Nanotechnology – a robust tool for fighting the challenges of drug resistance in non-small cell lung cancer

EGFR mutations and current problems in NSCLC treatment

The main reasons behind the limited success of TKI monotherapy in the suppression of lung cancer growth for an extended period are tumor heterogeneity, key signaling pathway alteration, and activation of alternate signaling, which effectively rescue the main inhibited pathway. The oncogenic significance of EGFR and the weak response to TKIs have been the focus of clinical interest for more than a decade, motivating the research community to look deeper into relevant explanations for therapeutic failure and suggest smart solutions for overcoming resistance.

There are five selective EGFR TKIs approved for the treatment of EGFR-mutated NSCLC, namely, gefitinib (GEF) and erlotinib (ERL) (first-generation reversible EGFR TKIs), afatinib (AF) and dacomitinib (DAC) (second-generation irreversible EGFR TKIs), and osimertinib (OS) (third-generation irreversible EGFR TKI). All of these drugs act as therapies of choice for NSCLC with EGFR-activating mutations [16-19].

First-generation EGFR TKIs are well-established molecularly driven therapies for lung cancer harboring specific types of activating EGFR mutations involved in the development of NSCLC (classical sensitive EGFR mutations, i.e., deletions in exon 19 and the single-point substitution mutation L858R in exon 21) [8,9]. Not all tumors with activating EGFR mutations will respond to EGFR TKI treatment. A subgroup of around 20–30% of patients harboring an activating mutation is intrinsically resistant to TKIs and shows weak clinical response, including those with wild-type EGFR NSCLC cancer. The establishment of clinical criteria for intrinsic resistance is still ongoing, and considerable efforts are made toward the estimation of the efficacy and optimal sequence of administration of different EGFR TKIs across TKI-sensitive patients with common and uncommon EGFR mutations [20]. The type of EGFR mutation influences the effectiveness of gefitinib and erlotinib across NSCLC tumors. For example, gefitinib is more efficient in treating NSCLC harboring the L858R mutation than in NSCLC expressing the G719S mutation [21]. Further, an approved treatment with EGFR TKIs for NSCLC with defined but uncommon mutations such as G719X, S768I, and L861Q is the second-generation TKI afatinib. Lastly, osimertinib is a third-generation TKI that is approved for EGFR T790M positive NSCLC with acquired resistance to first- and second-generation drugs [12,22,23]. Similar to traditional cytotoxic agents, acquired resistance to TKIs and early relapse are still significant limitations of this therapeutic approach. Following the initial pronounced response, after only 9 to 14 months around 50% of patients develop resistance to therapy with first- and second-generation TKIs as a result of T790M secondary mutation in exon 20 of EGFR [24]. Acquired resistance to TKIs is unavoidable and has already been documented even for the third generation of TKIs (target-dependent and target-independent molecular mechanisms of resistance to TKIs are presented in Table 1). Target-independent acquired resistance driven by cMET amplification after EGFR TKI treatment may be treated with crizotinib, a dual inhibitor of ALK and cMET, and brigatinib (multi-kinase inhibitor of EGFR, ALK, FLT3, and other kinases). This therapy shows effectiveness against mutant variants of EGFR and ALK that are resistant to common types of EGFR and ALK inhibitors [25].

Table 1: Molecular mechanisms of resistance to EGFR TKIs.

Target-dependent Secondary exon 20 mutation: EGFR T790 (40–55% of EGFR resistance cases) and EGFR amplification
Secondary mutations with low occurrence: D761Y (exon 19), L747S (exon 19), and T854A (exon 21) Tertiary mutation: EGFR C797S; L798I
Other tertiary mutations: WZ4002, L718Q, and L844V Secondary (T790M gatekeeper residue) and tertiary kinase domain (C797S) resistance mutation in the targeted kinase reduces drug affinity or prevents access of the TKI to the active site and reduces its efficacy. Resistance to gefitinib and erlotinib is evident when the T790 mutation is present, and the C797S mutation induces resistance to osimertinib. Target independent Bypass resistance (bypass the EGFR blockade)
MET gene amplification: Amplified MET causes phosphorylation of ERBB3. Even when phosphorylation of EGFR is inhibited by an EGFR TKI, activation of the PI3K/AKT pathway is maintained through ERBB3 or the ERBB3/ERBB2 duet.
HGF overexpression: HGF induces activation of the PI3K/AKT pathway through MET; this activation is independent of ERBB3 or EGFR).
HER2 amplification: HER2 forms heterodimers with EGFR to activate downstream signaling. Downstream signaling
mutations in BRAF and PI3K, KRAS, PTEN loss, NF-1 loss, and CRKL amplification, MAPK pathway activation by mutated KRAS or MEK
(less frequent events) Phenotypic alterations
epithelial-mesenchymal transition (ЕМТ), or small cell histologic transformation

