Wistar rats under the action of chemokines and migrate to the tumor region, making MSCs become carriers for the delivery of GBM therapeutic drugs. The researchers also observed that MSCs may promote tumor growth by releasing exosomes [102]. However, advances in cell membrane bionic carrier technology can address the shortcomings of natural cells as carriers. MSC and NSC membranes are employed as drug delivery vehicles, retaining protocell tumor-homing and BBB-crossing capabilities while enhancing their crossing and tumor targeting through specific modifications [89].

2.5. Passive Diffusion

Small, fat-soluble molecules can form transient pores in the phospholipid bilayer, allowing passive diffusion across the BBB [103], a process influenced by molecular properties that typically restrict the penetration of the majority of molecules, with only a small subset of drugs effectively treating CNS diseases [104]. These hydrophobic molecules exploit the enhanced permeability and retention (EPR) effect for intercellular delivery and passive accumulation in cancer tissues, with passive administration across the BBB occurring via paracellular and transcellular routes tailored to drugs with specific physicochemical properties [105]. Strategies to modulate BBB permeability are essential for passive endocytosis, as demonstrated by the efficacy of fluoroethyl-modified tyrosine kinase inhibitors [106] and the ARTPC nanoplatform loaded with TMZ and surface functionalized with ApoE (ApoE-ARTPC@TMZ), which utilizes LDLRs-mediated transcytosis for enhanced BBB permeation and anti-GBM activity in vivo [42]. In vivo studies have shown that targeted liposomes demonstrate enhanced circulation time, superior BBB permeation, and GBM accumulation, leading to significant anti-GBM effects and prolonged survival in an intracranial U251-TR mouse model [42] (Figure 7). Cationic liposomes selectively target tumor vasculature due to their affinity for the negatively charged BBB endothelial cells [107]. Conversely, non-targeted lipophilic systems may fail to penetrate the BBB due to its tight junctions. The use of drugs ( such as Mannitol ) or focused ultrasound (FUS) can disrupt BBB connections, thereby enhancing drug penetration [105]. Intravenous administration of microbubbles followed by ultrasound exposure induces localized BBB disruption, facilitating transient drug access to the brain parenchyma [108]. Furthermore, microbubble-mediated FUS has been shown to improve the targeting of drugs to brain tumors [109], with enhanced efficacy when combined with other delivery methods [108].

3. Strategies for NDDS to Bypass the BBB

While crossing the BBB is essential, there are alternative strategies to bypass it entirely, offering potential solutions for drug delivery to the brain.

3.1. Intranasal Administration

By administering drugs via the nasal route, it is possible to circumvent the BBB, evade first-pass metabolism, and achieve rapid onset of action [110]. ABC and SLC transporters in the nasal cavity facilitate neurotherapeutic efficacy [111]. This approach highlights intranasal delivery of diverse drugs and biopharmaceuticals [112]. Nanoparticles target brain tumors via olfactory and trigeminal nerves, crossing the olfactory epithelium [113]. In vivo studies show reduced peripheral distribution and prolonged survival in brain tumor rats [114]. Chitosan-manganese/gold nanoparticle hybrids enhance RNA delivery to brain regions [115]. However, nasal administration's limited targeted volume and specificity can cause toxicity. Combining routes, like microbubble-mediated FUS with nasal delivery, significantly increases targeted drug delivery in tumors [108] , promising more efficient drug delivery to the brain.

3.2. Convection-Enhanced Delivery

Convection-enhanced delivery (CED), a local administration method during neurosurgery [116] , which involves placing a catheter at the tumor site and using an external pump to establish a pressure gradient for direct drug delivery to the target tissue [117]. The infusion dose does not depend on molecular size and weight, improving drug spatial distribution and reducing systemic toxicity, potentially enhancing patient survival rates at 24 and 36 months [118] and median survival time [119]. Phase I trials for recurrent high-grade gliomas confirmed the feasibility and safety of intracerebral CED of carboplatin [120]. However, in a multicenter phase III study involving 276 patients with recurrent GBM, there was no significant difference in median survival when treating CED with Cintredekin besudotox (IL13-PE38QQR) and Gliadel wafers (a carmustine implant) [121]. The rich tumor vasculature, interstitial fluid pressure within tumors, limitations in catheter technology, and imaging for drug delivery hinder the reliability and reproducibility of this technique. Drug excretion/absorption and catheter placement issues affect tumor exposure [105]. With technological advancements, CED could become a glioma drug delivery technique, but further trials are needed to confirm its efficacy.

