Nanoparticle-Mediated Delivery of STAT3 Inhibitors in the Treatment of Lung Cancer

Lung cancer is a common malignancy worldwide, with high morbidity and mortality. Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor that not only regulates different hallmarks of cancer, such as tumorigenesis, cell proliferation, and metastasis but also regulates the occurrence and maintenance of cancer stem cells (CSCs). Abnormal STAT3 activity has been found in a variety of cancers, including lung cancer, and its phosphorylation level is associated with a poor prognosis of lung cancer. Therefore, the STAT3 pathway may represent a promising therapeutic target for the treatment of lung cancer. To date, various types of STAT3 inhibitors, including natural compounds, small molecules, and gene-based therapies, have been developed through direct and indirect strategies, although most of them are still in the preclinical or early clinical stages. One of the main obstacles to the development of STAT3 inhibitors is the lack of an effective targeted delivery system to improve their bioavailability and tumor targetability, failing to fully demonstrate their anti-tumor effects. In this review, we will summarize the recent advances in STAT3 targeting strategies, as well as the applications of nanoparticle-mediated targeted delivery of STAT3 inhibitors in the treatment of lung cancer.


Introduction
Lung cancer is one of the most common malignancies and the leading cause of cancerrelated deaths, with an estimated 2.09 million newly diagnosed cases in both genders and 1.76 million deaths worldwide [1]. Small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) are the two main pathological types of lung cancer, and NSCLC accounts for approximately 80% to 85% of all cases. NSCLC has three main subtypes, including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma [2]. Treatment of lung cancer varies greatly depending on the type and stage of the disease, including traditional chemotherapy, radiotherapy, targeted therapy, and immunotherapy. In recent years, targeted therapy has emerged as an important therapeutic strategy for the management of NSCLC. For example, epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) and anaplastic lymphoma kinase (ALK)/c-ros oncogene 1 (ROS1) inhibitors have replaced chemotherapy as the first-line treatment of lung cancer [3]. Although targeted therapies are initially effective, acquired drug resistance is usually inevitable due to cancer-driven genetic alterations, epigenetic alterations, and tumor heterogeneity [4]. Immunotherapy, such as immune checkpoint blockade (ICB), is a new treatment strategy that may improve the survival of lung cancer. However, the clinical benefits of ICB in the treatment of advanced lung cancer have proven to be limited and unsatisfactory, with an overall response rate (ORR) of approximately 10-20% [5]. Therefore, there is an urgent need to identify alternative strategies to improve existing treatments or to provide new treatments.
Signal transducer and activator of transcription 3 (STAT3), a key component of the Janus kinase (JAK)-STAT pathway, is found aberrantly activated in the majority of NSCLC patients [6]. STAT3 can be phosphorylated by multiple cytokines, interferons, and growth factors, and the activated STAT3 can be transferred to the nucleus and bind to the promoter and enhancer regions of target genes to modulate gene transcription [7]. Multiple studies have proven that increased phosphorylated STAT3 (pSTAT3) is usually associated with cell proliferation, invasion, and angiogenesis, leading to tumor progression, metastasis, drug resistance, and immune escape [8]. Moreover, the existence of a subpopulation of cells in tumors called cancer stem cells (CSCs) or tumor initiating cells (TICs), responsible for drug resistance, tumor metastasis, and relapse, is also reported to correlate with increasing levels of STAT3 activation [9]. Thus, targeting the STAT3 signaling pathway has emerged as a promising therapeutic strategy for lung cancers.
A variety of nanocarriers have been recently developed to deliver poorly water-soluble drugs, thus improving their bioavailability and targeting capability. Nanoparticles (NPs) with particle sizes of 10-100 nm can increase drug enrichment in tumor tissue and reduce its distribution in normal tissue through enhanced permeability and retention (EPR) effect and active targeting strategies (e.g., decoration of specific ligands) [10]. NPs-based drug delivery systems, such as inorganic NPs, polymeric NPs, micelles, dendrimers, and liposomes, have shown great potential in the diagnosis, imaging, and treatment of cancer. Some of these nanocarriers have been successfully used in the clinic for drug delivery, such as Abraxane ® (albumin-bound paclitaxel) and Doxil ® (liposomal doxorubicin).
In this review, we discuss and summarize the importance of the STAT3 signaling pathway in the progress of lung cancer and the recent progress in STAT3 targeting strategies. In addition, we also introduce the potential application of NPs-based targeted delivery of STAT3 inhibitors in the treatment of cancers, including lung cancer.

Role of STAT3 in Lung Cancer Cells
It is believed that multiple signaling pathways are related to the initiation and metastasis of lung cancer. STAT3, one of the numerous transcription factors among those signal pathways, is closely related to the occurrence and development of cancer and is considered a promising target for cancer treatment (Figure 1). Among the seven members of the STAT family (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6), STAT3 has attracted much attention because of its vital function in cell differentiation [11]. The structure of STAT3 is comprised of six functionally conserved domains, including the amino-terminal domain (NH2), coiled-coil domain (CCD), DNA-binding domain (DBD), linker domain, SRC homology 2 (SH2) domain, and carboxyl-terminal transactivation domain (TAD) [12]. Among them, the SH2 domain plays an important role in STAT3 activation by engaging the dimerization of two phosphorylated STAT3 monomers [13]. The activation of STAT3 can be induced by nearly 40 different polypeptide ligands, such as cytokines, growth factors, carcinogens, and environmental stress [11]. It is mainly activated by the direct phosphorylation of tyrosine (705) and serine (727) residues to form a STAT3 dimer, which is subsequently translocated into the nucleus to regulate target genes [12]. Under normal circumstances, the activation of STAT3 is negatively regulated by suppressors of cytokine signaling (SOCS) proteins, protein inhibitors of activated STATs (PIAS) proteins, and protein tyrosine phosphatases (PTPases) and is involved in mammary gland development and embryogenesis through interferon-gamma (IFN-γ) signaling, the cell cycle, and apoptosis [13]. However, the constitutive activation of STAT3 is Involved in many cellular processes, including survival, proliferation, invasion, angiogenesis, metastasis, and immunosuppression, all of which favor tumor initiation and progression [12,14].

Therapeutic Strategies Targeting STAT3
Given the important role of abnormally activated STAT3 in the occurrence and progression of lung cancer, targeted inhibition of STAT3 may become an effective therapeutic strategy for lung cancer. The ways targeting STAT3 may be direct or indirect, mainly including non-oncology drugs, natural products and derivatives, small-molecule inhibitors, nucleotide-based therapeutics, and agents regulating STAT3 upstream genes (Table 1).

Non-Oncology Drugs
Several known drugs that were not initially used in cancer treatment have shown significant anti-cancer activity by targeting the STAT3 signaling pathway. Antiparasitic drugs have been demonstrated to successfully inhibit the activation of the STAT3 pathway, selectively impairing the growth of lung cancer cells and eliciting lethal effects both in vitro and in vivo [28,[35][36][37]. Moreover, recent studies have shown that niclosamide (an antihelminthic drug) and its derivative (HJC0152) enhance the efficacy of immunotherapy in NSCLC by blocking the binding of pSTAT3 to the PD-L1 promoter, thus downregulating the expression of PD-L1 [28,35]. Other classical drugs exhibited anti-cancer effects by direct or indirect targeting STAT3. A recent study reported that high doses of acetaminophen (AAP), a commonly used antipyretic and analgesic drug, exhibited anti-CSCs activity in lung cancer and melanoma cells by directly binding STAT3 with an affinity in the low micromolar range [38]. In addition, dihydroartemisinin (DHA), a semisynthetic derivative of the herbal antimalarial drug artemisinin, can effectively inhibit STAT3 phosphorylation, downregulate myeloid cell leukemia-1 (Mcl-1) and survivin, and enhance ABT-263 (Bcl-2 inhibitor)-induced cytotoxicity [39]. Therefore, non-oncology drugs capable of targeting STAT3 may have therapeutic potential in lung cancer.

