Insights into Targeted and Stimulus‐Responsive Nanocarriers for Brain Cancer Treatment

Brain cancers, especially glioblastoma multiforme, are associated with poor prognosis due to the limited efficacy of current therapies. Nanomedicine has emerged as a versatile technology to treat various diseases, including cancers, and has played an indispensable role in combatting the COVID‐19 pandemic as evidenced by the role that lipid nanocarrier‐based vaccines have played. The tunability of nanocarrier physicochemical properties —including size, shape, surface chemistry, and drug release kinetics— has resulted in the development of a wide range of nanocarriers for brain cancer treatment. These nanocarriers can improve the pharmacokinetics of drugs, increase blood‐brain barrier transfer efficiency, and specifically target brain cancer cells. These unique features would potentially allow for more efficient treatment of brain cancer with fewer side effects and better therapeutic outcomes. This review provides an overview of brain cancers, current therapeutic options, and challenges to efficient brain cancer treatment. The latest advances in nanomedicine strategies are investigated with an emphasis on targeted and stimulus‐responsive nanocarriers and their potential for clinical translation.


Introduction
3] There are ≈330,000 incident cases of brain cancers and other central nervous system (CNS) cancers worldwide each year with efficient use in the clinic is still challenged by limitations such as poor blood-brain barrier (BBB) crossing, short half-lives, offtarget toxicities, and limited efficacy at systemically tolerated doses. [14]Drug delivery systems have been clinically approved to treat various types of cancers including but not limited to ovarian cancer, osteosarcoma, and squamous cell carcinoma. [15]They show great promise to treat brain cancers as well by enabling a more targeted delivery of therapeutic agents, including improving BBB crossing while avoiding systemic toxicity. [16]Compared to traditional nanocarriers, stimulus-responsive systems can further improve drug delivery to brain cancers by incorporating a triggered drug release using either externally-applied stimuli such as magnetic field, ultrasound (US), heat, and light, or internal stimuli which are based on unique hallmarks of the brain tumor microenvironment like acidic pH, upregulation of enzymes or oxidants, and hypoxia. [17]here are several comprehensive reviews that discuss the benefits of employing nanomedicine for brain cancer treatment and illuminate the various strategies and advances that hold promise in combating this devastating disease. [18,19]Our review article carves out a distinct niche by focusing on the innovative and rapidly evolving field of targeted and stimulus-responsive nanoparticles.By virtue of their responsiveness, these nanocarriers can selectively target and release therapeutic payloads precisely at the tumor site, minimizing collateral damage to healthy brain tissue.Furthermore, they can navigate the blood-brain barrier, a distinctive obstacle in the delivery of drugs to the brain, with a level of finesse that traditional drug delivery systems often lack.The real-time adaptability of these nanoparticles to environmental cues, such as pH or enzymatic activity within the tumor, enhances their potential for tailored and personalized treatment strategies.In this review, we will first give an overview of brain cancers and current therapeutic options, followed by introducing the different types of nanocarrier systems used for brain cancer drug delivery.Then we will focus on some promising examples for improved brain cancer treatment outcomes using either internal or external stimulus-responsive nanocarriers.Finally, the clinical translation status of these nanocarriers will be discussed.

Brain Cancers
Brain cancers cause excessive levels of morbidity and mortality and are one of the leading causes of death in children in developed countries. [20]Many risk factors have been identified and investigated, however often with inconclusive results due to small sample sizes in individual studies and differences between studies in subjects. [21,22]Two important risk factors that are known for brain cancer are ionizing radiation and genetic factors. [23]enome-wide association studies have identified heritable risk alleles within 7 genes that are associated with an increased risk of glioma, while, interestingly, a history of allergies or atopic disease(s) results in a decrease in risk. [22][26] However, as aforementioned, these studies have their limitations.
Despite the recent advances in cancer treatment, the survival rates of brain cancer patients are often low with a median survival time of a few months in the case of some gliomas. [27]Brain tumors can arise from different origins including nerves, dura, pituitary gland, or glial cells.The brain cannot expand to make space for a growing mass; therefore, growing tumors compress normal tissue and can cause symptoms like seizures, stumbling, difficulty when walking, weakness on one side of the body, and vomiting. [28]More than 120 types of brain tumors have been reported. [29]These were traditionally classified based on their histological characteristics by the World Health Organization.However, since the 2016 revised fourth edition, the classification increasingly relies on molecular biomarkers.This enables a more accurate diagnosis, which assists the clinician to adjust the treatment plan depending on the tumor type, size, and location. [30]reatment of brain tumors faces great challenges because of tumor heterogeneity, including multiple distinct sub-populations of cells with different biological responses. [31]Gaining more insight into these inter/intra patients' brain tumor heterogeneities is the key to the successful development of personalized treatments.

Current and Emerging Treatment Options
Treatment of brain cancers relies on several factors: 1) the type, location, and grade of the tumor, 2) the patient's age and health status, and 3) the risk of side effects. [32]Typically, the first line of treatment for brain tumors is surgery to resect the tumor with minimal damage to the surrounding healthy tissue. [33]In most cases, radiation therapy follows surgery to destroy any residual tumor cells.Radiation can cause side effects such as nausea and swallowing difficulties.To minimize side effects, radiation is targeted to the exact tumor site and patients often receive a radiotherapy immobilization mask to make the radiotherapy as accurate and effective as possible. [34]Recently, proton therapy is also emerging.Proton therapy has a clear dosimetry advantage with reduced neuro-cognitive disability and improved quality of life but has limited availability. [35]As a third line of treatment, chemotherapy using anticancer drugs is used to treat brain tumors.These drugs are usually administered intravenously or orally.However, as chemotherapeutics also damage healthy cells, this approach often causes side effects, such as nausea, vomiting, or loss of appetite, which limits the maximum tolerated dose. [36]emozolomide (TMZ) is the most commonly used chemotherapeutic agent for the treatment of brain tumors. [37]TMZ is an alkylating prodrug that methylates purine bases, resulting in structural damage to DNA.However, in patients who lack the O6methylguanine-DNA methyltransferase (MGMT) promoter, resistance to TMZ is observed over time. [38]The anti-angiogenic agent bevacizumab is also sometimes used as a second-line therapy in recurrent GBM.However, it is less effective compared to standard TMZ plus radiotherapy. [39]nother interesting U.S. Food and Drug Administration (FDA)-approved technology is alternating electrical fields, also known as tumor-treating fields.Extension of the survival time of patients with newly diagnosed GBM, recurrent GBM, and mesothelioma is suggested to occur through the induction of anti-proliferative effects via disruption of the mitotic spindle. [40]oreover, polymeric wafers have been introduced as an advantageous route for delivering chemotherapy drugs directly to brain cancer tumors. [41]For example, Gliadel, a poly-[bis(pcarboxyphenoxy propane) sebacic acid] implant has been approved by the FDA for delivery of carmustine. [41]This wafer has shown enhanced overall survival in patients with recurrent GBM as well as in patients with newly diagnosed GBM. [42]However, Gliadel is associated with some clinical challenges such as postoperative infection and cerebral edema. [43]ecently, oncolytic virus therapy has emerged as a promising avenue for the treatment of brain cancer, offering a novel approach to combat this challenging disease.Oncolytic viruses are designed to selectively infect and replicate within cancer cells due to various molecular and genetic differences between cancer cells and normal cells.This selectivity helps to minimize damage to healthy tissue.Once inside the cancer cell, the oncolytic virus replicates and causes cellular oncolysis which leads to the recruitment of proinflammatory/anti-tumoral immune mediators.G47∆ (Delytact, Daichii Sankyo) is an oncolytic herpes simplex virus that achieved successful outcomes in terms of survival benefit in GBM patients (phase II trial, UMIN000015995) and it is the first conditionally clinically approved oncolytic virus treatment in Japan. [44]Another oncolytic virus treatment called CAN-3110 was also recently trialed in GBM patients (NCT03152318).This treatment induced enrichment of tumor-infiltrating lymphocytes.In addition, oncolytic virus therapy can be used in combination with other cancer treatments.For example, DNX-2401 -an oncolytic adenovirus engineered to treat GBM-was recently trialed (CAPTIVE phase 1/2 clinical trial) in combination with pembrolizumab which met the safety endpoint and achieved survival benefits in some patients. [45]mmunomodulatory nanocarriers also hold great promise in the treatment of brain tumors, where the traditional therapeutic options are often limited by the BBB and the immune-privileged nature of the central nervous system.These nanocarriers can be engineered to carry antigens, immunomodulatory agents, or chemotherapeutic drugs while bypassing the BBB through decoration with specific targeting ligands.As an example, Yang et al. recently developed a glucosylated and multi-polyethylene glycol (PEG)ylated formulation for programmed death-ligand 1 (PD-L1) antibodies. [46]Glucose moieties target the glucose transporter-1 (GLUT1) which is overexpressed in the brain tumor vasculature.This results in enhanced localized accumulation in tumors that the PD-L1 antibodies are detached from the nanoconstruct and activated in the reductive microenvironment of tumor tissue due to the incorporation of redox-responsive PEG linkers.This tumor-specific activation mechanism leads to a significant reduction of immune-related adverse events compared to free antibodies.Immunomodulatory nanocarriers designed around activation via external stimuli have also been explored.Sun et al. developed a system called Trojan bacteria. [47]These attenuated bacteria were loaded with glucose polymer-modified indocyanine green (ICG) loaded silicon nanoparticles through interaction with bacteria-specific adenosine triphosphate (ATP)binding cassette transporter.Under 808 nm radiation, the photothermal effects of ICG lead to the lysis of bacteria carriers and the release of proinflammatory pathogen-associated molecular patterns in the glioma tumor microenvironment.This stimulus-responsive carrier was successfully validated in an orthotopic glioma murine model.As our understanding of the immune system deepens and nanotechnology continues to advance, the potential for immunomodulatory nanocarriers to revolutionize the field of medicine and significantly improve patient outcomes is becoming increasingly evident.
In summary, while there are currently some treatments in clinical use for brain cancers, there is still a pressing need for additional treatments that could offer better therapeutic outcomes to patients including targeted and stimulus-responsive delivery systems for chemotherapeutics, gene therapies, and/or immunomodulatory agents.

