Histidine-Rich Cell-Penetrating Peptide for Cancer Drug Delivery and

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Histidine-Rich Cell-Penetrating Peptide for Cancer Drug Delivery and its Uptake Mechanism Lei Zhang, Jiang Xu, Feng Wang, Yong Ding, Toby Wang, Grace Jin, Matthew Martz, Zhongzheng Gui, Pingkai Ouyang, and Pu Chen Langmuir, Just Accepted Manuscript • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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Histidine-Rich Cell-Penetrating Peptide for Cancer Drug Delivery and its Uptake Mechanism Lei Zhang,a,b,c,d,e,* Jiang Xu,b,c Feng Wang,c Yong Ding,b,c Toby Wang,b Grace Jin,c Matthew Martz,b,c Zhongzheng Gui,d,e Pingkai Ouyang a,* & P. Chena,b,c,* a

College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing

211816, China b

Department of Chemical Engineering and cWaterloo Institute for Nanotechnology, University of

Waterloo, Waterloo, 200 University Avenue West, Ontario, Canada, N2L 3G1 d

Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangsu

212018, China e

College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu

212018, China * Corresponding authors: Lei Zhang, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China, +86 511-85616716 [email protected] Pingkai Ouyang, Nanjing Tech University, 30 Puzhu Road South, Nanjing 211816, China, +86 25-58139386 [email protected] P. Chen, Quantum Nano Center 4622, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada, N2L 3G1, +1 519 888 4567 Ext. 35586 [email protected]

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ABSTRACT In this work, we report a drug delivery system based on the pH-responsive self-assembly and disassembly behaviors of peptides. Here, a systematically designed histidine-rich lipidated peptide (NP1) is presented to encapsulate and deliver an anti-cancer drug ellipticine (EPT) into two model cells: non-small cell lung carcinoma and Chinese hamster ovary cells. The mechanism of pH-responsive peptide self-assembly and -disassembly involved in the drug encapsulation and release process are extensively investigated. We found that NP1 could selfassemble as a spherical nano-complex (diameter = 34.43nm) in a neutral pH environment with EPT encapsulated and positively charged arginine amino acids aligned outward; and EPT is released in an acidic environment due to the pH-triggered disassembly. Furthermore, the EPTencapsulating peptide could achieve a mass loading ability of 18% (mass of loaded-EPT/mass of NP1) with optimization. More importantly, it is revealed that the positively charged arginine on the periphery of the NP1 peptides could greatly facilitate their direct translocation through the negatively charged plasma membrane via electrostatic interaction; instead of via endocytosis, which provides a more efficient uptake pathway. Keywords: pH-responsive, Self-assembly, Cell-penetrating peptide, Drug delivery, Uptake mechanism


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1. INTRODUCTION Nanotechnology offers new strategies for cancer therapy1-2 through the utilization of various nanomaterials and molecules such as mesoporous silica,3 nano-graphene,4 liposomes,5 proteins (or peptides).6-7 Among them, peptides have garnered attention for their ability to deliver anticancer drugs8 due to their simplicity in structure design,9 biocompatibility,10 and ease of synthesis.11 The morphology of a self-assembled peptide structure varies from fibers12-14 to tubes,15-17 ribbons,18-19 rods,20-21 to globules22-23, which provides many opportunities to explore and further the preclinical applications of drug encapsulation and target delivery.24-25 However, one severe limitation of peptides for anticancer drug delivery is their lack of efficiency in penetrating the cell membrane.26 The majority of the drug and their peptide carriers are prevented from entering the cell membrane directly, which causes most drug trapped in endosome through endocytosis for cellular uptake. Therefore, cell-penetrating peptides (CPPs) are strongly needed in order to enhance permeability, retention (EPR) effect, and intracellular delivery efficiency.27-31 Currently, there are more than 100 diverse CPPs with 5 to 40 amino acids in length that have been designed and synthesized. They can be classified into three subgroups based on their physical and chemical properties, i.e. cationic, amphipathic, and hydrophobic.31Besides, a previous study focusing on the function of arginine-rich CCPs such as the R9-Tat peptide (GRRRRRRRRRPPQ)32 has shown that they exhibit great potential in translocation ability.31 On the other hand, there are still challenges with targeting drugs to tumor microenvironments, controlling the stimulus sensitive activity of drug delivery system, and reducing side effects.33-34 By arranging of amino acids to form specific peptide sequences, CCPs can also become tumor targeting peptides.35 For example, iRGD, a tumor-penetrating peptide, can carry drugs into extravascular tumor tissue.36 Moreover, the application of peptide ligands in cancer chemotherapeutics has gained great attention.37-38 For instance, Chang et al. reported that a helical peptide serves as the selective dual inhibitor of MDM2 and MDMX, ATSP-7041, which effectively activates the p53 pathway in tumors in vitro and in vivo.39 In addition, multifunctional stimulation strategies were also developed to target the cancer cells.38 In our research group, the multifunctional pH-triggered peptide C8 was designed for selective anticancer activity.40 In addition, our research group designed a peptide NP1 to be an siRNA carrier as well as a potential cancer drug cargo.41-42 In this study, we found NP1 to be a pH-triggered cell-