Natural Compounds and Derivatives
Some researchers have explored the effects of natural compounds to develop anti-CSCs therapy. Curcumin (CUR) and its derivatives, one of the most commonly used natural compounds for cancer therapy, have been reported to inhibit the phosphorylation of STAT3 and its downstream genes, thereby inhibiting angiogenesis and tumor growth [40,41]. Terpenoids, widely distributed in nature, have been extensively studied for their biological activities, including anti-cancer effects. For example, Cucurbitacin I (a typical terpenoid) might inhibit the phosphorylation of STAT3 and enhance the phosphorylation of STAT1 in A549 lung adenocarcinoma cells by disrupting actin filaments [42]. Another natural triterpenoid extracted from Anemone Raddeana Regel, Raddeanin A, was able to inhibit the expression of pSTAT3 and STAT3, reduce the mitochondrial membrane potential, and promote apoptosis in A549 and H1299 lung cancer cells [43]. Other terpenoids, such as Ginkgolide C (GGC) and eupatolide isolated from Ginkgo biloba (Ginkgoaceae) leaf and Inula helenium, respectively, could effectively attenuate the phosphorylation of There is evidence that abnormally activated STAT3 has been found in a variety of cancers, including lung cancer, which leads to the inactivation of apoptotic pathways and the resistance of cancer cells to radiotherapy and chemotherapy [15][16][17]. The aberrant activation of STAT3 is usually associated with poor tumor differentiation, advanced clinical stage, lymph node metastasis, and drug resistance of lung cancer [13]. Recent studies have shown that microRNAs could promote proliferation, migration, invasion, anti-apoptosis, and angiogenesis of lung cancer cells by activating STAT3 [18][19][20]. Meanwhile, microRNAs could also inhibit epithelial-mesenchymal transition (EMT) and reverse drug resistance by inhibiting the STAT3 signaling pathway, thereby suppressing tumor growth and improving the therapeutic effect of chemotherapeutic drugs [21,22]. In addition, alterations of STAT3 activity using genetic and/or pharmacological methods may be effective in regulating immunosuppression in lung cancer cells, affecting the outcome of cancer immunotherapy [23]. Studies have shown that dysregulated IL-6 or JAK2 can reprogram the STAT3 pathway in metastatic tumor cells, induce recruitment of myeloid-derived suppressor cells (MDSCs) and polarized macrophages, and repress the infiltration of CD8+ T cells to evade host immunity in NSCLC [24][25][26]. On the contrary, inhibition of STAT3 can downregulate the expression of programmed cell death 1 ligand 1 (PD-L1) and enhance the infiltration of T cells into tumor tissues, exhibiting synergistic effect in combination with programmed cell death protein 1 (PD-1)/PD-L1 blockade [27,28].
It has been reported that STAT3 activation could not only stimulate a series of cascades associated with the development and progression of lung cancer but also serve as a key mediator to regulate the characteristics of lung cancer stem cells (LCSCs) [29]. Studies have shown that STAT3 can be phosphorylated by receptor tyrosine kinases (RTKs) such as EGFR and insulin-like growth factor 1 receptor (IGF1R), or non-RTKs such as c-Src and JAK, thereby maintaining the EMT-associated, CSC-like properties in cells [30][31][32]. For example, it was found that c-Src/IGF1R-mediated STAT3 activation was regulated by Tescalcin (TESC), which enhanced the expression of acetaldehyde dehydrogenase 1 (ALDH1) and thus reinforced CSC-like and radiation-resistant properties [11]. Another study showed that the elevation and activation of the Aryl hydrocarbon receptor (AhR) in NSCLC cells could induce JAK2/STAT3 phosphorylation, while the inhibition of JAK2/STAT3 signaling by pharmacologic approaches can attenuate the AhR-mediated stemness effects of NSCLC cells, indicating that the JAK2/STAT3 pathway plays a vital role in AhR-regulated NSCLC stemness [14], and targeting STAT3 has become a promising strategy for lung cancer treatment.
It is worth noting that STAT3 has also been reported to have a potential role in tumor suppression in addition to promoting the development of lung cancer. The activation of STAT3 in mice may reduce the occurrence and malignant progression of Kras (G12D)driven lung adenocarcinoma by controlling the expression of interleukin 8 (IL-8) induced by nuclear factor kappa-B (NF-κB), and inhibiting IL-8-mediated myeloid tumor infiltration and tumor vascularization [33]. Another study indicated that STAT3 might prevent the initiation of lung cancer by maintaining pulmonary homeostasis under oncogenic stress, but it may also facilitate the progression of lung cancer by promoting the growth of cancer cells at the same time [34]. Such contradictory conclusions make targeting STAT3 a complicated process, and the role of STAT3 in tumor initiation and progression must be carefully studied.

Therapeutic Strategies Targeting STAT3
Given the important role of abnormally activated STAT3 in the occurrence and progression of lung cancer, targeted inhibition of STAT3 may become an effective therapeutic strategy for lung cancer. The ways targeting STAT3 may be direct or indirect, mainly including non-oncology drugs, natural products and derivatives, small-molecule inhibitors, nucleotide-based therapeutics, and agents regulating STAT3 upstream genes (Table 1).

Non-Oncology Drugs
Several known drugs that were not initially used in cancer treatment have shown significant anti-cancer activity by targeting the STAT3 signaling pathway. Antiparasitic drugs have been demonstrated to successfully inhibit the activation of the STAT3 pathway, selectively impairing the growth of lung cancer cells and eliciting lethal effects both in vitro and in vivo [28,[35][36][37]. Moreover, recent studies have shown that niclosamide (an antihelminthic drug) and its derivative (HJC0152) enhance the efficacy of immunotherapy in NSCLC by blocking the binding of pSTAT3 to the PD-L1 promoter, thus downregulating the expression of PD-L1 [28,35]. Other classical drugs exhibited anti-cancer effects by direct or indirect targeting STAT3. A recent study reported that high doses of acetaminophen (AAP), a commonly used antipyretic and analgesic drug, exhibited anti-CSCs activity in lung cancer and melanoma cells by directly binding STAT3 with an affinity in the low micromolar range [38]. In addition, dihydroartemisinin (DHA), a semisynthetic derivative of the herbal antimalarial drug artemisinin, can effectively inhibit STAT3 phosphorylation, downregulate myeloid cell leukemia-1 (Mcl-1) and survivin, and enhance ABT-263 (Bcl-2 inhibitor)-induced cytotoxicity [39]. Therefore, non-oncology drugs capable of targeting STAT3 may have therapeutic potential in lung cancer.

