Construction of Supramolecular Drug-Drug Delivery System for Non

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Construction of Supramolecular Drug-Drug Delivery System for Non-Small Cell Lung Cancer Therapy Zhihao Zhang, Leilei Shi, Chenwei Wu, Yue Su, Jiwen Qian, Hongping Deng, and Xinyuan Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07565 • Publication Date (Web): 15 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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ACS Applied Materials & Interfaces

Construction of Supramolecular Drug-Drug Delivery System for Non-Small Cell Lung Cancer Therapy Zhihao Zhang, Leilei Shi, Chenwei Wu, Yue Su, Jiwen Qian, Hongping Deng*, and Xinyuan Zhu* School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China ABSTRACT Nanoscale drug delivery systems (DDSs) are generally considered to be an effective alternative to small molecular chemotherapeutics due to the improved accumulation in tumor site and enhanced retention in blood. Nevertheless, most of DDSs have low loading efficiency or even pose high threat to normal organs caused by severe side effects. Ideally, supramolecular drug-drug delivery system (SDDDS) made up of pure drugs via supramolecular interaction provides a hopeful solution for cancer treatment. Herein, we put forward a facile method to construct SDDDS via co-assembly of Gefitinib (GEF) and tripeptide tyroservatide (YSV), two kinds of chemotherapeutic pharmaceuticals for non-small cell lung cancer (NSCLC), via multiple intermolecular interactions, including hydrogen bond and π-π stacking. Through transmission electron microscope (TEM) and dynamic light scattering (DLS), GEF and YSV self-assemble into nanoparticles with regular morphology and uniform size, which facilitates the delivery of both drugs. In vitro studies demonstrate that the SDDDS is much more efficient in entering into cancer cells and inhibiting the proliferation of

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cancer cells compared with single GEF, YSV or GEF/YSV drug mixture. In vivo experiments show that the SDDDS can selectively accumulate in tumor tissue, resulting in much better drug efficacy without evident side effects. Considering the advantages of the SDDDS, we believe this strategy provides a promising route for enhanced anticancer therapy in nanomedicine. Keywords: Drug delivery, self-assembly, chemotherapy, nanoparticles, non-small cell lung cancer

1. Introduction Incessant development of effective therapeutic methods to treat fatal diseases like cancer is a long-term mission for researchers worldwide.1 Chemotherapy with small molecular anticancer drugs has achieved improved therapeutic effects in clinic, but often suffers from serious deficiencies, such as poor solubility, rapid clearance in blood and severe side effects.2-3 To overcome these defects, the development of various nanoscale drug delivery systems (DDSs) has drawn great attention, such as lipidosome,4-5 vesicle,6-7 hydrophilic polymers,8 and amphiphilic polymer nanoparticles.9-10 By means of chemical coupling or physical encapsulation of chemotherapeutics in the above carriers, these strategies show decreased side effects in vivo, as well as enhanced anticancer efficacy. Nonetheless, most of DDSs have low drug loading contents, and some of them would even be toxic or cause inflammation when they are degraded, metabolized and excreted in vivo.11-12 Recently, researchers put forward an innovative strategy to construct DDSs


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with reducing non-therapeutic carriers, which is coupling anticancer agents with amphiphilic molecular fragments via cleavable linkage.13-15 For instance, Lin and coworkers conjugated hydrophobic drug paclitaxel to assembly-inducing polypeptide to prepare amphiphilic prodrugs, which not only greatly improved the drug loading efficiency, but also reduced the amount of non-therapeutic carriers. Wang and coworkers coupled 7-ethyl-10-hydroxyl-camptothecin with oligomeric polyethylene glycol through GSH-responsive linkage, resulting in the formation of amphiphilic prodrugs, which could assemble into loading-efficiency-tunable nanodrugs. As a step forward, our group recently proposed an amphiphilic drug-drug conjugate (ADDC), which constructed DDSs completely without non-therapeutic carriers.16 Nanoparticles were formed after the self-assembly of ADDC in water, realizing self-delivery of drugs without non-therapeutic carriers in vivo. Although this strategy effectively improved the loading efficiency of drugs and enhanced the anticancer efficacy, the self-delivered system still relies on chemical modification of drugs. Moreover, researchers proposed quite a novel strategy to construct drug self-delivery systems by taking advantage of intramolecular or intermolecular interactions of different anticancer agents, which could further assemble into nanoparticles with enhanced therapeutic efficiency.17 For example, Liu and co-workers reported a facile method to construct therapeutic nanoparticles via peptide-tuned self-assembly of Ce6.18 Since multiple interactions exist between amphiphilic polypeptide and Ce6, the assembled nanoparticles exhibit tunable size,



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high loading efficiency and responsive release property. Then, they also constructed nanodrugs via self-assembly of a chemotherapeutic drug, doxorubicin, and a photosensitizer, Ce6,19 which combined photodynamic therapy with chemotherapy. This strategy is a valuable attempt for construction of therapeutic nanoparticles via supramolecular interactions. It can be imagined that the construction of supramolecular drug-drug delivery system (SDDDS) consisting of a molecular targeted anticancer drug and a polypeptide drug would achieve enhanced accumulation of drugs in tumor site and improved drug efficacy with minimal side effects. In this study, we report the development of SDDDS via Gefitinib (GEF) and tripeptide tyroservatide (YSV). Uniform GEF-YSV nanoparticles are generated after the co-assembly of GEF and YSV via hydrogen bond and π-π stacking to deliver these two chemotherapeutic drugs to tumor site through enhanced permeability and retention effect (EPR). The acidic tumor microenvironment could accelerate the release of free GEF and YSV due to the disruption of the multiple supramolecular interactions.20-21 GEF, the first clinically approved molecular targeted pharmaceutical for NSCLC,22 is a selective tyrosine kinase inhibitor for epithelial growth factor receptor (EGFR).23 GEF retards the growth and propagation of cancer by occupying the adenosine triphosphate (ATP) binding site of EGFR, which decreases the activity of the tyrosine kinase and blocks the signaling pathway of EGFR, inducing the apoptosis of cancer cells without causing severe side effects to normal cells.24 However, its poor aqueous solubility