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penetrating peptide, which could use for targeted cancer therapy. This peptide consists of one stearic acid, eight arginine amino acids, and sixteen histidine amino acids which initiated peptide self-assembly or -disassembly when the pH was above or below pH 6.0. Using ellipticine (EPT) as a model drug, our results suggest that NP1 can efficiently deliver drugs into cells via a direct translocation-uptake mechanism and the NP1-EPT exhibited cytotoxicity to non-small cell lung carcinoma and Chinese hamster ovary cells. Besides, the interfacial interaction between the nano-complex and cell membrane was investigated by the fluorescence-activated cell sorting (FACS) technique and confocal laser scanning microscopy. We also determined how each segment in NP1 affects its pH-triggered self-assembly and -disassembly. In short, the NP1 peptide is a promising anticancer drug cargo and a highly controllable tool for stimuli-responsive and targeted drug delivery, with a more efficient uptake pathway without endosomes. 2. MATERIALS AND METHODS Materials. Two peptides NP1 (Stearyl-HHHHHHHHHHHHHHHH-RRRRRRRR-NH2) and H16R8 (Acetyl-HHHHHHHHHHHHHHHH-RRRRRRRR-NH2) were designed in our research group and ordered from CanPeptide Inc. (Pointe-Claire, Canada) for synthesis. The anticancer agent ellipticine (EPT, 99.0% pure) was purchased from EMD Biosciences Inc. (La Jolla, CA, USA). Sample Preparation. The fresh NP1 (100 µM) and H16R8 (80 µM) peptide were dissolved in water (HyPure water, HyClone Laboratories Inc., Utah, USA), followed by a 10 min bath sonication (BRANSON 2510, VA, USA). The NP1 peptide solution was then diluted to different concentrations as needed. The pH of the NP1 solution was adjusted by using 0.01 M HCl and 0.01 M NaOH, and measured by a microprobe pH meter (Accumet, Canada). EPT was dissolved in THF of 1 mg/mL (Sigma-Aldrich, Oakville, ON, Canada). Aliquots of EPT-THF were then transferred to a vial and dried to form a thin film. The EPT-encapsulated NP1 nano-complex was generated by drop-wisely adding NP1 solution (80 µM) into the vial, followed by the magnetic string at 800 rpm overnight. An optimized molar ratio (0.375:1)42 of NP1 and EPT was applied here. Finally, the NP1-EPT solutions were diluted and the pH was adjusted per the use. Solutions of the only EPT (80 µM at pH 4.0 and 8.0) as control groups were prepared in HyPure water using the same protocols. Fluorescence Spectroscopy. An ANS fluorescence assay was used for determining the critical aggregation concentrations of NP1 peptide solution and NP1-EPT nano-complex solution. NP1


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solution (0.625, 1.25, 2.5, 5, 10, 20, 30, 40, 60, and 100 µM) of different pH (4.0, 5.6, 7.4, and 8.0) were prepared for the measurement. First, the NP1 peptide solution (40 µL) was added into the ANS solution (40 µL, 10 µM) for 10s vortex mixing. Second, the mixture was transferred to a quartz microcell and measured by a spectrofluorometer (Type QM4-SE, London, Canada). An excitation wavelength of 360 nm was used and the fluorescence spectrum was collected from 420 nm to 660 nm. The ANS fluorescence intensity at 475 nm was plotted against the concentration of NP1 peptide to determine the CAC of NP1 peptide. The mixture of ANS and HyPure water was measured as a baseline. The fluorescence intensity of EPT (213 µM) and NP1-EPT nano-complex (NP1: 80 µM, EPT: 213 µM) at pH 8.0 was measured with an excitation wavelength of 295 nm and an emission wavelength from 380 nm to 585 nm. In order to investigate interactions between NP1 and EPT during the co-assembly process, different concentrations of peptide (0, 1.25, 2.5, 5, 7.5, 10, 15, 30, 60, 80 and 90 µM at pH 8.0) were prepared (protocol described as above). The fluorescence intensity of EPT in the NP1-EPT nano-complex was measured and the values of the peak at 430 nm were collected for determining the critical aggregation concentration when NP1 and EPT interact with each other. Characterization of Drug Loading Capacity of NP1 Peptide. The amount of EPT encapsulated by NP1 (pH 8.0) in aqueous solution was determined by a UV-Vis absorption method described previously.43 In short, the EPT UV-Vis absorbance at the of concentration range from 2.5 µM to 80 µM (2.5, 5, 10, 20, 40, 80 µM) were measured, and then the peak values of 295 nm were used to obtain a standard calibration curve of EPT. The concentrations of EPT in unknown NP1-EPT nano-complex solutions are calculated by comparing its UV absorbance at 295nm to a known standard calibration curve. We plotted this loading capacity of NP1 against its concentration of NP1 to characterize the critical aggregation concentration of NP1 when it encapsulates EPT. Circular Dichroism Spectroscopy (CD). The secondary structure of NP1 peptide (80 µM) and NP1-EPT nano-complex (NP1: 80 µM, EPT: 213 µM) of various pH values were characterized by using a circular dichroism spectroscopy (Jasco Europe, Carmella, Italy). The NP1 solution (150 µL) was added into a 1 mm quartz cell (Hellma, Concord, Canada), and the spectra were collected from 190 nm to 260 nm with a 1 nm bandwidth at a scanning speed of 100 nm/min. We


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calculate the mean residue molar ellipticity (θ in deg cm! dmol!! ) of the peptide according to this equation below: Ellipticity θ =

(millidegrees×mean residue weight) (path length in millimetres×concentration of peptide in mgml!! )

