Nanoformulations of small molecule protein tyrosine kinases inhibitors potentiate targeted cancer therapy

Journal Pre-proofs Nanoformulations of Small Molecule Protein Tyrosine Kinases Inhibitors Potentiate Targeted Cancer Therapy Yanlong Yin, Xiao Yuan, Huile Gao, Qian Yang PII: DOI: Reference:

S0378-5173(19)30830-0 https://doi.org/10.1016/j.ijpharm.2019.118785 IJP 118785

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International Journal of Pharmaceutics

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30 August 2019 5 October 2019 10 October 2019

Please cite this article as: Y. Yin, X. Yuan, H. Gao, Q. Yang, Nanoformulations of Small Molecule Protein Tyrosine Kinases Inhibitors Potentiate Targeted Cancer Therapy, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.118785

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Nanoformulations of Small Molecule Protein Tyrosine Kinases Inhibitors Potentiate Targeted Cancer Therapy Yanlong Yin a, #, Xiao Yuan a, #, Huile Gao a, b, *, Qian Yang a, * a

The School of Pharmacy, College Key Laboratory of Sichuan Province for Specific

Structure of Small Molecule Drugs, Chengdu Medical College, Chengdu, 610500, China b

Key Laboratory of Drug-Targeting and Drug Delivery System of the Education

Ministry, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research, Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu, 610064, PR China

* Corresponding author: E-mail: [email protected], [email protected] (H. Gao); [email protected] (Q. Yang). # These

authors did equal contributions to this work

Keywords: small molecule tyrosine kinase inhibitors; signaling pathways; drug resistance; targeted delivery; combination therapeutic strategies

Abstract：Protein tyrosine kinases (PTKs) are closely related to tumor development and usually participate in apoptosis, DNA repair, and cell proliferation by activating signaling pathways. Therefore, PTKs have become the most promising targets for cancer therapy. In recent years, a large number of studies on the mechanism of tyrosine kinase activation have indicated that tyrosine kinase inhibitors (TKIs) have important clinical significance and application prospects as targeted anticancer drugs because they can effectively block certain cellular signaling pathways, inhibit tumor metastases and reduce tumor proliferation. Although the increasing emergence of anticancer drug resistance limits the clinical application of TKIs, emerging nanotechnology has made it possible to solve this problem. In this work, the state-of-art of small molecule protein tyrosine kinase inhibitors and the applications of drug delivery systems for TKIs are reviewed, and the potentials and challenges for future research of small molecule TKIs are addressed.

1. Introduction Cancer is a worldwide health problem with high morbidity and mortality, and the basis of malignancy is dysfunction of cellular signal transduction pathways caused by aberrant expression of genes, which leads to uncontrolled cell growth or proliferation. Therefore, these molecules and genes related to tumor occurrence, development, invasion, metastasis and apoptosis are targets for the new generation of anticancer drug development. Generally, protein tyrosine kinases (PTKs) are a family of enzymes that catalyze the phosphorylation of tyrosine residues in many important proteins. As mediators in cellular signaling pathways, PTKs participate in a series of cell functions, resulting in cell proliferation, metabolism, differentiation, migration, and ultimately apoptosis (Lemmon and Schlessinger, 2010; Levitzki and Mishani, 2006). However, abnormal tyrosine

kinase

activities

resulting

from

mutation,

overexpression

and

autocrine-paracrine stimulation have been demonstrated as the major causes of various cancers as well as other diseases (Tonks, 2006). PTK dysregulation is highly related to oncology, especially in chronic myeloid leukemia, gastrointestinal stromal tumors, non-small cell lung cancer, breast cancer, and renal cell carcinoma (Levitzki, 2013). Consequently, PTKs became an attractive target for cancer therapy, and compounds that can inhibit the kinase activity of PTKs are potential therapeutics to treat tumors. The proteins of the tyrosine kinase family are composed of two major kinases, receptor tyrosine kinases (RTKs) and nonreceptor tyrosine kinases (nRTKs). Generally, RTKs are well known as cell surface receptors that are considered pivotal regulators of critical cellular processes, and many of them, such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR), have emerged as prospective targets in cancer therapy. Moreover, RTKs are not only transmembrane receptors with extracellular domains but also intracellular enzymes with tyrosine kinase activity. Clinical cancer research has shown that these receptors and their ligands are closely related to various cancers, and excessive tyrosine phosphorylation signal transduction might be caused by the overexpression

of

relevant

growth

factors.

The

nRTKs

are

cytoplasmic enzymes without extracellular domains, including the Abelson murine

leukemia (ABL) family, SRC kinase family, focal adhesion kinase (FAK) family, Janus kinase (JAK) family and so on. The structure of the nRTK catalytic domain is tyrosine residues with specific ATP-binding sites, which are activated by phosphorylation. Activated nRTKs are critically important in the process of tumor genesis and growth via activation of downstream signaling and subsequently inducing proliferation and inhibiting apoptosis in tumor tissue (Cowan-Jacob, 2006). Hence, highly activated PTKs in malignant tumor cells but very low activity and expression in normal cells enable PTKs to serve as vital targets and be the focus of intensive attention in potential cancer fighting strategies (Robertson et al., 2000). Targeted inhibitors of PTKs would provide most cancer patients with acceptable tolerance and quality of life when compared with conventional cytotoxic agents. As a consequence, two approaches have been selected to block the abnormal proliferation signaling pathways in tumor cells by PTKs, monoclonal antibodies and small-molecule inhibitors of tyrosine kinase (TKIs). Monoclonal antibodies can specifically inhibit tumor cell growth via binding to cell-surface antigens such as the EGFR extracellular domain, which blocks signal transduction by competitive inhibition. Small molecular tyrosine kinase inhibitors (TKIs) have been developed as ideal candidates for target-specific cancer therapeutics due to their unique molecular structures, which interfere with specific intracellular tyrosine kinase domains, block the tyrosine phosphorylation process and inhibit the malignant tumor cell proliferation pathway. With the assiduous research by many scientists, hundreds of small molecule compounds were designed and synthesized as RTKs and nRTK inhibitors over the past three decades, and some have been approved by the FDA as the first-line therapy for different kind of cancer (Table 1). However, with the successful development of anticancer drugs, new problems have developed. The existing small inhibitors of tyrosine kinases have defects such as poor solubility, low oral bioavailability, as well as some severe adverse effects, which are major limitations in their clinical application; on the other hand, the gradual emergence of drug resistance during therapy urgently needs to be solved. The application of nanotechnology provides a prospective drug delivery strategy, in which the nanoformulations process obvious advantages. With the characteristics of small particle size, large surface area, high surface reactivity and active sites, and desirable adsorption capacity, nanomaterials applied as drug carriers have been reported to improve drug absorption and

bioavailability, enhance efficient targeting delivery, prolong circulation time, and reduce harmful side effects on normal tissues (Su et al., 2019). In this review, we summarized the application of small molecule TKIs available on the market and the advanced research of different nanoformulations for small molecule TKIs, as well as the combination therapeutic strategies in targeted cancer therapy. With discussing their comparative advantages and limitations, the potentials and challenges for future research of small molecule TKIs are addressed. Table 1.

