New insights into targeted therapy of glioblastoma using smart nanoparticles

The established method for radiographic characterization of GBM is magnetic resonance imaging (MRI), widely utilized for diagnosis and post-therapeutic management [21]. Additionally, to identify risk factors tests based on computed tomography (CT) or MRI, fundamental tools for glioma detection, should be incorporated [22].

The current WHO classification is notably intricate and advocates for an integrated diagnosis, considering both histopathological and molecular typing that incorporates genetic mutations and molecular markers [17, 23]. A conclusive diagnosis relies on histopathological examinations of the tumor or its parts, obtained intraoperatively, using conventional histological, cytological, and histochemical methods. In cases where neurosurgical lesion excision is not feasible, a fine-needle aspiration (FNA) biopsy is recommended [24, 25]. Glial fibrillary acidic protein (GFAP), part of the cytoskeletal protein family, is extensively expressed in astroglial and GBM cells. The absence of GFAP expression indicates significantly undifferentiated tumor cells but does not suggest tumor progression. Therefore, serum GFAP can be considered a candidate biomarker in diagnosing GBM [26].

Standard therapy for malignant gliomas involves the administration of chemotherapy, radiotherapy, and interventional surgical procedures [27]. Surgery followed by temozolomide-based chemoradiotehrapy is the standard treatment in the setting of early diagnosis. There is no standard of care in the case of relapse, but, according to the patient’s conditions, radiotherapy, surgery, and systemic treatment with chemotherapy or bevacizumab might be indicated [8, 28]. Nevertheless, the outcomes concerning survival extension and treatment response are inconsistent with both chemotherapy and radiotherapy [29].

In spite of progress in diagnosing and treating GBM, the prognosis, incidence, and mortality rates continue to be unfavorable [30].

MRI serves as the prevailing standard for diagnosing and monitoring newly identified and recurrent masses. The outcomes derived from MRI are crucial for pre-treatment characterization and assessing the response to therapy. However, challenges arise as conventional MRI faces difficulty distinguishing between primary tumors and metastases, as well as CNS masses, and determining true progression versus pseudoprogression. Radiological features of these conditions often overlap. Gliomas, metastatic lesions, and primary CNS lymphomas typically manifest as contrast-enhancing tumors surrounded by T2-hyperintense edema [31]. Furthermore, glioblastomas exhibit similarities in metabolite ratios, making cutting-edge imaging techniques insufficient. Another obstacle in diagnosis of glioma is the substantial intertumoral heterogeneity, challenging the idea that gliomas originate from a single cell [32]. Differential patterns of copy number alteration (CNA) have been suggested to influence tumor development [17].

A significant hurdle in glioblastoma treatment is tumor recurrence, with a median survival of 14.6 months for GBM patients undergoing conventional multimodal therapies. The progression-free survival (PFS) for recurrent GBM does not reach 24 weeks. Drawbacks in conventional GBM treatment, such as neurotoxic effects and unsatisfactory loading efficiency limit its therapeutic potential [30].

Additionally, the blood-brain barrier (BBB) plays a central role in restricting therapeutic strategies, as numerous drugs exhibit little or no solubility to cross this physical barrier [30]. The BBB, a specialized system in the brain’s vasculature, regulates molecular transport across the endothelial wall [33]. In many regions of the CNS, the vasculature comprises endothelial cells linked by tight junctions, situated alongside pericyte on a basement membrane. These cells, alongside neighboring neurons and microglia, facilitate cerebral immune responses, primarily aiming to safeguard this intricate organ [34]. The transportation of most molecules is orchestrated by different receptors within the BBB [35]. It is not uncommon for the BBB to undergo impairment due to specific pathologies, such as neoplasms. Brain masses can disrupt the normal functioning of the BBB [33], due to the positive regulatory effect they exert on angiogenesis [36].