Dynamic Light Scattering (DLS). The hydrodynamic diameter of diluted NP1-EPT nanocomplex particle was measured by a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The sample solution (40 µL) was added into a quartz microcell with a 3 mm light path. The scattering light intensities were then collected at an angle of 173°. Dispersion Technology software 5.0 was used to analyze the intensity-based size. Atomic Force Microscopy (AFM). The morphologies of EPT, NP1 peptide, H16R8 peptide, and NP1-EPT nano-complex were characterized on a Dimension Icon AFM (Bruker, Santa Barbara, USA). In general, 50 µL of sample solution was incubated on a cleaved mica surface for 5 min, followed by a 50 µL water flushing for three times. After drying, AFM images were taken at room temperature by using ScanAsyst-Air mode and the ScanAsyst-Air tips (Bruker, Santa Barbara, USA). Two different NP1 peptide samples (30 µM and 80 µM)) were compared to study the effect of peptide concentration on its morphology on the mica surface. Urea solution (8 µL, 8 M) was added into NP1 (800 µL, 80 µM) peptide solution at pH 8.0 and incubated for one day 24 h to investigate the effect of hydrogen bonding effect between histidine amino acids. In order to investigate the pH-responsive disassembly behavior of the NP1 peptide and the NP1-EPT nanoparticle, the pH of NP1 (80 µM) or NP1-EPT nano-complex(30 µM of NP1) solution was adjusted from 8.0 to 7.4, 5.6, and 4.0, or from 8.0 to 4.0, respectively. Transmission Electron Microscopy (TEM). The NP1 peptides at pH 4.0 and 8.0 were used for the TEM characterizations. The NP1 solution (10 µL, 80 µM) was incubated on a 400-mesh Formvar-coated copper grid (Canemco-Marivac, Canton de Gore, Canada) for 5 min, following by three-time 30 µL water washing flushing. The sample was then stained with uranyl acetate. After drying, a JEOL JEM 1200 EX TEMSCAN transmission electron microscope (JEOL, Peabody, USA), with an accelerating voltage of 80 kV, was used for imaging. Cell Cytotoxicity Assay. The non-small cell lung carcinoma (A549) and Chinese hamster ovary (CHO-K1) cells (ATCC, Manassas, USA) were cultured in F-12 Kaighn’s modification medium (F-12K, HyClone Laboratories Inc., Utah, USA) supplemented with 10% fetal bovine serum


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(FBS, HyClone Laboratories Inc., Utah, USA). The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. A549 cells (8,000 cells/well) were plated in a 96-well plate. After incubating for 24 h, the medium was removed and washed three-time with phosphate buffer saline (PBS, HyClone Laboratories Inc., Utah, USA). The EPT (80 µM), NP1 (30 µM), and NP1-EPT nanocomplex(NP1: 30 µM, EPT: 80 µM) solutions at pH 8.0 were diluted to 1/8 of its original concentration with Opti-MEM (HyClone Laboratories Inc., Utah, USA). Then a serial dilution was done (1, 2, 4, 8, 16, and 32 times) for the NP1-EPT nano-complex particle solution. In order to decrease the serum effect, after that, each diluted solution (60 µL) was added to cancer cells for a 3 h incubation. Second, 60 µL F-12K medium with 20% FBS was added into each well for another 48 h incubation, and all the solutions were removed and washed three times with PBS afterward. Finally, 100 µL Opti-MEM medium with CCK-8 reagent was added to each well for another 3 h incubation. The plates were then red to collect the fluorescence absorbance of 570 nm by a FLUOstar OPTIMA microplate reader. Cell viability was calculated as the ratio of the cell treated by NP1-EPT nano-complex over the non-treated one (negative control). We used an F-12K medium as a negative control, and EPT, NP1 as a positive control. Fluorescence-activated Cell Sorting (FACS) and Endocytic Inhibitor Treatment Protocol. In order to the characterization of drug uptake pathway, the uptake of NP1-EPT at pH 8.0 was measured. CHO-K1 cells (60,000 per well) were seeded into 24-well plate. After incubating for 24 h, we discarded the medium and rinsed the cells with PBS, which was then replaced by OptiMEM. NP1-EPT nano-complex solution (concentration: 8X samples used in Cytotoxicity Assay) at pH 8.0 was treated for 3 h incubation. Then we washed the cells using PBS five times to get rid of free nano-complex. After that, cells were then incubated with trypsin-ETDA (HyClone Laboratories Inc., Utah, USA) for 10 min for detaching. Then, 4% paraformaldehyde (PFA, Sigma-Aldrich, Oakville, ON, Canada) was added to collect the cells in suspension for FACS measurements. The F12-K medium, NP1 (pH 8.0), and EPT (pH 8.0) were used as the controls. The concentrations of EPT, NP1, and NP1-EPT were the same as the 8X samples used in the Cytotoxicity Assay.


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To investigate the uptake pathway of NP1-EPT at pH 8.0, cells were first incubated with various inhibitors (Table S1) for 1 h, then flushed by PBS 5 times before adding the NP1-EPT solution. The control was treated with the F12-K medium instead of the inhibitor. To investigate the effects of temperature on the uptake, the cells were incubated with NP1-EPT nano-complex of pH 8.0 for 3 h at 37 °C and 4 °C, respectively. Then medium served as the negative control. The Cellular uptake of all samples was determined using BD FACS Calibur Flow Cytometry (BD 208 Biosciences, Mississauga, Canada), and the data were analyzed by Flowjo software. Confocal Laser Scanning Microscopy (CLSM). CHO-K1 cells (80,000 per well) were seeded onto 14 mm coverslips in a 24-well plate and incubated to grow to ~50% confluency. After that, cells were washed three times by PBS and treated with the NP1-EPT solution at pH 8.0 (concentration: 8X samples used in Cytotoxicity Assay) with the Opti-MEM medium for 3 h incubation. In order to label endosomes, cells were washed three-time with ice-cold PBS and were stained by LysoTracker Deep Red (50 × 10−9 M, Life Technologies, Canada) for 15 min incubation. After that, the cells were fixed with fresh 4% PFA for 10 min at room temperature. To reduce fluorescence photobleaching, the coverslips were mounted on glass microscope slides with a drop of antifade mounting media

(Sigma-Aldrich Co., USA). The intracellular

localization was measured using a laser scanning confocal fluorescence microscope (LSM510Meta, CarlZeiss Inc., Thornwood, NY). EPT was excited with a 405 nm diode laser (15 mW) with emission setting at 528 nm. LysoTracker Deep Red was excited with a 633 nm laser (20 mW) and the emission was 679 nm. The images were analyzed by LSM Zen 2009 software. 3. RESULTS AND DISCUSSION 3.1 Characterizations and Mechanism of Self-Assembly of NP1 Peptide.


