*5.4. Targeting Downstream Pathway of EGFR*

A comprehensively studied downstream pathway of EGFR is the PI3K/AKT/mTOR, which is critically involved in regulating cell apoptosis, autophagy, proliferation, and metabolism. Dysregulation of the pathway, as mentioned earlier, played a prominent role in various cancers [92,93]. Therapeutic strategies targeting PI3K/AKT in GBM have given promising results in the in vitro and in vivo xenograft models; however, clinical safety and efficacy need to be proven. Sonolisib (PX-866), an irreversible PI3K inhibitor, inhibited the angiogenesis and invasion of GBM cells in vitro but did not induce the apoptosis of GBM cells; nevertheless, the drug caused cell cycle arrest. Sonolisib was well tolerated but showed disease progression in almost 73% of recurrent GBM patients in the phase II clinical trial [94]. Various other inhibitors such as XL765 (SAR245409) and GDC-0084, both PI3K and mTOR inhibitors, showed efficacy against GBM in the in vitro and in vivo models. However, the results as mentioned above lack the support from relevant clinical data [95].

Sirolimus (rapamycin), temsirolimus (CCI-779), and everolimus (RAD001), the mTOR inhibitors were evaluated in the various clinical phases and showed little efficacy in treating GBM patients. Everolimus showed very little effectiveness and a low survival rate in monotherapy and combination with temozolomide radiotherapy in a phase II clinical trial [96]. Sirolimus monotherapy and in combination with erlotinib [97] and temsirolimus [98] failed to show any effect in the treatment of GBM patients in a phase II clinical trial.

Other inhibitors such as vistusertib (AZD2014), palomid 529, and mTOR kinase inhibitor (CC-223) were dual inhibitors of mTORC1 and mTORC2. Vistusertib showed radiosensitization in GBM cell lines both in the in vitro and in vivo models, due to which the participants were recruited to phase I and II clinical trials [99] (clinical trial ID: NCT02619864). In a GBM xenograft model (U87MG cells), CC-223 exhibited an anti-tumor effect, while Palomid 529 exhibited anti-tumor activity in the orthotopic murine tumor model [100].

#### **6. Mechanism of Drug Resistance to EGFR–TKIs in Glioma**

Although the mechanism of drug resistance to EGFR–TKI in GBM remain unclear, few reports discussed the possible mechanisms in this regard. The absence of mutation in exons 19 and 21 of the TK domain was reported, especially in first-line EGFR-TKIs such as erlotinib and gefitinib. Their pharmacological actions were dependent on the modifications, as mentioned above [101]. Another possible mechanism mentioned was an alternative activating signal that compensated for the inactivation of EGFR signaling by EGFR–TKIs. In addition, the absence of EGFRvIII and loss of phosphatase and tensin homolog (PTEN) were the other determinants of resistance in certain studies [102].

The inhibition of mTOR, a downstream molecule of the PI3K/PTEN/AKT pathway, promoted the response of glioma cells to EGFR-TKIs in vitro [103,104]. Conversely, there was no responsiveness to erlotinib and no expression of EGFRvIII and PTEN in the phase II clinical trial with relapsed GBM patients [105]. In addition, a combination of the mTOR and EGFR–TKIs inhibitors (sirolimus) did not improve the patients' responsiveness in recurrent GBM patients [106]. On the other hand, erlotinib inhibited EGFR in EGFRvIII expressing U87 GBM cells and enhanced the expression of PDGFRα, thereby compensating the signaling pathway inhibited by erlotinib [97].

Despite the numerous studies on GBM treatment targeting EGFR, no therapeutic efficacy has been reported [107,108]. The therapeutic efficacy was minimal or nil in the case of first and second-generation EGFR inhibitors for the treatment of recurrent GBM [109,110]. The primary reasons for the above drugs' failure were their inability to cross the BBB and the requirement of a relatively high amount of drug concentrations in the brain [92], which in turn limited their usage. By overcoming the above-said limitations, effective therapy for GBM could be discovered [92].

*7.1. Organic Nanoparticles*

therapeutic outcomes.

7.1.1. Albumin Nanoparticles

### **7. Current Pharmaceutical Drug Targets in Glioma**

Despite the great activity of EGFR–TKIs, mAb, and chemotherapeutic agents, the therapeutic outcomes limited by BBB penetration in both preclinical and clinical studies have urged the thought of using TKIs-loaded nanoformulations in the management of GBM [111]. For example, lipophobic and less molecular weight drugs could not achieve specific delivery in tumor tissues and were characterized by a short circulation half-life [112]. Furthermore, compared to the other cancer types harboring EGFR amplification, clonal resistance was not observed in GBM after the EGFR inhibitor treatment. However, multiple failures and/or resistance such as the absence of exons 19 and 21 of the TK domain, an alternative activation of signals, rapid adaptive responses due to EGFR inhibitors, and the lesser ability of EGFR–TKI drugs to cross the BBB were reported in various studies [113].

