Antiangiogenic Targets for Glioblastoma Therapy from a Pre-Clinical Approach, Using Nanoformulations

Glioblastoma (GBM) is the most aggressive tumor type whose resistance to conventional treatment is mediated, in part, by the angiogenic process. New treatments involving the application of nanoformulations composed of encapsulated drugs coupled to peptide motifs that direct drugs to specific targets triggered in angiogenesis have been developed to reach and modulate different phases of this process. We performed a systematic review with the search criterion (Glioblastoma OR Glioma) AND (Therapy OR Therapeutic) AND (Nanoparticle) AND (Antiangiogenic OR Angiogenesis OR Anti-angiogenic) in Pubmed, Scopus, and Cochrane databases, in which 312 articles were identified; of these, only 27 articles were included after selection and analysis of eligibility according to the inclusion and exclusion criteria. The data of the articles were analyzed in five contexts: the characteristics of the tumor cells; the animal models used to induce GBM for antiangiogenic treatment; the composition of nanoformulations and their physical and chemical characteristics; the therapeutic anti-angiogenic process; and methods for assessing the effects on antiangiogenic markers caused by therapies. The articles included in the review were heterogeneous and varied in practically all aspects related to nanoformulations and models. However, there was slight variance in the antiangiogenic effect analysis. CD31 was extensively used as a marker, which does not provide a view of the effects on the most diverse aspects involved in angiogenesis. Therefore, the present review highlighted the need for standardization between the different approaches of antiangiogenic therapy for the GBM model that allows a more effective meta-analysis and that helps in future translational studies.


Introduction
Glioblastoma (GBM) presents the worst prognosis for affected patients among all malignant tumors [1]. Regarding GBM treatment, advances in the pathophysiologic knowledge of disease in the last few decades have allowed the development and improvement of new therapeutic approaches in light of limited conventional therapeutics. Tumor resection followed by concomitant temozolomide administration and radiotherapy, in addition to occasional use of the bevacizumab (Beva) adjuvant, has not yet achieved a significant improvement in patient survival [2][3][4], with the exception of only a few cases with specific factors such as treatment of younger patients and maximal safe tumor resection, which has allowed survival after five years and corresponds to 5.6% of cases of GBM [5,6]. GBM has a complex character, showing high genetic heterogeneity with subclones existing in the same population of tumor cells [7].
The basic principle of antiangiogenic therapy is the disruption of blood supply to tumor cells, thus depriving the tumor of nutrients and oxygen and inhibiting the uncontrolled growth of tumor cells and their microenvironment [8][9][10][11].
When the tumor grows to a thickness of more than 2 mm, the cells in the nucleus are farther away from the blood supply, and the exposure of cancer cells to certain hypoxic stimuli [9,11] is the main trigger for initiating a series of reactions involving diverse molecular processes; for example, expression of hypoxia-inducible factors (HIF) that stimulate the expression of vascular endothelial growth factors (VEGFs) by tumor cells [8,9,12], in addition to communication with its auxiliary cells that stimulate the formation of other growth factors, such as platelet-derived growth factors (PDGFs) [12][13][14][15] and epidermal growth factors (EGFs) [10,16,17]. In chemotactic communication, inflammatory cells are recruited, such as tumor-associated macrophages (TAMs), which, in turn, stimulate a chain reaction of continuous tumor growth, intensifying the signaling of hypoxia to tumor cells, which further stimulates the expression of HIF and, mainly, VEGF [18][19][20][21][22]. All these growth factors in intercellular communication form several fronts of action that, in a dependent or independent manner, favor the formation of new blood vessels from pre-existing vessels [8][9][10]. Throughout the links of growth factors with the receptors present in endothelial cells that make up blood vessels, there is the activation of receptors, which are mostly transmembrane kinase proteins that, when activated, trigger a series of intracellular reactions to stimulate endothelial cell migration, proliferation, and differentiation as well as modulate vascular support cells [23][24][25]. For this migratory process to occur, it is necessary to have basement membrane degradation of the brain parenchyma extracellular matrix (ECM) through the stimulation of matrix metalloproteinases (MMPs) [11,26]. In all these processes of migration, proliferation, invasion, and remodeling, there is the ubiquitous action of calcium-dependent cell adhesion molecules, such as cadherins [27], or calcium-independent, such as integrins [28][29][30][31]. All of these described elements involved in angiogenesis are potential targets for therapeutic strategies, for example, the monoclonal antibody Beva that targets VEGF-A [32,33], or the tyrosine kinase inhibitor sorafenib (SFN) that targets the VEGF and PDGF receptors [34].