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Figure 1. (A) Molecular structure of NP1 peptide. (B) The schematic illustration of the self-assembly of NP1 peptide according to the status of histidine amino acid. (C) Secondary structures of NP1 (80 µM) at pH 4.0 and pH 8.0. (D) 3D AFM image of NP1 (30 µM) on mica surface at pH 8.0 and corresponding (E) 2D AFM image. (F) AFM image of NP1 (30 µM) at pH 4.0, an insert is the height information profiled by the red line.


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As designed, this NP1 molecule contains a hydrophobic stearic acid tail, histidine, and arginine segments (Figure 1A). In this peptide, histidine with an imidazole side chain can serve as a hydrogen donor or acceptor:44 deprotonated when pH > pKa (6.0) and protonated when pH < pKa (6.0).44-45 This property of pH-dependent hydrogen bonding (or positively charged) leads to a behavior of pH-triggered NP1 self-assembly/disassembly (Figure 1B). The hydrophobic aliphatic stearic acid tail in the C-term of NP1 could encapsulate anti-cancer drugs and form nano-complex, due to hydrophobic interactions.42, 46 The eight arginine segments at the N-term, which are always be positively charged could provide an electrostatic repulsive force to prevent aggregation of peptide molecules.8,

47

More importantly, arginine can also facilitate NP1

nanoparticles to penetrate the cell membrane.31, 48-49 Details of self-assembly behaviors of NP1 peptide were further characterized. First, the secondary structure of NP1 peptide was determined by CD spectra (Figure 1C), showing two totally different morphologies in the two situations: mainly beta-turn and beta-strand when histidine is deprotonated (pH 8.0) and the random coil of the protonated case (pH 4.0). Threedimensional measurement of NP1 (30 µM) at pH=8.0 was done by AFM (Figure 1D, E), showing a height of 2.11 ± 0.42 nm and a diameter of 19.43 ± 2.38 nm (Figure S1A) when histidine was deprotonated. When pH=4.0, no specific assembled structure of NP1 (30 µM) was detected of the protonated case, and a thin pattern was observed with a thickness of 1.17 ± 0.13 nm instead, indicating the single layer coating on the mica surface (Figure 1F).


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Figure 2. AFM image of NP1 (80 µM) at (A) pH 8.0 and (B) pH 4.0, and (C, D) corresponding TEM images. (E) ANS fluorescence binding assay was used to determine the CAC of NP1 peptide at (E) pH 8.0, with standard peptide solutions of concentrations ranging from 0 to 100 µM. (F) The CAC was ~ 9 µM of NP1 at pH 8.0 and no self-assembly for pH 4.0 case.

With an increase of peptide concentration from 30 to 80 µM, more self-assembling globular structures were detected (Figure 2A) for the deprotonated case. Even though we increased the concentration from 30 to 80 µM, no structure was formed for the protonated case; only a smooth surface is shown in the AFM image (Figure 2B). Similar morphology transition from a thin specific pattern to a smooth surface has been reported for surfactants such as tetradecyltrimethylammonium bromide (C14TAB) on the mica surface.50 The interaction between protonated NP1 peptide molecules with sixteen positively charged histidine amino acids and negatively charged mica surface is most likely an electronic interaction. As a result of the crystal logically smoothness of mica surface, peptide molecules prefer to coat in an ordered way, to form the pattern. As more and more molecules coming in, a multi-layered of the peptide was formed, which exhibited as a smoother surface. For the situation of a deprotonated case, NP1 peptide already self-assembled as nanoparticles in the solution and attached on the mica surface and showed a globular morphology. The self-assembly structure also can be further confirmed by TEM (Figure 2C), in which NP1 peptide assembled as nanoparticles with (Figure S1A) a diameter of 11.87 ± 1.78 nm when histidine was deprotonated (80 µM at pH 8.0). It should be noted that the size of NP1 peptide nanoparticles measured by TEM (11.87 ± 1.78 nm) is smaller than the one by AFM (19.43 ± 2.38 nm); which is due to the AFM technology amplifies the size of nanoparticles during the scanning.51 Due to the free/un-self-assembled peptide (80 µM at pH 4.0), no specific structure was observed (Figure 2D), which is similar to our AFM observation (Figure 2B). A schematic representation of how NP1 peptide self-assembly is shown in Figure 1B. In addition, strength of interaction of the self-assembled peptides could be evaluated by critic aggregation concentration (CAC).44 Various concentration was applied to determine the CAC of NP1 by using ANS assay (details in Materials and Methods).44 No obvious variation of ANS intensity was detected when the concentration of protonated NP1 peptide (Figure S1B) increased. However, a burst intensity increase was observed in the deprotonated case (Figure 2E). Evidently, peptide could self-assemble with a low CAC (~9 µM) of deprotonated NP1, while could not self-assemble of protonated NP1 (Figure 2F).


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3.2 Characterizations and Mechanism of Disassembly of NP1 Peptide.

Figure 3. AFM Morphologies, secondary structures and CACs of NP1 (80 µM) at different pH. (A-D) AFM imaging, pH= 8.0, 7.4, 5.6, and 4.0, respectively. (E) The secondary structure of NP1. (F) The CACs of NP1.

