cancers Review Advances in Preclinical/Clinical Glioblastoma Treatment: Can Nanoparticles Be of Help? Daniel Ruiz-Molina 1 , Xiaoman Mao 1 , Paula Alfonso-Triguero 1,2 , Julia Lorenzo 2,3 , Jordi Bruna 4 , Victor J. Yuste 5,6 , Ana Paula Candiota 2,3,7, * and Fernando Novio 1,8, * 1 Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST, Campus UAB, Bellaterra, 08193 Barcelona, Spain 2 Institut de Biotecnologia i de Biomedicina, Departament de Bioquimica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain 3 Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain 4 Neuro-Oncology Unit, Bellvitge University Hospital-ICO (IDIBELL), Avinguda de la Gran Via de l’Hospitalet, 199-203, L’Hospitalet de Llobregat, 08908 Barcelona, Spain 5 Instituto de Neurociencias, Universitat Autònoma de Barcelona (UAB), Campus UAB, 08193 Cerdanyola del Vallès, Spain 6 Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas (CIBERNED), Campus UAB, 08193 Cerdanyola del Vallès, Spain 7 Centro de Investigación Biomédica en Red: Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 08193 Cerdanyola del Vallès, Spain 8 Departament de Química, Universitat Autònoma de Barcelona (UAB), Campus UAB, 08193 Cerdanyola del Vallès, Spain * Correspondence: anapaula.candiota@uab.cat (A.P.C.); fernando.novio@icn2.cat (F.N.); Tel.: +34-93-581-4126 (A.P.C.); +34-93-737-3630 (F.N.) Simple Summary: As one of the most lethal human cancers, glioblastoma treatment is a real challenge because of several resistance mechanisms, including limited drug entry into the central nervous system through the blood–brain barrier and the vast heterogeneity of this family of tumors. In Citation: Ruiz-Molina, D.; Mao, X.; the development of precision medicine, various nanoconstructs are being proposed to cross the Alfonso-Triguero, P.; Lorenzo, J.; BBB, specifically target GB tumors, release the therapeutic cargo in a controlled manner, and reduce Bruna, J.; Yuste, V.J.; Candiota, A.P.; therapeutic resistance. This review summarizes the different families of nanoparticles and approaches Novio, F. Advances in Preclinical/ followed so far pursuing these aims. Clinical Glioblastoma Treatment: Can Nanoparticles Be of Help? Cancers Abstract: Glioblastoma multiforme (GB) is the most aggressive and frequent primary malignant 2022, 14, 4960. https://doi.org/ tumor in the central nervous system (CNS), with unsatisfactory and challenging treatment nowadays. 10.3390/cancers14194960 Current standard of care includes surgical resection followed by chemotherapy and radiotherapy. Received: 30 August 2022 However, these treatments do not much improve the overall survival of GB patients, which is Accepted: 28 September 2022 still below two years (the 5-year survival rate is below 7%). Despite various approaches having Published: 10 October 2022 been followed to increase the release of anticancer drugs into the brain, few of them demonstrated Publisher’s Note: MDPI stays neutral a significant success, as the blood brain barrier (BBB) still restricts its uptake, thus limiting the with regard to jurisdictional claims in therapeutic options. Therefore, enormous efforts are being devoted to the development of novel published maps and institutional affil- nanomedicines with the ability to cross the BBB and specifically target the cancer cells. In this context, iations. the use of nanoparticles represents a promising non-invasive route, allowing to evade BBB and reducing systemic concentration of drugs and, hence, side effects. In this review, we revise with a critical view the different families of nanoparticles and approaches followed so far with this aim. Copyright: © 2022 by the authors. Keywords: glioblastoma; preclinical model; nanoparticles; drug delivery; brain cancer; BBB Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// 1. Glioblastoma Treatment: State-of-the-Art creativecommons.org/licenses/by/ Glioblastoma (GB) is a devastating tumor representing more than 50% of primary 4.0/). malignant brain tumors [1], and has been classified as having the highest malignant grade Cancers 2022, 14, 4960. https://doi.org/10.3390/cancers14194960 https://www.mdpi.com/journal/cancers Cancers 2022, 14, x FOR PEER REVIEW 2 of 26 1. Glioblastoma Treatment: State‐of‐the‐Art Cancers 2022, 14, 4960 2 of 25 Glioblastoma (GB) is a devastating tumor representing more than 50% of primary malignant brain tumors [1], and has been classified as having the highest malignant grade (4) behavior by the World Health Organization (WHO) [2,3]. Its expected median overall (4) behavior survival by the15 is around World months Health evenOrganization (WHO) after aggressive [2,3].The therapy. Its expected standardmedian treatmentoverall en‐ survival is around 15 months even after aggressive therapy. The standard tails surgical resection and radiotherapy, combined with oral temozolomide (TMZ) chem‐ treatment entails surgical resection otherapy and radiotherapy, [4,5], as validated combined in a randomized with trial clinical oral in temozolomide (TMZ) chemother- 2005 [6]. Its therapeutic mech‐ apy [4,5], as validated in a randomized clinical trial in 2005 anism involves methylation of the guanine O6 position and subsequent DNA [6]. Its therapeutic mechanism double‐ involves strand methylation rupture, leadingoftothe guanine a cell O6 position cycle arrest and celland subsequent death DNA double-strand [7,8]. Additional benefits of TMZ can be immunogenic cell damage and exposure of calreticulin immunogenicTMZ rupture, leading to a cell cycle arrest and cell death [7,8]. Additional benefits of can signals be On [9]. immunogenic the negative cell damage side, TMZand mustexposure of calreticulin be administered at highimmunogenic signals [9].periods doses and prolonged On the negative side, TMZ must be administered at high doses and prolonged periods due to its due to its low yield crossing the blood–brain barrier (BBB), short half‐life, inducing side low yield crossing the blood–brain barrier (BBB), short half-life, inducing side effects [10,11], effects [10,11], and an intrinsic resistance to DNA repair [12,13]. Alternatively, second‐line and an intrinsic resistance to DNA repair [12,13]. Alternatively, second-line treatments treatments involving nitrosourea‐based drugs such as lomustine (lomus) or carmustine involving nitrosourea-based drugs such as lomustine (lomus) or carmustine were ap- were approved by the US Food and Drug Administration (FDA) and European Medicines proved by the US Food and Drug Administration (FDA) and European Medicines Agency Agency (EMA) [14,15]. These drugs are also effective alkylating agents inducing cross‐ (EMA) [14,15]. These drugs are also effective alkylating agents inducing cross-linking linking between DNA strands and protein carbamylation that prevent cell cycle develop‐ between DNA strands and protein carbamylation that prevent cell cycle development [16]. ment [16]. However, remarkable side effects may appear, such as myelosuppression, he‐ However, remarkable side effects may appear, such as myelosuppression, hepatic toxicity, patic toxicity, and pulmonary fibrosis [17]. In addition, the antiangiogenic drug Bevaci‐ and pulmonary fibrosis [17]. In addition, the antiangiogenic drug Bevacizumab (BVZ, a zumab (BVZ, a recombinant humanized monoclonal antibody acting through inhibition recombinant humanized monoclonal antibody acting through inhibition of the vascular of the vascular endothelial growth factor A) was also FDA approved for tumor recurrence endothelial growth factor A) was also FDA approved for tumor recurrence [18], yet clinical [18], trialsyet clinical trials combining unlikecombining unlike schedules/combinatory schedules/combinatory treatments [19] treatments and immune [19] and im‐ checkpoint mune checkpoint inhibitors (ICIs) [20] revealed disappointing inhibitors (ICIs) [20] revealed disappointing efficacy results (Figure 1). efficacy results (Figure 1). First-line (TMZ) Figure1.1. First‐line Figure (TMZ)and andsecond-line (lomus, second‐line carmustine (lomus, and BVZ) carmustine and drugs BVZ)for glioblastoma drugs treatment. for glioblastoma treatment. Finally, platinum (Pt) complexes appear as a potential fourth line of GB treatment. These agentsplatinum Finally, are known to form (Pt) DNA adducts complexes appear asthata avoid bothfourth potential cell replication line of GBand transcrip- treatment. These agents are known to form DNA adducts that avoid both cell replication anddiverse tion [21], inducing DNA damage and cell apoptosis with good effectiveness for tran‐ cancers [22]. scription [21], However, inducing DNAinconsistent damageresults and cellareapoptosis obtained with in thegood treatment of GB patients effectiveness for di‐ mainly verse due to[22]. cancers systemic toxicities However, [23] occurring inconsistent resultsbefore effectiveindrug are obtained concentrations the treatment of GBreach pa‐ the tumor [24–26]. Other antiangiogenic agents, such as integrins, transmembrane tients mainly due to systemic toxicities [23] occurring before effective drug concentrations receptors overexpressed on both GB and tumor-associated endothelial cells [27], or tyrosine kinase reach the tumor [24–26]. Other antiangiogenic agents, such as integrins, transmembrane inhibitors likewise represent complementary therapeutic approximations. However, none receptors overexpressed on both GB and tumor‐associated endothelial cells [27], or tyro‐ of these therapeutic approaches showed relevant advantages after being tested in phase sine kinase inhibitors likewise represent complementary therapeutic approximations. 