Natural Compounds and Derivatives
Some researchers have explored the effects of natural compounds to develop anti-CSCs therapy. Curcumin (CUR) and its derivatives, one of the most commonly used natural compounds for cancer therapy, have been reported to inhibit the phosphorylation of STAT3 and its downstream genes, thereby inhibiting angiogenesis and tumor growth [40,41]. Terpenoids, widely distributed in nature, have been extensively studied for their biological activities, including anti-cancer effects. For example, Cucurbitacin I (a typical terpenoid) might inhibit the phosphorylation of STAT3 and enhance the phosphorylation of STAT1 in A549 lung adenocarcinoma cells by disrupting actin filaments [42]. Another natural triterpenoid extracted from Anemone Raddeana Regel, Raddeanin A, was able to inhibit the expression of pSTAT3 and STAT3, reduce the mitochondrial membrane potential, and promote apoptosis in A549 and H1299 lung cancer cells [43]. Other terpenoids, such as Ginkgolide C (GGC) and eupatolide isolated from Ginkgo biloba (Ginkgoaceae) leaf and Inula helenium, respectively, could effectively attenuate the phosphorylation of STAT3 and its upstream kinases, demonstrating significant inhibition of NSCLC tumor growth [44,45].
Although many natural compounds have shown therapeutic effects on lung cancer by inhibiting STAT3 activation, their molecular mechanisms remain to be further studied. Ma et al. investigated the anti-cancer effect and underlying mechanisms of Proscillaridin A (PSD-A), a cardiac glycoside component of Urginea maritima. They found that PSD-A was able to inhibit both constitutive and inducible STAT3 activations and reduce STAT3-DNA binding activity, which was associated with increased expression of SH2 domain-containing protein tyrosine phosphatase 1 (SHP-1), decreased phosphorylation of Src, and binding of PSD-A with the STAT3 SH2 domain. This was the first in-depth illustration of the molecular mechanism by which PSD-A inhibits STAT3 [46].
Since the aberrant activation of STAT3 was found to be related to EGFR-TKI resistance in lung cancer, some natural compounds were proved to successfully reduce cell growth and induce apoptosis in EGFR-TKI-resistant NSCLC cells through the suppression of STAT3 activity, showing their potential as novel therapeutics for lung cancer patients with EGFR-TKI resistance [47]. Interestingly, pterostilbene alone, which is isolated from Pterocarpus marsupium (PM) heartwood, was not able to induce anti-proliferative effects in EGFR-mutated NSCLC cells. However, pterostilbene plus osimertinib reversed osimertinibinduced phosphorylation of STAT3, Yes-associated protein 1 (YAP1), and CUB domaincontaining protein-1 (CDCP1), abrogating the resistance pathways activated by single osimertinib treatment in EGFR-mutated NSCLC [48].

Small Molecule Inhibitors
Recently, a lot of efforts have been made to develop specific and potent STAT3 small molecule inhibitors. The SH2 domain plays a pivotal role in the STAT3 signaling cascade, so targeting the SH2 domain of STAT3 will prevent the dimerization and transcriptional activity of STAT3 [49]. Therefore, Rangappa et al. performed high-throughput virtual screening using a cheminformatics platform to search for STAT3 inhibitors and identified 2-Amino-6-[2-(Cyclopropylmethoxy)-6-Hydroxyphenyl]-4-Piperidin-4-yl Nicotinonitrile (ACHP) as an effective inhibitor. It was found that ACHP interacted with the SH2 domain of STAT3 and significantly inhibited the phosphorylation of STAT3 at Tyr705, resulting in the apoptosis of NSCLC cells [50]. Another novel STAT3 inhibitor, W2014, characterized by the core structure of imidazopyridine, was able to occupy the sub-pockets of the SH2 domain and bind to STAT3 protein with high affinity. W2014 not only exhibited potent anti-tumor activities but also sensitized drug-resistant NSCLC cells to gefitinib by inhibiting aberrant STAT3 signaling in vitro and in vivo [49]. Moreover, Napabucasin (BBI608) and LL1, two newly developed small molecule inhibitors, were able to significantly inhibit the self-renewal of LCSCs by targeting the SH2 domain of STAT3 [51][52][53][54].
In addition to chemotherapeutic drugs and EGFR-TKI inhibitors, the combination of STAT3 inhibitors and other small molecular inhibitors has also achieved promising therapeutic effects on lung cancer. The STAT3 inhibitors combined with rapidly accelerated fibrosarcoma (RAF) inhibitor or ALK inhibitor effectively suppress the survival of Kras-mutated or ALK-rearranged lung cancer cells by abrogating the activation of mitogen-activated extracellular signal-regulated kinase (MEK)/extracellular regulated protein kinases (ERK) signaling pathway [55,56].
In addition to the SH2 domain, there has been a growing interest in developing specific inhibitors that can target the DBD of STAT3. A new small molecule, (E)-2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl) phenol (MMPP), was found to regulate cell cycle and apoptosis-related genes by directly binding to the hydroxyl residue of threonine 456 in the DBD of STAT3, leading to G1-phase cell cycle arrest and apoptosis. In addition, MMPP showed comparable or better anti-tumor activity than docetaxel or cisplatin [57].
Although STAT inhibitors, alone or in combination with other drugs, have achieved promising effects, no STAT3 inhibitor has been approved for the treatment of lung cancer, likely due to their low bioavailability and off-target toxicities. Therefore, the exploration of more efficacious STAT3 inhibitors and other effective STAT3 targeting strategies should be inspired.

Therapeutic RNA Molecules
In recent decades, nucleic acid-based therapeutics have been extensively studied because of their ability to target a broader group of proteins and their ability to be engineered to expand their applicability compared with small molecular drugs [58]. Therapeutic RNA molecules, including antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), and long intergenic non-coding RNA (lncRNAs), have been developed for STAT3-targeted lung cancer therapy. ASOs are short single-or double-stranded oligonucleotides that complement the target mRNA they hybridize to modulate protein expression [58]. Recently, an oligonucleotide STAT3 decoy (CS3D) was used to treat mice previously exposed to the tobacco carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which contained a sequence of double-stranded STAT3 DNA response elements and interrupted the STAT3 signaling by binding to STAT3 dimers, preventing them from initiating transcription at the native DNA binding site of STAT3. CS3D successfully reduced oncogenic signaling in the airway epithelium by reducing the expression of STAT3 and its downstream genes and favored a lung microenvironment with alleviated immunosuppression by reducing pulmonary M2 macrophages and MDSCs cells [59].
siRNA are short, double-stranded RNA molecules which could silence the expression of target mRNAs through an RNA-induced silencing complex (RISC) with highly efficiency and specificity. siRNA-based therapy has gained much attention in cancer treatment, and many siRNA-based drugs have already entered clinical trials [58]. For lung cancer, many studies have demonstrated that using siRNA to suppress STAT3 expression could successfully repress cancer development, reverse drug resistance (chemotherapeutic drugs and TKIs), and improve immunotherapy by inhibiting proliferation, migration, and ROS production of cancer cells as well as decreasing the expression of PD-L1 on immune cells [60][61][62].
Double-strand miRNAs, which mimic naturally occurring miRNAs, could replenish the function of altered miRNAs by binding to the 3 -untranslated region (3 -UTR) of target mRNAs, leading to mRNAs degradation and suppression [58]. Recent studies showed that transfecting cells with miRNAs not only inhibited the proliferation of NSCLC cells and reversed cisplatin resistance but also activated CD8 + T cells in a STAT3/PD-L1-dependent manner by binding to STAT3 3 -UTR [21,63].
In addition, lncRNAs have been demonstrated to regulate various stages of gene expression, positively or negatively correlated with the clinical outcomes of NSCLC. Over-or underexpression of target lncRNA that regulate the phosphorylation of STAT3 significantly inhibited the proliferation of NSCLC cells and reversed the resistance of afatinib to NSCLC cells [64,65].
The specificity and effectiveness of therapeutic RNA molecules make them popular therapeutic agents for diseases such as lung cancer. However, the rapid degradation of RNA molecules during circulation in vivo has become a key issue in RNA therapeutics. The most widely used transfection reagents are cationic lipid (Lipo 2000) or polymer (polyethylenimine), which might induce high cytotoxicity. Therefore, designing safe and effective delivery methods to protect RNA molecules from degradation and improve in vivo transfection efficiency is very important in RNA-based cancer therapy.