Approved small molecular tyrosine kinase Inhibitors for cancer therapy IC50

Compound

Kinase target

Disease

Gefitinib (Iressa)

EGFR

NSCLC

14.6

Erlotinib (Tarceva)

EGFR

NSCLC, pancreatic cancer

2

Icotinib (Conmana)

EGFR

NSCLC

45

Lapatinib (Tykerb)

EGFR, HER2

Breast cancer

10.8, 9.2

Neratinib (Nerlynx)

EGFR, HER2

NSCLC, breast cancer

92, 59

Afatinib (Gilotrif)

EGFR, HER2

NSCLC, breast cancer, squamous cell carcinoma of the head and neck

0.5, 14

Imatinib(Glivec,Gleevec )

Abl, PDGFR, Kit

CML, CMML, GIST

600, 100, 100

Dasatinib (Sprycel)

Abl, SRC, Kit, PDGFR

CML resistant to imatinib

<10

Nilotinib (Tasigna)

Abl, Kit, PDGFR

CML resistant to imatinib

<30

Sunitinib (Sutent)

VEGFR1-3, PDGFR, Kit FLT3, RET, CSF1R

GIST resistant to imatinib, Advanced RCC

<100

Sorafenib (Nexavar)

VEGFR1-3, PDGFR, Kit, FLT3, BRAF

Advanced RCC hepatocellular carcinoma

<100, 57, 68,33,6

Pazopanib (Votrient)

VEGFR1-3, Kit

Advanced RCC

<150

PDGFR,

(nmol/L)

2. Classifications of Small Molecule Tyrosine Kinases Inhibitor (TKIs) 2.1 Epidermal Growth Factor Receptor (RGFR) Kinase Inhibitors

The epidermal growth factor receptor (EGFR) family is a significant target for cancer targeting therapy, specifically for non-small cell lung cancer (NSCLC) and breast cancer, and RTKs on the cell membrane are coded for by the oncogene c-ErbB. The EGFR family comprises four receptors with similar molecular structures, including EGFR (HER1/ErbB1), HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). As transmembrane receptor tyrosine kinases, activated EGFR transduces crucial signal pathways in the process of tumorigenesis and eventually induces a range of cellular responses, such as tumor cell differentiation, proliferation, invasion, metastasis and apoptosis (Ranson, 2004). Moreover, as a result of EGFR activation, vascular endothelial growth factor (VEGF) can also be stimulated, inducing tumor angiogenesis, adhesion, invasion and metastasis (Ferrara et al., 2003).

Figure 1. The scheme for Tyrosine kinase inhibitors binding to the targets and anti-cancer mechanism through suppressing EGFR signal pathway acting on cycle progression.

Numerous preclinical studies have demonstrated that aberrant EGFR signaling by overexpression of EGFR is linked with tumorigenesis. Furthermore, a high proportion of cancers in the head and neck, lung, breast, prostate, bladder, kidney, and colon have been found to be associated with dysregulation of EGFR signaling pathways (Rocha-Lima et al., 2007). Many therapeutic agents inhibit the activity of EGFR, and their ligands have been under development since the beginning of this century. Small molecular EGFR protein tyrosine kinase inhibitors (EGFR-TKIs) have become the most advanced chemotherapeutic drugs in anticancer drug development. These

molecules can covalently link with the ATP binding site of the receptor tyrosine kinase to form the active conformation in which the activation loop is phosphorylated and consequently inhibit the phosphorylation of tyrosine kinase (Fig. 1) (Liu and Gray, 2006). Most small molecule EGFR inhibitors are based on a 4-anilinoquinazoline structure, some of which are reversible inhibitors (e.g. the EGFR kinases inhibitors gefitinib/Iressa and erlotinib/Tarceva; the EGFR/Her-2 dual kinase inhibitor lapatinib/Tykerb; Chinese me-too drug icotinib/Conmana), while others are irreversible inhibitors (neratinib/Nerlynx and afatinib/Gilotrif) (Fig. 2A). 2.1.1 First generation EGFR kinase inhibitors Gefitinib, a well-tolerated oral EGFR-TKI, was approved in 2003 for advanced NSCLC patients who failed with regular chemotherapy (cisplatin or docetaxel) (Cohen et al., 2003; Herbst et al., 2004). Due to its high selectivity for EGFR tyrosine kinase, gefitinib showed obvious inhibition of EGFR transmembrane receptor autophosphorylation and could alleviate complications and induce tumor regressions in patients with persistent NSCLC after radiochemotherapy (Kris et al., 2003). As a first-line TKI for EGFR-positive patients, gefitinib dramatically increased progression-free survival and the overall survival rate with acceptable tolerance, which was superior to cytotoxic agents (Maemondo et al., 2010). Erlotinib (Tarceva) was approved as a first-line treatment for NSCLC by the FDA in 2004 (Dowell et al., 2005). On a cellular or molecular level erlotinib can bind with to ATP site of the activated EGFR tyrosine kinase and then produce specific, reversible inhibition of EGFR kinase activity and elicit antiproliferative effects and induction of tumor cell apoptosis (Zhou et al., 2011). In addition to the effects in NSCLC, erlotinibop has also been proven effective as a first-line treatment in gastroesophageal cancer (Dragovich et al., 2006), and has shown antitumor effects in other EGFR-positive cancers, including pancreatic cancer (Van Cutsem et al., 2009), ovarian carcinoma (Nimeiri et al., 2008), breast cancer (Dickler et al., 2009), and hepatocellular carcinoma (Thomas et al., 2007). Besides these EGFR kinase inhibitors above, the me-too drug icotinib (Conmana), approved by Chinese regulators in 2011, is a reversible EGFR kinase inhibitor. Although icotinib showed significant activity in inhibiting A431 tumor cell proliferation and tumor xenograft growth, no evident difference in clinical efficacy and safety was found as the other two reversible inhibitors (gefitinib and erlotinib)