Blood-Brain Barrier: The Main Challenge for Brain Cancer Drug Delivery
Due to the unique characteristics of brain cancers, including their anatomical location, they are challenging to cure.Any collateral damage during surgery or radiation therapy might result in significant cognitive impairments and reduced quality of life. [32,48]he use of chemotherapeutics is further complicated as brain tissue is protected by the BBB, which regulates the homeostasis of CNS through the neurovascular unit. [49]The BBB protects the brain from pathogens and toxins and only lets specific compounds like small lipid-soluble molecules and nutrients enter the brain.The BBB consists of different types of cells, including endothelial cells (ECs), pericytes (PCs) which are responsible for regulating blood flow, neurons, and astrocytes (ACs) which connect the brain capillary and neurons.There are three kinds of junctions in the BBB, namely, adherent junctions, tight junctions, and gap junctions. [50]Tight junctions are the main contributor to the high trans-endothelial resistance of the BBB. [51]This is further augmented by the ACs and PCs encapsulating the blood capillary ECs. [52]everal pathological changes can affect the function of the BBB in brain cancers, which results in a tumor-specific delivery pattern of chemotherapeutic agents. [53]As a result of excessive vascularization of the tumor tissue, part of the BBB transforms into the blood-brain tumor barrier (BBTB) which often refers to a potentially more leaky BBB with disrupted physiological functions in brain tumors. [54]BBTB capillaries are larger, dilated, and have thicker basal membranes.Loss of astrocytic end feet is also often observed in BBTB.In contrast to the normal BBB, the BBTB also contains large vacuoles and pinocytic vesicles. [54]However, this disruption is highly heterogeneous. [55] better understanding of these cellular and molecular features of the BBB and BBTB can lead to improvements in the treatment of brain tumors. [56]In this context, nanosized drug delivery systems are emerging as an effective and noninvasive system to treat brain tumors.Surface functionalization of nanocarriers with targeting ligands can mediate the BBB crossing via receptor-mediated transcytosis which enhances the local concentration of therapeutic payload in brain tumor tissue. [57]Nanocarriers can also be delivered to brain cancers using other routes of administration such as the intranasal route.The latter can bypass the BBB where olfactory epithelium serves as a gateway for CNS and peripheral circulation.For example, the spatiotemporal biodistribution of gold nanorods (AuNRs) following intranasal administration and their subsequent brain uptake has been demonstrated. [58]The results obtained from autoradiography of sagittal brain sections indicated that radioisotope-labeled AuNRs entered the brain through the olfactory bulb and diffused to various other regions of the brain within a time frame of approximately one hour.Microbubble (MB)-assisted, low-frequency, focused US (FUS) has also shown promise in enhancing the delivery of nanocarriers to the brain by temporarily opening up the BBB via the US-induced MB cavitation. [59]The different strategies for targeting the BBB using nanocarriers will be discussed in the next section.

Targeting Brain Cancers Using Nanocarriers
Nanocarriers have emerged as versatile toolboxes with unique properties that can be harnessed for a variety of purposes, including drug delivery, imaging, and sensing [60] (Figure 1).Both organic and inorganic nanocarriers have gained significant use in medical treatment and diagnosis.These drug delivery systems can enhance drug stability, target specific cells or tissues, and control release kinetics, thereby improving therapeutic efficacy and minimizing side effects. [61]Their tunable properties and versatile surface modifications enable precise drug encapsulation and delivery, in other words enabling precision medicine. [62]In addition, stimulus-responsive nanomaterials can be used that respond to environmental cues, making them ideal for controlled drug delivery. [63]Targeting methods for these nanocarriers can be passive -taking advantage of the enhanced permeability and retention (EPR) effect in some tumor tissues, or active -using ligands such as antibodies to selectively deliver nanocarriers to specific cells or tissues. [64]

Passive and Active Targeting of Nanocarriers
Nanocarriers can promote the delivery of anticancer drugs into tumors via two mechanisms: passive targeting and active targeting.This results in a higher level of localized accumulation of tumor tissue, and fewer side effects. [65]Passive targeting is usually attributed to the EPR effect caused by the leaky vasculature of most tumors and resulting in increased accumulation of nanocarriers into tumor tissue by ≈20-30%. [66]Unlike normal blood vessels, the vasculature in tumors is irregularly formed and has gaps between endothelial cells. [67]This structural abnormality leads to increased permeability, allowing for the passive diffusion of molecules and nanocarriers into the tumor interstitium. [68]he EPR effect is further enhanced by the impaired lymphatic drainage within the tumor.Lymphatic vessels, responsible for the removal of excess fluid and waste products, are often dysfunctional in tumors.As a result, the accumulation of substances within the tumor microenvironment is prolonged, promoting the retention of drugs or nanocarriers. [69]The EPR effect has been IL-13R2 Interleukin 13 receptor [111]   Internalized-arginylglycylaspartic acid cyclic peptide (iRGD) Neuropilin-1-mediated receptors [112]   KDKPPR Des-Arg9-Kallidin (B1L) NRP-1 receptor B1 receptor [113, 114]   Antibodies Anti-EGFRvIII EGFR receptor [115]   Cetuximab (Erbitux, IMC-C225) EGFR receptor [116]   Single-chain Fv (scFv), M25, M58, and M89 Unknown receptor [117]   Nimotuzumab EGFR receptor [118]   Small molecules Folic acid Folate-receptor [119]   L-Histidine LAT1 receptor [109]   extensively studied and harnessed for targeted drug delivery. [68]lthough the EPR effect is a well-established strategy in some cancers, it is more complex and difficult to exploit for brain cancers due to the presence of the BBB and BBTB. [70]ctive targeting can be consecutive to passive targeting or happen independently. [71]Here, nanocarriers are decorated with targeting ligands, such as antibodies or peptides, to target overexpressed receptors on the surface of the BBB and tumor cells. [72]n ideal nanocarrier would first follow receptor-mediated transcytosis in brain microvascular endothelial cells followed by receptor-mediated endocytosis in brain cancer cells.
Several specific transporters and receptors are expressed on the endothelial cells of the BBB, facilitating the transport of various molecules across the brain.For example, transferrin binds to the transferrin receptor to facilitate the transport of iron across the BBB [73] and insulin is transported by the insulin receptor. [74]lucose transporters, specifically GLUT1, are also highly ex-pressed in the endothelial cells of the BBB. [75]Other present receptors are the choline transporter-like protein 1, [76] adenosine receptor, [77] and the low-density lipoprotein receptor-related protein 1 (LRP1). [78]An overview of these receptors and ligands that have been exploited for nanocarrier delivery is presented in Table 1.

Types of Nanocarriers
Various nanocarriers have been exploited for the delivery of anticancer drugs to brain cancers in recent years.Nanocarriers are prepared using diverse materials that can be tailored to improve their pharmacokinetic properties and drug release profiles.[122][123] The main categories of nanocarriers -including polymeric nanocarriers, lipid-based nanocarriers, and inorganic nanocarrierswill be discussed next.

Polymeric Nanocarriers
Polymeric systems have shown great promise for brain drug delivery.They present many advantages including ease of preparation, controlled drug release, relatively high storage stability, and possible functionalization with targeting ligands and imaging probes. [124,125]Polymeric systems can also be engineered to prolong the circulation time of drug payload and maximize brain uptake. [126]Loading of anticancer drugs to polymeric nanocarriers is achieved using different methods such as surface adsorption, covalent bonding, or physical encapsulation within the polymeric core of the nanocarrier. [126] promising class of polymeric nanocarriers are block copolymer micelles prepared using amphiphilic or oppositely charged copolymers. [127]Block copolymer micelles possess a hydrophilic shell that can modulate their pharmacokinetic properties. [128]hey are usually employed to deliver water-insoluble drugs and can be prepared at a wide range of sizes; extending from 10 to 200 nm. [129]Various active targeting strategies have been explored, to facilitate transport across the BBB, using block copolymer micelles encouraged by the ease of surface functionalization. [130]FDA-approved poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), and their derivatives are the most extensively used polymers in brain drug delivery research due to their biocompatibility and low toxicity. [131]In a recent study by Chen et al., [132] transferrin receptor-targeting T12 peptidemodified PEG-PLA micelles were utilized to deliver paclitaxel (PTX) to glioma.The hydrophobic PTX was loaded into the PLA core for enhanced crossing of the BBB and increased uptake by glioma in mice.
Polymersomes are vesicular polymeric nanocarriers with an enclosed aqueous cavity and a shell of self-assembling amphiphilic block copolymers. [133]This structure allows them to encapsulate both hydrophilic and hydrophobic drugs, with versatile drug-loading capabilities.The stable and tunable membrane structure, alongside the ability to attach targeting ligands, are among the key advantages of polymersomes, especially for applications that require co-delivery of multiple therapeutics and enhanced stability in biological environments.Polymersomes have been extensively studied for the treatment of brain cancer. [134]In another recent work, ANG2decorated polymersomes were prepared to co-deliver small interfering RNA (siRNA) and TMZ to GBM. [135] In this study, polymersomes were designed by self-assembly of ANG2 decorated poly(ethylene glycol)-block-poly-(2,2,3,3-tetrafluoropropyl methacrylate) di-block copolymers and poly(ethylene glycol)block-poly-(2,2,3,3-tetrafluoropropyl methacrylate)-block-poly[(N-(3-methacrylamidopropyl) guanidinium) tri-block polymers.The presence of a fluorinated hydrophobic layer provided stability to the self-assembled polymeric system and helped in the encapsulation of TMZ.On the other hand, cationic guanidinated polymer chains assembled themselves within the aqueous interiors and encapsulated negatively charged siRNA.Upon uptake by glioma cells, the delivered siRNA increased sensitivity to TMZ by suppressing the expression of retinoblastoma binding protein 4. Consequently, the downregulation of this protein effectively regulated the DNA-damage repair process related to TMZ treatment, specifically targeting the MGMT pathway.
Dendrimers represent another category of polymeric drug delivery systems.They are described as highly branched macromolecules with a 3D tree-like shape. [136]Their precisely defined structure allows for controlled drug encapsulation and release.Dendrimers can be functionalized with various groups for targeting specific cells or tissues, and their small size offers a high capability to penetrate biological barriers. [137,138]The surface of a dendrimer can be designed with amines, hydroxyl, or carboxyl groups to create positive, neutral, or negative surface charges. [66]The most common example of dendrimers is based on poly(amidoamine) (PAMAM) which is often modified with PEG to reduce its toxicity and prolong its blood circulation time. [139]Anticancer drugs, such as PTX and Doxorubicin (DOX), can be loaded into the core via hydrogen bonds, hydrophobic, or electrostatic interactions.Covalent conjugation to the surface functional group can be also employed. [140]Dendrimerbased nanocarriers have shown promise in targeting neuroinflammation and brain tumors. [141,142]For example, mixedsurface PAMAM dendrimers containing 10% terminal amine groups [143] demonstrated high capability to cross the BBB.The dendrimer possessed favorable positively charged surface properties, enabling its cellular uptake, potentially through a process known as adsorptive endocytosis.Moreover, when administered via carotid injection, the dendrimer was able to successfully traverse the BBB and enter the brain tissue.
Nanogels are defined as physically or chemically cross-linked polymeric nanosized hydrogels. [144,145]These nanocarriers have a high water content, are biocompatible, and have outstanding mechanical features such as high fracture strength, high toughness, and high resistance to wear. [146]Due to their tunable physicochemical properties, they are promising systems for drug delivery. [147]Nanogels are often prepared with functional groups to facilitate the covalent conjugation of drugs. [148]It is possible to the size of nanogels (50-200 nm) to promote nanocarrier diffusion across the BBB. [144]Furthermore, nanogels possess the ability to enhance the solubility of hydrophobic drugs by incorporating them either within their outer shell or by attaching them into the gel structure. [148]Recently, She et al. [149] reported a hypoxia-degradable zwitterionic phosphorylcholine nanogel for delivery of DOX to GBM.The prepared nanogels showed acceptable biocompatibility and blood serum anti-fouling characteristics in mice with enhanced BBB crossing.The cell membranemimicking structure of the phosphorylcholine polymers led to increased accumulation in GBM tissue.

