Biomimetic nanotherapeutics for targeted drug delivery to glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is an aggressive brain tumor with poor prognosis and high mortality, with no curative treatment to date as limited trafficking across the blood–brain barrier (BBB) combined with tumor heterogeneity often leads to therapeutic failure. Although modern medicine poses a wide range of drugs that are otherwise efficacious in treating other tumors, they often do not achieve therapeutic concentrations in the brain, hence driving the need for more effective drug delivery strategies. Nanotechnology, an interdisciplinary field, has been gaining immense popularity in recent years for remarkable advancements such as nanoparticle (NP) drug carriers, which possess extraordinary versatility in modifying surface coatings to home in on target cells, including those beyond the BBB. In this review, we will be highlighting recent developments in biomimetic NPs in GBM therapy and how these allowed us to overcome the physiological and anatomical challenges that have long plagued GBM treatment.


| INTRODUCTION
Glioblastoma multiforme (GBM) is the most prevalent and aggressive brain malignancy in adults, accounting for nearly half of malignant central nervous system (CNS) tumors with no curative treatment to date. [1][2][3] The median overall survival (OS) of GBM patients is 15 months, with only 5.5% of patients surviving beyond 5 years from initial diagnosis. 4 The current standard treatment follows the STUPP protocol which comprises maximal tumor tissue safe resection followed by concurrent chemo-and radiotherapies for 6 weeks and thereafter a 6-month regime of oral temozolomide (TMZ). 5 Unlike surgery elsewhere in the body where en-bloc resection can be performed, in the brain, due consideration has to be given to balance both oncological and functional outcomes when resecting infiltrative lesions like GBM. Not uncommonly, the risk of inflicting irrevocable neurological damage precludes complete exenteration of the entire tumor during GBM surgery. In addition, while necessary for controlling tumor progression, chemoradiation in itself causes numerous undesirable side effects that are not well tolerated by most patients. Furthermore, nearly all GBM cases recur with a more aggressive form of tumor, often with a poorer prognosis and lowered median OS of 2-9 months, 6 driving the need to develop more effective treatment strategies to improve patients' overall prognosis.
Glioblastoma multiforme is a grade IV primary brain malignancy arising from astrocytes, a type of glial cell which supports neurons in the CNS. Astrocytes are the most prevalent cells in the brain, outnumbering neurons by more than five-fold and serving an equally vital function. Beyond providing nutrients to neurons, they play key roles in regulating the blood-brain barrier (BBB) and maintaining interstitial fluid homeostasis-a crucial factor to facilitate synaptic transmission. 7 The BBB consists of astrocytes anchoring pericytes and extracellular matrices to the basement membrane of an endothelial cell layer that forms tight junctions (TJs), which are responsible for regulating the movement of substances across the BBB. The diffusion of small lipophilic molecules is facilitated by important transmembrane proteins within TJs, which include zona occludens (ZO), claudins, and junctional adhesion molecules. 8 Retrograde movement of substances back into the systemic circulation is facilitated by efflux transporters present within the BBB, which include P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and organic anion transporting peptide (OATP). 9 Astrocytes play a supportive, yet critical role by releasing soluble factors such as vascular endothelial growth factors (VEGFs), matrix metalloproteinases (MMPs), endothelins, angiopoietin-1, and glial-derived neurotrophic factor, which are essential for the maintenance and modulation of the BBB structure. These aspects allow the BBB to inhibit paracellular transport of around 98% of small molecules and nearly 100% of macromolecules. 10 Though its protective role is vital, the BBB's highly selective nature unfortunately creates a formidable barrier impeding the delivery of drugs to the CNS. This is not necessarily the case with GBM however, as the rerouting of vasculature to the growing tumor often destabilizes the BBB.
The "leaky barrier," or blood-brain tumor barrier (BBTB), allows macromolecules designed for targeted drug delivery to bypass the normally impregnable BBB. Unfortunately, high-grade gliomas rapidly infiltrate surrounding tissues where the BBB is not yet compromised, making the BBTB as a sole delivery route via the enhanced permeability and retention (EPR) effect an unreliable strategy. 11 The EPR effect refers to the ability of molecular compounds to accumulate and retain in the vascularized areas of the tumor. There is a clear need to identify ways to enhance drug delivery across the BBTB and fortunately, recent studies have shown that a solution capitalizing on this unique therapeutic avenue may be found in nanoparticles (NPs).
Nanoparticles range in size from 1 to 100 nm, and can be synthesized using inorganic, polymeric, or organic materials. 12 Critically, they are able to carry a therapeutic payload and efficiently home in on their target cells. Multiple molecular subtypes of NPs exist, including lipidbased, polymeric, and metallic variants. 13 This allows flexibility in surface modification and enables them to accommodate a wide range of therapeutics, making them ideal vehicles for drug delivery. NPs such as the metallic subtypes are also synergistic with other treatment adjuncts, such as photodynamic therapy. 14 Hence, it is no surprise that NPs have become an increasingly popular choice for drug delivery in the newly developing paradigm of GBM treatment. 15 In this review, we discuss current GBM therapeutic strategies and their associated challenges, as well as demonstrate how NPs could be a novel approach to overcome the limitations in conventional GBM treatment. In Figure 1a, we outline the different ways NPs have been enhanced using biomaterials to increase uptake at the BBB and GBM tumor cells. Combinatorial therapeutics involving synergy between biomimetic NPs and other adjuncts will also be discussed, in hopes that we can present a novel perspective on the potential future of GBM treatment. In addition to TMZ resistance, GBM's angiogenic tendencies also pose a significant hurdle to treatment. 23 Angiogenesis refers to the formation of new blood vessels which is critical for tumor growth. As any tumor mass enlarges and outgrows its blood supply, cells at the periphery become hypoxic and undergo necrosis. The production of VEGF to promote angiogenesis therefore becomes essential for tumor survival. 24 In GBM, the abundant secretion of proangiogenic factors such as VEGF facilitates the extensive microvascular proliferation and rapid tumor growth, contributing to a more aggressive and fatal disease. 25 Bevacizumab, an immunotherapeutic drug, binds to VEGF and prevents it from binding to its receptor. This reduces neovascularisation, normalizes vasculature, and eliminates GBM tumor cells that express VEGF. 26,27 It is most commonly used in patients experiencing GBM recurrence as phase II trials have demonstrated improvement in progression-free survival (PFS) in this select group. 28 Unfortunately, bevacizumab is still not part of standard GBM therapy as it has not shown to demonstrate any significant improvement in OS. 29,30 It is also not curative as tumor mutations lead to activation of alternative angiogenic pathways, or reduce reliance on angiogenesis for proliferation. 31 Another major factor limiting the efficacy of current GBM treatment is the highly heterogenous nature of the tumor. Even within the same tumor mass, there can be multiple clonal and subclonal populations, each marked by distinct genetic alterations. 32 Most recurrent GBM tumors also differ from the initial lesion. This limits the efficacy of single-target therapies. Mutations can affect a variety of genes regulating critical pathways, such as MEK/ERK and PI3K/AKT/mTOR signaling channels. 33 EGFR in particular is often overexpressed or constitutionally activated. This has led to the exploration of EGFR inhibitors such as erlotinib and gefitinib as anti-GBM therapies, given their success in the treatment of EGFR-positive lung cancers. However, despite their initial positive outlook, these drugs have yet to demonstrate significant improvement in OS and were instead found to cause unacceptable toxicities during GBM treatment. Their inability to reach therapeutic concentrations in the brain also contributed to their low therapeutic value. 34 In addition, the Notch signaling pathway has been found to be associated with the establishment and maintenance of cancer stem cell niches, leading to gamma secretase inhibitors (GSIs) being assessed as potential GBM therapeutics.