Agents Regulating the STAT3 Upstream Gene
Since STAT3 activity was found to be associated with many signal pathways, targeting its upstream regulators may be an alternative strategy to inhibit STAT3. Recently, cyclin-dependent kinase (CDK) family members, which were supposed to regulate the cell cycle, were demonstrated to promote RNA synthesis in genetic processes by regulating the STAT3 signaling pathway [66]. Therefore, CDK inhibitors successfully reduced the expressions of pSTAT3 and transcriptional target genes such as cyclin B1 and IL-6, leading to apoptosis of lung squamous cell carcinoma (LUSC) cells and inhibition of tumor growth in patient-derived xenograft (PDX) models [66,67]. In addition, the DNA damage-induced apoptosis suppressor (DDIAS) has been reported to promote the progression of lung cancer through the regulation of the STAT3 pathway, and DDIAS inhibitors were found to suppress the activation of c-Jun NH(2)-terminal kinase (JNK) or interfere with DDIAS/STAT3 binding [68,69]. Other targets, such as Ataxia Telangiectasia Mutated (ATM), have been reported to upregulate PD-L1 expression via the JAK1, 2/STAT3 pathway, mediating the cisplatinresistance in lung cancer cells [70]. Chen and colleagues found that the inhibition of ATM using specific ATM inhibitor CP466722 or siRNA reduced JAK/STAT3 signaling and PD-L1 expression, thus inhibiting the EMT and metastatic potential of cisplatin-resistant lung cancer cells [70]. A newly discovered heat shock protein 90 (Hsp90) inhibitor, NCT-80, bound directly to the C-terminal ATP-binding pocket of Hsp90, disrupted the interaction between Hsp90 and STAT3, and degraded the STAT3 protein, thereby reducing CSC-like phenotypes of NSCLC cells and their sublines with acquired resistance to anti-cancer drugs [71].
In addition, STAT3 is also negatively regulated by several regulators. Overexpression of miR-218 exhibited an anti-CSCs effect in lung cancer by directly targeting the 3'-UTR of mRNAs of the IL-6 receptor and JAK3 gene [22]. In addition, Herman et al. found that protein tyrosine phosphatase receptor-T (PTPRT) negatively regulated STAT3 function by dephosphorylating STAT3 (Tyr705), and the silence of PTPRT using siRNA in NSCLC led to the increased expression of pSTAT3 (Tyr705) and STAT3 target genes such as cyclin D1 and Bcl-XL [72].

Nanoparticle-Based Delivery of STAT3 Inhibitors in the Treatment of Lung Cancer
Although the above STAT3 inhibitors have shown good therapeutic potential in preclinical trials of cancers such as lung cancer, they still face problems such as poor solubility and bioavailability, low tumor targeting, off-target toxicity, easy degradation (therapeutic RNA molecules), and drug resistance during the process of clinical translation.
Nanoparticles (NPs) are generally believed to increase drug concentrations in cancer cells, protect RNA from degradation, and reduce toxicity to normal cells through passive (enhanced permeability and retention effect, EPR effect) or active targeting strategies. A variety of nanocarriers, including lipid nanoparticles (LNPs)/liposomes, inorganic NPs, polymeric micelles, and extracellular vesicles (EVs), have shown great potential in the diagnosis and treatment of a variety of cancers that rely primarily on the oncogenic STAT3 signaling pathway ( Figure 2). However, the development of nanocarriers for lung cancertargeted delivery of STAT3 inhibitors is quite challenging due to several hurdles presented by the lungs' anatomy and pathology/physiology that need to be carefully addressed. The applications of NPs-mediated STAT3 inhibitors delivery in other cancers can be useful as guidance (Table 2). In addition, the challenges in lung cancer-specific drug delivery have also been summarized.

Nanoparticle-Based Delivery of STAT3 Inhibitors in the Treatment of Lung Cancer
Although the above STAT3 inhibitors have shown good therapeutic potential in preclinical trials of cancers such as lung cancer, they still face problems such as poor solubility and bioavailability, low tumor targeting, off-target toxicity, easy degradation (therapeutic RNA molecules), and drug resistance during the process of clinical translation.
Nanoparticles (NPs) are generally believed to increase drug concentrations in cancer cells, protect RNA from degradation, and reduce toxicity to normal cells through passive (enhanced permeability and retention effect, EPR effect) or active targeting strategies. A variety of nanocarriers, including lipid nanoparticles (LNPs)/liposomes, inorganic NPs, polymeric micelles, and extracellular vesicles (EVs), have shown great potential in the diagnosis and treatment of a variety of cancers that rely primarily on the oncogenic STAT3 signaling pathway ( Figure 2). However, the development of nanocarriers for lung cancertargeted delivery of STAT3 inhibitors is quite challenging due to several hurdles presented by the lungs' anatomy and pathology/physiology that need to be carefully addressed. The applications of NPs-mediated STAT3 inhibitors delivery in other cancers can be useful as guidance (Table 2). In addition, the challenges in lung cancer-specific drug delivery have also been summarized.

LNPs/Liposomes
LNPs, typically surrounded and stabilized by lipid bilayers with an aqueous (liposomes), oil, solid, or amorphous core (for nucleic acid delivery), are one of the most common nanoparticle formulations used to deliver anti-cancer drugs or genes because of their stable drug encapsulation and enhanced delivery efficiency [73]. The initial success of several liposome/LNPs-based drugs has fuelled further clinical investigations [74]. The im-