In order for these therapeutic agents to exert their antiangiogenic and antitumor effects, it is necessary that they penetrate the brain-blood barrier (BBB) [35,36]. One of the ways to penetrate the BBB is through local administration of therapeutic agents exercised by the technique of convection enhanced delivery (CED), in which a specific drug, through catheters connected with an infusion pump, is implanted directly into the developed tumor or in the parenchyma surrounding the tumor mass [37]. This technique is commonly applied in preclinical studies in GBM orthotopic models [38]; however, it has technical limitations and complications that make this type of therapy ineffective in clinical studies [39], requiring the development of structures that are safely administered in systemic circulation, are able to cross the BBB, and allow effective bioavailability of anti-tumor/anti-angiogenic drugs [35,36,40]. In this regard, a method that has been remarkable and promises to improve the delivery of bioactive compounds is the coupling of different types of nanostructured materials, such as a peptide based on polymeric structures [41,42], lipid-based liposomal formulations [43][44][45][46][47], and nanoshells [48]. These nanoformulations have the potential to provide clinically minimally invasive and targeted delivery of drugs with proven antiangiogenic effects or whose therapeutic potential is being pre-clinically tested [35,36,40].
One of the major scientific gaps in therapeutic strategies aimed at angiogenesis is how to effectively prevent the tumor from becoming nourished while also preventing GBM recurrence. It is necessary to highlight the actions of these nanoformulations on the various processes involved in angiogenesis, especially those that have multiple directions and assume different therapeutic front lines, so that they can interfere in a more comprehensive and effective way in the stages of angiogenesis, whether in triggering the cascade of hypoxia; in the release of growth factors; in dilation, permeability, proliferation, and migration of angiogenic blood vessels; in supporting cells; or also in the actions of metalloproteinases in the ECM, among others.
Of the six studies that used rats as the model, the Sprague-Dawley species was used in 50% of these studies [54,60,61], two studies (33%) [38,65] used the Nude athymic species, and the study by Hekmatara [68] used Wistar rats. Rats were male in four studies (67%) [38,54,60,68], and only the study by Banerjee [61] used female rats. The minimum rat age was four weeks in the study by Hu [60], and the maximum age was 27 weeks in the study by Banerjee [61].
Tumors were induced in all of the selected studies by the intracranial route, and in most studies, the tumor cells were implanted in the right cerebral hemisphere of rodents, with the exception of the studies by Sousa [32] and Lin [56] that used the left cerebral hemisphere. Specific brain regions were reported in the selected studies; in mice, 29% [34,41,51,52,59,62] implanted in the right striatum, followed by 5% each in the right frontal lobe [57], parenchyma [63], right caudate nucleus-putamen [43], right hippocampus [66], and right basal ganglia field [67]. In the study by Hekamtara [68], tumors were administered in the right lateral ventricle, and by Saito [38] in the striatum.

Antiangiogenic Therapeutic Process for Glioblastoma
The therapeutic process of antiangiogenesis applied to the GBM models was analyzed in terms of the type of therapy, the therapeutic target, the route of drug administration, drug dose and the frequency used, the vehicle associated with the drug, and the time between induction of GBM and the outcome, focusing mainly on the efficiency in reducing the size of the tumor over time. The techniques applied for this evaluation are shown in Table 4.
The therapy type most reported (56%) in the selected studies for nanoformulation administration was the Drug Delivery system [32,41,43,45,50,51,53,54,56,[58][59][60][61][62]68]. Three of these studies [45,58,61] specified as Targeted Drug Delivery, in which the nanoparticle was directed at a specific target molecule, and Dual-Targeting Drug Delivery was used in four studies [41,51,59,60]. This therapeutic type represents a new method of improving the action of anti-tumor drug delivery, exploiting the targets for various kinds of receptors expressed on the surface of or inside tumor cells.
Systemic therapy was used in 5 studies (18%) [55,57,64,66,67], in which functionalized nanoparticles with different molecules were used such as proteins, peptides, and tumor markers. CED is a method of direct delivery and was used in three (11%) of the selected studies [34,38,52].
PDT and Photothermal Therapy (PTT) modalities use photosensitizers to promote a therapeutic effect and were used in two (7%) studies [48,63]. The study by Bechet [63] used PDT, which, with the combined action of photosensitizer and visible light, resulted mainly in the formation of reactive oxygen species (ROS) and oxygen singlets (1O 2 ). The study by Day [48] used PTT combined with the exposure of infrared light in the gold-based nanoparticles, which produced enough heat in the tumor region to induce cell death by protein denaturation and cell membrane rupture.
The study by Janic [65] applied a Ferumoxides-Protamine Sulfate (FePro) nanoparticulate contrast agent for cell therapy, and they reported the benefits of using endothelial progenitors cells (EPCs) in clinical applications as an alternative method to inhibit tumor vascular growth. The study by Costa [44] used a therapeutic approach based on indirect epigenetic modulation, using miR-21, which is associated with antiangiogenic chemotherapy and shows promising results.