However, only a limited number of mutations are covered by clinical therapy. In addition, some novel therapeutic approaches against resistant tumors have failed due to the heterogeneity of the progression of genetic alteration and the resulting complexity of resistance mechanisms [23]. Enormous efforts have been made in finding a way forward from this standstill, and evidence has been derived that no single drug can treat the broad spectrum of molecular alterations in NSCLC. Considering the fact that multiple mechanisms are involved in the reactivation of the EGFR signaling pathway, targeting multiple constituents within the EGFR cascade or targeting parallel pathways to prevent cross talk between multiple growth factor receptors have emerged as valid approaches that could be used to tackle cancer resistance and maximize the efficacy of EGFR inhibition. Presently, a combinatorial therapeutic strategy is believed to be a rational approach to combat the complexity of resistance and continuous cancer mutations. Co-delivery of TK inhibitors with anticancer drugs, immunotherapy, or gene-specific therapeutics to disrupt key resistance pathways, reactivate p53-mediated apoptosis, or inhibit cellular drug efflux are only a few examples of strategies used to fight cancer resistance mechanisms successfully [23,24]. In addition, co-delivery of anticancer therapy using surface-engineered nanoparticles for tumor targeting may alleviate some of the unwanted effects on off-site targets and increase the therapeutic concentration at the site of action as well as efficacy and safety of the current therapy for lung cancer treatment. Co-delivery of combined therapeutic agents at the right time and at the right place using smart nanotools to exert a simultaneous effect on multiple signaling pathways, leading to the avoidance or combating of resistance as well as the prevention of side effects, is the theoretical rationale behind the use of designed nanoparticles (NPs) [26-28].

Advances in the therapeutic approaches used for overcoming NSCLC resistance

Combinatorial treatments are designed with the goal of exerting additive or synergistic inhibitory effects on the proliferation and survival mechanisms on which the cancer cells are heavily dependent. The efficacy of existing small molecules in synergistic combinations for relevant genetic mutations in resistant cancers has been evaluated in many research and clinical studies, with promising results in some types of mutant lung cancers. A plethora of multimodal treatments for the co-administration of: (i) conventional cytotoxic agents with signaling pathway inhibitor/s, (ii) inhibitors of two or more signaling pathways within a signaling network, (iii) inhibitors of multiple targets within a single pathway exerting synergistic effects, and (iv) cytotoxic or molecular targeting agents with small interfering RNA (siRNA) for silencing the mutating genes at protein and messenger RNA (mRNA) level, have made their way to clinical therapy or are under evaluation for their efficacy and safety in many research studies and several clinical trials. The synergistic effects of combination therapy depend on the status and the type of genetic alteration; as such, the most potent will be the one showing synergistic or additive effects on oncogene pathways essential for cell survival.

Conventional cytotoxic agents with signaling pathway inhibitor/s

EGFR signal transduction pathways can be roughly divided into a pro-survival arm with the PI3K-mTOR-AKT cascade and a proliferative arm with the Ras-Raf-Mek-Erk cascade. Enhanced kinase activity on mutated EGFR with exon 19 deletion is associated with upregulated c-MYC levels through the Ras-Raf-Erk pathway, promoting angiogenesis via hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) signaling [29-31]. According to preclinical data, a combination therapy consisting of erlotinib and cisplatin targets angiogenesis and manifests synergistic and additive antitumor activity via downregulation of the c-MYC–HIF-1α–VEGF signaling pathway in mutated NSCLC with exon 19 deletions (Figure 1) [32-34]. Several randomized clinical studies have also reported on the increased effectiveness of combined chemotherapy/EGFR TKI treatments in patients with NSCLC bearing an EGFR mutation. Therefore, to prevent or delay the emergence of acquired resistance to EGFR TKIs, adding carboplatin and pemetrexed to gefitinib is recommended as a first-line option in patients with EGFR-mutated tumors [35-40].

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Figure 1: Examples of combinatorial therapy for (left) EGFR-dependent and (right) EGFR-independent TKI resistance. The upward-pointing black arrows indicate upregulation/increase, while the downward-pointing black arrows indicate inhibition/decrease. The black tick marks indicate a beneficial effect. The red x mark indicates a delayed or prevented event.