| CURRENT PARADIGM OF GBM TREATMENT
Unfortunately, despite being well-tolerated in a combination therapy with TMZ and radiotherapy, GSIs showed inconsistent BBB penetration, especially at sites where the BBB remained intact. 35 Figure 2i). 44 In the same vein, a study examining the use of MRgFUS for pre-resection chemotherapy in high-grade glioma patients demonstrated higher chemotherapeutic drug concentrations in sonicated compared to unsonicated tissues. 45 Furthermore, no adverse side effects as a result of the procedure were observed.
Despite its small sample size, this study highlights the potential of MRgFUS in opening the BBB and enhancing drug delivery to the brain. Nevertheless, more statistically-powered studies evaluating MRgFUS are required before it can be implemented in GBM patients. Studies centered on harnessing the extent and duration of BBB disruption are also paramount as an indefinite opening of the BBB will predispose the brain to infection or drug-induced cerebral toxicity. 46,47

| GBM-targeted NPs
Over the years, many types of NPs have been developed to enhance permeability across the BBTB and concentrate drug delivery at target sites to minimize systemic toxicity. 48  This resulted in temporary disruption of endothelial TJs followed by activation of the paracellular transport pathway, hence promoting movement of all circulating agents across the BBB. 53 Recently, extraordinary advancements in magnetic technology, such as magnetguided fish-shaped microrobots that could release chemotherapeutic drugs in response to a decreased pH, which simulates the acidic TME, have also been developed ( Figure 2iii). 54 If a magnetic field strong enough to penetrate the cranium and its contents to exert effects on the BBB could be developed, this novel technique could usher in a new paradigm of drug delivery to the brain. 55 Despite the immense potential of these NPs in treating GBM, their efficacy remains limited by their susceptibility to clearance by the reticuloendothelial system (RES) due to the presence of numerous  56 Therefore, development of NPs with lower immunogenicity and greater biocompatibility is crucial for improved NP delivery to the brain.