VEGF and its receptors are the most common angiogenic markers. Of the studies that evaluated VEGF expression 50% quantified decreased expression [32,57,58]. The studies by Kuang [58] and Souza [32] obtained similar results for the expression of endogenous VEGF determined by VEGF mRNA analysis by quantitative polymerase chain reaction (qPCR) (49-50%) after 9 and 10 days, respectively. Souza [32] also analyzed VEGF extracellular expression by ELISA, showing that modulation of VEGF caused by Beva-loaded Poly(d,l-lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) at the intracellular level, although lower, had the desired effect of considerably inhibiting VEGF extracellular expression. In the study by Kuang [58], DGL-PEG-T7 [dendrigraft poly-l-lysines polyethylene glycol-peptide T7 (sequence His-Ala-Lle-Tyr-Pro-Arg-His)]/shVEGF could inhibit VEGF mRNA due to the T7 peptide that binds transferrin receptor (TfR) on the surface of the tumor cell, and the shVEGF subunit in the nanocomplex allowed DOX to inhibit tumor growth and angiogenesis.     Another classic endothelial cell marker for angiogenic blood vessels used in 19 of the selected studies [34,[43][44][45][50][51][52][53][54]56,57,[59][60][61][62][64][65][66] was CD31, in which 47% of the studies quantified results after therapy with different nanostructured materials applied [34,43,44,50,52,54,61,62]. The study by Wu [50] had the greatest reduction in CD31 expression with Lut with folic acid modified poly(ethylene glycol)-poly(e-caprolactone) (Lut/Fa-PEG-PCL) application (89% compared to the control group), significantly inhibiting the neovasculature of the GL261 tumor and playing an important role in inhibiting tumor cellular growth. A similar effect was observed in the study by Séhédic [52], with an 80% decrease in CD31 expression after seven days of lipid nanocapsules (LNC) (12G5-LNC 188 Re) application, in which clinical improvement was accompanied by locoregional effects on the tumor development including hypovascularization and stimulation of the recruitment of bone-marrow-derived TAM precursors (CD11b+ myeloid cells) or CD68-positive cells together with NOSII or Arg1 indicated the presence of pro-and anti-angiogenic macrophages, respectively, in which CD68 + / NOS-II was found inside towards the external part of the tumor, while CD68 +/Arg1 was exclusively present in the peripheral area of the tumor [52]. With the same therapeutic time (7 days), in the study by Feng [62], peptide (ACGLSGLGVA) with NP-PTX (CooP-NP-PTX) demonstrated great potential to improve anticancer activity and avoid the drawbacks of anti-angiogenic therapy alone with a reduction of 69% to 70% of CD31 expression. This reduction was also reported in the study by Bernarjee [61], after 15 days of application, the PTX-loaded solid lipid nanoparticles (SLN) modified with Tyr-3-octreotide (TOC) (PSM) demonstrated great potential as a dual target for tumor neovasculature and tumor cells due to TOC (in PSM surface) interaction with SSTR2 expressed in endothelial cells of the neovasculature-improving the PTX antiangiogenic effects. The study by Xu [54] also reported a similar reduction in CD31 expression, although 21 days after the end of treatment, the DTX-NPs-dBECM (decellularized brain extracellular matrix) complex displayed effective anti-angiogenic effects. With less efficiency in the ability to reduce CD31 expression (19-21%), the study by Clavreul [34] showed that lipic nanocapsules with SFN (SFN-LNCs) decreased the proportion of proliferating cells and the tumor vessel area 7 days after the end of treatment, inducing an early increase in tumor blood flow and vascular normalization process. The study of Varreault [43] also showed reduced tumoral blood vessel density 21 days after the end of treatment, suggesting restoration of the vessel architecture to a more normal state.
Another immunoassay reported in the selected studies was Western Blot (WB). The study by Lin [56] used WB to detect the presence of SPARC and gp60, two binding proteins of albumin that are shown to be expressed in blood vessels. The study by Saito [38] established the expression of blood vessels by laminin labeling and recognized the inhibition of Akt protein phosphorylation, which is related to the antiangiogenic activity. In addition to this, liposomal TPT decreased the expression of both proteins when compared to free TPT [38]. The study by Lin [57] reported the detection of VEGF expression by WB when performing a tissue analysis, despite having detected CD31 and CA IX by IHC. The ELISA immunoassay was reported in only one study [32] to detect VEGF protein levels.
The four studies [34,43,48,53] that evaluated angiogenesis by image techniques all evaluated blood perfusion in different ways. The laser speckle contrast images (LSCIs) recorded a 58% decrease in vascular permeability/flow after treatment with CARD-B6 (nanoparticles with B6 loading three drugs) [53]. MRI perfusion recorded a 24% decrease in blood flow due to the action of SFN-LNC [34], similar to the application of Dynamic Contrast-Enhanced-MRI. This made it possible to calculate the 85% reduction in Ktrans, which expresses a transfer volume constant between the vasculature and tissue compartment, by the action of Irinophore C TM [43].
In front of all results obtained in the systematic review, Figure 2 schematically shown the antiangiogenic nanoformulations for glioblastoma therapy from a pre-clinical approach and the angiogenic process in the tumor microenvironment, as well as the pie charts of the main results found in the systematic review, and the quantification of antiangiogenic efficiency appointed by each study.