2–3 trials [28–32]. However, none of these therapeutic approaches showed relevant advantages after being Challenges of crossing natural barriers: BBB represents a vasculature for preserving tested in phase 2–3 trials [28–32]. brain homeostasis and neurological functions, being permeable to only 2–3% of small Challenges of crossing natural barriers: BBB represents a vasculature for preserving molecules (large molecules are excluded). Additionally, it has functional efflux pumps for brain homeostasis and neurological functions, being permeable to only 2–3% of small mol‐ clearance of toxic, metabolic, and other waste products. The BBB consists of a neurovas- ecules (large molecules are excluded). Additionally, it has functional efflux pumps for cular unit (NVU) that includes a basement membrane, pericytes, astrocytes, and capillary clearance of toxic, metabolic, and other waste products. The BBB consists of a neurovas‐ endothelial cells connected to each other by tight junctions. However, when cancer cells cular unit (NVU) that includes a basement membrane, pericytes, astrocytes, and capillary displace endothelia from the other NVU cells, the BBB breaks down and both passive endothelial cells connected and active transport to each are altered, otherrise giving by tight to thejunctions. However, blood–tumor barrierwhen cancer (BTB). cells Although displace endothelia from the other NVU cells, the BBB breaks down and the BTB is more permeable than the BBB, its permeability to molecules including drugs both passive and active transport arerepresenting is heterogeneous, altered, giving rise to a great the blood–tumor challenge for therapy.barrier The (BTB). BTB isAlthough accompanied the by a decrease in the expression of tight junctions, and increased secretion of vascular endothelial growth factor (VEGF) from tumor cells, as well as a breakdown of the basal membrane. Transport occurs to a very limited extent mostly in disrupted BBB. In a similar Cancers 2022, 14, 4960 3 of 25 way, passive diffusion is uncommon and only for small lipophilic compounds. Worth of mention, endogenous molecules pass through specific transporters. Cell-mediated and adsorption-mediated transport occur as well, but receptor-mediated transport is the most efficient mechanism for drug delivery, being one of the strategies used in nanomedicine approaches. Some of the receptors found on the luminal side of the BBB are transferrin receptor (TfR), insulin and insulin-like growth factor receptor, low-density lipoprotein receptor (LDLR), low-density lipoprotein receptor-related protein 1 and 2 (LRP1 and LRP2), scavenger receptor class B type I (SR-B1), leptin receptor, and lactoferrin receptor. More recently, nicotinic acetylcholine receptors (nAChRS) and diphtheria toxin receptor have been also described [33]. All in all, systemic toxic effects may arise before effective drug concentrations are reached within the tumor, and this fact limits chemotherapy efficacy in GB treatment. This is mostly, though not exclusively, due to difficulties in crossing the BBB, reason why different approaches have been designed to overcome this barrier while minimizing side effects [34]. These strategies include methods to increase the BBB permeability [35–37], use of biodegradable polymeric implants in the tumor bed [38], or more aggressive ap- proaches, such as the use of a catheter to deliver drugs directly into the brain through convection-enhanced delivery (CED) [39–41]. Gliadel® wafer was also approved by the US FDA back in 2003 as a local implant depot of active ingredient carmustine (BCNU) to treat malignant and recurrent high-grade gliomas (HGG). Soft materials, such as films, fibers, and (hydro)gels, have also been investigated as local implanting depots for dif- ferent therapeutic agents mostly in preclinical studies [42–45]. An interesting example is that of Zhao et al., who reported the codelivery of paclitaxel (PTX) and TMZ through a photopolymerizable hydrogel able to synergistically inhibit tumor growth and prevent GB recurrence after surgical resection (Figure 2). The drug combination showed high tolerability and the suppression of tumor growth more efficiently than the administration of single drugs in a U87MG orthotopic tumor model [46]. However, in spite of these pioneering and successful results, formulations that need local device placements for drug delivery are not very attractive from a clinical point of view. These approaches require a new neurosurgical intervention at the relapsing tumor scenario, in patients with in general poor life expectancy and usually handicapped neurologic status. These devices can also hamper neuroimaging, a key point in the assessment of treatment response. In addition, such devices may trigger new local serious adverse events, which the physicians are not familiarized with, as spotlighted by the use of carmustine wafers. These inconveniences preclude these patients to be candidates for other clinical trials, limiting its attractiveness with disappointing clinical performances [47,48]. All in all, the development of innovative Cancers 2022, 14, x FOR PEER REVIEW 4 of 26 approaches that ensures proper and efficient chemotherapeutic treatments still represents an urgent challenge nowadays. Figure2. Figure 2. Codelivery Codelivery of of PTX PTX and and TMZ TMZ through through aa photopolymerizable photopolymerizable hydrogel hydrogel for for the the postresection postresection treatment of glioblastoma. Reproduced from Ref. [46] with permission of the copyright holder. treatment of glioblastoma. Reproduced from Ref. [46] with permission of the copyright holder. 2. Nanoparticles for Glioblastoma One of the most promising approaches to increase the intravenous (i.v.) local delivery of drugs into the brain has been the use of nanoparticles (NPs) [49–53]. A schematic rep‐ resentation of the NP characteristics that define their efficiency for drug release is shown in Figure 3. Due to their small size and large surface area, nanoparticles can increase the solubility and bioavailability. Cancers 2022, 14, 4960 Figure 2. Codelivery of PTX and TMZ through a photopolymerizable hydrogel for the postresection 4 of 25 treatment of glioblastoma. Reproduced from Ref. [46] with permission of the copyright holder. 2. Nanoparticles for Glioblastoma 2. Nanoparticles for Glioblastoma One of the most promising approaches to increase the intravenous (i.v.) local delivery One of drugs of the into the most brainpromising approaches has been the to increase the use of nanoparticles intravenous (NPs) (i.v.) [49–53]. A local delivery schematic rep‐ of drugs into the brain has been the use of nanoparticles (NPs) [49–53]. resentation of the NP characteristics that define their efficiency for drug release A is schematic shown representation of the NP characteristics that define their efficiency for drug release is shown in Figure 3. Due to their small size and large surface area, nanoparticles can increase the in Figure 3. Due to their small size and large surface area, nanoparticles can increase the solubility and bioavailability. solubility and bioavailability. Figure 3. Key nanoparticle (NP) characteristics conditioning drug release and BBB crossing, classi‐ Figure 3. Key nanoparticle (NP) characteristics conditioning drug release and BBB crossing, classified fied in function of their composition (synthetic or natural), capacity for drug uploading, size, shape, in function of their composition (synthetic or natural), capacity for drug uploading, size, shape, charge, and possibility of surface functionalization to increase their biocompatibility and therapeu‐ ticcharge, and efficacy. possibility of Reproduced surface from functionalization Ref. [53] to increase with permission their biocompatibility of the copyright holder. and