3.3. Intracavitary/Intrathecal Drug Administration

Intrathecal drug administration, bypassing the BBB to access the ventricular system, elevates brain drug concentrations but risks tissue damage [122,123]. Compared to intraventricular injection, it's less harmful, extensively studied for delivering large molecules in stroke and neurodegenerative models [124], and a promising avenue for future anti-tumor nanomedicine delivery.

4. Progress in Clinical Trials

Preclinical research is a pivotal step in the drug development process, providing a solid data foundation for clinical trials and drug marketing by comprehensively assessing the safety, efficacy, and pharmacokinetic properties of drugs in animal models and in vitro systems. This phase of research not only aids in optimizing drug candidates and reducing the risks associated with clinical trials but also ensures that new drugs meet stringent safety and efficacy standards before marketing and comply with regulatory requirements. This chapter will focus on the progress of clinical trials of nanomedicine drug delivery systems in the treatment of GBM, systematically analyzing key data on drug safety, PK, and pharmacodynamics, and discussing the advantages and disadvantages of different drug delivery systems, as well as challenges and solutions encountered in clinical trials.

Building upon the robust foundation laid by preclinical research, the field of nanomedicine has made inroads into clinical applications, as evidenced by the favorable outcomes of Phase I clinical trials. Table 2 offers a comprehensive overview of pivotal trials that have utilized NDDS based on liposomal formulations, polymeric nanoparticles, and inorganic nanoparticles. These nanocarriers have been the subject of considerable interest and investigation [125]. Notably, the clinical progression of nanoliposomal irinotecan, assessed in trials NCT00734682, NCT02022644, NCT03086616, and NCT03119064 since 2008, underscores the extensive exploration of nanomedicine in GBM treatment. A notable Phase I trial among recurrent GBM patients aimed to ascertain the maximum tolerated dose and preliminary therapeutic effects of pegylated nanoliposomal irinotecan in conjunction with TMZ. Despite the study's premature conclusion due to unmet efficacy expectations in an interim analysis [126], it yielded critical insights for the development of advanced treatment modalities. Another Phase I trial investigated the safety, PK, and maximum tolerated dose of intravenously administered nal-IRI, considering the UGT1A1*28 genotype. The findings suggest that intravenous nal-IRI is well-tolerated and that genotype does not significantly alter the drug's PK or maximum tolerated dose [127]. The next step involves exploring the efficacy and safety of nal-IRI under CED though the outcomes of this trial have yet to be published (NCT02022644). The results of these clinical trials are anticipated to provide crucial information for the clinical translation of nanomedicine therapies for GBM. Furthermore, Phase II clinical trials are ongoing to investigate the efficacy and safety of other liposomal NDDS. For instance, the combination of pegylated liposomal doxorubicin (PLD) and TMZ has demonstrated superior outcomes compared to radiotherapy alone in preliminary studies [128]. Although the sample size was limited and lacked randomized controls, this finding warrants further investigation.

To enhance the targeting efficiency of NDDS, chemical modification has emerged as a strategy aimed at precisely targeting GBM cells, thereby bolstering the safety and efficacy of treatment. For instance, Kasenda et al. reported the effective drug delivery capability of anti-EGFR doxorubicin-loaded immunoliposomes in tumor tissues with a compromised BBB [131]. EGFR-Erbitux receptor EnGeneIC Dream Vector with mitoxantrone (EEDVsMit), an innovative nanocellular therapy, has demonstrated good tolerability and no dose-limiting toxicity observed in clinical trials [133] (Evans et al., 2024). This advancement offers novel therapeutic strategies for pediatric refractory tumors, particularly those with positive EGFR expression. Additionally, glutathione, as a targeting ligand, has been conjugated to polyethylene glycol liposomes, a strategy designed to enhance drug delivery efficiency to the brain. Glutathione pegylated liposomal doxorubicin (2B3-101) completed Phase I/II clinical trials in 2014 (NCT01386580).