To further understand the self-assembly mechanism, a disassembly behavior of NP1 nanostructure was investigated at various pH conditions. First, in an alkaline condition, we observed NP1 peptides mostly formed a globular structure at pH 8.0 and 7.4 (Figure 3A, B). However, more and more globular NP1 transformed as a monomer state when the solution turned acidic (pH 5.6 and pH 4.0, Figure 3C, D), due to the protonation of histidine. In addition, the secondary structure of various pH values was explored (Figure 3E). Results show that two characteristic peaks of NP1 at pH 7.4 (positive peak at ~205 nm and negative peak at ~218nm) were converted to opposite peaks when pH decreased (negative peaks at~200 nm (pH 6.8), ~198 nm (pH 6.4), ~196 nm (pH 6.0), and ~195 nm (pH 5.6); positive peak at ~221 nm). These changes indicate that the secondary structure of peptide gradually transfer from beta-strand to random coil when pH decreases. Besides, the CAC of NP1 (which represents the strength of the self-assembly) is determined to be ~ 9 µM of pH 7.4 and more than 60 µM of pH 5.6 (Figure 3F, S1C, D), which are consistent with our AFM observations. Moreover, the disassembly process was observed when we broke the hydrogen bonding in the nanostructure by adding urea (Figure S2E).52 Our previous study related to the interaction between NP1 and HOPG surface by molecular dynamics simulations showed that in the deprotonated case of NP1, no hydrogen bonding was observed between two peptide molecules,


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however, lots of hydrogen bonds form and break during the time of the deprotonated case. The protonated NP1 peptide finally forms ultra-thin parallel strips on HOPG surface, otherwise, the compact and thickness structure was observed of the deprotonated case in the AFM results (Manuscript Submitted). This finding shows the important role of the hydrogen bonding of NP1 peptide in the architecture building and controlling. Another interesting observation is that deprotonated H16R8 (NP1 without stearic acid) peptide coated on the mica surface as a thin layer (Figure S2B) with a pattern similar to the case of NP1 at pH 4.0. The globular structure did not happen because no hydrophobic driven force contributes by stearic acid to promote the selfassembly. Previously studies reported that without less than eight charged amino acid in the Nterm, similar peptide amphiphiles prefer to form a nanofibers structure.53-54 The enough charged arginine amino acids, together with the specific histidine amino acid might be the reason to form the globular structure, not the fibers. In general, the roles of each sergeant of the peptide sequence in the self-assembly and disassembly behavior were investigated. It indicates that stearic acid contributes to the hydrophobic force to drive the nanostructure formation; deprotonated histidine provides the hydrogen bonding to interact with the peptide molecules to enhance the forming of the nanostructure and the protonated histidine triggers the disassembly due to the huge reclusive force induced; the arginine could avoid the endless growth of the nanostructure and possibly drive to form the globular structure instead of the fibers. When the globular structure formed, the hydrophobic core also could provide the hydrophobic drugs loading, and the positively charged arginine outward could also provide the cell membrane penetration ability, which is useful to carry drugs into the cell membrane plasma. Importantly, the low pH-triggered disassembly ability could be applied to drug target delivery purpose to release the drug by disassembling the nanostructure in the cancer environment, which is more acid than normal cells.55-56 3.3 Encapsulation of Ellipticine by Peptide NP1 and pH-Triggered Release


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Figure 4. (A) The scheme of encapsulation of ellipticine by NP1 and pH-triggered release. AFM images show the morphology of the NP1-EPT nano-complex at (B) pH 8.0 and (C) pH 4.0. (D) The secondary structure of EPTencapsulated NP1 when co-assembled with EPT at pH 8.0 and pH 4.0. (E) The fluorescence of EPT and EPTencapsulated NP1 at different pH. The concentrations of NP1 and EPT are 80 µM and 213 µM, respectively.

Based on the secondary structure and morphology study, the pH 4.0 and 8.0 cases were chosen for the following drug encapsulation study. A typical encapsulation of EPT by NP1 was achieved by tuning pH from 4.0 to 8.0 since the deprotonated histidine of NP1 drives the formation of nano-complex and the core of NP1 nanoparticles could provide a hydrophobic interaction to attract the drug. The release of EPT is induced by disassembly of NP1 by changing the pH from 8.0 to 4.0, reversely (Figure 4A-C) We furthered measured the EPT loading capacity (Details in the Materials and Methods). As shown in Figure S3A, B, the strand curve of EPT was first obtained as below: 𝑌 = 0.03235𝑋 + 0.09867 Where Y is the EPT UV-Vis absorption at 295 nm, X is the concentration of EPT. We determined the amount of EPT loaded by NP1 using the standard curve, and it is 14.35 µM EPT in 80 µM NP1, 8.90 µM EPT in 60 µM NP1, and 2.23 µM EPT in 30 µM NP1. The 80 µM NP1 case was used for the further study (Figure S3C, D). In the AFM images of the pH 8.0 case (deprotonated histidine), co-assembled EPT-NP1 nanocomplex were observed, which mostly globular structure, however, not as uniform as only


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deprotonated NP1 nanoparticles (compare Figure 4B and Figure 1E). Due to the extremely hydrophobic properties of EPT (Figure S4A),57 one possible explanation is that although most EPT is encapsulated by the stearic acid hydrophobic core, some EPT could interact with the histidine with the imidazole side chain by π-π stacking. This interaction between histidine and EPT may disarrange the hydrogen bonding of the self-assembled deprotonated histidine. The size and height of the nano-complex are 34.43 ± 11.14 nm and 12.17 ± 3.46 nm, which are larger than those of only NP1 nanostructure (Figure S3A). The schematic representation of the NP1 coassembly with EPT is shown in Figure 4A. As we expected, the co-assembling nano-complex could be disassembled when we adjusted the pH from 8.0 to 4.0, forming the similar structure as the pronated NP1 on mica surface (Compare Figure 4C with Figure 1F). In addition, for the protonated case, some aggregations occurred, it should be due to a weak interaction between the stearic acid of protonated NP1 molecules and drug EPT. For comparison, the blank deprotonated and protonated EPT are shown in Figure S4D-E. It should be noted that most of the deprotonated EPT could not be dispersed into the aqueous solution and already be washed during the AFM sample preparation.58 However, we still observed thick particles in the deprotonated case and thin pattern of the protonated cases (Compare Figure S4D-E with Figure 4B-C). Furthermore, the secondary structure of self-assembled peptide and EPT was investigated, which is similar to the deprotonated NP1 (Compare Figure 4D with Figure 1C). It appeared that beta-turn is the dominating formation, which indicates a co-assembly behavior of peptide and EPT happened. However, the beta-standard of the peptide was lost when co-assembling with EPT, which confirmed our hypothesis again that the histidine might interact with the EPT, and cause the slight change of secondary structure. On the other hand, the secondary structure changed to the random coil when we adjusted the pH from 8.0 to 4.0, which indicates the protonated histidine triggered the nano-complex of peptide and EPT to disassemble, and most EPT could be released. In order to confirm the pH-trigged drug loading and releasing behavior, the fluorescence of deprotonated and protonated EPT were recorded during the pH changing process. As shown in Figure 4E, at pH 8.0, a peak of ~430 nm (Peak of deprotonated EPT) was observed for the NP1EPT nano-complex, there is no peak of the deprotonated EPT since it is insoluble in water,58 which indicates NP1 peptide encapsulated EPT in the aqueous environment. Moreover, the peak of ~430 nm for the nano-complex disappeared when we adjusted the pH from 8.0 to 4.0, yet