(Tan et al., 2012; Shi et al., 2013). Moreover, short half-life is the main disadvantage of icotinib. Nevertheless, it still has potential interest and prospects in China and other Asian countries. The results of oncogene study showed that EGFR mutations among Asian patients with NSCLC occurred much more frequently than those of Western NSCLC patients (Gazdar, 2009), and icotinib was demonstrated the effectivity and sensitivity for patients with EGFR mutations (Wheeler et al., 2010). 2.1.2 The EGFR and HER-2 dual tyrosine kinases inhibitor lapatinib Lapatinib (Tykerb) is a reversible EGFR and HER-2 dual tyrosine kinase inhibitor and was approved by the FDA in 2002 (Xia et al., 2002). It was demonstrated to have acceptable binding affinity to EGFR (Cys 773) and HER2 (Cys 805), which are key sites against EGFR mutations that are resistant to gefitinib and erlotinib (Howe and Brown, 2011). Phase I clinical study results showed that its safety, tolerability and pharmacokinetics were acceptable for patients with EGFR-expressing and/or HER2-overexpressing metastatic cancers (Burris et al., 2005). Modest clinical activity of lapatinib monotherapy for HER2-positive metastatic breast cancer patients was observed in a phase II study (Burstein et al., 2008b). Furthermore, the therapeutic effects of lapatinib in combination with the anti-HER2 monoclonal antibody trastuzumab are better than single-targeting agent treatment for the gene-positive breast cancer, which would benefit from dual blockade (Konecny et al., 2006). Combined with capecitabine, lapatinib showed its superiority and obvious improvement in the progression time for advanced breast cancer patients previously treated with anthracycline, taxane, and trastuzumab. No increase in serious toxic effects associated with the combination treatment was observed, which supports the role of lapatinib in the treatment of HER2-positive breast cancer (Geyer et al., 2006). 2.1.3 Second-generation irreversible EGFR TKIs Among patients with NSCLC in America, approximately 10% possess activating mutations in EGFR (Villaflor and Salgia, 2013). Unfortunately, patients acquired mutations while on therapy, which led to patient resistance to the available drugs. Nearly all EGFR mutations eventually develop resistance to reversible EGFR inhibitors (gefitinib or erlotinib) after 10-13 months (Paz-Ares et al., 2010), including and the EGFR T790M point mutation. Strategies to overcome resistance include developing oral irreversible inhibitors targeting EGFR T790M. The advantage of

these irreversible kinase inhibitors is that they could covalently bind to the cysteine residue (Cys) of EGFR and HER2 receptors, perpetually blocking the mutated versions of the tyrosine kinases, and consequently reduce autophosphorylation in the tumor cells. Nevertheless, the irreversible inhibitors had limited success, which was associated with unavoidable toxicity due to the suppression of wildtype EGFR. Neratinib (Nerlynx) is the first irreversible EGFR kinase inhibitor against the T790M resistance mutation (Bose and Ozer, 2009). In preclinical studies, neratinib significantly inhibited HER2-overexpressing BT474 cell xenografts (Tsou, 2005). Moreover, NSCLC cells overexpressing HER2 were sensitive to neratinib (Minami et al., 2007). However, concurrent inhibition of wildtype EGFR leads to the most common toxicity associated with neratinib. Therefore, the clinical therapeutic outcome of neratinib has been limited, especially for patients with prior benefit from first-generation EGFR kinase inhibitors (Burstein et al., 2010; Sequist et al., 2010). Diarrhea, the dose-limiting toxicity, became the biggest obstacle for neratinib to achieving minimum plasma concentration for EGFR T790M inhibition (Wong et al., 2009). Afatinib (Gilotrif), an oral irreversible multiple ErbB blocker, is approved under the FDA’s priority review program for the frontline treatment of NSCLC in patients harboring EGFR mutations (Li et al., 2008; Solca et al., 2012). In a dose-escalation study, afatinib showed acceptable tolerance with an acceptable safety profile (Marshall et al., 2013). The results of the clinical trial indicated that afatinib presents a much higher level of activity in EGFR mutation NSCLC, especially for patients with erlotinib, gefitinib, and other chemotherapy failure (Miller et al., 2012; Wu et al., 2014). Therefore, afatinib is a valuable new option for EGFR mutation patients.

Figure 2. The chemical structure of A). EGFR tyrosine kinase inhibitors; B). Bcr-Abl tyrosine kinase inhibitors; C). Multitargeted tyrosine kinase inhibitors.

2.2 Bcr-Abl tyrosine kinase inhibitors Chronic myeloid leukemia (CML) is a type of hematological abnormality characterized by malignant proliferation of myeloid stem cells. The Bcr-Abl oncogene, found in 95% of patients with CML, has been suggested to be the underlying cause of this disease. In addition, the molecular characteristic of CML is the Bcr-Abl fusion gene produced by oncogene translocation. The fusion protein BCR-ABL, which is encoded by the Bcr-Abl fusion gene, has persistently activated Bcr-Abl tyrosine kinase phosphorylation, which then plays a definite role in the development of CML (Druker and Lydon, 2000). Before molecular targeted therapeutic agents were used as a first-line treatment for CML, allogeneic bone marrow transplantation was the standard therapy option. However, BMT had been limited by waiting for a suitable

donor to become available and also had substantial morbidity and mortality. For these reasons, Bcr-Abl tyrosine kinase inhibitors were under development as potent and selective therapeutic options for CML (Fig. 2B). When the first Bcr-Abl TKI was approved by the FDA in 2001 as a new therapeutic approach for CML, it opened the door to a new era of molecular targeted anticancer drug development. The availability of TKIs has drastically changed the treatment of CML and dramatically improved the long-term survival rates of patients. This new revolution in therapy provides patients a fundamentally good quality of life and enables the malignant hematopathy to be chronic disease with control. 2.2.1 Imatinib Imatinib (Gleevec/Glivec) was the first TKI to enter the clinical setting in 2001 and has been approved as the first-line treatment for CML and gastrointestinal stromal tumors (GIST) by the FDA (Capdeville et al., 2002). As a competitive ATP inhibitor, imatinib selectively inhibits the kinase activity of Bcr-Abl, c-Kit and PDGF-R, which are responsible for the proliferative signaling of tumor cells (Mol et al., 2004; Nagar et al., 2003). In clinical study, imatinib was found to be highly effective for patients with CML and considerably improved their prognosis, when compared with that of cytotoxic drug chemotherapy (Hughes et al., 2003; O'Brien et al., 2003). Unfortunately, resistance to imatinib became the biggest obstacle in its clinical efficacy (Soverini et al., 2006). The mechanism for drug resistance was reported to involve mutations of the Bcr-Abl kinase and Bcr-Abl genes (Gambacorti-Passerini et al., 2003). Subsequent research indicated that mutations of BCR-ABL kinase in the ATP-binding site altered the structure of the protein tyrosine kinase, which might reduce the binding affinity of imatinib and Bcr-Abl kinase (Shah et al., 2004). Consequently, the second-generation small molecular Bcr-Abl kinase inhibitors were developed to address the resistance to imatinib (Weisberg and Manley, 2007). In addition, the combination of ABL1 inhibitor (ABL001) and the second-generation TKIs was reported as a promising treatment strategy which could eradicate the CML without recurrence (Wylie et al., 2017). 2.2.2 Nilotinib Nilotinib, an aminopyrimidine derivative, selective Bcr-Abl kinase inhibitor, was shown to be more than 30-fold more effective than imatinib in vitro and could