Lipid-Based Systems
Lipid-based drug delivery systems, especially liposomes and solid lipid nanocarriers (SLN), have been used extensively in the delivery of a wide range of drugs and biomolecules for several applications. [150]Lipid-based nanocarriers have recently played a key role as non-viral nucleic acid delivery systems that have been approved for clinical use. [151]They can efficiently deliver nucleic acid payloads.The functional component of these lipid nanocar-riers is usually an ionizable lipid, which helps efficient encapsulation of nucleic acids, delivery to cells, and endosomal escape. [152]iposomes are vesicular nanocarriers composed of a phospholipid bilayer enclosing an aqueous core.Due to its amphiphilic nature, both hydrophobic and hydrophilic drugs can be loaded into the phospholipid bilayer and aqueous core of liposome, respectively. [153]Liposomes also provide stabilization of drugs against the external environment and modulated release kinetics of drug payload.Surface functionalization of liposomes with targeting ligands and imaging probes is facilitated by the versatile availability of commercially supplied functional phospholipids.However, long-term storage stability and possible leakage of loaded drugs represent an ongoing challenge, [154] which could be mitigated through lyophilization or coating with polymeric nanomaterials.157] A recent study by Straehla et al. [158] presented a novel brainpenetrant ANG2-functionalized liposome exploring the LRP1mediated transcytosis (Figure 2).Layer-by-layer coating of liposome core was employed using poly-(L-arginine) and poly-(Laspartic acid), which was further functionalized with ANG2.This layer-by-layer surface functionalization process was suggested to create a denser and more uniform coating on the nanocarrier surface, hence reducing the potential for nonspecific interactions with endothelial cells of the BBB.First, the ANG2-functionalized liposomes presented a significantly higher association with GBM spheroids, compared to bare liposomes (Figure 2b,c).Then, an innovative microfluidic model of vascularized GBM was used to investigate the nanocarrier permeability across the BBB and into GBM cells simultaneously.This BBB-GBM model features a GBM spheroid embedded within a network of brain blood vessels, recapitulating the in vivo infiltrative phenotype of GBM (Figure 2d).The increased permeability, observed from ANG2 nanocarriers near the GBM tumor, suggested that the ANG2 peptide is effectively targeting the overexpressed receptors on the tumor vasculature (Figure 2e).Further, cisplatin (CDDP)-loaded NPs were examined for GBM chemotherapy (Figure 2f-h).Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of extracted regions from the BBB-GBM devices revealed elevated levels of Annexin V, Caspase 3, and Caspase 7 transcripts in GBM tumors treated with ANG2 CDDP NPs, compared to other CDDP formulations.There were no major differences in the expression of apoptosis-related genes far from the tumor for the different CDDP formulations.To assess the predictive capability of the BBB-GBM device, the same CDDP nanocarriers formulations were tested in an orthotopic xenograft model using patient-derived GBM cells (Figure 2i,j).Tumor volume was measured using MRI before and after a short dosing period, mirroring the timeframe of the in vitro studies.Animals treated with ANG2 CDDP NPs exhibited slower tumor growth compared to those treated with equivalent doses of free CDDP, aligning with the enhanced NP accumulation observed in vitro.
In another work, Rosenblum et al. [159] designed lipid nanocarriers to encapsulate Cas9 messenger ribonucleic acid (mRNA) and single-guide RNA (sgRNA) by using ionizable cationic properties of the lipids from a novel ionizable amino lipid library for brain cancer treatment.For therapeutic genome editing, the research group investigated the potential of cationic lipids loaded with polo-like kinase 1 (PLK1) sgRNA compared to control green fluorescent protein (GFP) targeting sgRNA loaded into cationic LNPs.PLK1 is a kinase essential for mitosis, and its absence leads to cell cycle arrest at the G 2 -M phase and cell death in dividing cells.In this research, in vitro experiments demonstrated gene editing rates of up to 98% in various cancer cell types, while in vivo studies showed ≈80% gene editing.By targeting the PLK1 gene, cationic LNPs effectively inhibited tumor growth and increased survival in aggressive cancer models in mice.Administration of a single or double dose of sgPLK1-cationic LNPs resulted in significant gene editing of the PLK1 gene, induction of apoptosis in vivo, extended median survival by ≈50%, and improved overall survival in mice with GBM tumors by 30%.Importantly, PLK1 gene editing induced a robust G 2 -M cell cycle arrest 48 h later, whereas the control sgGFP-cationic LNPs had no impact on the cell cycle profile.This demonstrates the specific and potent effect of PLK1 gene editing using sgPLK1-cationic LNPs as a therapeutic approach.
Kadiyala et.al. developed a high-density lipoprotein (HDL) nanoparticle for docetaxel (DTX) and cholesterol-modified CpG (a toll-like a receptor agonist). [160]This nanoformulation is an interesting platform from the drug delivery point of view as HDL formulations are naturally targeted to tumor tissues through interaction with scavenger receptors.Intratumoral administration of DTX-CpG nanoformulation in an orthotopic GBM murine model was associated with enhanced efficacy and limited systemic toxicity.Due to the high tolerated dose of HDL formulations and well-established safety profile, this formulation offers great advantages from the translational point of view.
SLNs are generally composed of biocompatible solid lipids encapsulating lipophilic drugs in a matrix core.They show superior tolerability, improved storage stability compared to liposomes, and the feasibility of scale-up.SLNs have been used to mediate site-specific drug delivery, achieve sustained drug release profiles, enhance drug bioavailability, and stabilize drugs during storage. [161]Several research groups have investigated the use of SLNs for brain tumor delivery. [162]A study by Kuo et al. [163] explored the use of SLNs, dual-conjugated to lactoferrin and tamoxifen (TX), for the delivery of carmustine across the BBB.The conjugated lactoferrin was employed to mediate transcytosis across the BBB and into GBM through receptor-mediated signaling pathways.The incorporation of TX, a selective estrogen receptor modulator, was used to prevent drug efflux.In a Transwell BBB model, the dual-conjugated SLNs showed ≈10 times higher permeability coefficient of carmustine compared to the bare SLNs.The dual-conjugated SLNs extended the release of carmustine and showed the highest growth inhibitory effect in U-87 MG cancer cells, compared to the different controls.

