Cascade Dual-Targeted Nanocarrier for Enhanced Alectinib Delivery to ALK-positive Lung Cancer
Fanjun Zenga,b, Bin Xua,Hongyuan Zhub, Shaowei Wua, Guoqing Liaoa, Daipeng Xiea, Luyu Huanga, Guibing Qiaoa,b,c, Xianzhu Yang,c Haiyu Zhoua,b,c,* a
Abstract
Alectinib is a highly efficacious inhibitor for the treatment of anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC) in the clinic; however, serious adverse events (AEs) occurred in 44.0% of patients. Herein, we explored magnetic/TAT dual-targeted nanocarriers as delivery systems for alectinib. Magnetic targeting efficiently enhanced the extravasation of alectinib-loaded nanoparticles from vessels into tumor tissue, while TAT targeting reactivated in the tumor tissue significantly improved tumor cellular uptake of the nanocarrier. As a result, this dual-targeted polymeric nanocarrier exhibited superior therapeutic effects and induced tumor shrinkage in vivo. Meanwhile, this dual-targeted nanocarrier also minimized alectinib-induced hepatotoxicity, providing an efficient strategy to extend the application of alectinib for NSCLC patients.
Keywords: dual-targeted nanocarrier; cascade targeting effect; alectinib-loaded nanoparticles; cancer therapy; ALK-positive NSCLC;
1. Introduction
Lung cancer is one of the main leading causes of cancer-related deaths, and NSCLC comprised approximately 85% of cases.1 Traditional platinum-based chemotherapeutics together with other agents, such as pemetrexed, gemcitabine, docetaxel, and vinorelbine, increased the 5-year survival rate from 5% to 14% but has plateaued.2 Over the past two decades, with the development of genetic and biomarker testing methods, molecular targeted therapy, which targets specific mutations (ALK, epidermal growth factor receptor (EGFR), tyrosine kinase inhibitors, etc.), have realized revolutionary therapeutic benefits.3-5 To date, numerous inhibitors have been developed and widely used for select NSCLC patients.6-9 For instance, crizotinib and alectinib, as first- and second-generation ALK inhibitors, efficiently prolonged the median progression-free survival (PFS) of ALK-positive NSCLC patients to 10.9 and 34.8 months, respectively.10,11 Despite great progress, serious adverse events (AEs, grades 3–5) occurred in 55.0% and 44.0% of patients with crizotinib and alectinib treatment, respectively.11 In the protocol mandated for these AEs, dose reduction, interruption, or discontinuation is usually performed; however, the therapeutic benefit is hindered accordingly.12-15 Therefore, the exploration of an effective strategy to address this dilemma is urgently needed.
In recent decades, nanoscale drug delivery systems have been widely employed to deliver chemotherapy drugs, and these nanocarriers have reduced drug toxicity in the cases of several clinically approved nanocarrier-based drugs,16-18 such as DOXIL®, Abraxane®, and Onivyde®.19, 20 In addition, nanocarriers improved drug accumulation at the tumor sites through passive and/or active targeting.21-24 As such, nanocarriers could be applicable to address the dilemma of crizotinib and alectinib-based therapies. Unfortunately, nanocarriers are rarely used as delivery systems for small molecular targeted inhibitors.
As reported, alectinib is a lipophilic inhibitor with very low aqueous solubility. Herein, we explored a cascade dual-targeted polymeric nanocarrier (named DATAT-MNCA) as a delivery system of alectinib to treat ALK-positive NSCLC. This alectinib-loaded nanocarrier DATAT-MNCA was capable of efficiently extravasating vessels into tumor tissue under the guidance of magnetic targeting. Subsequently, the slightly acidic tumor microenvironment reactivated the masked TAT targeting of DATAT-MNCA, which significantly improved tumor cellular uptake of the alectinib-loaded nanocarrier. As a result, the cascade dual-targeted polymeric nanocarrier DATAT-MNCA exhibited great therapeutic effects. More interestingly, the cascade dual-targeted nanocarrier strategy also minimized the side effects in the mouse model. Collectively, this study provides new avenues to elevate the therapeutic outcome and minimize AEs of alectinib for ALK-positive NSCLC treatment.