therapeutic efficacy. Reproduced from Ref. [53] with permission of the copyright holder. The other main advantages of their use are: The other main advantages of their use are:  They can endorse BBB diffusion [54] and increase the delivery of higher chemother‐ • They can endorse BBB diffusion [54] and increase the delivery of higher chemothera- apeutic doses (typically restricted by systemic toxicity) to the brain parenchyma peutic doses (typically restricted by systemic toxicity) to the brain parenchyma [55,56], [55,56], facilitated in some specific cases with the use of targeting receptors [57]; facilitated in some specific cases with the use of targeting receptors [57];  They can incorporate additional fluorescent/MRI/radioactive compounds that allow • They can incorporate additional fluorescent/MRI/radioactive compounds that allow the non‐invasive monitoring of its biodistribution [58]; the non-invasive monitoring of its biodistribution [58]; • They Theyconfer conferchemical chemicalprotection protectiontotothe thedrug drugand anda atheoretical theoreticalcontrol controlover overthe therelease release upon activation with a stimulus that minimize undesired side effects upon activation with a stimulus that minimize undesired side effects [59]; [59]; • They Theyincrease increasesolubility solubilityofofhydrophobic hydrophobicdrugs drugswhile whilefavoring favoring a aproper properbiodistribu‐ biodistribu- tion [60], evading the mononuclear phagocyte system (MPS) tion [60], evading the mononuclear phagocyte system (MPS) [61–64]; [61–64]; • They can combine different additional therapeutic approaches, such as, not exclusively, radiotherapy sensitization, immune cells stimulation, or induction of heat/radical oxygen species (ROS) [65]; • They can benefit from the well-known enhanced permeability and retention effect (EPR) to access (and remain on) tumor tissues; • Nanocapsulation increases the half-life activity, for instance in the case of TMZ-loaded chitosan NPs from 1.8 to 13.4 h [66]. All these advantages have raised an increasing interest for the development of nanopar- ticles for brain diseases, as reflected by the increasing number of publications and citations in the area for the last 10 years (Figure 4a). Specifically, for glioblastoma diagnosis and/or treatment (Figure 4b), the 195 articles published in 2010 contrast with more than 400 pub- lished in 2021, and allow predicting an upward trend for next years. The data shown in Figure 4b reflects the number of articles mentioning “nanoparticles + glioblastoma” and corresponding citations consulted in the WoS webpage until 15 September 2022. The re- lated publications show different families of NPs that have been used for diagnosis and/or treatment of diseases in the central nervous system (CNS) [65,67–70], mainly polymeric and and/or treatment (Figure 4b), the 195 articles published in 2010 contrast with more than 400 published in 2021, and allow predicting an upward trend for next years. The data shown in Figure 4b reflects the number of articles mentioning “nanoparticles + glioblas‐ toma” and corresponding citations consulted in the WoS webpage until September 15, Cancers 2022, 14, 4960 2022. The related publications show different families of NPs that have been used for 5di‐of 25 agnosis and/or treatment of diseases in the central nervous system (CNS) [65,67–70], mainly polymeric and lipid‐based NPs [71,72]. Drug encapsulation can take place by ei‐ ther physical entrapment, covalent linking or surface adsorption combined with passively lipid-based NPs [71,72]. Drug encapsulation can take place by either physical entrapment, [73–75] or actively [76–79] design‐dependent targeting. Other frequently used carriers are covalent linking or surface adsorption combined with passively [73–75] or actively [76–79] vesicles (lipidic, micellar, or exosomes) [80–84], mesoporous silica [85], metal nanoparti‐ design-dependent targeting. Other frequently used carriers are vesicles (lipidic, micellar, cles [86,87], carbon dots [88], nano‐implants [89], or dendrimers [90]. Overall, the use of or exosomes) [80–84], mesoporous silica [85], metal nanoparticles [86,87], carbon dots [88], NPs for GB therapy [91] aims to increase the efficacy of drugs both at preclinical and clin‐ nano-implants [89], or dendrimers [90]. Overall, the use of NPs for GB therapy [91] aims to ical stages [92]. increase the efficacy of drugs both at preclinical and clinical stages [92]. Figure 4. Number of publications and citations in the last 10 years related to (a) nanoparticles used Figure 4. Number of publications and citations in the last 10 years related to (a) nanoparticles used forfor brain brain diseases diseases andand (b)(b) nanoparticles nanoparticles developed developed forfor glioblastoma glioblastoma treatment. treatment. 3. Preclinical Studies Preclinical studies enable researchers to model drug biological effects and predict treatment outcomes (efficacy) as well as toxicity and adverse events (safety) in human patients. While different comprehensive studies have already been reported [93–95], in this review, we aim to launch a critical comparison of the different examples so far described in the literature; most of them summarized in Table 1. As it can be seen there, the experimental design is highly variable across different studies, which makes comparisons extremely difficult. On top of that, nanoparticle batch variations and aspects such as endotoxin amounts are relevant, especially in the case of intravenous administrations [96]. Considering such scattering of experimental parameters, particular attention has been paid to the following relevant aspects: 1. Orthotopic GB studies. Glial tumors are characterized by their heterogeneity and their immunosuppressive tumor microenvironment, which can be hardly replicated in a heterotopic model (e.g., subcutaneous). Such “niches”, comprising all components of a tumor as well as its interaction with tumor microenvironment, must be considered as it might play a role in the therapeutic efficacy [97]. Thus, useful translational studies of relevance in subsequent clinical cases should replicate as faithfully as possible the human situation. Cancers 2022, 14, 4960 6 of 25 2. Animal and gender model. Regarding species, circa two-thirds of the studies were performed in mice, while the remaining ones have used rats as experimental subjects. With respect to gender of the preclinical subjects, it is worth mentioning that it was not detailed in almost one-third of the studies, and only one of the mentioned papers included representation of both sexes. As for the rest, males are slightly more represented than females, but it is still quite balanced. Overall, it was reported that glioblastoma growth and aggressiveness may vary between males and females [98], so a lack of this information in part of the published studies can lead to a biased information [99]. 3. Administration schedule. Administration schedule and methodology used is quite different along the studies shown in Table 1. Regarding the therapy starting point, circa one-third of the studies have started therapy ranging 1–6 days post cell inoc- ulation. The remaining studies are distributed equally around days 7–10 post cell inoculation or later time points. The administration schedule was probably the most variable, both from the interval point of view and the final number of administrations (in general, intravenous). It was already shown that the administration protocol may strongly influence outcomes [100,101]. Thus, discrepancy in this factor may help to explain the differences in the results obtained. 4. Immune system. Undesirable interactions between the immune system and nanopar- ticles can take place, due to either immunostimulation or immunosuppression [102], removing at least part of the administered nanoparticles before their delivery to the target area. Thus, selection of immunocompetent (i.e., mice/rats bearing gliomas originating from their same species) versus immune-deprived (i.e., PDX models or xenograft inoculated with cell lines from human origin) models represents an impor- tant step. Moreover, specific pathogen free (SPF) husbandry is a common practice applied in laboratories conducting preclinical experiments, and SPF mice have an immature immune system when compared with wild strains [103]. Therefore, the im- mune system of SPF mice does not adequately reflect that of clinical subjects, neither do the results [104]. Immunocompetent, syngeneic/isogeneic murine models may be of help, but even in this case, certain cell targets or metabolic pathways may be different from human counterparts, since they will bear murine tumors. The most advisable model would be humanized PDX, although those are more complex and challenging than the already-established murine models [105,106] 5. Animal age. This is a relevant factor that governs baseline immunity and affects the hormone levels (sexual maturity) of the individuals; both impact the disease evolution. However, in most cases indicated in Table 1, the age of individuals is not given, so it was inferred from the standard growth charts. Having this limitation in mind, circa 50% were within 4–6 weeks of age, and the remaining ones were around 7–10 weeks of age, with one study going above (14–18 weeks). These values would rather correspond to early adolescence [107], while glioblastoma incidence increases with age, with circa 50% of the cases diagnosed in patients equal or more than 65 years old [108]. 