overcome most imatinib-resistant Bcr-Abl mutations (O'Hare et al., 2005). In addition to Bcr-Abl, nilotinib can also inhibit PDGFR and c-Kit kinase (Verstovsek etal., 2006; Weisberg et al., 2005). Furthermore, nilotinib has significant clinical efficacy at well-tolerated doses, can provide an alternative therapeutic choice for CML patients after imatinib failure and can also be approved as a frontline therapy for patients in the chronic phase of CML (Kantarjian et al., 2011). 2.2.3. Dasatinib Dasatinib, an oral dual SRC/ABL kinase inhibitor with a broad specificity, has been approved in 2006 as an effective therapeutic agent for CML patients with imatinib failure and resistance (Kantarjian et al., 2006). Dasatinib binds to Bcr-Abl both in the active and inactive formation, even in the majority of kinase mutants with imatinib resistance. Therefore, dasatinib shows greater binding affinity for Bcr-Abl kinase in vitro, which confers much more potent inhibition than that of imatinib and nilotinib (Lombardo et al., 2004). In clinical trials, dasatinib demonstrated substantial hematologic and cytogenetic activity with CP-CML. Importantly, continuing improved response rates and satisfying progression-free survival indicated that dasatinib is a potent therapeutic approach for CML patients after imatinib failure (Baccarani et al., 2006; Khorashad et al., 2008). In addition to SRC/ABL kinase, dasatinib is a bioavailable inhibitor of various key kinases that target the immune system, including Tec and Btk, suggesting that dasatinib might display potential immunosuppressive effects for immunological disorders (Hantschel et al., 2007). 2.3 Multitargeted tyrosine kinase inhibitors After the PTKs had been proven to be targets for tumor therapy, numerous small molecule compounds have been designed and synthesized and have demonstrated their inhibition of the tyrosine kinase domain in vitro and in vivo. However, reduced activity and drug resistance became the biggest obstacles in the clinical efficacy of those TKIs targeting just one receptor or signaling pathway. As a consequence, a strategy to improve the clinical efficacy of TKIs focused on the development of multitargeted small molecular inhibitors or combination therapeutic approaches with different single targeted drugs, which would inhibit tumor cell proliferation by blocking multiple signaling pathways or the same signaling pathway at multiple steps. Multitargeted TKIs are more convenient and less complex for clinical

use and have become a new research trend. Therefore, a number of small molecule multitargeted TKIs have been developed, and some of them have recently reached clinical settings (Fig. 2C). 2.3.1 Sunitinib Sunitinib is a multitargeted TKI that was proven to inhibit VEGFR, PDGFR, c-KIT tyrosine kinase, colony-stimulating factor 1 (CSF-1) and Fms-like tyrosine kinase-3 receptor (FLT3) (Abrams et al., 2003; Murray et al., 2003; O'Farrell et al., 2003; Osusky et al., 2004). The results from clinical trials included disease control, progression-free survival, superior survival and tolerability, which guaranteed its clinical efficacy and safety, indicating that sunitinib was regarded as an effective therapeutic approach for patients with GIST after failure of imatinib (Demetri et al., 2006). Furthermore, due to its ability to inhibit VEGFR and PDGFR, which have been demonstrated to be crucial for tumor cell proliferation and tumor angiogenesis, sunitinib is considered a potent option for metastatic breast cancer monotherapy (Burstein, 2008a), and has also been proven as a second-line treatment for advanced gastric cancer therapy (Bang et al., 2011). 2.3.2 Sorafenib Sorafenib, an oral broad-spectrum multikinase inhibitor, showed potent activity in inhibiting tumor growth in various human xenograft models, including breast, colon, ovarian, pancreatic, and thyroid cancers and melanoma in preclinical studies (Wilhelm et al., 2006). On the one hand, sorafenib can suppress other members of the RAF protein family, wildtype BRAF and V599E BRAF; on the other hand, significant inhibition of neovascularization and tumor progression was demonstrated against some receptor tyrosine kinases, including VEGFR-2, PDGFR-and VEGFR-3 (Wilhelm et al., 2008; Wilhelm et al., 2004). A phase 3, double-blind, randomized clinical trial of sorafenib demonstrated that it was effective for patients with advanced renal cell carcinoma (RCC) after chemotherapy failure, especially for improving the progression-free survival of patients; however, the toxic effect of this treatment was unavoidable (Escudier et al., 2007). Sorafenib was approved as a first-line treatment for patients with advanced hepatocellular carcinoma for efficacy and safety (Cheng et al.,

2009), and the overall survival in the sorafenib group was significantly improved by at least 3 months compared to that of the placebo group (Llovet et al., 2008). 2.3.3 Pazopanib Pazopanib (Votrient), a pyrimidine multikinase inhibitor targeting VEGFR, PDGFR, and c-Kit, was first approved by the FDA in 2009 (Bukowski et al., 2010). The phase III trial data demonstrated that pazopanib monotherapy for metastatic renal-cell carcinoma

patients

displayed

remarkable

reinforcement

in

progression-free

survival and overall survival, which was similar to that of sunitinib (Motzer et al., 2013). Pazopanib is also approved as a novel systemic therapy option for patients with metastatic nonadipocytic soft-tissue sarcoma when conventional chemotherapy failed, and the group with pazopanib treatment had a prolonged progression-free survival compared with that of the placebo group (Van der Graaf et al., 2012).

3. Nanotechnology for TKI delivery Despite the remarkable activity of TKIs, the limited therapeutic outcome in preclinical and clinical studies has spurred the rethinking of TKIs formulations in the aspect of pharmaceutics. For example, apatinib is limited by its poor solubility and oral bioavailability (only approximately 10% in dogs), and hepatobiliary disorder, anorexia and some skin diseases frequently occur with the therapeutic dose of gefitinib (Kola Srinivas et al., 2017). Besides, small molecule TKIs also lack of selectivity and develop inevitable acquired resistance (e.g. the EGFR-T790M mutations were proved to be the main reason for EGFR TKIs resistance, as well as the resistance of imatinib was involved with mutations of the Bcr-Abl kinase and Bcr-Abl genes), when applied with conventional administration (Brugger and Thomas, 2012; Gambacorti-Passerini et al., 2003). Consequently, drug delivery systems have been introduced for the delivery of approved agents to improve their therapeutic outcomes and alleviate the adverse effects during tumor therapy. Among them, nanoparticles with various structures and properties served as the desirable carriers for small molecule TKIs have been invented and becoming as the promising approach to solve these above problem (Fig. 3).

Figure 3. A). Schematic illustration of the small molecule TKIs nanoformulations by various nanocarriers would be the promising approach to improve the therapeutic outcomes during clinic application. B-D). The publications about the research of small molecule TKIs (B) or small molecule TKIs plus delivery (C) or small molecule TKIs plus nano in the past two decades (D).