LNPs/Liposomes
LNPs, typically surrounded and stabilized by lipid bilayers with an aqueous (liposomes), oil, solid, or amorphous core (for nucleic acid delivery), are one of the most common nanoparticle formulations used to deliver anti-cancer drugs or genes because of their stable drug encapsulation and enhanced delivery efficiency [73]. The initial success of several liposome/LNPs-based drugs has fuelled further clinical investigations [74]. The improved therapeutic outcomes of Doxil ® (a liposome formulation of doxorubicin) and the recent approval by the U.S. Food and Drug Administration (FDA, Silver Spring, MD, USA) for LNP-loaded mRNA vaccines used to prevent COVID-19 have made LNPs a promising drug delivery system for various diseases [75]. Due to uniform particle size distribution, LNPs have exhibited superior biodistribution in vivo, which has established them as outstanding drug carriers for lung cancer treatment [76]. Moreover, since the main components of LNPs (e.g., phospholipids and cholesterol) are very similar to pulmonary surfactants in mammals, several highly biocompatible and biodegradable inhaled LNPs are promising candidates for pulmonary drug delivery [77]. Studies indicated that inhaled LNPs could increase the therapeutic effect of the drug and decrease the systemic toxicity simultaneously because they are able to restrict the drug effect in the pulmonary system for a prolonged time [78].
Villanueva and colleagues reported the potential of STAT3 decoy-loaded cationic lipid microbubbles (STAT3-MB) combined with ultrasound-targeted microbubble cavitation (UTMC) in the treatment of head and neck squamous cell carcinoma (HNSCC). The STAT3 decoy was loaded via charge-charge interaction. The formed STAT3-MB combined with UTMC treatment promoted the delivery of cell-impermeant oligonucleotides exclusively to sites exposed to the ultrasound beam, significantly inhibiting tumor growth and prolonging the survival in CAL33 tumor-bearing mice compared to the negative control groups, which was associated with the downregulation of the expression of target genes Bcl-xL and Cyclin-D1 at the RNA transcription and protein levels [79]. In order to achieve better tumor targeting ability and efficient cell transfection, surface modification of liposomes/LNPs by covalently conjugating specific ligands has gained much attention. A study reported that α5β1 integrin receptor selective liposomes prepared via conventional thin-film hydration method containing RGDK-lipopeptide simultaneously delivered a small molecule STAT3 inhibitor (WP1066) and STAT3 siRNA to brain tumors. It was found that WP1066/STAT3-siRNA-loaded liposomes were internalized in glioblastoma cells via integrin α5β1 receptors and selectively accumulated in brain tissues of glioblastoma-bearing mice, thus significantly improving the overall survival of orthotopically-established glioblastoma-bearing mice [80]. Solid lipid nanoparticles (SLNs) are another promising delivery system for small molecules and genes due to their good biocompatibility and physical stability. A recent study developed and evaluated the use of cationic SLNs for delivery of RNA interference (RNAi)-mediating plasmid DNA to downregulate STAT3 in cisplatin-resistant lung cancer cells. Cationic SLNs were prepared by a modified hot microemulsion method, and these cSLN:plasmid DNA complexes successfully encoded anti-STAT3 short hairpin RNA, reduced STAT3 expression, and improved the sensitivity of the cisplatin-resistant Calu1 cell line to cisplatin (Figure 3) [81].
Since STAT3 activation is associated with immune suppression, inhibition of STAT3 activation by different strategies has shown promising results in cancer immunotherapy. For example, ablation of STAT3 in mice could induce potent anti-tumor immunity by increasing the production of IL-12 and tumor necrosis factor α (TNFα), reducing the production of IL-10, and inducing M1-like reprogramming of murine macrophages [82]. To this end, Møller et al. reported a type of long-circulating liposomes (CA-LCL-αCD163), which were passively inserted lipidated CD163 (markers of M2-polarized macrophages) into the liposome lipid bilayer and packed with STAT3 inhibitor corosolic acid (CA). The CA-LCL-αCD163 liposomes were able to target macrophages with high CD163 expression, inhibit IL-6-induced STAT3 activation, and induce the production of pro-inflammatory cytokines, resulting in reprogramming tumor-associated macrophages (TAMs) from a tumor-supporting (M2-like) phenotype towards a tumoricidal (M1-like) phenotype [82].
Another study prepared doxorubicin-loaded, cholesterol-free CA liposomes (DOX/CALP) based on PEGylated liposomal doxorubicin (DOXIL®) by replacing its cholesterol with CA. They found that DOX/CALP displayed higher in vitro cellular uptake and tumor spheroid permeation, as well as stronger anti-tumor cytotoxicity, compared to doxorubicin-loaded cholesterol liposomes (DOX/LP). In addition, the pSTAT3 level in the DOX/CALP group was significantly suppressed, and fewer intratumoral macrophages were observed in the DOX/CALP group, further suggesting that CALP as a functional delivery nanocarrier has some advantages over classic liposomes, and hence could enhance the efficacy of chemotherapeutic drugs [83]. In addition to STAT3 inhibitory drug delivery, targeting TAMs with STAT3 siRNA-loaded LNPs to modify their function responsible for M2 polarization could also be used to reverse the tumor-promoting function of TAMs. Harashima et al. fabricated a type of pH-sensitive LNPs (CL4H6-LNPs) for targeted delivery of STAT3 siRNA to TAMs. The silencing of STAT3 and hypoxia-inducing factor 1α (HIF-1α) led to an increase in levels of infiltrated macrophages (CD11b + cells) and M1 macrophages (CD169 + cells) in the tumor microenvironment (TME), achieving novel macrophage-based cancer immunotherapy [84].
delivery, targeting TAMs with STAT3 siRNA-loaded LNPs to modify their function re-sponsible for M2 polarization could also be used to reverse the tumor-promoting function of TAMs. Harashima et al. fabricated a type of pH-sensitive LNPs (CL4H6-LNPs) for targeted delivery of STAT3 siRNA to TAMs. The silencing of STAT3 and hypoxia-inducing factor 1α (HIF-1α) led to an increase in levels of infiltrated macrophages (CD11b + cells) and M1 macrophages (CD169 + cells) in the tumor microenvironment (TME), achieving novel macrophage-based cancer immunotherapy [84].
Studies also revealed that the activation of STAT3 was strongly associated with the expression of PD-L1 in multiple cancers, and inhibition of STAT3 can reduce the expression of PD-L1, resulting in the improved therapeutic effect of checkpoint inhibitors [26]. Li et al. synthesized a novel IL-20 receptor subunit alpha (IL20RA)-targeted liposomal NP that encapsulates the STAT3 inhibitor stattic (NP-Stattic-IL20RA) to inhibit breast cancer. They demonstrated that IL20RA could promote the stemness of breast cancer cells via the JAK1-STAT3-SOX2 signaling pathway and regulate the expression of PD-L1 to modulate the immune microenvironment. NPs-Stattic-IL20RA combined with anti-PD-L1 antibody effectively inhibited the stemness of cancer cells and improved the tumor immune microenvironment, resulting in an increase in the efficacy of chemotherapy [85]. Recently, a pHresponsive liposome (Liposome-PEO, LP) loaded with apatinib (AP) and cinobufagin (CS-1) and coated with a hybrid membrane (R/C) (LP-R/C@AC NPs) was prepared for combined treatment of gastric cancer. LP-R/C@AC efficiently killed tumor cells by inhibiting the vascular endothelial growth factor receptor 2 (VEGFR2)/STAT3 pathway and reverse tumor immunosuppression by inhibiting the expression of PD-L1 and matrix metalloproteinase 9 (MMP-9), showing the dual advantages of targeting tumor cells and immune escape [86].  Studies also revealed that the activation of STAT3 was strongly associated with the expression of PD-L1 in multiple cancers, and inhibition of STAT3 can reduce the expression of PD-L1, resulting in the improved therapeutic effect of checkpoint inhibitors [26]. Li et al. synthesized a novel IL-20 receptor subunit alpha (IL20RA)-targeted liposomal NP that encapsulates the STAT3 inhibitor stattic (NP-Stattic-IL20RA) to inhibit breast cancer. They demonstrated that IL20RA could promote the stemness of breast cancer cells via the JAK1-STAT3-SOX2 signaling pathway and regulate the expression of PD-L1 to modulate the immune microenvironment. NPs-Stattic-IL20RA combined with anti-PD-L1 antibody effectively inhibited the stemness of cancer cells and improved the tumor immune microenvironment, resulting in an increase in the efficacy of chemotherapy [85]. Recently, a pH-responsive liposome (Liposome-PEO, LP) loaded with apatinib (AP) and cinobufagin (CS-1) and coated with a hybrid membrane (R/C) (LP-R/C@AC NPs) was prepared for combined treatment of gastric cancer. LP-R/C@AC efficiently killed tumor cells by inhibiting the vascular endothelial growth factor receptor 2 (VEGFR2)/STAT3 pathway and reverse tumor immunosuppression by inhibiting the expression of PD-L1 and matrix metalloproteinase 9 (MMP-9), showing the dual advantages of targeting tumor cells and immune escape [86].