6. Tumor volume. This is an essential parameter, since preclinical tumors are usually variable even when experiments are performed by experienced researchers. This value was reported in circa 60% of the studies included in this review, while it is unclear whether it was not performed or not reported in the remaining studies. Additionally, it is definitely not the same for any therapeutic agent to fight a large established or an early, low-volume, starting tumor [109]. Moreover, based on our experience, we should not assume that the whole cohort will have tumors with comparable volumes. 7. Therapeutic efficacy. Some articles report euthanization of mice and postmortem evaluation of tumors, which may definitely inform about the immediate action of the agent, but ideal situations may imply non-invasive assessment of tumor disappear- ance or growth arrest at long-term follow-up procedures. Cancers 2022, 14, 4960 7 of 25 Table 1. Nanomedicine-based drug delivery systems tested in orthotopic preclinical glioma models. Tumor Dose and Evaluation of Immunocompetent/ Administration Therapy Volume/Presence Drug(s) Type of NPs Animal Model Age Administration Targeting Antitumor Refs Immunodeprived Schedule Starting Point Estimated at Route Effect/Site Arrival Starting Point U87-TL-bearing 5–10 mg/kg Every 3 days, Angiopep-2 + In vivo TMZ Liposomes BALB/c male 4–6 wk immuno-deprived Day 1 p.i. yes [110] intravenous 5 times in total anti-CD133 mAb bioluminiscence nude mice U87-bearing NCR nude mice/GL261- 100 µL Every day In vivo TMZ + JQ1 Liposomes 6 wk Both Day 14 p.i. yes Transferrin [111] bearing intravenous during 5 days bioluminiscence C57/BL6 male mice TMZ-resistant 5–10 mg/kg Every 3 days, In vivo TMZ + ART Liposomes U251-TR GB 5–6 wk immune-competent Day 8 p.i. no ApoE peptide [112] intravenous 5 times in total bioluminiscence nude mice C6-bearing 10 mg/kg Every 2 days, Histopathology at TMZ Albumin NP BALB/c and 5–6 wk Both Day 5 p.i. no Sinapic acid [113] intravenous 8 times in total endpoint KM mice GL261-bearing 5 mg/kg Every 2 days, Histopathology at TMZ Lactoferrin NP 5 to 10 wk * immune-competent Day 3 p.i. no Lactoferrin [114] C57/BL6 mice intravenous 4 times in total endpoint GL261-bearing β Polymer-lipid 10 mg/kg Every 2 days, T2w MRI/ TMZ + siTGF C57/BL6 male n.d. immune-competent Day 8 p.i. no Angiopep-2 [115] hybrid NP intravenous 3 times in total Prussian staining mice C57BL/6 mice erythrocytemem- bearing brane 5 mg/kg Every 2 days In vivo and ex vivo TMZ + OTX015 orthotopic 3–4 wk Immune-competent Day 20 p.i. no ApoE peptide [116] camouflaged intravenous 5 times in total fluorescence GL261-Luc nanoparticle tumor Carmustine + F98-bearing Every 4 T1 and T2w 6.43–19.29 mg/kg O6- Polymeric NP Fischer 344 male 7 wk * immune-competent days, 3 times in Day 5 p.i. no ———– MRI/histopathology [117] intravenous Benzylguanine rats total at endpoint F98-bearing ICR Carmustine + Overall male mice and 6.43–19.29 mg/kg Every 4 days, O6- Polymeric NP 5–6 wk * Both Day 5 p.i. no iRGD survival/In vivo [17] F98-bearing intravenous 3 times in total Benzylguanine fluorescence male nude mice Magnetic targeting + C6-bearing 0.5–13 mg/kg transient T1 and T2 * w Carmustine Magnetic NP Sprague-Dawley 14–18 wk immune-competent intravenous, via Single dose Day 17 p.i. yes [118] ultrasound- MRI/Histology male rats jugularvein mediated BBB disruption Cancers 2022, 14, 4960 8 of 25 Table 1. Cont. Tumor Dose and Evaluation of Immunocompetent/ Administration Therapy Volume/Presence Drug(s) Type of NPs Animal Model Age Administration Targeting Antitumor Refs Immunodeprived Schedule Starting Point Estimated at Route Effect/Site Arrival Starting Point U87-bearing In vivo fluores- 1 mg/kg Carmustine Micelles BALB/c male 5–6 wk * immune-deprived Single dose Day 14 p.i. yes T7 peptide cence/postmortem [119] intravenous nude mice brain fluorescence BT325-bearing 2 mg/kg Every 3 days, In vivo Carmustine Micelles BALB/c nude n.d. immune-deprived Day 14 p.i. yes Pep-1 + borneol [120] intravenous 5 times in total bioluminiscence mice U87-bearing 1.2–13 mg/kg 10 consecutive Lomustine Nanocapsules female CD-1 5–6 wk * Immuno-deprived Day 7 p.i. yes ——- T2w MRI [121] intravenous days nude mice 3–5 mg (calculated to Cisplatin, F98-bearing Overall Liposomes n.d. Immune-competent body surface Single dose Day 10 p.i. no ——- [122] Oxaliplatin male Fischer rats survival/ICP-MS area), intracarotid 101/8-bearing 5 mg/kg, Every 5 PMAA-PEG mAb anti-Cx43 + Cisplatin female Wistar 9–10 wk * Immune-competent intravenous days, 3 times in Day 5 p.i. yes T2w MRI [123] Nanogel mAb anti-BSAT1 rats (femoral) total F98-bearing female Fischer344 2–5 mg/kg, Every 7 days, Cisplatin PAA-PEG NP 8–9 wk * Immune-competent Day 14 p.i. yes --– T1w MRI [124] rats/9L-bearing intravenous 3 times in total Sprague- Dawleyrats Transient C6-bearing Gelatin-heparin 2 mg/kg, Every 2–3 days, ultrasound- Cilengitide Sprague Dawley 8–10 wk * Immune-competent Day 7 p.i. yes T1 and T2w MRI [27] NP intravenous 8 times in total mediated BBB male rats disruption Magnetic targeting + C6-bearing T2w MRI and 2 mg/kg, Twice a week, transient Cilengitide Liposomes Sprague Dawley 8–10 wk * Immune-competent Day 7 p.i. yes fluorescence imag- [125] intravenous 8 times in total ultrasound- male rats ing/Histology mediated BBB disruption U87-bearing 15.2 µmoles/kg, Every 2 days, Transferrin + Overall sur- Erlotinib + DOX Liposomes nude female and n.d. Immune-deprived Day 10 p.i. no [29] intravenous 3 times in total Penetratin vival/Histopathology male mice 2–4 times a U87-bearing 10–100 mg/kg, Lapatinib Albumin NP 4–6 wk Immune-deprived week, for Day 8 p.i. no --- Histopathology [30] BALB/C mice intravenous 2 weeks Cancers 2022, 14, 4960 9 of 25 Table 1. Cont. Tumor Dose and Evaluation of Immunocompetent/ Administration Therapy Volume/Presence Drug(s) Type of NPs Animal Model Age Administration Targeting Antitumor Refs Immunodeprived Schedule Starting Point Estimated at Route Effect/Site Arrival Starting Point U87-EGFRwt- Methacrylamide 5 mg/kg, Every other day, Choline In vivo Nimotuzumab bearing female 5 wk Immune-deprived Day 3 p.i. yes [126] NP intravenous 9 times in total analogues bioluminiscence mice U87-bearing Regorafenib nude mice 1.5 mg/kg, Not specified, Peptide T12 + In vivo +Disulfi- Albumin NP 4–6 wk Both Day 10 p.i. yes [127] GL261-bearing intravenous 5 times total mannose bioluminiscence ram/cooper C57/BL6 mice Cediranib PEG-bilirrubin C6-bearing male 1.7–3.6 mg/kg, Every 2 days, n.d. Immune-deprived Day 10 p.i. no D-T7 peptide Histopathology [128] +Paclitaxel NP Balb/c mice intravenous 6 times in total U87-bearing Every 3 days or 4 or 10 mg/kg, Day 3 or day 5 Camptothecin Polymeric NP athymic nude 8 wk Immune-deprived every 5 days, no Adenosine Overall survival [129] intravenous p.i. mice 3 times in total GL261-bearing 10–20 mg/kg Every 7 days, In vivo Camptothecin Polymeric NP 10 wk Immuno-competent Day 8 p.i. yes --- [130] C57 albino mice intravenous, 3 times in total bioluminiscence U87, GBM43, or Twice a week, GBM6- 1 mg/kg, In vivo Topotecan Liposomes 6 wk Immune-deprived up to 6 times in Day 6–8 pi.i yes --- [131] bearingFemale intravenous bioluminiscence total athymic mice U251-bearing “non-leaky” Every 7 to Overall survival, Irinotecan Liposomes Rag2 female 7–10 wk 25–100 mg/kg Day 21 p.i. no --- [132] immune-deprived 14 days histopathology mice U251-bearing In vivo Irinotecan + NOD.CB17- 25–50 mg/kg, Every 7 days, fluorescence, Liposomes 7–10 wk Immuno-deprived Day 14 p.i. yes --- [133] TMZ SCIDfemale intravenous 3 times in total overall survival, mice histopathology Twice a U87-bearing Overall survival, Irinotecan Liposomes 7–9 wk * Immune-deprived 50 mg/kg week, 4 times in Day 5 p.i. no --- [134] male nude rats histopathology total 3.5 mg, GS2-bearing intranasal 0.01 Every 7 days, Unclear15–30 In vivo Irinotecan Liposomes