Discussion
In this systematic review, we showed different antiangiogenic therapies using nanoformulations in the GBM model. In the analysis of antiangiogenic therapy, the tumor cell type was considered as well as the GBM animal model, the characteristics of nanoformulations to reach therapeutic targets, and how they interfered with the angiogenic process.
An adequate GBM animal model requires a genetic pattern that resembles human GBM; has genetic, epigenetic, and phenotypic intratumoral heterogeneity; an adequate microenvironment in relation to immunocompetence; presence of the blood-brain barrier (BBB); and interactions between tumor cells and healthy cells that are reproducible [70]. Therefore, many of the selected studies used human U87-MG tumor cells [32,34,38,52,53,[55][56][57][58][59][62][63][64]66,67], in which the tumor has a diffusely invasive infiltration pattern into normal brain parenchyma, resistance to therapy, and high recurrence rate [70]. Studies also used rat C6 tumor cells [41,51,54,60,61], in which the tumor mimics several features of human GBM including a high mitotic index, focal tumor necrosis, parenchymal invasion, and neoangiogenesis [71,72]. The GBM orthotopic model was induced mainly in immunosuppressed animals (e.g., nude or SCID) because this immunodeficient condition allows a greater possibility of the growth of human (xenogeneic) tumor cells after implantation, preventing their rejection [73]. GBM subcutaneous model is widely used in the literature due to its technical ease and high productivity, but it is not recommended for use in exploring the brain's infiltrative behavior and lacks adequate brain microenvironments, which are important factors in the study of angiogenesis [74].
In order to improve the efficiency of antiangiogenic therapy applied to the GBM model, nanoformulations were developed to target the different processes involved in angiogenesis. The hypoxic cascade is the main driver of sprouting angiogenesis through the expression of HIF-1, a heterodimer composed of HIF-1α and HIF-1β. Whereas HIF-1β is constitutively expressed, the level of HIF-1α is low under normoxic conditions [11]. Due to hypoxia, increased HIF-1α expression results in the increase of mRNA VEGF and sequential expression of VEGF in GBM tumor cells [8,9,12]. Only the study by Lin [57] used this first stage of the angiogenic process as a therapeutic target, interfering in the expressions of HIF-1α, CA IX, and VEGF in the hypoxia cascade. The study by Gao [41] evaluated the antiangiogenic process and detected the accumulation of HIF-1α expression in the GBM orthotopic model induced in BALB/c nude mice with C6 cells, as an angiogenic marker. Increased HIF-1α expression induces CA IX expression, a promissory endogenous hypoxia-related cell surface enzyme [75,76]. There is a need for tumor cells to adapt to the highly acidic extracellular microenvironment caused by increased metabolic production of CO 2 and lactic acid, in which CA IX catalytic activity is inhibited by low pH and is half-maximal (i.e., the pK) at pH~6.8 [75,76]. The expression of CA IX in GBM cases should be further investigated since its expression has shown to be more correlated with the prognosis of esophageal and gastric adenocarcinomas than the expression of VEGF [76]. Another protein upregulated by HIF-1α is CXCR4, a chemokine receptor for SDF-1α also known as CXCL12, whose expression occurs in hypoxic tumoral microenvironment conditions and vascular ischemia [69,77]. The studies by Séhédic [52] and Janic [65] used the CXCRL12 (SDF-1α) chemokine receptor, i.e., CXCR4, as their main target, which activates different signaling pathways including phosphatidylinositol-3 kinase (PI3K)/Akt and MAP-kinases [78]. In adults, the SDF-1/CXCR4 axis plays a role in GBM development, tumor cell proliferation, and invasiveness via activation of matrix metalloproteinases (MMPs) [78]. The study by Janic [65] applied cell therapy with umbilical cord EPCs carrying the magnetic agent Ferumoxides-Protamine Sulfate. EPCs have neovascularization potential due to the attraction to CXCR4 receptors, resulting from the hypoxia process present in the tumor microenvironment, activating this signaling pathway [79][80][81]. Thus, it is relevant to use EPCs labeled with magnetic nanoparticles as MRI probes for monitoring the neovascularization process [65]. In septic patients, EPC expression of CXCR-4 and high serum concentrations of VEGF, SDF-1α, and Ang-2 were associated with probability of survival, showing that the SDF-1/CXCR4 axis plays a crucial role in homing EPCs in the course of sepsis [80].