In the research on strategies to enhance the targeting of NDDS, utilizing biological vectors, particularly human cells, has become a key innovation for achieving precise localization of GBM cells and enhancing the safety and efficiency of treatment. Genetically engineered NSCs that express cytosine deaminase (CD) can efficiently convert 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU) in the tumor microenvironment. Portnow et al.'s study first demonstrated the safety of single intracranial injection of CD-NSCs after oral administration of 5-FC in recurrent high-grade GBM patients [134]. Subsequent studies further evaluated the safety of repeated intracranial injection of CD-NSCs and explored the appropriate dose when combined with Leucovorin, laying the foundation for Phase II clinical trials [135]. This strategy significantly enhances the targeting of treatment by bypassing the BBB, optimizing the concentration of drugs in the affected area, and reducing systemic side effects. NSCs as an ideal biological vector demonstrate the potential to deliver a variety of anti-cancer therapies to the tumor region. For example, in clinical trials using NSCs to deliver oncolytic adenovirus (NSC-CRAd-S-pk7) to newly diagnosed malignant GBM patients, good safety was observed in the dose-escalation phase, and partial tumor volume reduction and other positive responses were observed in some patients [136]. Another clinical trial uses hCE1m6-NSCs expressing carboxylesterase and irinotecan to treat recurrent high-grade GBM to inhibit tumor growth (NCT02192359), aiming to determine the recommended II phase dose, with the results yet to be released. Additionally, monocyte antigen carrier cells have shown potential in the treatment of newly diagnosed GBM with an unmethylated MGMT gene promoter, such as the MT-201-GBM vaccine, which is made from a patient's own cells loaded with CMV pp65-LAMP mRNA and has been in clinical trials since August 2023. To determine its maximum tolerated dose (NCT04741984). Meanwhile, antigen-MRNA-loaded dendritic cells (DCS) have also entered clinical trials using autologous DC cells as mRNA delivery carriers to target Survivin for GBM of the brain (NCT06524063). This trial is currently in progress and the results have not yet been published. Therefore, combining the dual strategy of genetic engineering technology and biological vectors is expected to further promote the development of the field of GBM treatment and bring hope to more patients.

Lastly, inorganic nanoparticles composed of magnetically responsive materials, such as superparamagnetic iron oxide nanoparticles (NanoTherm®), have shown potential in prolonging overall survival in patients with recurrent GBM [129]. In 2013, NanoTherm® became the first nanotechnology-based cancer treatment device to receive CE mark approval. Furthermore, a new clinical trial (NCT06271421) is evaluating the efficacy and tolerability of the NanoTherm treatment system in recurrent GBM via Cyclic hyperthermia. Meanwhile, other types of inorganic nanoparticles, like NU-0129 (NCT03020017), a nanomedicine delivery system centered on gold nanoparticles and covalently conjugated with small interfering RNA (siRNA) oligonucleotides, target the BCL2L12 gene in GBM cells. This system is being explored through clinical trials for its potential as a brain-penetrating precision therapy [130]. Additionally, a Phase I/II clinical trial investigating AGuIX nanoparticles combined with radiotherapy and temozolomide in newly diagnosed GBM patients has shown that AGuIX nanoparticles significantly enhance the clinical potential of GBM treatment when used in conjunction with radiotherapy and temozolomide [132].