male athymic 6 wk Immune-deprived to 1 mg, CED 30 yes --- [135] 3 times in total p.i. bioluminiscence rats mg/kg, intravenous U87-bearing Cetuximab + Irinotecan + 30 mg/kg, Every 3 days, In vivo Liposomes Balb/c nude 6–8 wk Immune-deprived Day 11 p.i. yes Magnetic [136] Cetuximab intravenous 3 times in total bioluminiscence mice targeting Cancers 2022, 14, 4960 10 of 25 Table 1. Cont. Tumor Dose and Evaluation of Immunocompetent/ Administration Therapy Volume/Presence Drug(s) Type of NPs Animal Model Age Administration Targeting Antitumor Refs Immunodeprived Schedule Starting Point Estimated at Route Effect/Site Arrival Starting Point U87-bearing Ex vivo 2 mg/kg, Every 3 days, Day 6 or day 15 DOX Liposomes male Balb/c n.d. Immune-deprived no MC + DA7R (postmortem) [137] intravenous 5 times in total p.i. nude mice bioluminiscence 100 µL with a Overall survival, concentration of U87-bearing Every 3 days, Ex vivo DOX Liposomes n.d. Immune-deprived 0.01 µM (total Day 10 p.i. no CB5005 peptide [138] nude mice 5 times in total (postmortem) dose 10 mg/kg), bioluminiscence intravenous C6-bearing male DOX + pH-sensitive 0.33–1 mg/kg, Unclear Sprague- 8–10 wk * Immune-competent Day 7 p.i. yes —- T1w MRI [139] Curcumin coreshell NP intravenous schedule DawleyRats GL261luc- In vivo bearing Every 3 days, DOX +1-MT i MSNs 6 wk Immune-competent 2.5 mg/kg Day 5 p.i. yes iRGD bioluminiscence, [140] C57BL/6 female 5 times in total MRI mice Legumain Every 2 days, DOX + HCQ responsive gold C6-bearing mice n.d. unclear 2.5–15 mg/kg Day 10 p.i. No —- Overall survival [141] 5 times in total NP TMZ: temozolomide; CD133: transmembrane glycoprotein overexpressed in cancer stem cells; mAb: monoclonal antibody; JQ1: bromodomain inhibitor; ART: artesunate; iRGD: cyclic integrin-targeting peptide; PMAA: poly (methacrylic acid); OTX01: epigenetic bromodomain inhibitor; PEG: poly (ethylene glycol); PAA: poly (aspartic acid); Cx43: membrane protein connexin 43; BSAT1: brain-specific anion transporter 1; EGFR: endothelial growth factor receptor; MC + D A7R: myristic acid-modified neuropilin-1 and vascular endothelial growth factor-targeting peptide; PDCP-NP: pH-sensitive core-shell nanoparticles; 1-MT: 1-methyltryptophan; DSPE-PEG: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethyleneglycol)-2000; SPION: superparamagnetic iron oxide nanoparticle; HCQ: hydroxychloroquine; MSNs: mesoporous silica nanoparticles; CREKA: fibronectin- targeting peptide. wk: weeks. n.d.: not defined. p.i.: post-inoculation. CED: Convection-enhanced Delivery. * Not detailed, only body weight provided: age estimated according to the body weight charts provided from Jax, Janvier, or Charles River webpages for any specified model. Cancers 2022, 14, x FOR PEER REVIEW 12 of 26 Cancers 2022, 14, 4960 11 of 25 3.1. Representative Examples 3.1. Representative Examples All the studies shown in Table 1 prolonged the survival of orthotopic GB rodent mod‐ All the els [110,142]. studies shown Representative in Table examples are1 detailed prolonged the survival of orthotopic GB rodent next. models [110,142].(TMZ). Temozolomide Representative examples The efficacy are detailed of diverse next. nanocarriers has been TMZ‐loaded Temozolomide (TMZ). The efficacy of diverse TMZ-loaded evaluated in GB orthotopic animal models. For instance, TMZ‐loaded nanocarriers liposomeshascoated been eval- with the BBB‐targeting angiopep‐2 and the anti‐CD133 monoclonal antibody were admin‐with uated in GB orthotopic animal models. For instance, TMZ-loaded liposomes coated the BBB-targeting istered in adult BALB/c angiopep-2 nude mice and the anti-CD133 instilled with humanmonoclonal antibody glioma stem cells were [110].administered The me‐ in adult BALB/c nude mice instilled with human glioma stem dian survival time doubled (49.2 days) when compared to the non‐structured TMZ cells [110]. The (23.3 median survival time doubled (49.2 days) when compared to the non-structured days), using the same doses (10 mg/kg). Similar nanoparticles based on transferrin‐func‐ TMZ (23.3 days), using the tionalized same doses PEGylated (10 mg/kg). liposomes wereSimilar nanoparticles also combined withbased on transferrin-functionalized the epigenetic drug JQ1 that PEGylated liposomes were also combined with the epigenetic blocks the interaction between transcriptional proteins, aiming to sensitize drug JQ1GB thattowards blocks the interaction between transcriptional proteins, aiming to sensitize TMZ. Accordingly, murine glioma‐bearing mice treated for 5 days extended median GB towards TMZ. sur‐ Ac- cordingly, murine glioma-bearing mice treated for 5 days extended median survival while vival while reducing systemic side effects (Figure 5) [111]. reducing systemic side effects (Figure 5) [111]. Figure Figure (a) Schematic 5. Schematic 5. (a) of a of a PEGylated PEGylated dualdual drugdrug (TMZ (TMZ + JQ1)-loaded + JQ1)‐loaded liposome liposome that that can becanfunc‐ be func- tionalized (i.e., transferrin, folate, and hyaluronic acid (HA)) to enhance transport across the BBBBBB tionalized (i.e., transferrin, folate, and hyaluronic acid (HA)) to enhance transport across the and and targeting targeting to glioma to glioma cells;cells; (b) Kinetics (b) Kinetics of drug of drug release release fromfrom liposomes liposomes loaded loaded withwith bothboth JQ1 JQ1 and and TMZ; (c) Bioluminescent TMZ; (c) Bioluminescent images of GL261 images micemice of GL261 taken on day taken 0 and on day dayday 0 and 5 following initiation 5 following of of initiation treatment with free drug formulations (free drug), drugs loaded in PEG‐Cy5.5 treatment with free drug formulations (free drug), drugs loaded in PEG-Cy5.5 liposomes (PEG-NP liposomes (PEG‐NP drug), or transferrin‐PEG‐Cy5.5 drug), or transferrin-PEG-Cy5.5 liposomes (Tf‐NP liposomes drug); (Tf-NP (d) quantification drug); (d) quantificationof average dailydaily of average body body weights weights of mice after 96 h of free drug and Tf-NP treatment course (Student t-test for n = 5; ** p p≤≤0.01; of mice after 96 h of free drug and Tf‐NP treatment course (Student t‐test for n = 5; ** 0.01; **** p ≤ 0.0001). Reproduced from Ref. [111] with permission of the copyright holder. **** p ≤ 0.0001). Reproduced from Ref. [111] with permission of the copyright holder. Recently, TMZ Recently, TMZ was wasencapsulated encapsulated in ApoE‐functionalizedliposomes in ApoE-functionalized liposomes based based on ar‐ on artesunate- tesunate‐phosphatidylcholine (ARTPC). This nanosystem was presented as phosphatidylcholine (ARTPC). This nanosystem was presented as a delivery nanocarrier a delivery nanocarrier for dual for dual therapeutic therapeutic agents (ART/TMZ) agents (ART/TMZ) to TMZ‐resistant to TMZ-resistant U251-TRU251‐TR GB inGB in vivo. vivo. In vitro In vitro studies studies showed showed synergistic synergistic DNADNA damage damage and and induction induction of apoptosis. of apoptosis. Moreover, Moreover, in vivo in vivo studies studies demonstrated demonstrated an effective an effective BBBBBB crossing crossing andanddeepdeep intracranial intracranial tumortumor pen‐ penetration. etration. The targeted The targeted combination combination liposomes liposomes resulted resulted in a significant in a significant decreasedecrease of U251‐ of U251-TR glioma TR and glioma and improved the survival of mice, reducing systemic TMZ‐induced improved the survival of mice, reducing systemic TMZ-induced toxicity [112]. toxicity [112]. Administration of albumin-based NPs coated with a polymer bearing sinapic acid (BBB-delivery shuttle) and encapsulating TMZ to orthotopic C6-bearing BALB/c mice Cancers 2022, 14, 4960 12 of 25 also increased the median survival time while decreasing peripheral toxicity [113]. En- capsulation of TMZ within lactoferrin and administration to orthotopic GL261-bearing C57/BL6 mice also yielded similar results [114]. Lastly, the encapsulation of TMZ in polymer-lipid hybrid nanoparticles decorated with both the targeting peptide angiopep-2 and the small interfering RNA against tumor growth factor β (siTGF-β) demonstrated synergism in modifying the immune microenvironment of GB and improving the effects of the drug [115]. In a recent study, TMZ was coencapsulated with an erythrocyte membrane camouflaged nanoparticle decorated with ApoE peptide [116]. The treatment using the obtained nanoconstructs in mice bearing orthotopic GL261 GB resulted in notable tumor inhibition and extended survival time with few side effects. The efficacy of the treatment was ascribed to several factors, namely, the improved GB targeting due to the greatly enhanced blood circulation time and blood–brain barrier penetration, the synergistic effect in