2. Materials and methods
2.1. Materials
Alectinib was purchased from Yuanye Bio-Technology Co. Ltd. (Shanghai, China). Hydrophobic oleic acid functionalized Fe3O4 with a diameter of 10 nm was provided by Nanjing Nanoeast Biotech Co., Ltd. (Nanjing, China). PEG-b-PHEP, TAT-PEG-b-PHEP, and DATAT-PEG-b-PHEP were provided synthesized at Kelan Biotechnology Co., Ltd. (Guangzhou, China).
2.2. Preparation of alectinib-loaded nanoparticles TAT-NCA and DATAT-MNCA.
To prepare alectinib-loaded TAT-NCA, 2.0 mL DMSO containing 10.0 mg TAT-PEG-b-PHEP and 2.0 mg alectinib was added into 10.0 mL of ultrapure water with ultrasonication; the solution was further sonicated for 1 h under ultrasonication at room temperature, transferred into a dialyzed bag (molecular weight cutoff (MWCO) of 14000 Da) and dialyzed against ultrapure water for 48 h. The solution was passed through a 0.45 m filter membrane to obtain the nanoparticle TAT-NCA.
To prepare alectinib-loaded nanoparticles DATAT-MNCA. 4.0 mL DMSO solution containing the 1.0 mg DATAT-PEG-b-PHEP, 2.0 mg alectinib, and 1.0 mg Fe3O4 was added into 10.0 mL of ultrapure water, and the performed following the similar method as described above to obtain the nanoparticle DATAT-MNCA.
To determine the alectinib content, the alectinib-loaded nanoparticles were freeze-dried, and DMSO was added to dissolve alectinib. Subsequently, the contents of alectinib in these solutions was determined by a UV spectrophotometer (UV-2600, Shimadu, Japan), with reference to a standard curve of alectinib (0.0625-2.0 g/mL) at λex = 380 nm. And, the contents of Fe3O4 in the DAT-MNCA was determined by inductively coupled plasma-optical emission spectrometry (ICP-OES; iCAP 7200 ICP-OES (iCAP 7200 ICP-OES, Thermo Fisher, USA). The encapsulation efficiency of alectinib and Fe3O4 were calculated according to the following formulae:encapsulation efficiency (%)= (amount of alectinib or Fe3O4 in nanoparticle)/total amount of alectinib or Fe3O4 added)×100%In addition, DiD-labeled TAT-NCA and NCA or DAT-MNCA were fabricated by adding DiD (0.2 mg) to the preparation.
2.3. The size and morphology of nanoparticle
The size of TAT-NCA was determined by dynamic light scattering (DLS, Malvern Zetasizer Nano ZS90, Worcestershire, UK), while the morphology of TAT-NCA was observed by transmission electron microscopy (TEM, JEOL-2010, Tokyo, Japan).
2.4. Stability of nanoparticle
The nanoparticle TAT-NCA (1.0 mg/mL) was dispersed in PBS containing 10% FBS, and the size change of TAT-NCA were recorded by DLS at each predetermined time points.
2.5．In vitro drug release
The nanoparticle TAT-NCA (3.0 mL, 75.0 g) was added to a dialysis bag (MWCO of 14000 Da) and suspended in PB buffer (20 mL, 0.02 M; pH 7.4, pH 6.5, or pH 5.5) with gentle shaking in a shaker incubator at 37 C. External PB buffer was collected at predetermined time points, the external PB buffer was replaced with fresh PB buffer, and the collected PB buffer was freeze-dried and redissolved in DMSO to determine alectinib contents by UV–Vis spectrophotometry as described above.
2.6. Cell culture
The human lung adenocarcinoma cell line H2228 obtained from American Type Culture Collection (ATCC, Rochville, MD, USA) were cultured in complete RPMI 1640 medium with 10% FBS and 1% penicillin/streptomycin in a 5% CO2 incubator.