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hinders the circulation to reach its effective blood drug concentration. On the other hand, as polypeptide drug, tripeptide YSV proves to have low toxicity to normal cells. Through interrupting cell cycle and suppressing the activity of histone deacetylase, it is remarkably effective in inhibiting NSCLC.25-27 Because of their divergent drug function mechanisms, it is highly possible that the nanoparticles combining these drugs can produce enhanced or synergetic effect against NSCLC.16

2. Experiments Materials. All reagents are commercially available and used without any purification. Tripeptide tyroservatide (YSV, 95%) was purchased from GL Biochem (Shanghai) Ltd. Gefitinib (GEF, 99%) was obtained from Meilun Biotech (Dalian, China). Dimethyl sulfoxide (DMSO) and sodium hydroxide (NaOH) were purchased from Adamas. 1,1,2,2-tetrachloroethane-d2, dimethyl sulfoxide-d6 were obtained from Acros. RPMI-1640 medium, F-12K medium, Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), 0.25% pancreatin and penicillin-streptomycin liquid (100×) were purchased from Gibco. CellLight®

Early

Endosomes-GFP

*BacMam

2.0*,

CellLight®

Late

Endosomes-GFP *BacMam 2.0* and LysoSensor Green DND-189 were purchased from Thermo Fisher Scientific (USA). MTT, Dulbecco’s phosphate buffered saline (PBS) and nile red (NR) were obtained from Sigma-Aldrich (USA). The Annexin V-FITC apoptosis detection kit for flow cytometry was obtained from Invitrogen. All antibodies for Western Blot were purchased from



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Abcam. Dialysis tubes (MWCO, 1000 Da or 3500 Da) were purchased from Lvniao Tech. Corp. (China). All consumable items used for cell experiments were purchased from Sangon Biotech (China). Methods. The ultraviolet-visible (UV-vis) absorption spectra were measured with a Thermo EV300 UV-vis spectrophotometer. TEM (B-TEM, Tecnai G2 Spirit Biotwin) at 200 kV acceleration voltage was used to analyze the morphology of the nanoparticles. Nanoparticles suspensions (0.4 mg/mL) were dropped on copper grids with amorphous holey-carbon film covering, which were then dried overnight at 25 oC for observation. DLS was performed with a Malvern Zetasizer Nano S apparatus equipped with a 633 nm laser (4.0 mW) to analyze the size and distribution of the nanoparticles at 25 oC (0.4 mg/mL, scattering angle = 90°). Variable-temperature 1H nuclear magnetic resonance (NMR) was performed with dimethylsulfoxide-d6 and 1,1,2,2-tetrachloroethane-d2 as solvents at 298 K, 313 K and 328 K (Mercury plus 400 spectrometer, 400 MHz, Varian, U.S.A). Ultrafiltration method was used to calculate the encapsulation efficiency (EE). The amount of unencapsulated drugs in aqueous solution was measured using UV-vis absorption spectra. Based on the following equation, the EE for GEF and YSV were determined: EE% =

ℎ

− ℎ

× 100 ℎ

Based on the following equation, the drug loading content (DLC) for GEF and YSV were determined: DLC% =

ℎ ℎ

× 100 ℎ ℎ


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Preparation of YSV-GEF Nanoparticles. 15 mg YSV was firstly dissolved in 2 mL aqueous solution of 1 M NaOH, to which 50 µL GEF dissolved in DMSO (40 mg/mL) was added. The mixture was dispersed by ultrasonic dispersion for 1 h before 2 hours’ stirring at room temperature. After that, dialysis was performed (MWCO = 1000 Da) against super-pure water for 3 h to eliminate DMSO and NaOH. Every other hour, the external water was renewed. GEF and YSV which failed to assemble were removed by centrifugation. Lyophilization of the resulting nanoparticles was performed by a lyophilization apparatus before performing further characterizations. In Vitro Drug Release from the GEF-YSV Nanoparticles. The property of responsive and controlled release of the GEF-YSV nanoparticles was confirmed by dialysis method. 1 mg GEF-YSV nanoparticles were suspended in 2 mL PBS, which was then loaded into a 3500 Da MWCO dialysis bag. The pH of PBS and acetate buffered solution were adjusted to 7.4 and 5.0, respectively. A third PBS (pH = 7.4) was supplemented with 10% FBS. The dialysis bags were immersed in these solutions at 37 oC and gently shaken in a shaker. At preselected time, 3 mL of the buffer solutions were sampled in each group, and subsequently supplemented with fresh buffer solution with the original contents respectively. The released GEF was calculated by measuring the absorbance at 331 nm via UV-vis spectrometer, and the released YSV was calculated by integrating the peak area through HPLC. Cell Cultures. RPMI-1640, F-12K and DMEM mediums, supplemented with 10%