Consequently, the novel drug delivery systems (NDDS) were employed for the specific delivery of FDA-approved drugs to increase its therapeutic outcomes and reduce the adverse effects during GBM treatment. Among the above-mentioned NDDS, the NPs (Figure 3) with various structures and properties to serve as the desirable carriers for anti-cancer drugs were invented [114,115]. In addition, the systems appear to be promising approaches to solve the existing problems in the management of GBM [114]. NPs-based systems have many unique benefits. Firstly, NPs can be loaded with the hydrophilic and hydrophobic drugs simultaneously, which results in an enhanced solubility and anti-cancer effect when employed with the suitable combinations of medicines in carriers. Secondly, the uniform particle size distribution and surface modifications enabled passive or active cancer targeting and resulted in improved drug availability in the tumor region. Lastly, the NPs as drug carriers also aided the sustained and controlled drug release at a specific region. The augmented drug release profiles with extended circulation time permitted improved pharmacokinetics and decreased the dose-dependent toxicity of therapeutic agents [116,117]. Hence, the subsequent portion reviews the benefits of various classes of NPs used in EGFR-targeted drug delivery to manage glioma. *Pharmaceutics* **2020**, *12*, x 11 of 28 properties to serve as the desirable carriers for anti-cancer drugs were invented [114,115]. In addition, the systems appear to be promising approaches to solve the existing problems in the management of GBM [114]. NPs-based systems have many unique benefits. Firstly, NPs can be loaded with the hydrophilic and hydrophobic drugs simultaneously, which results in an enhanced solubility and anti-cancer effect when employed with the suitable combinations of medicines in carriers. Secondly, the uniform particle size distribution and surface modifications enabled passive or active cancer targeting and resulted in improved drug availability in the tumor region. Lastly, the NPs as drug carriers also aided the sustained and controlled drug release at a specific region. The augmented drug release profiles with extended circulation time permitted improved pharmacokinetics and decreased the dose-dependent toxicity of therapeutic agents [116,117]. Hence, the subsequent portion reviews the benefits of various classes of NPs used in EGFR-targeted drug delivery to manage glioma.

**Figure 3.** Different nanodelivery systems that facilitate BBB penetration and target-specific action in glioma. **Figure 3.** Different nanodelivery systems that facilitate BBB penetration and target-specific action in glioma.

serum albumin-based paclitaxel (PTX) nanoparticles exhibited superior anti-tumor activity by the prolongation of survival and pro-apoptotic effect, as depicted by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis, thereby serving as a novel strategy for treating GBM (Figure 4A,B) [119]. Furthermore, radioiodine cross-linking anti-EGFR (cetuximab) and bovine serum albumin (BSA) polycaprolactone (PCL) nanoparticles were also useful to induce tumor regression, which in turn enhanced the cytotoxicity on tumor cells and limited the adverse effects of chemical agents [120]. Thus, it can be stated that albumin NPs exhibited an improvement in

Due to the greater biodegradability and low immunogenicity of serum albumin, it has been identified as a suitable nanocarrier for the cancer management in recent years. In addition, the ability

#### *7.1. Organic Nanoparticles*

#### 7.1.1. Albumin Nanoparticles

Due to the greater biodegradability and low immunogenicity of serum albumin, it has been identified as a suitable nanocarrier for the cancer management in recent years. In addition, the ability of binding or absorbent proteins around the NPs was showcased as the foremost prominent factor for prolonged circulation time and phagocytosis [118]. As an endogenous substance, albumin might inhibit therapeutics drugs from unnecessary stability interaction and targeting efficiency. The human serum albumin-based paclitaxel (PTX) nanoparticles exhibited superior anti-tumor activity by the prolongation of survival and pro-apoptotic effect, as depicted by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis, thereby serving as a novel strategy for treating GBM (Figure 4A,B) [119]. Furthermore, radioiodine cross-linking anti-EGFR (cetuximab) and bovine serum albumin (BSA) polycaprolactone (PCL) nanoparticles were also useful to induce tumor regression, which in turn enhanced the cytotoxicity on tumor cells and limited the adverse effects of chemical agents [120]. Thus, it can be stated that albumin NPs exhibited an improvement in therapeutic outcomes.

Tsutsui et al. demonstrated bio-nano capsules (BNCs) as an efficient way to deliver drugs to brain tumors in Gli36 cell lines. BNCs are composed of a hepatitis B surface antigen, small interfering ribonucleic acid (siRNA), genes, chemical components, and proteins that selectively target brain tumors. BNCs, when conjugated with an EGFR antibody, were capable of recognizing EGFRvIII, which in turn was overexpressed in various human malignancies. EGFRvIII was reported to be overexpressed in the variability of human malignancies of epithelial origin, particularly in gliomas. As mentioned above, the reports indicated BNC's potential as a means to achieve tumor targeting delivery [121].

The intravenous (i.v.) administration of T7 peptide modified core–shell NPs (T7-LPC/siRNA NPs) consisting of protamine/chondroitin sulfate/siRNA/cationic liposomes assembled layer by layer followed by modification using T7 peptide resulted in siRNA targeted delivery. T7-LPC/siRNA NPs, when compared with PEG-LPC/siRNA NPs, showed increased fluorescence intensity in microvascular endothelial cells of the brain (BMVECs) and U87 glioma cell lines. The NPs resulted in the downregulation of expression of EGFR protein in U87 glioma cells in vitro. The accumulation of NPs was more specific to the tumor tissues and penetrated the deep region ascertained by the co-culture model of BMVECs and U87 cells and in vivo imaging. The reports also confirmed that the NPs demonstrated the most prolonged survival period and highest down-regulated expression of EGFR, thereby showing the potential of siRNA delivery for the targeted therapy of GBM [122].