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another peak of ~540 nm (Peak of protonated EPT) appeared, which is very similar to the only EPT at pH 4.0. Combining the AFM and fluorescence results, we conclude that NP1 peptide could co-assemble with/encapsulate the EPT at pH 8.0, and dis-assemble with/release the drug at 4.0 by switching its deprotonated histidine to protonated ones. Previously, our lab reported the co-assembly delivery for EPT and siRNA, determined the 𝜁potential of NP1-EPT (80 µM of NP1, and the co-assembly molar ratio is NP1: EPT = 0.375:1) is ~38.5 mV.42 However, the positively 𝜁-potential indicates the positively charged arginine is outward the surface and the high surface charge may cause serum protein adsorption and hemolysis.59-60 The PEGylated and DEGylated nanoparticles were synthesized in our lab to improve the serum protein adsorption and hemolysis issue. It the previous report, NP1 peptide could interact with negatively charged siRNAs by positively charged arginine and successfully deliver it into cells by the endocytic mechanism.41 In our case, a drug was loaded into the hydrophobic core and histidine part, with the positively charged arginine outward. The hydrodynamic diameter of noncomplex is much higher (~117.9 nm) than AFM result with low polydispersity index. It might be hydrophilic arginine outward increases the intensity-based diameter of NP1-EPT detected by DLS (Figure S4B). As shown in Figure S4C, the zeta potential of the nano-complex is 37.9 ± 5.481 mV (n=3). In order to decrease the serum effect, for the in vitro experiment in this manuscript and our previous work,42 we added the nano-complex without serum into cells and function for 3 h. After that, we completed the medium with 20% FBS to make the final FBS concentration 10% for the overnight incubation and function. Here we propose a four steps NP1-EPT co-assembly mechanism: i) the stearic acid segments drive the co-assembly or -disassembly of peptide and EPT by the hydrophobic interaction; ii) the deprotonated histidine segments provide the hydrogen bonding to form the uniform globular nanostructure or interacts with EPT by π-π stacking to form NP1-EPT nano-complex; iii) the arginine segments consist of a positively charged shell to avoid aggregations, finally the NP1 nanostructure or NP1-EPT nano-complex formed; iv) the protonated histidine could disassemble the nano-complex which induces the drug release. The out layer of the arginine amino acids in the system might promote the nano-complex directly translocate into the plasma membrane with no nano-complex trapped into the endosome, for a more efficient uptake.30 Furthermore, the positively charged nano-complex prefer more negatively charged cancer cells, which might be used for the target delivery.55 Additionally, the


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pH-triggered disassembly and drug release might provide a stimulated strategy for a more effective drug delivery.61 3.4 Characterizations and Mechanism of Direct Translocation Intracellular uptake of NP1EPT Nano-complex

Figure 5. (A) FACS results show the cellular uptake of the EPT(green), NP1(dark blue), NP1-EPT-pH 8.0(sky blue), and two-times diluted NP1-EPT-pH 8.0(orange) in CHO-K1 cells. (B) CHO-K1 cellular uptake of NP1-EPTpH 8.0 treated with inhibitors to inhibit various endocytosis ways. (E) Temperature effects of the NP1-EPT-pH 8.0 cellular uptake on CHO-K1. (B), (D) and (F) are the corresponding statistic results of (A), (C) and (E), n = 3.

Uptake mechanisms and intracellular pathways of nano drug carriers are of great interest for the design of a high-performance delivery system for cancer therapy. To further understand how the NP1-EPT nano-complex goes through the cell membrane, fluorescence-activated cell sorting and confocal microscopy techniques were used. First, the uptake of NP1-EPT nano-complex and two-times diluted NP1-EPT nano-complex were investigated along with two controls: deprotonated EPT and NP1. The peak of the NP1EPT shift to right, with the intensity ~24, indicates the efficient uptake of NP1-EPT (Figure 5A, B), while there is no shift of the NP1 and EPT case, indicating no uptake of them. Also, we