Inorganic Systems
Inorganic nanocarriers have gained great attention due to their unique optical, electronic, and magnetic features attributed to their high surface-to-volume ratio. [164,165] Copyright 2022, The Authors.
MSNPs consist of hundreds of empty channels arranged in a highly ordered 2D or 3D porous structure.Their ordered porous structure provides a large surface area and high drugloading capacity.This allows for efficient encapsulation and controlled release of therapeutic agents.Furthermore, MSNPs can be easily functionalized with targeting ligands and imaging agents, enabling precise delivery and monitoring of therapeutic interventions. [166]Among the unique properties of MSNPs are their high chemical, mechanical, and thermal stability.Besides, MSNP pore diameters can be adjusted in the range of 2-50 nm with pore volumes reaching 0.9 cm 3 g −1 , and surface areas exceeding 700 m 2 g −1 . [167,168]For example, Asn-Gly-Arg (NGR)-modified, polydopamine-coated, DOX-loaded MSNPs were used for targeted pH-responsive drug delivery to brain gliomas. [169]This targeted drug delivery system demonstrated improved anti-angiogenesis and anti-glioma efficacy in vivo.In another study, DOX-loaded ultra-small porous silica nanocarriers coated with lactoferrin -whose receptors are overexpressed on both BBB and GBM-were able to selectively cross the BBB in an in vitro assay and showed improved penetration in U-87 tumor spheroids, compared to uncoated particles. [170]agnetic nanoparticles (MNPs) have emerged for both therapeutic and diagnostic applications such as MRI and magnetic particle imaging.Their core size and surface coating can be readily adjusted to control their physicochemical properties and several iron oxide nanocarrier formulations have been approved for clinical applications, although with varying levels of success. [171,172]NPs can be divided into two categories: i) superparamagnetic iron oxide (SPIO) and ii) ultra-small superparamagnetic iron oxide (USPIO), and are sized between 10 to 100 nm. [173,174]Due to their magnetic properties, MNPs can potentially be directed using external magnetic fields.For example, an electromagnet enabled enhanced brain tumor targeting of a long-circulating PEG-cross-linked starch MNP. [175]In addition, Shevtsov et al. developed a hybrid SPIO system containing a chitosan-dextran coating.As compared to dextran-coated nanocarriers, chitosandextran-conjugated NPs were internalized more efficiently by U-87, C6 glioma, and HeLa cells. [176]uNPs are a good example to illustrate the multifunctional properties of inorganic nanocarriers tailored for brain cancer therapy and imaging. [177,178]There are several reasons why AuNPs are becoming increasingly popular in this area of research. [179]AuNPs have high optical properties due to the localized surface plasmon resonance, [180] potential as a radiosensitizer, a flexible surface chemistry that allows for the addition of functional groups, [181] and last but not least, the ability to control particle size and shape during synthesis.Epidermal growth factor peptide-targeted gold nanocarriers were investigated for delivering photodynamic therapy (PDT) agents, specifically silicon phthalocyanine (a second-generation photosensitizer), to brain cancer cells.This nanocarrier reduced the metabolic activity of tumor cells two-fold more efficiently than silicon phthalocyanine, according to an MTT assay. [182]Ds are semiconductor-based nanomaterials with a size between 2 and 10 nm. [183]Their optical features are directly dependent on their size.QDs are bright, resistant to photobleaching, and have a high surface-to-volume ratio which makes them a potential candidate as a diagnostic as well as a nanocarrier for drug delivery. [184]QDs can also be coated with PEG or other polymers to prolong their circulation half-life. [185]The first generation of cadmium and selenium QDs had some safety concerns as released cadmium ions are highly toxic, but this was addressed via substitution by other elements, like silicon, carbon, or another cadmium-free combination. [185]A biofunctionalized ZnS quantum dot was coated with carboxymethyl cellulose and loaded with DOX electrostatically. [186]In vitro, these nanohybrids were non-cytotoxic and were effectively taken up by brain cancer cells.Li et al. reported large amino acid-mimicking carbon QDs (LAAM CQDs) for selectively imaging and delivering drugs to tumors without harming normal tissue. [187]LAAM CQDs refer to a cluster of CQDs featuring paired -carboxyl and amino groups on their edges, enabling them to engage in multivalent interactions with large neutral amino acid transporter 1.Through this research, the researchers discovered that a specific type of LAAM CQDs called LAAM TC-CQDs, which were synthesized using 1,4,5,8-tetraminoanthraquinone and citric acid, possessed near-infrared fluorescence and photoacoustic imaging capabilities.Notably, these LAAM TC-CQDs demonstrated the ability to image and transport chemotherapy drugs to tumors, including those located in the brain.
CNTs also have unique properties that can be exploited for brain cancer treatment such as high surface area and high aspect ratio. [188]Generally, CNTs are divided into two categories singlewalled nanotubes and multi-walled nanotubes (MWNTs). [189,190]he diameters of these nanotubes range between 1 and 100 nm, and they are usually capped with half of a fullerene molecule at both ends.A CNT is a cylinder composed of one or more coaxial graphite layers with a diameter in the nanometers range. [188]Moreover, their ability to be functionalized with a wide range of bio/chemical species opens up numerous medical applications. [191]The common synthesis methods of CNTs are plasma-based synthesis methods that consist of arc discharge and laser ablation techniques. [188]Anticancer drugs can be loaded either by covalent conjugation using a linker, [192] or non-covalently to the surface via - stacking, hydrophobic interaction, or electrostatic adsorption. [193]Anionic, cationic, and non-ionic functionalized MWCNT (fMWCNT) were used to investigate CNT mass internalization into and transportation across the BBB. [194]his interesting work demonstrated the effect of surface charge on CNT efficacy for brain cancer treatment.Although cationic and non-ionic fMWCNT showed high endothelial cell association, they were not able to cross the BBB, in contrast to anionic fMWCNT.PEGylated oxidized MWCNTs decorated with ANG2 were used to deliver DOX to gliomas. [195]In glioma-bearing mice, the targeted MWCNTs showed more effective anti-glioma properties, compared to free DOX.Their biocompatibility and low toxicity make them a promising vehicle for delivering drugs to brain tumors.
pSiNPs have unique properties due to their high surface area, porous structure, and their intrinsic biodegradation into nontoxic orthosilicic acid.PSiNPs can be synthesized with a variety of sizes and their surface chemistry can be modified to allow for targeted delivery of therapeutic agents. [196]Targeted pSiNPs have shown the ability to cross the BBB and accumulate in brain tumors. [197,198]pSiNPs can be loaded with a variety of drugs and imaging agents, such as chemotherapy drugs, siRNA, and  [198] Copyright 2018, Springer Nature.
201] In one study, we investigated the potential use of polyethyleneimine (PEI)-capped pSiNPs for siRNA delivery to GBM (Figure 3). [198]PEI is a cationic polymer that can complex the negatively charged siRNA molecules to prevent its degradation and promote cellular uptake.In vitro studies using U-87 cells showed that the PEI-capped pSiNPs efficiently delivered siRNA to silence the multidrug resistance-associated protein 1 (MRP1) gene, which is overexpressed in GBM and contributes to drug resistance and tumor proliferation.We observed a significant decrease in cell proliferation and increased cell death following treatment with the siRNA-loaded PEI-pSiNPs, compared to untreated cells or cells treated with non-capped siRNA-loaded pSiNPs.In vivo studies, conducted with a mouse model of GBM, showed a significant decrease in MRP1 expression and tumor proliferation in mice treated with the siRNA-loaded pSiNPs, compared to untreated mice or mice treated with non-targeting siRNA-loaded pSiNPs.To assess the proliferation potential of GBM cells with MRP1 knockdown, the expression of Ki67 in GBM tumors was also analyzed.Analogous to in vitro studies, mice treated with PEI-pSiNP/siRNA exhibited reduced Ki67 expression in their tumors compared to the control group (Figure 3e).This reduction in Ki67 was observed at both 48 and 72 h after treatment, indicating sustained retardation in proliferation even after the restoration of nascent MRP1 mRNA and protein levels.
Recently, we also reported a nanocarrier of pSiNPs with an antisense oligonucleotide (AON) as a targeted gene delivery plat-form for GBM treatment. [197]Carbodiimide coupling was utilized to conjugate amine-terminated AON to undecylenic acidfunctionalized SiNPs (UnSiNPs) (Figure 4).Their internalization was investigated in both the U-87 GBM cell line and hCMEC/D3 endothelial cells.The transmission electron microscopy (TEM) images revealed that the nanocarriers formed clusters within membrane-wrapped vesicles, providing evidence of their internalization in the specific targeted cell lines.The selective uptake of Cy-labelled AON@pSiNPs was examined using confocal microscopy imaging.As a control, HEK293, a human embryonic kidney cell line, was used and a notable disparity in fluorescence intensity was observed (Figure 4d).Given the increased internalization of AON@pSiNPs in GBM cells and hCMEC/D3, their ability to permeate across the BBB was further examined using a microfluidic BBB-GBM model.Compared to the conventional Transwell BBB model, the microfluidic BBB model better mimics the physiological complexity of the BBB by allowing fluid shear stimulus, and simulating blood flow conditions (Figure 4e).Importantly, the microfluidic device enables direct visualization of nanocarrier permeability across the BBB under flow conditions, by means of confocal microscopy.Notably, hCMEC/D3 cells exposed to AON@pSiNPs exhibited significantly higher Cy5 fluorescence intensity, compared to cells exposed to UnpSiNPs.IVIS fluorescence imaging of an orthotopic GBM mouse model revealed that, after 6 h of treatment, mice treated with AON@pSiNPs exhibited a higher Cy5.5 fluorescence signal in the brain compared to mice treated with UnpSiNPs, as confirmed by ex vivo brain images (Figure 4f).iii.Schematic of the nanoparticle perfusion into the blood channel under shear stress.Insets are showing the selective binding of AON@pSiNPs to hCMEC/D3 cells in the blood channel following 3 h perfusion under shear stress.f,g) Brain uptake and biodistribution study of AON@pSiNPs in an orthotopic mouse model of GBM, following intravenous administration of Cy5.5-labelled nanocarrier using IVIS imaging.Reproduced with permission. [197]Copyright 2022, American Chemical Society.AP-1-conjugated liposome DOX Repeated pulsed high-intensity FUS [296]   Piezoelectric nanocarriers Nulin-3a Combination of chemotherapy treatment with chronic piezoelectric stimulation [297]   Cationic lipid-polymer siRNA US-targeted MBs destruction [230]   CDDP conjugated gold nanocarrier CDDP Enhancement in BBB permeability using FUS, and GNP-drug delivery to the brain [298]   Magnetic Hydrogel polymeric nanocarrier -Heating upon exposure to AMF leads to killing of M059K GBM cells [299]   Lipid-based magnetic nano-vector TMZ Enhanced drug release after exposure to AMF [255]   Polymeric nanocarrier Salinomycin Site-specific magnetic targeting using an external magnetic field [300]   SPIO nanocarrier -Receptor-mediated BBB trespassing and T2-weighted MR imaging [110]   Polymeric nanocarrier Oleic acid Applying an external magnetic field to enhance permeation across the BBB [254]   Light Photosensitizer nanocarrier -Photodynamic therapy [263]   Hybrid nanocarrier DOX Controlled release under NIR laser and receptor-mediated endocytosis [265]   pSiNPs ICG Photodynamic therapy [264]   Copolymer nanocarrier -Folic-acid-mediated targeting and photodynamic therapy [266]

Stimulus-Responsive Systems
Recently, numerous studies have investigated the use of stimulus-responsive systems for brain cancer delivery (Table 2, Table 3).Stimulus-responsive nanocarriers can enhance drug release in diseased sites in response to either an internal stimulus in the disease microenvironment or an externally applied stimulus.[204][205] Examples of internal stimuli include overexpressed tumor-specific enzymes, acidic pH in the tumor microenvironment, and increased levels of reactive oxygen species.Externally applied stimuli include magnetic field, FUS waves, light, radiofrequency pulses, and hyperthermia.Besides the localized drug release effect, external stimuli such as FUS waves can induce transient opening of BBB which can potentially lead to enhanced drug delivery.Moreover, the anti-tumor effects of drugs can be synergized with the biological effects of external stimuli.As an example, induced localized heating of photothermal and magnetic systems have reported to synergize with chemotherapeutic agents.The next section will discuss the latest advances in stimulus-responsive systems for brain cancer therapy in detail.