#### 7.1.2. Immunoliposomes (IL) and Solid Lipid Nanoparticles (SLNs)

The sustained and targeted drug release profiles of the immunoliposomes (ILs) and solid lipid nanoparticles (SLNs) nanosystems enabled the enhanced cancer cell inhibition and decreased the adverse effects throughout the tumor therapy [123]. Lipids (phospholipids) were utilized to manufacture NPs due to their safety and biocompatibility [124]. The dual targeting SLN loaded with etoposide (ETP) containing mAb for insulin receptors and anti-EGFR was used to treat GBM. The dual-functionalized SLNs crossed the BMVECs/HA (human astrocytes), which is an in vitro model for BBB, and showed enhanced cytotoxicity against U87MG cells [125], thus proving its potential against GBM. In another study, the Cetuximab (C225)-immunoliposomes (ILs) encapsulating boron anion were constructed by using novel maleimido–Polyethylene Glycol (PEG)–cholesterol for the targeted delivery of boron compounds to EGFR (+) glioma cells for boron neutron capture therapy (BNCT). It was concluded that the prepared ILs could serve as an efficient delivery vehicle for the BNCT of glioma [126].

*Pharmaceutics* **2020**, *12*, x 12 of 28

**Figure 4.** (**A**) Schematic representation of preparation and mechanism of action of albumin nanoparticles (NPs); (**B**) Fluorescent microscopic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay images of in vivo anti-cancer efficacy of SP–HSA–PTX NPs (Green: TUNEL-stained apoptosis cells. Blue: 4′,6-diamidino-2-phenylindole (DAPI)-labeled nucleus, Yellow dashed lines: boundary between (N) normal brain and (G) glioma section). Reprinted with permission from [119], Elsevier, 2018. **Figure 4.** (**A**) Schematic representation of preparation and mechanism of action of albumin nanoparticles (NPs); (**B**) Fluorescent microscopic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay images of in vivo anti-cancer efficacy of SP–HSA–PTX NPs (Green: TUNEL-stained apoptosis cells. Blue: 40 ,6-diamidino-2-phenylindole (DAPI)-labeled nucleus, Yellow dashed lines: boundary between (N) normal brain and (G) glioma section). Reprinted with permission from [119], Elsevier, 2018.

Tsutsui et al. demonstrated bio-nano capsules (BNCs) as an efficient way to deliver drugs to

brain tumors in Gli36 cell lines. BNCs are composed of a hepatitis B surface antigen, small interfering ribonucleic acid (siRNA), genes, chemical components, and proteins that selectively target brain tumors. BNCs, when conjugated with an EGFR antibody, were capable of recognizing EGFRvIII, which in turn was overexpressed in various human malignancies. EGFRvIII was reported to be overexpressed in the variability of human malignancies of epithelial origin, particularly in gliomas. As mentioned above, the reports indicated BNC's potential as a means to achieve tumor targeting delivery [121]. The intravenous (i.v.) administration of T7 peptide modified core–shell NPs (T7-LPC/siRNA NPs) consisting of protamine/chondroitin sulfate/siRNA/cationic liposomes assembled layer by layer followed by modification using T7 peptide resulted in siRNA targeted delivery. T7-LPC/siRNA NPs, when compared with PEG-LPC/siRNA NPs, showed increased fluorescence intensity in microvascular endothelial cells of the brain (BMVECs) and U87 glioma cell lines. The NPs resulted in Quantum dot immunoliposome (QD-IL), a hybrid nanoparticle, was targeted toward EGFR to treat GBM. QD-ILs were taken up efficiently by the malignant cells. In addition, QD-ILs served as imaging methods proven in both the in vitro and in vivo models. Furthermore, the NPs were also employed in ligand-directed delivery that allowed targeted drug delivery to the desired site to achieve efficient treatment for GBM [127]. Moreover, the anti-tumor effect of the combination of bevacizumab (Bev) and gemcitabine (GM) loaded IL (Bev-GM-IL) in a xenograft mice model (XMM) showed that the combinational therapy is better than monotherapy. This is due to the synergistic activity of two different drugs on GBM stem cells. Likewise, the combinational treatment extended the mean survival time of XMM. Altogether, the above results suggested that the combination of Bev-GM-IL offered promising outcomes in the treatment of GBM [128].

the downregulation of expression of EGFR protein in U87 glioma cells in vitro. The accumulation of NPs was more specific to the tumor tissues and penetrated the deep region ascertained by the coculture model of BMVECs and U87 cells and in vivo imaging*.* The reports also confirmed that the NPs demonstrated the most prolonged survival period and highest down-regulated expression of EGFR, thereby showing the potential of siRNA delivery for the targeted therapy of GBM [122]. In another study, doxorubicin (DOX) and vincristine (VCR) were loaded with T7 and <sup>D</sup>A7R dual peptides-modified liposomes (T7/ <sup>D</sup>A7R-Ls) to treat glioma. The in vivo (Figure 5) results of T7/ <sup>D</sup>A7R-Ls showed improved glioma localization compared with mono ligand-modified liposomes or the free drug. In conclusion, the dual-targeting, co-delivery approach delivered a potential method for successful brain drug delivery in the glioma treatment [129].