the inhibition of cellular DNA repair and enhanced TMZ sensitivity in tumor cells, and the enhanced antitumor immune responses by inducing immunogenic cell death. Nitrosoureas. The best therapeutic success of NPs was reached by combining ni- trosoureas with other drugs [17,117] in comparison to monotherapy [118–120]. On top of that, higher doses can be administered as encapsulation, and solubility is increased in comparison to free drugs, prolonging in this way the survival of tumor-bearing mice decreasing/abrogating myelosuppressive effects [121]. Platinum-based drugs. Lipoplatin and Lipoxal, which are liposomal formulations con- taining cisplatin and oxaliplatin, respectively, were administered to F98 glioma-bearing male Fischer rats and demonstrated boosted anticancer activities compared to free drugs [122]. PE- Gylated poly (methacrylic acid) nanogels loaded with cisplatin and superficial monoclonal antibodies were also i.v. administered to glioma-bearing female Wistar rats, and the results indicated that nanogels exhibited enhanced tumor growth inhibition and increased the life expectancy of animals in comparison with other investigated formulations [123]. Moreover, an enhanced delivery efficiency and survival of animals, while reducing off-target toxicity, was obtained by using MRI-guided ultrasound after i.v. administration in glioma-bearing rats facilitating BBB crossing [124]. EGFR inhibitors. Administration of cilengitide-loaded NPs in C6-bearing Sprague– Dawley rats with BBB disrupted by transient focused-ultrasound [27], or directed with magnetic targeting [125] attained a median survival of up to three times that detected for free cilengitide, with no pathological damage to major organs. In another example, the authors demonstrated that the coencapsulation of erlotinib with DOX in liposomes functionalized with transferrin and cell-penetrating peptides was able to enhance its thera- peutic effect [29]. Other approaches involving the encapsulation—within NPs—of tyrosine kinase inhibitors, such as nimotuzumab, cediranib, or regorafenib [126–128], endorsed BBB crossing and glioma cellular uptake, therefore increasing the therapeutic effectiveness. Topoisomerase inhibitors. Polymeric NPs and liposomes decreased secondary effects of topoisomerase inhibitors, such as camptothecin and its derivatives topotecan and irinote- can [129–136], while providing a significant median survival increase [136]. Nanoliposomal formulations encapsulating irinotecan were particularly interesting when tested in preclini- cal studies with male athymic rat models [135] using three different administration routes, i.e., i.v., CED, and intranasal administration (IN). All of them were tolerated by non-tumor- bearing animals even though CED and IN allowed for a 4-fold and 100-fold dose range increase, respectively. Afterwards, the experiments were repeated on tumor-bearing ani- mals showing significant survival benefits, though this strongly depended on parameters such as infusate volume, addition rate, and number of infusions. The conclusion suggests that a single treatment of CED had the same effect on overall survival than multiple IV treatments. Interestingly, IN administration showed significantly increased survival of animals and demonstrated the promising potential of this non-invasive approach of deliv- ery bypassing the BBB using nanoliposomal formulations for the treatment of brainstem gliomas. These excellent results led to human clinical trials (NCT03086616, finished last October 4th), with no results reported yet. Cancers 2022, 14, 4960 13 of 25 Finally, DOX-loaded PEGylated liposomes covered with myristic acid modified DA7R were i.v. administered to intracranial U87-bearing male nude mice showing therapeutic effect [137]. If the DOX-loaded PEGylated liposomes are decorated with the CB5005 peptide, similar results are obtained with remarkable cell and nucleus permeabilities, resulting in a significant increase of the survival times [138]. Finally, different examples of NPs combining DOX with a curcumin-loaded dual-layer pH-sensitive NPs [139], 1-methyltryptophan (immune checkpoint inhibitor) in mesoporous silica [140], or hydroxychloroquine-loaded legumain-responsive gold nanoparticles [141] have been reported and substantiated their efficiency to overcome BBB, while enhancing synergistic effects. Other interesting explorative studies can be found in literature. For example, an innovative development consisted in biomimetic designs including angiopeptide-2 func- tionalized red blood cell membrane (Ang-M) camouflaging of NPs carrier containing copper chelators (Dp44mT), administered to Cu-enriched orthotopic U87MG-Luc GB for glioblastoma treatment. The principal objective of this study was to induce the generation of tumor-toxic reactive oxygen species (ROS) by selective targeting of copper present in cancer cells. The experimental results indicated that the NPs actively targeted and tra- versed the BBB, delivering Dp44mT specifically to GB cells and substantially prevented orthotopic GB growth, leading to maximal increases in median survival time [143]. An- other interesting development was based in the encapsulation of Plk1 inhibitor volasertib (Vol) in an angiopep-2 functionalized polymersome prepared from poly(ethylene glycol)- b-poly(L-tyrosine)-b-poly(L-aspartic acid). The in vitro assays determined an eight-fold better antitumor activity, and in vivo studies demonstrated an effective suppression of tumor growth in orthotopic U87 MG GB mice, increasing survival rates and reducing toxicity in comparison with free Vol [144]. It is worth to mention that nanomedicines are attractive systems, but the currently developed nanomedicines often suffer from poor serum stability or premature drug re- lease. In this sense, the development of liposomes or polymeric nanoparticles based on crosslinking strategies to employ stimuli-responsive chemical bonds were performed [145]. These improvements allow holding a robust nanostructure during systemic circulation, releasing the payloads spatiotemporally in a controlled manner in response to particular tumoral conditions. Apart from the later representative examples, there are several other nanosystems based in inorganic nanoparticles with potential to be used in GB treatment. Metallic nanoparticles are the most used approach for GB among inorganic nanoparticles, since they have shown high internalization ability, with the potential to be used as radiotherapy enhancers and as agents for brain imaging, presenting a good strategy as theragnostic probes. Some examples are related to metal oxide [146,147], gold [148], and silver [149] nanoparticles. Despite efforts made with metallic nanoparticles, few studies have validated BBB crossing for in vivo GB treatment [150], suggesting that more studies are necessary with this type of nanoparticles. Mesoporous silica nanoparticles (MSNs) are also inorganic NP with emerging uses in GB treatment due to their drug loading capacity, low toxicity, excellent biodistribution, and ease for surface functionalization, leading to tumor targeting, among others [151]. Several studies have shown that MSNs might have GB therapeu- tic potential. However, the lack of in vivo studies limits the projection of these type of nanoparticles [152,153]. Interestingly, carbon allotropes, such as carbon nanotubes (CNTs), graphene-based nanomaterials (GBNs), and quantum dots (QDs), have also been tested for GB treatment. CNTs garnered superior properties such as hydrophilicity, biocompatibility, and specificity, and a great variety of chemotherapeutics were encapsulated for GB treat- ment [154–156]. GBN, graphene oxides (GO), and reduced graphene oxides (rGO) can be functionalized with specific molecules on their surface to enhance BBB crossing [157,158], as well as different drugs with activity against GB [159], indicating that functionalization could significantly improve the BBB transport and GB targeting specificity. Finally, QDs have demonstrated to be nanoconstructs able to be functionalized with different targeting molecules and drugs, and showed remarkable cytotoxicity against GB cells, displaying Cancers 2022, 14, x FOR PEER REVIEW 15 of lated for GB treatment [154–156]. GBN, graphene oxides (GO), and reduced graphene o ides (rGO) can be functionalized with specific molecules on their surface to enhance BB Cancers 2022, 14, 4960 14 of 25 crossing [157,158], as well as different drugs with activity against GB [159], indicating th functionalization could significantly improve the BBB transport and GB targeting spe ficity. Finally, QDs have demonstrated to be nanoconstructs able to be functionalized wi different synergistic effects owing targeting molecules combinations to the dual-drug and drugs, and[160–162]. showed remarkable However,cytotoxicity as in most against G cells, displaying studies with inorganic synergistic nanoparticles, effects the lack of inowing to the dual‐drug vivo studies combinations makes it difficult [160–162]. How to evaluate ever, the real potential as in most of these studies with nanosystems inorganic for the nanoparticles, treatment of GB. the lack of in vivo studies makes difficult to evaluate the real potential of these nanosystems for the treatment of GB. 3.2. Targeting and Theranostic Beyond the3.2. Targetingsummarized examples and Theranostic in Table 1, another area of activity is the decoration of nanoparticles with Beyond the examples moieties binding BBBsummarized in TableSince target proteins. 