Ultrasound (US)-Responsive Systems
US-responsive systems are usually comprised of air-entrapping nanocarriers that release drugs in response to externally applied sound waves (20 kHz-1GHz).Usually, US waves are often described by their frequency, which can be expressed as the ratio between speed and wavelength. [206]US waves for medical purposes can be classified based on their frequencies into low frequency (20-200 kHz), medium frequency (0.7-3 MHz), and high-frequency waves(>3 MHz). [207]S-responsive nanocarriers can be formulated in different forms such as MB, nanobubbles, nanodroplets, liposomes, emulsions, and micelles. [208]For drug delivery applications, USresponsive systems offer some advantages such as biocompatibility, low cost, and simultaneous diagnostic imaging potentials with high spatial resolution. [209,210]Low-frequency pulses possess high tissue penetration ability and low attenuation which facilitates deep tissue imaging, as in the case of CNS imaging. [211]oreover, US-induced cavitation exposure can increase drug permeation through the cell membrane, if cells are located in the proximity of air-filled nanocarriers. [212]This enhanced cell permeation effect can be attributed to several mechanisms such as sonoporation, cellular sonication, cavitation, and localized hyperthermia. [213,214]Sonoporation introduces shear stress on the cell membrane and hence creates transient non-lethal holes in the membrane for more efficient transport of therapeutics drugs into the cell. [213,215,216]Cavitation augments the sonoporation effect by introducing MBs (exogenous) to the cellular microenvironment. [217]A cavitation effect occurs when bubbles form in a liquid if the ultrasonic intensity is sufficient, and the bubbles grow continuously as the ultrasonic wave expands, and then collapse violently, resulting in a variety of biological and thermal effects. [217]MBs, nanobubbles, and perfluorocarbon nanodroplets are the most common bubbles that produce cavitation in the US. [218]The cavitation process includes two different types namely, inertial cavitation and non-inertial (stable) cavitation.Internal cavitation happens at higher acoustic pressures when an MB quickly expands and violently collapses in the medium, while stable cavitation occurs at lower acoustic pressures without the violent collapse of MBs. [219]The US energy can be also converted into heat energy resulting in localized hyperthermia. [220]Figure 5 summarizes the different biological effects associated with the MB cavitation process under US stimulus. [221]igh-intensity FUS was first reported to modulate the BBB permeability by Bakay et al. in 1950. [222]Transcranial FUSassisted treatment of brain diseases was further investigated by Vykhodtseva et al. in 2003, when FUS combined with MBs was able to safely open the BBB. [223]In the years since then, several studies have demonstrated that FUS can enhance the delivery of nanomedicines to the brain by assisting the opening of the BBB.The US-assisted drug brain delivery technology and associated nanomedicines are referred to as US-assisted brain delivery nanomedicines.The technology includes using FUS alone to open the BBB to facilitate the introduction of drugs or nanomedicines into the brain or using FUS combined with the cavitation effect of MBs to penetrate the BBB.Despite the advantages of US-responsive systems, their efficient use is challenged by the effects of US energy on cell integrity.High US energy would potentially damage the cell membrane and cause the denaturation of proteins and DNA. [224]FUS-assisted treatment of brain cancers has also been applied in diffuse intrinsic pontine glioma, which is also referred to as diffuse midline glioma.This subtype of pediatric brain tumor is characterized by its intact BBB with resistance to most of the current treatment options. [225]ere, FUS-induced BBB opening has been explored in combination with chemotherapy and is now being investigated in clinical trials for pediatric brain tumor treatment. [226]n another study, nanodroplets were developed by Cheng et al. [227] comprising a perfluoropentane core and a poly(ethylene glycol)-poly(lactic acid-glycolic acid) shell.By applying ultrasonic sound pressure of 1.0 MPa, the ethidium bromide overflow induced by nanodroplets opened the BBB in a small central area and did not cause tissue damage, whereas by applying MBs, an overflow of ethidium bromide was caused in a broader area.A study by Lu et al. [228] reported the preparation of cilengitideloaded nanocarriers (CGT-NP) and their use for GBM treatment through combined US-targeted microbubble destruction (UTMD) and CGT nanotherapy.UTMD caused transient, reversible, and localized BBB disruption facilitating CGT-NP passage through the BBB and consequent accumulation and retention in brain tumor tissue.The biodistribution data in a rat GBM model showed around threefold increase in accumulation of CGT in brain tumor when CGT-NP was combined with UTMD, compared to free CGT.Moreover, the median survival period was extended from 30 to 80 days.Recently, USresponsive drug-loaded organic piezoelectric nanocarriers were also used efficiently to treat GBM. [229]Here, nutlin-3a-loaded ApoE-functionalized nanocarriers were remotely activated with US-based mechanical stimulations.This externally applied stimulus induced drug release and locally delivered anticancer electric signals leading to cell apoptosis and reduction of cell invasiveness in drug-resistant GBM cells.
Recently, another group [230] prepared a cationic lipid-polymer hybrid nanocarriers (Cat LPH) which are co-loaded with rhodamine B and Cy5-siRNA.As a result, the median particle size increased from 40 to 50 nm, and surface charge decreased from +26 to +11 mV (Figure 6a).The experimental protocol for brain uptake study of Cat LPH is presented in Figure 6b, where a 0.5 MHz transducer with standard exposure settings (10-ms bursts, every 1 s for 1 min, at 475-kPa peak negative pressure in water) was used.Ex vivo fluorescence scans of the excised healthy mouse brain showed around twelve-fold increase in the extravasation of Cat LPH in the FUS-treated region, as compared to the non-FUS region (Figure 6c).Moreover, around a five-fold increase in Cat LPH accumulation in FUS-treated tumors was achieved, as compared to control (Figure 6d,e).This variation in the observed accumulation compared to healthy brains aligns with the diverse characteristics of the brain tumor microenvironment.The system used enabled precise targeting Figure 5. Schematic cavitation processes of microbubbles (MB) under US stimulus and the subsequent biological effects on the BBB.At low acoustic pressure, MBs undergo stable cavitation with cycles of compression and expansion resulting in push-and pull-interaction and radiation force effects on the BBB.Carrier protein-/receptor-mediated transcytosis and caveolin-mediated endocytosis are upregulated, while tight junction proteins are downregulated in response to stable cavitation.At relatively high acoustic pressures, MBs tend to collapse producing microjets, shock waves, sonoprinting, and sonoporation.These forces can induce cellular membrane perforation and large-scale opening of the BBB.Reproduced with permission. [221]Copyright 2022, Elsevier.
at submillimeter levels and allowed for real-time monitoring of MB behavior by detecting MB acoustic emissions during sonication.To confirm the delivery of siRNA in medulloblastoma cells, fluorescence in situ hybridization was conducted.The researchers proceeded to evaluate the impact of the delivered therapeutic siRNA on smoothened gene (SMO) knockdown and cell apoptosis in medulloblastoma brain tumors.Immunohistochemistry analysis demonstrated a decrease in the expression of constitutively activated SMO protein, which is responsible for driving the Sonic Hedgehog signaling pathway and tumorigenesis in this particular model of Sonic Hedgehog medulloblastoma.This reduction in SMO protein expression indicated the effective knockdown of SMO (Figure 6f,g).To ensure that the observed apoptosis was not caused by rhodamine B-LPH toxicity, the researchers analyzed GL261 GBM tumors treated with non-targeting Cy5-siRNA.They found comparable levels of cell apoptosis between FUS-treated and non-FUS-treated tumors 8 h after administering LPH: Cy5-siRNA nanocarriers (Figure 6h).Copyright 2021, American Association for the Advancement of Science.

Thermo-Responsive Systems
Thermo-responsive nanocarriers are formulated using nanomaterials whose physicochemical properties are altered in response to mild hyperthermia (40-45 °C), leading to the release of drug payload. [231,232]In most cases, this localized mild hyperthermia is generated using external sources of energy such as radiofrequency thermal ablation, microwave, alternating magnetic field, light, or high-frequency US waves. [233]Heated water has been also investigated as a source of heat energy through thermal conduction, however, its tissue penetration depth is limited to 3-5 mm. [234][237] Thermo-sensitive liposomes are usually formulated through the assembly of phospholipids with average gel-to-liquid crystalline transition temperatures slightly above the physiological temperature such as 1,2-dipalmitoyl-snglycero-3-phosphocholine (≈41 °C), where mild hyperthermia can increase the permeability of the phospholipid bilayer through phase transition from gel to liquid-crystalline state to release its payload.

Magnetic-Responsive Systems
The multifunctional properties of magnetic nanomaterials offer great potential for targeted drug delivery and diagnostic imaging of brain cancer.Magnetic nanocarriers can be actively directed to cancer tissue under the effect of an externally applied magnetic field.An alternating magnetic field (AMF) can also be used to generate localized hyperthermia for tumor-specific drug release of thermo-responsive nanocarriers. [238,239]242] Enhanced colloidal stability, optimized suspension characteristics, and improved biocompatibility can be achieved by employing a protective layer made up of ceramics, polymers, or highly charged/sterically hindered molecules around magnetic nanocarriers. [243]The most common example of a magnetic nanocarrier is based on an iron oxide core (magnetite) and a biocompatible (polymeric) surface coating [244] that increases colloidal stability and enables the covalent/electrostatic binding of anticancer drugs and targeting moieties. [245,246]Besides iron oxide nanocarriers, nickel-based and gadolinium-based nanocarriers have been also investigated. [247,248][251] For example, Tan et al. [110] reported the synthesis of a SPION covalently modified with an interleukin-6 receptor targeting peptide to enhance the transport through the BBB and recognition of low-grade gliomas where the SPION was used for nanocarrier T2-weighted MRI.In hyperthermiaassisted therapy, AMF offers the advantage of high tissue penetration, which is comparable to US, and is less harmful than ionizing X-ray radiations. [252,253]Jani et.al [254] reported a study with the aim to create polymeric nanocarriers with a magnetic core.This magnetic core was produced using an altered seed synthesis technique that involved the use of tris(acetylacetonate) iron (III) [Fe(acac) 3 ] for targeting GBM, by applying a field of 1.5 T. Analysis of iron content demonstrated that cells cultured under an AMF exhibited higher levels of iron, compared to cells cultured without the AMF.
Another recent study by Tapeinos et al. [255] prepared biomimetic lipid-based SPIONs for the delivery of TMZ to GBM.The novel nanocarrier showed a slow and sustained release profile of TMZ of ≈3.3% at pH 7.4 over 7 days.In an acidic environment of pH 4.5, a slightly enhanced release of 8.2% was obtained.Further, exposure to AMF resulted in a significantly higher release up to 65.8% at pH 7.4 and 63.3% at pH 4.5 in response to the generated hyperthermia.This study proves the applicability of therapeutic payload release control using magnetothermal stimulation.
In another interesting study, Huang et al. developed a formulation based on adipose-derived stem cells loaded with a nano-assembly of poly(-glutamic acid-co-distearyl -glutamate) with poly(lactic-co-glycolic acid), PTX and iron oxide nanoparticles. [256]The adipose derived stem cells are recruited into glioma tumors via interaction with chemokine receptors.Also, the incorporation of iron oxide NPs provides stimuliresponsive PTX release upon induction of high frequency magnetic field.In vivo biodistribution data confirmed higher brain tumor tissue accumulation of Cy5.5 labeled particles loaded in adipose-derived stem cells compared to free nanoparticles.The high apoptosis rate was observed upon induction of a high frequency magnetic field which confirms stimuli responsive release of PTX.