Inorganic Nanoparticles
Inorganic NPs, including gold/silver NPs, mesoporous silica NPs, and magnetic NPs, have been extensively explored in cancer theranostics over the past two decades due to their advantages of facile preparation, excellent biocompatibility, and wide surface conjugation chemistry [87]. In addition, these inorganic NPs, including gold NPs and magnetic NPs with minimal toxicity, good stability, and powerful imaging properties, are widely used in lung cancer diagnosis, acting as nanoprobes in computed tomography (CT) or magnetic resonance imaging (MRI) for molecular imaging of lung cancer in the clinic [88]. However, the potential cellular toxicity and adverse effect of magnetic NPs should not be ignored; thus, the size, concentration, and exposure time must be carefully understood [89].
Gold NPs are widely used for drug delivery because of their easy synthesis, high surface volume, and functionalization [90]. A study reported the synthesis of curcumin-loaded or curcumin/paclitaxel co-loaded gold NPs for the treatment of triple-negative breast cancer [90]. The results demonstrated that gold NPs loaded with curcumin with/without paclitaxel exhibited anti-cancer and anti-metastatic properties by downregulating the expression of STAT3 and downstream genes (MMPs, VEGF, and Cyclin D) [90]. Another study reported the development of layer-by-layer assembled gold NPs (LbL-AuNP) containing anti-STAT3 siRNA and imatinib mesylate (IM) to treat melanoma. Notably, LbL-AuNP prepared using sequential adsorption of natural polyelectrolytes, chitosan, and sodium alginate resulted in a positive charged surface, which could be utilized for iontophoresis therapy to enhance skin penetration in the local treatment of melanoma at an early stage. The topical iontophoretic application of dual-drug loaded LbL-AuNP significantly inhibited tumor growth and STAT3 expression in mouse melanoma models, compared with the control treatments [91].
Iron oxide is another commonly used material for the synthesis of inorganic NPs. Superparamagnetic iron oxide nanoparticles (SPIONs) composed of superparamagnetic magnetite (Fe 3 O 4 ) or maghemite (Fe 2 O 3 ) at certain sizes are considered to be highly efficient nanocarriers for anti-cancer therapeutics [92]. However, non-specific binding to serum proteins and rapid clearance from the bloodstream are major challenges for the application of SPIONs. Niaragh and colleagues coated SPIONs with positively charged chitosan derivatives such as trimethyl chitosan (TMC) and thiolated chitosan (ChT) to improve the stability of SPIONs and siRNA loading potential. In addition, Hyaluronate (HA) and TAT peptide were conjugated on the surface of SPIONs to facilitate their tumor tissue penetration and tumor cellular uptake. These HA-conjugated TAT-chitosan-SPION (SPION-TMC-ChT-TAT-H) NPs successfully co-delivered STAT3/HIF-1α siRNA, and significantly inhibited STAT3/HIF-1α gene-driven tumor proliferation, migration, and metastasis [93].
In addition to metal NPs, inorganic, nonmetallic materials have attracted considerable attention in the field of drug delivery. For example, silica NPs have been successfully used for gene and drug delivery, owing to their ability to improve the stability of protected substances in their cores without interfering with their chemical and physical properties [94]. One study developed a type of SiO 2 NPs (ZnAs@SiO 2 ), which encapsulated arsenic trioxide (ATO) by a "one-pot" reverse emulsification approach (Figure 4) [95]. The ZnAs@SiO2 NPs reduced the expression of stemness markers (CD133, Sox-2, and Oct-4) and EMT markers (E-cadherin, Vimentin, and Slug) by inhibiting the STAT3 signaling pathway and thus in-hibited tumor spheroid formation in vitro and tumor initiation and metastasis in vivo [95]. Calcium phosphate NPs (CaP) are also utilized for gene delivery with negligible cytotoxicity and superior biodegradability. Furthermore, CaP dissolves in acidic endosomes and helps the cargo be released into the cytosol through the endosome rupture [96]. Li et al. proposed a novel hybrid vesicle with inorganic CaP as the kernel and with HA modification on the surface (CaP@HA), for targeted delivery of STAT3-decoy ODNs. They demonstrated that the STAT3-decoy ODNs-loaded CaP@HA vesicles effectively suppressed the expression of STAT3 and its downstream target gene mucin 4 (MUC4), which could interfere with the interaction of Trastuzumab (TRAZ) and human epidermal growth factor receptor 2 (HER2), thereby efficiently reversing TRAZ resistance in anti-HER2 therapy [96]. Similarly, Ke et al. proposed another inorganic kernel of CaP as the core of reconstituted low-density lipoprotein (LDL) nanovehicles (CaP@LDL) for targeted delivery of STAT3-decoy ODNs to reverse the resistance of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The results showed that CaP@LDL nanovehicles possessed LDL-mimicking pharmacokinetics, which enabled them to efficiently deliver STAT3 decoy-ODNs to overcome TRAIL resistance by blocking the expression of STAT3 and downstream anti-apoptotic target genes (Bcl-2, Bcl-xl, and Mcl-1) [97].

Polymeric Micelles
Polymeric micelles with core-shell structures have been extensively applied for the delivery of small molecules, therapeutic genes, antibodies (Abs), and RNA-based therapeutics due to their stable structure, good biocompatibility, high drug loading, outstanding pharmacokinetics, and preferential tumor accumulation [98]. Despite the passive tar-