#### 7.1.2. Immunoliposomes (IL) and Solid Lipid Nanoparticles (SLNs) The sustained and targeted drug release profiles of the immunoliposomes (ILs) and solid lipid 7.1.3. Polymeric Nanoparticles

nanoparticles (SLNs) nanosystems enabled the enhanced cancer cell inhibition and decreased the adverse effects throughout the tumor therapy [123]. Lipids (phospholipids) were utilized to manufacture NPs due to their safety and biocompatibility [124]. The dual targeting SLN loaded with etoposide (ETP) containing mAb for insulin receptors and anti-EGFR was used to treat GBM. The The comparative evaluation of the other conventional nanocarriers, the polymeric NPs, exhibited promising benefits in the biomedical applications due to their improved solubility, biocompatibility, and biodegradability. The biodegradation through circulation in vivo can be eluded efficiently, and the elimination half-life of the drugs is also prolonged after polymeric NPs encapsulation [130]. Additionally, the polymeric NPs with an applicable particle size distribution could passively help accumulate medicines in the tumor region by improved permeability and retention time [131,132]. Moreover, the retention time of encapsulated drugs in the tumor region could also be precisely regulated by various strategies [133]. For all of these advantages, the surface of polymeric NPs could be altered by selecting specific ligands to achieve active targeting delivery [134]. A study demonstrated the delivery

BNCT of glioma [126].

of curcumin using poly (D, L-lactic-co-glycolic acid) (PLGA) NPs tagged with an EGFRvIII, which was internalized by EGFRvIII overexpressed GBM cells leading to the enhanced photodynamic toxicity of curcumin [135]. peptides-modified liposomes (T7/DA7R-Ls) to treat glioma. The in vivo (Figure 5) results of T7/DA7R-Ls showed improved glioma localization compared with mono ligand-modified liposomes or the free drug. In conclusion, the dual-targeting, co-delivery approach delivered a potential method for successful brain drug delivery in the glioma treatment [129].

In another study, doxorubicin (DOX) and vincristine (VCR) were loaded with T7 and <sup>D</sup>A7R dual

*Pharmaceutics* **2020**, *12*, x 13 of 28

dual-functionalized SLNs crossed the BMVECs/HA (human astrocytes), which is an in vitro model for BBB, and showed enhanced cytotoxicity against U87MG cells [125], thus proving its potential against GBM. In another study, the Cetuximab (C225)-immunoliposomes (ILs) encapsulating boron anion were constructed by using novel maleimido–Polyethylene Glycol (PEG)–cholesterol for the targeted delivery of boron compounds to EGFR (+) glioma cells for boron neutron capture therapy (BNCT). It was concluded that the prepared ILs could serve as an efficient delivery vehicle for the

Quantum dot immunoliposome (QD-IL), a hybrid nanoparticle, was targeted toward EGFR to treat GBM. QD-ILs were taken up efficiently by the malignant cells. In addition, QD-ILs served as imaging methods proven in both the in vitro and in vivo models. Furthermore, the NPs were also employed in ligand-directed delivery that allowed targeted drug delivery to the desired site to achieve efficient treatment for GBM [127]. Moreover, the anti-tumor effect of the combination of bevacizumab (Bev) and gemcitabine (GM) loaded IL (Bev-GM-IL) in a xenograft mice model (XMM) showed that the combinational therapy is better than monotherapy. This is due to the synergistic activity of two different drugs on GBM stem cells. Likewise, the combinational treatment extended the mean survival time of XMM. Altogether, the above results suggested that the combination of Bev-

**Figure 5.** In vivo anti-glioma effect of doxorubicin (DOX) and vincristine (VCR)-loaded T7/<sup>D</sup>A7R-LS immunoliposomes. (**A**) Distribution of Cy5.5 in the mice brain bearing intracranial C6 glioma determined by a CLSM; (**B**) MRI of normal and pathological brains at 16 d after inoculation. Reprinted from [129], Taylor and Fransis Group, 2017. **Figure 5.** In vivo anti-glioma effect of doxorubicin (DOX) and vincristine (VCR)-loaded T7/ <sup>D</sup>A7R-LS immunoliposomes. (**A**) Distribution of Cy5.5 in the mice brain bearing intracranial C6 glioma determined by a CLSM; (**B**) MRI of normal and pathological brains at 16 d after inoculation. Reprinted from [129], Taylor and Fransis Group, 2017.