Light-Responsive Systems
Light-responsive drug delivery is characterized by its high spatiotemporal control of drug release to tumor cells with minimal effects on nearby healthy cells. [257,258]Nevertheless, its clinical translation is hindered by some challenges including biocompatibility, stability, and the need to incorporate guide-wires to deliver light to deep tissue. [259]Novel light source modalities are also required.Inorganic nanomaterials such as transition metal nanocarriers and gold nanomaterials have shown satisfactory photothermal conversion efficiencies and excellent photostability, however, they show poor biodegradability and potential longterm toxicity. [260]hotodynamic therapy, a combination of a photosensitizer, light, and molecular oxygen, represents a promising strategy for GBM treatment when compared to chemotherapy and radiotherapy.The light energy, produced by laser irradiation, initiates the photosensitizer excitation inside tumor cells and subsequent photo-oxidative reactions. [261,262]Numerous nanocarriers have been designed for photosensitizer delivery to maximize its accumulation in tumor tissue and minimize diffusion to healthy tissues.Near-infrared light (NIR) is most commonly applied, encouraged by its relatively high tissue penetration with minimal attenuation and refraction by endogenous chromophores.
The photodynamic therapeutic effect of a cholesterol-BSA nanocarrier containing a mitochondria-targeting photosensitizer (4-carboxy-butyl)-triphenyl phosphonium conjugated to pheophorbide-a) was assessed for GBM. [263]An increase in cellular uptake of these nanocarriers in both U-87 MG cells and brain endothelial cell lines was evident.Nanocarriers showed significant anti-tumor photodynamic efficacy upon administration to an orthotopic GBM mouse model with a fiber optic cannula at 671 nm laser irradiation.In another work, Kan et al. [264] prepared a SIWV peptide-functionalized GBM homing, and ICG incorporated pSiNPs (SIWV-pSiNP(ICG), as shown in Figure 7.This nanoformulation consists of three main components: 1. porous pSiNPs as a nanocarrier known for their biocompatibility and substrate-incorporating efficacy, 2. ICG as a photosensitizer for PDT, and 3. SIWV peptide with multifunctional properties such as BBB penetration and GBM homing ability.SIWV-pSiNP(ICG) successfully crossed the BBB and entered the GBM cells through caveolin-mediated endocytosis.Upon NIR irradiation at 808 nm, the incorporated ICG produced reactive oxygen species (ROS), promoting the degradation of the pSiNP nanocarrier, and leading to further release of ICG and generation of ROS (Figure 7d).Following promising in vitro results, biodistribution and anti- Step-by-step synthesis process of pSiNPs, their loading with ICG, and subsequent functionalization with GBM-targeting peptide.c) TEM images of pSiNP and SIWV-pSiNP(ICG).Insets show digital photographs of dispersions in glass vials.d) Fluorescence intensity plots ( em : 810-860 nm) of SIWV-pSiNP(ICG) (left) and emission intensity of the reactive oxygen species (ROS) sensing probe (DMA) under 808 nm laser exposure (100 mW cm −2 ) (right), in comparison to free ICG.e) Experimental protocol of photodynamic therapeutic efficacy using GBM cancer xenograft mice.f) Ex vivo imaging of different organs after 2 h of intravenous administration of different formulations.g) Tumor volume comparisons showing significant shrinkage of tumors in response to PDT with SIWV-pSiNP(ICG).Reproduced with permission. [264]Copyright 2022, American Chemical Society.
cancer efficacy studies in a GBM xenograft mouse model were conducted (Figure 7e-g).Analysis of fluorescence intensity after 2 h of circulation revealed that SIWV-pSiNP(ICG) exhibited strong intensity in GBM xenografts, while undesirable accumulation of pSiNP(ICG) in the liver decreased significantly.Survival curves consistently supported the superior therapeutic ef-ficacy of SIWV-pSiNP(ICG), as evidenced by a significant reduction in GBM tumor size in the SIWV-pSiNP(ICG)-treated group.The average volume of GBM tumors in the SIWV-pSiNP(ICG)treated group (60.3 ± 15.3 mm 3 ) was smaller compared to the tumor volumes in the PBS, ICG, and pSiNP(ICG) treated groups (Figure 7g).
In another study, Zhong et al. developed a cRGD-targeted, photothermal-responsive, AuNR/PEG-poly(-caprolactone) hybrid nanocarriers for delivery of DOX. [265]NIR irradiation caused a phase transition of poly(-caprolactone), resulting in increased drug release.Moreover, in a U-87 MG glioma xenograft, this nanocarrier was able to completely inhibit tumor growth in the presence of NIR light.Targeted photothermal therapy of C6 glioma brain cancer cells by folate-conjugated gold-photoactive polymer nanocarriers was also reported by Mahadavian et al. [266] In this work, acrylic copolymers were modified with spiropyran and imidazole groups, and Au 3+ ions were immobilized to achieve photo-stimulus-responsive nanocarriers.The polymer nanocarrier was functionalized with folic acid as a tumor cell targeting agent and spiropyran groups were altered to zwitterionic merocyanine isomers and conjugated to the nanocarrier, increasing the photogeneration of ROS.

pH-responsive Systems
pH-responsive nanocarriers have been widely investigated for ondemand drug release in the acidic microenvironment of tumor tissue (pH = 6.4-6.8). [267,268][271][272] For example, Zhao et al. [271] prepared pHresponsive peptide-modified, DOX-loaded liposomes as a gliomatargeted system to increase activity in glioma-bearing mice models.In this study, researchers employed a tumor-specific pHresponsive peptide known as H 7 K(R 2 ) 2 as a targeting ligand.This peptide has the ability to respond to the acidic pH environment found in gliomas and possesses cell-penetrating peptide characteristics.pH-sensitive liposomes were chosen as the carrier system, which can also respond to the acidic pH environment in gliomas.Enhanced activity of liposomes in C6 tumor-bearing mice and U-87 MG orthotopic tumor-bearing nude mice was confirmed.In another study, PTX-loaded acid-sensitive nanofibrous scaffolds with four different release rates were prepared from acetylated dextran (Ace-DEX) or PLA, which was then confirmed as a platform for therapeutic options against GBM. [272]Broome and co-workers, [273] developed a pH-responsive micelle of distearoyl phosphoethanolamine-PEG-2000-amine and N-palmitoyl homocysteine for TMZ delivery to GBM via receptor-mediated endocytosis.In a recent study, [109] the development of dualtargeted PLGA polymeric nanocarriers for the delivery of docetaxel to GBM has been reported (Figure 8a-c).PLGA-PEG block copolymers were modified with both ANG2 and histidine to promote BBB trafficking and tumor cell uptake, respectively.This two-level surface decoration of the nanocarriers was achieved using a lower molecular weight PEG (2K) for histidine attachment and a higher molecular weight PEG (5K) for ANG2 attachment.Moreover, ANG2 was conjugated using a pH-sensitive linker which is cleavable in the acidic microenvironment of the endosomes of the endothelial cells, triggering structural rearrangement of nanocarriers and exposure of histidine for tumor targeting.An increased in vitro BBB permeability of the ANG2decorated nanocarriers was demonstrated in a Transwell assay.In vivo studies also confirmed the enhanced nanocarrier accumula-tion in the brain and increased anti-tumor efficacy in an orthotopic GBM model.
Quader et al. developed a pH-triggered micellar system based on poly(ethylene glycol)-poly(-benzyl L-aspartate). [274]Desacetyl vinblastine hydrazide, a derivative of vinblastine was conjugated to a ketone-functionalized copolymer.High pH-dependent release in acidic pH was confirmed for the system, in vitro.Hydrophobic segments of the copolymer attached to the therapeutic payload are arranged in the interior compartment of the micelle which limits premature hydrolysis of pH-sensitive linkers upon systemic administration.To monitor the in vivo stability profile of the micellar system, polymers were labeled with Alexa 594 and Alexa 647 dyes (1:1 ratio), and the stability was monitored based on Förster resonance energy transfer.The micellar structure was stable for 24 h after administration.In another study, a pH-sensitive micellar formulation for epirubicin called NC-6300 showed satisfactory efficacy outcomes in combination with immune checkpoint blockade in both phosphatase and tensin homolog (PTEN) positive and negative orthotopic glioma models.This system was based on PEG-poly(aspartatehydrazide-epirubicin) block copolymers. [275]

Oxidation/Reduction-Responsive Systems
Oxidation/reduction-responsive nanocarriers are designed based on the difference in the redox potential between brain tumors and healthy tissue.Tumor tissue is associated with higher intracellular concentrations of GSH in comparison to the extracellular environment. [276]Examples of redox-responsive functional groups include diselenide bond, disulfide bond, and thioether bond. [277]A study by Lu et al. [112] investigated the use of iRGD-labelled polymeric micelles for the delivery of CPT to glioma.CPT was linked to the polymer with a glutathionesensitive disulfide linker, followed by modification with the iRGD ligand (Figure 8d-f).The self-assembled polymeric micelles (≈100 nm) were co-loaded with photosensitizer IR780 for chemophotodynamic combination therapy.
Jiang et al. [278] formulated an ANG2-targeted, redoxresponsive, virus-mimicking polymersome (PS) to deliver saporin (SAP), a highly potent natural protein toxin (Figure 9a).TEM imaging confirmed that ANG-PS-SAP exhibited a vesicular structure with spherical morphology and a size of ≈76 nm (Figure 9b).In vitro release studies showed minimal protein release under physiological conditions within 24 h, while over 80% of SAP was released under reductive conditions (Figure 9c).Blank ANG-PS and PS showed no cytotoxicity in U-87 MG-Luc cells, while ANG-PS-SAP effectively inhibited cell growth.Nontargeted PS-SAP exhibited enhanced antiproliferation activity compared to free SAP but had lower potency than ANG-PS-SAP.These results confirmed that ANG-PS-SAP selectively targeted and released SAP to U-87 MG-Luc GBM cells.BBB crossability was enhanced for ANG20-PS (20% ANG2 density), comparable to ANG30-PS and significantly better than ANG10-PS and PS (Figure 9e).The transport ratio of ANG20-PS decreased when the BBB monolayer was pretreated with free ANG2, supporting LRP1-mediated enhancement of BBB transcytosis.Flow cytometry demonstrated that both ANG20-PS and ANG30-PS exhibited over two-fold higher uptake by U-87 MG cells than  and c) successful conjugation to the targeting ANG2 peptide.Adapted with permission. [109]Copyright 2023, Wiley-VCH GmbH, Weinheim.Schematic presentation of d) the synthesis reaction to prepare a disulfide bond-conjugated polymer of camptothecin (CPT) and PEG with further conjugation to the iRGD, and e) the polymer self-assembly into polymeric micelles that target glioma cells by the iRGD targeting peptide and glutathione (GSH)-induced disassembly.f) TEM images of the nanocarriers before (1), and after (2) treatment with 10 mm of GSH for 12 h confirming the GSH-responsive property.Adapted with permission. [112]Copyright 2020, Elsevier.
ANG10-PS and PS (Figure 9f), indicating that ANG2 facilitated BBB passage and cellular uptake by GBM cells.Pretreatment of U-87 MG cells with free ANG2 markedly decreased the uptake of ANG20-PS, further confirming a receptor-mediated mechanism of ANG20-PS uptake by U-87 MG cells.In vivo, real-time imaging demonstrated a robust presence of DiR fluorescence in GBM tumors when administered with ANG20-PS and ANG30-PS (Figure 9g).The therapeutic efficacy of ANG-PS-SAP against GBM was evaluated in mice with orthotopic U-87 MG-Luc tumors.Bioluminescence imaging demonstrated that ANG-PS-SAP successfully inhibited tumor progression (Figure 9h).Quantitative analysis of bioluminescence intensity revealed that mice treated with ANG-PS-SAP had approximately four-and seven-folds lower tumor bioluminescence compared to those treated with PS-SAP and PBS, respectively, (Figure 9h) and significantly improved survival times (Figure 9i).
In another study, PTX-loaded human serum albumin NPs were prepared and stabilized via disulphide bonds. [88]Using the SP peptide as a redox-responsive component, it was demonstrated that drug delivery to BBB endothelial cells was significantly improved.The higher intra-tumoral drug concentrations were translated into significant anti-tumor activities, in vivo.and i) survival rate plots.Reproduced with permission. [278]Copyright 2018, Wiley.