1, another passive area of activity targeting drugis the decor delivery systems tionhave of nanoparticles with moieties not shown clinical binding value due to a BBB lack target proteins.many of selectivity, Since devel- passive targetin drug delivery opments are focused on active systems havedrug targeting not shown deliveryclinical value due nanosystems to to a lack selective achieve of selectivity, man treatment of GB. These nanoconstructs are designed to exploit differences in receptors to developments are focused on active targeting drug delivery nanosystems orachieve s antigen expression on the surface of tumor cells in comparison to normal cells. Common in rece lective treatment of GB. These nanoconstructs are designed to exploit differences active targetingtors or antigen vectors includeexpression on the surface small molecules, proteins,ofpeptides, tumor cells andinaptamers, comparison suchto as normal cel Common active targeting vectors folic acid, transferrin, and the RGD peptide [163]. include small molecules, proteins, peptides, and a tamers, such as folic acid, transferrin, and the RGD peptide [163]. However, special care must be considered, since most of the time such proteins are However, not exclusively expressed special at the care brain must be considered, microvasculature. since most Therefore, of the alternative finding time such proteins a not exclusively expressed at the brain microvasculature. Therefore, finding alternati strategies to target the BBB are required to increase drug efficacy [164]. In fact, targeting strategies to target the BBB are required to increase drug efficacy [164]. In fact, targetin of liposomes towards the glioma peritumoral area dates to 2012 [165], with several other of liposomes towards the glioma peritumoral area dates to 2012 [165], with several oth examples being reported since then. For instance, Kim et al. reported siRNA and small examples being reported since then. For instance, Kim et al. reported siRNA and sm drug-loaded cationic liposomes functionalized with an antibody recognizing the transferrin drug‐loaded cationic liposomes functionalized with an antibody recognizing the transfe receptor, resulting in improved effect when compared to free TMZ (see Figure 6) [166,167]. rin receptor, resulting in improved effect when compared to free TMZ (see Figure The treatment of U87 or TG98 xenografts with TMZ-containing NPs decorated with transac- [166,167]. The treatment of U87 or TG98 xenografts with TMZ‐containing NPs decorat tivator of transcription (TAT) peptides and/or imaging contrast agents exhibited enhanced with transactivator of transcription (TAT) peptides and/or imaging contrast agents exhi effect on features such ited as tumor enhanced volume, effect Ki67 staining, on features such as apoptosis, and CD133 tumor volume, staining,apoptosis, Ki67 staining, as an well as increased survival [168]. CD133 staining, as well as increased survival [168]. Figureof6.BBB Figure 6. Schematic Schematic of BBB crossing crossing ability ability of drug‐loaded of drug-loaded cationic liposomes cationic liposomes and cancer and cancer cell cell int nalization induced by surface functionalization with antitransferrin receptor (TfR) antibody fra internalization induced by surface functionalization with antitransferrin receptor (TfR) antibody ment designed to target TfR. Reproduced from Ref. [167] with permission of the copyright holde fragment designed to target TfR. Reproduced from Ref. [167] with permission of the copyright holder. Further experimental designs involved emulsions encapsulating CPT, tetraethylene glycol (TEG), and α-lipoic acid (ALA), which released the active drug upon enzymatic degradation [169]. In vitro assays using U87 MG glioma cells revealed a notable intracellu- lar uptake via cell membrane penetration, whereas in vivo studies demonstrated that the NPs were able to cross the BBB and to increase survival rates in a U87 xenograft model. Alternatively, endogenous exosomes are more stable than liposomes, and thus, their use may improve forthcoming drug delivery approaches. Beyond targeting, the development of theranostic NPs, such as iron oxide NPs (IONPs) that contribute not only to therapy, but Cancers 2022, 14, 4960 15 of 25 are also able to act as MRI imaging agents, has been explored. For example, IONPs coated with chitosan, PEG, and polyetheleneimine (PEI) exhibited T2 MRI contrast and siRNA delivery that fruitfully knocked down Ape1 expression, an enzyme that is crucial in the base excision repair pathway [170]. Alternatively, Mu et al. described IONPs decorated with gemcitabine (GEM), hyaluronic acid, and chlorotoxin (CTX), enabling GB cell targeting [171], even though hindering its infiltrative nature, which currently represents a challenge for thorough surgical resec- tion [172,173]. PEG-coated IONPs have also been functionalized with paclitaxel (PTX), fluorescein (FL), and CTX to eliminate in vitro MGMT-resistant GB tumor cells [174]. Despite the relevant scientific advances, the protective effect of the BBB poses chal- lenges to the clinical treatment of gliomas, even when its integrity is disrupted as in GB. Within the most recent progresses, the development of biomimetic drugs/materials with homologous binding and immune escape functions has been regarded as the most promis- ing alternative. Accordingly, future research should pay attention to the latest progress in basic research, including structural characteristics, composition, and permeability of BBB in gliomas. Additionally, researchers should identify and evaluate the factors that may affect the in vivo behavior of a specific nanosystem, such as the biodegradability of the utilized nanoparticles and aspects related to the protein corona on the nanoparticle surface, which can vary depending on the tumor type, tumor environment conditions, and even the administration route chosen for treatment. Although many nanosystems are designed in order to minimize this impact, it must be considered, and the take-home message may be that in vitro experiments are not able to totally mimic in vivo conditions affecting the protein corona. 4. Clinical Studies The average rate of translation from preclinical models to clinical cancer trials is less than 8% [175], mostly because the lack of: (i) standardization protocols, (ii) experimental details regarding cohorts, and (iii) best practice standards. A summary of them is given in Table 2. Table 2. Nanoparticle-based systems containing drugs currently undergoing clinical trials for glioma treatment. TMZ: temozolomide; anti-TfR: antitransferrin. Clinical Trials.Gov Name Drug Particle Type Targeting Moieties Identifier Onyvide® Irinotecan PEGylated liposomes --- NCT03119064 NL CPT-11 Irinotecan PEGylated liposomes --- NCT00734682 DOX (combined with Caelix® PEGylated liposomes --- NCT00944801 prolonged TMZ) 2B3-101 DOX PEGylated liposomes Glutathione NCT01386580 C225-ILs-Dox DOX Liposomes Cetuximab NCT03603379 Magforce, Inc. (Berlin, Iron oxide Nanotherm® --- --- Germany) (Approv. nanoparticles 2013) P53 plasmid (combined NCT02340156 SGT-53 cationic liposomes anti-TfR antibody with oral TMZ) NCT03554707 SGT94-01 RB94 plasmid Liposomes anti-TfR antibody NCT01517464 nucleic acids targeting (NU-0129) --- gold nanoparticles NCT03020017 BCL2L12 gene DaunoXome® --- Liposomes --- (Zucchetti et al.) [176] NCT02861222 Myocet® --- Liposomes --- (Chastagner et al.) [177] Cancers 2022, 14, 4960 16 of 25 Among them, worth to mention are the aminosilane-coated superparamagnetic iron oxide NPs, termed NanoTherm® , which generate heat upon application of an external alternating magnetic field that induces thermal ablation of the tumor tissue [178]. Accord- ingly, in a non-randomized phase 2 trial, the use of these NPs doubled the median overall survival of patients (up to 13.4 months) [65,179] with recurrent