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FBS and 100 U/mL penicillin as well as 100 µg/mL streptomycin, were used in HCC827 cells, A549 cells and NIH-3T3 cells, respectively. The condition for cell cultures is 37 oC, 5% CO2 in humid atmosphere. Cell Uptake. Nile red (NR), a red fluorescent molecule, was co-assembled into nanoparticles to study the cell uptake behavior of the nanoparticles. Briefly, NR dissolved in DMSO was instilled into the solution of nanoparticles while stirring, followed by the formation of NR-loaded GEF-YSV nanoparticles. HCC827 cells were incubated in 6-well plates, with each well containing 2×105 cells and RPMI-1640 cell culture medium (3 mL) supplemented with 10% FBS. After cell adherence, HCC827 cells were incubated with the nanoparticles for 0.5 h, 1 h, 2 h, 4 h and 6 h. Flow cytometry (FCM) and confocal laser scanning microcopy (CLSM) were used to investigate the cell internalization behaviors of the nanoparticles in HCC827 cells. For FCM, HCC827 cells were rinsed with fresh PBS (pH = 7.4) and digested using pancreatin, which were then characterized with BD LSRFortessa FCM. A total of 1.0×104 gated events per sample were recorded. For CLSM, at predetermined intervals, HCC827 cells were rinsed by cool PBS twice. The resulting HCC827 cells were fixed by formalin at 25 oC and washed by PBS twice. Finally, the nuclei of the resulting HCC827 cells were stained by 100 µL DAPI (10 µg/mL) and 0.1% Triton X-100. After 15 min, the resulting samples were rinsed by fresh PBS (pH = 7.4) before they were sealed, which were then analyzed using LEICA TCS SP8 fluorescence microscope. For early endosome staining, late endosome staining and lysosome staining, HCC827 cells (2×104


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cells per well) were stained with CellLight® Early Endosomes-GFP *BacMam 2.0*, CellLight® Late Endosomes-GFP *BacMam 2.0* and LysoSensor Green DND-189, respectively. Live cell imaging was performed after 4 hours’ culture of HCC827 cells with 30 µM NR-loaded nanoparticles. In Vitro Anticancer Activity of Nanoparticles. Non-small lung cancer HCC827 and A549 cells, normal NIH-3T3 cells were used to evaluate the anticancer efficiency of the nanoparticles. HCC827, A549 and NIH-3T3 cells were incubated in 96-well plates, with each well containing 1×104 cells and 200 µL corresponding medium. After cell adherence, the cells were incubated with GEF, YSV, drug mixture and nanoparticles, respectively, with different concentrations and cultured for 3 days. Subsequently, 20 µL 5 mg/mL MTT solution was supplemented to each well. Four hours later, cell culture mediums were extracted, following which the same volume of DMSO was added. The cell viability was calculated by measuring the absorbance at 490 nm with a BIO-RAD Model 680 microplate reader. Cell Apoptosis. HCC827 cells were cultured in 6-well dishes, with each well containing 6×105 cells and 4 mL cell culture medium. After cell adherence, cells were incubated with GEF, YSV, drug mixture and nanoparticles at the same concentration (GEF 1 µg/mL, YSV 11.25 µg/mL), as well as PBS (control). After one day, HCC827 cells were detached by pancreatin, rinsed twice by fresh PBS (pH =7.4) and suspended in binding buffer. Then, the resulting cell suspensions were labeled with Alexa Fluor FITC-conjugated annexin-V and PI. Cell apoptosis



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was studied using FCM. Western Blot Analysis. HCC827 cells were incubated in 6-well dishes, with each well containing 1×106 cells and 4 mL cell culture medium. After cell adherence, cells were treated with nanoparticles (12.25 µg/mL) and RPMI-1640 medium (control) respectively for 36 h. Then, the resulting cells were detached by pancreatin and rinsed with cool PBS. The cellular proteins were extracted in Laemmli buffer and a bicinchoninic protein assay kit (Pierce, USA) was applied to quantify the protein contents. Equal amount of protein (20µg/lane) were added to sodium dodecyl sulfate-polyacrylamide gels and then moved to 0.45 µm polyvinylidene fluoride membranes, which were then treated with nonfat milk powder dispersed in tris-buffered saline supplemented with 0.1% Tween-20 and probed with antibodies against β-actin (loading control), PI3K and AKT (1:1000 dilution), p-PI3K and p-AKT, followed by HRP-conjugated (HRP = horseradish peroxidase)

anti-mouse

immunoglobulin-G

(IgG,

1:5000

dilution).

Chemiluminescent HRP Substrate S13 was used to visualize the protein bands, which were then analyzed with the alphaEaseFC (Alpha Innotech, USA). Western blot analysis was repeated 3 times. Animal and Tumor Models. The protocols of all the following animal experiments were checked and ratified by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University. Balb/c female nude mice (5 weeks) were obtained from Chinese Academy of Science (Shanghai, China). The mice were injected hypodermically in the right flank (for in vivo anticancer