Inhibitors of two or more signaling pathways within a signaling network

EGFR and VEGF share common downstream signaling, although they may function independently during oncogenesis. Increased VEGF levels, which have been confirmed in cancers with acquired resistance, lead to the preservation of tumor growth when the tumor is under attack from EGFR TKIs. Because of the interplay of mechanisms, combining EGFR TKIs and VEGF inhibitors seems to be a rational approach to combat tumor resistance and increase the efficacy of anti-tumor therapy [41-45]. Several trials that compared the efficacy and safety of EGFR TKIs from the first, second or third generation combined with anti-angiogenic therapy, such as small-molecule inhibitors of VEGFR-2 (vandetanib, nintedanib, axitinib, and cediranib) or anti-VEGF/VEGFR monoclonal antibodies (ramucirumab, which is specific for the VEGFR-2 extracellular domain, or bevacizumab, a VEGF-A inhibitor), have been conducted to evaluate the efficacy of combination therapy in mutated NSCLC (Figure 1). Results from preclinical and clinical trials point to substantially improved progression-free survival in patients with EGFR-mutant NSCLC receiving combined EGFR TKIs and angiogenic therapy. However, this combined therapeutic approach has been characterized by an increased incidence of adverse reactions of grades 3–5 [41,46-53]. Nanomedicines can be used as tools for improved localization of combination therapy at the site of action, improving the outcome and decreasing the adverse effects.

Inhibitors of multiple targets within a single pathway exerting synergistic effects

Amplification, overexpression, and mutation of MET and HER2 are heavily involved in EGFR TKI resistance development, and the cross talk of these receptors is a way to avoid TK inhibition in many cancers (Figure 1). Hence, these oncogenic drivers are legitimate therapeutic targets in NSCLC with off-target mechanisms of acquired resistance to first- and second-generation TKIs. Various MET TKIs, among them crizotinib and the more selective savolitinib, tepotinib, and capmatinib, may be good candidates for EGFR TKI/MET TKI combination therapy (Figure 1). This approach shows improved clinical outcomes over chemotherapy or MET TKI monotherapy in patients with advanced EGFR-mutant NSCLC who acquired MET amplification or MET overexpression during EGFR TKI treatment [54-62].

Further, substantial evidence for the efficacy of EGFR TKIs combined with HER2-targeted therapy in patients with developed EGFR TKI resistance due to HER2 amplification can be found in the literature [63-65]. Patients with HER2 gene mutations showing resistance to EGFR TKIs may be sensitive to novel, more selective HER2 inhibitors (poziotinib and pyrotinib), HER2 targeting agents such as anti-HER2 antibodies, and small-molecule EGFR tyrosine kinase inhibitor dual therapy (Figure 1). The administration of target-specific antibody–drug conjugates (cytotoxic chemotherapy) has also been characterized by positive responses regardless of HER2-specific mutation sites. In oncogene-addicted cancers (HER2-mutant cancer, oncogene-driven EGFR-positive, ALK-positive, or RET-positive NSCLC), current studies do not encourage the use of immune checkpoint inhibitors, with the exception of KRAS-mutated cancers [61,64,65].

Despite the encouraging results of antibody–drug conjugates, acquired resistance to these agents might eventually develop following the initial positive response. Various mechanisms of acquired resistance in patients with HER2-positive locally advanced breast cancer or metastatic breast cancer involving factors crucial for their mechanism of action are reported across the literature [66,67]. This suggests that such acquired resistance might also become a common problem in advanced NSCLC treatment [68]. Some of the potential factors of resistance, such as poor internalization, defective intracellular trafficking of the HER2 antibody–drug conjugates, masking of the HER2 epitope, high rate of recycling, and the effect of upregulated drug efflux pumps, may be resolved by novel nanomedicines designed to interact with the tumor cells in a variety of ways with the goal of overcoming the limitations of the conjugates [68,69].

Cytotoxic or molecular targeting agents with siRNA

Targeting homologous mRNA sequences in cells and knockdown of receptors involved in cell survival and proliferation using RNA interference downregulates receptor protein expression, inhibits cell growth, and induces apoptosis. The effect obtained by siRNA is not influenced by the receptor alteration status and significantly decreases the gene's oncogenic potential. Chen et al. compared the efficacy of TKIs in NSCLC cells harboring different mutations with combined therapy consisting of TKIs (gefitinib, erlotinib, and afatinib) and EGFR-specific siRNA. The authors noted that combined therapy with the potent irreversible EGFR/HER TKI afatinib and EGFR-specific siRNA resulted in enhanced growth inhibition and apoptosis due to the inhibitory effect of the EGFR-specific siRNA on the overall EGFR oncogenic activity, including the downstream TKI resistance mutations (Figure 1) [70]. Lu et al. tested the efficacy of combined siRNA treatment with gefitinib in several NSCLC cell lines (A549, H1975, and H1993). The A549 cell line carried wild-type EGFR and KRAS mutations, H1975 cells expressed L858R/T790M EGFR, and H1993 cells harbored MET amplification. Therefore, three types of siRNA were used as EGFR–scFv–arginine nonamer peptide fusion protein complexes, namely siRNA for KRAS, EGFR, and MET gene expression silencing (Figure 1). The synergistic effect of gefitinib and scFv antibody-mediated siRNA delivery for silencing the expression of resistance-related genes was evidenced by a significant reduction in cell growth and increased rate of apoptosis compared to the cells treated with siRNA only. Furthermore, considering that these cell lines are EGFR-positive TKI-resistant NSCLC cells, a synergistic effect of gefitinib and siRNA may be regarded as proof of restored sensitivity of EGFR-positive NSCLC to gefitinib as a result of silencing the expression of resistance-related genes [71]. Therapy consisting of EGFR siRNA combined with EGFR TKIs and anti-EGFR monoclonal antibodies can additively enhance growth factor inhibition in vitro, maintaining its biological efficacy in cells and xenograft models with different mutation statuses [70,72-74].