Lei Wang et al. (2015) prepared the angiopep-2 (ANG)-modified PLGA/DOX/siRNA NPs, which inhibited the cells by inducing apoptosis and silenced the EGFR pathway in U87MG cells. The NPs were capable of penetrating the BBB, thus resulting in the enhanced accumulation of drugs in brain in vivo. Animal studies not only demonstrated the co-delivery of DOX and EGFR SiRNA but also prolonged the life span of GBM-bearing mice [136]. Chengkun et al. (2019) utilized the Golgi phosphoprotein 3 (GOLPH3) nanobody to construct an angiopep-2 (A2)-modified cationic lipid PLGA NPs (A2-N) targeting the Ge and GOLPH3 siRNA (siGOLPH3). The NPs not only penetrated the BBB but also silenced the expression of GOLPH3 mRNA and enhanced the expression of EGFR and pEGFR upon entering glioma cells. In addition, the above-mentioned NPs acted as a combinational anti-tumor therapy in vitro and in vivo [137]. In vivo imaging revealed that the T7-LPC/siRNA NPs penetrated the deeper regions of the tumor. Furthermore, the accumulation was more in the brain, which was an added advantage compared to PEG-LPC/siRNA NPs. The group also demonstrated the enhanced survival period by down-regulating the expression of EGFR in mice, and therefore, it can serve as a potential target for treating GBM [119]. In another study, C225 was conjugated to TMZ-loaded PLGA NPs (C225–TMZ–PLGA–NPs) by cross-linking chemistry to target the EGFR receptor. Furthermore, in vitro cellular uptake and the in vivo evaluation of PLGA–NPs, TMZ–PLGA–NPs, and C225–TMZ–PLGA–NPs were conducted. In addition, the results

of cell cytotoxicity, apoptosis in U-87MG, SW480, and SK-Mel 28 cancer cell lines confirmed that the C225-PLGA-NPs can be utilized as a versatile nanocarrier for the management of EGFR overexpressing cancers [138].

#### 7.1.4. Dendrimers

The dendrimers are hyper-branched macromolecules that exhibit advantages over the conventional carriers (liposome, polymeric NPs etc.) such as enhanced stability in the blood circulation and ability to accommodate various ligands due to its chemosynthetic approach rather than self-assembly through non-covalent interaction [139,140]. In addition, the structure, size, and molecular weight of dendrimer have resemblance with bio-structures and proteins (insulin, hemoglobin and cytochrome), which makes them employable in various fields as gene delivery, immunodiagnosis, and encapsulation of drugs [141,142]. Dendrimer-based drug delivery employing polyamidoamine (PAMAM) was also explored for its application in GBM therapy [143,144].

An antisense oligonucleotide (ASODN) delivery of conjugates of folate–PAMAM (FA-PAMAM) inhibited the C6 cell growth in glioma. The coupling of folic acid to the surface amino groups of PAMAM dendrimers and ASODNs (ASODN: FA-PAMAM) corresponded to rat EGFR in the ratio of 16:1. The ASODN:FA-PAMAM combination suppressed EGFR and C6 cell growth expression, thus enhancing the survival time [145].

Cetuximab (C225) could be covalently linked to methotrexate (MTX) by the 5th generation (G5) of PAMAM dendrimers via its fragment crystallizable (Fc) region (C225–G5–MTX) to target EGFR and EGFR variant III (EGFRvIII). Competitive binding assay (CBA) demonstrated that C225–G5–MTX exhibited a higher affinity for the EGFR-expressing rat glioma cell line (F98EGFR) than the wild-type rat glioma cell line (F98WT). Subsequently, the improved distribution of <sup>125</sup>I bio-conjugate of C225-G5-MTX noticed in F98EGFR was six-fold greater than F98WT cells, thereby contributing to specific molecular targeting the GBM treatment. The animal models that received C225-G5-MTX and C-225 or MTX exhibited 15- and 19.5-day survival rates, respectively. Correspondingly, the results were non-significant between the control and test animals [146].

PAMAM dendrimer and Tat peptide were fabricated to bacterial magnetic NPs (Tat–BMPs–PAMAM), which were then complexed with the siRNA expression plasmid of human EGFR (psiRNA–EGFR) through electrostatic interplay (Tat–BMPs–PAMAM/psiRNA-EGFR). The conjugate offered promising results in reducing tumor growth and suppressing the expression of oncoproteins. In addition, the conjugate could serve as a possible targeted gene delivery for GBM [147].

Recently, an angiopeptide-2 (Ang2) and low-density lipoprotein receptor-relative protein-1 (LRP1) was conjugated with PAMAM to improve BBB penetration glioma sites. Furthermore, PAMAM was concurrently functionalized with an EGFR-targeting peptide (EP-1) to achieve specificity and improved affinity to target EGFR. The above results showed the potential of the dual drug-loaded PAMAM in the treatment of gliomas by improving BBB penetration and specific EGFR targeting efficiency, both in vitro and in vivo (Figure 6) [148].

All the above-mentioned experiments concluded that the dendrimer-based NPs could be utilized as extensive drug delivery carriers to target and treat various CNS cancer cells by BBB penetration with the backing of targeting ligands.

type rat glioma cell line (F98WT). Subsequently, the improved distribution of 125I bio-conjugate of C225-G5-MTX noticed in F98EGFR was six-fold greater than F98WT cells, thereby contributing to specific molecular targeting the GBM treatment. The animal models that received C225-G5-MTX and C-225 or MTX exhibited 15- and 19.5-day survival rates, respectively. Correspondingly, the results were

PAMAM dendrimer and Tat peptide were fabricated to bacterial magnetic NPs (Tat–BMPs– PAMAM), which were then complexed with the siRNA expression plasmid of human EGFR (psiRNA–EGFR) through electrostatic interplay (Tat–BMPs–PAMAM/psiRNA-EGFR). The conjugate offered promising results in reducing tumor growth and suppressing the expression of oncoproteins.