VEGF expression is the main product of hypoxia related to the angiogenesis process, which was approached in some studies [32,57] as the therapeutic target. The study by Souza [32] used Beva, a well-established monoclonal antibody that acts by directly inhibiting extracellular VEGF, linked in polymeric nanoparticles [(Poly(d,l-lactic-co-glycolic) acid] to improve the antibody-based therapeutic delivery and reduce off-target toxicity, a strategy that resulted in a greater (around 50%) antiangiogenic effect analyzed by VEGF protein and endogenous mRNA VEGF. These polymeric nanoparticles are used as carriers for different drugs such as in chemotherapy, which are either adsorbed on the surface or encapsulated within the nanoparticles. These improve the release kinetics, the compatibility with some active agents, have no oxidation issues (as with phospholipids), and enhance shelf-life [82]. Other studies [63,68] also detected VEGF in antiangiogenic therapy but did not quantify the protein expression after therapy. The study by Bechet [63] showed an intense decrease of Ki67 and VEGF protein expressions in the tumor tissue immediately after interstitial photodynamic therapy (iPDT), and the study by Hekmatara [68] showed that DOX in solution led to a slight decrease in necrosis and microvascular proliferation, whereas DOX bound to polysorbate 80-coated poly(butyl cyanoacrylate) nanoparticles drastically decreases necrosis and led to the complete disappearance of microvascular proliferation. DOX is an antibiotic applied as chemotherapeutic medication and is synthesized by Streptomyces peucetius, which inhibits topoisomerase II (topo II) through multifactorial mechanisms involved in the cytotoxic response. It is clinically effective in the management of hematological malignancies and solid tumors [83]. This antibiotic was used in some of the selected studies, but it was linked with different formulations, such as in polylactic acid nanoparticles linked with the CRKRLDRNC peptide to direct the nanoformulation to IL-4R [51]; with shVEGF, an anti-VEGF gene drug [58]; and with polydopamine-coated mesoporous silica functionalized with Asn-Gly-Arg (NGR) targeting CD13 [59], which is expressed exclusively on the angiogenic endothelium and not on normal vasculature [84].
VEGF angiogenic effects are mediated by three receptor tyrosine kinases (RTKs): VEGFR-1 and VEGFR-2, which play major roles in physiological as well as pathological angiogenesis, including tumor angiogenesis, and VEGFR-3, which can regulate angiogenesis in early embryogenesis but mostly functions as a critical regulator of lymphangiogenesis [85]. These VEGF receptors were used as therapeutic targets in three of the selected studies [34,48,63]. Using nanoformulations for external therapy, the study by Day [48] targeted VEGF linked to nanoshells and applied PTT to achieve an effect in the endothelial cell. They showed a decrease of 42% vessel density, evaluated by intravital microscopy after 3 days of treatment, as well as disruption of tumor vessels, predominantly in the tumor and at its periphery, but not in the adjacent normal brain. The study by Béchet [63] administered the photosensitizer chlorin conjugate with a heptapeptide (ATWLPPR), specific for the VEGFR and its membrane-bound coreceptor neuropilin-1 (NRP-1), for iPDT; they showed vascular disruption, edema in both the tumor and brain-adjacent tumor areas, and also a large decrease in VEGF expression.
The heptapeptide (ATWLPPR) targeting the NRP-1 protein was also used in the study by Hu [59], in which this peptide was associated with CGKRK forming the dual-decorated nanoparticulate (designated AC-NP) to achieve a dual-targeting effect for angiogenic blood vessels and the tumor microenvironment. Targeting RTKs receptors, the study by Clavreul [34] used SFN conjugated with LNCs applied locally through CED. This technique associated with LNCs has the advantage of bypassing the BBB and results in a greater volume of disponible drug, meaning promising results were yielded in orthotopic GBM models [52,86]. This strategy is also commonly used with PTX [87] as well as anti-EGFR and anti-Galectin-1 siRNAs [88]. SFN is a multikinase inhibitor chemotherapy agent that acts on endothelial cell-surface RTKs (VEGFR-2, VEGRF-3, PDGFR-β, c-kit, and Flt-3). Recently, novel cross-family interactions between VEGF, PDGF, and their receptors were discovered, proposing a new mechanism for understanding anti-angiogenic drug resistance, and this presents an expanded role of growth factor signaling with significance in health and disease [89]. The structural difference in both RTKs is that PDGFRs have five extracellular immunoglobulin-like (IgG-like) domains [90] and VEGFRs have seven extracellular IgG-like domains [91].
The abundant secretion of VEGF by tumor cells promotes the formation of poorly developed blood vessels and with inadequate coating of endothelial supporting cells, i.e., the pericytes. Transforming the conditions of the blood vessels associated with the tumor into vessels properly covered with perycites is one of the main objectives of the application of nanoformulations and another therapies applied to combat aggressive tumors that favor angiogenesis. Goel [92] theorized the vascular normalization process, in which the restoration of normal vasculature condition is due to the greater effectiveness of antiangiogenic factors rather than proangiogenic factors. This normalized vasculature would result in increasing tumor perfusion, which would cause a reduction and consequent absence of hypoxia conditions. Despite the importance of pericytes in the process of vascular normalization, none of the selected studies analyzed the influence of the respective therapies on these cells. However, some therapies analyzed, such as the application of CARD-B6 [53], associated their therapeutic success with the reduction of blood flow. Likewise, the suggested restoration of vessel architecture to a more normal state provoked by I Irinophore C TM was associated with reduced blood flow expressed by Ktrans values [43]. Blocking VEGFR2, which can be achieved with SFN [34] and through DC101, a VEGFR-2 monoclonal antibody, promotes Ang-1/Tie2 signaling, which correlates with the recruitment of pericytes through blood vessels [93]. The relatively narrow normalization window depends on the dosage of VEGF inhibitors, that varies with tumor type, schedule and the signaling inhibitor [8,94]. It is important that the process of VEGF inhibition don't be excessive, but that a balance be achieved between the proangiogenic and antiangiogenic factors, since the total inhibition of VEGF signals represents a hypoxic picture for tumor cells, favoring to a risk of metastatic spread [95]. In addition, vascular normalization would promote decreased permeability of proangiogenic factors by tightening cell-cell junctions and decreasing cell adhesion of endothelial cells to extracellular tumor matrix and adjacent cells mediated by signaling of integrins [8,96].