The formulations of nanoparticles include protein nanoparticles, polymeric nanoparticles liposomes and lipid nanoparticles, inorganic nanoparticles, metal nanoparticles, etc. Nanoparticle-based systems have some unique advantages. Firstly, nanoparticles could co-load the hydrophilic and hydrophobic therapeutic agents simultaneously, which not only benefit with improving the solubility of hydrophobic drugs, but also enhance anticancer effect when employ with the appropriate drug combinations in delivery systems. Secondly, the controllable particle size distribution and surface modification enable the passive or active tumor targeting and result in increased drug accumulation in tumor region. Finally, nanoparticles as drug carrier also enable the sustained and controlled drug release. The optimized release profiles with

prolonged

circulation

lifetime

permit

possibility

for

improving

the

pharmacokinetic and reduced the dose-relative toxicity of chemical drugs (Donahue et al., 2019; Zhao et al., 2019). Therefore, the following section reviews the application of different types of nanoparticles in TKIs delivery. 3.1 Organic nanoparticles 3.1.1 Albumin nanoparticles In recent years, serum albumin has been regarded as an ideal nanocarrier due to its high biodegradability and low immunogenicity. The “protein corona”, consisting of

binding proteins or adsorbent proteins around the nanoparticles, was considered as the mainly influence factors for the phagocytosis and retention during circulation time (Nguyen and Lee, 2017). As the endogenous substances, albumin could prevent the small molecule TKIs from unwanted interaction and fast elimination in vivo, and the albumin-based nanoformulations exhibit superior antitumor activity, not only for inhibiting primary breast cancer but also suppressing the migration of tumor cells (Fig. 4) (Shargh et al., 2016; Wan et al., 2015). Moreover, the strategy of covalently anchoring chemotherapeutic drugs to albumin was developed to improve drug stability in circulation (Ruan et al., 2018). Furthermore, crosslinking nanobodies and albumin nanoparticles is also a useful way to induce tumor regression (Heukers et al., 2014). The enhanced cytotoxic effect on cancer cells and alleviated adverse effect of chemotherapeutic agents in treatments demonstrated that albumin nanoparticles exhibited improved therapeutic effects.

Figure 4 A). The schematic photograph and TEM image of lapatinib-loaded human serum albumin nanoparticles(LHNPs); B). The Scanning electron micrograph of cancer proliferation in vitro and tumor growth curves with lapatinib solution (LS) and LHNPs treatment; C). The H&E staining of tumor slides after LS and LHNPs treatment; D). Immunofluorescence staining of

tumor slides after LHNPs treatment. Adapted from (International Journal of Pharmaceutics, 2015, 484, 16-28.). Copyright (2015) with permission from Elsevier Science.

3.1.2 Liposomes and lipoprotein nanoparticles Erlotinib was considered the first-line treatment for NSCLC and was associated with mutations in the EGFR gene (Villaflor et al., 2013). The conventional formulations of erlotinib available on the market are tablets, and while it is convenient for patients, the dose-related adverse effects are problematic. Lipids (such as phospholipids) are used in the preparation of nanoparticles due to their benign biocompatibility and high safety (Zhao et al., 2015). The integrity of the nanoparticle structure can be maintained because the liposomes encapsulate the outer layers of the nanoparticles. Compared with traditional administration, a liposome-based KTI delivery strategy presents the advantages of high drug loading capacity and surface ligand modification, which could lead to desirable therapeutic efficacy in targeted therapy of NSCLC (Lu et al., 2019). Similarly, liposome nanosystems are utilized for delivery of the multitarget tyrosine kinase inhibitor XL184 (cabozantinib). The sustained drug release and enhanced tumor region targeting profiles of the liposome nanosystem enable the dramatically increased tumor inhibition effect and reduce dose-associated adverse effects during renal cell carcinoma therapy (Fig. 5) (Kulkarni et al., 2016). Moreover, the EGFR and HER2 dual inhibitor lapatinib-incorporated lipoprotein nanoparticles (LTNPs) were reported to accumulate in the tumor region by the prolonged circulation time and the enhanced permeability and retention (EPR) effect. When compared with the lapatinib suspension, LTNPs arrest BT-474 cancer cells in the G0/G1 phase more effectively, indicating potential for targeted breast cancer therapy (Zhang et al., 2014).

Figure 5.

The scheme for XL-184 liposomes assembly and the tumor inhibition behavior in vitro

and in vivo. Adapted from (Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 1853–1861). Copyright (2016) with permission from Elsevier Science.

3.1.3 Polymeric Nanoparticles To improve the solubility and bioavailability of TKIs, nanoparticles with sustained drug release properties are employed as an effective route for TKI delivery. Compared with the other conventional nanocarriers, polymeric nanoparticles exhibit some advantages in biomedical applications owing to their biocompatibility and biodegradability. When encapsulated in polymeric nanoparticles, the degradation of chemotherapeutic drugs during circulation in vivo can be avoided effectively, and the elimination half-life is also prolonged (Naahidi et al., 2013). In addition, polymeric nanoparticles with an appropriate size distribution can accumulate in the tumor region passively by EPR effect (Ni et al., 2017b; Alves Rico et al., 2017). Moreover, retention time in the tumor region can also be delicately regulated by the variable design (Hu et al., 2018). Above all, the surfaces of polymeric nanoparticles can be modified by a variety of specific ligands to achieve active targeting delivery (Deng et al., 2012; Zhou and Dai, 2018).

For instance, imatinib, as the first generation of

small molecule TKIs in the treatment of CML, was limited by the solubility and toxicity on normal tissue, as well as the drug resistance. The imatinib-loaded PLGA nanoparticles with Nrp1-targeting modification displayed the inhibition effect on the Treg cells of tumor microenvironment, which was benefit for the tumor immunotherapy (Ou et al., 2018). Besides, the polycaprolactone (PCL)-based copolymer PCEC were introduced to encapsulated gefitinib, and the obtained gefitinib nanoparticles exhibited a narrow particle size distribution and desired drug loading efficiency, which were benefit to increase the drug accumulation in tumor via EPR effect and reduce the dose-related toxicity. PEGylation of carriers decreased the adsorption of serum proteins on the surface of nanoparticles, resulted in the prolonged circulation time and improved antitumor efficacy (Ni et al., 2017b). 3.2 Inorganic nanoparticles 3.2.1 Silica nanoparticles Mesoporous silica nanoparticles are commonly utilized as multifunctional nanocarriers due to their mesoporous structure and high surface area. As a result, drugs can be deposited in the porous structure of nanoparticles and acquired high drug-loading efficiency. Similar to other kind of nanoparticles, mesoporous silica nanoparticles with surface modification could enhance the intracellular uptake.

Moreover, nanoparticles can also exhibit improved biocompatibility and reduced cytotoxicity by adjusting the particle size, shape and surface charge (Mekaru et al., 2015; Tang et al., 2012). In addition, mesoporous silica nanoparticles could load with contrast agents for MRI imaging (Ni et al., 2017a). Hence, mesoporous silica nanoparticles provide an opportunity to increase the poor solubility and stability of TKIs by deposited in the porous structure, and the sustained drug release profiles also indicate the potential to reduce the toxicity in long-term administration of TKI therapy (Wang et al., 2015). For instance, the amphiphilic polymer cyclodextrin was introduced to modify the surface of silica nanoparticles for sorafenib delivery, which resulted in the improved solubility of the hydrophobic drug and controlled drug release in vivo (Correia et al., 2015). On the other hand, nucleic acid delivery for gene therapy also employed porous nanoparticles as vectors due to effective cell internalization and tumor targeting effect. Silica nanoparticles modified with lactobionic acid (LA) were reported as the carriers with enhanced positive tumor targeting efficacy for VEGFR-targeted siRNA (siVEGFR) and sorafenib loading, and the

obtained

nanosystem

showed

synergistic

inhibition

of

hepatocellular

carcinoma (HCC) (Fig.6) (Zheng et al., 2018).