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efficacy assay) or in the oxter region (for small animal in vivo optical imaging and biodistribution analysis) with 200 µL cell suspension containing 5×106 HCC827 cells. Experiments began after the tumors grew to about 50 mm3. In Vivo Optical Imaging and Biodistribution Analysis. HCC827 tumor-bearing mice were divided into 4 groups at random and intravenously injected via tail vein with 200 µL free Cy5.5, Cy5.5-loaded single GEF, Cy5.5-loaded single YSV, and Cy5.5-loaded nanoparticles dissolved in PBS with 0.1% DMSO as co-solvent respectively. The fluorescence distribution was detected at preselected time using a small animal in vivo imaging system with λex = 690 nm. To access the in vivo distribution of the GEF-YSV nanoparticles, the HCC827 tumor-bearing mice were intravenously injected via tail vein with 200 µL GEF-YSV nanoparticles (loading Cy5.5) (10 mg/kg) dissolved in PBS. Mice injected with Free Cy5.5 dissolved in PBS with 0.1% DMSO as co-solvent were used as control. Mice were executed according to the protocol of the IACUC at predetermined intervals after injection, following which the tissues of the mice (tumor, liver, spleen, lung, kidney and heart) were excised. The above mentioned tissues were washed with 0.9 % sodium chloride solution and wiped by absorbent paper. These tissues’ weights were recorded and then the tissues were stored at -80

o

C, following which

homogenization was performed. Cy5.5 in different tissues were collected using extraction method (DCM: MeOH = 4: 1). The bottom layer was blow-dried using a Termovap Sample Concentrator, and then dried under vacuum in darkness for 24 h. Afterwards, the samples were redissolved in MeCN. The amount of Cy5.5 was



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determined by recording the fluorescent emission intensity at 700 nm with a fluorescence spectrometer. In Vivo Anticancer Efficacy Assay. The HCC827 tumor-bearing nude mice were divided into 5 groups at random, with each group having 5 mice. The tumor-bearing mice were intravenously injected via the tail vein with PBS, GEF (1 mg/kg), YSV (11.25 mg/kg), drug mixture (1 mg/kg, 11.25 mg/kg) and nanoparticles (12.25 mg/kg) once every 2 days for 22 days in total. In the course of therapy, the volume and weight of the tumors were measured using vernier caliper and platform scale before every drug administration. The tumor volume was calculated by applying the following formula: 1 ! = × ℎ ! × ℎ !# 2 After 22 days, mice in all groups were executed according to the protocol of the IACUC and the tissues of mice (liver, spleen, lung, kidney, heart and tumor) were excised with tumors weighed and photographed. In addition, the tumors and other organs were chopped into pieces, fixed in 4% polyoxymethylene and embedded in the paraffin. Afterwards, the tumors were sliced up for H&E and TUNEL staining. Other organs were sectioned for H&E staining. Statistical Analysis. All data represent at least three parallel experiments. Individual data points were compared by Student’s t test. In all cases, p < 0.05 was regarded statistically significant. 3. Results and Discussion Preparation and Characterization of GEF-YSV nanoparticles. A wide range


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of polypeptides have been found to be able to assemble into nanoparticles with various morphologies and sizes.28-30 According to our investigation, amphiphilic polypeptide YSV can generate nanoparticles with a size of ca.193 nm (Figure S1). Thus, amphiphilic YSV was used as the polypeptide to co-assemble with hydrophobic GEF into SDDDS nanoparticles due to its intermolecular interactions with GEF, including hydrogen bond and π-π stacking (Scheme 1). The nanoparticles were conveniently prepared by adding GEF into the NaOH solution of YSV under stirring. The resulting suspension was dialyzed against ultrapure water to eliminate DMSO and NaOH. By regulating the co-assembly ratio of GEF and YSV, the best one was found to be 1: 7.5 for GEF and YSV with higher encapsulation efficiency (EE) for both drugs (Table S1). More than 50 % of GEF and ca. 75% of YSV were encapsulated into the nanoparticles as determined by UV-vis absorption spectra. Therefore, this feed ratio was chosen to perform all of the following experiments, with a true ratio of 1:11.25 (GEF: YSV) in the nanoparticles. Based on this ratio, the DLC was calculated to be 8.2% for GEF and 91.8% for YSV. The morphology, size and size distribution of the nanoparticles were characterized by TEM and DLS. It was reported that nanoparticles with a size of 30-200 nm could favorably accumulate in cancer cells via EPR.31 Figure 1 shows that the size of nanoparticles is about 140 nm in diameter with almost regular spherical morphology. On the other hand, the average hydrodynamic radius of the nanoparticles is about 180 nm, which is slightly bigger than that of TEM. The inconsistency of the results can be explained



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by the fact that DLS measures the hydrodynamic size that corresponds to the core and the swollen corona of the nanoparticles; but TEM only gives a size of the core in a dried state, since the corona has too low electronic density to be observed.32 The reason why the size of GEF-YSV nanoparticles is slightly smaller than that of the YSV nanoparticles is probably because the encapsulation of GEF makes the nanoparticles more compact, which results in the increased electron density, thus increasing the contrast in TEM image.33 Then, variable-temperature 1H-NMR spectra of the GEF-YSV nanoparticles were performed to study the hydrogen bond interaction between GEF and YSV. GEF and YSV with equivalent molar quantities were solved in a mixed solvent (1,1,2,2-tetrachloroethane-d2 : dimethyl sulfoxide-d6 =5 : 1). Figure S2 shows that the chemical shift of the hydrogen of the NH in GEF shifts to high magnetic field (from 8.65 to 8.45 ppm) step by step as the temperature raises from 25 oC to 55 oC. It has been reported that the gradual shift of NH can be ascribed to the disruption of hydrogen bond.21, 34-36 Moreover, the chemical shift of NH returns to its original position (8.65 ppm) once the temperature returns to 25 oC from 55 oC, indicating the reversibility of the hydrogen bond interaction. The hydrogens of NH2 of YSV exhibit similar property in the region of 7.3 to 7.4 ppm. These results suggest that hydrogen bond interaction does exist between GEF and YSV. It is worth noting that π-π stacking also plays a part, as proved by the obvious redshift of GEF absorbance wavelength in GEF-YSV nanoparticles (Figure S3).37 DLS and TEM images of GEF-YSV nanoparticles in RPMI-1640 medium are shown in Figure S4 to prove the stability