| BIOMIMETIC NANOPARTICLES
As with most drugs, the efficacy of traditional NPs is limited by their relative inability to pass through the BBB, with research showing that only 0.7% of the administered NP dose is successfully delivered to tumors in the brain. 57 Hence, novel NP designs that can bypass the BBB and specifically target GBM tumor cells are imperative for effective therapy. The integration of biomaterials mimicking characteristics of biological molecules and cells has been shown to increase NP uptake at the BBB through natural ligand-receptor interactions. 58 These biological mimics, also referred to as biomimetic NPs, are significantly less immunogenic and also possess greater specificity for GBM tumor cells expressing biomimetic complements. 59 Table 1 highlights the advantages of using biomimetic NPs compared to traditional NPs, focusing on the increased stability, higher drug loading capacity and enhanced safety profile conferred by biomimetic NPs. Clinical trials have also been implemented for several NPs, but many require further work to fully elucidate its performance ( Table 2).
As illustrated in Figure 1b  was also added to enable these NPs to target GBM tumor cells with increased EGFR expression. 75 In addition, short peptides derived from naturally occurring proteins can be used as ligands to enhance specificity of biomimetic NPs for drug delivery. Low-density lipoprotein receptors (LDLRs) are often overexpressed in GBM and are responsible for the movement of many molecules across the BBB. An apolipoprotein B derived peptide, ApoB29 mer peptide, is a ligand that binds with high affinity to LDLRs and was used in a study by Seo et al. 76 The study showed that ApoB29-conjugated gold NPs penetrated the BBB more efficiently compared to bare gold NPs and preferentially accumulated in the tumor microenvironment (TME), demonstrating the potential of ApoB29 mer peptide as a ligand to increase NP specificity for GBM tumor cells.
In another study, albumin-based NPs were conjugated with a short peptide that can bind to transferrin receptors on the BBB and GBM tumor cells. 77

| Cell membrane-based biomimetic NPs
The key advantage of NPs coated with natural cell membranes is their hypoimmunogenic physicochemical properties as they are able to evade immune surveillance by the RES, thereby increasing their time in circulation. 79 To achieve this, various source cells including anucleate cells, prokaryotes, and eukaryotes, can be used to tailor-make biomimetic NPs to suit a variety of purposes. 80 80 This shows the potential of NK cell membranes in helping NPs traverse the BBTB and achieve better therapeutic outcomes.

| Cancer cell membrane-coated biomimetic NPs
Cancer cells have also been explored as potential sources of cell membranes to synthesize membrane-coated NPs. The tumor specific proteins used to coat these NPs include cell adhesion molecules such as cadherins, selectins, and integrins, which enable homologous binding to the source cell line to bestow a potent homing effect. 86  which recognize HDL scavenger receptors in the BBB and on GBM tumor cells. 89 HDLs are small particles involved in reverse cholesterol transport, 64 and are ideal for drug delivery due to their small size and nanodisc configuration which enables them to diffuse through dense tumors and accumulate at high concentrations. 12 Synthetic HDL nanodiscs loaded with cytosine-phosphate-guanine (CpG), a toll-like receptor 9 (TLR9) agonist, were formulated to deliver docetaxel to the GBM TME in GBM-bearing mice in vivo. 65 The HDL nanodiscs solely accumulated in the tumor with minimal amounts found in other organs, thus demonstrating the homing ability of these nanodiscs.
Besides the cytotoxic effect of docetaxel, tumor cell death was also This shows how the combination of natural transporters and NPs can work synergistically to enhance drug delivery to GBM tumor cells.
The numerous examples listed above have highlighted improved antitumoral outcomes with biomimetic NPs when compared to traditional NPs. However, despite all these promising developments in the field of biomimetic NPs, translation from preclinical to clinical setting remains a work in progress. Henceforth, more effective targeting strategies that augment current biomimetic NPs are imperative.

| STRATEGIES TO FURTHER ENHANCE NP-MEDIATED GBM TUMOR KILLING
As mentioned previously, efficacy of most drugs including NPs, is often reduced due to inadequate BBTB penetration and the tricky characteristics of solid tumors, especially tumor heterogeneity. Current GBM treatment is also limited by the immunosuppressive TME, irregular vasculature causing an uneven distribution of systemically administered drugs, 100 as well as the natural clumping of tumor cells restricting NP access. 101 The following strategies are aimed at overcoming some of these limitations.