Enzyme-Responsive Systems
Enzyme-responsive systems deliver anticancer drugs through enzymatic degradation of the nanocarrier matrix or a linker, in response to high local concentrations of specific enzymes at the target tumor site. [279]Overexpression of enzymes like proteases, phosphatases, and glycosidases has been reported in cancerous and inflammatory tissues. [280]On-demand drug release from nanocarriers is triggered by enzyme-induced structural change, macroscopic deformation, charge switching, or covalent bond breakage in the nanocarrier. [281]Enzymes as endogenous stimuli are not associated with potential safety concerns linked to external stimuli such as magnetic field, US, or light.Furthermore, most enzymatic reactions which enzyme-responsive carriers are designed around, are fast, efficient, and highly specific.These properties potentially lead to more controlled spatiotemporal release of therapeutic payload and less pronounced off-target effects. [282]uan et al. developed an interesting nanocarrier approach based on legumain-induced aggregation that enhanced nanocarrier retention in brain tumors. [283]Two types of AuNPs were used with different ligands.Through legumain-catalyzed hydrolysis (overexpressed in brain cancers), one ligand exposed its 1,2thiolamino groups leading to in vivo click cycloaddition with a second AuNP bearing 2-cyano-6-aminobenzothiazole.This resulted in the formation of AuNPs aggregates that could effectively block nanocarrier exocytosis.In combination with DOX delivery via a pH-responsive hydrazone bond, this dual-particle system led to increased survival in C6-glioma-bearing mice, compared to either of the single particles.Another study reported the design of a two-stage enzyme-responsive LNP for the delivery of siRNA to GBM. [284] ANG2 was conjugated to the surface of the LNP to promote BBB crossing while the positively charged LNPs were covered with a negatively charged PEGylated lipopeptide.This cleavable lipopeptide has a recognition sequence for matrix metalloproteinases -commonly expressed in the tumor microenvironment.Proteolytic cleavage reverses the surface charge from weakly negative to positive, favoring cell endocytosis and the release of siRNA for effective silencing.

Hypoxia-Responsive Systems
287] Hypoxia-responsive ionizable liposomes, prepared by Liu et al., [288] were applied to treat GBM through delivering therapeutic siRNA.This nanocarrier demonstrated an increase in cellular uptake under hypoxic and low-pH conditions and inhibited glioma tumor growth in vivo.This was achieved by employing a hypoxia-responsive lipid containing nitroimidazole groups.Nitroimidazoles are converted to aminoimidazole, containing positively charged amino groups, by malate dehydrogenase under hypoxic conditions leading to enhanced uptake of liposomes under hypoxic conditions.Another study reported a multifunctional hypoxia-responsive lipid polymer nanocarrier combining chemotherapy, PDT, and photothermal nanocarrier. [289]This formulation consisted of four functional components: A) ANG2 for nanocarrier targeting gliomas, B) ICG for imaging-guided surgery and combining PDT and photothermal therapy.Upon irradiation with an 808 nm laser, ICG also created a hypoxic environment.C) Poly(nitroimidazole) 25 as the hydrophobic and hypoxia-responsive component -resulting in the release of DOX.D) DOX to kill residual glioma cells post-operatively.

ROS-Responsive Systems
Reactive oxygen species generation is an inevitable by-product of cell oxidative metabolism, which play a role in the pathophysiology of brain cancer. [290]The mechanisms of drug release mediated by ROS-responsive nanocarriers include ROS-induced carrier solubility change, cleavage, or ROS-induced prodrug linker cleavage.[293] The concentration of ROS in cancer cells has been reported 100 times higher than in normal cells. [294] ROS-responsive polymeric siRNA nanocarrier has been utilized by Shi and co-workers [295] for GBM therapy.Herein, a combination of electrostatic, hydrogen bond, and hydrophobic interactions were employed in the nanomaterial design tailored for improving physiological stability.The nanocarrier was modified with ANG2 peptide to actively traverse the BBB. This anocarrier showed an active ROS response and efficient siRNA release upon treatment with H 2 O 2 .The charge and hydrogen bond competition caused further nanocarrier decomposition after the hydrophobic interactions were lost, resulting in the effective release of encapsulated siRNA.The results showed that PLK1 siRNA and VEGFR2 siRNA co-loaded ANG-3I-nanocarriers effectively reduced glioma growth as shown by the significant reduction in Luc-bioluminescence, with limited body weight loss.On the other hand, non-targeting and single gene-targeting counterparts showed weaker glioma growth inhibition.

Clinical Trials of Nanocarriers for Brain Cancer Treatment
Nanocarrier drug delivery systems first reached the clinics in the early 1990s. [306]Since then, nanomedicine research has evolved alongside the increasing demands for improved therapeutic options. [306]Many nanocarriers have reached clinical trials and have been approved for a variety of indications in the current clinical landscape.In the context of brain cancer treatment, several examples are listed in Table 4, covering different types of formulations.For example, the brain-penetrant RNA interferencebased spherical nucleic acids, which comprise AuNPs cores covalently conjugated to the siRNA, have recently found their way into clinical trials. [307]They were first preclinically evaluated for their safety, pharmacokinetics, intratumoral accumulation, and genesuppressive activity.At the safety assessment stage, no grade 4 or 5 toxicities were identified.The uptake of these nanocarriers into glioma cells was associated with a reduction in the expression of the tumor-associated Bcl2L12 protein.Based on these results, these nanoconjugates are being considered as a potential brainpenetrant precision medicine approach for the treatment of GBM systemically.The other example of nanocarriers that have been tried in the clinical stage is rhenium-186 nanoliposomes [308,309] as an alternative to external beam radiation. [310]Rhenium-186 nanoliposomes allow for the delivery of beta-emitting radiation of high specific activity with significant retention in the tumor.In a Phase 1 dose-escalation study, rhenium-186 nanoliposomes were administered via convection-enhanced delivery to recurrent glioma and were well tolerated, without dose-limiting toxicity, and no treatment-related serious adverse events, despite significantly higher absorbed doses than typically delivered by external beam radiation therapy. [308]In a second clinical study, rhenium-186 nanoliposomes were administered using multiple catheters to limit treatment time. [309]Results were promising within a 13patient subset receiving a radiation dose over 100 Gy with an average 487-day survival with 6 of 13 patients still alive.While, for patients receiving lower radiation doses (<100 Gy), the average survival was only 167 days with no patients remaining alive.
In another clinical trial, the capacity of EGFR-targeted immunoliposomes to deliver cargo to brain tumor tissue in patients with relapsed GBM harboring an EGFR amplification has been studied. [311]The researchers evaluated how well anti-EGFR immunoliposomes containing DOX performed in patients with GBM multiforme.The findings indicated that minimal to no amounts of DOX were detected in the cerebrospinal fluid, suggesting that the anti-EGFR ILs loaded with DOX cannot effectively pass through the BBB.However, significant levels were detected in GBM tissue 24 h after the application, showing that the disruption of BBB integrity present in high-grade gliomas might leads liposome delivery into tumor tissue.There were no safety concerns identified.The median duration of progression-free survival was 1.5 months, while the median overall survival was 8 months.Notably, a patient who underwent surgery experienced an exceptionally prolonged period of remission, indicating that the administration of treatment before the main therapy might positively influence the outcome.

Conclusion and Future Perspectives
Despite the success of many targeted and stimuli-responsive nanocarriers in preclinical settings, the number of clinical trials for these nanocarriers in the context of brain cancers is very limited, let alone clinically approved nano-products.First of all, this translational gap should be acknowledged and second a road map should be designed to address this gap based on the wealth of preclinical data generated through years by the nanomedicine and antibody-drug conjugate (ADCs) research communities.In recent years, the success of COVID-19 vaccines based on nanodelivery platforms for mRNA proved how extensive formulation know-how about in vitro and in vivo fate of nanomedicine accumulated over many years can be applied to address the unmet clinical need of rapid vaccine development during the pandemic.Nonetheless, it should be noted that delivery of therapeutics payloads to extrahepatic organs such as CNS, especially through systemic administration, is far more challenging than the localized delivery of an RNA payload in COVID-19 vaccines.Simultaneously, insights can be learned from the advancements made in ADCs.Where years of refining stimuli-responsive linkers, enhancing stability, and judiciously selecting highly potent drugs have collectively led to a recent coming of age of ADCs in the area of oncology. [313]n light of these challenges, several important areas demand focused attention.First, nanomedicine and specifically-stimuli responsive carriers have a tendency toward overengineering during the formulation design phase.The incorporation of too many formulation components and the use of sophisticated/multi-step synthesis methods imposes substantial technical difficulties for chemistry, manufacturing, and control (CMC) processes.While production of such formulations can be achieved for small-scale animal studies, scaling up and reproducible production for early-stage clinical trials will be challenging and sometimes impossible.In summary, we would argue that the simplest stimuli-responsive delivery formulation is almost always the best.In addition, a smart and rational selection of therapeutics payloads for targeted and stimuli-responsive delivery systems that will most benefit from stimuli-responsive nanocarriers is necessary.A substantial proportion of studies are limited to a few commonly used small molecule chemotherapeutics such as PTX and DOX.While these therapeutic payloads play a crucial role in some treatment regimens for brain cancers, there is an opportunity to explore alternative and more innovative payloads based on both formulation design and the pathophysiology of brain cancers.Along this line, RNA therapeutics such as mRNA and siRNA candidates offer great potential.A third reason contributing to the existing translation gap lies in the inadequacy of current in vivo models used for the investigation of biodistribution and efficacy of brain cancers-targeted nanomedicine.The commonly used orthotopic models, developed over a few weeks via intracranial injection of rapidly proliferating cell lines, lack fidelity in recapitulating the clinical pathophysiology of brain tumors.These models often exhibit a high level of disruption in BBB, potentially leading to an overrepresentation of accumulation and efficacy of tested nanocarriers.However, there have been encouraging breakthroughs in oncology in vivo models and state-of-the-art preclinical imaging platforms.As an example, a combination of humanized mice and slow-growing primary patient-derived cancer cells can potentially generate more clinically relevant orthotopic brain tumor models.While the associated costs with such models are high at the moment, the ongoing expansion and refinement of these platforms suggests the prospect of reduced costs in the foreseeable future.
Understandably, most nanocarriers for brain delivery are based on intravenous (IV) administration.However, still achieving the required therapeutic dose in brain tumor tissue is challenging and off-target distribution in organs such as the liver, spleen, and lungs imposes safety challenges for early-stage clinical trials.To bypass these shortcomings, in some cases, IV systemic administration can be replaced by intra-tumoral administration during surgery.Along this line, the promotion of interdisciplinary collaborations between the nanomedicine research community and end users such as neurosurgeons and other clinicians can provide innovative avenues for the administration of nanocarriers.In addition, recent successes with intranasal delivery of therapeutics to CNS introduce another potential alternative route of administration.