Since the clinical application of Nanomedicine delivery system over a decade ago, numerous Phase I/II clinical trials have been conducted to comprehensively evaluate their safety and efficacy, yet the outcomes of most of these trials remain unpublished. Among the published results, the strategy of encapsulating drugs within liposomes and delivering them via passive diffusion mechanisms has demonstrated more frequent application compared to other administration routes. Despite summarizing eight strategies to overcome the BBB from preclinical studies, this preference may be attributed to the generally good liposolubility of first-line drugs commonly used in GBM treatment, coupled with the unique advantages of liposomes as carriers, including high stability, low leakage rates, drug sustained-release properties, and excellent biocompatibility. Building upon this foundation, scientists have further delved into introducing ligands into liposomal formulations to enhance drug targeting. Based on the outcomes of two reported clinical trials, targeted immunoliposomes have shown potential in delivering cytotoxic drugs and potentially immunomodulatory molecules to GBM tissues, albeit without achieving the anticipated significant clinical improvement. However, the exceptional long-term remissions observed in individual patients provide positive signals for this novel therapeutic strategy, hinting at its potential positive impact on treatment outcomes [131]. The utilization of cells or cell membranes as drug delivery vehicles constitutes a prominent option in clinical translation, with notable achievements particularly evident in studies employing neural stem cells (NSCs) as carriers, which have yielded published clinical outcomes. The dual-pronged strategy of combining gene engineering technologies with NSCs as biological vectors has yielded more abundant clinical translation results compared to other cell types. This may stem from the unique advantages possessed by NSCs that are not shared by other cell types. For instance, NSCs exhibit superior BBB permeability, precise migration and tracking capabilities towards tumor regions, and differentiation potential that aids in the repair and regeneration of brain tissue, alongside relatively low immunogenicity. In contrast, the use of red blood cells as drug carriers is hindered by limitations such as burst release characteristics and the absence of specific receptors for targeted delivery.

Given the challenges faced in GBM chemotherapy, the ongoing proliferation of clinical trial projects signifies the gradual progress in the clinical translation of nanomedicines. In the future, the key to nanomedicine development will lie in optimizing drug delivery systems, enhancing drug targeting and efficacy, and actively exploring combination strategies with other treatment modalities (such as immunotherapy and radiotherapy) to achieve more pronounced therapeutic effects.

5. Technical Challenges and New Strategies

The BBB, comprising endothelial cells, pericytes, astrocytes, basement membrane, tight junctions, and adherens junctions among endothelial cells [104,137] (Figure 2a). The intact BBB safeguards the brain from toxins while restricting drug entry. Only small, lipophilic molecules (< 500 daltons) can cross it [138]. The coordinated action of three cell types constructs a robust "city wall" for brain protection, utilizing membrane receptors, ion channels, and transport proteins as "delivery channels". To deliver drugs, leveraging these channels is key. However, efflux pumps on cell membranes pose a challenge. In gliomas, BBB integrity deteriorates with tumor progression, influenced by malignancy grade [83]. Elevated malignancy correlates with heightened metabolic demands, yet nutrient and oxygen scarcity induce local hypoxia, fostering abnormal angiogenesis and BBB dysfunction [139]. Despite variable BBB integrity, local disruptions do not markedly affect drug concentrations in tumor tissues [140,141]. Consequently, BBB penetration is essential for effective anti-glioma drug design.

The main barriers for therapeutic drugs to penetrate the BBB encompass four aspects: "physical barrier", "transportation barrier", "metabolic barrier", and "immune barrier".

(1)

Physical barrier". This encompasses the anatomical and functional features of BBB endothelial cells, forming a vital anatomical gateway for targeted brain drug delivery. The lipid bilayer membrane of these cells exhibits lipophilicity and hosts receptors, carrier proteins, and other components that regulate molecular trafficking from the bloodstream to brain tissue. High molecular weight drugs (>500 daltons) often fail to traverse this barrier [140]. Tight and adherens junctions between endothelial cells maintain BBB integrity, preventing unrestricted substance exchange.

(2)

"Transportation barrier". Functionally, the surface of the BBB endothelial cells is negatively charged,, impeding negatively charged compounds from entering neurons. Endothelial membranes express specific transporters regulating substrate influx/efflux, preventing unauthorized bloodstream substances from crossing [142]. Pericytes and astrocytes encapsulate BBB endothelial cells, creating resistance that allows only small molecules (e.g., water, gases, lipids) to diffuse passively. Large, charged, polar, hydrophilic molecules (amino acids, glucose, drugs) rely on luminal membrane transport proteins/receptors [137]. ATP-driven efflux pumps (P-glycoprotein) limit toxin/drug permeability, reducing CNS exposure [104,143], impacting drug efficacy, exacerbating side effects, and challenging drug action in brain tissue.