Temozolomide (TMZ), which is known as the gold standard of treatment, promotes tumor progression and angiogenesis by alteration of IL8/CXCL2/CXCR2 signaling. Urbantat et al. investigated modifications in the signaling pathway during the recurrence of human glioblastoma multiforme and explored the specific impact of TMZ. They also established a combination therapy involving TMZ and CXCR2 antagonization to evaluate its effectiveness and tolerability. The study revealed a significant reduction in the infiltration of tumor-associated microglia (TAM), with high TAM infiltration in primary tumors correlating with reduced overall survival (OS). Moreover, more patients exhibited IL8 expression, and TMZ therapy maintained the expression of CXCL2. In rodents, the combination therapy demonstrated enhanced anti-tumoral effects. Additionally, the combinatorial therapy confers promise for overcoming CXCR2-mediated resistance [37]. Although focal radiotherapy increases the average survival, it may cause cognitive impairment, DNA damage and other severe systemic side effects. In studies, the anti-angiogenic drug bevacizumab was found to result in prolonged PFS when administrated in combination with chemotherapy for newly diagnosed GBM, albeit, the non-significant effect on OS as observed in phase III trials [38]. Chinot et al. assessed the impact of incorporating bevacizumab into the radiotherapy–temozolomide regimen in glioblastoma treatment. The inclusion of bevacizumab alongside radiotherapy–temozolomide was not accompanied by enhanced survival for glioblastoma patients. However, bevacizumab was associated with more frequent adverse events compared to the placebo [39]. Lack of specificity, unwanted cytotoxicity and multi-drug resistance (MDR) stand among the most impactful challenges associated with the current chemotherapeutic regimens [40]. Generally, Surgery, radiotherapy, and chemotherapy as existing treatments for GBM ultimately increase the patient’s survival by only a few months [7].

Various computational models have been created to capture different facets of glioblastoma, and these simulation tools can be employed to forecast tumor expansion, evaluate the impact of disrupting molecular pathways in specific brain regions, and comprehend the considerable heterogeneity within the tumor microenvironment [43, 44]. In general, these models can be categorized into distinct groups, ranging from simplified models that simulate only tumor volume growth to intricately detailed models that encompass numerous genetic or proteomic processes implicated in the development and progression of glioblastoma [45]. As researchers gain a deeper understanding of the biological complexity, modeling strategies have progressed to offer insights into glioblastoma across various scales (tissue, cellular, and molecular) [46] encompassing essential glioma behaviors like vascularization, diffusion, and invasion capacity [47]. Improved modeling contributes to more effective therapy optimization tailored to each cancer patient [42]. Molecular signaling pathway analysis aids in quantitatively identifying potential cancer targets, and mathematical models of mass transport phenomena enable predictions regarding drug delivery to the brain, assisting in experimental design [48].

Numerous bioinformatic studies have been carried out to uncover new targets for regulating GBM progression. Leveraging public genomic databases like TCGA, REMBRANDT, Gravendeel, KEGG, and CGGA, researchers have sought to identify therapeutic targets and construct predictive nomograms [49]. Ongoing investigations aim to discover novel biomarkers for identifying molecular pathways [50], employ advanced genome editing technologies such as CRISPR-Cas or CRISPR-Cas9 to overcome chemotherapeutic resistance, utilize targeted miRNAs to silence genes promoting autophagy, and explore the use of plant-derived bioflavonoids to inhibit autophagy and enhance the therapeutic efficacy of TMZ in GBM [41].

The field of nanotechnology has brought about a revolution in the conventional methods of treating, diagnosing, and managing gliomas. This transformation is largely attributed to late progresses in bioengineering, improved drug accessibility, and the ability to specifically target cancer cells by accumulating and entrapping them [30]. Nanomaterials (NMs), such as metal and polymer-based nanoparticles [51] are being increasingly used in the field of cancer theranostics [52], owing to their small size, large surface area, specific structural features, binding affinity, cell membrane or tissue penetration capability, and long elimination half-life in the circulation [53‐55]. The high surface-to-volume ratio of nanoparticles enables them to deliver small biomolecules such as nucleic acids, proteins and drugs to the target site and increase the efficacy of therapeutic agents [53].

Various methods have been demonstrated to enhance the transport of drugs across the BBB, with many of these techniques involving the disruption of the BBB. However, this disruption compromises the integrity of the cerebral microvasculature. One promising approach is the NP-based delivery of anticancer agents. Polymer and lipid NPs are commonly utilized as nanovehicles for delivering anticancer therapeutics. Figure 1 represents new approaches in GBM targeted therapy [56].

Nanovectors, whether hollow or solid, serve as nanoparticles with diverse applications in anticancer drug delivery, targeting moieties, and detection, thereby reducing toxic effects. These nanotechnological devices have garnered interest for their potential in cancer drug delivery and imaging [66, 70]. Nanovectors can be categorized into different generations [71]. The first generation non-specifically targets surface receptors on tumor cells [72]. An example is albumin-bound Paclitaxel used in breast cancer chemotherapy [73]. The second generation of nanoparticle technology focuses on active targeting, designed to identify and target specific biological molecules present on cancer cells. This approach incorporates high-affinity ligands and specific antigens on the surfaces of nanoparticles [74]. The ongoing development of the third generation involves a multi-stage strategy [75]. In the initial stage, biodegradable silicon microparticles with pores are designed to navigate the circulatory system and recognize endothelium specific to the disease. The subsequent stage comprises various nanoparticles loaded within the first-stage particles, released specifically toward the tumor mass. These nanoparticles, each smaller than 20 nm, can easily traverse interendothelial junctions and carry diverse payloads for both therapy and imaging, presenting a promising direction for future applications in cancer treatment [76].