2.7. Cellular uptake of TAT-NCA by H2228 cells
H2228 cells were co-cultured with DiD-labeled TAT-NCA or NCA at a DiD dosage of 2.5 μg/mL. After 4 h of incubation, the H2228 cells were rinsed with PBS and fixed with 4% paraformaldehyde. Afterwards, DAPI was added to stain the nucleus. The samples were observed by confocal laser scanning microscopy (CLSM). In addition，H2228 cells were co-cultured with DiD-labeled TAT-NCA or NCA at a DiD dosage of 2.5 μg/mL. After 4 h of incubation, cells were collected by trypsinization, washed with PBS and suspended in 300 μL of PBS. The intracellular fluorescence intensity was quantitated by flow cytometer (BD Accuri C6 plus, BD Bioscience, USA).
2.8. Cytotoxicity assay
The H2228 cells were incubated with TAT-NCA or free alectinib at a series of concentrations ([AT] = 0.01, 0.05, 0.25, and 1.25 μg/mL). And, the viability of these treated H2228 cells was determined by MTT (Sangon Biotech, Shanghai, China) assay after further incubation for 72 h.
2.9. Annexin V/PI staining
H2228 cells were incubated with TAT-NCA and free alectinib ([AT] = 1.25 μg/mL). After 72 h of coculture, the cells were trypsinized, washed, and further stained with an Annexin V-FITC apoptosis detection kit (Thermo Fisher, Waltham, USA) following the manufacturer’s instructions, and then analyzed by flow cytometry (BD FACS CantoTM).
2.10. Animal and tumor models
Female BALB/c nude mice at the age of 6~8 weeks was supported by Hunan SJA Laboratory Animal Co., Ltd. The animal experiments were carried out according to the Guide for the Care and Use of Laboratory Animals. All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the South China University of Technology, and approved by the Animal Ethics Committee of South China University of Technology. H2228 tumor-bearing BALB/c-nude mice were prepared by hypodermic injection of 5106 cells into the mammary fat pads of each mouse.
2.11. Biodistribution of DATAT-MNCA.
To investigate the in vivo distribution, the mice bearing H2228 tumors were injected intravenously with DATAT-MNCA ([DiD] = 0.5 mg/kg), and a small magnet was attached to the tumor site for magnetic guidance (MG). The In-Vivo Xtreme imager (Bruker, Germany) was employed to investigate the biodistribution of DATAT-MNCA. The mice were sacrificed at 72 h postinjection, and the major organs and tumor tissue were excised for imaging.
2.12. Antitumor efficacy
H2228 tumor-bearing mice were injected of 200 L PBS, free alectinib, DATA-/MNCA, and a small magnet was attached to the tumor site for the DATAT-MNCA plus MG group. All treatments were administered through intravenous injection every 3 days, and the dosage of alectinib was 3.0 mg/kg. Tumor size and weight were measured every 3 days. After the last treatment, mice were sacrificed, and the tumors and major organs were collected.
2.13. TUNEL and Ki67 staining
The harvested tumors were fixed in 4% formalin buffer, processed routinely into paraffin for preparation of tissue slides. The slides were stained for the terminal transferase dUTP nick-end labeling (TUNEL) and Ki67 assay.
2.14. Toxicity estimation of the nanoparticle
Healthy BALB/c mice were administered DATAT-MNCA and free alectinib via intravenous injection at various doses ([AT] = 1.0, 3.0, 5.0, and 10.0 mg/kg). The body weights of the mice were measured daily during the study. Mice were sacrificed 14 days after administration, and blood and livers were collected for histological characterization and blood chemistry analyses and hematoxylin and eosin (H&E) staining, respectively.