Recently, an angiopeptide-2 (Ang2) and low-density lipoprotein receptor-relative protein-1 (LRP1) was conjugated with PAMAM to improve BBB penetration glioma sites. Furthermore, PAMAM was concurrently functionalized with an EGFR-targeting peptide (EP-1) to achieve

In addition, the conjugate could serve as a possible targeted gene delivery for GBM [147].

non-significant between the control and test animals [146].

targeting efficiency, both in vitro and in vivo (Figure 6) [148].

**Figure 6.** (**A**) Schematic representation of fabrication of the dual drug loaded polyamidoamine (PAMAM) dendrimers in the treatment of gliomas by improving BBB penetration; (**B**) Assessment of the affinity and specificity of peptide EP-1 toward EGFR; (**C**) In vitro evaluation of biocompatibility and anti-tumor efficacy of the dual drug-loaded PAMAM dendrimers; (**D**) Flow cytometry evaluation for intracellular uptake of different DOX-loaded dendrimers. Reprinted from [148] Ivyspring International Publisher, 2020. \**p* < 0.1, \*\**p* < 0.01, \*\*\**p* < 0.001, \*\*\*\**p* < 0.0001 (Student's *t*-test). **Figure 6.** (**A**) Schematic representation of fabrication of the dual drug loaded polyamidoamine (PAMAM) dendrimers in the treatment of gliomas by improving BBB penetration; (**B**) Assessment of the affinity and specificity of peptide EP-1 toward EGFR; (**C**) In vitro evaluation of biocompatibility and anti-tumor efficacy of the dual drug-loaded PAMAM dendrimers; (**D**) Flow cytometry evaluation for intracellular uptake of different DOX-loaded dendrimers. Reprinted from [148] Ivyspring International Publisher, 2020. \* *p* < 0.1, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 (Student's *t*-test).

#### All the above-mentioned experiments concluded that the dendrimer-based NPs could be utilized as extensive drug delivery carriers to target and treat various CNS cancer cells by BBB *7.2. Inorganic Nanoparticles (NPs)*

#### penetration with the backing of targeting ligands. 7.2.1. Silica NPs

The mesoporous silica nanoparticles (MS-NPs) are frequently employed as multifunctional nanocarriers to treat cancer cells owing to its mesoporous structure and enormous surface area. As a result, the active components can be dumped in the porous structure of NPs to obtain the maximum amount of drug-loading, and the surface modification of MS-NPs could also increase the intracellular uptake. Additionally, the alteration of carriers' particle size, surface charge, and shape could increase the biocompatibility and minimize the cytotoxicity of MS-NPs [149]. In addition, MS-NPs could be encapsulated with contrast agents for MRI imaging. Hence, MS-NPs exhibit a chance to improve the solubility and stability of anti-EGFR drugs by being deposited in the porous structure.

Furthermore, prolonged drug release profiles resulted in decreased cytotoxicity in long-period cancer therapy [150,151]. The properties mentioned above were ensured by developing DOX magnetic (Fe3O4) NPs, encapsulated in polyethylene glycol (PEG), to functionalize the porous silica shell and treat cancer cells [152]. Likewise, multi-targeted oleic acid (OA)–MNPs were developed. Reports confirmed promising outcomes about the in vitro and in vivo efficacy in treating human cancer cells (HeLa). The study results stated that the MS-NPs formulations were more predominant than the placebo or free drugs and could overcome the drawbacks mentioned above of the conventional treatment approaches [153]. The synthesized and functionalized DOX magnetic MS-NPs were fabricated with PF-127 and then conjugated with transferrin (Tf) to enhance BBB penetration and achieve sustained release at the specific site. The Tf-loaded NPs resulted in improved BBB permeability (Figure 7). Thus, the prepared Tf nanocarriers could be considered as potential candidates in the treatment of brain tumors [154].

candidates in the treatment of brain tumors [154].

*7.2. Inorganic Nanoparticles (NPs)*

7.2.1. Silica NPs

The mesoporous silica nanoparticles (MS-NPs) are frequently employed as multifunctional nanocarriers to treat cancer cells owing to its mesoporous structure and enormous surface area. As a result, the active components can be dumped in the porous structure of NPs to obtain the maximum amount of drug-loading, and the surface modification of MS-NPs could also increase the intracellular uptake. Additionally, the alteration of carriers' particle size, surface charge, and shape could increase the biocompatibility and minimize the cytotoxicity of MS-NPs [149]. In addition, MS-NPs could be encapsulated with contrast agents for MRI imaging. Hence, MS-NPs exhibit a chance to improve the

Furthermore, prolonged drug release profiles resulted in decreased cytotoxicity in long-period cancer therapy [150,151]. The properties mentioned above were ensured by developing DOX magnetic (Fe3O4) NPs, encapsulated in polyethylene glycol (PEG), to functionalize the porous silica shell and treat cancer cells [152]. Likewise, multi-targeted oleic acid (OA)–MNPs were developed. Reports confirmed promising outcomes about the in vitro and in vivo efficacy in treating human cancer cells (HeLa). The study results stated that the MS-NPs formulations were more predominant than the placebo or free drugs and could overcome the drawbacks mentioned above of the conventional treatment approaches [153]. The synthesized and functionalized DOX magnetic MS-NPs were fabricated with PF-127 and then conjugated with transferrin (Tf) to enhance BBB

solubility and stability of anti-EGFR drugs by being deposited in the porous structure.