Polymeric Micelles
Polymeric micelles with core-shell structures have been extensively applied for the delivery of small molecules, therapeutic genes, antibodies (Abs), and RNA-based therapeutics due to their stable structure, good biocompatibility, high drug loading, outstanding pharmacokinetics, and preferential tumor accumulation [98]. Despite the passive targeting ability of polymeric micelles under the EPR effect, facile methods were used to modify their surfaces with specifically targeting ligands, which could target overexpressed receptors on lung cancer cells (e.g., EGFR and CD44 receptors) in order to achieve improved tumorspecific targeting [99,100]. Moreover, these targeting ligands could partially inhibit the function of overexpressed receptors and regulate over-activated pathways, thus improving drug action in tumor-specific lung tissues [99].
Micellar formulations containing cationic polymers, such as poly-L-lysine (PLL) and poly(ethylenimine) (PEI), were able to deliver siRNA via electrostatic interaction, thus reducing RNA degradation and enhancing intracellular accumulation [101]. For example, researchers developed cholesterol-modified dicer-substrate siRNA (Chol-DsiRNA) Polyplexes, which were formed by the encapsulation of STAT3 siRNA with 50 poly-L-lysine residues and 5 kDa polyethylene glycol. Chol-DsiRNA Polyplexes demonstrated improved anti-tumor efficacy with good tolerance by efficiently inhibiting STAT3 [102]. Another study prepared the dual-targeting system by electronic self-assembly, which was composed of folic acid-conjugated carboxymethyl chitosan for targeting and cationic chitosan derivatives for STAT3 siRNA package (FA-OCMCS/ N-2-HACC/siSTAT3). The NPs dramatically reduced STAT3 expression in M2 macrophages and Lewis lung cancer cells and shifted the phenotype of macrophages from M2 to M1, resulting in the suppression of tumor growth [103].
The unique core-shell structure of micelles is able to co-deliver two or more therapeutic agents through self-assembly, which is considered an effective combination therapy strategy to overcome drug resistance, tumor metastasis, and immunosuppression. A study developed a micellar delivery system (PEG-PLA NPs) based on FDA-approved poly(ethylene glycol) (PEG)-poly(lactic acid) (PLA) for the co-delivery of Erlotinib (ELTN, EGFR-TKI) and fedratinib (FDTN, JAK2 inhibitor). A synergistic anti-cancer effect was achieved by PEG-PLA NPs in ELTN-resistant NSCLC by downregulating the expression levels of proteins in the JAK2/STAT3 signaling pathway, including pEGFR, pJAK2, pSTAT3, and survivin [104]. PEG-PLA was also utilized to co-deliver gefitinib (Gef) and Cyclosporin A (CsA), and the results showed that CsA formulated in NPs sensitized Gef-resistant NSCLC to Gef treatment by inactivating the STAT3/Bcl-2 signaling pathway ( Figure 5) [105]. Li and colleagues developed a multifunctional nanocomplex to simultaneously deliver paclitaxel (PTX) and STAT3 siRNA (siSTAT3) to inhibit tumor growth and prevent metastasis. PTX and siSTAT3 were encapsulated into the synthesized polyethyleneimine-polylactic acidlipoic acid (PPL) micelles through hydrophobic or electrostatic interaction, respectively. Furthermore, the negatively charged HA was coated on the surface of the drug-loaded nanocomplex (HA/ siSTAT3 PPL PTX ) in order to effectively enter CD44-overexpressed 4T1 cells via an active targeting mechanism. HA/ siSTAT3 PPL PTX exhibited superior anti-tumor efficacy and effectively reduced the lung metastasis of 4T1 cells by silencing the expression of STAT3 and pSTAT3 [106]. Another study reported the development of a pulmonary delivery system (FM@PFC/siRNA) based on perfluorocarbon (PFC) nanoemulsions for co-delivery of C-X-C motif chemokine receptor 4 antagonist (FM) and anti-STAT3 siRNA. The FM@PFC/siRNA nanoemulsions inhibited both CXCR4 and STAT3 signaling, induced apoptosis and anti-invasive activity, and overcame the immunosuppressive TME, achieving good efficacy in lung metastatic tumor models [107].
anti-tumor nanovaccine (SVMAV) using an unsaturated fatty acid docosahexaenoic acid (DHA)-conjugated antigen and R848 (a Toll-like receptor 7/8 agonist) to encapsulate stattic (STAT3 inhibitor). The obtained SVMAV efficiently migrated into lymph nodes and primed CD8 + T cells to exert neoantigen-specific killing by promoting antigen uptake of dendritic cells (DCs), stimulating DCs maturation, and enhancing antigen cross-presentation, and finally achieved a robust anti-tumor effect in primary and lung metastasis models of melanoma [111].  Studies have shown that siRNA delivery by polymeric micelles though electrostatic interaction is not very stable, resulting in inefficient transfection efficiency and high variability [108]. In addition, cationic NPs are toxic and potentially capable of inducing immunogenicity in animals and humans. Furthermore, siRNAs are susceptible to being cleared by the kidneys because excess cationic components could make NPs easy to disassemble at the glomerular basement membrane [109]. To solve these problems, researchers used dithiothreitol (DTT) to reduce the disulfide-protecting groups of siRNA at the 3 end of the sense strand, and the obtained siRNA was reacted with Pluronic F108 functionalized with pyridyl disulfide groups. This covalent conjugation of siRNA with Pluronic F108 provides a stable nanoparticle formulation with efficient siRNA loading, achieving consistent target-specific gene knockdown [108]. Shi et al. reported a new class of cation-free polymeric micellar spherical nucleic acid (SNA), which can deliver both STAT3 siRNA and temozolomide (TMZ) in a controlled release manner. The siRNA-disulfide-poly (N-isopropylacrylamide) (siRNA-SS-PNIPAM) diblock copolymer could self-assemble to form SNAs, cross the blood-brain barrier, and enter brain tumor cells through a scavenger receptor-mediated mechanism, achieving a remarkable synergistic effect against TMZ-resistant tumors [109].
Micelles for targeted STAT3 delivery may potentially modulate immunosuppressive TME, leading to improvements in immunotherapy. Jiang and colleagues synthesized a pH-responsive copolymer PEG-poly(lysine-DMMA)-poly(phenylalanine) to co-encapsulate two prodrugs, Gemcitabine-C18 and NI-HJC0152 (STAT3 inhibitor). NI-HJC0152 responded to the progressively intensive hypoxia in tumor tissue to yield parental HJC0152 that inhibits STAT3, leading to the reversal of tumor immunosuppression through modulating TAM polarization, recruiting cytotoxic T lymphocytes, and reducing regulatory T cells. In addition, inhibition of STAT3 also downregulated the expression of cytidine deaminase (CDA) and α-smooth muscle actin (α-SMA), thus relieving the resistance of gemcitabine [110]. Another study constructed a self-assembling vehicle-free multi-component anti-tumor nanovaccine (SVMAV) using an unsaturated fatty acid docosahexaenoic acid (DHA)-conjugated antigen and R848 (a Toll-like receptor 7/8 agonist) to encapsulate stattic (STAT3 inhibitor). The obtained SVMAV efficiently migrated into lymph nodes and primed CD8 + T cells to exert neoantigen-specific killing by promoting antigen uptake of dendritic cells (DCs), stimulating DCs maturation, and enhancing antigen cross-presentation, and finally achieved a robust anti-tumor effect in primary and lung metastasis models of melanoma [111].

Extracellular Vesicles
Studies have demonstrated that multiple cell types can excrete extracellular vesicles (EVs) with phospholipid bilayer membrane-bound structures, and EVs have shown great potential as drug delivery vehicles due to their nano-sized structure and ability to transport bioactive cargos between cells or tissues [112]. According to formation mechanism and typical size, Evs are mainly classified into three categories: exosomes (30-150 nm), microvesicles (MVs), or microparticles (MPs) (100-1000 nm) and apoptotic bodies (500-2000 nm) [113]. Compared with other nanocarriers, EVs have some advantages in lung cancer treatment, such as low toxicity, low immunogenicity, the ability to cross biological barriers, and the realization of multifunction through chemical or genetic modifications [112]. For example, exosomes modified with CD47 protein on the surface were able to evade phagocytosis by macrophages, leading to prolonged circulation time [114]. Furthermore, exosomes delivered from brain endothelial cells had the ability to cross the blood-brain barrier, exhibiting the potential ability to treat lung cancer with brain metastasis [115]. However, the acquirement of abundant exosomes with high quality is quite costly, and the lack of standardized methods to isolate, purify, and store exosomes has limited large-scale production and clinic translation [116]. Therefore, there is an urgent need to establish standard protocols to ensure the mass and consistent production of exosomes.
EVs from various origins hold great promise in cell-free anti-cancer treatment. For example, mesenchymal stem cell (MSCs)-derived EVs have shown unique advantages as carriers for anti-cancer drugs due to their lower immunogenicity and tumor migration capacity [117]. Qian et al. prepared EVs from human umbilical cord MSCs (huc-MSCs) transfected with adenovirus encoding Lipocalin-type prostaglandin D2 synthase (L-PGDS). EVs-L-PGDS inhibited the phosphorylation of STAT3 and the expression of downstream stem cell markers (Oct4, Nanog, and SOX2), thus inhibiting in vitro cancer cell proliferation and in vivo tumor growth [117]. Neural stem cell (NSCs)-derived exosomes have also been reported as vehicles for delivering oligonucleotide therapeutics (CPG-STAT3 antisense oligonucleotides, CpG-STAT3 ASO) to the glioma microenvironment, as NSCs have been shown to traffic into hypoxic areas of gliomas and secondary brain metastases. The results demonstrated that CpG-STAT3 ASO encapsulated NSCs/EV significantly activated glioma-associated myeloid cells and inhibited tumor progression in mice [118]. In addition, plant-derived nanovesicles for drug delivery have been discussed in recent years due to their safe and cost-efficient characteristics. Chen et al. obtained cucumber-derived nanovesicles (CsDNVs) at high yield and low cost, which may be natural nanocarriers that contain Cucurbitacin B (CuB, STAT3 inhibitor). They demonstrated that these CsDNVs enhanced the anti-cancer effects of CuB by improving its bioavailability [119]. Notably, owing to the remarkable capability for penetration of the BBB, exosomes might improve the prognosis of glioblastoma (GBM). Ye and colleagues prepared Angiopep-2 (An2)conjugated (STAT3) siRNA-loaded exosomes (Exo-An2-siRNA) derived from human M1 macrophages. Exo-An2-siRNA could boost BBB permeation and GBM targeting by exploiting the tumor-homing characteristic of M1 macrophages and specifically targeting (low-density lipoprotein receptor-related protein 1) LRP-1 ligands at the surfaces of both GBM cells and BBB endothelial cells, resulting in the favorable inhibition of the proliferation of orthotopic U87MG xenografts ( Figure 6) [120].