GB. Thus, NanoTherm® was approved in 2010 by the European Medicines Agency (EMA), though patients were re-irradiated as an adjunctive therapy to nanoparticles. Another example of inorganic nanocarriers suitable to reach clinical trials (NCT03020017, phase I, active) are gold NPs functionalized with nucleic acids targeting the gene BCL2L12 associated with GB tumor growth [180]. Beyond these two inorganic nanocarriers, most clinical trials are based on liposomal systems. FDA-approved Onyvide® consists of a PEGylated irinotecan liposomal formula- tion, aimed to treat metastatic pancreatic cancer, and already investigated in four clinical trials (NCT03119064, NCT00734682, NCT02022644, and NCT03086616), either alone or combined with TMZ and administered via i.v. or CED. Another PEGylated liposomal doxorubicin formulation (Caelyx® , Doxil® ) has been tested following the Stupp regimen [6] in the RNOP-09 phase II clinical trial (NCT00944801); however, this results in a non-reliable median OS, most likely owing to the insufficient sample size (n = 63) [181,182]. DOX-bearing PEGylated liposomal formulations combining additional glutathione (NCT01386580; phase I/II clinical trial) or cetuximab (NCT03603379, phase I, recruiting) were also analyzed. Other liposomes were focused on gene-based therapies, such as the plasmid-loaded cationic li- posome bound to anti-TfR antibodies (SGT-53), which, combined with TMZ, were able to finalize a phase II clinical trial (NCT02340156) for recurrent GB treatment. The same system lately joined a phase I trial (NCT03554707) to treat CNS tumors in children. Moreover, a related formulation (SGT94-01), based on the RB94 gene encapsulated in a liposome and bearing antitransferrin receptor single chain antibody fragment targeted to tumor cells, was tested on solid tumors (NCT01517464). 5. Summary and Future Perspectives Up to now, satisfactory results increasing median survival have been obtained using NPs for the treatment of GB in preclinical models, endorsed by the potential of NPs to increase drug availability with both lower doses and side effects. Functionalization of NPs with targeted receptors and/or proteins overexpressed on GB cells have also demonstrated in vivo capabilities regarding the specific delivery of drugs to tumor cells. However, only a few of them (less than 8%) have reached Phase I/II clinical trials and almost none of them have progressed to phase III or even to randomized phase II trials, which is surprising considering the successful preliminary preclinical results. This could be at least partially attributed to aspects related to the lack of standardization in preclinical studies. In other words, the use of NPs still must face development and planification challenges before its complete clinical implementation becomes true. Other issues to be solved are effective BBB crossing, minimizing side effects, and avoiding inappropriate biodistributions. Additionally, human biological diversity and disease heterogeneity should also be considered, as well as age and gender aspects, at least in preclinical models [183]. Future strategies should also reconsider the physicochemical and colloidal properties of the NPs, which are currently still not fully agreed. In other words, it would be highly recommended to carry out detailed and systematic preclinical studies that helps us to unequivocally determine and universally agree on basic aspects, such as dose ranges, administration schedules, pharmacokinetics and absorption, biodistribution, metabolism, and excretion, before reaching any clinical trial. Nanoformulations should also prove sufficient long shelf life and stability during long-term storage and clinical administration. On top of that, alternative and more effective administration pathways should be fully studied to avoid physiological barriers (i.e., BBB). In this context, intranasal drug administration represents a non-invasive route to directly targeting the brain. In the last few years, a few nanomedicines have been developed for this type of nose-to-brain transport Cancers 2022, 14, 4960 17 of 25 that bypasses the BBB and reduces systemic side effects. The preclinical and clinical studies using this administration route predict a breakthrough advancement in the fight against GB and have demonstrated the benefits of intranasal administration versus classical routes. In fact, our research groups have initiated this type of preclinical studies with very promising results, which encourage the developing of advanced and improved nanomedicines for GB treatment [184,185]. 6. Conclusions Current treatment strategies for glioblastoma are clearly unsuccessful, resulting in low patient survival rates and poor prognosis. Nanomedicine is able to afford different tools for improving diagnosis and especially to enable efficient targeting and transport of drugs across the BBB. Although many nanoformulations are currently being evaluated in cell lines and animal models, only a few have reached the clinical trial stage, and none of them proved to be an effective replacement for the present standard of care. The possible reasons behind this deceiving outcome are the hurdles due to the BBB barrier and the immunosuppressive glioblastoma environment as well as its heterogeneity. However, recent results are providing hope about the nanomedicine potential for achieving a better percentage of long-term survivors, provided some relevant experimental aspects are taken into account. Present advances in glioblastoma therapy may help to understand the molecular mechanisms underlying tumor progression and the potential role of new nanomedicine approaches. This review has discussed the advantages and limitations of current develop- ments and aimed to pave the way for research in the forthcoming years. There is presently no consensus about the most effective nanoformulations for brain cancer treatment, and variability in nanoparticle size, composition, targeting ability or mechanisms of drug deliv- ery. This situation makes it extremely hard to compare different studies. In this scenario, an effort to standardize in vitro/in vivo studies is urgently needed. Moreover, the ongoing clinical trials and future studies will hopefully shed some light on the optimal nanoparticle design for improving outcome in brain tumor patients. Thus, the major challenge of next years will be to optimize the existing nanoformulations and/or to work towards additional improvements, including the investigation of new administration routes, to enhance the translatability of therapies. Author Contributions: D.R.-M., J.L., F.N. and A.P.C. contributed to the conceptualization, methodol- ogy, validation, writing original, draft preparation, writing review and editing. P.A.-T., X.M., J.B. and V.J.Y. contributed to the draft preparation, editing. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by grant RTI2018-098027-B-C21, RTI2018-098027-B-C22, and PID2020-113058GB-I00 funded by MCIN/AEI/10.13039/501100011033, by ERDF A way of making Europe and by PI21/00181 from the Instituto Carlos III. This work was also supported by “Met- alfármacos multifuncionales para el diagnóstico y la terapia”, through grant RED2018-102471-T, funded by MCIN/AEI/10.13039/501100011033. The ICN2 and IDIBELL are funded by the CERCA programme/Generalitat de Catalunya. The ICN2 is supported by the Severo Ochoa Centres of Excellence programme, grant SEV-2017-0706, funded by MCIN/AEI/10.13039/501100011033. COST is kindly acknowledged for stimulating scientific discussion and financial support via the Cost Action CA 17140 “Cancer nanomedicine from the bench to the bedside”. A.P.C. received funding from the ATTRACT project funded by the EC under Grant Agreement 777222. This work was also supported by the Centro de Investigación Biomédica en Red-Bioingeniería, Biomateriales y Nanomedicina (CIBER- BBN [http://www.ciber-bbn.es/en], CB06/01/0010), an initiative of the Instituto de Salud Carlos III (Spain). The PhD program of X.M. was supported by the La Caixa-Severo Ochoa International PhD Fellowship. Conflicts of Interest: The authors declare no conflict of interest. Cancers 2022, 14, 4960 18 of 25 References 1. Stavrovskaya, A.A.; Shushanov, S.S.; Rybalkina, E.Y. Problems of Glioblastoma Multiforme Drug Resistance. Biochemistry 2016, 81, 91–100. [CrossRef] [PubMed] 2. Louis, D.N.; Perry, A.; Reifenberger, G.; von Deimling, A.; Figarella-Branger, D.; Cavenee, W.K.; Ohgaki, H.; Wiestler, O.D.; Kleihues, P.; Ellison, D.W. The 2016 World Health Organization Classification of Tumors of the Central Nervous System: A Summary. Acta Neuropathol. 2016, 131, 803–820. [CrossRef] 3. 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