Figure 1 .
Figure 1.Schematic illustration of brain cancer drug delivery showing different types of nanocarriers with a focus on stimuli-responsive and targeted systems.Created using Biorender.com(2023).

Figure 2 .
Figure 2. ANG2-functionalized liposomes represent a promising class of brain-penetrant nanocarriers for GBM treatment.a) Schematic illustration of layer-by-layer coating of core liposomes with an anionic pPLD coat and its further conjugation to ANG2.b) Fold change in mean fluorescence intensity from GBM spheroids after 12 min incubation with different fluorescent-labelled liposomal formulations, as presented in confocal microscopy imaging (c).d) Generation of the BBB-GBM model in a microfluidic device, where a preformed spheroid of GBM-22 with PC is transferred into a suspension of induced pluripotent stem cell-derived endothelial stem cells (ECs), PCs, and ACs in fibrinogen.The addition of thrombin then induces self-assembly of the vascular networks.The inset shows confocal imaging of GBM spheroid (GFP in green) infiltrating into the BBB microvascular network (CD31 in red).e) Liposome permeability in BBB vascular networks without GBM spheroid (no), near, and far from a GBM spheroid (n = 6).f) Confocal imaging of BBB-GBM before and after 4 days of treatment with free CDDP, CDDP-loaded bare liposomes (Bare CDDP nanocarrier), or CDDP-loaded ANG2functionalized liposomes CDDP nanocarrier).g) Cell viability assays using Sytox staining of regions near GBM, far from GBM, or at GBM spheroids following treatment with different formulations of cisplatin.h) Heatmap of cell death gene-expression level at two different regions of interest.i) Timeline of in vivo efficacy study of cisplatin-loaded nanocarriers in an orthotopic model of GBM with insets showing magnetic resonance imaging (MRI) of brain tumor before and after treatment dosing.j) Tumor volume change after different treatments with a dotted line representing the median tumor volume change for the ANG2-functionalized liposomes group.Adapted under the terms of the CC BY-NC-ND license.[158]Copyright 2022, The Authors.

Figure 3 .
Figure 3. PEI-capped pSiNPs (PEI-pSiNP) for siRNA efficient delivery to GBM silencing MRP1 gene and inhibiting cancer proliferation.a) TEM images of pSiNPs and PEI-capped pSiNPs.b) Confocal microscopy imaging of m-cherry expressing U-87 GBM cells showing cellular uptake of fluorescein labeled pSiNPs (Green).In vivo study of MRP1 silencing effects in a GBM tumor-bearing mouse model using c) qRT-PCR, and d) immunoblotting for MRP1 mRNA and B-actin expression levels at 48 and 72 h post-intravenous injection.e) Immunohistochemistry of Ki67 expression in GBM tumors in mice after different treatments.Reproduced with permission.[198]Copyright 2018, Springer Nature.

Figure 4 .
Figure 4.Antisense oligonucleotide-modified pSiNPs (AON@pSiNPs) as a targeted gene delivery platform for GBM treatment.a) TEM image of AON@pSiNPs.b) dynamic light scattering (DLS) analysis of undecylenic acid-modified pSiNPs and AON@PSiNPs in H2O.c) Cryo-TEM images showing internalization of AON@PSiNPs into U87 and hCMEC/D3 cells.d) Confocal microscopy images showing cellular uptake of AON@pSiNPs in U-87, hCMEC/D3, and HEK293 cells.e) Study of AON@pSiNPs BBB permeability in an innovative BBB-GBM microfluidic model.i.Digital photo of the microfluidic device which is composed of 8 subunits with each unit having 3 channels.ii.Optical microscopy image showing the blood channel, brain channel, and medium channel with an interconnecting array of microchannels.iii.Schematic of the nanoparticle perfusion into the blood channel under shear stress.Insets are showing the selective binding of AON@pSiNPs to hCMEC/D3 cells in the blood channel following 3 h perfusion under shear stress.f,g) Brain uptake and biodistribution study of AON@pSiNPs in an orthotopic mouse model of GBM, following intravenous administration of Cy5.5-labelled nanocarrier using IVIS imaging.Reproduced with permission.[197]Copyright 2022, American Chemical Society.

Figure 6 .
Figure 6.MBs-enhanced FUS (MB-FUS) for effective delivery of siRNA-loaded cationic nanocarriers in brain tumors.a) Hydrodynamic size distribution (by number) and zeta potential measurements of the before (Cat LPH), and after loading with Cy5-siRNA (Cat LPH: Cy5-siRNA).Insets show the morphology of nanocarriers as per TEM imaging.b) Schematic of the in vivo protocol to investigate the delivery of Cat LPH into the brains of either healthy immunocompetent mice (c), or GL261 orthotopic glioma mouse model (d,e) using MB-FUS.c) Ex vivo fluorescence scans of excised healthy mouse brains showed a twelve-fold increase in Cat LPH extravasation into the FUS-treated regions (bottom), compared to the non-FUS region (top).d) Contrast-enhanced T1-weighted MR images (top) and ex vivo fluorescence scans (bottom) of brains in GL261 orthotopic glioma mice before and after MB-FUS.e) Level of Cat LPH extravasation in brains of GL261 orthotopic glioma mouse, showing around five-fold increased uptake following MB-FUS.f-h) In vivo therapeutic efficacy of LPH: SMO-siRNA in a medulloblastoma mouse tumor model.f) SMO protein immunostaining in medulloblastoma tumors showing substantial protein knockdown in the MB-FUS treated group (g).SMO is presented in brown, and nuclei in purple.h) Tumor cell apoptosis (CC3-positive signal) after non-therapeutic LPH: Cy5-siRNA or therapeutic LPH: SMO-siRNA administration in the presence or absence of MB-FUS.Reproduced with permission.[230]Copyright 2021, American Association for the Advancement of Science.

Figure 7 .
Figure 7. PDT of GBM using targeted ICG-loaded pSiNPs.a) Schematic illustrations of the proposed GBM-homing peptide-functionalized, ICG-loaded pSiNPs (SIWV-pSiNP(ICG)) design and their biological mechanism of action.b)Step-by-step synthesis process of pSiNPs, their loading with ICG, and subsequent functionalization with GBM-targeting peptide.c) TEM images of pSiNP and SIWV-pSiNP(ICG).Insets show digital photographs of dispersions in glass vials.d) Fluorescence intensity plots ( em : 810-860 nm) of SIWV-pSiNP(ICG) (left) and emission intensity of the reactive oxygen species (ROS) sensing probe (DMA) under 808 nm laser exposure (100 mW cm −2 ) (right), in comparison to free ICG.e) Experimental protocol of photodynamic therapeutic efficacy using GBM cancer xenograft mice.f) Ex vivo imaging of different organs after 2 h of intravenous administration of different formulations.g) Tumor volume comparisons showing significant shrinkage of tumors in response to PDT with SIWV-pSiNP(ICG).Reproduced with permission.[264]Copyright 2022, American Chemical Society.

Figure 8 .
Figure 8. Stimulus-responsive polymeric nanocarriers designed using acid-cleavable and oxidation-reduction sensitive properties.a) Schematic illustration of a dual ligand decorated polymeric PLGA nanocarrier for targeted delivery of docetaxel to GBM. 1 H NMR analysis confirmed the b) acid-cleavable property of the used polymer,and c) successful conjugation to the targeting ANG2 peptide.Adapted with permission.[109]Copyright 2023, Wiley-VCH GmbH, Weinheim.Schematic presentation of d) the synthesis reaction to prepare a disulfide bond-conjugated polymer of camptothecin (CPT) and PEG with further conjugation to the iRGD, and e) the polymer self-assembly into polymeric micelles that target glioma cells by the iRGD targeting peptide and glutathione (GSH)-induced disassembly.f) TEM images of the nanocarriers before (1), and after (2) treatment with 10 mm of GSH for 12 h confirming the GSH-responsive property.Adapted with permission.[112]Copyright 2020, Elsevier.

Figure 9 .
Figure 9. Redox-responsive, ANG-functionalized, virus-mimicking polymersomes for SAP toxin delivery (ANG-PS-SAP) to GBM. a) Graphical presentation of the nanocarrier design.b) TEM images of ANG-PS-SAP.c) SAP in vitro release profiles showing a redox-responsive release behavior in the presence of 10 mm GSH at pH 7.4 and 37 °C.d) Cell viability (MTT) assay in U87-MG GBM cells showing effective inhibition of cell growth from ANG-PS-SAP.e) BBB permeability study of Cy5-labelled PS, with different ANG densities (10-30%), was conducted using a Transwell model of immortalized endothelial cell line.f) Flow cytometry histograms showing enhanced uptake of ANG20-PS and ANG30-PS in U-87 MG cells, based on Cy5 signals.g) In vivo fluorescence imaging of orthotopic U-87 MG-Luc GBM-bearing nude mice.at 24 h postinjection of DiR-loaded PS (I) and ANG-PS with varying ANG densities (II: 10%, III: 20%, IV: 30%).Therapeutic efficacy study of ANG-PS-SAP as per h) bioluminescence images of U-87 MG-Luc GBM-bearing nude mice, and i) survival rate plots.Reproduced with permission.[278]Copyright 2018, Wiley.

Table 1 .
An overview of BBB and GBM targeting ligands and their respective receptors.

Table 2 .
Summary of external stimulus-responsive nanocarriers for GBM therapy.

Table 3 .
Summary of internal stimulus-responsive nanocarriers for GBM therapy.

Table 4 .
List of clinical trials based on nanocarrier type/formulation for brain cancer treatment.[312]