**Figure 7.** Synthesis of magnetic mesoporous nanoparticles using polymer and subsequently conjugated with ligands to achieve the sustained release of the drug at the specific targeted site. **Figure 7.** Synthesis of magnetic mesoporous nanoparticles using polymer and subsequently conjugated with ligands to achieve the sustained release of the drug at the specific targeted site.

#### 7.2.2. Magnetic Nanoparticles (MNPs) 7.2.2. Magnetic Nanoparticles (MNPs)

Magnetic nanoparticles (MNPs) specifically created an interest in the biomedical application and research due to various advantages: separation of molecules, gene/drug delivery, magnetic resonance imaging (MRI), and hyperthermic tumor treatment [155]. Among the several magnetic nanocarriers, super-paramagnetic iron oxide (SPIO) has been commonly utilized owing to its promising biocompatibility and magnetic properties. For example, an improved survival rate was noticed in animal models with C225-IONPs compared to pure C225 in the treatment of GBM [156]. In addition, SPIO and peptide nanoprobe were effectively combined and demonstrated for specific molecular Magnetic nanoparticles (MNPs) specifically created an interest in the biomedical application and research due to various advantages: separation of molecules, gene/drug delivery, magnetic resonance imaging (MRI), and hyperthermic tumor treatment [155]. Among the several magnetic nanocarriers, super-paramagnetic iron oxide (SPIO) has been commonly utilized owing to its promising biocompatibility and magnetic properties. For example, an improved survival rate was noticed in animal models with C225-IONPs compared to pure C225 in the treatment of GBM [156]. In addition, SPIO and peptide nanoprobe were effectively combined and demonstrated for specific molecular MRI and sensitive optical imaging (SOI). Both in vitro and in vivo MRI and SOI showed that the nanoprobe was useful for targeting GBM with desirable biosafety [157]. In another study, MNPs were employed by convection-enhanced delivery (CED) in the brain to target the EGFRvIII xenografts GBM model. Then, MRI was conducted to evaluate brain targeting and the delivery of conjugated MNPs after CED. The accomplishment of a human clinical trial containing a direct injection of MNPs into recurrent GBM for thermotherapy proved the safety, efficacy, and feasibility in the patients [158]. In a recent report, pazopanib was loaded in MNPs, which stimulated the ultrasound's drug release. The enhanced drug distribution in the non-small cell lung cancer (NSCLC) region resulted in improved therapeutic outcomes [159].

#### 7.2.3. Noble Metal Nanoparticles (NM-NPs)

Commonly, the Gold (Au) NPs are known as noble metal NPs, which were comprehensively studied for biomedical applications due to their lower toxicity, distinctive electronic, and optic properties that prompted cellular destruction with an application of radiation [160] or light [161]. In addition, AuNPs could be loaded with organic molecules (antibodies), which enhanced the accumulation of AuNPs within specific cancer tissues or lesions [162]. AuNPs were loaded with malondialdehyde-modified low-density lipoprotein (MDA-LDL) antibodies by distinct chemistries for drug recognition and capture from the biological system [163]. Furthermore, 40 nm AuNPs with mAb targeting the EGFR acted by random adsorption to treat oral squamous cancer. The antibody-loaded AuNPs accumulation into the tumor region enhanced cancer cell death by photothermal therapy [161]. In addition, the 5 nm AuNPs were surface modified with EGFR antibodies and functionalized using GM for targeting GBM cells [164]. In another study, Au nanocubes (AuNCs) with pH or temperature sensitivity were prepared for erlotinib or DOX encapsulation. Firstly, the drug-loaded AuNCs could accumulate more at the cancer tissue; the particular acidic microenvironment of cancer initiated erlotinib's release, precisely. Finally, the controlled release of DOX by near-infrared (NIR) laser

irradiation improved the therapeutic effect of AuNCs-based nano carrier in A431 cancer cell lines [165]. In addition, the in vivo activity of the Au–C225 conjugate resulted in a similar effect compared to free C225, concluding that the site-specific conjugation to the AuNP did not affect the biological action of the EGFR antibody, thereby signifying the value of the intended functionalization approach. The opportunity to yield accurate AuNP–Immunoglobuin G (IgG) conjugates creates novel paths to assay the Au–C225 conjugate for cancer therapy, either for sensitizing tumor cells to external radiation [166]. Based on the promising results of the developed NM-NPs provided a novel way for the delivery of chemotherapeutic and TKIs drugs.