Further advances in the multimodal combination therapy approach

KRAS proteins operate as guanosine diphosphate (GDP)/guanosine triphosphate (GTP) molecular switches in response to activated transmembrane receptors such as EGFR. The KRAS mutation occurs at a frequency of around 30% in NSCLC, with the KRAS p.G12C mutation being the most frequent variant. Mutated KRAS cannot return to its inactive GDP form, which triggers EGFR-independent activation of several downstream effectors [75-77]. The binding of KRAS-GTP to several effectors, among them PIK3K and RAF kinases, triggers activation of downstream AKT and mTOR (PIK3K), which regulates apoptosis, metabolism, and translation, as well as MEK and ERK signaling (RAF kinases), which influences cell cycle progression and proliferation [78]. Therefore, it is expected that KRAS-mutated tumors would not respond to EGFR TKIs. Patients with KRAS-mutant NSCLC can benefit from direct KRAS inhibitors, such as sotorasib, which lock KRAS in its inactive GDP-bound form. However, a heterogeneous resistance pattern during KRASG12C inhibitor treatment has been noticed after an initial positive response [78-80]. Co-targeting of upstream signaling (suppression of receptor tyrosine kinases) and downstream signal inhibition by targeting the RAF-MEK-ERK signaling cascade are tested in clinical studies as relevant approaches to delay resistance and improve KRASG12C inhibitor efficacy [81,82]. Clinical data from CodeBreak 100/101 revealed promising efficacy with long-lasting anti-tumor effects when a programmed cell death protein 1 (PD-1) antibody was administered alongside a KRASG12C inhibitor, suggesting that PD-1 inhibition produces a synergistic effect with sotorasib and enhances CD8-positive T-cell infiltration, which causes an inhibition of tumor growth [83-87]. In addition, there is substantial evidence that the co-delivery of siRNA that shows specific binding to mRNA of the most commonly occurring KRAS missense mutations together with a chemical EGFR inhibitor may efficiently reduce mutant KRAS-induced effects and may contribute to overcoming resistance in the treatment of NSCLC [72,88,89].

Many of the obstacles to the co-delivery of combined therapies can be resolved by nanomedicines as tools for the targeted delivery of high concentrations of anticancer drugs at their site of action. Although designated as molecularly targeted therapies, the targeting of receptors by EGFR TKIs and other receptor inhibitors is not absolute. Once the EGFR TKIs are absorbed from the gastrointestinal tract and distributed in the body, they interact with EGFR signaling pathways of many normal cells influencing their proliferation, differentiation, migration, and apoptosis. This leads to side effects, including rash, erythema, diarrhea, gastrointestinal perforations, ocular lesions, and hematological disorders. Using nanomedicines as a vehicle for the administration of TKIs may alleviate the aforementioned problems of conventional administration and (i) improve their pharmacokinetic profile, (ii) increase tumor targeting potential and localization at the tumor site, (iii) decrease the exposure of healthy tissues to the drug, (iv) minimize off-site targets and side effects, (v) even bypass, reduce, or reverse the multidrug resistance mechanisms, and/or (vi) overcome acquired resistance and sensitize mutant NSCLC cells to EGFR TKIs through the synergistic action of combined therapy against various multiple anti-tumor targets [20,22,90].