Figure 6 A). Sorafenib-loaded silica nanoparticles (SO/siVEGF@MSN-LA NPs) was fabricated and enhanced intracellular uptake for gene therapy; B). The TEM images of MSN-NH2 (left) and

SO/siVEGF@MSN-LA NPs (right); C). and D). Intercellular transfection efficiency of siVEGF by various nanoparticles delivery. Adapted from (European Journal of Pharmaceutical Sciences, 2018, 111, 492-502). Copyright (2018) Elsevier Science.

3.2.2 Magnetic Nanoparticles Among the various magnetic nanomaterials, superparamagnetic iron oxide (SPIO or Fe3O4) has been widely used due to its favorable biocompatibility and magnetic properties. On the one hand, SPIO is usually co-loaded with therapeutic drugs as vehicles for physical targeting (Tom et al., 2018). For example, dasatinib-loaded Fe3O4 magnetic micelles were utilized enhance tumor-targeting therapy in triple-negative human breast cancer with an external magnetic field (Sabra et al., 2019). Moreover, SPIO also serves as a probe for magnetic resonance imaging (MRI) as a noninvasive diagnostic tool in biomedical applications (Bárcena et al., 2009). Hsu et al. constructed magnetic nanoparticles (FeDC-E NPs) loaded with SPIO and erlotinib for intracellular uptake tracking. Notably, the FeDC-E NPs were displayed significant cytotoxicity to CL1-5-F4 EGFR-overexpressing cancer cells. In contrast, the biocompatibility of this nanoformulation to normal cells was observed (Ali et al., 2016; Hsu et al., 2018). In a recent report, the multitargeted TKI pazopanib was encapsulated in a magnetic responsive nanobubble system which could stimulate drug release triggered by ultrasound. The improved drug distribution in the tumor region resulted in enhanced therapeutic outcome (Hamarat Şanlıer et al., 2019). 3.2.3 Calcium carbonate Nanoparticles Calcium carbonate (CaCO3) nanoparticles as a novel inorganic nanoparticles have caused extensive public concern in the field of targeting drug systems due to biocompatibility and slow biodegradability. Moreover, is easy to acquire and has been applied to various fields (Maleki Dizaj et al., 2015). Most importantly, CaCO3 nanoparticles displayed pH-dependent properties due to their physical and chemical properties. As the pH decrease, the dilution rate of CaCO3 becomes faster. Accordingly, the nanoparticles are able to as the responsive nanosystem for drug delivery to enhance the therapeutic effect, because the pH value in tumor microenvironment is lower than that of normal tissue (Kamba et al., 2013). Based on the above advantages, CaCO3 are employed as carriers for TKIs (sorafenib) delivery via LbL assembly method to enhance the anti-tumor efficiency. In this study,

sorafenib was adsorbed by the CaCO3 core, and then ionic polyelectrolytes was introduced for surface layer assembling through electrostatic interaction. The obtained nanoformulation maintained the antiangiogenic activity of Sorafenib, and in addition with the improved solubility, stability and avoiding rapid degradation in vivo (Poojari et al., 2016). Moreover, in order to overcome the drug resistance, sorafenib and microRNA-375 (miR-375) were co-loaded by CaCO3 nanoparticles with lipid coating via a water-in-oil reverse emulsion method (Fig. 7). The rapid dissociation of CaCO3 nanoparticles in acidic environment resulted in the responsive release of sorafenib and miR-375 in the tumor region, particularly in tumor cell cytoplasm (Zhao et al., 2018). Furthermore, the autophagy inhibition of MiR375 enhanced the chemotherapeutic efficiency of sorafenib, which implied that combining sorafenib with miR375 was a potential strategy for combination therapy of HCC (He et al., 2011).

Figure 7. A). The scheme for calcium carbonate nanoparticles (miR-375/Sf-LCC NPs) and intracellular release behavior for multitargeting mechanism; B). Tumor growth curves of miR-375/Sf-LCC treatment which exhibited favorable antitumor effect; C). TEM imaging of tumor tissue slides with miR-375/Sf-LCC and NC/Sf-LCC treatments; D). The biodistribution of miR-375/Sf-LCC, which showed more extensive distribution in tumor. Adapted from (Acta Biomaterialia, 2018, 72, 248-255). Copyright (2018) with permission from Elsevier Science.

3.3 Noble Metal Nanoparticles Noble metal nanoparticles, such as gold nanoparticles (AuNPs), have been of interest in recent years as potential nanocarriers in precision cancer therapy with the

advantages of controllable shape and dimension, low cytotoxicity and surface plasmon resonance (LSPR) properties for biomedical imaging and photothermal convention (Grzelczak et al., 2008; Sarkar et al., 2017; Yu and Zheng, 2015). Based on these outstanding properties, the development of noble metal nanoparticles provides a new route for the delivery of small molecule TKIs. Briefly, Feng et al. designed Au nanocubes (AuNCs) with pH or temperature responsiveness for erlotinib or doxorubicin loading, respectively. On one hand, When the drug-loaded AuNCs accumulated at the tumor region, the specific acidic microenvironment of the tumor triggered the release of erlotinib precisely. On the other hand, the sequential release manner of doxorubicin by NIR laser irradiation, further enhanced the therapeutic outcome of AuNCs-based nanoformulation in A431 tumor model (Fig. 8) (Feng et al., 2019). In addition, afatinib-conjugated AuNPs not only present improved biocompatibility and anticancer efficacy, but also attenuate drug-related inflammation, which reduced the adverse effect of the TKIs obviously (Cryer et al., 2019).

Figure 8. A). The scheme of synthesis procedure for pH-responsive Au nanocubes (PAAu) and thermal-responsive Au nanocubes (PNAu), and the synergistic therapeutic mechanism of Erlotinib, doxorubicin and PTT; B). TEM images of Au nanocubes, PAAu and PNAu; C). Tumor growth curve of pAAu-Erl + pNAu-Dox treatment; D). The photo of A431 tumor-bearing mice with different treatment and H&E analysis of tumor slice after various nanocubes treatment. Adapted from (Biomaterials, 2019, 217, 119327). Copyright (2019) Elsevier Science.

Silver nanoparticles possess notable properties of antiviral, anti-inflammatory and antibacterial, and also can be used as cytotoxic agents to induce cancer cell apoptosis (Ge et al., 2014). By linking silver nanoparticles with imatinib, the IMAB-AgNPs exhibited slow and sustained drug release profiles and induced MCF-7 tumor cell apoptosis effectively (Sadat Shandiz et al., 2017).