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found it is a concentration-dependent uptake when we diluted the NP1-EPT two-times, the uptake intensity decreased to ~13, with the peak shift to left compared with the NP1-EPT (Figure 5A, B). Furthermore, various chemical endocytosis inhibitors were used to study the uptake mechanism of NP1-EPT nano-complex; and the concentration of inhibitors, along with the according blocked pathway are summarized in Table S1. In general, the clathrin-mediated pathway could be blocked by chlorpromazine by avoiding forming the endocytosis sag coated with clathrin.62 Macropinocytosis and phagocytosis pathway could be inhibited by EIPA (5-(Nethyl-N-isopropyl) amiloride) by inhibiting the Na+/H+ exchangers.63 Both of the macropinocytosis and caveolae-mediated endocytosis could be inhibited by the methyl-βcyclodextrin (MβCD) by depleting cholesterol, which is required for the two pathways.62 The microtubule polymerization involved the lysosome trafficking could be disrupted by the nocodazole.64 As shown in Figure 5C, D, no changes of the uptake intensities when treated with the inhibitors, indicate our nano-complex did not go through the cell membrane by the endocytosis pathway. In addition, the uptake of NP1-EPT complex was found to be temperature independent as shown in Figure 5E, F. As we reported previously, EPT in DMSO go through the cell membrane by passive diffusion, it is a concentration-dependent and temperature-independent pathway.65 Here we also checked how the inhibitor affect the uptake of EPT (in DMSO) and found there was almost no effect on the EPT uptake (Figure S6A). Similar to the EPT (in DMSO), the deprotonated NP1-EPT go through the cell membrane into the plasma by the direct translocation pathway.


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Figure 6. (A) Intracellular delivery of NP1-EPT-pH 8.0 in CHO-K1 cells. EPT and lysotracker are shown in green and red, respectively. (B) The schematic representation of the process of how NP1-EPT goes through the cell membrane. (C) Cell cytotoxicity of the EPT, NP1, and different-times diluted NP1-EPT-pH 8.0 on A549 and CHOK1 cells.

Confocal microscopy images in Figure 6A show that cells treated with the labeled Lysotracker Red revealed a punctate pattern with red color and the deprotonated EPT delivery by deprotonated NP1 revealed a punctate distribution with green color. The majority of deprotonated EPT appeared to be outside the endosomal/lysosomal vesicles as demonstrated by its extensive colocalization with the LysoTracker Red, which is visualized as yellow punctuate fluorescence. The similar confocal microscopy results (Figure S6B) were observed in the EPT only (in DMSO). Confocal microscopy images confirm the direct translocation uptake mechanism involved in the uptake of NP1-EPT nano-complex. The Figure 6B and Figure S6C present the process of how EPT-NP1 and EPT (in DMSO) directly translocate through the cell membrane, respectively. As a result, the cancer drug EPT will not be trapped into the endosome and could be easier to release and function. For comparison, the endocytosis pathway of siRNA delivery was


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determined when using NP1 as a carrier.41 The negatively charged siRNA exhausted the positively charged arginine, and no enough positively charge to interact with the negatively charged membrane to act as a penetration tool. The previous study focusing on the function of arginine-rich CCPs such as the R9-Tat peptide (GRRRRRRRRRPPQ)32 has shown that they exhibit great potential in translocation ability.31 In EPT drug delivery case, all the positively charged arginine exposed outward and function as a perpetration peptide to penetrate the cell membrane. In addition, researchers reported that the size of the nanoparticles affects the translation pathway,66 and the smaller size, the higher chances to directly penetrate the cell.67 In our case, the NP1-EPT nano-complex is ~34.43 nm, which is sufficiently small to penetrate the cell membrane. The cells cytotoxicity of NP1-EPT nano-complex (pH 8.0) was measured. After the uptake of NP1-EPT, it presented toxicity to A549 and CHO-K1 cell lines (Figure 6C). The neutral EPT (in water) and NP1 peptide at pH 8.0 were applied as the control and showed low toxicity to the cells. Optical images of the NP1-EPT at pH 8.0 treated A549 and CHO-K1 cells are shown in Figure S7: more pink means more dead cells. And NP1-EPT (NP1: 1.79 µM, EPT: 10 µM) could kill ~more than ~85% of the cancer cells. The IC50 are ~ 0.60 µM and ~0.48 µM of EPT on CHO-K1 and A549 cells, respectively. In the future, the NP1-EPT nano-complex should be tested in the tumor model, the disassembly behavior which is triggered by the low pH environment might help target cancer cells (where the pH is lower than the normal cells55-56) and releasing a drug to specific tumor environment to reduce the side effect. At the tumor microenvironment, the NP1 will be disassembled. The drug ellipticine will be released in the tumor microenvironment. We investigated how the ellipticine itself transport into the CHO-K1 and A549 cells and found it is a passive transport and cholesterol (or other elements) in the cell membrane might affect the transport.57 Here one possible advantage is that the arginine-rich penetrating peptide might promote penetrate extravascular tumor tissue, which is worth for further investigation. 4. CONCLUSIONS In this work, a systemically designed cell-penetrating peptide, NP1, was used to deliver the cancer drug ellipticine via a direct translocate uptake mechanism. It is demonstrated that the pHresponsive self-assembly (or disassembly) of NP1 is the trigger of encapsulation (or release) of


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drug EPT. Moreover, this outward positively charged NP1-EPT could interact with the negatively charged cell membrane to directly translocate it without being trapped into the endosome to inhibit the viability of cancer cells A549 and CHO-K1. Our stimuli-responsive peptide is a highly efficient tool for drug encapsulation and delivery, and it has great potential for future applications such as nanomedicine, targeted cancer therapy, and cosmetics industry. ASSOCIATED CONTENT Supporting Information: Figure S1 to S7 and Table S1. AUTHOR INFORMATION Corresponding Author [email protected] (L.Z) [email protected] (P.O) [email protected] (P.C) Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Research Chairs program, the Canadian Foundation for Innovation (CFI) for funding, and Prof. Guy Guillemette for providing circular dichroism spectroscopy. REFERENCES (1) Farokhzad, O. C.; Langer, R. Impact of nanotechnology on drug delivery. Acs Nano 2009, 3 (1), 16-20. (2) Shi, J.; Votruba, A. R.; Farokhzad, O. C.; Langer, R. Nanotechnology in drug delivery and tissue engineering: from discovery to applications. Nano Lett 2010, 10 (9), 3223-3230. (3) Alderton, G. K. Nanotechnology: Improving drug delivery with algae. Nat. Rev. Cancer 2016, 16 (1), 5-5. (4) Yang, K.; Feng, L.; Liu, Z. Stimuli responsive drug delivery systems based on nano-graphene for cancer therapy. Adv. Drug Deliv. Rev. 2016. (5) Pattni, B. S.; Chupin, V. V.; Torchilin, V. P. New developments in liposomal drug delivery. Chem. Rev. 2015, 115 (19), 10938-10966. (6) Hertel, S. P.; Winter, G.; Friess, W. Protein stability in pulmonary drug delivery via nebulization. Adv. Drug Deliv. Rev. 2015, 93, 79-94.