Integrins are key regulators of communication between cells and their microenvironment. They are directly involved in the interplay between pro-angiogenic growth factors and their receptors, thereby exercising varied biological functions such as cell adhesion, migration/invasion, proliferation, survival, and apoptosis [11,97]. Integrins are a set of heterodimeric transmembrane glycoproteins made up of different α and β subunits, and αvβ3-Integrin has received much attention as a potential anti-angiogenic target because it is upregulated in tumor-associated blood vessels [11]. Agents targeting αvβ3-integrin are now showing some success in phase III clinical trials for the treatment of GBM, but the exact function of this integrin in tumor angiogenesis is still relatively unknown [98]. αvβ3-Integrin recognizes the Arg-Gly-Asp (RGD) peptide present in ECM proteins such as fibronectin, vitronectin, von Willebrand factor, osteopontin, and laminin, contributing to information exchange between the intracellular molecules and ECM proteins [11]. The synthesis of peptides and peptidomimetics coupled to RGD improved drug delivery to tumor vessels in varied studies [41,45,55] that sought to target antitumor agents to heparin [55], EPR [45], and DTX [41], and different types of encapsulation were also developed. The study by Wang [55] used a heparin-based polymer conjugated with cRGD and SWLAYPGAVSYR (SWL; S = serine, W = tryptophan, L = leucine, A = alanine, Y = tyrosine, P = proline, and V = valine) peptides to promote selective affinity for the overexpressed integrin and EphA2 tyrosine kinase receptor, respectively, reaching 56% simultaneous inhibition of endothelial-lined blood vessels and 82% vasculogenic mimicry. VEGFR-2 plays a key role in vasculogenic mimicry formation, neovascularization, and tumor initiation by glioma stem-like cells [99], and EphA2 and its receptors are significantly involved in blood vessel formation and remodeling during the vascular development of cancers [25]. EphA2 may regulate vessel sprouting during developmental angiogenesis independently via inhibition at both the gene and protein levels of VEGFR-2, without affecting VEGF expression by GBM cells [25]. The study by Zhang [45] modified the surface of liposomes with a thiolated cyclic pentapeptide derivative containing RGD conjugated with DSPE-PEG2000-maleimide, aiming at a nanoformulation capable of crossing the BBB and target GBM neovasculature. The study by Gao [41] developed a nanoformulation with interleukin-13 and RGD to target DTX in GBM neovasculature; this nanoformulation prevented HIF1α accumulation in the GBM site. Another study [48] used αvβ3-Integrin as the therapeutic target but did not specify the RGD application. Stimulation of proliferation and endothelial cell migration in the angiogenic process, when mediated by VEGF, occurs mainly via the αvβ3 integrin subtype present in these cells [11] as the therapeutic target of nanoformulations with RGD [41,45,55]. In addition, αvβ3-Integrin helps during the activation of MMPs, contributing to remodeling the ECM that facilitates endothelial cell migration [11]. Therefore, it is important, but not indispensable, for tumor angiogenesis [11], and blocking αvβ3 results in a drastic reduction in angiogenesis due to the inhibition of downstream signaling via the PAK/Src/Akt-pathway [100].
In GBM, overexpressed MMP helps tumor cells to survive, grow, and metastasize in distant sites [101], participating in the disruption and tumor neovascularization. Thus, the angiogenic response can be directly or indirectly mediated by MMPs through modulating the balance between pro-and anti-angiogenic factors [102]. Only the study by Séhédic [52] targeted a nanoformulation to MMP as an angiogenic marker, specifically MMP9.