Surface-engineered nanoparticles for lung tumor targeting and co-delivery of combinatorial therapy

Simultaneous delivery of combinatorial inhibitors with the goal of targeting multiple constituents within a single pathway or different oncogenic pathways in therapeutic concentrations at the tumor site, preferentially in the tumor cell, is essential for the efficacy of the therapy. Adequate concentrations might not be achieved with conventional dosage forms mainly due to the poor localization of the free drug molecules at the site of action and the differences in bioavailability and pharmacokinetic parameters. Notably, the failure of delivery at the right time and at the right place contributes to severe systemic toxicities and ineffectiveness. Successful translation of scientific knowledge of the mechanisms of resistance combined with nanotechnology as a tool for targeted delivery may bring improvements in the efficacy of anticancer drugs and may aid in elucidating the beneficial synergistic combinations regarding lung cancer subtype treatment. Nanomedicines have the potential for (i) multivalent targeting and co-delivery of agents to endothelial cells, tumor microenvironment, and tumor cells, (ii) delivering large payloads of active substances with different physicochemical properties, such as small-molecular drugs and siRNA, to the site of action, and (iii) limiting drug resistance [91]. Nanotherapy can change the landscape of clinical lung cancer treatment by mitigating the risk of therapeutic failure due to the non-coordinated co-delivery of therapeutic agents and off-target side effects. However, despite substantial progress, a precise control of the in vivo trajectories of the nanosystems is still beyond our reach. Some of the promising approaches and design considerations in the engineering of tumor-homing nanoparticles will be discussed below with an emphasis on increased lung tumor tissue localization.

Current approaches for overcoming biological barriers and improved drug targeting

Looking back at almost half a century of research on drug targeting, experimental evidence shows that efficient tumor localization and intracellular delivery may still be very challenging. However, an improved understanding of the mechanisms involved in angiogenesis, tumor–stroma interactions, molecular heterogeneity between cancers, genetic and epigenetic alterations, and cancer marker expression has not only improved current therapeutic plans for cancer patients but has had an impact on the design approaches of the nanotools for cancer imaging and anticancer drug delivery. In recent years, new platforms to enhance the low tumor targeting capacity of nanomedicines using biomimetic targeting motifs, multifunctional and multistage nanomicelles and polymer nanoparticles, and nanostructured lipid nanocarriers, combined with precision oncology research to identify additional targetable biomarkers, have emerged. Some have been applied in the co-delivery of clinically relevant combinations of molecularly targeted drugs, chemotherapeutic agents, and siRNA.

Historically, the most promising first-generation, passive targeting, stealth polymer NPs for anticancer drug/gene delivery are hydrophobic core–hydrophilic shell NPs including (i) self-assembled kinetically stable amphiphilic block copolymer core–shell NPs, (ii) polymer–polypeptide hybrid core–shell NPs, and (iii) polymer–lipid hybrid core–shell NPs additionally decorated with ligands for overexpressed receptors on cancer cells [92]. Traditionally selected overexpressed cancer cell surface markers for the active targeting of NPs include ανβ3 integrin, aminopeptidase N (CD13), lymphocyte homing receptor (CD44), programmed death ligand-1 (CD274), folate receptor protein, nucleolin receptor, epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), human epidermal growth factor receptor 2 (HER2), luteinizing hormone-releasing hormone (LHRH) receptor, and somatostatin receptors (SSTRs) [93].

Due to the complexity of the problem of specific targeting, secondary to the many different types of barriers in the body, incorporating several functionalities to address diverse barriers might improve the targeting efficacy of nanomedicines. Various solutions have been proposed to improve mononuclear phagocytic system (MPS) evasion, extravasation at the tumor site, and diffusion through the dense collagen matrix of the solid tumors. Biomimetic, multifunctional, and multistage targeted nanoscale delivery systems with improved potential for intratumor and intracellular localization, as well as sub-cellular targeting, capable of tackling several body barriers and tumor heterogeneity more efficiently, have been designed to address the problems of efficient targeting [94].