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(27) Maeda, H.; Nakamura, H.; Fang, J. The EPR effect for macromolecular drug delivery to solid tumors: Improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo. Adv. Drug Deliv. Rev. 2013, 65 (1), 71-79. (28) Boisguérin, P.; Deshayes, S.; Gait, M. J.; O'Donovan, L.; Godfrey, C.; Betts, C. A.; Wood, M. J.; Lebleu, B. Delivery of therapeutic oligonucleotides with cell penetrating peptides. Adv. Drug Deliv. Rev. 2015, 87, 52-67. (29) Komin, A.; Russell, L.; Hristova, K.; Searson, P. Peptide-based strategies for enhanced cell uptake, transcellular transport, and circulation: Mechanisms and challenges. Adv. Drug Deliv. Rev. 2016. (30) Ruoslahti, E. Tumor penetrating peptides for improved drug delivery. Adv. Drug Deliv. Rev. 2016. (31) Wang, F.; Wang, Y.; Zhang, X.; Zhang, W.; Guo, S.; Jin, F. Recent progress of cellpenetrating peptides as new carriers for intracellular cargo delivery. Journal of Controlled Release 2014, 174, 126-136. (32) Futaki, S. Membrane-permeable arginine-rich peptides and the translocation mechanisms. Adv. Drug Deliv. Rev. 2005, 57 (4), 547-558. (33) Venditto, V. J.; Szoka, F. C. Cancer nanomedicines: so many papers and so few drugs! Adv. Drug Deliv. Rev. 2013, 65 (1), 80-88. (34) Wang, A. Z.; Langer, R.; Farokhzad, O. C. Nanoparticle delivery of cancer drugs. Annual review of medicine 2012, 63, 185-198. (35) Jiang, T.; Zhang, Z.; Zhang, Y.; Lv, H.; Zhou, J.; Li, C.; Hou, L.; Zhang, Q. Dual-functional liposomes based on pH-responsive cell-penetrating peptide and hyaluronic acid for tumortargeted anticancer drug delivery. Biomaterials 2012, 33 (36), 9246-9258. (36) Sugahara, K. N.; Teesalu, T.; Karmali, P. P.; Kotamraju, V. R.; Agemy, L.; Greenwald, D. R.; Ruoslahti, E. Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs. Science (New York, N.Y.) 2010, 328 (5981), 1031-1035. (37) Amin, M.; Mansourian, M.; Koning, G. A.; Badiee, A.; Jaafari, M. R.; ten Hagen, T. L. Development of a novel cyclic RGD peptide for multiple targeting approaches of liposomes to tumor region. Journal of Controlled Release 2015, 220, 308-315. (38) Gu, F. X.; Karnik, R.; Wang, A. Z.; Alexis, F.; Levy-Nissenbaum, E.; Hong, S.; Langer, R. S.; Farokhzad, O. C. Targeted nanoparticles for cancer therapy. Nano today 2007, 2 (3), 14-21. (39) Chang, Y. S.; Graves, B.; Guerlavais, V.; Tovar, C.; Packman, K.; To, K.-H.; Olson, K. A.; Kesavan, K.; Gangurde, P.; Mukherjee, A. Stapled α− helical peptide drug development: A potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (36), E3445-E3454. (40) Lu, S.; Bennett, W.; Ding, Y.; Zhang, L.; Fan, H. Y.; Zhao, D.; Zheng, T.; Ouyang, P. K.; Li, J.; Wu, Y. Design and Characterization of a Multifunctional pH‐Triggered Peptide C8 for Selective Anticancer Activity. Advanced healthcare materials 2015, 4 (17), 2709-2718. (41) Chu, D.; Xu, W.; Pan, R.; Ding, Y.; Sui, W.; Chen, P. Rational modification of oligoarginine for highly efficient siRNA delivery: structure–activity relationship and mechanism of intracellular trafficking of siRNA. Nanomedicine: Nanotechnology, Biology and Medicine 2015, 11 (2), 435-446. (42) Chu, D.; Xu, W.; Pan, R.; Chen, P. Co-delivery of drug nanoparticles and siRNA mediated by a modified cell penetrating peptide for inhibiting cancer cell proliferation. RSC Adv. 2015, 5 (26), 20554-20556. (43) Lu, S.; Wang, H.; Sheng, Y. B.; Liu, M. Y.; Chen, P. Molecular binding of self-assembling peptide EAK16-II with anticancer agent EPT and its implication in cancer cell inhibition. Journal of Controlled Release 2012, 160 (1), 33-40. (44) Fung, S. Y.; Yang, H.; Sadatmousavi, P.; Sheng, Y.; Mamo, T.; Nazarian, R.; Chen, P. Amino Acid Pairing for De Novo Design of Self-Assembling Peptides and Their Drug Delivery Potential. Adv Funct Mater 2011, 21 (13), 2456-2464. (45) Hansen, A. L.; Kay, L. E. Measurement of histidine pKa values and tautomer populations in invisible protein states. Proc. Natl. Acad. Sci. U.S.A. 2014, 111 (17), E1705-E1712.


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TOC


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Histidine-Rich Cell-Penetrating Peptide for Cancer Drug Delivery and

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