The angiogenic process involves many signaling pathways, intracellular signaling pathways mediated by the growth factor of RTKS and intracellular proteins, as well as extra-cellular signaling pathways such as the notch/delta, ephrin/Eph receptor, roundabout/slit, and netrin/UNC (uncoordinated) receptor families, providing many opportunities for therapeutic intervention [103]. The study by Wang [55] developed a nanoformulation coupled with SWL that interfered in the EphA2 signaling pathway mediated angiogenesis process in GBM. This process occurs via the recruitment of phosphoinositide 3-kinase (PI3K) that stimulates downstream molecules of the Vav family of guanine nucleotide exchange factors (GEFs) and Rac1-GTP. Thus, the EphA2 signaling pathway promotes crosstalk with pro-angiogenic molecules such as growth factor receptors and adhesion molecules such as integrins and cadherins [104]. Other signaling pathways were also reported in the nanoformulation development [34,50,52,55,61,65], aiming to interfere in the regulation of tumor and angiogenic activities, mainly in signaling pathways involved with kinase enzymes. When activated by the phosphorylation process, they create a signal by transferring a phosphate group to a protein substrate. The study by Clavreul [34] used a multi-kinase inhibitor that interfered with the intracellular downstream serine/threonine kinases Raf-1, B-Raf, and mutant B-Raf through the application of SFN-loaded lipid nanocapsules, which intensively decreased the proportion of proliferating cells and tumor vessel area, and induced an early increase in tumor blood flow and vascular normalization, due to the correction of the structure and functionality of blood vessels, in which the increase in blood flow associated with tumor reduction prevents tumor cells from acquiring an aggressive phenotype by eliminating hypoxia, in addition to favoring a better effect of chemotherapeutic and radiotherapeutic agents [8]. Inhibition of the RAF1 gene by MiR-7-5p is associated with inhibition of vascular endothelial cell proliferation because Raf1 activates growth factor signaling downstream of the epidermal growth factor receptor (EGFR), a major drug target in GBM [105]. This miRNA is frequently downregulated in GBM microvasculature [106].
Microvessel tumor density and histopathological growth patterns during the angiogenic process can be analyzed using lectins such as WGA or the application of antibodies against specific antigens [9]. Most of the selected studies used the CD31 marker present in endothelial cells for this purpose [34,[43][44][45][50][51][52][53][54]56,57,[59][60][61][62][64][65][66], which can also detect antibodies against CD34 [55,58], von Willebrand factor [65], αvβ3-Integrin, or type IV collagen [9]. Detection of specific antigens ex vivo is relatively easy, either through chromogenic detection or fluorescent detection, but both methods have significant differences in their applicability. Through the IF, it is possible that several targets are detected simultaneously, with different fluorescence spectra, and targets are abundant or scarce. The study by Verreault [43] performed double detection of CD31 and Collagen IV after Irinophore C TM administration and they quantified different structures involved in the angiogenic process: the extent of discontinuous basement membrane, the fraction of pericyte-uncovered blood vessels in tumor tissue, the blood vessel diameter, and the proportion of empty basement membrane sleeves that indicates the regression of pre-existing blood vessels suggesting a more homogenous distribution of blood across the entire tumor. It is also useful in epitope detection when the fluorescent liposomes used in nanoformulations are detected using the fluorochrome DiI applied to them, resulting in a higher dynamic range, as seen in Zhang's work [45]. However, a clear disadvantage of IF compared to chromogenic detection of molecular targets is sensitivity. IHC benefits from signal amplification via indirect chromogenic detection, but it is not able to effectively mark multiple therapeutic targets simultaneously. As a result of this, the study by Séhédic [52] marked the CD68, NOSII, and Arg1 samples independently, differentiating TAMs of the antiangiogenic macrophages but without performing a quantification process. The study by Lin [57] detected several epitopes in isolation by IHC, providing more details on the action of CRLX101 (Cerulean Pharma, Cambridge, MA, USA) nanoformulation on the angiogenic process. In addition, the CD31 + endothelial cells were evaluated via the signaling cascade through the marking of CA IX, a promising endogenous hypoxia-related cell surface enzyme associated with increased necrosis [75,76]; this is considerably less common than ex vivo detection of the HIF1α marker, which was used in the study by Gao [41]. IHC was also the most frequent technique used for the detection of VEGF expelled by tumor cells [57,63,68], which was also detected by WB [57] and ELISA [32]. The study by Wu [50] showed a direct correlation between the VEGF protein level and the VEGF mRNA by transcriptomic analysis, which was also detected in the study by Kuang [58]. Transcriptomics can analyze different phases of the angiogenic process using qPCR, microarray, or RNA sequences [9]. By qPCR technique vascular signatures of genes originating from the tumor implantation and the stroma can be differentiated, due to a low overlap, with the help of bioinformatics analysis [9,107]. Blood perfusion evaluations were the focus when analyzing the antiangiogenic therapeutic process using different image techniques [34,43,48,53]. Results showed a decrease in proliferating cells and tumor vessel area after SFN-LNCs treatment [34], no blood flow existed in the tumor region following treatment with CARD-B6 [53], vessel dilation and hemorrhaging within the tumor exposed to VEGF-NSs and PEG-NSs [48], and restored basement membrane architecture (BMA) and reduced blood vessel diameter (BVD) of the tumor vasculature, suggesting restoration of the vessel architecture to a more normal state after Irinophore C TM treatment, occurring vascular normalization [43].