Multifunctional stimuli-responsive nanosized drug delivery carriers: Mixed-layer and multilayered nanocarriers with bioresponsive and cleavable layers, possessing different functional properties for improving the enhanced permeability and retention (EPR) effect, diffusion in the tumor microenvironment, cellular internalization and subcellular targeting, were synthesized by click coupling reactions or arranged by self-assembly and co-assembly of block copolymers. These carriers may challenge different barriers after bioresponsive cleavage of the above functionalities. One recently published example involves a micellar structure composed of a polycaprolactone (PCL) core, a mixed poly(2-dimethylamino)ethyl methacrylate/poly(ethylene oxide) (PDMAEMA(TPP+)/PEO) middle layer, and a PEO corona. The system was self-assembled using poly(ethylene oxide)-poly(ε-caprolactone)-b-poly(ethylene oxide) (PEO113-b-PCL70-b-PEO113) and poly(2-(dimethylamino)ethyl methacrylate)-b-poly(ε-caprolactone)-b-poly(2-(dimethylamino)ethyl methacrylate (PDMAEMA20(TPP+)-b-PCL70-b-PDMAEMA20(TPP+)) block copolymers. PEO-PCL blocks were linked using acetal groups to enable the cleavage of the PEO blocks from the NP surface in the acidic environment of a tumor and the lysosomes, exposing the PDMAEMA layer decorated with triphenylphosphonium (TPP) ligand to the environment. The TPP lipophilic cation is characterized by a large hydrophobic surface area, which facilitates its permeation throughout phospholipid bilayers, lysosomal escape due to the proton sponge effect, and further accumulation within mitochondria [95]. Barthel et al. developed mixed-layer ABC triblock terpolymer mixed PEO shell nanomicelles of a size below 30 nm, based on poly(ethylene oxide)-b-poly(allyl glycidyl ether)-b-poly(tert-butyl glycidyl ether) (PEO-b-PAGE-b-PtBGE). The PAGE segment can be subsequently modified using thiolene chemistry to introduce positive charges [−NH2 (cysteamine, ENT)], negative charges [−COOH (3-mercaptopropionic acid, ECT)], and active targeting ligands [thiogalactose residues (EGT)] for fine-tuning the charges in the shell in different biological environments either for higher uptake or reduced toxicity. In brief, the ABC triblock terpolymers comprised identical A and C segments. Yet, different functionalities in the middle PAGE block (B) are directly correlated to the combination and the number of functionalities and, therefore, easily adjusted to optimize the systems for different target sites, which is especially promising for nucleic acid delivery [96]. One example of multifunctional, multilayer, bioresponsive lipid polymer nanoparticles with a cleavable layer as a vessel for the co-delivery of erlotinib and bevacizumab was recently published by Pang and co-workers. Clinical studies point to the serious toxicity of conventional application, which might be mitigated with nanotools for co-delivery of therapy for the dual inhibition of VEGF and EGFR pathways. The designed nanocarrier was composed of a polycaprolactone core with bevacizumab and erlotinib, coated with a phospholipid layer, with anchors composed of hyaluronic acid–adipic acid hydralazide–poly(ethylene glycol) (HA-ADH-PEG). Hyaluronic acid-decorated lipid polymer hybrid nanoparticles (LPH NPs) specifically target overexpressed CD44 at the NSCLC cells. In the acidic tumor environment, the pH-responsive linker between HA and PEG is hydrolyzed, leading to the cleavage of the HA layer. This, in turn, decreases the NP size and enables faster tumor diffusion, improved internalization, and drug release at the site of action. These nanocarriers exhibited a high degree of tumor homing, low toxicity, and efficient tumor inhibition in vitro and in a tumor mouse model [97]. An additional example of multilayered cleavable pH-responsive nanoparticles for KRAS mutated cancer is described in Table 2.

Table 2: Examples of various nanoparticles for the co-delivery of combinatorial therapy for resistant lung tumor treatment.

Type Co-delivered agents Description and outcome Ref. polymer NPs (PLGA-PEIa) paclitaxel (PTX) + Stat3 siRNA increased sensitivity of NSCLC cells to paclitaxel due to the silencing of STAT3
[98] polymer NPs (MPEG-PLAb) ERL + cilengitide (Cilen; integrin αvβ3 inhibitor) Cilen reversed EGFR resistance to ERL by inhibition of integrin αvβ3, the activator of galectin-3/KRAS/RalB/TBK1/NF-κB [99] polymer NPs (PEG-PLAc) ERL + fedratinib (FDTN; JAK2 inhibitor) re-sensitization of EGFR resistant cancer to ERL due to suppression of JAK2/STAT3 and disruption of EGFR/JAK2/STAT3 axis [100] pH-responsive polymer NPs (DOX-PEI+Bcl2 siRNAd) doxorubicin (DOX) + Bcl2 siRNA suppression of Bcl2 (a key regulator among the anti-apoptotic proteins) resulted in DOX enhanced antitumor efficacy [101] complex polymer micelles (P85-PEI/TPGSe) paclitaxel (PTX) + survivin shRNA (shSur) down-regulation of survivin, enhanced paclitaxel-induced apoptosis and cell arrest in the G2/M phase [102] micelleplexes (pH-responsive cationic micellar nanoparticles; PDMA-b-PDPAf) paclitaxel (PTX) + Bcl-2 siRNA suppression of Bcl2 (a key regulator among the anti-apoptotic proteins) resulted in enhanced antitumor efficacy of PTX [103] multilayered PAA cleavable pH-responsive nanoceria (FA-PAA-PNCg) doxorubicin + ganetespib ganatespib synergizes and accelerates therapeutic efficacy of DOX via ROS production [104] multistage solid lipid NPs (SLNs) loaded in microspheres afatinib (AFT) + paclitaxel (PTX) synergistic effect of afatinib and PTX due to inactivation of p70 s6 kinase by PTX and inactivation of PI3K/AKT/mTOR [105] liposomes (PEGylated lipo-DTX/siRNA NPsh) docetaxel (DTX) + Bcl-2 siRNA suppression of Bcl2 (a key regulator among the anti-apoptotic proteins) resulted in enhanced DTX antitumor efficacy [106] nanostructured lipid carriers (LHRH-decorated NLC-PTX-siRNAi) paclitaxel (PTX) + gefitinib (GEF) + siRNA targeted to EGFR i) suppression of EGF tyrosine kinase signaling pathways, ii) prevention of EGF receptor protein synthesis, and iii) induction of cell death by the microtubule-stabilizing drug paclitaxel lead to an enhanced therapeutic effect in EGFR TKI resistant cancer [107] polymer-coated magnetic NPs (FA-PAA-Pt(MCO)2(Pt) + ganetespibj) platinum cyanoximate complex + ganetespib ganetespib effectively suppressed KRAS mutated cancer cells when co-delivered with Pt-agents and prevented multidrug resistance [108] PEG-modified metallic NPs (IR780@INPs-CTXk) cetuximab + IR780 (sonodynamic therapy) combinatorial treatment compensates for cetuximab-resistant mutations due to the ROS-generating potential of IR780, which promotes cell apoptosis and inhibits proliferation [109] PEG-modified photoresponsive metallic nanocarriers gefitinib + CuS (photodynamic switching) re-sensitization to gefintinib as a result of the inactivation of bypass signaling in gefitinib resistant tumors due to increased ROS levels which downregulate expression of IGF1R and its downstream AKT/ERK/NF-kB signaling [110]