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of the nanoparticles in cell culture. To further confirm the stability of the nanoparticles, DLS measurements were performed at predetermined intervals (3, 6, 8, 12, 15 days). These results demonstrate that the nanoparticles would remain stable in PBS and cell culture RPMI-1640 medium at 37 oC at least for 15 days. In Vitro Drug Release of the GEF-YSV Nanoparticles. The standard curves of GEF and YSV were firstly measured with UV-vis spectroscopy and HPLC (Figure S6). At 37 oC, dialysis was used to study the release profile of the GEF-YSV nanoparticles when the pH is 7.4 (phosphate buffered solution with or without FBS) or 5.0 (acetate buffered solution), the reason of which is that pH=5.0 is usually used as a satisfactory simulation of the slightly acidic microenvironment of tumor site, which has been adopted by many researchers. Although the actual pH of tumor microenvironment is not as low as 5.0, this particular pH could provide insight into the following cell and animal experiments.19, 21, 35-36 Figure 2 shows that the releases of GEF and YSV are relatively slow when the pH is 7.4 (phosphate buffered solution with or without FBS), indicating the GEF-YSV nanoparticles are relatively stable at physiological pH or in simulated physiological environment. In contrast, the releases of GEF and YSV are accelerated at slightly acidic condition (pH = 5.0). The cumulative release of drug is up to 65% for GEF and 75% for YSV in 10 hours, respectively. Under acidic condition, the hydrogen bond between GEF and YSV is broken, resulting in the disassembly of the nanoparticles and the accelerated release of both drugs.21, 35-36 The minor difference of the release rate between pH = 7.4 and pH = 5.0 is quite



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common

among

supramolecular

DDSs.18-19

SDDDS

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constructed

by

supramolecular interaction is not as stable as that constructed by chemical conjugation or physical encapsulation by amphiphilic carriers16, 20, 32, resulting in higher accumulative release content even in physiological situations. However, the accelerated release in lower pH would still render the nanoparticles selectivity towards cancer cells, since the pH of cancer microenvironment is lower than that of normal cells.38 Cell Internalization. The cell uptake behavior of the GEF-YSV nanoparticles (loading NR) was studied using CLSM and FCM. Since the nanoparticles are not fluorescent, nile red, a hydrophobic red fluorescent dye, was used to label the SDDDS, endowing the nanoparticles with fluorescent property. In term of CLSM, HCC827 cells were cultured with the fluorescent nanoparticles (30 µM) for 0.5, 1, 2, 4, 6 h at 37 oC. DAPI was applied as a selective dye to stain the nuclei of HCC827 cells. Figure 3A shows that only faint red fluorescence could be observed in 30 min, indicating only a few nanoparticles entered cells. Fluorescent brightness has a progressive increase with the extension of incubation time. As it can be seen from the merged image, the nanoparticles locate mainly in nuclei. To further study the cell internalization behavior of GEF-YSV nanoparticles, lysosome staining, as well as early endosome staining and late endosome staining were performed. Early endosomes were stained by CellLight® Early Endosomes-GFP *BacMam 2.0*, late endosomes were stained by CellLight® Late Endosomes-GFP *BacMam 2.0*, and lysosomes were stained by LysoSensor


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Green DND-189. The staining experiments mentioned above were performed according to the manufacture’s protocols. Live cell imaging was performed after NR-loaded GEF-YSV nanoparticles (30 µM) were incubated with HCC827 cells for 4 h. As shown in Figure S7, majority of the NR-loaded GEF-YSV nanoparticles were localized in early/late endosome and lysosomes after 4 h incubation, which is evidenced by co-localization of red fluorescence with the green fluorescence of CellLight® Early Endosomes-GFP *BacMam 2.0*, CellLight® Late Endosomes-GFP *BacMam 2.0* and LysoSensor Green DND-189, respectively. These results indicate that the GEF-YSV nanoparticles are effectively internalized by cells through endocytosis. Additionally, cell internalization of the nanoparticles was also characterized by FCM. HCC827 cells were cultured with fluorescent nanoparticles (30 µM) at 37 oC for 0.5, 1, 2, 4, 6 h. Cells cultured with RPMI-1640 medium were used as controls to eliminate autofluorescence interference. As shown in Figure 3B, the fluorescent value has a progressive increase with the elongation of incubation time. Only weak fluorescence could be observed in the cells for 30 min incubation, while the fluorescence became stronger when cells were incubated for longer period, indicating more nanoparticles entered cells. Both results confirm that the nanoparticles could be internalized efficiently by HCC827 cells. In Vitro Cytotoxicity. GEF is a selective tyrosine kinase inhibitor for epithelial growth factor receptor (EGFR). GEF retards the growth and propagation of cancer by occupying the adenosine triphosphate (ATP) binding site of EGFR, which