(3)

"Metabolic barrier". Various drug-metabolizing enzymes, such as CYP450 enzymes, have been documented within the endothelial cells of the brain [104].

"Immune barrier". Neurovascular units, comprising pericytes and astrocytes, regulate tight junctions, waste clearance, vascular function, and neuroimmune responses, forming an 'immune barrier' that constitutes the BBB.

The main challenges in BBB drug delivery involve physical, transportation, metabolic, and immune barriers. Rational design of drug delivery systems, particularly nanocarriers, can overcome metabolic and immune barriers by reducing enzymatic reactions, phagocytosis, and immune clearance, enhancing drug stability [144]. From the perspective of morphology and function of the BBB, there are two types of coping strategies to overcome the BBB: across nd bypassing BBB. This review emphasizes how nanodrug delivery systems penetrate the BBB, focusing on overcoming physical and transportation obstacles.

6. Conclusions and Perspective

The BBB plays a pivotal role in influencing the efficacy of GBM treatment agents. NDDS have ushered in a new era for brain drug delivery, potentially overcoming the BBB to improve the treatment outcome of GBM patients. Preclinical studies reveal that, compared to drug substance, NDDS, through rational design, exhibit diverse delivery modalities and superior delivery efficiency. Encapsulation and specific chemical modifications enhance drug stability and targeting, thereby significantly mitigating toxicity and side effects, advancing towards clinical translation. Nevertheless, the number of NDDS transitioning into clinical practice remains limited. In contrast, emerging therapies such as tumor immunotherapy and cellular therapy are progressively being explored, posing additional challenges to the widespread clinical application of NDDS. Consequently, continued technological innovation, aimed at markedly improving the clinical efficacy of NDDS, is imperative for achieving their broad clinical transformation. Another challenge that lies ahead is the complexity and heterogeneity of GBM, which further requires researchers to integrate knowledge from multiple disciplines and undergo a paradigm shift in the strategic development of NDDS-specific to drive NDDS from the laboratory to the clinic [145].

In clinical trials, in order to improve the clinical efficacy of the NDDS, researchers tend to adopt the strategy of combination therapy [132], aiming to achieve a synergistic effect in three primary avenues, thereby attaining a "1+1>2" augmentation: 1) Theranostic NDDS. Endowing nanomedicines with dual functions of therapy and imaging provides a robust tool for early tumor diagnosis, treatment monitoring, and efficacy assessment. A feasible approach being the co-assembly of magnetic resonance imaging contrast agents and therapeutic drugs within NDDS, enabling real-time navigation for multimodal theranostics [146]. 2) Combination of therapeutic modalities. For instance, utilizing CAR-T cells and CAR-neutrophils for targeted drug delivery, the integration of chemotherapy and immunotherapy mitigates off-target effects and prolongs the survival of tumor-bearing mice [37,92,93]. 3) Design of multi-drug synergistic NDDS. The combined delivery of sicPLA2 and metformin based on exosomes selectively targets GBM energy metabolism for antitumor effects, demonstrating potential for personalized treatment in GBM patients [147]. However, these strategies are currently in preclinical research. Despite the aforementioned challenges, recent clinical trials of GBM using nanomedicine as a therapeutic modality indicate the gradual expansion of nanochemotherapy's clinical application in GBM treatment.

Looking ahead, further advancements in NDDS will require interdisciplinary collaboration across fields such as nanotechnology, neurobiology, and clinical medicine. Future research should focus on optimizing nanoparticle design for specific therapeutic applications, improving patient-specific targeting, and refining drug release mechanisms. Additionally, well-designed clinical trials are crucial for evaluating the safety, efficacy, and long-term outcomes of these innovative delivery systems. By addressing these challenges, NDDS can move closer to realizing their full potential in GBM therapy, ultimately leading to more effective treatments with fewer side effects and better patient outcomes.