aPLGA-PEI NPs covered with a PEI corona; bmethoxy poly(ethylene glycol)–poly(ʟ-lactide) NPs; cpoly(ethylene glycol)–poly(lactic acid) NPs; ddoxorubicin–polyethyleneimine conjugate via a pH-sensitive linker mixed with Bcl2 siRNA; ePluronic P85–polyethyleneimine conjugate and ᴅ-α-tocopheryl–polyethylene glycol 1000 succinate complex NPs; fpH-responsive poly(2-(dimethylamino) ethyl methacrylate)-b-poly(2-(diisopropylamino) ethyl methacrylate) NPs; gfolic acid-decorated polyacrylic acid-coated pH-responsive cerium oxide NPs; hPEGylated cationic liposome complex with Bcl-2 siRNA; iluteinizing hormone-releasing hormone decapeptide-decorated multifunctional nanostructured carriers prepared by self-assembly of liquid/solid lipids, surfactants, and cationic lipids; jfolate-ligated PEGylated polyacrylic acid-coated magnetic NPs with encapsulated platinum cyanoximate complex and ganetespib; kPEG-modified (distearoyl-glycero-phosphorylethanolamine-PEG-COOH) iron tetroxide core nanoparticles loaded with IR780 and decorated with cetuximab.

Multistage drug delivery systems: Multistage nanocarriers address the issue of heterogenous barriers by the use of different groups of particles carrying various functional modalities. Tasciotti et al. proposed a multistage delivery system composed of stage-1 mesoporous silica particles with improved deposition in the vascular endothelium, optimized for crossing the endothelial barrier through intravascular gaps or fenestrations or actively by a transcytotic mechanism, loaded with one or more types of stage-2 nanoparticles. The payload of drug/diagnostic agent-loaded nanoparticles optimized for improved interaction with various cancer cells, including lung cancer cells, is released over time at the tumor site, showing facilitated diffusion through tumor tissue due to their smaller size and specific surface engineering [111]. Wong et al. developed a multistage system with facilitated tumor diffusive transport composed of 100 nm gelatin nanoparticles, capable of releasing 10 nm NPs from their surface, triggered by protease degradation after tumor homing by the EPR effect [112]. Mesoporous silica vesicles (MSVs; dav = 3 μm) with high affinity to tumor vasculature were also described by Blanco et al. as a platform for the triggered release of various therapeutic nanoscale vectors (liposomes, gold nanoshells, and microbots) and imaging contrast nanoparticles (quantum dots and iron oxide), after homing into the tumor environment [113]. Xu et al. described an injectable nanoparticle generator (iNPG) showing substantial natural tumor tropism designed as aminopropyltriethoxysilane (APTES)-functionalized nanoporous silica particles loaded with a poly(ʟ-glutamic acid) pH-cleavable linker–doxorubicin conjugate, which self-assembles into NPs after its release from the iNPG [114]. Li et al. designed a multistage nanocarrier for NSCLC targeting, composed of icotinib-loaded amphiphilic chitosan micelles with hyaluronic acid–doxorubicin NPs layered by electrostatic adsorption upon the micelle surface. Hyaluronic acid was used for CD44 targeting (a receptor that is often overexpressed on the surface of lung tumor cells), as well as for the optimization of biodistribution, improved tumor homing potential, and cell internalization of the nanocarriers. Due to the enhanced tumor accumulation, reduced accumulation at the off-site targets, and same-place/same-time delivery of therapeutic concentrations of both drugs at the site of action, an optimal synergistic effect of the active substances and efficient tumor inhibition was achieved [115]. Lv et al. prepared multifunctional dendrimer nanoscale complexes composed of anti-EGFR aptamer-modified poly(amidoamine) (PAMAM) loaded with erlotinib and chloroquine (CQ) for NSCLC treatment. These cationic nanoparticles showed high condensation capacity for survivin-small hairpin RNA (survivin-shRNA), which was trapped by electrostatic interactions in the cavity between several assembled nanoparticles (AP/ES+CQ NPs; AP = amine-terminated PAMAM dendrimers modified with anti-EGFR aptamer; ES = erlotinib and survivin-shRNA; Figure 2, Figure 3). The nanocomplexes demonstrated pH-dependent selective survivin-shRNA release in the acidic environment after endosomal escape and disassembly to single PAMAM nanoparticles showing continuous release of erlotinib and chloroquine. Chloroquine has a dual effect on the efficacy. It improves vascular barrier integrity and together with PAMAM, facilitates endosomal escape. Down-regulation of survivin reverses EGFR TKI resistance in T790M mutant NSCLC cells and sensitizes the tumor to erlotinib. The synergy of survivin and EGFR downregulation coupled with decreased angiogenesis results in significant inhibition of proliferation and improved induction of apoptosis [116].