### **8. Current Clinical Studies of Nanoformulations**

After promising results of preclinical investigations, the organic and inorganic nanoformulations have entered the clinical trials to assess the tolerability, safety, pharmacokinetics, and efficacy for the treatment of GBM [167]. Table 3 summarizes the list of current clinical trials on EGFR loaded nanoformulation for the treatment of GBM. Firstly, the researchers have a newly developed EnGeneIC delivery vehicle (EDV). This inorganic nanocarrier exploits antibody-targeted, transporting active anti-cancer drugs into EGFR-expressing cancer cells (EnGeneIC, Lane Cove West, Australia). In a phase I/II study, the recurrent GBM adult patients were dosed of up to 5 <sup>×</sup> <sup>10</sup><sup>9</sup> to determine the safety and possible dose of EGFR–EDV–DOX (NCT02766699) [168]. The effect of anti-EGFR targeted DOX loaded into C225-decorated Immunoliposomes (ILs) (C225–ILs–Dox) is being evaluated in a phase I clinical trial (NCT03603379) [169]. Analogously, DOX–trastuzumab consisting of PEGylated liposomes has completed a phase I/II clinical trial (NCT01386580). The phase I clinical study of cationic liposomes loaded with cancer suppressor gene p53 and TMZ (SGT–53–TMZ) was conducted to observe minimal side effects with a 6-month progression-free survival (PFS) and overall survival (OS) rate in patients with advanced solid tumors (NCT02340156) [170]. The phase I/II clinical trial was conducted to estimate side effects and a suitable dose of EGFR-bispecific antibody armed T cells (EGFR-Bi-T) in GBM patients' treatment. In phase I trials, the patients received EGFR-Bi-T intrathecal (IT) injection twice per week for four weeks to determine the efficacy and toxicity profile. In phase II trials, the patients received EGFR-Bi-T IT twice weekly for four weeks and then i.v. over 15–30 min twice weekly for two weeks (NCT02521090).


**Table 3.** List of Current Clinical Studies of Nanoformulations.

#### **9. Future Perspectives**

The results of EGFR–TKIs drugs in clinical research showed that the molecularly targeted medicines combined chemotherapy could achieve the highest therapeutic outcome compared to free active components. However, the low specific inhibition and drug resistance during treatment were difficulties in developing targeted molecular active agents. Novel signaling transduction anti-cancer drugs based on modified therapy could minimize or overcome drug resistance. Remarkably, nanotechnology's benefits for the currently available chemotherapeutic and TKI agents provided alternative strategies for improving therapeutic results: low solubility and BBB penetration, and prolonging the drug accumulation in the cancer region, decreasing the side effects triggered by non-specific distribution. The nanocarriers' design has been moved forward via its technological upgradations, yet the nanoplatform fails to attain comprehensive clinical interpretation. Thus, the nanocarrier delivery approach is intended to assure better chances of success in a clinical trial [172].

Moreover, the clinical trial evidence (Table 3) was intended to determine the safety and efficacy of nanoformulations for the GBM treatment that have been happening since the beginning of the twenty-first century. Hence, the results of clinical studies have not yet been published, which contribute to paving the way for the clinical interpretation of nanotherapies for GBM. The researchers have recently considered utilizing natural compounds such as polyphenols and cannabinoids to target EGFR and its downstream pathway in various cancer cell lines [173–178]. The brain's microenvironment, prominently advanced as per our understanding, targeting GBM cells with Janus kinases/signal transducer and activator of transcription proteins (JAK/STAT) inhibitors via combinational approaches could boost immunity with the reduced oncogenic effect of the GBM cancer cells [179,180].

#### **10. Conclusions**

The clinical trials of the nano-based formulations of chemotherapeutic and/or TKI drugs in combination are awaited. After an extensive literature search, it could be stated that novel approaches to treat GBM using mono or combinational therapy of polyphenols and anti-EGFR drugs to target multi signaling pathways (EGFR, JAK/STAT, and PI3K/AKT/mTOR) would help overcome the multiple failures of EGFR–TKI drug trials. Furthermore, the novel nano-based combinational therapy might positively reverse chemotherapeutic and TKI drug-induced resistance.

**Author Contributions:** Conceptualization, S.P., D.V.G. and S.B.C.; methodology, S.P., D.V.G. and S.B.C.; software, S.P., M.G.S., A.M. and M.M.; validation, D.V.G., S.B.C., N.N. and S.H.N.; formal analysis, S.P.; investigation, D.V.G., S.B.C.; resources, S.P.; data curation, S.P., writing—original draft preparation, S.P., writing—review and editing, S.P., M.G.S. and M.M.; visualization, D.V.G.; supervision, D.V.G. and S.B.C.; project administration, D.V.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work received no external funding.

**Acknowledgments:** This work was supported by JSS Academy of Higher Education and Research, Mysore, Karnataka, India. S.P. is thankful to Department of Biotechnology (DBT), India for providing Junior Research Fellowship for his research work. Authors are thankful to Edit n Stat for Proofreading this Manuscript.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

© 2020 by the authors. 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 (http://creativecommons.org/licenses/by/4.0/).

*Article*