4 TKI-based Combinatory Therapeutic Strategies 4.1 TKIs combined with Phototherapy In numerous studies, small molecule TKIs have been successfully encapsulated by nanocarriers. However, the therapeutic outcomes are still far from satisfactory. Nanoparticles not only have excellent potential for tumor region accumulation and controlled drug release behavior but also employ other therapeutic methods, especially phototherapy by multifunctional nanoparticles (Peng et al., 2019b). Photo-chemo combinational therapy has been of interest for the enhancement of chemotherapeutic outcomes by laser-irradiated photothermal conversion (Yang et al., 2018a; Yang et al., 2019b). Nanoparticles that exhibit strong absorption in the near-infrared region were chosen for laser-induced thermal ablation, which could significantly induce necrosis in tumor cells (Huang et al., 2019; Li et al., 2018; Luo et al., 2018). In addition, the near-infrared (NIR) dye can also be employed as a photosensitizer; when coloaded with antiangiogenic agents in liposomes, the optimized drug delivery system can utilize photothermal therapy combined with chemotherapy (Fig. 9) (Yang et al., 2018b). Meanwhile, light-triggered hyperthermia can promote drug release and diffusion in the tumor region, resulting in an elevated antitumor effect. Correspondingly, when photothermal therapy is utilized in albumin nanoparticles, hyperthermia can cause protein denaturation and apoptosis, generating a synergistic effect to kill tumor cells (Callaghan et al., 2016). However, the biomedical applications of photothermal therapy combined with TKIs is still limited by the penetration depth of the laser, and hyperthermia causes acute inflammation.

Figure 9. A). The scheme of sunitinib and IR780-coloaded liposomes and NIR-induced enhanced anti-angiogenesis by active tumor targeting; B). The biodistribution of Lip-IR780-Sunitinib in 4T1 tumor-bearing mice; C). The tumor growth curves and D). the mean tumor weights of various nanoparticles treatments. Adapted from (Nanomedicine-Nanotechnology, Biology and Medicine, 2018, 14, 2283-2294). Copyright (2018) Elsevier Science.

On the other hand, photodynamic therapy (PDT) is known as a noninvasive treatment modality, utilizing photosensitizers to release toxic reactive oxygen species (ROS) when exposed to lasers, which can kill tumor cells with high precision, safety and control (Zhu et al., 2018a). However, the execution of PDT mainly relies on photosensitizers (PSs) that promote NIR adsorption and increase the generation of active oxygen altering the tumor hypoxic microenvironment (Liu et al., 2012; Rai et al., 2010). Accordingly, the low selectivity and toxicity of PSs limits the application of PDT. Alternatively, a targeted delivery strategy using nanoparticles could reduce the dose-related toxicity of PSs (Zhu et al., 2018b). Furthermore, it has been reported that the inhibition of EGFR signaling could enhance PDT efficacy by inducing apoptosis (Edmonds et al., 2012). A nanotheranostic agent (ECMI) with ICG (photosensitizers) and erlotinib (EGFR TKI) was employed for synergistic photodynamic chemotherapy. The ZnO QD-capped MSNs enabled the pH and redox responsive release of ICG in the tumor region, resulting in imaging-guided PDT therapy. Furthermore, combined with the targeted molecular agent erlotinib, the enhancement of the anticancer therapeutic effect was achieved in EGFR-mutated lung cancer (Fig. 10) (Zhang et al., 2019).

Figure 10 A). The scheme for preparation of ECMI nanotheranostic agent and the mechanism of photodynamic-chemotherapy. The loaded drugs were released in tumor sites and PDT was

triggered by external NIR laser, which experts synergistic effect; B). The atomic force microscope (AFM) image and TEM image of ECMI; C). Tumor growth curve after intravenously injected various agents for 21 days treatment; D). Immunohistochemistry staining of tumor slice after treatment with or without laser irradiation, which demonstrated that NIR could suppress expression of EGFR. Adapted from (Chemical Engineering Journal. 2019, 372, 483–495.). Copyright (2019) Elsevier Science.

4.2 TKIs Combined with other Chemotherapeutics Several approved small molecule agents targeting receptor tyrosine kinases still display limited efficacy in the clinic. Although the combination effect of TKIs and conventional chemotherapeutic drugs has been demonstrated by a series of clinical studies, poor solubility and low bioavailability are still drawbacks for these drugs (Leo et al., 2008; Satoh et al., 2014). To improve the therapeutic outcome and minimize chemoresistance, drug delivery systems were employed for TKI-based combination chemotherapy (Blanco et al., 2015). For example, sorafenib and curcumin were coloaded into polymer nanoparticles for hepatocellular carcinoma (HCC) therapy (Cao et al., 2015), gefitinib-loaded and doxorubicin-conjugated PLA-PEG

nanoparticles

displayed

synergistic

effects

during

combination

chemotherapy (Zhou et al., 2017), and a temperature-sensitive hydrogel system combining lapatinib with paclitaxel was reported to achieve controlled drug release for the treatment of breast cancer (Hu et al., 2015). Notably, a D-T7 peptide-modified

PEGylated bilirubin nanoparticle with cediranib and paclitaxel codelivery was fabricated to overcome the blood brain barrier for glioma-targeting therapy. Moreover, light- and ROS-stimulated in situ drug release properties achieved desirable antiangiogenesis and antiglioma effects (Fig. 11) (Yu et al., 2019). These research results demonstrate that combination chemotherapy not only alleviated the dose-related adverse effects during treatment but also obviously enhanced the therapeutic outcome by inhibiting angiogenesis and inducing tumor cell apoptosis. In conclusion, combination chemotherapy provides the potential to further improve antitumor efficacy and reverse chemoresistance.

Figure 11 A) The scheme for glioma-targeted nanoparticles design and the in-situ drug release property for improvement of antiangiogenesis; B). The fluorescence imaging of D-T7 peptide modified BRNPs (TBRNPs), which displayed long retention time in tumor regions; C). The curves of body weight and survival rate with various treatment. Reprinted (adapted) with permission from (ACS Appl. Mater. Interfaces 2019, 11, 176−186.). Copyright (2019) American Chemical Society