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further decreases the activity of the tyrosine kinase and blocks the signaling pathway of EGFR.27 To study the toxicity of the GEF-YSV nanoparticles to different cell lines, EGFR-mutated non-small cell lung cancer cells HCC827 cells, non-EGFR-mutated non-small cell lung cancer cells A549 cells, and normal cells NIH-3T3, were incubated with GEF, YSV, drug mixture and SDDDS nanoparticles at different concentrations for 72 h. Cells cultured with PBS were used as the control group. Figure 4 shows that nanoparticles exhibit the best efficacy to cell proliferation among other experiment groups especially at low concentrations, with 50% cellular growth inhibition (IC50) of 1.19 µg/mL for HCC827 cells, 21.43 µg/mL for A549 cells and 15.59 µg/mL for NIH-3T3 cells. The disparate IC50 for different cell lines can be illustrated by the fact that HCC827 cells are EGFR mutated, while A549 and NIH-3T3 cells are not.39 As molecular targeted anti-tumor agent for EGFR, GEF can fully exert its efficacy in HCC827 cells. On the other hand, due to the lack of EGFR in A549 cells and NIH-3T3 cells, GEF is quite ineffective in inhibiting cell proliferation, which means the GEF-YSV nanoparticles could decreases side effect on normal cells like NIH-3T3. It is worth noting that even though GEF/YSV mixture has the same dosage as nanoparticles, its efficacy is not as good as nanoparticles in EGFR-mutated HCC827 cells, because nanoparticles can be more easily internalized by HCC827 cells, resulting in the release of free drugs to exert the therapeutic effects.40 Cell Apoptosis. It has been widely accepted that most small molecular anticancer


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agents take effect by inducing apoptosis.16 Here, to make sure whether the observed cytotoxicity to cancer cells was due to apoptosis, the Annexin-FITC/PI double-staining assay method was performed. Briefly, HCC827 cells were cultured with 1 µg/mL GEF, 11.25µg/mL YSV, GEF/YSV drug mixture(1µg/mL + 11.25µg/mL), GEF-YSV nanoparticles (containing 1µg/mL GEF and 11.25µg/mL YSV) and RPMI-1640 medium (control) for 24 h, followed by FITC Annexin/PI staining. The samples were analyzed using FCM. Figure 5 shows that the percentages of apoptotic HCC827 cells are 55.89%, 73.75%, 74.94% and 81.35% after treatment with GEF, YSV, GEF/YSV drug mixture and nanoparticles, respectively. These results can be explained by the fact that GEF-YSV nanoparticles can be effectively internalized and retained in HCC827 cells compared with free drugs or drug mixture which easily diffuse out of cells, resulting in more drugs in tumor cells to exhibit higher efficacy to induce apoptosis.41 The results above demonstrate that the nanoparticles could take effect by inducing apoptosis, as well as induce a higher percentage of apoptosis compared with single drugs or drug mixture. Pharmacological Mechanism Evaluation. The pharmacological mechanism of the nanoparticles is still not clear, even though in vitro cytotoxicity shows that the nanoparticles containing both GEF and YSV have much better efficacy than free drugs or drug mixture on HCC827 cells. GEF is known to restrain the proliferation of cancer cells by regulating the PI3K/AKT pathway.42 Base on this knowledge, western blot analysis was conducted to confirm whether the



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nanoparticles containing GEF and YSV still take effect by regulating PI3K/AKT pathway. To be more specific, proteins were extracted after the incubation of HCC827 cells with GEF-YSV nanoparticles for 36 h. HCC827 cells cultured in RPMI-1640 medium were used as the control. To quantify the proteins contents, the cellular proteins were extracted in Laemmli buffer and quantified with a bicinchoninic acid protein assay kit. Equal quantities of proteins (20 µg per lane) were used for western blot analysis. Figure 6 shows that the relative expressions of phosphorylation of PI3K (p-PI3K) and phosphorylation of AKT (p-AKT) were remarkably decreased in the groups in which HCC827 cells were incubated with the GEF-YSV nanoparticles, indicating that the nanoparticles exert anticancer effect by inhibiting EGFR signaling pathway. In Vivo Optical Imaging and Biodistribution Analysis. To evaluate the in vivo targeting ability and biodistribution of the GEF-YSV nanoparticles, Cy5.5 was co-assembled with GEF, YSV and the GEF-YSV nanoparticles respectively, endowing them with near-infrared fluorescence property. The real-time imaging of free Cy5.5 (control), Cy5.5-loaded single GEF, Cy5.5-loaded single YSV and Cy5.5-loaded GEF-YSV nanoparticles in the HCC827 tumor-bearing nude mice were conducted at 0.5 h, 1 h, 2 h, 6 h, and 8 h. Figure 7A shows that most free Cy5.5 is accumulated in liver. The fluorescence signals increase to the strongest at 2 h after injection, and then decrease from 2 h to 8 h. At 8 h, the near-infrared fluorescence in the HCC827 tumor-bearing mice is almost invisible due to the fact that free Cy5.5 tends to be eliminated from blood real fast. In contrast,


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Cy5.5-loaded nanoparticles maintain higher fluorescence signals up to 8 h, indicating better blood retention for the nanoparticles. In comparison with free Cy5.5, Cy5.5-loaded nanoparticles could be selectively accumulated in tumor tissues, especially at the first 2 h. These data demonstrate that the nanoparticles have prominent in vivo targeting ability. Figure S9 shows the in vivo optical imaging result of Cy5.5-loaded single GEF and Cy5.5-loaded single YSV. Owing to the hydrophobicity, GEF and Cy5.5 failed to assemble into nanoparticles. As a result, it shows poor targeting ability to tumor, which is very similar with that of free Cy5.5. Interestingly, on the part of YSV, due to its ability to co-assemble with hydrophobic molecules, nanoparticles were formed after the assembly between YSV and Cy5.5 (Figure S8). So, it shows improved targeting ability than that of Cy5.5-loaded single GEF. Moreover, the HCC827 tumor-bearing mice were executed at 1 h, 2 h and 6 h after being injected with GEF-YSV nanoparticles (loading Cy5.5) to evaluate the distribution of drugs in tumor and other organs. Figure 7B shows that there are initially a large portion of nanoparticles accumulated in tumor, liver, spleen, lung and kidney. Nevertheless, the concentrations of drugs in other organs decrease more rapidly than that in tumor at 6 h, resulting in more nanoparticles in tumor tissue to exert long-term toxicity towards tumor. In contrast, for mice injected with free Cy5.5, the concentration of Cy5.5 is considerably lower in tumor as well as other organs. It mainly accumulates in liver and spleen, which decreases sharply with the elongation of time. It can be ascribed to the fact that small molecules tend