3. Results and discussion
3.1. Preparation and characterization of alectinib-loaded TAT targeted nanocarrier.
The TAT peptide, which is derived from human immunodeficiency viruses and has the excellent targeting capability, has been widely used to decorate nanocarriers to enhance cellular uptake.25 As such, we used the TAT-functioned amphiphilic diblock polymer TAT-PEG-b-PHEP to fabricate a TAT-targeted nanocarrier to encapsulate lipophilic alectinib (Figure 1A). The resultant TAT-NCA displayed an average diameter of approximately 122.0 nm and exhibited a regular and spherical morphology (Figure 1B), and the drug encapsulation efficiency of alectinib into TAT-NCA was approximately 44.9%. Furthermore, the stability of TAT-NCA nanoparticles was also evaluated. As shown in Figure 1C, after coincubation in PBS containing 10% FBS, TAT-NCA demonstrated a slight size increase within 24 h, suggesting good colloidal stability. Furthermore, we also evaluated alectinib release from TAT-NCA at various pH values. As shown in Figure 1D, alectinib release from TAT-NCA showed a pH-dependent profile. For instance, approximately 36.0%, 43.8%, and 51.3% alectinib were released from TAT-NCA at pH 7.4, pH 6.5, and pH 5.5, respectively, suggesting that alectinib release could be accelerated in tumor cells.
3.2. Evaluation of the in vitro anticancer effect
According to our design, TAT decoration significantly enhanced tumor cellular uptake of TAT-NCA (Figure 2A). To verify this, the nanoparticle NCA without TAT decoration was used as a control formulation, and both TAT-NCA and NCA were labeled by DiD. H2228 cells were coincubated with DiD-labeled TAT-NCA and NCA for 4 h, and then observed by CLSM. As shown in Figure 2B, only H2228 cells showed a slight intracellular fluorescence signal after incubation with NCA without TAT. In contrast, the intracellular red fluorescence signal was dramatically improved in the TAT-NCA group, which clearly confirmed that the TAT decoration significantly enhances tumor cellular uptake of alectinib-loaded nanoparticles TAT-NCA. In addition, the enhanced tumor cell uptake of the TAT decorated nanoparticles was further corroborated by flow cytometry. The H2228 cells incubated with TAT-NCA were much stronger fluorescence signals than the cells which incubated with TAT-NCA (Figure 2C and 2D), further corroborating the TAT peptide-mediated enhanced cellular uptake of alectinib-loaded nanoparticles TAT-NCA.
The enhanced cellular uptake of TAT-NCA by H2228 cells should elevate the therapeutic outcome. To demonstrate this, we evaluated the anticancer activity of TAT-NCA and free alectinib against H2228 cells. Figure 3A indicated that treatment with either TAT-NCA or free alectinib exhibited dose-dependent anticancer activity, and the cell viabilities of TAT-NCA were much lower than those of free alectinib at all concentrations (Figure 3A). For instance, at an alectinib concentration of 0.05 μg/mL, the cell viability of the free alectinib group was 86.6%, while only 46.9% of H2228 cells survived after treatment with TAT-NCA, showing the superior therapeutic outcome of TAT-NCA over free alectinib.
In addition, the cell apoptosis of TAT-NCA against H2228 cells was further evaluated. After treatment with TAT-NCA at an alectinib concentration of 1.25 μg/mL for 72 h, the apoptosis of these treated cells was analyzed. As shown in Figure 3B and 3C, free alectinib induced 50.7% cell apoptosis (early and late apoptotic cells), while TAT-NCA nanoparticles increased the apoptosis rate to 80.08%. These results collectively demonstrated that TAT-NCA efficiently elevates the therapeutic efficacy against ALK-positive NSCLC cells via enhanced cellular uptake of NCA with the TAT decoration.