4.3 TKIs Combined with Immunotherapy

During targeted cancer treatment, TKI-based combination chemotherapy displayed a more satisfactory therapeutic outcome than conventional chemotherapy. However, some patients still suffered from tumor recurrence and metastasis, even if the chemotherapy was effective initially. The investigations of immuno-oncology have unveiled the complexity of tumor invasion and metastasis after chemotherapy. Alternatively, increasing immune activation has become the most promising strategy for future targeted tumor therapy (Yang et al., 2019a). Recent studies have demonstrated that the tumor microenvironment (TME) essentially contributes to the progression and metastasis of cancer (Peng et al., 2019a). The polarization of tumor-associated macrophages (TAMs) promotes related cytokines, subsequently resulting in DC maturation and T cell differentiation, which are considered a potential route for regulating TME immunity (Mantovani et al., 2017). During targeted therapy of NSCLC, the EGFR-T790M mutations became the main reason for TKI resistance. To overcome chemoresistance, TME remodeling and TAM targeting therapeutic strategies were developed for targeted TKI delivery (Klemm and Joyce, 2015). Yin et al. utilized the PD-L1 nanobody to construct a novel TAM-specific liposome for simvastatin and gefitinib codelivery. The regulation of TAM-related immunity, combined with antiangiogenesis effects, present enhanced antitumor efficacy for the treatment of gefitinib-resistant NSCLC (Fig. 12) (Yin et al., 2018). Moreover, ibrutinib-loaded sialic acid (SA)-modified nanocomplexes were designed and fabricated for targeted delivery of ibrutinib to TAMs. The TAM-targeting delivery of ibrutinib with prolonged circulation time and enhanced tumor accumulation enabled effective Bruton’s tyrosine kinase inhibition, thereby resulting in active antitumor immunity (Qiu et al., 2019). The combination of erlotinib and anti-PD-L1 antibody-based immunotherapy resulted in dramatically improved tumor inhibition efficacy by the erlotinib-induced normalization of tumor vasculature and antibody-modulated TME (Chen et al., 2017). In recent years, Immune Checkpoint Blocking (ICB) has attracted attention for its remarkable clinical efficacy in cancer immunotherapy. However, the efficiency and prognosis of immunotherapy by immune checkpoint inhibitor used alone is still far from satisfactory. The regulatory T (Treg) cells induced immunosuppression is considered as the principal obstacle in immunotherapy. Although imatinib was failed in the treatment of CML due to mutations of BCL-ABL kinase, it was reported to impair the suppressive functions Treg cell. Therefore, the imatinib-loaded PLGA

nanoparticles with Nrp1-targeting modification was designed for Treg cells targeting and downregulating Foxp3 expression in Treg cell. When combined with the cytotoxic T-lymphocyte antigen-4 antibody ( α -CTLA-4), the intratumoral cytotoxic CD8+ T cells were further activated, resulted in long-term antitumor effect (Ou et al., 2018).

Figure 12 A). The antitumor evaluation in vivo: tumor growth curve, photograph of tumors, tumor weight variation and tumor inhibition ration; B). In vivo ultrasound imaging of blood vessels in the tumor areas; C). Immunofluorescence staining for the co-localization of liposomes and PD-L1 in tumor slides and the western bolt analysis of PD-L1 expression in tumor and TAM; D). Immunofluorescence staining for identification of TAM repolarization (the marker of M1Φ was iNOS, and the marker of M2Φ was CD206). Reprinted (adapted) with permission from (Small, 2018, 14, 201802372). Copyright (2018) Wiley Online Library.

5. Perspectives and challenges Since the beginning of the twentieth century, the discovery of tyrosine kinase inhibitor drugs has made enormous progress, and several small molecule TKIs have been developed as targeted molecular therapy approaches for cancer. Some of these drugs are approved by the FDA and show clinical advantages in monotherapy or in combination with other drugs, while other drugs are still undergoing clinical trials. According to clinical research results, targeted molecular drugs combined with

conventional chemotherapy can achieve the maximum therapeutic effect; however, reduced selective inhibition during treatment and the emergence of tumor resistance have become obstacles to the development of targeted molecular drugs. New signal transduction anticancer agents based on personalized therapy may overcome or delay drug resistance, but the molecular mechanisms associated with multidrug resistance remain to be further studied. Notably, the application of nanotechnology for small molecule TKI delivery has provided an alternative route for the promotion of therapeutic outcomes. The enhancement of therapeutic efficacy by TKIs delivery approaches is focused on improving the poor solubility and oral bioavailability, prolonging the circulation time, enhancing accumulation in the tumor region, and reducing the adverse effects caused by nonspecific distribution. Despite the significant advances in new treatment options, the current nanoparticle-based TKI delivery strategies still have some challenges for further clinical applications. Time- and spacial-controlled release strategies for TKIs delivery systems are required for further inducing cell-specific uptake and intracellular endosomal escape of therapeutic molecules, and the responsive or “on-demand” release manner is crucial for multidrug delivery. Moreover, the development of a multifunctional delivery system should focus not only on improving selective activity with minimized adverse effects of TKIs but also on controlling acquired drug resistance. For instance, nanosystems for TME regulation, TAM-targeting immunotherapy, and combinational therapy would be promising strategies to reverse TKI resistance.

Declaration of interests The manuscript was written through the contributions of all authors, and no conflicts of interest to declare for this work.

Funding Sources

This work was financially supported by the National Natural Science Foundation of China (grant numbers NSFC31600811 and NSFC31871008); the Scientific Research Foundation for the Returned Overseas Chinese Scholars, Sichuan Province, China; the China Postdoctoral Science Foundation (grant numbers 2018M643486); the Research Project of Innovative Research Team in Chengdu Medical College (grant number CYTD18-05). References

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Graphical Abstract

Nanoformulations of Small Molecule Protein Tyrosine Kinases Inhibitors Potentiate Targeted Cancer Therapy Yanlong Yin a, #, Xiao Yuan a, #, Huile Gao a, b, *, Qian Yang a, * a

The School of Pharmacy, College Key Laboratory of Sichuan Province for Specific

Structure of Small Molecule Drugs, Chengdu Medical College, Chengdu, 610500, China b

Key Laboratory of Drug-Targeting and Drug Delivery System of the Education

Ministry, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research, Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu, 610064, PR China

* Corresponding author. E-mail: [email protected], [email protected] (H. Gao); [email protected] (Q. Yang). # These

authors did equal contributions to this work

Protein tyrosine kinases (PTKs) have become the most promising targets for cancer therapy. Tyrosine kinase inhibitors (TKIs) have been developed as targeted molecular therapy approaches for cancer, and the nanoparticle-based TKIs delivery strategies are introduced to improve their therapeutic. This work is focused on the research status of small molecule TKIs and the applications of drug delivery systems for TKIs. The strategies for TKIs nanoformulation-based combination therapeutic strategies are discussed as well.

Nanoformulations of Small Molecule Protein Tyrosine Kinases Inhibitors Potentiate Targeted Cancer Therapy Yanlong Yin a, #, Xiao Yuan a, #, Huile Gao a, b, *, Qian Yang a, * a

The School of Pharmacy, College Key Laboratory of Sichuan Province for Specific

Structure of Small Molecule Drugs, Chengdu Medical College, Chengdu, 610500, China b

Key Laboratory of Drug-Targeting and Drug Delivery System of the Education

Ministry, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research, Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu, 610064, PR China

Conflict of Interest： The manuscript was written through the contributions of all authors, and no conflicts of interest to declare for this work.

Corresponding Author * E-mail: [email protected], [email protected] (H. Gao); [email protected] (Q. Yang). # These

authors did equal contributions to this work