A persisting challenge in the brain cancer theranostics is the BBB, which consists of endothelial cells, astrocytes, and the basement membrane lying in between that together form a structural and functional barrier to protect the brain parenchyma from potentially hazardous compounds in the blood [56]. BBB limits cerebral drug delivery, which is of particular importance at the periphery of tumors, where tumor cells invade the neighboring intact tissue [56]. Small-sized nanoparticles may cross the BBB barrier and deliver drugs to the target site [90]. The advantage of neutral and anionic small-size nanoparticles (20–70 nm) is that they cause less neurotoxicity [91]. In contrast, metal nanoparticles (e.g., copper, silver and aluminum) may be more neurotoxic [54]. Following the change in the function and organization of the BBB due to the increase in the severity of GBM malignancy, the blood brain tumor barrier (BBTB) is formed. BBTB limits the penetration of drug delivery systems [92]. Proper release of drugs is also necessary for effective treatment. Following the aggressive invasion of GBM, the migration of cancer cells to the neighboring tissues of the brain occurs. After tumor surgery, the migrating cancer cells may become recurrent GBM adjacent to the original tumor area [92]. Most current topical delivery systems that bypass the BBB cover only a small area near the delivery site, a shortcoming that needs to be addressed [93]. Also, low drug release may bring about high toxicity at local delivery sites [94]. Another major barrier against the efficacy of anticancer treatment is MDR [95]. Inherent resistance to chemotherapy drugs exists in certain cancer cells, while others acquire this trait through mutations during the carcinogenesis process [96]. Multi-Drug Resistance (MDR) arises due to the upregulation of the ATP-binding cassette (ABC) transmembrane transporter superfamily. Among the frequently overexpressed members of ATP-ABC, MDR-associated protein-1 (MRP1/ABCC1), breast cancer resistance proteins (ABCG2), and P-glycoprotein (P-gp/ABCB1) are notable. The heightened expression of these proteins reduces the effective intracellular concentration of chemotherapeutic agents in an ATP-dependent manner. Another mechanism leading to MDR involves the redistribution of drug molecules away from the target site, facilitated by non-ABC drug transporters like lung resistance protein (LRP; major vault protein). Additionally, the cytotoxic effects of chemotherapeutic drugs directed at tumor cells can be countered by detoxification mechanisms. Noteworthy drug-metabolizing enzymes that contribute to drug inactivation and exhibit increased expression in malignant cells include Glutathione-S-transferases, cytochrome P4503A, and aldehyde dehydrogenase-related phase II [97].

Poor penetrability often compromises the efficacy of treatment [54]. In contrast to this, though, in targeted drug delivery the therapeutic agent accumulates at the target site via the circulation. Based on the mechanism of delivery, targeted therapy can be classified into two major categories: (1) Passive targeting: in which the therapeutic particles are intercepted as a result of physiological phenomena such as enhanced permeability and retention (EPR) effect at tumor tissue; and (2) Active targeting: in which the therapeutic agent is modified by a specific ligand, the receptor of which is amply expressed at the target site. Combination of both classes, as in modification of particles with certain morphological features, would result in better delivery compared to either class alone. As such, application of passive targeting strategy, in the case of GBM that is inherently associated with BBB-induced inaccessibility, would render the treatment ineffective [98]. Owing to a feature termed ‘controlled release reservoir’ nanoparticles have been shown to be quite effective at releasing therapeutic agents within a good proximity of the target site. However, certain criteria must be met before clinical adoption of these nanoparticles such as biocompatibility, since a primary goal in targeted therapy is to avoid the adverse events caused by conventional therapy in the first place [99].

Jallouli and colleagues investigated the permeability of 60 nm porous nanoparticles with maltodextrin backbones, comparing cationic and neutral variants, in an in vitro BBB model known for its correlation with in vivo observations. Neutral NPs were observed to traverse endothelial cells through caveolae-dependent transcytosis, potentially mediated by glucose transporter (GLUT-1) and/or lectins. Both cationic and neutral NPs successfully traversed the BBB model via lectin-dependent transcytosis, although the efficiency of cationic NP transcytosis was lower [100].