3.3. In vivo biodistribution of magnetic/TAT dual-targeted nanocarrier.
The obviously improved therapeutic efficacy of TAT-NCA in vitro inspired us to perform its in vivo effect on tumor growth inhibition. However, it is well known that TAT peptide-decorated nanoparticles are rapidly eliminated by the reticuloendothelial system.26 In addition, TAT inefficiently increased nanocarrier extravasation from vessels into tumor tissue.27 To address these issues, the 2,3-dimethylmaleic anhydride (DA)-modified TAT-PEG-b-PHEP diblock copolymers DATAT-PEG-b-PHEP was used,28 and the hydrophobic magnetic Fe3O4 nanoparticles were coencapsulated into nanocarrier. The resultant alectinib/Fe3O4 co-loaded nanocarrier was denoted as DATAT-MNCA. For the DATAT-MNCA, The Fe3O4-mediated magnetic targeting could efficiently enhance the extravasation of nanoparticles from vessels into tumor tissue, and reactivated TAT targeting in the tumor tissue would improve tumor cellular uptake of the nanocarrier, achieving a reasonable cascade of magnetic and TAT targeting effects (Figure 4A). The successful encapsulation was confirmed by UV–Vis and TEM measurements (Figure 4B). And, the encapsulation efficiencies of alectinib and Fe3O4 were approximately 40.7% and 25.6%, respectively. To demonstrate the dual-targeting effect, the biodistribution of DiD-labeled DATAT-MNCA was investigated in mice bearing H2228 tumors. A small magnet was attached at the tumor site and kept for 24 h to realize magnetic targeting. The fluorescence intensity of tumor sites gradually increased within 24 h after the injection of DATAT-MNCA with or without a magnetic targeting effect (Figure 4C). With MG, the fluorescence intensities of DiD were obviously higher than those of the bare DATAT-MNCA group at 6 h postinjection. Moreover, at 72 h postinjection, the major organs and tumor tissues of the mice were collected for imaging. As shown in Figure 4D and 4E, the fluorescence signal of tumor tissue in the DATAT-MNCA plus MG group was obviously stronger than that in the bare DATAT-MNCA group.
3.4. In vivo therapeutic effect and safety evaluation of magnetic/TAT dual-targeted nanocarrier.
Furthermore, the in vivo anticancer activity of DATAT-MNCA with a dual-targeting cascade effect was evaluated. The mice bearing H2228 tumors were administered DATAT-MNCA with or without MG, and free alectinib was used as a control. The injection dose of alectinib was 3 mg/kg. As shown in Figure 5A, free alectinib slightly inhibited the growth of H2228 tumors compared to the PBS group. Treatment with bare DATAT-MNCA efficiently inhibited tumor growth. Furthermore, treatment with DATAT-MNCA plus MG induced tumor shrinkage, and the tumor growth rate was decreased to 70.15% at the end of therapy (Figure 5B). Moreover, the images (Figure 5C) and weights (Figure 5D) of the tumor tissue further supported the best anticancer efficacy of DATAT-MNCA plus MG. Moreover, the immunohistochemical staining (Figure 5E) also clearly showed that the highest level of tumor cell apoptosis (green) and lowest percentage of tumor cell proliferation (Ki67-positive, brown) were found in the DATAT-MNCA plus MG group, which further verified it has the best anticancer efficacy against ALK-positive NSCLC tumors.
In addition, to evaluate the potential AEs of this formulation, the in vivo safety was determined. DATAT-MNCA and free alectinib were administered at various doses, and the change in the body weight was first examined. As shown in Figure 6A, treatment with free alectinib at an injection dose of 3.0 or 5.0 mg/kg induced an obvious decrease in body weight, and the mice died immediately when the injection dose increased to 10.0 mg/kg. In contrast, we did not observe obvious body weight loss following treatment with DATAT-MNCA, even at an injection dose of 10.0 mg/kg, and no mice died at any injection dose.
In addition to body weight, several serum markers were analyzed to evaluate the safety of DATAT-MNCA. In clinical practice, alectinib-induced hepatotoxicity is one of the major concerns. Obviously, the free alectinib treatment induced a marked increase in alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and direct bilirubin (DBIL) at all three injection doses and exhibited a dose-dependent tendency. Urea (UREA), creatinine (CREA), and uric acid (UA) also exhibited moderate increases in the free alectinib treatment group at all three injection doses. In stark contrast, treatment with DATAT-MNCA did not affect these kidney and liver function markers, even at the highest alectinib dose of 10.0 mg/kg, showing the excellent safety of DATAT-MNCA.
4. Conclusion
In summary, we successfully explored the alectinib-loaded nanocarrier DATAT-MNCA with cascade magnetic and TAT targeting capabilities. The magnetic targeting capability efficiently enhanced the extravasation of DATAT-MNCA into tumor tissue; subsequently, the slight tumor acidic microenvironment reactivated the masked TAT targeting of DATAT-MNCA to significantly improve tumor cellular uptake. As a result, this cascade dual-targeted nanocarrier DATAT-MNCA treatment initiated tumor shrinkage in the ALK-positive NSCLC tumor model, exhibiting superior therapeutic effects. More importantly, this dual-targeted nanocarrier cascade also minimized the alectinib-induced hepatotoxicity effect in a mouse model.