| Combinatorial antigen targeting with CAR-T cells and NPs
Tumor specificity is a key, yet often limiting factor in the efficacy of biomimetic NPs, especially in a highly heterogeneous tumor like GBM. One antigens that have been employed in CAR design. 104 Other newly developed targets include ganglioside 2, B7-H3, chlorotoxin, and CD70. 104,105 These molecules have all been clinically verified as safe targets of CAR-T therapy for GBM, but only two CAR-T therapies specific to CD19+ B cell malignancies are currently approved by the FDA. 106  oxalate to release energy, which is subsequently utilized by the photosensitiser chlorin e6 for the generation of singlet oxygen to eliminate GBM cells. 112 This underscores how homologous targeting increases the specificity of biomimetic NPs and can further enhance therapeutic outcomes by utilizing TME-responsive substances to generate cytotoxic particles.
Another example would be the creation of GBM cell membranecoated NPs enveloping a pH degradable acetal grated dextran inner core loaded with TMZ and cisplatin. 113 Under physiological conditions (pH 7.4), less than 20% of the drugs was released from the NPs due to their stable configuration. However, at pH 6.5 and pH 5.0 which simulate the acidic TME of GBM tissue, the percentage of drug release increased to 54% and 76% respectively within 24 h. This allows for controlled drug release at GBM cells and reduces the risk of off-target cytotoxicity. The synergistic combination of homologous targeting and TME-sensitive elements is hence able to concentrate drug delivery at the target tissues and enhance GBM tumor killing.

| Aptamers to complement biomimetic NPs
Another strategy to increase target specificity of biomimetic NPs is the usage of aptamers, which are single-stranded oligonucleotides that possess different configurations and bind to target molecules such as membrane-bound proteins. 114 These ligand-receptor interactions lead to internalization of the aptamer-containing molecule. 115 This phenomenon has been beautifully demonstrated by Liu

| ECM-digesting enzyme-coated biomimetic NPs
After extravasating from blood capillaries in the BBB, NPs enter an interstitial space containing interstitial fluid that bathes the parenchymal cells of the brain and spinal cord. 122 126 These conditions are associated with stromal thickening and lead to an irregular distribution of NPs throughout the tumor, impairing subsequent endocytosis-mediated uptake of NPs into GBM cells. 127 Besides capitalizing on ligand-receptor interactions to improve NP uptake into GBM cells, the transport of NPs through the interstitial space can also be enhanced by breaking down the dense ECM using ECM-digesting enzymes such as collagenase and hyaluronidase. 128 In a study on GBM-bearing mice, human adipose- Human recombinant hyaluronidase has also been successfully added to red blood cell membrane-coated NPs and this conjugation was observed to enhance NP diffusion in the hyaluronic acid-rich matrix surrounding PC3 prostate cancer cells. 131 This can potentially be applied to GBM as well to increase NP distribution. This is especially true given that GBM is a highly proliferative tumor with a florid stroma and fibrous matrix. The incorporation of ECM-digesting enzymes into biomimetic NP formulation is hence likely to enhance drug distribution within the tumor core.

| Robust drug delivery method
The combination of biomimetic NPs with other specific types of therapy can also selectively enhance different therapeutic effects. PLGA nanovectors encapsulating silver NPs were coated with chlorotoxin (CTX) to target matrix metalloproteinase-2 (MMP-2) and chloride channel-3 (CIC-3), both of which are highly expressed on GBM cells. 132 When these NPs were used in conjunction with radiation therapy, it was associated with improved targeting of scattered GBM cells as ionizing radiation damages the integrity of the BBB and induces MMP-2 expression. 132 This synergistic combination resulted in significantly delayed residual and metastatic tumor growth vis-à-vis monotherapy with biomimetic NPs alone, and shows how various strategies can be simultaneously employed for their synergistic effects to overcome limitations of GBM monotherapy.

| CONCLUSION
Despite significant progress in the treatment of other cancers over the years, current treatment strategies for GBM often fall short of treatment goals. The current array of therapeutic tools remains inadequate, and is often blunted by acquired drug resistance by rapidly evolving GBM cells. To effectively pioneer a new paradigm of GBM treatment, overcoming the major obstacles faced today is paramount.
One of which is the impermeable BBB, which precludes the penetration of several therapeutic drugs into the CNS. The need for improved drug delivery methods across the BBB so that better therapeutic strategies can be devised is clear, and biomimetic NPs are an extremely attractive proposition, especially if combined with other strategies such as CAR-T technology and ECM-digesting enzymes.
The increased tumor specificity and decreased immunogenicity conferred by these particles could usher in a new age of improved therapeutic outcomes. Although further studies and enhancements are necessary to optimize these NPs for clinical use, nanotechnology is undoubtedly a field that possesses a wealth of untapped potential that could radicalize the treatment of GBM.