Challenges in Lung Cancer-Targeted Drug Delivery
Although intravenous injection of nanomedicine is commonly used in other cancers, inhalation of nano-delivery systems is an additional administration route for lung cancer due to the unique anatomical and physiological characteristics of the lungs. However, due to the complex molecular and biochemical composition of the lung tissue, different biological barriers to drug delivery in lung cancer should be taken into consideration when designing efficient strategies for nanotechnology in lung cancer.
The presence of mucus in the respiratory system is the key mechanical barrier of the pulmonary region [121]. Mucins could form complex mesh by interacting with other mucin molecules and glycans in the mucins could provide negative charge, allowing NPs of different sizes and positive charges to be deposited in the mucus layer [121][122][123]. The pulmonary surfactant is one of the crucial chemical barriers for NPs to overcome before reaching the pneumocytes. Studies indicate that proteins in pulmonary surfactants prefer to bind magnetic NPs [124]. Moreover, hydrophilic NPs are easily trapped by the surfactant layer [125]. Proteolytic enzymes (e.g., cathepsin H) are another chemical barrier as they are responsible for the hydrolysis of protein and peptides of the NPs [126,127].
In addition, the complex tumor microenvironment (TME) is another biological bar-

Challenges in Lung Cancer-Targeted Drug Delivery
Although intravenous injection of nanomedicine is commonly used in other cancers, inhalation of nano-delivery systems is an additional administration route for lung cancer due to the unique anatomical and physiological characteristics of the lungs. However, due to the complex molecular and biochemical composition of the lung tissue, different biological barriers to drug delivery in lung cancer should be taken into consideration when designing efficient strategies for nanotechnology in lung cancer.
The presence of mucus in the respiratory system is the key mechanical barrier of the pulmonary region [121]. Mucins could form complex mesh by interacting with other mucin molecules and glycans in the mucins could provide negative charge, allowing NPs of different sizes and positive charges to be deposited in the mucus layer [121][122][123]. The pulmonary surfactant is one of the crucial chemical barriers for NPs to overcome before reaching the pneumocytes. Studies indicate that proteins in pulmonary surfactants prefer to bind magnetic NPs [124]. Moreover, hydrophilic NPs are easily trapped by the surfactant layer [125]. Proteolytic enzymes (e.g., cathepsin H) are another chemical barrier as they are responsible for the hydrolysis of protein and peptides of the NPs [126,127].
In addition, the complex tumor microenvironment (TME) is another biological barrier in the drug delivery of lung cancer. One of the most critical stromal cells in TME of lung cancer, namely cancer-associated fibroblasts (CAFs), has been demonstrated to play a key role in remodeling the tumor stroma and increasing the stiffness of the extracellular matrix (ECM), which might restrict the diffusional movement of NPs in tumor cells [128,129]. Macrophages in the TME, part of the clearance system, were found to be able to engulf, degrade, and remove NPs, thus affecting the number of NPs entering the tumor site [121]. Furthermore, the engulfment of NPs might induce the release of pro-inflammatory cytokines by macrophages, resulting in the activation of the immune system [130].

Conclusions and Perspectives
Many studies have proved the importance of the STAT3 signal pathway in the initiation, progression, and metastasis of lung cancer. Several drugs targeting STAT3 have achieved remarkable therapeutic effects and are expected to become potential treatments for lung cancer. Different therapeutic strategies, including natural compounds, new small molecule inhibitors, and gene therapies, have shown effective targeting of STAT3. However, it should be pointed out that the low bioavailability of drug candidates and rapid degradation of RNA drugs have limited their application in cancer treatment. In addition, as STAT3 is widely expressed in various cells and tissues to regulate the self-renewal of normal stem cells and to participate in mammary gland development and embryogenesis, the potential adverse effects of STAT3 inhibitors on normal cells cannot be ignored.
Various nanotechnologies have been explored to improve drug solubility, prolong circulation time in vivo, and preferentially deliver drugs to tumor sites through the EPR effect. To date, different types of nanocarriers have been attempted for the targeted delivery of STAT3 inhibitors in the treatment of cancer. Although their results are promising in some preclinical trials, there are still some challenges to be addressed during the process of clinical translation. Firstly, as the application of nanoparticles in the lungs is challenging due to several biological barriers, as discussed above, there is an urgent need to design new and more efficient nanoparticles to achieve ideal therapeutic outcomes. The material, composition, size, and charge of nanoparticles, as well as the cross-talk between tumor and stromal cells, should be carefully considered in order to avoid rapid clearance of nanoparticles, enhance drug penetration, and increase tumor response to therapeutic agents. Secondly, more extensive studies and investigations are necessary to examine the safety and biocompatibility of nanoparticles with long-term metabolism in vivo. Thirdly, as STAT3 activation is involved in drug resistance and immunosuppression, NPs-based co-delivery of STAT3 inhibitor and chemotherapeutic drug/EGFR-TKIs/immune adjuvant is considered a promising strategy for combination therapy. To ensure the safety and synergistic effect of these NPs, it is necessary to carry out detailed and complete studies on their pharmacokinetic, biodistribution, and pharmacodynamic parameters. Fourthly, STAT3 inhibitor-loaded nanosystems have also been prepared for the eradication of LCSCs by targeting the markers (such as CD133 [131] and CD44 [132]) of LCSCs. However, there are no specific markers to accurately identify LCSCs, and it is unclear whether these markers will change in different environments. Moreover, as LCSCs and normal stem cells usually share the same self-renewal signaling pathways, such treatment may also lead to the inhibition of signaling cascades in normal cells and the exhaustion of the normal resident stem cell population. Finally, the preclinical application of most NP-delivered STAT3 inhibitors is currently limited to in vitro cancer cell lines or CDX models. Therefore, it is necessary to validate their enhanced anti-tumor efficacy in more clinically relevant tumor models, such as PDX models, and ultimately translate these preclinical outcomes into clinical applications.
In summary, lung cancer remains one of the most common cancers in both men and women, and the efficiency of conventional treatment has quickly leveled off, so there is an urgent need for more effective treatment strategies. As we have summarized, the STAT3 pathway plays a key role in tumorigenesis, metastasis, drug resistance, and cancer stem cell maintenance of lung cancer. Scientists have developed various intervention strategies with STAT3 as the therapeutic target, including natural compounds, small molecules, and RNA-based therapeutics. They have also attempted to apply multiple nanotechnologies for targeted delivery of STAT3 inhibitors, which significantly improves their bioavailability and tumor targeting capabilities and shows great potential for lung cancer treatment in preclinical trials. We expect that nanotechnology-based targeted delivery of STAT3 inhibitors can simultaneously eliminate both lung cancer cells and cancer stem cells, which will lead to new treatment strategies for lung cancer and will significantly improve patient survival if translated into clinical applications in the near future.
Author Contributions: Conceptualization, Q.F. and K.X.; writing-original draft preparation, Q.F.; writing-review and editing, K.X.; supervision, K.X.; funding acquisition, K.X. All authors have read and agreed to the published version of the manuscript.  Table 2 are available at http://www.clinicaltrials.gov (accessed on 20 September 2022).