Collectively, this study provides new avenues to improve the therapeutic outcome and minimize the AEs of alectinib for ALK-positive NSCLC treatment. Overall, this study provides a promising approach to improve the therapeutic outcome and minimize the AEs of alectinib for ALK-positive NSCLC treatment.
References
1. S. RL, M. KD and J. A, CA Cancer J. Clin., 2020, 70, 7-30.
2. F. R. Hirsch, G. V. Scagliotti, J. L. Mulshine, R. Kwon, W. J. Curran, Jr., Y. L. Wu and L. Paz-Ares, Lancet, 2017, 389, 299-311.
3. K. Takeuchi, M. Soda, Y. Togashi, R. Suzuki, S. Sakata, S. Hatano, R. Asaka, W. Hamanaka, H. Ninomiya, H. Uehara, Y. Lim Choi, Y. Satoh, S. Okumura, K. Nakagawa, H. Mano and Y. Ishikawa, Nat. Med., 2012, 18, 378-381.
4. C. M. Della Corte, G. Viscardi, R. Di Liello, M. Fasano, E. Martinelli, T. Troiani, F. Ciardiello and F. Morgillo, Mol. Cancer, 2018, 17, 30.
5. D. B. Doroshow and R. S. Herbst, JAMA Oncol., 2018, 4, 569-570. View Article Online
6. F. Casaluce, A. Sgambato, P. Maione, A. Rossi, C. Ferrara, A. Napolitano, G. Palazzolo, F. Ciardiello and C. Gridelli, Target Oncol., 2013, 8, 55-67.
7. B. J. Solomon, T. Mok, D. W. Kim, Y. L. Wu, K. Nakagawa, T. Mekhail, E. Felip, F.Cappuzzo, J. Paolini, T. Usari, S. Iyer, A. Reisman, K. D. Wilner, J. Tursi, F. Blackhall and P. Investigators, N. Engl. J. Med., 2014, 371, 2167-2177.
8. V. Noronha, V. M. Patil, A. Joshi, N. Menon, A. Chougule, A. Mahajan, A. Janu, N. Purandare, R. Kumar, S. More, S. Goud, N. Kadam, N. Daware, A. Bhattacharjee, S. Shah, A. Yadav, V. Trivedi, V. Behel, A. Dutt, S. D. Banavali and K. Prabhash, J. Clin. Oncol., 2020, 38, 124-136.
9. P. A. Brown, B. Shah, A. Fathi, M. Wieduwilt, A. Advani, P. Aoun, S. K. Barta, M. W. Boyer, T. Bryan, P. W. Burke, R. Cassaday, P. F. Coccia, S. E. Coutre, L. E. Damon, D. J. DeAngelo, O. Frankfurt, J. P. Greer, H. M. Kantarjian, R. B. Klisovic, G.Kupfer, M. Litzow, A. Liu, R. Mattison, J. Park, J. Rubnitz, A. Saad, G. L. Uy, E. S. Wang, K. M. Gregory and N. Ogba, J. Natl. Compr. Canc. Netw., 2017, 15, 1091-1102.
10. S. Peters, D. R. Camidge, A. T. Shaw, S. Gadgeel, J. S. Ahn, D. W. Kim, S. I. Ou, M. Perol, R. Dziadziuszko, R. Rosell, A. Zeaiter, E. Mitry, S. Golding, B. Balas, J. Noe, P. N. Morcos, T. Mok and A. T. Investigators, N. Engl. J. Med., 2017, 377, 829-838.
11. D. R. Camidge, R. Dziadziuszko, S. Peters, T. Mok, J. Noe, M. Nowicka, S. M. Gadgeel, P. Cheema, N. Pavlakis, F. de Marinis, B. C. Cho, L. Zhang, D. Moro-Sibilot, T. Liu, W. Bordogna, B. Balas, B. Muller and A. T. Shaw, J. Thorac. Oncol., 2019, 14, 1233-1243.