Currently, a wide range of natural compounds has been recognized for the important role in the improvement in the efficacy of chemotherapeutics in combination therapy of GBM, highlighting the relative nontoxicity of the natural compounds when used in combination therapy, requiring lower dosage level, and showing as a potential solution for the multidrug resistance question that is a major cause of failure in cancer chemotherapies [108]. The PTX and DTX were the more representative drugs derived from plants, representing 36 and 18%, respectively of the selected studies that used drugs in the nanoformulations. These taxanes acting as anti-mitotic agents by promoting polymerization of microtubules and reducing depolymerization [109], as also as a radiation sensitized used for inhibiting the GBM growth and proliferation when associated with radiation therapy. In addition, the plant-based medicines used in angiotherapy, mainly GBM therapy reported in the selected studies already are well described in the literature, regarding its antiangiogenic and antimetastatic action that occurs targeting different molecular pathways, for example, the inhibiting VEGF production and the expression of HIF-1by taxanes, inhibition of the action of Topoisomerase I by alkaloids, suppressing the production of plasminogen activator (PA) and PA inhibitor-1 by flavonoids, among other targets of angiogenesis pathways [110][111][112]. The selected studies that used these natural drugs associated with nanoformulations [38,41,43,50,53,54,56,57,59,61,62], showed that the nanocarriers overcame the biochemical/biophysical barriers, allowing a supply of medicines throughout the tumor region through the BBB. The properties of the nanocarriers can be modified for selective and controlled release of drugs with minor effects [113]. The search for better responses in the treatment of GBM makes these studies that associate natural drugs and nanoformulations extremely promising.
Analysis of the studies included in this review showed that the applications of nanoformulations with drug delivery strategies aimed at interfering in the angiogenic process in pre-clinical GBM models were very diverse in almost all aspects addressed, making it difficult to define the best antiangiogenic strategy. The tumor models of the studies were induced with different cell lines and subjected to various chemotherapeutic agents coupled with nanoformulations designed to achieve different therapeutic targets and exert equally diverse mechanisms. On the other hand, in relation to angiogenesis, there is little variety in the methods of analyzing the therapeutic effects of nanoformulations on the various processes involved in angiogenesis, both in global and specific ways such as the process of hypoxia, recruitment of inflammatory cells, angiogenic growth factor production, basement membrane degradation, endothelial cell migration, and proliferation, differentiation, and modulation of vascular support cells. Therefore, the present review highlighted the need for standardization between different antiangiogenic therapy approaches for the GBM model. This would allow for more effective analyses, especially meta-analyses, and it would help in future translational studies. Currently, only the biodegradable polymeric nanoformulation of carmustine wafers is used in clinical practice into the ressection cavity, presenting controversial results until recently, although meta-analyzes have shown increased patient survival compared to control groups, increasing the effectiveness of traditional therapy [114].

Search Strategy
This systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Guideline [49]. We performed a search for indexed articles published prior to February 2020 in the databases: PubMed, Scopus, and Cochrane using the following search criteria: (Glioblastoma OR Glioma) AND (Therapy OR Therapeutic) AND (Nanoparticle) AND (Antiangiogenic OR Angiogenesis OR Anti-angiogenic). Then, we applied the following boolean operators (DecS/MeSH) and keywords sequence in the search of each database: PubMed: (((((((((angiogenesis) OR antiangiogenesis) OR angiogenic) OR antiangiogenic) OR anti-angiogenic) OR anti-angiogenesis)) AND ((((glioblastoma) OR glioma)) OR gliomas)) AND ((nanoparticle) OR nanoparticles)) AND ((((((((((((therapy) OR therapeutic) OR therapeutics) OR therapies) OR theranostic) OR theranostics) OR nanotherapeutic) OR nanotherapy) OR nanotherapeutics) OR nanotherapies)) OR treatment). Cochrane: "glioma" in Title Abstract Keyword OR "glioblastoma" in Title Abstract Keyword AND "angiogenesis" in Title Abstract Keyword AND "therapy" in Title Abstract Keyword AND "nanoparticle" in Title Abstract Keyword (Word variations have been searched).

Inclusion Criteria
This review included only original articles written in English that had used: (i) antiangiogenic therapy using nanoparticles, (ii) detection of the effect of therapy using angiogenic markers, (iii) therapies for glioma or GBM, and (iv) in vivo studies with intracranial induction of glioma or GBM.

Exclusion Criteria
Reasons for excluding studies were as follows: (i) nanodiagnosis, (ii) review articles, (iii) book chapters, (iv) protocols, (v) editorials/expert opinions/notes, (vi) letters/communications/conference abstracts, (vii) publications in languages other than English, (viii) studies that did not analyze angiogenesis, (ix) studies with tumor induction in the flank/subcutaneous, (x) in vitro studies only, (xi) use of tumors other than glioma or GBM, and (xii) non-angiogenic targets. The analysis of the articles and the preparation of the tables were carried out by consensus. In each case of disagreement, a fifth independent and senior author (L.F.G.) decided by data addition or subtraction. The final inclusion of studies in this review was in agreement with all authors.

Data Analysis
All results were described and presented using the percentage distribution for all variables analyzed in the tables.