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to be cleared more quickly in blood. These results show that the nanoscale property endows the GEF-YSV nanoparticles with passive targeting ability, through which the SDDDS nanoparticles are favorably accumulated in tumor site. In Vivo Antitumor Efficacy. Cells experiments and small animal imaging results demonstrate that the nanoparticles have improved biodistribution and efficient accumulation. To evaluate whether these positive results will result in better therapeutic efficacy, HCC827 tumor-bearing mice were intravenously injected with PBS (control), GEF, YSV, GEF/YSV drug mixture and the GEF-YSV nanoparticles via the tail vein. Figure 8A and Figure 8D shows that after the course of therapy, the tumor volumes of the mice injected with PBS increased from ~50 mm3 to ~750 mm3, indicating no antitumor efficacy. The volumes of the tumor of the mice treated with GEF or YSV alone increase from ~50 mm3 to ~460 mm3 and ~420 mm3 respectively, showing that single anticancer agents have moderate effect in inhibiting the growth of tumor. Since GEF and YSV can exert enhanced effect in inhibiting the proliferation of HCC827 cells, the tumor volume of this group increases from ~50 mm3 to ~350 mm3. In contrast, due to the improved biodistribution and efficient accumulation of the nanoparticles, mice treated with the GEF-YSV nanoparticles underwent only minor growth of tumor from ~50 mm3 to ~180 mm3. Owing to the fact that the dosages of free GEF, free YSV and drug mixture are relatively small, the body weights of HCC827 tumor-bearing mice in all five groups keep steady in the early course of treatment and increase a little bit in later stage, showing no obvious difference (Figure 8B).


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At the end of the treatment, mice in all groups were executed and the tissues of mice (liver, spleen, lung, kidney, heart and tumor) were excised. Figure 8C shows the tumor inhibitory rate (TIR) of GEF, YSV, GEF/YSV drug mixture and GEF-YSV nanoparticles, which is determined by the growth rate of tumor weight compared with the control group. Base on the weight of the excised tumors of the mice injected with PBS, 27%, 29%, 52% and 72% were calculated to be the TIR of GEF, YSV, GEF/YSV drug mixture and GEF-YSV nanoparticles respectively, which further proves that the GEF-YSV nanoparticles have the best drug efficacy in preventing the growth of tumor among all groups. To further evaluate the drug efficacy of the GEF-YSV nanoparticles, H&E staining of tumor sections was performed to observe tumor tissue morphology (Figure 8E). Large nuclei and spindle shapes are obvious in PBS group, indicating rapid cancer proliferation. In other groups, the tumor cellularity decreases prominently, and the nuclei shrinkage and fragmentation are observed, especially for nanoparticles treated group. Meanwhile, tissue necrosis is observed in the nanoparticles treated group, too. The result of H&E staining demonstrates that the GEF-YSV nanoparticles are more effective in prohibiting the proliferation of tumor than other formulations. Furthermore, TUNEL was conducted to evaluate the apoptosis-inducing effect of the nanoparticles (Figure 8E). The sections of tumor tissue of all groups were stained with the in situ cell death detection kit. Cells with green fluorescence in microscopic field are apoptosis cells. It can be seen from the results that the tumor tissues which are taken from nanoparticles



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treated group show the highest percentage of apoptosis among all groups, which conforms to the result of the in vitro cell apoptosis experiment. The result of TUNEL also suggests that the SDDDS nanoparticles are effective in inducing cancer cells apoptosis. Furthermore, H&E staining of livers, spleens, lungs, kidneys and hearts after treating with different formulations was performed to evaluate the safety of the GEF-YSV nanoparticles (Figure S10). Due to the molecular targeting ability of GEF and low toxicity of polypeptide drug YSV, the livers, spleens, lungs, kidneys and hearts of experimental groups show no discernible pathologic lesion compared with those of the control group. Therefore, the release of drugs in liver tissue, as well as other organs, would not cause new problems. 4. Conclusions In summary, SDDDS with uniform morphology and size distribution has been constructed through co-assembly of GEF and YSV via supramolecular interactions. Compared with free drugs or GEF/YSV drug mixture, the SDDDS nanoparticles prove to have improved cell internalization, enhanced selectivity and also exhibit much better drug efficacy both in vitro and in vivo. More importantly, the SDDDS is straightforwardly fabricated and requires no sophisticated chemical modification, effectively avoiding the undesirable changes for the structure and property of drugs, thus providing a hopeful way for mass production of nanoparticle drugs. Considering the advantages of the SDDDS mentioned above, we believe this strategy provides a feasible method for


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enhanced anticancer therapy with minimal side effect.

ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org. The encapsulation efficiency of GEF and YSV in the self-assembled SDDDS nanoparticles is shown in Table S1, the TEM image and DLS measurement of YSV nanoparticles are shown in Figure S1, the variable-temperature 1H-NMR spectra of the GEF-YSV nanoparticles is shown in Figure S2, the UV spectra of GEF, YSV and GEF-YSV nanoparticles in water-dimethyl sulfoxide mixture is shown in Figure S3, the TEM image and DLS measurement of GEF-YSV nanoparticles in RPMI-1640 medium are shown in Figure S4, the influence of storage on diameter of GEF-YSV nanoparticles is shown in Figure S5, standard curves of GEF and YSV are shown in Figure S6, CLSM of intracellular distribution of Nile-Red-loaded GEF-YSV nanoparticles after incubation in HCC827 cells stained with CellLight® Early Endosomes-GFP *BacMam 2.0*, CellLight® Late Endosomes-GFP *BacMam 2.0*, and LysoSensor Green DND-189 are shown in Figure S7, TEM images of Cy5.5-loaded GEF-YSV nanoparticles and Cy5.5-loaded single YSV are shown in Figure S8, In vivo near-infrared images of Cy5.5-loaded single GEF and Cy5.5-loaded single YSV are shown in Figure S9, H&E staining images of liver, spleen, lung, kidney and heart after treating with different formulations are shown in Figure S10. AUTHOR INFORMATION Corresponding Author



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*Email: [email protected] *Email: [email protected] Notes:

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work is financially supported by National Basic Research Program of China (2015CB931801), National Key Research and Development Plan of China (2016YFA0201500), and National Natural Science Foundation of China (51473093).

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Scheme 1. Graphical representation for the construction and self-delivery of supramolecular drug-drug delivery system (SDDDS) to cancer cells. SDDDS is conveniently fabricated via co-assembly of GEF and YSV through multiple intermolecular interactions, including hydrogen bond and π-π stacking. SDDDS nanoparticles exhibit prolonged retention in blood and improved accumulation in tumor tissue, which further inhibits the proliferation of cancer cells via apoptosis.



A

B 40

Number (%)

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Dh = 180 nm

30 20 10 0

1

10 100 Size (d/nm)

1000

Figure 1. (A) TEM images and (B) DLS measurement of GEF-YSV nanoparticles (0.4 mg/ml) in aqueous solution.


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Cumulative release of drugs (%)

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100 80 60 40

GEF(pH=7.4) GEF(pH=7.4 with 10% FBS) GEF(pH=5.0) YSV(pH=7.4) YSV(pH=7.4 with 10% FBS) YSV(pH=5.0)

20 0 0

10

20 30 Time (h)

40

50

Figure 2. In vitro GEF and YSV release profiles of GEF-YSV nanoparticles under different conditions (pH = 5.0 and pH =7.4 containing FBS or not).



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Figure 3. (A) Cell internalization of GEF-YSV nanoparticles encapsulating Nile Red observed by CLSM (HCC827 cells) for 0.5 h, 1 h, 2 h, 4 h and 6 h. Scale bar = 50 µm. (B) FCM histogram profiles of HCC827 cells incubated with GEF-YSV nanoparticles encapsulating Nile Red for 0.5, 1, 2, 4, 6 h.


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100 80

B

HCC827 cells Gefitinib Peptide Drug Mixture Nanoparticle

60 40 20 0 10 20 30 40 50 60 Concentration (ug/mL)

A549 cells Gefitinib Peptide Drug Mixture Nanoparticle

100 80 60 40 20 0

C

NIH-3T3 cells

100

Cell Viabilty (%)

A Cell Viabilty (%)

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Cell Viability (%)

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80

Gefitinib Peptide Drug Mixture Nanoparticle

60 40 20 0

0

10 20 30 40 50 60 Concentration (ug/mL)

0 10 20 30 40 50 60 Concentration (ug/mL)

Figure 4. Cell viability of (A) HCC827 cells, (B) A549 cells and (C) NIH-3T3 cells after incubation with GEF, YSV, GEF/YSV drug mixture or GEF-YSV nanoparticles at different concentrations for 72 h.



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Figure 5. Apoptosis analysis of HCC827 cells incubated with GEF, YSV, GEF/YSV drug mixture or GEF-YSV nanoparticles by FCM. In each graph, the lower right region represents the early stage of apoptotic cells and the upper right region represents the late stage of apoptotic cells. The percentages in each graph represent the ratio of the cells in this region.


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Figure 6. (A) Expression level and (B) proteins relative expression level of p-PI3K, PI3K, p-AKT and AKT in HCC827 cells induced by GEF-YSV nanoparticles (1 µg/mL for GEF and 11.25 µg/mL for YSV). Control group was HCC827 cells incubated with RPMI-1640 medium. β-actin is used as the loading control.



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Figure 7. In vivo biodistribution of Cy5.5-loaded nanoparticles. (A) In vivo near-infrared images of Cy5.5-loaded nanoparticles and free Cy5.5. (B) Tissue distribution of Cy5.5 after mice being intravenously injected with Cy5.5-loaded nanoparticles and free Cy5.5 (10 mg/kg).


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Figure 8. The variation of (A) tumor volumes and (B) body weights of HCC827 tumor-bearing mice in different groups in the course of treatment. (C) The tumor inhibition rate (TIR) after treatment with PBS, GEF, YSV, GEF/YSV drug mixture or GEF-YSV nanoparticles. (D) The photographs of excised tumor after HCC827 tumor-bearing nude mice were treated with PBS, GEF, YSV, GEF/YSV drug mixture and the GEF-YSV nanoparticles. (E) The H&E and TUNEL stained tumor tissue sections. For H&E staining analysis, cell nuclei were stained by DAPI. For TUNEL analysis, green fluorescence represents apoptotic cells.



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For Table of Contents Only


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Construction of Supramolecular Drug-Drug Delivery System for Non

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