These results imply that surface charge could influence binding to and passage through endothelial cells, making both cationic and neutral porous NPs potential candidates for brain drug delivery. A common method to modify NP surfaces is PEGylation, which involves the conjugation of PEG. PEGylation has been proven to reduce opsonization, resulting in decreased uptake by the reticuloendothelial system (RES) and prolonged circulating half-lives of PEGylated NPs [101]. In a study conducted by Zhao et al., a GBM mouse model was employed to demonstrate the safety and efficacy of PEGylated PAMAM dendrimer NPs when conjugated with the CREKA peptide. These PEGylated NPs exhibited prolonged in vivo circulation compared to uncoated NPs, alleviated the inherent toxicity of PAMAM, and achieved deep penetration into GBM tissue [102]. Gref et al. designed sterically stabilized nanospheres using amphiphilic diblock or multiblock copolymers. These nanospheres featured a hydrophilic PEG coating and a biodegradable core encapsulating various drugs. Hydrophobic drugs, such as lidocaine, were successfully entrapped up to 45 wt%, and the release kinetics were influenced by the physico-chemical characteristics of the polymer. The PEG-coated particles demonstrated a significant reduction in plasma protein adsorption compared to non-coated ones, with varying protein amounts over time. The nanospheres displayed prolonged blood circulation times and reduced liver accumulation, depending on the molecular weight and surface density of the PEG coating. Furthermore, they could be freeze-dried and redispersed in aqueous solutions, showcasing good shelf stability. This approach introduces the possibility of tailoring “optimal” polymers for specific therapeutic applications [103].

A multitude of in vivo investigations, encompassing both rodents in good health and a mouse model with breast cancer, provide substantial evidence affirming that PEGylated nanoparticles efficiently prolong the circulation of nanocarriers in the bloodstream and improve the stability of NP formulations when contrasted with their uncoated counterparts [104].

Moreover, bioactive compounds present in seeds, vegetables, and fruits possess antioxidant, anti-inflammatory, and anticancer attributes that could enhance the well-being of cancer survivors during chemotherapy or other treatments. The integration of these compounds into nanocarrier-based drug delivery systems for addressing GBMs presents a potential therapeutic approach for this type of tumor. This strategy aims to enhance targeting precision, elevate bioavailability, and minimize side effects by improving drug internalization into cells. Simultaneously, it reduces the likelihood of off-target organ accumulation [105].

A mostly commonly lab-synthesized nanoscale particle [112], magnetic iron oxide (either γ-Fe₂O₃ or Fe₃O₄) nanoparticles are the most extensively used type of NPs in the field of cancer theranostics, as they are both reactive to magnetic currents and well-tolerated by the patients [109]. As their therapeutic effects are temperature-dependent [113], IOMNPs have been used at different temperatures in several investigations [114]. Size and surface functionality play an important role in the pharmaceutical applications of IOMNPs [115]. Nanoparticles with a size larger than 200 nm are readily filtered by the reticuloendothelial system (RES). Excessively small particles (< 8 nm), on the other hand, are easily eliminated from the body through excretion in the urine [116] and their blood circulation time is reduced. Particles with a size of 10–40 nm (including very small IOMNPs) persist the longest in blood circulation [114], and can be stabilized at the target size by applying an external magnetic field, which in turn mitigates the required dose and potential adverse effects. As the therapeutic success highly depends on the composition of the outer coating, polymer layers, capsules, particles or vesicles have been proposed for use as the outer layer. Surface modifications of these particles are carried out using organic polymers and metals or inorganic oxides [117].

With IOMNPs, higher levels of efficacy are attained with homogeneous dispersion of the NPs in an aqueous media along with the addition of functional groups, which can be used for attachment of targeting units [116]. Considering the reactive area of IOMNPs and their capability to cross biological barriers, they stand among the NPs of choice for clinical application [116]. In this context, anticancer agents such as doxorubicin, docetaxel, 5-fluorouracil, gemcitabine and methotrexate can be encapsulated within IOMNPs [117, 118]. Studies show that IONPs are able to stimulate immune effects mediated by T cells against tumors. Once they are accumulated at the tumor site, IOMNPs can generate heat under an external alternating magnetic field and kill cancer cells, so they enhance immune function in the Tumor microenvironment by releasing pro-inflammatory cytokines [119]. IOMNPs can activate NADPH oxidases, induce the formation of reactive oxygen species and promote an imbalance in redox homeostasis, which renders them a highly effective tool for killing of malignant cells [120].