12. T. Hida, H. Nokihara, M. Kondo, Y. H. Kim, K. Azuma, T. Seto, Y. Takiguchi, M. Nishio, H. Yoshioka, F. Imamura, K. Hotta, S. Watanabe, K. Goto, M. Satouchi, T. Kozuki, T. Shukuya, K. Nakagawa, T. Mitsudomi, N. Yamamoto, T. Asakawa, R. Asabe, T. Tanaka and T. Tamura, Lancet, 2017, 390, 29-39.
13. K. Nakagawa, T. Hida, H. Nokihara, M. Morise, K. Azuma, Y. H. Kim, T. Seto, Y. Takiguchi, M. Nishio, H. Yoshioka, T. Kumagai, K. Hotta, S. Watanabe, K. Goto, M. Satouchi, T. Kozuki, R. Koyama, T. Mitsudomi, N. Yamamoto, T. Asakawa, M. DOI: 10.1039/D0BM00970AHayashi, W. Hasegawa and T. Tamura, Lung Cancer, 2020, 139, 195-199.
14. S. I. Ou, S. M. Gadgeel, F. Barlesi, J. C. Yang, L. De Petris, D. W. Kim, R. Govindan, A. M. Dingemans, L. Crino, H. Lena, S. Popat, J. S. Ahn, E. Dansin, E. Mitry, B. Muller, W. Bordogna, B. Balas, P. N. Morcos and A. T. Shaw, Lung Cancer, 2020, 139, 22-27.
15. J. C. Yang, S. I. Ou, L. De Petris, S. Gadgeel, L. Gandhi, D. W. Kim, F. Barlesi, R. Govindan, A. C. Dingemans, L. Crino, H. Lena, S. Popat, J. S. Ahn, E. Dansin, S. Golding, W. Bordogna, B. Balas, P. N. Morcos, A. Zeaiter and A. T. Shaw, J. Thorac. Oncol., 2017, 12, 1552-1560.
16. G. M. Ngandeu Neubi, Y. Opoku-Damoah, X. C. Gu, Y. Han, J. P. Zhou and Y. Ding, Biomater. Sci., 2018, 6, 958-973.
17. J. Wolfram and M. Ferrari, Nano Today, 2019, 25, 85-98.
18. H. S. El-Sawy, A. M. Al-Abd, T. A. Ahmed, K. M. El-Say and V. P. Torchilin, ACS Nano, 2018, 12, 10636-10664.
19. H. J. Zhang, Onco. Targets Ther., 2016, 9, 3001-3007.
20. G. Ruan and S. S. Feng, Biomaterials, 2003, 24, 5037-5044.
21. M. Ovais, S. Mukherjee, A. Pramanik, D. Das, A. Mukherjee, A. Raza and C. Chen, Adv. Mater., 2020, 32, 2000055.
22. B. T. Mai, S. Fernandes, P. B. Balakrishnan and T. Pellegrino, Acc. Chem. Res., 2018, 51, 999-1013.
23. L. L. Zhang, C. G. Jiang, F. J. Zeng, H. Y. Zhou, D. D. Li, X. Y. He, S. Shen, X. Z. Yang and J. Wang, Biomater. Sci., 2020, 8, 2255-2263.
24. D. Rosenblum, N. Joshi, W. Tao, J. M. Karp and D. Peer, Nat. Commun., 2018, 9, 1410.
25. Y. Z. Huang, Y. F. Jiang, H. Y. Wang, J. X. Wang, M. C. Shin, Y. Byun, H. He, Y. Q. Liang and V. C. Yang, Adv. Drug Deliv. Rev., 2013, 65: 1299-315.
26. Y. Qian, Y. Wang, F. Jia, Z. Wang, C. Yue, W. Zhang, Z. Hua and W. Wang, Biomaterials 2019, 188, 96-106.
27. K. Kurrikoff, U. Langel, Expert Opin Drug Deliv, 2019, 16, 1183-1191.View Article Online
28. D. D. Ling, Y. C, Ma, J. Z. Du, W. Tao, X. J. Du, Y. Z. Yang and J. Wang, DOI: 10.1039/D0BM00970ANano Lett., 2017, 17, 2871-2878.