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Figure 2: (A) Gefitinib-loaded gelatin-A NPs functionalized with a cetuximab-siRNA conjugate (Ab-SiRNA-GelGEF NPs). 1: Gelatin-A nanoparticle, 2: cetuximab–KRASG12C-specific siRNA conjugate, and 3: gefitinib [117]. (B) Multifunctional dendrimer nanocomplex. 1: poly(amidoamine) (PAMAM) nanoparticle, 2: erlotinib, 3: anti-EGFR aptamer, 4: chloroquine, and 5: survivin sh-RNA trapped in the cavity between PAMAM nanoparticles structured together in a dendrimer nanocomplex (AP/ES+CQ NPs; AP = amine-terminated PAMAM dendrimers modified with anti-EGFR aptamer; ES = erlotinib, and survivin-shRNA) [116]. (C) Cetuximab–cationic gelatin–specific siRNA delivery system (CTB-cGel-siRNA conjugate). 1: Cetuximab, 2: cationic gelatin, and 3: KRASG12C-specific siRNA [118].

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Figure 3: Nanotools for reversal of EGFR TKI resistance by RNAi. Favorable cell internalization was mediated by anti-EGFR aptamer/EGFR interaction (AP/ES+CQ) and cetuximab–EGFR interaction (Ab-siRNA-GelGEF NPs; CTB-cGel-siRNA conjugate). Efficient transfection was enabled by endosomal escape facilitated by (i) endosomal buffering and the proton sponge effect of chloroquine and PAMAM dendrimer (AP/ES+CQ), (ii) proton sponge effect of Ab-siRNA-GelGEF NPs, and (iii) proton sponge effect the CTB–cationic gelatin–siRNA conjugate. 1: Early endosome and 2: late endosome. This figure contains a modified version of “Liposome” by rafeequemv1 CC BY 3.0. This figure contains a modified version of "Antibodies IgG" by LucasPresoto is licensed under CC BY 4.0.

Biomimetic drug delivery systems: The natural tropism of biomimetic materials for improved tissue localization has been proven to be a valuable tool in lung cancer targeting. Anselmo et al. evaluated the cell hitchhiking approach in targeting using red blood cell–polystyrene NP (200 and 500 nm) complexes (RBCsNP complexes) [119]. Compared to the free NPs, the delivery of RBCsNP complexes to the lungs, that is, the first capillary bed downstream of the IV injection of the NPs, was five-fold increased, which makes RBCsNP complexes very useful for lung tissue targeting. Once in the lung microcirculation, the RBC-bound NPs are mechanically detached from the RBCs when the RBCs are squeezed through the tiny capillaries of the air–blood barrier and transferred to the endothelium by nonspecific interactions. When decorated with vascular endothelium-specific ligands, as in the case of RBCs anti-PECAM mAb-coated polystyrene NPs, the complex showed a 760-fold increase in the lung-to-liver distribution ratio compared to nonspecific NPs. Different studies envision RBCsNPs complexes as a highly performant platform for augmented NP localization, which can be easily translated to drug delivery systems for lung and brain targeting

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